Organochlorine pesticides in serum and adipose tissue of pregnant women in Southern Spain giving birth by cesarean section

Science of the Total Environment 372 (2006) 32 – 38 www.elsevier.com/locate/scitotenv

Organochlorine pesticides in serum and adipose tissue of pregnant women in Southern Spain giving birth by cesarean section M. Jimenez Torres a , C. Campoy Folgoso b , F. Cañabate Reche b , A. Rivas Velasco a , I. Cerrillo Garcia a , M. Mariscal Arcas a , F. Olea-Serrano a,⁎ a

Department Nutrition and Food Science, School of Pharmacy, University of Granada, Spain b Department Pediatrics, University of Granada, Spain Received 21 June 2005; received in revised form 27 June 2006; accepted 4 July 2006 Available online 14 August 2006

Abstract Since the appearance of DDT, increasingly potent insecticides have been developed to overcome the resistance developed by insects to successive products. Pesticides are also used in public health programs to control disease vectors. Despite legislation to control the use of certain products, they repeatedly appear in the adipose tissue, milk and serum of human populations. The present study determined the presence of organochlorine molecules in the adipose tissue, serum, and umblical cord of women giving birth by cesarean section in order to establish a possible correlation in organochlorine molecule content between these biological compartments and to examine fetal exposure to molecules with hormonal effects. Presence of nine organochlorines was detected by GC/ECD and confirmed by GC/MS. Highly significant differences ( p b 0.000) were observed between adipose tissue and maternal serum in concentrations of lindane, HCB, DDE, DDD, and endosulfan but not ( p N 0.5) in concentrations of endosulfan II or endosulfan sulfate. Only DDE concentrations differed ( p b 0.001) between maternal serum and umbilical cord serum. An association between pp'DDE and op'DDT was observed in maternal serum ( p b 0.094). An association in pp'DDE and pp'DDD content was found between adipose tissue and umbilical cord serum, and in pp'DDT content between adipose tissue and maternal serum. Results obtained indicate that exposure can be measured solely in serum when relatively high concentrations of pesticides are present. © 2006 Elsevier B.V. All rights reserved. Keywords: Organochlorine compounds; Umbilical cord serum; Adipose tissue

1. Introduction New products to which humans and animals must adapt appear daily. Since the appearance of DDT, there ⁎ Corresponding author. Fatima Olea Department Nutrition and Food Science. Facultad de Farmacia, Campus de Cartuja Universidad de Granada 18071 Granada, Spain. Tel.: +34 958 24 28 41; fax: +34 958 24 95 77. E-mail address: [email protected] (F. Olea-Serrano). 0048-9697/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2006.07.009

have been continuous efforts to find increasingly potent insecticides to overcome the resistance developed by insects to successive products. Many of these chemical substances present in the environment can alter the hormonal homeostasis of living beings. More than ten groups of substances belonging to different chemical families have been reported to behave in vivo as natural estrogens. Chemical synthesis products are currently preferred, and their utilization in agriculture has led to a marked increase in agricultural production. Pesticides

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are also used in public health programs to control diseases transmitted by intermediary vectors or hosts. The use of pesticides in health and agriculture has very varied effects: the benefits derived from the systematic destruction of parasites that affect the health of plants and humans must be set against the effects of these biocide chemical compounds on animal species and humans themselves (Olea et al., 1996). Despite legislation to control the use of certain products, they repeatedly appear in the adipose tissue, milk, and serum of animal and human populations (Albers et al., 1996; Huisman et al., 1995; Pandit et al., 2002; Moysich et al., 2002). Because organochlorine pesticides accumulate in the adipose tissue, blood/serum, and milk, any of these media are suitable for estimating human impregnation by these xenobiotics, and studies have demonstrated the presence of polychlorinated biphenyls (PCBs), dioxins, DDT, and HCH in all of them (Atuma et al., 1998; Hura et al., 1999, Campoy et al., 2001; Turusov et al., 2002). The concentration in blood depends on the continual accumulation of organochlorine molecules and their low metabolic degradation and excretion, and all of these compounds have been shown to have a long half-life (Asplund et al., 1994; Patandin et al., 1999; Lopez-Carrillo et al., 2001). The distribution between adipose tissue and serum is of the order of 200 to 1 depending on the molecule in question and is a function of the lipid content of the two compartments, which is around 90% in the former versus 0.4% in the latter (Luotamo et al., 1991; Wolff et al., 1993; Rivas et al., 2001). The present study determined organochlorine molecules in the adipose tissue, serum, and umbilical cord of women giving birth by cesarean section. The aim was to establish a possible correlation in organochlorine molecule content between these biological compartments and to examine fetal exposure to molecules with hormonal effects.

