ABSTRACT
A validated and accredited analytical method of inductively coupled plasma–mass spectrometry was used to determine the levels of 22 trace elements in 52 equine meat samples collected during 2015. Greater amounts of Zn, Fe, and Ca were found with mean values of over 25 µg g−1. Levels of non-essential trace elements, that is, Pb, Cd, Hg, and As, were generally low (mean values lower than 11.3 ng g−1). Equine gender and geographic origin of meats (Italy and Poland) were compared, with no significant differences being found, whereas equine meats could be differentiated from bovine through a multivariate approach. As regards trace element accumulation, evaluated considering slaughter age, Zn, Fe, Ca, and Cr showed greater increase. Finally, a good correlation was obtained between two pairs of trace elements, Zn/Fe (r = 0.82) and Ca/Fe (r = 0.87).
Introduction
Some trace elements, such as iron (Fe), manganese (Mn), zinc (Zn), cobalt (Co), selenium (Se), molybdenum (Mo), and copper (Cu), play an important role in biological systems; consequently, they are defined as essential micronutrients, as an adequate amount of each needs to be consumed in order to maintain normal physiological functions.[Citation1–Citation5] Other trace elements, which have no biological role, are defined as non-essential trace elements. Some of these are associated with toxic health effects on humans and animals.[Citation6] These well-known environmental pollutants[Citation7], better known as heavy metals, are characterised by atomic weights higher than sodium, specific gravity of over 5.0,[Citation8] and high affinity with biological tissues, from which they are only slowly eliminated.[Citation9,Citation10] Among heavy metals, lead (Pb), cadmium (Cd), and mercury (Hg) are considered the most harmful, due to their bioaccumulation in some foodstuffs, and their high toxicity.[Citation11–Citation16]
Over recent years, it has become possible to study the trace element exposure pattern for humans, found in different diets in different countries. This approach, known as the total diet study (TDS), has recently been introduced, both in the European Union and in the USA.[Citation17,Citation18] For example, a recently published complete study related to TDS in France stated that some elements such as Li, Cr, and Mn are mostly taken in by consuming fish products, fats, and nuts/oilseeds, respectively; while the most hazardous heavy metals, that is, Pb, Hg, and Cd are linked, as expected, to seafood consumption.[Citation4,Citation19]
Regarding meat and meat products, as indicated by Doyle and Spaulding,[Citation20] the essential trace elements present at highest concentrations are Zn and Fe, with levels up to 33.9 and 130.0 µg g−1 wet weight, respectively, in sheep muscle. Pb is the most present heavy metal, and its concentration may reach 340 ng g−1 wet weight in cattle muscle. Concerning different types of meat (pork, beef, and chicken), Lei et al.[Citation5] reported that beef meat is the richest in Fe, Mn, and Zn (mean levels equal to 43.9, 0.848, and 53.5 µg g−1 wet weight, respectively), while, among heavy metals, Cd and Pb concentrations were higher in chicken meat (mean levels equal to 0.818 and 35.8 ng g−1, respectively).
As regards the trace element profile of equine meat, bibliographical data are lacking. Indeed, most trace element characterisations in meat have been developed for beef, pork, and chicken, the most popular meats consumed worldwide. However, in recent years, the consumption of equine meat has gradually increased, especially in western European countries, as an alternative red meat.[Citation21] Moreover, the food safety problem related to heavy metal accumulation in equine liver and kidney is well-known.[Citation22–Citation25] Indeed, in 2005, the Italian Ministry of Health issued Memorandum No. DGVA/IX/35232/P, in which the sale of liver and kidney of equine origin was banned due to the high non-compliance rates for Cd in liver samples. At the same time, the memorandum strongly suggested implementing periodic monitoring plans for equine organs and meats.
This study aimed to provide a contribution to the evaluation of the trace element profile in equine meats. Measuring the levels of 9 essential trace elements (Mo, V, Co, Se Mn, Cu, Zn, Fe, and Ca) and 13 non-essential trace elements (Pb, Cd, Hg, As, U, Sr, Sn, Tl, Sb, Cr, Ni, Be, and Al), out of a total of 52 equine meat samples, highlighted the following points: (1) evaluation of essential trace element profile/nutritional aspects, and comparison with meats of other species; (2) assessment of food safety aspects related to non-essential trace elements; (3) statistical comparison between geographical and gender groups, evaluation of trace elements accumulation and correlations between pairs of elements; (4) evaluation of inductively coupled plasma–mass spectrometry (ICP-MS) as a tool for identifying meat species. Finally, in order to follow up the above Italian Ministry of Health memorandum, the 52 equine liver samples were analysed, and their suitability for human consumption was evaluated. The analyses were carried out using a validated and accredited analytical method based on ICP-MS.
