Peroxisomal proteomic approach for protein profiling in blue mussels (Mytilus edulis) exposed to crude oil

Peroxisomal proteomic protein profiles of exposure to marine pollution have been recently introduced in biomonitoring experiments. However, laboratory experiments to study the independent effect of common pollutants are needed to define a minimal protein expression signature (PES) of exposure to a specific pollutant. The aim of this study was to obtain PESs in blue mussels (Mytilus edulis) exposed to two different crude oil mixtures for future application in biomonitoring areas affected by oil spills. In the study, peroxisome-enriched fractions from digestive gland of M. edulis (L., 1758) were analysed by two-dimensional fluorescence difference electrophoresis (DIGE) and mass spectrometry (MS) after 3 weeks of exposure to crude oil mixtures: crude oil or crude oil spiked with alkylated phenols (AP) and extra polycyclic aromatic hydrocarbons (PAH) in a laboratory flow-through system. A minimal PES composed by 13 protein spots and unique PESs of exposure to the two different mixtures were identified. A total of 22 spots from the two-dimensional maps that had shown a significant increase or decrease in abundance in each of the exposed groups exposed were analysed. The hierarchical clustering analysis succeeded in discriminating the exposed groups from the control groups based on the unique PES. The PESs obtained were consistent with protein patterns obtained in previous field experiments. The results suggest that the protein profiles obtained by peroxisomal proteomics could be used to assess oil exposure in marine pollution assessments.     

Identification of proteomic signatures of exposure to marine pollutants in mussels (Mytilus edulis)

Bivalves and especially mussels are very good indicators of marine and estuarine pollution, and so they have been widely used in biomonitoring programs all around the world. However, traditional single parameter biomarkers face the problem of high sensitivity to biotic and abiotic factors. In our study, digestive gland peroxisome-enriched fractions of Mytilus edulis (L., 1758) were analyzed by DIGE and MS. We identified several proteomic signatures associated with the exposure to several marine pollutants (diallyl phthalate, PBDE-47, and bisphenol-A). Animals collected from North Atlantic Sea were exposed to the contaminants independently under controlled laboratory conditions. One hundred and eleven spots showed a significant increase or decrease in protein abundance in the two-dimensional electrophoresis maps from the groups exposed to pollutants. We obtained a unique protein expression signature of exposure to each of those chemical compounds. Moreover a set of proteins composed a proteomic signature in common to the three independent exposures. It is remarkable that the principal component analysis of these spots showed a discernible separation between groups, and so did the hierarchical clustering into four classes. The 14 proteins identified by MS participate in alpha- and beta-oxidation pathways, xenobiotic and amino acid metabolism, cell signaling, oxyradical metabolism, peroxisomal assembly, respiration, and the cytoskeleton. Our results suggest that proteomic signatures could become a valuable tool to monitor the presence of pollutants in field experiments where a mixture of pollutants is often present. Further studies on the identified proteins could provide crucial information to understand possible mechanisms of toxicity of single xenobiotics or mixtures of them in marine ecosystems.     

石油烃污染对海洋浮游植物群落的短期毒性效应

海上溢油和船舶含油废水排放等石油污染给海洋生态环境带来严峻考验。为了解石油污染对海洋浮游植物的短期群落效应, 于实验室对采自浙江省乐清湾海域的四季浮游植物进行了14天的微宇宙实验研究。通过测定不同浓度原油水溶性成分(water accommodated fraction, WAF)胁迫下浮游植物群落各种类的细胞密度, 发现原油WAF对浮游植物群落的种类数、多样性、均匀度和优势种组成及优势度均有极显著影响。原油污染组的种类数均低于对照组, 多样性指数也总体上低于对照组, 但均匀度指数无明显规律。WAF对各季节浮游植物群落优势种的影响存在季节、浓度梯度和种类差异: 1)在高浓度WAF (≥2.28 mg·L^-1)胁迫下, 各季节浮游植物群落中中肋骨条藻(Skeletonema costatum)的优势度均呈升高趋势, 除秋季外, 长菱形藻(Nitzschia longissima)在其余季节的优势度均显著下降, 另外秋季实验中微小原甲藻(Prorocentrum minimum)的优势度呈先升高后下降的趋势, 秋冬季斜纹藻(Pleurosigma sp.)和念珠直链藻(Melosira moniliformis)的优势度呈下降趋势; 2)在低浓度WAF (≤1.16 mg·L^-1)胁迫下, 除秋季中肋骨条藻仍为绝对优势种外, 其余季节该种微藻的优势度均呈下降趋势, 春、夏季长菱形藻的优势度逐渐升高, 而冬季则是念珠直链藻的优势度不断增加。原油WAF影响下, 可能r策略种将逐渐取代k策略种, 导致浮游植物群落趋于小型化。     

