Vet. World, 2012, Vol.5(7):412-416 RESEARCH Risk analysis and exposure assessment of Ochratoxin A in Serbia

Aim: The aim of this study was to check the current status of OTA contamination of the Serbian pork and chicken meat in naturally exposed animals which are part of chain production. The knowledge of these factors, their importance and significance, can be used as a tool from the food safety point of view to predict if OTA content in pork and chicken tissues poses a risk to human health and deleterious effects on animals\' health and wellbeing. 
 
Materials and Methods: A total of 540 samples of pigs blood, kidney, liver and chicken gizzard were randomly selected from slaughtered pigs (n=90) and chicken (n=90) and analyzed for the presence of ochratoxin A (OTA) by High-performance liquid chromatography (HPLC-FL). 
Results: In porcine tissue samples, of the 90 liver samples, 26.6% contained OTA in the range of 0.22-14.5 ng/g. The incidence of OTA in serum and kidneys were very similar (31% and 33.3%, respectively), with a maximum concentration of 220.8 ng/mL, and 52.5 ng/g, respectively. Majority of chickens\' tissues samples, were not found to contain measurable amounts of OTA. Moreover, the OTA levels found in analysed tissues were low in general. 
 
Conclusion: The results of this preliminary research indicate that the content of OTA in the examined tissues are far below the values that represent hazard to the health of consumers. However, the results of this study show that in Serbia, consumers are nevertheless frequently exposed to ochratoxins.


Introduction
considered to be the most contaminated samples, meat and meat products are also frequently contaminated Undesirable substances such as mycotoxins can with OTA due to carry-over effect.Kidney is the most be present in plants and derived products thereof.contaminated organ, followed by pig blood, liver, and Depending on the nature and the concentration levels meat from animals fed with contaminated feed [5][6][7][8]. of the compound, these might be of concern for human Through the food chain OTA can appear in human and/or animal health.From the more than 300 fluids, such as plasma [9], urine [10] and milk [11].mycotoxins isolated and described to the present day, Exposure to OTA is a potential human health OTA is recognized as a contaminant with a strong hazard because of the frequent exposure, long-half life nephrotoxic activity [1].The International Agency for in the organism and its carcinogen properties in Research on Cancer has classified OTA as possibly experimental animals.The risk is well recognized, but carcinogenic for humans (group 2B) [2], also at present it has not been quantified accurately.In teratogenic and carcinogenic effects have been some Eastern European countries (in particular described in some animal species [3]. Bulgaria, Romania, Serbia, Croatia, Bosnia and Ochratoxin A is a secondary metabolite of fungi Herzegovina) OTA has been associated with a high such as Penicillium verrucosum and Aspergillus incidence of a kidney disease known as Balkan ochraceus that occur in a number of food commodities endemic nephropathy [12].Consumption of foods like cereals, coffee, dried fruit, pulses, and derivates contaminated with OTA during pregnancy and/or products [4].Apart from cereals products, that are childhood is suspected of inducing lesions in testicular DNA which, during puberty, can be promoted to nm (=5550).After suitable dilutions by watertesticular cancer [13].Indeed, rates of incidence of methanol-acetic acid (50:49:1 v/v/v), the working testicular cancer in 20 countries correlated solution was used to prepare the external calibration significantly with the per-capita consumption of curve.A working standard OTA for HPLC was coffee and pig meat.The dietary intake of OTA by the prepared daily just before starting the injection of a population of EU member states has been assessed and series of samples.Other reagents were of HPLC grade.the Joint FAO/WHO Expert Committee on Food All chemicals were of HPLC grade, reagent grade or Additives (JEFCA) has recently established a chemically pure.provisional tolerable weekly intake of 100 ng/kg b.w.

Extraction and clean-up for ochratoxin analyses:
per week [14].
Serum (0.8 mL) was extracted according to Curtui and Moreover, poultry and pork meat comprises a Gareis (2001) [15] with 15% trichloroacetic acid (0.2 substantial portion of the Serbian diet (17.4 kg and 16 mL) and dichloromethane (1 mL), by vigorous kg, respectively, per person in 2009).The aim of this vortexing for 30 s in a 2 mL safe-lock polypropylene study was to check the current status of OTA conical-bottom centrifuge tube.The mixture was contamination of the Serbian pork and chicken meat in allowed to stand for 24 h at room temperature, and then naturally exposed animals which are part of chain centrifuged at 14,000 × g for 10 min.The lower production.The knowledge of these factors, their dichloromethane phase was carefully withdrawn by a importance and significance, can be used as a tool Pasteur pipette and transferred to a 1.5 mL safe-lock from the food safety point of view to predict if OTA polypropylene conical bottom centrifuge tube.The content in pork and chicken tissues poses a risk to acidic phase and the compact precipitate layer formed human health and deleterious effects on animals' between the two phases were re-extracted with health and wellbeing.dichloromethane (0.5 mL) for 30 s on a vortex mixer and then centrifuged for 5 min at 14,000 × g.The

