Wild Boar ( Sus scrofa L.) as the Biomonitor of Cadmium and Lead Pollution in the republic of North Macedonia

Until the outbreak of African swine fever in almost the entire Europe and consequently in the Republic of North Macedonia (2022)


INtrODUCtION
Regardless of whether it comes from natural processes on the Earth's surface or from human activities, elements and compounds that are released into the environment and cause damage to the biosphere are treated as pollutants (Kłos et al. 2018).About 13,000 tons of cadmium (Cd) are produced annually worldwide, mainly intended for chemical stabilizers, nickel-cadmium batteries, pigments, metal coatings and alloys (Flora et al. 2008).Through the soil and the water, cadmium compounds can be easily adsorbed by plants.With the increased accumulation of toxic metals in the plants and the soil, the risk of their transfer to herbivorous wild mammals, game and livestock may increase (Bilandzić et al. 2010).Lead (Pb) is a highly toxic metal, which with its wide distribution, mainly as a result of human activities (mining, production and combustion of fossil fuels), can cause environmental contamination and health problems in many parts of the world (Jaishankar et al. 2014).The exposure to lead contamination can be manifested in lead-induced encephalopathy, gastroenteritis, and peripheral nerve degeneration (Lénárt et al. 2023).For the domestic and the wild animals and for the humans, acute or chronic exposure to Pb and Cd may have severe consequences because these heavy metals accumulate in almost all tissues, mainly in liver and kidneys as target organs (Satarug et al. 2003, Kramárová et al. 2005, Pandey and Madhuri 2014).
The Republic of North Macedonia, as part of the former Yugoslavia, was an important producer of metals such as copper, steel, ferroalloys, lead, zinc, cadmium, nickel, silver and gold, with long-term mining and mining-related activities contributing greatly to the pollution of the environment (Barandovski et al. 2020)

Wild Boar (Sus scrofa L.) as the Biomonitor of Cadmium and Lead Pollution in the republic of North Macedonia
Macedonia (Rogan et al. 2010, Stafilov et al. 2010, Stafilov 2017, Barandovski et al. 2020, Stafilov et al. 2023), where the concentration of heavy metals was measured in soils and mosses, offer a good contribution to getting to know the situation regarding the representation of heavy metals in this country.On the other hand, very little is known about the exposure of game (herbivores and omnivores) to heavy metals as a direct consumer of food from nature, which is often contaminated around critically endangered areas (industrial zones and mines).The anthropogenic pollution and the environmental areas naturally enriched in toxic heavy metals where game animals reside can increase the risk of contaminants entering the food chain and induce elevated level of them in game (Lehel et al. 2016).The diet is considered to be the most important pathway of metal transfer from soil and plants to the tissues of animals (Stankovic et al. 2014).
A well-known and commonly used bioindicator in modern research is wild boar (Sus scrofa L.) (Bilandzić et al. 2009, Bilandzić et al. 2010, Danieli et al. 2012, Gasparik et al. 2012, Lénárt et al. 2023).Wild boar is a potentially good bioindicator for monitoring of heavy metals in the environment because it is an opportunistic omnivore ingesting both flora and fauna (invertebrates, vertebrates, crops (Lee and Lee 2019) and mushrooms (Brzezicha-Cirocka et al. 2016)).Wild boars have a characteristic way of diet; they search for food by browsing or grazing (grasses, herbs, stems, leaves), on the surface of the ground (fruits and fungi) or below the surface of the ground by rooting (roots, invertebrates, rhizomes) (Bueno et al. 2011, Ballari andBarrios-García 2014).They have relatively long lifespan and thus long period of accumulating chemicals (Bilandzić et al. 2010).Also, they are relatively mobile and have the large effective population sizes (Frantz et al. 2012), so they can cover a large area in search for food and thus provide an early warning of adverse toxic effects in the ecosystem as a whole.Wild boars are widely distributed in the world and can be found in Eurasia, the southern part of Asia, and some of the islands od Indonesia (Lénárt et al. 2023).
The presence of wild boar in the last years in European areas has increased (Jori et al. 2021).In the Republic of North Macedonia, wild boars are represented in the entire territory of the country, while the last decade their number has increased drastically.This is confirmed by data from the State Statistical Office, where the number of the population in 2010 was estimated to be around 2,872 individuals, and in 2022 it was estimated to be around 9,400 individuals (MakStatDatabase 2021a).Since the largest number of hunters in North Macedonia are focused on wild boar hunting, in recent years the number of wild boars harvested during the hunting seasons has increased dramatically (in 2010, about 472 were shot, and in 2022, about 2,800 individuals) (MakStatDatabase 2021b), which makes biomonitoring with this species much easier.The good reflection of environmental pollution in wild boar tissues demonstrated in numerous papers in the last decade and the relevance for the consumers of boar meat (transmission to humans) are just a few of the reasons why we decided to investigate the occurrence of Pb and Cd in the liver of wild boar as the biomonitor of the pollution in the Republic of North Macedonia.