Material and methods
Sampling and chemicals
A total of 52 samples of equine meat (loin), from equines of both genders, were collected from slaughter houses in the Apulia region of Italy, during March–May 2015. The samples were characterised by a slaughter age varying from 4 to 61 months. In addition to equine meats of Italian origin (13 samples), 39 samples of Polish origin were also taken into consideration. Descriptions of the samples collected in this study, in terms of gender, breeding geolocation, and slaughter age are reported in .
Table 1. Description of the samples collected in this study.
as Along with meat samples, during slaughter, a liver sample was collected from each equine. These samples were analysed in order to evaluate both liver contamination levels and the correlation with accumulation in muscle. HNO3 (68% v/v), H2O2 (30% v/v), and ultrapure water were purchased from Romil Ltd. (Cambridge, UK); element standard solutions (U, Hg, Pb, Cd, As, Sr, Sn, V, Ni, Cr, Mo, Co, Cu, Zn, Ca, Mn, Fe, Tl, Sb, Be, Se, and Al) (1000 mg L−1) were supplied by CPA Ltd. (Stara-Zagora, Bulgaria); ultrapure argon (N 60), anhydrous ammonia, and methane (N 55) were purchased from AIR Liquide S.p.A. (Milan, Italy).
Experiments
This study was carried out using a modified and integrated version of the UNI EN 15763:2009 reference method[Citation26] as the analytical method for determining trace elements. This procedure was accredited by the Italian organization for laboratory accreditation ACCREDIA, in 2010, and is currently adopted for this type of analytical determinations at the Chemistry Department of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, in Foggia, Italy. About 500 g of each sample was collected and pretreated by separating the muscle from the subcutaneous fat. The samples were homogenized in a commercial blender (model Mixer B-400, BUCHI Labortechnik AG, Flawil, Switzerland), with ceramic blades to prevent the release of metals into the sample. Then, 1.0 ± 0.0001 g of homogenized sample was weighed using an analytical balance (Mettler Toledo s.p.a., Novate Milanese, Milan, Italy) and then mineralized by microwave-assisted acid digestion. Mineralization was accomplished using an Ethos-One Microwave Reaction System (Milestone s.r.l. Sorisole, Bergamo, Italy), following the UNI EN 13805:2014 reference procedure.[Citation27] Briefly, 1.0 g of homogenized sample was weighed in a Teflon vessel, 6 mL of HNO3 (68% v/v) and 2 mL of H2O2 (30% v/v) were added and the vessels were placed into the microwave reaction system. Total sample digestion was achieved using the following program: up to 120°C in 15 min and constant for 10 min; up to 190°C in 15 min and constant for 20 min; cooling stage (30 min) to reach room temperature. After digestion, the solution was transferred into 50 mL polypropylene disposable tubes and filled to the mark by adding ultrapure water for subsequent analysis by ICP-MS.
An inductively coupled plasma mass spectrometer (PerkinElmer Inc., model Elan DRC II, MA, USA) equipped with a concentric nebulizer (Meinhard Associates, Golden, USA), a baffled cyclonic spray chamber (Glass Expansion, Inc., West Melbourne, Australia), and a quartz torch with a quartz injector tube (2 mm i.d.) was used for trace element quantification. The operational parameters were as follows: radio frequency power: 1200 W; plasma gas (Ar) flow rate: 15 L min−1; nebulizer gas (Ar) flow rate: 0.97 L min−1; sample flush: 60 s; sample flush speeding: 32.0 rpm; read delay: 20 s; read delay and analysis speeding: 20 rpm; wash: 45 s; wash speeding: 32 rpm; dwell time: 50 ms; sweeps/reading: 20. Rhodium and bismuth (both at 200 ng mL−1) were used as internal standards, added to standard and sample solution by on-line mixing.
