Evolutionary approach for pollution study: The case of ionizing radiation.

Estimating the consequences of environmental changes, specifically in a global change context, is essential for conservation issues. In the case of pollutants, the interest in using an evolutionary approach to investigate their consequences has been emphasized since the 2000s, but these studies remain rare compared to the characterization of direct effects on individual features. We focused on the study case of anthropogenic ionizing radiation because, despite its potential strong impact on evolution, the scarcity of evolutionary approaches to study the biological consequences of this stressor is particularly true. In this study, by investigating some particular features of the biological effects of this stressor, and by reviewing existing studies on evolution under ionizing radiation, we suggest that evolutionary approach may help provide an integrative view on the biological consequences of ionizing radiation. We focused on three topics: (i) the mutagenic properties of ionizing radiation and its disruption of evolutionary processes, (ii) exposures at different time scales, leading to an interaction between past and contemporary evolution, and (iii) the special features of contaminated areas called exclusion zones and how evolution could match field and laboratory observed effects. This approach can contribute to answering several key issues in radioecology: to explain species differences in the sensitivity to ionizing radiation, to improve our estimation of the impacts of ionizing radiation on populations, and to help identify the environmental features impacting organisms (e.g., interaction with other pollution, migration of populations, anthropogenic environmental changes). Evolutionary approach would benefit from being integrated to the ecological risk assessment process.

Population transcriptogenomics highlights impaired metabolism and small population sizes in tree frogs living in the Chernobyl Exclusion Zone.

BACKGROUND: Individual functional modifications shape the ability of wildlife populations to cope with anthropogenic environmental changes. But instead of adaptive response, human-altered environments can generate a succession of deleterious functional changes leading to the extinction of the population. To study how persistent anthropogenic changes impacted local species' population status, we characterised population structure, genetic diversity and individual response of gene expression in the tree frog Hyla orientalis along a gradient of radioactive contamination around the Chernobyl nuclear power plant. RESULTS: We detected lower effective population size in populations most exposed to ionizing radiation in the Chernobyl Exclusion Zone that is not compensated by migrations from surrounding areas. We also highlighted a decreased body condition of frogs living in the most contaminated area, a distinctive transcriptomics signature and stop-gained mutations in genes involved in energy metabolism. While the association with dose will remain correlational until further experiments, a body of evidence suggests the direct or indirect involvement of radiation exposure in these changes. CONCLUSIONS: Despite ongoing migration and lower total dose rates absorbed than at the time of the accident, our results demonstrate that Hyla orientalis specimens living in the Chernobyl Exclusion Zone are still undergoing deleterious changes, emphasizing the long-term impacts of the nuclear disaster.

Strengths of ecosystem services concept for radiation protection.

The successful ecosystem services concept, defined as the benefits people obtain from ecosystems is still not really reflected in the current approaches for protecting public and environment against radiation promoted by the International Commission on Radiological Protection or other similar approaches. Yet some recent thoughts from international organizations lead us to believe that an eco-based approach could be more promoted in the coming years in environmental radiation protection field. The French Institute for Radiation Protection and Nuclear Safety has identified different fields of application of this concept into radiation protection, in line with its integrated approach of radiological risks management. As the ecosystem services approach makes it possible to highlight biophysical and socio-economic approaches of the impacts of ionizing radiation on ecosystems, it represents a subject of primary importance for future works conducted by IRSN. However, the operationality of the ecosystem services concept is the subject of many debates. In many situations, scientists have not yet fully understood how radioactive contamination could affect ecosystem services, and how to articulate with certainty cause and effect relationships between state of an ecosystem and provision of services. In addition, the concept is also accompanied by contradictory perceptions of the status of humans in ecosystems. To solve these knowledge gaps and uncertainties, it is necessary to acquire robust data on the impacts of radiation on ecosystems both under experimental and realistic conditions, and to integrate all potential consequences (direct and indirect, ecotoxicological but also economic and cultural).

Ionizing radiation affects the demography and the evolution of Caenorhabditis elegans populations.

