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Journal of Food Protection, Vol. 60, No. 11, 1997, Pages 1381-1385 Copyright ©, International Association of Milk, Food and Environmental Sanitarians

Mycotoxins of clavatus: Toxicity of Cytochalasin E, Patulin, and Extracts of Contaminated Barley Malt

lERESA-MARIA LOPEZ-DIAZI and BRIAN FLANNIGAN2* Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/11/1381/2301759/0362-028x-60_11_1381.pdf by guest on 26 September 2021 IDepartment of Food Hygiene and Technology, University of Leon, 24071 Leon, Spain, and 21nternational Centre for Brewing and Distilling, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, U.K.

(MS# 96-338: Received 26 November 1996/Accepted 6 March 1997)

ABSTRACT malting process before separation of dried malt kernels for the food, brewing, and distilling industries from the rootlets Brine shrimp and conductimetric Saccharomyces cerevisiae (or "culms") used in animal feeds (22). bioassays were used to investigate the toxicity of green barley malt Patulin is produced by a number of Penicillium and prepared at 16 and 25°C from grain inoculated with a strain of Aspergillus and was first investigated as a potential Aspergillus clavatus known to produce the cytochala- sin E and patulin during malting. Pure cytochalasin E was broad-spectrum antibiotic, but it shows moderately high considerably more toxic to brine shrimp larvae than patulin acute toxicity to experimental rodents, chicks, and (30). (LCso < 0.5 versus >30 Ilg ml~I). In contrast, patulin significantly Cytochalasin E is one of around 40 chemically related inhibited growth-related change in conductance of S. cerevisiae cytochalasins, which are variously produced by a wide range cultures at a concentration of 10 Ilg ml ~ 1, but cytochalasin E had no of molds. In addition to A. clavatus (8, 18), A. terreus (15) I effect at 80 Ilg ml- . Extracts of both 16 and 25°C malts and Rosellinia necatrix (3) produce cytochalasin E. The contaminated with A. clavatus were toxic to brine shrimp larvae, chemical structure of cytochalasin E was first described by but had only limited inhibitory effects on the growth of S. Aldridge et al. (3), but subsequently revised (4) and later cerevisiae. Since concentrations of cytochalasin E in contaminated malts produced at 16°C are below or close to the limits of detection, amended by Btichi et al. (8), who isolated the from the presence of other fungal metabolites toxic to brine shrimps in glutinous rice contaminated with A. clavatus and other such malts is indicated. molds and implicated in Reye's syndrome in Thailand. Cytochalasin E is acutely toxic to rats, mice, and guinea pigs Key words: Aspergillus clavatus, cytochalasin E, patulin, malting, (17) and highly toxic to chick embryos (32). As with other toxicity cytochalasins, it inhibits cytoplasmic cleavage and movement in mammalian cell cultures (9). It also inhibits Aspergillus clavatus is best known as the cause of the glucose absorption by mammalian cells (16). allergic respiratory disease, malt worker's lung (14). How- Various bioassays have been employed for assessing the ever, this species is also toxigenic, producing a range of toxicity of microbiologically contaminated foods and feed- mycotoxins, and has caused mycotoxicoses among stock fed stuffs. One such bioassay, employing brine shrimp (Artemia on malting and brewing by-products (14). However, the salina L.) larvae, was first used as a screening system for particular mycotoxins in the by-products involved in these fungal by Brown et al. (7) and Brown (6). It has since episodes have never been identified. The germination phase been widely used to test numerous mycotoxins (5, 11, 12, of malt production from barley, wheat, and other cereals 19-21, 28, 29) and extracts of fungi (10, 31). A variety of provides a substrate with the high water activity (aw) microorganisms have also been used in bioassays, and a necessary for the growth of A. clavatus (14) and the aw of rapid impedimetric method employing the yeast Hansenula 0.99 or greater indicated by laboratory experiments to be fabianii was developed for the assay of (1, 2, necessary for production of one of the mycotoxins, patulin 25). Flannigan and Pearce (14) employed a conductance (26). Recent laboratory investigations have shown that method to test the toxicity of extracts of culms from an patulin and cytochalasin E are produced by A. clavatus on outbreak of mycotoxicosis in stock and of artificially the germinating grain during malting of barley and wheat inoculated spent grains to broth cultures of Saccharomyces (22). In addition, it appears that approximately one-fifth of cerevisiae. both toxins may survive kilning, the final stage of the In the present paper, the toxicity of cytochalasin E and patulin and extracts of green malt prepared from barley inoculated with A. clavatus in a brine shrimp mortality test

