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Dec 22, 2011 - ever, biota samples like fish require extensive sample clean-up prior to analysis ... the US Environ- mental Protection Agency (EPA) advisory methods on frequency .... 2.2% and 8.0% while the recoveries for the OCPs were good. RSDs ..... 405–416. Hong, J., Kima, H.Y., Kim, D.G., Seo, J., Kimb, K.J., 2004.
Chemosphere 86 (2012) 1066–1071

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Simultaneous determination of organochlorine pesticides and bisphenol A in edible marine biota by GC–MS V.A. Santhi, T. Hairin, A.M. Mustafa ⇑ Shimadzu-UMMC Centre of Xenobiotic Studies, Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e

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Article history: Received 27 May 2011 Received in revised form 21 November 2011 Accepted 21 November 2011 Available online 22 December 2011 Keywords: Organochlorine pesticides Endosulfan Bisphenol A Marine biota Coastal waters GC–MS

a b s t r a c t A study to assess the level of organochlorine pesticides (OCPs) and bisphenol A (BPA) in edible marine biota collected from coastal waters of Malaysia was conducted using GC–MS and SPE extraction. An analytical method was developed and validated to measure the level of 15 OCPs and BPA simultaneously from five selected marine species. It was observed that some samples had low levels of p,p0 -DDE, p,p0 DDT and p,p0 - DDD ranging from 0.50 ng g1 to 22.49 ng g1 dry weight (d.w) but significantly elevated level of endosulfan I was detected in a stingray sample at 2880 ng g1 d.w. BPA was detected in 31 out of 57 samples with concentration ranging from below quantification level (LOQ: 3 ng g1) to 729 ng g1 d.w. The presence of OCPs is most likely from past use although there is also indication of illegal use in recent times. The study also reveals that BPA is more widely distributed in coastal species caught off the coast of the most developed state. The potential health risk from dietary intakes of OCPs and BPA from the analysed fish species was negligible. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Organochlorine pesticides (OCPs) are a group of persistent organic pollutants (POPs) which are commonly found in the aquatic environment (Wania and MacKay, 1996; Sabljic, 2001; Imo et al., 2007; Guan et al., 2009). They are harmful to the human health and can result in detrimental effect to the environment due to their high persistence and bioaccumulative properties (Fung et al., 2005). A number of them are known to act as endocrine disrupting agents that can affect the reproductive system of animals and humans (Anway et al., 2005; Ottinger et al., 2005; Tiemann, 2008). OCPs are easily absorbed in the fatty tissues of living organisms and can accumulate significantly in marine biota such as fish and mussels (Sabljic and Protic, 1982; Yang et al., 2006). In Malaysia, OCPs were widely used since 1950s for agriculture maintenance and pest control which continued well into the 90s. Bisphenol A (BPA) is the common name for 4,40 -dihydroxy-2, 2-diphenylpropane, and is a high volume chemical used extensively in the production of polycarbonate and epoxy resins (Staples et al., 1998). In addition, it may be found in paints, flame retardants (mainly tetrabromobisphenol A), unsaturated polyester resins and plastic products among others. The hydrolysis of the ester bonds of BPA based polymer and the non-polymerised monomer residues has led to its widespread contamination (Ballesteros-Gomez et al., 2009). While environmental monitoring of BPA in rivers, ⇑ Corresponding author. Tel.: +60 3 79492103; fax: +60 3 79674791. E-mail address: [email protected] (A.M. Mustafa). 0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2011.11.063