MAtErIALS AND MEtHODS the Study Area
The Republic of North Macedonia is a country situated in the central part of the Balkan Peninsula, bordering Serbia to the north, Bulgaria to the east, Greece to the south and Albania to the west, and it has a total area of 25,713 km 2 .The country is mountainous and has deep basins and valleys with three large lakes and is bisected by the Vardar River.It has a water area of 857 km 2 , while its land area is 24,856 km 2 .The country has 16 mountains higher than 2,000 m, but most of the surface is between 500 and 1,000 meters above sea level.The highest point (2,764 m) in the country is the peak Golem Korab of Mount Korab and the lowest point (44 m) marks the area at the crossing point of the Vardar River (Barandovski et al. 2020).
In North Macedonia, the state establishes hunting grounds as basic spatial units for game management according to hunting law (2009).However, in the general hunting management plan for the Republic of North Macedonia (period 1997-2016), according to Trpkov and Maletic (1997), the total territory of the country is divided into 11 hunting areas as broader categories for game management.This division was made for the purpose of implementing a unique hunting policy and appropriate measures for protection, breeding and promotion of game management.In fact, the hunting area represents an ecological unit with a large area, conditioned by a complex of environmental factors and the vital characteristics of game populations, where it resides throughout the year and has the greatest impact on the environment.Given the mobility of game, especially large game, and its relatively large radius of action during all seasons to satisfy life's needs, such an organization is fully justified, especially when planning for the joint implementation of hunting policies in the hunting grounds that are part of the same hunting area, especially for the shooting.Also, when monitoring certain types of big game at the hunting ground level, wrong data on the numerical condition of the game are often obtained because the hunting ground is a small spatial unit, so there is a fear of the possibility of counting the same game in two or three hunting grounds which border.
Therefore, this study was carried out on the same 11 hunting areas that cover the territory of the entire country, namely: Pelagonisko hunting area (1) including the municipalities of Prilep, Bitola, Demir Hisar and Kruševo, with a total area of 408,279 ha; Ohridsko-Prespansko hunting area (2) including the municipalities of Resen, Ohrid, Struga and Debar, a total area of 183,743 ha; Kičevsko-Brodsko hunting area (3) including the municipalities of Kičevo and Makedonski Brod, a total area of 173,624 ha; Pološko hunting area (4) including the municipalities of Gostivar and Tetovo, a total area of 168,717 ha; Skopsko-Kumanovsko hunting area (5) including the municipalities of Skopje and Kumanovo, a total area of 276,105 ha; Sredno-Vardarsko hunting area (6) including the municipalities of Veles and Sveti Nikole, a total are of 220,525 ha; Krivorečansko hunting area (7) including the municipalities of Kratovo and Kriva Palanka, a total area of 109,524 ha; Bregalnčko hunting area (8) including the municipalities of Kočani, Vinica, Probištip and Štip, a total area of 214,435 ha; Vlainsko-Maleševsko hunting area (9) including the municipalities of Delčevo, SEEFOR 14(2): 235-243 237 Berovo, Pehčevo and Makedonska Kamenica, a total area of 139,233 ha; Strumičko hunting area (10) including the municipalities of Strumica and Radoviš, a total area of 168,695 ha; Dolno-Vardarsko hunting area (11) including the municipalities of Kavadarci, Negotino, Valandovo and Gevgelija, a total area of 282,772 ha.

Sampling
From 11 hunting areas (number 1 to 11) covering the whole territory of the Republic of North Macedonia, during the regular hunting season, which starts on 1 October and lasts until 31 January every year (between 2016 and 2022), a total number of 608 liver samples of wild boar (aged between 2 and 4 years) harvested by the active hunters were collected.The collected samples do not have a normal numerical arrangement in the hunting areas.From hunting area number 1, 143 samples were collected, from number 2 -106 samples, from number 3 -57, from number 4 -50, from number 5 -17, from number 6 -15, from number 7 -57, from number 8 -3, from number 9 -26, from number 10 -53 and from number 11 -81.After collection, the samples were individually packed in polyethylene bags and transferred to the laboratory in refrigerated bags.The tissue samples were frozen and stored at -20°C until analysis.During sampling operations, special care was taken to avoid tissues near the bullet pathway; all tissue samples were taken from >40 cm away from the areas of bullet damage (Dobrowolska andMelosik 2008, Danieli et al. 2012).

