Marilena ; Muscarella

Marco Iammarino

Donatella Nardiello

Sonia Lo Magro

Carmen Palermo

Diego Centonze
email: centonze@unifg.it

Domenico Palermo

Sonia Lo Magro

Palermo

Validation of a confirmatory analytical method for the determination of aflatoxins B 1 , B 2 , G 1 and G 2 in foods and feed materials by HPLC with on-line 4 photochemical derivatization and fluorescence detection 6

Keywords: affinity columns, HPLC, Method validation Aflatoxins, HPLC, photochemical derivatization, validation, Regulation (EC) No

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Validation of a confirmatory analytical method for the determination of aflatoxins B1, B2, G1 and G2 in foods and feed materials by HPLC with on-line photochemical derivatization and fluorescence detection

Aflatoxins B 1 , B 2 , G 1 and G 2 (AFB 1 , AFB 2 , AFG 1 and AFG 2 ), secondary metabolites of mould fungi Aspergillus flavus and Aspergillus parasiticus, are a group of structurally related 50 mycotoxins, which exhibit acute and chronic toxicity including mutagenic, carcinogenic and teratogenic effects in several organisms [START_REF] Harwig | Mycotoxins of possible importance in diseases of Canadian farm animals[END_REF]Munro, 1975, Bullerman, 1979, Dickens 52 and Jones, 1963, Garner and[START_REF] Garner | Chemical Carcinogens and DNA[END_REF]. The International Agency for Research on Cancer (IARC) classified aflatoxins B 1 , B 2 , G 1 and G 2 as group 1 of human carcinogens (IARC, 54 1993). The widespread occurrence of the Aspergillus mould means that several agricultural commodities are vulnerable to contamination with aflatoxins. The storage of these products 56 under high moisture and increased temperature conditions (25-30°C) enhances the natural occurrence of aflatoxins in a great variety of foods for animal and human consumption (Peraica 58 et al., 1999, Eaton and[START_REF] Eaton | The Toxicology of Aflatoxins[END_REF], such as cereals, corn, peanuts, pistachios, almonds and beans.

60

The European Union (EU) has set a maximum level (ML) of 2 µgkg -1 for AFB 1 and 4 µgkg -1 for 62 total aflatoxins (B 1 , B 2 , G 1 , and G 2 ) in nuts, dried fruit and cereals intended for direct human consumption (Regulation (EC) No. 1881[START_REF] Eurachem Guide | Quantifying Uncertainty in Analytical Measurement[END_REF]. In baby food products, more restrictive limits 64 (0.10 µgkg -1 for AFB 1 ) have been established (Regulation (EC) No. 683/2004). In agreement with the Directive (EC) No. 100/2003, a maximum residue limit of 20 µgkg -1 for AFB 1 has been 66 fixed in cereals for animal consumption (Italian Ministerial Decree 149/2004, Italian Official Journal n.139, June 2004). [START_REF] Trucksess | Enzyme-linked immunosorbent assay of total aflatoxins B1, 470 B2, and G1 in corn: follow-up collaborative study[END_REF]Stack, 1994, Park et al.,1989), and chromatographic 74 methods based on RP-HPLC and fluorescence detection (FLD) with chemical pre-or postcolumn [START_REF] Wilson | Use of MycoSep multifunctional cleanup column for liquid 482 chromatographic determination of aflatoxin in agricultural product[END_REF][START_REF] Espinosa | Quantification of aflatoxins in corn distributed in the city of Monterrey, Mexico. 468[END_REF][START_REF] Roch | The development and validation of a solid phase extraction/HPLC method for the determination of aflatoxins in groundnut meal 450[END_REF], Saad et al., 76 1995, Garcia-Villanova et al., 2004[START_REF] Davis | Improved fluorometric-iodine method for determination 376 of aflatoxin in corn[END_REF][START_REF] Tuinstra | Rapid determination of aflatoxin B 1 in dutch feeding stuffs 472 by high-performance liquid chromatography and post-column derivatization[END_REF][START_REF] Niedwetzki | Determination of aflatoxins in food by use of an automatic work 436 station[END_REF][START_REF] Ahmed | Selection of a Suitable Method for Analysis of Aflatoxins in Date Fruits[END_REF][START_REF] Holcomb | Analysis of Aflatoxins (B 1 , B 2 , G 1 , and G 2 ) in Rodent Feed by HPLC Using Postcolumn Derivatization and Fluorescence Detection[END_REF] 78 [START_REF] Garner | Analysis of United Kingdom 404 purchased spices for aflatoxins using an immunoaffinity column clean-up procedure followed by high-performance liquid chromatographic analysis and post-column derivatization with 406 pyridinium bromide perbromide[END_REF][START_REF] Chan | Simultaneous determination of aflatoxins and ochratoxin A in food using a fully automated immunoaffinity column clean-up 374 and liquid chromatography-fluorescence detection[END_REF], Va´zquez et al., 1999[START_REF] Cepeda | 370 Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis[END_REF] or on-line electrochemical [START_REF] Kok | Determination of aflatoxins in 424 cattle feed by liquid chromatography and post-column derivatization with electrochemically generated bromine[END_REF][START_REF] Kussak | Determination of Aflatoxins in Hot Chilli Products by Matrix Solid-Phase Dispersion and Liquid Chromatography[END_REF][START_REF] Dorner | Performance of two immunochemical assays in the 380 analysis of peanuts for aflatoxin at 37 field laboratories[END_REF], Scholten and Spanjer, 80 1996[START_REF] Tavčar-Kalcher | Validation of the procedure for the 460 determination of aflatoxin B 1 in animal liver using immunoaffinity columns and liquid chromatography with postcolumn derivatization and fluorescence detection[END_REF] derivatization of analytes.

82

Although ELISA methods are extensively used for rapid qualitative screenings of aflatoxins, they are not useful in providing a definitive confirmation of the toxins and an accurate 84 quantitative determination. The present trend is the use of HPLC for the determination of aflatoxins because of characteristics of specificity, high sensitivity and simplicity of operation.

86

Additionally, the chromatographic determination allows quantifications of each toxin individually, obtaining more information than methods that give only a total quantification of 88 aflatoxins. This is particularly important for food analysis where the determination of AFB 1 is required.

90

The highly conjugated and rigid aflatoxin moieties give rise to native fluorescence 92 characteristics, therefore small structural modifications can have a strong influence on the fluorescence properties; for instance aflatoxins G 2 and B 2 are more fluorescent than B 1 and G 1 94 that are required to be converted into more highly fluorescent derivatives. Several methods of derivatization are available, including pre-column treatment with trifluoroacetic acid (TFA) 96 (Garcia-Villanova et al, 2004) and post-column derivatization with iodine [START_REF] Davis | Improved fluorometric-iodine method for determination 376 of aflatoxin in corn[END_REF] (Va´zquez et al., 1999[START_REF] Cepeda | 370 Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis[END_REF] and pyridinium hydrobromide perbromide [START_REF] Garner | Analysis of United Kingdom 404 purchased spices for aflatoxins using an immunoaffinity column clean-up procedure followed by high-performance liquid chromatographic analysis and post-column derivatization with 406 pyridinium bromide perbromide[END_REF][START_REF] Chan | Simultaneous determination of aflatoxins and ochratoxin A in food using a fully automated immunoaffinity column clean-up 374 and liquid chromatography-fluorescence detection[END_REF]. However, these methods are laborious and 100 present a number of disadvantages, such as handling of toxic reagents, instability of the derivatives, a low day-to-day reproducibility. An alternative approach is based on post-column 102 electrochemically generated bromine [START_REF] Cepeda | 370 Postcolumn excitation of aflatoxins using cyclodextrins in liquid chromatography for food analysis[END_REF][START_REF] Kok | Determination of aflatoxins in 424 cattle feed by liquid chromatography and post-column derivatization with electrochemically generated bromine[END_REF][START_REF] Kussak | Determination of Aflatoxins in Hot Chilli Products by Matrix Solid-Phase Dispersion and Liquid Chromatography[END_REF][START_REF] Dorner | Performance of two immunochemical assays in the 380 analysis of peanuts for aflatoxin at 37 field laboratories[END_REF][START_REF] Scholten | Determination of Aflatoxin B 1 in Pistachio Kernels and Shells. 454[END_REF] that do not give a good chromatographic peak 104 resolution and allows a less extent of automation, since any accidental interruption of the mobile phase flux determines a serious damage to the electrochemical cell. Nowadays, an on-line 106 photochemical derivatization system is available [START_REF] Joshua | Determination of aflatoxins by reversed-phase high-performance liquid chromatography with post-column in-line photochemical derivatization and fluorescence 422 detection[END_REF][START_REF] Papadopoulou-Bouraoui | Comparison of Two Post-Column Derivatization Systems, Ultraviolet Irradiation and Electrochemical Determination[END_REF], Sobolev and Dorner, 2002[START_REF] Sobolev | Simple, Rapid, and Inexpensive Cleanup Method for Quantitation of 458 Aflatoxins in Important Agricultural Products by HPLC[END_REF][START_REF] Waltking | Liquid Chromatographic Analysis of Aflatoxin Using Post-Column Photochemical 480 Derivatization: Collaborative Study[END_REF][START_REF] Arranz | Determination of Aflatoxin B 1 in Medical Herbs: Interlaboratory Study[END_REF] 108 [START_REF] Zang | Hitachi HPLC aflatoxin photochemical post-column derivatization[END_REF], but its potential has not fully investigated. It has been reported that the on-line photochemical derivatization coupled to fluorescence detection guarantees an 110 enhancement of efficiency and performances of the HPLC methods [START_REF] Papadopoulou-Bouraoui | Comparison of Two Post-Column Derivatization Systems, Ultraviolet Irradiation and Electrochemical Determination[END_REF][START_REF] Zang | Hitachi HPLC aflatoxin photochemical post-column derivatization[END_REF]. Differently from chemical pre-and post-column derivatization 112 techniques, the photochemical derivatization does not involve any additional chemicals and, consequently, no auxiliary pump is required, ensuring better results without any control of the

Materials and Methods

Chemicals.

