Survey of trichothecene mycotoxins in grains and animal feed in Croatia by thin layer chromatography

Food Control 17 (2006) 733–740 www.elsevier.com/locate/foodcont

Survey of trichothecene mycotoxins in grains and animal feed in Croatia by thin layer chromatography Marijana Sokolovic´ *, Borka Sˇimpraga

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Croatian Veterinary Institute, Poultry Centre, Heinzelova 55, 10000 Zagreb, Croatia Received 22 February 2005; received in revised form 7 May 2005; accepted 9 May 2005

Abstract Trichothecene mycotoxins are common contaminants of cereal grains and animal feed worldwide. The toxins are toxic to both human and animals. The objectives of this study were to determine the occurrence of trichothecenes in grains and animal feed in Croatia. Total of 465 samples were collected during the seven-year period (1998–2004) from manufactures and small holders farm storage facilities. The samples were analyzed by thin layer chromatography, which proved to be fast, reliable and inexpensive method. T-2 toxin, diacetoxyscirpenol and deoxynivalenol were detected in 16.8%, 27.6% and 41.2%, respectively. The amount of toxins ranged between 0.05 and 3.4 mg/kg. The majority of animal feed samples was poultry feed. Only small number of it contained T-2 toxin and diacetoxyscirpenol levels greater than the Croatian regulatory levels for poultry feed. Positive samples were in correlation with evidenced clinical symptoms of toxicosis in poultry. Since trichothecenes are frequently isolated from animal feed and grains in Croatia, they could have significant economic and safety implications in animal production. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Trichothecenes; Feed; Grains; TLC; Croatia

1. Introduction Trichothecenes are a group of over 170 mycotoxins produced mainly by fungi of the genus Fusarium (Krska, Baumgartner, & Josephs, 2001; Langseth & Rundberget, 1998). The toxins occur worldwide in a wide variety of food, feed and other commodities. They are often found in cereal grains, especially in the temperate regions of America, Asia and Europe (Creppy, 2002; DÕMello & Macdonald, 1997). It has been evidenced that grains can be contaminated at any stage from pre harvest to post harvest and during storage.

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Corresponding author. Tel.: +385 1 2441 394; fax: +385 1 2441 396. E-mail addresses: [email protected] (M. Sokolovic´), borkas@ inet.hr (B. Sˇimpraga). 1 Tel.: +385 1 2441 392; fax: +385 1 2441 396. 0956-7135/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2005.05.001

Recent European study on occurrence of Fusarium toxins provided results covering 12 different trichothecenes. Fifty seven percent of the samples from 11 countries were positive for deoxynivalenol (DON). Twenty percent of the samples were positive for T-2 toxin and only 4% were positive for diacetoxyscirpenol (DAS) (EC, 2003). Similar study was done by Joint FAO/ WHO Expert Committee on Food Additives (JECFA, 2001). It has been shown that DON was also a frequent contaminant of cereal grains. The incidence of contamination of grain samples was 11% for T-2 toxin and 14% for HT-2 toxin, while no data were available for DAS. A survey done by Pavicˇic´, Brlek, and Nemanicˇ (1998) revealed that Fusarium species are the most frequently isolated fungi from grains in Croatia, indicating on possible economic importance of trichothecenes in Croatia. T-2 toxin and DAS belong to a group of the most acute toxic trichothecenes (type A), but have lower incidence in feed. Deoxynivalenol (type B), at the other hand

