Occurrence of different trichothecenes and deoxynivalenol-3-ß-D-glucoside in naturally and artificially contaminated Danish cereal grains and whole maize plants.

Fusarium mycotoxins such as deoxynivalenol (DON) can occur in cereals conjugated to glucose and probably also to other sugars. These conjugates, which are often referred to as "masked mycotoxins", will not be detected with routine analytical techniques. Furthermore, it is suspected that the parent toxin may again be released after hydrolysis in the digestive tracts of animals and humans. Today, our knowledge of the occurrence of these compounds in cereal grains is limited. In this paper, a LC-MS/MS method for the simultaneous determination of DON, deoxynivalenol-3-ß-D-glucoside (DON-3-glucoside), 3 acetyl-DON, nivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, and T-2 toxin in naturally (n = 48) and artificially (n = 30) contaminated cereal grains (wheat, barley, oat, rye triticale) is reported. The method has also been applied to whole fresh maize plant intended for production of maize silage (n = 10). The samples were collected from the harvest years 2006-2010, The results show that DON-3-glucoside and DON co-occurred in cereal grains and, especially in several of the highly contaminated samples, the concentration of the glucoside can be relatively high, corresponding to over 37 % of the DON concentration. The DON-3-glucoside levels in both the naturally and in the artificially grain inoculated with Fusarium were second only to DON, and were generally higher than those of the other tested trichothecenes, which were found at low concentrations in most samples, in many cases even below the detection limit of the method. This argues for the importance of taking DON-3-glucoside into account in the ongoing discussion within the European Community concerning exposure re-evaluations for setting changed values for the tolerable intake for DON. Our results indicate that, in the naturally contaminated grains and in the Fusarium infested cereal grains (winter and spring wheat, oat, triticale), the concentration level of DON-3-glucoside is positively correlated to the DON content. When the DON concentration is high, then the content of DON-3-glucoside will most probably also be high and vice versa.

Fusarium head blight of cereals in Denmark: species complex and related mycotoxins.

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.

Effects of microbial inoculants on corn silage fermentation, microbial contents, aerobic stability, and milk production under field conditions.

The present study aimed to investigate the effects of 2 corn silage inoculation strategies (homofermentative vs. heterofermentative inoculation) under field conditions and to monitor responses in silage variables over the feeding season from January to August. Thirty-nine commercial dairy farms participated in the study. Farms were randomly assigned to 1 of 3 treatments: control (nonactive carrier; Chr. Hansen A/S, Hørsholm, Denmark), Lactisil (inoculation with 1 x 10(5)Lactobacillus pentosus and 2.5 x 10(4)Pediococcus pentosaceus per gram of fresh matter; Chr. Hansen A/S), and Lalsil Fresh (inoculation with 3 x 10(5)Lactobacillus buchneri NCIMB 40788 per gram of fresh matter; Lallemand Animal Nutrition, Blagnac, France). Inoculation with Lactisil had no effects on fermentation variables and aerobic stability. On the contrary, inoculation with Lalsil Fresh doubled the aerobic stability: 37, 38, and 80+/-8h for control, Lactisil, and Lalsil Fresh, respectively. The effect of Lalsil Fresh on aerobic stability tended to differ between sampling times, indicating a reduced difference between treatments in samples collected in April. Lalsil Fresh inoculation increased silage pH and contents of acetic acid, propionic acid, propanol, propyl acetate, 2-butanol, propylene glycol, ammonia, and free AA. The contents and ratios of DL-lactic acid, L-lactic acid relative to DL-lactic acid, free glucose, and DL-lactic acid relative to acetic acid decreased with Lalsil Fresh inoculation. Lalsil Fresh inoculation increased the silage counts of total lactic acid bacteria and reduced yeast counts. The Fusarium toxins deoxynivalenol, nivalenol, and zearalenone were detected in all silages at all collections, but the contents were not affected by ensiling time or by inoculation treatment. The effect of inoculation treatments on milk production was assessed by collecting test-day results from the involved farms and comparing the actual milk production with predicted milk production within farm based on test-day results from 2007 and 2008. The average milk production of lactating cows at test days during the study (January to September 2009) was 30.7+/-0.5 kg of energy-corrected milk/d. Milk production was 104.6+/-0.7% of the predicted yield and did not differ among treatments. In conclusion, the present study showed that homofermentative inoculants might not compete efficiently or might not deviate sufficiently from the epiphytic flora on whole-crop corn to affect fermentation in standard qualities of corn silage. Heterofermentative inoculation increased aerobic stability and numerous fermentation variables. None of the treatments affected milk production, and more-stable corn silage seemed to have a similar production value as compared with less-stable homofermented silage. Heterofermented silage can be evaluated for its properties to limit aerobic silage deterioration in the feed chain.

