a silviculture application of the glyphosate-based herbicide visionmax to wetlands has limited...

Environmental Toxicology

A SILVICULTURE APPLICATION OF THE GLYPHOSATE-BASED HERBICIDE VISIONMAX TOWETLANDS HAS LIMITED DIRECT EFFECTS ON AMPHIBIAN LARVAE

CHRISTOPHER B. EDGE,*y DEAN G. THOMPSON,z CHUNYAN HAO,§ and JEFF E. HOULAHANyyBiology Department, University of New Brunswick, Saint John, New Brunswick, Canada

zGreat Lakes Forestry Center, Canadian Forest Service, Sault Ste. Marie, Ontario, Canada

§Laboratory Services Branch, Ontario Ministry of the Environment, Etobicoke, Ontario, Canada

(Submitted 27 February 2012; Returned for Revision 12 June 2012; Accepted 15 June 2012)

Abstract—Herbicides are commonly used in agriculture and silviculture to reduce interspecific competition among plants and therebyenhance crop growth, quality, and volume. Internationally, glyphosate-based herbicides are the most widely used herbicides in both ofthese sectors. Laboratory and mesocosm studies have demonstrated that some formulations are toxic to amphibian larvae belowconcentrations that approximate predicted maximal or ‘‘worst-case’’ exposure scenarios. However, field studies have not found evidenceof toxicity at these concentrations. The authors conducted a replicated field experiment involving 10 naturalized wetlands split inhalf with an impermeable plastic barrier to assess the direct toxicity of a glyphosate formulation commonly used in silviculture(VisionMAXTM). The herbicide formulation was applied directly to the surface of one side of each wetland at one of two target aqueousexposure rates (high¼ 2,880, low¼ 550mg acid equivalents [a.e.]/L), and the other side was left as an untreated control. The survivaland growth of green frog larvae (Lithobates clamitans) were assessed for two years following herbicide treatment. The herbicide did nothave a negative impact on survival or growth of L. clamitans larvae at either treatment level. In fact, mean larval abundance was typicallygreater in the treated sides than in control sides within the year of herbicide application. These results indicate that typical silviculture useof VisionMAX poses negligible risk to larval amphibians, likely because the combined effects of sorption and degradation in naturalwetlands limit the exposure magnitude and duration. Environ. Toxicol. Chem. # 2012 SETAC

Keywords—Glyphosate Amphibian Whole-system experiment Roundup Long-term experimental wetlands area

INTRODUCTION

Herbicides are used in Canadian forestry, typically veryshortly after harvest, to enhance regeneration success throughreduction of interspecies competition among crop trees andearly successional plants. Worldwide and in Canada, glyphosate-based herbicides dominate the silviculture use sector [1–3].These herbicides have generally been considered to pose littlerisk to wildlife, including fish, amphibians, and other aquaticorganisms [3–6]. A fundamental reason for this is the plant-specific mode of action for glyphosate itself [7,8], and com-prehensive risk assessments have indicated that these herbicidescould be used with minimal risk to the environment [4].

A common approach in risk assessment involves comparingpredicted worst-case environmental exposure concentrations,commonly referred to as predicted environmental concentrationor expected environmental concentration values, to mediantoxicity values (e.g., median lethal concentration [LC50]) derivedfrom laboratory and mesocosm studies. Typical worst-case(wetland 15 cm deep with no intercepting vegetation over-sprayed at the maximum label rate) predicted environmentalconcentration values range from 1,430 [9] to 4,486 [4,10]mgacid equivalents (a.e.)/L (based on a one-time application of theannual maximum total for any cropping situation, 6.73 kg a.e./ha).Standardized laboratory toxicity testing of formulated glyph-osate products with amphibian species typically yields 48- or96-h LC50 values in the range of 1,000 to 4,500mg a.e./L for avariety of formulations [10–13]. With some recent studies

reporting LC50 values at lower concentrations than thoseused in earlier risk assessments, the lowest reported 96-hLC50 values for anuran species approximates 800mg a.e./L[10,13]. Furthermore, many other laboratory and mesocosmstudies [14–20] have found that exposure to glyphosate for-mulations at concentrations of glyphosate below those predictedunder worst-case exposure scenarios results in increased mor-tality, suggesting the potential for widespread direct acutetoxicity to sensitive amphibian larvae. However, a monitoringstudy conducted under typical Canadian silviculture operationalscenarios showed that the upper 99% confidence interval (CI) ofaqueous concentrations in wetlands was well below worst-caseexposure scenarios [9]. Directly oversprayed wetlands showedmean concentrations of 330mg a.e./L (upper 99% CI, 550mga.e./L), and wetlands adjacent to spray blocks showed meanconcentrations of 177mg a.e./L (upper 99% CI, 390mg a.e./L).Thompson et al. [9] concluded that these results suggest verylimited potential for direct acute toxic effects under real-worldexposure scenarios.

