Odette L. Shotwell

Measurements of airborne aflatoxins during the handling of contaminated corn

Sample of airborne dust generated the handling of aflatoxin-contaminated corn were collected and analyzed to assess potential exposures of farmers and other agricultural workers to these mycotoxins. Using high volume total dust samplers and a high volume Andersen sampler, downwind dust samples were collected on glass filter when contaminated corn was transferred by augers from a storage bin into a wagon and back into the storage bin. The aflatoxin B1 content of the 15 dust samples ranged from 12.5 to 204.3 ppb, with an average of 138 ppb; the aflatoxin B2 content ranged from 1.1 to 41.6 ppb, with an average of 24.6 ppb. The B1 and B2 levels of contamination in the bulk corn were 223.9 and 17.5 ppb, respectively. The gravimetric dust concentration in the air ranged from 7 mg/m3 to 417 mg/m3. The samples taken with an Andersen sampler indicate the dust is relatively coarse with only approximately 17% less than 7 micrometer. An analysis of the dust from each stage showed higher levels of aflatoxins in the larger first-stage particles than in the finer particles on the succeeding stages. The results of this study indicate that the dust generated when handling contaminated commodities also may be contaminated and represent a potential inhalation hazard. This fact, coupled with the extreme toxicity and carcinogenicity previously demonstrated in animal studies, suggests that appropriate measures be taken to prevent worker exposure during handling of contaminated materials.

Aflatoxin and Mold Flora in North Carolina in 1977 Corn Crop

In a 1977 survey, 187 of 238 maize samples from the Piedmont and Coastal Plain area contained aflatoxin; of these 139 were above the 20 p.p.b. guideline. Aflatoxin G1 was found in 32 samples from the Piedmont/mountain area. The highest level of aflatoxin found was 3622 p.p.b. In general, the higher the percentage of insect-damaged maize the higher the aflatoxin levels found. The sample with the most insect damaged maize (11%) showed a level of 2208 p.p.b., and all samples with 4.5% or more insect damage contained aflatoxin. In general, the greater the infection of maize by Aspergillus flavus the higher the level of aflatoxin, with 7 samples having 40-44 undamaged kernels out of 50 infected. A. niger was found in 10.9% of the samples, but there was no positive correlation with the amounts of aflatoxin found. Strs. of Penicillium funiculosum series isolated from S. Carol. were found again in 25.3% of the total kernels examined. Few other P. or A. spp. were seen. Maize germination was high in most samples. Assuming that most of the aflatoxin G1 was produced by A. parasiticus, a definite ecological correlation was found between G1 and a specific area, i.e. the Piedmont/mountain area.

Zearalenone in cereal grains

Zearalenone, a secondary metabolite with estrogenic properties, is produced by severalFusarium species that colonize cereal grains in the field and in storage. Recently, there have been reports of zearalenone contamination in corn, oats, barley, wheat, and grain sorghum. Corn and grain sorghum were examined for contamination due to obvious mold damage. Wheat, corn, and sorghum have been examined to determine the incidence of zearalenone in grains moving through commercial channels and stored on farms and at country elevators. Other grains such as oats and barley were analyzed because of associated estrogenic disturbances in farm animals. Stepsin procedures for the determination of zearalenone are extraction of a representative sample, partial purification of the extract by column chromatography, alkali treatment, or liquid-liquid partitioning, and subsequent measurement of the isolated toxin. Zearalenone is measured in partially purified extracts by thin layer chromatography (TLC), gas liquid chromatography (GLC), and high pressure liquid chromatography (HPLC). Confirmation of zearalenone contamination can be accomplished by gas chromatography-mass spectroscopy (GC-MS). Multitoxin screening procedures have been deveoped for zearalenone in combination with one or more of the following mycotoxins: aflatoxin, T-2 toxin, diacetoxyscirpenol, patulin, ochratoxin, penicillic acid, citrinin, penitrem A, and sterigmatocystin.

Utilization of zearalenone-contaminated corn for ethanol production.

