Article pubs.acs.org/est
Mycotoxins in the Environment: I. Production and Emission from an Agricultural Test Field Judith Schenzel,† Hans-Rudolf Forrer,† Susanne Vogelgsang,† Konrad Hungerbühler, and Thomas D. Bucheli†,* †
Agroscope Reckenholz-Tanikon, Research Station ART, CH-8046 Zurich, Switzerland Institute for Chemical and Bioengineering; Swiss Federal Institute of Technology (ETH), CH-8093 Zurich, Switzerland
‡
S Supporting Information *
ABSTRACT: Mycotoxins are secondary metabolites that are naturally produced by fungi which infest and contaminate agricultural crops and commodities (e.g., small grain cereals, fruits, vegetables, and organic soil material). Although these compounds have extensively been studied in food and feed, only little is known about their environmental fate. Therefore, we investigated over nearly two years the occurrence of various mycotoxins in a field cropped with winter wheat of the variety Levis, which was artificially inoculated with Fusarium spp., as well as their emission via drainage water. Mycotoxins were regularly quantified in whole wheat plants (0.1−133 mg/kgdry weight, for deoxynivalenol), and drainage water samples (0.8 ng/L to 1.14 μg/L, for deoxynivalenol). From the mycotoxins quantified in wheat (3-acetyl-deoxynivalenol, deoxynivalenol, fusarenone-X, nivalenol, HT-2 toxin, T-2 toxin, beauvericin, and zearalenone), only the more hydrophilic ones or those prevailing at high concentrations were detected in drainage water. Of the total amounts produced in wheat plants (min: 2.3; max: 292 g/ha/y), 0.5−354 mg/ha/y, i.e. 0.002−0.12%, were emitted via drainage water. Hence, these compounds add to the complex mixture of natural and anthropogenic micropollutants particularly in small rural water bodies, receiving mainly runoff from agricultural areas.
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ng/L and 3 mg/ha for ZON,7−9 resulting in a frequent (DON) and occasional (ZON) detection of these two mycotoxins in river waters.9,10 These results demonstrated that the runoff from agricultural fields is one significant source of mycotoxins in surface waters. Other mycotoxins, like nivalenol (NIV), or 3acetyl-deoxynivalenol (3-AcDON), are produced in similar amounts as DON and ZON, and exhibit similar or even higher water solubility than DON. Therefore, it is likely that further mycotoxins are introduced into the aqueous environment. In fact, Schenzel et al.11 observed three more mycotoxins, that is, 3-AcDON, NIV, and beauvericin (BEA), in the drainage water from a wheat field by using a multiresidue screening method for various classes of mycotoxins. The aim of this study was to investigate systematically and over two growing seasons the production of a diverse set of mycotoxins on agricultural plots cropped with wheat, and to quantify the amounts emitted via drainage water discharge. Therefore, an experimental field was repeatedly cultivated with winter wheat of the variety Levis. To ensure disease and mycotoxin production the crop was artificially infected with four Fusarium species (F. avenaceum, F. crookwellense, F. graminearum, F. poae) commonly found in wheat. For almost
INTRODUCTION During cultivation, agricultural commodities are at risk of infection from a number of different fungi, and of contamination with their toxic metabolites, called mycotoxins. These fungi include species of Fusarium, Alternaria, Cladosporium, and Claviceps. Especially, crops like small grain cereals and maize could contain a number of potential mycotoxins at harvest. Thus far, several hundred different mycotoxins have been discovered, exhibiting great structural diversity, which results in widely varying chemical and physicochemical properties.1 Fumonisins, trichothecenes, and zearalenone (produced by Fusarium species), and ergot alkaloids (produced by Claviceps species) are most prominent due to their frequent occurrence and their severe effects on animal and human health.2,3 Mycotoxins have been ranked as the most important chronic dietary risk factor, higher than synthetic contaminants, plant toxins, food additives, or pesticide residues.4 Thus, much research has been carried out on the analysis of mycotoxins in food and feed matrices, human and husbandry animal exposure, and related health effects.3,5,6 However, the environmental exposure to mycotoxins has been scarcely investigated. Recent studies on the occurrence of two Fusarium produced mycotoxins, deoxynivalenol (DON) and the estrogenic zearalenone (ZON), in plants, soil, and drainage water of an infected wheat and maize field reported concentrations in drainage water and corresponding emission rates of 20−5000 ng/L and 600 mg/ha for DON, and up to 35 © 2012 American Chemical Society
