Metal concentrations of tadpoles in experimental ponds

Environmnrd

Pollution, Vol. 91, No. 2, pp. 14%159, 1996 E1swie.r Science Ltd printed in Great Britain

0269-7491(95)00057-7

ELSEVlER

METAL CONCENTRATIONS EXPERIMENTAL

OF TADPOLES PONDS

IN

Donald W. Sparling & T. Peter Lowe National Biological Service, Patuxent Environmental Science Center, 11510 American Holly Drive, Laurel, MD 20708, USA (Received 20 January

1995; accepted 29 June 1995)

Abstract Anuran tadpoles are found in a variety of habitats, many of which are acidified or have high ambient concentrations of metals from anthropogenic sources. A few studies that have been conducted on metals in tadpoles demonstrate that they can contain high concentrations of some metals but have not demonstrated clear relationships between ambient conditions and metal concentrations. This study examines the influence of soil, water treatment, amphibian species, and body portion analyzed on metal concentration in tadpoles. In northern cricket frogs, gray treefrogs, and green frogs, concentrations of Al and Fe exceeded 10000 ug.g-’ and Mg and Mn exceeded 1000 pg g-l. Body concentrations of Ba, Be, Fe, Mg, Mn, Ni, Pb, and Sr increased with soil concentrations. Acidtfication reduced body concentrations of Be and Sr, and pH correlated with Be, Mg, and Sr. Gray treefrogs had significantly lower concentrations of most metals compared to northern cricket frogs, possibly because of dtrerences in microhabitats and soil ingestion. More than half of most metals was sequestered in the gut coil of green frog tadpoles, probably mixed with soil. Depending on bioavailablity, many of the metals in gut coils and whole bodies of these tadpoles could be potentially toxic to predators.

Aluminum is one of the metals with greatest concern in acidified environments (Sparling and Lowe, in press). Aluminum is the most common metal and the third most common mineral in the earth’s crust. Under circumneutral conditions its bioavailability is low but at pH < 5.5, Al becomes increasingly soluble and available (Driscoll and Schecher, 1990). Acid deposition can leach Al from watersheds into streams and ponds where it can reach toxic concentrations. Anuran populations are of concern in acidified areas for several reasons. First, there seems to be a worldwide decrease in amphibian populations (Baringa, 1990; Pechmann and Wilbur, 1994). Second, amphibian larvae are sensitive to elevated aqueous concentrations of several metals including Al, Cu, Cd, Fe, Pb, and Zn (Freda, 1991). Third, tadpoles consume detritus or periphyton, are often closely associated with benthic habitats, and tend to accumulate metals in relation to ambient concentrations (Hall and Mulhem, 1984; Birdsall et al., 1986). Both adult frogs and tadpoles also accumulate some metals at higher than ambient concentrations (Niethammer et al., 1985). Only a few studies have related metal concentrations in tadpoles with those in sediments and we have been unable to find any which specifically examined the effects of acidification or water treatment on body burdens of metals. The purpose of this study was to describe metal concentrations in anuran tadpoles collected from ponds that have been experimentally treated with acid and Al. Specifically, we determined: (i) if relationships occur between soil and body metal concentrations; (ii) if water chemistry associated with acidification or Al additions affects metal concentrations in tadpoles; and (iii) if there are differences in metal concentrations in tadpoles due to species or to part of body examined.

INTRODUCTION Anthropogenic metal contamination of freshwater wetlands through runoff, acid mine drainage, atmospheric deposition, and similar activities is a serious, widespread problem with demonstrably negative effects on wetland quality and biota. Acidic deposition, which is prevalent through the northeastern United States and eastern Canada, compounds the problem of metal contamination because reduced pH increases the solubility of many metals such as aluminum, increasing their mobility in soils and their bioavailability in water (Campbell and Stokes, 1985; LaZerte, 1986). However, metal toxicity is complex and varies with the concentration of ligands such as dissolved organic carbons (DOC), pH, and the presence of other metals that may either act synergistically or competitively (Campbell and Stokes, 1985; Freda et al., 1989; Freda, 1991; Spry and Wiener, 1991).

METHODS Pond conslmction and characteristics

This study occurred during 1991 as part of a larger investigation on the effects of acidification and Al additions in a complex of 24 constructed ponds in central Maryland, USA. The ponds were built between June 149

D. W. Spading, T. P. Lowe

150

Table 1. Metal concentrations @g g-l dry weight) in soils of experimental ponds prior to flooding, 1990

Metal

Soil Type Loam

Clay x N

Al B Ba

12 21675 4.0 101

z Cr cu Fe Mg Mn Ni Pb Sr V Zn

0.77 0.71 22.5 10.1 18850 1821 255 12.5 23.2 8.4 38.5 44.2

SD 1468 1.1 11 0.04 0.43 2.5 0.9 592 122 35 1.0 2.4 1.2 2.3 4.0

X

12 9 740 1.2 48.9 0.46 0.18 12.1 8.7 8 025 727 64.8 5.9 31.0 5.9 21.9 20.7

SD 1213 0.6 6.7 0.03 0.19 3.1 1.6 1564 128 16.4 0.9 10.7 0.8 3.1 3.0

P

0.0001 0.0001 0.0001 0.165 0.0001 0.0001 0.043 0.0001 0.0001 0.0001 0.0001 0.053 0.0001 0.0001 0.0001

