Heavy Metal Levels in Ribbon Snakes (Thamnophis sauritus) and ...

Arch Environ Contam Toxicol 53, 647–654 (2007) DOI: 10.1007/s00244-006-0175-3

Heavy Metal Levels in Ribbon Snakes (Thamnophis sauritus) and Anuran Larvae from the Mobile-Tensaw River Delta, Alabama, USA J. Albrecht, M. Abalos, T. M. Rice Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688, USA

Received: 7 August 2006 /Accepted: 21 January 2007

Abstract. The Mobile-Tensaw River Delta (MTD) drains more than 75% of the state of Alabama and leads into Mobile Bay and the Northern Gulf of Mexico. Although it is a relatively healthy watershed, the MTD is potentially impacted by inputs of contaminants such as heavy metals. The levels of lead, copper, cadmium, and mercury were measured in whole body samples of Eastern Ribbon Snakes (Thamnophis sauritus) collected from the MTD. Lead, copper, and cadmium levels were also measured in anuran larvae (Rana catesbeiana, R. clamitans, and Hyla cinerea). These organisms were chosen because they are abundant in the MTD and are underrepresented in environmental contaminant biomonitoring studies. Ribbon snakes had significantly lower levels of lead, copper, and cadmium compared to whole body levels in anuran larvae, indicating that these metals were not biomagnifying through upper trophic levels. Copper and mercury levels were significantly correlated with age/growth indices in ribbon snakes. Although detectable levels of all metals were found in anuran larvae and ribbon snakes, these levels appear to be less than body burdens that would be associated with toxic effects. Populations of ribbon snakes in our particular collection sites within the MTD appear to be at minimal risk of exposure to toxic levels of metals. However, the MTD contains low- and high-impact areas, and other populations within this watershed could be at higher risk of exposure to heavy metals. We found the Eastern Ribbon Snake to be an excellent snake model for contaminant biomonitoring because of its abundance, reasonable size, and ease of collection.

The Mobile-Tensaw River Delta (Mobile and Baldwin Counties, Alabama) is one of the nationÕs largest river deltas. The drainage basin of the Mobile-Tensaw Delta (MTD) covers more than 75% of the state of Alabama and eventually leads into Mobile Bay and the Northern Gulf of Mexico. Much of this river delta remains in a natural state, but the area is subjected to a variety of potential insults from development,

Correspondence to: T. M. Rice; email: [email protected]

agriculture, forestry, and industry. The Mobile River in particular is potentially exposed to environmental toxicants such as heavy metals. For example, the Stauffer Chemical Company (now owned by Zeneca, Inc., and Akzo Nobel Chemicals, Inc.) has plants along streams in Axis and Bucks, Mobile County, Alabama, that feed into the Mobile River. These sites have been added to the U.S. Environmental Protection AgencyÕs (EPAÕs) National Priority List as contaminated areas requiring remediation (US EPA 1989a, 1989b, 1993, 1995a, 1999). Heavy metals are among the contaminants that have been released at these sites. Additionally, the Olin Corporation chlor-alkali plant in McIntosh, Alabama (Washington County, north of Mobile) lies along the Tombigee River, which feeds into the MTD. This area has also been added to the U.S. EPAÕs National Priority List (US EPA 1995b). The citizens of McIntosh held a town meeting in June 2005 to express concerns over the release of mercury from waste brine piles created by the plant (Tolkkinen 2005). In measurements of heavy metals and other toxicants in wild organisms, fish receive much attention because of their popularity as food and sport. However, it is important to measure toxicant levels in organisms that are more abundant or occupy important ecological positions in the MobileTensaw Delta. Although uncommonly studied in toxicology, anurans (Order Anura, frogs, and toads) and snakes make excellent candidates as bioindicators of ecological health. These organisms are extremely abundant in subtropical habitats such as the MTD (Conant and Collins 1998). Anuran larvae process high amounts of organic material and thus could acquire metals from food as well as from contaminated water. Both anurans and snakes occupy important trophic positions as both predator and prey across the aquatic and terrestrial interface. As prey items, anuran larvae and snakes could potentially transfer metals to higher trophic levels such as game fish and wading birds. Furthermore, some anurans and snakes have smaller home ranges compared to large game fish, birds, and mammals, and would therefore be more effective bioindicators of local inputs of toxicants into an area (Dunson et al. 1992; Pechmann and Wilbur 1994; Campbell and Campbell 2001). Snakes are additionally of interest because reptiles are underrepresented in toxicology studies compared to all other vertebrate taxa (Hopkins 2000; Campbell and Campbell 2001).

