Yolanda Saavedra
email: ysaavedra@intecmar.org

Juan Blanco

Aurora González

Anatomical distribution of heavy metals in the scallop Pecten maximus

Keywords: Heavy metals, Anatomical distribution, Scallops

This paper studies the anatomical distribution of Hg, Cd, Pb, Cr, Ni, As, Ag, Cu and Zn in the scallop Pecten maximus and the possible implications in terms of shellfish management. Six organs were analysed: the mantle, gills, foot, digestive gland, kidney and gonad. On the basis of their anatomical distribution, two groups of metals were able to be distinguished: the first included Pb, Hg , Ni, Zn and Ag; and the second comprised the four other metals studied. The metals in the first group preferentially accumulated in the kidney (except for Pb), with generally much lower concentrations in the other organs. The metals in the second group accumulated mainly in the digestive gland. As and Cu were included in the second group, but they also had particular inter-organ distribution characteristics. Among the edible organs of the scallop, only the adductor muscle contained important proportions of one metal, arsenic (which is very likely accumulated as a non-toxic derivative). A selective evisceration of the metal rich non-edible organs may therefore be considered a reliable measure to be taken with a view to reduce the metal content of scallops used for human consumption. This could be especially relevant for Cd which is accumulated in high concentrations in the digestive gland.

Introduction

The king scallop Pecten maximus is an important resource in Galicia as well as in other areas of EU and Canada and therefore requires close monitoring of its content of substances that could have a harmful effect on human health, such as heavy metals. Other pectinids are known to accumulate large amounts of cadmium, even from unpolluted areas [START_REF] Stewart | Environmental Requirements of the sea scallop (Placopecten Magellanicus) in Eastern Canada and its response to human impacts[END_REF][START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF][START_REF] Francesconi | Cadmium in the saucer scallop, Amusium balloti, from western Australian waters: concentrations in adductor muscle and redistribution following frozen storage[END_REF]. If the ability of the king scallop to accumulate heavy metals is high, then the exploitation strategy of this species should take into account both the accumulated and the environmental metal concentrations.

Moreover, if the metal accumulation capability of this species is very high, as suggested by previous studies, then in some cases, its use as a resource for human consumption would be linked to the ability to eliminate or reduce such contaminants by means of specific treatments.

As the different parts of the soft tissues of this species vary in economic value, one of the possible treatments would be the elimination of the most toxic parts, provided that they are the ones with the lowest economic value. This approach has recently been used by the EU to minimise the effects of the accumulation of domoic acid, the main biotoxin responsible for Amnesic Shellfish Poisoning [START_REF] Fernández | Management of shellfish resources[END_REF].

Previous studies have dealt with metal distribution in various groups of scallop organs, but they did not separate all the possible fractions, taking into account only a reduced sub-set of the dangerous metals [START_REF] Topping | Heavy metals in shellfish from Scottish waters[END_REF][START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF]. Only a few studies on metal distribution in scallop soft tissues have separated the kidney to analyse metals [START_REF] Khristoforova | Heavy metals in soft tissues of the scallop chlamys rosealbus[END_REF][START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF] even though this organ may be considered an important accumulator. The objective of this study was to check whether the organs in scallops differ in their ability to accumulate metals and, as a consequence of this, to find the target organs to monitor metals in this species.

Material and Methods

Sample collection and treatment

One sample of 36 scallops Pecten maximus was collected in June of 2000 from the Ría de Arousa (NW of the Iberian Penindula) (Figure 1) to study metals in different organs. The scallops in the sample were pooled into 3 batches of 12 individuals each. The soft tissues extracted from scallops of each batch were carefully dissected to obtain the mantle, gills, foot, digestive gland, kidney and gonad (Figure 2). The organs were lyophilised and pulverised in a mixer mill of zirconium oxide. A 0.5 g portion of triturated samples was digested with nitric acid by heating in a microwave oven. The analytical determination of copper and zinc concentrations in the digest was performed by flame atomic absorption spectrometry.

Cadmium, lead, chromium, nickel, arsenic and silver were analysed by electrothermal atomic absorption spectrometry with Zeeman background correction and STPF (Stabilised Temperature Platform Furnace) conditions. Mercury was determined by flow injection system-cold vapour atomic absorption spectrometry. More detailed methodology was described elsewhere [START_REF] Saavedra | A simple optimized microwave digestion method for multielement monitoring in mussel samples[END_REF].

Validation of the techniques used was performed by intralaboratory quality control using two certified reference materials (Table 1) The main factors responsible for the variation in metal concentrations as regards the different organs examined were studied by means of Principal Component Analysis. All the statistical analyses were carried out using the Minitab 15 statistical package.

Results and Discussion

Target organs. Metal concentrations in different organs of the scallops

There is a high degree of organ specificity to the different metals studied, with the exception of arsenic (Table 2). The nine metals may be classified into two groups on the basis of their anatomical distribution, using Principal Component Analysis (Fig. 3). The first group includes metals that are closely related to the first principal component (PC1), and is characterised by preferential accumulation in the kidney. The second group includes the metals that are more closely related to PC2, where preferential accumulation takes place in the digestive gland. As neither of the two organs is included in the edible portion of the scallops, comprising the muscle and gonad, it would follow that the edible organs contained very low concentrations of metals, with the highest concentrations of metals being found in either the digestive gland or the kidneys, except for arsenic.

Arsenic

Scallops had similar concentrations in the gills and digestive gland, and showed the lowest dispersion among organs. Arsenic would therefore seem to exhibit a different organ affinity than other metals, as it is more evenly distributed throughout the body. Arsenic distribution in Pecten maximus has not been studied to date, but the concentrations found were similar to those in Patinopecten yessoensis (Shiomi et al. 1988), who also observed no marked differences in arsenic concentration among tissues. In Pecten maximus from Galicia the digestive gland contains by far the highest cadmium concentrations. Other work studying cadmium in organs of this species from different areas also pointed to the digestive gland as a target organ [START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF][START_REF] Topping | Heavy metals in shellfish from Scottish waters[END_REF][START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF] in scallops from the Irish Sea, Scottish waters and Plymouth area, respectively.

Most studies with other pectinids suggest that this is a general characteristic of this group of molluscs (Adamussium colbecki and Pecten jacobaeus, [START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF]Placopecten magellanicus, Greig et al., 1978;[START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF]Chlamys rosealbus, Khristoforova et al., 1989), with the possible exception of Mizuhopecten yessoensis in which [START_REF] Chelomin | Cadmium-induced alterations in essential trace element homoeostasis in the tissues of scallop Mizuhopecten yessoensis[END_REF] reported the kidney as being an important organ of cadmium accumulation. Other bivalves would also appear to concentrate cadmium preferentially in the digestive gland, but the difference in concentration as compared to other organs is much lower than in pectinids.

Different levels of this metal in the digestive gland have been found in the studied areas.

