Global Metabolite Profiling Reveals Transformation Pathways and

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Global Metabolite Profiling Reveals Transformation Pathways and Novel Metabolomic Responses in Solea senegalensis after Exposure to a Non-ionic Surfactant Diana Á lvarez-Muñoz,†,‡ Raghad Al-Salhi,† Alaa Abdul-Sada,† Eduardo González-Mazo,‡ and Elizabeth M. Hill*,† †

School of Life Sciences, University of Sussex, Brighton BN1 9QJ, United Kingdom Faculty of Marine and Environmental Sciences, University of Cadiz, Cadiz 11010, Spain

‡

S Supporting Information *

ABSTRACT: Alcohol polyethoxylate (AEO) surfactants are widely used in household and industrial products, but the health effects arising from short-term exposure to sublethal concentrations are unknown. A metabolomic approach was used to investigate the biotransformation and effects of exposure to sublethal concentrations of hexaethylene glycol monododecylether (C12EO6) in juvenile sole, Solea senegalensis. After 5 days, C12EO6 was rapidly metabolized in the sole by oxidation, glucuronidation, and ethoxylate chain shortening. C12EO6 exposure at either 146 or 553 μg L−1 resulted in significant metabolite disruption in liver and blood samples, including an apparent fold increase of >106 in the circulating levels of C24 bile acids and C27 bile alcohols, disturbance of glucocorticoid and lipid metabolism, and a 470-fold decrease in levels of the fatty acid transport molecule palmitoyl carnitine. Depuration resulted in rapid elimination of the surfactant and normalization of metabolites toward pre-exposure levels. Our findings show for the first time the ability of metabolomic analyses to discern effects of this AEO on metabolite homeostasis at exposure levels below its no effect concentrations for survival and reproduction in juvenile fish. The pronounced alteration in levels of liver metabolites, phospholipids, and glucocorticoids in S. senegalensis in response to surfactant exposure may indicate that this contaminant could potentially impact a number of health end points in fish.

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INTRODUCTION Alcohol polyethoxylates (AEOs) are a major class of non-ionic surfactants, which are mainly used in household and industrial applications, such as laundry detergents, cleaning agents, cosmetics, and textile lubricants.1 They can also be used as wetting agents in drilling muds and dispersants in oil spill events.2 AEOs have the general formula CH3(CH2)n(OCH2CH2)yOH, and commercial products are usually mixtures comprising a range of alkyl chain lengths and an average molar number of ethylene oxide groups. For many applications, commercial AEO mixtures comprise alkyl groups, where n is between 12 and 18 and y, the EO composition, is between 2 and 20.1 The global consumption of AEOs in 2009 was estimated to be 612 000 tonnes, raising concerns of the environmental risk of AEO exposure in aquatic systems.3 After domestic and/or industrial use, surfactants are released either directly or via wastewater treatment plants into surface waters. Concentrations of AEO mixtures in wastewater influents can be between 0.5 and 5 mg/L,4−6 but they can be effectively removed from wastewater treatment plants as a result of significant aerobic biodegradation, resulting in effluent and surface water concentrations of generally 100 mg/L depending upon the length of the alkyl chain and the ethoxylate (EO) content.1 For the pure homologue C12EO4 or the mixture C12−13EO6.5, the LC50 is 1.3 mg/L.1 A no effect concentration (NOEC) of 0.88 mg/L has Received: Revised: Accepted: Published: 5203