A standard solution of aflatoxins B 1 , B 2 , G 1 and G 2 with a total certified 136 concentration of 5 µgmL -1 corresponding to a concentration of 2.0 µgmL -1 each for B 1 and G 1 and of 0.5 µgmL -1 each for B 2 and G 2 , was supplied by Riedel-de Haën (Sigma-Aldrich 138 Laborchemikalien, Seelze, Germany). Water, methanol and acetonitrile of HPLC grade were purchased from Baker (Deventer, Holland). Sodium chloride (≥99.0%), Na 2 HPO 4 •2H 2 O 140 (≥99.0%), NaH 2 PO 4 •H 2 O (≥99.0%) and TWEEN 20® were purchased from Sigma-Aldrich. All chemicals were of ACS grade. A 500 µgL -1 stock solution of aflatoxins standard, containing 200 142 µgL -1 of AFB 1 , 50 µgL -1 of AFB 2 , 200 µgL -1 of AFG 1 and 50 µgL -1 of AFG 2 , were prepared in acetonitrile and stored in amber flask at -20°C up to 6 months. Working standard solutions were Validation procedure. In consequence of different legal limits for aflatoxins in foodstuffs and 184 cereals for animal consumption, different sets of standard solutions and spiked samples were used for foodstuffs (wheat bran, almonds and pistachios) and materials for feed (durum wheat, 186 maize and oats). The linearity test in foodstuffs was performed by three series of analyses on three different days, by injecting four standard solutions of aflatoxins B 1 and G 1 each at 188 concentrations of 0.25, 0.5, 1.0 and 2.0 µgL -1 and aflatoxins B 2 and G 2 each at concentrations of 0.0625, 0.125, 0.25 and 0.5 µgL -1 . Likewise, method linearity in feed materials was determined 190 by using working standard solutions of AFB 1 at concentration of 2.5, 5.0, 10 and 20 µgL -1 . The method selectivity was tested by the analysis of 20 independent blank samples of foods (7 of 192 wheat bran, 7 of almonds and 6 of pistachios) and 20 samples of cereals (7 of durum wheat, 7 of maize and 6 of oats), found negative by ELISA screening. The absence of interfering peaks in fortified with AFB 1 at concentration of 0.5, 1.0 and 1.5 times the ML in foodstuffs (i.e., 1.0, 2.0 198 and 3.0 µgkg -1 ) and aflatoxins B 2 , G 1 and G 2 at concentration of 0.625, 1.25 e 1.875 times the ML of the sum of aflatoxins B 1 , B 2 , G 1 and G 2 (i.e., 1.0, 2.0 and 3.0 µgkg -1 for B 1 and G 1 , and 200 0.25, 0.5 and 0.75 µgkg -1 for B 2 and G 2 ). As far as the analysis of feed materials is concerned, only two sets of blank durum wheat samples (six replicates each) fortified with AFB 1 at 202 concentration of 0.5, 1.0 and 1.5 times the ML in cereals for animals (i.e., 10, 20 and 30 µgkg -1 ) were analysed. The experiments, corresponding to a total number of 54 and 36 for food samples Calibration curves and limits of detection and quantification. The goodness-of-fit of the data to the calibration curve is obtained in terms of response factor distribution (signal-to-234 concentration ratio, y i /x i ) whose reference range is y/x mean ±10%. Furthermore, any systematic instrumental bias can be ruled out since the intercept includes the zero value at 95% confidence 236 level. The calibration parameters evaluated for each aflatoxin by using standard solutions are reported in Table 1. Limits of detection and quantification were calculated according to the 238 following equations [START_REF] Miller | Commission Decision 2002/657/EC of a confirmatory method for aflatoxin M 1 in milk based on immunoaffinity columns and high performance liquid chromatography with fluorescence 434 detection[END_REF]: LOD=3.3 s a /b and LOQ=10 s a /b, where s a is the standard deviation of the intercept and b is the slope of the regression line, obtained from the 240 calibration curve. The limit of quantification for AFB 1 of 0.11 µgL -1 corresponding to a value of 0.22 µgkg -1 in matrix is satisfactory with respect to the allowed maximum content of 2 µgkg -1 242 (Regulation (EC) No. 1881/2006). Fit of the data to the calibration curve of AFB 1 in cereals for feeds gave the linear regression equation y = (1.002±0.006) x + (0.09±0.06) with a correlation 244 coefficient of 0.9999, where y is the peak area expressed in luminescence unit (LU) and x is the concentration of AFB 1 in µgL -1 . The obtained limit of quantification of 1.2 µgkg -1 is noticeably Furthermore, Table 4 shows the results of repeatability and within-laboratory reproducibility 254 determined for AFB 1 in spiked durum wheat (cereal for feeds). Regulation (EC) No. 401/2006 issued that the recommended values of experimental RSD for each concentration level must be 256 lower or equal than value derived by Horwitz equation, which provides the expected RSD% only on the basis of the concentration, independently of the matrix and analytical method used. 258 Nevertheless, for mass fractions lower than 120 µgkg -1 , the RSD% can be predicted in a more restrictive manner by the Thompson equation [START_REF] Thompson | Recent trends in inter-laboratory precision at ppb and sub-ppb 464 concentrations in relation to fitness for purpose criteria in proficiency testing[END_REF], which provides a 260 recommended value of RSD r of 15 % and a reference inter-laboratory reproducibility of 23%.

As can be seen in Table 2 and4 have been calculated by using the same equations previously described, ranged from 0.06 to 0.3 µgkg -1 and from 0.2 to 0.8 µgkg -1 , respectively, with LOD and LOQ for AFB 1 of 0.2 µgkg -1 and 284 0.6 µgkg -1 , respectively. As expected, taking into account the loss of analyte during extraction and cleanup steps, LODs and LOQs are higher than those determined by calibration curves of 286 standard solutions, but still considerably lower than legal limits.

Ruggedness (major changes).

The method ruggedness under conditions of major changes was 288 evaluated by using the Youden experimental design as reported elsewhere (Muscarella et al., 2007). Two independent Youden tests were performed for different sets of real samples: calculated standard deviation of difference S Di (0.09 µgkg -1 for almonds and pistachios) lower than the estimated method precision at the ML (0.21 µgkg -1 ). Similar considerations are valid 298 for maize sample whose standard deviation of difference S Di (1.40 µgkg -1 ) resulted slightly lower than method precision (1.45 µgkg -1 ). In the case of oats matrix, test F (performed at n-1 300 degrees of freedom and 95% confidence level) demonstrated that S Di (2.36 µgkg -1 ) was not statistically different from the reproducibility standard deviation (1.45 µgkg -1 ) at the ML, 302 therefore the variation of the matrix has no effect on the method performances. [START_REF] Nmkl) | Estimation and expression of 438 measurement uncertainty in chemical analysis[END_REF]. In this work we have used the bottom-up method together with validation data 308 obtained from each step of the analytical procedure [START_REF] Hund | Operational definitions of uncertainty[END_REF]. On the basis of uncertainties propagation law, for each aflatoxin the concentration relative uncertainty has been 310 calculated by the equation: A relative expanded measurement uncertainty was calculated by using a coverage factor k of 2, corresponding approximately to a 95% confidence level (Nordic Committee on Food Analysis, 318 [START_REF] Nmkl) | Estimation and expression of 438 measurement uncertainty in chemical analysis[END_REF] obtaining values of 8.4%, 12.9 %, 9.9 % and 14.2 % for aflatoxins B 1 , B 2 , G 1 and G 2 , in foodstuffs. In the validation on cereals for animal feeds, the relative expanded 320 measurement uncertainty calculated for AFB 1 was 7.6 %. These results were compared with the method of analysis is "fit-for-purpose". This function, which gives the acceptable maximum uncertainty level of the method, is calculated by the following equation: 324

Measurement uncertainty. The evaluation of uncertainty of analytical results is compulsory for

( ) ( ) ( ) ( ) ( ) ( ) u u u u 2 2 f 2 w V C + + =
( )         +       = 2 2 αC 2 LOD Uf
where C is the concentration of interest and α is a C dependent constant factor. Measurement 326 uncertainties evaluated for AFB 1 in food samples and feed materials (8.4% and 7.6%, respectively) are considerably lower than corresponding Uf values (20% at C = 2 µgkg -1 , α = 328 0.2 and 21 % at C = 20 µgkg -1 , α = 0.2, respectively). The above results confirm that method is "fit-for-purpose".

68

  Due to the significant health risks associated with the presence of aflatoxins in foods and to 70 satisfy the rigorous legal requirements too, it is important to have efficient techniques for the detection of aflatoxins in foods and animal feeds. Several procedures for the determination of 72 developed, comprising immunological methods, such as enzyme linked immunosorbent assays

  114 derivatization reaction. Nevertheless, to the best of our knowledge chromatographic methods for the determination of aflatoxins, exploiting the photochemical derivatization and fluorescence 116 detection, have been never validated according to the recent European Commission guidelines. 118 In the present work, the procedure for the determination of aflatoxins B 1 , B 2 , G 1 and G 2 in foods and materials for feed, based on immunoaffinity column sample cleanup and liquid 120 chromatography coupled to photochemical derivatization and fluorescence detection, has been submitted to the validation procedure following the Regulation (EC) No. 882/2004.