Materials and methods
pooled dichloromethane extract was evaporated to Samples collection: Pilot study was carried out dryness at 40 °C under a gentle nitrogen flow.The during 2009 and 2010.Swine and poultry commercial remaining residue was reconstituted in methanol (500 farms in the northem and central agricultural area of µL) and transferred to a HPLC vial.The limit of Serbia were randomly selected.Pigs and chicken were detection (signal/noise: 3/1) was estimated at 0.1 selected randomly in slaughterhouses that are ng/mL of OTA, and average recovery was 95%.important for overall meat production, at the end of Kidney, gizzard and liver analyses were performed fattening week, by random method.About 50 mL of by the method of Matrella et al. [16], which briefly blood per pig was sampled by jugular puncture.Blood includes a double extraction with acidic ethyl acetate.samples remained at room temperature for 24 h to The organic phase was removed and extracted with allow for clotting, and were subsequently centrifuged 0.5M NaHCO , pH 8.4.The aqueous extract was 3 at 3,000 × g for 20 min.Serum was decanted and acidified to pH 2.5 with 7M orto-phosphoric acid.stored at -20 °C prior to analysis.Whole liver, kidney OTA was finally back extracted into ethyl acetate (3 and gizzard (chickens) were sampled from each mL).The organic phase was evaporated to dryness animals.No preservatives were added.A total of 540 under N steam, reconstituted in 500 µL of mobile samples of serum samples (pigs), liver, kidneys and 2 phase and a 50 µL of aliquot was injected.The gizzard (chickens) from slaughtered pigs (n=90) and detection limit for OTA in organs was 0.01 ng/g with chicken (n=90) were collected.61% (C.V. =14.5%) mean recovery from fortified Chemical and Reagents: Ochratoxin A crystalline samples at 3 ng/g (n = 3).material was purchased from Sigma (St. Louis, MO, Chromatographic conditions (HPLC): An aliquot USA).Stock concentrated solution was prepared in of 20 µL for serum samples, 50 µL for kidney, gizzard toluene-acetic acid (99:1 v/v) at a final concentration and liver samples, and for the standards 10 µL, were of 1 mg/mL and kept in security conditions at -20 ºC, injected onto a Waters Symmetry Shield RP (Reversed wrapped in aluminum foil, because OTA gradually phase) 18, high pressure liquid chromatography breaks down under ultraviolet light.The OTA working column (length and inner diameter 150×4.6 mm, solution was prepared by diluting the stock solution particle size 5 µm) on a Waters Alliance 2695 HPLC with toluene-acetic acid (99:1 v/v) to ~10 µL/mL.The system.The OTA was eluted with 4% acetic acid and actual concentration of OTA was calculated using the acetonitrile (32:68 v/v) at 25 °C and a flow rate of 1 mL/min.absorbance by the UV spectrophotometer set at 333 Measurements were performed by fluorescence from chicken farms located in the different area of detection at wavelengths of 334 nm (excitation) and Serbia, OTA was reported in 23 (25.6%), 17 (18.9%)and 15 (16.7%) samples, respectively.The data in 460 nm (emission), detector gain was set to 10.For Table 1 show that majority (215) of these samples did more accuracy, 40 µL of the serum samples, was renot contain ochratoxin A and the rest largely had injected in the case of the samples with an amount of concentrations close to the detection limit.OTA levels OTA near the detection limit. in liver were slightly higher than those in kidneys and Statistical analysis: Descriptive data are presented gizzard, but this difference was not statistically as means, standard deviation (SD), and range, for significant (p < 0.

05). continuous variables. Statistical differences in the
The average level of OTA in liver was 0.41 ± mean levels of OTA contamination across the three 0.92 ng/g (range 0.14-3.9ng/g), whereas OTA average groups of positive samples were determined by onevalue in kidneys and gizzard were 0.36 ± 1.18 ng/g way ANOVA (p<0.05).Significance was set at p<0.05.

