Metal Contaminants in Fish: Blood as a Potential Non-lethal Monitoring Tool.

The use of fish to monitor metal contamination is well established, but existing studies often focus on internal tissues that require the sacrifice of organisms. Developing non-lethal methods is thus a scientific challenge to enable large scale biomonitoring of wildlife health. We explored blood as a potential non-lethal monitoring tool for metal contamination in brown trout (Salmo trutta fario) as a model species. First, we investigated differences in metal contamination loads (i.e., Cr, Cu, Se, Zn, As, Cd, Pb and Sb) in different blood components (whole blood, red blood cells and plasma). Whole blood was reliable to measure most metals, implying that blood centrifugation is not necessary, thus minimizing sample preparation time. Second, we measured the within individual distribution of metals across tissues (whole blood, muscle, liver, bile, kidney and gonads) to test if blood could be a reliable monitoring tool compared to other tissues. Results show that the whole blood was reliable compared to muscle and bile to measure the levels of metals such as Cr, Cu, Se, Zn, Cd and Pb. This study opens the possibility for future ecotoxicological studies in fish to use blood instead of internal tissues to quantify some metals, thus reducing the negative impacts of biomonitoring on wildlife.

Adaptive plastic responses to metal contamination in a multistress context: a field experiment in fish.

Wild populations often differ in their tolerance to environmental stressors, but intraspecific variability is rarely taken into account in ecotoxicology. In addition, plastic responses to multiple stressors have rarely been investigated in realistic field conditions. In this study, we compared the responses to metal contamination of gudgeon populations (Gobio occitaniae) differing in their past chronic exposure to metal contamination, using a reciprocal transplant experiment and an immune challenge mimicking a parasite attack to test for potential effects of multiple stressors across biological levels. We measured fish survival and traits involved in metal bioaccumulation, oxidative stress, immunity, cell apoptosis, and energy management to decipher underpinning physiological mechanisms across biological levels (i.e., gene expression, cell, organism). Fish from the two replicate High Contamination sites had higher survival when transferred into contaminated sites, suggesting a local adaptation to the contaminated site, possibly explained by higher levels of detoxification and antioxidant capacity but with potential higher apoptosis costs compared to their naïve counterparts. We found no evidence of co- or maladaptation to the immune stressor, suggesting no specific costs to face pathogens. In the emerging field of evolutionary ecotoxicology, this study underlines the need to consider intraspecific variability to better understand the effects of pollution in heterogeneous populations.

Changes in fish skin microbiota along gradients of eutrophication in human-altered rivers.

The skin microbiota plays a major role in health of organisms but it is still unclear how such bacterial assemblages respond to changes in environmental conditions and anthropogenic perturbations. In this study, we investigated the effects of the eutrophication of freshwater ecosystems on the skin microbiota of fish. We sampled wild gudgeon Gobio occitaniae from 17 river sites along an eutrophication gradient and compared their skin microbiota diversity and composition, using a 16s rRNA gene metabarcoding approach. Results showed a tendency for higher taxonomic and phylogenetic diversity in highly eutrophic sites linked to the presence of suspended organic matters. We also highlighted significant links between eutrophication and skin microbiota taxonomic composition and beta-diversity. In contrast, skin microbiota characteristics did not correlate with host factors such as age or sex, although microbiota beta-diversity did vary significantly according to host parasite load. To conclude, our study highlights the importance of environmental factors, especially eutrophication, on the diversity and composition of skin mucus bacterial communities. Because changes in the skin microbiota may induce potential deleterious consequences on host health and population persistence, our results confirm the importance of accounting for host-microbiota interactions when examining the consequences of anthropogenic activities on aquatic fauna.

Intraspecific variability of responses to combined metal contamination and immune challenge among wild fish populations.

