Co-Exposure of Phenanthrene and the cyp-Inducer 3-Methylchrysene Leads to Altered Biotransformation and Increased Toxicity in Fish Egg and Larvae.

Polycyclic aromatic hydrocarbons (PAHs) have frequently been suspected of governing crude oil toxicity because of similar morphological defects in fish. However, PAH concentrations are often not high enough to explain the observed crude oil toxicity. We hypothesize that one PAH can enhance the metabolism and toxicity of another PAH when administered as a mixture. Early life stage Atlantic haddock (Melanogrammus aeglefinus) were in this study exposed to phenanthrene in the presence and absence of 3-methylchrysene that is known to induce the metabolic enzyme cytochrome P450 1A via cyp1a gene expression. Uptake, metabolism, and multiple toxicity endpoints were then measured in a time-course study up to 3 days post-hatching. Passive dosing provided aqueous concentrations ≈180 µg/L for phenanthrene and ≈0.6 µg/L for 3-methylchrysene, which resulted in tissue concentrations ≈60 µg/g ww for phenanthrene and ≈0.15 µg/g ww for 3-methylchrysene. The low concentration of 3-methylchrysene led to the elevated expression of cyp1a but no toxicity. Levels of phenanthrene metabolites were 5-fold higher, and morphological defects and cardiotoxicity were consistently greater when co-exposed to both compounds relative to phenanthrene alone. This work highlights the metabolic activation of PAH toxicity by a co-occurring PAH, which can lead to excess toxicity, synergistic effects, and the overproportional contribution of PAHs to crude oil toxicity.

Discovery of polycyclic aromatic acid metabolites in fish exposed to the petroleum compounds 1-methylphenanthrene and 1,4-dimethylphenanthrene.

Most of the polycyclic aromatic hydrocarbons (PAHs) in petroleum are alkylated (alkyl PAHs), still the metabolism of these alkyl PAHs to the expected acid products (polycyclic aromatic acids; PAAs) has yet to be demonstrated in oil-exposed fish. Should these compounds be discovered in fish as they have in ragworm, rodents, and humans, they could present an indicative biomarker for assessing oil pollution. In this study, the ability to biotransform alkyl PAHs to PAAs was examined on Atlantic haddock (Melanogrammus aeglefinus). Exposure to phenanthrene, 1-methyphenanthrene or 1,4-dimethylphenanthrene was performed via intraperitoneal injection. An Ion Mobility Quadrupole Time-Of-Flight Mass Spectrometer (IMS-Q-TOF MS) was used in exploratory analysis of extracted bile samples. Acquisition of four-dimensional information by coupling liquid chromatography with the IMS-Q-TOF MS and in-silico prediction for feature prioritization in the data processing workflow allowed several tentative identifications with high degree of confidence. This work presents the first detection of PAAs in fish and suggests the importance of investigating alkyl PAHs in ecotoxicological studies of oil-polluted fish environments.

Cardiac toxicity of phenanthrene depends on developmental stage in Atlantic cod (Gadus morhua).

Complex mixtures like crude oil, and single components such as Phenanthrene (Phe), induce cardiotoxicity by interfering with excitation-contraction coupling. However, recent work has demonstrated that the timing of pollutant exposure during embryogenesis greatly impacts the degree of cardiac dysfunction caused. Here, we aimed to clarify the temporal dependence of Phe toxicity and the downstream effects of cardiac dysfunction using Atlantic cod (Gadus morhua). Phe (nominal concentration, 1.12 µmol/L), or the L-type­calcium channel blocker Nicardipine (Nic) (nominal concentration, 2 and 4 µmol/L), were individually applied to cod embryos either during cardiogenesis (early) or after the onset of cardiac function (late). Phe toxicity was highly dependent on the timing of exposure. Exposure after the onset of cardiac function (i.e. late) caused more severe cardiac and extracardiac abnormalities at 3 days post hatching (dph) than early exposure. Late Phe exposure resulted in a smaller ventricle, eliminated ventricular contraction, and reduced atrial contraction. In contrast, early Phe exposure did not have an effect on cardiac development and function. This temporal difference was not as evident in the Nic treatment. Early Nic exposure created similar morphological phenotypes to the late Phe exposure. The two treatments (early Nic and late Phe) also shared a cardiofunctional phenotype, comprised of eliminated ventricular, and reduced atrial, contraction. These data suggest that extracardiac abnormalities, such as the craniofacial deformities seen after late embryonic exposure to cardiotoxic oil components and mixtures, are mostly downstream effects of cardiac dysfunction.