Ionizing radiation can reduce survival, reproduction and affect development, and lead to the extinction of populations if their evolutionary response is insufficient. However, demographic and evolutionary studies on the effects of ionizing radiation are still scarce. Using an experimental evolution approach, we analyzed population growth rate and associated change in life history traits across generations in Caenorhabditis elegans populations exposed to 0, 1.4, and 50.0 mGy.h-1 of ionizing radiation (gamma external irradiation). We found a higher population growth rate in the 1.4 mGy.h-1 treatment and a lower in the 50.0 mGy.h-1 treatment compared to the control. Realized fecundity was lower in both 1.4 and 50.0 mGy.h-1 than control treatment. High irradiation levels decreased brood size from self-fertilized hermaphrodites, specifically early brood size. Finally, high irradiation levels decreased hatching success compared to the control condition. In reciprocal-transplant experiments, we found that life in low irradiation conditions led to the evolution of higher hatching success and late brood size. These changes could provide better tolerance against ionizing radiation, investing more in self-maintenance than in reproduction. These evolutionary changes were with some costs of adaptation. This study shows that ionizing radiation has both demographic and evolutionary consequences on populations.

Male frequency in Caenorhabditis elegans increases in response to chronic irradiation.

Outcrossing can be advantageous in a changing environment because it promotes the purge of deleterious mutations and increases the genetic diversity within a population, which may improve population persistence and evolutionary potential. Some species may, therefore, switch their reproductive mode from inbreeding to outcrossing when under environmental stress. This switch may have consequences on the demographic dynamics and evolutionary trajectory of populations. For example, it may directly influence the sex ratio of a population. However, much remains to be discovered about the mechanisms and evolutionary implications of sex ratio changes in a population in response to environmental stress. Populations of the androdioecious nematode Caenorhabditis elegans, are composed of selfing hermaphrodites and rare males. Here, we investigate the changes in the sex ratio of C. elegans populations exposed to radioactive pollution for 60 days or around 20 generations. We experimentally exposed populations to three levels of ionizing radiation (i.e., 0, 1.4, and 50 mGy.h-1). We then performed reciprocal transplant experiments to evaluate genetic divergence between populations submitted to different treatments. Finally, we used a mathematical model to examine the evolutionary mechanisms that could be responsible for the change in sex ratio. Our results showed an increase in male frequency in irradiated populations, and this effect increased with the dose rate. The model showed that an increase in male fertilization success or a decrease in hermaphrodite self-fertilization could explain this increase in the frequency of males. Moreover, males persisted in populations after transplant back into the control conditions. These results suggested selection favoring outcrossing under irradiation conditions. This study shows that ionizing radiation can sustainably alter the reproductive strategy of a population, likely impacting its long-term evolutionary history. This study highlights the need to evaluate the impact of pollutants on the reproductive strategies of populations when assessing the ecological risks.

Unusual evolution of tree frog populations in the Chernobyl exclusion zone.

Despite the ubiquity of pollutants in the environment, their long-term ecological consequences are not always clear and still poorly studied. This is the case concerning the radioactive contamination of the environment following the major nuclear accident at the Chernobyl nuclear power plant. Notwithstanding the implications of evolutionary processes on the population status, few studies concern the evolution of organisms chronically exposed to ionizing radiation in the Chernobyl exclusion zone. Here, we examined genetic markers for 19 populations of Eastern tree frog (Hyla orientalis) sampled in the Chernobyl region about thirty years after the nuclear power plant accident to investigate microevolutionary processes ongoing in local populations. Genetic diversity estimated from nuclear and mitochondrial markers showed an absence of genetic erosion and higher mitochondrial diversity in tree frogs from the Chernobyl exclusion zone compared to other European populations. Moreover, the study of haplotype network permitted us to decipher the presence of an independent recent evolutionary history of Chernobyl exclusion zone's Eastern tree frogs caused by an elevated mutation rate compared to other European populations. By fitting to our data a model of haplotype network evolution, we suspected that Eastern tree frog populations in the Chernobyl exclusion zone have a high mitochondrial mutation rate and small effective population sizes. These data suggest that Eastern tree frog populations might offset the impact of deleterious mutations because of their large clutch size, but also question the long-term impact of ionizing radiation on the status of other species living in the Chernobyl exclusion zone.