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surfactant-spiked seawater for 5 days. After this period, half of the fish from each treatment were transferred to clean water for an additional 3 days. A total of 12 replicates per exposure concentration were sampled at the end of the exposure and depuration phases, and no mortality occurred in any treatments. Surfactant concentrations of C12EO6 in water were analyzed using the procedure described by Lara-Martin et al.21 (see the Supporting Information for details). Measured (daily) levels of C12EO6 in spiked seawater [mean ± 1 standard deviation (SD)] were 146 ± 25 μg L−1 (termed low dose) and 553 ± 72 μg L−1 (termed high dose). Fish were anaesthetized with 2-phenoxyethanol, and blood (120−250 μL) sampled from the caudal vein was combined with 300 μL of methanol to preserve the samples. Fish were killed by increasing the dose of anesthetic up to lethal levels, and all of the gill and liver tissues were removed from the fish and immediately frozen by immersion in liquid nitrogen. All of the samples were stored at −80 °C until analysis. Metabolite Extraction. Samples from liver and gills (low and high exposure) and blood (high exposure) were extracted to determine the bioconcentration of C12EO6 in fish tissues as well as to investigate changes in the fish metabolome and xenometabolome. Prior to extraction, an internal standard (20 ng of C16EO6) was added to the samples to monitor recovery during chemical profiling studies. Tissue samples were thawed (100 mg of liver or gills); 1 mL of methanol was added; and the tissues were disrupted with an ultrasonic probe (Microson XL2000, Misonix, Farmingdale, NY, 18 W for 1 min). Samples were centrifuged (3000g for 10 min); the supernatant was collected; and the remaining pellet was re-extracted with 0.2 mL of methanol. The methanol supernatants were combined and diluted with ultrapure water (23.5 mL) containing 1% glacial acetic acid for solid-phase extraction (SPE). SPE cartridges (200 mg of OASIS HLB, Waters, Elstree, U.K.) were conditioned with 10 mL each of ethyl acetate (EtAc), methanol (MeOH), and 1% acetic acid in ultrapure water. After sample loading, the cartridges were washed with acidified water (5 mL) and dried under vacuum for 10 min. Cartridges were eluted sequentially with ethyl acetate (4 mL) and methanol (4 mL). The eluates were evaporated separately under vacuum, reconstituted in 200 μL of methanol/water (50:50), filtered (0.2 μm) and stored at −20 °C until their analysis by mass spectrometry (MS). Frozen blood samples were thawed on ice and vortexed, and an aliquot (150 μL) was removed and centrifuged (3000g for 10 min). The supernatant was collected, diluted with water (50 μL), and filtered prior to MS analysis. Bioconcentration of C12EO6 in Sole. Analytical details for the targeted analysis of C12EO6 in sole are given in the Supporting Information. Chemical Profiling of Tissues. Samples were profiled using ultraperformance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) with an electrospray ionization (ESI) source operated in either positive or negative mode. Samples from liver and blood extracts were profiled in both −ESI and +ESI modes, and gill extracts were profiled in +ESI mode. Deuterated internal standards were added to the sample extracts to monitor UPLC-TOF-MS performance during profiling. Full details of the MS methods, quality control procedures, and multivariate analysis are given in the Supporting Information. Sample extracts (4 μL) were separated following a linear gradient with (A) 95% water, 5% methanol, and 0.2% formic acid and (B) 0.2% formic acid in methanol

been reported for C12−13EO6.5 for reproduction and survival in fish species.7 The mechanisms of toxicity of AEOs are thought to be similar to other non-ionic surfactants in that they have been shown to act as general narcotics, and their toxicity is dependent upon their hydrophobicity and generally increases with longer alkyl chains and fewer EO units.12 They have also been shown to interact with biological membranes, causing an increase in permeability and disruption of transmembrane solute transport.13 In one study, exposure of Xenopus laevis embryos to AEOs resulted in alterations in the structure of gill mitochondrial membranes, resulting in collapse of the electrochemical gradient and a decrease in oxygen consumption.14 There is little information on the effect of sublethal concentrations of surfactants on tissue biochemistry, and it is not clear whether exposure to concentrations below the NOEC can affect certain biochemical pathways, with potential effects on selected health end points. Recent studies revealed that rainbow trout exposed to a treated wastewater treatment effluent can accumulate complex mixtures of xenobiotics and surfactants, including AEOs with an alkyl chain length of C12− C15 and 1−8 EO units.15 In the same study, disruption of bile salt and lipid metabolism in fish was observed, but it remains unclear which components of the contaminant mixture bioconcentrating in the fish were responsible for the metabolite perturbations. The aim of this study was to investigate whether exposure to an AEO species resulted in metabolite changes in fish. A metabolomics approach was used to profile biochemicals in fish tissues and plasma using time-of-flight mass spectrometry (TOF-MS), which enables high-resolution detection of many biologically important molecules, such as steroids, bile acids, and lipid metabolites at the sub-nanogram level.16−18 The linear AEO, C12EO6, was used as the test compound because this is the most abundant AEO homologue in the composition of many cleaning products,1 is present in dispersant formulations,19 and is a common homologue detected in surface waters and sediments.8 The marine flat fish Solea senegalensis was selected as the target organism because it is commonly used in marine ecotoxicology studies and has been shown to rapidly accumulate C12EO6, reaching steady-state concentrations in juvenile whole fish after 2−3 days of exposure.20 Juvenile fish were exposed to C12EO6 at concentrations of 2- and 6-fold below the NOEC levels. The primary goal of this work was to investigate the nature of the endogenous metabolic changes in fish gills, liver, and blood as a result of surfactant exposure. In addition, profiling of the C12EO6 metabolites in the fish (i.e., the xenometabolome) allowed for the identification of biotransformation pathways, some of which have not been previously identified for AEOs. Our findings indicate that the AEO was extensively metabolized in line with rapid depuration and loss of the parent molecule from the tissues. In addition, a short-term sublethal exposure to the AEO homologue resulted in perturbations in metabolite concentrations in liver and blood, similar to those that had been previously associated with wastewater exposures.

