Crude oil exposure of early life stages of Atlantic haddock suggests threshold levels for developmental toxicity as low as 0.1 µg total polyaromatic hydrocarbon (TPAH)/L.

Atlantic haddock (Melanogrammus aeglefinus) embryos bind dispersed crude oil droplets to the eggshell and are consequently highly susceptible to toxicity from spilled oil. We established thresholds for developmental toxicity and identified any potential long-term or latent adverse effects that could impair the growth and survival of individuals. Embryos were exposed to oil for eight days (10, 80 and 300 µg oil/L, equivalent to 0.1, 0.8 and 3.0 µg TPAH/L). Acute and delayed mortality were observed at embryonic, larval, and juvenile stages with IC50 = 2.2, 0.39, and 0.27 µg TPAH/L, respectively. Exposure to 0.1 µg TPAH/L had no negative effect on growth or survival. However, yolk sac larvae showed significant reduction in the outgrowth (ballooning) of the cardiac ventricle in the absence of other extracardiac morphological defects. Due to this propensity for latent sublethal developmental toxicity, we recommend an effect threshold of 0.1 µg TPAH/L for risk assessment models.

DNA damage and health effects in juvenile haddock (Melanogrammus aeglefinus) exposed to PAHs associated with oil-polluted sediment or produced water.

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3-0.7 mg PAH/kg fish/day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.

Dispersants have limited effects on exposure rates of oil spills on fish eggs and larvae in shelf seas.

Early life stages of fish are particularly vulnerable to oil spills. Simulations of overlap of fish eggs and larvae with oil from different oil-spill scenarios, both without and with the dispersant Corexit 9500, enable quantitative comparisons of dispersants as a mitigation alternative. We have used model simulations of a blow out of 4500 m(3) of crude oil per day (Statfjord light crude) for 30 days at three locations along the Norwegian coast. Eggs were released from nine different known spawning grounds, in the period from March 1st until the end of April, and all spawning products were followed for 90 days from the spill start at April first independent of time for spawning. We have modeled overlap between spawning products and oil concentrations giving a total polycyclic hydrocarbon (TPAH) concentration of more than 1.0 or 0.1 ppb (µg/l). At these orders of magnitude, we expect acute mortality or sublethal effects, respectively. In general, adding dispersants results in higher concentrations of TPAHs in a reduced volume of water compared to not adding dispersants. Also, the TPAHs are displaced deeper in the water column. Model simulations of the spill scenarios showed that addition of chemical dispersant in general moderately decreased the fraction of eggs and larvae that were exposed above the selected threshold values.

Ecotoxicological mechanisms and models in an impact analysis tool for oil spills.

In an international collaborative effort, an impact analysis tool is being developed to predict the effect of accidental oil spills on recruitment and production of Atlantic cod (Gadus morhua) in the Barents Sea. The tool consisted of three coupled ecological models that describe (1) plankton biomass dynamics, (2) cod larvae growth, and (3) fish stock dynamics. The discussions from a series of workshops are presented in which variables and parameters of the first two ecological models were listed that may be affected by oil-related compounds. In addition, ecotoxicological algorithms are suggested that may be used to quantify such effects and what the challenges and opportunities are for algorithm parameterization. Based on model exercises described in the literature, survival and individual growth of cod larvae, survival and reproduction of zooplankton, and phytoplankton population growth are denoted as variables and parameters from the ecological models that might be affected in case of an oil spill. Because toxicity databases mostly (67%) contain data for freshwater species in temperate environments, parameterization of the ecotoxicological algorithms describing effects on these endpoints in the subarctic marine environment is not straightforward. Therefore, it is proposed that metadata analyses be used to estimate the sensitivity of subarctic marine species from available databases. To perform such analyses and reduce associated uncertainty and variability, mechanistic models of varying complexity, possibly aided by new experimental data, are proposed. Lastly, examples are given of how seasonality in ecosystems may influence chemical effects, in particular in the subarctic environment. Food availability and length of day were identified as important characteristics as these determine nutritional status and phototoxicity, respectively.

Development of Atlantic cod (Gadus morhua) exposed to produced water during early life stages: Effects on embryos, larvae, and juvenile fish.