1989 and April 1990. The complex was created by excavating basins in a sand-filled depression and lining each basin with impervious polyvinyl sheeting and 1820 cm of soil. Soils came from the same upland site and consisted of either an 0 or A horizon sandy loam (LOAM) with comparatively low metal concentrations, or a B horizon clay (CLAY) with higher metal concentrations (Table 1). Ponds had a mean area of 212 m2 and a maximum depth of 0.6 m. Further descriptions of the ponds and their construction are given in Sparling et al. (1995). Chemical treatment and analysis

Water treatments consisted of control (CONT), aluminum (ALUM), acid (ACID), and aluminum with acid (ALAC). Our initial experimental design randomly stratified treatments across soil types but rupture of a tank used for water treatments early in the study necessitated an unbalanced design. Every two weeks, 2 g of Al as A12(S0& were dissolved in 1150 litres of water from a neighboring pond in polyethylene tanks and gravity fed through polyvinyl chloride pipes into the ALUM and ALAC ponds. Reagent grade H2S04 was mixed with approximately 1300 litres water from the pond and added to ALAC and ACID ponds twice weekly as needed to maintain a target pH range of 4.85.3. Water treatments continued from late May to midOctober 1990 and late March to mid-October 1991. Water samples for metal analyses were collected in 500 ml, amber, high-density linear polyethylene bottles every three weeks and refrigerated at 4°C until they could be filtered, usually within 1-2 days. Samples were first filtered through 21 qualitative grade paper filters leached with 1.2 litres deionized water to remove larger suspended particles, then filtered through 0.2 pm cellulose acetate membranes protected by a borosilicate glass filter. A 200 ml aliquot of each filtered sample was

acidified to pH < 2 for metal analysis. US Fish and Wildlife Service protocols (Brown and Goodyear, 1987) were followed in preparing bottles for sample collection and storage. Samples for monomeric Al were collected by suspending prerinsed, closed, 1000 mwco dialysis tubes filled with deionized water in situ for 24 h. These samples were acidified (pH < 2.0) and stored. All samples for metal analyses were digested with nitric acid and analyzed with graphite furnace atomic absorption spectrophotometry (monomeric Al) or inductively coupled plasma spectrophotometry (ICP) (other metals). The methods for monomeric Al and other metal analyses primarily measured dissolved or small molecular forms of the metals which could pass through filters; total metal concentrations in water would be higher. Dissolved organic carbon concentrations were measured on an infrared carbon analyzer and alkalinity was measured on unfiltered water using potentiometric titration (APHA, 1989). Other water chemistry measurements including pH were made every week with electronic probes in situ. Prior to flooding in 1990 and following the main study in 1992, we collected composite soil samples from each basin and analyzed them for nutrients, metals, texture, and percent organic matter. Soil samples used for metal analyses were digested in nitric and perchloric acids and analyzed with ICP. Because this study occurred approximately midway between the two soil sampling periods and metal concentrations varied between the two periods (see below), we used the mean of the periods to estimate metal concentrations at the time of tadpole sampling. Tadpole trapping and analyses Gray treefrog (Hyla versicolor) and northern cricket frog (Acris crepitans) tadpoles were collected in early

August 1991 by seining each pond with a 1 cm mesh seine. Duplicate or triplicate composite samples consisting of 5-10 individuals of a single species were placed in acid-washed glass jars and set on wet ice to anesthetize the tadpoles and maintain sample freshness. Samples were frozen at -20°C and sent to a contract laboratory (Environmental Trace, Columbia MO’) for metal analyses. Each sample was freeze dried and an aliquot was digested in nitric and perchloric acid and analyzed for metal content with ICP. After finding very high metal concentrations in these species, we decided to determine how the metals were partitioned in tadpole bodies. It took several months to receive the analytical reports and sufficient numbers of northern cricket frog or gray treefrog tadpoles were no longer available. Therefore we used green frog (Rana da&tans) tadpoles that had been collected from the same ponds during June through July 1991 in clear acrylic funnel traps (Sparling et al., 1995) as models for metal partitioning. These samples were frozen, stored, subsequently thawed, and dissected. Separate composite samples of five tadpoles per pond were made of the ‘Mention of product or company name does not imply endorsement by the federal government.

