Bioaccumulation and biotransformation of pyrene and 1-hydroxypyrene by the marine whelk Buccinum undatum.

The fates of a phenolic contaminant and its hydrocarbon precursor have rarely been compared, especially in an invertebrate species. Two groups of Buccinum undatum were exposed to equimolar amounts of pyrene and 1-hydroxypyrene over 15 d through their diets. Tissue extracts from the muscle and visceral mass were analyzed by liquid chromatography with fluorescence and mass spectrometry detection. Nine biotransformation products were detected in animals from both exposures. These included 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1-glucuronide, pyrene glucose sulfate, two isomers each of pyrenediol sulfate and pyrenediol disulfate, and one isomer of pyrenediol glucuronide sulfate. These compounds represent a more complex metabolic pathway for pyrene than is typically reported. Diconjugated metabolites were as important in animals exposed to pyrene as in those exposed to 1-hydroxypyrene. Biotransformation products represented >90% of the material detected in the animals and highlight the importance of analyzing metabolites when assessing exposure. A mean of only 2 to 3% of the body burden was present in muscle compared with the visceral mass of both groups. The analytical methods were sufficiently sensitive to detect biotransformation products both in laboratory control whelks and in those sampled offshore. The tissue distribution of [(14)C]pyrene was also studied by autoradiography. Radioactivity was present primarily in the digestive and excretory system of the whelks and not in the gonads or muscle tissue.

Estrogenic compounds in seawater and sediment from Halifax Harbour, Nova Scotia, Canada.

Abstract-Samples of seawater and surface sediment were collected from seven locations around Halifax Harbour, Nova Scotia, Canada, and analyzed for the presence of the organic estrogenic contaminants, bisphenol A (BPA), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2). Samples were extracted using solid phase extraction (seawater) or sonication (sediments), followed by fractionation on a two-layer alumina/silica gel column prior to analysis by liquid chromatography-tandem mass spectrometry (LCMS/MS) with negative-ion electrospray ionization. Levels of the three compounds consistently ranked as BPA > E2 > EE2. The least potent compound and plasticizer BPA reached levels of up to 2.6 ng/L in seawater and 9.5 ng/g in sediments; the natural product E2 was detected at concentrations up to 0.57 ng/L and 0.86 ng/g; while the synthetic estrogen EE2 was in most cases below the method detection limit (0.14 ng/L and 0.28 ng/g). The highest levels were observed in the influent of a secondary treatment plant that discharges into the harbor, with concentrations of 32.4 ng/L for BPA and 5.3 ng/L for E2. Overall, the results indicate that these compounds readily associate with suspended particles rather than remaining in the soluble phase. Measurement of the octanol-water partition coefficient (log K(OW)) confirmed these results, with values of 3.41, 3.89, and 4.16 for BPA, E2, and EE2, respectively. Partitioning experiments using spiked field samples further confirmed these findings, with sorption directly related to sediment total organic content and following the order EE2 > E2 > BPA.

Comparison of the partitioning of pesticides relative to the survival and behaviour of exposed amphipods.

Pesticides sprayed on farmlands can end up in rivers and be transported into estuaries, where they could affect aquatic organisms in freshwater and marine habitats. A series of experiments were conducted using the amphipod Corophium volutator Pallas (Amphipoda, Corophiidae) and single pesticides, namely atrazine (AT), azinphos-methyl (AZ), carbofuran (CA) and endosulfan (EN) that were added to sediments and covered with seawater. Our goal was to compare the concentrations affecting the survival of the animals relative to potential attractant or repellent properties of sediment-spiked pesticides. The avoidance/preference of contaminated/reference sediments by amphipods was examined after 48 and 96 h of exposure using sediments with different organic carbon content. The octanol-water partition coefficients (log K(ow)) ranked the pesticides binding to sediments as EN > AZ > AT > CA. LC(50) and LC(20) covered a wide range of nominal concentrations and ranked toxicity as CA-AZ > EN > AT. Under the experimental set up, only EN initiated an avoidance response and the organic carbon normalised concentration provided consistent results. Using the present data with wide confidence limits, >20% of a population of C. volutator could perish due to the presence of EN before relocation or detecting CA or AZ in sediments by chemical analysis.

