Investigations into the uptake and effects of long-term cadmium and arsenic exposure on the earthworm Eisenia fetida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Wellington, New Zealand
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2020
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Massey University
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Abstract
Cadmium (Cd) and arsenic (As) are trace elements that differ in their chemistries but are both highly toxic, and common soil contaminants in agricultural land and contaminated sites. Their potential impacts range from adverse effects on soil-dwelling organisms to uptake into food and leading to human exposures. This study examines the uptake and effects of Cd and As individually and in mixtures primarily on the earthworm Eisenia fetida on up to three consecutive generations, and the potential for recovery when exposure ceases. Exposure ranges were selected to minimise mortality and permit reproduction. Key variables examined were contaminant concentrations and associated trace elements in worm tissue, growth, reproduction, and levels of gene expression.
In worms exposed to Cd-spiked soils, Cd accumulation was rapid. Three key factors determining [Cd] in worm tissue were exposure level, time, and the bioconcentration factor (BCF), which increased with decreasing soil [Cd]. Results indicate that for all exposure conditions, and given enough time, Cd accumulation will continue until a lethal tissue level is reached. This point may be either before or after reproduction has occurred (depending on circumstances), but indicates a need to re-examine standardised approaches to toxicity testing for cumulative and biologically persistent contaminants such as Cd. The biological half-life for Cd loss was 6.5 months. This implies that worms that have been exposed to elevated Cd for more than a few weeks would unable to eliminate much of the accumulated burden over their normal lifetimes.
Worms exposed to Cd took longer to reach sexual maturation, and at higher exposures, cocoon production progressively decreased from generation to generation. However, there were differences depending on the exposure level. At the lowest level (30 mg/kg), first generation worms returned to clean soil showed a large rebound effect, and by the third generation there was a recovery in cocoon production. By contrast, for higher (90 and 270 mg/kg) and longer (56 d and 84 d) exposures worms performed more poorly, suggesting that there is a tissue Cd threshold beyond which recovery becomes challenging. Evidence from gene expression results are consistent with the idea that this threshold corresponds to a point at which the Cd-sequestering protein metallothionein (MT) has reached saturation, as can also occur in human kidney tissue. Below this point, worms transferred to clean soils will recover. Above it, they will not.
As (spiked as arsenate, AsO₄³⁻) also accumulated in worm tissue with exposure concentration and time, but showed some distinct differences compared to Cd. Modest As exposure extended for longer than 28 d had the unusual effect of stimulating growth and causing excessive cocoon production, an effect likely to be missed in most standardised tests. The effects are not thought to be related to parasite suppression, because they were accompanied by large-scale changes to gene expression. Despite appearing beneficial, by the second generation it was clear that effects of the As exposure were overwhelmingly negative, both in terms of extremely low survival rates and the delayed growth of surviving earthworms.
Perhaps more notably, results for both the lower exposure condition (10 mg/kg soil As) and As-exposed worms returned to clean soils, suggest there are circumstances where As may promote its own uptake in a positive feedback loop. If correct such an effect may be linked to an increase in uptake of phosphate (PO₄³⁻) for cellular repair, with co-uptake of arsenate (AsO₄³⁻, which is isomorphous). A parallel mechanism is known for marine fish. Remarkably, results suggest that Cd exposure may have also caused an increase in As uptake, from soils that contained only natural [As]. Though a tentative finding, such an effect would be consistent with the idea that any contaminant that causes cellular damage in an invertebrate may trigger a need for more soil phosphate, presumably with some As co-uptake. This would also imply that many (presumed) single contaminant exposures whether in the laboratory or the field may in fact be As co-exposures.
Relevant to this, the adverse impacts of As and Cd co-exposure were found to be more severe than effects of exposure to either contaminant alone; despite the fact the lower amounts of each contaminant were taken up under the co-exposure condition. This result supports an argument that soil guideline values derived from single contaminant toxicity experiments may be insufficiently protective for soil invertebrates in many real-life settings.
Gene expression results were useful as an interpretive tool, with numbers and overlaps of differentially expressed genes being more useful than knowledge of the subset of named genes and their putative functions. Exposure to Cd or/and As triggered large-scale changes in gene expression, indicating ‘organism-wide’ biochemical responses and providing circumstantial evidence that supported particular interpretations, such as existence of an MT saturation-threshold, and the existence of substantive biochemical changes between lower and higher As exposures. Analysis of differentially expressed genes in common between Cd-only, As-only and co-exposure suggests existence of both similar and different impacts of toxicity under the three conditions.
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Appendix 1: Published paper DOI:10.1016/j.fct.2017.09.002 has not been removed in accordance with Elsevier's policy on article sharing: "Theses and dissertations which contain embedded PJAs as part of the formal submission can be posted publicly by the awarding institution with DOI links back to the formal publications on ScienceDirect" (https://www-elsevier-com.ezproxy.massey.ac.nz/about/policies/sharing).
Keywords
Eisenia foetida, Effect of chemicals on, Cadmium, Arsenic, Physiological effect