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has lower toxicity but is frequent contaminant of grains. According to the Croatian regulation (NN 26/98) maximally allowed quantity of T-2 toxin and diacetoxyscirpenol in complete and supplemental feed is 0.5 mg/kg for young chicken, pigs and calves and 1 mg/kg for adult poultry and pigs. Maximally allowed quantity of deoxynivalenol in complete and supplemental feed for pigs is 0.5 mg, while there are no regulations for feed for other animals. As for grains, maximally tolerable level of trichothecene mycotoxins is recently regulated only for deoxynivalenol in grains appointed for human consumption in a quantity of 2 mg/kg (NN 16/05). Toxicological evaluation of trichothecenes in animal feed has been extensively reviewed (Eriksen & Pettersson, 2004; Van Egmond, 2004). The clinical symptoms of intoxicated animals vary from acute mortality to reduced growth and productivity. They include necrotic changes in mouth and gastrointestinal tract, emesis, diarrhoea, anorexia, haematological and immunological alterations and sometimes even a lethal outcome. Chronic consumption of low levels of these mycotoxins, especially in combination with commercial productionÕs stress, may result in impaired immunity and decreased resistance to infectious diseases. Thin layer chromatography (TLC) method represents simple, rapid and economical method for semi-quantitative detection of many mycotoxins. The use of TLC plates has become the most widely used technique for detection, quantification and confirmation of identity. Although, the most significant advantage of this technique is the low cost of each analysis, quantification step suffers from a high level of a variation. It is classified as a semi-quantitative method because of visual detection of the developed spots on a plate. In comparison of TLC, enzyme-linked immunosorbent assays (ELISA) tests and high performance liquid chromatography (HPLC) methods for identifying and quantifying deoxynivalenol and other Fusarium toxins, TLC proved to be in very good agreement between levels of deoxynivalenol measured with ELISA and HPLC over a wide range of concentrations (Schaafsma et al., 1998). Determination of trichothecenes and metabolites down to the lg/kg level can be accomplished with more accurate and reliable methods. For example, gas chromatography with either electron-capture or mass spectrometric detection is the method of choice for detection of trichothecene mycotoxins. Other analytical techniques used for determination of trichothecene include high-performance liquid chromatography, supercritical fluid chromatography and immunochemical methods like enzyme-linked immunosorbent assay systems (Koch, 2004; Krska et al., 2001; Langseth & Rundberget, 1998; Mateo, Llorens, Mateo, & Jimenez, 2001). Since trichothecenes are a group of closely related compounds, analytical methods are used for determination of various trichothecenes simultaneously (Razzazi-Fazeli, Bo¨hm, Jarukamjorn, &

Zentek, 2003). However, some steps in analytical methods may vary depending on the group of analyzed trichothecenes (Josephs, Derbyshire, Stroka, Emons, & Anklam, 2004). The choice of analytical method depends upon proposed tolerable daily intake (TDI), established legislative limit and limit of detection. Furthermore, the criteria include the level of contamination, precision, linearity, quantification, as well as limited instrumentation and low costs. In many laboratories in Croatia that do not have expensive equipment at their disposal, thin layer chromatography still represents important method for screening purposes. The aim of this study was to evaluate the natural occurrence of T-2 toxin and DAS in grains and animal feed in Croatia over a seven-year period. Additional analyses were made in last few years for DON. Unfortunately, number of analyzed samples is insufficient for evaluation of DON occurrence in specific samples. However, results can implicate on a need for more accurate information on the exposure to DON.

2. Experimental 2.1. Samples A total of 465 samples of grains and animal feed were collected during the period from 1998 to 2004. Samples of grains and feed from each storage bin and load leaving manufactures and smallholder farm storage facilities were taken regularly at the time of filling. Five percent of feed samples were examined because of suspicion on mycotoxin contamination. All samples were collected in the presence of authorized doctor of veterinary medicine according to the method of taking a representative sample (NN, 106/99). In order to achieve reasonably representative samples, primary large samples of approximately 10 kg were composed of several samples collected from different part of storage lots. The primary samples were homogenized and quartered to obtain a 1 kg of laboratory sample. All collected samples were stored at 4 °C prior analysis and thoroughly ground for analysis. 2.2. Standard solutions Mycotoxin standards were purchased form Sigma (St Luis, MO, USA). All chemicals were purchased from Merck (Darmstadt, Germany). Stock solution of individual toxin was prepared weighing 1 mg of each toxin and dissolving in 1 ml methanol (HPLC grade). Stock solutions were stored in freezer. Working standard solution was prepared using adequate aliquots of each individual standard stock solution and diluting with a suitable solvent. Each working standard was prepared prior analysis. The exact concentrations of the standard