Cadmium concentrations in the digestive gland of Galician scallops are similar to those found in scallops from the Irish Sea (Pecten maximus, [START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF] and Antarctica (Adamussium colbecki, [START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antartic scallop Adamussium colbecki: role of metallothionein in heavy metal homeostasis and detoxication[END_REF]). The highest concentrations recorded to date have been found in scallops from English Channel [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF], three times above the levels reported here . The lowest values have been reported in Scottish [START_REF] Topping | Heavy metals in shellfish from Scottish waters[END_REF] and Adriatic [START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF]) scallops. It is important to consider that the differences cited in cadmium concentration may be due not only to different environmental levels of this metal, but also to the interspecific variation in the capacity of the scallops examined to concentrate cadmium in the digestive gland. This interspecific variation has been suggested by [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF] to explain differences in cadmium concentrations between Pecten maximus and Chlamys opercularis collected from the same area; and [START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF], who attributed a greater capability of cadmium accumulation in the digestive gland to Adamussium colbecki rather

Chromium and copper

In addition to cadmium, the digestive gland is a site of selective chromium and copper deposition, which is in agreement with previous studies [START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF][START_REF] Vashchenko | Assessment of the state of the japanese scallop Mizuhopecten yessoensis in Alekseeva Bight (Peter the Great Bay, Sea of Japan) based on morphological and biochemical parameters[END_REF][START_REF] Zaroogian | Copper accumulation in the Bay Scallop, Argopecten irradians[END_REF]. Notwithstanding, these metals are less selectively deposited than cadmium. The kidney is the second most important organ for chromium accumulation and gonads have copper concentrations similar to those of the kidney.

Zinc

The resulting data would point to the kidney as the target organ for zinc. Zinc concentrations in the kidney have been shown to be higher that those found in other organs of Pecten maximus in other studies with the same species [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF]. However, the gonad has also been reported as an important organ, -the principal organ, in fact-of zinc concentration [START_REF] Topping | Heavy metals in shellfish from Scottish waters[END_REF]. Notwithstanding, the metal concentrations reported by [START_REF] Topping | Heavy metals in shellfish from Scottish waters[END_REF], as in most of the studies, referred to mixed viscera (including the digestive gland and kidney), and the organ having the highest concentration of metals was unknown. Although the great capability of the kidney to concentrate zinc is also a characteristic of other scallop species [START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF] and bivalves [START_REF] George | Metabolism of zinc in the mussel, Mytilus Edulis (L.): a combined ultrastructural and biochemical study[END_REF]Pirie, 1980, Ahn et al., 1996), few differences between the gonad and kidney were reported by [START_REF] Karaseva | Accumulation of heavy metals in gonads and somatic organs of bivalve mollusks[END_REF] in females of Mizuhopecten yessoensis and most zinc concentrations appeared in the gills of other scallop species [START_REF] Khristoforova | Heavy metals in soft tissues of the scallop chlamys rosealbus[END_REF][START_REF] Khristoforova | Metal concentration in tissues of the scallop Mizuhopecten yessoensis of different age[END_REF].

Zinc renal concentrations in Pecten maximus found in this study were lower than those from the English channel and Scottish waters, but higher than those in other scallops, which would

Mercury

Our results point to the kidney as a target organ for mercury accumulation. The digestive gland was also found to contain higher concentrations than the rest of the tissues. As far as we know, this is the first study dealing with the anatomical distribution of Hg in Pecten maximus, so there are no other data available to support our findings. In other Pecten species, P. alba, [START_REF] Walker | Effects of length, locality and tissue type on mercury and cadmium content of the commercial scallop, Pecten alba Tate, from Port Phillip Bay, Victoria[END_REF] found the highest levels of mercury in the inedible viscera which contains the digestive gland, among other organs.

Lead

This metal was concentrated preferentially in the kidney followed by the digestive gland. The concentration found in this latter organ was more than half as much as that found in kidney. [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF], studying scallops from Plymouth, also found a preferential concentration in the kidney, but the concentrations he found in this organ were one hundred times higher that those in digestive gland, instead of less that double, as found in this study, despite the fact that the scallops had similar total lead concentrations. Other forms of Pb or a different physiological status might be involved in this difference.

Silver

In this study the kidney displayed the most important silver concentrations and the digestive gland, foot and gonads had concentrations in the same order of magnitude as the kidney.

Several other studies have pointed to the digestive gland as the main silver accumulator organ for Pecten maximus [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF][START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF] and the foot has been shown as one of the highest silver accumulator organs in Chlamys varia by [START_REF] Martoja | Effects de la contamination expérimentale par l´argent chez Chlamys varia l. (bivalve, pectinidé). Données quantitatives, histologiques et microanalytiques[END_REF].

Notwithstanding, it is important consider that total concentrations in our study are smaller that

Nickel

Here the results showed that nickel is selectively accumulated in the kidney, supporting the findings of [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus and Chlamys opercularis[END_REF] who, in the same species, reported higher kidney than digestive gland concentration factors. In other studies, where the kidney was not separated as an individual fraction for nickel analysis, the most important nickel concentrations were found in the digestive gland of Pecten maximus [START_REF] Segar | The distribution of the major and some minor elements in marine animals[END_REF], the digestive gland and gills of Chlamys rosealbus [START_REF] Khristoforova | Heavy metals in soft tissues of the scallop chlamys rosealbus[END_REF], the visceral mass and gonads of Placopecten magellanicus [START_REF] Greig | Trace metals in sea scallops, Placopecten magellanicus, from Eastern United States[END_REF] and the gills of Mizuhopecten yessoensis [START_REF] Khristoforova | Metal concentration in tissues of the scallop Mizuhopecten yessoensis of different age[END_REF].

Organ contributions-relative importance of metals in different tissues.

As cited above, the highest concentrations of metals are found either in the kidneys or digestive gland. The importance of each organ in determining the metal concentration of the whole soft tissues of a bivalve depends not only on the concentration but also on the weight of each specific organ. Taking this into account, the adductor muscle (the largest organ) appears as the principal contributor for mercury (38% of the total metal burden), nickel (38%), arsenic (56%) and zinc (36%) (Table 3). The digestive gland contained by far the greatest amount of cadmium (84% of total cadmium). Similar percentages have been reported in most studies on different scallop species [START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF][START_REF] Francesconi | Cadmium in the saucer scallop, Amusium balloti, from western Australian waters: concentrations in adductor muscle and redistribution following frozen storage[END_REF]. The digestive gland was also the main contributor for chromium, copper, silver and lead; but, in the case of silver and lead, gonad and muscle contributions, respectively, were similar to those of the digestive gland. Thus, the digestive gland and muscle are the organs which may play a key role as being toxic or harmful in terms of human consumption in relation to the total metal body burden, especially for cadmium and arsenic, respectively. Notwithstanding, arsenic in the muscle probably does not pose a real problem for human consumption, since arsenobetaine, a nontoxic form of arsenic, was found to be the major fraction of this metal in scallop speciation studies (Shiomi et al., 1988[START_REF] Lai | Arsenic speciation in scallops[END_REF]. Consequently, in view of the anatomical distribution of the metal contents, especially cadmium, it would seem that the elimination of the non-edible body fractions could substantially reduce the elements harmful to human consumption and this might be a feasible way to reduce the incidence of these metals in pectinid fisheries. Furthermore, if this approach (evisceration of scallops) were taken into account by EU legislation on the established Cd legal limits (Commission Regulation EC No 1881/2006), it might be a solution for scallops marketing.

Conclusions

Two groups of metals were found on the basis of their anatomical distribution: one group was preferentially accumulated in the digestive gland and the other, in the kidney. As is the only metal homogeneously distributed among the different organs.

