Wageha A Awad

Khaled Ghareeb

Josef Böhm

Jürgen Zentek
email: zentek.juergen@vetmed.fu-berlin.de

deoxynivalenol in animal feed and the effectiveness of microbial 2 biodegradation -a review 3 4 5 6

Keywords: Mycotoxins, Mycotoxins -trichothecenes Trichothecenes, Deoxynivalenol, detoxification, microbial degradation, review 56 57 58

Trichothecenes are a group of mycotoxins mainly produced by fungi of the Fusarium genus. 28 Deoxynivalenol (DON) is one of the most abundant and important trichothecenes in food and 29 feed and it is of a great importance among these contaminants because of its frequent 30 occurrence in toxicologically relevant concentrations worldwide. Since the toxin production 31 depends strongly on environmental conditions such as temperature and humidity, a Fusarium 32 toxin contamination can not be avoided completely. Therefore, exposure to this toxin is a 33 permanent health risk for both humans and farm animals. As cereal crops are commonly 34 contaminated with DON and animal diets consist mainly of cereals, it can be assumed that 35 animals are frequently exposed to DON contaminated feeds. Many strategies can be 36 undertaken to reduce the toxic effect of DON. In addition to the general necessity for 37 minimizing all risk factors that might influence the contamination of cereals with DON, such 38 as the so-called field toxins before harvest, several post harvest strategies can be applied to 39 counteract possible deleterious effects of this mycotoxin in farm animals. Another approach 40 for decontamination in feedstuffs is the use of adsorbent materials. Adsorbent materials may 41 bind mycotoxins in the gastrointestinal tract and reduce the absorption and systemic toxicity. 42

It was shown that some adsorbents are suitable to alleviate the toxic effects of specific 43 mycotoxins, but its efficacy against trichothecenes is practically zero. Therefore, alternative 44 strategies to reduce animal and human health risk are needed. The use of microbial additives 45 is a method, which uses microorganisms having the capability to detoxify mycotoxins by 46 metabolism or degradation prior to their resorption in the gastrointestinal tract. DON has been 47

reported to be completely transformed to de-epoxy-DON by ruminal and intestinal microflora. 48

Eubacterium BBSH 797 was capable of DON degradation and counteracted the toxic effects 49 of DON in animals. This review focuses on the efficacy of microbial feed additives in 50 ameliorating the toxic effects of DON. According to the results of experiments to date, it 51 appears that microorganisms are the main living organisms applicable for this mycotoxin 52 biodegradation. However, the use of this approach depends on its effectiveness from both the 53 practical and economic standpoints.

Introduction 59

No region of the world escapes the problem of mycotoxins and a higher percent of the world's 60 crops are annually affected by mycotoxins [START_REF] Lawlor | Mycotoxin management[END_REF]. Whether grain is 61 produced in temperate, sub-tropical or tropical climates, if rainfall and humidity are high 62 during the harvest season, infection of the grain by fungi is likely. Food and feed are usually 63 contaminated by more than one mycotoxin, because a certain strain of moulds may produce 64 different mycotoxins. Residues of some mycotoxins may also be found in food of animal 65 origin (meat, milk, eggs, and cheese) as the consequence of contaminated feed. Mycotoxins 66 are toxic compounds, and some of them are also mutagenic, genotoxic, carcinogenic, or 67 teratogenic. In mild climates, the most frequent toxicogenic moulds in food and feed are the 68 species of the genera Fusarium and Penicillium. 69 70 A complex of closely related species of the genus Fusarium is responsible for destructive 71 action and results in very economically important diseases of cereal crops (Fusarium head 72 blight, FHB1, of wheat and barley) and maize (Fusarium ear rot). In years with climatic 73 conditions that favour the development of the fungi, these infections can reach epidemic 74 proportions [START_REF] Mcmullen | Scab of wheat and barley: A re-emerging disease 665 of devastating impact[END_REF]. Diseases caused by Fusarium do not only severely 75 reduce yield, but also results in the contamination of grain with unacceptable high amounts of 76 mycotoxins, a problem of world-wide importance. A toxin class of particular concern to 77 human and animal health are trichothecenes, sesquiterpenoid epoxides, which are potent 78 inhibitors of eukaryotic protein synthesis. Depending on the concentration and the substitution 79 pattern of the trichothecene, translational initiation, elongation or termination are 80 preferentially inhibited [START_REF] Cundliffe | Inhibition of initiation, elongation, and termination of 570 eukaryotic protein synthesis by trichothecene fungal toxins[END_REF]. Animal exposure to DON (vomitoxin) 81 has numerous adverse health effects, with neural, gastrointestinal tract and immune system 82 being the most sensitive organs (Canady, et al., 2001). Low to moderate dose of trichothecene 83 cause gastrointestinal irritation or necrosis, haematological disorders, diarrhoea, vomiting and 84 feed refusal and decreased body weight gain. Whereas, the exposure to higher dose levels of 85 Therefore, many attempts have been made to find ways to detoxify contaminated feedstuffs in 92 order to cope with the problem. Different physical and chemical methods have been 93

recommended for detoxification of mycotoxin contaminated food and feed. Nevertheless, 94 only a few of them (e.g. destruction of aflatoxin by ammonia treatment) have been accepted 95 for practical use. Although preventive strategies to reduce the formation of mycotoxins are 96 applied, there is increasing awareness that it is sometimes very difficult to control mycotoxin 97 contaminations in the field and during storage. Therefore, detoxification strategies have been 98

developed to protect livestock animals against mycotoxin contaminated feed. Many 99 specialists are of the opinion that the best approach for decontamination should be 100 degradation by selected microorganisms, giving a possibility of removal of mycotoxins under 101 mild conditions, without using harmful chemicals and without substantial losses in nutritive 102 value and the palatability of detoxified food or feed. 103 104 According to results of experiments, it appears that microorganisms are the main organisms 105 applicable for mycotoxin biodegradation. A review of the literature revealed that DON is 106 degraded by Eubacterium sp. DSM 11798 which transforms DON into its metabolite DOM-1 107 the non-toxic de-epoxide of DON [START_REF] Binder | 552 Screening for deoxynivalenol-detoxifying anaerobic rumen microorganisms[END_REF][START_REF] Binder | Microbiological 538 degradation of deoxynivalenol and 3-acetyl-deoxynivalenol[END_REF]. [START_REF] Fuchs | Structural characterization of metabolites 602 after the microbial degradation of type A trichothecenes by bacterial strain BBSH 797. 603[END_REF] also 108 reported that the Eubacterium sp. (DSM 11798) is capable of detoxifying A-Trichothecenes. 109

Additionally, [START_REF] Awad | Effect of addition of a 518 probiotic microorganism to broiler diets contaminated with deoxynivalenol on 519 performance and histological alterations of intestinal villi of broiler chickens[END_REF] found that Eubacterium BBSH 797 is beneficial in 110 counteracting the toxicity of DON in commercial broilers at the gut level. DON has been 111

reported to be completely transformed to de-epoxy-DON after incubating for 96 h with the 112 content of the large intestine of hens [START_REF] He | Microbial transformation of deoxynivalenol (vomitoxin). 614[END_REF]. Enzymatic detoxifications of 113 mycotoxins in feed additives are a promising approach for the reduction of mycotoxin 114 contamination of feed and food. Therefore, this review summarizes the current state of 115 knowledge in the field of DON detoxification with special reference to the microbial 116 degradation. 117 118

Pre-harvest and post-harvest prevention strategies 119

It is important for producers to realize that good agricultural practices (GAP) represent the 120 primary line of defense against contamination of cereals with mycotoxins, followed by the 121 implementation of good manufacturing practices (GMP) during the handling, storage, 122 processing, and distribution of cereals for human food and animal feed. A complementary 123 management system to consider in the future is Hazard Analysis Critical Control Point 124 (HACCP) system, which is used to identify and control hazards within the production and 125 processing system. Emphasis should be placed on the fact that the planting, preharvest and 126 postharvest strategies for a particular crop will depend on the climatic conditions of that 127 particular year, taking into account the local crops, and traditional production conditions for 128 that particular country or region. 129 130 Therefore, it is understandable that much research has been conducted in an attempt to 131 salvage mycotoxin-contaminated products and to avert health risks associated with the toxins. 132

Whatever decontamination strategy is used, it must meet some basic criteria [START_REF] Park | Controlling aflatoxin in food and feed[END_REF]133 Beaver, 1991;[START_REF] Pomeranz | Fusarium head blight (scab) in cereal 690 grain[END_REF]. The mycotoxin must be inactivated (destroyed) by 134 transformation to non-toxic compounds, fungal spores and mycelia should also be destroyed. 135

