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Metal Levels in Southern Leopard Frogs from the Savannah River Site: Location and Body Compartment Effects
Environmental Research Section A 86, 157}166 (2001) doi:10.1006/enrs.2001.4245, available online at http://www.idealibrary.com on
Metal Levels in Southern Leopard Frogs from the Savannah River Site: Location and Body Compartment Effects Joanna Burger* and Joel Snodgrass* *Division of Life, Consortium for Risk Evaluation with Stakeholder Participation (CRESP), and Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, New Jersey, 08854-8082; and -Department of Biology, Towson University, Towson, Maryland 21252 Received January 20, 2000
both to preserve ecological integrity (Karr, 1991, 1993) and to protect human health (DiGiulio and Monosson, 1996). In ecological systems, natural chemical cycles and movement of contaminants occur rapidly in aquatic systems. Whereas it may take decades or longer for chemicals to move through terrestrial systems, once they enter an aquatic system, they move rapidly through the food chain, potentially reaching human consumers within days or weeks, rather than years. Indicators are often selected to assess levels and effects of toxics in ecosystems (O’Connor and Dewling, 1986; Hunsaker et al., 1990; NRC, 1991, 1993; Kremen, 1992), largely because of the overwhelming dif7culty of assessing even a small fraction of the biota. This has led to the examination of species that are high on the food chain (O’Connor and Dewling, 1986; Peakall, 1992; Burger, 1995), such as 7sh-eating birds and predatory mammals. In aquatic systems, however, the top trophic level is often composed of predatory amphibians or 7sh, which are appropriate bioindicators of these systems. In some ephemeral aquatic systems, particularly those with a short hydroperiod, the body burdens of heavy metals in larval amphibians, such as tadpoles, are being used as indicators of metal pollution (Carlson and Adriano, 1993). One potential problem with the use of tadpole body burdens as bioindicators is that tadpoles ingest sediment, and analysis of whole bodies may overestimate the concentration of contamination in tissues if some of the sediment (with heavy metals) remains in the digestive tract and these metals are not incorporated into tissues. Burger and Snodgrass (1998) showed that, in bullfrog (Rana catesbeiana) tadpoles, the variance in concentrations of arsenic, chromium, lead, manganese, mercury, selenium, and cadmium was explained only by body compartment (body, tail, whole body);
Tadpoles have been proposed as useful bioindicators of environmental contamination; yet, recently it has been shown that metal levels vary in different body compartments of tadpoles. Metals levels are higher in the digestive tract of bullfrog (Rana catesbeiana) tadpoles, which is usually not removed during such analysis. In this paper we examine the heavy metal levels in southern leopard frog (R. utricularia) tadpoles from several wetlands at the Savannah River Site and test the null hypotheses that (1) there are no differences in metal levels in different body compartments of the tadpoles, including the digestive tract; (2) there are no differences in heavy metal levels among different wetlands; and (3) there are no differences in the ratio of metals in the tail/body and in the digestive tract/body as a function of metal or developmental stage as indicated by body weight. Variations in heavy metal levels were explained by wetland and body compartment for all metals and by tadpole weight for selenium and manganese. In all cases, levels of metals were higher in the digestive tract than in the body or tail of tadpoles. Metal levels were highest in a wetland that had been remediated and lowest in a wetland that was never a pasture or remediated (i.e., was truly undisturbed). Although tadpoles are sometimes eaten by Ash and other aquatic predators, leopard frogs usually avoid laying their eggs in ponds with such predators. However, avian predators will eat them. These data suggest that tadpoles can be used as bioindicators of differences in metal levels among wetlands and as indicators of potential exposure for higher-trophic-level organisms, but that to assess effects on the tadpoles themselves, digestive tracts should be removed before analysis. 2001 Academic Press
INTRODUCTION
It is increasingly clear that as a society we are interested in assessment of the health of ecosystems, 157
0013-9351/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
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for arsenic, chromium, and lead, in tadpoles held for 2 days without being fed metal concentrations decreased slightly. Moreover, for fresh tadpoles, the digestive tract contained signi7cantly higher concentrations of all metals than either the body or the tail, probably re8ecting metals absorbed to sediment particles in the gut (Burger and Snodgrass, 1998). Thus, analysis of whole tadpoles may not give a realistic picture of exposure because of averaging across compartments with very different levels of metals. Before frogs and other amphibians can be used effectively as bioindicators it is important to know whether metal levels consistently differ among body compartments for different species. In this paper we examine the levels of heavy metals in leopard frog (R. utricularia) tadpoles from the Savannah River Site (SRS), a Department of Energy (DOE) Site in South Carolina. We wanted to con7rm that (1) heavy metals vary by body compartment and (2) levels of all metals are signi7cantly higher in the digestive tract than in the whole body. Further, we test the null hypothesis that there are no differences in heavy metals in different wetlands, three of which are considered reference sites (Schalles et al., 1989). If the three sites are all truly reference sites, with the same atmospheric deposition history and no DOE site contamination for the nearly 50 years of their occupation, then levels should be similar in the tadpoles from the various sites. We expected that metal levels would be higher in wetland 28 because it was a remediated site (Kirkman et al., 1996). We also examine whether the ratio of metals in different body compartments varied by metal. The isolated wetlands on SRS have a diverse community that includes ranid frogs, sirens, predaceous diving beetles, snakes, and dragon8y larva. Of these, amphibians are the most likely to accumulate body burdens because toxicants can readily enter their body through their diet and through absorption across their semipermeable skin. In the egg and tadpole stages, amphibians live entirely in wetlands and obtain all their nutrients from the aquatic environment, making them useful as a tool for the assessment of contaminants in wetland habitats and as sentinels of potential effects (Cooke, 1981). However, the distribution of contaminants in different body parts and the relative contribution of stomach contents to the body burden must be known for several species before metal concentrations can be interpreted or before they can be used effectively as a biomonitoring tool. This study was initiated partly because of the 7ndings of mouth part deformities in bullfrog tadpoles on SRS wetlands associated with coal ash
deposits, which are highly contaminated with selenium, among other elements (Rowe et al., 1996, 1998). Further, Raimondo et al. (1998) demonstrated de7cits in predator avoidance and elevated maintenance costs of tadpoles exposed to coal ash. MATERIALS AND METHODS
With appropriate state permits, tadpoles were collected from four Carolina bays within the Savannah River Site in 1997 and brought to the Savannah River Ecology Laboratory on the SRS where they were frozen. The Savannah River Site is a 780-km2 nuclear production and research facility of the U.S. Department of Energy, located in west-central South Carolina (33.1@N, 81.3@W) and bounded on the southwest side by the Savannah River (Fig. 1). Vegetation communities on the SRS are typical of the upper coastal plain of South Carolina (Workman and McLeod, 1990). Some of the cooling reservoirs for the reactors (no longer active) were contaminated with 137 Cs (radiocesium) and smaller amounts of other
FIG. 1. Map of the Savannah River Site, showing the locations of the wetlands studied.
