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

Évaluation des effets de mélanges de contaminants stables et radioactifs sur le nématode C. elegans à l’aide d’outils d’écotoxicologie prédictive.

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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 μg 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 μg L-1 of 233U). 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 μg 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 μg L-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 233U than DU exposure, while the presence of 233U 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. © 2008 Elsevier B.V. All rights reserved

Water toxicity and metal contamination assessment of a polluted river: the Upper Vistula River (Poland)

International audienceIn aquatic systems, the bioavailability of an element to microorganisms is greatly influenced by its chemical speciation. The goal of this work was to assess metal toxicity to a green algae (Pseudokirchneriella subcapitata) and a bacterium (Vibrio fisheri) as a function of size fractionation and chemical speciation (using the program MINTEQA2) in contaminated water of the Upper Vistula River. Water samples were collected at 1 reference site, 4 polluted sites and one polluted site on the Vistula's main tributary, the Przemsza River. Toxicity measurements were performed on unfiltered samples and, total dissolved (<1.2 μm), and truly dissolved (<1 kDa) fractions. Trace metal (Cd, Co, Cr, Cu, Mn, Pb, Zn) concentrations were measured in these samples and also in the colloidal fraction (1 kDa–1.2 μm). At the reference site, the low metal concentrations were in agreement with the absence of measurable toxicity. In the polluted section of the river, free metal concentrations were largely below the potential toxic levels for bacteria, which was in agreement with the absence of toxicity. Although Zn2+ was at potentially toxic-level concentrations in total dissolved and truly dissolved fractions in the polluted riverine section, toxicity for algae was observed, only in truly dissolved fractions from two stations. The absence of toxicity in most samples was related to metal association with particles and with low molecular weight ligands as well as the presence of organic ligands (phenol). The reason for toxic effects in two ultrafiltered samples is not clear, but may be related to the elimination of the colloidal organic fraction and thus the eradication of its protective effect occurring in natural samples

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 31 mGy h-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 31 mGy h-1 than in the control. Reproduction was affected through early release and reduced size of broods after 15 days of exposure at 31 mGy h-1 (broods 3-5), resulting in a 21% fecundity decrease at 31 mGy h-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. © 2007 Elsevier Ltd. All rights reserved

Sensitivity evaluation of the green alga Chlamydomonas reinhardtii to uranium by pulse amplitude modulated (PAM) fluorometry

International audienceAlthough ecotoxicological studies tend to address the toxicity thresholds of uranium in freshwaters, there is a lack of information on the effects of the metal on physiological processes, particularly in aquatic plants. Knowing that uranium alters photosynthesis via impairment of the water photo-oxidation process, we determined whether pulse amplitude modulated (PAM) fluorometry was a relevant tool for assessing the impact of uranium on the green alga Chlamydomonas reinhardtii and investigated how and to what extent uranium hampered photosynthetic performance. Photosynthetic activity and quenching were assessed from fluorescence induction curves generated by PAM fluorometry, after 1 and 5h of uranium exposure in controlled conditions. The oxygen-evolving complex (OEC) of PSII was identified as the primary action site of uranium, through alteration of the water photo-oxidation process as revealed by F0/Fv. Limiting re-oxidation of the plastoquinone pool, uranium impaired the electron flux between the photosystems until almost complete inhibition of the PSII quantum efficiency (F'q/F'm, EC50=303±64μgUL-1 after 5h of exposure) was observed. Non-photochemical quenching (qN) was identified as the most sensitive fluorescence parameter (EC50=142±98μgUL-1 after 5h of exposure), indicating that light energy not used in photochemistry was dissipated in non-radiative processes. It was shown that parameters which stemmed from fluorescence induction kinetics are valuable indicators for evaluating the impact of uranium on PSII in green algae. PAM fluorometry provided a rapid and reasonably sensitive method for assessing stress response to uranium in microalgae. © 2013 Elsevier B.V

Development of biochemical methods to estimate the subcellular impact of uranium exposure on Chlamydomonas reinhardtii

