Paulo Vale
email: pvale@ipimar.pt

Susana S Gomes

Jorge Lameiras

Susana Margarida Rodrigues

Maria João Botelho

Maurice Laycock

Paulo ; Inrb / L-Ipimar

U-Amb Gomes

Susana Lameiras

Jorge Rodrigues

Susana Botelho

Maria João Laycock

M V Laycock

Assessment of a new LFIC assay for the okadaic acid group of toxins using naturally contaminated bivalve shellfish from the Portuguese coast

Keywords: lateral flow immunochromatography, LFIC, LC-MS, okadaic acid, acyl esters, hydrolysis

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

contained toxins in excess of this limit, and about 40% of the samples contained residual levels between 16-160 µg OA equiv./kg [START_REF] Vale | Two decades of marine biotoxin monitoring in bivalves from Portugal (1986-2006): a review of exposure assessment[END_REF]. The recent Regulation EC 854/2004 requires a laboratory control system to verify food business operators' compliance with the requirements for the end product at all stages of production, processing and distribution in order to verify that the levels of marine biotoxins do not exceed safety limits (European Commission, 2004b).

Liquid chromatography methods, such as HPLC coupled with fluorescence detection (HPLC-FLD)

or mass spectrometry (LC-MS), are very expensive for laboratories processing a limited number of samples, while antibody methods offer the best approach for the development of an easy-to-use, reliable and inexpensive test kit for toxin detection. Some antibodies towards okadaic acid have been reported and used in the commercial ELISA format. A Japanese kit, 'DSP-Check', has been exhaustively used to study contamination of Portuguese shellfish [START_REF] Vale | Comparison between HPLC and a commercial immunoassay kit for detection of okadaic acid and esters in Portuguese bivalves[END_REF][START_REF] Vale | Monitoring DSP toxins with immunoassay, LC-MS and mouse bioassay[END_REF]. Another type of test for these toxins was developed recently that may be suitable for use without laboratory equipment in the field, and that can be applicable to as few as one sample [START_REF] Laycock | First report of a new rapid assay for diarrhetic shellfish poisoning toxins[END_REF]. In aquaculture facilities located far from official control laboratories, the time involved in sample transportation constitutes a key factor in the overall efficiency of the food safety control scheme.

The Jellett Rapid Testing (JRT) lateral flow imunochromatography (LFIC) test for OA group toxins is a competitive immuno-assay on a test strip contained in plastic cassette as shown in Figure 1.

Polyclonal antiserum was obtained from rabbits injected with okadaic acid conjugated to a carrier protein via the carboxyl group. Conjugate pads were prepared with mixed antiserum and colloidal gold. The test line on the nitrocellulose membrane consisted of bound okadaic acid and the control line of the same carrier protein used to raise the polyclonal antibodies. To carry out a test, a methanolic extract is diluted with the running buffer, which is supplied with the tests. The composition and dilution with the buffer is designed to provide optimum conditions for the reactions to occur so that the test line is absent or faint with okadaic acid or its analogues at the regulatory and dinophysistoxin 2 (DTX2) were detected similarly with 50% reduction in test line color intensity at 5 nM for the solutions applied to test strips [START_REF] Laycock | First report of a new rapid assay for diarrhetic shellfish poisoning toxins[END_REF]. In the absence of toxin in the sample the antibody coated colloidal gold particles bind to okadaic acid on the test line and form a visible line. A visible control line indicates that the sample composition was suitable for immune reactions to occur.

This LFIC test was assessed against LC-MS with several naturally contaminated bivalves from the Portuguese coast.

Materials and methods

Bivalve samples were collected from the Portuguese coast, and belonged to the regular coastal monitoring for marine biotoxins (Figure 2). Species sampled from estuarine/lagunar environment:

blue mussel (Mytilus galloprovincialis), common cockle (Cerastoderma edule), clams (Tapes decussatus, Venerupis senegalensis), razor clams (Solen marginatus), oyster (Crassostrea spp.)

and the main species from offshore environment: clams (Donax spp. and Spisula solida).

A sample consisted of at least 30 shellfish, except for oysters, where only 12 were used, and razor clams, where 20 shellfish were used. The samples were dissected and whole flesh pooled and homogenised.

Two different batches of LFIC prototypes were acquired from Jellett Rapid Testing Ltd. (Nova Scotia, Canada), designated here as Prototype A and Prototype B, and assayed with different solvent extractant and sample preparation.

Prototype A assessment

Prototype A was assessed with bivalve samples harvested during 2005, and involved minimal sample preparation with dilution of the methanolic extract after alkaline hydrolysis for OA and DTX2 esters with the running buffer provided with the kit.

LFIC assay

Tissue homogenates (5 g) were extracted with 20 ml 80% aqueous methanol. Aliquots of 2 ml of supernatant were hydrolysed with 250 µl 1.0 M NaOH (aq) for 45 min at 40ºC, neutralised with 250 µl 1.0 M HCl (aq), and 125 µl of this mixture were diluted with 400 µl running buffer solution and 100 µl transferred to the test strips. Tissue homogenates (5 g) were extracted with 20 ml 80% aqueous methanol. Aliquots of 2 ml of supernatant were hydrolysed with 400 µl 1.0 M NaOH (aq) for 45 min at 40ºC and neutralised with 425 µl 1.0 M HCl (aq). This mixture was washed with 2 ml of hexane, extracted twice with 2 ml of dichloromethane, the combined dichloromethane fractions dried in a vacuum evaporator, resuspended in 500 µl 80% methanol and 2.5 µl injected into the LC-MS system.

LC-MS preparation

Prototype B assessment

Prototype B was assessed with bivalve samples harvested from September 2006 until April 2007, and involved evaporation by nitrogen stream of the residual methanol in the extract after alkaline hydrolysis, followed by resuspension with the running buffer.

LFIC assay

Tissue homogenates (1 g) were extracted with 3 ml methanol. Aliquots of 1 ml of supernatant were hydrolysed with 125 µl 2.5 M NaOH (aq) for 40 min at 70ºC, neutralised with 125 µl 2.5 M HCl (aq).

An aliquot of 25 µl was dried with a nitrogen stream in an Eppendorf tube, ressuspended with 100 µl running buffer solution and the whole contents transferred to the test strips.

LC-MS preparation

Tissue homogenates (2 g) were extracted with 18 ml 90% aqueous methanol. Aliquots of 2 ml of supernatant were hydrolysed with 200 µl 2.5 M NaOH (aq) for 40 min at 70ºC and neutralised with 210 µl 2.5 M HCl (aq). This mixture was washed twice with 2 ml of hexane, filtered by a disposable 0.22 µm filter, and 5.0 µl injected into the LC-MS system.

LC-MS analysis

LC-MS analysis for Prototype A assessment

The final extracts were separated for 9 minutes on a Merck Lichrospher-100 RP-18 column (5 µm, to be identical to OA, and later confirmed with a non-commercial standard.

LC-MS analysis for Prototype B assessment

Separation and LC-MS conditions were the same as above. Extraction with dichloromethane was not carried and the extracts contained high levels of NaCl from the hydrolysis reagents. In order to correct for signal suppression in the MS system, calibration standards were also prepared with a similar concentration of NaCl by diluting a stock solution of 0.96 µg OA/ml in pure methanol with a solution consisting of 2.5 M aqueous NaCl and methanol (1:5 v/v). Four calibration solutions were prepared (31-241 ng/ml).

Trials of sample preparation by LC-MS

In order to compare different hydrolysis procedures a similar solvent to tissue ratio was used.

Tissue hydrolysis:

Tissue homogenate (1 g) was hydrolysed with 125 µl 2.5 M NaOH for 40 min at 70ºC and then neutralised with 125 µl 2.5 M HCl. Extraction was performed with 3 ml of methanol. The supernantant was filtered and injected in the LC-MS system.

Extract hydrolysis:

Tissue homogenate (1 g) was extracted with 3 ml of methanol. A 1.0 ml aliquot of the supernatant was hydrolysed as above. The hydrolysate was filtered and injected in the LC-MS system.

Results and discussion

The materials and methods for the manufacture of the test strips were essentially the same for both prototype batches used in this paper. Concentrations of buffer solution components varied only slightly. The main differences between the prototypes were in sample preparation. The methanol concentration for extraction and to limit residual methanol on the test strip were critical in achieving the required sensitivity.

Although DTX1 did not occur in the bivalves used for these studies, and is uncommon in commonly exist. The maximum contribution of total DTX2 in the first set of species is commonly 40% of the total of toxins found; while in the second set of species this proportion might increase during the decay of the Dinophysis acuta blooms [START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins between blue mussel and common cockle: a phenomenon originating from the complex toxin profile of Dinophysis acuta[END_REF][START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins, part II: offshore bivalve species[END_REF].

Prototype A -raw data:

One hundred samples were tested with the first batch of JRT prototypes and results are summarised in Figure 3. Overall, 9 % of the results corresponded to false positive assays (traces),

i.e., samples with low levels of toxins that were classified as containing OA analogues at unsafe levels. Although the theoretical limit of detection (LOD) for this test was reported as 80 µg/kg [START_REF] Laycock | First report of a new rapid assay for diarrhetic shellfish poisoning toxins[END_REF], the most serious drawback found with this prototype, was a high percentage (20 %) of false negatives, i.e., samples containing toxins in excess of the current maximum allowable level in the EU of 160 µg OA equivalents/kg but not detected with the test. All samples with OA group toxins levels between 480 up to 6590 µg OA equiv./kg were classified as positive.

From past experience, these samples were judged as capable of inducing from mild to severe intoxications [START_REF] Vale | Two decades of marine biotoxin monitoring in bivalves from Portugal (1986-2006): a review of exposure assessment[END_REF]. Overall, an experimental high LOD was observed with the protocol employed with this prototype, compromising a correct detection of positive samples. According to the kit producer, this was attributable to the residual methanol in the final extract. The second batch of prototypes, was assayed resorting to prior evaporation of methanol.

Prototype B

JRT raw data:

With the second JRT batch 450 samples were tested and no false negatives were found (Figure 4).

All samples above 160 µg OA equiv./kg were correctly detected and classified as positive (n=25).

The most detrimental drawback with the protocol employed with this prototype, was a high percentage of false positives (45 % in total). From these, 37% in the range below 50 µg OA equiv./kg, i.e., the cut-off programmed in the method, and 79% in the range of 50-160 ug/kg

The detection of too many false positives is cumbersome, particularly when low levels of toxins occurred. This will require food business operators' to unnecessarily further test by a confirmatory method in a centralised laboratory a high percentage of the samples. Extracts of bivalves from clams (Tapes decussatus, Venerupis senegalensis) and oysters (Crassostrea spp.) gave fewer false positives (Table 1, Figure 3). Cockles (Cerastoderma edule) and the clams Scrobicularia plana gave low percentages of false positives in the south and SW coasts, but these were higher from the NW coast. Mussels (Mytilus spp.) had the highest percentage of false positives, which also seemed dependent on geographic location. The clams Donax spp. had a high percentage of false positives from the south and SW coast. No commercial harvest of this offshore species takes place on the NW coast, so no data are available for this species at this location. Comparison Hydrolysis temperature used in Prototype A followed for precautionary approach a mild temperature by lack of detailed information on the stability of the ester. This was corrected at the time of Prototype B, as it was found that the reaction was incomplete at 40ºC for 45 min [START_REF] Rodrigues | Optimisation of DSP esters determination and assessment of its cytotoxicity[END_REF].