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
radionuclides from 1954 to 1964, in addition to mercury, which most likely originated from Savannah River water pumped through the reservoir (Clay et al., 1980). Whereas there has been extensive study of radionuclides and mercury on the cooling reservoirs of SRS (Clay et al., 1980; Potter et al., 1989; Brisbin, 1993; Kennamer et al., 1993; Colwell et al., 1996; Burger et al., 1997), heavy metals and selenium from the wetlands that we studied have not been examined. The wetlands from which we collected tadpoles are referred to as wetlands 10, 27, 28, and 66 (Schalles et al., 1989). We use these numbers here to refer to these wetlands because other authors have used them in the past. All of the wetlands are considered depression wetlands in which water level 8uctuations are controlled by precipitation and vary among years. All wetlands have similar hydroperiods involving winter and spring recharge, summer draw down, and eventual drying in late summer or early fall. Vegetation in the wetlands is generally characterized by herbaceous and emergent species. Kirkman et al. (1996) provides information on past land use within and size of wetlands. Wetlands range in size from 1.59 ha (10) to 11.65 ha (66). Wetland 10 was not disturbed by agricultural practices before 1951 (when the site was established). Wetlands 27 and 66 were pasture before 1951. Wetland 28 is a remediated CERCLA site that was previously contaminated by metals and radionuclides and was cultivated for row crops before 1951. Remediation activities involved removal of sediments and planting of vegetation and were complete in the late 1980s. Tadpoles were collected with dip nets. They were collected as part of a study on species diversity in these wetlands (Snodgrass et al., 2000); so sample sizes re8ect these considerations. Thus, we did not have the same number of tadpoles available from each wetland. Tadpoles were euthanized by slow cooling, stored in plastic bags, labeled by species and collection location, and frozen (!20@C) before being shipped to the Environmental and Occupational Health Sciences Institute laboratory at Rutgers University. In the laboratory all tadpoles were thawed and weighed. The digestive tract was removed from all tadpoles; the tail and body sections were separated, weighed again, and analyzed separately. We de7ne the body as the whole tadpole minus the tail. In the tables, whole body refers to the whole tadpole, which consists of the body, tail, and digestive tract. Since we analyzed the metals in the individual body compartments, we reconstructed the levels in the whole
body by multiplying the weight of the compartment by the metal levels (see Table 2). All tadpoles were at stage 25 (after Gosner, 1960). Tissues were digested in 70% nitric acid in a microwave vessel for 10 min under 130 pounds per square inch and then diluted in deionized water (EPA, 1981). The sediment in the digestive tracts of the tadpoles did not digest, although some metals may have been removed from the sand during the extraction process. Mercury was analyzed by a cold vapor technique, and all other metals were analyzed by graphite furnace atomic absorption. All concentrations are expressed in lg/kg (parts per billion) on wet weights obtained from air-dried specimens (except where noted in the tables) and are rounded to three signi7cant 7gures. Mercury detection limits were 0.2 lg/kg; detection limits for other metals were 0.7 lg/kg (selenium) and 0.15 lg/kg (lead) or lower. All specimens were run in batches that included a standard calibration curve and spiked specimens. The accepted recoveries ranged from 87 to 105%, and batches with recoveries less than 87% were rerun. Further quality control measures included periodic blind analysis of an aliquot from a large sample of known concentrations and blind runs of duplicate samples during the analysis for each metal. In all cases, there was no signi7cant difference between duplicate samples (correlations of 0.9 or higher for all metals). Regression procedures on log-transformed data were used to determine whether wetland, tadpole body compartment, and tadpole weight contributed to differences in metal levels (PROC GLM; SAS, 1985). The procedure adds the variable that contributes the most to the R2, then adds the next variable that increases the R2 the most, and continues until all signi7cant variables are added. Wilcoxon tests were used to examine differences between groups (Siegel, 1956); Wilcoxon is a nonparametric analysis of variance based on ranks and is more conservative than parametric ANOVAs. Kendall q correlations were used to examine the relationships between the concentrations of different metals in body and tail. RESULTS
All of the leopard frog tadpoles had nondetectable levels of mercury; mercury is not discussed further in this paper. Models Explaining Variations in Metal Levels Despite the low sample sizes in some wetlands, 42 to 67% of the variation in metal levels was explained
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TABLE 1 Models Explaining Variations in Concentrations (Wet Weight, ppb) of Six Metals Found in Leopard Frog Tadpoles (with All Body Compartments, Including Digestive Tract)
Model F df r2 P Factors entering, s2 (P) Wetland Compartment Wetland;compartment Recombined weight
Lead
Cadmium
Arsenic
Selenium
Manganese
Chromium
10.4 11,165 0.45 0.0001
9.19 11,163 0.42 0.0001
19.4 11,162 0.61 0.0001
14.8 11,165 0.54 0.0001
23.7 11,155 0.67 0.0001
17.9 11,162 0.59 0.0001
25.4 (0.0001) 2.55 (0.06) NS NS
16.5 (0.0001) 6.54 (0.0003) NS NS
51.7 (0.0001) 5.58 (0.001) NS NS
8.58 (0.0003) 6.90 (0.0002) 3.73 (0.002) 10.8 (0.001)
9.4 (0.0002) 43.6 (0.0001) 22.32 (0.04) 5.38 (0.02)
27.2 (0.0001) 18.1 (0.0001) NS NS
Note. NS, not signi7cant.
by wetland and body compartment (section); weight also entered the models for selenium and manganese (Table 1). Locational Differences in Metal Levels There were differences in heavy metal levels in whole tadpoles (including all parts) from the different wetlands for all metals (Table 2). In general, metals were highest in wetlands 28 and 66 (see Fig. 1). Levels were highest for manganese, followed by lead and chromium, and were lowest for cadmium, arsenic, and selenium. In most cases, these differences persisted for each body compartment (Table 2). Body Compartment Differences in Metal Levels There were differences in metal levels in body compartments (Fig. 2). Metals levels were signi7cantly higher in the digestive tract than in the other body compartments for all metals (s2 tests, P(0.0001 for all metals). There were signi7cant differences between the tail and the body only for lead (P(0.001) and manganese (P(0.02); levels were higher in the body than in the tail (refer to Fig. 2). Ratios of Metals in Different Body Compartments One important aspect of the use of tadpoles for biomonitoring is to understand whether the ratio of metals in different body compartments is constant for different metals and whether this ratio is the same for different species. Generally the ratios are higher for the digestive tract/body than for the tail/body (Fig. 3). There is a great deal of variability among individuals, however, in the amounts of lead
in the body, tail, and digestive tract, as re8ected by the ratios between them (refer to Fig. 3). Figure 3 also shows the similar ratios for the bullfrog, computed from Burger and Snodgrass (1998). In general, there are greater differences in the ratios for leopard frog than for bullfrog (except for cadmium). It is possible that the amount of metal in the body compared to that in the tail is a function of development; as tadpoles age (and become heavier), they deposit more metal into their tails. To examine this possibility, we computed the correlation of the ratio of the amount of metal in the tail to the amount of metal in the body against weight (a surrogate for age). We predicted that the amount in the tail should increase with age if they are storing the metals there. However, for many metals there was no relationship, and where there was, it was negative for 7ve of six signi7cant correlations for the tail/body compartment ratio (Table 3). That is, the relative amount of metals in the tail for leopard frog decreased with tadpole weight for manganese and chromium. However, this ratio increased with weight for selenium, suggesting that the tadpoles put relatively more selenium in their tail as they aged. They also had more in their digestive tract as they aged (Table 3). We also computed these correlations for bullfrogs (computed from Burger and Snodgrass, 1998). Except for selenium in bullfrogs, there was no signi7cant relationship between the ratio of metals in the digestive tract/body and the weight (Table 3). Correlations among Metals There were a number of signi7cant correlations among metal levels in all compartments for all
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
TABLE 2 Concentration Value (Mean ⴞ SE; Wet Weight, ppb) for Six Metals in Tissues from Leopard Frog Tadpoles
Sample Whole body Arsenic Cadmium Chromium Lead Manganese Selenium Body Arsenic Cadmium Chromium Lead Manganese Selenium Tail Arsenic Cadmium Chromium Lead Manganese Selenium Digestive tract Arsenic Cadmium Chromium Lead Manganese Selenium
66
10
Wetland 28
27
8
7
3
38
167$32.6 149 A 131$18.7 126 A,B 1,840$277 1,710 A 3,180$669 2,790 B 41,400$2,770 40,800 B 397$27.3 391 A
27.4$6.84 22.6 C 38.4$2.91 37.8 C 137$8.73 136 C 256$33.2 243 D 13,600$950 13,400 C 152$18.7 146 C
206$50.7 194 A 267$94.3 234 A 1,710$303 1,650 A 5,450$1,120 5,240 A 146,000$;a
69.1$6.57 60.3 B 104$19.2 80.2 B 394$54.6 300 B 983$78.9 882 C 153,000$11,400 143,000 A 238$21.3 224 B
25.1 (0.0001)
49.7$18.5 37.7 A 108$29.2 86.4 A 537$79.2 501 A 1,771$751 297 A 35,000$3,910 33,900 B 377$29.8 370 A
17.4$4.93 12.1 B 30.9$3.39 29.9 B 82.1$9.57 79.0 C 180$64.6 58.9 A 16,600$1,790 16,100 C 91.9$28.5 66.0 C
40.4$4.39 39.9 A 100$37.2 85.2 A 261$59.5 245 B 783$773 38.8 A 20,700$1,570
35.3$3.54 29.1 A 57.2$12.8 41.3 A,B 167$15.3 148 B 611$115 234 A 166,000$21,100 137,000 A 204$34.4 174 B
5.37 (NS)
106$29.5 81.3 A 94.9$21.9 86.3 A 979$233 782 A 592$264 69.0 B 41,800$6,330 38,400 A 429$57.5 405 A
15.0$5.45 13.7 B 22.7$2.57 21.8 B 52.8$8.81 49.1 C 53.4$15.4 30.9 B 6,340$577 6,200 B 152$15.1 148 B
30.2$3.08 29.9 B 229$191 90.8 A 3,650$3490.0 641 A 1,660$1,140 1,010 A 841,000$62,700 319$16.4 318 A
31.9$2.80 28.4 B 94.2$41.3 46.5 A,B 175$29.0 132 B 157$29.1 45.0 B 105,000$17,500 65,900 A 188$8.31 181 B
388$109 315 A 160$13.2 156 B 3,850$428 3,640 A 6,580$1,300 2,820 A,B 50,400$6,330 49,200 B 439$30.4 432 A
70.0$19.4 48.9 C 76.4$14.3 69.3 C 386$40.1 375 B 747$132 653 B 19,000$1,830 18,600 C 284$55.7 217 B
504$144.0 465 A 466$266 334 A 2,610$1,280 1,480 A,B 137,000$3,370 12,900 A 173,000$62,200 140,000 A 270$68.4 251 B
135$14.0 115 B 154$35.6 118 B,C 863$129 507 B 2,120$173 1,720 B 212,000$19,100 186,000 A 309$12.0 300 A,B
274$39.9 268 B
259$30.5 255 A,B
Wilcoxon s2 (p)
Overall 56
24.3 (0.0001) 26.6 (0.0001) 31.3 (0.0001) 27.5 (0.001) 23.5 (0.0001)
10.8 (0.01) 26.1 (0.0001) 5.43 (NS) 28.6 (0.0001) 26.0 (0.0001)
9.93 (0.02) 15.6 (0.001) 28.1 (0.0001) 7.25 (0.06) 18.7 (0.0001) 26.5 (0.0001)
22.4 (0.0001) 16.5 (0.0009) 20.3 (0.0002) 25.1 (0.0001) 29.4 (0.0001) 11.3 (0.01)
83.7$9.11 63.6 107$15.6 81.1 640$98.2 383 1,370$200 911 116,000$11,700 84,800 250$17.6 230 35.2$3.53 27.4 62.8$10.0 45.4 210$23.5 165 694$130 212 119,000$17,000 75,500 215$26.2 173 40.3$5.79 30.1 92.5$30.9 46.8 461$193 165 287$81.1 69.9 82,100$13,100 44,500 225$15.5 205 183$24.9 129 162$28.8 121 1,350$197 697 3,200$481 1,710 164,000$17,000 116,000 323$13.3 301
Note. Geometric means and Duncan Multiple Range values given below each mean. Difference in letters across represent signi7cant differences in metal concentration with a Duncan Multiple Range test. Wetlands 10, 28, and 66 are considered reference sites. ? Only one value to analyze; no standard error available.
