Occurrence of Azoxystrobin, Propiconazole, and SelectedOther Fungicides in US Streams, 2005–2006

William A. Battaglin & Mark W. Sandstrom &

Kathryn M. Kuivila & Dana W. Kolpin &

Michael T. Meyer

Received: 26 March 2010 /Accepted: 23 September 2010# Springer Science+Business Media B.V. (outside the USA) 2010

Abstract Fungicides are used to prevent foliar dis-eases on a wide range of vegetable, field, fruit, andornamental crops. They are generally more effectiveas protective rather than curative treatments, andhence tend to be applied before infections take place.Less than 1% of US soybeans were treated with afungicide in 2002 but by 2006, 4% were treated. Likeother pesticides, fungicides can move-off of fieldsafter application and subsequently contaminate sur-face water, groundwater, and associated sediments.Due to the constant pressure from fungal diseasessuch as the recent Asian soybean rust outbreak, andthe always-present desire to increase crop yields, there

is the potential for a significant increase in the amountof fungicides used on US farms. Increased fungicideuse could lead to increased environmental concen-trations of these compounds. This study documentsthe occurrence of fungicides in select US streamssoon after the first documentation of soybean rust inthe US and prior to the corresponding increase infungicide use to treat this problem. Water sampleswere collected from 29 streams in 13 states in 2005and/or 2006, and analyzed for 12 target fungicides.Nine of the 12 fungicides were detected in at least onestream sample and at least one fungicide was detectedin 20 of 29 streams. At least one fungicide wasdetected in 56% of the 103 samples, as many as fivefungicides were detected in an individual sample, andmixtures of fungicides were common. Azoxystrobinwas detected most frequently (45% of 103 samples)followed by metalaxyl (27%), propiconazole (17%),myclobutanil (9%), and tebuconazole (6%). Fungicidedetections ranged from 0.002 to 1.15 μg/L. There wasindication of a seasonal pattern to fungicide occur-rence, with detections more common and concen-trations higher in late summer and early fall than inspring. At a few sites, fungicides were detected inall samples collected suggesting the potential forseason-long occurrence in some streams. Fungicideoccurrence appears to be related to fungicide use inthe associated drainage basins; however, current useinformation is generally lacking and more detailedoccurrence data are needed to accurately quantifysuch a relation. Maximum concentrations of fungi-

Water Air Soil PollutDOI 10.1007/s11270-010-0643-2

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11270-010-0643-2) containssupplementary material, which is available to authorized users.

W. A. Battaglin (*) :M. W. SandstromUS Geological Survey,Denver, CO, USAe-mail: wbattagl@usgs.gov

K. M. KuivilaUS Geological Survey,Sacramento, CA, USA

D. W. KolpinUS Geological Survey,Iowa City, IA, USA

M. T. MeyerUS Geological Survey,Lawrence, KS, USA

cides were typically one or more orders of magni-tude less than current toxicity estimates forfreshwater aquatic organisms or humans; however,gaps in current toxicological understandings of theeffects of fungicides in the environment limit theseinterpretations.

Keywords Fungicide . Soybean rust . Toxicity .

Water quality

1 Introduction

1.1 Problem

Fungicides are used in the US to prevent foliardiseases on a wide range of crops, and like otherpesticides, can be transported from the fieldswhere they are applied and contaminate theenvironment. Recent studies have detected fungi-cides and their primary transformation products(TPs) in streams, precipitation, groundwater, andbed sediment (McConnell et al. 1998; Wauchope etal. 2004; Scribner et al. 2006). Fungicide use isincreasing in agricultural and nonagricultural sectorsbut the potential for a rapid increase in fungicide usedue to soybean rust may be one of the mostsignificant current causes. In some areas of the US,this may represent the first time that fungicides havebeen included in crop management practices.

Soybean rust is a devastating disease caused bytwo fungal pathogens: Phakopsora pachyrhizi (Asianspecies) and Phakopsora meibomiae (New Worldspecies). Asian soybean rust (soybean rust) wasdetected for the first time in the US in November2004, in Louisiana. The concern for its arrival,however, preceded that date (Livingston et al. 2004).Subsequently, soybean rust has spread to an everwider geographic area across the US (Sconyers et al.2006; US Government Accountability Office 2006;US Department of Agriculture 2007). In 2005,detections of soybean rust were made in nine statesand 131 counties (Fig. 1); in 2006, 15 states and 231counties reported detections; and in 2007, 19 statesand 335 counties (some as far north as Iowa), oneprovince of Canada, and two municipalities inMexico (US Department of Agriculture 2007).

The extent to which soybean rust will affectfuture US soybean production is unknown. The US

Department of Agriculture has developed a planto minimize the impact of soybean rust and toactively monitor for this disease (US Department ofAgriculture 2004, 2007). Annual yield losses of50% or greater in the southern US and a least 10%in the upper Midwest have been predicted (Dorranceet al. 2005).