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solutions were verified by UV absorbance measurements on a spectrophotometer. Occasional comparisons of stock solutions were made with fresh standard. 2.3. Analytical methods 2.3.1. TLC method for detection of T-2 toxin and diacetoxyscirpenol The samples were analyzed for T-2 toxin and diacetoxyscirpenol by thin layer chromatography methods proposed by Romer, Boiling, and MacDonald (1978) and Sano, Asabe, Takitani, and Ueno (1982) with slight modifications. The procedure was used as follows: fifty grams sample was weighted into 500 ml glass-stopper flask. Two hundred and fifty millilitres of methanol– water (125:125, v/v) was added, and shaken for 30 min. One hundred and twenty five millilitres of filtered extract was mixed with 180 ml of 30% ammonium sulphate and 24 g of diatomaceous earth (Acid-washed Celite 545, chemically analyzed for purity). Aliquot of 250 ml of sample filtrate was transferred to a separatory funnel (500 ml), and 20 ml of chloroform was added and shaken slowly. After layers were separated, the bottom layer was transferred in the second separatory funnel filled with 100 ml mixture of 1% potassium hydroxide and 0.02 N potassium-chloride solution. The procedure was repeated with addition of 20 ml of chloroform to the first separatory funnel. Collected chloroform layer was filtered and evaporated to dryness under vacuum at 60 °C. Residue was dissolved in 3 ml of methanol. Thirty microlitres of the concentrated sample and spike extract, alongside with 10, 20, 30 and 40 ll of 100-lg/ml working standard were spotted on TLC plates SIL G-25 (Macherey-Nagel, Germany). Plates were developed in toluene–ethyl acetate–formic acid (100:50:15, v/v/v) until solvent front was 1 cm from the top of the plate (90 min). The plate was sprayed with 4% nicotinamide in the mixture of acetone–ethanol (83.3:16.7, v/v), dried at the air and heated at 160 °C for 15 min. The cooled plate was sprayed with 2-acetylpyridine-n-hexane (3:100, w/v) and 2 N potassium hydroxide–ethanol– water (11.2:96:6, w/v/v). Dried plate was further sprayed with formic acid–ether (30:100, w/v) and heated at 100 °C for 5 min. The toxins were identified as fluorescent spots at the same height (Rf value) as standard spots and compared with known amounts of the toxin standards under long wave UV light (MinUVIS-UV 254/366, Desaga). In each analytical batch we have analyzed internal blank samples to check the interference. The accuracy of measurements was assessed through recovery of additions of known amounts of the toxin to blank samples of finely ground corn and wheat. The spike sample was mixed well and treated as a sample through the rest of the analysis. Positive samples were tested twice alongside with appropriate spike samples to confirm the presence and quantity of mycotoxins.