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compartment and that the digestive tract has signi7cantly higher levels of all metals than the body. There were also signi7cant differences in metal levels among wetlands even though the sample sizes were low for some sites. These 7ndings are discussed below, along with the generality of the ratios of metal levels in different body compartments. Locational Differences in Metal Levels
FIG. 2. Metal levels (ppb) in different body compartments of leopard frogs collected at the Savannah River Site.
leopard frogs combined (Table 4). In general, lead was signi7cantly correlated with most other metals in most body compartments, as was chromium. Nearly all correlations were positive; as one metal increased, the other did also. The correlations, although signi7cant, were not high, except for lead and arsenic, lead and chromium, and arsenic and chromium in the digestive tract and in the whole body (Table 4). DISCUSSION
The results of this study con7rm our pervious 7ndings (Burger and Snodgrass, 1998) that there were signi7cant differences in metal levels by body
FIG. 3.
Three wetland sites where we collected tapoles on the Savannah River Site are considered reference sites (Schalles et al., 1989). There is a considerable amount of ecological research conducted on the SRS by the personnel of the Savannah River Ecology Laboratory, and these sites are considered reference sites because they have not been impacted by the industrial and radiological research activities of the Department of Energy. Presumably, these sites experience the same amount of atmospheric deposition and receive no point-source contamination from SRS itself. This suggests that contamination should be similar in all three wetlands examined. Hydroperiod, however, varies, although all four wetlands dry down eventually during the summer. Thus, the cycle of drying down and re7lling, which results in an increase in some metals through increases in bioavailability (in mercury, for example), should also be similar among the wetlands. However, we found signi7cant differences in the levels of all metals in the tadpoles from the
Ratio of metals in the tail/body and digestive tract/body for bullfrogs and leopard frogs collected at the Savannah River Site.
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
TABLE 3 Correlation of Body Weight to Two Body Compartment Metal Ratios in Species of Tadpoles (Wet Weight, ppb) Bullfrog
Leopard frog
Tail/body compartment ratio, r2 (P) Arsenic !0.18 (0.06) Cadmium NS Chromium NS Lead NS Manganese !0.25 (0.009) Selenium !0.20 (0.04)
NS NS !0.17 (0.06) NS !0.30 (0.002) 0.31 (0.0006)
Digestive tract/body compartment ratio, r2 (P) Arsenic NS Cadmium NS Chromium NS Lead NS Manganese NS Selenium NS 0.32
NS NS NS NS NS (0.0003)
Note. NS, not signi7cant.
wetlands, even given the small sample sizes. Overall, metal levels were generally highest in the remediated site and lower in the reference sites. However, there was variation even within the reference sites. Metal levels were generally highest in tadpoles from wetlands 28 and 66 and lowest in wetland 10. We suggest that these differences may be due to land use differences before the DOE bought the land in the early 1950s. Wetland 10 was a herbaceous wetland when the site was established; it was not previously used for agriculture (Kirkman et al., 1996). Wetlands 27 and 66 were pastures in 1951, but since DOE occupation, they have undergone suc-
cession to herbaceous/forested wetlands. The suggestion that agricultural processes nearly 50 years ago are still affecting contaminant levels in these wetlands is intriguing and requires further study. Wetland 28, the wetland where the whole body metal levels in tadpoles were the highest (except for selenium and manganese), was a contaminated site that had undergone remediation, which included removal of all sediments and planting of vegetation (Kirkman et al., 1996). It suggests, however, that not all contamination was removed. Additionally, this wetland was cultivated before the site establishment, making it dif7cult to assess the sources of metals in this wetland. Overall, levels were relatively high for lead, manganese, and chromium. Lead arsenate was a common insecticide 50 years ago, and manganese was used as fungicide, but manganese is also an abundant element in soil (Parmeggiana, 1983). The great variations in lead and chromium levels among wetlands suggests that these sites cannot be equally considered reference sites for places on site that are identi7ed as being contaminated by DOE. This is especially true of wetland 28, but since these observations are based on few tadpoles, they require further study. In such a study, other remediated sites should be examined. Comparison of Metals in Leopard Frog and Bullfrog Tadpoles It is surprising that the levels of lead, cadmium, and manganese were higher in leopard frog than in bullfrog tadpoles, given that the bullfrogs were
TABLE 4 Correlations (Kendall-s) Values between Six Metal Concentrations (Wet Weight, ppb) in Bodies and Isolated Tissues of Leopard Frog Tadpoles Arsenic
Arsenic Cadmium Chromium Lead Manganese Selenium
; NS 0.40 (0.0001) 0.23 (0.01) NS NS
Arsenic Cadmium Chromium Lead Manganese Selenium
; (0.004) (0.0001) (0.0001) (0.06) NS
0.36 0.92 0.99 0.25
Note. NS, not signi7cant.
Cadmium
Chromium
Lead
Manganese
Selenium
Whole body above diagonal; body compartment below diagonal 0.23 (0.09) 0.71 (0.0001) 0.64 (0.0001) ; 0.34 (0.02) NS 0.25 (0.006) ; 0.65 (0.0001) NS 0.20 (0.04) ; NS NS 0.19 (0.05) NS 0.42 (0.0001) 0.18 (0.05)
NS NS NS NS ; NS
0.22 (0.09) NS 0.33 (0.02) NS NS ;
Tail above diagonal; digestive tract below diagonal 0.25 (0.0001) 0.31 (0.0005) 0.20 (0.03) ; 0.34 (0.0002) NS 0.41 (0.001) ; NS 0.37 (0.004) 0.92 (0.0001) ; 0.24 (0.07) NS 0.26 (0.05) NS NS !0.24 (0.06)
NS NS NS 0.18 (0.06) ; NS
0.16 (0.07) 0.39 (0.0001) 0.37 (0.0001) NS NS ;
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BURGER AND SNODGRASS
examined from a wetland known to be contaminated with arsenic, chromium, lead, and selenium (Burger and Snodgrass, 1998). Whereas the differences in cadmium were slight (107 ppb vs 67 ppb), they were larger for lead (1370 ppb vs 775 ppb) and manganese (116,000 ppb vs 3840 ppb). Even when we eliminate the tadpoles from wetland 28, where we had only three, the levels of lead in leopard frogs (1220 ppb vs 775 ppb) are still higher. We expected that bullfrog tadpoles would have higher levels than leopard frog tadpoles because bullfrog tadpoles are older and larger at the same developmental stage; thus, they have had longer to accumulate metals. Further, they were collected in a known contaminated wetland that is fenced to prevent human exposure (Burger and Snodgrass, 1998). There could be several reasons for these differences: (1) leopard frog tadpoles may absorb metals in the digestive tract more readily than bullfrog tadpoles; (2) once absorbed, leopard frog tadpoles may bioaccumulate contaminants to a greater extent than bullfrog tadpoles; and (3) the metal levels in the sediments in these ‘‘reference wetlands’’ (Schalles et al., 1989) may be higher than previously thought. The differences could not be due solely to sediment levels, since the lead levels in the digestive tract were higher for bullfrogs than for leopard frogs (about 9000 ppb vs 3200 ppb). However, manganese levels were 34,000 ppb in the digestive tracts of bullfrog tadpoles, compared to 164,000 in leopard frog tadpoles. These differences suggest that far more study of potential species differences in absorption and uptake is required before tadpoles can usefully be used as bioindicators. Body Compartment Differences in Metal Levels Across all leopard frog tadpoles, there were higher levels of all metals, except manganese, in the tail than in the body. There were also higher levels in the digestive tract than in the body. The concentrations of lead in the tail were 11 times higher than those in the body, and the concentrations in the digestive tract were 112 times higher. These data indicate that the tadpoles ingest high levels of lead in sediment, and although it is absorbed into the body, a higher percentage is stored in the tail than in the body. Storing lead in the tail may well be a mechanism for ridding the body of high levels, but during absorption of the tail, the tadpole receives a large dose at one time, which could prove problematic. Lead is also a neurotoxin in tadpoles, where it causes learning de7cits (Nixdorf et al., 1997).
