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In Vivo Tracing Uptake and Elimination of Organic Pesticides in Fish Muscle Jianqiao Xu, Junpeng Luo, Jingwen Ruan, Fang Zhu, Tiangang Luan, Hong Liu, Ruifen Jiang, and Gangfeng Ouyang* MOE Key Laboratory of Aquatic Product Safety/KLGHEI of Environment and Energy Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, 510275 Guangdong, China S Supporting Information *

ABSTRACT: Bioconcentration factors (BCFs) measured in the laboratory are important for characterizing the bioaccumulative properties of chemicals entering the environment, especially the potential persistent organic pollutants (POPs), which can pose serious adverse effects on ecosystem and human health. Traditional lethal analysis methods are timeconsuming and sacrifice too many experimental animals. In the present study, in vivo solid-phase microextraction (SPME) was introduced to trace the uptake and elimination processes of pesticides in living fish. BCFs and elimination kinetic coefficients of the pesticides were recorded therein. Moreover, the metabolism of fenthion was also traced with in vivo SPME. The method was time-efficient and laborsaving. Much fewer experimental animals were sacrificed during the tracing. In general, this study opened up an opportunity to measure BCFs cheaply in laboratories for the registering of emerging POPs and inspecting of suspected POPs, as well as demonstrated the potential application of in vivo SPME in the study of toxicokinetics of pollutants.

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INTRODUCTION Persistent organic pollutants (POPs) have aroused worldwide concern because of their prolonged and serious adverse effects on ecosystem and human health.1−8 As reported previously, POPs impart tremendous threats on human beings’ nervous system, immune system, endocrine, reproduce, and development; and POPs are probably carcinogenic.1−4 Moreover, due to the long-range transport properties, POPs are omnipresent on the earth even in the most remote districts, where biota are under the stress of POPs.5−8 Unfortunately, the scope of POPs is still extending nowadays within the commercial chemicals. In 2009, the perfluorinated compounds were labeled as emerging POPs. As the exact number of potential POPs within the existing commercial chemicals is still unknown,9,10 quantitative structure−activity relationship (QSAR) is widely adopted to screen potential POPs within chemicals in commerce.9−13 For the final ascertaining of the potential POPs and registering of the ascertained POPs in regulation frameworks, experimental studies such as detection in the environment, measuring BCFs or BAFs, and toxicity testing are also inevitable. As we well know, experiment derived bioconcentration factors (BCFs) based on standardized guideline such as OECD 305 are widely accepted to characterize the bioaccumulative features of chemicals including the suspected POPs;14 but, on the other hand, measurement conducting remains a challenge because it can be much consumptive of experimental animal, human labor, and money expense. © 2014 American Chemical Society

Sample preparation in a typical analytical process is considered to be the most time-consuming step.15 In addition, animal sacrifice with traditional lethal sampling and tedious cleanup steps after sampling raise the money expense for bioanalysis.16 Conversely, the novel nonlethal bioanalytical methods without tedious sample preparation procedures can be the solutions, such as in vivo fluorescence mapping,17 biosensors,18 and microdialysis,19 as well as solid-phase microextraction (SPME).16,20 SPME simplifies the sample preparation procedure by integrating sampling, isolation, and preconcentration into one step,21,22 and it was soon employed for in vivo analysis after its conception because of its minimized morphology and its slight invasiveness to living systems.16,23 Although SPME is the latest appearing in vivo analysis method, it is thought to be a competitive alternative to other in vivo analysis methods for its feasibility to a wide variety of analytes with different polarities (an impressive example is its potential application in metabolomics studies),24,25 its simple analysis procedures,20 and probably the simplest and cheapest devices.20,26 Moreover, pre-equilibrium SPME was developed to cut down the sampling durations to improve analysis efficiency, catch instantaneous Received: Revised: Accepted: Published: 8012

February 21, 2014 June 13, 2014 June 16, 2014 June 16, 2014 dx.doi.org/10.1021/es5009032 | Environ. Sci. Technol. 2014, 48, 8012−8020

