Arch. Environ. Contam. Toxicol. 23, 339-350 (1992)

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E nvironmental

C o n t a ma nidn a t i o n | oxicology

© 1992Springer-VerlagNew York Inc.

Effects of Acute and Chronic Acidification on Three Larval Amphibians that Breed in Temporary Ponds Christopher L. Rowe*, Walter J. Sadinski**, and William A. Dunson* *Department of Biology, The Pennsylvania State University, 208 Mueller Laboratory, University Park, Pennsylvania 16802, USA and **Lawrence Berkeley Laboratory, 1 Cyclotron Rd., Berkeley, California 94720, USA

Abstract. This study explored the effects of acute (7 days) and

chronic (4 months) exposure to pH 4.2 on three species of larval amphibians, Ambystoma jeffersonianum, Ambystoma maculatum, and Rana sylvatica. Acute tests were conducted in 24 impermeable enclosures in three temporary ponds. Total dissolved aluminum was higher in acidified enclosures in comparison with controls (pH 4.2, [AI] ~ 10-30 p,M and pH >4.7, [A1] ~ 5-15 IxM, respectively). Greater mortality ofA. jeffersonianum occurred at pH 4.2 than at pH >4.7, whereas survival ofA. maculatum and R. sylvatica were unaffected by pH. Mean wet masses ofR. sylvatica were significantly lower at pH 4.2 than at pH >4.7, but mean wet masses of surviving A. jeffersonianum and A. maculatum were not influenced by pH. There were no pH-related differences in body sodium concentration in larval R. sylvatica. Chronic acidification of mesocosms to pH 4.2 ([AI] ~ 16 p~M) (controls = pH >6, [All ~ 0.1 IxM) resulted in total mortality of A. jeffersonianum. Survival of A. maculatum and R. sylvatica were not associated with pH, but survival ofA. maculatum was low at both pH levels. Time to metamorphosis was longer for R. sylvatica maintained at pH 4.2, but not for A. maculatum. No differences in wet masses at metamorphosis were observed for R. sylvatica or A. maculatum. These results indicate that short and long term acidification of temporary wetlands could dramatically affect amphibians which rely upon them as breeding sites, either by causing mortality or by decreasing growth rates.

Concern has increased regarding apparent declines in populations of amphibians (Barinaga 1990, Blaustein 1990). This is a complex issue since natural fluctuations in population sizes must be separated from effects of stresses induced by anthropogenic causes (Pechmann et al. 1991). Habitat degradation can occur potentially via acidification of breeding sites resulting from the burning of fossil fuels. Amphibian species differ in physiological tolerance to low pH (Freda 1986), which leads to differing reproductive success in low pH environments and subsequent alterations of pond communities. Acidification of breeding sites can also affect amphibians indirectly through the food web.

Temporary ponds in Pennsylvania are small wetlands that partially fill in autumn with rainfall and reach maximum volume in early spring due to snowmelt and rainfall. These ponds usually dry in mid to late summer. Over the course of 3-4 months, these wetlands are used heavily as breeding sites by 5 species of amphibians (Ambystoma jeffersonianum, Ambystoma maculatum, Rana sylvatica, Pseudacris crucifer, Notophthalamus viridescens) and numerous invertebrates. In the southeastern U.S., similar ponds may have up to 20 species of breeding amphibians. These ponds collect surface and subsurface runoff and generally have no surface outlets, thereby concentrating contaminants derived from precipitation. Sadinski (1991) reported declines in pH associated with rainfall events and a negative relationship between total seasonal precipitation and median pH's of temporary ponds in central Pennsylvania. Thus, temporary ponds are ideal for studying the effects of atmospheric pollutants on breeding amphibians (Freda et al. 1991). Differential tolerances to low pH exist among amphibian species (see review by Freda 1986). Acidic conditions affect embryos primarily by preventing expansion of the vitelline membrane during development (Dunson and Connell 1982; Freda and Dunson 1985b), whereas low pH affects larval amphibians mainly by disrupting sodium and chloride balance (McDonald et al. 1984; Freda and Dunson 1984, 1985a). Laboratory LCso'S for embryos of the amphibians studied here (wood frogs, Rana sylvatica; Jefferson salamanders, Ambystoma jeffersonianum; spotted salamanders Ambystoma maculatum), were reported to be pH 4.10, 4.31, and 4.51 respectively (Sadinski 1991). Because larvae may be more tolerant than embryos to acidic conditions (Freda 1986) and this trend in tolerance was established under rather reductionist conditions, rigorous field testing was necessary. Previous studies of chronic acidification in Florida (Warner et al. 1991, in press) used two species of tree frogs (Hyla gratiosa and H. femoralis) in varying densities in simulated ponds. Low pH (pH 4.3 and 4.6) resulted in decreased survival and size at metamorphosis for both species and decreased body sodium concentrations in H. gratiosa. A prior investigation of chronic effects on the three amphibians studied here which included a different invertebrate predator and did not consider color morph of A. maculatum egg masses resulted in lower

C.L. Rowe et al.

340

survival of both A. jeffersonianum and A. maculatum at pH 4 . 2 than at pH > 6 (Sadinski 1991). These studies indicate that abiotic factors m a y work independently or interactively with biotic factors to alter the populations of a m p h i b i a n s in temporary ponds (Sadinski and Dunson, in press). The goal of this study was to e x a m i n e effects of reduced pH at two temporal extremes (7 days and 4 months) on R. sylvatica, A. jeffersonianum, and A. maculatum. Acute acidification was undertaken in order to simulate acidification that can occur following significant rain events (Sadinski 1991) and was conducted in enclosures e m b e d d e d in three temporary ponds. T w o responses (% survival and wet mass) were m e a s u r e d for all three species at the end of the experiment. Surviving R. sylvatica were also analyzed for concentrations of body sodium. The chronic portion of this study was conducted in semi-field m e s o c o s m s and modelled a scenario in which the pH of temporary ponds remains low o v e r the entire larval period o f the amphibians. Percent survival to, time to, and wet mass at m e t a m o r p h o s i s were measured for all three species.

