Arch Environ Contam Toxicol (2009) 56:557–565 DOI 10.1007/s00244-008-9201-y

Effects of Copper Sulfate on Growth, Development, and Escape Behavior in Epidalea calamita Embryos and Larvae E. Garcı´a-Mun˜oz Æ F. Guerrero Æ G. Parra

Received: 6 March 2008 / Accepted: 5 July 2008 / Published online: 26 August 2008 Ó Springer Science+Business Media, LLC 2008

Abstract Epidalea calamita embryos at Gosner stages 3 and 19, and larvae at Gosner stage 25, were exposed to different copper sulfate concentrations, ranging from 0.05 to 0.40 mg Cu L-1, in 96-h acute toxicity tests. Embryonic and larval mortality, development, growth, and larval escape behavior were evaluated. LC50 at 96 h obtained at Gosner stages 3, 19, and 25 were 0.22, 0.08, and 0.11 mg Cu L-1, respectively. Embryonic and larval developments were delayed after 96 h of copper sulfate exposure. Growth was also affected and individuals in control treatments grew to twice the size of those exposed to copper concentrations over 0.2 mg Cu L-1 during the experiments initiated at Gosner stage 19. Escape behavior was altered after 96 h of copper sulfate exposure; larvae showed shorter distances moved and abnormal displacement types. However, after 4 days of recovery process, most of the larvae showed normal escape behavior. For amphibians that develop in temporary wetlands, increased development time, lower size, and altered escape behavior might have repercussions on the number of individuals that can successfully complete metamorphosis and, consequently, on recruitment.

Typical procedures used in agricultural areas include the use of different toxic substances that presumably may have significant consequences on the surrounding ecosystem. Most of these substances have a negative effect on aquatic ecosystems, and the degree of impact depends on their E. Garcı´a-Mun˜oz
F. Guerrero
G. Parra (&) Departamento de Biologı´a Animal, Biologı´a Vegetal y Ecologı´a, Universidad de Jae´n, Campus de las Lagunillas s/n, 23071 Jae´n, Spain e-mail: [email protected]

chemical characteristics [e.g., persistence time and solubility (Rand et al. 1995)]. In recent years, ecotoxicological studies have substantially increased efforts to understand the effect of intensive agricultural pollution on aquatic ecosystems (Van Dam et al. 1998). The use of acute toxicity data is essential for the ecotoxicological assessment of chemicals (Hall et al. 1998). However, sublethal and chronic effects associated with exposure may be more significant in mediating ecological effects and can be used to detect effects on growth, development, and reproduction (Linder and Grillitsch 2000; Marcial et al. 2005). Moreover, individual response studies (e.g., mortality, growth rate, behavior) are essential to an integrative approach for understanding multiple stressor impacts on natural populations (Sih et al. 2004). In southern Spain different toxic substances are used in intensive olive tree agriculture, including copper sulfate, used to control Cycloconium oleaginum (Fungi) during spring and autumn (Junta de Andalucı´a 2008). This metal has two oxidation states: Cu1+ and Cu2+. The Cu2+ ion is the most environmentally relevant to aquatic systems and is generally considered the most toxic form to aquatic life (Hall et al. 1998). Exposure to high concentrations of copper can affect populations and individuals at morphological, physiological, biochemical, or genetic levels (Troncoso et al. 2000), so toxicological studies with different species present in wetlands which are surrounded by this kind of agriculture are needed. Amphibian populations are sensitive to pollutants not only during the terrestrial adult phase, but also during aquatic embryonic and larval stages (Murphy et al. 2000). Many amphibian species breed opportunistically in a range of aquatic habitats, some of which are embedded in agricultural landscapes. Consequently, their eggs and larvae may be exposed to environmental contaminants at some

