Airborne mycotoxins in dust from grain elevators

Workers in grain elevators are exposed to grain dust and may therefore have an increased risk of inhalatory contact with mycotoxins. To study the mycotoxin burden of such environments, settled grain dust samples (n=35) were collected from several locations of a total of 13 grain elevators in Germany, and analysed for ochratoxin A (OTA, detection limit 0.01 ng/g), deoxynivalenol (DON, detection limit 15 ng/g), and zearalenone (ZEA, detection limit 6 ng/g), respectively. Cytotoxicity of these samples was assessed by a MTT bioassay with a swine kidney target cell line. Additionally, the airborne dust concentration of these locations was determined. Nearly all settled dust samples contained OTA (96%), DON (100%), and ZEA (100%) with median concentrations of 0.4 ng/g, 416 ng/g, and 126 ng/g, respectively. Cytotoxic effects in varying degrees from weakly to highly toxic were caused by crude extracts of 86% of the dust samples. However, cytotoxicity did not correlate with mycotoxin levels in these samples and thus indicated the presence of cytotoxic compounds of unknown origin. Based on the mycotoxin findings in settled dust samples and the airborne dust concentrations, the average airborne mycotoxin concentrations were estimated to be 0.002 ng/m³ (OTA), 2 ng/m³ (DON), and 1 ng/m³ (ZEA), respectively. The relevance of these findings for occupational health was assessed by comparison with WHO recommendations for the maximum tolerable daily (oral) intake (TDI). Even in a worst case scenario, the calculated inhalatory intake was far below the TDI values. However, considering the uncertainties resulting from different exposure pathways, namely oral ingestion versus inhalation, further research should primarily address the problem of how adequate assessment criteria for airborne exposure to mycotoxins could be established. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Effects of forage composition and growing season on methane emission from sheep in the Inner Mongolia steppe of China

Understanding the effects of dietary composition on methane (CH₄) production of sheep can help us to understand grassland degradation resulting in an increase of CH₄ emission from ruminant livestock and its resulting significance affecting CH₄ source/sink in the grazing ecosystem. The objective of this study was to investigate the effect of forage composition in the diet of sheep in July and August on CH₄ production by sheep in the Inner Mongolia steppe. The four diet treatments were: (1) Leymus chinensis and Cleistogenes squarrosa (LC), (2) Leymus chinensis, Cleistogenes squarrosa and concentrate supplementation (LCC), (3) Artemisia frigida and Cleistogenes squarrosa (AC), and (4) Artemisia frigida, Cleistogenes squarrosa and concentrate supplementation (ACC). CH₄ production was significantly lower in July than in August (31.4 and 36.2 g per sheep-unit per day, respectively). The daily average CH₄ production per unit of digestive dry matter (DM), organic matter (OM) and neutral detergent fiber (NDF) increased by 10.9, 11.2 and 42.1% for the AC diet compared with the LC diet, respectively. Although concentrate supplementation in both the AC and LC diets increased total CH₄ production per sheep per day, it improved sheep productivity and decreased CH₄ production by 14.8, 12.5 and 14.8% per unit of DM, OM and NDF digested by the sheep, respectively. Our results suggested that in degraded grassland CH₄ emission from sheep was increased and concentrate supplementation increased diet use efficiency. Sheep-grazing ecosystem seems to be a source of CH₄ when the stocking rate is over 0.5 sheep-units ha⁻¹ during the growing season in the Inner Mongolia steppe. Shiping Wang and He Zhou contributed equally to this work.     

The effect of plant species,weather variables and chemical composition of plant material on decomposition in a tropical grassland

