Estimated daily intake of pesticides and xenoestrogenic exposure by fruit consumption in the female population from a Mediterranean country (Spain)

Food Control 21 (2010) 471–477

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Estimated daily intake of pesticides and xenoestrogenic exposure by fruit consumption in the female population from a Mediterranean country (Spain) S. Iñigo-Nuñez a,*, M.A. Herreros a, T. Encinas b, A. Gonzalez-Bulnes b,c a

Direccion General de Ordenacion e Inspeccion, Comunidad de Madrid, 28037 Madrid, Spain Dpto. Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain c Dpto. Reproduccion Animal INIA, 28040 Madrid, Spain b

a r t i c l e

i n f o

Article history: Received 3 February 2009 Received in revised form 13 July 2009 Accepted 20 July 2009

Keywords: Pesticides Fruit consumption Dietary intake Xenoestrogens Spain

a b s t r a c t The presence and concentrations of a total of 100 pesticides in apple samples (n = 30) and 65 in orange juice samples (n = 19) were determined in markets in Madrid (Spain). The presence of at least one pesticide residue was detected in 87% (26 of 30) of samples of apples and 16% (3 of 19) of orange juice samples; orange juices contained only residues from a single pesticide (organophosphates), whilst nearly 75% (19 of 26) of apples showing residues contained more than one compound (organochlorines, organophosphates, carbamates, pyrethroids and others). However, overall, the estimated daily intakes (EDIs) of the different pesticides, from fruit consumption, in Spanish female population were negligible; although is concerning that prepubertal girls accounted for the highest percentages. The analysis of the estimated estrogenic intake also showed minor exposure to pesticides; in this case, the highest intake occurring in perimenopausal women, while the lowest intake happened at childbearing age. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The hypothesis of endocrine-disrupting chemicals or contaminants (EDCs), the existence of various chemical contaminants with deleterious effects on reproductive outputs of humans and animals, through an endocrine-like action, was put forward in 1992 (Colborn & Clement, 1992). These authors suggested that numerous xenobiotic chemicals released into environment, food products and drinking water by the human activity, had the potential to disrupt the endocrine system of wildlife and humans at ecologically relevant concentrations. Thus, a world-wide concern regarding possible association of human health problems with exposure to EDCs arose (US Environmental Protection Agency, 1997; EU Commission, 1997, 1999a). The interest being mainly focused on the activity of the so called xenoestrogens (Mueller, 2004); due to their possible links with idiopathic infertility (Foster, 2003; Massaad et al., 2002) and other hormone-related diseases like breast cancer and endometriosis (Davis et al., 1993; Rudel, 1997). Pesticides of different chemical structure (e.g. organochlorines, organophosphates and carbamates) have been reported as xenoestrogens (Fénichel & Brucker-Davis, 2008; Singleton & Khan, 2003). The endocrine-disrupting action was firstly established in orga* Corresponding author. Tel.: +34 91 205 23 08; fax: +34 91 205 12 13. E-mail address: [email protected] (S. Iñigo-Nuñez). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.07.009

nochlorines (Bustos, Denegri, Diaz, & Tchernitchin, 1988; Gaido et al., 1998). More recently, the organophosphates chlorpyrifos and diazinon, the pyrethroid cypermethrin and other compounds such as thiabendazole have been also reported as showing estrogenic activity (Andersen, Vinggaard, Rasmussen, Gjermandsen, & Bonefeld-Jorgensen, 2002; Kojima, Katsura, Takeuchi, Niiyama, & Kobayashi, 2004; Kojima et al., 2005). Chronic intake of pesticides through food consumption during lifespan may have a negative impact on reproductive health of both males (Balash, Al-Omar, & Abdul, 1987; LeBlanc, Bain, & Wilson, 1997) and females (Ahlborg et al., 1995; Hunter & Kelsey, 1993; Zumbado et al., 2005), particularly if exposure occurs during vulnerable periods of development. Specifically for female population, the critical phases for estrogenic exposure (known as ‘‘windows of vulnerability”) are the period from adrenarche to first ovulatory cycles and even up to first pregnancy, as well as later in perimenopausal periods (EU Commission, 1999b). Exposure to pesticides through consumption of fruits is almost continuous, either as a result of direct treatment or due to environmental or cross contamination. Fruits are usually subjected to preand post-harvest treatments. Organophosphates, carbamates and pyrethroids are routinely applied to fruit crops for broad spectrum insect control (Rawn, Roscoe, Krakalovich, & Hanson, 2004; Rawn et al., 2006); organochlorines and other compounds are mainly