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time during development (Bridges and Boone 2003). Moreover, amphibians absorb many toxic substances through the epithelium, so eggs and newly metamorphosed amphibians are more sensitive to poor water quality than later age classes (Vitt et al. 1990). Because of their sensitivity to habitat degradation, amphibians have been regarded as bioindicators of aquatic and agricultural ecosystems (Schuytema and Nebeker 1999; Pollet and Bendell-Young 2000; Marco et al. 2001; Tejedo 2003) and have been used as typical test animals in evaluating the effects of chemicals on aquatic and agricultural ecosystems (Cooke 1973, 1977; Sundaram 1995). Recent studies have hypothesized that chemical contaminants are, in some way, responsible for amphibian declines (Sparling et al. 2001; Marco 2002; Blaustein et al. 2003; Greulich and Pflugmache 2003; Davidson 2004; Relyea 2005). The consequences of chemical stressors, such as pesticides, heavy metals, acidification, and nitrogen-based fertilizers, on amphibians can be either lethal or sublethal (Sparling et al. 2000). The sublethal effects of contaminants on amphibians include hampered growth, developmental delays, malformations, disruption of metamorphosis, and behavioral changes, which could lead to developmental and behavioral abnormalities (Bridges 1997, 2000; Diana et al. 2000; Herkovits et al. 2002; Bridges and Boone 2003). Our main objective was to examine the effects of copper sulfate on the survival, development, growth, and behavior of natterjack toad (Epidalea calamita, Laurenti 1768) embryos and larvae. This species is widely distributed in the Iberian Peninsula and uses small and temporary wetlands for reproduction (Tejedo and Reques 1997). Given that in Spain more than 60% of wetlands have disappeared (Casado and Montes 1995), it is important to assess the potential use of remaining wetlands which could be degraded as a result of agricultural pollution.

Materials and Methods Test Organisms Four E. calamita egg clutches (*500 eggs) were collected in ‘‘El Ardal,’’ an inland temporary shallow ecosystem, situated in the Alto Guadalquivir region (Guerrero et al. 2006), southern Spain. This wetland has no known history of pollution. Egg masses were brought into the laboratory immediately after collection and placed in an aquarium (20 L) with water from a wetland without any known pollution history (pH, 7.2–7.8; alkalinity, 170–250 mg L-1). The eggs were maintained at 20°C on a 12-h light: 12-h dark cycle in a temperature-controlled chamber. The eggs and resulting larvae were allowed to develop to Gosner (1960) stages 3, 19, and 25 before acute toxicity testing. A

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subsample of the individuals used in the following toxicity experiments at Gosner stages 19 and 25 (n = 30, respectively) was used to calculate initial total length (mean values ± SE: Gosner stage 19, 4.52 ± 0.28 mm; Gosner stage 25, 6.55 ± 0.41 mm). Acute Toxicity Tests Copper sulfate (CuSO4 5H2O; Sigma) was used to prepare concentrations for use in acute toxicity tests. To assure the accuracy of the dose, copper concentrations in each experimental vessel were analyzed with a photometer (Filterphotometer PF11; Macherey-Nagel) following a colorimetric technique (detection limit: 0.04 mg Cu2+ L-1). Copper was not detected in wetland water (pH, 7.2–7.8; alkalinity, 170–250 mg L-1), and this water was used to prepare the different test concentrations. Final test concentrations were as follows: embryo tests at Gosner stage 3 (0.10, 0.20, 0.30, and 0.40 mg Cu L-1); embryo tests at Gosner stage 19 (0.05, 0.08, 0.10, and 0.20 mg Cu L-1); and larval test at Gosner stage 25 (0.07, 0.10, 0.13, and 0.16 mg Cu L-1). Different concentrations were used at specific stages in order to obtain the specific mortality rates necessary for LC50 calculations (see below). Ten individuals (either embryos or larvae) were placed in each glass vessel (15-cm [) filled with 1000 mL of test solution. Six replicates for each concentration, including control treatments (without copper), were maintained in a temperature-controlled chamber at 20°C and with a 12-h light:12-h dark cycle. Individuals were visually examined once every 24 h, and dead embryos and larvae were removed. Embryos were considered dead when they turned gray and larvae were considered dead when they did not respond to gentle touching. The experiments concluded after 96 h of exposure. LC50 values were estimated using linear functions relating log-transformed copper concentration to probittransformed mortality (Abel and Axiak 1991). To obtain the parameters of these lineal functions with statistic significance, a wide range of larval mortality values is necessary. As a consequence of the different mortality range observed in the developmental stage test, a different range of copper concentrations was used. Developmental Stage and Growth At the end of the experimental period (96 h), embryonic and larval stages and larval total length were recorded for each surviving individual. Individuals were placed in petri dishes with a 1-cm depth of water. Then they were photographed individually against a grid paper using a Canon S40 camera. Images were downloaded onto a computer and magnified onscreen. Total lengths (from snout to tail tip) were measured using the ruler function (accurate to 0.01 mm) in Adobe

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Photoshop 5.5. Stage differences among concentrations were analyzed with the nonparametric Kruskal-Wallis test. Larval length differences were analyzed with one-way ANOVA and Tukey post hoc test. Analyses were performed on logtransformed larval length data.

