Accumulation of Toxicants in Tadpoles of the Common Frog (Rana temporaria) inHigh Mountains

R. Hofer,1 R. Lackner,1 G. Lorbeer2

1 Institut f�r Zoologie und Limnologie, Universit�t Innsbruck, Technikerstrasse 25, A-6020 Innsbruck, Austria2 Umweltbundesamt, Spittelauer L�nde 5, A-1090, Wien, Germany

Received: 2 September 2004 /Accepted: 7 March 2005

Abstract. We analyzed the accumulation of inorganic andorganic toxicants in tadpoles from nine populations of thecommon frog (Rana temporaria) at different altitudes of theHohe Tauern National Park in Austria). Lead and cadmiumconcentrations in tadpoles increased with the acidity and me-tal-to-HCO3

– ratio in the water exceeding those found in tad-poles from agricultural areas. Organochlorines (DDTs,polychlorinated biphenyls, lindane) and polyaromatic hydro-carbons were present in all of the populations, but only DDTsaccumulated significantly compared with the concentrations intheir diet.

At Alpine low altitudes, populations of the common frog(Rana temporaria) progressively decreased or even disap-peared because of the destruction of breeding sites and habitatfragmentation during the last century (Landmann et al. 1999).At and above the timberline, however, the common frog isabundant, reaching high population densities even at altitudesup to 2,400 to 2,600 m (Grossenbacher 1997). With increasingaltitude, the risk of predation decreases, but physical andchemical factors become more important for the survival oftadpoles and frogs. At high altitudes, the tadpoles at theembryonic stage are the most threatened. Egg clutchesdeposited at the shallow edges of the ponds often desiccatebefore the embryos are hatched. Acid runoff during thesnowmelt period or heavy rain is another risk in ponds ofcrystalline catchments (Faber 2000). Acidic and low-alkalinewater may also increase the mobilization of metals from theground and the uptake of toxicants of geogenic and atmo-spheric origin as demonstrated in fish from high mountainlakes (Kçck et al. 1995; Kçck and Hofer 1998; Vives et al.2004). Because of their complex living cycle between aquaticand terrestrial habitats and their permeable skin, amphibiansare sensitive to environmental changes (Duellman and Trueb1994). During the last decades, a global decrease of amphib-

ians has been observed, caused by different, often additivefactors including the increase of toxicants even in pristineareas (Green 1997). Toxicologic investigations of amphibianlarvae have focused on the effects of metals and pesticides ongrowth, development, and behavior (e.g., Steele et al. 1999;Diana et al. 2000; Fordham et al. 2001; Westerman et al.2003), but only very small amounts of data on bioaccumula-tion under natural conditions have been published (e.g.,Grillitsch and Chovanec 1995; Burger and Snodgrass 1998,2001). Despite their short life span, tadpoles are suitable bio-indicators because they are typical substrate feeders with highingestion rates of potentially contaminated food. The goal ofthis study was to investigate the contamination of frog tadpolesin an almost undisturbed mountain environment by geogenicand atmospheric deposited inorganic and organic toxicants.

Material and Methods

The Hohe Tauern National Park in Austria is the largest protected areain Central Europe and covers almost 1,800 km2 of valleys andmountains with elevations between 1,200 and 3,798 m. Tadpoles fromnine sites at different parts (Salzburg, Osttirol, and K�rnten) andaltitudes (1,465 to 2,150 m) of the national park were investigatedduring comparable warm and dry summer periods (Table 1).Water samples of each site were analyzed after the limnologic

routine methods of Wathne (1996). Water pH was determined with anIngold pH electrode, which is recommended for water of low-ionicstrength. For the analyses of cadmium and lead, the water was filteredthrough a 0.45-lm cellulose acetate membrane and acidified imme-diately after sampling. Dissolved reactive aluminum was analyzed infiltered samples (Dougan and Wilson 1974).

Tadpoles

At each site, approximately 400 tadpoles at the developmental stagesbetween 32 and 37 (Gosner 1960) were killed with MS222, and theintestinal tracts and carcasses were collected separately in acid andchloroform–cleaned glass tubes. The filled intestine made up 33% to45% of the total weight. The samples were cooled in ice, transferred tothe laboratory, frozen, and freeze-dried until constant weight.Correspondence to: R. Hofer; email: rudolf.hofer@uibk.ac.at

Arch. Environ. Contam. Toxicol. 49, 192–199 (2005)DOI: 10.1007/s00244-004-0188-8

Cd and Pb

Dried samples were digested in a microwave oven using ultra-pureHNO3 and H2O2 and analyzed twice by way of graphite furnaceatomic absorption spectrometry. Detection limits were 0.05 and 0.5lgÆg–1 dry matter for Cd and Pb, respectively. The aluminum con-centration was determined in the ash of samples, which was dissolvedin 0.1N HCl. The supernatant was diluted and analyzed photometri-cally (Dougan and Wilson 1974). Metal analyses of the intestine alsoincluded the mineral portion of ingested sediments.

