Antonio J Signes

Kasturi ; Mitra

Francisco M Burló

Angel A Carbonell-Barrachina

Antonio ; Signes

Miguel Universidad

Hernández

Ramakrishna

Miguel Hernández

Angel ; Universidad

Keywords: FAAS, Market basket survey, Metals analysis -AAS, Risk assessment Additives/Contaminants: Environmental contaminants, Environmental contaminants -Soils, Heavy metals -arsenic Food Types: Cooked foods, Rice arsenic speciation, cooked food, cooking water, organic arsenic, 25 total arsenic

Introduction 1 2

The world's two biggest cases of groundwater arsenic contamination and the 3 worst sufferings of people have been in Asia; in order of magnitude, these are 4 Bangladesh and West Bengal, India [START_REF] Rahman | Arsenic 34 groundwater contamination and sufferings of people in North 24-Parganas, 35 one of the nine arsenic affected districts of West Bengal, India[END_REF]. Several million people 5 in West Bengal consume water with arsenic concentrations which exceed by up 6 two orders of magnitude the threshold value of 10 µg L -1 recommended by the 7 WHO [START_REF] Norra | Impact of irrigation with As rich groundwater on soil and crops: A 30 geochemical case study in West Bengal Delta Plain, India[END_REF]. Arsenic contamination of groundwater and illnesses of 8 people have been reported in nine districts out of a total of 18 districts in West 9

Bengal (Roychowdhury et al. 2003), including North-24-Parganas where this 10 study was located. Groundwater is the main source for drinking, cooking and 11 other household purposes in these arsenic-affected districts. Even the 12 agricultural system is mostly groundwater-dependent. In this way, a large 13 amount of arsenic deposits on the irrigated lands.

15

Recently, it has been considered that foods are responsible for an important part 16 of arsenic intake, and studies on total arsenic (t-As) in food obtained from arsenic could introduce a substantial bias into the estimation of risks for the 20 population of arsenic endemic areas [START_REF] Díaz | Contribution of water, bread, and vegetables (raw and cooked) to 6 dietary intake of inorganic arsenic in a rural village of Northern Chile[END_REF]).

22

In most arsenic-contaminated areas of West Bengal, the residents depend 23 heavily on rice for their caloric intake (about 70 % of total), suggesting that if 24 their rice is arsenic polluted it will become an important dietary source of this

30

Correct estimation of arsenic intake should consider not only the arsenic content 31 of the raw product but also the content of the contaminant in the product in the the high total arsenic contents found in the studies on total arsenic contents in 1 cooked foods from contaminated areas [START_REF] Concha | Metabolism of inorganic arsenic in children 32 with chronic high arsenic exposure in northern Argentina. Environmental 33 Health Perspectives 106:355-359. cooking on total and inorganic contents[END_REF][START_REF] Bae | 26 Arsenic in cooked rice in Bangladesh[END_REF] 2 Roychowdhury et al. 2003;Sengupta et al. 2006). In many areas of West 3 Bengal, rice is washed and/or cooked with a substantial amount of water, which 4 is sometimes contaminated with arsenic. The actual amount of arsenic in cooked 5 rice could be either increased, by chelation of arsenic in water by binding on rice 6 grains, or decreased if water-soluble arsenic is released from rice into the water 7 to be discarded [START_REF] Bae | 26 Arsenic in cooked rice in Bangladesh[END_REF]).

9

In the literature there are data for total arsenic contents in raw and cooked rice

22

The aim of the present study was to determine total and inorganic arsenic 23 contents in cooked rice using arsenic polluted cooking water. Two factors were 24 assayed in the cooking water: 1) arsenic speciation (arsenite, arsenate, 25 methylarsonate, or dimethylarsinate) and 2) total arsenic concentration (50, 26 250, or 500 µg L -1 ). Besides, the contribution of cooked rice and drinking water 27 to the daily intake of total and inorganic arsenic of the inhabitants of an arsenic-

28

affected rural village of West Bengal was evaluated.

30

Materials and Methods

32

Instrumentation

33

For arsenic speciation analysis, a high-performance liquid chromatography

12

Determination of total arsenic was performed with a Unicam Model Solaar 969 13 atomic absorption spectrometer equipped with a continuous hydride generator 14 Unicam Solaar VP90 (AAS-HG).

16

Other equipment used included a hot air oven (Selecta, Barcelona, Spain) with a 17 maximum temperature of 250 ºC, a grinder (Moulinex, Valencia, Spain), a

22

Christ Alpha 2-4, Osterode, Germany).

24

Reagents 25 Deionized water (18 MΩ cm) was used for the preparation of the reagents and 26 standards. All glassware was treated with 10% v/v HNO 3 for 24 h and then rinsed 27 three times with deionized water before use.

29

All chemicals were of, at least, pro analysis quality. Commercial standards of were obtained from Panreac (Barcelona, Spain), while CH

21

The number of interviewees was set at 115 (60 male and 55 female), with ages I). As expected boro rices contained higher t-As and i-As 5 concentrations than aus and aman samples; these higher contents are related to 6 the use of higher volume of arsenic polluted groundwater for irrigation of the

25

The first experiment was conducted to investigate effect of a) arsenic species 26 and b) arsenic concentration on the arsenic content in the cooked rice. The rice 27 was cooked using deionized water or deionized water spiked with different levels

28

(50, 250 or 500 µg L -1 ) of only one of the following four arsenic species: arsenite, 29 arsenate, methylarsonate (MA), or dimethylarsinate (DMA).

31

The food survey carried out in this study showed that none of the three methods

32

described above was the most popular in the studied rural village; the most 33 popular rice cooking method was in between methods "a" and "c" (it is a hybrid 34 in the evolution of method "a" towards method "c"). The unwashed rice is boiled 35 with a volume of water 1.5-4 times the weight of rice; the wash step is omitted. Thus, the rice (250 g) was added to boiling water (750 mL) and kept under this 1 heat conditions until cooked; the remaining water was discarded. No additional 2 ingredients were employed.

4

The second experiment was carried out to investigate the effect of arsenic 5 speciation in the cooking water on the arsenic speciation in the cooked rice. Rice 6 was cooked in the same way as described for the first experiment; cooking water 7 was spiked with four arsenic species (arsenite, arsenate, MA, or DMA) but only 8 one arsenic concentration was studied 250 µg As L -1 .

10

Once cooked, all rice samples were frozen at -20 ºC and then freeze-dried. The 11 lyophilized samples were ground in a domestic apparatus, and the resulting 12 powder was vacuum-packed and kept in the freezer at -20 ºC until analysis.

13

Total arsenic concentrations were measured in dry raw rice, wet cooked rice, 14 cooking water, and discarded starched water.

16

Quantification of total arsenic 17 A 0.250-g portion of lyophilized rice sample was weighed and digested using the

27

The certified reference materials (rice flour = NIST SRM 1568a, and bush,

4

The arsenic species (arsenite, arsenate, MA, and DMA) were determined in the 5 water extract using HPLC-HG-AAS. Separation of the arsenic compounds was 6 carried out in ca. 15 min in the anion-exchange column, using a 25 mM 7 phosphate buffer (pH 6.0) as mobile phase at 1.1 mL min -1 flow rate. The elution 8 order was arsenite, DMA, MA, and arsenate. 9 10 50 µL of sample were injected in the HPLC system, following the instrumental 11 and analytical conditions described in Table II. Under these conditions, the 12 retention times were 3.1, 4.2, 5.1 and 8.6 min for arsenite, DMA, MA, and 13 arsenate, respectively. 0.03 mg kg -1 ); accuracy for bush, branches and leaves (GBW07603), found value 1 = 1.18 ± 0.03 mg kg -1 (certified value = 1.25 ± 0.10 mg kg -1 ). 2 3 Arsenic speciation. An estimate of the instrumental detection limit (IDL) for 4 each of the four arsenic species was calculated based on 3 times the standard 5 deviation of peak area measurements for replicate 50 µL injections of an arsenic 6 standard containing 2.0 µg L -1 each of arsenite, arsenate, MAA and DMA. The IDL 7 estimates were 1.7, 1.5, 1.1, and 1.4 µg L -1 , respectively. Finally, estimates of 8 the method detection limits (MDLs) were calculated using the IDLs multiplied by 9 a dilution factor of 10 (0.5 g of rice diluted to a final volume of 5 mL). The MDLs 10 were 17, 15, 11, and 14 µg As kg -1 fresh matter for arsenite, DMA, MA and 11 arsenate, respectively. The average fortification recoveries (for a 0.75 µg As 12 spike on 10 mL of a 20 µg L -1 standard) through the method were 94%, 89%, 13 90%, and 92% for arsenite, DMA, MA, and arsenate. No significant 14 reduction/oxidation reactions were observed in these fortification studies.

16

There are no food certified reference materials available for arsenic species 17 and/or inorganic arsenic. The quality criterion adopted for testing the current 18 analytical method, therefore, was the overlapping between the ranges of I-As

24

Finally, the total arsenic concentration (sum of the four studied arsenic species)

25

of the NIST SRM 1568a, 0.268 ± 0.028 mg kg -1 , was compared with the certified 26 value of the material, 0.290 ± 0.030 mg kg -1 , and shows the goodness of the 27 analytical method used.

29

1 st Experiment "total arsenic in cooked rice"

30

The ratio of added water to raw rice was 3:1 (750 mL: 250 g), which is typical of 31 the cooking habits in West Bengal and Bangladesh [START_REF] Misbahuddin | Consumption of arsenic through cooked rice[END_REF]).

32

According to [START_REF] Bae | 26 Arsenic in cooked rice in Bangladesh[END_REF] the water:rice ratio ranges from 3.2:1 to 4.0:1 in 33 Bangladesh, which was significantly higher than the ratio used, for example, in Tables III-IV show the effect of t-As concentration and As speciation initially 1 present in the cooking water on the t-As concentration in the cooked rice. In 2 general, no effect of t-As concentration (Table III) or As speciation (Table IV) 3 was found on the volumes of absorbed water in the cooked rice (absorbed water 4 was measured by drying cooked rice and raw rice at 70ºC until constant weights 5 and subtracting these two water contents); although the rice cooked with 6 arsenite retained significantly more water (639 ± 17 mL) than the others (mean 7 of 533 ± 17 mL).

9

As will be discussed later, results from our calculations suggest that cooked rice 10 could be an important source of As if it is boiled using As-contaminated water.

