Ecotoxicology (2008) 17:598604 DOI 10.

1007/s10646-008-0233-x

Sediment copper bioavailability to freshwater snails in south Florida: risk implications for the Everglade snail kite (Rostrhamus sociabilis plumbeus)
Robert A. Frakes Timothy A. Bargar Emily A. Bauer

Accepted: 3 June 2008 / Published online: 5 August 2008 Springer Science+Business Media, LLC 2008

Abstract Many properties being acquired as part of the Comprehensive Everglades Restoration Plan (CERP) are heavily contaminated with copper. Estimated copper bioaccumulation in the Florida apple snail (Pomacea paludosa) has led to the prediction of risk to the Everglade snail kite (Rostrhamus sociabilis plumbeus) at some CERP projects. Field study results presented in this paper examine the relationship between copper levels in sediments, snails, and other biota. Copper concentrations in all biota (snails, aquatic vascular plants, and periphyton) were strongly correlated with those in sediments. No correlation with water copper concentrations was evident. Mean copper concentrations in snails ranged from 23.9 mg/kg at the reference site to 732 mg/kg at a high copper site. Calculated biota-sediment accumulation factors (BSAFs) ranged from 36.7 to 7.0 over the range of copper levels in sediments. BSAFs were highest at low copper levels in sediments and declined sharply as copper levels in sediment increased. Risk for the snail kite is discussed in light of the results of this study. Keywords Comprehensive Everglades Restoration Plan Copper Bioaccumulation Everglade snail kite Florida apple snail

Introduction The Comprehensive Everglades Restoration Plan (CERP) has been developed by a consortium of state and federal
R. A. Frakes (&) T. A. Bargar E. A. Bauer Environmental Contaminants Program, U.S. Fish and Wildlife Service, 1339 20th Street, Vero Beach, FL 32960, USA e-mail: robert_frakes@fws.gov

agencies to reduce nutrient loading into the Everglades Ecosystem and meet the increasing water demands in south Florida. Decades of wetland drainage, agricultural activity, and development in south Florida have resulted in loss and degradation of the regions aquatic ecosystems. The CERP intends to improve water quality entering the Everglades and meet increasing water supply demands through the creation of storm water treatment areas (STAs) and water storage reservoirs (U.S. Army Corps of Engineers and South Florida Water Management District 1999). Since the development and funding of CERP, thousands of acres of agricultural land, including many active citrus groves, have been acquired for conversion to STAs and reservoirs. Many of the citrus groves have received heavy copper (Cu) treatments for years, resulting in extensive copper contamination in the soil (Alva et al. 1993). Over 36% of the states orange acreage and over 83% of the states grapefruit acreage are treated with copper compounds (USDA 2006). Copper in the form of copper hydroxide or copper sulfate is applied to orange groves at rates ranging from 1.0 to 2.5 pounds of active ingredient per acre, one or two times per year, for a total of approximately 650,000 pounds per year. Grapefruit groves are treated at slightly higher rates (1.22.8 pounds per acre) and at a greater frequency (up to four times per year), resulting in approximately 440,000 pounds applied annually (USDA 2006). Therefore, much of the land acquired for CERP is likely to be signicantly contaminated by copper. Inundation of these lands will release unknown amounts of copper into aquatic ecosystems, making it available for uptake by aquatic and aquatic-dependent organisms. Recently, a prospective ecological risk assessment performed for a large (8,000 acre) citrus grove predicted that foraging Everglade snail kites (Rostrhamus sociabilis

