Transgenerational DNA Methylation Changes in Daphnia magna Exposed to Chronic γ Irradiation.

Our aim was to investigate epigenetic changes in Daphnia magna after a 25-day chronic external γ irradiation (generation F0 exposed to 6.5 µGy·h-1 or 41.3 mGy·h-1) and their potential inheritance by subsequent recovering generations, namely, F2 (exposed as germline cells in F1 embryos) and F3 (the first truly unexposed generation). Effects on survival, growth, and reproduction were observed and DNA was extracted for whole-genome bisulfite sequencing in all generations. Results showed effects on reproduction in F0 but no effect in the subsequent generations F1, F2, and F3. In contrast, we observed significant methylation changes at specific CpG positions in every generation independent of dose rate, with a majority of hypomethylation. Some of these changes were shared between dose rates and between generations. Associated gene functions included gene families and genes that were previously shown to play roles during exposure to ionizing radiation. Common methylation changes detected between generations F2 and F3 clearly showed that epigenetic modifications can be transmitted to unexposed generations, most likely through the germline, with potential implications for environmental risk.

A dynamic energy-based model to analyze sublethal effects of chronic gamma irradiation in the nematode Caenorhabditis elegans.

Understanding how toxic contaminants affect wildlife species at various levels of biological organization (subcellular, histological, physiological, organism, and population levels) is a major research goal in both ecotoxicology and radioecology. A mechanistic understanding of the links between different observed perturbations is necessary to predict the consequences for survival, growth, and reproduction, which are critical for population dynamics. In this context, experimental and modeling studies were conducted using the nematode Caenorhabditis elegans. A chronic exposure to external gamma radiation was conducted under controlled conditions. Results showed that somatic growth and reproduction were reduced with increasing dose rate. Modeling was used to investigate whether radiation effects might be assessed using a mechanistic model based upon the dynamic energy budget (DEB) theory. A DEB theory in toxicology (DEB-tox), specially adapted to the case of gamma radiation, was developed. Modelling results demonstrated the suitability of DEB-tox for the analysis of radiotoxicity and suggested that external gamma radiation predominantly induced a direct reduction in reproductive capacity in C. elegans and produced an increase in costs for growth and maturation, resulting in a delay in growth and spawning observed at the highest tested dose rate.

Population-level modeling to account for multigenerational effects of uranium in Daphnia magna.

As part of the ecological risk assessment associated with radionuclides in freshwater ecosystems, toxicity of waterborne uranium was recently investigated in the microcrustacean Daphnia magna over a three-generation exposure (F0, F1, and F2). Toxic effects on daphnid life history and physiology, increasing over generations, were demonstrated at the organism level under controlled laboratory conditions. These effects were modeled using an approach based on the dynamic energy budget (DEB). For each of the three successive generations, DEBtox (dynamic energy budget applied to toxicity data) models were fitted to experimental data. Lethal and sublethal DEBtox outcomes and their uncertainty were projected to the population level using population matrix techniques. To do so, we compared two modeling approaches in which experimental results from F0, F1, and F2 generations were either considered separately (F0-, F1-, and F2-based simulations) or together in the actual succession of F0, F1, and F2 generations (multi-F-based simulation). The first approach showed that considering results from F0 only (equivalent to a standard toxicity test) would lead to a severe underestimation of uranium toxicity at the population level. Results from the second approach showed that combining effects in successive generations cannot generally be simplified to the worst case among F0-, F1-, and F2-based population dynamics.

Adverse outcome pathways (AOPs) for radiation-induced reproductive effects in environmental species: state of science and identification of a consensus AOP network.

BACKGROUND: Reproductive effects of ionizing radiation in organisms have been observed under laboratory and field conditions. Such assessments often rely on associations between exposure and effects, and thus lacking a detailed mechanistic understanding of causality between effects occurring at different levels of biological organization. The Adverse Outcome Pathway (AOP), a conceptual knowledge framework to capture, organize, evaluate and visualize the scientific knowledge of relevant toxicological effects, has the potential to evaluate the causal relationships between molecular, cellular, individual, and population effects. This paper presents the first development of a set of consensus AOPs for reproductive effects of ionizing radiation in wildlife. This work was performed by a group of experts formed during a workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. The work presents a series of taxon-specific case studies that were used to identify relevant empirical evidence, identify common AOP components and propose a set of consensus AOPs that could be organized into an AOP network with broader taxonomic applicability. CONCLUSION: Expert consultation led to the identification of key biological events and description of causal linkages between ionizing radiation, reproductive impairment and reduction in population fitness. The study characterized the knowledge domain of taxon-specific AOPs, identified knowledge gaps pertinent to reproductive-relevant AOP development and reflected on how AOPs could assist applications in radiation (radioecological) research, environmental health assessment, and radiological protection. Future advancement and consolidation of the AOPs is planned to include structured weight of evidence considerations, formalized review and critical assessment of the empirical evidence prior to formal submission and review by the OECD sponsored AOP development program.