1.2 Fungicide use

Fungicide use on crops in the US was estimated to be52,000 metric tons per year in 2002 (the most recentyear with estimates of US fungicide use; Gianessi andReigner 2006). Some fungicides such as chlorothalo-nil have been in use for decades (first registered in1966), whereas others such as azoxystrobin wererecently introduced (first sales in 1996). Fungicidesare used in the US to prevent foliar diseases on a widerange of crops (Meisterpro 2007). Typically, fungi-cides are more effective when used as protectiverather than curative treatments. The primary crops onwhich fungicides are applied in terms of mass ofactive ingredient (circa 2002) are grapes (20,360metric tons per year (mt/y)), tomatoes (4,680 mt/y),apples (3,310 mt/y), potatoes (3,270 mt/y), and citrus(3,180 mt/y). Other crops for which more than 50% ofthe planted acreage is generally treated with afungicide include artichokes, brussel sprouts, cab-bage, cucumbers, melons, peaches, peanuts, pump-kins, and sugarbeets (Gianessi and Reigner 2006).Fungicides are also used as biocides (i.e., woodpreservatives); as antifungal agents for humans, pets,or livestock; and as a treatment for breast tumors(Zarn et al. 2003; Kahle et al. 2008).

The use of fungicides on corn and soybeans wasminor prior to 2002. In 2002, less than 1% of USsoybean acres were treated with a fungicide (USDepartment of Agriculture 2009a). In 2006, this usewas already starting to increase, with 4% of soybeansreceiving a fungicide application, most commonlypyraclostrobin or azoxystrobin (US Department ofAgriculture 2009a). Fungicide use on corn was sominor that it was not reported by the US Departmentof Agriculture (2009a) in 2002 or in 2005 (the mostrecent year with a survey of agricultural chemical useon corn).

Another recent trend in crop management practicesis to use fungicide treatments to increase yields ofsoybean and corn crops. Results of field trials suggest

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that “preventative” fungicide use can increase soy-bean yields by as much as 20% (Bradley and Sweets2008; Yang et al. 2008), and corn yields by 5% ormore (Syngenta FarmAssist 2009). Other researchers,however, suggests more evidence is needed beforesuch claims can be substantiated (Ortiz-Ribbing et al.2008; US Environmental Protection Agency 2009b).

In 2006, four fungicides were registered fortreatment of soybean rust in the US: azoxystrobin,boscalid, chlorothalonil, and pyraclostrobin (USEnvironmental Protection Agency 2007), and fiveother fungicides were granted emergency exemptionsfor treatment of soybean rust: myclobutanil, propico-nazole, tebuconazole, tetraconazole, trifloxystrobin.By 2009, 14 fungicides were registered for treatmentof soybean rust; the nine previously mentioned pluscyproconazole, flusilazole, flutriafol, metconazole,and prothioconazole (West Virginia Department ofAgriculture 2009). For the 12 target fungicide for thisstudy (azoxystrobin, boscalid, chlorothalonil, cyproco-nazole, metalaxyl, metconazole, myclobutanil, propico-nazole, pyraclostrobin, tebuconazole, tetraconazole, andtrifloxystrobin) use increased about 25% from 4,210

metric tons in 2002–5,520 metric tons in 2007 (Agro-Trak® agrochemical usage data (AgroTrak® is aregistered trademark of dmrkynetec, St. Louis, MO,USA)). All 12 target fungicides are also registered foruse on other crops or turf.

Therefore, there are many characteristics offungicides that result in increasing usage and shiftingspatial/temporal patterns in the US including: fungaldiseases are more common than any other plantdiseases; uses transcend agricultural sectors andinclude domestic and industrial sectors; agriculturaland other treatments are often protective as well ascurative; the recent onset of soybean rust hasresulted in many new fungicides being registeredand used; fungicides are used on an increasinglywide variety of vegetable, fruit, and grain crops;recent usage has been related to increased yields inotherwise healthy crops; and fungicide resistancewill continue to challenge agriculture and impactusage rates and patterns sometimes resulting in theneed for larger doses to kill resistant organisms andother times restricting or eliminating the use of afungicide.

Fig. 1 Location of study sites and counties in the US with detected soybean rust in 2005 and 2006

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1.3 Environmental Fate

Characteristics of the different fungicides determinehow they are used in a pest management program(Tenuta et al. 2007). Protective fungicides, such aschlorothalonil, pyraclostrobin, and azoxystrobin, pre-vent fungi from successfully colonizing on hosttissue, but have little effect on colonies alreadyestablished on host tissue. Thus, these fungicideswould be applied before the onset of soybean rust forpreventative purposes. Curative fungicides, such aspropiconazole, tebuconazole, and myclobutanil, caninhibit or stop existing fungal infections by limitingspore germination, helping slow disease development.Thus, these fungicides would be applied after fungihave successfully colonized on a plant to cure or limitfurther development of such fungal infections.

An increase in fungicide use either in response todiseases such as soybean rust, to increased yieldpotential, or any of the other reasons noted abovemay lead to increased transport of fungicides andfungicide TPs to US water resources. Fungicideshave a wide range of physical properties whichaffect their occurrence and fate in the environment(supplemental material, Table S1). For example,azoxystrobin or propiconazole are expected to bemobile in the environment because they are watersoluble, slow to degrade in soil and water, unlikelyto volatilize, and unlikely to partition to soils orsediment in aquatic systems. Other fungicides suchas chlorothalonil or pyraclostrobin, are not as likelyto be mobile in the environment because they areless water soluble, degrade more rapidly in soil andwater, and would be expected to partition rapidly tosoils or sediment in aquatic systems. In addition,fungicides can enter streams by way of wastewaterdischarge or storm water runoff in urbanized areas(Kahle et al. 2008).

