Maria Antonia Carolina Mariana Gláucia Fernanda Mirella Eduardo Tadeu Adriano

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Food Additives & Contaminants: Part B

Surveillance

ISSN: 1939-3210 (Print) 1939-3229 (Online) Journal homepage: http://www.tandfonline.com/loi/tfab20

Co-occurrence and distribution of deoxynivalenol,

nivalenol and zearalenone in wheat from Brazil

Maria Antonia Calori-Domingues, Carolina Maria Gil Bernardi, Mariana

Sartori Nardin, Gláucia Vendramini de Souza, Fernanda Gregório Ribeiro dos

Santos, Mirella de Abreu Stein, Eduardo Micotti da Gloria, Carlos Tadeu dos

Santos Dias & Adriano Costa de Camargo

To cite this article: Maria Antonia Calori-Domingues, Carolina Maria Gil Bernardi, Mariana Sartori Nardin, Gláucia Vendramini de Souza, Fernanda Gregório Ribeiro dos Santos, Mirella de Abreu Stein, Eduardo Micotti da Gloria, Carlos Tadeu dos Santos Dias & Adriano Costa de Camargo (2016): Co-occurrence and distribution of deoxynivalenol, nivalenol and zearalenone in wheat from Brazil, Food Additives & Contaminants: Part B

To link to this article: http://dx.doi.org/10.1080/19393210.2016.1152598

Accepted author version posted online: 17 Feb 2016.

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Publisher: Taylor & Francis

Journal: Food Additives & Contaminants: Part B

DOI: 10.1080/19393210.2016.1152598

Co-occurrence and distribution of deoxynivalenol, nivalenol and zearalenone in wheat from Brazil

Maria Antonia Calori-Dominguesa, Carolina Maria Gil Bernardia, Mariana Sartori Nardina, Gláucia Vendramini de Souzaa, Fernanda Gregório Ribeiro dos Santosa, Mirella de Abreu Steina, Eduardo Micotti da Gloriaa, Carlos Tadeu dos Santos Diasb, Adriano Costa de Camargoa

a

Department of Agri-Food Industry, Food & Nutrition, bDepartment of Exact Sciences, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Av. Pádua Dias 11, P.O. Box 9, CEP 13418-900, Piracicaba, SP, Brazil * Author to whom correspondence should be addressed; E-mail: macdomin@usp.br

Abstract

Fusarium mycotoxins deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) were investigated in wheat from the 2009 and 2010 crop years. Samples (n = 745) from commercial fields were collected in 4 wheat producing regions (WPR) which differed in weather conditions. Analyses were performed using HPLC-DAD. Contamination with ZEN, DON and NIV occurred in 56, 86 and 50%, respectively. Also mean concentrations were different: DON = 1046 µg kg

-1

, NIV <100 µg kg-1 and ZEN = 82 µg kg-1. Co-occurrence of ZEN, DON and NIV was observed in 74% of the samples from 2009 and in 12% from 2010. Wet/cold region WPR I had the highest mycotoxin concentration. Wet/moderately hot region WPR II had the lowest mycotoxin levels. Furthermore, the mean concentration of each mycotoxin was higher in samples from 2009 as compared with those from 2010. Precipitation during flowering or harvest periods may explain these results.

Keywords: Fusarium mycotoxin; HPLC- DAD; cereal; contamination; wheat producing region.

Introduction

Wheat is one of the most important food crop. It is ingredient of different types of food products such as bread, pasta, biscuit and wheat bran. In the last years, the wheat consumption in Brazil was about 10.3 million ton, which demonstrates its economic importance. However, for the same period, the wheat production was about 5.5 million tons (Companhia Nacional de Abastecimento, 2015). The safety of the wheat produced worldwide deserves attention. The climatic conditions present in wheat producing areas from Brazil and the main producing countries allows for the occurrence of important diseases in this crop, among them the Fusarium head blight (FHB), also known as scab. FHB is an economically devastating disease of small cereal crops, specially wheat and barley.

The most important pathogenic fungus in wheat is Fusarium graminearum (Gibberlla zeae). However, other species may also be important, depending on the planting region (Bottalico & Perrone, 2002). Presence of F. graminearum and related species in wheat is an economical issue, which reflects in production losses and requires much investment to its control. Investigations carried out in several parts of the world demonstrated the presence of

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mycotoxins in wheat and its products (EFSA-CONTAM, 2011; Tanaka el al., 1988), which are produced by these fungi, namely deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN). Contamination with mycotoxins may also occur in other food products such as oilseeds, milk and cereals (de Camargo et al., 2012; Peluque et al., 2013; Santili et al., 2015).

