Keywords: Chromatography -HPLC, Screening -microbial screening, Screening assays Additives/Contaminants: Veterinary drug residues -sulphonamides sulphamethazine, residues, detection, rabbit tissues, microbial inhibition tests, HPLC

The aim of the present study was to evaluate three microbial inhibition tests (MIT) based on the inhibition of the growth of the test organism, (a) the Four Plate Test (FPT) containing Bacillus subtilis BGA; (b) the Screening Test for Antibiotic Residues (STAR) containing Bacillus stearothermophilus var. calidolactis ATCC 10149, and (c) the Premi ® Test containing Bacillus stearothermophilus var. calidolactis. The tests were used for determining sulphamethazine (SMZ) residues in edible tissues of rabbits after its oral administration up to day 15 of the withdrawal period (WP). A solvent extraction procedure was used to enhance the capability of the tests to detect SMZ residues at or below maximum residue limit (MRL), and para-aminobenzoic acid (PABA) was employed to identify SMZ residues already in the first stage of the residue screening. The presence of SMZ residues in the samples was confirmed and quantified by a validated HPLC method. The Premi ® Test detected SMZ residues in the muscle and heart tissue up to day 9 of the WP and in the liver, lungs and kidneys up to day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits up to day 8 of the WP. The kidneys were positive up to day 5 of the WP, the liver until day 4 of the WP and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. By using the solvent extraction procedure, SMZ residues were detected in the muscle extract up to day 10 of the WP and the muscle was positive until day 6 of the WP.

No detection sensitivity was observed by using the FPT. After the solvent extraction, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscle was positive until day 3 of the WP. No positive results were detected after the addition of PABA into/onto the agar medium. PABA at the concentration of 10 µg.ml -1 completely reversed the inhibitory activity of SMZ and enabled reliable identification of SMZ in the examined samples. By using the HPLC, SMZ was detected in the muscle samples until day 10 of WP (0.02 mg.kg -1 ) and in the liver until day 12 of the WP (0.09 mg.kg -1 ). The results obtained by the HPLC method and the limit of detection (LOD) of screening tests for SMZ (FPT 0.4 µg.ml -1 , STAR 0.2 µg.ml -1 , Premi ® Test 0.05 µg.ml -1 ) allowed us to state that the most suitable screening tests for the detection of SMZ residues in the edible tissues of rabbits at the level corresponding to MRL of 0.1 mg.kg -1 , established for sulphonamides, are the Premi ® Test and the STAR in conjunction with the solvent-extraction procedure.

Introduction

Sulphonamides are one of the oldest groups of pharmacologically active substances used in veterinary medicine to date. Their discovery in 1935 signified the beginning of a new era in the treatment of a wide range of bacterial diseases and a number of protozoan infections. In rabbits, sulphonamides have been used for the treatment of coccidiosis. Because of concerns related to sulphonamide residues in food products of animal origin, they must be administered only in recommended concentrations and their respective withdrawal times must be observed.. Council Regulation (EEC) No. 2377/90 establishes the MRL of 0.1 mg.kg -1 for sulphonamides (all compounds of the sulphonamide group) in foods of animal origin. The MRL is defined as the maximum concentration of residue resulting from the use of veterinary medicinal products (expressed in mg.kg -1 or µg.kg -1 on a fresh weight basis), which is legally permitted by the Community as acceptable in or on food [START_REF] Ec | Council Regulation (EEC) No. 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin[END_REF]. There is a requirement set by the Council Directive 96/23/EC to screen live animals and animal products for residues of antimicrobial drugs [START_REF] Ec | Council Directive 96/23/EC of 29 April 1990 establish on measures to monitor certain substances and residues thereof in live animals and animal products[END_REF].

MIT are widely used in the screening of antimicrobial residues in food-producing animals and their animal products. MIT are based on inhibiting of the growth of test organisms sensitive to many antibiotics and sulphonamides. Among these tests, the FPT [START_REF] Bogaerts | A standardised method for the detection of residues of antibacterial substances in fresh meat[END_REF], the STAR developed at the Community Reference Laboratory in Fougères (France) and the Premi ® Test developed by DSM Gist B.V. (The Netherlands) have been most widely used in the residue screening.

The FPT and STAR are plate agar diffusion tests in which the presence of an antibacterial substance is expressed by the production of an inhibition zone. The FPT consists of plates with Bacillus subtilis BGA (at pH 6.0, 7.2 and 8.0) and Micrococcus luteus ATCC 9341 (at pH 8.0). For the detection of the presence of sulphonamide residues, the FPT prescribes the Bacillus subtilis BGA at pH 7.2. The STAR consists of plates with Bacillus subtilis BGA (at pH 7.2), Kocuria varians ATCC 9341 (at pH 8.0), Bacillus cereus ATCC 11778 (at pH 6.0), Escherichia coli ATCC 11303 (at pH 8.0) and Bacillus stearothermophilus var. calidolactis ATCC 10149 (at pH 7.4). For the detection of the presence of sulphonamide residues, the STAR prescribes Bacillus stearothermophilus var. calidolactis ATCC 10149. In order to enhance the sensitivity of the above test organisms to sulphonamides, trimethoprim (TMP) is incorporated in the agar medium at a concentration of 0.05 µg.ml -1 and 0.005 µg.ml -1 , respectively. The Premi ® Test is an agar diffusion test in ampoules in which the presence of an antibacterial substance in concentration exceeding the LOD is indicated by the absence of colour change from purple to yellow of the indicator (bromocresol purple) in the ampoule.

The test organism Bacillus stearothermophilus var. calidolactis is very sensitive to many antibiotics and sulpha compounds.

An ideal screening method would detect all licensed antimicrobials at or below their MRLs. Both techniques are capable of detecting a range of analytes at or below the MRL concentration. Stead et al. (2004) evaluated an alternative solvent extraction technique for enhancement of the capability of the Premi ® Test to detect a wider range of drugs at or bellow ½MRL in a variety of matrices. They found that the use of the solvent extraction procedure enables effective concentration of the extract to provide increased sensitivity to reach ½MRL limits that otherwise would not be achievable.

In practice, the samples showing a positive response are subjected to further investigations based on chromatographic methods with specific detectors. Because the final confirmation step can be preceded by preliminary identification and confirmation of sulphonamides by PABA, the post-screening detection of sulphonamides already in the first stage of the residue screening represents an important step of distinguishing the sulphonamides from other classes of veterinary drugs. In the presence of PABA, sulphonamides became weak and lose their antibacterial activity due to antagonistic competition for the synthesis of hydro-folic acid [START_REF] Gudding | The suitability of some media and peptones for sulfonamide testing[END_REF][START_REF] Gudding | An improved bacteriological method for the detection of sulfonamide residues in food[END_REF][START_REF] Ferrini | The Combinated Plates Microbial Assay (CPMA) technique for the detection and presumptive identification of β-lactam, sulfonamide, streptomycin and tetracycline residues in meat[END_REF]Stead et al. 2004;Kožárová et al. 2005;Janošová et al. 2007a;[START_REF] Janošová | A comparison of the sensitivity of antibiotic residue screening methods to sulphonamide standards and their presumptive identification by para-aminobenzoic acid[END_REF].

In food analysis, according to the Commission Decision 2002/657/EC, only chromatographic methods can be used for confirmation [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results[END_REF]. The principal approach of the HPLC analysis involves extraction, sample clean-up and HPLC analysis steps. The choice of method used in the laboratory is often dictated by the availability of suitable expertise, facilities, and equipment. However, all analytical methods used in the testing of official samples taken pursuant to Directive 96/23/EC must fulfil criteria set by the Commission Decision 2002/657/EC [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results[END_REF]. SMZ has been observed to be effective against coccidiosis in rabbits when it is administered orally via drinking water for 3 consecutive days at the dose of 2 g.l -1 . To avoid its residues in the edible tissues, a 15-day withdrawal period must be observed before the rabbits are slaughtered for human consumption (Sovík et al. 2005). Because SMZ remains to be the sulphonamide group most widely used in veterinary medicine, the objective of this paper was to determine which of the three aforementioned MIT used in practice today -FPT, STAR and Premi ® Test -is most sensitive and suitable for the detection of SMZ residues in the edible tissues of rabbits at the level of concern. To detect the SMZ residues at or below the MRL established, we applied an improved screening technique based on multi-residue solvent extraction. PABA at the concentration of 10 µg.ml -1 was used to confirm the presence of SMZ residues already during the first stage of residue screening. Finally, the level of SMZ residues in the edible tissues of rabbits was determined by validated HPLC analysis.

All four methods mentioned above have been officially approved for screening foodproducing animals and their products for residues of veterinary drugs in Slovakia (CH 6.2. 2004;CH 12.16. 2004;CH 12.18. 2006;CH 12.19. 2006).

Materials and methods

Preparation of standard solutions

The stock solution (1000 µg.ml -1 ) of the SMZ standard was prepared by dissolving 10 mg of SMZ (Sulfamethazine sodium salt, Sigma S 5637) powder in 2.4 ml ammonia (Lachema, Czech Republic), and diluting it to 10 ml with sterile deionised water. The working solutions of SMZ were prepared by serial dilutions with sterile deionised water to the concentrations of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 1 µg.ml -1 . Control solutions of SMZ (20 µg.ml -1 for the FPT and 2 µg.ml -1 for the STAR) were used to verify that the operating conditions were systematically respected. The TMP stock solution for the STAR was prepared by dissolving 10 mg of TMP standard (Trimethoprim, Fluka 92131) in 1 ml 5% acetic acid (Merck, Germany) and diluting with sterile distilled water to the concentration of 0.5 µg.ml -1 . The TMP stock solution for the FPT was prepared by dissolving 10 mg of TMP standard in 10 ml ethanol (Frukona, Slovakia) and diluting it with sterile distilled water to the concentration of 5 µg.ml -1 . The stock solution of PABA (1000 µg.ml -1 ) was prepared by dissolving 10 mg PABA (p-aminobenzoicacid, Sigma A 9878) powder in 10 ml distilled water. All stock and working solutions of respective standards were stored in a refrigerator at 4°C.

Origin and pre-preparation of incurred samples

Sixteen rabbits (meat line HY+) 16-weeks old, weighing between 2.60 kg-2.90 kg, were obtained for residue analysis from a farm (Hylapa s.r.o., The Slovak Republic). The rabbits were supplied SMZ (Sulfadimidin PG plv. sol. a.u.v., Pharmagal, The Slovak Republic) in drinking water for 3 consecutive days at a dose of 2 g.l -1 of water. After withdrawal of the drug, one rabbit per one day was slaughtered up to day 15 of the WP. The respective samples of muscle, heart, liver and lung were withdrawn and stored at -20°C until analysis. Six untreated rabbits served as sulphamethazine-free control.

