Pesticides and Polychlorinated Biphenyls in Milk and Dairy Products in Croatia: A Health Risk Assessment.

The aim of this study was to evaluate contamination levels and the frequency of detection of organochlorine (OCPs) and organophosphate pesticides (OPPs), pyrethroids, carbamates and polychlorinated biphenyls (seven PCB congeners) in a total of 534 samples of cow's, sheep's and goat's milk and dairy products from Croatia. Concentrations above the limit of quantification (LOQ) were measured for fourteen OCPs, nine OPPs, six pyrethroids, one carbamate and PCBs with a total of 172 results, and no concentrations exceeded the maximal residue levels defined by the European Union. The mean concentrations of pesticides and the sum of seven PCBs were determined in the ranges 0.92-17.4 µg/kg and 1.38-2.74 µg/kg. Pesticides were quantified in 27% of samples, and seven PCBs were quantified in 5.23% of samples. Among the three pesticide groups, the highest numbers of quantified results were found for OCPs (12.1-20.8%). The highest frequencies of detection were found for the sum of 4,4'-dichlorodiphenyltrichloroethane and its isomers (DDTs), hexachlorobenzene and seven PCBs. The sum of seven PCBs was quantified within the range of 3.3-6.67% of samples per milk type and dairy products. Among the OPPs, the highest frequency of detection was found for chlorpyrifos in cow's milk. Based on the estimated daily intakes, chronic risk characterisation showed no risk for adults or ten-year-old children for the consumption of cow's milk and dairy products.

Distribution of Pesticides and Polychlorinated Biphenyls in Food of Animal Origin in Croatia.

Pesticides and polychlorinated biphenyls (PCBs) are persistent environmental pollutants. When entering the food chain, they can represent a public health problem due to their negative effects on health. In this study, concentrations of organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), pyrethroids, carbamates, and PCBs-a total 73 compounds-were determined in a total of 2268 samples of fat tissues (beef, pork, sheep, goat, poultry, game, horse, rabbit) and processed fat, meat, and processed meat products collected in Croatia during an 8-year period. In fatty tissues, 787 results exceeded the limits of quantification (LOQ): 16 OCPs, eight OPPs, six pyrethroids, one carbamate, and seven PCBs. The most positive results in fat samples were found for OCPs, with a frequency of quantification in the range of 57.5-87.5%. Hexachlorobenzene (HCB) and dichlorodiphenyldichloroethylene (DDE) were quantified in the highest percentages, in the ranges of 5.5-66.7% and 5.4-55.8%. Concentrations above the MRL values were determined for chlorpyrifos in pork fat and for resmethrin in six fat samples and one pâté. In 984 samples of meat and meat products, only 62 results exceeded the LOQ values. The highest frequency of quantification was determined for OCPs (25 samples), of which 40% were DDT isomers (60% DDE). Frequency quantifications of PCBs in fat samples were between 7.23 and 36.7%. An evaluation of the health risk assessment showed that the consumption of fat, meat, and meat products does not pose a threat to consumer health, since all EDI values were well below the respective toxicological reference values.

Seasonal Occurrence of Aflatoxin M1 in Raw Milk during a Five-Year Period in Croatia: Dietary Exposure and Risk Assessment.

This study's objective was to estimate the seasonal occurrence of aflatoxin M1 (AFM1) in cow's milk between winter 2016 and winter 2022 and to assess dietary exposure and risk assessment for the adult Croatian population. In total, 5817 cow milk samples were screened for AFM1 concentrations using the enzyme immunoassay assay (ELISA). For confirmation purposes of AFM1 concentration above the European Union maximum permitted level (MRL), ultra high-performance liquid chromatography with tandem mass spectrometry was performed. In 94.7% of milk samples, AFM1 levels were below the detection limit (LOD) of the ELISA test. For 3.47% of samples, the AFM1 was between the LOD and MRL values. Only 1.87% of all samples exceeded the MRL. The mean value of elevated AFM1 in different seasons ranged between 59.2 ng/kg (autumn 2017) and 387.8 ng/kg (autumn 2021). The highest incidences of positive AFM1 were determined in autumn and winter and the maximum (6.4%) was in winter 2019/2020. The largest percentage of positive samples (69.7%) was found in central Croatia. The estimated daily intakes for positive samples ranged between 0.17 and 2.82 ng/kg body weight/day. Risk assessment indicated a high level of concern during autumn and winter, especially for consumers of large amounts of milk.