Materials and Methods Study Area The study of effects of acute reduction in pH on larval amphibians was carried out from May 15-22, 1991, in three temporary ponds in Rothrock State Forest (Barrville topographic quadrangle, Pennsylvania). This forest is located in a mountainous region of sandstone bedrock dominated by deciduous vegetation (oak-hickory type). The pHs of the three ponds used in this study, as well as of 32 others in the immediate area, have been closely monitored for several years (Sadinski 1991).

The three study ponds all had mean seasonal pHs >4.7, above embryonic lethal limits for the amphibian species tested (Sadinski 1991). Each pond provided areas of relatively equal depth (20.8 m) within which the enclosures were placed.

Acute Test--Enclosure Design Eight watertight enclosures were constructed in each of 3 ponds (Bar 6, H, C3). They were constructed of FDA approved (for food storage) polyethylene 55 gallon drums, polyethylene drum liners, fiberglass window screening (2 mm mesh), and PVC pipe (Figure 1). The drums had the tops and bottoms removed and the remaining cylinders were cut into thirds. These drum sections served as the lower part of the enclosure; the rigidity of the material allowed this section to be driven several inches into the substrate. The drum liners were placed inside the drum sections, with several inches rolled up around the outside of the drum section in order to form a seal. Approximately 24 inches of liner extended above the top of the drum section and was supported by a PVC frame. Fiberglass window screening was used both as a top and a bottom for each enclosure. Screening was laid across the top of each enclosure and secured to the liner with wooden clothespins.This lid provided a barrier to birds and insects while allowing passage of light, precipitation, and gases. Screen bottoms were placed underneath, prior to the enclosures being driven into the sediment. This kept any macrofauna in the sediments and any vegetation from entering the enclosure. This was necessary to eliminate any structural heterogeneity among enclosures within and between ponds. By carefully lifting the entire enclosure and allowing the water to drain out, the larval amphibians were trapped on the screen bottom and could be collected quite easily. The bottom of each enclosure was covered to a depth of approximately 1.5 cm with leaves collected from the pond bottom. Samples of leaves from each pond were mixed together and any exogenous larvae were removed prior

Screen lid

PVC frame

Drum liner

Drum section

Screen bottom

Fig. 1. Enclosure used in acute acidification experiment. The screen lid was cut oversize and attached to the drum liner with wooden clothespins; the screen bottom was cut oversize and pulled up to the top of the drum section, where it was attached with clothespins and metal binder-clips (not illustrated)

Acute and Chronic Acidification on Larval Amphibians

placement of subsamples of leaves into enclosures. This provided roughly equivalent refugia and periphyton for each enclosure.

Acute Test--The Amphibian Assemblage One day prior to the start of this experiment (May 14), R. sylvatica and A. jeffersonianum were added to all enclosures, whereas A. maculatum were added on the following day (Day 1, May 15). R. sylvatica (stage 30-33; Gosner 1960) and A. jeffersonianum (stage 44-46; Harrison 1969) were collected from two temporary ponds in the study area (mean pH 5.2), pooled, mixed, and indiscriminately subsampled before distribution to individual enclosures. A. maculatum (stage 4 2 4 4 ; Harrison 1969) were hatched in the laboratory from egg masses collected from lower altitude temporary ponds (mean pH 5.4), approximately 16 km from our study ponds. Volumes of enclosures varied among ponds due to different mean water depths, but there was little variation within each pond. Densities of larvae were based on the volumes of enclosures and ranges of densities found in the field (Sadinski 1991). A summary of numbers of larvae added per enclosure per pond and several abiotic characteristics of the ponds is given in Table 1.

Acute Test--Experimental Protocol Four of the 8 enclosures in each pond were selected at random to be maintained at low pH (4.2); the remaining 4 were not adjusted, so that pH's remained greater than pH 4.7. The pH of each of the enclosures was measured between 8:00 am and 11:00 am daily using an Orion model 290A portable pH meter with an Orion model 91-57BN triode electrode, pHs inside low pH enclosures drifted ± 0.3 pH units in 24 h, but averaged 4.19 (SD 0.12) following initial adjustment on Day 1. pH was adjusted when it drifted above 4.25 or below 4.15. Concentrated H 2 S O 4 o r KOH was added to a 50 ml aliquot of water taken from the enclosure being adjusted, mixed, and poured back into the enclosure. On Day 7, amphibians were removed from enclosures, counted in the field, and returned to the laboratory where they were weighed as a group on a Mettler AE 160 balance to the nearest 0.1 mg. Seven enclosures were found to be contaminated with exogenous R. sylvatica larvae which had congregated along the sides of enclosures and were trapped when enclosures were removed. These enclosures were excluded from all analyses pertaining to R. sylvatica. There was no evidence of A. jeffersonianum or A. maculatum contamination in any enclosure. Ten larvae of R. sylvatica were randomly selected from each enclosure, blotted dry, and weighed as a group for analyses of body sodium. Each group was then placed in a plastic bag, covered with deionized water, and frozen for storage. Prior to analysis, larvae were thawed and placed in tared beakers. Beakers were placed in an oven at 60°C for 6 days to dry. Dried larvae were weighed and dissolved in concentrated nitric acid. The resulting solution was diluted with deionized water and analyzed for sodium concentration in an

341

air-acetylene flame on a Perkin-Elmer model 2280 atomic absorption spectrophotometer. At the termination of the experiment, water samples were collected for analyses of total dissolved aluminum. Samples for only 12 of the 24 enclosures (all enclosures in pond H, 2 pH 4.2 and 2 pH >4.7 in pond C3) were collected due to a logistical error. Samples were filtered through 0.10 Ixm Millipore cellulose-nitrate filters and analyzed by the catechol-violet method (Dougan and Wilson 1974) on a Beckman model 25 spectrophotometer.