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plumbeus) may be adversely affected if the property was converted to a reservoir, as planned by CERP (URS 2003). The source of risk to the snail kite would be copper accumulated by the kites primary dietary item, the Florida apple snail (Pomacea paludosa). This risk prediction has signicant implications for CERP, because the Everglade snail kite is federally listed as an endangered species afforded protection under the Endangered Species Act of 1973 (Federal Register notice: 32 FR 4001, March 11, 1967). Costs of remediation of tens of thousands of acres of copper-impacted soils would be considerable, while shifting to an alternative land cover type (such as unimproved pasture or natural lands) for construction of CERP projects would have other environmental consequences. Perhaps the area of greatest uncertainty in copper risk assessment for the snail kite is the use of bioconcentration factors (BCFs), bioaccumulation factors (BAFs), or biotasediment accumulation factors (BSAFs) to predict copper concentrations in apple snails for a given concentration in soils or sediments. There are a few studies in the literature of copper uptake by snails and other mollusks. Laskowski and Hopkin (1996) found that garden snails (Helix aspersa) were very efcient at accumulating copper, with concentrations in soft tissue exceeding the concentrations in their food. In contrast, Anderson et al. (1978) found no difference in copper levels in snails and other biota at two sites with differing metal inputs on the Fox River in Illinois. This was postulated to be due to physiological regulation. Anderson (1977) found that copper concentrations in the bodies of freshwater clams were similar to those in sediments and much higher than those in water. Sediments in the Apalachicola River, Florida contained about three to four times higher concentrations of copper than freshwater clams (Corbicula manilensis). There was no signicant correlation between sediment and clam residues of copper (Elder and Mattraw 1984). Bioaccumulation of metals by snails and bivalves appears to be extremely situation dependent, and it is difcult to extrapolate results across species and ecosystems (Elder and Collins 1991). Little research has been conducted investigating copper contamination in the Florida apple snail. Winger et al. (1984) investigated copper concentrations in water, sediments, plants, and apple snails along a gradient in a canal entering Loxahatchee National Wildlife Refuge. The canal had been receiving periodic treatments with copper-based aquatic herbicides (Komeen, Grifn Corporation, Valdosta, GA; Cutrine-plus, Applied Biochemists, Germantown, WI). The range of copper concentrations found in apple snails (1782 mg/kg) was about four times higher than that in sediments. This paper presents the results of a eld study designed to assess copper concentrations in freshwater snails collected from wetlands in close proximity to citrus groves

and in a reference area. The primary purpose of the study was to attempt to determine the relationship between sediment copper concentrations and snail tissue copper concentrations in copper-impacted aquatic habitats in south Florida. The primary focus was on the Florida apple snail, although other snails were also included. A secondary purpose was to examine the correlation between copper concentrations in snails and those in other matrices (water, periphyton, and vascular plants) that are possible contributors to copper body burdens in the snails. Risk for the snail kite is then discussed in light of the results of this study.

Methods Snail, sediment, water, periphyton, and vascular aquatic plant samples were collected from ve different copperimpacted locations in south Florida, plus a reference location. Three locations (TMC1, TMC2, TMC3) were located along the upper reaches of Ten Mile Creek in St. Lucie County. This section of Ten Mile Creek drains a large citrus growing area and is adjacent to citrus groves along much of its length. Another sample location (SB) was at the Sunrise Boys citrus grove in southern Martin County. The sample location at Sunrise Boys was a small pond surrounded by grapefruit groves. The nal copperimpacted sample location (ARC) was at ARCCO, a citrus grove in northern St. Lucie County. The sampling location at ARCCO was a canal that drains adjacent groves. Water Conservation Area 3 (WCA3) was selected as the reference location. WCA3 is a large wetland area north of Everglades National Park that is distant from any citrus operations and receives no known anthropogenic copper inputs. Biota, sediment, and water samples were collected from three to six randomly selected subsites at each location. Each subsite was separated from the others by at least 10 m to minimize the possibility that a snail had recently spent time in more than one subsite. Most of the subsites were much greater than 10 m apart. Darby et al. (1997) found that Florida apple snails moved on average about 5 m per day and that 62% moved less than 10 m in a week. Therefore, subsites were considered to be relatively independent statistical units, although the possibility that some snails had recently been exposed to more than one subsite cannot be ruled out. Except for snails, one sample of each matrix was collected from each subsite. Depending on abundance, anywhere from one to four snail samples were collected at each subsite, most commonly two or three. When more than one snail was collected at a subsite, the geometric mean concentration was used to represent that subsite. While apple snails were the primary interest in this study,

