Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Transformation and plant availability of copper in pasture soils A Thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Soil Science at Massey University, Palmenton North, New Zealand o MasseyUniversity Md. Afiqur Rahman Khan 2001 II ABSTRACT The response of pasture to copper (eu) fertilisers in most soils is very short-lived necessitating frequent applications of eu fertilisers. The short-term response to eu by plants is attributed to the ready adsorption of eu by organic matter and other soil components. eu distribution among these different fractions and the relative availability of these fractions for plant uptake, are fundamental to an understanding of the transformation of eu in soil. As eu has not been routinely analysed in the past, there is no standard soil test extractant in New Zealand. The use of single chemical extractions in routine soil analysis is a fast and simple way to evaluate the availability of soil nutrients to plants. Farmers require accurate information on the length of time that eu applications remain fully effective in order to supply the eu required for the grazing animal. Pasture provides the main source of eu for grazing animals. There is a need to define the rates of change in the effectiveness of eu fertiliser over the range of soil and climatic conditions encountered in New Zealand. The specific objectives of the study were: (i) to investigate the effect of soil components on the sorption and desorption of added and native eu in soils; (ii) to examine the soil and fertiliser properties that influence the effectiveness of eu topdressing in terms of increasing eu uptake by pastures; (iii) to determine the transformation of Cu added through fertiliser applied to soils; (iv) to quantify the forms of Cu in soils using a sequential fractionation procedure; (v) to identify the forms of eu in various soil test extractants and to assess the efficiency of these soil test extractants in predicting Cu uptake; (vi) to estimate the effects of N and P fertilisers on the uptake of native Cu by ryegrass; (vii) to examine at the residual effectiveness of two Cu source fertilisers as influenced by N fertiliser, lime and EDTA additions; and (viii) to evaluate the seasonal influence on the availability of native and added Cu to pasture. Copper sorption and desorption isotherms were determined for a number of soils (M an aw atu , Tokomaru, Ramiha, Ngamoka and Mangamahu) before and after the removal of various soil components. A series of glasshouse and field trials were carried out using three Cu sources, five soils and four Cu levels. The dry matter yields of ryegrass and Cu concentration in the herbage were monitored over a number of harvests. The soil was collected from the glasshouse trial at various intervals and analysed for different fractions (exchangeable, organic, oxide and residual) and were III extracted with vanous soil test extractants. Copper extracted from the soils was correlated with the eu concentration in the herbage. A second glasshouse trial with two soils, four levels of nitrogen (N) and five levels of phosphate fertiliser was conducted. The dry matter (DM) yield and the Cu concentration in the ryegrass were measured. The effects of N fertiliser, lime and EDTA addition on the availability of residual Cu was investigated in a separate glasshouse trial. A series of field trials were conducted, in the same paddocks, to examine the effect of season on the uptake of Cu from two eu sources. The differences in the chemical characteristics of the soils resulted in some variation in the sorption and desorption of Cu between the soils. Soil pH, organic carbon, iron and aluminium oxides play a major role in the sorption and desorption of Cu in soils. Organic matter and oxides are important in adsorption reactions, but differences exist in their relative importance. Increasing levels of Cu increased the Cu concentration in plants. Sources of Cu fertiliser have a significant effect on DM yield, and Cu concentration at all harvests. Soil pH, organic matter, CEC and clay content correlated with Cu concentration in plants. Cu uptake in grasses decreased with time after fertiliser application. Organic and oxide bound eu contributed >80% of total Cu in all the soils. The organically bound eu fraction was highest in soils with high levels of organic matter. Both the organically bound and the oxide bound fractions of Cu decreased with time after fertiliser application, indicating a possible decrease in the availability of Cu. Soil exchangeable, organic and oxide bound fractions of Cu were correlated with soil organic matter, CEC and clay content. Both the organic and oxide bound Cu were correlated with plant Cu uptake. The major forms of Cu extracted by the soil test reagents include organically bound, followed by oxide bound, residual and exchangeable forms. The ratios of different forms of Cu strongly suggest that Cu is residing mainly in the organic fonn and increases in this order: exchangeable < residual < oxide < organic. The efficiency of chemical extractants in extracting the eu from the soil followed: TEA-DTPA > Mehlich-3 > Mehlich-l > O.02M SrClz > O.lM HCI > 1 .0M NH4N03 > O.OIMCaCh > O.IM NaN03 > O.OIM Ca(N03)2. Increasing levels of both N and P fertilisers increased both the DM yield and the uptake of native Cu. Increasing levels of N increased both the DM yields and the Cu IV concentration in soils with residual eu. The effect on eu concentration persisted beyond the first cut only at the highest N addition. Increasing levels of lime increased the DM yield of pasture, but decreased the eu concentration in pasture at the highest level of lime addition. Increasing levels of EDT A increased the eu concentration in soils and thereby increased the eu concentration in the pasture. The application of 1 000 kg lime ha·' and 50 kg N ha-I was very effective in enhancing plant availability of residual eu in soils, but EDT A increased the plant available eu to toxic levels. The highest application rate of lime and N fertiliser decreased the exchangeable and free eu in the Ngamoka soil, but EDT A showed the opposite effect. In the field experiment eu levels have no significant effect on DM yield during all seasons. The field study shows differences in seasonal response to added eu. Increasing levels of eu increased the eu concentration in pasture. Types of eu fertiliser have a significant effect on eu concentration. The differences in pasture growth and eu concentrations in plants seasonally could be attributed to the differences in air and soil temperature, soil moisture content and solar radiation patterns within the trial period. Adsorption and de sorption reactions are likely to be the major factors controlling the availability of eu to plants. The major forms of eu that can be extracted by soil test extractants are the organically bound, followed by oxide bound, residual and exchangeable forms. Organic and oxide bound eu were the main sources of plant available eu. The uptake of native eu and residual eu from soils showed that N and lime at 50 kg N ha-' and 1 000 kg lime ha-1 levels increased the eu concentration, and EDTA also increased the plant available eu to toxic levels. The effect ofN, lime, and EDT A on the availability of residual eu in ryegrass needs further investigation. Both the glasshouse and field trials indicate that eu uptake is internally regulated by the growth of pasture and externally affected by the transformation of eu in soils. v DEDICATED to my BELOVED PARENTS VI ACKNOWLEDGMENTS It is the heartfelt gratitude that I acknowledged the many people who have made the completion of this study possible. Firstly and most importantly to Associate Professor Nanthi S. Bolan for his unfailing help and advice, wholehearted guidance, patience and friendship during my study period. To Dr. Alec D. Mackay for his very helpful guidance and encouragement. His advice was often keenly sought throughout the study. To Associate Professor M. 1. Hedley, Dr. A. Palmer and Dr. P. Loganathan for their valuable guidance and suggestions. To AgResearch for allowing the collection of soil samples and Massey University Pasture Growth Research Unit for providing the facility for the field trial. Members of the Soil and Earth Science Group, particularly to Messrs L. D. Currie, James A. Hanly, B. Toes, 1. Furkert, Ross J. Wallace and Mrs. Anne West and Mrs. Glenys C. Wallace for their help with field and laboratory works. To Mr. Mike Bretherton for his willing assistance in solving computer related problems. To Mr. Malcolm Boag for his comprehensive proof reading of the thesis. The staff and postgraduate students, past and present, of the Soil and Earth Science Group, all of whom contributed in a variety ways. To my wife, Salma and son, Sami and daughter, Anisa for their patience and encouragement throughout the study. To the Academic Board and University Council for awarding the Helen E Akers and Johannes August Anderson Ph D Scholarships. To New Zealand Society of Soil Science for awarding the Summit Quinphos Bursary "1 999". And to Mankind Trading Company and Massey University Research Fund (MURF) for providing the financial support for the analytical costs of my research. VII TABLE OF CONTENTS ABSTRACT .................................................................................................................... n ACKN"OWLEDGMENTS ........................................................................................... VI TABLE OF CONTENTS ............................................................................................ VII LIST OF FIGURE-S ................................................................................................... XVI LIST OF TABLES ............................................................................................... ...... xx.I LIST OF PLATES .................................................................................................. XXIV CHAPTER 1 GENERAL INTRODUCTION ........... ............. 1 1 . 1 1 .2 1 .2 . 1 1 .2.2 1 .2.3 1 .2.4 IN"TRODUCTION . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . 1 OBJECTIVES OF VARIOUS EXPERIMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Adsorption and desorption of eu in pasture soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Plant availability of Cu from different Cu fertilisers in pasture soils . . . 3 Transfonnation and plant uptake of Cu in different soils . . . . . . .. . . . . .... . . . . . 3 Effect of nitrogen, phosphorus fertilisers, lime, and EDT A on the availability of native Cu and residual Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 .2 .5 Seasonal response of copper availability in pasture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 CHAPTER 2 LITERATURE REVIEW ................................ 6 2. 1 IN"TRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . ... . . . . . . . . . . . .. . . ... ..... . . . 6 2.2 SOURCES OF COPPER IN" SOILS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 6 2.2. 1 Soil parent materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.2 Ore minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 7 2.2.3 Fertilisers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2.2.4 Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2.2.5 Sewage sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 2.2.6 Composts and agricultural wastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2.2.7 Wood preservatives . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2.2.8 Atmospheric deposition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2.3 COPPER IN" SOILS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . .. 1 2 2.3 . 1 Total Cu in soils . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2 2 .3 .2 Fonns of soil Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 2.3.2. 1 2.3.2.2 2.3.2.3 2.3.2.4 2.3.2.5 2.3.2.6 2 .3 .3 2.3.3. 1 2.3.3.2 2.3.3.3 2.3.3.4 VIII Soil solution Cu ................. .. .. ... . ......... ..... . . . ... ............ . ............ ...... .. . 14 Exchangeable Cu . ... .. .. .. .... ........... . . .......... . . . ....... . .... .................. .. .... 17 Specifically adsorbed Cu ........ . .. . .. . . . ......... .. ............ ............... ... ... . ... 17 Occluded Cu ....................... . . . . ............ . . .. . . .... . ... . ... .. ....................... .. 17 Cu in the structure of silicate clay and primary minerals .... .. ....... . 18 Cu associated with biomass .......... . .. . .... .. . . . ..... . ... . ....... . ........ .... . ... ... 18 Determination of soil Cu by a sequential fractionation scheme . . . . . . . . . 1 9 Exchangeable fraction .... . ...... ................. . .......... ............ ...... ......... .. 23 Organic fractions . ......... . . ...... , . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Oxide fractions ... .......... ........... ...... .. . ........ . ........... ...... ................ . ... . 24 Residual fractions .......... . ...... ........... . ... . ... ........................ .......... . . ... 24 2 .3 .4 Determination of total Cu ......... .. . .. ......... .. ...... . . . .............................. 24 2 .3 .5 Factors affecting forms ofCu in soils . ... ..... ...... . ..... ..... . .. ..... . . .... ...... . 25 2.3.5. 1 Cu addition to soils . ... ................... . ........... ... . ....... ...... ..................... 26 2.3.5.2 Soil pH ........ . .................................................................................... 26 2.3.5.3 Organic matter amendments ......... . ..... . . ..... . ........ ........... . ........ ... .... . 27 2.3.5.4 Effect of contact time ..... ....................................................... .. . ... .... . 27 2.4 REACTIONS OF COPPER IN SOILS .......... . ........ . .. ...... .... ........... ...... .. . 28 2.4. 1 Cu adsorption and fixation by soils ... .... ........ ..... .. . . ..... . ................ ... . 29 2.4. 1. 1 Formation ofCu complexes in soils .. ..... .... ....... ........................ .. .... 32 2.4. 1.2 Adsorption ofCu by soil components . . . . .. .................. ................... .. 33 2.4.1.2.1 Sorption by organic matter . ... ....... . ..... . .. . . . . . .... .. . . . . . ... . . . . . . ..... . .... .. . ..... . . ... . . . ....... 33 2.4.1.2.2 Sorption by hydrous oxides ... . ... . . . . ........... ........ . . . . . .... . . ...... . .... .... . . . ...... .. . . . ... .... 35 2.4.1.2.3 Sorption by silicate clay rninerals . . ... . . . ........ . . . ... . ... . ........... ... . ... ... . . . . . . . . . . ....... . . . 36 2.4.2 Desorption ofCu .................... . ........... ..... ............ .. . ....... ............. ..... 37 2.4.3 Cu chelate equilibria in aerobic and anaerobic soils .... .... . ......... ....... 38 2 .5 PLANT AVAILABILITY OF COPPER IN SOILS . . .... . .. ... . .. ........... . ...... 39 2.5 . 1 Availability of native Cu ............. .................................. ............ . ...... 39 2 .5 .2 Availability of fertiliser Cu .. . .. . . . . .... .. .. . . .... ..... . . . ...... . . . .. .... . . . .... ..... .... 40 2 .5 .3 Cu uptake and translocation ... .. . ..... . ......... . ....... .. . . .... . .... . .... .. .... ...... .. 41 2.5.3. 1 Cu absorption by plant roots ......... ...... .................... ... .......... . . .. .. .... 41 2.5.3.2 Translocation ofCu in plants .. .. . . . .. . ..... . . . . . . ... .. .... .. .. . .. . ... . .... ........ . . . . 42 2.5.3.3 Interaction with other ions and nutrients . . . ......... ... .. . ........ . . .. . . . . ..... . 43 2.5 .4 Effect ofCu fertiliser on DM yield and Cu concentration . ..... .......... . 45 2 .5 .5 Residual effectiveness of applied Cu ... .............. ........ . . .. .................. . 47 IX 2 .5 .6 Biochemical functions ofCu .... . . . ..................................................... 47 2 .5 .7 Deficiency and toxicity ofCu in plants . . .... ............ ....... . .................. 48 2 .5 .8 Copper and animal health .. ...... ..... ...... .......... ......... . ................. � ....... 49 2 .5 .9 Soil and plant tests for Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . 50 2.5.9.1 Water soluble Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 2.5.9.2 Exchangeable Cu .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 2.5.9.3 Complexed, chelated and adsorbed Cu . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.6 ENVIRONMENTAL FACTORS AFFECTING REACTIONS AND THE AVAILABILITY OF COPPER IN SOILS .......... . ..................... . .......... ........... .... 54 2.6. 1 Soil moisture content and redox reaction . ..................... .................... 54 2.6.2 Seasonal variation and soil temperature .......................................... . 55 2.6 .3 Radiation ................... ......... . . ........................................................... 56 2.6.4 Plant and other organisms .... . ............................................... ... ......... 57 2.6.4. 1 Mycorrhizae . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 CHAPTER 3 ADSORPTION AND DESORPTION OF COPPER IN PASTURE SOILS ........................................... 60 3 . 1 INTRODUCTION ............. . .... . .............. .............. ..... . .... . ......................... 60 3 . 1 . 1 Cu adsorption ..... .......... . . . .... .. .... .............................. . . ................ ...... 60 3 . 1 .2 Cu desorption ........ . ......... . . .. . ... . . . ....... ........ . .. ................ ................... 61 3 .2 MATERIALS AND METHODS ....... .. .......... ......... ....... .. ............ ... ......... 62 3 .2 . 1 3 .2 .2 3 .2 .3 3 .2.4 3.2 .5 3 .2 .6 3 .2 .7 3 .2 .8 3 .2 .9 3 .2 . 1 0 Soils used ................... ............... . ............... ......... .. ........................... 62 Soil physical and chemical analysis ....................... . .. ................... .... 63 Time-dependent experiment on Cu sorption ............................ ......... 63 pH dependent copper sorption isotherm for soils .......... . ....... . ........... 64 Copper sorption isotherm for soil components ... ............ . . .... ......... . .. 64 Fractionation of soil Cu .. . . ... ............... . . . . ........... . .............. ............... 65 Desorption of native and added Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Desorption study for incubated soil . . ... . . .. . .............. . . . . .... .............. . ... 66 Desorption study at different pH levels .... ......... ..... ...... . ................... 67 Extractable soil Cu ............ ..................... . ........ . . .... ......................... . . 67 3.2.10.1 O. 04M EDTA extracting solution . .... ......... ... .... .. . .. . . . . ... . ...... . . .. . ...... 67 3.2. 10.2 Mehlich-1 extracting solution ...... .. .. ... ... . . . . ... ... . .. ............ . ....... ... . .. . 67 3.2. 10.3 Mehlich-3 extracting solution .. . ...... . . ... ... ... ....... ... . ....... . .. . . . . . . . ..... .. . 68 x 3.2. 10.4 0. 1M HCI extractant ..... . . . ...................... ...... . . . .......... ...... ................ 68 3.2. 10.5 DTPA extracting solution ..................... ........... ......... ...... ... ............. 68 3.3 3 .3 . 1 3 .3 .2 3 .3 .3 RESULTS AND DISCUSSION ... .. ....... ......... . . ....... . . .... ........ ....... ........... 68 3.3.3. 1 3.3.3.2 3.3.3.3 3.3.3.4 3.3.3.5 3.3 .4 3.3.4. 1 3.3.4.2 3 .3 .5 3.3.5. 1 3.3.5.2 3.3.5.3 General description of soils ... .. .................. . ...... . . ....... ...... .. . .. ... ......... 68 Soil properties .. ........ . . .. . . ........................... .. . ............. .. ........... .. ........ 69 Adsorption ....................................................................................... 70 Copper sorption as a function of time .................. ........................... 70 Adsorption isotherms for the original soils .... ................................. 72 Copper sorption affected by soil properties ............. ...................... . 73 Soil components and Cu sorption ............ .... .... ............................... 74 pH dependent adsorption isotherms for different soils ...... ............. 79 Distribution coefficients .... ..... ...... .... ... .... ............. . '" . . . . . . . . . . . . . . . . . . . . . . . . 84 Distribution coefficients for the original soils ..... ......................... .. 84 Distribution coefficients from the pH dependent experiment ......... 85 Desorption ........ .............................................. ................... .... , . ... . .. . . 86 Desorption of native Cu ...... . ....... .......... . .................. ..................... .. 86 Desorption of added Cu .............. .... .................. ............................ .. 89 Effect of contact time on desorption of added Cu ................ . ....... . . . 92 3.3.5.4 pH dependent desorption for different soils ............. .... .... ............ ... 93 3.4 CONCLUSION AND FURTHER STUDy ......... ...... ......... ................ ... . . 96 CHAPTER 4 PLANT AVAILABILITY OF COPPER FROM DIFFERENT COPPER FERTILISERS IN PASTURE SOILS . 4. 1 4.2 4.2. 1 4.2.2 4.2.3 4.2.4 4.2.5 4.2.6 ................................................................... 98 INTRODUCTION ................................ .... .... . . .... .............. .......... . .......... . . 98 MATERIALS AND METHODS . ... ............. . ........ .. ......... . . .... ...... . . ........ .. 99 Soil collection and preparation ............. .................. ... ....................... 99 Copper fertilisers used ....................... .................... ................ .......... 99 Plant growth experiment . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . 1 00 Grass and soil sample preparation ........... ........ ................... ......... ... 102 Ryegrass samples for Cu analysis . . . ... . .. . . .. . ... ... .. .... ...... . . ..... . . ... ...... 102 Chemical Analysis .... ..... ........................ . . ...... . .......... .. . . ................. 102 4.2 .7 Statistical analysis . ........ . ...... .............. ...... ... .................. .. ............... 103 4.3 RESULTS AND DISCUSSION .............. . . ...... . .................... ................. 103 XI 4.3 . 1 Initial soil characteristics . .... . . . ........... ........................ ........ ............ 1 03 4.3.2 Dry matter yield ................................... ..... .................................... . 103 4.3.2. 1 Effect of the soil types ......... ........... ......... .... .... ... . ...... . ... ... ...... ....... 103 4.3.2.2 Effect of types of Cu fertiliser . . ..... ... ............... ..... ......... . . ... ... ........ 105 4.3.2.3 Effect of fertiliser rates ................................ . ..... ......... .... . . ............. 105 4.3.3 Copper concentration in rye grass ....... ............................................ 107 4.3.3. 1 Effect of the soil types .. ........ ........................... ........... . .... .............. 107 4.3.3.2 Effect of types of Cu fertiliser ........... . .... ......... .......... . ... . . . ............. 110 4.3.3.3 Effect of fertiliser rates ........... ... ..... .............. ........... ...... ... .......... ... 111 4.3.4 Copper uptake ...... .......................................................................... 1 1 2 4.3.4.1 Effect of the soil types ...... ...... .. . .. . ........... ..... ................. ... .... ...... .. . 112 4.3.4.2 Effect of types of Cu fertiliser .... . . ............. . . ............... ...... ............. 114 4.3.4.3 Effect of fertiliser rates . ................ ............. ............. . ...... . .. .. ......... .. 115 4.3 .5 Recovery ofCu fertilisers .......................................... .................... 1 1 6 4.4 CONCLUSION AND FURTHER STUDy ........................... .............. .. 1 1 8 CHAPTER 5 TRANSFORMATION AND PLANT UPTAKE OF COPPER IN SOILS ..................................................... 119 5 . 1 5 .2 INTRODUCTION ........... ..... ...... ........... ................ ............................ .... 1 1 9 MATERIALS AND METHODS ............ .................... ......... ........... ....... 120 5 .2 . 1 5 .2 .2 5 .2 .3 5 .2 .4 5 .2 .5 5 .2 .6 5 .2 .7 Soil sampling from pots ................... . .... . ..... ....................... . ....... . ... 120 0.005 M EDTA-extractable Cu ... .................................... ... . ....... . ... 1 20 Total soil Cu .................................................................................. 1 20 Fractionation of soil eu .......... .......................... ................. ..... . ..... . 120 Total Cu of particle size fractions ................ ....................... ............ 1 2 1 Chemical analysis . .............. . .............................................. ........... . 1 2 1 Statistical analysis ...................................................... ........ ............ 1 2 1 5.3 RESULTS AND DISCUSSION ...... .......... ........................ .................... 1 2 1 5 .3 . 1 Recovery of Cu by fractionation ... ................................. ..... ........... 1 2 1 5.3. 1. 1 Recovery of native Cu . . ....... . .. ... ...... ...... .... . . . ............... .......... .. ...... 121 5.3. 1.2 5.3.2 5.3.2. 1 5.3.2.2 Recovery of applied Cu during fractionation .. . . ...... . ... ....... . ...... . . . . 122 Distribution of native Cu .................................... . ........................... 1 23 Fractionation of control soil . . . .... .. ... . ............. . ..... . . .. ............. ...... . . 123 Total Cu in particle size fractions . ........ ... ........ ................ . ... . ...... .. 124 XII 5 .3 .3 Distribution of applied Cu . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25 5.3.3. 1 Exchangeable Cu .... . . . .... ... .............. . . . . . . . . ..... . .... .... . ... ...... . . ... . ... ... . . 125 5.3.3.2 Organic bound Cu ...... . .. .. ..... . ....... ... ... ... ..... . . ...... .... . . ... . . .. .... .. . ....... 128 5.3.3.3 Oxide bound Cu . ... ... ... . ... . ....... ........... . ......... ... . . . ..... . . . . ....... ... . .. . . ... 131 5.3.3.4 5 .3 .4 5 .3 .5 5 .3 .6 Residual Cu . . . . ....................... ........................................................ 134 Total sum of Cu fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 1 36 E ffect of soil properties on fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Plant availability to Cu fractions . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . .. .... . . . . . . . . . . . . . . 142 5 .4 CONCLUSION AND FURTHER STUDy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 144 CHAPTER 6 SOIL TEST TO PREDICT THE AVAILABILITY OF COPPER .......................................... 145 6. 1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.2 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 6.2 . 1 Soils and soil analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 6.2.2 6.2.2. 1 6.2.2.2 6.2.2.3 6.2.2.4 6.2.2.5 6.2.2.6 6.2.3 6.2.4 6.2.5 6.2.6 6.2.7 Single soil test extractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Methods for M1, M3, O.lM HCl, and TEA-DTPA extractants ... . . 147 0.01 M Ca(N03h extractant . ....... ... . . ... ................ . . . . . . ..... . .. . . . ... .... .. 148 0.01 M CaCh extractant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 148 0. 1 M NaND3 extractant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 149 1 M NH4ND3extractant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 0.02 M SrCl2 extractant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Fractionation ofCu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 149 Chemical form study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 Speciation of Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 Chemical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 6.3 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 6.3 . 1 6.3.2 6.3 .3 6.3.4 Soil characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 Fractionation of Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 1 S oil test extractants (Ml, M3 and TEA-DTPA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 54 E4traction of Cu by soil test reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 55 6.3.4. 1 Effect of soil type on Cu concentration . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 6.3.4.2 Effect of types of fertiliser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 XIII 6.3.4.3 Effect of level of residual Cu ......................................................... 156 6.3 .5 Speciation ofCu ............................................................................ 1 57 6.3.6 Ratios of fractions in the soil test extractants ...................... ............ 1 6 1 6.3 .7 Chemical forms in the soil test extractants .................................. ... 163 6 .3 .8 Relationships between the extractants ............................................ 1 67 6.3.9 Relationships between the extractants and plant Cu concentration .. 169 6 .4 CONCLUSION AND FURTHER STUDy ........................................... 1 7 1 CHAPTER 7 EFFECT OF NITROGEN AND PHOSPHORUS FERTILISER ON THE AVAILABILITY OF NATIVE COPPER ......................... .................................... 173 7. 1 INTRODUCTION ................................................................................. 1 73 7.2 MATERIALS AND METHODS ........................................................... 1 74 7.2 . 1 Soils .............................................................................................. 1 74 7.2.2 Fertilisers ....................................................................................... 1 74 7.2.3 Plant growth experiment ................................................................ 1 74 7.2.4 Grass sample preparation ............................................... ................ 1 75 7 .2.5 Ryegrass samples for Cu analysis .................................................. 1 75 7.2.6 Chemical Analysis ......................................................................... 1 75 7.2 .7 Statistical analysis ......................... ................................................. 1 75 7.3 RESULTS AND DISCUSSION ............................................................ 1 76 7.3 . 1 Initial soil ........................................... .................................. ......... 1 76 7 .3 .2 Effect of soil types on dry matter yield and Cu concentration ......... 1 76 7.3 .3 Effect ofN on dry matter yield, Cu concentration and Cu uptake ... 1 76 7.3 .4 Effect ofP on dry matter yield, Cu concentration and Cu uptake .... 1 78 7 .4 CONCLUSION AND FURTHER STUDy ........................................... 1 79 CHAPTER 8 FERTILISER COPPER EFFECT OF LIME, EDTA AND NITROGEN ON THE AVAILABILITY RESIDUAL .................................................................. 180 8. 1 INTRODUCTION ................................................................................. 1 80 8.2 MATERIALS AND METHODS ........................................................... 1 8 1 8.2. 1 Soil collection and preparation ... . ........................................ ........... 1 81 8.2.2 Plant growth experiment and treatments ......................... , . . . . . . . . . . . . . . . 1 8 1 XIV 8.2.3 Harvesting and soil sampling ...................................................... ... 1 83 8.2.4 Soil analysis .................................................... . . ............. ................ 1 83 8.2.5 Ryegrass samples for Cu analysis .... ........................ . ..................... 1 83 8 .2 .6 Chemical Analysis ...................................... .................... ............... 1 84 8 .2 .7 Statistical analysis .......................................................................... 1 84 8.3 RESULTS AND DISCUSSION ..... .................. ... .................................. 1 84 8 .3 . 1 8 .3 .2 8.3.2.1 8.3.2.2 8.3.2.3 8.3 .3 8 .3 .4 8 .3 .5 Soil characteristics ..................................... .................................... 1 84 Dry matter yield and Cu concentration ...... . . ..... .............................. 1 84 Effect of N fertiliser ......................... ............................ . ................. 184 Effect of lime ....... . ......................................................................... 184 Effect of EDTA .................... ... ................................................... .... 185 Effect of soil types on DM yield and Cu concentration .................. 1 86 Effect on exchangeable Cu ................ . ........... ................................. 1 88 Effect of fertiliser Cu sources and levels of residual Cu on DM yield and Cu concentration .............................................. .. ..................................... 190 8 .3 .6 Effect of soil test extractants on plant Cu concentration ................. 1 92 8.4 CONCLUSION AND FURTHER STUDy ........................................... 1 96 CHAPTER 9 SEASONAL RESPONSE OF COPPER AVAILABILITY IN PASTURE ......................................... 197 9. 1 INTRODUCTION .. .................................................................... ... ........ 197 9.2 MATERIALS AND METHODS ... ........................... ............................ . 1 98 9.2. 1 Field trial ....................................................................................... 198 9.2.1.1 9.2.1.2 9.2.1.3 9.2.1.4 9.2.1.5 9.2.1.6 9.2.2 9.2.3 Experimental site ................................ ........ ...................... ....... ..... . 198 Soil and pasture sampling ...... . . ......... ...................................... ...... 199 Soil moisture, soil temperature and climate data ... . . ................ .... 200 Fractionation of Cu and soil analysis .................. ......................... 200 Pasture analysis ........... . . ....... .... . .. ................. ....... ........ ........ ......... 200 Chemical analysis . .................. ....... ........................................... .... 200 Incubation study ....................................................................... ..... 201 Statistical analysis ............. ........................................................ ..... 201 9 .3 RESULTS AND DISCUSSION ......... ... ........... . .................................... 201 9.3 . 1 Climatic data ................. . .... . . ... ......... ....... .... .................................. 201 9 .3 .2 Pasture growth rate .................. .. .. ... ............................... ................ 202 xv 9.3.3 Pasture Cu concentration ....................... ...................................... .. 206 9.3 .4 Pasture Cu uptake ............. ............. .......... ...................................... 2 1 1 9 .3 .5 Effect of soil temperature and moisture on Cu concentration in soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 6 9 .3 .6 Seasonal effect on soil pH ........ . ................ .............. ....................... 2 1 7 9.3 .7 Effect on exchangeable Cu ........................................ ..................... 2 1 8 9 .3 .8 Fractionation of soil Cu ................. .............................................. .. 2 1 9 9.4 CONCLUSIONS ...... ......................................... .................................... 22 1 CHAPTER 10 SUMMARY, CONCLUSION AND FURTHER STUDY ............................................................ 222 1 0. 1 LITERATURE REVIEW .......................................... ..... ....................... 222 1 0.2 ADSORPTION AND DESORPTION OF COPPER IN PASTURE SOILS . .............................................................................................................. 223 1 0.2. 1 Adsorption study ............ ............................................................... 223 1 0.2 .2 Desorption study ............................................................................ 224 10.3 PLANT AVAILABILITY OF DIFFERENT COPPER SOURCES IN PASTURE SOILS .................................... .............. .......................... ................. 225 1 0.4 TRANSFORMATION AND PLANT UPTAKE OF COPPER IN DIFFERENT SOILS ............. ............... ............. ................................................ 225 1 0.5 SOIL TESTS TO PREDICT THE AVAILABILITY OF COPPER ........ 227 1 0.6 EFFECT OF LIME, EDTA, NITROGEN AND PHOSPHATE FERTILISERS ON THE AVAILABILITY OF COPPER. . ............... ... ............. 228 1 0.6. 1 Effect of nitrogen and phosphorus fertilisers on the availability of native copper. ................... ....... ....................... ..................... .. ... ..................... 228 1 0.6.2 Effect of lime, EDTA and nitrogen fertiliser on the availability of residual copper . ........ .................. ....... ............................................................ 229 1 0.7 SEASONAL RESPONSE OF COPPER AVAILABILITY IN PASTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 1 0.8 SUGGESTION FOR FUTURE STUDy ................... ............................. 232 REFERENCES ... ..... ... ... ......... ... ... ... ......... ... ............... 233 XVI LIST OF FIGURES Figure 1 . 1 The flow diagram / structures of the thesis . ..................................... . . ... . . .. .... 5 Figure 2. 1 Development of variable surface positive charge through the dissociation or association ofH+ on a mineral surface (Bolan et aI., 1 999) . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . 29 Figure 2.2 Complex formation of Cu ions by humic acid according to pH (Van Dijk, 1 97 1 ) . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . ... . .. . . 34 Figure 3 . 1 Time dependent Cu adsorption isotherms. The data were fitted to Mitscherlich growth function, Y= A(I-Bx), where Y= amount sorbed, X= time, A and B are constants . .... . .................. ............. .......... .... ............... ............... ..... . .... . . 71 Figure 3.2 Copper adsorption isotherms for the different soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Figure 3.3 Copper adsorption isotherm for different soil components of the Manawatu soil. . . . . . .. . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 75 Figure 3 .4 Copper adsorption isotherm for different soil components of the Tokomaru soil. . . . . . . . .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . 76 Figure 3 .5 Copper adsorption isotherm for different soil components of the Ramiha soil. ............................................. .............................................................................. 76 Figure 3.6 Copper adsorption isotherm for different soil components of the Ngamoka soil. . . .. . . . ....... ................ .................... . .......................... ... .... . .. .. . . .......... . ............... 77 Figure 3 .7 Copper adsorption isotherm for different soil components of the Mangmahu soil. ........................................................ . ...................... .. . . . .. ......................... ..... . 77 Figure 3 .8 Effect of soil pH level on Cu sorption of the Manawatu soiL .. .... . .......... .... 80 Figure 3.9 Effect of soil pH level on Cu sorption of the Tokomaru soil. .............. ........ 8 1 Figure 3 . 1 0 Effect o f soil pH level on eu sorption ofthe Ramiha soil. ........................ 8 1 Figure 3 . 1 1 Effect o f soil pH level on Cu sorption of the Ngamoka soil. ............... ...... 82 Figure 3 . 1 2 Effect of pH levels on equilibrium solution Cu concentrations at various input concentrations (mg L-1) (a) Manawatu, (b) Tokomaru, (c) Ramiha and (d) Ngamoka soils. Data are means ± SE, n=2 . ... ... ... .................. ... .............. . ............ 83 Figure 3 . 1 3 Freundlich constant (K) for Cu adsorption by different soils at various pH values ....................................................... . .. ............................................ ...... ...... 84 Figure 3 . 1 4 Distribution coefficient (K!) for Cu adsorption by different soils at various equilibrium concentration levels. Data are means ± SE, n=2 . . ............. . . . . _ . . . . . ...... 85 Figure 3 . 1 5 Distribution coefficient (Ko) of Cu sorption at different input concentrations by (a) M anaw atu, (b) Tokomaru, (c) Ramiha and (d) Ngamoka soils at different XVII pH levels. [Input concentrations (mg Cu L-I ) : + 1 00; +200;. 400; A600; .... 1 000) . Data are means ± SE, n=2 . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 Figure 3 . 1 6 Desorption of native Cu from two soils (Manawatu and Ngamoka) . . . . . . . . . . 88 Figure 3 . 1 7 Desorption of native and added Cu at (a) 2 hrs and (b) 24 hrs desorption period in Manawatu and Ngamoka soil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Figure 3 . 1 8 Cumulative desorption of native and added eu from the (a) Manawatu soil and (b) Ngamoka soil incubated with added Cu for different periods. Desorption was carried out using two de sorption periods [(i) 2 and (ii) 24 hours) . . . . . . . . . . . . . . . . . . 93 Figure 3 . 1 9 Effect of pH on Cu adsorption and desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Figure 3 .20 Cumulative desorption of Cu at two pH (5 and 8) and two sorption levels (30 and 50 mg L-I) in different soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Figure 4. 1 Effect of different soils on dry matter yield (g porI) of rye grass at different harvests. Data are means ± SE, n=48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . 1 04 Figure 4.2 Effect of fertiliser type on dry matter yield at different harvests. Data are means ± SE, n=80; control n=20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 05 Figure 4.3 Effect of soil types on Cu uptake after fertiliser addition. Data are means ± SE, n=48 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . 1 1 4 Figure 4.4 Effect of sources of Cu on eu uptake following fertiliser addition. Data are means ± SE, n=80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... . . . . . . . . . . . . . . . . . . 1 1 5 Figure 4.5 Cumulative recovery of Cu at (a) 50 mg Cu kg-I soil, (b) 1 00 mg Cu kg-I soil and (c) 200 mg Cu kg-I soil of different Cu fertilisers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 17 Figure 5 . 1 Comparison of total native Cu with the sum of individual fractions for each soil. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . ..... 1 22 Figure 5 .2 Comparison of total applied Cu determined by tri-acid extraction with the sum of individual fractions 28 days after application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Figure 5.3 Effect of sampling periods on exchangeable Cu concentration (mg kg- I ). Data are means ± SE, n=12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . � . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 26 Figure 5 .4 Effect of fertilisers on exchangeable eu concentration (mg kg"l ) at various sampling periods. Data are means ± SE, n=20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Figure 5 .5 Effect of treatment levels on exchangeable Cu concentration (mg kg"l ) at various sampling periods. Data are means ± SE, n=1 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Figure 5 .6 Effect of treatment levels on organic bound Cu (- - - -) and oxide bound eu (--) concentration (mg kg-I ) at various sampling periods. Data are means ± SE, n=1 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1 XVIII Figure 5 .7 Effect of fonns of Cu fertilisers on oxide bound Cu concentrat.ion (mg kg' l ) at various sampling periods. Data are means ± SE, n=20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 33 Figure 5 .8 Effect of treatment levels on oxide bound Cu concentration (mg kil ) at various times of soil sampling following fertiliser application. Data are means ± SE, n=1 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34 Figure 5 .9 Effect of fonns of Cu fertilisers on residual Cu concentration (mg kg'l ) at various sampling periods. Data are means ± SE, n=20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 35 Figure 5 . 1 0 Effect of treatment levels on residual Cu concentration (mg kg'l) at various sampling periods following fertiliser application. Data are means ± SE, n=1 5 . . . 1 36 Figure 5 . 1 1 Sum of fractions of Cu (mg Cu kg' l ) at different levels (a) 50 mg Cu kg,l , (b) 1 00 mg Cu kg' l , (c) 200 mg Cu kg' l following the fertiliser application. [(+Manawatu; + Tokomaru; eRamiha; .A Ngamoka; and �Mangamahu)] . Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 37 Figure 5 . 1 2 Relationship between soil organic carbon and different fractions of Cu: (a) exchangeable Cu; (b) organically bound Cu; (c) oxide bound Cu; and (d) residual Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 39 Figure 5 . 1 3 Relationship between CEC and different fractions of Cu: (a) exchangeable Cu; (b) organically bound Cu; (c) oxide bound Cu; and (d) residual Cu . . . . . . . . . . . . . 1 40 Figure 5 . 1 4 Relationship between clay content and different fractions of Cu: (a) exchangeable Cu; (b) organically bound Cu; (c) oxide bound Cu; and (d) residual Cu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 1 Figure 5 . 1 5 Correlation of (a) soil exchangeable Cu, (b) organically bound Cu, (c) oxide bound Cu and (d) residual Cu on plant uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 43 Figure 6. 1 Concentration of different fractions of Cu extracted by soil tests at different levels of Cu application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 64 Figure 6.2 Percentage of different fractions of Cu (a) Manawatu soil and (b) Ngamoka soil extracted by the soil test extractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 66 Figure 6.3 Correlation between Cu in soil test extractants and (a) exchangeable (b) organic bound Cu (c) oxide and (d) residual Cu fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67 Figure 6.4 The relationship between the concentrations of soil eu extractable by (a) M1 and M3, (b) M1 and TEA-DTPA (c) and M3 and TEA-DTPA extractants from the two soils that received 0 to 200 mg Cu kg' l soil from two different eu fertilisers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69 XIX Figure 6 .5 Linear correlations of eu in the ryegrass with the amounts of extractable Cu determined by using the (a) M1 , (b) M3 and (c) TEA-DTPA extracting procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 7 1 Figure 7. 1 Effect of N levels on (a) dry matter yield, (b) Cu concentration and (c) Cu uptake. Data are means ± SE, n=3 . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 77 Figure 7 .2 Effect of P levels on (a) dry matter yield, (b) Cu concentration and (c) Cu uptake. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 79 Figure 8 . 1 Effect of N fertilisers, lime and EDTA on DM yield and Cu concentration at different harvests for both soils. The optimum ( ) and the toxic (- - - -) levels of Cu in the plant are indicated in the figure. Data are means ± SE, n=48 . . . . . . . . . . 1 86 Figure 8.2 Effect of soil types on DM yield and Cu concentration as affected by N fertilisers, lime and EDT A addition at various level of Cu from different sources. Data are means ± SE, n=24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 87 Figure 8 .3 Effect of soil types on exchangeable Cu as affected by (a) Nitrogen, (b) Lime, and (c) EDTA. Data are means ± SE, n=24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Figure 8 .4 Effect of Cu fertiliser sources on DM yield [(a) CuO, (b) CUS04) and Cu concentration [(c) CuO, (d) CUS04) as affected by lime, Nitrogen or EDTA. Data are means ± SE, n=24 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9 1 Figure 8 . 5 Effect of residual Cu levels on Cu concentration either treated with N (a), lime (b) or EDTA (c). Data are means ± SE, n=12 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 92 Figure 8 .6 Relationship between Cu concentration in p lants and Cu extracted by (a) M1 , Cb) M3 and Cc) TEA-DTPA extractants as affected by EDTA. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 92 Figure 8 .7 Concentration of Cu extracted by (a) Mehlich 1 (Ml ), (b) Mehlich 3 (M3) and (c) TEA-DTPA extractions prior to and after the addition of lime . . . . . . . . . . . . . . . 1 93 Figure 8 .8 Relationship between soils exchangeable Cu as affected by N (a, d), lime (b, e), and EDTA (c, f) additions and Cu extracted by the M l , M3 and TEA-DTPA extracting procedures prior to and after lime application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 94 Figure 8 .9 Relationship between Cu concentration in plants and (a) exchangeable eu and (b) free Cu in soil solution with addition ofN (+) and lime (e) . . . . . . . . . . . . . . . . 195 Figure 8 . 1 0 Relationship between Cu concentration in plants and (a) excha:qgeable Cu and (b) free Cu in soil solution with addition ofEDTA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 95 xx Figure 9. 1 Weather data for soil temperature (.), air temperature (+), radiation (_), gravimetric soil moisture CT) and rain fall (+ ) during different trial periods. CData are mean ±SE for the different parameters at each trial period) . . . . . . . . . . . . . . . . . . . . . . . . . . 202 Figure 9.2 Pasture growth rates as influenced by different levels of Cu fertiliser at different periods after fertiliser application. Data are means ± SE, n=3 . . . . . . . . . . . . . . 203 Figure 9 .3 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture (c) and solar radiation (d) on pasture growth rates. Data are means ± SE, n=3 . . . 206 Figure 9.4 Effect of fertiliser Cu sources on Cu concentration at 5 kg Cu ha- I level. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Figure 9.5 Effect of seasonal responses to added Cu on Cu concentration in pasture. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 Figure 9.6 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture (c), and solar radiation (d) on Cu concentration. Data are means ± SE, n=3 . . . . . . . 2 10 Figure 9.7 Relationship between pasture growth rate and Cu concentration . . . . . . . . . . . . . . . 2 1 1 Figure 9.8 Effect of seasonal responses to added Cu on total Cu uptake in pasture. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 12 Figure 9.9 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture Cc), and solar radiation (d) on Cu uptake. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . 2 1 5 Figure 9. 1 0 Relationship between pasture growth rate and Cu uptake . . . . . . . . . . . . . . . . . . . . . . . 2 1 6 Figure 9. 1 1 Soil pH and exchangeable Cu at different trial periods. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 8 Figure 9. 12 Effect of (a) soil moisture and (b) soil temperature on exchangeable Cu at different trial periods. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 9 Figure 9 . 1 3 Fractionation of soils native Cu and Cu added as (CUS04) at different trial periods. Data are means ± SE, n=3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 1 XXI LIST OF TABLES Table 2. 1 Copper content of major rock types (mg kg- I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Table 2.2 Typical Cu concentrations in soils from various parent materials (Viets, 1 962) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Table 2.3 Copper ore minerals (Hignett and McClellan, 1 985) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Table 2.4 The principal copper materials used in Cu fertilisers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Table 2 . 5 Copper minerals and Cu containing fertiliser materials (Netzer and Beszedits, 1 979) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Table 2.6 Ranges and mean concentrations of Cu in surface soils calculated on the world scale (Kabata-Pendias and Pendias, 1 992) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Table 2 . 7 Copper in the natural soil solution of different soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 Table 2 .8 Effect o f pH o n solution composition of Cu, expressed as percent in solution (Harter, 1 983) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 Table 2.9 Various fractionation schemes used by different authors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Table 2 . 1 0 Comparison of the relative extracting abilities of sequential copper fractionation schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Table 2. 1 1 Total soil Cu recovered from soils by three methods of digestion . . . . . . . . . . . . . . 25 Table 2. 1 2 Adsorption of Cu in soils and soil components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 Table 3 . 1 The soils and experimental conditions used in the various adsorption and desorption experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Table 3.2 Sequential fractionation scheme for Cu in soil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Table 3 .3 Initial soil properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 Table 3 .4 Freundlich equation describing the adsorption of Cu in different soil components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Table 3 .5 Percent contribution of the organic matter and oxide components to Cu adsorption at two initial concentration levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Table 3 .6 Freundlich equation describing the adsorption of Cu at various pH levels in different soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 Table 3 .7 Cumulative desorbed native soil Cu and different extractable Cu concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 Table 3 .8 Soil pH at various intervals of de sorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1 Table 4. 1 Sources of Cu used as fertilisers in the plant growth experiment. . . . . . . . . . . . . . . . 1 00 Table 4.2 Correlations of dry matter yield with soil properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04 XXII Table 4.3 Effect of fertiliser rates on dry matter yield (g porI) of ryegrass at different harvests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07 Table 4.4 Effect of soil types on Cu concentration (mg kg-I ) in rye grass at different harvests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 0 Table 4 .5 Correlations of Cu concentration in rye grass with soil properties . . . . . . . . . . . . . . . 1 1 0 Table 4.6 Effect of fertiliser type on Cu concentration (mg kg- I ) of rye grass at different harvests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1 Table 4.7 Effect of fertiliser rates on Cu concentration (mg kg-I ) of ryegrass at different harvests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2 Table 4 .8 Correlations of copper uptake with soil properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 Table 4.9 Effect of fertilisers and treatment levels on Cu uptake by ryegrass . . . . . . . . . . . . . 