Assessment of exposure to ionizing radiation in Chernobyl tree frogs (Hyla orientalis).

Ionizing radiation can damage organic molecules, causing detrimental effects on human and wildlife health. The accident at the Chernobyl nuclear power plant (1986) represents the largest release of radioactive material to the environment. An accurate estimation of the current exposure to radiation in wildlife, often reduced to ambient dose rate assessments, is crucial to understand the long-term impact of radiation on living organisms. Here, we present an evaluation of the sources and variation of current exposure to radiation in breeding Eastern tree frogs (Hyla orientalis) males living in the Chernobyl Exclusion Zone. Total absorbed dose rates in H. orientalis were highly variable, although generally below widely used thresholds considered harmful for animal health. Internal exposure was the main source of absorbed dose rate (81% on average), with 90Sr being the main contributor (78% of total dose rate, on average). These results highlight the importance of assessing both internal and external exposure levels in order to perform a robust evaluation of the exposure to radiation in wildlife. Further studies incorporating life-history, ecological, and evolutionary traits are needed to fully evaluate the effects that these exposure levels can have in amphibians and other taxa inhabiting radio-contaminated environments.

Lack of impact of radiation on blood physiology biomarkers of Chernobyl tree frogs.

BACKGROUND: Human actions have altered natural ecosystems worldwide. Among the many pollutants released to the environment, ionizing radiation can cause severe damage at different molecular and functional levels. The accident in the Chernobyl Nuclear Power Plant (1986) caused the largest release of ionizing radiation to the environment in human history. Here, we examined the impact of the current exposure to ionizing radiation on blood physiology biomarkers of adult males of the Eastern tree frog (Hyla orientalis) inhabiting within and outside the Chernobyl Exclusion Zone. We measured the levels of eight blood parameters (sodium, potassium, chloride, ionized calcium, total carbon dioxide, glucose, urea nitrogen, and anion gap), physiological markers of homeostasis, as well as of liver and kidney function. RESULTS: Levels of blood physiology biomarkers did not vary in function of the current exposure of tree frogs to ionizing radiation within the Chernobyl Exclusion Zone. Physiological blood levels were similar in frogs inhabiting Chernobyl (both in areas with medium-high or low radiation) than in tree frogs living outside Chernobyl exposed only to background radiation levels. CONCLUSIONS: The observed lack of effects of current radiation levels on blood biomarkers can be a consequence of the low levels of radiation currently experienced by Chernobyl tree frogs, but also to the fact that our sampling was restricted to active breeding males, i.e. potentially healthy adult individuals. Despite the clear absence of effects of current radiation levels on physiological blood parameters in tree frogs, more research covering different life stages and ecological scenarios is still needed to clarify the impact of ionizing radiation on the physiology, ecology, and dynamics of wildlife inhabiting radioactive-contaminated areas.

Dose-dependent genomic DNA hypermethylation and mitochondrial DNA damage in Japanese tree frogs sampled in the Fukushima Daiichi area.

The long-term consequences of the nuclear disaster at the Fukushima Daiichi Nuclear Power Plant (FDNPP) that occurred on March 2011, have been scarcely studied on wildlife. We sampled Japanese tree frogs (Dryophytes japonicus), in a 50 -km area around the FDNPP to test for an increase of DNA damages and variation of DNA methylation level. The ambient dose rate ranged between 0.4 and 2.8 µGy h-1 and the total estimated dose rate absorbed by frogs ranged between 0.3 and 7.7 µGy h-1. Frogs from contaminated sites exhibited a dose-dependent increase of global genomic DNA methylation level (5-mdC and 5-hmdC) and of mitochondrial DNA damages. Such DNA damages may indicate a genomic instability, which may induce physiological adaptations governed by DNA methylation changes. This study stresses the need for biological data combining targeted molecular methods and classic ecotoxicology, in order to better understand the impacts on wildlife of long term exposure to low ionizing radiation levels.

