Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): do mussels "integrate" biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (alpha-HCH, aldrin, dieldrin, p,p'-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.

Field validation of antioxidant enzyme biomarkers in mussels (Perna viridis) and clams (Ruditapes philippinarum) transplanted in Hong Kong coastal waters.

Green-lipped mussels, Perna viridis, and Manila clams, Ruditapes philippinarum were sourced from "clean" sites in the Hong Kong region, depurated in a laboratory using uncontaminated filtered seawater for 8 days, and transplanted to a suspected gradient of chemically polluted sites in Hong Kong. After 14- and 28-days of field exposure, several antioxidant parameters including glutathione S transferase (GST), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH) were quantified in gill and hepatopancreas tissues. Whole body tissue concentrations of polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons (PHCs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs) were determined in pooled site samples. Chemical analyses indicated that: (a) clams had higher levels of PAHs, PHCs, DDTs and PCBs, whereas mussels had higher hexachlorocyclohexane (HCHs) and there was no difference between species for dieldrin and remaining OCs; (b) Kat O should not be continued as a "clean" reference site for Hong Kong, because of the levels of contaminants measured and (c) PAH concentrations in the current survey were similar to those previously measured. Toxicological conclusions were: (a) antioxidant responses were different between species; (b) CAT and GST have highest utility in clams for field use in Hong Kong, whereas CAT in both gill and hepatopancreas tissue showed most potential in mussels; (c) significant induction of antioxidant responses over day 0 (excluding GPx in both tissues, and GST in mussel hepatic tissue); (d) groups of contaminants do not consistently induce antioxidant responses and (e) organochlorines and PCBs correlated significantly with CAT and GST in clam hepatopancreas and with CAT in mussel gill and hepatic tissue. Multivariate statistical techniques indicated little relationship between the site patterns for antioxidant responses and the contaminant gradients identified in body burden analysis.

Uptake and depuration of PAHs and chlorinated pesticides by semi-permeable membrane devices (SPMDs) and green-lipped mussels (Perna viridis).

Semi-batch seawater experiments were conducted to follow the uptake and release of selected PAHs (anthracene, fluorathene, pyrene and B[a]P) and organochlorine pesticides (alpha-HCH, aldrin, dieldrin, p,p'-DDT) in semi-permeable membrane devices (SPMDs) and green-lipped mussels (Perna viridis). Mathematical models were applied to describe the uptake and elimination curves of the contaminants for SPMDs, and kinetic parameters, such as uptake rate constants, and equilibrium triolein/water partitioning coefficients were calculated. SPMD data showed a good fit to estimate rate constant and partition coefficient equations, but only those contaminants which partitioned mainly in the dissolved phase (alpha-HCH and dieldrin) were well explained for mussels. Poor conformity of the other contaminants indicated mussels uptake by routes other than diffusion, such as ingestion of algae. An apparent equilibrium state was only noted for alpha-HCH in mussels. Aldrin was not detected in mussels in the first few days of exposure, indicating potential metabolism of this compound. B[a]P was not detected in the triolein of SPMDs, which suggests that the membrane may act as a reservoir. Loss of spiked B[a]P from the triolein was evident in a depuration experiment, which may indicate transfer to the membrane. Rate constants for mussels were higher than those for SPMDs, but the reverse was true for partition coefficients. Overall, mussels and SPMDs had similar uptake rates for all compounds in this study, excluding p,p'-DDT and dieldrin. Contaminant elimination took place more rapidly in mussels, implying that SPMDs are better candidates for detecting episodic discharge of organic contaminants.

Micronucleus induction in gill cells of green-lipped mussels (Perna viridis) exposed to mixtures of polycyclic aromatic hydrocarbons and chlorinated pesticides.

Different groups of green-lipped mussels (Perna viridis) were exposed to the same net amount of a genotoxicant mixture of four polycyclic aromatic hydrocarbons ([PAHs]; anthracene, fluoranthene, pyrene, and benzo[a]pyrene) and four organochlorine pesticides ([OCs]; alpha-hexachlorocyclohexane (HCH), aldrin, dieldrin, and p,p'-DDT) for four weeks under different regimes that simulated various scenarios of fluctuating toxicant levels in the marine environment. Micronucleus (MN) formation in gill cells was studied at the end of each week. Micronucleus frequencies increased with continual addition of genotoxicants, and did not diminish significantly under conditions of either gradually decreasing concentrations or cessation of exposure for one to two weeks, suggesting that the MN response may persist over relatively long exposure periods. An almost two-fold higher mean MN frequency was recorded in a chronic exposure group than in an acute group that had received the same net nominal dose of genotoxicants, indicating that chronic exposure may lead to a greater genotoxic impact than acute exposure. The results suggested that in field studies, MN response should be monitored at multiple time points in order to elucidate the effects of potentially fluctuating toxicant levels. Finally, MN formation was positively correlated with both nominal contaminant levels and tissue levels of the genotoxicants. These findings suggest that MN responses can be a sensitive indicator of exposure to relatively low levels of genotoxicants and that MN response in mussel gill cells can be a stable biomarker of genotoxicity.

A comparison of polycyclic aromatic hydrocarbon and petroleum hydrocarbon uptake by mussels (Perna viridis) and semi-permeable membrane devices (SPMDs) in Hong Kong coastal waters.

The ability of mussels (Perna viridis) and semi-permeable membrane devices (SPMDs) to accumulate polycyclic aromatic hydrocarbons (PAHs) and petroleum hydrocarbons (PHCs) from five sites in Hong Kong's coastal waters was compared. Mussels consistently had higher levels of contaminants, but their utility was limited at one highly polluted site due to mortality. Mussels and SPMDs ranked sites differently in terms of individual contaminant levels. Although SPMDs overcome many of the disadvantages of using living organisms to measure contaminants in marine waters, they cannot be used as "mimics" due to different PAH and PHC accumulation patterns.

Biofouling confounds the uptake of trace organic contaminants by semi-permeable membrane devices (SPMDs).

The outer layers of layflat, low density polyethylene plastic tubing (the principal component of semi-permeable membrane devices, SPMDs) were biofouled at a clean site in Hong Kong coastal waters for periods of 1-4 weeks. Following pre-fouling, triolein was added to the SPMDs and, along with control (unfouled) devices, they were exposed to a range of organochlorine pesticides (alpha-HCH, aldrin, p,p'-DDT) and PAHs (anthracene, fluoranthene and benzo(a)pyrene) under laboratory conditions. Results showed that the uptake of contaminants by SPMDs was severely reduced by as much as 50% under fouling conditions in comparison to unfouled controls. The ultimate utility of SPMDs as passive monitors is thus reduced, although alternative measures, such as the use of permeability reference compounds may compensate, and allow for realistic evaluations of dissolved environmental concentrations in aquatic environments. However, due to the complexities involved in such procedures--especially as they need to be conducted on a case-by-case basis-the utility of SPMDs appears to be limited for estimates of bioavailability unless necessary calibrations are undertaken within each environment that the sampler is used.