J. Albrecht et al.

Tensaw River ll M oun dA

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Alabama

Basin Negro

Te ns a

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Ri ve

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Mobile River

The present study was conducted to determine levels of the heavy metals lead, copper, cadmium, and mercury in Eastern Ribbon Snakes (Thamnophis sauritus) collected from habitats in the Mobile-Tensaw River Delta. Lead, copper, and cadmium were also measured in anuran larvae collected in the area because they are the primary prey items of ribbon snakes (Carpenter 1952; Rossman 1963). Eastern Ribbon Snakes were chosen as the snake model organism because they are one of the most abundant snake species within the MTD (Nelson 2004), are large enough to provide sufficient biomass for analyses, and are nonvenomous. Furthermore, no prior information exists regarding any toxicant levels in this species, so the results presented here expand the limited database of environmental contaminants in snakes (Campbell and Campbell 2001). Comparisons of the metal levels between anuran larvae and snakes were made to assess the potential risk that ribbon snakes face from ingesting metal-laden larvae. Comparisons of metal levels between snakes and prey have been conducted previously only by Hopkins et al. (1999) and Hopkins et al. (2002). Finally, we evaluated ribbon snakes as a potential reptile model to be used in studies of the overall health of the MTD.

Mo bile iver R

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Materials and Methods Study Site and Species Collected Nine Eastern Ribbon Snakes, Thamnophis sauritus, were hand-collected from the Shell Mound Conservation Area within the MTD (Figure 1) between August 2004 and August 2005. Only specimens between 30 and 90 cm total length were used, and no obviously gravid females were collected. The specimens were held in the laboratory with only water for 7 days after collection to purge their digestive systems. Then they were sacrificed by cervical dislocation with a finepoint scissors to ensure instantaneous and painless death. The specimens were weighed, measured (total and snout-vent length), and then frozen at )30
C for later processing. A sample of five bronze frog larvae (Rana clamitans), and four green treefrog larvae (Hyla cinerea) were collected from temporary pools in the Shell Mound Conservation Area (Figure 1) on August 16, 2004 and August 13, 2005, respectively. The R. clamitans larvae were at approximately Gosner stage 26–28, while the H. cinerea larvae were near final metamorphosis at Gosner stage 40–42 (Gosner 1960). A sample of three bullfrog larvae (Rana catesbeiana, Gosner stage 26–28) was collected within a floating mat of stargrass (Hypoxis sp.) from the Basin Negro off the Tensaw River (Figure 1) on June 23, 2005. All larvae were held for 24–48 h to purge their digestive systems, then weighed and subsequently frozen whole at )30
C.

0

2km

Fig. 1. The Mobile-Tensaw River Delta, Alabama, USA. Rana clamitans and Hyla cinerea larvae, and Thamnophis sauritus were collected from the Shell Mound Conservation Area. Rana catesbeiana larvae were collected in the Basin Negro off the main Tensaw River

acid-washed centrifuge tube and stored at room temperature. This process allowed us to use the whole animal, so that abundant tissue was available for replicate readings or multiple chemical analyses. Furthermore, our method required no metal parts, which could lead to contamination of the sample. Dry weights of larvae and snakes were required to express heavy metal concentrations on a dry-weight basis. For larvae, two larvae were weighed wet, and then placed in a drying oven at 65
C for a period of 48 h. They were then reweighed. The dry weight was approximately 9% of total wet weight; this factor was used to convert the wet weight of acid-extracted samples to a dry weight. For snakes, a preweighed sample of homogenized dried tissue (approximately 1.30 g) was placed in a drying oven set at 65
C for 48 h, and then reweighed. The dry weight was approximately 95% of total wet weight of the homogenate.