11

According to the experimental data, 1000 g of cooked rice will correspond to 12 roughly 309 g of raw rice, and about 691 g of contaminated water. Therefore, 13 this cooked rice will provide an additional and substantial burden of As to that 14 coming from the drinking water.

16

The amount of As in the cooked rice was 2.2-20.0 % higher than predicted (from 17 raw rice and absorbed water) for initial As concentrations of 250 and 500 µg As

29

On the other hand, when the initial As concentration was 50 µg As L -1 , the As These experimental observations indicate that the concentration of As through 32 loss of water in the cooking process is not a good explanation. Besides, another 33 experimental parameter sustaining this statement is that the ratio of As in initial 34 water to that in the discarded water was close to 1 in samples cooked with water 35 containing 250 and 500 µg As L -1 (implying no As concentration due to water 36 evaporation), while it was 0.4 for the initial concentration of 50 µg As L -1 .

12

Arsenic speciation of raw rice demonstrated that arsenic was mainly present as 13 inorganic forms, 206.8 µg arsenite kg -1 rice plus 39.7 µg arsenate kg -1 , and 25.5 14 µg DMA kg -1 were also found. Considering that 0.250 kg of raw rice were used in 15 our cooking simulations, the total amounts of 51.7 µg arsenite, 9.9 µg arsenate 16 and 6.4 µg DMA were incorporated into the cooking system from the raw rice.

17

Besides, a total of 187.5 µg of each species (arsenite, arsenate, MA, and DMA)

18

were incorporated from the spiked cooking water in each of the different 19 simulations. In summary, a total of 255.5 µg arsenic was added to the cooking 20 system from both rice and spiked cooking water.

22

A control cooking experiment using arsenic-free water was carried out and

23

showed that cooking caused an oxidation of arsenite to arsenate, and a complete 24 transformation of the initial DMA into MA and inorganic arsenic. Arsenic was 25 present in this cooked rice as 110.4 µg arsenate kg -1 , 76.4 µg arsenite kg -1 , and 26 10.8 µg MA kg -1 .

28

Data on Table V showed that when arsenic was present in the cooking water as 29 inorganic forms (arsenite or arsenate), it was mainly present in the cooked rice 30 as inorganic forms as well. However, about 10 % of the final arsenic in the 31 cooked rice was present under the form of non-added species; for example,

8

In general, from this section dealing with As speciation in the cooked rice it can 9 be concluded stating that arsenic will be present in the cooked rice in the same 10 form it was initially found in the cooking water and raw rice.

12

Estimation of arsenic intake and evaluation of risks 13

From the data provided by the 24 h recall questionnaire administered to the 14 inhabitants of the village in 24-North Parganas district [quantity of water (mL), 15 rice ingested (g)] and the t-As and i-As concentrations spiked in the cooking 16 water and found in raw and/or cooked rice analyzed (Tables III andIV), the 17 intakes of t-As and i-As were calculated, and, expressed as µg As day -1 , are 18 shown in Table VI. According to the food habits survey, the average daily water 19 intake for adult (mean of males and females) was 2.5 L. This result is relatively

26

Our results proved that the general assumption that As is present in the drinking 27 water and foods mainly under inorganic forms, arsenite and/or arsenate, is 28 correct; the As intakes estimated in this study show that the daily i-As intake 29 represents between 82 and 99 % of the daily T-As intake. This is due to the 30 types of foods analyzed, water and rice, as indicated earlier, i-As are the major 31 species. Bengal also provide data for t-As intake from water and from raw and cooked 17 food. Assuming that at least 50 % of the t-As in food sample is i-As, the 18 maximum intake obtained from water and foods was 708 µg day -1 in adult males, 19 which is 4.7 times greater than the TDI. In this case, the daily intake contributed 20 by foodstuffs (rice, vegetables, and spices) was 189 µg, 27 % of the daily t-As 21 intake.

33

23

In the present study, the daily arsenic intakes obtained from water plus rice were AsIDW=arsenic intake from drinking water; AsICR=arsenic intake from cooked rice; DAsI=Total arsenic daily intake.

25 metalloid(

 25 Watanabe et al. 2001). As rice is cultivated in arsenic contaminated 26 soils under anaerobic conditions (at which arsenic is highly available for plant 27 uptake), arsenic concentration in rice is high compared to other crops and 28 regions (Carbonell-Barrachina et al. 1998; Abedin et al. 2002; Meharg 2004).

32 form

 32 in which it is consumed by the population (raw or cooked). Studies have33shown changes in arsenic concentration after cooking of seafood (Devesa et al.

10(

  Roychowdhury et al. 2003; Islam et al. 2004; Norra et al. 2005), but few values 11 have been reported for inorganic arsenic (Ackerman et al. 2005; Laparra et al.

15 (

 15 12 2005). However, knowledge of inorganic arsenic contents is essential for an 13 evaluation of health risks. Inorganic arsenic has been classified by the 14 International Agency for Research on Cancer (IARC) as a carcinogen to humans Díaz et al. 2004; Tsuda et al. 1992). Nevertheless, there are no prior references 16 to evaluate the influence of cooking on individual arsenic species in rice from 17 arsenic affected areas such as West Bengal. Up to our knowledge there are only 18 two studies dealing with speciation of arsenic in cooked rice, the first one carried 19 out by Laparra et al. (2005) using Spanish rice and the second one carried out 20 by Ackerman et al. (2005) using North-American rice.

34( 4 NaBH 4

 44 HPLC) system consisting of a Varian 9012 ternary pump (Varian, San Fernando, 35 CA, USA), a Rheodyne 7125 injector and a 50 µL loop for sample introduction, 36 was used. Separations of arsenic species were performed on a Hamilton PRP X-exchange column (10 µm, 250 mm × 4.1 mm i.d.; Hamilton, Reno, 1 NV, USA). A guard column packed with the same material (12-20 µm; 25 mm × 2 2.3 mm i.d.) preceded the analytical column. Hydride generation of volatile 3 arsines prior to the detection was performed adding on-line solutions of HCl and by means of a Gilson Minipuls 3 peristaltic pump. The quantification of 5 arsenic was performed on a hydride generation system (PSA 10.044, PS 6 Analytical, Kent, U.K.) using an atomic fluorescence spectrometer system (AFS) 7 (PSA 10.044 Excalibur, PS Analytical) equipped with a boosted-discharge hollow 8 cathode lamp (Photron Pty. Ltd., Victoria, Australia). The analogical signal output 9 was connected to a computer equipped with chromatographic software (PS 10 Analytical).

18 mechanical

 18 shaker Vibromatic (J.P. Selecta S.A., Barcelona, Spain), a centrifuge 19 (Heraeus BioFuge, Heraeus Instruments, Hanau, Germany), a sand bath (Falc, 20 Treviglio, Italy), model BS 70 with a maxim temperature of 200 ºC, a muffle 21 furnace (Hobersal, Barcelona, Spain) and a lyophilizer (B. Biotech International,

22

  above 12 years old to below 60 years. The interviewees selected were mainly 23 farmers and housewives who normally eat at home, buy or get their food from 24 local markets and/or farms, cook themselves and take drinking water from 25 surrounding tube wells. People working at nearby cities were not included in the 26 survey; they are not representative of endemic arsenic area because they eat 27 frequently at the city.

1

  Commercial rice samples were collected in farms surrounding the previously 2 cited rural village of the North 24-Parganas district. Rice samples from different 3 periods of the year and varieties were analyzed for total and inorganic arsenic 4 concentrations (Table

7

  boro rice plants (grown during summer time) compared to aus and aman plants.

8

  After carefully study of information summarized in TableI, authors decided to 9 select one of the boro varieties, the khitish rice (IET-4094), for their experiments 10 because boro rices are those representing the highest potential health risks for are three common methods of cooking rice (Sengupta et al. 15 2006): a) The traditional method still used by more than 90% of the villagers in 16 Bengal delta: raw rice is washed until the washings become clear (5-6 times), 17 washings are discarded and then the rice is boiled in excess water (5-6 times 18 the weight of raw rice) until cooked, finally discarding the remaining water 19 (discard water) by tilting the pan against the lid before serving the rice; b) the 20 rice is washed as in "a" and boiled with water of a volume 1.5-2 times the 21 weight of rice until no water is left to discard; c) unwashed rice is boiled with 22 water 1.5-2 times the weight of rice; the wash and discard steps are both 23 omitted. This is the contemporary method.

  18ashing method previously described by[START_REF] Muñoz | Total and inorganic arsenic in fresh and processed fish products[END_REF]. Calibration 19 standards were prepared using the same HCl concentration of the samples and 20 certified materials. The instrumental conditions used for arsenic determination by 21 HG-AAS were as follows: reducing agent: 1.4 % (m/v) NaBH 4 in 0.4 % NaOH, 5 22 mL min -1 ; HCl solution: 10 % (v/v), 10 mL min -1 ; carrier gas: argon, 250 mL 23 min -1 flow rate; and for atomic absorption spectrometry, wavelength: 193.7 nm; 24 spectral bandpass: 0.5 nm; hollow cathode lamp current setting 8 mA; 25 air/acetylene flame with a fuel flow rate of 0.8 L min -1 .

28 branches

 28 and leaves = GBW07603) used for testing this analytical method were 29 provided by CYMIT Química, S.L. (Barcelona, Spain) and produced by the 30 National Institute of Standards and Technology of U.S.A. and the Institute of 31 Geophysical and Geochemical Exploration of China, respectively.

32 33

 32 Quantification of arsenic species 34The method used for the extraction of the arsenic species was that described by 35[START_REF] Heitkemper | Determination of total 12 and speciated arsenic in rice by ion chromatography and inductively 13 coupled plasma mass spectrometry[END_REF]. Dried and milled rice sample (0.5 g) was treated with 36 3 mL of 2 M TFA. The mixture was allowed to stand for 6 h at 100 ºC in a 60 mL 37 HDPE centrifuge tube. The mixture was centrifuged and the supernatant 1 was collected and diluted to volume with deionized water. The TFA extracts were 2 filtered through a 045 µm nylon syringe filter prior to analysis by HPLC-HG-AFS.

  Figure 1 shows the separation obtained in: a) a 50 µg L -1 14 standard of arsenite, DMA, MA, and arsenate, b) the certified material NIST SRM 15 1568a, rice flour, c) raw rice, and d) rice cooked using arsenite-polluted water.16 17External calibration was accomplished using standard concentrations of 1, 10,

18 20 ,

 20 30, 40, and 50 µg L -1 of each of the four arsenic species studied (arsenite, 19 DMA, MA and arsenate).

