Heavy metals in meat of Finnish city rabbits

Levels of cadmium, chromium, lead, copper, manganese and zinc in city rabbits were determined to evaluate the edibility of the meat. Mean concentrations of toxic metals were 0.011 mg/kg for cadmium and 0.037 mg/kg for lead. Cadmium and lead concentrations were below the admissible maximum levels set by the EU. Concentrations of toxic metals in the meat were sufficiently low as to assume that consumption of the meat does not pose a health risk for human health. Investigation of polycyclic aromatic hydrocarbons, pesticides and other environmental pollutants is required before the meat can be declared to be completely safe.


Introduction
The European rabbit (Oryctolagus cuniculs) is not indigenous to Finland but many pet rabbits have escaped and become wild. The first rabbits appeared in Helsinki (the capital of Finland) in the 1970s. Since 2000, the rabbit population has been growing fast and new areas of Helsinki have been colonised. In 2008, the population was about 7000. The rapid increase in numbers has been linked to the warmer and shorter winters with less snow. One rabbit can reproduce three to four times a year, which means that the female produces an average of 20 pups each year. Common habitats are parks, gardens, allotments and cemeteries (Leikas and Nummi 2009). European rabbits are adept at modifying their behaviour according to habitat (Thompson and King 1994). They eat the bark of woody plants in the winter, causing the plants to die, and they dig holes into the ground. The rabbits are now being hunted by the municipal council authority.
The aim of our study was to investigate whether city rabbits can be considered edible for humans, with particular regard to the metal content of their meat. Because the rabbits are living in the city centre their diet could easily be contaminated with metals from motor vehicles and industry. Cadmium and lead concentrations are of special interest because EC regulations specify acceptable limits for their concentration in meat (Commission Regulation [EC] No 1881/2006and 629/2008. Humans are exposed to cadmium through natural occurrence and from industrial and agricultural sources. Food is the main source for the non-smoking members of the population. Cadmium is primarily toxic to the kidney, especially to the proximal tubular cells where it accumulates over time, and may cause renal dysfunction. The International Agency for Research on Cancer has classified cadmium as a human carcinogen (Group 1, EFSA 2009). The main exposure to lead of the general non-smoking adult population comes from food and water. Lead is a classic long-term, cumulative poison. It can damage nervous connections and cause blood (anaemia) and brain (encephalopathy) disorders. Evidence of carcinogenicity of lead comes from both epidemiological and experimental studies (WHO 2000).
Chromium, copper, manganese and zinc are of interest for their nutritional value as essential trace elements. Trivalent chromium is an essential element in human nutrition, influencing carbohydrate, lipid and protein metabolism by tuning the action of insulin. Copper is an essential component of many enzymes (e.g. cytochrome-C-oxidase and superoxide dismutase) and proteins. Manganese is a component of arginase, pyruvate carboxylase and superoxide dismutase and plays a role as co-factor of important enzyme systems. Zinc is essential for growth and development, testicular maturation, neurological function, wound healing and immunocompetence (EFSA 2006).
With a view to establishing edibility, we determined the levels of cadmium, chromium, copper, lead, manganese and zinc. Measurements were made by inductively coupled plasma-mass spectrometry (ICP-MS), which allows the measurement of more than one element at a time. Samples were prepared by microwave-assisted acid digestion (Taylor et al. 1994(Taylor et al. , 1995Lo´pez-Alonso et al. 2007;Gerber et al. 2009).

Materials and method
Analysis of heavy metals Samples of muscle tissue were taken from 44 wild rabbits (Oryctolagus cuniculus), mostly caught in Helsinki city parks, between September 2008 and January 2009 and stored at À18 C until analysis.
Ultrapure water of resistance 18 M/cm was obtained from a Milli-Q Academic purification device (Millipore S.A.S., Molsheim, France). Super purity nitric acid (67%-69%) was from ROMIL Ltd (Cambridge, UK). The multi-element standard (ICP calibration mix FS 9: copper, zinc 100 mg/L; cadmium, chromium, manganese, nickel, lead, selenium 10 mg/L; arsenic 1 mg/L) and the standard solutions for iridium, germanium and rhodium (all 1000 mg/L) were from ROMIL Ltd. Iridium, germanium and rhodium were used as internal standards and an aqueous solution was prepared in 2% nitric acid at a concentration of 1 mg/L each. Hydrogen peroxide (30%) was provided by J. T. Baker (Deventer, Netherlands). As a reference material, NIST standard reference material 8414, bovine muscle powder was used.
Representative samples and reference material of approximately 0.5 g were weighed into microwave vessels, and 3 mL of nitric acid and 2 mL of hydrogen peroxide were added. The vessels were sealed and subjected to the microwave (CEN, Mars 5 Microwave Reaction System, Matthews, NC, USA) program described in Table 1. After digestion, 1 mL of the internal standard was added to each sample. The samples were diluted with ultra-pure water to a volume of 100 mL.
Samples were measured by ICP-MS (Thermo Scientific, XSeries 2, Bremen, Germany) under the operating conditions listed in Table 2. Determinations were performed with use of the aqueous multi-element standard in 2% nitric acid covering concentrations from 0 to 500 mg/L.

