The Comet Assay, a Sensitive Biomarker of Water Quality Improvement Following Adoption of Beneficial Agricultural Practices?

Numerous actions have been undertaken by farmers to attenuate the impact of agricultural activities on aquatic ecosystems. The identification of biomarkers that respond quickly to water quality improvement could facilitate the assessment of adopted alternative practices and help maintain mobilization among stakeholders. We evaluated the potential of the comet assay, a biomarker of genotoxic effects, using a freshwater mussel, Elliptio complanata, as a model animal. The frequency of DNA damage was assessed in hemocytes of mussels collected from a pristine habitat and caged for 8 weeks in the Pot au Beurre River, a tributary of the fluvial Lake St.-Pierre (Quebec, Canada) impacted by agricultural activities. We found that the level of DNA damage naturally induced in mussel hemocytes was low and showed very limited variations over time. Compared with these baseline levels and to laboratory controls, we observed a doubling in DNA alterations in mussels exposed to agricultural runoff in the third branch of the Pot au Beurre River. The genotoxic response was significantly lower in mussels caged in the first branch of the Pot au Beurre River, where longer stretches of shoreline have been restored as buffer strips. Glyphosate, mesotrione, imazethapyr, and metolachlor were the main discriminant pesticides between these two branches. Metolachlor was found in sufficient concentrations to induce DNA damage, but it is more likely that the observed genotoxicity was the result of a "cocktail effect," that is, the cumulative contribution of coexisting genotoxicants including the above-mentioned herbicides and ingredients in their formulation. Our findings suggest that the comet assay is a sensitive tool for the early detection of changes in water toxicity following the adoption of agricultural beneficial practices. Environ Toxicol Chem 2023;42:2201-2214. © 2023 Crown copyright and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article is published with the permission of the Controller of HMSO and the King's Printer for Scotland.

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.

An investigation of physiological effects of the Deepwater Horizon oil spill on a long-distance migratory seabird, the northern gannet.

Exposure to oil can have long-term impacts on migratory birds. Following the 2010 Deepwater Horizon blowout in the Gulf of Mexico (GOM), we investigated potential impacts of oil exposure on a population of northern gannets (Morus bassanus) that breed on Bonaventure Island (Québec, Canada) and winter in GOM and along the U.S. Atlantic coast (AC). Blood and feather samples were collected from adults previously equipped with geolocators to determine wintering locations. Parent and alkylated polycyclic aromatic hydrocarbons (PAHs); trace metals; stable isotopes of carbon, nitrogen, and hydrogen; and immune, thyroid, steroid, retinoid, and genetic endpoints were measured. PAH and trace metal concentrations did not differ between gannets using different wintering sites. Feather stable isotope values varied significantly between birds from different wintering locations. Gannets wintering in GOM showed higher feather corticosterone and plasma thyroid hormone levels, which may indicate increased energetic demands and/or greater exposure to environmental stressors.

Cumulative effects of municipal effluent and parasite infection in yellow perch: A field study using high-throughput RNA-sequencing.

Multiple metabolic, immune and reproductive effects have been reported in fish residing in effluent-impacted sites. Natural stressors such as parasites also have been shown to impact the responses of organisms to chronic exposure to municipal effluent in the St. Lawrence River (Quebec, Canada). In order to comprehensively evaluate the cumulative impacts of anthropogenic and natural stressors on the health of yellow perch, differential mRNA transcription profiles were examined in juvenile females collected from effluent-impacted and upstream sites with low or high infection levels of the larval trematode Apophallus brevis. Transcriptomics was used to identify biological pathways associated with environmental exposure. In total, 3463 isoforms were differentially transcribed between sites. Patterns reflecting the combined effects of stressors were numerically dominant, with a majority of downregulated transcripts (68%). The differentially expressed transcripts were associated with 27 molecular and cellular functions ranging from cellular development to xenobiotic metabolism and were involved in the development and function of 13 organ systems including hematological, hepatic, nervous, reproductive and endocrine systems. Based on RNA-seq results, sixteen genes were measured by qPCR. Significant differences were observed for six genes in fish exposed to both stressors combined, whereas parasites and effluent individually impacted the transcription of one gene. Lysozyme activity, lipid peroxidation, retinol-binding protein and glucose-6-phosphate dehydrogenase were selected as potential biomarkers of effects to study specific pathways of interest. Lipid peroxidation in perch liver was different between sites, parasite loads, and for combined stressors. Overall, results indicated that juvenile yellow perch responded strongly to combined parasite and effluent exposure, suggesting cumulative effects on immune responses, inflammation and lipid metabolism mediated by retinoid receptors. The present study highlight the importance of using a comprehensive approach combining transcriptomics and endpoints measured at higher levels of biological organization to better understand cumulative risks of contaminants and pathogens in aquatic ecosystems.