“Survival in air” of the blue mussel Mytilus edulis L. as a sensitive response to pollution-induced environmental stress

Mussels, Mytilus edulis, were exposed for periods of 6 weeks at various locations in Dutch coastal waters during 1989 and 1990. “Survival in air” showed to be a sensitive response parameter for indicating pollution induced environmental stress in transplanted mussels sampled from eight field sites. Increased tissue contaminant levels, especially PCBs and PAHs, correlated with a reduced survival time during aerial exposure. Three weeks exposure of mussels in the laboratory to 1 μg · 1⁻¹ PCBs affected the aerial survival time negatively. Laboratory experiments did not indicate that lowered salinity influences the “Survival in air” response after sufficient acclimation (15 days), facilitating the use of this response parameter in both marine and estuarine waters.     

Proteomics-based method for the assessment of marine pollution using liquid chromatography coupled with two-dimensional electrophoresis

Using a proteomic approach, we have developed a new method for the assessment of marine pollution that generates highly reproducible protein expression patterns and it is simple and scalable. The protocol is based on applying liquid chromatography (LC) coupled with two-dimensional electrophoresis (2-DE) to analyze changes in the protein expression pattern after exposure to marine pollution. The digestive gland of the sentinel "blue mussel" (Mytilus edulis) was batch-processed through a simple cell fractionation followed by ion-exchange chromatography and 2-DE. The selection of ligands, elution method, and small volume design was carefully considered to define a protocol that could be mainly robotized. A pilot study with samples collected from different Gothenburg harbor areas indicated that the clean area could be distinguished from the polluted ones based on a protein expression pattern (PES) composed of 13 proteins. Principal component analysis (PCA) and hierarchical clustering confirmed that the PES was sufficient to discriminate polluted and unpolluted areas and to provide a spatial gradient from the polluted source. Several proteins from the PES were identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS), and they are involved in beta-oxidation, amino acid metabolism, detoxification, protein degradation, organelle biogenesis, and protein folding. In the near future, this methodology could show potential advantages to assess marine pollution and could become a stable platform to elucidate ecotoxicological questions.     

Induction of a putative monooxygenase of crabs (Carcinus spp.) by polycyclic aromatic hydrocarbons

As part of a programme to develop biomarker assays for polycyclic aromatic hydrocarbons (PAHs) in marine invertebrates, two species of crabs, Carcinus maenas and Carcinus aestuarii were exposed to benzo(a)pyrene (B(a)P) or crude oil. Microsomes were prepared from the midgut gland (hepatopancreas), examined by gel electrophoresis and Western blotting and assayed for B(a)P monooxygenase activity. In early experiments there was evidence of protein degradation and results were inconsistent and inconclusive. However, when steps were taken to minimize this in subsequent experiments, including the inclusion of four protease inhibitors in the homogenization buffer, there was consistent evidence for an increase of proteins of estimated molecular weight 45-60 kDa, and particularly of a distinct band at c. 48 kDa, following exposure to PAH at levels down to 0.1 ppm in ambient water. In C. aestuarii the increase in this band was found to coincide with an 8-12-fold increaseof B(a)P monooxygenase activity in midgut gland microsomes. These results suggest that one or more forms of cytochrome P450 may be induced by PAHs in these species. However, Western blotting using antibodies raised to vertebrate P450s, and representing four different gene families, failed to recognize any proteins in either the PAH-treated samples or in the controls. The isolation and characterization of induced protein, and the production of antibodies may provide the basis for a biomarker assay to measure a response to environmental PAHs in crabs.     