In field experiments, limited or no effects of aquatic expo-sure to glyphosate formulations have been observed on larvalamphibian survival, growth, development, and avoidancebehavior [9,21]. More recently, studies involving direct over-spray of juvenile, semiaquatic amphibians also showed nosignificant effects on survival, liver somatic index, or bodycondition [22]. These results appear to contradict those oflaboratory and mesocosm studies, which commonly findadverse effects on larval survival [10–20] or sublethal effectson growth and development [12,23,24]. Direct comparisonsbetween studies are difficult owing to differences in test meth-odology, formulations tested, and exposure regimes. Toxicity isknown to vary substantially among formulations, with LC50values ranging from 800 to 16,100mg a.e./L [10,11], as well as

Environmental Toxicology and Chemistry# 2012 SETAC

Printed in the USADOI: 10.1002/etc.1956

All Supplemental Data may be found in the online version of this article.* To whom correspondence may be addressed

([email protected]).Published online 25 July 2012 in Wiley Online Library

(wileyonlinelibrary.com).

1

among species [11–13,17,18,23,25]. Differences among for-mulations are likely due to additives and surfactants, which arenot reported on product labels and can increase the toxicity of aformulated product. For glyphosate-based herbicides this is ofparticular importance because the majority of toxicity of theseformulations has been attributed to additives and surfactants, inparticular the polyetholxylated tallowamine (POEA) surfactant[10,11,16,26]. The differences among formulations limit ourability to make generalized conclusions about the toxicity ofglyphosate-based herbicides, because such risk assessmentsmust be conducted on a product-specific basis with full con-sideration given to the associated use pattern and label spec-ifications.

The effects of glyphosate-based herbicides on amphibianlarvae have been well studied in laboratories and mesocosms,but there are few studies on the effects of these herbicides innatural systems. Whole-system experiments are a critical com-ponent of ecological research because they test hypothesesunder realistic environmental conditions, incorporating mostor all of the complexity found in nature, and they can be carriedout at environmentally relevant spatial and temporal scales[27,28]. One limitation of whole-system experiments is thatthey tend to suffer from low power to detect small but poten-tially biologically meaningful effect sizes, using traditionalvalues of alpha (e.g., 0.05). Low power results from thecombination of low replication and high natural variabilityamong experimental units. Despite the problems associatedwith low power and natural variability, decision makers areoften more strongly persuaded by results from large-scale,whole-system manipulations than by small-scale, laboratoryor mesocosm studies [27,29], owing largely to increased extra-polative uncertainty.

This manipulative ecosystem study was designed to assessthe potential risk of the commercial glyphosate-based herbicideformulation VisionMAXTM (Monsanto) to amphibian larvae ata concentration at the upper end of those measured in theenvironment and at a concentration expected under a worst-case direct overspray scenario. In this experiment, we tested twoalternate hypotheses: (1) the abundance, growth, and develop-ment of free-swimming larval green frogs (Lithobatesclamitans) are directly affected by exposure to the herbicideat either test concentration within the year of treatment; (2) theabundance, growth, and development of free-swimming larvalgreen frogs (L. clamitans) are affected by exposure to theherbicide at either test concentration one year after herbicideapplication.

MATERIALS AND METHODS

Site description

The field site was located in the Long Term ExperimentalWetlands Area (LEWA) on Canadian Forces Base Gagetown(CFB Gagetown) in southeastern New Brunswick, Canada(458400N, 668290W), a 1,200-km2 military installation primarilyused for ground training. The study area is approximately6 km2; it was mechanically cleared of trees and the topsoilwas pushed into berms in 1997 and 1998. This created alandscape of elevated treed ridges and open areas, with hun-dreds of small wetlands ranging from <1 ha to several hectaresin surface area, which have become naturalized to varyingextents since their creation. No prior chemical applicationwas made to this area [30]. Within this area, we chose 10semipermanent or permanent wetlands that were relativelysmall (<1 ha), had no permanent inflows or outflows, had

relatively homogenous macrophyte cover, and were knownbreeding sites for L. clamitans. During the late summer of2008 each wetland was split in half using an impermeableplastic barrier constructed from 30-mil (0.76-mm) black high-density polyethylene (Poly-Flex; Geomembrane Lining Sys-tems). A pocket at the bottom of the barrier was filled withgravel and buried 10 cm in the substrate. The barrier itselfextended approximately 2m beyond the high-water mark ofeach wetland. Barriers were supported by rebar posts locatedapproximately every 3m to maintain the top edge 10 to 100 cmabove the water surface.

A permanent staff gauge was hammered into the sediment atthe deepest point in each wetland half. To calculate the volumeof each half, water depth was measured every 2 or 4m alongtransects located 2 or 4m apart (depending on the surface areaof the wetland) running perpendicular to the barrier. The initialvolume of the wetland half was calculated by summing thevolume of each cube determined using the average water-depthmeasure at each of the four vertices as the depth. Subsequentwater-depth measurements at the permanent staff gauge wereused to estimate the volume of each wetland half at any pointin time.