Metal concentrations of tadpoles in experimental ponds

151

TnbIe 2. Metal concentration (mg Iitre-r), dIs&ved organIe earboo eoneentratIon (IQ IItre-‘), pH, and aRalini@’in W&H of experimental ponds as a function of soil type and treatment, MarckJuly 1991 Loam

Clay

Metal CONT

rib Al SD Ba SD Fe SD Mg SD Mn SD Sr SD DOC SD PI-I SD ALK’ SD

4 0.02 0.02 0.05 0.02 0.67 1.05 2.50 1.04 0.65 0.52 0.02 0.01 9.51 0.98 6.3 0.16 21.81 5.59

P”

ALUM

ACID

ALAC

CONT

ALUM

ACID

ALAC

3 0.02 0.02 0.06 0.03 0.92 1.17 2.76 1.47 0.62 0.56 0.02 0.02 10.26 1.76 6.2 0.11 27.81 10.02

2 0.03 0.02 0.06 0.02 0.48 0.82 2.83 0.65 1.82 1.17 0.02 0.02 7.50 0.45 5.4 0.12 3.15 0.52

3 0.04 0.04 0.06 0.02 0.45 0.63 2.48 1.12 1.69 0.60 0.02 0.02 8.06 1.58 5.3 0.13 3.76 0.97

2 0.03 0.01 0.06 0.01 0.75 0.63 2.37 1.22 0.21 0.22 0.01 0.01 11.50 1.27 6.0 0.21 14.05 8.74

3 0.04 0.04 0.05 0.02 1.31 2.52 1.99 1.12 0.18 0.12 0.01 0.01 11.39 1.70 6.0 0.30 16.98 11.68

4 0.04 0.03 0.06 0.02 0.57 0.80 2.12 0.97 0.75 1.04 0.02 0.01 10.23 1.33 5.1 0.06 2.38 0.51

3 0.03 0.01 0.06 0.01 0.28 0.46 2.03 0.96 0.67 0.41 0.02 0.01 9.92 1.36 5.1 0.04 2.65 1.25

Soil

Trtmnt

0.075 0.049 0.0002

0.0002

0.003 0.004

0.059

0.003

0.0001

0.074

0.0001

‘Results of ANOVA using soil, treatment (Trtmnt), and soilx treatment interaction; missing p values and those for interaction terms were far from statistical significance (D > 0.20) and therefore are not reported. bNumber of ponds. “Alkalinity in mg of Ca litre-‘.

stomach and intestinal tract to vent including contents (hereafter referred to as gut coil) and all other body parts (hereafter referred to as body). Each tissue sample was rinsed in deionized water and refrozen. Composite samples were sent to the same contract laboratory and processed as for northern cricket and gray treefrog tadpoles. Soil concentrations in northern cricket frogs and gray treefrogs were estimated by measuring the acidinsoluble ash content using the method described by Beyer et al. (1994). Fresh or frozen specimens were not available so we used tadpoles that had been preserved in formalin. Because this preservative may alter the dry weights of soft-bodied organisms, the percent acidinsoluble ash should only be used for comparative purposes. Statistical analysis The following metals were quantified in water, soil, and tissues: Al, B, Ba, Be, Cd, Cr, Cu, Fe, Mg, Mn, MO, Ni, Pb, Sr, V, and Zn. Because of slight changes in standard requests for metal analyses by the contract laboratory, As was measured in water and tissues only; Se was only measured in tissues. We report only those metals which had > half of their concentrations exceeding detection limits. There is no universally accepted method of accommodating values below detection limits. Therefore, we used techniques similar to those of Newman et al. (1989), and determined that substituting detection limits/2 for values c detection limits provided estimates with the smallest bias in means and closest approximations to actual variances.

Three types of univariate statistical techniques were used (SAS, 1986): analysis of variance (ANOVA) to determine if tadpole and ambient concentrations differed according to the experimental design; correlations to further establish relations between specific ambient conditions and tadpole concentrations; and paired ttests for specific contrasts in tadpole concentrations due to soil or body part examined. When the ANOVAs involved repeated measures, we used the mean square error of pond nested within treatment and soil type as the error variance. RESULTS Ambient chemistry The pH of the acidified ponds increased from 4.8-5.7 in March 1991 to 5.3-6.0 in April as temperatures increased, and declined to 5.e5.2 in July. The pH of non-acidified ponds in March 1991 ranged from 6.5569 but declined to 6.&6.5 by July when the tadpoles were collected (Sparling et al., 1995). Many of the metal concentrations in water were below detection limits for ICP. Of the metals with > half of their aqueous concentrations above detection limits (Table 2) Mg and Sr differed due to soil type. Despite the addition of Al to ALAC and ALUM ponds, neither total dissolved or monomeric Al concentrations differed among treatments. This may be due to the high levels of DOC in the ponds which can bind with Al. Manganese and DOC concentrations, and pH differed between soil types and treatments. Alkalinity was lower in acidified ponds than in circumneutral ones.

