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Environmental Pollution 172 (2013) 275e282

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Environmental Pollution

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Effects of chlorimuron ethyl on terrestrial and wetland plants: Levels of, and timeto recovery following sublethal exposure

David Carpenter, Céline Boutin*, Jane E. AllisonEnvironment Canada, Science & Technology Branch, National Wildlife Research Centre, 1125 Colonel By Drive, Carleton University, Ottawa, Ontario, Canada K1A 0H3

a r t i c l e i n f o

Article history:Received 16 March 2012Received in revised form17 August 2012Accepted 8 September 2012

Keywords:Chlorimuron ethyl herbicideNon-target plantsPlant reproductionRecoverySublethal doses

* Corresponding author.E-mail address: Celine.Boutin@ec.gc.ca (C. Boutin)

0269-7491/$ e see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.envpol.2012.09.007

a b s t r a c t

Current pesticide registration guidelines call for short-term testing of plants; long-term effects onvegetative parts and reproduction remain untested. The aims of our study were to determine level ofrecovery and recovery times for plants exposed to the sulfonylurea herbicide chlorimuron ethyl usingdata collected from single species, doseeresponse greenhouse experiments. The nine terrestrial andeight wetland species tested showed variable levels of recovery and recovery timeframes. Many species(six terrestrial and five wetland) were vegetatively stunted at sublethal doses and were reproductivelyimpaired. Full recovery did not occur at all doses and maximum recovery times varied from 3 to 15 weeksin this controlled environment. In a complex community, affected species may be displaced by tolerantspecies, through interspecific competition, before they fully recover. It is plausible that individual pop-ulations could be diminished or eliminated through reduced seedbank inputs (annuals and perennials)and asexual reproduction (perennials).

Crown Copyright � 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

The increased usage of pesticides worldwide calls for thoroughmonitoring of the effects of these chemicals on unintended targetswithin the environment. Herbicide drift into natural areas affectswild plant communities in the short-term (de Jong et al., 2008; deSnoo and van der Poll, 1999; Kleijn and Snoeijing, 1997; Marrset al., 1989). Furthermore, in the long-term, susceptible speciesmay be displaced through loss of valuable resources to moreresilient competitors resulting in community changes (Boutin andJobin, 1998; Gove et al., 2007; Peterson et al., 2006). Currentregulatory methods rely heavily on the use of crops as indicatorsof the risk of herbicide drift on wild vegetation (OECD, 2006;USEPA, 2012). Though toxicology results for crops and wildspecies yield similar short-term outcomes in terms of plantbiomass (Carpenter and Boutin, 2010; White and Boutin, 2007),little attention has been paid to long-term recovery or to repro-duction (but see Carpenter and Boutin, 2010; Riemens et al., 2008,2009). In addition, crops bred as vigorous producers but poorcompetitors cannot adequately represent natural systems wherespecies coexist and demonstrate wide ranges of sensitivity tocontaminates.

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012 Published by Elsevier Ltd. All

Variability in the susceptibility of plant species to herbicides iswidely known and documented. From a community standpoint,this variability would be extremely important in predicting how thebiodiversity structure of the community may shift if affected byherbicide spray drift (Aavik and Liira, 2010; Krauss et al., 2011).Greenhouse experiments can be used to evaluate individual leveleffects of herbicides on plants by maintaining consistent parame-ters for potentially confounding variables (i.e. weather, light,herbivory, etc.); however, this is also a limiting factor, as naturalenvironments are themselves highly variable, and plants (exceptcrops) grow within complex species communities. Simplified arti-ficial microcosm studies conducted in greenhouses that haveincluded several species show variable results in short-termexperiments. For example, two species tested singly in potsdemonstrated more sensitivity to the herbicide mecoprop thanwhen tested in competition (Damgaard et al., 2008). Conversely,species growing in mesocosms were more sensitive to atrazine andglyphosate thanwhen grown in individual pots (Dalton and Boutin,2010). Riemens et al. (2008) showed there was little relationshipbetween plants grown in mixture or individually. Nonetheless,baseline knowledge of how groups of individual species respondfollowing exposure can provide key information on what mightoccur within the context of a natural environment. This informa-tion can be strengthened by examining species for longer timesafter exposure and by taking into account both vegetative andreproductive effects over the course of the trial.

rights reserved.

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282276

Toxicological analyses of the effects of herbicides on plants relyon data collection at set endpoints, often not focusing on repeatablemeasures of plant health over time. Previous work with wildvegetation exposed to the herbicide glufosinate ammonium foundvegetative recovery following exposure to low doses. However,reproductive endpoints did not always recover to similar levels(Carpenter and Boutin, 2010). Of considerable importance is thetime required for recovery, since plants that recover faster at anygiven dose would likely have advantages over those that are slowerat, or incapable of, recovering.