sewage effluents and industrial sources have been widely performed in many countries (Suzuki et al., 2004; Höhne and Püttmann, 2008; Stasinakis et al., 2008 and Chen et al., 2010), studies on its level in biota especially fish are limited (Belfroid et al., 2002; Basheer et al., 2004; Mita et al., 2011). Human exposure through dietary intake is the major route of uptake of both OCPs and BPA (Yim et al., 2005; Vandenberg et al., 2007; Ballesteros-Gomez et al., 2009). Aquatic organisms accumulate OCPs by diffusion through biological membranes in direct contact with water (bioconcentration) or by ingestion of food and sediment (biomagnification) (Barriada-Pereira et al., 2008). However, biota samples like fish require extensive sample clean-up prior to analysis due to its high fat content. In recent years, solidphase extraction (SPE) has become an essential technique in reducing the usage of solvent and processing time. It is widely used for both the extraction of BPA in liquid food and cleaning up of crude extracts after solvent extraction (Ballesteros-Gomez et al., 2009). Malaysia is among the countries with the highest fish consumption in the world with per capita consumption of 53 kg in 2005 and this number is expected to further increase in the near future (Ahmed et al., 2011). In this study, we analysed the level of OCPS and BPA in marine samples collected from locations along the Malaysian shores. A simplified method combining both SPE and liquid–liquid extraction (LLE) was developed and validated to extract 15 OCPs (hexachlorobenzene (HCB), heptachlor, aldrin, cis and trans chlordane, dieldrin, mirex, endosulfan 1, endosulfan sulphate, DDT and its metabolites) and BPA. To the best of the authors’ knowledge, this is the first time that these compounds were

V.A. Santhi et al. / Chemosphere 86 (2012) 1066–1071

extracted simultaneously. The method was then applied to measure the concentration of these contaminants in five species of marine fish and squid commonly consumed in Malaysia. In addition, adverse health effects were assessed using the US Environmental Protection Agency (EPA) advisory methods on frequency of fish consumption.


malabaricus) and squid (Loligo sp.). Individual fish from each location was used as a single sample except for squid samples which consists of pooled 3–4 individuals. Squid samples from site 6, 7, 8, 9 and 12 were purchased from fish landing sites. Edible parts, mostly muscles were homogenised and freeze dried at 40 °C. 2.3. Chemicals and reagents

2. Materials and methods 2.1. Sampling location Peninsular Malaysia is located to the south of Thailand and north of Singapore (Fig. 1). It faces the South China Sea to the east and The Straits of Malacca to the west. With an extensive coastline of 2068 km, fishery is an important industry here. In 2009, the fisheries sector produced 1 870 000.81 tonnes of fish with a value of US 2801 million. 2.2. Sample collection Edible fish and squid samples were purchased from wet markets near known fishing sites in Peninsular Malaysia from June 2009 to August 2009. The sampling location will to a certain extent reflect the location of the fish dwelling place and the source of pollution. Sampling locations and fish species are presented in Table 1. Five species of fish and squid with high commercial value selected for this study are mackerel (Scomberomorus commerson), sea bass (Lates calcalifer), stingray (Dasyatis sp.), red snapper (Lutjanus

Analytical standards BPA and internal standard BPA-d16 were supplied by Supelco (Bellefonte, USA). A mix of OCPs standards containing aldrin, cis-chlordane, trans-chlordane, dieldrin, endosulfan 1, endosulfan sulphate, heptachlor, p,p0 -DDD, p,p0 -DDE and p,p0 -DDT was purchased from Accustandard (New Haven, CT) while pure standards of o,p0 -DDD, o,p0 -DDE, o,p0 -DDT and HCB was obtained from Supelco (USA). Internal standards 13C-p,p0 -DDT and BPA-d16 were from Dr. Ehrenstorfer (Germany) and Supelco (USA) respectively. All solvents (hexane, acetone, ethyl acetate and acetonitrile) used were of pesticide grade and purchased from Supelco (USA). Standard mixture (100 lg mL1) of OCPs and BPA were prepared in ethyl acetate and stored at 20 °C. A working standard of 1 lg mL1 in ethyl acetate was prepared daily. ‘‘Sole’’ brand mineral water (Italy) was used for liquid–liquid extraction. The silyl-derivatization agent bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% of trimethylchlorosilane (TMCS) was purchased from Fluka (Buchs, Switzerland). SPE cartridges, C18 (1 g, 6 mL) and NH2 (1 g, 6 mL) from Strata (Phenomenex) were used for solid phase extraction. Sodium chloride and anhydrous sodium sulphate, used as a drying agent were from Merck (Germany).

Fig. 1. Map of Peninsular Malaysia where samples were obtained. The numbers on the map indicate the sites mentioned in Table 1.