Processing of Animal Samples Larvae were processed whole, whereas the snakes had to be homogenized. Frozen snakes were thawed and then cut into approximately 5-cm sections using a nonstick (Teflon)-coated cleaver. The resulting sections were placed in a mortar bowl with liquid nitrogen and pulverized. This coarse homogenate was then dried overnight and pulverized further into a flour-like state. The weight of this dry homogenate was approximately 25% of the original wet weight of the whole animal. The final dry homogenate powder was placed into an

Extraction of Pb, Cu, and Cd from Larvae and Snakes Using a modification of EPA Method 3051 (US EPA 1994a), individual R. clamitans > 0.50 g wet weight, or 0.25 g of dry homogenized T. sauritus, were placed a 45-ml Teflon insert with 5 ml concentrated, ultrapure nitric acid. Teflon inserts were capped, placed into Parr microwave digestion bombs (Parr Instrumental Company, Moline,

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Heavy Metals in Ribbon Snakes in Alabama

Table 1. Percent recovery of heavy metals from replicate readings from NIST standard reference materials and replicates of one Thamnophis sauritus homogenate specimen Sample type

Sample size

Lead

Copper

Cadmium

Mercury

Oyster 1566b Mussel 2976 T. sauritus #5

5 replicates 3 replicates 4 replicates

91% 48% 25%

78% 93% 108%

58% 67% 77%

NA 103% NA

Four replicates of Thamnophis sauritus #5 homogenate were each spiked with a solution of 500 lg/L Pb and Cu, and 100 lg/L Cd. Standard reference materials and snake homogenate were of similar matrix (dry powder). All samples were prepared for metal extraction and analysis by modifications of US EPA standard methods (1994a, 1994b). NA not analyzed. Table 2. Mean (€ 1 SE) levels of heavy metals in anuran larvae (N = 12) and Thamnophis sauritus (N = 9) collected from the Mobile Tensaw River Delta, Alabama, from August 2004–August 2005 Species

Sample size

Lead

Rana clamitans Rana catesbeiana Hyla cinerea Larvae totals Thamnophis sauritus

5 3 4 12 9

1.19 0.65 1.32 1.10 0.35

Copper € € € € €

0.26 0.23 0.71 0.25a 0.12b

14.36 6.08 10.74 11.26 3.79

€ € € € €

4.13 0.90 0.69 1.87a 0.49b

Cadmium

Mercury

1.10 2.59 1.88 1.73 1.62

NA NA NA NA 0.58 € 0.12

€ € € € €

0.22 1.25 0.40 0.35a 0.88a

Metal levels of three species of anuran larvae (Rana clamitans, R. catesbeiana, Hyla cinerea) were similar and so data were combined for comparisons to T. sauritus. All metal levels are in lg metal/g dry weight, based on analysis of whole-body homogenates. Different letters indicate significant differences between anuran larvae and snakes, based on StudentÕs t-tests, within a particular metal. NA not analyzed. IL, USA), and placed in a microwave. Samples were digested for 3 min at 700 W. The digestate was diluted to 10% of original volume by the addition of 45 ml deionized water. Heavy metals (Pb, Cu, Cd) were analyzed by Varian SpectrAA220 graphite furnace atomic absorption spectrophotometer (Varian, Inc., Palo Alto, CA, USA) at the University of South Alabama. Detection limit was approximately 0.01 lg metal/g dry weight.

4.02 € 0.33 lg Cu/g, 0.82 € 0.16 lg Cd/g, 0.061 € 0.004 lg Hg/g). A sample weight of 0.25 g was used, equivalent to 0.50 g wet weight of material. As a further determination of recovery of Pb, Cu, and Cd, we processed four additional replicates from a single T. sauritus homogenate. With these four replicates, we also processed four replicates that were each spiked with a solution of 500 lg/L Pb and Cu, and 100 lg/L Cd. Recovery from standard reference materials and spiked snake samples was variable among the four metals (Table 1). However, we are confident that our levels reasonably represent actual levels.