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few could be found at some locations. At those locations, other snail species (family viviparidae) were collected as surrogates for apple snails. Four of ve snail samples collected from TMC1, and all collected from TMC3, were viviparidae snails. At some of the subsites at two locations (ARC and TMC3), no suitable snails of any species could be found. All snails were collected by dip net from submerged and emergent vegetation and placed into whirlpack bags. Once in the laboratory, snails were removed from their shells, rinsed with de-ionized (DI) water, and placed into tared plastic vials. Periphyton samples were collected by dip net, triple-rinsed with DI water, and placed into Ziploc bags. Vascular plant samples were collected by snipping with scissors, placed into Ziploc bags, and triple-rinsed with DI water. Only the portion below the water surface was collected, as this is the portion that would be ingested by apple snails. Sediment samples were collected using a stainless steel spoon. Several spoonfuls were collected within a 1 m radius, composited in a stainless steel bowl, and thoroughly homogenized. A portion from this composite was then transferred into a 250 ml, precleaned plastic jar. Approximately 250 ml surface water grab samples were collected into a precleaned plastic bottle and preserved with 10% nitric acid. All biota, sediment, and water samples were collected within an area of approximately four square meters. All samples were labeled and placed on ice for transport back to the laboratory. All tissue and sediment samples were held in the laboratory at approximately 20C until analysis. Water samples were held at 5C until analysis. All samples were processed via microwave digestion in nitric and hydrochloric acid (modication of EPA method 3051) (EPA 1994). Copper concentrations were quantied by inductively coupled plasma mass spectrometry and, except for water, are reported in this paper on a dry weight (dw) basis. Tests for normality or lognormality on pooled data sets for each matrix were performed using EasyFit 3.2 (Mathwave Technologies, Dnipropetrovsk, Ukraine). Mean concentrations reported are geometric means. Copper concentrations in matrices from different locations were compared by the Tukey-Kramer procedure for unequal sample sizes. Since pooled data sets t a lognormal distribution better than a normal distribution, tests of statistical signicance were performed on natural-log transformed data with a signicance level (a) of 0.05 using Systat 12 (Systat Software, Inc., San Jose, California, USA). Data for apple snails and viviperidae snails were pooled for the regression analyses. Inclusion of the viviparidae snail data with the apple snail data in the regression analyses greatly extended the range of sediment concentrations

that could be included in the analysis. Regression analyses were performed on the paired snailsediment or snailplant data obtained from each subsite, except for periphyton where the site-wide means were used. Subsite data were preferred for regression analysis because there was considerable variability in sediment copper concentrations within some sites, and most subsites were separated from one another by a distance unlikely to be traveled by a snail in a short time. Thus, the subsite was considered to be the more likely source of recent exposure to the snail, rather than the entire site. Regression analyses were performed using the least squares linear regression module in Systat 12. Some statistical outliers identied by the regression software were eliminated from the regression analyses. BSAFs for each site were calculated as the ratio of the geometric mean copper concentration in snail tissue and the geometric mean copper concentration in sediment, on a dry weight basis. Predicted BSAFs were calculated by using the snail-sediment regression equation to predict a mean snail concentration for each site and then dividing this by the mean sediment concentration.

Results A total of 30 subsites were sampled among the six locations (six each at WCA3, SB, ARC, and TMC3, and three each at TMC1 and TMC2). A total of 49 snail samples (40 apple snails and nine viviperidae) were collected at 22 subsites. No snails could be found at eight subsites. Apple snail abundance appeared to be lower at locations with greater copper concentrations in sediments, suggesting possible sensitivity of apple snails to copper contamination in the habitat. At the two most highly contaminated sites (TMC1 and TMC3), almost no apple snails could be found, necessitating the collection of viviperidae snails instead. Copper concentrations in sediments ranged from less than 1 mg/kg to over 300 mg/kg (Table 1). WCA3 (the reference location) had the lowest copper concentration in sediments, while TMC1 and TMC3 had the highest. Sediments from TMC2 were not signicantly different from the reference location. Copper concentrations in sediments from SB, ARC, TMC1, and TMC3 were signicantly elevated compared with the reference location. Copper was below the limit of detection (3 lg/l) in all water samples from WCA3, in four samples from SB, and in one sample from TMC2. Copper was detectable in water in all samples from the more highly impacted sites (ARC, TMC1, and TMC3). The presence of so many non-detections in water at the reference and low-copper sites precluded making any meaningful statistical comparisons among locations. However, in the other matrices (periphyton, vascular plants, and snails), copper concentrations at all ve