Carotenoid distribution in wild Japanese tree frogs (Hyla japonica) exposed to ionizing radiation in Fukushima.

The nuclear accident in the Fukushima prefecture released a large amount of artificial radionuclides that might have short- and long-term biological effects on wildlife. Ionizing radiation can be a harmful source of reactive oxygen species, and previous studies have already shown reduced fitness effects in exposed animals in Chernobyl. Due to their potential health benefits, carotenoid pigments might be used by animals to limit detrimental effects of ionizing radiation exposure. Here, we examined concentrations of carotenoids in blood (i.e. a snapshot of levels in circulation), liver (endogenous carotenoid reserves), and the vocal sac skin (sexual signal) in relation to the total radiation dose rates absorbed by individual (TDR from 0.2 to 34 µGy/h) Japanese tree frogs (Hyla japonica). We found high within-site variability of TDRs, but no significant effects of the TDR on tissue carotenoid levels, suggesting that carotenoid distribution in amphibians might be less sensitive to ionizing radiation exposure than in other organisms or that the potential deleterious effects of radiation exposure might be less significant or more difficult to detect in Fukushima than in Chernobyl due to, among other things, differences in the abundance and mixture of each radionuclide.

Adaptation costs to constant and alternating polluted environments.

Some populations quickly adapt to strong and novel selection pressures caused by anthropogenic stressors. However, this short-term evolutionary response to novel and harsh environmental conditions may lead to adaptation costs, and evaluating these costs is important if we want to understand the evolution of resistance to anthropogenic stressors. In this experimental evolution study, we exposed Caenorhabditis elegans populations to uranium (U populations), salt (NaCl populations) and alternating uranium/salt treatments (U/NaCl populations) and to a control environment (C populations), over 22 generations. In parallel, we ran common-garden and reciprocal-transplant experiments to assess the adaptive costs for populations that have evolved in the different environmental conditions. Our results showed rapid evolutionary changes in life history characteristics of populations exposed to the different pollution regimes. Furthermore, adaptive costs depended on the type of pollutant: pollution-adapted populations had lower fitness than C populations, when the populations were returned to their original environment. Fitness in uranium environments was lower for NaCl populations than for U populations. In contrast, fitness in salt environments was similar between U and NaCl populations. Moreover, fitness of U/NaCl populations showed similar or higher fitness in both the uranium and the salt environments compared to populations adapted to constant uranium or salt environments. Our results show that adaptive evolution to a particular stressor can lead to either adaptive costs or benefits once in contact with another stressor. Furthermore, we did not find any evidence that adaptation to alternating stressors was associated with additional adaption costs. This study highlights the need to incorporate adaptive cost assessments when undertaking ecological risk assessments of pollutants.

A long-term copper exposure in a freshwater ecosystem using lotic mesocosms: Invertebrate community responses.

A lotic mesocosm study was carried out in 20-m-long channels, under continuous, environmentally realistic concentrations of copper (Cu) in low, medium, and high exposures (nominally 0, 5, 25, and 75 µg L-1 ; average effective concentrations <0.5, 4, 20, and 57 µg L-1 respectively) for 18 mo. Total abundance, taxa richness, and community structure of zooplankton, macroinvertebrates, and emerging insects were severely affected at Cu treatment levels of 25 and 75 µg L-1 . Some taxa were sensitive to Cu, including gastropods such as Lymnaea spp. and Physa sp., crustaceans such as Chydorus sphaericus, Gammarus pulex, and Asellus aquaticus, rotifers such as Mytilina sp. and Trichocerca sp., leeches such as Erpobdella sp., and the emergence of dipteran insects such as Chironomini. Other taxa appeared to be tolerant or favored by indirect effects, as in Chironimidae larvae, the emergence of Orthocladiinae, and the zooplankter Vorticella sp., which increased in the 25 and 75 µg L-1 treatments. After approximately 8 mo of Cu exposure, the macroinvertebrate community in the high treatment was decimated to the point that few organisms could be detected, with moderate effects in the medium treatment, and very slight effects in the low-Cu treatment. Subsequently, most taxa in the high-Cu exposure began a gradual and partial recovery. By the end of the study at 18 mo, macroinvertebrate taxa richness was similar to control richness, although overall abundances remained lower than controls. After 18 mo of copper exposure, a no-observed-effect concentration at the community level for consumers was set at 5 µg L-1 (4 µg L-1 as average effective concentration), and a lowest-observed-effect concentration at 25 µg L-1 (20 µg L-1 as average effective concentration). Environ Toxicol Chem 2017;36:2698-2714. © 2017 SETAC.