In order to prevent the formation of new toxins, the food or feed material should retain its 136 nutritive value and remain palatable, the physical properties of raw material should not 137 change substantially, and the process must be economically feasible. Principally there are 138 three possibilities to avoid harmful effects of contamination of food and feed caused by 139 mycotoxins: (1) prevention of contamination; (2) decontamination of mycotoxin-containing 140 food and feed; and (3) inhibition of absorption of mycotoxin from the digestive tract. The 141 theoretically soundest approach of prevention is without doubt the breeding of cereals and 142 other feed plants for resistance to mould infection and, consequently, mycotoxin production. 143

Particularly in breeding wheat and corn, significant improvement of resistance has been 144 achieved [START_REF] Mesterhazi | Progress in breeding wheat and corn not susceptible to is infection by 675 Fusaria. In Fusarium mycotoxins taxonomy, and pathogenecity[END_REF][START_REF] Medianer | Breeding wheat and rye for resistance to Fusarium diseases[END_REF]. According to data reported in a review paper 145 [START_REF] Medianer | Fusarien in Getreide-Bedeutung von Pflanzenbau und 671 Resistenzzüchtung zur Verminderung von Ertragsverlusten und einer Kontamination mit 672 Mykotoxinen[END_REF] in Germany, about 25% of the area under wheat production 146 is represented by resistant varieties. Despite progress in breeding, full resistance has not been 147 achieved. Another applied way of prevention is the inhibition of the growth of moulds and 148 their production of mycotoxins. First of all, well-selected harvesting, storage-and processing 149 methods may be a successful tool in the prevention of mould growth. A practical guide to the 150 prevention of mycotoxin contamination of grain and animal feedstuffs by fusaria was 151 developed and published by Trenholm et al. (1988, Figure 1) [START_REF] Reid | Crop rotation and nitrogen effects on maize susceptibility to 700 gibberella (Fusarium graminearum) ear rot[END_REF], and balanced fertilization based on soil analysis is required. 164

Removing old seed heads, stalks, and other debris that may serve, or may potentially serve, as 165 substrates for the growth of mycotoxin-producing fungi. 166 167 However, even the best management practices can not eliminate the risk of contamination 168 over years are in favour of mycotoxins. Climatic conditions, such as temperature and 169 humidity, are not under human control, but they may be crucial in contamination with 170 moulds. Fungicides applied before blossoming decrease contamination with Fusarium and the 171 related production of mycotoxins [START_REF] Cvjetkovic | The control of Fusarium on wheat by fungicides and its 574 influence on mycotoxins[END_REF]. Delayed harvest 172 particularly favours contamination with Fusarium. Therefore, early harvest followed by 173 drying of the corn can help avoiding increased mycotoxin contamination. During harvest it is 174 of utmost importance to use appropriate harvesting equipment that is correctly adjusted to 175 avoid damage of the kernel, since those damaged kernels might be predisposed to infection 176 during storage. 177 178 Obviously damaged and high moisture parts should be removed from the harvest as they pose 179 a great risk of contamination. Mechanically damaged and shrivelled grains are regularly 180 contaminated by moulds, and mouldy grains can partially be removed by separators (Miller, 181 2001). The humidity of grain and the relative air humidity are very important in processing 182 and transport [START_REF] Additives | Mycotoxin prevention and decontamination -a case study on 703 maize[END_REF]. The kernel must be desiccated as soon as possible, 183 and optimal humidity maintained. It is particularly important not to allow increases in 184 humidity after desiccation, as they strongly favour contamination. It is well accepted that 185 grains should be dried to 15% humidity or lower before storage. The final safe moisture 186 content depends on the crop and the climatic conditions under which the commodity is stored. 187

Avoiding mycotoxin contamination depends primarily on moisture control. If the product is 188 dry and also kept dry further on, no deterioration of mycotoxin contamination will take place. (Korunic, 1990). Insect and rodent control is important not only because 193 their activity in stored products creates favourable microclimates for fungal growth but also 194 because they damage kernels and therefore predisposes these kernels to fungal manifestation. 195 Adequate Storage should be without delay, good and clean storage facilities, insect control, 196 moisture of grains and humidity control, temperature control, insect and rodent control, 197 chemical preservatives, antifungal agents (acids, mould inhibitors) and the hygiene in silos 198 may decrease the growth of toxicogenic moulds [START_REF] Willson | Storage of cereal grains 764 and their products[END_REF]. Antifungal 199 agents should be used to supplement good management practice rather than as a substitute for 200 safe, clean handling practices of mixed feed. It should be emphasised that at the end of the 201 storage period grains can not be less contaminated with mycotoxins than at the beginning. 202

The entrance of mycotoxins in the nutritional chain can be avoided by storing only high 203 Mycotoxins that were already present on the field can not be influenced either by a correct 214 method during storage or by using mould inhibitors, and the decontamination in later stages 215 of food production is difficult, thus it increases the cost of production and the results are not 216 always satisfactory. Because mycotoxin contamination often is unavoidable, developing and 217 implementing efficient detoxification methods are very important. According to FAO (Scott, 218 1998), the decontamination process must: 1. Destroy, inactivate or remove the mycotoxin 2. 219 Not produce or leave toxic, carcinogenic or mutagenic residues in final products or in food 220 products obtained from animals fed decontaminated feed; 3. Retain the nutritive value and 221 acceptability of the product; 4. Do not substantially alter important technological properties, [START_REF] Trenholm | Two physical methods for the 743 decontamination of four cereals contaminated with deoxynivalenol and zearalenone[END_REF], density 239 segregation of contaminated kernels from non contaminated kernels using water and saturated 240 sodium chloride solution [START_REF] Babadoost | Field contamination of 523 sorghum with zearalenone and deoxynivalenol in North Carolina. Density segregation to 524 remove mycotoxins[END_REF] or water and 30% sucrose solution (Huff 241 and Hagler, 1982), and food processing practices such as milling [START_REF] Lee | Effect of milling on 648 decontamination of Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone in 649 Korean wheat[END_REF]Sietz et al., 242 1986;[START_REF] Scott | Distribution of the 723 trichothecene mycotoxin deoxynivelenol (Vomitoxin) during milling of naturally 724 contaminated hard red spring wheat and its fate in baked products[END_REF][START_REF] Young | Effect of mllling and baking 784 on deoxynivalenol (Vomitoxin) content of eastern Canadian wheats[END_REF], cleaning or washing (Sietz et al., 1986;Scott et 243 al., 1983), and baking [START_REF] Abbas | Effect of cleaning, milling and baking 506 on deoxynivalenol in wheat[END_REF]Sietz et al., 1986;[START_REF] Tanaka | Residues of Fusarium 739 mycotoxins, nivalenol, deoxynivalenol, and zear-alenone, in wheat and processed food 740 after milling and baking[END_REF]Young et al., 244 1984;[START_REF] El-Banna | Fate of mycotoxins during processing of foodstuffs 587 II-Deoxynivalenol (Vomitoxin) during making of Egyptian bread[END_REF]. Rinsing grain with water or sodium carbonate water solution 245 could lower the concentration of mycotoxin DON in wheat and maize [START_REF] Voss | Comparative subchronic toxicity 757 studies of nixtamalized and water-extracted Fusarium moniliforme culture material[END_REF]. 246

Density segregation decreases the level of contamination of DON in corn and wheat. 247

