Impacts of chemical stress, season, and climate change on the flounder population of the highly anthropised Seine estuary (France).

The main objective of this study was to improve our knowledge on the responses of fish populations to multistress (diffuse pollution and warming waters) in estuaries. Adult flounders were caught in two estuaries in the Eastern English Channel: the heavily polluted Seine estuary vs the moderately contaminated Canche estuary. Fish samplings were conducted in January just before the reproduction period, and in July when gonads were at rest. The overall rise in coastal winter water temperatures detected over the Channel impairs the flounder's phenology of reproduction in the two estuaries, inducing a delay of maturation process and probably also spawning. The higher liver histopathology index in Seine vs Canche could be the consequence of the fish exposition to a complex cocktail of contaminants in a strongly industrialized estuary. Higher levels of neurotoxicity, gill lipid peroxidation, and liver EROD activity were observed in Seine vs Canche. Furthermore, a possible impairment in mitochondrial metabolism was suggested in the Seine flounder population. We confirmed in this study the potential role of two membrane lipids (sphingomyelin and phosphatidylserine) in the resistance towards oxidative stress in Seine and Canche. Finally, we suggest that the Seine flounder population (and possibly the connected Eastern English Channel flounder populations over the French Coast) could be seriously impacted in the future by multistress: higher winter temperatures and chemical contamination.

A "Population Dynamics" Perspective on the Delayed Life-History Effects of Environmental Contaminations: An Illustration with a Preliminary Study of Cadmium Transgenerational Effects over Three Generations in the Crustacean Gammarus.

We explore the delayed consequences of parental exposure to environmentally relevant cadmium concentrations on the life-history traits throughout generations of the freshwater crustacean Gammarus fossarum. We report the preliminary results obtained during a challenging one-year laboratory experiment in this environmental species and propose the use of population modeling to interpret the changes in offspring life-history traits regarding their potential demographic impacts. The main outcome of this first long-term transgenerational assay is that the exposure of spawners during a single gametogenesis cycle (3 weeks) could result in severe cascading effects on the life-history traits along three unexposed offspring generations (one year). Indeed, we observed a decrease in F1 reproductive success, an early onset of F2 offspring puberty with reduced investment in egg yolk reserves, and finally a decrease in the growth rate of F3 juveniles. However, the analysis of these major transgenerational effects by means of a Lefkovitch matrix population model revealed only weak demographic impacts. Population compensatory processes mitigating the demographic consequences of parental exposure seem to drive the modification of life-history traits in offspring generations. This exploratory study sheds light on the role of population mechanisms involved in the demographic regulation of the delayed effects of environmental toxicity in wild populations.

Natural variability and modulation by environmental stressors of global genomic cytosine methylation levels in a freshwater crustacean, Gammarus fossarum.

To improve the assessment of aquatic organism responses to environmental stressors, there is an interest in studying epigenetic marks in addition to other validated biomarkers. Indeed, the epigenetic marks may be influenced by the surrounding environment. Non-model invertebrates such as gammarids are sentinel organisms representative of the diversity of natural stream communities. Despite their ecologically relevance, the epigenetic responses have been to date poorly documented in these species. The present study explores the measurement of the global cytosine methylation level in the genome of the freshwater crustacean Gammarus fossarum. In a first step, natural variability of global cytosine methylation level (basal level) was assessed by studying the effect of sex, age and sampling site of organisms. Results showed a significant effect of age and sampling site. In a second step, effects of water temperature and food starvation were studied. For both factors, a hypermethylation was observed after 1 month of exposure. In a third step, gammarids were exposed to a range of environmentally relevant cadmium concentrations (0.05-5 µg/L) in order to assess the effect of a chemical stress. Whatever the cadmium concentration used, a significant hypomethylation was observed after 14 days followed by a trend for hypermethylation after 1 month of exposure. These results are the first ones dealing with the 5C-methylation status in gammarids. The results constitute potential markers of environmental stresses in relevant sentinel species widely used in ecotoxicological studies.