Results and discussion
All samples were prepared and measured as duplicates, together with at least one blank and one reference sample. LOD (mean blank value þ 3 Â SD) and LOQ (2 Â LOD) were calculated from results of 12 blanks. LOD and LOQ for each element and the trueness relative to the standard reference material are summarised in Table 3. The results for copper, manganese and zinc were above the LOQ in all 44 samples; for cadmium, results were above the LOQ for 24 samples. For chromium, results were above the LOQ for 31 of the samples, whereas for lead the values were above the LOQ in only 21 of the samples. Metal concentrations in the rabbit samples are summarised in Table 4. All individual results are given in the database. Mean concentrations of toxic metals were for cadmium 0.011 mg/kg and for lead 0.037 mg/kg in rabbit meat. Concentrations were below the maximum admissible levels in meat (cadmium 0.05 mg/kg and lead 0.10 mg/kg) stipulated in the Commission Regulations (EC) No 1881/2006 and 629/2008 except for one rabbit for which the level of cadmium was above 0.05 mg/kg. Our results were much lower than the levels reported by Eira et al. (2005;3.81 mg/kg lead and 0.04 mg/kg cadmium) in the muscle tissue of wild rabbits in Portugal. Our value for cadmium was higher and our value for lead lower than values measured by Vena¨la¨inen et al. (1996) (0.003 mg/kg for cadmium and 0.05 mg/kg) for lead in the muscle tissue for European hares in the industrial areas of Finland. Concentrations in muscle of rabbits in Poland (0.005 mg/kg for cadmium and 0.020 mg/kg for lead) reported by Falandysz (1991) were lower than our results. Compared to the levels in muscle of rabbits in New Zealand reported by Krelowska-Kulas et al. (1994;0.013 mg/kg for cadmium and 0.054 mg/kg for lead) the cadmium concentrations in our study were quite similar and our lead concentrations were slightly lower. The samples in New Zealand were collected from a nonindustrial area. The levels of cadmium and lead are a little higher but still comparable with the levels in meat of several farmed species (Table 4). The level of lead in Galician pigs is noticeably lower (Lo´pez-Alonso et al. 2007). In comparing the results, it should be remembered that farm animals live under controlled conditions and are fed diets with low level of metals.
The other elements (chromium, copper, manganese and zinc) are interesting for their nutritional aspect. No Allowed Daily Intake (ADI) value has been published for chromium. The Societies for Nutrition of Germany (DGE), Austria (Ö GE) and Switzerland (SEG) jointly established an adequate daily intake of 30-100 mg/day for adults (D-A-CH 2000). On the other basis, the results in Table 5 suggest that rabbit meat could provide 6.2%-43% of their adequate daily intake of  Mean dietary copper intakes of adults in different European countries have been estimated with a range of 1.0-2.3 mg/day for males and 0.9-1.8 mg/day for females. An EU population reference intake (PRI) of 1.1 mg/day for adults was established in 1992 (EFSA 2006). Calculated daily intakes of copper in Finland in 2007 were 1.6 mg (men, 25-64 years), 1.4 mg (men, 65-74 years), 1.3 mg (women, 25-64 years) and 1.2 mg (women, 65-74 years) (National Public Health Institute, KTL 2008). According to the results in Table 5, it would be possible to cover 4.0-8.5% of the PRI for copper with rabbit meat. Rabbit meat is not such a good source of copper as pig (Lo´pez-Alonso et al. 2007) or lamb (Gerber et al. 2009) (Table 4).
The EU Scientific Committee for Food estimated in 1993 that 1-10 mg/day manganese was an acceptable range of intake (EFSA 2006). With these values, the results in Table 5 indicate that only 0.2%-5.2% of the acceptable intake per day could be met with rabbit meat. While rabbit meat is a poor source of manganese, mean value of 0.329 mg/kg is comparable to the levels in farmed species (Table 4). Note that the results for manganese ranged widely from 0.069 to 1.24 mg/kg, so that the mean value can only be considered indicative.
The European PRI for zinc for adult males and females is 9.5 mg/day and 7.0 mg/day, respectively (estimated 1993(estimated , EFSA 2006. Calculated daily intakes of zinc in Finland in 2007 were 13.6 mg (men, 25-64 years), 12.3 mg (men, 65-74 years), 10.0 mg (women, 25-64 years) and 9.4 mg (women, 65-74 years) (National Public Health Institute, KTL 2008). On the basis of the results given in Table 5, males could cover 15.5%-19.9% and females 12.8%-15.8% of the PRI for zinc. Rabbit meat appears to be a good source for zinc. Given the range from 7.14 to 29.7 mg/kg, levels are fully comparable with the levels of zinc in meat of other animal species, 7-42.5 mg/kg (Table 5).

Conclusions
Helsinki City rabbits are fully edible from the point of view of metal content. In terms of toxicity, there is no risk for humans. In nutritional value, rabbit meat is comparable to the meat of farmed species.
The contents of the essential trace elements determined are relatively low but not as low as in chicken (Gerber et al. 2009). Before the meat of city rabbits can be considered safe for human consumption, investigation will need to be made of polycyclic aromatic hydrocarbons, pesticides and other environmental pollutants.