Food safety aspects of primary environmental ...

Environ Sci Pollut Res (2017) 24:25372–25382 DOI 10.1007/s11356-017-0206-9

RESEARCH ARTICLE

Food safety aspects of primary environmental contaminants in the edible tissues of roe deer (Capreolus capreolus) József Lehel 1

&

Dóra Zwillinger 1 & András Bartha 2 & Katalin Lányi 1 & Péter Laczay 1

Received: 30 March 2017 / Accepted: 12 September 2017 / Published online: 20 September 2017 # Springer-Verlag GmbH Germany 2017

Abstract The muscle, liver, kidney and fat samples of 20 roe deer of both sexes originating from a hunting area in central Hungary were investigated for the presence of heavy metals such as As, Cd, Hg and Pb, and their contents were evaluated for possible health risk to consumers. Both As and Hg were found at a level below the limit of detection (< 0.5 mg/kg wet weight) in all samples. The median of the measured Cd concentrations was significantly higher in both the kidney and the liver (p = 0.0011) of bucks than of does. In bucks, Cd levels exceeded the respective maximum limits laid down in the European legislation in four kidney and three muscle samples, whereas in does, the measured concentrations were below the respective limits in all samples. The detected amounts of Pb exceeded the maximum limits in the kidney of one buck and eight does, in the liver of two bucks and six does, in the muscle of six bucks and nine does, whereas in all fat tissues of both bucks and does. The concentration of Pb (p = 0.02) was significantly greater in the kidney of does compared to roebucks. Based on data obtained from the present study, the consumption of organs and tissues of the investigated roe deer could be objectionable from foodtoxicological point of view and may pose risk to the high consumers of wild game due to their cadmium and lead contents. Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-0206-9) contains supplementary material, which is available to authorized users. * József Lehel [email protected]

1

Department of Food Hygiene, University of Veterinary Medicine, István u. 2, Budapest H-1078, Hungary

2

Department of Animal Hygiene, Herd Health and Veterinary Ethology, University of Veterinary Medicine, Budapest, Hungary

Keywords Environmental contaminants . Food chain safety . Roe deer . Edible tissues . Risk assessment

Introduction The accumulation of toxic heavy metals has significant human health, ecological and biological consequences. Therefore, they got special attention within the scope of chemical pollution issues (Csathó 1994). Several researches are also focused on their distribution in the ecosystem, their accumulative properties and harmful effects in the last years. A wide variety of researches was carried out to determine how they get into the soil and ground water, what form they bind to and mobilise, which plant and plant parts can accumulate them, as well as how it is possible to enter the soil-plant-animal-human food chain (Farsang 2011). Several metals including heavy metals can be found as natural components in the environment; however, they can primarily get into the foods of animal origin and the body of human consumer due to the anthropogenic activities (e.g. industrial and agricultural processes, household use, transport, waste incineration). Some heavy metals such as cadmium, lead and mercury may accumulate in the tissues of animals, including wild games, intended for human consumption in such concentrations that have potential harmful effect to human health, despite the fact that their amounts in feed are safe to animals (NRC 2005). The liver, kidney and spleen are the primary organs for the accumulation of cadmium, lead and mercury. However, significant amounts may be detected in the muscles, and they can also be present in milk. Organic mercurial can be accumulated in all tissues including muscles. Therefore, human and animal health should also be considered during the calculation of their tolerable concentrations in the feed and water of livestock animals (NRC 2005).