According to Pitt et al. (2014), the effects of DON consumption in animals can be noted by feed refusal, while gastrointestinal upset is the most prevalent effect in humans. The toxicity of NIV has been studied in in vitro and in vivo, demonstrating immunotoxicity, haematotoxicity/myelotoxicity and reproductive toxicity (EFSA-CONTAM, 2013). Exposure to a diet with high NIV content has been associated with an increased incidence of oesophageal cancers in certain regions of China (Hsia et al., 2004), but this toxin is unlikely genotoxic. ZEN causes numerous toxic effects in animals, specially related to the reproductive system. ZEN is a nonsteroidal estrogenic mycotoxin which mimesis the effects of females’ oestrogen hormone. In concentrations higher than 1 mg kg-1, ZEN may affect the fertility at several steps such as conception, ovulation and fetal development (CAST, 2003). Pigs are usually affected and there are few epidemiological studies suggesting the estrogenic effect in humans. However, ZEN was considered as the cause for precocious puberty development in children from Porto Rico during the years of 1978-1981 (Saenz de Rodriguez, 1984; Saenz de Rodriguez, et al., 1985). In Italy, Massart et al. (2008) investigated the occurrence of estrogenic mycotoxins in 32 girls with symptoms of central precocious puberty and detected high serum concentrations of ZEN (933.7 pg mL-1) and alpha-zearalenol (106.5 pg mL-1) in six of them, thus suggesting a possible relationship between environmental mycoestrogen exposure and the development of the disease.

Several countries limit the levels of mycotoxins in grains and cereals due to their negative health effects in humans. The European Commission has limited the maximum levels of DON for unprocessed wheat to 1250 μg kg-1

(for durum wheat it is 1750 μg kg-1_{). In the case of ZEN the limit is 100 μg kg}-1

(European Commission, 2006b). In Brazil, the legislation has set the maximum level of DON and ZEN to 3000 and 400 µg kg-1, respectively. These levels, which are for unprocessed wheat, are expected to be in effect in 2017 (Brasil, 2013). To date no legislation addresses the limit for NIV contamination neither in Brazil nor in the European Community. As mentioned before, NIV is unlikely to be genotoxic. However, immunotoxicity and haematotoxicity have been reported. Therefore, the Panel on Contaminants in the Food Chain (CONTAM Panel) has established a tolerable daily intake (TDI) of 1.2 µg kg-1 body weight (bw) per day (EFSA-CONTAM, 2013). The TDI for ZEN is 0.25 μg kg-1

bw per day (EFSA-CONTAM; 2011); whereas for DON and its acetyl derivatives it is 1.0 μg kg-1

bw per day (EFSA-CONTAM, 2013).

The occurrence of DON, NIV and ZEN in cereals was reported by Tanaka et al., (1988). Their study showed the occurrence of these mycotoxins in cereal samples obtained from 19 countries. Ever since, several works were conducted and all of them evidenced the presence of DON, NIV and ZEN in cereals and their by-products (EFSA-CONTAM, 2013). Investigations on the presence of Fusarium mycotoxins in different cereals as well as in their by-products are frequent, specially in regions with higher production such as North America and European Union. On the other hand, South America has limited data available and most studies have focused only on DON, which is the main Fusarium toxin present in wheat and/or its by-products in countries such as Argentina, Brazil and Uruguay (Pinto et al., 2013).

The policy makers’ concern about mycotoxin-contaminated food is legit and has been growing through the years. According to Calori-Domingues et al. (2007), 94% of 50 wheat samples obtained in several locations of Southeast Brazil tested positive for DON. Furthermore, two samples from Paraná, which is the state with the highest wheat production in Brazil, had concentrations of 3327 and 4573 µg kg-1, respectively, supporting the need for continuous monitoring of Fusarium mycotoxin contamination of wheat in Brazil. Only the permanent monitoring will provide the necessary data to establish regional food safety guidelines in accordance with the international standards. In summary, wheat mycotoxin contamination reflects in economic losses, which are related to yield crop decreases, lower quality grains, effects in animal productivity upon contaminated wheat consumption and lower competitivity in national and international markets. Thus, wheat producers, millers and food and feed chain must manage the level of mycotoxin contamination along several steps of production to provide better quality products for human and animal consumption (Miller et al., 2014).