Two lots of yellow corn, severely damaged byFusarium fungi and contaminated with 8.0 and 33.5 ppm zearalenone, respectively, were used for ethanol fermentations. Substrate corn (5-kg samples) was processed in a laboratory procedure similar to that used by the fermentation industry. Stillages obtained were 7.0 to 9.0% ethanol. Ethanol was recovered by distillation, residual grain solids by filtration, and solubles by concentration. No zearalenone could be detected in the ethanol fraction. Zearalenone in the original corn was concentrated in the residual solids and solubles, which are generally used for animal feed. Treatment with formaldehyde significantly reduced the level of zearalenone in fermentation solids. Ammonium hydroxide was a much less effective agent for toxin degradation.

Aflatoxin in corn

Abstract and SummaryLow incidence and levels of aflatoxin were identified in corn of all grades grown in the Midwest in 1964, 1965, and 1967. Later surveys indicate that corn grown in southern regions is subject to invasion byAspergillus flavus and subsequent aflatoxin formation. This mycotoxin is formed either in the field or in storage. In the field, such factors as insect damage and weather conditions probably influence aflatoxin formation. In storage, temperatures must be above 25 C and moisture levels above 16% if toxin is to form. Aflatoxin formed in a hot spot in stored corn in the Midwest when temperatures rose early in the summer and when the grain became wet because of leaks in the storage building. Analytical methods to detect and determine aflatoxin fall into three categories: presumptive tests indicating the presence ofA. flavus and the possible occurrence of aflatoxin, rapid screening tests establishing the presence or absence of the toxin, and quantitative procedures determining toxin levels. Detoxification methods being studied include ammoniation and roasting. Ammoniated corn is being fed domestic animals to determine whether it has adverse effects and whether toxic compounds are transmitted in animal tissues.

Aflatoxin Occurrence in 1973 Corn at Harvest. II. Mycological Studies

SUMMARY Since aflatoxin is formed in corn in the field before harvest, our objectives were to determine at harvest (a) the amount of Aspergillus flavusinfected corn kernels, (b) the amount of A. flavus spores on the surface of corn, (c) the total amount of fungus-infected kernels, (d) the occurrence of A flavus spores in and on insects from corn reported in the first paper of this series, and (e) the correlation between A. flavus infection and occurrence of aflatoxin. The corn was collected at harvest from seven counties in northeastern South Carolina and dried to less than 13% moisture as quickly as possible. Of the 152 aflatoxin-positive samples, 120 showed one or more kernels internally infected with A. flavus and of the 145 aflatoxin-negative samples, 59 showed infection. Of the 297 samples examined, 276 had one or more kernels with surface contamination of A. flavus spores, and in 75 of the samples every kernel was contaminated. When kernels were surface disinfected, 224 samples (50 kernels each) showed 100% internal mold contamination. One or more kernels of 185 samples were infected with A. flavus; this number represents 60% of the total samples. Of the 375 insects collected and examined for A. flavus from the corn samples, 247 showed A. flavus present. Of the 85 rice weevils, 78 were carrying A. flavus spores and of the other 290 insects, 165 were contaminated. Besides A. flavus, the predominant infecting fungi internally were two species of Penicillium and Fusarium. Members of the Mucorales were rarely seen. The occurrence of Aspergillus flavus Link ex Fries and A. parasiticus Speare growing on corn plants in the field before harvest has rarely been recorded (Taubenhaus, 1920). From 0.02 to 0.09% infection of corn kernels before harvest was reported by Tuite (1961) from Indiana. The highest single instance had a 22% infection by A. flavus. In 1970, Tuite and Caldwell (1971) found an average incidence of 0.4% kernel infection before harvest in corn also in Indiana. The A. flavus incidence in southern Indiana counties was 1.2% as compared to 0.2% in northern counties. Rambo et al. (1974) reported a 0.03 to 0.08% incidence of

Isolation and purification of deoxynivalenol and a new trichothecene by high pressure liquid chromatography