  144prepared by dilution in mobile phase and stored at -20°C when not in use; these solutions were stable for at least 4 weeks. Phosphate buffer salinum (PBS) was prepared by dissolving 9.0 g of 146 sodium chloride, 2.85 g of Na 2 HPO 4 •2H 2 O, 0.55 g of NaH 2 PO 4 •H 2 O and 1.0 mL of TWEEN mL of water. The pH value was adjusted to 7.2 with concentrated hydrochloric acid 148 or NaOH 1M and the volume made to 1L with water.Instrumentation. Chromatographic separations were performed on a HPLC system, Agilent 150 Technologies SL 1200 Series (Waldbronn, Germany) composed of a binary pump equipped with micro vacuum degasser, thermostated autosampler, column compartment and fluorescence 152 detector (model G1321A). On-line photochemical derivatization was performed using a commercially available system UVETM LCTech GmbH (Dorfen, Germany) placed between the 154 separation column and the fluorescence detector, which consisted of a 254 nm low-pressure mercury lamp and a 1 mL knitted reaction coil, fitted around the UV lamp. All the separations 156 were performed by using a ZORBAX Eclipse XDB C 18 column (150 × 4.6 mm i.d., particle size 5 µm, Agilent Technologies) operating at a flow-rate of 1.0 mLmin -1 in isocratic elution with a 158 mixture of water, methanol and acetonitrile (55:15:30, v/v/v). The injection volume was 20 µL and the column temperature was set at 40°C. The fluorescence detection was carried out at a λ exc 160 and λ em of 365 and 435 nm, respectively. The system was interfaced, via network chromatographic software (Agilent ChemStation), to a personal computer for instrumentation 162 control, data acquisition and processing. Sample preparation. Samples of wheat bran, almonds and pistachios were bought in local 164 stores; durum wheat, maize and oats were supplied by farms chosen among the major cereal producers in the province of Foggia (Italy). Both sample extraction and cleanup procedure were 166 those recommended by LCTech and slightly modified in order to simplify the analytical protocol and to reduce reagents and chemical wastes. The method did not require vacuum or 168 pumping devices ensuring better recoveries and reproducibility. For each matrix, from a sample weight of 1 kg, an amount of 15 g of ground sample was suspended in 30 mL of 80 % methanol 170 and vortexed for few min. For the validation measurements, samples were spiked, prior to the extraction, with the proper amounts of mixed aflatoxin standard solutions to give required 172 filtration with paper (Whatman No. 40), 2 mL of the mixture were added to 8 mL of PBS solution and passed through the immunoaffinity columns Aflaclean TM (LCTech 174 GmbH, Dorfen, Germany) for sample clean-up. After washing with 10 mL of water, the fraction containing the aflatoxins was slowly eluted with 2 mL of methanol by gravity at a flow rate 176 lower than of 0.5 mL min -1 . The eluate was evaporated to dryness at 40 °C under nitrogen stream (Dubnof Bath BSD/D). Finally, the residue was solubilized in 2 mL of the mobile phase, 178 filtered by an Anotop 10 LC (0.2 µm, 10 mm, Whatman) and then injected. The analytical procedure resulted in a 2-fold dilution of aflatoxins B 1 , B 2 , G 1 and G 2 , evaluated against a 180 known concentration added to a blank real sample. The stability of purified extracts was high enough to allow autosampler overnight injections. No significant change in concentration or 182 purity of aflatoxins within 8 hours of analysis was observed.

  194the retention time-window of interest was checked for each analyte within the ± 2.5% retention time range. Precision, recovery and both detection and quantification limits of the method were 196 determined by performing tests on three sets of blank wheat bran samples (six replicates each)

  204and animal feed materials, respectively, were performed in different days with the same instruments but different operators.206Results and Discussion208 Method Validation. The proposed method has been validated according to Regulation (EC) No. 882/2004, a document about official controls performed to ensure the verification of compliance 210 with feed and food laws, animal health and animal welfare rules, which demonstrates the conformity of the analytical performances with criteria established in Regulation (EC) No. 212 401/2006. Therefore, through validation procedure, linearity, selectivity, recovery, precision, detection and quantification limits, ruggedness of the method and measurement uncertainty have 214 been evaluated. Selectivity towards interferences. Figure 1 shows, as an example, the fluorescence enhancement 216 attained by on-line photochemical derivatization for a wheat bran sample fortified with AFB 1 and AFG 1 at 3.0 µgkg -1 and AFB 2 and AFG 2 at 0.75 µgkg -1 . Through the post-column UV 218 irradiation, AFB 1 and AFG 1 are converted by the reaction with water contained in the mobile phase to fluorescent hemiacetals, which are similar compounds to those obtained by 220 derivatization with TFA (Garcia-Villanova et al., 2004). This photochemical modification determines an increase of the response sensitivity of AFB 1 and AFG 1 , while a very slight 222 intensities has been observed for AFB 2 and AFG 2 . Use of both the immunoaffinity sample cleanup and post-column photochemical derivatization permitted an 224 efficient elimination of possible endogenous matrix interfering substances. In pre-validation experiments the probable interfering compounds, ochratoxin A and fumonisins B 1 and B 2 , 226 common contaminants of cereals, nuts and dried fruit, have been tested. These compounds showed retention times significantly different from those of aflatoxins or undetectable signals 228 under the adopted experimental conditions. Chromatograms of Figure 2, obtained for blank and spiked samples with aflatoxins B 1 , B 2 , G 1 and G 2 , demonstrate the selectivity of the method 230 toward other naturally present compounds, since at the retention times of the aflatoxins no interfering peaks were observed.232

  246

  maximum content of 20 µgkg -1 set for feed materials(Italian Ministerial Decree 149/2004, Italian Official Journal n.139, June 2004). 248 Precision and recovery. Precision results have been previously processed by the Shapiro-Wilk test to verify the normal distribution. Afterwards, ANOVA one way test was performed in order 250 to verify the homogeneity of the concentration mean values evaluated on three different days. In Table 2 and 3 the results of repeatability (RSD r ) and within-laboratory reproducibility (RSD R ) 252 are given for each aflatoxin at the three contamination levels in spiked wheat bran samples.

  , RSD r % values are in agreement with the calculated 262 repeatability by Thompson equation. In absence of within-laboratory reproducibility reference values, our experimental data have been compared with the repeatability maximum permitted 264 limit of 30%, which has been calculated as 2 fold the RSD r recommended value, by using a similar approach proposed for the calculation of the maximum permitted value of Horwitz RSD R 266 (Regulation (EC) No. 401/2006). As shown in

  from 76.4 to 94.6% for aflatoxins B 1 and G 1 and from 79.5 to 92.8% for 272 aflatoxins B 2 and G 2 . These results are in agreement with Regulation (EC) No. 401/2006 that establishes recoveries in the range 70-110% and 50-120% for concentrations in the range 1-10 274 µgkg -1 and less than 1 µgkg -1 , respectively. In the analysis of cereals for animal feeds, mean recovery data higher than 72.6 % were obtained. It was not possible to compare experimental 276 mean recoveries with reference values since so far no legal criteria have been set for these matrices. 278 Assessment of LODs and LOQs in real samples has been calculated for each analyte by mean the fit of the data to calibration curves obtained by plotting the fluorescence signals of 18 spiked 280 wheat bran samples (six replicates evaluated in the three different working sessions at each validation level) as a function of the nominal fortification level. LOD and LOQ values, which

  282

  290almonds and pistachios (foodstuffs) and maize and oats (feed materials) spiked at the AFB 1 maximum level. The seven factors chosen as variables for the Youden test were the matrix and 292 six fictitious factors; the use of a fictitious variable means no variation of analysis conditions. In this case the Youden experimental design requires eight independent experiments: four with the 294 matrix of validation (e.g. wheat bran and durum wheat) and four with each testing matrices (almonds, pistachios, maize and oats). Analysis of foods for human consumption gave a 296

  304 laboratories accredited according to ISO 17025 (UNI CEI EN ISO/IEC 17025/2000), and several methods for the determination of this parameter have been proposed (EURACHEM 306 Guide, 1995; EURACHEM/CITAC GUIDE CG 4, 2000; Nordic Committee on Food Analysis,

  312where ū indicates the relative uncertainty, C is the analyte concentration in the sample, V f is the volume of the final extract, and w is the sample weight. The determination of ū has been 314 performed by considering four sources of uncertainty: (a) preparation of the standard; (b) method reproducibility; (c) method recovery; (d) calibration curve.

  316

  Uf), reported in the Regulation (EC) No. 401/2006, to evaluate if the 322

330Figure 1 .Figure 2 .

 12 photochemical derivatization, has been validated. Photochemical derivatization results

  Figure 1 839x1187mm (150 x 150 DPI)

  ,

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

Table 3

 3 In Table5mean recoveries of aflatoxins evaluated at each fortification level in spiked wheat 270 bran and durum wheat samples are reported. As can be noticed, the recovery values obtained for

	, all RSD R values resulted well below of

Table 1 .

 1 Performance parameters for aflatoxins in foodstuffs.

				y = a + bx a
		Aflatoxin	b ± SD	a ± SD	r b	LOD c	LOQ c
		B 1	0.87 ± 0.01	0.02 ± 0.01	0.9998	0.04	0.11
		B 2	2.31 ± 0.04	0.02 ± 0.01	0.9996	0.01	0.04
		G 1	0.368 ± 0.009	0.014 ± 0.009	0.9991	0.08	0.24
		G 2	0.87 ± 0.01	0.005 ± 0.004	0.9996	0.02	0.05
	2
		a y is the signal in lumiscence unit (LU); x is the value of concentration in µg L -1 . b Correlation coefficient. c
	4	Values expressed in µg L -1 .
	6
	8
	10
	12
	14
	16
	18
	20
	22
	24
	26
	28
	30
	32
	34
	36
	38
	40
	42
	44
	46
	48

Table 2 .

 2 Repeatability for the determination of aflatoxins in spiked wheat bran samples.

	50
	Aflatoxin Operator	Contamination Level (µg kg -1 )	Determined Concentration Mean ± SD (n = 6)	RSD r (%)	a
		1		1.05 ± 0.04	3.5
		2	1	1.0 ± 0.1	9.8
		3		1.05 ± 0.06	5.5
		1		1.9 ± 0.1	7.1
	B 1	2	2	2.0 ± 0.2	10
		3		1.9 ± 0.1	4.9
		1		3.05 ± 0.14	4.8
		2	3	3.00 ± 0.08	2.7
		3		3.1 ± 0.3	9.5
		1		0.27 ± 0.06	11
		2	0.25	0.25 ± 0.02	9.1
		3		0.26 ± 0.03	10
	B 2	1 2	0.50	0.46 ± 0.03 0.49 ± 0.05	7.0 9.8
		3		0.47 ± 0.03	5.9
		1		0.77 ± 0.06	7.4
		2	0.75	0.75 ± 0.02	3.0
		3		0.76 ± 0.07	9.6
		1		1.10 ± 0.08	7.7
		2	1	1.0 ± 0.1	10
		3		1.0 ± 0.1	9.2
		1		1.8 ± 0.2	11
	G 1	2	2	2.0 ± 0.2	9.0
		3		1.9 ± 0.1	6.2
		1		3.1 ± 0.2	7.0
		2	3	3.0 ± 0.1	4.4
		3		3.0 ± 0.3	9.2
		1		0.27 ± 0.02	8.8
		2	0.25	0.24 ± 0.04	15
		3		0.27 ± 0.04	15
		1		0.46 ± 0.06	13
	G 2	2	0.50	0.51 ± 0.07	14
		3		0.47 ± 0.05	11
		1		0.77 ± 0.07	9.0
		2	0.75	0.74 ± 0.05	6.6
		3		0.77 ± 0.08	11
	52
	a Relative standard deviation under repeatability conditions.
	54
	56

Table 3 .