Assessment of long term ecotoxicity of urban stormwaters using a multigenerational bioassay on Ceriodaphnia dubia: A preliminary study.

Standardized ecotoxicity bioassays show some limits to assess properly long-term residual toxicity of complex mixture of pollutants often present at low concentration, such as stormwaters. Among invertebrate organisms used for ecotoxicity testing, the microcrustacean Ceriodaphnia dubia (C. dubia) is considered as one of the most sensitive, especially regarding reproduction impairment as a toxicity endpoint. Consequently, this work explores the interest to perform a multigenerational assay based on the study of the reproduction of C. dubia to assess long-term ecotoxicity of complex mixture, using stormwater samples. With this in mind, a battery of standardized bioassays (Daphnia magna mobility, Pseudokirchneriella subcapitata population growth, Heterocypris incongruens growth and one generation C. dubia reproduction inhibition assays) was performed in parallel to a three generation C. dubia reproduction inhibition assay on 2 stormwater samples. Results highlighted that while all standardized bioassays failed to reveal residual toxicity in the stormwater samples, the C. dubia multigenerational assay exhibited an higher sensitivity than the previous ones. No adverse effect was observed for the first exposed generation, but an increase in mortality and a reproduction disturbance was obtained in the second and third exposed generation depending of the sample. Further experiments are now needed to optimize the exposure protocol of this multigenerational assay.

Gammarus fossarum as a sensitive tool to reveal residual toxicity of treated wastewater effluents.

Wastewater treatment plants (WWTPs) are one of the main sources of freshwater pollution eventually resulting in adverse effects in aquatic organisms. Treated effluents can contain many micropollutants at concentrations often below the limit of chemical quantification. On a regulatory basis, WWTP effluents have to be non-toxic to the aquatic environment, wherefore not only chemical abatement but also ecotoxicological evaluation through relevant bioassays is required. Standardized bioassays currently used are often not sensitive enough to reveal a residual toxicity in treated effluents. Therefore, attention must be paid to the development of better-adapted approaches implementing more sensitive organisms and relevant endpoints. In this study, the toxicity of two differently treated effluents (activated sludge treated effluents with and without ozonation) towards the ecologically relevant species Gammarus fossarum was evaluated. Organism fitness traits such as reproduction and sperm DNA integrity were followed in exposed organisms. In complement, enzymatic biomarkers were measured indicating the presence of neurotoxic compounds (acetylcholinesterase activity), the presence of pathogens likely to increase the toxic effects of chemical compounds (phenol-oxidase activity), and the presence of toxic compounds inducing detoxification mechanisms (glutathione-S-transferase activity). Enzymatic activities were not modified, but significant sub-lethal effects were observed in exposed organisms. In both effluents, females showed a retarded molt cycle, a reduced fecundity and fertility, and >90% of developed embryos exhibited developmental malformations. In addition, a slight but significant genotoxic effect was measured in gammarid sperm. In a whole, no difference in toxicity was found between both effluents. Coupling reproduction impairment and genotoxicity assessment in Gammarus fossarum seems to be a valuable and sensitive tool to reveal residual toxicity in effluents containing a mixture of micropollutants at very low concentrations. Finally, a direct relationship between the observed toxic responses and the quantified micropollutant concentrations could not be evidenced.

An integrative approach to assess ecological risks of surface water contamination for fish populations.

Contamination of aquatic ecosystems is considered as one of the main threats to global freshwater biodiversity. Within the European Water Framework Directive (EU-WFD) a particular attention is dedicated to assess ecological risks of surface water contamination and mitigation of chemical pressures on aquatic ecosystems. In this work, we evaluated ecological risks of surface water contamination for fish populations in four EU-WFD rivers through an integrative approach investigating three Lines of Evidence (chemical contamination, biomarker responses as early warning signals of contamination impacting individuals and ecological analyses as an indicator of fish community disturbances). This work illustrates through 4 case studies the complementary role of biomarkers, chemical and ecological analyses which, used in combination, provide fundamental information to understand impacts of chemical pressures that can affect fish population dynamics. We discuss the limitations of this approach and future improvements needed within the EU-WFD to assess ecological risk of river contamination for fish populations.