1 1 6 Table 5 . 1 Fractions of native Cu (mg kg-I ) in the whole soil. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 24 Table 5 .2 Concentration of total native Cu in whole soil and particle size fractions . . . 125 Table 5 .3 Effect of soil types on organically bound Cu concentration (mg kg- I ) at various sampling periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29 Table 5 .4 Effect of fertilisers on organically bound Cu concentration (mg kg-I ) at various sampling periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 30 Table 5 . 5 Effect of soil types on oxide bound Cu concentration (mg kg-I ) at various sampling periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 32 Table 5 .6 Effect of soil types on residual Cu concentration (mg kg- I ) at various sampling periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . _ . _ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 35 Table 5 .7 Correlations between soil properties and Cu concentration in the different fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 38 Table 6. 1 Comparison of methods used in determination of extractable Cu . . . . . . . . . . . . . . . 148 Table 6.2 Effect of treatment combinations on various forms of Cu by sequential fractionation procedure at 295 days after . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 53 Table 6.3 Copper concentration in soil test extractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 5 Table 6.4 Effect of soils and types of fertiliser on extractable soil Cu (mg kg-I ) . . . . . . . . 1 56 Table 6.5 Effect ofCu levels on extractable soil Cu (mg kg-I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57 Table 6.6 Copper species in soil test extractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Table 6.7 Copper species in chemical fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 60 Table 6 .8 Copper fractions in the soil test extractants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 62 Table 6.9 Correlation coefficient of different extractants with plant available eu . . . . . . 1 70 Table 7. 1 Effect of soil types on DM yield, Cu concentration and uptake . . . . . . . . . . . . . . . . . . 1 76 XXIII Table 9. 1 Time frame of the different trial periods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Table 9.2 Effect of fertiliser levels on Cu concentration (CUS04 and CuO) . . . . . . . . . . . . . . . 208 Table 9.3 Mean Cu concentration in soil solution at various levels of soil temperature and moisture contents for four different soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 17 XXIV LIST OF PLATES Plate 4. 1 Plant growth experiment with different levels of CUS04 fertiliser (Ballantrae high fertility and Ballantrae low fertility known as Ngamoka and Mangamahu soil, respectively) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1 Plate 4.2 Effect of soil types on plant growth experiment (Ballantrae low fertility known as Mangamahu soil) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1 Plate 8 . 1 Glasshouse trial ofN effects on residual Cu availability in ryegrass . . . . . . . . . . . 1 82 Plate 8 .2 Glasshouse trial of lime effects on residual Cu availability in ryegrass (Ballantrae high fertility known as Ngamoka soil) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 82 Plate 8 .3 Glasshouse trial of EDTA effects on residual Cu availability in ryegrass . . . 1 83 Plate 9. 1 Location of the field trial. . . . . . . . . . . . . ; . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 99 Chapter 1 General introduction 1 CHAPTER 1 GENERAL INTRODUCTION 1.1 INTRODUCTION Copper (Cu) is one of the most important essential elements for plants, animals and humans. eu was confirmed as an essential element for plants in the 1 930s. In nature, Cu forms sulphides, sulphates, carbonates and other compounds, and also occurs under reducing environments as the native metal. eu is associated with soil organic matter, oxides of iron and manganese, soil silicate clays and other minerals (McLaren et al. , 1983b; Shuman, 1 99 1 ; Swift and McLaren, 1 99 1 ; McBride, 1 994). eu in the soil solution occurs as free ionic eu and organically complexed species (Hodgson et al. , 1 966; Baker and Senft, 1 995; Fotovat and Naidu, 1 997; McBride et al. , 1 998). The bioavailability of eu is related to the chemical potential of the respective species in the soil solution. The level and distribution of total and extractable eu in the soil profile varies with soil type and parent material. eu is specifically adsorbed or fixed in soils, making it one of the trace elements that moves the least. Higher concentrations of eu in the surface horizon of a soil is an indication of increased retention of eu added from various sources. eu is added as smelter ash, fertilisers, sewage sludges and other wastes, fungicides or bactericides, manures from pigs and poultry, fed selected eu containing compounds for increased feed efficiency and greater growth rates. Generally eu concentrations in plants range from 5 to 20 mg kg-I , which may extend from 1 to 30 mg kg-I . Cu accumulation differs among plant species and cultivars. Therefore, it is not possible to give single values for eu deficiency or toxicity concentrations. eu in plants functions as part of the prosthetic group of enzymes, and as a facultative activator of enzyme systems. In animal nutrition, eu deficiency is almost entirely confined to the grazing animal due to Iow levels of eu in the herbage/forage. Or to the normal to be low normal levels of Cu accompanied by elevated levels of Mo, S and Fe, that are sufficient to limit eu absorption and retention. In many parts of the world, under a wide range of soil and climatic conditions, the occurrence of eu deficiency in grazing animals may be attributed to: (i) inherently low soil and herbage eu concentrations, such as in the area of the falling disease of cattle and neonatal ataxia of lambs in western Australia; (ii) Chapter 1 General introduction 2 dual deficiency of Cu and Co on coastal calcareous-leached soils of granite origin, such as in Florida and southern Australia; and (iii) conditioned deficiency by the use of lime (CaC03) and from a high intake of antagonists to Cu metabolism, such as Mo in the pumice and peat soils of New Zealand and the humic peat soils of Europe (Underwood, 1 98 1 ) . In New Zealand, the pastoral system is legume based and nitrogen fertiliser is used in pastures for encouraging out of season pasture growth. Phosphate fertilisers are regularly used in legume based pasture systems. Application of a' high N supply may cause rapid plant growth and accentuate Cu deficiency by exhausting the Cu in the soil solution (Alloway and Tills, 1 984; Robson and Reuter, 1 98 1 ). Reuter et al. ( 1981b) reported that interaction between P and Cu was indirect and positive in its effect on growth in subterranean clover, and when the P supply increased from a marginally deficient level to an adequate level, there was depressed Cu absorption and an accentuated Cu deficiency. There has been some conflict of opinion as to the best means of correcting the Cu deficiency in stock. The effectiveness of pasture topdressing with Cu depends on the nature of the pasture and on soil properties. The residual effects of Cu topdressing also differs between soils. It has often been observed that in most New Zealand soils, the response to Cu fertilisers in pasture is very short lived. In Cu deficient soils, application of Cu fertilisers increases the Cu uptake by plants only for the first few harvests and the Cu concentration in the herbage decreases to the background level within a short time (Sherrell and Rawnsley, 1 982; Willimott, 1 995; Khan et al., 1 996). Copper added to the soil is rapidly immobilised and plant utilisation is very dependent on exploitation of the soil by roots. There is a need to define the rates of decline in the effectiveness of Cu fertilisers over the range of conditions encountered. This information is required so that the needs of crops, pastures and the grazing animal can be met and agricultural production maintained. Cu in the soil solution occurs mainly as Cu organic matter complexes. There is stil l insufficient knowledge concerning the reactions controlling the availability of Cu to plants, although it is generally assumed that adsorption and desorption reactions control the levels of Cu in the soil solution. Chapter 1 General introduction 3 Therefore it is difficult to predict changes in the supply of plant available eu in field situations from the existing knowledge of the chemistry of eu in soils. In this study the adsorption and desorption reactions of eu in a range of pasture soils were examined. The effects of different eu sources as fertilisers, other nutrients (nitrogen and phosphorus), soil amendments (lime and EDTA) on the plant availability and transformation of eu were examined. Finally the seasonal responses on the availability of eu in pasture plants is examined under field conditions. The overall structure of this thesis is presented in Figure 1 . 1 . 1 .2 OBJECTIVES OF VARIOUS EXPERIMENTS A number of laboratory, glasshouse and field experiments were conducted with the following objectives. 1.2.1 Adsorption and desorption ofCu in pasture soils • To determine the adsorption capacity of a range of pasture soils. • To examine the effect of pH on eu adsorption. • To quantify the contribution of the different soil components to eu adsorption. • To examine the effects of pH, the incubation period of eu with soil and the contact time on the desorption of eu. 1.2.2 Plant availability of Cu from different Cu fertilisers in pasture soils. • To examine the effectiveness of a range of eu fertilisers on plant growth and the raising of pasture eu concentration. 1.2.3 Transformation and plant uptake of Cu in different soils • To determine the transformation of eu fertilisers with time, which might account for the changes in eu availability to plants. • To evaluate the p lant availability of eu from the various soil eu fractions. Chapter 1 General introduction 4 • To evaluate the efficiency of different extracting reagents for the detennination of Cu availability in pasture soils. 1.2.4 Effect of nitrogen, phosphorus fertilisers, lime; and EDTA on the availability of native Cu and residual Cu. • To evaluate the effects of nitrogen and phosphorus fertilisers on the uptake of native Cu by ryegrass in two contrasting soils. • To determine the residual effectiveness of fast and slow release Cu fertilisers as influenced by N fertiliser, lime and EDT A addition. 1.2.5 Seasonal response of copper availability in pasture • To examine the seasonal effect on the ability of two different Cu fertilisers (CUS04 and CuO) in raising the Cu concentration in pasture and the transfonnation of Cu in soils. Chapter I General introduction 5 Figure 1 . 1 The flow diagram / structures of the thesis. Chapter 1 � The importance of Cu in pasture and the grazing animals General Introduction is discussed in relation to the adsorption, desorption of Cu, transfonnation and the plant availability of Cu fertilisers. Chapter 2 The current understanding of the soil, pasture, and General review of literature r----. fertiliser management factors that affect Cu concentration in plants, soils and animals and to � summarise the literature on sources, forms, adsorption and fixation, complex fonnation and availability of Cu in Chapter 3 soils and plants. Adsorption and desorption of Cu in pasture soils � The adsorption and desorption reactions of Cu in a range � of soils are reported. Chapter 4 Availability of Cu depends on soil pH, organic matter Plant availability of Cu from -+ and clay content. Copper uptake in the grass decreased different Cu fertilisers in soils. with increasing time after fertiliser application. � ..- Chapter 5 The organic bound fraction Cu increased and oxide Transformation and plant uptake of -+ Cu in soils fraction decreased with time after fertiliser application. � • Soil organic matter plays important role on the an Chapter 6 extraction of Cu by using various soil test extractants. Soil tests to predict the availability ..... TEA-DTPA and M3 are more efficient than other of Cu extractants used for the measurement of Cu availability in � soils. " Chapter 7 & 8 � Increasing additions of N and P fertilisers increased the The availability of Cu as affected by soil amendments (N, P, lime and untake of native Cu. EDTA). � +- Low level of N and lime, and increasing levels of EDT A increased the concentration of Cu in plants grown in Chapter 9 soils with residual Cu. Seasonal responses on the transformation and availability of Copper uptake was forced by growth related factors such Cu. � as soil temperature, soil moisture, radiation and air � temperature and also by changes of Cu forms in soils. Chapter 10 The ability of Cu uptake was internally regulated by the Summary and Conclusion f--+ growth of pasture shoot and externally affected by the transfonnation of Cu in soils. Chapter 2 Literature review 6 CHAPTER 2 LITERATURE REVIEW 2.1 INTRODUCTION The objective of this review is to describe the current understanding of the soil, pasture, and fertiliser management factors that affect Cu concentration in plants, soils and animals. And to summarise the literature on the Cu sources of additions to soils, the forms of Cu in soils, adsorption and desorption of Cu in soils, and organic binding and plant availability of Cu in soils. 2.2 SOURCES OF COPPER IN SOILS 2.2.1 Soil parent materials Typical Cu contents of the major rock types are given in Table 2. 1 . The average Cu concentration in the earth's crust ranges from 24 to 55 mg kg-I and the average Cu range for soils is 20 to 30 mg kg-I . Aubert and Pinta ( 1 977) and McBride ( 1 98 1 ) however reported that C horizon soils derived from a parent material of crystalline schists (garnet, granulite) containing an intrusion of no rite with 1 000 mg kg-I Cu, contain up to 1 00 mg kg-I Cu. Typical Cu concentrations found in soils derived from different parent materials are presented in Table 2.2. Copper is translocated with clays and tends to be most abundant when the clay content is high (Fagbami et aI. , 1 985). Fractionation (Shuman, 1 979) and sorption studies (McBride, 1 98 1 ; McLaren et aI. , 1 983b) have shown that Cu may be tightly adsorbed on AI, Mn, and Fe oxides. Soils derived from volcanic ash and pumice may be deficient in plant available Cu (Nyandat and Ochiing, 1976). Table 2.1 Copper content of major rock types (mg kg-I). Rock type Average concentrations Range References Igneous rocks - 1 0- 1 00 Krauskopf ( 1972) Limestone and dolomite 6 0.6- 1 3 Cox ( 1979) Shale and clay 35 23-67 Cox ( 1 979) Earth's crust 70 - Parker ( 198 1 ) Soils 30 2-250 Bowen ( 1 979) Chapter 2 Literature review 7 Table 2.2 Typical Cu concentrations in soils from various parent materials (Viets, 1962). Soils Parent materials Peat Peat (Histosols) Sandy soil Drift (Aresols, Podzols) Sandy soil Granite Silty clay loams Shales (Gleysols, Cambisols) Clays Clay developed on clay (Gleysols) Loams Developed on basalt (Cambisols) Humic loams Developed on chalk Organic- rich loams Loess (Chemozems) Pumice soil Pumice (Lithosols, Arenosols) Tropical soil Ferralsols 2.2.2 Ore minerals Concentrations (mg kg" ) 1 5-40 2- 1 0 1 0 40 rocks 1 0-27 rock 40- 1 50 7-28 1 - 1 00 3-25 8- 1 28 In nature, Cu occurs both in the free state and in combination with other elements in a host of minerals. Copper usually occurs as the metal or associated with sulphur in most rock types. Two distinct assemblages of sulphides are recognised. The magnetic sulphides include Cu-Fe sulphides and are represented by chalcopyrite, the most important mineral of this group. The second assemblage is characterised by the complex sulphides and sulpho salts, and is the source of up to 70% of the world's copper production. Schroeder (1 979) reported that cha1copyrite (CuFeS2) is the most abundant. Although Cu occurs in approximately 1 50 minerals, only few are considered important ore minerals (Table 2.3). Chapter 2 Literature review 8 Table 2.3 Copper ore minerals (Hignett and McClellan, 1985). Group Minerals Chemical formula Complex sulphides Chalcopyrite CuFeS2 Bomite CuSFeS4 Enargite CU3AsS4 Tetrahedrite (Cu, Fe) 12Sb4S I3 Simple sulphides Chalcocite CU2S Covellite CuS Oxides Cuprite CU20 Carbonates Malachite CuC03. Cu(OHh Azurite 2CuC03. Cu(OHh 2.2.3 Fertilisers A wide range of Cu materials are used to supply Cu to soils. The various sources of Cu used for crop production are presented in Table 2.4. The most usual Cu fertiliser is CUS04. 5H20, although other compounds, mixtures and chelates are also used (Table 2.5). Hydrated CUS04 is compatible with most fertiliser materials. Man made inputs of Cu to land are very diverse. Soil levels of Cu are affected by soil and crop treatments including fungicides and fertilisers; not used exclusively to rectify Cu deficiencies, such as livestock manures, sewage sludges and atmospheric deposition. Table 2.4 The principal copper materials used in Cu fertilisers. Material Formula Cua g kg-I Copper sulphate pentahydrate CuS04.5H2O 250 Copper sulphate monohydrate CuS04.H2O 350 Basic copper sulphate Cll4(OH)6S04 530 Copper carbonate CUC03 550 Copper hydroxide Cu(OHh 300 Copper oxide CuO 250-750 a -I The g kg copper lIsted IS that of the generally marketed grade, and not necessarily that of the pure compound. Chapter 2 Literature review 9 Table 2.5 Copper minerals and Cu containing fertiliser materials (Netzer and Beszedits, 1979). Source Cuprite Tenorite Covellite Cha1cocite Cha1copyrite Malachite Azurite Chalcanthite Copper sulphate monohydrate Basic copper sUlphate Copper nitrate Copper acetate Copper oxalate Copper oxychloride Copper ammonium phosphate Copper chelate Copper chelate Copper polyflavanoids Copper-sulphur frits Copper-glass fusions Sewage sludge Animal manures (no Cu supplements) Animal manures (With Cu supplements) 2.2.4 Pesticides Fonnula CU20 CUO CuS CU2S CuFeS2 CuC03. Cu(OHh 2CuC03• CU(OH)2 CuS04. 5H20 CUS04' H2O CUS04' 3Cu(OHh CU(N03)2. 3H2O Cu(C2H302). H2O CUC204.O.5H2O CuClz·2CuO.4H2O CU(NH4)P04. H2O Na2Cu EDTA NaCu EDTA - - - - - - Cu (g kg-I) Water solubility 890 Insoluble 750 Insoluble 660 Insoluble 800 Insoluble 350 Insoluble 570 Insoluble 550 Insoluble 250 Soluble 350 Soluble 1 30-530 Insoluble - Soluble 320 Slightly 400 Insoluble 520 Insoluble 320 Insoluble 1 30 Soluble 90 Soluble 50-70 Soluble Varies Varies Varies Varies 0.4- 1 0 Slightly 0.02-0.00 Slightly 0.6- 1 .9 Slightly Copper compounds have been used for many years as fungicides and bactericides_ In the past, CuS04.5H20 was mixed with lime to control plant pathogens, recently CU(OH)2, has been used in fairly large amounts in sprays (Bordeaux mixture) as a blanket preventive treatment (Walsh et al., 1 972; Florez-V elez et aI. , 1996). Copper sprays have been recommended for disease control. These sprays can enrich Cu in the soil by as much as 1 1 .2 to 1 6.8 kg ha-! yr-! . Copper toxicity may develop in sandy soils, Chapter 2 Literature review 10 because they have a low CEC « 5 cmol kg-I) and often contain <1 % organic matter. Continued overuse of Cu containing fungicides and bactericides can result in the accumulation of Cu in soils to phytotoxic levels (Delas et aI., 1 960; Lepp et ai., 1 984). 2.2.5 Sewage sludge Copper concentrations in sewage sludge vary widely. Page ( 1 974) and Sommers ( 1977) have reported significant levels of Cu in sludge. Sewage sludge contains appreciable amounts of Cu, but application of sludge to soils generally results in only slight to moderate increases in the Cu content of plants (CAST, 1 976). In Pennsylvania, USA it has been found that many sludges from waste water treatment plants cannot meet the 1 000 mg kg-I maximum threshold limit for Cu, but can meet a 1 200 mg kg- I Cu limit. Maximum Cumulative metal loading of soils from sludges is regulated in many countries through resource management legislation. In New Zealand, the New Zealand Dairy Board has recently introduced restrictions on the use of sewage sludge in pastures soils. But recent studies have showed that the addition of bio-solid composts increases the formation of organic-metal complexes and thereby reduces the bioavailability of heavy metals (Bolan et al., 200 1). New Zealand guidelines for the application of sewage sludge to soils contains maximum annual permissible applications of Cu to land and maximum allowable concentrations in soils are 1 2 kg ha-I and 1 40 mg kg-I , respectively (Tiller et al., 2000). The regulated level of Cu of 60 ppm in the plough layer ( 1 5 cm) is intended to prevent long-term adverse effects on biological processes essential for soil fertility; as well as possible adverse health effects to sheep and cattle on treated pastures (Hornick et aI. , 1 976 and Baker et aI. , 1985). Because of the relatively high concentrations of Cu and other metals, compared to non-sludge treated soils, sewage sludge should be analysed before use as a soil amendment. Under no conditions is it considered practical to use sewage sludge as a soil conditioner without consideration of the metal and pathogen concentrations in the sludge. In case the resultant loading of Cu and other metals to the soil damage the crops grown. A Cu rich sludge (6000 mg kg-I on a dry basis with 2300 mg kg-1 extractable in 0.005 M EDTA) addition to land at 1 25 t ha- 1 in the first year and 3 1 t ha-1 in each of 3 subsequent years, raised the total soil Cu from 30 to 600 mg kg-I and the 0.05 EDTA extractable Cu from 8 to 280 mg kg, of which 1 80 mg kg-I was still EDTA extractable after 4 years (Stevenson and Fitch, 1 98 1 ). Chapter 2 Literature review 1 1 While sewage sludges are capable of substantially increasing the soi l levels of eu, there have been no reports of plant toxicity's to sludge eu, when grown in fertile limed soils. Soil organic matter appears to be the dominant factor controlling eu retention (Harter, 1 986). Thus, the rate of decomposition of sludge organic matter becomes a prime consideration for sludge treated soils. 2.2.6 Composts and agricultural wastes Garcia et al. ( 1995) and Ciavatta et at. ( 1993) reported that the concentration of eu in sewage sludge increases during composting. Japenga et at. ( 1992) pointed out that complex formation involving high molecular weight dissolved organic matter is the most important driving force for heavy metal solubilisation. Barbera ( 1987) found that the application of compost to agricultural soils increased both the total and available concentrations of metals in the soils. Arnesen and Singh ( 1998) investigated three sources of organic material (cow manure, pig manure and peat) on the extractability of Cu in soil, and Cu uptake by wheat and barley plants. They reported that increasing rates of all three organic materials increased the Cu concentration. The increase in Cu uptake by plants suggests that a change in the speciation of Cu in the soil solution may have enhanced its plant availability. 2.2. 7 Wood preservatives The wood preservative industry is one of the major markets for eu (Loebenstein, 1 993). McLaren et al. ( 1 994) examined the leaching of Cu, Cr, and As (CCA) from the timber treatment solution through free draining coarse textured surface and subsurface soils (Typic Ustipsarnment and Udic Ustochrept) using undisturbed soil lysimeters. Cumulative amounts of Cu leached through the lysimeters ranged from 4 to 30% of the total Cu applied (90 mg Cu) from a CCA solution. The amounts of eu leached from the CCA solution were generally higher than those recorded from sewage sludge addition, which is attributed to the presence of eu in relatively immobile forms in the latter (McLaren et aI., 1 994). Chapter 2 2.2.8 Atmospheric deposition Literature review 12 Atmospheric inputs of C u to soils from both rain and dry deposition vary considerably according to the proximity of industrial emissions containing Cu, and the type and quantities of wind-blown dust. In the UK, the total annual deposition of Cu from dust was found to vary between 100 g ha- I and 480 g ha-I . While crop removal was estimated at 50 to 1 00 g ha-1 , the deposition of dust was not sufficient to correct Cu deficiency in crops or livestock (Shorrocks and Alloway, 1987). The lack of response to this deposition was attributed either to the form of Cu or to soil adsorption or both. Duce et al. ( 1 976) estimated the annual natural fluxes of Cu to the atmosphere to be 1 4.3x 106 kg. This is in reasonable agreement with Nriagu's ( 1 979) estimate of the total quantity of atmospheric emission of 3 .2x 1 06 t Cu (about 1 % of the 307x l 06 t produced) since 3800 BC. This amount is about three orders of magnitude greater than the present day atmospheric Cu burden. 2.3 COPPER IN SOILS 2.3.1 Total Cu in soils Gilbert ( 1 952) reported that the total Cu content in normal agricultural soils is expected to range from 1 to 50 mg kg-I . Copper content of the worlds soils has been characterised by Aubert and Pinta ( 1 977) according to various climatic zones as follows: a) temperate and boreal regions - total Cu ranges from trace to several hundred mg kg-I , (lowest content of 8 mg kg-I was reported on the fluvioglacial and alluvial sands of the lower Volga Valley in the USSR; some of the highest values, 50 to 200 mg kg- I in the USSR are found on the Kola Peninsula); b) arid and semi arid region soils, in these regions generally contain from average to high total Cu; and c) tropical humid regions soils have fluctuating total Cu contents, with values from trace to 200-250 mg kg-1 • The data in Table 2 .6 gives concentrations of eu in uncontaminated surface layers of the major soil groups. Chapter 2 Literature review 1 3 Table 2.6 Ranges and mean concentrations of Cu in surface soils calculated on the world scale (Kabata-Pendias and Pendias, 1992). Name of soils Podzols (sandy soil) Cambisols (silty and loamy soil) Rendzinas Kastanozems and Chemozems Histosols (organic soil) 2.3.2 Forms of soil Cu Range (mg kg·1dry wt.) Mean (mg kg' I dry wt.) 1 - 70 1 3 4 - 1 00 23 6.8 - 70 23 6.S - 140 24 1 - 1 1 3 16 Total Cu in soils includes six 'pools', classified according to their physico-chemical properties. The pools include: soluble ions, inorganic and organic complexes in the soil solution; exchangeable Cu; stable organic complexes in humus; Cu adsorbed by hydrous oxides of Mn, Fe and AI; Cu adsorbed on the clay-humus colloidal complex; and crystal lattice-bound Cu in soil minerals. When dilute salt solutions such as O.O IM CaCh are used to extract soils, the soil solution and exchangeable Cu are considered as one fraction (Shorrocks and Alloway, 1987) . In surface soils, the total Cu concentration in the soil solution is normally only 0.01 to 0.6 J.1M, owing to its high affinity for sorption by organic and inorganic colloids (Shorrocks and Alloway, 1 987). The dissolved and exchangeable forms are of special importance in plant nutrition. Cu in the soil solution, exchangeable, specifically adsorbed and organically bound Cu are considered in equilibrium. They represent the available forms for plant uptake. Oxide­ bound and residual forms of Cu are relatively unavailable to plants (McLaren and Crawford, 1 973a) . The amount of Cu in any given form can vary with time, and the extractants used. McLaren and Crawford ( 1 973a) found that the distribution of eu between the main soil constituents could be influenced considerably by the presence of free manganese oxide. They found that the bulk of the 'available' soil eu reserves reside in the organically bound fraction. Mathur and Levesque (1 983) concluded that the organically bound Cu could be the dominant form in Histosols. Apparently, enrichment of the soil with Cu, from either organic or inorganic sources, can be reflected by increases in most fractions, with the exception of the residual form. Most of the increases occurring in the organic fraction. In some cases, however, eu associated Chapter 2 Literature review 14 with F e and Mn oxides may constitute the major fraction of total Cu i n soils (Kuo et al., 1 983). 2.3.2. 1 Soil solution Cu Although Cu is one of the least mobile heavy metals in soils, this metal is abundant in the soil solution of all types of soil. Concentration of Cu in the soil solution obtained by different techniques from different soils varies from 3 to 420 j.lg L-1 , which corresponds to Cu concentrations of 0.047 to 6.58 J.1M (Table 2 .7) . Williams ( 1 98 1 ) reported that soil solution Cu concentrations ranged from 3 j.lg L-1 to 240 j.lg L-1 in soils receiving regular additions of Cu. Soil solution Cu can be classified into three categories. • Inorganic complexes of Cu. • Cu complexed with soluble organic matter. • Uncomplexed Cu or ionic Cu. The stability constants of the inorganic Cu complexes show that the formation of significant quantities of these complexes does not occur in most soils (Lindsay, 1 972). Gardea-Torresdey et al. (1 996) observed that the amount of Cu bound to soluble organic matter is dependent on the solution pH. Only hydroxy and carbonate species are expected to exist in significant quantities in the soil solution. The solubility of Cu2+ becomes negligible around pH 7.0 - 8.0, while above this pH Cu carbonate complexes and CuOW become significant (McBride, 1 98 1 ). Mattigod and Sposito ( 1 977) observed that the soluble carbonate forms would be the dominant inorganic Cu species in highly alkaline soils. Nitrate and phosphate species would not be expected to contribute significantly to total inorganic Cu in the soil solution. McBride ( 1 98 1 ) mentioned that, among the anions (nitrate, chloride, sulphate, and phosphate) in soil solutions, sulphate and chloride may form complexes with Cu2+ in saline soils. Mattigod and Sposito ( 1977) concluded that the major inorganic form of complexed Cu2+ in neutral and alkaline soil solutions would be CUC03. In highly alkaline soils, Cu(OH)l- and CU(C03){ become the predominant soluble species. McBride and Bouldin ( 1 984) estimated that at least 99.5% of the Cu in the soil solution of a surface loamy soil (5.3% organic matter content) was in an organically complexed form . Hodgson et al. ( 1 966) observed that as the pH was raIsed there was little change in the total concentration of Cu in the soil solution, but the partitioning between uncomplexed and complexed Cu Chapter 2 Literature review 1 5 changed. At low pH, a higher proportion o f Cu was present as the uncomplexed ion and with increasing pH, soluble Cu-organic complexes became more signIficant. They showed that the log ratio of complexed Cu to uncomplexed Cu was positively correlated to the organic matter in solution (r = 0.88, p < 0.01 ) . As illustrated in Table 2.8, hydrated forms of Cu ions are unlikely to exist in aqueous systems with a pH below about 6.0. As the pH approaches neutrality, however, Cu is rapidly hydrolysed to Cu(OH)2o. Harter ( 1 983) reported that the amount of Cu(OHh species in the soil solution at pH 6 accounted for only 2% of the Cu2+ ion, but at pH 8 for 92% of the Cu2+ Ion. Brlimmer et al. ( 1 986) and McBride ( 1989) consider that at pH values close to 7, more than 99% of the Cu in solution is complexed by organic matter. Wu et al. (2000) investigated the activity of Cu2+ ions and analysed the dependence on pH and other soil properties. In saturated soil extracts, the activity of free Cu2+ ions varied from 4.78 x I 0-8 to 1 1 .4 x 1 0-8 M, which constituted 0.002 to 0.008% of total soil Cu. The activity ofCu2+ ion in soils fell in the range of 1 0.9 x 1 0-1 1 to 4.0 x l 0-9 M, which accounted for 0. 1 to 7.8% of the dissolved Cu. The dominant species of Cu in soil solutions is Cu2+ and CuOH+ ions, which are about 1 0 times greater than CuHC03+. They obtained a highly significant correlation coefficient between soil properties (PH and DOe) and the activities of Cu2+ and CuOH+, which implies that the concentration of free Cu2+ ions in the soil system is strongly pH dependent. Chapter 2 Literature review 1 6 Table 2.7 Copper i n the natural soil solution of different soils. Techniques used for Soils/Country Copper concentration References obtaining soil solution (�g L-1) Suction from soil paste 30 series of soil, USA 40 Bradford et a/. ( 1 97 1 ) Pump off O.OIN CaCl2 5 series of soil, New 3 - 1 8 Hodgson et a/. ( 1 965) solution Yorlc, USA Centrifugation Contaminated soil 28 - 1 35 Kabata-Pendias ( 1 972) do Three field soil, Japan 37 Yamasaki et al. ( 1 975) do Contaminated soil, 3.04 - 7.42 Wu et a/. (2000) Canada Ceramic plate or porous Cup Two forest soils, 1 8 - 27 Heinrichs and Mayer suction Germany ( 1980) do Contaminated soil, 14 - 44 Itoh and Yumura Japan ( 1 979) Zerotention lysimeter Temperate forest soil. 1 . 8 - 22 Bergkvist et a/. ( 1 989) Unspecified technique Different paddy soils, <1 - 3 Tiller ( 198 1 ) Australia do Contaminated soil, 29 - 1 1 6 Upitis and Gubar Russia ( 1 986) Centrifugation Dutch forest and 1 5 - 1 50 Romkens and Salomons arable soils ( 1998) Ion exchange resin 1 1 different soils, 5.2 - 74.4 Fotovat and Naidu Australia ( 1 997) Centrifugation Dutch sandy soils 46- 1 1 7 Romkens et a/. ( 1 999) Deionised water Surface soil, USA 60 - 420 Reddy et a/. ( 1 995) Table 2.8 Effect of pH on solution composition of Cu, expressed as percent in solution (Harter, 1983). Soil pH 4.0 5.0 6.0 7.0 8.0 Cul+ 1 00 100 96 33 1 CuOH+ - - 2 7 1 Cu(OHhU - - 2 56 92 - Chapter 2 Literature review 1 7 2.3.2.2 Exchangeable Cu Copper held by electrostatic attraction (non specific adsorption or ion exchange mechanisms) onto exchange sites in soils is termed as exchangeable Cu. Exchange sites comprises the negatively charged surfaces of clays and iron, aluminium and manganese oxides and also the functional groupings of organic matter (Hodgson, 1 963). Variable charge of clay minerals results from the deprotonation of hydroxyl groups, while isomorphous substitution of ions in the clay structure accounts for the permanent charge associated with these materials. The negative charge on iron, aluminium and manganese oxides results from the deprotonation of surface hydroxyl groupings (Parfitt, 1 980). Similarly, the dissociation of protons from mainly carboxyl and phenol functional groups of organic matter contributes to its negative charge (Parfitt, 1 980). 2.3.2.3 Specifically adsorbed Cu Specific adsorption of Cu is a term, which refers to the formation of stable complexes between Cu ions and specific functional groups at the surface of organic and inorganic soil colloids. Copper can be "specifically" adsorbed by layer silicate clays, oxides of Fe, Mn, and AI, and organic matter. In specific adsorption, ions are held much more strongly by the surface charge, as these ions penetrate the co-ordination shell of the structural atoms and are bonded by covalent bonds via 0 atoms (Cu-O-Fe or Cu-O-AI bonds) or OH groups to the structural cations (Huang, 1 980; Wu et aI., 1 999). Stevenson and Fitch ( 198 1 ) observed that organic colloids and clays play a major role in Cu retention by soil. In most mineral soils, Cu may be bound as clay-metal-organic complexes, since in these soils, organic matter is intimately bound to clay. Specific adsorption of Cu is measured in the presence of a weak electrolyte solution of one of the major soil cations (calcium or magnesium) thereby minimising the occurrence of non­ specific (exchange) reactions (Swift and McLaren, 1 99 1 ) . 2.3.2.4 Occ/uded Cu Soil oxides have a reactive surface capable of adsorbing certain amounts of Cu. Over time the oxide component of soils builds up, and is capable of occluding increasing amounts of Cu. Warnant et al. ( 198 1 ) used Tamm's reagent (ammonium oxalate and oxalic acid) to extract Fe, Al and Cu associated with oxidic material . In both the A and Chapter 2 Literature review 1 8 B horizons there was a positive linear relationship between either the amount of iron or aluminium extracted and the amount of Cu extracted. McLaren and Crawford (1 973b) also observed that acid oxalate-extractable Cu was correlated {r=0.44, p6) 1 . 1 1 x 1 0-4 to l . l lx 1 0·' Choi et al. ( 1 999) M Cu (ll) Orthic humic gleysols 0.005 M CaC12 2.74 -6.74 o and 200 mg Cu L· as Yuan and CuC12 Lavkulich ( 1 997) Typic Fragiochrept 0.02 M CaClz 4 - 7 300 �Cu Cavallaro and McBride ( 1984) Raplic podzol, Fimic O.O IM Ca(N03h 2 - 8 0.00 1 - 100 mg Cu L-' as Welp and Anthrosol, Eutric Cu(N03h Briimmer ( 1999) Cambisol and Calcaric Regosol Various soil 0. 1 M CaCh and 4 - 6 10· to 1 0 .... M Cavallaro and variable McBride ( 1 978) Goethite, O.OOIM to 4 - 1 0 O.OOOIMCu Spark et al. ( 1997) (X-alumina, silica and 0 . 1 M NaCl kaolinite Georgia, kaolinite 0. 1 M NaCl and 4 - 6 10.0 to 1 0'> M McBride ( 1 978) - variable () mdIcates Cu hydrOXIde formatIOn. Chapter 2 Literature review 32 2.4.1.1 Formation of Cu compleXes in soils A number of studies have shown that Cu2+ in soil solution, especially at higher pH, exists primarily in a fonn complexed with soluble organic matter (Hodgson et al., 1 966). In soils amended with organic residues, such as sewage sludge, organically complexed Cu may be expected to be the predominant fonn (Behel et al., 1983). Stevenson and Fitch ( 1 98 1 ) reported that, based on a commonly used soil Cu fractionation scheme, organically bound Cu accounts for about 20% to 50% of the total soil Cu. The amount of organically complexed Cu in solution generally increases when soil pH is 7 or higher, because of the greater solubility of soil organic matter at higher pH values (McBride and Blasiak, 1 979), while the concentration of free ionic Cu at a higher pH is much less, usually in the range of 10-9 to 1 0-8 M (McBride and Blasiak, 1979). Organic enriched surface horizons usually contain higher concentrations of Cu than the deeper horizons that generally contain less organic matter. Thus complexing of Cu by organic matter, in the fonn of humic and fulvic acids, has long been recognised as an effective mechanism of Cu retention in soils (Mc Bride, 1 98 1 ) . Stevenson and Fitch ( 198 1 ) provided the following evidence for the complexing of Cu2+ by humic and fulvic acids. • The inability of K+ and other monovalent cations to replace adsorbed Cu2+ from mineral and organic soils. • The correlation between Cu2+ retention and humus content. • The ability of chelating agents to extract Cu, while solubilising part of the soil humus. • The selective retention of Cu2+ by humic and fulvic acids in the presence of cation­ exchange resin. The complexing ability of humic and fulvic acids is due to their high oxygen-containing functional groups, such as carboxyl, phenolic hydroxyl, and carbonyls of various types. Because of the high acidity and relatively lower molecular weights, metal complexes with fulvic acids are more mobile than those of humic acids. Fulvic acids are also more efficient in complexing metals than humic acids, and so metal ions may also be more available to plants than humic acid-complexed metals. Colloidal organic matter in soils Chapter 2 Literature review 33 provides ' specific' adsorption sites for eu, thereby causing eu deficiency in organic soils. The fraction of soil eu associated with organic matter should be expected to be high in soils rich in organic matter. Stevenson and Fitch ( 1 98 1 ) summarised the following effects on soils of the formation of Cu-organic matter complexes: • The soil solution Cu concentration can be decreased by clay-humus complexes, and by the formation of insoluble complexes with humic acids. Soluble ligands may be of considerable importance in transforming solid phase forms of Cu into dissolved forms. • In high pH soils (eg. calcareous soils) complex formation will promote maintaining Cu in dissolved forms. • In the presence of excess Cu, organic complexes may reduce the concentration of Cu2+ to non-toxic levels. • Natural complexing agents may be involved in the transport and mobility of Cu in soils. 2.4. 1.2 Adsorption of Cu by soil components The major soil components involved in the adsorption of Cu include soil organic matter, hydrous oxides of Fe, Al and Mn, and clays. The Cu adsorption ability of soils is affected by the pH, the amount of organic matter, Fe and Al oxides and clay minerals (McLaren et al., 1 983b; Stevenson et al., 1 993 ; Wu et al. , 1 999). Soil pH is the major factor for the control of Cu sorption, and many authors confirmed that the sorption of Cu onto soils increases with an increase in pH (Cavallaro and McBride, 1 978; Kuo and Baker, 1 980; Harter, 1 983, McLaren et al. , 1 983a; Hue et al., 1997; Choi et al. , 1 999). 2.4. 1.2.1 Sorption by organic matter Organic matter plays a major role in the retention of Cu in soils (Stevenson and Fitch, 1 98 1 ; Kabata-Pendias and Pendias, 1 992, McBride, 1 994). The organic fraction, in particular, seems to be a source of specific Cu sorption sites in pasture soils (McLaren and Crawford, 1 973a; Kadlec and Keoleian, 1 986), perhaps because the ion is unique in its ability to form inner sphere complexes at a wide range of pH levels (McBride, 1 98 1 ). Chapter 2 Literature review 34 Organic complex formation could lead to an increased mobility in the soil, since Cu is known to form stable complexes with fulvic acid (Kabata-Pendias and Pendias, 1 992). The complexing of metals by organics can also affect retention by mineral surfaces. Goh et al. ( 1 986) demonstrated increased retention of Cu by Al precipitation products, when tannic acid was present during precipitation. They attributed the increased sorption capacity to exposed edges as well as to the presence of carboxylate and phenolic groups. Small amounts of organics in solution have been shown to increase solution Cu concentrations, above that which can be expected for ionic Cu in equilibrium with inorganic sorption sites or precipitated forms (McLaren et al., 1 98 1 ). The complexing of metals by carboxylate groups is commonly proposed as a mechanism of retention (Figure 2.2). Boyd et al. ( 1 98 1 ) found from electron spin resonance evidence that Cu2+ is sorbed to humic acid by forming two inner-sphere bonds with oxygen atoms. Subsequent infrared evaluation of Cu-humic acid complexes indicated that the two bonds were unlikely to result from a complex with a single carboxylate group. They proposed the formation of a chelate ring of either two adjacent carboxylate groups or a carboxylate and an adjacent phenolic OH group, with an oxygen atom from each group fonning a single bond with Cu2+. COO- OH Low pH + Cu2+ � High pH COO � CuOH(H20)x-1 + H+ / o Figure 2.2 Complex formation of Cu ions by humic acid according to pH (Van Dijk, 1971). Chapter 2 Literature review 35 2.4.1.2.2 Sorption by hydrous oxides According to Jenne ( 1 968) hydrous oxides play a significant role in the control of Cu concentrations in soil solution. Reports are readily available of Cu retention by hydrous Fe oxide (Okazaki et al., 1 986), hydrous Mn oxides (Kabata-Pendias, 1 980) and hydrous Al oxides (Shuman, 1 977; Barrow, 1 986). Retention of Cu ions by oxide surfaces is inversely dependent on the degree of crystalinity (Okazaki et ai., 1 986) and, since the oxides have variable charge, the extent to which retention occurs is dependent on the pH of the solution (Barrow, 1 987; Bolan et al., 1 999) . Predictably, surface conditions strongly influence Cu sorption by hydrous oxides. Cavallaro and McBride ( 1 984) found that treatment of clays for the removal of organics tended to either enhance or have little effect on sorption and fixation of Zn and Cu. They suggested that the oxide component of soil clay was more significant than the organic component in metal sorption and fixation, and that oxide fOTIns in general and organic coatings have an affinity for metals (Lion et aI., 1 982). In studying the effect of phosphate on the sorption of Cu by Al hydroxide, McBride ( 1985) observed decreased sorption of Cu, when phosphate was present. Using electron spin resonance, he demonstrated that the sorbed phosphate blocked the co-ordination of Cu2+ to the surface AIOH group. McBride et al. ( 1 998) conducted adsorption at two pH ranges (5 to 5 .5 and 6.5 to 7.0) using a suspension of natural organic matter and iron oxide (FeOOH) that were equilibrated with a wide range of concentrations of added Cu (50 to 5000 mg kg-I ). Both FeOOH and organic matter adsorbed >98% of the added Cu up to an addition level of 1 000 mg kil . At higher Cu additions, adsorption efficiency of the oxide decreased markedly, when the pH was 5 .5 or lower. Whereas the organic matter still adsorbed >98% of the Cu. Across the range of pH and Cu loading studied, the Cu2+ activity in the organic matter was generally one to two orders of magnitude lower than with the oxide, even after long equilibration periods. It appears that under slightly acidic conditions in soils, organic matter is more likely to limit Cu2+ activity and phytotoxicity than Fe oxides. Two mechanisms for the adsorption of Cu on goethite were proposed by Padmanabham ( 1 983). Chapter 2 Literature review 36 ( 1 ) Specific adsorption on positive surface sites H + H 2+ /OH / O - Cu ", ' + Cu2+ � "", I + H+ Fe Fe / , ~ / 1 ~ OH2 OH2 Adsorption of Cu by this mechanism involves low bonding energy, and over time, adsorbed Cu may isomorphously substitute into the oxide lattice. At low pH a higher proportion of Cu is present as Cu2+. In this valence state Cu is similar in size to Fe(III) and is able to be easily fixed or occluded in the oxide lattice. (2) Specific adsorption on neutral sites OH OH � I H � I H Fe �OH Fe �O / / \ 0 CU2+� 0> /CU + 2H+ '" /Fe �OH Fe � I H I H OH OH Hydrolysis occurring at the oxides surface results in the adsorption of Cu2+ and the release of two protons. 2.4.1.2.3 Sorption by silicate clay minerals Cu sorption by silicate clay minerals has been shown to be an important retention site for this ion (Kabata-Pendias, 1980). Shuman ( 1 980) pointed out that clays are capable of sorbing both Cu and Zn in excess ofCEC. Wakatsuki et at. ( 1 975) found that Cu was Chapter 2 Literature review 37 sorbed by kaolinite clays at solution Cu activities lower than required for oxide I hydroxide precipitation. They interpreted this to indicate that Cu is retained at specific sorption sites on clays. While Kabata-Pendias and Pendias ( 1992) maintain that the ability of soil minerals to remove Cu from solution is dependent on the mineral surface charge, the relationship between CEC and Cu is not always straight forward. Okazaki et al. ( 1 989) reported a direct relationship between the Si02 I Ah03 ratio of synthetic aluminosilicates and Cu adsorption. Jenne ( 1 968) argued that retention is more a function of hydrous Mn and Fe oxides on clay surfaces than on the properties of the surfaces themselves. 2.4.2 Desorption of Cu Presently it is generally accepted that soil solution concentrations of micronutrients are regulated by sorption-desorption reactions at the surfaces of soil colloidal material (Swift and Mc Laren, 1 99 1 ). Hogg et al. ( 1 993) reported the availability of soil Cu to plants is dependent on desorption in the soil solution of Cu from the surfaces of soil colloidal materials. They found that the proportion of added Cu desorbed was reduced substantially by increasing the contact period to 1 2 weeks before desorption. The longer the contact period the greater the amount of Cu that was irreversibly sorbed. The nature of the slow reactions that result in a decrease in the desorption of added Cu remains open to conjecture. With increased time of sorption, there is a slow redistribution of Cu ions to more strongly binding or less accessible sites, possibly involving diffusion into extremely small pores and inter particle spaces. Desorption of native soil Cu was increased by increasing the temperature at which desorption was carried out. They concluded that the amount of Cu that can desorb readily from soil depends not only on the total amount of labile Cu in the soil, but also on soil pH, temperature, and in the case of added Cu, on to the length of time since the addition of the Cu to the soil. Soil acidification enhances Cu desorption from the solid phase, thereby increasing its concentration in the soil solution (Basta and Tabatabai, 1 992). This enhanced desorption may intensify the bioavailability of Cu. Cu uptake by plants is a function of the activity of the soil solution Cu2+, which is controlled by the concentration of total soluble Cu, the inorganic and organic ligands and by soil pH. A decrease in pH enhances Cu2+ absorption by plants, because of the increased solution activity of Cu2+ associated with the dissolution of soil minerals, the decrease in organic complexing and solid phase Chapter 2 Literature review 38 adsorption of Cu (Baker and Senft, 1 995). McLaren et al. ( 1 990) found that initially less than 10% of the Cu adsorbed by the soil was desorbed and after three months of soil contact, only a negligible amount « 1%) of the adsorbed Cu could be desorbed. Temminghoff et al. ( 1 994) studied the effect of pH on Cu desorption from a sandy soil, and complex formation by dissolved organic fractions. They observed Cu desorption and its pH dependency could be modelled with two species of the Freundlich equation. As the soil pH decreased, the amount of Cu desorbed from the soil increased. The amount of Cu bound to solid organic carbon was almost equal to Cu bound by dissolved organic carbon. 2.4.3 Cu chelate equilibria in aerobic and anaerobic soils Copper is a strongly chelated metal, as indicated by the low value of free Cu compared with chelated forms. Diethylenetrinitrilopentaacetic acid (DTPA), especially, and N-(2- Hydroxyethyl)ethylenedinitrilotriacetic acid (HEDTA), ethylenedinitrilotetraacetic acid (EDTA) and trans- l ,2-Cyc1ohexylenedinitrilotetraacetic acid (CDTA) are relatively effective chelators for Cu in alkaline soils, in which Ca is the main competing cation. Experimental studies using DTP A and EDT A in soils provide support for the prediction of Cu (Norvell and Lindsay, 1 972). Competition from Fe restricts the chelation of Cu at low pH, but HEDT A has the potential to chelate Cu effectively under moderately acidic conditions. Ethylenediiminobis(2-hydroxyphenyl)acetic acid (EDDHA) appears to have some limited potential as a chelator for Cu under alkaline conditions. However, experimental results indicate that Cu2+ solubility in soils is generally too low to allow effective chelation, when Cu-EDDHA itself is added (Aboulroos, 198 1 ). N-(2- Hydroxyethyl)iminodiacetic acid (HIDA) and nitrilotriacetic acid (NTA) have the greatest abi lity to chelate Cu under moderately acidic conditions. At low pH, none of the ligands is particularly effective, and soluble organic materials in the soil solution may provide similar levels of complex formation as noted by Norvell ( 1 972). The experimental studies using EDTA and DTPA show that competition from Fe2+ and Mn2+ can limit the stability of chelated Cu and Zn in soils under reducing conditions (Reddy and Patrick, 1 977). The relative effectiveness of many ligands is not altered by changing redox conditions. For example, HEDTA is the dominant chelator for several Chapter 2 Literature review 39 metals at all redox levels. The most prominent among these ligands is EDDHA, which becomes a much better chelating ligand for other metals as the Fe3+ concentration declines with increased reduction. This effect is most obvious for Cu2+ because Cu­ EDDHA chelates are also reasonably stable. EDDHA is one of the most effective chelators for Cu2+ under reducing conditions, but it is one of the least effective chelators under strong oxidising conditions. 2.5 PLANT AVAILABILITY OF COPPER IN SOILS The availability of Cu to plants is commonly measured by two methods: • Plant uptake • Chemical extraction from soils. Plants are grown in the field or more commonly in a glasshouse, where environmental conditions can be monitored if not controlled. In order to obtain a measurement of Cu uptake, which can be usefully compared by chemical assessment, all limitations to plant growth must be removed. This includes adequate fertilisation with all nutrients (except Cu), and control of the moisture content and temperature of the soil. Soil Cu can be measured by chemical extraction, and then related to Cu uptake, by the Cu content of the experimentally grown plants. 2.5. 1 Availability ofnative Cu Copper in soil is strongly held on inorganic and organic exchange sites and in complexes with organic matter. For this reason a large proportion of the total Cu content of soils is not available for plant uptake. The proportion of the total Cu taken up by plants has been found to be greater in mineral soils than in peats (Kabata-Pendias, 1 963). Deficiencies in crops may be due to an inherently low total Cu content of a soil, or to only a small amount being in an available form. Although Cu held on exchange sites is not readily available to plants, cation exchange for Cu2+ and CuOH+ can take place and is best affected by H+ (Mengel and Kirkby, 1 978). Some organic forms of Cu in soils are thought to be more available than others. It has been shown that in organic soils the availability of Cu depends not only on the concentration in the soil solution, but also on the form in which the Cu is present Chapter 2 Literature review 40 (Mercer and Richmond, 1 970). Kubota et al. ( 1963) reported that the Cu concentration increased in the soil solution with increasing wetness in two soils from New Hampshire, but decreased with increasing soil moisture in two soils from Nevada. It has been observed in Britain that Cu deficiency is more severe in dry, sunny years than when dull, moist conditions occur (Caldwell, 197 1 ). The application of Cu-free fertilisers to soils may lead to Cu deficiency in crops or aggravate it. It was observed in several published reports that nitrogenous and phosphatic fertiliser compounds lead to an increase in Cu deficiency in cereal crops (Caldwell, 1 97 1 ; Touchton et al., 1 980; Robson and Reuter, 1 98 1 ). Kubota ( 1983) reported that low Cu levels in grass is likely to result in Cu deficiency in ruminant animals, especially where the soil has an elevated Mo level. Brennan ( 1 993) reported that for either previously or currently applied Cu, the application of nitrogenous fertilisers decreased the eu concentration (lsd (p=0.05) = 0.25) in yellow brown earth soils, except on the plot currently receiving Cu. This is mainly attributed to increased plant growth, diluting the Cu concentration in the plants. 