100

MORTALITY (%)

80

Escape Behavior Larvae at Gosner stage 25 were used in escape behavior tests. Initially, larvae were exposed to 0, 0.06, 0.08, and 0.10 mg Cu L-1 during 96 h. During this period (exposure phase) no behavior analysis was carried out. After this period, surviving larvae were placed in 40 9 30 9 10-cm trays with clean water and then another 96-h period (recovery phase) started. During the recovery phase larvae were visually examined at the beginning of this phase (time 0) and every 24 h during the 96-h recovery phase. The following traits were recorded in each observation during the recovery phase. –

–

–

Number of stimuli: A small stimulus with a needle at the tail base was given to each larva until displacement was obtained. The number of stimuli necessary to obtain this displacement was recorded. Distance moved: Once larval displacement was obtained, the distance moved was computed using grid paper under the recovery tray. Movement type: Once larval displacement was obtained, each movement type was registered and differentiated.

Two-way repeated-measures ANOVAs were used to confirm differences in number of stimuli and distance moved throughout recovery processes, using the time as one factor (with five levels: 0, 24, 48, 72, and 96 h) and the concentration (with four levels: control and 0, 0.06, 0.08, and 0.10 mg Cu L-1) as the other. In order to analyze the possible impact of larval size on the distance moved, the residuals obtained after performing the regression of distance moved against total length were analyzed. A chi-square analysis of the contingency table was used to confirm differences in displacement type. The contingency table was set up with the different displacement types as column data and time in recovery as raw data. SPSS v.15.01 was used for statistical analysis of data at the 5% significance level.

60

40

Gosner stage 3 Gosner stage 19 Gosner stage 25

20

0 0.0

0.1

0.2

0.3

0.4

0.5

-1

CONCENTRATION (mg Cu L ) Fig. 1 Epidalea calamita embryonic and larval mortality obtained with the acute toxicity tests after 96 h of exposure to different copper concentrations developed at different development stages (Gosner stages 3, 19, and 25)

Table 1 Summary of regression parameters obtained in the acute toxicity tests on Epidalea calamita embryos and larvae exposed to different copper concentrations, LC50 values, and 95% confident limits (CL) Gosner stage

Regression parameter b

F

n

p

7.19

3.26

145.76

21

\0.05

0.22 (0.35–0.13)

19

9.26

3.93

21.20

16

\0.05

0.08 (0.12–0.05)

25

10.79

5.98

92.15

28

\0.05

0.11 (0.14–0.08)

3

a

LC50 (CL) (mg Cu L-1)

Note: Regression parameters (y = a + bx) were obtained with logtransformed copper concentration (x) vs. mortality expressed in probit (y)

copper concentration and mortality showed a typical sigmoid form (Fig. 1). After 96 h total mortality resulted at concentrations higher than 0.30 mg Cu L-1 for Gosner stage 3 and higher than 0.20 mg Cu L-1 for Gosner stages 19 and 25. The LC50’s values indicated that individuals at Gosner stages 19 and 25 appeared to be more sensitive to copper than individuals at Gosner stage 3 (Table 1). Development Stages and Growth

Results Larval Mortality No mortality occurred in any control vessels during the 96h experimental period. Moreover, during the first 48 h of exposure no mortality occurred among Gosner stage 3 embryos. Curves describing the relationships between

E. calamita individuals exposed to copper sulfate showed delayed development (Fig. 2). Among embryos at Gosner stages 3 and 19 significant differences were found between the stages reached by larvae in control treatments and those reached by larvae from other treatments (Kruskal–Wallis test, v2 = 21.11, df = 3, and p\0.05 and v2 = 23.809, df = 3, and p\0.05, respectively, in embryos at Gosner stages 3 and

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14

A

25

A 12 10

20

8 15

a

6 10

4

5

2 CONTROL 0.10

0.20

0

0.30

ab

CONTROL

0.10

b

b

0.20

0.30

14

B

B 12

LENGTH(mm)

GOSNER STAGE

25 20 15 10

a

10 b

8

b

b b

6 4 2

5

0 CONTROL

0.05

0.08

0.10

0.15

CONTROL 0.05

0.08

0.10

0.20

14

C

25

12

a

C

ab abc

10

bc

20

c

8 6

15

4 10

2 5

0 CONTROL 0.07 CONTROL 0.07

0.10

0.13

0.16

CONCENTRATION (Cu mg L-1)

0.10

0.13

0.16 -1 CONCENTRATION (Cu mg L )

Fig. 2 Gosner stage reached at the end of the exposure period (96 h) by Epidalea calamita individuals that started the acute toxicity tests at Gosner stage 3 (a), Gosner stage 19 (b), and Gosner stage 25 (c). Dotted line indicates initial Gosner stage

Fig. 3 Total length (mean ± SE) reached at the end of the exposure period (96 h) by Epidalea calamita individuals that started the acute toxicity tests at Gosner stage 3 (a), 19 Gosner stage (b), and Gosner stage 25 (c). Different letters denote statistical differences. Dotted line indicates initial total length

19,). The delay in development is higher at the highest concentrations in both embryo tests. In the experiments initiated at Gosner stage 25 no statistical differences were found (Kruskal–Wallis test, v2 = 3.843, df = 4, p = 0.428).

E. calamita larval growth was affected by copper sulfate exposure (Fig. 3). Control treatments showed the highest mean larval sizes at the end of all the experiments. Analyses of variance indicated that there were significant

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561

12

N° STIMULI

control 0.06 0.08 0.10

2

1

DISTANCE MOVED (cm)

3

10 CONTROL 0.06 0.08 0.10

8 6 4 2 0

0

24

48

72

0

96

24

48

72

96

TIME IN RECOVERY (h)

TIME IN RECOVERY (h) Fig. 4 Number of stimuli (mean ± SE) necessary to obtain a displacement during the recovery processes (from 0 to 96 h) in Epidalea calamita larvae that came from different origin treatments (control and 0.06, 0.08, and 0.01 mg Cu L-1)

Fig. 5 Distance moved (mean ± SE) after a stimulus during the recovery processes (from 0 to 96 h) by Epidalea calamita larvae that came from different origin treatments (control and 0.06, 0.08, and 0.10 mg Cu L-1)

differences between exposure levels for all stages (one-way ANOVA: Gosner stage 3, F3,19 = 7.66, p \ 0.05; Gosner stage 19, F4,31 = 44.45, p \ 0.05; Gosner stage 25, F4,19 = 5.79, p \ 0.05).

observed between distance moved and larval size (r = 0.12, p = 0.5429, p = 0.46; n = 40). The recovery process was tested using a two-way repeated-measures ANOVA which confirmed differences in distance moved in larvae at the beginning versus the end of the recovery period and in all treatments with interaction (Table 2). Once the displacement was obtained, larval movements were differentiated and the following categories were classified: type 1, normal movement in straight line; type 2, abnormal movement in zigzag; and type 3, abnormal movement in circles. Larvae from control treatments always presented displacement type 1. As the recovery time increased, the percentage of individuals within the displacement type 3 decreased (Fig. 6), and completely disappeared after 96 h in recovery. The most frequent displacement type at the end of the recovery period was type 1, irrespective of the treatment from which the larvae had come. The chi-square analysis of contingency table indicated that the displacement type shown by the larvae was dependent on the time in recovery (0.06 mg Cu L-1, v2 = 23.12, df = 8, p = 0.003; 0.08 mg Cu L-1, v2 = 20.32, df = 8, p = 0.009; 0.10 mg Cu L-1, v2 = 19.05, df = 8, p = 0.015). Therefore the recovery processes were corroborated.

Escape Behavior No mortality occurred during recovery phase (96 h). All larvae from control treatments needed just one stimulus to react, while the larvae from the other treatments needed more stimuli (Fig. 4). At the end of 96 h in recovery, all larvae from all treatments reacted to the first stimulus (Fig. 4). The recovery process was tested using a two-way repeatedmeasures ANOVA. The results confirm differences between the number of stimuli necessary to obtain displacement in larvae at the beginning and at the end of the recovery phase and in all treatments with interaction (Table 2). Larvae from control treatments always reached the maximum distance moved (Fig. 5). No relation was Table 2 Summary of the two-way repeated-measures ANOVA results, for the effect of time in recovery and copper concentration on (A) number of stimuli necessary to obtain displacement and (B) distance moved during the escape behavior tests (A) No. of stimuli

(B) Distance moved

F-value

p-value

F-value

p-value

Concentration

15.353

0.000

173.676

0.000

Time

24.145

0.000

47.104

0.000

2.774

0.003

8.393

0.000

Main effect

Interaction Concentration time

Discussion Toxic metals such as cadmium, lead, copper, and zinc have been detected regularly in the drainage basins of the main Spanish rivers (Tajo, Guadalquivir, and Ebro rivers; MMA 2000) owing to industrial and agricultural activity. Moderately polluted Spanish rivers show high copper