However, it should be noted that appearance and weight of a particular kernel do necessarily 248 indicate mycotoxin contamination. These methods are limited by the cost of seed drying, and 249 they are used only before wet milling and brewing. Milling was not effective in removing 250 DON from the naturally contaminated wheat [START_REF] Lee | Effect of milling on 648 decontamination of Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone in 649 Korean wheat[END_REF]. Cleaning or washing (Seitz et 251 al., 1986;[START_REF] Scott | Effects of experimental 728 flour milling and breadbaking on retention of deoxynivalenol (vomitoxin) in hard red 729 spring wheat[END_REF], and baking [START_REF] Seitz | Cleaning, milling, and 732 baking tests with hard red winter wheat containing deoxynvalenol[END_REF][START_REF] Young | Effect of mllling and baking 784 on deoxynivalenol (Vomitoxin) content of eastern Canadian wheats[END_REF]Abbas et al., 252 1988;[START_REF] Tanaka | Residues of Fusarium 739 mycotoxins, nivalenol, deoxynivalenol, and zear-alenone, in wheat and processed food 740 after milling and baking[END_REF]El-Banna et al., 1986), whilst not a decontamination method per se, 253 dilution of contaminated grain with clean grain has also been used to reduce the toxicity of 254 contaminated feedstuffs. Density segregation, cleaning, and baking have been largely 255 unsuccessful in completely removing the DON from flour fractions or from whole wheat 256 [START_REF] Tanaka | Residues of Fusarium 739 mycotoxins, nivalenol, deoxynivalenol, and zear-alenone, in wheat and processed food 740 after milling and baking[END_REF]. Most mycotoxins are heat resistant, and high temperatures are not used 257 in the decontamination of cereals and other agricultural products. However, autoclave 258 treatment of DON-contaminated maize and wheat with sodium bisulphite was shown to be 259 general is negatively influenced by those treatments and even toxic by-products cannot be 293 excluded. Thus, there is no practical application of these methods. 294 295 For a decontamination method to be commercially feasible it needs to be effective without 296 creating new toxins, introducing new toxic compounds, or altering the nutritional value or 297 other desirable parameters of food or feed; it should be simple and inexpensive and use 298 existing technology (Young, 1986). 299 300 solution can adsorb most of the mycotoxins efficiently, whereas differently activated 309 charcoals have less and even no effects against mycotoxicosis. This might be due to the fact 310 that activated charcoal is a relatively unspecific adsorbent and hence, essential nutrients are 311 also adsorbed particularly if their concentrations in the feed are much higher compared to 312 those of the mycotoxin. Most studies related to the alleviation of mycotoxicosis by the use of 313 adsorbents are focused on aluminosilicates, mainly zeolites and hydrated sodium calcium 314 aluminosilicates (HSCAS), and aluminosilicate-containing clays, all consisting of aluminates, 315 silicates and some interchangeable ions, mainly alkali metal and alkaline earth metal ions 316 [START_REF] Barrer | Shape-selective sorbents based on clayminerals -a review[END_REF][START_REF] Mumpton | La roca magica. Uses of natural zeolites in agriculture and industry. 682[END_REF]. 317 318 319

Efficacy of different adsorbents for the binding of the Fusarium mycotoxin

In several studies, hydrated sodium calcium aluminosilicates (HSCAS) have proven to be the 320 most promising adsorbents. However, while good and scientifically explained results were 321 obtained for counteracting aflatoxins [START_REF] Ramos | In vitro aflatoxin adsorption by means of a montmorillonite 697 silicate. A study of adsorption isotherms[END_REF][START_REF] Scott | Industrial and farm detoxification processes for mycotoxins[END_REF], adsorption of 322 other mycotoxins was limited (e.g. zearalenone, ochratoxin A, fumonisin) or even failed (e.g. 326 Some types of activated carbons have been shown to adsorb DON from aqueous solutions of 327 the toxin with a saturation limit of ca. 2 µg DON/mg activated carbon [START_REF] Galvano | Activated carbons: In 606 vitro affinity for ochratoxin A and deoxynivalenol and relation of adsorption ability to 607 physicochemical parameters[END_REF]. 328

In addition, [START_REF] Avantaggiato | Evaluation of the intestinal absorption of 509 deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding 510 efficacy of activated carbon and other adsorbent materials[END_REF] found that the dietary inclusion of activated carbon 329 produced a significant reduction in the intestinal mycotoxin (DON) absorption. 330 331 Although the different methods used at present for detoxification of Fusarium mycotoxins are 332 to some extent successful, they have big disadvantages with limited efficacy and possible 333 losses of important nutrients (Chung et al., 1990;[START_REF] Kubena | Efficacy of a Hydrated Sodium Calcium Aluminosilicate to Reduce the Toxicity of 643 Aflatoxin and Diacetoxyscirpenol[END_REF] and are normally 334 expensive. Most of the commercially available mycotoxin-binders failed in sequestering 335

Fusarium mycotoxin in vitro. Only a small number of adsorbent materials possessed the 336 ability to bind more than one mycotoxin. Cholestyramine was proven to be an effective binder 337 for fumonisins and zearalenone in vitro, which was confirmed for zearalenone in experiments 338 using a dynamic gastrointestinal model and for fumonisins by conducting in vivo 339 experiments. No adsorbent materials, with the exception of activated carbon, showed relevant 340 ability in binding deoxynivalenol and nivalenol. Many scientists in the field are of the opinion 341 that the best solution for decontamination should be detoxification by biodegradation, giving 342 a possibility for removal of mycotoxins under mild conditions without using harmful 343 chemicals and without substantial losses in nutritive value and palatability of the 344 decontaminated food or feed. 345 346

Microbial Biodegradation of Fusarium mycotoxin deoxynivalenol 347

The elimination of trichothecenes from contaminated feedstuffs is an unsolved problem and, 348 once contamination has occurred in grains, few strategies can be adopted for limiting adverse 349 effects in livestock (WHO, 2001). Investigation of the biodegradability of mycotoxins has 350 become an area of great interest. Efforts have been done to screen microorganisms from 351 different origins, which could detoxify trichothecene mycotoxins in vitro and in vivo. This 352 approach is defined as biodegradation or biotransformation that leads to less toxic products. 353

The use of microorganism in the in the field of mycotoxin detoxification is not new research 354 approach [START_REF] Schatzmayr | Detoxification of mycotoxins by biotransformation[END_REF]. Some microorganisms with mycotoxin degradation 355 activity were isolated; the first was Flavobacterium aurantiacum with the ability to detoxify Gliocladium roseum detoxified zearalenone by opening the ring structure with subsequent 359 decarboxylation in yields ranging between 80 and 90% (El-Sharkawy and [START_REF] El-Sharkawy | Microbial cleavage of zearalenone[END_REF]. It 360 is known, that the 12, 13-epoxide ring is responsible for trichothecenes toxic activity and 361 removal of this epoxide group entails a significant loss of toxicity. 362

Of the microorganisms screened for their ability to degrade DON, some have been found to 363 be able to convert DON to the far less toxic compound (Table 1). 364 365Environmental microorganism 366 DON is the most common mycotoxin produced on cereal crops and is chemically stable but 367 does not appear to accumulate in agriculture soils, implying the possibility of biological 368 mineralization of DON in nature [START_REF] Völkl | Microbial detoxification of 754 mycotoxin deoxynivalenol[END_REF]. Among 1285 microbial cultures 369 obtained from farmland soils, cereal grains, insects and other sources, one mixed culture was 370 able to transform DON into 3-keto-DON [START_REF] Völkl | Microbial detoxification of 754 mycotoxin deoxynivalenol[END_REF]. The culture was also capable of 371 transforming 3ADON, 15ADON and FUS. However, the microorganisms responsible for the 372 biotransformation have not been identified. In addition, mixed soil microorganisms have been 373

reported as transforming DON and 3ADON into five unidentified metabolites that showed 374 toxicity reduction in the bioassay with Saccharomyces cerevisae [START_REF] Binder | Microbiological 538 degradation of deoxynivalenol and 3-acetyl-deoxynivalenol[END_REF]. With 375 an enrichment strategy that used DON as the sole carbon source in a medium containing 376 mineral salts. Agrobacterium-Rhizobium strain E3-39 was isolated from soil samples (Shima 377 et al., 1997). The strain transformed DON into 3-keto DON under aerobic conditions. The 378 transformation activity of the strain was considerably persistent and the enzyme(s) 379 responsible for the microbial transformation was found to be in cells-culture, cell-free filtrate, 380 but not in the cell extract [START_REF] Shima | Novel 735 detoxification of the trichothecene mycotoxin deoxynivalenol by a soil bacterium isolated 736 by enrichment culture[END_REF]. Six agricultural soils capable of transforming 381 DON into several products were obtained after enhancement using F. graminearum infected 382 mouldy maize. These enhanced soils transformed DON by more than 87%, and two of them 383 completely removed DON from culture media after incubation at 28 °C for 3 days (Zhou, 384 2008). A bacterium strain Barpee was isolated from one of the soil samples and showed 385 strong DON transforming activity. Under aerobic conditions, the bacterial strain transformed (Kiessling et al., 1984). However, DON was not transformed 393 (Kiessling et al., 1984). In contrast, the microorganisms in the rumen fluid from a dairy cow 394 transformed DON into 3a, 7a, l5-trihydroxytrichothec-9, 12-dien-8-one (de-epoxy DON), 395 which has been named DOM-1 [START_REF] Yoshizawa | Structure of a novel metabolite from deoxynivalenol, a 770 trichothecene mycotoxin, in animals[END_REF]. The deepoxidation of DON by 396 rumen fluid was also reported by [START_REF] Cote | Production of de-epoxynivalenol a 566 metabolite of deoxynivalenol, by in vitro rumen incubation[END_REF] and [START_REF] He | Microbial transformation of deoxynivalenol (vomitoxin). 614[END_REF]. It appeared that 397 microorganisms from rumen fluid could have both deacetylation and deepoxidation functions, 398 transforming 3ADON to DON and DOM-1 [START_REF] King | Transformation of deoxynivalenol 627 (vomitoxin) by rumen microorganisms[END_REF]. These transformations by 399 rumen fluid were also observed when animal feed containing DON and 3ADON was used in 400 a test [START_REF] Binder | Microbiological 538 degradation of deoxynivalenol and 3-acetyl-deoxynivalenol[END_REF]. 401 402Eubacterium strain BBSH 797 is by far the most intensively studied bacterial isolate that is 403 capable of transforming trichothecenes. The strain was isolated from the bovine rumen fluid 404 and its primary function was to transform DON into DOM-1 in vitro and in vivo (Binder et 405 al., 1998;[START_REF] Schatzmayr | Detoxification of mycotoxins by biotransformation[END_REF]. A BBSH 797 based commercial feed additive product is 406 available on the market. 407 408 Bacterial mixtures originating from the rumen of a fistulated dairy cow showed both 409 deacetylation and deepoxidation functions (Swanson et al., 1987a, b). Microbial 410 transformations were obtained after the tested mycotoxins were incubated within the bacterial 411 suspensions at 4.9 x 107 per ml anaerobically at 38 °C for 24 or 48 hours. DON was 412 transformed into DOM-1 413 414

