Using chiral liquid chromatography quadrupole time-of-flight mass spectrometry for the analysis of pharmaceuticals and illicit drugs in surface and wastewater at the enantiomeric level.

This paper presents and compares for the first time two chiral LC-QTOF-MS methodologies (utilising CBH and Chirobiotic V columns with cellobiohydrolase and vancomycin as chiral selectors) for the quantification of amphetamine, methamphetamine, MDA (methylenedioxyamphetamine), MDMA (methylenedioxymethamphetamine), propranolol, atenolol, metoprolol, fluoxetine and venlafaxine in river water and sewage effluent. The lowest MDLs (0.3-5.0 ng L(-1) and 1.3-15.1 ng L(-1) for river water and sewage effluent respectively) were observed using the chiral column Chirobiotic V. This is with the exception of methamphetamine and MDMA which had lower MDLs using the CBH column. However, the CBH column resulted in better resolution of enantiomers (R(s)=2.5 for amphetamine compared with R(s)=1.2 with Chirobiotic V). Method recovery rates were typically >80% for both methodologies. Pharmaceuticals and illicit drugs detected and quantified in environmental samples were successfully identified using MS/MS confirmation. In sewage effluent, the total beta-blocker concentrations of propranolol, atenolol and metoprolol were on average 77.0, 1091.0 and 3.6 ng L(-1) thus having EFs (Enantiomeric Fractions) of 0.43, 0.55 and 0.54 respectively. In river water, total propranolol and atenolol was quantified on average at <10.0 ng L(-1). Differences in EF between sewage and river water matrices were evident: venlafaxine was observed with respective EF of 0.43 ± 0.02 and 0.58 ± 0.02.

Influence of halogen salts on the production of the ochratoxins by Aspergillus ochraceus Wilh.

The first report of the biological production of bromo ochratoxin B by Aspergillus ochraceus Wilh. is presented as well as a study of the influence of potassium bromide, potassium iodide, potassium fluoride, and potassium chloride on the production of ochratoxin A and ochratoxin B. Potassium fluoride and potassium iodide inhibited the growth of the fungus, whereas potassium chloride substantially stimulated the production of ochratoxin A in shaken solid substrate fermentation on whole wheat or shredded wheat, generally giving a high yield of ochratoxins. Increasing levels of potassium bromide led to a decline in ochratoxin A production and an increase in bromo-ochratoxin B, ochratoxin B, and 4-hydroxy ochratoxin B. Nevertheless, A. ochraceus was much less versatile in the bromo analogues than other fungi, which produce metabolites containing chlorine. Analysis included aminopropyl solid-phase extraction column cleanup followed by quantitative analysis on reversed-phase HPLC using fluorescence detection and employing N-(5-chloro-2-hydroxybenzoyl)phenylalanine as an internal standard.

Enumeration of fungi in barley.

Estimation of fungal contamination of barley grain is important as certain fungi can proliferate during the malting process. The following factors which may affect the enumeration of fungi were evaluated: dilution versus direct plating, presoaked versus unsoaked grain, five culture media: potato dextrose agar (PDA), acidified Czapek-Dox agar (ACA), pentachloronitrobenzene agar; (PCNB) dichloran rose bengal chloramphenicol agar (DRBC) and malt salt agar; two disinfectants' ethanol/water (80:20 v/v) and sodium hypochlorite (3.5% w/v in H2O). Two barley samples, one having a high incidence of storage fungi and one with a high incidence of field fungi were used and most fungi were identified to species level. Results showed that direct plating was superior to dilution plating for assessing the mycoflora of barley. Unsoaked grain gave significantly higher counts than presoaked grain in the case of Alternaria alternata, Rhizopus oryzae, Epicoccum nigrum and Mucor spp. Presoaked grain resulted in higher counts of Penicillium spp. Chlorine disinfection resulted in significantly higher counts of Aspergillus flavus, Eurotium spp. and Penicillium spp. Ethanol disinfection resulted in higher counts of Mucor spp., Phoma sorghina, Rhizopus oryzae and Aspergillus restrictus. PDA and ACA, in general gave some what better results than DRBC for both field and storage fungi. PCNB consistently gave the highest Fusarium counts. More than thiry fungal genera were found in the two samples.

