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Mycotoxins zearalenone and vomitoxin (4-deoxynivalenol) are often joint contaminants of grains infested by micromycetes of the genus Fusarium. Toxic effects of both mycotoxins on experimental organisms and farm animals are well known, but we have not found any literary reference to toxic effects of the combination zearalenone and vomitoxin. Embryotoxic effects of zearalenone, vomitoxin and combinations of various doses of zearalenone with constant addition of vomitoxin were studied in a three-day chick embryo. The objective of the study was to determine the coaction of vomitoxin on zearalenone embryotoxicity. Thermostat-incubated fertile eggs of White Leghorn hens were candled after three-day incubation, the shell above the embryo was removed, and within the embryotoxicity range zearalenone, vomitoxin and various doses of zearalenone with constant addition of 2 micrograms vomitoxin were applied to morphologically normal embryos. The groups of ten embryos were applied mycotoxins and their combinations in 10 microliters of their solutions to amnions using a special glass micropipette. Control group comprised twenty embryos which were applied 10 microliters of solvents used, 1% NaHCO3 and 10% ethanol. The eggs were covered with glass plates and their incubation was going on until the eighth day of their development. The embryos that died during incubation were discarded. On the eighth day of development, surviving embryos were taken out from the eggs and malformations of head, orofacial region, body wall, limbs and heart were determined microscopically. Tab. I shows total numbers of dead and malformed embryos after application of the particular doses of zearalenone, vomitoxin, their combinations and control solvents. The embryotoxicity range started at a dose of 5 to 20 micrograms per embryo. Zearalanone did not have any teratogenic effects on chick embryos. Applications of high doses of zearalenone (100 and 30 micrograms) instantly caused arrhythmia, atrio-ventricular dissociation or even heart stoppage. The beginning of the embryotoxicity range for vomitoxin was found to be within the narrow range of 1 to 3 micrograms per embryo. Among malformations, only a defect of the interventricular septum of the heart was found in 4% of the cases. The combined embryotoxic effects of zearalenone and vomitoxin were of additive, and mostly embryolethal nature. Among the malformations searched for, only 5% of the embryos exhibited a defect of the interventricular septum of the heart. Due to the good prediction fitness of chick embryo that has been proved by estimates of mycotoxin toxicity to mammals it is possible to suppose that toxic effects of the frequently occurring combinations of zearalenone and vomitoxin in fusarium-infected feeds will also be of additive nature for farm animals.

Recent data from deep uranium mines in Czechoslovakia indicated that in addition to radon daughter products, miners are also exposed to chemical mutagens. Mycotoxins were identified as a possible source of mutagenicity present in the mines. Various methods of biomonitoring were used to examine three groups of miners from different uranium mines. Cytogenetic analysis of peripheral lymphocytes, unscheduled DNA synthesis (UDS) in lymphocytes, and lipid peroxidation (LPO) in both plasma and lymphocytes were studied on 66 exposed miners and 56 controls. Throat swabs were taken from 116 miners and 78 controls. Significantly increased numbers of aberrant cells were found in all groups of miners, as well as decreased UDS values in lymphocytes and increased LPO plasma levels in comparison to controls. Molds were detected in throat swabs from 27% of miners, and 58% of these molds were embryotoxic. Only 5% of the control samples contained molds and none of them was embryotoxic. The following mycotoxins were isolated from miners' throat swab samples: rugulosin, sterigmatocystin, mycophenolic acid, brevianamid A, citreoviridin, citrinin, penicilic acid, and secalonic acid. These data suggest that mycotoxins are a genotoxic factor affecting uranium miners.