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Table 1 Summary of sampling sites and fish species purchased along the coast of Peninsula Malaysia. Site


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total sample

Kuala Besar Pulau Kambing Cendering Kuantan Pandan Salak Tinggi Morib Klang Kuala Selangor Selayang Kuala Pari TanjungMalim Bukit Mertajam Mergong Kampung Bakau

Number of samples Mackerel


Sea bass

Red snapper


2 2 1 1 – – – – – 2 1 – – – – 9

2 – – 1 2 – – 1 – – 1 – 1 – 1 9

– 2 – 2 1 – – 3 – – 1 – – 1 – 10

1 – 1 2 – – – 1 – 3 2 – – – – 10

– – – 1 2 3 2 – 3 – – 4 – 2 – 17

All glassware was washed with laboratory detergent and thoroughly rinsed with acetone and hexane before use. Care was taken not to use plastic products to avoid contamination.

2.4. Extraction method Freeze-dried sample (1.5 g) spiked with 30 ng each of 13C-p,p0 DDT and BPA-d16 was extracted twice with 15 mL of acetonitrile by vortexing for 5 min and centrifuging at 8000 rpm for 10 min at 0 °C. The combined acetonitrile layer was then stored at 20 °C for 30 min to allow for lipid precipitation. Freezing lipid filtration has previously been used to eliminate large amounts of lipid from biological sample in the determination of organochlorine pesticides (Hong et al., 2004) and phenols (Ahn et al., 2007). Most of the lipid will be precipitated on the glass surface while the compound of interest will be dissolved in the solvent. The solvent was collected and the precipitated lipid on the glassware surface was further extracted with another 10 mL of acetonitrile to improve recovery of the target compounds. The combined solvent was then concentrated to a few mL by rotary evaporator (Buchi, Switzerland) and made to 10 mL. This was later applied to a Strata C18 cartridge (1 g, 6 mL) conditioned with 15 mL of acetonitrile and positioned above an extraction flask containing 150 mL of water and 7 g of NaCl for subsequent liquid–liquid extraction. After sample loading, the cartridge was washed with 5 mL of acetonitrile. Both the eluant and wash solvent collected in the extraction flask was extracted with 30 mL of hexane: ethyl acetate (1:1). The flask was shaken at 145 rpm for 5 min and the organic layer collected. This step was repeated twice. Anhydrous sodium sulphate was added to the collected organic layer to remove trace amounts of water. It was then concentrated to less than 1 mL at 30 °C by rotary evaporator. Further clean up was carried out using Strata NH2 cartridge (1 g, 6 mL) which was rinsed with 6 mL acetone: hexane (1:1, v/v) and conditioned with 20 mL hexane: acetone (9:1, v/v). The sample was loaded onto the cartridge and eluted with 3  4 mL of hexane–acetone (9:1, v/v). This eluant (Fraction 1) contains the OCPs. Further desorption of BPA and BPA-d16 (Fraction 2) was carried out using 3  4 mL of hexane–acetone (2.5: 7.5, v/v). Both fractions were dried under a gentle stream of nitrogen and stored at 80 °C before analysis. Fraction 1 was dissolved in 100 ll of hexane for analysis and 2 ll was injected to the GC–MS for pesticide analysis. Meanwhile, Fraction 2 was reconstituted with 100 ll of acetone: hexane (1:1) and 20 ll of BSTFA + 1% TMS before being derivatized at 75 °C for 40 min. One microlitre was injected to the GC–MS for BPA analysis.