Preparation of extract from the rabbit muscle by solvent extraction

10 g homogenized rabbit muscle was extracted with 25 ml acetonitrile/acetone (70:30, v/v) (Acetonitrile HPLC, Labscan, Ireland; Acetone p.a., Lach-Ners, Czech Republic) on a mechanical shaker LT2 (Votice, Czech Republic) for 15 min. The extract was centrifuged at 4500 rpm for 10 min in a centrifuge (Jouan BR 4I, France) at 4°C. The supernatant was removed and evaporated to near dryness under a stream of nitrogen in an evaporator (Turbo-Vap LV, Zymarck, Germany) at 50°C. The residue was suspended in 1 ml Nutrient Agar No. 1 (pH 6.8) (Imuna, The Slovak Republic).

Media and strains

FPT: Test Agar pH 7.2 (Merck, dehydrated medium 15787, Germany), Bacillus subtilis BGA (Merck 10649, Germany); STAR: Test agar DST pH 7.4 (Oxoid, dehydrated medium CM 261, England), Bacillus stearothermophilus var. calidolactis ATCC 10149 (Merck 1.11499, Germany). Both media were prepared according to the manufacture's instruction.

Premi ® Test: Premi ® Test kits, Bacillus stearothermophilus var. calidolactis (DSM, The Netherlands), ready to use.

Preparation of test plates

FPT: Test Agar pH 7.2 was seeded with the test strain Bacillus subtilis BGA to obtain the final concentration of 5x10 4 spores.ml -1 in the agar medium. To obtain the final concentration of TMP in agar medium (0.05 µg.ml -1 ), 1 ml of TMP solution (5 µg.ml -1 ) was added to 100 ml of agar medium. STAR: Test agar DST pH 7.4 was seeded with the test strain Bacillus stearothermophilus var. calidolactis ATCC 10149 to give a final concentration of 5x10 6 spores.ml -1 in the agar medium. To obtain a final concentration of TMP in agar medium (0.005 µg.ml -1 ), 1 ml of TMP solution (0.5 µg.ml -1 ) was added to 100 ml of agar medium.

Finally, 5 ml of the seeded media were transferred into Petri dishes 90 mm in diameter. Premi ® Test: 100 µl of respective working solutions of SMZ standard was pipetted onto the agar in the ampoule. Ampoules were incubated in the DSM heating block incubator for approximately 3 h at 64±0.5°C. Each working solution of SMZ standard was tested four-fold.

Determination of LODs

Detection of SMZ residues in incurred rabbit samples

FPT, STAR: A cylindrical core 8 mm in diameter and 2 cm long was removed from each frozen sample using a cork borer. To avoid a doubtful result, each cylindrical core was heat pre-treated in a water bath at 71°C for 20 min. 2 mm slices were cut with a lancet from both end of each core, and were discarded. Eight 2 mm slices were then cut from the remaining rabbit tissue. The slices and the filter paper discs moistened with 100 µl of extract were placed in parallel onto the surface of agar medium in Petri dishes. The plates were incubated as follows: the plates seeded with Bacillus subtilis BGA (FPT) at 30˚C for 18-24 h, and the plates seeded with Bacillus stearothermophilus var. calidolactis ATCC 10149 (STAR) at 55°C for 12-15 h. Premi ® Test: Juice from rabbit tissues obtained by the heat pre-treatment of the samples in a volume of 100 µl was pipetted onto the agar in the ampoule and allowed to stand at room temperature for 20 min for pre-diffusion. After pre-diffusion, the juice was flushed away by washing the test twice with demineralised water. The ampoules were incubated in the DSM heating block incubator for approximately 3 h at 64±0.5°C.

Presumptive identification of SMZ residues by PABA

FPT, STAR: The plates used for the confirmatory purposes were prepared by adding 1ml of stock solution of PABA to 100 ml of respective agar medium to obtain the confirmatory PABA concentrations 10 µg.ml -1 in the agar medium.

Premi ® Test: 100 µl of stock solution of PABA from the concentration of 100 µg.ml -1 was placed onto the agar in the respective ampoules and allowed to stand at room temperature for 20 min for pre-diffusion. After pre-diffusion, the PABA was discharged from the ampoules and both the working solutions of SMZ standard and examined tissue juice were further tested using the Premi ® Test procedure mentioned above.

Reading the test results

FPT, STAR: After incubation, the plates were evaluated and the diameters of clear inhibition zones around the tissue samples and surrounding the filter paper discs moistened with respective solutions of SMZ standard and residue extracts were measured in mm using a digital caliper (Mitutoyo, Japan). The SMZ control disk should present a zone of inhibition of 6 mm according to the FPT and 5±1,5 mm according to the STAR. The samples are considered positive exhibiting an inhibition zone 2 mm or more according to the FPT and 4 mm or more according to the STAR.

Premi ® Test: The results were read when the negative control changed colour from purple to yellow. A yellow colour indicated absence of SMZ residues and a purple colour indicated the presence of SMZ residues at or above the LOD of the Premi ® Test. In the case of doubtful results, the ampoules were incubated a few minutes longer for a clearer result or the samples were retested. In the presence of PABA, the yellow colour indicated that PABA inhibited the antibacterial effect of SMZ and confirmed the presence of SMZ residues. 100 µl of sterile distilled water was used as a negative control.

Confirmation of the SMZ residues by HPLC

Solvents and reagents

All solvents and reagents were analytical or HPLC grade. Acetonitrile, methanol, n-hexane, and acetic acid were obtained from Merck (Darmstadt, Germany), ethyl acetate, anhydrous sodium sulphate, and sodium acetate from Lachema (Brno, Czech Republic), and deionized and redistilled water was prepared on Milli-Q Plus (Millipore, France).

Sample extraction and sample clean-up

50 g homogenized tissue sample (muscle, liver) was extracted with 50 ml of ethyl acetate on a mechanical shaker for 15 min. The extract was filtered through a filtrate paper with 20 g anhydrous sodium sulphate. The extraction was repeated once more with 50 ml ethyl acetate for 10 min. Both filtrates were collected in a 100 ml round-bottom flask and evaporated to dryness in a rotary vacuum evaporator (Laborota 4000, Heidolph, Merck, Germany) at 40°C.

The residue was re-dissolved in 1 ml methanol and 1 ml of mobile phase, and transferred to a centrifuge tube. The flask was washed twice with 2 ml n-hexane, and 4 ml n-hexane was added to the centrifuge tube. The mixture was centrifuged twice at 3500 rpm for 10 min (Jouan BR 41, France). The lower layer was filtered through a disposable syringe filter unit and transferred to the vial. 25 µl volume of the solution was injected into the HPLC system.

HPLC analysis

Samples were analysed by a TSP liquid chromatograph (Thermo Separation Products, USA) equipped with a variable UV-VIS detector UV 3000 HR (Hewlett-Packard, USA).

The separation was performed on a LiChroCART RP-18e column (125-4, 5 µm) (Merck, Germany) using an acetonitrile-acetate buffer (pH 4,6) (25:75, v/v) as a mobile phase at a flow-rate of 0,8 ml.min -1 at the temperature of 40°C. The detection was conducted at 275 nm.

Results

The initial experiment was undertaken to detect the LOD of the screening methods for SMZ by determining the MIC of the SMZ standard for the respective test strains of screening methods. Because the MIC is defined as the lowest concentration of the respective antibiotic standard which completely inhibits the growth of the sensitive test organism, the diameters of the inhibition zones (FPT, STAR) and the colour change which represented the positive or negative results (Premi ® Test) were evaluated. In an attempt to confirm the presence of SMZ residues already during the first stage of residue screening, PABA at the concentration of 10 µg.ml -1 was used. This PABA concentration was taken into consideration according to the results obtained in our preliminary studies (Kožárová et al. 2005;Janošová et al. 2007a). All the results are summarized in Table I. Our results showed that the MIC of SMZ was 0.4 µg.ml -1 for FPT, 0.2 µg.ml -1 for STAR, and 0.05 µg.ml -1 for Premi ® Test /A/. By adding PABA to the agar medium (FPT, STAR) or onto the top of the agar medium (Premi ® Test), the absence of the inhibition zones or yellow colour was observed /B/.

The results of the screening for the presence of SMZ residues in the edible tissues of rabbits after its oral administration up to day 15 of the withdrawal period are presented in Tables II, III, and IV. Tables II andIII show the mean diameters of the inhibition zones (mm±SD /standard deviation/) produced by SMZ residues around the examined rabbit tissues and discs moistened with the extract from the rabbit muscle. Table IV shows the sensitivity of the test towards SMZ residues presented in the juice from rabbit tissue. By adding PABA to the agar medium (FPT, STAR) or onto the top of the agar medium (Premi ® Test), no positive results were detected. PABA at the concentration of 10 µg.ml -1 completely reversed the inhibitory activity of SMZ and identified reliably the presence of SMZ in the examined samples. Because all results with plates B or ampoules B were identical (0 /FPT, STAR/, -/Premi ® Test/), Tables II, III, and IV contain the results observed at the screening of SMZ residues in edible tissues of rabbits only with plates A. No detection sensitivity presented by the absence of the inhibition zones around the examined rabbit tissues was observed when using the FPT (Table II. /A/). When using the solvent extraction, the FPT detected the presence of SMZ residues in examined rabbit muscle tissue until day 8 of the WP. Because the FPT designated the sample positive at the inhibition zone of 2 mm and more, the muscle was positive until day 3 of the WP, and the mean diameter of the inhibition zone was 2.83±0.13 mm. According to the results obtained by screening of the SMZ standard, the residual concentration of SMZ in the positive muscle tissue was 0.4-0.5 µg.g -1 .

The STAR detected SMZ residues in the examined rabbit tissues (lungs, liver and kidneys) until day 8 of the WP. No SMZ residues were detected in the muscle and heart (Table III. /A/). Because the STAR rated the sample positive if the inhibition zone was 4 mm and greater, the liver was positive until day 4 of the WP (mean inhibition zone 4.21±0.08 mm), the lungs were positive until day 3 of the WP (mean inhibition zone 4.01±0.48 mm), and the kidneys were positive until day 5 of the WP (mean inhibition zone 4.35±0.25 mm).

According to the results obtained by screening of the SMZ standard, the residual concentration of SMZ in the presented positive rabbit tissue was in the range of 0.4-0.5 µg.g - 1 . After isolation of SMZ residues from the muscle by solvent extraction, the SMZ residues were detected in the muscle extract until day 10 of the WP and the muscle was positive until day 6 of the WP (mean inhibition zone 4.32±0.19). According to the results obtained by screening of the SMZ standard, the residual concentration of SMZ in the presented positive rabbit muscle tissue was 0.4-0.5 µg.g -1 .

The Premi ® Test detected SMZ residues in all examined matrices. By using the fluid expression procedure, the muscle and heart were positive until day 9 of the WP, and the liver, lungs and kidneys until day 10 of the WP (Table IV /A/). The positive results manifested by the maintenance of the violet colour of the agar medium imply the presence of SMZ residues at or above the LOD of the method. The negative results manifested by decolouration of the agar medium imply the absence of SMZ or presence of its residues below the LOD of the method. According to the results obtained by screening of the SMZ standard we can assume that the residual concentration of SMZ in the presented positive rabbit tissues was 0.05 µg.ml - 1 .

