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Environmental Pollution xxx (2010) 1e8
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Environmental Pollution
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Acute and chronic toxicity of Roundup WeathermaxÒ and IgniteÒ 280 SL to larval
Spea multiplicata and S. bombifrons from the Southern High Plains, USA
Simon K. Dinehart a, *, Loren M. Smith a, Scott T. McMurry a, Philip N. Smith b, Todd A. Anderson b,
David A. Haukos c
a
b
c
Department of Zoology, Oklahoma State University, Stillwater, OK 74078, USA
The Institute of Environmental and Human Health, Texas Tech University, Box 41163, Lubbock, TX 79409, USA
U.S. Fish and Wildlife Service, MS 2125, Texas Tech University, Lubbock, TX 79409, USA
Roundup WeatherMAXÒ may cause extensive mortality among larval New Mexico and Plains spadefoots; it is unlikely that exposure to IgniteÒ
280 SL would result in extensive mortalities.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2010
Received in revised form
3 May 2010
Accepted 5 May 2010
Pesticides have been implicated in widespread amphibian declines. We assessed acute and chronic
toxicity of two widely used herbicides to larval New Mexico (Spea multiplicata) and Plains (S. bombifrons)
spadefoots from cropland and native grassland playas. Roundup WeatherMAXÒ (WM) toxicity estimates
(48- and 216-h LC50; 48-h LC1) for both species were similar to environmental concentrations expected
from accidental overspray. Chronic (30-day) exposure to WM at predicted environmental concentrations
(2.0 and 2.8 mg glyphosate acid equivalents/L) reduced survival of both species. IgniteÒ 280 SL (IG)
toxicity estimates (48-h LC50 and LC1) for both species were above predicted environmental concentrations of 1.0 mg glufosinate/L. Chronic exposure to predicted environmental concentrations of IG did
not reduce survival of either species. Toxicity test results suggest that at predicted environmental
concentrations IG would not cause extensive mortalities among larval New Mexico and Plains spadefoots. However, WM may cause extensive mortality among larvae of these species.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Amphibian decline
Glyphosate
Herbicide
Roundup
1. Introduction
Amphibian populations are declining worldwide (Wyman,
1990; Stuart et al., 2004). Many of these declines result from
habitat destruction (e.g., Wyman, 1990). Chemical contamination of
remaining habitat may also threaten amphibians (Howe et al.,
2004). Because wetlands tend to concentrate chemicals applied in
the surrounding landscape (Anderson and D’Apollonia, 1978),
amphibian larvae may be exposed to pesticides (Semlitsch, 2003).
Glyphosate and glufosinate herbicides are used worldwide (Howe
et al., 2004; Lee et al., 2005) to control weeds in various habitats
(e.g., forests, farmland) (Lee et al., 2005; Relyea, 2005a). Most
formulations contain two main components: the active ingredient
and surfactants. Glyphosate herbicides commonly contain glyphosate
and a polyethoxylated tallowamine (POEA) surfactant (Giesy et al.,
2000). Glufosinate formulations contain glufosinate-ammonium
and a surfactant, often sodium polyoxyethylene alkyether sulfate
(Koyama and Goto, 1997).
* Corresponding author.
E-mail address: simon.dinehart@okstate.edu (S.K. Dinehart).
Glyphosate and glufosinate bind tightly to soil (Giesy et al.,
2000; Lee et al., 2005) and runoff is usually limited (Malone
et al., 2004; Lee et al., 2005). However, herbicides may enter
wetlands via spray drift (Johansson et al., 2006), accidental overspray, or runoff (Faber et al., 1998). Glyphosate herbicides are predicted to reach concentrations of 2.8 mg glyphosate acid
equivalents (ae)/L in aquatic habitats due to accidental overspray
(Giesy et al., 2000), though the highest concentration reported is
1.7 mg glyphosate ae/L (Horner, 1990 in Giesy et al., 2000). Accidental overspray with glufosinate herbicides is predicted to result
in aquatic concentrations of 1.0 mg glufosinate/L (Faber et al., 1998).
Time for 50% dissipation (DT50) of glyphosate in aquatic environments is 1e2 weeks (Goldsborough and Brown, 1993; Giesy et al.,
2000), while that for glufosinate is 6e9 weeks (Faber et al., 1998).
The POEA surfactant persists longer in aquatic environments than
glyphosate with a DT50 of 3e4 weeks (Banduhn and Frazier, 1974 in
Giesy et al., 2000). Little information is available concerning environmental fate of surfactants in glufosinate herbicides (Cox, 1996).