Comparing the data for leopard frogs with the data for bullfrogs from a different wetland on SRS (Burger and Snodgrass, 1998), lead, cadmium, and manganese levels were higher and arsenic, selenium, and chromium levels were lower in leopard frogs than in bullfrogs. Furthermore, there is no consistent pattern in relative contaminant levels in the body and tail. This is, the levels of lead were about twice as high in the tadpoles of leopard frog than in the tadpoles of bullfrog, but the ratio of lead in the tail/body was less than 1 for bullfrog and it was 11 for leopard frog. Levels of cadmium were similar in the two species; yet, the ratio of cadmium in the digestive tract/body was 5.8 for leopard frogs and 34.9 for bullfrogs. That some metals are at much higher concentrations in the digestive tract than in the whole body and that some metals are in higher concentrations in the tail than in the body indicates that metals are not being similarly absorbed in the digestive tract and that once in the body, they are not necessarily stored in the tail. Thus, these data suggest that the toxicodynamics of metals within tadpoles may be species speci7c and not merely dependent on their stage of development nor on the relative levels of contaminant exposure. This assumes that exposure can be partly surmised from the relative levels in the digestive tract, which in all cases contained some sediment. Tadpoles are 7lter feeders on sediment and are thus highly exposed to substances that concentrate in sediments (Hall and Mulhern, 1984). Implications for Biomonitoring Amphibians, particularly frogs, are ideal as bioindicators because they are readily available in high numbers, they are easy to work with in the 7eld and laboratory, they have behaviors that can be monitored, they are representative of freshwater environments, and they have a life cycle that often includes both an aquatic and a terrestrial stage (Cooke, 1981; Burger and Snodgrass, 1998). They are microphagous 7lter feeders, making them particularly sensitive to contaminants that concentrate in sediments (Hall and Mulhern, 1984). For tadpoles, it is critical to understand where heavy metals are concentrating, to understand the potential effects on the frogs themselves and on the organisms that consume them. The present study, in conjunction with our previous study on bullfrogs from only one wetland on SRS, indicates that tadpoles do accumulate heavy metals and selenium, that these levels vary in different wetlands, and that accumulated metal levels differ in the tail and in the body.
METAL LEVELS IN SOUTHERN LEOPARD FROGS
Variations in metal levels among body parts (digestive tract, tail, and body) are clearly important for the health and well-being of the tadpole, but may not complicate their use as a bioindicator if the desired objective is to use tadpoles to biomonitor metal concentrations within given wetlands. However, the functional metal concentrations (that is, those that may affect the growth, physiology, and behavior of the tadpole) are more dif7cult to determine because of the differences among the whole tadpole, body, tail, and digestive tract. Our data suggest that determination of whether tadpoles have metal concentrations that are suf7cient to affect their growth and survival requires examination of levels in their body separate from levels in the digestive tract. To assess the food chain effects, however, it may also be critical to know the levels of contaminants in the tadpole tissues, rather than just in the soil or other material in the digestive tract, which might not be bioavailable to animals that consume tadpoles. Whereas tadpoles are abundant and easily captured, caution must be used in employing them as bioindicators without taking into account the relative contribution of the tadpole body versus the digestive tract. ACKNOWLEDGMENTS We thank the South Carolina Department of Natural Resources for permits to collect frogs. This project was funded by the Consortium for Risk Evaluation with Stakeholder Participation (CRESP; through the Department of Energy Cooperative Agreement, AI DE-FC01-95EW55084, DE-FG-26-00NT 40938), NIEHS Grant ESO 5022, the Environmental and Occupational Health Sciences Institute, and Financial Assistance Award No. DE-FC09-96SR 18546 from the U.S. Department of Energy to the University of Georgia Research Foundation. We thank J. Ackerman, D. McElroy, and G. Thomas for 7eld assistance, and C. McCreedy, T. Shukla, M. McMahon, S. Shukla, and C. Dixon for laboratory assistance. During this study, we followed an animal welfare protocol approved by the University of Georgia Institutional Animal Care and Use Committee (A930026) and Rutgers University (97-017).
165
Potential hazards to doves and hunters. Environ. Res. 76, 173}186. Carlson, C. L., and Adriano, D. C. (1993). Environmental impacts of coal combustion residues. J. Environ. Qual. 22, 227}247. Clay, D. L., Brisbin, I. L., Jr., Bush, P. B., and Provost, E. E. (1980). Patterns of mercury contamination in a wintering waterfowl community. Proc. Annual Conf. Southeastern Assoc. Fish Wildl. Agencies 32, 309}317. Colwell, S. V., Kennamer, R. A., and Brisbin, I. L., Jr. (1996). Radiocesium patterns in wood duck eggs and nesting females in a contaminated reservoir. J. Wildl. Manage. 60, 186}194. Cooke, A. S. (1981). Tadpoles as indicators of harmful levels of pollution in the 7eld. Environ. Pollut. 25, 123}133. DiGuilio, R. T. and Monoson, E. (1996). ‘‘Interconnections between Human and Ecosystem Health.’’ Wiley, Chichester, UK. EPA (Environmental Protection Agency). (1981). ‘‘Interim Methods for Sampling and Analysis of Priority Pollutants in Sediments and Fish Tissue.’’ EPA 600/4-81-055, Cincinnati, OH. Gosner, K. L. (1960). A simpli7ed table for staging anuran embryos and larvae with notes on identi7cation. Herpetology 16, 183}190. Hall, R. J., and Mulhern, B. M. (1984). Are anuran amphibians heavy metal accumulators? In ‘‘Vertebrate Ecology and Systematics: A Tribute to Henry S. Fitch’’ (R. A. Seigel, L. E. Hunt, J. L. Knight, L. Malaret, and N. L. Zuschlag, Eds.), pp. 123}133. Museum of Natural History, Univ. of Kansas, Lawrence, KS. Hunsaker, C., Carpenter, D., and Messer, J. (1990). Ecological indicators for regional monitoring. Bull. Ecol. Soc. Am. 71, 165}172. Karr, J. R. (1991). Biological integrity: A long-neglected aspect of water resource management. Ecol. Appl. 1, 66}84. Karr, J. R. (1993). Protection of ecological integrity: An urgent societal goal. Yale J. Int. Law 18, 287}306. Kennamer, R. A., McCreedy, C. D., and Brisbin, I. L., Jr. (1993). Patterns of radiocesium contamination in eggs of free-ranging wood ducks. J. Wildl. Manage. 75, 716}724. Kremen, C. (1992). Assessing the indicator properties of species assemblages for natural areas monitoring. Ecol. Appl. 2, 203}217. NRC (National Research Council). (1991). ‘‘Animals as Sentinels of Environmental Health Hazards.’’ National Academy Press, Washington, DC. NRC (National Research Council). (1993). ‘‘Issues in Risk Assessment.’’ National Academy Press, Washington, DC. Nixdorf, W. L., Taylor, D. H., and Isaacson, L. G. (1997). Use of Bullfrog tadpoles (Rana catesbeiana) to examine the mechanisms of lead neurotoxicity. Am. Zool. 37, 363}368.
REFERENCES
O’Connor, J. S., and Dewling, R. T. (1986). Indices of marine degradation: Their utility. Environ. Manage. 10, 335}343.
