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Natural occurrence of trichothecenes and zearalenone in Argentine wheat
Food Conrrol, Vol. 6. No. 4, pp. 201-204, 1995 Copyright 0 1995Elsevier Science Ltd Printed in Great Britain. All rights reserved 09%7135/95 $10.00 + 0.00
095%7135(95)00021-6
PAPER
Natural occurrence of trichothecenes and zearalenone in Argentine wheat Nora Quiroga, *? Silvia Resnik to Ana Pacin ,*** Elena Martinez,§ Ana Pagano,? Isabel Riccobene? and Susana NeiraS* * A survey of the natural occurrence of trichothecenes and zearalenone in samples from Buenos Aires and Santa Fe provinces, Argentina, was carried out in six different crops during harvest. Among 1056 samples analysed, 524 (49.62%) were contaminated with trichothecenes and zearalenone. Deoxynivalenol was detected in all of the contaminated samples, 3-Acetyl DON in 22 samples and zearalenone in 78. In 1986, T-2 toxin (20 samples), HT-2 toxin (18), neosolaniol (16) and diacetoxyscirpenol(26) were also found. The results suggest that when meteorological conditions _favour contamination it is necessary to carry out strict controls on harvest time. Keywords: trichothecenes,
zearalenone, natural occurrence, wheat
INTRODUCTION Wheat is the most important cereal in Argentina both from the point of view of local consumption and its high earning power as an export commodity. The infestation of wheat with the Fusarium species and its consequent contamination with trichothecenes and zearalenone have been amply studied in many countries. Accumulated data have revealed the natural occurrence of trichothecenes in, among others, Canada (Trenholm et al., 1981; Scott, 1983), Japan (Tanaka et al., 1985), USA (Wood and Carter, 1989), Korea (Lee et al., 1986), Poland (Perkowski et al., 1990), The Netherlands (Tanaka et al., 1990), UK (Tanaka et al., 1986), and New Zealand (Agnew et al., 1986). Examination of *Municipalidad de Olavarria. ‘Universidad Nacionat de1 Centro. *Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires. %Jniversidad de Buenos Aires. **Universidad National de Lujhn. Address for correspondence: S. Resnik, Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina. Received 9 December 1994; revised 14 April 1995; accepted 16 May 199.5.
these surveys reveals a significant variation in the occurrence of trichothecenes within regions. Adverse climatic conditions at critical periods in the growth cycle have been reported to produce a favourable environment for the growth of Fusarium spp. capable of producing these mycotoxins. Trichothecenes are able to cause, both in man and animals (Pacin, 1988; Bhat et al., 1989), immunosupression, haematological changes of varying degree and digestive disorders (Ueno, 1983). Zearalenone can produce estrogenic syndromes of different degrees (Kuiper-Goodman et al., 1987). To evaluate the occurrence of contamination by trichothecenes and zearalenone in wheat, we analysed samples of wheat harvested in the main production area in Argentina over a six year period.
MATERIALS AND METHODS Samples Samples of different
crops were collected during the
Food Control 1995 Volume 6 Number 4
201
Trichothecenes and zearalenone in wheat: N. Quiroga et a/.
Table 1 Number of samples analysed each year Year
Number of samples
1985” 1986 1989 1990 1991 1992
123 261 102 159 189 222
‘These samples were collected after the 1985 harvest from different storages in only one area of Buenos Aires province.