Unexpected Interaction with Dispersed Crude Oil Droplets Drives Severe Toxicity in Atlantic Haddock Embryos

The toxicity resulting from exposure to oil droplets in marine fish embryos and larvae is still subject for debate. The most detailed studies have investigated the effects of water-dissolved components of crude oil in water accommodated fractions (WAFs) that lack bulk oil droplets. Although exposure to dissolved petroleum compounds alone is sufficient to cause the characteristic developmental toxicity of crude oil, few studies have addressed whether physical interaction with oil micro-droplets are a relevant exposure pathway for open water marine speices. Here we used controlled delivery of mechanically dispersed crude oil to expose pelagic embryos and larvae of a marine teleost, the Atlantic haddock (Melanogrammus aeglefinus). Haddock embryos were exposed continuously to two different concentrations of dispersed crude oil, high and low, or in pulses. By 24 hours of exposure, micro-droplets of oil were observed adhering and accumulating on the chorion, accompanied by highly elevated levels of cyp1a, a biomarker for exposure to aromatic hydrocarbons. Embryos from all treatment groups showed abnormalities representative of crude oil cardiotoxicity at hatch (5 days of exposure), such as pericardial and yolk sac edema. Compared to other species, the frequency and severity of toxic effects was higher than expected for the waterborne PAH concentrations (e.g., 100% of larvae had edema at the low treatment). These findings suggest an enhanced tissue uptake of PAHs and/or other petroleum compounds from attached oil droplets. These studies highlight a novel property of haddock embryos that leads to greater than expected impact from dispersed crude oil. Given the very limited number of marine species tested in similar exposures, the likelihood of other species with similar properties could be high. This unanticipated result therefore has implications for assessing the ecological impacts of oil spills and the use of methods for dispersing oil in the open sea.     

Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii

Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers.     

Interactive effects of benzo(a)pyrene and cadmium and effects of di(2-ethylhexyl) phthalate on antioxidant and peroxisomal enzymes and peroxisomal volume density in the digestive gland of mussel Mytilus galloprovincialis Lmk

Exposure of marine animals to certain organic and metal pollutants is thought to enhance reactive oxygen species (ROS) production with concomitant alterations of antioxidant defence mechanisms. Some of these organic pollutants cause peroxisome proliferation, a process resulting also in possible enhanced production of ROS. The aim of this study was to investigate the effects of two organic xenobiotics, benzo(a)pyrene (B(a)P) and di(2-ethylhexyl)phthalate (DEHP), as well as the effects of cadmium (Cd), on antioxidant and peroxisomal enzymes and on peroxisomal volume density in the digestive gland of mussel, Mytilus galloprovincialis Lmk., experimentally exposed for 21 days. Special attention was paid to the interactive effects of organic and metal compounds by exposing one group of mussels to a mixture of B(a)P and Cd. Exposure of mussels to Cd caused a decrease in superoxide dismutase (SOD) activity, in Mn-SOD protein levels and in volume density of peroxisomes. B(a)P exposure significantly increased catalase and glutathione peroxidase (GPX) and inhibited Mn-SOD after 21 days of exposure. B(a)P also caused a slight increase in acyl-CoA oxidase (AOX) activity and peroxisomal volume density after 21 days of exposure. Cd tended to inhibit changes provoked by B(a)P, indicating that responses to organic xenobiotics can be modulated by concomitant exposure to metal contaminants. Exposure to DEHP increased catalase and AOX and inhibited SOD activity and Mn-SOD protein levels. In conclusion, peroxisome proliferation, measured as an increase of the peroxisomal enzymes catalase and AOX (up to 1.53-fold for AOX), is a specific response to organic contaminants such as B(a)P and DEHP, whereas Cd does not cause peroxisome proliferation. Thus, peroxisome proliferation may be a specific biomarker of organic pollutants in mussels. Both organic and metal pollutants inhibited SOD activity and protein levels (up to 0.21-fold for Mn-SOD protein levels), the latter offering potential as general marker of pollution.     

