Ecotoxicological Studies in Amphibian Populations of Southern Ontario

J. Great Lakes Res. 24(1 ):45-54 Internat. Assoc. Great Lakes Res., 1998

Ecotoxicological Studies in Amphibian Populations of Southern Ontario Katherine A. Gillan, Bruce M. Hasspielert , Ronald W. Russell, Khosrow Adeli t , and G. Douglas Haffner* Departments of Biological Sciences and tChemistry & Biochemistry Great Lakes Institute for Environmental Research University of Windsor 304 Sunset Ave. Windsor, Ontario N9B 3P4 ABSTRACT. In order to evaluate the relative exposure and stress of environmental contaminants on amphibian populations of Southern Ontario, two species offrogs, Rana pipiens and Rana clamitans, were collected from nine sites and analyzed for polychlorinated biphenyls (PCBs) and pesticides. Sediment samples were also collected, and analyzed for PCBs, pesticides, and polycyclic aromatic hydrocarbons (PAHs). Biota-sediment accumulation factors (BSAFs) were calculated for PCBs and pesticides at all sitesfor both species offrogs. BSAFs ranged from 33.28 ± 16.16 to 1.06 ± O.Ofor leopardfrogs and from 23.02 ± 7.89 to 0.42 ± 0.0 for green frogs. Sediment extracts were further tested for cytotoxicity and genotoxicity on a leopard frog embryo cell line. The Neutral Red Uptake bioassay was used to measure cytotoxicity and a DNA break bioassay was used to test genotoxicity. Cytotoxicity was evident in four of the nine sites, Cornwall, Brighton, Ancaster, and Ojibway, at 200 g sediment equivalents per liter of culture medium. Genotoxicity, expressed as F-values, ranged from 0.921 ± 0.052 to 0.975 ± 0.004, indicating that sediment extracts were not causing significant genotoxic stress. INDEX WORDS: factor (BSAF).

Green frog, leopard frog, genotoxicity, cytotoxicity, biota-sediment accumulation

INTRODUCTION

both as a predator and as a prey (Wright and Wright 1933, Duellman and Trueb 1986). Many amphibian species occupy spatially discontinuous habitats, and the relative abundance of species is affected by local colonizations and extinctions (Pechmann and Wilbur 1994). The physiology and somewhat complicated life cycle of amphibians make them susceptible to environmental degradation, and therefore amphibians have the potential to be biological indicators of ecosystem health (Barinaga 1990, Vitt et al. 1990). Home ranges of amphibians are typically small, with green frog home range sizes varying from 20 to 200 m 2 (Martof 1953). This site fidelity and the fact that amphibians are relatively long lived (Duellman and Trueb 1986) result in an excellent system to develop in situ causeeffect models for toxic chemicals. Chemical uptake in amphibians can occur via absorption through the skin (particularly when hibernating in sediments), and from the ingestion of water and food. The aim of this study is to quantify the importance of bioaccumulation of persistent chemicals in amphibians and to determine the rela-

There is an increasing concern about the apparent global decline of amphibian species, and environmental contamination has been suggested as a possible cause (Russell et al. 1995, Barinaga 1990, Phillips 1990). Effects of exposure of amphibian species to xenobiotic chemicals include uncoordinated activity (Cooke 1970), developmental anomalies (Osborn et al. 1981, Cooke 1979, Cooke 1972), increased predation (Cooke 1971), cancer (Busbee et al. 1978), and death (Sanders 1970). Other potential causes contributing to amphibian declines include habitat destruction, acid precipitation, and increased UV-b radiation (Barinaga 1990). To determine the relative importance of contaminant stress is difficult as a result of the interactive and interdependent nature of the factors related to the decline of amphibians (Russell et al. 1995). Amphibians play an important role in both aquatic and terrestrial food webs in that they act

"'Corresponding author. E-mail: [email protected]

45

Gillan et al.