As just mentioned, a continuous survey on the contamination of food and feed products with mycotoxins must be conducted to provide information to regulatory agencies and producers. As far as we know, the present study evaluated the highest number of samples (745) in the last decade in Brazil. According to Cunha et al. (2006), there are four different wheat producing regions in Brazil, which are defined by their weather conditions (temperature and rainfall) and geography (altitude). The samples from the present study were collected in all wheat producing regions. Furthermore, all three Fusarium mycotoxins were evaluated. The same samples were used to provide data about the co-occurrence of DON, NIV and ZEN in wheat produced in Brazil. In summary, the present study was designed to (i) investigate the mycotoxin contamination (DON, NIV and ZEN) in wheat producing regions from Brazil and ii) provide the Brazilian mycotoxin contamination map of wheat produced in the country.

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Material and methods

Chemicals and reference materials

Acetonitrile (Merck, Darmstadt, Germany) and methanol (Tedia, Fairfield, USA) were HPLC grade. Certified mycotoxin standards were purchased from Biopure (Romer Labs, Tulln, Austria). Mix 2 (B-Trichothecenes) containing DON and NIV (BRM 002016) and ZEN (BRM S02029). Wheat reference material for DON (MO9451A) was provided by Romer Labs (Tulln, Austria). Wheat reference material for ZEN (TR-Z100) was purchased from Trilogy Analytical Laboratory (Washington, MO, USA).

Sampling

A total of 745 samples were collected from commercial fields of wheat (crop years 2009 and 2010). The samples were collected considering the adaptation regions for wheat production in Brazil. This is defined taking into account the ecological characteristics of these regions, which is based on the rainfall during the growing season for each producing region according to Cunha et al. (2006) in a publication from the Brazilian Agricultural Research Corporation - Embrapa. The cities, grouped by region and producing state can be visualized in Figure. 1. The number of samples collected in each region was defined based on its representability in terms of production and/or growing area. Therefore, the highest number of samples (about 83%) was collected in the states of Paraná and Rio Grande do Sul, which together represent about 85% of the national wheat production and about 90% of the total growing area (Companhia Nacional de Abastecimento, 2015). The samples (about 3-5 kg each) were collected in grower associations, milling facilities and silos, following the local legislation for wheat grading “Instrução Normativa Nº 7” from Ministry of Agriculture, Livestock and Supply (Brasil, 2001). The samples were collected at 3 different moments. If the samples were already stored in milling facilities and silos, they were collected in at least 25 different points from a total of up to 100 tons. During wheat transfer from trucks to milling facilities and silos, subsamples of 100g grams were collected every 10 min (at least 25 subsamples every 100 tons). Samples were also collected from trucks (at least 11 subsamples every 50 tons). After each step, the subsamples were homogenized and about 3-5 kg from the aggregate sample were sent for mycotoxin analysis. The wheat-sampled lots represented about 20% of the annual wheat production in Brazil. The samples were ground and subsampled using RAS disk mill (Romer Labs Inc., MO, USA), ground again in a MA 090 hammer mill (Marconi, São Paulo, Brazil) using a 0.85 mm screen, homogenized and stored at -18oC until analyses.

Extraction procedure

Ground wheat samples (25 g) were extracted with 100 mL of acetonitrile:water (84:16, v/v) for 60 min in a MA 139 shaker (Marconi, São Paulo, Brazil). The solution so obtained was filtered using a folded qualitative filter paper. To evaluate ZEN, a portion of the filtrate was applied to a multifunctional cleanup using a MycosepTM 224 column (Romer Labs Inc., MO, USA) to purify the extract (Silva & Vargas, 2001). The first 2.5 mL (0.6245 g) of MycosepTM 224 purified extract was collected and transferred to a 10 mL glass vial and evaporated until dryness at 50oC (under air flow) using a MA 4006 dry block heating system (Marconi, São Paulo, Brazil). To investigate DON/NIV the purification of a second portion of the filtrate was conducted using a multifunctional MycosepTM 225 column (Berthiller et al., 2005; Mateo et al., 2002). 5.0 mL (1.25g) MycosepTM 225 column extract was collected and the drying step was conducted as described for ZEN. The ZEN dry residue was reconstituted in 600 µL of methanol:water (7:3, v/v) and NIV/DON dry residue was reconstituted in 500 µl of methanol:water (25:75, v/v), which was followed by filtration using a Millex-GV (0.22 µm x 13 mm) filter (Millipore, Bedford, MA, USA) before HPLC injection. Aliquots of each extract (50 µL) were injected into the HPLC equipment.