Body burden of contaminants and biological effects in mussels: an integrated approach.

An investigation of contaminants and biological effects in mussels from Halifax Harbour, Nova Scotia, Canada, focused on a 6 km section in the central most industrialized core of the harbour, where a site was previously identified as highly contaminated. The aim of the study was to compare the body burden of mussels in terms of polycyclic aromatic hydrocarbons (PAH), dichlorophenyltrichloroethane (DDT family), coprostanol and elements, relative to biological parameters such as condition indices, sex ratio, survival time in air, and to biochemical indicators of mussels' health analysed in gills, digestive gland and gonad tissues. These markers are total sugar and lipid content of gonads, mitochondrial electron transport activity in digestive gland and gonad tissues, lipid peroxidation in gill, digestive gland and gonad tissues, and heme oxidase activity in the digestive gland. At the north western end of the area, near a major sewage effluent, shorter survival time, higher oxidative stress and metabolism, gonad electron transport activity, levels of coporostanol, PAH, p,p'-dichlorophenyldichlo-roethylene (p,p'-DDE), Ag, Cu, Fe and P were observed. At the opposite south eastern end, longer survival time, higher lipid content, lowest condition indices and concentrations of coprostanol, PAH and p,p'-DDE, but higher concentrations of Sn and Cd, were detected. On-going improvements to sewage treatment in Halifax Harbour, including construction of sewage treatment plants that will discharge into deeper parts of the central harbour, should improve inter-tidal mussels' health in our study area.

A non-lethal chemically based approach to investigate the quality of harbour sediments.

A non-lethal chemically based approach was used to investigate the quality of harbour sediments receiving combined road runoff and sewage effluents. A previous investigation of the behaviour of the amphipod Corophium volutator linked polycyclic aromatic hydrocarbons (PAH) in sediments corresponding to the probable effects levels listed in the sediment quality guidelines of the Canadian Council of the Marine Environment to a sediment avoidance response. Since the amphipods did not biotransform contaminants, bioaccumulation was the only fate pursued to examine the bioavailability of PAH. For five Halifax Harbour sediments, a relationship was established between the threshold effects level representing the amphipods' avoidance response and the bioaccumulation of PAH. A body burden of 0.3-1.1 mumol/kg (wet weight) was determined for the sum of abundant parental PAH in amphipods exposed to sediments that initiated the behavioural effect. PAH were much more available from spiked sediments than from field sediments, with biota-sediment accumulation factors of 2.2-7.8 compared to <0.01-0.3, respectively. Animals exposed to PAH-spiked sediments avoided contaminated sediments when their body burden was up to seven times higher than observed with field sediments. This latter result and two exposures to sediments collected further away from sewage discharges point to a role for unidentified chemicals in the body burden and behaviour relationship. Further research is warranted to develop this promising assessment tool.

Behavioral response of Corophium volutator relative to experimental conditions, physical and chemical disturbances.

The preference/avoidance behavioral response of a widely used amphipod in toxicity tests, Corophium volutator, was investigated in relation to the presence of anthropogenic physical or chemical materials in sediments. Exposure conditions, including the density of amphipods, the depth of sediments, amount of overlying water, and exposure time, were examined for their influence on amphipods' preference for field sediments and avoidance of coarse sand. It was shown that these variables did not affect the response; thus, conditions similar to published standard toxicity tests were chosen. A gradient of sediments spiked with potential habitat disturbances that can be found on a beach or in contaminated sediments, such as those in harbors, were tested. These substances included sand, seaweed, burned wood, coal, crankcase oil, and diesel oil. To enhance the interpretation of results and decrease the variability observed when tests were conducted at different times over the summer, exposures were performed over a gradient of spike material in reference sediments. We conclude that physical obstacles added to reference sediments lead to less correlation with the behavioral response than observed with chemical interferences. Amphipods' behavior ranked harbor sediments similarly to previous studies concerning the health of intertidal mussels collected in proximity to the sediments sites. For five sites, preference of reference sediments was observed until the level of polycyclic aromatic hydrocarbons in diluted harbor sediments reached the Canadian Council of Ministers of the Environment sediment quality guidelines.