Extraction of Mercury from Snakes Samples from the nine dried snake homogenates (no anuran larvae were available) were also put through an acid extraction method using EPA Method 7470A (US EPA 1994b), to be analyzed for total mercury concentrations. Approximately 0.25 g of dry homogenate was placed in 300 ml BOD bottles with concentrated 5 ml of aqua regia (1:3 ultrapure nitric acid and ultrapure hydrochloric acid) and 5 ml ultrapure water. Samples were heated for 2 min in a 95
C water bath. After cooling, 15 ml of potassium permanganate and 50 ml of ultrapure water were added. The samples were placed back in the water bath for 30 min. Then the BOD bottles were capped; the cap sealed while the bottles cooled. Samples were stored at 4
C until they were sent to Analytical Chemical Testing Labs (Mobile, AL) for analysis. Total mercury was analyzed through cold-vapor atomic absorption. Stannous chloride was added to the samples, which produced mercury gas. Gaseous mercury was moved via a carrier gas across the beam of a hollow cathode mercury lamp. Detection limit was approximately 0.092 lg Hg/g dry weight.

Statistical Methods StudentÕs t-tests were used to make comparisons between heavy metal levels of snakes and anuran larvae. For snakes only, correlation analysis was conducted between snout–vent length, weight, or body condition, and metal level within each of the metals. Body condition for each snake was calculated as the residuals from a linear regression of log10 snout–vent length · log10 weight (Reading 2004; Aubret and Bonnet 2005; Aubret et al. 2005). Additional correlations within snake samples were conducted to make comparisons among individual levels of all four metals. This analysis was conducted to determine whether individual levels of one metal corresponded relatively with those of the other metals. All analyses were considered significant at a = 0.050.

Results Recovery Efficiency and Accuracy To monitor recovery efficiency of heavy metals from tissues, standard reference materials from the National Institute of Standards and Technology (NIST) were processed with the above methods. These consisted of five oyster tissue samples (NIST #1566b: 0.308 € 0.009 lg Pb/g, 71.6 € 1.6 lg Cu/g, 2.48 € 0.08 lg Cd/g) and three mussel tissue samples (NIST #2976: 1.19 € 0.18 lg Pb/g,

Measurable levels of heavy metals were detected in all larvae and snakes. Levels of Pb, Cu, and Cd were very similar for all three species of anuran larvae, so data for all three species were combined for comparisons to levels in T. sauritus. Larvae contained significantly greater levels of Pb and Cu compared to snakes (df = 19, t > 2.40, p < 0.05; Table 2). No such differences between larvae and snakes were observed for Cd levels (df = 19, t = 0.13, p = 0.447).

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A

individuals with high levels of one metal did not necessarily have high levels of another metal.

Whole Body Concentration (µ g Cu/g dry weight)

8 7 6 5

Discussion

4 3 2 1 0 10

20

30

40

50

60

70

Snout-vent Length (cm)

Whole Body Concentration (µ g Hg/g dry weight)

B

2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 10

20

30

40

50

60

70

Snout-vent Length (cm)

Whole Body Concentration (µ g Hg/g dry weight)

C

2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 0.40 0.20 0.00 0

10 20 30 40 50 60 70 80

Body Weight (g) Fig. 2. Linear relationship between (A) whole-body copper levels and snout–vent length; (B) whole-body mercury levels and snout–vent length; or (C) whole-body mercury levels and dry weight, in nine Thamnophis sauritus collected from the Shell Mound Conservation Area of the Mobile-Tensaw River Delta, August 2004–August 2005. Correlations for all three relationships were significant (p < 0.05)