Monitoring protein expression by proteomics: human plasma exposed to benzene

Low levels and long term exposure to benzene is associated with hematotoxicity including aplastic anemia, acute myelogenous leukemia, and lymphoma. Current biomonitoring methods such as urinary phenol, S-phenylmercapturic acid, and trans-trans muconic acid were found to be unreliable as analytical methods to detect benzene exposure. Therefore, to search for a specific protein for biomonitoring benzene exposure, we investigated plasma proteins from workers (n = 50) at a printing company who were exposed to benzene, by two-dimensional gel electrophoresis. The protein profiles are significantly different (p < 0.05) between benzene exposed and unexposed groups, as identified by matrix-assisted laser desorption ionization/time of flight mass spectrometry and confirmed by Western blot analyses. T cell receptor beta chain (TCR beta), FK506-binding protein, and matrix metalloproteinase-13 were expressed only in benzene exposed workers. In addition, interleukin-4 receptor alpha chain and T cell surface glycoprotein CD1b precursor were found to be up-regulated in the plasma of benzene exposed workers. When we treated Jurkat cells with benzene (10 microM-10 mM), TCR beta expression was increased in the membrane more than 6-9-fold compared to untreated cells. In addition, the amount of TCR beta released into the culture media, at benzene concentrations greater than 50 microM, increased up to 10 mM. Therefore, TCR beta levels in plasma could be used as a biomarker and a possible therapeutic target for benzene exposure.     

Long-Term Effects of Crude Oil on Uptake and Respiration of Glucose and Glutamate in Arctic and Subarctic Marine Sediments

The effects of crude oil on uptake and respiration (mineralization) of glucose and glutamate in marine sediments were investigated. After the sediments were treated with crude oil, they were replaced at or near the collection site by scuba divers. These sediments remained in situ until they were retrieved for analysis. Glucose and glutamate uptake rates were found to decrease, and the percent respired was found to increase in Arctic and subarctic marine sediments that had been exposed to fresh crude oil. These same changes were also observed when “weathered” crude oil was used and when untreated sediments were overlaid with oiled sediments. When the kinetics of glutamate uptake were determined, both the maximum potential uptake rate and the turnover time were significantly affected. A comparison between the proportion of glucose taken into the cells and that respired as CO₂ indicated that crude oil affected biosynthetic mechanisms. A study of sediments that had been exposed to crude oil for at least 5 months showed that glutamate transport into the cells was affected more extensively than biosynthetic mechanisms. In the initial months of exposure, bacterial concentrations and total adenylate concentrations were found to decrease in the presence of crude oil. Our data suggest that secondary productivity in the marine environment could be adversely affected by the presence of crude oil in marine sediments.     

Effects of hypoxia and petroleum on the genotoxic and morphological parameters of Hippocampus reidi

Hypoxia events are common in many aquatic systems, which may be a natural event or provoked by anthropogenic actions, as well as accidents involving oil occurring throughout the world are frequent. Thus, through the possibility of occurrence of these two situations in same place the purpose of this study was to evaluate if damage caused by crude oil on genotoxic and morphological parameters in the marine fish species Hippocampus reidi will be aggravated by events of severe hypoxia. Sea horses were exposed during 8h to the following conditions: crude oil (OIL), severe hypoxia (HYP), association of severe hypoxia and crude oil (HYP+OIL) and normoxia without contaminant (CONT). An increase in micronuclei observed in OIL and HYP+OIL groups indicates that the crude oil exposure was a determining factor in the micronuclei induction and hypoxia did not intensify this result. In comet assays, both petroleum and hypoxia provoke DNA damage. The most frequent histopathology in the control groups and in those exposed to OIL and HYP+OIL groups were: hypertrophy and capillary dilation; hypertrophy and hyperplasia; hypertrophy, epithelial "lifting" and epithelial hyperplasia. An elongation of the lamellae was observed in fish from the two groups exposed to hypoxia, probably due to the fact that these groups required a greater flow of blood in the gills to increase the efficiency of gas exchange, since they were in a hypoxic environment. In summary, the micronuclei test and comet assay can be used as a good biomarker of contamination by petroleum. The association of hypoxia with crude oil in some aspects may exacerbate the responses of fish, in the light of the increase in DNA damage and the alterations in thickness of the gill epithelium.     

Experimental oil exposure of Ascophyllum nodosum (L.) Le Jolis

The effect of weathered, crude oil on Ascophyllum nodosum was tested in an outdoor experiment and in a laboratory experiment. Adult A. nodosum plants which were exposed to oil in an outdoor basin for 3 days showed reduced growth for a certain period of time, after which growth recovered. Also, some necrotic damage was observed. Three-week-old and 1-year-old germlings placed under the adult plants showed no significant reduction in number compared to control plants. In the laboratory experiment some of the 1-month-old germlings were initially exposed to oil for 6 min, some for 1 h, some for 3 h and some for 5 h. After 3 weeks in running, filtered water oil-exposed germlings had reached the same length as those in the control groups, and no effect of exposing the germlings to oil for different lengths of time at the start of the experiment was found. The results indicate that a limited oil pollution of weathered, crude oil may reduce growth and biomass of adult plants for a certain period of time. However, this need not be detrimental to a population with good recruitment from germlings or vegetative shoots growing under a dense canopy layer.     