Experimental treatments and chemical applications

A split-wetland experimental design was employed, withone side of each wetland randomly assigned as the control andthe other as the treatment. In the few wetlands with suspectedtransient flow patterns (three wetlands in spring), the inflow sidewas designated as the control and the outflow side as the treatedside. This ensured that if the integrity of the barrier wascompromised, the control side of the wetland would not becontaminated by the treatment side. The experimental treat-ments were generated by a one-time, direct application of theformulated glyphosate-based herbicideVisionMAX (540 g a.e./L)at one of two concentrations directly to the water surface, witheach concentration replicated in five wetlands. The first was alow target aqueous glyphosate concentration (550mg a.e./L),designated as low. This is the upper 99th percentile concen-tration measured in forest wetlands that were directly over-sprayed during an operational monitoring study [9]. The secondwas a high target aqueous glyphosate concentration (2,880mga.e./L), designated as high. This is the concentration expected ina wetland 15 cm deep with no intercepting vegetation over-sprayed at the maximum label rate (7.9 L/ha). These two targetconcentrations represent a probabilistic estimate of the highestconcentrations likely to be observed in forestry wetlands underan inadvertent overspray scenario (low) and a reasonable worst-case overspray scenario (high).

The herbicide was applied to all experimental wetlands onAugust 17, 2009, under an approved pesticide research author-ization (2009 0593) granted by the Pest Management Regu-latory Agency of Health Canada. The timing of treatmentscoincided with the typical startup of operational herbicide sprayprograms throughout the boreal and Acadian forest regions ofCanada. To ensure that the aqueous target application rates wereachieved and emergent vegetation was exposed, two targetedspray applications were made to each wetland. In an attempt toattain the nominal aqueous concentrations as described above,wetland volumes were calculated immediately before herbicideapplication, and the volume of herbicide required to achieve thedesired concentration was mixed in approximately 3 L ofwetland water. The dilute spray mixture was applied directlyto the surface of the wetland using a backpack sprayer (Flow-master; Root-Lowell Manufacturing). In addition, to ensure that

2 Environ. Toxicol. Chem. 31, 2012 C.B. Edge et al.

the emergent vegetation within the treated half of each exper-imental wetland was directly exposed to the herbicide, anadditional spray was applied directly to the emergent vegetation(dominant vegetation was Typha latifolia), using a fine mist. Onthe control side, the same volume of wetland water (3 L) wasapplied with a separate but identical backpack sprayer (vehiclecontrol) to ensure equivalent physical disturbance on both thecontrol and treated sides of each wetland.

The integrity of the impermeable barriers was inspected inthe spring and fall of all years and periodically throughout thesummer of both years to ensure there was limited water mixingbetween wetland halves. If the integrity of the barrier wascompromised, sediment or sandbags filled with sediment wereplaced against and on the barrier to form an effective seal. Themajority of potential breaches occurred in the spring aftersnowmelt, and in these cases the barriers were repaired withintwo weeks. With frequent inspections and repairs, we arerelatively confident that there was limited mixing of waterbetween wetland halves. Samples taken from control sidesshortly after herbicide application confirmed minimal contam-ination (see Results section).

Validation of gyphosate and aminomethylphosphonic acid(AMPA) aqueous exposure concentrations

Aqueous glyphosate concentrations were measured imme-diately after herbicide application on day 0 and 1, 3, 5, 7, and14 d after herbicide application in the treatment side of wetlandsand 1 d after application in the control sides. Aqueous concen-trations were estimated based on pooling and mixing 50-mlsubsamples from each of five locations within each wetland halfand taking a 50-ml aliquot from the pooled sample. Sampleswere placed in uniquely labeled, sterile plastic centrifuge tubes,capped, and stored in the dark on ice prior to placement infrozen storage at the end of each sampling day.

Field samples were stored and shipped under frozen con-ditions to an OntarioMinistry of the Environment laboratory forthe determination of glyphosate and its metabolite AMPAusing a recently published method [31]. Briefly, the analyticalmethod is based on liquid chromatography/tandem mass spec-trometry (LC/MS-MS) and involves chromatographic separa-tion of analytes via reversed-phase and weak anion-exchangemixed-mode chromatography on an Acclaim1 WAX-1 column.Aqueous samples are directly injected with no sample concen-tration or derivitization steps. By monitoring two channels foreach analyte in multiple reaction monitoring scan mode, theanalytic technique achieves true positive identification. Anisotopic form of glyphosate (13C,15N-glyphosate) was utilizedas an internal standard. The instrument detection limits forglyphosate and AMPA were 1 and 2mg a.e./L, respectively.

Species description

Green frogs (L. clamitans) were chosen as the focal speciesfor this study because they are very common throughout thestudy site, are widespread throughout the Acadian and GreatLakes Forest regions, and occur well into the boreal transitionand southern boreal forests of eastern Canada. Lithobatesclamitans breed in semipermanent to permanent wetlands dur-ing the summer (June–July). They require one full year to reachmetamorphosis, overwintering during their first year as larvae inwetlands of more northern climates, including all of Canada.The breeding phenology and development period are wellsuited to studies investigating potential longer-term impactsof a herbicide that is typically applied in the late summer orearly fall.