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2. Material 2.1. Sample selection Seventy-two women volunteers scheduled for birth by cesarean section and aged from 18 to 35 years were selected at random from two distinct geographical settings: El Ejido (recruitment site, Hospital del Poniente), an area of intensive agriculture; and Granada (Clínico University Hospital), a city. All of the women gave their written informed consent and the study was approved by the ethical committee of each hospital. During the cesarean delivery, samples of panniculus adiposus, maternal blood (by venipuncture), and umbilical vein blood were obtained and immediately stored at − 70 °C. 2.2. Reagents Standards:,Lindane, methoxychlor, endosulfan I and II, p-p′-DDT and o-p′-DDT (Sulpeco, Bellefonte, PA); o, p′-DDD and p-p′dichlorobenzophenone (Dr Ehrenstorfer Lab., Ausburg, Germany); p,p′-DDE (Chem Service, West Chester, PA); endosulfan sulfate (Hoechst Schering AgrEvo, Frankfurt, Germany) were used, always with purity higher than 99%. Stock standard solutions, 100 μg mL− 1, were prepared by exact weighing and dissolution in n-hexane. Working standard solutions were prepared by appropriate dilutions and stored at 4 °C. Pesticide quality solvents: n-hexane, methanol, diethyl ether, and 2-propanol were supplied by Panreac (Barcelona, Spain). Reagent grade concentrated sulfuric acid was also supplied by Panreac. Organic free water was prepared by distillation and then by Milli-Q SP treatment (Millipore Corporation, Billerica, MA). Alumina Merck 90 (70–230 mesh n°1097; Merck KGaA, Darmstadt, Germany) and silica Sep-Pak (Wat 051900; Waters

Table 1 Analytical parameters of the gas chromatography/electron capture method

HCB Lindane Endosulfan I p,p′ DDE p,p′DDD Endosulfan Sulfate o,p′DDT Endosulfan II p,p′DDT Methoxychlor

RT (min)

Calibration curve

R2

Linearity of detector (ng/mL)

Recovery (%)

LOQ (ng/mL)

8.30 8.74 11.04 11.29 11.41 11.87 12.13 12.61 12.71 13.81

y = 6.9741x + 0.0575 y = 5.9075x + 0.0344 y = 4.9822x + 0.0125 y = 6.4316x + 0.0190 y = 0.3551x + 0.0039 y = 4.8177x + 0.0031 y = 3.6420x + 0.0291 y = 4.7905x + 0.0082 y = 4.5472x + 0.0332 y = 2.1599x + 0.0115

0.9816 0.9804 0.9982 0.9924 0.9921 0.9999 0.9959 0.9996 0.9908 0.9971

1.0–50 1.0–100 0.5–150 1.0–200 1.0–500 0.5–20 1.0–200 2.0–200 1.0–100 1.0–50

99.20 99.90 99.79 94.18 105.97 100.03 95.03 93.99 97.73 97.00

1.0 1.0 0.5 1.0 1.0 0.5 1.0 2.0 1.0 1.0

RT = retention time; LOQ = limit of quantification.

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Table 2 Organochlorine pesticides determined in 72 adipose tissue samples (ng/g of fat) Pesticides

N° of positive samples (%)

Range

Minimum

Maximum

Mean

SD

Lindane pp′DDE pp′DDD o-p′ DDT p-p′ DDT Methoxychlor HCB Endosulfan I Endosulfan II Endosulfan sulfate

36 (50) 67 (93.0) 18 (25.0) 14 (19.4) 9 (12.5) 3 (4.1) 67 (93.0) 10 (13.8) 19 (26.4) 12 (16.6)

403.15 5192.85 783.16 366.55 79.77 711.85 5779.63 331.15 141.44 57.24

4.22 45.95 .00 1.60 16.51 106.00 74.08 3.03 12.01 7.23

407.37 5238.80 783.16 368.15 96.28 817.85 5853.71 334.18 153.45 64.47

113.82 2240.84 315.85 53.89 60.62 347.73 1290.39 75.46 51.68 23.95

93.31 1254.39 247.79 94.52 28.38 407.19 1057.93 112.24 35.52 16.67

SD = standard deviation.

