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Monitoring and identification of fungal toxins in food products, animal feed and cereals in Tunisia
J. stored Prod. Res. Vol. 24, No. 4, pp. 199-206, Printed in Great Britain. All rights reserved
1988
0022-474X/88$3.00+ 0.00 Copyright 0 1988Pergamon Press plc
MONITORING AND IDENTIFICATION OF FUNGAL TOXINS IN FOOD PRODUCTS, ANIMAL FEED AND CEREALS IN TUNISIA H. BACHA,’ R. HADIDANE,’ E. E. CREPPY,~ C. REGNAULT,’ F. ELLOUZE’ and G. DIRHEIMER’.* ‘Laboratoire de Biochimie, Facultt de Pharmacie et de M&de&e Dentaire, Monastir, Tunisie and ‘Laboratoire de Toxicologic et de Biologie Moleculaires, Institut de Biologie Moleculaire et Cellulaire du C.N.R.S., Universite Louis Pasteur, 15, rue Descartes. 67084 Strasbourg, France (Received 28
July
1988)
Abstract-The occurrence in Tunisia of several mycotoxin contaminations (aflatoxins (B, , B,, G, , G,), ochratoxin A, citrinin and sterigmatocystin) in cereals, food products and animals feed is reported. There appears to be a connection between climatic factors, local customs, storage conditions and mycotoxin contamination. Ochratoxin A appeared to be absent from local cereals, and was detected only in imported cereals and in “Premix” (a component of animal feeds). Citrinin was only encountered in samples collected in dirty locations where hygiene prescriptions were not observed.
INTRODUCTION
Mycotoxicoses are becoming increasingly implicated in human and animal pathology, both in industrial and developing countries (Senti, 1977). In the latter, malnutrition may be an aggravating factor for mycotoxigenic effect (Frayssinet and Lafont, 1976). The proliferation of toxigenic fungal species depends on several factors: temperature (Moreau, 1974; Le Bars, 1976) humidity (Golumbic and Kulik, 1969) and oxygen vs carbon dioxide ratio (Landers et al., l967). In addition to these climatic factors, particular social conditions and behaviour, such as methods of preservation of food products, traditional feeding, and environmental pollution, may play a signifiant role. In all of these respects, Tunisia was considered to provide a favoura.ble situation for toxigenic mould proliferation. Previous reports have been made of mycotoxin contamination in food and feeds in Tunisia (Boutrif et al., 1977a, b). In the 1970’s, many horses, cattle and poultry died in Tunisia. The causes of these deaths conld be related to those of similar deaths of animals in Denmark, Yugoslavia and Poland in which mycotoxins were implicated (Krogh et al., 1977; Pepeljnjak et al., 1982). The aim of this istudy was to verify whether cereals, which are the principal component of animal and human food in Tunisia, contain mycotoxins; local people usually consume semolina, flour and noodles obtained from durum wheat and tender wheat, and cattle are fed on industrial food containing oats, maize, wheat bran, soybean oil cake and “Premix” (vitamins + trace elements + fishmeal). The present paper reports the results of the analysis of samples taken from the main points of production, storage and distribution of cereals all over the country and also from samples of animal feeds. MATERIALS
Cereal samples ‘were withdrawn from vertical cells in silos and from warehouses in the northern part of the country, the only cereal producing region, and from centres receiving imported cereals. The components of animal feeds were taken from factory stocks. Mycotoxin standards (aflatoxins (B,, B,, G, , G2), ochratoxin A, citrinin, patulin, sterigmatocystin) were obtained from Sigma Chemical Co. (St Louis, MO., U.S.A.) and thin layer chromatography plates (Silicagel 60) from Merck A.G. (Darmstadt, F.R.G.). Other chemicals or reagents were obtained from Sigma Co. or Merck A.G. and were of the purest grade available. *Author for correspondence. 199
H. BACHA et al.
200
METHODS Sampling Sampling was undertaken under conditions which cannot be directly related to USDA or TDRI sampling plans, with regard to the variable characteristics of storage of cereals. In the case of cereals stored in horizontal bulk, which was the most usual method, samples were taken from three different points, at the surface, at the side and at the low front of the bulk, the different samples from each stock being mixed to obtain the final sample used for analysis. For cereals stored in closed containers, separate samples from the top and bottom of the container were mixed to obtain the final sample. In the case of cereals stored in sacks, the sample for analysis was a mixture of two samples from two different sacks, one sample being taken from the upper part and another from the lower part of the stack. Analyses were made mainly in respect of large stocks of cereals, for distribution all over the country, and imported cereals. Twelve storage locations were visited, with capacities ranging from 13,000 to 30,000 tons. About 50 samples were collected. Observations were made regarding the particular maintenance, preservation and storage conditions (Table 2). Samples were analysed to detect aflatoxins (B, , B,, G, , G2), ochratoxin A, citrinin, patulin and sterigmatocystin. Extraction, purification and separation techniques were adapted from Patterson and Roberts (1979) and modified as described in Fig. 1. Extraction and purijication Samples of 100 g were pulverized and stirred with 250 ml of chloroform and 25 ml of 0.1 M phosphoric acid for 30 min. The mixture was decanted and the solution filtered and the solid then washed three times with chloroform and 0.1 M phosphoric acid (10 : 1, v/v); the washes were pooled with the first extract and chloroform then evaporated. The remaining acqueous phase was defatted three times with n-hexane. The defatted aqueous solution was made alkaline with 25 ml 0.1 N NaHCO, and extracted three times with chloroform. Two phases were obtained (I and II). The alkaline aqueous solution (I) was brought to pH 3 with concentrated HCl and the latter used for monitoring the presence of ochratoxins and citrinin by chloroform extraction. 100 g pulverized l l l
Discard residue
Extraction -/
sample extraction with decantation mixture filtration
250 ml CHCI, 25ml H,PO, 0.1 M
Filtrate l l
evaporation of CHCI, defatting with n-hexane
Discard hexane 1 Aqueous
phase . alkalinizations with 25 ml 0.1 N NaHCO, l extraction with chloroform
1 Aqueous
l
Purification
l
d
1 Chloroformic
I
phase
I Chloroformic
II
evaporation of CHCI, . dissolution in 1 ml acetonitrile
l
I Acetonitrile
phase l
phase
acidification with HCI extraction with CHCI,
reduction of the volume to 0.1 ml
phase . dialysis against acetone-water (3:7 v/v)
I Acetone-water
phase extraction with CHCI, l reduction of the volume to 0.1 ml l
I Final extract
Separation i
I
i i::r.::in:
Thin layerchromatography.