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.

Genotoxic and immunotoxic potential effects of selected psychotropic drugs and antibiotics on blue mussel (Mytilus edulis) hemocytes.

The potential toxicity of pharmaceuticals towards aquatic invertebrates is still poorly understood and sometimes controversial. This study aims to document the in vitro genotoxicity and immunotoxicity of psychotropic drugs and antibiotics on Mytilus edulis. Mussel hemocytes were exposed to fluoxetine, paroxetine, venlafaxine, carbamazepine, sulfamethoxazole, trimethoprim and erythromycin, at concentrations ranging from µg/L to mg/L. Paroxetine at 1.5 µg/L led to DNA damage while the same concentration of venlafaxine caused immunomodulation. Fluoxetine exposure resulted in genotoxicity, immunotoxicity and cytotoxicity. In the case of antibiotics, trimethoprim was genotoxic at 200 µg/L and immunotoxic at 20 mg/L whereas erythromycin elicited same detrimental effects at higher concentrations. DNA metabolism seems to be a highly sensitive target for psychotropic drugs and antibiotics. Furthermore, these compounds affect the immune system of bivalves, with varying intensity. This attests the relevance of these endpoints to assess the toxic mode of action of pharmaceuticals in the aquatic environment.

Vitellogenin-like protein measurement in caged Gammarus fossarum males as a biomarker of endocrine disruptor exposure: inconclusive experience.

A vitellogenin (Vg) mass spectrometry-based assay was recently developed to actively biomonitor and assess the exposure of the amphipod Gammarus fossarum to endocrine-disrupting chemicals in freshwater hydrosystems. This paper focuses on the appropriate use of this biomarker, which requires good knowledge of its basal level in males and its natural variability related to intrinsic biotic and environmental abiotic factors. To obtain the lowest biomarker variability, we first studied some of these confounding factors. We observed that the spermatogenesis stage did not have an impact on the Vg level, allowing flexibility in the choice of transplanted gammarids. In the second part of the study, males were transplanted in two clean stations for 21 days, with results indicating a spatial and temporal variability of Vg levels. These Vg changes could not be correlated to environmental factors (e.g., temperature, pH and hardness of waters). Vg induction was then assessed in 21 stations having various levels of contamination. Inductions were observed for only two of the impacted stations studied. Under reference and contaminated conditions, a high interindividual variability of Vg levels was observed in caged organisms, severely limiting the sensitivity of the biomarker and its ability to detect a significant endocrine-disruptor effect. This may be explained by unidentified environmental factors that should later be determined to improved the use of Vg as a biomarker in male G. fossarum. Moreover, as discussed in this paper, recent advancements regarding the pleiotropic functions of the Vg gene in some species may complicate the application of this biomarker in males of invertebrate species.

In situ feeding assay with Gammarus fossarum (Crustacea): Modelling the influence of confounding factors to improve water quality biomonitoring.

In situ feeding assays implemented with transplanted crustacean gammarids have been claimed as promising tools for the diagnostic assessment of water quality. Nevertheless the implementation of such methodologies in biomonitoring programs is still limited. This is explained by the necessity to improve the reliability of these bioassays. The present study illustrates how modelling the influence of confounding factors could allow to improve the interpretation of in situ feeding assay with Gammarus fossarum. We proceeded in four steps: (i) we quantified the influence of body size, temperature and conductivity on feeding rate in laboratory conditions; (ii) based on these laboratory findings, we computed a feeding inhibition index, which proved to be robust to environmental conditions and allowed us to define a reference statistical distribution of feeding activity values through the data compilation of 24 in situ assays among diverse reference stations at different seasons; (iii) we tested the sensitivity of the feeding assay using this statistical framework by performing 41 in situ deployments in contaminated stations presenting a large range of contaminant profiles; and (iv) we illustrated in two site-specific studies how the proposed methodology improved the diagnosis of water quality by preventing false-positive and false-negative cases mainly induced by temperature confounding influence. Interestingly, the implementation of the developed protocol could permit to assess water quality without following an upstream/downstream procedure and to compare assays performed at different seasons as part of large-scale biomonitoring programs.