2.5.2 Availability offertiliser Cu Copper deficiencies can be corrected by applying Cu to plant foliage, or to the soil by either band or broadcast application procedures. Use of soil application procedures for the correction of Cu deficiency is usually justified on the basis of a relatively high residual value for soil-incorporated Cu (Gartrell, 1980). Copper added to soil is rapidly immobilised, and plant utilisation is mainly dependent on plant roots being in the vicinity of the added Cu. Generally finely ground powders are more rapidly effective than coarser materials, which have a greater residual effect. Placement of fertiliser may have an influence on the effectiveness of the fertiliser. McLaren and Williams ( 198 1 ) found that surface applied Cu had little influence on the yield o f rye grass and red clover, when compared to soil incorporated fertilisers. Cu added to the soil as chelates have been found to be more effective than CUS04, due to a greater solubility and mobility in the soil (Gartrell, 1 98 1 ; McLaren and Williams, 1 98 1 ). Wallace and Mueller ( 1 973) showed that chelated Cu increased the concentration of Cu in bush beans (Phaseolus vulgaris) compared with the equivalent rate of CUS04 fertiliser. Chapter 2 Literature review 41 The general use of CUS04 as a Cu source reflects its high water solubility, and wide availability (Allow ay and Tills, 1 984; Karamanos et al. , 1 986). The same rate ofCuS04 and CuO were recommended for correcting Cu deficiency by either band or broadcast Cu application (Mengel, 1 980; Vitosh et al., 1 98 1 ). The suitability of CuO when broadcast has been found to depend on particle size. CuO broadcast on to the soil surface at 5 kg Cu ha-I , and worked into the soil, did not correct Cu deficiencies in canola, barley, or wheat during the year of application, but corrected the deficiencies in the following years (Karamanos et al., 1 986). The lack of Cu response to CuO was mainly due to the low water solubility of the coarse, granular CuO, which ranged in particle diameter from <0.2 to 3 .0 mm. Coarse Cu carriers are also ineffective where inadequate contact occurs between the roots and the applied Cu (Gartrell, 1 98 1 ). Seed treatment is recognised as being less effective than either soil application or foliar sprays (Gartrell, 1 98 1 ). 2.5.3 Cu uptake and translocation 2.5.3.1 Cu absorption by plant roots Copper in biological systems is predominantly present as Cu2+, which has great significance in terms of Cu absorption by plant roots from the soil solution, because most organic chelates bind Cu2+ strongly (Graham, 1 98 1 ). In the soil solution, up to 98% of the Cu is complexed to low molecular weight organic compounds (Hodgson et al., 1 966). The Cu2+ ion has a high affinity for peptide N and S, and binds quite strongly to proteins, especially proteins high in cysteine residues. Also due to its affinity for carbonyl, carboxyl, sulfhydryl, and phenolic groups, all of which are found in cell walls, there is a strong and specific adsorption of Cu to cell walls that is not easily desorbed (Harrison et al., 1 979). It should also be noted that because Cu2+ can be reduced to Cu + in a range of physiological redox potentials, the significance of the absorption of Cu + by roots should not be ignored (Graham, 1 98 1 ). There is considerable evidence that the free Cu2+ ion is the absorbed species. A number of researchers have demonstrated that Cu is absorbed more rapidly from Cu2+ solutions than from solutions of Cu complexed to synthetic chelators such as DTPA or EDTA (Harrison et al., 1 979; Wallace, 1 980). Also, Goodman and Linehan ( 1 979), using electron paramagnetic resonance spectrometry to study root absorption, presented evidence consistent with the dissociation of Cu2+ from EDTA during absorption from a Cu2+-EDTA solution. Chapter 2 Literature review 42 Graham ( 1 98 1 ) has suggested that Cu2+ uptake could be facilitated by a carrier type protein that can bind Cu2+ on either side of the plasma membrane. This would have a greater probability of releasing Cu inside the cell due to the lower electrochemical potential for Cu in this compartment. Thus Cu2+ uptake could occur by a passively driven, carrier type membrane protein. Loneragan ( 1 98 1 ) concluded that the absorption of Cu2+ and Zn2+ is a metabolically active process at least at normal soil levels. This conclusion is supported by the finding that absorption is reduced by metabolic inhibitors. Since higher activities of either Zn2+ or Cu2+ in soil or solution cultures are antagonistic to plant uptake of other ions, it is usually recognised that both ions are absorbed in the same way. Other cations such as Ca+, K+ and NH/ reduce the Cu absorption. However, it is likely that these ions affect the uptake of Cu2+ via differential complexing, and other surface effects, which determine the soil solution activity of Cu2+ and membrane permeability. 2.5.3.2 Translocation of Cu in plants Gilkes ( 1 98 1 ) reported that the rates of absorption of Cu is the lowest of the essential elements, and there are large genetic differences among plant species and cultivars within species. Baker and Low ( 1 970) concluded that the Cu in the environment of the rhizosphere is almost all organically complexed by root exudates and soil humus. But uptake and translocation are functions of the activity of Cu2+ in true solution at the active absorption sites. According to published literature (Gupta, 1 979; Baker and Low, 1 970; Dragun and Baker, 1 982), while root absorption of Cu includes specific adsorption onto cell walls of the root free space, the limiting step of transport across the plasmalema involves an electrochemical gradient relating the activity of ci+ in solution outside the root to that of the cytoplasm of the cortical cells. Because of the plant component, the absorption process is controlled by the plant species and cultivars, it becomes obvious that Cu uptake is a function of the activity Cu2+ at the absorption site outside the plasmalema. The translocation of Cu2+ within plants occurs in both the xylem and phloem, where the metal is bound by organic nitrogen compounds such as amino acids. Loneragan ( 198 1 ) reported that the concentrations of 1 .5 to 2.0 J.LM ci+ in the xylem and 3-140 J.LM in the Chapter 2 Literature review 43 phloem. Much of the Cu2+ associated with plant roots may not be translocated into the shoot even when a deficiency occurs in an aerial part. In the shoot, N metabolism appears to control the binding and transport of Cu2+. Cu is a relatively immobile element in plants. Green leaves may accumulate high concentrations of Cu2+, and subsequently do not release it to younger leaves and other tissues, such as inflorescences, despite their deficiency. Cu concentration in young leaves of wheat is important in diagnosing Cu deficiency (Snowball and Robson, 1 984), and is now being used to provide fertiliser recommendations for farmers (Gartrell et al., 1 979). If Cu deficiency is diagnosed before flowering, a foliar application of Cu can be made without much yield loss (Graham, 1 975). 2.5.3.3 Interaction with other ions and nutrients The uptake, metabolism and accumulation of Cu by organisms can be affected by the action of other ions (Pellegrini et al., 1 993; Shuttleworth and Unz, 1 993). A number of variables must be considered in studies of the role of Cu in ecological and physiological functions. For example, metal ions may be competing for binding sites on proteins such as metallothionein (Witkowska et aI. , 1 991). The complexity of metal to metal interactions has an important implication for bioassays and the toxicity testing of mixtures since multiple elements and compounds often co-occur in industrial and/or agricultural residues (Mullick and Konar, 1991) . The important interactions of Cu with other elements include copper-iron, copper-molybdenum and copper-zinc and with copper-N and P fertilisers. Copper-iron Copper-iron antagonism is indicated as Cu chlorosis. High levels of Cu in plants decreases the Fe content in the chloroplast (Reilly and Reilly, 1 973). Fe, on the other hand, reduces Cu absorption from soil solutions, especially in peat soils. The optimal CulFe ratio varies for different plant species. The toxic effect ofCu can be decreased by the addition of Fe. However a synergistic effect of Cu on Fe absorption by rice seedlings was reported by Lidon and Henriques ( 1 993). Copper-cadmium Copper-cadmium interactions reported by some authors as both antagonistic and synergistic in the elements uptake by roots. Synergism may be a secondary effect of damage to membranes due to the imbalance in proportion of the metals. Khan et al. Chapter 2 Literature review 44 (1 996) reported that increasing the rate of Cu fertiliser application had no significant effect on Cd concentration in a mixed pasture. Copper-Manganese Studies by Lidon and Henriques ( 1 993) using rice seedlings have shown a decrease in net uptake by the roots and translocation to the shoots of several elements, including Zn, Fe and Mn, with an increase in the external concentration of Cu. An increased level of external Cu has been reported to affect the Zn, Fe and Mn metabolism in plants (Bowen, 1 98 1 ; Alva and Chen, 1 995). Copper-Molybdenum Copper-Molybdenum interactions are closely related to N metabolism. Cu interferes with the role of Mo in the enzymatic reduction ofN03. The mutual antagonism existing between these elements is highly dependent on plant species, and the type of N nutrition. Copper aggravates Mo deficiency in plants, especially those using N from N03. Copper-Zinc A number of researchers have observed mutually competitive interactions between Cu and Zn (Bowen, 1 979; Luo and Rimmer, 1 995). Bowen ( 1 979) found that Cu and Zn competitively inhibit the uptake of the other, and proposed that both ions were absorbed by the same transport system. High Zn concentrations in the soil accentuate Cu deficiency (Halder and Mandal, 1 98 1 ). The effect is not primarily due to dilution effects or reduced translocation of Cu from roots to tops. Since Cu and Zn seem to be absorbed through the same mechanism, each competitively inhibits uptake of the other (Giordano et aI., 1 974). Studies of Lidon and Henriques ( 1 992b and 1 993) found a decrease in net uptake of Cu by the roots due to an increase in the translocation to the shoots of Cu, and other elements, including Zn, Fe and Mn, with an increase in the external concentration of Cu in rice (Oryza sativa L.) seedlings. In contrast, Beckett and Davis ( 1 978) working with nutrient solutions found that Cu had little effect on the amount of Zn uptake in barley or vice versa. Kabata-Pendias and Pendias ( 1992) described that the Zn-Cu interaction as an antagonism effect and also observed that addition of large amounts of Zn decreased the uptake of Cu in the plant top. Copper-Nitrogen and Phosphorus Several published works reported that nitrogenous and phosphatic fertiliser compounds induce a Cu deficiency in cereal crops (Caldwell, 1 97 1 ; Touchton et al., 1 980). In citrus, highly concentrated long-term applications of P fertilisers have resulted in, and Chapter 2 Literature review 45 intensified Cu deficiencies (Olsen, 1 972). Copper deficiency on initially infertile soils was enhanced after the soil N supply was elevated by leguminous pastures (Gartrell, 1981 ) . Brennan (1 993) reported that for either previously or currently applied Cu, an application of nitrogenous fertilisers decreased the Cu concentration in yellow brown earth soils, except on the plot currently receiving Cu. Kumar et a!. ( 1 990) reported that N and Cu were found to have a mutUally antagonistic effect on each other's concentration in the wheat plant. The antagonism was greater with NH4 + sources than N03- compounds. Soil P also reduces the concentration of plant Cu. This may be due to plant dilution effects as a result of added P increasing plant growth without a consequent increase in Cu uptake (Robson and Reuter, 1 98 1 ). The P absorbed or precipitated the metals to form insoluble complexes, and therefore make both the Cu and P unavailable to plants (Lindsay, 1 979; Ross, 1 994). In subterranean clover, the interaction between P and eu was indirect and positive on its effect on growth, and when the P supply was increased from a marginally deficient level to an adequate level, there was depressed Cu absorption and an accentuated Cu deficiency (Reuter et a!., 198 1 b). Varvel ( 1 984) found that applied Cu competed with both the uptake of P by wheat plants and also the translocation of P from root to shoot. Plants given no Cu application contained 6.5 g P kg-I DM, addition at Cu rates of 3 .4 to 2 1 .5 kg Cu ha-I decreased the content of P to an average of 3 .4 g P kg-I DM. Availability of P in the presence of Cu, and vice versa, was thought to be decreased due to the formation of insoluble phosphate compounds in the soil (Halder and Mandal, 1 982). 2.5.4 Effect of Cu fertiliser on DM yield and Cu concentration Copper concentrations in forage and pasture crops depend on the soil availability of Cu, plant species, stage of growth, time of year, and lime and fertiliser applications. Legumes tend to take up larger amounts of Cu than grasses. In some cases, non-crop species or weeds may also contribute to the increased dietary intake of Cu by grazing livestock. Kubota ( 1 983) found that mixed pasture herbage rarely contains more than-2a mg Cu kg- I and usually less than 1 a mg Cu kg- I . However, grazing animals may ingest up to I a times more Cu in the form of soil than in the herbage. Soil ingestion commonly Chapter 2 Literature review 46 ranges from 1 - 1 0% of the dry matter intake of grazing cattle and up to 30% in sheep (Thomton, 1 979). Wells ( 1 957) recorded that the pasture Cu concentration ranged from 3.5 to 1 8 mg Cu kg-I . He also found that pasture species had marked differences in Cu concentration. The Cu concentration in white c lover, red clover and ryegrass was 1 0.6 mg kil , 19. 1 mg kg-1 and 4.0 mg kg- I , respectively, when these plants were grown on the same soil. Reuter et al. ( 1 98 1 a) recorded that the eu concentration in subterranean clover declined from 3 .9 mg kg-1 at 26 days after sowing to 1 .6 mg kg- 1 at 98 days. Sherrell and Rawnsley ( 1 982) found that an application of 2 to 4 kg Cu ha-1 as copper sulphate increases herbage Cu concentration from 5 to 1 2 mg kil within 4 weeks and then it decreases markedly to 8 mg kg- l over the next 9 to 1 0 months. He also observed that the clovers tended to take up larger amounts of Cu. Willimott ( 1 995) found that an application of 5 , 1 0 kg Cu ha- I as copper sulphate increased the herbage Cu concentration up to three months after the Cu application, and then decreased back to the initial levels nine months after application. Gilkes and Lim-Nunez ( 1 979) observed that increasing levels of Cu promoted Cu uptake in wheat. Jarvis ( 1 978) found that with increasing concentrations of Cu in solution, the Cu concentration in the roots increased much more rapidly than in the shoots of ryegrass. He also found that in most species Cu concentrations seldom exceeded 30 mg Cu kg- I even when very large additions of Cu (953 mg Cu kg- I of soil or 1 0 mg Cu L-I of solution) were made to either soils or solution cultures. Rasheed and Seeley ( 1 966) found that the Cu concentration in legume shoots increases more rapidly than in grasses. Davis and Beckett ( 1 978) estimated that the upper critical tissue concentration of Cu in rye grass was 2 1 mg kg-I . Similarly Davis and Carlton-Smith ( 1 984) recorded that the upper critical foliar concentration of Cu in ryegrass was 22 mg CU kil . Reuter et al. ( 1 98 1 a) noted that with increasing rates of Cu application (ranging from 0 to 533 mg Cu kg- I) , the Cu concentration increased in both plant tops and roots. Plenderleith and Bell ( 1 984) found that with increasing additions ofCu (4 to 600 mg Cu kg-I soil) the Cu concentration in tropical grasses increased from 1 7 to 27 mg kg-I . Alva Chapter 2 Literature review 47 and Chen ( 1 995) observed that the concentration ofCu in shoots increased linearly both in Cleopatra mandarin and Swingle citrumelo citrus rootstock seedlings with an increase in external Cu concentration. 2.5.5 Residual effectiveness of applied Cu The residual effects of Cu application have been recorded in a number of publications (Forbes, 1 978; Brennan et a!., 1 980; Gartrell, 1 980; Sherrell, 1 989; Levesque and Mathur, 1 986). B6langer et a!. ( 1 986) observed that residual Cu had a positive effect on the Cu content of an oat crop grown in the soil four years later. Cox ( 1 992) reported that the residual effect of Cu fertilisation with time· varied between soils. An application of 4.48 kg Cu ha-I increased the extractable Cu above the initial concentration for 9 to 27 years, the average time for the five soils tested was about 16 years. Gartrell ( 1 980) observed that Cu applied four years earlier increased the yield of wheat up to three times more than current applications banded with the seed. Copper applied 1 2 years earlier was still more effective at increasing the Cu uptake of wheat crops than from current fertiliser applications. Soil analysis revealed that all applied Cu remained in the top 1 0cm up to four years after the initial application. Brennan et al. ( 1 980) observed in an incubation study that when Cu fertiliser was thoroughly mixed with soil, slow reactions between added Cu and the soil decreased the residual effectiveness of the Cu fertiliser. Although the residual effectiveness of soil applied Cu has been clearly demonstrated, there is little information available relating to the change in the form and distribution of soil Cu with time. Sherrell ( 1 989) reported that the effect of CUS04 applied to an established stand of lucerne (Medicago sativa L.) on a yellow brown pumice soil was measured over a 4 year period. Dry matter yield was not affected, but plant and soil Cu concentrations were increased by the Cu application. Extractable soil Cu indicated that the residual effect of a Cu application might last for a considerable time. 2.5.6 Biochemicalfunctions ofCu Extensive studies have been made on the form and behaviour of Cu in plants. All the findings described in a number of publications can be summarised as follows: • Copper is mainly complexed with organic compounds of low molecular weight and with proteins. Chapter 2 Literature review 48 • Copper occurs in compounds with no known functions as well as in enzymes having vital functions in plant metabolism. • Copper plays a significant role in several physiological processes:- photosynthesis, respiration, carbohydrate distribution, N reduction and fixation, protein metabolism, and cell wall metabolism. • Copper influences water permeability of xylem vessels and thus controls water relationships. • Copper controls the production of DNA and RNA, and its deficiency greatly inhibits the reproduction of plants (reduced seed production and pollen sterility). • Copper is involved in the mechanisms of disease resistance. The resistance of plants to fungal diseases is likely to be related to an adequate Cu supply. There is also evidence that plants with enriched Cu concentrations are susceptible to some diseases. These phenomena may indicate that the role of Cu in disease resistance is an indirect one. 2.5. 7 Deficiency and toxicity oleu in plants Gartrell ( 1 98 1 ) reported that peats and mucks have commonly produced Cu deficient crops throughout Northern and Western Europe, some Midwestern and Eastern states of the United States, New Zealand and Australia. Bames and Cox ( 1 973) reported that in general, soils most commonly found to be deficient in Cu are poorly drained mineral soils, and mineral soils that are high in organic matter. Lucas and Knezek ( 1 972) indicated that the total Cu in the soil should exceed 4 to 6 mg kg-1 in a mineral soil and 20 to 30 mg kg-1 in organic soils to sustain maximum yield of Cu responsive crops. Robson and Reuter ( 1 98 1 ) reported that in most plant species, Cu deficiency is characterised by chlorosis, necrosis, · leaf distortion, and terminal dieback, with symptoms occurring first in young shoot tissues. Once absorbed, Cu is poorly translocated in plants. Hence, the terminal growth, of most plants, is the first to be affected. Specific symptoms often depend on plant genotypes and the stage of the deficiency. Copper is required in trace amounts for various metabolic processes in plants, at higher concentrations Cu is toxic to plant growth. Published reports on Cu toxicity to plants include the effects on photosynthesis (Lidon and Henriques, 1 991) and nitrogen Chapter 2 Literature review 49 metabolism (Weber et al., 1 991 ). Copper toxicity most frequently occurs where Cu containing fungicides have long been in use (Lepp et al., 1 984). Plants affected by Cu toxicity are slow growing and frequently exhibit symptoms typical of Fe deficiency. Heavy metals such as Cu tend to accumulate in the roots and in turn, affect the growth of the whole plant. Lidon and Henriques ( 1 992a and 1 993) observed that the Cu concentration in roots increased linearly with increasing external Cu concentrations. Decreased translocation of Cu to the above ground parts from the roots has been suggested as a mechanism to withstand Cu toxicity. Beckett and Davis ( 1 977) found that at high levels, Cu depressed plant growth in barley. Alva and Chen (1 995) observed that with an increase in external Cu concentration, the shoot and root dry weight of citrus plants decreased significantly. Luo and Rimmer ( 1 995) observed Cu and Zn interactions on barley plant growth, and concluded that the growth of barley was controlled principally by the amounts of both added Zn and Cu. The effect of the added Cu was to increase the toxicity of the added Zn. Plenderleith and Bell ( 1984) conducted an experiment to evaluate the growth response of 1 2 sub-tropical grasses with additions of Cu and Zn. They observed that depending on the species, the Cu and Zn concentration associated with a 50% yield reduction ranged from 1 7 to 27 mg kg-1 and 475 to 1 925 mg kg-1 , respectively. Characteristic symptoms of Cu toxicity include Cu induced chlorosis and root malformation, which are the most common symptoms. The growth depression of sensitive plants was observed at 15 to 20 mg kg- 1 Cu in tissues (Kloke et aI., 1 984) and 1 0 % yield decrease is most likely at 1 0 to 30 mg kg-1 Cu (Macnicol and Beckett, 1 985). 2.5.8 Copper and animal health There are very limited data available to relate soil extractable Cu levels to animal health. Peverill et al. ( 1 988) suggested that EDT A extractable soil Cu levels between 0.5 and 1 .0 mg kg-1 are considered low for animal health, but noted that critical levels could vary with pasture species grown on soil types with varying soil properties. A poor relationship between soil extractable Cu and Cu levels in grazing animals has often been observed (McDonald and Mahoney, 1 982). Chapter 2 Literature review 50 There are several reasons for this poor relationship. The Cu concentrations in pasture plant species vary considerably with soil type (Gartrell, 198 1 ; Sherrell arid Rawnsley, 1982) and with seasonal climatic conditions (Gartrell, 1 98 1 ; Reay and Waugh, 1983; McFarlane et al. , 1 997). Generally, a Cu concentration of 5.0 mg kg-I of dry matter appears adequate for the Cu nutrition of animals (Underwood, 1 977). However, if Cu deficiency is diagnosed in animals, plant analysis is required to identify the cause of the deficiency. The cause of the deficiency could be low Cu concentrations in the plants as a result of low soil Cu, or with interactions in the animal involving Mo or S in the plants. Where there are high concentrations of Mo relative to Cu in the pasture plants, Cu is less available to the animal and Cu deficiency may occur. McFarlane et al., ( 1 997) suggest that if Mo levels in pasture are high (>1 0 mg kg-I dry matter) a direct Cu supplement in the diet is required. Only the relationships between soil extractable Cu and Cu concentration in the plant can be defined by soil analysis. 2.5.9 Soil and plant tests for Cu Common chemical extractions used to predict plant available Cu extract from one or more of three main pools: - • Water soluble Cu. • Exchangeable Cu. • Complexed, chelated or adsorbed Cu. Water and neutral salts have often been used as extractants to simulate the ability of plant roots to absorb Cu from the soil solution. The amount of Cu extracted by these extractants is small and does not give any indication of the capacity of the soil to supply further Cu to the soil solution. Research shows that accurate analysis of low Cu concentrations was difficult and contamination hard to avoid. Therefore extractants, like strong acids, were chosen, which extracted more Cu than normally required by plants. Dilute acid solutions usually remove micro nutrients from the soil solution and from exchange sites on clays and organic matter. Strong acids should be avoided because they generally extract micronutrients from non-labile solid phases (primary minerals), which are unavailable to the growing plants. However the suitability of dilute acid extractants is confined to acidic soils, because they generally are not sufficiently buffered to extract meaningful levels of micronutrients from calcareous soil. An Chapter 2 Literature review 5 1 extensive database relating acid extractable levels o f most micronutrients to crop response for USA soils exists, because of their extensive field and laboratory studies. The most commonly used dilute acids are Mehlich 1 and 0 . 1 M HCl. Of all the extractants used, chelating agents show the most promise. This is due to the fact that Cu extracted by chelating agents is a combination of solution exchangeable, and also specifically adsorbed forms of soil Cu. Thus, chelating agents give a good indication of the labile pool of Cu. 2.5. 9.1 Water soluble Cu The concentration of water soluble Cu in soils is very small. Gupta and Mackay ( 1 966) found that water-soluble Cu ranged from 0.09 to 0.46 mg Cu kg-I in some mineral soils. Miller et al. ( 1 986) found less than 0. 1 mg Cu kg- I soil water soluble Cu in all but one of the soils used in the study. Fiskell and Leonard ( 1 967) grew citrus on a severely Cu deficient soil. Levels of water soluble Cu were highly correlated (r=0.67, pO.O I ). The correlation between wheat and barley plant Cu concentrations and concentrations of Cu in the soil extracted with 1 .0 M NH4N03 was found to be significant, indicating that a weaker extractant gave a better assessment of plant available Cu in soils with variable properties (Arnesen and Singh, 1 998). Ammonium acetate is the other main reagent used to extract exchangeable Cu from soils. Several workers have found ammonium acetate extractable Cu correlated to plant uptake (Chand and Singh, 198 1 ; Selvarajah et al., 1 982; Sedberry et al., 1 988). Ma and Chapter 2 Literature review 52 Dren ( 1 998) observed that the addition of CaC03 increased the soil pH from 7.08 to 7.68 and decreased the exchangeable Cu concentration. 2.5.9.3 Complexed, ch elated and adsorbed Cu Copper that is comp lexed, chelated or adsorbed by iron and aluminium oxides, organic matter and clay minerals can be extracted by different types of reagents. Single chemical extractions have been extensively evaluated over the years, as a method for the estimation of the amount of plant available micronutrients in soils (Cox and Kamprath, 1 972; De Abreu et a!. , 1 996). They have been used with varying degrees of success for diagnosing micronutrient deficiency and toxicity in soils. In neutral and alkaline soils, TEA-DTPA [0.005 M DTPA in 0.01 M CaCh solution buffered by O. IM TEA (HOCH2CH2)3N at pH 7.3] has been used to extract available Zn, Fe, Mn and Cu (Lindsay and Norvell, 1 978). Chelated based extractants are usually well buffered near neutrality (pH=7.3) to avoid dissolution of any carbonate minerals that could release occluded and unavailable micronutrients. The ionic strength of the extracting solution is maintained near 0.01 M to promote flocculation, and to regulate chelate activity. Direct transfer of an extractant that is buffered at a pH appropriate for calcareous soils to acidic soils is inappropriate, and can succeed only if the pH and buffering capacity of the reagents are modified. The Mehlich (Ml ; 0.05 N HCI and 0.025 N H2S04) method was employed to measure soil micronutrients by a number of Soil Testing Laboratories in the United States (Hanlon and DeVore, 1 989). Because Ml was found unsuitable for neutral and alkaline soils, the method was modified as M2 and then as M3 (0.2 M glacial acetic acid, 0.25 M NH4N03, 0.0 1 5 M N&F, 0.0 13 M RN03 and 0.00 1 M EDTA) so that it could be used over a wide range of pH conditions to measure both macro and micronutrients (Mehlich, 1984). The use of extractions in routine soil analysis, such as M l , M3, O. IN HCI, 0.01 M Ca(N03h, 0.0 1 M CaCb, 0. 1 M NaN03, 1 M Nli4N03, TEA-DTPA and ammonium acetate salt solution, could be a fast and simple way to evaluate availability of macro and micronutrients to plants. The TEA-DTPA extraction method, developed by Lindsay and Norvell ( 1 978), seems to be the most appropriate method for the extraction of Chapter 2 Literature review 53 available Cu in soils (Shuman, 1 986; Agrawal, 1 992; Singh et al., 1 994), but other multinutrient extractants such as Morgan, M1 , M3, and ammonium bicarbonate (AB) DTPA and ammonium acetate procedures (Raij , 1 994) have been preferred for use with the ICP-AES technique in routine analysis. Very few studies have been done comparing the extraction of Cu from soil, and the actual absorption of Cu by plants. Reed et al. ( 1993) evaluated the Cu availability for corn using the M3 extracting reagent, and found this procedure to be promising to detect the deficiency and toxicity levels of Cu in soils. On the other hand, Walworth et al. (1 992) observed that neither the DTPA nor the M3 procedures were effective in detecting Cu availability for broccoli and potatoes cultivated in soils from Alaska. Similar results were also observed by Makarim and Cox ( 1 983) for corn, wheat and soybeans. Such information indicates that the best procedure for determining Cu availability in all soils has yet to be determined. Minnich et al. ( 1 987) measured Cu2+ activity on saturated soil extracts, and related it to the accumulation of Cu in young snap beans (Phaseolus Vulgaris L.). They found that the Cu concentration in both shoots and roots increased with measured Cu2+ activity. The efficiency of extractants for the dissolution of soil Cu has been well researched. The choice of extractant is dependent upon several factors: • Pool of eu to be extracted. • Soil pH. • Presence of free calcium carbonate. • Organic matter. Many reagents remove far greater amounts of eu from the soil than the plant is able to. However, of all the extractants tested, the chelating agents such as DTPA and EDTA are the most promising for use in non-calcareous soils. Good correlations have been obtained between EDTA and TEA-DTPA extractable soil eu and plant eu under a variety of conditions. These types of extractants have largely superseded the dilute salt and acid extractants, which do not appear to give reliable estimations of plant available eu. Chapter 2 Literature review 54 2.6 ENVIRONMENTAL FACTORS AFFECTING REACTIONS AND THE AVAILABILITY OF COPPER IN SOILS This section will review the specific effects of environmental factors on the solubility of eu in soils. In addition, it will focus on the effects of temperature, light, and soil moisture on the uptake, translocation, and metabolism of eu. The seasonal pattern of pasture Cu concentration may vary with location, soil type, climate, pasture species and management practices. 2. 6.1 Soil moisture content and redox reaction Ponnamperuma ( 1 972) reviewed the chemistry of a submerged soil . After submersion, the aerobic soil becomes anaerobic and undergoes changes in soil pH, organic matter decomposition and microbial activity. Other important changes also occur, but the aforementioned effects are relevant to Cu availability. The effects on soil eu mobility in anaerobic soils result from the following: � In acid and alkaline soils pH tends to stabilise around pH 7.0. � Iron and manganese oxides are reduced to release Cu. • Decomposition of organic matter is slower than in aerobic soils, but results in a large number of strongly complexing organic compounds. Ponnamperuma ( 1972) in his review suggested that the overall effect of soil chemical reduction is to increase the mobility of eu in the soil. However, it appears that acid soils with higher amounts of organic matter undergoing soil reduction, will have less 'plant­ available ' Cu. Complexing of Cu by soluble organic matter will increase, firstly as a function of soil pH, and secondly due to the decomposition of organic matter. Beckwith et al. ( 1 975) grew rice plants in both flooded and well drained soils. There was very little change in yield between treatments. They found that, although EDTA-extractable eu increased with flooding, the uptake of eu by rice shoots was decreased. This trend persisted from sowing to harvest maturity, so did not appear to be related to the stabilisation of soil reduction changes. Soil solution Cu levels were too low to be accurately measured, so no speculation could be made about the influence of soluble organic complexing. Chapter 2 Literature review 55 Williams and McLaren ( 1 982) incubated dry and moist soils, containing 5 mg Cu kg-] soil, for a total of 43 weeks. The amount of EDTA extractable Cu decreased with time in all soils. Recovery of added Cu by EDTA ranged from 87% to 67% for dry soils and 67% to 45% for moist soils. The decrease in extractability being greatest for the moist soils. This was found to be correlated to the manganese and iron oxide content of the soils. Ageing of amorphous oxides, which are consider to be attacked by EDT A, results in crystallisation of the oxide structure. Hence EDTA extractable Cu decreases with time. Under moist conditions, diffusion of Cu to sites that are not attacked by EDT A is faster. Sims and Patrick ( 1 978) observed that a large proportion of Cu solubilised by soil reduction would not remain in the water soluble fraction, but would be complexed by organic material and become unavailable to plants. Nambiar ( 1 977) concluded that rye grass (Lolium multiflorum Lam.) absorbed significant amounts ofCu from nearly air­ dry soil, when the roots had access to subsoil water. Reddy et al. ( 1 98 1 ) stated that the moisture content of a soil did not influence the concentration of Cu in the soil solution or affect its availability to plants. In both cases, aerobic soils were studied and these are expected to behave differently to anaerobic soils. Williams ( 1 98 1 ) suggested that the concentration of soil solution Cu is independent of the soil moisture content if both solid and solution phase Cu are in true equilibrium. In conclusion, it appears that the influence of soil redox conditions on the availability of Cu to plants is complex. This is mainly because a number of different soil chemical properties change when a soil becomes reduced, and it becomes difficult to separate the effect of a single property. 2.6.2 Seasonal variation and soil temperature It was reported that the variation III season affects Cu concentration III plants. Hemmingway ( 1 962) obtained a higher concentration of Cu in clover and grasses in the autumn, but Reddy et al. ( 198 1 ) obtained higher pasture Cu concentrations in winter both in glass house and field experiments. Whereas Metson et al. ( 1 979) observed no seasonal influences on Cu concentration in pasture species. Several scientists (Hogg and Moore, 1 976; Reddy et al., 1 98 1 ; Merry et al. , 1 986) observed that increasing soil temperature caused an increase in the availability of Cu to plants. Reddy et al. ( 198 1 ) grew subterranean clover in a lateritic podzol and a calcareous sandy soil . They imposed two soil temperature ( 1 2 QC and 22 QC) and moisture levels. They obtained the higher dry matter yield under the higher temperature and moisture regime. The Cu Chapter 2 Literature review 56 concentration in clover was higher at 22 QC compared to 1 2 Qc. This was irrespective of the soil moisture content. The greater availability of Cu to clover plants at 22 QC was related to the higher amount of Cu extracted by calcium chloride at 22 Qc. Brennan et al. ( 1980, 1984) found in their incubation studies that increasing the soil temperature up to, but not more than 35 QC decreased the relative effectiveness of Cu fertilisers on plants. Incubation periods were maintained for up to 1 20 days, and it was suggested that incubation of warm soils promoted diffusion reactions of Cu into the soil colloids. It appears that seasonal variations in the Cu concentration of pasture are related to soil moisture content, although laboratory studies could not substantiate this. Copper availability seems to be at a peak in the autumn and in the winter seasons with transient changes in soil temperature having little effect. When different temperatures are imposed over longer periods of time several effects are thought to occur: • Increased rate of mineralisation of organic matter with increasing temperature. • Adsorption of Cu by soil colloids and diffusion into the soil materials may be influenced by temperature. • Equilibrium between solid and solution phases may be established more rapidly under higher soil temperatures (Beckwith, 1 963). 2. 6.3 Radiation Light does not seem to have any major effects on the frequency of Cu deficiency under field conditions. However, Graves and Sutc1iffe ( 1 974) showed that eu deficiency slowed down the rate of flower initiation and development in chrysanthemum (Chrysanthemum morifolium Ramat.), which required a short photoperiod for flowerbud initiation. The severity of eu deficiency, unlike that of Zn, in subterranean clover (Trifolium subterranean L.) was unaffected by a reduction in light intensity under green house conditions (Millikan, 1 953). The effects of radiation on eu uptake by plants are mediated through its effect on plant growth. The effects, of temperature on crop development and solar radiation on bio­ mass accumulation, combine to impose well-defined limits on potential crop yields under various environmental conditions. The frequency of solar radiation defines the Chapter 2 Literature review 57 maximal limit on crop yield, because intercepted solar radiation provides the energy for photosynthetic fixation of C02. By assuming an upper limit to the efficIency of CO2 fixation (or crop biomass accumulation) per unit of intercepted solar radiation, an estimate can be calculated for the limit on crop yield, based on the amount of solar radiation during the growing season. Spaeth et at. ( 1 987) were able to account for much of the yield variability between seasons, as a consequence of differences in temperature and incident solar radiation. The radiation use efficiency (RUE) and the harvest index were held constant under all conditions in their calculation. Both the pattern of leaf area development (as determined by temperature) and incident solar radiation in these environments influenced the amount of intercepted radiation, which in turn accounted for differences in yield between seasons in nonstressed soybeans grown in Japan. Yields of nonstressed spring wheat in Israel were examined in terms of environmental variation in temperature and solar radiation (Amir and Sinclair, 1 99 1 ). Woledge and Dennis ( 1 982) observed that the photosynthetic rate of leaves was twice as high at 1 5°C than at 5°C for perennial ryegrass (Lolium perenne L.) and white clover (Trifolium pratense L) grown at different temperatures. Ryegrass and clover had similar photosynthetic rates, which responded similarly to temperature. Reay and Waugh ( 1 983) observed Cu concentration fluctuated monthly in seasonal variation trials. They also observed that the Cu concentration, which fluctuated without seasonal trends in ryegrass leatblades, correlated with the organic-N concentration. 2.6.4 Plant and other organisms There is considerable evidence to include the plant as an external factor acting on the soil. The influences the plant exerts on the availability of Cu have been reviewed by Wilkinson ( 1 972) and Marschner ( 1 986). These effects are outlined as below: • Absorption of ions from the soil solution by plant roots results in a change in the concentration gradient in the rhizosphere. • During plant respiration, oxygen is absorbed and carbon dioxide is evolved into the rhizosphere. There is a subsequent decrease in pH resulting in a greater proportion of Cu2+ available for plant uptake. • Roots are able to exude various substances, which include: H+, OH-, HC03 and CO2. These are all capable of changing soil pH. Organic and amino acids are also released Chapter 2 Literature review 58 and may complex solution Cu. Organic exudates are thought to be involved in the release of exchangeable and specifically adsorbed Cu from the solid phase of the soil. • The growth of bacterial and fungal populations are stimulated in the rhizosphere and are believed to accelerate the release of Cu from the solid phase of soils. Nielsen ( 1 976) obtained samples of soil solution by suction from a calcareous peat soil in which barley plants were growing. As expected, the levels of Cu in the soil solution remained constant, when no plants were grown. In the presence of barley plants, Cu in the soil solution increased. Williams ( 198 1 ) suggested that some portion of the organic material solubilised in the presence of plants would be due to root exudates. Linehan et al. ( 1 985) displaced the soil solution by centrifugation, from pots containing barley plants. The concentration of Cu in the rhizosphere was found to be greatest during early plant development and decreased up to harvest. Microbial activity was considered to be involved. The pattern of change of the Cu concentration in the rhizosphere with time was similar to that of a changing soil biomass. It was suggested that enhanced Cu levels in the rhizosphere could also be related to soil pH changes. Stevenson and Fitch ( 1 98 1 ) found that the decomposition of crop residues and organic wastes by microorganisms may lead to the release of significant quantities of Cu. Complex formation may reduce the concentration of Cu2+ to a non-toxic level, when excess Cu2+ is present. 2.6.4.1 Mycorrhizae Mycorrhizae are naturally occurring associations between certain soil fungi and plant roots in which the fungi colonise the root tissue. But also act as extensions of the plant root system, in which hyphae external to the root absorb and transport water and various nutrients back to the root zone. Kothari et al. ( 1 99 1 ) reported that mycorrhizal plants normally contain significantly higher levels of P, and various micronutrients, most notably Zn and Cu. This effect may partly be due to the enhanced vigour of mycorrhizal plants, but mycorrhizae have also been shown to take up and transport metals to the roots where they are absorbed and translocated to the plant shoot. Timmer and Leyden ( 1 980) concluded that the reduction of plant Cu concentration at high P levels resulted from a reduced exploitation of the soil by mycorrhizae. Chapter 2 Literature review 59 Baker and Senft ( 1 995) concluded that the activity of Cu2+ involved in uptake by plant roots is a function of the soil solution activity of Cu2+ as modified by mycorrhizal effects. The uptake of Cu, Zn and P is enhanced by fungi associated with roots, known as vesicular-arbuscular mycorrhizae, whose hyphae penetrate the root at one end with the other end extending several centimetres into the soil . It is not known whether the beneficial effects of mycorrhizae result from an increased activity of ions or to a greater effective root surface area. While the contribution of mycorrhizal fungi to plant element uptake were quantified in detail for several plant species under a controlled environmental conditions (George et al. , 1 995), the quantification of the mycorrhizal contribution to plant growth under field conditions is difficult, because control plants cannot be maintained with mycorrhiza­ free root systems. Indirect evidence, however, indicates benefits due to mycorrhizal infection on low-P soils are large enough to be measurable, albeit to varying magnitudes (George et al., 1 994; Abbott et al., 1 995). Chapter 3 Adsorption and de sorption of copper in pasture soils CHAPTER 3 3.1 INTRODUCTION 3.1.1 Cu adsorption ADSORPTION AND DESORPTION OF COPPER IN PASTURE SOILS 60 The chemical behaviour of cations and anions in the soil is primarily governed by retention and release reactions of solute within the soil matrix. The retention and release reactions in soils include precipitation/dissolution, ion exchange, and adsorption I de sorption reactions (Amacher et al., 1 986). Retention may be due to precipitation and lor adsorption, and depends on factors such as the nature of the ion, the nature of the mineral and organic constituents of the soil, and the composition of the soil solution. For adsorption, cations are held either through electrostatic attraction, thus giving rise to ion exchange with the surrounding ions, or by specific adsorption by surface bonding with organic and mineral substrates. Surface sorption is considered the most important mechanism that regulates the concentrations of Zn, Cu and Cd in the soil solution (Boekhold et al., 1 99 1 ; Guadalix and Pardo, 1 995). Several studies of heavy metal adsorption by individual soil components such as organic matter (humic and fulvic acids), silicate minerals (montmorillonite, kaolinite, illite etc) and sequioxides (iron, aluminium or manganese oxide) have indicated relatively strong bonding and high capacities of various materials for adsorption (Barrow et aI. , 1 98 1 ; Fu et al., 1 99 1 ; Oden et aI. , 1 993). Bivalent transition metal cations exhibit a similar pH dependent sorption behaviour; many authors have reported that the amount of sorbed metal ions increases with the raising of solution pH within a certain range (Elliott et. aI. , 1 986; Guadalix and Pardo, 1 995). Soil is complicated by the heterogenous nature of its components. Thus interaction among different soil components is likely. In soils, eu is adsorbed on the surface of clay minerals, Fe and Mn oxides, and organic matter; it can precipitate with sulphide, carbonate, and hydroxide ions and complex with soluble organic molecules (McBride, 198 1 ; Baker, 1 990). Although organic matter and oxides are important in adsorption reactions, differences exist in their relative importance. The Cu adsorption ability of Chapter 3 Adsorption and desorption of copper in pasture soils 61 soils is affected by pH, organic matter, iron and aluminium oxides, and clay minerals (McLaren et al., 1 983b; Stevenson et aI. , 1 993; McBride, 1 994; Luo and Chistie, 1 996). pH is a major factor controlling the sorption of Cu, and many authors confirm that the sorption of Cu onto soils increases with an increase in soil pH (Cavallaro and McBride, 1 978; Kuo and Baker, 1 980; Harter, 1 983; King, 1 988). Whatever the processes may be, retention in soils greatly determines the mobility and the bioavailability of Cu, and it is therefore essential to improve our knowledge of these processes in soils, which vary in their chemical characteristics. The measurement of adsorption and mobility of Cu in soils with different compositions is required for determining the loading capacity of Cu in a given soil. 3.1.2 Cu desorption To predict the fate and mobility of Cu in soils and to develop effective remediation strategies to overcome Cu deficiency, and toxicity in pasture soils and orchards, which receive frequent applications of Cu fertilisers and pesticides, information on desorption is required. Surface application of high levels of Cu to pasture soils through fertilisers and effluents with high concentrations of Cu may result in Cu toxicity in the grazing animaL Although rare, Cu phytotoxicity has been encountered in soils receiving long­ term additions of sewage sludge, industrial wastes and repeated applications of Cu fertiliser and pesticides (Tisdale et al., 1 993). It is generally accepted that Iow Cu concentrations typically found in the soil solution are regulated by adsorption and de sorption phenomena associated with various soil colloidal materials. Soil acidification enhances Cu desorption from the solid phase, thereby increasing its concentration in the soil solution (Basta and Tabatabai, 1 992). This enhanced de sorption may intensify the bioavailability of eu. Copper uptake by plants and animals is a function of the activity of soil solution Cu2+, which is controlled by the concentration of total soluble Cu, inorganic and organic Iigands and by soil pH. A decrease in pH enhances Cu2+ absorption by plants, because of the increased solution activity of Cu2+ associated with the dissolution of soil minerals, decreased organic bonding and in solid phase adsorption of eu (Baker, 1 990). The adsorption of eu by soils and its relationship with plant availability have been examined by many authors. In contrast, very little Chapter 3 Adsorption and desorption of copper in pasture soils 62 information is available on de sorption of eu. Such information is required in order to improve our ability to predict the release of both native eu from soils, and the release of eu added to soils as fertiliser or as a pollutant. In terms of plant and ultimately animal nutrition, it is the supply of eu from the solid phase into solution, which determines the availability of eu for plant uptake. Objectives : The objectives of the experiments reported in this chapter are: • To determine time-dependent eu adsorption by five soils. • To determine the adsorption capacity of soils with widely differing properties. • To examine the effect of pH on eu adsorption. • To quantify the contribution of different soil components to eu adsorption. • To examine the effect of pH and incubation period lengths on eu de sorption in the various soils. 3.2 MATERIALS AND METHODS 3.2.1 Soils used Five different soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu) were used for the adsorption and desorption study. The soils used for different adsorption and desorption experiments, and the experimental conditions are given in Table 3 . 1 . Chapter 3 Adsorption and desorption of copper in pasture soils 63 Table 3.1 The soils and experimental conditions used in the various adsorption and desorption experiments. SI Experiments Soils Experimental condition No I Time-dependent experiment Manawatu, Tokomaru, Sorption at 1 0 mg Cu L-1 shaken for on Cu sorption Ramiha, Ngamoka and Mangamahu 2 pH dependent Cu sorption Manawatu, Tokomaru, isotherm for soil. Ramiha and Ngamoka 3 Cu sorption isotherm for soil Manawatu, Tokomaru, and soil components Ramiha, Ngamoka and Mangamahu 4 Extractable soil Cu Manawatu and Ngamoka 5 Desorption of native Cu Manawatu and Ngamoka 6 Desorption of added Cu Manawatu and Ngamoka 7 Effects of contact times on Manawatu and Ngamoka desorption of added Cu and native Cu. 8 pH dependents desorption of Manawatu, Tokomaru, soil. Ramiha and Ngamoka 3.2.2 Soil physical and chemical analysis 30- 1 20 minutes Sorption at 5-50 mg Cu L-1 shaken for 2 hrs. Sorption at 5-50 mg Cu L- shaken for 2 hrs. Extraction was done with M l , M3, DTPA, O. lM HCI and 0.04M EDTA. Desorption of native Cu equilibrated for 2 hrs. Desorption in 50 mg Cu kg-I soil equilibrated for 2 and 24 hrs. Soils incubated with Ca(N03)2 and 50 mg Cu kg-I for different periods. Desorption for both were equilibrated for 2 and 24 hrs contact periods. Desorption at 30 and 50 mg Cu L-1 equilibrated for 2 and 24 hrs contact periods at pH 5 and 8 . Soil particle size (sand, silt and clay) analysis was completed after oxidising the organic matter in the soil samples with aliquots of 30% hydrogen peroxide. Soil pH ( 1 :2.5 H20), cation exchange capacity (by IM �OAc (PH 7) extraction), Olsen P (by 0.5 M NaHC03 extraction), sulphate (by 0.04 M Ca(H2P04h extraction), organic carbon (by IN K2Cr207 extraction) were measured according to Black ( 1965). Acid oxalate­ extractable Fe and AI, and exchangeable K, Ca, Mg and Na were detennined for the initial soil samples by the standard methods (Blackmore et a/., 1987). 3.2.3 Time-dependent experiment on Cu sorption Duplicate soil samples of 1 .0g were weighed into 50 ml centrifuge tubes. Twenty ml of 1 0 mg Cu L-1 [as Cu(N03)2] solution was added and the tubes were shaken in an end­ over-end shaker for 30, 60, 90, 120 and 1 80 minutes at 20°C. The samples were then centrifuged at 77 1 9 g and filtered through Whatman No. 44. The concentration ofCu in Chapter 3 Adsorption and desorption of copper in pasture soils 64 the filtrate was measured by flame atomic absorption spectrophotometry (F-AAS). The Cu sorbed was calculated as the difference between the amount added and that remaining in the equilibrium solution. 3.2.4 pH dependent copper sorption isotherm for soils Copper sorption isotherms at pH 5.0, 6.0, 7.0 and 8 .0 were obtained for each soil as follows: 1 .0 g of each soil was suspended in 20 ml of 0.01 M Ca(N03)2 solution. The above pH values were achieved by adding O.OlM HCI or sodium hydroxide (NaOH) and allowed to equilibrate for 48 hrs. Various concentrations (0, 5 , 1 0, 20, 30, and 50 mg Cu L-1 ) of Cu were added as Cu(N03h. The soil suspension was equilibrated for 24 hrs before centrifuging and filtering. The Cu in the filtrate was determined by F-AAS. The Cu sorbed was calculated as the difference between the amount added and that remaining in the equilibrium solution. 3.2.5 Copper sorption isotherm for soil components Copper adsorption isotherms were obtained for the original soil samples together with subsamples of the same soils treated to remove the organic matter, iron and aluminium oxides. Organic matter was removed by using 0.7M sodium hypochlorite at pH 8.5 and iron and aluminium oxides were removed by using a mixture of 0.2 M ammonium oxalate, 0.2 M oxalic acid and 0. 