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used as post-harvest treatments for fungi control, especially in fruits intended for direct human consumption (Kupferman, 1984; Kupferman, 1986; Northwest Horticultural Council, 1992; Park, Peterson, Zhao, & Coats, 2004). Fruits (and derivatives like fruit juices) are a traditional part of the Mediterranean diet. The consumption of fruits in Spain (1792 g per person and week; Ministerio de Agricultura, 2004) is higher than in other countries like United Kingdom (1206 g per person and week; Defra, 2003) and the most demanded fruits are orange and apples (Consumer, 2008). In the region of Madrid, fruit consumption is above the Spanish average, 2027 g per person and week (http://www.mapa.es/es/alimentacion/pags/consumo) and, thus, becomes a good model for evaluation of pesticide contamination. The objectives of current study were: (a) to determine types and levels of pesticides in fruits placed on the market in a Mediterranean country (Spain), and (b) to estimate the subsequent daily intake in female consumers of different ages and, thus, their risk of exposure. 2. Materials and methods 2.1. Sampling Sampling was performed in retailers, the final stages of the food chain, where posterior pesticide treatments are not likely and where the consumers have direct access to foodstuffs. The two fruits with higher consumption, oranges and apples, were chosen for testing; the study of pesticide residues in oranges was performed using juice, since this fruit is always peeled before consumption. Thus, in the framework of an official surveillance programme, 30 samples of apples and 19 of orange juice were taken in dealers of Madrid region. Composite samples of apples were obtained, each one containing up to 10 units, to weight at least 1 kg. The samples of orange juice weighted at least 1 l. 2.2. Analysis of pesticide residues The presence and concentrations of a total of 100 pesticides in non-peeled apple samples and 65 in orange juice samples were measured by using multiresidue methods; choice of pesticides was based in its routinely use in the field. Compounds included organochlorines (7), organophosphates (37 in apples, 32 in juice), carbamates (17 in apples; 0 in juice), pyrethroids (12) and other pesticides (27 in apples; 14 in juice), as detailed in Table 1. Analytical methods were based in chromatography. In brief, for chromatographic analysis, samples were firstly extracted and purified with 1 ml of NaOH (1 M) solution, 500 ll of acetonitrile and 2 ml of n-hexane. The mixture was vigorously vortex-mixed for 2 min and centrifuged at 2500 g for 5 min. The upper phase was transferred to another glass test tube and the lower phase was extracted again with 2 ml of n-hexane. The upper organic phase was added to the first one and dried under nitrogen atmosphere at 40 °C. For liquid chromatography, the dried extracts were reconstituted in 250 ll of acetonitrile, vortex, mixed for 20 s and transferred to auto-sampler vials. The chromatographic system consisted of Spectra-physic Series (Thermo Scientific, Essex, UK) components including a pump (P100), an auto-sampler (AS1000) and a variable UV wavelength detector (UV100) set at 210 nm. For gas chromatography, the dried extracts were derivatized with 50 ml TMSI, heated up to 80 °C in a water bath for 30 min and, thereafter, left to cool down at room temperature before 1 ml hexane and 1 ml distilled water were added and stirred at each step. After the phases were separated, the organic phase was collected in a vial with anhydrous sodium sulfate for elimination of residual water

and 1 ml of this being injected into the chromatograph. The chromatographic analysis was performed with a Hewlett–Packard Series II 5890 gaschromatograph, equipped with a nickel detector of electron capture. The injector and detector temperatures were 250 °C and 260 °C, respectively; the carrier gas was nitrogen at 1 atm of pressure. All chemicals were of HPLC grade and obtained from Sigma–Aldrich (Madrid, Spain). Limit of detection (LOD) ranged from 0.01 to 0.04 lg/g according to each respective compound. 2.3. Data analysis 2.3.1. Estimation of daily intake The ‘‘estimated daily intake” (EDI) of pesticides was calculated, in agreement with international guidelines (FAO, 2002; WHO, 1997), using the following equation:

EDI ¼ R  C  EP  PF=BW

lg=kg body weight=day

ð1Þ

where R denotes the mean concentration of the residue in a food commodity (lg/g); C represents the daily consumption (g/person/ day); EP indicates the edible portion (from 0 to 1) and PF refers to the processing factor for this food commodity (from 0 to 1). In this study, since the proportion of non-detected results was greater than about 80%, two simple estimates of the mean concentration were calculated (WHO, 1995) by setting all non-detectable results to LOD (upper bound) and to zero (lower bound). Thus a range of EDI, expressed in lg/kg body weight/day for a better comparison between groups, was obtained for each pesticide. Average food consumption and mean body weights in female population were extrapolated and analyzed from individual data (Comunidad de Madrid, 1994; Ministerio de Sanidad y Consumo, 1994) and grouped into the four female sub-populations that are more sensitive to estrogenic exposure (prepubertal, pubertal, childbearing and perimenopausal ages; EU Commission, 1999b), as detailed in Table 2. With regard to calculation of the edible portion, it was established in 0.89 for apples (FAO/WHO, 2003) and 1 for orange juice. Finally, the effects of processing factors were not taken into account in any case (PF = 1). The EDI was compared with the acceptable daily intake (ADI), meaning the daily dosage of a chemical which, during the entire lifetime, appears to be without appreciable risk on the basis of all the facts known at the time (FAO/WHO, 1965). 2.3.2. Estimation of the xenoestrogenic exposure by pesticides intake The xenoestrogenic exposure by fruit consumption was extrapolated by determining the estrogenic activity from in vitro assays, as previously described (Fang et al., 2000). Extrapolation was performed in the 4 main estrogenic pesticides (chlorpyrifos, diazinon, cypermethrin and thiabendazole; Kojima et al., 2004, 2005). The estimated daily intake of each one was converted into molarity (mol/kg body weight/day), in order to compare them, within pesticides and within age groups, with levels showing estrogenic activity by in vitro assays. 3. Results 3.1. Pesticide residues The presence of at least one pesticide residue was detected in 87% (26 of 30) of samples of apples and 16% (3 of 19) of orange juice samples. Orange juices contained only residues from one pesticide, whilst nearly 75% (19 of 26) of apples showing residues contained more than one compound per sample (Fig. 1). A total of 19 different compounds were detected in apples (two organochlorines, seven organophosphates, three carbamates, two pyrethroids

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S. Iñigo-Nuñez et al. / Food Control 21 (2010) 471–477 Table 1 Highlight of the different pesticides analyzed in apples and orange juices in current study. Organochlorines

Organophosphates

Carbamates

Others

Captan + folpet Chlorobenzilate Chlorothalonil Dichlofuanid Dicofol (op´ + pp´) a-Endosulfan + b-endosulfan + endosulfan sulfate HCH (a + b + d)

Acephate Azinphos ethyl Azinphos methyl Bromophos ethyl Carbophenothion Chlorpyrifos Chlorpyrifos methyl Chlorfenvinphos Coumaphos Diazinon Dichlorvos Dimethoate Disulfoton Ethion Fenamiphos Fenitrothion Fenthion Formothion Phosphamidon Malathion Mecarbam Methamidophos Methidathion Mevinphos Omethoate Parathion ethyl Parathion methyl Phorate Phosalone Phosmet Pyrazophos Pirimiphos methyl Profenofos Pyridafenthion Thiometon Tolclofos methyl Triazophos

Aldicarb Bendiocarb Butocarboxim Butoxycarboxim Carbaryl Carbofuran Carbosulfan Dithiocarbamates (CS2) Ethiofencarb Methiocarb Methomyl Oxamyl Pirimicarb Carbendazim + benomyl Promecarb Propoxur Thiodicarb

Azoxystrobin Benalaxyl Bromopropylate Buprofezin Captafol Chlozolinate Diphenylamine Fenarimol Fluorochloridone Hexaconazole Imazalil Iprodione Kresoxim-methyl Metalaxyl Myclobutanil Nuarimol Ofurace Penconazole Prochloraz Procymidone Propyzamide Tetradifon Thiabendazole Tolylfluanid Triadimefon Triadimenol Vinclozolin