 3 Within-laboratory reproducibility for the determination of 58 aflatoxins in spiked wheat bran samples. RSD R , relative standard deviation under within-laboratory reproducibility conditions. 62

	60
	Aflatoxin	Contamination Level (µg kg -1 )	Determined Concentration Mean ± SD (n = 18)	RSD R (%)	a
		1	1.0 ± 0.1	9.9
	B 1	2	1.9 ± 0.2	11
		3	3.0 ± 0.2	6.8
		0.25	0.26 ± 0.03	12
	B 2	0.50	0.47 ± 0.05	11
		0.75	0.76 ± 0.06	7.6
		1	1.05 ± 0.15	14
	G 1	2	1.9 ± 0.3	15
		3	3.0 ± 0.2	8.1
		0.25	0.26 ± 0.05	20
	G 2	0.50	0.48 ± 0.09	19
		0.75	0.76 ± 0.08	10

a

Table 4 .

 4 Repeatability and within-laboratory reproducibility for the determination of 64 aflatoxin B 1 in spiked durum wheat samples.

	Page 25 of 28		Food Additives and Contaminants
	66
		Aflatoxin	Contamination Level (µ µ µ µg kg -1 )	Operator Determined Concentration Mean ± ± ± ± SD	RSD a (%)
				Repeatability (n = 6)
			10	1 2	10 ± 1 10.0 ± 0.8	10 7.9
		B 1	20	1 2	20 ± 1 20.0 ± 1.5	6.1 7.4
			30	1 2	29.8 ± 1.6 30 ± 2	5.2 5.7
			Within-laboratory Reproducibility (n = 12)
			10		9.9 ± 0.9	9.6
		B 1	20		20.2 ± 1.5	7.2
			30		29.9 ± 1.6	5.5
	68	a RSD, relative standard deviation under repeatability (RSD r ) and within-laboratory reproducibility
		(RSD R ) conditions.
	70

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Table 5 .

 5 Recovery of aflatoxins in spiked wheat bran (foodstuffs) and durum wheat samples (feed materials).

	72
	Aflatoxin	Contamination Level (µ µ µ µg kg -1 )	Recovery ± ± SD ± ± (%)	Recommended Range (%)
			Foodstuffs (n = 18)
		1	76.4 ± 6.5
	B 1	2	83.0 ± 9.8	70-110
		3	92.2 ± 8.3
		0.25	79.5 ± 11.6
	B 2	0.50	82.1 ± 9.9	50-120
		0.75	92.8 ± 8.0
		1	80.4 ± 9.6
	G 1	2	83.4 ±11.1	70-110
		3	94.6 ± 8.2
		0.25	89.2 ± 16.9
	G 2	0.50	82.0 ± 10.7	50-120
		0.75	87.7 ± 10.1
		Feed materials (n = 12)
		10	75.7 ± 6.3
	B 1	20	74.3 ± 4.7	N.A. a
		30	72.6 ± 3.9

a N.A., Not Available; a legal value has not been established.

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Kerstin Kuhn
email: ker.kuhn@gmail.com

Bernhard Nowak

Andreas Behnke

Prof Albrecht Seidel

Alfonso Lampen

Prof.Dr Gernot Grimmer

Effect-based and chemical analysis of polycyclic aromatic hydrocarbons in smoked meat -a practical food monitoring approach

Keywords: Screening -Calux, Screening -cell culture, Screening assays

screening method. We recently adapted the in vitro chemical-activated luciferase expression 6 (CALUX) assay as a rapid, sensitive and inexpensive screening technique for compounds 7 such as dioxins, polychlorinated biphenyls and PAHs. The aim of the present study was to 8 apply a practical approach under realistic conditions. Custom-made meat samples produced 9 under defined conditions with different PAH levels were analysed using this bioassay and 10 GC/MS to determine the influence of different smoking conditions (temperature and duration) 11 on PAH levels. We found that cold smoking for up to six hours did not result in strong PAH 12 contamination, whereas hot (65 °C) and longer smoking times caused a considerable increase 13 in both the bioassay response and the levels of 31 individually determined PAHs. The 14 response in the effect-based bioassay was in good agreement with the values of chemical 15 analysis. The bioassay made it possible to determine accurately the degree of contamination. 16

Our results show that this assay is suitable for high throughput screening for unknown levels 17 of toxicologically relevant PAHs in meat samples and is sensitive enough to differentiate 18 between different PAH levels generated under various smoking conditions. Effect-based 19 screening techniques therefore provide a new instrument for official food monitoring. 20 environment or from food processing (Gomaa 1993). In general, PAHs are formed as 28 complex mixtures during heat treatment processes such as smoke curing, barbecuing, deep 29 frying, or drying and they contaminate food by direct contact with these combustion products. 30 Contamination of food during the smoking process is highly variable and depends on the 31 composition of the wood, the type of generator (internal or external), oxygen accessibility, the 32 temperature of smoke generation and smoking time (European Union 2002;[START_REF] Simko | Factors affecting elimination of polycyclic aromatic hydrocarbons from 353 smoked meat foods and liquid smoke flavorings[END_REF]. 33

At the moment, PAHs in smoked meat samples are determined by costly, time-34 consuming chemical analysis. Toxic equivalence factors [START_REF] Nisbet | Toxic equivalency factors (TEFs) for polycyclic aromatic 344 hydrocarbons (PAHs)[END_REF] or potency 35 equivalency factors [START_REF] Collins | Potency Equivalency Factors for 300 Some Polycyclic Aromatic Hydrocarbons and Polycyclic Aromatic Hydrocarbon 301 Derivatives[END_REF] for individual PAHs as to their carcinogenic potency 36 relative to B[a]P are used for risk assessment of PAH mixtures in smoked meat and other 37 heat-treated food items. We adapted an effect-based screening assay using the in vitro 38 chemical-activated luciferase expression (CALUX) assay [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF]). This bioassay 39 can evaluate the total activity of the sample compounds that interact with the aryl 40 hydrocarbon receptor (AhR). The AhR mediates most of the toxic effects of dioxins, 41 polychlorinated biphenyls and PAHs [START_REF] Guillen | Polycyclic Aromatic Hydrocarbons in Diverse Food[END_REF]. The total carcinogenic 42 potential is in good accordance with AhR-mediated activities of individual PAHs both in vitro 43 [START_REF] Machala | Aryl hydrocarbon receptor-341 mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in 342 vitro reporter gene assay[END_REF]in vivo (Shimada et al. 2002). Following the binding of the ligand 44 to the receptor, the resulting ligand-receptor complex is activated and translocated to the 45 nucleus, where it binds to a dioxin-responsive element (DRE) and stimulates the transcription 46 of target genes including those of PAH-metabolising enzymes such as cytochrome P450 1A1 47 [START_REF] Ms | EFSA Data Collection on Polycyclic Aromatic 310 Hydrocarbons in Food -A Report from the Unit of Data Collection and Exposure on a 311[END_REF]. The CALUX assay is based on a genetically engineered 48 cell line (rat hepatoma H4IIE cells) which contains a stable transfected vector including the 49 firefly luciferase gene under the transcriptional control of several DREs. This transgenic cell 50 line responds to ligand exposure with the induction of the luciferase, which in the presence of 51 its substrate produces a luminescent signal proportional to the cell's response. Because of its 52 specifity to AhR, this bioassay does not allow identification of the chemicals that are 53 responsible for the enhanced signal but it does provide information about the effect of the 54 specific compounds in a meat sample on the receptor. 55

Effect-directed analysis (EDA) is routinely used for screening of environmental 56 contaminants occurring in soil, water and air [START_REF] Giesy | Cell bioassays for 320 detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in 321 environmental samples[END_REF], Vondracek et al. 2001) and it 57 should also be possible to use it as a pre-screening method to test smoked meat samples, 58 contaminants and the potency of both known and unknown ingredients. Such a pre-screening 59 method can handle a high sample throughput and analyse large numbers of samples for the 60 presence of potentially toxic compounds, thus improving food monitoring and consumer 61

safety. 62

In a previous study we showed that higher molecular weight PAHs, particularly B[a]P 63 and dibenzo[a,h]anthracene (DBA), could be detected by the CALUX assay (Kuhn et al. 64 2008). The potency of the meat samples to cause a defined biological response mediated by 65 the AhR activity of the PAHs present in the sample was expressed relative to the response of 66 the two standards, benzo[k]fluoranthene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 67 [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF]. The biological potency of the contaminated meat samples was expressed 68 as induction equivalents (IEQs). 69

The aim of our present study was to apply a practical approach using an effect-based 70 screening assay under real-life conditions with custom-made meat samples with different 71 degrees of PAH accumulation. The robustness of this method was investigated both in terms 72 of the influence of smoking conditions (temperature and duration) on PAH levels and in 73 regard to their distribution in the smoked meat sample. The robustness of the bioassay was (for 3 or 6 h) and products smoked hot (for 1 or 3 h). The raw pork bellies were deboned and 82 the meat was rubbed with a mixture of curing salt and white pepper to yield final 83 concentrations in the meat of 2% and 0.2%, respectively. The bellies were packed in 84 polyamide-polyethylene foil under vacuum and stored for two weeks at 7 °C. The salt was 85 removed by washing and the bellies were dried for two days at 7 °C. Two types of indirect 86 smoking were applied: cold (18 °C) and hot (65 °C). Smoke was generated in a separate 87 chamber from which the smoke was passed onto the meat. The pork bellies were then stored 88 at 7 °C for four days before sampling. The unsmoked pork bellies were prepared (deboned, 89 rubbed, packed, washed) and stored in the same way and served as negative controls. 90

