At-sea application of the comet assay to a deep-sea fish

Given the go ahead, deep-sea mining operations are likely to continue for decades on a substantial spatial scale and the resulting sediment plumes combined, are likely to extend beyond the licenced mining areas, and could lead to the chronic exposure of deep-sea organisms to a mixture of metals, even mobile species, such as fish, that could conceivably display avoidance behaviour. The metal concentrations, often substantially below lethal doses, mean that individual mortality is too blunt a measure to allow assessment of "serious harm". Commonly used cellular biomarkers of exposure in ecotoxicology include DNA damage using the Comet assay. True deep-sea ecotoxicological studies with fish are rare and to our knowledge, there are no published data or method optimizations for deep-sea fish. Coryphaenoides ssp. were collected during SMARTEX expedition 1 (Feb/Mar, 2023) to the Clarion Clipperton Zone (CCZ) in the Eastern Pacific Ocean using a baited trap deployed between 4580-4,732m depth for 24-48 h. Blood and gill tissue were removed and processed for the Comet assay. In order to reduce artefactual DNA damage from cryopreservation observed previously, two sets of samples were prepared: a cryopreservative (10% DMSO) was added to one set of samples and stored at -80 degrees C; the second set was used to perform a Comet assay within hours of collection. A custom-built gimble table enabled horizontal electrophoresis at sea after which Comet assay slides were dried and stored at room temperature until further analysis. The Comet assay was also assessed in freshly sampled and frozen rainbow trout cells as a proxy control in order to evaluate potential artefacts from the collection and sampling procedure of the deep-sea fish. The blood samples processed at sea had a significantly reduced level of DNA damage compared to the frozen samples. There was no significant difference between the fresh deep-sea and rainbow trout samples. However, the freshly prepared gill samples in Coryphaenoides ssp. showed substantial artefacts, possibly as a consequence of barotrauma. These results represent the first effort at establishing baseline DNA damage data for deep-sea fish, an essential component in understating and quantifying the impact of deep-sea mining.