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Figure 1. In vivo SPME sampling of freely swimming fish (A) with the custom-made 44 μm PDMS fiber (B) or the custom-made 165 μm PDMS fiber (C).

species, and circumvent the necessity of reaching equilibrium (especially in unstable systems).16,24,25 In the present study, a simple and time-efficient in vivo SPME method was proposed to trace the uptake and elimination kinetics of organic pollutants in fish dorsal-epaxial muscle. With much less fish sacrifice, the uptake and elimination of two groups of pesticides, organochlorine pesticides (OCPs, previously documented to be highly bioaccumulative and persistent in biota) and organophosphorus pesticides (OPPs, previously documented to be slightly bioaccumulative and persistent in biota), were both traced in tilapias (Oreochromis mossambicus) and pomfrets (Piaractus brachypomus). Notably, the BCFs determined with the new method were much close to the database values. Moreover, as metabolism is one of the major elimination pathways in fish, and it is crucial for understanding the toxicity of pollutants, the method was also used to trace the metabolism of fenthion in individual tilapias in this study.

Helixmark (Carpinteria, CA, USA), and stainless steel wires (127 and 480 μm in diameter, medical grade) were purchased from Small Parts (Miami Lakes, FL, USA). Fiber Preparation. Two kinds of PDMS fibers for GC-MS detection (44 μm in thickness) and LC-MS/MS detection (165 μm in thickness) were made in the laboratory to replace the expensive commercial fibers. Custom-made 44 μm thick PDMS fibers were prepared as follows: a piece of stainless steel wire with a length of 3 cm was cut and sonicated in acetone and deionized water for 15 min successively, to remove the impurity. Before a piece of well-cut PDMS tubing (1.0 cm) was worn on the stainless steel wire, the stainless steel wire was dried at room temperature and coated with a thin layer of epoxy glue for about 1 cm at one end. The custom-made fiber was dried in the air at room temperature for 24 h until the glue was solidified completely. The prepared PDMS fibers were conditioned in nitrogen flow at 250 °C for 15 min prior to use. The thicker fibers were prepared according to ref 26 without any further modification and conditioned in methanol for 1 h prior to use. In Vivo SPME. Fish was anaesthetized in dechlorinated municipal water containing 0.1% eugenol until loss of vertical equilibrium. The dorsal-epaxial muscle of the fish was pierced with a 26 gauge hypodermic needle to a depth of approximately 1.4 cm (or a 22 gauge hypodermic needle for deploying the thicker fibers). Subsequently, the needle was removed, and the custom-made SPME fiber was deployed in the punched hole. Two parallel samplings on both sides of the dorsal-epaxial muscle were conducted at each sampling point for mutual reference. The fish was then placed in fresh water to resume vertical balance (as shown in Figure 1A). At the end of the sampling interval, the fish was reanaesthetized, and the fiber was removed. The total extraction duration was controlled to be 10 min (thinner fiber) or 20 min (thicker fibers). The fiber was then rinsed with deionized water and dried with a Kimwipe tissue. The thinner fibers were assembled to a recycled SPME fiber assembly for being directly introduced to GC-MS for analysis. While the thicker fibers were immersed in 100 μL of methanol for desorption for 1 h on vibration (600 rpm), 50 μL of fenitrothon solution (100 ng·mL−1) was added as internal standard to calibrate the ionization efficiency before LC-MS/ MS analysis.