Chronic Test--Design of Simulated Ponds The study of effects of chronic acidification was carried out in mesocosms between April 18 and August 13, 1991. The design consisted of 1 factor (pH) with 2 levels (pH 4.2, pH >6) arranged in two randomized blocks of 12 pools each. The composition of the biotic assemblage was held constant. Mesocosms were erected in a field at the Rock Springs Agricultural Research Area of the Pennsylvania State University, and consisted of blue polyethylene wading pools which held 260 L of soft stream water. This design was a modification of that used by Sadinski (1991). Soft water (conductance = 25 txS/cm, [Na +] = 26 IxM, [Mg ++] = 66 IxM, [K +] = 17 p.M, [Ca --+] = 38 IxM) from the headwaters of Roaring Run in Rothrock State Forest (upstream of PA route 26), approximately 13 km from the experimental site, was used to fill the pools on April 2. Pools were covered with fiberglass window screening (2 mm mesh) to prevent immigration or emigration of animals. Metamorph traps made of screening and 7 L polyethylene containers were attached along the pool edges to capture metamorphosing amphibians and adult newts (Sadinski 1991). Shade cloth (60% shade) was erected over the pools on June 6 (Day 48) in order to simulate shading due to trees surrounding natural ponds.

Chronic Test--Experimental Protocol Four 3.8 L buckets full of leaf litter from a nearby wooded area were added to each pool at the time of filling (April 2). Leaf litter provided refugia and nutrients for organisms in the pools. On April 11, 6 g of crushed rabbit chow (Alphapet, no added limestone) were added to each pool to provide additional nutrients. Filamentous algae, leaves from the bottom of ponds (with attached periphyton), and algae from among communal egg masses ofR. sylvatica were collected from three ponds of various pHs (pH range 4.3-5.2). Each type of algae from the three ponds was mixed thoroughly. Subsamples of 30 ml of each type of algae and three leaves were added to all pools on April 12. Between April 16 and April 20, other organisms were collected from nearby temporary ponds and added to the simulated pond assemblage. Equal volumes of zooplankton (thoroughly mixed from three ponds of pH 4.3-5.5), 14 fairy shrimp (Eubranchipus holmani), 100 mosquito larvae (Culex and Anopholes spp.), and two adult backswimmers (No-

Table 1. Biotic and abiotic parameters associated with acute acidification experiment. Rs, Aj, and Am represent the numbers ofR. sylvatica, A. jeffersonianum, and A. maculatum larvae added per enclosure (8 enclosures/pond). Mean volumes of enclosures were based upon depth measurements made within each enclosure prior to addition of any animals, pH values were measured in surrounding water at the time of initiation of the experiment, pH's inside the closures were subsequently adjusted to 4.20 or left at ambient levels. Ion concentrations were measured on samples collected on April 24, 1991, just prior to the experiment

Pond

Rs

Aj

Am

Mean volume (L) ± std. dev.

pH

Na (~M)

Mg (~M)

K (~M)

Ca (~M)

Bar6 H C3

44 44 29

8 8 5

20 20 13

88.62 ± 9.92 82.86 ± 15.86 68.49 ± 9.03

5.35 4.82 4.70

20 18 14

36 30 25

16 10 13

17 9 29

C.L. Rowe et al.

342

tonecta undulata) of undetermined sex were added to each pool. Densities of the above organisms, except that of N. undulata, were based