1 M ascorbic acid. The measurement of adsorption after the removal of various soil components provides useful quantitative information on the relative importance of each different soil component on Cu sorption. Samples of the original soils and their subsamples, after the removal of the different components (organic matter removed and organic matter plus oxides removed), were equilibrated with a 1 0 ml solution containing known amounts of Cu as Cu(N03h; 0.01 M Ca(N03h was used as the background electrolyte and a range of Cu concentrations (0, 5, 10, 20, 30, 40 and 50 mg Cu L-1) were used. Equilibration was carried out in centrifuge tubes on an end-over-end shaker for 24 hrs at 20°C. The time dependent experiment indicated that more than 90% of Cu adsorption occurred within a 2 hrs period. The samples were then centrifuged at 77 1 9 g and filtered through Whatman No. 44 and the Cu was determined in the filtrate. Cu in the soil filtrate was analysed using F-AAS. Cu adsorbed was calculated from the initial and the final solution Cu concentrations. Chapter 3 Adsorption and desorption of copper in pasture soils 3.2.6 Fractionation of soil Cu 65 Fractionation of soil Cu was carried out according to the sequential extraction method described by McLaren and Ritchie ( 1993). The soil Cu is fractionated into four forms: (i) exchangeable Cu; (ii) organically bound Cu; (iii) Cu associated with iron oxide; and (iv) residual Cu (Cu remaining after the removal of the first thee fractions) (Table 3 .2). Hydrochloric acid was used instead of hydrofluoric acid for the measurement of the residual fraction. Step A (Exchangeable Cu): A known weight (5 .0 g) of the finely ground soil samples were placed into the 50 ml polyethylene centrifuge tubes with 35 ml O .OIM Ca(N03)2. The suspension was shaken in an end-over-end shaker for 24 hrs at 20°C and then centrifuged for five minutes at 77 19 g in the Sorvall RC 5C automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42. Step B (Organic matter bound Cu): A known weight ( 10.0 g) of the finely ground soil samples were placed into the 250 ml polyethylene centrifuge bottles with 20 ml 0.7M sodium hypochlorite (NaOCI); NaOCl was adjusted to pH=8.5 immediately before using. The suspension was then boiled in a water bath for 30 minutes and stirred occasionally. After the organic matter extraction and water wash, the soil was air dried, crushed and passed through 400,um sieve. The water washed NaOCl solution was condensed in a water bath and filtered through Whatman No. 42. Step C (Cu associated with iron oxide): A known weight ( 1 .0 g) of the finely ground soil samples from step B was placed into the 250 ml polyethylene centrifuge bottles and mixed with 25 ml of a mixture containing 0.2M ammonium oxalate [�)2C204.H20], 0.2M oxalic acid [H2C204], and O . lM ascorbic acid [C6Hs06] . The suspension was then boiled in a water bath for 30 minutes and stirred occasionally. After the iron oxide Cu extraction and water wash, the soil was air dried and crushed. The water washed solution was condensed in a water bath and filtered through Whatrnan No. 42. Step D (Residual Cu): The soil remaining after the removal of iron oxide Cu (step C) was used to determine residual Cu by wet digestion with a mixture ofHN03, HCI04 and HCI at the ratio (by voluine) of 5 :5 :7 . The Cu in the filtrate was measured by F-AAS. Chapter 3 Adsorption and desorption of copper in pasture soils 66 Table 3.2 Sequential fractionation scheme for Cu in soil. Step Fraction Extractant Soil Solution Conditions g rnl l . Exchangeable Cu O.O IM calciwn nitrate 5 35 Shaken 24 hrs 2 . Organic bound Cu 0.7M sodiwn hypochlorite l OA 20 Boiling in a water (pH=8.5) bath 30 min, stirred occasionally 3 . Copper associated with 0.2 M ammonium oxalate lIS 25 Boiling in a water iron oxides 0.2 M oxalic acid bath 30 min, stirred 0. 1 M ascorbic acid occasionally 4 . Residual Cu Concentrated nitric, perchloric le 25 Digested at 1600 C and hydrochloric acid A, D · l., Separate sample from step 1 . 1 g from step 2 after drying and gnndmg. ReSIdue from step 3 . 3.2. 7 Desorption of native and added Cu Desorption of Cu from the soil samples was measured repeatedly using 1 .0 g duplicate soil samples, which were pre-equilibrated for 2 hrs and 24 hrs with a 20 ml solution containing known amounts of Cu [as CU(N03)2 in 0.01 M Ca(N03)2] . Equilibration for the desorption step was carried out in centrifuge tubes on an end-over-end shaker for 2 hrs and 24 hrs at 20°C. After each 2 hrs and 24 hrs equilibration period, the samples were centrifuged, and the supematant decanted and filtrated through Whatman No. 42 filter paper. The soil was resuspended with fresh 0.01 M Ca(N03)2, and the procedure repeated as necessary. The pH of the de sorption solution was measured at the end of each 2 hrs and 24 hrs desorption period. Cu in the soil extractant was analysed using F­ AAS. Cumulative Cu de sorption was calculated from the Cu concentrations in each successive equilibration solution. 3.2.8 Desorption study for incubated soil Two soils (Manawatu and Ngamoka), which represent similar pH values but varying organic matter content, were chosen for the desorption study. The soil was mixed with eu fertiliser to give 50 mg eu kg-1 soil and incubated for different periods (control, 0, 8 and 28 days). The desorption of eu was measured following the method described in 3 .2.7. Chapter 3 Adsorption and de sorption of copper in pasture soils 3.2. 9 Desorption study at different pH levels 67 The soil that received 30 and 50 mg Cu L-1 during the sorption experiment at pH 5.0 and 8 .0 were subsequently used in the desorption study. In the earlier study in which desorption at low levels of Cu (5- 1 0 mg Cu L-1 ) was examined, measurement of low levels of Cu in the desorption solution becomes difficult, especially in soils with a high organic matter content. So in this experiment, high Cu addition rates were selected to minimise the difficulties and uncertainties in the measurement of low Cu levels in the solution by F-AAS. The centrifuged soils from the sorption experiment (Section 3 .2 .4) were resuspended in 20 ml of 0.01 M Ca(N03)2 at pH 5 .0 or 8.0, and were equilibrated on an end-over-end shaker for two hours. Subsequently, the soil suspension was centrifuged, filtered and the Cu in the supernatant was determined by F-AAS. The desorption procedure was repeated 1 0 times for each samples. 3.2.10 Extractable soil Cu The amount of Cu extracted by various soil test reagents was measured; so a comparison could be made with the amounts removed during the desorption experiment. 3.2.10.1 0.04M EDTA extracting solution 0.5 g duplicate soil samples (Manawatu and Ngamoka) were equilibrated with a 35 ml solution containing 0.04 M EDTA. Equilibration was carried out in centrifuge tubes on an end-over-end shaker for 2 hrs and 24 hrs at 200 C; after each equilibration period, the samples were centrifuged, and the supernatant solutions decanted and filtered through Whatman No. 42 filter paper. Copper in the extractant was measured by F-AAS. 3.2. 10.2 Mehlich-1 extracting solution The Mehlich- l solution was prepared to contain 0.05 N HCI and 0.025 N H2S04 (Mehlich, 1 953). For Mehlich- l extraction, 20 ml of the extractant was added to 5 g of soil in a 50 ml centrifuge tube, shaken for ten minutes and filtered through a Whatman No. 40 filter paper. Chapter 3 Adsorption and desorption of copper in pasture soils 68 3.2.10.3 Mehlich-3 extracting solution The Mehlich-3 solution was prepared to contain 0.2 M glacial acetic acid, 0.25 M NH4N03, 0.0 1 5 M �F, 0.01 3 M HN03 and 0.001 M EDTA (Mehlich, 1 984). For Mehlich-3 extractions, 25 ml of the extractant was added to 2.5 g of soil in a 50 ml centrifuge tube, shaken for ten minutes and filtered through a Whatman No. 40 filter paper. 3.2.10.4 0.1M HCI extractant A known weight (8.0 g) of the finely ground soil was placed into 50 ml polyethylene centrifuge tube with 20 ml O. l M HCI (Haynes and Swift, 1 985). The suspension was then shaken in an end-over-end shaker for 2 hrs at 20° C. Then centrifuged for three minutes at 301 5 g in the Sorvall RC 5C automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42. 3.2.10.5 DTPA extracting solution The DTPA extracting solution was prepared to contain 0.005 M DTPA, O.O lM CaCh, and O. l M TEA and adjusted to pH 7.3 (Lindsay and Norvell, 1 978). Ten grams of air dried soil was placed in a 125 ml conical flask, and 20 ml of the DTP A extracting solution was added. Each flask was covered with stretchable Parafilm and secured upright on a horizontal shaker with a stroke of 8.0 cm with a speed of 1 20 cycles/min. After two hours of shaking, the suspensions were filtered by gravity through Whatman No. 42 filter paper. The filtrates were analysed for Cu using F-AAS with the appropriate standards. 3.3 RESULTS AND DISCUSSION 3.3.1 General description of soils Manawatu silt loam: This soil sample was collected from the Massey University Fruit Crop unit. The Manawatu soil is a dark brown fine sandy loam, alluvium, slightly plastic, and weathered fluvial recent soil, quartzo-feldspathic, mixed greywacke and argillite. These soils are characterised by dark greyish brown friable fine sandy loam or Chapter 3 Adsorption and desorption of copper in pasture soils 69 silt loam with moderately developed nut structured topsoils overlying olive brown firm fine sandy loam with weakly developed nut structured subsoils. Tokomaru silt loam: This soil sample was collected from the Massey University Dairy Farm unit 4. It is formed on fairly thick deposits of loess of fine sandy loam texture. It is classified as a Central Yellow Grey Earth. It is a dark greyish brown silt loam with weakly to moderately developed nut structure and some fine brown mottles at topsoils. Tokomaru silt loam is a poorly drained soil, which experiences wet conditions in winter, and relatively dry conditions in summer. Ramiha silt loam: This soil sample was collected from the Massey University Farm (Tuapaka). It is a strongly leached Yellow Brown Earth formed from loess and solifluction material and slope deposits. It is a dark greyish brown friable silt loam with strongly developed nut structured topsoils. Angular greywacke stones may occur through the profile, and slightly weathered greywacke occurs at variable depths ranging from 25 cm to over 90 cm but on average at about 60 cm. Ngamoka silt loam (Ballantrae High Fertility Soil) : The soil sample was collected from the Ballantrae hill area and related steepland. It is classified as Central Yellow Brown Earth or steep land intergrades to Yellow Grey Earth formed from a mixture of parent materials, including sandy siltstones, silty sandstones and silty mudstones. These soils are characterised by deep well structured silt loam topsoils overlying yellowish brown friable to slightly firm subsoils. Ngamoka soils are strongly leached and the topsoil is strongly to moderately acid, and the temperature regime is mesic or frigic. Ngamoka soil is highly weathered, rich in illite, vermiculite and montmorillonite, with dark-coloured topsoils and low « 60%) base saturation. Mangamahu steepland soil (Ballantrae Low Fertility soil) : This soil was obtained from Ballantrae hill country area and is classified as a steep land soil associated with Central Yellow Brown Earth and intergrades to Yellow Grey Earth. Dark brown fine sandy loam topsoil with moderately developed fine nutty structure. Parent material is silty sandstone. Subsoil contains some pale yellowish brown mottles. Mangamahu soils occur on the steep faces. Profiles are in general shallow with a fine sandy loam. 3.3.2 Soil properties The highest and the lowest pH values were recorded for the Manawatu soil (PH 6.0) and the Mangamahu (PH 4.8) soil, respectively (Table 3.3). The Tokomaru and the Chapter 3 Adsorption and desorption of copper in pasture soils 70 Ngamoka soils have the same pH (5.6) value. The data presented in Table 3.3 indicates that the Manawatu soil contained the highest amount of Olsen P (5 1 .9 mg kg-I ) and the lowest amount ( 1 0.5 mg kg-I ) was in the Mangamahu soil. The Ngamoka soil contained the highest amount (5 .85%) of organic carbon and the lowest amount (2.91 %) was in the Manawatu soil. The CEC of the Ngamoka soil was twice as high (26 cmol kg-I ) as that of the Mangamahu soil ( 1 3 cmol kg-I ) . The Manawatu and the Mangamahu soils contained higher amounts of exchangeable Cu than the other soils. Clay content was significant in all soils, ranging from 1 78 g kg-! in the Manawatu and 255 g kg-! in Ngamoka soil. The Ramiha soil contained the highest amount of both Al and Fe. The Manawatu and the Tokomaru soils contained the lowest amounts of Al and Fe. Table 3.3 Initial soil properties Parameters Manawatu Tokornaru Ramiha Ngamoka Mangamahu Soil classification Typic Typic Andic Umbric Typic Udifluvent Fragiaqualf Haplumbrept Dystrochept Distrochepts Particle size (g kg-I) Sand 203 1 97 129 1 97 207 Silt 606 575 597 548 497 Clay 178 2 10 256 256 262 Organic carbon% 2.9 3.4 5.6 5.9 4.9 pH 6.0 5.6 5 .3 5 .6 4.8 Olsen P (mg kg-I) 5 1 .9 1 6.8 1 9.5 24.3 1 0.5 S04 (mg kg-I) 5.0 6.0 1 4 . 8 1 3 . 3 6.8 CEC (cmo1 kg-I ) 1 7 .0 1 3 .0 23.0 26.0 20.0 K (cmol kg- I) 0.92 0.24 0.84 1 .56 0.3 1 Ca (cmol kg·') 9.5 4.4 9. 1 7.4 3 .5 Mg (cmol kg· ') 1 .4 1 1 . 1 8 1 .67 1 .64 1 .4 Na (cmol kg-I) 0.09 0. 1 6 0. 1 7 0.09 0.20 Acid oxalate Al (%) 0.09 0 . 1 2 1 .9 0.42 0.23 Fe (%) 0.3 1 0.28 1 .2 0.38 0.28 Excbangeable Cu (mg kg·' ) 0.54 0.3 1 0.5 1 0.33 0.47 Total Cu (mg kg· ') 33.39 14.86 26.75 23.38 1 7.69 3.3.3 Adsorption 3.3.3.1 Copper sorption as a function of time Copper sorption by the soils was rapid in the beginning, this fast rate of sorption generally took place within a few minutes. Later on the rate of sorption slowed down, this might be due to the diffusion of Cu into the soil aggregates. In all soils, sorption of Cu increased with time (Figure 3 . 1 ). The effect of time on sorption varied among the Chapter 3 Adsorption and desorption of copper in pasture soils 7 1 soils. The sorption percentage ranged from 86% in the Ngamoka soil to 95% in the Ramiha soil within 30 minutes of equilibration at 200 mg eu L-1 • In the Ngamoka soil the sorption increased from 86% to 92% within 3 hrs (Figure 3 . 1) . Initial eu ( 10 mg eu L-1 ) concentration (and loading rates) in this study was affected by sorption. Hue et al. ( 1 997) reported that at a lower loading rate of 30 mg eu L-1 , between 98% and 99.9% of the added eu was sorbed within one hour. At a lower rate more than 99% of the added eu was sorbed within 2 hrs. The time required for the maximum sorption was relatively consistent in all soils. The results indicated that a quasi stationary state was achieved within half an hour of shaking in all soils. However, the Mitscherlich function indicated that more than 90% of sorption occurred within 2 hours, and hence in order to give sufficient contact time to ensure an equilibrium system, a 2 hours shaking time was adopted for all subsequent adsorption studies. 1 00 80 - '""" I Cl � � u 60 Cl .s "0 Q) + .0 Manawatu; Y=96.3284(1 -0.8738X) L-e (J) • "0 40 Tokomaru; Y=92.3229( 1 -0.8934X) co - c e Ramiha; Y=96.91 78(1 -0.87 15X) � e E � Ngamoka; Y=90.3072( 1 -0.8989X) <{ 20 T Mangamahu; Y=92. 7929( 1 -0.8938X) o o 20 40 60 80 1 00 1 20 140 160 1 80 Time (minutes) Figure 3.1 Time dependent Cu adsorption isotherms. The data were fitted to Mitscherlich growth function, Y= A(1-Bx), where Y= amount sorbed, X= time, A and B are constants. Chapter 3 Adsorption and desorption of copper in pasture soils 72 3.3.3.2 Adsorption isotherms for the original soils Copper adsorption isotherms for the original soils are shown in Figure 3 .2. The solution Cu concentrations used in determining the isotherms were much higher than would exist in the soil solution under normal field conditions. These high concentrations were used mainly because the measurement of Cu at low concentration was difficult, especially in pasture soils with high organic matter. In some recently developed orchard soils the eu concentration in the soil solution are found to be very high. The data were fitted to the Freundlich equation: Y =KCN (Eq. 3 . 1 ) where, Y i s the amount of eu sorbed (mg Cu kg-I soil), C i s the concentration of eu (mg L-1 ), and K and N are constants. K is termed the Freundlich unit-capacity coefficient, and N is a joint measure of both the relative magnitude and diversity of energies associated with a particular sorption process. Both K and N are used to characterise eu sorption by soils. - � I 0) ..:.:: 0) E - " Q) ..a ... 0 Vl -0 co ... Q) Cl. Cl. 0 () 1 000 800 600 400 200 o + Manawatu. y = 1 30.05C 0.45 . R2 = 0.97 • Tokomaru. y = 7 1 .89C 0.38 . R2 = 0.97 • Ramiha. y = 138.26C 0.41 . R2 = 0.99 • Ngamoka. y = 81 .1 1 C 0.47 . R2 = 1 .0 A Mangamahu. y = 70.72C 0.40 . R2 = 0.98 50 1 00 1 50 200 Equil ibrium solution Cu concentration (mg L -1 ) Figure 3.2 Copper adsorption isotherms for the different soils. 250 Chapter 3 Adsorption and desorption of copper in pasture soils 73 3.3.3.3 Copper sorption affected by soil properties The sorption of Cu followed: Ramiha > Manawatu > Ngamoka > Tokomaru soil > Mangamahu. The Ngamoka and Ramiha soils contained higher amounts of organic matter and the Manawatu soil contained the highest percentage of silt. The difference in sorption between the soils is attributed to the difference in the chemical characteristics of the original soils. Higher pH values, organic matter, silt and clay content of the Ramiha and Manawatu soils contributed to the higher sorption capacity of these soils. The Manawatu soil had the highest pH value of all the soils and sorbed the higher amount of Cu. Elzinga et al. ( 1 999) reviewed the batch sorption data for Cu from the literature with a wide variety of soils, experimental conditions, and metal concentrations ranging over five orders of magnitude. They used multiple linear regressions and reported that the inorganic and organic complex formation increases with organic matter, soil pH, and CEC. In this study it was observed that Cu sorption as measured by the sorption constant K, correlated with soil properties, such as silt content (r=0.60), organic carbon (r=0.25) and soil pH (r=0.53). Soil textures, particularly the clay and silt fractions, have often been found to be positively correlated with the Cu sorption capacities of soils (Dhillon et al. , 1 98 1 ). A correlation coefficient of >0.73 was observed between adsorption of Cu and soil pH for the pH dependent Cu sorption study. Welp and Briimmer ( 1 999) conducted a correlation analysis and observed that pH was the main factor controlling the partitioning of the metals between the solid and the liquid phase for 8 of 1 0 metals. They also reported that sorption capacity decreases with decreasing soil pH. The Tokomaru soil has a lower K value, indicating less affinity for Cu sorption than the other soils. This may be due to the very low CEC value of this soil. In this study, insignificant correlation between adsorption of Cu and CEC of the soil was observed. The influence of CEC on Cu sorption has also been established by others (Kuo and Baker, 1 980; Zachara et al., 1 992; Elzinga et a!. , 1 999; Wu et aI., 1 999). The very low sorption capacity shown by the Tokomaru soil may be attributed to a low net surface negative charge density. Copper sorption increased as the organic carbon content of the soils increased, except for the Manawatu soil, which had the highest pH. Organic carbon has been reported to increase Cu sorption, often so strongly that Cu deficiency is common in organic soils (Harter, 199 1 ; Stevenson, 1 99 1 ). This retention of Cu by Chapter 3 Adsorption and desorption of copper in pasture soils 74 organic matter appears to be the predominant mechanism involved, confirming the results described by Ritchie and Jarvis ( 1 986). The effect of pH and organic matter on Cu sorption can be explained by the changes in charge characteristics on soil surfaces (Elliott et aI., 1 986). 3.3.3.4 Soil components and Cu sorption To investigate the sorption of Cu by various soil components, the extraction scheme described earlier (Section 3 .2.6) was used to remove the various soil components. Copper sorption was measured for the whole soil (Step 1 ). The whole soil was extracted with NaOCI to remove organic matter and the Cu sorption for these sub samples was measured (Step 2). The remaining soil was extracted to remove the oxide components, and the Cu sorption for these subsamples, after the removal of organic matter and oxides was measured (Step 3). The data for Cu sorption isotherms for the whole soil and the sub samples after the removal of organic matter and iron and aluminium oxides were fitted to the Freundlich equation. From this equations the sorption at different equilibrium concentrations were estimated for the soil samples. The sorption values for the organic matter component are estimated by subtracting the Cu sorption values of step 2 from those of step 1 . Similarly the sorption values for the oxide component are estimated by subtracting the Cu sorption values of step 3 from those of step 2. The sorption values for step 3 give the sorption for the residue component. Copper sorption isotherms for the soil components of the different soils are presented in Figures 3 .3-3 .7 . The K value calculated from the Freundlich equation for the different components are presented in Table 3 .4, and show that the organic matter alone is responsible for most of the retention of Cu in all the soils. Oxide components have a significant contribution for retention of Cu only in the Ramiha, Ngamoka and Mangamahu soils. Copper is adsorbed by various soil constituents, including organic matter and iron oxide (Sims, 1986; Lindsay, 1 99 1 ). Analysis of the relative importance of mineral and organic components of multiphase soil systems suggests that organic matter has an intrinsically stronger affinity for Co, Cu and Cd than the common silicate or oxide minerals (McLaren et al., 1 983b; Zachara et aI., 1992). Chapter 3 Adsorption and desorption of copper in pasture soils 75 Table 3.4 Freundlich equation describing the adsorption of Cu in different soil components Name of soils Manawatu Tokomaru Ramiha Ngamoka Mangamahu 1 000 800 - ..-, C> � C> ,S 600 "0 Q) .0 L-a en "0 co 400 ID a. a. a U 200 Freundlich equation for Cu adsorption Whole soil Organic y= 1 30.05 CO.4�; R2 =0.97 y= 79.35 CO.4�; R2 =0.97 y= 7 1 .89 CUll; RL =0.97 y= 23.5 1 CV.4/; RL =0.9 1 y= 1 3 8.26 CO.41; R2 =0.99 y= 120.32 CU.3U; R2 =0.95 y= 8 1 . 1 1 CU·4.; Rl =0.99 y= 68.07 CV·l'; Ri =0.94 y= 70.72 CU•4U; Ri =0.98 y= 22.87 CV·4'; Ri =0.96 + Soil • Organic matter • Oxides lA Residual • • o 1 00 200 300 Equilibrium solution Cu concentration (mg L-1 ) Oxide y= 1 .23 Co.n; R2 =0.99 y= 0.43 CV.Yl; Rl =0.99 y= 5 .58 CO.7�; R2 =0.99 y= 5 .84 CU.ll; RL =0.99 y= 5 .63 CV·OI ; Ri =0.95 400 Figure 3.3 Copper adsorption isotherm for different soil components of the Manawatu soil. Chapter 3 1 000 800 � ...... , Cl .>0:: Cl .s 600 "0 Q) .D CS peat soil > organic soil > sandy loam soil. In the alum shale soil almost all of the added Cu was retained and this was the case for the peat soil and the organic soil, but only 49% of the added Cu was adsorbed in the sandy loam soil. Copper retention by the Ramiha, Ngamoka and Mangamahu soils is also dominated by both the organic matter and oxide components. These three soils contain relatively higher amounts of Al and Fe, which may contribute to sorption by the oxide component, in comparison with the Manawatu and Tokomaru soils. The organic fractions seem to be a source of sites for specific Cu sorption (McLaren and Crawford, 1 973a), perhaps because the Cu ion is unique in its ability to form inner-sphere complexes at a wide range of pH levels (McBride, 1 98 1 ). Conversely, organic complex formation could lead to increased mobility in the soil, since Cu is known to form stable soluble complexes with fulvic acid (Kabata-Pendias and Pendias, 1 992). Sorption onto organic matter, clay minerals and metal oxides in soils is the main mechanism of heavy metal removal from the soil solution (McBride, 1 989; Scheidegger and Sparks, 1 996). Reports are readily available on Cu retention by hydrous iron oxide (Okazaki et al., 1 986), hydrous manganese oxides (Kabata-Pendias, 1 980) and hydrous aluminium oxides (Shuman, 1 977; Barrow, 1 986). Retention of Cu ions by oxide surfaces is inversely dependent on the degree of crystallinity (Barrow et. aI., 1 98 1 ; Okazaki et al., 1 986). Since the oxides have variable charge, the extent to which retention occurs is dependent on the pH of the solution (Barrow, 1 987; Bolan et al., 1 999). Cavallaro and McBride ( 1 984) found that treatment of the clays for the removal of organics tended to either enhance or have little effect on sorption and fixation of Zn and Cu. They suggested that oxide components of soil clay are more significant than the organic component in metal sorption and fixation. Wu et al. (1999) suggested that Cu sorption may involve the formation of Cu-O-AI bonds for sites on the lateral edges at 2 : 1 phyllosilicates. This chemisorption process is Chapter 3 Adsorption and de sorption of copper in pasture soils 79 likely to be controlled by the nature and quantity of surface hydroxyl groups. However, the results of their study indicate that chemisorption on variable charge sites cannot account for all of the observed immobilisation of Cu on the inorganic soil clay component. To date, however httle is known about the relative contribution of the soil clay component to Cu sorption. Table 3.5 Percent contribution of the organic matter and oxide components to Cu adsorption at two initial concentration levels. Soils At 1 0 mg L-1 initial solution At 50 mg L-1 initial solution concentration concentration Organic Oxides Organic Oxides Manawatu 88 6 86 8 Tokomaru 83 12 74 22 Ramiha 7 1 24 55 40 Ngamoka 55 30 48 38 Mangamahu 46 42 44 48 3.3.3. 5 pH dependent adsorption isotherms for different soils Copper sorption isotherms measured at different pH levels for the soils are presented in Figure 3 .8 - 1 1 . In all soils, Cu sorption increased with increasing pH. Freundlich constant (K) values increased with increasing pH in all soils (Table 3 .6). Except for the Ramiha soil, large variations in K values were obtained within the pH range. There is no doubt that changes in soil pH by several units will also change the sorption capacity (Figures 3 .8- 1 1 ) of the soil for trace metals as has been shown in many studies for minerals and soils (Boekhold et aI., 1 99 1 ; Duker et aI. , 1 995; Yuan and Lavkulich, 1 997). The greatest increase in sorption occurred at pH 8.0 in all soils, perhaps because for most components with variable charge, such as organic molecules and oxide minerals, charge reversal from positive to neutral to negative occurs at this pH; and the retention of Cu, mostly as Cu2+, is much stronger by negatively charged surfaces than positively charged ones (Hue et al., 1 997). A practical implication of this effect is that Cu concentrations in the soil solution, can be regulated by changing soil pH; raising soil pH to 7 .0 or slightly above increases Cu sorption and thus reduces its potential toxicity to crops. In higher pH regions, Cu2+ may precipitate as Cu(OHh. Harter ( 1 983) reported that the amount of Cu(OHh at pH 6 accounted for only 2% of Cu2+ but at pH 8 for 92% Chapter 3 Adsorption and desorption of copper in pasture soils 80 of Cu2+. In this study, it was difficult to distinguish between Cu2+ adsorbed and Cu2+ precipitated after soil suspensions reached the equilibrium state. The amount of Cu sorbed tends to be low if the indigenous Cu is high and organic C is low. The pH dependent characteristics of metal sorption by soils and soil materials has been reported by Barrow ( 1 986), Naidu et al. ( 1 994), and Pardo ( 1 997). Table 3.6 Freundlich equation describing the adsorption of Cu at various pH levels in different soils. Soil pH 5.0 6.0 7.0 8.0 ---..-I Cl .::s:. :::J () 1000 800 Cl 600 ,S "C Q) .0 L- e en "C ro C :::J e E « 400 200 Manawatu y= 36.00 C�.Q) R2 =0.99 y= 80.04 Cv.)) R2 =0.98 y= 94.64 CV•/J R2 =0.97 y= 1 1 3 . 1 3 Cv .•• R2 =0.99 Freundlich equation for Cu adsorption Tokomaru Rarniha y= 5 .29 C�· .. • y= 69. 1 7 C'"'' R2 =0.97 R2 =0.94 y= 8.94 Cv ... J y= 8 1 .50 Cv.,o R2 =0.95 R2 =0.99 y= 66.49 Cv., y= 8 1 .77 Cv.o� R2 =0.99 R2 =0.96 y= 95.29 Cu.JO y= 89.58 Cv.o, R2 =0.84 R2 =O.87 + + pH 5, Y = 36.0CO.65, R2=0.99 • pH 6, Y = 80.04Co.55, R2=0.98 • pH 7, Y = 94.64Co.73, R2=O.97 .. pH 8, y = 1 13 .13Co.84, R2=0.89 Ngamoka y= 10.27 Cv. to R2 =0.93 y= 49.3 1 Cu.JO R2 =0.95 y= 59.78 Cv.JO R2 =0.92 y= 127 .84 C'''' R2 =0.87 o 13 25 38 50 63 75 88 100 1 13 1 25 Equilibrium concentration (mg L-1 ) Figure 3.8 Effect of soil pH level on Cu sorption of the Manawatu soil. Chapter 3 - ..-, Cl � ::J <..> 1000 800 Cl 600 5 "0 Q) .0 .... a Cl) "0 ro c: ::J a E <{ 400 200 o o Adsorption and desorption of copper in pasture soils • + + pH 5, Y = 5.29Co.94 , R2 = 0.97 pH 6, Y = 8.941 CO. 93 , R2 = 0.95 pH 7, Y = 66.485Co.57 , R2 = 0.84 50 100 150 Equilibrium concentration (mg L -1 ) 200 Figure 3.9 Effect of soil pH level on Cu sorption of the Tokomaru soil. ......... ..­ I Cl .::s:. ::J o 1 000 800 Cl 600 5 "0 Q) .0 L- a Cl) -g 400 c: ::J a E <{ 200 o o • + + pH 5, Y = 69.17Co.so , R2 = 0.94 • pH 6, Y = 81 .50Co.S6 , R2 = 0.95 _ pH 7, Y = 81 .77Co.62 , R2 = 0.96 � pH 8, Y = 89.58Co.63 , W = 0.87 � � n 100 1� 1� Equi l ibrium concentration (mg L-1 ) 175 Figure 3 . 10 Effect of soil pH level on Cu sorption of the Ramiha soil. 8 1 Chapter 3 .-.. ..­I C) ..lI::: :::l () 1000 800 C) 600 E '-' "0 Q) .0 '-o en "0 co -c: :::l o E • .::.:. ....J '-" 75 Q) ..:! co > 50 � 25 0 4 5 6 7 8 Soil pH Figure 3 .13 Freundlich constant (K) for Cu adsorption by different soils at ¥arious pH values. 3.3.4 Distribution coefficients 3.3.4. 1 Distribution coefficients for the original soils Distribution coefficients �) were calculated from the amount of eu adsorbed per unit eu concentration in soil solution at various equilibrium concentrations and it ranged from 12 to 1 09, 2.5 to 27, 1 0 to 1 2 1 , 6 to 49 and 2.8 to 3 1 L kg-I for the Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu soils, respectively. The Freundlich isotherms for eu sorption showed that the shape of the curve was dependent on the concentration of the equilibrium solution and the soil types. The l«I values decreased with increasing solution concentration (Figure 3 . 14). Zehetner and Wenzel (2000) observed that distribution coefficients �) ranged from 0.8 to 1 544 L kg-I for eu and that l«J values decreased with increasing metal concentrations applied. Adsorption constant (K) values correlated well with soil pH (F0.53) and organic carbon (FO.25) for the original soils. Chapter 3 Adsorption and desorption of copper in pasture soils 85 1 25 + Manawatu 1 00 • Tokomaru • Ramiha .-.. .. Ngamoka � 75 I 0> ..::.c: I> Mangamahu ---l --- "0 50 � 25 o o 50 1 00 1 50 200 250 Equilibrium Cu concentration (mg L -1 ) Figure 3.14 Distribution coefficient 1 7% Cu desorbed from both soils) and 0.04M EDTA (4.7% and 1 8% Cu desorbed from the Manawatu and Ngamoka soils) extractable Cu produced higher amounts of desorbable Cu than other soil test extractants after 1 0 desorption equilibration in both soils. These two extractants probably remove different proportions of ions from the labile pool. For instance, 0.04M EDTA may preferentially remove some ions from organic matter, whereas O. I M HCI may remove the same ions preferentially from mineral surfaces. Both the O. I M HCI (Haynes, 1 997) and 0.04M EDTA extractants (Sims and Johnson, 1 99 1 ) have been shown to be successful in predicting soil Cu availability to plants. The de sorption data would however suggest that these reagents overestimate the amounts of readily plant available Cu in soils. DTP A and EDT A reagents extract Cu predominantly from the organic fraction of the soil, much of which is probably not directly available to plants. Desorption of Cu into a dilute Ca(N03)2 solution will depend not only the total amount of labile Cu in the soil but also other factors such as the relative proportion of soil components responsible for the retention of Cu in the soil, and soil pH. The Manawatu and Ngamoka soils desorbed 0.86 and 0.33 mg kg-I cumulative native Cu, respectively during 1 0 successive 2 hrs desorption periods (Table 3 .7). These amounts are much less than the total native Cu in these soils. In soil systems, it is difficult to attribute the loss of exchangeability of Cu to any single mechanism. The sorption of Cu on oxides is an inner-sphere complex that does not obey the reversible mass action relationship predicted by simple cation exchange (McBride, 1 989). In mineral soil with regards to the organic matter, it has been stated that in soils the retaining ability for Cu is predominantly controlled through CEC, rather than the Chapter 3 Adsorption and desorption of copper in pasture soils 88 chelating ability (Cavallaro and McBride, 1 984). The Ngamoka soil contains a higher CEe and more organic matter than the Manawatu soil, and this should represent an important contribution to the total number of Cu sorption sites. Thus, the sorption of eu onto the organic matter may explain the more plant available form of eu, that complexed with this soil component. -.... I C) � C) E - :::s o "tJ Q) Q. � o IJ) Q) "C Q) > .. ca "3 E 1 .0 0.5 :::s 0.0 o + Manawatu • Ngamoka + + + + o 5 1 0 Number of desorption periods Figure 3 .16 Desorption of native Cu from two soils (Manawatu and Ngamoka). Table 3.7 Cumulative desorbed native soil Cu and different extractable Cu concentrations. Soil Desorbed Soil test extractable Cu Desorbed as a percentage of soil test Cu mg kg-' extractable Cu (%) mg kg-1 M ! M3 TEA- O. !M O.04M M ! M3 TEA- O. !M O.04M DTPA HCI EDTA DTPA HC! EDTA Manawatu 0.86 1 7.4 1 9.3 1 6.7 4.8 1 8.4 4.9 4.4 5.2 1 7.9 4.7 Ngamoka 0.33 3.6 9.6 6.2 1 .8 1 . 8 9 . 1 3 .5 5 .4 1 8.2 1 8.3 In both soils, a significant portion of the sorbed Cu was apparently strongly bonded to the soil. Nevertheless, whether the surface reaction is genuinely irreversible or simply very slow in the backward direction is unclear (Sparks, 1 989). Pardo (2000) carried out a laboratory experiment to evaluate the sorption and de sorption by three soils with contrasting characteristics. The soil with a high native pH of 6.4 and CEe sorbed more Chapter 3 Adsorption and desorption of copper in pasture soils 89 eu than the other two soils. He observed that the differences in the reversibility of the sorption reaction, in response to a reduction of the solution eu concentration resulted from the differences in physico-chemical characteristics of the soil matrix. In the present study it was observed that Cu desorption values in the Ngamoka soil were low compared to the Manawatu soil, indicating that Cu is strongly bound to organic matter and oxides in the former soil. The quantitative differences observed in the extractability of sorbed Cu between the soils indicated that soil properties (organic matter, oxides and soil pH), which enhanced Cu sorption, contributed at the same time to the slowing down of the backward reaction. Temminghoff et al. ( 1994) studied the effect of pH on Cu desorption from a sandy soil and on complex formation by dissolved organic fractions. They observed that as the soil pH decreased, the amount of Cu desorbed from the soil increased. The amount of eu bound to solid organic carbon was almost equal to Cu bound by dissolved organic carbon. Cavallaro and McBride ( 1984) have also shown that desorption of Cu from soil clays decreases with an increase in soil pH. The fact that the de sorption of Cu from the soil labile pool is influenced by pH has an important implication for the uptake of soil Cu by plants. Soil organic matter and the oxide components play an important role in the retention of Cu. Desorption data will not necessarily provide an effective measure of soil eu availability. Many other factors, such as physico-chemical properties of the soil (soil pH, organic matter, oxides and clay), the plant itself and its influence on the root environment also affect Cu uptake. 3.3.5.2 Desorption of added Cu Two contrasting soils were used for this study. The soil samples were incubated for 0, 8 and 28 days with 50 mg Cu kil soil. Subsequent successive desorptions from the ° day incubated soil resulted in large increases in the amounts of Cu desorbed, compared with samples where only native Cu was present (Figure 3 . 1 7). The Manawatu soil desorbed more Cu than the Ngamoka soil after 2 hrs and 24 hrs desorption periods. It suggests that soil organic matter complexes eu with time. The proportion of added Cu desorbed during 1 0 desorption periods were extremely low, ranging from 2.5% in 24 hrs to 6% in 2 hrs cumulative desorption periods (Figure 3 . 1 7). The increase in soil pH (Table 3 .8) during desorption periods decrease the cumulative desorption. The low proportion of added Cu desorbed from the soils agrees with the results for native eu in these two soils. The sorption study (Section 3 .3 .3 .4) revealed that the soil organic matter and Chapter 3 Adsorption and de sorption of copper in pasture soils 90 oxides were the major components involved in Cu sorption in three soils (Ramiha, Ngamoka and Mangamahu). McLaren et al. ( 1 983b) demonstrated that the amount of Cu desorbed from the soil component (humic acid and soil oxide) was very small. Hogg et al. ( 1 993) provided evidence for the existence of slow reactions between the added Cu and the soil that reduce the ability of the Cu to desorb back into the soil solution. Narwal and Singh ( 1995) studied adsorption on four soils and reported that Cu adsorption among the soils followed: alum shale > peat soil > organic soil > sandy loam soil. Desorption of Cu by 0.005M CaCh was <1 % in the alum shale, peat soil and organic soil but in the sandy loam soil the maximum amount desorbed was 14%. This suggests that a near complete irreversibility of adsorbed Cu in all soils with the exception of sandy soils. Wu et al. ( 1 999) studied Cu desorption on various clay fractions and reported that 39% of adsorbed Cu was desorbed from the fine clay after five de sorption cycles, while 27% and 25% of the adsorbed Cu was desorbed from the medium and coarse clays, respectively. They also observed organic matter associated with coarse clay had a strong Cu retention affinity. Copper retained on permanent charge sites of 2 : 1 phyllosilicates by electrostatic attraction should be readily desorbed by mass action. Therefore, the irreversible sorption of Cu after the removal of organic matter in fine and medium clay samples suggests that ion exchange is not a major mechanism for retention of Cu on smectite in the O.O I M CaCh, pH 6.0 system. Sorption of Cu by a range of soil components has been shown to be largely irreversible, or only slowly reversible (McLaren et aI., 1 983b; McBride et al. , 1 984). The irreversibility or reversibility behaviour of Cu sorption varies with different factors. McBride ( 1 99 1 ) reported that activation energies for desorption may be much larger than those for sorption, and rates of sorption at ambient temperature are likely to be much faster than desorption rates. Thus the low proportions of added Cu desorbed in this and other studies may reflect, in part, non-equilibrium conditions caused by slow de sorption rather than true irreversibility. Barrow ( 1 985) has also mentioned that initial sorption reactions may be followed by slower reactions that would render a proportion of the sorbed Cu unavailable for immediate equilibrium with the soil solution. It is uncertain, however, whether such reactions are likely to be of importance for a relatively short de sorption period of 24 hrs. The proportion of added Cu desorbed is higher in 2 hrs cumulative desorption periods than 24 hrs periods due to higher sorption at 24 hrs desorption Chapter 3 Adsorption and desorption of copper in pasture soils 9 1 periods. Although such small proportions o f the added eu were desorbed during 1 0 desorption equilibrations, it should be noted that at the end o f this period, e u was still being desorbed. It might be expected that, with time, greater amounts of the added eu could be desorbed back into solution and therefore be considered to remain available for plant uptake. .,.... 4.0 l� , Native 0) ..:.: (a) 2 hrs, Manawatu :::l Added () • 0) -S • c 0 , • '';:; 2.0 -i e-o I (/) ID "0 ID 1 .2: � tu "S E I :::l 0.0 () 0 5 1 0 ....... ..- 4.0 l l') 2 h .:.:: ::J () 3.0 3.0 • 0> ..s c:: 0 � 2.0 2.0 0 C/l <1> '0 <1> > 1 .0 1 .0 � ::J E ::J () 0.0 0.0 0 3 5 8 1 0 0 3 5 8 1 0 4.0 4.0 • o day added Cu � a(ii ) ..- b(ii) '0> • 8 day added Cu .:.:: ::J A 28 day added Cu () 3.0 3.0 0> + o day native ..s c:: 0 8 day native 0 Cl. 2.0 2.0 D. 28 day native .... 0 C/l <1> '0 �::: <1> .� 1 .0 1 .0 ]! ::J E ::J � () 0.0 0.0 0 3 5 8 1 0 0 3 5 8 1 0 Number of desorption periods Number of desorption periods Figure 3.18 Cumulative desorption of native and added Cu from the (a) Manawatu soil and (b) Ngamoka soil incubated with added Cu for different periods. Desorption was carried out using two desorption periods [(i) 2 and (ii) 24 hours] . 3.3. 5.4 pH dependent desorption for different soils Copper desorption in various soils (Manawatu, Tokomaru, Ramiha and Ngamoka) at two pH levels is illustrated in Figure 3 . 1 9 . Only a small portion of Cu was desorbed, Chapter 3 Adsorption and desorption of copper in pasture soils 94 even after 1 0 consecutive extractions at a 1 :20 soil-to-solution ratio. At 30 mg Cu L-1 addition, the desorption decreased from 35%, 29%, 1 8% and 1 0% at pH 5 to 1 3 %, 7%, 3% and 5% at pH 8 for the Tokomaru, Ngamoka, Manawatu and Ramiha soils, respectively. Increasing sorption of Cu by soil as pH increased results in a decreased desorption (Figure 3 . 1 9) . A similar result was reported by Hue et al. ( 1 997) and they obtained relatively higher desorption (3 0%) at pH 5 . 0 than at pH 8.0 ( 1 0%). They also observed that more Cu was sorbed in soils with high organic carbon and low indigenous Cu. Atanassova and Okazaki ( 1 997) examined the Cu adsorption and desorption under acid conditions by soil c lay fractions in O.O I M Ca(N03)2 and found a considerable amount o f sorbed Cu could be solubilised by decreasing pH values to 4; 39% was desorbed in the PIano sol c lay and 45% was desorbed in the Gleyic Arcisol c lay. 1 00 1 00 30 mg Cu Kg- 1 soil - Manawatu "0 � Tokomaru -0 (l) ID ..0 ..0 .... Ramiha is 0 (f) if) (l) -0 50 l:I 50 - Ngamoka ().) "0 "0 Q) "0 "0 « "0 � « <> >? <> 0 0 5 8 5 8 1 00 50 mg Cu Kg-1 soil 1 00 l 50 mg Cu Kg-1 soil "0 "0 (l) (IJ ..0 ..0 <5 Cl (f) '" (l) "0 50 "0 50 (I) "0 "0 (l) "0 "0 « "0 >? « " � " 0 0 5 8 5 8 Soil pH Figure 3.19 Effect of pH on Cu adsorption and desorption. The effect of pH and the level of sorbed Cu on the de sorption of Cu by the four soils is i l lustrated in Figure 3 . 20. Desorption increased with increasing levels of added Cu in Chapter 3 - .------------------------------- Adsorption and de sorption of copper in pasture soils 95 the soil (Figure 3 .20). The Tokomaru soil contains lesser amounts of soil organic matter, oxides, and CEC, resulting in largest amounts of desorption of added Cu at both pH levels. The desorption of Cu decreased with increasing soil pH (Figure 3.20). The Ramiha soil contains higher amounts of organic matter, oxides, and CEC, and has the highest :KJ value at pH 5 .0, resulting in the lowest desorption of Cu, but at the high pH level the Manawatu soil showed the lowest desorption. The Ngamoka soil resulted in a higher desorption of Cu at both pH levels, which may be related to the disruption and reformation of organo-mineral associations induced by wetting and re-drying in the incubation process, and also due to the low base saturation of this soil. The differences of desorption behaviour in these soils was due to the speciation of Cu at two levels of pH in the different soils. Cavallaro and McBride ( 1 984) have also shown that desorption of Cu from soil clays decreases with an increase in soil pH. A number of studies have shown that Cu2+ in soil solution, especially at higher pH, exists primarily in a form complexed with soluble organic matter (Hodgson e t al. , 1 966; Fotovat and Naidu, 1 997; Wu et al. , 2000). The amount of organically complexed Cu in solution generally increases above pH 7.0 because of a greater solubility of soil organic matter at higher pH (McBride and Blasiak, 1 979), while the concentration of free ionic Cu2+ at higher pH is much lower, usually in the range of 10-9 to 10-8 M (McBride and Blasiak, 1 979). It has been estimated that hydrolysis products of Cu [CuOH+ and CU2(OHh2+] are the most significant species below pH 7, while above pH 8 anionic hydroxy complexes of Cu become important. Solubility of metals in soils and mineral oxide surfaces as a function of pH is often dictated by the presence of organic and inorganic ligands (McBride, 1 989; Sparks, 1 995; Harter and Naidu, 1 995). In this study, it was observed that the irreversible retention of Cu might be the result of complex formation of Cu at high pH. Quantification of the plant availability of Cu in soil suspensIOns reqmres the determination of labile Cu (ions in the solid and solution phases in equilibrium with free Cu forms in the soil solution) and the activity of Cu2+ in the soil solution of acid soils and Cu(OH)2 in neutral or alkaline soils. It suggests that the de sorption of Cu from the soil labile pool is affected by pH, which has an important implication for the uptake of soil Cu by plants. Chapter 3 Adsorption and desorption of copper in pasture soils 96 ........ 200 200 .,.- pH =5.0, 30 mg Cu L-1 pH =8.0, 30 mgCu L-1 , Ol � ::::l I + Manawatu () I Ol 1 50 1 50 I • Tokomaru I E I ---- I • Ramiha c 0 li 1 00 1 00 -i ... Ngamoka ..... 0 I C/) I � (]) 1 u (]) 50 50 > i(j :5 1 E I ::::l 0 0 () 0 3 5 8 1 0 0 3 5 8 1 0 ........ 400 400 .- -, pH =8.0, 50 mg Cu L-1 I I pH =5.0, 50 mg Cu L-1 Ol � ::::l '1 () I Ol 300 l 300 ,S I C I 0 � 'a 200 , 200 ..... I 0 C/) (]) I u (]) 1 00 J 1 00 > � ::::l E ::::l 0 0 () 0 3 5 8 1 0 0 3 5 8 1 0 Number of desorption periods Number of desorption periods Figure 3.20 Cumulative desorption of Cu at two pH (5 and 8) and two sorption levels (30 and 50 mg L-l) in different soils. 3.4 CONCLUSION AND FURTHER STUDY • Sorption capacity increased with time and reached the maximum level within 2 hours in all soils. • Adsorption of eu varied amongst the five soils, which vary in their physico­ chemical properties. • The Freundlich equation adequately described the eu adsorption data. • The highest value of adsorption constant (K) for eu was observed in the Ramiha soil and the lowest in the Mangamahu soil . • The order of eu adsorption followed: Ramiha > Manawatu> Ngamoka > Tokomaru Mangamahu. Chapter 3 Adsorption and desorption of copper in pasture soils 97 • Copper retention by the Ramiha, Ngamoka and the Mangamahu soils is mostly determined by both the organic matter and oxide components. • Organic matter is the principal component for Cu retention in all soils. • In all soils, Cu concentration in solution decreased and Cu sorption increased with an increasing pH level. • Desorption of native Cu from all soils was generally low, but increased with an increasing concentration of added Cu in solution. • The cumulative desorption of added Cu from the soils decreased with increasing pH. • In the Manawatu soil, 4 .7 % ofO .04M EDTA extractable Cu could be desorbed after 1 0 desorption equilibrations with Ca(N03h, while for the Ngamoka soil 1 8% of the extractable Cu could be desorbed. • The proportion of added Cu desorbed during 1 0 de sorption periods was extremely low, ranging from 2 .5% in 24 hrs to 6% in 2 hrs. • Desorption was 24% (at pH 5 .0) and 1 0% (at pH 8 .0) of the sorbed Cu for both soils. Adsorption and desorption reactions are likely to be the major factors controlling the availability of Cu to plants. In this chapter, a basic knowledge of the type of surfaces that will adsorb Cu and the relative amounts of Cu retained under given conditions has been acquired. The availability of Cu from different Cu source as fertilisers in different pasture soils will be discussed in the next chapter. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils CHAPTER 4 4.1 INTRODUCTION PLANT AVAILABILITY OF COPPER FROM DIFFERENT COPPER FERTILISERS IN PASTURE SOILS 98 Copper is essential for both plants and animals. Copper is involved in the functioning of a wide range of enzymes, and is required in very small amounts by both plants and animals . Copper levels are low in some New Zealand soils (Wells, 1 957 ; Haynes and Swift, 1 983 ; McLaren et at., 1 984). This can limit pasture growth (During, 1 972) and the Cu supply to the grazing animal (Go old and Smith, 1 975) . Deficiency of Cu occurs when the Cu concentration is < 4 mg kg- l in pasture and <0.3 mg L-1 in animal blood. In animal nutrition, Cu deficiency is almost entirely confined to grazing animals due to low levels of eu in the herbage/forage, or because of the normal to low levels of Cu accompanied by elevated intakes of Mo, S and Fe that are sufficient to limit Cu absorption and retention. Willimott ( 1 995) reported that Cu deficiency in grazing ruminants is widespread and occurs on the YeHow Brown Pumice soils and YeHow Brown Earths of the Wairoa district, resulting in problems with brittle bones in lambs. Copper fertiliser is the second most frequently used trace element in Northland, New Zealand. Many of the Yellow Brown Earths, sand and peat soils have a requirement for Cu either to aid clover vigour, as is the case on some of the West Coast sands or to address low animal eu levels (Jorulson, 1 995). Korte et al. ( 1996) reported widespread Cu deficiency in grazing animals in New Zealand costing the farming industry annually several million dollars in animal remediation. Good correlation between herbage Cu concentration and liver eu concentration of grazing Romney sheep has often been observed (Grace et at. , 1 998 ; Knowles et al., 1 998). There are conflicting opinions to the best means of correcting Cu deficiency in livestock. Copper deficiency in grazing animal is corrected either through application of Cu fertilisers or by the use of Cu bullets. Copper sulphate is the most commonly used Cu fertiliser in New Zealand. A wide range of Cu sources have been examined for their effectiveness as fertilisers, when applied to soils or foliage (Gilkes, 1 98 1 ) . Pasture topdressing with CUS04 has been the major measure to prevent the primary Cu Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 99 deficiency (Grace et al. , 1 998). The effectiveness of pasture topdressing with Cu (CUS04' 5H20) depends on the nature of the pasture and soil properties (Sherrell and Rawnsley, 1 982). The residual effects of Cu topdressing also differ between soils and the types of Cu fertilisers. There is a need to define the rates of change in the effectiveness of Cu fertiliser with time over the range of soils and cl imatic conditions encountered. This information is required so that the needs of crops , pastures and the grazing animal can be met and agricultural production maintained. Copper in the soil solution occurs mainly as copper-organic matter complexes. Little is known about Cu availability and the rates of absorption by plants from different Cu organic complexes. Therefore it is difficult to predict changes in the supply of plant available Cu in field situations from the existing knowledge of the chemistry of Cu in soils. Objectives: The objectives of the experiment reported in this chapter are as follows: • To examine the effectiveness of a range of Cu fertilisers for plant growth and raising pasture Cu concentration. • To monitor the transformation of Cu in soils. 4.2 MATERIALS AND METHODS 4.2.1 Soil collection and preparation Soil samples were collected from different locations (Chapter 3 ; Section 3 .3 . 1 ) and air dried. The soil samples were ground and passed through 6 .0 mm sieve to remove the weeds and stones. One kg (dry weight basis) of soil was mixed with the required amount of fertiliser and was placed in pots. The soil in the pots was maintained at 80% field capacity for equilibration. 4.2.2 Copper fertilisers used The different Cu sources used as fertiliser in this experiment are given in Table 4. 1 . The CUS04 and CuO are granular fertilisers and CU(OH)2 is a powder. To maintain the uniformity of particle size, the fertilisers were ground to a 1 50-400 Ilm particle size range and used in the experiment. The solubility data indicates that CUS04 is more Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 00 readily soluble than the other two fertilisers (Table 4. 1 ). The fertiliser rates used were: 0, 50, 1 00 and 200 mg Cu kg- I soil. Assuming a bulk density of 1 .0 tonne m-3 and a depth of incorporation of 1 cm, the rates are equivalent to 0, 5 , 1 0, 20 kg Cu ha- I . Table 4.1 Sources of Cu used as fertilisers in the plant growth experiment. Fertiliser types Copper content Solubility (g kg" !) water (g m-3) Copper 250 1 00 sulphate Copper 308 0.04 hydroxide Copper oxide 250 0. 1 0 4.2.3 Plant growth experiment In Supplied by Ravensdown Fertilisers, New Zealand. Mankind Trading Co, New Zealand. BOP Fertilisers Ltd, New Zealand. A modified split plot design was used in this glasshouse trial. The treatments were five soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu) as main plot and three Cu sources [CUS04, Cu(OHh and CuO] at four levels (0, 50, 1 00 and 200 mg Cu kg- ! soil) as sub-plot (Plate 4. 1 ). The pots were placed randomly on each trolly (block) by using a random number table (plate 4.2). The treatments were replicated four times. There were 240 (60 x 4) pots in this experiment. Two weeks after fertiliser application, fifty seeds of ryegrass (Lolium perenne cv. Super Nui) were sown in each pot. The pots were covered with brown paper until germination. The first Cu-free nutrient solution was given 2 1 days after sowing time. The Cu-free nutrient solution was given twice a week. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 0 1 Plate 4.1 Plant growth experiment with different levels o f CUS04 fertiliser (Ballantrae high fertility and Ballantrae low fertility known as Ngamoka and Mangamahu soil, respectively). Plate 4.2 Effect of soil types on plant growth experiment (Ballantrae low fertility known as Mangamahu soil). Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 02 4.2.4 Grass and soil sample preparation The grass was harvested at 68, 96, 1 25 , 1 53 and 1 8 1 days after sowing. The plants were cut to a height of approximately 3 .0 cm above the surface of the soil. The samples were dried at 70 DC in a forced air oven. The dry weights of the grass samples were recorded and the samples were then ground using a coffee grinder and kept in airtight polyethylene bags for chemical analysis. A soil sample was collected from each pot using a soil corer at 0, 8 , 28, 1 06, 1 63 and 1 9 1 days intervals. The soil samples were air dried, ground and passed through a 2 .00 mm sieve. The soil samples were analysed for exchangeable, organic , oxide and residually bound Cu. The results of Cu transformation in the soils are presented in Chapter 5 . 