Interactions of an Insecticide with Larval Density and

Predation in Experimental Amphibian Communities

MICHELLE D. BOONE* AND RAYMOND D. SEMLITSCH
105 Tucker Hall, Division of Biological Sciences, University of Missouri, Columbia, MO 652117400, U.S.A.

Abstract: This study examines the effects of the short-lived insecticide carbaryl, a neurotoxin, on amphibian
communities experiencing natural stresses of competition and predation. Tadpoles of three species ( Wood-
houses toad [ Bufo woodhousii], gray treefrog [ Hyla versicolor], and green frog [ Rana clamitans]), representing
a commonly encountered assemblage in Missouri, were reared in outdoor polyethylene pond mesocosms. We
determined the effects of initial tadpole density (low or high), predation (newts [ Notophthalmus viridescens]
absent or present), chemical exposure (0, 3.5, or 7.0 mg/L carbaryl), and their interactions on body mass, lar-
val period, and survival to metamorphosis. Green frogs in high-density ponds did not reach metamorphosis,
but metamorphs in low-density ponds and tadpoles in high-density ponds were not significantly affected by
treatments or their interaction. Carbaryl reduced survival to metamorphosis in toads and treefrogs and in-
creased mass at metamorphosis in treefrogs. Effects of carbaryl varied with predator environment and initial
larval density. Interactions of carbaryl with predator and with density may result in an indirect effect of car-
baryl causing increased food resources through the elimination of zooplankton populations that may com-
pete for similar resources. Our results indicate that differences in biotic conditions influenced the potency of
carbaryl and that even low concentrations induce changes that may alter community dynamics in ways not
predicted from single-factor, laboratory-based studies.

Interacciones de un Insecticida con la Densidad Larval y la Depredacin en Comunidades Experimentales de Anfibios

Resumen: Este estudio examina los efectos de un insecticida de vida corta (carbaryl, un neurotxico) en com-
unidades de anfibios que se encuentran experimentando estreses naturales de competencia y depredacin.
Los renacuajos de tres especies (el sapo casa de madera [ Bufo woodhousii], la rana arborcola gris [ Hyla ver-
sicolor] y la rana verde [ Rana clamitans]), representantes de un ensamblaje comn en Missouri, fueron man-
tenidas en mesocosmos de estanques de polietileno. Se determinaron los efectos de la densidad inicial de los
renacuajos (baja o alta), la depredacin (tritones [ Notophthalmus viridescens] ausentes o presentes), la ex-
posicin qumica (0, 3.5 o 7.0 mg/L de carbaryl) y sus interacciones sobre la masa corporal, el perodo lar-
val, y la supervivencia a la metamorfosis. Las ranas verdes en estanques con altas densidades no alcanzaron
la metamorfosis, pero los metamorfos en los estanques con baja densidad y los renacuajos en estanques con
alta densidad no fueron afectados significativamente por los tratamientos ni por sus interacciones. El car-
baryl redujo la supervivencia a la metamorfosis e increment la masa corporal durante la metamorfosis de
las ranas arborcolas. Los efectos del carbaryl variaron dependiendo del ambiente de depredacin y de la
densidad larval inicial. Las interacciones de carbaryl con el depredador y con la densidad pueden resultar en
un efecto indirecto del carbaryl al ocasionar un incremento en los recursos alimenticios mediante la elimi-
nacin de poblaciones de zooplancton que podran competir por recursos similares. Nuestros resultados indi-
can que las diferencias en las condiciones biticas influyen en la potencia del carbaryl y hasta podran al-
terar las dinmicas comunitarias de formas no predecibles a partir de estudios de laboratorio basados en un
solo factor.

* email mdb7ef@mizzou.edu
Paper submitted October 15, 1999; revised manuscript accepted March 22, 2000.
228