Amphibian responses

Amphibian larvae in each wetland half were sampled twicein 2009, once (August 13–14) before herbicide application, andonce (October 8–12) after herbicide application. In 2010,wetlands were sampled twice (May 31–June 1 and September25–October 1). This sampling schedule included one completelarval cohort of L. clamitans exposed to the herbicide in 2009 aslarvae and up until the time of metamorphosis during June andJuly of 2010. The second sampling period in 2010 included oneestimate of abundance and body size of L. clamitans larvae thathatched from eggs that were laid in wetlands one year afterherbicide application (i.e., eggs that were laid in June andJuly 2010).

Amphibian larvae were sampled by dipnetting along trans-ects. Transects were 3m apart and ran parallel to the barrier ineach wetland half. Along each transect one sweep (�1.5m) wastaken with a dipnet every 3m. The number of transects andsweeps varied with wetland size. All captured amphibian larvaewere counted and identified to species. First-year L. clamitanslarvae were photographed in a Petri dish containing 0.5 cm ofwater on a 0.5- or 1.0-cm grid with a digital camera (CanonPowershot A95). Subsequently, body size was measured usingdigital image analysis software (ImageJ; U.S. National Insti-tutes ofMental Health). In 2010, the snout–vent length (SVL) ofL. clamitans larvae was measured with a ruler. This was donebecause second-year larvae were too large to be accuratelyphotographed and measured in small Petri dishes.

Statistical analyses

To determine if the herbicide application resulted in twodistinct treatment groups, the mean log-transformed aqueousglyphosate concentrations over the first 14 d of exposure werecompared with a one-factor (treatment level, low or high)repeated measures analysis of variance (RM-ANOVA). Wealso compared the time-weighted average (TWA) aqueousglyphosate concentrations over the first 14 d between treatmentgroups with a t test. To characterize the length of the exposure,we calculated the time to 50% dissipation (DT50) of glyphosatein the water column using a first-order reaction rate. Larvalabundance was calculated as the number of animals capturedper unit effort (sweep). For the majority of end points we used abefore–after/control–impact design. To investigate effects onsurvival, we compared the percentage of change in abundanceof larvae between the first and second sampling periods in 2009(about two months after herbicide application), abundance atmetamorphosis in 2010, and abundance of first-year larvae in2010. To investigate effects on growth rate, we compared thepercentage of change in body size between the first and secondsampling periods in 2009, the SVL of larvae prior to meta-morphosis in 2010, and the body size of first-year larvae in2010.

Effects on larval abundance and growth were assessed usingseparate split-plot ANOVAs with treatment level as the among-plot factor (low or high) and side as the within-plot factor(control or treatment). Although our analyses of aqueousglyphosate concentrations indicated that two treatment groupswere achieved, there was some variation in measured concen-tration both within and between treatment groups (see Resultssection). Due to this variation, we also analyzed the data vialinear regression of response variables against the TWA overthe first 14 d of exposure to check for a dose–response relation-ship. For these analyses, the response variable was calculated bysubtracting the control value from the treatment value for each

Effects of VisionMAX on amphibians in whole wetlands Environ. Toxicol. Chem. 31, 2012 3

wetland (y¼mxþ b; where y¼T–C and x¼TWA). In ourinterpretation of the results we place more emphasis on theANOVA results because the regression analyses are post hocexploratory analyses as the experiment was not specificallydesigned to assess a dose–response relationship. Results ofthese analyses should be considered hypotheses that requirevalidation through experiments in which dose and exposurerates are intentionally varied. Abundance data were log-trans-formed to meet the assumption of equal variance prior toanalyses, and all percentage data were arcsine square root–transformed prior to analyses. For each statistical test wedetermined the power to detect a large effect size defined byCohen [32] with an alpha of 0.05. Means are reported�SEunless otherwise stated. All statistics were performed using thenlme or base package, where appropriate, in R (R Foundationfor Statistical Computing).

RESULTS

Glyphosate exposure

On day after treatment (DAT) 0 mean aqueous glyphosateconcentrations were 776.2� 211.1mg a.e./L in the treated sidesof wetlands receiving the low treatment level and 3,100.2�823.3mg a.e./L in the treated sides of wetlands receiving thehigh treatment level. Despite the fact that aqueous concentra-tions were approximately four times higher in the high treat-ment than the low treatment on DAT 0 and DAT 1, there was nodifference in aqueous glyphosate concentrations among the twotreatment levels over the first 14 d of exposure (F1,8¼ 3.015,p¼ 0.12), but the effect of time was significant (F5,5¼ 55.79,p< 0.0001). Glyphosate dissipated rapidly in all wetlands(Fig. 1), and after 14 d the mean aqueous concentration inthe treated side of all wetlands was 2.019mg a.e./L and belowdetection limits (1mg a.e./L) in two of the wetlands receivingthe low treatment level and one of the wetlands receivingthe high treatment level. The calculated half-life of glyphosatewas relatively short in both the low treatment (mean¼ 1.88 d,min¼ 1.53 d, max¼ 2.34 d) and the high treatment (mean¼1.47 d, min¼ 1.19 d, max¼ 2.01 d). Because of this rapiddissipation we conducted t tests with equal variances on log-transformed aqueous glyphosate concentrations on each day.On DAT 0 and DAT 1, the aqueous glyphosate concentrationswere higher in wetlands receiving the high treatment level thanthose receiving the low treatment level (DAT 0 t8¼ 3.065,p¼ 0.015; DAT 1 t8¼ 3.778, p¼ 0.0054), but there wasno difference on subsequent days (p> 0.05 in all cases).