According to [START_REF] Toth | Polycyclische Kohlenwasserstoffe in geräuchertem Schinken und Bauchspeck[END_REF] PAH accumulation takes place in the outer part of a 91 meat sample. We therefore used two sampling regions, the "middle" and "edge". The middle 92 samples were taken more than 1 cm from the edge of the pork belly. The smoke thus reached 93 the pork belly only from the top and the bottom, but not from the sides. The edge samples 94 were taken from the outer 1 cm of the pork belly. 95

All 30 samples of pork belly (100 g each) were minced and blended in a chopper 96 (Rondo 1000, Tefal, Offenbach, Germany), packed in aluminium boxes, frozen and stored at 97 -18 °C until the extractions were performed. 98

Five 10-g aliquots (four for the bioassay and one for the GC-MS) were collected from 99 each of the 30 pork belly samples and prepared for further analysis. Although a recently 100 published study [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF] showed that one aliquot would be sufficient for meat 101 monitoring, we prepared more to ensure reproducibility of the bioassay. All aliquots were 102 Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany, which also 120 performed the GC-MS analyses on an Agilent 6890N GC with an Agilent 5973N MSD 121 (Agilent, Santa Clara, CA, USA). The analyses were made with slight modifications 122 according to a previously published procedure implementing an isotope dilution technique 123 (Grimmer and Bohnke 1975;[START_REF] Grimmer | Atmospheric emission of polycyclic aromatic 329 hydrocarbons in sampling areas of the German Environmental Specimen Bank. Method 330 for the precise measurement of gaseous and particle-associated polycyclic aromatic 331 hydrocarbons in the sub-nanogram range using deuterated internal standards[END_REF]

Sample extraction and cleanup procedure 128

The methodology used for the extraction of the PAH of the pork belly samples and for the 129 cleanup procedure has been described previously [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF]. Briefly, this includes an 130 alkaline hydrolysis followed by a two-step liquid-liquid partition with cyclohexane and N,N-131 dimethylformamide and a final cleanup through a silica gel column. Prior to use, all glassware 132 and equipment were rinsed with acetone and cyclohexane. 133

The purified extract was further concentrated under a gentle stream of N 2 , and 100 µl of 134 dimethylsulfoxide were added just prior to the complete evaporation of the solvent. Samples 135 intended for analysis by GC-MS were dissolved instead in 1 ml of a mixture of cyclohexane 136 and toluene (9:1, vol/vol). The samples were concentrated and an aliquot was used for GC-137

MS analysis. 138

The CALUX bioassay 139 A rat hepatoma cell line (H4IIE), stably transfected with the luciferase reporter gene plasmid 140 pGudLuc1.1 [START_REF] Garrison | Species-specific 317 recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-318 p-dioxin-like chemicals[END_REF], was provided by Prof. Dr. A. Brouwer (BDS, Amsterdam, 141

The Netherlands). All media and supplements for cell culture were purchased from PAA 142 (Cölbe, Germany). As described previously [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF]), the cells were grown at a 143 density of 30,000 per well in 96-well culture plates (Nunc, Wiesbaden, Germany) in 200 µl 144 DMEM/F-12 with 10% FBS (Mycoplex®, PAA) and 1% glutamine. The cell layer reached 145 90% confluence 24 h after seeding and 50 µl of culture medium containing the test extracts or 146 the standard dilutions were added to the medium in the wells. Exposures were performed in 147 quadruplicate. Solvent control, TCDD calibration standards (between 0.3 pM and 300 pM) 148 and one pork belly sample with a known grade of PAH contamination were included on each 149 well plate. The final concentration of DMSO in each well was 0.1% (vol/vol). 150

The measurement of the IEQ relative to the TCDD standard was carried out as 151 previously described [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF]. In brief, the cells were harvested after 6 hours of 152 incubation in cell lysis reagent and transferred to a white 96-well plate (Luminunc, Nunc, Wiesbaden, Germany). Luminescence was measured using a multi-label Mithras LB 940 154 reader (Berthold, Bad Wildbad, Germany) immediately after automatic injection of assay 155 buffer and luciferin solution. Results were converted into x-fold induction of the mean of the 156 negative samples (the control). The sigmoid dose-response standard curve was fitted using 157 this ligand binding function of SigmaPlot 9.01 (Systat, San Jose, CA, USA): 158

Hillslope x EC x f ) (log 50 10 1 min max min ) ( - + - + = 159
The extracts were interpolated on this curve. Results were expressed as TCDD-IEQ and 160 converted into nanograms per kilogram of meat. The limit of detection was set to the DMSO 161 response plus three times the standard deviation (SD); the limit of quantification (LOQ) was 162 set to the DMSO response plus ten times SD. 163

Statistical evaluation 164

As described recently [START_REF] Kuhn | Determination of 336 polycyclic aromatic hydrocarbons in smoked pork by effect-directed bioassay with 337 confirmation by chemical analysis[END_REF], certain parameters were calculated and evaluated in 165 every assay for quality assurance: curve fitting was carried out with R² greater than 0.985. 166

The results of a culture plate were excluded if the results for the 3-and 10-pM dilutions 167 from 3% to 37%). The pork belly samples were extracted four times, and the reproducibility 180 (mean coefficient of variation of the IEQ) was 24% (3% to 55%). 181

We manufactured a total of 30 pork belly samples: 5 unsmoked, 13 cold smoked (for 3 182 to 6 h) and 12 hot smoked (for 1 to 3 h). The total number of samples includes four from the 183 preliminary trial (two cold smoked for 1 or 3 h and stored not in aluminium boxes but in 184 plastic boxes and two hot smoked for 1 h and additionally cold smoked for 1 h). Those four 185 samples are included in the overview presented in table 1, but not in the more detailed 186 presentation in the figures. 187

The effect-based bioassay evaluates the total activity of the specific compounds in the 188 meat samples interacting with the AhR. The induction equivalents measured by effect-based 189 analysis correspond to the degree of smoking. There were significant differences in the 190 averaged bioassay results (p < 0.01) between the three smoking procedures: unsmoked (28 ng 191 TCDD-IEQ/kg), cold smoked (248 ng TCDD-IEQ/kg) and hot smoked (661 ng TCDD-192 IEQ/kg). This bioassay thus basically categorises the pork belly samples according to 193 smoking technique. The maximum bioassay value was 52 ng TCDD-IEQ/kg for the 194 unsmoked meat samples; 429 for the cold smoked samples and 1,346 for the hot smoked 195 samples. Figure 1 shows the mean IEQ and the median of the pork belly samples including 196 the distribution according to smoking time. The average TCDD-IEQ for samples cold smoked 197 3 h was 221; for samples cold smoked 6 h, 282; for samples hot smoked for 1 h, 476; and for 198 those hot smoked for 3 h, 1,093. Significant differences (p < 0.05) were found between 199 unsmoked samples and all smoked samples and between those hot smoked for 1 h and those 200 hot smoking led to a significant increase in IEQ levels (p < 0.05). 203

PAHs are generated as complex mixtures and their profiles in food are variable. Table 1 204 shows the levels of 31 individual PAHs in these pork belly samples, including the 16 "EFSA- substantially to the overall PAH-induced AhR-mediated activity [START_REF] Machala | Aryl hydrocarbon receptor-341 mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in 342 vitro reporter gene assay[END_REF]. For 236 this reason, those PAHs were the focus of this study. Figure 3 shows the amounts of these 237 compounds in the meat samples. The increases in the individual compounds depending on 238 smoking technique are obvious, while the relative proportions of the individual PAHs 239 remained relatively constant at every treatment level. However. it is questionable to apply 240 conclusions drawn for one PAH to others. A better tool for food risk monitoring and analysis 241 is the estimation of the total carcinogenic potential of a smoked meat sample. 242

We determined the distribution of PAHs in the meat samples more precisely by 243 subdividing the pork belly samples according to sampling location. Figure 4 shows the 244 induction equivalents of the two types of cut meat samples: those taken from the middle and 245 from the edges of the pork belly. There were no significant differences (p = 0.6) in the 246 unsmoked samples depending on the sampling zone. The differences between the two 247 sampling zones became more pronounced with increasing smoking temperature and duration. 248

The induction equivalents detected for the edge zone samples differed significantly (p < 0.01) 249 from those of the middle zone when the samples were subjected to hot smoking for one hour. 250 There appeared to be an even more pronounced difference between sampling zones between 251 the meat samples subjected to hot smoking for three hours, but no statistical significance 252 In summary, it appears that cold smoking of up to six hours did not cause strong 255 contamination of the pork belly. In fact, there was no indication of any influence of cold 256 smoking for up to six hours, but the results for hot smoking at 65 °C were very different. In 257 particular, the duration of hot smoking seems to have substantially influenced the level of 258 PAH contamination, which was clearly detected by the screening test. These findings are 259 supported by those of Lijinsky [START_REF] Lijinsky | Production of Carcinogenic Polynuclear Hydrocarbons in the 339 Cooking of Food[END_REF] striking results were found for the samples smoked hot for three hours: here, the IEQs 279 determined by bioassay were noticeably higher than the calculated theoretical values so that 280 true positive results are easily detectable. 281

In conclusion, the results of our study provide further confirmation that the CALUX 282 bioassay is a practicable pre-screening method for detecting AhR-active compounds such as 283

PAHs in smoked meat. The toxic potential of the total meat sample can thus be determined 284 using this method, although the specific agents still would have to be confirmed by chemical 285 analysis. Large numbers of samples can thus be pre-screened and analysed for potential non-286 compliant samples. Further chemical analyses could then be limited to those samples. Effect-287 based screening tests like this bioassay have been proven to be sensitive in a range much 288 lower than the European threshold value for B[a]P of 5 µg kg-1 smoked meat and also lower 289 than the former German threshold value of 1 µg kg-1. This study lends further support to the 290 conclusion that effect-based analysis applied as pre-screening method is suitable for food 291 monitoring and risk control. 292 "Material and Methods". All samples were prepared in the same manner; then some were 369 cold smoked at 18 °C (for 3 or 6 hours), hot smoked at 65 °C (for 1 or 3 hours), or remained 370 unsmoked (LOQ = 6 ngkg -1 TCDD-IEQ) 371