For the nine T. sauritus specimens, whole-body Cu levels were inversely correlated with snout–vent length (r = 0.693, p = 0.038; Fig. 2A), but not with weight or body condition (p > 0.050). Total Hg levels were positively correlated with snout–vent length (r = 0.866, p = 0.003; Fig. 2B) and with weight (r = 0.785, p = 0.012; Fig. 2C), but not with body condition (p > 0.050). No significant correlations between snake length, weight, or body condition, and Pb or Cd levels were observed. We also found no relationships among pairwise correlations of Pb, Cu, Cd, nor Hg levels (p > 0.050);

Detectable levels of heavy metals were found in the three species of anuran larvae collected during this study from the MTD. All three species had essentially equivalent levels of these metals, despite the fact that the R. clamitans and H. cinerea larvae were collected from a temporary terrestrial pool, whereas the R. catesbeiana samples were collected from a permanent basin (Basin Negro) connected to the main Tensaw River channel. Analysis of water from both temporary pools and the main river channels (Rice, unpublished data) indicate that metal levels were within U.S. EPA guidelines for protecting aquatic life (US EPA 2005). Anuran larvae would be exposed to metals not only in water (Rice et al. 2001) but also from ingested food and sediment (Jennet et al. 1977; Hall and Mulhern 1984; Birdsall et al. 1986; Sparling and Lowe 1996). We have no information on the levels of metals in sediments from the MTD. Even accounting for the variability in our recovery of Pb and Cd, the levels of metals in larvae we collected from the MTD were well below those levels described in previous studies from other ecosystems in presumed contaminated sites. Within Pb mining areas in Missouri, Gale et al. (1973) measured whole body concentrations of 36.0–1590 lg Pb/g, 8–48 lg Cu/g, and 1.1–3.0 lg Cd/g dry weight, in ‘‘tadpoles.’’ Jennet et al. (1977) measured whole-body concentrations of 22–4139 lg Pb/g and 15–260 lg Cu/g dry weight in ‘‘tadpoles’’ from similar habitats. Although some of our Cu and Cd values approached these levels from Missouri mining areas, our Pb values were well below those of Gale et al. (1973) and Jennet et al. (1977). Birdsall et al. (1986) measured whole body concentrations of 0.07–270 lg Pb/g dry weight in R. catesbeiana and R. clamitans larvae collected along highway drainages with high sediment levels of Pb. More recently, Hopkins et al. (1999) reported that R. catesbeiana, R. clamitans, and B. terrestris larvae collected from Savannah River, South Carolina reference areas had 6.33– 29.10 lg Cu/g and 0.11–0.38 lg Cd/g dry weight, compared to larvae from polluted coal-ash basins that had 13.79–26.18 lg Cu/g and 0.15–1.28 lg Cd/g dry weight. Larvae from the MTD in the present study had similar Cd values, but lower Cu levels, compared to larvae from the polluted ash basins studied by Hopkins et al. (1999). To further estimate risk to larvae in the MTD from metal contamination, an evaluation of toxic effects in conjunction with tissue levels should be assessed from previous research. Only a few such studies have been conducted in either field or laboratory-exposed larvae. Gillilland et al. (2001) measured levels of Pb, Cu, and Cd from R. clamitans larvae collected in southwestern Michigan at or below levels in the present study. These investigators also surveyed larvae for malformations at these study sites, but found no deformed animals among the 783 specimens collected (Gillilland et al. 2001). Hopkins et al. (2000) collected R. catesbeiana larvae from coal ash-basins in the Savannah River, South Carolina. These investigators observed increased incidence of malformations, as well as decreased response to prodding, in specimens with metal levels of >29.07 lg Cu/g and >1.59 lg Cd/g dry weight. These