The analysis of acidic and basic non-steroidal anti-inflammatory drugs in milk and muscle samples: a comprehensive analytical approach using UHPLC-MS/MS.

In animal husbandry for food production, the administration of non-steroidal anti-inflammatory drugs (NSAIDs) as anti-inflammatory, analgesic, and antipyretic agents, has seen an increase due to raised awareness of animal welfare issues. Residues of NSAIDs may be present in animal products that are intended for human consumption, and since some may pose a certain risk to human health, there is a need to monitor NSAID residues at low levels via routine and targeted surveillance. In analytical chemistry, NSAIDs are usually differentiated using their acid-base properties. Within this study, a method for simultaneous analysis of 27 NSAIDs, including both groups, in milk and muscle samples in 12.5 min is described. Sample processing consisted of enzymatic hydrolysis and acetonitrile extraction, followed by a clean-up of the extract by SPE, and measurement by UHPLC-MS/MS. The in-house validation study (alternative approach), covering trueness, precision, sensitivity, decision limit (CCα), detection capability (CCß) and matrix effect, was designed and evaluated with the help of validation software to meet the demands of regulatory compliance. The method recovery for milk and muscle matrix was in the range of 98.1% to 106.5% and 98.8% to 102.7%, whereas the CCß as the parameter for screening analysis, ranged from 0.07 to 46.7 µg/kg and 1.19 to 69.7 µg/kg, and the CCα, as the parameter for confirmatory analysis, from 0.11 to 56.7 µg/kg and 1.12 to 518.6 µg/kg, respectively. The occurrence of NSAID residues in milk and muscle samples was assessed using the developed method within the Croatian National Residue Control Plan, revealing quantifiable residues for diclofenac, ketoprofen, and salicylic acid mostly in milk samples. The most abundant NSAID in analysed samples was salicylic acid, which may be introduced into the food chain and be present in various types of matrices due to its natural occurrence in plants as a phytohormone.

Difference in pesticides, trace metal(loid)s and drug residues between certified organic and conventional honeys from Croatia.

Quality and safety of food, including honey, is one of the leading priorities regarding residues of anthropogenic chemicals with proven adverse health effects. In total, 61 honey samples of known botanical origin were collected in period 2018-2019 from Croatian registered organic and conventional beekeepers (N = 16 organic and N = 45 conventional honey samples). Eleven trace metal(loid)s (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn; quantitated by ICP-MS), 24 antibiotics (LC-MS/MS, ELISA, microbiological inhibition test), six indicator PCBs (GC-MS/MS) and 121 pesticides (LC-MS/MS, GC-MS/MS) originating from environment and/or beekeeping practice were measured to assess possible differences in contaminant residues between organic and conventional honeys. All honey samples had contaminant residues below the legal maximum levels and are considered safe for consumers. However, 2/16 organic and 34/45 conventional honeys contained one or two synthetic acaricides (most frequently coumaphos), while other pesticides, antibiotics and PCBs were not quantified. Also, organic honey contained lower levels of coumaphos, amitraz and amitraz metabolite N-(2,4-dimethylphenyl) formamide than conventional honeys, on average. Higher levels of Cr (p = 0.006) were detected in organic compared to conventional chestnut honeys. This study pinpointed beehive disease control treatment as prominent pesticide residue source, which was to some extent reduced in organic honeys. Quantified pesticide and metal(loid) levels were within range or lower than reported in recent literature regarding honey of the same botanical origin.

Evaluation of Element Concentrations in Beef and Pork Meat Cuts Available to the Population in the Croatian Capital.