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2.3.2. TLC method for detection of deoxynivalenol Deoxynivalenol was identified by the modified twodimensional TLC method described by Tanaka, Hasegawa, Matsuki, and Ueno (1985). In brief, twenty grams of thoroughly grinded sample was mixed with 200 ml of acetonitrile–water (75:25, v/v) and shaken for 30 min. One hundred and twenty five millilitres of filtered sample were transferred to a separatory funnel and mixed with 100 ml of n-hexane. The two layers were allowed to separate, and the bottom layer was transferred to a flask along with 100 ml of ethanol and evaporated on rotary vapor. The residue was dissolved in 6 ml of methanol and transferred to a Florisil column. Small ball of glass wool was placed in bottom of column tube, previously washed with methanol and n-hexane. N-hexane was added to the column to cover the half. The chromatographic column was packed with 45 g of sodium sulphate–florisil–sodium sulphate (10:20:15, w/w/w) and conditioned by releasing the n-hexane down to the first layer. Six millilitres of the sample and spike filtrate were transferred to the column. Two hundred millilitres of nhexane and 250 ml of chloroform–methanol (225:25, v/v) were added for rinsing of the column. The filtrate was evaporated to dryness. Residues were dissolved in 2 ml methanol. Ten microlitres of sample extract and 10, 20, 30 and 40 ll of 10-lg/ml working standard were spotted on HPTLC Kieselgel 60 plate F-254 (Macherey-Nagel, Germany). The plate was developed by two-dimensional technique using toluene–ethyl acetate–formic acid (5:4:1) as a first solvent system and chloroform–methanol (7:1) as a second. Developed plate was sprayed with 5% aluminium chloride and heated at 90 °C for 10 min. Deoxynivalenol was observed as blue fluorescent spot under long wave UV light at Rf approximately 0.6 (MinUVIS-UV 254/366, Desaga). Spots were well resolved from background fluorescent spots. The concentrations of the toxin were estimated by visual comparison with known amounts of the standards. Spiked samples were prepared according to the described methodology for T-2 toxin and diacetoxyscirpenol. Response was linear relative to standard concentrations. In a case of samples with higher concentrations, quantity was determined after second chromatography using different spike sample.

3. Results All samples were analyzed in duplicates, in series of 8–10 samples with additional spike sample for recovery test, fortified with 0.1 mg/kg T-2, DAS and DON. In a case of highly contaminated samples, higher concentrations of toxin were used for spiking. The results of the contaminated samples were corrected for the recovery of the test sample from each series. The mean recoveries were 85% for T-2, 90% for DAS and 93% for DON with

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mean coefficients of variation of 11.3%, 8.1% and 9.1%, respectively. The detection limits were 0.1 mg/kg for T-2 toxin and DAS, and 0.01 mg/kg for DON. The overall incidence of T-2 toxin, DAS and DON were 16.8%, 27.6% and 41.2%, respectively (Table 1). All of the occurrence data show wide variation in concentrations in most of the grains tested during this

seven-year period. The percentage of samples positive for T-2 toxin ranged between 3% and 66.7% (Table 2) whereas the highest incidence was detected in grains in 2004 (66.7%). The incidence of DAS was permanently slightly higher during the whole survey (9.1–63.6%) and the highest incidence was in 2004 as well (Table 3). The percentage of samples positive for DON varied

Table 1 Occurrence of trichothecene mycotoxins in grains and feeds in Croatia during the seven-year period (1998–2004) Year

Toxin detected

1998

T-2 DAS

1999

No. of samples analyzed

No. (%) of samples positive

Toxin concentrations (mg/kg) of positive samples Average

Range

Median

83 79

2 (2.4) 16 (20.3)

0.10 0.20

0.1 0.1–0.4

0.1 0.2

T-2 DAS

103 95

11 (10.7) 32 (33.7)

0.10 0.40

0.1 0.1–0.5

0.1 0.2

2000

T-2 DAS

79 73

13 (16.5) 20 (27.4)

0.15 0.15

0.1–0.2 0.1–0.5

0.1 0.2

2001

T-2 DAS DON

47 47 5

11 (23.4) 9 (19.1) ND

0.20 0.25 ND

0.1–0.5 0.1–0.4 ND

0.2 0.1 ND

2002

T-2 DAS DON

50 50 7

7 (14) 11 (22) ND

0.15 0.25 ND

0.1–0.5 0.1–1.2 ND

0.2 0.15 ND

2003

T-2 DAS DON

40 38 13

12 (30) 8 (21.1) 3 (23.1)

0.10 0.10 0.08

0.1–0.7 0.1–0.5 0.05–0.63

0.15 0.13 0.1

2004

T-2 DAS DON

27 28 26

16 (59.3) 17 (60.7) 18 (69.2)

0.36 0.10 2.25

0.1–0.52 0.1–0.4 0.1–3.44

0.15 0.1 0.5

ND—not detected.