Laboratory and mesocosm experiments indicate that exposure
to glyphosate herbicides at environmentally expected concentrations may pose a risk to amphibian larvae (Howe et al., 2004;
0269-7491/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.envpol.2010.05.006
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S.K. Dinehart et al. / Environmental Pollution xxx (2010) 1e8
Relyea, 2004, 2005b; Relyea et al., 2005). However, amphibians
vary widely in their sensitivity to glyphosate herbicides (Mann and
Bidwell, 1999; Howe et al., 2004). Mann and Bidwell (1999)
reported 48-h LC50 values ranging from 2.9 to 16.1 mg glyphosate
ae/L. To our knowledge, no previous studies have investigated the
toxicity of glufosinate herbicides to amphibians.
The Southern High Plains (SHP) of Texas and New Mexico
contain numerous playa wetlands (Bolen et al., 1989). Because
amphibians in this region rely on playas for continued persistence
(Haukos and Smith, 1994), activities which degrade playas pose
a serious threat to amphibians. One of the most widely applied
herbicides in Texas is glyphosate (National Pesticide Use Database,
2004). Agricultural formulations (e.g., Roundup WeatherMAXÒ) are
frequently used to control weeds in herbicide-resistant cotton
(Blair-Kerth et al., 2001; National Pesticide Use Database, 2004).
IgniteÒ 280 SL, a glufosinate herbicide used with FiberMaxÒ LibertyLinkÒ cotton, is an alternative to glyphosate formulations
(Carter, 2005). Both herbicides are applied during spring and
summer (Bayer CropScience LP, 2005; Monsanto Company, 2005).
SHP amphibians are likely exposed to agrochemicals (Venne
et al., 2008) because herbicide application coincides with
breeding and larval development periods (Garrett and Barker, 1987;
Anderson et al., 1999). It is therefore important to determine
whether larval amphibians in playas are affected by common
pesticides. Another relevant question is whether animals inhabiting cropland and native grassland playas vary in terms of pesticide
sensitivity. This phenomenon has been demonstrated among
invertebrates (Brausch and Smith, 2009) and amphibians (Bridges
and Semlitsch, 2000). Amphibians from cropland and grassland
playas differ in terms of community structure (Gray et al., 2004;
Ghioca and Smith, 2008; Ghioca-Robrecht et al., 2009), body size
(Gray and Smith, 2005), and immunological development
(McMurry et al., 2009). However, no previous studies have investigated whether landuse related differences in pesticide sensitivity
exist among SHP amphibians.
Therefore, we examined acute and chronic toxicity of a glyphosate [Roundup WeatherMAXÒ (WM)] and glufosinate herbicide
[IgniteÒ 280 SL (IG)] to New Mexico and Plains spadefoot larvae
(Spea multiplicata and S. bombifrons, respectively) from SHP playas.
To assess acute toxicity, we determined 48-h LC50 and LC1 values for
larvae of each species from playas embedded in cropland and
native grassland. In addition, 216-h LC50 values were calculated
because traditional LC50 values may ignore post-exposure mortality
and underestimate toxicity to field populations (Zhao and
Newman, 2004; Jones et al., 2009). Because animals may experience prolonged exposure under field conditions (Goldsborough
and Brown, 1993; Faber et al., 1998; Giesy et al., 2000), we
assessed whether chronic (30-day) exposure to predicted environmental concentrations of these herbicides affected survival of
New Mexico and Plains spadefoot larvae from cropland and
grassland playas.
2. Materials and methods
2.1. Larval collection and housing
Playas were selected based on landuse within the surrounding watershed and
the availability of Spea spp. larvae. These species are very common anurans in the
SHP (Anderson et al., 1999). Playas were considered “cropland” if 75% of the
surrounding watershed was cultivated, and “grassland” if 75% was native grassland. Selected cropland playas were in Floyd, Hockley, and Terry counties, while
grassland playas were in Briscoe and Floyd counties, TX. Sampled playas were
separated by 1 km because most anurans do not disperse further from their natal
sites (Sinsch, 1990). Larvae were collected from three cropland playas on 19 May
2007 and two grassland playas on 20e21 May 2007. Larvae were collected from two
grassland playas due to the limited availability of spadefoot larvae in this landuse.
New Mexico and Plains spadefoots were collected, held, and tested as a mixed
species culture because protein electrophoresis is required to differentiate larvae of
these species (Simovich and Sassaman, 1986).
Larvae were transported to Texas Tech University (TTU), Lubbock, TX in buckets
containing playa water. Animal research procedures were approved by the TTU
Institutional Animal Care and Use Committee (protocol no. 06018-06). Larvae were
held in a climate controlled room on a 14 he10 h light-dark photoperiod throughout
acclimation and toxicity testing. This photoperiod approximates that near the center
of the SHP during MayeSeptember. Larvae were housed in 39.7 L glass aquaria filled
with aerated, aged tap water from the same source used during toxicity tests (Mann
and Bidwell, 1999). Larvae were acclimated to laboratory conditions for nine days,
during which time pulverized rabbit food was provided ad libitum (Relyea et al.,
2005). Larvae were observed daily during acclimation and exhibited no obvious
signs of stress (i.e., <5% died during acclimation).