In this experiment, we seek to further explore the long-termeffects associated with exposure to sublethal doses of herbicidesonwild plant species from different habitats. The major aims of ourstudy were: (1) to evaluate the effects of the sulfonylurea herbicidechlorimuron ethyl on the vegetative and reproductive health ofterrestrial andwetland plants and (2) to determine the levels of andtime to recovery for plants exposed to increasing doses of theherbicide.

2. Materials and methods

2.1. General experimental setup

Experiments were conducted in the greenhouses of the National WildlifeResearch Centre (Environment Canada), Ottawa, ON, between July 2010 andDecember 2011. Average temperature ranged from 17 � 3 to 39 � 5 �C andphotosynthetic active radiation ranged from 285 (cloudy day) to 1951 mmolphotons m�2 s�1 (sunny day). Biological control agents were used to minimize theeffects of greenhouse pests (aphids, fungus gnats, thrips and whiteflies): Aphi-doletes aphidimyza (Rondani) Diptera, Encarsia formosa Gahan, Hippodamia con-vergens Guerin, Hypoaspis miles (Berlese) Acari, and Neoseiulus cucumeris(Oudemans) Acari.

Nine terrestrial and eight wetland plant species were tested during the course ofthe experiment (Table 1). Species were classified based on habitat informationprovided in Gleason and Cronquist (1991) and Oldham et al. (1995). All seeds weresurface sown in small trays on top of 3:1 (by volume) Pro-mixMPV soil:horticulturalsand for germination. Trays of seeds requiring stratification (Table 1) were firstplaced into a dark 2e4 �C refrigerator before being moved into the greenhouses.Species without stratification requirements were soil sown only after the stratifiedspecies from their habitat had completed their stratification periods. Approximately120 seedlings of each species were transplanted singly into 10 � 10 � 9 cm potscontaining the 3:1 soil:sand mixture when sufficient numbers had germinated andhad mature cotyledons. In total, 2040 plants were grown for all experimental work(1080 terrestrial and 960 wetland).

Table 1Names and characteristics of plant species tested in the chlorimuron ethyl experimentperformed in a dark 2e4 �C refrigerator with seeds placed on top of the 3:1 soil:sand m

Code Common name

Terrestrial speciesAnagallis arvensis L. AA Scarlet pimpernelAsclepias syriaca L. AS Common milkweedCapsella bursa-pastoris (L.) Medik. CBP Shepherd’s purseCentaurea cyanus L. CC Bachelor’s buttonChenopodium album L. CA LambsquartersCleome serrulata Pursh CS Rocky Mountain beeElymus canadensis L. EC Canada wildryeHelianthus strumosus L. HS Paleleaf woodland suLobelia inflata L. LI Indian tobacco

Wetland speciesAsclepias incarnata L. AI Swamp milkweedElymus virginicus L. EV Virginia wildryeEupatorium maculatum L.a EM Spotted joe pye weeGlyceria striata (Lam.) Hitchc. GS Fowl mannagrassLycopus americanus Muhl. ex W. Bartram LA American water horePanicum clandestinum L.b PC DeertonguePolygonum hydropiper L. PH Marshpepper knotwPolygonum pensylvanicum L. PP Pennsylvania smartw

a Synonym: Eupatoriadelphus maculatus (L.) King & H. Rob.b Synonym: Dichantelium clandestinum (L.) Gould.

2.2. Herbicide information and application

The sulfonylurea herbicide chlorimuron ethyl (Classic� 25DF; DuPont CanadaCompany, Mississauga, Ontario) (hereafter chlorimuron) was used for all experi-ments. It is a selective post-emergence herbicide used to control broad-leaf plants insoybean fields (Claus, 1987). Chlorimuron acts by inhibiting the enzyme acetolactatesynthase, thereby stopping plant cell growth and division. It is readily absorbedthrough leaves and easily transported throughout the plant (OMAFRA, 2009).

Eight 500 ml solutions of chlorimuron were prepared following a geometricprogression of 1.95 assuming a 100% application rate of 9 g active ingredient (ai) perhectare (0.09, 0.18, 0.34, 0.67, 1.31, 2.54, 4.95, 9.63 g ai ha�1) as indicated by theproduct label. One milliliter of Agral 90� (non-ionic surfactant) was added to eachsolution as recommended on the label to improve efficacy. Once made, all solutionswere used within one week, after which fresh batches were prepared.