HPLC- Photodiode Array Detector (PDA) and chromatography conditions

The identification and quantification of mycotoxins was performed on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of a LC-20 AT pump equipped with a FCV-10AL quaternary valve, a SIL-20A autosampler, a DGU-20A5 degasser, a CTO-20A column oven maintained at 40°C, a photodiode array detector SPD M20A and the software LC Solution v. 1.21. The ZEN chromatographic separation was conducted with an ODS Purospher column (4.0 x 250 mm x 5 µm, Merck, Darmstadt, Germany) and its respective guard column. The mobile phase consisted of (A) acetonitrile:water (48:52, v/v) and (B) acetonitrile:water (90:10, v/v) at a flow rate of 1.5 mL min-1

. The optimized gradient elution procedure, adapted from Schollenberger et al. (2006), was as follows: 0-9 min 100% (A); 9.1-13 min 100% (B); 13.1-18 min 100% (A). The compound was detected at 236 nm. The UV spectral data was used for confirmation (peaks ≥ 10 µg kg-1

). The chromatographic separation of DON/NIV was carried out with an Hypersil ODS column (4.6 x 250 mm x 3 µm, Thermo, Bellefonte, PA, USA) and its respective guard column. The mobile phase consisted of (A) acetonitrile:water (8:92, v/v) and (B) acetonitrile:water (90:10, v/v) and the flow rate was adjusted to 0.8 mL min-1. The optimized gradient elution procedure was as follows: 0-12 min 100% (A); 12.1-21 min 100% (B)

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21.1-36.0 min 100% (A). The detection was recorded at 219 nm. Certified mycotoxin standards (100.4 µg mL-1 NIV plus 100.3 µg mL-1 DON in acetonitrile and 100.8 µg mL-1 ZEN in acetonitrile) were used to prepare the standard curve. Dilutions of NIV/DON were prepared in methanol:water (25:75, v/v) to achieve concentrations from 0.12 to 5.0 ng µl-1 (corresponding to 50 to 2000 µg kg-1) and dilutions of ZEN standard solutions were prepared in methanol:water (70:30, v/v) using concentrations in the range of 0.008-0.121 ng µl-1 (corresponding to 7.7-116 µg kg-1). Aliquots (50 µL) were injected in triplicate into the HPLC using the aforementioned conditions. The standard curve for each mycotoxin was constructed using the mean area of the peak against the concentration. The linearity of the curve was verified through the coefficient of determination.

In house validation of analytical methods

Selectivity, linearity, accuracy and precision were used to evaluate the analytical methods. The retention time and UV spectral data of standard solutions and samples were the parameters used for selectivity. Regression coefficients of the standard curve plotted graphs (r2) were used for linearity. The concentrations of DON, NIV and ZEN in wheat samples and those found in blank samples fortified with three different concentrations of each mycotoxin were used to calculate the recovery rates and the relative standard deviation of repeatability (RSDr). Recovery rates and the relative standard deviation expressed the accuracy and precision of the methods, respectively. The accuracy and precision were also studied by using reference materials: DON was evaluated in wheat (MO9451A – Romer Labs, Tulln, AU) provided during an interlaboratory test organized by the supplier. Therefore, the mentioned reference material is not available in the market. Wheat, TR-Z100, Batch number Z-W-3303 (Trilogy Analytical Laboratory, Washington, MO, USA ) was used to evaluate ZEN. At the time of analyses, any NIV reference material (e.g. wheat or cereal) was available in the market. The limit of detection (LOD) was defined as the lowest mycotoxin concentration detected in a blank wheat sample associated with the spectral data of each mycotoxin (DON, NIV or ZEN). The limit of quantification (LOQ) was defined as the lowest concentration at which we would be able to quantify each mycotoxin according to the accuracy and precision (RSDr) required by the performance criteria established by Commission Regulation (EC) No. 401/2006 (European Commission, 2006a). The recommended NIV values for performance criteria were not stablished by any regulation at the time of analyses; however, as an arbitrary definition, the recommended values for DON were also applied to NIV.

Statistical analysis

To accomplish the homogeneity assumption of ANOVA, the original data were transformed applying the optimal power Box-Cox transformation (Box & Cox, 1964), non-detected values were assumed as half the LOD and values below LOQ were assumed as half the LOQ (Santili et al., 2015). The transformations used for mycotoxins were: ZEN= x-0.2; DON= x0.1; NIV= x-0.8, where x is the concentration in µg kg-1 of each mycotoxin. The results were submitted to analysis of variance (ANOVA) and the differences in contamination were compared by Tukey test (p<0.05) and SAS software version 9.2.