2.5. Gas chromatograph–mass spectrometer (GC–MS) conditions The GC–MS instrument used in this study was a GC system coupled with quadrupole mass spectrometer (GCMS-QP2010 model, Shimadzu Corporation, Japan) equipped with an AOCi automatic sampler. It was operated using electron ionisation in the selected ion monitoring (SIM) mode. The target compounds were separated using a BP-1 (SGE, Australia) capillary column (length: 30 m; i.d: 0.25 mm; film thickness 0.25 lm). Identification was based on retention time and relative intensities of quantification and specific confirmatory ions. Although the GC–MS analytical condition for OCPs could be used to analyse both the OCPs and BPA simultaneously, the authors found some of the pesticide compounds degrading during the derivatization process necessary for BPA analysis. It was then decided to analyse these compounds separately. For OCPs analysis, helium was used as the carrier gas at a constant flow rate of 2.25 mL min1. Ion source temperature was set at 230 °C, injection temperature at 250 °C and interface temperature at 270 °C. The GC temperature programme was as follows: initial temperature 70 °C (held for 1 min), increased to 160 °C at a rate of 15 °C min1 and thereafter increased to 190 °C at a rate of 2 °C min1. Finally the temperature was increased to 320 °C at a rate of 15 °C min1 and held for 5 min. Meanwhile, for BPA analysis, the GC was operated at a programmed oven temperature from 80 °C (held for 1 min) to 250 °C at a rate of 15 °C min1, thereafter to 300 °C at a rate of 30 °C min1 (held for 7 min). The injection temperature was set at 280 °C while interface temperature was maintained at 300 °C. Ion source temperature was set at 200 °C while Helium flow rate was a constant 1.45 mL min1. 2.6. Statistical analysis The results from the method development and validation experiments were compared for any significant differences using one-way analysis of variance (ANOVA) using SPSS for Windows, Version 15.0 (SPSS Inc., Chicago, IL, USA). Differential statistics was used to calculate mean, median, standard deviation and range of OCPs and BPA concentration. 2.7. Quality control and quality analysis A reagent blank (carrying out the whole extraction procedure without a sample) was run to rule out false positives due to contamination from instrument, apparatus, solvents or chemicals used. Spiked blank sample was also used to evaluate the extraction


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efficiency and as QC sample. While no OCPs were present in the reagent blank, trace level of BPA was detected. Peak area ratios of the compounds were in the range of the calibration curve while samples with high concentration were diluted and analysed. For the quantification, calibration curves obtained using matrix matched standards were used to ensure bias-free analytical results. All the results were expressed on a dry weight basis.

3. Results and discussions The correlation coefficient (R2) for each chlorinated pesticide and BPA was more than 0.993 for the selected range. The limit of detection (LOD) was set as the lowest concentration with a signal-to-noise ratio (S/N) of at least 3, while the limit of quantification (LOQ) was 10 times S/N. The LOQs ranged from 0.50 ng g1 to 10.00 ng g1 and were adequate for the monitoring of the compounds studied. Recoveries at low (5 ng g1 for all compounds except endosulfan 1, 10 ng g1), medium (40 ng g1) and high (80 ng g1) levels ranged between 81.9% and 111.4% and between 51.6% and 56.0% for OCPs and BPA respectively. Although BPA recovery was rather low, it was accepted as the RSD was between 2.2% and 8.0% while the recoveries for the OCPs were good. RSDs for both the intra and interday precision were less than 16.52%. A typical total ion chromatogram of the OCPs extracted from the spiked fish is given in Fig. 2. Interferences from some lipids found




during the GC–MS analysis at low concentrations of OCPs were overcome by selecting specific ions in the selected ion monitoring (SIM) mode. There were no lipid interferences observed during BPA analysis (Fig. 3). 3.1. OCP concentration in fish Only limited data was available on contamination of OCPs in Malaysian marine biota (Monirith et al., 2003). Our study revealed that the OCPs contamination in the fish samples was generally low (Table 2). Among the detected OCPs were p,p0 - DDD, p,p0 -DDE, p,p0 DDT and endosulfan 1 while HCB, heptachlor, aldrin, dieldrin, mirex, trans-chlordane, cis-chlordane, endosulfan sulphate, o,p0 -DDD, o,p0 -DDE and o,p0 -DDT were not detected. While mackerel had the highest level of total DDTs (p,p0 DDD + p,p0 -DDE + p,p0 -DDT) at 39.47 ng g1, it was detected more frequently in red snapper and sea bass at 60% and 50% respectively while none were detected in stingray and squid samples. The most prevalent OCP, p,p0 -DDE was found in 12 samples. In this study, all three species containing DDTs were at a higher tropic level than stingray and squid. Bioaccumulation of OCPs normally occurs with increasing tropic level due to its reduced removal efficiency and increased lipid content (LeBlanc, 1995). It was also dependant on sampling location as higher levels of DDTs and residual level of p,p0 -DDT, the main isomer of technical DDT were mostly detected in fish collected from northern Malaysia, caught from South China Sea water. While the presence of DDTs is most likely from its past use in agriculture and vector eradication programme in Malaysia (Leong et al., 2007), recent studies on sediment, water and fish from its neighbouring countries suggest recent input of p,p0 -DDT (Sudaryanto et al., 2007; Poolpak et al., 2008; Samoh and Ibrahim, 2009). Meanwhile only two samples had quantifiable levels of endosulfan 1, both limited to the stingray species. The sample from Klang in Selangor has significantly elevated level of endosulfan 1