152

D. W. Spading, T. P. Lowe

Table 3. Mean metal concentrations (pg g-’ dry wt) in northern cricket frog tadpoles from experimental ponds as a function of soil types and treatments, 1991 Metals

Clay Soils CONT

nb Al As Ba Be Cr CU Fe Mg Mn Ni Pb Se Sr V Zn

4 16920 4880 22.0 3.8 169 60 0.44 0.21 17.9 6.1 11.2 3.6 22 900 8 860 1940 230 2335 3448 9.4 3.8 13.4 13.6 54.8 51.5 25.8 7.2 28.0 6.4 78.0 50.1

ALUM 2 12970 6 850 18.8 13.5 296 212 0.25 0.04 15.3 8.9 9.8 1.4 20 880 4 920 1720 340 425 89 4.6 1.7 9.7 6.1 49.8 10.9 38.8 19.3 23.5 9.6 103.1 86.2

ACID 2 13480 1870 14.2 8.3 153 23 0.38 0.04 15.7 2.4 15.0 1.1 19750 2 500 1700 150 530 362 10.0 1.6 8.5 1.7 43.8 4.0 12.5 1.4 24.2 2.0 61.6 0.9

Loam Soils ALAC

CONT

ALUM

ACID

2 14 670 5 270 22.6 14.9 160 39 0.19 0.08 14.5 4.9 12.5 3.3 30 170 4 580 1820 190 1072 899 3.7 3.2 6.7 5.9 74.0 17.9 12.7 2.1 24.7 7.1 66.9 9.1

2 13 550 3 760 15.5 11.9 143 52 0.40 0.22 16.5 3.7 15.7 2.1 14050 2110 1660 250 252 158 7.3 1.9 9.8 9.6 37.0 4.8 27.3 12.8 27.5 5.3 80.4 12.1

3 12710 3 220 19.8 10.3 110 16 0.30 0.11 16.1 3.7 12.6 1.9 18400 3 450 1510 120 785 497 5.4 2.8 17.1 12.6 44.3 9.0 17.9 3.2 23.2 4.6 69.7 5.1

4 13070 6 320 18.2 8.2 114 43 0.13 0.07 17.7 5.1 12.8 1.2 20 270 4830 1430 170 271 142 4.6 1.4 19.7 10.0 43.1 7.6 16.8 5.0 24.1 9.1 59.0 8.7

ALAC

106L20 3 820 11.5 8.2 73 38 0.15 0.07 12.3 4.1 11.1 4.8 14690 5210 1230 370 382 315 2.4 1.6 15.1 12.7 31.8 14.6 10.8 3.8 20.2 6.5 59.6 35.1

Soil

Trtmnt

SxT

0.004

0.014

0.001 0.017

0.008

0.054

0.002 0.044 0.062

0.089

0.173

0.028 0.105

0.009

0.033

“Based on ANOVA on soil, treatment (Trtmnt) and soilx treatment ’ were not included to increase clarity of-table. bNumber of ponds in which species was found.

interaction

The two types of soils differed in most of the metals analyzed (Table 1). Aluminum, B, Ba, Cd, Cr, Cu, Fe, Mg, Mn, Ni, Sr, V, and Zn were substantially higher in CLAY than in LOAM. Lead concentrations were higher in the LOAM soils than in CLAY. The two sampling periods differed in that 1990 concentrations of Al, Be, Cr, Fe, Ni, Pb, and V were 7-22% less than in 1992 whereas Mn was 23% higher in 1990 (p < 0.05 for each metal). The soils did not attain the high organic, partially decomposed surface layer typical of welldeveloped aquatic sediments.

CONT or ALUM ponds. Tadpole concentrations of Be, Fe, and Sr showed significant interactions between soils and treatments. A small (n = 5) subsample of tadpoles showed that whole bodies were approximately 32% acid-insoluble ash. Concentrations of Be, Fe, Mg, Mn, and Pb in northern cricket frog tadpoles correlated positively with those of soils (Table 4). However, none of the metal concentrations in tadpoles correlated with those in water. Water pH correlated positively with tadpole concentrations of Be, Mg, and Sr. Alkalinity and DOC correlated positively with tadpole Sr. Gray treefrog tadpoles were only collected from 17 of the 24 ponds. None were found in CLAY-ACID ponds, so we pooled ALAC and ACID treatments for our analyses of variance. The metals with the highest concentrations included Al, Fe, Mg, and Mn (Table 5). Manganese, Sr, and V concentrations in gray treefrogs differed between soil types. Acidification decreased tadpole concentrations of Sr. Approximately 17% of the dry weight of this species (n = 6) was acid-insoluble ash. Iron, Pb, and Sr concentrations in soils correlated positively with those in gray treefrogs but none of the

Metal concentrations treefrog tadpoles

in northern cricket and gray

Northern cricket tadpoles were collected from 21 of the 24 ponds. Aluminum, Fe, Mg, and Mn concentrations tended to be at least an order of magnitude above the other metals (Table 3). Based on ANOVA, concentrations of Ba, Be, Fe, Mg, Mn, and Se differed between soil types with tadpoles from CLAY ponds having higher concentrations of these metals than those from LOAM ponds. Tadpoles in ALAC and ACID treatments had lower Be and Sr concentrations than those in

(SxT).

