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Serum levels of organochlorine pesticides in healthy adults from five regions of Spain
Chemosphere 76 (2009) 1518–1524
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Serum levels of organochlorine pesticides in healthy adults from five regions of Spain Paula Jakszyn a, Fernando Goñi b,c, Arsenio Etxeandia d, Asunción Vives e, Esmeralda Millán f, Raul López c, Pilar Amiano c,g, Eva Ardanaz c,h, Aurelio Barricarte c,h, M. Dolores Chirlaque c,i, Miren Dorronsoro c,g, Nerea Larrañaga c,g, Carmen Martínez c,j, Carmen Navarro c,i, Laudina Rodríguez k, M. José Sánchez c,j, M. José Tormo c,i, Carlos A. González a, Antonio Agudo a,* a
Unit of Nutrition, Environment, and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (ICO), IDIBELL, L’Hospitalet de Llobregat 08907, Spain Laboratorio de Salud Pública de Guipúzcoa, 20013 San Sebastian, Spain CIBER Epidemiología y Salud Pública (CIBERESP), 20013 San Sebastian, Spain d Laboratorio de Salud Pública de Vizcaya, 48010 Bilbao, Spain e Laboratorio Unificado Donostia, Hospital N.S. Aranzazu, San Sebastian, Spain f Departamento de Química Aplicada, Universidad del País Vasco, Facultad de Química, San Sebastián, Spain g Dirección de Salud de Guipúzcoa, 20013 San Sebastian, Spain h Instituto de Salud Pública de Navarra, 31003 Pamplona, Spain i Consejería de Sanidad y Consumo, 30008 Murcia, Spain j Escuela Andaluza de Salud Pública, 18080 Granada, Spain k Consejería de Salud y Servicios Sanitarios de Asturias, 33001 Oviedo, Spain b c
a r t i c l e
i n f o
Article history: Received 29 January 2009 Received in revised form 28 May 2009 Accepted 29 May 2009 Available online 7 July 2009 Keywords: Pesticides Organochlorine compounds Serum levels Biomarkers
a b s t r a c t The aim of this study was to measure of serum levels of p,p0 -dichlorodiphenyl trichloroethane (p,p0 -DDT), p,p0 -dichlorodiphenyl dichlorethylene (p,p0 -DDE), b-hexachlorocyclohexane (b-HCH), and hexachlorobenzene (HCB) in healthy adults in Spain. Furthermore, we also analyzed these levels according to dietary, other lifestyle factors and anthropometric characteristics. We measured the concentrations of such organochlorine pesticides (OCPs) in serum samples collected during 1992–1996 from 953 subjects aged 35– 64 years, they were residents of five Spanish regions, they were randomly selected from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. OCPs were determined by means of gas chromatography with electron-capture detection (GC-ECD). The most frequent compound found in serum was p,p0 -DDE, present in 98% of the samples, followed by HCB and b-HCH, found in 89% and 77% of samples, respectively, while p,p0 -DDT could be measured only in 26% of subjects. The geometric means of serum concentrations (ng/g lipid) were 822 for p,p0 -DDE, 167 for b-HCH, and 379 for HCB. The concentrations of all OCPs were positively associated with age and body mass index, and decreased along the period of blood collection. No association was found between OCPs levels and dietary factors. The concentrations of p,p0 -DDE and b-HCB were higher in Murcia, one of southern regions, most likely associated with intensive past use of pesticides related to agricultural practices, while higher levels of HCB were found in Navarra, located in the north, maybe due to industrial use rather than agricultural application. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Organochlorine pesticides (OCPs) are a group of synthetic chemicals effective against a variety of insects, many of which are highly persistent in the environment. Among them, p,p0 -dichlo-
* Corresponding author. Unit of Nutrition, Environment, and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (ICO). Av. Gran Via 199-203, 08907 L’Hospitalet de Llobregat, Spain. Tel.: +34 932607401; fax: +34 932607787. E-mail address: [email protected] (A. Agudo). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.05.048
rodiphenyl trichloroethane (p,p0 -DDT) and its metabolite p,p0 dichlorodiphenyl dichlorethylene (p,p0 -DDE), b-hexachlorocyclohexane (b-HCH, the isomeric form of HCH with longest half-life), and hexachlorobenzene (HCB) are some of the organochlorine compounds most commonly found (WHO, 2003). Although OCPs are primarily environmental pollutants, they are very resistant to degradation and highly lipophilic, and consequently they become part of the food chain, where they tend to bioaccumulate, mainly in fatty foods. Thus, the main source of exposure in the general population is the diet (WHO, 2003). The current background body burden of OCPs is still of concern because
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of their estrogenic properties with potential adverse effects both to the environment and human (Safe, 2004). On the other hand, even though there is sufficient evidence of carcinogenicity for experimental animals, the epidemiological evidence in humans is inadequate for the three groups of compounds, and they are classified by the International Agency for Research on Cancer in the group 2B, as possibly carcinogenic to humans (IARC, 1987; IARC, 1991; IARC, 2001). However, the health effects of chronic exposure to OCPs on the general population at the current levels are still unknown. Because of its persistence and bioaccumulation, as well as growing concern about adverse effects, a list of twelve organochlorine compounds were the objects of international restrictions, defined by the treaty called Stockholm Convention, aimed to eliminations of such compounds (UNEP, 2005). DDT and b-HCB are included in this list, but HCH is not covered by the Stockholm Convention, so the use of HCH in parts of the world is continuing. In Spain, although most OCPs were banned during the 1970s and the levels of such compounds seems to have decreased, most OCPs are often found in food, and detectable concentrations of some of these have been observed in a high proportion among the population (Porta et al., 2008). Most OCPs can be measured in relatively small amounts of serum, and because of their long half-life, often of about 10 years, these levels may be considered good indicators of long-term exposure. However, comprehensive monitoring in the general population in Spain is scarce, and most studies focused on specific groups exposed because of occupation or residence (Porta et al., 2008). The aim of our study was to measure the serum levels of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB in healthy adults belonging to the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Furthermore, we also aimed to analyze these levels according to dietary and other lifestyle factors and anthropometric measurements.
2. Methods 2.1. Study population The EPIC-Spain cohort consisted of 15 632 men and 25 806 women aged 29–69 years, with different social and education levels. Detailed description of methods and population has been published elsewhere (González et al., 2004). Briefly, participants were recruited between 1992 and 1996 among healthy volunteers from five Spanish regions, three from the North (Asturias, Navarra, and Guipúzcoa) and two from the South (Murcia and Granada). At recruitment all the participants provided information on diet and other lifestyle factors, anthropometric measurements, and a sample of 30 mL of blood. All participants gave their informed consent and the study was approved by the Ethical Review Board. For this study, a random sample of 200 subjects from each centre was selected, stratified according to the age and sex structure of the Spanish population, aged 35–64 years. Biological material was not available for 14 individuals, or was of poor quality or insufficient amount to obtain valid measurement in another 33 individuals; thus, the final population of study included 953 subjects. 2.2. Diet and lifestyle questionnaires and anthropometry The usual food intake during the preceding year, taking into account seasonal variations, was estimated by personal interview using a computerized dietary history questionnaire, developed and validated specifically for the EPIC study in Spain (EPIC Group of Spain, 1997). The questionnaire was structured according to occasions of food intake and included a list of more than 600 foods and beverages including local recipes. A questionnaire adminis-
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tered by an interviewer was used to collect information on sociodemographic characteristics, and work and leisure physical activity. The section dealing with current or past occupational exposures included an item asking whether the subject had ever worked in agriculture; those who answered yes to this question were also asked whether this job involved manipulation of pesticides. For women reproductive history was collected as well; this section gathered information about each live birth, including date of childbirth, whether the woman breastfed the newborn (either as exclusive or as complementary nourishment), and duration of breastfeeding. Thus, we estimated the cumulative duration of breastfeeding by adding up weeks or months of lactation periods from different newborns, as well as the time in years elapsed since cessation of the last period of breastfeeding. Weight and height were also taken at recruitment following standardized procedures, and were then used to compute the body mass index (BMI) as kg m2. 2.3. Analytical methods Blood samples were divided into 0.5 mL aliquots of serum, plasma, concentrated red blood cells, and buffy coat, and stored in liquid nitrogen at 196 °C. An improved method for the determination of organochlorine pesticides was used to measure serum levels of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB (Goñi et al., 2007). The method required that low volume of serum (500 lL) and 48–96 samples per day be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Quantification was carried out by gas chromatography with electron-capture detector (GC-ECD). A mass spectrometer detector (GC–MSD) was used for quantitative and qualitative confirmation. Intra-day relative standard deviation of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB varied from 1% to 11%, depending on compound. Inter-day relative standard deviation (measured over a yearlong period) was <15% in all cases. Total cholesterol (TC) and triglycerides (TG) were determined enzymatically by developing colored compounds that were measured spectrophotometrically (Wahlefeld, 1974; Wiebe and Bernert, 1984). Total serum lipids (TL) were calculated from total cholesterol and triglycerides (all of them expressed as g/L) by applying the formula TL = 2.27 TC + TG + 0.623 (Phillips et al., 1989). Then, we used this result to report the serum pesticides on lipid basis. The limit of quantification (LOQ) was 0.4 ng/mL on serum basis; for subjects whose values were below the detection level, a concentration of 0.2 ng/mL (half the LOQ) was assigned. 2.4. Statistical analysis The serum levels of each pesticide were expressed as lipid-corrected concentrations in ng/g lipid. Since the distributions of concentrations were right skewed, the variables were transformed using natural logarithms. For descriptive purposes, apart from the geometric mean, the median and the 75th percentile, with their corresponding 95% confidence intervals (CI), were reported for the whole sample and separately for men and women. Given the sampling framework, all the means reported may be considered standardized according to the age–sex structure of the Spanish population 35–64 years old. Analysis of covariance was used to examine how the log-transformed serum concentration of each OCP varied by demographic, anthropometric, dietary and lifestyle variables (Armitage and Berry, 1994). Geometric means of lipidcorrected concentrations of each OCP were calculated from multiple linear regression models with log-transformed concentration (as ng/g lipid) as the dependent variable; these geometric means are adjusted by all the covariates included in the model. The linear
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trend for ordered variables was assessed by the significance of the coefficient including in the model such variables using the numerical value corresponding to each category. Two-way interactions between the main variables were assessed by means of the likelihood ratio test (LRT), comparing the likelihood of the model with the corresponding interaction terms and the model containing terms for the main effects only (Armitage and Berry, 1994). Regression modeling was not applied to p,p0 -DDT, given the high proportion (73.6%) of subjects with values below the LOQ. Since the total lipids and individual components of lipids may not behave the same way in models, regression modeling was also performed using the volume concentration of OCPs (as lg/ml) and including the concentration of triglycerides and cholesterol as covariates. Nevertheless, as this approach yielded the same results as the one using the lipid-corrected concentration only the latter is presented. All the statistical analyses were carried with the release 10 of STATA (StataCorp, 2007). 3. Results Overall, serum samples from 953 subjects aged 35–64 years old from five Spanish regions were analyzed to quantify the four selected pesticides. Table 1 shows the main socio-demographic characteristics and other factors of the study population. The mean age was 49 years (48.7 for women and 49.4 for men). About half of subjects had overweight (BMI 25–30) or were obese (BMI > 30), and about one third had not completed the primary school or lacked formal education. Almost one quarter of subjects had ever worked in agriculture, but less than 10% had ever manipulated pesticides; in both cases the proportions were substantially higher for men. Among women, more than three out of four had ever lactated.
