Unraveling the mechanistic effects of crude oil toxicity during early life stages of cold-water marine teleosts

The EGGTOX project has investigated the susceptibility of oil pollution of eggs of 6 important species of marine fish: cod, haddock, pollock, polar cod, herring, and halibut. The results are clear for all the species that have been tested. Fish embryos (eggs) are extremely sensitive to oil pollution and all the different species have serious developmental effects with oil doses that are likely to occur following an accidental oil release. But there are also distinct species differences where haddock and polar cod are the most sensitive species. For these species, we found lethal effects down to 30 ug oil/L, which is 6-20 times lower than for the other species tested. This is because the eggshells of haddock and polar cod can accumulate microscopic oil drops from the water, and thus they are more heavily exposed than the eggs of the other species which only absorb the water-soluble components of oil. The EGGTOX project is a large international collaboration among researchers from Norway, USA, England, France, and Denmark. Similar exposure systems have been established in 4 different laboratories: two in Norway (at SINTEF in Trondheim, and at Havforskningsinstituttet on Austevoll) and two in USA (at NOAA in Seattle, Washington and Newport, Oregon). These similar exposure systems provide capacity for comparisons between numerous species. The experiments are standardized and consist of a short oil exposure of three days. Thereafter the fish eggs are transferred to clean seawater and allowed to hatch into larvae (10-40 days). Some of the studies (haddock and cod) were conducted with and without UV to study phototoxicity. For haddock and polar cod, long-term studies were also performed, where the fish were allowed to reach juvenile stage (4-6 months). All experiments exposed the fish eggs to both dispersed oil (where the oil components are in both droplet form (microdroplets < 20 um) and oil dissolved in water). In EGGTOX, we seek to understand the underlying biological mechanisms for oils toxicity and to identify precisely which oil components cause serious effects. It is known that the heart is especially vulnerable to oil pollution, and both the function and development of the heart are affected. This leads to a series of secondary effects from the loss of proper circulation, such as deformities of the spine and jaw which in turn lead to reduced swimming ability and food intake. These results can eventually result in death during the larval stage. Polycyclic aromatic hydrocarbons (PAHs) are known to be among the most toxic substances in oil, and we find strong correlations among PAH water concentrations, the amounts of PAHs absorbed into the eggs (body burden), and the damaging effects in fish eggs and larvae. The oil exposure results in acute mortality only with relatively high doses, or in exposures in combination with UV. But even at very low doses, embryonic development is disturbed leading to high mortality at the larval stage. Modelling of the haddock egg toxicity data shows acute mortality with PAH doses at LD50 = 2,8 ug PAH/L (LD50 is the concentration that is lethal to 50 % of the experimental animals), while the delayed mortality for 20-day-old haddock larvae is LD50 = 0,25 ug PAH/L. Our results also show that oil doses that do not result in lethal effects (as low as 10 ug oil/L, 0.1 ug PAH /L)) can still cause effects in behavior (swimming speed and orientation). Such behavior changes can affect survival in the wild. We recommend therefore to use effect doses at 0.1 ug PAH/L in further risk modelling studies. We have further fractioned the oil mixture and studied which fractions and single PAHs are most toxic. Our results clearly show that none of the fractions or single PAHs can single-handedly explain the toxicity that is observed with exposure to the whole oil mixture. Both our studies and several other studies show that the amount of PAHs in oil mixtures is strongly correlated to mortality and deformities. That is, even though the PAHs cannot explain the toxicity themselves, the toxicity of the oil mixture can still be estimated by measuring PAHs. Regardless, future work should focus on the development of new methods to study which other toxic compounds the oil mixture contains, and which compounds might be a better measure of toxicity. For now, however, it is the amount of PAHs that seems to correlate best with toxicity. We recommend that PAHs continue to be used to model oil toxicity after oil spills.The results from the EGGTOX project have provided solid, data-based, tolerance limits for oil pollution on the eggs of marine fish, which are now used in risk modelling of oil spills in Lofoten and Vesterålen and other marine ecosystems in the north.

Resultatene fra EGGTOX prosjektet har gitt et solid datagrunnlag for tålegrenser for oljeforurensning på egg fra marine fisk som nå brukes til risikomodellering av oljeutslipp i Lofoten og Vesterålen og andre marine økosystem i nord. Prosjektet har gitt en økt forståelse hvordan oljeforurensning påvirker organismen, samt påvist at de alvorlige effektene av oljeforurensning ikke kan tilegnes enkeltkomponenter eller enkeltfraksjoner av oljen. Resultatene fra EGGTOX inngår i data materialet til rapporten fra FAGLIG FORUM FOR NORSKE HAVOMRÅDER; "Risiko for og beredskap mot akutt forurensning - endringer og utviklingstrekk, M-1304, 2019"

Oil spills can have significant adverse impacts on spawning fish populations due to the toxicity of crude oil-derived chemicals to sensitive early life history stages. Despite increasing knowledge in this area, there are still important unanswered questions relating to responses of different fish species, the identity of the precise chemicals causing toxicity, and exact mechanisms of action. This project builds on novel findings in these areas from the recently completed RCN-funded OIL-HADDOCK Project (No. 234367; 2014-16). Filling these data gaps will improve assessment of ecological risk and environmental damage related to future oil spills in highly productive fish habitats, and lead to the development of state-of-the-art tools for monitoring fish health in areas of oil extraction. In OIL-HADDOCK we identified genes that may serve as the basis for new biomarkers of several adverse outcomes stemming from exposure of fish embryos and larvae to crude oil. The project also provided new data and hypotheses relating to the role of polycyclic aromatic hydrocarbons (PAHs) as drivers of crude oil toxicity to developing fish. In this project, we will further advance oil toxicity science in seven key Norwegian fish species through three specific aims. These are: (1) Validate new AOP molecular biomarkers identified in haddock across multiple species using qPCR and whole-mount in situ hybridization. The results will determine the extent to which the new tools can accurately diagnose disrupted cardiac function, lipid metabolism, and osmoregulation from oil exposure. (2) Identify the precise cardiotoxic components of crude oil. We will specifically test the hypothesis that bioactivated metabolites of tricyclic PAHs are more toxic than their corresponding parent compounds. (3) Verify a mechanism of cardiotoxic action using structure-activity relationships and related screening tools developed for human drug discovery and pharmaceutical safety.