Organochlorines

The freeze-dried, homogenized samples (2 g) were spiked with car-bon-13–labeled surrogates [p,p-DDE; p,p-DDT; polychlorinated bi-phenyls (PCBs) 28, 52, 101, 138, 153, and 180; and gamma–halogenated cyclic hydrocarbon] and extracted with 300 ml n-hexaneby Soxhlet for 24 hours. Cleanup was carried out with Florisil (U.S.Silica) and conditioned with 6% water. Elution was performed with150 ml n-hexane/toluene (9:1). After concentration, another cleanupstep on silica oxide (3g) covered by sodium sulfate was carried outwith 40 ml n-hexane and 50 ml n-hexane/toluene (65:35) as the sol-vent. After another concentration step before gas chromatography–mass spectrometry (GC-MS) analysis, decachlorobiphenyl as aninternal standard was added. The GC-MS analysis was carried outusing a Fisons GC Top/Voyager GC-MS System with El using a se-lected ion–monitoring mode (Thermo Electron). A fused silica cap-illary column (60 m x 0.25 mm i.d; 0.25-lm film thickness) of cross-linked DB-5 MS (J&W) was used. The operating conditions were asfollows: column temperature, programmed from 90�C to 190�C at6�CÆmin-1 and held for 5 minutes, then from 190�C to 330�C at5�CÆmin–1 and held at 330�C for 10 minutes. Injection temperaturewas 270�C; transfer line temperature was 330�C; and source tem-perature was 255�C. Quantification was done by external calibrationincluding the recovery rates of the labeled surrogates. Recovery rateswere >70%, and the detection limit was 0.5lgÆkg–1dry weight.

PAHs

The freeze-dried, homogenized samples (2 g) were spiked with deu-terated fluoranthene and deuterated benzo(a)pyrene and extractedwith 150 ml n-hexane by Soxhlet for 8 hours. Cleanup was performed

with solid-phase extraction using columns containing 1 g silica and0.5 g cyanoprpoylsilene conditioned with n-hexane. Elution wasperformed with 7.5 ml n-hexane and dichlormethane (1:1). The ex-tract was concentrated and the solvent replaced with 1 ml acetonitrilebefore high-pressure liquid chromatography analysis. PAHs wereseparated on a PAH 16-Plus (20 x 3.0 mm; thermostated at 30�C)using a gradient of acetonitrile in water at 0.5 mlÆmin–1 and detectedby a programmable fluorescence detector. Quantification was done byexternal calibration including the recovery rates of the added surro-gates. Fluoranthene was corrected with the recovery of deuteratedfluoranthene, and all other analytes were corrected with the recoveryof deuterated benzo(a)pyrene. Recovery rates were >70%, and thedetection limit was 5lgÆkg-1 dry matter.

Parameters of Oxidative Stress

The livers of 20 tadpoles were shock-frozen in liquid nitrogenimmediately at the sampling site, transferred to the laboratory, andkept at –80�C until further use. Each liver was homogenized in 0.2 ml10% metaphosphoric acid and centrifuged. Glutathione (GSH) andglutathione disulfide (GSSG) were analyzed by HPLC (Hofer et al.2001). MS222 has proven to be a suitable anesthetic for this type ofanalysis in fish (Lackne 1998, unpublished results).

Morphology

Tadpoles from each site (40 to 60) were killed in MS222 and fixed informalin and ethanol (4%/35%). In the laboratory, body lengths weremeasured and developmental stages determined (Gosner 1960).

Statistical Analyses

The chemical data of the tadpoles represent the means of two inde-pendent measurements from pooled samples. The correlations be-tween variables were calculated with the Spearman rank order (squarecoefficient tables) and Pearson product-moment correlation coeffi-cient (where a linear relationship is assumed). Nonlinear correlationwas calculated after logarithmic transformation. The differences ofbody lengths at stages 36 and 37 were tested with one-way analysis ofvariance (ANOVA).