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MATERIALS AND METHODS Animals. All the immature tilapias and pomfrets (length 13 ± 1 cm, weight 41 ± 2 g) used were purchased from a local fishery. Tilapias and pomfrets were chosen because they were two common edible fish species in Guangdong Province of China; inspecting the bioaccumulative properties of pesticides in these two species was of great importance for guaranteeing the food safety of local citizens. Tilapias and pomfrets were kept in aerated 50 L aquaria with 30 L of dechlorinated tap water for 2 weeks before experiment. The water quality (pH, dissolved oxygen, and temperature) was monitored daily. All the animal experiments were approved by the Animal Ethical and Welfare Committee of Sun Yat-sen University. Chemicals and Supplies. Propetamphos, fenthion, and quinalphos were purchased from Accustandard (New Haven, CT, USA). Hexachlorobenzene (HCB), heptachlor, and aldrin were purchased from Dr. Ehrenstorfer (Augsburg, Germany), and cis-chlordane and trans-chlordane were purchased from Cerilliant (Round Rock, TX, USA). HPLC grade methanol was purchased from Anpel (Shanghai, China). Two kinds of polydimethylsiloxane (PDMS) tubings (i.d. 0.212 mm, o.d. 0.40 mm and i.d. 0.31 mm, o.d. 0.64 mm) were purchased from 8013

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Table 1. Sampling Rates (Mean ± SD, μg·min−1; n = 6) of OPPs and OCPs in Tilapias and Pomfrets with 44 μm PDMS Fibers and the Physicochemical Parameters of the Analytesa Rs (tilapia) propetamphos fenthion quinalphos HCB heptachlor aldrin trans-chlordane cis-chlordane

378.6 159.5 478.7 387.4 122.5 176.4 96.6 130.2

± ± ± ± ± ± ± ±

46.4 7.8 135.5 95.8 9.6 40.7 24.0 16.5

Rs (pomfret)

LogPow

solubility (mg·L−1)

± ± ± ± ± ± ± ±

2.51 4.08 3.04 5.86 5.86 6.75 6.26 6.26

110 4.2 17.8 0.005 0.056 0.027 0.1* 0.1*

842.9 377.9 568.8 60.5 213.7 39.3 51.5 48.9

229.0 73.6 117.2 8.5 75.1 9.3 8.4 7.2

a

The octanol−water partition coefficients (LogPow) of the analytes were estimated with EpiWin (version 1.67). The solubility of the analytes was referred to the database of University of Hertfordshire.

180 °C at a rate of 25 °C·min−1, held at 180 °C for 1 min, then increased to 230 °C at a rate of 3 °C·min−1, held at 230 °C for 2 min; increased to 250 °C at a rate of 10 °C·min−1. The total run time was 22 min. Ions chosen for quantification were as follows: propetamphos m/z 138, 194, and 236; fenthion m/z 153, 169, and 278; quinalphos m/z 146, 157, and 298; HCB m/ z 142, 214, 249, and 284; heptachlor m/z 100, 272, and 337; aldrin m/z 66, 263, and 293; cis-chlordane m/z 237, 272, and 373; trans-chlordane m/z 237, 272, and 373. HPLC/MS/MS analysis was based on an Agilent 1260 high performance liquid chromatography (Agilent Technologies, CA, USA) coupled to an AB Sciex Triple Quad 4500 triplequadrupole tandem mass spectrometer (MS/MS) using an ESI source in positive ion mode (Applied Biosystems/MDS Sciex, MA, USA). A Zorbax SB-C18 column (2.1 mm × 150 mm, 3.6 μm, Agilent Technologies, CA, USA) was used for separation. An aqueous solution containing 10 mM ammonium formate (A) and methanol (B) were used for gradient elution. The flow rate was set at 300 μL·min−1, and an 80% A gradient was applied for the first 2 min. This was ramped to 10% A in 0.5 min, held for 8.5 min, then retained to 80% A in 0.1 min, held for another 10 min for reconditioning. Transitions monitored were as follows: fenthion m/z 279.1/246.9; fenthoxon m/z 263.0/230.9; fenthion sulfoxide m/z 294.9/279.8; fenthoxon sulfoxide m/z 279.0/263.9; fenitrothion, m/z 278.0/245.9. Quality Assurance and Quality Control. Two grams of blank fish muscle samples was spiked at two different concentrations (100 ng·g−1 and 500 ng·g−1) to evaluate the performance of SPME method in terms of precision and recovery (extraction efficiency). The RSDs ranged from 11.8% to 18.9% for OCPs and 16.2% to 21.4% for OPPs (n = 6). The amounts extracted from the spiked muscle ranged from 0.3‰ to 0.5‰ for OCPs and 0.8‰ to 1.4‰ for OPPs.