20 21

 2021 Information on the certified material NIST SRM 1568a (rice flour) was used for 22 testing this analytical method.23 24Statistical analyses 25All data were subjected to analysis of variance (ANOVA) and the Tukey least 26 significant difference multi-comparison test to determine significant differences 27 among samples (arsenic species and/or arsenic concentration). The statistical 28 analyses were done using SPSS 12.0 (SPSS Science, Chicago, USA). The analytical characteristics for the total arsenic methodology 34 were as follows: detection limit, 7 µg kg -1 ; precision 2 %; accuracy for rice flour35(NIST SRM 1568a), found value = 0.29 ± 0.04 mg kg -1 (certified value = 0.29 ± 36

19

  found in a certified rice flour sample (NIST SRM 1568a): 0.082 ± 0.09 mg kg -1 20 and those reported in a previous study carried out by Heitkemper et al. (2001), 21 0.083 ± 0.06 mg kg -1 using the same method of extraction for the arsenic 22 species.

34

  Japan, 1.3:1.

18 L

 18 -1 , respectively. These results might suggest either that As in the water is 19 chelated by rice grains, or that As becomes concentrated during the cooking 20 process, because of evaporation. 21 22 Díaz et al. (2004) stated that the total arsenic contents in raw food can alter in 23 various ways during cooking treatments, with a consequent effect on the intake 24 of this contaminant. Cooking treatments such as boiling and frying can alter total 25 arsenic content by a) concentration of arsenic through loss of water, volatiles, 26 and, to a lesser extent, certain macronutrients (carbohydrates, lipids, and 27 proteins) and b) loss of As through solubilization.

11 1

 11 Data on TableIVshows a significantly higher As concentration in the rice 2 cooked using polluted cooking water containing arsenite. The mechanisms for 3 capturing t-As and inorganic arsenic (i-As) might be related to the incorporation 4 of water into food during cooking. This statement is supported by the fact that 5 significantly more water was absorbed by the rice cooked with arsenite (639 ± 17 6 mL) compared to the others (532 ± 11 mL). The high water retention capacity of 7 the rice might be due to the high starch content in this cereal (≅ 90 %), which 8 incorporates a large quantity of water during its gelatinization in the boiling 9 process (Tinarelli 1989).

10 11 2

 11 nd Experiment "arsenic speciation in cooked rice"

32 when1

 32 As was initially present in the cooking water as arsenite, 23.8 ± 0.4 µg of 33 arsenite were transformed in arsenate. On the other hand, if As was initially 34 present in the cooking water under organic forms (MA or DMA), no 35 transformation between organic species occurs; however, a significant amount of 36 arsenite was found.To date, the only previous reference found dealing with the contents of i-As in 2 cooked foods is the study carried out by[START_REF] Díaz | Contribution of water, bread, and vegetables (raw and cooked) to 6 dietary intake of inorganic arsenic in a rural village of Northern Chile[END_REF] in Chile, with a 3 maximum concentration of 1.58 µg g -1 ww in maize boiled in water containing 4 0.572 µg As mL -1 . The lack of data concerning i-As content in cooked samples 5 from arsenic endemic areas shows the novelty of the results reported in the 6 present work.

20 low

 20 compared to results provided by Roychowdhury et al. (2003), who found that 21 adult males, adult females and children (<10 years of age) consumed 4, 3, and 2 22 L, respectively, at Murshidabad district, West Bengal. Besides, a mean rice intake 23 of about 450 g for adults was used for our calculations, while Roychowdhury et 24 al. (2003) found rice intakes of about 750 g.

Díaz 8 %

 8 et al. (2004) studied the contribution of water, bread, and vegetables to the 34 dietary intake of arsenic in a rural village of Northern Chile. They studied these 35 items in two different periods, in which the water used by the population for 36 drinking and cooking purposes contained 0.572 (first period) or 0.041 µg mL -1 ). The foods studied contribute to 5 % (first period) or 30 % 1 (second period) of the total As intake. Consequently, Díaz et al. (2004) 2 concluded that the significance of the intake of As from food increases as the 3 concentration of As in water decreases. This result should be taken into account 4 in non-arsenic endemic areas. The main difference between the study by Díaz et 5 al. (2004) and the present study carried out in West Bengal is the amount of rice 6 being consumed by the Indian population, 450 g day -1 . With these initial 7 considerations, only rice consumption will contribute 45.0 %, 38.6 %, and 37.2 to the final total As intake for cooking water containing 50, 250, and 500 µg L - 9 1 , respectively (TableVI).

  weight of 70 kg for adult people in West Bengal, the reference 12 intakes stated by the FAO/WHO are equivalent to 150 µg i-As day -1 for adult 13 people (Díaz et al. 2004; WHO 1989).

14 15 In

 15 India, the studies carried out by Roychowdhury et al. (2002-2003) in West 16

  24 227, 1018, and 1989 µg As day -1 for initial As concentrations in the cooking 25 water of 50, 250, and 500 µg As L -1 , respectively, which are 1.5, 6.8, and 13.3 26 times greater than the TDI. Similar figures are obtained when inorganic species 27 are considered (Table VI); 229, 1024, and 2000 µg As day -1 for initial As 28 concentrations in the cooking water of 50, 250, and 500 µg As L -1 , respectively, 29 which are 1.5, 6.8, and 13.3 times greater than the TDI.

32 Our7(

 32 results suggest that rice cooked with arsenic-contaminated water might be 33 an important source of arsenic, especially inorganic arsenic, and that the 34 conditions of the cooking process (ratio rice:water and volume of water 35 discarded) could affect the amount of this element in final cooked rice. A dose-36 response association between arsenic exposure and any health effects might 37 risk of arsenic if the intake of this element from 1 sources other than drinking water is not included. Finally, if cooking water 2 containing low levels of arsenic can be provided to villagers, even if their 3 vegetables are still contaminated with arsenic, cooked items will have an arsenic 4 concentration lower than expected due to migration of some the arsenic to the 5 cooking water. 6 This paper has been produced with the financial assistance of the European Union 9 Development of a low cost technology for in-situ treatment of groundwater for 10 potable and irrigation purposes, TIPOT, ASI/B7-301/2598/24-2004/79013), MEC 11 (CTM2005-2425-E), and Caja de Ahorros del Mediterráneo (CAM-2006). The 12 contents of this document are the sole responsibility of UMH and can under no 13 circumstances be regarded as reflecting the position of the European Union 14 and/or CAM. for mankind. Applied Organometallic Chemistry 6:309-322. 16 Watanabe C, lnaoka T, Kadono T, Nagano M, Nakamura S, Ushijima K, 17 Murayama N, Miyazaki K, Ohtsuka R. 2001. Males in rural Bangladeshi

18 communities

 18 are more susceptible to chronic arsenic poisoning than 19 females: analyses based on urinary arsenic. Environmental Health 20 Perspectives 109:1265-70.

21 WHO. 1989 .Food

 211989 Evaluation of Certain Food Additives and Contaminants; 33rd Report 22 of the Joint FAO/WHO Expert Committee on Food Additives; Technical Page 16 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

3Figure 1 .3

 1 FIGURE CAPTIONS

Table I .

 I Total arsenic concentration in raw rice samples procured. All samples 1 were from an arsenic affected area of North-24-Parganas district, West Bengal.

	2 3 Sample Cultivar (number of samples) Khitish, Initial Evolution Trial IET-4094 (3) Jaladhi-2, BAKU (3) Aditya Initial Evolution Trial IET-7613 (3) Biraj, CNM-539 (3) Khitish, Initial Evolution Trial IET-4094 (3) Ratna, IET-1411 (3) Khitish, Initial Evolution Trial IET-7328 (3) F o r P e F o r P e Food Additives and Contaminants Type of Rice Boro † Boro † Aus ‡ Aus ‡ Aman γ Aman γ Aman γ e r e r	Mean t-As (µ µ µ µg kg -1 ) 272 ± 20 410 ± 11 178 ± 14 125 ± 12 166 ± 16 133 ± 14 177 ± 16	Mean i-As (µ µ µ µg kg -1 ) 247 ± 7 407 ± 8 165 ± 7 116 ± 13 140 ± 9 120 ± 11 163 ± 5
	R R
	e e
	v i e v i e
	w w
	O n l O n l
		y y

† Boro: irrigation done by groundwater during summer time (November to June).

4 ‡ Aus: prekharif (April to September). γ Aman: irrigation done by rainwater 5 (kharif: June to December). 6 Page 17 of 24 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table III .

 III Effect of the arsenic concentration (50, 250, or 500 µg L -1 ) present in 1 the cooking water on the variables under study in the cooking of rice.

	Page 19 of 24	Food Additives and Contaminants
	2
		Variable		As in Cooking Water (µ µ µ µg L -1 )	Mean ± ES †
		Absorbed Water in Cooked Rice (mL)
				50	548 ± 15 a ‡
				250	583 ± 16 a
		Discarded Water (mL) F o r		500 50 250	548 ± 15 a 202 ± 15 a 167 ± 16 a
		As in Cooked Rice (µ µ µ P µg kg -1 ) e e r		500 50 250 500	202 ± 15 a 227 ± 22 a 874 ± 24 b 1642 ± 22 c
		As in Discarded Water (µ µ µ µg L -1 ) R	50	128 ± 14 a
		250 e	234 ± 14 b
		As Retention in Cooked Rice (%)	500 50 v i e	492 ± 14 c (-)30,1 ± 2,5 a ‡
				250 500	w	80,2 ± 2,6 c 91,4 ± 2,5 c
	3 4 5	† Mean ± ES: Mean value of three replicates ± standard error. ‡ Values with the same letters were not significantly different at p<0.05 for the variable studied (Tukey multiple range test). O n l
				y
				20

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table IV .

 IV Effect of the arsenic species (arsenite, arsenate, MA, or DMA) present 1 in the cooking water on the variables under study in the cooking of rice.