Brisbin, I. L. (1993). Birds as monitors of radionuclide contamination. In ‘‘Birds as Monitors of Environmental Change’’ (R. W. Furness and J. J. D. Greenwood, Eds.), pp. 144}178. Chapman & Hall, London. Burger, J. (1995). A risk assessment for lead in birds. J. Toxicol. Environ. Health 45, 369}396. Burger, J., and Snodgrass, J. (1998). Heavy metals in bullfrogs (Rana catesbeiana) tadpoles: Effects of depuration before analysis. Environ. Toxicol. Chem. 17, 2203}2209. Burger, J., Kennamer, R. A., Brisbin, I. L., Jr., and Gochfeld, M. (1997). Metal levels in mourning doves from South Carolina:
Parmigiani, L. (1983). ‘‘Encyclopedia of Occupational Health Safety.’’ Int. Labor Of7ce, Geneva. Peakall, D. B. (1992). ‘‘Animal Markers as Pollution Indicators.’’ Chapman & Hall, London. Potter, C. M., Brisbin, I. L., Jr., McDowell, S. G., and Whicker, F. W. (1989). Distribution of 137Cs in the American coot (Fulica americanus). J. Environ. Radioact. 9, 105}113. Raimondo, S. M., Rowe, C. L., and Congdon, J. D. (1998). Exposure to coal ash impacts swimming performance and predator avoidance in larval Bullfrogs (Rana catesbeiana). J. Herpetol. 32, 289}292.
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Rowe, L. C., Kinney, O. M., Flori, A. P., and Congdon, J. D. (1996). Oral deformities in tadpoles (Rana catesbeiana) associated with coal ash deposition: Eeffects on grazing ability and growth. Freshwater Biol. 36, 723}730. Rowe, L. C., Kinney, O. M., Nagle, R. D., and Congdon, J. D. (1998). Elevated maintenance costs in an Anuran (Rana catesbeiana) exposed to a mixture of trace elements during the embryonic and early larval periods. Physiol. Zool. 71, 27}35. SAS (Statistical Analysis Systems). (1985). ‘‘SAS Users’ Guide.’’ Statistical Institute, Cary, NC. Schalles, J. F., Sharitz, R. R., Gibbons, J. W., Leversee, G. J., and Knox, J. N. (1989). ‘‘Carolina Bays of the Savannah River Plant.’’ Savannah River Plant National Environmental Research Park Program, Savannah River Ecology Laboratory, Aiken, SC.
Siegel, S. (1956). ‘‘Nonparametric Statistics.’’ McGraw}Hill, New York. Snodgrass, J. W., Ackerman, J. W., Bryan, A. L., and Burger, J. (1999). In8uence of hydroperiod, isolation and heterospeci7cs on the distribution of aquatic salamanders (Sirens and Amphiuma) among depression wetlands. Copeia 1999, 107}113. Snodgrass, J. W., Bryan, A. L., and Burger, J. (2000). Development of expectations of larval amphibian assemblage structure in southeastern depression wetlands. Ecol. Appl., in press. Workman, S. W., and McLeod, K. W. (1990). ‘‘Vegetation of the Savannah River Site: Major Community Types.’’ Publication SRO-NERP-19, Savannah River Ecology Laboratory, Aiken, SC.
Metal Levels in Southern Leopard Frogs from the Savannah River Site: Location and Body Compartment Effects Joanna Burger* and Joel Snodgrass* *Division of Life, Consortium for Risk Evaluation with Stakeholder Participation (CRESP), and Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, New Jersey, 08854-8082; and -Department of Biology, Towson University, Towson, Maryland 21252 Received January 20, 2000
both to preserve ecological integrity (Karr, 1991, 1993) and to protect human health (DiGiulio and Monosson, 1996). In ecological systems, natural chemical cycles and movement of contaminants occur rapidly in aquatic systems. Whereas it may take decades or longer for chemicals to move through terrestrial systems, once they enter an aquatic system, they move rapidly through the food chain, potentially reaching human consumers within days or weeks, rather than years. Indicators are often selected to assess levels and effects of toxics in ecosystems (O’Connor and Dewling, 1986; Hunsaker et al., 1990; NRC, 1991, 1993; Kremen, 1992), largely because of the overwhelming dif7culty of assessing even a small fraction of the biota. This has led to the examination of species that are high on the food chain (O’Connor and Dewling, 1986; Peakall, 1992; Burger, 1995), such as 7sh-eating birds and predatory mammals. In aquatic systems, however, the top trophic level is often composed of predatory amphibians or 7sh, which are appropriate bioindicators of these systems. In some ephemeral aquatic systems, particularly those with a short hydroperiod, the body burdens of heavy metals in larval amphibians, such as tadpoles, are being used as indicators of metal pollution (Carlson and Adriano, 1993). One potential problem with the use of tadpole body burdens as bioindicators is that tadpoles ingest sediment, and analysis of whole bodies may overestimate the concentration of contamination in tissues if some of the sediment (with heavy metals) remains in the digestive tract and these metals are not incorporated into tissues. Burger and Snodgrass (1998) showed that, in bullfrog (Rana catesbeiana) tadpoles, the variance in concentrations of arsenic, chromium, lead, manganese, mercury, selenium, and cadmium was explained only by body compartment (body, tail, whole body);
Tadpoles have been proposed as useful bioindicators of environmental contamination; yet, recently it has been shown that metal levels vary in different body compartments of tadpoles. Metals levels are higher in the digestive tract of bullfrog (Rana catesbeiana) tadpoles, which is usually not removed during such analysis. In this paper we examine the heavy metal levels in southern leopard frog (R. utricularia) tadpoles from several wetlands at the Savannah River Site and test the null hypotheses that (1) there are no differences in metal levels in different body compartments of the tadpoles, including the digestive tract; (2) there are no differences in heavy metal levels among different wetlands; and (3) there are no differences in the ratio of metals in the tail/body and in the digestive tract/body as a function of metal or developmental stage as indicated by body weight. Variations in heavy metal levels were explained by wetland and body compartment for all metals and by tadpole weight for selenium and manganese. In all cases, levels of metals were higher in the digestive tract than in the body or tail of tadpoles. Metal levels were highest in a wetland that had been remediated and lowest in a wetland that was never a pasture or remediated (i.e., was truly undisturbed). Although tadpoles are sometimes eaten by Ash and other aquatic predators, leopard frogs usually avoid laying their eggs in ponds with such predators. However, avian predators will eat them. These data suggest that tadpoles can be used as bioindicators of differences in metal levels among wetlands and as indicators of potential exposure for higher-trophic-level organisms, but that to assess effects on the tadpoles themselves, digestive tracts should be removed before analysis. 2001 Academic Press
INTRODUCTION
It is increasingly clear that as a society we are interested in assessment of the health of ecosystems, 157
0013-9351/01 $35.00 Copyright 2001 by Academic Press All rights of reproduction in any form reserved.
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for arsenic, chromium, and lead, in tadpoles held for 2 days without being fed metal concentrations decreased slightly. Moreover, for fresh tadpoles, the digestive tract contained signi7cantly higher concentrations of all metals than either the body or the tail, probably re8ecting metals absorbed to sediment particles in the gut (Burger and Snodgrass, 1998). Thus, analysis of whole tadpoles may not give a realistic picture of exposure because of averaging across compartments with very different levels of metals. Before frogs and other amphibians can be used effectively as bioindicators it is important to know whether metal levels consistently differ among body compartments for different species. In this paper we examine the levels of heavy metals in leopard frog (R. utricularia) tadpoles from the Savannah River Site (SRS), a Department of Energy (DOE) Site in South Carolina. We wanted to con7rm that (1) heavy metals vary by body compartment and (2) levels of all metals are signi7cantly higher in the digestive tract than in the whole body. Further, we test the null hypothesis that there are no differences in heavy metals in different wetlands, three of which are considered reference sites (Schalles et al., 1989). If the three sites are all truly reference sites, with the same atmospheric deposition history and no DOE site contamination for the nearly 50 years of their occupation, then levels should be similar in the tadpoles from the various sites. We expected that metal levels would be higher in wetland 28 because it was a remediated site (Kirkman et al., 1996). We also examine whether the ratio of metals in different body compartments varied by metal. The isolated wetlands on SRS have a diverse community that includes ranid frogs, sirens, predaceous diving beetles, snakes, and dragon8y larva. Of these, amphibians are the most likely to accumulate body burdens because toxicants can readily enter their body through their diet and through absorption across their semipermeable skin. In the egg and tadpole stages, amphibians live entirely in wetlands and obtain all their nutrients from the aquatic environment, making them useful as a tool for the assessment of contaminants in wetland habitats and as sentinels of potential effects (Cooke, 1981). However, the distribution of contaminants in different body parts and the relative contribution of stomach contents to the body burden must be known for several species before metal concentrations can be interpreted or before they can be used effectively as a biomonitoring tool. This study was initiated partly because of the 7ndings of mouth part deformities in bullfrog tadpoles on SRS wetlands associated with coal ash
deposits, which are highly contaminated with selenium, among other elements (Rowe et al., 1996, 1998). Further, Raimondo et al. (1998) demonstrated de7cits in predator avoidance and elevated maintenance costs of tadpoles exposed to coal ash. MATERIALS AND METHODS
With appropriate state permits, tadpoles were collected from four Carolina bays within the Savannah River Site in 1997 and brought to the Savannah River Ecology Laboratory on the SRS where they were frozen. The Savannah River Site is a 780-km2 nuclear production and research facility of the U.S. Department of Energy, located in west-central South Carolina (33.1@N, 81.3@W) and bounded on the southwest side by the Savannah River (Fig. 1). Vegetation communities on the SRS are typical of the upper coastal plain of South Carolina (Workman and McLeod, 1990). Some of the cooling reservoirs for the reactors (no longer active) were contaminated with 137 Cs (radiocesium) and smaller amounts of other
FIG. 1. Map of the Savannah River Site, showing the locations of the wetlands studied.
