Assessing tritium internalisation in zebrafish early life stages: Importance of rapid isotopic exchange

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.