Partitioning of polycyclic aromatic hydrocarbons between water and particles compared to bioaccumulation in mussels: a harbour case.

Water and particles from three sites located in Halifax Harbour, near and further away from combined municipal sewage effluents (CMSE) were analysed for parental and alkylated polycyclic aromatic hydrocarbons (par and alkPAH). Bioavailability of PAH was compared for inter-tidal mussels collected at the same sites in April and November. The PAH fingerprint determined over 9 sampling times covering a period of 19 months differed more between phases (water and particles) and seasons, than between sites. In the spring, more alkPAH associated with diesel and gasoline were detected in the soluble phase, along with more bioaccumulation of alkPAH in inter-tidal mussels. A broader number of parPAH were detected in mussels collected in the fall. The mean sum of dissolved alkPAH concentrations was higher in water at the site closer to raw CMSE than at the other two sites and particles of that site. However, lowest bioconcentration factors (BCF) were determined in mussels of this more contaminated site. Similar biota-particle accumulation factors (BPAF) were determined for parPAH in mussels from the three sites, all lower than the BCF of alkPAH. The study indicates that sewage treatment plants will reduce the amount of parPAH especially larger than fluoranthene and pyrene from being deposited in the harbour; that alk naphthalenes and fluorenes present in water will continue to be discharged; that the disturbance of sediments can make particle-bound PAH available to mussels.

Depletion of metronidazole in brook trout (Salvelinus fontinalis).

Metronidazole (MNZ), which is effective in the treatment of intestinal infections in fish, is also a suspected carcinogen and has been banned in numerous jurisdictions for use in any food-producing animal, including fish. Few reports have been published on the depletion of MNZ in fish. A depletion study was therefore undertaken using MNZ in feed provided to trout under controlled conditions. The water was maintained at 17.5 ± 0.9°C throughout the medication and depletion periods in the study. Following a 20-day acclimatisation period in the holding tanks, the trout (approximately 150-200 g bodyweight at the start of the study) were subjected to two separate medication and withdrawal periods: (A) 5 day medication/5 day withdrawal and (B) 5 day medication/16 day withdrawal. This simulated a potential multiple dosing in an aquaculture setting. In both medication periods, the trout were dosed with medicated feed containing 3 g MNZ kg(-1) fish. Fish were sacrificed in accordance with accepted animal care protocols and tissue samples were analysed by UPLC-MS/MS. Analyte concentrations in trout muscle ranged from a high of 27,000 ± 10,000 ng g(-1) for MNZ and 830 ± 570 ng g(-1) for MNZ-OH on day 1 of withdrawal period A to a low of 1.8 ± 2.3 ng g(-1) for MNZ and < 0.4 ng g(-1) for MNZ-OH on day 16 of withdrawal period B. The results demonstrate that when using the UPLC-MS/MS method, residues of MNZ may be detected in fish treated with MNZ after 16 days of withdrawal.

Chemical contaminants and biological indicators of mussel health during gametogenesis.

Mytilus edulis were collected intertidally from three locations in Halifax Harbor, Nova Scotia, on five occasions during spring and summer 2000. Bioindicators of health (lipid content), condition and gonad indices (CI and GI), and sex ratio, as well as vitellins, were compared with the bioaccumulation of polycyclic aromatic compounds (PACs) including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), coprostanol, and metals. Twice as many male as female mussels were collected from a downtown site (M8) close to numerous raw sewage effluents and a naval dockyard. Males from M8 had a high lipid content, and females had a delayed production of vitellins. These mussels also displayed the highest levels of PACs, coprostanol, Ag, and Sn. Coprostanol and silver are sewage markers in sediments, and their presence in mussels confirms exposure to sewage effluents. Female mussels were more abundant in an area outside the industrialized part of the harbor that had higher marine traffic (M14); displayed higher levels of vitellins in gonads; had similar time trends for CI and GI; and had some similar metals compared with mussels from M8. The lowest variability in biomarkers was observed at a site in a mostly residential arm of the harbor (M12), which was expected to be more pristine based on an earlier investigation. Compared to mussels in M14, the mussels of M12 had the lowest condition indices and PCB concentrations and low but similar levels of lipids, PACs, and coprostanol. They also displayed the highest concentrations of Cd, Cu, Pb, and Hg, and females had the highest gonad indices early in the season.