Few studies have investigated the occurrence offungicides in surface water. Studies in agriculturalregions of Germany, however, found fungicides to becommonly present in streams with concentrations upto 30 μg/L (Berenzen et al. 2005; Liess and Von DerOhe 2005). In samples from Swiss Midland lakes,fluconazole, propiconazole, and tebuconazole werefrequently detected at with concentrations up0.009 μg/L (Kahle et al. 2008). In samples fromselect US streams chlorothalonil was infrequentlydetected, whereas its major transformation product 4-

hydroxy-chlorothalonil was detected more frequently(Scribner et al. 2006; Battaglin et al. 2008).

The potential water quality effect of fungicide useto combat soybean rust in Indiana was recentlyassessed using a modeling approach (Deb et al.2010). Under a worst-case scenario, they simulatededge-of-field concentrations of 14 fungicides andcompared them with concentrations of concern forchronic exposure to humans from drinking water andto fish in aquatic habitats (Plotkin et al. 2009a,2009b). They concluded that most fungicides hadthe potential to occur in edge-of-field runoff at greaterthan 1 μg/L concentrations, and that several, includ-ing chlorothalonil, pyraclostrobin, and tebuconazole,had the potential to occur in runoff at concentrationsthat are above their chronic exposure thresholds (Debet al. 2010).

1.4 Effects of Fungicides in the Environment

Fungicides are used to kill fungi and (or) their spores.Fungicides can be chemical or biological and functionsystemically or via contact with plant surfaces. Allfungicides are at risk of losing all or part of theireffectiveness due to development of fungicide resis-tant fungi (Brent and Hollomon 1998). Modes ofaction, whether the product functions systemically oron contact, the potential for onset of resistance,compound-specific behavior in the environment, andother factors all are taken into account by manufac-turers when providing guidance on use of fungicidesand related formulations. In turn, this accounts for theamount and timing of fungicide use and relatedpathways to the environment.

In the environment, fungicides can be toxic to fish,invertebrates, and other non-target organisms at awide range of concentrations. Most toxicity estimatesfrom the US Environmental Protection Agency’sECOTOX database (US Environmental ProtectionAgency 2009a), and many from other publishedsources (Bartlett et al. 2002; Belden et al. 2007;Berenzen et al. 2005; Birge et al. 2000; Liess and VonDer Ohe 2005; Ochoa-Acuna et al. 2009; Plotkin etal. 2009a, 2009b) are 50th-percentile effect concen-tration or 50th-percentile lethal concentrations. There-fore, confounding factors such as effects of mixtures,novel or other modes of action (e.g., hormonally activeprocesses), chronic exposures to non-target organismsas well as beneficial and indigenous fungal communi-

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ties, and the potential for fungicide resistance patternsare not accounted for in present paradigms.

Toxicity estimates for the 12 fungicides (and someindividual fungicides) assessed span more than fourorders of magnitude from less than 10 to more than10,000 μg/L (Fig. 2). In some cases, estimates of afungicide’s toxicity to a class of organisms varied bymore than two orders of magnitude (i.e., azoxystrobinand diatoms). The toxicity of some fungicides toDaphnia magna increased by an order of magnitudeor more with an increase in exposure duration from24 to 96 h (Ochoa-Acuna et al. 2009). The wide rangeof toxicity estimates for fungicides as well as thelimitations they impose on interpretations of environ-mental occurrence data was not unexpected as similarresults for other pesticides have been documented(Kerby et al. 2009).

Toxicological data for amphibians and many othernon-target aquatic species are limited. For example,no data were found that described the toxicity of thetarget fungicides analyzed for in this study tonaturally occurring beneficial fungi such as thosefrom the class Trichomycetes (Lichtwardt et al. 2001;White et al. 2006). Some fungicides exhibitendocrine-disrupting activities in mammals and sev-

eral other toxic modes of action have been described(Zarn et al. 2003; Taxvig et al. 2007a, b; Maltby et al.2009; Sancho et al. 2009). A recently conductedfungicide risk assessment (Maltby et al. 2009)indicated there was “...no appropriate toxicity datafor fungi...”, but that there also was no evidence thattoxicity estimates based on acute data from non-fungalspecies were not adequately protective. The targetfungicides are similarly toxic to aquatic organisms asare the herbicides and insecticides that have tradition-ally been used in corn and soybean production(Battaglin and Fairchild 2002). Finally, some pesticidemixtures can have adverse effects on aquatic commu-nities even when no individual compound occurs attoxic levels (Castillo et al. 2006; Hayes et al. 2006;Relyea 2009). The toxicological effect of mixtures offungicides, like all pesticides, is poorly understood.

1.5 Objectives

The objective of this study was to collect baselinedata on the occurrence of 12 target fungicides inselect US streams soon after the first documentationof soybean rust in the US and prior to the corre-sponding increase in fungicide use to control and treat

Fig. 2 Concentrations of 12fungicides in 103 watersamples collected in 2005and 2006, and fungicidetoxicity to selected aquaticorganisms and humans

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this problem. These data were used to identify spatialand seasonal patterns in fungicide occurrence and toinvestigate potential relations between fungicideoccurrence and the use of those products in theassociated drainage basins. This is the first study tolook for azoxystrobin, boscalid, cyproconazole, metal-axyl, metconazole, pyraclostrobin, tetraconazole, andtrifloxystrobin in streams across the US.