The following elements/isotopes were detected: 9Be, 27Al, 44Ca, 51V, 52Cr, 55Mn, 56Fe, 59Co, 60Ni, 63Cu, 66Zn, 75As, 78Se, 88Sr, 98Mo, 111Cd, 118Sn, 120Sb, 202Hg, 205Tl, and 238U. In order to eliminate the intrinsic variability of lead isotope distribution and to improve signal sensitivity, the sum of 206Pb, 207Pb, and 208Pb was counted. In order to minimize isobaric interference, the dynamic reaction cell (DRC) system was used, employing ammonia gas (100%, high purity) at 0.5 mL min−1 for the determination of Al, As, Co, Cr, Cu, Fe, Mn, Ni, V, and Zn, and methane gas at 0.5 mL min−1 for the determination of Se. Instrument calibration was performed by standard addition into the mineralized and diluted solution: for each element, five addition levels, including not-added level, were used. The addition levels for each element were as follows: U, Hg, Tl, Be (0.010–0.020–0.050–0.20 ng mL−1); Sb (0.10–0.20–0.50–2.0 ng mL−1); Pb, Cd, As, V, Ni, Se, Co, Mo, Sn, Cr (1–2–5–20 ng mL−1); Al, Cu, Mn (5.0–10–25–100 ng mL−1); Ca, Zn, Fe, Sr (40–80–200–1000 ng mL−1). Linearity was verified by adopting five calibration levels (as described above). Good linearity was observed in the calibration range set for each element, with a determination coefficient (R2) of over 0.9971. The goodness-of-fit of the calibration curve was checked using Mandel’s test. Limit of quantification (LOQ) values for the method, for each element, were determined by blank determination assays, as 10 times standard deviation of 20 blank replicates. All validation parameters for the analytical procedure are reported in . Two replicates of each sample were analysed, and trace element concentrations evaluated as the mean of both measurements. A certified material (NIST 1577b – bovine liver) was analysed during each working session for quality assurance purposes. This method has already been successfully applied for a recently published monitoring of heavy metals in seafood.[Citation15] The entire procedure described above was used for analysing both meat and liver samples.
Table 2. Validation parameters of ICP-MS analytical method.
Statistical analysis
For each trace element, mean concentrations and standard deviations were calculated and compared. Comparisons between groups of samples (male/female and Italy/Poland) were carried out using the T-test. Pearson’s r correlation coefficient was computed in order to evaluate the correlation between each pair of metals. The accumulation trends, in relation to slaughter age, were evaluated by considering the slope of regression lines. Finally, principal component analysis (PCA) was performed, together with the T-test, to compare meats of equine and bovine origin.
Results and discussion
A concentration equal to the LOQ for each element (see ) was assigned when a sample showed an unquantifiable amount of that element. This approach, known as upper-bound, was indicated by the Italian Institute of Health in the document “Rapporti ISTISAN 04/15,” as a protective measure, relating to human health and the environment.[Citation28] All results obtained by analysing 52 samples of equine meat, subdivided into non-essential trace elements and essential trace elements,[Citation29] are reported in .
Table 3. Trace elements in 52 samples of equine meat with statistical descriptors: range, average, and standard deviation (SD).
Essential trace elements and nutritional aspects
Concentration levels for most of the elements were quantified in all samples. Only V and Mn were not detected in 4 and 9 samples, respectively. Zn, Fe, and Ca were the elements with highest levels, with mean values of 48.1, 25.1, and 53.9 µg g−1, respectively. Cu was also present at high concentrations, with mean values of 1.13 µg g−1. These results were comparable with those reported in literature.[Citation30–Citation32] Indeed, in equine meat of the same cut type (loin) Seong et al.[Citation32] found mean concentrations of 59.2 µg g−1 (Ca), 25.4 µg g−1 (Fe), 34.8 µg g−1 (Zn), and 1.72 µg g−1 (Cu). It should be noted that the present results were in good agreement with the literature data for three of the four elements, with the exception of Zn, probably due to feeding and/or geographical factors.
At lower concentrations, among essential trace elements, mean Se and Mn concentrations of 85.2 and 82.0 ng g−1, respectively, were quantified; Mo, V, and Co showed the lowest concentrations (14.7, 4.5, and 1.9 ng g−1, respectively). Considering the most important trace elements, from a nutritional point of view, namely Ca, Zn, Fe, and Cu, and considering as reference data published by Williams,[Citation33] the first impression was that levels of Zn and Cu in the equine meats were comparable to those in beef, lamb, veal, and mutton. Ca levels were lower than in veal, lamb, and mutton, whereas Fe, which is usually correlated with the nutritional advantages of equine meat consumption, was only slightly higher than in beef, veal, and lamb, but lower than in mutton (). Using Reilly[Citation34] as reference data, Se levels recorded in this survey were comparable to levels in beef and lower than in chicken and pork. Regarding Mn, levels in this study seem lower than those for ovine meats and comparable to those of pork.[Citation20]
Figure 1. A comparison of Ca, Fe, Zn, and Cu levels among meats of different origin.