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Fig. 6 Percentage of Epidalea calamita larvae with different dis-c placement type in each origin treatment (control and 0.06, 0.08, and 0.10 mg Cu L-1) during the recovery processes. Type 1, normal movement in straight line (white bars); type 2, abnormal movement in zigzag (light-gray bars); type 3, abnormal movement in circles (darkgray bars)

100

0h

80 60 40

-1

123

20 0 100

24 h

80 60 40 20

DISPLACEMENT TYPE (% LARVAE)

concentrations (30–60 mg L ) (Armengol et al. 1993). The copper concentration measured in 2005 in the Guadalquivir basin ranged from 0.001 to 0.007 mg Cu L-1 (Mendiguchı´a 2005). In the upper zone of the Guadalquivir basin, where olive cultivation represents 78% of the total agriculture area, some wetlands surrounded by this human activity have shown cooper concentrations around 0.04 mg Cu L-1 (Garcı´a-Mun˜oz, unpublished data). It is interesting to note that the regular dose used in olive cultivation is in the range of 1875–3750 mg Cu L-1 (De Lin˜a´n 1997). Taking this into account, copper currently poses a high risk to E. calamita in wetlands where this species breeds and that are surrounded by this type of intensive agriculture. The results obtained in this study show that E. calamita embryos and tadpoles are sensitive to copper sulfate exposure. The lowest LC50-96 h obtained in this study (0.08 mg Cu L-1) is similar to the LC50-96 h obtained in the copepod Arctodiaptomus salinus [LC50-96 h = 0.06 mg Cu L-1 (Parra et al. 2005)], but lower than the copper resistance obtained in other aquatic organisms, such as the rainbow trout [LC50-96 h = 0.88 mg Cu L-1 (Bridges et al. 2002)] or the water flea [LC50-96 h = 0.30 mg Cu L-1 (Khangarot and Battish 1994)]. Available toxicity literature on amphibians exposed to metals indicates that copper sulfate LC50-96 h values calculated in tadpoles and embryos ranged from 5.38 to 0.04 mg Cu L-1 (Linder and Grillitsch 2000). Our results suggest that E. calamita larvae are among the most sensitive species, while the embryonic phase showed a slightly higher tolerance, probably associated with protection by egg envelope. That could be the reason why we had to use a wider range of copper concentrations in the embryo experiments. Ra¨sa¨nen et al. (2003) reported that the jelly envelope is not the most important means of protecting R. temporaria embryos from UV-B radiation, as other factors are responsible for the high UV-B radiation tolerance of embryos. However, lower sensitivity to environmental pollutants in embryos with a jelly envelope has also been found (Marquis et al. 2006). Copper sulfate exposure induces negative effects on development. At the end of the experiments initiated at Gosner stage 19, all larvae from control treatments reached Gosner stage 24, while the maximum concentration tested (0.2 mg Cu L-1) interrupted development completely. Moreover, copper sulfate exposure also determines a reduction in E. calamita growth rate. Even at the lowest copper sulfate concentration, tadpoles showed smaller sizes

0 100

48 h

80 60 40 20 0 100

72 h

80 60 40 20 0 100

96 h

80 60 40 20 0

control

0.06

0.08

ORIGIN TREATMENT

0.10

Arch Environ Contam Toxicol (2009) 56:557–565

than the size obtained in control vessels. Similar results were found by Lande´ and Guttman (1973) in Rana pipiens tadpoles, which showed a significant weight decrease under 0.06 and 0.16 mg Cu L-1. Reduced growth rate is correlated with longer developmental time and smaller size at metamorphosis (Breden and Kelly 1982). That effect on growth and development might have important repercussions on the conservation of this species. An increase in development time affects the total of individuals which can complete their metamorphosis successfully, since this species uses small and temporary wetlands that suffer rapid desiccation processes (Sinsch 1998), and fitness later in life is directly related to size at metamorphosis (Semlitsch et al. 1988). The limited extension of these habitats is normally exploited through a short larval development and a small metamorphic size, as has been mentioned above (Salvador and Garcı´aParı´s 2001). However, high larval mortality rate in E. calamita (80–90%) has been reported associated with premature wetland desiccation (Salvador and Garcı´a-Parı´s 2001). Further, more than 80% of adults return to the same wetland for reproduction, which shows the value of these habitats in the biology of this species. So, this sublethal effect could have consequences on recruitment. If factors such as copper sulfate exposure induce an increase in developmental time and reduce growth rate, then the probability of reaching metamorphic size before wetland desiccation decreases, thereby generating a reduction in recruitment to the terrestrial environment and, consequently, a reduction in the number of potential breeding individuals for the population. The effects of a contaminant in their aquatic phase could carry over into effects on the terrestrial phase via the loss of recruits to the terrestrial habitat (Rowe et al. 2001). Maximizing survival and size at metamorphosis in the dynamic and unpredictable environment of a temporary pond poses a major challenge to amphibians (Griffiths 1997). So, wetland desiccation, together with any other factor, such as the presence of toxic substances that lengthen the premetamorphic stage, lead to a decrease in recruitment possibilities. The results shown in this study must be considered over the experimental conditions designed. This information can help to understand the effects of toxic substances on the aquatic ecosystem. However, more complex toxicity tests, such microcosm and mesocosm studies, will provide more useful information for assessing the impact of a stressor on the ecosystems (Hall et al. 1998). The experiments initiated in Gosner stage 25 showed less apparent results, because the experimental period (96 h) is not long enough for this development phase. Similarly to other amphibians, E. calamita larvae remainin Gosner stage 25 longer, while their weight increases (Garcı´a-Mun˜oz, unpublished data). However, copper negative effects occur during these experiments, as was observed through the large-scale skin epithelial damage detected (Garcı´a-

