Seasonal variation in cytochrome P450 immunopositive protein levels, lipid peroxidation and genetic toxicity in digestive gland of the mussel Mytilus edulis.

The relationship between cytochrome P450 1A- and 2E-immunopositive proteins, lipid peroxidation and DNA strand breaks (SBs) was studied in Mytilus edulis digestive gland at different seasons and at different sites around the UK coast. Cytochrome P4501A (CYP1A)-immunopositive protein and DNA strand breaks were generally lowest in December but there was no correlation between PAH exposure (indicated by chemical measurement and CYP1A-immunopositive protein expression) and DNA strand breaks which was highest at the relatively non-polluted site (Port Quin). As with CYP1A, CYP2E1-immunopositive protein was maximal at most sites in May. Lipid peroxidation, in contrast, did not alter markedly throughout the year. In conclusion, DNA strand breakage was not correlated with any of the above parameters although it did correlate with "scope for growth" as did the inverse of PAH levels. The study highlights the need to establish the relative contribution of DNA damage and DNA repair processes to the production of DNA strand breaks and emphasises the need to consider seasonal variation in interpretation of biomarkers.

The role of algae (Isochrysis galbana) enrichment on the bioaccumulation of benzo[a]pyrene and its effects on the blue mussel Mytilus edulis.

The role of algal concentration in the transfer of organic contaminants in a food chain has been studied using the ubiquitous model polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel (Mytilus edulis) as the primary consumer. The effect of algal concentration on BaP uptake by M. edulis was determined by feeding M. edulis daily with I. galbana which had previously been kept in the presence of BaP for 24 h. Four combinations of concentrations of algae and BaP were used to give final exposure concentrations of 30,000 or 150,000 algal cells ml(-1) in combination with either 2 or 50 microg BaP l(-1). BaP concentrations were determined fluorometrically in rest tissues (excluding digestive glands) and digestive gland microsomal fractions of M. edulis after 1, 7 and 15 days exposure, and also in isolated algae. Potentially toxic effects of BaP on M. edulis were examined in terms of blood cell lysosomal membrane damage (neutral red dye retention assay) and induction of digestive gland microsomal mixed-function oxygenase (MFO) parameters [BaP hydroxylase (BPH) and NADPH-cytochrome c (P450) reductase activities]. BaP bioaccumulation in rest tissues (and to a lesser extent in digestive gland microsomes) of M. edulis increased with both increasing BaP and algal exposure concentrations, and over time, producing maximal bioconcentration factors in rest tissues after 15 days exposure to 150,000 algal cells ml(-1) and 50 microg BaP l(-1) of 250,000. The five-fold higher concentration of algae increased BaP bioaccumulation by a factor of approximately 2 for 50 microg BaP l(-1) at day 15. Blood cell neutral red dye retention time decreased linearly with increasing log(10) tissue BaP body burden, indicating an increased biological impact on M. edulis with increasing BaP exposure possibly due to a direct effect of BaP on blood cell lysosomal membrane integrity. An increase was seen in NADPH-cytochrome c reductase activity, and indicated in BPH activity, with 1 but not 7 or 15 days exposure to BaP, indicating a transient response of the digestive gland microsomal MFO system to BaP exposure.

QSARs for the sublethal responses of marine mussels (Mytilus edulis).

The marine environment is contaminated with many organic compounds, some of which induce deleterious responses in biota. Biological impact can be assessed by measuring the physiological responses of mussels, though the task of establishing which of the bioaccumulated compounds cause the observed effects is complex. To facilitate this task, quantitative structure-activity relationships (QSARs) for the physiological responses are being established. In this paper, the responsiveness of ciliary feeding to alkanes and benzene-substituted alkanes is described and compared with a QSAR established previously for aromatic compounds. Most of the test compounds with aqueous solubilities greater than 70 micrograms dm-3 were toxic to feeding activity when bioaccumulated to similar concentrations, whereas compounds of lower solubility were less toxic. The only exceptions were the polyaromatic hydrocarbons pyrene and fluoranthene, which were less toxic than predicted from their solubility. These data are consistent with the hypothesis that the toxicity cut-off is due to solubility-related phenomena, the effect perhaps being enhanced for aromatic hydrocarbons dosed near to their solubility limits, by sequestration of crystals within the mussel tissues. These observations indicate that many organic contaminants detected by chemical analysis of mussels have no direct effect on filter feeding, whereas the less frequently determined volatile compounds are toxic.

Hydrocarbon humps in the marine environment: synthesis, toxicity, and aqueous solubility of monoaromatic compounds.

A recent study has shown that some monoaromatic hydrocarbon constituents of the so-called unresolved complex mixtures (UCMs) or gas chromatographic humps, which are widespread in the marine environment, are toxic to the mussel Mytilus edulis. Here we describe the synthesis and toxicological assessment of 6-cyclohexyltetralin, 7-cyclohexyl-1-methyltetralin, and 7-cyclohexyl-1-n-propyltetralin, which contain structural features consistent with some monoaromatic UCM hydrocarbons. The compounds were all toxic to M. edulis when measured in the assay used previously to determine the toxicity of a monoaromatic UCM. The aqueous solubilities of the hydrocarbons in fresh and seawater at different temperatures were determined and found to range from about 10 to 110 microg/L (10-60 microg/L in seawater at 15 degrees C). Further studies of the aromatic UCM composition of a wide range of oils and oil residues are required to determine whether such alkylated compounds as 7-cyclohexyl-1-methyltetralin and 7-cyclohexyl-1-n-propyltetralin or their analogues are widespread in oils. If these aromatic compounds prove to be important in UCMs, toxicity experiments should be conducted with other biological end points and monitoring studies of pollutant hydrocarbons should probably include measurement of aromatic UCM hydrocarbons.

Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells.

Coastal zones are among the most productive and vulnerable areas on the planet. An example of impact on these fragile environments was shown in the case of the "Sea Empress" oil tanker, which ran aground in the Bristol Channel in 1996, spilling 72,000 tons of "Forties" crude oil. The objective was to investigate the sub-lethal cellular pathology and tissue hydrocarbon contamination in marine mussel populations, 4 months after the initial spill, using the neutral red retention (NRR) assay for lysosomal stability in blood cells. NRR was reduced in mussels, and indicative of cell injury, from the two sites closest to the spill in comparison with more distant and reference sites. Lysosomal stability was inversely correlated with polycyclic aromatic hydrocarbon concentrations in mussel tissues. Reduced lysosomal stability has previously been shown to contribute to impaired immunocompetence and to autophagic loss of body tissues. The use of this type of technique is discussed in the context of cost-effective, ecotoxicological tools for Integrated Coastal Zone Management.

Measurement of stress effects (scope for growth) and contaminant levels in mussels (Mytilus edulis) collected from the Irish Sea.

The objective of this research was to quantify the impact of pollution along the coastlines of the Irish Sea. Pollution assessment was based on the combined measurement of scope for growth (SFG), and chemical contaminants in the tissues of mussels (Mytilus edulis) collected from 38 coastal sites around the Irish Sea during June-July in 1996 and 1997. On the UK mainland coast, the SFG showed a general trend with a significant decline in water quality in the Liverpool and Morecambe Bay region. High water quality was recorded along the west coast of Wales, as well as southwest England and northwest Scotland (clean reference sites outside the Irish Sea). Along the coast of Ireland there was a similar trend with reduced SFG within the Irish Sea region. SFG was generally low north of Duncannon and then improved north of Belfast. The poor water quality on both sides of the Irish Sea is consistent with the prevailing hydrodynamics and the spatial distribution of contaminants associated with urban/ industrial development. The decline in SFG of mussels on both sides of the Irish Sea was associated with a general increase in contaminant levels in the mussels. Certain contaminants, including PAHs, TBT, sigmaDDT, Dieldrin, gamma-HCH, PCBs, and a few of the metals (Cd, Se, Ag, Pb), showed elevated concentrations. Many of these contaminants were particularly elevated in the coastal margins of Liverpool Bay, Morecambe Bay and Dublin Bay. A quantitative toxicological interpretation (QTI) of the combined tissue residue chemistry and SFG measurements indicated that at the majority of coastal sites, c. 50 to > 80% of the observed decline in SFG was due to PAHs as a result of fossil fuel combustion and oil spills. TBT levels were highest at major ports and harbours, but these concentrations only made a minor contribution to the overall reduction in SFG. At no sites were individual metals accumulated to concentrations that could cause a significant effect on SFG. The study identified many sites where the observed reduction in SFG was far greater than predicted from the limited number of chemical contaminants analysed, thus indicating the presence of additional 'unknown toxicants'. Sewage (containing domestic, agricultural and industrial components) appears to be an important contributor to reduced SFG and linear alkylbenzenes (LABs) and As may provide suitable 'sewage markers'. There was a highly significant positive correlation between SFG and As (P < 0.001). This relationship may be due to reduced As uptake by algal food material and mussels at sites with elevated P04 concentrations (e.g. at sites with sewage inputs). Phosphate is a known competitive inhibitor of As accumulation, at least in algae. The results highlight that further research is required on 'sewage markers' in mussels. The SFG approach therefore provides a rapid, cost-effective and quantitative measure of pollution impact, as well as a means of identifying the causes through a QTI of tissue contaminants levels. It also serves to identify the presence of unidentified toxicants and areas that require further study.

Role of physiological energetics in ecotoxicology.

1. The role of physiological energetic measurements combined with chemical analyses of contaminants in body tissues of mussels in fundamental toxicological studies and pollution monitoring programmes is outlined. 2. Important features of this toxicological approach are briefly reviewed, including aspects of bioaccumulation, sensitivity, quantitative concentration-response relationships, QSARs, mechanistic interpretation, ecological relevance, integration of the consequences of multiple mechanisms of toxicity and effects of contaminant mixtures and application to laboratory and field studies. 3. This review focuses particularly on recent advances in understanding and predicting the effects of complex mixtures of contaminants.

Aromatic hydrocarbon "humps" in the marine environment: unrecognized toxins?

Gas chromatographic analysis of the hydrocarbons of environmental samples often reveals that unresolved complex mixtures (UCMs) or gas chromatographic "humps" of aliphatic and aromatic hydrocarbons are most abundant--yet little consideration seems to have been given to the possible toxicological impacts of hydrocarbon "humps". Here we show, using a well-accepted bioassay, that monoaromatic components of a UCM of hydrocarbons from a crude oil elicit a sublethal toxic response in a typical marine pollution indicator organism (the mussel, Mytilus edulis). Furthermore, coastal U.K. mussels shown previously to have unexplained impaired health contained high concentrations of UCMs, including monoaromatic UCMs. These findings may have important implications for our understanding of the toxicological sublethal effects of oil residues in the environment. Given the relatively resistant nature of UCM hydrocarbons, the effects of both acute oil spills and more chronic discharges may need further consideration.