46

tive genotoxic and/or cytotoxic threat of sediment bound chemicals to amphibians. Chemical accumulation was measured by determining biota-sediment accumulation factors (BSAFs) for two species of frogs (Rana pipiens and Rana clamitans) from nine sites along the north shore of the lower Great Lakes. To determine the potential stress of chemical mixtures in the sediment, sediment extracts were screened using two in vitro bioassays with a cultured frog embryo cell line (Rana pipiens). The first is an assay for cytoxicity based on the ability of healthy, intact cells to uptake a membrane impermeable dye, Neutral Red (Ali et al. 1994). The second assay is based on the quantification of genotoxicity, monitored as DNA single strand breaks (SSB) (Hasspieler et al. 1995). The formation of DNA SSB may result from exposure to a variety of genotoxic agents including PAHs, aromatic amines, quinones and nitroaromatic compounds (Hasspieler et al. 1995). METHODS Sample Collection A total of 18 leopard frogs (Rana pipiens) and 26 green frogs (Rana clamitans) was collected at nine

sites along the north shore of the lower Great Lakes (Fig. 1). At all sites, 4 hours were spent in search of frogs in order to assess relative abundance. Each frog was wrapped in hexane-rinsed aluminum foil and stored at -20°C until preparation for gas chromatographic (GC) analysis. Composite sediment samples were collected at each of the nine sites and stored in hexane-rinsed amber jars at -20°C until preparation for GC analysis. Sample Preparation Tissues were prepared for gas chromatography according to the method of Lazar et al. (1992). Individual frogs were ground whole by mortar and pestle in 20 g anhydrous sodium sulfate then added to a 0.025 x 0.60 m glass column containing 10 g anhydrous sodium sulfate and 70 mL 1: 1 dichloromethane:hexane. After 1 hour, the column was eluted with 250 mL of 1: 1 dichloromethane:hexane solution. Two milliliters of extract were removed for gravimetric lipid determination. The extract was concentrated to 2 mL by rotary evaporation and added to a 0.0 I x 0.55 m glass column containing 40 g activated Florisil® (60/100 mm mesh) and 3 g anhydrous sodium sulfate for cleanup. The column was eluted with 50 mL hexane to yield Fraction I followed by elution with 50 mL of 15% dichloro(1)

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Ecotoxicological Studies in Amphibian Populations TABLE 1.

47

Chemicals analyzedfor by gas chromatography from frog tissue and sediment samples.

Fraction I (Tissue and sediment) 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB) 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) pentachlorobenzene (QCB) hexachlorobenzene (HCB) octachlorostyrene (OCS) trans-nonachlor p,p'-DDE mirex PCBs (inc!. mono-ortho substituted)

Fraction II (Tissue only) X-hexachlorocyclohexane (x- HCH) ?-hexachlorocyclohexane (?-HCH) ("lr hexachlorocyclohexane ("lr-HCH, lindane) oxychlordane trans-chlordane cis-chlordane cis-nonachlor p,p'-DDT p,p'-DDD

Fraction II (Sediment only)* naphthalene benzo[g,h,ilperylene acenaphtylene dibenzo[a,hlanthracene acenaphtene indeno[ 1,2,3-c,dlpyrene fluorene benzo[alpyrene phenanthrene benzo[klfluoranthene anthracene benzo[blfluoranthene fluoranthene chrysene/triphenylene pyrene benzo[alanthracene *This Fraction is also known to contain coplanar PCBs (Lazar et al, 1992)

methane:hexane to yield Fraction 11. The fractions were collected separately and concentrated to 10 mL for gas chromatography. Sediment samples were extracted as described previously (Ali et al, 1993). A polycyclic aromatic hydrocarbon (PAH) fraction was obtained by eluting the Florisil® column first with hexane to remove PCBs and pesticides, and then with 1: 1 dichloromethane:hexane. This second fraction was evaporated to dryness and resuspended in acetone. A 1.0 g aliquot of dry sediment sample was weighed into a 15 mL glass beaker and left for combustion into a muffle furnace at 450 DC for 24 hours. Total organic content was determined by the difference in weight (Hakanson and Jansson 1983). Table 1 summarizes the chemicals quantified in frogs and sediment. Gas Chromatography Gas chromatographic analysis of tissue samples Fractions I and II and sediment samples Fraction I was performed on a Hewlett-Packard (HP) 5890/ECD equipped with an HP-3396 integrator, an HP-7673A autosampler, and a 30 m x 0.25 mm DB5 column. Injection was splitless at 250 DC, and