Received: Revised: Accepted: Published: 13067
April 19, 2012 October 18, 2012 November 12, 2012 November 12, 2012 dx.doi.org/10.1021/es301557m | Environ. Sci. Technol. 2012, 46, 13067−13075
Environmental Science & Technology
Article
Centraalbureau voor Schimmelcultures, Utrecht, NL): F. avenaceum CBS 121289, CBS 121294, and CBS 121290; F. crookwellense CBS 121293; F. graminearum CBS 121291, CBS 121292, and CBS 121296; F. poae CBS 121298, CBS 121297, and CBS 121299. Fungal suspensions (equal amounts of each strain) were prepared with the nonionic detergent Tween 20 (Promega AG, Dübendorf, Switzerland) at 0.0125% to obtain final concentrations of 2.5 × 105 conidia/mL, which were subsequently applied on each subplot. The control subplot received a treatment with distilled water (Tween at 0.0125%) only. In the control plot only the slightly contaminated straw from the control subplot itself was laid out again after the harvest, leading to a potential reinfection of the wheat plants with Fusarium species. In all subplots, the final suspensions were applied at a total volume of 730 L/ha using a back-pack sprayer (width 1.5 m, 3 bar, Birchmeier M125, Birchmeier Sprühtechnik AG, Stetten, Switzerland) covering the entire plot surface. Please note that the chosen experimental setup was not designed to investigate and statistically evaluate the relationship between Fusarium spp. infection, chemotypes and agricultural and climatic influences (for this widely investigated field of research, we refer to the specialized literature, for example, refs 16−20), but to ensure the presence of a wide variety of mycotoxins, and to obtain a prominent input signal of these compounds into the agro-environment, allowing studying their emission and fate via drainage water. The lower field (SI Figure S1,II) was used in 2011 only. In contrast to the upper part, the whole field was ploughed, and the seedbed was prepared in early march using a rotary harrow. Afterward, spring wheat (Triticum aestivum L.) of the variety Rubli was planted. Here, no artificial infection was conducted, and no wheat straw was laid out on the lower field. Therefore, this field serves as an additional control treatment. A seed health test (SHT) was conducted to determine which species and to which extent the species was responsible for an incidence of Fusarium spp. infection.21 Briefly, grains were surface-sterilized (10 min, 1% chloramine T) and 100 grains were placed on potato dextrose agar (PDA) (10 grains/plate) and incubated for 6 days at 19 ± 1 °C with a photoperiod of 12 h dark/12 h near-UV light. Colonies of Fusarium species and M. nivale/majus were identified according to Leslie and Summerell.22 Fungal incidences in each sample were calculated in percentage. Field Sampling, Sample Preparation, and Extraction. The wheat plants from the individual subplots of the upper field were sampled on July 29, 2010, that is, immediately before the harvest, and at two times from mid of June 2011 until harvest at July 16, in 2011. Each sample consisted of 5−10 wheat plants that were collected manually from just above the ground level from randomly selected locations of a given subplot. Sampling in the lower field in 2011 was conducted concomitant with the upper one, and in the same manner from the whole field. Samples were processed and analyzed as described in Schenzel et al.23 Briefly, collected wheat plant samples including stems, leaves and entire ears were freeze-dried, ground and homogenized prior to extraction. The whole-wheat plant samples were extracted using a simple solid−liquid extraction approach with 20 mL of MeCN/Milli-Q water (80:20, v/v) for 2 h on a SM 30 orbital shaker at 200 rpm (Edmund Bühler GmbH, Hechingen, Germany). The extract was filtered and further applied to a cleanup procedure using a Varian Bond Elut Mycotoxin cartridge. Finally, 1 mL of the extract was reduced to 100 ± 5 μL, further reconstituted in 900 μL of Milli-
two years, mycotoxins were monitored regularly in whole wheat samples, and continuously in drainage water. To the best of our knowledge, this is the first time that such an approach is used to examine the risk of environmental exposure to several classes of mycotoxins. Additionally, the ecotoxicological relevance of the presence of these compounds in drainage waters will be briefly evaluated. Finally, in a companion publication, the exposure of river water by mycotoxins is presented in detail, and the relative contribution of diffuse (i.e., Fusarium infected wheat crop8,9), and point sources (i.e., wastewater treatment plants12,13) is assessed.
