Environmental Toxicology and Chemistry, Vol. 25, No. 12, pp.

3164–3170, 2006
! 2006 SETAC
Printed in the USA
0730-7268/06 $12.00 ! .00

TOXICITY OF SIX PESTICIDES TO COMMON FROG (RANA TEMPORARIA) TADPOLES

MARKUS JOHANSSON,*† HENNA PIHA,‡ HENRIK KYLIN,§ and JUHA MERILĆ

†Department of Population Biology Evolutionary Biology Centre, Uppsala University, Norbyvägen 18 D, SE-75236 Uppsala, Sweden
‡Ecological Genetics Research Unit, Department of Bio- and Environmental Sciences, Box 65, FI-00014 University of Helsinki, Finland
§Department of Environmental Assessment, Swedish University of Agricultural Sciences, P.O. Box 7050, SE-750 07 Uppsala, Sweden

( Received 16 December 2005; Accepted 5 June 2006)

Abstract—Amphibian species inhabiting agricultural areas may be exposed to pesticides during their aquatic larval phase. We
tested the toxicity of six commonly used pesticides on Rana temporaria spawn and tadpoles. In acute tests, tadpoles were exposed
to relatively high concentrations of azoxystrobin, cyanazine, esfenvalerate, MCPA ([4-chloro-2-methylphenoxy] acetic acid), per-
methrin, and pirimicarb for 72 h. Chronic exposure tests were performed from fertilization to metamorphosis with azoxystrobin,
cyanazine, and permethrin at concentrations similar to those found in surface waters in agricultural areas in Sweden. The most
lethal pesticides in the acute exposure were azoxystrobin, permethrin, and pirimicarb. Also, negative effects on the growth of the
tadpoles were observed with azoxystrobin, cyanazine, and permethrin. The chronic exposure at lower pesticide concentrations did
not result in increased mortality or impaired growth. However, we found a positive effect of permethrin on growth and size at
metamorphosis. The results suggest that the examined pesticides can inflict strong negative effects at high concentrations but have
no or relatively weak effects on R. temporaria spawn or tadpoles at concentrations found in Swedish surface waters.