Temperature and water quality were monitored daily during acclimation. These
variables
were
similar
among
all
aquaria
(mean
1
S.E.):
temperature ¼ 21.96 0.04 C, pH ¼ 8.37 0.02, dissolved oxygen ¼ 8.06 0.08 mg/L,
ammonia ¼ 2.1 0.1 mg/L. An 80% water change (Meyer and Di Giuliuo, 2003)
occurred whenever ammonia exceeded 1 mg/L (i.e., nearly daily) so that ammonia
levels would not exceed those noted during similar studies (2.8e4.2 mg/L; Relyea,
2004).
2.2. Acute toxicity methods
Tests procedures were based on American Society for Testing and Materials
(ASTM) Guidelines (ASTM, 2003). WM and IG were obtained from retail outlets
(Table 1). To facilitate accurate preparation of test concentrations, stock solutions
[WM, 330 g glyphosate/L; IG, 93.3 g glufosinate/L] were created by diluting
formulated herbicides with aged tap water.
Test chambers consisted of 18.95 L glass compartments created by dividing
37.9 L aquaria in half with a glass divider secured with silicone Aquarium Sealant
(Perfecto Manufacturing, Inc., Noblesville, IN). Fifteen liters of tap water was added
to each compartment and allowed to age for 48 h. This water volume ensured that
biomass loading limits (0.5 g/L) would not be exceeded (ASTM, 2003).
Test concentrations were prepared immediately before tests by pipetting the
appropriate amount of stock solution into compartments. Solution in each
compartment was gently mixed with a clean glass rod. Larvae from cropland playas
were exposed to eight herbicide concentrations (WM e 0.75, 1.5, 2.25, 3, 4.5, 6, 7.5,
10 mg glyphosate/L; IG e 0.5, 2.5, 3.75, 5, 7.5, 10, 12.5, 15 mg glufosinate/L) and
a control (aged tap water). The highest dose of each herbicide was excluded from
tests with grassland larvae due to their limited availability. The range of WM
concentrations was based on previously determined amphibian LC50 values for
glyphosate herbicides (Mann and Bidwell, 1999; Howe et al., 2004), and was
weighted toward lower concentrations more likely to include the actual LC50 value.
Preliminary toxicity tests with Spea spp. larvae during summer 2006 indicated that
the above range of IG test concentrations was appropriate.
Acute toxicity tests with cropland and grassland larvae commenced 29 May and
31 May 2007, respectively. Larvae received food until transferred to test compartments because developing amphibian larvae are sensitive to starvation (Mann and
Bidwell, 1999). All larvae used for toxicity tests were Gosner-stage 29e30 (Gosner,
1960). Twenty randomly selected Spea spp. larvae from each playa were blotted
dry and weighed.
Larvae from each playa were randomly allocated to test compartments. Cropland
compartments contained three larvae from each of three cropland playas for a total
of nine larvae. Grassland compartments contained four larvae from each of two
grassland playas; the ninth Spea spp. larva was randomly selected from grassland
holding tanks. Food was withheld during the first 48 h of testing (Mann and Bidwell,
1999; ASTM, 2003). Four replicates (for each herbicide concentration and controls)
were used, and test compartments were randomly arranged.
Table 1
List of ingredients (by percent composition) in formulated herbicides used during
larval Spea multiplicata and S. bombifrons (New Mexico and Plains spadefoot,
respectively) acute and chronic toxicity testing.
Herbicide
formulation
Ingredients
Percent
WMa
Glyphosate (potassium salt form)
Other Ingredients
48.8
51.2
IGb
Glufosinate-ammonium
Other Ingredients
24.5
75.7
a
Roundup WeatherMAXÒ (WM) is manufactured by Monsanto Company, St.
Louis, MO and contains 660 g glyphosate/L (equivalent to 540 g glyphosate acid
equivalents/L).
b
IgniteÒ 280 SL (IG) is manufactured by Bayer CropScience LP, Research Triangle
Park, NC and contains 280 g glufosinate-ammonium/L.