Statistical Analyses

upon observations in temporary ponds over the past several years (Sadinski 1991). The density ofN. undulata was chosen based on the range of densities reported by Streams (1987). Larvae of R. sylvatica and A. jeffersonianum and adults of the red-spotted newt (Notophthalamus viridescens) were collected from 3 temporary ponds on April 17. Samples of each species were thoroughly mixed together and subsampled indiscriminately prior to distribution to pools. On April 18, 135 larvae of R. sylvatica (stage 25-28; Gosner 1960), 24 larvae of A. jeffersonianum (stage 40-42; Harrison 1969), and 2 preweighed adult (1 male, 1 female) N. viridescens were added to each pool. Due to low numbers ofA. maculatum larvae available in the local temporary ponds at this time, addition of this species to mesocosms occurred later from a sample of wild larvae hatched in the laboratory. A. maculatum commonly produces egg mass jellies of 2 distinct color morphs: an opaque (white) morph in which the embryos are hidden in the jelly and a clear morph in which embryos are plainly visible through the jelly. In order to identify any effects of egg mass color on the responses measured, egg masses of the two morphs were hatched separately and distributed such that each pool received larvae of only 1 morph. Over the period of April 30-May 8 (Days 13-21 ), A. maculatum (stage 40, Harrison 1969) were added until a total of 60 larvae per pool was reached. Equal numbers of larvae were added to each pool on days that additions took place. Monitoring and adjustment of pH in the pH 4.2 treatments began 1 day after filling with water (prior to the addition of any animals) and continued daily until the end of the experiment on August 13. High pH pools were checked for pH occasionally throughout the season, but were not adjusted, pH was checked during late morning to mid afternoon daily when pHs should have been near their highest values due to photosynthetic activity (Keeley and Sandquist 1991). An Orion 290A portable pH meter with an Orion model 91-57BN triode electrode was used for pH measurements and adjustments. Since these electrodes leak small amounts of KCI and AgC1, a 100 ml sample of water was removed from each pool for each pH reading, and then discarded, pHs were adjusted by adding concentrated H2SO 4 or KOH to a 150 ml aliquot of water removed from the pool. This aliquot was then gently stirred into the pool and allowed to diffuse for several minutes prior to taking another pH reading. Stirring was limited to the upper 10-20 cm of the water column to ensure that leaf litter on the pool bottom was disturbed as little as possible. High-pH pools were stirred in the same manner so that any disturbance to the assemblage was similar across all treatments. Upward adjustment in pH with KOH was seldom necessary. Drift in pH was rarely more than 0.15 units per day throughout the course of the experiment. Water samples were collected from each pool on May 24 for analysis of concentrations of Na + , Mg ++ , K + , and Ca + +. These samples were filtered through 0.45 l,zm Nalgene cellulose-nitrate filters and analyzed on a Perkin-Elmer Model 2280 atomic absorption flame spectrophotometer. Samples for analysis of NOx, P042, and SO42 were collected on June 17 and filtered as above. Samples were analyzed using a Hach dr/2 spectrophotometer (NO,,), a Hach dr/100 calorimeter (SO42), and a Beckman model 25 spectrophotometer (po42). Samples for aluminum analysis were collected on June 5 for pools in Block 1 and June 12 for pools in Block 2. These samples were filtered through 0.10 Ixm Millipore cellulose-nitrate filters and analyzed within 4 h of collection using the catechol violet method (Dougan and Wilson 1974). On May 30, the first metamorphs (R. sylvatica) were captured. R. sylvatica metamorphs were defined as metamorphs after having shown emigration behavior (captured in traps) and having a remaining tail of less than 2 mm. Captured R. sylvatica having a tail greater than 2 mm were returned to the pools from which they emigrated. Metamorphs of A. jeffersonianum and A. maculatum were defined as those captured in the traps. Three pools contained larval A. maculatum at the end of the experiment. These larvae were excluded from calculations. Wet masses of metamorphs were measured to the nearest 0.1 mg in the laboratory using a Mettler AE 160 balance.

Statistical tests for all parameters were carried out using Minitab version 7.1 statistical software (Minitab-State College, PA). We used the general linear model approach to analysis of variance, since it is effective for both balanced and unbalanced experimental designs. We employed the Tukey-Kramer method for complex comparisons, which is designed for use with unequal ni's (Day and Quinn 1989). Comparison of presence of N. viridescens to survival of larvae was done using linear regression. All data were checked for normality by assessing the correlation between normal scores and the data (raw and transformed), which is essentially equivalent to the Shapiro-Wilk test (Minitab 1989). Data were checked for homogeneity of error variances by visually comparing plots of standardized residuals against factor levels for raw and transformed data. Similar distributions of standardized residuals at each factor level indicated that error variances were similar. Small n~'s precluded more elaborate tests of model assumptions. In order to satisfy the assumptions of the statistical models, transformations of some of the raw data were necessary. All proportional data were transformed via the arcsine transformation (Y' = 2 arcsin ~/Y; Neter et al. 1990). Data for wet masses from each experiment were transformed to 1/(wet mass) 2 prior to analyses. In order t o maintain experiment-wise Type I error rates at e~ = 0.05, Type I error rates for tests on each response were adjusted downward by dividing by the number of tests carried out in the experiment (Bonferroni procedure for comparing multiple single degree of freedom tests, Rice 1989; Neter et al. 1990). These adjustments gave critical a ' s of 0.0071 and 0.0062, respectively for the acute and chronic acidification experiments. Unless otherwise noted, all results are reported as X _+ SD.

Results Acute Test At the end of this experiment, concentrations of total dissolved a l u m i n u m were significantly higher (P < 0.001) in pH 4.2 enclosures than in pH > 6 enclosures and higher (P < 0.001) in pond C3 than in pond H (Table 2). Percent survival and m e a n wet mass for amphibians exposed to 2 p H ' s for 7 days are s h o w n in Figures 2 and 3. Significantly fewer A. jeffersonianum survived at pH 4 . 2 than at pH > 4 . 7 , whereas survival of R. sylvatica or A. maculatum were unaffected by pH (Table 3). There was no relationship between survival and pond for R. sylvatica or A. maculatum, although there was a significant effect of p o n d on survival o f A . jeffersonianum. Percent survival (pooled across all treatments) for A. jeffersonianum was lowest in pond Bar 6 and highest in pond

Table 2. Mean concentrations of aluminum in 12 field enclosures following a acute acidification experiment

Pond

Treatment pH

n

H

>4.7

4

H

4.2

4

C3

>4.7

2

C3

4.2

2

Mean (in ixM) (s.d.)

Mean (in i,zg/L) (s.d.)

4.6 (0.4) 10.1 (1.3) 14.6 (0.5) 30.0 (5.5)

123 (10) 273 (36) 384 (13) 797 (147)

Acute and Chronic Acidification on Larval Amphibians

Chronic Test

i00 9O >

80

u] 60 oJ ©

50 40 30

o~ (D

BO 10

pH 4.2 >4.7 A. j e f f e r s o ~ z i a n u m

4.2 >4.7 R. s y l v a t i c a

4.2 >4.7 A. r t ~ a c u l a t u ~

Fig. 2. Mean percent survival of three amphibians following acute acidification (means -+ standard deviations)

0.6

0.5

5q ~3 -~

0.4

0.3

O.2 0.1

0,0

343

pH 4.2 >4.7 A. j e f f e r s o n i a n u m

4.2 >4.7 R. s y l v a t i c a

4.2 >4.7 A maculatum

Fig. 3. Mean wet masses of three amphibians following acute acidification (means -+ standard deviations)