Deoxynivalenol (3,7,15-trihydroxy-12,l3-epoxytrichothec-9-ene-8-one) was extracted from corn with methanol/water (80:20, v/v) and purified by liquid:liquid partitioning and by preparative high pressure liquid chromatography (HPLC). This procedure was used to prepare mg quantities of toxin from field-inoculated corn for reference standards. Analysis of the isolated deoxynivalenol by analytical HPLC, gas liquid chromatography (GLC) and gas liquid chromatography/mass spectroscopy (GLC/MS) indicated the presence of a second compound similar to deoxynivalenol. This compound comigrates with deoxynivalenol on thin layer chromatography plates in chloroform/methanol (90:10, v/v), but can be separated by HPLC on a reverse-phase C8 column with methanol/water (10:90, v/v). GC/MS of the compound and the trimethylsilyl ether derivative gave parent ions of m/e 280 and 424, respectively. These data and NMR data indicate that the compound is 3,15-dihydroxy-12,13-epoxytrichothec-9-ene-8-one, a previously unreported trichothecene.

Aflatoxin M1 removal from aqueous solutions by Flavobacterium aurantiacum

In liquid cultures growing and stationary phase cells ofFlavobacterium aurantiacum NRRL B-184 eliminated aflatoxin M1. Toxin concentrations of 15µg/ml and 37.5µg/ml interfered with bacterial growth, and at the higher level 4.4µg M1 was removed from the growth medium by a milligram (dry weight) of bacteria. Toxin was completely removed from the liquid medium by incubating 5 × 1010 resting cells per milliliter with 8µg/ml of aflatoxin M1 for 4 h. Attempted recovery of M1 from cells following incubation of the bacteria with the toxin demonstrated that the M1 was essentially nonextractable. Bacterial cells also removed aflatoxin M1 from toxin-contaminated milk.

Destruction of zearalenone in contaminated corn

Several chemical and physical treatments were investigated as possible methods for destroying zearalenone in contaminated corn. An ammoniation process which significantly lowers aflatoxin levels had no effect on zearalenone contamination in yellow corn. Also, treatments of propionic acid, acetic acid, hydrochloric acid, sodium bicarbonate and hydrogen peroxide failed to reduce toxin levels. High-temperature treatment (150 C) had no effect on zearalenone. Formaldehyde, in vapor form from paraformaldehyde crystals or in aqueous solutions, destroyed significant quantities of zearalenone in naturally contaminated yellow corn meal and in spiked corn grits and animal feed. Samples treated with aqueous formaldehyde must be dried at 50 C or more to cause effective destruction of zearale-none. Levels as high as 10 ppm zearalenone in animal feed and 8 ppm in ground corn were reduced to less than 0.5 ppm with formal-dehyde. Ammonium hydroxide and formaldehyde partially des-troyed zearalenone in highly contaminated ground corn. Levels as high as 33.5 ppm were reduced to 12 ppm by 3% ammonium hydroxide and to 2.1 ppm by 3.7% formaldehyde. No treatment used in this study significantly reduced zearalenone levels in whole-kernel corn.

Hydrocarbons in haemolymph from healthy and diseased Japanese beetle larvae

Abstract The hydrocarbons in extractable lipids of haemolymph from healthy and diseased larvae of the Japanese beetle ( Popillia japonica ) have been characterized. Haemolymph contains at least 21 saturated hydrocarbons having from 21 to 27 carbon atoms. Normal, monomethyl-branched, and dimethyl-branched alkanes with even and odd carbon numbers were identified. Tricosane (12%), 11-methyltricosane (19%), 9,13-dimethyltricosane (27%), and 11-methylpentacosane (11%) are the major hydrocarbons. Haemolymph from third instar larvae contains at least six minor hydrocarbons that could not be obtained in sufficient quantities for structure identification. The total amount of hydrocarbons per ml is reduced in haemolymph from larvae infected with Bacillus popilliae ; however, the relative concentration of each hydrocarbon is not significantly changed as a result of infection.