Trials of sample preparation for LC-MS

The protocol for assaying Prototype B came with a suggestion from the JRT company of hydrolysing the homogenated bivalve matrix directly, instead of hydrolysing the methanolic extract, as commonly performed by research groups around worldwide [START_REF] Yasumoto | Polyether toxins produced by dinoflagellates[END_REF][START_REF] Vale | Comparison between HPLC and a commercial immunoassay kit for detection of okadaic acid and esters in Portuguese bivalves[END_REF][START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shelfish: single-laboratory validation and interlaboratory study[END_REF]. The advantage was supposed to be to avoid excessive tissue dilution. A short trial of hydrolysis carried out directly in tissue homogenates was compared with the customary preparation of hydrolysates from methanol supernatants, and is shown in Figure 6. Almost no OA group toxins were detected when hydrolysis was carried out directly in tissue homogenates, in particular in bivalve species where acyl esters comprise the majority of the toxin fraction, such as cockles, clams and razor clams. In mussels (Mytilus spp.) and Donax clams (Donax spp.), where a fair proportion of free toxins commonly exists, toxicity could be detected when hydrolysis was carried out in homogenates, and was attributed to the free toxins already present in these bivalve species. Due to the very low recovery of free toxins recognisable by the antibody, this preparation was discontinued.

Conclusions

Prototype A protocol was incapable of classifying as positive many samples that by LC-MS showed the presence of OA analogues in the range of 160-480 µg/kg. However, highly toxic samples, with the potential to cause human outbreaks of gastroenteritis, were always detected.

Prototype B protocol classified as positive all samples above 160 µg/kg as detected by LC-MS.

However, matrix effects with the second prototype were found to be a major drawback and led to false positives, particularly for mussels, due to compounds in shellfish extracts interfering with the antibodies and reducing the test line intensity.

Matrix effects seemed to be geographically and species dependent. Generally, contamination with marine bioactive compounds, such as some commonly studied marine biotoxins, occurs predominantly at the NW coast [START_REF] Vale | Two decades of marine biotoxin monitoring in bivalves from Portugal (1986-2006): a review of exposure assessment[END_REF]. Mussels eliminate several marine biotoxins (eg: okadaic acid group, saxitoxins, yessotoxins) at slower rates than other estuarine species such as cockles, clams, razors and oysters [START_REF] Artigas | Prolonged toxicity of Scrobicularia plana after PSP events and its relation to Gymnodinium catenatum cyst consumption and toxin depuration[END_REF][START_REF] Gomes | ELISA Screening for yessotoxins in Portuguese shellfish[END_REF][START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins, part II: offshore bivalve species[END_REF]. From offshore species, Donax clams eliminate OA group toxins particularly slowly, but may contain lower concentrations of yessotoxins than the clam Spisula solida [START_REF] Gomes | ELISA Screening for yessotoxins in Portuguese shellfish[END_REF][START_REF] Vale | Differential dynamics of dinophysistoxins and pectenotoxins, part II: offshore bivalve species[END_REF]. This finding led to the supposition that matrix effects might result from ingested compounds, rather than the species itself.

Further work on this kit is required before it would be fit for purpose, either in end-product testing or in monitoring programmes.

  Assessment of a new LFIC assay for the okadaic acid group of toxins using naturally contaminated bivalve shellfish from the Portuguese coast Introduction Bivalves molluscs from the Portuguese coast are seasonally exposed to Dinophysis acuminata and D. acuta blooms, which contaminate these shellfish with okadaic acid (OA) and dinophysistoxin-2 (DTX2) toxins above the current EU regulatory limit of 160 µg OA equivalents/kg (European Commission, 2004a). Between the years 2003 and 2006, about 20% of samples tested by LC-MS

  extract of 160 µg/kg of whole tissue. Okadaic acid, dinophysistoxin 1 (DTX1)

125 x 2

 2 mm) protected by a guard column packed with the same material (4 x 4 mm) using a mobile phase of acetonitrile:0.05% acetic acid (65:35, v/v) at a flow rate of 200 µL/min. The LC effluent was diverted to waste during the first 2 minutes of separation and then was introduced into the mass spectrometer. Selected ion monitoring (SIM) was used to record the signals from the [M-H]¯ ions at: m/z 803.5 (for OA and DTX2) and 817.5 (DTX1). All LC-MS equipment used (in-line degasser, quaternary pump, autosampler, column oven and 1946A single-quadrupole mass detector) was from 1100-Series from Hewlett-Packard (HP).For calibration, an OA certified reference material (CRM-OAb) from the Institute for Marine Biosciences (NRC, Canada) was used. A working solution of 0.96 µg/ml in methanol was used to point calibration curve every 6-12 samples by variable injection volumes (0.5, 2.5, 5.0 µl). Due to the close structural relation between OA and DTX2, peak area response was assumed

  Portuguese shellfish, nevertheless its presence was always screened. The OA equivalents reported always refer to the sum of OA and DTX2 obtained after hydrolysis. Samples tested during 2005 or 2006/2007 were contaminated by OA alone or simultaneously with OA and DTX2 (data not shown). In bivalve species such as common cockle (Cerastoderma edule), clams (Tapes decussatus, Venerupis senegalensis, Spisula solida), razors (Solen marginatus), oyster (Crassostrea spp.) acyl esters comprise greater than 96% of the toxin fraction, while in mussels (Mytilus spp.) and Donax clams (Donax spp.), a similar proportion of free and esterified toxins can

  cockles from Aveiro lagoon, shows false positives might appear from autumn through spring (Figure5).The use of 100% methanol, although suited for OA and DTX2 esters, might contribute simultaneously to higher recovery of interfering compounds. As the shellfish homogenate is ca. 80% water, both the initial solvent composition and the sample/solvent ratio are important for the efficiency of a single extraction. Therefore the 5g + 20 mL 80% MeOH in the prototype A equates to a final solvent strength of ca 67% MeOH, whereas in the prototype B the extraction with 1g + 3mL MeOH equates to a solvent strength of ca 79% MeOH. A commercial ELISA kit (DSP-Check) was used successfully in the Portuguese monitoring program between 2003 and 2005 for measuring these toxins. It used an 80% aqueous methanol followed by hexane washing and liquid partitioning into dichloromethane, followed by vacuum concentration[START_REF] Vale | Monitoring DSP toxins with immunoassay, LC-MS and mouse bioassay[END_REF]. For an efficient extraction of acyl esters, increase of the methanol concentration in the extraction solvent was recommended by[START_REF] Mcnabb | Multiresidue method for determination of algal toxins in shelfish: single-laboratory validation and interlaboratory study[END_REF]. In 2006, a 90% methanol extraction (final solvent strength of ca 83%) was adopted in order to improve recovery of OA and DTX2 acyl esters in shellfish samples from the Portuguese monitoring program for lipophilic toxins led to a high percentage of false positives, despite the continued use of the liquid-liquid clean-up, leading to discontinue the use of this ELISA kit in the monitoring programme (unpublished data).

Figure 1 .

 1 Figure captions:

Figure 2 .

 2 Figure 2. Localization of main inshore and offshore bivalve production areas along the Portuguese coastline. Est. = Estuary; Lag. = Lagoon; NW = northwest offshore coast; SW = southwest offshore coast. Offshore areas are numbered from north to south.

Figure 3 .Figure 5 .

 35 Figure 3. Detection of OA group toxins in methanolic extracts of bivalves by Prototype A: results grouped according to the current EU regulatory limit and 3 times the limit. Samples were harvested between May 2005 and September 2005. (N = number of samples; ND = not detected)

Figure

  Figure 6. Concentration of OA group toxins found by LC-MS in different bivalves after: hydrolysis

  Fig. 1.

Table 1 .

 1 Distribution of Prototype B false positives, with OA group toxins levels below 50 µg OA equiv. /kg, by geographic area and species.

				Nº of samples	False
				tested	positives
	Coastline	Coastline			(%)
		Lagunar/Estuarine
	NW+SW+South Ruditapes+Venerupis+Crassostrea 41	5.0
	SW+South SW+South NW NW	Cerastoderma+Scrobicularia F Mytilus Cerastoderma+Scrobicularia o r Mytilus	50 20 85 81	14.0 35.0 37.6 72.8
	SW+South SW+South	Offshore Spisula + others P e Donax	32 20	9.4 45.0
	NW	Spisula	r e	13	53.8
			R
			e
				v i e
				w
					O n l
					y
					1

6. Concentration of OA group toxins found by LC-MS in different bivalves after: hydrolysis carried out in tissue homogenate prior to methanol extraction (tissue) or, hydrolysis carried in the methanolic extract (extract).

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

Acknowledgements

Programme "Safety, Surveillance and Quality of Bivalve Molluscs" (QCAIII/med.4/MARE Programme) supported this work. We acknowledge the biotoxin laboratory technicians for preparing the bivalve homogenates.


Castro Barrilero

Elena ; Crea

Keywords: Nitrate, Lettuce, Sewage sludge, Wastewater

the third crop season, a significant increase in nitrate content was observed in lettuce 1 from organic treatments. Nitrate concentration in lettuce from irrigated treated 2 wastewater was higher than control plants, although significant differences were not 3 found.

Juan, Spain. Six plots of 36 m 2 each, were set up in agricultural land. A drip irrigation 13 system was used to water all plots: five plots with drinking water and one plot with 14 wastewater from Wastewater Treatment Plant (WWTP) from an Activated Sludge 15 system. One drinking water irrigated plot was not fertilized and was considered as a 16 control. Five different treatments were applied to the soil: Three organic mixtures 17 (sewage sludge, sewage sludge mixed with pine bark and municipal solid waste with 18 composted sludge) and a conventional fertilizer were applied to the soil in each of four [START_REF]Servicio de Asesoramiento a la Fertilización[END_REF] plots irrigated with drinking water. The last plot was irrigated with treated wastewater. 20 These treatments were tested for their effects on plant growth and nitrate concentration 21 in vegetable tissue. An increase in fresh weight in the lettuce was linked to the dosage 22 of sewage sludge. The highest nitrate level was observed in the sewage sludge treatment 23 in all crops and seasons, although in general, all values were below the maximum limits 24 established by the European Commission for nitrate content in fresh romaine lettuce. In 25

Introduction 6
Nitrogen is one of the principal nutrients that determine crop production. Nitrogen 7 deficiency lowers production, but an excess can be harmful to people and the 8 environment. This is due to the fact that nitrate is one of the most important nitrogen 9 compounds in the soil for plant nutrition, it is very soluble and can be transported in 10 water to the aquifers. Generally, high quantities of nitrogen are applied to horticultural 11 crops using both inorganic and organic fertilisers. In addition, there is a high potential 12 of groundwater nitrate pollution due to the low efficiency of nitrogen utilization by this 13 type of crop. Excessive fertilization can lead to cumulative levels of nitrate exceeding 14 the maximum levels recommended for human consumption [START_REF] Ramos | El abonado nitrogenado de los cultivos hortícolas[END_REF]. 15 16 Nitrate in vegetables, as well as in water and generally other type of foods is relatively 17 non-toxic, but approximately 5% of all ingested nitrate in saliva and the gastrointestinal 18 tract is converted into nitrite, which is more toxic (Santamaria 2006). Nitrite interacts 19 with haemoglobin, causing blood to be less efficient in transporting oxygen and 20 resulting in a condition known as methaemoblobinaemia [START_REF] Merino | Levels of nitrate in 21 Swedish lettuce and spinach over the past 10 years[END_REF]. Nitrite may 21 also combine with secondary or tertiary amines to form N-nitroso compounds. These 22 compounds have been shown to produce cancer in a wide range of laboratory animals 23 (Zeegers et al. 2006). An acceptable daily intake (ADI) for 0-3.7 mg kg -1 body weight 24 nitrates contributing approximately 80-92% of the average daily intake [START_REF] Prasad | Nitrate-N determination in leafy vegetables. Study of the 13 effects of cooking and freezing[END_REF]. 5