Fig. 1. Extraction. purification and separation method for mycotoxin
Fina’ ~~“c0t-W
analysis
of contaminated
cereals.
Fungal Table
toxins in Tunisia
201
1.Minimum detection levels and extraction yields of mycotoxins from contaminated and mycotoxin supplemented samples of cereals
Mycotoxins Aflatoxin B, Allatoxin B, Aflatoxin G, Aflatoxin G, Ochratoxin A Citrinin Patulin Steriematocvstin
Minimum detection level: pg on TLC
Yields (%)
&kg (ppb)
0.3 0.37 0.3 0.35 6.4 10.5 10.8 I8
28 26 25 25 22 28 23 -
I .07 1.42 I.20 I .40 30 38.57 46.95 -
The chloroformic solution (II) was evaporated and the residue dissolved in 1 ml of acetonitrile and dialysed aga:inst 100 ml of an acetone-water solution (30:70, v/v). Eventually mycotoxins dialyse out. The acetone-water solution was then extracted three times with chloroform and later used for monitoring most mycotoxins. Separation This was performed by unidimensional ascending thin-layer chromatography on Silicagel plates. The solvent systems used were those described by Patterson and Roberts (1979) for ochratoxins and aflatoxins, Stubblefield (1979) for citrinin, and Egon and Miiller (1977) for sterigmatocystin. The samples containing aflatoxins, after chromatography in a first direction using chloroform-acetone (9 : 1, v/v), were submitted to a second dimensional chromatography using toluene-ethylacetate-formic acid (6: 3 : 1, v/v/v). Identification This was performed by U.V.light illumination at 254 and 366 nm, by comparison of the migration of the samples with mycotoxin standards. Identification was achieved by the use of two chemical treatments which modify specifically the fluorescence of different samples: spraying of the plates with a sulphuric acid-water solution (1: 1, v/v) or a p-anisaldehyde-acetic acid-sulphuric acid solution (5:90:5, sv/v/v) (Schuller et al., 1967; Fremy and Corbion, 1979). Yields of mycotoxin extractions Aliquots (0.1-30 pg) of the various mycotoxins (aflatoxins B, , B,, G, , G,; ochratoxin A; patulin; citrinin; sterigmatocystin) were added to non-contaminated wheat. Controls were processed as already described. The lower detection levels were defined as the minimal amounts detectable under U.V.light after thin-layer chromatography (Table 1). The detected spots of mycotoxins were eluted for the Silicagel with methanol and the concentrations determined spectrophotometrically according to the molar extinction coefficifznt (aflatoxin B, : E = 21,800 at 360 nm; aflatoxin B,:c = 24,000 at 362 nm; aflatoxin G, : 6 = 17,700 at 362 nm; aflatoxin G,: 6 = 19,300 at 362 nm; ochratoxin A: 6 = 5,550 at 333 nm; citrinin: 6 = 16,100 at 332 nm and sterigmatocystin: 6 = 15,200 at 325 nm). The extraction yields were determined by comparing the amounts of mycotoxins extracted with the added one (Table 1). The extraction yield of ochratoxin A was also assessed using [‘Hlochratoxin A (C.E.A., Saclay, France). About 1 nmol of [3H]ochratoxin A (specific activity: 800 cpm/pmol) was added to the sample, radioactivity countings being performed at each step of purification. The yield of the purification was determined by the radioactivity after purification vs the radioactivity before purification. RESULTS
The minimum detection levels for mycotoxins (aflatoxins B, , B,, G, , G2), ochratoxin A, citrinin, patulin and sterigmatocystin are given in Table 1. We were able to detect approximately l-l .4; 30 and 47 pgg/kg of allatoxins, ochratoxin A and sterigmatocystin respectively. Several mycotoxins were detected in the cereal samples analysed (Table 2). Only ochratoxin A was determined quantitatively, while the fluorescence intensities of all other spots were estimated with ( + ) symbols.
202
H. BACHA et al.
+ +i”
+ +
-I-
+
+
+
+
(83) (83)
El Aroussa Bou Arada
idem idem Storage in bulk? Damp and dirty environment Insect infestation idem idem idem i&m Stnraee in hIIlk? Bad maintenance Damp and dirty environment idem Mixture of several harvests Storage in open and humid tanks* Storage in open and humid tanks* and outside in sacks Clean environment Mixture of several harvests Storage in open and humid tanks* and outside idem Storage in bulk? Damp and very dirty environment Storage in bulk? Bad maintenance Very dirty environment Storage m bulkf m closed store
‘Tank: Cereals stored in large metallic containers within a store. tBulk: Cereals stored in bulk: conventional warehouse.
