Article pubs.acs.org/est
Effects of Nereis diversicolor on the Transformation of 1‑Methylpyrene and Pyrene: Transformation Efficiency and Identification of Phase I and II Products Linus M. V. Malmquist,*,† Jan H. Christensen,‡ and Henriette Selck† †
Department of Environmental, Social and Spatial Change, Roskilde University, Universitetsvej 1, P.O. Box 260, DK-4000 Roskilde, Denmark ‡ Analytical Chemistry Group, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark S Supporting Information *
ABSTRACT: Transformation of nonsubstituted and alkyl-substituted polycyclic aromatic hydrocarbons (PAHs) by the benthic invertebrate Nereis diversicolor was compared in this study. Pyrene and 1-methylpyrene were used as model compounds for nonsubstituted and alkyl-substituted PAHs, respectively. Qualitative and quantitative analyses of metabolites and parent compounds in worm tissue, water, and sediment were performed. Transformation of 1-methylpyrene generated the benzylic hydroxylated phase I product, 1-pyrenecarboxylic acid that comprised 90% of the total metabolites of 1-methylpyrene, and was mainly found in water extracts. We tentatively identified 1-methylpyrene glucuronides and 1-carbonylpyrene glycine as phase II metabolites not previously reported in literature. Pyrene was biotransformed to 1-hydroxypyrene, pyrene-1-sulfate, pyrene-1glucuronide, and pyrene glucoside sulfate, with pyrene-1-glucuronide as the most prominent metabolite. Transformation of 1methylpyrene (21% transformed) was more than 3 times as efficient as pyrene transformation (5.6% transformed). Because crude oils contain larger amounts of C1−C4-substituted PAHs than nonsubstituted PAHs, the rapid and efficient transformation of sediment-associated 1-methylpyrene may result in a high exposure of water-living organisms to metabolites of alkyl-substituted PAHs, whose toxicities are unknown. This study demonstrates the need to consider fate and effects of substituted PAHs and their metabolites in risk assessments.
■
INTRODUCTION
pyrolytic input, proportions of alkylated PAHs are normally much lower (often less than 1% of the total PAHs)10,12 but can also have relatively high concentrations of alkylated PAHs. For example, Rogge et al. found that as much as 56% of the total PAHs was alkyl-substituted in diesel exhaust gas.13 Microbial transformation processes are considered one of the most important routes of PAH degradation at contaminated sites.14 Microbial degradation rates decrease with increasing degree of alkylation: C0 > C1 > C2 > C3 > C4 etc., most likely due to steric hindrances of aromatic ring oxidation.4,14−17 The lower transformation rates and high relative concentrations of substituted PAHs compared to nonsubstituted PAHs in PAHpolluted sediments demonstrate the persistent nature of
Most studies on fate and effects of crude oil in the environment focus on nonsubstituted polycyclic aromatic hydrocarbons (PAHs) such as naphthalene, phenanthrene, pyrene, and benzo(a)pyrene, which are all part of the U.S. EPA priority pollutant list.1 Even though crude oil contains higher concentrations of C1−C4-substituted PAHs than nonsubstituted PAHs,2−5 none of the PAHs on this list are alkyl substituted. Studies addressing whether substituted PAHs are more or less persistent and toxic than nonsubstituted PAHs are scarce in the literature.6,7 Also, effects of PAHs are not trivial, as toxicity not only relates to the PAHs themselves, but often also to their metabolites. For example, the vicinal diol-epoxide is the carcinogenic promoter for benzo(a)pyrene as reviewed by, e.g., Myers et al.8 In crude oil as much as 98% of the total PAHs are alkyl substituted,5 and in petrogenic contaminations of sediments as great as 70% of the PAHs are alkylated.4,9−11 In relation to © 2013 American Chemical Society
Received: Revised: Accepted: Published: 5383
July 6, 2012 April 22, 2013 April 23, 2013 April 23, 2013 dx.doi.org/10.1021/es400809p | Environ. Sci. Technol. 2013, 47, 5383−5392
Environmental Science & Technology
■