Results and Discussion

The method performance characteristics are summarized in Table 1. The results showed that the analytical methods fit well the purpose of monitoring DON, NIV and ZEN in wheat samples. The LOD for ZEN was associated with the spectral confirmation (10 µg kg-1). Since the accuracy and precision (RSDr) at this concentration fit the criteria established by Commission Regulation (EC) No. 401/2006 (European Commission, 2006a) the same concentration was also considered as the LOQ. The recovery values (RSDr) were in accordance with Regulation 401/2006 (European Commission, 2006a), which determines the RSDr for DON and ZEN. The RSDr for NIV have not been established thus far. However, in the present study, the RSDr for DON was also applied to NIV. As mentioned before, the limit of detection (LOD) was defined as the lowest mycotoxin concentration detected in a blank wheat sample associated with the spectral data. A peak was detected at the same retention time of ZEN in a concentration of 3 µg kg-1 (3:1 signal-to-noise ratio) which would be the LOD. However, the spectral confirmation was possible only at 10 µg kg-1. Since accuracy and precision (RSDr) at 10 µg kg-1 fit the criteria as established by Regulation (EC) No. 401/2006 (European Commission, 2006a) it was also considered as the LOQ.

Distribution of mycotoxin contamination per crop year

The distribution of DON, NIV and ZEN concentration/incidence was evaluated in each crop year as percentage of positive samples (>LOD) and grouped by concentration range in Tables 2 and 3. The great variability among samples collected in different regions reflects in a high standard deviation. However, one should bear in mind that our study reports the reality of the natural contamination. Additionally, this results lend further support on previous studies

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(Bertuzzi et al., 2014; Bianchini et al., 2015), which substantiates the need of permanent evaluation of mycotoxin contamination in food and feed. The concentration of all mycotoxins was higher in samples from 2009 as compared with those from 2010. It is known that the weather conditions (precipitation and temperature), specially during the flowering time, influence the infection of the plant with Fusarium and, in turn, the mycotoxin production. Thus, mycotoxin concentrations may be different depending on the year. According to a publication from the Brazilian Agricultural Research Corporation (Torres et al., 2009), in 2009 the weather conditions during wheat flowering favoured the occurrence of wheat diseases, among them the FHB, which may have allowed for the occurrence of mycotoxins in some Brazilian regions of wheat production. Conversely, according to another report from researchers from the Brazilian Agricultural Research Corporation (Fernandes & Tibola, 2011), the incidence of FHB was low during the year of 2010.

Occurrence and concentration of ZEN

The incidence of ZEN (LOD>10 µg kg-1) was higher in samples from 2009 (85%) as compared with the incidence found in samples from 2010 (27%). Likewise, the mean ZEN concentration was 142 µg kg-1 in 2009 and 22 µg kg-1 in 2010 (Table 2). Only 4.6% of the samples from 2009 had concentrations higher than 400 µg kg-1, which is the maximum limit (ML) for ZEN in wheat for further processing, expected to be in effect in 2017 in Brazil (Brasil, 2011, 2013). Few studies have addressed the occurrence of ZEN in wheat from Brazil. Recently, Almeida-Ferreira et al. (2013) reported no ZEN contamination (LOD = 40 µg kg-1) in cracked wheat samples (n = 109) collected in several cities from Paraná state which, as mentioned before, is the greatest wheat producer in Brazil. However, our results evidenced the occurrence of ZEN in wheat grown in Brazil. Furthermore, specially for samples from 2009 the concentrations were higher than the ML that will be in effect in Brazil in 2017 (Brasil, 2011, 2013). In 2009, 51% of the samples had ZEN contamination in the range of 100-400 µg kg-1 and 4.6% of the samples showed a concentration > 400 µg kg-1 (Table 2). According to the study conducted by Geraldo et al. (2006), 79% of the F. graminearum isolates from Southern Brazil, which is associated with scab disease in wheat (ears and seeds), were ZEN producing strains. Evaluating the worldwide ZEN contamination of foodstuffs and animal feeds, Zinedine et al. (2007) observed that the predominant feature of ZEN distribution in cereal grains and animal feed is its occurrence with other Fusarium toxins including trichothecenes.

Occurrence and concentration of DON and NIV

Table 3 shows the contamination distribution range of DON and NIV. The contamination incidence, the mean and the median are also reported. It is worth noting that the original data is provided in Tables 2, 3 and 5; however, statistical analysis were performed using optimal power Box-Cox transformation as indicated before (statistical analysis section). DON and NIV incidence was dependent on the crop year and the concentration was higher in wheat samples from 2009 as compared with ones from 2010. However, only samples from 2009 showed concentrations of DON higher than 3000 µg kg-1, which will be the ML in Brazil expected to be in effect in 2017 (Brasil, 2011, 2013). Contamination higher than 3000 µg kg-1 accounted for 12% in samples from 2009. Furthermore, concentrations as high as 8500 µg kg-1 were detected. Additionally, 215 wheat samples from 2009 (58%) and 30 from 2010 (8%) would not fit the ML set by the European Community for DON in raw cereals, which is 1250 μg kg-1