Summary The decomposition of litter and roots ofChenopodium album, Desmostachya bipinnata and mixed grass samples for a period of 402 days and ofDichanthium annulatum andSesbania bispinosa for a period of 278 days was studied in a tropical grassland. Litter bags positioned at midcanopy height, soil surface and at five cm depth below the soil surface and root bags placed at 5, 15, 25 and 35 cm depths belowground were used. For the total study period, the cumulative weight loss in litter bags was: Chenopodium=76–100%; Desmostachya=33–98%; Dichanthium=26–96%; mixed grass=43–99% and Sesbania=25–99%. The weight loss in root bags was: Chenopodium=93–100%; Desmostachya=47–56%; Dichanthium=71–87%; mixed grass=61–82%; Sesbania=87–100%. The nature of plant species affected decomposition rates. The position of litter/root bags also affected the decomposition rates. The mean relative decomposition rates of litter as well as of root material were found to be highest in rainy season and lowest in winter months. Rainfall, particularly the frequency of rainfall, was an important factor affecting decomposition rates. The litter species characterized by highest concentration of nitrogen, ash, acid detergent cell wall component and lowest concentration of carbon, cellulose and lignin, decomposed rapidly. In the case of roots, the material having high nitrogen, carbon, cellulose and ash content and low C/N ratio and lignin content decomposed rapidly.     

Mycobiota and mycotoxins in malted barley and brewer's spent grain from Argentinean breweries

Aims: To evaluate mycobiota and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁), G₂ (AFG₂) and fumonisin B₁ (FB₁) contamination in different malted barley types and brands and brewer’s grain collected from a major Argentinean brewery. Methods and Results: Total fungal counts were performed using the plate count method. Aflatoxin B₁, AFB₂, AFG₁, AFG₂ and Zearalenone (ZEA) analyses were performed by thin‐layer chromatography (TLC). Fumonisin B₁ was determined by HPLC. Eighty‐three percentage of the malted barley (100% M1, 50% M2 and 100% M3) and 61% of brewer’s grain samples had a count >1 × 10⁴ CFU g^?1. Yeasts were isolated from all malt and brewer’s grain samples. Genera containing some of the most important mycotoxin producer species –Fusarium ssp., Aspergillus ssp., Penicillium ssp. and Alternaria ssp. – were isolated from the analysed samples, along with other environmental saprophytic fungi such as Geotrichum ssp., Mucorales and Cladosporium ssp. All samples were contaminated with 104–145 μg kg^?1 FB₁. Eighteen per cent of brewer’s grain samples were contaminated with 19–44·52 μg kg^?1 AFB₁. Aflatoxin B₂, AFG₁, AFG₂ and ZEA were not detected in any of the analysed samples. Conclusions: Fungal and mycotoxin contamination in malt and brewer’s grain is an actual risk for animal and human health. Significance and Impact of the Study: This study may be useful for assessing the risk of mycotoxins in Argentinean beers and especially in animal feeds.     

Quantification of the mycotoxins patulin and ochratoxin A by stable isotope dilution assays

For analysis of trace compounds, stable isotope dilution assays (SIDAs) have gained increasing importance in the past years. This methodology is based on the use of stable isotopically labelled analogues of the analytes as internal standards (IS). To take the mycotoxins patulin and ochratoxin A as examples, the benefits of SIDAs were demonstrated both for foods and for clinical analyses. Regarding PAT, an isotopomer labelled with¹³C was used as IS and enabled quantitation of the mycotoxin in tissues and blood. By applying this technology, a fast passive diffusion into tissue was proven with the model of the perfused rat stomach. Furthermore, rapid degradation of PAT was observed when it was reacted with blood, which was attributed to the formation of PAT-GSH adducts detected by LC-MS/MS. For OTA, a SIDA was based on the use of [²H₅]-OTA as the IS and proved to be more accurate when compared to alternative methods such as HPLC-FD or ELISA. In contrast to PAT, OTA was detectable in human blood and urine samples. Under the assumption that the majority of OTA is circulating in blood, an urinary excretion rate of about 1% of the whole body content per day was calculated. Presented at the 27^th Mykotoxin-Workshop, Dortmund, Germany, June 13–15, 2005 Financial support: in part by a grant from the Deutsche Forschungsgemeinschaft (Ry, 19/4-1)     

Combined effects of selected Penicillium mycotoxins on in vitro proliferation of porcine lymphocytes

The in vitro effect of combinations of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from six piglets. Dose–response curves for each mycotoxin and mycotoxin combinations were generated. The combined effects of toxin pairs based on IC₂₀ were illustrated in isobole diagrams and statistically calculated. OTA and CIT elicited a synergistic effect. Four toxin pairs elicited additive effects, four pairs less–than–additive effects and six pairs independent effects. Thus, the majority of toxin pairs tested produced lower combined effects than an additive effect. The results indicate that the sum effect of all toxins is less than that from the summation of concentrations of the individual compounds, adjusted for differences in potencies.     