Chicken digesta microorganisms 415

A very effective deepoxidation activity was achieved when DON was mixed with the digesta 416 of the large intestines of chickens; DON was completely transformed to DOM-1 (He et al., 417 1992). It was shown that the deepoxidation function of the intestinal microorganisms of 418 chickens may vary greatly among the samples collected from different chicken varieties, 419 individuals and intestinal regions. The variation was also noticed in DON deepoxidation 420 activity by microorganisms from swine intestine, either no activity [START_REF] He | Microbial transformation of deoxynivalenol (vomitoxin). 614[END_REF] or with 421 activity [START_REF] Kollarczik | In vitro transformation of the Fusarium mycotoxins 630 deoxynivalenol and zearalenone by the normal gut microflora of pigs[END_REF]. 422

423 In general, several authors described the de-epoxidation reaction of ruminal or intestinal flora 424 [START_REF] Kollarczik | In vitro transformation of the Fusarium mycotoxins 630 deoxynivalenol and zearalenone by the normal gut microflora of pigs[END_REF][START_REF] He | Microbial transformation of deoxynivalenol (vomitoxin). 614[END_REF][START_REF] Yoshizawa | Structure of a novel metabolite from deoxynivalenol, a 770 trichothecene mycotoxin, in animals[END_REF]Yoshizawa et al., , 1994)). However, Binder et al. 425 (2000a) were the first to isolate a pure bacterial strain, which was able to biotransform the 426 epoxide group of trichothecenes into a diene [START_REF] Fuchs | Structural characterization of metabolites 602 after the microbial degradation of type A trichothecenes by bacterial strain BBSH 797. 603[END_REF]. Deoxynivalenol, the best 427 known trichothecene is enzymatically reduced by an epoxidase of Eubacterium DSM 11798 428 to the less toxic metabolite deepoxy-deoxynivalenol (DOM-1, Figure 1). The degradation of 429 the epoxide group is carried out by reductive cleavage of the toxic 12, 13 epoxy ring. This 430 strain was isolated out of bovine rumen fluid; its mode of action was proven in vitro and also 431 in vivo. The detoxifying strain Eubacterium BBSH 797 was the first microbe used in a 432 mycotoxin deactivating feed additive (Binder et al. 2000 b). [START_REF] Awad | Effects of deoxynivalenol 514 on general performance and electrophysiological properties of intestinal mucosa of broiler 515 chickens[END_REF][START_REF] Awad | Effect of addition of a 518 probiotic microorganism to broiler diets contaminated with deoxynivalenol on 519 performance and histological alterations of intestinal villi of broiler chickens[END_REF] The rumen and intestinal microflora detoxify DON by reductive deepoxidation under strictly 439 anaerobic conditions [START_REF] King | Transformation of deoxynivalenol 627 (vomitoxin) by rumen microorganisms[END_REF][START_REF] Kollarczik | In vitro transformation of the Fusarium mycotoxins 630 deoxynivalenol and zearalenone by the normal gut microflora of pigs[END_REF]. Biological degradation of 440 DON under aerobic conditions appears to be much more difficult to achieve (Karlovsky 441 1999). 442 443The investigations in this field (Line and Bracket 1995a, b;[START_REF] Line | Evidence for Degradation of Aflatoxin B1 by 652 Flavobacterium Aurantiacum[END_REF] concentrated on 444 possible mechanisms of aflatoxin degradation by Flavobacterium Aurantiacum. The first 445 important question which must be answered is: whether the bacteria actually degrade the 446 toxin or whether the disappearance of the toxin resulted from adsorption to the cells. Without 447 this knowledge, no real advantage can be taken of this microbial method of removing the 448 toxin (Figure 2). 449 Different strains of lactic acid bacteria (LAB) are able to bind the main Fusarium mycotoxins 450 (DON), but biodegradation did not appear to be the mode of action, as no toxin derivatives 451 were observed and removal was not impaired in nonviable bacteria. The binding ability of 452 selected strains could be used to decrease the bioavailability of toxins in contaminated silages 453 [START_REF] Niderkorn | Binding of Fusarium mycotoxins by 685 fermentative bacteria in vitro[END_REF]. resistant to mycotoxins should be investigated. Investigations of several bacteria and yeasts 460 have shown that differences may be found both in sensitivity and selectivity of these 461 microorganisms against mycotoxins [START_REF] Madhyastha | Comparison of toxicity 661 of different mycotoxins to several species of bacteria and yeasts: use of Bacillus Brevis in 662 a disc diffusion assay[END_REF], e.g. Bacillus brevis was 462 sensitive to eight mycotoxins including zearalenone and ochratoxin-A, but had not affected by 463 high concentrations of trichothecene toxins. Kluyveromyces marxianus was sensitive to all 464 trichothecene toxins, but this yeast was not inhibited by other mycotoxins. The study of the 465 mechanisms involved in resistance (e.g. selective uptake through membranes, decomposition 466 by selective enzymes, blocking by complex formation etc.) may be helpful in finding 467 applicable microbes. The control of degradation products, the effects of detoxification on the 468 nutritive and organoleptic properties are, in every case, a decisive part of such research and 469 potential applications. 470 471 [START_REF] Schatzmayr | Detoxification of mycotoxins by biotransformation[END_REF] reported that for an effective use of mycotoxin-degrading 472 microorganisms as animal feed additives certain prerequisites have to be fulfilled, such as 473 rapid degradation of mycotoxins into non-toxic metabolites under different oxygen conditions 474 and in a complex environment, safe to use, and stabile in the gastrointestinal tract at different 475 pH levels. In addition, the choice of the biodegradation approach depends on its effectiveness 476 in practice and its economics. Anaerobic microorganisms isolated from animal guts are 477 generally suitable for developing feed additives [START_REF] Zhou | Microbial transformation of trichothecene mycotoxins[END_REF], which will act in the 478 intestines of the targeting animals. Survival and adaptation of the microorganisms in the 479 animal guts are key factors for successful detoxification. Additionally, [START_REF] Cheng | Toxicity of different Fusarium mycotoxins 559 on growth performance, immune responses and efficacy of a mycotoxin degrading 560 enzyme in pigs[END_REF]

  189 190 During long-term storage, the kernel is exposed to oscillations in temperature and humidity, 191 and insects of species Sithohilus, Tribolium, Trogodernma, Oryzaephilus can be vectors for 192

  quality products in silos, and by strictly observing good agricultural practices. Minimize 204 insect damage and fungal infection in the vicinity of the crop by proper use of registered 205 insecticides, fungicides and other appropriate practices within an integrated pest management 206 program. Control weeds in the crop by use of mechanical methods or by use of registered 207 herbicides or other safe and suitable weed eradication practices. Irrigation is a valuable 208 method of reducing plant stress in some growing situations. Excess precipitation during 209 anthesis (flowering) makes conditions favourable for dissemination and infection by 210 Fusarium sp.; thus irrigation during anthesis and during the ripening of the crops, specifically 211 wheat, barley, and rye, should be avoided. 212 213

  applicable in regions where less-stringent protocols exist. Detoxification efforts 227 have been mainly focused on the aflatoxins and many strategies to decontaminate aflatoxin 228 contaminated crops and products have been reported and reviewed. Therefore, this paper 229 gives an overview of methods used in industrial Fusarium toxin decontamination of food and 230 feed. It also addresses the possibilities of the use of adsorbents as farm animal feed additives. 231 232 Physical Methods of detoxification 233 There are several physical methods of decontamination of agricultural products known as the 234 removal of damaged grains or of a part of the contaminated crop, washing procedures, 235 radiation, ultrasound and extraction with organic solvents. The removal of damaged parts of a 236 crop (usually mould-contaminated) is possible when contamination is uneven or partial. 237 238 Physical methods have included sieving and dehulling

  of physical and chemical treatments of feed prompted the search for 303 other solutions concerning the mycotoxin hazard. Consequently, techniques were investigated 304 based on deactivation of mycotoxins directly in the gastrointestinal tract of animals (in vivo). 305 306 Several adsorbent products have been tested for their effectiveness to detoxify DON 307 contaminated feed, but the results have been unsatisfactory. Activated charcoal in aqueous 308