Results
standard deviation is due to two samples in kidneys, A total of 540 tissues samples were analysed, and one in gizzard that showed a concentration of OTA 270 samples per pigs and 270 chickens, for the up to 7.02 ng/g and 9.5 ng/g, respectively (Fig. 2).All presence of OTA.The incidence of contamination, on the analyzed tissues samples were below the limit serum, liver, kidneys and gizzard, by OTA is established by JECFA (10 ng/g) [14].Only one sample summarized in Table 1, Figure 1 and 2. Of the 90 liver, of gizzard contained OTA (9.94 ng/g), close to the kidneys and gizzard samples, respectively, originating maximum permissible levels of this toxin proposed by N-total number of analyzed samples, n-number of samples <LOD (limit of detection, see Materials and methods), X arithmetic mean (conc.below LOD are regarded as zero) in all of analyzed samples, X -arithmetic mean in samples  body and it is frequently found in human blood due to In the case of the OTA content in pigs tissues, its long elimination half-life (about 35 days in serum), OTA was detected in most frequently (33,4%) in as a consequence of its binding to plasma proteins, its enterohepatic circulation and its reabsorption from kidney samples, whereas OTA was present in 31.2%urine [19].This makes of ochratoxin A the most and 26.7% of serum and liver samples, respectively.detected mycotoxin in human blood all over the world With regards to the concentrations of OTA, the highest [20].Therefore, public health authorities in Serbia pay level (220.8 ng/mL), was detected in serum samples.
attention to ochratoxin A particularly by monitoring One samples of kidneys had 52.5 ng/g OTA, while food and feedstuffs.At present, Serbia has not a maximum level of OTA in liver was 14.5 ng/g (Fig. 1).maximum tolerable limits for ochratoxin A in meat and In 2.2% samples of kidneys, OTA levels was meat products, particularly pork meat.However, there considerably higher and greatly exceed the is a tendency of authorities in Serbia to adopt the permissible levels of this toxins established in Serbia European Union limits for this mycotoxin.and included those proposed (10 ng/g) by the Scientific Committee on Food [17], and JECFA [14].

Conclusion Discussion
The study provides the necessary knowledge information for updating the level of OTA exposure The exposure assessment was performed by a for the target products.Despite the limited number of point-estimate deterministic approach, which is an samples examined in the present study, it is important evaluation built by combining concentration single to notice that even the low levels of OTA concentration levels, or mean contamination values, of analytes with found in tissues of slaughtered animals, may food consumption data and divided by the body contribute to the OTA daily intake.On the basis of the weight.In this study the Tolerable Daily Intake (TDI), risk assessment carried out in this study, the exposure calculated as nanograms of ochratoxin A per kilogram to OTA, due to the consumption of pork and chicken of body weight per week (ng/kg bw/daily), was products, is to be considered not a major concern.evaluated using the equation; CM x P/M.Therefore, it could be concluded that data arising from CM is the concentration of mycotoxin in tissues this study, adequate to align national legislation with (ng/g), P is the meat consumption (g/day), M is the the EU provisions.However, it should be remembered mass of "average consumer" (60 kg body weight) [18].
when align that factors such as climate conditions Results found in this study suggest that, in during harvest, practices for grain/feed storage etc general, OTA contamination in chicken meat have influence on the ochratoxin A level in edible originating from different part of Serbia is low, hence tissues.Additionally, the study emphasized the role of for the consumer the contribution to the total intake of management practices in the prevention of mycotoxin ochratoxin A from chicken products is very small prevalence in animls farms.To protect consumer compared to other sources (0.28 ng/kg b.w. to 0.32 health and to reduce economic losses, surveillance and ng/kg b.w).Of 270 chicken tissues samples tested, control of mycotoxins in food and feed has become a measurable amounts of OTA were found in 55 (20.3%) major objective for producers, regulatory authorities tissues samples, wheres incidence of OTA in pigs and researchers worldwide.tissues were higher, which accounts for 30.3% of all samples.Comparison the data obtained in this trial Acknowledgements with other recently published data for the occurrence This study was funded by the Ministry of of OTA in pig edible tissues shows that the found Education and Science, Belgrade, Serbia (project code levels are comparable with levels in other European TP-31008).We are grateful to the Ministry for their countries, therefore the estimated daily intake of OTA understanding and support to veterinary development.through pork meat by a 60 kg adult is 0.46 ng/kg b.w.
Competing interest to 0.90 ng/kg b.w.respectively.
Currently, Ochratoxin A is receiving increasing Authors declare that they have no Competing interest.attention for its toxic effects on human health and high detected, C.V.-coeff. of variation (x100).

Figure 1 .Figure 2 .
Figure 1.Distribution of OTA content in tissues of slaughtered pigs (ng/mL, ng/g), N-number of samples, LOD (limit of detection, see Materials and methods) Figure 2. Distribution of OTA content in tissues of slaughtered chicken (ng/g), N-number of samples, LOD (limit of detection,see Materials and methods)

Table - 1
. Incidence and the mean concentration of ochratoxin A in tissues of slaughtered animals