The adductor muscle and digestive gland were the organs that contributed in the greatest proportion to the total metal burden and therefore to metal concentration. Notwithstanding, the adductor muscle was only of primary importance in the case of arsenic, having, therefore, little repercussion on human health, as this metal appears to be found in scallops in a mostly non-toxic form. From a practical point of view, the presence of the maximum concentrations of metals, especially cadmium, in non-edible organs such as the kidney and digestive gland, together with the fact that the two most valuable organs of the scallop Pecten maximus for human consumption -the adductor muscle and gonad-were found to concentrate the metals at substantially lower levels than other organs, with the sole exception of arsenic, which was accumulated in only slightly lower concentration , opens the possibility of using selective evisceration of scallops to improve their quality for human consumption. Foot 0.7 0,5+0,05 0,3+0,1 0,6+0,1 0,8+0,2 1,0+0,2 0,5+0,01 3,3+0,5 0,7+0,2 0,3+0,0 Kidney 0.4 3,1+0,3 0,05+0,0 2,7+1,2 1,0+0,2 4,8+0,5 0,4+0,01 2,4+0,1 1,1+0,1 18,7+4,2

F

  , CRM 278R: trace metals (except Ni and Ag) in mussel tissue from BCR (Community Bureau Reference) and NIST 1566 b: trace metals (Ni and Ag) in oyster tissue from NIST (National Institute of Standards and Technology).

  Pecten jacobaeus to explain higher values in the Antarctic as compared to Mediterranean areas.

  made by[START_REF] Mauri | Heavy metals in the Antartic scallop Adamussium colbecki[END_REF] that the kidney is a strong zinc accumulator in the genus Pecten .

  other studies we have cited and therefore, the difference in environmental availability may affect the anatomical distribution of this metal in the organisms under study.

  Sampling location (grey area) of scallops in the Ría de Arousa (NW Spain) 122x112mm (300 x 300 DPI) Loading plot of metal concentrations and the organs of more specific accumulation on the first two principal components. 370x251mm (96 x 96 DPI) Page 19 of 19 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

Table 1 .

 1 Metal concentrations (mg Kg -1 ) in certified reference materials CRM 278 R (As, Cd, Cr, Cu, Hg, Pb and Zn) and NIST 1566 b (Ni and Ag).

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

Table 2 .

 2 Metal concentrations in the organs of Pecten maximus. Results were expressed as mean and standard deviation of three batches.

	Organ	Hg	Cd	Metal concentration (mg kg -1 , dry weight) Pb Cr Ni As Ag	Cu	Zn
	Mantle 0.082+0.010 3.6+0.3 0.91+0.28 1.4+0.2	1.0+0.2 9.9+0.5 0.31+0.02	4.5+0.2	95+11
	Gills	0.15+0.01 6.1+1.0 1.7+0.6	1.8+0.4	2.1+0.2 16+0.3	0.37+0.03 7.6+0.51	213+37
	Gonad	0.070+0.003 2.6+0.2 1.1+0.1 0.82+0.31 1.1+0.1 8.5+0.9 0.57+0.06	15+1	223+16
	Digestive gland	0.24+0.01	120+8	4.8+0.4	5.2+0.6	3.3+0.3 15+0.9	0.67+0.04	28+2	210+24
	Muscle 0.054+0.001 1.3+0.2 0.45+0.08 0.20+0.13 0.70+0.14 10+0.1 0.055+0.002 1.8+0.4	94+5
	Kidney	0.70+0.05 1.8+0.1 7.5+2.2	2.7+0.8	14+2	11+1	1.5+0.10	18+1	7737+1459
	Foot	0.064+0.003 5.6+1.3 0.95+0.10 1.2+0.1	1.6+0.4 8.3+0.4	1.1+0.1	6.5+1.0	75+8

Table 3 .

 3 Contribution of each organ of scallop to total weight and metal content. Results were expressed as mean and standard deviation.

	Organ	% W	%Hg	%Cd	%Pb	%Cr	%Ni	%As	%Ag	%Cu	%Zn
	Muscle	58	37,6+0,8 6,1+0,2 24,5+1,8 11,0+5,1 38,2+5,2 55,6+1,1 13,8+0,2 16,2+3,6 35,8+2,4
	Mantle	15	15,2+1,2 4,6+0,1 12,9+2,4 21,7+2,2 15,0+1,5 14,3+0,8 20,3+0,5 11,0+0,9 9,6+0,8
	Gonad	11	8,6+1,0 2,3+0,3 11,1+1,7 9,1+4,1	11,0+1,3 8,6+0,8 26,1+0,6 26,1+3,6 15,6+1,1
	Digestive gland	8.6	24,5+1,6 83,9+0,4 39,0+3,1 45,8+3,5 27,4+7,2 11,9+0,9 24,9+0,3 38,2+2,2 11,8+1,8
	Gills	5.8	10,4+0,6 2,9+0,4 9,1+2,5 10,7+0,4 11,8+3,2 8,6+0,2	9,2+0,1	6,9+0,2 8,1+1,7

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


Antonio J Signes

Kasturi ; Mitra

Francisco M Burló

Angel A Carbonell-Barrachina

Antonio ; Signes

Miguel Universidad

Hernández

Vivekananda Ramakrishna

Miguel Hernández

Angel ; Universidad

A Signes

EFFECT OF COOKING METHOD AND RICE TYPE ON ARSENIC CONCENTRATION IN COOKED RICE AND ESTIMATION OF ARSENIC DIETARY INTAKE IN A RURAL VILLAGE OF WEST BENGAL, INDIA

Keywords: Metals analysis -AAS, Total diet studies Additives/Contaminants: Environmental contaminants, Heavy metals -arsenic Food Types: Drinking water, Rice atab rice, Calcutta, inorganic arsenic, North 24-Parganas, total arsenic

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Arsenic-contaminated groundwater from tube wells is the main source for drinking, cooking and other household purposes in West Bengal (India) and for that reason groundwater is the most important source of arsenic (As) intake. However, recently it has been considered that foods are responsible for an important part of As intake too, and studies on total As (t-As) in food obtained from As-endemic areas have increased in recent years [START_REF] Roychowdhury | Survey of arsenic and other heavy metals in food composites and drinking water and estimation of dietary intake by the villagers from an arsenic-affected areas of West Bengal, India[END_REF]. Furthermore Signes et al. (2008a) suggested that rice cooked with As-contaminated water can be an important As source, especially inorganic As (i-As), and that the conditions of the cooking process could affect the amount and/or speciation of this element in cooked rice.

Most of the population of West Bengal depends heavily on rice for their caloric intake, suggesting that if their rice is contaminated with As it will become an important dietary source of this metalloid [START_REF] Watanabe | Males in rural Bangladeshi communities are more susceptible to chronic arsenic poisoning than females: analyses based on urinary arsenic[END_REF]. Koichi et al. (2007) studied arsenic intake via water and food in a population living in an Asaffected area of Bangladesh and reported that the average As intake from cooked rice represented about 56% of the daily arsenic intake [START_REF] Ohno | Arsenic intake via water and food by a population living in an arsenic-affected area of Bangladesh[END_REF].