The regulatory limits for tissue nitrate concentrations in fresh romaine lettuce are as 6 follows (in each case values are presented for crops grown either under cover or in the 7 open air, on a fresh weight basis): 4500 and 4000 mg NO 3 -kg -1 for lettuces harvested 8 from 1st October to 31 st March; 3500 and 2500 mg NO 3 -kg -1 for lettuces harvested 9 form 1 st April to 30 st September (European Commission 2005). 10 11 Accumulation of nitrates in vegetables, especially lettuce, has been shown to be affected 12 by several factors (genetic, environmental and agricultural). Studies have identified 13 nitrogen fertilization and light intensity as the major factors that influence nitrate 14 content in lettuce [START_REF] Maynard | Nitrate accumulation in 15 vegetables[END_REF], Tamme 2006). Photosynthetic activity depends 15 on the intensity of light and if this intensity is reduced, the transformation of the 16 absorbed nitrates into amino acids and proteins will decrease. In conditions of low 17 luminosity the accumulation of nitrate will be higher and there will be a higher 18 concentration of nitrates in the plants. Due to this, the maximum limits allowed are 19 higher in crops grown in autumn -winter or in a greenhouse [START_REF] Gunes | Effects of partial replacement of nitrate by NH 4 -5 N, urea-N and amino acid-N in nutrient solution on nitrate accumulation in 6 lettuce (Lactuca sativa, L.)[END_REF]. 20 21 On the other hand, among the possibilities of water resource availability, the re-22 utilization of water is considered a non-conventional resource that can increase the 23 utility of water. Reu-se for agricultural irrigation is the most common application in 24 practically the whole world [START_REF] Qadir | Crop and irrigation management strategies for saline-sodic 15 soils and water aimed at environmentally sustainable agriculture[END_REF]). However, water re-use should be 25 planned for increasing water flow levels available in a specific area, substitute flows, 1 reduce the negative impact of waste and improve the integrated management of water 2 [START_REF] Iglesias | Present and future of wastewater reuse in Spain[END_REF]. Within the water treatment process, it is impossible to exclude 3 the generation of a solid waste, called sludge, for which an efficient application must 4 also be found and described following the specific norms for this waste product. This 5 material can be an important source of nutrients for the soil [START_REF] Mena | Bioremediation of sewage sludge by 19 composting[END_REF]. Using 6 this material as a fertilizer can benefit the environment by turning wastes into valuable 7 resources; otherwise, the sludge would have to be disposed of by landfilling, storage in 8 lagoons, incineration, or ocean dumping. On the other hand, heavy metals found in 9 sewage sludge may sometimes present environmental problems. Several practices, 10 similar to those used with other organic fertilizers, will maximize the benefits of using 11 sewage sludge while minimizing the risks [START_REF] Dai | Characteristics of sewage sludge and distribution of 18 heavy metal in plants with amendment of sewage sludge[END_REF]. 12 13 Nitrogen in sludge is in the form of ammonium or organic nitrogen, with only a small 14 fraction in the nitrate form. All of the nitrogen in the ammonium and nitrate forms is 15 immediately available to the plant. Organic nitrogen in sludge must be mineralized 16 before it can be assimilated by crops. Research shows that 10 to 50 percent of organic 17 nitrogen will become available the first year after application, depending on the type of 18 treatment used in producing the sludge [START_REF] Pascual | Estudio de la aplicación de lodos de depuradora en el área rural 4 mediterránea[END_REF]. 19 20 In this study the nitrate concentration was compared in lettuce leaves treated with 21 different fertilizers including organic and inorganic fertilizers and irrigated with 22 different types of water, including treated wastewater. This allowed us to examine the 23 cumulative effect of these treatments on nitrate content and the influence of the season 24 in which crops were cultivated. In addition, the residual effect of sewage sludge on 25 central Spain (39º 24' N; 3º 12' W, altitude 644m). This treatment plant purifies 10 wastewater using the biological system of Activated Sludge. This is a sewage treatment 11 process in which air or oxygen is forced into sewage liquid to develop a biological floc 12 which reduces the organic content of the sewage. The origin of wastewater was mixed: 13 70% of domestic origin and 30% industrial from two industrial zones. Most of these 14 industries are related to cheese and wine manufacturing. Treated wastewater is reused 15 for the conservation of the Natural Reservation of Alcázar de San Juan, composed of 16 two steppe lagoons. To prepare for this use, at the end of the wastewater treatment 17 process nitrogen and phosphorus are eliminated from the treated water. The solid waste 18 from water purification is re-used by some farmers in the area as organic amendment for 19 cereals and grapevines. 20 Six plots of 36 m 2 each, were set up in agricultural land. Two types of water treatment 1 were considered: drinking water in five plots and treated wastewater in the last plot. 2 3 One drinking water irrigated plot was not fertilized (neither organic nor inorganic 4 fertiliser) and was considered the control (DW). The irrigated, treated wastewater plot 5 did not receive fertilizer treatment (WW). In the other three plots, high doses of sewage 6 sludge were used: 40 t ha -1 of air-dried sewage sludge (SS), 40 t ha -1 of air-dried sewage 7 sludge mixed with pine bark (SSPB) in a 3:1 ratio (three parts sludge and one part pine 8 bark) and 40 t ha -1 municipal solid waste with composted sludge (MSWC) in each. In 9 the last plot a conventional fertilizer dose adapted to crop needs (1000 kg ha -1 ) was 10 used. The treatments were carried out a week before the transplant and no other type of 11 treatment was performed during the crop growth period. 12 13

F
The plant selected was Lactuca sativa L. var. "Líbano" (lettuce) and planting was 14 carried out every season with a density of 60,000 plants ha -1 due the fact that this crop is 15 considered one of the highest accumulators of nitrates in the leaves [START_REF] Merino | Levels of nitrate in 21 Swedish lettuce and spinach over the past 10 years[END_REF]. [START_REF] Maynard | Knott's Handbook for vegetables growers[END_REF] Dates of transplanting and harvesting, crop duration and climatic characteristics in each 17 crop season are described in Table 1. 18 19 Drip irrigation was used regularly to avoid drought stress in each plot. To calculate the 20 water needs for this crop we followed the methodology proposed by the FAO 21 was applied to study the cumulative effect. In the other half, no treatment was applied to 4 evaluate the residual effect. 5

Sampling 6

Treated wastewater and drinking water were analysed once a month while the crop was 7 in the field. Significant differences between both types of water (drinking and treated 8 wastewater) were recorded (Table 2). The composition of the two types of irrigation 9 water did not vary significantly over the study period, so the mean values ± standard 10 deviation for the chemical properties are presented. Electrical conductivity (E.C.), 11 carbonate, bicarbonate, total phosphorus, nitrogen, nitrite, sulphate, chloride, inorganic 12 carbon (IC), organic total carbon (TOC), total carbon (TC), sodium, potassium, 13 magnesium, copper, iron and manganese have significantly higher values in treated 14 wastewater than in drinking water. However, nitrate content was significantly lower in 15 treated wastewater. [START_REF] Maynard | Knott's Handbook for vegetables growers[END_REF] 17 Dried sewage sludge, sewage sludge mixed with pine bark and municipal solid waste 18 with composted sludge were analysed once a crop season before incorporating them into 19 the soil. Chemical characteristics of the organic mixtures are given in Table 3. In each crop season, when plants were estimated to have reached their highest 1 commercial size they were harvested by cutting the stems at the soil level. Ten plants 2 from each plot were harvested randomly for nitrate determination. 3

Analytical methods 4

Immediately after harvest the fresh weight of lettuces was measured. The leaves were 5 washed off with doubly-distilled water and blotted dry with absorbing paper. To carry 6 out nitrate extraction, samples of 300 g of fresh lettuce were taken and dried in a forced 7 air oven (Memmert, model 800) to 70 ºC for 48 h. The dried leaves were then ground in 8 a mill. A quantity of 100 mg of milled sample was added 20 ml of doubly-distilled 9 warm water (80 ºC) and the mix was shaken for 30 min, using a mechanical agitator 10 (Heidolph Unimax, mod. 2010). The resulting solution was filtered through borosilicate 11 membrane filters of 0.45 mm porosity. Nitrate content in the solution was determined 12 by ionic chromatography using a module of chemical suppression and conductivity 13 detection using the ISO method (UNE-EN 10304-1 1995). 14 15 Soil and sewage sludge samples (0.5 g dry weight) were prepared for analysis with acid 16 digestion in 4 ml of HNO 3 , 0.25 ml of H 2 O 2 and 2 ml of HF by applying the 17 temperature program according to Milestones Cookbook of microwave application 18 notes for MDR technology. Samples were analysed with the following techniques: pH 19 with a previously calibrated CRISON GLP22 pH meter and 1:2 w v -1 suspension of soil 20 in water [START_REF] Peech | Hydrogen ion activity[END_REF]); organic matter using the Walkley Black method (Allison 1965); 21 electrical conductivity (Bower & Wilconx 1965); total N by the Kjeldahl procedure 22 [START_REF] Bremmer | Total nitrogen[END_REF]); soil and sewage sludge available P using the Olsen method (Olsen & 23 Dean 1965); Na and K using the atomic emission spectroscopy method (MAPA, 1994), 24 Ca and Mg, using the atomic absorption spectroscopy method (MAPA, 1994), Fe, Zn 1 and Mn using the DTPA extraction method [START_REF] Lindsay | Development of a DTPA soil test for zinc, iron, 10 manganese and copper[END_REF]. 2 3 Wastewater samples were prepared for analysis with a previous acid digestion applying [START_REF] Gunes | Effects of partial replacement of nitrate by NH 4 -5 N, urea-N and amino acid-N in nutrient solution on nitrate accumulation in 6 lettuce (Lactuca sativa, L.)[END_REF] the temperature program according to Milestones Cookbook of microwave application 5 notes for MDR technology. Wastewater and drinking water samples were analysed with 6 atomic emission method to determinate Na (limit of detection (LOD): 0.05 mg l -1 ) and 7 K (LOD: 0.01 mg l -1 ); atomic absorption spectroscopy method to determine Fe (LOD: 1 8 µg l -1 ), Al (LOD: 3 µg l -1 ), Cu (LOD: 2.5 µg l -1 ) and Mn content (LOD: 0.15 µg l -1 ) 9

according APHA-AWWA-WPCF (1998); and ionic chromatography method to 10 determine chloride (LOD: 2 mg l -1 ), sulphate (LOD: 1 mg l -1 ), nitrate content (LOD: 1 11 mg l -1 ). Other chemical parameters such as N-Kjeldahl, total P, BOD 5 , COD, Electrical 12 conductivity, Carbonate and Bicarbonate were determined according APHA-AWWA-13 WPCF (1998). No significant changes were recorded for pH and MgO in the soil throughout the study 1 in any of the plots that had received any type of treatment (Table 4). However, 2 significant increases in organic matter, phosphorus and copper in organic treatment 3 plots (SSPB, SS and MSWC) were obtained. A significant increase in nitrogen in these 4 plots was observed. The highest content of this nutrient at the end of the study was in 5 the SS treatment plot, mainly due to the sewage sludge dose applied and to the sewage 6 sludge mineralization during the study. These results have been reported in other studies 7