Figure 2: Mean (column), SD and median (cross) of total 16 EFSA-priority PAHs in 26 372 samples of pork belly, determined with GC-MS as described in "Material and Methods". All 373 samples were prepared in the same manner; then some were cold smoked at 18 °C (for 3 or 6 374 hours), hot smoked at 65 °C (for 1 or 3 hours), or remained unsmoked. 375 [START_REF] Machala | Aryl hydrocarbon receptor-341 mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in 342 vitro reporter gene assay[END_REF]

  analysis of 31 individual PAHs which were determined in every meat 75 sample by gas chromatography-mass spectrometry (GC-MS)

  same manner, with particular care taken to assure equal reaction and storage -dimethylformamide, spectroscopic grade acetone (CAS No. 67-64-1; 107 Merck, Darmstadt, Germany) and Pestilyse® grade methanol (CAS No. 67-56-1; Roth, 108 Karlsruhe, Germany). TCDD (CAS No. 1746-01-6; LGC Promochem Wesel, Germany) was 109 dissolved in spectrophotometric grade dimethylsulfoxide (DMSO, CAS No. 67-68-5; Acros 110 Organics, Geel, Belgium). The final concentration of TCDD was 300 nM. Silica gel 60 (0.06 111 -2 mm) was purchased from Roth (Karlsruhe, Germany). All other chemicals used were of 112 high purity grade. 113 For the identification and quantification of individual PAHs, an internal standard 114 mixture was added at the beginning of the extraction to the samples intended for GC-MS 115 analysis. This mixture included the deuterated or fluorinated PAHs d 8 -naphthalene, d 8 -116 acenaphthylene, d 10 -acenaphthene, 2-fluorofluorene, d 10 -phenanthrene, d 10 -fluoranthene, d 10 -117 pyrene, d 12 -benzo[a]anthracene, d 12 -benzo[b]fluoranthene, d 12 -benzo[a]pyrene, indeno[1,2,3-118 cd]pyrene and d 12 -benzo[ghi]perylene. The internal standard mixture was provided by the 119

  and carried out in the single-ion 124 monitoring (SIM) modus; quantification was obtained by comparing the area responses of the 125 target ions in relation to the area responses of the internal standards after a three-point

SCOOP

  deviated from the expected values by more than three times SD or if those of one known meat 168 extraction sample deviated by more than two times SD. 169 All calculations were made using Microsoft Excel 11.0 (Microsoft, Redmond, WA, 170 USA) and SAS 8.02 (SAS Institute Inc., Cary, NC, USA) software. 171For PAH levels determined by GC-MS to be less than LOQ, we used half of the LOQ 172 value for statistical calculations as recommended by the task group for scientific cooperation 173

  Additionally, the median is marked in Figure1. It is notable that the duration of 201 cold smoking seemed to have little effect on the results of the bioassay, whereas one hour of 202

  in the levels of the lower molecular weight PAHs depending on 227 different smoking treatments and found significant differences (p < 0 01) for fluorene and 228 anthracene, but this was not the case for acenaphthene, phenanthrene, fluoranthene and 229 pyrene. This supports the hypothesis that determination of the lower molecular weight PAHs 230 in smoked meat products may not be useful for risk analysis. 231 The SCF has classified B[a]P as a marker compound for the presence and 232 carcinogenicity of PAHs in food (European Union 2002) based on the fact that B[a]P levels 233 remain fairly constant in relation to those of B[a]A, benzo[b+j+k]fluoranthene (B[b+j+k]F), 234 indeno[1,2,3-cd]pyrene (IP) and DBA. With the exception of B[a]A, these PAHs contribute 235

  here due to the small number of samples. This unusual technique was 253 used only once in order to induce a high PAH contamination. 254

  and by the EFSA statistical report of 260 2007, which showed increased concentrations of B[a]P with increasing temperatures 261 (European Union 2007). The EFSA also reported low B[a]P levels resulting from the use of 262 liquid smoke or from filtering the smoke before application (European Union 2007). The 263 EFSA found no correlation between smoking time and B[a]P levels (European Union 2007). 264 This was most likely due to the combined effects of smoking time and smoking temperature, 265 as cold smoking is normally carried out for a much longer time than hot smoking. 266 Machala et al. (2001) cited two types of induction equivalent factors (IEFs) of PAHs 267 relative to TCDD as standard substance in the CALUX assay. Those investigators derived 268 IEFs from EC 50 or EC 25 values in 6-h exposure assays. Figure 5 shows the IEQs we 269 determined in relation to the theoretical IEQs of the smoked meat samples, which are 270 calculated using the GC-MS values of the samples and the IEFs of 17 PAHs cited by Machala 271 (fluorene, anthracene, fluoranthene, pyrene, B[a]A, B[b]F, B[k]F, B[j]F, B[a]P, DBA, IP, 272 benzo[e]pyrene, chrysene, benzo[ghi]perylene, cyclopenta[c,d]pyrene, 5-methylchrysene, 273 benzo[c]phenanthrene). It is striking that the IEQs determined from the bioassay of the 274 unsmoked samples were lower than the calculated IEQs; these products can thus easily be 275 considered safe and the likelihood of false positive results is minimal. On the other hand, our 276 results for the cold smoked and the samples smoked hot for 1 hour are in good agreement 277 with the IEQs calculated using Machala's induction equivalent factors. However, the most

Figure 1 :

 1 Figure 1: Mean (column), SD and median (cross) of TCDD -induction equivalents (TCDD-367

Figure 3 :Figure 4 :Figure 5 :

 345 Figure 3: Content and SD of 7 PAHs in 26 samples of pork belly, determined with GC-MS: 376

  ), SD and median (cross) of TCDD -induction equivalents (TCDD-IEQ) of 26 samples of pork belly, determined with the CALUX bioassay as described in "Material and Methods". All samples were prepared in the same manner; then some were cold smoked at 18 °C (for 3 or 6 hours), hot smoked at 65 °C (for 1 or 3 hours), or remained unsmoked (LOQ = 6 ngkg-1 ), SD and median (cross) of total 16 EFSA-priority PAHs in 26 samples of pork belly, determined with GC-MS as described in "Material and Methods". All samples were prepared in the same manner; then some were cold smoked at 18 °C (for 3 or 6 hours), hot smoked at 65 °C (for 1 or 3 hours), or remained unsmoked. 194x143mm (72 x 72 DPI) Content and SD of 7 PAHs in 26 samples of pork belly, determined with GC-MS: benzo[a]anthracene (B[a]A), total benzo[b+j+k]fluoranthene (B[b+j+k]F), benzo[a]pyrene (B[a]P), indeno[1,2,3cd]pyrene (IP) and dibenzo[a,h]anthracene (DBA). 213x133mm (72 x 72 DPI) (TCDD-IEQ) and SD of 26 samples of pork belly, determined with the CALUX bioassay. Samples were taken from both the middle (left column) and the edges (right column) of the pork belly after the smoking process. There were significant (p < 0.01) differences between sampling locations for the samples hot smoked for 1 h (LOQ = 6 ngkg-1 TCDD-IEQ) 213x137mm (72 x 72 DPI) Mean and SD of calculated IEQs, using the contents measured with GC-MS and the induction equivalent factors (IEFs) of 17 PAHs published by Machala et al. 2001, who derived IEFs from EC50 or EC25 values in 6 h exposure assays with CALUX. Comparable to them, the means and SD of measured IEQs are shown. 213x106mm (72 x 72 DPI)

Table 1 .

 1 Mean, median and maximum levels (µg kg -1 ) of 31 PAHs, measured with GC-MS as described in "Material and Methods" in 30 pork belly samples: 5 unsmoked, 13 cold smoked (for 3 or 6 h) and 12 hot smoked (for 1 or 3 h). EFSA-priority list PAHs are indicated with an asterisk.

	, who

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uk Food Additives and Contaminants

Polycyclic aromatic hydrocarbons (PAHs) are a widespread group of environmental

contaminants, many of which, including the representative compound benzo[a]pyrene

(B[a]P), are known to exert teratogenic, mutagenic and carcinogenic effects(Guillen and

[START_REF] Guillen | Polycyclic Aromatic Hydrocarbons in Diverse Food[END_REF][START_REF] Shaw | Prediction and monitoring of the carcinogenicity of polycyclic 346 aromatic compounds (PACs). (Springer). 347 polycyclic aromatic hydrocarbons and polychlorinated biphenyls in genetically engineered 351 C57BL/6J mice[END_REF]. PAHs in food may result from their uptake from a contaminated

in an FPC100 smoking chamber (Fessmann, Winnenden, Germany) using beech wood

shavings. Five types of samples were produced: unsmoked products, products smoked cold

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The repeatability of the CALUX bioassay (variability of extraction samples analysed in the

samples was calculated from at least four assays in independent runs (different days, different

plates); the average variation coefficient between the results of these plates was 16% (ranging

hydrocarbons in smoked food products and commercial liquid smoke flavourings. Food

Addit.Contam. 10(5):503-521.

protein foods, oils and fats by gas chromatography. J Assoc Off Anal Chem. 58(4):725-

dependent induction of liver and lung cytochromes P450 1A1, 1A2, and 1B1 by

Acknowledgements


Mariusz Rudy
email: mrudy@univ.rzeszow.pl

Correlation of lead, cadmium and mercury levels in tissue and liver samples with age of cattle

Keywords: Heavy metals, Heavy metals -arsenic, Heavy metals -cadmium, Heavy metals -mercury Food Types: Animal, Meat beef, heavy metals, coefficients of correlation .manuscriptcentral.com/

Correlation of lead, cadmium and mercury levels in tissue and liver samples with age of cattle