El Fahs (83)
(83)
El Aroussa
TW,
DW,
(83)
El Aroussa
DW,.
El Fahs (83)
Jendouba (83) El Aroussa (83)
TW, DW,
DW,
Bou Salem (83) Bou Salem (83) Boo Salem (83) Bou Salem (83) Jendouba (83)
DW, TW TW, TW5DW,
%ja (83) %ja (83) Bou Salem (83)
+i +
+ +
+
+ + c
+
++
+ ++
+
+ +
++
I
+ ++ ++ +
+
+++
I
++ +++ +++ ++
+
++ ++
+ ++ + ++
+++
(+) +
A+ + +
+
ti+
+
++
Aliment no. 7
no. 2 Aliment no. 4 Aliment no. 5
AIiment
for chicken
Local oats Local soybean imported maize Imported “Premix”
Samples
Megrine Djebel Jelloud Mateur B&ja Bon Salem Jendouba El Aroussa Boll Arada Ef Fahs
Locations
l/i
f 8,Dou
Fish meal Trace &merits Vitamins Oats, salts Wheat bran, oats Oats, corn, wheat bran, Soybean cake, salts Oats. wheat bran.
Composition
Table 4. Occurred
--
X2 315
f/l
I/E
515
IIS
ARa 0,
j/f
515 2/z 315
l/2 l/4
Affa G,
J/j
r/s ti’2 IiS
214 vs
OTA
VI
l/f
Citrinin
Mycotoxins detected; frequency (a/b) ..-. l.ll~ ~ _--. .l_ll_
212 5i5
w
212 114 115
Afla B,
-
.
-..._.
Atla B,
_ Afla B,
-_
Afla G,
OTA
~~
~
Citrinin
--
112 114
_
_-_” Slerigmatocystin
..__-l^l__.
Sterigmatocystin
Mycotoxins detected (level) ~~~ ----. .-I- _I___
Afla G,
^-~~
of mvcotoxins in animal feed oroducts and comoosite animal feeds
212 51s
1wXKl
w
f6,ooo
112 l/4 w l/3
ABa B,
18,ooo t7*000
20,GQo 25,000 14,500 13,ooo
Capacity (tons)
^.. .-.--
Table 3. Occurrence and frequency of mycoiaxins in different storage locations
Fungal
toxins
in Tunisia
205
The following mycotoxins were detected: aflatoxin B, and G, respectively at very high levels in 55 and 41% of the samples analysed, aflatoxin B, and G, respectively in 55 and 52% of analysed samples, at lower levels than aflatoxins B, and G, , ochratoxin A and citrinin in 7% of the analysed samples at variable levels (34-360 pg/kg, especially for ochratoxin A) and trace amounts of sterigmatocystin in 7% of samples analysed (Table 3). The analysis of different samples of animals feeds (nos l-5 in Table 4) and their main components (maize, oats, soybean oil cake, wheat bran and “Premix”) gave the following results: very high levels of ochratoxin A contamination were found (maize-320 ,ugg/kg, “Premix”-360 pgg/kg and feed no. 5-140 ,ug/kg usually associated with lesser degrees of aflatoxin B2 contamination (Table 4). DISCUSSION
Aflatoxins B,, B,, G, and G,, all produced by AspergiZZus~uvus Link, are normally detected together in the same positive samples of cereals or animal feeds, but this was not verified in all cases, because the physiologial conditions of the moulds and specificity of the substrate can favour one or other forms of aflatoxin, e.g. B, or G, (Hesseltine, 1976). The lack of B, and (or) G, in some samples containing B, and (or) G2 (which are respectively metabolites of El, and G,) could be explained by the almost total conversion of B, and G, respectively into B, and G, (Diener and Davis, 1969). The minimum detection level of aflatoxins in our test was about 0.14 pg/kg. The amounts of mycotoxins detected are probably underestimates because of the low recoveries, and the fact that the samples analysed were not collected in a statistically representative manner due to accessibil:ity to the bulk lots, and the problem of minimum detection levels. Indeed, the minimum amounts of mycotoxins we were able to detect are higher than those reported by Patterson and Roberts (1979) so that the traces we observed could even mean a higher level of contamination. Mycotoxins were found to be contaminating cereals in Tunisia. All these cereals come from the northern part of the country, a region with a high humidity level (125 rain days/year) and temperatures ranging from 6 to 45°C with an average of about 25-30°C. These climatic factors potentiate hazards associated with the specific storage and preservation conditions of the cereals. Thus, aflatoxins I),, B,, G, and G2 were predominantly detected in places where humidity was high, and citrinin was encountered only in connection with hygiene problems in dirty locations. On the contrary, in the case of cereals stored under good conditions and protected from humidity, no mycotoxin contamination occurred (samples DW,, TW, , Table 2). Ochratoxin A appeared to be absent from the local cereal production. It was found only in imported cereals, and essentially in wheat (180 pg/kg) and maize (320 pgg/kg). The latter constitutes the major constituent of composite animal feeds and could represent the origin of the heavy contamination detected in the composite animal feed no. 5 (Table 4). It is well establ.ished that mycotoxins in cereals, food and feeds cause damage in humans and animals (Elling, 1977; Richard et al., 1975; Wyllie and Moorehouse, 1978; Galtier et al., 1974). A positive correlation between the presence of mycotoxins in food products with several diseases observed in the s,ame area has been established (Hadidane et al., 1985). In view of the importance of mycotoxins to the economy and public health, it is considered that systematic controls of both local and imported cereals, food and feed products should be established in Tunisia and other developing countries. Acknowledgemenrs-This work was supported by grants from the “Ministere Tunisien de 1’Enseignement Superieur et de National de la Sante et de la Recherche la Recherche Scientilique” (Contrat no. PC 5/83/FPMO), the “Institut Medicale”-INSERM (Contrat CRL 85: 2009) and the “Centre National de la Recherche Scientifique” (Contrat PIREN, ATP Alimentation-Nutrition no. 82). H. Bacha and R. Hadidane acknowledge the short-term fellowships given by the Direction des Relations et de la Cooperation Internati’onale du CNRS. REFERENCES Boutrif E., Jemmali M., Pohland Chem. 60, 747-748.