  433 indicated that the Eubacterium sp. DSM 11798 was capable of completely compensating for 434 the adverse effect of the trichothecene deoxynivalenol in poultry. 435 436 Please inset Figure 1 about here 437 438

  of microorganisms with the degrading or binding properties may lead to the 458 detection of more efficient and more applicable bacteria. First of all, the microorganisms

  Figure 1. Microbial transformation (Eubacterium sp.) of deoxynivalenol to non-toxic metabolites

Table 1 . Microbial Biodegradation of mycotoxin deoxynivalenol

 1 methods described above may become the technology of choice, as enzymatic reactions offer 494 a specific, irreversible, efficient and environmentally friendly way of detoxification that 495 leaves neither toxic residues nor any undesired by-products. The isolation and 496 characterization of microorganisms that are able to biotransform mycotoxins is promising for 497 the practical application of biotechnology, in addition to the decontamination processes taking 498 place directly in the intestinal tract of animals. Finally, a technology must be developed that is 499 suitable for economically feasible treatment of cereals and legumes. Providing high quality 500 and safe animal products is vital and the research priority must be to eliminate toxic residues 501

	480
	481	reported that the MDE product (mycotoxin degrading enzyme) with esterase, epoxidase and
	482	peptidase activity could block epoxide formation in DON metabolism and esterase
	483	transformed ZON into a non-toxic metabolite in pigs. The feed additive was capable of
	484	counteracting the adverse effects on animal performance
	485
	486
	487	Conclusion
	488	The scope for mycotoxin decontamination is still very limited therefore the elimination of
	489	mycotoxins from foods is a big problem worldwide wide. Fusarium mycotoxin
		deoxynivalenol contamination seems to be an important issue in animal nutrition especially in

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

and 5. Destroy fungal spores and mycelia which could, under favourable conditions,

proliferate and form new toxins. In addition to these criteria, the process (es) should be

readily available, easily utilized, and inexpensive. These latter attributes facilitate

trichothecenes, like deoxynivalenol) under field conditions[START_REF] Friend | Effect of adding 598 potential vomitoxin (deoxynivalenol) detoxicants of a F. graminearum inoculated corn 599 supplement to wheat diets fed to pigs[END_REF] Kubena et al.

aflatoxins[START_REF] Ciegler | Microbial detoxification of aflatoxin. 563[END_REF].[START_REF] Wegst | Bacterial degradation of ochratoxin A[END_REF] provided evidence of the

degradation of ochratoxin A by the aerobic bacterium Phenylobacterium immobile.

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Europe and North America. As a preventive measure, it is possible to use agents that can

inactivate mycotoxins, particularly substances with a high absorption capacity, and specific

enzymes, which can break down the chemical structure of the mycotoxins. The biological

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Mariusz Rudy
email: mrudy@univ.rzeszow.pl

Dependences between the age and the level of bioaccumulation of heavy metals in tissues and the chemical composition of wild boars' meat

Keywords: GFAAS, Metals analysis -AAS Additives/Contaminants: Heavy metals, Heavy metals -arsenic, Heavy metals -cadmium, Heavy metals -mercury Food Types: Animal products -meat Meat of wild boars, heavy metals, coefficients of correlation

Dependences between the age and the level of bioaccumulation of heavy metals in tissues and the chemical composition of wild boars' meat

Mariusz Rudy

To cite this version:

Introduction

Currently among consumers there is increased interest in meat from animals kept in conditions as close as possible to the natural ones. Such a requirement is undoubtedly fulfilled by game characterized by high nutritional value and special sensory properties, desired by consumers [START_REF] Daszkiewicz | Charakterystyka mięsa łań jelenia szlachetnego (Cervus elaphus L.) oraz zmian jego jakości w czasie dojrzewania w modyfikowanej atmosferze[END_REF][START_REF] Soriano | Proteolysis, physicochemical characteristics and free fatty acid composition of dry sausages made with deer (Cervus elaphus) or wild boar (Sus scrofa) meat: A preliminary study[END_REF][START_REF] Vergara | Conservation of Cervus elaphus meat in modified atmospheres[END_REF].

However, animals living in the natural ecosystem often show higher levels of contamination by xenobiotics compared to farm animals and can constitute a bigger hygienic-toxicological threat. Integration of these animals with the environment for the whole (relatively long) period of life means these species can be usedas an excellent indicator of the level of contamination of their natural habitat.

In the research reported here the content of such elements as lead, cadmium, mercury and arsenic in tissues of wild boars was determined because they constitute a real danger for health due to high toxicity. These metals can produce neuro-toxic, carcinogenic effects, they disturb functioning of cardiovascular system, osseous system, and organism's management of minerals. Moreover, ions of these elements do not undergo biodegradation, thus they circulate in the environment and accumulate in tissues of living organisms.

Among the tests on wild mammals, numerous publications concern the load of their tissues and organs with heavy metals indicating the practical value of these tests for the assessment of quality of the environment. Some animals are good bio-indicators such as the hare (Lepus europaeus) [START_REF] Baikov | Heavy metals in hare organs as bioindikators for environment pollution[END_REF][START_REF] Bukovjan | Hygienic-medicinal aspects of game and consumable organs of some kinds of free-living animals[END_REF][START_REF] Milanov | Biological indication of the heavy metals pollution with the help of the hare (Lepus europaeus)[END_REF][START_REF] Paukert | The hair of the Common hare (Lepus europaeus Pall.) and of the Common vole (Microtus arvalis Pall.) as indicator of the environmental pollution[END_REF], deer, elk, reindeer [START_REF] Frøslie | Levels of trace elements in liver from Norvegian moose, reindeer and red deer in relation to atmospheric deposition[END_REF][START_REF] Niemi | Heavy metals in muscle, liver and kidney from Finnish Elk in 1980-91 and 1990[END_REF][START_REF] Venäläinen | Heavy metals in tissues of hares in Finland, 1980-82 and 1992-93[END_REF] and wild boar [START_REF] Falandysz | Some toxic and trace metals in big game hunted in the northern part of Poland in 1987-1991[END_REF][START_REF] Falandysz | Metale w mięśniach, wątrobie i nerkach zwierząt łownych z rejonu Polski północnej[END_REF][START_REF] Krupa | Ocena zanieczyszczenia metalami cięŜkimi tkanek zwierząt dziko Ŝyjących w południowo-wschodnim regionie Polski[END_REF][START_REF] Kryński | SkaŜenie narządów i tkanek saren i dzików metalami cięŜkimi[END_REF][START_REF] Kryński | SkaŜenie zwierzyny grubej metalami cięŜkimi -aspekty poznawcze i praktyczne[END_REF][START_REF] Michalska | Zawartość metali w tkankach dzików, saren i jeleni w regionie wielkopolskim[END_REF][START_REF] Monkiewicz | Rozmieszczenie ołowiu w tuszy dzików w zaleŜności od odległości od rany postrzałowej[END_REF][START_REF] Racz | Perspectives in the central nervous system toxicity of methyl mercury[END_REF].

Many authors [START_REF] Gufler | Investigation of the lead, cadmium and mercury contents in the organs and muscules of roe deer and chamois in southern Tyrol[END_REF][START_REF] Holm | Investigation of roe deer -criteria for use as a bioindicator in specimen banking[END_REF][START_REF] Kottferova | Distribution of Cd and Pb in the tissues and organs of free-living animals in the territory of Slovakia[END_REF][START_REF] Pokorny | Lead, cadmium, and zinc in tissues of roe deer (Capreolus capreolus) near the lead smelter in the Koroska Region (Northern Slovenia)[END_REF][START_REF] Szkoda | Pierwiastki toksyczne w tkankach zwierząt łownych[END_REF] noted higher concentrations of cadmium and lead in tissues of game (roe deer, wild boar) than the levels of these elements found in other tests conducted on farm animals such as: pigs [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-1988[END_REF][START_REF] Larsen | Monitoring the content and intake of trace elements from food in Denmark[END_REF][START_REF] Niemi | The lead, cadmium and mercury concentrations in muscle, liver and kidney from Finnish pigs and cattle during 1987-1988[END_REF], cattle [START_REF] López | Toxic and trace elements in liver, kidney and meat from slaughtered in Galicia (NW Spain)[END_REF][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken, and in pig and cow liver in Finland during 1991[END_REF] Niedziółka et al. 2004;[START_REF] Rudy | The analysis of correlations between the age and the level of bioaccumulation of heavy metals in tissues and the chemical composition of sheep meat from the region in SE Poland[END_REF]. However, in the case of the contents of these metals in tissues of horses, many researchers [START_REF] Baldini | Cadmium in organs and tissues of horses slaughtered in Italy[END_REF][START_REF] Znamirowska | Level of accumulation of selected heavy metals in horse tissue as a function of age[END_REF][START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF][START_REF] Weyermann | Cadmiumbelastung verbraucherrelevanter Muskulatur beim Pferd[END_REF] found that the maximum permissible levels for these toxicants were exceeded, especially such cases occurred in the material obtained from older animals.