* Author for correspondence. Tel: + 131 451 3457; Fax: + 131 451 3009; and in an indirect conductimetric assay of growth of the E-mail: [email protected] yeast S. cerevisiae is examined. 1382 LOPEZ-DIAZ AND FLANNIGAN

MATERIALS AND METHODS Statistical analysis Statistical significance (P:S 0.01) of differences between Toxin solutions means of different treatments under comparison were determined Standard solutions of pure cytochalasin E and patulin (Sigma, by Student's t test. Poole, Dorset, U.K.) were prepared and diluted in ethanol (Spectro- soL, Merck, Lutterworth, Leics., U.K.). RESULTS AND DISCUSSION

Malting extracts Toxicity of cytochalasin E and patulin For preparation of extracts from green malt (germinated The results of the brine shrimp bioassay (Table 1) barley), triplicate 25-g samples of malting quality barley were indicate that cytochalasin E can be considered a substance steeped, inoculated with Aspergillus clavatus ACM2, drained, and very toxic to the larvae. Mortality rates at 16 h were incubated in 300-ml Erlenmeyer flasks at 16 and 25°C for 7 days significantly lower than at 24 h, but nevertheless the LC in (22). Together with an uninoculated control, two green malts were 50 1 both sets of tests was <0.5 Ilg ml- . There are no previous Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/11/1381/2301759/0362-028x-60_11_1381.pdf by guest on 26 September 2021 prepared from inoculated barley at both temperatures. The contents of each flask were then extracted at room temperature with 100 ml reports on the toxicity of cytochalasin E in brine shrimp of dichloromethane for 30 min in an orbital shaker operating at 160 assays, but our results indicate that this cytochalasin is rpm and filtered through Whatman No. 1 filter paper and then among the mycotoxins that are most toxic to shrimp larvae. anhydrous sodium sulfate. After rotary evaporation of the filtrate to It appears to be more toxic than most of the mycotoxins from dryness (22), the residue was dissolved in 1 ml of ethanol aspergilli and penicillia previously tested in brine shrimp (SpectrosoL), and the undiluted solution and tenfold serial dilu- bioassays (11, 20), including Bl and Gj, , tions were used in toxicity tests. citreoviridin, mycophenolic acid, A, and sterig- matocystin. However, the toxicity of cytochalasin E to brine Brine shrimp bioassay shrimp larvae does not appear to be as great as that of some Dry eggs (0.5 g) of Artemia salina L. were hatched in 750 ml Fusarium toxins, viz., the trichothecenes T-2 (LC50, com- of natural sea water incubated at 28°C with continuous forced puted from LD50 for toxin-loaded filter-paper discs in wells, aeration for 30 to 32 h. Aliquots (0.1 ml) of sea water containing 30 approximately 0.091lg ml-1), HT-2 (0.30), and diacetoxyscir- to 50 larvae were then added to triplicate wells of disposable penol (0.26), although it is markedly higher than some other microtiter plates to which 5 to 20 III of toxin solution or 10 III of malting extract, tenfold serial dilutions, or ethanol (controls) had trichothecenes, such as nivalenol, deoxynivalenol, and 3- previously been added and dried in an air current at room acetyldeoxynivalenol, and the estrogenic compound zearale- temperature (20 to 30 min) in a fume hood. The plates were covered none (31). with porous plastic film and incubated at 27 ± 1°C. After 16 and 24 In tests in which the larvae were exposed to the toxin for 1 h, the dead larvae were counted under a stereoscopic microscope; 24 h, the LC50 for patulin