Particle-facilitated pesticide leaching from differently structured soil monoliths.

The leaching of soil particles and surface applied 14C-labeled glyphosate and pendimethalin from intact soil columns (height: 50 cm; diameter: 30 cm) were investigated, and the relative significance of particle-facilitated pesticide transport was quantified. Investigations were performed with a recently plowed (four columns) and an untilled (five columns) sandy loam soil. Leaching was driven by three irrigation events (15 mm h(-1); 2 h each). Samples of the leachate were filtered immediately (within 1.5 minutes) using 20 nm filters, and the 14C-pesticide content was determined for filtered and unfiltered samples. Pesticide leaching was driven by preferential water flow in macropores. For the plowed structure, 68+/-10% of the leached glyphosate (average of 6 events+/-std.) was bound to particles whereas significantly less glyphosate was bound to particles in leachate from minimally disturbed columns (17+/-12%). Thus, the results suggest that soil structure affected the mode of transport of glyphosate. It is likely that glyphosate sorbed strongly when applied on recently plowed soil (Kd=503 L kg(-1) for the soil), and that it could be mobilized and transported independently of soil particles more easily when applied on the minimally disturbed soil covered in part with crop residues (Kd<1 L kg(-1) for straw). Significantly less amounts of soil particles were leached from minimally disturbed (119-247 mg) than from recently plowed (441-731 mg) columns. The significance of particle-facilitated pendimethalin leaching could not be accurately quantified due to disagreement between control measurements based on both 14C-activity and chemical analyses.

Drift of 10 herbicides after tractor spray application. 2. Primary drift (droplet drift).

In the present study the primary drift of 10 herbicides was investigated in five field experiments, and the amount deposited per surface area was quantified outside the application area using simple passive dosimeters. In addition, samples for measuring a possible background value were taken upwind of the sprayed field. Deposits of spray drift were common to all spray equipment and spray was detected up to 150 m off-target. There were deposits of 0.1-9% of the applied amount close to the sprayed field (up to 2 m). But 3m from the spraying zone deposits were reduced to 0.02-4%. The amounts decreased exponentially when moving away from the field. The differences in drift could be described mainly by the different drop sizes, the wind velocity, the formulation and the filtering effect of vegetation on the sampling area. The tendency of the active ingredients to evaporate could also have an, although less important, influence on the drift. This is a factor, which ought to be exposed to a further study. The findings supported that it is the physical properties of the spray and the conditions of application (i.e. equipment and meteorology) that are the primary determinants of primary drift rather than the chemical property of the pure active ingredients.

Drift of 10 herbicides after tractor spray application. 1. Secondary drift (evaporation).