Perinatal mortality in lambs of ewes exposed to cultures of Diplodia maydis (= Stenocarpella maydis) during gestation.

During 1987 when cob rot was rife, perinatal losses were reported in flocks and herds that had been exposed to diplodiosis. The affected lambs or calves were either stillborn or died soon after birth. Dosing trials at the Onderstepoort Veterinary Research Institute involving 82 ewes revealed that 66% of the offspring of dams exposed to cultures of Diplodia maydis (= Stenocarpella maydis) in the second trimester of pregnancy and 87% of lambs of ewes exposed in the third trimester were born dead or died soon afterwards. A status spongiosus similar to that of the field cases was present in the white matter of the brains of all the affected lambs. The lambs of ewes that had shown nervous signs and those of ewes that had not shown such signs were equally affected. The experiments also clearly demonstrated that foetuses were much more susceptible to diplodiosis than the adults, e.g. in the third trimester 87% of the lambs were affected compared to only 44% of the dams. No perinatal mortalities were recorded in the group exposed to the culture in the first trimester. Ewes developed significant resistance to intoxication after initial exposure to the culture and both the length of gestation and birth mass of the affected lambs were less than those of their untreated counterparts.

Toxicity and moniliformin production by four recently described species of Fusarium and two uncertain taxa.

Four recently described species. Fusarium nygamai, F. dlamini, F. beomiforme and F. napiforme and two uncertain taxa, F. nygamai from millet in Africa and Fusarium species from rice with Bakanae disease, were tested for toxicity and moniliformin production. Cultures grown on autoclaved corn were fed to groups of four one-day-old ducklings for 14 days. Isolates that caused the death of 3 or 4 out of 4 ducklings were considered to be toxic and analyzed for moniliformin. All 15 isolates of F. dlamini tested were nontoxic. The other taxa contained some isolates that were toxic to ducklings and produced moniliformin in corn cultures. This is the first report of moniliformin production by F. beomiforme (200-890 micrograms/g), and F. napiforme (16-388 micrograms/g), and by F. nygamai not obtained from millet in Africa (15-874 micrograms/g). The highest production of moniliformin was obtained from the 19 isolates of F. nygamai from millet in Africa (4300-18,200 micrograms/g) and the 15 isolates from rice with Bakanae disease (2300-19,300 micrograms/g). The taxonomic position of these two uncertain taxa should be re-evaluated.

T-2 toxin production by Fusarium acuminatum isolated from oats and barley.

Oats grain from South Africa was frequently found to be infested by toxic strains of Fusarium acuminatum, as was one barley sample. All 11 toxic strains tested produced T-2 toxin (0.8 to 2,600 mg/kg), and 6 of 11 strains produced diacetoxyscirpenol (0.6 to 8.4 mg/kg). This is the first record of T-2 toxin-producing Fusarium isolates from Africa and of the production of large amounts of T-2 toxin at relatively high (25 degrees C) temperatures.

Moniliformin production and toxicity of different Fusarium species from Southern Africa.

Four new moniliformin-producing species of Fusarium were found, viz., F. acuminatum, F. concolor, F. equiseti, and F. semitectum. Isolates of F. acuminatum and F. concolor produced large amounts of moniliformin (3.4 and 9.5 g/kg, respectively), whereas isolates of the other three species yielded less than 30 mg/kg. The production of moniliformin by isolates of F. oxysporum and F. avenaceum from southern Africa is described. All 14 toxic isolates of F. oxysporum produced moniliformin. Most isolates of F. fusarioides and all six isolates of Fusarium moniliforme va. subglutinans tested produced moniliformin, as did 28 of 36 toxic isolates of F. moniliforme. A number of F. moniliforme isolates produced greater than 10 g/kg, and one isolate yielded 33.7 g/kg in corn after incubation for 5 weeks at 25 degrees C. Moniliformin production in the field in corn ears was shown by inoculating plants with known moniliformin-producing isolates of three Fusarium species. Yields of up to 645 mg/kg were recorded. Isolates of F. acuminatum, F. equiseti, F. fusarioides, and F. moniliforme were found that were highly toxic to ducklings but which did not produce moniliformin.

Inhibitory effect of molybdenum and vanadium salts on aflatoxin B1 synthesis by Aspergillus flavus.