Conservation Biology, Pages 228238

Volume 15, No. 1, February 2001
Boone & Semlitsch
Introduction
Rachel Carsons Silent Spring (1962) raised public
awareness of the ecological consequences of wide-
spread pesticide use by noting the many anecdotal inci-
dents associated with organochlorine application: dead
robins littering lawns across the countryside, repro-
ductive failure among carnivorous birds and farm ani-
mals, and a high incidence of disease in humans after
chemical exposure. Many of the 63,000 pesticides em-
ployed today in the United States ( Ramade 1988) are
benign in comparison with the early synthetic pesti-
cides that came into use after World War II (Rand 1995).
Because todays pesticides are relatively short-lived and
do not typically bioaccumulate (Rand 1995), the con-
sequences of rampant pesticide use are presumed triv-
ial or even a necessary trade-off for adequate crop pro-
duction, economic profit, and a healthy appearance of
lawns and crops. Although current pesticides are rou-
tinely tested for ecological risk, one of Carsons con-
cerns in 1962 is still relevant today: The chemicals may
have been pretested against a few individual species,
but not against living communities (Carson 1962). Al-
though safety standards are set to minimize mortality
risks to nontarget wildlife, the effect of low levels of
contaminants on communities has not been explored
sufficiently and may have implications for conservation
of species vulnerable to pesticide exposure. This data
lacuna is due in part to limited overlap in research
among conservation biologists and toxicologists, despite
shared goals of protecting organisms in nature (Hansen
& Johnson 1999).
The effects of pesticides on natural community dy-
namics may be especially noteworthy for amphibians in
light of recent concerns about amphibian declines in
both disturbed and pristine environments (Blaustein
1994; Corn 1994). These declines are suspected to stem
from a number of causes such as habitat destruction or
alteration, introduction of nonnative species, fungal and
virus outbreaks, chemical pollutants or the interaction
of these factors (Corn 1994; Dodd 1997; Carey et al.
1999). Identifying the main cause for extinctions on a lo-
cal or regional scale is further complicated by differen-
tial responses among species. While abundance of many
amphibian species appears to be declining, others are
expanding their historical ranges (e.g., Rana catesbei-
ana and R. pipiens) or show no sign of decline (Corn
1994). This observation suggests that some species are
more vulnerable to environmental stressors than others.
Because amphibian abundances are naturally variable in
time and space (Pechmann et al. 1991), however, differ-
entiating natural variation from human-induced declines
may be difficult.
Whether or not contaminants contribute to declines,
the presence of chemicals could alter community func-
tionand thereby affect biodiversitybecause many
Effects of Insecticide on Amphibians 229
amphibians breed within or around agricultural areas
that are routinely exposed to pesticides. The effects of
pesticides on natural amphibian populations are unclear
because few toxicological data exist for amphibians. Be-
cause environmental levels are often not great enough
to induce direct mortality, the effect of sublethal con-
taminants may be more relevant. Recently, studies have
considered important sublethal effects of chemicals on
behavior (Little et al. 1985; Little et al. 1993; Bridges
1997) and long-term, chronic effects to chemical expo-
sure at different life stages of organisms (Marian et al.
1983).
Although some have suggested that amphibians may
be more vulnerable to pesticides than organisms typi-
cally used for toxicity testing (Bishop & Pettit 1992), re-
cent data suggest that amphibians may be less vulnera-
ble to contaminants (Mayer & Ellersieck 1986; Bridges
1999; Bridges & Semlitsch 2000). If amphibians are less
sensitive than the organisms used to set safety standards,
it might be presumed that they would be protected un-
der the Federal Insecticide, Fungicide, and Rodenticide
Act (Touart 1995). This is not necessarily true. Most of
the studies conducted to determine toxicity are short-
term, acute toxicity studies that take place on a 96-hour
time scale. Such studies are favored because they can be
done quickly and cheaply on a number of model organ-
isms with adequate replication and maximum control.
Many of these studies focus on the dose required to
elicit a response (usually mortality), and the data are
condensed to a value, or set of values, used to set admis-
sible environmental levels (Rand et al. 1995).
Laboratory studies often examine the effects of chemi-
cals in isolation of factors known to be important in nat-
ural communities. What is effective in a glass jar for 96
hours may be less so in an environment where tempera-
ture varies and where predators and competitors are
present; therefore, single-factor, acute laboratory tests
may not be sufficient to predict effects in natural envi-
ronments. For this reason, we should not assume that
contaminant levels that are safe in the laboratory are also
innocuous in the environment.
To ensure that federal levels are acceptable, it is fun-
damental to conduct exploratory studies that incorpo-
rate factors important in real communities. Therefore,
mesocosm studies may offer important advantages over
laboratory-based studies (Fairchild & Little 1993; La Point
& Fairchild 1994; Rowe & Dunson 1994) in determining
the possible effects of a chemical on amphibian diversity
and population longevity. We examined the effects of
the short-lived insecticide carbaryl on anurans from hatch-
ing through metamorphosis in environments where the
competitive and predatory intensities differed. Our main
objectives were to determine whether species showed
differential susceptibility to carbaryl and whether car-
baryls effect depended on conditions in the biotic envi-
ronment (competitive density and predator presence).
Conservation Biology
Volume 15, No. 1, February 2001
230 Effects of Insecticide on Amphibians
Methods
Species Collection and Chemical Contaminant
Three species of anurans (Bufo woodhousii, Hyla versi-
color, and Rana clamitans) used in this study can oc-
cur sympatrically in the Midwest region of the United
States and represent component and common species of
natural communities found in this area. In mid-June of
1997, eggs from 10 amplexed pairs of gray treefrogs (H.
versicolor) and three egg masses of the green frog (R.
clamitans) were collected at the Baskett Wildlife Area, a
relatively pristine site near Ashland, Missouri (Boone
County), and one egg mass of Woodhouses toad ( B.
woodhousii) was collected in a flooded agricultural
field near Jefferson City, Missouri (Callaway County),
along the floodplain of the Missouri River. Eggs were
hatched in the laboratory at 2325 C and held until tad-
poles were free-swimming. To homogenize genetic vari-
ation, we pooled egg masses by species before using
them in our experiment. Adult red spotted newts (Noto-
phthalmus viridescens; male 2.43 0.10 g, mean 1 SE;
female 2.46 0.12 g, mean 1 SE), which are ubiqui-
tous tadpole predators, were collected in early July 1997
at Three Creeks Conservation Area near Ashland, Mis-
souri (Boone County).
Carbaryl (1-naphthyl N-methyl carbamate) is a short-
lived (hours to weeks) carbamate insecticide that inhib-
its acetyl cholinesterase (Cox 1993; Beyers & Meyers
1996), thereby disrupting nervous-system function. Car-
baryl affects organisms similarly to other carbamate and
organophosphate insecticides; it is therefore representa-
tive of these classes of pesticides (Rand 1995) and may
serve as a model chemical stressor. Carbaryl can enter
aquatic systems from direct application or runoff from
agricultural lands (Cox 1993). The persistence and po-
tency of carbaryl varies with soil type, rainfall, pH, tem-
perature, and ultraviolet-B radiation (Guerrant et al.
1970; Wauchope & Haque 1973; Gibbs et al. 1984; Zaga
et al. 1998; Boone & Bridges 1999). Carbaryl affects
anuran feeding and swimming behavior at sublethal
levels (Marian et al. 1983; Bridges 1997) that are con-
sistent with field-application levels (4.8 mg/L and 3.5
mg/L; Norris et al. 1983 and Peterson et al. 1994, re-
spectively).
Experimental Design
Amphibian communities were created in 36 polyethyl-
ene ponds (1.85 m diameter; 1480 L volume) to which
1000 L of well water, 1 kg of leaf litter, and plankton
from natural ponds (500 mL of plankton/pond at six dif-
ferent times) were added in late May 1997. The ponds
were located in a fenced field at the University of Mis-
souri Research Park in Boone County, Missouri. Screen-
mesh lids covered each pond to exclude unwanted
Conservation Biology
Volume 15, No. 1, February 2001
Boone & Semlitsch
predators and colonists; these lids provided some shade
to ponds.
We manipulated three factors in a fully crossed design
with three replicates: initial larval density (low, 102 tad-
poles, or high, 306 tadpoles), predation (absent or present
[1 male and 1 female newt]) and chemical concentration
(0, 3.5, or 7.0 mg/L carbaryl). Free-swimming tadpoles
(Gosner developmental stage 25; Gosner 1960) were
randomly assigned to ponds. Our chosen densities were
representative of tadpole densities in natural communi-
ties (144238 per 1000 L; Morin 1983; Petranka 1989).
Numbers of tadpoles of each species added to low- and
high-density ponds, respectively, were 27 and 81 Wood-
houses toads and 45 and 135 gray treefrogs on 24 June
1997, (day 0) and 30 and 90 green frogs on 25 June
1997, for a total of 102 (low-density) and 306 (high-den-
sity) tadpoles per pond. Tadpole densities were rela-
tively high to ensure that some individuals of each spe-
cies survived in the predator treatments. Newts were
added to predator treatments on 4 July 1997.
Carbaryl was added as liquid Sevin (which contains
21.3% carbaryl) by mixing it with 5 L of pond water and
pouring the mixture evenly across the pond surface
with a watering can. Ponds were not stirred, both to
minimize disturbance and because direct application in
a natural environment would occur similarly. Chemical
concentrations were chosen based on expected environ-
mental concentrations and followed a 50% dilution se-
ries: control, 0 mg/L; sublethal level, 3.5 mg/L, expected
environmental level; and worst-case level, 7.0 mg/L,
known to be near the LC50 for some anuran species
(Boone & Bridges 1999; Bridges 1999). Chemical treat-
ments were applied on 9 July 1997. At the time of addi-
tion, the ponds had an average water temperature of
30 C, pH of 8.0, and 3.47 0.19 mg/L dissolved oxy-
gen. Chemical analyses were not conducted on the wa-
ter, but analyses from another experiment in these
ponds indicated that the chemical has a half-life of ap-
proximately 4 days (Boone & Semlitsch 2000).
Response Variables and Statistical Analyses
Ponds were searched daily for metamorphs (Gosner de-
velopmental stage 42; Gosner 1960). Metamorphs were
removed from each pond and kept in the laboratory un-
til tail resorption (usually 2 days), at which time they
were towel-dried and weighed to the nearest milligram.
The study was terminated on days 121122 of the exper-
iment (2324 October 1997). Ponds were drained and
thoroughly searched for any remaining tadpoles and
metamorphs. Nearly all ponds in our experiment still
contained green frog tadpoles at that time.
Days to metamorphosis, mass at metamorphosis, and sur-
vival to metamorphosis were used to measure the response
of anurans to treatments. Statistical analyses were per-
Boone & Semlitsch Effects of Insecticide on Amphibians 231