		Food Additives and Contaminants	Page 20 of 24
	2
	Variable	As Species	Mean ± ES †
	Absorbed Water in Cooked Rice (mL)	Arsenite ¥	639 ± 17 a ‡
			Arsenate	496 ± 17 b
			MA	562 ± 18 b
	Discarded Water (mL) F o r	DMA Arsenite Arsenate	540 ± 17 b 111 ± 17 a 254 ± 17 b
		P e	MA DMA	188 ± 18 b 210 ± 17 b
	As in Cooked Rice (µ µ µ µg kg -1 ) e r	Arsenite Arsenate	1001 ± 25 a 855 ± 25 b
		MA DMA R	915 ± 27 ab 886 ± 25 b
		e
	As in Discarded Water (µ µ µ µg L -1 )	Arsenite Arsenate MA v i e	230 ± 16 a 298 ± 16 b 308 ± 17 b
			DMA	w	302 ± 16 b
	As Retention in Cooked Rice (%)	Arsenite Arsenate MA DMA	56,4 ± 2,9 a ‡ 41,8 ± 2,9 b 50,0 ± 3,1 ab 40,4 ± 2,9 b n O l
	3	† Mean ± ES: Mean value of three replicates ± standard error. y
	4	¥ MA=methylarsonate; DMA=dimethylarsinate. ‡ Values with the same letters
	5	were not significantly different at p<0.05 for the variable studied (Tukey
	6	multiple range test).
				21

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Table V .

 V Content of arsenic species (arsenite, arsenate, MA, and DMA) in cooked 1 rice. In the present cooking process, water containing only one arsenic species, † A total of 255.5 µg of As were added to each system (187.5 µg from cooking 6 water and 68.0 µg from raw rice). From this total amount 236.5, 231.9, 242.6, 7 and 234.7 µg were found after cooking in the arsenite, arsenate, MA, and DMA

	Page 21 of 24		Food Additives and Contaminants
	2
	3 4 5	at 250 µg L -1 , and a cooking ratio of water:rice of 3:1 (750 mL water:250 g rice) were used. Initial As Species As Species Added As (%) Found As (%) Arsenite Arsenite 93.6 (rice+water) † 89.9 Arsenate 3.9 (rice) 10.1 MA 0 0 DMA 2.5 (rice) 0 Arsenate Arsenite 20.2 (rice) 9.9 Arsenate 77.3 (rice+water) 90.1 MA 0 0 DMA 2.5 (rice) 0 MA Arsenite 20.2 (rice) 6.9 Arsenate 3.9 (rice) 12.4 MA 73.4 (water) 80.7 DMA 2.5 (rice) 0 F o r P e e r R e v i e w	O n l y
		DMA	Arsenite	20.2 (rice)	7.6
			Arsenate	3.9 (rice)	10.7
			MA	0	0
			DMA	75.9 (rice+water)	81.7
	8			22
	9	systems, respectively.

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Table VI .

 VI Daily dietary arsenic intake from water and cooked rice. It is assumed that each people drinks daily about 2.5 L of water 1 and eats daily about 0.450 kg of cooked rice.

	2
	TOTAL ARSENIC
	As in drinking/cooking	AsIDW	AsIDW	As in cooked	AsICR	AsICR	DAsI
	water (µg L -1 )	(µg As day -1 )	(% TAI)	rice (µg kg -1 )	(µg As day -1 )	(% TAI)	(µg As day 1 )
	50	125	55.0	227	102	45.0	227
	250	625	61.4	874	393	38.6	1018
	500	1250	62.8	1642	739	37.2	1989
	INORGANIC ARSENIC
	As in drinking/cooking	AsIDW	AsIDW	As in cooked	AsICR	AsICR	DAsI
	water (µg L -1 )	(µg As day -1 )	(% TAI)	rice (µg kg -1 )	(µg As day -1 )	(% TAI)	(µg As day 1 )
	50	125	54.6	231	104	45.4	229
	250	625	61.0	887	399	39.0	1024
	500	1250	62.5	1667	750	37.5	2000

arsenic endemic areas have increased in recent years[START_REF] Díaz | Contribution of water, bread, and vegetables (raw and cooked) to 6 dietary intake of inorganic arsenic in a rural village of Northern Chile[END_REF]

Roychowdhury et al. 2003). Failure to consider the contribution of food intake of

NaAsO 2 (sodium meta-arsenite) and Na 2 HAsO 4 ⋅7H 2 O (sodium hydrogen arsenate)

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content in the cooked rice was about 30 % lower than expected (TableIII).

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Hotta N, Harada M, Inomata S. 1992. Inorganic arsenic: A dangerous

Report Series 776; Geneva (Switzerland): WHO.

Acknowledgements

intake by the villagers from an arsenic-affected area of West Bengal, India.


Nigel G Halford
email: nigel.halford@bbsrc.ac.uk

Nira Muttucumaru

Tanya Y Curtis

Martin A J Parry

Genetic and agronomic approaches to decreasing acrylamide precursors in crop plants

Progress in developing genetic and agronomic approaches for reducing the levels of the principal precursors of acrylamide, asparagine and sugars, in crop plants is reviewed. The factors that affect asparagine and sugar accumulation, particularly in cereal seeds and potato tubers, are described. Asparagine levels appear to be the key parameter in determining acrylamide formation in processed wheat flour and agronomic strategies for reducing asparagine accumulation in wheat grain are reviewed. Sulphur availability has been shown to be particularly important, with sulphur deprivation causing a dramatic increase in grain asparagine levels and acrylamide risk. Nitrogen availability is also a factor, with increasing nitrogen availability causing grain asparagine levels and acrylamide risk to rise. In potato, attention has been focussed on sugars, and there has been some success in reducing sugar accumulation in stored potatoes by genetic modification, with a resultant reduction in acrylamide formation. However, the wisdom or otherwise of this dogma is discussed. Other possible genetic targets for manipulation or development as genetic markers in breeding programmes are reviewed. Plant breeders and farmers are encouraged to exploit the varietal differences in acrylamide risk that have already been identified and to develop good agronomic practice to reduce the levels of acrylamide precursors in cereals and potato.

Introduction

Foods with the highest levels of acrylamide are carbohydrate-rich and include those derived from potato, wheat, maize and other cereals, as well as coffee. A number of proposals have been put forward to lower acrylamide levels by changing processing methods; they include reducing cooking times and temperatures [START_REF] Amrein | Potential for acrylamide formation in potatoes: data from the 2003 harvest[END_REF][START_REF] Surdyk | Effects of asparagine, fructose, and baking conditions on acrylamide content in yeast-leavened wheat bread[END_REF][START_REF] Taubert | Influence of processing parameters on acrylamide formation during frying of potatoes[END_REF] and lowering the pH [START_REF] Amrein | Potential for acrylamide formation in potatoes: data from the 2003 harvest[END_REF][START_REF] Jung | A novel technique for limitation of acrylamide formation in fried and baked corn chips and in French fries[END_REF][START_REF] Rydberg | Investigations of factors that influence the acrylamide content of heated foodstuffs[END_REF]. However, the Maillard reaction, which is responsible for acrylamide formation, also generates desirable flavour and colour compounds in heated food, so reducing the cooking time and temperature, or lowering the pH may reduce colour and flavour development. There must, therefore, be a limit to what can be achieved by these methods.

The use of asparaginase to convert asparagine to aspartic acid has been successful in reducing acrylamide formation in some foodstuffs [START_REF] Zyzak | Acrylamide formation mechanism in heated foods[END_REF] but it has not yet been approved for commercial use or shown to be practical or cost-effective in large-scale food production and is only likely to work in those foods that allow take-up and mixing-in of the enzyme.

The use of raw materials with low sugar or asparagine content is an alternative or complementary solution and this makes the problem of acrylamide in food a plant and agronomic sciences issue as well as a food processing one. This article reviews progress in this area and the prospects for developing new varieties and agronomic strategies that help to meet the challenge of reducing dietary intake of acrylamide. First we must consider the factors that affect the accumulation of the principal precursors for acrylamide, asparagine and sugars, in plants.

Factors controlling asparagine accumulation

Asparagine accumulates in all plant tissues, but in the context of acrylamide formation the asparagine content of the edible parts of plants (e.g. seeds and tubers) is of greatest In potato tubers, asparagine is the dominant free amino compound, typically accounting for approximately one third of the total free amino acid pool [START_REF] Eppendorfer | Free and total amino acid composition of edible parts of beans, kale, spinach, cauliflower and potatoes as influenced by nitrogen fertilisation and phosphorus and potassium deficiency[END_REF]. In contrast, in cereal grains asparagine usually accounts for less than 10 % of the total free amino acids [START_REF] Lea | Asparagine in plants[END_REF]. However, the amount and concentration is influenced by both genetic and environmental factors. In general, asparagine accumulates when the rates of protein synthesis are low and there is a plentiful supply of reduced nitrogen.

Whilst asparagine accumulates during normal physiological processes, such as seed germination, its accumulation can also be induced by both biotic and abiotic stresses. This can occur either as a result of inhibition of protein synthesis or by direct effects on asparagine metabolism.

Nutrient Supply

In both cereals and potatoes nitrogen availability correlates positively with both the free asparagine and the total free amino acid content, thus asparagine concentration increases but its contribution to the total free amino acid pool remains relatively static (De Wilde et al. 2006, Winkler and[START_REF] Winkler | Amino acid composition of the kernel proteins in barley resulting from nitrogen fertilization at different stages of development[END_REF]. When plants are grown with a plentiful supply of nitrogen, deficiencies in potassium, sulphur, phosphorus and magnesium, but not molybdenum, stimulate accumulation of asparagine [START_REF] Possingham | The effect of mineral nutrition on the content of free amino acids and amides in tomato plants. I. A comparison of the effects of deficiencies of copper, zinc, manganese, iron and molybdenum[END_REF][START_REF] Stewart | Accumulation of amino acids and related compounds in relation to environmental stress[END_REF][START_REF] Rabe | Increased arginine biosynthesis during phosphorus deficiency[END_REF][START_REF] Rufty | Phosphorus stress effects on the assimilation of nitrate[END_REF][START_REF] Almeida | Evidence that P deficiency induces nitrogen feedback regulation of symbiotic nitrogen fixation in white clover (Trifolium repens L.)[END_REF][START_REF] Nikiforova | Effect of sulphur availability on the integrity of amino acid biosynthesis in plants[END_REF]. However sulphur deficiency leads to the greatest asparagine accumulation and in conditions of severe sulphur deprivation asparagine can accumulate to up to 50 % of the total free amino acid pool [START_REF] Shewry | The effects of sulphur starvation on the amino acid and protein compositions of barley grain[END_REF][START_REF] Baudet | Changes in accumulation of seed nitrogen compounds in maize under conditions of sulphur deficiency[END_REF], Muttucumaru et al., 2006). This is discussed in more detail later in the review. Exposure to toxic metals can cause accumulation of some amino acids, including asparagine. For example, cadmium, a major pollutant worldwide, has been shown to induce asparagine accumulation in both lettuce and lupins (Costa and[START_REF] Costa | Water relations, gas exchange and amino acid content in Cdtreated lettuce[END_REF][START_REF] Costa | Influence of cadmium on soluble carbohydrates, free amino acids, protein content of in vitro cultured Lupinus albus[END_REF]. Asparagine can bind to or react with cadmium, copper, lead and zinc to generate less toxic compounds [START_REF] White | Metal complexation in xylem fluid II. Theoretical equilibrium model and computational computer program[END_REF][START_REF] Bottari | Asparagine as a ligand for cadmium (II), lead (II) and zinc (II)[END_REF][START_REF] Smirnoff | Nitrogen assimilation and zinc toxicity to zinc-tolerant and non-tolerant clones of Deschampsia cespitosa (L.) Beauv[END_REF]

Pathogen attack

Fungal and bacterial attack can lead to very large increases in asparagine accumulation. For example, an 800-fold increase in asparagine concentration has been reported in tomato leaves infected with the bacteria Pseudomonas syringea [START_REF] Perez-Garcia | Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment[END_REF] and a similarly large increase has been reported in cocoa infected with the basidiomycete fungus Crinipellis perniciosa [START_REF] Scarpari | Biochemical changes during the development of witches' broom: the most important disease of cocoa in Brazil caused by Crinipelis perniciosa[END_REF].