Heavy Metals in Ribbon Snakes in Alabama

Cd levels were within those of anuran larvae from the MTD, but Cu levels were higher. For evidence from laboratory studies, Rice et al. (1999, 2002) observed that R. catesbeiana larvae exhibited decreased growth and increased respiration behaviors only at levels of Pb > 750 lg/L in water and body concentrations >200 lg Pb/g dry weight. For Cu, Ferreira et al. (2004) determined that R. catesbeiana larvae accumulated 100,000 lg Cu/g dry weight and exhibited 50% mortality at 2.40 mg Cu/L exposure for 96 h. These body levels were considerably higher than those reported in the present study. Finally, for Cd, Loumbourdis et al. (1999) determined that R. ridibunda larvae accumulated 14.00 lg Cd/g dry weight and exhibited 30% mortality after 15 d of exposure to 12.50 mg Cd/L. These levels of Cd were below those measured in larvae from MTD. Although the above information is indirect evidence, we are confident that toxic effects in anuran larvae from heavy metal exposure would be expected only after body accumulation of levels much higher than those measured in the present study from the MTD. Therefore, we see little indication, based on the current data, that anuran larvae in these locations within the MTD are accumulating levels of metals that should be of concern. Our areas of collection (Basin Negro and Shell Mounds Conservation Area) are areas of the MTD relatively far from point sources of contamination and so could serve as future reference areas. However, the MTD encompasses a large ecosystem of both low and high impact areas, and larvae from other areas, particularly along the Mobile River, could be at higher risk of exposure to heavy metals. We measured levels of heavy metals and total Hg well above detection limits in T. sauritus from the MTD. To our knowledge, the present study is the first report of any contaminant levels in T. sauritus. The levels of these metals in the present study were within the ranges reported for other snake species (review Campbell and Campbell 2001). However, most of these prior studies measured toxicant levels in separate organs such as liver or kidney, rather than in whole body samples as we did. Heinz et al. (1980) measured Hg in wholebody homogenates (excluding stomach contents) of Thamnophis sirtalis from the Pilot Island, Michigan. T. sirtalis is a close relative of T. sauritus. Levels were reported in wet weight; using our dry:wet weight ratio of 25%, levels of Hg were 0.56–1.64 lg Hg/g dry weight, within the range of levels in the T. sauritus samples from the MTD. Winger et al. (1984) measured Pb, Cd, and Hg levels in whole-body homogenates of Natrix (currently Nerodia) sp. from the Appalachicola River ecosystem. Nerodia sp. share similar life history characteristics with T. sauritus, and these species co-occur in the MTD. Levels reported by Winger et al. (1984) were in wet weight; using our dry:wet weight ratio of 25%, the levels of Winger et al. (1984) were 0.40–3.48 lg Pb/g, 0.04–0.20 lg Cd/g, and 0.52–1.52 lg Hg/g dry weight. These levels of Cd were lower, but Pb and Hg levels were within those measured here in T. sauritus from the MTD. Niethammer et al. (1985) measured Pb and Cd from whole-body homogenates (excluding skin and gastrointestinal tract) of Nerodia sipedon from Pb mining areas in Missouri. As with Heinz et al. (1980) and Winger et al. (1984), Niethammer et al. (1985) reported their results in wet weight. Using our dry:wet weight ratio, levels of Pb would be 0.76–56.40 lg Pb/g dry weight (Cd was below detection limit). Even taking into account our conversions from wet to