0 14 & 15

9 12


1400000 16



7 1


2 3



1000000 13 8


800000 600000 400000










Retention time (min) Fig. 2. Total ion chromatograms of blank fish extracts after clean-up with C18 and NH2 cartridge (A) and extracted spiked sample matrix with 80 ng g1 of OCPs (B). Peak identities as follows: (1) HCB, (2) heptachlor, (3) aldrin, (4) trans-chlordane, (5) o,p0 -DDE, (6) endosulfan 1, (7) cis-chlordane, (8) dieldrin, (9) p,p0 -DDE, (10) o,p0 DDD, (11) p,p0 -DDD, (12) o,p0 -DDT, (13) endosulfan sulphate, (14) 13C-p,p0 -DDT, (15) p,p0 -DDT and (16) mirex.

BPA-d16 200000 0 13.1






Retention time (min) Fig. 3. Total ion chromatograms of blank fish extracts for BPA after clean-up with C18 and NH2 cartridge (A) and extracted sample (B).


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Table 2 Levels of detected OCPs in the fish samples in ng g1 dry weight (d.w). Sample no 1SB 2SB 3SB 4SB 5SB 6SB 7SB 8SB 9SB 10SB 11MK 12MK 13MK 14MK 15MK 16MK 17MK 18MK 19MK


p,p0 -DDD

p,p0 -DDE

p,p0 -DDT

Endosulfan 1

Sample no


ND 1.94 5.63 0.96 0.96 ND ND ND 6.40 ND ND ND ND ND ND 7.13 22.5 ND ND



20RS 21RS 22RS 23RS 24RS 25RS 26RS 27RS 28RS 29RS 30SR 31SR 32SR 33SR 34SR 35SR 36SR 37SR 38SR


p,p0 -DDD

p,p0 -DDE

p,p0 -DDT

Endosulfan 1


3.70 ND 7.45 5.23 ND ND 4.96 10.1 ND ND ND ND ND ND ND ND ND ND ND



SB = sea bass, MK = mackerel, RS = red snapper, SR = stingray and ND = not detected. a Only results for fish samples are shown.



Concentration (ng g-1)





0 1





11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55

Sample No. 1

Fig. 4. Levels of BPA (ng g squid.

d.w) in individual samples. Sample identities as follows: (1–10) sea bass, (11–19) mackerel, (20–29) red snapper, (30–38) stingray and (39–57)

at 2880 ng g1. Although it could be due to contamination, Abdullah (1995) has previously referred to the high levels of endosulfan 1 and endosulfan 2 (5130 ng g1 and 1700 ng g1 respectively) detected in a species of fresh water fish from Malaysia. Moreover stingray’s feeding habits and habitat near coastal, brackish water could contribute to the elevated level. The Selangor River has previously been reported to have levels of endosulfan ranging from 125.1 to 1848.7 ng L1 and from 100.0 to 151.1 ng L1 during dry and rainy seasons respectively (Leong et al., 2007). Furthermore, a recent report suggests it is still brought in illegally to be used as pesticide in the paddy field (Chiew, 2009). 3.2. BPA concentration in fish BPA was detected in a total of 31 samples including samples with levels below the limit of quantification (Fig. 4). The highest frequency of BPA detection was in sea bass and squid samples at 77.8% and 76.5% respectively while lower frequency of detection