Although a few subjects were recruited during the last quarter of 1992 or first quarter of 1996, most blood samples were more or less evenly distributed during the 3-year period 1993–1995. The most frequent compound found in serum was p,p0 -DDE, present in 98% of the samples, followed by b-HCH and HCB, found in 89% and 77% of samples, respectively (Table 2). On the contrary, p,p0 -DDT could be measured only in about one quarter of subjects, and thus its median and geometric means were not computed. The geometric means of serum concentrations (ng/g lipid) were 822, 167 and 379 for p,p0 -DDE, b-HCH and HCB, respectively; except for p,p0 -DDE, the concentrations were higher for women than for men. The serum concentrations of the three selected pesticides according categories of selected variables are shown in Table 3; the corresponding geometric means are mutually adjusted by sex, age, centre, BMI and year of blood collection. As already stated, the levels were higher among women except for p,p0 -DDE. On the other hand, the serum levels of all the compounds analyzed significantly increased with age and with BMI, and decreased with the year of blood collection. Noticeably, although the recruitment of subjects did not last over a long period, there was a substantial decrease ranging from the 13% for p,p0 -DDE to 33% for b-HCH and almost 50% for HCB for samples taken with an average difference of 3 years. There are two clear geographical patterns, depending on the compounds considered. One of the northern regions (Asturias) shows the lowest level for the three pesticides. However, for p,p0 DDE and b-HCH the highest levels were observed in Murcia, followed by Granada, both in the Mediterranean coast at the south of Spain. On the contrary, for HCB, Navarra had concentration remarkably higher than any other centre, and levels were also elevated in Guipúzcoa, both in the north and near to each other. The
Table 1 Main characteristics of the study population. Men (479) N Centre Asturias Guipúzcoa Navarra Granada Murcia
Women (474) (%)
N
Total (953) (%)
N
(%)
98 94 99 89 99
(20.5) (19.6) (20.7) (18.6) (20.7)
92 97 95 94 96
(19.4) (20.5) (20.0) (19.8) (20.3)
190 191 194 183 195
(19.9) (20.0) (20.4) (19.2) (20.5)
Age group (years) 35–44 45–54 55–64
185 159 135
(38.6) (33.2) (28.2)
190 161 123
(40.1) (34.0) (25.9)
375 320 258
(39.3) (33.6) (27.1)
Body mass index (kg m2) 625 25–30 >30
72 280 126
(15.1) (58.6) (26.4)
136 193 145
(28.7) (40.7) (30.6)
208 473 271
(21.8) (49.7) (28.5)
Year of blood collection 1992–93 1994 1995–96
160 178 141
(33.4) (37.2) (29.4)
141 186 147
(29.7) (39.2) (31.0)
301 364 288
(31.6) (38.2) (30.2)
125 164 67 39 81 145 68
(26.3) (34.5) (14.1) (8.2) (17.0) (30.3) (14.2)
177 206 28 25 37 76 17
(37.4) (43.6) (5.9) (5.3) (7.8) (16.0) (3.6)
302 370 95 64 118 221 85
(31.8) (39.0) (10.0) (6.7) (12.4) (23.2) (8.9)
106 362
(22.6) (77.4)
Educational level No formal education Primary school Secondary school Technical degree University degree Ever worked in agriculture Ever manipulated pesticides Breastfeeding Never breastfed Ever breastfed
Proportions calculated over the subjects with valid information. Missing information: body mass index, one man; educational level, three men and one woman; breastfeeding six women. The ‘never breastfed’ category includes 58 nulliparous women and 48 parous women who never breastfed.
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P. Jakszyn et al. / Chemosphere 76 (2009) 1518–1524 Table 2 Serum concentrations (ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort. Na
(%)
Geometric meanb (95% CI)
Median (95% CI)
p75 (95% CI)
Men (479) p,p0 -DDTc p,p0 -DDE b-HCH HCB
129 473 417 371
(26.9) (98.7) (87.1) (77.5)
– 888.8 155.4 300.6
– (827.0–955.1) (140.7–171.7) (272.7–331.4)
– 936.5 193.5 377.7
(852.2–1000.0.1) (176.1–220.5) (359.8–402.7)
42.9 1454.6 363.4 585.6
(36.7–66.1) (1317.3–1617.6) (330.2–392.6) (554.7–631.8)
Women (474) p,p0 -DDTc p,p0 -DDE b-HCH HCB
123 461 435 358
(25.9) (97.3) (91.8) (75.5)
– 759.7 180.5 479.1
– (702.1–822.1) (162.7–200.3) (438.5–523.4)
– 789.1 248.8 554.2
(744.2–861.7) (221.1–277.6) (520.5–585.9)
44.9 1333.4 408.8 790.8
(41.4–60.5) (1238.4–1415.6) (381.8–454.1) (742.3–851.8)
Total (953) p,p0 -DDTc p,p0 -DDE b-HCH HCB
252 934 852 729
(26.4) (98.0) (89.4) (76.5)
– 822.1 167.4 379.0
(779.2–867.2) (155.8–179.9) (354.3–405.4)
– 857.9 221.0 462.5
(802.6–912.5) (196.0–243.0) (434.1–495.2)
43.9 1372.3 387.9 693.3
(41.4–54.8) (1311.0–1471.1) (364.5–416.5) (657.6–735.6)
a b c
Number and proportion of subjects whose serum levels could be measured (value above the LOQ of 0.4 ng/mL on serum basis). Geometric means computed over all subjects assigning 0.2 ng/mL to those with levels below the LOQ. For p,p0 -DDT less than 30% of subjects had quantifiable serum levels (above LOQ), and thus the geometric mean and the median are not shown.