Method of Analyzing
Approximately 1 g of homogenized samples (measured with accuracy of ±0.01 g) were digested with 5 mL HNO 3 (67% v/v, purity for atomic absorption, Merck, Darmstadt), 1 mL of hydrogen peroxide (30%, analytical grade, Merck Darmstadt) with high-performance microwave oven (model Ethos Up, Milestone Srl, Sorisole, Italy) according to EN 13805:2002(CEN 2002a).In brief, the temperature was ramped for 20 min to 220°C, after which samples were held at the same temperature for 15 min.During the digestion the potency was automatically adjusted by temperature and pressure control in each digestion vessel.Digested and cooled samples were diluted to the final volume of 25 mL with deionized water with Milli-Q quality.
Analysis of Pb and Cd was conducted by electrothermal atomic absorption spectrometer (ETAAS) with Zeeman background correction, model AAnalyst 600 (Perkin Elmer, Waltham, Massachusetts), and using 0.005 mg Pd (NO 3 ) as matrix modifier for each atomization of Pb and Cd (CEN 2003).The selected wavelengths for Pb and Cd measurement were 283.3 nm and 228.8 nm, respectively.Furnace programs for ETAAS determination were optimized for liver matrix (Table 1).Calibrations were prepared from commercial solutions of Pb and Cd (1,000 mg•L -1 ) in 2% HNO 3 (Carl Roth GmbH, Karlsruhe, Germany).For recovery and precision validation, reference material offal liver FAPAS test material 07199 was used.

Method Performance Characteristics
The quality assurance of the ETAAS method was performed by validation in accordance with the requirements proscribed in Commission Regulation (EC 2007).The obtained method of linearity range was 3.716-50 µg•L -1 , 0.319-6.0µg•L -1 for Pb and Cd, respectively, with R 2 >0.99, with limits of quantification of 12.39 µg•L -1 for Pb and 1.06 µg•L -1 for Cd.Method precision expressed as relative standard deviation was 6.90% and 8.56% for Pb and Cd, respectively, and the recovery was 104.25% (Pb) and 102.07%(Cd).Internal quality control was performed by running a reagent blank sample and reference material with every batch of samples.External quality control was assured by satisfactory participation in proficiency tests organized by FAPAS and the European Union Reference Laboratory for metals, EURL-MN.Matrix liver was within the ETAAS method scope which was accredited according to EN ISO/ IEC 17025:2017.

Statistical Analysis
Statistical analysis was performed using the Statistica software version 14 (StatSoFT STATISTICA Software) for descriptive analysis and for analysis of variance.To examine differences between sampling areas we used the twoway analysis of variance (ANOVA) test.All statements of significance were based on the 0.05 level of probability (p≤0.05).