formed on these responses to test the effects of chemi-
cal concentration, predator presence, density, and their
interactions for Woodhouses toad and the gray tree-
frog. Because few green frogs reached metamorphosis
in high-density treatments, density was dropped as a
treatment effect, and only green frog metamorphs from
low-density ponds were used in analyses. We deter-
mined the effects of predator, chemical, and their inter-
action on survival, tadpole mass, and Gosner develop-
mental stage (Gosner 1960) for green frog tadpoles in
high-density ponds (these tadpoles were not likely to
metamorphose before winter). We determined the ef-
fects of density, predator, chemical, and their interac-
tions on total pond survival (tadpoles and metamorphs)
for green frogs. We used analyses of variance (ANOVA,
SAS Institute 1988) to determine the effects of treatment
on days to metamorphosis, mass at metamorphosis, and
survival for each species. Days and survival to metamor-
phosis were used as covariates (they were not corre-
lated: r 2 0.16, p 0.1803) for mass at metamorpho-
sis, and survival alone was used as a covariate for days to
metamorphosis (because survival and mass were corre-
lated: r 2 0.41, p 0.0004). Because species were
reared together, they were not statistically independent,
although we analyzed them as such. We believe, how-
ever, that this was the best and most parsimonious
method to determine significant treatment effects within
communities. The proportion that survived to metamor-
phosis was angularly transformed, and mass at meta-
morphosis and days to metamorphosis were log-trans-
formed before analyses to normalize the data and stabilize
variances (Snedecor & Cochran 1980). We used Scheffes
multiple comparison tests for pair-wise comparisons of
main effects.
We determined the effects of chemical, larval density,
and the interaction of these factors on newts by measur-
ing the change in mass from the beginning to the end of
the experiment. Analyses were completed by ANOVA
(SAS Institute 1988). The presence of newt larvae was
noted at the end of the experiment.
Results
Effects on the Anurans
Because few green frogs metamorphosed in high-density
ponds, data on metamorphs in low-density ponds and
tadpoles from high-density ponds were both analyzed
separately. For green frog metamorphs in low-density
ponds, neither chemical, predator, nor their interaction
significantly affected responses to metamorphosis (Ap-
pendix). There were no significant effects of predator,
chemical, or the interaction of these factors on survival,
stage, or tadpole mass for green frog tadpoles in high-
density ponds (Appendix). The total survival of tadpole
and metamorphosed green frogs was negatively affected
by density (F2,23 20.56, p 0.0001) but not by chemi-
cal, predator, or any interactions. In low-density ponds,
38.6% of the green frogs (tadpoles and metamorphs) sur-
vived to the end of the experiment, whereas 69.2% of
the frogs (tadpoles and metamorphs) in high-density
ponds survived.
Survival of toads decreased with increasing density
and with chemical level (Table 1). A density-by-chemical
interaction affected toad survival (Appendix). At the
high chemical level, survival in both high- and low-den-
sity ponds was lower than in controls; yet at the low
concentration, survival increased in low-density and de-
creased in high-density ponds (Fig. 1). Increased density
significantly reduced mass at metamorphosis (Table 1),
but other treatments and their interactions did not affect
mass (Appendix). Length of the larval period was mar-
ginally affected by predator presence (Appendix), with
toads metamorphosing from ponds one day earlier, on
average, in the presence of predators (Table 1).