Effect of dietary sodium bicarbonate supplementation on the toxicokinetics of ochratoxin A in pigs

The mycotoxin ochratoxin A (OA) is regarded as a causative agent for endemic nephropathy in farm animals and humans. Reabsorption of OA along the nephron results from nonionic diffusion and by carrier-mediated mechanisms indicating that urine alkalinization may help to accelerate OA excretion and thus reduce its toxicity. The aim of the present study was to investigate the effect of a dietary sodium bicarbonate (NaHCO₃) supplementation as a means to increase urinary pH on the systemic availability and excretion of OA in pigs. Dietary supplementation of 2% NaHCO₃ increased urinary pH (5.7±0.2 to 8.3±0.1) and daily urine volume (1108±276 to 2479±912ml) significantly. The systemic availability of OA and its dechloro-analog Ochratoxin B (OB) in the NaHCO₃ group calculated as the area under the serum concentration-time curve (AUC) was reduced to 75 and 68%, respectively, of the control (P<0.05). This effect was mainly due to an accelerated elimination of OA and OB in the urine. The faster renal elimination might be explained by a reduced reabsorption of the ochratoxins by nonionic diffusion, and other H⁺-dependent mechanisms. Thus, urinary alkalinization might be an efficient means to partially reduce the toxic effects and carry-over of OA in pigs. Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.     

Results of long-term field studies into the effect of strobilurin containing fungicides on the production of mycotoxins in several winter wheat varieties

In total, 23 field trials have been performed during the past 5 years to assess the effect of strobilurin containing fungicides on mycotoxin production in winter wheat. In 85% of the plots, the treatment with strobilurins at growth stages before blossom increased the content of deoxynivalenol in comparison with untreated controls. This effect was intensified by treatments at BBCH stage 45–49 and could be demonstrated in a great number of various cultivars. Application of strobilurin containing fungicides at early growth stages seems to be a risk factor for mycotoxin formation in wheat, especially under favourable conditions forFusarium infections. Presented at the 26^th Mykotoxin-Workshop in Herrsching. Germany, May 17–19, 2004     

Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness

Aims: To examine Saccharomyces cerevisae strains with previously reported beneficial properties and aflatoxin B₁ binding capacity, for their ability to remove ochratoxin A (OTA) and zearalenone (ZEA) and to study the relation between cell wall thickness and detoxificant ability of yeast strains. Methods and Results: A mycotoxin binding assay at different toxin concentrations and the effect of gastrointestinal conditions on mycotoxin binding were evaluated. Ultrastructural studies of yeast cells were carried out with transmission electronic microscopy. All tested strains were capable of removing OTA and ZEA. Saccharomyces cerevisiae RC012 and RC016 showed the highest OTA removal percentage, whereas RC009 and RC012 strains showed the highest ZEA removal percentages. The cell diameter/cell wall thickness relation showed a correlation between cell wall amount and mycotoxin removal ability. After exposure to gastrointestinal conditions, a significant increase in mycotoxin binding was observed. Conclusions: All tested Saccharomyces cerevisiae strains were able to remove OTA and ZEA, and physical adsorption would be the main mechanism involved in ochratoxin A and ZEA removal. Gastrointestinal conditions would enhance adsorption and not decrease mycotoxin–adsorbent interactions. Significance and Impact of the Study: Live strains with mycotoxin binding ability and beneficial properties are potential probiotics that could be included in animal feed. Previous and present results suggest that the RC008 and RC016 strains are very promising candidates for functional feed product development.     