Pyrethroids Acrinathrin Alphamethrin Bifenthrin Cyfluthrin Cypermethrin Deltamethrin Fenpropathrin Fenvalerate Flucythrinate Fluvalinate Lambda-cyhalothrin Permethrin

Table 2 Mean body weight and food consumption in Spanish female sub-populations grouped by age and considering lower and upper bounds. Female sub-populations (range; years old)

Body weight (range; kg)

Prepubertal (6–10) Pubertal (11–14) Childbearing (25–44) Perimenopausal (45–55) a

24.65–37.51 41.73–53.27 57.40–60.41 63.80

Food consumption (range; g/person/day) Applesa

Orange juice

211–260 206–238 195–259 322

84–117 111–130 35–49 36

Data calculated considering an edible percentage of 89%.

4% 4% 4% 27%

1 compound 2 compounds

12%

3 compounds 4 compounds 5 compounds

15%

6 compounds 7 compounds

nylamine and chlorpyrifos (detected in around 20% of the samples). The highest concentrations corresponded to compounds used in post-harvest treatments (diphenylamine, 1.3 lg/g, thiabendazole 0.6 lg/g, and imazalil, 0.6 lg/g). In all the cases, pesticides concentrations found in our study were below the Maximum Residue Limits (MRL) set by the European Union legislation (EU Council, 1990). The pesticides found in orange juice were mainly the organophosphates chlorpyrifos (5.3% of the samples) and diazinon (10.5% of the samples), as shown in Fig. 2.

34% Fig. 1. Relative percentage of samples of apples showing residues from one or more pesticides.

and five of other categories), while only two organophosphates were found in orange juice (Fig. 2). In apples, the most abundant pesticides were captan and folpet (detected in more than 40% of the samples) and phosalone, diphe-

3.2. Estimated daily intake The ‘‘estimated daily intake” (EDI) was calculated and compared to the ‘‘acceptable daily intake” (ADI) for all the detected pesticides; except for dithiocarbamates, since no ADI has been established for these compounds. The EDI in female population ranged from 0 to 2.48 lg/kg body weight/day, depending on the pesticide and the consumers group of age (Table 3, presenting data

S. Iñigo-Nuñez et al. / Food Control 21 (2010) 471–477 50

50

40

40

% Samples > LOD

30 20 10 0

30 20 10

Organochl.

Carbamate

Pyret.

Chlorpyrifos

Thiabendazole

Procymidone

Iprodione

Imazalil

Diphenylamine

Deltamethrin

Cypermethrin

Pirimicarb

Dithiocarbamates

Phosmet

Organophosphorus

Carbendazim + Benomyl

Phosalone

Fenitrothion

Diazinon

Chlorpyrifos methyl

Chlorpyrifos

Azinophos methyl

Chlorothalonil

Captan + Folpet

0 Diazinon

% Samples > LOD

474

Organoph.

Others

0,04

1,4

1,2 0,03 Concentration (ug/g)

Concentration (ug/g)

1

0,8 0,6

0,4

0,02

0,01 0,2

Orgchlor.

Organophosphorus

Carbamate Low er bound

Diazinon

0 Chlorpyrifos

Thiabendazole

Procymidone

Iprodione

Imazalil

Diphenylamine

Deltamethrin

Cypermethrin

Pirimicarb

Dithiocarbamates

Carbendazim + Benomyl

Phosmet

Phosalone

Diazinon

Fenitrothion

Chlorpyrifos methyl

Chlorpyrifos

Azinophos methyl

Chlorothalonil

Captan + Folpet

0

Organophosphorus Pyret. Upper bound

Others Minimum

Maximum

Fig. 2. Frequencies of contamination (percentage of samples above limit of detection; upper panels) and concentration (lg/g; lower panels) of different pesticides in apples (left hand) and orange juice (right hand).

from the Joint FAO/WHO Meetings on Pesticide Residues and the Joint FAO/WHO Committee on Food Additives). The highest absolute intakes corresponded to captan and folpet, followed by diphenylamine and thiabendazole. Considering the range of ages of the female population, the highest relative contributions to ADI were in prepubertal subpopulation. In this group of women females, the pesticides with a higher contribution to ADI were diazinon (around 13% of ADI), followed by azinophos methyl (6%), chlorpyriphos and fenitrothion (5%), phosmet (4%), and phosalone (3%); chlorpyrifos methyl, captan + folpet, imazalil and deltamethrin contributed to 2% of ADI; procymidone represented 0% of ADI, and the remaining pesticides provided 1% of ADI (Table 3).