Table 1. Characterization of the sampling sites and the total body length (means and SE) of tadpole populations

Site code Sampling site Altitude (m) Remarkable water parametersTotal body length in mmat stages 36 and 37

SalzburgA Ofner Alm (Hollersbachtal) 1465 46.7 € 0.5 (18)B Hintermoos (Hollersbachtal) 2009 Ptot, conductivity, Ca 36.7 € 0.5 (39)C Postalm (Obersulzbachtal) 1690 32.9 € 0.3 (25)

OsttirolD Auge Gottes (Innergschlçß) 2150 pH, conductivity, Ca 39.9 € 0.4 (41)E Seichenbrunn (Debanttal) 1686 32.6 € 0.4 (22)F Pfauenauge (Arvental) 2080 Al 35.5 € 0.3 (38)

K�rntenG Winkler Almsee 2020 39.5 € 0.6 (22)M4 Jamnigh�tte (Tauerntal) 1770 pH, Pb, Cd, Al, Ca, DOC 30.3 € 0.2 (42)M17 Laschgboden (Tauerntal) 2110 pH, Pb, Cd, Al, P, Ca, DOC 43.4 € 0.4 (40)

Number of specimens in parantheses.

Toxicants in Tadpoles of the Common Frog (Rana temporaria) 193

Results

Water Analysis

All ponds investigated contained soft water of neutral to acidcondition (pH 7.3 to 5.0) with alkalinity between 2 and 183lequ.l–1 and a conductivity of 5 to 46 lS.cm-1 (Table 2).Ponds with the most extreme conditions were situated in theTauerntal near Malnitz (sites M4 and M17) with high con-centrations of DOC and metals and low pH. Nevertheless,these ponds contained dense populations of tadpoles. Leadconcentrations in the water were correlated with those of Cdand Al (Table 3).

Growth of Tadpoles

The total length of tadpoles of comparable developmentalstages (stages 36 and 37) was site specific (p <0.001) with thesmallest specimens at sites C, E, and M4 (<33 mm) and well-growing (>43 mm) populations at sites A and M17 (Table 1).Neither water chemistry nor the contamination of tadpoles hadnegative effects on their size (Table 3). The correlation be-tween sizes at stages 36 and 37 and lindane in the carcassesand Cd in the intestines cannot be explained.

Accumulation of Toxicants

Compared with body tissues (carcasses), the intestinal tracts(intestines with contents) contained Pb one order of magnitudehigher concentrations, whereas for Cd, the ratio between car-casses and intestines was variable (Table 4). The highestconcentrations of Pb and Cd in the tadpole carcasses and in thewater were measured in frog populations at Tauerntal (sitesM4 and M17) and Innergschlçß (site D). Pb and Cd correlatedpositively in tadpole carcasses (Figure 1), but no significantcorrelation of these metals was found in tadpole intestines(Table 3). A correlation between the metal concentrations incarcasses and intestines was seen for Pb but not for Cd (Fig-ure 2).Metal accumulation in tadpole carcasses significantly in-

creased at low pH (Figure 3) and higher metal-to-HCO3 ratiosin the water (Figure 4). Al accumulated in tadpoles, but theconcentration in carcasses did not correlate with any otherparameters (Tables 3 and 4). The high concentration of alu-minum found in tadpole intestine derived mainly from themineral portion of the diet.The organochlorine load was relatively low because of the

young age of the tadpoles (approximately 2 months) and theabsence of significant lipid stores, which is the typical sink forlipophilic substances (Table 5). Nevertheless, there was asignificant accumulation of DDT fractions in the bodiescompared with the diet (filled intestine), with remarkable highconcentrations (in particular, the fraction of non degradedp,p,DDT) found in populations from Hollersbachtal (sites Aand B). Almost uniform concentrations and no evidence ofaccumulation (no differences between intestines and carcasses)were found for lindane and PCBs. PCBs 28 (3.0 to 5.6 lgÆkg-1T

able2.

Analysisofpond

waterobtained

onthedaythattadpolesweresampled

Site

code

Cond

(lS)

pHSO4

(mgl)

Cl

(mg/l)

Na

(mg/l)

K (mg/l)

Mg

(mg/l)

Ca

(mg/l)

Alk[HCO3]

(leq)

Ptot

(lg/l)

Pdis

(lg/l)

DOC

(mg/l)

NO3-N

(lg/l)

NH4-N

(lg/l)

DN

(mg/l)

Al

(lg/l)

Cd

(lg/l)

Pb(lg/l)

