Jocelyne Hellou

Behavioural ecotoxicology, an “early warning” signal to assess environmental quality

BackgroundIn 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.DiscussionThe 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.ConclusionsRecent 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.

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.

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.

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.

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.

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. Johns 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. Johns harbor, while trace amounts of estrone was measured in some mussels collected from Halifax harbor.

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.

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.

Bioaccumulation and biotransformation of 1-hydroxypyrene by the marine whelk Neptunea lyrata

In the marine environment, organisms can be exposed to oxidised forms of polycyclic aromatic hydrocarbons. Bioaccumulation and biotransformation of these derivatives has rarely been investigated and would lead to a better understanding of the overall fate of polycyclic aromatic hydrocarbons and that of other phenolic contaminants. The marine whelk Neptunea lyrata was exposed to 1-hydroxypyrene through its diet over 35 days. Extracts from the muscle and visceral mass of each animal were analysed by liquid chromatography with fluorescence detection. The quantified compounds included 1-hydroxpyrene and the phase II metabolites pyrene sulphate, pyrene glucuronide and one isomer of pyrenediol disulphate. The hydroxylated hydrocarbon was highly retained with 78% of the exposure amount recovered primarily from the visceral mass of the whelks, while the muscle accounted for 4% of the body burden. Whelks efficiently biotransformed 1-hydroxypyrene with a mean of 81% of the compound detected as phase II metabolites. The novel biotransformation product, pyrenediol disulphate, accounted for the largest proportion of the 1-hydroxypyrene derivatives detected at body burdens below 200u2009ng. At higher body burdens, bioaccumulation increased. Control animals showed trace levels of pyrene derivatives with 76% represented by metabolites. This study highlights the importance of investigating the multiple fates of reactive chemicals in order to interpret exposure.

Determining the bioavailability of contaminants and assessing the quality of sediments: Part 2: Behavioural response of snails, Ilyanassa obsoleta, towards contaminated harbour sediments

Background, Aims and ScopeOur study determined if and when an avoidance or attraction behavioural response would be displayed by Ilyanassa obsoleta when presented with a choice between harbour and reference sediments. Our goal was to develop a non-lethal approach that could be used in future assessments of the environmental quality of field sediments.Materials and MethodsTwenty snails were exposed in 7 L tanks to different amounts of Halifax Harbour sediments mixed with reference sediment placed in one half of a tank and reference sediments in the other. Sediments were covered with seawater, while the top half of the tank was exposed to air. One level of exposure was placed in triplicate tanks where the positioning of the snails was of 20:0, 0:20 and 10:10, in half:half of a tank. Details regarding the investigation of the set up conditions and results of exposures to specific chemicals or sediment extracts are outlined in Part 1.ResultsThe distinct avoidance of contaminated sediments and the preference for reference sediments were observed between 24 and 72 hrs of exposure, which was more pronounced with increasing percentage of harbour sediments mixed in reference sediments.DiscussionA decrease in avoidance was associated with a slight increase in temperature and longer exposure time and is interpreted as potential conditions favouring the degradation of contaminants and an increase in food. Part 1 of our study demonstrated the important role associated with the availability of food in reference sediments and a general lack of attraction to three biologically derived compounds known to be present in sewage discharges, i.e. a fatty acid methyl ester, cholesterol and coprostanol. The repelling properties of solvent extracts of a harbour sediment demonstrated that non polar chemicals were implicated in the avoidance response and would likely be reflecting the role of lipophilic contaminants in the avoidance response.ConclusionsIt is proposed that the behaviour towards harbour sediments represents a balance between the avoidance of contaminants and the attraction of food. The present study provides a robust green assessment tool that is easy to set up to assess the quality of sediments.Recommendations and PerspectivesThe interpretation of field observations regarding the abundance of animals during environmental assessments must consider both nutritional and toxicological causes. Additional laboratory exposures are needed to determine the cause of the movement of snails away from harbour sediments, i.e. which specific chemicals play a role in the behavioural response and at which level of exposure. The degradation of contaminants should also be pursued to better understand the fate of organic compounds in this habitat.