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MATERIALS AND METHODS Fish Exposure. S. senegalensis (body weight of 3−6 g) were purchased from the aquaculture facilities of the University of Cadiz. Fish were held in a continuous flow-through seawater system in 72 L tanks at a temperature between 18 and 19 °C, pH 8.41−8.47, salinity 37‰, and dissolved oxygen between 60 and 70%. Fish were exposed in duplicate tanks per treatment, which consisted of either controls (seawater only) or 5204

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Figure 1. PCA score plots of the chemical profiles of (A) liver, (B) blood, and (C) gills of S. senegalensis exposed to C12EO6. Tissue (SPE ethyl acetate fraction) and blood extracts were profiled by UPLC-TOF-MS in +ESI mode. Fish, 12 replicates per treatment, were exposed to 0 μg L−1 (controls), 146 μg L−1 (low dose), and 553 μg L−1 (high dose) surfactant for 5 days. A further 12 replicates per treatment were held for an additional 3 days in clean water (termed control, low, and high depuration). The percentages of explained variation (R2X) for the first two components (t1 and t2) are displayed on the relative axes.

using an UPLC BEH C18 column (100 × 1 mm, 1.7 μm particle size). Mass spectra were collected in full-scan mode from m/z 100 to 1000; spectral peaks were deconvoluted and aligned; and the data sets were normalized to total spectral area for each sample, mean-centered, and pareto-scaled. Data sets were analyzed using principal component analysis (PCA), followed by partial least-squares discriminant analysis (PLSDA), to further explore differences between treatment classes. The explained variation (R2X/R2Y) and the predictive ability (Q2X/Q2Y) parameters of the PCA/PLS-DA models were examined to investigate the performance of the models. The discriminatory variables (i.e., metabolite markers) that influenced the discrimination between two classes (i.e., data sets from control and surfactant-exposed fish) were detected using orthogonal partial least-squares to latent structures (OPLS) models, followed by analysis of the “S” plots.22 Metabolite identities were determined from their accurate mass, isotopic fit, and collision-induced dissociation (CID) MS. Statistical Analyses. Significant differences in metabolite concentrations between the different treatment classes were determined using Student’s t test for parametric data and the Mann−Whitney U test for non-parametric data (identified as such by the Kolmogorov−Smirnov test). To avoid false positives associated with multivariate data, a Bonferroni correction value was used at a false discovery rate of 5%.

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from 48 to 67%, and suggest that the parent surfactant was rapidly metabolized and/or eliminated from the organism. Multivariate Analyses of the Fish (Xeno)metabolome. Data sets from liver, gill, and blood samples were modeled by PCA to study the separation between the control and surfactant-treated fish after 5 days of exposure. Examples of the results of the scores plots from extracts of liver and gills (SPE ethyl acetate fraction) and blood are shown in Figure 1 (analyses in +ESI mode) and Figure S1 of the Supporting Information (analyses in −ESI mode). The results of the scores plots showed clear separation between control and surfactantexposed fish in the liver, gill, and blood samples, indicating that there were significant differences between the tissue and blood biochemistry of control and exposed fish at the end of the exposure phase of the experiment. The effects of depuration were also apparent from the PCA models. In liver and gills, a 3 day depuration period resulted in a shift in the scores plot of the low-dose exposure group toward the controls, but this change was less apparent in the high-dose exposure group. In blood samples, the scores plots revealed a similar shift of samples from the depurated high-dose group toward that of the reference population. In these data sets, the differences in clustering of samples between the different exposure treatments were mainly generated by the influence of loading variables (MS signals) associated with the parent C12EO6 compound and its transformation products (see further work below). All of the PCA models showed poor predictive ability (Q2X < 0.34), and the only outliers were detected in the PCA model of the liver −ESI data set (see Figure S1B of the Supporting Information). After the three outliers were removed, remodeling of the data set did not improve the predictive ability of the model. However, the data sets from control fish and those exposed to low and high doses of C12EO6 were remodeled by PLS-DA, and examination of the model parameters revealed a higher explained variation (R2Y > 0.92) and predictive ability (Q2Y > 0.71) for most samples (see Table S2 of the Supporting Information). To determine which metabolites influence discrimination between the sample classes, control and surfactant exposure groups from the data sets were modeled using OPLS-DA, and the “S” plot was used to detect the discriminatory marker metabolites (for example, see Figure S2 of the Supporting Information). The significance of each marker signal was assessed from the p value of the control versus low- or highdose exposure groups. From these analyses, it was apparent that many of the main markers that significantly contributed to the separation of the classes were the same in all of the extracts of the liver, gill, and blood samples from the surfactant-exposed