lay between 30 and 50 Ilg ml- the surviving larvae were killed by adding 10 III of chloroform to (Table 1), i.e., two orders of magnitude higher than for each well and the mortality calculated as percentage of dead larvae cytochalasin E; 100% mortality was obtained only at toxin of the total (20). Each experiment was carried out at least thrice. concentrations greater than 150 Ilg ml-1• In tests employing toxin-loaded filter-paper discs, Harwig and Scott (20) re- Disc diffusion bioassays ported a mortality of 20% among larvae exposed to discs Disc diffusion agar-plate tests in which duplicate filter-paper loaded with 10 Ilg of patulin, equivalent to 100 Ilg toxin discs loaded with 10 Ilg of cytochalasin E were applied to plates ml-1• In the present study, the mortality at this concentration seeded with broth cultures of yeasts (24) and, for comparison, was >80%. However, Harwig and Scott terminated their test suspensions of molds (27) in duplicate tests were incubated at 25°C for several days. The yeasts were Debaryomyces hansenii NCYC 10, Kluyveromyces marxianus NCYC100 and NCYC15l, and Saccha- TABLE 1. Percentage mortality among brine shrimp larvae romyces cerevisiae NCYCll08. The molds were the producing exposedfor 16 and 24 h to cytochalasin E and patulin A. clavatus ACMl-ACM4, Aspergillus fumigatus P48, A. versicolor IMI016139, and Penicillium expansum IMI039761. Toxin % Mortality concentration (mean :<: SD, n 2: 3) after: (llglm1of Saccharomyces bioassay Toxin sea water) 16 h 24 h For assay of toxicity of pure toxins and extracts of green malt, 300-ml Erlenmeyer flasks containing 100 ml of modified wort Cytochalasin E >5 100 100 broth (Malthus Instruments Ltd., Bury, Lancashire, U.K) were 1 81.5 ± 5.2 96.5 ± 2.5 inoculated with Saccharomyces cerevisiae NCYC 1108 and incu- 0.5 69.9 ± 7.4 97.4 ± 0.9 bated on an orbital shaker (ca. 160 rpm) at 28°C. After 24 h, 90 ml 0.1 7.6 ± 0.8 18.3 ± 6.4 of broth was inoculated with the resulting culture and incubated for 0.05 2.9 ± 2.4 7.9 ± 5.6 4 h under the same conditions. This log-phase culture was then <0.01 0 0 adjusted to an absorbance of 0.10 to 0.15 at 570 nm with fresh broth Patulin >150 nda 100 and 0.2-ml aliquots were used to inoculate quadruplicate cells of a 100 nd 83.4 ± 6.5 microbiological growth analyzer (Malthus, U.K.) containing 2 ml 50 nd 62.3 ± 18.1 of wort broth amended with 20 III of ethanolic solution (standards 30 nd 15.7 ± 11.3 or extracts) or ethanol (controls). The cells were incubated in the 10 nd 2.7 ± 4.8 thermostatically controlled water bath of the analyzer at 26°C for <5 nd 0 35 h, with the conductance of the culture in each cell being read and recorded at 6-min intervals. Each test was performed twice. a nd, not determined. A. CIA VATUS AND MALTING EXTRACT TOXICITY 1383 at 16 h, and as the results for cytochalasin show (Table 1), 100 extending the duration of the test to 24 h is likely to increase the mortality. In other studies, LCso values of 75.0 (28) and 80 1 ,..... 90.3 Ilg rnl- (11) have been quoted. rJ:l ::l.. In none of the yeast or mold cultures tested in disc ~ 60 Cl) diffusion bioassays were inhibition zones observed round u the discs loaded with 10 Ilg of cytochalasin E. The negative a 40 •..u results obtained with the yeasts exposed to this mycotoxin ::l "'0 Q are in line with earlier reports for eight other non- 0 20 mycotoxins, including patulin (24). Although U growth of Kluyveromyces marxianus was inhibited by a 0 range of trichothecenes, including verrucarin A at a concen-