Chlorimuron application was performed using a track spray-booth (de VriesManufacturing, Hollandale, MN, USA) outfitted with a TeeJet 8002E flat-fan spraynozzle (Spraying Systems, Wheaton, IL, USA). The system was calibrated prior toherbicide application to ensure that 6.75 ml m�2 of solution was delivered ata pressure of 206.84 kPa. Plants were sprayed when seedlings had reached the fourto six true leaf stage. To prevent potential biases during measurements, plants wererandomly assigned ID tags. The experimental setup consisted of 6 replicates � 9doses (including controls)� 2 treatments (short- and long-term) for each species. Intotal, 108 plants of each species were tested: half for short-term biomass assessmentat four weeks and the remainder for long-termmeasurements. Short- and long-termplants were sprayed at the same time (see exceptions below) with the samechlorimuron preparations. To ensure uniformity of growing conditions, plants ofa given species were randomized by dose/treatment within blocks in the green-house, and were regularly rotated. In addition, plants from a given habitat weregrown within the same greenhouse unit and effort was made to spray all specieswithin the same timeframe.

Measurements of initial seedling height/length and basal diameter (Capsellabursa-pastoris) were taken prior to spray. In order to ensure size uniformity withindoses and treatments, plants were grouped by size across a given replicate (doseswere statistically similar in starting height/diameter: ANOVA or KruskaleWallisanalyses; p > 0.05 for each species). Due to unequal seedling growth, short-termplants of Eupatorium maculatum were sprayed one week later than their corre-sponding long-term plants to allow smaller seedlings to reach the four to six trueleaf stage. To maintain size homogeneity, one replicate from each dose in the short-term treatment was discarded (n ¼ 5 plants per dose). In addition, an outbreak ofroot-rot within the seedlings of Polygonum hydropiper resulted in an insufficientnumber of plants to test both short- and long-term effects without seed re-stratification; therefore short-term biomass was not assessed.

2.3. Assessing short-term effects on plant biomass

To assess short-term effects of chlorimuron on plant biomass, all plants withinthe short-term treatment were harvested exactly four weeks after herbicide expo-sure. All above-ground plant material was harvested by cutting the plants at the soil

. Plant code is used in subsequent figures. For stratified species, stratification wasixture.

Family Lifespan Stratificationtime (Months)

Primulaceae Annual/Biennial NoneAsclepiadaceae Perennial 1Brassicaceae Annual NoneAsteraceae Annual NoneChenopodiaceae Annual None

plant Capparaceae Annual 1Poaceae Perennial 1

nflower Asteraceae Perennial 2Campanulaceae Annual None

Asclepiadaceae Perennial 1.5Poaceae Perennial None

d Asteraceae Perennial 1.5Poaceae Perennial 1.5

hound Lamiaceae Perennial NonePoaceae Perennial 1.5

eed Polygonaceae Annual 1.5eed Polygonaceae Annual 1.5

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282 277

and removing any necrotic (dead/dry) tissue resulting from herbicide exposure.Plants were bagged separately and dried in a drying oven at approximately 70 �C forat least three days prior to being weighed.

2.4. Assessing level of recovery and long-term effects on plant health

Due to the anticipated length of the study, the long-term plants from both theterrestrial and wetland communities were supplemented with additional nutri-ents during the experiment. These fertilizations were performed when controlplants exhibited early signs of nutrient stress (i.e. discoloration) in order tomaintain plant growth rates and prevent stunting that would bias measurements.For the terrestrial plants, 50 mL of a prepared solution consisting of 2.5 mL/L of20e20e20 ‘‘All Purpose Plant Food’’ fertilizer (Optimum Hydroponix) wasapplied at approximately 32 and 77 days after exposure to all plants, with theexceptions of C. bursa-pastoris and Chenopodium album which were harvestedbefore the second fertilization occurred due to their shorter lifespans. Similarly,all wetland plants were fertilized after approximately 35 days with the abovementioned fertilizer and again with 50 mL of a 5 mL/L solution of 15e30e15“Botanix Flowering Plant Fertilizer” after approximately 56 days due to controlplants exhibiting signs of phosphorous stress (reddening of leaves). Elymus vir-ginicus, Lycopus americanus, Panicum clandestinum, Polygonum pensylvanicum,and P. hydropiper all received an additional fertilization with the 20e20e20fertilizer at approximately day 88, while E. virginicus received a final fertiliza-tion on day 132.

A summary of all measured parameters can be found in Table 2. Vegetativegrowth measurements were recorded weekly for all long-term plants, corre-sponding to the day when plants were sprayed. Maximum height was recordedfor all herbs/dicots (from the soil to the highest point excluding leaves) andgrasses/monocots (from the soil to the highest point including leaves). Due toAnagallis arvensis’ mat-forming growth pattern, the alternate measurement oflongest branch length was recorded instead of height. In addition, sinceC. bursa-pastoris forms rosettes, basal diameter was also recorded for thisspecies.