A. E. and Campbell
A. D. (1977a)
Aflatoxins
in Aleppo
pine nuts. J. Ass. ofl analyf.
H. BACHA et at.
206
Boutrif E., Jemmali M., Campbell A. D. and Pohland A. E. (1977b) Aflatoxin in Tunisian foods and foodstuffs. Annis Nutr. Aliment. 31, 431-434.
Diener U. L. and Davis N. D. (1969) Atlatoxin formation by As~rg~iius~uv~. In A~atoxin (Edited by L. A. Goldblatt), pp. 13-54. Academic Press, New York. Egon B. G. J. and Mbller T. E. (1977) Screening method for the detection of aflatoxins, ochratoxin, patulin, sterigmatocystin and zearalenone in cereals. J. Ass. 05 analyr. Chem. 60, 1369-1371 I Elling F. (1977) Morphological aspects of mycotoxic nephropathy. In Mycotoxins in Human and Animal Health (Edited by J. V. Rodricks, C. W. Hesseltine and M. A. Mehlman), pp. 4999506. Pathotox Publishers, Park Forest South, Illinois. Frayssinet C. and Lafont P. (1976) Les Mycotoxines: Problemes d’actualiti. Cah. Nutr. Diet. (Mycotoxines), 2, 7-12. Fremy J. M. and Corbion 8. (1979) Analyse mycotoxicologique des aliments. Arms Fatsif: Expert Chim. 72, 53-64. Galtier P., More J. and Bodin G. (1974) Toxines d’Aspergillus ochraceus W. III. Toxicid aigue de l’ochratoxine A chez le rat et la souris adultes. Ann/s Rech. Vet. 5, 233-247. Golumbic C. and Kufik M. M, (1969) Fungal spoilage in stored crops and its control. In AJIaruxin (Edited by L. A. Goldblatt), pp. 307-332. Academic Press, New York. Hadidane R., Regnault C., Bouattour H., Ellouze F., Bacha H., Creppy E. E. and Dirheimer G. (1985) Correlation between alimentary mycotoxin contamination and specific diseases. Human Toxicol. 4, 491-501. Hesseltine C. (1976) Mycotoxins other than aflatoxins. Proc. Int. Biodegradation Symp. (Edited by J. Sharpley and A. Kaplan) pp. 607-623. Applied Science Publishers Kingston, London. Krogh P., Hald B., Pldstina R. and Ceovic S. (1977) Balkan (endemic) Nephropathy and foodborn ochratoxin A: Preliminary results of a survey of foodstuffs, Acta path. microbial. stand. R 85, 238-240. Landers K. E., Davis N. D. and Diener U. L. (1967) Influence of atmospheric gas on aflatoxin production by Aspergillus flavus in peanuts. Phytopathology 57, 1086-1090. Le Bars J. (1976) Ecologic des Moisissures Toxinogenes. Cab. Nutr. Diet. (~ycotoxines), 2, 23-27. Moreau C. (1974) In Mo~is~res Toxiques dans I’Alimentation, p. 471. Masson et Cie, Paris. Patterson D. S. P. and Roberts B. A. (1979) Mycotoxins in animal feedstuffs: sensitive thin-layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone and T-2 toxin, J. Ass. ofl analyt. Chem. 62, 1265-1267. Pepeljnjak S., Blaievic N. and Culjak K. (1982) Contamination with moulds and occurrence of ochratoxin A in smoked meat products from endemic nephropathy region of Yugoslavia. In Mycotoxins and Phycotoxins, Vth Int. IUPAC Symp. Mycotoxins and Phycotoxins, Vienna, pp. 102-105. Technical University, Vienna. Richard J. L., Thurston J. R., Deyoes B. L. and Booth G. D. (1975) Effects of ochratoxin A and aflatoxin on serum proteins, complement activity and antibody production to Brucella abortus in Guinea pigs. Appt. Microbial. 29, 27-30. Schuller P. L., Gchlunizen ‘I., Werringloer J. and Marquardt M. (1967) Aflatoxin B, und Histamin in Wein. Arzneim. Forsch. 17, 888-890.
Senti F. R. (1977) Conference on Mycotoxin in Human and Animal Health. In Mycoroxjns in Humun and Animal Heu~th (Edited by J. V. Rodricks, C. W. Hesseltine and M. A. Mehlman), pp. 779-789. Pathotox Publishers, Park Forest South, Illinois. Stubblefield R. D. (1979) Thin-layer chromatographic determination of citrinin. J. Ass. ofi analyt. Chem. 62, 201-202. Wyllie T. D. and Moorehouse L. G. (1978) Mycotoxic fungi, Mycotoxins, Mycotoxicoses. In Encyclopedic Handbook, Mycotoxicoses of Domestic and Laboratory Animals, Poultry and Aquatic Invertebrates, Vol. 2. Marcel Dekker, New York.