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Table 1 Geomean [range] of copper concentrations (mg/kg dw) in sediment, water, periphyton, vascular plant, and snail samples collected from Water Conservation Area 3 (WCA3), as well as three sites on Ten Mile Creek (TMC), ARRCO (ARC), and Sunrise Boys (SB) Location WCA3 TMC2 SB ARC TMC1 TMC3
a b c d e f

na 6 3 6 6 3 6

Sediments 1.6 [0.93.4] 3.9 [1.913.1] 12.5 [6.820.1]* 36.2 [13.7110]* 68.1 [35.8133]* 65.2 [15.6302]*

Waterb (lg/l) \3c 3.7 [1.57.6] 2.5d [1.513] 3.7 [2.015.7] 5.4 [3.89.5] 1.7 [1.32.1]
d

Periphyton 1.1 [0.71.7] 23.5 [1630]* 20.5 [1332.2]* 20.4 [16.327.7]* 70.7 [53.6113]* 93.7 [41.9335.9]*

Vascular plants 0.7 [0.50.97] 2.8 [1.73.9]* 3.0 [1.95.9]* 1.7 [1.24.0]* 6.4 [4.411]* 6.8 [3.29.8]*

Snails (total no. collected) 23.9 [1156.7] (14) 118 [94.4179]* (9) 299 [183988]* (14) 205 [192218]* (2) 401 [313507]* (5)e 732 [6021,138]* (5)f

Number of subsites sampled at each location. Except for snails, one sample was collected at each subsite No statistical analyses performed due to the number of samples in which copper was not detected No copper detected in any sample. Reported value is method detection limit (MDL) Copper undetected in one or more samples; MDL was used for computing means for non-detections Four of ve snail samples were viviparidae All snails were viviparidae

* Mean is signicantly different from the reference location (a = 0.05)

impacted sites were signicantly elevated compared with the reference location. Patterns of relative copper levels in biota generally followed those in sediments. An exception to this pattern occurred at the ARC location. Although sediments at ARC contained three to nine times more copper than SB and TMC2, this difference was not reected in periphyton or vascular plant samples. Mean copper concentrations in snails ranged from 23.9 mg/kg at the reference site to 732 mg/kg at TMC3 (Table 1). The mean value for the reference site in this study agrees well with concentrations reported by Winger et al. (1984) from the interior of Loxahatchee National Wildlife Refuge (1721 mg/kg). The highest copper concentration detected in any snail tissue (1,138 mg/kg) was in a viviparidae snail collected from TMC3, while the highest detected in an apple snail was 988 mg/kg at SB. Copper concentrations in periphyton also varied over a wide range, from about 1 mg/kg at the reference site, to a sample from TMC3 that contained 335 mg/kg. At most sites, copper concentrations in periphyton were roughly equal to those in sediment. On the other hand, copper concentrations in vascular plants (about 110 mg/kg) were much lower than those in sediments and the other biota. Vascular plant

concentrations fell within the same range as those reported by Winger et al. (1984) for a south Florida canal. Copper concentrations in sediments, periphyton, vascular plants, and snails were positively correlated (Table 2). The relationship between copper concentrations in snails and sediments at the subsite level is shown in Fig. 1. The best t was obtained with a logarithmic model which produced a highly signicant correlation coefcient of
Snail tissue [Cu] mg/kg dw
900 800 700 600 500 400 300 200 100 0 0.1 1 10 100 1000
y = 117.64Ln(x) - 16.946 R 2 = 0.8233

Sediment [Cu] mg/kg dw

Fig. 1 Scatter plot of individual subsite copper concentrations in snails and sediments collected from ve locations in south Florida (solid diamonds represent Florida apple snails, open diamonds represent viviperidae snails)

Table 2 Regression statistics of copper concentrations in south Florida snailsa with copper concentrations in sediments, periphyton, vascular plants, and water Model Sediments Periphyton Vascular plants Water
a b