Measurements of reproduction varied according to species (Table 2). Healthyfruit/pod production was recorded weekly for A. arvensis, C. bursa-pastoris andLobelia inflata after onset of production. To prevent loss, very ripe fruit were cut fromthe plants, counted, bagged and dried for final biomass determination. Seeds of P.pensylvanicum and P. hydropiper were collected and counted weekly as they devel-oped. Spikes of P. pensylvanicumwere shaken into a bucket allowing the ripe seeds tofall off while seeds of P. hydropiper were removed with forceps. Seeds of Elymuscanadensis and E. virginicus were collected when the floral spike matured (yel-lowed), and seed counts taken only at the end of the experiment. Due to the diffi-culty in counting flowers or collecting seeds in the highly productive C. album, seedproduction was only measured at the end of the experiment. Floral parts were cut,bagged, and dried and were then sieved to separate out the seeds. The seeds werethen weighed to obtain total seed weight per plant. The small size of both the seedsand flowers of L. americanus made it unpractical to take weekly counts. Instead, thetotal number of floral nodes with open flowers was used as a surrogate measure forreproduction.

Due to a lack of pollinators within the greenhouses, flowers of both Centaureacyanus andHelianthus strumosus failed to produce viable seeds. For this reason, thealternate approaches of counting dried flower head production over time

Table 2Summary of plant variables evaluated.

Species Measures of vegetative growth

ST biomass LT biomass H

Terrestrial Anagallis arvensis x x xAsclepias syriaca x x xCapsella bursa-pastoris x x xCentaurea cyanus x x xChenopodium album x x xCleome serrulata x x xElymus canadensis x x xHelianthus strumosus x x xLobelia inflata x x x

Wetland Asclepias incarnata x x xElymus virginicus x x xEupatorium maculatum x x xGlyceria striata x x xLycopus americanus x x xPanicum clandestinum x x xPolygonum hydropiper x xPolygonum pensylvanicum x x x

‘x’ indicates that the variable was measured/recorded in the experiment for that species

(C. cyanus) or counting seedhead florets (H. strumosus e end of experiment only)were used as indicators of potential reproductive outputs. Similarly, flowers/fruitof Cleome serrulata often aborted shortly after production, therefore only themeasurement of inflorescence length was a viable character for reproduction.Since all plants of Glyceria striata failed to flower, tiller/stem counts were used asan indicator of asexual reproduction. Reproductive parameters could not beassessed for one terrestrial and three wetland species due to failures to flower(Asclepias syriaca and Asclepias incarnata), inconsistencies in flowering(P. clandestinum), or due to potential biases introduced as a result of insect damage(E. maculatum).

Long-term plants were harvested for biomass, as per the short-term plants,when the control plants had finished reproducing and/or began senescing. Allpreviously collected plant materials (fruits, seeds, etc.) were also bagged and driedfor inclusion in long-term biomass.

2.5. Statistical analysis

Statistical analyses were performed in Systat 13 (Version 13.00.05).

2.5.1. Determination of inhibition concentrationsInhibition concentrations (IC50) resulting in a 50% decrease in a given variable as

compared to the controls were calculated through non-linear regression models(Environment Canada, 2005). If either the assumption of normality of residuals(ShapiroeWilk Test) or homogeneity of variance (Levene’s Test) could not be met,even with data transformations, then the linear interpolation method for sublethaltoxicity, also known as the inhibition concentration approach (ICPIN), was used(Norberg-King, 1993). These analyses were performed for all vegetative and repro-ductive parameters measured in the short- and long-term treatments (seeSupplementary Table A for species specifics).

2.5.2. Evaluation of time to recoveryANOVA models (or non-parametric KruskaleWallis tests) were used to

determine the length of time required for vegetative or reproductive parametersto recover by comparing the doses to the unsprayed controls. In this sense, wedefine recovery as re-attaining expected levels of height or reproduction in rela-tion to the non-exposed (control) plants at the same time interval. Each parameterwas assessed individually, starting either at week 0 (day of spray) or at the onset ofproduction (i.e. fruits or seeds) and continuing at weekly intervals until the end-date harvest. For all analyses, the model assumptions of homogeneity of variance(Levene’s test) and normality of residuals (either ShapiroeWilk test orKolmogoroveSmirnov test) were verified; if the assumptions failed then the non-parametric KruskaleWallis test was performed. Post-hoc comparisons wereconducted to determine which doses were statistically, negatively different fromthe controls. For parametric ANOVA analyses, Dunnett’s one-sided post-hoc testwas chosen since it assumes an alternate hypothesis of less than the controls andcan compare a set of doses to only one specific mean (i.e. controls) thus mini-mizing the number of comparisons. When KruskaleWallis analyses were per-formed, the ConovereInman multiple comparison test was used. Both types ofanalyses were performed for each species in accordance with the modelassumptions for a given week. Parameters were considered recovered at a givendosagewhen there was no longer an observed, consistent negative difference fromthe controls (p-value � 0.05).

Measures of reproduction

eight/Length Diameter Seeds Fruit Flowers Tillers

x

x xx

xx

x xx

x

x x

xx

xx

. ST, short-term; LT, long-term.