Table 2 Occurrence of T-2 in cereal grains and feeds in Croatia Year

Sample

No. of samples analyzed

No. (%) of samples positive

Toxin concentrations (mg/kg) of positive samples Average

Range

Median

1998

Grains Feed

16 67

ND 2 (3%)

ND 0.1

ND 0.1

ND 0.1

1999

Grains Feed

38 65

4 (10.5) 7 (10.8)

0.1 0.1

0.1 0.1

0.1 0.1

2000

Grains Feed

30 49

9 (30) 4 (8.2)

0.11 0.13

0.1–0.2 0.1–0.2

0.1 0.1

2001

Grains Feed

19 28

4 (21.1) 7 (25)

0.13 0.31

0.1–0.2 0.1–0.5

0.1 0.2

2002

Grains Feed

21 29

2 (9.5) 5 (17.2)

0.15 0.24

0.1–0.2 0.1–0.5

0.15 0.2

2003

Grains Feed

23 17

4 (17.4) 8 (47.1)

0.35 0.19

0.1–0.7 0.1–0.5

0.3 0.15

2004

Grains Feed

6 21

4 (66.7) 12 (57.1)

0.41 0.2

0.1–0.52 0.1–0.5

0.51 0.1

ND—not detected.

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Table 3 Occurrence of diacetoxyscirpenol in grains and feeds in Croatia Year

Sample

No. of samples analyzed

No. (%) of samples positive

Toxin concentrations (mg/kg) of positive samples Average

Range

Median

1998

Grains Feed

15 63

2 (13.3) 14 (22.2)

0.1 0.18

0.1 0.1–0.4

0.1 0.2

1999

Grains Feed

29 66

9 (31) 23 (34.9)

0.22 0.22

0.1–0.4 0.1–0.5

0.2 0.2

2000

Grains Feed

27 46

7 (25.9) 13 (28.2)

0.19 0.19

0.1–0.4 0.1–0.5

0.2 0.2

2001

Grains Feed

19 28

4 (21.1) 5 (17.9)

0.18 0.22

0.1–0.4 0.1–0.4

0.1 0.1

2002

Grains Feed

21 29

5 (23.8) 6 (20.7)

0.19 0.48

0.1–0.4 0.1–1.2

0.15 0.4

2003

Grains Feed

22 16

2 (9.1) 6 (37.5)

0.3 0.17

0.1–0.5 0.1–0.4

0.3 0.13

2004

Grains Feed

6 22

3 (50) 14 (63.6)

0.13 0.21

0.1–0.2 0.1–0.4

0.1 0.13

Table 4 Occurrence of deoxynivalenol in grains and feeds in Croatia Year

Sample

No. of samples analyzed

No. (%) of samples positive

Toxin concentrations (mg/kg) of positive samples Average

Range

Median

2001

Grains Feed

2 3

ND ND

ND ND

ND ND

ND ND

2002

Grains Feed

– 7

– ND

– ND

– ND

– ND

2003

Grains Feed

7 6

1 (14.3) 2 (33.3)

0.1 0.34

0.1 0.05–0.63

0.1 0.34

2004

Grains Feed

5 21

3 (60) 15 (71.4)

2.7 0.42

1.5–3.44 0.1–1.05

3.17 0.4

ND—not detected.

up to 71.4%, but considering the rather small number of samples these data cannot be interpreted as the actual situation in the field conditions (Table 4).