Corporation, Milford, MASS), were used for analysis of adipose tissue and clean-up, respectively. 2.3. Chromatographic conditions 2.3.1. Gas Chromatography (GC/ECD) The chromatograph apparatus used was a Varian3350 ECD Electron Capture Detector (63Ni) (Walnut Creek, CA), a split/splitless injector operated in the splitless mode (0.01 min), with Millennium Chromatography Manager Software, and a Varian CP8944 chromatographic column (VF-5ms; 30 m × 0.25 mm i. d. × 0.25 μm film thickness). 2.3.2. Working conditions Injector and detector temperatures were 250° and 300°, respectively. Temperature column was programmed from 130 °C (1 min) to 150° at 20 °C/min; 150 °C to 200° at 10 °C/min; 200 °C to 260° at 20 °C/min, (20 min). The carrier and auxiliary gas was nitrogen at a flow rate of 30 mL/min and 40 mL/min, respectively (Rivas et al., 2001). 2.3.3. GC and Mass Spectrometry (GC/MS) Saturn 2100T Varian equipment with Varian injector 1177 and CP5860 WCOT fused silica column

(30 m × 0.25 mm) was used. The computer controlling the system contained a specially created library for the target analytes under our experimental conditions. Working conditions were: injector temperature 250 °C; initial column temperature 50 °C (2 min), 30 °C/min to 185 °C (5.5 min), 2 °C/min to 250 °C (32.5 min), and 30 °C to 300 °C (6.67 min). The carrier gas used was helium (purity 99.999%) with an injector flow of 1mL/ min Manifold, transfer-line, and trap temperatures were 50, 230, and 200 °C, respectively. 2.3.4. GC–ECD analysis Optimal chromatographic conditions were established to separate analytes of interest. Identification of each analyte was based on the mean retention time, established as the mean of retention times in 10 measurements ± three times the standard deviation (SD). Linearity: Calibration curves were obtained from standard solutions. The linearity range is shown in Table 1. Sensitivity: The limit of detection in GC–ECD is designated by the IUPAC as 3-fold the standard deviation of the blank and the limit of quantification (LOQ) as 10-fold the standard deviation of the blank (Long and Wineffordner, 1983) (Table 1). Recovery: Recovery in the extraction was determined by fortifying 10 aliquots of 4 mL of blank medium to an intermediate

Table 3 Organochlorine pesticides determined in maternal sera (ng/mL serum) Pesticides (ng/mL)

N of positive samples (%)

Range

Minimum

Maximum

Mean

SD

Lindane HCB p,p′DDE p,p′DDD o,p′ DDT p,p′ DDT Endosulfan I Endosulfan II Endosulfan sulfate

33 (45.8) 72 (100) 71 (99) 17 (23.6) 41 (56.9) 36 (50) 44 (61) 43 (59.7) 18 (25.0)

4.40 129.21 228.89 106.88 28.08 37.34 6.02 195.89 146.27

.00 2.11 3.54 9.41 .00 3.59 .39 5.68 4.17

4.40 131.32 232.43 116.29 28.08 40.93 6.41 201.57 150.44

1.26 20.05 31.87 44.47 3.16 10.51 1.27 76.38 34.17

1.19 22.65 34.69 32.28 4.60 7.18 1.08 52.60 34.96

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Table 4 Organochlorine pesticides determined in chord sera (ng/mL chord serum) Pesticides (ng/mL)

N° of positive samples (%)

Range

Minimum

Maximum

Mean

SD

Lindane HCB p,p′DDE p,p′DDD o,p′ DDT p,p′ DDT Endosulfan I Endosulfan II E. sulfate

45 (62.5) 65 (90.2) 71 (99.0) 12 (16.6) 24 (33.3) 27 (37.5) 44 (61.0) 23 (31.9) 28 (38.8)