2 Methods and Materials

2.1 Study Sites and Sample Collection

Study sites in the southern and central US (Fig. 1)were selected on the basis of estimates of soybeanacreage, expected fungicide use, watershed area, andthe availability of ancillary data (such as streamflow).Our objective was to select sites with a range ofdrainage areas representing a range in hydrologicconditions, some of which have a substantial portion(more than 25%) of that drainage area in soybeanproduction. Sites were also selected where fungicideuse was (e.g., Georgia and Mississippi) and was not(e.g., Iowa) a common crop management practice.

A total of 103 water samples (not including qualityassurance (QA) samples) from 29 streams across 13states were collected between 2005 and 2006 (Ta-ble 1). A majority of sites (20 of 29) had multiplesamples collected, with as many as four samplecollected in a given year. Study sites ranged indrainage area from 61 to 221,000 km2. Drainagebasin area in soybean production ranged from 0% to41% (Table 1). Most samples were collected insummer and early fall during the time when fungi-cides would typically be applied for the prevention ofsoybean rust or be used on other crops, but somesamples were collected as early as April or as late asNovember. A few samples were collected duringrunoff events, but these events were not specificallytargeted for this study. All samples were collectedusing standardized protocols (Wilde et al. 1999) bywading or from bridges using the equal-width-increment method (Shelton 1994).

2.2 Analytical Methods

Several modifications (Sandstrom, oral communica-tion) to an analytical method (Zaugg et al. 1995) were

made to measure the concentrations of 11 fungicidesat sub μg/L concentrations in filtered water samples.In 2005, the method was first modified to measure sixfungicides (azoxystrobin, chlorothalonil, myclobuta-nil, propiconazole, pyraclostrobin, and tebuconazole)in water. In 2006, the method was further modified toinclude five more fungicides (boscalid, cyprocona-zole, metconazole, tetraconazole, and trifloxystrobin).Cis- and trans-propiconazole were analyzed separate-ly but results were reported as the sum of these twoisomers. Concentrations of one other fungicide (metal-axyl) were measured using another method (Sandstromet al. 2001). The fungicides were isolated from 1-Lwater samples into a 0.5-g octadecyl-bonded silicasolid-phase extraction column and eluted with 2 ml ofethyl acetate. After solvent evaporation and exchangeto toluene, the extract was analyzed by gas chroma-tography, with selected ion-monitoring mass spec-trometry. The laboratory reporting level (LRL;Childress et al. 1999) for the 11 fungicides rangedfrom 0.004 to 0.010 μg/L (Fig. 2).

Most (94 of 103) samples also were analyzed for63 moderate use pesticides and pesticide TPs usingsolid-phase extraction and gas chromatography/massspectrometry (Sandstrom et al. 2001), and selectedsamples (25) were analyzed for two chlorothalonildegradation products: 4-hydroxy-chlorothalonil and1-amide-4-hydroxy-chlorothalonil using a methoddescribed in Scribner et al. (2006). From theseanalyses, only concentrations of the fungicide metal-axyl are included in this interpretation.

QA samples consisted of laboratory reagentblanks, laboratory reagent spikes, laboratory dupli-cates, field matrix spikes, field blanks, and fieldduplicates. There were no detections of target fungi-cides in the 27 laboratory reagent blanks or the tenfield blanks. Eight field duplicates were collectedallowing for a total of 91 possible comparisons offungicides concentrations. The presence or absenceof fungicides was confirmed in 98% of thesecomparisons. In two instances at concentrations nearthe reporting level, there was a detection of afungicide in the field duplicate sample, but not inthe corresponding environmental sample. The abso-lute difference in measured detections ranged from0.004 to 0.108 μg/L, with the average differencebeing 0.024 μg/L.

Field and laboratory matrix spikes were preparedto evaluate the effects of sample matrix on fungicide

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Table 1 Study sites for soybean rust study, 2005–2006 (km2)

Site no. Station name USGS stationidentificationnumber

Drainagearea (km2)