4. Discussion European study on occurrence of Fusarium toxins revealed that 57% of the samples from 11 countries (11022) were positive for DON. A high frequency of DON was found in maize (89%) and wheat (61%). Only 3490 (from 8 countries) and 1886 (from 3 countries) samples were analyzed for the presence of T-2 toxin and DAS, with incidence of 20% and 4%, respectively. Maize (28%), wheat (21%) and oats (21%) were most frequently contaminated with T-2 toxin (EC, 2003). In the study done by JECFA (2001), deoxynivalenol was detected in oats (834 samples, 68% positive), barley (1662

samples, 59% positive), wheat (11,444 samples, 57% positive), maize (5349 samples, 41% positive), and in many other grains and processed food products. The mean concentrations were 4–760 lg/kg for oats, 4–9000 lg/ kg for barley, 1–5700 lg/kg for wheat and 3–3700 lg/ kg for maize. Data on the concentrations of T-2 toxin were available on 8918 samples of grains, including barley, maize, oats, rice, rye and wheat. Samples had been collected in Europe, Brazil, China, Finland, Germany, Norway and Sweden. The incidence of contamination was 11% for T-2 toxin and 14% for HT-2 toxin; and occasionally simultaneous high concentrations of the two toxins were found. Annual variations were reported in the levels of contamination of barley, oats and wheat. The mean concentrations in data sets in which positive samples of T-2 and HT-2 were found are 0.1–21 mg/kg and 0.4–15 mg/kg in barley, 1.3–6.0 mg/kg and 2.4– 14 mg/kg for maize, 2.3–26 mg/kg and 3.7–20 mg/kg

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for oats and 0.1–60 mg/kg and 20 mg/kg for wheat, respectively. There was no information about DAS occurrence at all. Da¨nicke (2002) reviewed the natural occurrence of mycotoxins in relation to toxicological relevance for poultry. The results showed that concentrations of T-2 toxin and DAS in grains and mixed feed are commonly low and seldom reach levels higher than 0.5 mg/kg. Harmful levels of T-2 toxin and DAS, which could cause toxicosis in poultry, are rare and sporadic. The contamination levels presented in our survey are similar to aforementioned results covering reports from European countries. T-2 toxin and DAS were commonly found in grains and feed with a number of positive samples up to 60.7%. In the year 1999, 31% of grain and 34.9% of feed samples were contaminated with DAS. T-2 toxin was detected in only 10.5% and 10.8% of grains and feed, respectively. Higher contamination of grains in the year 2000 (30%) is not in correlation with contamination of feed with T-2 toxin as it is seen for diacetoxyscirpenol (Table 3). The highest contamination of grains was in the year 2004, while feed was heavily contaminated in both 2003 and 2004. Correlation of contaminated feed and grains has been evidenced for deoxynivalenol. Since regulation of maximal mycotoxin limits for grains intended for animal consumption does not exist in Croatia, a rather small number of samples was examined, predominantly maize (80%). Other tested grains were wheat, barley, oat and soy. Percentage of positive, even in such a small number of samples, with almost adequate correlation to contamination of feed samples urge for regular screening. Most of the examined feed samples were poultry feed. Although deoxynivalenol has the most potent effects in pigs, it is also necessary to avoid use of poultry feed with a dietary concentration greater than 5 mg/kg because of adverse effects on health and performance of animals (Da¨nicke, 2002). Unfortunately, neither regulatory, nor guidance level for DON in poultry feed in Croatia exist. Furthermore, data for DON does not cover the whole sevenyear period, but detected concentrations between 0.05 and 3.44 mg/kg can draw attention to possible higher contamination of grains and feed samples in Croatia. Some of the tested samples were positive for zearalenone, too (results not shown). Overall incidence of zearalenone in grains and feed was 34.1% (30 positive samples of 88 examined). F. graminearum is considered as the most potent source of deoxynivalenol and zearalenone in grains. In survey done by Pepeljnjak and Sˇegvic´ (2004) during the last 6 years there was high incidence of Fusarium (70%) species on non-harvested and stored grains. F. graminearum, F. sporotrichoides and F. tricinctum were detected in 10% of examined samples. Evaluation of toxigenic potential of isolated strains showed production of T-2 toxin in only 3.2% of F. tricinctum strains. Production of zearalenone was found in 14.8% of F. graminearum strains, and only 3.2% in strains of