108.18 117.01 391.36 130.47 8.50 24.57 76.29 293.32 154.25

.43 1.72 3.74 9.60 .00 3.50 .00 6.16 5.95

108.61 118.73 395.10 140.07 8.50 28.07 76.29 299.48 160.20

3.95 21.40 24.03 55.39 2.18 9.00 3.18 66.05 31.83

15.98 23.93 48.11 37.40 1.95 6.26 11.33 78.50 35.62

point on the calibration curve (100 ng/mL), using 4,4′ dichlorobenzophenone as internal standard. 2.4. Analysis of serum from umbilical or maternal vein Half of the same volume of methanol was added to 2–4 mL of serum and the solution was shaken for 5 min. The extraction of organochlorines was performed using 10 mL ethyl ether/hexane (1:1 v/v) centrifuged for 15 min at 3000 g. The organic phase was obtained and the extraction procedure was repeated twice more. The organic phases were evaporated and completely dried under a gentle stream of nitrogen. To this residue, 0.5 mL of concentrated sulfuric acid was added and centrifuged for 10 min at 3000 rpm. The acid residue was extracted twice more with the addition of 1 mL

hexane. The three organic phases were collected and dried in a gentle stream of nitrogen. The dry residue was redissolved in 1 mL of hexane and cleaned up (Rivas et al., 2001; Moreno Frias et al., 2004). 2.5. Analysis of adipose tissue Alumina Merck 90 (70–230 mesh n°1097) was dried for 4 h at 600 °C. 2 g of alumina was weighed, brought to 5% hydration with distilled water, and used to fill a Pyrex glass chromatograph of 6 mm internal diameter. For extraction of organochlorines, 0.2 g of adipose tissue sample was weighed and homogenized in a potter with 2 mL of hexane. It was eluted through the Pyrex chromatographic column with 20 mL hexane and the elute was collected in a flask with round base. The

Table 5 Application of the Spearman correlation to contents of the endosulfan group of pesticides in the adipose tissue, maternal serum, and umbilical cord serum

Adipose Endosulfan I tissue Endosulfan II E. sulfate Maternal Endosulfan I serum Endosulfan II E. Sulfate Chord Endosulfan I serum Endosulfan II E. sulfate

Adipose tissue

Maternal serum

Chord serum

Endosulfan. Endosulfan Endosulfan I II sulfate

Endosulfan Endosulfan Endosulfan I II sulfate

Endosulfan Endosulfan Endosulfan I II Sulfate

1.000 0.400 p b 0.600 – – − 0.429 p b 0.397 − 0.086 p b 0.872 − 0.800 p b 0.200 − 0.090 p b 0.848 − 0.400 p b 0.600 0.800 p b 0.200

1.000 – 0.134 p b 0.449 0.136 p b 0.689 − 0.050 p b 0.799 0.242 p b 0.426 0.002 p b 0.994

1.000 – 0.460 p b 0.041 0.055 p b 0.829

1.000 – − 1.000 p b 0.000 0.410 p b 0.186 0.615 p b 0.033 0.500 p b 0.667 − 0.134 p b 0.648 0.714 p b 0.071 − 0.200 p b 0.747

1.000 – 0.571 p b 0.180 0.100 p b 0.798 −0.500 p b 0.667 −0.524 p b 0.183 0.500 p b 0.667 0.400 p b 0.600

1.000 – 0.879 p b 0.000 0.323 p b 0.081 0.286 p b 0.344 −0.050 p b 0.835

1.000 – − 0.308 p b 0.306 0.119 p b 0.779 − 0.800 p b 0.010

1.000 – 0.214 p b 0.645

1.000 –

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Table 6 Application of the Spearman correlation to contents of the DDT group of pesticides in the adipose tissue, maternal serum, and umbilical cord serum Adipose Tissue p,p DDE Adipose p,p tissue DDE p,p DDD o,p′ DDT p,p′ DDT Maternal p,p′ serum DDE p,p′ DDD o,p′ DDT p,p′ DDT Cord p,p′ serum DDE p,p′ DDD o,p′ DDT p,p′ DDT

Maternal Serum

Chord Serum

p,p DDD o,p′ DDT p,p′ DDT p,p′ DDE

p,p′ DDD

o,p′ DDT p,p′ DDT p,p′ DDE

p,p′ DDD

o,p′ DDT p,p′ DDT

1.000 – 0.800 p b 0.200 − 1.000 p b 0.000 0.154 p b 0.542 0.200 p b 0.747 − 0.228 p b 0.588 − 0.607 p b 0.148 0.173 p b 0.491 1.000 – 0.551 p b 0.257 0.714 p b 0.071