Biology-based modeling to analyze uranium toxicity data on Daphnia magna in a multigeneration study.

Recent studies have investigated chronic toxicity of waterborne depleted uranium on the life cycle and physiology of Daphnia magna. In particular, a reduction in food assimilation was observed. Our aims here were to examine whether this reduction could fully account for observed effects on both growth and reproduction, for three successive generations, and to investigate through microscope analyses whether this reduction resulted from direct damage to the intestinal epithelium. We analyzed data obtained by exposing Daphnia magna to uranium over three successive generations. We used energy-based models, which are both able to fit simultaneously growth and reproduction and are biologically relevant. Two possible modes of action were compared - decrease in food assimilation rate and increase in maintenance costs. In our models, effects were related either to internal concentration or to exposure concentration. The model that fitted the data best represented a decrease in food assimilation related to exposure concentration. Furthermore, observations of consequent histological damage to the intestinal epithelium, together with uranium precipitates in the epithelial cells, supported the assumption that uranium has direct effects on the digestive tract. We were able to model the data in all generations and showed that sensitivity increased from one generation to the next, in particular through a significant increase of the intensity of effect, once the threshold for appearance of effects was exceeded.

Impact of microcystin-producing cyanobacteria on reproductive success of Lymnaea stagnalis (Gastropoda, Pulmonata) and predicted consequences at the population level.

Our previous studies showed that microcystin (MC)-accumulation in the gastropod Lymnaea stagnalis and effects on life-history traits (survival, growth, and fecundity) varied according to age, exposure pathway (MC-producing cyanobacteria or dissolved MC), and presence or not of additional non-toxic food. This study investigated effects of exposure to MC-producing cyanobacteria or to dissolved MC of parent and of parent and egg masses of L. stagnalis on hatching success, duration of embryonic development and neonate survival. Secondly, the potential impact of MC-producing cyanobacterial proliferations (blooms) on L. stagnalis population growth, depending on bloom frequencies and recovery duration of life traits after exposure, was evaluated using a modelling approach. Experimental results showed that embryonic development was shortened in case of parent exposure to toxic cyanobacteria. Parent and eggs exposure to dissolved MC extended embryonic development and reduced hatching percentage, suggesting a permeability of egg masses to MC. Whatever exposure, neonate survival was reduced. Neonates exposed to cyanobacteria accumulated MCs 24 h after hatching, suggesting very early cyanobacteria ingestion. Modelling results showed that L. stagnalis population growth was influenced by the recovery time of life-history traits after exposure. When setting the latest at 6 weeks according to previous experiments, a frequency of one to four blooms per year strongly affected population dynamics and induced up to a 80-weeks delay compared to control in time required for populations to grow from 1 to 1000 individuals. Results are discussed in terms of impact of intoxication pathways on parents, eggs and neonates, and on population dynamics of L. stagnalis.

Effects of waterborne uranium on survival, growth, reproduction and physiological processes of the freshwater cladoceran Daphnia magna.

Acute uranium toxicity (48 h immobilisation test) for Daphnia magna was determined in two different exposure media, differing in pH and alkalinity. LC(50) varied strongly between media, from 390+/-40 microgL(-1)U at pH 7 to 7.8+/-3.2 mgL(-1)U at pH 8. According to the free ion activity model uranium toxicity varies as a function of free uranyl concentration. This assumption was examined by calculating uranium speciation in our water conditions and in those reported in the literature. Predicted changes in free uranyl concentration could not solely explain observed differences in toxicity, which might be due to a competition or a non-competitive inhibition of H(+) for uranium transport and/or the involvement of other bioavailable chemical species of uranium. Chronic effects of uranium at pH 7 on mortality, ingestion and respiration, fecundity and dry mass of females, eggs and neonates were investigated during 21-day exposure experiments. A mortality of 10% was observed at 100 microgL(-1)U and EC(10) for reproduction was 14+/-7 microgL(-1)U. Scope for growth was affected through a reduction in feeding activity and an increase in oxygen consumption at 25 microgL(-1)U after 7 days of exposure. This had strong consequences for somatic growth and reproduction, which decreased, respectively, by 50% and 65% at 50 microgL(-1)U after 7 days and at 25 microgL(-1)U after 21 days. Uranium bioaccumulation was quantified and associated internal alpha dose rates from 2.1 to 13 microGyh(-1) were estimated. Compared to the toxicity of other alpha-emitting radionuclides and stable trace metals, our results confirmed the general assumption that uranium chemical toxicity predominates over its radiotoxicity.

