In vitro assessment of the genotoxicity and immunotoxicity of treated and untreated municipal effluents and receiving waters in freshwater organisms.

Municipal wastewater effluent is one of the largest sources of pollution entering surface waters in the Laurentian Great Lakes. Exposure to wastewater effluent has been associated with impaired immune systems and induction of genotoxicity to aquatic animals. Due to habitat degradation and environmental pollution linked to industrial development and population growth, several regions of the Great Lakes have been designated Areas of Concern (AOCs). In this study, we assessed the effect of extracts of sewage influent, (treated) effluent and receiving surface waters from the Hamilton Harbour AOC and the Toronto and Region AOC (Ontario, Canada) on the phagocytic immune response of rainbow trout (Oncorhynchus mykiss) kidney leukocytes and the genotoxicity (DNA strand breaks) of these extracts on freshwater mussel (Eurynia dilatata) hemocytes. We identified and quantified numerous chemicals present in the various samples extracted for exposure. In freshwater mussels, extracts from Hamilton Harbour AOC induced DNA damage with the most frequency (12 out of 28 samples) regardless of sample type, reflecting past and present industrial activities. In contrast, extracts from Toronto and Region AOC induced DNA damage infrequently (2 out of 32 (summer) and 5 out of 32 (fall) samples, respectively) and from different WWTPs at different times. None of the extracts induced any significant effect on phagocytosis of rainbow trout kidney leukocytes. The present study indicates that despite overall improvements to effluent quality, treatment of influent by WWTPs may not result in a corresponding improvement of the genotoxicity of effluents. In vitro bioassays are useful and cost-effective rapid-screening tools for preliminary assessments of contamination of aquatic ecosystems.

Evaluation of uptake of the cytostatic methotrexate in Elliptio complanata mussels by LC-MS/MS.

Aquatic organisms are continuously exposed to emerging contaminants coming from urban effluents of wastewater treatment plants. The contamination of surface water by those effluents poses a number of environmental risks, and pharmaceuticals are part of this class of effluent contaminants. Various classes of pharmaceuticals are not treated by wastewater treatment plants and anticancer drugs are part of them. The chemotherapy drug methotrexate (MTX) is an emerging contaminant and its growing use with the increase in cancer cases worldwide raises potential risk to aquatic organisms exposed to effluent discharges. However, chemical analyses in exposed freshwater aquatic organisms for ecotoxicological studies are rarely available and no studies have been done yet to accompany ecotoxicological data of exposed filter-feeding organisms. The purpose of this study was to develop a specific and sensitive analytical LC-MS/MS method for the quantification of methotrexate uptake in mussels exposed at different concentrations of the drug. A solid/liquid extraction followed by solid phase extraction (SPE) using an MCX phase purification scheme was optimized. The optimal recovery of 65% and matrix effect of 38% allowed to achieve a limit of quantification of 0.25 ng g-1, with an accuracy of 99-106%, a precision of no more than 3% RSD, and linearity ranging from 0.25 to 25 ng g-1. This methodology was tested with mussels exposed for 96 h at different concentrations (4 to 100 µg L-1) of MTX. The data revealed tissue uptake at concentrations ranging from 0 to 2.53 ng g-1. This suggests that this drug has low uptake potential and this methodology could be used to examine tissue levels of this drug in organisms continuously exposed to urban pollution.

Occurrence and ecotoxicity of cytostatic drugs 5-fluorouracil and methotrexate in the freshwater unionid Elliptio complanata.

Municipal effluents continuously release cytostatic drugs with unknown consequences in aquatic organisms. The purpose of the study was to examine the sublethal toxicity of 2 commonly-found cytostatic drugs 5-fluouracile (5-FLU) and methotrexate (MTX) to endemic Elliptio complanata freshwater mussels. The mussels were exposed of each drugs at 0, 4, 20 and 100 µg/L for 96 h t 15 °C. After the exposure period, glutathione S-transferase (GST) and dehydrofolate reductase (DHFR) activities, DNA damage and lipid peroxidation (LPO) were determined. The drugs were detected in mussel tissues with no evidence of accumulation with either drugs. The drug 5-FLU gave a larger spectrum of effects than MTX such as increased DHFR, decreased LPO and DNA strand breaks (repair activity) suggesting that the mussels were metabolically hindered and reduced DNA repair activity. The drug MTX only increased DHFR activity in the gonad. Hence, the data suggest that these drugs are biologically active in freshwater mussels and based on the reported maximum levels of these drugs in municipal effluents, the observed effects are likely in sessile freshwater mussel species downstream urban sources of pollution.