1.000 – 0.298 p b 0.403 0.450 p b 0.224 − 0.051 p b 0.844 0.800 p b 0.200 0.033 p b 0.932 − 0.607 p b 0.148

1.000 – 0.220 p b 0.290 0.086 p b .592 0.400 p b 0.600 0.261 p b 0.348 − 0.014 p b 0.955

1.000 – 0.214 p b 0.645 0.900 p b 0.037

1.000 – 0.564 p b 0.028

1.000 0.052 p b 0.839 0.455 p b 0.102 0.450 p b 0.224 0.040 p b 0.750 0.288 p b 0.279 0.019 p b 0.910 0.099 p b 0.590 − 0.022 p b 0.862 0.564 p b 0.071 0.133 p b 0.567 − 0.200 p b 0.337

1.000 – 0.400 p b 0.600 0.490 p b 0.075 − 1.000 p b 0.000 0.314 p b 0.544 0.200 p b 0.606 − 0.147 p b 0.615 0.500 p b 0.667 0.300 p b 0.624 0.200 p b 0.704

1.000 – 0.533 p b 0.139 1.000 – − 0.500 p b 0.667 0.679 p b 0.094 − 0.317 p b 0.406 0.200 p b 0.800 − 0.600 p b 0.400 − 0.800 p b 0.200

1.000 – 0.216 p b 0.406 0.042 p b 0.799 0.177 p b 0.301 0.179 p b 0.139 − 0.294 p b 0.354 0.225 p b 0.301 0.309 p b 0.124

organic extract was concentrated under reduced pressure at 40° in a vacuum concentrator to a volume of 1 mL, taking care to avoid dryness. Finally, the extract was purified.

1.000 – 0.168 p b 0.328 0.200 p b 0.800 − 0.309 p b 0.385 0.024 p b 0.931

1.000 – 0.399 p b 0.199 0.464 p b 0.022 0.402 p b 0.038

1.000 –

used to compare data from maternal venous serum and adipose tissue. Where the value was zero, half of the limit of quantification was used in the analysis. 3. Results

2.6. Clean-up In all extractions, the organic extracts obtained were purified with the silica Sep-Pak (Wat 051900) after prior treatment of the cartridge with 2 mL hexane The extract was eluted with 10 mL hexane and then with 10 mL hexane: methanol: isopropanol (45:40:15; v/v/v). Both eluates were collected and dried in a stream of nitrogen. The dry residue was dissolved in 1 mL hexane, labeled with the p-p′dichlorobenzophenone internal standard, and analyzed using GC/ECD. Results were confirmed with GC/MS. 2.7. Statistical analysis An inferential statistical study was performed on the results obtained in the different biological media. Because the pesticides analyzed in these samples did not show a normal distribution, the Mann–Whitney test was

The presence of 10 organochlorines was shown by GC/ECD and confirmed by GC/MS. Results of the analysis of 72 samples of adipose tissue, maternal serum, and umbilical cord serum are shown in Tables 2, 3, and 4. The LOQ in GC–ECD ranged from 0.5 to 2 ng/mL for the molecules studied, and the recovery in the extraction ranged from 94% for endosulfan II to 106% for p,p′ DDD (Table 1). Highly significant differences ( p b 0.001) were found between serum and adipose tissue samples in concentrations of lindane, HCB, DDE, DDD, and endosulfan I but not in concentrations of endosulfan II or endosulfan sulfate ( p N 0.5). There were no significant differences (Mann–Whitney test, p N 0.5) between maternal serum and umbilical cord serum in lindane, HCB, DDD, op DDT, ppDDT, endosulfan I, endosulfan II, or endosulfan sulfate content, but a significant difference ( p b 0.001) in DDE content was observed.