Metal accumulation,filtration and O(2) uptake rates in the mussel Perna perna (Mollusca: Bivalvia) exposed to Hg(2+), Cu(2+) and Zn(2+)

Tissue metal concentrations, filtration and oxygen uptake rates were investigated for Perna perna (Bivalvia: Mollusca) during exposure to Hg(2+), Cu(2+) and Zn(2+) (50 microg/l for 24 days, and 24 days recovery with no metal). Hg and Cu tissue levels increased with exposure time, reaching maximum levels after 24 days (87.5 microg Hg/g dry mass and 45 microg Cu/g dry mass, respectively). Zn levels peaked after 4 days exposure (to 233 microg Zn/g dry mass) and stabilized thereafter. Accumulated metal was rapidly lost from tissues when mussels were returned to uncontaminated seawater, suggesting that tissue concentration data may be of limited use in biomonitoring situations where environmental metals fluctuate to low levels. Filtration rates fell below control rates during Hg(2+) exposure, and became elevated again during the recovery period. Cu(2+) and Zn(2+) exposure had little effect on filtration, but suppressed oxygen uptake. During recovery, oxygen uptake of Cu(2+) and Zn(2+) exposed mussels was elevated above the controls. Filtration and oxygen uptake rates were not correlated, but rather responded in different ways to metal pollution. While these physiological responses of P. perna may be of limited use in biomonitoring, they could indicate how populations may respond to marine pollution.     

Human Risk Assessment for Nonylphenol

This article presents a risk assessment for human exposure to nonylphenol (NP). We critically reviewed and assessed all relevant full-text publications based on a variety of data quality attributes. Two categories of data, environmental monitoring and biomonitoring from exposed individuals, were used to estimate human exposure to NP. Environmental monitoring data included the measurement of NP in food, water, air, and dust. From these data and estimates of human intake rates for the sources, exposures were estimated from each source and source-specific Margins of Exposure (MOEs) calculated. However, the nature of the populations studied prevented the calculation of aggregate exposure calculations from these data. Rather, the most reliable estimates of aggregate exposure to NP were those derived from biomonitoring studies in exposed individuals. Using the daily absorbed dose estimates for NP, MOEs were calculated for these populations. The MOEs were based on the use of a No-Observed-Adverse-Effect-Level (NOAEL) for sensitive toxicological endpoints of interest, that is, systemic and reproductive toxicity from continuous-feeding more than 3.5 generations (13 mg/kg/day). The MOEs were all greater than 1000 (ranging from 2863 to 8.4 × 10⁷), clearly indicating reasonable certainty of no harm for source-specific and aggregate (based on biomonitoring) exposures to NP.     

Life Cycle Assessment of Greenhouse Gas Emissions from Marine Fuels: A Case Study of Saudi Crude Oil versus Natural Gas in Different Global Regions

The understanding of the greenhouse gas (GHG) emissions dimension in discussing the future of marine fuels makes it important to advance the current life cycle assessment (LCA) practice in this context. Previous LCA studies of marine fuels rely on general LCA models such as GREET and JEC well‐to‐wheels study. These models do not fully capture the various methane losses in the fuel supply chain. The primary goal of this LCA study is to compare the GHG emissions of heavy fuel oil and marine gas oil produced from Saudi crude oil to liquefied natural gas (LNG) in different global regions. A sensitivity analysis was performed to show how results may vary with non‐Saudi crudes. A secondary goal was to advance LCA of marine fuels by utilizing, for the first time, a set of bottom‐up engineering models that enable detailed analysis of specific oil and gas projects worldwide. The results show particular cases where LNG use in marine applications has a significant countereffect in terms of climate change compared to conventional marine fuels produced from a low‐carbon‐intensity crude oil. When the results are calculated based on a 20‐ versus 100‐year methane global warming potential, LNG appears noncompetitive for climate impact in marine applications.     