(

Lettuce plant growth 13

In the first crop season and after harvesting, the average weight of fresh lettuce from the 14 SS plot (846 g) was significantly higher than in all treatments (Figure 1). Significant 15 differences among the fresh weight of plants from FER, SSBP and MSWC treatments 16 plots were not observed. Lettuce fresh weight from DW and WW treatment plots was 17 very low (below 100 g), indicating that growth was very deficient due to the lack of 18 important nutritional elements [START_REF] Maynard | Knott's Handbook for vegetables growers[END_REF]. 19 20 In the second crop season solar radiation was higher, although temperatures were 21 similar to those in the previous crop season. Also, in the second crop season, an increase 22 in weight was produced in all treatments except in plants from DW plot. As in the first 23 crop season, lettuce growth from the SS treatment plot was significantly higher. During 24 the third crop season (autumn-winter), characterized by the lowest temperatures and the 1 least daily sunlight, reduced plant growth was observed in all treatments. An exception 2 to this was for lettuce from the SSPB treatment plot, where significant differences in 3 fresh weight were not observed between the three crop seasons. 4 5 It is possible to affirm that an increase in fresh weight in the lettuce is linked to the dose 6 of sewage sludge. These results have been reported in other studies [START_REF] Mañas | Aptitud agronómica del agua residual depurada y lodos generados en 12 una Estación Depuradora de Aguas Residuales de lechos bacterianos[END_REF]) in 7 which increasing the dose of sludge leads to an increase in the weight of fresh matter. In 8 this study the quantity contributed in the SS treatment was 10 t ha -1 higher than in SSPB 9

treatments. 10

Nitrate content in lettuce 11

A total of ten lettuces from each treatment were analysed in each crop season. The 12 maximum average value found was 1832 mg kg -1 in the SS treatment plot in the third 13 crop season, which is below the maximum limits for levels of nitrate in lettuce 14

established by European Commission (2005). 15 16

In the first cultivation period the median nitrate content in lettuces leaves from the SS 17 treatment plot (940 mg kg -1 ) was significantly higher than the concentration observed in 18 lettuces from the FER (387 mg kg -1 ) and SSPB (439 mg kg -1 ) treatment plots, where 19 significant differences were not observed (Figure 2). 20 21 In the second crop season an increase in nitrate concentrations was observed in all 22 treatments but it was only significant in SSPB treatment. The plants from the SS and 23 SSPB treatment plots presented significantly higher nitrate content (1262 mg kg -1 and 24 1220 mg kg -1 ) (Figure 3). Significant differences between lettuces from DW, WW, FER 1 and MSWC treatment plots were not observed. European regulatory limits were not 2 exceeded for any treatment. However, Figure 3 shows that the nitrate content in a 3 sample from the SSBB treatment reached the limit without exceeding it (2477 mg kg -1 ). 4

It is important to emphasize the low nitrate concentration in lettuces from DW and WW 5 treatments, as these plants had deficient growth. 6 7 In the last crop season, nitrate concentration in lettuces from the SS treatment was 8 significantly higher (1832 mg kg -1 ). [START_REF] Pavlou | Effect of organic and inorganic 7 fertilizers applied during successive crop seasons on growth and nitrate 8 accumulation in lettuce[END_REF] reported similar results in 9 lettuce treated with organic fertilizer during the fall crop season. Significant differences 10 between the plants treated with SSPB and MSWC were not observed. Nitrate content in 11 lettuces from the FER treatment was lower than nitrate levels in plants treated with 12 organic fertilizer (Figure 4). 13 14 When crop seasons are compared, an increase in nitrate content in lettuce leaves was 15 observed from one crop season to another. This was consistent for all treatments, 16 although significant differences among DW, WW and FER treatments were not 17 observed. These results show that the continued application of SS, SSPB and MSWC 18 treatments for the three cultivation periods increased the nitrate concentration in 19 vegetable tissue in each crop season, although in the last crop season the reduction of 20 hours of sunlight could influence the increase in nitrate content in lettuce. Byrne et al. 21 (2004) occur in treatments DW, FER and WW in which an increase was produced, but it was 1 not significant. For these three treatments and specifically for the FER treatment, an 2 adjusted dose based on the requirements of the crop was applied for the three crop 3 seasons. No significant differences in nitrate concentration from the FER treatment were 4 observed between crop seasons, despite the reduction in hours of sunlight in the third 5 crop season. It can therefore be stated, as in other studies [START_REF] Pavlou | Effect of organic and inorganic 7 fertilizers applied during successive crop seasons on growth and nitrate 8 accumulation in lettuce[END_REF]), that the 6 nitrate concentration of romaine lettuces leaves depends more on the fertilizer type, 7 dose, and repeated applications (accumulative effect) than on the time of the year in 8 which the crop is grown. [START_REF] Brand | Organic agriculture: does it enhance or reduce the 7 nutritional value of plant foods?[END_REF] showed that the production method 9 (organic or conventional farming) has great importance in obtaining low nitrate levels in 10 vegetables. 11 12 The residual effect of the SS and SSPB treatments (Figure 5) was that plants treated 13 with SS in the first crop season showed a significant decrease in nitrates during the 14 second and third crop seasons. Plants treated with SSPB showed an increase in nitrate 15 levels during the second and third crop seasons, even when no extra treatments were 16 applied during these second and third crop seasons. The nitrate content in lettuce 17 samples harvested from the third crop season was 833 mg kg -1 , higher than was 18 observed in lettuces treated with FER treatment during the third crop season. 19

Conclusions 20

In this study, nitrate content in romaine lettuce leaves was shown to depend on fertilizer 21 type and dose rather than on crop season. The hours of sunlight in winter are lower than 22 in spring, so crop development in autumn-winter requires a longer time in the field than 23 spring crops. In this study, the total daily hours of sunshine in both seasons was 24 approximately equal, meaning that lettuce cultivation in different seasons does not 1 affect the nitrate concentration in vegetable tissue. Irrigation with treated wastewater 2 from the WWPT of Alcázar de San Juan did not produce high nitrate levels in lettuce 3 leaves, mainly due to the elimination of a high quantity of nitrogen from the water, 4 which avoids eutrophication when it is re-used. 5 6 On the other hand, high application rates of sewage sludge produces an increase in N, P 7 and organic matter reserves in the soil, which will be converted into chemical forms 8 available to crops over time. This does not occur when treated wastewater is used. 9 10 Town Hall of Alcázar de San Juan for financial support through the project: "Aptitud 13 para uso Agricola del Agua y Lodos Procedentes de la Depuradora de Aguas Residuales 14 de Alcázar de San Juan". The authors would like to thank to the laboratory of the 15 Environment Science Centre (CSIC) and specifically Dr. Alfredo Polo for help with the 16 Zeegers M, Selen R, Kleinjans J, Goldbohm A, Van 4: Chemical characteristics of the soil before transplanting in the first crop season 3 (B) and after harvesting in the third crop season (A). For the same parameter, averages 4 followed by different letters denote significant differences according to Fisher's 5 multiple range test (LSD; p< 0.05). treatments and maximum level (ML winter = 4000 mg kg -1 ). Different letters denote 15 significant differences according to Fisher's multiple range test (LSD) (p<0.05). The dot 16 represents the mean value. 17

6 Centro 9

 69 , M. P. MAÑAS & J. DE LAS HERAS 5 Regional de Estudios del Agua (CREA). Universidad de Castilla-La Mancha. 7 Ctra. Las Peñas, km3.2. 02071. Albacete. Spain. 8 Abstract 10 A romaine type lettuce (Lactuca sativa L.) was cultivated during three crop seasons 11 (spring 2005, spring 2006 and autumn-winter 2006) in the same soil in Alcázar de San 12

3

  of 0-0.07 mg kg -1 body weight for nitrite was established by the 1 European Commission Scientific Committee on Food (SCF 1995; Speijers 1996). 2 It has been estimated that vegetables constitute a major source of human exposure to 4

For

  the experiments, treated wastewater and sludge from the WWPT of Alcázar deSan 21 Juan were air-dried, and drinking water from urban underground supply were used.

(

  Doorenbos & Pruitt 1984), which calculates the Evapotranspiration of Reference (ETo) 22 using the Penman-Monteith method and applies the crop coefficient (Kc). Since the ETo 23 and Kc values are known, Evapo-transpiration, and therefore water needs, could be 24 and third crop seasons, SS and SSPB treatment plots were divided 2 into two subplots. In one half the same dose of treatment as in the previous crop season 3

20 21

 20 Before transplanting in the first crop season and after harvest in the third crop season 22 soil was sampled for characterization from ten samples at random inside each plot, at a 23 depth of 15 cm, with three repetitions (S.A.F., 2005). 24 25

14 15

 14 Statistical design 16Data were subjected to ANOVA treatments and the method used to discriminate among 17 the means was Fisher's least significant difference (LSD) for p<0.05. To ensure that 18 data came from a normal distribution, standardized skewness and standardized kurtosis 19 values were checked. Percentage values considered were transformed by arcsine.

  De las Heras et al. 2005). Few changes in DW and WW plots were recorded during the 8 study. Only K 2 O content increased in the plot irrigated with treated wastewater after 9 harvesting in the third crop season, and in both plots there was a decrease in the C:N 10 ratio for the same time period. Micronutrient content (Fe, Mn, Cu and Zn in organic 11 treatment plot and DW and WW plots) was relatively constant throughout the study. 12

  chemical analysis. Thanks to Stefanie Kroll and Steven Watkinson for reviewing the 1965. Organic Carbon. In: Black CA, Evans DD. White JL, Ensminger L, 21 Clark FE (Eds.), Methods of Soil Analysis. American Society of Agronomy, 22 Madison, Wisconsin, pp: 1367-1378. 23 Committee On Food), Opinion on nitrate and nitrite, expressed on 22 1 September 1995 (Anex 4 to Document III/5611/95, ed by European 2 Commission, Brussels, p 20. 3 Speijers GJA. 1996. Nitrate in Toxicological evaluation of certain food additives and 4 contaminants in food; ed by World Health Organization, Food Additives Series 5 35, Geneva, pp 325-360. 6 Tamme T, Reinik M, Roasto M, Juhkam K, Tenno T, Kiis A., 2006. Nitrates and nitrites 7 in vegetables and vegetable-based products and their intakes by the Estonian 8 population. Food Additives and Contaminants 23 (4): 355-361. 9 UNE-EN 10304-1. 1995. Determinación de iones fluoruro, cloruro, nitrito, ortofosfato, 10 bromuro, nitrato y sulfato disueltos, por cromatografía en fase líquida. 11

Figure 1 : 3 Figure 2 : 7 8Figure 3 : 12 13Figure 4 :

 13273124 Figure 5 17

18

 18

Figure 5 :

 5 Figure 5: Residual nitrate content of fresh lettuce in second and third crop season SS 19and SSPB treatment and maximum levels (ML summer = 2500 mg kg -1 , ML summer = 20 4000 mg/kg -1 ). Different letters for the same treatment denote significant differences 21

Table 1 :

 1 den Brandt P. 2006. Nitrate intake 12 does not influence bladder cancer risk: the Netherlands Cohort study. 13 Cultivation practices and climatic conditions during the crop seasons. 4