P”

All of the missing p values were > 0.20 and

153

Metal concentrations of tadpoles in experimental pon&

Table 4. Correlation coefficients (r) and signilkance levels (p) between tadpole metal concentrations and the corresponding metal concentration in soil and water, water pH or water DOC; only metals that had at least one s&Scant (p < 0.05) or near significant (p < 0.10) correlation are included=

r

Northern cricket frog tadpoles Be 0.424 Fe 0.449 Mg 0.724 Mn 0.421 Ni 0.535 Pb 0.681 Sr 0.381 Gray treefrog tadpoles Al -0.435 Ba 0.461 Be -0.167 Fe 0.442 Mg 0.344 Mn 0.462 Pb 0.750 Sr 0.511 V -0.465 ~~ “p values > 0.20 are omitted for clarity.

r

P

r

NA -0.079 0.043 -0.068 NA NA 0.143

0.518 0.029 0.565 0.295 0.359 0.119 0.713

0.016

-0.121 -0.375 0.141 -0.169 -0.197 -0.114 0.468

-0.115 NA NA -0.079 0.043 -0.068 NA 0.143 NA

0.145 0.366 0.518 0.029 0.565 0.295 0.119 0.713 0.284

P

r

0.056 0.041 0.0002 0.057 0.012 0.0007 0.088 0.081 0.062 0.045 0.176 0.062 0.0005 0.036 0.060

Water DOC

Water pH

Water concentration

Soil level

Metal

P

0.007 0.197 0.110 0.0003

P

0.093

0.032

0.519 -0.271 -0.121 -0.375 0.141 -0.169 -0.114 0.468 -0.420

0.103 0.016 0.007 0.194 0.0003

0.033

0.093

0.032 0.058

Table 5. Mean metal concentrations (pg g-’ dry wt) in gray treefrog tadpoles from experimental ponds as a function of soil type and treatment, 1991 -__ Metal

Clay soils CONT

nb Al As Ba Be Cr CU Fe Mg Mn Ni Pb Se Sr V Zn

4

8 450 3010 11.3 4.9 123 29 0.21 0.08 9.5 3.9 10.9 1.4 21560 6010 1610 280 4618 3614 7.1 1.2 5.2 3.7 42.7 12.3 31.4 7.7 15.9 4.7 71.9 10.6

Loam soils

ALUM

ALAC

1 3 460

2 4170 1840 7.6 0.6 93.9 3.8 0.22 0.25 6.2 1.8 12.0 0.0 21400 2 970

1.9 140 0.05 4.0 7.4 9120 1400 511 2.0 1.1 20.0 51.5 7.0 59.4

1390 160 2 338 2 505 3.0 0.1 17.5 16.2 55.5 34.6 16.7 6.0 8.0 4.2 62.1 4.1

“Results of ANOVA based on soil and treatment significant 0, > 0.20) and are not included. 6Number of ponds in which species was found.

CONT

ALUM

2 11470 1330 14.0 5.5 93.3 36.7 0.38 0.04 14.8 2.3 12.0 0.0 12 180 1010 1470 210 330 267 7.5 0.8 25.4 14.9 24.2 5.6 16.6 5.4 21.8 2.3 70.6

3 8 950 4 700 9.8 4.1 89.6 25.4 0.18 0.09 9.8 4.0 11.0 3.3 14250 6610 1330 180 350 540 5.0 2.0 22.0 15.2 28.2 12.6 17.6 3.7 17.2 7.1 65.6

6.8

9.0

(Trtmnt). All of the interaction

Pa

_

ACID 3 7 050 2 590 9.2 2.1 58.5 20.9 0.19 0.05 8.6 3.2 12.6 2.4 14 190 6640 1200 90 112 84 4.4 2.2 17.2 9.6 26.9 14.5 13.7 2.8 14.4 4.3 60.1 4.8

ALAC 2 8 140 2 630 9.5 0.6 97.4 78.5 0.14 0.05 9.3 3.2 11.8 2.1 16 520 3 020 1310 130 268 20 4.6 1.4 26.2 7.1 34.0 10.1 12.2 1.4 17.7 4.5 59.1

Soil

Trtmnt

0.137

0.121

0.151

0.163

0.174

0.009 0.100 0.089 0.115 0.012

0.008

0.070 0.088

6.9

terms and the missing p values were far from

154

D. W. Spading,

2ooo r

Fe

28000r

0

T. P. Lowe

Clay

Loam

90

75

[r

60

g E &

45

30

Clay

Loam

Clay

Loam

Matrix

?? GTF 0

NCF

?? Soil

Fig. 1. Metal concentrations (mg kg-’ dry wt) in whole bodies of gray treefrog tadpoles (GTF), northern cricket frog tadpoles (NCP), and soils from ponds, 1991. metal concentrations in water correlated with tadpole concentrations (Table 4). Tadpole concentrations of Be, Mg, and Sr correlated positively with water pH. DOC concentrations correlated positively with tadpole concentrations of Al, Sr, and V. We used paired r-tests between soil metal concentrations within a pond and the respective tadpole concentrations to determine if metal uptake was exclusively passive. For northern cricket frogs, body concentrations of Ba, Fe, Sr, and Zn were higher than those in either soil type (Fig. 1). One tadpole sample from CLAY ponds had a very high value of Mn which resulted in a high variance estimate. When that sample was excluded from analysis, Mn concentrations in bodies (x = 690*495 pg g-r) became statistically higher than those in clay soils (x = 250*35 pug g-l, p = 0.017). Concentrations of Be and Pb were consistently lower in tadpoles than in soils. Similarly, gray treefrogs had concentrations of Mn, Sr, and Zn that were higher than soil concentrations regardless of type. Their Be, Pb, and V concentrations were lower than those in either type of soil.