In West Bengal the agricultural system is mostly groundwater-dependent producing a large amount of arsenic deposit on the irrigated lands [START_REF] Signes-Pastor | Arsenic biochemistry as affected y phosphorus fertilizer addition, redox potential and pH in a West Bengal (India) soil[END_REF].

Besides, rice is cultivated in As-contaminated soils under anaerobic conditions (at which As is highly available for plant uptake) increasing its As concentration to levels higher than other crops [START_REF] Meharg | Arsenic in rice -understanding a new disaster for South-East Asia[END_REF]).

The As concentration of cooked rice is known to increase with the As concentration of cooking water but the effects of cooking methods have not been defined in detail. It is assumed that if cooking As-free water could be provided to villagers, cooked items will have an As concentration lower than expected due to migration of some As to discarded washing and cooking waters (Signes et al. 2008b). In fact, [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF] tested the three major rice cooking procedures from this area. Using low-As containing water (As < 3 µg l -1 ) in all three methods, they concluded that the traditional method, in which rice is washed until clear, cooked with a rice:water ratio of about 1:6 and with extra water being discarded, removed up to 57% of the As from rice containing As 203-540 µg kg -1 . The second method, in which rice is washed as in the traditional method but is cooked with a lower rice:water ratio (1:1.5-2) and no water is discarded, removed about 28% of rice As. Finally, the contemporary method, in which the rice was unwashed and no water was discarded, did not modify the rice As content [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF].

In this paper the effects of: a) rice cooking methods and b) rice type on t-As concentration in cooked rice are reported. The two most popular rice types, atab and boiled (Signes et al. 2008b), were cooked using the three most popular cooking methods in the Indian subcontinent [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF]. The two types of rice under study were from the block of Barasat in North 24-Parganas, where 70% of its tube wells have As concentrations above 50 µg l -1 , maximum permissible limit of As in drinking water established by the World Health Organization in 1984 [START_REF] Chowdhury | Groundwater Arenic Concentration in Bangladesh and West Bengal, India[END_REF], although the current provisional guideline by WHO was established in 10 µg l -1 (WHO 2006). The cooking experiments were carried out as previously described by [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF] but using cooking water with an As concentration of 40 µg l -1 , which is more representative of this specific district of West Bengal. Finally, an estimation of the comparative importance of cooked rice (obtained by three different cooking methods) and drinking water (containing 40 µg As l -1 ) to the total As daily intake was studied. Other equipment used included a sand bath (Falc, Treviglio, Italy) model BS 70 with a maxim temperature of 200ºC, a muffle furnace (Horbesal, Barcelona, Spain) model 12 PR/300 series 8B with a maximum temperature 1200ºC, a grinder (Moulinex, Valencia, Spain), and a hot air oven (Selecta, Barcelona, Spain) with maximum temperature of 250ºC.

Reagents

Deionized water was used for preparation of the reagents and standards. All glassware was treated with 10% v/v HNO 3 for 24 h and then rinsed three times with deionized water before use.

All chemicals were of, at the least, pro analysis quality. Commercial standard salt from NaAsO 2 (Panreac, Barcelona, Spain) was used for the preparation of standards. Magnesium nitrate, MgNO 3 and MgO (Panreac, Barcelona, Spain) were used in the ashing solution and HNO 3 65% (Merck, Germany) during the digestion of samples. Hydrochloric acid, HCL 37% (Merck, Germany), IK 99% (Retapur, Leuven, Belgium) and ascorbic acid 99% (Panreac, Barcelona, Spain) were used as reducing agents. As a reducing solution for AsH 3 generation, NaBH 4 (Panreac, Barcelona, Spain) solutions were prepared daily.

Study area

The study was conducted in a village of North 24-Parganas district, under the block Barasat, approximately 25 km from Calcutta (India). The total area of the village is 5.0 km 2 , with 22270 people living in residential area of the village with the remaining area being cultivated land. The average annual income of the villagers is 350 US$ annum -1 . The main source of water for the village is 100, mainly shallow, wells and tube wells, for irrigation purposes. The village was chosen as the model village in our study because it was known that 70 % of its tube wells had As concentrations above 50 µg l -1 and it is highly affected by As contamination in the groundwater.

Rice samples

Two different rice (Oryza sativa L.) types (according to their dehusk processing)

were selected for this study, atab and boiled rice, which are obtained by drydehusking (only mechanical dehusking) or wet-dehusking (includes soaking in water, light boiling, and mechanical dehusking), respectively (Signes et al. 2008b).

Both of rice types were from the same boro rice variety, khitish rice (Initial Evolution Trial IET-4094); authors decided to use this variety because boro rice varieties represent the highest potential health risks for humans (plants are irrigated with As contaminated water during the dry season).

Cooking methods

Worldwide there are three common methods of cooking rice [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF]):

1) The traditional method (A) still used by more than 90% of the villagers in the Bengal delta: raw rice is washed until the washings become clear (five to six times), the washings are discarded and then the rice is boiled in excess water (five to six times the weight of the raw rice) until cooked, finally discarding the remaining water (discarded water) by tilting the pan against the lid before serving the rice. 2) The intermediate method (B): The rice is washed as above and boiled with water of a volume 1.5 times to twice the weight of rice until no water is left to discard.

3) The contemporary method (C): unwashed rice is boiled with a volume of water 1.5 to twice the weight of rice; the wash and discard steps are both omitted.

Thirty six samples of rice were analyzed for t-As (18 of atab rice and 18 of boiled rice), and 12 samples (6 of atab rice and 6 of boiled rice) were cooked following each one of the three cooking methods under study (traditional, intermediate, and contemporary). In all cooking steps, water containing 40 µg As l -1 was used.

Quantification of t-As

Raw and cooked rice samples were dried in an air-oven (Selecta) for 72 h at 70 ºC.

Then, samples were ground in a domestic grinder (Moulinex) and the resulting powder was vacuum-packed and kept in the freezer at -18 ºC until analysis. Total

As in washing and discarded waters was analyzed immediately after carrying out the cooking simulations.

Raw rice, washing water, discarded water, and cooked rice were analyzed for t-As by Hydride Generation Atomic Absorption Spectrometry (HG-AAS). A 1-g portion of homogenized dried and ground rice sample or a 5-ml water (washed or discarded) sample was weighed and digested using the method previously described by [START_REF] Muñoz | Total arsenic in the fresh and processed fish products[END_REF]. Calibration standards were prepared using the same HCl concentration of the samples and certified materials. The instrumental conditions used for As determination by HG-AAS were as follows. Reducing agent: 1.4% (m/v) NaBH 4 in 0.4% NaOH, 5 ml min -1 ; HCL solution: 10 % (v/v), 10 ml min -1 ; carrier gas: argon, 250 ml min -1 flow rate; and for atomic absorption spectrometry, Exploration of China, respectively. These two materials were already dried and powdered, so they were subjected to the same exact digestion process than rice or water samples without any previous preparation.

Statistical analyses

All data were subjected to analysis of variance (ANOVA) and the Tukey leastsignificant difference multi-comparison test to determine significant differences among samples (cooking method and type of rice). The statistical analyses were performed using SPPS 14.0 (SPSS Science, Chicago, USA).