Percentage ofdrainage area insoybeans in 2002

Number ofsamples in2005

Number ofsamples in2006

Iowa

1 Turkey River at Garber, IA 05412500 4,002 21.7 1 1

2 Wapsipinicon River near De Witt, IA 05422000 6,050 28.9 1 1

3 Skunk River at Augusta, IA 05474000 11,170 28.6 1 1

4 Little Sioux River near Turin, IA 06607500 9,132 35.9 1 0

5 Boyer River at Logan, IA 06609500 2,260 36.7 1 0

6 S. Fork Iowa R. NE of New Providence, IA 05451210 580 36.1 1 3

7 Iowa River at Wapello, IA 05465500 32,380 32.4 0 2

Illinois

8 Sangamon River at Monticello, IL 05572000 1,420 41.0 1 4

Indiana

9 Sugar Creek at New Palestine, IN 394340085524601 240 36.5 0 3

Ohio

10 Auglaize River near Fort Jennings, OH 04186500 860 36.7 0 3

North Carolina

11 Neuse River at Kinston, NC 02089500 6,972 5.9 2 3

12 Contentnea Creek at Hookerton, NC 02091500 1,900 11.5 2 3

Nebraska

13 Maple Creek near Nickerson, NE 06800000 953 27.5 1 1

14 Platte River at Louisville, NE 06805500 221,000 3.9 0 3

Minnesota

15 Little Cobb River near Beauford, MN 05320270 337 31.8 1 4

Mississippi

16 Bogue Phalia nr Leland, MS 07288650 1,250 30.9 3 4

17 Yazoo R bl Steele Bayou nr Long Lake, MS 07288955 34,590 12.0 4 4

Georgia

18 Lime Creek near Cobb, GA 02350080 159 0.6 2 4

19 Sope Creek near Marietta, GA 02335870 76 0.0 2 1

20 Chattahoochee River near Whitesburg, GA 02338000 6,290 0.0 2 0

Alabama

21 Tombigbee R bl Coffeeville L&Dnear Coffeeville, AL

02469762 47,700 1.5 2 3

22 Flint River at Brownsboro, AL 03575100 971 3.9 1 3

23 Cahaba Valley Creek at Cross CRRD at Pelham, AL

0242354750 66 0.0 2 0

Kentucky

24 Bayou de Chien near Clinton, KY 07024000 178 34.7 1 1

Missouri

25 Locust Creek near Unionville, MO 06900900 201 10.2 1 0

26 Medicine Creek near Harris, MO 06899950 497 10.0 1 0

South Carolina

27 Gills Creek at Columbia, SC 02169570 154 0.8 3 1

28 Cow Castle Creek near Bowman, SC 02174250 61 2.9 2 4

29 Edisto River nr Givhans, SC 02175000 7,070 1.5 3 4

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recovery. In 2005, 16 matrix spike samples from 13streams were analyzed. At three sites, duplicatematrix spikes were prepared, one spike being addedat the collection site and one spike added at thelaboratory. Calculated recoveries of fungicides instream matrix spike samples are shown in Table S2.Calculated spike recoveries were not used in the 2005summary statistics for three fungicides in one samplebecause the concentrations in the environmentalsamples were close to the spike concentration level(ratio of spike/environmental <1.5). In 2005, medi-an recoveries of azoxystrobin (215%) and myclo-butanil (177%) were found to be elevated(Table S2). This was in contrast to the recoveriesof chlorothalonil (86%), cis-propiconazole (109%),and trans-propiconazole (90%) that were closer to100%. The recoveries in spike samples indicate thatsome fungicides have a high bias from the samplematrix which could translate to a positive bias in thecorresponding environmental samples. This, how-ever, only effects measured concentrations and doesnot introduce false-positives in the data. Because thebias was higher in stream samples than in reagentwater, the cause is most likely the stream samplematrix. Similar enhancement in observed chromato-graphic response was observed for pesticide residuesin a matrix extract when compared with the sameconcentration in a matrix-free solution (Poole 2007).The matrix increases the transfer of fungicides fromhot vaporizing injectors by reducing the thermalstress for labile compounds and by masking activesites in the injector responsible for the adsorption ordecomposition of polar pesticides.

In 2006, 21 matrix spike samples from 12 streamswere analyzed, 15 using a spike mixture thatcontained ten fungicides, and six using a spikemixture with four fungicides. All samples were spikedin the field into filtered water samples. Expected spikeconcentrations were 0.1 μg/L. Calculated recoveriesof fungicides in stream matrix spike samples areshown in Table S2. Calculated spike recoveries werenot used in the 2006 summary statistics for threesamples because the concentrations in the environ-mental samples were close to the spike concentrationlevel (ratio of spike/environmental <1.5). The calcu-lated spike recoveries had high positive bias and werequite variable with median values for azoxystrobin(268%), boscalid (310%), and pyraclostrobin (399%;Table S2). These fungicides have chemical structures

that cause them to be susceptible to thermal stress ofthe hot injector and active sites in the injector or gaschromatograph (GC) column. Co-extracted matrixcomponents block active sites in the GC, reducinglosses of these susceptible analytes, and results inhigher analyte signals compared to the matrix-freecalibration solutions (Mastovska et al. 2005).

The matrix spike recovery results indicate that trueconcentrations of azoxystrobin, boscalid, and pyra-clostrobin in the streams might be two to five timeslower than reported concentrations. However, thefungicide concentrations reported here were notcorrected for enhanced recoveries because matrixspikes results were not available for every site andsample, and the sample matrix and resulting bias wasexpected to be different for each sample. The elevatedspike recoveries have no impact on the detection ofthe target fungicides. Consequently, interpretation ofconcentration data is qualified and limited in thispaper to general perspective on the range andmaximum concentrations encountered. With detectionfrequencies these data provide adequate indications ofthe presence and magnitude of the target fungicides inthe environments sampled.

In some cases, fungicide concentrations that wereless that the LRL were reported. These values wereflagged by the laboratory with an “E” code whichindicates that the sample met all retention time andmass spectral acceptance criterion (Focazio et al.2008). These values are displayed graphically andused for the calculation of detection frequency, butmay not be appropriate for other statistical uses.