We compared species differences in metal concentrations by using only those ponds in which both species were found (n = 16). Northern cricket frogs had higher concentrations of Al (p = O.OOOl),As (p = O.OOOl),Ba (j = 0.019), Cr (p = O.OOOl),Fe QI = 0.026), Mg (JJ = 0.008), Se @ = 0.009), and V Cp = 0.0001) than gray treefrogs. Partitioning of metals in green frog tadpoles Weighted averages of metals in whole bodies of green frog tadpoles were undistinguishable from those in gray treefrog tadpoles and substantially lower than those in northern cricket frogs (Table 6). Metal concentrations in green frog gut coils were generally higher than those in body tissues. However, gut coils accounted for 34% of the body mass, so 3495% of the total burden for a metal was present in gut coils. Of the 15 metals analyzed in green frogs, positive correlations between body and gut coil concentrations occurred for Cd (r = 0.833, p = O.OlO), Mn (r = 0.697,~ = 0.055), Ni (r = 0.829,~ = 0.01 l), and V (r = 0.844, p = 0.008). Magnesium concentrations between the two body parts were negatively correlated (r = -0.777, p = 0.023).

Metal concentrations of tadpoles in experimental ponds

155

Ni

Pb

Loam

Clay

Loam

Matrix

?? GTF 0 NCF ?? Soil

Fig. 1. -

contd

Table 6. Metal concentrations (pg g-’ dry wt), mass, and percent moisture of bodies and gut coils of green frog tadpoles collected from experimental ponds, 1991

Measure x Al B Ba Be Cd Cr cu Fe Mg Mn Ni Pb Sr V Zn % Moisture Dry mass

Gut coil

Body

634 2.0 90 0.07 1.9 1.7 6.7 1590 921 405 4.1 3.1 16.7 11.0 58.1 88.1 31.0

Percentb

x

416 1.4 27 0.02 0.6 0.5 2.1 820 105 368 4.8 0.3 3.9 12.1 8.9 0.8

5 38 4.5 14 41 13 37 8 49 29 31 35 66 25 54

20 840 6.3 210 0.81 8.4 2 21.5 33450 1890 1990 16.4 19.2 11.0 50.6 99.3 78.8

16.1

66

SD

16.3

Soil concentration SD 5 900 3.1 50 0.15 8.1 5.5 4.0 6 850 349 1400 4.2 14.7 12.1 16.4 21.1 1.2

X

15720 N.M.” N.M. 0.56 B.D. 16.6 9.9 15070 1320 206 9.3 21.5 5.9 30.1 36.2

SD

Results of paired t-test@ Body/Gut

6 870

0.05 7.3 1.8 6440 670 111 3.5 9.2 1.6 8.6 17.0

8.9

“Based on paired t-tests between body and gut, body and soil, and gut and soil concentrations. clarity. bPercent of total metal or other measurement due to body (less gut coil). ‘N.M. = not measured. B.D. = below detection levels.

0.0001 0.003 0.0001 0.0001 0.04 0.0001 0.0001 0.0001 0.0001 0.008 0.0001 0.008 0.0001 0.002 0.0001

Body/Soil

Gut/Soil

0.0004

0.131

0.0001

0.006

0.0006 0.015 0.0006 0.165 0.125 0.019 0.0008 0.0001 0.004 0.009

0.137 0.0001 0.0002 0.019 0.006 0.0008 0.0001 0.012 0.0001

0.0001 p values >0.20 were omitted for

156

D. W. Spading, T. P. Lowe

Be

l.clL

0

0.8 0.6 -

PH 70 60 50

5.00

Sr 0

5.25

5.50

5.75

6.00

0

6.25

6.50

6.75

7.00

PH

5.00

5.25

5.50

5.75

6.00

6.25

6.50

6.75

7.00

PH Species -o- GTF -o-NCF

Fig. 2. Relationship between Be, Mg, and Sr concentrations in gray treefrog (GTF) and northern cricket frog (NCF) tadpoles and water pH in ponds, 1991. Values along vertical axis are mg kg-’ metal concentrations. Dotted and solid lines are regressions to highlight trends.

Except for Mg and Mn, metals in body were lower than those in soils and there was little relation between body and soil concentrations. Gut coils had higher concentrations of Be, Cu, Fe, Mg, Mn, Ni, Sr, V, and Zn than soils. Manganese (r = 0.805, p = 0.016) and Zn (r = 0.778, p = 0.023) correlated between soil and gut coil.

