Food and Chemical Toxicology 75 (2015) 156–165

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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x

Risk assessment for children exposed to DDT residues in various milk types from the Greek market Ioannis N. Tsakiris a, Marina Goumenou b, Manolis N. Tzatzarakis c, Athanasios K. Alegakis c, Christina Tsitsimpikou d, Eren Ozcagli e, Dionysios Vynias c, Aristidis M. Tsatsakis c,* a TEI of Western Macedonia, Florina Branch, Department of Agricultural Products Management and Quality Control, TermaKontopoulou, 53100 Florina, Greece b European Food Safety Authority, Parma, Italy c Laboratory of Toxicology, Department of Medicine, University of Crete, Voutes 71003, Heraklion, Greece d General Chemical State Laboratory of Greece, 16, An. Tsocha Str., 11521, Ampelokipi, Athens, Greece e Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Beyazit–Istanbul 34116, Turkey

A R T I C L E

I N F O

Article history: Received 29 September 2014 Accepted 11 November 2014 Available online 20 November 2014 Keywords: DDT Organochlorine pesticides Exposure assessment Risk assessment GC-MS Milk

A B S T R A C T

The occurrence of residues of DDT and its metabolites was monitored in 196 cow milk samples of various pasteurized commercial types collected from the Greek market. Residue levels were determined by GCMS analysis. In 97.4% of the samples at least one DDT isomer or one of the DDT metabolites was detected, in levels not exceeding the maximum permitted residue level by the EU. Hazard Index for both carcinogenic and non-carcinogenic effects was estimated under two assumptions: a) using DDT concentrations from positive samples and b) imputing LOD/2 as an arbitrary concentration for negative samples. No statistically significant differences in detected or summed residue (p > 0.05) concentrations between different milk types were observed, with the exception of specific metabolites of DDT in some milk types. Exposure assessment scenarios were developed for children aged 1, 3, 5, 7 and 12 years old based on estimated body weights and daily milk consumption. Hazard Indices for non-carcinogenic effects were below 0.109 covering also carcinogenic effects according to WHO approach. The cancer risk values for carcinogenic effects according to the US EPA Cancer Benchmark Concentration approach, ranged from 0.4 to 18. For both effects the highest values were calculated for the 1- to 3-year-old age groups. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Organochlorine pesticides (OCPs) are typically characterized by high vapor pressures and lipophilicity. They are persistent and highly stable under most environmental conditions. Additionally, their fat

Abbreviations: ADI, Acceptable Daily Intake; CBC, Cancer Benchmark Concentration; CI, confidence of interval; DDD, dichlorodiphenyldichloroethane (metabolite of DDT); DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (metabolite of DDT); DDT, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane; DDTs, sum of DDT and its metabolites; EDI, Estimated Daily Intake; GC-MS, gas chromatography–mass spectrometry; HI, Hazard Index; IRIS, Integrated Risk Information System; JMPR, Joint FAO-WHO Meeting of Pesticides Residues; MRLs, Maximum Residues Levels; OCPs, organochlorine pesticides; PTDI, Provisional Tolerable Daily Intake; RfD, Reference Dose; TCN, 1,2,3,4-tetrachloronaphthalene; UHT, Ultra High Temperature (milk treatment). * Corresponding author. Toxicology Laboratory, Medical School, University of Crete, Heraklion, 71409, P.O. Box 1393, Crete, Greece. Tel.: +30 (2810) 542098 or 394679; fax: +30 (2810) 394688 or 542098. E-mail address: [email protected] (A.M. Tsatsakis). http://dx.doi.org/10.1016/j.fct.2014.11.012 0278-6915/© 2014 Elsevier Ltd. All rights reserved.

solubility promotes bioaccumulation through the food chain (Pardio et al., 2003). OCP residues are regarded as severe environmental contaminants and they are toxic to both humans and animals. OCPs are also considered as endocrine-disrupting chemicals and carcinogenic compounds (Stoker et al., 2011). Dichlodiphenyl-trichloroethane (DDT), a previously widely used OCP, is officially classified according to EU regulation as suspected of causing cancer (Carc 2) and responsible for causing damage to organs through repeated and prolonged exposure (Annex VI of Regulation 1272/2008/EC), while the Integrated Risk Information System (IRIS) of United States Environmental Protection Agency (USEPA) has classified DDT and some of its major metabolites as probable human carcinogens (Group B2) (Luo et al., 2014). Despite the fact that the production and usage of the great majority of OCPs has been banned in Europe (Regulation 528/2012/EU) and substantially reduced worldwide, their residues may still be detected in food products from different regions (e.g. Heck et al., 2007; Tsatsakis et al., 2008). Human exposure to OCPs occurs mainly through the food chain. OCPs, including DDT, are among the most common persistent organic