A24.9

7.07

3.15

0.11

0.25

1.05

0.22

3.59

140

12.1

3.5

0.88

136

80.539

229

0.008

0.053

B7.8

6.61

0.22

0.12

0.20

0.87

0.25

0.88

73193.5

32.4

3.33

014

0.308

448

0.008

0.405

C16.9

6.91

1.16

0.21

0.23

1.11

0.21

2.35

123

10.3

7.9

1.10

106

210.439

374

0.001

0.292

D5.3

5.51

0.60

0.18

0.09

0.23

0.16

0.66

1931.8

18.2

3.63

03

0.549

240

0.010

0.403

E45.6

7.27

6.08

0.19

0.90

1.22

0.79

5.79

283

12.6

7.9

1.24

724

0.335

348

0.004

0.516

F42.7

6.55

1.65

0.18

0.14

1.16

1.54

5.17

372

106.5

74.7

1.32

036

0.480

861

0.019

0.751

G16.9

6.78

2.93

0.18

0.90

0.74

0.35

1.57

7916.5

7.1

4.85

755

0.735

246

0.010

0.211

M4

10.6

4.96

0.22

0.42

0.30

0.26

0.26

0.74

270.9

18.5

12.01

01

0.686

1229

0.028

2.418

M17

10.9

5.85

0.74

0.67

0.34

1.79

0.29

0.65

63214.7

58.8

7.79

09

0.807

1121

0.027

2.109

Cond=conductivity,25�C.

Ptot=totalphosphorous.

Pdis=dissolvedphosphorous.

Doc

=Dissolved

organiccarbon.

194 R. Hofer et al.

dry carcass) and 153 (1.1 to 3.0 lgÆkg–1) were the dominantcongeners of PCB. The correlation between lindane and theconcentrations of Pb and Al in the water cannot be explained.Among the PAHs, fluoranthene attained concentrations be-tween 19 and 39.5 lgÆkg–1 dry carcass or intestine.Benzo(b)fluoranthene, benzo(k)fluoranthene, benz(a)pyrene,and indeno(1,2,3-c,d)pyrene were below the detection limits.The concentration of GSH in tadpole livers, an important

protectant against oxidative stress, and that of GSSG, theoxidation product of GSH, was similar in most of the popu-lations (Table 6). Only in tadpoles from sites A and M17,which were the best-growing populations, was the GSSG-to-GSH ratio (the most powerful indicator of oxidative stress)significantly lower. However, no significant correlations werefound with toxicants accumulated in tadpole tissues, but anegative correlation was found with the total length of tadpolesat stages 36 and 37 (r = –0.79; p <0.05).

Discussion

The expected pH of most of the ponds of the Hohen Tauern isbetween 6 and 7 because their buffering capacity is low.During the sampling period in the summer, only two pondsinvestigated (sites D and M4) contained water with pH <5.8,the natural pH of pure water in equilibration with atmosphericcarbon dioxide. In pristine mountain areas, most of the acid isof atmospheric origin (Skjelkvale and Wright 1998). Particu-larly in very small ponds, the typical habitat of R. temporariatadpoles, the pH can decrease suddenly to <pH 4 (even to pH2.3) during rainy periods (Faber 2000), and sphagnum mosses,if present, stabilize the acid (Olsson et al. 1987). The soil ofthe catchment is an additional source of acids by releasinghumic and fulvic acids into ponds and giving the water abrownish color. Tadpoles at the embryonic stage are the mostsensitive at critical (median lethal tolerance or LC50) pH levels

Table 3. Spearman rank order correlations between toxicants in the water, gut, and carcassa

Water Carcass

Alk Cd Pb Al Cd/HCO3 Pb/HCO3 Tadpole bodylength (mm)

Cd Pb Al Lindan p, p-DDE Intestine Cd

WaterCd )0.45 –Pb )0.30 0.76 –Al )0.28 0.61 0.88 –Cd/HCO3 – – 0.53 0.33 –Pb/HCO3 – 0.57 – 0.58 0.68 –

TadpoleBody length 0.12 )0.11 )0.45 )0.57 0.07 )0.21 –CarcassCd )0.92 0.50 0.45 0.29 0.78 0.86 0.05 –Pb )0.82 0.66 0.60 0.40 0.85 0.80 0.07 0.95 –Al 0.47 )0.27 0.23 0.17 )0.53 )0.25 )0.67 )0.33 )0.35 –Lindane 0.33 )0.48 )0.74 )0.88 )0.14 )0.55 0.62 )0.28 )0.31 )0.48 –p,p)DDE )0.22 0.41 0.12 )0.13 0.53 0.19 0.79 0.28 0.43 )0.66 0.36 –

IntestineCd 0.27 )0.06 )0.21 )0.24 )0.03 )0.33 0.83 )0.18 0.01 )0.50 0.43 0.65 –Pb )0.83 0.59 0.48 0.31 0.79 0.82 0.28 0.91 0.95 )0.44 )0.22 0.62 0.13

a Bold correlations are significant at p < 0.05.