4.2.5 Ryegrass samples for Cu analysis A known weight (0.4 g) of the finely ground grass samples was placed in 50 ml acid­ washed conical flasks. Approximately 1 0 m1 of Aristar grade RN03 (69%) was added to each conical flask. A reflux funnel was placed on top of the conical flasks and left for 1 6 hrs in a fumehood. The samples were then digested for at least 4 hrs at 1 50 °C (until the brown fuming stopped). The reflux funnels were removed and the temperature was increased slowly to 250 Dc. The samples were boiled until just dry and then allowed to cool. The volume of the digest was made up to 1 0 ml with 1% RN03. The digest aliquots were then stored for chemical analysis. 4.2.6 Chemical Analysis Copper in the plant digests and in the soil extracts was analysed using flame atomic absorption Spectrophotometry (F-AAS). All glassware was acid washed (2M HCl), rinsed with deionised water, and oven dried before use. To check the reproducibility of the analy6cal procedure, three blanks were included in each batch of 50 samples of grass and of soil. Certified herbage and soil samples were also analysed in parallel with the unknown samples. All results are expressed on an oven-dry weight basis. - - - ------------- Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 03 4.2. 7 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS). Significant differences between treatment means were evaluated using analysis of vanance (ANOV A), performed by using the SAS GLM procedure (SAS Institute, 1 996). 4.3 RESULTS AND DISCUSSION 4.3.1 Initial soil characteristics The initial soil characteristics are discussed in chapter 3 (Section 3 .3 .2) . The soils varied in their Olsen P, organic matter content, CEC, and pH values. In general all five soils used in this experiment were slightly acidic and are optimum in soil Cu. 4.3.2 Dry matter yield 4.3.2.1 Effect of the soil types The dry matter (DM) yields were significantly different among the soils at all harvests (Figure 4. 1 ) . The highest dry matter yield was obtained from the Manawatu silt loam and the lowest from the Tokomaru silt loam at harvest 1 . The low DM yield of the Tokomaru soil was due to the low fertility of this soil as indicated by the low Olsen P, organic matter, soil pH and CEe. The high dry matter yield of the Manawatu soil was due to a high fertility status resulting from the recent alluvial origin of the soil. A good correlation between DM yield at harvest 1 and Olsen P (r=0.90), soil pH (r=0.54), c lay (r= -0. 3 1 ) and silt (r=0.34) was obtained. At harvest 2 correlations were obtained between DM and CEC (r=0.46) and Olsen P (r=0.39), and for harvest 5 with CEC (r=0.29) (Table 4.2). All significant correlations (Table 4 .2) are in accord with the suggestion that the yields were influenced by the physical and chemical status of the soils. The DM yields increased in the Tokomaru soil up to harvest 3 and then decreased. The DM yields decreased at harvests 4 and 5 due to insect infestation, and to high temperatures in the glasshouse. At harvest 4, the Mangamahu soil produced the highest DM yield, which was statistically similar to the Manawatu and Ramiha soils, the Tokomaru soil produced the lowest DM yield, which was statistically similar to the Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 04 Ngamoka soil . At harvest 5, the Ramiha soil produced the highest DM yield, which was significantly more than all the other soils. The Tokomaru soil produced the lowest DM yield, which was also significantly different from the others. In this study, there was no response in DM yield with eu fertiliser additions. King ( 1 974) and Gartrell ( 1 98 1 ) obtained a good response to the application o f eu to sand plain soils and sodic soils for wheat crops in Western and Southern Australia. In Australia, inherently the soil eu is very low, which may be the reason for a eu response in dry matter yield. 1 0 + Manawatu <) Tokomaru 8 • Ramiha ......... • Ngamoka ..-I (5 7 0. .. Mangamahu .9 -0 m 5 ':;" "-Cl> � co E 3 C-o 2 0 67 1 00 133 1 67 200 Days fol lowing fertiliser appl ication Figure 4 . 1 Effect of different soils on dry matter yield (g porI ) of rye grass at different harvests. Data are means ± SE, 0=48. Table 4.2 Correlations of dry matter yield with soil properties. Soil properties Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 Olsen P 0.90** 0.39** 0.22* 0.02 0.03 pH 0.54** 0 . 1 2 0.003 0. 1 5 0.04 Organic carbon 0.07 0.08 0.00 1 0.0 1 2 0. 1 6 CEe 0.03 0.46** 0.05 0.05 0.29* Silt 0.34* 0.08 0. 1 2 0.004 0.05 Clay -0.3 1 * 0.004 0.01 0.03 0.08 ** and * denote significance at 99% and 95% level, respectIvely. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 05 4.3.2.2 Effect of types of eu fertiliser There was no significant yield response observed with the addition of Cu fertilisers when compared to control. The fertiliser type caused a different effect on DM yield in harvests 1 , 2 and 4 (Figure 4.2). At harvest 1 , CUS04 produced the lowest DM yield, which was significantly different to the other two fertilisers. CUS04 is a readily soluble fertiliser. Thus, the decrease in yield might be due to a quick release of Cu from CUS04 causing yield depression at higher levels of Cu. At harvest 2, Cu(OHh produced the highest DM yield, which was similar to CuO, but higher than the CUS04 fertiliser. CU(OH)2 produced the highest DM yields at each harvest, this may be due to the liming effect of the Cu(OHh fertiliser. It contains 30% lime as an inert material. 8 -. 6 ..... , - 0 Cl.. .9 "0 ID 4 '>, ..... Q) t::: C'V E C-o 2 o + Control + CuS04 It Cu(OH)2 ;6;; CuO 50 1 00 1 50 200 Days following ferti liser application Figure 4.2 Effect of fertiliser type on dry matter yield at different harvests. Data are means ± SE, n=80; control n=20. 4.3.2.3 Effect of fertiliser rates Increasing fertiliser rates resulted in a significant decrease in DM yield in all harvests (Table 4.3) . At harvest 1 , the eu level of 50 mg Cu kg-I produced the highest DM yield, but it was statistically similar to the other treatments except the 200 mg Cu kg- ! Chapter 4 Plant availability of copper from different c opper fertilisers in pasture soils 1 06 treatment leveL The Cu level of 200 mg Cu kg-l produced the lowest DM yield and it was significantly different to the other treatments. A similar trend was also observed at all other harvests. During ( 1 972) reported that as little as 1 1 kg CUS04 ha-1 can depress pasture growth. Carter and Day ( 1 977) found that at a rate of 2 kg Cu ha-1 (8 kg CUS04 ha- I ) pasture yield was depressed in field trials in South Australia. Khan et al. ( 1 996) in an earlier field trial showed that ryegrass yields did not respond to Cu application, indicating that ryegrass is less sensitive to eu than legumes. Copper concentration in legumes increased with increasing levels of Cu up to 5 kg Cu ha-1 , but grasses did not show any response with increasing levels of Cu. This may reflect the requirement of Cu by rhizobia for nitrogen fixation (van der Elst et aI., 1 96 1 ). Yield responses to Cu occur only where the Cu concentration in plants is below 4 mg kg-J (Sherrell and Rawnsley, 1 982). Forbes ( 1 978), in a survey of luceme on Yellow Brown Pumice soils found very few stands with <4 mg kg-1 eu, indicating widespread DM yield response to Cu is unlikely to occur in these soils. Copper sulphate fertiliser at 200 mg Cu kg-1 soil produced the lowest DM yield in all harvests, and it was significantly different from the other treatments (Table 4.3) . The treatment at 200 mg Cu kg-1 soil for the CU(OH)2 and CuO fertilisers also produced significantly lower DM yields than other treatment levels. This indicates that the higher eu levels might have caused toxicity, and thereby depressed the growth of rye grass. The effect of eu levels on yield depression was less pronounced for the CU(OH)2 and CuO fertilisers than for the CUS04 fertiliser. Beckett and Davis ( 1 978) found that the high levels of Cu depressed plant growth in barley. Alva and Chen ( 1 995) observed that with an increase in external eu concentrations, the shoot and root dry weights of citrus plants decreased significantly. Plenderleith and Bell ( 1 984) conducted an experiment to evaluate the growth response of 1 2 sub-tropical grasses with additions of Cu and Zn. They observed that depending on the species the Cu and Zn concentrations in the plants was associated with a 50% yield reduction ranging from 1 7 to 27 mg kil and 475 to 1 925 mg kg-l , respectively. Heavy metals such as eu tend to accumulate in the roots and in turn, affect the growth of the whole plant. Lidon and Henriques ( l 992b and 1 993) observed that the eu concentration in roots increased linearly with increasing external Cu concentrations. Decreased translocation of eu to the above ground parts from the roots has been suggested as a mechanism to withstand eu toxicity. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 07 Table 4.3 Effect of fertiliser rates on dry matter yield (g pori ) of ryegrass at different harvests. Fertilisers Treatments Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 (mg Cu kg' l soil) Control Control 5 .79a 7.20a 6.55a 3 .05a 2.07a CUS04 50 5 . 8 1a 6.64a 5 .99a 2.56abc 1 .43bc 1 00 5 .35a 6 . 1 8a 6 . 1 8a 1 .7 Ode 1 .27bcd 200 2.32c 2 .59c 3.23c 1 .22e 0.75d Cu(OH)2 50 6. 1 1 a 6.92a 6.43a 2.78ab 1 .42bc 1 00 5 .43a 6.30a 5 .76a 2.44bc 1 .56ab 200 4.09b 4.22b 4.27b 1 . 74d 1 .36bcd CuO 50 5 .90a 6.78a 6.00a 2 .27bc 1 .09bcd 100 5 .48a 6. 1 9a 5 .83a 2 . 1 7cd 1 .33bcd 200 4.47b 4.29b 3 .98bc 1 .2 1 e 0.9 1 cd * Treatment means followed by the same letter WIthin a colwnn are not slgruficantly different at the 5% level. 4.3.3 Copper concentration in ryegrass 4.3.3.1 Effect of the soil types The eu concentration in ryegrass was significantly different among soils (Table 4.4). The ryegrass grown in the Manawatu silt loam soil contained the highest eu concentration at all harvests. The Ramiha soil contained the lowest amount of eu at harvest 1 . At harvest 2, the Tokomaru and Ngamoka soils ryegrass contained the lowest concentration of eu and was significantly different to the others . The rye grass of the Ngamoka soil contained the lowest concentration of eu and was significantly different to the others at harvest 3 . There was a general trend of decrease in the eu concentration in all soils after the first harvest (Table 4.4), but the eu concentration increased at harvests 4 and 5 . The decrease in eu concentration in ryegrass with time is due to a decreasing eu concentration in the soil resulting from changes in soil pH in the rhizosphere (Linehan et al. , 1 985), and reactions of eu with soil constituents (Brennan et al., 1 980, 1 983), A positive correlation between eu concentration in plants and soil pH (r=0 . 1 8) ; a positive correlation between eu concentration in plants and Olsen P (r=0.42) ; and a negative correlation between eu concentration in plants and organic matter (r= -0.54) and c lay content (r= -0.49) were observed for all harvests. A positive Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 08 correlation between the Cu concentration in plants and Olsen P was obtained at each harvest. The availability of Cu to plants is largely dependent on soil pH, organic matter and the c lay content of the soil . A positive relationship between the Cu concentration and soil pH was observed up to harvest 3 (Table 4 .5) . This may be attributed to the effects of pH on the mobility of the eu in soils and its absorption by roots. There is evidence to suggest that the mechanisms, for the absorption of eu by roots, are affected by changes in soil pH. However, in the previous chapter 3 (Section 3 . 3 .3 .5), as pH is increased towards neutrality, the mobility of Cu in solution is reduced as the result of increased sorption. There are marked increases in the gradients of sorption isotherms as the pH of the soil i s raised. At low concentrations of Cu, the gradients of the sorption isotherms are highly correlated with soil pH, but not with some of the other soil properties thought to be important in controlling mobility (Jarvis, 1 98 1 a). Copper availability is reduced at a soil pH above 7 and is most readily available below pH 6 (Lucas and Knezek, 1 972; Locascio, 1 978). The role of organic matter in the incidence of eu deficiency, complex formation, and sorption of Cu have been reported by many authors (McLaren and Crawford, 1 973b; Harter, 1 983 ; Shorrocks and Alloway, 1 987; McBride, 1 99 1 ; Wu et al., 2000). The relative sorption capacity of different soil components for Cu (Chapter 3) indicates that, among soil constituents, organic matter and oxides are the most important for the sorption of Cu. Nielsen ( 1 976) obtained samples of soil solution by suction from a calcareous peat soil in which barley plants were grown. As expected, the levels of Cu in the soil solution remained constant when no plants were grown. In the presence of barley plants, Cu in the soil solution increased. Williams ( 1 98 1 ) suggested that some portion of the organic material solubilised in the presence of plants would be due to root exudates. Linehan et al. ( 1 985) displaced the soil solution by centrifugation in the presence of the barley plants. The concentration of eu in the root rhizosphere was found to be maximum during early plant development and decreased up to harvest. Microbial activity was considered to be involved in the transformation of Cu in soil. The pattern of change of Cu concentration in the rhizosphere with time was similar to that of the changing soil biomass. It was suggested that enhanced Cu levels in the rhizosphere could also be related to soil pH changes. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 09 The increase in plant Cu concentration in all soils except the Ramiha soil at harvests 4 and 5 was due to a lower growth rate and high temperatures. The grass grown in the Ramiha soil contained the lowest concentration of Cu at harvests 4 and 5 and was significantly different to the others. The Ngamoka and Ramiha soils are rich in organic matter and oxides, Cu complex fonnation occurs more rapidly in these two soils, resulting in Cu being unavailable for plant uptake. Ryegrass grown in the soil with the lowest organic matter (Manawatu) contained the highest Cu concentration at each harvest, while rye grass grown in the Ramiha and Ngamoka soils with the higher amounts of organic matter contained the lowest concentration of Cu at later harvests. This is consistent with the more rapid immobilisation of Cu into the organic fraction in the Ramiha and Ngamoka soils. Cu in soil is strongly held on inorganic and organic exchange sites and in complexes with organic matter. For this reason a large proportion of the total Cu content of soils is not available for uptake by plants . The proportion of the total Cu taken up by plants has been found to be greater in mineral soils than in peats (Kabata-Pendias, 1 963). Since most of the Cu added to pasture soils is associated with organic matter, a large proportion of the Cu taken up by plants must originate from soluble organic complexes. The dissociation of Cu2+ from the organic ligand apparently must occur prior to plant uptake (Goodman and Linehan, 1 979) . The ease with which the dissociation and subsequent absorption of Cu by roots takes p lace will therefore depend upon the complexing ligand and this, in turn, will depend on the particular soil in which the plant i s growing, as well as conditions within that soil. Stevenson and Fitch ( 1 98 1 ) contended that organic colloids and clay minerals play a major role in Cu retention by soil. In most mineral soils, Cu may be bound as c lay-metal-organic complexes, since in these soils, organic matter is intimately bound to c lay. A significant negative correlation between Cu concentration and soil clay was obtained at each harvests (Table 4.5) . As well as reactions with organic matter, applied Cu may have been rendered unavailable to plants by reactions with clays (McBride, 1 98 1 ), and sesquioxides (Grimme, 1 968). Dragun and Baker ( 1 982) conducted a glasshouse experiment to detennine the physical and chemical properties of soils, which control Cu availability to corn seedlings using 1 6 Northeastern United States soils and obtained a good relationship between Cu availability and soil pH, organic matter, CEC and percent clay. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 0 Table 4.4 Effect of soil types on Cu concentration (mg kg-I) in ryegrass at different harvests. Soils Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 Manawatu 33 .43a 3 1 .62a 20.46a 25 .68a 33.34a Tokornaru 27.77ab 22 .47c 1 8.65b 22. 1 6b 23 .25b Rarniha 25 .30b 24.92b 12 .74d 1 2 .96c 12 .08c Ngamoka 28 .63ab 22.8 1c 1 1 .62e 22.23b 2 1 .20bc Mangarnahu 29.57ab 25.2 1b 15 .30c 22 .44b 26.38b *Treatment means followed by the same letter within a column are not slgruficantly different at the 5% level. Table 4.5 Correlations of Cu concentration in ryegrass with soil properties. Soil properties Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 Olsen P 0.55** 0.70** 0.28* 0.2 1 * 0.34* pH 0.20* 0. 1 8* 0.22* 0 . 14 0. 1 5 Organic carbon -0.42** -0. 3 1 * -0.98** -0.38** -0.63** CEC 0.09 0.04 -0.80** -0. 1 6 -0.28* Silt 0.001 0. 1 8* 0. 12 0.04 0.0 1 Clay -0.45** -0.42** -0.8 1 ** -0.32* -0.45** ** and * denote Significance at the 99% and 95% levels, respectively. 4.3.3.2 Effect of types ofCufertiliser The fertiliser types resulted in a significant effect on Cu concentration in rye grass (Table 4.6). The application of CUS04 fertiliser resulted in the highest concentration of Cu at harvest 1 and it was significantly different from CuO. This might be attributed to the quick releasing characteristics of CUS04 (Gilkes, 1 98 1 ). At harvest 2, the application of CU(OH)2 resulted in the highest concentration of Cu in plants, and was significantly higher than CuO. At harvests 3 and 4, the highest Cu concentration was observed in CuO treated ryegrass, and it was significantly higher than the Cu(OHh and CUS04 fertilisers (Table 4.6). The higher residual effect of Cu(OH)2 and CuO fertilisers might be due to the slow release characteristics of these two insoluble fertilisers. The suitability of CuO for broadcasting depends on particle size. CuO broadcast on to the soil surface at 5 kg Cu ha- I , and worked into the soil, did not correct Cu deficiencies in canola, barley, or wheat during the year of application, but corrected these deficiencies in following years (Karamanos et al. , 1986). The lack of a Cu response to CuO was Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils I I I mainly due to the low water solubility of the coarse, granular CuO, which ranged in particle diameter from <0.2 (powder) to 3 .0 mm. Coarse Cu carriers are also ineffective, where inadequate contact occurs between the plant root and the applied Cu (Gartrell , 1 98 1 ) . The Cu concentration decreased with time from the first harvest and again it increased at harvests 4 and 5 resulting in low dry matter yields. Willimott ( 1 995) found that application of 5 and 1 0 kg Cu ha-l as copper sulphate increased the herbage Cu concentration up to three months after the Cu application, and then decreased back to the initial 1evels nine months after application. Table 4.6 Effect of fertiliser type on Cu concentration (mg kg-I) of ryegrass at different harvests. Fertilisers Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 CUS04 33 .23a 25 .65ab 1 5 . 1 0b 2 1 .36b 26.29a Cu(OHh 29.32a 26. 1 9a 15 .68b 1 8 . 04c 20.45b CuO 24.26b 24.39b 1 6,48a 23 .88a 24.55a . . * Treatment means followed by the same letter Wlthin a column are not slgmficantly dlfferent at the 5% level. 4.3.3.3 Effect of fertiliser rates The increasing levels of Cu increased the Cu concentration in ryegrass (Table 4.7). CUS04 at the 200 mg Cu kg- l soil level resulted in the highest Cu concentration at harvest 1 , which was significantly different from all other treatments. Cu(OHh fertiliser at the 1 00 and 200 mg Cu kg- ] soil levels and the CuO fertiliser at the 200 mg Cu kg-] soil level contained statistica1ly similar amounts of Cu but significantly different from the others at harvest 1 . Cu(OHh and CuO fertilisers at the 200 mg Cu kg-I soil level contained the highest concentration of Cu in ryegrass, which was significantly higher than the other treatment at harvest 2 . The CuO fertiliser at the 200 mg Cu kg"l soil level contained the highest amount of Cu, which was significantly higher than the other treatments at harvests 3 , 4 and 5 . This reflected the low solubility characteristics of the CuO fertiliser. The treatment levels of 1 00 and 200 mg Cu kg- ! of soil resulted in statistically similar concentrations of Cu in Cu(OH)2 at harvest 1 . Reuter et al. ( 1 9 8 1 a) noted that the increasing rates (ranged from 0 to 533 mg Cu kg- ] ) of Cu application increased the Cu concentration in plant tops and roots. Plenderleith and Bell ( 1 984) Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 12 found that with increasing additions of Cu (4 to 600 mg Cu kg-I soil) the Cu concentration in tropical grasses increased from 1 7 to 27 mg kg-I . Reuter et al. ( 1 98 1 a) recorded that the Cu concentration in subterranean clover declined from 3 .9 mg kg-I at 26 days after sowing to 1 .6 mg kg- I at 98 days. Sherrell and Rawnsley ( 1 982) found that applications of 2 to 4 kg Cu ha-I as copper sulphate increased herbage Cu concentrations from 5 to 1 2 mg kg- l within 4 weeks and then it decreased markedly to 8 mg kg-l over the next 9 to 1 0 months. They also observed that clover tended to take up larger amounts of Cu and its persistence in maintaining higher Cu concentration that was greater than grasses. Khan et al. ( 1 996) observed that applications of 2, 5 and 1 0 kg Cu ha- I as CUS04 increased the pasture Cu concentrations ranging from 9.98 to 1 5 .89 mg kg-I and 6 .5 1 to 6.69 mg kg-I at 73 and 1 87 days after fertiliser application, respectively. Table 4.7 Effect of fertiliser rates on Co concentration (mg kg-I ) of rye grass at different harvests. Name of Treatments Harvest I Harvest 2 Harvest 3 Harvest 4 Harvest S fertilisers (mg Cu kg' ! soil) Control Control 12 .48d 12 . S2h 6.4 1 f 6. 1 Sg 6.6Sf CUS04 SO 23 .S2c 23 .47fg 1 2.38e 1 2 .36f 1 3 .46e 1 00 41 .94b 32.08bc 1 8.04d 29 .39d 39.43c 200 S4.98a 34.S2ab 23.S7c 37.5Sb 4S.63ab Cu(OH)2 SO 23.78c 26.20ef 1 2.80e 1 3 .03f l S .28e 100 38.48b 29.44cd 17.3Sd 20. 12e 1 9. 1 Sde 200 42.56b 36.S8a 26. 1 Sb 32 .8Sc 40.7 1bc CuO SO 2 1 .97c 22.04g 13 .80e 1 3. 80f 1 6.S0e 1 00 2S .80c 26.62de 1 7.08d 28 .96d 24.68d 200 36.80b 36.36a 28 .62a 46.60a SO.38a * Treatment means followed by the same letter WIthin a column are not sIgruficantly dIfferent at the S% level. 4.3.4 Copper uptake 4.3.4.1 Effect of the soil types There was a general trend of decreasing Cu uptake with increasing time after fertiliser application from the 96 days harvest (Figure 4.3) . The Manawatu soil showed a greater rate of decline in Cu uptake in comparison to the other soils. The differences in the rate Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 3 of decline may be due to differences in soil pH, Olsen P, organic matter, oxides and clay minerals of these soils. The decrease in Cu uptake is due to adsorptionJdesorption processes, which control the amount and rate of release of Cu for plant uptake. This applies both to native soil Cu and Cu applied as fertiliser. Results obtained for Cu sorption by soil components (Section 3 .3 .3 .4) and the pH dependent adsorption­ desorption study in Chapter 3 (Section 3 .3 .3 . 5 and 3 . 3 . 5 .4) explain the reasons for the reduction in Cu uptake. Reduction in eu uptake was due to the stronger adsorption of Cu to organic ligands or mineral surfaces. McLaren et al. ( 1 990) reported that the adsorption of Cu is strongly influenced by the amount of added Cu and soil pH. They found that initially less than 1 0% of the Cu adsorbed by the soil was desorbed, and after three months of soil contact, only a negligible amount « 1%) of the adsorbed Cu could be desorbed. Tiwari and Kumar ( 1 982) conducted an experiment with rice plants in soils where the pH values ranged from 3 to 8, and they observed that Cu in the rice seedlings was negatively correlated with soil pH (r= -0. 83, p < 0.0 1 ). It was suggested that the decrease in Cu uptake with increasing pH is related to the formation of insoluble inorganic Cu compounds, especially carbonates and hydroxides. Adsorption measurements in Chapter 3 (Section 3 .3 .3 . 5 ) showed similar trends. A positive correlation between Cu uptake and soil pH (r=0.44), a positive correlation between Cu uptake and Olsen P (r=0.93), and negative correlations between Cu uptake and organic carbon (r= -0.20) and clay content (r= -0.42) were obtained for harvest 1 (Table 4.8). Significant positive correlations existed between Cu uptake and Olsen P at each harvest. Increasing levels of P increased the Cu uptake (Section 7.4.3 ; Figure 7 .2c). P status (Olsen P) of the soil is directly related to dry matter yield, causing a lift in Cu uptake. There was a good correlation between the time after fertiliser application and Cu uptake in different soils. The linear model equations relating negative Cu uptake with time after fertiliser application explained 95%, 9 1 %, 82%, 88% and 87% of the variation in Cu uptake in the Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu soils, respectively. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 4 300 -+- Manawatu ---+- Tokomaru 250 -e- Ramiha -. "<'""" --.- Ngamoka I - 0 200 � a. Mangamahu Cl E - Q) 1 50 � cu - a. ::::I '- Q) 1 00 a. a. 0 U 50 0 50 75 1 00 1 25 1 50 1 75 200 Days following ferti lizer addition Figure 4.3 Effect of soil types on Cu uptake after fertiliser addition. Data are means ± SE, n=48. Table 4.8 Correlations of copper uptake with soil properties. Soil properties Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 Olsen P 0.93** 0.76** 0.62** 0.25* 0.23* pH 0.44** 0.28* 0. 1 3 0.00 1 0.002 Organic carbon -0.20* 0.03 -0.28* 0.07 -0. 1 9* Silt 0.27* 0.29* 0. 1 7 0.03 0 .009 Clay -0.42** -0. 1 8* -0.36** 0.06 0. 1 2 ** and * denote slgruficance at the 99% and 95% level, respectIvely. 4.3.4.2 Effect of types ofCufertiliser Figure 4.4 shows a general trend of decreasing Cu uptake of all fertilisers from 96 days after fertiliser addition. There was a greater decline in Cu uptake between harvests 1 and 2 for the CUS04 than for the other two fertilisers. For each type of fertiliser, increasing the time of contact between the soil and Cu source led to a decline in the Cu uptake in all soils. The r values relating Cu uptake and time after ferti liser application are 0.94, 0.90 and 0 .87 for CUS04, Cu(OHh, and CuO, respectively. The reasons for this decline Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 5 may be related to the changes in the distribution of Cu in the soil (See chapter 5), and to the decrease in dry weight of the shoots during each growth period. On average, Cu(OHh and CuD showed the highest and the lowest Cu uptake, at each harvest. This low response to CuO is mainly related to the slow release characteristics and low solubility of this source. 200 1 50 1 00 50 o I + • CuS04 Cu(OH)2 CuO 50 1 00 1 50 200 Days fol lowing fertil izer addition Figure 4.4 Effect of sources of Cu on Cu uptake following fertiliser addition. Data are means ± SE, n=80. 4.3.4.3 Effect of fertiliser rates Increasing levels of fertiliser up to 1 00 mg Cu kg- I increased the Cu uptake at all harvests (Table 4 .9) . Cu uptake was the highest at the 1 00 mg Cu kg-I level for both CUS04 and Cu(OHh but not for CuO. Gilkes and Lim-Nunez ( 1 979) observed that increasing levels of Cu promoted Cu uptake in wheat. A Cu level of 200 mg Cu kg- l with CUS04 fertiliser showed a greater decrease in Cu uptake at each harvest, it might be due to a low growth rate of ryegrass at this level . Minnich et al. ( 1987) conducted a glasshouse trial in a Mardin silt loam amended with either CUS04 (75-300 mg Cu kil ) or sludge ( 1 20 g kg-I ) . They found a good correlation between soil solution Cu2+ activity and Cu accumulation in young snapbean plants. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 6 Table 4.9 Effect of fertilisers and treatment levels on Cu uptake by ryegrass. Fertilisers Treatments Harvest 1 Harvest 2 Harvest 3 Harvest 4 Harvest 5 Control Control 75 . 1 c 94.3d 43 .0e 20.7c 1 3 .7d CUS04 50 1 36.8b 147.9c 74 .9d 33 .8b 1 6.2dc 1 00 254.7a 1 99.3a 1 1 3 .8a 32.0b 45 .2ab 200 1 26. 1 b 94.7d 79.6d 28 . 1 bc 3 1 . 1 bc Cu(OHh 50 143 . 1 b 1 72 . 1 abc 82.6dc 34 .6b 23 .7dc 1 00 233.9a 193 .7ab 1 06.8ab 44.3a 27 .4dc 200 1 62.3b 1 73.2abc 1 03 .0ab 46.9a 57.9a CuO 50 1 23 .8b 1 43 .8c 77.9d 28.3bc 1 7.8dc 1 00 1 40.5b 1 65 .6bc 96.3bc 34. 1b 26.4dc 200 1 7Ub 147.5c 85 .8dc 27. lbc 50.6a * Treatment means followed by the same letter Wlthm a column are not slgmficantly different at the 5% level. 4.3.5 Recovery of Cu fertilisers The apparent percentage recovery of applied Cu fertiliser (Ri) was calculated for each fertiliser Cu source: Ri = (Ct - Cc) x 1 00 Ca (Eq 4. 1 ) Where Ct i s the cumulative shoot Cu uptake from a Cu fertiliser treatment with different fonus of Cu; Cc is the cumulative shoot eu uptake from a nil eu fertiliser; Ca is the rate of Cu applied per pot. The results indicated the apparent average recovery of Cu is very low, ranging from 0 .26 to 1 .23 for CUS04, from 0 .44 to 1 .36 for Cu(OHh and from 0.48 to 0.97 for CuO at different eu levels in harvest 1 . The apparent recovery of Cu at the 5 0 mg eu kg-! rate was higher than the 200 mg Cu kg-l rate of fertiliser application (Figure 4.5). This might be related to the higher DM yield of ryegrass at 50 mg Cu kg- ! level. The CU(OH)2 fertiliser showed a higher recovery at 50 and 200 mg Cu kg-! rates than the other two fertilisers. At 1 00 mg eu kiJ soil level CUS04 showed the highest recovery of eu. The CuO fertiliser resulted in the lowest recovery rate, except at the 200 mg Cu kil soil level, which is due to the lower rate of dissolution. All fertilisers showed a decline in the rate of recovery with time after fertiliser application. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 1 1 7 5 5 5 --+-- CuS04 (a) (b) (c) � -+ -+- Cu(OH)2 oR 4 4 4 e.... � -e- CuO Q) 8 3 3 3 l!! :::l () (I.) 2 2 2 > r ? .� ::; E :::l () 0 0 0 I 0 50 1 00 1 50 200 0 50 1 00 1 50 200 0 50 1 00 1 50 200 Days following sowing Figure 4.5 Cumulative recovery of Cu at (a) 50 mg Cu kg-l soil, (b) 1 00 mg Cu kg-l soil and (c) 200 mg Cu kg-l soil of different Cu fertilisers. The concentration of Cu in plant shoots, as well as absorption by roots, is influenced by soil properties. The transport of Cu from root to shoot, specially in Gramineae is restricted (Jarvis, 1 978). This is an important phenomenon resulting in large proportions of the absorbed Cu being immobilised in roots even when plants are suffering from acute Cu deficiency (Jarvis and Robson, 1 982). Such restricted transport may be partially responsible for the low recovery in the shoots of Cu added to soils as fertilisers (Gilkes and Sadleir, 1 978). When rye grass was grown under controlled environmental conditions in 2 1 soils with wide ranging properties, from 49 to 79 percent of the total Cu absorbed was retained in the roots at the last of six harvests of the shoots (Jarvis and Whitehead, 1 98 1 ) . Variation of Cu concentration in the shoots of plants grown in a particular soil between harvests was almost as great as that between plants grown in all soils (Jarvis and Whitehead, 1 98 1 ) . A similar variation of Cu concentration at various harvests was observed in this study. Other studies (Chapter 7) show that the change in Cu concentration has been related to the supply of N or P from the soil. The soil pH and organic matter influenced the variation in Cu concentration in plants with time after fertiliser application, but the maj or variation in Cu concentration in shoots could often be accounted for by the variation o f plant growth and the N and P content of the soil. Chapter 4 Plant availability of copper from different copper fertilisers in pasture soils 4.4 CONCLUSION AND FURTHER STUDY 1 1 8 • Soils have a significant effect on DM yield and on Cu concentration at all harvests. • High Cu level (200 mg Cu kg- ! soil) caused a significant decrease in dry matter yield, but at levels of 0, 50 and 1 00 mg Cu kg-l soil produced similar yields. • Increasing levels of Cu increased the Cu concentration at all harvests. • Source of Cu in fertilisers has a significant negative effect on DM yield and a positive effect on Cu concentration at all harvests. • Copper sulphate showed significantly lower DM yields and significantly higher Cu concentration than the other two fertilisers at harvest 1 . • Copper hydroxide showed significantly higher DM yields, lower Cu concentration and higher Cu uptake than the other two fertilisers. • Except for harvests I and 2, copper oxide resulted m significantly higher Cu concentration at all other harvests. • Copper concentration and uptake are correlated with soil pH, organic matter and clay content of the soil. • Copper uptake in ryegrass decreased with time from the first harvest. • Copper uptake is directly related to DM yield of ryegrass . • Recovery of eu was highest at lower levels of fertiliser applied. • Copper hydroxide demonstrated the highest recovery of Cu than other two fertilisers Relationships between soil properties and plant Cu concentration will be difficult to formulate until more is known about the fraction of Cu in the soil solution and the extent to which they may be utilised by plants. The results of transformation of Cu and its relationship with soil properties and plant uptake are presented in chapter 5 . Chapter 5 CHAPTER 5 Transfonnation and plant uptake of copper in soils TRANSFORMATION AND PLANT UPTAKE OF COPPER IN SOILS 5.1 INTRODUCTION 1 1 9 It has been demonstrated in the previous glasshouse trial (Chapter 4), using different sources of Cu as fertilisers, that the availability of eu to plants declines with increasing time of contact between the soil and eu fertilisers. The rate of decline in the availability of Cu depends on the soil organic matter, iron and aluminium oxides, which control the availability of eu in soils. A similar decline in the availability was also reported from a long term eu fertiliser field experiment (Brennan et al., 1 986). This observed decline in eu availability has been interpreted as resulting from slow reactions, which convert a proportion of applied eu into forms with lower availability. Knowledge of the eu distribution in different fractions and the relative availability of these fractions for plant use is fundamental to an understanding of the transformation of eu in soil. The distribution of nutrient forms in soils is not always experimentally clear, fractionation provides insight into their forms and their availability. The proportions of the different eu fractions in soils vary considerably, depending on the soil characteristics and the fractionation technique used. The contribution of various soil eu fractions to plant available Cu in different soils i s not well understood. In this chapter a chemical fractionation scheme have used to examine the different forms of eu present in soils obtained from the previous glasshouse (Chapter 4) experiment. Objectives: The main objectives are as follows: • To determine the transformation of eu fertilisers with time, which might account for the changes in eu availability to plants. • To evaluate the plant availability of the various fractions of eu. In this chapter the distribution of native eu and eu added through fertiliser addition is discussed. The effect of eu sources on the distribution of Cu fractions is also discussed. Chapter 5 Transformation and plant uptake of copper in soils 5.2 MATERIALS AND METHODS 5.2.1 Soil samplingfrom pots 1 20 The soil samples were collected from the previous glasshouse trial pots by using a mini­ corer at 0, 8, 28, 1 06 1 63 and 1 9 1 days intervals. The treatments for the glasshouse trial included three Cu sources [CUS04, Cu(OHh and CuO], five soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu) and four Cu levels (0, 50, 1 00 and 200 mg kg" I soil). The soil samples were air dried, ground and passed through 2 .00 mm sieve. The soil was analysed for total Cu and various fractions of Cu that included exchangeable Cu, organically bound Cu, oxide bound Cu and residual Cu (Table 3 .2). 5.2.2 0.005 M EDTA-extractable Cu Soil samples ( 1 0 g) in triplicate were extracted for 1 6 hrs on an end-over-end shaker with 25 ml 0 .005 M EDTA (disodium salt of ethylene diaminetetraacetic acid) in 0 .01 M Ca(N03h. The samples were then centrifuged at 77 1 9 g for 10 minutes and filtered through Whatman No. 42. The Cu concentration in the filtrate was measured by flame atomic absorption spectrophotometry (F-AAS). 5.2.3 Total soil Cu Soil samples ( l .Og) in triplicate were used for the determination of total Cu. The original soil samples and residual fractions remaining after the removal of oxide bound Cu (step C ; Table 5 . l ) were used to determine total Cu by wet digestion with a mixture of HN03, HCI04 and HCl at the ratio (by volume) of 5 : 5 : 7 (Bolan and Hedley, 1 987). The Cu in the filtrate was measured by F-AAS. 5.2.4 Fractionation of soil Cu Fractionation of soil Cu was carried out according to the sequential extraction method described by McLaren and Ritchie ( 1 993). Hydrochloric acid was used instead of hydrofluoric acid on the residual fraction. Details of the methods are described in Section 3 .2 .6 . Chapter 5 Transformation and plant uptake of copper in soils 5.2.5 Total Cu o/particle size/ractions 1 2 1 The particle size fractionation of the bulk soil was carried out as described in Section 3 .2 .2 . The individual fractions (sand, silt and clay) were digested by wet digestion with a mixture of RN03, HCI04 and HCl at the ratio (by volume) of 5 :5 :7 (Bolan and Hedley, 1 987) . The Cu in the filtrate was measured by F-AAS. 5.2.6 Chemical analysis Copper in the soil extracts was analysed usmg flame atomic absorption spectrophotometry (F-AAS). Details of this technique and other precautions are discussed in section 4.2 .6 . 5.2. 7 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS). As discussed in section 4.2 .7 . 5 .3 RESULTS AND DISCUSSION 5.3.1 Recovery o/Cu by /ractionation 5.3.1 .1 Recovery 0/ native eu To examine the robustness of the fractionation procedure, the sum of the individual fractions was plotted against the total soil Cu, as measured by tri acid digestion (Figure 5 . 1 ). Five soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu) were used for the measurement of total Cu and the various fractions of Cu. Results show a satisfactory agreement (r2=0.98) between the total soil Cu values and the sum of the Cu extracted from the individual fractions. The sum of the individual fractions resulted in the recovery of more than 80% total eu. The soil with a high amount of total Cu (>30 mg kg- l ) resulted in a lesser recovery of Cu in the individual fractions. This may be due to losses during each step of the extraction procedure and / or errors, which occurred during the measurement of the high concentrations of Cu in the F-AAS procedure. Chapter 5 40 30 (/) § 20 U ro � ::l () '0 E ::l Cl) 10 o o _.- -- --�--------- / Transformation and plant uptake of copper in soils 1 : 1 line / / / 1 0 20 30 / Total Cu concentration (mg kg-1 ) 40 1 22 Figure 5. 1 Comparison of total native Cu with the sum of individual fractions for each soil. Data are means ± SE, n=3. 5.3. 1.2 Recovery of applied eu during fractionation The soils treated with Cu from the glasshouse trial (Chapter 4) were used for this fractionation study. Soil samples were collected at 0, 8, 28, 1 06 1 63 and 1 9 1 day intervals after fertiliser application. The measured independent total eu versus sum of Cu recovered from the different fractions is presented in Figure 5 .2 . A good relationship (r2=0.99) was obtained between the measured independent Cu values and the sum ofCu extracted from the individual fractions. The percentage of total Cu extracted by the individual fractions was more than 80% (Figure 5 .2). Chapter 5 Transfonnation and plant uptake of copper in soils 1 23 250 1 : 1 l ine / . / 225 • • / • • � 200 • . / • .....-.. •• / """" • I 1 75 / . Cl � / .... Cl E / ......, 1 50 ::J / () • / "- 0 1 25 .. . JIv (f) • c: -4 • 0 :;:: / '-' 1 00 / . co .... ... • '+- �. '+- 0 � � E 75 • / .. ::J Cl) '" .. .-\ 50 /. / / 25 /. 0 0 25 50 75 1 00 1 25 1 50 1 75 200 225 250 Measured independent total Cu (mg kg-i ) Figure 5.2 Comparison of total applied Cu determined by tri-acid extraction with the sum of individual fractions 28 days after application. 5.3.2 Distribution of native Cu 5.3.2.1 Fractionation of control soil The fractionation data for the native Cu, 0.005 M EDTA extractable Cu and the total eu in the whole soil are presented in Table 5 . 1 . The Manawatu and the Tokomaru soils contained the highest and the lowest concentrations of total native eu, respectively. The Manawatu and the Mangamahu soils contained the highest and the lowest amounts of 0.005M EDTA extractable Cu, respectively. I observed that the native Cu was present mainly in the residual (43 .2%), organic bound (28 .5%) and oxide bound (26.0%) fractions (Table 5 . 1 ). Only a small proportion of the total eu was present in the exchangeable fraction (2.2%). Chapter 5 Transformation and plant uptake of copper in soils 124 Table 5.1 Fractions of native Cll (mg kg-I) in the whole soil. Soils name Exchangeable Organic Oxide Residual Sum of Total 0.005 M total EDTA Manawatu 0.54 5 .07 8.44 14.2 28.25 33 .39 9.21 Tokomaru 0.3 1 3 .02 2 .98 5 .68 1 1 .99 1 4.86 1 .64 Ramiha 0.5 1 7.86 4.97 8 .41 2 1 .75 26.75 2.28 Ngamoka 0.33 6.52 5 . 3 1 6.85 1 9.0 1 23.38 1 .38 Mangamahu 0.47 5.27 3 .68 6.94 16.36 1 7.69 1 . 1 2 5.3.2.2 Total Cu in particle size fractions The total eu concentrations in the whole soil and in the particle size fractions of the soils are presented in Table 5 .2 . Sands contributed the lowest amount of eu in all soils compared to the silt and clay fractions. Most soils contained higher amounts of eu in the clay fractions. Exception being the Manawatu and Ramiha soils. Silt contains the highest concentration of eu in the Manawatu and Ramiha soils. Silt and clay contributed most of the eu in these five soils. The humus-clay complexes have large specific surface areas and surface charges to which eu is attracted. The higher contents of eu in the fine particle fractions compared to those of the bulk soil were due to the larger specific area and surface charge of the fine particles. Shuman ( 1 985) reported that eu was found mainly in the silt, clay and crystalline Fe oxide fractions of 16 acid topsoils. According to the sum of eu concentration in the partic le size fractions, the Manawatu and the Tokomaru soils resulted in the highest and the lowest amounts of total eu, respectively. The trend in total eu concentration in soils is similar to the sequential fractionation of native eu (Section 5 .3 .2 . 1 ) . Stevenson and Fitch ( 1 98 1 ) concluded that organic colloids and clays play a major role in eu retention by soils. In most mineral soils, eu may be bound as clay-metal-organic complexes, since in these soils, organic matter is intimately bound to clay. Chapter 5 Transformation and plant uptake of copper in soils 1 25 Table 5.2 Concentration of total native Cu in whole soil and particle size fractions. Soils name Particle size % Particle fractions size Manawatu Sand 20.34 Silt 60.64 Clay 17 .82 Whole soil 98.80 Tokornaru Sand 19.72 Silt 57.45 Clay 2 1 .02 Whole soil 98. 1 9 Ramiha Sand 1 2.93 Silt 59.67 Clay 25.62 Whole soil 98.22 Ngamoka Sand 19.68 Silt 54.83 Clay 25.57 Whole soil 1 00.0 Mangarnahu Sand 20.37 Silt 49.7 Clay 26.23 Whole soil 96.30 5.3.3 Distribution of applied Cu Copper Copper content Contribution concentration (mg) in different particle size (%) (m�kg- l ) particle size 3 1 .58 6.42 1 3 .20 56.99 34.56 7 1 .03 43.04 7.67 1 5 .76 48 .65 8.38 1 .65 8.79 14. 1 1 8. 1 1 43 . 1 0 43.04 9.05 48. 1 1 1 8. 8 1 1 6.7 2 . 1 6 6.58 29.87 1 7.82 54.35 50.01 12 . 8 1 39.07 32.80 49.7 9.78 23.05 27.42 1 5 .03 35 .43 68.9 1 7.62 4 1 .52 42.43 1 6.4 3 .34 9.70 24.9 1 2.38 35 .94 7 1 .36 1 8.72 54.36 34.43 Copper applied to soils reacts with inorganic and organic compounds within the soils (McBride 1 98 1 ; J ames and Barrow, 1 98 1 ) . The effect of these reactions on the availability of eu to plants is not well understood. The effectiveness of a eu fertiliser applied for promoting growth and supplying eu to plants is strongly affected by the period of contact between the soil and the eu fertiliser (Brennan et al., 1 980). The mean concentrations of individual fractions in the samples from the pots at each application rate are described in the following sections. The mean recoveries of the applied eu from the various fractions showed that the oxide and organic fractions are the major fractions holding eu. 5.3.3. 1 Exchangeable Cu The Tokomaru and the Ramiha soils contained the highest and the lowest concentrations of exchangeable eu, respectively, at the various sampling times and were significantly different from the other soils. This difference in exchangeable eu is related to the variation in soil organic matter among the soils. The Tokomaru and the Ramiha soils of Chapter 5 Transformation and plant uptake of copper in soils 1 26 contained the lowest and the highest organic matter, respectively. The exchangeable Cu increased from fertiliser application till 1 06 days after fertil iser addition and then decreased gradually in the Ramiha and the Ngamoka soils (Figure 5 .3). Generally most incubation studies showed that the concentration of exchangeable Cu in soils decreased with increasing time after fertiliser application. But in this study, the large increases in exchangeable Cu at 1 06 days might be attributed to high temperature (� 30 °C) in the glasshouse, root development and higher microbial activity in soil. McGrath et al. ( 1 988) examined the effect of soil organic matter on the concentrations of Mn, Zn and Cu in soil solution and their extractability from soil . They observed that O.O IM CaCh extractable exchangeable Cu was the smallest fraction, and it decreased with increasing levels of soil organic matter. 1 5 -+- Manawatu -+- Tokomaru 1 3 --e-- Ramiha ..--.. ----At.:- Ngamoka T""" I 0) -17- Mangamahu .::.:: 1 0 0) E .......- c .2 8 ro � - c Q) (.) c 0 5 (.) :J 0 3 o o 25 50 75 1 00 1 25 1 50 1 75 200 Days following fertil izer addition Figure 5.3 Effect of sampling periods on exchangeable Cu concentration (mg kg-I). Data are means ± SE, n=12. Fertiliser Cu sources have a significant effect on exchangeable Cu measured for 0, 8, 28 and 1 63 days soil samplings. CUS04 fertiliser resulted in the highest exchangeable Cu at the beginning of the experiment due to higher solubility characteristics. CuO fertiliser resulted in the highest concentration of exchangeable Cu for soil samples collected at 1 06, 1 63 and 19 1 days after fertiliser application. The Cu(OH)z resulted in the lowest Chapter 5 Transformation and plant uptake of copper in soils 1 27 concentration of exchangeable Cu in soil samples collected at all sampling times. This is due to the lower solubility ofCu(OH)2 . The results presented in Figure 5 .4 show that the amount of exchangeable Cu for CUS04, Cu(OHh and CuO fertilisers were 8 . 1 , 6 . 8 and 8 .8 mg kg- I , respectively and constituted 5.4, 6 . 1 and 5 .3% of total Cu in soils at 1 06 days after fertiliser application. 1 2 1 0 --- ...... I 0) 8 � 0) E '-" c 0 :;::; 6 C\l 10- "E Q) CJ C 0 4 CJ ;:) u 2 o o I + • CuS04 Cu(OH)2 CuO 33 67 1 00 1 33 1 67 Days following fertilizer appl ication 200 Figure 5.4 Effect of fertilisers on exchangeable Cu concentration (mg kg-I ) at various sampling periods. Data are means ± SE, n=20. The treatment levels had significant effect on exchangeable Cu. High Cu levels (200 mg eu kg- 1 soil) caused a significant increase in the exchangeable Cu (Figure 5 . 5) . At 1 06 days, the exchangeable Cu increased sharply due to high temperatures (� 30 QC) in the glasshouse. The amount of exchangeable Cu for 50, 1 00, and 200 mg Cu kil soil levels were 2 .6, 7 .8 and 1 3 . 3 mg kg-I , respectively, and constituted 3 .6, 5 .9 and 6.3% of total Cu in soils at 1 06 day after fertiliser application. Reddy et al. ( 1 9 8 1 ) observed that the concentration of CaClz extractable Cu increased as soil temperature increased from 1 2 to 22 Qc. Williams and McLaren ( 1 982) reported that both extractable Cu and soluble organic matter increased when the temperature was raised from 1 0 to 30 Qc. They suggested that this extra Cu originated from the organic matter. Chapter 5 .......... '<"" � Cl -S c o � L.-e (]) (.) c o (.) ::l () 20 1 7 1 3 1 0 7 3 Transformation and plant uptake of copper in soils --+- Control -+- 50 mg kg-1 soil -e- 1 00 mg kg-1 soil -.- 200 mg kg-1 soil (]) :0 ro (]) Cl c ro .c: (.) x ill � � o � • • • � I I I I o 33 67 1 00 1 33 1 67 200 Days following ferti lizer application 1 28 Figure 5.5 Effect of treatment levels on exchangeable Cu concentration (mg kg-I) at various sampling periods. Data are means ± SE, n=15. 5.3.3.2 Organic bound Cu The Ramiha and the Tokomaru soils contained the highest and the lowest concentration of the organically bound Cu fractions, respectively. The organically bound fraction Cu decreased with time after fertiliser application (Table 5 .3) . The organically bound eu fraction was often found to be higher in organic soils compared with mineral soils. Since Cu is mostly associated with the organic fraction. It is therefore important that Cu complexing by organic ligands and subsequent release by microbes should be emphasised in chemical fractionation studies (Sims, 1 988). Luo and Christie ( 1 996) observed that the addition of Cu to soils significantly increased eu in the adsorbed fractions and decreased it in the organic fractions. The Ramiha and Ngamoka soils are rich in organic matter compared to the other soils. eu added to organic soils, or added in sewage sludge or manures will revert to the more insoluble and plant unavailable fractions with time. Cu that is added to peat soils changed to more and more non­ reactive forms, causing less replacement of weakly held metals (Mathur and U�vesque, 1 988) . Cu has a strong ability to bind to organic matter. Chang et at. ( 1 984) observed that in untreated soil, eu was highest in the sulphide residual fraction, but with sewage sludge addition, the eu level in the carbonate and organic fractions increased more than Chapter 5 Transfonnation and plant uptake of copper in soils 1 29 In the other fractions. Liang et al. ( 1 99 1 ) observed that most of the added Cu accumulated in forms strongly bound to sesquioxides, organic matter and clay minerals in twenty-seven Saskatchewan soils. McLaren and Ritchie ( 1 993) reported that organically bound and iron oxide bound Cu fractions accounted for most of the applied Cu, irrespective of the rate of application. Table 5.3 Effect of soil types on organically bound Cu concentration (mg kg-I) at various sampling periods. Soils o days 8 days 28 days 1 06 days 1 63 days I 9 1 days M anawatu 52.49bc 85 .06b 76.57a 50. 1 8b 1 2 .88b 6.05b Tokomaru 40.26c 64.92c 5 1 .6 1 b 20.58c 1 9.46b 9.22b Ramiha 77.99a 1 06.7a 96. 76a 8 1 . 1 3a 66.03a 55 . I l a Ngamoka 67.3ab 85 .66b 95 .96a 74.05ab 66. 1 6a 44. 1 2a Mangamahu 6 1 .59b 8 1 . 52bc 88 .2 1 a 53 .6 1 ab 48.42a 22.70a * Treatment means followed by the same letter WIthin a column are not sIgmficantly dIfferent at the 5% level. Fertiliser caused a significant effect on the organically bound Cu fraction in soil samples collected at different times after fertiliser application (Table 5 .4). All the fertilisers increased the organically bound Cu up to 8 days except CU(OH)2, then decreased with time after fertiliser application. The results presented in Table 5 .4 show that the Cu concentration in the organic fraction for CUS04, CU(OH)2 and CuO fertilisers were 58 .7, 42.7 and 66.2 mg kg- I , respectively, and constituted 39.4%, 38 .0% and 40. 1 % of the total Cu in the soils, at 1 06 days after fertiliser application. Stevenson and Fitch ( 1 98 1 ) reported that, based on a commonly used soil Cu fractionation scheme, organically bound Cu accounts for 20% to 50% of the total soil Cu. Luo and Christie ( 1 996) observed that the added Cu was present mainly in the adsorbed state (45%), iron­ manganese oxide bound (35%), organic matter bound ( 1 5%) and residual fraction ( 1 0%). Chapter 5 Transformation and plant uptake of copper in soils 1 30 Table 5.4 Effect of fertilisers on organically bound eu concentration (mg kg-1) at various sampling periods. Fertilisers o days 8 days 28 days 1 06 days 1 63 days 1 9 1 days CUS04 62.05a 84.28b 73 .66b 5 8.77ab 38 .01 25 .66 CU(OH)2 47 .28b 64.85c 76.00b 42. 76b 39.76 28. 1 0 CuO 70.47a 1 05 . 1 9a 95 .80a 66. 1 9a 43 .85 34. 7 1 * Treatment means followed by the same letter WIthin a column are not sIgruficantly dIfferent at the 5% level. Increasing levels of Cu increased the eu concentration in the organic fraction of all the soils. At the highest application rate, the added Cu appeared to be distributed evenly between the organic matter and oxide fractions (Figure 5 .6). Ma and Uren ( 1 998) reported that the proportions of recently added Cu in extractable fractions decreased markedly with time. Miller et al. ( 1 987) observed that when Cu-enriched manure was added to soils the Cu initially existed in specifically adsorbed fonus, but after 1 2 days, some was found in the Mn and amorphous Fe oxides, indicating a shift away from plant availability. Schalscha et al. ( 1 999) reported that added Cu increased exchangeable Cu in soils only slightly, however, significant increases in the carbonate, reducible (associated with oxide fonu) and oxidisable (bound to organic matter) fractions were observed. Chapter 5 Transformation and plant uptake of copper in soils 1 3 1 1 50 I -+- 50 mg kg-1 I - & -e- 1 00 mg kg-1 1/ 1 25 " --.--- " 200 mg kg-1 � I " 0) .:::.::. " 0) E 1 00 " .......- " c 0 " :.;:::::; � co .... 75 ...... c Q) u c 0 u 50 - 11-.... " Q) 0.. 0- -0 - � -0 "- 25 �- o I o 25 50 75 1 00 1 25 1 50 1 75 200 Days following fertilizer application Figure 5.6 Effect of treatment levels on organic bound Cu (- - - -) and oxide bound Cu (--) concentration (mg kg-I) at various sampling periods. Data are means ± SE, n=15. 