Wild organisms are increasingly exposed to multiple anthropogenic and natural stressors that can interact in complex ways and lead to unexpected effects. In aquatic ecosystems, contamination by trace metals has deleterious effects on fish health and commonly co-occurs with pathogens, which affect similar physiological and behavioral traits. However, the combined effects of metal contamination and parasitism are still poorly known. In addition, the sensitivity to multiple stressors could be highly variable among different fish populations depending on their evolutionary history, but this intraspecific variability is rarely taken into account in existing ecotoxicological studies. Here, we investigated i) the interactive effects of metal contamination (i.e., realistic mixture of Cd, Cu and Zn) and immune challenge mimicking a parasite attack on fish health across biological levels. In addition, we compared ii) the physiological and behavioral responses among five populations of gudgeon fish (Gobio occitaniae) having evolved along a gradient of metal contamination. Results show that single stressors exposure resulted in an increase of immune defenses and oxidative stress at the expense of body mass (contamination) or fish swimming activity (immune challenge). Multiple stressors had fewer interactive effects than expected, especially on physiological traits, but mainly resulted in antagonistic effects on fish swimming activity. Indeed, the immune challenge modified or inhibited the effects of contamination on fish behavior in most populations, suggesting that multiple stressors could reduce behavioral plasticity. Interestingly, the effects of stressors were highly variable among populations, with lower deleterious effects of metal contamination in populations from highly contaminated environments, although the underlying evolutionary mechanisms remain to be investigated. This study highlights the importance of considering multiple stressors effects and intraspecific variability of sensitivity to refine our ability to predict the effects of environmental contaminants on aquatic wildlife.

Dose- and time-dependent effects of an immune challenge on fish across biological levels.

Due to global changes, fish are increasingly exposed to immune challenges associated with disease outbreaks in aquatic ecosystems. Adjustments in physiology and behavior are generally critical to maintaining homeostasis after an immune challenge, but there is limited knowledge on the specific thresholds and dynamics of responses across levels of biological organization in fish. In this study, we tested how different concentrations of an antigens mixture (phytohemagglutinin and lipopolysaccharide) affected innate immunity with potential consequences on oxidative stress, energy reserves, body condition, and behavior across time, using the common gudgeon (Gobio sp.) as model species. The immune challenge induced a transitory increase in lytic enzyme activity (i.e., lysozyme) and local immune response (i.e., skin swelling) 2 days after the antigen injection. The available energy stored in muscle was also reduced 4 days after injection, without inducing oxidative stress at the cellular level. Overall, the immune challenge induced limited costs at the molecular and cellular levels but had strong effects at the whole organism level, especially on behavior. Indeed, fish swimming activity and sociability were affected in a dose- and time-dependent manner. These results suggest that immune challenges have dose-dependent effects across levels of biological organization and that behavior is a key response trait to cope with pathogen-induced immune costs in the wild, although fitness consequences remain to be tested.

Direct and indirect effects of multiple environmental stressors on fish health in human-altered rivers.

Freshwater fish face multiple challenges in human-altered rivers such as trace metal contamination, temperature increase and parasitism. These multiple stressors could have unexpected interactive effects on fish health due to shared physiological pathways, but few studies investigated this question in wild fish populations. In this study, we compared 16 populations of gudgeon (Gobio occitaniae) distributed along perturbation gradients in human-altered rivers in the South of France. We tested the effects of single and combined stressors (i.e., metal contamination, temperature, parasitism) on key traits linked to fish health across different biological levels using a Structural Equation Modelling approach. Parasitism and temperature alone had limited deleterious effects on fish health. In contrast, fish living in metal-contaminated sites had higher metal bioaccumulation and higher levels of cellular damage in the liver through the induction of an inflammatory response. In addition, temperature and contamination had interactive negative effects on growth. These results suggest that trace metal contamination has deleterious effects on fish health at environmentally realistic concentrations and that temperature can modulate the effects of trace metals on fish growth. With this study, we hope to encourage integrative approaches in realistic field conditions to better predict the effects of natural and anthropogenic stressors on aquatic organisms.

Health indicators and contaminant levels of a critically endangered species in the Gironde estuary, the European sturgeon.