Keywords—Amphibians Chronic exposure Acute toxicity Sublethal effects

INTRODUCTION
Agricultural practices affect natural habitats in several
ways, such as through land conversion, increased fragmenta-
tion, and agrochemical contamination [1–5], and have there-
fore been suggested as potential agents in the global amphibian
decline [6–8]. Much of the interest on amphibian declines is
currently focused on the role of pesticides, which is shown in
several recent studies. For example, Hayes et al. [3] found that
pesticide exposure at field concentrations can cause femini-
zation of male frogs, Sparling et al. [9] demonstrated increased
endocrine activity in amphibians exposed to pesticides in the
field that may explain declines in some Californian ranid pop-
ulations, and Relyea [10] detected significant disruption in
species richness in aquatic communities including both sec-
ondary positive and direct negative effects in the amphibian
community at ecologically relevant pesticide concentrations.
Studies like these have highlighted the importance of research
on the effects of amphibian exposure to novel stressors (e.g.,
pesticides) at levels as low as those found in the field. Pesticide
concentrations detected in the field usually do not cause mor-
tality [3], and therefore ecotoxicological studies should also
focus on sublethal responses. Pesticide concentrations found
in the environment have been shown to have negative effects
on growth, development, immune responses, and behavior of
tadpoles [11–13]. Reduced size at metamorphosis can decrease
the fitness of individuals via reduced survival until maturity
[14–16]. Moreover, some of the potential negative effects of
pesticides may become apparent only when present with other
environmental stressors, such as predation [17–20].
The common frog, Rana temporaria, is the most wide-
spread anuran species in Europe, inhabiting various kinds of
environments from northern Spain to subarctic Fennoscandia
* To whom correspondence may be addressed
(mochm@bredband.net).
3164
and to the Ural Mountains in the east [21]. It breeds in early
spring in the shallow water of the littoral zone of lakes and
smaller water bodies. It is an explosive breeder, and the spawn
is aggregated from a few up to several thousands of clutches,
consisting of 700 to 3,000 eggs each [22]. Larval development
from fertilization until metamorphosis takes 40 to 80 d, de-
pending on population origin, water temperature, and com-
petition [22,23]. Because of its abundance, it is an important
species in European ecological communities. This, together
with the biphasic life cycle and permeable skin of amphibians,
makes it potentially a good bioindicator of chemical pollution
[24]. Despite this, there has been little interest in studying the
effects of pesticides in R. temporaria in recent years (see, e.g.,
[25]), even if the literature produced from the 1970s to the
early 1990s is rather extensive (e.g., [26–29]). Moreover, next
to nothing is known about its response to low concentration
levels in the range that can be found in the field. Many am-
phibians, including R. temporaria, breed in ponds in which
the larval development takes place during the spring and sum-
mer months. As much of the pesticide application is likely to
coincide with the aquatic larval phase, tadpoles can be exposed
to pesticides through contaminated surface water after heavy
rains or pesticide spray drift into the breeding ponds adjacent
to fields. Thus, exposure studies during the aquatic phase is a
highly relevant approach. However, amphibians are relatively
long lived, and virtually nothing is known about long-term
effects of pesticide exposure in adult amphibians or at am-
phibian community level.
The aim of this study was twofold: first, to investigate the
potential effects of six commonly used pesticides on survival
and development of R. temporaria tadpoles in acute laboratory
experiments, and, second, to assess the influence of chronic
exposure from fertilization to metamorphosis for the three most
potent pesticides from the acute test at concentrations relevant
to field conditions in Sweden.
Toxicity of six pesticides to tadpoles
Table 1. Summary of selected pesticides and some of their important characteristics. A " azoxystrobin, C " cyanazine, E " esfenvalerate,
MCPA " (4-chloro-2-methylphenoxy) acetic acid, Per. " permethrin, Pir. " pirimicarb, F " fungicide, H " herbicide, I " insecticide
Pesticide Type KOC Log KOW
A F 207–767 2.5
C H 73–413 2.2
E I !200 000 6.2
MCPA H High mobility Low
Per. I 14,000 6.5
Pir. I 54–5,600 1.7
a
Measured field concentrations in Sweden, within the Swedish monitoring program (http://www.mv.slu.se/Vv/Datavskap/dv"program.html).
b
Median lethal concentration.
MATERIALS AND METHODS
Study populations
The frogs forming the parental generation for the experi-
ments were collected from ponds in Tvedöra (55$42%N,
13 $27 % E), southern Sweden (acute test), and Gullsmyra
(60$07%N, 16$56%E), central Sweden (chronic test), in April
2001.
Animal husbandry and laboratory procedures
Four amplexing pairs per population were transported to a
laboratory in Uppsala, where they were artificially crossed
following the procedures (with some modifications [30]) out-
lined by Berger et al. [31]. Artificial crossings were used to
ensure that all eggs from a given clutch were full sibs and that
they were not exposed to pesticides prior to the experiments.
All tests were performed in the laboratory at 15$C with
indoor lighting of 17:7-h light:dark cycle, using reconstituted
soft water made from deionized water and salts (NaHCO3, 48