Environ Sci Pollut Res (2017) 24:25372–25382

Currently, the basic legal regulation in the European Union on contaminants occurring in meat and offal of food-producing animals (bovine animals, sheep, pig, poultry, horse, fishes, crustaceans, live bivalves and cephalopods) is contained in Regulation No. 1881/2006/EC setting the maximum levels for certain contaminants in foodstuffs (Table 1). However, the concentrations of environmental contaminants are not regulated in the organs and tissues of game animals or in industrially processed game meat products (Commission Regulation 2006). Although the average annual consumption of game meat is generally low in Europe, it may be much higher where hunting is a cultural and heritage tradition until now and the hunting grounds were preserved. In addition, the popularity of venison is increasing among the consumers due to its features: healthy, redder and less crumbly than farm animal meat and provides specific intense taste (Ramanzin et al. 2010). In addition to the direct food safety issues, conclusions can be drawn from the heavy metal levels observed in game animals about the general environmental loads the given territory may face. The aim of the present study was to determine the concentrations of primary contaminants of geogenic and/or environmental origin (arsenic-As, cadmium-Cd, lead-Pb, mercuryHg) in edible tissues (muscle, liver, kidney and fat) of roe deer (Capreolus capreolus) and to evaluate their food safety risk to human consumers.

Materials and methods Location of the study and sampling Twenty roe deer (ten males, ten females) were sampled from the hunting area near the town of Ecser (GPS 47° 26′ 39.54″; 19° 19′ 0.6″) with a total area of 3500 ha. It is located in Pest county of Central Hungary region, about 22 km far from Budapest as the crow flies. There are industrial activities (wastewater treatment plant, air pollution due to Ferihegy Airport, logistics warehouses, chemical plants dealing with pesticides and fertilisers), busy roads and railway lines within about 5–10 km distance from the hunting area. Sampling was performed during the regular hunting season for deer, 15 April to 30 September, 2014 for roebuck and 1 Table 1 Maximum limits for lead and cadmium in foodstuffs expressed in mg/kg wet weight (Commission Regulation 2006)

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October, 2014 to the end of February, 2015 for doe (National Regulation 2004). Four samples were taken from each animal without external contamination: the muscle (musculus biceps femoris), liver, kidney and abdominal fat around the kidney and omentum. Each sample, about 1–3 g, was collected in a well-identifiable plastic container. All samples were transported to the analytical laboratory in a portable cooler and stored at −18 °C until laboratory analysis. The average age of the animals was 2.8 years (4.7 years for roebuck and 0.9 year for doe). The youngest deer was 0.5 and the oldest one 7 years old. Estimation of the age was performed by a professional hunter based on the wear of mandibular teeth. Analytical method Chemicals used The samples were digested by a mixture of high-purity concentrated nitric acid (69 m/m%, Aristar; VWR International Ltd., Debrecen, Hungary) and hydrogen peroxide (30 m/m%, Normapur, VWR International Ltd.). Laboratory glassware and plastic tools were cleaned by 0.15 M hydrochloric acid (37 m/m%, Aristar, VWR International Ltd.) and then rinsed with deionised water produced by Purite Select Fusion 160 BP water purification system (Purite Ltd., UK). Calibrations were performed by ICP multi- (Perkin Elmer Inc., USA, Shelton) and monoelement (VWR International) standards. 99.99996% purity argon gas was used for measurements (Messer Hungarogáz Kft., Hungary). Standards for quality control (QC) were prepared from standard bovine liver (NIST-1577C, NIST, USA). Preparation of sample Half gram from each organ and tissue sampled was weighed into a Teflon vessel, 5–5 mL of both nitric acid and hydrogen peroxide was added to it, and then the digestion process was started in a microwave digestion system (CEM MARS6, CEM Corporation, USA). The parameters of the process were as follows: ramp 35 min; temperature 200 °C; hold 50 min; and energy 1700 W. Then the sample was filled up to 25 mL with deionised water, and the concentration of heavy metals

Food

Lead

Cadmium

Meat of bovine animals, sheep, pig, poultry (excluding offal) Offal of bovine animals, sheep, pig, poultry Fats and oils, including milk fat Liver of bovine animals, sheep, pig, poultry, horse Kidney of bovine animals, sheep, pig, poultry, horse