Table 3 Serum concentrations (multivariate adjusted means as ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort according to selected variables. Geometric mean (95% confidence interval)a p,p0 -DDE Sex
Centre
Age group (years)
Body mass index (kg/m2)
Year of blood collection
Breastfeeding,b years since breastfeeding a b c
Men Women p-Value Asturias Guipúzcoa Navarra Granada Murcia p-Value 35–44 45–54 55–64 p-Trend 625 25-30 >30 p-Trend 1993-94 1994 1995-96 p-Trend Never Yes, <10 Yes, P10 p-Valuec
888.0 759.7 0.014 608.1 664.5 750.2 957.4 1287.2 <0.001 716.6 835.5 981.9 <0.001 632.4 840.9 964.7 <0.001 887.8 812.4 768.9 0.001 715.2 683.2 791.9 0.71 (0.40)
HCB
b-HCH (828.4–952.0) (708.5–814.7) (544.6–679.0) (595.3–741.8) (672.6–836.7) (855.3–10,716) (1154.4–1435.3) (662.5–775.1) (767.3–909.7) (893.3–1079.4) (569.1–702.7) (784.1–901.8) (879.6–1058.0) (813.2–969.3) (750.1–879.7) (703.0–841.0) (614.7–832.2) (559.5–834.2) (723.8–866.4)
156.0 180.5 0.005 107.3 135.5 162.1 170.8 325.2 <0.001 119.1 179.9 253.4 <0.001 120.2 159.1 237.6 <0.001 201.9 170.0 136.0 <0.001 165.4 116.2 203.1 0.18 (0.08)
(142.7–170.5) (165.1–197.4) (93.2–123.5) (117.7–156.0) (141.0–186.4) (147.9–197.3) (282.9–373.7) (107.7–131.6) (161.3–200.6) (224.5–286.0) (105.1–137.6) (145.5–174.0) (211.1–267.3) (180.4–225.8) (153.6–188.3) (121.3–152.5) (136.8–200.1) (90.4–149.2) (181.4–227.4)
300.6 479.1 <0.001 259.2 485.1 722.2 310.3 273.8 <0.001 282.4 402.8 539.7 <0.001 265.9 364.4 533.5 <0.001 477.2 427.8 256.1 <0.001 413.6 260.1 566.8 <0.001 (<0.001)
(278.0–325.0) (442.9–518.2) (229.0–293.5) (428.7–549.0) (638.8–816.5) (273.4–352.2) (242.3–309.5) (258.6–308.5) (366.0–443.2) (485.2–600.3) (236.1–299.3) (336.8–394.1) (480.9–591.8) (432.3–526.7) (391.1–467.8) (231.6–283.2) (358.1–477.6) (215.1–314.5) (520.4–617.4)
Geometric means (95% CI) adjusted by centre, sex, age, body mass index and year of blood collection. Includes 106 women who never breastfed and 362 who ever breastfed, classified according to time since breastfeeding: 61 less than 10 years, 301 ten years or more. In brackets, p-value for the comparison of means by time since breastfeeding (<10 versus 10 or more years), excluding those who never breastfed.
serum level of pesticides for women who ever breastfed their children were not different from those who did not so. Among women who had lactated the average duration of breastfeeding was approximately 1 year (median 7.5 months), but the levels of OCPs were not related to duration. However, the levels were lower for those who had breastfed within the last 10 years, as compared with those who lactated more than 10 years ago, mainly for bHCH and HCB, although only for the latter the difference was statistically significant. There were no differences in the pesticide serum levels regarding previous work in agriculture or occupational exposure to pesticides (results not shown). Finally, we checked all two-way interactions between the main demographic and anthropometric variables, most of them being nonsignificant.
There was only a significant interaction between gender and age group for p,p0 -DDE and HCB. For p,p0 -DDE, the serum concentrations were higher among men in subjects ageing 35–44 years and 45–54 years, but it was the opposite in older subjects (55– 64 years). Regarding HCB, the increasing concentration with age was attenuated among men, yielding a p-trend = 0.08. The levels of pesticides, adjusted by age, sex, centre, BMI and year of recruitment, were not associated with any dietary factor. These analyses were performed both with the dietary factors as continuous variables in grams per day, and with the intake categorized in quartiles. Comparisons for all food groups were carried out, but only selected groups of foods from animal origin are presented in Table 4. Although these products are thought to be the main
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Table 4 Serum concentrations (multivariate adjusted means as ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort according to quartiles of intake of selected food groups. Food group
Fish and fish products
White fishb
Blue fishb
Meat and meat products
Dairy products
Foods from animal originc
Intake (grams/day)
634.7 34.8–56.0 56.1–84.6 P84.7 p-Trend 66.1 6.2–21.8 21.9–41.2 P42.4 p-Trend 62.3 2.4–12.8 12.9–27.3 P27.4 p-Trend 686.9 87.0–123.9 124.0–172.6 P172.7 p-Trend 6178.4 178.5–268.9 270.0–387.9 P388.0 p-Trend 6392.9 393.0–497.8 497.9–622.5 P622.6 p-Trend
Geometric meana p,p0 -DDE
b-HCH
HCB
847.6 787.9 806.1 846.6 0.64 1081.4 767.4 760.3 716.1 0.10 819.7 865.6 790.5 812.8 0.54 831.7 826.1 806.0 822.9 0.71 876.9 829.4 852.9 734.3 0.10 901.7 797.0 843.7 751.2 0.23
187.3 172.2 148.5 165.3 0.46 209.7 164.9 143.8 158.0 0.70 189.5 179.8 144.7 160.6 0.54 186.8 177.9 149.6 159.2 0.97 163.2 173.2 168.9 165.9 0.85 175.8 166.0 182.9 148.3 0.88
403.8 370.5 347.6 397.1 0.82 316.3 331.6 441.3 448.4 0.57 444.7 374.7 349.3 354.6 0.31 375.7 404.7 403.1 337.1 0.20 380.0 370.8 352.2 416.3 0.90 377.1 382.2 402.4 356.1 0.36
a Geometric means adjusted by centre, sex, age, body mass index and year of blood collection. b Classification based on the fat content (per 100 g) of fish: white fish <4 g, blue fish P4 g. c Includes intake of fish and fish products, meat and meat products, dairy products and eggs.
source of organochlorine pesticides in the general population, the geometric means across quartiles of intake of fish, meat or dairy products did not show any clear pattern; neither there was a difference in concentrations when in the type of fish was considered nor when all foods from animal origin were grouped into a single variable. We also explored the variation in serum levels of pesticides by dietary factors adjusting by energy intake, as well as including interaction terms for age and gender in the model, but the results remained unchanged. 4. Discussion In this study we have shown that detectable concentrations of three selected pesticides or their metabolites (p,p0 -DDE, b-HCH and HCB) have been observed in 80–100% of the serum samples of healthy adults from five regions of Spain collected in 1993– 1995, while p,p0 -DDT was detected in less than 30% of subjects. One of the limitations in our data set is the fact that the cohort was not selected as a representative sample of the general population; however, it includes a broad study base, with subjects from different geographical areas, with a broad range of educational levels and occupations, and an age–sex structure similar to Spanish population aged 35–64 years. Thus the serum levels reported may be seen as indicators of serum level pesticides in the adult population of Spain. On the other hand, a relatively high proportion of blood samples were nonfasting (38%), but nonfasting samples are not likely to introduce a bias when lipid adjustment is per-
formed (Phillips et al., 1989); actually, we reported the OCPs serum levels as ng of the compound per gram of blood lipids. We consistently observed a positive correlation between age and blood levels of pesticides both in men and women. This phenomenon has been often observed (WHO, 2003), and can be seen as a function both of age itself and of a birth cohort effect: older people had a greater chance for high levels of exposure to these compounds before they were banned, whereas they also had a longer time to accumulate the compounds or their metabolites in their body. This highlights as well the relevance of the period of blood collection, as environmental exposure is assumed to decrease over time. We had a single measurement for each subject, so that individual patterns over time cannot be analyzed; nevertheless, we clearly observed a decrease in serum levels by year of blood collection in our population. Even for a relatively short period there such as 3 years in average a decrease in serum concentrations was found for all compounds, ranging from 13% for DDE to 50% for HCB. This measurement of exposure to banned pesticides seems to point out that measures have being reinforced in different parts of Spain in such a way as to progressively lower serum concentrations of OCPs in general populations. We compared our results with those from studies that reported lipid-adjusted concentrations of OCPs whose blood collection took place approximately during the same period with a clear reference to the age of subjects. Moreover, we restricted comparison to results from samples of general population, not selected on the basis potential exposure due to occupational or residential sources. Measurable levels of p,p0 -DDT were detected in 27% of our samples, similar to the 30% found in New Zealand (Bates et al., 2004); and higher than the 10% in the USA (CDC, 2005). In Sweden, 96% of men and about 80% of women had detectable serum levels of p,p0 -DDT (Glynn et al., 2000, 2007), while all individuals had measurable levels in Japan (Masuda et al., 2005). However, among subjects with measurable levels, exposure in our population tended to be relatively high; for instance, the maximum concentrations (ng/g lipid) were 124 in Sweden and 157 in Japan, lower than the percentile 95 in our study (256.5 ng/g lipid). Regarding p,p0 -DDE, the level of 822 ng/g lipid in our population were comparable to the concentration of 919 ng/g lipid measured in 1996–1997 in of New Zealand (Bates et al., 2004), but was consistently higher than levels within the range 200–600 found in Sweden (Glynn et al., 2000, 2007), the USA (CDC, 2005) and Japan (Masuda et al., 2005). As for b-HCH, only the Japanese, with concentration above 600 (Masuda et al., 2005) had higher levels than the subjects from our population, with 167 ng/g lipid; much lower values (below 40) were found in populations from Sweden, the USA or New Zealand (Glynn et al., 2000, 2007; Bates et al., 2004; CDC, 2005). Similarly, our concentration of serum (HCB of 379 ng/g lipid) was consistently higher than the levels below 80 in Sweden and Japan (Glynn et al., 2000, 2007; Masuda et al., 2005) and even undetectable in the USA (CDC, 2005) and New Zealand (Bates et al., 2004). The only Spanish study from a representative sample of general population was carried out in the Canary Islands (Zumbado et al., 2005); they measured DDT/DDE in serum samples collected in 1998 from subjects aged 6–75 years. Overall, 47% of population had detectable levels of p,p0 -DDT and about 100% of p,p0 -DDE. The DDT levels seem to be higher than in our population: the 75th percentile was 242 ng/g lipid, while in our study it was 42.9. On the contrary, they observed substantially lower DDE levels, with a median of 118 as compared with 858 ng/g lipid in our study. They found significantly higher level of DDT in islands with largest farming areas of intensive agriculture with plastic greenhouses than in the rest of the islands, where agriculture is mostly of the traditional type. A remarkable result of our study is the clear geographical pattern, with southern regions showing a significantly higher levels