Toxicity of representative mixture of five rare earth elements in juvenile rainbow trout (Oncorhynchus mykiss) juveniles.

Rare earth elements (REEs) are contaminants of increasing interest due to intense mining activities for commercial purposes and ultimately released in the environment. We exposed juvenile rainbow trout (Oncorhynchus mykiss) to a representative mixture of the five most abundant REEs for 96 h at concentrations similar found in lakes contaminated by mining activities at 0.1, 1, 10, and 100X whereas the 1x mixture contained cerium (Ce, 280 µg/L), lanthanum (La, 140 µg/L), neodymium (Nd, 120 µg/L), praseodymium (Pr, 28 µg/L), and samarium (Sm, 23 µg/L). We investigated the expression of 14 genes involved in oxidative stress, DNA repair, tissue growth/proliferation, protein chaperoning, xenobiotic biotransformation, and ammonia metabolism in the liver. In addition, DNA damage, oxidative stress (lipid peroxidation or LPO), inflammation (cyclooxygenase or COX activity), detoxification mechanisms (glutathione-S-transferase activity or GST), and labile zinc were determined in gills. The data revealed that genes involved in oxidative stress-catalase (cat), heat shock proteins 70 (hsp70), and glutamate dehydrogenase (glud) were upregulated while glutathione S-transferase (gst) and metallothionein (mt) gene expressions were downregulated. The mixture was genotoxic and increased labile Zn in gills of exposed trout. These changes occurred at concentrations 600 times lower than the LC50 for this mixture indicating effects below the 1X concentration. Based on principal component analysis and concentration-dependent reponses, the following sublethal effects were considered the most important/significant: DNA strand breaks (genotoxicity), labile Zn, cat, gst, hsp70, sparc, mt, and glud. These effects of fish juveniles are likely to occur in environments under the influence of mining activities.

T lymphocyte-proliferative responses of harbor seal (Phoca vitulina) peripheral blood mononuclear cells (PBMCs) exposed to pharmaceuticals in vitro.

The ubiquity of pharmaceuticals in the aquatic environment and the accumulation in organisms of lower trophic levels have been documented. The immunotoxicity of these xenobiotics has however been little investigated. This study assessed the effects of pharmaceuticals on the immune responses of harbor seal lymphocytes. Peripheral blood mononuclear cells isolated from harbor seal pups were exposed to varying concentrations of 17α-ethinyl estradiol (250-50,000µg/L), naproxen (500-100,000µg/L), carbamazepine (500-100,000µg/L), erythromycin (750-150,000µg/L) and binary mixtures thereof in vitro. All individual compounds and mixtures inhibited lymphocyte proliferation. Mixture effects were non-additive and predictive values overestimated the inhibition of proliferation. Male pups were more sensitive to erythromycin exposure. Comparison with the sensitivity of the 11B7501 cell line showed a higher sensitivity of pups to individual compounds and the inverse trend for mixtures. Based on our results, we hypothesize that pharmaceuticals may have the potential to interrupt immune functions in harbor seals.

Immunotoxic effects of single and combined pharmaceuticals exposure on a harbor seal (Phoca vitulina) B lymphoma cell line.

The potential risk of pharmaceuticals in the environment to top-predators is still largely unknown. In this study, we assessed the immunotoxic effects of ten pharmaceuticals individually and as mixtures on a harbor seal (Phoca vitulina) B lymphoma cell line. A significant reduction in lymphocyte transformation was observed following an exposure to 12,500µg/L 17α-ethinyl estradiol and 25,000µg/L naproxen. Exposure to 12,500µg/L 17α-ethinyl estradiol decreased the percentage of cell in the G0/G1 phase of the cell cycle while increasing the percentage of cells in the S phase. Carbamazepine exposure increased the amount of cells in the G2/M phase. Binary mixtures showed synergistic effects in lymphocyte transformation, cell cycle and apoptosis assays. Concentrations inducing toxic effects in the cell line were similar to those affecting fish in previous studies. A reduction of functional activities of the immune system may lead to altered host resistance to pathogens in free-ranging pinnipeds.