0.10 0.50 0.10 – –

0.05 – – 0.50 1.00

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Environ Sci Pollut Res (2017) 24:25372–25382

was measured after twofold dilution. Solution of yttrium (1 mg/L) was used as internal standard (VWR International Ltd.), and solution of gold (0.25 mg/L) was applied for stabilisation of Hg content (VWR International Ltd., UK, Leicestershire). The preparation of blank and quality control (QC) samples was performed by the same method. Analytical measurements The concentration of heavy metals was measured by a Perkin Elmer Optima 8300 DV type (Perkin Elmer, USA) inductively coupled plasma optical emission spectrometer (ICP-OES). Calibration curves were ranged between 0 and 200 mg/kg. Limit of detection (LOD) was 0.05 mg/kg for Cd, 0.2 mg/kg for Pb and 0.5 mg/kg for As and Hg. Internal quality control of the measurements was ensured by at least ten consecutive measurements of QC samples with known heavy metal concentration. After discarding the extremes, the standard deviation (SD) of data was established, that must have remained within the ± 15% of the nominal concentration value in order to accept the QC measurement. The certified Cd content of the reference sample was above the LOD of method, thus it was measured directly. The certified values of As, Hg and Pb were below the relevant LODs; therefore, these parameters were checked by spiking the QC samples to contain additional 0.75 mg/mL from the elements. The calculated values were obtained by subtracting the values measured for the 0.75 mg/mL standard solutions from the values measured for the spiked QC samples (Table 2). The same internal standard was used every time. Spiked QC samples were subjected to the same sample preparation process than all the other samples.

Statistical analysis Statistical processing of the results was performed by Microsoft Excel and R program (version 3.3.2.). In samples where the concentration of metals was below the LOD, half of the LOD value was applied. Pb concentration of the tissues of both sexes was compared by Mood’s median test because the number of samples below LOD was different in the investigated tissues/organs. Table 2 Results of QC measurement (mg/mL)

Cd concentration in the muscle and fat samples of roebuck and does was analysed statistically by Fisher exact test and in the livers and kidneys by Mood’s median test. The various statistical methods were chosen because the results of all samples of muscle and fat in does were below the LOD. The concentrations of As and Hg were below the LOD in all samples of both sexes; therefore, they were not analysed statistically. The dietary weekly exposure was calculated based on the determined concentration of heavy metals in the samples and a standard portion of 300 g for the muscle, 100 g for the liver and 50 g for the kidney and fat consumed by a human consumer (EMEA 2001). These portions were then divided with an average body weight of man (60 kg) and multiplied with 7 (days/week). This calculation was used for comparison of weekly exposure of Pb to provisional tolerable weekly intake (PTWI) using the previously established value that can be considered indicative of possible health hazards even today. However, the legal regulation gives provisional tolerable monthly intake (PTMI) for Cd, thus the mentioned counting was multiplied with 4 (number of weeks) (JECFA-776 1989; JECFA-959 2011; JECFA-960 2011). Then, the detected concentrations were compared to the regulated maximum limits (Commission Regulation 2006).

Result and discussion The average concentrations (mean ± SD) of As, Cd, Pb and Hg measured in the samples of the liver, kidney, muscle and fat tissues of both sexes (roebuck, doe), the median and the range of data are presented in Table 3. The results are expressed in mg/kg wet weight (w.w.). Results obtained from our study were compared to similar findings of the scientific literature presented in Table 4. The detected concentrations of As and Hg were below the LOD (< 0.5 mg/kg) in all samples (muscle, liver, kidney and fat) of both sexes. Kidney The median of Cd concentration (0.94 mg/kg w.w.) was below the maximum limit (1.0 mg/kg) in the kidney of roebucks, but

Element

Certified value

Measured value

Spiked QC samples

LOD

Measured/ calculated value

Recovery (%)

As Cd Hg Pb

0.0190 0.0970 0.0050 0.0630

n.d. 0.095 ± 0.006 n.d. n.d.