DISCUSSION Our data are consistent with other studies and demonstrate that differences in tadpole metal concentrations are due to soil types, species, and body part examined. Less dramatic effects may be due to water chemistry such as pH and DOC. Few studies have been conducted on metal concentrations in tadpoles and many of the metals that we analyzed in this study have not been reported previously. Of those metals which are comparable, most of our concentrations were consistent with previous findings. For example, our concentrations were similar to those reported by Hall and Mulhern (1984) in whole bodies of bullfrog (Rana catesbeiana) and green frog tadpoles from the same research center as ours, but from different ponds. Our Pb concentrations were less than those in tadpoles from Pb-contaminated ponds studied by Gale et al. (1973) (36-1590 pg g-‘) and

Jennett et al. (1977) (4139 pg g-l) and in roadside ditches of Maryland and Virginia (2&250 pg g-‘) (Birdsall et al., 1986). Zinc concentrations were also less than that found by Gale et al. (1973) (16&1090 pg g-l) and Jennett et al. (1977) (2808 pg g-i) in contaminated sites. Effects of ambient conditions on tadpole metals The pH of precipitation in our study area averages between 3.8 and 4.5, which is adequate to leach Al, Cd, Zn, Ni and other metals from surface soils to lower horizons (Nelson and Campbell, 1991). In contrast, Pb has an affinity to organic matter and DOC (Driscoll et al., 1988) which accounts for its higher concentration in the loam soils with a greater organic matter content. Thus the difference in metal concentrations between the loam and clay soils probably was due to a combination of leaching from acidic deposition, greater cation exchange capacity of clay soils (Stumm and Morgan, 1981), and adsorption onto organic matter. Because the soils surrounding our ponds were not treated, we ascribe the increase in metal concentrations of pond soils to leaching from surface soils caused by acidic precipitation. Soils provided the bulk of metals for northern cricket frog and gray treefrog tadpoles. Many of the metal concentrations in tadpoles differed between soil types and several correlated with soil concentrations. In contrast, aqueous concentrations of most metals were

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below detection limits and did not correlate with body concentrations. The DOC concentrations in the ponds were relatively high and many of the metals may have been bound to DOC. For example, DOC > 5 mg litre-’ can virtually eliminate toxic species of Al from water (Freda et al., 1989) which probably explains why we did not observe differences in aqueous Al due to treatment. The high concentrations of many metals in gut coils of green frog tadpoles compared to body concentrations and the percent body weight due to acid-insoluble ash in gray treefrog and northern cricket frog tadpoles further demonstrate that much of the total body burden of metals may be present in ingested soil. The differences we observed in metal concentrations between northern cricket frogs and gray treefrogs may be related to habitat partitioning and diet within our experimental ponds. While observing and collecting the species, we perceived that gray treefrogs more frequently used open water in the middle of the ponds whereas northern cricket frogs favored the near shore, shallow waters where they would be in greater contact with soils. Closer contact with the bottom could lead to greater ingestion of metal-laden soils. Documented evidence for microhabitat preferences by northern cricket frog tadpoles could not be found, but Lawler (1989) concluded that gray treefrog tadpoles were pelagic. Our limited data on soil ingestion supports this in that gray treefrogs had approximately half of the acid-insoluble ash as northern cricket frogs. The problems we had in collecting sufficient numbers of tadpoles in all of the ponds were consistent with results of a study that examined tadpole abundance in the same set of ponds (Sparling et al., 1995). Northern cricket frogs were less abundant in acidified ponds than in circumneutral ones (p < 0.05) and less in ponds with loam soils than in those with clay (p < 0.04). Similarly, gray treefrogs were less abundant in acidified ponds than in circumneutral ponds within the clay soil type but did not differ among treatments in those with loam. Although we were able to show population effects due to acidification, our treatments resulted in pH levels that were higher than those used to show toxic effects in laboratory studies (e.g. Freda and McDonald, 1990; Rowe et al., 1992) and higher than those reported in some field investigations of amphibians (e.g. Dale et al., 1985; Glooschenko et al., 1992). Therefore, we may not have observed as strong of a relationship between acidification and body concentrations of metals as might occur under more stressful situations. However, in the current study, acidification decreased Be, Mg, and Sr in both northern cricket frog and gray treefrog tadpoles. Aqueous Mg is a major contributor with Ca to acid neutralizing capacity. Through the summer pH, alkalinity, and Mg concentrations decreased in all ponds but significant differences only occurred in acidified ponds. The decline in treated ponds reduced the availability of Mg to aquatic organisms. Aqueous concentrations of Sr and Be also decreased through time but their rates did not differ among treatments. It is possible that acidi-