Glutathione, glutathione S-transferase, and glutathione conjugates, complementary markers of oxidative stress in aquatic biota.

Contaminants are ubiquitous in the environment and their impacts are of increasing concern due to human population expansion and the generation of deleterious effects in aquatic species. Oxidative stress can result from the presence of persistent organic pollutants, metals, pesticides, toxins, pharmaceuticals, and nanomaterials, as well as changes in temperature or oxygen in water, the examined species, with differences in age, sex, or reproductive cycle of an individual. The antioxidant role of glutathione (GSH), accompanied by the formation of its disulfide dimer, GSSG, and metabolites in response to chemical stress, are highlighted in this review along with, to some extent, that of glutathione S-transferase (GST). The available literature concerning the use and analysis of these markers will be discussed, focusing on studies of aquatic organisms. The inclusion of GST within the suite of biomarkers used to assess the effects of xenobiotics is recommended to complement that of lipid peroxidation and mixed function oxygenation. Combining the analysis of GSH, GSSG, and conjugates would be beneficial in pinpointing the role of contaminants within the plethora of causes that could lead to the toxic effects of reactive oxygen species.

Behavioural ecotoxicology, an "early warning" signal to assess environmental quality.

BACKGROUND: In this review, the position of behavioural ecotoxicology within the available means to assess the status of marine environments is described as filling the gap for the needed "early warning" signals. A few examples of studies performed since the 1960s are discussed to highlight the sensitivity of these approaches in investigating the effects of chemicals, including priority pollutants and emerging contaminants, relative to conventional toxicity tests measuring survival. DISCUSSION: The advantage of the behavioural response is due to the integration of biochemical and physiological processes that reflect changes at higher levels of organisation with ecological relevance. Avoidance often represents a behavioural symptom easily detected in many animals exposed to contaminants and would be a useful test to explore more widely. This rapid response would reflect a defence mechanism protective against further exposure and the potential development of more pronounced deleterious effects, whilst in some cases, escape could lead to the relocation of a species with negative consequences. An investigation of the avoidance behaviour of mud shrimp, Corophium volutator, along with the chemical analyses of sediments and amphipods to assess the quality of harbour sediments is summarised. The body burden of the amphipods was 1,000 times lower than the one associated with narcosis, emphasizing the sensitivity of this endpoint. The application of this acute toxicity test is briefly compared to additional work that involved intertidal mussels collected in the field. CONCLUSIONS: Recent research undertaken with mud snails, Ilyanassa obsoleta, and harbour sediments confirmed the usefulness of the escape behaviour as an assessment tool. However, the limits of the state of knowledge regarding the fate of contaminants in species with the ability to metabolise contaminants is further discussed along with directions to be pursued to address questions arising from the reviewed literature.

GC-MS/MS measurement of natural and synthetic estrogens in receiving waters and mussels close to a raw sewage ocean outfall.

In recent times, there has been an increased concern over the appearance of human estrogens in marine ecosystem and their effects on the marine habitat. Discharge of raw sewage has been identified as one of the most important sources of human estrogens in the marine environment. Therefore, we have developed a gas chromatography-(ion-trap) mass spectrometry/mass spectrometry method for the analysis of natural estrogens estrone (E1), and 17beta-estradiol (E2) and synthetic estrogens 17alpha-ethynylestradiol (EE2) and diethylstilbestrol (DES) in sewage effluents, seawater and mussels. Recovery of target analytes from mussels (n=3) was above 60% with RSD ranging from 8% to 13%. For aqueous samples (n=3) recoveries were above 80% with RSD ranging from 3% to 7%. Method detection limits for the target analytes ranged from 0.1ngg(-1) to 1.0ng/g for mussel sample analysis and from 0.5ngL(-1) to 1.2ngL(-1) for water sample analysis. The usefulness of the method was demonstrated by analyzing environmental samples from St. John's and Halifax, Canada, where raw sewage is directly discharged into the harbors. Estrone and 17 beta-estradiol were found at 1.5ngL(-1) and 1.8ngL(-1) in seawater samples collected from St. John's harbor, while trace amounts of estrone was measured in some mussels collected from Halifax harbor.