Table 1. Least-square means (1 SE) in mass, days, and survival to metamorphosis for Woodhouses toad and gray treefrogs for larval
density, predator, and carbaryl treatments.*

Density Predator Carbaryl (mg/L)

Species Response low high absent present 0 3.5 7

Woodhouses toad mass 0.2365a 0.1727b 0.2129a 0.1963a 0.1995a 0.1977a 0.2166a
(0.0112) (0.0100) (0.0115) (0.0109) (0.0130) (0.0116) (0.0177)
days 19.97a 20.09a 20.98a 19.06b 19.24a 19.56a 21.26a
(0.9745) (0.8937) (0.6745) (0.6424) (0.7868) (0.7070) (1.0534)
survival 0.5093a 0.2047b 0.4129a 0.3011a 0.4861a 0.4434a 0.1415b
(0.0560) (0.0490) (0.0543) (0.0508) (0.0611) (0.0576) (0.0735)
Gray treefrog mass 0.4419a 0.3371b 0.3940a 0.3849a 0.3856a 0.3767a 0.4059b
(0.0113) (0.0113) (0.0099) (0.0099) (0.0179) (0.0135) (0.0225)
days 24.44a 25.60a 24.82a 25.23a 24.00a 24.07a 27.01a
(1.1108) (1.1108) (0.7156) (0.7156) (1.2758) (0.9630) (1.5956)
survival 0.5864a 0.6506a 0.6177a 0.6193a 0.7778a 0.6969b 0.3809c
(0.0250) (0.0250) (0.0250) (0.0250) (0.0306) (0.0351) (0.0306)
*Differing letters indicate significant differences within main treatment effects according to Scheffes multiple comparison tests.

