Rodolphe Gilbin

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 <or=25 microgL(-1). Using a (14)C-labelled food technique, the study showed that uranium affected carbon assimilation in F0 at concentrations of 25 and 75 microgL(-1) (34 and 80% reduction respectively) and as low as 10 microgL(-1) in F1 and F2 (40 and 36% reduction respectively). Consequences were strong for both somatic growth and reproduction and increased in severity across generations. Maximum size was reduced by 12% at 75 microgL(-1) in F0 and 23% at 25 microgL(-1) in F2. Reduction in 21-day fecundity ranged from 27 to 48% respectively at 25 and 75 microgL(-1) in F0 and from 43 to 71% respectively at 10 and 25 microgL(-1) in F2. Growth retardation caused a delay in deposition of first brood of 1.3 days at 75 microgL(-1) in F0, of 1.9 days at 25 microgL(-1) in F1 and of 5 days at 25 microgL(-1) in F2. Differences in respiration rates and egg dry mass between the control and exposed daphnids were mainly an indirect result of uranium effect on body size. The observed increase in toxic effects across generations indicated the necessity of carrying out multigeneration tests to assess environmental risk of uranium in daphnids.

Issues and practices in the use of effects data from FREDERICA in the ERICA Integrated Approach

The ERICA Integrated Approach requires that a risk assessment screening dose rate is defined for the risk characterisation within Tiers 1 and 2. At Tier 3, no numerical screening dose rate is used, and the risk characterisation is driven by methods that can evaluate the possible effects of ionising radiation on reproduction, mortality and morbidity. Species sensitivity distribution has been used to derive the ERICA risk assessment predicted no-effect dose rate (PNEDR). The method used was based on the mathematical processing of data from FRED (FASSET radiation effects database merged with the EPIC database to form FREDERICA) and resulted in a PNEDR of 10 microGy/h. This rate was assumed to ascribe sufficient protection of all ecosystems from detrimental effects on structure and function under chronic exposure. The value was weighed against a number of points of comparison: (i) PNEDR values obtained by application of the safety factor method, (ii) background levels, (iii) dose rates triggering effects on radioactively contaminated sites and (iv) former guidelines from literature reviews. In Tier 3, the effects analysis must be driven by the problem formulation and is thus highly case specific. Instead of specific recommendations on numeric values, guidance on the sorts of methods that may be applied for refined effect analysis is provided and illustrated.

The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio).

In this study, we investigated the effects of the radioactive metal uranium (U) on the embryonic development, hatching success, growth rate, and survival of juvenile zebrafish (Danio rerio). We studied the effects of depleted uranium (20-500 microg L(-1) of DU), inducing mainly chemical toxicity due to its low specific activity, and the combined effects of chemical and radiological toxicity by using a higher specific activity uranium isotope (20 and 100 microg L(-1) of (233)U). Results showed that early life stages are significantly affected by uranium exposure through both chemical and combined (chemical and radiological) toxicity. Experiments showed significant effects of U on hatching success starting at the concentration of 250 microg L(-1) of DU, causing a 42% delay in median hatching times relative to control. Furthermore, a reduction of growth (decrease in body length and weight) was observed followed by a high mortality of pro-larvae stage (up to 100% at DU concentrations of 250 microgL(-1) upon a 15 day exposure). Bioaccumulation measurements highlighted that U was mainly localised in the chorion but penetrated in the embryo inside eggs at a higher concentration. The effects differed depending on the isotopic composition of the uranium: sublethal defects in the tail detachment process were more pronounced for (233)U than DU exposure, while the presence of (233)U specifically affected embryo development and led to higher mortality rates of the prolarvae. The results from this study showed that the early life stages of zebrafish seems to be more sensitive to uranium contamination than more mature stages, and underline the importance of including pro-larval stages into toxicity tests in order to improve the relevancy for environmental risk assessments.

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.

Increased effects of internal alpha irradiation in Daphnia magna after chronic exposure over three successive generations

A 70-day experiment was performed with Daphnia magna exposed to waterborne Am-241 on a range of concentrations (from 0.4 to 40 Bq ml(-1)) in order to test chronic effects of internal alpha irradiation on respiration, somatic growth and reproduction over three successive generations. Changes in Am-241 concentrations were followed in the water and in daphnid tissues, eggs and cuticles. Corresponding average dose rates of 0.3, 1.5 and 15 mGy h(-1) were estimated. This study confirmed that oxygen consumption increased significantly in the first generation (F0) after 6 days of exposure to a dose rate >or=1.5 mGy h(-1). Consequences were limited to a reduction in body length (5%) and dry mass of females (16%) and eggs (8%) after 23 days of exposure, while mortality and fecundity remained unaffected. New cohorts were started with neonates of broods 1 and 5, to examine potential consequences of the reduced mass of offspring for subsequent exposed generations. Results strongly contrasted with those observed in F0. At the highest dose rate, an early mortality of 38-90% affected juveniles while survivors showed delayed reproduction and reduced fecundity in F1 and F2. At 0.3 and 1.5 mGy h(-1), mortality ranged from 31 to 38% of daphnids depending on dose rate, but was observed only in generation F1 started with neonates of the brood 1. Reproduction was affected through a reduction in the proportion of breeding females, occurring in the first offspring generation at 1.5 mGy h(-1) (to 62% of total daphnids) and in the second generation at 0.3 mGy h(-1) (to 69% of total daphnids). Oxygen consumption remained significantly higher at dose rates >or=0.3 mGy h(-1) than in the control in almost every generation. Body size and mass continued decreasing in relation to dose rate, with a significant reduction in mass ranging from 15% at 0.3 mGy h(-1) to 27% at 15 mGy h(-1) in the second offspring generation.