I.N. Tsakiris et al./Food and Chemical Toxicology 75 (2015) 156–165

pollutants detected in dietary milk (e.g. Losada et al., 1996; Tsakiris et al., 2013a); they accumulate in fat-rich food products including dairy products, such as cheese, butter and milk (e.g. Salem et al., 2009). Quantitative exposure assessment is now widely used to estimate human exposure to xenobiotics through the consumption of food and therefore to provide a quantitative estimate of possible risks to human health. Risk assessment outputs are the scientific basis for risk management decisions and option analysis (FAO/WHO, Food Standards Programme, Codex Alimentarius Commission, 2008). The objectives of the present study were to evaluate children’s exposure to DDTs (DDT and its metabolites) via dietary milk consumption in Greece, and to assess the respective potential risks to children’s health in terms of cancer and non-cancer effects. 2. Materials and methods 2.1. Sampling A total of 196 samples of dietary cow milk from different market outlet points were collected. The characteristics of the milk samples are presented in Fig. 1. Of the 196 samples, 154 (79%) were conventionally produced while 42 (21%) were organic (certified by Greek certification bodies) and 30 (15%) were milk formula products specifically for children older than 12 months. Pasteurized milk samples represented 51% of the total samples while the rest were Ultra High Temperature (UHT) processed. The majority of samples (89%) were bottled in Tetrapack® aseptic packages. Sampling was conducted according to European Commission Directive 2002/ 63/EC. All samples were transported at 2–4 °C to the laboratory and were analyzed at the same day of sampling; additional portions for replicate and confirmatory analysis were stored deep-frozen. 2.2. Analytical method 2.2.1. Reagents and materials Analytical grade standards (opDDE, ppDDE, opDDD, ppDDD, opDDT, ppDDT) were purchased from Dr. Ehrenstorfer Laboratories (Augsburg, Germany). All solutions were stored at −20 °C. Hexane (Riedel-deHaen, Sigma-Aldrich, Laborchemikalien, D-30926) and dichloromethane (Fluka, Riedel-deHaen, Laborchemikalien, D-30926) were of pro analysis grade. Hydrochloric acid 37% and concentrated sulfuric acid 95–97%, were analytical grade reagents (Merck).

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2.2.2. Sample extraction procedure Milk treatment was based on a slightly modified previously published method (Campoy et al., 2001). Briefly, 5 ml of milk were placed in a glass vial. 2.5 ml methanol and 0.1 g of sodium oxalate were added. The solution was shaken for 5 min and 5 ml of ethyl ether/hexane (1:1 v/v) were added for extraction. The extracted samples were centrifuged for 5 min at 3000 rpm and the organic phase was separated. The extraction procedure was repeated twice. The collected organic phases were concentrated under a gentle stream of N2 to a final volume of 1 ml. A volume of 0.5 ml of concentrated sulfuric acid was added and centrifugation was performed for 5 min at 3000 rpm. The acid residue was extracted twice with 1 ml of hexane. The organic phases were collected and hexane was evaporated to dryness. 2.2.3. Clean up step The dry residue was re-dissolved in 1 ml of hexane cleaned up on solid phase extraction (SPE) columns, packed with 500 mg of acidified silica and 250 mg anhydrous Na2SO4 (analytical grade). The modified silica gel was prepared as follows: 27 ml of concentrated sulfuric acid were added drop wise into 50 g of anhydrous silica gel (60–200 Mesh; Merck), while the mixture was stirred to ensure good homogeneity. The acidified silica was stirred for another 30 minutes. Both sodium sulfate and acidified silica gel were heated at 120 °C for 3 hours before use. The SPE cartridges were activated by addition of 2 ml of hexane:dichloromethane (4:1 v/v) and 2 ml of hexane. The elution solvent was hexane:dichloromethane (1:1 v/v). The final eluate was concentrated to dryness under a gentle nitrogen stream and 100 μl of a solution (0.1 ppm) of 1,2,3,4-tetrachloronaphthalene (TCN) internal standard was added and transferred to a GC-MS vial. 2.2.4. GC-MS analysis Electron ionization mass spectrometric analysis of milk extracts was performed on a GC-2010 Shimadzu system equipped with a BPX5 capillary (SGE), 30 m × 0.25 mm i.d. × 0.25 μm (Supelco). Pure helium with a column flow of 1 ml/ min was used as a carrier gas. Two microliters of each analyte solution were injected into the system in splitless mode and analyzed under the following conditions: The column temperature was initially held at 60 °C for 1 min, raised to 180 °C at 15 °C/ min, held for 1 min, raised to 250 °C at 4 °C/min, held for 1 min and finally raised to 300 °C at 25 °C/min, where it remained stable for 2 min. The injector, interface and ion source temperatures were set at 270 °C, 310 °C and 230 °C. The mass spectrometer was operated in the selected ion-monitoring mode (SIM). 2.2.5. Assay validation For the recovery estimation of the extraction method, blank milk samples were spiked with each analyte at concentration levels of 0.5, 1.0, 2.5 and 5.0 ng/ml. The mean recoveries for opDDE, ppDDE, opDDD, ppDDD, opDDT and ppDDT were 63.5%, 62.3%, 60.3%, 70.2%, 80.2% and 81.7% respectively.