Determination of mycobiota and mycotoxins in pig feed in central Argentina

Aims: To evaluate the mycobiota and natural levels of aflatoxins, fumonisins and zearalenone present in compound feed and home‐corn grains intended for fattening pigs. Methods and Results: Total fungi, Fusarium and Aspergillus species occurrence were examined. Aflatoxins and zearalenone were detected by thin‐layer chromatography and fumonisins by high‐pressure liquid chromatography. Fungal counts were generally higher than 1 × 10⁵ colony forming units (CFU) ml^?1. Aspergillus flavus, Aspergillus parasiticus and Fusarium verticillioides were the most prevalent species. FB₁ and FB₂ were detected in all feed and corn samples. Aflatoxin B₁ was detected in 33·33% of initial and growing feed and in 44·44% of final feed samples. It was not detected in corn samples. All feed and corn samples were negative for AFB₂, AFG₁, AFG₂ and ZEA presence during all growing stages tested. Conclusions: Fungal counts at all growing periods exceeded the levels proposed as feed hygienic quality limits. Aflatoxin levels in all feeds and fumonisin levels in many samples were higher than the established regulations. Significance and Impact of the Study: The presence of mycotoxins indicates the existence of contamination. This fact requires periodic monitoring to prevent the occurrence of mycotoxicosis in animal production, to reduce the economic losses and to minimize hazards to human health.     

内蒙古锡林郭勒草地的草种组分与放牧压的关系研究

通过了采样调查,对内蒙古锡林河流域的放牧草地群落的组成成分,现存生物量,物种多样性等与放牧强度的关系进行了研究。结果表明,距离定居点越远放牧强度越弱,出现了以羊草(Leymus chinensis)和大针茅(Stipa grandis)为优势种的群落。距离定居点越近放牧强度越强,出现了以糙隐子草(Cleistogenes squarrosa)和寸苔草(Carex korshinskyi)为优势种的群溶。单位样方内(1m^2)地上部的现存生物量在强放牧压下的127．3g和较弱放牧压下的303g之间变化。物种多样性的变化则独立于放牧强度差异,在3．13至3．64之间波动。为了判断放牧草地的牧草的生长状态,采用了生长状态质量指数(Stand quality index：SQI)来评价了实验样地群落的生长状态。在定居点的内部,SQI为278,而在离定居点4公里远的样地,指数则变为667。禁牧13年的原退化样地的SQI值从420上升至624。     

牦牛放牧率和放牧季节对小嵩草高寒草甸土壤养分的影响

经过两年牦牛放牧试验表明,夏季草场不同土壤层有机质、有机碳、全磷、全氮和速效磷的含量及0～20cm土壤层营养因子的平均含量与放牧率呈线性回归关系,速效氮含量与放牧率呈二次回归关系;冬季草场不同土壤层有机质、有机碳和全氮与放牧率呈线性回归关系,全磷、速效磷和速效氮的含量及0～20cm土壤层营养因子的平均含量与放牧率呈二次回归关系。随着放牧率的逐渐增加,夏季草场不同土壤深度速效氮含量也逐渐减小,当放牧率达到1.07、1.08和1.22头·hm-2时,0～5、5～10、10～20cm土壤速效氮含量依此达到最小;冬季草场不同土壤层全磷、速效磷和速效氮含量逐渐增加,当放牧率达到0.81、0.78和1.00头·hm-2,1.03、1.09和1.03头·hm-2,1.36、1.35和1.30头·hm-2时,0～5、5～10、10～20cm土壤层全磷、速效磷和速效氮含量分别依此达到最大。在夏季草场,0～20cm土壤层速效氮平均含量达到最小放牧率是1.07头·hm-2;冬季草场0～20cm土壤层全磷、速效磷和速效氮含量达到最大放牧率分别是0.90、0.83和1.21头·hm-2。     

Effects of ozone on species composition in an upland grassland

Northern hemispheric background concentrations of ozone are increasing, but few studies have assessed the ecological significance of these changes for grasslands of high conservation value under field conditions. We carried out a 3-year field experiment in which ozone was released at a controlled rate over three experimental transects to produce concentration gradients over the field site, an upland mesotrophic grassland located in the UK. We measured individual species biomass in an annual hay cut in plots receiving ambient ozone, and ambient ozone elevated by mean concentrations of approximately 4 ppb and 10 ppb in the growing seasons of 2008 and 2009. There was a significant negative effect of ozone exposure on herb biomass, but not total grass or legume biomass, in 2008 and 2009. Within the herb fraction, ozone exposure significantly decreased the biomass of Ranunculus species and that of the hemi-parasitic species Rhinanthus minor. Multivariate analysis of species composition, taking into account spatial variation in soil conditions and ozone exposure, showed no significant ozone effect on the grass component. In contrast, by 2009, ozone had become the dominant factor influencing species composition within the combined herb and legume component. Our results suggest that elevated ozone concentrations may be a significant barrier to achieving increased species diversity in managed grasslands.     