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282278

3. Results

3.1. Comparison of the short- and long-term inhibition effects ofchlorimuron

3.1.1. Terrestrial speciesBased on the determination of IC50 values for short-term vari-

ables, five terrestrial species had detectable 50% reductions in bothbiomass and height following chlorimuron exposure: C. bursa-pas-toris,C. cyanus,C. serrulata,H. strumosus and L. inflata (Fig.1a, see alsoSupplemental graphs). Biomass was generally the more sensitivevariable based on the calculated IC50 values. Biomass and height

Fig. 1. Calculated IC50 values (g ai ha�1) for plant vegetative and reproductive endpointswetland wild plant species. Values listed as ‘>9.63’ indicate that the IC50 exceeded the rangbiomass; LTB, long-term biomass; STH, short-term height/length; LTH, long-term height/lencodes and to Table 2 for LTR parameter measured. Information on the models used for the Imaterial (Table A). Detailed species graphs can be seen in the Supplemental figures (Fig. A

of the four remaining terrestrial species: A. arvensis, A. syriaca,C. album and E. canadensis were unaffected in the short-term.

When long-term biomass and height were considered (seeTable 3 for harvest times), there was an overall trend towardshigher IC50s as compared to the corresponding short-term variableindicating recovery, with three exceptions: C. bursa-pastoris (1.53 to0.97 g ai ha�1), C. serrulata (4.46 to 3.74 g ai ha�1), and A. arvensis(>9.63 to 8.08 g ai ha�1). Asclepias syriaca, C. album andE. canadensis remained unaffected in terms of vegetative endpoints.

Long-term reproduction was found to be a very sensitive IC50endpoint for three species. For C. bursa-pastoris the calculated IC50for reproduction (pods) was slightly lower than that for short-term

following exposure to chlorimuron ethyl for (a) nine terrestrial/upland and (b) eighte of doses tested in the experiment (i.e. no 50% effect). Plant variables: STB, short-termgth; and LTR, long-term reproduction (varies with species). Refer to Table 1 for speciesC50 calculations, confidence intervals and R2 values can be found in the Supplemental).

Table 3Time to recovery (weeks) of various plant growth and reproduction parameters as determined through ANOVA (or KruskaleWallis) analyses of weekly data. All numbers reflectthe time (in weeks) when affected plants no longer consistently differ from the controls. Time for reproductive parameters to recover is listed in reference to the timemeasurements began, i.e. ‘þ2’ would indicate a two week delay from when the parameter was first assessed for plants to recover. ‘þ’ indicates that the parameter did notrecover during the experiment at the given dose; ’NE’ indicates no observed statistical effect.

Week measurementsbegan

Total timegrown (weeks)

Weeks until recovered

Dose (g ai ha�1) and [% of label rate]

0.09[1%]

0.18[1.95%]

0.34[3.8%]

0.67[7.4%]

1.31[14.5%]

2.54[28.2%]

4.95[55%]

9.63[107%]

Terrestrial speciesAnagallis arvensisStem length 0 18.5 NE NE 5 5 7 15 þ þFruit production 6 18.5 NE NE þ2 þ4 þ2 þ þ þ

Asclepias syriacaHeight 0 12 NE NE NE NE NE NE 4 4

Capsella bursa-pastorisRosette diameter 0 7 NE 2 3 þ 7 þ þ þHeight 0 7 NE 2 5 6 6 þ þ þPod production 5 7 NE þ1 þ1 þ þ þ þ þ

Centaurea cyanusHeight 0 14 NE NE 5 5 5 7 7 7Seedhead production 8 14 NE NE þ1 NE þ1 þ3/þ5b þ þ

Chenopodium albumHeight 0 8 NE NE 5 5 4 5 6 6Seed production (weight)a 8 only 8 NE NE þ NE NE NE þ þ

Cleome serrulataHeight 0 14 NE NE 4 3 4/6b 9/þb þ þInflorescence lengtha 14 only 14 NE NE NE NE NE NE þ þ

Elymus canadensisHeight 0 18 NE NE NE NE NE NE NE NETiller production 0 18 NE NE NE NE NE NE NE NESeed productiona 18 only 18 NE NE NE NE NE NE NE NE

Helianthus strumosusHeight 0 16.5 NE NE NE 4 4 þ þ þFloret productiona 16.5 only 16.5 NE NE NE NE NE þ þ þ

Lobelia inflataHeight 0 13 NE 4 4 5 6 þ þ þFruit production 4 13 NE NE NE þ1/þ4b þ5 þ8/þb þ þ

Wetland speciesAsclepias incarnataHeight 0 12 NE NE NE 5 6 þ þ þ

Elymus virginicusHeight 0 23 NE NE NE NE NE NE NE NETiller production 0 23 NE NE NE NE NE NE NE NESeed productiona 23 only 23 NE NE NE NE NE NE NE NE