Combining different assays and chemical analysis to characterize the genotoxicity of waters impacted by textile discharges.

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg-modified comet assay in the RTL-W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg-modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559-571, 2016. © 2016 Wiley Periodicals, Inc.

Proposal to optimize ecotoxicological evaluation of wastewater treated by conventional biological and ozonation processes.

A mixture of urban and hospital effluents (50% v/v) was evaluated for ecotoxicity with an advanced bioassay battery. Mixed effluents were tested before any treatment, after biological treatment alone, and after biological treatment followed by a tertiary ozonation (15 mg O3/L). Laying a high value on the continuance of organisms' fitness, essential to preserve a healthy receiving ecosystem, the main objective of this study was to combine normalized bioassays with newly developed in vivo and in vitro tests in order to assess alteration of embryo development, growth and reproduction, as well as genotoxic effects in aquatic organisms exposed to complex wastewater effluents. Comparison of the bioassays sensitivity was considered. Contrary to the lack of toxicity observed with normalized ecotoxicity tests, endpoints measured on zebrafish embryos such as developmental abnormalities and genotoxicity demonstrated a residual toxicity in wastewater both after a biological treatment followed or not by a tertiary O3 treatment. However, the ozonation step allowed to alleviate the residual endocrine disrupting potential measure in the biologically treated effluent. This study shows that normalized bioassays are not sensitive enough for the ecotoxicological evaluation of wastewaters and that there is a great need for the development of suitable sensitive bioassays in order to characterize properly the possible residual toxicity of treated effluents.

Characterization of a genotoxicity biomarker in three-spined stickleback (Gasterosteus aculeatus L.): Biotic variability and integration in a battery of biomarkers for environmental monitoring.

As a large array of hazardous substances exhibiting genotoxicity are discharged into surface water, this work aimed at assessing the relevance of adding a genotoxicity biomarker in a battery of biomarkers recently developed in the model fish three-spined stickleback (Gasterosteus aculeatus). First the confounding influence of gender, body length, and season (used as a proxy of age and of the fish reproductive status, respectively) on the level of primary DNA damage in erythrocytes was investigated in wild sticklebacks. Then, the genotoxity biomarker was included in a large battery of biomarkers assessing xenobiotic biotransformation, oxidative stress and neurotoxicity, and implemented in five sites. Gender, age and reproductive status did not influence DNA damage level in fish from the reference site. A significant relationship between the level of primary DNA damage and fish length (as a proxy of age also correlated to the season) was highlighted in the contaminated site. Among all biomarkers investigated in the field, the level of DNA damage was one of the four most discriminating biomarkers with EROD, catalase activity and the level of lipid peroxidation representing together 75.40% of the discriminating power in sampled fish. The level of DNA damage was correlated to the EROD activity and to the level of peroxidation, which mainly discriminated fish from sites under urban pressure. Finally, Integrated Biomarker Response indexes (IBRv2), which were calculated with the whole biomarker response dataset exhibited higher values in the Reveillon (9.62), the Scarpe and Rhonelle contaminated sites (5.11 and 4.90) compared with the two reference sites (2.38 and 2.55). The present work highlights that integration of a genotoxicity biomarker in a multiparametric approach is relevant to assess ecotoxicological risk in freshwater aquatic organisms.

Field evidence of reproduction impairment through sperm DNA damage in the fish nase (Chondrostoma nasus) in anthropized hydrosystems.

This work aims to explore in the field the relationship between the integrity of sperm DNA and the quality of offspring as a possible cause of the decline of a feral fish population through reproduction impairment. Mature nase (Chondrostoma nasus) were caught during the breeding season in three locations (A-C) of the Rhône River basin and gametes collected by stripping. Sampling locations were chosen according to the following gradient of contamination due to human activities on the watershed: A≤B

Automated Lab-on-a-Chip Technology for Fish Embryo Toxicity Tests Performed under Continuous Microperfusion (µFET).