RESULTS

Analytical quality assurance

The analytical characteristics for the t-As methodology were as follows: detection limit, 4 µg kg -1 (calculated according to the European Standard prEN 13804, which establishes that the detection limit is numerically equal to three times the standard deviation of the mean of blank determination); precision 2 % (calculated as mean relative standard deviation obtained from six independent analyses of certified material NIST SRM 1568a); accuracy for rice flour (NIST SRM 1568a), found value = 0.29 ± 0.04 mg kg -1 (certified value = 0.29 ± 0.03 mg kg -1 ); accuracy for bush, branches and leaves (GBW07603), found value = 1.18 ± 0.03 mg kg -1 (certified value = 1.25 ± 0.10 mg kg -1 ). Table I shows the results from a mass balance experiment dealing with the effects of the cooking method on t-As concentration in atab rice. The raw atab rice increased its t-As content by 27.6%, 37.8% and 42.2%, when it was cooked with As contaminated water (40 µg l -1 ) using traditional, intermediate or contemporary methods, respectively. Rice washing reduced t-As content in atab rice by 4.5-11.6%.

Table II shows the results from a mass balance experiment studying the effects of the cooking method on t-As concentration in boiled rice. The raw boiled rice decreased its t-As by 12.7% when it was cooked by the traditional method but increased its t-As by 15.9% or 23.5%, when it was cooked by the intermediate or contemporary methods, respectively. Rice washing reduced t-As by 9.1-16.4%.

Cooked boiled rice had higher t-As concentration than cooked atab rice (390 ± 12 µg kg -1 and 283 ± 11 µg kg -1 ), independently of the cooking method used (Table III). No significant differences were found in t-As in the water coming from washing atab or boiled rices; however, t-As was higher in water discarded at the end of the cooking method when boiled rice was used.

Rice cooked by the traditional method had significantly lower t-As (258 µg kg -1 ) than rice samples cooked by the intermediate or contemporary methods; no significant differences were found between rice samples cooked by these two methods (mean of 376 µg kg -1 ) (Table IV).

DISCUSSION

t-As in cooked rice

The results reported here have shown that there is a strong dependence of the t-As content in the cooked rice on the cooking protocol and on the type of rice being used. The dependence of t-As content in the cooked rice on the cooking method was previously reported by [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF]. These researchers, however, used almost As-free cooking water in their experiments (< 3 µg As l -1 ) and concluded that the traditional and intermediate methods removed 57% and 28% of the t-As content initially present in raw rice; the contemporary method did not remove any arsenic from raw rice [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF]). In the present experiment the t-As concentration in cooking water was higher (40 µg l -1 ) than in Sengupta's experiment and under these experimental conditions all cooking methods increased significantly the t-As content in cooked rice compared to raw rice. However, cooking rice by the traditional method always implied the lowest As concentration in the final rice. [START_REF] Sengupta | and cooked rice to an estimation of the dietary intake of inorganic arsenic in a rural village of West Bengal, India[END_REF] showed that when rice was cooked by this traditional method, the washing water was able to remove an important amount of the metalloid, about 25-27%; a similar pattern was reported for the discarded water, which caused an As reduction of about 29.9-30.1%.

Consequently, it seems that the traditional method of cooking rice is the best one to reduce As intake, because the rice washing and cooking with a high volume of water reduces the As concentration in the final cooked rice compared to other methods; this is even true when As contaminated water is still used (40 µg As l -1 ).

As previously described by Signes et al. (2008b) t-As concentrations were always higher in boiled rice samples than in atab rice; this is without any doubt link to usage of As-contaminated water in the dehusking operations of the wet method.

The atab rice is obtained from paddy rice exclusively by mechanical dehusking while boiled rice is obtained after heating wet paddy rice before its mechanical hulling.

Daily arsenic intake

The parameter most commonly used for the evaluation of As risk assessment is the Provisional Tolerable Weekly Intake (PTWI) established by FAO/WHO at the level of 15 µg i-As week -1 kg -1 body weight [START_REF] Who | Evaluation of certain food additives and contaminants[END_REF]. This parameter can be easily transformed into the tolerable daily intake (TDI) as follows: TDI = PTWI/7 days = 2.14 µg i-As day -1 kg -1 body weight, and if we assumed that adults in West Bengal have a mean body weight of approximately 70 kg, the TDI can be established at 150 µg i-As day -1 . According to [START_REF] Roychowdhury | Survey of arsenic in food composites from an arsenic affected area of West Bengal, India[END_REF], the mean average daily intakes for an adult in West Bengal are 712.5 g of cooked rice and 4 l of water [START_REF] Roychowdhury | Survey of arsenic in food composites from an arsenic affected area of West Bengal, India[END_REF]. According to Signes et al. (2008a) approximately 95% of the As present in cooked rice will be present as i-As; so t-As can be considered equal to i-As for the below described estimations of As intakes.

Table V summarizes the estimation made in this study for the daily intake of t-As from rice cooked using the three studied cooking methods. Initial As concentrations in raw atab rice and cooking water were 185 µg kg -1 and 40 µg l -1 ; after cooking this type of rice following the traditional, intermediate and contemporary methods, the daily intakes could be estimated in 168, 182, and 187 µg day -1, respectively. If these amounts are added to the As intake coming from the drinking water, total daily i-As intakes could be established at 328, 342, and 347 µg day -1 , for traditional, intermediate and contemporary cooking methods. Similar patterns but higher figures were obtained for boiled rice (initial concentration of 315 µg kg -1 ), 356, 420, 437 µg day -1 , respectively.

In both cases (atab and boiled rice) daily As intakes were higher than the WHO TDI of 150 µg i-As day -1 . In fact, considering just cooked rice will provide figures higher than this recommended maximum intake. When atab rice was used, the daily As intake was 2.1-2.3 times higher than TDI and when boiled rice was used the daily As intake was 2.3-2.9 times higher than TDI.

  arsenic (t-As) was performed with a Unicam atomic absorption spectrometer, model Solaar 969, equipped with a continuous hydride generation Unicam Solaar VP90 (AAS-HG).

  .7 nm; spectral bandpass: 0,5 nm; hollow cathode lamp current setting 8 mA; air/acetylene flame with a fuel flow rate of 0.8 l min -1 .The certified reference materials (rice flour=NIST SRM 1568a; and bush, branches and leaves=GBW07603) used for testing this analytical method were provided by CYMIT Química S.L. (Barcelona, Spain) and produced by the US National Institute of Standards and Technology and the Institute of Geophysical and Geochemical

  Potable and Irrigation Purposes, TIPOT, ASI/B7301/2598/24-2004/79013), the Spanish Government MEC (CTM2005-2425-E), Valencian Government GV (AE05/056), and Caja de Ahorros del Mediterráneo(CAM-2007). The contents of this paper are the sole responsibility of UMH and can under no circumstances be regarded as reflecting the positions of the European Union, Spanish/Valencian governments and/or CAM.

Table I .

 I Mass balance table in atab rice as affected by the cooking method.