Mariusz Rudy

Introduction

Among farm animals the ruminants are most exposed to contamination of environment by heavy metals owing to the system of pasture feeding of these animals in the summer period and hay feeding in the winter period. Metals penetrate into grasses most often from the soil and are also present in hay fodder. The differences in the level of contamination of internal organs and muscles by heavy metals depend, among other things, on the species and age of animals [START_REF] Hecht | Toxische Schwermetalle in Fleisch und Innereien verschiedener Tierarten[END_REF][START_REF] Krełowska-Kułas | Zanieczyszczenia chemiczne surowców i Ŝywności pochodzenia zwierzęcego[END_REF][START_REF] Studziński | StęŜenie ołowiu, kadmu, miedzi i cynku w wątrobie, nerkach, mięśniach szkieletowych i mózgowiu jagniąt i owiec dorosłych[END_REF]) and also on the degree -2 -of contamination in the region where the animals were bred and their feeds [START_REF] Barghigiani | Mercury levels in agricultural products of Mt. Amiata (Tuscany, Italy)[END_REF][START_REF] Sikora | Kadm i ołów w mięśniach oraz wątrobie królików z terenu aglomeracji krakowskiej[END_REF][START_REF] Śebrowska-Rasz | Zanieczyszczenia chemiczne w tkankach zwierząt i Ŝywności pochodzenia zwierzęcego[END_REF]. The degree of contamination by heavy metals of the tissue of rabbits [START_REF] Sikora | Kadm i ołów w mięśniach oraz wątrobie królików z terenu aglomeracji krakowskiej[END_REF] and sheep [START_REF] Studziński | StęŜenie ołowiu, kadmu, miedzi i cynku w wątrobie, nerkach, mięśniach szkieletowych i mózgowiu jagniąt i owiec dorosłych[END_REF], has been studied among three age groups. Other researchers [START_REF] Hecht | Toxische Schwermetalle in Fleisch und Innereien verschiedener Tierarten[END_REF][START_REF] Krełowska-Kułas | Zanieczyszczenia chemiczne surowców i Ŝywności pochodzenia zwierzęcego[END_REF][START_REF] Barghigiani | Mercury levels in agricultural products of Mt. Amiata (Tuscany, Italy)[END_REF][START_REF] Śebrowska-Rasz | Zanieczyszczenia chemiczne w tkankach zwierząt i Ŝywności pochodzenia zwierzęcego[END_REF] have been concerned with contamination by heavy metals of tissues of other species of animals, but did not include the influence of age on the level of accumulation in tissues. In practice cattle are slaughtered more often at the age from 1,5 to 2 years but nevertheless, meat is also obtained from a fewyear-old or even several-year-old animals (e.g. cows).

The aim of our research was to determine the level of accumulation of selected heavy metals (Pb, Cd, Hg, As) in meat and liver of cattle. The animals were divided into four age groups which allowed the analysis of statistical-mathematical correlations between the age of the animals and contamination of meat and liver by these elements. Additionally, the content of the above-mentioned toxicants in tissues of these animals slaughtered in the years 2002 -2006, presented in the paper, allows assessment of the degree of contamination by these elements during this period.

Materials and methods

The research was carried out in the years 2002-2006 on cattle in the region of south-eastern Poland (the areas of Podkarpackie and Lubelskie provinces), coming from the farming areas, with majority of small scale commodity production, practically with no industry. The region of south-east Poland has very good conditions for production of food of high health and nutritional value, since as much as 95% of soils are of the first class purity in respect of the content of heavy metals [START_REF] Sucha | Stan środowiska w województwie rzeszowskim w 1993 roku Wojew. Inspekt. Ochrony Środ[END_REF]. The concentration of the heavy metals, both in soil as well as in drinking water of this region was within the standard.

For the research there were selected clinically healthy animals, of similar slaughter weight within an age group. The cattle came from individual farms and were fed traditionally (with farm's own fodder).

The slaughter of cattle was carried out in one of meat plants of south-eastern Poland in accordance with the methodology applied by the meat industry. The samples for tests were Group I -young slaughter cattle from 1.5 to 2 years, Group II -middle cattle from 2 to 6 years, Group III -older cattle from 6 to 12 years, Group IV -old cattle over 12 years Samples for determining the content of heavy metals, i.e. arsenic, cadmium, lead and mercury was taken (at least at 100 g from the same part of each tissue) from a part of the longissimus muscle of back (m. longissimus dorsi) at the level of the last thoracic vertebrae, and samples of liver from the tail lobe. The taken material was lyophilized (FreeZone6L, Labconco) and homogenized .

Samples were mineralized with pressure-microwave method (mineraliser MARS XPRESS, CEM Corporation) in the presence of the mixture of nitric acid and hydrogen peroxide (6/1) according to PN-EN 13805:2003. Simultaneously the whole procedure was carried out for a reagent blanks. The digested solutions were diluted (in such a way so that the measured value of concentration of a given element should be possibly in the centre of the operating range) and there were determined in them: As by atomic absorption spectrometry with generation of hydrides HGAAS according to PN-EN 14627:2005, Pb and Cd by atomic absorption spectrometry with atomization in graphite furnace FGAAS (SpectrAA 880Z, Varian) according to PN-EN 14084:2004, Hg by cold-vapours with the method of atomic absorption spectrometry according to PN-EN 13806:2003. The content of heavy metals in tissues was expressed in mg/kg of fresh mass, taking into account all quantitative processes during preparation of the material and considering reagent blanks .

The blanks were in contact with homogenizer, equipment and containers for similar time as samples. A strict analytical quality control programme was employed during the study, the results of which are presented in table 1. The limits of detection, expressed as a concentration Results were subjected to statistical-mathematical analysis which included:

1. Calculation of basic statistical measures of position (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).

Calculation of the values of coefficients of simple correlation for the purpose of

determining the correlation between the content of examined metals in meat and liver of cattle.

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

Results and discussion

Variability of the chemical composition of beef depends on the sex, degree of musculature and de-fatting of carcasses [START_REF] Salvini | w:] Banca dati di composizione degli alimenti per studi epidemiologici in Italia[END_REF][START_REF] Williams | Effect of production systems on performance, body composition and lipid and mineral profiles of soft tissue in cattle[END_REF]) and on age, breed or category of cattle [START_REF] Florek | The quality of meat from carcasses of young bulls and heifers classified according to the EUROP system[END_REF][START_REF] Litwińczuk | Variability of beef chemical composition with regard to some factors determining it. Part II. Protein and fat content[END_REF].

From among farm animals, cattle are the species most susceptible to the effect of heavy metals, especially lead -the element which often is the cause of poisoning with these animals [START_REF] Monkiewicz | Rozmieszczenie ołowiu w tuszy dzików w zaleŜności od odległości od rany postrzałowej[END_REF][START_REF] Szkoda | StęŜenia pierwiastków śladowych w tkankach bydła w Polsce[END_REF]. Susceptibility of cattle to the toxic effect of Pb results, among other things, from a long period of pasture feeding during the year.

Particularly dangerous is pasturing cows in roadside ditches due to considerable contamination of their flora with lead. The quantities of metals (Cd, Pb, Hg and As) in muscles and internal organs of farm animals provide an accurate reflection of the level of these elements in the environment of the animals' breeding and confirm usability of cattle as test organisms of contamination of the production environment [START_REF] Krupa | Badania bioakumulacji toksycznych metali cięŜkich w mięśniach i narządach wewnętrznych zwierząt gospodarskich z południowo -wschodniego makroregionu Polski[END_REF].

In table 2 the content of heavy metals in tissues of cattle in the years 2002-2006 is given. The data show that the content of cadmium in a muscle fluctuated from 0.012 mg/kg in 2002 to 0.018 mg/kg in 2005, a 50% increase (differences statistically significant P≤0.05). The content of mercury both in the muscle as well as in the liver in this period did not exceed 0.001 mg/kg. In the tissues of cattle the presence of arsenic was not detected. In turn, the (table 2) were twice the average concentration of domestic level -0.027 mg Pb/kg, determined in 1990 for muscles of this species of animals by śmudzki et al. (1991). Also the average content of Pb in the liver of cattle in the tested population in all analyzed years turned out to be higher than the level of 0.135 mg/kg determined by these authors (table 2). In the case of the content of mercury, both in muscles as well as in the liver, higher values (about 0.003 mg/kg) than in this paper were obtained by other authors [START_REF] Krupa | Badania bioakumulacji toksycznych metali cięŜkich w mięśniach i narządach wewnętrznych zwierząt gospodarskich z południowo -wschodniego makroregionu Polski[END_REF][START_REF] Szkoda | StęŜenia pierwiastków śladowych w tkankach bydła w Polsce[END_REF].

The content of heavy metals in tissues of cattle of different age are presented in table 3. The data show that the content of lead in meat of cattle was similar in groups I and II and fluctuated around 0.051 -0.054 mg/kg. The quantity of this element increased in comparison with group I by 23% in group III and by 47% in group IV. Statistically significant differences were confirmed between groups I and IV, II and IV, and also I and III. However, the content of cadmium in meat (0.009 mg/kg) was the lowest in group I and increased in successive age groups respectively by: 44%; 55% and 133% (high correlation r=0.67). -6 -Statistically significant differences were confirmed only for groups I and IV. In turn, the concentration of mercury in meat and in the liver in particular age groups was lower than 0.001 mg/kg. In the analyzed age groups of cattle there was not stated the presence of arsenic in meat or in the liver. In the case of the content of lead and cadmium in the liver the lowest level was stated in group I respectively: 0.125 mg/kg and 0.123 mg/kg. The concentration of these metals in the liver increased together with age of animals respectively by: 3%; 15% -in group II, 13%; 64% -in group III, and 43%; 126% -in group IV (very high correlation r≥0.80). It should be also noted that for cadmium in the liver there was exceeded the permissible level (0.5 mg/kg) in two samples in group IV and in one case in group III.