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dry weight, the levels of Pb in N. sipedon reported by Niethammer et al. (1985) were considerably higher than those in T. sauritus from the MTD, and are a potential indication of the different levels of Pb contamination between Missouri Pb mining areas (Niethammer et al. 1985) and the MTD. More recently, Burger (1992) measured metals in center body sections of hatchling P. melanoleucus from New Jersey. Levels of Pb, Cd, and Hg were 0.405–0.927, 0.043–240, and 0.051– 0.317 lg/g dry weight, respectively. These levels were similar to the levels found in the present study with T. sauritus. Clips from the tails of snakes have been proposed as a method of collecting tissue samples without requiring the sacrifice of the whole animal (Hopkins et al. 2001). The procedure requires a 2–3-cm section, which typically provides 0.05–0.50 g of sample depending on the species and size (Hopkins et al. 2001; Rainwater et al. 2005; Burger et al. 2006). Tail clips contain multiple types of tissues and can therefore serve as a surrogate comparison of whole-body toxicant levels, as were measured in the present study. Rainwater et al. (2005) used tail clips to measure metals in Agisktrodon piscivorus from a U.S. EPA Superfund site in Northeast Texas; accounting for dry:wet weight conversions, levels of Hg ranged from 0.492 to 0.860 lg/g dry weight. These levels of Hg were within the range for whole-body levels in T. sauritus from the MTD. Burger et al. (2006) also used tail clips to measure metals in three species of snakes (Nerodia fasciata, Nerodia taxispilota, A. piscivorus) from impacted areas within the Savannah River Site, South Carolina. Mean levels of Pb, Cu, and Hg for the three species were similar and ranged from 2.0 to 3.0, 2.0 to 3.0, and 0.6 to 0.9 lg/g dry weight, respectively. Although levels of Cu and Hg were similar to those in the present study, levels of Pb were higher than in T. sauritus from the MTD. Even when accounting for the variability in recovery of Pb and Cd from our samples, the above comparisons with studies from other areas indicate that the T. sauritus collected from the MTD (present study) appear to have accumulated metal levels more like those of snakes with little exposure time (hatchlings, Burger 1992) or from low-impacted reference areas (Heinz et al. 1980; Winger et al. 1984), and lower than the levels in snakes collected from impacted areas (Niethammer et al. 1984; Rainwater et al. 2005; Burger et al. 2006). Therefore, we expect that T. sauritus from within the Shell Mound Area of the MTD are at low risk of accumulating metals of levels high enough to be of concern. However, as discussed with the anuran larvae samples above, the MTD is a large watershed with potentially impacted sites away from our collection area. Therefore, we cannot conclude that T. sauritus throughout the entire MTD ecosystem are safe from exposure to heavy metals. We observed differences in accumulation of Cu and Hg according to growth and age indices such as snout–vent length and weight. These types of changes in metal accumulation with size/age have been observed for Cd, as well as for metals other than Pb, Cu, and Hg. Hopkins et al. (1999) observed a significant positive relationship with liver selenium (Se) and weight in N. fasciata collected from the Savannah River, South Carolina. In contrast, Burger et al. (2005) observed a significant negative relationship with Se between either blood or skin, and mass or length, in N. sipedon collected from sites in eastern Tennessee. Burger et al.Õs (2005) observations of Se levels with age/size were similar to our observations of

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decreasing Cu levels with length in T. sauritus. Burger et al. (2005) proposed that loss of Se with growth or age might be indicative of changes in homeostatic control of this essential trace metal. Such age/growth-related changes in homeostasis might also explain our observations of decreased Cu in larger/ older T. sauritus, because, like Se, Cu is an essential trace metal in vertebrates. Burger et al. (2005) also observed positive relationships with blood Cd and length in male but not female N. sipedon, as well as a positive relationship with skin manganese (Mn) and weight. Collectively, these previous studies and the present one indicated that metal levels in snakes vary with age/size, and that levels in different tissues (blood vs. liver vs. skin) can have different associations with age/size (e.g., Se: Hopkins et al. 1999 vs. Burger et al. 2005). The T. sauritus specimens collected in the present study had lower levels of Pb, Cu, and Cd compared to levels in anuran larvae. Metals such as Pb, Cu, and Cd are not known to biomagnify through food webs, unlike more persistent organic contaminants (Wright and Welbourn 2002; Pattee and Pain 2003; Yu 2005). However, other than the present study, few comparative studies have been conducted to examine metal levels between snakes and prey items. Hopkins et al. (1999) observed that Cu levels were higher, and Cd levels were similar, in N. fasciata livers compared to whole-body samples of anuran larvae and fish collected from coal-ash basins. In a related study, Hopkins et al. (2002) observed that N. fasciata accumulated increasing levels of Cd in liver when fed metalcontaminated fish prey. Therefore, based on this one study by Hopkins et al. (2002), there is some potential for snakes to increase their metal loads by ingesting contaminated prey items. Unlike other metals, Hg does biomagnify in its organic methylated form and would therefore be found at higher levels in top predators (Morel et al. 1998). Unfortunately, we did not have the opportunity to measure mercury in anuran larvae due to insufficient sample sizes, and no other authors have conducted such a study. Any attempt to estimate risk of toxicity from metal exposure in T. sauritus from the MTD should be conducted in the same manner as the risk to anuran larvae, discussed above. Levels of metals in snakes should be evaluated with measured toxic effects. Both Wolfe et al. (1998) and Bazar et al. (2002) made anecdotal reports of no observable effects in snakes fed Hgcontaminated diets. Hopkins et al. (1999) observed that N. fasciata collected from coal-ash polluted areas accumulated metals, including Cu, in the liver and had standard metabolic rates that were 33% higher than reference snakes. As a followup to these studies, Hopkins et al. (2002) fed N. fasciata prey from a contaminated area; detectable levels of Cu and Cd were measured in the livers of these specimens, but no effects on growth or metabolic indices were demonstrated. The above studies indicate that snakes can potentially withstand high intake and accumulation of metals with minimal adverse effects; this conclusion was supported by Hopkins et al. (2002). However, given the paltry amount of research on toxicological effects in snakes, we concur with the conclusions of Hopkins et al. (1999, 2002), that future studies on snakes should include both information on toxicant accumulation as well as some measure of biological impact from these toxicants. Furthermore, we agree with Burger et al. (2006) that reports of

