Detecting the effects of chronic metal exposure on benthic systems: Importance of biomarker and endpoint selection.

Understanding metal toxicity to benthic systems is still an ecotoxicological priority and, although numerous biomarkers exist, a multi-biomarker and endpoint approach with sediment as the delivery matrix combined with life-history relevant exposure timescales is missing. Here we assess potential toxicity by measuring a suite of biomarkers and endpoints after exposing the ecologically important polychaete Alitta(Nereis)virens to sediment spiked with environmentally relevant concentrations of copper and zinc (and in combination) for 3, 6 and 9 months. We compared biomarker and endpoint sensitivity providing a guide to select the appropriate endpoints for the chosen time frame (exposure period) and concentration (relevant to Sediment Quality Guidelines) needed to identify effects for benthic polychaetes such as A. virens. Target bioavailable sediment and subsequent porewater concentrations reflect the global contamination range, whilst tissue concentrations, although elevated, were comparable with other polychaetes. Survival reduced as concentrations increased, but growth was not significantly different between treatments. Metabolic changes were restricted to significant reductions in protein after 9 months exposure across all copper concentrations, and reductions in lipid at high copper concentrations (3 months). Significant changes in feeding behaviour and increases in metallothionein-like protein concentration were limited to the medium and high copper and zinc concentrations, respectively, both after 6 months exposure. Despite data highlighting A. virens' metal tolerance, DNA damage and protein concentrations are the most sensitive biomarkers. Copper and zinc cause biomarker responses at concentrations routinely found in coastal sediments that are characterised as low contamination, suggesting a reappraisal of the current input sources (especially copper) is required.

Identifying conserved polychaete molecular markers of metal exposure: Comparative analyses using the Alitta virens (Annelida, Lophotrochozoa) transcriptome.

Polychaetes are vital for evaluating the effects of toxic metals in marine systems, and sensitive molecular biomarkers should be integral to monitoring efforts. However, the few polychaete markers that exist are inconsistent, even within the same species, failing to identify gene expression changes in metal-exposed animals incurring clear metabolic costs. Comparing previously characterised polychaete metal-responsive genes with those of another carefully selected species could identify biomarkers applicable across polychaetes. The ragworm Alitta virens (Sars, 1835) is particularly suited for such comparisons due to its dominance of fully saline coastal areas, widespread distribution, large biomass, and its phylogenetic position relative to other polychaete 'omic' resources. A transcriptome atlas for A. virens was generated and an RNASeq-qPCR screening approach was used to characterise the response to chronic exposures of environmentally relevant concentrations of copper and zinc in controlled mesocosms. Genes presenting dramatic expression changes in A. virens were compared with known metal-responsive genes in other polychaetes to identify new possible biomarkers and assess those currently used. This revealed some current markers should probably be abandoned (e.g. Atox1), while others, such as GST-Omega, should be used with caution, as different polychaete species appear to upregulate distinct GST-Omega orthologues. In addition, the comparisons give some indication of genes that are induced by metal exposure across phylogenetically divergent polychaetes, including a suite of haemoglobin subunits and linker chains that could play conserved roles in metal-stress response. Although such newly identified markers need further characterisation, they offer alternatives to current markers that are plainly insufficient.

Chronic exposure to copper and zinc induces DNA damage in the polychaete Alitta virens and the implications for future toxicity of coastal sites.

Copper and zinc are metals that have been traditionally thought of as past contamination legacies. However, their industrial use is still extensive and current applications (e.g. nanoparticles and antifouling paints) have become additional marine environment delivery routes. Determining a pollutant's genotoxicity is an ecotoxicological priority, but in marine benthic systems putative substances responsible for sediment genotoxicity have rarely been identified. Studies that use sediment as the delivery matrix combined with exposures over life-history relevant timescales are also missing for metals. Here we assess copper and zinc's genotoxicity by exposing the ecologically important polychaete Alitta virens to sediment spiked with environmentally relevant concentrations for 9 months. Target bioavailable sediment and subsequent porewater concentrations reflect the global contamination range for coasts, whilst tissue concentrations, although elevated, were comparable with other polychaetes. Survival generally reduced as concentrations increased, but monthly analyses show that growth was not significantly different between treatments. The differential treatment mortality may have enabled the surviving worms in the high concentration treatments to capture more food thus removing any concentration treatment effects for biomass. Using the alkaline comet assay we confirm that both metals via the sediment are genotoxic at concentrations routinely found in coastal regions and this is supported by elevated DNA damage in worms from field sites. However, combined with the growth data it also highlights the tolerance of A. virens to DNA damage. Finally, using long term (decadal) monitoring data we show stable or increasing sediment concentrations of these metals for many areas. This will potentially mean coastal sediment is a significant mutagenic hazard to the benthic community for decades to come. An urgent reappraisal of the current input sources for these 'old pollutants' is, therefore, required.

Effects of a POEA surfactant system (Genamin T-200(®)) on two life stages of the Pacific oyster, Crassostrea gigas.

Surfactants used in herbicide formulations are generally considered inert with no toxic effects on animals. Polyethoxylated tallow amines (POEAs) are non-ionic surfactants used in many herbicide formulations to promote the penetration of the active matter into plant cuticles. The present study aimed to assess the toxicity of a POEA surfactant system, the Genamin T-200®, on two larval stages of the Pacific oyster, Crassostrea gigas. The embryotoxicity of Genamin T-200® was quantified after 36 hr of exposure, considering both arrested development and abnormalities in D-shaped larvae. The ability of pediveliger larvae to metamorphose was studied after 24 hr exposure to Genamin T-200®. According to the European toxicity classification, the present results suggest that Genamin T-200® could be considered very toxic to embryo larval development, with an EC50 of 262 µg/l, and toxic to metamorphosis processes with an EC50 of 3,027 µg/l. The high toxicity of glyphosate-based formulations compared to the active ingredient and its by-product appears to be due primarily to surfactants.