The European sturgeon, Acipenser sturio, is a highly endangered species that almost disappeared in the last decades. Thanks to yearly restocking of the population, this species is still found in the Gironde estuary (France), where juveniles grow during several years before leaving to the ocean. The aims of this study were to evaluate the pressure exerted on these fish by known organic and inorganic contaminants during their stay at the Gironde estuary, and to get information on the fish's health in this context. Monthly captures over the year 2014 provided 87 fish from the cohorts 2012 and 2013 mainly, and from cohorts 2008, 2009, and 2011, all fish born in hatchery. We report the very first analyses of contaminant levels and of biological markers measured in the blood of these fish. Low inorganic contamination was found, composed of seven metals mainly Zn (< 5 µg mL-1), Fe (< 1.5 µg mL-1), Cu (< 0.8 µg mL-1), Se (< 0.8 µg mL-1), As (< 0.25 µg mL-1), Co (< 0.14 µg mL-1), and Mn (< 0.03 µg mL-1). Concerning persistent organic contaminants, the sum of seven PCBs varied from 1 to 10 ng g-1 plasma, that of eight OCPs from 0.1 to 1 ng g-1, and that of eight PBDEs from 10 to 100 pg g-1. Higher levels of contaminants were measured during spring as compared to summer. The sex steroid hormone plasma levels (estradiol, testosterone, and 11-ketotestosterone) were quite low, which was predictable for juveniles. The transcription of reproduction-involved genes (EstR, AR, LHR, sox9) in blood cells was demonstrated for the first time. Some of them were correlated with organic contaminant levels PCBs and OCPs. Other gene transcriptions (sodCu and bax) were correlated with PCBs and OCPs. However, the DNA damage level measured here as comet tail DNA and micronuclei ratio in red blood cells were in the very low range of the values commonly obtained in fish from pristine areas. The data presented here can serve as a reference base for future monitoring of this population of sturgeons.

Combined effects of temperature increase and immune challenge in two wild gudgeon populations.

In the context of global changes, aquatic ecosystems are increasingly exposed to multiple stressors that can have unexpected interactive effects on aquatic organisms. Among these stressors, the occurrence of heat waves and pathogens is changing rapidly in freshwater rivers, but their combined effects on fish health are still understudied. In this study, we experimentally tested the crossed effects of increased temperature (mimicking a heat wave) and a standardized immune challenge (mimicking a parasite attack) on wild gudgeon (Gobio occitaniae) physiology and behaviour across biological levels from molecules to the whole individual. We also investigated the potential variation of sensitivity among populations by comparing two wild populations from contrasted thermal regimes. Combined stressors (i.e. temperature increase and immune challenge) had contrasted effects on fish physiology and behaviour compared to single stressors, but only at the individual level. In particular, the immune challenge inhibited the effect of the temperature on fish behaviour (activity, exploration and foraging) but amplified the negative effect of temperature on fish survival. No interactions were found at other biological levels. This study thus shows that it is essential to consider biotic stressors such as pathogens to better anticipate the effects of global changes on aquatic organisms. In addition, there was a high variability of response between the two gudgeon populations, suggesting that future studies should take into account population variability to better predict the responses of aquatic wildlife to current and future stressors.

Stress responses in fish: From molecular to evolutionary processes.

In the context of global changes, fish are increasingly exposed to multiple stressors that have cascading effects from molecules to the whole individual, thereby affecting wild fish populations through selective processes. In this review, we synthetize recent advances in molecular biology and evolutionary biology to outline some potentially important effects of stressors on fish across biological levels. Given the burgeoning literature, we highlight four promising avenues of research. First, (1) the exposure to multiple stressors can lead to unexpected synergistic or antagonistic effects, which should be better taken into account to improve our predictions of the effects of actual and future human activities on aquatic organisms. Second, (2) we argue that such interactive effects might be due to switches in energy metabolism leading to threshold effects. Under multiple stress exposure, fish could switch from a "compensation" strategy, i.e. a reallocation of energy to defenses and repair to a "conservation" strategy, i.e. blocking of stress responses leading to strong deleterious effects and high mortality. Third, (3) this could have cascading effects on fish survival and population persistence but multiscale studies are still rare. We propose emerging tools merging different levels of biological organization to better predict population resilience under multiple stressors. Fourth (4), there are strong variations in sensitivity among populations, which might arise from transgenerational effects of stressors through plastic, genetic, and epigenetic mechanisms. This can lead to local adaptation or maladaptation, with strong impacts on the evolutionary trajectories of wild fish populations. With this review, we hope to encourage future research to bridge the gap between molecular ecology, ecotoxicology and evolutionary biology to better understand the evolution of responses of fishes to current and future multiple stressors in the context of global changes.