	14	Environmental Health Perspectives 114 (10): 1527-1531.

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	1 1 1
	2 2	DW	WW	FER	SS FER	SSPB SSPB	MSWC	SS	MSWC
	F o r P e e r R e v i e w DW COD (mg l -1 ) < 10 BOD 5 (mg l -1 ) Undetected pH 8.1 ± 0.3a E.C. (microscm -1 ) 624 ± 30.2a Carbonate (mg l -1 ) Undetected Bicarbonate (mg l -1 ) 180 ± 15.6a T. Phosphorus (mg l -1 ) 0.3 ± 0.05a pH 6.8 6.9 E.C. (mmhos cm -1 ) 9.3 6.6 7.8 ± 0.5a 7.9 ± 0.1a 7.7 ± 0.1a 7.7 ± 0.2a 60.2 ± 65.8 WW 8 ± 5.5 7.8 ± 0.6a 2085 ± 602b 43.8 ± 28.5 396 ± 98.9b 7.4 11.5 Organic matter (%) 29.1 73 7.9 9.2 ± 1.2a 9.6 ± 1.2a 8.7 ± 1.1a 8.5 ± 1.1a C:N 4 38 5 N (%) 10 4 1.1 0.9 0.8 ± 0.05a 0.9 ± 0.2a 0.6 ± 0.1a 1.2 ± 0.2a 0.6 ± 0.1a 0.8 ± 0.1a 0.6 ± 0.1a 2.5 ± 0.3b 3.1 ± 0.4b 7.8 ± 0.8a 8.5 ± 0.4a 2 ± 0.05a P 2 O 5 (mg kg -1 ) 120000 5289 4566 989 K 2 O (mg kg -1 ) 170000 3622 1485 6526 Na (mg kg -1 ) 3622 989 0.04 ± 0.01a 0.1 ± 0.02a 0.1 ± 0.01a 0.1 ± 0.01a 0.05 ± 0.01a 0.1 ± 0.01b 0.1 ± 0.01b 0.1 ± 0.01a 0.2 ± 0.03b 0.3 ± 0.04b 3199 3.3 ± 2.1b N-Kjeldahl (mg l -1 ) Undetected 20.8 ± 15.5 N-Ammoniacal (mg l -1 ) Undetected CaO (%) 1.1 0.4 1.4 MgO (%) 2 0.2 0.2 27.5 ± 1.8a 18.3 ± 1.3a 103 ± 5.7a 148 ± 14.9a 114 ± 4.a 27.5 ± 3.5a 14.4 ± 5.3a 141 ± 18.3b 822 ± 106b 1259 ± 162b 0.3 9.4 ± 9.4 Nitrite (mg l -1 ) < 0.01 2.2 ± 1.1 Nitrate (mg l -1 ) 27.9 ± 0.9b 6.9 ± 6.9a Sulphate (mg l -1 ) 73.6 ± 3.1a 301 ± 101b Chloride (mg l -1 ) 53.5 ± 5.9a 227 ± 28.7b IC (mg l -1 ) 46.2 ± 2.6a F o r Zn (mg kg -1 ) 259 108 300 Cu (mg kg -1 ) 109 23 210 Fe (mg kg -1 ) 2000 5421 4007 9301 Mn (mg kg -1 ) 8.6 45 178 F 769 ± 50.3a 830 ± 12.6a 699 ±10.2a 506 ± 22.5a 647 ±15.8a 628 ± 81a 1388 ± 179b 605 ± 78a 398 ± 51.4a 414 ± 53.4b o r 0.7 ± 0.05a 0.8 ± 0.02 2.8 ± 0.2b 2.5 ± 0.2a 2.7 ± 0.1b 0.8 ± 0.10a 0.7 ± 0.1a 1.9 ± 0.3a 2.2 ± 0.28a 1.7 ± 0.22a 135 ± 24.8b TOC (mg l -1 ) 0.3 ± 0.5a 33.9 ± 38.5b Na (mg l -1 ) 20.9 ± 1.2a 318 ± 103b K (mg l -1 ) 1.9 ± 0.1a 106 ± 34.3b Ca (mg l -1 ) 85.7 ± 12a 138 ± 74.8a Mg (mg l -1 ) 32.3 ± 13.3a 54.7 ± 21.8b Zn (mg l -1 ) <0.2 0.4 ± 0.2 P e e r Table 3: Chemical characteristics of sewage sludge (SS), sewage sludge + pine bark B A B Organic matter (%) 3 pH 4 5 6 7 8 9 A B N (%) A 10 11 B P 2 O 5 (mg kg -1 ) A 12 13 14 15 B K 2 O (mg kg -1 ) A B CaO (%) A 16 17 18 (SSPB), municipal solid waste compost (MSWC) and fertilizer (FER). 19 20 B 0.1 ± 0.01a 0.1 ± 0.01a 0.1 ± 0.01a 0.1 ± 0.01a 0.1 ± 0.01a MgO (%) A 0.1 ± 0.01a 0.1 ± 0.01a 0.04 ± 0.01a 0.04 ± 0.01a 0.1 ± 0.01a P e B 25136 ± 1643a 25592 ± 56a 24883 ±3492a 33252 ± 1003b 24494 ±1655a Fe (mg kg -1 ) A 24295 ± 3133a 22279 ± 2873a 21013 ± 2710a 19114 ± 2465a 24329 ± 3137a 25986 ± 3351a 7.9 ± 1a 8.6 ± 1.1a 1.8 ± 0.2a 3.2 ± 0.4b 0.1 ± 0.01a 0.3 ± 0.03b 135 ± 17.4a 446 ± 57.5b 1506 ±194b 853 ± 110a 3.3 ± 0.1b 1.5 ± 0.2a 0.1 ± 0.01a 0.1 ± 0.01a 21339 ±2752a e B 212 ± 21.3a 196 ± 5.6a 195 ± 25.6a 205 ± 13.5b 220 ± 22.1b 153 ± 19.8a Mn (mg kg -1 ) A 180 ±23.3a 178 ± 24a 172 ± 22.1a 167 ± 21.6a 145 ± 18.7a 180 ± 23.2a r Cu (ug l -1 ) 2.4 ± 2.7a 144 ± 112b Mn (ug l -1 ) 0.4 ± 1.2a 102 ± 128b Fe (ug l -1 ) 8.2 ± 4.7a 2314 ± 2427b 3 R e B 37.5 ± 3.8a 38.5 ± 5.1a 40.5 ± 5.6b 56.5 ± 2a 47 ± 4.7a 41 ± 5.3b Zn (mg kg -1 ) A 31.8 ± 4.1a 30.1 ± 3.4a 30.2 ± 3.9a 49 ± 6.3a 54.1 ± 6.9a 10 ±1.3a R B 11 ± 1.1a 11 ± 2a 11.5 ± 2.3a 11 ± 0.3a 11.5 ± 1.2a 12.7 ± 1.6a Cu (mg kg -1 ) A 13 ± 1.7a 13 ± 1.7a 13.5 ±1.7a 22.5 ± 2.9b 21.7 ± 2.8b 53.8 ± 6.9b e 4 Table 2: Chemical characteristics of drinking water and wastewater. Different letters 5 mean significant differences among columns (p<0.05) w w 6 v i e v B 12 ± 0.8b 7 ± 1b 5 ± 0.6a 9 ± 1a 10 ± 0.6b 9 ± 1.2b C:N A 4 ± 0.5a 4 ± 0.5a 4 ± 0.5a 7 ± 0.9a 6 ± 0.8a 7 ± 0.9a i e 2
						O n l O n l O n l
							y y y

Table

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Acknowledgements 11

The authors are grateful to the municipally-owned company "Aguas de Alcázar" and 12


Franz Berthiller

Christian Hametner

Pamela Krenn

Wolfgang Schweiger

Roland Ludwig

Gerhard Adam

Rudolf Krska

Rainer Schuhmacher

Keywords: mycotoxins, mass spectrometry, nuclear magnetic resonance, zearalenone

Preparation and characterization of the conjugated

Fusarium mycotoxins zearalenone-4O-β-D-glucopyranoside, α-zearalenol-4O-β-D-glucopyranoside and β-zearalenol

Introduction

Zearalenone (ZON) is an estrogenic mycotoxin produced by several Fusarium spp. Both its toxicological relevance (JECFA, 2000) and analytical methods for its determination in cereals (Krska and Josephs) have been reviewed. Living plants have an amazing capability to transform xenobiotics into more polar and usually less toxic conjugates. Since several Fusarium spp. excrete mycotoxins like ZON or deoxynivalenol into the tissue of infected host plants, these compounds are also the subject of such biotransformations [START_REF] Berthiller | Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry[END_REF]. About 20 years ago, zearalenone-4O-β-D-glucopyranoside (ZON4G) was identified by [START_REF] Engelhardt | Transformation of the Fusarium mycotoxin zearalenone in maize cell suspension cultures[END_REF] as a transformation product of ZON in maize cell suspension cultures.

Shortly thereafter, [START_REF] Gareis | Cleavage of zearalenoneglycoside, a "masked" mycotoxin, during digestion in swine[END_REF] proved, that ZON4G can be cleaved during digestion in swine, releasing its estrogenic precursor -ZON. The term "masked mycotoxin" was coined around that time, describing a compound that is usually not detected by routine analytical methods, but still maintains its toxicity for mammals due to reactivation of the mycotoxin after hydrolysis of the glucoside in the digestive tract. Previously, ZON4G was also identified and characterized with 1 H-and 13 C-NMR as a transformation product from ZON by Rhizopus spp. [START_REF] Kamimura | Conversion of zearalenone to zearalenone glycoside by Rhizopus sp[END_REF]. Chemical synthesis (Zill et al., 1990;[START_REF] Grabley | Glycosylation of Mycotoxins[END_REF]) yielded ZON4G from ZON in high purity with yields up to 80% for use as analytical standard. 1 H-NMR spectra were shown in both publications, which were matching with that from the Rhizopus biotransformation product. A small survey of the occurrence of ZON4G along with ZON in wheat was conducted, showing that ZON4G was detected in levels of about 10% of the ZON contamination in wheat (Schneweis et al., 2002). [START_REF] Engelhardt | Metabolism of mycotoxins in plants[END_REF] et al., 2006). Poppenberger et al. (2006) identified the A. thaliana UDP-glucosyltransferase (UGT) UGT73C6, that is mainly responsible for the glucosylation of ZON, αZOL and βZOL, cloned and expressed the encoding gene into Saccharomyces cerevisiae. The resulting yeast expression system was used to convert ZON into ZON4G. It was briefly shown before by our group that chemical reduction of ZON4G can yield both αZOL4G and βZOL4G in a ratio of about 1:1 [START_REF] Krenn | Production of zearalenone-4-glucoside, α-zeraralenol-4glucoside and β-zearalenol-4-glucoside[END_REF].

Here, we report an optimized procedure to obtain ZON4G after fermentation of ZON treated yeast, expressing UGT73C6. Furthermore, αZOL4G and βZOL4G were produced by chemical reduction of ZON4G with sodium borohydride, purified by preparative HPLC and crystallized. Both substances were fully characterized for the first time using various MS/MS and NMR techniques.

Materials and methods

Fermentation of ZON treated yeast expressing UGT73C6

Saccharomyces cerevisiae strain YGZA515 [START_REF] Poppenberger | Detoxification of the Fusarium mycotoxin[END_REF] was transformed with plasmid pBP918 (Poppenberger et al., 2006). The 2µ-LEU2 plasmid harbors the Arabidopsis thaliana glucosyltransferase UGT73C6 with a c-myc epitope tag behind the string constitutive ADH1 promoter. The transformed strain was streaked out on SC-LEU plates. A single colony was picked, suspended in 30 mL SC-LEU medium and grown overnight under shaking at 30°C. To this pre-culture, 100 mL SC-LEU medium was added to replenish essential medium components (OD 600 = 0.05 after the dilution) and again grown overnight. This was repeated two-times by adding 400 mL SC-LEU (OD 600 = 0.1) at the third and 3000 mL SC-LEU (OD 600 = 0.25) at the fourth day where the liquid culture reached a final OD 600 of 0. [START_REF] Krenn | Production of zearalenone-4-glucoside, α-zeraralenol-4glucoside and β-zearalenol-4-glucoside[END_REF], the yeast for inoculation was collected already at OD 600 of 0.8 well before it reached the stationary growth phase. The bioreactor was loaded with 28 L SC-LEU medium, containing a surplus of the carbon source (+20 g/L glucose) and inoculated with 2 L of the yeast culture. The cell suspension was stirred at 600 rpm at a constant pH value of 4.9 at 30°C. Oxygen saturation was kept above 90% using pressurized air. The optical density values were measured every 2 h to determine the transition from exponential growth to the stationary growth phase, which happened about 11 h after the inoculation. At this point, the cell suspension was immediately harvested, cooled to 4°C and the biomass separated from the medium in five subsequent centrifugation steps (6000×g, 8 min). The cell pellet was resuspended in 300 mL SC-LEU medium and used as biocatalyst for ZON bioconversion in a 7

L bioreactor (MBR Bioreactor AG, Wetzikon, Switzerland) controlled by an IMCS-2000 unit.