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MATERIALS AND METHODS Field Site Description, Instrumentation, and Cultivation. Wheat cultivation and drainage water sampling took place on two fields located near to the research station Agroscope ART Reckenholz, North of Zurich, Switzerland (47°257̀ 4” N, 8°308̀ 5” E). The two adjacent but separately drained fields of 0.2 ha exhibit a gentle slope of 1−2°. The top soil of both of these test sites was classified according to the World reference base for soil resources and shows characteristics of a medium to heavy textured gleyic cambisol with 31% clay, 30% sand, 39% silt, and an organic carbon fraction of 2%.14 A detailed list of all soil parameters is given in the Supporting Information (SI) (Table S1). Both test fields were drained by two long and two short drainage tubes, which individually connect to a main drainage tube with a diameter of 15 cm (SI Figure S1). The drainage tubes are located in a depth of 80−90 cm. The groundwater table depth ranged from 100− 125 cm and thus was permanently below the drainage system.8 Both drainage tubes ended in sampling ducts, which were equipped with flow meters and automated samplers (7612 ISCO with a 730 bubbler module, both from Teledyne Isco Inc. Lincoln, NE) for flow proportional sampling of discharged drainage water. Precipitation data were gathered by the meteorological station (Reckenholz 443 m above sea level, 47°254̀ 0” N, 8°310̀ 4” E, MeteoSwiss, approximately 300 m from the field site) in 10 min intervals and are depicted in Figure S2 in the SI. To study the potential production and runoff of mycotoxins from an agricultural field, the following conditions favorable for mycotoxin producing Fusarium species infestation15 were chosen for the upper part of the test field (SI Figure S1,I): (i) winter wheat (Triticum aestivum L.) was cultivated repeatedly over the entire investigation period from 2008 until 2011, (ii) a wheat variety (Levis), which is highly susceptible to Fusarium graminearum infestation, was selected, (iii) no soil cultivation (“no-till”) or a superficial tillage with a temporary removal of the straw was performed and wheat straw was spread back onto the surface of the original field plots, and (iv) during flowering in 2009, 2010, and 2011 the winter wheat was artificially infected with four different Fusarium species including F. avenaceum, F. crookwellense, F. graminearum, and F. poae. The fields were cultivated using standard farming conditions, except that no fungicides were applied. Sowing dates for wheat were October 29th, and October 12th in 2009, and in 2010, respectively. In particular, inoculations of the upper field were carried out with the different Fusarium species spore suspensions applied individually on four different subplots of equal size (11 × 22 m; space between plots ≥0.5 m, SI Figure S2). The four different species comprised the following strains isolated by Agroscope ART from different sites in Switzerland (all deposited at the 13068
dx.doi.org/10.1021/es301557m | Environ. Sci. Technol. 2012, 46, 13067−13075
13069
3.9 3.9
3.8 1.9
3.9 4.9
3.8
93/92 103/99
88/86 103/99
89/88 28/13
18/12
F. avenaceum
F. crookwellense
F. graminearum
29.07.2010 16.06.2011 30.06.2011 14.07.2011 29.07.2010 16.06.2011 30.06.2011 14.07.2011 29.07.2010 16.06.2011 30.06.2011 14.07.2011 29.07.2010 16.06.2011 30.06.2011 14.07.2011 29.07.2010 16.06.2011 30.06.2011 14.07.2011
nd 0.2 0.4 0.03 nd 0.3 0.4 nd nd 0.5 0.3 0.2 nd 0.5 0.2 0.1 nd 0.4 0.1 0.1 ± 0.01b
±0.01b
±0.03b
±0.03b
HT-2 [mg/kgdw] nd 0.04 0.10 0.02 nd 0.05 0.12 nd nd 0.03 0.1 0.07 nd 0.1 0.1 0.03 nd 0.1 0.1 0.03 ± 0.02b
±0.02b
±0.03b
±0.06b
T-2 [mg/kgdw]
type A trichothecenes
0.3 nd 0.08 0.06 0.1 nd 0.2 nd 0.2 nd nd 0.11 1.8 0.2 3.1 2.1 nd nd nd nd ± 0.1b