Effects of chronic external gamma irradiation on growth and reproductive success of Daphnia magna.

Aquatic invertebrates (water flea Daphnia magna) were exposed to low dose rates of external gamma radiation (from 0.4 to 31mGyh(-1)) over a 23-day period (i.e. 5 broods). Gamma radiation caused changes in neither survival nor somatic growth. Mass-specific respiration rate was significantly lower at 31mGyh(-1) than in the control. Reproduction was affected through early release and reduced size of broods after 15 days of exposure at 31mGyh(-1) (broods 3-5), resulting in a 21% fecundity decrease at 31mGyh(-1) compared to the control. A decreased resistance of neonates to starvation was observed in relation to dose rates. Possible mechanisms of gamma radiotoxicity for daphnid reproduction and implications for radioprotection are discussed.

Population modelling to compare chronic external radiotoxicity between individual and population endpoints in four taxonomic groups.

In this study, we modelled population responses to chronic external gamma radiation in 12 laboratory species (including aquatic and soil invertebrates, fish and terrestrial mammals). Our aim was to compare radiosensitivity between individual and population endpoints and to examine how internationally proposed benchmarks for environmental radioprotection protected species against various risks at the population level. To do so, we used population matrix models, combining life history and chronic radiotoxicity data (derived from laboratory experiments and described in the literature and the FREDERICA database) to simulate changes in population endpoints (net reproductive rate R0, asymptotic population growth rate λ, equilibrium population size Neq) for a range of dose rates. Elasticity analyses of models showed that population responses differed depending on the affected individual endpoint (juvenile or adult survival, delay in maturity or reduction in fecundity), the considered population endpoint (R0, λ or Neq) and the life history of the studied species. Among population endpoints, net reproductive rate R0 showed the lowest EDR10 (effective dose rate inducing 10% effect) in all species, with values ranging from 26 µGy h(-1) in the mouse Mus musculus to 38,000 µGy h(-1) in the fish Oryzias latipes. For several species, EDR10 for population endpoints were lower than the lowest EDR10 for individual endpoints. Various population level risks, differing in severity for the population, were investigated. Population extinction (predicted when radiation effects caused population growth rate λ to decrease below 1, indicating that no population growth in the long term) was predicted for dose rates ranging from 2700 µGy h(-1) in fish to 12,000 µGy h(-1) in soil invertebrates. A milder risk, that population growth rate λ will be reduced by 10% of the reduction causing extinction, was predicted for dose rates ranging from 24 µGy h(-1) in mammals to 1800 µGy h(-1) in soil invertebrates. These predictions suggested that proposed reference benchmarks from the literature for different taxonomic groups protected all simulated species against population extinction. A generic reference benchmark of 10 µGy h(-1) protected all simulated species against 10% of the effect causing population extinction. Finally, a risk of pseudo-extinction was predicted from 2.0 µGy h(-1) in mammals to 970 µGy h(-1) in soil invertebrates, representing a slight but statistically significant population decline, the importance of which remains to be evaluated in natural settings.

DNA alterations and effects on growth and reproduction in Daphnia magna during chronic exposure to gamma radiation over three successive generations.

This study examined chronic effects of external Cs-137 gamma radiation on Daphnia magna exposed over three successive generations (F0, F1 and F2) to environmentally relevant dose rates (ranging from 0.007 to 35.4 mGy h(-1)). Investigated endpoints included survival, growth, reproduction and DNA alterations quantified using random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). Results demonstrated that radiation effects on survival, growth and reproduction increased in severity from generation F0 to generation F2. Mortality after 21 days at 35.4 mGy h(-1) increased from 20% in F0 to 30% in F2. Growth was affected by a slight reduction in maximum length at 35.4 mGy h(-1) in F0 and by reductions of 5 and 13% in growth rate, respectively, at 4.70 and 35.4 mGy h(-1) in F2. Reproduction was affected by a reduction of 19% in 21 day-fecundity at 35.4 mGy h(-1) in F0 and by a delay of 1.9 days in brood release as low as 0.070 mGy h(-1) in F2. In parallel, DNA alterations became significant at decreasing dose rates over the course of F0 (from 4.70 mGy h(-1) at hatching to 0.007 mGy h(-1) after ∼21 days) and from F0 to F2 (0.070 mGy h(-1) at hatching to 0.007 mGy h(-1) after ∼21 days), demonstrating their rapid accumulation in F0 daphnids and their transmission to offspring generations. Transiently more efficient DNA repair leading to some recovery at the organism level was suggested in F1, with no effect on survival, a slight reduction of 12% in 21 day-fecundity at 35.4 mGy h(-1) and DNA alterations significant at highest dose rates only. The study improved our understanding of long term responses to low doses of radiation at the molecular and organismic levels in a non-human species for a better radioprotection of aquatic ecosystems.