RESULTS AND DISCUSSION

Surfactant Bioconcentration and Depuration in Fish Tissues. Bioconcentration factors (BCF values) in sole were calculated as the ratio of the surfactant concentration measured in the tissue or blood compared to the surfactant concentration in the exposure water. After 5 days of exposure, the bioconcentration of the surfactant was highest in blood (9.9 L kg−1) and liver (4.1−4.8 L kg−1) and lowest in gills (1.1−2.4 L kg−1), indicating rapid uptake and transport of the parent compound in the circulatory system (see Table S1 of the Supporting Information). Previous work has shown that exposure of S. senegalensis to C12EO6 under the same conditions resulted in steady-state concentrations within the fish after 2−3 days of exposure, with a BCF for the whole fish that was 10-fold higher than that reported for liver in our study.20 Generally, the BCF values reported for AEOs in whole fish are low and reported to be between 13 and 50 L kg−1 for homologues such as C12EO8 and C13EO8.23 The elimination percentages were calculated from the decrease in the concentration of the surfactant in the tissues at the end of the 3 day depuration phase (see Table S1 of the Supporting Information). The rates of elimination were very similar for all three tissues, ranging 5205

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Table 1. Metabolites of C12EO6 Identified in Blood of S. senegalensis after a 5 Day Exposure and Removal Following Depurationa p value difference between control and exposed fishb

observed value

RT (min)

experimental molecular formula of ion

theoretical mass

Δ (ppm)

identity of metabolite

665.3724 489.3404 561.3251 363.3111 605.3514 407.3372 451.3634 649.3774 275.2593 319.2848

6.92 8.17 11.04 11.04 11.17 11.17 11.28 11.39 12.98 12.98

C30H58O14Na C24H50O8Na C26H50O11Na C20H43O5 C28H54O12Na C22H47O6 C24H51O7 C30H58O13Na C16H35O3 C18H39O4

665.3724 489.3403 561.3251 363.3110 605.3513 407.3373 451.3635 649.3769 275.2586 319.2848

0.0 0.2 0.0 0.3 0.2 −0.2 0.2 −0.2 2.5 0.0

hydroxylated C12EO6 glucuronide hydroxylated C12EO6 C12EO4 glucuronide C12EO4 C12EO5 glucuronide C12EO5 C12EO6 C12EO6 glucuronide C12EO2 C12EO3

2.0 3.9 7.4 2.0 7.4 7.4 7.4 7.4 5.0 1.0

× 10−4 × 10−2 × 10−7 × 10−4 × 10−7 × 10−7 × 10−7 × 10−7 × 10−3 × 10−3

percent change after depuration in comparison to exposure levels (%) ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↑ ↓ ↓

40.1 23.5 50.9 29.6 94.1 26.5 98.8 9.7 99.2 52.5

All metabolites were detected in blood, liver, and gills after a 5 day exposure to either 146 or 553 μg L−1 C12EO6. Metabolite levels in blood continued to increase (↑) or decrease (↓) after a 3 day depuration period. Full details of metabolite identifications are given in Table S3 of the Supporting Information. RT = retention time on UPLC. bp values were calculated between the metabolite signals in blood of control and exposed (high dose, 553 μg L−1) fish; values in bold are significant after a Bonferroni correction for the false discovery rate. a

fish but were not present in controls, and these markers were identified as biotransformation products of the surfactant. Identification of Surfactant Metabolites. A variety of C12EO6 metabolites were identified from their accurate mass composition and elemental composition in blood, liver, and gill samples (see Table S3 of the Supporting Information). Many structures were present in +ESI as the [M + H]+ ion or the Na adduct, and their chemical composition was confirmed with an error between −0.2 and 2.5 ppm. Additional ions were also present as adducts of NH4 or K (data not shown). Surfactant metabolites were reanalysed by UPLC-QTOF-MS (CID) to obtain fragmentation data. Fragments arose mainly from the loss of the glucuronide moiety and cleavage of the ethoxylated chain, and structures that were conclusively identified are given in Table 1 (see the Supporting Information for an explanation of fragment data). Three different metabolic pathways were identified in the biotransformation of C12EO6 in the fish, namely, phase I and II biotransformation and shortening of the ethoxylated chain (see Figure S3 of the Supporting Information). The phase I biotransformation comprised hydroxylation (likely to be ω oxidation on the terminal methyl group) of the parent compound, a common metabolism of AEOs.24 The parent compound was also metabolized by shortening the number of ethoxymer groups in the ethylene oxide chain, and C12AEO metabolites between 2 and 5 EO units were identified. The mechanism of EO chain shortening in fish remains to be investigated. It may have arisen via β oxidation of the ethoxylate chain; however, chain-shortened carboxylated intermediates were not detected in this study. Shortening of the polyoxyethylene moiety through direct cleavage of ethoxylate units has been previously reported but only in bacteria and after central fission of the parent molecule to the ethylene glycol moiety.25 Many of the surfactant metabolites were further transformed by phase II conjugation, and glucuronide conjugates of C12EO6, hydroxylated C12EO6, C12EO5, and C12EO4 were identified in blood, liver, and gill extracts of exposed fish. All of the surfactant metabolites detected in liver and blood extracts were present in gill extracts during both the exposure and depuration periods, which suggested that this tissue may be a significant route of excretion of C12EO6 and its metabolites, alongside urine and feces. During depuration, the concentrations of all metabolites of C12EO6 in the blood decreased (Table 1), with