No positive results implied by the presence of inhibition zones or by maintenance of violet colour of the agar medium were observed in all examined rabbit tissues obtained from SMZ-free control group.

HPLC analysis

The presence of SMZ residues in the rabbit tissues (muscle, liver) throughout the 15 days of the WP were consequently confirmed and quantified by HPLC. The LOD of the method for SMZ was 0.004 mg.kg -1 and the respective limit of quantification (LOQ) for SMZ was 0.010 mg.kg -1 . Recoveries of SMZ from spiked control sample at the concentration from 0,3 mg.kg -1 to 3 mg.kg -1 ranged between 83% and 88%. SMZ residues were detected with UV-VIS detector at 275 nm at the retention time between 6.958 and 7.731 min. The mean residue concentrations of SMZ (mg.kg -1 ) in the examined samples are shown in Table V.

Table V shows that SMZ residues were detected in both examined matrices and the decrease in SMZ level was gradual and very slow. The highest residue concentrations were found on the first day of the WP. On this day, the mean residue concentration of SMZ in the liver was 0.56 mg.kg -1 , and in the muscle it was 0.42 mg.kg -1 . SMZ residues in the liver of rabbits were detectable up to day 12 of the WP and the mean SMZ residue concentration was 0.09 mg.kg -1 . On day 13 of the WP, SMZ residues were below the LOD of the method. SMZ residues in the rabbit muscles were detectable up to day 10 of the WP and the mean SMZ residue concentration was 0.02 mg.kg -1 . On the day 11 of the WP, SMZ residues were below the LOD of the method. No residues of SMZ were found in the muscles and liver obtained from the SMZ-free control group.

Discussion

The presence of contaminants and veterinary drug residues in products of animal origin is a matter of concern for public health [START_REF] Danko | Xenobiotics and their relation to health[END_REF][START_REF] Koréneková | Occurence of selected trace elements in cattle meat[END_REF][START_REF] Lukáč | Effects of trace elements on the immune system[END_REF]. The strategy of official residue control is based on two sequential steps: screening and confirmation. According to the Commission Decision 2002/657/EC a screening method means method that is used to detect the presence of a substance or class of substances at the level of interest. Screening methods have the capability for a high sample throughput and are used to sift large number of samples for potential non-compliant results. They are specifically designed to avoid false compliant results. Confirmatory method means method that provides full or complementary information enabling the substance to be unequivocally identified and, if necessary, quantified at the level of interest. The level of interest means the concentration of substance or analyte in a sample that is significant for determination of its compliance with legislation [START_REF] Ec | Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results[END_REF].

Live animals and animal products are often screened for antibiotic and sulphonamide residues in the first stage of residue screening by using the microbial inhibition tests. There is have been most widely used and investigated. The published data show that their sensitivity is not equal for all compounds of the sulphonamide group. Their sensitivity to sulphonamides were often affected by differences in test media, depth of agar layers, pH value of the agar medium, addition of TMP or dextrose to the agar medium, temperature, preparation and application of the standard solution, animal tissues, etc. [START_REF] Bogaerts | A standardised method for the detection of residues of antibacterial substances in fresh meat[END_REF][START_REF] Hrdlička | The residues of inhibitory substances in the times of slaughter animalscomparison of microbiological methods of agar diffusion[END_REF]Heitzman 1994;[START_REF] Koenen-Dierick | Optimization of an antibiotic residue screening test, based on inhibition of Bacillus subtilis BGA, with experimental design[END_REF][START_REF] Okerman | Evaluation of the European Four-Plate Test as a tool for screening antibiotic residues in meat samples from retail outlets[END_REF][START_REF] Okerman | Evaluation of the European Four-Plate Test as a tool for screening antibiotic residues in meat samples from retail outlets[END_REF][START_REF] Korsrud | Bacterial Inhibition Tests Used to Screen for Antimicrobial Veterinary Drug Residues in Slaughter Animals[END_REF][START_REF] Hussein | Experimental design for the microbiological Four-plate test for the detection of sulphadimidine residues at the level of the concern[END_REF][START_REF] Gaudin | Validation of a microbiological method: the STAR protocol, a five-plate test, for the screening of antibiotic residues in milk[END_REF][START_REF] Dey | Calf antibiotic and sulfonamide test (CAST) for screening antibiotic and sulfonamide residues in calf carcasses[END_REF][START_REF] Kožárová | Comparison study of estimation of detection sensitiveness of microbial inhibitory tests used for screening of sulphonamides in practise[END_REF]Janošová et al. 2007b;Pikkemaat et al. 2008).

PABA specifically inhibits the bacteriostatic activity of sulphonamides. Many authors investigated PABA at its various concentrations (from 0.1 µg.ml -1 to 100 µg.ml -1 ) for identification of sulphonamide residues at the first stage of their residue screening [START_REF] Lin | Simultaneous determination of residual sulphonamides in the presence and absence of p-aminobenzoic acid by high-performance liquid chromatography[END_REF][START_REF] Ferrini | The Combinated Plates Microbial Assay (CPMA) technique for the detection and presumptive identification of β-lactam, sulfonamide, streptomycin and tetracycline residues in meat[END_REF][START_REF] Braham | A rapid assay for detecting sulfonamides in tissues of slaughtered animals[END_REF]Stead et al. 2004;Kožárová et al. 2005, Janošová et al. 2007a[START_REF] Janošová | A comparison of the sensitivity of antibiotic residue screening methods to sulphonamide standards and their presumptive identification by para-aminobenzoic acid[END_REF]. All authors found that in the absence of PABA, sulphonamides produced inhibition zones on the test plates, whereas in its presence, no inhibition zones or reduction of their diameters were observed. The confirmatory PABA concentration was often conditioned by respective TMP concentration in the agar medium.

Chemical denaturation of tissues assists with release of drug residues. Stead et al. (2004) improved screening technique for rapid detection of antimicrobial residues in animal food products using of a solvent extraction procedure. The use of the solvent extraction technique coupled to the Premi ® Test provides an enhanced test capable of detecting a wider range of drugs at or below maximum residue limit concentrations. A solvent ratio of 70:30 v/v acetonitrile/acetone was found experimentally to be optimum for extracting the widest range of analytes including sulphonamides. Bearing in mind these consideration, the aim of our study was to evaluate three MIT used in practise today, the FPT containing Bacillus subtilis BGA, the STAR containing Bacillus The LODs of all tested MIT for SMZ were determined by detecting the MIC of SMZ standard which inhibited the growth and multiplication of the test organism with the respective method. The test organism Bacillus stearothermophilus var. calidolactis showed higher sensitivity to SMZ than the test organism Bacillus subtilis BGA. According to our results, the LOD of MIT for SMZ were as follows: FPT 0.4 µg.ml -1 , STAR 0.2 µg.ml -1 , and Premi ® Test 0.05 µg.ml -1 . The Premi ® Test detected SMZ residues (positive results) in the muscles and heart tissues until day 9 of the WP, and in the liver, lungs and kidneys until day 10 of the WP. The STAR detected SMZ residues in the edible organs of rabbits until day 8 of the WP. The kidneys were positive until day 5 of the WP, the liver until day 4 of the WP, and the lungs until day 3 of the WP. No SMZ residues were detected in the muscle and heart. The FPT detected no SMZ residues in any examined matrices.

The use of the solvent extraction significantly improved the existing screening methodology of plate agar diffusion methods and confirmed the data presented by the author mentioned above. Mixture of acetonitrile/acetone at a solvent ratio of 70:30 v/v, enabled extraction of SMZ residue from "negative" muscle tissues and enhanced the capability of the STAR and the FPT for detecting SMZ residues at the level of interest. By using the STAR, SMZ residues were detected in the muscle extract until day 10 of the WP. The muscles were positive until day 6 of the WP. By using the FPT, SMZ residues were detected in the muscle extract until day 8 of the WP and the muscles were positive until day 3 of the WP. PABA, at the concentration of 10 µg.ml -1 completely inhibited the antibacterial effect of SMZ in all examined matrices. The absence of inhibition zones around the examined matrices on PABA containing agar and visible decolourization of the agar medium from purple to yellow after adding PABA to the ampoule indicated the presence of SMZ residues in the samples. These results confirmed that PABA is an efficient and reliable agent for presumptive identification of SMZ residues in screen-positive samples.

The choice of method for confirmatory residue analysis is often dictated by the availability of suitable facilities and equipment. The HPLC method used in our study was found to be suitable for reliable detection of SMZ residues in the edible tissues of rabbits bellow the MRL level of 0.1 mg.kg -1 . The LOD and LOQ of the method for SMZ were 0.004 mg.kg -1 and 0.010 mg.kg -1 , respectively. The SMZ was detected in the muscle samples until day 10 of WP at the residual concentration of 0.02 mg.kg -1 , and in the liver until day 12 of the WP at the residual concentration of 0.09 mg.kg -1 .

With regard to the results obtained by the HPLC method we can conclude that the most suitable MIT for the detection of SMZ residues in the edible tissues of rabbits at the level of interest is the Premi ® Test followed by the STAR coupled with the solvent extraction procedure. The solvent extraction step markedly influenced the results of both plate agar diffusion methods, the STAR and the FPT, and positively improved their detection capability.

Conclusions

The residue-monitoring process is critical to the protection of the consumer's health. It must be effective and match the patterns of use for all classes of drugs applied in animal-production system, including sulphonamides. MIT are highly valuable in the first stage of residue screening. The purpose of these screening methods is to give a quick result whether the residue is either not present in the sample or is at or bellow the level of interest. With regard to their excellent practicality and throughput, they allow us to classify a large number of samples as being either "negative" or "potentially positive". All potentially positive samples are then subjected to a confirmatory test.

Because SMZ is still widely used for the treatment of coccidiosis in all food-producing animals, SMZ residues remain an important consideration for consumers. Thus the objective of the present work was to detect which of the three aforementioned MIT, used in practise today, the FPT, the STAR and the Premi ® Test, is most suitable for relevant detection of SMZ residue in the edible tissues of rabbits without any risk for consumers. Our results showed that the commercially produced Premi ® Test is by far the most suitable MIT for the detection of SMZ residues bellow the level of the MRL 0.1 mg.kg

  FPT, STAR: Filter paper discs having a diameter of 12,7 mm (Schleicher & Schull No. 2668, Germany) moistened with 100 µl of respective working solutions of SMZ standard were placed in parallel on the surface of the agar medium in the Petri plates. The plates were : the plates seeded with Bacillus subtilis BGA (FPT) at 30˚C for 18-24 h, and the plates seeded with Bacillus stearothermophilus var. calidolactis ATCC 10149 (STAR) at 55°C for 12-15 h.

  of such tests with various test organisms used worldwide. The LOD of the MIT for a given antibacterial depends primarily on the innate sensitivity of the test organism. In sulphonamide residue screening, four test organisms, namely Bacillus subtilis BGA, Bacillus megaterium ATCC 9885, Bacillus stearothermophilus var. calidolactis, and Bacillus pumilus

  The samples showing positive responses are subjected to further investigations by quantitative physico-chemical analysis. Chromatographic methods are being developed to confirm the findings of the screening methods. To determine sulphonamide residues in different matrices, several chromatographic methods based on HPLC with ultraviolet, fluorescence or mass spectrometry detection have been used in practise.

  var. calidolactis ATCC 10149, and the Premi ® Test containing Bacillus stearothermophilus var. calidolactis, for determining SMZ residues in the edible tissues of rabbits after its oral administration up to day 15 of the withdrawal period at the level of concern. To obtain reliable results already in the first stage of residue screening and to identify the presence of SMZ residues in the examined rabbit samples, both the screening procedure based on solvent extraction step and the post-screening identification of SMZ by PABA were used. The confirmatory analysis was undertaken by validated HPLC method.

