New, sensitive high-performance liquid chromatography method for the determination of slaframine in plasma and milk.

Slaframine was derivatized precolumn with fluorescamine. The derivatized slaframine was chromatographed isocratically using HPLC on a Hamilton PRP-1 C18 polymeric column with fluorescence detection. By using fluorescent derivatization, sensitivity was increased 100-fold over previously reported GC methods. A liquid-liquid partition was used to extract slaframine from plasma with a 95% recovery and a CV% of 8.4. A solid-phase extraction was used to extract slaframine from milk with a 91% recovery and a CV% of 9.8.

Cocklebur toxicosis in cattle associated with the consumption of mature Xanthium strumarium.

Cockleburs (Xanthium spp.) are herbaceous annuals with worldwide distribution. Toxicoses are usually associated with the consumption of the seedlings in the cotyledon stage, which contain a high concentration of the toxic principle, carboxyatractyloside. The seeds are also known to contain the toxin, but it has long been assumed that the spiny capsule would deter their consumption. Six of 70 yearling calves died while being fed round bale hay composed predominantly of foxtail and mature cocklebur plants with burs. Clinical signs ranged from acute death to hyperexcitability, blindness, tense musculature, and spastic gaits with heads held high and ears erect. Some symptomatic calves would stumble, fall to lateral recumbency, convulse, and later recover. Overall, the herd was very uneasy. Prominent gross lesions were ascites and a firm, pale liver with a mottled hemorrhagic pattern on cut surface. The rumen contained numerous intact burs and well-ruminated grass. Histological examination of the liver revealed marked centrolobular degeneration and necrosis with associated hemorrhage and congestion. Brain lesions were present. Plant and tissue samples were analyzed for carboxyatractyloside with various results. Samples of rumen contents, urine, and burs contained 100-200 ppm, 0.1-0.05 ppm, and 0.1 ppm, respectively. Based on the history, clinical signs, pathological lesions, and chemical analyses, cocklebur toxicosis associated with consumption of mature Xanthium strumarium in hay was confirmed.

Evaluation and application of a bioluminescent bacterial genotoxicity test.

The Mutatox test (commercial name for the bioluminescent bacterial genotoxicity test) has been shown to be a good alternative to the Ames test. The test uses dark mutants of luminous bacteria (Vibrio fischeri) and determines the ability of various genotoxic agents to restore the luminescence by inducing mutation. It provides a rapid screening test which can be used to assay the genotoxicity of large numbers of pure and complex compounds. The test is completed in 1 day, and by serially diluting the compound, dose response data plus toxicity data can be generated for a number of samples simultaneously. For the direct assay (without exogenous metabolic activation), the positive controls selected were 3,6-diaminoacridine (proflavine) and N-methyl-N-nitro-nitrosoguanidine. For the S-9 assay, which incorporated the microsome fraction (S-9) from rat liver as an exogenous metabolic activation system, the positive controls selected were aflatoxin B1 and benzo(a)pyrene. This study also indicated that methyl-imidazo-quinoline and tryptophan pyrolysates were genotoxic in the presence of S-9 activation, aflatoxin B1 epoxide and fumonisin B1 showed direct genotoxic activity, and aflatoxin B2 and ochratoxin A were not genotoxic.

Thin layer chromatographic determination of citrinin.

A thin layer chromatographic (TLC) method is described for the determination of citrinin in feeds. Citrinin is extracted from feed with methanol and water, the mixture is made alkaline with 10% sodium carbonate, and the aqueous solution is filtered and extracted with chloroform to remove most of the interfering materials. The aqueous layer is acidified with 2N HCl and extracted with chloroform. The chloroform extract is concentrated and spotted on a thin layer chromatographic (TLC) plate which is developed in chloroform-acetone-ethanol-water (60 + 40 + 10 + 1). The citrinin is viewed under ultraviolet light after TLC. Either visual or fluorodensitometric quantitation is used. Recoveries of citrinin from various feed samples spiked at levels of 2.0--5 micrograms/g were 75--92%. The proposed method can detect 0.5 micrograms/g feed, including corn, silage, ready mixed feeds, and feed pellets.

Retention and distribution of aflatoxin in tissues of chicks fed aflatoxin-contaminated poultry rations amended with soil.