2.3 Fungicide Use Estimates

The total annual use of selected fungicides on allcrops by county circa 2002 was calculated from dataprovided in Gianessi and Reigner (2006). An area-weighted sum algorithm programmed in a geographicinformation system (Battaglin and Goolsby 1998) wasused to estimate the amounts of fungicides used orareas of soybeans harvested within drainage basinsassociated with the sampling sites. The algorithmaccounts for cases where an entire county is within asingle drainage basin and where only a portion of acounty is within a drainage basin. The total annualuse of 12 fungicides (Table S1) on all crops by statefor 2002–2007 was prepared by the USGS usingAgroTrak® agrochemical usage data (AgroTrak®

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is a registered trademark of dmrkynetec, St. Louis,MO, USA).

3 Results and Discussion

3.1 Fungicide Occurrence

Nine of the 12 fungicides analyzed were detected in atleast one sample (Fig. 2, Table 2), at least onefungicide was detected in 58 (56%) of the 103samples, and at least one fungicide was detected in20 of 29 streams. Two or fewer samples were collectedat eight of the nine streams with no fungicidedetections. As many as five fungicides were detectedin an individual sample, and mixtures of fungicideswere common. Azoxystrobin was the most frequentlydetected fungicide (45%, 1.13 μg/L maximum con-centration; Table 2) followed by metalaxyl (27%,0.067 μg/L maximum concentration); propiconazole(reported as the sum of the concentrations of trans- andcis-propiconazole; 17%, 1.15 μg/L maximum concen-tration) and myclobutanil (9%, 0.032 μg/L maximumconcentration). Tebuconazole, pyraclostrobin, trifloxy-strobin, and chlorothalonil were infrequently detectedin this study. Boscalid, cyproconazole, and metcona-zole were not detected in any samples.

Azoxystrobin was found in samples from 17 sitesin 11 states (Fig. 3a) with 11 of these sites havingdetections in more than one sample. Its relativelywidespread occurrence may be due to its high watersolubility and its rapidly increasing use in a widevariety of agricultural and non-agricultural settings(US Environmental Protection Agency 1997; Gianessiand Reigner 2006; US Department of Agriculture2009b). The maximum concentration of azoxystrobin(1.13 μg/L) from this study (Table 2) was less thanthat reported in German streams (11.1 and 29.7 μg/L;Berenzen et al. 2005). Azoxystrobin was detected inone sample from Indiana, where simulation modelssuggested that it could occur in runoff there under aworst-case scenario (Deb et al. 2010). There wassignificant direct correlation (Spearman’s rho) be-tween the maximum azoxystrobin concentration at asite (Fig. 3a) and estimates of the use of azoxystrobinin the upstream drainage basins (p=0.007).

Metalaxyl was found in samples from ten sites ineight states (Alabama, Indiana, Iowa, Minnesota,Mississippi, North Carolina, Ohio, and South Caro-

lina). The maximum concentration of metalaxyl fromthis study was 1.15 μg/L (Table 2). Metalaxyl use inUS agriculture was not quantified for 2002 (Gianessiand Reigner 2006), but use in 1997 was estimated tobe ∼300 metric tons. Only 0.8 metric ton of metalaxyluse in the US was reported by AgroTrak® in 2006.Metalaxyl is used alone or in mixtures with otherfungicides as seed treatments for a wide variety ofcrops including soybeans, corn, and vegetables.Metalaxyl has many non-agricultural uses such as onturf and non-food crops, for which use estimates arenot available.

Myclobutanil was found in samples from eightsites in five states (Fig. 3b). Myclobutanil wasdetected at least once at both sites from Mississippiand at all three sites in South Carolina. Myclobutanilwas detected more than once in a year at only one site,the Neuse River, NC, USA. There was no correlationbetween the maximum myclobutanil concentration at asite (Fig. 3) and estimates of the use of myclobutanil inthe upstream drainage basins (p=0.58).

Cis- or trans-propiconazole was found in samplesfrom four sites in three states (Fig. 3c). The maximumconcentration of propiconazole (1.15 μg/L) from thisstudy (Table 2) was greater than that reported forSwiss lakes (0.0019 μg/L; Kahle et al. 2008). Therewas significant direct correlation between the maxi-mum propiconazole concentration at a site (Fig. 3)and estimates of the use of propiconazole in theupstream drainage basins (p=0.047).

Tebuconazole was found in samples from threesites in two states (Fig. 3d). The maximum concen-tration of tebuconazole (0.115 μg/L) from this study(Table 2) was less than that reported in Germanstreams (9.1 μg/L; Berenzen et al. 2005), but greaterthan that reported for Swiss lakes (0.001 μg/L; Kahleet al. 2008). Tebuconazole was detected more thanonce in a year at only one site, Bogue Phalia, MS,USA. There was no correlation between the maxi-mum tebuconazole concentration at a site (Fig. 3) andestimates of the use of tebuconazole in the upstreamdrainage basins (p=0.66).

Chlorothalonil was only detected in two samplesfrom two sites in Georgia even though it was used inall 13 states, and the rate of its use was much greaterthan for the other fungicides (Table S1; Gianessi andReigner 2006). Chlorothalonil physical properties anddegradation characteristics are such that it is seldomobserved in surface-water samples (Battaglin et al.