3.3. Estimation of the xenoestrogenic exposure by pesticides intake The estimated estrogenic intake (EEI) from exposure to pesticides in female population was determined both by the range of age and the compound (‘). Overall, the highest intake occurred in perimenopausal women, while the lowest intake happened at

childbearing age; the lowest ingested pesticide was cypermethrin (6.2  1010 mol/body weight/day in childbearing ages), the highest was thiabendazole (1.3  107 in perimenopausal women). The highest intake for chlorpyrifos, cypermethrin and thiabendazole (4.1  108; 2.3  108 and 1.3  107 mol/body weight/ day, respectively) was at least 100-fold below levels of established in vitro estrogenic activity (7.5  106; 8.1  106 and 2.2  105 mol/kg, respectively). For diazinon, the maximum intake (2.3  108 mol/kg body weight/day) was 2000-fold below estrogenic activity in vitro (4.6  104 mol/kg).

4. Discussion In the current study, the presence of residues in apples (87%) was higher than previously reported Spanish and European averages (47% and 59%, respectively; EU Commission, 2006). On the other hand, in orange juice, current data were similar to the mean percentage in Europe, but significantly lower than in the rest of Spain (43%). In our sampling, orange juices contained only residues from one pesticide (either chlorpyrifos or diazinon), whilst nearly

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Table 3 Acceptable daily intake (ADI in lg/kg body weight/day), pesticide estimated daily intake in female sub-populations grouped by age and percentage of ADI in prepubertal population (ADI-P). JMPR is the Joint FAO/WHO Meetings on Pesticide Residues; JECFA is the Joint FAO/WHO Committee on Food Additives. Group

Compound

ADI (lg/kg bw/day) (source; year)

Estimate of pesticide dietary intake in female sub-populations (ranges; lg/kg bw/day) Prepubertal

Pubertal

Childbearing

Perimenopausal

Organochlorines

Captan + folpet Chlorothalonil

100 (JMPR; 1995) 30 (JMPR; 1992)

1.34–2.48 0.05–0.28

0.97–1.17 0.04–0.13

0.76–1.01 0.03–0.11

1.13–1.19 0.04–0.13

2 1

Organophosphorus

Azinophos methyl Chlorpyrifos Chlorpyrifos methyl Diazinon Fenitrothion Phosalone Phosmet

5 (JMPR; 1991) 10 (JMPR; 1999) 10 (JMPR; 1992) 2 (JMPR; 1993) 5 (JMPR; 2000) 20 (JMPR; 2001) 10 (JMPR; 1998)

0.01–0.32 0.08–0.47 0.01–0.22 0.01–0.25 0.03–0.25 0.18–0.57 0.07–0.41

0.00–0.15 0.06–0.22 0.01–0.10 0.01–0.13 0.03–0.12 0.13–0.27 0.05–0.19

0.00–0.13 0.05–0.19 0.01–0.09 0.00–0.09 0.02–0.10 0.10–0.23 0.04–0.17

0.01–0.15 0.07–0.22 0.01–0.11 0.01–0.11 0.03–0.12 0.15–0.27 0.06–0.20

6 5 2 13 5 3 4

Carbamates

Carbendazim + benomyl

0.03–0.43

0.02–0.20

0.02–0.18

0.03–0.21

ADI-P (%)

Pirimicarb

30 (JMPR; 1995) 100 (JMPR; 1995) 20 (JMPR; 1982)

0.00–0.21

0.00–0.10

0.00–0.08

0.00–0.10

1 0 1

Pyrethroids

Cypermethrin Deltamethrin

20 (JECFA; 2004) 10 (JMPR; 2000)

0.01–0.32 0.00–0.21

0.01–0.15 0.00–0.10

0.00–0.13 0.00–0.09

0.01–0.15 0.00–0.10

1 2

Others

Diphenylamina Imazalil Iprodione Procymidone Thiabendazole

80 (JMPR; 1998) 30 (JMPR; 2001) 60 (JMPR; 1995) 200 JMPR; 1989) 100 (JECFA; 1992)