Liquid Extraction. Liquid extraction methods derived from China national standards (GB/T 5009.20-2003, GB/T 5009.162-2003) were used to determine the concentrations of OPPs and OCPs in fish muscle; for details, see SI Text. Uptake and Elimination Tracing. Elimination Tracing. Several tilapias or pomfrets were kept in 30 L of dechlorinated tap water spiked with OPPs or OCPs stock solutions (1000 mg· L −1 , dissolved in methanol) for 24 h. The nominal concentrations were 5 μg·L−1 for each OCP or 50 μg·L−1 for each OPP. At the end of exposure, the pollutant concentrations in fish muscle were measured with in vivo SPME, and then the fish were transferred to 30 L of nonspiked dechlorinated tap water for in vivo elimination tracing. Water was changed with fresh water daily. The elimination of OCPs was traced for 15 days, and OCP concentrations in fish muscle were measured every fifth day. The elimination of OPPs was traced for 12 h, OPP concentrations in fish muscle were measured after being taken out from the spiked water for 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 h. Uptake Tracing. Dechlorinated tap water spiked with OPPs or OCPs stock solutions (1000 mg·L−1, dissolved in methanol) was used for exposure. The nominal concentrations at the beginning were 0.5 μg·L−1 for each OCP or 50 μg·L−1 for each OPP. Five tilapias or five pomfrets were kept in 30 L of the spiked tap water for uptake tracing. In order to keep the aquatic concentrations steady, two-thirds of the polluted water (20 L out of 30 L) was changed with fresh water and then spiked with two-thirds of the initial spiking amounts, twice a day. OCPs in fish muscle were measured with in vivo SPME every fifth day, and OPPs in fish muscle were measured with in vivo SPME every second day. The aquatic concentrations were also measured with SPME at the same time the muscle concentrations were measured. The external standard calibration method was used for quantification. Instrumental Analysis. GC/MS analysis was performed on an Agilent 6890N gas chromatograph coupled to an Agilent 5975 mass spectrometer with an electron ionization (EI) source (Agilent Technologies, CA, USA). A HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent Technologies, CA, USA) was used for separation. Ultrapure helium was employed as the carrier gas. The inlet temperature was 250 °C, and the oven temperature programs were as follows. For OPPs, the column temperature was initially 100 °C for 2 min, ramped to 180 °C at a rate of 30 °C ·min−1, and held at 180 °C for 2 min, then increased to 200 °C at a rate of 3 °C·min−1, held at 200 °C for 4 min, increased to 250 °C at a rate of 30 °C ·min−1, held for 3 min. The total run time was 26.7 min. For OCPs, the column temperature was initially 80 °C, held for 1 min, increased to

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RESULTS AND DISCUSSION Determination of Sampling Rates of SPME Fibers in Living Fish. Pre-equilibrium SPME sampling of living fish was calibrated with the sampling-rate calibration method.26 Prior to the dynamic tracing, the sampling rates of the SPME fiber from sample matrix were preliminarily calculated according to the following equation26 n Rs = Cs·t (1) where Rs was the defined sampling rate, n was the extracted amount of the analyte in a fiber determined by injecting a series of standard solutions into the analytical instrument, Cs was the concentration in sample matrix (determined with liquid 8014

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Figure 2. Elimination tracing of OPPs and OCPs in the dorsal-epaxial muscle of tilapias and pomfrets. (a) Elimination of OCPs in dorsal-epaxial muscle of tilapias (n = 3), (b) elimination of OCPs in dorsal-epaxial muscle of pomfrets (n = 4), (c) elimination of OPPs in the dorsal-epaxial muscle of tilapias (n = 3), (d) elimination of OPPs in the dorsal-epaxial muscle of pomfrets (n = 5), the inset was a magnified view of the elimination curve of propetamphos. Error bars were the standard deviations.