  -1 . The STAR detected SMZ residues closely above the MRL level, and the FPT above the MRL level. This indicates that the MIT containing Bacillus stearothermophilus var. calidolactis the test organism are more sensitive to SMZ than those with Bacillus subtilis BGA. When the edible tissues of rabbits were screened by classical methodologies of the STAR and the FPT, no SMZ residues were detected in the rabbit muscles. The alternative screening technique based on solvent extraction of the test detection capability and improved positive results of both less sensitive MIT. Because the MIT are sensitive to a wide range of antimicrobial compounds, the use of PABA in post-screening investigation of the screen-positive samples allowed us to identify the presence of SMZ residues in the examined samples and choose the method suitable for confirmatory analysis. HPLC analysis of muscle and liver samples of rabbits confirmed the presence of SMZ residues in both examined matrices.The results obtained allowed us to conclude that the Premi ® Test and the STAR with the test organism Bacillus stearothermophilus var. calidolactis ATCC 10149 in combination with the alternative screening technique based on the solvent extraction procedure, can be used as the most suitable screening tests for detection of SMZ residues in the edible tissues of rabbits at the level corresponding to MRL of 0.1 mg.kg -1 . The utilisation of PABA for the presumptive identification of SMZ residues in the screen-positive samples constitutes an impressive challenge to employ efficient and cost-effective means of controlling SMZ residues in foods.

  De Wasch K, Van Hoof J. 1998. Detection of antibiotics in muscle tissue with microbiological inhibition tests: effects of the matrix. Analyst 123: 2361-2365. Pikkemaat MG, Oostra-van Dijk S, Schouten J, Rappaline M, Van Egmond HJ. 2008. A new microbiological screening method for the detection of antimicrobial residues in slaughter animals: the Nouws antibiotic test (NAT-screening). Food Control 19: 781-789. Sovík L, Hederová J, Bíreš J, Gajdoš M. et al. 2005. Vademecum of veterinary preparations and disinfestation aids. Bratislava: Pro-Trade. Stead S, Sharman JA, Tarbin JA, Gibson E, Richmond S, Stark J, Geijp E. 2004. Meeting maximum residue limits: an improved screening technique for rapid detection of antimicrobial residues in animal food products. Food Additives and Contaminants 21:216-221.

Table I .

 I LOD of screening methods for SMZ determined by the MIC of SMZ standard presented as the lowest mean diameter of the inhibition zones (mm ± SD) or positive results (A) and presumptive identification of SMZ with PABA at the concentration of 10 µg.ml -1 (B).

		Plate/			Concentration of SMZ (µg.ml -1 )
	MIT	Ampoule 0.05 0.1	0.2	0.3	0.4	0.5	1
	FPT	A	0	0	0	0	2.62 ± 0.26 3.09 ± 0.17 3.67 ± 0.14
		B	0	0	0	0	0	0	0
	STAR Premi ® Test	A B A B F 0 0 + -o r 0 1.09 ± 0.48 2.17 ± 0.75 3.17 ± 1.17 4.00 ± 0.63 5.00 ± 0.63 0 0 0 0 0 0 + + + + + + ------
	Bold concentrations represent the LOD of respective MIT for SMZ; + positive results; All data are the mean ± SD of six observations. P
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Table II .

 II The mean diameters of the inhibition zones (mm ± SD) observed at the screening of SMZ residues in edible tissues of rabbits after its oral administration up to the day 15 of the withdrawal period using the FPT with Bacillus subtilis BGA at pH 7.2. without the addition of PABA into the agar medium; Bold numerals represent the positive results; All data are the mean ± SD of four observations.

	Day of WP	Plate Muscle	Muscle extract	Heart	Liver	Lungs Kidneys
	1.	A	0	8.04 ± 1.07	0	0	0	0
	2.	A	0	4.79 ± 0.35	0	0	0	0
	3.	A	0	2.83 ± 0.13	0	0	0	0
	4. 5. 6. 7. 8. 9.-15. Control sample A -plates F A A A A A A A o r 0 0 0 0 0 0 0 P e e 1.73 ± 0.26 1.72 ± 0.15 1.64 ± 0.12 1.46 ± 0.23 0.75 ± 0.60 0 0 r R	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0	0 0 0 0 0 0 0
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Table III .

 III The mean diameters of the inhibition zones (mm ± SD) observed at the screening of SMZ residues in edible tissues of rabbits after its oral administration up to the day 15 of the withdrawal period using the STAR with Bacillus stearothermophilus ATCC 10149 at pH 7.4. without the addition of PABA into the agar medium; Bold numerals represent the positive results; All data are the mean ± SD of four observations.

	Day of WP Plate Muscle Muscle extract Heart	Liver	Lungs	Kidneys
	1.	A	0	9.91 ± 0.55	0	8.12 ± 1.78 5.85 ± 0.67 8.75 ± 0.39
	2.	A	0	8.16 ± 1.04	0	5.39 ± 0.67 4.17 ± 0.63 6.53 ± 0.20
	3.	A	0	7.93 ± 1.01	0	4.46 ± 0.53 4.01 ± 0.48 5.59 ± 1.30
	4. 5. 6. 7. 8. 9. 10. 11.-15. Control sample A A A A A A A A A o r 0 0 0 0 0 0 0 0 0 P e 6.65 ± 1.39 5.01 ± 0.57 4.32 ± 0.19 2.36 ± 0.38 2.31 ± 0.28 2.26 ± 0.90 1.29 ± 0.51 0 0 A -plates F e r	0 0 0 0 0 0 0 0 0	4.21 ± 0.08 2.42 ± 0.49 4.45 ± 0.71 2.54 ± 0.66 1.,28 ± 0.25 4.35 ± 0.25 2.01 ± 0.51 1.08 ± 0.23 3.05 ± 0.26 1.66 ± 0.13 0.71 ± 0.84 2.53 ± 0.19 1.57 ± 1.08 0.63 ± 0.76 1.95 ± 0.27 0 0 0 0 0 0 0 0 0 0 0 0
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Table IV .

 IV Positive and negative results observed at the screening of SMZ residues in edible tissues of rabbits after its oral administration up to the day 15 of the withdrawal period using the Premi ® Test.

					Food Additives and Contaminants
	Day of WP	Ampoule	Muscle	Heart	Liver	Lungs	Kidneys
	1.		A			+	+	+	+	+
	2.		A			+	+	+	+	+
	3.		A			+	+	+	+	+
	4. 5. 6. 7. 8.	A A A A A o F r		+ + + + +	+ + + + +	+ + + + +	+ + + + +	+ + + + +
	9. 10.		A A P	+ -	+ -	+ +	+ +	+ +
	11.-15. Control sample	A A	--e e r	--	--	--	--
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A -ampoule without the addition of PABA onto the agar medium; + positive results;negative results; All data are the mean ± SD of four observations. Page 21 of 22 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table V .

 V SMZ-residue concentrations (mg.kg -1 ± SD) in the rabbit muscle and liver after its oral administration up to the day 15 of the withdrawal period detected by the HPLC.Bold numerals represent the days of the WP when the lowest residue levels were measured; ND -not detectable.

	Day of WP	Muscle	Liver
	1.	0.42 ± 0.046	0.56 ± 0.021
	2.	0.38 ± 0.011	0.49 ± 0.032
	3.	0.33 ± 0.033	0.41 ± 0.017
	4.	0.33 ± 0.015	0.39 ± 0.009
	5.	0.27 ± 0.007	0.39 ± 0.011
	6.	0.25 ± 0.004	0.31 ± 0.013
	7.	0.21 ± 0.025	0.27 ± 0.015
	8.	0.18 ± 0.016	0.25 ± 0.008
	9.	0.15 ± 0.002	0.21 ± 0.050
	10.	0.02 ± 0.004	0.21 ± 0.070
	11.	ND	0.19 ± 0.006
	12.	ND	0.09 ± 0.003
	13.-15.	ND	ND

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

Acknowledgements

This study was supported by VEGA grant No 1/3491/06.


H Barton
email: mfbarton@cyf-kr.edu.pl

Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow (Poland). Part 4: Copper

Keywords: Exposure, FAAS, Metals -uptake, Risk assessment Additives/Contaminants: Copper, Food contact materials, Trace elements (nutritional), Trace elements (toxic) drinking water, copper intake, contamination, children, health protection

The aim of this study was to assess exposure of children to copper (Cu) from household drinking water (DW). The DW samples were collected from 1997-2004 in ca. 650 households and preschools by a double sampling method (morning -W1, evening -W2). The study group comprised ca. 300 children (5-7 years old) living in Krakow (urban, peripheral) and rural areas in southern Poland. Cu concentrations were determined by the Flame Atomic Absorption Spectrometry. There was no significant relationship between the sampling period to Cu concentrations, whilst statistically significant differences were found between urban and nonurban (rural, peripheral) sites and between morning and evening samples. Geometric means of Cu concentrations in evening DW (95% confidence interval, in µg l -1 ) were 8 (1-110), 20 (1-274) and 12 (0-364) in urban, peripheral and rural sites, respectively. DW contamination after overnight standing was comparable in all sites (average ca. 20 µg Cu l -1 ). The adopted threshold of 100 µg Cu l -1 was exceeded in evening DW by 3.6 % and 15% in urban and non-urban households, respectively. The exceedance of EC limit (2 mg l -1 ) was not significant. The predicted contributions of evening DW to Cu intake by children were as site's means 18-37 µg day -1 or 0.8-1.6 µg kg b.wt. -1 day -1 , but were two-three fold higher for morning DW. Polish RDA (1 mg Cu day -1 ) was exceeded for morning (evening) DW in 3.7(0.2) % of children, at mean intake 159(118) % of RDA. The observed copper levels and predicted intakes can be considered low and should not raise a nutritional or toxicological concern of age group studied.

Nevertheless, due to the overnight contamination, the former suggestion that stagnant portion of drinking water should be discarded, should remain valid.