Conservation Biology
Volume 15, No. 1, February 2001
232 Effects of Insecticide on Amphibians Boone & Semlitsch

Treefrog survival to metamorphosis was negatively af- Effects on the Newt Predator
fected by carbaryl (Table 1; Appendix). A density-by-
predator interaction demonstrated that, in the presence Chemical exposure decreased the growth of newts, but
of the newts, roughly the same proportion of individuals neither density nor density-by-chemical interaction af-
metamorphosed in low- and high-density ponds. In the fected mass. Except for two newts from low-chemical
absence of the newts, however, a higher proportion
metamorphosed from high-density ponds (Fig. 2a). A
density-by-chemical interaction showed that the strength
of carbaryls effect varied with density (Fig. 2b). Under
control conditions, low-density ponds produced propor-
tionally more individuals. With exposure to carbaryl,
however, this relationship was reversed at both chemi-
cal concentrations, with high-density ponds producing
proportionately more metamorphs than low-density ponds
at either exposure. Nevertheless, the high chemical level
reduced survival substantially in comparison to controls.
The mass of treefrog metamorphs was affected nega-
tively by density (Table 1; Appendix). A predator-by-
chemical interaction indicated that treefrogs weighed
less in the presence of a predator than in the absence of
a predator under control and low chemical conditions
( Fig. 3a). At the high chemical concentration, however,
larger metamorphs were produced on average when
reared in the presence of newts than in any other envi-
ronment. A density-by-chemical interaction showed that
mass at metamorphosis increased with chemical concen-
tration under high-density conditions and was reduced
under low chemical concentrations (Fig. 3b). The length
of the larval period for treefrogs was affected by density
but not by any other treatments or their interactions
(Appendix).

Figure 2. Proportion of treefrogs that survived to

Figure 1. Proportion of toads that survived to meta- metamorphosis in (a) low- and high-density ponds in
morphosis in ponds with low- and high-density across the presence or absence of newt predators and (b)
carbaryl treatments. Plotted values are least-square low- and high-density ponds across carbaryl treat-
means. ments. Plotted values are least-square means.

