Uranium transfer and accumulation in organs of Danio rerio after waterborne exposure alone or combined with diet-borne exposure.

Uranium (U) toxicity patterns for fish have been mainly determined under laboratory-controlled waterborne exposure conditions. Because fish can take up metals from water and diet under in situ exposure conditions, a waterborne U exposure experiment (20 µg L-1 , 20 d) was conducted in the laboratory to investigate transfer efficiency and target organ distribution in zebrafish Danio rerio compared with combined waterborne exposure (20 µg L-1 ) and diet-borne exposure (10.7 µg g-1 ). 233 Uranium was used as a specific U isotope tracer for diet-borne exposure. Bioaccumulation was examined in the gills, liver, kidneys, intestine, and gonads of D. rerio. Concentrations in the organs after waterborne exposure were approximately 500 ng g-1 fresh weight, except in the intestine (> 10 µg g-1 fresh wt) and the kidneys (200 ng g-1 fresh wt). No significant difference was observed between waterborne and diet-borne conditions. Trophic U transfer in organs was found but at a low level (< 10 ng g-1 fresh wt). Surprisingly, the intestine appeared to be the main target organ after both tested exposure modalities. The gonads (57% at 20 d) and the liver (41% at 20 d) showed the highest accumulated relative U burdens. Environ Toxicol Chem 2019;38:90-98. © 2018 SETAC.

Toxicokinetic and toxicodynamic of depleted uranium in the zebrafish, Danio rerio.

This study investigated the accumulation pattern and biological effects (genotoxicity and histopathology) to adult zebrafish (male and female) exposed to a nominal waterborne concentration of 20 µg L-1 of depleted uranium (DU) for 28 days followed by 27 days of depuration. Accumulation pattern showed that (i) DU accumulated in brain, (ii) levels in digestive tract were higher than those measured in gills and (iii) levels remained high in kidney, brain and ovary despite the 27 days of depuration period. Genotoxicity, assessed by comet assay, was significant not only during DU exposure, but also during depuration phase. Gonads, in particular the testes, were more sensitive than gills. The histology of gonads indicated severe biological damages in males. This study improved knowledge of ecotoxic profile of uranium, for which a large range of biological effects has already been demonstrated.

In situ effects of metal contamination from former uranium mining sites on the health of the three-spined stickleback (Gasterosteus aculeatus, L.).

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.

Effects of chronic exposure to environmentally relevant concentrations of waterborne depleted uranium on the digestive tract of zebrafish, Danio rerio.

Uranium is a naturally occurring element, but activities linked to the nuclear fuel cycle can increase background levels in the surrounding waters. For this reason it is important to understand how this affects organisms residing in the water column. The objective of this study was to assess histopathological effects of uranium on the gut wall of a widely used model organism: zebrafish, Danio rerio. To this end we exposed zebrafish to 84 and 420 nM depleted uranium for over a month and then examined the histology of intestines of exposed individuals compared to controls. The gut wall of individuals exposed to 84 and 420 nM of uranium had large regions of degraded mucosa. Using transmission electron microscopy (TEM) coupled to energy-dispersive X-ray spectroscopy microanalysis (EDX) we found that uranium induced a decrease in the amount of calcium containing mitochondrial matrix granules per mitochondria. This is suggestive of perturbations to cellular metabolism and more specifically to cellular calcium homeostasis. TEM-EDX of the gut wall tissue further showed that some uranium was internalized in the nucleus of epithelial cells in the 420 nM treatment. Fluorescent in situ hybridization using specific probes to detect all eubacteria was performed on frozen sections of 6 individual fish in the 84 nM and 420 nM treatments. Bacterial colonization of the gut of individuals in the 420 nM seemed to differ from that of the controls and 84 nM individuals. We suggest that host-microbiota interactions are potentially disturbed in response to uranium induced stress. The damage induced by waterborne uranium to the gut wall did not seem to depend on the concentration of uranium in the media. We measure whole body residues of uranium at the end of the experiment and compute the mean dose rate absorbed for each condition. We discuss why effects might be uncoupled from external concentration and highlight that it is not so much the external concentration but the dynamics of internalization which are important players in the game.

Former uranium mine-induced effects in caged roach: a multiparametric approach for the evaluation of in situ metal toxicity.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites; however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

Depleted uranium disturbs immune parameters in zebrafish, Danio rerio: an ex vivo/in vivo experiment.

In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 µg DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 µg DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 µg DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments. Cell mortality was also decreased at 20 µg DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases.

Effects of depleted uranium on oxidative stress, detoxification, and defence parameters of zebrafish Danio rerio.

In this study, we investigated the effects of depleted uranium (DU), the by-product of nuclear enrichment of uranium, on several parameters related to oxidative stress, detoxification, and the defence system in the zebrafish Danio rerio. Several parameters were recorded: phenoloxidase-like (PO) activity, reactive oxygen species (ROS) production, and 7-ethoxyresrufin-O-deethylase (EROD) activity. Experiments were performed on adult and larvae D. rerio. Adult fish were exposed for 28 days at 20 µg U/L followed by a 27-day depuration period. Eggs of D. rerio were exposed for 4 days at 0, 20, 100, 250, 500, and 1,000 µg U/L. Results showed that DU increased ROS production both in adult and in larvae even at the low concentrations tested and even during the depuration period for adult D. rerio. DU also modified PO-like activity, both in the D. rerio adult and larvae experiments, but in a more transient manner. EROD activity was not modified by DU, but sex effects were shown. Results are discussed by way of comparison with other known effects of uranium in fish. Overall, these results show that the mechanisms of action of DU in fish tend to be similar to the ones existing for mammals. These results encourage the development and use of innate immune biomarkers to understand the effects of uranium and, more generally, radionuclides on the fish immune system.