mg/L; CaSO4·2 H2O, 30 mg/L; MgSO4·7 H2O, 61 mg/L; and
KCl; Merck, Darmstadt, Germany; 2 mg/L [32]). Because of
the highly hydrophobic character of some of the pesticides
(azoxystrobin, esfenvalerate, and permethrin), stock solutions
for these pesticides were prepared with an organic solvent
(acetone). For the same reason, we used enameled containers
to prepare test concentrations and glass jars as experimental
containers for the tadpoles during the experiments. To test for
possible effects of the organic solvent, an additional control
group with the highest concentrations of acetone was used
(acute test: 330 #mol/L; chronic test: 33 #mol/L). In the chron-
ic tests, pesticide concentrations were measured with the gas
chromatography/mass spectrometry methods used in the
Swedish environmental monitoring program for pesticides in
surface water ([33], http://www-mv.slu.se/publ/type.asp) be-
fore and after a 3-d exposure cycle to evaluate precision of
the test solution preparation and the magnitude of degradation
of the active substance. The pesticide concentrations were
close to the targeted level before any tadpoles were subject to
the solution. At the next water change after 3 d, the concen-
trations of azoxystrobin and permethrin were 10 to 20% lower
than at the start, whereas the cyanazine concentration was
roughly the same. Hence, exposure at the intended concentra-
tions was achieved.
Pesticides
We used prior estimations of toxicity (LC50, i.e., the lethal
concentration for 50% of the exposed organisms) to select the
range of concentrations used in this study. Data from fish were
used when no previous studies on amphibians were found. The
Environ. Toxicol. Chem. 25, 2006 3165
LC50b
Field
concn.a Fish Amphibian
0.2 #g/L 0.47–1.6 mg/L —
0.9 #g/L 4 mg/L —
0.02 #g/L 0.13–0.1 #g/L 7.3 #g/L
10 #g/L 180 mg/L (72 h) 3.6 g/L
0.8 #g/L 0.21 #g/L 2.5 #g/L
0.5 #g/L 32–36 mg/L —
choice of pesticides in the study was based on pesticide toxicity
to aquatic organisms, absence of earlier studies, and that at
least one compound from the main types of agricultural pes-
ticides (herbicides, insecticides, and fungicides) should be in-
cluded. We also considered the annual usage and occurrence
in natural waters in Sweden as reported from the Swedish
environmental monitoring program (http://www.mv.slu.se/Vv/
Datavskap/dv"program.html). The properties of the selected
pesticides are presented in Table 1. The results from the acute
tests were used to set up the chronic tests.
Chemical analyses
The concentrations of the pesticides were measured before
and after a 3-d exposure cycle to evaluate precision of the test
solution preparation and the magnitude of degradation of the
active substance. Methods used for the neutral compounds
were the method organisk miljökemi (OMK) 51:5 and for
MCPA ([4-chloro-2-methylphenoxy] acetic acid) the method
OMK 50:8, in-house methods used in the Swedish environ-
mental monitoring program for pesticides in surface water with
accreditation from the Swedish Board of Accreditation (Boro̊s,
Sweden). In brief, neutral compounds were extracted from the
water solution with pesticide-grade dichloromethane (Labscan,
Stillorgan, County Dublin, Ireland) using terbutylazine-D5 (Dr.
Ehrehstorfer, Augsburg, Germany) as surrogate standard added
prior to extraction and ethione (Dr. Ehrehstorfer) as recovery
standard added after extraction. For the analyses of the py-
rethroids, deltamethrin (Dr. Ehrehstorfer) was used as addi-
tional surrogate standard.
For the quantification of MCPA, 2-(2,4,5-trichloro)-pro-
pionic acid (Dr. Ehrehstorfer) was used as surrogate standard
and ethione as recovery standard. The sample was buffered to
pH 8 with a phosphate buffer (Merck) to which tetrabutyl-
ammonium hydrogensulfate (Merck) was added. The aqueous
solution was extracted with dichloromethane containing pen-
tafluoro-benzylbromide (Merck) to convert the phenoxyacids
to pentafluorobenzyl esters prior to quantification.
Quantification was done on an Agilent 6890 gas chro-
matograph coupled to a 5973 mass selective detector (Agilent,
Kista, Sweden).
Acute tests
Each fertilized egg clutch was divided into four parts and
allowed to develop in 1-L jars filled with reconstituted soft
water at 15$C until larvae reached Gosner development stage
25 (G25), when the external gills were completely absorbed
[34]. To evaluate the acute toxicity of the pesticides and
achieve guidance for the design of the subsequent chronic tests,
tadpoles were exposed to azoxystrobin (0.03, 0.13, 0.5 mg/L),
3166 Environ. Toxicol. Chem. 25, 2006
cyanazine (0.75, 3, 12 mg/L), esfenvalerate (0.0003, 0.0013,
0.005 mg/L), MCPA (0.75, 3, 12 mg/L), permethrin (0.002,
0.008, 0.032 mg/L), and pirimicarb (Sigma-Aldrich Sweden
AB, Stockholm, Sweden) (1, 4, 16 mg/L) for 72 h.
To homogenize genetic variation among the four clutches,
they were mixed in a larger container from which five ran-
domly chosen G25 tadpoles (free from visible anomalies) per
pesticide, concentration, and replicate were placed in 1-L glass
jars containing 0.7 L test solution. Each treatment was repli-
cated five times. The control was replicated 15 times. The
same control was used for all pesticides. Tadpoles were fed
boiled spinach (Coop, Solna, Sweden) ad libitum. To keep
temperatures as constant as possible, containers were kept in
two separate water-filled aquaria (blocks) with a constant water
flow through a water-cooling unit. At the termination of the
experiment, tadpoles were killed with the anesthetic tricaine
methane sulfonate (MS 222; Sigma-Aldrich) and preserved in
