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Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

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IntroductionCalanus finmarchicus, a highly abundant copepod that is an important primary consumer in North Atlantic ecosystems, has a flexible life history in which copepods in the last juvenile developmental stage (fifth copepodid, C5) may either delay maturation and enter diapause or molt directly into adults. The factors that regulate this developmental plasticity are poorly understood, and few tools have been developed to assess the physiological condition of individual copepods.ResultsWe sampled a cultured population of C. finmarchicus copepods daily throughout the C5 stage and assessed molt stage progression, gonad development and lipid storage. We used high-throughput sequencing to identify genes that were differentially expressed during progression through the molt stage and then used qPCR to profile daily expression of individual genes. Based on expression profiles of twelve genes, samples were statistically clustered into three groups: (1) an early period occurring prior to separation of the cuticle from the epidermis (apolysis) when expression of genes associated with lipid synthesis and transport (FABP and ELOV) and two nuclear receptors (ERR and HR78) was highest, (2) a middle period of rapid change in both gene expression and physiological condition, including local minima and maxima in several nuclear receptors (FTZ-F1, HR38b, and EcR), and (3) a late period when gonads were differentiated and expression of genes associated with molting (Torso-like, HR38a) peaked. The ratio of Torso-like to HR38b strongly differentiated the early and late groups.ConclusionsThis study provides the first dynamic profiles of gene expression anchored with morphological markers of lipid accumulation, development and gonad maturation throughout a copepod molt cycle. Transcriptomic profiling revealed significant changes over the molt cycle in genes with presumed roles in lipid synthesis, molt regulation and gonad development, suggestive of a coupling of these processes in Calanus finmarchicus. Finally, we identified gene expression profiles that strongly differentiate between early and late development within the C5 copepodid stage. We anticipate that these findings and continued development of robust gene expression biomarkers that distinguish between diapause preparation and continuous development will ultimately enable novel studies of the intrinsic and extrinsic factors that govern diapause initiation in Calanus finmarchicus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-014-0091-8) contains supplementary material, which is available to authorized users.