0.48–1.02 0.19–0.52 0.01–0.32 0.06–0.30 0.29–0.89

0.35–0.48 0.14–0.24 0.01–0.15 0.04–0.14 0.21–0.42

0.27–0.41 0.11–0.21 0.00–0.13 0.03–0.12 0.17–0.36

0.41–0.49 0.16–0.25 0.01–0.15 0.05–0.14 0.25–0.42

1 2 1 0 1

Table 4 Estrogenic activity determined by in vitro test and estimated intake (mol/person/day) of estrogenic pesticides, grouped in female sub-populations by age, by consumption of apples and orange juices from Madrid marketplace. Compound

Molecular weight (g)

Estrogenic activity in vitro (M mol/kg)

Estimate of estrogenic daily intake in female sub-populations (ranges; mol/person/day) Prepubertal

Pubertal

Childbearing

Perimenopausal

Chlorpyrifos Diazinon Cypermethrin Thiabendazole

350.59 304.35 416.30 201.25

7.5  106 a 4.6  104 b 8.1  106 a 2.2  105 b

8.4  109–3.3  108 1.5  109–2.0  108 6.8  1010–1.9  108 5.2  108–1.1  107

8.2  109–3.0  108 1.6  109–2.1  108 6.6  1010–1.7  108 5.1  108–9.9  108

7.8  109–3.3  108 9.5  1010–1.8  108 6.2  1010–1.9  108 4.8  108–1.1  107

1.3  108–4.1  108 1.1  109–2.3  108 1.0  109–2.3  108 7.9  108–1.3  107

a b

Concentration of test compound showing 20% of the agonistic activity (ERa) of 1010 M E2 (17b-estradiol) (Kojima et al., 2004). Concentration of test compound showing a response of 10%, taking the E2 maximal response as 100%; the EC10 of E2 was 3.2  1012 M (Kojima et al., 2005).

75% of apples contained more than one compound. Other authors have also found similar data in commercial fruit juice (Picó & Kozmutza, 2007) and apples (Cesnik, Gregorcic, Bolta, & Kmecl, 2006; Stepán, Tichá, Hajslová, Kovalczuk, & Kocourek, 2005). These differences between apples and orange may be related to the fact that taking composite samples at the final stages of the food chain allows the mixing of apples from different origins. In addition, the presence of background levels of organophosphates (chlorpyrifos and diazinon) in apples may account for final results (Rawn et al., 2006). The presence of organophosphates insecticides and fungicides, such as the organochlorine captan, have been also reported as the most frequent residues in previous studies in apples (Stepán et al., 2005); however, previous reports (Cesnik et al., 2006) also indicated higher frequencies of other compounds like dithiocarbamates. The highest concentrations corresponded to compounds used in post-harvest treatments (diphenylamine, thiabendazole and imazalil). In all the cases, pesticides concentrations found in our study were below the Maximum Residue Limits (MRL) set by the European Union legislation (EU Council, 1990); in contrast to previously published data, though MRL was always exceeded in only a small percentage of samples (1.4%, Stepán et al., 2005; 2%, Cesnik et al., 2006). The presence of the organophosphates chlorpyrifos and diazinon has been also reported in previous studies in oranges; with some samples containing high levels of diazinon (Torres, Picó, Marín, & Mañes, 1997) and chlorpyrifos (Fernández, Picó, & Mañes, 2001; Ortelli, Edder, & Corvi, 2005). However, thia-

bendazole and imazalil have been also found (Fernández et al., 2001; Ortelli et al., 2005). A possible explanation for the differences between these results and current study may be related to the fact that oranges are peeled and squeezed for juice (Rasmusssen, Poulsen, & Hansen, 2003); thus, pesticides on the surface of the fruit can be significantly reduced in the oranges, resulting in lower levels in the commercial juice. The evaluation of the ‘‘estimated daily intake” (EDI) also showed that organophosphates reached the highest percentages of the EDI, representing 13% in the case of diazinon; the occasional exceeding of the acute dose for this group has been previously reported in children (Fenske, Kedan, Lu, Fisker-Andersen, & Curl, 2002). However, overall, the EDIs of the different products were negligible when expressed as percentage of the ADIs, which is similar to intakes estimated by other authors when considering only fruit consumption (Berrada, Fernández, Ruiz, Moltó, & Mañes, 2006; Poulsen, Hansen, Sloth, Christensen, & Andersen, 2007). In current report, prepuberal girls accounted for the highest percentages of ADIs, in agreement with recent data from Lorenzin, (2007), whilst analysis of women in childbearing age showed the lowest percentages. We have to keep in mind, when evaluating contribution to ADI, that fruits and derived products represent around 21% and 24% of the total diet of Spanish adult and young females, respectively (Comunidad de Madrid, 1994; Ministerio de Sanidad y Consumo, 1994). Nevertheless, studies considering the total diet showed that pesticide usually concentrate in fruits (Fenske et al., 2002;