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Tables 5 and 6 show the relationship between each product and its metabolites and between their presence in the serum and adipose tissue, respectively, and the significant associations found (Spearman correlation). 4. Discussion The aim of this study was not to develop new hypotheses on exposure to persistent organochlorine molecules but rather to contribute to the growing body of evidence that these contaminants are present in all human tissues in all regions of the world and to discuss the risk this may pose for future generations. It is widely accepted that high contamination levels in the environment and food have exposed a large part of the world's population to low-to-medium concentrations of these xenobiotics (Berry, 1992; National Research Council, 1993; Whitmore et al., 1994). However, the human health effects of exposure to organochlorine pesticides are not adequately understood. Although it was recently suggested that maternal exposure to environmental pollutants during pregnancy is related to ambiguous genitalia in newborn (Paris et al., 2006), it is not clear whether this observation, in a population exposed to relatively high levels of toxins, can be extrapolated to the population in general. Other researchers found no adverse birth outcomes in contaminated mothers (Khanjani and Sim, 2006). Most published reports to date have focused on detecting a relatively small number of pesticides in milk, serum and/or adipose tissue, usually DDT and its metabolites and some PCBs, and occasionally including lindane or endosulfan. The concentrations of pesticides observed in the present study were always within the range reported by other authors (Cooper et al., 2001; Muckle et al., 2001; Minh et al., 2001; Waliszewski et al., 2001), and confirm the high exposure of this study population to chemical substances with hormonal effect. All of the samples studied contained p,p′DDE and HCB molecules. These findings are consistent with worldwide reports that around 90–100% of the population have detectable concentrations of DDE (Aronson et al., 2000; Rivas et al., 2001; Campoy et al., 2001; Waliszewski et al., 2001; Botella et al., 2004; Cerrillo et al., 2005; Khanjani and Sim, 2006; Thomas et al., 2006). Endosulfan and metabolites were more frequently detected in maternal and umbilical cord sera than in adipose tissue, perhaps because of their lower liposolubility. o,p′DDT and p,p′DDT were somewhat more frequently detected in serum than in the adipose tissue. No methoxychlor was detected in serum and it was found in only 5% of the adipose tissue samples.

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It is of great interest to establish whether serum samples alone are adequate for the assessment of human exposure and risk, since the adipose tissue samples can only be obtained from individuals undergoing surgery and are a much less available source. In the present study, there were highly significant differences ( p b 0.000) between adipose tissue and maternal serum in concentrations of lindane, HCB, DDE, DDD, and endosulfan but not ( p N 0.5) in concentrations of endosulfan II or endosulfan sulfate. In the comparison between maternal serum and umbilical cord serum, concentrations of lindane, HCB, DDD, op DDT, ppDDT, endosulfan I, endosulfan II, and endosulfan sulfate did not significantly differ ( p N 0.5) but the concentration of DDE did ( p b 0.001). The passage of these molecules through the placental barrier and the potential exposure of the fetus are clearly demonstrated by the concentrations found in maternal and umbilical cord sera. There were slightly more positive samples in maternal versus umbilical cord sera, although more umbilical cord than maternal sera were positive for lindane (62.5% vs. 36.1%). The Spearman test confirmed significant associations within groups of pesticides (e.g., DDT group, endosulfan group), reflecting their transformation in the medium. Thus, there was a correlation in umbilical cord samples between endosulfan I and II (r = 0.460, p b 0.041) and in serum between endosulfan sulfate and endosulfan II (r = 0.879, p b 0.000). In the adipose tissue, associations were found between DDT and its metabolites, and there was a negative association between pp′DDT and pp′DDD (r = − 1.000 p b 0.000). The concentrations of each pesticide in the different media were also correlated, indicating diffusion of the molecules. Thus, a correlation in endosulfan II content was observed among the adipose tissue, maternal serum, and umbilical cord serum. There was an association between pp′DDE and op′DDT in maternal serum (r = 0.490, p b 0.075), and pp′DDT content in adipose tissue was associated with that in maternal serum (r = 0.679, p b 0.094). In chord serum, pp′DDE content was significantly associated with op′DDT (r = 0.464, p b 0.02) and pp′DDT (r = 0.402, p b 0.038. An association was also found between adipose tissue and umbilical cord serum in pp′DDE (r = 0.564, p b 0.071) and pp′DDD (r = 0.714, p b 0.071) content (Table 6). The mean values found and the associations established indicate that exposure parameters can be measured solely in serum when pesticides are present in relatively high concentrations. When concentrations are very low, measurement in adipose tissue is recommended given the ratio of concentrations between the two compartments. The finding that individuals are exposed to these molecules from the very beginning of life is of special

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