ethanol for later measurements.
Chronic tests
In the chronic experiment, tadpoles were exposed to two
lower concentrations from fertilization until metamorphosis.
The larvae were exposed to azoxystrobin (0, 1, 10 #g/L),
cyanazine (0, 10, 100 #g/L), and permethrin (0, 0.1, 1 #g/L),
which were the pesticides giving the most interesting responses
in the preceding acute tests. The same control (0) was used
for all pesticides. In the chronic test, 30 randomly selected
eggs from the four families were placed in a 1-L glass jar,
containing test solution (0.7 L), 6 h after fertilization. When
tadpoles had reached G25, one seemingly healthy tadpole from
each jar was randomly selected to remain in the experiment
and reared until metamorphosis in the same conditions as it
was reared in as embryo. Each treatment was replicated 20
times, and the jars were randomly placed in a shelf system.
Hence, each family was replicated five times per treatment.
Every 3 d, the test solutions were renewed, and replicate po-
sition was randomized. During the experiment, the tadpoles
were fed boiled spinach ad libitum. At metamorphosis (approx.
Gosner stage 42), the tadpoles were killed with anesthetic MS-
222 and preserved in ethanol for later measurement.
Response variables
In the acute experiment, mean dry weight, body length, tail
length, and survival were measured. The response variables
in the chronic experiment were body length, tail length, wet
weight, survival, age at metamorphosis (number of days from
fertilization), and growth rate (mg/d; defined as the body
weight at metamorphosis divided by days elapsed between
fertilization and metamorphosis). Wet weight was estimated
by first dabbing the anesthetized tadpole on a paper cloth to
remove excessive water and then measuring it with an ana-
lytical balance to the nearest 0.1 mg before preservation in
ethanol. Body and tail lengths were measured with digital
calipers to the nearest 0.01 mm. Tadpoles within each exper-
imental unit in the acute tests were dried together at 55$C and
weighed with an analytical balance to the nearest 0.01 mg.
Average dry weight was estimated by dividing with the number
of surviving tadpoles in each replicate. Survival rates were
estimated at termination of experiments.
In the chronic test, only the wet-weight measure is graph-
ically presented since all three size measures—body length,
tail length, and dry weight (acute)/wet weight (chronic)—were
M. Johansson et al.
highly correlated (acute test: r " 0.68–0.92; chronic test: r "
0.77–0.83).
Statistical analyses
Analyses of variance and covariance were used to test for
effects of the different pesticide treatments on weight, length
measures and age at metamorphosis, and growth rate until
metamorphosis using PROC MIXED in SAS# [35]. Block
(acute tests) and concentration were treated as fixed main ef-
fects, whereas family (chronic tests) was treated as a random
main effect. Since size and age at metamorphosis are correlated
in R. temporaria [23,36], the age at metamorphosis was in-
cluded as a covariate in the analysis to exclude possible size
effects due to exposure time and not the treatment concentra-
tion.
Survival at termination of experiments was analyzed with
logistic regression using generalized linear models, binomial
errors, and a log link function as implemented in PROC GEN-
MOD in SAS [35].
RESULTS
Chemical analyses
The chemical analyses showed that the starting concentra-
tions were within a few percent of the target concentrations,
and the concentrations at the end of a 3-d period were for
azoxystrobin 87 & 8% (mean and relative standard deviation),
cyanazine 96 & 5%, esfenvalerate 81 & 9%, MCPA 102 &
4%, permethrin 86 & 6%, and pirimicarb 91 & 7% of the
target concentrations.
Acute tests
Cyanazine showed the strongest negative effect on size and
strongly affected all three size endpoints (body length: F3,26
" 10.5, p " 0.0001; tail length: F3,26 " 12.1, p " 0.0001; dry
weight: F3,26 " 12.9, p " 0.0001; Fig. 1), but also pirimicarb
affected tadpole size negatively (tail length: F3,25 " 3.90, p "
0.021; dry weight: F3,25 " 3.43, p " 0.032; Fig. 1f ). Azox-
ystrobin concentration had an effect only in body length (F3,24
" 4.73, p " 0.0099; Fig. 1a), whereas the remaining three
pesticides, esfenvalerate, MCPA, permethrin, and acetone
(control) showed no effects of concentration in any of the size
parameters ( p ' 0.09).
Survival was generally high in all treatment combinations
except in the highest azoxystrobin treatment (0.5 mg/L), which
showed a strong negative effect of concentration ((23,25 " 86.8,
p " 0.0001; Fig. 1a). However, there was a weak but significant
main effect of concentration on survival also in permethrin
((23,25 " 14.2, p " 0.0026; Fig. 1e) and pirimicarb ((23,25 "
14.1, p " 0.0028; Fig. 1f ), whereas there was a nonsignificant
indication of an effect in cyanzine ((23,25 " 6.81, p " 0.078;
Fig. 1b). Tadpoles in treatments with esfenvalerate ((23,25 "
2.79, p " 0.42), MCPA ((23,25 " 4.83, p " 0.18), and the acetone
control ((21,27 " 0.032, p " 0.86) survived equally well across
the entire concentration range (Fig. 1).
Chronic tests
Permethrin was the only pesticide with a significant effect
on size at metamorphosis (body length: F2,46 " 4.46, p " 0.017;
tail length: F2,46 " 5.58, p " 0.0068; wet weight: F2,46 " 7.27,
p " 0.0018), and the metamorphs gradually increased in size
with increasing pesticide concentration (Fig. 2 and Table 2).
The other two pesticides and acetone (control) showed no
Toxicity of six pesticides to tadpoles Environ. Toxicol. Chem. 25, 2006 3167