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
radionuclides from 1954 to 1964, in addition to mercury, which most likely originated from Savannah River water pumped through the reservoir (Clay et al., 1980). Whereas there has been extensive study of radionuclides and mercury on the cooling reservoirs of SRS (Clay et al., 1980; Potter et al., 1989; Brisbin, 1993; Kennamer et al., 1993; Colwell et al., 1996; Burger et al., 1997), heavy metals and selenium from the wetlands that we studied have not been examined. The wetlands from which we collected tadpoles are referred to as wetlands 10, 27, 28, and 66 (Schalles et al., 1989). We use these numbers here to refer to these wetlands because other authors have used them in the past. All of the wetlands are considered depression wetlands in which water level 8uctuations are controlled by precipitation and vary among years. All wetlands have similar hydroperiods involving winter and spring recharge, summer draw down, and eventual drying in late summer or early fall. Vegetation in the wetlands is generally characterized by herbaceous and emergent species. Kirkman et al. (1996) provides information on past land use within and size of wetlands. Wetlands range in size from 1.59 ha (10) to 11.65 ha (66). Wetland 10 was not disturbed by agricultural practices before 1951 (when the site was established). Wetlands 27 and 66 were pasture before 1951. Wetland 28 is a remediated CERCLA site that was previously contaminated by metals and radionuclides and was cultivated for row crops before 1951. Remediation activities involved removal of sediments and planting of vegetation and were complete in the late 1980s. Tadpoles were collected with dip nets. They were collected as part of a study on species diversity in these wetlands (Snodgrass et al., 2000); so sample sizes re8ect these considerations. Thus, we did not have the same number of tadpoles available from each wetland. Tadpoles were euthanized by slow cooling, stored in plastic bags, labeled by species and collection location, and frozen (!20@C) before being shipped to the Environmental and Occupational Health Sciences Institute laboratory at Rutgers University. In the laboratory all tadpoles were thawed and weighed. The digestive tract was removed from all tadpoles; the tail and body sections were separated, weighed again, and analyzed separately. We de7ne the body as the whole tadpole minus the tail. In the tables, whole body refers to the whole tadpole, which consists of the body, tail, and digestive tract. Since we analyzed the metals in the individual body compartments, we reconstructed the levels in the whole
body by multiplying the weight of the compartment by the metal levels (see Table 2). All tadpoles were at stage 25 (after Gosner, 1960). Tissues were digested in 70% nitric acid in a microwave vessel for 10 min under 130 pounds per square inch and then diluted in deionized water (EPA, 1981). The sediment in the digestive tracts of the tadpoles did not digest, although some metals may have been removed from the sand during the extraction process. Mercury was analyzed by a cold vapor technique, and all other metals were analyzed by graphite furnace atomic absorption. All concentrations are expressed in lg/kg (parts per billion) on wet weights obtained from air-dried specimens (except where noted in the tables) and are rounded to three signi7cant 7gures. Mercury detection limits were 0.2 lg/kg; detection limits for other metals were 0.7 lg/kg (selenium) and 0.15 lg/kg (lead) or lower. All specimens were run in batches that included a standard calibration curve and spiked specimens. The accepted recoveries ranged from 87 to 105%, and batches with recoveries less than 87% were rerun. Further quality control measures included periodic blind analysis of an aliquot from a large sample of known concentrations and blind runs of duplicate samples during the analysis for each metal. In all cases, there was no signi7cant difference between duplicate samples (correlations of 0.9 or higher for all metals). Regression procedures on log-transformed data were used to determine whether wetland, tadpole body compartment, and tadpole weight contributed to differences in metal levels (PROC GLM; SAS, 1985). The procedure adds the variable that contributes the most to the R2, then adds the next variable that increases the R2 the most, and continues until all signi7cant variables are added. Wilcoxon tests were used to examine differences between groups (Siegel, 1956); Wilcoxon is a nonparametric analysis of variance based on ranks and is more conservative than parametric ANOVAs. Kendall q correlations were used to examine the relationships between the concentrations of different metals in body and tail. RESULTS
All of the leopard frog tadpoles had nondetectable levels of mercury; mercury is not discussed further in this paper. Models Explaining Variations in Metal Levels Despite the low sample sizes in some wetlands, 42 to 67% of the variation in metal levels was explained
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TABLE 1 Models Explaining Variations in Concentrations (Wet Weight, ppb) of Six Metals Found in Leopard Frog Tadpoles (with All Body Compartments, Including Digestive Tract)
Model F df r2 P Factors entering, s2 (P) Wetland Compartment Wetland;compartment Recombined weight
Lead
Cadmium
Arsenic
Selenium
Manganese
Chromium
10.4 11,165 0.45 0.0001
9.19 11,163 0.42 0.0001
19.4 11,162 0.61 0.0001
14.8 11,165 0.54 0.0001
23.7 11,155 0.67 0.0001
17.9 11,162 0.59 0.0001
25.4 (0.0001) 2.55 (0.06) NS NS
16.5 (0.0001) 6.54 (0.0003) NS NS
51.7 (0.0001) 5.58 (0.001) NS NS
8.58 (0.0003) 6.90 (0.0002) 3.73 (0.002) 10.8 (0.001)
9.4 (0.0002) 43.6 (0.0001) 22.32 (0.04) 5.38 (0.02)
27.2 (0.0001) 18.1 (0.0001) NS NS
Note. NS, not signi7cant.