(European Commission, 2006b). For NIV, no ML has been set thus far in Brazil, nor in the European Community. However, only one sample (0.3%) had a NIV concentration higher than 1250 µg kg-1 (Table 3). As explained for the occurrence of ZEN, the weather conditions during wheat flowering may have allowed for the occurrence of diseases such as FHB in 2009. This might have resulted in a higher incidence of Fusarium spp. toxins in some of the wheat producing regions. Despite the worst weather conditions for FHB disease during 2010, the percentage of samples that tested positive for DON was similar to samples from 2009. Conversely, the concentrations found in samples from 2010 were lower (Table 3). In fact, 57% of the samples were in the range of 101-1250 µg kg-1, whereas the samples from 2009 showed 58% of samples with concentrations higher than 1250 µg kg-1. Savi et al. (2014) reported DON in whole wheat grain from Southern Brazil (crop year 2012) in the range of 243.7 to 2281.3 µg kg−1, which is in good agreement with the results of 2010 from the present study. However, their data differ from our results from 2009, lending further support on our findings, which demonstrate the importance of investigating the presence of different mycotoxins in different crop years.

According to Del Ponte et al. (2012), the information about Fusarium mycotoxins in wheat from Brazil and South America has been focused on DON, which is the main Fusarium mycotoxin. These authors carried out a multi-year (2006–2008) regional epidemiological survey in Brazil. Their study evaluated 65 wheat samples obtained in the state of Rio Grande do Sul and DON was detected in 98% of the samples (LOD= 250 µg kg-1, mean concentration of 540 µg kg-1). Dos Santos et al. (2013) evaluated the occurrence of DON in 113 wheat samples from different regions of Paraná state, Brazil during 2008 and 2009. DON was detected in 66% of the samples (LOD= 177.1 µg kg-1) and the contamination ranged from 206 to 4732 µg kg-1. The study conducted by Calori-Domingues et al. (2007) had 50 wheat samples from Brazil and 50 imported. The samples were collected from May to December 2005. According to them, 94% (from Brazil) and 88% (imported) of the samples tested positive for DON (LOD=30 µg kg-1). Furthermore, the

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mean contamination was higher in samples from Brazil (332 µg kg-1), as compared with the imported ones (90 µg kg-1). Additionally, concentrations as high as 3327 and 4573 μg kg-1

were detected in two of the national wheat samples (4%), indicating the presence of regions with better weather conditions for fungi development and further toxin production. Samar and Resnik (2002) summarized the DON contamination for cereals and their products in research papers published in the decade 1990-2000 in several parts of the world. According to these authors, 62% of the samples tested positive for DON and the mean contamination was 531 μg kg-1

.

Regarding the NIV contamination found in the present study, from 371 wheat samples collected in 2009, 50% had contamination higher than 100 µg kg-1 and concentrations up to 1329 µg kg-1 were detected. Furthermore, 14 samples had contamination levels higher than 500 µg kg-1. Conversely, a maximum value of 278 µg kg-1 was detected from 374 samples collected in 2010 and only 18 (4.8%) had contamination higher than 100 µg kg-1. It is worth noting that no sample had NIV as the only toxin detected. Additionally, the concentration of DON was higher than that found for NIV in both years. According to Del Ponte et al. (2012), NIV concentrations were lower than DON with few cases showing the opposite trend, lending support to the findings of the present study.

Studies on NIV occurrence in wheat produced in Brazil are still scarce. However, some investigations on the role of mycotoxin production in fungi isolates from Fusarium suggested the occurrence of NIV contaminated grains. In fact, according to Martinelli et al. (2004), NIV was the main trichothecene produced by isolated strains of F. graminearum. Scoz et al. (2009) evaluated 82 fungal isolates from wheat kernel samples from commercial wheat fields affected by FHB collected in southern Brazil. These authors noted that 93% of the isolates were of the DON/15-ADON genotype and the remaining 7% were of the NIV genotype. Furthermore, Astolfi et al. (2012) also identified NIV genotypes in a considerable number of Fusarium isolates from wheat kernel samples from commercial wheat fields in different years and locations, which suggested the possible occurrence of NIV contaminated wheat. The study of Del Ponte et al. (2012), successfully demonstrated the NIV occurrence in wheat from Brazil by analyzing 66 samples. The present study, however, evaluated 745 samples; thus, due to the great contamination variability, which can be noted even in samples from the same lot, our contribution provides an important update on the actual NIV contamination in wheat produced in Brazil.