Eupatorium maculatumHeight 0 12 NE NE NE NE 3 3 þ þ

Glyceria striataHeight 0 12 4 4 5 5 7 þ þ þTiller production 0 12 NE NE NE NE þ þ þ þ

Lycopus americanusHeight 0 14 NE 6 5 3 8 8 9 10Floral nodes 8 14 NE NE NE NE þ3/þ5b þ þ þ

Panicum clandestinumHeight 0 14 NE NE NE NE NE NE NE NE

(continued on next page)

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282 279

Table 3 (continued )

Week measurementsbegan

Total timegrown (weeks)

Weeks until recovered

Dose (g ai ha�1) and [% of label rate]

0.09[1%]

0.18[1.95%]

0.34[3.8%]

0.67[7.4%]

1.31[14.5%]

2.54[28.2%]

4.95[55%]

9.63[107%]

Polygonum hydropiperHeight 0 23.5 NE NE NE 3 4 5 5 7Seed production 12 23.5 NE NE NE NE NE NE NE NE

Polygonum pensylvanicumHeight 0 17 NE 3 6 5 5 13 þ þSeed production 12 17 NE NE NE NE þ þ þ þ

a Parameter only measured at the end of the experiment. Weekly data not available.b Analysis results vary with week, therefore exact recovery timeframe unclear. Numbers refer to earliest and latest time to recovery.

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282280

height. In the cases of both A. arvensis and C. album, the calculatedIC50s for thevegetativemeasures indicated resistance (8.08 gaiha�1)or no effect (>9.63 g ai ha�1) respectively; however, the corre-sponding reproductive measures for these species were significantlylower at 1.92 (fruit) and 6.65 g ai ha�1 (seed mass) respectively.Results for C. serrulata and H. strumosus reproduction varied butnonetheless were on par with their vegetative measures.

Short-term biomass was the most sensitive overall IC50endpoint for three species (C. serrulata, H. strumosus and L. inflata),long-term reproduction was the most sensitive for three species(C. bursa-pastoris, A. arvensis and C. album), and no IC50 effects weredetectable for two species (A. syriaca and E. canadensis). Centaureacyanus was a unique case with 50% effects detected in the short-term but with total recovery in the long-term in terms of vegeta-tive and reproductive parts.

3.1.2. Wetland speciesShort-term effects, as determined by IC50, of chlorimuron

exposure on both height and biomass were observed for five out ofeight wetland species tested: A. incarnata, E. maculatum, G. striata,L. americanus, and P. pensylvanicum (Fig. 1b, see also supplementalgraphs). Similar to the trends observed for the terrestrial species,IC50s calculated based on long-term biomass and heights wereconsistently higher than those determined in the short-term. Noshort- or long-term 50% effects were observed for E. virginicus,P. clandestinum, or P. hydropiper (note: short-term biomass was notassessed for P. hydropiper).

In terms of reproduction, two species, L. americanus andP. pensylvanicum, that appeared to fully recover vegetatively in thelong-term (IC50s > 9.52 g ai ha�1) had detectable reductions inflower (IC50 ¼ 3.59) and seed production (IC50 ¼ 3.36), respec-tively, at the end of the experiment. The reproductive IC50 forG. striata (tiller production) was on par with calculated values forboth height and biomass, being between the IC50s for short- andlong-term. Nonetheless, for the above mentioned species, short-term biomass remained the most sensitive IC50 endpoint. NoIC50 effects on reproduction (seeds) were observed for eitherE. virginicus or P. hydropiper.

No significant hormesis was detected on any of the wetland orterrestrial species.

3.2. Level of recovery of plants exposed to sublethal doses ofchlorimuron

Table 3 illustrates the time to recovery for all 17 species. Onlythree species, the terrestrial grass E. canadensis and the wetlandgrasses E. virginicus and P. clandestinum experienced no negativeeffects of chlorimuron at any dose or time. The remaining 14 species

haddetectable effects for at least one vegetative (height or diameter)or reproductive parameter. Extreme effects include 0.09 g ai ha�1

(1%) doses for G. striata (height) and 0.18 g ai ha�1 (1.95%) doses forC. bursa-pastoris (height, diameter, pods), L. inflata (height),L. americanus (height) and P. pensylvanicum (height). Two species forwhich no effects were detected using IC50 analyses, A. syriaca andP. hydropiper, exhibited some initial reductions inheight as comparedto the controls at doses of 4.95 (55%) and 0.67 g ai ha�1 (7.4%)respectively. Both species eventually fully recovered: A. syriaca atfour weeks and P. hydropiper at seven weeks post-spray.