±0.01b
±0.01b
3-AcDON [mg/ kgdw] 4.4 nd 0.7 0.7 1.8 nd 1.9 0.1 3.2 0.2 2.1 107.0 30.6 133.0 37.4 1.1 nd 0.2 0.2 ±0.3b
± 3.0b
±0.08b
±0.01b
±0.2b
DON [mg/kgdw]
27,32
nd nd nd nd nd nd nd nd nd nd 0.7 0.2 nd nd nd nd nd nd nd nd ± 0.01b
FUS-X [mg/kgdw]
type B trichothecenes
± 0.05b
± 0.1b
± 0.8b
±0.04b
NIV [mg/kgdw] nd nd 0.3 0.2 0.2 nd 0.5 nd 9.8 6.5 11.9 3.9 0.7 0.3 0.4 0.9 0.9 nd 1.1 0.4
0.05 0.07 0.05 0.5 0.02 nd 0.02 0.06 0.03 nd 0.7 nd nd 0.4 0.6 0.4 0.05 2.2 0.4 ± 0.01b
±0.03b
±0.2b
±0.02b
±0.01b
BEA [mg/kgdw]
0.1 nd nd 0.03 0.2 nd nd nd 1.8 nd 0.09 0.5 5.7 nd 0.7 6.3 0.08 nd 0.04 0.3
±0.03b
± 0.2b
± 0.07b
±0.04b
ZON [mg/kgdw]
nd: not detected; dw: dry weight; Bold numbers indicate mycotoxins associated with the individual Fusarium species according to. Numbers in italics indicate the time of harvest (in 2011 the samples were collected two days prior to the harvest). bTriplicate measurements ± standard deviation of three measurements. cThe 1st number indicates the percentage of wheat grains infected with Fusarium spp. and the 2nd number the percentage of grains infected with the corresponding Fusarium species. A percentage of >100 is possible if two or more species are isolated from a single grain. In Table S6 for each Fusarium species the corresponding incidence is given in detail.
a
4.6 4.9
5/77/73
F. poae
4.8
68/-
control
yield [tdw/ha]
inc. fus [%]
subplot artificially infected with:
c
Table 1. Incidence of Wheat Grains Infected with Fusarium spp. [%], Crop Yield [tdw/ha], and Mycotoxin Concentrations [mg/kgdw] Detected in Whole Wheat Plants in All Subfields from the Upper Test Field Monitored at Harvest in 2010 and Several Times until Harvest 2011a
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dx.doi.org/10.1021/es301557m | Environ. Sci. Technol. 2012, 46, 13067−13075
Environmental Science & Technology
Article
Table 2. Mycotoxin Concentrations in Drainage Water Samples of Fusarium Species Infected Winter Wheat Crops in [ng/L] Collected from December 2009 until the End of October 2011 compound
no. of samples analyzed/above limit of quantification (% of detection)
min concentration [ng/L]
max concentration [ng/L]
average concentration (STD) [ng/L]a
3-AcDON DON NIV BEA ZON
411/79 (19%) 411/222 (54%) 411/179 (44%) 411/36 (9%) 411/14 (3%)
5.5 0.8 5.0 1.4 6.0
367.5 1114.5 71.3 10.4 48.4
46 (59) 75 (184) 13 (14) 2.3 (2.0) 12.6 (14)
a Average concentrations calculated solely from the analytes, which were quantifiable. STD = standard deviation calculated from all quantified analytes.
repeatedly detected (Tables 1 and 2). The remainder of this paper will therefore focus on these. Mycotoxins in Whole Wheat Plants. Artificial infection of the upper wheat field’s individual subplots (SI Figure S1,I) in June 14, 2010 and May 26, 2011 led to highly varying Fusarium species incidence ranging from five (control plot) up to 103% (F. graminearum subfield) for the sum of all Fusarium species (SI Table S6). Generally, seeds were mostly contaminated with the Fusarium species they were actually inoculated with, but the sum of all infections can be slightly higher than 100% due to some cross-contamination (Table 1, SI Table S6). Mycotoxin concentrations and patterns in wheat plants varied both over time and in between subfields (Table 1). In addition, the absolute amount of mycotoxins at harvest differed from plot to plot, as well as in between both years. Although largely deliberately provoked by artificial Fusarium infection, such variability is similar to the one taking place under natural conditions.25 Within all subfields and in both years of investigation, DON was by far the compound which was quantified with the highest concentrations of up to 133 mg/ kgdw, followed by NIV (