curves or n-t curves.31 The in vitro sampling rates were significantly different from the in vivo ones (Table S2 and Table 1). For in vivo analysis, the in vivo sampling rates were preferred, as both the determination of sampling rates and the quantitative analysis were conducted in living fish. Tracing Elimination of OPPs and OCPs in Living Fish. Pollutants in environmental waters can be taken by fish via dietary and gill. Simultaneously, the accumulated pollutants eliminate mainly through metabolism and/or fecal egestion.32 When the uptake rates of the pollutants are equal to the elimination rates, the steady state of the pollutants between fish and water is reached. Theoretically, the time to reach steady state is determined by the elimination kinetics based on the first order uptake and elimination kinetics, if supposing aquatic concentrations steady.32 As shown in eq 2

extraction (LE) in the present study), and t was the extraction duration. Theoretically, Rs is a function of the physicochemical properties of the sample matrix, fiber coating, and analyte, as well as the fiber dimension and matrix temperature.27 It was proven that the sampling rates were independent from Cs and t at the linear regime of extraction as supposed (Figures S1 and S2). In the present study, the sampling-rate calibration method also permitted the extraction durations as short as 10 or 20 min. Figure 1 is the picture of in vivo SPME sampling of the freely swimming fish (Figure 1A) with the custom-made PDMS fibers (Figure 1B and 1C). The in vivo sampling rates of three OPPs and five OCPs in both tilapias and pomfrets with the thinner PDMS fibers are listed in Table 1. Figure S3 presented equivalent analysis results between sampling-rate calibrated SPME (SR-SPME) and LE, which verified the feasibility and accuracy of the sampling-rate calibration method for quantification. The detection limits of OPPs and OCPs in living tilapias and pomfrets with the SPMEGC-MS method were presented in Table S1, which were between 1.8 and 15.5 ng/g. There was a plausible relationship between the sampling rates and the solubility of the two groups of pesticides, as the sampling rates of OPPs were generally larger than OCPs. However, the sampling rates did not positively correspond to their solubility among the three OPPs as well as the five OCPs (Table 1). The relationship between sampling rates and LogPow was generally consistent with the previous studies of passive sampling in aquatic matrices.28−30 Chemicals with higher LogPow were extracted by PDMS fibers more slowly. However, the sampling rates of HCB and aldrin in tilapias seemed to be too large to be fit to this relationship (Table 1). In vitro sampling rates could be derived from the slopes of the n-Cs

Cs =

Cwku (1 − e−ket ) ke

(2)

where Cs and Cw are the analyte concentrations in fish body and water respectively, and ku and ke are the uptake rate coefficient and the elimination rate coefficient, respectively. Steady state is reached when e−ket becomes approximately zero. Chemicals with higher ke reach steady state faster. In addition, the persistence of pollutants in living biota can be also characterized with the elimination rates. In the present study, tilapias and pomfrets were kept in OPPs polluted water, or OCPs polluted water in laboratory, for 24 h. Subsequently, the polluted tilapias and pomfrets were transferred to clean water for 12 h elimination tracing. Using SRSPME, the concentrations of OPPs and OCPs in fish dorsalepaxial muscle were plotted against tracing time in Figure 2. 8015