1988
0022-474X/88$3.00+ 0.00 Copyright 0 1988Pergamon Press plc
MONITORING AND IDENTIFICATION OF FUNGAL TOXINS IN FOOD PRODUCTS, ANIMAL FEED AND CEREALS IN TUNISIA H. BACHA,’ R. HADIDANE,’ E. E. CREPPY,~ C. REGNAULT,’ F. ELLOUZE’ and G. DIRHEIMER’.* ‘Laboratoire de Biochimie, Facultt de Pharmacie et de M&de&e Dentaire, Monastir, Tunisie and ‘Laboratoire de Toxicologic et de Biologie Moleculaires, Institut de Biologie Moleculaire et Cellulaire du C.N.R.S., Universite Louis Pasteur, 15, rue Descartes. 67084 Strasbourg, France (Received 28
July
1988)
Abstract-The occurrence in Tunisia of several mycotoxin contaminations (aflatoxins (B, , B,, G, , G,), ochratoxin A, citrinin and sterigmatocystin) in cereals, food products and animals feed is reported. There appears to be a connection between climatic factors, local customs, storage conditions and mycotoxin contamination. Ochratoxin A appeared to be absent from local cereals, and was detected only in imported cereals and in “Premix” (a component of animal feeds). Citrinin was only encountered in samples collected in dirty locations where hygiene prescriptions were not observed.
INTRODUCTION
Mycotoxicoses are becoming increasingly implicated in human and animal pathology, both in industrial and developing countries (Senti, 1977). In the latter, malnutrition may be an aggravating factor for mycotoxigenic effect (Frayssinet and Lafont, 1976). The proliferation of toxigenic fungal species depends on several factors: temperature (Moreau, 1974; Le Bars, 1976) humidity (Golumbic and Kulik, 1969) and oxygen vs carbon dioxide ratio (Landers et al., l967). In addition to these climatic factors, particular social conditions and behaviour, such as methods of preservation of food products, traditional feeding, and environmental pollution, may play a signifiant role. In all of these respects, Tunisia was considered to provide a favoura.ble situation for toxigenic mould proliferation. Previous reports have been made of mycotoxin contamination in food and feeds in Tunisia (Boutrif et al., 1977a, b). In the 1970’s, many horses, cattle and poultry died in Tunisia. The causes of these deaths conld be related to those of similar deaths of animals in Denmark, Yugoslavia and Poland in which mycotoxins were implicated (Krogh et al., 1977; Pepeljnjak et al., 1982). The aim of this istudy was to verify whether cereals, which are the principal component of animal and human food in Tunisia, contain mycotoxins; local people usually consume semolina, flour and noodles obtained from durum wheat and tender wheat, and cattle are fed on industrial food containing oats, maize, wheat bran, soybean oil cake and “Premix” (vitamins + trace elements + fishmeal). The present paper reports the results of the analysis of samples taken from the main points of production, storage and distribution of cereals all over the country and also from samples of animal feeds. MATERIALS
Cereal samples ‘were withdrawn from vertical cells in silos and from warehouses in the northern part of the country, the only cereal producing region, and from centres receiving imported cereals. The components of animal feeds were taken from factory stocks. Mycotoxin standards (aflatoxins (B,, B,, G, , G2), ochratoxin A, citrinin, patulin, sterigmatocystin) were obtained from Sigma Chemical Co. (St Louis, MO., U.S.A.) and thin layer chromatography plates (Silicagel 60) from Merck A.G. (Darmstadt, F.R.G.). Other chemicals or reagents were obtained from Sigma Co. or Merck A.G. and were of the purest grade available. *Author for correspondence. 199
H. BACHA et al.
200
METHODS Sampling Sampling was undertaken under conditions which cannot be directly related to USDA or TDRI sampling plans, with regard to the variable characteristics of storage of cereals. In the case of cereals stored in horizontal bulk, which was the most usual method, samples were taken from three different points, at the surface, at the side and at the low front of the bulk, the different samples from each stock being mixed to obtain the final sample used for analysis. For cereals stored in closed containers, separate samples from the top and bottom of the container were mixed to obtain the final sample. In the case of cereals stored in sacks, the sample for analysis was a mixture of two samples from two different sacks, one sample being taken from the upper part and another from the lower part of the stack. Analyses were made mainly in respect of large stocks of cereals, for distribution all over the country, and imported cereals. Twelve storage locations were visited, with capacities ranging from 13,000 to 30,000 tons. About 50 samples were collected. Observations were made regarding the particular maintenance, preservation and storage conditions (Table 2). Samples were analysed to detect aflatoxins (B, , B,, G, , G2), ochratoxin A, citrinin, patulin and sterigmatocystin. Extraction, purification and separation techniques were adapted from Patterson and Roberts (1979) and modified as described in Fig. 1. Extraction and purijication Samples of 100 g were pulverized and stirred with 250 ml of chloroform and 25 ml of 0.1 M phosphoric acid for 30 min. The mixture was decanted and the solution filtered and the solid then washed three times with chloroform and 0.1 M phosphoric acid (10 : 1, v/v); the washes were pooled with the first extract and chloroform then evaporated. The remaining acqueous phase was defatted three times with n-hexane. The defatted aqueous solution was made alkaline with 25 ml 0.1 N NaHCO, and extracted three times with chloroform. Two phases were obtained (I and II). The alkaline aqueous solution (I) was brought to pH 3 with concentrated HCl and the latter used for monitoring the presence of ochratoxins and citrinin by chloroform extraction. 100 g pulverized l l l
Discard residue
Extraction -/
sample extraction with decantation mixture filtration
250 ml CHCI, 25ml H,PO, 0.1 M
Filtrate l l
evaporation of CHCI, defatting with n-hexane
Discard hexane 1 Aqueous
phase . alkalinizations with 25 ml 0.1 N NaHCO, l extraction with chloroform
1 Aqueous
l
Purification
l
d
1 Chloroformic
I
phase
I Chloroformic
II
evaporation of CHCI, . dissolution in 1 ml acetonitrile
l
I Acetonitrile
phase l
phase
acidification with HCI extraction with CHCI,
reduction of the volume to 0.1 ml
phase . dialysis against acetone-water (3:7 v/v)
I Acetone-water
phase extraction with CHCI, l reduction of the volume to 0.1 ml l
I Final extract
Separation i
I
i i::r.::in:
Thin layerchromatography.