The aim of this study was to determine the concentrations of essential, trace, and toxic elements in beef and pork meat cuts available at markets and retail chains in the Croatian capital. Significant differences in the concentrations of Al, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Se, Pb, and Zn were found between bovine cuts (p < 0.01, all) and also between pork cuts (p < 0.01, all). A risk assessment using the estimated intakes based on the lowest and highest mean values of Al, Cr, Ni, and Pb in beef and pork showed low contributions to tolerable toxicological limits. However, consumers whose diets consist of large amounts of beef and pork kidneys may be at risk because the estimated intakes for Cd and Se exceeded the toxicological limits. Consumers of large quantities of beef mixed meat may be at risk due to higher values of estimated As intakes compared to health-based guidance values. Estimation based on the provisional maximum tolerable daily intake values for Cu, Fe, and Zn showed that beef and pork cuts can be considered safe for consumption. A comparison with data from other studies shows that the concentrations of the analyzed elements in beef and pork cuts vary considerably.

Estimation of the Withdrawal Time of Levamisole in Eggs after Oral Administration to Laying Hens.

This study was conducted to evaluate withdrawal time of levamisole in eggs after oral administration in laying hens at different doses. Sampling of eggs was conducted for 37 days after the end of treatment, and levamisole concentrations were measured by liquid chromatography-tandem mass spectrometry validated according to the Commission Decision 2002/657/EC. Estimated validation parameters were as follows: decision limit, 0.54 µg/kg; detection capability, 0.56 µg/kg; limit of detection, 0.04 µg/kg; limit of quantification, 0.15 µg/kg; accuracy (recovery), between 92.9 and 102.3%; precision (relative standard deviation), ≤4.62%; and within-laboratory precision (relative standard deviation), ≤5.19%. Levamisole residue levels were significantly higher in egg yolks than in egg whites. The highest levels of levamisole were detected on day 2 posttreatment in groups receiving 50 mg/kg of body weight (556.2 µg/kg in egg yolks and 166.5 µg/kg in egg whites). Significant elimination occurred within 5 days after the cessation of treatment in all groups, with an elimination half-life of 1.3 days. Levamisole was still detectable on day 30 after the end of treatment in egg whites (0.06 µg/kg) and on day 37 in egg yolks (0.06 µg/kg). The longest withdrawal time for levamisole in eggs (14.9 days) was determined in a group treated with 25 mg of levamisole per kg of body weight for two consecutive days. According to the results, oral treatment of laying hens with levamisole may result in noncompliant egg samples even 14 days after treatment.

Essential and toxic element concentrations in monofloral honeys from southern Croatia.

The concentrations of 24 elements in seven honey types (multifloral, heather, common heather, bearberry, sage, mandarin orange-blossom and honeydew) collected in southern Mediterranean regions of Croatia were determined using ICP-MS. Significant differences were found in the concentrations of Ag, As, Ba, Cu, Co, Fe, K, Mg, Mn, Mo, Na, Ni, Se, Sb, U and Th (p<0.05, all) among honeys. The highest element concentrations were determined in honeydew honeys, with the exception of multifloral (Ca, Cr, Mo, Se), common heather (Mg, Na), bearberry (Ba, Fe, Pb) and sage (Ag) honeys. Among the floral honeys, the highest concentrations were found in multifloral honey (Al, As, Be, Ca, Cr, Mn, Mo, Ni, Se, Th and U), common heather (Co, K, Mg, Na, V), sage (Ag, Cd, Cu), and bearberry (Ba, Fe, Pb, Sb, Zn). The results contribute to the evidence supporting the role of botanical origin on the elemental composition of honey.

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides, trimethoprim and dapsone in muscle, egg, milk and honey.

A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO3H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCß for sulfonamides ranged between 5.6 and 8.2 µg kg(-1) for eggs, between 11.1 and 69.9 µg kg(-1) for milk, between 64.7 and 87.9 µg kg(-1) for muscle, and between 2.7 and 5.3 µg kg(-1) for honey. CCß values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg(-1) and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg(-1) calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.

Lead Concentrations in Raw Cow and Goat Milk Collected in Rural Areas of Croatia from 2010 to 2014.