Table 4. Metal concentrations in tadpole carcass and filled intestine

Cd (lg.g)1 dry weight) Pb (lg.g)1 dry weight) Al (lg.g)1 dry weight)

Site code Carcass Intestine Carcass Intestine Carcass Intestine

SalzburgA 0.08 0.73 1.4 26.5 5.3 4,499B 0.20 0.68 2.1 33.5 8.1 11,115C 0.08 0.27 1.2 10.0 18.3 8,466

OsttirolD 0.43 0.41 4.0 50.0 11.8 5,044E 0.11 0.61 1.8 28.5 68.5 7,980F 0.05 0.42 1.3 19.5 24.5 10,574

K�rntenG 0.11 0.57 1.7 29.0 15.7 15,623M4 0.45 0.26 7.0 50.0 14.6 4,292M17 0.31 0.73 4.2 95.0 8.6 7,196

Toxicants in Tadpoles of the Common Frog (Rana temporaria) 195

between 4.0 and 4.5 (Bçhmer and Rahmann 1990). Sublethaleffects are seen in tadpoles even when pH is slightly below 5,leading to growth decrease and a delay of metamorphosis(Cummins 1986).Acidification increases the solubility of metals, specifically

Pb, and shifts the chemical speciation towards free ions, whichmay lead to an accelerated uptake of metals (Stripp et al.1990). Furthermore, low alkalinity and high temperature, as

seen in the shallow sun-exposed ponds inhabited by tadpoles,enhance metal uptake (Kçck and Hofer 1998). Some of theseeffects on the accumulation of Pb and Cd in tadpoles of theHohen Tauern, where high acidity or metal-to-HCO3 ratios ofthe water increase metal accumulation, are illustrated in Fig-ures 3 and 4. In contrast, humic acids may lead to lowerbioavailability for organisms because they form large, polar

Fig. 2. Correlation between cadmium and lead in the carcasses andfilled intestine of tadpoles (the Pearson product-moment correlationcoefficient is given).

Fig. 3. The effect of water pH on the accumulation of cadmium andlead in the carcasses of tadpoles (the Pearson product-moment cor-relation coefficient is given).

Fig. 1. Correlation between cadmium and lead in the carcasses oftadpoles (the Pearson product-moment correlation coefficient is gi-ven).

Fig. 4. The effects of the metal-to-HCO3 ratio in water on theaccumulation of cadmium and lead in the carcasses of tadpoles (thePearson product-moment correlation coefficient is given).

196 R. Hofer et al.

complexes with metals and organic toxicants (Hutchinson andSprague 1987; Haitzer et al. 1998). In tadpoles of the HohenTauern, however, no effect of dissolved organic carbon (DOC)on metal uptake could be observed.The direct anthropogenic impact on the environment of the

national park is small and limited by its use as pastures and formoderate tourism. Both types of land use alter the landscapebut do not contribute to a significant input of toxicants. Incontrast, the area receives globally distributed toxicants byway of wet and dry atmospheric deposition (Sucharova andSuchara 1998; Wania 1999), and the steep slopes of the HohenTauern facilitate the condensation of semivolatile chemicals(Wania and Mackay 1996). Metals that accumulated inorganisms are mainly of geogenic origin, although atmo-spheric deposition must also be considered (Nriagu 1979).Dependent on the chemical properties of toxicants and

environmental factors (Witeska and Jezierska 2003), aquaticorganisms absorb toxicants from their diet or at their surface.Dietary uptake dominates in almost insoluble chemicalsincluding highly lipophilic compounds. This may lead tobiomagnification along the food web (Gobas et al. 1999). Incontrast, skin and gills are the major site of the uptake of

metals and hydrophilic substances (Vogiatzis and Loumbour-dis 1997; McKim et al. 1985). Because of the dramaticreconstruction of organs during metamorphosis, accumulatedchemicals are remobilized within the animals and thus maycause damage during this period.To date, only a limited amount of data on the toxicant load of

natural populations of tadpoles have been published. Analysisof tadpoles of R. dalmatina, R. ridibunda, and Bufo bufo fromagricultural areas in Lower Austria revealed concentrations ofCd and Pb of 0.07 to 0.28 ppm and 2.45 and 5.70 ppm,respectively (Grillitsch and Chovanec 1995). The lowest con-centrations were found in R. dalmatina, the species most clo-sely related to R. temporaria. At least in some populations ofthe Hohen Tauern, the metal load, in particular cadmium, ishigher than that in related lowland species. These values alsoexceed those found in the carcasses of European minnows(Phoxinus phoxinus), age 5 to 7 years, from Alpine high-mountain lakes (Hofer et al. 2001). Similar concentrations ofCd and Pb as found in tadpoles of the Hohen Tauern have beenreported in tadpoles of R. utricularia in South Carolina (Burgerand Snodgrass 2001), whereas the contamination of R. cates-beiana tadpoles with Pb was lower, even at polluted sites (0.3