In the present study the evaporation of 10 herbicides was investigated during five field experiments, and the amount deposited per surface area was quantified inside the field using simple passive dosimeters consisting of microscope slides placed on plastic lids. On an average basis 90% of the applied amount reached the field. The accumulated evaporation from the microscope slides was largest during the first hours after application, and the losses in 24 h (from 0% for tribenuron-methyl, fluroxypyr-1-methylheptylester and phenmedipham to 80% for prosulfocarb) was similar to other studies of losses from plant surfaces. An indication of a diurnal difference in the evaporation was observed, probably caused by the differences in temperature or by global radiation. The evaporation did not generally correlate to the vapour pressure. The amounts collected at t=0 on the passive dosimeters were for all field experiments in the same range as the reported amounts applied to the field, and the passive-dosimeters method was found to be a good and reliable method for collection of sprayed pesticides. The advantage of this method was also that it was simple and cheap and easy to set up for screening of evaporation of pesticides from the field after spraying.

Sorption and mobility of metronidazole, olaquindox, oxytetracycline and tylosin in soil.

Laboratory studies were conducted to characterise four different antibiotic compounds with regard to sorption and mobility in various soil types. Distribution coefficients (Kd values) determined by a batch equilibrium method varied between 0.5 and 0.7 for metronidazole, 0.7 and 1.7 for olaquindox and 8 and 128 for tylosin. Tylosin sorption seems to correlate positively with the soil clay content. No other significant interactions between soil characteristics and sorption were observed. Oxytetracycline was particularly strongly sorbed in all soils investigated, with Kd values between 417 in sand soil and 1026 in sandy loam, and no significant desorption was observed. Soil column leaching experiments indicated large differences in the mobility of the four antibiotic substances, corresponding to their respective sorption capabilities. For the weakly adsorbed substances metronidazole and olaquindox the total amounts added were recovered in the leachate of both sandy loam and sand soils. For the strongly adsorbed oxytetracyline and tylosin nothing was detected in the leachate of any of the soil types, indicating a much lower mobility. Results from defractionation and extraction of the columns (30 cm length) showed that 60-80% of the tylosin added had been leached to a depth of 5 cm in the sandy loam soil and 25 cm in the sand soil.

Occurrence of pesticides in Danish shallow ground water.

Shallow ground water from four different catchment areas, two sandy and two clayey locations, were investigated for content of pesticides and -degradation products. The samples were taken from screens in extraction wells placed from 1.5 to 5 meters below surface 46 different compounds were included in the study, and the analysis were performed with liquid chromatography mass spectrometry (LC-MS) with detection limits below 0.01 microgram/L. In total more than 300 samples were analysed and the most frequently found compounds being atrazine and its degradation products, bentazone, MCPA, metamitron, isoproturon and simazine. 23 out of the 46 compounds were detected in one or more samples. Four different sulfonylurea herbicides were included in the study as they to some extent have substituted phenoxyacid herbicides in agricultural practice and in much lower dosage. Sulfonylurea herbicides did not occur in any samples.

Determination of acidic herbicides using liquid chromatography with pneumatically assisted electrospray ionization mass spectrometric and tandem mass spectrometric detection.

Liquid chromatography-pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS-MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS-MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 microgram/l or below, whereas detection limits were three-four times higher using MS-MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS-MS. Application of the method for detecting acidic herbicides residues in a "real-world" ground water sample is demonstrated.

Determination of polar pesticides in ground water using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization.

A method is described for determination of pesticides in ground water by liquid chromatography combined with mass spectrometry (LC-MS) using atmospheric pressure chemical ionization (APCI). The study demonstrates the sensitivity of the technique, with detection limits for water samples of about 0.001-0.005 microgram/l, corresponding to 50-300 pg injected. Performance data for the method such as recovery, precision and linearity are also reported. It is illustrated that the technique is applicable for many different types of pesticide structures, such as triazines, phenylurea herbicides, acetanilides, organophosphorus insecticides, etc. Twelve different pesticides and pesticide degradation products are included in the study. The optimization of inlet conditions with respect to sheath gas pressure, vaporizer temperature, capillary temperature and corona current is reported. Applicability of the method for analysis of "real-world" ground water samples is demonstrated.