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2008; Scribner et al. 2006). At one of the sites, LimeCreek, GA, USA the estimated use rate (total use inthe basin divided by drainage area) of chlorothalonilwas much larger (20,220 gm/km2) than at any othersite. The use rate was only 5.2 gm/km2 at the othersite with a detection of chlorothalonil (Sope Creek,GA, USA). The two degradation products of chlor-othalonil, 4-hydroxy-chlorothalonil and 1-amide-4-hydroxy-chlorothalonil, were not detected (LRL of0.050 μg/L) in any of the 25 samples that were analyzed.

Boscalid, cyproconazole, and metconazole werenot detected in any sample. Boscalid was firstregistered for use in the US in 2003 and is used ona wide variety of vegetable crops but not significantlyon soybeans (US Department of Agriculture 2009b).Some boscalid was used in Georgia, Illinois, Indiana,Minnesota, Missouri, and North Carolina in either2005 or 2006. Cyproconazole was first registered foruse in the US in 1988 as a wood preservative. It isused in some turf products and was registered for useon soybeans in 2008. There was no reported cypro-conazole use in 2005 or 2006 in any of the 13 stateswhere samples were collected (Fig. 1). Metconazolewas not registered for use on crops in the US in 2005and 2006 and is used internationally primarily onbananas (US Environmental Protection Agency2006a). Metconazole is currently (2009) approvedfor treatment of soybean rust but is not being

marketed, and there was no reported use in 2005 or2006 (AgroTrak®) in any of the 13 states wheresamples were collected.

3.2 Relations to Toxicity and Use

The concentrations of fungicides in US streamsmeasured in this study were in general one or moreorders of magnitude less than toxicity estimates forthose individual compounds on aquatic organisms orhumans (Fig. 2). Fungicides, however, appear tofrequently contribute to the mixture of pesticides thataquatic organisms in these habitats can be exposed to.Because fungicides typically are applied later in thegrowing season than are herbicides, their use in areasthat historically have not had fungicide applicationsmeans that non-target organisms are now potentiallyexposed via new pathways to these compound. Otherconfounding factors such as effects of mixtures, novelor other modes of action (e.g., hormonally activeprocesses), chronic exposures to non-target organismsas well as beneficial and indigenous fungal commu-nities, and the potential for fungicide resistancepatterns are not accounted for in present toxicological

Table 2 Summary of water quality analysis for selected fungicides, chlorothalonil degradation products in selected US streams 2005–2006 (μg/L)

Fungicide Number ofsamples

Number ofdetections

Number of siteswith detections(out of 29 possible)

Laboratoryreporting levelin μg/L

Mean ofdetectionsin μg/L

Maximumconcentrationin μg/L

Azoxystrobin 103 46 17 0.008 0.163 1.13

Boscalid 103 0 0 0.006 <0.006 <0.006

Chlorothalonil 103 2 2 0.004 0.031 0.033

Cyproconazole 103 0 0 0.006 <0.006 <0.006

Metalaxyl 94 25 10 0.007 0.017 0.067

Metconazole 103 0 0 0.006 <0.006 <0.006

Myclobutanil 103 9 8 0.004 0.012 0.032

Propiconazole 103 18 4 0.004 0.291 1.15

Pyraclostrobin 103 3 2 0.006 0.031 0.054

Tebuconazole 103 6 3 0.010 0.053 0.115

Tetraconazole 103 1 1 0.006 0.047 0.047

Trifloxystrobin 103 2 2 0.004 0.029 0.029

4-hydroxy-chlorothalonil 25 0 0 0.050 <0.050 <0.050

1-amide-4-hydroxy-chlorothalonil 25 0 0 0.050 <0.050 <0.050

Fig. 3 Estimated 2002 annual use rate and the potential dailyflux for: a azoxystrobin, b myclobutanil, c propiconazole, andd tebuconazole

b

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benchmarks. Therefore, the comparison of measuredfungicide concentrations with current toxicologicalbenchmarks provides some perspective on potentialenvironmental hazards many potential impacts remainincompletely quantified.

The lack of correlation between the use andoccurrence of some fungicides is likely the result oftwo factors: (1) insufficient number of samples toaccurately characterize fungicide occurrence and (2) alack of accurate fungicide use data by county on anational scale for 2005 or 2006. In particular,information on the use of newer fungicides is out-of-date at best (circa 2002), and does not account foremergency exemptions or non-agricultural use.

State-level fungicide use estimates from Agro-Trak® confirm that the circa 2002 county-level data(Gianessi and Reigner 2006) may not be sufficient foridentification of relations between fungicide occur-rence and use in 2005 and 2006. For example,azoxystrobin use is reported by AgroTrak® in both2005 and 2006 for all 11 states with reporteddetections including Iowa and Nebraska, which donot show use in the circa 2002 data (Fig. 3a).Likewise, myclobutanil and tebuconazole weredetected at both sites in Mississippi and use isreported by AgroTrak® in 2005 in Mississippi whennone is shown in the 2002 county-level data (Fig. 3b,d). Pyraclostrobin was detected in three samples fromtwo sites: Bogue Phalia, MS, USA and Bayou DeChien, KY, USA. Gianessi and Reigner (2006) do not

indicate any use of pyraclostrobin in 2002 inMississippi or Kentucky; however, the AgroTrak®data indicate that there was use of pyraclostrobin inboth states starting in 2003. Tetraconazole wasdetected in one sample from the Iowa River, IA,USA. Gianessi and Reigner (2006) do not indicateany use of tetraconazole in 2002 in Iowa; however,the AgroTrak® data indicate that there was use oftetraconazole in Iowa in 2005.