0.761 ± 0.018 – 0.759 ± 0.020 0.796 ± 0.020

0.500 0.050 0.500 0.200

0.0197 0.0953 0.0053 0.0637

103.5 98.3 106.7 101.1

n.d. not detectable

Environ Sci Pollut Res (2017) 24:25372–25382 Table 3 Sample

Liver

Concentration of heavy metals in the liver, kidney, muscle and fat tissue of roebuck and doe (mg/kg w.w.) Sex

Buck

Doe

Kidney

Buck

Doe

Muscle

Fat

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Concentration of metal (mg/kg w.w.) Cadmium

n

Lead

n

Mercury

n

Arsenic

n

Mean ± SD Median

0.13 ± 0.04 0.14

10

0.90 ± 2.02 0.27

10

< 0.5

10

< 0.5

10

Range

0.06–0.17

Mean ± SD Median

0.03 ± 0.02 0.03

10

< 0.5

10

< 0.5

10

Range Mean ± SD Median Range Mean ± SD Median

< 0.05–0.07 1.03 ± 0.52 0.94 0.26–1.78 0.21 ± 0.20 0.18

10

< 0.5

10

< 0.5

10

10

< 0.5

10

< 0.5

10

< 0.20–6.64 10

10

10

2.23 ± 3.77 0.68 < 0.20–10.70 0.28 ± 0.26 0.21 < 0.20–0.95 0.68 ± 0.30 0.72

Range

< 0.05–0.75

Buck

Mean ± SD Median Range

0.04 ± 0.02 0.03 < 0.05–0.06

10

0.40 ± 0.29 0.39 < 0.20–0.86

< 0.20–1.17 10

< 0.5

10

< 0.5

10

Doe


< 0.05

10

30.41 ± 91.57 1.32 < 0.20–291.0

10

< 0.5

10

< 0.5

10

Buck


0.05 ± 0.02 0.05 < 0.05–0.08

10

2.93 ± 6.26 1.02 0.38–20.60

10

< 0.5

10

< 0.5

10

Doe

Mean ± SD Median

< 0.05

10

11.57 ± 27.73 0.68

10

< 0.5

10

< 0.5

10

Range

0.25–88.10

n number of sample

40% of the samples exceeded this limit. However, it was 21% (0.21 ± 0.20 mg/kg w.w.) lower than the limit in does. Based on the scientific literature, generally, higher Cd concentrations are present in the kidney than in the liver or the muscle meat of roe deer and other game animals or ruminants (Pompe-Gotal and Crnić 2002, Jarzyńska and Falandysz 2011). It can also be seen in our study results (kidney 0.62 ± 0.57 mg/kg w.w., liver 0.08 ± 0.06 mg/kg w.w.). Significant part of the Cd in bucks was found in the kidney (83%), followed by the liver (10%), fat (4%) and the muscle (3%) (Fig. 1). In our study, the ratio of liver:kidney of Cd is lower than 1 (0.13) that indicates the long-term exposure of extremely low dose (Bilandzić et al. 2009). Similar results compared to our ones were reported in the kidney of red deer (Cervus elaphus) (0.69 ± 0.25 mg/kg w.w., Čelechovská et al. 2008; 0.94 ± 1.80 mg/kg w.w., Lazarus et al. 2005). In comparison to our results, lower level of Cd was measured in roe deer of north-western Spain (0.197 ± 0.24 mg/kg d.w., Hermoso de Mendoza García et al. 2011), whereas higher concentrations of Cd were found in the kidney of roe deer (2.05 ± 0.30 μg/g w.w.) and red deer (1.64 ± 0.16 μg/g w.w.)

in north-western part of Poland (Wieczorek-Dąbrowska et al. 2012), and practically similar results were also reported previously from Slovakia (2.63 ± 2.24 mg/kg w.w., Kottferova and Korénekova 1998) and for the northern part of Poland (1.10– 0.50 mg/kg w.w., Falandysz 1994). Also higher accumulation potential of Cd was reported in the kidney of red deer (12 mg/kg dry weight (d.w.), Jarzyńska and Falandysz 2011), roe deer (4.91 mg/kg w.w., Pompe-Gotal and Crnić 2002) and reindeer (Rangifer tarandus) (7.69 mg/ kg w.w., Robillard et al. 2002). The concentration of Cd was highly increased in the kidney of roe deer at industrial environment (39.6 mg/kg w.w.); however, it was gradually reduced at more distant area (1.58 mg/kg w.w.) (Durkalec et al. 2015). Also, very high Cd concentrations (22.7 mg/kg w.w. with a maximum of 76.0 mg/kg w.w.) were measured in kidneys of roe deer shot near a lead smelter in Slovenia (Pokorny and Ribarič-Lasnik 2000). Based on the heavy metal content of different organs, the kidney exhibited the highest concentration factor (CF = 60.8–71.4) due to the high accumulation of Cd (Durkalec et al. 2015). In our study, the detected amount of Cd was significantly higher in the kidney of roebuck (p = 0.0011) than of does.