This work aims at determining early effects of uranium on the green microalga Chlamydomonas reinhardtii. First, the global effect on growth rate inhibition of exponentially-growing cultures was assessed on favourable conditions for uranium bioavailability (e.g. pH=5), EC50-24hrs equals roughly to 150 nM whatever the uranium isotopic composition considered (depleted U or 233U). Then, the sensitivity of different parameters representative of (i) oxidative stress (GSH/[GSH + 0.5 GSSG] ratio) (ii) metal detoxifying (phytochelatins production) and (iii) photosynthetic activity (chlorophyll fluorescence) was tested. Setting assay of different forms of glutathione and phytochelatins by HPLC was firstly optimised with cadmium-contaminated cells. This assay completed by chlorophyll fluorescence and algal growth was subsequently applied on samples contaminated by 150 nM of depleted uranium or 233U. No phytochelatin was produced in our experimental conditions. No difference of GSH/[GSH + 0.5 GSSG] ratio was shown between control and contaminated algae. This result suggests that the algae could be stressed before contamination due to culture condition. Chlorophyll fluorescence measurement showed photosynthetic activity inhibition after 24 hrs, in the same way for depleted uranium and 233U. Thus, the effect observed on the photosynthetic activity could be mainly attributed to the chemical toxicity of the metal

Subcellular fractionation and chemical speciation of uranium to elucidate its fate in gills and hepatopancreas of crayfish Procambarus clarkii

International audienceKnowledge of the organ and subcellular distribution of metals in organisms is fundamental for the understanding of their uptake, storage, elimination and toxicity. Detoxification via MTLP and MRG formation and chelation by some proteins are necessary to better assess the metal toxic fraction in aquatic organisms. This work focused on uranium, natural element mainly used in nuclear industry, and its subcellular fractionation and chemical speciation to elucidate its accumulation pattern in gills and hepatopancreas of crayfish Procambarus clarkii, key organs of uptake and detoxification, respectively. Crayfish waterborne exposure was performed during 4 and 10d at 0, 30, 600 and 4000μgUL-1. After tissue dissection, uranium subcellular fractionation was performed by successive ultracentrifugations. SEC-ICP MS was used to study uranium speciation in cytosolic fraction. The uranium subcellular partitioning patterns varied according to the target organ studied and its biological function in the organism. The cytosolic fraction accounted for 13-30% of the total uranium amount in gills and 35-75% in hepatopancreas. The uranium fraction coeluting with MTLPs in gills and hepatopancreas cytosols showed that roughly 55% of uranium remained non-detoxified and thus potentially toxic in the cytosol. Furthermore, the sum of uranium amount in organelle fractions and in the non-detoxified part of cytosol, possibly equivalent to available fraction, accounted for 20% (gills) and 57% (hepatopancreas) of the total uranium. Finally, the SEC-ICP MS analysis provided information on potential competition of U for biomolecules similar than the ones involved in endogenous essential metal (Fe, Cu) chelation. © 2012 Elsevier Ltd

Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells

International audienceAluminium is a toxic metal whose genotoxicity has been scarcely studied in aquatic species and more generally in mammals. Recently, human and ecological disaster caused by the discharge of red mud in Hungary has revived questions about the toxicity of this metal particularly for the environment. On the contrary, cadmium is a highly toxic metal whose genotoxicity has been well characterized in various mammalian cells. However on non-human cells, little is known about its impact on DNA damage and repair.In this study, the genotoxic potential of both metals on embryonic zebrafish cells ZF4 was analyzed and particularly the impairment of the major DNA double strand breaks (DSB)-repair pathway, i.e. non-homologous end-joining (NHEJ).To this aim, DNA single strand breaks (SSB) and DSB were evaluated using the comet assay and the immunodetection of γ-H2AX proteins, respectively, in AlCl3 or CdCl2 exposed ZF4 cells. These exposures result in the production of DSBs a few hours after incubation. The DNA-PK kinase activity, essential for NHEJ, is more affected by the presence of aluminium than cadmium. Altogether our data provide evidence of the high toxicity induced by aluminium in zebrafish and indicates the pertinence of genotoxicity evaluation in organisms living in contaminated water. © 2012 Elsevier B.V