harvest from farm areas in Buenos Aires province and south of Santa Fe. The number of samples was proportional to the total production of each area (Table I). Sampling was carried out following the procedure described by Apro et al. (1987). During harvest, as the grain flowed into the trucks, portions of about 50 g were taken at regular time intervals. All the portions corresponding of the same batch were mixed and quartered to give primary samples of 10 kg. These primary samples were homogenized and quartered to give 3 kg samples. These were then ground to pass through a 14 mesh sieve and quartered to give subsamples of about 1 kg which were ground to pass a 20 mesh sieve. From each subsample, approximately 200 g was taken and stored at -20°C prior to analysis. Analytical methodology The method used to quantify DON involves sample extraction with acetonitrile-water (84+16) cleanup using a disposable column of charcoal, celite and alumina, and detection by thin-layer chromatography after spraying with an aluminium chloride solution (Eppley et al., 1986; Trucksess et al., 1984). In order to select the acetonitrile to be used for the extraction, two acetonitriles were evaluated: Merck No 30 and Merck No.800015. DON was added to a ground sample of wheat to obtain five levels of concentration (50, 70, 100, 150 and 300 &kg). Quadruplicated analyses were made for each level, using 40 ml of the sample extract. Both acetonitriles had the same average recovery but Merck No 30 gave sharper TLC spots allowing a better quantification of DON. The Trucksess et al. method (1984) was also employed for the analysis of 3-Acetyl DON, T2-toxin, HT-2, DAS, neosolaniol and zearalenone. Two TLC plates were used for each sample, one for Group B trichothecenes (DON and 3-Acetyl DON) and zearalenone and the other for Group A toxins. The plates (Merck No 5553) were developed with benzene:acetone (3:2). Zearalenone and trichothecenes of group B exhibit blue fluorescence at 364 nm when the plate was treated with 15% aluminium chloride solution and heated for 6 min at 120°C. Trichothecenes of group A exhibit blue fluorescence at the same UV wave when the plate was sprayed with a sulphuric acid solution (20%) followed by heating at 130°C for 10 min (Takitani and Asabe,
202
Food Control 1995 Volume 6 Number 4
1983). Rf values, detection limits and approximate recovery percentages are presented in Table 2. Toxin confirmation was made by TLC using other solvent systems. In 1990, 1991 and 1992, duplicated analyses were made using two subsamples of 50 g, while in the other years only one subsample of 100 g was analysed. Statistical methodology To assess the equality of mean annual contamination of DON over positive samples, a one-way analysis of variance (ANOVA) was performed. The model is of the form:
where Z is the number of years compared and IZ~is the number of positive samples in year i. The main assumption underlying model (1) is that the measurement errors, eii, are independent and identically distributed with zero location parameter and homogeneous and constant scale parameter. An additional hypothesis is the normal or Gaussian distribution of errors. Under normality, the test to be performed is the test based on the sum of squared residuals which led to F-statistics (Scheffk, 1959). If the hypothesis of normality is rejected, a free distribution or nonparametric test as, for example the Kruskall-Wallis test, must be used (Conover, 1971). To test the hypothesis of normality, the Shapiro-Wilk statistic was used (Shapiro and Francis, 1972).
RESULTS
AND CONCLUSIONS
Tables 3-6 summarize the analytical data on the contamination of mycotoxins in Argentine wheat. Average levels of DON in positive samples in the last four years are similar to each other and lower than the levels in 1985 and 1986 (Table 3). The last years have also shown lower contamination frequencies than 1985 and 1986. It should be pointed out that, in 1985, samples were collected after the harvest and with varying storage periods.
Table 2
Rf values, detection limits and toxins recovery
Toxins
Rf benceneacetone (3:2)
Limits of detection (&kg)
Recovery (%)
Zearalenone” DON 3-Acetyl DON T-2 toxin HT-2 toxinb DAS Neosolaniol
0.80 0.22 0.54 0.52 0.19 0.49 0.26
80 30 100 500 500 500 500
50 90 90 20 25 50 80
aFor a contamination level greater than 100 &kg, the recovery percentage was 60%. bFor a contamination level greater than 1000 /.&kg, the recovery percentage was 80%.
Trichothecenes
Table 3
Contamination
by DON
in different
wheat
and zearalenone
harvests Over positive
Harvest year
Contaminated samples/total
1985 1986 1989 1990 1991 1992
881123 1791261 231102 104/159 471189 831222
Table 4 vests
Contamination
Harvest year
Contaminated samples/total
1986 1989 1990 1991 1992
141261 2/102 o/159 6/189 01222
Contamination
Harvest year
Contaminated samples/total
1986 19XY 1940 1991 lYY2
65126 1 01102 01159 13/189 01222
Contamination
hy 3-Acetyl
DON
Average
Median
Minimum
Maximum
570.9 329.5 47.5 145.8 54.7 75.3
798.0 480.5 210.8 223.0 219.X 201.3
390 400 195 200 195
260 50 100 30 54 71.5
1730 2400 400 672.5 515 50s
in different
wheat
har-
Total average
Over positive
sample
yglkg
Average
Median
40,8 15,5 ND 4,8 ND
760.4 792.5 ND 147.2 ND
547.5 792.5 ND 153.3 ND
&kg
by zearalenone in different Total average
with
wheat
harvests
&kg
Over positive samples &kg Average Median
109.0 ND ND 16,6 ND
441.1 ND ND 240.7 ND
other
Toxin
Contaminated samples/total
T-2 HT-2 Neosolaniol Diacetoxyscirpenol
20/26 1 181261 161261 26/261
&kg
/&kg
360.0 ND ND 190.0 ND
ND: not detected. Table 6 harvest
samples
Total average
ND: not detected. Table 5
in wheat: N. Quiroga et al.