The reactor was loaded with 5 L SC-LEU medium and the yeast-cell slurry and stirred (600 rpm) at 30°C. During the conversion, the pH value was kept constant at 5.0 and the oxygen saturation was regulated (80%) using compressed air. After 20 min, 25 mg ZON (Biopure, Tulln, Austria) was dissolved in 1 mL acetone and added to the actively growing biocatalyst.

After 2.5 h, another dose of 25 mg ZON was added and the stirrer speed increased to 1000 rpm thereafter. The bioconversion was stopped after 21 h and the cells were removed by centrifugation. The obtained 5 L of clear supernatant were mixed with 500 mL methanol (to assure complete solution of ZON4G) and used for the purification of ZON4G.

Purification of ZON4G

ZON4G was isolated from the yeast supernatant using Bakerbond Octadecyl Polar Plus (1000 mg; J. min hold time at the same composition was followed by a rapid change to the starting conditions, which were held for another 5 min to re-equilibrate the column.

Reduction of ZON4G to αZOL4G and βZOL4G

A 10-fold molar excess of sodium borohydride was used to reduce ZON4G to both αZOL4G and βZOL4G. Therefore, 10 mg ZON4G and about 3 mg fresh sodium borohydride were dissolved in 10 mL methanol and stirred for about 30 min (until no more gas bubbles emerged). The solution was pipetted into 90 mL of a 1% aqueous acetic acid solution to quickly stop the reaction and to avoid degradation of the products due to the alkaline conditions in the reaction mixture. Enrichment was again achieved using SPE columns. 4

Bakerbond Octadecyl Polar Plus (1000 mg; J.T. Baker, Phillipsburg, USA) columns were conditioned with 20 mL methanol, equilibrated with 2x20 mL methanol/water (10/90, v/v).

Aliquots of 25 mL reaction mixture were applied per column and each column was subsequently washed with 50 mL methanol/water (10/90, v/v), before eluting αZOL4G and βZOL4G with 30 mL acetonitrile. The eluted fractions were pooled, evaporated with a rotavapor and re-dissolved in 5 mL acetonitrile/water (20/80, v/v). αZOL4G and βZOL4G

were then separated using preparative HPLC using the conditions described above.

MS/MS experiments

A Q TRAP LC-MS/MS system (Applied Biosystems, Foster City, CA, USA) with a TurboIonSpray (ESI) source was used to characterize the purified compounds. 5 mg/L solutions of ZON4G, αZOL4G and βZOL4G were prepared in methanol and directly infused into the ionsource using a 1 mL syringe with at flow rate of 10 µL/min. The source was used solely in the negative ionization mode at a temperature of 250°C and 20 scans were summed for a final spectrum. MS parameters were: curtain gas 20 psi (138 kPa), nebulizer gas 20 psi (138 kPa), auxiliary gas 65 psi (448 kPa), ionspray voltage -4200 V, interface heater on, declustering potential -30 V, entrance potential -10 V. For the full scan mode quadrupole 1 was set to scan between 300 and 1000 amu within 1 s. The enhanced product ion scan mode was used to acquire MS/MS spectra between 100 and 500 amu. For this mode, the following further parameters were set: scan rate 250 amu/s, Q0 trapping on, linear ion trap fill time 20 ms, collision activated dissociation gas low, collision energy -5 eV.

NMR spectroscopy

1 H-and 13 C-NMR spectra were obtained using an Avance DRX 400 NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) at 300 K using a 5 mm inverse broadband Zgradient probehead. About 4 mg of crystalline ZON4G, αZOL4G and βZOL4G were dissolved in deuterated methanol for analysis. The spectrometer was operated at 400.13 MHz for 1 H and 100.62 MHz for 13 C measurements and the spectra were referenced to the respective solvent peaks.

Results and discussion

Production of ZON4G, αZOL4G and βZOL4G A total of 50 mg ZON was used in two doses to produce ZON4G with yeast, expressing UGT73C6. As fermentation took place in a 5 L scale, the concentration of ZON was always kept below 10 mg/L. Concentrations above 10 mg/L proved to be toxic for the yeast strain, resulting in a drastic loss of productivity. The fermentation yielded 24 mg ZON4G. Besides, also 10 mg βZOL and minor amounts of αZOL, βZOL4G, αZOL4G and unfermented ZON were found in the fermentation supernatant. These side products were presumably formed due to the activity of alcohol dehydrogenases of the used yeast strain and further conjugation of αZOL and βZOL. After purification, 21 mg of pure ZON4G were obtained. Purity was determined by LC-UV at 200 nm as shown previously [START_REF] Krenn | Production of zearalenone-4-glucoside, α-zeraralenol-4glucoside and β-zearalenol-4-glucoside[END_REF] and was estimated to be 99.7% (data not shown). It has to be pointed out that certain impurities (e.g. salts or inorganic materials) that do not have a chromophore are not detected by the used method.

Although the used yeast strain can principally be used to convert αZOL directly into αZOL4G and βZOL into βZOL4G, we decided to produce the latter compounds by chemical reduction from ZON4G. This way only ZON was needed as reactant. After purification 4.1 mg αZOL4G (estimated purity 99.8%) and 4.5 mg βZOL4G (estimated purity 99.6%) were obtained from 10 mg ZON4G.

MS/MS characterization

The fullscan (LC-)MS scans of ZON4G (Figure 2), αZOL4G (Figure 3) and βZOL4G (Figure 4) showed various MS-peaks in negative ionization mode. The negative ionization mode was used exclusively, since it yielded far higher signal intensities compared to the positive mode.

Besides the expected [M-H] -ions, also chlorine, formiate and acetate adducts were observed in the mass spectra. We suspect that the adduct ions are arising due to unavoidable contaminations of our mass spectrometer, which is often used with several different solvents and buffers for other methods. Characteristic [2M-H] -ions of high intensities were formed for all three glucosides. In-source-decay yielded the deprotonated ions of the unconjugated mycotoxins ZON, αZOL and βZOL respectively, after neutral loss of the glucose moieties (-162 amu). Most interestingly, an uncommon [M+90-H] -adduct could be seen at various intensities for ZON4G, αZOL4G and βZOL4G. We believe this adduct to be a C 4 H 10 O 2 molecule, formed from methanol, as shown before in negative ESI spectra of glycolipids [START_REF] Ii | Observation of a novel adduct-ion in negative ion electrospray mass spectrometry of neutral glycolipids[END_REF].

MS/MS spectra were acquired for the three glucosides using the minimum possible collision energy of 5 eV, as harsher fragmentation conditions resulted in MS/MS spectra that no longer showed the selected [M-H] -precursor ions. The major ions in the MS/MS spectra (depicted as inlays in Figures 234) clearly were [ZON-H] -in case of ZON4G, [αZOL-H] -in case of αZOL4G and [βZOL-H] -in case of βZOL4G. Minor peaks corresponded to losses of water or CO 2 from the non-glucosylated compounds. Higher collision energies (data not shown) yielded MS/MS spectra that were not distinguishable from those obtained for ZON, αZOL and βZOL respectively.

NMR characterization

1 H and 13 C-APT NMR spectra were recorded for all glucosides. The identity of ZON4G was confirmed by the complete accordance of its NMR data with those published earlier by [START_REF] Kamimura | Conversion of zearalenone to zearalenone glycoside by Rhizopus sp[END_REF]. Additionally, full structure characterizations and complete signal assignments were carried out for the two isomeric zearalenol derivatives by means of 2D NMR methods: proton-proton (DQF-COSY) and proton-carbon (HSQC) direct correlations as well as proton-carbon long range correlation (HMBC). Thus, the attachment of the glucoside to position 4 of the zearalenols was shown by a long range correlation of the glucosidic proton to the C-4 carbon signal, and the β configuration of the sugar unit could be proven by the 7.5

Hz coupling constant between H-1'' and H-2''. The stereochemical assignment of the -OH group within the macrocyclic ring (α-vs. β-isomer) was done by comparing the spectra of our products with those of the two isomeric non-conjugated zearalenols, which were produced by Burckhardt and Ley ( 2002) via total synthesis. Full 1 H and 13 C assignments of the zearalenol-4O-β-D-glucopyranosides are given in Table 1, the spectra are shown in Figures 5 and6.

Conclusions

It is currently unknown to which extent conjugates of mycotoxins occur in food and feed.

Here we biotechnologically and chemically produced three glucosidic conjugates of Fusarium mycotoxins. All three compounds, ZON4G, αZOL4G and βZOL4G were obtained in acceptable yields and high purities. MS scans showed the expected nominal molecular mass of the produced compounds, while NMR characterization additionally proved the position of the glucose group and the stereochemical assignments of the glucose and hydroxyl groups of αZOL4G and βZOL4G. These substances will be used as standards for a survey of conjugated mycotoxins in cereals and cereal-based food using LC-MS/MS.

F

  Preparation and characterization of the conjugated Fusarium mycotoxins zearalenone-4O-β-D-glucopyranoside, α-zearalenol-4O-β-D-glucopyranoside and β-zearalenol-4Oβ-D-glucopyranoside by MS/MS and 2D-NMR

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  Structures and numbering of αZOL4G (R1=H, R2=OH), and βZOL4G (R1=OH, R2=H) ) and 13 C-APT (bottom) NMR spectra of αZOL4G ) and 13 C-APT (bottom) NMR spectra of βZOL4G

Table 1 .

 1 1 H and 13 C NMR shifts of zearalenol-4O-β-D-glucopyranosides (ppm)

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

Acknowledgements

We thank the Christian Doppler Society, the Austrian Science Fund (FWF project L225) and the UK Food Standards Agency for financial support.


N Waegeneers
email: nadia.waegeneers@var.fgov

J.-C Pizzolon

M Hoenig

L De Temmerman

Accumulation of trace elements in cattle from rural and industrial areas in Belgium

Keywords: Copper, Heavy metals -arsenic, Heavy metals -cadmium, Lead Food Types: Animal products -liver, Animal products -kidney, Animal cadmium, lead, arsenic, zinc, copper, kidney, liver, meat

Trace element concentrations were determined in meat, kidney and liver tissue of cattle, which had resided more than 18 months in areas historically contaminated by emissions from the non-ferrous metal industry or in regions with normal background metal contamination levels in Belgium. Trace element levels were determined by inductively coupled plasma-mass spectrometry. Lead, arsenic and cadmium concentrations in meat were low for all cattle. The concentrations in kidneys were respectively 1.8-, 2.2-and 2.5-fold higher in cattle from the contaminated areas compared to animals from reference areas. The European maximum level for cadmium in kidney was exceeded by 75% of the kidneys from contaminated areas and 47% of the kidneys from reference areas. Lead, arsenic and cadmium concentrations in bovine livers from contaminated areas were increased by about a factor of 2.3. Zinc levels in livers and kidneys were within normal ranges, but 20% higher in the contaminated areas compared to reference areas. Copper levels in livers were high in all areas. Dairy cattle accumulated significantly higher levels of cadmium in kidneys and of lead in liver and kidneys than did beef cattle.