Basisb Subsite Mean Subsite Subsite

r-square 0.82 0.88 0.68 \0.01

Slope 117.6 6.58 75.3 -4.7

Intercept -16.9 44.1 2.7 332.4

p value \0.001 \0.01 \0.001 0.83

Logarithmic Linear Linear Linear

Primarily Florida apple snails but including some viviparidae Regressions were performed on the individual subsite data (subsite) or the site-wide means (mean)

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R. A. Frakes et al. Table 3 Biota-sediment accumulation factors (BSAFs) for copper in snails from six locations in south Florida Location WCA3 TMC2 SB ARC TMC1 TMC3
a

0.82. Copper in snails also correlated well with copper concentrations in vascular aquatic plants from the same subsites (r2 = 0.68, simple linear regression). On the other hand, copper in snails was not very well predicted by copper levels in periphyton at the subsite level (r2 = 0.27). However, this relationship was greatly improved by examining site-wide mean copper levels in snails and periphyton. In this case, snails and periphyton showed a strong linear relationship (r2 = 0.88). No relationship was evident between copper concentrations in snails and water (r2 \ 0.01). Figure 2 shows a plot of the site-wide mean copper concentrations in all biota versus sediment concentrations. As a rough generalization from this gure, copper levels in snails were about one order of magnitude higher than those in periphyton, and about two orders of magnitude higher than vascular plants, for a given sediment concentration. Biota-sediment accumulation factors (BSAFs) were calculated from site-wide mean copper concentrations in snails and sediments and compared with predicted BSAFs calculated using the regression equation in Fig. 1 (Table 3). Good correspondence was found between predicted and observed BSAFs, except at WCA3. Predicted biota-sediment accumulation factors (BSAFs) ranged from 36.7 at TMC2 to 7.0 at TMC1. BSAFs were highest at low copper levels in sediments and declined sharply as copper levels in sediment increased. McGeer et al. (2003) also
1000.0

Actual BSAFa 14.7 30.2 24.0 5.7 5.9 11.2

Predicted BSAFb 24.8 36.7 22.5 11.2 7.0 7.3

Calculated using mean sediment and snail copper concentrations for each location from Table 1 Calculated using regression equation from Fig. 1

found an inverse relationship between BCF/BAF and exposure concentration.

Discussion Apple snails are herbivores feeding on periphyton and submerged vascular plants in the water column, indicating that they are primarily exposed to surface water. However, they are known to release from substrates and drop to the bottom as a defense mechanism (Turner et al. 2001). In addition, Darby et al. (1997) monitored apple snails in wetlands during dry down conditions and found that they were either near (13 cm) or on the bottom when stranded in isolated, shallow water pools. Therefore, apple snails are exposed to both surface water and sediments, but the relative exposure time in each is uncertain. This study found a strong correlation between copper concentrations in snails and those in sediments, but no relationship with those in surface water. A surprising initial nding of this study was that copper concentrations in snails were poorly correlated with those in periphyton, since periphyton are an important component of the apple snails diet (Turner et al. 2001). Notten et al. (2005) found that copper transfer from food (leaves) was more important than transfer from soil in a terrestrial snail (Cepaea nemoralis). Other studies have found food to be an important source of metals to snails (Laskowski and Hopkin 1996; Gomot and Pihan 1997). Closer inspection of our data revealed that, although a weak correlation existed at the subsite level, a very strong relationship between snails and periphyton was evident when the means for the sites were compared. The obvious explanation for this observation is that oating periphyton masses are not stationary, as are sediment and vascular plants, but can move by wind and water currents from one part of a site to another. Therefore, a periphyton sample may not be representative of copper concentrations at the subsite where collected, but may have recently been exposed to ambient

snail

100.0

peri

Biota [Cu] mg/kg dw

10.0

vasc

1.0

0.1 0.00

20.00

40.00

60.00

80.00

Sediment [Cu] mg/kg dw

Fig. 2 Plot of mean copper concentrations in biota versus sediment copper concentrations (circles snails, open squares periphyton, triangles vascular plant)