Effects of temperature on the development and growth of winter wheat roots

Winter wheat was sown on 2 dates with 3 levels of nitrogen fiertiliser (0, 50 and 200 kg N ha⁻¹) in one year and on 2 sites in a followign season. Shoot and root development and growth were measured between emergence and anthesis in the first season and emergence and 7 mainstem leaves in the second. Differences in temperature and light regime led to significant differences in shoot and root development and growth between sowing dates. A thermal time-scale, based on soil surface or air temperatures, with a base of 0°C, adequately described the production of mainstem leaves and nodal root axes over all treatments. Autumn applied nitrogen had little effect on development. Shoot growth and green area index increased exponentially with thermal time prior to spring nitrogen application and the completion of canopy development. Early-sown crops had larger root systems than late-sown crops prior to winter and this divergence was retained until anthesis. The relationship between root growth and thermal time was little better than with days after sowing and was not improved by either varying the site of temperature measurement or the base temperature used for calculation. Differences in soil texture and drainage, between sites, led to significant changes in root length distribution. Although spring applied nitrogen generally increased root length, its effects were inconsistent. There was a curvilinear relation between root length and the amount of photosynthetically active radiation (PAR) intercepted; this relation was unaffected by sowing date or nitrogen treatment. The amount of root produced per unit PAR decreased as the season progressed, reflecting the decrease in the proportion of total dry matter partitioned to the root system.     

Influence of water activity, temperature and time on mycotoxins production on barley rootlets

AIMS: The objective of this study was to determine the ochratoxin (OT) and aflatoxin (AF) production by three strains of Aspergillus spp. under different water activities, temperature and incubation time on barley rootlets (BR). METHODS AND RESULTS: Aspergillus ochraceus and Aspergillus flavus were able to produce mycotoxins on BR. Aspergillus ochraceus produced ochratoxin A (OTA) at 0.80 water activity (a(w)), at 25 and 30 degrees C as optimal environmental conditions. The OTA production varies at different incubation days depending on a(w). Aflatoxin B(1) (AFB1) accumulation was obtained at 25 degrees C, at 0.80 and 0.95 a(w), after 14 and 21 incubation days respectively. Temperature was a critical factor influencing OTA and AFB(1) production. CONCLUSIONS: This study demonstrates that BR support OTA and AFB(1) production at relatively low water activity (0.80 a(w)) and high temperatures (25-30 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: The study of ecophysiological parameters and their interactions would determine the prevailing environmental factors, which enhance the mycotoxin production on BR used as animal feed.     

不同冷季型牧草品种的产草量及品质比较分析

采用随机区组试验设计,对引进的8个冷季型牧草品种进行产草量和营养成分的比较分析.结果表明:4次刈割的总鲜重和总干重在品种间差异极显著,且以蓝天堂黑麦草(7号)最高.品种间的粗蛋白含量差异显著,其中第1次刈割以奇可利(3号)最高,蓝天堂黑麦草(7号)居第2位;第2次仍以奇可利(3号)最高,特高黑麦草(1号)和蓝天堂黑麦草(7号)居其次;第3次以紫花苜蓿(8号)最高,其次是多年生黑麦草(4号);第4次以奇可利(3号)最高,多年生黑麦草(4号)居第2位;粗纤维、粗脂肪和粗灰分在品种间的差别较小.综合产草量和品质两方面考虑,蓝天堂黑麦草(7号)可作为本地首选推广的冷季型牧草品种,奇可利(3号)和多年生黑麦草(4号)可作为备选推广的牧草品种.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone