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

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 MERILA¨†

†Department of Population Biology Evolutionary Biology Centre, Uppsala University, Norbyva¨gen 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), permethrin, 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

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, depending on population origin, water temperature, and competition [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 amphibians, including R. temporaria, breed in ponds in which the larval development takes place during the spring and summer 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 amphibian 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.

INTRODUCTION

Agricultural practices affect natural habitats in several ways, such as through land conversion, increased fragmentation, and agrochemical contamination [1–5], and have therefore 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 feminization 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 populations, and Relyea [10] detected significant disruption in species richness in aquatic communities including both secondary 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 mortality [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 widespread anuran species in Europe, inhabiting various kinds of environments from northern Spain to subarctic Fennoscandia * To whom correspondence may be addressed ([email protected]). 3164

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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 A C E MCPA Per. Pir. a b

Type

KOC

Log KOW

F H I H I I

207–767 73–413 !200 000 High mobility 14,000 54–5,600

2.5 2.2 6.2 Low 6.5 1.7

Field concn.a 0.2 0.9 0.02 10 0.8 0.5

#g/L #g/L #g/L #g/L #g/L #g/L

LC50b Fish

Amphibian

0.47–1.6 mg/L 4 mg/L 0.13–0.1 #g/L 180 mg/L (72 h) 0.21 #g/L 32–36 mg/L

— — 7.3 #g/L 3.6 g/L 2.5 #g/L —

Measured field concentrations in Sweden, within the Swedish monitoring program (http://www.mv.slu.se/Vv/Datavskap/dv"program.html). Median lethal concentration. MATERIALS AND METHODS

Study populations The frogs forming the parental generation for the experiments were collected from ponds in Tvedo¨ra (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]) outlined 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 chronic 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) 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. 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 concentrations 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 concentrations 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

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 pesticides (herbicides, insecticides, and fungicides) should be included. 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 miljo¨kemi (OMK) 51:5 and for MCPA ([4-chloro-2-methylphenoxy] acetic acid) the method OMK 50:8, in-house methods used in the Swedish environmental monitoring program for pesticides in surface water with accreditation from the Swedish Board of Accreditation (Boro˚s, 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 pyrethroids, deltamethrin (Dr. Ehrehstorfer) was used as additional surrogate standard. For the quantification of MCPA, 2-(2,4,5-trichloro)-propionic 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 tetrabutylammonium hydrogensulfate (Merck) was added. The aqueous solution was extracted with dichloromethane containing pentafluoro-benzylbromide (Merck) to convert the phenoxyacids to pentafluorobenzyl esters prior to quantification. Quantification was done on an Agilent 6890 gas chromatograph 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),

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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 randomly 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 replicated 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 position 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 MS222 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 analytical 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 experimental 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 graphically presented since all three size measures—body length, tail length, and dry weight (acute)/wet weight (chronic)—were

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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 effects, 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 included as a covariate in the analysis to exclude possible size effects due to exposure time and not the treatment concentration. 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 GENMOD in SAS [35]. RESULTS

Chemical analyses The chemical analyses showed that the starting concentrations 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 ). Azoxystrobin 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

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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 pesticide 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 treatments ( p ' 0.078; Fig. 2 and Table 2). As indicated in the analyses of size and age at metamorphosis, 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 negative effects on growth and survival in the highest pesticide concentration: In the azoxystrobin (not significant) and pirimicarb treatments (Fig. 1a and f ), the upper end of the concentration 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 possible 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. However, the response was counterintuitive since the size at metamorphosis 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

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Fig. 2. Pesticide concentration response curves of tadpole growth from fertilization to metamorphosis (mg/d; diamonds), age at metamorphosis (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 permethrin is relatively low (2.5 annual tons) in Sweden as compared 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 nonexistent; 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

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7.03 mg/L [40]. Both studies used tadpoles that were significantly 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 concentrations had little or no effect, it cannot be ruled out that they may have negative effects on amphibians during other circumstances (i.e., in the field). For example, field studies and mesocosm 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. Several 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# (Monsanto, 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 fieldrelevant 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 reasons, 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

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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 concn. (#g/L)

Tail length (SE) (mm)

Body length (SE) (mm)

Weight (SE) (g)

Age (SE) (d)

Growth (SE) (mg/d)

Survival

A1 A 10 C 10 C 100 P 0.1 P1 Control Ac 33 #mol/L

35.27 (0.77) 34.37 (0.77) 34.96 (0.72) 35.99 (0.78) 35.92 (0.71) 37.34 (0.69) 34.60 (0.75) 34.51 (0.76)

17.24 (0.23) 17.02 (0.23) 17.33 (0.19) 17.59 (0.21) 17.29 (0.22) 17.98 (0.22) 17.14 (0.23) 17.05 (0.21)

0.91 (0.039) 0.88 (0.039) 0.91 (0.036) 0.92 (0.037) 0.92 (0.039) 1.03 (0.038) 0.84 (0.038) 0.87 (0.038)

47.7 (1.7) 48.7 (1.7) 49.5 (1.2) 48.5 (1.3) 48.6 (1.6) 50.5 (1.5) 49.5 (1.7) 52.5 (1.4)

18.2 (1.2) 18.0 (1.2) 18.2 (0.8) 19.1 (0.9) 18.5 (1.1) 20.0 (1.0) 16.5 (1.0) 16.9 (1.0)

0.9 0.9 1 1 0.95 0.95 0.95 0.95

contamination. The main source of pesticides to both Tvedo¨ra and Gullsmyra is atmospheric deposition, and this is likely higher in Tvedo¨ra 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 Tvedo¨ra 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 relatively low responses found in the chronic tests, this was obviously 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 studies employing finer tools to detect pesticide effects at the sublethal level and utilizing a multiple-stressor experimental setup to mimic natural situations are warranted. Acknowledgement—We thank Jacob Ho¨glund, Anssi Laurila, Maarit Pahkkala, Fredrik So¨derman, 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. REFERENCES 1. Hecnar SJ. 1995. Acute and chronic toxicity of ammonium-nitrate fertilizer to amphibians from southern Ontario. Environ Toxicol Chem 14:2131–2137. 2. Davidson C, Shaffer HB, Jennings MR. 2002. Spatial tests of the pesticide drift, habitat destruction, UV-B, and climate-change hypotheses for California amphibian declines. Conserv Biol 16: 1588–1601. 3. Hayes TB, Collins A, Lee M, Mendoza M, Noriega N, Stuart AA, Vonk A. 2002. Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proc Natl Acad Sci U S A 99:5476–5480. 4. Lehtinen RM, Galatowitsch SM, Tester JR. 1999. Consequences of habitat loss and fragmentation for wetland amphibian assemblages. Wetlands 19:1–12. 5. Vos CC, Chardon JP. 1998. Effects of habitat fragmentation and road density on the distribution pattern of the moor frog Rana arvalis. J Appl Ecol 35:44–56. 6. Green DM. 2003. The ecology of extinction: Population fluctuation and decline in amphibians. Biol Conserv 111:331–343. 7. Houlahan JE, Findlay CS, Schmidt BR, Meyer AH, Kuzmin SL. 2000. Quantitative evidence for global amphibian population declines. Nature 404:752–755. 8. Blaustein AR, Wake DB, Sousa WP. 1994. Amphibian declines— Judging stability, persistence, and susceptibility of populations to local and global extinctions. Conserv Biol 8:60–71. 9. Sparling DW, Fellers GM, McConnell LL. 2001. Pesticides and amphibian population declines in California, USA. Environ Toxicol Chem 20:1591–1595. 10. Relyea RA. 2005. The impact of insecticides and herbicides on

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