trichothecenes
Total average
in 1986 wheat
pg/kg
Over positive sample &kg Average Median
12.7 21.8 10.6 78.9
165.3 316.4 172.5 792.5
too 205 190 945
The frequency of 3-Acetyl DON contamination is far less than the frequency of DON (Table 4). It must be noticed that 3-Acetyl DON was found in samples contaminated with DON. The frequency (24.9%) and average over positive samples of zearalenone (441.14 pg/kg) are high in 1986 (Table 5). The contamination values of DON in 1985 and 1986 could be due to the floods which occurred in 1985 and which probably also affected the following harvest. These events could have also influenced the contamination averages and frequencies of 3-Acetyl DON and zearalenone. The 1986 wheat harvest was also contaminated with various trichothecenes as shown in Table 6. It is important to point out that the analytical
I80
methodology was developed by Trucksess et al. (1984) only for DON and the average recoveries are low for other mycotoxins (Table 2). Deoxynivalenol was detected in all of the contaminated samples and so this mycotoxin could be an indicator of the possibly finding other trichothecenes and/or zearalenone in wheat. For these reasons we analysed the DON results in more detail. To evaluate the hypothesis that the mean contamination of positive samples in the last four years was similar, it was necessary to apply a statistical test of hypothesis. As previously stated, the parametric F-test of analysis of variance relies strongly on the assumption of normality. To test this hypothesis the Shapiro-Wilk test was performed. The plot of rankits (expected values of the order statistics if the sample is drawn from a normal population) versus the order statistics of the sample is presented in Figure 1. If the variables were normally distributed this plot should result in a straight line, except for random variation. As can be observed, this was not the case and the approximate Shapiro-Wilk statistic was 0.8965, whose P-value is less than 0.01 (Shapiro and Francis, 1972). So the hypothesis of normality was rejected and a non-parametric test of analysis of variance, the Kruskall-Wallis test, was performed. The value of the statistic was 0.3018, which led to a P-value of 0.9597. As a consequence, the hypothesis of equality of the mean annual contamination of DON over positive samples in the last four years could not be rejected. The maximum levels of contamination by DON 800
r
-3
-2
-1
0
I
2
0.8965
257 cases
3
Rankits ApproximateShapiro-Wilk
Figure
1
ShapirwWilk
Rankit
Plot of DON
Food Control 1995 Volume 6 Number 4
203
Trichothecenes and zearalenone in wheat: N. Quiroga et al.
during these last years are acceptable values for consumption and for this reason intoxication risk due to ingestion of foodstuffs manufactured with this wheat could be discarded, if the post-harvest handling was appropriate. However, the results suggest that, when meteorological conditions favour contamination (as in 1985/86) it is necessary to carry out strict controls prior to storage.
ACKNOWLEDGEMENTS The authors acknowledge the financial support from Consejo National de Investigaciones Cientificas y TICnicas, Universidad de Buenos Aires, Comisidn de Investigaciones Cientificas de la Provincia de Buenos Aires and Fundaci6n Cargill. Thanks are also extended to Merck Quimica Argentina for donating chemicals.