Drought and salt stress

Asparagine shows a characteristic linear increase in plants subjected to drought stress.

However, asparagine as a percentage of the total free amino acids may fall as proline often accumulates to a much greater extent [START_REF] Good | The effects of drought stress on free amino acid accumulation and protein synthesis in Brassica napus[END_REF]. In most species, accumulation of asparagine and proline under salt stress is similar to that under drought [START_REF] Garthwaite | Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl-into the shoots[END_REF].

Factors controlling sugar accumulation

Carbon is, of course, derived by plants from atmospheric CO 2 by photosynthesis and generally sugar availability is dependent upon that process. The major harvested organs of crop plants, such as seeds and tubers, however, are not themselves photosynthetic; they are 6 carbon sinks that derive all of their carbon from the leaves in the form of sucrose. The first effect of a change in the supply of sucrose to the developing seed or tuber is probably one of yield, rather than of concentration, so we would predict that it would have a relatively small effect on acrylamide formation, although there is little data to support this hypothesis. The principal determinant of acrylamide formation will be what happens to the sucrose when it arrives at the sink organ, in other words how it is partitioned between different end products and intermediates, including reducing sugars such as glucose, fructose and maltose that can participate in the Maillard reaction.

A large proportion of the sucrose that is imported into a potato tuber or cereal seed is incorporated into starch, oils or storage proteins and stored. On seed germination or tuber sprouting these storage compounds are remobilised and sugars are released for use in providing energy and the raw materials for growth. Prevention of sprouting during storage and the exclusion of sprouted material from the food chain may therefore be important in reducing acrylamide risk, depending on the type of food that the material is destined for and the nature of the processing that it will undergo.

A complicating factor in the subject of sugar metabolism in plants is the fact that sugars themselves are key signalling metabolites and the signals that they initiate have profound effects on enzyme activity and gene expression, thereby influencing the flux of compounds through metabolic pathways. In this way, sugar levels affect and are affected by processes such as photosynthesis, the cell cycle, biotic and abiotic stresses from the environment, nutrient availability and the partitioning of resources within the plant. This subject is reviewed elsewhere [START_REF] Halford | Regulation of carbon and amino acid metabolism: roles of sucrose nonfermenting-1-related protein kinase-1 and general control nonderepressible-2related protein kinase[END_REF]Paul, 2003, Halford, 2006) and we will not review it further here, but it must be remembered that changing sugar levels can have profound and wide-ranging effects on primary and secondary metabolism.

Agronomic strategies for reducing the levels of acrylamide precursors

Effects of sulphur availability

Sulphur (S) is an essential macronutrient required by plants for growth. Inorganic sulphate is acquired from the soil and uptake is mediated by specialised sulphate transporters at the root-soil interface. It is subsequently assimilated into the amino acids cysteine and methionine and a variety of other metabolites.

Ironically, sulphur nutrition of crops benefited in the past from an otherwise undesirable by-product of the industrial revolution, namely atmospheric SO 2 , generated predominately from the burning of fossil fuels. While being environmentally damaging in other ways, the deposition of this atmospheric sulphur in the form of 'acid rain' helped to maintain the sulphur content of soils used for crop production. 'Old-fashioned' fertilizers such as ammonium sulphate and superphosphate (figure 1) also contained sulphur, although they were marketed principally as sources of nitrogen and phosphate. This meant that sulphur nutrition was of little practical concern in the UK. However, in the latter part of the last century there was a huge switch from the burning of coal as a fuel to low sulphur or sulphurfree alternatives such as natural gas. At the same time, fertilizers such as ammonium sulphate and superphosphate had been replaced by fertilisers based on nitric acid, better sources of nitrogen but containing no sulphur, and increasing crop yields were resulting in a more rapid removal of soil minerals, including sulphur. As a result, by the end of the century increasing areas of land were beginning to become sulphur deficient [START_REF] Zhao | Sulphur assimilation and effects on yield and quality of wheat[END_REF]) and the Home Grown Cereals Authority (www.hgca.com) estimates that 23 % of the United Kingdom is now at high risk from sulphur deficiency for cereal cultivation. Sulphur deficiency has also been reported in Northern and Western Europe, Australia and New Zealand [START_REF] Zhao | Crop responses to sulphur fertilisation in Europe[END_REF][START_REF] Blair | Sulphur fertilisers: A global perspective[END_REF]. The relevance of all of this to the acrylamide issue arises from the effect of sulphur deprivation on asparagine accumulation. A study undertaken in 1983 demonstrated that grain harvested from barley grown under conditions of sulphur deprivation contained a reduced amount of total protein and a high content of aspartic acid and asparagine [START_REF] Shewry | The effects of sulphur starvation on the amino acid and protein compositions of barley grain[END_REF][START_REF] Shewry | Nutritional control of storage protein synthesis in developing grain of wheat and barley[END_REF]. More recently, Muttucumaru and co-workers reported that wheat grown under conditions of severe sulphate depletion showed dramatic increases in the concentration of free asparagine in the grain of up to thirty times compared with samples receiving the normal levels of sulphate fertilizer. The effect was observed both in plants grown in pots (figure 2), where the levels of nutrients were carefully controlled, and in plants grown in field trials on soil with poor levels of natural nutrients (figure 3). When flours from the sulphate-deprived wheat were heated at 160 °C for 20 minutes levels of acrylamide between 2600 and 5200 µg per kg were found compared with 600 to 900 µg per kg in wheat grown with normal levels of sulphur fertilization.

It is unlikely that any wheat would be grown in the UK or other developed country under conditions of complete sulphur deprivation without a farmer intervening because the effect on the growth of the plants and grain yield would be so obvious (figure 4). However, it is possible that plants growing around the margins of fields where sulphur application is required due to poor soil nutrient retention could be severely sulphur deprived if fertilizer application did not reach them. The entry of grain from such plants into the food chain might explain why food processors have made anecdotal reports of unpredictable spikes of acrylamide formation affecting some batches of their products. Furthermore, the results of Muttucumaru and co-workers showed that even flours from wheat grown in the field with an intermediate level of sulphur application (10 kg sulphur per hectare) contain more acrylamide after processing than flours from wheat grown with an application of 40 kg sulphur per hectare (figure 3) [START_REF] Muttucumaru | The formation of high levels of acrylamide during the processing of flour derived from sulfate-deprived wheat[END_REF]. Perhaps significantly, the higher level of acrylamide was above the 1000 ppb mark that has been used as a de facto limit for acrylamide content by German regulatory authorities and which could be adopted in other countries should they decide to follow Germany's lead (Germany is the only country to have set such a limit to date).

Clearly, the importance of maintaining sulphur levels for cereal cultivation must be stressed to farmers. However, it should be noted that while the effect of sulphur deprivation on asparagine accumulation appears to be widespread in plants, its link with acrylamide formation has not been demonstrated for other types of crop plants. In potato, for example, while sulphur deprivation is to be avoided because of its effect on plant development and yield, which is as severe as it is for wheat (figure 4), there are indications that the relationship between sulphur feeding and acrylamide formation in processed tuber material is more complicated than it is for wheat grain (unpublished data). It would therefore be premature to issue advice to potato farmers concerning sulphur feeding and the acceptability of their product with respect to acrylamide formation.

Effects of nitrogen availability

In a recently published study it was reported that the application of nitrogen (N) fertiliser to wheat resulted in an increase in the levels of free amino acids and protein content in the grain [START_REF] Claus | Influence of agronomic factors and extraction rate on the acrylamide contents in yeast-leavened breads[END_REF]. Neither of these is a novel finding (see [START_REF] Shewry | Nutritional control of storage protein synthesis in developing grain of wheat and barley[END_REF] for a review of work in this area) but the study of Claus and co-workers showed that the increase in amino acid levels resulted in an increase in acrylamide formation in the flour after heating. A simple schematic of the steps in the Maillard reaction is shown in figure 5 and reminds us that amino acids are involved both in the formation of the carbonyl compound intermediates and in the conversion of those intermediates to flavour and colour compounds or, in the case of asparagine, acrylamide [START_REF] Mottram | Acrylamide is formed in the Maillard reaction[END_REF][START_REF] Stadler | Acrylamide from Maillard reaction products[END_REF] ). An increase in any amino acid can therefore drive the reaction by participating in the first step and this as well as an increase in asparagine itself is the likely explanation for the effect of nitrogen on acrylamide formation.

The fact that increasing nitrogen supply also increases protein content is important because clearly this will mop up some of the free amino acids. However, this will not occur if sulphur is in short supply because sulphur deprivation results in a decrease in the synthesis of the major seed storage proteins [START_REF] Shewry | The effects of sulphur starvation on the amino acid and protein compositions of barley grain[END_REF][START_REF] Shewry | Nutritional control of storage protein synthesis in developing grain of wheat and barley[END_REF]. The effects of an increase in nitrogen supply are therefore likely to be exacerbated by sulphur deprivation, and vice versa.

The effect of increasing nitrogen supply is relatively small compared with that of sulphur deprivation.

Furthermore, while incorporating sulphur into their fertilizer applications will no doubt incur some cost to the farmer, that cost might be offset somewhat by an increase in the yield and/or quality of the product. In contrast, reducing the application of nitrogen will give an initial saving in fertilizer costs but this will be more than offset by a reduction in yield and/or quality. There are other reasons for reducing the use of nitrogen fertilizer, of course, notably the impact that nitrogen run-off has on water quality. However, the issue is a controversial one and farmers are likely to be reluctant to reduce nitrogen application.