Embryotoxic effects of 25 mycotoxins were investigated in two-, three- and four-day chick embryos; the results were evaluated on the eighth day of development. The embryotoxicity ranged from 0.0001 to 0.1 microgram per embryo in T-2 toxin, aflatoxin B1, G1, B2 and M1, cytochalasin E, ochratoxin A and PR-toxin; from 0.1 to 1.0 microgram per embryo in sterigmatocystin, aflatoxin G2, vomitoxin (4deoxynivalenol), patulin, rubratoxin B, secalonic acid D, mycophenolic acid, and from 1.0 to 100 micrograms per embryo in penicillic acid, cyclopiazonic acid, tenuazonic acid, citrinine, brevianamide A, zearalenone, fusaric acid, griseofulvin, kojic acid and 8-methoxypsolaren. Acute cardiotoxic effects were observed in PR-toxin, patulin, rubratoxin B, penicillic acid, citrinine and zearalenone. Teratogenic effects with a spectrum of different embryonal malformations occurred in T-2 toxin, ochratoxin A, PR-toxin, patulin, secalonic acid D, mycophenolic acid and citrinine. The embryotoxic effects demonstrated in chick embryos correlated with the well-know literary data on mammals. Considering the different chemical composition and biological effects of mycotoxins, we suppose that the embryotoxicity test of chick embryos will also be suitable for testing other biologically active substances in the environment.

The toxinogenicity of 720 fungal isolates was evaluated using the Chisk Embryotoxicity Screening Test (CHEST, phase I). Three hundred and sixteen (43.9%) isolates produced some of the 23 identified mycotoxins, unidentified toxic metabolites were produced by 170 (23.6%), and 234 (32.5%) isolates showed no signs of toxinogenicity. The 24 h lasting chick embryo assay proved very suitable for detecting fungal products with general cytotoxic potential where it yielded results consistent with those obtained with cell cultures and higher organisms. Simplicity, rapidity, and modest laboratory equipment belong to the advantages of this reliable and non expensive screening procedure.

The activity of 20 mycotoxins was tested by the recently developed initiator tRNA acceptance assay for carcinogens. With the exception of citrinin, all compounds carcinogenic for rodents stimulated the charging of tRNA with L-methionine. In three out of four non-carcinogenic mycotoxins the test was negative. Five carcinogenic mycotoxins were negative in mutagenicity tests but positive in the acceptance assay indicating that also non-mutagenic carcinogens may be detected by the latter procedure.

Embryos of White Leghorn Fowls incubated for 40 h were injected subgerminally with 19 mycotoxins dissolved in 30% ethanol to provide a range of doses. Embryonic death as well as the incidence of caudal-trunk abnormalities were determined after a further 24 h incubation. Of the substances tested, the maximum toxic effects were exerted by T-2 toxin and diacetoxyscirpenol which produced 100% embryonic mortality at doses as low as 0.01 microgram. 100 micrograms of griseofulvin, on the other hand, were needed to achieve the same effect. Abnormal development of the caudal trunk was observed after T-2 toxin and diacetoxyscirpenol (0.001 micrograms each) and griseofulvin (10 micrograms) administration. Comparison of these data with results published for both cell and tissue culture techniques and for classical rodent acute toxicity tests reveals a high predictive value for the Chick Embryotoxicity Screening Test (CHEST I), at least for mycotoxins.

The fungus Fusarium tricinctum, producing T-2 toxin, was isolated from mouldy maize silage. T-2 toxin was identified by TLC and by a bioassay. Consumption of mouldy silage resulted in a disease in a dairy cow, which had to be killed. The patho-anatomical findings obtained in the slaughtered cow generally suggested trichothecene poisoning. It was derived from these findings that the disease was due to the presence of trichothecenes in the feed.

Contamination with microscopic fungi was studied in broiler complete feed mixtures in 1983-1984. The contamination levels ranged from 10(1) to 10(5) of fungus units per gram of feed (mostly around 10(3) units per gram). Sixty species of microscopic fungi of sixteen genera were isolated from the feeds. The genera Aspergillus and Penicillium occurred most frequently (97% of samples), followed by Mucor (93%), Cladosporium (79%), Rhizopus (52%) and Fusarium (41%). Of the 64 isolates of ten species of microscopic fungi, 42 (66%) were toxicogenic, and out of 49 such strains, 27 (55%) were toxic.