5.3.3.3 Oxide bound eu The Ramiha and the Tokomaru soils contained the highest and the lowest concentrations of the oxide bound eu fraction, respectively (Table 5 .5) . In most of the soils, oxide bound eu initially increased and then decreased with time after fertiliser application. The observed changes with time in the distribution of applied Cu between fractions are thus most probably a result of a redistribution of eu from organic sites to sites associated with strongly crystalline iron oxide materials. The organically bound and the oxide bound Cu fraction values were often related to soil organic matter content. An increase in the organic eu and a decrease in the oxide fraction with organic matter additions have been observed, indicating a possible increase in bioavailability (McGrath et aI., 1 988). McLaren et al. ( 1 983b) were able to induce some redistribution of eu between components of a humic acid/soil oxide! montmorillonite system in a laboratory study. The impetus for redistribution of eu is most likely to be the slow movement of eu into solid oxide materials. Such movement has been observed by several researchers, and is known to exhibit extremely limited reversibility (Swift and McLaren, 1 99 1 ). Chapter 5 Transformation and plant uptake of copper in soils 1 32 Such movement is due to diffusion into lattice structures, or by penetration of extremely small pores and inter-particle spaces. The net result i s a reduction in the concentration of Cu adsorbed at the surface of the oxide. This, in turn, would lead to the establishment of new equilibria between the soil solution and surface adsorbed forms of Cu including the Cu adsorbed by soil organic matter (McBride, 1 99 1 ). Increases in the oxidation of organic matter generally resulted in the redistribution of Cu from the exchangeable and organic fractions into the Fe oxide fractions, reducing plant availability (Sims and Patrick, 1 978) . eu concentration is usually high in the carbonate fraction of alkaline soils and in the Fe-oxide fraction of acid soils, probably due to occlusion and strong adsorption (Sposito et al., 1 982; Kuo et al. , 1 983 ; S ims, 1 986). Table 5.5 Effect of soil types on oxide bound Cu concentration (mg kg'I ) at various sampling periods. Soils o days 8 days 28 days 1 06 days 1 63 days 1 9 1 days Manawatu 64. 1 9ab 94.78ab 86. 14a 78 .82a 54.9 1bc 35 .S9b Tokomaru 54.62b 75 .68c 57 .2b 43 .67b 46.52c 1 8 .03b Ramiha 76.22a 99.4 1 a 94.2a 85 .9 1 a 70.8ab 59.63a Ngamoka 70.86a 85 .5 1 abc 97.99a 86.46a 80.57a 58.77a Mangamahu 63 .97ab 80.02bc 88 .35a 63 .42ab 60.87bc 37 .01b *Treatment means followed b y the same letter within a c olumn are not s ignificantly different at the 5% level. Fertiliser Cu sources have a significant effect on the oxide bound fraction of Cu. CuO resulted in the highest oxide bound Cu fraction of all the various time samples (Figure 5 . 7) . The amounts of oxide bound Cu for CUS04, CU(OH)2 and CuO fertilisers were 75.7, 56 .3 and 82 .9 mg kg' 1 , respectively and constituted 50.7%, 50. 1 % and 50.3% of total Cu in the soils, respectively, at 1 06 days after fertiliser application. This indicated that the majority of total Cu is held within the oxide mineral structure. Chapter 5 ,........ ...... I 0) .:::s:. 0) 1 00 E c o :;::::; � .- c ro .)::: 50 :::> () 0 E 25 :::> Cl) 0 0 50 1 00 1 50 200 300 'I (b) I T (C) I 1 50 200 1 00 1 00 50 200 0 50 1 00 1 50 200 0 Days following the fertiliser application 50 1 00 1 50 200 Figure 5 . 1 1 Sum of fractions of Cu (mg Cu kg-I) at different levels (a) 50 mg Cu kg­ I , (b) 1 00 mg Cu kg-I , (c) 200 mg Cu kg-! following the fertiliser application. [(+Manawatu; +Tokomaru; eRamiha; A.Ngamoka; and TMangamahu)] . Data are means ± SE, n=3. 5.3.5 Effect of soil properties on fractionation The mean concentrations of soil eu present in the different fractions decreased in the order: oxide bound > organic > residual > exchangeable. Limura ( 1 993) examined the distribution of eu in non-contaminated and contaminated paddy soils irrigated with the river water from mines. He reported an accumulation of eu in fractions, which reacted with the soil solution ( i .e . exchangeable, inorganically and organically bound fractions). Mullins et al. ( 1 982a) observed that most of the added Cu was accumulated as organically bound and sesquioxide occluded eu fractions. In most of the soils in the Chapter 5 Transformation and plant uptake of copper in soils l 38 present study, exchangeable Cu, organically bound Cu and oxide bound Cu increased initially and then decreased with increasing time after fertiliser application. The amount of organically bound Cu is dependent on the soil organic matter and the maximum amount was obtained in soils with high organic matter levels (Ramiha and Ngamoka). A good correlation between the Cu concentration in the different fractions and soil organic matter, CEC, clay content and soil pH was obtained (Table 5 . 7) . Soil pH showed a negative relationship with organically bound Cu (r= -0.05) and residual Cu (r= -0.2 1 ) . An increase in pH has often been shown to cause a redistribution of Cu from the exchangeable and organic fractions to the Mn oxide and Fe oxide fractions (Sims and Patrick, 1 978). Thus Cu lost from the exchangeable fractions change into the less soluble and less bio-available fractions. The effects of pH on Cu transformation have been reported by McLaren and Crawford (1 973a) and Jarvis ( 1 98 1 b). These workers and our previous study (Section 3 .3 . 3 . 5 ) showed that Cu adsorption was related to soil pH, which was primarily attributed to a greater association with soil organic matter and oxides. Table 5.7 Correlations between soil properties and Cu concentration in the different fractions. Soil properties Copper fractions Exchangeable Organic Oxide Residual pH 0.07 -0.05 0.0004 -0.2 1 * Organic carbon -0.07 0.30** 0. 1 7 0.30** CEC -0. 1 1 0 .33** 0.28* 0.26* Clay -0.03 0.20* 0.07 0.27* ** and * denote slgruficance at the 99% and 95% levels, respectively. Soil organic matter shows a negative relationship with exchangeable Cu (r= -0.07) and a significant positive relationship with the organically bound Cu (r=0.30), oxide bound (r=0. 1 7) and residual Cu (r=0.30) fractions (Figure 5 . 1 2) . Since Cu is associated mostly with organic matter, it could be expected that organic matter additions would cause a redistribution of Cu among the fractions, especially to increase the organic fraction of Cu. However this has not always been found by others (Shuman, 1 988b). Elsokkary and Lag ( 1 978) reported that the organic and oxide fractions were related to soil organic matter. A decrease in both the organic and oxide bound Cu fractions found by the Chapter 5 Transfonnation and plant uptake of copper in soils 1 3 9 present study indicates a possible decrease in bio-availability of Cll. Iwasaki et al. ( 1 997) investigated the depth wise distribution of Cu in selected agricul tural soils and in a natural soil, near an abandoned Cu mine. They also observed Cll was mainly adsorbed onto organic matter and I or occluded by oxides and hydroxides of Fe and Mn. -- 1 5 ...- • 0> .:.::: 0> 1 3 E - (J') c: 1 0 0 :.;::; u 8 � '+- '0 (J') 5 .5: .... 3 Q) Cl.. Cl.. 0 u 0 2 -- 1 25 .,..... , 0> .:.::: 0> 1 00 E - (J') c: 75 0 :.;::; u m .... 50 - '0 (J') .5: ..... 25 E - Q) ..lI::: co 250 • 200 1 50 • • Q. 1 00 :::l • • .... Q) a. a. o U ---- .,-- I .-o a. 0> g Q) ..lI::: $ a. :::l l-Q) a. a. o U 50 o o • • • • • 5 (a) • 1 0 1 5 250 l R2=0.43 200 • 1 50 1 00 50 o • • ./ �. : �) I I o 1 7 33 50 67 83 1 00 Copper in soil fractions (mg kg-1 ) 250 200 1 50 1 00 50 o o 250 l 200 � 1 50 1 00 50 o o • • • • • 25 • • • • • • • • • 50 • • 75 • • ..-----.-. • •• • • 5 • • 1 0 (b) 1 00 (d) 1 5 Copper i n soil fractions (mg kg-1 ) Figure 5.1 5 Correlation of (a) soil exchangeable Cu, (b) organically bound Cu, (c) oxide bound Cu and (d) residual Cu on plant uptake. The Cu concentration and Cu uptake by ryegrass varied with the amounts of Cu in the fractions (i .e organic, oxides, total, EDTA extractable and clay). A significant relationship between EDTA-extractable Cu and plant Cu concentration (r=O.20) and Cu uptake (r=O. I S) was obtained. The sum of the fractions of Cu strongly correlated with the measured total Cu. This indicates that the distribution of Cu in the individual fractions in the soils studied was dependent on the total Cu content of the particular soil. Copper associated with oxides and organic matter is more important to Cu availability than Cu in the other fractions. The changes to applied Cu observed in this study confirmed when compared with, the results of plant Cu uptake carried out using the same soils in chapter 4. In this experiment, the availability of applied Cu for uptake by rye grass declined with time of contact between the soil and the applied Cu was Chapter 5 Transformation and plant uptake of copper in soils 1 44 observed. Brennan et al. ( 1 986) reported that the decline in plant eu uptake by wheat plants with applied eu resulted from slow reactions between eu and the soil in the organic and oxide fractions. 5.4 CONCLUSION AND FURTHER STUDY • The mean concentrations of eu present in the different fractions decreased in the order: oxide bound > organic > residual > exchangeable. • The organically bound eu fraction was highest in soils with high levels of organic matter. • The Ramiha and Tokomaru soils contained the highest and the lowest concentrations of organically bound eu, respectively. • Both the organically bound and oxide bound eu fractions decreased with time after fertiliser application. • The sum of the eu fractions declined with time indicating the formation of a tightly bound fraction not extractable with triacid digestion. • Both the organic and oxide bound eu were strongly correlated with plant eu uptake. • The availability of applied eu for uptake by ryegrass declined with time of contact between the soil and the applied eu. The most important application of the fractionation technique is to examme eu redistribution among the fractions caused by changes in soil properties. eu tends to shift from less to more soluble fractions in response to decreasing soil pH and organic matter content. Selective sequential extraction techniques for determining eu forms in soils have not been used extensively. The proportion of eu that can be extracted by soil test extractants varied with the soil matrix. Soil testing for micronutrients is, in general, a well-established practice, based on fundamental principles of soil chemistry, and verified by field and glasshouse research. The plant responses to eu are rare and occur in quite specific situations (high organic matter soils). As a result, eu soil tests have not received much attention, and extractable eu is measured by the same procedures used for other cations. An attempt was made to identify the various forms of eu extracted by different soil test reagents. This could help to identify the plant available forms of eu and to examine the efficiency of various extractants to predict the availability of eu in pasture soils; these are presented in chapter 6. Chapter 6 CHAPTER 6 6.1 INTRODUCTION Soil test to predict the availability of copper SOIL TEST TO PREDICT THE AV AILABILITY OF COPPER 1 45 The use of single chemical extractions in routine soil analysis can be a fast and simple way to evaluate the plant availability of soil nutrients to plants. These have been used, with varying degrees of success, for diagnosing micronutrient deficiency and toxicity in soils. Some of the common extractants used for cations include: Mehlich- l , Mehlich-3, O. lM HCI, 0 .0 1 M Ca(N03)z, TEA-DTPA, 0.02 M SrCh, 0 .01 M CaCh, 0. 1 M NaN03 and 1 M NH4N03 salt solution. The TEA-DTPA extraction method, developed by Lindsay and NorvelI ( 1 978), seems to be the most appropriate method for extraction of available Cu from soils (Shuman, 1 986; Agrawal, 1 992; S ingh et al. , 1 994) , but other multinutrient extractants such as Morgan, Mehlich- l , Mehlich-3 , and AB-DTPA and ammonium acetate (Raij , 1 994) have been preferred for use with an Inductively Coupled Plasma-Atomic Emission Spectrophotometry (ICP-AES) technique in routine analysis. Very few studies have compared the extraction of Cu from soils by soil test extractants, with the actual absorption of Cu by the plants. The Mehlich- l (M1 ; 0.05 N HCI and 0.025 N H2S04) method was employed to measure soil micronutrients at a number of Soil Testing Laboratories in the United States (Hanlon and DeVore, 1 989). Because M l was found unsuitable for neutral and alkaline soils, the method was modified as M2 and then as M3 (0.2 M glacial acetic acid, 0.25 M NH4N03, 0.0 1 5 M NH4F, 0.0 1 3 M HNO) and 0.001 M EDTA) so that it could be used over a wide range of soils to measure both macro and micronutrients (Mehlich, 1 984). Alva ( 1992) reported a good correlation between the concentrations of Zn, Fe, Mn and Cu extractable by neutral NH40Ac, M l , M3 and AB-DTPA in soils from citrus groves in Florida. Reed et al. ( 1 993) evaluated the Cu availability for corn using the Mehlich-3 reagent and found this procedure very promising in detecting the deficiency and toxicity levels of Cu in soils. On the other hand, Walworth et at. ( 1 992) observed that neither the DTPA nor the Mehlich-3 procedures were effective in detecting Cu availability for broccoli and potatoes cultivated in soils from Alaska. Similar results were also observed by Makarim and Cox Chapter 6 Soil test to predict the availability of copper 1 46 ( 1 983) for corn, wheat and soybeans. These studies indicate that the suitability of a chemical extractant to predict the availability of Cu depends on both the soil and the plant species. As plant available Cu in soils has not been routinely determined in the past, there is no standard soil test for Cu in New Zealand. The extraction procedures are standardised or undergoing standardisation in several European countries. 0.01 M CaCh solution has been recommended as an extractant in The Netherlands (Houba et al., 1 990), 0. 1 M NaN03 solution in Switzerland (VSBo, 1 986) and Germany (DIN, 1 995) and IM NH4N03 solution in Germany (DIN, 1 995). Correlations between the quantities o f metal extracted by these extractants and the metal absorbed by plants have been used to justify the use of these extraction methods (Lebourgh et al. , 1 996). The absorption of trace metals by plants depends on their concentration, chemical speciation, and particularly on their activity in the soil solution (Brlimmer et al., 1 986). Micronutrients in soils generally exist in several forms, including free ions in the soil solution, exchangeable, organic, precipitated and residual. Various sequential extraction procedures have been developed to estimate quantities of micro nutrients in each of these forms (Sposito et aI. , 1 983 ; Shuman, 1 985). The forms and proportion of micronutrients that can be extracted by a soil test reagent depend on the type of extractant used. Elucidation of the forms of micronutrients extracted by different soil test reagents could help to identify the p lant available form of Cu. In this study a sequential extraction procedure, described by McLaren and Ritchie ( 1 993), was used to partition Cu into the various fractions (exchangeable, organic, oxide and residual) in two soils, which varied in organic matter content. The concentration of these fractions was also measured, after removal by the three most commonly used soil test extractants (M1 , M3 and TEA-DTPA). This chapter will discuss the application of sequential extraction to measure the forms of Cu extracted by various soil tests, these tests are used to predict the plant availability of Cu in soils. Objectives: The main objectives are as follows: • To identify the forms of Cu extracted by different soil tests. Chapter 6 Soil test to predict the availability of copper 1 47 • To evaluate the efficiency of different extraction reagents for the determination of Cu availability in pasture soils. 6.2 MATERIALS AND METHODS 6.2.1 Soils and soil analysis The soil samples were collected immediately after the termination of the glasshouse experiment (Chapter 4), in which the effect of a slow (CuO) and a fast release (CUS04) Cu sources, applied at four Cu levels (0, 50, 1 00 and 200 mg kg- I ) to two contrasting soils (Manawatu and Ngamoka) on Cu uptake by ryegrass was examined. The soil samples were air dried, ground and passed through 2 mm sieve. The soil was extracted with various extractants for the measurement of Cu. 6.2.2 Single soil test extractants 6.2.2.1 Methods for M1, M3, O.lM Hel, and TEA-DTPA extractants Soil samples were extracted either by Mehlich- l (0.05 N HCl and 0 .025 N H2S04; Mehlich, 1 953), Mehlich-3 (0.2 M glacial acetic acid, 0.25 M NH4N03, 0.0 1 5 M NH4F, 0.0 1 3 M RN03 and 0.00 1 M EDTA; Mehlich, 1 984), O . l M HCl (Haynes and Swift, 1 985) or TEA-DTPA [0.005 M DTPA, O.O IM CaCb and O. l M (HOCH2CH2)3N (TEA); adjusted to pH 7.3 ; Lindsay and Norvell, 1 978] reagents. The detail methods for Mehlich- l , Mehlich-3, O. l M HCI, and DTPA extracting solution are described in Chapter 3 (Sections 3 .2 . 1 0.2-5). The methods for the other extraction reagents used in this study are presented below. For each extraction, the soil suspension was shaken for the times as shown in Table 6 . 1 , centrifuged, and the supematant solution filtered using Whatman filter papers. All extractions were done in duplicate. Chapter 6 Soil test to predict the availability of copper 1 48 Table 6.1 Comparison of methods used in determination of extractable Cu SI No Extractants Soil Volume Shaker Shaking Centrifuge Whatrnan weight rnl time time Ircf (g) No. (g) 1 Mehlich- l 5 20 End-over-end 1 0 rnin 3rnin13015 40 2 Mehlich-3 2 .5 25 End-over-end 1 0 min 3rnin13015 40 3 O.O l M HCI 8 20 End-over-end 2 hrs 3rnin1301 5 42 4 O.O l M Ca(N03h 5 35 End-aver-end 24 hrs 5rnin177 19 42 5 0.005M TEA 1 0 20 Horizontal 2 hrs 1 20 cycles 42 DTPA Irnin 6 0.02M SrCl2 3 35 Horizontal 30 rnin 120 cycles 42 Irnin 7 0.0 1 M 5 35 End-aver-end 2 hrs 5rnin1301 5 42 CaCl2 8 O. lM 5 35 End-over-end 2 hrs 5rnin1301 5 42 NaN03 9 1 M NH4N03 5 35 End-aver-end 2 hrs 5minl3015 42 6.2.2.2 0.01 M Ca (N03)2 extractant Known weights (5.0 g) of the finely ground soil were placed into the 50 ml polyethylene centrifuge tubes with 35 ml O.O IM Ca(N03)2 . The suspension was then shaken in an end-over-end shaker for 24 hrs at 20° C and then centrifuged for five minutes at 77 1 9 g in the Sorvall RC 5C automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42. 6.2.2.3 0. 01 M CaCl2 extractant Known weights (5 .0 g) of the finely ground soil were placed into the 50 rol polyethylene centrifuge tubes with 35 ml O .OIM CaCh. The suspension was then shaken in an end­ over-end shaker for 2 hrs at 20° C and then centrifuged for five minutes at 301 5 g in the Sorvall RC 5C automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42 . Chapter 6 Soil test to predict the availability of copper 1 49 6.2.2.4 0.1 M NaN03 extractant Known weights (5 . 0 g) of the finely ground soil were placed into the 50 ml polyethylene centrifuge tubes with 35 ml O. lM NaN03 . The suspension was then shaken in an end­ over-end shaker for 2 hrs at 20° C and then centrifuged for five minutes at 30 1 5 g in the Sorvall RC 5C automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42. 6.2.2. 5 1 M NH4N03 extractant Known weights (5 . 0 g) of the finely ground soil were placed into the 50 ml polyethylene centrifuge tubes with 3 5 ml IM NH4N03. The suspension was then shaken in an end­ over-end shaker for 2 hrs at 20° C and then centrifuged for five minutes at 30 1 5 g in the Sorvall RC SC automatic superspeed refrigerated centrifuge and the supematant was filtered through Whatman No. 42. 6.2.2. 6 0. 02 M SrCh extractant The SrCh extracting solution was prepared to contain 0 .02 M SrCh and 0 .05M citric acid. Three grams of air dried soil was placed in a 125 ml conical flask, and 35 ml of the SrCh extracting solution was added. Each flask was covered with stretchable Parafilm and secured upright on a horizontal shaker with stroke of 8 . 0 cm with a speed of 1 20 cycles/min. After thirty minutes of shaking, the suspensions were filtered by gravity through Whatman No. 42 filter paper. The filtrates were analysed for Cu using atomic absorption spectrophotometry with appropriate standards . 6.2.3 Fractionation of Cu Fractionation of soil Cu was carried out according to the sequential extraction method described by McLaren and Ritchie ( 1 993) . Details of the methods are described in section 3 .2 .6 . Chapter 6 - - - -- - --- Soil test to predict the availability of copper 6.2.4 Chemical form study 1 50 The fractionation of eu was measured for the original soil samples after the termination of the glasshouse study. To investigate the forms of eu extracted by the single soil test reagents, the sequential extraction scheme described earlier was used to remove the various forms of eu in four steps. First the whole soil was extracted with the different reagents (step 1 ). A sample of the original soil was extracted to remove the exchangeable Cu and part of this soil was treated with the 3-soil test extractants (step 2). A sample of the original soil was extracted to remove both exchangeable and organically bound eu. A part of this sample was extracted with the 3 soil test reagents (step 3) . A sample of the original soil was extracted to remove the exchangeable, organically bound and oxide bound Cu fractions. This soil sample was extracted with the 3 soil test reagents (step 4). The difference in eu concentration in the soil test extractants between steps 1 and 2 represents exchangeable eu, between steps 2 and 3 the organic form, between steps 3 and 4 the oxide form and step 4 represents the residual form of eu in the soil test extractants. All extractions were done in duplicate. The relationship between the concentration of the individual forms of eu extracted by the sequential extraction procedure, and that extracted by the soil test extractants was examined using the SAS program. 6.2.5 Speciation ofCu The concentrations of both the total eu and the free ionic eu were measured in the soil test extractants, and in the various chemical fractions of eu·. The difference between the total and ionic Cu was considered as complexed Cu. The total eu was measured using AAS and the ionic eu was measured by using an Orion 9629 cupric ion selective electrode (ISE). The ion selective electrode was calibrated each day with activity buffers. The electrode is known to measure free Cu2+ activity reasonably accurately in solutions of chelating organic l igands, although unstable results were observed with highly stable Cu complexes (eg. EDTA, DTPA) (Neshkova and Sheytanov, 1 985). - - - ----------------- Chapter 6 6.2. 6 Chemical analysis Soil test to predict the availability of copper 1 5 1 Copper in the soil extracts was analysed usmg flame atomic absorption Spectrophotometry (F-AAS) . Details of the other procedures are discussed in section 4.2.6. 6.2. 7 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS) . Details are discussed in section 4.2 .7 . 6.3 RESULTS AND DISCUSSION 6.3.1 Soil characteristics Both the soils are slightly acidic with the Ngamoka soil (5.9% organIc carbon) containing a two fold higher amount of organic carbon than the Manawatu soil (2.9% organic carbon). The Ngamoka soil also contained a higher amount of Olsen P ( 1 24 mg kil ) than the Manawatu soil (52 mg kg-I ) . The Manawatu soil is a recent soil, weakly weathered, alluvium, quartzo-feldspathic, mixed greywacke and argillite; the Ngamoka soil is highly weathered with a mineralogy rich in illite, vermiculite and montmorillonite, and a low « 60%) base saturation. The initial soil properties were discussed in Chapter 3 (Section 3 .3 .2). 6.3.2 Fractionation of eu The concentration of Cu in individual fractions and the measured total Cu from the soils are presented in the Table 6.2. For both soils, Cu concentration was lower in the exchangeable fraction and higher in the oxide bound fraction. The mean concentrations of eu present in the different fractions (Table 6 .2) decreased in the order: oxide bound > organic > residual > exchangeable. The present study revealed that a substantial proportion (>80%) of the Cu is present in the oxide and organic fractions and only a small amount is present in the exchangeable and residual fractions. Similar results were reported by Liang et al. ( 1 99 1 ), who observed that most of the added Cu accumulated in forms strongly bound to sesquioxides (26-54%), organic matter ( 1 0-36%) and clay - � --�-�-----------�� Chapter 6 Soil test to predict the availability of copper 1 52 minerals ( 1 5-36%) in seven Saskatchewan soils. McLaren and Ritchie ( 1 993) reported that the organically bound and iron oxide bound Cu fractions accounted for most of the applied Cu, irrespective of the rate of application. The sequential fractionation of the whole soil showed a significant difference in the exchangeable and organic fractions between the soils. Increasing levels of fertiliser addition resulted in a significant increase in the Cu concentration in all fractions. The effect was more pronounced for the exchangeable, organically bound and oxide bound Cu fractions. CuO fertiliser resulted in a higher extraction of exchangeable Cu (Table 6.2) at 295 days after fertiliser application, which was significantly different to the CUS04 fertiliser. This may be due to the slow releasing characteristic of CuO fertiliser. Chapter 6 Soil test to predict the availability of copper 1 53 Table 6.2 Effect of treatment combinations on various forms of Cu by sequential fractionation procedure at 295 days after fertiliser application. Soil Fertilisers Levels Fractions Sum ofCu Total Cu (mg Cu kg- I ) fractions (mg kg- I ) (mg kg- I) Exchangeable Organic Oxide Residual (mg kg- I) (mg kg-I ) (mg kg- ' ) (mg kg' l) Manawatu CUS04 0 0.5 5 . 1 8 . 4 1 4. 5 2 8 . 5 33.4 5 0 2 . 6 2 6 . 6 2 6.4 6 , 1 6 1 . 8 62.9 1 00 4 . 6 5 1 . 2 34.4 6.7 9 7 . 0 1 1 8 . 0 200 6 . 8 8 9 . 9 5 7 . 2 6 . 6 1 60 . 5 1 87 . 1 CuO 5 0 3 . 3 3 1 .7 3 7 . 2 6 . 1 7 8 .4 86.3 1 00 1 0. 6 5 3 , 7 4 5 .4 8 . 2 1 1 8 , 0 1 4 6 . 6 200 23 . 3 1 03 . 4 77.4 8 . 8 2 1 3 . 0 2 04 . 4 Ngamoka CUS04 0 0.3 6 . 5 5 . 4 6 . 9 1 9 . 1 23 .4 5 0 1 . 7 2 5 .4 3 0 . 6 3 . 9 6 1 . 6 76.9 1 00 2 . 3 5 1 . 6 4 5 . 1 4 . 8 1 03 . 8 1 03 . 9 2 0 0 6 . 3 73 .7 9 4 . 8 7 .6 1 8 2 . 5 1 89 . 6 CuO 5 0 2 . 3 l O A 3 8 . 1 3 . 2 5 4 . 1 60.9 1 00 4 . 4 2 8 . 0 72.4 8 . 2 1 1 3 . 0 1 07 . 5 2 0 0 1 8 . 8 7 1 .6 8 6 . 0 1 1 .9 1 88 . 4 2 1 1 . 6 Chapter 6 Soil test to predict the availability of copper 6.3.3 Soil test extractants (Ml, M3 and TEA-DTPA) 1 54 Higher amounts of Cu were extracted by the soil test extractants from the Manawatu soil than the Ngamoka soil (Table 6 .3). This may be due to the lower level of organic matter in the former soil. In pasture soils, regular application of Cu fertiliser would result in an increase in extractable Cu. Among the three soil test reagents used, M3 and TEA-DTPA extracted significantly greater amounts of Cu than M l (Table 6.3) . The quantity of Cu extracted by TEA-DTPA, M3 and M l accounted for 45%, 44%, 39% of the total Cu, respectively, as measured by tri-acid digestion. Zhu and Alva ( 1993) reported that the amount of Cu extracted by AB-DTPA, M3 and MI accounted for 38 to 61 %, 54 to 66% and 0 to 47% of the total Cu, respectively, for a range of soils. According to Sims ( 1989), Mehlich-3 extracted 50% more Cu than Mehlich- l . Garcia et al. ( 1 997) compared TEA-DTPA, AB-DTPA, EDTA and M3 in the extraction of Cu, Zn, Mn and Fe; M3 yielded the highest extraction for the four micronutrients. It is possible to infer that the acid condition of M3 could cause the dissolution of these cations from their solid phase increasing their activity in the soil solution. Thus, the formation of metal-chelates is also increased. It was also reported that M3 without EDTA extracted only 36% of the Cu removed by M3+EDTA, illustrating the importance of EDTA (Mehlich, 1 984). Results presented in Table 6 .3 indicate that TEA-DTP A extracted a higher amount of Cu than the M 1 and M3 soil test extractants. The soil with a high level of organic matter (Ngamoka) released significantly less Cu than the soil with a low level of organic matter (Manawatu) to the TEA-DTPA and M3 extractants. TEA-DTPA extracted the highest amounts of both native Cu and Cu added through fertiliser application. Increasing levels of Cu addition resulted in a significant increase in Cu concentration in all soil test extractions. CuO fertiliser accounted for a higher release of Cu than CUS04 with all soil test extractants. This may be due to the longer residual effect of this fertiliser. Chapter 6 Soil test to predict the availability of copper 1 55 Table 6.3 Copper concentration in soil test extractants. Soils Fertilisers Treatment levels Copper concentration in soil tests (mg Cu kg- I) (mg Cu kg- I) Mehlich- 1 Mehlich-3 TEA-DTPA Manawatu CUS04 0 1 7.7 20.2 2 l .5 50 20.2 29.0 26.9 1 00 46.5 53.9 59.2 200 88 .9 98.2 1 07 CuO 50 30.8 37.3 38.2 1 00 79.3 68.5 72.6 200 95 . 1 1 08 1 1 7 Ngamoka CUS04 0 4.50 1 0.4 8. 1 0 S O 27.3 39.3 42.0 1 00 59.7 45 . 1 44. 1 200 67.9 84.6 90.9 CuO SO 28.3 20.0 20.0 1 00 56.8 59.5 55.3 200 95 .7 9 1 .9 95.7 6.3.4 Extraction of Cu by soil test reagents 6.3.4.1 Effect of soil type on Cu concentration Results presented in Table 6.4 show that higher amounts of Cu were extracted from the Manawatu soil than the Ngamoka soiL The difference in the amount of Cu extracted between the soils was significant for O. IM HCI, O.02M SrCh, and O. IM NaN03 solutions. The Manawatu soil contained a lesser amount of organic matter than the Ngamoka soiL With extracting reagents of an acidic nature produced a higher amount of Cu in the Manawatu soil . As a practical test to assess the bioavailability of the metal in soils, extraction with an unbuffered electrolyte solution has been recommended (Novozamsky et aI., 1 993; Lebourgh et aI. , 1 998). M3 extracted the highest amount of Cu. The mean concentrations of Cu in the different extractants decreased in the order: Mehlich-3 > TEA-DTPA > Mehlich- l > O.02M SrCh > O . IM HCI > 1 .0M NH4N03 > O .OIMCaCh > O_ IM NaN03 > O.O IM Ca(N03)2 . Chapter 6 Soil test to predict the availability of copper 1 56 Table 6.4 Effect of soils and types of fertiliser on extractable soil Cu (mg kg-I). Extractants Soils Fertilisers Manawatu Ngamoka CUS04 CuO Mehlich- l 45 . 8 32 .5 37 .5 40.8 Mehlich-3 52.2 4 1 . 3 44.0 49.5 O. IM HCl 1 8 .3a 5 .9b 1 l . 1 1 3 . 1 O.O IM Ca(N03)2 2 .2 2.2 2 . 1 2 .3 TEA-DTPA 48.8 37. 1 4 1 .2 44.6 0 .02M SrCh 37. 1 a 2 1 .9b 27 .5 3 1 . 5 O.O IM CaCh 2.4 2 . 5 2 . 5 2.4 O. IM NaN03 2 .6a 2 . 1 b 2 .3 2 .4 1 M �N03 4. 1 3 . 9 3 . 7 4.3 * Treatment means followed by the same letter WIthin a row are not slgruficantly dIfferent at the 5% level. 6.3.4.2 Effect of types of fertiliser Copper oxide showed a higher amount of extractable Cu in all extractants except the O.O IM CaCh solutions (Table 6.4). However the difference in the concentration of extractable Cu was not significant. The extraction was carried out after the termination of the glasshouse trial (Chapter 4), the Cu was incorporated into the soil 295 days before starting this study. Higher amounts of extractable Cu at 295 days after fertiliser application reflected the low solubility characteristics of CuO fertiliser. 6.3.4.3 Effect of level of residual Cu Increasing the rate of residual Cu in the soil increased the extractable Cu in all extractants. TEA-DTPA extracted most Cu. The mean concentrations of Cu present in the different extractants followed: TEA-DTPA > Mehlich-3 > Mehlich-l > 0.02M SrCh > O . IM HCl > 1 .0M NH4N03 > O .OIMCaCh > O. 1M NaN03 > O .O IM Ca(N03h (Table 6 .5) . The use of single extraction reagents has some limitations. The results presented in the Table 6 .5 show that the extraction reagents 0.02M SrCh and O . IM Hel also have extracted significant quantities of total Cu as measured by tri acid digestion. Chapter 6 Soil test to predict the availability of copper 1 5 7 Table 6.5 Effect o f Cn levels on extractable soil Cn (mg kg-\ Treatments M I M3 O. IM O.O IM TEA- O.02M O.O IM O. IM I M (mg kg- I) HCI Ca(N03h DTPA SrCI2 CaCI2 NaN03 NH4N03 0 IUOc I 5 .30d 2 .80b 0.58c I 4.80d 7.07c 0.67d 0.84d 0.B9d 50 26.65c 3 1 .40c 5 .73b I . I Bc 3 1 .70c 14.B2c l A I c I A9c 2.30c 1 00 60.57b 56.70b l 3 .64b 2.43b 57.BOb 30.23b 2 .97b 2 .74b 4.36b 200 86.90a 95.BOa 26.23a 4.55a 102 .70a 65.76a 4.70a 4.2Ba 8.34a * Treatment means followed by the same letter within a colwnn are not SIgnificantly dIfferent at the 5% level. 6.3. 5 Speciation ofCu In this section the speciation of Cu with only for three soil test extractants (M 1 , M3 and TEA-DTPA) is discussed. In both soils, more than 98% of the Cu extracted by the three soil test extractants was in a complexed form and only a small amount of Cu remained as the ionic form. The Manawatu soil gave a significantly higher concentration of free Cu2+ than the Ngamoka soil. Brlirnmer et al. ( 1 986) and McBride ( 1 989) considered that at pH values close to 7, more than 99% of the Cu in solution is complexed by organic matter. Fotovat and Naidu ( 1 997) in their speciation studies of Cu reported that <5 % of the total Cu is present as free Cu in the soil extract Among the soil test extractants, TEA-DTPA extracted the highest level of complexed Cu. TEA-DTPA being a strong complexing agent not only enhanced the solubility of the complexed Cu, but also reduced the re sorption of Cu during extraction due to surface coordination. Among the soil test extractants, TEA-DTPA produced a significantly lower amount of free Cu2+ (Table 6 .6) . All extractants showed a significant increase in free Cu2+ with increasing levels of fertiliser addition. The acid condition of M 1 and M3 could cause the dissolution of Cu2+ from the solid phase enhancing the concentration in the soil solution. According to Baker and Senft ( 1 995), in acid conditions, the main form is free Cu2+, and CuOH+, CUC03 and CU(OH)2 occur in alkaline soil solutions. Wu et al. (2000) observed that the activity of the Cu2+ ion in soils fell in the range of 1 0.9x l O- 1 1 to 4.0x l O-9 M, which accounted for 0. 1 -7.8% of the dissolved Cu. In saturated soil extracts, the concentration of dissolved Cu varied from 4.78 x 1 0-8 to I I .4x 1 0-8 M, which constituted 0.002 to 0.008% of total soil Cu. The dominant species Chapter 6 Soil test to predict the availability of copper 1 5 8 of Cu in soil solution are the Cu2+ and CuOH+ ions, which are about 1 a times more common than CuHC03 +. They found that the activity of Cu2+ was governed significantly by the factors of soil pH, Ca2+ concentration, CEC and DOC. The highly significant correlation coefficient between any two of Cu2+ activity, pH, and DOC, implying that the concentration of free Cu2+ ions in soil system was strongly pH dependent. This is due to the strong complexing of Cu by organic matter, and also due to the high pH (7 .3) value of the solution. McBride et al. ( 1 998) concluded that EDTA, while dissolving and dispersing substantial amounts of high molecular weight organic matter, also displaces most of the bound Cu2+ from organic complexes by chelating the metal. The high efficiency of Cu extraction by EDT A must be attributed in part to the disruption of organic aggregation structures by the chelating agent, thereby making surface bound and occluded Cu accessible to extraction. The use of extraction with chelating agents may overestimate the bioavailability of trace elements in soils, where organic matter limits the activity of trace metals. There was no significant difference in free Cu2+ measured in the extractant of the exchangeable and organically bound Cu fractions. Among the chemical fractions of Cu, the exchangeable Cu fraction maintained a higher concentration of free ionic Cu2+ than the other chemical fractions (Table 6 . 7) . This is expected because the Ca2+ competes more strongly with the exchangeable fraction [extracted with a.aIM Ca(N03)z] and it represents the ionic species of Cu adsorbed to cation exchange sites (Temminghoff et aI., 1 994). The Manawatu soil showed a higher amount of free Cu2+ both in the Ml and M3 extractants, but the Ngamoka soil contained a higher amount of free Cu2+ in the TEA­ DTP A extractant for the organic fractions of Cu. A higher concentration of free Cu2+ was measured in the M3 extractant in soils with or without the addition of Cu. Increasing levels of Cu addition resulted in a significant increase in free Cu2+ in all the soil test extractants for exchangeable and organically bound Cu fractions. Chapter 6 Soil test to predict the availability of copper 1 59 Table 6.6 Copper species in soil test extractants. Soils Fertilisers Treatment levels Copper concentration in soil tests (mg Cu kg- ' soil) Mehlich- 1 Mehlich-3 TEA-DTPA Free Cu Complexed Free Cu Complexed Free Cu Complexed (mg L- ' ) Cu (%) (mg L- ' ) Cu (%) (mg L- ' ) Cu (%) Manawatu CUS04 0 0.02 99.8 0.08 99.5 0 .0 1 99.9 50 0.03 99.8 0. 1 0 99.6 0.02 99.9 1 00 0.03 99.9 0. 1 3 99.7 0.02 99.9 200 0.04 99.9 0. 1 5 99.8 0 .03 99.9 CuO 50 0.03 99.9 0. 1 1 99.6 0.02 99.9 1 00 0.03 99.9 0. 1 3 99.8 0.02 99.9 200 0.04 99.9 0. 1 6 99.8 0.03 99.9 Ngamoka CUS04 0 0.01 99.7 0.04 99.5 0 .01 99.8 50 0.02 99.9 0.08 99.7 0.0 1 99.9 1 00 0.02 99.9 0. 1 1 99.6 0.02 99.9 200 0.03 99.9 0. 1 3 99.8 0.02 99.9 CuO 50 0.02 99.9 0.09 99.5 0 .01 99.9 1 00 0.02 99.9 0. 1 2 99.7 0 .0 1 99.9 - 200 0.03 99.9 0. 1 5 99.8 0.02 99.9 - Chapter 6 Soil test to predict the availability of copper 1 60 Table 6.7 Copper species in chemical fractions. Copper concentration in soil fractions Soils Fertiliser Exchangeable Organic bound Cu Free Cu Complexed M l M3 TEA-DTPA (mg LO !) Cu (%) Free Cu Complexed Free Cu Complexed Free Cu Complexed (mg LO !) Cu (%) (mg LO ! ) Cu (%) (mg LO !) Cu (%) Manawatu CUS04 0.02 99. 1 0.02 99.9 0.0 1 99.9 0.02 99.9 0.04 98.4 0.02 99.9 0.02 99.9 0.03 99.8 0.07 98.4 0.02 99.9 0.03 99.9 0.04 99.9 0. 1 1 98.4 0.02 99.9 0.04 99.9 0.04 99.9 CuO 0.03 99. 1 0 .02 99.9 0.02 99.9 0.04 99.8 0.08 99.2 0.02 99.9 0.02 99.9 0.05 99.9 0. 1 0 99.5 0.03 99.9 0.04 99.9 0.05 99.9 Ngamoka CUS04 0.02 98.5 0.02 99.7 0.01 99.8 0.02 99.6 0.03 98. 1 0.02 99.9 0.01 99.9 0.03 99.8 0.06 97.3 0.02 99.9 0.02 99.9 0.05 99.9 0 . 1 0 98 .4 0.02 99.9 0 .04 99.9 0.05 99.9 CuO 0.03 98.6 0.02 99.8 0.02 99.8 0.04 99.6 0.08 98.2 0.02 99.9 0.02 99.9 0.05 99.8 0 . 1 0 99.4 0.03 99.9 0.04 99.9 0.06 99.9 Chapter 6 Soil test to predict the availability of copper 6.3.6 Ratios offractions in the soil test extractants 1 6 1 The amounts o f Cu extracted by M l , M3 and TEA-DTPA from the whole soil and the percentage of different Cu fractions in these soil test extractants are presented in Table 6 .8 . M3 extracted a higher amount of exchangeable Cu than M l and TEA-DTPA. The M3 and TEA-DTPA removed higher amounts of organically bound Cu than M l . Again M l extracted a higher amount of oxide bound Cu than M3 and TEA-DTPA. TEA­ DTP A extracted higher amounts of residual Cu. The ratios of exchangeable, organic, oxide bound and residual Cu fractions in the different extractants were 1 : 1 6 : 1 2 :4, 1 : 1 1 :6 :2 and 1 : 55 :3 1 : 1 3 for M l , M3 and TEA-DTPA, respectively. It clearly indicates that TEA-DTPA extracted more of the organic form of Cu than Ml and M3 . Cu treatment levels have no significant effect on the ratios of the Cu fractions removed by the soil test extractants. The ratios of exchangeable, organic, oxide bound and residual forms of Cu in M 1 , M3 and TEA-DTPA for the Manawatu soil were 1 :20:25 :4, 1 : 1 5 :8 :2 and 1 :56: 3 5 :8, respectively, and the values for the Ngamoka soil were 1 : 1 4 :6 :4, 1 : 9 : 5 :2 and 1 :55 :26: 1 7, respectively. The ratios of exchangeable, organic, oxide bound and residual forms of Cu in M 1 , M3 and TEA-DTPA are 1 : 1 2 :9 :2 , 1 : 1 2 : 5 :2 and 1 :60:28 : 1 0, respectively for CUS04 and 1 : 1 5 : 1 2 :4, 1 : 8 : 6 :0. 1 6 and 1 : 5 1 : 3 3 : 1 5 , respectively, for the CuO fertiliser. The ratio of the different fractions for M3 was closer to that for M l . Again TEA-DTPA showed a higher amount of Cu in the organic fractions compared with M l and M3 . This can be explained by the fact that the solution contains a complexing agent that extracts the Cu complexed with organic matter, an important pool of soil Cu. CUS04 showed more organic form Cu than the CuO fertiliser, which may be due to quicker releasing and complex formation of CUS04 with organic matter. The ratios of different forms of Cu strongly suggest that the Cu is residing in soils mainly in the organic form and decreased in order: organic > oxide > residual > exchangeable. This result strongly supports the adsorption study (Section 3 . 3 . 3 .4), where Cu retained on organic components in all soils and on both the organic and oxide components for Ramiha, Ngamoka and Mangamahu soils. Chapter 6 Soil test to predict the availability of copper 1 62 Table 6.8 Copper fractions in the soil test extractants. Soils Fertilisers Treatment Mehlich I Mehlich 3 TEA-DTPA levels Whole soil Different fractions (%) Whole Different fractions (%) Whole Different fractions (%) (mg Cu kg·1 (mg kg· l ) Exchan- Organic Oxide Residual soil (mg Exchan- Organic Oxide Residual soil (mg Exchan- Organic Oxide Residual soil) geable kg· l) geable kg· l ) geable Manawatu CUS04 0 1 7 .7 \ .86 2 8 . 8 5 2 . 8 1 6 . 6 2 0 . 2 4 . 5 5 57.9 25.7 1 \ . 8 2 \ . 5 0.98 49.7 35.3 1 3 .9 5 0 20.2 2 . 8 2 3 6 . 1 54.4 6.53 29.0 4. 1 4 69.6 20.0 6. 1 7 26.9 1 . 08 52.4 38.2 8.5 1 00 46.5 2.54 47.5 45. 1 4.90 5 3 . 9 4. 1 9 64.7 27.6 3 . 3 6 59.2 0.9 1 63.0 32.0 4.0 200 88.9 2.68 5 8 . 2 34.0 4.98 98.2 5 .24 6 8 .4 22.6 3 .76 1 07 . 2 1 .76 66.3 26.2 5 . 7 CuO 5 0 30.8 2.05 3 \ ,4 54.5 1 1 . 8 3 7.3 3 . 5 7 5 7 . 9 29.7 8.74 38.2 0.73 6 1 . 2 3 1 . 1 6.96 1 00 79.3 1 . 90 4 1 . 6 5 2 . 5 3 . 93 68.5 4.45 3 6 . 0 5 5 . 3 4. 1 9 72.6 0.95 49. 1 45.3 4. 59 200 95. 1 2 . 3 1 49. 1 4 5 . 7 3 . 3 2 1 08 . 5 3 . 99 56.4 36.4 3 . 1 9 1 1 7 1 .47 54.2 42. 1 2. 1 1 Ngamoka CUS04 0 4.5 9.78 5 7 . 7 4.44 27. 1 1 0.4 8.65 1 6. 3 6 5 . 9 9 . 0 8 . 1 2.35 57.2 24.8 1 5 .4 5 0 27.3 3 .74 5 8 . 2 3 3 .7 4 . 3 39.3 5 . 1 1 74.8 5 . 9 1 4. 1 42.0 0.83 69.2 25 .0 4 . 8 1 00 59.7 2.48 60.6 24.6 1 2. 1 4 5 . 1 5 . 1 0 77.8 3 . 0 1 4. 0 44. 1 1 . 54 60.7 1 4 .7 23 . 1 200 67.9 3 . 64 5 0 . 0 40.6 5 . 66 84.6 5 . 84 65 . 1 24 .5 4.4 90.9 1 . 24 60.8 3 1 .6 6. 1 CuO 5 0 2 8 . 3 1 .9 1 72.7 6.8 1 8 .5 20.0 5 . 25 65.5 6.2 23.3 20.0 1 . 1 0 40.5 22.4 36.0 1 00 56.8 2 . 94 39.9 3 6.0 2 1 . 1 59.5 5 .90 49.9 26.8 1 7. 1 5 5 . 3 1 . 46 47.7 24.4 26.4 200 95.7 3 .07 44.6 43.3 8 . 9 9 \ .9 6.43 5 3 . 1 3 3 . 3 7. 1 95.7 1 .49 49. 5 38.5 1 0 .4 Chapter 6 Soil test to predict the availability of copper 6.3. 7 Chemical forms in the soil test extractants 1 63 The quantities of exchangeable, organic, oxide and residual forms of Cu extracted by M l , M3 and TEA-DTPA were measured at each fractionation step. The soil test extraction of the whole soil showed significant differences in the organic fractions between soils. The results indicated that the organically bound Cu fractions are the major form extracted by the three soil test extractants. M l , M3 and TEA-DTPA extracted 54%, 66% and 69% of the organic bound Cu. The corresponding values for oxide bound are 4 1 %, 32% and 38% and for exchangeable eu are 22%, 45% and 26%, respectively. A substantial amount of exchangeable eu is removed by plants, as measured by the soil test extractants. This is due to high temperatures during the growth periods in the glasshouse. This suggests that residual Cu in clay mineral structures can be weathered and released slowly as more readily available forms of eu during the growing season, resulting in increases in exchangeable eu. It also suggests that as the amount of eu loading in soils is increased, more Cu i s associated with non-residual fractions, which increases the potential for mobility and bioavailability of eu. All the three soil test extractants removed significantly higher amounts of oxide bound Cu from the Manawatu soil than the Ngamoka soil. It was also observed that TEA­ DTP A extracted the highest amount of native eu. The extraction capacities of the reagents tested are in agreement with the extraction mechanism proposed for each method, and with the chemical forms of eu in the soil. Dilute acids, such as those employed by the M l procedure, apparently induce only a partial solubilisation of the Cu containing solids. However chelating agents, such as DTP A or EDT A, reduce the Cu activity in the solution by forming complexes, thus promoting further dissolution of the solid labile forms of eu. As organic matter may keep up to 45% of the total Cu complexed in the soil (McLaren and Crawford, 1 973a; Haynes and Swift, 1 984), it is reasonable to assume that the methods which show a preference for extracting organically bound Cu present a greater extraction capacity than the dilute acids (Ml ). Increasing levels of eu through fertiliser application significantly increased the concentration of Cu in all forms (except the residual form) extracted by the soil test extractants (Figure 6 . 1 ) . Chapter 6 Soil test to predict the availability of copper r­ '0') "" 0') E 80 :::l 40 () .5::1 c (\) � o CUS04 -exchageable _ Mehlich 1 � Mehlich 3 � TEA-DTPA o 1 00 o 1 00 � 8 0 1 CUS04- 45% of the exchangeable Cu was extracted by M3, while an average of 24% was extracted by TEA-DTPA and M l . M3 and TEA­ DTPA extractants removed more of the organic form of eu than M 1 . An average of > 67% of the total organic form of Cu was extracted by M3 and TEA-DTPA and the corresponding value for M l was 54%. More than 32% of the oxide form of eu was extracted by M3, while an average 40% of the oxide form Cu was extracted by TEA­ DTPA and M l (Figure 6 .2) . A considerable amount (>63%) of the residual form of Cu was also extracted by M l and M3, which was less than that extracted by TEA-DTPA (72%). Similar results were reported by Zhu and Alva ( 1 993) who found 62% and 33% of the total organically bound Cu was extracted by M3 and M l . Both extractants removed >30% of the precipitated form ofCu and 62% of the residual form ofCu. Chapter 6 1 00 ......... 75 eft -- -0 .& <.> � 50 -x Q) '- Q) 0-0-0 () 25 0 1 00 ........ 75 eft ---- -0 Q) ...... <.> (tj "- 50 ...... x (]) "-(]) 0-0- 0 () 25 o Soil test to predict the availability of copper (a) Manawatu soil (b) Ngamoka soil I Exch. Organic Form of eu - � I Oxide Mehlich 1 Mehlich 3 TEA-DTPA Residual 1 66 Figure 6.2 Percentage of different fractions of Cu (a) Manawatu soil and (b) Ngamoka soil extracted by the soil test extractants. According to the regression analysis presented in Figure 6.3, Cu extracted by TEA­ DTPA, M3 and M l was strongly correlated with the exchangeable, organic and oxide fractions of the sequential extraction. Zhu and Alva ( 1 993) reported significant linear correlation between Cu extractable by sequential fractions (exchangeable, organic and precipitated) and Cu extracted by M l , M3 and AB-DTPA. Exchangeable eu and organically bound Cu have been suggested as the plant available fonns (McLaren and Crawford, 1 973a). Cl) ::l o .;:: ro > cv 0.. 0.. o U Cl) ::l o .;:: ro > .!: cv 0.. 0.. o U Chapter 6 Soil test to predict the availability of copper 1 67 (a) Exchangeable 1 00 (b) Organic form Cu 20 + Mehlich 1 + •• 15 • Mehlich 3 75 • TEA-DTPA + • • 1 0 50 5 25 0 0 0 2 5 5 0 7 5 1 00 1 25 0 25 50 75 1 00 1 25 1 00 ] 12 (c) Oxide form Cu (d ) Residual Cu 75 1 0 50 25 o �---,-----,----.----.---, o 25 50 � 1 00 1 25 o 25 50 75 1 00 1 25 Copper in soil test extractants (mg kg-1 ) Copper in soil test extractants (mg kg-1 ) Figure 6.3 Correlation between Cu in soil test extractants and (a) exchangeable (b) organic bound Cu (c) oxide and (d) residual Cu fractions. 6.3.8 Relationships between the extractants A highly significant linear relationship was found between the concentrations of Cu extractable by M l and M3 (r2=0.9 1 ), M l and TEA-DTPA (r2=0.89), and M3 and TEA­ DTPA (r2=0.99). The slope of the regression equation was close to 1 (Figure 6.4) indicating each reagent extracted from the same pool, but by different mechanisms. In contrast Zhu and Alva ( 1 993) showed that the amount of Cu extractable by M3 was greater than that extractable by M l . More than 50% of the total Cu in the soils used by Zhu and Alva ( 1 993) was in the organically bound form. In the current study, Cu was added as inorganic CUS04 or CuO. In the M3 extraction solution, EDT A as a chelating agent to remove organically bound forms of Cu (Mehlich, 1 984; Alva, 1 992). This Chapter 6 Soil test to predict the availability of copper 1 68 explains why Cu extractable by M3 was much greater than that extracted by M 1 in the study by Zhu and Alva ( 1 993) . The M3 and TEA-DTPA extractants appear to be very similar with respect to their capacity to extract soil Cu, probably this c lose relationship is due to the fact that most of the soils used in this study have a low pH. Walworth et al. ( 1 992) compared M3 and TEA-DTPA in the extraction of Cu, Zn, Mn, and Fe. They observed r values between TEA-DTPA and M3 of 0 .85 , 0.97 and 0.95 for Cu, Zn, and Mn, respectively. Mozaffari et al. ( 1 996) also reported a highly significant relationship (r2=0.97) between the concentrations of Cu extractable by M3 with that by AB-DTPA from several soils representing the major soil series used for citrus production. The DTP A soil test, developed for near neutral and calcareous soils by Lindsay and Norvell ( 1 978), illustrates the evolution of a soil test extractant from theoretical principles derived from soil chemistry to verification through green house and field calibration studies. The DTP A extractant was selected because it offered the most favourable combination of stability constants necessary to simultaneously extract four micronutrient cations (Fe, Mn, Cu and Zn) . The buffered pH and presence of soluble Ca2+ prevents excessive dissolution of CaC03, avoiding the release of unavailable micronutrients occluded by this solid phase. It should be noted that at pH 7.3 , 70 to 80% of the buffering capacity provided by TEA has been consumed. Therefore, using the DTP A extractant on acidic soils, is likely to result in neutralisation of the remaining buffer capacity, resulting in an unpredictable extraction pH (Norvell, 1 984). Chapter 6 o -- � ------------ Soil test to predict the availability of copper 50 1 00 150 Mehlich 3 o 50 1 00 1 50 TEA-DTPA Extractable Cu (mg kg-1 ) o 1 69 50 1 00 1 50 TEA-DTPA Figure 6.4 The relationship between the concentrations of soil Cu extractable by (a) Ml and M3, (b) Ml and TEA-DTPA (c) and M3 and TEA-DTPA extractants from the two soils that received 0 to 200 mg Cu kg-t soil from two different Cu fertilisers. 6.3. 9 Relationships between the extractants and plant Cu concentration Considering that the Cu concentration in the shoot of the plant should be an adequate indicator of the availability of this element in the soil, it was of interest to perform a regression analysis comparing the plant Cu concentration with the amount of extractable Cu from the soil for each extraction reagent (Table 6.9) . Nearly similar correlation coefficient (r) values were obtained between Cu extracted by the different soil test extractant reagents and the Cu concentration in the plants. This similarity can be explained by the fact that M3 and TEA-DTPA solutions contain the complexing agents, which extract the Cu cornplexed by organic matter, an important soil pool of Cu. Similar results were observed by Walworth et al. ( 1 992), who obtained a correlation coefficient (r) of 0.73 for Cu extracted by both the M3 and TEA-DTPA reagents. Haddad and Evans ( 1 993), comparing extractants using clover as the test plant, found TEA-DTPA and AB-DTPA were comparable and better than M3 in predicting Cu concentration in the clover plant. Gimenez et al. ( 1 992) reported that DTPA and HCl extractable soil Cu were found to give higher correlations with coffee seedlings for Cu concentrations in both sandy and clay soils. - - - -- - ------ Chapter 6 Soil test to predict the availability of copper 1 70 All the extractants explained > 90 % variation found in the Cu concentration in the shoot (Figure 6 .5) . It can also be noted from this figure, that high correlation coefficients were found, indicating that the soil analysis for evaluation of the Cu availability to plants is feasible. Singh et al. ( 1 994) observed an r value of 0.994 between the Cu concentration in plants and the Cu extracted by the TEA-DTPA procedure for two gramineae plant species grown in polluted soils. De Abreu et al. ( 1 996) observed that the extraction of available Cu from a soil decreased in the order: M3 > TEA-DTPA > M l and strongly correlated this with the Cu concentration in the aerial part of the wheat plants grown; the r values between plant Cu concentration and soil test Cu were 0. 80, 0. 74 and 0.45 for M3, TEA-DTPA and Ml , respectively. Grundon and Best ( 1 982) evaluated DTPA as a soil test for Cu on 3 5 soils in Australia, and noted responses by wheat either as grain yield or increased grain Cu, where DTP A extractable Cu was .::t:; 25 j 25 ~ 25 0> .s rA c 20 20 20 .Q "§ 1 5 1 5 � 1 5 C ID <..) C 0 1 0 1 0 1 0 <..) ::l (a) (b) () (c) C 5 5 5 (\) 0: 0 0 0 0 25 50 75 1 00 1 25 0 25 50 75 1 00 1 25 0 25 50 75 1 00 125 Soil extractable Cu (mg kg-1 ) Figure 6.5 Linear correlations of Cu in tbe ryegrass witb tbe amounts of extractable Cu determined by using tbe (a) Ml , (b) M3 and (c) TEA-DTPA extracting procedures. 