Two studies have been published concerning the effect of nitrogen on potato: De Wilde and co-workers ( 2006) studied how potatoes were affected by nitrogen levels in the soil and found that levels of reducing sugars rose in nitrogen-deprived potatoes by up to 100 %, resulting in an increase in acrylamide in French fries of up to 65 % compared with adequately fertilized potatoes. On the other hand, Amrein and co-workers (2003) found no correlation between the amount of nitrogen fertilization and acrylamide formation. Potato tubers have to be stored for long periods to enable a year-round supply for consumers and the food industry. The tubers are chilled to prevent sprouting but storage at 4 ºC, which is very effective at preventing sprouting, results in the phenomenon of cold sweetening in which starch is broken down and reducing sugars accumulate. This difficult problem predates the acrylamide issue because, as well as being precursors for acrylamide, sugars cause blackening during frying. It is also complicated by the end-use of the potatoes in that potatoes that are produced specifically for crisp production are allowed to be sprayed at harvest with chemical sprout suppressants such as chloropropham (CIPC). This enables the potatoes to be stored at 12 ºC, a temperature high enough for cold sweetening not to occur.

There are concerns regarding residues of these chemicals in the food product (Food Standards Agency, 2006) and the maximum residue limit (MRL) for CIPC has been reduced recently to 10 ppm. However, the practice is allowed for crisping potatoes because the potatoes are always peeled before use and the MRL is not exceeded. Not so for the fresh potato market and for that reason the use of chemical sprouting suppressants on potatoes destined for retail is not allowed; a balance therefore has to be achieved between sprouting control and the prevention of cold sweetening. Clearly, the discovery that sugars are precursors for acrylamide formation makes this already intractable problem all the more important.

Sprouting is also a major factor for acrylamide formation in cereal grain, as it leads to degradation of starch and proteins, and the release of sugars and asparagine. Claus and coworkers ( 2006) reported a 500 % increase in acrylamide formation, from 54.5 ppb to 273.3 ppb, in bread prepared from sprouted grain compared with non-sprouted grain and recommended that sprouted grain should not be used for bakery products. The implications of this for the malting and brewing industries were not investigated but high levels of acrylamide have not been reported for beers or spirits. Theoretically, any of the environmental factors described in the previous sections on asparagine and sugar accumulation that are under human control could be the target of changes in agronomic practice to reduce acrylamide formation. However, while it is likely that maintaining potassium, phosphorus and magnesium availability would help to avoid increases in asparagine accumulation there is little or no information available on how significant or cost-effective this would be. Clearly, some environmental factors, such as rainfall, are out of human control. A possible but controversial strategy would be for food processors to avoid using material produced under environmental conditions, such as drought, if the increase in asparagine content brought about by growth in those conditions showed a clear link with acrylamide risk.

Genetic targets

Varietal differences

Several groups have reported that the asparagine content of tubers from different potato cultivars grown at the same site vary within a narrow range [START_REF] De Wilde | Influence of storage practices on acrylamide formation during potato frying[END_REF][START_REF] Matsuura-Endo | Effects of storage temperature on the contents of sugars and free amino acids in tubers from different potato cultivars and acrylamide in chips[END_REF][START_REF] Olsson | Tuber components affecting acrylamide formation and colour in fried potato: variation by variety, year, storage temperature and storage time[END_REF]. Although other authors have reported much greater (almost 50 fold) differences for purchased tubers of different varieties it is impossible to assess whether this is due to genotype or other factors. Indeed, a frustration in reviewing this data is that many of the studies have relied on the analysis of potatoes purchased from the retail sector, with no knowledge of the time that the tubers have been in storage or the conditions in which they have been kept. Rigorous, systematic studies are therefore still required to determine genotype differences in asparagine and free amino acid content. Plant breeding and food processing companies may have undertaken such studies themselves but there is little hard data in the public domain.

Much attention is now being focussed on the sugar content of different potato varieties. Amrein and co-workers (2003) reported that seventeen potato cultivars grown in Switzerland in 2002 showed large differences in their potential for acrylamide formation and that this was primarily related to their sugar contents. It was concluded that acrylamide formation in potato products could be reduced substantially by selecting cultivars with low concentrations of reducing sugars. However, while we agree that sugar levels are a key parameter we believe that the relationship between sugars, asparagine and other amino acids in the determination of acrylamide risk in potato products is more complicated than this study indicated, and that the advice to concentrate solely on sugar levels is premature.

In contrast, the advice that asparagine content is the key parameter in cereal grain appears to be sound and significant differences between varieties have been reported. For example, Claus and co-workers (2006) reported an approximately five-fold difference in flour asparagine content for different varieties, from 4.90 mg per 100g (Terrier) to 24.9 mg per 100g (Transit). This correlated with an acrylamide content in heated flour ranging from 153.7

µg per kg to 624.1 µg per kg. Similarly, Taeymans and co-workers ( 2004) reported a study of the asparagine content of different European wheat varieties harvested in 2002. The level ranged from 163 to 664 mg per kg. However, large differences between samples of the same cultivar indicated that environmental factors were also having a significant effect.

Clearly, a reduction in acrylamide risk in wheat products could be achieved already, simply by growing cultivars that do not accumulate very high levels of asparagine in the grain. The wide variation in asparagine content also suggests that this key parameter should be amenable to plant breeding.

Genes involved in asparagine biosynthesis and amino acid signalling

Biosynthesis of asparagine, as shown in figure 6, involves the initial assimilation of ammonia to produce glutamine, a reaction mediated by the enzymes glutamine synthetase and glutamate synthase [START_REF] Lea | Glutamate synthase and the synthesis of glutamate in plants[END_REF]. Glutamine then provides the nitrogen required for asparagine biosynthesis, a reaction which is catalysed by the enzyme asparagine synthetase.

Asparagine synthetase also catalyses the adenosine triphosphate-dependent reaction between glutamine and aspartate to generate glutamate. Another key enzyme involved in asparagine metabolism is asparaginase: this enzyme catalyses the hydrolysis of asparagine, resulting in the generation of aspartate and ammonia. Clearly, glutamine synthetase, asparagine

synthetase and asparaginase are all potential candidate genes either for manipulation by genetic modification or mutagenesis, or for the development of genetic markers for breeding programmes.

Genes involved in amino acid signalling could also be useful targets for manipulation.

One such gene is general control nonderepressible-2 (GCN2), a protein kinase known to be involved in both the control of protein synthesis and the induction of expression of amino acid biosynthesis genes [START_REF] Zhang | Molecular cloning of an arabidopsis homologue of GCN2, a protein kinase involved in co-ordinated response to amino acid starvation[END_REF].

Genes involved in sugar metabolism

Plants have two enzymes that can cleave sucrose; these are invertase, which cleaves the sucrose molecule to produce glucose and fructose, and sucrose synthase, which in the presence of UDP cleaves sucrose to form UDP-glucose and fructose (note that under physiological conditions the equilibrium of this reversible reaction very much favours cleavage over synthesis, despite the name of the enzyme). In potato tubers, sucrose synthase is the predominant enzyme for sucrose metabolism [START_REF] Morrell | Sugar metabolism in developing tubers of Solanum tuberosum[END_REF]) and appears to be involved in supplying the starch biosynthetic pathway (figure 7), while invertase is principally involved in supplying hexoses for flux into glycolysis. Hence, over-expression of invertase together with glucokinase results in a dramatic reduction in starch accumulation and an increase in glucose and glucose phosphates [START_REF] Trethewey | Combined expression of glucokinase and invertase in potato tubers leads to a dramatic reduction in starch accumulation and a stimulation of glycolysis[END_REF]. In contrast, upregulation of sucrose synthase gene expression together with ADP-glucose pyrophosphorylase, another enzyme in the starch biosynthetic pathway, results in an increase in starch and a decrease of up to 80 % in glucose concentration [START_REF] Mckibbin | Production of high starch, low glucose potatoes through over-expression of the metabolic regulator, SnRK1[END_REF]. The latter was achieved by over-expressing a metabolic regulator, SNF1-related protein kinase-1 (SnRK1), which controls the flux of carbon into the starch biosynthetic pathway in potato tubers (figure 7).

One word of caution concerning sugar levels in potatoes as a target is that the major crisp and French fry manufacturers already use very low sugar varieties but still have a significant acrylamide problem. Furthermore, there must be a sugar level below which colour and flavour formation is not acceptable and if significant levels of acrylamide are still formed then other strategies will have to be adopted. However, the development of varieties that can be stored at 4 ºC without accumulating sugars is a sensible target and success in this area has already been reported by Rommens and co-workers (2006), who used RNA inhibition techniques to reduce the expression of the starch-associated R1 and phosphorylase-L ( PhL)

genes in variety Russet Ranger. The enzymes encoded by these genes are both involved in the degradation of starch during cold storage. Glucose levels in the transgenic lines were up to 80 % lower than in the controls after three months of storage at 4 ºC and acrylamide levels after processing were less than 200 ppb in one transgenic line compared with almost 1200 ppb in unmodified Russet Ranger controls.

This result appears to be a clear indication that a genetic approach to reducing acrylamide risk is a viable strategy and one worth pursuing.

Conclusions

Acrylamide is widely used in industry; it is also familiar to biochemists as a constituent of gels used for the electrophoretic separation of protein, DNA and RNA molecules. Its familiarity probably made the discovery that it is present in many cooked foods all the more shocking for the science community, the food industry and regulatory authorities. Reassurance can be derived from the fact that the presence of acrylamide in foods does not represent a new risk to consumers. Furthermore, its discovery prompted an immediate reaction from the food industry to modify their processes where possible to reduce its formation. Food safety is therefore being improved, an example of how science and technology can benefit consumers. Nevertheless, the levels of acrylamide in foods such as French-fried potatoes, breakfast cereals, cereal-and potato-based snacks and crispbreads are still commonly several hundred ppb.

Estimates of the intake levels of acrylamide from the diet are much lower than the levels that are known to cause toxicological effects in rodents and to date no epidemiological study has found a link between dietary exposure to acrylamide and cancer risk [START_REF] Mucci | Dietary acrylamide and cancer of the large bowel, kidney, and bladder: absence of an association in a population-based study in Sweden[END_REF][START_REF] Pelucchi | Fried potatoes and human cancer[END_REF]. The position taken by most regulatory authorities to date, therefore, which has been to state that they wish to see dietary intake of acrylamide reduced rather than to set limits for the presence of acrylamide, would appear to be a sensible one.