Fig. 1. Extraction. purification and separation method for mycotoxin
Fina’ ~~“c0t-W
analysis
of contaminated
cereals.
Fungal Table
toxins in Tunisia
201
1.Minimum detection levels and extraction yields of mycotoxins from contaminated and mycotoxin supplemented samples of cereals
Mycotoxins Aflatoxin B, Allatoxin B, Aflatoxin G, Aflatoxin G, Ochratoxin A Citrinin Patulin Steriematocvstin
Minimum detection level: pg on TLC
Yields (%)
&kg (ppb)
0.3 0.37 0.3 0.35 6.4 10.5 10.8 I8
28 26 25 25 22 28 23 -
I .07 1.42 I.20 I .40 30 38.57 46.95 -
The chloroformic solution (II) was evaporated and the residue dissolved in 1 ml of acetonitrile and dialysed aga:inst 100 ml of an acetone-water solution (30:70, v/v). Eventually mycotoxins dialyse out. The acetone-water solution was then extracted three times with chloroform and later used for monitoring most mycotoxins. Separation This was performed by unidimensional ascending thin-layer chromatography on Silicagel plates. The solvent systems used were those described by Patterson and Roberts (1979) for ochratoxins and aflatoxins, Stubblefield (1979) for citrinin, and Egon and Miiller (1977) for sterigmatocystin. The samples containing aflatoxins, after chromatography in a first direction using chloroform-acetone (9 : 1, v/v), were submitted to a second dimensional chromatography using toluene-ethylacetate-formic acid (6: 3 : 1, v/v/v). Identification This was performed by U.V.light illumination at 254 and 366 nm, by comparison of the migration of the samples with mycotoxin standards. Identification was achieved by the use of two chemical treatments which modify specifically the fluorescence of different samples: spraying of the plates with a sulphuric acid-water solution (1: 1, v/v) or a p-anisaldehyde-acetic acid-sulphuric acid solution (5:90:5, sv/v/v) (Schuller et al., 1967; Fremy and Corbion, 1979). Yields of mycotoxin extractions Aliquots (0.1-30 pg) of the various mycotoxins (aflatoxins B, , B,, G, , G,; ochratoxin A; patulin; citrinin; sterigmatocystin) were added to non-contaminated wheat. Controls were processed as already described. The lower detection levels were defined as the minimal amounts detectable under U.V.light after thin-layer chromatography (Table 1). The detected spots of mycotoxins were eluted for the Silicagel with methanol and the concentrations determined spectrophotometrically according to the molar extinction coefficifznt (aflatoxin B, : E = 21,800 at 360 nm; aflatoxin B,:c = 24,000 at 362 nm; aflatoxin G, : 6 = 17,700 at 362 nm; aflatoxin G,: 6 = 19,300 at 362 nm; ochratoxin A: 6 = 5,550 at 333 nm; citrinin: 6 = 16,100 at 332 nm and sterigmatocystin: 6 = 15,200 at 325 nm). The extraction yields were determined by comparing the amounts of mycotoxins extracted with the added one (Table 1). The extraction yield of ochratoxin A was also assessed using [‘Hlochratoxin A (C.E.A., Saclay, France). About 1 nmol of [3H]ochratoxin A (specific activity: 800 cpm/pmol) was added to the sample, radioactivity countings being performed at each step of purification. The yield of the purification was determined by the radioactivity after purification vs the radioactivity before purification. RESULTS
The minimum detection levels for mycotoxins (aflatoxins B, , B,, G, , G2), ochratoxin A, citrinin, patulin and sterigmatocystin are given in Table 1. We were able to detect approximately l-l .4; 30 and 47 pgg/kg of allatoxins, ochratoxin A and sterigmatocystin respectively. Several mycotoxins were detected in the cereal samples analysed (Table 2). Only ochratoxin A was determined quantitatively, while the fluorescence intensities of all other spots were estimated with ( + ) symbols.
202
H. BACHA et al.
+ +i”
+ +
-I-
+
+
+
+
(83) (83)
El Aroussa Bou Arada
idem idem Storage in bulk? Damp and dirty environment Insect infestation idem idem idem i&m Stnraee in hIIlk? Bad maintenance Damp and dirty environment idem Mixture of several harvests Storage in open and humid tanks* Storage in open and humid tanks* and outside in sacks Clean environment Mixture of several harvests Storage in open and humid tanks* and outside idem Storage in bulk? Damp and very dirty environment Storage in bulk? Bad maintenance Very dirty environment Storage m bulkf m closed store
‘Tank: Cereals stored in large metallic containers within a store. tBulk: Cereals stored in bulk: conventional warehouse.