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copper concentrations elsewhere on the site. Vascular plants, on the other hand, are anchored by their roots to one place and therefore showed a stronger correlation with snails and sediments collected at the same subsite. Copper concentrations in water were low, usually less than 10 lg/l. Even though it was difcult for the analytical laboratory to detect and accurately quantify copper in water at some sites, it was still apparent from the data that grab water samples bore no relationship to copper concentrations in biota or sediment, at either the subsite or site-wide level. While a review by McGeer et al. (2003) found no signicant relationship between water-born copper concentrations and copper concentrations in algae, it is reasonable to assume that copper concentrations in biota and water should be positively related. It is likely that dissolved copper concentrations in surface water change rapidly in response to rainfall events and other factors, relative to sediments and biota. A time-weighted average of a series of water samples collected over days or weeks might provide a more useful index than the single water sampling event performed in this study. The relative inability to nd apple snails at high-copper locations may be an indication of copper toxicity to the apple snail at levels currently found in many locations in south Florida. In fact, copper has been effectively used as a molluscicide (Howard et al. 1964). Winger et al. (1984) concluded that aquatic plant control using copper-containing herbicides was not responsible for decreased Florida apple snail populations in canals since the acute toxicity values for dissolved copper were considerably greater than dissolved copper concentrations they found in canal waters. However, copper concentrations in sediments in this study were signicantly higher than those examined by Winger and colleagues. In addition, chronic exposure to copper can result in reduced snail survival at levels much lower than acute toxicity values (Reed-Junkins et al. 1997). The critical snail body burden (CSBB), the copper concentration in the apple snail above which adverse impacts to the Everglade snail kite are expected, has been estimated to be about 450 mg/kg (URS 2003). Copper concentrations in eight individual snail samples exceeded this level. Six of the snail samples were viviparidae (507 1,138 mg/kg) while the other two were apple snails (988 and 611 mg/kg). The mean concentration at TMC3 (732 mg/kg) also exceeded the CSBB. Therefore, it is reasonable to expect that snails in wetlands constructed on properties with signicant copper contamination may accumulate sufcient copper to represent a risk to snail kites. However, most of the snails with high copper levels collected in this study were viviparidae, which are not known to be consumed by snail kites. Based on a risk assessment done for the South Florida Water Management District by URS Corporation (URS

2003), in 2003, the Fish and Wildlife Service recommended an interim soil screening value of 85 mg/kg for protection of the snail kite. Copper bioaccumulation in apple snails was predicted based on a summary of biotasediment accumulation factors (BSAF) for other invertebrates (Bechtel Jacobs 1998). A conservative BSAF of 5.25 was selected, representing the 90th percentile of BSAFs for all invertebrates. Using the regression equation from Fig. 1, BSAFs calculated for this study ranged from 36.7 to 7.0. Compared with the data from Bechtel Jacobs (1998), snails from this study had a greater propensity to accumulate copper than the vast majority of other invertebrates. The results obtained in this study can be used to reevaluate the interim soil screening value using data specic for snails from this area. A CSBB of 450 mg/kg substituted in the equation shown in Fig. 1 results in a screening value of 53 mg/kg, considerably lower than the current screening value. Based on a database of more than 400 soil samples from citrus groves, approximately 30% of the citrus property acreage being acquired for CERP would exceed this screening value (FWS, unpublished data). The data collected in our eld study indicate the current screening value may overestimate the copper concentration in the soil at which risk for the snail kite is possible. Properties acquired for incorporation within CERP need to be prospectively evaluated for impacts to the future wetland community. Current prospective risk assessments on the acquired properties are performed with uncertainties that take on increased signicance, since risk has been predicted for a federally-listed endangered species on a large portion of the properties. The accumulation of copper by apple snails predicted in previous risk assessments as a major uncertainty has been conrmed by this study. In fact, data from the eld indicate that accumulation factors used in previous assessments, then thought to be conservative, may in fact have been too low, and that currently used guidelines and action levels for copper should be reevaluated. In addition to the eld studies, this reevaluation will draw on laboratory microcosm studies that are currently under way to further rene our understanding of the behavior of copper residues in citrus soils and its food chain bioavailability.
Acknowledgements The South Florida Water Management District (District) permitted the authors to access District properties for sampling purposes. Loxahatchee National Wildlife Refuge provided an airboat and driver for sampling at Water Conservation Area 3.