C3 (Bar 6 x 25.0 + 29.1; H x 40.6 -+ 28.2; C3 ~ 52.5 + 36.9). There was no significant interactive effect of pH*pond for any species. Mean wet mass at 7 days was significantly lower at pH 4.2 (X = 0.381 + 0.89 g) relative to pH >4.7 (X = 0.513 -+ 0.065 g) for R. sylvatica (Table 4). Pond and the pH*pond interaction also significantly affected mean wet mass of R. sylvatica. R. sylvatica were largest in pond H and smallest in pond Bar 6 (H, 0.523 -+ 0.055 g; C3, 0.390 -+ 0.109 g; Bar6, 0.363 -+ 0.064 g). There were no significant effects of pH or pond on mean wet mass ofA. jeffersonianum orA. maculatum. Concentrations of body sodium in larvae ofR. sylvatica from all enclosures were not significantly different among pH's or ponds (Table 5).

Chemical analyses of water from mesocosms (Table 6) showed that concentrations of Mg ++, K +, and Ca ++ were higher than initial concentrations. Nitrate and phosphate concentrations were quite variable and do not seem to be related to the pH of the water. Sulfate was below detection limits in pH > 6 treatments but ranged from 1062-1666 IxM in the pH 4.2 treatments. This difference between treatments is due obviously to the use of H2SO 4 to acidify the water in pH 4.2 mesocosms. Higher [A1] in pH 4.2 pools is likely related to solubilization of aluminum in acidic water. Thus the effects of our nominal pH treatments represent the combined action of all of these constituents, not just pH. Survival of A. jeffersonianum to metamorphosis was significantly influenced by the pH of the water in the mesocosms. No A. jeffersonianum larvae survived to metamorphosis in the mesocosms at pH 4.20, whereas 8% of those in pools at high pH survived (Figure 4). Survival of R. sylvatica and A. maculatum was not affected by pH; survival ofA. maculatum did not differ between egg mass morphs (Table 7). Survival ofA. maculatum was extremely low overall in this experiment (3.9% survival over all treatments). Time to metamorphosis was significantly affected by pH for R. sylvatica (Table 8). All surviving R. sylvatica had metamorphosed by the 69th day of the experiment in pH > 6 mesocosms, whereas it was not until the 77th day that all surviving R. sylvatica in pH 4.2 mesocosms metamorphosed (Figure 5). pH and egg mass morph did not affect time to metamorphosis for A. maculatum, although initial metamorphs were captured 18 days later in pH 4.2 treatments than in pH > 6 treatments (Figure 6). pH*morph interaction was not calculated for A. maculatum due to complete mortality of the white morph at pH 4.2. No comparison of time to metamorphosis of A. jeffersonianum was made due to complete mortality of this species in the pH 4.2 pools. Wet mass at metamorphosis was measured for all 3 species, although comparisons based upon pH could be made only for R. sylvatica and A. maculatum (Figure 7). There were no significant differences in wet mass at metamorphosis between pH's for R. sylvatica or A. maculatum or between egg mass morphs for A. maculatum (Table 9). No interaction of pH*morph was calculated due to total mortality of white morph A. maculatum at pH 4.2. Thirty one of the 48 N. viridescens added to the mesocosms escaped under the screen tops during the experiment. At the end of the experiment, 14 pools contained zero N. viridescens (seven pH 4.2 pools, seven pH > 6 pools), two pools contained one male (one pH 4.2 pool, one pH > 6 pool), two pools contained one female (one pH 4.2 pool, one pH > 6 pool), five pools contained one male and one female (two pH 4.2 pools, three pH > 6 pools), and one pH > 6 pool contained two males and one female due to immigration of one male. Since N. viridescens captured in metamorph traps were sexed and recorded before being returned to the pools, we calculated the minimum number of days that at least one N. viridescens was present in each pool (newt-days). There were no differences in survival due to presence of newts for R. sylvatica or A. maculatum, or for A. jeffersonianum at pH > 6 (no comparison was possible at pH 4.2) (Table 10). N. undulata fared well in all treatments, actually propagating in 19 of the 24 pools. The total number ofN. undulata present

344

C.L. Rowe et al.

Table 3. Analysis of variance for survival during acute acidification experiment (critical c~ = 0.0071) Source

D.F.

Seq. SS

pH Pond pH*Pond Error

1 2 2 18

11.703 2.935 0.539 2.917

pH Pond pH*Pond Error

1 2 2 11

0.064 0.034 0.009 0.545

pH Pond pH*Pond Error

1 2 2 18

0.014 0.513 0.558 3.071

Adj. SS a. A. jeffersonianum 11.703 2.935 0.539 2.917 b. R. sylvatica 0.009 0.024 0.009 0.545 c. A. maculatum 0.014 0.513 0.558 3.071

Adj. MS

F

P

11.703 1.467 0.270 0.162

72.22 9.05 1.66

6mean (s.d.)

30 (10) 15 (8)

281 (35) 224 (44)

69 (21) 73 (15)

299 (12) 272 (22)

2.78 (5.49) 0.77 (2.26)

0.04 (0.03) 0.02 (0.03)

1422 (172) 0 (0)

16.19 (5.28) 0.12 (0.43)

60 "~ >

5o

~

40

20 c~

pH 4.2 >6 A jeffersonianum

4.2 >6 R. s y l v a t i z a

4.2 >6 A. m a c u Z a t u m

Fig. 4. Mean percent survival for three amphibians following chronic acidification (means -+ standard deviations), regardless of egg mass morph ofA. maculatum

is likely that this species suffered from predation from surviving A. jeffersonianum at both levels of pH. Survival of A. jeffersonianum was lowest in pond Bar 6 and highest in pond C3. Since enclosures were completed first in C3 and last in Bar 6, it may be that greater handling times resulted in additional stress to animals added to enclosures in Bar 6. Aluminum concentrations in pH 4.2 enclosures in pond C3 were within the range known to ameliorate effects of pH 4.2 on 3 week old tadpoles, and concentrations of aluminum in pH >4.7 enclosures in this pond are slightly toxic (4.7, resulting in greater overall survival of A. jeffersonianum in this pond.