DNA damage in Gammarus fossarum sperm as a biomarker of genotoxic pressure: intrinsic variability and reference level.

In the perspective of a biomonitoring application for assessing genotoxicity of freshwater ecosystems, the Comet assay has recently been developed on spermatozoa in the amphipod Gammarus fossarum, in order to propose a sensitive and reliable genotoxicity biomarker in an ecologically relevant freshwater species. The appropriate use of a genotoxicity biomarker requires good knowledge of its basal level and its natural variability related to intrinsic biotic and environmental abiotic factors. We propose a procedure for which the lowest biomarker variability related to methodological and intrinsic biotic factors is obtained and a reference value of biomarker basal response taking into account its spatio-temporal changes has been defined. A strong impact of spermatogenesis status and exposure time on the response to genotoxicant pressure was observed. These reports led us to select a standard organism, i.e., the mature male gammarid in precopula. No effect of temperature and conductivity on baseline DNA damage was observed in the laboratory for the tested range (6-24 °C and 300/600 µS cm⁻¹). Similarly, no spatio-temporal change relative to season or the physico-chemical characteristics of the water was recorded during the field survey. On the basis of these results, a reference level with maximal threshold values has been proposed for the standard gammarid.

Linking genotoxic responses in Gammarus fossarum germ cells with reproduction impairment, using the Comet assay.

Germ cells perform a unique and critical biological function: they pass down DNA that will be used for the development of the next generation. Thus there is an increasing need to understand how the adult exposure to genotoxicants could show negative impact on the offspring of aquatic organisms. Hence this work addresses the question of the consequences of germ cell DNA damage resulting from parental exposure on reproduction quality in the freshwater crustacean Gammarus fossarum, a high ecologically relevant species. Initially, the sensitivity response of mature oocytes and spermatozoa to two model genotoxicants, MMS and K(2)Cr(2)O(7) was compared by implementing the Comet assay after the exposure of these gammarids in the laboratory and after the exposure of caged organisms in the field. Spermatozoa appeared significantly more susceptible than the oocytes to genotoxicants whatever were the exposure conditions. Secondly, a significant correlation between the level of damage to the sperm DNA of exposed parents and the abnormality rate in embryos that had developed in non-contaminated water were demonstrated. Interestingly, this relationship bridges the biomarker response measured in germ cells at molecular level and its consequences at individual level for the subsequent generation. Moreover, reproduction defects were observed for a level of DNA damage exceeding a minimal threshold, which could have significant consequences for the population dynamics of this high ecologically relevant species.

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

Genotoxicity assessment in the amphipod Gammarus fossarum by use of the alkaline Comet assay.

Many xenobiotics and newly developed substances released in the aquatic environment have been found genotoxic for living organisms. There is interest in developing biomarkers of genotoxicity in different phyla and the need to increase our understanding of the impact of genotoxic insult on invertebrates, particularly on crustaceans. Freshwater invertebrates and particularly amphipods are highly relevant species ecologically. However, genotoxic responses of such species are rarely studied, whereas understanding these responses is becoming an urgent concern. The aim of this study was to develop and optimize the Comet assay in the freshwater invertebrate Gammarus fossarum by use of different cell-types: haemocytes, oocytes and spermatozoa. In a first step, the Comet assay was performed on these three cell types after exposure to the model genotoxicant methyl methanesulfonate (MMS) in vitro and in vivo. Results showed a clear dose-response relationship for all tissues, a low variability and a high sensitivity of the response, demonstrating the effectiveness of the Comet assay to detect genotoxic insult in amphipods. In a second step, to explore the potential of this technique for use in ecotoxicological studies with amphipods, these organisms were exposed to five known or suspected genotoxic compounds. The results demonstrated the possibility to use the freshwater amphipod G. fossarum in environmental genotoxicity studies with the Comet assay.