	Food Additives and Contaminants	Page 14 of 18
	As in boiled rice (µg/kg) i 185 185 185 washings Method A B C As in Weight of Weight of raw rice (g) ii 12 12 12 washings t-As in washings t-As in raw rice (µg) iii = (i × ii)/1000 2.220 2.220 2.220 As in cooked rice As in washings (µg kg -1 ) iv 40 40 0 Weight of Weight of washings (g) v 60 60 0 cooked rice (dry) t-As in cooked rice t-As in washings (µg) vi = (iv × v)/1000 2.400 2.400 0.000 As in discarded As in cooking water (µg kg -1 ) vii 40 40 40 water Weight of Weight of cooking water (g) viii 60 24 24 discarded water (µg/kg) (g) (µg) (µg/kg) (g) (µg) (µg/kg) (g) x Xi xii = (x × xi)/1000 xiii xiv xv = (xiii × xiv)/1000 xvi xvii 47 57 2.679 236 12 2.832 108 14 44 57 2.508 255 12 3.060 0 0 0 0 0.000 263 12 3.156 0 0 F o r P e e r R e v i e w O n l y	t-As in cooking water (µg) ix = (vii × viii)/1000 2.400 0.960 0.960 t-As in discarded water (µg) xviii = (xvi × xvii)/1000 1.512 0 0

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Table II .

 II Mass balance table in boiled rice as affected by the cooking method.

	Page 15 of 18						Food Additives and Contaminants
	Method	As in boiled rice	Weight of raw rice	t-As in raw rice	As in washings	Weight of washings	t-As in washings	As in cooking water	Weight of cooking water	t-As in cooking water
			(µg/kg)	(g)	(µg)		(µg kg -1 )	(g)	(µg)	(µg kg -1 )	(g)	(µg)
			i	ii	iii = (i × ii)/1000	iv	v	vi = (iv × v)/1000	vii	viii	ix = (vii × viii)/1000
	A		315	12	3.780		40	60	2.40	40	60	2.400
	B		315	12	3.780		40	60	2.40	40	24	0.960
	C As in washings (µg/kg)	315 Weight of washings (g) F 12 o t-As in washings 3.780 (µg) r	0 cooked rice As in (µg/kg)	0 Weight of cooked rice (dry) (g)	0.00 t-As in cooked rice (µg)	40 As in discarded water (µg/kg)	24 Weight of discarded water (g)	0.960 t-As in discarded water (µg)
	x 53		Xi 57	xii = (x × xi)/1000 3.021 P		xiii 275	xiv 12	xv = (xiii × xiv)/1000 3.300	xvi 133	xvii 17	xviii = (xvi × xvii)/1000 2.261
	49 0		56 0		2.744 0.000 e e r		365 389	12 12	4.380 4.668	0 0	0 0	0 0
					R
					e
						v i e
							w
							O n l
								y

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Table III .

 III Effect of rice type (atab or boiled) on As concentrations (µg kg -1 ) in cooked rice, washing water, and discarded water.Values with the same letters were not significantly different at p<0.05 for the variable studied (Tukey multiple range test).

		Food Additives and Contaminants	Page 16 of 18
		Variable	Type of rice	Mean ± ES
		As in cooked rice (µg kg -1 )	Atab rice	283 ± 11 b †
		As in Washings (µg kg -1 ) F o r	Boiled rice Atab rice	390 ± 12 a 46 ± 1 a
			Boiled rice	50 ± 2 a
	†	P As in discarded water (µg kg -1 ) Atab rice Boiled rice e e r	109 ± 9 b 146 ± 9 a
			R
			e
			v i e
			w
			O n l
				y

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Table IV .

 IV Effect of cooking method (traditional, intermediate, or contemporary) onAs concentrations (µg kg -1 ) in cooked rice, washing water, and discarded water.

	Variable	Cooking method	Mean ± ES
	As in Cooked rice (µg kg -1 )	Traditional	258 ± 14 b †
		Intermediate	365 ± 14 a
		Contemporary	387 ± 15 a
	As in Washings (µg kg -1 )	Traditional	49 ± 1 a
		Intermediate	47 ± 1 a
		Contemporary	-
	As in Discarded Water (µg kg -1 )	Traditional	127
		Intermediate	-
		Contemporary	-

†

Values with the same letters were not significantly different at p<0.05 for the variable studied (Tukey multiple range test).

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ACKNOWLEDGEMENTS

The paper was produced with the financial assistance of the European Union (Development of a Low Cost Technology for In-situ Treatment of Groundwater for

 V. Daily dietary arsenic intake from water and cooked rice as affected by the cooking method. It is assumed that each person drinks daily about 2.5 L of water and eats daily about 712.5 g of cooked rice. The arsenic concentration in the drinking and cooking water was 40 µg L -1 .

As in raw rice

As in cooked rice AsICR AsIDW DAsI (µg kg -1 ) (µg day -1 ) (µg day -1 ) (µg day AsICR, arsenic intake from cooked rice; AsIDW, arsenic intake from drinking water; DAsI, total arsenic daily intake; Mtd, method.


Gabrijela Tavčar-Kalcher
email: gabrijela.tavcar@vf.uni-lj.si

Katarina Pavšič-Vrtač

Anton Vengušt

Day

Analyst

Validation of the procedure for the determination of maduramicin in concentrates, premixes, and feeds by liquid chromatography

Keywords: Chromatography -HPLC, Extraction, Health significance, In-house validation Additives/Contaminants: Feeding, Veterinary drug residues Maduramicin, Concentrate, Premix, Feeds, Liquid chromatography, In-house validation

Validation of the procedure for the determination of maduramicin in concentrates, premixes, and feeds by liquid chromatography

Gabrijela Tavcar-Kalcher, Katarina Vrtac, Anton Vengust

Introduction

Maduramicin is a carboxylic acid ionophore for the treatment of chicken coccidiosis. It is registered in EU as a feed additive for broiler and turkey feeds. It is contained in feeds at the concentration level of 5 mg kg -1 . Premixes and concentrates marketed in Slovenia contain 1 g kg -1 and 1 % of maduramicin, respectively. To control the presence of maduramicin in concentrates, premixes, and feeds, analytical methods with performance characteristics complying with requirements of European regulation (European Parliament and Council, 2004) are needed. De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF], described two possibilities for the determination of maduramicin with liquid chromatography exist. The first method [START_REF] Gliddon | Determination of maduramicin ammonium in poultry feedstuffs by high-performance liquid chromatography[END_REF][START_REF] Markantonatos | Derivatization and HPLC/fluorescence quantitation of maduramicn ammonium in feed and premixes at levels down to 5 ppm[END_REF]) includes pre-column derivatisation with dansylhydrazine and fluorescence detection and the second method (De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF]) includes post-column derivatisation and the detection at 520 nm. While De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF] carried out the validation of the later one, in our laboratory, the procedure for the determination of maduramicin in concentrates, premixes, and feeds based on the methods of [START_REF] Gliddon | Determination of maduramicin ammonium in poultry feedstuffs by high-performance liquid chromatography[END_REF] and [START_REF] Markantonatos | Derivatization and HPLC/fluorescence quantitation of maduramicn ammonium in feed and premixes at levels down to 5 ppm[END_REF] was introduced and validated. An attempt was made to obviate the drawbacks of the procedure indicated by De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF] and the validation of the procedure was performed in line with Decision 2002/657/EC (European Commission 2002). In view of the absence of suitable regulations, it seems to be a reasonable starting point, although it provides rules for analytical methods used for the determination of residues in products of animal origin. The purpose of the present work is to report the performance characteristics of the procedure and to provide the comparison with data presented by De Jong (2004). Nevertheless, the procedure might be an alternative for the determination of maduramicin avoiding the use of post-column derivatisation device.