3.3 Seasonal Occurrence

At seven sites, four samples were collected during2005 or 2006. Results from these sites (Table 1; onesite had four samples in both 2005 and 2006) wereused to determine if there is a seasonal pattern tofungicide occurrence or concentration as previouslydocumented for herbicides (Thurman et al. 1991;Battaglin et al. 2005; Scribner et al. 2005). At thethree sites in warmer climates with moderate to highsoybean production (Yazoo River, Bogue Phalia, andCow Castle Creek) fungicides are detected throughoutthe growing season (Fig. 4a) with the highestconcentrations occurring in July, August, or Septem-ber (Fig. 4b). At the more northern sites (SangamonRiver and Little Cobb River) and at sites with littlesoybean production (Lime Creek and Edisto River)fungicide detections are more sporadic but generallymore common and at greater concentration duringJuly, August, and September (Fig. 4).

Fig. 4 The a number of target fungicides detected and b total concentration of target fungicides, by month for selected site in 2005and 2006

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Results from these sites suggest that in many USstreams one should expect a seasonal pattern tofungicide occurrence, and that detection of fungicidesis more likely in late summer and early fall than inspring. However, fungicides may occur throughoutthe growing season in agriculturally impacted streamsin warmer climates suggesting the potential forseason-long exposure of aquatic organisms to fungi-cide in these watersheds.

4 Summary

Results from this study indicate that fungicidesoccurred in streams with moderate frequency priorto their widespread use to control soybean rust orincrease crop yields in the southern and central US.This study collected baseline data on the occurrenceof 12 fungicides in 29 US streams soon after the firstdocumentation of soybean rust in the US and prior tothe corresponding increase in fungicide use to controland treat this problem. These data were used to assessspatial and seasonal patterns in fungicide occurrenceand to investigate relations between the occurrence offungicides in US streams and the use of thoseproducts in the associated drainage basins. Nine ofthe 12 fungicides were detected in at least one sample.At least one fungicide was detected in 56% of thesamples and in 20 of 29 streams. As many as fivefungicides were detected in an individual sample, andmixtures of fungicides were common. Azoxystrobinwas detected most frequently (45% of samples)followed by metalaxyl (27%), and propiconazole(17%). Myclobutanil, tebuconazole, pyraclostrobin,trifloxystrobin, chlorothalonil, and tetraconazole wereinfrequently detected during this study. Boscalid,cyproconazole, and metconazole were not detectedin any samples. Fungicide concentrations ranged from0.002 to 1.15 μg/L. Generally, fungicides occurredmore frequently and in higher concentrations in thesouthern US where their use on soybeans and othercrops was more common and widespread, butdetections also occurred where fungicide use onsoybeans was not reported and fungicide use on othercrops was not common suggesting that non-agricultural uses are a potential source of fungicideto the environment. The concentrations of fungicidesin US streams measured in this study were in generalone or more orders of magnitude less than toxicity

estimates for those individual compounds on aquaticorganisms or humans; however, there are limitationsin available toxicological benchmarks. There is someevidence of a seasonal pattern to fungicide occur-rence, with detections more likely and concentrationshigher in late summer and early fall than in spring. Ata few sites in Mississippi and South Carolina,fungicides were detected throughout the growingseason. Fungicide occurrence in US streams appearto be positively correlated to the use of those productsin the associated drainage basins; however, moredetailed monitoring data and more accurate useinformation are needed to quantify these relations.

It is not possible to determine if any of the fungicidedetections from this study were explicitly the result oftreatments to cure or prevent soybean rust, because allof the detected compounds have more than one cropon which they are used and more than one disease thatthey treat. It is clear that some of the detections were inareas where soybean rust eventually spread to in either2005 or 2006. However, these results show that manyof the target fungicides are being introduced to theaquatic environment in low-level mixtures. It is alsoclear that fungicide occurrence will likely increase inthe environment in response to increases in fungicideapplications to prevent fungal disease outbreaks suchas soybean rust and to increase crop yields. Finally,because this occurrence assessment was done prior toany major outbreak of soybean rust in the Central US,the results will provide powerful comparative infor-mation for future studies.

Acknowledgments This study was supported by the USGSToxic Substances Hydrology Program and conducted with thelogistical support of USGS Water Science Centers in Alabama,Georgia, Illinois, Indiana, Iowa, Kentucky, Minnesota, Mis-sissippi, Missouri, Nebraska, North Carolina, Ohio, and SouthCarolina. The authors would especially like to acknowledgeUSGS personnel who collected samples: Richard Coupe,Angela Crain, David Dupre, James Fallon, Jeffery Frey,Thomas Harris, Brian Hughes, Steve Kalkhoff, Michael Wood-side, and Ron Zelt. The use of trade names or product names inthis report does not constitute endorsement or recommendationby the USGS.

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