Transmission of DNA damage and increasing reprotoxic effects over two generations of Daphnia magna exposed to uranium.

This study aimed to examine the mechanisms involved in the transgenerational increase in Daphnia magna sensitivity to waterborne depleted uranium (DU) under controlled laboratory conditions. Daphnids were exposed to concentrations ranging from 2 to 50 µg L(-1) over two successive generations. Genotoxic effects were assessed using random amplified polymorphic DNA and real time PCR (RAPD-PCR). Effects on life history (survival, fecundity and somatic growth) were monitored from hatching to release of brood 5. Different exposure regimes were tested to investigate the specific sensitivity of various life stages to DU. When daphnids were exposed continuously or from hatching to deposition of brood 5, results demonstrated that DNA damage accumulated in females and were transmitted to offspring in parallel with an increase in severity of effects on life history across generations. When daphnids were exposed during the embryo stage only, DU exposure induced transient DNA damage which was repaired after neonates were returned to a clean medium. Effects on life history remained visible after hatching and did not significantly increase in severity across generations. The present results suggest that DNA damage might be an early indicator of future effects on life history.

Modelling population-level consequences of chronic external gamma irradiation in aquatic invertebrates under laboratory conditions.

We modelled population-level consequences of chronic external gamma irradiation in aquatic invertebrates under laboratory conditions. We used Leslie matrices to combine life-history characteristics (duration of life stages, survival and fecundity rates) and dose rate-response curves for hatching, survival and reproduction fitted on effect data from the FREDERICA database. Changes in net reproductive rate R0 (offspring per individual) and asymptotic population growth rate λ (dimensionless) were calculated over a range of dose rates in two marine polychaetes (Neanthes arenaceodentata and Ophryotrocha diadema) and a freshwater gastropod (Physa heterostropha). Sensitivities in R0 and λ to changes in life-history traits were analysed in each species. Results showed that fecundity has the strongest influence on R0. A delay in age at first reproduction is most critical for λ independent of the species. Fast growing species were proportionally more sensitive to changes in individual endpoints than slow growing species. Reduction of 10% in population λ were predicted at dose rates of 6918, 5012 and 74,131 µGy·h⁻¹ in N. arenaceodentata, O. diadema and P. heterostropha respectively, resulting from a combination of strong effects on several individual endpoints in each species. These observations made 10%-reduction in λ a poor criterion for population protection. The lowest significant changes in R0 and λ were respectively predicted at a same dose rate of 1412 µGy h⁻¹ in N. arenaceodentata, at 760 and 716 µGy h⁻¹ in O. diadema and at 12,767 and 13,759 µGy h⁻¹ in P. heterostropha. These values resulted from a combination of slight but significant changes in several measured endpoints and were lower than effective dose rates calculated for the individual level in O. diadema and P. heterostropha. The relevance of the experimental dataset (external irradiation rather than contamination, exposure over one generation only, effects on survival and reproduction only) for predicting population responses was discussed.

Effects of chronic uranium exposure on life history and physiology of Daphnia magna over three successive generations.

Daphnia magna was exposed to waterborne uranium (U) at concentrations ranging from 10 to 75 microgL(-1) over three successive generations (F0, F1 and F2). Progeny was either exposed to the same concentration as mothers to test whether susceptibility to this radioelement might vary across generations or returned to a clean medium to examine their capacity to recover after parental exposure. Maximum body burdens of 17, 32 and 54 ng U daphnid(-1) were measured in the different exposure conditions and converted to corresponding internal alpha dose rates. Low values of 5, 12 and 20 microGy h(-1) suggested that radiotoxicity was negligible compared to chemotoxicity. An increasing sensitivity to toxicity was shown across exposed generations with significant effects observed on life history traits and physiology as low as 10 microgL(-1) and a capacity to recover partially in a clean medium after parental exposure to