Transfer of marine mercury to mountain lakes.

Stocking is a worldwide activity on geographical and historical scales. The rate of non-native fish introductions have more than doubled over the last decades yet the effect on natural ecosystems, in the scope of biologically mediated transport and biomagnification of Hg and Hg-isotopes, is unknown. Using geochemistry (THg) and stable isotopes (N, Sr and Hg), we evaluate natal origin and trophic position of brown trout (Salmo trutta fario), as well as mercury biomagnification trends and potential pollution sources to three high-altitude lakes. Farmed trout show Hg-isotope signatures similar to marine biota whereas wild trout shows Hg-isotope signatures typical of fresh water lakes. Stocked trout initially show Hg-isotope signatures similar to marine biota. As the stocked trout age and shifts diet to a higher trophic level, THg concentrations increase and the marine Hg isotope signatures, induced via farm fish feed, shift to locally produced MeHg with lower δ202Hg and higher Δ199Hg. We conclude that stocking acts a humanly induced biovector that transfers marine Hg to freshwater ecosystems, which is seen in the Hg-isotopic signature up to five years after stocking events occurred. This points to the need of further investigations of the role of stocking in MeHg exposure to freshwater ecosystems.

Adaptive response under multiple stress exposure in fish: From the molecular to individual level.

Aquatic systems are subjected to various sources of stress due to global changes, such as increasing temperature and pollution. A major challenge for the next decade will be to evaluate the combined effects of these multiple stressors on organisms and ecosystems. For organisms submitted to chemical, biological or physical stressors, the capacity to set up an efficient adaptive response is a fundamental prerequisite for their long-term survival and performance. In this study, goldfish (Carassius auratus) were subjected to individual and combined pesticide mixtures and increased temperatures to evaluate their adaptive response in multistress conditions from the molecular to the individual level. Fish were exposed for 16 days to a mixture of pesticides at environmental relevant concentrations (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin and tebuconazole) and at two temperatures (22 °C and 32 °C). Three major physiological traits of the stress response were measured: the hormonal response (i.e. plasma cortisol), the metabolic balance from molecular to individuals' levels (metabolomics, cellular energy allocation, energy reserves and global condition indexes), and the cellular defense system induction (SOD, CAT and GST). Results show that (1) environmentally relevant concentrations of pesticides lead to significant responses in fish at all biological levels; (2) the metabolic response depends on the nature of stress (thermal vs. chemical); and (3) fish may be unable to set up an efficient adaptive response when chemical and thermal stresses were combined, with adverse outcomes at the individuals' level.

Proteome response of fish under multiple stress exposure: Effects of pesticide mixtures and temperature increase.