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p,p0 -DDE and b-HCH. In the Murcia region, due to geographical and climatological reasons, agriculture has been the main resource for decades, with the intensive use of insecticides such as DDT in the past years and lindane more recently. The levels of DDT, lindane (c-HCH) and b-HCH in fat samples from patients residents in Murcia (Molina et al., 2005) were significantly higher in areas of intensive horticultural cultivation using traditional irrigation or extensive dry farming and greenhouse cultivation, both with high or moderate requirements of pesticides. On the contrary, the highest concentrations of HCB were observed in Navarra, located in the north. HCB had many uses in agriculture and industry in the past, but most of them have been discontinued after 1970. However, it may still be generated in the manufacture of chlorinated products including solvents, aromatic compounds and pesticides, and it may be present as contaminant in some other pesticides, as well as in emissions from waste incinerators (WHO, 2003). Nevertheless, we have not identified specific settings in this region which could explain its high HCB level as compared with other regions within Spain. One study measured several OCPs in serum samples collected in 1994 among inhabitants of a village (Flix, Catalonia, Spain), located in the vicinity of an organochlorine compound factory (Sala et al., 1999). They reported levels consistently higher than ours, with median 16.5 ng/ml, as compared the 4.7 ng/mL in Navarra. These results are expressed per volume of serum; thus, the influence of fasting or any other factor related to lipid content in blood cannot be accounted for. We observed a clear positive association between BMI and serum levels of the three analyzed OCPs. This is consistent with results of most cross-sectional studies, although the pattern may differ according to temporality of exposure (Wolff et al., 2005). During the uptake phase the correlations tend to be positive by simple dilution, as higher BMI implies larger adipose tissue. On the other hand, it has been proposed that BMI alters pharmacokinetics of the organochlorine compounds, so that the half-life is longer in obese than in lean individuals. Thus, as elimination occurs slowly among obese persons, after two or more half-lives the body burden becomes greater in subjects with higher BMI and the correlation shifts to positive (Wolff et al., 2005). Thus, strong positive correlations observed in our study suggest distant cessation of exposure at high levels of OCPs. Moreover, temporality of exposure may also explain the results observed among women regarding breastfeeding, often considered as the primary way of excretion of most of the organochlorine compounds. We did not observe an association of any OCPs with lactation overall. However, given the age of women from our population, the time elapsed between last breastfeeding and blood collection was in average 17 years. When we stratified parous women according to time since lactation, lower concentrations were observed among women who had breastfed within the previous ten years, although the difference was significant only for HCB. This suggests that OCPs intake subsequent to breastfeeding may have reduced its influence on serum levels after one or two half-lives and the effect of lactation is overcome by the accumulating effect of age. It is accepted that the major source of exposure to OCPs in general population is diet, mainly foods from animal origin. We did not find any relationship between OCPs levels and intake of any specific food item, or groups of them. For instance, the contribution of the main three groups of foods from animal origin (meat, fish and dairy products) to the explained variance of OCPs serum levels (R-square) was only of about 1%. Few studies have analyzed the relationship between serum levels of OCPs and dietary factors in the general population. One study carried out in a sample of elderly population in Germany reported a positive correlation between beef and lamb intake and serum levels of total DDT, b-HCH and HCB; b-HCH was also negatively associated with fruit consumption (DeVoto et al., 1998). Two American studies performed such anal-
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ysis in women included as controls in case-control studies of breast cancer. In one of them (Laden et al., 1999), serum DDE could not be predicted by dietary factors, including consumption of meat, fish, poultry, dairy products, vegetables, fruit and grain; in this study, fish intake was positively associated with PCB concentrations. The other American study (Moysich et al., 2002) found that serum DDE was positively associated with intake and HCB was negatively associated with consumption of red meat; for both compounds there was a weak positive association with fruit intake. The authors acknowledged that it was difficult to provide an explanation to such findings and that they needed to be further explored. Finally, a study in volunteers of a rural area from Japan (Hanaoka et al., 2002), weakly positive associations were found for rice and milk in relation with b-HCH, and for fish and rice in relation with HCB; no association was observed between dietary factors and DDE + DDT serum levels. The absence of association between diet and levels of OCPs could be related to the fact that the levels of such compounds in food supply have been decreasing over time; furthermore, subject’s diet may have changed during the years before assessment. Thus, levels of OCPs in blood could be related to diet in distant past rather than to current diet. Error measurement in dietary assessment based upon subject’s recall can never be ruled out; on the other hand, although we analyzed also specific food items, not only food groups or subgroups, lack of the relevant food items could, to some extent, explain our findings. Moreover, levels of OCPs in food may vary substantially depending on the source of the food; thus, our inability to discriminate between foods that were truly contaminated and those that did not could in part explain these results. In conclusion, we have reported relatively high levels of OCPs in serum samples of Spanish healthy adults collected during 1992– 1996. For DDE and b-HCB, the concentrations were higher in southern regions, most likely associated with intensive past use of pesticides related to agricultural practices. As for HCB, the serum levels were particularly elevated in one of the northern regions; this could be due to industrial use rather than agricultural application of such compound. We have not identified specific settings in this region as potential source of HCB contamination, and thus the high level of serum HCB in this population remains unexplained. No association was found between OCPs levels and dietary factors. The decrease in serum levels of OCPs according to the period of blood collection, together with the positive relationship with age and BMI suggest that exposure to such compounds is decreasing. However, the serum levels of b-HCH and especially of HCB are substantially higher than in most Western countries, and the health effects of chronic exposure to the current levels are still unknown. Therefore, it seems worthy further assessment both of body burden and potential health effects of such compounds in our population.
Acknowledgements Sources of financial support: this project was funded by the Health Research Fund (FIS) of the Spanish Ministry of Science and Innovation (Exp. 021598). The EPIC study received financial support from the European Commission (Agreement SO 97 200302 05F02); the participating Regional Governments; the Red Temática de Investigación Cooperativa de Centros de Cáncer (RTICCC, C03/10), the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation; and the International Agency for Research on Cancer (Agreement AEP/93/02). Some authors (PJ, CAG, AA) are members of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a Network of Excellence of the 6th EU Framework Programme (FP6, FOOD-CT-2005-513 943).
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Laden, F., Neas, L.M., Spiegelman, D., et al., 1999. Predictors of plasma concentrations of DDE and PCBs in a group of US women. Environ. Health Perspect. 107, 75–81. Masuda, Y., Haraguchi, K., Kono, S., Tsuji, H., Päpke, O., 2005. Concentrations of dioxins and related compounds in the blood of Fukuoka residents. Chemosphere 58, 329–344. Molina, C., Falcón, M., Barba, A., Cámara, M.A., Oliva, J., Luna, A., 2005. HCH and DDT residues in human fat in the population of Murcia (Spain). Ann. Agric. Environ. Med. 12, 133–136. Moysich, K.B., Ambrosone, C.B., Mendola, P., et al., 2002. Exposures associated with serum organochlorine levels among postmenopausal women from western New York State. Am. J. Ind. Med. 41, 102–110. Phillips, D.L., Pirkle, J.L., Burse, V.W., Bernert, J.T., Henderson, L.O., Needham, L.L., 1989. Chlorinated hydrocarbon levels in human serum: effects of fasting and feeding. Arch Environ. Contam. Toxicol. 18, 495–550. Porta, M., Puigdomènech, E., Ballester, F., et al., 2008. Studies conducted in Spain on concentrations in humans of persistent toxic compounds. Gac. Sanit. 22, 248– 266 (article in Spanish). Safe, S., 2004. Endocrine disruptors and human health: is there a problem. Toxicology 205, 3–10. Sala, M., Sunyer, J., Otero, R., Santiago-Silva, M., Camps, C., Grimalt, J., 1999. Organochlorine in the serum of inhabitants living near an electrochemical factory. Occup. Environ. Med. 5, 152–158. StataCorp. 2007. Stata Statistical Software: Release 10. StataCorp LP, College Station, TX. UNEP (United Nations Environment Programme Chemicals), 2005. Ridding the World of POPs: A Guide to the Stockholm Convention on Persistent Organic Pollutants. United Nations Environment Programme Chemicals, Geneva, Switzerland. Wahlefeld, A.W., 1974. Triglycerides determination after enzymatic hydrolysis. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, second ed. Academic Press Inc., New York, pp. 1831–1835. WHO (World Health Organization), 2003. Health Risks of Persistent Organic Pollutants from Lon-range Transboundary Air Pollution. The Regional Office for Europe of the World Health Organization, Copenhagen, Denmark. Wiebe, D.A., Bernert Jr., J.T., 1984. Influence of incomplete cholesteryl ester hydrolysis on enzymic measurements of cholesterol. Clin. Chem. 30, 352–356. Wolff, M.S., Britton, J.A., Teitelbaum, S.L., Eng, S., Deych, E., Ireland, K., et al., 2005. Improving organochlorine biomarker models for cancer research. Cancer Epidemiol. Biomarkers Prev. 14, 2224–2236. Zumbado, M., Goethals, M., Alvarez-León, E.E., Luzardo, O.P., Cabrera, F., SerraMajem, L., Domínguez-Boada, L., 2005. Inadvertent exposure to organochlorine pesticides DDT and derivatives in people from the Canary Islands (Spain). Sci. Total Environ. 339, 49–62.