the exception of C12EO6 glucuronide and its hydroxylated product, indicating ongoing metabolism of the parent compound at the end of the exposure period. Identification of Endogenous Metabolites. The ions corresponding to the metabolites of C12EO6 were removed from the data sets of the treatments corresponding to the 5 day exposed fish, and the data were remodeled using PLS-DA (see Table S2 of the Supporting Information). Examination of the model parameters from gill extracts (SPE ethyl acetate and methanol fractions) and liver extracts (SPE methanol fraction only) revealed poor explained variation (R2Y < 0.43) and predictive ability (R2Y < 0.26). Further modeling of these data sets using OPLS-DA and examination of the “S” plots from control and low- or high-dose treatments did not show any discriminatory metabolites (data not shown). Hence, this finding indicated that surfactant metabolites were responsible for the class separation of these groups in previous models of the gill and liver (methanol fraction) extracts (see Table S2 of the Supporting Information). Examination of the chromatograms from these extracts also confirmed that surfactant metabolites were present in both the ethyl acetate and methanol fractions from SPE, although the majority of the signal of each metabolite was eluted in the ethyl acetate fraction. In contrast, extracts of blood and liver (ethyl acetate fraction from SPE) resulted in models with high explained variation and predictive ability (R2Y > 0.86, and Q2Y > 0.54), indicating that metabolites other than surfactant-derived compounds were influencing separation between the treatment classes. OPLSDA modeling of the data sets and “S” plot analyses revealed a number of endogenously derived metabolites, which were either up- or downregulated in surfactant-exposed fish (see Table S3 of the Supporting Information). The structures of many of these metabolites were identified from QTOF-MS fragmentation (see Table S4 of the Supporting Information), and their fold change in blood after exposure and depuration is given in Table 2. Four different bile salts were identified, whose levels significantly increased in blood of sole exposed to surfactant (Table 2). Bile salts can be divided into different classes depending upon their side-chain structure. In the sole, two C24 bile acids, taurocholic acid and hydroxytaurocholic acid, and two sulfated C27 bile alcohols were detected. Taurocholic acid 5206

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5.43 6.19 6.23 7.26 6.55

9.19 9.77 17.94

22.36

25.27

12.39

530.2780 547.2938 514.2838 531.2991 409.2227

363.2163 512.2972 820.5710

778.5579

832.6029

400.3425

C26H44NO8S C27H47O9S C26H44NO7S C27H47O8S C22H33O7 C21H31O5 C23H47NO9P C43H83NO11P C41H81NO10P C45H87NO10P C23H46NO4

− H]− − H]− − H]− − H]− + HCOO]−

[M + H]+ [M + HCOO]− [M + CH3COO]−

[M + CH3COO]−

[M + CH3COO]−

[M + H]+

[M [M [M [M [M

formula of ion

ion species

palmitoyl carnitine

PC (18:0/18:1)

PC (16:0/16:0)

cortisol lyso PC (14:0) PC (16:0/hydroxy 18:1)

hydroxytaurocholic acid scymnol sulfate? taurocholic acid cyprinol sulfate tetrahydrocortisone

chemical identity blood blood blood blood liver (low) liver (high) blood blood liver (low) liver (high) liver (low) liver (high) liver (low) liver (high) blood

↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↑ ↑ ↓ ↓ ↓ ↓ ↓

233 × 106 ± 790 × 105 ± 170 × 107 ± 350 × 106 ± 9.6 ± 0.3 55.7 ± 2.5 6.0 ± 0.1 2.3 ± 0.1 220 ± 3 471 ± 12 3.2 ± 0.1 6.8 ± 0.2 2.1 ± 0.1 7.1 ± 0.3 471 ± 29 4.9 7.7 3.4 6.9

× × × × 106 105 107 106

detected in blood (high dose) or apparent fold change after surfactant liver (low/high dose) exposure (mean ± SE)a 1.0 3.0 1.0 3.0 2.2 9.0 1.0 2.0 1.0 1.0 4.9 1.0 2.2 1.0 1.0