Effects of uranium on the metabolism of zebrafish, Danio rerio.

The increasing demand for nuclear energy results in heightened levels of uranium (U) in aquatic systems which present a potential health hazard to resident organisms. The aim of this study was to mechanistically assess how chronic exposure to environmentally relevant concentrations of U perturbs the complex interplay between feeding, growth, maintenance, maturation and reproduction throughout the life-cycle of an individual. To this end we analysed literature-based and original zebrafish toxicity data within a same mass and energy balancing conceptual framework. U was found to increase somatic maintenance leading to inhibition of spawning as well as increase hazard rate and costs for growth during the early life stages. The fish's initial conditions and elimination through reproduction greatly affected toxico-kinetics and effects. We demonstrate that growth and reproduction should be measured on specific individuals since mean values were hardly interpretable. The mean food level differed between experiments, conditions and individuals. This last 'detail' contributed substantially to the observed variability by its combined effect on metabolism, toxic effects and toxico-kinetics. The significance of this work is that we address exactly how these issues are related and derive conclusions which are independent of experimental protocol and coherent with a very large body of literature on zebrafish eco-physiology.

The effects of radionuclides on animal behavior.

Concomitant with the expansion of the nuclear industry, the concentrations of several pollutants, radioactive or otherwise, including uranium, caesium, cadmium and cobalt, have increased over the last few decades. These elemental pollutants do exist in the environment and are a threat to many organisms. Behavior represents the integration of all the anatomical adaptations and physiological processes that occur within an organism. Compared to other biological endpoints, the effects of pollutants on animal behavior have been the focus of only a few studies. However, behavioral changes appear to be ideal for assessing the effects of pollutants on animal populations, because behavior links physiological functions with ecological processes. The alteration of behavioral responses can have severe implications for survival of individuals and of population of some species. Behavioral disruptions may derive from several underlying mechanisms: disruption of neuro-sensorial activity and of endocrines, or oxidative and metabolic disruptions. In this review, we presented an overview of the current literature in which the effects of radioactive pollutants on behavior in humans, rodents, fish and wildlife species are addressed. When possible, we have also indicated the potential underlying mechanisms of the behavioral alterations and parameters measured. In fried, chronic uranium contamination is associated with behavior alterations and mental disorders in humans, and cognitive deficits in rats. Comparative studies on depleted and enriched uranium effects in rats showed that chemical and radiological activities of this metal induced negative effects on several behavioral parameters and also produced brain oxidative stress. Uranium exposure also modifies feeding behavior of bivalves and reproductive behavior of fish. Studies of the effects of the Chernobyl accident shows that chronic irradiation to 137Cs induces both nervous system diseases and mental disorders in humans leading to increased suicides, as well as modification of preferred nesting sites, reduced hatching success and fecundity in birds that live in the Chernobyl zone. No significant effect from caesium exposure was shown in laboratory experiments with rats, but few studies were conducted. Data on radioactive cadmium are not available in the literature, but the effects of its metallic form have been well studied. Cadmium induces mental retardation and psychomotor alterations in exposed populations and increases anxiety in rats, leading to depression. Cadmium exposure also results in well-documented effects on feeding and burrowing behavior in several invertebrate species (crustaceans, gastropods, annelids, bivalves) and on different kinds of fish behavior (swimming activity, fast-start response, antipredatory behavior). Cobalt induces memory deficits in humans and may be involved in Alzheimer's disease; gamma irradiation by cobalt also decreases fecundity and alters mating behavior in insects. Collectively, data are lacking or are meagre on radionuclide pollutants, and a better knowledge of their actions on the cellular and molecular mechanisms that control animal behavior is needed.

Transfer modelling and toxicity evaluation of the effluent from an installation of cleansing and uranium recovery using a battery of bioassays.

On July 7, 2008, a leak of effluent from an Installation of Cleansing and Uranium Recovery (Tricastin, France) led to the spillage of uranium in a stream. The acute toxicity of the effluent was evaluated, and compared to the toxicity of uranium nitrate in bioassays using several organisms: Chlamydomonas reinhardtii, Daphnia magna, Chironomus riparius and Danio rerio. A sediment bioassay was also performed on C. riparius using water and sediment sampled along the river. Results showed that effluent EC(50) 72 h was 0.65 mg U/l for algae and LC(50) 48 h was 1.67 mg U/l for daphnia, while values obtained for uranium nitrate were higher. The LC(50) 96 h of effluent to C. riparius was 22.7 mg U/l, similar to value for uranium nitrate; the sediment collected was not toxic to C. riparius larvae. The LOEC of effluent and uranium nitrate on HT(50) of D. rerio were similar (0.03 mg U/l), but larvae were more sensitive to uranium nitrate than to effluent. Our results suggest that other substances contained in the effluent could potentially be toxic to wildlife in association with uranium. In parallel, the modelling of the transfers based on uranium measurements in the surface water was used to fill data gaps and assess the impact along the river. These results provided an estimate of exposure conditions that occurred along the river. This approach allowed us to see that the risk to ecosystem during this incident was certainly low and concerned a short period of time, but it could have existed at least for some species.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.