tration of 0.005 Ilg per disc (23), it was not affected by -20 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/11/1381/2301759/0362-028x-60_11_1381.pdf by guest on 26 September 2021 0 10 20 30 40 nontrichothecene mycotoxins (24), even at concentrations as Time (h) high as 200 Ilg per disc (23). The relative toxicity of cytochalasin E with respect to FIGURE 2. Effect of patulin on conductance of broth cultures of s. patulin in the Saccharomyces bioassay was the reverse of cerevisiae incubated at 26°C in a Malthus microbiological growth that in the brine shrimp tests. As indicated by comparison of analyzer (0, 1 Jlg of toxin per ml of medium; e, 10 Jlg ml-1; 0, the change of conductance in the medium and controls in 20 Jlg ml-1; _,40 Jlg ml-1; *, control). representative experiments, cytochalasin E had no appre- ciable effect on growth, even at 80 Ilg ml-1 (Fig. 1). according to the scale of Harwig and Scott (20) "very toxic" However, patulin showed an inhibitory effect even at a (mortality 90 to 100% at 16 h). Extracts of uninoculated concentration of 10 Ilg ml-1 (Fig. 2). Whilst little or no barley were "nontoxic" (mortality <5%). In 16-h tests, all difference was observed in the time for the conductance lO-fold dilutions (Table 3) of extracts from corresponding value of the medium to reach the arbitrary level of - 50 IlS in malts prepared in two trials at both 16 and 25°C were of a the presence of cytochalasin E at the concentrations tested a similar order of potency, being "toxic" (mortality 50 to dose-related retardation of growth in the presence of patulin 89%). In parallel 24-h tests, percentage mortality was on was confirmed, with the difference from ethanol controls average nearly 25% greater than in 16-h tests. There was no being statistically highly significant at concentrations of 10 substantial difference in toxicity to larvae between extracts Ilg ml-1 or greater (Table 2). Differences between duplicate of malts prepared at different temperatures within individual tests were not statistically significant. malting trials, and the toxicity of corresponding malts in different trials was similar (Table 3). Toxicity of malting extracts In contrast to the brine shrimp bioassay, the Saccharo- Although not shown in Table 3, toxic substances in myces bioassay did not reveal any toxicity in the malts 10-111aliquots of undiluted ethanolic extracts (equivalent to prepared from inoculated barley. The conductance curves 250 mg of grain prior to steeping) from all green malts (not shown) were similar to those for corresponding uninocu- prepared from barley inoculated with A. clavatus on average lated controls. The mean times for the conductance of caused 97% mortality among larvae, and were therefore cultures in medium supplemented with extracts of green malts prepared at 16 and 25°C to fall to -50 IlS were close

100 TABLE 2. Effect of cytochalasin E and patulin on change in 80 conductance of broth cultures ofS. cerevisiae incubated at 26°C in ,..... a Malthus microbiological growth analyzer rJ:l ::l.. 60 ~ Toxin Time (h) Cl) concentration to -50 flS Mean u a 40 (flg/mlof (mean:<: SD, difference •.. Toxin medium) n = 6) from control (h) .gu Q 20 0 Cytochalasin E 1 17.70 ± 0.53 +0.10 U 10 17.69 ± 0.56 +0.09 0 20 17.73 ± 0.51 +0.13 80 18.00 ± 1.35a +0.40 -20 Patulin 1 18.54 ± 1.00 +0.94 0 10 20 30 40 10 22.38 ± 1.35 +4.78b Time (h) 20 24.65 ± 2.71 +7.05c 40 33.88 ± O.73a + 16.28b FIGURE 1. Effect of cytochalasin E on conductance of broth Control 0 17.60 ± 0.52 NAd cultures ofS. cerevisiae incubated at 26°C in a Malthus microbio- logical growth analyzer (0,80 Jlg of toxin per ml of medium; *, an = 3. control). b P < 10-6; c P < 10-5; dNA, not applicable. 1384 LOPEZ-DlAZ AND FLANNIGAN