The first report of NIV wheat contamination in Argentina was carried out by Pinto et al. (2008). From FHB affected wheat samples (n = 19) collected in the main producing regions during the years of 2001 and 2002, these authors reported co-occurrence DON and NIV in two samples and the NIV contaminations of these samples were 50 and 100 µg kg-1. According to Tanaka et al. (1988), from 222 wheat samples 50% had a mean concentration of 127 µg kg-1. In a study conducted in the European Community (Schothorst & van Egmond, 2004), from 2170 wheat samples 14% tested positive for NIV. In Poland, Tomczak et al. (2002) reported that the NIV contamination for 129 samples with FHB collected from two regions in 1998 and 1999 was in the range of 30-85% (LOD = 10 µg kg-1). The mean concentration was in the range of 80-1080 µg kg-1 and contaminations as high as 14200 µg kg-1 were reported. The European Food Safety Authority report showed the data on NIV contamination from 2001 to 2011. Several samples were investigated (n = 13164) in 18 European countries comprising 3846 food, 1707 feed and 7611 samples of unprocessed grains. Oats, maize, barley, wheat and their products had the highest mean contaminations. Furthermore, unprocessed grains had higher contamination than that found in grains for human consumption (EFSA-CONTAM, 2013).

The co-occurrence of ZEN, DON and NIV for each crop year is presented in Table 4. It is possible to suggest that the climatic conditions in the year of 2009 allowed for the plant disease, higher mycotoxin production and co-occurrence of more than one mycotoxin in several samples. DON, which was the mycotoxin with higher incidence in 2009, was the only mycotoxin found in 1.6% of the samples, whereas the co-occurrence of ZEN, DON and NIV was noted in 74%. In samples from 2010 the occurrence of DON as the only mycotoxin found was 46% and the presence of all three mycotoxins was 12% in that year. A synergistic effect has been suggested regarding the presence of more than one mycotoxin, which can cause even more health issues for humans and animals upon consumption of mycotoxin-contaminated food or feed (Segvic Klaric, 2012). The data from the present study is in agreement with the literature. According to the study from European Food Safety Authority, 6% of the samples demonstrated co-occurrence of NIV and DON and, in general, the concentrations of DON were higher than those found for NIV (EFSA-CONTAM, 2013). Del Ponte et al. (2012) also observed the predominant co-contamination with DON and NIV as 56 out of 65 samples evaluated tested positive for both. Co-occurrence of mycotoxins is an indication of human exposure to these contaminants upon consumption of wheat based food and feed. The effect of co-exposure on human and animal health remains unclear (Lindblad et al., 2013). However, the data from the present study clearly demonstrate the co-occurrence of DON, NIV and ZEN as a real problem for the wheat-milling sector, which must be addressed.

Evaluation of contamination in each wheat producing region (WPR) of Brazil

The mean concentration of ZEN, DON and NIV in all four WPRs (Cunha, et al., 2006) from Brazil is shown in Table 5. The lower contamination was found in the WPR IV, whereas the higher contaminations were detected in both WPR I and II, which did not differ from each other. The higher rainfall (data now shown) in the WPR I during the flowering time may have influenced the fungi infection and further mycotoxin production. The climatic conditions in

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Brazil allows for fungi growth and mycotoxin production. Control programs for mycotoxin contamination should be mandatory to avoid supply restriction and economic losses. Therefore, the data on the mycotoxin contamination will be helpful for the wheat producing chain. Furthermore, with the adequate control, the processing industry and final consumer will also have benefits due to a better feedstock and, in consequence, final product. Besides that, the present study shows valuable information for regulatory agencies and future research, which must be focused on wheat safety for food and feed manufacture.

Conclusions

In summary, the present study evaluated 745 wheat samples from seven states of Brazil (crop years 2009 and 2010). The contamination with ZEN, DON and NIV occurred in 56, 87 and 50% of the samples, respectively. The co-occurrence of all mycotoxins was observed in 43% of the samples. The mean concentration of ZEN, DON and NIV was higher in 2009 than in 2010. Their respective maximum concentrations were 82, 1046 and 88 µg kg-1 in 2009, whereas in 2010 they were 1057, 8501 and 1329 µg kg-1. The concentrations of ZEN and DON were higher than the maximum limit of Brazil for only 2.3 and 5.9% of the samples, respectively. The WPR I and II had the highest contamination for all mycotoxins and the WPR IV had the lowest one.