Aquatic systems can be subjected to multiple stressors, including pollutant cocktails and elevated temperature. Evaluating the combined effects of these stressors on organisms is a great challenge in environmental sciences. To the best of our knowledge, this is the first study to assess the molecular stress response of an aquatic fish species subjected to individual and combined pesticide mixtures and increased temperatures. For that, goldfish (Carassius auratus) were acclimated to two different temperatures (22 and 32°C) for 15 days. They were then exposed for 96h to a cocktail of herbicides and fungicides (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin and tebuconazole) at two environmentally relevant concentrations (total concentrations of 8.4µgL-1 and 42µgL-1) at these two temperatures (22 and 32°C). The molecular response in liver was assessed by 2D-proteomics. Identified proteins were integrated using pathway enrichment analysis software to determine the biological functions involved in the individual or combined stress responses and to predict the potential deleterious outcomes. The pesticide mixtures elicited pathways involved in cellular stress response, carbohydrate, protein and lipid metabolisms, methionine cycle, cellular functions, cell structure and death control, with concentration- and temperature-dependent profiles of response. We found that combined temperature increase and pesticide exposure affected the cellular stress response: the effects of oxidative stress were more marked and there was a deregulation of the cell cycle via apoptosis inhibition. Moreover a decrease in the formation of glucose by liver and in ketogenic activity was observed in this multi-stress condition. The decrease in both pathways could reflect a shift from a metabolic compensation strategy to a conservation state. Taken together, our results showed (1) that environmental cocktails of herbicides and fungicides induced important changes in pathways involved in metabolism, cell structure and cell cycle, with possible deleterious outcomes at higher biological scales and (2) that increasing temperature could affect the response of fish to pesticide exposure.

Multistress effects on goldfish (Carassius auratus) behavior and metabolism.

Crossed effects between climate change and chemical pollutions were identified on community structure and ecosystem functioning. Temperature rising affects the toxic properties of pollutants and the sensitiveness of organisms to chemicals stress. Inversely, chemical exposure may decrease the capacity of organisms to respond to environmental changes. The aim of our study was to assess the individual and crossed effects of temperature rising and pesticide contamination on fish. Goldfish, Carassius auratus, were exposed during 96 h at two temperatures (22 and 32 °C) to a mixture of common pesticides (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin, and tebuconazol) at two environmentally relevant concentrations (total concentrations MIX1 = 8.4 µg L(-1) and MIX2 = 42 µg L(-1)). We investigated the sediment reworking behavior, which has a major ecological functional role. We also focused on three physiological traits from the cellular up to the whole individual level showing metabolic status of fish (protein concentration in liver and muscle, hepatosomatic index, and Fulton's condition factor). Individual thermal stress and low concentrations of pesticides decreased the sediment reworking activity of fish and entrained metabolic compensation with global depletion in energy stores. We found that combined chemical and thermal stresses impaired the capacity of fish to set up an efficient adaptive response. Our results strongly suggest that temperature will make fish more sensitive to water contamination by pesticides, raising concerns about wild fish conservation submitted to global changes.

New insight into pesticide partition coefficient Kd for modelling pesticide fluvial transport: application to an agricultural catchment in south-western France.

Pesticides applied on crops are leached with rainfall to groundwater and surface water. They threat the aquatic environment and may render water unfit for human consumption. Pesticide partitioning is one of the pesticide fate processes in the environment that should be properly formalised in pesticide fate models. Based on the analysis of 7 pesticide molecules (alachlor, atrazine, atrazine's transformation product deethylatrazine or DEA, isoproturon, tebuconazole and trifluralin) sampled from July 2009 to October 2010 at the outlet of the river Save (south-western France), the objectives of this study were (1) to check which of the environmental factors (discharge, pH, concentrations of total suspended matter (TSM), dissolved organic carbon (DOC) and particulate organic carbon (POC) could control the pesticide sorption dynamic, and (2) to establish a relationship between environmental factors, the partition coefficient Kd and the octanol/water distribution coefficient Kow. The comparison of physico-chemical parameters values during low flow and high flow shows that discharge, TSM and POC are the factors most likely controlling the pesticide sorption processes in the Save river network, especially for lower values of TSM (below 13mgL(-1)). We therefore express Kd depending on the widely literature-related variable Kow and on the commonly simulated variable TSM concentration. The equation can be implemented in any model describing the fluvial transport and fate of pesticides in both dissolved and sorbed phases, thus, Kd becomes a variable in time and space. The Kd calculation method can be applied to a wide range of catchments and organic contaminants.

Lead-induced DNA damage in Vicia faba root cells: potential involvement of oxidative stress.

Genotoxic effects of lead (0-20µM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10µM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.

Expression of the heat shock protein Hsp70 in chloride target cells of mayfly larvae from motorway retention pond: a biomarker of osmotic shock.