In the research of [START_REF] Krupa | Pozostałości pestycydów i niektórych metali cięŜkich (Cd, Pb, As i Hg) w tkankach bydła[END_REF] concerning the concentration of cadmium in tissues of cattle of the region of south-eastern Poland the differences in quantities of this metal in muscles and organs of cows and young cattle turned out to be statistically irrelevant. The level of accumulation of Cd in tissues of young cattle and cows at that time was respectively: in muscles 0.016 and 0.021 mg/kg, in the liver 0.262 and 0.331 mg/kg. And in kidneys it was significantly higher than in muscles and in the liver, constituting respectively: 0.836 and 1.212 mg/kg. In other research of this author [START_REF] Krupa | Badania bioakumulacji toksycznych metali cięŜkich w mięśniach i narządach wewnętrznych zwierząt gospodarskich z południowo -wschodniego makroregionu Polski[END_REF]) on bulls of the age of 18 -22 months there were obtained higher values of the content of cadmium in meat (0.023 mg/kg), and also of the content of mercury in meat (0.003 mg/kg) and in the liver (0.0037 mg/kg) than in this paper (table 3) for the same age group of animals. In turn, this author obtained in cattle smaller quantities of lead in the liver (0.077 mg/kg) and the concentration of lead in meat (0.049 mg/kg) and cadmium in the liver (0.115 mg/kg) at similar level as in this paper. The same author in the research [START_REF] Krupa | Ocena zawartości niektórych metali cięŜkich w tkankach bydła i koni z regionu południowo-wschodniej Polski[END_REF][START_REF] Krupa | Pozostałości pestycydów i niektórych metali cięŜkich (Cd, Pb, As i Hg) w tkankach bydła[END_REF] concerning the assessment of accumulation of lead in tissues of young cattle and cows of the region of south-eastern Poland did not observe significant differences in respect of the quantity of Pb in muscles and kidneys, however, significantly more of this metal accumulated in livers of older specimens. During the research presented in quoted papers there also was not stated a dependence on this factor despite noting some differentiation of the level of Hg with younger and older specimens. [START_REF] Wałkuska | StęŜenie ołowiu, kadmu, miedzi i cynku w mózgowiu, płynie mózgowo-rdzeniowym, wątrobie, Ŝółci i krwi u bydła z okolic Lublina[END_REF], however, indicate definitely higher concentration of lead in the liver of young beef cattle (0.64 mg/kg) and cows (0.77 mg/kg), and lower with calves (0.018 mg/kg). [START_REF] Salisbury | Multielement concentrations in liver and kidney tissues from five species of Canadian slaughter animals[END_REF] this population the following ones respectively: 0.07 and 0.11 mg/kg Pb, and for calves a bit higher since amounting to 0.10 and 0.15 mg/kg. Similar as in this paper (table 3) content of lead (0.050 mg/kg) and cadmium (0.004 mg/kg) in meat of young slaughter cattle was obtained by [START_REF] Doganoc | Lead and cadmium concentrations in meat, liver and kidney of Slovenian cattle and pigs from 1989 to 1993[END_REF]. However, a lower quantity of these elements respectively: (0.005 -0.015 mg/kg) and (0.001 -0.002 mg/kg) in beef was obtained by other authors (Gonzales-Weller 2006;[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] Alonso | Toxic and trace elements in liver, kidney and meat from slaughtered in Galicia (NW Spain)[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][START_REF] Tahvonen | Lead and cadmium contents in pork, beef and chicken, and in pig and cow liver in Finland during 1991[END_REF]. In turn, López et al.

(2002) found a bit higher quantities of Pb, Cd and As in beef than in veal respectively: 0.017; 0.009 mg Pb/kg, 0.0014; 0.0013 mg Cd/kg and 0.005; 0.004 mg As/kg.

In the research of [START_REF] Krupa | Ocena zawartości niektórych metali cięŜkich w tkankach bydła i koni z regionu południowo-wschodniej Polski[END_REF] on concentration of arsenic in tissues of cattle there was stated its presence at the level of thousandth parts of mg As/kg. In this research there were not shown significant differences between the quantity of this element in muscles and in kidneys and age. However, this factor decided on significantly higher level of arsenic in livers of older animals (0.0097 mg/kg) than in young cattle (0.0065 mg/kg). In turn, with the cattle of Krośnieńskie and Rzeszowskie provinces examined by the same team of authors [START_REF] Krupa | Pozostałości pestycydów i niektórych metali cięŜkich (Cd, Pb, As i Hg) w tkankach bydła[END_REF], and slaughtered at the age of 13-18 months and at the age of 7-11 years, a slightly higher level of this metal was stated in muscles (0.0056 mg/kg) and in the liver (0.0112 mg/kg) of young specimens. With cows there was a bit less of arsenic, respectively: 0.0045 and 0.0086 mg/kg. The reverse dependence occurred in contamination of kidneys by this metal, in which the quantities of arsenic were higher than in meat and in the liver, with young cattle amounting on average to 0.0102 mg/kg, and in cows 0.0119 mg/kg [START_REF] Krupa | Pozostałości pestycydów i niektórych metali cięŜkich (Cd, Pb, As i Hg) w tkankach bydła[END_REF].

Correlative dependences between the content of heavy metals in tissues of cattle were presented in table 4. The data show that the biggest dependence occurred between the content of lead in the liver and in meat (r = 0.95), between the concentration of cadmium in the liver and in meat (r = 0.91), and also between the quantity of Cd and Pb in meat (r = 0.93). However, a bit lower correlative dependencies occurred in the following pairs Cd in liver -Pb in liver (r = 0.89), Pb in liver -Cd in meat (r = 0.87), and also Hg in liver -Hg in meat (r = 0.62). śarski et al. (1994) assessing the content of mercury in tissues of young cattle obtained the strongest relation between the content of Hg in liver and muscles (r = 0.62). [START_REF] Krupa | Badania bioakumulacji toksycznych metali cięŜkich w mięśniach i narządach wewnętrznych zwierząt gospodarskich z południowo -wschodniego makroregionu Polski[END_REF], however, stated that in tissues of cattle the significant correlations occurred most often between the content of cadmium and lead and the content of mercury and -8 -arsenic. From among the essential coefficients of correlation the highest correlation (r from 0.80 to 0.87) was obtained by this author between the content of lead and cadmium in muscles. In turn, he noted a significant but negative correlation between the content of cadmium and the content of mercury in tissues of cattle (r = from -0.40 to -0.48).

Conclusions

The contamination of meat of cattle by Cd and Pb clearly depends on the age of these animals. In muscles and in the liver of the animals arsenic was not detected above 0.001 mg/kg. However, the average concentration of mercury in these tissues was similar (≤0.001 mg/kg) in all age groups of cattle. Three samples exceeded the maximum permissible concentration of cadmium in the liver of cattle of age groups III and IV. The livers from these age groups should be used for consumption only to a limited extent. Between the content of lead and cadmium in the liver and the concentration of these elements in muscles there occurred very high positive correlative dependence. nd -presence not stated:

-As over 0.001 mg/kg; * -differences significant at P≤0.05.

** -differences significant at P≤0.01.

  . Each year of research used 20 head of cattle (5 years x 20 samples of meat and 20 samples of liver = 100 samples of meat and 100 samples of liver). The age of animals was determined on the basis of teeth and purchase documentation. Cattle of black-and-white lowland breed (pre-slaughter weight 480 -600 kg), in each age group (4 groups x 20 samples of meat and 20 samples of liver = 80 samples of meat and 80 samples of liver) were divided into 4 age groups:

  , 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, arsenic and NIST -RM 8414 bovine muscle powder from the National Institute of Standards and Technology for the determination of mercury.

  content of Pb in beef liver in the years 2002-2004 was about 0.147 mg/kg. The concentration of this element in the liver slightly increased in 2005 by about 6% and by 25% in 2006 (however, differences statistically significant P≤0.05 occurred only between the years 2002 and 2006). The situation was a bit different in the case of concentration of Cd in the beef liver. The content of this metal slightly increased in successive years. It should be also noted that the quantity of this element exceeded the permissible level 0.5 mg/kg (Commission Regulation 466/2001/EC) in as many as four samples of liver. The average concentrations of lead in muscle of cattle in the years 2002 -2006

  , assessing in the years 1982-1989 in this respect the livers and kidneys of young fattening beef cattle in Canada, present as average values for

Table 1 .

 1 Results of the analytical quality control programme used in the determination of cadmium (Cd), lead (Pb), mercury (Hg) and arsenic (As) in bovine samples

	Page 11 of 14		Food Additives and Contaminants
	Quality characteristics	Pb	Cd	As	Hg
	Detection limit in
	samples (mg/kg)
	Muscle	0.006	0.001	0.001	0.0001
	Liver	0.012	0.002	0.001	0.0001
		Certified Reference Material BCR -185R (Bovine Liver)		NIST -Reference Material 8414 (Bovine Muscle Powder)
	Measured mean ± SD (mg/kg)	0.163 ±0.008	0.526 ± 0.019	0.029 ± 0.003	0.0045 ± 0.0005
	Certified mean ± SD (mg/kg)	0.172 ± 0.009	0.544 ± 0.017	0.033 ± 0.002	0.005 ± 0.003
	Recovery (%)	94.8	96.7	87.9	90.0
	Relative standard deviation (%)	5	4	10	11

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Table 2 .

 2 The content of heavy metals in meat and liver of cattle in the years2002-2006 (mg/kg)

	Specification	x	2002 x min	x max	x	2003 x min	x max	x	2004 x min	x max	x	2005 x min	x max	x	2006 x min	x max	The significance of differences
	Meat:
	Pb	0.060	0.047	0.069	0.062	0.052	0.072	0.062	0.051	0.073	0.066	0.057	0.089	0.060 0.041 0.086
	Cd	0.012	0.006	0.015	0.015	0.007	0.021	0.016	0.007	0.019	0.018	0.009	0.031	0.015 0.005 0.034	2002<2005*
	Hg	<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
	As	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
	Liver:
	Pb	0.147	0.079	0.209	0.148	0.083	0.271	0.148	0.071	0.302	0.156	0.087	0.389	0.183 0.097 0.445	2002<2006*
	Cd	0.159	0.024	0.434	0.172	0.098	0.478	0.189	0.121	0.594	0.200	0.098	0.558	0.191 0.082 0.378
	Hg	<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 0.002
	As	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd	nd
	Explanation:
	nd -presence not stated:
	-As over 0.001 mg/kg;

* -differences significant at P≤0.05. Page 12 of 14 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y

Table 3 .

 3 The content of heavy metals in meat and liver of cattle of different age (mg/kg)

	Groups

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