× × × × × × × × × × × × × × ×

10−6 10−6 10−6 10−6 10−5 10−6 10−6 10−4 10−6 10−5 10−5 10−6 10−5 10−6 10−6

p value after exposureb ↓ ↓ ↑ ↓ ↑ ↑ ↑ ↓ ↓ ↓ ↑ ↑ ↑ ↑ ↑ 11.8 5.1 36.4 57.0 74.7 5.3 75.2 5.7 70.2 55.6 59.3 74.4 29.5 88.3 99.2

percent change after depuration in comparison to exposure levels (%)

a

The mean ± standard error (SE) of the apparent fold change was calculated from a comparison of the UPLC-TOF-MS signal of the metabolite in extracts from fish exposed to C12EO6 compared to control fish held in clean water. Metabolite levels increased (↑) or decreased (↓) after surfactant exposure. RT = retention time on UPLC. PC = glycerophosphatidylcholine. Full details of metabolite identifications are given in Tables S3 and S4 of the Supporting Information. Cortisol, taurocholic acid, palmitoyl carnitine, and tetrahydrocortisone coeluted with authentic standards. Tetrahydrocortisone did not coelute with a standard of the isobaric metabolite 11β-17α-21-hydroxy-5β-pregnane-3,20-dione. Other compounds were annotated from the fragmentation patterns. Phospholipids were also detected in +ESI mode as sodium adducts. bThe p value was calculated between levels of the metabolite signal in control and exposed fish; values in bold are significant after a Bonferroni correction for the false discovery rate.

RT (min)

observed ion (m/z)

Table 2. Metabolite Perturbations Identified in Blood or Liver of Sole after a 5 Day Exposure to C12EO6 and Following a 3 Day Depuration

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by 471-fold after surfactant exposure, which provides further evidence of alteration of lipid metabolism. Palmitoylcarnitine is an ester drivative of carnitine, which facilitates the transfer of C14 long fatty acids from cytoplasm into mitochondria during the oxidation of fatty acids in the liver and other tissues. Its reduction may indicate depletion of C14 fatty acid or carnitine substrate, increased β oxidation of fatty acids, or inhibition of carnitine palmitoyl transferase, a target of many different xenobiotic drugs.35 It is not clear whether these observed alterations in lipid homesostasis are a direct result of C12EO6 exposure or a consequence of the high concentrations of plasma bile salts, which can induce similar metabolite changes.36 In addition, levels of an oxidized PC (16:0/hydroxy 18:1) in liver increased between 220−471-fold. The nonenzymatic free-radical-mediated peroxidation of lipids is associated with oxidative stress and production of ROS.37 AEO surfactants have been shown to induce oxidative stress genes in bacterial systems,38 and it is possible that metabolism of C12EO6 or other endogenously derived metabolites produced in response to surfactant exposure have increased free radical production in liver cells. After a 3 day depuration period, the levels of all metabolites, with the exception of taurocholic acid, showed some recovery back to pre-exposure concentrations. For instance, levels of cortisol and palmitoylcarnitine in the blood increased by 75− 99% after depuration compared to concentrations directly after exposure (Table 2). This indicated the potential of the organism to recover from short-term exposure to C12EO6 in response to the rapid clearance of the surfactant and most of its metabolites from the blood. This study reveals a number of effects of short-term AEO exposure on metabolite levels, including elevated plasma concentrations of bile acids, with the potential for downstream effects in nuclear receptor signaling and alteration of lipid and glucocorticoid homeostasis. Alteration of lipid homeostasis and increased levels of circulating bile salts are some of the metabolomic changes detected in fish exposed to a final treated wastewater effluent.15 The same study revealed that a wide variety of non-ionic and anionic surfactant molecules bioconcentrated in the fish after effluent exposure. This observation together with the present study that shows the same metabolite perturbations in response to AEO exposure raises the question as to whether the mixtures of surfactants present in aquatic environments pose a risk to fish health. Importantly, our study reveals marked changes in metabolite profiles in response to C12EO6 exposure, which, for the lowdose exposure, was 6 times lower than the reported NOEC concentrations for a similar AEO for reproduction and survival in fish.39,40 In these studies, a 30 day NOEC of 0.88 mg/L was reported for C12−13EO6 and the survival of juvenile fathead minnow (Pimephales promelas) and bluegill sunfish (Lepomus macrochirus).39 In further work, a NOEC of 0.73 mg/L was determined for the effect of C9−11EO6 on egg production and larval survival in fathead minnow.40 Assuming that similar NOEC values for effects of C12EO6 on egg production and survival apply to sole, then findings of metabolomic effects at concentrations below the NOEC may indicate possible other effects on fish physiology than these selected end points. A number of other studies have revealed metabolomic perturbations at exposures below the NOEC measured for classical or phenotypic end points.41,42 Together, these studies indicate the potential of metabolomic analysis to identify biochemical changes that may affect the long-term health of the organism