Produced water (PW) contains numerous toxic compounds of natural origin, such as dispersed oil, metals, alkylphenols (APs), and polycyclic aromatic hydrocarbons (PAHs). In addition, PW also contains many different chemicals which have been added during the oil production process. In the study described here, cod were exposed to real PW collected from an oil production platform in the North Sea. This was done in order to best recreate the most realistic field-exposure regime in which fish will be affected by a wide range of chemicals. The biological effects found in this study therefore cannot be assigned to one group of chemicals alone, but are the result of exposure to the complex chemical mixture found in real PW. Since APs are well known to cause endocrine disruption in marine organisms, we focused our chemical analysis on APs in an attempt to better understand the long-term effects of APs from PW on the biology of fish. In this study, cod were exposed to several concentrations of real PW and 17ß-oestradiol (E(2)), a natural oestrogen, at different developmental stages. Cod were exposed to PW either during the embryo and early larvae stage (up to 3 months of age) or during the early juvenile stage (from 3 to 6 months of age). Results showed that, in general, APs bioconcentrate in fish tissue in a dose and developmental stage dependent manner during PW exposure. However, juveniles appeared able to effectively metabolise the short chain APs. Importantly, PW exposure had no effect on embryo survival or hatching success. However, 1% PW clearly interfered with the development of normal larval pigmentation. After hatching most of the larvae exposed to 1% PW failed to begin feeding and died of starvation. This inability to feed may be linked to the increased incidence of jaw deformities seen in these larvae. In addition, cod exposed to 1% PW, had significantly higher levels of the biomarkers vitellogenin and CYP1A in plasma and liver, respectively. No similar effects were seen in cod exposed to either 0.1% or 0.01% PW.

Biomarker candidate discovery in Atlantic cod (Gadus morhua) continuously exposed to North Sea produced water from egg to fry.

In this study Atlantic cod (Gadus morhua) were exposed to different levels of North Sea produced water (PW) and 17beta-oestradiol (E(2)), a natural oestrogen, from egg to fry stage (90 days). By comparing changes in protein expression following E(2) exposure to changes induced by PW treatment, we were able to compare the induced changes by PW to the mode of action of oestrogens. Changes in the proteome in response to exposure in whole cod fry (approximately 80 days post-hatching, dph) were detected by two-dimensional gel electrophoresis and image analysis and identified by MALDI-ToF-ToF mass spectrometry, using a newly developed cod EST database and the NCBI database. Many of the protein changes occurred at low levels (0.01% and 0.1% PW) of exposure, indicating putative biological responses at lower levels than previously detected. Using discriminant analysis, we identified a set of protein changes that may be useful as biomarker candidates of produced water (PW) and oestradiol exposure in Atlantic cod fry. The biomarker candidates discovered in this study may, following validation, prove effective as diagnostic tools in monitoring exposure and effects of discharges from the petroleum industry offshore, aiding future environmental risk analysis and risk management.

Candidate biomarker discovery in plasma of juvenile cod (Gadus morhua) exposed to crude North Sea oil, alkyl phenols and polycyclic aromatic hydrocarbons (PAHs).

In this study we have investigated protein changes in plasma of juvenile Atlantic cod (Gadus morhua) induced by crude North Sea oil and North Sea oil spiked with alkyl phenols and polycyclic aromatic hydrocarbons, a surrogate produced water composition. Using a proteomic approach, we identified 137 differentially expressed proteins at different levels of crude oil exposure. Many of the induced protein changes occurred at low levels of exposure. The results obtained with protein expression profiles after exposure to oil and surrogate produced water indicate effects on fibrinolysis and the complement cascade, the immune system, fertility-linked proteins, bone resorption, fatty acid metabolism as well as increased oxidative stress, impaired cell mobility and increased levels of proteins associated with apoptosis. Although the number of individuals and samples in this study is limited within each treatment group, the protein changes observed in this study represent a first screening for potential biomarker candidates in cod plasma reflecting potential effects of crude oil and produced water exposure on fish.

Protein responses in blue mussels (Mytilus edulis) exposed to organic pollutants: a combined CYP-antibody/proteomic approach.

Polyclonal antibodies were raised against highly conserved, trans-metazoan sequences of cytochrome P450 (CYP) families 2 and 4 and used to investigate responses in the common blue mussel (Mytilus edulis) exposed to various organic contaminants. The results were evaluated by means of cross-reacting proteins on Western blots of both one- and two-dimensional electrophoresis gels, and by scanning spectroscopy measurements of total CYP content. Furthermore, a proteomic approach was applied aimed at elucidating exposure-related protein changes in a more general term. Identities of isolated proteins were searched by means of peptide mass fingerprints obtained from MALDI-TOF MS analyses. The results demonstrated that both antibodies rendered several cross-reactive bands when probed on Western blots. The most obvious cross-reaction of the CYP2 antibody was with a strongly expressed protein of size approximately 57kDa, pI 4.5-4.6, whereas the CYP4 antibody cross-reacted with a protein of size approximately 55kDa, pI 5.6. However, expression of cross-reacting proteins did not change as a result of the exposures, and resulted only in small and insignificant fluctuations in total CYP content. As a contrast, silver-stained 2DE gels showed that several microsomal proteins were affected in individuals exposed to diallylphthalate as well as crude oil, with and without a spike of alkylphenols and PAHs. Mass spectrometry based analyses of excised, trypsin-digested spots did so far not decipher the identities of the proteins affected by the exposures, nor of those cross-reacting with CYP2 and CYP4 antibodies. This study has underlined the power of the proteomic approach in environmental toxicology, although protein identification was not successful. The missing identities of the proteins cross-reacting with the CYP2- and CYP4-antibodies does not enable a clear conclusion as to whether or not these peptides actually represent CYP iso-enzymes.