Copper (Cu) is present in natural waters at trace level and enhanced concentrations, if observed, usually come as contaminant during water treatment, from supply systems and interior pipelines made from metallic copper and fitting/pipes from brass (WHO 2006). Some known factors that affect copper levels in household drinking water are acidity, low hardness, chlorine, elevated temperature and long standing time in pipes.

Free Cu cation (Cu +2 ) is a clear oxidant contributing to oxidative stress [START_REF] Attri | Erythrocyte metabolism and antioxidant status of patients with Wilson disease with hemolytic anemia[END_REF] and involved in mutations [START_REF] Hussain | Increased p53 mutation load in nontumorous human liver of Wilson disease and hemochromatosis: oxyradical overload diseases[END_REF]. Copper [START_REF] Solfrizzi | Macronutrients, aluminium from drinking water and foods, and other metals in cognitive decline and dementia[END_REF], among other transition metals, is suspected of involvement in pathogenesis of neuro-degenerations, including Alzheimer's disease [START_REF] Sparks Dl Friedland | Trace copper levels in the drinking water, but not zinc or aluminum influence CNS Alzheimer-like pathology[END_REF]). High Cu (and iron) intakes were associated with an overall increased risk of colorectal cancer [START_REF] Senesse | High Dietary Iron and Copper and Risk of Colorectal Cancer: A Case-Control Study in Burgundy, France[END_REF]).

Copper in drinking water affects taste above 2.5 mg l -1 and colour above 5 mg l -1 (WHO 2006). Common acute health effects of Cu in pure DW observed over 3 mg l -1 , are gastrointestinal symptoms, but in children the effects were observed at lower Cu levels. The presence of food, juice or even a flavour weakened the gastrointestinal effects. The symptoms are modulated by sex, concentration and time of exposure [START_REF] Araya | Community-based randomized double-blind study of gastrointestinal effects and copper exposure in drinking water[END_REF]). As a strong bactericide Cu +2 can disturb normal intestinal flora but this may be not a case for complexed Cu.

Recent studies suggest that free dietary Cu cation can bypass the liver and enter by itself the circulation inducing some toxic events [START_REF] Brewer | The risks of free copper in the body and the development of useful anticopper drugs[END_REF]).The study in Finland have reported that copper in DW may be associated with increased risks of heart attack [START_REF] Kousa | Geochemistry of ground water and the incidence of acute myocardial infarction in Finland[END_REF].

A potential indirect toxicity associated with Cu-redox activity should also be considered.

In the presence of antioxidants copper-induced redox events in organism propagated pro-oxidant activity [START_REF] Bagnati | When and why a water-soluble antioxidant becomes pro-oxidant during copper-induced low-density lipoprotein oxidation: a study using uric acid[END_REF]). [START_REF] Asplund | Measurement of ascorbic acid (vitamin C) induced hydroxyl radical generation in household drinking water[END_REF] and [START_REF] Jansson | Vitamin C (ascorbic acid) induced hydroxyl radical formation in copper contaminated household drinking water: role of bicarbonate concentration[END_REF] have shown that Cu +2 in drinking water produced, in the presence of vitamin C, hydroxyl radicals. In household tap water samples and commercial bottled water, Cu produced high concentration of H 2 O 2 and this possibly can also occur in homemade or commercial beverages. Problems associated with copper in wine (Thoukis and Amerine 1956) and beer [START_REF] Pohl | Determination and fractionation of metals in beer: A review[END_REF]) have also been observed.

Copper homeostasis is more effective than many others element nutrients and involves controlling processes of Cu absorption and excretion, but the range of tolerance, especially exposure, is not much known. Availability of Cu in humans from a typical diet is 30-40% [START_REF] Wapnir | Copper absorption and bioavailability[END_REF], but vary significantly between 17-97%. Availability is dose-and concentration-dependent and increases for Cu deficient diets. Cu absorption from drinking water is generally greater than that in the presence of food. Dietary factors (e.g. phytates, fiber, ascorbic acid, alcohol, carbohydrates ), elemental supplements (e.g. Zn, iron, calcium) and some other elements (tin, molybdenum known as 'anti-copper', tungsten) negatively interfere with Cu absorption [START_REF] Pekelharing | Iron, copper and zinc status in rats fed on diets containing various concentrations of tin[END_REF][START_REF] Hou | Antitumor and antiinflammatory effects of tetrathiotungstate in comparison with tetrathiomolybdate[END_REF], and can induce a copper deficiency [START_REF] Prasad | Hypocupremia induced by zinc therapy in adults[END_REF].

Total dietary Cu intakes by the adult population in EU were in the range 0.8-1.8 mg day - 1 , while intakes for children ranged broadly from 0.7 to 1.5 mg day

Materials and methods

The study area and population were described previously (e.g. Part 3, Barton 2008). A group of households from 2004 was also included. Briefly, in the period 1997-2004 nearly 700 households and pre-schools were investigated, of which about 650 was included in this study.

Drinking water (DW) samples were collected in households by double sampling method, at the first use in the morning (W1) and in the evening (W2) after daily use.

Analytical characterisation

Metal analyses were performed by means of flame atomic absorption spectrometry (F-AAS) using a Perkin-Elmer instrument (5100ZL) with burner of 10 cm length. Hollow-cathode lamp (HCL) and slit 0.7 nm were used. Injection rate was ca. 1 ml/min. The absorbance averaged over time was applied as an analytical signal and three successive signals per each measurement were collected. Each sample was run in duplicate and the mean of successive runs at the relative standard deviation not exceeding 5% was accepted as an estimate. For each 10 samples, the blank and internal quality control standard was run and the result was used for slow sensitivity drift correction. The other analytical parameters were as follows: analytical wavelength -324.8 nm, analytical signal -time averaged absorbance (3 sec), calibration range: 0-1000 µg l -1 , air : acetylene ratio flow rates (in liter/minute) -10 : 2. Analytical characteristics of the method: at the precise stabilization of working parameters the limit of detection of 2 µg l -1 was achieved; practical (safe) parameters for routine analyses were: limit of detection (LOD, n=21) was 4 µg l -1 and limit of quantitation (LOQ) -14 µg l -1 ; precision (RSD) estimated for Cu standards at levels: 100 µg l -1 -1.9 %, 500 µg l -1 -1.6 %, 1000 µg l -1 -1.1 %; validation: certified reference material (CRM, IMEP-12 test sample, year 2001, EC-JRC-IRMM, Geel, Belgium): obtained 212±3 µg l -1

(certified 217±4 µg l -1 ).

Calibration, post-measurement re-calibration and data processing

The non-linear curves were observed for Cu within the calibration range. The collected signals were subjected to post-measurement processing as described previously [START_REF] Barton | Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow, Poland. Part 3: Aluminium[END_REF]) consisting of: were applied. Since populations were not sufficiently uniform in type of distribution, both, geometric means (GM) and/or median (MED) values were commonly included, if not stated otherwise. The range of values, which included of 95% of population (95% confidence interval, CI) were estimated primarily from lognormal distribution profile as GM*10 (±1.96 * logSDg) (or alternatively from frequencies as 2.5 and 97.5 percentiles for median, if stated). The t-test (for normal distribution profile) or Kruskal-Wallis and median χ2-test (at poor normality or inhomogeneity) were commonly used and for multiple tests, the more rigorous criteria of significance (higher value of a p-level) was considered.

Results

Metal concentrations in drinking water

Among ca. 650 households investigated, the maximum metal concentration was 3.08 and 1.19 mg l -1 in W1 and W2 samples, respectively. Generally Cu concentrations were not related to the study period. In U-sites a small drop in the mean Cu concentrations after the year 2000 was noticed, e.g. for W2 (GM in µg l -1 ) (6 vs. 11, p<0.001). This was not further included as a critical parameter of the population under present study.

Overall distribution of Cu concentration for U and NU-sites is shown in Figure 1 [Insert Figure 1 about here] Cu concentration distributions showed not quite uniform profiles. The frequency profiles were close to lognormal after exclusion of outliers below LOD, but this was less significant for U-subset. The frequency maximum for NU sites was at higher concentration than for U sites.

The differences between U and NU sites were statistically significant at least at p<0.05 for both, W1 and W2. A weaker difference was seen for Cu in morning water samples (W1). Many W1 (W2) samples fell below LOD, i.e. 5% (12%) of total, and 3% for both samples from the same household. The 90 th percentiles (P90 in µg l -1 ) were for W1 (W2) 172 [START_REF] Zimoch | The water production subsystem failures affects of Krakow's water supply system[END_REF] and 264(169), for U and NU sites, respectively, while 10 th percentiles (P10) were below 7 µg l -1 . Mean ratio of P90 to median, GM and arithmetic mean was 2.6, 9.3 and 11.0, respectively. The arbitrarily adopted threshold of 100 µg Cu l -1 was exceeded for W2 in 4 % and 15% of U and NU samples, respectively, while for W1 series, site's exceedances were comparable and were 20% on average. The exceedances in U and R sites significantly decreased for higher threshold, as for example, the value 500 µg Cu l -1 was exceeded by 0.5, 1.3 and 0.8 % of W2 samples in U, P and R-site, respectively.

In Figure 1, the weakly resolved minor modes of maximum at 58 µg l -1 (U2) and 182 µg l -1 (NU2) suggested presence of the domains of enhanced Cu, for which contributions to sites were 13% and 17%, respectively. Due to right-tailed profiles the significance of tested distributions was weaken, and from the same reason, the percentiles above median were I.

[Insert Table I about here]

The Table I contains detailed data for mean metal concentrations in W1, W2 samples for U, P and R sites, as well as for the combined NU sites. Overall mean values for Cu were also given. W2 samples showed the lowest Cu concentration at U sites, and were about two-threefold higher for NU (P, R) sites. Similar relations were observed in the morning samples (W1).

The exceedances the 1 mg l -1 limit were 1.4% or below.

The related parameters (differences, ratios) were calculated for the major fraction of households (fraction 'a'), where increase in metal concentration was observed in the first draw of water sample (W1) and which contributed by of 70%. This effect was parameterised by difference (D 1,2 ), ratio (R 1,2 ) and relative difference (D 1,2% ) between morning and evening metal concentration (W1, W2) as described earlier.

The parameter values, D 1,2 and D 1,2% , were similar in all sites and mean values (GM)

were 20 µg l-1 and 63%, respectively. For selected households (40% of total households), where the absolute D 1,2 values exceeded the limit of quantitation (LOQ), the mean D 1,2 was (GM) 73 µg

l -1 .

Metal concentration in morning and evening samples

Distribution of metal concentrations in DW samples collected by double sampling was shown in Figure 2, where concentrations in morning samples (W1) were plotted versus evening samples (W2). Each point represents one household.