TABLE 3. Percentage mortality among brine shrimp larvae yielded amounts close to the limit of detection. At 25°C, the exposed to extracts of green malt prepared barley inoculated with concentrations with that strain were again very low and with A. clavatus a second no cytochalasin E was detected, but with ACM2 % Mortality" (mean ± SD; n = 3) and another strain the cytochalasin was present in concentra- of 10-1 dilutions of malt extracts in: tions similar to or greater than those of patulin. It has been suggested (22) that, since grain is usually malted at 16°C in Malting Malting trial I after: Malting trial 2 after: u.K., patulin rather than cytochalasin may have been temperature (aC) 16h 24h 16 h 24h associated with a mycotoxicosis in stock fed on malt screenings. On the other hand, cytochalasin may have been 16 60.0 ± 12.3 82.4 ± 8.7 51.8 ± 1.7 76.7 ± 6.4 of importance in episodes associated with malt produced at 25 65.9 ± 7.4 88.9 ± 5.7 57.3 ± 7.9 85.3 ± 8.7 25°C or above in Israel and South Africa (22). Bearing in mind the low toxicity of patulin to brine a Undiluted extracts, 97.2 ± 2.2%; mortality of extracts of uninocu- 1ated grain, 0 to 5.0%. shrimps and the previously reported absence of cytochalasin Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/11/1381/2301759/0362-028x-60_11_1381.pdf by guest on 26 September 2021 E in green malt produced at 16°C from ACM2-inoculated barley (22), it can be suggested that the toxicity of the 16°C to each other (Table 4). Except for the extract of one malt contaminated malt to Artemia (Table 3) may have been due prepared at 25°C, which extended the time significantly in to tremorgens or other toxins which A. clavatus can produce relation to the extract of its uninoculated control (Table 4), (14). Alternatively, synergy between toxins may have ac- no differences between and among extracts of inoculated counted for the toxicity. Since cytochalasin E has little effect and uninoculated controls at 16 and 25°C and ethanol on Saccharomyces growth, the slight growth retardation controls were statistically significant (P < 0.01). In only caused by extracts of the green malt produced at 16°C two cases were duplicate conductance tests significantly appears to be in line with the previous report of greater different. specific production of patulin at the lower temperature (22). Although no statistical difference in toxicity of extracts Although cytochalasin E inhibits cytoplasmic cleavage of green malt to S. cerevisiae was observed in this investiga- and cell movement in mammalian cell cultures (9), the tion, in other malting experiments carried out under similar evidence from the conductimetric (Fig. 1, Table 2) and disc conditions with barley and wheat (22), visible growth of A. diffusion tests indicates that eukaryotic microorganisms may clavatus-inoculated samples was more profuse at 25 than at be unsuitable for the bioassay of cytochalasin E and other 16°C, and ergosterol measurements of fungal biomass cytochalasins. However, the brine shrimp bioassay appears greater. In that study, patulin was found in all green malts to be a very sensitive test for cytochalasin E, and could also produced from inoculated barley, but concentrations were be used as a screen for toxicity due to this mycotoxin and lower in malt prepared at 25 than at 16°C in the case of two other toxic metabolites, but not patulin, associated with the strains (including ACM2) and equal to or higher than at presence of A. clavatus in malt. For patulin and any other 16°C in the other two. Based on mean values for ergosterol mycotoxins, such as some trichothecenes, which may affect and patulin, and irrespective of strain, specific production of yeast viability and growth (13), the conductimetric bioassay the toxin was greater at 16°C, the temperature cited as being for Saccharomyces growth would be appropriate. optimum for patulin production (26). Cytochalasin E could not be found in green malt produced at 16°C from barley inoculated with ACM2 and two other strains, but a fourth ACKNOWLEDGMENTS

The authors wish to acknowledge that this work was carried out while TABLE 4. Effect of extracts of green malt prepared from barley T.-M. L.-D. was in receipt of a Post-Doctoral Fellowship from the Spanish inoculated with A. clavatus on change in conductance of broth Ministry of Science and Education. cultures of S. cerevisiae incubated at 26°C in a Malthus microbio- logical growth analyzer REFERENCES