References
Alva AK, Graham JH, Tucker DPH (1993) Role of calcium in amelioration of copper phytotoxicity for citrus. Soil Sci 155(3):211218

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604 Anderson RV (1977) Concentration of cadmium, copper, lead, and zinc in six species of fresh water clams. Bull Environ Contam Toxicol 18(4):492496 Anderson RV, Vinikour WS, Brower JE (1978) The distribution of cadmium, copper, lead and zinc in the biota of two freshwater sites with different trace metal inputs. Holarct Ecol 1(4):377384 Bechtel Jacobs Company (1998) Biota sediment accumulation factors for invertebrates: review and recommendations for the Oak Ridge reservation. BJC/OR-112. Prepared by Bechtel Jacobs Company LLC for U.S. Department of Energy, Ofce of Environmental Management, Oak Ridge National Laboratory, Oak Ridge, TN Darby PC, Valentine-Darby PL, Bennetts RE, Croop JD, Percival HF, Kitchens WM (1997) Ecological studies of apple snails. Special publication SJ98SP6. Florida Cooperative Wildlife Research Unit, Gainesville, FL Elder JF, Collins JJ (1991) Freshwater molluscs as indicators of bioavailability and toxicity of metals in surface-water systems. Rev Environ Contam Toxicol 122:3779 Elder JF, Mattraw HC (1984) Accumulation of trace elements, pesticides, and polychlorinated biphenyls in sediments and the clam Corbicula manilensis of the Apalachicola River, Florida. Arch Environ Contam Toxicol 13(4):45369 Gomot A, Pihan F (1997) Comparison of the bioaccumulation capacities of copper and zinc in two snail subspecies (Helix). Ecotoxicol Environ Saf 38(2):8594 Howard TE, Halvorson HN, Walden CC (1964) Toxicity of copper compounds to the snail vector hosts of the agent of schistosome dermatitis, in water of differing hardness. Am J Hygiene 80:3344 Laskowski R, Hopkin SP (1996) Accumulation of Zn, Cu, Pb and Cd in the garden snail Helix aspersa: implications for predators. Environ Pollut 91:289297 McGeer JC, Brix KV, Skeaff JM, DeForest DK, Brigham SI, Adams WJ, Green A (2003) Inverse relationship between

R. A. Frakes et al. bioconcentration factor and exposure concentration for metals: implications for hazard assessment of metals in the aquatic environment. Environ Toxicol Chem 22:10171037 Notten MJ, Oosthoek AJ, Rozema J, Aerts R (2005) Heavy metal concentrations in a soil-plant-snail food chain along a terrestrial soil pollution gradient. Environ Pollut 138(1):17890 Reed-Junkins DK, Farris JL, Cherry DS, Heath AG, Cairns J (1997) Functional responses in Leptoxis praerosa to increasing metal concentration and exposure duration. Environ Toxicol Chem 16:16661676 Turner RL, Hartman MC, Mikkelsen PM (2001) Biology and management of the Florida apple snail. Report for the Florida Fish and Wildlife Conservation Commission URS Corporation (2003) Berry Labelle-Congen Grove ecological risk assessment. Job no. 10582-145-024. URS Corporation, Boca Raton, FL U.S. Army Corps of Engineers, South Florida Water Management District (1999) Central and Southern Florida project comprehensive review study. Final integrated feasibility report and programmatic environmental impact statement. U.S. Army Corps of Engineers, Jacksonville, FL U.S. Department of Agriculture (2006) Agricultural chemical usage, 2005 fruit summary. National Agricultural Statistics Service, Washington, DC U.S. Environmental Protection Agency (1994) Test methods for evaluating solid waste, physical/chemical methods, 3rd edn. SW846, method 3051, revision 0, September, 1994. U.S. Government Printing Ofce, Washington, DC Winger PV, Imlay MJ, McMillan WE, Martin TW, Takekawa J, Johnson WW (1984) Field and laboratory evaluation of the inuence of copper-diquat on apple snails in southern Florida. Environ Toxicol Chem 3:409424

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