and its hydroxylated metabolite are commonly found in teleost fish and higher vertebrates.26 One of the C27 alcohols (m/z 531.2991) gave the same fragmentation pattern and relative retention time as that of cyprinol sulfate previously detected in the cyprinid fish Rutilus rutilus.15 Cyprinol sulfate is a predominant bile acid of cypriniformes but has been detected in other noncyprinid fish, such as sturgeon and trout.27,15 Analysis of the other sulfated C27 alcohol detected in sole revealed an empirical formula consistent with six hydroxy groups on the sterol nucleus and was tentatively identified as scymnol sulfate. This hexahydroxylated bile alcohol is normally the predominant bile acid of the elasmobranchii cartilaginous fish, such as sharks and rays, but, to date, has not been reported in teleost fish.26 After 5 days of surfactant exposure, the levels of the four bile salts in sole blood significantly increased by >106fold (Table 2). Following a 3 day depuration period (which resulted in elimination of 99% of the parent C12EO6 burden in the fish), levels of plasma taurocholic acid were still increasing, despite reductions in concentrations of the other 3 bile salts of between 5 and 50% in comparison to the levels immediately after surfactant exposure. These differences in response to depuration may reflect dissimilar mechanisms of synthesis or secretion of the individual bile acids. For instance, the increase in bile salts in the blood after surfactant exposure could be due to a number or combination of factors: alteration of hepatocyte cell membranes resulting in an increase in permeability and release of bile salts into the circulatory system, modulation of bile acid synthesis and metabolism, or alteration of the enterohepatic recirculation as a compensatory response to liver inflammation. The toxicological effects, if any, of such a high increase in the levels of circulating bile salts in the fish are currently unknown and remain to be evaluated. In vertebrates, some bile salts can act as signaling molecules and activate a number of nuclear receptors that play critical roles in the regulation of xenobiotic, lipid, and carbohydrate metabolism.28 In mammalian models, an increase in plasma concentrations of bile salts has previously been shown to cause liver damage and toxicity.16 It has been previously reported that the exposure of sole to surfactants enhances the production of reactive oxygen species (ROS), with subsequent damage to macromolecules and histological alterations.29 Conversely, some bile salts, such as scymnol, may have a hepatoprotective effect by scavenging free radicals and ROS formed during xenobiotic metabolism.30 The levels of some glucocorticoid steroids were also significantly reduced in response to surfactant exposure (Table 2). Levels of cortisol decreased by 6-fold in the blood, and levels of its major metabolite, tetrahydrocortisone, were also reduced by 10- and 56-fold in liver in response to low and high doses of surfactant, respectively. Cortisol has an important role in immune function and osmoregulation of teleost fish,31 and its release involves the coordinated activation of the hypothalamic−pituitary−interrenal (HPI) stress axis. Exposure to a number of chemicals, including metals,32 pharmaceuticals,33 and polycyclic aromatic hydrocarbons (PAHs),34 have also been previously reported to result in an attenuated release of cortisol. Further studies are needed to determine whether exposure to AEOs can affect end points relevant to immune function or cortisol- associated stress response in fish. Surfactant exposure in S. senegalensis also resulted in a 2−7fold reduction in levels of a number of phosphatidylcholine (PC) species, including lyso PC C14:0 in blood and C16:0/ 16:0 and C18:0/18:1 phospholipid species in liver (Table 2). Furthermore, blood levels of palmitoylcarnitine were reduced 5208