[Insert Figure 2 about here] The two cut-off lines refer to the aesthetic limit of metal concentration in drinking water in force in the period of sample collection. The lines divide the plot into four sectors: sector Arefers to the households for which metal concentration fell below the limit for both, the evening and morning samples; B1 or B2 -morning or evening samples exceeded the limit, respectively;

C -both concentrations were over the limit. Percentages of households in particular sectors are

given in an inserted box in Figure 2. The line designed as 1:1 represents equality of concentrations in the morning and evening. For both elements the most points lay over the line 1:1, what shows higher metal concentrations in the water after stagnancy period, i.e.

contamination. Influence of the internal pipeline system on copper concentrations The metal concentration was increased in stagnant water in 70% of households (major fraction 'a'). The effect of contamination shown in Figure 4A was not linear and was accelerated for higher background (in W2) metal concentration. Contamination pattern -'a' (higher plot)exhibited plateau within the concentration range 30-150 µg l -1 at the D 1,2 value ca. 170 µg l -1 .

Metal concentrations in water from the major water treatment plants

The concentration range at plateau approximately fit to second mode of distribution profile As shown in the upper plot 'a' in Figure 4B, the percentage of Cu from contamination (D 1,2% ) increased with the decrease in Cu W2, i.e. background Cu concentration. This means that in these cases the most Cu present in DW came from interior pipeline system. Deposition process was negligible for low concentrations and was strongly accelerated with increase in Cu in W2. The relative yield of deposition was not strongly dependent on Cu concentration and can be approximated as constant of ca. 60% for Cu concentrations over 50 µg l -1 in W2.

Predicted copper intake with drinking water by children

Mean predicted metal intakes with drinking water W1 and W2 by 6-year-old children in selected subgroups are shown in Table II. The intakes were calculated using approximated adequate water intake (1.5 litre day -1 ) and anthropometric data of study group of children. Relative intakes were expressed as a percent of lower value of safe range of RDA for Polish population(1 mg day - 1 ). The intake values were calculated separately for both, W1 and W2 samples. The identification of a so-called 'risk group', i.e. households with exceedance of the concentration limits (risk group 1) and exceedance of RDA (risk group 2) was also preformed.

[Insert Table II about here]

The relationships between metal intakes followed the same patterns as the metal concentrations and all relations between different data subsets can be transposed from concentrations to intakes, except for those expressed per body weight. The Cu intake for different sites ranged (GM, in µg day -1 ) between 45-88 and 18-37, for W1 and W2 samples, respectively. Percentiles 90th for W1(W2) were 431(105) and 504(285) in µg day -1 , for U and NU sites, respectively, or alternatively 16(5) and 25(12) µg kg b.wt. -1 day -1 , respectively.

Relative intakes were between (GM) 1.8-3.7 % RDA for W2 and were over two times higher for W1. The 'risk' group 1 derived from exceedance of the Cu concentration aesthetic limit accounted for 1.7% of children group. Exceedance of this limit in total study group was not much different (0.9 %) but this group included also other households (e.g. preschools).

The exceedance of the intake over the RDA of lower limit (1 mg day -1 ) was found for 3.7% of children based on data for W1 (risk group 2 in Table II). The higher value of the RDA range (1.5 mg day-1) was exceeded for 2.4% of children. None of children exceeded both RDA levels for W2. Both exceedances were slightly higher than these derived from aesthetic concentration limit. For both risk groups and both RDA's, the mean predicted intakes were within the range 159-222 % of RDA.

Discussion

Copper levels and diversity among sites

Evaluation of the results of Cu concentrations using double sampling method in this study can be summarized in the following points:

-Cu concentrations and exceedances of the quality limits were low.

-mean concentrations in major sites and exceedance of the limits increased in the following order: urban < rural < peripheral. The parameters for the later two sites (rural and peripheral) differed weakly giving rationale for joint non-urban category.

Overall mean site's concentrations for evening sample series can be considered low and differences as mild, even though statistical significance was achieved. It is consistent with rare usage of copper materials for water pipeline systems except commonly used brass taps. Spread private buildings are less uniform and thus rural and peripheral sites exhibit higher Cu level in DW.

Water contamination with copper

For all sites, over three-fold increase (R value was 20 µg l -1 or 73 µg l -1 for effects exceeding LOQ.

The contamination pattern exhibits general increase in relation to background Cu level. In the middle Cu concentration range in W2 a characteristic plateau of mean Cu contamination 170 µg l -1 was observed (Figure 1).

The similar plateau was observed in the case, e.g. of cadmium [START_REF] Barton | Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow (Poland). Part 2: Cadmium (year 1997-2001)[END_REF]). This range can be assigned to the presence of copper-containing parts at the end of pipeline, for which absolute contamination was not Cu W2 concentration-dependent. Lets assume that initial magnitude of Cu (in W2), i.e. present due to contributions from treatment plus contamination between supplier and building (in production, storage, transport) depends on water corrosivity.

Then Cu dissolution would be kinetically controlled, and at the constant over-night period should be positively related to Cu W2, taken as an index of water corrosivity. Except for the plateau range, the rise of contamination is really observed, suggesting that corrosivity of water itself may be one of determinants of metal concentration increase during stagnancy. However in the plateau range, one may suggest that the process can be diffusion controlled, thus weakly dependent on water corrosivity. The mechanism can be associated with metal exchange in the presence of porous (e.g. corroded) residues or biofilm layers on copper/brass surface (Cale et al.

2007

). In such a case, a longer stagnancy period may enlarge the height and width of the plateau.

Alternatively this can also be due to a saturation of end portion of DW with Cu ions during stagnancy period. This is possible for small value of volume to surface ratio in the end-part of pipeline (small pipeline diameter, possibly tap from brass). .

It is worth to emphasise that absolute overnight contamination values (D 1,2 ) were not different among three sites studied (see Table I) and can be considered constant. This was probably due to constant period of stagnation in this study and common copper containing endpart of pipeline systems, i.e. brass taps. 'True' contamination, however, which can be identified (ca. 60%, Figure 4B).

Diversity of DW Cu among suppliers

Among five major water suppliers in urban area of Kraków the Cu concentrations in household water differed significantly. The highest background contamination (in W2) was associated with the slowest exchange rate in storage source, as can be deduced from report of [START_REF] Zimoch | The water production subsystem failures affects of Krakow's water supply system[END_REF]. The other factors, that is, significantly different hardness and mineral composition, can also contribute to these differences, as shown by suppliers' data of water Metallic copper and PVC tubing is also sometimes used as ending part of low pressure water supply systems in private houses. The area covered by WTP supplying water from source A can be evaluated as relatively new built, thus internal pipelines are expected to be less corroded, which is in agreement with the lowest Cu. For deep well, (source E) the highest contamination may be due to low exchange rate as described by [START_REF] Zimoch | The water production subsystem failures affects of Krakow's water supply system[END_REF].

Exceedance the limits

The exceedance of EC limit for Cu in DW was found for one household in morning sample. This is not a significant exceedance and is consistent with the whole conclusion that the Cu concentrations in all sites and sources can be considered low as well against safety limits as in comparison to other geographical EU regions (Zietz et al. 2003a, Pettersson and[START_REF] Pettersson | Daily intake of copper from drinking water among young children in Sweden[END_REF]. For example, Cu in stagnant DW over 0.5 mg l -1 was found in about 10% of households in the region of Germany [START_REF] Zietz | Copper concentrations in tap water and possible effects on infant's health -Results of a study in Lower Saxony, Germany[END_REF]. In the regions where copper pipelines are prevailing, this limit (0.5 mg l -1 ) was exceeded by significant part of households. In this study, however, this limit was exceeded by 0.5-1.3%. The more restrictive threshold 100 µg l -1 was exceeded by 3.6 % of urban and 15% of non-urban households. This limit corresponded to minor domain of enhanced Cu in NU sites (point NU2 in Figure 1). This is probably due to a variety of individual supply systems in private houses, in comparison to uniform urban large

Cu intakes

Evening DW (W2) can supply on average, 18-37 µg l -1 Cu daily for children. These values contributed to Polish reference (RDA) value of 1 mg day -1 for 6-year-old children from different sites by 1.8-3.7 % RDA. The intakes were the highest in peripheral areas of Kraków (88 µg day -1 from W1, 37 µg day -1 from W2). Similar estimation was obtained for adults in Spain 53 µg day -1 (Velasco-Reynold et al. 2008).

For 1.7% children from the risk group 1 (Table 2), DW taken in the morning (W1) for whole-day food preparation may introduce 222% of recommended daily Cu. Similarly, 2.4% of children may be at risk of exceedance of the higher limit of the RDA range (1.5 mg day -1 ) by about 148% with W1, on average. It is worth to note that exceedance estimates based on concentration safety limit (1 mg l -1 ) and Polish RDAs (1-1.5 mg day -1 ) are quite comparable as applied to the study group of children.

The above results suggest that the risk of Cu overload from DW can be considered marginal. These estimations have confirmed the other Polish studies in elderly [START_REF] Kaluza | Contribution of food products and drinking water to the intake of minerals by elderly (in Polish)[END_REF]) that DW is not a significant Cu contributor. Drinking water can contribute by less than 4% of RDA for age group studied, however, for morning, not flushed portion of water, the contributions can be two-three fold higher, and near 9% for peripheral sites (Table II). Since nutritional value of DW (W2) as a source of such small amount of Cu can be considered low, the increase in Cu intake, due to an overnight contamination does not seem to introduce significant advantage. On the other hand, such a moderate contamination with Cu did not introduce also any significant risk for health of population. Taking into account, however, that during stagnancy period the pattern of contamination by toxic metals (cadmium, lead, see previous parts of the series) is similar , the advantage of intake of such an additional Cu via morning DW cannot be thus discussed. Consequently, the current results do not weaken the previous suggestion that discarding stagnant portion of water should prevent additional intake of these toxic metal by small children. This is especially applicable if morning portion of water might be taken for whole-day formula-based food preparation for child.

The above suggestion is not new and was regarded to be applicable to households with copper-made pipelines/fittings supply systems. It was included in the WHO(2006) and Australian DW guidelines (ADWG 2004), to prevent excessive exposure: : "water that has been in stagnant contact with copper pipes and fittings should not be used in the preparation of food or drink, however, copper levels can be effectively reduced by flushing the taps...", besides, this was pertinent only to premises with a history of copper corrosion. It was also considered in the studies of copper exposure from DW (Pettersson and Rasmussen 1999).

General comments

In the light of the background literature information regarding magnitude of reference values and limits, the evaluation of Cu concentrations in this paper perhaps were performed somewhat excessively, than may be suggested by metal levels found. This is because healthbased criteria for safe metal levels in drinking water, especially for copper, are still being debated, and new data argue for its lowering. There are several potential factors which may influence the risk of copper overload and other adverse health effects due to drinking water Cu.

First, there is a possibility of Cu overload due to DW and diet combination. Some foods rich in Cu, as oyster and livers (especially goose or duck), sesame seeds, wheat bran and germ, can easy overload individual UL in combination with high DW Cu. As mentioned above, however, availability of copper is generally lower from food, and especially for Cu several food constituents were found to decrease absorption. Also, a strong adaptive response of humans to higher dietary Cu may weaken for some extent the impact of Cu overload [START_REF] Harvey | Adaptive responses in men fed low-and high-copper diets[END_REF]).