Pacin, A. (1988) Evaluacidn de Riesgo de Contaminacidn por Tricotecenos en la Poblacidn. Tesis de doctorado. Facultad de Medicina, Universidad de Buenos Aires Perkowsky, J., Plattner, R.D., Golinski, P., Vesonder, R.F. and Chelkowski, J. (1990) Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, 4,7 dideoxynivalenol, and zearalenone in Polish wheat. Mycotoxin Res. 6,7-12
Scott, P.M. (1983) Trichothecene Problems in Canada. VI. Toxicoses Natural Occurrence and Control. In Trichothecenes. Chemical, Biological and Toxicological Aspects. Edited by Y. Ueno. Elsevier, pp. 218-220. Shapiro, S. and Francis, R. (1972) An approximate analysis of variance test for normality. J. Amer. Stat. Assoc. 67, 215-216 Scheff6, H. (1959) The Analysis of Variance. J. Wiley, New York, USA Takitani, S. and Asabe, Y. (1983) TLC analysis of trichothecenes mycotoxins. IV Analysis. In Trichothecenes. Chemical, Biological and Toxicological Aspects. Edited by Y. Ueno. Elsevier, pp 113-120 Tanaka, T., Hasegawa, A., Matsuki, Y., Matsui, Y., Lee, U.S. and Ueno, Y. (1985) Co-contamination of the Fusarium mycotoxins, nivalenol, deoxynivalenol and zearalenone, in scabby wheat grains harvested in Hokkaido, Japan. J. Food. Hyg. Sot. Japan. 26,519-522
REFERENCES
Tanaka, T., Hasegawa, A., Matsuki, Y., Lee, U.S. and Ueno, Y. (1986) A limited survey of Fusarium mycotoxins nivalenol, deoxynivalenol and zearalenone in 1984 UK harvested wheat and barley. Food. Addit. Contam. 3, 247-252
Agnew, M.P., Poole, P.R., Lauren, D.R. and Ledgard, S.F. (1986) Presence of zearalenone and trichothecenes mycotoxins in Fusarium-infected New Zealand-grown wheat. NZ Vet. J. 34, 176-177
Tanaka, T., Hasegawa, A., Yamamoto, S., Lee, U.S., Sugiura, Y., and Ueno, Y. (1988) Worldwide contamination of cereals by the Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone. 1. Survey of 19 countries. J. Agric. Food. Chem. 36,
Apro, N., Resnik, S. and Ferro Font& C. (1987) Obtenci6n de muestras representativas para el an&lisis de micotoxinas. Anafes Asociacidn Quimica Argentina 75, 501-510 Bhat, R.V., Ramakrishna, Y., Beedu, S.R. and Munshi, K.L. (1989) Outbreak of trichothecene mycotoxiocosis associated with consumption of mould-damage wheat products in Kashmir Valley, India. The Lance& 35-37 Conover, W.J. (1971) Practical Nonparametric Statistics. Wiley, New York, USA Eppley, R.M., Trucksess, M.W., Nesheim, S., Thorpe, C.W., and Pohland, A.E. (1986) Thin-layer chromatographic method for determination of deoxynivalenol in wheat: collaborative study. J. Assoc. Off. Anal. Chem. 69,37-40
Kuiper-Goodman, T., Scott, P.M. and Watanabe, H. (1987) Risk assessment of the mycotoxin zearalenone. Reg. Toxicol. Pharm. 7,253-306 Lee, U.S., Jang, H.S., Tanaka, T., Hasegawa, A., Oh, Y.J., Cho,
C.M., Sugiura, Y. and Ueno, Y. (1986) Further survey on the Fusarium mycotoxins in Korean cereals. Food. Addit. Contam. 3, 253-261
204
Food Control 1995 Volume 6 Number 4
979-983
Tanaka, T., Yamamoto, S., Hasegawa, A., Aoki, N., Besling, J.R., Sugiura, Y. and Ueno, T. (1990) A survey of the natural occurrence of Fusarium mycotoxins deoxynivalenol, nivalenol, and zearalenone in cereals harvested in the Netherlands. Mycopathologia 110, 19-22 Trenholm, H.L., Cochrane, W.P., Cohen, H., Elliot, J.I., Farnworth, E.R., Friend, D.W., Hamilton, R.M.G., Neish, G.A. and Standish, J.F. (1981) Survey of vomitoxin contamination of the 1980 white winter wheat crop in Ontario, Canada. JAOCS 992-994
Trucksess, M.W., Nesheim, S. and Eppley, R.M. (1984) Thin layer chromatrographic method for deoxynivalenol (DON) vomitoxin in wheat and corn. J. Assoc. Off. Anal. Chem. 67, 40-43 &no, Y. (1983) General Toxicology. V. Toxicology. In Trichothecenes. Chemical, Biological and Toxicological Aspects. edited by
Y. Ueno. Elsevier, Amsterdam, pp. 135-146 Wood, G.E. and Carter, L. (1989) Limited survey of deoxynivalenol in wheat and corn in the United States. J. Assoc. Off. Anal. Chem. 72, 3S40.