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Serum levels of organochlorine pesticides in healthy adults from five regions of Spain Paula Jakszyn a, Fernando Goñi b,c, Arsenio Etxeandia d, Asunción Vives e, Esmeralda Millán f, Raul López c, Pilar Amiano c,g, Eva Ardanaz c,h, Aurelio Barricarte c,h, M. Dolores Chirlaque c,i, Miren Dorronsoro c,g, Nerea Larrañaga c,g, Carmen Martínez c,j, Carmen Navarro c,i, Laudina Rodríguez k, M. José Sánchez c,j, M. José Tormo c,i, Carlos A. González a, Antonio Agudo a,* a
Unit of Nutrition, Environment, and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (ICO), IDIBELL, L’Hospitalet de Llobregat 08907, Spain Laboratorio de Salud Pública de Guipúzcoa, 20013 San Sebastian, Spain CIBER Epidemiología y Salud Pública (CIBERESP), 20013 San Sebastian, Spain d Laboratorio de Salud Pública de Vizcaya, 48010 Bilbao, Spain e Laboratorio Unificado Donostia, Hospital N.S. Aranzazu, San Sebastian, Spain f Departamento de Química Aplicada, Universidad del País Vasco, Facultad de Química, San Sebastián, Spain g Dirección de Salud de Guipúzcoa, 20013 San Sebastian, Spain h Instituto de Salud Pública de Navarra, 31003 Pamplona, Spain i Consejería de Sanidad y Consumo, 30008 Murcia, Spain j Escuela Andaluza de Salud Pública, 18080 Granada, Spain k Consejería de Salud y Servicios Sanitarios de Asturias, 33001 Oviedo, Spain b c
a r t i c l e
i n f o
Article history: Received 29 January 2009 Received in revised form 28 May 2009 Accepted 29 May 2009 Available online 7 July 2009 Keywords: Pesticides Organochlorine compounds Serum levels Biomarkers
a b s t r a c t The aim of this study was to measure of serum levels of p,p0 -dichlorodiphenyl trichloroethane (p,p0 -DDT), p,p0 -dichlorodiphenyl dichlorethylene (p,p0 -DDE), b-hexachlorocyclohexane (b-HCH), and hexachlorobenzene (HCB) in healthy adults in Spain. Furthermore, we also analyzed these levels according to dietary, other lifestyle factors and anthropometric characteristics. We measured the concentrations of such organochlorine pesticides (OCPs) in serum samples collected during 1992–1996 from 953 subjects aged 35– 64 years, they were residents of five Spanish regions, they were randomly selected from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. OCPs were determined by means of gas chromatography with electron-capture detection (GC-ECD). The most frequent compound found in serum was p,p0 -DDE, present in 98% of the samples, followed by HCB and b-HCH, found in 89% and 77% of samples, respectively, while p,p0 -DDT could be measured only in 26% of subjects. The geometric means of serum concentrations (ng/g lipid) were 822 for p,p0 -DDE, 167 for b-HCH, and 379 for HCB. The concentrations of all OCPs were positively associated with age and body mass index, and decreased along the period of blood collection. No association was found between OCPs levels and dietary factors. The concentrations of p,p0 -DDE and b-HCB were higher in Murcia, one of southern regions, most likely associated with intensive past use of pesticides related to agricultural practices, while higher levels of HCB were found in Navarra, located in the north, maybe due to industrial use rather than agricultural application. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Organochlorine pesticides (OCPs) are a group of synthetic chemicals effective against a variety of insects, many of which are highly persistent in the environment. Among them, p,p0 -dichlo-
* Corresponding author. Unit of Nutrition, Environment, and Cancer, Cancer Epidemiology Research Program, Catalan Institute of Oncology (ICO). Av. Gran Via 199-203, 08907 L’Hospitalet de Llobregat, Spain. Tel.: +34 932607401; fax: +34 932607787. E-mail address: [email protected] (A. Agudo). 0045-6535/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2009.05.048
rodiphenyl trichloroethane (p,p0 -DDT) and its metabolite p,p0 dichlorodiphenyl dichlorethylene (p,p0 -DDE), b-hexachlorocyclohexane (b-HCH, the isomeric form of HCH with longest half-life), and hexachlorobenzene (HCB) are some of the organochlorine compounds most commonly found (WHO, 2003). Although OCPs are primarily environmental pollutants, they are very resistant to degradation and highly lipophilic, and consequently they become part of the food chain, where they tend to bioaccumulate, mainly in fatty foods. Thus, the main source of exposure in the general population is the diet (WHO, 2003). The current background body burden of OCPs is still of concern because
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of their estrogenic properties with potential adverse effects both to the environment and human (Safe, 2004). On the other hand, even though there is sufficient evidence of carcinogenicity for experimental animals, the epidemiological evidence in humans is inadequate for the three groups of compounds, and they are classified by the International Agency for Research on Cancer in the group 2B, as possibly carcinogenic to humans (IARC, 1987; IARC, 1991; IARC, 2001). However, the health effects of chronic exposure to OCPs on the general population at the current levels are still unknown. Because of its persistence and bioaccumulation, as well as growing concern about adverse effects, a list of twelve organochlorine compounds were the objects of international restrictions, defined by the treaty called Stockholm Convention, aimed to eliminations of such compounds (UNEP, 2005). DDT and b-HCB are included in this list, but HCH is not covered by the Stockholm Convention, so the use of HCH in parts of the world is continuing. In Spain, although most OCPs were banned during the 1970s and the levels of such compounds seems to have decreased, most OCPs are often found in food, and detectable concentrations of some of these have been observed in a high proportion among the population (Porta et al., 2008). Most OCPs can be measured in relatively small amounts of serum, and because of their long half-life, often of about 10 years, these levels may be considered good indicators of long-term exposure. However, comprehensive monitoring in the general population in Spain is scarce, and most studies focused on specific groups exposed because of occupation or residence (Porta et al., 2008). The aim of our study was to measure the serum levels of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB in healthy adults belonging to the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Furthermore, we also aimed to analyze these levels according to dietary and other lifestyle factors and anthropometric measurements.