The second risk factor which would be seriously considered are the health consequences Finally, growing popularity of self-supplementation is considered to be a risk factor for Cu overload. However, there is a commonly accepted "silent" assumption that dietary copper, as a natural constituent of food bound to natural ligands and copper from inorganic compounds, are the same or similar in terms of nutrition value. Many studies of copper have applied arbitrarily inorganic copper salts for experiments. Recent studies argue against such an approach. The natural levels and forms are factors affecting the human body, which should be seriously considered as a reference, before the modifications to humans are introduced, as history shows e.g. for lead or asbestos. Moreover, mild disturbances, caused especially by factors involved in multiple interactions with biological system, may be difficult to identify, as possibly shown by the example of aluminium. The same disturbances can be true for mild overload with nutrients over the needs, when homeostatic systems are not strong. As to copper, a very recent study suggests possible perturbation in Cu homeostasis introduced by free Cu ions which can bypass the liver and directly enter the circulation inducing toxicity [START_REF] Brewer | The risks of free copper in the body and the development of useful anticopper drugs[END_REF]).

Since DW is an everyday, whole-life factor affecting whole populations, the drinking water quality should certainly be based on long-term, not on acute health implications. Drinking water must be considered as one of the primary factors of human environment [START_REF] Frimmel | Drinking water, the most essential and not replaceable foodstuff -legal situation and assessment[END_REF], undoubtedly safe, and its quality should be high and committed overall to the most sensitive population, and especially the newborns and children. Owning to the civilisational progress and replacement of individual sources by common large water distribution systems, the water quality can be easier and better controllable than food. Thus, the author believes that the criteria for water quality and safety should be more restrictive then these for food.

Conclusions

This study showed that copper levels in drinking water were low in comparison to guideline limits. Slightly higher Cu was observed in non-urban sites but differentiation between sites was not distinct. Rural areas were weakly distinguishable from peripheral sites, supporting rationale for common, non-urban category. The contamination with Cu during over-night stagnation was common and to a similar extent for all sites, suggesting that this portion of copper arose mainly from corrosion of brass taps. According to current safety criteria, Cu was present in drinking water at safe levels and did not introduce significant risk of exceedance of the DW quality limits or nutritional criteria for 6-year old children. DW can be a minor contributor of Cu nutrient, below 4% of Polish RDA. The risk of copper RDA overload from drinking water can affect less than 4% population of 4-6-years old children using morning portion of DW or below 1% for evening one, independently on site.

It was also found that drinking water, which was contaminated with copper during overnight stagnation in pipelines, may increase potential metal intakes by 2-3 times or more.

Since this process was associated with the increase in concentrations of other toxic elements, the previously suggested recommendation for discarding a stagnating water portion from or nearby the thinnest end-part of pipeline remains valid. # Per cent of samples exceeding the limit of 1 mg l -1 . § one W1 sample exceeded current EC limit of 2 mg l -1 .

F

! Quartile range 4 -83 µg l -1 ; 95%CI: 1 -690 µg l -1 . 1180 † metal intake was calculated at assumed water consumption 1.5 L day -1 by 6-years-old children and the lower value of safe range of Polish RDA ( Cu ) for 4-6 year old children -1 mg day -1 , GM -geometric mean; MED -median; P90 (P10) -90 th (10 th ) percentile ; SDg -geometric standard deviation; P2.5, P97.5 -95% confidence interval for median was calculated as 2.5 -97.5 percentiles; ‡ number of children (in parentheses, percent in total group); * minimum and maximum; ¶ the children derived via risk criteria from whole population: 1) at DW Cu concentration > 1 mg l -1 , 2) at Cu intake over the lower RDA value (1 mg day -1 ); ¶1 For 5 children (2.4%) the predictions with W1 exceeded the higher value of the RDA range -1.5 mg day -1 -at mean (GM) intake 148% of RDA.

F

  Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow (Poland). Part 4: Copper Introduction Copper (Cu) is both, an essential micronutrient or toxic element. It is a structural component and catalytically active centre of key enzymes and plays a crucial role in human body. Cu is required for infant growth, host defence mechanisms, bone strength, red and white cell maturation, iron transport and brain development. Serious Cu deficiency is rare and occurs mainly in young infants, however, it has also been diagnosed in malnourished children and in adults (Olivares and Uauy 2005). Dietary deficiency and excessive exposure to copper including drinking water and supplements is the major concern of the world, regional and domestic organizations or task committees and was discussed in the recent documents [WHO (2007), JECFA(2006), NRC(2000), EC(2006)]

uncertainty): 1 )

 1 Intake [µg day -1 ] = (1.5±0.25 l of daily water requirement) * (Cu concentration [µg l -1 ] ±10 %), or 2) Intake [µg day -1 kg body weight -1 ]= (1.5±0.25 l of daily water requirement) * (Cu concentration [µg l -1 ] ±10 %) [body weight ±0.5 kg] -1 . The relative intake was calculated as percentage of Polish RDA values for 4-6 year-old children (safe range: 1.0-1.5 mg day -1 , Ziemlanski et al.1998).The cumulative uncertainty of the metal intake was approximated as a sum of particular relative contributions calculated with the total differential of the equations and adjusted to the coverage of 95% (Ellison et al. (ed.) 2001). The cumulative uncertainty of daily Cu intake was estimated at 53% (intake 1) or 57% of intake[START_REF] Araya | Community-based randomized double-blind study of gastrointestinal effects and copper exposure in drinking water[END_REF]. Major contribution to uncertainty (ca. 60% of total) arose from water intake.The exceedances of metal concentrations were evaluated based on the Polish limits for DW obligatory during the period of sample collections ( 1 mg l -1 (PMHa)) or EC (EC 1998) and WHO (1993) standard limit -2 mg l -1 -introduced in Poland in 2002 and harmonized with EC from 2007 (PMHc).

Definitions

  The following parameters were derived from metal concentrations in W1 (W1 Cu) and W2 (W2 Cu): R 1,2 -morning-to-evening sample concentration ratio, called coefficient of contamination D 1,2 (abbreviated by parameter D) -difference of the above concentrations, called contamination. Positive and negative D values are associated with contamination and deposition of Cu, respectively. D 1,2% (abbreviated by parameter D%) -relative change of concentration, percentage of metal amount attributed to water contamination (or deposition), here: 100% * (W1-W2)/maximum(W1;W2) Statistics Statistical analysis was performed using Statistica for Windows (StatSoft, inc.) packages. Both non-parametric (for concentrations) and parametric tests (for logarithmic values, if applicable)

  higher than upper values of respective confidence values calculated from standard deviation. Mean metal concentrations in W1 and W2 samples and derived parameters for major sites were collected in Table

  Mean metal concentrations in tap water collected in households (W1, W2) according to the suppliers are shown in Figure3. Drinking water in Kraków is supplied from five major water treatment plants and areas covered by these suppliers were shown in the previous part of the series[START_REF] Barton | Predicted intake of trace elements and minerals via household drinking water by 6-year-old children from Krakow, Poland. Part 3: Aluminium[END_REF], see also[START_REF] Zimoch | The water production subsystem failures affects of Krakow's water supply system[END_REF]. To the largest urban supply area 'A', DW is supplied from the Raba River, whose soft waters originate from the Tatry Mountains. The other sources are local rivers (B-D) with hard water and a deep well (E). The percentage of contribution of a particular source to the total water consumption is also shown under the plot.[Insert Figure3about here] Mean Cu concentrations in water from suppliers A-E in W1 (W2) were within the range 18-29 (6-16) µg l -1 . Differences among all these sources analysed together were statistically insignificant, neither in W1 nor W2 samples as estimated by Kruskal-Wallis test. However, significantly different Cu concentrations were for selected pairs of sources. Significant differences were found between W1 and W2 samples for every source.Weighted mean of Cu concentrations calculated using contributions of particular supplier 9 µg l -1 , in W1 and W2 series, respectively. These values reflect average water Cu potentially consumed by population of Krakow.

Figure 4 (

 4 Figure 4 (A, B) show the effect of overnight water stagnancy in pipelines on the changes in

(Figure 1 )

 1 indicating presence of a domain of households at excessive and specific contamination. On the contrary, for one-third of total households an opposite change was observed, i.e. decrease in concentration indicating possible deposition of Cu (lower plots, fraction 'b'). This minor fraction corresponded to points below line 1:1 in Figure2and contributed by 27%. Application of criteria of exceedance of the limit of quantitation reduced of households of fraction 'b'.

  method used with certainty, is that at the concentration change higher than LOQ. This was found only for 40% households, for which mean (GM) contamination was 73 µg l -1 .The relative indices (D 1,2% , R 1,2 ) of DW contamination by Cu from internal pipelines were comparable to these of cadmium or lead. This suggested that some of general corrosion mechanisms might be common for all these metals. The obtained relationships bring one nearer the explanation of the mechanisms of this contamination on population scale, and need separate discussion. The copper corrosion and related phenomena which can affect water quality in pipelines are a research focus and were recently widely described (e.g. Critchley et al. 2004, Edwards 2004, Shi and Taylor 2007, Zhang et al. 2008).The second minor pattern of changes in Cu concentration during stagnancy, a deposition (b), contributes from approximately one third of samples. Significant part of these samples lied close to the line 1:1, and thus was due to random analytical imprecision and spread around the line 1:1 in Figure2. Significant effect (higher than LOQ) is found for 13% households. The Cu deposition on pipelines may hypothetically involve electrochemical metal exchange (e.g. with zinc or iron), co-precipitation, binding by organic matter on surface and bacterial absorption processes. The evidence of main influence of pipeline on deposition suggested lack of effect of Cu concentration on percentage of Cu deposition, which was constant over the 50 µg l -1 in W2

  ). Lower contamination was associated with markedly lower chloride concentration in water. On contrary, the water of the lowest hardness from source A was the least contaminated for both flushed and after stagnation. Several factors are known to enhance copper corrosion (NRC 2000, Shi and Taylor 2007), which involves physical, chemical, electrochemical and biological processes (e.g.Critchley et al. 2004). For low water hardness, however, an enhanced contamination was expected. One of possible explanations may be associated with a kind, quality and age of pipelines. In this geographical region of Poland, zincplatted iron-steel and brass for pipes were the most often used materials for internal pipelines.

  houses,. This differentiation and possible trends in Cu levels should be observed in a longer time scale.

  of free copper cations in drinking water and possibly in home made beverages, involving Fenton mechanism[Jansson et al. 2003 and related papers]. This may also include other potential interactions with food and can be expected to proceed also in the alimentary tract with food and even with body cells on duct wall and thus should be of serious concern. The similar reactivity should occur with the processed foods widely introduced to human diet. Combination of food additives (including vitamin C) and free Cu from e.g. DW is another problem to be taken into account.