Fig. 1. Pesticide concentration response curves of body length (dashed-dotted line), tail length (dashed line), dry weight (full line), and survival
(filled circles) after acute exposure of (a) azoxystrobin (0, 0.03, 0.13, 0.5 mg/L), (b) cyanazine (0, 0.75, 3, 12 mg/L), (c) esfenvalerate (0, 0.0003,
0.0013, 0.005 mg/L), (d) MCPA ((4-chloro-2-methylphenoxy) acetic acid) (0, 0.75, 3, 12 mg/L), (e) permethrin (0, 0.002, 0.008, 0.032 mg/L),
and ( f ) pirimicarb (0, 1, 416 mg/L) during 72 h. – $ – dry weight; – · – · $ – · – · body length; – – – $ – – – tail length; $ survival.

effects of concentration in any of the size parameters ( p '
0.20).
Survival was generally high for all three pesticides in all
concentrations (90–100%), and there were no significant pes-
ticide effects on survival (azoxystrobin: (23,25 " 0.49, p " 0.61;
cyanazine: (23,25 " 0.76, p " 0.47; permethrin: (23,25 " 0.35, p
" 0.71; Fig. 2 and Table 2).
Age at metamorphosis was not affected by any of the treat-
ments ( p ' 0.078; Fig. 2 and Table 2).
As indicated in the analyses of size and age at metamor-
phosis, in which there were effects on size but not on age of
the permethrin concentration, there was a weak but significant
positive effect of permethrin concentration on growth rate
(F2,50.1 " 3.49, p " 0.038; Fig. 2c and Table 2). No other
treatment showed any effect on growth rate ( p ' 0.15; Fig.
2 and Table 2).
DISCUSSION
Many of the acute toxicity treatments showed strong neg-
ative effects on growth and survival in the highest pesticide
concentration: In the azoxystrobin (not significant) and pirim-
icarb treatments (Fig. 1a and f ), the upper end of the con-
centration range accounted for most of the variation in size.
Likewise, the highest concentration accounted for most of the
effect on tadpole survival in azoxystrobin and permethrin (Fig.
1a and e). However, in the cyanazine (Fig. 1b) treatment, which
had the strongest effect on size, tadpole size decreased more
or less linearly with increasing concentration. The reason why
only the highest concentration had a negative effect in many
of the treatments is because we expected a higher toxicity.
Even if an effect, as in cyanazine, for the entire concentration
range is desirable, these results give a valuable indication about
the no-observed-effects concentration.
Also, the esfenvalerate and MCPA treatments indicate a
lower acute toxicity than expected, as no effects were observed
for the entire concentration range. At least for esfenvalerate,
this is surprising when compared with previous studies on
amphibians [37] in which mortality were observed at lower
concentrations than used in this study. As indicated by the
MCPA LC50 (120 h) in Xenopus (3.6 g/L) [38], very high
concentrations may be needed to observe any effect. Thus, a
lack of response for this pesticide was rather expected, and
we consider exposure at higher concentrations irrelevant in a
study with an ecological purpose. In the case of esfenvalerate
(and also permethrin) with highly hydrophobic properties, the
dosage may pose problems to an accurate exposure, but pos-
sible water chemistry adjustments lie beyond the technical
abilities of this study.
Permethrin was the only pesticide treatment that showed
any effects at exposure to field-relevant concentrations. How-
ever, the response was counterintuitive since the size at meta-
morphosis gradually increased with increasing concentration.
The response to an increasing concentration of a chemical
stressor is expected to be the opposite (e.g., [11]) as in the
3168 Environ. Toxicol. Chem. 25, 2006
Fig. 2. Pesticide concentration response curves of tadpole growth
from fertilization to metamorphosis (mg/d; diamonds), age at meta-
morphosis (days; squares), wet weight at metamorphosis (g; triangles),
and survival after exposure at field relevant concentrations of (a)
azoxystrobin (0, 1, 10 #g/L), (b) cyanazine (0, 10, 100 #g/L), and
(c) permethrin (0, 0.1, 1 #g/L). # " growth; □ " age; ! " weight;
$ " survival.
acute test, in which mortality increased significantly in the
highest concentration (32 #g/L). However, the usage of per-
methrin is relatively low (2.5 annual tons) in Sweden as com-
pared to for example, MCPA (439 annual tons)—and as it
binds hard to soil (KOC " 14,000), it is unlikely to be found