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Tracing Uptake of OPPs and OCPs in Living Fish. Tilapias and pomfrets were kept in OPPs or OCPs polluted water to tracing the uptake of the pollutants in fish dorsalepaxial muscle. The aquatic concentrations were also measured with SPME at the same time the muscle concentrations were measured (Figure 3). Unlike the tracing of the elimination processes, the sampling intervals during the uptake tracing of OPPs were long enough for reaching steady state, which was also demonstrated by the phenomena that the concentrations of OPPs in fish muscle closely followed the fluctuations of the aquatic concentrations (Figure 3c and 3d). The aquatic concentrations of OCPs fluctuated during the uptake tracing, while the concentrations of HCB seemed to increase along the tracing course in fish dorsal-epaxial muscle, and other OCPs increased in the first 15 days and also fluctuated in the last 10 days (Figure 3a and 3b), which corresponded to the results of elimination tracing that the steady state of OCPs might not be reached in the first 15 days. Previous endeavors were also focused on continuous inspecting of endogenous and exogenous substances in living fish with any other techniques, i.e. collection of secretion, excretions, or biopsy samples; implantable biosensors and microdialysis.20 However, these techniques might not be as simple, time-efficient, or effective as the presently applied in vivo SPME technique. First, collection of specific samples from living fish to indicate contaminant levels in fish body might impart severe invasiveness on fish, and sample preparation was often inevitable and remained to be the traditional methods.33,34 Implantable biosensors were only suitable for the electrochemically active analytes, and it was not easy to prepare a highly sensitive and selective biosensor.35 For microdialysis, the nonpolar pollutants were likely to be partitioned into the semipermeable membranes rather than the dialysates, and long-term tracing might be troublesome due to the need of syringe pump and tubings.20 BCF Values. According to the rapid elimination kinetics of the OPPs, the steady state of OPPs might be reached at each sampling point during the uptake tracing course. Therefore, the BCF values of OPPs were figured out as the mean values of all sampling points (Table 2). Nevertheless, the steady state of OCPs might not be reached at the end of uptake tracing according to the tardive elimination kinetics of OCPs. Actually, steady state is hard to be fully reached for hyperhydrophobic compounds; in the OECD 305 guideline, 80% steady state is requested.14 The ratios of OCP concentrations in fish and concentrations in water on the 20th day were recorded as the pseudo-BCFs of OCPs (Table 2), where 60−90% steady state might be reached except for HCB according to the first order elimination kinetic model. However, mathematically, the difference between the BCFs figured out under 80% steady state and 60% steady state is only 0.2 logarithmic unit, which could not be too significant to adopt the pseudo-BCFs to characterize the bioaccumulative properties of these OCPs. The last time point was excluded because only one tilapia and two pomfrets survived on the 25th day probably due to the toxic effects. The BCFs figured out were all at comparable levels to the previously recorded values (except for propetamphos), although different fish species and different tissues might be inspected (Table 2). The relative standard derivatives of the BCFs ranged from 16.7% to 51.4% (determined with five fish). In most cases, pollutant concentrations in whole fish body were used to calculate BCFs; however, concentrations in muscle

During the 12 h tracing of OPPs, propetamphos, fenthion, and quinalphos could not be detected in tilapias after 3 h, 8 h, and 2 h, respectively (Figure 2c); while all three OPPs in pomfrets were still above the determination limits at low levels at the last sampling point (Figure 2d). For both species, more than 80% of each accumulated OPP was eliminated during the first 2 h in the dorsal-epaxial muscle (Figure 2c and 2d). By contrary, the elimination of OCPs was relatively tardive. No more than 60% of the initially accumulated OCPs were eliminated in the first 5 days of elimination tracing, and the concentrations of HCB in tilapias and pomfrets were nearly unchanged during the tracing (Figure 2a and 2b). The analytical process should be fast enough to catch the rapid elimination processes of OPPs, while the traditional lethal analysis methods such as LE and SPE were quite tardive;15 important information about the fast changing species might be missed in the tardive processes of sample preparation.16 By contrast, the time-efficient SR-SPME with a sampling duration of only 10 min sufficiently identified the rapid elimination occurring within 2 h. Moreover, OPPs were isolated from the fish muscle, which guaranteed accurate determination of fast changing species without additional enzyme quenching steps. In the case of a first order elimination kinetics, the elimination rate coefficients of OPPs and OCPs could be estimated by plotting the natural logarithm concentrations against the tracing time, and the slopes are the elimination rate coefficients. Data of all sampling points were used for modeling (Table 2); linear correlation coefficients (R2) were in the range Table 2. ke and BCFs of the OPPs and OCPsf tilapia propetamphos fenthion quinalphos HCB heptachlor aldrin trans-chlordane cis-chlordane