rESULtS AND DISCUSSION
The concentrations of lead and cadmium found in livers of wild boars, collected during the period of 7 years on 11 different hunting areas in North Macedonia, are presented in tables 2 and 3.According to Commission Regulation (EC) no.1881/2006(EC 2006)), Food and Veterinary Agency of the Republic of North Macedonia have established maximum levels of Cd and Pb in liver of bovine animals, sheep, pigs, poultry and horses, which amounts to 0.5 mg•kg -1 (EC 2006).In this study, no statistically significant difference in hepatic Pb was found between 11 hunting areas locations.The mean concertation for Pb, looking at all hunting areas, ranged from 0.154 to 0.722 mg•kg -1 , and mean concentration for all in total was 0.225 mg•kg -1 .Compared to other studies, somewhat lower values were found in Croatia (Bilandzić et al. 2009), where the mean values ranged from 0.061 to 0.202 mg•kg -1 .Quite similar mean values were obtained in a study carried out in Italy (Danieli et al. 2012), with the mean value of 0.329 mg•kg -1 .However, somewhat higher values for Pb were found in Poland (Durkalec et al. 2015), with the mean value of 0.083 to 0.903 mg•kg -1 .Significantly higher values than those in our study were obtained in Southern Spain (Santiago et al. 1998), with mean value of 0.44 to 5.05 mg•kg -1 .The higher values obtained in Poland and Southern Spain compared to those in our study are probably a result of the choice of locations where the research was conducted.Namely, in Poland, the research was conducted in 3 locations, 2 of which are known as industrial areas, and the third location was taken as a reference area.In Southern Spain, the situation was similar, each of the four zones that were the subject of research had its own sources of possible pollution.In this study, the 11 hunting ground locations on which the research was carried out are of such a setting that they cover the entire territory of the country, and not only the known industrial facilities where higher values can be expected.
Looking at all locations, out of the total number of samples (n=608), in 18% of samples Pb was not detected (<LODs).Seven percent of samples (41 of 608) exceeded the maximum levels for Pb in liver of domestic animals (0.5 mg•kg -1 ).The maximum measured concentrations of Pb in individual locations ranged from 0.443 to 4.338 mg•kg -1 , and the maximum measured value of 4.338 mg•kg -1 was obtained from a sample at hunting area no. 7, which is 8.5 times higher than the maximum allowed concentration for lead.This is most likely due to contamination from Toranica, which is present in this area with a production capacity of 700,000 tons of ore per year.This is confirmed also by the results of the study conducted during the moss biomonitoring of air pollutinon where high contents of Pb and Zn were discovered (average values of 60 and 75 mg•kg -1 of moss) in the area very close to the source of pollution (Angelovska et al. 2014).Moreover, this hunting area is bordered by two more lead and zinc mines, , Zletovo and , Sasa, which are characterized by high deposition of Pb and Zn, especially near the source of pollution (Balabanova et al. 2014, Balabanova et al. 2017) .This has also been confirmed by one of the latest studies conducted in the Republic of North Macedonia (Stafilov et al. 2023) in which the distribution of various chemical elements was determined in soils and vegetables from gardens in the region of Probištip, North Macedonia (with the main point being the Zletovo mine).The researchers obtained the results in which the lead content in 8 of 19 soil samples studied was higher than the target value (85 mg•kg -1 ) according to the Dutch standards, N -number of samples above the limit of detection; N < LODs -number of samples bellow the limit of detection; N > Max.l. -number of samples exceeding maximum levels of Cd/Pb in liver of bovine animals, sheep, pigs, poultry and horses, set out by law; % of samples from the total number of samples is presented in brackets.