in concentrations of 32 #g/L in the field. The increased size
in the chronic permethrin treatment is hard to explain without
further experiments. Comparable studies on chronic exposure
of permethrin and other pyrethroids are more or less nonex-
istent; however, acute studies are more numerous. Two studies
on Rana catesbiana tadpoles report highly differing results of
permethrin toxicity with LC50s (96 h) of 115 #g/L [39] and
M. Johansson et al.
7.03 mg/L [40]. Both studies used tadpoles that were signif-
icantly larger than in the present study (LC80 [72 h] " 30 #g/
L; Fig. 1e), which may be indicative of the different LC values.
A Slovakian study that exposed R. temporaria tadpoles to
cypermethrin, another pyrethroid pesticide, revealed a higher
toxicity in cypermethrin than permethrin, as the detected
LC50s (48 h) were as low as 6.5 #g/L [41]. Also, sublethal
effects, EC50 (96 h) " 10 #g/L, in several Ranid species were
reported in a study by Berril et al. [42], and in concert with
the result of the present study, one can conclude the acute
toxic effects of permethrin in amphibians at very low water
concentrations.
No significant effect in any response variable was observed
in the other chronic pesticide treatments. Hence, the general
conclusion is that azoxystrobin, cyanazine, and permethrin
have no or small effects on the studied tadpole life-history
characteristics within the chronic test’s concentration range.
The absence of response is likely related to the concentration
level, but, then again, these higher concentrations are unlikely
to be found in the field.
Despite the fact that these pesticides at the tested concen-
trations had little or no effect, it cannot be ruled out that they
may have negative effects on amphibians during other circum-
stances (i.e., in the field). For example, field studies and me-
socosm experiments pose a much broader and ecologically
relevant stress regime, including, predators and competition
for food, and may reveal direct and indirect pesticide toxicity
that would have been undetected in laboratory studies of a
single species [10,43,44]. Also, laboratory studies with a more
complex treatment structure have shown that multiple stressors
may have synergistic or cumulative effects on tadpoles. Sev-
eral examples of pesticides have shown synergistic detrimental
effects on tadpoles when combined with a predatory cue, such
as Carbryl [18,19], Malathion [17,20], and Roundup# (Mon-
santo, St. Louis, MO, USA) [20]. Moreover, weakened immune
response in relation to pesticide exposure has been shown by
Kiesecker [45], who revealed a connection between pesticide
exposure and trematode-related hind-limb deformities in Rana
sylvatica in the field, and Christin [12], who showed reduced
lymphocyte formation during a nematode infection in juvenile
Rana pipiens when exposed to a mixture pesticides at field-
relevant levels. Both these studies conclude that atrazine, a
triazine pesticide and chemically related to cyanazine, could
be responsible for the adverse effects. However, a key issue
in these studies is the exposure to multiple stressors, which
was necessary to provoke the adverse effect. Hence, because
of the chemical resemblance with atrazine, a similar response
of cyanzine at ecologically relevant levels could have appeared
if additional stressors of some kind had been present. Also,
competition between individuals as well as hydroperiod length
can interact with a pesticide and significantly reinforce the
environmental stress level [44].
The interpretations may seem obscured by the fact that the
tadpoles used in the acute and chronic tests, for logistic rea-
sons, had different origins. However, we do not think so. First,
the purpose of the acute test was mainly to select pesticides
for the chronic tests and to chose the chronic exposure dose.
Second, although the tadpoles used for the acute tests come
from a region with more intensive agriculture than the tadpoles
used for the chronic tests, the site from where the frogs were
collected is situated in a large military training field with no
agriculture or use of pesticides. Analysis of surface and
groundwater from the area confirms that there is no pesticide
Toxicity of six pesticides to tadpoles Environ. Toxicol. Chem. 25, 2006 3169

