Article pubs.acs.org/est
Polycyclic Aromatic Acids Are Primary Metabolites of Alkyl-PAHsA Case Study with Nereis diversicolor Linus M. V. Malmquist,*,†,‡ Henriette Selck,‡ Kåre B. Jørgensen,§ and Jan H. Christensen† †
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark ‡ Department of Environmental, Social and Spatial Change, Roskilde University, Universitetsvej 1, P.O. Box 260, DK-4000 Roskilde, Denmark § Faculty of Science and Technology, University of Stavanger, 4036 Stavanger, Norway S Supporting Information *
ABSTRACT: Although concentrations of alkylated polycyclic aromatic hydrocarbons (alkyl-PAHs) in oil-contaminated sediments are higher than those of unsubstituted PAHs, only little attention has been given to metabolism and ecotoxicity of alkyl-PAHs. In this study we demonstrated that metabolism of alkyl-PAHs primarily forms polycyclic aromatic acids (PAAs). We generalize this to other alkyl-PAHs, based on literature and the present study of the metabolism of 1-methylphenanthrene, 3,6-dimethylphenanthrene, and 1-, 2-, 3-, and 6-methylchrysene related to their unsubstituted parent PAHs. Also, we observed that body burdens and production of PAAs was related to the position of the methyl group, showing the same isomer specific preferences as for microbial degradation of alkyl-PAHs. We detected a high production of PAAs, and larger metabolism of alkyl-PAHs than their unsubstituted parent PAHs. We therefore propose that carboxylic acid metabolites of alkyl-PAHs have the potential of constituting a new class of contaminants in marine waters that needs attention in relation to ecological risk assessments.
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INTRODUCTION Alkylated polycyclic aromatic hydrocarbons (Alkyl-PAHs) comprise up to 98% of the total PAH fraction of crude oil, and after spills in the aquatic environment the proportion of alkyl-PAHs in sediments remains high (up to 70% of the total PAH fraction).1−5 Despite the high proportions of alkylated- to unsubstituted PAHs, fate and effect-studies of PAHs in relation to benthic macrofauna and sediments have primarily focused on unsubstituted PAHs, whereas studies of alkyl-PAHs are scarce.6,7 We have previously shown that sediment-associated 1methylpyrene (1mP) exposed to the benthic invertebrate Nereis diversicolor results in an effective metabolism of 1mP to phase I and II metabolites, at a rate five times higher than that of the unsubstituted PAH pyrene.8 The phase I metabolite 1pyrenecarboxylic acid (1PCA), comprised 90% of the total detected metabolites. It is well-known that oil degradation in aerobic environments generates polar oxygenated hydrocarbons, and hazardous effects of the oxygenated fractions of oil have recently caught much attention. This includes toxicological examinations of oxygenated metabolites of spilled oil,9−12 and input-related studies, especially from extraction of oil sands, where process-affected waters contain high concentrations of oxygenated hydrocarbons in the form of naphthenic and aromatic acids.13−16 Naphthenic and aromatic © 2015 American Chemical Society
acids occur naturally in oil sands because the oil has been exposed to severe bacterial oxidation.15 Some naphthenic and aromatic acids have been shown to be acutely toxic to Daphnia magna in water exposures, and in vitro tests have shown genotoxic and estrogenic effects.13,14,17 In addition, the polar fraction of sediment-associated oil, and otherwise polluted sediments, have been shown to induce effects such as mutagenicity, estrogenic effects, and DNA damage, and to create reactive oxygen species (ROS) in standard laboratory in vitro tests.18−20 Because alkyl-PAHs are found in high concentrations in sediments, and some aromatic acids are known to be toxic, the overall aim of this study was to examine whether the efficient metabolism of alkyl-PAHs to water-soluble polycyclic aromatic acids (PAAs), as observed for 1-methylpyrene by Malmquist et al.,8 can be generalized to include other alkyl-PAHs present in petroleum products, and thereby represent a new class of potential contaminants in marine waters. We investigated the effect of macrobenthos on metabolism of alkylated and unsubstituted PAHs to elucidate (a) the transformation potential of alkyl-PAHs relative to unsubstituted PAHs, and Received: September 9, 2014 Accepted: April 1, 2015 Published: April 1, 2015 5713
DOI: 10.1021/acs.est.5b01453 Environ. Sci. Technol. 2015, 49, 5713−5721
Article
Environmental Science & Technology
widespread organism present at high densities in aquatic environments (up to 3700 individual adults m−2), and (iii) is capable of feeding on deposits as well as filter feeding. N. diversicolor irrigates the burrow with overlying oxygenated water leading to an increase in the oxygen level in the surrounding sediment which will lead to increased microbial PAH degradation.27 In addition, N. diversicolor is known to have high tolerance to PAHs, and has as such been used in several studies on metabolism of oil and PAHs.28−34
(b) the effect of presence of macrobenthos on metabolite production. PAH uptake and PAA production was analyzed in mesocosms with or without presence of the polychaete N. diversicolor, after exposure to sediment-associated phenanthrene, 1-methylphenanthrene, 3,6-dimethyphenanthrene, chrysene, 1-methylchrysene, 2-methylchrysene, 3-methylchrysene, 5-methylchrysene, or 6-methylchrysene, respectively. The methylated phenanthrenes and chrysenes were chosen because the presence of these in a typical crude oil is more than 10 times higher than their nonalkylated parent1 (phenanthrene: 2.65 μg/g crude oil, C1-phenanhtrenes: 32.96 μg/g crude oil, C2-phenanthrenes: 134.72 μg/g crude oil, chrysene: 0.50 μg/g crude oil, and C1-chrysenes: 4.25 μg/g crude oil). Studies of alkyl-PAH isomers have shown that certain positions of methyl substitution favor microbial oxidation whereas other positions render the PAH almost resistant to degradation.2,21−24 It is a general trend that isomers with methyl substitution in the β-position degrade more rapidly compared to isomers with methyl substitution in the α-position (see Figure 1 for chemical structures). Examples of microbial
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MATERIALS AND METHODS Chemicals. Phenanthrene, 1- and 9-methylphenanthrene, 3,6-dimethylphenanthrene, 9-hydroxyphenanthrene, 4-phenanthrenecarboxylic acid, 1-pyrenecarboxylic acid, chrysene, 5methylchrysene, phenanthrene-d10, anthracene-d10, pyrene-d10, fluoranthene-d10, chrysene-d12, and benz(a)anthracene-d12 were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-, 2-, 3-, and 6-methylchrysene and 3-chrysenecarboxylic acid were synthesized at the University of Stavanger.35 All organic solvents (acetone, acetonitrile, methanol, n-pentane, isooctane, and dichloromethane) were LC-MS grade. Water for ultrahigh performance liquid chromatography (UHPLC) analyses was glass distilled and used for a maximum of 3 days. The internal standard solution for GC-MS analysis of sediments was prepared from pyrene-d10, phenanthrene-d10, and chrysene-d12 in isooctane. The recovery standard solution was prepared from anthracene-d10, fluoranthene-d10, and benz(a)anthracene-d12 in isooctane. Experimental Section. Two separate exposure setups were employed. In experiment 1, N. diversicolor and sediment (sieved in situ to