The fish embryo toxicity (FET) biotest has gained popularity as one of the alternative approaches to acute fish toxicity tests in chemical hazard and risk assessment. Despite the importance and common acceptance of FET, it is still performed in multiwell plates and requires laborious and time-consuming manual manipulation of specimens and solutions. This work describes the design and validation of a microfluidic Lab-on-a-Chip technology for automation of the zebrafish embryo toxicity test common in aquatic ecotoxicology. The innovative device supports rapid loading and immobilization of large numbers of zebrafish embryos suspended in a continuous microfluidic perfusion as a means of toxicant delivery. Furthermore, we also present development of a customized mechatronic automation interface that includes a high-resolution USB microscope, LED cold light illumination, and miniaturized 3D printed pumping manifolds that were integrated to enable time-resolved in situ analysis of developing fish embryos. To investigate the applicability of the microfluidic FET (µFET) in toxicity testing, copper sulfate, phenol, ethanol, caffeine, nicotine, and dimethyl sulfoxide were tested as model chemical stressors. Results obtained on a chip-based system were compared with static protocols performed in microtiter plates. This work provides evidence that FET analysis performed under microperfusion opens a brand new alternative for inexpensive automation in aquatic ecotoxicology.

Exposure to runoff from coal-tar-sealed pavement induces genotoxicity and impairment of DNA repair capacity in the RTL-W1 fish liver cell line.

Coal-tar-based (CTB) sealcoat, frequently applied to parking lots and driveways in North America, contains elevated concentrations of polycyclic aromatic hydrocarbons (PAHs) and related compounds. The RTL-W1 fish liver cell line was used to investigate two endpoints (genotoxicity and DNA-repair-capacity impairment) associated with exposure to runoff from asphalt pavement with CTB sealcoat or with an asphalt-based sealcoat hypothesized to contain about 7% CTB sealcoat (AS-blend). Genotoxic potential was assessed by the Formamido pyrimidine glycosylase (Fpg)-modified comet assay for 1:10 and 1:100 dilutions of runoff samples collected from 5 h to 36 d following sealcoat application. DNA-repair capacity was assessed by the base excision repair comet assay for 1:10 dilution of samples collected 26 h and 36 d following application. Both assays were run with and without co-exposure to ultraviolet-A radiation (UVA). With co-exposure to UVA, genotoxic effects were significant for both dilutions of CTB runoff for three of four sample times, and for some samples of AS-blend runoff. Base excision repair was significantly impaired for CTB runoff both with and without UVA exposure, and for AS-blend runoff only in the absence of UVA. This study is the first to investigate the effects of exposure to the complex mixture of chemicals in coal tar on DNA repair capacity. The results indicate that co-exposure to runoff from CT-sealcoated pavement and UVA as much as a month after sealcoat application has the potential to cause genotoxicity and impair DNA repair capacity.

Tamoxifen ecotoxicity and resulting risks for aquatic ecosystems.

Tamoxifen, a drug used to treat cancer, is regularly found in hydrosystems at concentrations of several hundred ng L(-1). To characterize its ecotoxicity, we implemented a battery of bioassays on organisms belonging to 3 different trophic levels: Pseudokirchneriella subcapitata, Chlorella vulgaris and Chlamydomonas reinhardtii, for primary producers, Daphnia magna (immobilization, grazing and reproduction) for primary consumers, and Danio rerio for secondary consumers (embryotoxicity test). In view of the results obtained and the ecotoxicity values of tamoxifen available in the literature, we established a PNEC (Predictive No Effect Concentration) equal to 81 ng L(-1) for continental water. This PNEC allowed us to calculate Risk Quotients (RQ) for 4 continental hydrosystems in 4 different countries in which measures of tamoxifen had already been performed on surface waters. In two of the situations studied, RQs were higher than 1, reaching a maximum of 2.6. These results show the need to deepen the characterization of ecotoxicological risks linked to the discharge of tamoxifen in surface waters. In addition, we propose applying this approach to other drug residues detected in the environment.