Fig. 1. Characteristics of milk samples collected from Greek market during 2009–2010 and analyzed for DDTs.

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I.N. Tsakiris et al./Food and Chemical Toxicology 75 (2015) 156–165

The limits of quantification (LOQ) and detection (LOD) were estimated by analyzing decreasing concentrations of spiked solutions and defined as the peaks that gave a signal to noise ratio of 10 and 3, respectively. The LOD value for DDTs ranged from 0.007 to 0.011 ng/ml, whereas LOQ ranged from 0.024 to 0.036 ng/ ml. The intra-day precision was evaluated in spiked milk samples at levels of 0.5, 1.0 and 2.5 ng/ml, analyzed in fourfold for each concentration. The mean intraday precision (% relative standard deviation, RSD) for the above selected concentrations was 18%, 20%, 17%, 20%, 18% and 20% for opDDE, ppDDE, opDDD, ppDDD, opDDT and ppDDT, respectively. 2.3. Statistical analysis Milk samples are categorized and analyzed into the following categories: life span (expiration date < = 4 days vs >4 days), lipid content (full >2.5%, semiskimmed 1.5–2.5%, skimmed LOD) of positive samples were used and case 2 where LOD/2 was used as an arbitrary concentration for negative samples. All calculations were made using 50% percentile (median) and 95% percentile concentrations. 2.5.1. Non-cancer effects In recent bibliography there are several approaches for the estimation of human health risk from organochlorine pesticide residues in food. Pardio et al. (2003) estimated the long-term risks of organochlorine pesticides by comparing the EDI with ADI, while for non-cancer hazard EDI was compared with Reference Dose (RfD). Wang et al. (2011) was focused on non-cancer effects following the same procedure with Pardio. Jeong et al. (2014) compared EDI with oral RfD and Tolerable Daily Intake (TDI) in order to assess the potential health risks of organochlorine pesticides via food consumption to infants. In order to estimate the children’s risk of non-carcinogenic effects from exposure to dietary milk through consumption, the EDI was compared with a) the WHO Provisional Tolerable Daily Intake (PTDI) for DDTs of 0.01 mg/kg bw/day, which is also used by EFSA (EFSA, 2006; Larsen, 2006) and b) the US EPA Reference Dose (RfD) of 0.0005 mg/kg bw/day (EPA, 2014a http://www.epa.gov/iris/subst/0147.htm). 2.5.2. Cancer effects The WHO Provisional Tolerable Daily Intake (PTDI) for DDTs of 0.01 mg/kg bw/ day, which also used also by EFSA (EFSA, 2006), is based on the lowest relevant NOAEL for developmental effects (1 mg/kg bw per day in rats) covering Joint FAO-WHO Meeting of Pesticide Residues (JMPR) estimation of lowest relevant NOAEL for carcinogenicity in rats (6.2 mg/kg bw per day) (INCHEM, 2014 http://www.inchem.org/ documents/jmpr/jmpmono/v00pr03.htm#_00032320). So cancer effects are covered from the same WHO approach for the non cancer effects, as presented above. According to the EPA approach for carcinogenic effects the Estimated Daily Intake (EDI) was compared with the Cancer Benchmark Concentration (CBC). For the calculation of CBC, the acceptable cancer risk is set to one per million for a lifetime of exposure, while the value of cancer slope was set to 0.34, 0.34 and 0.24 per mg·kg−1·d−1 for DDE, DDT and DDD sums, respectively, according to the information taken from IRIS database (EPA, 2014b, http://www.epa.gov/iris/subst/0328.htm, EPA, 2014a, http:// www.epa.gov/iris/subst/0147.htm, and EPA, 2014c, http://www.epa.gov/iris/subst/ 0347.htm, respectively). In each case cancer risk was divided by cancer slope factor for the calculation of CBC (CBC = 10−6/slope factor).

3. Results and discussion 3.1. DDT occurrence The levels of DDT and its metabolites in the milk samples were lower than the Maximum Residue Levels (40 μg/kg for sum of DDTs) specified in EU Commission Regulation (EC) No 149/ 2008. Of the 196 samples, 191 (97.4%) were positive for DDT residues with mean value of 2.1 ng/ml. Results are summarized in Table 1. Levels of DDD, DDE and their metabolites did not differ significantly between the sample categories, with some exceptions presented below. Summed DDT residues (opDDT + ppDDT) were significantly lower in conventional milk samples compared to organic samples (p = 0.032). In a similar study in Italy, only one sample (out of 78) of organic milk was found with detectable residues of ppDDE (LOD 0.2 μg/l), and in general no differences between organic and conventional samples were observed (Ghidini et al., 2005). In a recent study in Mexico, DDT and its metabolites were detected at concentrations

Risk assessment for children exposed to DDT residues in various milk types from the Greek market.

The occurrence of residues of DDT and its metabolites was monitored in 196 cow milk samples of various pasteurized commercial types collected from the...
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