Table 5. Concentrations of organochlorides in the carcass and filled intestine

p,p-DDE(lgÆkg)1 dry weight)

p,p-DDD + p,p-DDT(lgÆkg)1 dry weight)

p,p-DDT(lgÆkg)1 dry weight)

Lindane(lgÆkg)1 dry weight)

Site code Carcass Intestine Carcass Intestine Carcass Intestine Carcass Intestine

SalzburgA 2.7 <1 27.5 7.2 1.7 <1 5.3 3.5B 2.3 <1 27.0 9.0 1.0 <1 4.4 7.0C <1 <1 <5 <5 <1 <1 3.2 2.8

OsttirolD 3.4 1.4 8.2 6.8 <1 <1 4.9 2.9E 1.0 <1 <5 <5 <1 <1 2.6 2.5F 1.7 <1 <5 <5 <1 <1 3.7 3.5

K�rntenG 1.6 <1 4.5 6.9 <1 <1 3.8 4.9M4 1.6 1.3 <5 <5 <1 <1 2.8 2.6M17 5.5 1.4 8.3 <5 <1 <1 3.0 3.7

Table 6. GSH, GSSG, and GSSG-to-GSH ratios in tadpoles liversa

Site code GSH (lMÆg-1protein) GSSG (lMÆg)1protein) (GSSG/GSH)*103

SalzburgA 13.1 (11.8/14.2) 0.06 (0.05/0.09) 4.9 (3.6/6.3)B 11.8 (10.2/16.2) 0.26 (0.08/0.41) 25.2 (6.2/33.6)C 10.3 (8.4/11.6) 0.14 (0.09/0.17) 13.4 (8.1/21.3)

OsttirolD 15.3 (12.6/17.5) 0.24 (0.17/0.32) 13.7 (9.8/21.7)E 9.0 (7.0/9.7) 0.16 (0.14/0.17) 18.3 (16.4/22.8)F 10.6 (9.3/12.7) 0.18 (0.10/0.22) 13.8 (9.5/26.3)

K�rntenG 13.2 (9.9/15.3) 0.26 (0.21/0.34) 21.9 (18.7/24.3)M4 13.2 (11.5/15.9) 0.36 (0.25/0.44) 24.4 (20.8/34.1)M17 15.6 (13.3/17.5) 0.11 (0.09/0.15) 6.6 (5.9/10.9)

a Median and quartiles of 12 to 20 specimens in parentheses.High GSSG-to-GSH ratios are indicators of oxidative stress.GSH: Glutathione.GSSG: Glutathione disulfide.

Toxicants in Tadpoles of the Common Frog (Rana temporaria) 197

lgÆg–1 dry weight of tadpole tails; Burger and Snodgrass 1998).In contrast to the high gill toxicity of Al in fish (Wood andMcDonald 1987), tadpoles are less sensitive. The concentrationof dissolved Al in some ponds of the Hohen Tauern was ex-tremely high, exceeding 1 mgÆL–1. Metamorphosis of R.temporaria tadpoles exposed to 0.8 mgÆL–1 Al under extremelyacidic conditions of pH 4.4 (close to lethal pH values) and inthe absence of DOC was delayed, and the size at which theyattained metamorphosis was lower (Cummins 1986); evendoubled concentrations caused mortality only in the smallestspecimens. Olsson et al. (1987) found scoliosis, skin damage,and disturbed feeding behavior when embryos of R. temporariaand R. arvalis were exposed to aluminum concentrations of 0.8and 1.6 mgÆL–1, respectively. Although tadpoles at sites M4 andM17 lived under similar conditions, none of these changescould be observed in the field that might be related to theprotecting action of DOC.Tadpoles of the Hohen Tauern contained low concentra-