Nonetheless, regulatory authorities may feel obliged to take stronger action if consumer and/or scientific concern increases and plant breeders and farmers should be preparing for such an eventuality. Unfortunately, there is little indication that they are.

We have already identified one agronomic factor, sulphur nutrition, that has a clear, unambiguous effect on acrylamide risk in cereals and the importance of avoiding sulphate deficiency should be made clear to farmers. It is also evident that considerable reductions in acrylamide risk could be achieved simply by variety selection, in both potato and cereals.

Figure 2 .Figure 3 .Figure 4 Figure 7 .

 2347 Figure 2. Analysis of wholemeal flour from three varieties of winter wheat grown under

bag of superphosphate (a mixture of dihydrogen phosphate and hydrated calcium sulphate) from the mid-20 th century depicting an image of Sir John Bennett Lawes of Rothamsted, who began manufacturing superphosphate in 1841.

  More research is required before advice is given on other agronomic and genetic targets, but there is enough evidence available already to justify the conclusion that genetic and agronomic approaches could play a significant part in mitigating the acrylamide problem. The manufacturing process involved dissolving first bones and later mineral phosphates in sulphuric acid, and superphosphate was therefore a good source of sulphur. The move to more modern fertilizers based on nitric acid has contributed to sulphur deficiency.

	Figures.
	Figure 1. A
	F o r F o r F o r F o r F o r F o r F o r F o r
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	e e e e e e e e
	e r e r e r e r e r e r e r e r
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	O n l O n l O n l O n l O n l O n l O n l O n l
	y y y y y y y y

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Acknowledgements

Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. Nira Muttucumaru is financially supported by the BBSRC in conjunction with the United Kingdom's Food Standards Agency. Tanya Curtis is financially supported by the Home Grown Cereals Authority of the United Kingdom.


eastern Poland in 2003

Keywords: Mycotoxins -trichothecenes, Volatiles wheat, cultivation systems, trichothecenes, ergosterol, trichodiene, ATP

Fusarium toxins and total fungal biomass indicators in naturally contaminated wheat samples from north-

Introduction

In Poland, as in other EU countries, various fungal diseases affect wheat, but special attention is paid to Fusarium head blight (FHB). In the literature there is a shortage of convincing data about the influence of cultivation systems on the severity of its disease symptoms [START_REF] Teich | Epidemiology of wheat (Triticum aestivum L.) scab caused by Fusarium spp[END_REF][START_REF] Champeil | Influence of cropping system on Fusarium head blight and mycotoxin levels in winter wheat[END_REF][START_REF] Lemmens | The effect of nitrogen fertilization on Fusarium head blight development and deoxynivalenol contamination in wheat[END_REF][START_REF] Perkowski | Occurrence of mycotoxins in cereals, plants, foods and feeds in Poland[END_REF]). In north-eastern Poland, organic farming is particularly important because of the low level of environmental contamination and the possibility of growing healthy food (Grodzińska et al. 1999, Łukanowski and[START_REF] Łukanowski | Occurrence of Fusarium on grain and Hades of Winter wheat cultivated in orgnic, integrated, conventional systems and monoculture[END_REF]. Literature data reporting contamination by fungal metabolites of organically grown cereals are contradictory [START_REF] Elmholta | Fungi in Danish soils under organic and conventional farming[END_REF]Labouriau 2005, Pussemier et al. 2006). However, some authors point to the significant influence of 2 environmental conditions (weather in particular) on their concentrations. [START_REF] Malmauret | Contaminants in organic and conventional foodstuffs in France[END_REF] reported that concentrations of Fusarium toxins were higher in wheat and barley seed from organic farms, but their results showed no conclusive evidence whether conventional products were more or less safe than organic ones. Apart from Fusarium toxins, also fungal biomass in seed is important. Fungal biomass is usually estimated on the basis of ergosterol or chitin content [START_REF] Abramson | Relationship among deoxynivalenol, ergosterol and Fusarium exoantigens in Canadian hard and soft wheat[END_REF][START_REF] Ekblad | Chitin and ergosterol combined to measure total and living fungal biomass in ectomycorrhizas[END_REF]. Also ATP level is measured on the surface of kernels, because it is correlated with levels of some other fungal metabolites [START_REF] Williams | Methodological developments in food mycology[END_REF]), but it reflects the level of the whole microbial biomass. Seed colonization by fungi often affects its vigour and consequently also sowing value. Infection by pathogens of the genus Fusarium often disturbs or even completely inhibits seed germination (Eudes et al. 1997, Packa andKoczowska 1987).

This study was aimed to compare concentrations of fungal metabolites in wheat seed from conventional farms (with more or less intensive use of chemical protection) and organic farms. The comparison involved a complex of fungal metabolites: concentrations of selected Fusarium toxins (deoxynivalenol, 3-acetyldeoxynivalenol, the sum of 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, T-2 toxin and zearalenone), trichodiene (their precursor), and microbial biomass indicators (fungal ergosterol and general microbial ATP). Differences in the studied characters between four cultivation systems and correlations between concentrations of the studied metabolites were analysed.

Material and methods

Samples

The analysis involved 32 grain samples (of 1000 g each) of winter and spring wheat from conventional farms (20 samples) and organic farms (12 samples) located in 7 districts in north-western Poland (Figure 1). Conventional farms were divided into 3 groups: CON I, CON II and CON III. In CON I (9 samples from 6 districts), complete antifungal protection and pesticides were applied. CON II (6 samples from 6 districts) included large fields (over 30 ha) subject to a lower level of chemical protection (fungicidal protection against main foliar diseases, first of all mildew and leaf septoriosis), while CON III (5 samples from one district Ostróda) was an experimental farm Bałcyny, where neither fungicides nor pesticides were used. Production in organic farms conformed to the rules applied in this cultivation 3 system (the seed had the Polish ECO certificate). Production in conventional farms followed the rules of the currently recommended intensive methods of cereal cultivation.

Determination of major A-and B-trichotecenes and zearalenone by LC-MS

Deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), the sum of 3acetyldeoxynivalenol and 15-acetyldeoxynivalenol (AcDONs), nivalenol (NIV), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), HT-2 toxin, T-2 toxin and zearalenone (ZON) were determined by liquid chromatography -triple quadrupole mass spectrometry as described previously by [START_REF] Berthiller | Rapid simultaneous determination of major type A-and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry[END_REF]. 10 g of the ground samples were extracted with 40 mL acetonitrile/water (84/16, v/v) and purified using MycoSep 227 columns (RomerLabs, Tulln, Austria). 4 mL of the purified extracts were evaporated to dryness and dissolved in mobile phase (methanol/water 20/80, v/v containing 5 mM ammonium acetate). An Agilent (Waldbronn, Germany) 1100 HPLC system was used in conjunction to a QTrap MS/MS from Applied Biosystems (Foster City, USA). Atmospheric pressure chemical ionisation was used in negative mode for B-trichothecenes and in positive mode for A-trichothecenes. 25 µl were injected onto a Thermo Aquasil RP-C18 column (4.6 x 100mm, 3µm), thermostated to 30°C.

The multiple reaction monitoring mode was used for quantification of the samples. This method was carefully optimized for the quantification of selected Fusarium mycotoxins in cereal. Its limits of quanitification (LOQs) are 3 µg/kg DON, 13 µg/kg 3-AcDON, 4 µg/kg AcDONs, 18 µg/kg NIV, 6 µg/kg FUS-X, 1 µg/kg DAS, 4 µg/kg HT-2, 1 µg/kg T-2 and 3 µg/kg ZON. The limits of detection (LODs) were about 3 times lower than the LOQs.

Determination of ergosterol

Samples containing 100 mg of ground grains were placed into 17 mL culture tubes, suspended in 2 mL of methanol, treated with 0.5 ml of 2 M aqueous sodium hydroxide and tightly sealed. Then the culture tubes were placed within 250 mL plastic bottles, tightly sealed and placed inside the microwave oven (Model AVM 401/1WH, Whirlpool, Sweden) operating at 2450 MHz and 900 W maximum output. Samples were irradiated (370 W) for 20 s. and after ca. 5 min. an additional 20 s. After 15 min contents of culture tubes were neutralized with 1 M aqueous hydrochloric acid, 2 mL MeOH were added and extraction with pentane (3 x 4 mL) were carried out within the culture tubes. The combined pentane extracts were evaporated to dryness in a nitrogen stream. Prior to analysis samples were dissolved in 4 mL of MeOH, filtered through 13 mm syringe filters with 0.5 µm pore diameter (Fluoropore

Determination of trichodiene

Volatile metabolites were extracted from grain using solid phase microextraction method (SPME). Grain samples (8 g) were placed in 20 mL vials and extracted by means of headspace SPME during 30 min at 40˚C with 100 µm polydimethylsiloxane (PDMS) fibre. min. The helium flow rate was held constant at 0.7 mL/min. Quantitative analysis of trichodiene was performed in selected ion monitoring (SIM) mode, using following ions for trichodiene detection: m/z 67, 93, 108, 109. The amount of trichodiene was estimated comparing the area of its total ion current (TIC) peak with internal standard (tridecane, 25 ng in pentane) and expressed as their ratio (RU). Trichodiene was identified by comparing its mass spectrum with literature data [START_REF] Jeleń | Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes[END_REF].

Determination of ATP

Adenosine-5'-triphosphate (ATP) measurement was carried out using luciferase enzyme.

ATP was extracted from 1.00 g grain samples using 8 mL boiling Tris buffer (0.1 molar solution, pH -7.75). 100 µL of this extract were mixed with 100 µL reagent containing luciferin and luciferase (LuminATE (QM), Celsis, Netherlands). The emitted light was quantified by Lumac Biocounter M 1500 luminometer and readout as Relative Light Units (RLU). The emitted light correlates with the amount of ATP in the sample.

Seed vigour test

The test was performed in two replicates, according to the guidelines of the International Seed Testing Association [START_REF] Hampton | Handbook of vigour test methods. 3rd EditionZurich: ISTA[END_REF]. Subsamples of 50 surface-sterilized kernels each were placed on strips of filter paper (50 cm × 25 cm). Next the strips were rolled up and deposited in beakers containing a small amount of sterile distilled water and kept in a phytotron in darkness at 22ºC. After 7 days, proportions of non-germinated and abnormally germinated grains and of grains with Fusarium mycelium were assessed.

Statistical analyses

The significance of differences in mean values was evaluated with the use of Student's t-test, and linear correlation analysis was made for all studied parameters.