Time (h) Mean Mean 1. Adak, G. K., J. E. L. Corry, and M. O. Moss. 1987. Use of to -50 flS difference from difference impedimetry to detect trichothecene mycotoxins. 1. Screen for (mean ± SD; uninoculated from ethanol susceptible microorganisms. Int. J. Food Microbiol. 5: 1-13. Malt n 2: 6) control (h) control (h) 2. Adak, G. K., J. E. L. Corry, and M. O. Moss. 1987. Use of impedimetry to detect trichothecene mycotoxins. I. Limits of sensitiv- we ity of 4 microorganisms to T-2 toxin and the effects of solvent and test Inoculated 1 18.66 ± 1.69 + 1.59 +2.06 medium. Int. J. Food Microbiol. 5:15-27. Inoculated 2 18.13 ± 1.40 + 1.06 +1.53 3. Aldridge, D. c., B. F. Burrows, and W. B. Turner. 1972. The structure Uninoculated 17.07 ± 1.20 NAG +0.47 of the fungal metabolites cytochalasin E and F. J. Chern. Soc. Chern. 2ye Commun. ee:148-149. 4. Aldridge, D. C., D. Greatbanks, and W. B. Turner. 1973. Revised Inoculated 3 17.65 ± 0.58 + 1.78b + 1.05 structures for cytochalasins E and F. J. Chern. Soc. Chern. Commun. Inoculated 4 17.18 ± 0.89 + 1.31 +0.58 ee:551-552. Uninoculated 15.87 ± 1.14 NA -0.73 5. Bijl, J., D. Dive, and C. Van Petghem. 1981. Comparison of some Ethanol (control) 16.60 ± 1.21 NA NA bioassay methods for mycotoxins studies. Environ. Pollut. ser. A 26: 173-182. a NA, not applicable. 6. Brown, R. F. 1969. The effect of somemycotoxins on the brine shrimp b Statistically significant (P < 0.003). Artemia salina. J. Am. Oil Chern. Soc. 46: 119. A. eLA VATUS MYCOTOXIN AND MALTING EXTRACT TOXICITY 1385