tions of organic toxicants originating from atmosphericdeposition, except those of DDT compounds in two popula-tions of the Hollersbachtal (sites A and B). However, thepredominance of primary p,p-DDT fractions, compared withthe more degraded p,p-DDE in other tadpole populations andin fish from high mountain lakes, is noteworthy (Hofer et al.2001). DDE concentrations in tadpoles of Hyla regilla fromthe Sierra Nevada and Canada are at least one order ofmagnitude higher than in those found in the current investi-gation (Datta et al. 1998; Russell et al. 1997). Tadpoles ofthe Hohen Tauern contained lindane concentrations one orderof magnitude lower than tadpoles of R. dalmatina, R. ridib-unda, and B. bufo from an agricultural area (Grillitsch andChovanec 1995).GSSG-to-GSH ratios, the most powerful indicator of oxi-

dative stress (Lackner 1998), in tadpole livers did not correlatewith toxicologic parameters, but they did correlate negativelywith the growth (body length) of tadpoles of comparable stage.The livers of these poorly growing populations contained al-most no glycogen and a high density of melanomacrophages(Hofer, unpublished results). Because the same histologicsymptoms were observed in laboratory experiments with tad-poles to which a limited diet was offered, we conclude that themacrophages induced by decreased food resources are thereason for oxidative stress in the tadpole populations of theHohen Tauern.

Acknowledgments. Supported by the National Park Hohe Tauern.

References

Bçhmer J, Rahmann H (1990) Influence of surface water acidificationon amphibians. In: Hanke W (ed) Biology and physiology ofamphibians. Fortschr Zool 38:287–309

Burger J, Snodgrass J (1998) Heavy metals in bullfrog (Rana cates-beiana) tadpoles: Effects of depuration before analysis. EnvironToxicol Chem 17:2203–2209

Burger J, Snodgrass J (2001) Metal levels in southern leopard frogsfrom the Savannah River site: Location and body compartmenteffects. Environ Res 86:157–166

Cummins CP (1986) Effects of aluminium and low pH on growth anddevelopment of Rana temporaria tadpoles. Oecologia 69:248–252

Datta S, Hansen L, McConnell L, Baker J, LeNoir J, Seiber N (1998)Pesticides and PCB contaminants in fish and tadpoles from theKaweah River Basin, California. Bull Environ Contam Toxicol60:829–836

Diana SG, Resetarits WJ Jr, Schaeffer DJ, Beckmen KB, Beasley VR(2000) Effects of atrazine on amphibian growth and survival inartificial aquatic communities. Environ Toxicol Chem 19:2961–2967

Duellman WE, Trueb L (1994) Biology of amphibia. John HopkinsUniversity Press, Baltimore, MD, p 670

Faber H (2000) Short-term acidification pulses in alpine breedingponds. Biota 1:17–18

Fordham CL, Tessari JD, Ramsdell HS, Keefe TM (2001) Effects ofmalathione on survival, growth, development, and equilibriumposture of bullfrog tadpoles (Rana catesbeiana). Environ ToxicolChem 20:179–184

Gobas FAPC, Wilcockson JB, Russell RW, Haffner GD (1999)Mechanism of biomagnification in fish under laboratory and fieldconditions. Environ Sci Technol 33:133–141

Gosner KL (1960) A simplified table for staging anuran embryos andlarvae with notes on identification. Herpetologica 16:183–190

Green, (eds) (1997) Amphibians in decline. Society for the Study ofAmphibians and Reptiles, Saint Louis, MO, p 338

Grillitsch B, Chovanec A (1995) Heavy metals and pesticides inanuran spawn and tadpoles, water, and sediment. Toxicol EnvironChem 50:131–155

Grossenbacher K (1997) Rana temporaria Linnaeus, 1758. In: GascJP, Cabela A, Crnobrnja-Isailovic J, Dolmen D, Grossenbacher K,Haffner P, et al. (eds) Atlas of amphibians and reptiles in Europe.Paris Societas Europaea Herpetologica and Museum Nationald�Histoire Naturelle. Paris, France, pp 158–159

Haitzer M, Hçss S, Traunspruger W, Steinberg C (1998) Effects ofdissolved organic matter (DOM) on the bioconcentration of or-ganic chemicals in aquatic organisms. A review. Chemosphere37:335–1362

Hofer R, Lackner R, Kargl J, Thaler B, Tait D, Bonetti L, et al. (eds)(2001) Organochlorine and metal accumulation in fish (Phoxinusphoxinus) along a north-south transect in the Alps. Water Air SoilPollut 125:189–200

Hutchinson NJ, Sprague JB (1987) Reduced lethality of Al, Zn and Cumixtures to American flagfish by complexation with humic sub-stances in acidified soft waters. Environ Toxicol Chem 6:755–765