Results and discussion

The wheat grain samples classified into 4 different groups (CON I, CON II, CON III and ECO) markedly differed in the analysed parameters: HT-2 toxin, DON, 3-AcDON, NIV, sum of the most important group B trichothecenes in grain (DON+NIV together), ERG, TRICH, ATP and seed vigour parameters (non-germinated and abnormally germinated grains as well as grains with Fusarium mycelium). The results are presented in Tables 1 and2 (except of ZEA concentrations because their were below the limit of detection).

The grains were characterized by a low proportion of infestation by fungi of the genus Fusarium, ranging from 0 to 4.0%, but differences between the groups were not significant.

The proportion of grains with Fusarium mycelium was positively correlated with the proportion of abnormally germinated grains, although value of Pearson correlation coefficient was not significant, because during vegetation Fusarium is not the only factor influencing abnormal grain germination, but also harvest conditions, kernel damage as well as preinfestation changes. Additionally, since these were productive farms, an accurate determination of the above mentioned factors was difficult. A significant correlation was found between the proportion of non-germinated grains and the proportion of abnormally germinated grains. Mean values of both these parameters were the lowest in CON I, and the highest in ECO. The mean concentration of trichothecene toxins in CON I and CON II can be regarded as low, and it is comparable with results reported by [START_REF] Pettersson | Mycotoxins in Scandinavian cereals[END_REF][START_REF] Perkowski | Natural occurrence of Fusarium mycotoxins in Polish cereals[END_REF]). In those groups, NIV+DON concentration reached 43.0 µg kg -1 (CON I) and 30.0 µg kg -1 (CON II). In organically grown wheat, the NIV+DON level was similar, but the concentration of DON was higher than the concentration of NIV. In comparison with CON I, CON II and ECO, significant differences were found in CON III (conventional but lacking chemical protection), where NIV+DON content was over 14 times higher than the mean for the other groups (635.2 µg kg -1 versus 44.3 µg kg -1 ), and the major contributor was DON.

Concentrations of trichodiene (TRICH), which is a precursor of trichothecenes [START_REF] Jeleń | Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes[END_REF], were the highest in samples containing the highest levels of trichothecenes (CON III), while the lowest in organically grown wheat seed. A positive correlation (r=0.788, P=0.05) was observed between TRICH level and concentration of NIV+DON in seed.

In this study, with the use of so wide-ranging wheat materials, concentrations of ERG (ergosterol, indicator of fungal biomass) and ATP (indicator of microbial biomass in the outer parts of the grain) were measured. To be exact, [START_REF] Reid | Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass, and mycotoxin accumulation[END_REF] examined disease severity, ergosterol content, fungal DNA content and mycotoxin (DON and fumonisin B 1 ) concentration in silk maize ears channel-inoculated with F. graminearum and F. moniliforme.

The authors reported that although biomass may be the best predictor of disease ratings, the DNA analysis demonstrated that this biomass was not always produced by the expected Fusarium sp. However, the above experiments were carried out for maize. Similar conclusions were indicated by [START_REF] Marfleet | The relationship between fungal biomass, ergosterol and grain spoilage[END_REF] as well.

The highest ERG level was 2.39 mg/kg (CON III), but it reached a similar value in ECO. These values were significantly higher than in CON I and CON II, but still were below the threshold of 3 mg/kg reported by Schnürer [START_REF] Schnürer | Ergosterol levels and mould colony forming units in Swedish grain of food and feed grade[END_REF] for seed showing no symptoms of fungal colonization. In CON I, the lowest ERG level was recorded, and ATP content was also low (2153 RLU). It is interesting that such a low ATP concentration was recorded in all analysed samples of this group. This value was about 8-fold lower than in organically grown wheat seed (16128 RLU). It is probably a result of the lack of chemical protection in organic farming, which is favourable for microbial growth in the outer parts of the grain. The lack of chemical protection and high mineral fertilization combined with all other agronomic practices used in conventional cultivation in CON III could favour fungal growth at the expense of other microorganisms. This was reflected in a high ERG and mycotoxins concentration and relatively low ATP content. Because of the varied cultivation systems, only a low positive correlation between ERG and ATP content was found when all four groups were analysed jointly. When the groups were analysed separately, the highest correlation between these characters (r=0.726, significant at P<0.01) was recorded in CON I, which seems to be the most common in that part of Poland.

In conclusion, results of this study show that wheat seed in north-eastern Poland in 2003 was characterized by a low level of colonization by fungi of the genus Fusarium, but considerable differences were observed depending on cultivation system. Seed samples from organic farms contained similar amounts of Fusarium toxins but higher levels of ERG and ATP than conventionally grown and chemically protected seed samples did. A slightly higher DON content in ECO samples in comparison to CON II samples, and especially CON I samples, and a very low NIV content suggest that while observing the principles of organic farming, especially appropriate crop rotation, the absence of chemical protection does not necessarily have to led to increased concentrations of these metabolites in grain, which was also emphasized by [START_REF] Champeil | Influence of cropping system on Fusarium head blight and mycotoxin levels in winter wheat[END_REF]. At the conventional cultivation system cereals, especially wheat and maize, account for a very large share of crops in crop rotation.

Necrotrophic pathogens from genus Fusarium develop dynamically on post-harvest residue of these crops, which promotes the accumulation of FHB symptoms in the successive years, especially in wheat following maize. [START_REF] Dill-Macky | The Effect of Previous Crop Residues and Tillage on Fusarium Head Blight of Wheat[END_REF] reported that in case of wheat grown after soy FHB symptoms occur in a much lesser degree. The application of ploughing additionally limits the incidence of this disease, as post-harvest residue is transferred to deeper soil layers. Thus it seems that the application of an appropriate crop rotation scheme in organic farming (with papilionaceous plants), together with comprehensive mechanical soil cultivation reduces the risk of pathogenic species from genus Fusarium to such an extent that a lack of chemical protection does not have a significant effect on an increase of Fusarium toxin concentrations in wheat kernels. It is possible that a significantly higher dry matter content (including that of nutrients and minerals) in tissues of plants grown in the organic farming system (Kouba 2003) is also accompanied by a higher concentration of phytochemical substances exhibiting fungistatic action connected with resistance to fungal pathogens. It is of interest that conventional farming with no fungicides applied (CON III) resulted in a drastic increase of Fusarium toxin concentrations in kernels. This result is especially important in view of the increasingly promoted and implemented sustainable

  Millipore, Ireland) and evaporated to dryness into N 2 stream. The sample extract was dissolved in 1 mL of MeOH and 50 µL was analysed by HPLC technique.Separation was achieved on 150 x 3.9 mm Nova Pak C-18, 4 µm column and eluted with methanol/acetonitrile (90:10) at a flow rate of 0.6 mL /min. Ergosterol was detected with Waters 486 Tunable Absorbance Detector set at 282 nm. Quantification of ERG was made by a comparison of peak areas with those of external standards of ERG. Confirmation of ERG was achieved by a comparison of retention times with the external standard and by coinjection every tenth sample with an ergosterol standard.

  The analyses were run on a gas chromatograph mass spectrometer (Hewlett Packard GC 6890, MS 5972 A), using an HP -5MS (0.25 mm x 30 m) capillary column. The injection port temperature was 250 • C, the transfer line temperature was 280 • C and the analyses were performed with programmed temperature: initial temperature 40 • C hold 3 min, from 40 • C -200 • C at 10 • C/min, 200 • C -280 • C at 20 • C/min, the final temperature being maintained for 3

  in which chemical crop protection is reduced, at the simultaneous maintenance of major elements of the conventional farming system (e.g. the application of mineral fertilizers) and at the same time placing more emphasis on crop rotation and mechanical tillage. The most intensive agronomic practices resulted in significantly lower concentrations of ERG, ATP and TRICH, as compared to the other systems of cultivation.The highest levels of ergosterol were detected in samples from conventional cultivation lacking chemical protection. These results form the basis for further research on this subject.

Table 2 .

 2 DAS were not detected in the samples (LOQ: 1 µg/kg) FUS-X was detected only in sample 15 at 12 µg/kg (LOQ: 6 µg/kg) "-" means that no peak was found Results in brackets are below the limit of quantitation Mean values of analysed characters in wheat seed samples from four cultivation systems Values marked with the same letters within columns do not differ significantly (P>0.05). Figure 1. Location of sites of seed samples collection on a map of Poland.

	Page 13 of 16				Food Additives and Contaminants
	13	4	26	-	-	26	0.79	3.649	16199	4.5	7.0	1.5
	14	6	53	-	(13)	66	0.55	1.319	12706	3.5	4.5	0.5
	15	-	17	-	-	17	0.46	2.385	17262	6.0	8.0	3.5
						CON III
	16	-	784	18	27	811	1.83	6.293	16642	2.0	4.5	0.0
	17	-	480	-	43	523	2.9	3.961	14893	3.0	2.5	2.0
	18	66	607	19	(7)	614	2.01	7.866	10944	1.5	3.0	0.5
	19	-	190	-	-	190	3.34	8.581	5663	4.0	1.0	1.5
	20	-	997	37	41	1038	1.87	3.285	10009	1.5	1.5	0.0
						ECO
	21	-	-	-	28	28	3.34	1.528	10010	2.0	6.5	5.0
	22	8	-	-	13	16	3.26	1.262	10477	3.5	3.5	2.5
	23	-	-	-	-	0	2.01	1.404	12740	4.5	9.5	2.0
	24	-	380	-	-	380	2.95	4.843	9772	2.5	10.5	1.5
	25	-	12	-	-	12	1.65	0.644	15843	4.5	9.5	2.0
	26	-	-	-	-	0	2.44	0.799	14828	3.5	7.5	1.5
	27	-	7	-	-	7	0.92	2.009	14287	2.5	7.0	3.5
	28	-	60	-	-	60	2.88	0.472	22062	10.5	7.5	1.0
	29	-	-	181	-	0	2.84	1.410	31273	3.5	2.0	0.5

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Packa D, Koczowska I. 1987. The effect of Fusarium culmorum and Fusarium nivale metabolites on germination, seedling growth and cytological disturbances of rye, wheat and

Acknowledgments

This work was realized within a programme of Austrian-Polish scientific and technical cooperation no. 7/2004.

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

Table 1. Concentration of trichothecenes (µg kg -1 ) and ergosterol (ERG, mg kg -1 ) and amount of trichodiene (TRICH), adenosine triphosphate (ATP) and vigour test parameters for 32 examined wheat grain samples obtained from different cultivation systems.