Nonetheless, the mean TWA exposure over the first 14 dwas higher in the wetlands receiving the high (628.36�164.90mg a.e./L) treatment level than in those receivingthe low (126.98� 37.50mg a.e./L) treatment level (t8¼ 3.78,p¼ 0.0054). These values indicated that the aqueous glyphosateconcentrations approximated nominal target concentrationsbetween DAT 0 and DAT 1 and rapidly declined to valuesthat approximated detection limits within 14 d of herbicideapplication. Differences between the two treatment groups onDAT 0 and DAT 1 and in TWA indicated that two distincttreatment groups were achieved, but there was considerablevariation both between and within treatment groups. The powerfor the RM-ANOVA to detect a large effect size [32] was 0.703,and the power of each t test was 0.201. Despite the low power ofthe t tests to detect large differences, significant differenceswere detected at DAT 0 and DAT 1, implying that there werelarge differences between the low and high treatments.

Glyphosate was detected on the control sides of fourwetlands (two low and two high) at low concentrations(mean¼ 7.34, min¼ 4.65, max¼ 10.80mg a.e./L) on DAT 1.This contamination may have been due to spray drift over theimpermeable barrier during application, leakage around thebarrier, or cross-contamination during sampling or samplehandling. Irrespective of the mechanistic explanation, theexceedingly low levels of contamination documented on controlsides in these four cases are not likely to be biologically relevantbecause detected levels were two orders of magnitude below thelowest concentrations that result in mortality in laboratory andmesocosm studies and one order of magnitude below the lowestvalue (48.7mg a.e./L) measured on the treatment side of any ofthe wetlands on DAT 1.

Amphibian survival

Although the study design incorporated 10 wetlands, larvaewere only caught on both the treated and control sides of sevenwetlands (four low and three high) prior to herbicide applicationduring 2009 and first-year larvae were only caught in sixwetlands (three low and three high) during the second samplingperiod one-year post herbicide application. Only those wetlandsin which larvae were captured were used in analyses. In allwetlands the abundance of L. clamitans larvae declined betweenthe first and second sampling periods within the year ofherbicide application. However, there was no statistically sig-nificant difference between the treatment and control sides inthe mean percentage of change in abundance of L. clamitanslarvae between wetlands sides in either the low or high treat-ment wetlands (p¼ 0.166) (Fig. 2 and Table 1). Furthermore,there was no relationship between the difference in larvalabundance between the treatment and control sides of wetlandsand TWA (df¼ 5, r2¼ –0.17, p¼ 0.74; Supplemental Data,Fig. S1A). One year postherbicide application there was nostatistically significant difference between the treatment andcontrol sides in the abundance of premetamorphic larvae ineither the low or high treatment wetlands (p¼ 0.728) (Fig. 3and Table 1). Nor was there a relationship between TWAglyphosate exposure and the difference in the abundance ofpremetamorphic larvae between the treatment and control sidesof wetlands (df¼ 5, r2¼ 0.122, p¼ 0.23; Supplemental Data,Fig. S2A). Mean abundance of newly hatched larvae in June andJuly 2010 (about one year posttreatment) was higher on thetreated sides than the control sides of wetlands (p¼ 0.039), butthere was no difference in the response between the low andhigh treatment levels (p¼ 0.68) (Fig. 4 and Table 1). However,there was no statistically significant relationship between the

Fig. 1. Mean aqueous glyphosate concentrations (� SE) measured in thetreatment sides of wetlands in which glyphosate was applied at two nominaltarget concentrations (low [L]¼ 550mg a.e./L, high [H]¼ 2,880mg acidequivalents [a.e.]/L) measured for 14 d after application.


difference in the abundance of newly hatched larvae betweenthe treatment and control sides and TWA (df¼ 4, r2¼ 0.27,p¼ 0.16; Supplemental Data, Fig. S3A).