Introduction

Industrial and agricultural processes have resulted in the release of toxic trace elements into the environment, although for some specific elements relatively high concentrations can naturally occur in the soil. Cadmium, lead and arsenic are causing most concern because of their transfer through the food chain, bearing a potential risk to animals and humans [START_REF] Friberg | Handbook on the toxicology of metals[END_REF][START_REF] López-Alonso | Interactions between toxic and essential trace metals in cattle from a region with low levels of pollution[END_REF]. The major sources of toxic trace elements in agricultural soils include atmospheric deposition [START_REF] De Temmerman | Heavy metal content of arable soils in northern Belgium[END_REF], animal manure, agrochemicals and mineral fertilizers [START_REF] Nicholson | Heavy metal contents of livestock feeds and animal manures in England and Wales[END_REF].

Cattle originating from farms located within a 20-km radius around zinc refinery plants in the Netherlands, had 2.5 times higher average cadmium levels and 1.5 times higher average lead levels in organs when compared to controls [START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF].

Cadmium and lead levels in calves from a polluted area in northern Spain, were significantly higher than those in calves raised in a rural area, whereas arsenic levels in no case differed between calves from both areas [START_REF] Miranda | Effects of moderate pollution on toxic and trace metal levels in calves from a polluted area of northern Spain[END_REF]. In Belgium, some rural areas have been contaminated by emissions of the non-ferrous metal industry [START_REF] Lauwerys | Health effects of environmental exposure to cadmium. Objectives, design and organization of the Cadmibel Study: a crosssectional morbidity study carried out in Belgium from 1985 to 1989[END_REF]. This has led to increased trace element levels in soils and humans [START_REF] Staessen | Renal function and historical environmental cadmium pollution from zinc smelters[END_REF][START_REF] Cornelis | Use of the IEUBK model for determination of exposure routes in view of site remediation[END_REF]. Trace element concentrations in tissues of animals raised in these regions have, however, not yet been studied.

The objective of this study was to determine toxic and trace element concentrations in tissues of cattle that resided for more than 18 months in areas close to non-ferrous metal plants, and to compare the results with cattle from areas at ambient metal levels in the environment.

Materials and methods

Sampling

Kidneys, livers and muscle tissue of bovine animals were sampled at random by the Belgium Federal Agency for the Safety of the Food Chain (FASFC) in 2005. The samples were taken as part of the official control program of the FASFC in three contaminated areas and in regions with normal background metal levels in the environment, which will be further called the reference area. The contaminated areas were (i) site A -a rural area in the north-eastern part of the Kempen region near three primary zinc smelters, of which one has been closed since the 1970s, (ii) site B -a rural area south of the city of Antwerp near a precious metals refining facility, which processes secondary raw materials and wastes to extract and refine non-ferrous metals such as lead, copper and precious metals, and (iii) site C -a rural area in the south of the Antwerp Kempen region near a copper refining facility and a cobalt production plant (Figure 1). The so-called reference area consists of several rural areas throughout Belgium, away from point sources of toxic and trace elements. Belgium is divided in 10 major agricultural regions, from which seven were included in the current research (Zandstreek, Zandleemstreek, Leemstreek, Kempen, Weidestreek, Condroz and Ardennen). The division is based on differences in soil geochemical properties.

[insert Figure 1 [START_REF] Maudoux | Food safety surveillance through a risk based control programme: approach employed by the Belgian Federal Agency for the Safety of the Food Chain[END_REF]. In total, 99 animals from the reference area and 53 animals from the contaminated areas were investigated. Each bovine animal in Belgium is obligatory marked by an ear tag and has a bovine passport. Together with each sample, a copy of the passport was received, containing information on the date of birth, sex and type of the animal and on the farms where the animal resided. Farms where the animals had resided for more than 18 months, were contacted and the farmers were asked to further participate voluntarily.

A questionnaire was filled out with the farmers, rendering information about, amongst others, the feed supplied to the animals. In total, 19 farmers in the reference area (covering 20 animals) and 34 farmers in the contaminated areas (covering 38 animals) were visited.

Analytical methods

The animal tissues were sampled by officially appointed veterinarians and according to the rules outlined in the control program of the FASFC. The animal tissues were immediately deep frozen and stored in a freezer until analysis. At the time of analysis, the tissues were partially thawed and mixed in a home mixer to homogenize the samples. Consequently, the kidney cortex and medulla, which contain unequal trace element concentrations, were homogeneously mixed and metal concentrations are expressed on the whole organ. For each tissue, two homogenized sub-samples were mineralised by acid digestion (nitric acid) in pressure vessels of an X-PRESS microwave digestion system (CEM Corporation, Matthews, NC, USA). Trace element concentrations in all digests were quantified by inductively coupled plasma-mass Fish tissue) and one laboratory control material. In case of deviations from a set of common criteria for tolerable variations of blanks, and for values obtained for the reference materials, the analyses in a batch had to be repeated. The analyses of As, Cd and Pb in meat and organs were performed under accreditation, following the 17025 ISO norm. The limit of quantification (LOQ) for the analyzed trace elements was calculated as 10 times the standard deviation of 10 procedure blanks, multiplied with the dilution factor. The LOQ values, expressed on a fresh weight basis, equalled to 0.002 mg Cd kg -1 , 0.006 mg Pb kg -1 , 0.030 mg As kg -1 , 0.04 mg Cu kg -1 and 0.90 mg Zn kg -1 , respectively.

Statistical analysis

Statistical analyses were performed with UNISTAT Statistical Package, Version 5.5.06 (UNISTAT Ltd, London, UK). Measured values were used for samples below the LOQ, unless stated otherwise, so that distributions would not be distorted. The normal distribution of data was verified by the Kolmogorov-Smirnov test with Lilliefors correction. Basic statistics were calculated on untransformed data. Data that were lognormally distributed, were log-transformed before further analysis. Data were analysed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (α = 0.05). Two animals from the reference areas were excluded from statistical analysis because they were highly contaminated with metals and the source of contamination could not be traced.

Results and discussion

Many animal studies have shown that in chronic exposure experiments, the largest amounts of Cd are found in the liver and the kidneys (e.g. [START_REF] Kotsonis | The relationship of metallothionein to the toxicity of cadmium after prolonged administration to rats[END_REF]. Muscles, on the other hand, contain a relatively small part of the total body Cd [START_REF] Neathery | Cadmium-109 and methyl mercury-203 metabolism, tissue distribution and secretion into milk of cows[END_REF]. Lead is distributed to both soft tissues (blood, kidney, liver, …) and mineralising systems (bones, teeth). Among soft tissues, the liver and kidney contain the highest Pb concentrations in mammals while muscles contain the lowest concentrations. In the current study, As, Cd, Pb and Cu concentrations in meat were low compared to their respective concentrations in kidney and liver (Table I), which is in line with the earlier findings stated above. In the reference areas, the majority (57-82%) of the As, Cd and Pb concentrations in meat were below the LOQ, while in the contaminated areas 91% of the As concentrations, 28% of the Cd concentrations and 77% of the Pb concentrations were below the LOQ. Mean Cd and Cu concentrations in meat were significantly higher in the contaminated areas when compared to the reference area. For As, Pb and Zn, no significant differences could be observed between both areas. Although mean Cd concentrations are significantly different between the contaminated and the reference area, the Cd concentrations in all meat samples were far below the European maximum level of 0.05 mg kg -1 fresh weight (EU, 2006) and fell within previously reported ranges in other countries (e.g. [START_REF] Langlands | Cadmium concentrations in liver, kidney and muscle in Australian sheep and cattle[END_REF][START_REF] Jorhem | Lead, cadmium, arsenic, and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88[END_REF][START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF][START_REF] Olsson | Cadmium and zinc in kidney, liver, muscle and mammary tissue from dairy cows in conventional and organic farming[END_REF]. The Pb concentrations in meat samples from the reference and contaminated areas were as well far below the European maximum level of 0.1 mg kg -1 fresh weight (EU, 2006). Arsenic showed a higher accumulation in meat than did Cd and Pb, whereas an opposite tendency could be seen in liver and kidney (Table I). Arsenic, Cd and Pb concentrations were higher in kidney than in liver. It is known that kidney Cd concentrations exceed the liver concentration following prolonged exposure [START_REF] Kotsonis | The relationship of metallothionein to the toxicity of cadmium after prolonged administration to rats[END_REF], except at very high exposures [START_REF] Bernard | Significance of cadmium levels in blood and urine during long-term exposure of rats to cadmium[END_REF]. In case of repeated exposure, liver Cd levels increase rapidly and a re-distribution of Cd to the kidney occurs over a period of time. The major source of renal Cd in chronic exposure is derived from hepatitic Cd, which is transported in the form of Cd-metallothionein [START_REF] Chan | Mobilization of hepatic cadmium in pregnant rats[END_REF].

[Insert Table I around here] Within the reference area there were no significant differences in As and Cd concentrations in kidney between the different agricultural regions within the reference area. The geometric mean values for As in kidney ranged from 0.026 to 0.044 mg kg -1 while the Cd concentrations ranged from 0.661 to 0.894 mg kg -1 between the seven agricultural regions. The Pb concentrations in kidney were significantly different between the Zandstreek on the one hand (geometric mean: 0.246 mg kg -1 ) and the Kempen, Weidestreek, Condroz and Ardennen on the other hand (range of geometric means: 0.077-0.122 mg kg -1 ). The Zn concentrations in kidney differed significantly between the Ardennen (geometric mean: 20 mg kg -1 ) and the Kempen and Weidestreek (geometric mean: 14 and 15 mg kg -1 respectively). There were no significant differences in Cd or Zn concentrations in liver within the reference area. The geometric means for Cd in liver ranged from 0.116 to 0.190 mg kg -1 , those for Zn ranged from 28 to 41 mg kg -1 . The Pb concentrations in liver significantly differed between the Weidestreek (geometric mean: 0.025 mg kg -1 ) and the Zandstreek, Zandleemstreek and Leemstreek (of geometric means: 0.060-0.090 mg kg -1 ). Mean As, Cd, Pb and Zn concentrations in I). The mean concentrations in liver were 2.2-, 2.3-and 2.4-fold higher for As, Cd and Pb, respectively, in the contaminated areas when compared to the reference area. The average Cd concentration in livers of cattle around several zinc refinery plants in the Kempen area (The Netherlands) was 0.370 mg kg -1 fresh weight, whereas it was 0.170 mg kg -1 fresh weight in a control area [START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF] original data given on a dry weight basis, recalculated to fresh weight by means of the given dry matter content of liver). These values are comparable to the data found in the current study (0.446 and 0.190 mg Cd kg -1 fresh weight). The mean concentrations in kidneys were 1.8-fold, 2-fold and 2.5-fold higher for Pb, As and Cd, respectively, in the contaminated areas when compared to the reference area. Average Cd concentrations in kidneys in the Dutch Kempen area were 0.83 and 2.04 mg kg -1 fresh weight in the control and polluted area respectively [START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF], which is slightly lower than 1.14 and 2.86 mg Cd kg -1 fresh weight found in this study. Such pronounced Cd concentrations in kidneys have not been found in other EU countries so far (Antoniou et al., 1989;[START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF][START_REF] Miranda | Cadmium levels in liver, kidney and meat in calves from Asturias (North Spain)[END_REF][START_REF] Olsson | Cadmium and zinc in kidney, liver, muscle and mammary tissue from dairy cows in conventional and organic farming[END_REF]. These Cd levels in kidneys do cause a lot of concern as they frequently exceed the European maximum level (EU, 2006). In the contaminated areas, 75% of the kidneys were above the European maximum level of 1 mg kg -1 fresh weight and should not enter the food chain. The situation for kidneys in the reference area also causes concern as 47% of the samples exceed the European maximum level. Cadmium concentrations in livers exceeded less frequently the European maximum level of 0.5 mg kg -1 fresh weight, although still 25% of the livers from the contaminated areas and 4% of the livers from the reference area did not comply with the legislation. The Pb concentrations were above the European maximum level of 0.5 mg kg -1 fresh weight for 19% of the kidney samples from the contaminated areas, 8% of the kidney samples from the reference area, 6% of the liver samples from the contaminated areas and 1% of the liver samples from the reference area. The age of the animals is an important factor in the accumulation of trace elements, and especially Cd, in organs [START_REF] Andersen | Cadmium and zinc in kidneys from Danish cattle[END_REF]Antoniou et al., 1989). There was, however, no significant difference in the mean age of animals from the contaminated areas when compared to the reference area, neither were there any significant differences in age within the reference area (Table II). Zinc levels in livers and kidneys were within normal ranges [START_REF] Korsrud | Trace element levels in liver and kidney from cattle, swine and poultry slaughtered in Canada[END_REF][START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF][START_REF] López-Alonso | Toxic and trace elements in liver, kidney and meat from cattle slaughtered in Galicia (NW Spain)[END_REF] and references therein), but 20% higher in the contaminated areas compared to other regions. Zinc is known to be widely distributed in body tissues, but reaches its highest concentrations in liver, bones, muscles and kidneys (Eisler, 1973). Liver copper levels varied widely among all animals (1.9-395 mg kg -1 fresh weight; Table I) and mean levels were high compared to literature data as well in the reference areas as in the contaminated areas [START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF]López Alonso et al., 2000 and references therein).