The effects of the elements zinc, manganese, iron, copper, molybdenum, and vanadium, added in various salt forms, on mycelial weights and aflatoxin B1 accumulation in the mycelium of Aspergillus flavus were investigated in liquid shake cultures. Ammonium heptamolybdate, when added to a complete medium at concentrations of 50-100 mg/L, appreciably reduced aflatoxin B1 accumulation without affecting growth of the fungus. Sodium molybdate and sodium monovanadate also reduced aflatoxin B1 yields without affecting mycelial growth but to a lesser extent. The addition of zinc sulphate stimulated aflatoxin B1 production in all media used. The influence of the other trace elements on aflatoxin production depended on the level of trace elements present in the basal medium. In general, manganese chloride had a stimulatory effect, whereas copper sulphate depressed yields. Mycelial levels of aflatoxin had peaked and then declined before mycelial dry weights had reached maximum. High yields of aflatoxin B1 were obtained in media having a final pH as low as pH 2.8.

Toxic aspergilli from pistachio nuts.

Pistachio nut samples taken during various stages of development from orchards in Iran, showed that contamination with fungi occurred mainly during the later stages of nut development. Members of the genera Aspergillus and Penicillium occurred most frequently. Of the Aspergilli, the species A. niger, A. flavus and A. fischeri var. spinosus occurred most frequently, followed by A. terreus, A. tamarii and A. nidulans. Twenty-two isolates comprising 13 species were tested for toxicity to ducklings. Isolates of known toxic fungi included A. flavus, A. niger, A. parasiticus, A. ochraceus, A. versicolor, A. nidulans and A. terreus. The toxicity of A. fischeri var. spinosus is reported. Chemical analysis showed that all isolates of A. flavus and A. parasiticus produced aflatoxin B1, the isolates of A. versicolor and A. nidulans produced sterigmatocystin while the toxic isolate of A. ochraceus did not produce ochratoxins.

Toxicity of Diplodia maydis to laboratory animals.

Ten strains of Diplodia maydis isolated from commercial corn samples and grown on whole yellow corn at 25 degrees C for 6 weeks were toxic to ducklings and rats. The degree of toxicity depended on the incubation period and temperature. Minimum incubation periods of 3 to 4 weeks and 6 weeks were necessary to cause mortality in ducklings and rats, respectively. Cultures incubated at 31 and 25 degrees C were much more toxic than those kept at 16 and 20 degrees C. Heat treatments at 80 to 90 degrees C destroyed most of the toxicity of moldy meal. Mild lesions of a similar histopathological nature were found in subclinically and lethally poisoned rats. These included toxic myocarditis, enteritis, focal renal tubular necrosis, degeneration and peripheral necrosis of the islets of Langerhans, and a generalized venous congestion.

Production of sterigmatocystin by Aspergillus versicolor and Bipolaris sorokiniana on semisynthetic liquid and solid media.

Higher yields of sterigmatocystin were obtained with Aspergillus versicolor than with Bipolaris sorokiniana both in liquid and on solid media. The optimum temperature for sterigmatocystin production by A. versicolor was 27 to 29 degrees C and 23 degrees C for B. sorokiniana. In liquid shake cultures, production of sterigmatocystin by B. sorokiniana was negligible, whereas maximal production by A. versicolor was 210 mg/liter. On solid substrates, the highest yields (8 g/kg) were obtained with A. versicolor on still cultures of whole corn supplemented with Soytone.

Onyalai--the possible involvement of a mycotoxin produced by Phoma sorghina in the aetiology.

A number of species of fungi were isolated from millet (Pennisetum typhoides Staph and Hubb) and grain sorghum (Sorghum vulgare Pers) obtained from the households of patients suffering from onyalai. Several proved to be toxic to rats and chickens, including Phoma sorghina (Sacc.) which was the predominating fungus. Cultures of P. sorghina grown on maize and wheat were added to standard rations at concentrations of 5-30%. When fed to day-old New Hampshire chickens, the majority died within 4 days, exhibiting traces of blood on the beak and the cloaca. Rats given 10% or more of the mouldered material in the diet developed thrombocytopenia after 14 days which was followed by haematuria, epistaxis, melaena, and death. Pathological, including histological examination, revealed extensive damage to the fascular system which resulted in widespread haemorrhages.