Biodegradation of endocrine disrupting compounds in harbour seawater and sediments.

The biodegradation of three endocrine disrupting compounds was examined using samples of seawater and sediment collected from Halifax Harbour, Nova Scotia, Canada, an urbanized harbour impacted by over two centuries of anthropogenic contamination. Flask experiments, where the samples were mixed to form a slurry were used to monitor the aerobic biodegradation of the synthetic plasticizer bisphenol A (BPA), the natural hormone 17beta-estradiol (E2), and the pharmaceutical and contraceptive ethinylestradiol (EE2). Degradation rates followed the order E2>EE2>BPA with half-lives of up to 1, 5 and 14 days in seawater, respectively. A rapid initial degradation rate for all three compounds with no apparent lag phase indicated the ability of the microbial community to readily catabolise the chemicals. The formation of unidentified non-persistent intermediate metabolites was observed during the E2 degradation experiments. These degradation rates are more rapid and complete than reported in previous studies, indicating the adaptation of native microbial communities to these contaminants.

Analysis of pyrene metabolites in marine snails by liquid chromatography using fluorescence and mass spectrometry detection.

As part of a study of the metabolism of aromatic compounds in marine gastropods, a sensitive and selective method was developed to detect, identify and quantify pyrene (PY) and four of its metabolites in tissues: 1-hydroxypyrene (PYOH), pyrene sulfate (PYOS), pyrene glucuronide (PYOG) and pyrenediol disulfate (PYDS). Liquid chromatography (LC) with fluorescence detection was first used to detect the PY derivatives in the visceral mass of whelks exposed to PYOH. The identification of metabolites was accomplished through a combination of retention time and spectral matching with standards, enzymatic hydrolysis, solid phase extraction and LC coupled with electrospray ionization mass spectrometry. In addition to four known PY derivatives, two novel metabolites were identified as pyrenediol glucuronide sulfate and a second isomer of PYDS. The methanol extraction of metabolites from tissue gave excellent mean recoveries, ranging from 67 to 97%, for the available standards PY, PYOH, PYOS and PYOG spiked in both the muscle and visceral mass of Buccinum spp. The mean recoveries of a surrogate standard, 2-hydroxyfluorene, spiked in all tissue samples were 100% and 95% for visceral and muscle tissue samples, respectively. The method limits of detection for these compounds were all below 0.2 ng/g of wet tissue, low enough to detect metabolites in reference animals. Results from the application of this method to the quantitative analysis of biotransformation products in the visceral mass of the whelk Neptunia lyrata exposed to PYOH contaminated food are also presented. This method will be useful to apply to the analysis of PY metabolites in soft tissues of other animals.

Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada.

Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

Aerobic co-metabolism of sulfur, nitrogen and oxygen heterocycles by three marine bacterial consortia.

Bacterial samples were collected from three marine beaches in coastal Newfoundland, Canada, and enriched by growth on 1-methylnaphthalene. The most prominent bacterial cell type for each consortium was isolated in a serial dilutions test, and a substrate utilization profile was obtained for each using the Biolog MicroStation System. Each bacterial community was tested for its ability to co-metabolize sulfur heterocycles (benzothiophene: BT, 3-methylbenzothiophene: 3-MBT, and dibenzothiphene: DBT), a nitrogen heterocycle (carbazole: CARB), and an oxygen heterocycle (dibenzofuran: DBF). Co-metabolism of the starting material was determined using gas chromatography-mass spectroscopy (GC-MS), and formation of products was investigated by GC-MS and Fourier transform infrared (FTIR) spectroscopy. Bacterial growth was monitored turbidimetrically to determine the dry weight (microgram) of cells/ml. The 2-ringed heterocycles were co-metabolized faster and to a greater extent than the 3-ringed compounds. Co-metabolism of BT was not statistically different from that for 3-MBT and, likewise, a comparison of the 3-ringed heterocycles showed no significant differences in degradation rates. Statistical examination showed that no one culture demonstrated a significantly greater ability to co-metabolize the heterocycles studied. This study represents the first comprehensive investigation of the ability of local bacteria to co-metabolize a range of aromatic compounds and provides a preliminary understanding of their fate in sediments should contamination by these compounds occur.