Quantitative proteomics of heavy metal stress responses in Sydney rock oysters

Currently, there are few predictive biomarkers in key biomonitoring species, such as oysters, that can detect heavy metal pollution in coastal waterways. Several attributes make oysters superior to other organisms for positive biomonitoring of heavy metal pollution. In particular, they are filter feeders with a high capacity for bioaccumulation. In this study, we used two proteomics approaches, namely label-free shotgun proteomics based on SDS-PAGE gel separation and gas phase fractionation, to investigate the heavy metal stress responses of Sydney rock oysters. Protein samples were prepared from haemolymph of oysters exposed to 100 μg/L of PbCl(2), CuCl(2), or ZnCl(2) for 4 days in closed aquaria. Peptides were identified using a Bivalvia protein sequence database, due to the unavailability of a complete oyster genome sequence. Statistical analysis revealed 56 potential biomarker proteins, as well as several protein biosynthetic pathways to be greatly impacted by metal stress. These have the potential to be incorporated into bioassays for prevention and monitoring of heavy metal pollution in Australian oyster beds. The study confirms that proteomic analysis of biomonitoring species is a promising approach for assessing the effects of environmental pollution, and our experiments have provided insights into the molecular mechanisms underlying oyster stress responses.     

Long-term feeding effects of Catha edulis leaves on blood constituents in animals

In this study, the long-term (6 months) biochemical effects of varying levels of Catha edulis leaves on the plasma concentration of glucose, triglycerides, cholesterol, HDL-cholesterol, total protein, albumin, uric acid, urea and creatinine were examined. Our results demonstrated a significant decrease in plasma cholesterol throughout the treatment period by all levels of C. edulis leaves tested. This significant decrease in plasma cholesterol was halved at the end of the treatment period and corresponded with a significant increase in plasma HDL-cholesterol and a significant decrease in plasma glucose and triglycerides concentrations. Moreover, C. edulis treatment increased plasma uric acid significantly, in a time-dependent manner with the higher concentrations (20% and 30%) of C. edulis leaves. Only plasma albumin was decreased significantly at the end of the treatment period, with no significant effect on plasma total protein. This also coincided with a significant, dose-dependent decrease in plasma urea at month 6, with no significant effect on plasma creatinine concentration.     

Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers

The response of the glutathione S-transferase (GST, EC 2.5.1.18) and related proteins of Mytilus edulis to environmental pollution load was assessed. Mussels were reciprocally transplanted between an industrial estuary (Douglas), a rural estuary (Youghal) and a m arine site (Bantry). In addition, m ussels were sam pled along a pollution gradient in an estuary receiving leather tannery effluent (Colligan). These latter m ussels were previously shown to be subject to oxidative stress resulting from the discharges. GST specific activity of cytosolic extracts from the gill and digestive gland tissues was determ ined for all anim als. Specific activity was shown to vary significantly in anim als from different sites, with highest specific activity always observed in sam ples (local and transplanted) taken from the industrial site. By com parison, the m ussels exposed to tannery discharges displayed no significant alteration in GST specific activity. Total intracellular glutathione (GSH) was also determ ined for sam ples taken from the Douglas and Youghal estuaries but no correlation with pollution load was observed. Using FPLC analysis, we observed no specific effect on the relative levels of the GST and the individual GST related proteins in gill or digestive gland sam ples from local or reciprocally transplanted anim als from Douglas or Youghal. The increase in GST specific activity observed in samples from the industrial estuary are indicative of a possible, specific inductive agent at this site. The results from the tannery site, by com parison, indicate that general oxidative stress does not result in elevated G ST specific activity in M. edulis.     

Effects of crude oil on marine microbial communities in short term outdoor microcosms

To assess the effects of crude oil spills on marine microbial communities, 10 L outdoor microcosms were manipulated over an exposure period of 8 days. The responses of microbial organisms exposed to five crude oil concentrations in 10 to 10,000 ppm (v/v) were monitored in the microcosms. The abundance of microalgae and copepods decreased rapidly upon the addition of crude oil at concentrations over 1,000 ppm, whereas the total density of heterotrophic bacteria increased dramatically at the higher concentrations. Bacterial diversity, determined by denaturing gradient gel electrophoresis, was increased at higher concentrations. In particular, the intensity of the bands representing Jannaschia sp. and Sulfitobacter brevis increased with the addition of oil. These results indicate that crude oil spills with concentrations over 1,000 ppm seriously affected the structure of the microbial communities.