In situ visualization of carbonylation and its co-localization with proteins, lipids, DNA and RNA in Caenorhabditis elegans.

All key biological macromolecules are susceptible to carbonylation - an irreparable oxidative damage with deleterious biological consequences. Carbonyls in proteins, lipids and DNA from cell extracts have been used as a biomarker of oxidative stress and aging, but formation of insoluble aggregates by carbonylated proteins precludes quantification. Since carbonylated proteins correlate with and become a suspected cause of morbidity and mortality in some organisms, there is a need for their accurate quantification and localization. Using appropriate fluorescent probes, we have developed an in situ detection of total proteins, DNA, RNA, lipids and carbonyl groups at the level of the whole organism. In C. elegans, we found that after UV irradiation carbonylation co-localizes mainly with proteins and, to a lesser degree, with DNA, RNA and lipids. The method efficiency was illustrated by carbonylation induction assessment over 5 different UV doses. The procedure enables the monitoring of carbonylation in the nematode C. elegans during stress, aging and disease along its life cycle including the egg stage.

Effects of radionuclide contamination on leaf litter decomposition in the Chernobyl exclusion zone.

The effects of radioactive contamination on ecosystem processes such as litter decomposition remain largely unknown. Because radionuclides accumulated in soil and plant biomass can be harmful for organisms, the functioning of ecosystems may be altered by radioactive contamination. Here, we tested the hypothesis that decomposition is impaired by increasing levels of radioactivity in the environment by exposing uncontaminated leaf litter from silver birch and black alder at (i) eleven distant forest sites differing in ambient radiation levels (0.22-15µGyh(-1)) and (ii) along a short distance gradient of radioactive contamination (1.2-29µGyh(-1)) within a single forest in the Chernobyl exclusion zone. In addition to measuring ambient external dose rates, we estimated the average total dose rates (ATDRs) absorbed by decomposers for an accurate estimate of dose-induced ecological consequences of radioactive pollution. Taking into account potential confounding factors (soil pH, moisture, texture, and organic carbon content), the results from the eleven distant forest sites, and from the single forest, showed increased litter mass loss with increasing ATDRs from 0.3 to 150µGyh(-1). This unexpected result may be due to (i) overcompensation of decomposer organisms exposed to radionuclides leading to a higher decomposer abundance (hormetic effect), and/or (ii) from preferred feeding by decomposers on the uncontaminated leaf litter used for our experiment compared to locally produced, contaminated leaf litter. Our data indicate that radio-contamination of forest ecosystems over more than two decades does not necessarily have detrimental effects on organic matter decay. However, further studies are needed to unravel the underlying mechanisms of the results reported here, in order to draw firmer conclusions on how radio-contamination affects decomposition and associated ecosystem processes.

Toxicity of CeO2 nanoparticles on a freshwater experimental trophic chain: A study in environmentally relevant conditions through the use of mesocosms.