A total of 249 cow and 33 goat milk samples were collected in rural areas of Croatia during the period 2010-2014. Lead concentrations in milk samples were analyzed by graphite furnace-atomic absorption spectroscopy. Mean Pb concentrations in milk ranged from (µg/kg): cow 10.8-12.2; goat 9.33-60.0. The highest Pb level of 131 µg/kg in cow milk was measured during 2014. There were no significant differences in Pb levels between cow and goat milk and also in goat milk among the analysed years. However, significant differences were found in cow milk among years. The highest Pb was determined in 2011 (157 µg/kg in goat milk). The calculated estimated weekly intakes of Pb concentrations for cow and goat milk contribute only 1.37 % and 1.84 % to the provisional tolerable weekly intake. Therefore, the consumption of milk from both species should not pose a consumer health risk.

Distribution of sulfamonomethoxine and trimethoprim in egg yolk and white.

The distribution of sulfamonomethoxine (SMM) and trimethoprim (TMP) in egg yolk and white was measured during and after administration of a SMM/TMP combination in laying hens in doses of 8 g l(-)(1) and 12 g l(-)(1) in drinking water for 7 days. The SMM concentration reached maximal levels on day 2 of the post-treatment period for both doses (µg kg(-)(1)): 5920 and 9453 in yolk; 4831 and 6050 in white, in doses 1 and 2, respectively. Significant differences in the SMM and TMP concentrations between yolk and white in post treatment period were found. SMM dropped below the LOD (1.9 µg kg(-1)) in yolk after day 16 and 19 for doses 1 and 2. TMP reached maximal levels on day 3 after drug administration for doses 1 and 2 (µg kg(-)(1)): 6521 and 7329 in yolk, 1370 and 1539 in white. TMP residues were measured above LOD (0.3 µg kg(-)(1)) in yolk for both doses on day 37 post-treatment.

Deposition and depletion of maduramicin residues in eggs after oral administration to laying hens determined by LC-MS.

The coccidiostat maduramicin has been approved as a feed additive for chickens and turkeys, although it is prohibited for use in laying hens. In the present study, laying hens were divided into three groups and fed for 14 days with medicated feed containing maduramicin, at three different concentrations: 50, 100 and 500 µg kg(-1). Eggs were collected during treatment and for 26 days after the end of feeding with medicated feed. Maduramicin residues were found exclusively in egg yolk, with the highest concentration in egg yolk of 459 µg kg(-1) for the highest dose. The maximum concentration of maduramicin in whole egg was 16.6 µg kg(-1) for the group receiving feed containing the maximum permitted level of maduramicin in feed (50 µg kg(-1)). The half-life of elimination of maduramicin, calculated for post-treatment days 1-10, was 6.5 days. Twelve days after drug administration, the concentration of the maduramicin in egg yolk for Group 3 (fed with 500 µg kg(-1) maduramicin) still exceeded 20 µg kg(-1), while the concentrations for Groups 1 and 2 were 1.2 and 2.7 µg kg(-1), respectively.

Tylosin content in meat and honey samples over a two-year period in Croatia.

A total of 646 meat and 96 honey samples were examined over a 2-year period for the presence of tylosin residues. ELISA method used was validated according to the criteria of Commission Decision 2002/657/EC established for qualitative screening methods. The CCß values were 32.1 µg kg⁻¹ in muscle and 24.4 µg kg⁻¹ in honey. The recoveries from spiked samples ranged from 66.4-118.6%, with a coefficient of variation between 12.6% and 18.6%. All the investigated samples showed no presence of tylosin. Calculated estimated daily intakes show exposure levels lower than the acceptable daily intakes set by World Health Organization.

Feed additives diclazuril and nicarbazin in egg and liver samples from Croatian farms.

In total 307 egg and 275 liver samples were examined for nicarbazin and 365 eggs for diclazuril over a 30-month period. Enzyme-linked immunosorbent assay methods used for quantification were validated according to European Commission Decision 2002/657/EC. Non-compliant samples were confirmed by LC-MS/MS. Mean diclazuril concentrations in egg samples were 0.31 µg kg⁻¹, which is below the MRL. In only one egg sample, 2.26 µg kg⁻¹ was determined by enzyme-linked immunosorbent assay, although confirmation by LC-MS/MS gave a value of 1.6 µg kg(-1). Mean nicarbazin levels determined were 1.85 µg kg⁻¹ in egg and 21.1 µg kg⁻¹ in liver samples. Four samples, one egg and three livers, yielded elevated concentrations of nicarbazin, but only in the egg sample the LC-MS/MS method confirmed nicarbazin (106 µg kg⁻¹) above the MRL value.