was found in mackerel (22.2%), red snapper (50.0%) and stingray (44.4%). Squid samples had the highest and broadest range of BPA level (not detected (ND)-729 ng g1, median: 42.2 ng g1) while a narrower range of BPA levels were found in stingray (ND-19.85 ng g1), red snapper (ND-27.8 ng g1), sea bass (ND59.0 ng g1) and mackerel (ND-69.0 ng g1). However, Mita et al. (2011) recently suggested that BPA accumulation in fish muscle was not dependant on its size or species. It is suspected that the wide range of BPA levels in this study is due to the widely dispersed sampling location. Generally the west coast of peninsular Malaysia is more developed and urbanised compared to the east coast. Fish and squid samples collected from locations in Selangor, the most urbanised and industrialised state in Malaysia had average level of 10.50 ng g1 (n = 10) and 257.3 ng g1 d.w. (n = 8) respectively. These levels however, were lower than the levels found in fish and squid bought from supermarkets in Singapore, an industrial country at 65.6 ng g1 and 118.9 ng g1 wet weight (w.w) respectively (Basheer et al., 2004) but comparable to the

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levels (less than 7 ng g1 (w.w)) in fish caught off the coast of Italy (Mita et al., 2011). 3.3. Food regulations and risk assessment The detected level of total DDTs in this study were lower than the maximum residue limits (MRL) of 5.0 lg g1 for fish and fishery products set by the US Food and Drug Administration (FDA) and the default MRL of 0.01 lg g1 under the Reg. (EC) 296/2005. Overall, total DDTs level in the fish samples were less than 50% of the default MRL. However, endosulfan level in the stingray sample exceeded the default MRL by more than 40-fold. Meanwhile, monthly consumption limits of a 0.217 kg meal size were calculated using the reference dose (RfD) and the highest detected level (w.w basis) to represent a worst case scenario for a Malaysian weighing 60 kg. Using the RfD of 5E-04 mg kg1 d1 for total DDTs, 6E-03 mg kg1 d1 for endosulfan and 5E-02 mg kg1 d1 for BPA, the fish consumption limits per month for chronic health effects were 855 meals for total DDTs, 116 meals for endosulfan and >1000 meals for BPA while consumption limit for carcinogenic health effects for total DDTs is 51 meals. In addition, the estimated daily intake (EDI) of these contaminants for a Malaysian using the fish consumption rate of 137 g d1 (FAOSTAT, 2007) was below the guideline values. These guideline values were calculated using RfDs and the average body weight. The results show the risk of adverse health effects from dietary intakes of OCPs and BPA from the studied fish species are negligible. 4. Conclusion In this study the level of past contaminant, OCPs and emerging contaminant, BPA in edible marine biota was investigated using a novel method by GC–MS. Results showed that while the levels of OCPs detected were mostly lower than the regulatory level, there were some suggestion of new input of p,p0 -DDT and endosulfan 1. BPA was detected in 31 samples with the highest level and frequency found in the squid samples. Overall, the health risk associated with exposure to OCPs and BPA via seafood consumption was negligible. Acknowledgements This study was funded by University Malaya research Grant No. PS190-2009C. The authors would like to thank Dr. Nobumitsu Sakai for valuable inputs during the preparation of the manuscript and the United Nations University, Tokyo, Japan for providing the 13 C-p, p0 -DDT standard. References Abdullah, A.R., 1995. Environmental pollution in Malaysia: Trends and prospects. Trends Anal. Chem. 14, 191–198. Ahmed, A.F., Mohamed, Z., Ismail, M.M., 2011. Determinants of fresh fish purchasing behaviour among Malaysian consumers. Curr. Res. J. Soc. Sci. 3, 126–131. Ahn, Y.G., Shin, J.H., Kim, H.Y., Khim, J., Lee, M.K., Hong, J., 2007. Application of solidphase extraction coupled with freezing-lipid filtration clean-up for the determination of endocrine-disrupting phenols in fish. Anal. Chim. Acta. 603, 67–75. Anway, M.D., Cupp, A.S., Uzumcu, M., Skinner, M.K., 2005. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 308, 1466–1469. Ballesteros-Gomez, A., Rubio, S., Perez-Bendito, D., 2009. Analytical methods for the determination of bisphenol A in food. J. Chromatogr. A 1216, 449–469. Barriada-Pereira, M., Iglesias-Garcia, I., Gonzalez-Castro, M.J., Muniategui-Lorenzo, S., Lopez-Mahia, P., Prada-Rodriguez, D., 2008. Pressurized liquid extraction and microwave-assisted extraction in the determination of organochlorine pesticides in fish muscle samples. J. AOAC Int. 91, 174–180.


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