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in influent and effluent water samples and sludges of sewage treatment plants by a generic solid-phase extraction protocol. Analyst 2000, 125 (10), 1733−1739. (7) Belanger, S. E.; Dorn, P. B.; Toy, R.; Boeije, G.; Marshall, S. J.; Wind, T.; Van Compernolle, R.; Zeller, D. Aquatic risk assessment of alcohol ethoxylates in North America and Europe. Ecotoxicol. Environ. Saf. 2006, 64 (1), 85−99. (8) Lara-Martin, P. A.; Gonzalez-Mazo, E.; Brownawell, B. J. Multiresidue method for the analysis of synthetic surfactants and their degradation metabolites in aquatic systems by liquid chromatographytime-of-flight-mass spectrometry. J. Chromatogr., A 2011, 1218 (30), 4799−4807. (9) Petrovic, M.; Fernandez-Alba, A. R.; Borrull, F.; Marce, R. M.; Mazo, E. G.; Barcelo, D. Occurrence and distribution of nonionic surfactants, their degradation products, and linear alkylbenzene sulfonates in coastal waters and sediments in Spain. Environ. Toxicol. Chem. 2002, 21 (1), 37−46. (10) Lara-Martin, P. A.; Gomez-Parra, A.; Gonzalez-Mazo, E. Sources, transport and reactivity of anionic and non-ionic surfactants in several aquatic ecosystems in SW Spain: A comparative study. Environ. Pollut. 2008, 156 (1), 36−45. (11) van Compernolle, R.; McAvoy, D. C.; Sherren, A.; Wind, T.; Cano, M. L.; Belanger, S. E.; Dorn, P. B.; Kerr, K. M. Predicting the sorption of fatty alcohols and alcohol ethoxylates to effluent and receiving water solids. Ecotoxicol. Environ. Saf. 2006, 64 (1), 61−74. (12) Meng, Y. B.; Lin, B. L. A feed-forward artificial neural network for prediction of the aquatic ecotoxicity of alcohol ethoxylate. Ecotoxicol. Environ. Saf. 2008, 71 (1), 172−186. (13) Muller, M. T.; Zehnder, A. J. B.; Escher, B. I. Membrane toxicity of linear alcohol ethoxylates. Environ. Toxicol. Chem. 1999, 18 (12), 2767−2774. (14) Cardellini, P.; Ometto, L. Teratogenic and toxic effects of alcohol ethoxylate and alcohol ethoxy sulfate surfactants on Xenopus laevis embryos and tadpoles. Ecotoxicol. Environ. Saf. 2001, 48 (2), 170−177. (15) Al-Salhi, R.; Abdul-Sada, A.; Lange, A.; Tyler, C. R.; Hill, E. M. The xenometabolome and novel contaminant markers in fish exposed to a wastewater treatment works effluent. Environ. Sci. Technol. 2012, 46 (16), 9080−9088. (16) Want, E. J.; Coen, M.; Masson, P.; Keun, H. C.; Pearce, J. T. M.; Reily, M. D.; Robertson, D. G.; Rohde, C. M.; Holmes, E.; Lindon, J. C.; Plumb, R. S.; Nicholson, J. K. Ultra performance liquid chromatography-mass spectrometry profiling of bile acid metabolites in biofluids: Application to experimental toxicology studies. Anal. Chem. 2010, 82 (12), 5282−5289. (17) Bedair, M.; Sumner, L. W. Current and emerging massspectrometry technologies for metabolomics. TrAC, Trends Anal. Chem. 2008, 27 (3), 238−250. (18) Viant, M. R.; Sommer, U. Mass spectrometry based environmental metabolomics: A primer and review. Metabolomics 2012, 9, S144−S158. (19) Committee on Effectiveness of Oil Spill Dispersants. National Research Council. Using Oil Spill Dispersants on the Sea; National Academy Press: Washington, D.C., 1989. (20) Alvarez-Munoz, D.; Gomez-Parra, A.; Gonzalez-Mazo, E. Influence of the molecular structure and exposure concentration on the uptake and elimination kinetics, bioconcentration, and biotransformation of anionic and nonionic surfactants. Environ. Toxicol. Chem. 2010, 29 (8), 1727−1734. (21) Lara-Martin, P. A.; Gomez-Parra, A.; Gonzalez-Mazo, E. Development of a method for the simultaneous analysis of anionic and non-ionic surfactants and their carboxylated metabolites in environmental samples by mixed-mode liquid chromatography-mass spectrometry. J. Chromatogr., A 2006, 1137 (2), 188−197. (22) Wiklund, S.; Johansson, E.; Sjostrom, L.; Mellerowicz, E. J.; Edlund, U.; Shockcor, J. P.; Gottfries, J.; Moritz, T.; Trygg, J. Visualization of GC/TOF-MS-based metabolomics data for identification of biochemically interesting compounds using OPLS class models. Anal. Chem. 2008, 80 (1), 115−122.

and the need for health end points linked to the relevant mode of action studies.43,44 Conversely, it is possible that the metabolomic effects detected in our study may not have resulted in deleterious changes in fish physiology, and certainly the levels of most metabolites, with the exception of taurocholic acid, showed a trend of returning to pre-exposure levels during a short depuration period. However, when the intensity of changes in concentrations of many of the metabolites is taken into account, the possibility of physiological effects arising from prolonged exposure to this surfactant cannot be excluded.

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ASSOCIATED CONTENT

S Supporting Information *

Materials and methods, results, BCF values in liver, gills, and blood of Senegalese sole exposed to C12EO6 for 5 days and the percent depuration after a further 3 days in clean water (Table S1), PCA score plots of the chemical profiles of (A) blood and (B) liver of S. senegalensis exposed to C12EO6 (Figure S1), performance parameters of multivariate PLS-DA models for datasets from sole exposed to control, low, or high doses of C12EO6 for 5 days (Table S2), example of an OPLS-DA of the chemical profile of blood from S. senegalensis exposed to C12EO6 (Figure S2), surfactant and endogenously derived metabolites detected in blood, liver, and gill samples of S. senegalensis exposed to C12EO6 (Table S3), biotransformation pathways of C12EO6 in S. senegalensis (Figure S3), and UPLCQTOF-MS (CID) analyses of endogenously derived metabolites detected in sole after exposure to C12EO6 (Table S4). This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +44-1273-678382. Fax: 44-1273-877586. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially funded by the Spanish petrochemical company PETRESA (reference OT 143/02) and the European Regional Development Fund in the framework of the INTERREG IV A France (Channel)−England Programme (DIESE Project).

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REFERENCES

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