The effects of silty clay loam on aflatoxin B1 (AFB1) retention and distribution were investigated when added to diets of chicks fed aflatoxin-contaminated rations. One hundred 14-d-old White Leghorn chicks were fed a control ration (clean corn), a low aflatoxin-contaminated ration (120 ng AFB1/g), a high aflatoxin-contaminated ration (700 ng AFB1/g), or a high aflatoxin-contaminated ration (700 ng AFB1/g) + 10% or 25% soil. Livers, crops and breast muscles in each group were pooled and analyzed for AFB1 and metabolites. The addition of soil significantly reduced the AFB1 levels in the livers, although the reduction was less when 10% soil was fed compared with the 25% soil feeding. AFB1 concentrations in the crops of chicks fed high aflatoxin-contaminated ration without soil was statistically indistinguishable from the chicks in the other groups. AFB1 concentrations were significantly reduced in the breast muscles of chicks fed 10% or 25% soil compared to chicks fed the high aflatoxin-contaminated ration without soil. Aflatoxin B2 was detected only in livers, crops and breast muscles from chicks fed aflatoxin-contaminated ration without soil. Aflatoxin M1 (AFM1) was detected only in livers and crops of chicks fed aflatoxin-contaminated ration without soil and only in breast muscles of chicks fed the low aflatoxin-contaminated ration and high aflatoxin-contaminated ration + 25% soil. AFM1 concentration was significantly higher in the crops of chicks fed high aflatoxin-contaminated ration without soil.

Effect of soil on aflatoxin tissue retention in chicks when added to aflatoxin-contaminated poultry rations.

The effects of silty clay loam soil on aflatoxin B1 (AFB1) absorption were investigated when added to the diets of chicks fed aflatoxin-contaminated rations. Sixty 1-d-old White Leghorn chicks were fed a control ration (< 5 ng AFB1/g), a low aflatoxin-contaminated ration (55 ng AFB1/g), a high aflatoxin-contaminated ration (4,488 ng AFB1/g), or high aflatoxin-contaminated rations (4,488 ng AFB1/g) + 25% or 50% soil. Livers in each group were pooled and analyzed for AFB1 and metabolites. The addition of soil significantly reduced the levels of AFB1 in the livers, although the reduction was less when 25% soil was fed compared with the 50% soil feeding.

Detoxification of aflatoxin contaminated corn by methanol extraction.

Corn containing 500 ppb aflatoxin was decontaminated by methanolic extraction. Ninety percent of the aflatoxin was removed in one three-minute blending with a solvent to corn ratio of 4:1. Recycling methanol for additional extractions could be done by removal of aflatoxin by carbon or distillation of the methanol. Methanol costs per bushel of corn without recycling would probably be expensive. By recycling, the cost is estimated to be ten to twenty cents per bushel in a laboratory size experiment. A source of ethanol on the farm could make the process more feasible. Chicks fed decontaminated corn showed no signs of aflatoxin toxicity. Fatty degenerated livers were produced in chicks fed the nondetoxified corn. Other mycotoxins which are not affected by the ammoniation process used to decontaminate corn, can be removed by this process.

Determination of extractable mirex in whole blood.

A method for the determination of free mirex in blood has been developed in which whole blood is extracted with acetone-hexane (9+91), passed through Na2SO4, concentrated, and analyzed by electron capture gas-liquid chromatograpohy (GLC). Results were highest for fresh blood. Recoveries were verified by determining 14C-labeled mirex before and after extraction. Little mirex was detected in the samples after extraction. Hydrolysis of residual blood indicates that a metabolite of mirex is released. The possible metabolite has a GLC retention time and mass spectrum which resemble hydroxy metabolites of mirex.

Effects of Phosphate Solutions on Aflatoxin Production in a Synthetic Medium and in Frankfurters †.

Mold growth, sporulation, and aflatoxin B1 and G1 production were studied in Sabouraud dextrose agar (SDA) and frankfurters inoculated with Aspergillus flavus or Aspergillus parasiticus . Each of four phosphates, sodium polyphosphate glassy (SPG), sodium acid pyrophosphate (SAPP), tetrasodium pyrophosphate (TSPP), and Brifisol 414 (a blend of SPG, SAPP, and TSPP) were incorporated into the SDA (1 or 2%) or used as dipping solutions (5%) for the frankfurters. In SDA at 30°C, significant (P < 0.05) reductions in aflatoxin B1 and G1 production by A. flavus and A. parasiticus occurred when 1% SPG, 1% TSPP, 1% Brifisol 414, and 2% SAPP were present. In frankfurters, A. flavus B1 aflatoxin production was increased with SAPP and TSPP.

Distribution and Effects of Aflatoxin in Chicken Tissues After Feeding Radiolabeled (14C) Aflatoxin B1.

The effects of single oral administration of 1 mg of 14C-aflatoxin B1 to 3-wk-old chickens and 10 mg of non-radioactive aflatoxin to 6-wk-old chickens were determined. Analyses were made of the distribution of 14C in the blood, organs, tissues and feces. No toxic effects were observed on chickens fed 1 mg of aflatoxin B1 during 72 h but those fed 10 mg of non-radioactive aflatoxin exhibited both altered physical signs and pathological findings. Both the 14C-aflatoxin studies and chemical analyses for the parent compound indicated that most of the aflatoxin was excreted in the feces within 48 h and of the amount retained, most was found in the liver.

Evaluation of a commercial immunoassay system for sulfamethazine and aflatoxin detection in biological fluids and feeds.