Please cite this article in press as: Dinehart, S.K., et al., Acute and chronic toxicity of Roundup WeathermaxÒ and..., Environ. Pollut. (2010),
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Test compartments were checked every 6 h. Moribund larvae were euthanized
by immersion in 1% MS-222 (Howe et al., 2004) and preserved at 80 C for
subsequent species differentiation. Larvae were considered moribund if they
exhibited non-responsiveness to prodding or loss of coordinated movement (ASTM,
2003). After 48 h, all remaining larvae were transferred to plastic containers filled
with clean aged tap water (Relyea, 2004). Each container held 5 L of water and
additional water was added daily to maintain a constant level. Larvae received
pulverized rabbit food ad libitum. Larvae were observed every 6 h for seven days to
quantify delayed mortality (ASTM, 2003). Moribund larvae were euthanized and
preserved. An 80% water change occurred after four days or if ammonia levels surpassed 1 mg/L. After seven days, all remaining larvae were euthanized and
preserved. Larvae were identified as New Mexico or Plains spadefoots via protein
electrophoresis (Simovich and Sassaman, 1986). Temperature, pH, dissolved oxygen,
and ammonia levels were quantified initially and every 48 h in four randomly
selected low, medium, and high herbicide concentration test compartments or
associated plastic containers and four control compartments or containers (ASTM,
2003) (Table 2). Soon after tests started, water samples (50 mL) were collected
from these same test compartments and preserved in pre-cleaned environmental
sample vials at 20 C for subsequent analytical determination of glyphosate and
glufosinate concentrations.
2.3. Acute toxicity statistical analyses
Initial weight data for Spea spp. larvae were heterogeneous (Levene’s test:
F1,98 ¼ 24.01, P < 0.001) and non-normally distributed; standard data transformations (i.e., log, natural log, square root) did not correct these problems.
Therefore, weight data were compared with a Wilcoxon two-sample test (PROC
NPAR1WAY, SAS Version 9.2, SAS Institute, Cary, NC).
Forty-eight and 216-h LC50 values and associated 84% confidence intervals were
determined via probit analysis (PROC PROBIT, SAS Version 9.2). LC1 values were
determined using the same technique (Table 3). LC1 values are a preferable indicator
of low effect concentrations because they are not strongly influenced by experimental design (Crane and Newman, 2000). All LC values were calculated based on
nominal concentrations because analytical chemistry indicated that dosing solutions were prepared accurately (Section 3.1). Because only a single LC50 value was
determined for each specieselanduse combination, 48- and 216-h LC50 values were
compared by examining 84% confidence intervals for overlap (Jones et al., 2009). The
same criteria was used to compare LC50 values between species and landuses. This
approach was adopted because simulations indicate that hypothesis tests using 84%
confidence intervals have a type I error rate of 0.05 (Payton et al., 2003).
2.4. Chronic toxicity methods
Table 2
Water quality during Roundup WeatherMAXÒ (WM) and IgniteÒ 280 SL (IG) acute
and chronic (30-day) toxicity tests with larval Spea multiplicata and S. bombifrons
(New Mexico and Plains spadefoot, respectively) from playa wetlands embedded in
cropland or grassland. Acute water quality means were calculated for the first 48 h
and for the subsequent 168 h post-exposure monitoring period.
Dissolved
oxygen
(mg/L)
Ammonia
(mg/L)
Acute water quality (mean 1 S.E.)
48-h
Crop
19.00 0.04
8.67 0.01
Grass
22.28 0.09
8.75 0.01
8.35 0.03
7.96 0.09
0.3 0.1
0.2 0.0
168-h
Crop
Grass
8.43 0.04
8.55 0.01
7.36 0.06
7.35 0.07
0.2 0.0
0.3 0.0
Chronic water quality (mean 1 S.E.)
WM
Crop
22.39 0.10
8.51 0.01
Grass
22.65 0.11
8.50 0.01
7.33 0.05
7.55 0.07
0.1 0.0
0.1 0.0
IG
Crop
Grass
7.18 0.06
7.20 0.06
0.2 0.0
0.1 0.0
22.09 0.11
22.17 0.09
22.51 0.08
22.52 0.08
Larval care prior to chronic tests was the same as for acute tests. Larvae used for
chronic toxicity tests were Gosner-stage 29e30 (Gosner, 1960). Nine larvae were
allocated to each compartment in the same fashion as during acute tests. Each group
of nine larvae was weighed to quantify initial mean mass. Test compartments were
checked every 6 h. Moribund individuals were euthanized and preserved. Larvae
were provided pulverized rabbit food, with food levels constant among all
compartments. An 80% water change occurred every four days and herbicides were
reapplied (i.e., static-renewal). Survival was monitored for 30 days, after which all
remaining larvae were euthanized and preserved. Larvae were subsequently identified as New Mexico or Plains spadefoots (Simovich and Sassaman, 1986).
For each landuseeherbicide combination, water quality was measured as
described above in four randomly selected control, low, and high herbicide
concentration test compartments initially and every 48 h thereafter during chronic
toxicity tests (Table 2). Fifty milliliter water samples were collected and preserved as
described above.