Roe deer

Species

Reindeer

Red deer

Roe deer

Species

Table 4

Fat

Durkalec et al. 2015 Bąkowska et al. 2016

0.460 ± 0.017 μg/g w.w.

Hermoso de Mendoza García et al. 2011 Wieczorek-Dąbrowska et al. 2012

0.445 ± 0.018 μg/g w.w.

0.134 ± 0.047 μg/g w.w.

Čelechovská et al. 2008

Present study Falandysz 1994 Kottferova and Korénekova 1998 Pokorny and Ribarič-Lasnik 2000 Pompe-Gotal and Crnić 2002

0.006 ± 0.008 mg/kg w.w. 0.05 μg/g w.w. 0.19–20 ng/g w.w.

0.171 mg/kg w.w.

0.05–6.69 mg/kg w.w. 0.18 ± 0.34 mg/kg d.w. 0.48 μg/g d.w.

0.14 ± 0.43 mg/kg w.w. 0.48 ± 0.21 mg/kg w.w.

0.176 mg/kg w.w. 0.06 mg/kg w.w.

0.099 ± 0.022 mg/kg w.w. 0.221 ± 0.230 mg/kg d.w.

15.40 ± 64.88 mg/kg w.w. 0.170 ± 0.340 mg/kg w.w. 0.12 ± 0.03 mg/kg w.w. 0.05 ± 0.03 mg/kg w.w.

0.30 mg/kg w.w.

1.300 ± 1.451 mg/kg d.w.

2.399 ± 1.583 mg/kg d.w.

0.271 ± 0.116 μg/g w.w.

7.25 ± 20.06 mg/kg w.w.

Fat

Reference

0.05 ± 0.02 mg/kg w.w.

Lazarus et al. 2014

0.160 ± 0.159 mg/kg w.w.

0.060 ± 0.033 mg/kg w.w.

7.69 μg/g w.w.

1.639 ± 0.157 μg/g w.w.

0.94 ± 1.80 mg/kg w.w. 2.28–5.91 mg/kg w.w. 12 ± 8 mg/kg d.w.

39.6 mg/kg w.w.

0.62 ± 0.57 mg/kg w.w. 1.100 ± 0.500 mg/kg w.w. 2.63 ± 2.24 mg/kg w.w. 22.73 ± 8.92 mg/kg w.w. 4.905 ± 6.395 mg/kg w.w. 0.685 ± 0.246 mg/kg w.w. 0.197 ± 0.236 mg/kg d.w. 2.054 ± 0.297 μg/g w.w.

0.087 mg/kg w.w.


Kidney

1.13 μg/g w.w.

0.147 ± 0.016 μg/g w.w. 0.152 mg/kg w.w.

0.05–0.50 mg/kg w.w. 0.7 ± 0.39 mg/kg d.w.

0.08 ± 0.06 mg/kg w.w. 0.170 ± 0.130 mg/kg w.w. 0.21 ± 0.10 mg/kg w.w. 3.92 ± 0.88 mg/kg w.w. 0.568 ± 0.502 mg/kg w.w. 0.221 ± 0.226 mg/kg w.w. 0.016 ± 0.028 mg/kg d.w. 0.245 ± 0.025 μg/g w.w. 0.511 mg/kg w.w. 0.12 mg/kg w.w.


Liver

Lead

0.001 ± 0.001 mg/kg w.w. 0.01 μg/g w.w.