pomfret

ke

LogBCFa

ke

LogBCFa

LogBCFb

0.82 0.29 0.99

0.7 1.6 0.8 4.0 3.7 4.1 3.8 3.8

1.04 0.46 0.88

0.7 1.7 1.6 3.6 3.4 4.1 3.7 3.8

2.2d 1.0−2.3c 1.5d 3.1−4.5 3.7−4.2 3.2−4.3e 3.6−4.6 3.6−4.6

0.06 0.11 0.07 0.05

0.07 0.12 0.07 0.07

a

BCFs obtained in the present study. bBCFs from the database of the US Environmental Protection Agency. cBCFs from the database of the University of Hertfordshire. dBCF from the database of the US National Library of Medicine. eBCF from the database of the US Agency for Toxic Substances & Disease Registry. The ke value for HCB in tilapias was absent because no apparent elimination of HCB was observed in tilapias and pomfrets. fThe units of ke for OPPs and OCPs were h−1 and d−1, respectively.

of 0.6 to 0.88. The rate coefficients of OCPs were much smaller than those of OPPs, which was consistent with the previous conclusions that OCPs were much more persistent in biota than OPPs. Accordingly, the elimination kinetics indicated that 80% steady state (it is required BCFs should be calculated under at least 80% steady state in the OECD 305 guideline) of OPPs between fish muscle and water would be reached several hours after the fish being kept in spiked water at steady concentrations. On the other hand, the distribution of OCPs between fish dorsal-epaxial muscle and water would take more than 10 days to reach steady state, so the uptake tracing should cover a longer duration. 8016

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Figure 3. Uptake tracing of OPPs and OCPs in the dorsal-epaxial muscle of tilapias and pomfrets. (a) Uptake of OCPs in dorsal-epaxial muscle of tilapias (n = 5), (b) uptake of OCPs in dorsal-epaxial muscle of pomfrets (n = 5), (c) uptake of OPPs in dorsal-epaxial muscle of tilapias (n = 5), (d) uptake of OPPs in dorsal-epaxial muscle of pomfrets (n = 5). Error bars were the standard deviations (SDs), while the SDs of water analysis were too smaller to be presented in the figures.

and LC-MS/MS detection.26 The sampling rates of fenthion and its metabolites with the thicker fiber were listed in Table S3. Being kept in fenthion polluted water (initial 50 μg·L−1), fenthoxon, fenthion sulfoxide, and fenthoxon sulfoxide were detected, while fenthion sulfone and fenthoxon sulfone could not be detected in the dorsal-epaxial muscle of tilapias. The enzymes in tilapia might not be able to oxidize the sulfoxides to sulfones. The metabolism of fenthion in the other two fish species in the previous reports was consistent with the present study; fenthion sulfone and fenthoxon sulfone were not detected in homogenized whole goldfish (Carassius auratus) after in vivo metabolism37 and were also not detected in specific tissues of rainbow trout (Oncorhynchus mykiss) after in vitro metabolism.39 However, fenthoxon sulfoxide was also not detected in homogenized whole goldfish37 and could only be detected in liver microsomes after rainbow trout was acclimated to hypersaline environments.39 The aquatic concentrations of fenthion were also recorded (Figure S4) each time the fish concentrations measured during the first 6 h (uptake phase). The tilapias were then kept in fresh water for another 6 h (elimination phase). The distribution of fenthion between fish

were used in this study. Arnot and Gobas argued that lipid contents should be presented to improve the data quality, if BCFs were measured with specific tissues.36 In this study, lipid contents in dorsal-epaxial muscle of pomfrets and tilapias were (1.92 ± 0.10)% (n = 3) and (0.90 ± 0.04)% (n = 3), respectively. The BCF of each pesticide was closed between tilapias and pomfrets (Table 2). Besides, there was a clear relationship between the obtained BCFs and estimated LogPow. Chemicals with higher LogPow values presented higher BCF values. It was reasonable that more hydrophobic chemicals were more bioaccumulative in fish. From this aspect, the BCFs of propetamphos in tilapias and pomfrets reported in this study seemed to be more reasonable than the database value. Metabolism of Fenthion in Living Tilapias. Metabolism is one of the major elimination paths of pollutants in living biota,32 and the toxicology of the pollutants in biota was also related to the biotransformation kinetics of the parent substances and the toxicity of the parent substances and metabolites.37,38 In the present study, thicker PDMS fibers (165 μm) were used to extract fenthion and the metabolites in three individual tilapias, which were followed by solvent desorption 8017