The toxicity of CeO2 NPs on an experimental freshwater ecosystem was studied in mesocosm, with a focus being placed on the higher trophic level, i.e. the carnivorous amphibian species Pleurodeles waltl. The system comprised species at three trophic levels: (i) bacteria, fungi and diatoms, (ii) Chironomus riparius larvae as primary consumers and (iii) Pleurodeles larvae as secondary consumers. NP contamination consisted of repeated additions of CeO2 NPs over 4 weeks, to obtain a final concentration of 1 mg/L. NPs were found to settle and accumulate in the sediment. No effects were observed on litter decomposition or associated fungal biomass. Changes in bacterial communities were observed from the third week of NP contamination. Morphological changes in CeO2 NPs were observed at the end of the experiment. No toxicity was recorded in chironomids, despite substantial NP accumulation (265.8 ± 14.1 mg Ce/kg). Mortality (35.3 ± 6.8%) and a mean Ce concentration of 13.5 ± 3.9 mg/kg were reported for Pleurodeles. Parallel experiments were performed on Pleurodeles to determine toxicity pathways: no toxicity was observed by direct or dietary exposures, although Ce concentrations almost reached 100 mg/kg. In view of these results, various toxicity mechanisms are proposed and discussed. The toxicity observed on Pleurodeles in mesocosm may be indirect, due to microorganism's interaction with CeO2 NPs, or NP dissolution could have occurred in mesocosm due to the structural complexity of the biological environment, resulting in toxicity to Pleurodeles. This study strongly supports the importance of ecotoxicological assessment of NPs under environmentally relevant conditions, using complex biological systems.

Transgenerational Adaptation to Pollution Changes Energy Allocation in Populations of Nematodes.

Assessing the evolutionary responses of long-term exposed populations requires multigeneration ecotoxicity tests. However, the analysis of the data from these tests is not straightforward. Mechanistic models allow the in-depth analysis of the variation of physiological traits over many generations, by quantifying the trend of the physiological and toxicological parameters of the model. In the present study, a bioenergetic mechanistic model has been used to assess the evolution of two populations of the nematode Caenorhabditis elegans in control conditions or exposed to uranium. This evolutionary pressure resulted in a brood size reduction of 60%. We showed an adaptation of individuals of both populations to experimental conditions (increase of maximal length, decrease of growth rate, decrease of brood size, and decrease of the elimination rate). In addition, differential evolution was also highlighted between the two populations once the maternal effects had been diminished after several generations. Thus, individuals that were greater in maximal length, but with apparently a greater sensitivity to uranium were selected in the uranium population. In this study, we showed that this bioenergetics mechanistic modeling approach provided a precise, certain, and powerful analysis of the life strategy of C. elegans populations exposed to heavy metals resulting in an evolutionary pressure across successive generations.

Pollution breaks down the genetic architecture of life history traits in Caenorhabditis elegans.

When pollution occurs in an environment, populations present suffer numerous negative and immediate effects on their life history traits. Their evolutionary potential to live in a highly stressful environment will depend on the selection pressure strengths and on the genetic structure, the trait heritability, and the genetic correlations between them. If expression of this structure changes in a stressful environment, it becomes necessary to quantify these changes to estimate the evolutionary potential of the population in this new environment. We studied the genetic structure for survival, fecundity, and early and late growth in isogenic lines of a Caenorhabditis elegans population subject to three different environments: a control environment, an environment polluted with uranium, and a high salt concentration environment. We found a heritability decrease in the polluted environments for fecundity and early growth, two traits that were the most heritable in the control environment. The genetic structure of the traits was particularly affected in the uranium polluted environment, probably due to generally low heritability in this environment. This could prevent selection from acting on traits despite the strong selection pressures exerted on them. Moreover, phenotypic traits were more strongly affected in the salt than in the uranium environment and the heritabilities were also lower in the latter environment. Consequently the decrease in heritability was not proportional to the population fitness reduction in the polluted environments. Our results suggest that pollution can alter the genetic structure of a C. elegans population, and thus modify its evolutionary potential.

Energy-based modelling to assess effects of chemicals on Caenorhabditis elegans: a case study on uranium.