Acknowledgement

The authors are thankful to São Paulo Research Foundation - FAPESP (grant# 2009/ 09912-7, grant #2010 / 02088-4, grant #2010/09565-2, grant #2011/20489-9 and grant #2012/17683-0) and to National Council of Research – CNPq (grant# 2010/2331 - PIBIC). We are also thankful to Luiz Carlos Rodrigues for kindly providing the illustration service used in this manuscript.

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Table 1. Performance characteristics for DON, NIV and ZEN (5 replicates per data point).

Mycotoxin

(µg kg

)

Recovery

(%)

RSDr

(%)

DON

LOD

50

-

-

LOQ

100

78

7.3

Spiked wheat samples

200

86

15

500

84

14

800

84

1.1

Reference Material

(MO9451A)

1009

78

-

NIV

LOD

50

-

-

10

LOQ

100

71

10

Spiked wheat samples

260

66

12

650

68

9.3

1050

67

15

ZEN

LOD

10*

-

-

LOQ

10

81

15

Spiked wheat samples

25

93

15

50

94

16

100

89

4.2

200

91

15

Reference Material

(TR-Z100)

98

106

-

* explanation in the results and discussion paragraph.

11

Table 2. Occurrence and distribution of ZEN in wheat samples from the 2009 and 2010

crop years.

Contamination (µg

kg

)

ZEN 2009

ZEN 2010

n

(%)

n

(%)

< LOD

54 (15)

274 (73)

≤ 100

111 (30)

83 (22)

> 100 to < 400

189 (51)

17 (4.6)

> 400

17 (4.6)

0 (0)

Incidence (%)

85

27

Mean (µg kg

)

142 (±147)

22 (±43)

Median (µg kg

kg)

119

< 10

Highest (µg kg

)

1057

345

Total

371

374

< 10µg kg

.

Standard deviations are shown between parenthesis.

c

Different letters in the same row show that results are different (p < 0.05).

12

Table 3. Occurrence and distribution of DON and NIV in wheat samples from the 2009 and

2010 crop years.

DON 2009

DON 2010

NIV 2009

NIV 2010

n

(%)

n (%)

n

(%)

n

(%)

< LOD

37 (8.9)

64 (17)

84 (22.7)

288 (77)

≤ 100

9 (3.5)

68 (18)

101 (27)

68 (18)

> 100 to < 1250

110 (30)

212 (57)

185 (50)

18 (4.8)

> 1250 to <

3000

171 (46)

30 (8.0)

1 (0.3)

0 (0.0)

> 3000

44 (12)

0 (0)

0 (0)

0 (0.0)

Incidence (%)

90

83

77

23

Mean (µg kg

)

1690

(±1434)

407 (±485)

140 (±166)

36 (±34)

Median (µg kg

)

1478

209

102

< LOD

Highest (µg kg

)

8501

2419

1329

278

Total

371

374

371

374

< 50µg kg

.

Standard deviations are shown between parenthesis.

c

Different letters in the same row show that results are different within the same toxin (p <

0.05).

13

Table 4. Co-occurrence of ZEN, DON and NIV in wheat samples (number and

percentages) from 2009 and 2010 crop years.

Mycotoxins

2009

2010

No mycotoxin

34 (9.2%)

63 (17%)

Only ZEN

3 (0.8%)

1 (0.3%)

Only DON

6 (1.6%)

171 (46%)

DON + NIV

14 (3.8%)

40 (11%)

ZEN + DON

41 (11%)

53 (14%)

ZEN + DON + NIV

273 (74%)

46 (12%)

Table 5. Mean concentrations (

μg kg

) and incidences (%) of ZEN, DON and

NIV in wheat samples from different producing regions.

Mycotoxin

WPR I

WPR II

WPR III

WPR IV

ZEN

111 a

(±162)

82 ab

(±100)

65 b

(±98)

<10 c

(±1,7)

DON

1230 a

(±1201)

1182 a

(±1285)

893 b

(±1251)

144 c

(±682)

NIV

<100 a

(±127)

<100 a

(±160)

<100 a

(±102)

<100 b

(±67)

ZEN

66

60

52

4.0

DON

97

92

86

18

NIV

53

55

51

8.0

Total

242

242

211

50

Different letters in the same row show that results are different (p < 0.05).

14

Fig. 1 - Wheat producing regions (WPR) in Brazil. Cities where samples were collected are marked in black. WPR I: wet/cold region in areas with the highest altitutes; WPR II: wet/moderately hot region in areas with the lowest altitudes; WPR III: moderately dry/hot region; WPR IV: dry/hot region with ocasional water and thermal stress; NPR: non-producing regions, i.e. where wheat growing is not recommended. Figure taken from Cunha et al. (2006).