6.4 CONCLUSION AND FURTHER STUDY The result of the present study demonstrated that the major forms of Cu that can be extracted by M l , M3 or TEA-DTPA soil test extractants are the organically bound and oxide bound Cu. Increasing levels of Cu application significantly increased the exchangeable, organic and oxide bound forms of Cu in each extractant. The chemical forms of Cu extractable by M l , M3 or TEA-DTPA from pasture soils varied with the organic matter. As expected that the soil test extractants extracted higher levels of organically bound Cu for soils with high levels of organic matter than soils with low level of organic matter. Soil test extractants removed higher amounts of oxide bound Cu from soils treated with CuO than those treated with CUS04. The ratios of the different fractions for M3 was closer to that for M l . M3 and TEA-DTPA were more effective than M l in extracting the organically bound form of Cu. The TEA-DTPA and M l extract ants were equally effective in extracting the oxide bound form of Cu. Again TEA-DTPA and M l were more efficient than M3 in extracting the residual form of Cu. Further, the M3 showed a higher amount of free Cu than TEA-DTPA and M l . M3 demonstrated a greater increase of Cu from the exchangeable form and organic complexes due to the dual activity of EDTA and acids for the different fractions, and it is best suited for predicting the available Cu in pasture soils. The ratios of the different forms of Cu in the soil test extractants strongly suggest that the Cu is residing in the soil - - - ------------------ Chapter 6 Soil test to predict the availability of copper 1 72 mainly in the organic form and decreased in the order: organic > oxide > residual > exchangeable. TEA-DTPA yields a higher concentration of Cu than the other test extrantants. By means of the correlation equations, the amounts extracted using one method may be compared with the results obtained by means of any of the other tests having a larger data base or calibration curves for ryegrass . TEA-DTPA, M3, and M l extracted large amounts of Cu, when compared with the total Cu content measured by tri-acid digestion. The soils that have received recent Cu additions, TEA-DTPA, M3 and M l extractants are effective for extracting Cu. The efficiency in extracting the Cu from the soil, followed: O .OIM Ca(N03h < O . lM NaN03 < O .OlMCaCh < I .OM �N03 < O . IM HCI < O.02M SrCh < Mehlich- l < Mehlich-3 < TEA-DTPA. That Cu in soils is not available for plant uptake as it is strongly complexed with organic matter is shown in this study. The chemical forms of Cu extractable by soil test extractants from pasture varied considerably with organic matter. An attempt has been made to examine the effect of soil amendments (N and P fertilisers, lime and EDT A) on the uptake of both native and residual Cu. These results will be discussed in the next chapters. Chapter 7 Effect of soil amendments on the availability of native copper CHAPTER 7 7.1 INTRODUCTION EFFECT OF NITROGEN AND PHOSPHORUS FERTILISER ON THE AVAILABILITY OF NATIVE COPPER 1 73 Copper in soils is strongly held on inorganic and orgamc exchange sites, and it complexes with organic matter. For these reasons a large proportion of the total Cu content of a soil is not available for uptake by plants. The proportion of the total copper taken up by plants has been found to be greater in mineral soils than in organic soils. Copper deficiencies in crops may be due to either an inherently low total Cu content of the soil or to only small amounts being in an available form. In New Zealand, the pastoral system is legume-based and nitrogen fertiliser is used in pastures for encouraging out of season pasture growth. Phosphate fertilisers are also regularly used in legume based pasture systems. Copper requirements have tended to increase with increases in soil fertility, due to the growing of legumes, regular applications of superphosphate and the use of nitrogenous fertilisers (Gartrell, 1 98 1 ). It has been observed in several published reports that the application of N and P fertiliser compounds leads to increases in the Cu deficiency in cereal crops (Caldwell, 197 1 ; Touchton et al. , 1 980). In citrus, highly concentrated long term applications of P fertilisers have resulted in and intensified Cu deficiencies (Olsen, 1972). Application of a high N supply may cause rapid plant growth and accentuate Cu deficiency by exhausting the Cu supply in the soil solution (Robson and Reuter, 1 98 1 ; Alloway and Tills, 1 984) . Copper deficiency in infertile soils was enhanced after the soil N supply was elevated by the use of leguminous pasture species (Gartrell, 1 98 1 ). Brennan ( 1 993) reported high levels ofN in soils diluted the Cu concentration in wheat plants. In soils with a sufficient Cu supply, Cu levels in plants have been shown to increase with increasing N levels. Gladstones et al. ( 1 975) observed that the Cu concentration in shoots of a number of cereals and pasture grasses increased linearly with N concentration. Kumar et al. ( 1 990) reported that N and Cu were found to have a - - - ------------------- Chapter 7 Effect of soil amendments on the availability of native copper 1 74 mutually antagonistic effect on each other 's concentration in the wheat plant. The antagonism was greater with NH4 + sources than with N03 - compounds. Increasing levels of soil P also tend to reduce the concentration of plant Cu. This may be due to a plant dilution effect as a result of added P increasing plant growth without a consequent increase in Cu uptake (Robson and Reuter, 1 98 1 ). The P added induces the absorption and precipitation of metal ions to form insoluble complexes and therefore makes both the Cu and P unavailable to plants (Lindsay, 1 979; Ross, 1 994). In subterranean clover, the interaction between P and Cu on growth was indirect and positive, and when the P supply increased from a marginally deficient level to an adequate level, there was depressed Cu absorption and an accentuated Cu deficiency (Reuter et al., 1 98 1 b). The present study aims to evaluate the effects of N and P fertilisers on the uptake of native Cu by ryegrass in two contrasting soils. 7.2 MATERIALS AND METHODS 7.2.1 Soils Two contrasting soils (Manawatu and Ramiha) were used in this experiment. The soil samples were ground and passed through 6 .0 mm sieve. One kg of soil was mixed with the required amount of N and P fertilisers and placed in pots. The soil in the pots was maintained at 80% field capacity for equilibration. 7.2.2 Fertilisers There were four levels ofN added as urea (NH2-CO-NH2) and five levels ofP added as monocalcium phosphate [Ca(H2P04).H20] . Potassium and sulphur fertilisers were applied as a basal dose. 7.2.3 Plant growth experiment A completely randomised factorial design was used in this glasshouse trial. The treatments included two soils (Manawatu and Ramiha), four levels of nitrogen (0, 50, Chapter 7 Effect of soil amendments on the availability of native copper 1 75 1 00 and 200 mg kg-l soil) and five levels of phosphate fertiliser (0, 5 0, 1 00, 1 50 and 200 mg kg-! soil). The treatments were replicated three times. There were 1 20 (2 x 4 x 5 x 3) pots in this experiment. The treatments were placed randomly in each tray by using a random number table. After equilibration with water, forty seeds of ryegrass were sown in each pot. The pots were covered by brown paper until germination. The first Cu free nutrient solution was given after 25 days from sowing. The nutrient solution was applied twice a week. 7.2.4 Grass sample preparation The ryegrass was harvested at 90 days after sowing. The plants were cut to a height of approximately 2 .5-3 .0 cm above the surface of the soil . The samples were dried at 70 °C in a forced air oven. The dry weights of the ryegrass samples were recorded and the samples were then ground using a coffee grinder and kept in airtight polyethylene bags for chemical analysis. 7.2. 5 Ryegrass samples for Cu analysis The method of the preparation of the ryegrass samples for chemical analysis IS described in section 4 .2 .5 . 7.2. 6 Chemical Analysis Copper in the plant digests and the soil extracts was analysed following the methods described in section 4 .2 .6 . 7.2. 7 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS). Details of which are discussed in section 4.2 .7 . --------- - -- -- - Chapter 7 Effect of soil amendments on the availability of native copper 7.3 RESULTS AND DISCUSSION 7.3.1 Initial soil 1 76 The initial soil properties discussed in Chapter 3 (Section 3 .3 .2) indicate that the Ramiha soil contained higher amounts (5 .62%) of organic carbon than the Manawatu soil (2 . 9 1 %). 7.3.2 Effect of soil types on dry matter yield and Cu concentration Results presented in Table 7 . 1 show significant differences in dry matter yield and also Cu uptake between the two soils. The Ramiha soil shows a higher amount of dry matter yield, a lower Cu concentration, but a higher Cu uptake. The variation in shoot Cu concentrations indicated that soil Cu was less available from the high organic matter content Ramiha soil; the low plant Cu concentration in this soil may also be due to a dilution effect as a result of greater dry matter production. A similar observation was made by Soon et al. ( 1 997) and demonstrated that the Cu concentration is less in a Black Solodic soil rich in organic matter. There was a sharp difference in Cu uptake between the two soils. Table 7.1 Effect of soil types on DM yield, Cu concentration and uptake. Soils Dry matter yield Cu concentration Cu uptake (g porI ) (mg kg-I ) (mg pori ) Manawatu 1 .08b 8 . 1 0 ns 8 .85b Ramiha 1 .9 1 a 7 .26 1 1 . 74a * Treatment means followed by the same letter wlthm a column are not slgmficantly different at the 5% level and ns means not significant. 7.3.3 Effect of N on dry matter yield, Cu concentration and eu uptake The dry matter (DM) yield increased with increasing levels of N (Figure 7 . 1 a). At the highest level of N there was a slight decrease in yield, but it was not significantly different from the other N levels. Increasing levels of N increased the Cu concentration (Figure 7 . 1 b) and Cu uptake (Figure 7. 1 c) in both soils. Gladstones et al. ( 1 975) Chapter 7 Effect of soil amendments on the availability of native copper 1 77 observed that the eu concentration in shoots of a number of cereals and pasture grasses increased linearly with N concentration. Filipek-Mazur ( 1 99 1 ) found N application increased eu content in herbage. Buttigieg et al. ( 1 989) reported those N rates of 250 and 500 kg N ha-I year- I significantly increased the eu concentration pasture. A number of reasons could be attributed to an increase in eu concentration and eu uptake with increasing levels of N application (Hill et aI., 1 978; Robson and Reuter, 1 98 1 ) : (i) An increase in root growth due to N is likely to cause greater exploitation of the soil volume resulting in increased uptake of eu; this is known as the priming effect; (ii) an increase in N uptake increases the amino acid concentration in roots leading to greater translocation of eu from root to shoot. Bineev et al. ( 1 985) confirmed that free amino acids in soils form chelates with eu and thereby facilitate the migration of the trace element in the soil plant system. More recently, it was found that free nicotianamine and histidine are the major eu transporters in tomato and chicory xylem sap (Pich and Scholz, 1 996; Liao et al., 2000). High levels of N however, markedly reduce the rate of translocation of eu from older leaves to meristems. The coupling of eu movement to leaves to N movement may account for relatively high critical concentration of eu in the tops of plants found at high fertiliser rates (Loneragan et al., 1 980) . Soon et al. ( 1 997) conducted a study to quantify the variation in eu and P uptake among some common Canadian wheat cultivars due to N fertiliser and the environment. Nitrogen fertiliser produced four times as much variability in eu concentration and uptake as did the genotype. There was a strong correlation between N and shoot eu concentration (r=0.70, P=O.OI ) . 4.0 -+- Manawatu soil 1 0 1 5 .... -e-- Ramiha soil 'Cl .... .>0:: 1:; 3.0 Cl 1:; Cl. E Cl. .9 c Cl 1 0 '0 .Q .s � 2.0 '§ Q) 5 .>0:: C .l!! Q; Q) Cl. u := c: :J co 0 Q; 5 E u � 1 .0 Q; Cl. Cl. 0 (a) Cl. 0 Cl. (b) u (c) 0 u 0.0 0 0 0 1 00 200 0 1 00 200 0 1 00 200 N level (mg kg-1soil) Figure 7.1 Effect of N levels on (a) dry matter yield, Cb) Cu concentration and (c) Cu uptake. Data are means ± SE, n=3. Chapter 7 Effect of soil amendments on the availability of native copper 7.3.4 Effect of P on dry matter yield, Cu concentration and Cu uptake 1 78 Increasing levels of P significantly increased the DM yield in the Ramiha soil (Figure 7 .2a) due to the low Olsen P . Increasing rates of P fertil isers increased the available P status (Olsen P) of the soil and thereby increased the dry matter yield. Morton et al. ( 1 995) and Morton and Roberts ( 1 999) found a good relationship between soil Olsen P and relative pasture production for volcanic ash soils. The addition of P fertiliser decreased the concentration of Cu in plants (Figure 7 .2b), but there was no significant difference in the Cu concentration between different P levels. This effect was more pronounced in the Ramiha soil than the Manawatu soil. This may be due to the dilution of Cu in plant tissue through the promotion of plant growth by phosphate fertil isers (Loneragan et al. , 1 979) and the complexing of CuH2P04 + at a pH range of 5 . 8 to 6 . 8 (Lindsay, 1 979). Wallace et al. ( 1 978) showed that at high pH increasing solution P decreased the uptake of Zn, Cu, and Mn by soybean and bush bean, whereas at low pH it resulted in an increase in their uptake. Ahumada and Schalscha ( 1 995) observed that the addition of P decreased sorption of eu in soils due to complex formation with H2P04-. Increasing levels of P increased the Cu uptake (Figure 7 .2c). Maciejewska ( 1 992) observed that P application increased the Cu uptake by perennial ryegrass (Lolium perenne) . Both transport to shoot and uptake rate of Cu increased with the increase in P supply. The proportion of eu retained in roots (65%) did not vary with differential Cu uptake at the various P levels. eu and P concentrations in roots were positively correlated (Gill et al., 1 992). Timmer and Leyden ( 1 980) concluded that the reduction in plant eu concentration at high P levels resulted from a reduced exploitation of the soil by mycorrhizae. De Iorio et al. ( 1 996) demonstrated that the addition ofP has a positive interaction in lettuce with high levels of eu. - - -- ------------------------------ Chapter 7 Effect of soil amendments on the availability of native copper 3.0 l � I g I -9 2.0 -I ::Q .� I Qj :)::: � 1 .0 / f C-a ;::-- 1 5 l � � .- � f 1 0 / � (a) Q) () § 5 () Qj � � o (,) -+-- Manawatu -e- Ramiha (b) 0.0 -11-----,---,----,--, o 1 00 200 o 1 00 200 P level (mg kg-1 soil) o 1 79 1 00 200 Figure 7.2 Effect of P levels on (a) dry matter yield, (b) Cu concentration and (c) Cu uptake. Data are means ± SE, n=3. 7.4 CONCLUSION AND FURTHER STUDY • Soils have a significant effect on DM yield and also on Cu concentration. • Increasing levels ofN increased DM yield and Cu concentration. • Increasing levels of P increased the DM yield, but decreased Cu concentration. • Copper uptake was affected by the growth of ryegrass through the addition of both N and P fertilisers. The effect of lime, nitrogen fertiliser and EDT A on the availability of residual Cu is discussed in next chapter. Chapter 8 Effect of soil amendments on the availability of residual copper CHAPTER 8 EFFECT OF LIME, EDTA AND NITROGEN FERTILISER ON THE AVAILABILITY RESIDUAL COPPER Publication arising from this chapter: 1 80 Khan, M. A. R., N. S . Bolan and A. D. Mackay 2000. Effect of liming and nitrogen fertiliser on the plant availability of residual Cu in pasture soils. In Proceedings of the Second International Conference on Geospatial Information in Agriculture and Forestry, Lake Buena Vista, FL, USA. pp. I I-266 274. 8. 1 INTRODUCTION The results in chapters 3, 4, 5 and 6 indicated that copper (eu) is very strongly bound to soil organic matter, and hence the plant availability of eu added through fertiliser is likely to decline with increasing time of contact between the soil and the eu fertiliser. Farmers require accurate information on the length of time that a eu application remains fully effective in order to supply the Cu required for the grazing animal. Nitrogen fertiliser is used in pastures for encouraging out of season pasture growth. Lime i s used to overcome the problems associated with soil acidification. The application ofN fertilisers to pasture soils marginally supplied with eu may induce a eu deficiency in plants. Kriihmer and Sattelmacher ( 1 997) reported that the increased eu uptake with increasing N levels might be explained by the release of amines into the root apopIast and the rhizosphere, particularly under eu sufficient conditions. These may mobilise eu by the formation of soluble eu amine complexes. Among the various soil properties, pH has the largest effect on the adsorption of cations by variable--charge surfaces. The net negative charge on variable-charge surface materials increases with increasing pH; as a consequence, the adsorption of cations increases. In soils, where lime is applied, both the pH and the ea concentration in the soil solution increase. Whereas an increase in pH can decrease the adsorption, an increase in Ca concentration has the opposite effect on the adsorption eu, the resultant effect of hming on eu adsorption is likely to depend on the concentration of Ca in the soil solution. Bivalent transition metal cations exhibit a similar pH dependent sorption behaviour; and many authors have reported that the amount of sorbed metals increases with the raising of solution pH within a certain range. The effect of pH on adsorption is - - - -------------- Chapter 8 Effect of soil amendments on the availability of residual copper 1 8 1 confounded by the effect o f Ca added through liming (Fries en et aI., 1 980). The amount of Cu in the soil solution decreases with increasing pH because of stronger Cu adsorption (Lindsay, 1 974; Section 3 . 3 . 3 . 5) . Chelating compounds, such as EDT A are generally used to enhance the mobilisation of metal ions in soils. Like the natural complexing agents (humic and fulvic acids) that enhance the migration of metals, synthetic chelating agents (eg. EDTA, DTPA etc .) combine with trace metals to increase the total levels of these metals in the soil solution, thereby increasing the nutrient availability by increasing both diffusion and the mass flow of these metals to plant roots (Lindsay, 1 974). The residual value of Cu fertilisers as affected by N fertiliser, lime and EDT A was investigated in a glasshouse trial with the following obj ectives. • To detennine the residual effectiveness of fast and slow release Cu fertilisers as influenced by N fertiliser addition. • To examine the availability of residual Cu to plants in response to changes in soil pH through liming. • To evaluate the availability of residual Cu to plants through chelating agents. 8.2 MATERIALS AND METHODS 8.2.1 Soil collection and preparation Soil samples were obtained at the tennination of a glasshouse plant growth experiment (Chapter 4). From that trial, two contrasting soils (Manawatu and Ngamoka) treated with both slow (CuO) and fast release (CUS04) Cu sources, at four Cu levels (0, 5 , 1 0 and 20 kg Cu ha- I) were chosen for this experiment. Two hundred and fifty-gram soil samples were p laced in pots and the soil in the pots was maintained at 80% field capacity for equilibration. 8.2.2 Plant growth experiment and treatments A factorial design was used in this glasshouse trial. The treatments included two soils (Manawatu and Ngamoka), two Cu sources (CUS04 and CuO), four residual Cu levels (0, 5 , 1 0 and 20 kg Cu ha-I ) , three levels ofN fertiliser (0, 50 and 1 00 kg N ha- I ; Plate 8 . 1 ), three levels of lime (0, 1 000, 2000 kg ha- I ; Plate 8 .2) and three levels ofEDTA (0, Chapter 8 Effect of soil amendments on the availability of residual copper 1 82 1 0 and 20 kg ha-\ Plate 8 .3). The treatments were replicated three times. N fertiliser was applied as a solution 45 days after sowing. Lime and EDT A were incorporated 1 00 days prior to sowing. After equilibration with water, twenty-five seeds of ryegrass (Lolium perenne cv. Super Nui) were sown in each pot. The first eu-free nutrient solution was given 20 days after sowing. The nutrient solution was applied twice a week. Plate 8. 1 Glasshouse trial of N effects on residual Cu availability in ryegrass. Plate 8.2 Glasshouse trial of lime effects on residual Cu availability in ryegrass (Ballantrae high fertility known as Ngamoka soil). Chapter 8 Effect of soil amendments on the availability of residual copper 1 83 Plate 8.3 Glasshouse trial of EDTA effects on residual Cu availability in ryegrass. 8.2.3 Harvesting and soil sampling The ryegrass was harvested 60 and 90 days after sowing. The plants were cut to a height of approximately 2 . 5-3 .0 cm above the surface of the soil . The samples were dried at 70 QC in a forced air oven. The dry weights of the pasture samples were recorded, the samples were then ground using a coffee grinder and kept in airtight polyethylene bags for chemical analysis. The soil samples were collected at the end of experiment (after 1 06 days). The soil samples were air dried, ground and passed through 2.00 mm sieve. The soil samples were analysed for total, exchangeable and free ionic Cu. 8.2.4 Soil analysis Soil pH ( 1 :2 . 5 H20) was measured using a glass electrode. Total Cu was determined by wet digestion (Section 5 .2 . 3 ) . Soil samples were extracted either by Mehlich- l (Section 3 .2 . 1 0.2), Mehlich-3 (Section 3 .2 . 1 0. 3 ) or TEA-DTPA (Section 3 .2 . 1 0. 5 ) For the measurement of exchangeable Cu and ionic Cu, the soil samples were extracted with O .O IM Ca(N03k The filtrates were analysed for total Cu and free Cu2+ using F-AAS and a cupric electrode, respectively, with the appropriate standards. 8.2.5 Ryegrass samples for Cu analysis The method of the preparation of the plant samples for Cu analysis is described in Chapter 4 (Section 4 .2 . 5) . Chapter 8 Effect of soil amendments on the availability of residual copper 8.2.6 Chemical Analysis 1 84 Copper in the plant digests and soil extractants was analysed using F-AAS. Details of other procedures are discussed in Section 4.2.6. 8.2. 7 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS). Details are discussed in section 4 .2 .7 . 8.3 RESULTS AND DISCUSSION 8.3.1 Soil characteristics Both the soils are slightly acidic with the Ngamoka soil (5 .9% organic carbon) containing twice the amount of organic carbon compared to the Manawatu soil (2 .9% organic carbon). Soil characteristics are discussed in Chapter 3 (Section 3 .3 .2). 8.3.2 Dry matter yield and Cu concentration 8.3.2. 1 Effect of N fertiliser Figure 8 . 1 shows that the nitrogen treatment at 1 00 kg N ha- l produced the highest DM yield and Cu concentration at both harvests and it was significantly (p < 0.05) different from the control treatment. The 1 00 kg N ha- l level contained the highest amount of eu at each harvest, but it was not significantly different from the 50 kg N ha-! treatment. Similar results were obtained by Krahmer and Sattelmacher ( 1 997) who reported that increasing rates of N fertiliser increased the eu concentration in plants under eu sufficient conditions. 8.3.2.2 Effect oflime The lime treatment at 2000 kg ha- 1 produced the highest DM yield in both harvests and it was significantly (p< 0.05) different from control. The effect of lime levels on increasing yield was more pronounced at the higher application rate in the Manawatu Chapter 8 Effect of soil amendments on the availability of residual copper 1 85 soil . Thomson ( 1 982) and Edmeades et a!. ( 1984) found a correlation (r=0.6) between lime and pasture production. Increases in DM yield with increasing rates of lime were also obtained by others (Hogg et a!. , 1 988; Redente and Richards, 1 997; Stout et al. , 1 997). The lime level of lOOO kg ha-I soil resulted in the highest amount of Cu concentration at harvest 1 and that was significantly different from the other treatments (Figure 8 . 1 ) . This was attributed to the alleviation of a calcium deficiency. The application of lime to soils significantly increased soil pH values. The lime level of 2000 kg ha- 1 decreased Cu concentration at all harvests. Cu concentration in the ryegrass was found to be higher at the lower level than at the higher levels of lime (Levesque and Mathur, 1 983) . The reduction in plant Cu concentration in response to liming tended to be greater where the soil pH was relatively high. The decrease in the concentration of Cu in the plant resulting from an increase (0 .6 unit) in soil pH due to the addition of lime is in general agreement with the findings of others. Hooda et at. ( 1 997) observed that the Cu content of carrots and spinach produced on limed soils was decreased by 28% and 27%, respectively, compared to those grown on unlimed soils. Alva et at. ( 1993) reported that the addition of Ca as both lime and gypsum lowered Cu uptake in citrus plant thus ameliorating Cu toxicity. 8.3.2.3 Effect ofEDTA Dry matter yield decreased with increasing levels of EDT A. Addition of EOT A resulted in an increase in Cu concentration in ryegrass (nearly 3 fold) resulting in phytotoxicity (Figure 8 . 1 ) . Albasel and Cottenie ( 1985) reported that chelating agents increased the uptake of Cu in perennial ryegrass (Lolium perenne L) and Italian ryegrass (Lolium multiflorum), when humic podzols soils are contaminated with 40 mg Cu kg-1 • A high concentration of Cu is toxic to plants, because it directly or indirectly affects the metabolic processes such as respiration, photosynthesis, CO2 fixation, and gas exchange (Van Asche and Clij sters, 1 990; Ouzounidou et aI., 1 997). Increasing levels of residual Cu increased the Cu concentration in shoots. Cu concentrations in plants from the Manawatu soil were significantly higher, than those from the Ngamoka soil with the addition of EDTA. Hunter ( 1981 ) reported that when seedling maize roots were immersed for one hour in a Cu solution (0. 1 3 mM CUS04) growth almost completely stopped due to the Cu treatment. Chapter 8 4.0 2 .0 0.0 - 4.0 , Cd ...::: ..... '-' '"Cl c:; ';;" 2.0 ..... .8 ..... Cd S C;- Cl 0.0 4.0 2.0 0.0 � I I -----------� -- - - - Effect of soil amendments on the availability of residual copper � Cut 1 _ Cut 2 0 50 1 00 Nitrogen (kg ha- l ) 0 1 000 2000 Lime (kg ha- l ) ,-., -, /;)I) � /;)I) S '-' .:: , S ..... Cd ... ..... .:: a> () .:: o () :::l u 40 "' 20 0 0 50 1 00 Nitrogen (kg ha- I ) 40 20 0 0 1 000 2000 Lime (kg ha- l ) 40 20 l -1_. � 0 0 1 0 20 0 1 0 20 EDT A (kg ha- l ) EDTA (kg ha- I ) 1 86 Figure 8.1 Effect of N fertilisers, lime and EDTA on DM yield and Cn concentration at different harvests for both soils. The optimum ( ___ ) and the toxic (- - - -) levels of Cu in the plant are indicated in the figure. Data are means ± SE, n=48. 8.3.3 Effect of soil types on D M yield and Cu concentration Both the DM yield and eu concentration for the Manawatu soil were significantly higher than for the Ngamoka soil at each harvest (Figure 8 .2) with the addition of N fertiliser. The effectiveness of liming in reducing the Cu content in ryegrass varied between the soils. The Ngamoka soil contains more organic matter and lime solubilises the organically bound Cu. Addition of lime caused an increase (0.6 unit) in soil pH. The eu concentrations measured for ryegrass grown in both soils were similar at harvest 2 . -- - - ------------------ Chapter 8 Effect of soil amendments on the availability of residual copper 1 87 Brennan et al. ( 1 980) found no specific relationship between the decline in the availability of eu and soil pH, clay content, or sesquioxide content. On the other hand, King ( 1 988) found both Fe oxide and c lay content are important in determining the loading rate of heavy metals in soils. Soil pH affects the distribution of eu in soils. McLaren et al. ( 1 983a) reported an increase in eu sorption as pH increased, primarily due to a greater association with soil oxides. In general, lime applications reduce plant uptake of most metals (except Mo and Se), at least to some extent. This is due to the precipitation of metals in soils as insoluble hydroxides and carbonates, and to increase sorption resulting from increased charge density on pH dependent sites. -..- 2 o 'm 2 .I::. o 2 o o 50 1 00 N rate (kg ha-1 ) o 1 000 2000 Lime rate (kg ha-1 ) o 1 0 20 EDTA rate (kg ha-1 ) ..... , Ol ..>c Ol E - c .2 co .... E c 0 <..> :::l () 60 � M anawatu 40 - Ngamoka 20 0 0 50 1 00 N rate (kg ha-1 ) 60 40 20 0 0 1 000 2000 Lime rate (kg ha-1 ) 60 40 20 0 0 1 0 20 EDT A rate (kg ha-1 ) Figure 8.2 Effect of soil types on DM yield and Cu concentration as affected by N fertilisers, lime and EDT A addition at various level of Cu from different sources. Data are means ± SE, n=24. Chapter 8 Effect of soil amendments on the availability of residual copper 8.3.4 Effect on exchangeable Cu 1 88 Both N and lime applications decreased the soil exchangeable eu of the Ngamoka soil but the Manawatu soil showed an opposite effect (Figure 8 .3a and 8 .3b). EDTA increased the amount of exchangeable Cu in both soils (Figure 8 .3c) . The increase in pH due to liming resulted in a decrease in exchangeable eu. The Ngamoka soil binds Cu strongly due to a high amount of organic matter. Again the Ngamoka soil showed less exchangeable Cu with increasing rates of EDT A addition compared to the Manawatu soil . These differences were attributed to the differences in the characteristics of the two soils. Marschner et al. ( 1 995) reported that as a result of a pH increase the water soluble fraction of Cu was reduced by 30% by lime amendment. Guster et al. ( 1 983) indicated that liming increased the soil pH and reduced the exchangeable Cu. Komisarek et al. ( 1 990) reported that increasing lime rates reduced the water soluble and exchangeable Cu contents. Ma and Uren ( 1 998) observed that additions of CaC03 increased the soil pH from 7.08 to 7 .68 and decreased the exchangeable Cu concentration. Soil properties, primarily pH and organic matter content, influence the severity of Cu toxicity (Alva et al. , 1 995; Mozaffari et al. , 1 996). Rhoads et al. ( 1 989) conducted two experiments with increasing Cu rates from 44 to 2800 mg kil in the pH rage of 5 .3 to 6 .6 . In both experiments, the growth of tomato p lants decreased significantly with an increase in the eu rate above 1 50 mg kil at soil pH levels below 6 .5 . At soil pH >6. 5 significant growth reduction occurred only when the Cu rate was increased to 330 mg kil . Other studies also showed that Cu concentration in the plant and eu uptake were inversely related to the soil pH. This is due to a reduction in the solubility of Cu, and its availability to plants with an increase in soil pH (Jahiruddin et al. , 1 986; Lindsay, 1 99 1 ). Alva et al. (2000) demonstrated that soil pH influences the phytotoxicity of Cu primarily due to differential distribution of the Cu chemical fonns. Exchangeable and sorbed Cu are the most phytotoxic fonns, and their concentration increases with decreasing soil pH. Since the phytotoxic effects of Cu are dependent on soil pH, the critical toxic levels of Cu in soils vary depending on the soil pH. This infonnation is important for an adequate management of topsoils to mitigate the Cu toxicity effects. The effects of lime on the extraction by different chemical test extractants at various rates o f residual Cu are Chapter 8 Effect of soil amendments on the availability of residual copper 1 89 discussed in this chapter. Increasing levels of EDT A increased the exchangeable Cu in soil solution. Laboratory studies conducted by Dragun et al. ( 1 976) revealed that corn plants grown in 10 mM CUS04 solution, in equilibrium with the soil solids, resulted in a substantial decrease in height compared to corn grown in solutions with Cu concentrations less than 1 0 mM. A far less dramatic decrease in plant height was observed for corn grown in 2 .5 mM Cu solution. Minnich et at. ( 1 987) conducted greenhouse pot experiments and found an increase in Cu accumulation by snapbeans (Phaseolus vulgaris L.) in response to increased amounts of Cu as a Cu salt and Cu enriched sludge amended soils. They reported that phytotoxicity was not apparent for any of plants during growth, however they noted that plants grown in 300 mg Cu kiI salt treatments had thick stunted root systems at harvest. While the Cu concentration in soil solution is in the normal range of O.O l -0.6�when the total soil content of Cu is 25-40 mg kg- I . The minimum concentration of Cu for specific adsorption has been defined as that concentration of an ion in solution, which causes a reversal of charge of colloids at infinite dilution. The concentration of Cu has been reported as 2.5 x 10-4 M for colloidal Si02 at pH 6 .5 and 5 x 1 0-5 M for kaolinite at pH 5 , this concentration may be toxic to most plant species (lames and Barrow, 1 98 1 ). Since the availability of Cu is affected by a number of factors including soil pH, clay content, and organic matter, it is not possible to use total or some measure of 'extractable' Cu2+ to predict the solution activity of Cu2+ affecting plants (Baker and Senft, 1 995) . Gimenez et al. ( 1 992) reported that the critical toxic levels of soil Cu was 1 . 0 and 3 . 0 mg kg-l , respectively, for the sandy and clayey soils used for growing coffee seedlings. In the present study, the Cu concentrations in soil solution ranging from 2 .8 to 20.2 mg kg- l with the addition of EDTA causes toxicity to ryegrass, when the soil pH is below 5 .6 . Walsh et al. ( 1 972) recorded > 20 mg kil Cu was extracted from soil with O. IN HCl or DTPA and > 15 mg kg- l Cu was extracted with EDTA; these levels resulted in significant yield reductions of snapbeans. - - - - ------------------- Chapter 8 Effect of soil amendments on the availability of residual copper 1 90 5 � 4 0) .s ::J 3 () Q) � 2 Q) 0) c:: � 1 u x UJ o _ Manawatu � Ngamoka (a) o 50 1 00 N rate (kg ha-1 ) 5 4 3 2 o (b) o 1 000 2000 Lime rate (kg ha-1 ) 1 5 1 0 5 o (c) o 1 0 20 EDT A rate (kg ha-1 ) Figure 8.3 Effect of soil types on exchangeabJe Cu as affected by (a) Nitrogen, (b) Lime, and (c) EDTA. Data are means ± SE, n=24. 8.3.5 Effect of fertiliser Cu sources and levels of residual Cu on DM yield and Cu concentration Fertil iser Cu sources have a significant effect on DM yield and Cu concentration in ryegrass (Figure 8 .4). The residual Cu level of 20 kg Cu ha-l for CuO fertiliser resulted in the highest concentration of Cu at all harvests and was significantly different from other levels for both N and lime application. This increase in dry matter yield with residual Cu is increased further by the influence of N treatment However, even in the absence of residual eu, dry matter yield was increased by the addition ofN fertiliser and liming, presumably as a result of alleviating the adverse effect of low soil pH such as aluminium and manganese toxicity on root growth and metabolism (Marschner, 1 986) . The alleviation of such conditions also resulted in an increased concentration of Cu in spite of the reduced solution concentration of eu brought about by N and lime addition. B6langer et at. ( 1 986) observed that residual Cu had positive effects on the Cu content of an oat crop grown in soils after four years of fertiliser application. Cox ( 1 992) reported that the residual effect of eu fertiliser use with time varied between soils. An application of 4.48 kg Cu ha- ! increased the soil extractable Cu above the initial Cu concentration for from 9 to 27 years, the average time across the five soils was about 1 6 years. Sherrel l ( 1 989) reported that the effect o f CUS04 applied to an established stand of lucerne (Medicago sativa L.) on a yellow brown pumice soil was measured over a period of four years. Extractable soil eu indicated that the residual effect of Cu application might last for a considerable time. Chapter 8 .,-, cu ..c - -0 Q) ';;;" '- lB co E � 0 ---.,... , cu ..c ....... - 3.0 2.0 1 .0 0.0 3.0 2.0 1 .0 0 .0 Effect of soil amendments on the availability of residual copper 1 9 1 - N fertiliser (a) CuO � Lime (b) CuS04 Levels of l ime or N or EDT A - 40 ...... , 0) .:x: 0) E 30 --- c: .9 co J:;:; 20 c: (J) u c: 0 u I.- 1 0 (J) 0.. 0.. 0 () 0 .;:::' 40 I 0) .:x: 0) §. 30 c: o :;;:;; � 20 'E (J) u c: o u 1 0 '-(J) 0.. 0.. o () 0 (c) CuO (d) CuS04 Levels of l ime or N or EDTA Figure 8.4 Effect of Cu fertiliser sources on DM yield [(a) CuO, (b) CUS04] and Cu concentration [(c) CuO, (d) CUS04J as affected by lime, Nitrogen or EDTA. Data are means ± SE, n=24. The residual eu rate significantly influenced eu concentration III plants with the addition of EDTA (Figure 8 .5c) . The concentration of eu in the shoots also increased with increasing rates of residual eu by the addition of lime and N fertiliser. The regression slope between the shoot eu concentration and residual eu rate was greater for the Manawatu soil than for the Ngamoka soil. The concentration of eu was much greater at high rates of residual eu. This is in agreement with typical plant responses to heavy metal toxicity conditions. Chapter 8 40 � '0> -" 0> 30 S c .Q � 20 c:: Ql U c 8 :::> () 1 0 E <1l a::: 0 0 Effect of soil amendments on the availability of residual copper 1 92 (a) Nitrogen + Control • 50 kg ha-1 • 50 1 00 1 50 200 40 (b) Lime + Control • 1000 kg ha-i 30 • 2000 kg ha-1 1 0 l 0 0 50 1 00 1 50 200 Copper level (mg Cu kg-1soil) 80 (e) EDTA + Control • 1 0 kg ha-1 60 • 20 kg ha-1 40 ::/ 2 0 0 0 50 1 00 1 50 200 Figure 8.5 Effect of residual eu levels on eu concentration either treated with N (a), lime (b) or EDTA (c). Data are means ± SE, n=12. 8.3.6 Effect o/soil test extractants on plant eu concentration The amount of plant available Cu, as measured by the three soil test extractants, is affected by N, lime and EDTA. The r values between Cu concentration in the plants and Cu in the soil test extractants are 0.84, 0.9 1 and 0.67 for M l , M3, and TEA-DTPA, respectively, as affected EDTA (Figure 8.6). The application of EDTA resulted in the highest amount of plant available Cu, as measured by the three soil test extractants. The tendency to form complexes between Cu and soluble organic matter increases with an increase in pH. Applications of high levels of Cu as either CUS04 or CuO increased the DTPA extractable Cu in both soils. o 25 50 75 1 00 1 25 60 l (b) 50 l 40 l 30 � \ •• -t;:. •• 2 0 J . " 1 0 I o I • • • • • • • o 25 50 75 1 00 1 25 Soil extractable Cu (mg kg-1 ) • • • • o 33 67 1 00 1 33 1 67 200 Figure 8.6 Relationship between Cu concentration in plants and Cu extracted by (a) Ml, (b) M3 and (c) TEA-DTPA extractants as affected by EDTA. Chapter 8 Effect of soil amendments on the availability of residual copper 1 93 Lime treatments of 2000 kg ha- ) reduced M l , M3 and TEA-DTPA extractable Cu by 33%, 50% and 23% respectively. In this experiment lime additions decreased the exchangeable Cu by 32%, 3 8%, 23% and 1 5% for the 0, 5 , 1 0 and 20 kg Cu ha- l levels (Figure 8 .7). Thus Cu lost from the exchangeable fractions changed into less soluble, less bioavailable forms as the pH was increased. This finding agreed with Sims ( 1 986), who reported that lime additions reduced exchangeable and sorbed Cu, and increased Cu in more tightly bound forms. The two major factors influencing soil solution Cu and plant Cu concentration in this trial were soil pH and residual Cu. Alva ( 1 992) reported a good correlation between the concentrations of Zn, Fe, Mn and Cu extractable by neutral NH40Ac, M l , M3 and AB-DTPA in soils from citrus groves in Florida. Reed et al. ( 1 993) evaluating the Cu availability for corn using the M3 extracting reagent, found this procedure very promising to detect the deficiency and toxicity levels of Cu in soils . .;::' 1 25 � E' 100 c g 75 � 'E � 50 c 8 03 25 a. a. o _ Before lime � After lime (a ) 1 25 1 00 75 50 (b) o 0 -'---t"�ipOL- o 50 1 00 1 50 200 0 50 1 00 1 50 200 1 25 1 00 75 50 25 o Residual Cu level (mg Cu kg -1 soil ) (c) o 50 1 00 1 50 200 Figure 8.7 Concentration of Cu extracted by (a) Mehlich 1 (Ml ), (b) Mehlich 3 (M3) and (c) TEA-DTPA extractions prior to and after the addition of lime. Correlation studies showed a positive relationship between the exchangeable Cu as affected by N, lime and EDT A additions and Cu extracted by the various extract ants (Ml , M3 and TEA-DTPA) before and after the application of lime (Figure 8 .8). Immobilisation of Cu increases with increasing organic matter (Stevenson and Fitch, 1 98 1 ) . Lime reduced the extractable Cu in all the extractants, but it was more pronounced in the M3 extractant. Chapter 8 Effect of soil amendments on the availability of residual copper --- '7 1 25 Cl .x. Cl .s 1 00 c .Q "§ C 75 Q) (.) c 8 50 --- (a) o 2 3 4 5 '7 1 25 l � ! (d ) .s 1 00 I c .Q "§ C 7 5 Q.l (.) c 8 50 ::J () Q) ::0 25 re U � x 0 LU o 1 2 3 4 5 Exch. Cu affected by N Extractable Cu before l ime addition 1 2 5 1 00 75 50 25 o (b) o 2 3 4 5 Extractable Cu after lime a ddition 1 25 1 00 75 50 25 o (e ) o 1 2 3 4 5 Exch. Cu affected by lime 1 25 1 00 75 50 25 o 1 25 1 00 75 50 25 o + o • 1 94 M1 (c) M3 TE-DTPA o 5 1 0 1 5 20 25 (f) o 5 1 0 1 5 20 25 Exch. Cu affected by EDTA Figure 8.8 Relationship between soils exchangeable Cu as affected by N (a, d), lime (b, e), and EDTA (c, 1) additions and Cu extracted by the Ml, M3 and TEA-DTPA extracting procedures prior to and after lime application. There were significant linear correlations between eu concentration in plants, and exchangeable (r=0.9 1 ) and free eu (r=0.87) in the soil solution for the lime treatment. And between eu concentration in plants, and exchangeable (r=0. 89) and free eu (r=0.79) in the soil solution for the nitrogen treated soil (Figure 8 .9) . Increasing levels of residual eu tended to increase the total, ionic and exchangeable eu concentrations in the soil solution. With the addition of EDT A the plant eu concentration showed a positive relationship with exchangeable eu (r=0.90) and free eu (r=0.94) (Figure 8 . 1 0) . A rise in soil pH due to lime application resulted in a decline in both ionic and exchangeable Cu concentrations and an increase in the proportion of Cu present as Chapter 8 Effect of soil amendments on the availability of residual copper 1 95 soluble organic complexes. It might therefore have been expected that plant Cu concentrations would decrease with an increase in soil pH. However plant Cu concentration was the highest at 1 000 kg ha -1 level . A possible reason for the increase of Cu is the displacement of Cu from cation exchange sites by large amounts of base cations (Cu2+) released into the soil solution during the dissolution of lime. 30 (a ) 30 (b) + + .,.- • I Ol • • � + + + • Ol • 1 • E 20 20 , + .. • I c: ft l / 0 :;::; cu '- + • .- c: (\) 1 0 1 0 u c: 0 + + Nitrogen, r2=O.79 U Nitrogen, r2=O.88 ::J • • Lime, r2=0 87 () Lime, r2=O.91 0 0 0.0 1 ,0 2 .0 3.0 0 .00 0.05 0. 1 0 0 . 1 5 Exchangeable C u (mg kg-1 ) F ree Cu (mg l-1 ) Figure 8.9 Relationship between Cu concentration in plants and (a) exchangeable Cu and (b) free Cn in soil solution with addition of N (+) and lime (e). 60 r2=0.90 60 r2=0.94 • • ---.. ....... • • / I Ol • ..::.:: • • • Ol E 40 • 40 -- • c: • 0 . '" :;::; • • /' � - • •• c: • .... (\) 20 • 20 u • c: • 0 u + ::J (a ) (b) () 0 0 0 1 0 20 30 40 0 ,0 1 .0 2.0 3 .0 Exchangeable Cu (mg kg- 1 ) Free Cu (mg L-1 ) Figure 8.10 Relationship between Cu concentration in plants and (a) exchangeable Cu and (b) free Cu in soil solution with addition of EDT A. Chapter 8 Effect of soil amendments on the availability of residual copper 8.4 CONCLUSION AND FURTHER STUDY 1 96 Both lime and N fertiliser increased the DM yield, but EDT A decreased the DM yield of ryegrass with increasing level of residual Cu. The lowest level o f N increased the Cu concentration and the highest level of lime decreased the Cu concentration. The Cu concentration in ryegrass increased with increasing levels of EDT A. Liming improves the soil condition by alleviating the detrimental effects of soil acidity. The effect of N on Cu concentration persisted beyond the first cut only at the highest N addition level. The present study revealed that the application of 1 000 kg lime ha- 1 and 50 kg N ha-1 was very effective in enhancing the plant availability of residual Cu in soils without any toxicity; but EDT A increased the plant available Cu to toxic levels. Residual Cu had a positive effect on Cu concentration in the herbage. There were positive correlations between Cu concentration in plants and Cu in the soil test extractants. The free and exchangeable Cu in soils were strongly correlated with plant Cu concentration. The highest application rate of lime and N fertiliser decreased the exchangeable and free Cu in soil, but EDT A showed the opposite effect. The plant availability of Cu to plants is often poorly related to the quantity of the element in the soil. Soil properties such as pH, organic matter, CEC, plant species, and environmental factors, such as soil water content, temperature and light, greatly influence the likelihood that a plant will exhibit Cu deficiency or toxicity symptoms. Changes in the environment o ften have a greater effect on Cu concentration in plants. The e ffects of seasonal responses on Cu availability and the transformation of Cu from two different sources of Cu fertiliser are discussed in the next chapter. Chapter 9 CHAPTER 9 Seasonal response of copper availability in pasture SEASONAL RESPONSE OF COPPER AVAILABILITY IN PASTURE Publication arising from this chapter: 1 97 Khan, M. A. R., N. S. Bolan and A. D. Mackay 2000. Seasonal variation of Cu availability in pasture. pp 1 7 1 - 1 86 . In Soil research-a knowledge industry for land-based exporter. L.D. Currie, M.J. Hedley, D.J. Home and P. Loganathan (Eds). Occasional Report No. 1 3 . Fertiliser and Lime Research Centre, Massey University, Palmerston North, 9.1 INTRODUCTION Pasture provides the main source of eu for grazing animals. There is a need to define the rates of change in the effectiveness of eu ferti lisers over the range of soil and climatic conditions encountered in New Zealand, Soil moisture, soil temperature, air temperatures and solar radiation are important parameters that affect the growth of plants. The metabolic disorders caused by eu in grazing animals may be due to excesses or deficiencies of eu in the diet. These disorders are sometimes associated with ingestion of normal amounts of Cu. Retention of Cu by the animal is influenced by Mo and S and these elements are closely implicated in the development of eu disorders. The timing of eu application may also influence pasture Cu concentrations and therefore, the availability of eu and the uptake of Cu by pasture and the grazing animal varies with the seasons. Little information has been published on the seasonal effect of eu applications on pasture and its impact on the overall eu concentration, and status in pasture and the grazing animaL There is no published work on seasonal responses to added Cu on pasture eu concentration. The concentration of nutrient ions extracted by various chemical extractants varied considerably over the course of a growing season under field conditions (Haines and Cleveland, 1 98 1 ; Schnitzer, 1 99 1 ; Barrow, 1 992) . Questions arise as to whether climatic and soil moisture conditions affect the extractabi lity of Cu. Soil samples for microelement extraction are often taken with little regard to field moisture content and stored under a variety of conditions (Shuman, 1 980). In order to study the effects of these practices on extractable Cu, four different soils were incubated under different soil moisture and temperature conditions. Chapter 9 Seasonal response of copper availability in pasture 1 98 It has often been observed that a lift in eu concentration in pasture occurs under field conditions, even after a long period of fertiliser application (A.D. Mackay, Personal communication). This is attributed mainly to the seasonal influences on pasture growth, and eu transformation in soils. Variation of pasture eu concentration due to seasonal responses to added eu is not fully understood. This information is required so that the needs of crops, pastures and the grazing animal can be met, and agricultural production maintained. In this study, the effectiveness of two different Cu fertilisers (CUS04 and CuO) in raising the eu concentration in pasture was examined at different times of the year in the Tokomaru soiL The variation of Cu transformation within the trial periods was also examined. 9.2 MATERIALS ANDMETHODS 9.2. 1 Field trial 9.2. 1.1 Experimental site The study area was at the Massey University Pasture growth unit (Plate 9. 1 ). The average annual rainfall for this area is 1 200 mm. The soil is a Yellow BrownlYellow Grey Earth intergrade and related (Typic Fragiaqualf). There were two eu fertiliser sources (CUS04 and CuO), each applied at 3 rates (0, 2, and 5 kg Cu ha- I) laid out in a randomised design, with three replicates. Seven trials were conducted and the dates of each trial are given in Table 9. 1 . Plots were of uniform size (1 m2) . Chapter 9 Seasonal response of copper availability in pasture 1 99 Plate 9.1 Location of the field trial. Table 9. 1 Time frame of the different trial periods. Trial numbers Date of fertiliser application Date of harvesting Seasons Trial 1 (T1 ) 1 9th March 29th April Autumn Trial 2 (T2) 29th April 1 0th June Autumn Trial 3 (T3) 1 0th June 22nd July Winter Trial 4 (T4) 22nd July 2na September Winter Trial 5 (T5) 2nd September 1 4th October Spring Trial 6 (T6) 1 4th October 24th November Spring Trial 7 (T7) 26th November 6th January Summer 9. 2. 1.2 Soil and pasture sampling A set of 5 soil cores (25 mm diameter) to a depth of 75 mm was collected from each plot prior to and 6 weeks after fertiliser application. The soil samples were air-dried and Chapter 9 Seasonal response of copper availability in pasture 200 the soil aggregates were crushed using a porcelain mortar and pestle. These samples were used for the extraction of Cu. Pasture samples were collected 6 weeks after fertiliser application. The pasture samples were cut using a standard 0 .09 m2 quadrate to the height of the quadrate edges using hand shears. This was approximately within 2 .S cm above the ground level. The samples were weighed and washed with reverse osmosis (RO) water and dried at 70°C in a forced air oven. The dry weights of the pasture samples were recorded and the samples were then ground using a coffee grinder and kept in airtight polyethylene bags for chemical analysis. 9.2.1.3 Soil moisture, soil temperature and climate data Soil samples were placed in an oven at 1 0SoC overnight and the gravimetric moisture content was recorded before and after each set of trials . A data logger was used to record soil temperature. Daily hourly rainfall, radiation and air temperature for the site were obtained from New Zealand Meteorological Services Ltd. 9.2.1.4 Fractionation of Cu and soil analysis Fractionation of soil Cu was carried out in T 2 , T 4, and T 7 soils according to the sequential extraction method described (McLaren and Ritchie, 1 993) in chapter 3 (Section 3 . 2 .6). Hydrochloric acid was used instead of hydrofluoric acid in the residual fraction. Soil pH ( 1 :2 . S H20) and the exchangeable soil Cu [O.O IM Ca(N03)z] were measured. 9.2. 1.5 Pasture analysis Methods for the preparation of rye grass samples for chemical analysis were described in section 4 .2 .S . 9.2.1. 6 Chemical analysis Copper in the plant digests and soil extractants was analysed using F-AAS. Details of these procedures are discussed in section 4.2 .6 . Chapter 9 Seasonal response of copper availability in pasture 201 9.2.2 Incubation study The effect of soil moisture content and temperature on the transformation of Cu was examined under controlled conditions. Four pasture soils (Manawatu, Tokomaru, Ramiha and Ngamoka) taken from the A horizons were air dried and passed through a 2 mm sieve. Soil samples were equilibrated with a solution containing known amounts of Cu as Cu(N03)2 . Soil samples were incubated at four temperatures (5, 1 0, 1 5 and 20°C) and at four moisture levels ( 1 0, 20, 30 and 40%) for four weeks in polythene bags. After incubation, the soil samples were air dried, passed through a 2 mm sieve and used for Cu analysis. 9.2.3 Statistical analysis Statistical analysis of the data was carried out by the Statistical Analysis System (SAS). Details are discussed in section 4.2.7. 9.3 RESULTS AND DISCUSSION 9.3. 1 Climatic data Mean average hourly rainfall for the experimental period (0. 1 0 mm) was above the average rainfall for the previous 1 0 years (0.07 mm) (Figure 9. 1 ). Gravimetric soil moisture content at (0- 1 50mm depth) increased from 1 30 g kg-l soil for Tl to 290 g kg· l soil for T3 and then decreased to 1 90 g kg-l soil for Ts and again increased for T7 (Figure 9. 1 ). Mean daily soil temperature (7S-mm depth) was highest (>1 50C) at the beginning (Tt ) and the end (T7) of the experiment and decreased to 6.4°C for T4. Mean average hourly air temperature was highest for Tt ( 1 5 .4°C) and it decreased to 7.90C for T4 (Figure 9. 1 ). Average daily solar radiation was lowest during T3 (5.65 MJ m-2 dail ) and highest during T7 ( 1 9. 87 MJ m-2 dail ). Chapter 9 Seasonal response of copper availability in pasture 202 20 25 0.2 1 300 ,-., 20 '7 ,-., I ..... E I � � U "Q ... Q C \ '--' N '-' 1 5 '15 -� � lo. :::I '"":I .5 0.1 r ";: 10 � 200 lo. � Col 1 0 c lo. I Q. ..... E .� 1: I � - :::I � � :a c::> r == 5 � c=:: 0.0 I I 0 0 I I I I I I 1 00 April July OctoberJanuary April July October January Trial periods Trial periods Figure 9.1 Weather data for soil temperature (e), air temperature (+), radiation (_), gravimetric soil moisture (T) and rain fall (+) during different trial periods. (Data are mean ±SE for the different parameters at each trial period). 9.3.2 Pasture growth rate Pasture growth was not affected by either Cu source on rates of Cu fertiliser application throughout the trial period. This is consistent with reports that pasture responses to Cu only occur when the Cu concentration in the pasture is <4 mg kg- l (Sherrell and Rawnsley, 1 982). There are very few reported incidences of Cu concentration <4 mg kg" 1 in New Zealand pastures. For example, for New Zealand pasture, the exceptions are peats « 3 mg kg- I ; ) and pumice soils « 4 mg kg- I ;) . Forbes ( 1 978), in a survey of lucerne on Yellow Brown Pumice soils observed very few stands with <4 mg kg-l Cu concentration in the herbage. Khan et al. ( 1 996) observed that Cu applied, as CUS04 fertiliser has no significant effect on pasture growth rates under field conditions. Pasture growth rate was the highest for T7 and the lowest for T3 (Figure 9 .2). Pasture growth rate closely followed temperature and solar radiation, with the exception of T 2, where the lift in pasture growth appears to be in response to an increase in soil moisture with a decline in soil temperature and solar radiation. Hunt and Halligan ( 1 98 1 ) grew perennial rye grass plants in nutrient solution under 6 levels of irradiance at 6 constant temperatures (7 to 33°C) to provide growth response curves to light and temperature over a range of growth conditions_ Growth analysis revealed that at 7, 1 0, 1 7 and 20°C, ;:::::- ·S '" -I QJ) .:..:: QJ) '-' � lo. :::I ..... '" ·S E -·S '" � .£: -� E .:;;: � lo. � Chapter 9 Seasonal response of copper availability in pasture 203 changes in leaf area ratio compensated for changes in the net assimilation rate over a range in irradiance for which the growth rate was maintained near maximum. 1 50 • Control • 2 kg Cu ha -1 • 5 kg Cu ha -1 T7 o April July October January Trial periods Figure 9.2 Pasture growth rates as influenced by different levels of Cu fertiliser at different periods after fertiliser application. Data are means ± SE, n=3. Growth rate was highest at 1 5 . 