2.5. Chronic toxicity statistical analyses
Weight variances were homogeneous (Levene’s test: WM F1,34 ¼ 0.31, P ¼ 0.58;
IG F1,34 ¼ 0.52, P ¼ 0.47) and normally distributed. Therefore, weight data were
compared with t-tests. Survival data were analyzed using generalized linear models
(PROC GENMOD, SAS Version 9.1) assuming a Poisson distribution and a log link
function (Littell et al., 2002). Number of surviving larvae was the response variable,
while treatment, landuse, and species were independent variables. Because data
contained many zeros, 0.0001 was added to each datum so that the GENMOD model
converged.
2.6. Analytical chemistry
A portion of acute test solution samples with nominal concentrations above
analytical detection limits (5 mg glyphosate or glufosinate/L) and controls were
analyzed to compare nominal and measured concentrations. Chronic controls and
high herbicide concentration samples were also analyzed even though nominal
concentrations were expected to be below detection limits. The concentration of
active ingredient (glyphosate or glufosinate) in test solutions was determined by gas
chromatography of the TMOA-derivatized products following Tseng et al. (2004).
3. Results
3.1. Analytical chemistry
Chronic toxicity tests began 29 and 31 May 2007, respectively. Test chambers
identical to those used during acute toxicity tests contained 15 L of tap water aged
for 48 h. Test solutions were prepared as previously described. A control (aged tap
water) and two treatments were established for each herbicide. WM test solutions
contained 2.8 mg glyphosate ae/L or 2.0 mg glyphosate ae/L. IG test solutions contained 1.0 mg glufosinate/L or 0.5 mg glufosinate/L. Six replicates (for each herbicide
concentration and controls) were used and compartments were randomly arranged.
Temperature
( C)
3
pH
8.44 0.01
8.48 0.01
Mean (SD) glyphosate concentration was 6.5 1.8 mg/L for the
6.0 mg/L acute test solution, and mean glufosinate concentration
was 15.4 4.8 mg/L for the 12.5 mg/L acute test solution. This
degree of variability (8% and 23% for glyphosate and glufosinate
samples, respectively) conforms to U.S. Environmental Protection
Agency (EPA) guidelines (<30% difference between measured and
nominal concentrations; U.S. EPA, 1996). No glyphosate or glufosinate was detected in control samples from acute tests or in any
chronic samples. Recoveries for check standards were 93% for
glufosinate and 95% for glyphosate.
3.2. WM and IG acute toxicity
No mortalities occurred in control compartments, or in the
lowest WM or IG treatment (0.75 mg glyphosate/L and 0.5 mg
glufosinate/L). We did not detect a significant difference in mean
body weight (1 S.E.) of Spea spp. larvae between landuse types
(z ¼ 1.30, P ¼ 0.19) although grassland larvae (0.29 0.02 g) were
16% heavier than cropland larvae (0.25 0.01 g).
We did not detect any difference between WM 48- and 216-h
LC50 values for Plains or New Mexico spadefoots, nor did we detect
any between landuse or species differences in WM 48- or 216-h
LC50 values (Table 3). Only IG 48-h LC50 values are presented (Table 3)
because they are identical to 216-h values. While IG LC50 values did
not differ between landuses, they varied between species (Table 3).
IG LC50 values for New Mexico spadefoots were 56 and 31% greater
than those for Plains spadefoots from grassland and cropland playas,
respectively.
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Table 3
Acute toxicity of Roundup WeatherMAXÒ (WM) and IgniteÒ 280 SL (IG) to larval Spea multiplicata and S. bombifrons (New Mexico and Plains spadefoot, respectively) from playa
wetlands embedded in cropland or grassland. Both 48- and 216-h (i.e., including post-exposure mortality) LC values and associated 84% confidence intervals were calculated
via probit analysis. Only 48-h values are given for IG because they are identical to 216-h values.
WM LC50 values (84% confidence intervals), mg glyphosate
acid equivalents (ae)/La
WM LC1 values, mg glyphosate acid
equivalents/L
n
48-h
216-h
48-h LC1
216-h LC1
S. bombifrons
Grass
Crop
208
175
2.03 (1.90e2.16)Bc*
1.85 (1.62e2.06)Bc*
1.99 (1.85e2.13)Bc*
1.65 (1.42e1.87)Ba*
1.09*
0.97*
1.00*
0.72*
S. multiplicata
Grass
Crop
80
113
2.30 (2.06e2.55)Bc*
2.11 (1.85e2.41)Bc*
1.93 (1.68e2.20)Bc*
2.11 (1.85e2.41)Bc*
1.32*
1.01*
0.82*
1.01*
IG 48-h LC values (mg glufosinate/L), S. bombifronsa
Grass
Crop
IG 48-h LC values (mg glufosinate/L), S. multiplicataa
n
LC50
LC1
n
LC50
LC1
181
142
3.55 (3.20e3.87)Bc
3.70 (3.26e4.14)Bc
1.41
1.75
107
146
5.55 (5.01e6.12)Bd
4.85 (4.38e5.32)Bd
2.78
4.20
*Asterisks indicate LC values within the range of predicted environmental concentrations of glufosinate (1 mg glufosinate/L) or glyphosate (2.8 mg glyphosate ae/L)
herbicides.