095%7135(95)00021-6
PAPER
Natural occurrence of trichothecenes and zearalenone in Argentine wheat Nora Quiroga, *? Silvia Resnik to Ana Pacin ,*** Elena Martinez,§ Ana Pagano,? Isabel Riccobene? and Susana NeiraS* * A survey of the natural occurrence of trichothecenes and zearalenone in samples from Buenos Aires and Santa Fe provinces, Argentina, was carried out in six different crops during harvest. Among 1056 samples analysed, 524 (49.62%) were contaminated with trichothecenes and zearalenone. Deoxynivalenol was detected in all of the contaminated samples, 3-Acetyl DON in 22 samples and zearalenone in 78. In 1986, T-2 toxin (20 samples), HT-2 toxin (18), neosolaniol (16) and diacetoxyscirpenol(26) were also found. The results suggest that when meteorological conditions _favour contamination it is necessary to carry out strict controls on harvest time. Keywords: trichothecenes,
zearalenone, natural occurrence, wheat
INTRODUCTION Wheat is the most important cereal in Argentina both from the point of view of local consumption and its high earning power as an export commodity. The infestation of wheat with the Fusarium species and its consequent contamination with trichothecenes and zearalenone have been amply studied in many countries. Accumulated data have revealed the natural occurrence of trichothecenes in, among others, Canada (Trenholm et al., 1981; Scott, 1983), Japan (Tanaka et al., 1985), USA (Wood and Carter, 1989), Korea (Lee et al., 1986), Poland (Perkowski et al., 1990), The Netherlands (Tanaka et al., 1990), UK (Tanaka et al., 1986), and New Zealand (Agnew et al., 1986). Examination of *Municipalidad de Olavarria. ‘Universidad Nacionat de1 Centro. *Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires. %Jniversidad de Buenos Aires. **Universidad National de Lujhn. Address for correspondence: S. Resnik, Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, 1428 Buenos Aires, Argentina. Received 9 December 1994; revised 14 April 1995; accepted 16 May 199.5.
these surveys reveals a significant variation in the occurrence of trichothecenes within regions. Adverse climatic conditions at critical periods in the growth cycle have been reported to produce a favourable environment for the growth of Fusarium spp. capable of producing these mycotoxins. Trichothecenes are able to cause, both in man and animals (Pacin, 1988; Bhat et al., 1989), immunosupression, haematological changes of varying degree and digestive disorders (Ueno, 1983). Zearalenone can produce estrogenic syndromes of different degrees (Kuiper-Goodman et al., 1987). To evaluate the occurrence of contamination by trichothecenes and zearalenone in wheat, we analysed samples of wheat harvested in the main production area in Argentina over a six year period.
MATERIALS AND METHODS Samples Samples of different
crops were collected during the
Food Control 1995 Volume 6 Number 4
201
Trichothecenes and zearalenone in wheat: N. Quiroga et a/.
Table 1 Number of samples analysed each year Year
Number of samples
1985” 1986 1989 1990 1991 1992
123 261 102 159 189 222
‘These samples were collected after the 1985 harvest from different storages in only one area of Buenos Aires province.