oven temperature was programmed from 100DC to 270 DC at 3 DC/min. Carrier gas was ultrapure He at 30 cm/s and makeup gas was Ar/CH 4 (95%/5%) at 50 mUmin. Gas chromatographic analysis of Fraction II from the sediment samples was performed on a Hewlett-Packard (HP) 5890/5970 GC/MSD equipped with an HP-7673A autos ampler, and a 30 m x 0.25 mm DB-5 column. Injection was splitless at 250 DC, and oven temperature was programmed from 100DC to nODc at 3DC/min. Carrier gas was ultrapure He at 30 cm/s. Cell Culture

ICR-134, leopard frog (Rana pipiens) embryo cell cultures were maintained at 25 DC in Leibovitz's L-15 (50%) medium, supplemented with 5% fetal bovine serum (FBS). Cells were subcultured by digestion with 0.25% (w/v) Trypsin/1 mM Na-EDTA for 1 min at 25 DC. Cells were then twice passed through a 20-gauge needle to separate the cells, which were then diluted with complete medium. Culture plates were inoculated with an appropriate number of cells in order to reach confluency within 48 hours. Culture medium was replaced with fresh medium every 2-3 days.

48

Gillan et al.

Cytotoxicity Assay Cytotoxicity was quantified using the Neutral Red (NR) uptake bioassay (Ali et aZ. 1994). Frog embryo cells were grown to near confluency in 96well tissue culture plates in complete medium. The cells were then treated for 1 hour with fresh medium containing various concentrations of sediment extract in acetone from all nine sites. Acetone alone in complete medium (to 1% (v/v) final concentration) was used as a negative control. Following the treatment period, media was removed and replaced with complete medium containing 50 mg/L Neutral Red. The cells were incubated for 1 hour to allow for the uptake of the dye into viable cells. The cells were then washed with a fixative (4% (v/v) formaldehyde, 1% (w/v) CaCI 2 ). The Neutral Red due was extracted by adding 0.1 mL 1% acetic acid/50% ethanol to each well. The plates were briefly shaken and absorbence was measured using a Bio-Tek microplate reader equipped with a 540 nm filter. All nine sites plus negative controls were assayed simultaneously.

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Alkaline Unwinding Assay for DNA SSB The analysis of DNA single strand breaks (SSB) followed the method of Hasspieler et aZ. (1995). Frog embryo cells were grown to near confluency in 24-well tissue culture plates containing complete medium supplemented with 50 nCi/mL eH]thymidine. The cells were then treated for 24 hours with 200 g sediment equivalents per L of sediment extract added to the culture medium. Solvent alone (1 % (v/v) final concentration) was used as a negative control. 9,1 O-phenanthrenequinone (PQ) and 4-nitroquinoline N-oxide (4NQ) were used as positive controls (Fig. 2). Following the treatment period, medium was replaced with 0.5 mL ice-cold phosphate buffered saline (PBS)/20 mM EDTA, followed by alkalinization with 0.5 mL O.IN NaOH for 30 min in darkness. Following alkaline unwinding, samples were neutralized with 0.5 mL O.IN HCl, followed immediately by the addition of 0.25 mL 2% (w/v) sodium lauryl sarcosinate (SLS), and 20 mM EDTA. Plates were then sonicated for lOs. Hydroxylapatite chromatography was performed to elute single-stranded (SS) DNA and doublestranded (DS) DNA separately. Cell lysates were combined with hydroxylapatite gel, prepared as a slurry containing 2.5 g gel suspended in 25 mL of 12 mM potassium phosphate (KP0 4 ) buffer, pH 7.0. The mixtures were incubated at 60°C for 10

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min and then poured into individual columns. Columns were washed 2 x 4 mL of 12 mM KP0 4 (60°C). Single-stranded DNA was eluted with 2 mL of 100 mM KP0 4 (60°C, pH 7.0) and DS DNA was eluted with 2 mL 500 mM KP0 4 (60°C, pH 7.0). For DNA quantification, 1 mL of each of the two DNA fractions was combined with 0.1 mL concentrated HCI and samples were digested at BO°C for I hour. Digested samples were subsequently combined with 5 mL scintillation cocktail and samples were analyzed by liquid scintillation counting. Data were expressed as F-values corresponding to radioactivity (disintegrations per minute) in the DS fraction divided by the sum of the radioactivities in the SS and DS fractions (F = DS/(DS+SS)). All nine sites plus negative controls were assayed simultaneously.

Ecotoxicological Studies in Amphibian Populations TABLE 2. Ontario.

49

Characteristics of the nine sites studied, along the north shore of Lake Erie and Lake.

Site Summerstown Cornwall Kingston Brighton Ancaster Longpoint Rondeau Hillman Marsh Ojibway

Type of Habitat shallow ditch large pond shallow creek large pond small pond large pond shallow creek large marsh large pond

Surrounding area agricultural public park conservation area agricultural agricultural provincial park provincial park conservation area provincial park

RESULTS In the majority of sites, green frogs were the most abundant, with the exception of Cornwall and Kingston. Leopard frogs, being somewhat more terrestrial than green frogs, revealed large differences in relative abundance from pond to pond and ranged from scarce to abundant. Sites included agricultural settings and areas adjacent to provincial parks (Table 2). Mean tissue concentrations of pesticides and Aroclor 1254: 1260 (l: I mixture of Aroclors 1254 and 1260) for each site are summarized in Table 3. No significant difference was found in tissue concentrations between frog species (Student's t-test, p > 0.05), however, within a site, leopard frogs were observed to have more variable concentrations when compared with green frogs This variability in tissue concentrations might be a function of the site to site dispersal ability of leopard frogs. Site to site variability was evident in that DDE concentrations were elevated at Ancaster, whereas PCB concentrations were highest at Hillman Marsh. Estimates of BSAFs ranged from 33 ± 16 to l.l±O.O in leopard frogs and from 23 ± 8 to 0.4 ± 0.0 for green frogs (Figs. 3a and 3b). These estimates of BSAFs are relatively high compared with other reported values for aquatic systems (Drouillard et al. 1996), but might be elevated due to the terrestrial nature and feeding habits of adult frogs. Concentrations of polycyclic aromatic hydrocarbons in sediment are summarized in Table 4. The highest levels of PAHs were measured at the Ojibway site (LPAH = 27 /lg/g O.c.), whereas the lowest levels were measured at the Rondeau site (LPAH = 3.2 /lg/g O.c.). The Neutral Red uptake bioassay revealed cyto-

Green Frog Abundance abundant few few abundant abundant abundant abundant abundant abundant

Leopard Frog Abundance none observed abundant few abundant abundant moderate abundant moderate abundant

tOXICIty as a significant stress at Cornwall, Brighton, Ancaster, and Ojibway (Fig. 4). Cytotoxicity was observed at the 200 gEq/L concentration for all four sites. There was little evidence, however, of genotoxic stress at any site. Table 4 summarizes F-values from the single-strand break (SSB) bioassay for genotoxicity. F-values ranged from 0.921 ± 0.052 to 0.975 ± 0.004. Negative control values were 0.935 ± 0.025. The responses of the cell line to standard compounds in the SSB bioassay for genotoxicity are illustrated in Figure 2. The cells responded in a dose-dependent manner to 4-nitroquinoline N-oxide (4NQ) with induction of SSB in the 0.3-2.5 /lM range. The cell line was also sensitive to SSB formation by the PAH-quinone, 9,1O-phenanthrenequinone (PQ), at concentrations above 2.5 mM (Fig. 2), indicating that the cell line possesses adequate capabilities for chemical activation resulting in genotoxic stress. This implies that this cell line would be useful in the detection of genotoxins whose activation pathways resemble that of 4NQ or PQ.