Survival--Acute Test

Wet Mass--Acute Test

It is clear from the results that larvae ofA. jeffersonianum were intolerant of pH 4.2, whereas R. sylvatica and A. maculatum were more tolerant. This agrees with observations that Jefferson salamanders cannot successfully reproduce in temporary ponds in central Pennsylvania in which the pH averages below 4.3-4.4 throughout embryonic and larval periods (Sadinski 1991, Rowe and Dunson unpub, obs.). In laboratory tests examining the effects on a complex of larval A. jeffersonianum, A. maculatum, and R. sylvatica of exposure to low pH for 3 and 7 days, survival ofA. maculatum and R. sylvatica was higher in pH 4.1 than in pH 4.5 and pH >5.5 treatments, due to increased mortality of predatory A. jeffersonianum at pH 4.1 (Sadinski 1991). This effect was not present in the field tests, as indicated by no differences in survival of A. maculatum or R. sylvatica due to pH. A. jeffersonianum and A. maculatum used in the study were at nearly the same developmental stage as those used by Sadinski (1991) (reported as "mid-40s;" Harrison 1969), although Sadinski's (1991) were collected approximately one month later in the season than ours, and may have been somewhat larger. R. sylvatica were somewhat more developed in our study than in Sadinski's (1991) (stage 28; Gosner 1960). It is likely that the additional size of R. sylvatica protected them to some extent from gape-limited predation by A. jeffersonianum. Survival of A. maculatum was lower than that ofR. sylvatica orA. jeffersonianum, regardless of pH. Since A. maculatum were significantly smaller than A. jeffersonianum it

R. sylvatica were significantly smaller following 7 days at pH 4.2 than at pH >4.7. This result seems surprising since the exposure was relatively short in duration. A reasonable explanation may be that a severe negative impact of low pH on periphyton occurred (Sadinski 1991), limiting the food supply for tadpoles. Algal components added to enclosures were derived from the same ponds in which enclosures were located, all of which had a pH of 4.7 or greater. Perhaps the algae in these ponds did not possess the tolerance to low pH needed to survive in pH 4.2 enclosures. Mean wet masses ofR. sylvatica were also affected by pond and pH*pond. Mean wet masses were lowest for larvae enclosed in pond Bar 6 and highest for those in pond H. Since the larvae were indiscriminately assigned to enclosures in each pond from a mixed sample, no initial bias in wet mass was present. Periphyton in different ponds may have varied in abundance or nutritional value for R. sylvatica, resulting in differential accruement of biomass for this herbivore. There were no differences in mean wet masses among the 2 pH levels for either of the salamander species, indicating that these carnivores did not suffer a serious food limitation, or that fasting did not influence mass. We expected that the few surviving A. jeffersonianum in pH 4.2 treatments would have been larger than those in pH > 4 . 7 due to reduction in intraspecific competition for food. Presumably the treatments did not differ in A. jeffersonianum density for a long enough period to produce a difference in mass.

C.L. Rowe et al.

346

Table 7. Analysis of variance survival to metamorphosis during chronic acidification experiment (critical ~ = 0.0062)

Source

D.F.

Seq. SS

pH Morph (Am) pH*Morph Error

1 1 1 20

0.945 0.414 4.00 × 10-4 3.055

pH Morph pH*Morph Error

1 1 1 20

0. 145 0.416 0.015 2.262

Adj. SS

a. R. sylvatica 0.945 0.223 4.00 x 10-4 3.055 b. A. maculatum 0.233 0.416 0.015 2.262

Adj. MS

F

P

0.945 0.223 4.00 × 10-4 0.153

6.19 0.00

0.022 0.241 0.962

2.06 3.67 0.13

0.166 0.070 0.721

1.46

0.233 0.416 0.015 0.113

Table 8. Analysis of variance for time to metamorphosis during chronic acidification test (critical a = 0.0062) Source

D.F.

Seq. SS

pH Morph pH*Morph Error

1 1 1 20

524.54 5.47 28.01 459.40

1 1 4

21.91 25.52 232.31

pH Morph Error

Adj. SS

a. R. sylvatica 524.54 5.47 28.01 459.40 b. A. maculatum 38.00 25.52 232.31

Adj. MS

F

P

524.54 5.47 28.01 22.97

23.41 0.24 1.22

4 . 7 treatments. A loss in body sodium in aquatic organisms is commonly associated with exposure to low pH (Packer and Dunson 1972, Freda and Dunson 1985a), although the extent to which this occurs is dependent upon several factors such as prior exposure to acidic environments and ambient cation concentrations (Freda and Dunson 1986a). Such a net loss was not seen in this study, indicating that these larvae were not stressed to the point where ionic regulation was affected. This is not surprising, since the embryonic LCs0 forR. sylvatica is pH 4.10 (Sadinski 1991) and larvae are more tolerant than embryos to low pH (Freda 1986).