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Figure 4. Metabolism of fenthion in a tilapia, the left sections of the figure divided by the vertical dash line represented the uptake phase, and the right sections represented the elimination phase. (a) exhibited both the parent substance and metabolites, while (b) exhibited the metabolites specifically for clear version.

and water nearly reached steady state after the first 6 h, and the BCFs of fenthion in three tilapias (values in the sixth hour) were only a little smaller than the value remarked above (Table 2 and Table S4). Among the three detected metabolites, it was observed from Figure 4 and Figure S5 that fenthion sulfoxide seemed to be the most persistent in fish dorsal-epaxial muscle apparently, followed by fenthoxon sulfoxide, and then fenthoxon. Fenthoxon could not be detected before the tracing ending (Figure 4 and Figure S5). The elimination kinetics of fenthion traced with the thicker fibers also corresponded well to the former one with the thinner fibers (Table S4 and Table 2). As OPPs eliminated rapidly in fish dorsal-epaxial muscle, in fact, the metabolism of fenthion was also very fast. Therefore, accurate measurement of the concentrations of fenthion and the metabolites in living fish should be guaranteed by quick quenching of enzyme degradation, otherwise, biotransformation might be still undergoing during sample preparation. SPME can quench enzyme degradation by isolating the analytes from the sample matrices at the same time of sampling.24,25 Application Considerations. As emerging POPs such as perfluorinated compounds and polybrominated diphenyl ethers are burgeoning along with human activities,9−13 measurement of BCFs in laboratory is necessary for final registering of the emerging POPs and inspecting the suspected POPs. However, traditional lethal analytical methods consume too much physical labor and too many experimental animals, therefore raising the money expense. Moreover, reducing animal sacrifice is also ethically preferred in animal experiments. In the present study, in vivo SPME technique was adopted to study the bioaccumulative properties of both persistent OCPs and less persistent OPPs in two freshwater fish species. Using in vivo SPME, no fish was killed for sample preparation during both the uptake and elimination tracing. However, in OECD 305, fish need to be killed at each sampling point. The sample preparation procedure with in vivo SPME was also much simpler than any other sample preparation methods, such as liquid extraction and solid phase extraction. Moreover, the cost of in vivo SPME was also dramatically cut down by using the custom-made fibers. Clearly, the present method is promising for recording BCFs and half-lives in laboratories. Furthermore, this method can also be used to trace the bioaccumulation of pollutants in wild animals (not only the aquatic but also the terrestrial animals) for environmental assessment along a certain time period. Even though in vivo SPME has long been found to be a simple and cheap method for wild fish analysis,26

it has not been applied to trace the variation of pollutants in wild animals over a certain time period. In addition, the present study also probably opened up an opportunity to study toxicokinetics cheaply and simply, as the metabolism of fenthion in fish dorsal-epaxial muscle during uptake and elimination phases was caught in individual fish. Individual differences could be excluded with in vivo SPME. However, the recent SPME sampling still covered certain durations, which was actually not suitable for tracing the more rapid biological processes.40 Fortunately, optimizing the configuration of SPME sampler can be a promising supplementary choice to shorten the sampling duration other than using the pre-equilibrium calibration methods, which also conserve high sensitivity.40,41

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ASSOCIATED CONTENT

S Supporting Information *

Liquid extraction method, Figures S1−S5, and Tables S1−S4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-20-84110845. E-mail: [email protected]. edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (21037001, 21225731), the NSF of Guangdong Province (S2013030013474), and the Research Fund for the Doctoral Program of Higher Education of China (20120171110003).

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