Amphibian growth

No statistically significant difference was observed in thechange in body size of larvae between the treatment and controlsides of wetlands receiving either the low or high treatmentlevel during their first year of growth in 2009 (p¼ 0.15) (Fig. 2and Table 1). Nor was there a relationship between the magni-tude of the difference in body size between the treatment andcontrol sides and TWA (df¼ 5, r2¼ –0.18, p¼ 0.77; Supple-mental Data, Fig. S1B). In 2010 there was no statistically

significant effect of either treatment level on the SVL ofpremetamorphic larvae (p¼ 0.31) (Fig. 3 and Table 1). Fur-thermore, there was no relationship between TWA and thedifference in body size of premetamorphic larvae betweenthe treatment and control sides (df¼ 5, r2¼ –0.20, p¼ 0.90;Supplemental Data, Fig. 2SB). No statistically significant dif-ference was observed in the body size of larvae that hatchedfrom eggs one year posttreatment in wetlands receiving eitherthe low or high treatment (p¼ 0.21) (Fig. 4 and Table 1). Norwas there a relationship between the difference in body size oflarvae that hatched from eggs one year posttreatment betweenthe treatment and control sides of wetlands and TWA (df¼ 4,r2¼ 0.28, p¼ 0.16; Supplemental Data, Fig. S3B).

Post hoc power analyses indicated that for the ANOVAmodels the power to detect a large effect [32] on end pointsmeasured in 2009 was 0.256, and for end points measured in2010 was 0.216. For the regressionmodels, the power to detect alarge effect [32] was 0.248 for end points measured in 2009, and0.202 for end points measured in 2010. Although the power ofthese tests was relatively low, for almost all end points thedirection of the effect was opposite to what would have beenexpected if the herbicide was causing mortality or reducedgrowth.

DISCUSSION

The general goal of the present study was to assess the directeffects of a glyphosate-based herbicide (VisionMAX), which iscommonly used in the Canadian silviculture sector, on larvae ofthe green frog (L. clamitans), which usually occurs in semi-permanent forest wetlands throughout its range in Canada. Ourresults show no statistically significant deleterious effects of thisherbicide at either the high or the low treatment level on any ofthe response parameters that were measured. However, due tothe low power of the statistical tests to detect effects on endpoints measured in either year of study, they do not present

Fig. 2. Meanpercentage of change (� SE) in abundance andbody size ofLithobates clamitans larvaebetween thefirst samplingperiod (August 13–14) and the lastsampling period (October 8–12) in 2009.The y axis for larval abundance is negative because abundance declined in allwetlands, so smaller declines imply positiveeffects of the herbicide. C¼ control; T¼ treatment; L¼ low; H¼ high.

Table 1. Critical values from split-plot analyses of variance, with treatment(two levels: high vs low) as the whole-plot factor and side (two levels:

control vs treatment) as the within-plot factor

Response variable Effect df F p

2009 change in abundance Treatment level 1,5 0.54 0.5Side 1,5 2.62 0.17Treatment� side 1,5 0.023 0.89

2010 abundance priorto metamorphosis

Treatment level 1,5 0.00046 0.98Side 1,5 0.14 0.73Treatment� side 1,5 0.76 0.42

2010 abundance offirst-year larvae


2009 change in body size Treatment level 1,5 0.27 0.63Side 1,5 2.81 0.15Treatment� side 1,5 0.12 0.75

2010 snout–vent lengthprior to metamorphosis


2010 body size offirst-year larvae



conclusive evidence of no effect; therefore, trends in the datashould be considered and given some weight. Trends in the datasuggest a positive effect of the herbicide at both treatmentlevels on larval abundance and growth during the first year ofgrowth within the year that the herbicide was applied toexperimental wetlands (2009). The increase in growth ratewas transient and not observed in premetamorphic animals ineither treatment level one year after the herbicide was applied(2010). The increases in abundance were also transient; prior tometamorphosis (2010) it was not observed in the wetlandsreceiving the low treatment level and was lower (althoughnot statistically significant) in the treated sides of the wetlands

receiving the high treatment level. Of all the response variablesmeasured, the abundance and SVL of animals just prior tometamorphosis may be considered to be the most ecologicallyrelevant. Body size at metamorphosis is an important fitnesscorrelate [33–35], and abundance at metamorphosis is the bestmeasure of overall larval survival. The strongest evidence for aneffect of herbicide application was the higher abundance ofnewly hatched larvae in the treated sides relative to the controlsides in 2010, one year posttreatment. This is likely due to anindirect effect of the herbicide as it occurred one year aftertreatment and was not consistent with an exposure–responserelationship.

Fig. 3. Mean abundance and snout–vent length (�SE) of premetamorphicLithobates clamitans larvae in 2010 thatwere exposed to the herbicide one year prior in2009. Larvae hatched from eggs laid in 2009 and completed the larval stages of their life cycle in 2010. C¼ control; T¼ treatment; L¼ low; H¼ high.

Fig. 4. Mean abundance and body size (� SE) of first-year Lithobates clamitans larvae in 2010. Larvae hatched from eggs laid in wetlands in 2010 one year afterherbicide application. C¼ control; T¼ treatment; L¼ low; H¼ high.