The measured contents of total mercury and arsenic in food and feedstuff indicate that these elements usually occur in low concentrations. Most frequently there are found as contaminants in seafood, however, arsenic is also found in tissues of other animals [START_REF] Jorhem | Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-1988[END_REF][START_REF] Krupa | Pozostałości pestycydów i niektórych metali cięŜkich (Cd, Pb, As i Hg) w tkankach bydła[END_REF]. In the case of mercury it is still the subject of interest of many researchers [START_REF] Dickman | Mercury in human hair and fish: is there a Hong Kong male subfertility connection?[END_REF][START_REF] Innis | Increased levels of mercury associated with high fish intake among children from Vancouver, Canada[END_REF][START_REF] Morita | The determination of mercury species in environmental and biological samples[END_REF][START_REF] Naqvi | Analysis of mercury in seafood by cold vapor atomic absorption spectroscopy[END_REF][START_REF] Nguyen | Mercury distribution and speciation in Lake Balaton, Hungary[END_REF][START_REF] Olivero | Mercury levels in muscle of some fish species from the Dique Channel, Colombia[END_REF][START_REF] Storelli | Survey of total mercury and methylmercury levels in edible fish from Adriatic Sea[END_REF] and both mercury as well as arsenic are taken into consideration in every program of monitoring tests.

In this connection the purpose of the work reported here was to determine the level of accumulation of selected heavy metals (Pb, Cd, Hg, As) in tissues of wild boars. The animals were divided into 3 age groups, which allowed the analysis of statistical-mathematical dependences between the age of animals and contamination of their tissues. Moreover, the chemical composition of meat in particular age groups of wild boars was also determined to look for dependences between the accumulation of heavy metals and the contents of water, protein, fat and ash in meat. It also allowed an understanding of nutritional-health dependences between the age of animals and basic chemical components of meat which in great measure shapes its quality and nutritional value. In addition, the contents of the abovementioned toxicants in tissues presented in the work allowed stating the degree of contamination of the habitat of wild boars analyzed in the years (tests in the years 2002 -2006).

Materials and methods

The Varian, Walnut Creek, CA, USA) according to PN-EN 14084:2004. Hg was determined by cold vapour atomic absorption spectrometry according to PN-EN 13806:2003. The levels of heavy metals in tissues were expressed in mg/kg of fresh mass, taking into account all quantitative processes during material preparation and reagent blanks. The hydride generation conditions for arsenic and for mercury determination:

As -wavelength 193.7 nm, slit 0.7 nm, cell temperature 900ºC, reducing agent 0.2% w/v NaBH 4 , sample injection volume 500 µL;

Hg -wavelength 253.7 nm, slit 0.5 nm, cell temperature 300ºC, reducing agent 0.2% w/v NaBH 4 , carrier 3% v/v HCl, sample injection volume 500 µL.

As a modifier of matrix at determining Cd and Pb there was applied: 1% NH 4 H 2 PO 4 + 0.3%

Mg(NO 3 ) 2 .
The determinations of selected elements in the analysed period were made by the same analyst, in the same laboratory and under the same conditions. Moreover, the applied analytical procedures were regularly verified in intra-and inter-laboratory tests by the analysis of control samples enriched with various concentrations of tested compounds and the analysis of certified reference materials. The laboratory also participated regularly in domestic and international tests of expertise.

In all cases, the blanks were treated in the same way as samples. A strict analytical quality control program was employed during the study, the results of which are presented in Table 1.

The limits of detection, expressed as concentration in the tissue, was based on the mean sample weight for that tissue type. Quality control of analytical measurements was performed using blank samples and the following certified reference materials: BCR-185R bovine liver from the Institute for Reference Materials and Measurements for the determination of lead, cadmium and arsenic, and NIST-RM 8414 bovine muscle powder from the National Institute of Standards and Technology for the determination of mercury.

The results were subjected to statistical-mathematical analysis which included: ). These parameters were calculated in order to characterize the determined quantities of water, protein, fat and ash in meat. And for the purpose of characterising the determined quantities of cadmium, lead, mercury and arsenic in tissues of the assessed animals there was calculated the arithmetic mean and the minimum and maximum values were given.

2. One-factor variance analysis which was checked by Tukey's confidence intervals, at to levels of significance P≤0.01 and P≤0.05. The null hypothesis in variance analysis was verified by the test formulated by Fisher Snedecor (test F).

3. Calculation of the value of coefficients of simple correlation for the purpose of determining interdependences between the content of tested metals in meat and liver of wild boars and dependences between the chemical composition of meat and the determined toxicants. There were also calculated coefficients of correlation to determine the correlation between the examined features and age of animals. Coefficients of correlation were calculated at the significance level P≤0.05.

Calculations were made basing on the program STATISTICA ver. 6.0.

Results and discussion

The contents of the basic components in meat of wild boars (protein 17-24%, fat 1.5-3.2%, water 71-74%, ash 1%), as with other animals, depends mainly on the age and place of taking the sample [START_REF] Korzeniowski | Charakterystyka chemiczna mięsa dzików[END_REF].

In the author's tests (Table 2) there were stated increase with age of the content of protein, fat and ash and decrease of the contents of water. Particularly high difference (as much as 186%)

occurs in the contents of fat between the animals up to 1 year and animals above 3 years.

Between these groups the difference in the contents of protein is 2 percentage units and in the contents of water 3.6 percentage units.

One of the methods of control of the quality of the environment and health and correct development of vertebrates, including mammals, are the eco-toxicological tests. One of essential elements of these tests is the analysis of tissues and organs of animals for the purpose of determining the amount of occurrence of heavy metals in them. Any quantitative changes in the environment are reflected in tissues of animals, but the sensitivity of these In the years 1996-1997 [START_REF] Krupa | Ocena zanieczyszczenia metalami cięŜkimi tkanek zwierząt dziko Ŝyjących w południowo-wschodnim regionie Polski[END_REF] in tissues of wild boars from South-East Poland showed much higher level Pb, Cd, Hg and As both in muscles and in liver -respectively: 0.104 mg/kg; 0.035 mg/kg; 0.018 mg/kg and 0.0041 mg/kg in muscles and 0.196 mg/kg; 0.562 mg/kg; 0.057 mg/kg and 0.0082 mg/kg in liver than in the author's tests (Table 3). Also the tests conducted in the eighties by other authors [START_REF] Falandysz | Ołów w przetworach z dziczyzny[END_REF][START_REF] Falandysz | Metale w mięśniach, wątrobie i nerkach zwierząt łownych z rejonu Polski północnej[END_REF] prove higher concentration of lead in muscles (0.14 -0.16 mg/kg) and in liver (0.25 -0.31 mg/kg) of wild boars than in this work. Still higher concentration of lead in muscle and liver of wild boars was showed by [START_REF] Kryński | SkaŜenie narządów i tkanek saren i dzików metalami cięŜkimi[END_REF] respectively: 0.24 mg/kg and 2.28 mg/kg. [START_REF] Falandysz | Metale w mięśniach, wątrobie i nerkach zwierząt łownych z rejonu Polski północnej[END_REF] towards the end of the eighties showed the contents of cadmium in muscles of wild boars (from North Poland) at the level of 0.01 mg/kg and in liver 0.11 mg/kg. In turn, [START_REF] Kryński | SkaŜenie narządów i tkanek saren i dzików metalami cięŜkimi[END_REF] stated a few times higher concentration of this element both in meat (0.13 mg/kg) and in liver (0.24 mg/kg) of wild boars from the area of South-West Poland. Similar contents of cadmium in liver was stated in the author's tests (Table 3) in the year 2002. In turn, in the tests of [START_REF] Szkoda | Pierwiastki toksyczne w tkankach zwierząt i mleku -ocena naraŜenia[END_REF] it was shown that a serious hygienic-toxicological problem is the contamination by lead of meat obtained from game. High concentrations of Pb in samples of muscles, reaching several tens mg/kg, at low contents of this element in kidneys and liver prove contamination of muscles by lead from bullets used for shooting. The number of individual samples of muscles of game, in which there was exceeded the current limit, i.e. 0.10 mg/kg, reached the level of 39%. Such a considerable percentage of samples with high concentration of lead is a special threat for human health even at low consumption of meat of game. Such a condition indicates the need of permanent inspection of venison in order to protect the consumer's health and commencement of efficient actions eliminating this problem. [START_REF] Krupa | Ocena zanieczyszczenia metalami cięŜkimi tkanek zwierząt dziko Ŝyjących w południowo-wschodnim regionie Polski[END_REF] in their tests obtained quite considerable degree of contamination by cadmium and mercury of internal organs of roe deer and wild boars. Therefore, the offal of these animals should be eliminated from foodstuff use. Only the level of contamination by arsenic and lead of tissues of roe deer and wild boars did not bring sanitary-toxicological reservations.