Commercial immunoassay systems produced by International Diagnostics to analyze for sulfa drugs and aflatoxin residues have been evaluated with milk, urine, feed and serum samples. Neither system produced satisfactory results with feeds. The sulfa test reproducibility was good enough to provide semiquantitative results on milk (40 ul sample) and urine, but should be regarded as qualitative on urine or feed. The reproducibility of the aflatoxin test was acceptable for semiquantitative use on serum, but should be regarded as qualitative on urine and semiquantitative on milk with a solid phase concentration. The overall reproducibility was at least +/- 10% if the test was used semiquantitatively. The sensitivity of the test was 10 ng for both sulfamethazine and aflatoxin B1. The range of the color change was very narrow (0-20 ng). The aflatoxin test was designed for aflatoxin B1 and was roughly twice as sensitive to B1 as to M1 aflatoxin. These procedures can be used on appropriate matrices at suitable sensitivities to rapidly screen samples for sulfa drugs and aflatoxin residues. The use of proper standards to demonstrate effectiveness in each matrix is very important.

Effects of exposure time, material type, and granular pesticide on glove contamination.

Chemical-resistant gloves are recommended for pesticide applicators to reduce their exposure to agricultural chemicals. In this research, three chemical-resistant glove materials-nitrile, neoprene, and barrier laminate-were studied in relation to contamination with granular terbufos and tefluthrin. Surfaces of specimens backed with alpha cellulose were contaminated with 300 mg of either granular terbufos or tefluthrin for 1-, 2-, 4-, 8-, 16-, and 24-h time periods in petri dishes in the laboratory. Residues were extracted using ethyl acetate for terbufos and iso-octane for tefluthrin in test tubes for 24 h. Analysis of extracts by gas chromatograph and statistical analysis of the data showed that contamination levels varied with the time of exposure, material type, and pesticide used. Pesticide was not detected in the alpha cellulose even after 24 h contamination time. A linear relationship was found between contamination level and exposure time for terbufos in the three materials, with longer exposure times causing higher contamination levels. Contamination of nitrile was significantly less than neoprene or barrier laminate. Exposed glove materials contained higher levels of contamination of terbufos than tefluthrin.

The effect on performance and biochemical parameters when soil was added to aflatoxin-contaminated poultry rations.

The effects of silty clay loam soil on the performance and biochemical parameters of chicks were investigated when the soil was added to aflatoxin B1 (AFB1)-contaminated diets. One hundred 14-d-old White Leghorn chicks were fed a control ration (clean corn), a low aflatoxin-contaminated ration (120 ng AFB1/g), a high aflatoxin-contaminated ration (700 ng AFB1/g), or high aflatoxin-contaminated rations (700 ng AFB1/g) +10% or 25% soil. Body weight, feed consumption and blood samples were monitored weekly. Decreased feed consumption, body weight gain and efficiency of feed utilization, increased SGOT and LDH activities, and cholesterol and triglyceride concentrations, and decreased uric acid concentrations and ALP activity were observed in the chicks fed the high aflatoxin-contaminated ration without soil. Hepatomegaly was prominent in chicks fed the high aflatoxin-contaminated ration without soil, and some livers had extensive hepatocyte vacuolation, hepatocellular swelling, fatty change and hydropic degeneration, and stained positive for fat accumulation. Addition of soil reduced the detrimental effects of AFB1 for some parameters, although the reduction was less when 10% soil was fed compared with the 25% soil feeding.

Cleanup of gloves contaminated with granular terbufos and tefluthrin.

Chemical-resistant gloves are used for protection from pesticides in farming operations. Cleanup of gloves after pesticide contamination was the focus of this research. Nitrile, neoprene, and barrier laminate glove specimens were exposed to 300 mg terbufos or tefluthrin granules for 3 or 30 min in petri dishes in a laboratory. Specimens were cleaned by flush with running water or LaunderOmeter washing with detergent. Following the cleanup treatments, specimens were dried and placed in test tubes with solvents to extract pesticide residue. Levels of contamination remaining were determined by gas chromatography. The residue remaining varied with exposure time, material type, cleanup method, and pesticide. Flush was more effective with the shorter exposure time. Tefluthrin was more effectively removed than terbufos. Barrier laminate was confirmed as a single-use material. Cleanup procedures reduced contamination in nitrile and neoprene, but findings show that these materials retained residue after cleanup.

Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

Twenty-eight of 54 isolates of Aspergillus flavus grown on autoclaved agricultural commodities such as wheat, rice and corn were found to produce the mycotoxin cyclopiazonic acid. Eighteen of the A. flavus isolates produced aflatoxin, and fourteen isolates produced both cyclopiazonic acid and aflatoxin. A preliminary screening of some aflatoxin-contaminated corn samples revealed for the first time the natural occurrence of cyclopiazonic acid in agricultural commodities.