Water Chemistry---Chronic Test Concentrations of ions in the mesocosms were higher on day 36 (May 24) (Table 6), than in the same water prior to filling the pools. Concentrations of Mg ++ and Ca ++ were above the ranges of average seasonal concentrations measured in temporary ponds in Rothrock State Forest, whereas concentrations of Na + and K + were within these ranges: ([Mg ++] range 20-96 IxM, [Ca ++] range 5-59 IxM, [Na +] range 15-84 IxM, [K +] range 25-88 IxM). Little evaporation had taken place in the pools by day 36, indicating that this was not the reason for the increase. Probably, the addition of rabbit chow and leaves to the pools introduced some cations, but not to the extent measured. Agricultural activities in the area around the pools probably contributed ions to the pools through airborne dust. Certain ions, especially Ca ++ , may ameliorate the effects of low

pH on aquatic organisms (Freda and Dunson 1984, 1985a, 1985b), although concentrations of 20-80 mg/L (499-1996 puM) calcium are detrimental to developing embryos of R. sylvatica (Freda and Dunson 1985b). Calcium levels in the mesocosms were well below levels reported to be detrimental. Thus, effects of low pH seen in this experiment were presumably less drastic than those that actually occur in acidic temporary ponds with lower concentrations of Ca ++ . Higher concentrations of aluminum in the pH 4.2 mesocosms also may have been a factor in determining the outcome of this experiment. However, increased concentrations of aluminum are most likely due to increased solubility of aluminum in pH 4.2 and therefore are a natural consequence of acidification. These concentrations were less than those observed in many local temporary ponds (Sadinski 1991; Rowe and Dunson unpub. obs.) and were within the range of concentrations known to ameliorate effects of low pH (Freda and McDonald 1990). This may indicate that the stresses incurred in this experiment may be less strenuous than those in natural ponds.

Survival to Metamorphosis and Wet Mass at Metamorphosis--Chronic Test Due to the complex nature of the mesocosm communities, pH, predation, and competition may have individually or interactively affected amphibians. Chronic exposure to pH 4.2 causes mortality in larval A. jeffersonianum and may affect community structure by removal of this predator (Sadinski 1991). Chronic acidification to pH 4.2 may also decrease survival and wet mass of larval A. maculatum, although not to the extent that A. jeffersonianum is affected (Sadinski 1991). Predation by N. viridescens may affect density and thus, competition, among

Acute and Chronic Acidification on Larval Amphibians

347

1.0

0,9 so rO_ L. 0

E

0.8 0.7 0.6

5 ~8

0.5 0.4

cO

0.3 0 ¢3 0 L

Q_

0.2 0.1

0.0

Fig. 5. Time to metamorphosis for R. sylvatica. Points are means for all replicates at each pH

Time (Days)

1.0

•

pH>6.0

0.9

0.8 13. L0

E

0.7

13

0.6

5 ~8

0.5

t-0

0.4

i0

0.5 0 13_ 0 L_ IX.

0.2

0.1

O.O

Time (Days)

amphibian larvae (Morin 1981, 1983; Morin et al. 1983; Wilbur et al. 1983; Wilbur 1987); predation by invertebrates may have similar effects (Morin et al. 1988; Sadinski 1991). Chronic exposure to pH 4.2 resulted in complete mortality of A. jeffersonianum. Observed mortality of this species during the acute acidification study suggests that A. jeffersonianum

Fig. 6. Time to metamorphosis for A. maculatum. Points are means for all replicates at each pH (regardless of egg mass morph)

may have been eliminated early from pH 4.2 mesocosms in the chronic study, before they could exert significant predatory effects on populations of larval R. sylvatica or A. maculatum. Upon introduction into the mesocosms, A. jeffersonianum had a distinct size advantage over A. maculatum and was expected to be an important predator on this species. Thus, early mortality

348

C.L. Rowe et al.

1.4 1.2 v cn LO ~5

1.0 0.8 0.6

//

0.4 0.2 N.A.

0.0

pH 4 . 2 >6 A. j e f f e r s o n i a n u m

42

>6

R. s y l v a t i c a

4.2

>6

A. 77zaculaturn

Fig. 7. Mean wet masses of three amphibians following chronic acidification (means + standard deviations), regardless of egg mass morph ofA. maculatum

ofA. jeffersonianum in pH 4.2 treatments should have resulted in greater survival ofA. maculatum. This did not occur; instead survival of A. maculatum and A. jeffersonianum were low regardless of treatment. Perhaps higher initial densities of A. jeffersonianum would have resulted in more of this species escaping predation by N. viridescens and N. undulata, ultimately resulting in greater predatory pressure on A. maculatum at pH > 6 . In a similar study, Sadinski (1991) reported low survival ofA. jeffersonianum at pH 4.2 and pH > 6 which were attributed to direct effects of pH 4.2 and probable predation by N. viridescens and larval predaceous diving beetles (Dytiscus verticalis). No significant relationships were found between newt-days and survival of A. jeffersonianum, A. maculatum, or R. sylvatica at either pH. N. viridescens is predatory upon larval amphibians, and therefore was expected to have negative impacts on survival. It is possible that early in the experiment amphibian larvae reached a size beyond which they were released from gape-limited predation by newts, although we have no evidence of this. The low proportion of A. maculatum surviving to metamorphosis in both pH treatments could be explained by predation by N. undulata, although this is purely supposition. N. undulata is predatory on larval amphibians (Cronin and Travis 1986)

and may have impacted not only on A. maculatum at both levels of pH, but also survival of A. jeffersonianum in the pH > 6 treatments. In a similar study that did not include N. undulata, Sadinski (1991 ) reported much higher survival ofA. maculatum (~62% survival at pH 4.2, ~ 7 1 % survival at pH >6) but low survival of A. jeffersonianum at pH > 6 . A. maculatum were smaller than A. jeffersonianum, and both were considerably smaller than R. sylvatica when added to the pools. This small size could have caused salamander larvae to be more susceptible to predation by backswimmers (Cronin and Travis 1986). There were no effects of pH on wet mass at metamorphosis for any amphibian species. It was expected that A. maculatum metamorphs would be smaller at pH 4.2 than at pH > 6 due to exclusion of predatory A. jeffersonianum larvae at pH 4.2. Low survival of A. maculatum presumably reduced competition in all treatments, eliminating a density-dependent effect on wet mass at metamorphosis. Negative impacts of acute exposure to pH 4.2 on wet mass ofR. sylvatica did not occur during chronic exposure. In contrast to the acute acidification experiment, algal components added to mesocosms were from samples mixed from ponds of pH 4.3-5.2. This mixture may have provided algae which was tolerant of pH 4.2, and thus presented an adequate food supply for developing R. sylvatica.