Concentration in wet weight
(mg and the highest lead content measured in soil was 478 mg•kg -1 , which is 5.6 times higher than the target value (the target values indicate the level at which there is a sustainable soil quality).In the Republic of North Macedonia, the regulations for the maximum allowed content of pollutants in the soil and vegetables have not yet been adopted, so in the previous studies regarding the soil and vegetables, the values are compared with the Dutch regulations.
The results of the occurrence of lead in vegetables and cereals showed that a larger number of samples (18 samples out of 38) had an elevated value that exceeds the maximum allowed levels of 0.10 mg•kg -1 for vegetables, 0.30 mg•kg -1 for leafy vegetables and 0.20 mg•kg -1 for cereals.In the mentioned study, the highest content of Pb was found in the barley samples with the concentration 6.65 mg•kg -1 , exceeding the maximum allowed content by 33 times.Since the wild boar does not know natural boundaries and obstacles, and since omnivores when searching for food can cover a large area, the occasional high values that exceed the maximum permitted limit may be the result of contamination right near such industries.
The mean concertation for Cd, looking at all hunting areas together, ranged from 0.177 to 0.464 mg•kg -1 , and the total mean concentration was 0.366 mg•kg -1 .Similar values were obtained in two studies conducted in Croatia, where the mean values ranged from 0.3 to 0.49 and 0.162 to 0.308 mg•kg -1 (Bilandzić et al. 2009, Florijancic et al. 2015), and to one study in Spain (0.17 to 0.34 mg•kg -1 ) (Santiago et al. 1998), while the lower mean value was obtained in Italy amounting to 0.084 mg•kg -1 (Danieli et al. 2012).From a total of 608 samples, cadmium was not detected in 50 samples or 8.2% of the total number of samples (Cd<LODs).Тhis study indicates location differences, so the mean Cd concentrations were significantly higher in Pelagonisko, Krivorečko and Ohridsko-Prespansko hunting areas than in the samples collected from Pološko hunting area (p=0.004both.).The most acceptable explanation for these differences is the availability and distance from pollutants and mining activities, although it is a fact that there is no large-scale pollutant in the Ohrid-Prespa hunting ground.However, Cd and Pb are transported over long distances by air, so the dry and wet deposition of the particles may be due to pollutants from greater distances.In the Krivoreško hunting area or near its borders, as mentioned previously, there are 3 potential sources of pollution on a huge scale.In the research conducted near the Zletovo mine (Stafilov et al. 2023), high concentrations of cadmium were found in soil samples (16 out of 19 exceeded the maximum allowed limits of 0.8 mg•kg -1 ) and vegetables (14 out of 19 samples exceeded the maximum allowed limits of 0.05 mg•kg -1 for vegetables, 0.20 mg•kg -1 for leafy vegetables, and 0.10 mg•kg -1 for cereals).In the vicinity of the Sasa and Toranica mines, high concentrations of cadmium in moss were detected, especially near the source of contamination (Angelovska et al. 2014, Balabanova et al. 2017).Compared to the area where much lower concentrations of cadmium were determined, near the eastern border of the Pološko hunting area, a polluter of this scale is the previously active high-melting ferrochromium plant, Jugohrom -Tetovo, which stopped working in 2016.In the research carried out in North Macedonia, and referring to the Tetovo region, in general this area is characterized as highly contaminated with Sc, Cr, Fe, Co, and Ni, as a consequence of the former operation of the ferrochromium plant, and not with cadmium (Barandovski et al. 2008).Also, some of the hunting grounds in this area border the Mavrovo National Park, and most of them today are already part of the newly formed Shar Planina National Park.Characteristic of the Pelagonian hunting area, which applies to all other hunting areas and can be seen from the obtained results, is the fact that the largest number of samples that exceed the maximum permissible limit for cadmium (N>0.5 mg•kg -1 ), i.e. 40 out of 111 samples or 6.5 out of 18.2%, were determined exactly in this area.Тhe major polluter of the environment in the region is the thermo-electric power plant REK Bitola.The average value of cadmium of 0.423 mg•kg -1 for this locality does not exceed the maximum permissible limit, but it is still significantly higher than the average value of the Polog hunting area.Our results correspond with the recent study (Stafilov et al. 2018)indicating that the concentrations of cadmium in the soil in the Bitola region do not exceed the maximum allowed according to Dutch Standards.Furthermore, compared to the average values of European soils, they are lower in relation to them, but some samples in the area of REK Bitola have slightly increased content over the target value ranging from 1.0 to 1.3 mg•kg -1 .Pollution of soil with some elements present in fly ash deposited in this region influence the appearance of high content of arsenic, cadmium, lead and zinc.
Table 3 shows Pb and Cd concentration in livers of wild boars during the period of 7 years (2016-2022) in North Macedonia.Тhe mean values for lead over the years ranged from 0.095 to 0.336 mg•kg -1 , оr the mean for all years was 0.225 mg•kg -1 .The highest mean value for lead (0.336 mg•kg -1 ) was measured in 2020.In the same year, the largest number of liver samples exceeding the maximum allowed limits (N>0.5 mg•kg -1 ) were determined, 20 out of 41 samples or 3.4 out of 6.7%.The maximum measured lead concentration in wild boar liver was 4.333 mg•kg -1 and it was measured in 2019.
In our study, results show that the mean lead values in 2019 and 2020 are significantly higher than the mean value determined in 2021 (p=0.0002).It is very difficult to explain and determine the specific reason why the mean value for lead was significantly lower in 2021 compared to the previous two years.Additional research is needed, where many more factors that could influence the differences that occur over the years will be monitored to determine the specific causes.
In general, the mean values for cadmium over the years have showed no statistically significant difference.Mean values ranged from 0.324 to 0.449 mg•kg -1 and mean value for all years was 0.366 mg•kg -1 .The maximum mean value for Cd (0.449 mg•kg -1 ) was measured in 2016.It can be indicated that cadmium is present in the environment, but its average amount over the years has remained stable and no changes in an upward or downward direction have been observed.Also, in each of the researched years, certain values exceeding the maximum allowed limits for North Macedonia were observed (N>0.5 mg•kg -1 ), namely 7 in 2016, 18 in 2017, 18 in 2018, 19 in 2019, 19 in 2020, This research received no external funding.

Concentration in wet weight
(mg N -number of samples above the limit of detection; N < LODs -number of samples bellow the limit of detection; N > Max.l. -number of samples exceeding maximum levels of Cd/Pb in liver of bovine animals, sheep, pigs, poultry and horses, set by law; % of samples from the total number of samples is presented in brackets. table 3. Lead (Pb) and cadmium (Cd) concentrations in the livers of wild boars for the period 2016-2022 in North Macedonia.

Figure 1 .
Figure 1.Hunting areas and pollutants of anthropogenic origin in North Macedonia.
. The studies conducted in North

table 1 .
Furnace programs for lead (Pb) and cadmium (Cd) slit with 0.7 nm.

table 2 .
•kg -1 ) Lead (Pb) and cadmium (Cd) concentrations in the livers of wild boars from 11 hunting areas of North Macedonia.