El Fahs (83)
(83)
El Aroussa
TW,
DW,
(83)
El Aroussa
DW,.
El Fahs (83)
Jendouba (83) El Aroussa (83)
TW, DW,
DW,
Bou Salem (83) Bou Salem (83) Boo Salem (83) Bou Salem (83) Jendouba (83)
DW, TW TW, TW5DW,
%ja (83) %ja (83) Bou Salem (83)
+i +
+ +
+
+ + c
+
++
+ ++
+
+ +
++
I
+ ++ ++ +
+
+++
I
++ +++ +++ ++
+
++ ++
+ ++ + ++
+++
(+) +
A+ + +
+
ti+
+
++
Aliment no. 7
no. 2 Aliment no. 4 Aliment no. 5
AIiment
for chicken
Local oats Local soybean imported maize Imported “Premix”
Samples
Megrine Djebel Jelloud Mateur B&ja Bon Salem Jendouba El Aroussa Boll Arada Ef Fahs
Locations
l/i
f 8,Dou
Fish meal Trace &merits Vitamins Oats, salts Wheat bran, oats Oats, corn, wheat bran, Soybean cake, salts Oats. wheat bran.
Composition
Table 4. Occurred
--
X2 315
f/l
I/E
515
IIS
ARa 0,
j/f
515 2/z 315
l/2 l/4
Affa G,
J/j
r/s ti’2 IiS
214 vs
OTA
VI
l/f
Citrinin
Mycotoxins detected; frequency (a/b) ..-. l.ll~ ~ _--. .l_ll_
212 5i5
w
212 114 115
Afla B,
-
.
-..._.
Atla B,
_ Afla B,
-_
Afla G,
OTA
~~
~
Citrinin
--
112 114
_
_-_” Slerigmatocystin
..__-l^l__.
Sterigmatocystin
Mycotoxins detected (level) ~~~ ----. .-I- _I___
Afla G,
^-~~
of mvcotoxins in animal feed oroducts and comoosite animal feeds
212 51s
1wXKl
w
f6,ooo
112 l/4 w l/3
ABa B,
18,ooo t7*000
20,GQo 25,000 14,500 13,ooo
Capacity (tons)
^.. .-.--
Table 3. Occurrence and frequency of mycoiaxins in different storage locations
Fungal
toxins
in Tunisia
205
The following mycotoxins were detected: aflatoxin B, and G, respectively at very high levels in 55 and 41% of the samples analysed, aflatoxin B, and G, respectively in 55 and 52% of analysed samples, at lower levels than aflatoxins B, and G, , ochratoxin A and citrinin in 7% of the analysed samples at variable levels (34-360 pg/kg, especially for ochratoxin A) and trace amounts of sterigmatocystin in 7% of samples analysed (Table 3). The analysis of different samples of animals feeds (nos l-5 in Table 4) and their main components (maize, oats, soybean oil cake, wheat bran and “Premix”) gave the following results: very high levels of ochratoxin A contamination were found (maize-320 ,ugg/kg, “Premix”-360 pgg/kg and feed no. 5-140 ,ug/kg usually associated with lesser degrees of aflatoxin B2 contamination (Table 4). DISCUSSION
Aflatoxins B,, B,, G, and G,, all produced by AspergiZZus~uvus Link, are normally detected together in the same positive samples of cereals or animal feeds, but this was not verified in all cases, because the physiologial conditions of the moulds and specificity of the substrate can favour one or other forms of aflatoxin, e.g. B, or G, (Hesseltine, 1976). The lack of B, and (or) G, in some samples containing B, and (or) G2 (which are respectively metabolites of El, and G,) could be explained by the almost total conversion of B, and G, respectively into B, and G, (Diener and Davis, 1969). The minimum detection level of aflatoxins in our test was about 0.14 pg/kg. The amounts of mycotoxins detected are probably underestimates because of the low recoveries, and the fact that the samples analysed were not collected in a statistically representative manner due to accessibil:ity to the bulk lots, and the problem of minimum detection levels. Indeed, the minimum amounts of mycotoxins we were able to detect are higher than those reported by Patterson and Roberts (1979) so that the traces we observed could even mean a higher level of contamination. Mycotoxins were found to be contaminating cereals in Tunisia. All these cereals come from the northern part of the country, a region with a high humidity level (125 rain days/year) and temperatures ranging from 6 to 45°C with an average of about 25-30°C. These climatic factors potentiate hazards associated with the specific storage and preservation conditions of the cereals. Thus, aflatoxins I),, B,, G, and G2 were predominantly detected in places where humidity was high, and citrinin was encountered only in connection with hygiene problems in dirty locations. On the contrary, in the case of cereals stored under good conditions and protected from humidity, no mycotoxin contamination occurred (samples DW,, TW, , Table 2). Ochratoxin A appeared to be absent from the local cereal production. It was found only in imported cereals, and essentially in wheat (180 pg/kg) and maize (320 pgg/kg). The latter constitutes the major constituent of composite animal feeds and could represent the origin of the heavy contamination detected in the composite animal feed no. 5 (Table 4). It is well establ.ished that mycotoxins in cereals, food and feeds cause damage in humans and animals (Elling, 1977; Richard et al., 1975; Wyllie and Moorehouse, 1978; Galtier et al., 1974). A positive correlation between the presence of mycotoxins in food products with several diseases observed in the s,ame area has been established (Hadidane et al., 1985). In view of the importance of mycotoxins to the economy and public health, it is considered that systematic controls of both local and imported cereals, food and feed products should be established in Tunisia and other developing countries. Acknowledgemenrs-This work was supported by grants from the “Ministere Tunisien de 1’Enseignement Superieur et de National de la Sante et de la Recherche la Recherche Scientilique” (Contrat no. PC 5/83/FPMO), the “Institut Medicale”-INSERM (Contrat CRL 85: 2009) and the “Centre National de la Recherche Scientifique” (Contrat PIREN, ATP Alimentation-Nutrition no. 82). H. Bacha and R. Hadidane acknowledge the short-term fellowships given by the Direction des Relations et de la Cooperation Internati’onale du CNRS. REFERENCES Boutrif E., Jemmali M., Pohland Chem. 60, 747-748.