Materials and methods

Apparatus

Universal laboratory oven (Memmert, Schwabach, Germany) was used for drying samples and the linear shaker IKA HS 501 digital (IKA Labortechnik, Staufen, Germany) was used for the extraction. Measurements were performed with an HPLC system Waters Alliance 2690 (Waters, Milford, MA, USA) equipped with a computer with a program Millennium for the system control and data processing, a column Luna C18, 5 µm, 150 x 4.6 (Phenomenex, Torrance, CA, USA), and UV/VIS detector Waters 2487. The mobile phase flow rate was 3 mL min -1 , the injection volume 20 µL and the column was kept at ambient temperature. The detection was carried out at λ = 254 nm.

Samples

The validation of the procedure was performed with the samples of a concentrate, a premix and a feed containing 1 %, 1 g kg -1 , and 5 mg kg -1 of maduramicin, respectively and samples of blank feeds. The concentrate was produced by Alpharma Animal Health (Fordingbridge, UK). The premix, intended for broilers and the feed for turkeys containing maduramicin were produced by Lek Veterina (Lipovci, Slovenia). As blank feeds, two commercially available finishers for broilers and two commercially available feedingstuffs for laying hens produced by Slovene feed plants were used. They were spiked with maduramicin prior to the extraction using a standard solution. The selected volume of a standard solution was applied to a

Chemicals and reagents

A certified standard of maduramicin sodium was obtained from Alpharma Animal Health (Willow Island, WV, USA). For the stock standard solution (1 mg mL -1 ), 100 mg of maduramicin standard was dissolved in 100 mL of acetonitrile. Working standard solutions with the concentrations of 0.5, 1.0, and 1.5 µg mL -1 were prepared diluting the stock standard solution with acetonitrile. Acetonitrile, trichloroacetic acid (Merck, Darmstadt, Germany), dansylhydrazine

(1-dimethylaminonaphthalene-5-sulfonylhydrazine) (Fluka, Buchs, Switzerland) and tetrabutyl hydrogensulfate (Aldrich, St. Louis, MO, USA) were of analytical or chromatography grade purity. Dansylhydrazine solution and trichloroacetic acid solution were prepared by dissolving 0.38 g of dansylhydrazine and 7.5 g of trichloroacetic acid, respectively in 50 mL acetonitrile. The acetonitrile-water mixture was made of 50 mL of deionised water made up to 500 mL with acetonitrile. For the mobile phase, 0.22 g of tetrabutyl hydrogensulfate was dissolved in 150 mL of deionised water and made up to 1 L with acetonitrile. For solid-phase extraction clean-up of samples, Florisil Sep-Pak columns, 3 mL, 500 mg (Waters, Milford, MA, USA) were used.

Analytical procedure

A modified procedure of [START_REF] Gliddon | Determination of maduramicin ammonium in poultry feedstuffs by high-performance liquid chromatography[END_REF] and [START_REF] Markantonatos | Derivatization and HPLC/fluorescence quantitation of maduramicn ammonium in feed and premixes at levels down to 5 ppm[END_REF] was used. A weighed portion of a sample (20.0 g of a ground feed sample, 2.0 g of a premix or 2.0 g of a concentrate) was extracted with 100 mL of acetonitrile for 1 hour using a linear shaker. Feed µL trichloroacetic acid solution were added and the mixture was stirred for 10 seconds. The mixture was transferred promptly onto the SPE column. The column was washed twice with two 5.0 mL portions of acetonitrile. All the liquid from the column was discarded. The analyte was eluted from the column with 5.0 mL of acetonitrile-water mixture. Maduramicin was determined by an HPLC method with UV detection under conditions described above.

The working standard solutions used for the preparation of the calibration curve were derivatised with dansylhydrazine and passed the SPE column in the same way as samples.

Validation procedure

For the linearity test as well as for the determination of LOD and LOQ, working standard solutions of maduramicin with concentrations from 0.1 µg mL -1 to 2 µg mL -1 , which corresponds to the maduramicin content in feeds from 0.5 mg kg -1 to 10 mg kg -1 , were prepared and HPLC measurements were performed. The identity of the analyte was examined by co-chromatography. Chromatographic measurements of a sample extract, obtained from a feed sample containing maduramicin and the sample extract with an addition of a standard solution of maduramicin were performed. For the within-laboratory reproducibility test, the concentrate, the premix, and the feed containing maduramicin were analysed by three analysts in three consecutive days. Each analyst prepared each sample in duplicates every day. Blank feeds were spiked with maduramicin at the concentration level of 1 mg kg -1 and prepared in duplicates as well (each of two finishers for broilers and one of two feedingstuffs for laying hens were prepared twice, and one feedingstuffs for laying hens was prepared three times). The design of experiments is presented in Table 1. The results of these experiments were used also for the repeatability test, for the evaluation of measurement uncertainty and for the determination of the recovery. For the determination of the recovery, blank samples were spiked also at the concentration level of 5 mg kg -1 .

Statistical procedures

In the present study, LOD was defined as a concentration giving a signal

) ( 3 0 0 b s b × +
, where between duplicate measurements and outliers [START_REF] Miller | Statistics and Chemometrics for Analytical Chemistry[END_REF]. The repeatability was expressed with the average differences between results of duplicate measurements obtained within the within-laboratory reproducibility test. It was compared to the values given as the maximum permitted difference between parallel determinations at similar concentration levels of other substances (European Commission 1999). To check the stability of test results, the stability of the recovery was used. It was followed over the period of one year at the spiking level of 5 mg kg -1 .

The expanded measurement uncertainties U (EA 2003) at the tested concentration levels were calculated from the within-laboratory reproducibility s W using coverage factor 2

( W s U × ± = 2
).

Results and discussion

The equation describing the calibration curve, constructed from six points in the range from 0.1 µg mL -1 to 2 µg mL -1 , which corresponds to the maduramicin content in feeds from 0.5 mg kg -1 to 10 mg kg -1 , was y = 6917x -2.4, where y is the peak area expressed in µVs and x is the concentration of maduramicin expressed in µg mL -1 . The correlation coefficient was 0.9987 and it was considered acceptable. LOD and LOQ calculated as mentioned above were 0.4 mg kg -1 and 1.0 mg kg -1 of maduramicin in feed, respectively. They are comparable to the values reported by De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF].

The identity of a peak obtained with the analysis of a feed containing maduramicin was confirmed with co-chromatography. From the chromatograms of an extract of a feed containing maduramicin and of the extract spiked by a standard solution of maduramicin presented in Figure 1, it is evident that only the height of the maduramicin peak was enhanced by the addition of the maduramicin standard, hence the peak can be assigned to maduramicin.

In Table 2, the results for the within-laboratory reproducibility expressed with the standard deviation (s W ) and the relative standard deviation (RSD W ), as well as the reference values are given. s W and RSD W values were calculated after eliminating results with too high difference between duplicates and outliers as mentioned above. In the case of the premix and the feed maduramicin at the concentration level of 1 mg kg -1 . The reason for a high rejection rate at the concentration level of 1 mg kg -1 was a small volume of a spike applied to a sample (20 µL).