[Insert Table II around here] Interactions between trace element uptake and accumulation may be synergistic or antagonistic. Cadmium raises for example Zn levels in kidneys and liver because of active transport from the blood and the formation of metallotioneins, which bind both Cd and Zn [START_REF] Thijs | Determination of serum zinc in a random population of four Belgian towns with different degrees of environmental exposure to cadmium[END_REF]. Tabel III presents Pearson correlation coefficients between trace element concentrations in kidney and liver. These correlations show indeed increased Zn levels with Cd accumulation in kidney and liver, both in the reference area as in the contaminated areas. Also Pb, Cu and Zn were positively Antagonistic interactions have been reported for Cd and Cu [START_REF] Rice | The impact of low level cadmium feeding on blood chemicals in male, Sprague-Dawley rats[END_REF].

Cadmium causes a depression in the serum level of ceruplasmin, the protein which is responsible for Cu transport. Such interactions might lead to a Cu deficiency, which might explain the very low liver Cu concentrations (< 5 mg kg -1 , which is definitive for Cu-deficiency) that have been found in some animals (Table I). Copper levels in liver were also negatively correlated with Pb in liver. This negative correlation was also observed by [START_REF] Spierenburg | Cadmium, zinc, lead, and copper in livers and kidneys of cattle in the neighbourhood of zinc refineries[END_REF]. It is not clear if a competition such as between Cu and Cd can also explain the negative correlation between Cu and Pb. Increasing trace element concentrations in kidneys were also accompanied by increased concentrations of the same element in liver, except for Cu. The liver is known to be the major storage organ for Cu.

[Insert Table III around here] Dairy cattle from the reference area accumulated significantly higher levels of Cd in kidneys than did beef cattle (Table IV). Similarly, the Pb concentration in kidneys and in livers of dairy cattle was significantly higher than its concentration in kidneys and livers of beef cattle. [START_REF] López-Alonso | Cadmium and lead accumulation in cattle in NW Spain[END_REF] found higher levels of Cd and Pb in livers of dairy cattle compared to beef cattle, but not in kidneys. They suggested that the differences in metal accumulation could be associated with a higher metal dietary intake and a higher hepatitic metabolism associated with milk production. Beef cattle in the study of [START_REF] López-Alonso | Cadmium and lead accumulation in cattle in NW Spain[END_REF] received a diet essentially based on pasture whereas the dairy cattle received a supplementation with corn silage and concentrates, in addition to pasture. Differences in feed between beef and dairy cattle in the current 12 study were less pronounced, although beef cattle received in general about 60% corn silage and 40% pasture (fresh pasture and silage) whereas beef cattle received in general 50% corn silage and 50% pasture. The diet was supplemented with concentrates, both for beef and dairy cattle. The ratio of concentrates/roughage differs between dairy and beef cattle. In Belgium, this ratio is about 40/60 for dairy cattle and 50/50 for beef cattle. Concentrates are generally purchased and therefore comply with the Belgian legislation concerning maximum allowable trace element levels. Roughage is, on the other hand, very often locally grown by the farmer and not submitted to any compliance control. Whether there are differences in the metal content of the roughage, will be further investigated. The major difference between beef and dairy feed is, however, the daily dry matter intake. The daily dry matter intake by dairy cattle is 16-20 kg, while it is only about 10 kg for beef cattle [START_REF] Pauwels | Ministerie van Middenstand en Landbouw, Bestuur Onderzoek en Ontwikkeling -DG 6[END_REF][START_REF] Römkens | Blootstelling en opname van cadmium door runderen in de Kempen: een modelstudie[END_REF]. Given the fact that differences in Cd accumulation between dairy and beef cattle are only significant in the kidney, suggests that the difference in dietary exposure is the most important reason for the larger Cd accumulation in dairy cattle kidneys. The higher Pb concentrations in liver and kidney of dairy cattle compared to beef cattle may be explained by a higher dietary exposure and by Pb mobilisation from bones during dairy cattle lactation, as suggested by [START_REF] López-Alonso | Cadmium and lead accumulation in cattle in NW Spain[END_REF].

[Insert Table IV around here] Cattle originating from site A had higher average Cd concentrations in kidney and liver than cattle from site C and the reference areas (Figure 2). Site A is well known to be contaminated with Cd [START_REF] Lauwerys | Health effects of environmental exposure to cadmium. Objectives, design and organization of the Cadmibel Study: a crosssectional morbidity study carried out in Belgium from 1985 to 1989[END_REF][START_REF] Staessen | Renal function and historical environmental cadmium pollution from zinc smelters[END_REF]. The average Cd concentrations in kidney and liver at site B were also higher than in the reference areas. There are no differences in Pb concentrations in kidney and liver of cattle from [START_REF] Mira | State of the Environment Report on the Flemish region of Belgium[END_REF]VMM, 2007). Copper concentrations were elevated in liver of cattle originating from site C, near a Cu refining plant, but not in kidney (Figure 3).

  about here] Besides sites A, B & C there are other contaminated areas in Belgium, but no samples were taken in those areas because of the their negligible cattle or milk production activities. The number of samples taken by the FASFC was determined in the official The methodology developed for the program is based on risk evaluation, statistical tools and current scientific knowledge

  -MS, VG PQ-ExCell, TJA, US). Each batch of 20 samples additionally included one procedure blank, one certified reference material (IAEA-407

  were all significantly higher in the contaminated areas than in the reference area (Table

  other in kidneys. Cadmium and Cu in kidneys were not correlated with each other in the reference area. In liver, Cd and Cu were negatively correlated.

  sites although the yearly average air Pb concentration in PM 10 dust at site B (173 ng m -3 in 2006) was increased compared to air Pb emissions at site A (33 ng m -3 in 2006) and ambient background concentrations (13 ng m -3 in 2006), and the yearly average air Pb deposition level at site B (274 µg m -2 d -1 in 2006) was increased compared to ambient background concentrations (8 µg m -2 d -1 in 2006)

[

  Insert Figure 2 around here] [Insert Figure 3 around here]ConclusionToxic and trace element concentrations have been measured in meat, kidney and liver samples of cattle from contaminated and reference areas in Belgium. Although As , Cd and Pb concentrations in meat were low in all areas, Cd and Pb concentrations in kidney and liver regularly exceeded the European maximum levels, both in the contaminated and the uncontaminated areas. Zinc levels in livers and kidneys were within normal ranges, while.liver copper levels varied widely among all animals and mean levels were high compared to literature data. Dairy cattle accumulated significantly more Cd in kidneys and Pb in liver and kidneys compared to beef cattle, which is probably due to a larger dietary exposure compared of dairy cattle and by Pb mobilisation from bones during lactation. It is important to pay attention to the fact that a considerable contamination of toxic elements takes place in cattle originating from Belgium, as heavy metals can be transferred through the human food chain and cause food safety problems.

  . Luchtkwaliteit in het VlaamseGewest -2006. Vlaamse Milieumaatschappij, Aalst. pp. 148 + bijlagen (in Dutch).

Figure 1 .

 1 Figure 1. Geographical location of the sampling areas. The contaminated sites A, B and C are

Figure 2 .

 2 Figure 2. Average cadmium and lead concentration in kidney and liver as a function of the

Figure 3 .

 3 Figure 3. Average copper concentration in kidney and liver as a function of the origin of the

  Fig.3

F

  Descriptive statistics of trace element concentrations in bovine kidney, liver and meat collected in a reference area (n = 97) and in polluted areas (n = 53). The concentrations are expressed on a fresh weight basis.

  LOQ = 32; † n < LOQ = 3 ; ‡ n < LOQ = 88; ¶ n < LOQ = 28; § n < LOQ = 70; ║ n < LOQ = 55; # n < LOQ = 80; ** n < LOQ = 48; † † n < LOQ = 15; ‡ ‡ n < LOQ = 41.

  Detailed information on the age of the animals in the seven agricultural regions of the reference area and of the animals from the three contaminated areas.

Table III .

 III Pearson correlations between trace element contents in organs for cattle from polluted areas and from the reference area.Correlation coefficients were calculated on log-transformed trace element concentrations in fresh kidneys and livers.

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	1 4.0 3.6 5.0 3.2 4.9 3.7 3.4 4.4 Kidney 0.59 *** Pb -0.24 ** 0.50 *** 0.52 *** 0.88 *** -0.36 *** 0.51 *** F o r P e e r R e v i e w 1.5-11.4 17 4.6 ± 2.7 1.6-11.6 26 3.8 ± 1.3 2.0-5.3 7 5.4 ± 2.1 2.4-8.3 10 3.9 ± 2.5 1.0-8.1 9 5.4 ± 2.4 1.6-9.7 12 4.0 ± 1.7 1.7-7.2 11 3.7 ± 1.7 2.0-7.2 12 4.9 ± 2.1 0.6-10.9 30 Cu Zn Cd 0.31 * 0.61 *** 0.75 *** 0.63 *** Cd -0.27 * 0.41 ** 0.60 *** 0.07 -0.50 *** 0.17 0.44 *** 0.73 *** -0.43 *** 0.82 *** -0.09 0.26 ** -0.57 *** 0.10 0.39 *** 0.58 *** -0.36 *** 0.14 -0.14 -0.21 * 0.32 *** 0.26 ** 0.47 *** 0.44 *** F Leemstreek Kempen Weidestreek Condroz Ardennen Site A Site B Site C Kidney-Cd Pb Cu Zn Liver-Cd Pb Cu Zn o r P Upper right half: cattle from polluted areas. Lower left half: cattle from the reference area. p < 0.05 e e r ** p < 0.01 R *** p < 0.001 e	Liver O n l y Pb 0.25 * 0.68 *** -0.33 ** 0.15 0.51 *** --0.32 *** 0.56 ***	Cu -0.27 * -0.33 ** -0.15 -0.16 -0.33 ** -0.21 -0.02	Zn 0.00 0.06 0.04 0.36 ** 0.06 0.30 * 0.19 -
	v i e
	w
	O n l			3
	y
		4

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Table IV .

 IV Cadmium and lead concentrations (in mg kg -1 fresh weight) in kidney and liver of dairy and beef cattle from the reference area.

		Cadmium	Lead
		Dairy cattle	Beef cattle	Dairy cattle	Beef cattle
	Kidney	1.21 *	0.62	0.31 *	0.11
	Liver	0.20	0.13	0.12 *	0.04

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