The ubiquitous free-living nematode Caenorhabditis elegans is a powerful animal model for measuring the evolutionary effects of pollutants which is increasingly used in (eco) toxicological studies. Indeed, toxicity tests with this nematode can provide in a few days data on the whole life cycle. These data can be analysed with mathematical tools such as toxicokinetic-toxicodynamic modelling approaches. In this study, we assessed how a chronic exposure to a radioactive heavy metal (uranium) affects the life-cycle of C. elegans using a mechanistic model. In order to achieve this, we exposed individuals to a range of seven concentrations of uranium. Growth and reproduction were followed daily. These data were analysed with a model for nematodes based on the Dynamic Energy Budget theory, able to handle a wide range of plausible biological parameters values. Parameter estimations were performed using a Bayesian framework. Our results showed that uranium affects the assimilation of energy from food with a no-effect concentration (NEC) of 0.42 mM U which would be the threshold for effects on both growth and reproduction. The sensitivity analysis showed that the main contributors to the model output were parameters linked to the feeding processes and the actual exposure concentration. This confirms that the real exposure concentration should be measured accurately and that the feeding parameters should not be fixed, but need to be reestimated during the parameter estimation process.

Rapid evolutionary responses of life history traits to different experimentally-induced pollutions in Caenorhabditis elegans.

BACKGROUND: Anthropogenic disturbances can lead to intense selection pressures on traits and very rapid evolutionary changes. Evolutionary responses to environmental changes, in turn, reflect changes in the genetic structure of the traits, accompanied by a reduction of evolutionary potential of the populations under selection. Assessing the effects of pollutants on the evolutionary responses and on the genetic structure of populations is thus important to understanding the mechanisms that entail specialization to novel environmental conditions or resistance to novel stressors. RESULTS: Using an experimental evolution approach we exposed Caenorhabditis elegans populations to uranium, salt and alternating uranium-salt environments over 22 generations. We analyzed the changes in the average values of life history traits and the consequences at the demographic level in these populations. We also estimated the phenotypic and genetic (co)variance structure of these traits at different generations. Compared to populations in salt, populations in uranium showed a reduction of the stability of their trait structure and a higher capacity to respond by acclimation. However, the evolutionary responses of traits were generally lower for uranium compared to salt treatment; and the evolutionary responses to the alternating uranium-salt environment were between those of constant environments. Consequently, at the end of the experiment, the population rate of increase was higher in uranium than in salt and intermediate in the alternating environment. CONCLUSIONS: Our multigenerational experiment confirmed that rapid adaptation to different polluted environments may involve different evolutionary responses resulting in demographic consequences. These changes are partly explained by the effects of the pollutants on the genetic (co)variance structure of traits and the capacity of acclimation to novel conditions. Finally, our results in the alternating environment may confirm the selection of a generalist type in this environment.

Use of diatom assemblages as biomonitor of the impact of treated uranium mining effluent discharge on a stream: case study of the Ritord watershed (Center-West France).

The rehabilitation of French former uranium mining sites has not prevented the contamination of the surrounding aquatic ecosystems with metal elements. This study assesses the impact of the discharge of treated uranium mining effluents on periphytic diatom communities to evaluate their potential of bioindication. A 7-month survey was conducted on the Ritord watercourse to measure the environmental conditions of microalgae, the non-taxonomic attributes of periphyton (photosynthesis and biomass) and to determine the specific composition of diatom assemblages grown on artificial substrates. The environmental conditions were altered by the mine waters, that contaminate the watercourse with uranium and with chemicals used in the pit-water treatment plants (BaCl2 and Al2(SO4)3). The biomass and photosynthetic activity of periphyton seemed not to respond to the stress induced by the treated mining effluents whereas the altered environmental conditions clearly impacted the composition of diatom communities. Downstream the discharges, the communities tended to be characterized by indicator species belonging to the genera Fragilaria, Eunotia and Brachysira and were highly similar to assemblages at acid mine drainage sites. The species Eunotia pectinalis var. undulata, Psammothidium rechtensis, Gomphonema lagenula and Pinnularia major were found to be sensitive to uranium effluents whereas Neidium alpinum and several species of Gomphonema tolerated this contamination. The relevance of diatoms as ecological indicator was illustrated through the changes in structure of communities induced by the discharge of uranium mining effluents and creates prospects for development of a bioindicator tool for this kind of impairment of water quality.