A. E. and Campbell
A. D. (1977a)
Aflatoxins
in Aleppo
pine nuts. J. Ass. ofl analyf.
H. BACHA et at.
206
Boutrif E., Jemmali M., Campbell A. D. and Pohland A. E. (1977b) Aflatoxin in Tunisian foods and foodstuffs. Annis Nutr. Aliment. 31, 431-434.
Diener U. L. and Davis N. D. (1969) Atlatoxin formation by As~rg~iius~uv~. In A~atoxin (Edited by L. A. Goldblatt), pp. 13-54. Academic Press, New York. Egon B. G. J. and Mbller T. E. (1977) Screening method for the detection of aflatoxins, ochratoxin, patulin, sterigmatocystin and zearalenone in cereals. J. Ass. 05 analyr. Chem. 60, 1369-1371 I Elling F. (1977) Morphological aspects of mycotoxic nephropathy. In Mycotoxins in Human and Animal Health (Edited by J. V. Rodricks, C. W. Hesseltine and M. A. Mehlman), pp. 4999506. Pathotox Publishers, Park Forest South, Illinois. Frayssinet C. and Lafont P. (1976) Les Mycotoxines: Problemes d’actualiti. Cah. Nutr. Diet. (Mycotoxines), 2, 7-12. Fremy J. M. and Corbion 8. (1979) Analyse mycotoxicologique des aliments. Arms Fatsif: Expert Chim. 72, 53-64. Galtier P., More J. and Bodin G. (1974) Toxines d’Aspergillus ochraceus W. III. Toxicid aigue de l’ochratoxine A chez le rat et la souris adultes. Ann/s Rech. Vet. 5, 233-247. Golumbic C. and Kufik M. M, (1969) Fungal spoilage in stored crops and its control. In AJIaruxin (Edited by L. A. Goldblatt), pp. 307-332. Academic Press, New York. Hadidane R., Regnault C., Bouattour H., Ellouze F., Bacha H., Creppy E. E. and Dirheimer G. (1985) Correlation between alimentary mycotoxin contamination and specific diseases. Human Toxicol. 4, 491-501. Hesseltine C. (1976) Mycotoxins other than aflatoxins. Proc. Int. Biodegradation Symp. (Edited by J. Sharpley and A. Kaplan) pp. 607-623. Applied Science Publishers Kingston, London. Krogh P., Hald B., Pldstina R. and Ceovic S. (1977) Balkan (endemic) Nephropathy and foodborn ochratoxin A: Preliminary results of a survey of foodstuffs, Acta path. microbial. stand. R 85, 238-240. Landers K. E., Davis N. D. and Diener U. L. (1967) Influence of atmospheric gas on aflatoxin production by Aspergillus flavus in peanuts. Phytopathology 57, 1086-1090. Le Bars J. (1976) Ecologic des Moisissures Toxinogenes. Cab. Nutr. Diet. (~ycotoxines), 2, 23-27. Moreau C. (1974) In Mo~is~res Toxiques dans I’Alimentation, p. 471. Masson et Cie, Paris. Patterson D. S. P. and Roberts B. A. (1979) Mycotoxins in animal feedstuffs: sensitive thin-layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone and T-2 toxin, J. Ass. ofl analyt. Chem. 62, 1265-1267. Pepeljnjak S., Blaievic N. and Culjak K. (1982) Contamination with moulds and occurrence of ochratoxin A in smoked meat products from endemic nephropathy region of Yugoslavia. In Mycotoxins and Phycotoxins, Vth Int. IUPAC Symp. Mycotoxins and Phycotoxins, Vienna, pp. 102-105. Technical University, Vienna. Richard J. L., Thurston J. R., Deyoes B. L. and Booth G. D. (1975) Effects of ochratoxin A and aflatoxin on serum proteins, complement activity and antibody production to Brucella abortus in Guinea pigs. Appt. Microbial. 29, 27-30. Schuller P. L., Gchlunizen ‘I., Werringloer J. and Marquardt M. (1967) Aflatoxin B, und Histamin in Wein. Arzneim. Forsch. 17, 888-890.
Senti F. R. (1977) Conference on Mycotoxin in Human and Animal Health. In Mycoroxjns in Humun and Animal Heu~th (Edited by J. V. Rodricks, C. W. Hesseltine and M. A. Mehlman), pp. 779-789. Pathotox Publishers, Park Forest South, Illinois. Stubblefield R. D. (1979) Thin-layer chromatographic determination of citrinin. J. Ass. ofi analyt. Chem. 62, 201-202. Wyllie T. D. and Moorehouse L. G. (1978) Mycotoxic fungi, Mycotoxins, Mycotoxicoses. In Encyclopedic Handbook, Mycotoxicoses of Domestic and Laboratory Animals, Poultry and Aquatic Invertebrates, Vol. 2. Marcel Dekker, New York.