Genotoxicity of acute and chronic gamma‐irradiation on zebrafish cells and consequences for embryo development

The effects of radiation on biological systems have been studied for many years, and it is now accepted that direct damage to DNA from radiation is the triggering event leading to biological effects. In the present study, DNA damage induced by acute or chronic irradiation was compared at the cellular (zebrafish [Danio rerio] cell line ZF4) and developmental (embryo) levels. Zebrafish ZF4 cells and embryos (at 3 h postfertilization) were exposed within ranges of acute doses (0.3-2 Gy/d) or chronic dose rates (0.1-0.75 Gy/d). DNA damage was assessed by immunodetection of γ-H2AX and DNA-PK (DNA double-strand breaks) and the alkaline comet assay (DNA single-strand breaks). Zebrafish embryo development and DNA damage were examined after 120 h. At low doses, chronic irradiation induced more residual DNA damage than acute irradiation, but embryo development was normal. From 0.3 Gy, a hyper-radiosensitivity phenomenon compared to other species was shown for acute exposure with an increase of DNA damage, an impairment of hatching success, and larvae abnormalities. These results suggest a dose-dependent correlation between unrepaired DNA damage and abnormalities in embryo development, supporting the use of DNA repair proteins as predictive biomarkers of ionizing radiation exposure. This could have important implications for environmental protection.

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.

Effects of uranium uptake on transcriptional responses, histological structures and survival rate of the crayfish Procambarus clarkii

This work aims to investigate the accumulation levels and effects (transcriptional responses, histopathology and survival rate) associated with a wide range of dissolved uranium (U) concentrations (0, 0.03, 0.6, 4 and 8 mg/L of U) on adult male crayfish Procambarus clarkii during 4 (T4) and 10 (T10) days of exposure. The follow-up of the crayfish mortality showed that P. clarkii was highly resistant to U. Increasing waterborne U concentrations led to increasing bioaccumulation in key crayfish organs and increasing histological damages. U distribution in tissues was also evaluated using transmission electron microscopy and showed the presence of a detoxified form of U in the gills epithelium in the shape of flakes. Expression levels of mitochondrial genes (cox1, atp6 and 12S gene) and genes involved in oxidative stress (sod(Mn) and mt) were examined together with the housekeeping gene 18S. atp6 and mt genes of P. clarkii were cloned and sequenced before analysis. Significant correlations were observed between U bioaccumulation and the down-regulation of both cox1 and sod(Mn) genes. This work provides a first U toxicogenomic and histopathological pattern of P. clarkii, identify U biomarkers and associate gene expression endpoints to accumulation levels. It also provides new insights into the mechanisms involved in U stress.

Internal distribution of uranium and associated genotoxic damages in the chronically exposed bivalve Corbicula fluminea

Uranium (U) internal distribution and involved effects in the bivalve Corbicula fluminea have been studied after direct chronic exposure (90 d, 10 μg.L-1). U distribution was assessed at the subcellular level (Metal Rich Granules -MRG-, pellets and cytosol fractions) in two main organs of the bivalve (gills and visceral mass). Micro-localisation was investigated by TEM-EDX analysis in the gills epithelium. DNA damage in gill and hemolymph samples was measured by the Comet assay. The 90-d exposure period led to a significant increase of U concentration in gills over time (× 5) and a large U quantity in subcellular granules in gills. Finally, a significant increase (× 2) in DNA damage was noted in exposed gills and haemocytes. This study shows that the accumulation levels and consequently the potential toxicity cannot be successfully predicted only on the basis of concentration in water or in tissues and subcellular fractions after chronic exposure.

Mitochondrial gene expression, antioxidant responses, and histopathology after cadmium exposure

The present study investigates cadmium effects on the transcription of mitochondrial genes of Procambarus clarkii after acute (0.05, 0.5, and 5 mg Cd/L; 4–10 days) and chronic exposures (10 μg Cd/L; 30–60 days). Transcriptional responses of cox1, atp6, and 12S using quantitative real‐time RT‐PCR were assessed in gills and hepatopancreas. Additionally, the expression levels of genes involved in detoxification and/or oxidative stress responses [mt, sod(Mn)] and enzymatic activities of antioxidants (SOD, CAT, GPX, and GST) were analyzed. The histopathological effects in hepatopancreas of crayfish were evaluated by light microscopy. Relationships between endpoints at different levels of biological organization and Cd bioaccumulation were also examined. Cd induced high levels of bioaccumulation, which was followed by mitochondrial dysfunction and histological alterations in both experiments. Moreover, perturbations in the defence mechanisms against oxidative stress tended to increase with time. Results also showed that molecular responses can vary depending on the intensity and duration of the chemical stress applied to the organisms and that the study of mt gene expression levels seemed to be the best tool to assess Cd intoxication.