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Tritium, a radioactive isotope of hydrogen of natural and anthropogenic origin, is ubiquitously present in the environment. Effluents of nuclear centers of production are significant anthropogenic sources. With the upcoming project of thermonuclear fusion, tritium releases in the environment may increase. It is therefore important to characterize the ecological risk linked to tritium. The effects of tritiated water (HTO) were therefore studied in zebrafish larvae exposed for 10 d to different dose rates, 1.1 × 102, 4.1 × 102, and 3.8 × 103 µGy/h for larvae corresponding, respectively, to a water contamination of 104, 105, and 106 Bq/mL of HTO. Those dose rates were higher than 10 μGy/h, which is the threshold recommended to start monitoring ecosystems where radiological contaminants are present. Mortality, embryo‐larval development, immune toxicity, genotoxicity, neurotoxicity, and alterations of tissues were investigated. The results showed that HTO exposure induced DNA damage and reactive oxygen species production and modulated the expression of genes involved in detoxification processes. Moreover, modifications of the muscular tissues (degradation of myofibrils at 4 d post fertilization and disorganization of mitochondria at later stages) were observed. The results differed with HTO dose rates and with developmental stages. These results will drive future research for the development of new HTO‐sensitive biomarkers and will allow us to progress in the characterization of the modes of action of tritium in fish. Environ Toxicol Chem 2020;39:648–658. © 2019 SETAC [ABSTRACT FROM AUTHOR]

Abstract Tritium (3H) is mainly released into the environment in the form of tritiated water (HTO) by nuclear power plants and nuclear fuel reprocessing plants. To better understand how organisms may be affected by contamination to 3H it is essential to link observed effects to a correct estimation of absorbed dose rates. Due to quick isotopic exchanges between 3H and hydrogen, 3H measurement is difficult in small organisms such as zebrafish embryo, a model in ecotoxicological assay. This work aimed to optimise tritium measurement protocol to better characterise internalisation by early life stages of zebrafish. Zebrafish eggs were exposed at one HTO activity concentration of 1.22 × 105 Bq/mL. This activity was calculated to correspond to theoretical dose rates of 0.4 mGy/h, where some deleterious effects are expected on young fish. A protocol for the preparation of biological samples was adapted from the method classically used to segregate the different forms of tritium in organisms. To deal with very quick isotopic exchanges of 3H with hydrogen, the impacts of washing by non-tritiated water as well as the bias induced by absorbed tritium around organisms on the measured activity concentration were studied. We managed to develop protocols to perform total tritium and total organically bound tritium (OBT) activity concentrations measurements in zebrafish eggs and larvae. The measurement of these both forms allowed the calculation of tissue-free-water-tritium (TFWT). To better understand total tritium internalisation, a study of total tritium kinetics from 4 hpf (hour post-fertilization) to 168 hpf was performed. OBT and TFWT were also assessed to complete the total tritium internalisation kinetics. The internalisation is a rapid phenomenon reaching a steady-state within 24 h after the beginning of contamination for total tritium and TFWT, with concentration factors and TFWT/HTO close to unity. OBT formation seemed to be slower. It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf). To verify absorbed dose rates at key developmental stages (24 hpf eggs and 96 hpf larvae), they were calculated from total tritium activity concentrations after exposure at 1.22 × 105 and 1.22 × 106 Bq/mL, as these two activity concentrations were used to assess effects of tritium in another part of the study. Dose rates calculated from total tritium activity concentrations measured in 24 hpf eggs and 96 hpf larvae were consistent with the nominal ones, which validates the robustness of the protocol developed in the present study. Highlights • Zebrafish eggs and larvae were exposed to HTO. • Total 3H, TFWT and OBT were measured in organisms after a protocol optimisation. • The kinetic for total 3H and TFWT show a rapid internalisation in organisms. • The OBT measurement show a low internalisation in zebrafish eggs and larvae. • Absorbed dose rates in organisms were consistent with theoretical ones. [ABSTRACT FROM AUTHOR]

Tritium ( 3 H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish ( Danio rerio ). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction ( tnnt2d , tnni2a.4, slc6a1a or atp2a1l ) and eye opacity ( crygm2d9 , crygmxl1 , mipb or lim2.3 ) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H 2 O 2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae. [ABSTRACT FROM AUTHOR]

"It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% for both stages (24 hpf and 96 hpf)." Should be read as "It appeared that OBT content in organisms was low with an OBT/TFWT ratio of about 8% and 14% at 24 hpf and 96 hphf respectively". [ABSTRACT FROM AUTHOR]