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Volume 15, No. 1, February 2001
Boone & Semlitsch
ponds (which may have escaped the ponds), all other
newts were recovered at the close of the experiment
(males, 2.46 0.14 g, mean 1 SE; females, 2.73
0.16 g, mean 1 SE). No newts were found dead after
the chemical addition in the ponds. Overall, newts
gained 0.14 0.12 g (mean 1 SE). Mass gained was
significantly affected by the chemical treatment (F2,12
4.41, p 0.0366), with mass decreasing as chemical in-
creased (Fig. 4). Newt larvae were found in 3 out of 16
pondsone control low-density pond and two low-
chemical, high-density ponds.
Figure 3. Mass at metamorphosis of treefrogs in (a)
absence or presence of predators and (b) low- and
high-density ponds across carbaryl treatments. Plotted
values are least-square means.
Effects of Insecticide on Amphibians 233
Discussion
Direct Effects of Chemical Exposure
One of the conundrums of amphibian population de-
clines is that only some species appear to be affected at
given ponds. The anuran species do not appear equally
susceptible to the chemical stressor carbaryl. Differ-
ences in susceptibility to stresses in general may explain
why some amphibian populations decline while others
appear unaffected. Susceptibility to a contaminant may
be mediated by a number of important life-history char-
acteristics relating to length of the larval period and time
of breeding. The two species most affected by carbaryl
in our experiment, toads and treefrogs, had relatively
short larval periods. Neither the green frogs that meta-
morphosed in low-density ponds nor the tadpoles that
did not metamorphosis in high-density ponds were af-
fected by the contaminant. Species such as the green
frog that have longer larval periods may be able to com-
pensate for any negative metabolic effects of contami-
nants over time. Species such as toads and treefrogs with
short larval periods may be more likely to avoid larval
exposure to contamination by simply reaching metamor-
phosis before the chemical is released into the environ-
ment. When exposure occurs during larval development
for such species, however, the effects may be more pro-
found, as was the case in our experiment.
The timing of exposure during development may be
critical in influencing the effects or efficacy of a contami-
nant. Although green frogs from low-density ponds did not
Figure 4. Change in mass of newts across carbaryl
treatments over the course of our experiment. Differ-
ent letters indicate significant differences according to
Scheffes multiple comparison tests. Error bars repre-
sent 1 SE. Plotted values are least square means.
Conservation Biology
Volume 15, No. 1, February 2001
234 Effects of Insecticide on Amphibians
appear to be affected by carbaryl, subsequent experi-
ments showed that when over-wintered green frog tad-
poles are exposed to carbaryl at similar levels, the chem-
ical negatively affects mass at metamorphosis (Boone &
Semlitsch, unpublished data). At the time of carbaryl ad-
dition, toads in our experiment were near metamorpho-
sis: therefore, it was unlikely that carbaryl exposure could
have affected mass or time to metamorphosis. Neverthe-
less, their survival was significantly reduced at the high-
chemical concentration, indicating that exposure late in
larval development could be detrimental, as others have
demonstrated (Marian et al. 1983), and could lead to re-
duced population size through reduced recruitment. Ex-
posure earlier in larval development has been shown to
yield increased survival at greater carbaryl concentra-
tions (Boone & Semlitsch, unpublished data). Timing of
exposure and chemical concentration therefore may be
key factors determining the effect of a chemical at the
population or community level.
The species most strongly influenced by carbaryl, and
other treatment effects, was the gray treefrog. The larval
period of this species was approximately twice as long as
that of the toads, and because chemical exposure oc-
curred earlier in development, the chemical was more
likely to alter mass at metamorphosis, length of the larval
period, and survival to metamorphosis. For gray treefrogs,
the high chemical concentration exerted an effect similar
to that of predators: survival was reduced and the remain-
ing individuals that reached metamorphosis were larger
in size due to reduced competition. Carbaryl had a direct
negative effect on survival but a positive effect on individ-
uals that survived, because greater mass at metamorpho-
sis was attained, which is correlated with increased over-
winter survival and greater reproductive success (Berven
& Gill 1983; Smith 1987; Berven 1988; Scott 1994). In nat-
ural pond communities, then, it might be difficult to dif-
ferentiate a chemical effect from a natural predator-prey
interaction because they have the same outcome.
Interactions of Carbaryl with Natural Biotic Stresses
A demonstration that carbaryl directly affects anuran mass
and survival to metamorphosis, while interesting, is not
unanticipated. Laboratory studies have shown that higher
levels of contaminants increase mortality (Mayer & Eller-
sieck 1986; Boone & Bridges 1999) and that behavior may
be altered in ways that suggest mass and survival to meta-
morphosis may be affected (Bridges 1999). Carbaryl, how-
ever, also interacted with biotic stresses known to influ-
ence natural populations, and to our knowledge such an
effect has not been documented at the community level.
Chemical-by-Density Interactions
Survival was affected by a chemical-by-density interac-
tion for both toads and treefrogs. For toads and treefrogs
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Volume 15, No. 1, February 2001
Boone & Semlitsch
reared in control conditions, low-density ponds produced
proportionately more individuals than high-density ponds.
Low-density ponds should be relatively favorable envi-
ronments, with abundant food resources compared with
high-density ponds; under low-density conditions then,
fewer individuals would be expected to die from starva-
tion. At low chemical levels, toad survival increased in low-
density conditions, whereas survival was 69% lower
than in control ponds in high-density conditions. A high
chemical dose could be disastrous for toad recruitment
regardless of density, but the effects of lower levels of
carbaryl may depend on the competitive environment.
Why survival of toads may be enhanced by a low chemi-
cal level near metamorphosis at low density relative to
controls is unclear, although physiological compensa-
tion may be a possible explanation.
For treefrogs, density also influenced the effect of car-