by wetland and body compartment (section); weight also entered the models for selenium and manganese (Table 1). Locational Differences in Metal Levels There were differences in heavy metal levels in whole tadpoles (including all parts) from the different wetlands for all metals (Table 2). In general, metals were highest in wetlands 28 and 66 (see Fig. 1). Levels were highest for manganese, followed by lead and chromium, and were lowest for cadmium, arsenic, and selenium. In most cases, these differences persisted for each body compartment (Table 2). Body Compartment Differences in Metal Levels There were differences in metal levels in body compartments (Fig. 2). Metals levels were signi7cantly higher in the digestive tract than in the other body compartments for all metals (s2 tests, P(0.0001 for all metals). There were signi7cant differences between the tail and the body only for lead (P(0.001) and manganese (P(0.02); levels were higher in the body than in the tail (refer to Fig. 2). Ratios of Metals in Different Body Compartments One important aspect of the use of tadpoles for biomonitoring is to understand whether the ratio of metals in different body compartments is constant for different metals and whether this ratio is the same for different species. Generally the ratios are higher for the digestive tract/body than for the tail/body (Fig. 3). There is a great deal of variability among individuals, however, in the amounts of lead
in the body, tail, and digestive tract, as re8ected by the ratios between them (refer to Fig. 3). Figure 3 also shows the similar ratios for the bullfrog, computed from Burger and Snodgrass (1998). In general, there are greater differences in the ratios for leopard frog than for bullfrog (except for cadmium). It is possible that the amount of metal in the body compared to that in the tail is a function of development; as tadpoles age (and become heavier), they deposit more metal into their tails. To examine this possibility, we computed the correlation of the ratio of the amount of metal in the tail to the amount of metal in the body against weight (a surrogate for age). We predicted that the amount in the tail should increase with age if they are storing the metals there. However, for many metals there was no relationship, and where there was, it was negative for 7ve of six signi7cant correlations for the tail/body compartment ratio (Table 3). That is, the relative amount of metals in the tail for leopard frog decreased with tadpole weight for manganese and chromium. However, this ratio increased with weight for selenium, suggesting that the tadpoles put relatively more selenium in their tail as they aged. They also had more in their digestive tract as they aged (Table 3). We also computed these correlations for bullfrogs (computed from Burger and Snodgrass, 1998). Except for selenium in bullfrogs, there was no signi7cant relationship between the ratio of metals in the digestive tract/body and the weight (Table 3). Correlations among Metals There were a number of signi7cant correlations among metal levels in all compartments for all
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
TABLE 2 Concentration Value (Mean ⴞ SE; Wet Weight, ppb) for Six Metals in Tissues from Leopard Frog Tadpoles
Sample Whole body Arsenic Cadmium Chromium Lead Manganese Selenium Body Arsenic Cadmium Chromium Lead Manganese Selenium Tail Arsenic Cadmium Chromium Lead Manganese Selenium Digestive tract Arsenic Cadmium Chromium Lead Manganese Selenium
66
10
Wetland 28
27
8
7
3
38
167$32.6 149 A 131$18.7 126 A,B 1,840$277 1,710 A 3,180$669 2,790 B 41,400$2,770 40,800 B 397$27.3 391 A
27.4$6.84 22.6 C 38.4$2.91 37.8 C 137$8.73 136 C 256$33.2 243 D 13,600$950 13,400 C 152$18.7 146 C
206$50.7 194 A 267$94.3 234 A 1,710$303 1,650 A 5,450$1,120 5,240 A 146,000$;a
69.1$6.57 60.3 B 104$19.2 80.2 B 394$54.6 300 B 983$78.9 882 C 153,000$11,400 143,000 A 238$21.3 224 B
25.1 (0.0001)
49.7$18.5 37.7 A 108$29.2 86.4 A 537$79.2 501 A 1,771$751 297 A 35,000$3,910 33,900 B 377$29.8 370 A
17.4$4.93 12.1 B 30.9$3.39 29.9 B 82.1$9.57 79.0 C 180$64.6 58.9 A 16,600$1,790 16,100 C 91.9$28.5 66.0 C
40.4$4.39 39.9 A 100$37.2 85.2 A 261$59.5 245 B 783$773 38.8 A 20,700$1,570
35.3$3.54 29.1 A 57.2$12.8 41.3 A,B 167$15.3 148 B 611$115 234 A 166,000$21,100 137,000 A 204$34.4 174 B
5.37 (NS)
106$29.5 81.3 A 94.9$21.9 86.3 A 979$233 782 A 592$264 69.0 B 41,800$6,330 38,400 A 429$57.5 405 A
15.0$5.45 13.7 B 22.7$2.57 21.8 B 52.8$8.81 49.1 C 53.4$15.4 30.9 B 6,340$577 6,200 B 152$15.1 148 B
30.2$3.08 29.9 B 229$191 90.8 A 3,650$3490.0 641 A 1,660$1,140 1,010 A 841,000$62,700 319$16.4 318 A
31.9$2.80 28.4 B 94.2$41.3 46.5 A,B 175$29.0 132 B 157$29.1 45.0 B 105,000$17,500 65,900 A 188$8.31 181 B
388$109 315 A 160$13.2 156 B 3,850$428 3,640 A 6,580$1,300 2,820 A,B 50,400$6,330 49,200 B 439$30.4 432 A
70.0$19.4 48.9 C 76.4$14.3 69.3 C 386$40.1 375 B 747$132 653 B 19,000$1,830 18,600 C 284$55.7 217 B
504$144.0 465 A 466$266 334 A 2,610$1,280 1,480 A,B 137,000$3,370 12,900 A 173,000$62,200 140,000 A 270$68.4 251 B
135$14.0 115 B 154$35.6 118 B,C 863$129 507 B 2,120$173 1,720 B 212,000$19,100 186,000 A 309$12.0 300 A,B
274$39.9 268 B
259$30.5 255 A,B
Wilcoxon s2 (p)
Overall 56
24.3 (0.0001) 26.6 (0.0001) 31.3 (0.0001) 27.5 (0.001) 23.5 (0.0001)
10.8 (0.01) 26.1 (0.0001) 5.43 (NS) 28.6 (0.0001) 26.0 (0.0001)
9.93 (0.02) 15.6 (0.001) 28.1 (0.0001) 7.25 (0.06) 18.7 (0.0001) 26.5 (0.0001)
22.4 (0.0001) 16.5 (0.0009) 20.3 (0.0002) 25.1 (0.0001) 29.4 (0.0001) 11.3 (0.01)
83.7$9.11 63.6 107$15.6 81.1 640$98.2 383 1,370$200 911 116,000$11,700 84,800 250$17.6 230 35.2$3.53 27.4 62.8$10.0 45.4 210$23.5 165 694$130 212 119,000$17,000 75,500 215$26.2 173 40.3$5.79 30.1 92.5$30.9 46.8 461$193 165 287$81.1 69.9 82,100$13,100 44,500 225$15.5 205 183$24.9 129 162$28.8 121 1,350$197 697 3,200$481 1,710 164,000$17,000 116,000 323$13.3 301
Note. Geometric means and Duncan Multiple Range values given below each mean. Difference in letters across represent signi7cant differences in metal concentration with a Duncan Multiple Range test. Wetlands 10, 28, and 66 are considered reference sites. ? Only one value to analyze; no standard error available.
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compartment and that the digestive tract has signi7cantly higher levels of all metals than the body. There were also signi7cant differences in metal levels among wetlands even though the sample sizes were low for some sites. These 7ndings are discussed below, along with the generality of the ratios of metal levels in different body compartments. Locational Differences in Metal Levels
FIG. 2. Metal levels (ppb) in different body compartments of leopard frogs collected at the Savannah River Site.
leopard frogs combined (Table 4). In general, lead was signi7cantly correlated with most other metals in most body compartments, as was chromium. Nearly all correlations were positive; as one metal increased, the other did also. The correlations, although signi7cant, were not high, except for lead and arsenic, lead and chromium, and arsenic and chromium in the digestive tract and in the whole body (Table 4). DISCUSSION
The results of this study con7rm our pervious 7ndings (Burger and Snodgrass, 1998) that there were signi7cant differences in metal levels by body
FIG. 3.
Three wetland sites where we collected tapoles on the Savannah River Site are considered reference sites (Schalles et al., 1989). There is a considerable amount of ecological research conducted on the SRS by the personnel of the Savannah River Ecology Laboratory, and these sites are considered reference sites because they have not been impacted by the industrial and radiological research activities of the Department of Energy. Presumably, these sites experience the same amount of atmospheric deposition and receive no point-source contamination from SRS itself. This suggests that contamination should be similar in all three wetlands examined. Hydroperiod, however, varies, although all four wetlands dry down eventually during the summer. Thus, the cycle of drying down and re7lling, which results in an increase in some metals through increases in bioavailability (in mercury, for example), should also be similar among the wetlands. However, we found signi7cant differences in the levels of all metals in the tadpoles from the
Ratio of metals in the tail/body and digestive tract/body for bullfrogs and leopard frogs collected at the Savannah River Site.
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METAL LEVELS IN SOUTHERN LEOPARD FROGS
TABLE 3 Correlation of Body Weight to Two Body Compartment Metal Ratios in Species of Tadpoles (Wet Weight, ppb) Bullfrog
Leopard frog
Tail/body compartment ratio, r2 (P) Arsenic !0.18 (0.06) Cadmium NS Chromium NS Lead NS Manganese !0.25 (0.009) Selenium !0.20 (0.04)
NS NS !0.17 (0.06) NS !0.30 (0.002) 0.31 (0.0006)
Digestive tract/body compartment ratio, r2 (P) Arsenic NS Cadmium NS Chromium NS Lead NS Manganese NS Selenium NS 0.32
NS NS NS NS NS (0.0003)
Note. NS, not signi7cant.
