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Monitoring of undesirable substances by the European Union indicates a presence of natural and anthropogenic pollutants in animal feed that may be of concern for the producers, as well as the veterinary services. Although the literature concerning toxicity of DDT (an insecticide widely used in the past) is extensive, less attention has been focused on the biological properties of DDE and its interactions with other contaminants. This study reports on the concentration profile of p,p’-DDE and two other ogranochlorines (p,p’-DDT, p,p’-DDD) in different tissues of immature gilts after 14, 28, and 42 days of oral exposure to p,p’-DDE alone (0.5mg·kg-1feed·day-1) and in mixture with naturally occurring mycotoxin zearalenone, ZEN (0.5+0.1mg·kg-1feed·day-1). The treatment resulted in a time© UNIVERSITY OF WARMIA AND MAZURY IN OLSZTYN INTRODUCTION Dichlorodiphenyltrichloroethane (DDT) is an organochlorine pesticide, used worldwide in the past to control insect vectors of infectious diseases (Rogan and Chen 2005). DDTwas also used as an insecticide to protect crops, including Poland, before it was banned in most countries in the 1970’s for its negative effects on humans, wildlife, and the environment. Despite the ban, this insecticide is still used as a cost-effective method to prevent malaria in some tropical regions (Bettinetti et al. 2011). DDT is a highly persistent pollutant. In the environment, it may last for many years, as it is slowly biodegraded to DDE (dichlorodiphenyltrichloroethylene) and DDD (dichlorodiphenyldichloroethane) in processes generally driven by the action of microorganisms in the soil (ATSDR 2002). Common and intensive use of DDT has resulted in worldwide pollution with this compound. It has been found in organisms living in deserts as well as in the depths of oceans (Turusov et al. 2002). Since the times of DDT extensive use, its residual levels in the environment have greatly declined. 2 ENVIRONMENTAL BIOTECHNOLOGY 11 (1) 2015 However, due to its persistence, the pesticide will be present at low concentrations for decades (Glynn et al. 2009; NTP 2011). Current human exposure to DDT and its metabolites is known to occur mainly through dietary intake, particularly consumption of contaminated fish or meat (ATSDR 2002; NTP 2011). Technical grade DDT is a mixture of different isomers: approximately 85% of the p,p-DDT isomer with o,p’-DDT and o,o’-DDT present in lesser amounts (ATSDR 2002). In mammals, the p,p’-DDT isomer is metabolized mainly to p,p’-DDE and p,p’-DDD by the microsomal cytochrome P450 system (Kitamura et al. 2002). Due to their high lipophilic properties (p,p’-DDD<p,p’-DDT<p,p’-DDE), these compounds are readily distributed to the body once absorbed and stored in the tissues in proportion to the tissues’ lipid content; and they leave the body very slowly. This bioaccumulation leads to increasing concentrations of the compounds at higher trophic levels (ASTDR 2002). The literature concerning the biological properties of DDT and its metabolites is extensive, and considerable attention has focused on their adverse effects on the development and function of the reproductive system of animals. It has been shown that these compounds have the ability to modulate endocrine function and influence gene expression after binding to nuclear receptors. For example, unlike p,p’-DDT, an environmental estrogen which binds the estrogen receptor and induces estrogenic effects, p,p’-DDE has been shown to be a weak estrogen receptor agonist but a potent androgen receptor antagonist (Kelce et al. 1995). In a study on fetal rats, exposure to p,p’-DDE led to reduced anogenital distance at birth and retained thoracic nipples on postnatal day, which are indicative of antiandrogen activity (Kelce et al. 1995). Poland is an important producer of pork in the European Union. Aside from cattle, the domesticated pig (Sus scrofa) is the most popular species in Polish production of farm livestock (CSO 2011). Health problems related to reproductive system dysfunction of unknown etiology are generally considered an important factor leading to increased costs of animal production. It is believed that the alimentary route of exposure to various endocrine disrupting compounds (EDCs), both natural (e.g. mycotoxins) and/or of anthropogenic origin (e.g. pesticides), may be responsible for occurrence of the disorders in the reproductive organs. Moreover, gilts are particularly susceptible to the hormone mimicking action of EDCs (Jakimiuk et al. 2009). In legislation, these animals are often considered as sentinel species with the lowest limits for undesirable substances in feed (e.g. PMARD 2012). Monitoring of undesirable substances in animal feed materials indicates a common presence of pollutants, including DDT and its metabolites (Nag and Raikwar 2011). As a result of the ban on meat and bone meal in feed production, fish meal has been introduced as a source of protein and fat for animals (Weiner et al. 2012). However, fish meal may be a potential source of