1 oC (except TI) and lowest at 9 °C air temperature (Figure 9.3a) . During TJ (March-April), the growth rate was very low, which may be attributed to the low soil moisture content ( 1 20 g kg-] soil). Pollock ( 1 990) has found that air temperature exerts a much larger effect on increasing plant growth than it does on the chemical reactions of metabolism. Low temperatures appear to increase cell wall rigidity, which impairs turgor-driven cell extension processes. Naturally expansion growth is a significant component of crop yield. However, in the absence of other constraining factors, and low temperature induced injury, yield may not be diminished by a short duration of low temperature exposure, only delayed. Sarathchandra et al. ( 1 989) observed greater immobilisation of nitrogen and build-up of microbial biomass at low soil temperatures (6- 1 0oC) than at higher soil temperatures ( 1 1 - 1 SoC), resulting in low pasture growth. Cossens and Brash ( 1 98 1 ) reported that low temperature was the main factor restricting the pasture growth rate in a 7 years study in Central Otago, New Zealand. Camp bell et al. ( 1 999) reported that the growth was increased by increasing temperature from 5 to 25°C of seven white clover cultivars in controlled environmental chambers. Chapter 9 Seasonal response of copper availability in pasture 204 Growth rate was the lowest at 7 .SoC and the highest at 1 5 . 1 °c (except T 1) soil temperature (Figure 9 .3b). Growth rate was lowest at the highest moisture level for T 3, which may be attributed to this period having the lowest air temperature and solar radiation. Baker and Younger ( 1 986) demonstrated the effect of soil temperature on the spring growth of perennial ryegrass swards at three contrasting sites. They observed a highly significant relationship existed between leaf extension rates and soil temperature at each site . Soil moisture decreased the growth rate at the highest level of moisture content (Figure 9 .3c). Roberts and Thomson ( 1 984) reported that pasture growth rates (mainly for Lolium perenne L and Trifolium repens L.) for 8 years for a site on the Waimate plains of South Taranaki, New Zealand under 28 day cutting intervals were highest in late spring and rapidly declined over summer. Autumn growth rates were reasonably constant, then declined through winter to the lowest growth rates in mid July. The greatest variation in growth rates between years occurred over the summer and early autumn. Soil moisture and rainfall data indicated that variable summer rainfall caused soil moisture levels to become the critical factor affecting growth rates. Moir et al. (2000) developed a climatic function model, which describes the interacting effects of soil fertility, soil moisture, and evapotranspiration on pasture production. Growth rates increased with increasing radiation (Figure 9 .3d) . During T I (March-April) although the radiation was at the optimum level, the growth rate was very low, which may be attributed to the low soil moisture content ( 1 20 g kg> ] soil) during this period. It is well known that light intensity and temperature influence the quality of pasture plants . The effects of temperature on crop development, and solar radiation on bio-mass accumulation combine to impose well defined limits to potential crop yields under various environmental conditions. The amount of solar radiation defines the maximal limit to crop yield because intercepted solar radiation provides the energy for photosynthetic fixation of CO2. By assuming an upper limit to the efficiency of CO2 fixation (or crop biomass accumulation) per unit of intercepted solar radiation, an estimate can be calculated for the limit to crop .yield based on the available solar radiation during the growing season. Woledge and Dennis ( 1 982) observed that the photosynthetic rate of leaves was twice as high at l SOC than at SOC for perennial Chapter 9 Seasonal response of copper availability in pasture 205 ryegrass (Lolium perenne L.) and white clover (Trifolium pratense L). Ryegrass and clover have similar photosynthetic rates, which responded similarly to temperature. Spaeth et al. ( 1 987) were able to account for much of the yield variability between seasons as a consequence of differences in temperature and incident solar radiation. The radiation use efficiency (RUE) and the harvest index were held constant under all conditions in their calculation. Both the pattern of leaf area development (as determined by temperature) and incident solar radiation in these environments influenced the amount of intercepted radiation, which in turn accounted for differences in yield between seasons in nonstressed soybeans grown in Japan. Yields of nonstressed spring wheat in Israel were examined in terms of environmental variation in temperature and solar radiation (Amir and Sinc1air, 1 99 1 ). Calculated yields across nine seasons, assuming a constant RUE were found to agree well with the observed yields ranging from 410 to 660 g m2 for nonstressed wheat crops. As with maize, the duration of various phases of wheat development were very sensitive to variations in temperature. With warming temperatures, crop development is rapid and the length of time needed for radiation interception is shortened. The greatest yields in spring wheat were achieved under environments with cool temperatures and high radiation (Amir and Sinc1air, 1 99 1 ). Maeda and Y onetani ( 1 9 8 1 ) investigated the light interception by an Italian ryegrass population in each season by measuring the light diminution coefficient (LDC). LDC was determined with a negative regression coefficient between the increase in leaf area index and the decrease in relative light intensity. A close positive correlation was recognised in the autumn and spring. Noble ( 1 972) reported that increasing solar radiation increased the pasture growth rate. Differences in solar radiation patterns between the seasons provide an explanation for the differences in growth rate . In this experiment pasture growth rates followed a strong seasonal pattern with the lowest growth rates in winter (T 3), and the highest in early summer (T 7), which was similar to an earlier field trial (Khan, 1 996) . Lambert et al. ( 1 986) found that poa and ryegrass have maximum growth rates in spring. But most of the low fertility grasses produce maximum growth in late spring and early summer. Similar results were observed by Del Pino Machado ( 1 994), who found that the pasture growth rates ranged Chapter 9 Seasonal response of copper availability in pasture 206 from 1 08 kg DM ha-1 day-l in early summer to less than 2 kg DM ha-I day" 1 in late winter. 1 25 100 75 50 25 o 1 25 1 00 75 50 25 o o 100 (a) 5 1 0 15 Air temperature (OC) (c) T i 20 I t. 1 50 200 250 300 Gravimetric soil moisture (g kg-1soil) 1 25 100 75 50 25 o o (b) I , 5 10 1 5 20 Soil temperature ClC) 125 i (d) o 5 10 1 5 20 Solar radiation (MJ m-2 day-I) Figure 9.3 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture (c) and solar radiation (d) on pasture growth rates. Data are means ± SE, n=3. 9. 3.3 Pasture eu concentration The field study shows differences in seasonal response to added copper. Pasture Cu concentration was highest for T4 for the CUS04 fertiliser and Ts for the CuO ferti liser (Figure 9.4). Cu fertiliser sources have a significant effect on the Cu concentration in pasture. CUS04 resulted in the highest concentration in all trials compared with the CuO fertiliser (Figure 9.4). This might be attributed to the quick release characteristic of the Chapter 9 Seasonal response of copper availability in pasture 207 CUS04 fertiliser (Gi1kes, 198 1) . The general use of CUS04 reflects its high water solubility and wide availability (Alloway and Tills, 1 984; Karamanos et aI., 1 986). The suitability of CuO for broadcasting has been found to depend on particle size. CuO broadcast onto the soil surface at 5 kg Cu ha-l , and worked into the soil did not correct Cu deficiencies in canola, barley, or wheat during the year of application, but corrected the deficiencies in the following years (Karamanos et aI., 1 986). The lack of Cu response to CuO treatment was mainly due to the low water solubility of the coarse, granular CuO, which ranged in particle diameter from <0.2 to 3 . 0 mm. Coarse Cu carriers are also ineffective where inadequate contact occurs between the roots and applied Cu (Gartrell, 1 98 1 ). A similar result was also observed in a previous glass house trial, where three different sources of Cu fertilisers [CUS04, Cu(OHh and CuO] were used in five different soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu) (Khan et al. , 1 998; Section 4 .3 .3 .2) . 25 -+-- Control ,-., -I -+-ell CuS04 � 20 ell -e-=: CuO '-' :: .� 15 - I c: a.. -:: CI.I 10 ... J :: Q ... a.. CI.I 5 Cl. Cl. ! Q U 0 I I April July October January Trial periods Figure 9.4 Effect of fertiliser Cu sources on Cu concentration at 5 kg Cu ha-1 level. Data are means ± SE, n=3. Increasing Cu fertiliser rates increased the Cu concentration in p asture (Table 9.2). Significant seasonal responses to Cu concentration in p asture with added fertiliser was observed (Figure 9 . 5) . The highest Cu concentration was observed in T4 and the lowest concentration in T 6. The maj or effect on Cu concentration was due to pasture growth. Chapter 9 Seasonal response of copper availability in pasture 208 Table 9.2 Effect of fertiliser levels on Cu concentration (CUS04 and CuO). Treatments Trial 1 Trial 2 Trial 3 Trial 4 Trial S Trial 6 Trial 7 Control 8 .40c 9 . S 2 b 7 .27c 8 .00c 1 0 .02c 8 .7Sb 8.94b 2 kg Cu ha· l 1 0.S4b 1 2 . 1 8ab 1 1 . 23b l S .62b 1 3 .3 7b 8 . 89b 9.82b S kg Cu ha" 1 2 . 3 6a 1 3 .03a I S . 5 8a 1 9 .29a 1 7 . S 8 a 1 0.76a 1 O.99a S kg Cu ha"l as CuO 9 . 8 Sbc 1 O.02b 8 .27c 1 0. 1 7c 1 1 .93bc 9.35b 9.26b * Treatment means followed by the same letter Wlthm a column are not slgmficantly dIfferent at the S% level. 25 I Tl 23 + T2 • T3 20 T4 • T5 1 8 *' T6 ""' - V T7 I � ,.:;: � 15 S '-' I: .: -eo:: ... 13 - I: a. '" I: 0 '" ... 10 4i Q- Q-c u 8 5 3 0 0 1 2 3 4 5 Added Cu (kg ha-I) Figure 9.5 Effect of seasonal responses to added Cu on Cu concentration in pasture. Data are means ± SE, n=3. The Cu concentration was highest at 7°C and lowest at 1 4. 5°C air temperature. The Cu concentration decreased with increasing air temperature (Figure 9 . 6a) . It is apparent that Chapter 9 Seasonal response of copper availability in pasture 209 substantial mobilisation of Cu occurs in the root zone from T 4 to T 7 due to the higher growth rate of the pasture. Merry et al. ( 1 986) observed that pasture plants grown at 22°C produced a higher DM yield and Cu concentration than plants grown at 1 2°C. But in this study, the highest eu concentration at low air temperatures was obtained due to a low pasture growth rate. The Cu concentration was highest at 6.4°C and lowest at 1 4. SoC soil temperature at 5 kg Cu ha-I level (Figure 9 . 6b). The Cu concentration was highest during winter and lowest during late spring and early summer. Reddy et al. ( 1 98 1 ) reported strong seasonal variations in Cu concentrations in pasture, the concentrations of eu were low in spring and early summer when compared to winter. They also observed that increasing the soil temperature from 1 2 to 22 °C increased both dry matter production and the concentrations of Cu in subterranean clover on lateritic podzolic soils. The variation in soil temperature during the trial periods, observed in the present study, is likely to affect the availability of organically bound Cu. Organic ally bound Cu in soil is present in both soluble and insoluble forms (Stevenson and Fitch, 1 9 8 1 ) . Mobilisation-immobilisation reactions are undoubtedly temperature dependent, and affect the solubility of soil solution Cu. Differences in soil temperature patterns between the seasons provide an explanation for the differences in Cu concentrations in plants. Reay and Waugh ( 1 983) observed Cu concentrations fluctuated monthly in seasonal variation trials. They also observed that Cu concentration, which fluctuated without seasonal trends in rye grass leafblades, correlated with organic-N concentration. Soil moisture content was highest at T 3 and lowest at T J . It suggested that excessive moisture content, low air and soil temperature may also lead to low Cu concentrations in the T3 period (Figure 9 . 6c). The concentration of Cu increased with advancing time until T 4 and then it gradually declined. Soil moisture increased until T 3, then it decreased and again increased till T7. Copper concentrations increased progressively to a maximum value in September (T 4) due to adequate soil moisture. The Cu concentration was not directly related to radiation, but there was a tendency for eu concentrations to decrease at the highest level of solar radiation (Figure 9.6d). Copper concentration decreased with increasing growth rate of p asture (Figure 9.7) . Chapter 9 Seasonal response of copper availability in pasture 2 1 0 25 20 15 10 5 o o 25 20 15 1 0 5 o 1 00 (a) 11 tT of I , 5 1 0 1 5 20 Air temperature (OC) (c) T i 150 200 250 300 Gravimetric soil moisture (g kg-Isoil) 25 (b) f T 20 15 · t 1. 10 5 o o 5 1 0 1 5 20 Soil temperature �C) 25 (d) 20 15 10 5 o o 5 10 1 5 20 Solar radiation (MJ m-2 day-I) Figure 9.6 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture (c), and solar radiation (d) on Cu concentration. Data are means ± SE, n=3. Chapter 9 Seasonal response of copper availability in pasture 25 • 20 • .- • - I • I:>Li •• • .,:;d • I:>Li • e "- 15 :: • • .Sl • .... ... ) . � • ;.. -:: -; ./!.. • • <11 C.I . ,. • • • • :: Q 10 • • '" t.., .. . ' ;.. • • <11 . . - • • • Q.. ., .. • Q.. • 0 • u 5 o o 25 50 75 100 125 150 Figure 9.7 Relationship between pasture growth rate and Cu concentration. 9. 3.4 Pasture eu uptake 2 1 1 Copper fertiliser sources have a significant effect on Cu uptake in pasture. Cu uptake in pasture increased with increasing levels of Cu fertiliser application. The highest Cu uptake was observed for T7 and the lowest uptake for T3 (Figure 9.8), The total Cu uptake by plants from T4 to T7 increased at all levels of Cu fertiliser application. The increase in Cu uptake in these trials could be caused by an increased growth rate. It is apparent that substantial mobilisation of Cu occurs in the root zone from T 4 to T 7 due to the higher growth rate of pasture. Chapter 9 60 I + • 50 • • 40 V """' - , '" .c � � � '" 30 - Q. ::I .... � Q. Q. Q U 20 1 0 o o Seasonal response of copper availability in pasture Tt T2 T3 T4 TS T6 T7 1 ! I 1 2 3 Added Cu (kg ha-1 ) 2 1 2 T T 4 5 Figure 9.8 Effect of seasonal responses to added Cu on totaJ Cu uptake in pasture. Data are means ± SE, n=3. Increasing air temperature increased Cu uptake. The Cu uptake was highest at 14.5°C and lowest at 7°C air temperature (Figure 9.9a) . Increasing soil temperature also increased Cu uptake (Figure 9.9b). The uptake of both native and added Cu by carrots growing in an acid soil under green house conditions was increased, when the soil temperature was increased from 8 to 20°C (MacMilIan and Hamilton, 1 97 1 ). Brennan et al. ( 1 984) conducted a glasshouse experiment in which wheat plants were grown in soil samples incubated with a Cu fertiliser at various temperatures for different periods. They observed that the dry matter yield and Cu uptake decreased when the incubation temperature increased from 1 0 to 30°C and with a longer period of incubation. Chapter 9 Seasonal response of copper availability in pasture 2 1 3 Root extension rates, size and morphology are strongly affected by soil temperature. An increasing temperature lowers the resistance of roots to water uptake, increases rates of ion diffusion and affects the permeability of membranes to cations and anions (Merry et al. 1 986). Over the range of soil temperatures encountered during the trials, microbial activity and organic matter breakdown could be expected to increase the rate of mineralisation. In field conditions, some of the fluctuations in the concentrations of Cu in the plants may be explained by the dilution caused by increased growth rates. Changes in soil and air temperature, and light intensity affect both the uptake of nutrients by roots and their subsequent translocation to the tops. Temperature affects a number o f factors that are important for metal ion absorption. These include the stability of the metal species, the ligands and the l igand-metal complexes as well as the solubility of the metal ions. The low root temperature limits plant growth through reduced rates of nutrient uptake. Engels and Marschner ( 1 996) reported that the net translocation rates of Fe and Cu at all root zone temperatures were markedly enhanced by increasing the temperature o f the shoot base. Accordingly, the concentrations of Fe and Cu in the shoot were not greatly affected by root zone temperatures, irrespective of the air temperature. These results indicate that the ability of roots to supply Fe and Cu to the shoot are internally regulated by the growth related shoot demand per unit o f roots. Soil moisture has a profound effect on Cu concentration, which decreased at the highest moisture level . Pasture Cu uptake reached a maximum at about 1 90-230 g kg-I soil moisture content (Figure 9. 9c). Increases in soil moisture lead to a marked change in the ion pair formation, free hydrated metal concentrations, and the formation of complexes (Fotovat et af., 1 997) . Soil moisture increased until TJ, then it decreased, and again soil moisture increased till January (T7) ' Copper concentrations in plants increased progressively to a maximum value in September (T4), due to optimum soil moisture content. At optimum soil moisture levels, the Cu concentrations are generally high in the root zone soil solution. It is likely that this mobilisation resulted from the biological production of chelating ligands. Differences in the extent of mobilisation of Cu in the root zone between pasture species growing on the same soil have been observed (Linehan et aI., 1 989). This may reflect differences in the exudation of ligands and lor differences in the micro flora in the root zone. It is clear that mobilisation within the root zone provides a reservoir of soluble micronutrients available for uptake by plants. Sinclair et af. ( 1 990) reported, from nutrient inflow calculations and estimates of the Chapter 9 Seasonal response of copper availability in pasture 2 1 4 volume o f the root zone, that the quantity o f Cu present i n the root zone solution is rapidly absorbed by the growing crop. They also reported that the micronutrients in soil solution are depleted more rapidly than would be expected from nutrient inflow rates. The rate of depletion is driven by nutrient uptake generated by plant growth, it will be modified and limited by the balance between mobilisation and immobilisation within the root zone, which is dependent on microbial activity controlling the flux of organic ligands. Wardle and Parkinson ( 1 990) found that the microbial biomass during the growing season was significantly positively related to soil temperature, but only when the effects of soil moisture variation had been corrected. Seasonal changes in nutrient concentrations in plants have often been attributed to differences in growth rate (Loneragan, 1 975) or changes in soil moisture content, which may affect transport processes in the soil (Wilkinson, 1 972). A decline in soil moisture content would reduce the rate of diffusion, and consequently the uptake of Cu by plants. Porter et al. ( 1 960) reported that the rate of diffusion decreases 6 and 25 fold at 1 and 1 5 atmospheres o f soil moisture potential, respectively. This, o f course, reduces uptake substantially, especially when roots are not growing rapidly enough to exploit new volumes of soil. Nambiar ( 1977) showed that ryegrass absorbed significant amounts of Cu from nearly air-dry soil, when the roots had access to subsoil water. A prolonged period of drying tended to cause a small decrease in the Zn and Cu concentrations of the shoot, particularly Cu. Cu uptake increased with increasing radiation (Figure 9 .9d). During TJ (March-April) although the radiation was at the optimum level, the growth rate was very low, which may be attributed to the low soil moisture content ( 1 20 g kg-J soil) during this period. A significant positive relationship was observed between Cu uptake and pasture growth rate (r2 = 0.83) (Figure 9 . 1 0) . Chapter 9 Seasonal response of copper availability in pasture 2 1 5 60 50 40 30 20 10 o o 60 50 40 30 20 10 -1 o 1 00 (a) 5 1 0 15 20 Air temperature (OC) (c) T 1 50 T 200 250 300 Gravimetric soil moisture ( g kg-1soil) 60 50 40 30 20 1 0 60 50 40 30 20 10 o (b) o (d) T t � * , - : 5 1 0 IS 20 Soil temperature (OC) o 5 1 0 1 5 20 Solar radiation (MJ m-2 day-I) Figure 9.9 Effect of air temperature (a), soil temperature (b), gravimetric soil moisture (c), and solar radiation (d) on Cu uptake. Data are means ± SE, n=3. Chapter 9 Seasonal response of copper availability in pasture 60 R2=0.83 - 50 - - - "'""' 40 - -- -I • -Cl! ..=: - et) '-' -� - -,.:..:: Cl! 30 -- - -Cl. -::: - I-. - -� - -Cl. Cl. • 0 20 .. u . - 10 o o 25 50 75 100 125 150 Figure 9.1 0 Relationship between pasture growth rate and Cu uptake. 9.3.5 Effect of soil temperature and moisture on eu concentration in soils 2 1 6 The effects o f different soil temperatures at various levels o f soil moisture for the different soils under incubation study are i llustrated in Table 9 .3 . The highest Cu extractability was achieved at l SoC soil temperature and 30% soil moisture content. Reddy et al. ( 1 98 1 ) observed that the concentration of CaCh extractable Cu increased with increasing soil temperature. Lindsay and Norvell ( 1 978) reported that an increase in temperature from 1 0 to 30°C during extraction increased DTP A extractable Mn, Zn, Cu and Fe. Williams and McLaren ( 1 982) reported that there was more extractable Cu and solubilised organic matter when temperature was raised from 1 0 to 30°C. They suggest that this extra Cu originated from the organic matter. Changes in soil temperature thus alter the rates and shift the equilibria of chemical reactions between metal ions and soil constituents (Barrow, 1 992). Haynes and Swift ( 199 1 ) have demonstrated that drying increases the amount of Cu, Fe, Zn, and Mn that can be extracted by EDT A, DTP A, and HCI. Oven drying usually has a more pronounced effect than air drying. They suggest that physical disruption and reformation of organo- Chapter 9 Seasonal response of copper availability in pasture 2 1 7 mineral associations i s the main cause for increases in micronutrient extractability. Han et al. (2001 ) observed in their incubation study that soil samples kept under saturated conditions had higher Cu concentrations in the less labile fractions than those subjected to wetting-drying cycles. Table 9.3 Mean Cu concentration in soil solution at various levels of soil temperature and moisture contents for four different soils. Soil temperature Copper concentration (0C) (mg kg- I ) 5 0 . 3 7 1 0 0.37 1 5 0 . 3 9 2 0 0.35 9.3. 6 Seasonal effect on soil pH Soil moisture Copper concentration (g kg- I ) (mg kg- I ) 1 00 0 . 3 2 2 00 0 . 3 3 300 0.4 1 400 0 . 34 The change in Cu concentration in plants varies from trial to trial, it always exhibits a increased or decreased value, which may be sustained for a period, but then decreased towards the low winter values (T3), and then progressively increased to a maximum value (T 4). Soil pH was monitored during each trial period. Soil pH decreased with decreasing air and soil temperature and then increased slowly when all climatic parameters except soil moisture increased. The lowest (PH 5 .0) and the highest (pH 5 .75) values were obtained at the beginning of T3 and T6, respectively (Figure 9. 1 1 ) . There were many changes in the root zone of the pasture including a substantial degree of acidification. After T 3, the exchangeable Cu in the soil decreased with increasing soil pH (Figure 9. 1 1 ) . However, pH has only a small effect on the mobilisation of Cu into the soil solution (Sanders, 1 982; Chapter 3), so it is likely that the mobilisation resulted from the biological production of chelating ligands. Natural complexing substances produced during microbial breakdown of organic matter have the ability to complex Cu into soluble, plant available forms (Stevenson and Filch, 1 9 8 1 ). Nielsen ( 1 976) reported that the plant root might also increase soluble Cu concentrations through an increase in soluble organic matter in the soil resulting from the release of root exudates. Scheffer and Schachtschabel ( 1 989) reported that metal speciation could be affected during a season by changes in soil pH through proton production and consumption by biological Chapter 9 Seasonal response of copper availability in pasture 2 1 8 processes, acid deposition and subsequent alteration of pH dependent chemical equilibria, ego dissolution-precipitation, adsorption-desorption phenomena and metal­ organic complex stabilities in the solid and solute phase. 6.0 • Soil pH 0.50 • -0.45 ..... I 5.5 I:>J: � I:>J: e 0.40 -1:1:: = Q.. 5.0 u - Q.I '0 :0 rrJ 0.35 c; Q.I I:>J: C c; 4.5 .c � 0.30 ;;< J,;o;i 4.0 0.25 I I April July October January Trial periods Figure 9.1 1 Soil pH and exchangeable Cu at different trial periods. Data are means ± SE, n=3. Soil pH has little influence on Cu distribution among the different Cu fractions in soils (Sims, 1 986; Mathur and Levesque, 1 988; Shuman, 1 986). EI-Kherbawy and Sanders ( 1 984) found that pH did not influence Cu in soil solution, but increasing the pH decreased Cu in the exchangeable fractions, making it less bio-available. Increasing soil pH redistributed Cu from the exchangeable and organic fractions to the less bio­ available Mn-oxide and Fe-oxide fractions (Sims and Patrick, 1 978). Thus Cu lost from the exchangeable fractions changed into less soluble, less bio-available fractions as the pH was increased. This finding was corroborated by Sims ( 1 986), who reported that lime additions reduced exchangeable and sorbed Cu, and increased Cu in the more tightly bound fractions. 9.3. 7 Effect on exchangeable Cu The exchangeable Cu in the field soil of the control treatment increased with increasing soil temperatures, but decreased with increasing soil moisture (Figure 9. 1 2). The soil Chapter 9 Seasonal response of copper availability in pasture 2 1 9 moisture content at 0-75mm depth was lowest ( 1 20 g kg-J soil) at TJ and highest (340 g kg-1 soil) at T3 . Soil moisture plays a greater role than temperature in the extractability of eu. Shuman ( 1 980) observed that an increase in temperature resulted in an increase in extractable eu, when soils were incubated at O-bar moisture. Plant roots release organic substances capable of complexing micronutrient cations, although these organic complexing ligands may have a transient existence because rhizosphere microorganisms uti lise them as carbon sources. Despite this such Iigands might, jf produced continuously, play a significant role in micronutrient mobilisation. The release by microorganisms of relatively small amounts of chelating ligands could thus mobilise significant quantities of micronutrient cations. 0.5 0.5 (b) - (a) "...., - ..... I I 01) 01) .::c .::c 01) ! 01) ! El 0.4 El 0.4 -- '-' ::I ::I U ! u ! � � ::c , . " , ::c ! - I! � � � 0.3 � 0.3 I 01) 01) = = � � ..= ..= � � ;0< ;0< � � 0.2 0.2 0 100 200 300 400 0 5 10 15 20 Gravimetric soil moisture (g kg-1soil) Soil temperature (OC) Figure 9.1 2 Effect of (a) soil moisture and (b) soil temperature on exchangeable eu at different trial periods. Data are means ± SE, n=3. 9.3.8 Fractionation o/soil eu Fractionation of soil eu for the control (0 kg eu ha-l ) and 5 kg eu ha-1 level at time 0 and 42 days after fertiliser application is presented in Figure 9. 1 3 . The study indicated that the substantial proportion (>80%) of eu is present in the oxide and organic fractions and only small amounts in the exchangeable and residual fractions. The mean concentrations of eu in the different fractions decreased in the order: oxide bound > organic > residual > exchangeable. Addition of eu fertiliser resulted in an increase in the concentration of eu in all the fractions, but the effect was more pronounced in the oxide and organically bound fractions. Chapter 9 Seasonal response of copper availability in pasture 220 The concentration of eu in the organically bound fractions was highest at T 4 and lowest at T7 . This may be attributed to higher microbial activity at T7 when the soil temperature was high. The reaction of freshly applied eu with soil constituents plays a major role in the availability of eu for pasture. This is often cited as a mechanism for reduced availability of eu fertiliser and the long-term residual effect of eu addition (Brennan et aI. , 1 983). McLaren and Ritchie (1 993) observed that the organicaIly bound and iron oxide bound eu fractions account for most of the applied eu in lateritic sandy soils of Western Australia, irrespective of the rate of application. Similarly Mullins et al. ( l982a) and Liang et al. ( 1 99 1 ) reported that most of the added eu remained as organically bound and sesquioxide occluded eu. Therefore, organic matter and oxide are the major components responsible for the adsorption of added eu. Schnitzer ( 1 99 1 ) reported that metal availability could be affected seasonally by losses and gains of binding sites with the fluctuation of the organic matter content, and changes of functional groups at the surfaces of organic matter due to decomposition and humification processes. The effect of freshly applied eu in the different trials showed a variation of eu concentration in the various fractions, particularly in the exchangeable and organic fractions, which indicates that ion exchange and the formation and dissolution of organic complexes resulted in the variation in eu availability during the different trial seasons. Chapter 9 Seasonal response of copper availability in pasture 2 2 1 8 (a) Trial 2 � - ContrOl, 0 day '0> � ,;,: 6 Control, 42 day Cl) g 5 kg Cu ha-1 ,0 day c;; .2 � � 4 5 kg Cu ha-1 ,42 day C '" u c: 0 u ID 2 0-D. 0 t) 0 8 -, i (b) Trial 4 """ '", ,;,: 6 '" g c: g � 4 C '" u c: 0 " 0; 2 0-D. 0 t) 0 8 (c) Trial 7 , '" ,;,: 6 Cl g c: .2 � 4 C '" u '" 0 u ID 2 D. 0- 0 t) 0 Exchangeable Organic Oxide Residual Forms of Cu Figure 9.13 Fractionation of soils native Cu and Cu added as (CUS04) at different trial periods. Data are means ± SE, n=3. 9.4 CONCLUSIONS The patterns of eu concentration in pasture were related to dry matter accumulation, reaching a maximum at T7, Differences in air and soil temperature, soil moisture content and solar radiation patterns within the trial periods appear to provide an explanation for the differences in pasture growth and Cu concentrations for a freshly applied eu fertiliser. From the present study, it can be concluded that the ability of plants to take up Cu was internally regulated by shoot growth and external ly affected by the transformation of eu in the soils, Chapter 1 0 CHAPTER 10 Summary, conclusion and further study SUMMARY I CONCLUSION AND . FURTHER STUDY Summary and conclusions 1 0. 1 LITERATURE REVIEW 222 The review of the literature revealed the current level of understanding of the soil, pasture, and fertiliser management factors that affect Cu concentration in plants, soils and animals (Chapter 2). Cu in plants functions as part of the prosthetic group of enzyme systems, and as a facultative activator of enzyme systems. Cu deficiency is almost entirely confined to grazing animals due to low levels of Cu in the herbage and/or by elevated intakes of Mo, S and Fe. Fractionation and sorption-desorption studies have shown that eu is strongly adsorbed by organic matter, and AI, Mn, and Fe oxides. Sandy soils low in organic matter and soils derived from volcanic ash are deficient in plant available eu. The effectiveness of pasture topdressing with Cu depends on the nature of the eu fertiliser and soil properties. Fractionation schemes provide an insight into the forms and distribution of eu and its availability. The proportions of the different Cu forms in soils vary considerably, depending on the soil and the fractionation technique used. Common chemical extractions such as Mehlich 1 , Mehlich 3, TEA-DTPA, 0. 1 M Hel, and 0.01 M Ca(N03)z are commonly used to predict the plant available eu with varying degrees of success. The availability of plant Cu declines with increasing time of contact between the soil and eu fertilisers. The rate of decline in the availability of eu depends on the constituents of soil and the solubility of the eu fertilisers. N and P fertiliser compounds tend to exacerbate the eu deficiency in cereal crops, especially when the eu levels in the soils are low. In New Zealand, the pastoral system is legume based, P fertilisers are used to enhance N fixation; N fertilisers are also used on pastures to encourage out of season pasture growth; and lime is used to maintain the soil pH. eu concentrations in forage and pasture crops depend on soil availability of eu, plant species, stage of Chapter 1 0 Summary, conclusion and further study 223 growth, time of year, and fertiliser application. The seasonal pattern of pasture Cu concentration differs with location, soil type, climate, pasture species and management practices. This study was conducted with the overall objective of quantifying the retention and transformation of Cu in soils and the availability of Cu to pasture. Laboratory studies investigated the adsorption and desorption of Cu in pasture soils. This was followed by a series of glasshouse and field trials with different Cu fertilisers, which examined the availability and transformation of Cu in pasture soils. Various chemical extractions were used to predict the plant available Cu. The effect of different amendments on the availability of residual Cu, and the seasonal response to plant availability of Cu through added fertiliser and transformation of Cu in the soil under field conditions were examined. 10.2 ADSORPTION AND DESORPTION OF COPPER IN PASTURE SOILS 10.2.1 Adsorption study The adsorption of Cu at various pH values was measured using five pasture soils (Chapter 3) . In all the soils, sorption of Cu increased with time. The differences in the sorption of Cu between the soils are attributed to the differences in the chemical characteristics of the soils. The Cu sorption followed: Ramiha > Manawatu > Ngamoka > Tokomaru > Mangamahu. The Ramiha, Ngamoka and Mangamahu soils are dominated by both organic matter and oxide components. These three soils also contained relatively high amounts of Al and Fe oxides. Higher pH values, organic matter, and silt and c lay content of the Manawatu and Ramiha soil s contributed to the higher sorption capacity of these soils. Copper sorption, as measured by the Freundlich equation sorption constants (K and N) was strongly correlated with soil properties, such as silt content, organic carbon and soil pH. To investigate the relative contribution of different soil components in the sorption of Cu, sorption was measured after the removal of various other soil components; organic matter and Al and Fe oxides are important in Cu adsorption. The relative importance of Chapter 1 0 Summary, conclusion and further study 224 organic matter and oxides on Cu adsorption decreased and increased, respectively, with increasing solution Cu concentrations. In all soils, Cu sorption increased with increasing pH. Freundlich constant (K) values increased with increasing soil pH in all soils. The solution Cu concentration decreased with increasing soil pH. The Tokomaru soil showed the highest solution Cu due to low levels of organic matter and CEe. 10.2.2 Desorption study The cumulative aIllounts of native and added soil Cu desorbed from two contrasting soils (Manawatu and Ngamoka) during 1 0 successive 2 hrs and 24 hrs de sorption periods showed that the differences in the desorbability of Cu were a result of differences in the physico-chemical properties of the soil matrix. The Ngamoka soil contains a higher amount of organic matter and demonstrated less desorbability of native Cu than the Manawatu soil . Soil samples were incubated for 0, 8 and 28 days with 50 mg Cu kg-I soil . Subsequent successive desorption procedures from day 0 incubated soil resulted in large increases in the amounts o f Cu desorbed, compared to samples where only native Cu was present. The Manawatu soil desorbed more Cu than the Ngamoka soil after both the 2 hrs and 24 hrs de sorption periods. This suggests that soil organic matter complexes Cu added through fertiliser, resulting in decreased desorption. The proportions of added Cu desorbed during 1 0 de sorption periods were low, ranging from 2 .5% in the 24 hrs to 6% in the 2 hrs desorption periods. In both soils, the desorption of Cu decreased with increasing contact times between the soil and the added Cu. The effect of contact time was more pronounced in the Ngamoka soil, which contains a higher level of organic matter. The de sorption of Cu decreased with increasing soil pH. The desorption decreased from 35%, 29%, 1 8% and 1 0% at pH 5 to 1 3%, 7%, 3% and 5% at pH 8 for the Tokomaru, Ngamoka, Manawatu and Ramiha soils, respectively. The irreversible retention of Cu might be the result of complex formation with Cu at high pH. The Tokomaru soil contains less a amounts of soil organic matter and a lower CEC resulting in a large amount of de sorption at both levels (600 and 1 000 mg Cu L-1 ) of added Cu. The Ramiha --- ---------------- Chapter 1 0 Sununary, conclusion and further study 225 soil contains a high amount of organic matter and a high CEC resulting in the lowest desorption of Cu among the soils at pH 5 .0 . 10.3 PLANT AVAILABILITY OF DIFFERENT COPPER SOURCES IN PASTURE SOILS The treatments used in the glasshouse experiment (Chapter 4) include: five soils (Manawatu, Tokomaru, Ramiha, Ngamoka and Mangamahu), three Cu sources [CUS04, Cu(OHh and CuO], and four Cu levels (0, 50, 1 00 and 200 mg kg-1 soil) replicated four times. The summary of results is as follows: • Soils have significant effect on DM yield and also on Cu concentration at all harvests. • Cu concentration and uptake correlated with soil pH, organic matter and the clay content of the soi l . • Increasing levels of Cu increased the plant Cu concentration at all harvests. • The high Cu level (200 mg Cu kg -1 soil) caused a significant decrease in dry matter yie ld, but at the levels of 0, 50 and 1 00 mg Cu kg-1 soil resulted in similar yields. • Cu sources as fertiliser have a significant effect on DM yield, and also on Cu concentration at all harvests. • CUS04 shows a significantly lower DM yield and higher Cu concentration than the other two fertilisers at harvest 1 . • Except for harvests 1 and 2, CuO resulted in significantly higher Cu concentrations at all harvests. • Cu uptake in the ryegrass decreased with increasing time of contact between the fertiliser and the soils. • Cu uptake is directly related to the DM yield of ryegrass. The recovery of Cu by plant uptake was highest for the Cu(OH)2 fertiliser. Recovery of Cu was the highest at the lower levels of fertiliser application. 10.4 TRANSFORMATION AND PLANT UPTAKE OF COPPER IN DIFFERENT SOILS Soils from the preVIOUS glasshouse trial (Chapter 4) were used to examme the transformation of Cu in soils (Chapter 5). The Manawatu and the Tokomaru soils Chapter 1 0 Summary, conclusion and further study 226 contained the highest and the lowest concentrations of total native Cu, respectively. The Cu in the soils was fractionated into its different fonns. The fractionation procedure achieved nearly complete (>80%) recovery of the total Cu from the soils. The mean concentrations of Cu present in the different fractions decreased in the order: oxide bound > organic > residual > exchangeable. All fractions declined with time after fertiliser application, indicating that the development of a strongly bound fraction that is not extractable with triacid digestion. The sand fractions contributed the lowest amount of Cu from all soils compared to the silt and c lay fractions. In the Manawatu and Ramiha soils the silt fraction contained the highest amount of Cu, but this was not the case with the other three soils, where the c lay fraction held the most Cu. The silt and clay fractions contributed the highest amounts of Cu in these five soils. The organically bound Cu was highest in the organic soils compared to the mineral soils. In most of the soils, organic and oxide bound Cu initially increased and then decreased from time of fertiliser application. Increasing soil organic matter showed a negative relationship with exchangeable Cu but a positive relationship with the other fractions. Increasing CEC and clay content showed a negative relationship with the exchangeable Cu, but a positive relationship with the other fractions. S ince a majority of soil eu is associated with organic matter, it would be expected that organic matter additions would cause a redistribution of eu, leading to an increase of the organically bound Cu. The concentration o f Cu in the different fractions was affected by the nature of the Cu fertiliser. Soon after fertiliser application, CUS04 resulted in the highest concentration of eu in the organic fraction. However with increasing time after fertiliser application the organic and the oxide bound Cu decreased in both (CUS04 and CuO) fertilisers. The relationship between the amounts of Cu in the individual fractions of different soils, and the plant Cu uptake, showed that organic and oxide bound eu were correlated with plant eu uptake. eu associated with oxides and organic matters are more important to Cu availability than Cu in the other fractions. The Cu concentration and total uptake by ryegrass varied directly with the amounts of eu in the different fractions (organic, Chapter 1 0 --------------- - Summary, conclusion and further study 227 oxides), total Cu, EDT A extractable Cu, CEC and clay content. The availability of applied Cu for uptake by ryegrass declined with time of contact between the soil and the applied Cu. A significant relationship was obtained between EDTA-extractable Cu and plant Cu concentration and Cu uptake. 1 0.5 SOIL TESTS TO PREDICT THE AVAILABILITY OF COPPER The efficiency of vanous soil test extractants (O. O IM Ca(N03)2, O. l M NaN03, O.OIMCaCh, 1 .OM NH4N03, O. l M HCI, O.02M SrCh, Mehlich- l , Mehlich-3, and TEA­ DTP A.) to predict the availability of Cu is discussed for two contrasting soils treated with two sources of Cu fertilisers (Chapter 6). CuO fertiliser resulted in higher concentrations of exchangeable Cu at 1 9 1 days after fertiliser application, which was significantly more than the CUS04 fertiliser. This may be due to the slow releasing characteristic of CuO fertiliser. It appears that the organic and oxide bound fractions are in equilibrium and they constitute the major potential plant available Cu pool in soils. The efficiency of various chemical reagents in extracting the Cu from the soil followed this order: TEA-DTPA > Mehlich-3 > Mehlich- l > 0.02M SrCh > O. l M HCI > l .OM NH4N03 > O .OIMCaCb > O . IM NaN03 > O .OIM Ca(N03)2 - H igher amounts of Cu were extracted by the soil test extractants from the Manawatu than the Ngamoka soil. This may be due to the low level of organic matter in the Manawatu soil. In pasture soils, high levels of Cu application have resulted in an increase in extractable soil Cu. The ratios of exchangeable: organic : oxide bound: residual forms of Cu in Mt , M3 and TEA-DTPA for the Manawatu soil are 1 :20:25 :4, 1 : 14 :8 :2 and 1 : 56 :35 :8 , respectively_ The values of the Ngamoka soil are 1 : 1 4 :6 :4, 1 :9 :5 :2 and I : 55 :26 : 1 7, respectively. The ratios of different forms of Cu suggest that the Cu is residing mainly in the organic form and it decreases in the order: organic > oxide > residual > exchangeable. In both soils, more than 98% of the Cu extracted by the three soil test reagents was in a complexed form and only a small amount of Cu remains as the ionic form. Manawatu soil had a significantly higher concentration of free Cu2+ than the Ngamoka soil. The M l , M3 and DTPA extracted 44%, 56% and 65% of the organically bound Cu from the Manawatu soil as compared to 63%, 77% and 72%, respectively from the Ngamoka Chapter 1 0 Summary, conclusion and further study 228 soil. M3 and TEA-DTPA extracted more organically bound eu than M l . TEA-DTPA and Ml extracted more oxide bound eu and TEA-DTPA and M3 extracted more residual eu and exchangeable eu. A large variation in the extractable eu was observed for the soil samples. There was a highly significant relationship between the concentrations of eu extracted by the three soil test extractants. This indicates that in soils that have received recent eu additions, M3, TEA-DTPA and M l extractants are equally effective for extracting eu from the soil . The determination of the coefficients obtained from the regression relationship between the amounts of eu extracted by M l , M3 and TEA-DTPA reagents suggests that the behaviour of extractants were similar. This similarity can be explained by the fact that M3 and TEA-DTPA solutions contain the complexing agents that extract the eu complexed with organic matter, an important soil pool of eu, whereas Ml may extract the same eu ions preferentially from mineral surfaces. 1 0.6 10. 6. 1 EFFECT OF LIME, EDTA, NITROGEN AND PHOSPHATE FERTILISERS ON THE AVAILABILITY OF COPPER. Effect of nitrogen and phosphorus fertilisers on the availability of native copper. Two contrasting soils (Manawatu and Ramiha) were used in a glasshouse experiment to examine the effect of N and P fertil isers on the availability of native Cu (Chapter 7). The Ramiha soil resulted in higher dry matter yields and a lower Cu concentration, but higher Cu uptake when compared to the Manawatu soil. The variation in Cu concentrations in rye grass between the two soils varied with differences in shoot growth and organic matter content of these soils. Increasing levels ofN increased the DM yield, eu concentration and Cu uptake. An increase in N uptake increases the amino acid concentration in the roots leading to greater translocation of eu from roots to shoot. Increasing levels of P significantly increased the DM yield and decreased the concentration of Cu in the plants but increased Cu uptake. The decrease in Cu concentration may be due to dilution of Cu in plant tissue through the promotion of plant growth by phosphate fertilisers. It is also possible that reduced exploitation of the soil by mycorrhizae at high P levels may also result in reduced Cu concentration in plants. Chapter 1 0 Sununary, conclusion and further study 229 10.6.2 Effect of lime, ED TA and nitrogen fertiliser on the availability of residual copper. Soil samples obtained at the termination of the first glasshouse plant growth experiment (Chapter 4) were used to examine the effect of lime, EDTA and N fertiliser on the uptake of residual Cu from two fertiliser (CUS04 and CuO) sources, applied at four Cu levels to two contrasting soils (Manawatu and Ngamoka) (Chapter 8) . The DM yields were significantly higher for the Manawatu soil than the Ngamoka soil at each harvest with the addition of N fertiliser and lime. Copper concentration was higher in the Manawatu soil with the addition of N fertiliser and EDT A, but lower with a lime application. Fertiliser Cu sources have a significant effect on DM yield and Cu concentration in ryegrass. CUS04 fertiliser resulted in higher dry matter yields and a lower Cu concentration. Lime and EDTA applications resulted in the highest and the lowest DM yields, respectively, but had the opposite effect on Cu concentration. The residual Cu level at 20 kg Cu ha- I for CuO fertiliser giving in the highest concentration of Cu at all harvests, and this was significantly different to the others at all harvests for N, lime and EDT A applications. The residual Cu rates significantly influenced the Cu concentration in plants with the addition of EDT A. Additions of N fertiliser and lime also resulted in an increase in the Cu concentration in shoots with increasing rates of residual Cu. The treatment level of 1 00 kg N ha-J produced the highest DM yield and Cu concentration at both harvests, and it was significantly (p < 0.05) different from the control treatment. The lime treatment at 2000 kg ha- I produced the highest DM yield at all harvests and it was significantly (p < 0.05) different from the control . The lime level at 1 000 kg ha- 1 resulted in the highest Cu concentration at harvest 1 , and that was significantly different from the other treatments. The lime level at 2000 kg ha-1 decreased Cu concentration at all harvests. Dry matter yield decreased with increasing levels of EDT A application. Additions of EDT A resulted in an increase in Cu concentration in ryegrass. Chapter 1 0 Summary, conclusion and further study 230 The amount of plant available Cu, as measured by the three soil test extractants, i s affected by N, lime and EDT A. Significant relationships between Cu concentration in plants and Cu in the soil test extractants were observed. Both N and lime applications decreased the exchangeable Cu and the free ionic Cu at the highest application rate in the Ngamoka soil, but EDTA increased the amount of exchangeable Cu resulting in phytotoxicity of ryegrass. The Ngamoka soil showed less of an increase in exchangeable Cu with increasing rates of EDTA addition compared to the Manawatu soil. Higher amounts of Cu was extracted from the Manawatu soil by the M3 and TEA­ DTPA extractants before hming. Lime reduced the extractable Cu by the M l , M3 and TEA-DTPA extractants, and it was more pronounced in M3. EDTA increased the amount of plant available Cu, as measured by the Mt , M3 and TEA-DTPA extractants. 1 0.7 SEASONAL RESPONSE OF COPPER AVAILABILITY IN PASTURE Two Cu fertiliser sources (CUS04 and CuO), each applied at 3 rates with three replications were used in the field experiment in the Tokomaru soil (Chapter 9). Seven trials were conducted at different times within the same paddock. Both sources and rates of Cu fertiliser application throughout the trial period did not affect pasture growth. Pasture growth rate was highest in summer and lowest in winter. Pasture growth rates closely followed temperature and solar radiation, with the exception of the autumn period where the lift in pasture growth appears to be in response to an increase in soil moisture. Cu fertiliser sources have a significant effect on Cu concentration in pasture. CUS04 resulted in higher Cu concentrations in all trials compared to the CuO fertiliser. This might be attributed to the quick releasing characteristic of CUS04 fertiliser. Application of Cu fertil isers increased the Cu concentration III pasture. The Cu concentration was highest at 6.4°C and lowest at 14.SoC soil temperatures at the 5 kg Cu ha-1 level. Soil moisture has a profound effect on Cu concentration and it decreased at the highest moisture level. There was a strong seasonal variation in Cu concentration in Chapter 1 0 Summary, conclusion and further study 23 1 the pasture; the concentrations of Cu were low in spring and early summer as compared to late winter. Copper fertiliser sources have significant effect on Cu uptake in pasture. Cu uptake in pasture increased with increasing levels of Cu fertiliser applied. The highest Cu uptake was noticed in summer and the lowest in winter. It is apparent that substantial mobilisation of Cu occurs in the root zone in late winter / early summer due to the higher growth rate of pasture during this periods. Increasing air and soil temperature, and solar radiation increase the Cu uptake. The concentration of Cu in the organically bound fractions was the highest in late winter and the lowest in summer. This may be attributed to increased microbial activity in summer due to high soil temperatures leading to increased mineralisation of organically bound Cu. The reaction of freshly applied Cu with other soil constituents plays a major role in the availability of Cu for pasture. This is often cited as a mechanism for reduced availability of Cu from fertiliser and the long-term residual effect of Cu application. The variation of soil temperature during the trial periods, observed in the present study, is likely to affect the availability of organically bound Cu. Organically bound Cu in soils is present in soluble and insoluble forms. Mobilisation­ immobilisation reactions are temperature dependent and affect the solubility of soil solution Cu. The effect of applied Cu in each trial showed variations in the Cu fractions, particularly in the exchangeable and organic forms, which suggests that reactions of Cu with soil organic matter causes differences in Cu availability in the different trial seasons. Differences in air and soil temperatures, soil moisture and radiation pattern within the trial periods provide an explanation for the differences in pasture growth and Cu concentrations for freshly applied Cu fertil iser. It can be concluded that the ability to take up Cu is internally regulated by the growth of plant shoots and externally affected by the transformation of Cu in soils. Chapter 1 0 Summary, conclusion and further study 232 1 0.8 SUGGESTION FOR FUTURE STUDY The total Cu content in soils is not available for plant uptake due to its strong complex formation with organic matter as observed in the different experiments. The chemical forms of Cu extracted by soil test reagents from pasture soils varied considerably with the organic matter content. Further studies should be carried out to determine the influence of other soil properties (eg. nature and amount of c lay minerals and forms of organic matter) on the estimation of Cu availability so that a better standardisation of the methods could be achieved. The concentration and Cu uptakes are strongly related to soil properties such as pH, organic matter, CEC, clay and silt content. Positive relationships generally existed between organic and oxide bound Cu and plant uptake, indicating that these two forms are the potential sources of plant available Cu. The uptake of native Cu and residual Cu from two contrasting soils showed that N and lime at (50 kg N ha" ! and 1 000 kg lime ha"! ) levels increased the Cu concentration, and EDTA also increased the plant available Cu to toxic levels. The effect on Cu concentration persisted beyond the first cut only at the highest N addition level. Low levels of lime induce the mineralisation of organic matter and so release the organically complexed Cu into the soil solution. Low levels of EDTA should enhance the optimum level of available Cu. The effect of N, lime and EDTA on the availability of residual Cu in ryegrass needs further investigation. The transformation of Cu under both glasshouse and field conditions resulted in a decrease in the organic forms of Cu with time after fertiliser application. 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