baryl. Addition of a low level of carbaryl resulted in
slightly more metamorphs in high-density than in con-
trol ponds, whereas low-density ponds produced 27%
fewer individuals than control ponds. Unlike the toads,
treefrogs in low-density conditions fared worse than
those in high-density ponds when exposed to a low
level of carbaryl that, interestingly, does not induce mor-
tality in laboratory tests (Boone & Bridges 1999; Bridges
1999). At the high concentration, treefrog survival was
further reduced in both high- and low-density ponds,
but high-density ponds still produced proportionately
more metamorphs.
The most parsimonious explanation for this interac-
tion is an indirect effect within the food web, although
the design of our experiment precludes differentiating
between direct and indirect effects. Once the ponds
were dosed with carbaryl, the zooplankton population
disappeared, and within a few days there was a notice-
able algal bloom (personal observation), a common re-
sult of insecticide use (Hanazato & Yasuno 1990; Fair-
child et al. 1992). Because tadpoles and zooplankton
feed on algae, removal of zooplankton could essentially
release the tadpoles from competition. Removal of zoo-
plankton may enhance survival in high-density ponds
relative to low-density ponds because the tadpoles in
food-limited environments (i.e., high-density) benefited
more from increased food because of a reduced likeli-
hood of death via starvation. The significant chemical-
by-density interaction bolsters this idea because it dem-
onstrates that treefrog mass increased with chemical
concentration, regardless of survival differences (because
survival was used as a covariate). Although the food sup-
ply would also increase in low-density conditions, an
augmented food supply would not necessarily be advan-
tageous if food was already abundant.
An increase in survival due to carbaryl addition may
appear beneficial, but overall survival at the high chemi-
cal level at either density is much lower than in control
ponds. Furthermore, an increase in food resources for
Boone & Semlitsch
anurans may result in a decreased food supply for other
species that feed on zooplankton. For instance, newts from
ponds that contained carbaryl in our experiment showed
a reduced mass relative to controls; other salamander
species that feed on zooplankton may likewise be nega-
tively affected (Taylor et al. 1988). Our study demon-
strates that even if environmental levels of a chemical do
not directly affect anurans, a chemical effect may rico-
chet throughout the community via food webs.
Chemical-by-Predator Interactions
Level of predation interacted with chemical concentra-
tion. Treefrog mass at metamorphosis was significantly
affected by a chemical-by-predator interaction with meta-
morphs from ponds with predators having a smaller
mass under low and no chemical conditions. Mass at
metamorphosis is often reduced in the presence of pred-
ators (Morin 1983) because of reduced time spent feed-
ing by tadpoles. At the highest chemical level, however,
treefrog tadpoles that were raised in the presence of
newt predators attained a greater size at metamorphosis
than those in the absence of predators. This difference
in mass cannot be associated with differences in survival
among predator treatments because survival was used as
a covariate. There was not a significant three-way inter-
action of density, predator, and chemical, which sug-
gests that an increase in food supply alone does not
explain the chemical-by-predator interaction. Predators
may have been less active or less effective at the high
chemical level (Bridges 1999), which may have led to in-
creased feeding activity in the tadpoles and therefore to
larger metamorph mass.
Conclusions
In addition to important natural factors such as pH and
pond hydroperiod (Dunson & Travis 1991; Semlitsch et
al. 1996) that are known to vary spatially and tempo-
rally, chemical contamination may be an influential an-
thropogenic factor for many amphibian populations on
short- and long-term scales. These factors may be con-
stant through time, such as the acidic pH of bog habi-
tats, or they may occur stochastically among years, such
as shortened hydroperiods in drought years. Likewise, a
communitys exposure to contaminants may be chronic
or variable, depending on a ponds location and relative
propinquity to managed land. Many amphibians breed in
habitats surrounded by agricultural landscapes or oc-
cupy forested habitats that may be vulnerable to direct
chemical exposure (e.g., attempts to reduce weeds in
plantation settings or to remove disease pests such as
spruce budworm). Pesticides may be applied multiple
times per season or only in years in which there is a dan-
ger of an outbreak of pests.
Effects of Insecticide on Amphibians 235
Even a short-lived chemical like carbaryl can affect
anurans exposed as tadpoles, despite larval periods that
last longer than chemical exposure. Species with shorter
larval periods may be more susceptible to contaminant
exposure because they have less time to recover from
contaminant effects. The timing of exposure during de-
velopment may also influence the relative importance of
the chemicals effect. If a chemical tends to reduce mass
at metamorphosis, then exposure later in the larval pe-
riod may have a less dramatic effect. Carbaryl alone had
direct negative effects on survival in toads and treefrogs
and mass at metamorphosis in treefrogs, both of which
could have important fitness consequences and could
lead to population declines over time when such occur-
rences are routine. And because the chemical interacted
with factors known to be important in the natural envi-
ronment, such as density and predation, we suggest that
studies considering the effect of a chemical alone may
not be adequate to determine what substances will have
no effect in the environment. Although many pesticides
in use today are relatively ephemeral and do not bioac-
cumulate, they can still affect interactions within the
community because current regulations focus on mortal-
ity and not community function.
Acknowledgments
This manuscript benefited from the thoughtful com-
ments of C. Bridges, J. Fairchild, S. James, E. Little, and
A. Welch. We thank C. Bridges, M. Parris, B. Sonderman,
N. Sullivan, and A. Boone-Sullivan for field assistance, J.
Fairchild for experimental and chemical advice, and A.
Welch for contributing treefrog tadpoles from her
crosses. This research was supported by the University
of MissouriColumbia and grant 82709501 from the
U.S. Environmental Protection Agency.
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Boone & Semlitsch Effects of Insecticide on Amphibians 237