wetlands, even given the small sample sizes. Overall, metal levels were generally highest in the remediated site and lower in the reference sites. However, there was variation even within the reference sites. Metal levels were generally highest in tadpoles from wetlands 28 and 66 and lowest in wetland 10. We suggest that these differences may be due to land use differences before the DOE bought the land in the early 1950s. Wetland 10 was a herbaceous wetland when the site was established; it was not previously used for agriculture (Kirkman et al., 1996). Wetlands 27 and 66 were pastures in 1951, but since DOE occupation, they have undergone suc-
cession to herbaceous/forested wetlands. The suggestion that agricultural processes nearly 50 years ago are still affecting contaminant levels in these wetlands is intriguing and requires further study. Wetland 28, the wetland where the whole body metal levels in tadpoles were the highest (except for selenium and manganese), was a contaminated site that had undergone remediation, which included removal of all sediments and planting of vegetation (Kirkman et al., 1996). It suggests, however, that not all contamination was removed. Additionally, this wetland was cultivated before the site establishment, making it dif7cult to assess the sources of metals in this wetland. Overall, levels were relatively high for lead, manganese, and chromium. Lead arsenate was a common insecticide 50 years ago, and manganese was used as fungicide, but manganese is also an abundant element in soil (Parmeggiana, 1983). The great variations in lead and chromium levels among wetlands suggests that these sites cannot be equally considered reference sites for places on site that are identi7ed as being contaminated by DOE. This is especially true of wetland 28, but since these observations are based on few tadpoles, they require further study. In such a study, other remediated sites should be examined. Comparison of Metals in Leopard Frog and Bullfrog Tadpoles It is surprising that the levels of lead, cadmium, and manganese were higher in leopard frog than in bullfrog tadpoles, given that the bullfrogs were
TABLE 4 Correlations (Kendall-s) Values between Six Metal Concentrations (Wet Weight, ppb) in Bodies and Isolated Tissues of Leopard Frog Tadpoles Arsenic
Arsenic Cadmium Chromium Lead Manganese Selenium
; NS 0.40 (0.0001) 0.23 (0.01) NS NS
Arsenic Cadmium Chromium Lead Manganese Selenium
; (0.004) (0.0001) (0.0001) (0.06) NS
0.36 0.92 0.99 0.25
Note. NS, not signi7cant.
Cadmium
Chromium
Lead
Manganese
Selenium
Whole body above diagonal; body compartment below diagonal 0.23 (0.09) 0.71 (0.0001) 0.64 (0.0001) ; 0.34 (0.02) NS 0.25 (0.006) ; 0.65 (0.0001) NS 0.20 (0.04) ; NS NS 0.19 (0.05) NS 0.42 (0.0001) 0.18 (0.05)
NS NS NS NS ; NS
0.22 (0.09) NS 0.33 (0.02) NS NS ;
Tail above diagonal; digestive tract below diagonal 0.25 (0.0001) 0.31 (0.0005) 0.20 (0.03) ; 0.34 (0.0002) NS 0.41 (0.001) ; NS 0.37 (0.004) 0.92 (0.0001) ; 0.24 (0.07) NS 0.26 (0.05) NS NS !0.24 (0.06)
NS NS NS 0.18 (0.06) ; NS
0.16 (0.07) 0.39 (0.0001) 0.37 (0.0001) NS NS ;
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examined from a wetland known to be contaminated with arsenic, chromium, lead, and selenium (Burger and Snodgrass, 1998). Whereas the differences in cadmium were slight (107 ppb vs 67 ppb), they were larger for lead (1370 ppb vs 775 ppb) and manganese (116,000 ppb vs 3840 ppb). Even when we eliminate the tadpoles from wetland 28, where we had only three, the levels of lead in leopard frogs (1220 ppb vs 775 ppb) are still higher. We expected that bullfrog tadpoles would have higher levels than leopard frog tadpoles because bullfrog tadpoles are older and larger at the same developmental stage; thus, they have had longer to accumulate metals. Further, they were collected in a known contaminated wetland that is fenced to prevent human exposure (Burger and Snodgrass, 1998). There could be several reasons for these differences: (1) leopard frog tadpoles may absorb metals in the digestive tract more readily than bullfrog tadpoles; (2) once absorbed, leopard frog tadpoles may bioaccumulate contaminants to a greater extent than bullfrog tadpoles; and (3) the metal levels in the sediments in these ‘‘reference wetlands’’ (Schalles et al., 1989) may be higher than previously thought. The differences could not be due solely to sediment levels, since the lead levels in the digestive tract were higher for bullfrogs than for leopard frogs (about 9000 ppb vs 3200 ppb). However, manganese levels were 34,000 ppb in the digestive tracts of bullfrog tadpoles, compared to 164,000 in leopard frog tadpoles. These differences suggest that far more study of potential species differences in absorption and uptake is required before tadpoles can usefully be used as bioindicators. Body Compartment Differences in Metal Levels Across all leopard frog tadpoles, there were higher levels of all metals, except manganese, in the tail than in the body. There were also higher levels in the digestive tract than in the body. The concentrations of lead in the tail were 11 times higher than those in the body, and the concentrations in the digestive tract were 112 times higher. These data indicate that the tadpoles ingest high levels of lead in sediment, and although it is absorbed into the body, a higher percentage is stored in the tail than in the body. Storing lead in the tail may well be a mechanism for ridding the body of high levels, but during absorption of the tail, the tadpole receives a large dose at one time, which could prove problematic. Lead is also a neurotoxin in tadpoles, where it causes learning de7cits (Nixdorf et al., 1997).
Comparing the data for leopard frogs with the data for bullfrogs from a different wetland on SRS (Burger and Snodgrass, 1998), lead, cadmium, and manganese levels were higher and arsenic, selenium, and chromium levels were lower in leopard frogs than in bullfrogs. Furthermore, there is no consistent pattern in relative contaminant levels in the body and tail. This is, the levels of lead were about twice as high in the tadpoles of leopard frog than in the tadpoles of bullfrog, but the ratio of lead in the tail/body was less than 1 for bullfrog and it was 11 for leopard frog. Levels of cadmium were similar in the two species; yet, the ratio of cadmium in the digestive tract/body was 5.8 for leopard frogs and 34.9 for bullfrogs. That some metals are at much higher concentrations in the digestive tract than in the whole body and that some metals are in higher concentrations in the tail than in the body indicates that metals are not being similarly absorbed in the digestive tract and that once in the body, they are not necessarily stored in the tail. Thus, these data suggest that the toxicodynamics of metals within tadpoles may be species speci7c and not merely dependent on their stage of development nor on the relative levels of contaminant exposure. This assumes that exposure can be partly surmised from the relative levels in the digestive tract, which in all cases contained some sediment. Tadpoles are 7lter feeders on sediment and are thus highly exposed to substances that concentrate in sediments (Hall and Mulhern, 1984). Implications for Biomonitoring Amphibians, particularly frogs, are ideal as bioindicators because they are readily available in high numbers, they are easy to work with in the 7eld and laboratory, they have behaviors that can be monitored, they are representative of freshwater environments, and they have a life cycle that often includes both an aquatic and a terrestrial stage (Cooke, 1981; Burger and Snodgrass, 1998). They are microphagous 7lter feeders, making them particularly sensitive to contaminants that concentrate in sediments (Hall and Mulhern, 1984). For tadpoles, it is critical to understand where heavy metals are concentrating, to understand the potential effects on the frogs themselves and on the organisms that consume them. The present study, in conjunction with our previous study on bullfrogs from only one wetland on SRS, indicates that tadpoles do accumulate heavy metals and selenium, that these levels vary in different wetlands, and that accumulated metal levels differ in the tail and in the body.
METAL LEVELS IN SOUTHERN LEOPARD FROGS
Variations in metal levels among body parts (digestive tract, tail, and body) are clearly important for the health and well-being of the tadpole, but may not complicate their use as a bioindicator if the desired objective is to use tadpoles to biomonitor metal concentrations within given wetlands. However, the functional metal concentrations (that is, those that may affect the growth, physiology, and behavior of the tadpole) are more dif7cult to determine because of the differences among the whole tadpole, body, tail, and digestive tract. Our data suggest that determination of whether tadpoles have metal concentrations that are suf7cient to affect their growth and survival requires examination of levels in their body separate from levels in the digestive tract. To assess the food chain effects, however, it may also be critical to know the levels of contaminants in the tadpole tissues, rather than just in the soil or other material in the digestive tract, which might not be bioavailable to animals that consume tadpoles. Whereas tadpoles are abundant and easily captured, caution must be used in employing them as bioindicators without taking into account the relative contribution of the tadpole body versus the digestive tract. ACKNOWLEDGMENTS We thank the South Carolina Department of Natural Resources for permits to collect frogs. This project was funded by the Consortium for Risk Evaluation with Stakeholder Participation (CRESP; through the Department of Energy Cooperative Agreement, AI DE-FC01-95EW55084, DE-FG-26-00NT 40938), NIEHS Grant ESO 5022, the Environmental and Occupational Health Sciences Institute, and Financial Assistance Award No. DE-FC09-96SR 18546 from the U.S. Department of Energy to the University of Georgia Research Foundation. We thank J. Ackerman, D. McElroy, and G. Thomas for 7eld assistance, and C. McCreedy, T. Shukla, M. McMahon, S. Shukla, and C. Dixon for laboratory assistance. During this study, we followed an animal welfare protocol approved by the University of Georgia Institutional Animal Care and Use Committee (A930026) and Rutgers University (97-017).
165
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