DISCUSSION Chemical concentrations did not vary significantly between frog species (Student's t-test, p > 0.05). This similarity in chemical concentrations suggests that the two species have equivalent exposures despite considerable differences in habitat selection and dispersal ability. Within a site, leopard frogs were observed to have the most variable chemical concentrations, perhaps related to their broader dispersal than green frogs. Site to site differences in chemical concentrations were significantly different for both species, which supports the

TABLE 3. Concentrations of organochlorines and Aroclor 1254:1260 (A 1254: 1260, 1:1 mixture) measured in green frog and leopard frog tissue (pg/kg, lipid) from nine sites along the north shore of the lower Great Lakes. Green Frogs Compound 1,2,3,5-TCB 1,2,3,4-TCB QCB HCB OCS trans-NA pp'-DDE Mirex pp'-DDT A1254:1260

Summerstown Cornwall Brighton Ancaster Longpoint Rondeau mean stdev mean stdev mean mean mean stdev mean stdev ND ND ND 0.3 ND ND 2.4 ND ND ND 0.4 ND ND 3.4 4.7 29 3.6 3.4 8.5 11 9.0 1.1 12 6.2 16 20 8.0 2.8 4.5 7.0 0.9 26 17.2 66 ND ND 8.4 ND 1.4 0.6 4.4 2.2 ND 15 4.4 19 1.7 11 7.0 13 7.5 19 174 105.9 754 412.0 252 179 26 135 ND ND ND ND 39 ND ND 22.6 48 5.7 16 2.5 59 1343 1639 885 310 92.7 1139 395 89.1 986 1071

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Ecotoxicological Studies in Amphibian Populations

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FIG. 3a. Mean biota-sediment accumulation factors for green frogs. Vertical bars represent standard error of the mean. (QCB = pentachlorobenzene; HCB = hexachlorobenzene; trans-NA = trans-nonachlor; PCBs follow IUPAC numbering system).

conclusion that frogs can be excellent biomonitors of local contaminant inputs/accumulation. Biota-sediment accumulation factors (BSAFs) were relatively high. These data do not allow an estimate of the relative importance of food, water, and sediment as exposure routes, but suggest that adult amphibians have a considerable capacity to accumulate chemical compared with other aquatic species. Adult amphibians have relatively high assimilation efficiencies, and these elevated BSAFs support the biomagnification model proposed by Gobas et al. (1989). As frogs are important prey items for birds, minks, and fish, this unique trophic level might represent a critical component linking aquatic and terrestrial ecosystems. It is interesting to note that the site to site variability in chemical concentrations observed in this study tended to reflect local inputs rather than diffuse source inputs, such as atmospheric deposition. The disappearance of amphibians also seems to be occurring at local scales, thus it is unlikely that large scale phenomena, such as atmospheric deposi-

FIG. 3b. Mean biota-sediment accumulation factors for leopard frogs. Vertical bars represent standard error of the mean. (QCB = pentachlorobenzene; HCB = hexachlorobenzene; trans-NA = trans-nonachlor; PCBs follow IUPAC numbering system).

tion and UVb, are important in regulating the relative abundance and composition of amphibian communities. The leopard frog embryo cell line responded in a dose-dependent manner to Fraction II sediment extracts, revealing significant cytotoxicity at concentrations of 200 gEq/L in four of the nine sites tested: Cornwall, Brighton, Ancaster, and Ojibway. Although these data suggest that sediment bound pollutants have the potential to cause stress, field estimates of abundance revealed viable populations. These somewhat contradictory results indicate that sediment bound pollutants, when made bioavailable and concentrated via the extraction process, have the potential to cause stress in short-term assays. Actual exposure to sediment bound pollutants would be significantly less than those used in the in vitro study. Although any single test cannot be used as a complete measure of "ecosystem health," these in vitro assays represent a novel tool for assessing chemical stress in aquatic and terrestrial systems. The observed response of the leopard frog embryo

TABLE 4. Polycyclic aromatic hydrocarbon (PAH) concentrations (/lglg, organic carbon) measured in sediment from nine sites along the north shore of the two lower Great Lakes, Lake Erie and Lake Ontario, and F-values (±SE), representing proportion of double-stranded DNA, in the single strand break bioassay for genotoxicity. PAH NA AL AE FL PHE AN FLT PY B(a)A C&T B(b)F B(k)F B(a)P IP D(ah)A B(ghi)P