2. Methods 2.1. Study population The EPIC-Spain cohort consisted of 15 632 men and 25 806 women aged 29–69 years, with different social and education levels. Detailed description of methods and population has been published elsewhere (González et al., 2004). Briefly, participants were recruited between 1992 and 1996 among healthy volunteers from five Spanish regions, three from the North (Asturias, Navarra, and Guipúzcoa) and two from the South (Murcia and Granada). At recruitment all the participants provided information on diet and other lifestyle factors, anthropometric measurements, and a sample of 30 mL of blood. All participants gave their informed consent and the study was approved by the Ethical Review Board. For this study, a random sample of 200 subjects from each centre was selected, stratified according to the age and sex structure of the Spanish population, aged 35–64 years. Biological material was not available for 14 individuals, or was of poor quality or insufficient amount to obtain valid measurement in another 33 individuals; thus, the final population of study included 953 subjects. 2.2. Diet and lifestyle questionnaires and anthropometry The usual food intake during the preceding year, taking into account seasonal variations, was estimated by personal interview using a computerized dietary history questionnaire, developed and validated specifically for the EPIC study in Spain (EPIC Group of Spain, 1997). The questionnaire was structured according to occasions of food intake and included a list of more than 600 foods and beverages including local recipes. A questionnaire adminis-
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tered by an interviewer was used to collect information on sociodemographic characteristics, and work and leisure physical activity. The section dealing with current or past occupational exposures included an item asking whether the subject had ever worked in agriculture; those who answered yes to this question were also asked whether this job involved manipulation of pesticides. For women reproductive history was collected as well; this section gathered information about each live birth, including date of childbirth, whether the woman breastfed the newborn (either as exclusive or as complementary nourishment), and duration of breastfeeding. Thus, we estimated the cumulative duration of breastfeeding by adding up weeks or months of lactation periods from different newborns, as well as the time in years elapsed since cessation of the last period of breastfeeding. Weight and height were also taken at recruitment following standardized procedures, and were then used to compute the body mass index (BMI) as kg m2. 2.3. Analytical methods Blood samples were divided into 0.5 mL aliquots of serum, plasma, concentrated red blood cells, and buffy coat, and stored in liquid nitrogen at 196 °C. An improved method for the determination of organochlorine pesticides was used to measure serum levels of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB (Goñi et al., 2007). The method required that low volume of serum (500 lL) and 48–96 samples per day be prepared by one analyst without special automatic equipment. Initial extraction was performed using 96-well solid-phase extraction disk plates and was followed by a clean-up with silica gel/sulfuric acid. Quantification was carried out by gas chromatography with electron-capture detector (GC-ECD). A mass spectrometer detector (GC–MSD) was used for quantitative and qualitative confirmation. Intra-day relative standard deviation of p,p0 -DDT, p,p0 -DDE, b-HCH and HCB varied from 1% to 11%, depending on compound. Inter-day relative standard deviation (measured over a yearlong period) was <15% in all cases. Total cholesterol (TC) and triglycerides (TG) were determined enzymatically by developing colored compounds that were measured spectrophotometrically (Wahlefeld, 1974; Wiebe and Bernert, 1984). Total serum lipids (TL) were calculated from total cholesterol and triglycerides (all of them expressed as g/L) by applying the formula TL = 2.27 TC + TG + 0.623 (Phillips et al., 1989). Then, we used this result to report the serum pesticides on lipid basis. The limit of quantification (LOQ) was 0.4 ng/mL on serum basis; for subjects whose values were below the detection level, a concentration of 0.2 ng/mL (half the LOQ) was assigned. 2.4. Statistical analysis The serum levels of each pesticide were expressed as lipid-corrected concentrations in ng/g lipid. Since the distributions of concentrations were right skewed, the variables were transformed using natural logarithms. For descriptive purposes, apart from the geometric mean, the median and the 75th percentile, with their corresponding 95% confidence intervals (CI), were reported for the whole sample and separately for men and women. Given the sampling framework, all the means reported may be considered standardized according to the age–sex structure of the Spanish population 35–64 years old. Analysis of covariance was used to examine how the log-transformed serum concentration of each OCP varied by demographic, anthropometric, dietary and lifestyle variables (Armitage and Berry, 1994). Geometric means of lipidcorrected concentrations of each OCP were calculated from multiple linear regression models with log-transformed concentration (as ng/g lipid) as the dependent variable; these geometric means are adjusted by all the covariates included in the model. The linear
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trend for ordered variables was assessed by the significance of the coefficient including in the model such variables using the numerical value corresponding to each category. Two-way interactions between the main variables were assessed by means of the likelihood ratio test (LRT), comparing the likelihood of the model with the corresponding interaction terms and the model containing terms for the main effects only (Armitage and Berry, 1994). Regression modeling was not applied to p,p0 -DDT, given the high proportion (73.6%) of subjects with values below the LOQ. Since the total lipids and individual components of lipids may not behave the same way in models, regression modeling was also performed using the volume concentration of OCPs (as lg/ml) and including the concentration of triglycerides and cholesterol as covariates. Nevertheless, as this approach yielded the same results as the one using the lipid-corrected concentration only the latter is presented. All the statistical analyses were carried with the release 10 of STATA (StataCorp, 2007). 3. Results Overall, serum samples from 953 subjects aged 35–64 years old from five Spanish regions were analyzed to quantify the four selected pesticides. Table 1 shows the main socio-demographic characteristics and other factors of the study population. The mean age was 49 years (48.7 for women and 49.4 for men). About half of subjects had overweight (BMI 25–30) or were obese (BMI > 30), and about one third had not completed the primary school or lacked formal education. Almost one quarter of subjects had ever worked in agriculture, but less than 10% had ever manipulated pesticides; in both cases the proportions were substantially higher for men. Among women, more than three out of four had ever lactated.
Although a few subjects were recruited during the last quarter of 1992 or first quarter of 1996, most blood samples were more or less evenly distributed during the 3-year period 1993–1995. The most frequent compound found in serum was p,p0 -DDE, present in 98% of the samples, followed by b-HCH and HCB, found in 89% and 77% of samples, respectively (Table 2). On the contrary, p,p0 -DDT could be measured only in about one quarter of subjects, and thus its median and geometric means were not computed. The geometric means of serum concentrations (ng/g lipid) were 822, 167 and 379 for p,p0 -DDE, b-HCH and HCB, respectively; except for p,p0 -DDE, the concentrations were higher for women than for men. The serum concentrations of the three selected pesticides according categories of selected variables are shown in Table 3; the corresponding geometric means are mutually adjusted by sex, age, centre, BMI and year of blood collection. As already stated, the levels were higher among women except for p,p0 -DDE. On the other hand, the serum levels of all the compounds analyzed significantly increased with age and with BMI, and decreased with the year of blood collection. Noticeably, although the recruitment of subjects did not last over a long period, there was a substantial decrease ranging from the 13% for p,p0 -DDE to 33% for b-HCH and almost 50% for HCB for samples taken with an average difference of 3 years. There are two clear geographical patterns, depending on the compounds considered. One of the northern regions (Asturias) shows the lowest level for the three pesticides. However, for p,p0 DDE and b-HCH the highest levels were observed in Murcia, followed by Granada, both in the Mediterranean coast at the south of Spain. On the contrary, for HCB, Navarra had concentration remarkably higher than any other centre, and levels were also elevated in Guipúzcoa, both in the north and near to each other. The
Table 1 Main characteristics of the study population. Men (479) N Centre Asturias Guipúzcoa Navarra Granada Murcia
Women (474) (%)
N
Total (953) (%)
N
(%)
98 94 99 89 99
(20.5) (19.6) (20.7) (18.6) (20.7)
92 97 95 94 96
(19.4) (20.5) (20.0) (19.8) (20.3)
190 191 194 183 195
(19.9) (20.0) (20.4) (19.2) (20.5)
Age group (years) 35–44 45–54 55–64
185 159 135
(38.6) (33.2) (28.2)
190 161 123
(40.1) (34.0) (25.9)
375 320 258
(39.3) (33.6) (27.1)
Body mass index (kg m2) 625 25–30 >30
72 280 126
(15.1) (58.6) (26.4)
136 193 145
(28.7) (40.7) (30.6)
208 473 271
(21.8) (49.7) (28.5)
Year of blood collection 1992–93 1994 1995–96
160 178 141
(33.4) (37.2) (29.4)
141 186 147
(29.7) (39.2) (31.0)
301 364 288
(31.6) (38.2) (30.2)
125 164 67 39 81 145 68
(26.3) (34.5) (14.1) (8.2) (17.0) (30.3) (14.2)
177 206 28 25 37 76 17
(37.4) (43.6) (5.9) (5.3) (7.8) (16.0) (3.6)
302 370 95 64 118 221 85
(31.8) (39.0) (10.0) (6.7) (12.4) (23.2) (8.9)
106 362
(22.6) (77.4)
Educational level No formal education Primary school Secondary school Technical degree University degree Ever worked in agriculture Ever manipulated pesticides Breastfeeding Never breastfed Ever breastfed
Proportions calculated over the subjects with valid information. Missing information: body mass index, one man; educational level, three men and one woman; breastfeeding six women. The ‘never breastfed’ category includes 58 nulliparous women and 48 parous women who never breastfed.
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P. Jakszyn et al. / Chemosphere 76 (2009) 1518–1524 Table 2 Serum concentrations (ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort. Na
(%)
Geometric meanb (95% CI)
Median (95% CI)
p75 (95% CI)
Men (479) p,p0 -DDTc p,p0 -DDE b-HCH HCB
129 473 417 371
(26.9) (98.7) (87.1) (77.5)
– 888.8 155.4 300.6
– (827.0–955.1) (140.7–171.7) (272.7–331.4)
– 936.5 193.5 377.7
(852.2–1000.0.1) (176.1–220.5) (359.8–402.7)
42.9 1454.6 363.4 585.6
(36.7–66.1) (1317.3–1617.6) (330.2–392.6) (554.7–631.8)
Women (474) p,p0 -DDTc p,p0 -DDE b-HCH HCB
123 461 435 358
(25.9) (97.3) (91.8) (75.5)
– 759.7 180.5 479.1
– (702.1–822.1) (162.7–200.3) (438.5–523.4)
– 789.1 248.8 554.2
(744.2–861.7) (221.1–277.6) (520.5–585.9)
44.9 1333.4 408.8 790.8
(41.4–60.5) (1238.4–1415.6) (381.8–454.1) (742.3–851.8)
Total (953) p,p0 -DDTc p,p0 -DDE b-HCH HCB
252 934 852 729
(26.4) (98.0) (89.4) (76.5)
– 822.1 167.4 379.0
(779.2–867.2) (155.8–179.9) (354.3–405.4)
– 857.9 221.0 462.5
(802.6–912.5) (196.0–243.0) (434.1–495.2)
43.9 1372.3 387.9 693.3
(41.4–54.8) (1311.0–1471.1) (364.5–416.5) (657.6–735.6)
a b c
Number and proportion of subjects whose serum levels could be measured (value above the LOQ of 0.4 ng/mL on serum basis). Geometric means computed over all subjects assigning 0.2 ng/mL to those with levels below the LOQ. For p,p0 -DDT less than 30% of subjects had quantifiable serum levels (above LOQ), and thus the geometric mean and the median are not shown.