a
For within-species (i.e., between landuse) comparisons, LC50 values that share a common uppercase letter are not significantly different due to overlapping 84% confidence
intervals. For between-species comparisons, LC50 values that share a common lowercase letter are not significantly different based on the same criteria.
3.3. Chronic toxicity
4. Discussion
Spea spp. larvae from grassland playas were significantly heavier
than
those
from
cropland
playas
for
WM
tests
(grassland ¼ 0.29 0.01 g, cropland ¼ 0.24 0.01 g; t34 ¼ 5.24,
P < 0.001). Survival differed among treatments (c22 ¼ 181.99,
P < 0.001), but not species (c21 ¼ 0.44, P ¼ 0.51) or landuse
(c21 ¼ 0.02, P ¼ 0.88). There was a speciesetreatment interaction
(c22 ¼ 7.26, P ¼ 0.027), so data were separated by species and the
analysis repeated. Survival of Plains spadefoots (n ¼ 182) differed
among treatments (c22 ¼ 112.97, P < 0.001), but not by landuse
(c21 ¼ 0.25, P ¼ 0.61). No treatmentelanduse interaction was
present (c22 ¼ 0.00, P ¼ 0.99). While control survival (mean % 1 S.
E.) was high (cropland ¼ 93.9 3.9, grassland ¼ 85.4 5.3), no
Plains spadefoots exposed to WM at 2.0 or 2.8 mg glyphosate ae/L
survived the 30-day exposure. All larvae exposed to 2.8 mg
glyphosate ae/L died within 2 days, while some larvae exposed to
2.0 mg glyphosate ae/L survived 11 days (Fig. 1).
Survival of New Mexico spadefoots (n ¼ 142) differed among
treatments (c22 ¼ 76.37, P < 0.001), but not by landuse (c21 ¼ 0.01,
P ¼ 0.92). Survival (mean % 1 S.E.) of larvae exposed to WM at 2.0
or 2.8 mg glyphosate ae/L was reduced compared to control
(cropland ¼ 100, grassland ¼ 95.8 4.2) larvae. No larvae exposed
to 2.8 mg glyphosate ae/L survived the 30-day exposure, and 100%
mortality occurred within five days (Fig. 2). A treatmentelanduse
interaction was present (c22 ¼ 6.18, P ¼ 0.046). Therefore, survival
was compared between landuses for each treatment. Survival of
control larvae and those exposed to 2.8 mg glyphosate ae/L did not
differ by landuse (control: c21 ¼ 0.030, P ¼ 0.85; 2.8 mg glyphosate
ae/L: c21 ¼ 0.00, P ¼ 0.99). Larvae from grassland (n ¼ 67) exhibited
greater survival than those from cropland (n ¼ 75) following
chronic exposure to 2.0 mg glyphosate ae/L (c21 ¼ 6.41, P ¼ 0.011).
While all cropland larvae exposed to 2.0 mg glyphosate ae/L died
within 18 days, mean survival of grassland larvae through 30 days
was 20.8 13.5% (Fig. 2).
Spea spp. larvae from grassland playas weighed more than those
from cropland for IG tests (grassland ¼ 0.31 0.01 g,
cropland ¼ 0.22 0.01 g; t34 ¼ 8.31, P < 0.001). Survival did not
differ between landuses (c21 ¼ 0.03, P ¼ 0.86), or species (x21 ¼ 0.00,
P ¼ 0.97) and treatments (c22 ¼ 0.14, P ¼ 0.93). Plains (n ¼ 168) and
New Mexico (n ¼ 156) spadefoot survival was not affected by
chronic exposure to IG at 0.5 or 1.0 mg glufosinate/L (Fig. 3).
4.1. Toxicity of IG and WM
To our knowledge, no studies have examined toxicity of
formulated glufosinate herbicides to amphibians. Our results
indicate that IG would be considered “moderately toxic” to New
Mexico and Plains spadefoot larvae (1 < LC50 10 mg active
ingredient/L) according to EPA acute toxicity categories (U.S. EPA,
2009). Because IG LC50 and LC1 values for both species were
above predicted environmental glufosinate herbicide concentrations, it appears unlikely that exposure to IG at predicted environmental concentrations would result in extensive mortality
among larvae of these species under field conditions. Acute toxicity
estimates indicate that IG is less toxic to Plains and New Mexico
spadefoot larvae than WM. This likely resulted from differential
toxicity of the active or “inert” (e.g., surfactants) ingredients in
these formulations (Mann and Bidwell, 1999; Howe et al., 2004).