Alkyl-phenanthrenes in early life stage fish: differential toxicity in Atlantic haddock (Melanogrammus aeglefinus) embryos.

Tricyclic polycyclic aromatic hydrocarbons (PAHs) are believed to be the primary toxic components of crude oil. Such compounds including phenanthrene are known to have direct effects on cardiac tissue, which lead to malformations during organogenesis in early life stage fish. We tested a suite of 13 alkyl-phenanthrenes to compare uptake and developmental toxicity in early life stage haddock (Melanogrammus aeglefinus) embryos during gastrulation/organogenesis beginning at 2 days post fertilization via passive dosing. The alkyl-phenanthrenes were tested at their solubility limits, and three of them also at lower concentrations. Measured body burdens were linearly related to measured water concentrations. All compounds elicited one or more significant morphological defects or functional impairment, such as decreased length, smaller eye area, shorter jaw length, and increased incidence of body axis deformities and eye deformities. The profile of developmental toxicities appeared unrelated to the position of alkyl substitution, and gene expression of cytochrome 1 a (cyp1a) was low regardless of alkylation. Mortality and sublethal effects were observed below the expected range for baseline toxicity, thus indicating excess toxicity. Additionally, PAH concentrations that resulted in toxic effects here were far greater than when measured in whole crude oil exposures that cause toxicity. This work demonstrates that, while these phenanthrenes are toxic to early life stage fish, they cannot individually account for most of the developmental toxicity of crude oil, and that other compounds and/or mixture effects should be given more consideration.

Co-exposure to UV radiation and crude oil increases acute embryotoxicity and sublethal malformations in the early life stages of Atlantic haddock (Melanogrammus aeglefinus).

Crude oil causes severe abnormalities in developing fish. Photomodification of constituents in crude oil increases its toxicity several fold. We report on the effect of crude oil, in combination with ultraviolet (UV) radiation, on Atlantic haddock (Melanogrammus aeglefinus) embryos. Accumulation of crude oil on the eggshell makes haddock embryos particularly susceptible to exposure. At high latitudes, they can be exposed to UV radiation many hours a day. Haddock embryos were exposed to crude oil (5-300 µg oil/L nominal loading concentrations) for three days in the presence and absence of UV radiation (290-400 nm). UV radiation partly degraded the eggs' outer membrane resulting in less accumulation of oil droplets in the treatment with highest oil concentration (300 µg oil/L). The co-exposure treatments resulted in acute toxicity, manifested by massive tissue necrosis and subsequent mortality, reducing LC50 at hatching stage by 60 % to 0.24 µg totPAH/L compared to 0.62 µg totPAH/L in crude oil only. In the treatment with nominal low oil concentrations (5-30 µg oil/L), only co-exposure to UV led to sublethal morphological heart defects. Including phototoxicity as a parameter in risk assessments of accidental oil spills is recommended.

Photo-enhanced toxicity of crude oil on early developmental stages of Atlantic cod (Gadus morhua).

Photo-enhanced toxicity of crude oil is produced by exposure to ultraviolet (UV) radiation. Atlantic cod (Gadus morhua) embryos were exposed to crude oil with and without UV radiation (290-400 nm) from 3 days post fertilization (dpf) until 6 dpf. Embryos from the co-exposure experiment were continually exposed to UV radiation until hatching at 11 dpf. Differences in body burden levels and cyp1a expression in cod embryos were observed between the exposure regimes. High doses of crude oil produced increased mortality in cod co-exposed embryos, as well as craniofacial malformations and heart deformities in larvae from both experiments. A higher number of differentially expressed genes (DEGs) and pathways were revealed in the co-exposure experiment, indicating a photo-enhanced effect of crude oil toxicity. Our results provide mechanistic insights into crude oil and photo-enhanced crude oil toxicity, suggesting that UV radiation increases the toxicity of crude oil in early life stages of Atlantic cod.