Responses of the European flounder (Platichthys flesus) to a mixture of PAHs and PCBs in experimental conditions.

A multibiomarker approach was developed to evaluate the juvenile European flounder responses to a complex mixture of 9 polycyclic aromatic hydrocarbons (PAHs) and 12 polychlorinated biphenyls (PCBs). Exposure was performed through contaminated food pellets displaying: (1) PAH and PCB levels similar to those detected in the heavily polluted Seine estuary, respectively in sediments and in flatfish and (2) ten times these concentrations. Several biomarkers of the immune system (e.g., lysozyme concentration and gene expression of complement component C3 and TNF-receptor), DNA damage (e.g., Comet assay), energetic metabolism (e.g., activity of cytochrome C oxidase), detoxification process (e.g., cytochrome P450 1A1 expression level: CYP1A1; betaine homocysteine methyl transferase expression level: BHMT) were investigated after 14 and 29 days of contamination, followed by a 14-days recovery period. After 29 days of contamination, the detoxification activity (CYP1A1 expression level) was positively correlated with DNA damages; the increase of the BHMT expression level could also be related to the detoxification process. Furthermore, after the recovery period, some biomarkers were still upregulated (i.e., CYP1A1 and BHMT expression levels). The immune system was significantly modulated by the chemical stress at the two concentration levels, and the lysozyme appeared to be the most sensitive marker of the mixture impact.

UV-induced Nucleotide Excision Repair (NER) and Photoreactivation Repair (PER) in two trout fish cell lines used in ecotoxicological assessment studies.

A better knowledge of DNA repair capacities in permanent fish cell lines would contribute to establish their interest in genotoxicity testing for environmental risk assessment studies including the effects of an increase in solar UV radiations on aquatic organisms. NER (Nucleotide Excision Repair) and PER (Photoreactivation Repair) are the two repair pathways of choice for UV-induced photo-lesions. In the present paper, these repair processes were characterized in the two rainbow trout cell lines, RTGill-W1 and RTL-W1 (liver), by means of a T4-modified comet assay which allowed to follow the cyclobutane pyrimidine dimers repair kinetics specifically. Both repair processes have been found in the cell lines, PER repairing much faster UV lesions than NER, and NER being slightly more efficient in the gill cell line than in the liver one.

DNA repair activity in fish and interest in ecotoxicology: a review.

The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O6-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities in fish could be the base for the development of new biomarkers of genotoxicity. Knowing the importance of the germ cell DNA integrity in the reproductive success of aquatic organisms, the DNA repair capacity of such cells deserve to be more studied, as well as DNA repair capacities of established fish cell lines. The limited amount of available data, which shows low/slow DNA repair capacities of fish cell lines compared with mammalian cell lines, concerned mainly the NER system; thus this point merits to be explored more deeply. Additionally, since some of the DNA repair systems appear more efficient in embryo larval stages, it would be of interest to consider embryonic cell lineages more closely.

Assessment of base-excision repair activity in fish cell lines: toward a new biomarker of exposure to environmental contaminants?

Amongst DNA-repair processes, base-excision repair (BER) is the major mechanism for removal of DNA-base lesions caused by environmental genotoxicants. BER has been proven to exist in fish but has not been investigated in fish cell-lines, although these constitute increasingly important tools in eco-toxicological assessment. The present study aims at highlighting BER capacity of RTL-W1 and RTG-W1, two trout cell lines used in eco-genotoxicity studies. This is realized by following the kinetics of strand-break repair after a short exposure to model genotoxicants-leading predominantly to BER-specific lesions-by means of the standard alkaline and Fpg-modified comet assays. Results show that both cell lines efficiently repair single-strand breaks and base-alkylation damages within 4h and 24h, respectively. Then, the study shows that after minor modifications of the protocol, the cell extract-based BERc assay can be used to evaluate the base-incision capacity of the cell lines and its variation after exposure of the cells to a model inhibitor of BER (3-aminobenzamide) and to environmental contaminants such as cadmium and tributyltin. This work provides a basis for the further development of DNA-repair activity in fish cell-lines as a new biomarker of genotoxicity.