7. Brown, R. F., J. D. Widman, and R. M. Eppley. 1968. Temperature- 20. Harwig, J., and P. M. Scott. 1971. Brine shrimp (Artemia salina L.) dose relationships with on the brine shrimp, Artemia salina. larvae as a screening system for fungal toxins. Appl. Microbiol. J. Assoc. Off. Anal. Chern. 51:905-906. 21:lOll-1016. 8. Buchi, G., Y. Kitaura, S. S. Yuan, H. E. Wright, J. Clardy, A. L. 21. Harwig, J., P. M. Scott, D. R. Stoltz, and B. J. Blanchfield. 1971. Demain, T. Glinsukon, N. Hunt, andG. N. Wogan. 1973. The structure Toxins of molds from decaying tomato fruit. Appl. Environ. Micro- for cytochalasin E, a toxic metabolite of Aspergillus clavatus. J. Am. bioI. 38:267-274. Chern. Soc. 95:5423-5425. 22. Lopez-Diaz, T.-M., and B. Flannigan. 1997. Production of patulin and 9. Carter, S. B. 1972. The cytochalasins as research tools in cytology. cytochalasin E by Aspergillus clavatus during malting of barley and Endeavour 31:77-82. wheat. Int. J. Food Microbiol. 35:129-136. 10. Davis, N. D., R. E. Wagener, G. Morgan-Jones, and U. L. Diener. 23. Madhyastha, M. S., R. R. Marquardt, A. A. Frohlich, and J. Borsa. 1975. Toxigenic thermophilic and thermotolerant fungi. Appl. Micro- 1994. Optimization of yeast bioassay for trichothecene mycotoxins. J. bioI. 29:455-457. Food Prot. 57:490-495. 11. Durackova, A., V. Betina, B. Hornikova, and P. Nemec. 1977. Toxicity 24. Madhyastha, M. S., R. R. Marquardt, A. Masi, J. Borsa, and A. A. of mycotoxins and other fungal metabolites to Artemia salina larvae. Frohlich. 1994. Comparison of toxicity of different mycotoxins to Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. II 132:294- several species of bacteria and yeasts: use of Bacillus brevis in a disc 299. diffusion assay. J. Food Prot. 57:48-53. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/11/1381/2301759/0362-028x-60_11_1381.pdf by guest on 26 September 2021 12. Eppley, R. M. Sensitivity of brine shrimp (Artemia salina) to 25. Moss, M. 0., and G. K. Adak. 1986. Microorganisms and microbio- trichothecenes. J. Assoc. Off. Anal. Chern. 57:618-620. logical rapid methods in the bioassay of mycotoxins. Int. Biodeter. 13. Flannigan, B. 1996. The microflora of barley and malt, p. 83-125. In 22:141-145. F. G. Priest and 1. Campbell (ed.). Brewing microbiology, 2nd ed. 26. Northolt, M. D., H. P. van Egmond, and W. E. Paulsch. 1978. Patulin Chapman & Hall, Ltd., London. production by some fungal species in relation to water activity and 14. Flannigan, B., and A. R. Pearce. 1994. Aspergillus spoilage: spoilage temperature. J. Food Prot. 41:885-890. of cereals and cereal products by the hazardous species A. clavatus, p. 27. Paterson, R. R. M., and P. D. Bridge. 1994. Biochemical techniques ll5-127. In K. A. Powell, J. Peberdy, and E. Renwick (ed.). Biology for filamentous fungi. CAB International, Wallingford, U.K. of aspergillus, Plenum, New York. 28. Reiss, J. 1972. Comparing investigations on the toxicity of some 15. Fujishima, T., M. Ichidawa, H. Ishige, H. Yoshino, H., J. Ohishi, and mycotoxins to the larvae of the brine shrimp (Artemia salina). S. Ikegami. 1979. Production of cytochalasin E by Aspergillus Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. I Orig. B terre us. Hakkokogaku Kaishi 57:15-19. 155:531-534. 16. Glinsukon, T., B. Kongsuktrakoon, C. Toskulkao, and S. Sophasan. 29. Schmidt, R. 1989. The application of Artemia salina L. bioassay for 1983. Cytochalasin E: inhibition of intestinal glucose absorption in screening of fusaria toxins, p. 121-130. In J. Chelkowski (00.) the mouse. Toxicol. Lett. 15:341-348. Fusarium mycotoxins, and pathology, Elsevier, Amster- 17. Glinsukon, T., R. C. Shank, G. N. Wogan, and P. M. Newberne. 1975. dam. Acute and subacute toxicity of cytochalasin E in the rat. Toxicol. 30. Scott, P. M. 1977. Penicillium mycotoxins, p. 283-356. In T. D. Appl. Pharmacol. 32:135-146. Wyllie and L. G. Morehouse (ed.), Mycotoxic fungi, mycotoxins, 18. Glinsukon, T., S. S. Yuan, R. Wightman, Y. Kitaura, G. Buchi, R. C. mycotoxicoses, vol. I. Mycotoxic fungi and chemistry of mycotoxins. Shank, G. N. Wogan, and C. M. Christensen. 1974. Isolation and Marcel Dekker, Inc., New York. purification of cytochalasin E and two tremorgens from Aspergillus 31. Suarez, G., J. Guarro, and M. A. Calvo. 1981. Toxicological study of clavatus. Foods Man l:ll3-ll9. fungi isolated from intended for human consumption. 19. Harrach, B., C. J. Mirocha, S. V. Pathre, and M. Palynusik. 1981. Mycopathologia 75:27-31. Macrocyclic trichothecene toxins produced by a strain of Stachybot- 32. Vesely, D., D. Vesela, and R. Jelinek. 1984. Use of chick embryo in rys atm from Hungary. Appl. Environ. Microbiol. 41:1428-1432. screening for toxic-producing fungi. Mycopathologia 88: 135-140.