Our results contrast with previously conducted laboratoryand mesocosm studies on other glyphosate-based herbicidesthat found substantial acute mortality in amphibian larvaeexposed to glyphosate-based herbicides at concentrationsapproximating those associated with the high treatment levelin this study (e.g., [10–18]). For L. clamitans specifically, LC50values range from 800 to 3,500mg a.e./L [10,12,13,23]. Theresults of the present study parallel the general lack of directtoxic effects on amphibian larvae observed for the glyphosate-based herbicide vision in previous field studies conducted innatural wetland systems [9,21] as well as a previous studyexamining the potential effects of VisionMAX on juvenilegreen frogs under simulated natural exposure scenarios [22].Although differences in species sensitivity, innate toxicity ofvarious formulations, and testing methodology make directcomparison of data difficult or impossible, the general trendof lower toxicity being observed in field studies compared tolaboratory and mesocosm studies is evident. We postulate thatthis difference is largely the result of higher exposure magni-tude and longer exposure duration in laboratory versus field testscenarios.

Laboratory studies are typically conducted as short-term(48–96 h) acute toxicity tests in which the exposure concen-tration is sustained at the chosen test level through replacementof the exposure medium. In contrast, in natural wetland systemsour monitoring data indicate that aqueous glyphosate concen-trations (and presumably any surfactants in the formulation)dissipate rapidly. This exposure regime means that larvae areonly exposed to concentration levels that result in mortality forbetween 24 and 72 h after herbicide application. This postulateis further supported by previous environmental fate studies,which demonstrate that the active ingredient glyphosate and atleast one of the surfactants commonly used in glyphosate-basedherbicide formulations (POEA) are rapidly dissipated in shal-low, biologically active aquatic environments [21,36], and sorbquickly and strongly to sediments [37,38] where they are likelyto be less biologically available. All wetlands used in this studyare characterized by clay or loam substrates and are relativelyshallow (20–100 cm), conditions that result in high surface areato volume ratios, warm water, and presumably high microbialpopulations and activity, all of which are conducive to rapiddegradation and dissipation of labile chemicals such as glyph-osate and many surfactants. Thus, in natural wetland systemssuch as those studied here, the magnitude of chemical exposurein the aquatic phase will diminish rapidly after treatment and theoverall duration is likely to be quite limited. This postulate isbased on the assumption that neither glyphosate nor associatedsurfactants are readily bioavailable following sorption tosediments. For glyphosate per se, this assumption is stronglysupported by its fundamental physicochemical characteristics(i.e., zwitterionic nature, high partition coefficient values fororganic carbon and soil), the general lack of phytotoxicityassociated with soil treatments, and the reduced toxicity toaquatic organisms where test systems include soils or sedi-ments. Unfortunately, parallel supporting information is notavailable for POEA and other surfactants.

Our results also contrast with mesocosm studies that dem-onstrate that a variety of glyphosate-based herbicides are toxicto amphibian larvae [16,17,25]. Aqueous concentrations ofglyphosate were verified in all of these studies, but concen-trations were not measured throughout the duration of the study.In one case [19], herbicide was added to mesocosms three timesat 7-d intervals. In that study, aqueous glyphosate concentra-tions were measured after the third application and indicated

minimal breakdown of the herbicide. It is likely that in thesemesocosm studies animals were exposed to relatively higherconcentrations for longer periods of time than in our field studyor in plausible real-world exposure scenarios. Another impor-tant aspect that may contribute to differential test results is thelack of inclusion of natural sediments in mesocosm test systemsor, in cases where they are included, that water volume tosorption surface area ratios are too low to be consideredrepresentative of natural shallow wetlands. Given all of theseconsiderations, it is likely that the differences between thepresent study and previous work are due to exposure durationand concentration. This hypothesis should be addressed byfuture work focused on understanding the effects of short-termacute exposures and by monitoring the concentrations of bothglyphosate and the surfactants in the aqueous and sedimentphases throughout studies.

The observation of higher larval abundance on the treatedsides relative to control sides contrasts with the majority ofpublished field studies on glyphosate-based herbicides to date.Field studies have been designed to test for direct effects ofherbicide exposure on larval survival and growth, using free-swimming larvae caged in situ [9,21]. Survival in control in situcages is often >80% because such cages are specificallydesigned to maximize survival by reducing other sources ofmortality, such as predation. Thus, they are not well suited todetect increased survival, which we observed in the presentstudy. Our results on L. clamitans larvae are similar to those ofanother whole-system experiment we conducted to investigatethe potential effects of another glyphosate-based herbicide,Roundup WeatherMAX, in an agricultural exposure scenario(C.B. Edge, University of New Brunswick, Saint John, NewBrunswick, Canada, unpublished data). That study also foundthat the abundance of L. clamitans larvae was higher in thetreated sides of split wetlands compared to the control sides,both when the herbicide was applied alone at a similar high rateas employed in the present study and when it was applied incombination with nutrient enrichment. Furthermore, in thepresent study the abundance of newly hatched larvae washigher on the treated sides of wetlands one year after herbicideapplication, and monitoring of aqueous glyphosate concentra-tions indicated that the herbicide was very unlikely to be presentat toxicologically significant levels at this time. Thus, weconclude that positive effects of treatment on the abundanceof green frog larvae one year posttreatment as observed in thepresent study are likely due to indirect effects mediated throughthe changes to the structure or function of the ecosystem such asdecreased predation or increased food supply. These effects arealmost certainly due to indirect pathways possibly related to thereduction in macrophyte cover.