Non-essential trace elements and food safety aspects
As expected, the levels of 13 non-essential trace elements monitored during this study were generally low. Al, Ni, and Sr were the elements detected at greater amounts (1036.8, 171.1, and 132.0 ng g−1, respectively); however, these three elements are not particularly toxic for humans at these levels, with the exception of some allergic reactions,[Citation35] also considering that Ni and Sr concentrations in some foodstuffs may be high. In particular, Ni levels may reach levels higher than 3000 ng g−1 in cocoa products and nuts,[Citation36,Citation37] while Sr concentrations in milk and dairy products are not negligible due to this element’s high chemical affinity with calcium.[Citation38,Citation39] Beryllium was lower than the LOQ in all muscle samples analysed and Sb was detected in only two samples, at low concentrations. By contrast, Sn and Cr were above the LOQ in most samples, but their mean concentrations (15.0 and 21.0 ng g−1, respectively) do not cause particular food safety concerns. Among non-essential trace elements, Pb, Cd, As, and Hg are considered the most harmful. The mean values of Pb and Cd were equal to 6.0 and 11.3 ng g−1, respectively. Taking into consideration the legal limits established for these two elements in equine and other meats (Regulation 1881/2006/EC), equal to 100 and 200 ng g−1, respectively,[Citation40] the contamination levels recorded herein may be considered as not significant. Contamination by As was assessed at a mean level corresponding to 6.8 ng g−1. This level is not harmful; indeed, the main sources of As exposure, that is, contaminated water and rice, are considered as unsafe only at concentrations higher than 100 ng g−1.[Citation41–Citation43] Hg was detected only in five samples with a mean concentration of 1.48 ng g−1 that may be considered not particularly significant, being only slightly higher than its LOQ (1.07 ng g−1). The same is true for other toxic trace elements such as U and Tl.
Comparison between groups
All results obtained both for essential and non-essential trace elements were compared, element-by-element, using the T-test. Two comparisons were carried out, considering equine gender and geographic origin of meats (Italy and Poland). As can be seen in and , in which the mean levels of each element are compared, none of the metals in this study varied significantly between male and female and Italian and Polish origin. As a preliminary result, it is possible to suppose that trace element accumulation in equine meats is not strictly correlated with animal gender and geographic origin.
Figure 2. Element-by-element comparison between meats from equine of different genders.
Figure 3. Element-by-element comparison between equine meats from different breeding geolocations.
Trace element accumulation and correlations
It is well-known that trace elements may accumulate in biological systems over several years. This is known as bioaccumulation.[Citation44] This characteristic of trace elements was confirmed in this study, after examination of their accumulation during equine growth, evaluated considering slaughter age (from 4 to 27 months). Among all metals determined, Zn, Fe, Ca, and Cr showed greater accumulation, as can be seen in the trends shown in . In particular, as a first approximation, the concentrations of Fe, Zn, and Cr can double in 23 months, while Ca may triple. In order to find similar behaviours, relating to accumulation, the correlation between each couple of elements was studied. This approach was necessary especially regarding correlations between essential and non-essential trace elements which could lead to difficult evaluations both in terms of nutritional value and food safety. As expected, toxic trace elements did not correlate with essential trace elements. A good correlation was obtained between two pairs of essential trace elements: Zn/Fe (r = 0.82) and Ca/Fe (r = 0.87) (see ). A good Zn/Fe correlation (r = 0.9017, p < 0.05) was also obtained by Sandu Stef and Gergen in chicken breasts.[Citation45] Regarding Ca/Fe correlation, Guang-zhi et al.[Citation46] found a similar result (r = 0.667, p < 0.01) in pork meat (Musculus longissimus dorsi).
Figure 4. Trace element accumulation.
Figure 5. Correlation between levels of Zn/Fe (A) and Fe/Ca (B).