harvest from farm areas in Buenos Aires province and south of Santa Fe. The number of samples was proportional to the total production of each area (Table I). Sampling was carried out following the procedure described by Apro et al. (1987). During harvest, as the grain flowed into the trucks, portions of about 50 g were taken at regular time intervals. All the portions corresponding of the same batch were mixed and quartered to give primary samples of 10 kg. These primary samples were homogenized and quartered to give 3 kg samples. These were then ground to pass through a 14 mesh sieve and quartered to give subsamples of about 1 kg which were ground to pass a 20 mesh sieve. From each subsample, approximately 200 g was taken and stored at -20°C prior to analysis. Analytical methodology The method used to quantify DON involves sample extraction with acetonitrile-water (84+16) cleanup using a disposable column of charcoal, celite and alumina, and detection by thin-layer chromatography after spraying with an aluminium chloride solution (Eppley et al., 1986; Trucksess et al., 1984). In order to select the acetonitrile to be used for the extraction, two acetonitriles were evaluated: Merck No 30 and Merck No.800015. DON was added to a ground sample of wheat to obtain five levels of concentration (50, 70, 100, 150 and 300 &kg). Quadruplicated analyses were made for each level, using 40 ml of the sample extract. Both acetonitriles had the same average recovery but Merck No 30 gave sharper TLC spots allowing a better quantification of DON. The Trucksess et al. method (1984) was also employed for the analysis of 3-Acetyl DON, T2-toxin, HT-2, DAS, neosolaniol and zearalenone. Two TLC plates were used for each sample, one for Group B trichothecenes (DON and 3-Acetyl DON) and zearalenone and the other for Group A toxins. The plates (Merck No 5553) were developed with benzene:acetone (3:2). Zearalenone and trichothecenes of group B exhibit blue fluorescence at 364 nm when the plate was treated with 15% aluminium chloride solution and heated for 6 min at 120°C. Trichothecenes of group A exhibit blue fluorescence at the same UV wave when the plate was sprayed with a sulphuric acid solution (20%) followed by heating at 130°C for 10 min (Takitani and Asabe,
202
Food Control 1995 Volume 6 Number 4
1983). Rf values, detection limits and approximate recovery percentages are presented in Table 2. Toxin confirmation was made by TLC using other solvent systems. In 1990, 1991 and 1992, duplicated analyses were made using two subsamples of 50 g, while in the other years only one subsample of 100 g was analysed. Statistical methodology To assess the equality of mean annual contamination of DON over positive samples, a one-way analysis of variance (ANOVA) was performed. The model is of the form:
where Z is the number of years compared and IZ~is the number of positive samples in year i. The main assumption underlying model (1) is that the measurement errors, eii, are independent and identically distributed with zero location parameter and homogeneous and constant scale parameter. An additional hypothesis is the normal or Gaussian distribution of errors. Under normality, the test to be performed is the test based on the sum of squared residuals which led to F-statistics (Scheffk, 1959). If the hypothesis of normality is rejected, a free distribution or nonparametric test as, for example the Kruskall-Wallis test, must be used (Conover, 1971). To test the hypothesis of normality, the Shapiro-Wilk statistic was used (Shapiro and Francis, 1972).
RESULTS
AND CONCLUSIONS
Tables 3-6 summarize the analytical data on the contamination of mycotoxins in Argentine wheat. Average levels of DON in positive samples in the last four years are similar to each other and lower than the levels in 1985 and 1986 (Table 3). The last years have also shown lower contamination frequencies than 1985 and 1986. It should be pointed out that, in 1985, samples were collected after the harvest and with varying storage periods.
Table 2
Rf values, detection limits and toxins recovery
Toxins
Rf benceneacetone (3:2)
Limits of detection (&kg)
Recovery (%)
Zearalenone” DON 3-Acetyl DON T-2 toxin HT-2 toxinb DAS Neosolaniol
0.80 0.22 0.54 0.52 0.19 0.49 0.26
80 30 100 500 500 500 500
50 90 90 20 25 50 80
aFor a contamination level greater than 100 &kg, the recovery percentage was 60%. bFor a contamination level greater than 1000 /.&kg, the recovery percentage was 80%.
Trichothecenes
Table 3
Contamination
by DON
in different
wheat
and zearalenone
harvests Over positive
Harvest year
Contaminated samples/total
1985 1986 1989 1990 1991 1992
881123 1791261 231102 104/159 471189 831222
Table 4 vests
Contamination
Harvest year
Contaminated samples/total
1986 1989 1990 1991 1992
141261 2/102 o/159 6/189 01222
Contamination
Harvest year
Contaminated samples/total
1986 19XY 1940 1991 lYY2
65126 1 01102 01159 13/189 01222
Contamination
hy 3-Acetyl
DON
Average
Median
Minimum
Maximum
570.9 329.5 47.5 145.8 54.7 75.3
798.0 480.5 210.8 223.0 219.X 201.3
390 400 195 200 195
260 50 100 30 54 71.5
1730 2400 400 672.5 515 50s
in different
wheat
har-
Total average
Over positive
sample
yglkg
Average
Median
40,8 15,5 ND 4,8 ND
760.4 792.5 ND 147.2 ND
547.5 792.5 ND 153.3 ND
&kg
by zearalenone in different Total average
with
wheat
harvests
&kg
Over positive samples &kg Average Median
109.0 ND ND 16,6 ND
441.1 ND ND 240.7 ND
other
Toxin
Contaminated samples/total
T-2 HT-2 Neosolaniol Diacetoxyscirpenol
20/26 1 181261 161261 26/261
&kg
/&kg
360.0 ND ND 190.0 ND
ND: not detected. Table 6 harvest
samples
Total average
ND: not detected. Table 5
in wheat: N. Quiroga et al.