ponds

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fication altered ion regulation in tadpoles (Freda and Dunson, 1985), but the low concentrations of these metals in water cast doubt on any biological relevance for the relationship between tissue concentrations and pH. None of the metals in water exceeded recommended values for wildlife health (Environmental Protection Agency, 1976). Selective and passive uptake of metals The uptake of metals by tadpoles may be passive in that whole body concentrations are similar to those in the environment (soil or water) or selective, in which case body concentrations are either significantly greater (selectively positive or accumulated) or lower (selectively negative) than ambient concentrations. For the present discussion, selective elimination of metals results in the same measurable effect and cannot be distinguished from selective uptake. In turn, metals in whole bodies may be partitioned into gut concentrations, only some of which may be assimilated, and tissue concentrations, which more accurately reflect selective assimilation. Northern cricket frogs accumulated Ba, Fe, Mn, Sr, and Zn in both soil types and Al, Cu, and Mg in loams but not in clays. Gray treefrogs had higher concentrations of Mn, Sr, and Zn in both soil types and Ba, Cu, Fe, and Mg in loams. Of these metals, Zn, Cu, Fe, Mg, and Mn are essential elements and their selective uptake is not surprising. Strontium behaves similarly to Ca in organisms (National Research Council, 1980). Our ambient concentrations of Ca were low (X = 3.9 f 1.5 mg litre-‘) which may have facilitated uptake of Sr. Beryllium and Pb were the only metals that appeared to be selected against by both species of tadpoles in both soil types. All of the metals showed at least one significant difference between body and soils. If green frogs can serve as a model for other species, more than half of the whole body metal load of tadpoles is in the gut coil. The gut appears to concentrate Be, Cd, Cu, Mg, Ni, Sr, V, Zn, and especially Fe and Mn compared to soils. Cadmium, Sr, and Zn were higher in body tissues than soils, indicating selective and positive assimilation of these metals. Tadpoles as indicators of ambient concentrations of metals Hall and Mulhem (1984) demonstrated that green frog and bullfrog tadpoles accumulated Pb, Zn, Cu, Co, Se, Sr, Fe, and Mn and suggested that they could be good indicators of metal contamination. Niethammer et al. (1985) compared metal concentrations in adult bullfrogs, muskrats (Onalatra zibethicus), northern water snakes (Nerodia sipedon), rough-winged swallows (Stelgidopteryx serripennis), and green herons (Butorides striatus). They determined that bullfrogs were the best species to monitor metal concentrations in soils because of the high concentration of metals in tissues and correspondence between soil and body concentrations. Based on our data, which extend over a narrow range of ambient metal concentrations compared to some

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polluted areas (e.g. Gale et al., 1973; Birdsall et al., 1986), we caution against placing too great a reliance on using tadpoles as bioindicators of environmental contamination. Significant correlations only occurred between some but not all of the metals measured. For some metals, ambient concentrations were substantially below those of whole bodies whereas other metals showed no differences or were higher in soils. There was poor correlation between tissue and soil concentrations in green frogs and virtually no relationship between water concentrations and body or tissue concentrations in any of the species tested. Moreover, significant differences in metal concentrations between gray treefrogs and northern cricket frogs show that the selection of species is important in determining a bioindicator. Tadpoles as a source of metal toxicity Some of the metal concentrations measured in our study are above toxic concentrations to potential predators. Metal toxicity is complicated by the form or species of the metal (e.g. ferric or ferrous ion; monomeric or complexed Al), presence of other factors such as ligands or competitors, interspecific variability, and health of the individual. Despite this, guidelines which can approximate toxicity have been developed for domestic species (National Research Council, 1974; National Research Council, 1980). To the extent that domestic animals such as horses, sheep, pigs, and chickens represent birds and mammals in general, animals whose diets consisted largely of tadpoles from these ponds would ingest potentially toxic concentrations of Al, Fe, Mn, Se, and V. Recent studies have shown that food, as well as water, may be an important route of As, Cd, Cu, Pb, and Se uptake in fish although recommended dietary levels are unavailable (Besser et al., 1993; Woodward et al., 1994). Recall that our ponds were essentially uncontaminated and reflect low ambient concentrations of these metals; tadpoles from contaminated sites could be expected to have much higher concentrations of metals. Some predators reduce their intake of metals and other contaminants when feeding on tadpoles by consuming only body tissues. For example, we have observed that small (carapace length 6-7 cm) painted turtles (Chrysemys picta) in captivity repeatedly eviscerate tadpoles with their claws and leave gut coils uneaten. Many species of vertebrates such as pisciverous birds and mammals consume tadpoles intact, however, and could be ingesting a toxic dose of metals, especially in contaminated areas. In summary, northern cricket frog, gray treefrog, and green frog tadpoles accumulated moderate to high concentrations of metals in these ponds. Soil concentrations of these metals are the major factors determining body concentrations. Acidity had some effect on tadpole concentrations of Be, Mg, and Sr. Species differences in metal concentrations may be related to microhabitat with northern cricket frogs which inhabit near-shore benthic habitats having higher metal concentrations

than the pelagic gray treefrog. Because of their ability to concentrate metals, significant correlations between body and soil concentrations for some metals, and wide distribution, tadpoles can serve as indicators of metal contamination. However, there is sufficient variation in metal uptake and between species to exercise caution in using tadpoles indiscriminately as bioindicators. Animals that feed extensively on tadpoles may ingest toxic concentrations of metals but some species apparently reduce this problem by avoiding the tadpole digestive system.

ACKNOWLEDGEMENTS The authors wish to thank the several volunteers from the Student Conservation Association for their hard work and diligence in collecting water and tadpole samples. R. K. Schreiber was helpful in obtaining funding for the study. N. Beyer graciously conducted the analyses on acid-insoluble ash. M. Beetham and the Chesapeake Biological Laboratory, University of Maryland, provided analytical support. D. Day and N. Federoff were extremely helpful in technical support. B. Williams and P. Pendergrass were essential in constructing the ponds. D. Clark and P. Albers reviewed earlier drafts of this manuscript.

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