Figure 1

 1 Figure 1 Distribution of Cu concentrations in household drinking water (evening, W2) from

Figure 2

 2 Figure 2 Two-dimensional distribution plot of Cu concentrations in morning (W1) vs. evening

Figure 3 Mean

 3 Figure 3 Mean Cu concentrations in household drinking water according to the major supply

Figure 4

 4 Figure 4 Change in Cu concentration in tap water after over-night standing as a function of

Figure 1 Figure 2 Figure 3 FoodFigureFigure

 123 Figure 1 Distribution of Cu concentrations in household drinking water (evening, W2) from two major sub-sites, urban (U) and non-urban (NU). 221x147mm (72 x 72 DPI)

  was set at (RDA) 0.34-1.3 mg day -1 (NRC 2000, and upper tolerable intake level (UL) for adults was established at 10 mg day -1 . However, there are some uncertainties because of limitations in currently available biomarkers used to assess copper status(Cockell et al. 2008). There is common consensus of the need of surveillance and monitoring programs to continue evaluations of actual exposures to Cu of populations (Doets et al. 2008). Recently (2008), an initiative to update nutrient recommendations and reference values for EC countries and establish actual table of Dietary Reference Values (DRV) has been undertaken by European Commission and European Food Safety Authority. pilot study on DW Cu in Krakow was published previously[START_REF] Barton | Contribution of drinking water to daily copper requirement of inhabitants of Krakow. A pilot study[END_REF]). The current study presents the results of analysis of Cu concentration in household drinking water collected by a double sampling method in the Krakow metropolitan and suburban areas, and sample rural areas in southern Poland. Results were evaluated based on safety and nutritional appraisal and focussing on intake predictions for 6-year-old children.

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

-1

[START_REF] Sadhra | Dietary exposure to copper in the European Union and its assessment for EU regulatory risk assessment[END_REF]

). Polish Recommended Dietary Allowance (RDA) values for copper were approximated between 0.4-2.7 mg day -1

[START_REF] Ziemlanski | Recommended dietary Allowances for the Polish population (energy, protein, fat, vitamins and minerals)[END_REF]

. Recent Dietary Reference Intakes (DRI) for North Americans DW at natural low Cu concentrations is not generally considered to be an important Cu source, as found by a study e.g. in Poland

[START_REF] Kaluza | Contribution of food products and drinking water to the intake of minerals by elderly (in Polish)[END_REF]

, however, DW can have significant contribution to Cu intake at the tails of the distribution of dietary intakes. In some geographical EU locations Cu is present in tap DW at enhanced concentrations

(Pettersson and

Rasmussen 1999,

[START_REF] Zietz | Copper concentrations in tap water and possible effects on infant's health -Results of a study in Lower Saxony, Germany[END_REF]

).

The Cu limit in DW according to North-American recommendation was 1.3 mg l -1 NRC 2000) and 2 mg l -1 is allowed in EU (EC 1998), and from 2002 in Poland (PMHb). This provisional limit, was recently re-qualified among WHO as a health-based guideline WHO 2006) based on a short-term acute gastrointestinal effects. Long-term exposure still remains unclear. Cu can be considered to be a nutrient of high risk of excessive intake, for which the margin between the RDA and UL is very narrow

[START_REF] Grossklaus | Derivation of Maximum Levels of Vitamins and Minerals Added to Foods Based on Risk Assessment[END_REF]

). Joint Food and Agricultural Organization/World Health Organization Expert Committee on Food Additives (JECFA) has placed copper in the priority list of food additives and contaminants proposed for evaluation for toxicity and intake, its use in food additives and from other sources and included in the plan of work of the rolling revision of the DW Guidelines. WHO (2007) agreed with the need to review the guideline value for copper considering its possible toxicity in the preparations for bottle-fed infants. The revisions of drinking water guidelines for EC are also under discussion

[START_REF] Hecq | Drinking Water Regulations[END_REF][START_REF] Schumann | Dietary reference intakes for trace elements revisited[END_REF]

.

A

  The study area comprised Kraków urban and suburban sites and rural areas of south eastern Poland. The study area was

	preparation of routine quality control standard water sample. All analytes were prepared in 0.5%
	nitric acid (v/v). Other details were described previously.
	F o r
	P
	e
	e r
	R
	these individual sampling sites are referred to as "households" later in the text. e
	Among households studied, a group of about 300 children (5-7 years old) was included in analysis of metal intake. The anthropometrical data of children between sites as well as current v i e
	and total study group (Barton 2005) were homogeneous. The characteristics of the current study w
	group of children were as follows (mean±standard deviation): number of children 294 from whom 45.2% were boys; age 6.6±0.6; body weight 24.1±4.5 kg; height 1.23±0.07 m; BMI O n l 16.0±2.4 kg m -2 ; adequate water intake (AWI) was 1.55±0.08 litre calculated according to Manz y
	et al. (2002).
	Chemicals and solvents
	Water used for chemical analyses was from double water purification system Milli-Ro & Milli-Q
	(Millipore), nitric acid 65% HNO3 was from Merck (Suprapure), and metal standards were also
	from Merck. ICP Multiple Element Standard I (toxic elements, Merck, Nr.15474) was used for

supplied by five major water treatment plants producing drinking water from different sources: the Raba (A) river in Dobczyce and local rivers: Rudawa (B), Dlubnia (C) and Sanka (D) and deep well in Mistrzejowice (E). The study sites were located in urban apartment houses or private houses (415), pre-schools (158) and other public buildings (52) in the urban (389) and sub-urban (159) area of Kraków and rural areas (73) in southern Poland. For greater simplicity,

Table I

 I Copper concentration in drinking water and related parameters †

		Group			Concentration (µg l -1 )			Related parameters for fraction'a': D 1,2 >0 (contamination) ‡ Exceedance
		or parameter	Morning water samples	Evening water samples	p-level ¶	Concentration	Ratio	Difference	(percent) #
										(W2)
			(standing water), W1		(flushed), W2		(W1 vs.W2)	(µg l -1 ) ‡		R 1,2	D 1,2 (µg l -1 )	D 1,2 %
			GM	N SDg	Quartiles	GM	N SDg Quartiles Concentrati	GM	N	GM	GM	AM	W1 W2
									ons
			(median)		(95%CI)	(median)		(95%CI)	(variances)	(median)		(median)	(median)	(median)
		Urban(U)	20 (17)	364 4.77	7 -58	8 (7)	389 3.85	3 -18	*** (**)	6 (6)	245 5.46 (3.97)	20 (19)	67 (75)	0.5	0
					1 -427 &			1 -110 &
		Peripheral(P)	36 (28)	145 4.49	12 -99	20 (14)	159 3.85	8 -39	*** (0.09)	16 (12)	86	3.14 (2.21)	22 (22)	54 (55)	1.4	0.6
					2 -687			1 -274
		Rural(R)	18 (16)	75 5.74	5 -57	12 (11)	73 5.84	3 -38	** (0.96)	9 (9)	43	3.52 (3.02)	16 (17)	60 (67)	1.3	0
					1 -561			0 -364
		p ¶ (Uvs.R)	0.92(0.19)			0.10 (**)				0.69 (***)		0.14 (*)	0.87 (0.85) 0.14 (1.0)
		p ¶ (Pvs.R)	0.20(0.12)			0.54 (**)				0.27 (**)		0.54 (0.73) 0.62(0.80) 0.36 (0.64)
	Non-urban(NU)	29 (25)	220 5.05	9 -91	16 (13)	232 4.52	1 -179	*** (0.15)	13 (12)	129 3.26 (2.44)	20 (20)	56 (59)	1.4 § 0.4
					1 -684 &			1 -317 &
		p ¶ (Uvs.NU)	* (0.55)			*** (0.05)				0.14 (0.06)		** (***)	0.97 (0.59)	** (0.56)
		Total	23 (19)	584 4.92	7 -66	10 (9)	621 4.28	4 -24	*** (**)	8 (8)	374 4.57 (3.34)	20 (19) !	63 (70)	0.9	0.2
					1 -519 &			1 -179
	†	W1, W2 -samples of drinking water collected in the morning and evening, respectively; AM -arithmetic mean; GM -geometric mean; N -number of samples; SDg -geometric standard
	deviation; CI -confidence interval calculated from SDg.
	¶ p-level for concentrations (and variances in parentheses). Notations for p-level, if explicit value is not given: p≥ 0.05 (n.s.), * -p<0.05, ** -p<0.01, *** -p<0.001.
	‡ the concentrations and values of related parameters are given for fraction 'a' (D 1,2 >0 i.e. W1>W2) -the major fraction of samples. Mean metal concentrations in W1 can be calculated from
	the equation: W1 = R 1,2 * W2.
	& 97.5 percentiles were W1(W2): U: 525 (124), NU: 724(422), Total: 583(243).

  Table II Predicted Cu intake via drinking water by 6-year-old children †

			% RDA	Cu intake (µg		Daily Cu intake (µg day -1 )
				kg b.wt. -1 day -1 )
	Group	N	GM (MED);		GM (MED)	GM (MED) SDg Quartiles P10 -	P2.5 -P97.5
		(N%) ‡	P90						P90
	W1
	Urban	165	4.5 (3.5); 43		1.9 (1.5)	45 (35)	4.66 14 -144 8 -431	4 -874
	Peripheral	79	8.8 (9.8); 53		3.7 (4.0)	88 (98)	4.56 26 -251 14 -534	9 -1762
	Rural	51	4.0 (3.9); 48		1.7 (1.6)	40 (39)	5.64 10 -132 5 -478	2 -1160
	Non-urban Total Risk group 1 ¶	130 295 o 6.5 (6.2); 50 5.3 (4.6); 46 r 5 (1.7) 222 (185)		2.7 (2.5) 2.2 (2.0) 84 (87)	65 (62) 53 (46) 2220 (1850) 1.51 5.17 18 -208 9 -504 4.92 16 -175 9 -462	2 -1462 3 -1160 1760 -4620*
	Risk group 2 ¶1 11 (3.7) W2	159 (146) P		60 (61)	1592 (1462) 1.53			1055 -4620*
	Urban	163	e 1.8 (1.8); 10	0.8 (0.8)	18 (18)	3.59	9 -37	4 -105	2 -193
	Peripheral Rural	79 52	3.7 (2.6); 36 e 1.6 (1.1) r 2.5 (2.2); 26 1.0 (1.0)	37 (26) 25 (22)	3.54 14 -77 6 -285 5.73 6 -107 11 -358	5 -650 2 -350
	Non-urban	131	3.1 (2.4); 29		1.3 (1.0)	31 (24)	4.40 13 -78 2 -257	2 -505
	Total Risk group 1 ¶ Risk group 2 ¶	294 none 1(0.2)	2.3 (2.0); 16 118		1.0 (0.9) R e e 23 (20) i v 51	4.04 10 -56 4 -165	2 -393
						w
						O n l
								y

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

Acknowledgements

The author wishes to acknowledge Maria Folta for laboratory and analytical assistance; students of mother University for partial assistance in sample collection; and Professor dr hab. Zofia Zachwieja for interest in this study and for comments.