In the tests conducted in the area of South Tyrol in the hunting season in 1995 Gufler et al.

(1997) stated definitely higher average contents of cadmium in kidneys of roe deer (1.333 mg/kg) in relation to the levels occurring in liver of these animals (0.120 mg/kg). In turn, in the tests conducted in the industrial areas of Slovenia the concentration of cadmium in liver of roe deer was at the level of 3.920 mg/kg, and in kidneys it was above 22 mg Cd/kg [START_REF] Pokorny | Lead, cadmium, and zinc in tissues of roe deer (Capreolus capreolus) near the lead smelter in the Koroska Region (Northern Slovenia)[END_REF].

The data concerning the contents of heavy metals in meat and liver of wild boars of different age are in Table 4. From these data it appears that the contents of lead in muscular tissue was varied depending on the age (very high correlation -r = 0.81). But the lowest contents of lead in meat 0.042 mg/kg was found in the youngest specimens. The amount of this element increased in subsequent age groups -by 26% and 95% respectively. In the case of the contents of cadmium in meat the amount of this metal also increased from the level of 0.008 mg/kg in group I by 75% in group II and by 112% in group III. Statistically significant differences (P≤0.01) for the contents of both metals in meat were confirmed only between the groups I and III. Also the contents of mercury in meat as well as in liver of wild boars increased together with their age. However, both for the contents of this element in liver as well as in meat there were not confirmed statistically significant differences. Also in no single case was exceeded the maximum permissible level in meat, both for the contents of lead, cadmium as well as mercury. In the tested samples of tissues there were also not stated the presence of arsenic above 0.001 mg/kg. High diversity in the contents of lead (0.01 -0.078 mg/kg) and cadmium (0.01 -0.10 mg/kg) in meat of wild boars coming from Pomorze (maritime region)

was obtained in his tests by [START_REF] Falandysz | Some toxic and trace metals in big game hunted in the northern part of Poland in 1987-1991[END_REF]. The same author in other tests [START_REF] Falandysz | Ołów w przetworach z dziczyzny[END_REF][START_REF] Falandysz | Metale w mięśniach, wątrobie i nerkach zwierząt łownych z rejonu Polski północnej[END_REF]) also obtained higher concentration of lead both in liver and meat of wild boars.

In the case of contents of lead and cadmium in liver of wild boars (Table 4) the amounts of these metals were considerably dependent on the age of these animals (high correlation -r = 0.76 and 0.83 respectively). The contents of Pb in the liver was lowest in group I -0.017 mg/kg and increased in successive age groups by 46% and 92% respectively (statistically significant differences P≤0.01 were confirmed between the groups I and III). Similarly, the lowest contents of Cd in liver 0.251 mg/kg was obtained by the youngest specimens. The contents of this metal increased in successive age groups by 46% and 90% respectively (statistically significant differences P≤0.01). Moreover, in the group of animals above 3 years in the case of two samples the maximum permissible level of this metal in liver (0.5 mg/kg) was exceeded, and the average concentration of cadmium in this organ of wild boars from group III approached this limit and was 0.478 mg/kg. [START_REF] Falandysz | Some toxic and trace metals in big game hunted in the northern part of Poland in 1987-1991[END_REF] in his tests also observed high diversity as to the amount of Pb (0.02 -1.30 mg/kg) and Cd (0.05 -0.68 mg/kg) in liver of wild boars. However, [START_REF] Krupa | Ocena zanieczyszczenia metalami cięŜkimi tkanek zwierząt dziko Ŝyjących w południowo-wschodnim regionie Polski[END_REF] showed much higher level of Pb, Cd, Hg and As in muscles and in liver of wild boars than the author of this work for all age groups of these animals (Table 4). Only in the case of concentration of lead in liver the specimens from group III obtained higher values. In turn, [START_REF] Holm | Investigation of roe deer -criteria for use as a bioindicator in specimen banking[END_REF] in samples of livers collected from roe deer shot in the area of East Saxony noted the following amounts of cadmium (0.390 mg/kg) and lead (0.040 mg/kg). On the other hand, [START_REF] Kottferova | Distribution of Cd and Pb in the tissues and organs of free-living animals in the territory of Slovakia[END_REF] in organs of roe deer and wild boars shot in the area of Slovakia stated the contents of lead at the level respectively: 0.120 mg/kg and 0.250 mg/kg in liver and 0.250 mg/kg and 0.670 mg/kg in kidneys of these species of animals.

In Table 5 there are presented the coefficients of simple correlation between the contents of heavy metals in tissues of boars. From these data it appears that together with the increase of the amount of lead in liver there increased the amount of lead in meat (r = 0.88) and of cadmium in meat (r = 0.82), and also that together with the increase of the content of cadmium in liver there increased the content of the following metals: Pb in meat (r = 0.78),

Cd in meat (r = 0.81) and Pb in liver (r = 0.75). Also together with the increase of the concentration of mercury in liver there increased the concentration of this element in meat (r = 0.62) and the concentration of lead in liver (r = 0.60). Also the increase of the amount of cadmium in meat caused the increase of lead in this tissue (r = 0.83). In the majority of cases these were statistically significant differences, which means that the level of accumulation of lead and cadmium in liver is a good indicator of contamination of muscular tissue by these metals in remaining muscles obtained from carcasses of wild boars.

Coefficients of simple correlation between the chemical composition of meat and the contents of heavy metals in meat and liver of wild boars are presented in Table 6. From these data it appears that the majority of coefficients of correlation were positive. The exception were the interdependences between the contents of water in meat and the concentration of lead, cadmium and mercury in meat. For these dependences there were noted negative coefficients (statistically insignificant). However, positive and significant correlations occurred between the contents of chemical fat in meat and the contents of lead in meat (r = 0.53) and in liver (r = 0.62) of wild boars, and also between the amount of ash in meat and the concentration of lead in liver (r = 0.52) of these animals. [START_REF] Sikora | Jakość i stopień chemicznego skaŜenia mięsa i organów wewnętrznych testowanych zwierząt rzeźnych z krakowskiej strefy ekologicznie zagroŜonej[END_REF] carried out analysis of coefficients of correlation between the contents of basic chemical components of meat and the contents of elements (Cd, Pb, Fe, Zn, Cu, Mn, Mg, Ca) in meat and liver of rabbits. From these tests it appears that between the contents of fat and the contents of analyzed elements, both in meat and liver, there were no significant coefficients of correlation. However, the analysis of interdependences with the contents of protein conducted by this author, in the analogous arrangement, confirmed significant coefficients of correlation (although not high). In the case of meat for Zn and Mg, and in the case of liver for Zn, Cu, Mn and Mg. This observation may confirm the assumption of 6) allows us to conclude that with increasing amounts of fat and ash in meat of wild boars that there is an increase in the contents of lead and cadmium, both in meat and in liver of these animals. In the case of the contents of protein in meat such dependences were not found.

Conclusions

The analysis carried out in the previous chapter allows stating that accumulation of lead and cadmium in muscles and in liver increased together with the age of wild boars. However, most frequently the statistically essential differences were found only between the group of the youngest and oldest animals. Moreover in no single case was exceeded the maximum permissible level in meat, both for the contents of lead, cadmium as well as mercury. In the tested samples of tissues of wild boars there were also not stated the presence of arsenic above 0.001 mg/kg. In the group of animals above 3 years in the case of two samples the maximum permissible level of cadmium in liver (0.5 mg/kg) was exceeded. There was found very high positive correlative dependence between the contents of lead and cadmium in liver and concentration of these elements in muscles. Which means that the level of accumulation of lead and cadmium in liver is a good indicator of contamination of muscular tissue with these metals in remaining muscles obtained from carcasses of wild boars. The level of tested contaminations in tissues of wild boars in the analysed period (years 2002 -2006) was most often diversified and it cannot be explicitly stated that in this period there increased contamination of natural habitat of these animals.