Table 3 Serum concentrations (multivariate adjusted means as ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort according to selected variables. Geometric mean (95% confidence interval)a p,p0 -DDE Sex
Centre
Age group (years)
Body mass index (kg/m2)
Year of blood collection
Breastfeeding,b years since breastfeeding a b c
Men Women p-Value Asturias Guipúzcoa Navarra Granada Murcia p-Value 35–44 45–54 55–64 p-Trend 625 25-30 >30 p-Trend 1993-94 1994 1995-96 p-Trend Never Yes, <10 Yes, P10 p-Valuec
888.0 759.7 0.014 608.1 664.5 750.2 957.4 1287.2 <0.001 716.6 835.5 981.9 <0.001 632.4 840.9 964.7 <0.001 887.8 812.4 768.9 0.001 715.2 683.2 791.9 0.71 (0.40)
HCB
b-HCH (828.4–952.0) (708.5–814.7) (544.6–679.0) (595.3–741.8) (672.6–836.7) (855.3–10,716) (1154.4–1435.3) (662.5–775.1) (767.3–909.7) (893.3–1079.4) (569.1–702.7) (784.1–901.8) (879.6–1058.0) (813.2–969.3) (750.1–879.7) (703.0–841.0) (614.7–832.2) (559.5–834.2) (723.8–866.4)
156.0 180.5 0.005 107.3 135.5 162.1 170.8 325.2 <0.001 119.1 179.9 253.4 <0.001 120.2 159.1 237.6 <0.001 201.9 170.0 136.0 <0.001 165.4 116.2 203.1 0.18 (0.08)
(142.7–170.5) (165.1–197.4) (93.2–123.5) (117.7–156.0) (141.0–186.4) (147.9–197.3) (282.9–373.7) (107.7–131.6) (161.3–200.6) (224.5–286.0) (105.1–137.6) (145.5–174.0) (211.1–267.3) (180.4–225.8) (153.6–188.3) (121.3–152.5) (136.8–200.1) (90.4–149.2) (181.4–227.4)
300.6 479.1 <0.001 259.2 485.1 722.2 310.3 273.8 <0.001 282.4 402.8 539.7 <0.001 265.9 364.4 533.5 <0.001 477.2 427.8 256.1 <0.001 413.6 260.1 566.8 <0.001 (<0.001)
(278.0–325.0) (442.9–518.2) (229.0–293.5) (428.7–549.0) (638.8–816.5) (273.4–352.2) (242.3–309.5) (258.6–308.5) (366.0–443.2) (485.2–600.3) (236.1–299.3) (336.8–394.1) (480.9–591.8) (432.3–526.7) (391.1–467.8) (231.6–283.2) (358.1–477.6) (215.1–314.5) (520.4–617.4)
Geometric means (95% CI) adjusted by centre, sex, age, body mass index and year of blood collection. Includes 106 women who never breastfed and 362 who ever breastfed, classified according to time since breastfeeding: 61 less than 10 years, 301 ten years or more. In brackets, p-value for the comparison of means by time since breastfeeding (<10 versus 10 or more years), excluding those who never breastfed.
serum level of pesticides for women who ever breastfed their children were not different from those who did not so. Among women who had lactated the average duration of breastfeeding was approximately 1 year (median 7.5 months), but the levels of OCPs were not related to duration. However, the levels were lower for those who had breastfed within the last 10 years, as compared with those who lactated more than 10 years ago, mainly for bHCH and HCB, although only for the latter the difference was statistically significant. There were no differences in the pesticide serum levels regarding previous work in agriculture or occupational exposure to pesticides (results not shown). Finally, we checked all two-way interactions between the main demographic and anthropometric variables, most of them being nonsignificant.
There was only a significant interaction between gender and age group for p,p0 -DDE and HCB. For p,p0 -DDE, the serum concentrations were higher among men in subjects ageing 35–44 years and 45–54 years, but it was the opposite in older subjects (55– 64 years). Regarding HCB, the increasing concentration with age was attenuated among men, yielding a p-trend = 0.08. The levels of pesticides, adjusted by age, sex, centre, BMI and year of recruitment, were not associated with any dietary factor. These analyses were performed both with the dietary factors as continuous variables in grams per day, and with the intake categorized in quartiles. Comparisons for all food groups were carried out, but only selected groups of foods from animal origin are presented in Table 4. Although these products are thought to be the main
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Table 4 Serum concentrations (multivariate adjusted means as ng/g lipid) of pesticides in healthy adults from the EPIC-Spain cohort according to quartiles of intake of selected food groups. Food group
Fish and fish products
White fishb
Blue fishb
Meat and meat products
Dairy products
Foods from animal originc
Intake (grams/day)
634.7 34.8–56.0 56.1–84.6 P84.7 p-Trend 66.1 6.2–21.8 21.9–41.2 P42.4 p-Trend 62.3 2.4–12.8 12.9–27.3 P27.4 p-Trend 686.9 87.0–123.9 124.0–172.6 P172.7 p-Trend 6178.4 178.5–268.9 270.0–387.9 P388.0 p-Trend 6392.9 393.0–497.8 497.9–622.5 P622.6 p-Trend
Geometric meana p,p0 -DDE
b-HCH
HCB
847.6 787.9 806.1 846.6 0.64 1081.4 767.4 760.3 716.1 0.10 819.7 865.6 790.5 812.8 0.54 831.7 826.1 806.0 822.9 0.71 876.9 829.4 852.9 734.3 0.10 901.7 797.0 843.7 751.2 0.23
187.3 172.2 148.5 165.3 0.46 209.7 164.9 143.8 158.0 0.70 189.5 179.8 144.7 160.6 0.54 186.8 177.9 149.6 159.2 0.97 163.2 173.2 168.9 165.9 0.85 175.8 166.0 182.9 148.3 0.88
403.8 370.5 347.6 397.1 0.82 316.3 331.6 441.3 448.4 0.57 444.7 374.7 349.3 354.6 0.31 375.7 404.7 403.1 337.1 0.20 380.0 370.8 352.2 416.3 0.90 377.1 382.2 402.4 356.1 0.36
a Geometric means adjusted by centre, sex, age, body mass index and year of blood collection. b Classification based on the fat content (per 100 g) of fish: white fish <4 g, blue fish P4 g. c Includes intake of fish and fish products, meat and meat products, dairy products and eggs.