Kçck G, Hofer R, Wçgrath S (1995) Accumulation of trace metals(Cd, Pb, Cu, Zn) in Arctic char (Salvelinus alpinus) from oligo-trophic Alpine lakes: Relation to alkalinity. Can J Fish Aquat Sci52:2367–2376

Kçck G, Hofer R (1998) Origin of cadmium and lead in clear soft-water lakes of high altitude and high latitude and their bioavail-ability and toxicity to fish. In: Braunbeck T, Hinton DE, Streit B(eds) Fish ecotoxicology. Birkh�user Verlag, Bern, Switzerland,pp 225–257

Lackner R (1998) ‘‘Oxidative stress’’ in fish by environmental pol-lutants. In: Braunbeck T, Hinton DE, Streit B (eds) Fish eco-toxicology. Birkh�user Verlag, Bern, Switzerland, pp 203–224

Landmann A, Bçhm C, Fischler D (1999) Bestandssituation undGef�hrdung des Grasfrosches (Rana temporaria) in Talbçden derOstalpen: Beziehungen zwischen der Grçße von Laichpopula-tionen und dem Landschaftscharakter. Z Tkologie Naturschutz8:71–79

McKim J, Schmieder P, Veith G (1985) Absorption dynamics of or-ganic chemical transport across gills and related octanol-waterpartition coefficient. Toxicol Appl Pharmacol 77:1–10

Nriagu JO (1979) Global inventory of natural and anthropogenicemissions of trace metals to the atmosphere. Nature 279:409–411

198 R. Hofer et al.

Olsson M, Hogstrand C, Dahlber A, Berglind SA (1987) Acid-shock,aluminium, and presence of Spagnum aurantiacum: Effects onembryological development in the common frog, Rana tempo-raria and the moor frog, Rana arvalis. Bull Environ ContamToxicol 39:37–44

Russell RW, Gillan KA, Hafner GD (1997) Polychlorinated biphenylsand chlorinated pesticides in southern Ontario, Canada, greenfrog. Environ Toxicol Chem 16:2258–2263

Skjelkvale BL, Wright RF (1998) Mountain lakes: Sensitivity to aciddeposition and global climate change. Ambio 27:280–286

Steele CW, Strickler Shaw S, Taylor DH (1999) Effects of sublethallead exposure on the behaviours of green frog (Rana calamitas),bullfrog (Rana catesbiana) and American toad (Bufo americanus)tadpoles. Mar Fresh Behav Physiol 32:1–16

Steinwascher K (1978) Interference and exploitation competi-tion among tadpoles of Rana utricularia. Ecology59:1039–1046

Stripp RA, Heit M, Bogen DC, Bidanset J, Trombetta L (1990) Traceelement accumulation in the tissues of fish from lakes with dif-ferent pH values. Water Air Soil Pollut 51:75–87

Sucharova I, Suchara I (1998) Atmospheric deposition levels ofchosen elements in the Czech Republic determined in theframework of the International Biomonitoring Program 1995. SciTotal Environ 223:7–52

Vives I, Grimalt JO, Catalan J, Rosseland BO, Battarbee RW (2004)Influence of altitude and age in the accumulation of organo-chlorine compounds in fish from high mountain lakes. EnvironSci Technol 38:690–698

Vogiatzis AK, Loumbourdis NS (1997) Uptake, tissue distribution,and depuration of cadmium (Cd) in the frog Rana ridibunda. BullEnviron Contam Toxicol 59:770–776

Wania F (1999) On the origin of elevated levels of persistent chem-icals in the environment. Environ Sci Pollut Res 6:11–19

Wania F, Mackay D (1996) Tracking the distribution of persistentorganic pollutants. Environ Sci Technol 30:390A–396A

Wathne B (ed) (1996): MOLAR—Project manual draft of September1996. NIVA Rep. No 0-96061, 182; NIVA, Oslo, Norway, p 182

West LB (1960) The nature of growth inhibitory material fromcrowded Rana pipiens tadpoles. Physiol Zool 33:232–239

Westerman AG, Wiggington AJ, Price DJ, Linder G, Birge WJ (2003)Integrating amphibians into ecological risk assessment strategies.In: Linder G, Krest SK, Sparling DW, (eds) Amphibian decline:An integrated analysis of multiple stressor effects. SETAC,Pensacola, FL, pp 283–313

Witeska M, Jezierska B (2003) The effects of environmental factors onmetal toxicity to fish (review). Fresenius Environ Bull 12:824–829

Wood CM, McDonald DG (1987) The physiology of acid/aluminiumstress in trout. Ann Soc R Zool Belg 117:399–410

Toxicants in Tadpoles of the Common Frog (Rana temporaria) 199