persistent, lipophilic contaminants. In this context, control of the pesticides’ residues at safe levels may constitute an issue for feed producers as well as veterinary services. Since contaminants rarely occur as single compounds, there is a need to investigate possible interactions of different compounds and understand their combined effects. Although p,p’-DDE is among the most frequently studied DDT metabolites and commonly detected isomers (ASTDR 2002), no research by now has been published describing the toxicokinetics of this compound in porcine tissues. Zearalenone (ZEN) is a mycotoxin commonly found in animal feed or feed material of plant origin. This natural contaminant is recognized as an environmental estrogen with associated hormone mimicking effects that disrupts the reproductive system of livestock, especially gilts (Jakimiuk et al. 2009). Current knowledge on the effects of the two pollutants in pigs and their possible interactions is poor. The present study reports on the concentration profile of p,p’-DDE and two other organochlorine compounds (p,p’-DDT, p,p’-DDD) in different tissues of immature gilts after 14, 28, and 42 days of oral exposure to p,p’-DDE alone and combined with ZEN at the maximum doses allowed by current legislation. The measurements were taken to examine the distribution of organochlorine pesticides between different porcine tissues and to evaluate DDE’s accumulation potential and its related human and animal health risks. MATERIALS AND METHODS The study was performed on female pigs (Polish Large White breed) aged 8 weeks (mean body mass 19±2kg) which were obtained from a commercial fattening farm in Bałdy (Poland). The animals were housed and handled in accordance with resolution No. 24/2009 of the Local Ethics Committee. The gilts were housed in pens for 1 week to allow them to adapt to their new environment. During the acclimation and the further exposure period, all pigs were fed with “blank feed” that was tested for the presence of background contamination, and found to be free of mycotoxins (aflatoxin, ochratoxin, ZEN, α-zearalenol, and deoxynivalenol) and organochlorine pesticides (p,p’-DDT, p,p’-DDE, and p,p’-DDD). Throughout the study, the animals were maintained indoors and had constant access to water. Doses of DDE and ZEN were chosen based on the current legislation limits. According to the Polish regulation (PMARD 2012), the maximum content of any combination of DDT, DDD, and DDE (expressed as ΣDDT) in all feed materials should not exceed 0.05mg·kg-1, except fats and oils for which the limit is 0.5mg·kg-1. Since fats and oils are also used in feed production, the dose of p,p’-DDE at 0.5mg·kg-1 was chosen as the theoretically maximum allowable concentration of this compound in animal feeding. As for the guidance value for ZEN, its concentration in complementary Woêny et al. Residue levels of DDE in pig tissues 3 and complete feeding stuffs intended for piglets and gilts should not exceed 0.1mg·kg-1 (EC 2006), thus this value was selected for the exposure. Prior to oral exposure, the gilts were divided into 3 groups: an untreated (control) group (n=9) and 2 treatment groups (n=9 each) that were exposed to i) p,p’-DDE at a dose of 0.5mg·kg-1feed·day-1 (group DDE) or ii) exposed to p,p’-DDE together with ZEN at a dose of 0.5+0.1mg·kg-1feed·day-1 (group DDE+ZEN). Analytical samples of p,p’-DDE (#123897; Sigma-Aldrich) and ZEN (#Z2125; Sigma-Aldrich) were administered to the treated groups daily per os with their first (morning) feeding. After 14, 28, and 42 days of the experiment, the gilts (n=3 from each group at respective timepoint) were randomly selected from their pens, weighted, then anesthetized with sodium pentobarbital (Biowet, Poland) and exsanguinated. Immediately after cardiac arrest, the animals were decapitated and the hypothalamus with the pituitary gland was excised; then fragments of the adrenal gland, the uterus (from the uterine horns), the ovary, the duodenum, the liver, and adipose tissue (the fatback) were collected. All samples were stored at -20°C for further analysis. Sample collection was always performed before the pigs’ first morning feeding (after fasting for 12h). Organochlorine pesticides (p,p’-DDE, p,p’-DDD, and p,p’-DDT) were extracted from the tissue samples using a hexane and acetone mixture with tissues’ fat. The extracts were cleaned with sulphuric acid. The purified extracts were analysed by capillary gas chromatography with electron capture detection (GLC-ECD), according to the procedure described below. The concentrations of the organochlorine pesticides were expressed in μg·kg-1. The analyses were performed with an Agilent Technologies chromatograph, model 6890 Plus equipped with 7683B series autosampler, a split-splitless injector in pulsed splitless mode, and a 63Ni-EC detector. Chromatographic separation was performed in an HP-5MS capillary column (60m×0.25mm ID×0.25μm film thic

Dichlorodiphenyldichloroethylene (DDE) residue limit exceeded in pig tissues after feed-borne exposure to maximum allowed concentration