source of organochlorine pesticides in the general population, the geometric means across quartiles of intake of fish, meat or dairy products did not show any clear pattern; neither there was a difference in concentrations when in the type of fish was considered nor when all foods from animal origin were grouped into a single variable. We also explored the variation in serum levels of pesticides by dietary factors adjusting by energy intake, as well as including interaction terms for age and gender in the model, but the results remained unchanged. 4. Discussion In this study we have shown that detectable concentrations of three selected pesticides or their metabolites (p,p0 -DDE, b-HCH and HCB) have been observed in 80–100% of the serum samples of healthy adults from five regions of Spain collected in 1993– 1995, while p,p0 -DDT was detected in less than 30% of subjects. One of the limitations in our data set is the fact that the cohort was not selected as a representative sample of the general population; however, it includes a broad study base, with subjects from different geographical areas, with a broad range of educational levels and occupations, and an age–sex structure similar to Spanish population aged 35–64 years. Thus the serum levels reported may be seen as indicators of serum level pesticides in the adult population of Spain. On the other hand, a relatively high proportion of blood samples were nonfasting (38%), but nonfasting samples are not likely to introduce a bias when lipid adjustment is per-
formed (Phillips et al., 1989); actually, we reported the OCPs serum levels as ng of the compound per gram of blood lipids. We consistently observed a positive correlation between age and blood levels of pesticides both in men and women. This phenomenon has been often observed (WHO, 2003), and can be seen as a function both of age itself and of a birth cohort effect: older people had a greater chance for high levels of exposure to these compounds before they were banned, whereas they also had a longer time to accumulate the compounds or their metabolites in their body. This highlights as well the relevance of the period of blood collection, as environmental exposure is assumed to decrease over time. We had a single measurement for each subject, so that individual patterns over time cannot be analyzed; nevertheless, we clearly observed a decrease in serum levels by year of blood collection in our population. Even for a relatively short period there such as 3 years in average a decrease in serum concentrations was found for all compounds, ranging from 13% for DDE to 50% for HCB. This measurement of exposure to banned pesticides seems to point out that measures have being reinforced in different parts of Spain in such a way as to progressively lower serum concentrations of OCPs in general populations. We compared our results with those from studies that reported lipid-adjusted concentrations of OCPs whose blood collection took place approximately during the same period with a clear reference to the age of subjects. Moreover, we restricted comparison to results from samples of general population, not selected on the basis potential exposure due to occupational or residential sources. Measurable levels of p,p0 -DDT were detected in 27% of our samples, similar to the 30% found in New Zealand (Bates et al., 2004); and higher than the 10% in the USA (CDC, 2005). In Sweden, 96% of men and about 80% of women had detectable serum levels of p,p0 -DDT (Glynn et al., 2000, 2007), while all individuals had measurable levels in Japan (Masuda et al., 2005). However, among subjects with measurable levels, exposure in our population tended to be relatively high; for instance, the maximum concentrations (ng/g lipid) were 124 in Sweden and 157 in Japan, lower than the percentile 95 in our study (256.5 ng/g lipid). Regarding p,p0 -DDE, the level of 822 ng/g lipid in our population were comparable to the concentration of 919 ng/g lipid measured in 1996–1997 in of New Zealand (Bates et al., 2004), but was consistently higher than levels within the range 200–600 found in Sweden (Glynn et al., 2000, 2007), the USA (CDC, 2005) and Japan (Masuda et al., 2005). As for b-HCH, only the Japanese, with concentration above 600 (Masuda et al., 2005) had higher levels than the subjects from our population, with 167 ng/g lipid; much lower values (below 40) were found in populations from Sweden, the USA or New Zealand (Glynn et al., 2000, 2007; Bates et al., 2004; CDC, 2005). Similarly, our concentration of serum (HCB of 379 ng/g lipid) was consistently higher than the levels below 80 in Sweden and Japan (Glynn et al., 2000, 2007; Masuda et al., 2005) and even undetectable in the USA (CDC, 2005) and New Zealand (Bates et al., 2004). The only Spanish study from a representative sample of general population was carried out in the Canary Islands (Zumbado et al., 2005); they measured DDT/DDE in serum samples collected in 1998 from subjects aged 6–75 years. Overall, 47% of population had detectable levels of p,p0 -DDT and about 100% of p,p0 -DDE. The DDT levels seem to be higher than in our population: the 75th percentile was 242 ng/g lipid, while in our study it was 42.9. On the contrary, they observed substantially lower DDE levels, with a median of 118 as compared with 858 ng/g lipid in our study. They found significantly higher level of DDT in islands with largest farming areas of intensive agriculture with plastic greenhouses than in the rest of the islands, where agriculture is mostly of the traditional type. A remarkable result of our study is the clear geographical pattern, with southern regions showing a significantly higher levels
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p,p0 -DDE and b-HCH. In the Murcia region, due to geographical and climatological reasons, agriculture has been the main resource for decades, with the intensive use of insecticides such as DDT in the past years and lindane more recently. The levels of DDT, lindane (c-HCH) and b-HCH in fat samples from patients residents in Murcia (Molina et al., 2005) were significantly higher in areas of intensive horticultural cultivation using traditional irrigation or extensive dry farming and greenhouse cultivation, both with high or moderate requirements of pesticides. On the contrary, the highest concentrations of HCB were observed in Navarra, located in the north. HCB had many uses in agriculture and industry in the past, but most of them have been discontinued after 1970. However, it may still be generated in the manufacture of chlorinated products including solvents, aromatic compounds and pesticides, and it may be present as contaminant in some other pesticides, as well as in emissions from waste incinerators (WHO, 2003). Nevertheless, we have not identified specific settings in this region which could explain its high HCB level as compared with other regions within Spain. One study measured several OCPs in serum samples collected in 1994 among inhabitants of a village (Flix, Catalonia, Spain), located in the vicinity of an organochlorine compound factory (Sala et al., 1999). They reported levels consistently higher than ours, with median 16.5 ng/ml, as compared the 4.7 ng/mL in Navarra. These results are expressed per volume of serum; thus, the influence of fasting or any other factor related to lipid content in blood cannot be accounted for. We observed a clear positive association between BMI and serum levels of the three analyzed OCPs. This is consistent with results of most cross-sectional studies, although the pattern may differ according to temporality of exposure (Wolff et al., 2005). During the uptake phase the correlations tend to be positive by simple dilution, as higher BMI implies larger adipose tissue. On the other hand, it has been proposed that BMI alters pharmacokinetics of the organochlorine compounds, so that the half-life is longer in obese than in lean individuals. Thus, as elimination occurs slowly among obese persons, after two or more half-lives the body burden becomes greater in subjects with higher BMI and the correlation shifts to positive (Wolff et al., 2005). Thus, strong positive correlations observed in our study suggest distant cessation of exposure at high levels of OCPs. Moreover, temporality of exposure may also explain the results observed among women regarding breastfeeding, often considered as the primary way of excretion of most of the organochlorine compounds. We did not observe an association of any OCPs with lactation overall. However, given the age of women from our population, the time elapsed between last breastfeeding and blood collection was in average 17 years. When we stratified parous women according to time since lactation, lower concentrations were observed among women who had breastfed within the previous ten years, although the difference was significant only for HCB. This suggests that OCPs intake subsequent to breastfeeding may have reduced its influence on serum levels after one or two half-lives and the effect of lactation is overcome by the accumulating effect of age. It is accepted that the major source of exposure to OCPs in general population is diet, mainly foods from animal origin. We did not find any relationship between OCPs levels and intake of any specific food item, or groups of them. For instance, the contribution of the main three groups of foods from animal origin (meat, fish and dairy products) to the explained variance of OCPs serum levels (R-square) was only of about 1%. Few studies have analyzed the relationship between serum levels of OCPs and dietary factors in the general population. One study carried out in a sample of elderly population in Germany reported a positive correlation between beef and lamb intake and serum levels of total DDT, b-HCH and HCB; b-HCH was also negatively associated with fruit consumption (DeVoto et al., 1998). Two American studies performed such anal-
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ysis in women included as controls in case-control studies of breast cancer. In one of them (Laden et al., 1999), serum DDE could not be predicted by dietary factors, including consumption of meat, fish, poultry, dairy products, vegetables, fruit and grain; in this study, fish intake was positively associated with PCB concentrations. The other American study (Moysich et al., 2002) found that serum DDE was positively associated with intake and HCB was negatively associated with consumption of red meat; for both compounds there was a weak positive association with fruit intake. The authors acknowledged that it was difficult to provide an explanation to such findings and that they needed to be further explored. Finally, a study in volunteers of a rural area from Japan (Hanaoka et al., 2002), weakly positive associations were found for rice and milk in relation with b-HCH, and for fish and rice in relation with HCB; no association was observed between dietary factors and DDE + DDT serum levels. The absence of association between diet and levels of OCPs could be related to the fact that the levels of such compounds in food supply have been decreasing over time; furthermore, subject’s diet may have changed during the years before assessment. Thus, levels of OCPs in blood could be related to diet in distant past rather than to current diet. Error measurement in dietary assessment based upon subject’s recall can never be ruled out; on the other hand, although we analyzed also specific food items, not only food groups or subgroups, lack of the relevant food items could, to some extent, explain our findings. Moreover, levels of OCPs in food may vary substantially depending on the source of the food; thus, our inability to discriminate between foods that were truly contaminated and those that did not could in part explain these results. In conclusion, we have reported relatively high levels of OCPs in serum samples of Spanish healthy adults collected during 1992– 1996. For DDE and b-HCB, the concentrations were higher in southern regions, most likely associated with intensive past use of pesticides related to agricultural practices. As for HCB, the serum levels were particularly elevated in one of the northern regions; this could be due to industrial use rather than agricultural application of such compound. We have not identified specific settings in this region as potential source of HCB contamination, and thus the high level of serum HCB in this population remains unexplained. No association was found between OCPs levels and dietary factors. The decrease in serum levels of OCPs according to the period of blood collection, together with the positive relationship with age and BMI suggest that exposure to such compounds is decreasing. However, the serum levels of b-HCH and especially of HCB are substantially higher than in most Western countries, and the health effects of chronic exposure to the current levels are still unknown. Therefore, it seems worthy further assessment both of body burden and potential health effects of such compounds in our population.
Acknowledgements Sources of financial support: this project was funded by the Health Research Fund (FIS) of the Spanish Ministry of Science and Innovation (Exp. 021598). The EPIC study received financial support from the European Commission (Agreement SO 97 200302 05F02); the participating Regional Governments; the Red Temática de Investigación Cooperativa de Centros de Cáncer (RTICCC, C03/10), the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation; and the International Agency for Research on Cancer (Agreement AEP/93/02). Some authors (PJ, CAG, AA) are members of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a Network of Excellence of the 6th EU Framework Programme (FP6, FOOD-CT-2005-513 943).
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