The aim of this work was to study, by an in toto immunohistochemical technique, the expression pattern of the heat shock protein, Hsp70, in the widely used bioindicator species Cloeon dipterum (Linnaeus 1761) (Ephemeroptera, Baetidae), living in a motorway retention pond. All sampling and measurements have been performed from March 2002 to March 2003. The water physicochemical analyses have revealed a large increase in Na(+) and Cl(-) concentrations after the de-icing road surface in winter related to motorway maintenance that correspond to an osmotic shock (from 3.1 to 105.7 mg L(-1) for Na(+) and from 3.5 to 193.9 mg L(-1) for Cl(-)). An expression of Hsp70 was observed in the chloride cells only during the osmotic shock. In contrast, the gill insertions were Hsp70 immunoreactive in specimens collected all along the year. For comparison, the expression of Hsp70 was investigated in specimens collected in a temporary pond. C. dipterum larvae living in this pond, not submitted to such osmotic shock, do not express Hsp70 neither in chloride cells nor in gill insertions. Likewise, the expression of Hsp70 was not detected in these structures during the drying period when the abiotic conditions become progressively stressful (elevation of temperature and anoxia). As chloride cells play a key role in osmoregulation, their functional integrity is crucial for the survival of the mayfly larvae in occasionally salty freshwaters. According to the well known protective role of the Hsp70 stress proteins, it is likely that the induction of Hsp70 may protect the chloride cells from osmotic shock injuries resulting from the increase in salinity. So, the Hsp70 induction in chloride cells is designed as a useful biomarker of osmotic shock. The in toto immunohistochemical detection of Hsp70 allows to characterize both the exposure situation and biological effects in target cells induced by stresses. This method could be used as a complementary qualitative approach in the biomarker actual concept. Finally, this investigation that combines this osmotic shock biomarker and this kind of bioindicator species would be a helpful tool for the monitoring of freshwater ecological systems.

Pesticide contamination of workers in vineyards in France.

In order to build tools to quantify exposure to pesticides of farmers included into epidemiological studies, we performed a field study in Bordeaux vineyards during the 2001 and 2002 treatment seasons to identify parameters related to external contamination of workers. In total, 37 treatment days were observed in tractor operators corresponding to 65 mixing operations, 71 spraying operations and 26 equipment cleaning. In all, four operators with backpack sprayers and seven re-entry workers were also monitored. We performed both detailed observations of treatment characteristics on the whole day and pesticide measurements of external contamination (dermal and inhalation) for each operation. The median dermal contamination was 40.5 mg of active ingredient per day for tractor operators, 68.8 mg for backpack sprayers and 1.3 mg for vineyard workers. Most of the contamination was observed on the hands (49% and 56.2% for mixing and spraying, respectively). The median contribution of respiratory route in the total contamination was 1.1%. A cleaning operation resulted in a 4.20 mg dermal contamination intermediate between a mixing (2.85 mg) and a spraying operation (6.13 mg). Farm owners experienced higher levels than workers and lower contaminations were observed in larger farms. The contamination increased with the number of spraying phases and when equipment cleaning was performed. Types of equipment influenced significantly the daily contamination, whereas personal protective equipment only resulted in a limited decrease of contamination.

Patterns of gene expressions induced by arsenic trioxide in cultured human fibroblasts.

Arsenic exposure is associated with several human diseases and particularly, with neoplasia. Although the mechanism of arsenic toxicity is not fully understood, several recent works pointed out the involvement of oxidative stress in arsenic-induced DNA damage that, in living cells, correlates with changes in gene expressions. In cultured human fibroblasts exposed for 24 h to micromolar arsenic concentrations, we studied, using real-time RT-PCR, the expression profile of a limited number of genes: genes coding for a stress protein (HSP70), transcription factors (cJUN, cFOS, ETR103, ETR101 and TTP) and cell cycle or DNA repair proteins (P21, GADD153). We observed that the expression profile of genes followed individual different patterns that can be summed up in early-transient gene expression by contrast to delayed gene expression.