Our results indicate that WM would also be considered
“moderately toxic” to New Mexico and Plains spadefoot larvae
according to EPA toxicity categories (U.S. EPA, 2009). WM LC50 and
LC1 values for these species were similar to predicted environmental glyphosate herbicide concentrations (2.8 mg glyphosate
ae/L). Because various environmental factors (e.g., predator cues e
Relyea, 2003; pH e Chen et al., 2004) modify herbicide toxicity,
further tests under more realistic conditions are needed to determine whether exposure to WM may result in extensive larval
mortality among these species under field conditions.
Mann and Bidwell (1999) investigated acute toxicity of three
glyphosate formulations to larvae of four species (Litoria moorei,
Lymnodynastes dorsalis, Heleioporus eyrei, Crinia insignifera).
RoundupÒ and TouchdownÒ were much more toxic (48-h LC50:
2.9e16.1 mg glyphosate ae/L) than Roundup BiactiveÒ (48-h
LC50 328 mg glyphosate ae/L). Our WM toxicity estimates (48-h
LC50: 1.85e2.30 mg glyphosate ae/L) for Plains and New Mexico
spadefoot larvae were lower than those reported by Mann and
Bidwell (1999). However, it is difficult to make comparisons
between these studies because different glyphosate formulations
were used, and toxicity of glyphosate formulations varies due to
differences in “inert” ingredients (Mann and Bidwell, 1999).
Several laboratory, static-renewal studies have investigated
effects of chronic glyphosate herbicide exposure (Howe et al., 2004;
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Fig. 1. Survival (mean 1 S.E.) of Spea bombifrons (Plains spadefoot) larvae (n ¼ 182) from cropland (squares, n ¼ 87) and grassland playas (circles, n ¼ 95) during chronic (30-day)
exposure to aged tap water, or Roundup WeatherMAXÒ at 2.0 or 2.8 mg glyphosate acid equivalents (ae)/L in a static-renewal system.
Relyea, 2004). That work demonstrated that chronic exposure at or
below 1.8 mg glyphosate ae/L reduced survival among larvae of
several North American species. Chronic toxicity results from our
study agree with previous work: survival of Plains and New Mexico
spadefoot larvae was greatly reduced by exposure to WM at predicted environmental concentrations. In contrast, survival of these
species was not reduced by chronic exposure to predicted environmental concentrations of IG.
4.2. Variation in sensitivity by landuse
Acute toxicity tests indicated no difference in sensitivity
between larvae from grassland and cropland playas for either
herbicide. Chronic tests showed that New Mexico spadefoots from
grassland playas exhibited greater survival than those from cropland following exposure to WM at 2.0 mg glyphosate ae/L. Because
New Mexico spadefoots from cropland and grassland playas displayed similar WM LC50 values and survival of grassland larvae was
relatively low and variable (mean % 1 S.E. ¼ 20.8 13.5), the
above difference in chronic response must be viewed cautiously.
Selective pressure favoring evolution of herbicide resistance
among spadefoot larvae from cropland playas may have been lacking. This would be the case if WM and IG do not commonly attain
environmental levels that affect fitness. Few data describing aqueous
herbicide levels in playas are available (Thurman et al., 2000), and
none exist for glyphosate or glufosinate. Even if environmental levels
of IG negatively impact spadefoot larvae, it is unlikely that sufficient
time has elapsed for resistance to develop. Brausch and Smith (2009)
concluded that herbicide-resistant fairy shrimp were likely exposed
for approximately 60 generations. Because glufosinate herbicides
were first registered in the U.S. in 1993 (U.S. EPA, 1993 in Cox, 1996),
less than 14 generations of New Mexico and Plains spadefoots had
potentially been exposed to IG at the time of this study.
Another possibility is that cropland spadefoot larvae were
unable to develop resistance to the mode of toxicity displayed by
WM or IG. Glyphosate-based herbicides are thought to be toxic
toward amphibians because included surfactants disrupt gill
structure and function (Swedmark et al., 1971; Abel, 1974; Mann
and Bidwell, 1999). The mechanism of glufosinate herbicide
toxicity toward amphibians is unknown. The only previous
example of within-species variation in pesticide sensitivity among
amphibians was observed for carbaryl (Bridges and Semlitsch,
2000), an insecticide with a different mode of toxicity than WM.
Carbaryl is a neurotoxic insecticide that disrupts acetylcholinesterase activity (Bridges, 1997).