Responses of juvenile European flounder (Platichthys flesus) to multistress in the Vilaine estuary, during a 6-month survey.

Physiological and genetic responses of age 0+ Platichthys flesus were investigated in the eutrophicated and moderately contaminated Vilaine estuary, during a 6-month survey. The main objective of this study was to explore the biological responses of fishes during their juvenile period in an estuarine system in order to detect a possible selective pressure induced by the environmental stress. Our results showed a general convergence in physiological responses along the survey: an increase in genotoxicity was associated with an increase in mRNA expression of ATPase and betaine homocysteine methyltransferase. These results could suggest an increase of cellular damage, energetic request, and detoxification rate related to the growing exposure time to stress. Considering the aging of the cohort, the genetic characteristics of the Vilaine flounder cohort came closer to the one observed in a highly stressed system, the Seine estuary, suggesting a potential selective pressure mainly induced by the chemical stress.

Variation patterns in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Platichthys flesus) in the Bay of Biscay.

The objective was to describe and model variation patterns in individual fish responses to contaminants among estuaries, season and gender. Two hundred twenty-seven adult European flounders were collected in two seasons (winter and summer) in four estuaries along the Bay of Biscay (South West France), focusing on a pristine system (the Ster), vs. three estuaries displaying contrasted levels of contaminants (the Vilaine, Loire and Gironde). Twenty-three variables were measured by fish, considering the load of contaminants (liver metals, liver and muscle persistent organic pollutants, muscle polycyclic aromatic hydrocarbons); the gene expression (Cyt C oxydase, ATPase, BHMT, Cyt P450 1A1, ferritin); the blood genotoxicity (Comet test); and liver histology (foci of cellular alteration-tumour, steatosis, inflammation, abnormal glycogen storage). Canonical redundancy analysis (RDA) was used to model these variables using gender, season and estuary of origin as explanatory variables. The results underlined the homogeneity of fish responses within the pristine site (Ster) and more important seasonal variability within the three contaminated systems. The complete model RDA was significant and explained 35 % of total variance. Estuary and season respectively explained 30 and 5 % of the total independent variation components, whilst gender was not a significant factor. The first axis of the RDA explains nearly 27 % of the total variance and mostly represents a gradient of contamination. The links between the load of contaminants, the expression of several genes and the biomarkers were analysed considering different levels of chemical stress and a possible multi-stress, particularly in the Vilaine estuary.

Impact of toxic cyanobacterial blooms on Eurasian perch (Perca fluviatilis): experimental study and in situ observations in a peri-alpine lake.

Due to the importance of young-of-the-year (YOY) perch in the peri-alpine regions where they are consumed, the microcystin (MC) contamination of YOY perch was analysed both in field (Lake Bourget, France) and experimentally using force-feeding protocols with pure MCs. In-situ, schools of YOY perch present in the epilimnion of the lake were never found in direct contact with the P. rubescens blooms that were present in the metalimnion. However, MCs were detected in the muscles and liver of the fish and were thus assumed to reach YOY perch through dietary routes, particularly via the consumption of MC-containing Daphnia. Force-feeding experiment demonstrates the existence of MC detoxification/excretion processes and suggests that in situ, YOY perch could partly detoxify and excrete ingested MCs, thereby limiting the potential negative effects on perch populations under bloom conditions. However, because of chronic exposure these processes could not allow for the complete elimination of MCs. In both experimental and in situ studies, no histological change was observed in YOY perch, indicating that MC concentrations that occurred in Lake Bourget in 2009 were too low to cause histological damage prone to induce mortality. However, Deoxyribonucleic acid (DNA) damages were observed for both the high and low experimental MC doses, suggesting that similar effects could occur in situ and potentially result in perch population disturbance during cyanobacterial blooms. Our results indicate the presence of MCs in wild perch, the consumption of this species coming from Lake Bourget is not contested but more analyses are needed to quantify the risk.