substituted PAHs and their likelihood to increase relative to the sum of PAHs over time. Hence, there is a need to understand the environmental fate and effects of this group of compounds.4,18 Filter and deposit feeders in the benthic zone, such as echinoderms, bivalves, oligochaetes, and polychaetes are exposed to PAHs, since hydrophobic compounds tend to adsorb to particles in the water phase and eventually accumulate in the sediment compartment. The polychaete Nereis diversicolor is an omnivorous surface deposit feeder, also capable of filter feeding,19 which has been found in densities up to more than 3000 individuals m−2. Accordingly, and due to a high tolerance to PAHs, N. diversicolor has served as a model species in several studies examining the fate of PAHs at contaminated sites.20−22 The polychaete bioturbates (particle mixing, irrigation, etc.) the sediment resulting in oxygenation of the sediment which leads to an increased microbial degradation of PAHs. For example, Christensen et al. found that the presence of N. diversicolor resulted in transformation of 25% of the sediment-associated radioactive-labeled pyrene during 42 days of exposure,20 and that the presence of N. diversicolor increased the loss of pyrene from the sediment by a factor of 3 compared to systems without worm presence. In general, biotransformation of PAHs initiated by cytochrome P450 (CYP450) mixed function oxygenase (phase I oxidation) is followed by conjugation of water-soluble groups (phase II conjugation) and active excretion of the transformation products.20−24 The biotransformation pathway for pyrene by N. diversicolor is well understood and described.21,25,26 In general, pyrene undergoes CYP450 oxidation at the 1-position, followed by conjugation of glucuronide, glucoside, or sulfate groups or combinations of these. This biotransformation route is likewise recognized for other aquatic invertebrates such as Capitella teleta, Arenicola marina, and Lumbriculus variegates.23,26−28 However, for 1methylpyrene there exist no ecotoxicological studies, and only a few studies on mice or in vitro cultures of cells and microsomes have to our knowledge been published. The primary transformation step (phase I) of 1-methylpyrene in mice and in vitro cultures can lead to several metabolites. The most abundant metabolites are 1-hydroxymethylpyrene (benzylic hydroxylation), 1-methyl-X-hydroxypyrene (aromatic hydroxylation), and 1-pyrenecarboxylic acid (double benzylic hydroxylation).29 To our knowledge, phase II metabolites of 1-methylpyrene have not yet been identified in either invertebrates or vertebrates. However, the sulfate conjugate, 1-sulfoxymethylpyrene, which is both carcinogenic and mutagenic, has been proposed as a metabolite of 1-methylpyrene in human and rat liver microsomes after exposure to 1-hydroxymethylpyrene.29−32 Studies of other substituted PAHs transformed in vitro (with liver microsomes from trout and humans) or by mussels and bacteria also show formation of phase I products, such as quinones, diols, and aromatic and benzylic hydroxylated products.7,17,33,34 This study is the first comparative study on transformation of nonsubstituted and alkyl-substituted PAHs in marine sediments. The specific aims were therefore to examine the biotransformation potential for 1-methylpyrene and relate this to that of pyrene, and to compare the effects of N. diversicolor on metabolism of 1-methylpyrene and pyrene in sediment microcosms.
Article
MATERIALS AND METHODS
Chemicals. Pyrene, 1-methylpyrene, 1-hydroxypyrene, 1pyrenecarboxylic acid, 4-phenanthrencarboxylic acid, pyrene− d10, and fluoranthene−d10 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pyrene-1-glucuronide was purchased from Chiron (Trondheim, Norway). All organic solvents (acetone, acetonitrile, methanol, n-pentane, isooctane, and dichloromethane) were HPLC grade. Water for ultrahighperformance liquid chromatography (UHPLC) analyses was glass distilled and kept for a maximum of 3 days. The internal and the recovery standard solutions for GC-MS analysis were prepared from, respectively, pyrene−d10 and fluoranthene−d10 in isooctane. Pretreatment. N. diversicolor were collected at Herslev beach in Roskilde fjord, Denmark, June 2010 (GPS coordinates: +55° 40′ 40.77″, +11° 59′ 9.64″). The size of the worms was from 0.40 to 0.92 g wet weight, with a mean of 0.61 g ww (n = 13). At the same site, sediment was collected and sieved to below 1000 μm. The sediment was sieved through a 500-μm mesh in the laboratory. The sediment was frozen (−20 °C) for a minimum of 24 h before start of the experiment to remove meiofauna, following standard procedures of e.g McElroy et al.35 and Selck et al.36 Seawater (