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Mun˜oz, unpublished data), which probably disturbs osmotic equilibrium and energy expenditure (Prosser 1991). Similar histological and morphological effects have been observed in other species exposed to similar and ´ lvarez et al. 1991; different toxins (Rowe et al. 2002; A Chen et al. 2007). Bosch (2003) reported a decrease in the activity of the immunological system in amphibian populations that have been exposed to pesticides, detected by an increase in parasite infections. Synergistic effects with other pesticides can also result in more adverse embryonic survival (Pe´rez-Coll and Herkovits 2006). All these factors doubtless increase further indirect larval mortality. Copper exposure induces a lower capacity and less efficiency in larval escape behavior. Larval capacity to escape is reduced because more stimuli are necessary to provoke a displacement. Furthermore, larvae are less efficient owing to the shorter distances and the abnormal movements during their escapes. Although the results presented in this study were obtained under laboratory conditions, any reduction in distance moved during escape could endanger larval survival and, consequently, population survival. In addition, the abnormal movement types described in exposed larvae implies an increase in metabolic cost (Rowe et al. 2002), which generates an imbalance due to the absence of benefit (the escape), so these types of behavior must be energetically inefficient. Many toxicants are known to alter the behavior of aquatic biota (Weis et al. 2001) including amphibians (see Marco and Blaustein 1999; Blaustein et al. 2003). Three general types of contaminant influences on behavior have been observed: (a) reduced predation rates, (b) increased susceptibility to predation, and (c) reduced susceptibility to predation (Fleeger et al. 2003). Our results belong to type b, because the copper toxicity alters the behavior of the prey so that the success of predatory attacks is increased. One of the most interesting results obtained in this study is the finding that there is a successful recovery process after 96 h. This process is affected by copper concentration, time in recovery, and the interaction concentration 9 time. Almost all larvae exposed to the higher concentration showed escape behavior similar to larve that came from control treatments after 96 h in recovery. The recovery could take place in the aquatic ecosystem when the toxic substance has a short shelf life or is degraded in nontoxic forms. The escape behavior test could be used to develop future tools to detect the degree of alteration of the wetland. The use of widely distributed and abundant species in toxicological studies has been criticized because their wide distribution may also be due to their higher resistance to pollution (Marco 2003). There is a wide variation in tolerance levels among amphibians, even between closely related species (Bridges and Semlitsch 2000; Bridges et al. 2002). Therefore, in order to protect a wider range of species in the community, legal copper sulfate restrictions

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must take into account toxicity information from the most sensitive species and not from the tolerant ones. Although conclusions drawn from studies on only a few species cannot reveal the full effects of potentially harmful chemicals on amphibians in general (McDiarmid and Mitchell 2000), our results could give a picture of what is happening in small wetlands and will provide increased understanding of the ecology of temporary wetlands (Blaustein and Schwartz 2001). However, further research on sublethal effects, comparative sensitivity to key pesticides, and field experiments needs to be conducted. Acknowledgments This study was supported by the Universidad de Jae´n (Project ‘‘Plan Propio 2006–2008’’). Our thanks go to the Consejerı´a de Medio Ambiente (Junta de Andalucı´a) for permission to take amphibian samples. We thank Dr. Tejedo and the two anonymous reviewers for their valuable comments to improve the manuscript.

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