In the present study herbicide application resulted in adramatic decrease in macrophyte cover that persisted throughone year posttreatment and a short-lived, transient negativeeffect on periphyton (J. F. Mudge, University of New Brunswick,Saint John, New Brunswick, Canada, unpublished data),zooplankton, and potential amphibian predators (L. F. Baker,University of New Brunswick, Saint John, New Brunswick,Canada, unpublished data) that did not persist for the durationof the study. In field and mesocosm studies, increases in theabundance of phytoplankton and periphyton have been previ-ously reported in response to exposure to glyphosate-basedherbicides [17,20,21]. Lithobates clamitans larvae are oppor-tunistic omnivores, and this increase in food resources couldresult in lower mortality. A logical hypothesis would be thatincreased food resources would result in an increase in growth


rate or body size at metamorphosis. This hypothesis is sup-ported by the results of the present study. Some evidence(a consistent large response) was observed in both years ofthe study for a positive effect of the high herbicide applicationrate on growth of larvae, although these effects were notstatistically significant in either year.

Although we did not detect a negative impact of herbicideapplication on the end points studied, the increase in abundanceof L. clamitans larvae may in turn result in a negative impact onother amphibian species. For instance, we observed significantpredation by L. clamitans larvae on wood frog (L. sylvaticus)egg masses (C.B. Edge, University of New Brunswick, SaintJohn, New Brunswick, Canada, unpublished data). An increasein the size or abundance of L. clamitans larvae could result insignificant predation of L. sylvaticus in wetlands where bothspecies are found together. Increasing the abundance of a largespecies that overwinters as larvae could result in increasedinterspecific competition on species that breed earlier in theyear due to priority effects [39]. Interspecific competitioncan result in decreased growth rates, increased developmentperiods, and smaller size at metamorphosis [40–43]. Thesesublethal effects on time to and size at metamorphosis canhave effects on fitness traits such as size at or age at firstreproduction [35,44]. The effects of an increase in the abun-dance of a superior competitor species on the abundanceof other species requires further investigation by monitoringcontaminated compared to similar noncontaminated systemsover long periods of time.

CONCLUSIONS

The present study provides little evidence that application ofthe glyphosate-based herbicide VisionMAX has a negativeeffect on L. clamitans abundance or growth. It does providesome evidence that the herbicide has a positive effect on theabundance and growth of L. clamitans larvae. These results,which occurred in both years of the study, contrast with those ofsome laboratory and mesocosm studies conducted with otherglyphosate-based herbicides. Differences among studies may beattributed to differences in the herbicide formulation tested ordifferences in realized exposure magnitude and duration. Themost plausible explanation for the lack of significant toxiceffects in the present study is that glyphosate (and presumablythe associated surfactants) dissipated very rapidly from thewater column. This corroborates prior reports from environ-mental fate studies conducted in shallow, biologically activenatural wetlands. The rapid sorption and dissipation of glyph-osate in these systems also provide a basis for suggesting thatthe observed positive long-term effects on abundance weremediated through indirect mechanisms associated with reducedmacrophyte cover, greater food availability, or reduced preda-tion. Combining the results of laboratory, mesocosm, and fieldstudies suggests that glyphosate-based herbicides are toxic toamphibian larvae at sufficiently high concentrations. However,in nature, amphibian larvae are exposed to these concentrationsfor a short period of time, so direct effects on mortality, growth,and development are likely to be small or nonexistent. Overall,the present study demonstrates that typical Canadian silvicul-ture use of the herbicide VisionMAX poses negligible risk toamphibian species.

SUPPLEMENTAL DATA

Figs. S1–S3. (319 KB DOC).

Acknowledgement—We thank L. Baker, J. Mudge, S. Melvin, B. Reinhart,J. Stewart, S. Sadeghi, C. Carpenter, A. Colquhoun, A. Carpenter, M. Gahl,L. Navarro-Martin, C. Robertson, and C. Lanctot for their help in the field.D. Chartrand assisted in the preparation of samples for glyphosateanalyses. V. Trudeau, K. Kidd, and B. Pauli assisted with project design.The project would not have been possible without access to CFB Gagetownprovided by the Department of Defense. Funding was provided by anNSERC-SGP to J. Houlahan, D. Thompson, K. Kidd, V. Trudeau, andB. Pauli; the Canadian Department of Defence (CFB Gagetown); theUniversity of New Brunswick; and the Canadian Forest Service (NaturalResources Canada). Animal-handling procedures were approved bythe University of New Brunswick Animal Care Committee, protocol2009-01-04.

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