F

  Dependences between the age and the level of bioaccumulation of heavy metals in tissues and the chemical composition of wild boars' meat

  of basic statistical measures of position (arithmetic mean) and scatter (standard deviation

  " is very variable, even within the same species. Within hundreds of millions of years the living organisms created excellent mechanisms registering the changes in physical and chemical parameters of atmosphere, water and soil. This ability enables reacting even for the most subtle changes in the surrounding environment, which cannot be detected even by the most sensitive measuring devices.The contents of heavy metals in tissues of wild boars in the years 2002-2006 is presented in Table3. From these data it appears that the contents of cadmium in meat was varied in individual years and fluctuated within the limits from 0.047 mg/kg in the year 2003 to 0.077 mg/kg in the year 2004. It was similar in the case of concentration of cadmium in meat whose amount fluctuated within the range from 0.012 mg/kg in the year 2005 to 0.015 mg/kg in the year 2002. Also the contents of mercury in this tissue fluctuated within the limits from 0.001 mg/kg in the year 2006 to 0.008 mg/kg in the year 2004. However, for the contents of these metals in muscular tissue there were not stated statistically significant differences between the years. In the analyzed samples of meat and liver of wild boars the presence of arsenic was not detected. But the presence of Pb in liver (0.145 mg/kg ) in the years 2002-2003 increased by approximately 11% in the years 2004-2005 and by 31% in the year 2006 (statistically significant differences P≤0.01 occurred between the years 2003 and 2006). In turn, the concentration of cadmium in liver of wild boars was the lowest in the years 2002 and 2006 (0.290 mg/kg) and increased in the years 2003 -2005 by: 15%; 33% and 44% respectively. However, statistically significant differences (P≤0.01) occurred only between the year 2002 and the years 2004 and 2005. It should be also emphasized that in the year 2005 for one sample there was exceeded the permissible amount of this element in liver (0.5 mg/kg). Also in liver of wild boars there was found the presence of mercury whose contents fluctuated within the range from 0.001 mg/kg in the year 2002 to 0.004 mg/kg in the year 2005.

  in the protein system and not in fat of animals. The data presented in this work (Table

  research was carried out between 2002 and 2006 on wild boars in south-eastern PolandThis procedure was simultaneously carried out for reagent blanks. The digested solutions were diluted so that the measured concentrations of a given element were, if possible, in the centre of the operating range. Arsenic was determined by hydride generation atomic absorption spectrometry (HGAAS) according toPN-EN 14627:2005. Pb and Cd were determined by graphite furnace atomic absorption spectrometry (GFAAS) (SpectrAA 880Z;

	and nutritional value (Sucha and Zawada 1994). Concentration of heavy metals both in soil
	and drinking water are within standard regulatory limits.
	Wild boars were shot by hunters from the nearby hunters' associations of South-East Poland.
	The size in each age group and in every year of tests was by 20 heads. The age of wild boars
	was determined on the basis of external appearance (colour, height, length of fang wear). On
	the basis of the results of earlier author's tests it was found that wild boars at the age of up to
	F o r 1 year have lower immunity in preventing accumulation of toxic elements in their tissues. And the biggest part of wild boars shot in Poland are animals at the age between 1 and 3 years, and in the second place in this respect are wild boars between 3 and six years of age. In this connection in this paper there were analysed tissues of wild boars from these age F o r
	P intervals. The test material constituted so wild boars which were divided into 3 age groups: P
	e e r e Group II -from 1 to 3 years, Group I -to 1 year, e r
	R Group III -over 3 years to 6 years. R
	e The samples for tests were meat and liver. For the purpose of determining the chemical e
	v i e w composition of meat a sample was taken from a part of longissimus muscle of back (m. longissimus dorsi) at the height of the last thoracic vertebrae. The content of water was v i e determined in accordance with the standard PN-ISO 1442:2000. Protein was determined with Kjeldahl's method, with the content of nitrogen converted into protein according to PN-75/A-w
	O n l 04018. The content of fat was determined with Soxhlet's method in accordance with recommendations of PN-ISO 1444:2000, and the total ash was determined in accordance with the guidelines contained in PN-ISO 936:2000. O n l
	y For determination of heavy metal levels, i.e. arsenic, cadmium, lead and mercury, samples y
	were taken (at least at 100 g from the same part of each tissue) also from a section of the
	longissimus back muscle (m. longissimus dorsi) at the level of the last thoracic vertebra;
	samples of liver were taken from the tail lobe. Sampled material was lyophilized
	(FreeZone6L; Labconco, Kansas, City, MO, USA), homogenized and then mineralized using
	the pressure-microwave method (MARS XPRESS, CEM Corporation, Matthews, NC, USA)
	in nitric acid/hydrogen peroxide mixture (6:1, v/v), according to PN-EN 13805:2003.
	5

(Podkarpackie and Lubelskie provinces), an area with small-scale mixed farming and practically with no industry. As 95% of the soils have very low heavy metal concentrations, the region has excellent potential for the production of high quality food with superior health

Table 1 .

 1 Analytical quality control program used in the determination of cadmium (Cd), lead (Pb), mercury (Hg) and arsenic (As) in wild boars samples

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 .

 2 Chemical composition of meat of wild boars of different age (%)

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	Composition of muscle tissue	x	I	S	Groups II x	S	x	III	S	r	x
	Water	76.90		4.50	75.10	5.10	73.30		4.10	-0.84* 75.10
	Protein	21.20		1.80	21.90	1.20	23.20		1.60	0.75* 22.10
	Fat	0.70		0.30	1.40	0.20	2.00		0.50	0.68*	1.13
	Ash	1.10		0.20	1.40	0.40	1.40		0.30	0.49	1.30
	* -values significant at P≤0.05

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Table 3 .

 3 The content of heavy metals in meat and liver of wild boars in the years2002-2006 (mg/kg)

	Specification	x	2002 xmin	xmax	x	2003 xmin	xmax	x	2004 xmin	xmax	x	2005 xmin	xmax	x	2006 xmin	xmax	The significance of differences
	Meat:
	Pb	0.060	0.039	0.072	0.047	0.041	0.071	0.077	0.047	0.093	0.057	0.047	0.087	0.062	0.045	0.083
	Cd	0.015	0.009	0.020	0.013	0.008	0.019	0.014	0.006	0.019	0.012	0.010	0.018	0.014	0.008	0.020
	Hg	0.005	0.001	0.008	0.002	0.001	0.006	0.008	0.002	0.009	0.007	0.003	0.010	0.001	0.001	0.001
	As	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001 <0.001 <0.001
	Liver:
	Pb	0.145	0.103	0.187	0.146	0.125	0.197	0.161	0.116	0.264	0.162	0.122	0.234	0.190	0.117	0.430	2003<2006*
	Cd	0.290	0.232	0.338	0.333	0.275	0.376	0.387	0.301	0.493	0.417	0.391	0.565	0.292	0.089	0.442	2002<2004,2005*
	Hg	0.001	0.001	0.001	0.003	0.001	0.005	0.001	0.001	0.001	0.004	0.001	0.006	0.002	0.001	0.002
	As	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001 <0.001 <0.001

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Table 4 .

 4 The content of heavy metals in meat and liver of wild boars of different age (mg/kg)

	Groups

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Table 5 .

 5 Coefficients of simple correlation between the content of heavy metals in meat and liver of wild boars

	Pb meat	-
	Cd meat	0.83*	-
	Hg meat	0.53	0.45	-
	As meat	-	-	-	-
	Pb liver	0.88*	0.82*	0.41	-	-
	Cd liver	0.78*	0.81*	0.48	-	0.75*	-
	Hg liver	0.45	0.53	0.62*	-	0.60*	0.43	-
	As liver	-	-	-	-	-	-	-	-
		Pb meat Cd meat Hg meat As meat Pb liver	Cd liver Hg liver As liver
	* -coefficients significant at P≤0.05

Table 6 .

 6 The coefficients of simple correlation between the chemical composition of meat and the contents of heavy metals in meat and liver of wild boars

	Specification	Pb meat	Cd meat Hg meat As meat Pb liver Cd liver Hg liver As liver
	Water	-0.15	-0.21	-0.02	-	0.20	0.17	0.03	-
	Protein	0.29	0.21	0.07	-	0.19	0.30	0.04	-
	Fat	0.53*	0.42	0.05	-	0.62*	0.49	0.05	-
	Ash	0.39	0.31	0.09	-	0.52*	0.45	0.09	-

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