Recovery times for all species varied. Terrestrial plants thatrecovered vegetatively did so within a median time of 6.5 weeks(low of 4; high of 15 weeks), though not at all doses. Only threespecies, A. syriaca, C. cyanus and C. album, recovered in terms ofheight at all doses, the remaining five recovered only up to the 1.31(14.5%) or 2.54 g ai ha�1 (28.2%) dosage depending on the species(Table 3). Similarly, median time to height recovery for affectedwetland species was sevenweeks (low of 3, high of 13 weeks), withtwo species fully recovering at all doses (L. americanus andP. hydropiper) and the remaining four only recovering up to doses1.31 (14.5%) or 2.54 g ai ha�1 (28.2%).

Of the 11 species that were affected and had a measureablereproductive parameter, four (C. serrulata, H. strumosus, L. inflata,P. hydropiper) exhibited equal recovery of both their vegetative(biomass/height) and reproductive parameters (Table 3) at equiv-alent doses by the end of the experiment. In the case of theremaining seven species (A. arvensis, C. bursa-pastoris, C. cyanus,C. album, G. striata, L. americanus, P. hydropiper) reproduction neverrecovered to the same levels (doses) as the vegetative measure. Forinstance both C. cyanus and C. album fully recovered in height at alldoses; however, reproduction only recovered up to and includingthe 2.54 g ai ha�1 (28.2%) dose.

4. Discussion

Herbicidal threats to wild vegetation are often only assessedusing IC50s (or IC25s) in short-term studies following outlinedprotocols (OECD, 2006; USEPA, 2012). These studies rely heavily onobservations of herbicidal effects on plant (usually crop) biomass.Due to limited timeframes, assessments of long-term effects onplant health and reproduction are not conducted. Previously withglufosinate ammonium we found that plants can recover biomassover time; however, we did not assess how quickly this occurs, orhow species compare in terms of recovery (Carpenter and Boutin,2010). In addition, we identified that reproduction was a moresensitive IC50 endpoint than vegetative parameters for severalspecies. In the present study, short-term biomass IC50s were foundto be good predictors of long-term effects on wild plants, and were

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282 281

generally more sensitive than long-term biomass IC50s (Fig. 1).Nonetheless, for some species, biomass recovery did not translateto comparable levels of reproductive recovery. Several studies havefound that short-term biomass is a sensitive endpoint (Boutin et al.,2000; Dalton and Boutin, 2010) while in other cases short-termendpoints underestimated herbicide sensitivity (Fletcher et al.,1995; Gove et al., 2007). Therefore caution must be taken whenonly assessing short-term biomass in phytotoxicity studies.Furthermore, although commonly used for regulatory purposes,IC50s are not sensitive enough to detect subtle phytotoxic effects,and hence may not fully reflect the outcome of sublethal exposurescenarios characteristic of herbicidal drift (Gove et al., 2007).

Delays in vegetative growth (biomass/height) in at least onedose were evident for 14 out of the 17 species evaluated (eightterrestrial and six wetland; Table 3), and ten experienced 50%reductions in short-term vegetative measures as determinedthrough IC50 analyses (Fig. 1). For all affected species, full vegeta-tive recovery eventually occurred up to the 1.31 g ai ha�1 (14.5%)dose or greater in these single-species tests; however, recoverytime was species dependent. Vegetatively stunted plants may haveutilized more resources earlier to repair/re-grow new tissues andwere likely able to catch up to the non-exposed plants (controls) asthese individuals shifted more resources towards reproduction.Nonetheless, though vegetative recovery was evident in thesesimplified tests, competition for light and space within a naturalcommunity would likely favour the more resistant, faster to recoverspecies over those that are more sensitive and slower to re-grow(i.e. terrestrial: A. syriaca vs. L. inflata; wetland: P. hydropiper vs.P. pensylvanicum).

Though some species did recover vegetatively to levelscomparable to the controls at low doses by the end of theexperiment, i.e. A. arvensis, C. cyanus and C. album (terrestrial) andL. americanus and P. pensylvanicum (wetland), reproduction wasfurther delayed. As was observed in this experiment, levels ofreproductive recovery failed to attain the levels of vegetativerecovery observed for seven of the 12 species for which repro-duction was measurable (Table 3). For these species, it appearsthat vegetative re-growth was an energetic trade-off for repro-duction. The production of new stems and leaves appeared tocome at a cost to flower, fruit, and seed production. Though it wasnot assessed in this experiment, seed health is another factor toconsider when determining true reproductive recovery. It hasbeen shown in multiple studies that herbicide exposure canhinder seed development, viability, and germination rates whenapplied to growing plants at different life stages (Rinella et al.,2001, 2010; Steadman et al., 2006) or when applied directly tosoils (Rokich et al., 2009).