Time to Metamorphosis--Chronic Test Time to metamorphosis was significantly longer at pH 4.2 than at pH > 6 for R. sylvatica, indicating that growth rates were slower as a direct or indirect result of pH 4.2 treatments, Increased time to hatching, decreased growth rates, and prolonged time to metamorphosis in amphibian larvae due to low pH have been reported (Freda and Dunson 1985a; Ling et al. 1986; Cummins 1989; Sadinski 1991). Delayed metamorphosis may be extremely important to these animals since temporary ponds are dependent upon precipitation as a water source and are therefore highly susceptible to rapid drying during periods of low rainfall and warm temperatures. In a period of 1 week these ponds may change from having roughly half their initial volumes of water to being completely dry (Rowe and Dunson unpub, obs.). This indicates that even a minimal delay in metamorphosis may drastically affect recruitment from these ponds. There was no difference in time to metamorphosis between treatments for A. maculatum, In a similar study, Sadinski (1991) found time to metamorphosis to be greater for A. maculatum larvae maintained at pH 4.2 than for those maintained at pH >6.0, regardless of the initial density of amphibians. This

Table 9. Analysis of variance for wet mass at metamorphosis during chronic acidification experiment (critical a Source

D.F.

Seq. SS

pH Morph(Am) pH*Morph Error

1 1 1 19

0.300 5.221 1.116 73.372

l 1 4

1.021 5.272 2.612

pH Morph Error

Adj. SS a. R. sylvatica 0.761 5.055 1.116 73.372 b. A. maculatum 0.018 5.272 2.612

=

0.0062)

Adj. MS

F

P

0.761 5.055 1.116 3.862

0.20 1.31 0.29

0.662 0.267 0.597

0.018 5.272 0.653

0.03 8.07

0.877 0.047

Note: No calculation of pH*Morph for A. maculatum due to complete mortality of white morph at pH 4.2

Acute and Chronic Acidification on Larval Amphibians

349

10. Results of linear regression comparing newt-days (X days) to survival (Y = 2*arcsin ~/proportion survived) of 3 amphibians during chronic acidification experiment (critical ~ = 0.0062). Am, Rs, and Aj refer to A. maculatum, R. sylvatica, and A. jeffersonianum, respectively Table

Species

pH

DF

Regression equation

R2

P

Am Am Rs Rs Aj

4.2 >6 4.2 >6 >6

11 ll 11 11 11

Y Y Y Y Y

22.0% 19.5% 16.1% 44.7% 4.6%

0. 124 0.150 0.196 0.018 0.504

discrepancy could be attributable to much lower survival of A. maculatum across all treatments in our study. It was expected that time to metamorphosis for A. maculatum would be related to density-dependent intraspecific competition for resources. In this scenario, exclusion of predatory A. jeffersonianum larvae by pH 4.2 treatments would result in greater survival of A. maculatum at pH 4.2 than at pH > 6 where A. jeffersonianum was not excluded. Thus, higher densities of A. maculatum at pH 4.2 than at pH > 6 would result in greater intraspecific competition at pH 4.2. A. maculatum larvae maintained at pH > 6 would benefit from reduced intraspecific competition by having faster growth rates (shorter time to metamorphosis) than those in pH 4.2 treatments. Low densities o f A . maculatum as a result of mortality in all treatments may have eliminated this effect.

Conclusions

Results of acute and chronic tests indicate that habitat acidification could indeed have strong negative impacts on amphibians which breed in temporary ponds. A. jeffersonianum is extremely intolerant of pH 4.2 environments, suffering significant mortality during acute exposure and complete mortality during chronic exposure to pH 4.2. R. sylvatica, while more tolerant to direct effects of low pH, may be affected indirectly by food limitations caused by acidic conditions. Decreased growth rates may result from acidic conditions and may severely impact populations if pond hydroperiods are shortened, as occurs in dry years or as may occur in a global warming scenario.

Acknowledgments. This study was funded by U.S. Environmental Protection Agency Grant #R-817206-01-0. Christopher Rowe was supported for part of this study by a teaching assistantship through the Department of Biology of The Pennsylvania State University. We wish to thank Ben Ruth for supplying the spotted salamander larvae for these experiments, and Mike Home, Hui-Chen Lin, and Tak-Cheung Lau for their advice during the preparation of this manuscript. Many thanks must go to the following technical assistants and undergraduates whose efforts played a major role in making this study possible: Jeff Trulick, Jolene Adkins, Mike Cowan, Jim Grazio, Jody Kasmir, Nancy Longo, Carl Mikan, Laura Mulrenan, Joe Paradise, Kristen Ritter, Gregson Vaux, Dave Wayland, Amy Whipple, Mike Zeiders, and Paula Zuchowski. "Although the research described in this article has been funded wholly or in part by the United States Environmental Protection Agency through award #R-817206-01-0 to Dr. William A. Dunson, it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred."

= -0.219 + 0.00388 (X) = 0.795 - 0.00583 (X) = 0.262 + 0.00656 (X) = 0.607 + 0.00698 (X) = 0.722 - 0.00254 (X)

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Manuscript received April 4, 1992 and in revised form May 29, 1992.