Table 2. Chronic experiment mean and standard error (SE) estimates for each endpoint (at metamorphosis). Weight " wet weight, Age " age
at metamorphosis, Growth " growth rate (age at metamorphosis divided by wet weight). A " azoxystrobin, C " cyanazine, P " permethrin,
Ac " acetone control

Pesticide Tail length Body length Weight Age (SE) Growth

concn. (#g/L) (SE) (mm) (SE) (mm) (SE) (g) (d) (SE) (mg/d) Survival

A1 35.27 (0.77) 17.24 (0.23) 0.91 (0.039) 47.7 (1.7) 18.2 (1.2) 0.9
A 10 34.37 (0.77) 17.02 (0.23) 0.88 (0.039) 48.7 (1.7) 18.0 (1.2) 0.9
C 10 34.96 (0.72) 17.33 (0.19) 0.91 (0.036) 49.5 (1.2) 18.2 (0.8) 1
C 100 35.99 (0.78) 17.59 (0.21) 0.92 (0.037) 48.5 (1.3) 19.1 (0.9) 1
P 0.1 35.92 (0.71) 17.29 (0.22) 0.92 (0.039) 48.6 (1.6) 18.5 (1.1) 0.95
P1 37.34 (0.69) 17.98 (0.22) 1.03 (0.038) 50.5 (1.5) 20.0 (1.0) 0.95
Control 34.60 (0.75) 17.14 (0.23) 0.84 (0.038) 49.5 (1.7) 16.5 (1.0) 0.95
Ac 33 #mol/L 34.51 (0.76) 17.05 (0.21) 0.87 (0.038) 52.5 (1.4) 16.9 (1.0) 0.95

contamination. The main source of pesticides to both Tvedöra
and Gullsmyra is atmospheric deposition, and this is likely
higher in Tvedöra than in Gullsmyra ([46]; http://www-mv.
slu.se/publ/type.asp). Therefore, if a difference in tolerance
has developed, it is more likely that the Tvedöra population
would be the one having the highest tolerance. Hence, the
main problem for the interpretation would be that we selected
too high of doses for the chronic tests. Considering the rela-
tively low responses found in the chronic tests, this was ob-
viously not the case.
In conclusion, exposure to these pesticides at concentrations
that are likely to exist in nature has low or no effect on the
response variables used in this study. Nevertheless, more stud-
ies employing finer tools to detect pesticide effects at the sub-
lethal level and utilizing a multiple-stressor experimental setup
to mimic natural situations are warranted.
Acknowledgement—We thank Jacob Höglund, Anssi Laurila, Maarit
Pahkkala, Fredrik Söderman, and Beatrice Lindgren. The experiments
were conducted with permission (C62/1) from the Ethical Committee
of Uppsala University. Our research was supported by the Swedish
Natural Research Council, Academy of Finland, and the University
of Helsinki Science Foundation.
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