Evaluation of ICP-MS as a tool for meat species identification
The identification of meat type and origin has become increasingly important over the last decade, for economic, religious, and ethical reasons, as well as for its food safety implications. Indeed, the addition of low-quality and cheaper meats and/or of mechanically separated meats, as well as deteriorating the overall quality of the product, may bring about some food safety concerns, as in the recent case of detection of equine meats in products with no equine meat declared on the label. As a consequence, in Europe, several monitoring plans have been drawn up, aimed at identifying residues of phenylbutazone, the most commonly used drug in racehorses.[Citation47]
Currently, several analytical techniques able to identify different animal species and other meat authenticity-related aspects are available. Depending on the type of meat and processing, different techniques can be applied such as hybridization and polymerase chain reaction, enzymatic assays and immunoassays, mass spectrometry, microscopy, spectroscopy, electrophoresis, electronic spin resonance, etc.[Citation48] With regard to trace elements, together with stable isotope ratios, the technique used in this study has previously been extensively applied to determine the geographic origin of meat.[Citation49] Few studies linking trace element profile to type of meat are available, and none of these report a comprehensive evaluation of this application. This part of the study focused on the potentiality of the technique employed in this work as a tool for meat species identification. Thirteen samples of equine meat and 13 samples of bovine meat, characterised by the same geographic origin (Apulia region, Italy) were compared, measuring the levels of 22 trace elements. PCA was performed using SIMCA (Umetrics, Sweden). Data were scaled using the unit variance scaling method. PCA was used as the first step for data reduction and visualization, in order to highlight any variation in the dataset. The principal components were rank ordered by the variability that they represent in the dataset, with the first principal component accounting for the greatest variability in the data and so on.[Citation50] The T-test was used to find elements that changed significantly between two meat species (p < 0.05). Thirteen trace elements (Zn, V, Sr, Mn, Al, Cu, Co, Fe, Tl, Cr, U, As, Ni) changed significantly (T-test, p < 0.05) between two species of meat. As a preliminary result, we observed that two different species of meat clustered separately along the first principal component (PC1) that accounts for 33.8% of total variance (). Moreover, if the comparison is developed by taking into account only eight trace elements (p < 0.005), it is possible to achieve almost complete separation (PC1 accounts for 61.4% of the total variance) between the two data populations, making maximum use of this potential survey tool ().
Figure 6. Comparison of equine and bovine meats – principal component analysis: 22 trace elements (A); 8 significant trace elements (p < 0.005) (B).
Among the trace elements with the greatest contribution to differentiation, it is interesting to underline that mean levels of some essential elements such as Zn, Fe, and Mn were about twice as high in equine meats as in bovine. Future studies will be able to evaluate this technique by analysing a representative number of samples, extending the field of application also to pork and chicken and confirming or modifying the number and type of trace elements to take into consideration as representative within the technique.
Accumulation in liver samples
Trace element levels in liver samples were substantially different from those found in muscle samples (). Indeed, taking into consideration the legal limit established for Cd (0.5 mg kg−1) and Pb (0.5 mg kg−1, but only for similar species), as reported in European Commission Regulation 1881/2006,[Citation40] 25 samples were non-compliant for Cd levels, while 3 were above the reference level for Pb. These results confirm the adequacy of the above Italian Ministry of Health Memorandum No. DGVA/IX/35232/P, relating to human consumption of equine liver, since contamination levels may be high, especially of Cd. Pearson’s correlation coefficient (r) was evaluated to verify the correlation between trace element accumulation in liver and muscle. No significant correlation was verified for all trace elements determined, particularly heavy metals (r < 0.34). This is further confirmation of equine meat safety, regardless of liver contamination levels.
Table 4. Trace elements in 52 samples of equine liver with statistical descriptors: range, average, and standard deviation (SD).
Conclusion
In this study, 52 samples of equine meats slaughtered in the Apulia region (Italy), during March–May 2015, were collected and then analysed in order to determine the levels of 22 trace elements (Mo, V, Co, Se Mn, Cu, Zn, Fe, Ca, Pb, Cd, Hg, As, U, Sr, Sn, Tl, Sb Cr, Ni, Be, and Al). The analyses were carried out with a validated and accredited analytical method using ICP-MS, and the amounts recorded were statistically elaborated in order to contribute to equine meat characterisation. Most essential trace elements were quantified in all samples, with Zn, Fe, and Ca being those with the highest levels, with mean values above 25 µg g−1. Cu also shows high concentrations (mean of 1.13 µg g−1). Regarding toxic trace elements, the levels of the 13 heavy metals monitored during this study were generally low. In particular, mean Pb and Cd values were 6.0 and 11.3 ng g−1, respectively, while mean As levels were 6.8 ng g−1. Hg was detected in only five samples with a mean concentration of 1.48 ng g−1. Equine gender and geographic origin of meats (Italy and Poland) were compared, highlighting insignificant differences. As preliminary result, a PCA approach was successfully tested as tool for differentiating equine from bovine meats. As regards element accumulation, evaluated considering slaughter age, Zn, Fe, Ca, and Cr showed the greatest increases. Finally, a good correlation was obtained between two pairs of essential trace elements, Zn/Fe (r = 0.82) and Ca/Fe (r = 0.87).
Declaration of interest
The authors Oto Miedico, Marco Iammarino, Marina Tarallo, and A. Eugenio Chiaravalle certify that they have not a financial or personal relationship with other people or organizations that could inappropriately influence or bias this paper.
Funding
The authors thank the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (Foggia, Italy) for providing financial support. Italian Ministry of Health (Rome, Italy) is gratefully acknowledged for the financing of Research Project code GR-2013-02358862.
Additional information
Funding
References
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