Chloramphenicol residues in muscle of rainbow trout following two different dose treatments.

Depletion of chloramphenicol (CAP) in muscle of rainbow trout was evaluated following 4 days of oral administration with two dosages (42 and 84 mg/kg/day). Sampling was conducted during treatment and for 35 days following the end of treatment. Analysis was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Concentrations observed during treatment were more than 300 µg/kg. A significant elimination occurred within 9 days after the cessation of treatment in both groups. Higher CAP levels were measured in the group treated with higher dose. CAP was not detectable after 13 and 15 after the end of treatment in both groups.

Lead content in multifloral honey from central Croatia over a three-year period.

Lead concentrations were analysed by atomic absorption spectrometry in multifloral honeys collected in central Croatia (Zagreb County) during a three-year period from 2009 to 2011. The mean levels of elements (µg/kg) in honey samples measured were: 90.8 in 2009, 189 in 2010 and 360 in 2011. Significant differences were observed, and Pb levels determined in 2009 were significantly lower than those in 2010 and 2011 (p < 0.05, both). In 2009 there was no concentration found above 300 µg/kg. However, in 2010 and 2011 levels exceeding 300 µg/kg were found in 28.6 % and 25 % of samples. Trace element levels of Pb determined in multifloral honey were generally higher than concentrations obtained from other geographical origins and neighbouring countries. These high concentrations of Pb may be related to the fact that the central region is becoming increasingly urban and the network of motorways is growing. Accordingly, the risk of positioning hives near zones of busy highways and railways is increasing. This underlines that particular attention should be paid to toxic Pb levels, due to their gradual increased during the study period.

Elimination of chloramphenicol in rainbow trout receiving medicated feed.

Chloramphenicol muscle residue levels in rainbow trout were determined after oral administration of 84 µg kg-1d-1 of chloramphenicol for four days. Samples were taken one day before treatment and for 43 days after the treatment was over. Chloramphenicol was analysed using an in-house enzyme linked immunoassay (ELISA) validated against the criteria of the Commission Decision 2002/657/EC. Validation parameters confirmed that the method was appropriate for the detection of chloramphenicol at levels below the minimum required performance limit (MRPL) of 0.3 µg kg-1. The highest chloramphenicol levels were observed on the first day after the treatment had ended (144.3 µg kg-1). Elimination was significant over the first seven days; significant differences were detected between days 1 and 3 (p<0.001), 3 and 5 (p<0.001), and 5 and 7 (p<0.05). Chloramphenicol levels dropped below MRPL to 0.17 µg kg-1 on day 9 after the end of treatment. From day 11 to 43, chloramphenicol residues were detectable in a range from 0.091 µg kg-1 (highest) to 0.011 µg kg-1 (lowest). Our results indicate that trout muscle tissue could be compliant with health requirements for consumption 10 days after withdrawal from chloramphenicol treatment.

Concentrations of trace elements in tissues of red fox (Vulpes vulpes) and stone marten (Martes foina) from suburban and rural areas in Croatia.

Trace elements concentrations (As, Cd, Cu, Pb and Hg) were determined in the liver, kidney and muscle of 28 red fox (Vulpes vulpes) and 16 stone marten (Martes foina) from suburban and rural habitats from Croatia. Rural and suburban habitats affected Cd and Hg levels in the muscle, liver and kidney of red fox. Significant differences in metal concentrations in the muscle, liver and kidney were detected among species. Suburban stone marten accumulated the highest levels of trace elements (mg/kg w.w.): in muscle 0.019 for Hg; in liver 0.161 for Cd, 36.1 for Cu and 0.349 for Pb; in kidney 1.34 for Cd and 0.318 for Pb. Values observed were higher than those found in suburban red fox and therefore, may represent an important bioindicator for the accumulation of toxic metals in urbanized habitats.