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Lorenzin, 2007); this food category bears the greatest number of compounds, but none persistent (Vicente et al., 2004). In agreement with this, the intakes based in complete meals from another Mediterranean country (Italy) reached higher averages in relation to ADI, but showed the same age distribution that our study (Lorenzin, 2007). Lower intakes were estimated from total diet in a Nordic country (Denmark, Juhler et al., 1999), maybe due to differences in using pesticides and food consumption. Overall, the estimated intake of estrogenic pesticides in females is not seem to be concerning for endocrine disruption. The estimation of xenoestrogenic risk in current study indicate that the exposure is low, but, moreover, we have to consider that the pesticides are metabolized and the final amounts at receptor levels are lower than at intake. However, there are several factors to be considered; currently, evaluation of health risks from mixtures of chemicals that act via estrogenic mechanisms is hampered (Borget, LaKind, & Witorsh, 2003). There are factors depending of the source, timing of exposure, the pesticide (and/ or the pesticide mixture) and the mechanisms of actions that have not been considered in current study but can modify the pesticides estrogenic effects. Firstly, we have to consider that fruits are only a part of total diet, where other foods may also act as sources of pesticide intake. The total intake of fruit is also very variable between individuals; variability in concentrations of single serving fruits (Fenske et al., 2002) may occasionally lead to higher levels of estrogenic exposure. Moreover, exposure to pesticides in fruits may be different through lifelong and, thus, pesticides intake in childrenhood might have important consequences (EU Commission, 1999a). Second, the pesticide or pesticide mixture, their kinetics, metabolism and action mechanisms, are crucial. In this way, the metabolites of thiabendazole, the compound with the highest intake, exhibits a lower estrogenic activity than the compound (Kojima et al., 2005). In mixtures, additivity, synergism and induction of estrogenicity may be considered. Besides additivity, there are many evidences of synergism, where the potency of two estrogenic compounds is higher than addition (LeBlanc et al., 1997). These effects are not only found when mixing pesticides with estrogenic activity. It is known that the estrogenic activity of some compounds (e.g. diazinon or thiabendazole) is increased when mixed with another non-estrogenic compound (Kojima et al., 2005); but, moreover, and estrogenicity may also occur when two or three pesticides without estrogenic activity are used together (Kortenkamp & Altenburger, 1999). Third, we have to be rational when analyzing in vitro results. There are differential mechanism of action and effects of xenoestrogens in vitro and in vivo. In vivo, there have been found interactions with more than one hormone receptor and modulation of multiple endocrine response pathways (Safe, 1998). Coincidentally, other works have shown a cross-talking between estradiol receptors and other receptors, such as the aryl hydrocarbon receptor (AhR); and even other receptors that bind natural dietary constituents, such as retinoic acid receptors (Safe et al., 2002). Both in vivo and in vitro data suggest that a differential interaction of receptor complexes containing different ligands (xenoestrogens or endogenous ovarian estrogens) with the multiple estrogen response elements present in mammalian systems (Stancel et al., 1995). In this way, chlorpyrifos and cypermethrin showed estrogenic activity in vitro on estrogen receptor a, but not on receptor b (Kojima et al., 2004). Finally, pesticides are not only showing estrogenic activity; in this sense, chlorpyrifos methyl has shown anti-androgenic activity, without estrogenic and anti-estrogenic activity (Kang et al., 2004). In conclusion, oranges and apples from Madrid markets showed traces of pesticides exposure; orange juices contained only residues from a single pesticide (organophosphates), whilst most of

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