RESPONSES OF THE DECAPOD CRUSTACEAN, CARCINUS MAENAS, FOLLOWING EXPOSURE TO ENVIRONMENTAL CONTAMINANTS. DEVELOPMENT AND APPLICATION OF MULTIPLE BIOMARKERS.

Abstract

Marine, coastal and estuarine ecosystems continue to be contaminated by anthropogenic chemicals. Improving our mechanistic understanding of the toxicity of environmental pollutants to aquatic organisms is therefore an important priority. This work is focussed on detecting and measuring the molecular, cellular and physiological responses of the shore crab Carcimis maenas following exposure to environmental chemicals, and uses biomarkers to do so. To better understand the response of C.maenas to polycyclic aromatic hydrocarbons (PAHs) a novel biomarker of exposure to this class of compounds was developed. Direct fluorimetric techniques revealed that the shore crab C.maenas eliminated metabolites of pyrene in the urine following waterbome exposure to this PAH in the laboratory. The levels of excreted metabolites were exposure concentration and time dependent. Levels of pyrene metabolites were also detected in the haemolymph and their levels were exposure concentration dependent. HPLC/F analysis of urine samples revealed metabolites were conjugates of 1-OH pyrene. Levels in urine determined by fluorimetry correlated well with HPLC/F and ELISA methods of analysis. Levels in haemolymph did not correlate well with the ELISA, due to sample matrix interference with the assay. Detection of urinary PAH metabolites following laboratory exposure is evidence that the parent PAH has been taken up, metabolised by the enzymatic machinery of the organism and eliminated. Whilst this is an important finding and illustrates the response of Cmaenas when faced with PAH exposure, confirmation of this phenomenon in the field is also required. Field trials revealed that comparable metabolites were eliminated in the urine following exposure to pyrogenic PAH discharges from a Norwegian aluminium smelter. The assay also provided evidence of exposure to petrogenic PAH in crabs from petroleum contaminated field sites and crabs exposed to crude oil. Both laboratory and field studies illustrated the potential for this exposure biomarker to be used for environmental monitoring. The responses of C.maenas following exposure to combinations of PAH, metal and pesticide were measured using biomarkers of exposure and effect. Increasing the number of contaminants did not affect the responses of certain biomarkers (metallothionein, micronucleus, antioxidant activity), which were not induced or remained largely unchanged. These biomarkers also showed no significant differences between exposed and control groups. Specific biomarkers of exposure (urinary PAH assay) and effect (carboxylesterase activity) responded in a concentration dependent manner when exposed to PAH and pesticide, but the level of their response was not altered upon addition of further contaminants at each concentration. The lack of induction or alterations in biomarker responses unfortunately provides little additional information on the complex mechanisms of toxicity in organisms following exposure to these contaminants. Shnilarly, investigations into the effects of environmental chemicals on endocrine mediated processes in C.maenas did not provide conclusive information on mechanisms of endocrine toxicity. Exposure to 20-hydroxyecdysone and its insecticidal analogue tebufenozide had no gross effects on the processes investigated in C.maenas (moulting, vitellogensis and locomotor activity) and consequently, no suitable endpoints were established as markers of endocrine disruption in this species. The quantitative Vg ELISA proved to be applicable for monitoring potential endocrine disruption in female C.maenas, but only after the temporal progression of vitellogenesis is understood. The present work has investigated and measured the responses of C.maenas following exposure to exogenous chemicals. This has resulted in a better understanding of PAH disposition, a field applicable PAH exposure biomarker and evidence for robust responses following exposure to multiple contaminants. It has also highlighted the need for greater understanding of endocrine mediated processes before endocrine disruption can be confirmed as a realistic mechanism of toxicity in this species.