trichothecenes
Total average
in 1986 wheat
pg/kg
Over positive sample &kg Average Median
12.7 21.8 10.6 78.9
165.3 316.4 172.5 792.5
too 205 190 945
The frequency of 3-Acetyl DON contamination is far less than the frequency of DON (Table 4). It must be noticed that 3-Acetyl DON was found in samples contaminated with DON. The frequency (24.9%) and average over positive samples of zearalenone (441.14 pg/kg) are high in 1986 (Table 5). The contamination values of DON in 1985 and 1986 could be due to the floods which occurred in 1985 and which probably also affected the following harvest. These events could have also influenced the contamination averages and frequencies of 3-Acetyl DON and zearalenone. The 1986 wheat harvest was also contaminated with various trichothecenes as shown in Table 6. It is important to point out that the analytical
I80
methodology was developed by Trucksess et al. (1984) only for DON and the average recoveries are low for other mycotoxins (Table 2). Deoxynivalenol was detected in all of the contaminated samples and so this mycotoxin could be an indicator of the possibly finding other trichothecenes and/or zearalenone in wheat. For these reasons we analysed the DON results in more detail. To evaluate the hypothesis that the mean contamination of positive samples in the last four years was similar, it was necessary to apply a statistical test of hypothesis. As previously stated, the parametric F-test of analysis of variance relies strongly on the assumption of normality. To test this hypothesis the Shapiro-Wilk test was performed. The plot of rankits (expected values of the order statistics if the sample is drawn from a normal population) versus the order statistics of the sample is presented in Figure 1. If the variables were normally distributed this plot should result in a straight line, except for random variation. As can be observed, this was not the case and the approximate Shapiro-Wilk statistic was 0.8965, whose P-value is less than 0.01 (Shapiro and Francis, 1972). So the hypothesis of normality was rejected and a non-parametric test of analysis of variance, the Kruskall-Wallis test, was performed. The value of the statistic was 0.3018, which led to a P-value of 0.9597. As a consequence, the hypothesis of equality of the mean annual contamination of DON over positive samples in the last four years could not be rejected. The maximum levels of contamination by DON 800
r
-3
-2
-1
0
I
2
0.8965
257 cases
3
Rankits ApproximateShapiro-Wilk
Figure
1
ShapirwWilk
Rankit
Plot of DON
Food Control 1995 Volume 6 Number 4
203
Trichothecenes and zearalenone in wheat: N. Quiroga et al.
during these last years are acceptable values for consumption and for this reason intoxication risk due to ingestion of foodstuffs manufactured with this wheat could be discarded, if the post-harvest handling was appropriate. However, the results suggest that, when meteorological conditions favour contamination (as in 1985/86) it is necessary to carry out strict controls prior to storage.
ACKNOWLEDGEMENTS The authors acknowledge the financial support from Consejo National de Investigaciones Cientificas y TICnicas, Universidad de Buenos Aires, Comisidn de Investigaciones Cientificas de la Provincia de Buenos Aires and Fundaci6n Cargill. Thanks are also extended to Merck Quimica Argentina for donating chemicals.
Pacin, A. (1988) Evaluacidn de Riesgo de Contaminacidn por Tricotecenos en la Poblacidn. Tesis de doctorado. Facultad de Medicina, Universidad de Buenos Aires Perkowsky, J., Plattner, R.D., Golinski, P., Vesonder, R.F. and Chelkowski, J. (1990) Natural occurrence of deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, 4,7 dideoxynivalenol, and zearalenone in Polish wheat. Mycotoxin Res. 6,7-12
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