Summerstown

Cornwall

Kingston

Brighton

Ancaster

Longpoint

Rondeau

Hillman Marsh

Ojibway

0.10 0.12

0.03 0.07 0.06 0.10 0.40 0.10 0.81 0.52 0.42 0.69 2.27 0.17 0.33 1.01 0.34 0.71

0.04 0.13 0.15 0.09 0.16 0.24 1.85 1.37 0.96 1.03 1.56 0.69 0.92 1.41 0.38 0.92

0.06 0.00 0.07 0.12 0.21 0.13 0.29 0.25 0.28 0.35 0.71 0.20 0.33 0.54 0.32 1.03

0.05 0.09 0.76 0.18 0.47 0.12 0.79 0.64 0.44 Q.53 0.99 0.44 0.54 0.96 0.33 0.83

0.03 0.06 0.42 0.09 0.19 0.07 0.33 0.23 0.21 0.25 0.57 0.23 0.20 0.47 0.18 1.77

0.04 0.09 0.05 0.10 0.19 0.10 0.27 0.21 0.23 0.25 0.56 0.16 0.22 0.27 0.25 0.27

0.04 0.10 0.07 0.20 0.86 0.19 0.69 0.50 0.47 0.53 0.81 0.40 0.55 0.74 0.31 0.61

0.05 0.13 0.07 0.50 1.62 0.36 2.73 2.00 1.76 1.62 3.40 1.62 2.62 4.85 0.91 3.29

O.ll 0.12 0.38 0.14 0.90 0.70 0.54 0.70 0.96 0.83 0.43 1.22 0.47 0.98

8.70 8.04 2:PAH F-VALUE 0.966 ± 0.005 0.962 ± 0.001

11.91 8.16 5.30 4.89 3.27 7.70 27.53 0.975 ± 0.004 0.966 ± 0.001 0.967 ± 0.006 0.970 ± 0.003 0.921 ± 0.052 0.942 ± 0.032 0.929 ± 0.039

*Contro1 = 0.935 ± 0.025 NA = Naphtalene AL = Acenaphthylene AE = Acenapthene FL = Fluorene

PHE = Phenanthrene AN = Anthracene FLT = Fluoranthene PY = Pyrene

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Ecotoxicological Studies in Amphibian Populations

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cell line to these extracts demonstrates the utility of such tests for screening environmental pollutant mixtures for toxicity to amphibian species. As a result of habitat destruction, frog communities have become severely fragmented. Isolated communities might in turn become vulnerable to genotoxic stress where deleterious mutations can be rapidly fixed into a population. The absence of a genotoxic response observed in this study might reflect the inability of these embryonic cells to metabolize the parent compounds. Furthermore, the chemicals tested in this fraction do not reflect the stress of less persistent compounds such as pesticides and herbicides. Genotoxicity has been observed in the same cell line after exposure to Fraction II sediment extracts from a railroad site in Sarnia, Ontario (F-value = 0.433 ± 0.220; Leadley 1995). Petras et aI. (1995) have observed genotoxic stress in adult frogs collected near the Ojibway site used in this study. Therefore, genotoxicity can be a very important stress on the gene pools of amphib-

ian species, especially those with limited dispersal abilities.

ACKNOWLEDGMENTS Special thanks are given to Rodica Lazar and the GLIER laboratory staff for the chemical analyses performed. This study was supported by an NSERC grant to G.D. Haffner.

REFERENCES Ali, F., Lazar, R., Haffner, G. D., and Adeli, K. 1993. Development of a rapid and simple genotoxicity assay using a brown bullhead fish cell-line: Application to toxicological surveys of sediments in the Huron-Erie corridor. 1. Great Lakes Res. 19:342-351. _ _, Hasspieler, B. M., Haffner, G. D., and Adeli, K. 1994. Human bioassays to assess environmental genotoxicity: Development of a DNA repair assay in HepG2 cells. Clinical Biochemistry 27(6):441-448. Barinaga, M. 1990. Where have all the froggies gone? Science 247: 1033-1034. Busbee, D. L., Guyden, J., Kingston, T., Rose, F. L., and

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Submitted: 6 August 1996 Accepted: 30 September 1997 Editorial handling: Derek Muir