Unfortunately, it was found out that the pipetting of such a small volume was not accurate enough. A bigger volume of a diluted standard solution was proved to be favourable. As it is obvious from Table 2, the experimental RSD W values are higher than RSD W values derived from Horwitz equation, used as references, but only in the case of the concentrate and the premix, HORRAT values (the ratio between the obtained RSD W and Horwitz value) were higher than 2, which is considered unacceptable [START_REF] Horwitz | Performance characteristics of methods of analysis used for regulatory purposes. Part II. Pesticide formulations[END_REF]. The reason for a poor reproducibility was supposed to be the off-line pre-column derivatisation of sample solutions as already mentioned by De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF]. Beside the within-laboratory reproducibility test no collaborative trial was performed.

The repeatability of the procedure was evaluated by means of differences between results of duplicate measurements. The average differences were 5.9 % at the concentration level of 1 % (concentrate), 7.1 % at the concentration level of 1 g kg -1 (premix), and 11 % with the feed containing maduramicin with the nominal concentration of 5 mg kg -1 and feed spiked at the concentration level of 1 mg kg -1 . They are lower than 15 % relative to the higher value, taken as the maximum permitted difference as mentioned above, thus they are deemed acceptable. The recoveries at the concentration levels of 1 and 5 mg kg -1 were calculated from six experiments carried out in duplicate at the concentration level of 1 mg kg -1 and from twelve experiments done in duplicate at the concentration level of 5 mg kg -1 . The recoveries were 93.4 % (ranging between 76 % and 118 %) and 99.7 % (ranging between 82 % and 118 %) at the concentration levels of 1 mg kg -1 and 5 mg kg -1 , respectively and the mean recovery was 96.6 %. To achieve acceptable recoveries, drying of feed samples at 60ºC prior to the extraction was introduced as mentioned above. It was found out in previous experiments that the presence of water in the extraction solvent affects the recovery crucially, what is in accordance with the finding of De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF]. The use of molecular sieve suggested by [START_REF] Gliddon | Determination of maduramicin ammonium in poultry feedstuffs by high-performance liquid chromatography[END_REF] was not found effective enough, so it was omitted and replaced by sample drying.

The stability of test results was tested following the recovery over the period of one year and presented by means of the Shewhart control chart (Figure 2). The oscillation of the recovery between 55 % and 135 % in the first part of the control chart might indicate a poor robustness of the method. However, after certain time, the recovery settled between 81 % and 119 %. In the study reported by [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF], the recovery fluctuated between 96 % and 105 %.

The measurement uncertainties determined at the concentration levels of 1 % (concentrate), 1 g kg -1 (premix), 5 mg kg -1 (feed) and 1 mg kg -1 (blank feed spiked with maduramicin) were ±0.2 %, ±0.3 g kg -1 , ±1.9 mg kg -1 , and ±0.3 mg kg -1 , respectively. They will be reported along the analytical results and taken into account for checking the compliance of samples. As mentioned above, the measurement uncertainties were calculated from the within-laboratory reproducibility. Since the mean recovery was close to 100 % and results of routine analysis

Conclusions

The method performance characteristics of LOD (0.4 mg kg -1 ), LOQ (1 mg kg -1 ), and repeatability (the average differences between results of duplicate measurements of 5.9 % at the concentration level of 1 %, 7.1 % at the concentration level of 1 g kg -1 , and 11 % with the feed containing maduramicin with the nominal concentration of 5 mg kg -1 and with the feed spiked at the concentration level of 1 mg kg -1 ) were comparable to those reported by [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF]. The correlation coefficient of the calibration curve (0.9987) and the withinlaboratory reproducibility expressed by RSD W of 9.2 %, 16 %, 18 %, and 17 % at the concentration levels of 1 %, 1 g kg -1 , 5 mg kg -1 , and 1 mg kg -1 , respectively indicate that the performance of the analytical procedure is not as good as the performance of the method reported by De [START_REF] Jong | Determination of maduramicin in feedingstuffs and premixtures by liquid chromatography: Development, validation, and interlaboratory study[END_REF]. Except RSD W of 9.2 % and 16 % in the case of the concentrate and the premix (HORRAT >2), the determined parameters correspond to adopted reference values, hence the procedure might nevertheless provide an alternative for the determination of maduramicin in concentrates, premixes and feeds avoiding the use of postcolumn derivatisation device. lower action limit (mean-3s W ), (c) lower warning limit (mean-2s W ), (d) upper warning limit (mean+2s W ), (e) upper action limit (mean+3s W ). The spiking level: 5 mg kg -1 .

F

  a ground sample and the spiked sample was kept for half an hour prior to the addition of the extraction solvent.

  prior to the extraction at 60ºC in the oven overnight. 25.0 mL of a sample extract was transferred to a beaker (the extracts of a concentrate and a premix were diluted 200-folds or 20-folds previously with acetonitrile). 600 µL dansylhydrazine solution and 1200

  of a calibration curve and the standard deviation of the intercept, respectively. As LOQ, a value of 2.5 times LOD was selected. The withinlaboratory reproducibility was expressed with the standard deviation s W and the relative standard deviation RSD W . RSD W values were compared to the values derived from the Horwitz equation, given as the reference value in Decision 2002/657/EC (European Commission 2002). Prior to the calculation of listed parameters, results of experiments were examined by the Cochran test and the Grubbs test to eliminate results with too high difference

  containing maduramicin, no measurement was eliminated either by the Cochran test or by the Grubbs test. With the concentrate, a result of one experiment done in duplicates was eliminated by the Grubbs test and with the feed spiked with maduramicin at the concentration kg -1 , results of two experiments were eliminated by the Cochran test and a result of one experiment done in duplicates was eliminated by the Grubbs test. Therefore, the results for s W and RSD W were calculated from nine experiments done in duplicates with the premix and the feed containing maduramicin, from eight experiments done in duplicates with the concentrate, and from six experiments done in duplicates with the feed spiked with

  be reported uncorrected for recovery, the contribution of recovery was not included in the measurement uncertainty.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Shewhart control chart for the recovery of maduramicin. (a) mean recovery, (b)

Table 1 .

 1 The design of experiments for the within-laboratory reproducibility test.

	Concentrate	Premix	Feed containing 5 mg kg -1	Feeds spiked at the level 1 mg kg
	o r
	P
	e
	e r
		R
		e
		v i e
			w
			O n l
			y

Table 2 .

 2 The within-laboratory reproducibility of measurements, expressed with the standard deviation (s W ) and relative standard deviation (RSD W ).

			Food Additives and Contaminants	Page 14 of 16
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	Nominal concentration	Determined conc. level	Number of repetitions	s W	RSD W (%)	Horwitz value (%)
	1 % (concentrate)	1.18 %	16	0.11 %	9.2	4.0
	1 g kg -1 (premix)	0.93 g kg -1	18	0.15 g kg -1	16	5.7
	5 mg kg -1 (feed) 5.14 mg kg -1	18	0.94 mg kg -1	18	13
	1 mg kg -1 (feed) 0.95 mg kg -1	12	0.16 mg kg -1	17	16

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