It is possible that weight differences influenced acute toxicity
estimates and chronic response of larvae to WM and IG. Mean
weight of grassland larvae used during chronic toxicity tests was
greater than cropland larvae. Larvae used for acute tests exhibited
the same trend. These weight differences, which are common
among Spea spp. larvae from playas (McMurry et al., 2009), may
have affected our ability to detect variation in herbicide sensitivity
between landuses. Larvae with greater mass may exhibit decreased
sensitivity because higher herbicide concentrations are needed to
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Fig. 2. Survival (mean 1 S.E.) of Spea multiplicata (New Mexico spadefoot) larvae (n ¼ 142) from cropland (squares, n ¼ 75) and grassland playas (circles, n ¼ 67) during chronic
(30-day) exposure to aged tap water, or Roundup WeatherMAXÒ at 2.0 or 2.8 mg glyphosate acid equivalents (ae)/L in a static-renewal system.
attain a critical body burden, or because a smaller surface area-tovolume ratio decreases herbicide uptake (Wojtaszek et al., 2004).
However, the relationship between larval mass and glyphosate
herbicide sensitivity is unclear. Mann and Bidwell (1999) noted that
H. eyrei, with mean mass approximately three times greater than
the other species tested, was also the least sensitive to RoundupÒ.
However, sensitivity of H. eyrei to TouchdownÒ herbicide (another
glyphosate formulation) was similar to that of the species with the
smallest mass (L. dorsalis). No clear relationship between these
variables was present among species displaying smaller differences
in mass (Mann and Bidwell, 1999). Another study found no
consistent relationship between larval mass and Roundup Original
MaxÒ sensitivity among nine amphibian species (Relyea and Jones,
2009). If greater larval mass conferred increased WM or IG resistance, grassland larvae should consistently display greater LC50
values and increased survival following chronic herbicide exposure.
Acute and chronic results do not consistently demonstrate this
relationship.
Another possibility is that water temperature differences
between cropland and grassland compartments influenced acute
toxicity estimates, thereby masking variation in herbicide sensitivity among grassland and cropland larvae. Mean water temperature during the 48-h exposure period was 3.28 C higher in
grassland compartments than cropland compartments. This likely
resulted from air temperature variation in the room that housed
experimental compartments. While this room is climate controlled,
ambient air temperatures respond measurably to exterior (i.e.,
environmental) temperatures due to the room’s design and location (i.e., one wall was subject to direct sunlight).
The relationship between water temperature and toxicity of
formulated glyphosate herbicides to aquatic organisms is unclear.
Tsui and Chu (2003) found no consistent relationship between the
toxicity of RoundupÒ to Ceriodaphnia dubia and water temperature.
Folmar et al. (1979) noted that a 10 C increase in water temperature doubled the toxicity of RoundupÒ (i.e., reduced LC50 values by
half) to Salmo gairdneri and Lepomis macrochirus. However, in three
out of eight cases, a 5 C increase in water temperature resulted in
a negligible increase or had no effect on toxicity. If WM or IG
toxicity was affected by the above difference in water temperature,
grassland larvae would likely display lower LC50 values than cropland larvae. Results indicate that grassland and cropland LC50
values were not significantly different for either herbicide. Also, the
above trend (i.e., grassland LC50 < cropland LC50) was not consistent for either herbicide.
4.3. Variation in sensitivity between species
No between-species differences in WM sensitivity were present.
However, acute toxicity estimates indicated that New Mexico spadefoots were less sensitive to IG than Plains spadefoots, irrespective
of landuse. Pesticide sensitivity is known to vary among amphibian
species (Mann and Bidwell, 1999; Howe et al., 2004; Jones et al.,
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5. Conclusion
Pesticide toxicity is one factor that may adversely affect
amphibian populations. We investigated acute and chronic toxicity
of two commonly applied agricultural herbicides among larval New
Mexico and Plains spadefoots from playas embedded in cropland or
grassland. Acute toxicity estimates indicate that IG was less toxic
than WM to larvae of both species. IG likely does not pose
a significant risk for mortality among larval New Mexico and Plains
spadefoots. Future studies should examine WM toxicity under
increasingly realistic exposure scenarios to determine whether this
herbicide may cause extensive mortality among larvae of these
species under field conditions.
Acknowledgments
We thank John Allen Jones, Edward Black, and Karlee Dinehart for
valuable assistance with these experiments. Funding was provided
by the Caesar Kleberg Foundation for Wildlife Conservation.
References
Fig. 3. Survival (mean 1 S.E.) of Spea bombifrons (n ¼ 168) and S. multiplicata
(n ¼ 156) (Plains and New Mexico spadefoot, respectively) larvae from cropland
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