Appendix
Summary of univariate analyses of covariance of proportion of amphibians surviving to metamorphosis, body mass at metamorphosis, and
larval period length for Woodhouses toad, gray treefrog, and green frogs.

Response variable Source of variation df Mean square F p

Woodhouses toad
survival density 1 0.9851 14.31 0.0012
predator 1 0.2030 2.95 0.1014
chemical 2 0.4367 6.34 0.0074
density by predator 1 0.0118 0.17 0.6834
density by chemical 2 0.3405 4.95 0.0180
density by predator by chemical 2 0.0108 0.16 0.8561
error 20 0.0688
mass survival (covariate) 1 0.0248 0.84 0.3715
larval period (covariate) 1 0.0131 0.44 0.5134
density 1 0.5045 17.07 0.0006
predator 1 0.2932 0.99 0.3325
chemical 2 0.0072 0.24 0.7855
density by predator 1 0.0240 0.81 0.3798
density by chemical 2 0.0001 0.00 0.9953
predator by chemical 2 0.0029 0.10 0.9058
density by predator by chemical 2 0.0371 1.26 0.3085
error 18 0.0296
larval period survival (covariate) 1 0.0002 0.02 0.8968
density 1 0.0057 0.45 0.5083
predator 1 0.0505 4.06 0.0583
chemical 2 0.0118 0.95 0.4039
density by predator 1 0.0004 0.03 0.8610
density by chemical 2 0.0001 0.01 0.9921
predator by chemical 2 0.0132 1.06 0.3652
density by predator by chemical 2 0.0123 0.99 0.3893
error 19 0.0124
Gray treefrog
survival density 1 0.0347 2.37 0.1367
predator 1 0.0005 0.03 0.8557
chemical 2 0.6261 42.76 0.0001
density by predator 1 0.0675 4.61 0.0422
density by chemical 2 0.1136 7.76 0.0025
predator by chemical 2 0.0011 0.07 0.9289
density by predator by chemical 2 0.0019 0.13 0.8779
error 24 0.0146
mass survival (covariate) 1 0.0343 2.74 0.1122
larval period (covariate) 1 0.0230 1.83 0.1896
density 1 0.4059 32.39 0.0001
predator 1 0.0030 0.24 0.6275
chemical 2 0.0087 0.70 0.5085
density by predator 1 0.0475 3.79 0.0645
density by chemical 2 0.0503 4.01 0.0327
predator by chemical 2 0.0653 5.21 0.0141
density by predator by chemical 2 0.0011 0.09 0.9169
error 22 0.0125
larval period survival (covariate) 1 0.0018 0.13 0.7260
density 1 0.1454 10.20 0.0040
predator 1 0.0029 0.20 0.6557
chemical 2 0.0104 0.73 0.4928
density by predator 1 0.0001 0.01 0.9334
density by chemical 2 0.0023 0.16 0.8494
predator by chemical 2 0.0001 0.01 0.9897
density by predator by chemical 2 0.0036 0.25 0.7779
error 23 0.0142
continued

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238 Effects of Insecticide on Amphibians Boone & Semlitsch

Appendix (continued)
Response variable Source of variation df Mean square F p
Green frog*
metamorphs from high-density ponds
survival predator 1 0.1007 1.32 0.2591
chemical 2 0.1110 1.464 0.2486
predator by chemical 2 0.0374 0.49 0.6166
error 30 0.0761
mass survival (covariate) 1 0.6523 13.05 0.0012
larval period (covariate) 1 2.7200 54.43 0.0001
predator 1 0.2557 0.51 0.4803
chemical 2 0.3010 0.60 0.5455
predator by chemical 2 0.0584 1.17 0.3254
error 28 0.0500
larval period survival (covariate) 1 2.9458 25.24 0.0001
predator 1 0.1775 1.52 0.2274
chemical 2 0.2823 1.36 0.2725
error 29 0.1167
tadpoles from low-density ponds
survival predator 1 0.0634 1.74 0.2112
chemical 2 0.0139 0.38 0.6906
predator by chemical 2 0.0292 0.80 0.4704
error 12 0.0363
mass survival (covariate) 1 0.0389 2.61 0.1405
larval stage (covariate) 1 2.2867 153.57 0.0001
predator 1 0.0000 0.00 0.9821
chemical 2 0.0180 1.21 0.3427
predator by chemical 2 0.0127 0.85 0.4582
error 9 0.0149
larval stage mass (covariate) 1 79.7550 142.11 0.0001
predator 1 0.1630 0.29 0.6017
chemical 2 0.4193 0.75 0.4984
predator by chemical 2 1.0168 1.81 0.2131
error 10 0.0142
*Data from high- and low-density ponds were analyzed separately because tadpoles in high-density ponds did not reach metamorphosis; there-
fore, data on metamorphosed green frogs from low-density ponds and tadpoles in high-density ponds were analyzed separately.

Conservation Biology
Volume 15, No. 1, February 2001