F. moniliforme. No data were available for deoxynivalenol or diacetoxyscirpenol. In study of Pavicˇic´ et al. (1998) the most prevalent species of the genus Fusarium were F. graminearum, F. sporotrichoides, F. poae, F. moniliforme and F. tricinctum. Due to the incidence of F. graminearum, high occurrence of zearalenone in grains, and specificity of climate in Croatia, it can be supposed that deoxynivalenol is often contaminant of grains and feed in Croatia as well. Significant variations of grains and feed contamination can be explained with evidenced climate changes. According to the reports of World Meteorological Organisation, the years 1998, 2004 and 2003 are classified as the warmest years since the year 1861. In Croatia, climate monitoring and assessment in the last seven years has proved that the years were extremely warm, high above the average. Because of frequent rains in spring, fall and winter, the years 1998, 2003 and 2004 are also classified as ‘‘highly humid’’. Aforementioned data infers that favourable conditions for the growth of moulds and subsequent toxin production might have occurred. Although the moisture content and temperature are the most critical factors, there are several other factors that can affect mould growth. They include mechanical injury, insect damage, mineral nutrition of the plant, chemical treatment, rapidity of drying, leakage in storage, hot spots, etc. (Birzele, Prange, & Kra¨mer, 2000; Llorens, Mateo, Hinojo, Valle-Algarra, & Jimenez, 2004; Mateo, Mateo, & Jimenez, 2002). However, for the biosynthesis of mycotoxins in grains, interaction of several factors is usually more important than any single risk factor alone (Mateo et al., 2002). During the whole survey only few samples contained DAS over 1 mg/kg. Those samples derived from farms with evidenced poultry health problems with indications of possible trichothecene poisoning (Konjevic et al., 2004). Experimental toxicosis caused by trichothecene mycotoxins in poultry is well documented. Wyatt, Colwell, Hamilton, and Burmeister (1973) reported oral necroses in the mouth of chickens by the doses of 1 mg/kg of T-2 toxin. Dietary concentration of 1–4 mg/kg of T-2 toxin and 1–2 mg/kg of DAS were reported to cause oral lesions, decreased feed intake and weight (Ademoyero & Hamilton, 1991; Chi et al., 1977). Abnormal feathering and neural disturbances were observed in chickens receiving dietary T-2 toxin levels of 4 mg/kg or higher (Wyatt et al., 1973; Wyatt, Hamilton, & Burmeister, 1975). Feather abnormalities were also observed in broiler chickens fed with dietary DAS at concentrations of 2 mg/kg and above (Parkhust, Hamilton, & Ademoyero, 1992). In laying hens, T-2 toxin and DAS in dietary concentrations of 2 mg/kg each induced oral lesions and decreased significantly egg production and food intake (Diaz, Squires, Julian, & Boermans, 1994). In general, concentrations of detected mycotoxins in our survey are far below the one that induces disease

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with typical clinical symptoms. Nevertheless, simultaneous presence of toxigenic fungi and low dietary levels of mycotoxins in field conditions can cause subtle and ill-defined changes that are mainly characterized as decreased feed intake and gain rate as well as an increased risk to infectious diseases. Regarding to low dietary level of mycotoxins, Hamilton (1984) also pointed at the fact that any level of mycotoxins carries risk of economic losses and that is impossible to define a safe level under field conditions. As a result of toxicological evaluations of trichothecenes, some guidance levels have already been proposed in several countries (Eriksen & Pettersson, 2004; Van Egmond, 2004).

5. Conclusions The results obtained in this survey revealed that trichothecene mycotoxins are frequent contaminants of cereal grains and feed in Croatia. These results also emphasize the need for regular screening of even greater number of samples for several mycotoxins. Also, it would also be of great importance to precisely investigate the distribution of Fusarium species in Croatia, as well as their toxigenic potential. Further improvements are needed in sampling procedures as well as in analytical methods for trichothecenes, with the respect to recovery, accuracy and precision of measurements.

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