Reductions in seed production will likely have negative impactson a habitat’s seedbank. Seedbank contributions are vital for allspecies that must invest into the community each year to maintainfuture recruitment opportunities. Failure to flower or to produceseeds may result in future population declines. If this occurs overmultiple years (following successive spray events), populations ofhighly susceptible species may be greatly reduced (Crone et al.,2009; Marrs et al., 1991) or replaced by tolerant, reproductiveones. Short-lived susceptible annuals such as C. bursa-pastoriswould be at a major disadvantage since they would likely notrecover within their lifespans, and would hence not contribute tothe seedbank. Gove et al. (2007) found that the glyphosate sensitivespecies in a short-term greenhouse experiment were also those lessabundant inwoodland communities adjacent to intensively farmedfields as compared to woodlands abutting fields with lower agro-chemical inputs.

As our results demonstrate, herbicide susceptibility is speciesdependent, as has been shown in countless herbicide studies

(Gove et al., 2007 and references therein; Marshall, 1989). Moreimportantly, recovery capacity and recovery time are highlyvariable amongst species. Though plants recover, the process canbe slow, and this may hinder a plant’s ability to compete withthose that are immune or recover faster. For instance, it could bespeculated that E. canadensis and A. syriaca (terrestrial) orE. virginicus and P. hydropiper (wetland) may have competitiveadvantages over more susceptible species within their habitatsdue in part to their resistance to chlorimuron. This may allowthem to monopolize resources (Crone et al., 2009 and referencestherein; Kleijn and Snoeijing, 1997), at the expense of the affectedspecies thus further hindering the recovery of the more sensitivespecies. Early reductions in growth and slow recoveries of thespecies affected at lower doses (i.e. C. bursa-pastoris, L. inflata, L.americanus, and P. pensylvanicum) would likely provide opportu-nities for more resistant species to occupy spaces for light andto monopolize nutrient reserves. To fully address these ideas,community and mesocosm experiments are required in orderto understand how subtle susceptibilities and delays invegetative and reproductive growth can influence natural speciescompositions.

Chlorimuron is primarily used for the control of broad-leavedspecies, with minimal effects on grasses (Claus, 1987). For threeout of the four grasses tested in this study, no reductions in vege-tative growth or reproduction were observed. Our results aretherefore relatable to past research that identified long-term shiftsin plant community structure towards an increased presence ofgraminoids following the application of broad-leaf selectiveherbicides (Thilenius et al., 1974; Tomkins and Grant, 1977).Nonetheless, the remaining grass, G. striata, for reasons unknown,was one of the more sensitive species studied. This further stressesthe need to test a variety of species from at risk habitats anddemonstrates that caution will always be needed when makinggeneralities about given species groups.

The current single-species approach for conducting phytotox-icity assessments cannot directly address many of the community-related factors (i.e. competition, phenologies, etc.) that can be bestexamined in field or mesocosm studies. Nonetheless, in thisexperiment we strove to test a range of species from similar habi-tats, within the same timeframe to simulate communities of speciesthat may exist together. Though previous research into linkingsingle-species experiments with community-mesocosm tests haveproduced mixed results in terms of predicting species’ responses(Dalton and Boutin, 2010; Damgaard et al., 2008; Pfleeger et al.,2012; Riemens et al., 2008), it is clear that individual species dorespond differently and that shifts in community structure andreductions in diversity do occur following exposure (de Snoo, 1995;Marrs and Frost, 1997). While we cannot empirically state that thespecies examined will respond similarly in the field, it can beassumed that the high variability observed amongst species interms of herbicide sensitivity and levels/times of vegetative andreproductive recovery would also exist within a diverse naturalcommunity. As such, resistant, fast recovering species would likelybe at a competitive advantage.

In summary, levels of recovery and timeframes were variableamongst species within each habitat. Many of the study species (sixterrestrial and five wetland) were vegetatively and reproductivelystunted at or below the 7.4% label application rate (0.67 g ai ha�1),a realistic drift scenario according to Gove et al. (2007). Manyspecies eventually recovered with time. However, it took thesespecies three to fifteen weeks following exposure to regain vege-tative levels comparable to non-exposed plants in a simplifiedenvironment (one plant per pot, no direct competition), and asa result reproduction was further delayed and reduced. Ina complex community it is plausible that the affected species may

D. Carpenter et al. / Environmental Pollution 172 (2013) 275e282282

be displaced by tolerant species through interspecific competitionbefore they can fully recover or reproduce. Though they wouldlikely not be eliminated from the community, individual pop-ulations could be diminished as a result of a lack of contributions tothe seedbank or reduced asexual reproduction from affected indi-viduals. This could have many repercussions as it has been docu-mented that high plant diversity is required to maintain bothecosystem services (Isbell et al., 2011) and biodiversity at manyother trophic levels (Boutin et al., 2011; Scherber et al., 2010).

Acknowledgements

This study was funded by the Pesticide Science Funds of Envi-ronment Canada. The authors would like to thank Jessica Parsons,Montse Bassa Etxaurren, Philippe Thomas and Paula Smith forgreenhouse technical assistance.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.envpol.2012.09.007.

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