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toxicant bioaccumulation in snakes should be conducted in a standardized manner, ideally on a dry weight rather than wet weight basis because water weight can vary considerably among individuals. We measured metal levels in whole-body homogenates of T. sauritus, whereas most other studies measured metal levels in separate tissues such as liver, kidney, and blood (Campbell and Campbell 2001). There are advantages to measuring metal levels only in tissues rather than the whole snake. Unlike a small anuran larvae or a fish in which even the whole body provides only a small amount of material for extraction, even organs from small specimens of T. sauritus provide a great deal of biomass for study. Metals often accumulate to high degrees in certain organs (liver, kidney, bone); this accumulation might not be detected under whole-body homogenization. Finally, to measure whole-body levels of contaminants in a >30-g snake, the whole body would need to be homogenized before extraction. However, there are advantages to measuring toxicant levels in an entire snake specimen. Whole-body analysis provides enough material to analyze replicates of a single animal for quality assurance, to be used for analysis of multiple inorganic and organic analytes, and to allow comparisons to prey items (anurans, fish), which are typically analyzed as whole body. We suggest that future studies, including our own, collect data on both separate tissues (e.g., liver, kidney) and the rest of the snake carcass, so that metal levels could be reported both in organs and whole body. In this manner, new results could be compared to a wide range of previous studies on snakes, as well as to contaminant levels in other organisms. The Eastern ribbon snake, Thamnophis sauritus, appears to be an ideal reptile model for continued surveys of toxicant loading in the MTD. This species is abundant in the MTD (Nelson 2004) and is easily collected by simple hand capture. There is little risk of decreasing the population in the Delta as a whole because this species is so abundant. They are nonvenomous and are of a reasonable size, so there is no danger to the collector. Furthermore, T. sauritus is an important trophic link in the Delta. They are predators upon aquatic animals such as anuran larvae and small fish. They are, in turn, prey for birds and alligators. The carnivorous diet of T. sauritus and other snakes makes them useful animals in examining areas that are contaminated with heavy metals that are transferred by way of the food chain (Hopkins 2000). Our results in the present study indicate that T. sauritus inhabiting the Shell Mound Conservation Area appear to be at minimal risk of exposure to heavy metals, and this area would make a useful reference point of future biomonitoring of snakes. However, we make no assumptions about the health of specimens from other areas of the MTD. Acknowledgments. We would like to thank Gabe Langford and Joel Borden for 2004 collections, Dr. Robert Naman and ACT Laboratories for mercury analysis, Dr. Gene Cioffi for analytical consultation, and Dr. David Nelson for reviewing drafts of this manuscript. Support for this research was provided through a University of South Alabama Center for Undergraduate Research (UCUR) award to J. Albrecht, a grant from the Mobile National Estuary Program, and a College of Arts and Sciences Summer Research award to T.M. Rice.

Heavy Metals in Ribbon Snakes in Alabama

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