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3Tetra Tech, Inc., USA ... estimating Hg concentrations in other tissues in south Florida raccoon populations. ... the endangered Florida panther (Roelke et al.,.
Ecotoxicology, 13, 207–221, 2004  2004 Kluwer Academic Publishers. Manufactured in The Netherlands.

Retrospective Study of Mercury in Raccoons (Procyon lotor) in South Florida D. B. PORCELLA,1 E. J. ZILLIOUX,2 T. M. GRIEB,3 J. R. NEWMAN4 AND G. B. WEST2 1 Environmental Science and Management, USA 2 Florida Power and Light Co., USA 3 Tetra Tech, Inc., USA 4 Pandion Systems, Inc., USA Accepted 15 April 2003

Abstract. Museum and recent collections of raccoon hair were used to assess whether temporal or spatial trends existed in MMHg distributions in south Florida. The hypothesis that MMHg in raccoon hair had remained the same since 1947 could not be rejected. Some sampling regions showed increases while others did not. However, large differences existed between sites, amounting to a factor of 20 for raccoons collected during 2000 and during the period prior to 1960 (museum samples). Raccoon feeding behavior and the production of MMHg most probably accounted for the spatial differences. Large differences in MMHg concentrations existed in different tissues ranging in order of hair, liver, kidney, muscle, heart, brain, and blood in their respective ratios to blood: 96:10:6:5:4:2.5:1. Liver Hg is 7% MMHg, while hair Hg is 99% MMHg. These associations appear largely regulated by metabolic processes. Speciation of Hg is very important for gaining an understanding of ecosystem and organism Hg dynamics. Further work is needed to establish whether Se plays a role in Hg sequestration and whether hair Hg is a good surrogate for estimating Hg concentrations in other tissues in south Florida raccoon populations. Keywords: mercury; monomethylmercury; raccoons; trends; accumulation; feeding

Introduction The consumption of fish having high concentrations of mercury (Hg) poses health risks to both humans and wildlife. To assess the magnitude of these potential risks, scientists have investigated the many biogeochemical processes that affect Hg accumulation in fish and other aquatic biota (e.g., Grieb et al., 1990; Hudson et al., 1994; Harris and Bodaly, 1998). Mercury in the environment has complex cycles that involve rapid transport between air, water, soil, and biota, and transformation between three dominant chemical forms in the environment: inorganic Hg ions (HgII), elemental Hg (Hg0), and monomethyl-Hg (CH3Hgþ or MMHg). Greater than 95% of the Hg in fish

flesh is MMHg (Bloom, 1992). Sediment and ice cores provide valuable perspective on inputs of Hg into ecosystems, but inputs do not equate to the amount of Hg in fish. Hudson et al. (1994; also, USEPA, 1997; Harris and Bodaly, 1998) proposed models to relate Hg inputs to fish Hg concentrations. Historic patterns of Hg in biota and other archives (cores of lake sediments, peat bogs, ice) help quantify the relationship between loading (input of Hg) and bioaccumulation (EPRI, 1996). Long-term monitoring records of Hg in the environment are rare, especially in relation to Hg in biota. Some exceptions are noted with respect to avifauna. Berg et al. (1966) found roughly constant levels within bird species in Sweden from the period 1840–1940, but relate a marked increase in

208 Porcella et al. Hg levels after 1940 to widespread use of alkyl-Hgcompounds as seed dressings. Following a ban in 1966, levels decreased in terrestrial food webs but birds linked to aquatic food webs showed little or no decrease through the late 1970s (Lindberg and Odsjo, 1983). Newton et al. (1993) showed a decline from levels observed in 1967 in continuous and long-term records of Hg in birds from the UK. Monteiro and Furness (1997) inferred increases in bird feather Hg estimates using museum samples. Because of the abundance and distribution of raccoons (Procyon lotor) in museum samples and in present-day environments of concern, this species was chosen as an archive that might reveal historic patterns of Hg exposure in south Florida (Newman et al., 1994). The hypothesis that raccoons in south Florida may be a vector for Hg to the endangered Florida panther (Roelke et al., 1991) was also a consideration in the use of this species. Cumbie (1975a) showed that raccoons provided a satisfactory archive for Hg. Furthermore, most of the Hg input or loading in south Florida comes from atmospheric deposition (95% according to Stober et al., 1996; Atkeson and Parks, 2001) and most temporal records of atmospheric deposition suggest fairly even distribution throughout south Florida (Guentzal et al., 2001). The range of bulk deposition in 1996 for sites in south Florida, south of Lake Okeechobee, varied by a factor of 1.3 from 17 to 22 lg/m2-year; the site at Crawl Key – an area considerably south of the Everglades – was 12 lg/m2-year. Thus, it was hypothesized that raccoon hair Hg might provide an historical record indicating the role of deposition in Hg bioaccumulation.

Two hypotheses were formulated: (1) the hypothesis that a difference existed between the MMHg concentrations in older samples and present-day samples: (2) the hypothesis that site differences existed. In addition, the influences of other factors on MMHg accumulation were evaluated: life-stage, age, size, sex, concentrations in other tissues, other elements (Se, Zn), tanning, and drying of hair. The 1939 museum samples had been tanned using dilute organic acids (bark extracts) and this procedure was suspected of removing MMHg.

Methods and materials Collection of samples. Raccoon hair samples were collected from animals collected in south Florida over short intervals of time from 1939 to 2000. The hair samples were taken from museum specimens prior to 1960, from archived samples collected by Roelke et al. (1991), and from animals trapped during 2000. Roelke et al. did not analyze raccoon hair for Hg; available specimens were analyzed after obtaining nine hair samples from raccoons trapped in 1990 by Roelke et al. (1991). All hair (212 samples), blood (24 samples) and a select group of other tissue samples (55, from 5 tissues in 11 animals) were analyzed during the year 2000 for MMHg. Other characteristics were measured for a subset of samples. After determining collection sites of available samples contained in museums, five corresponding regions were identified for collection of modern samples (Table 1, Fig. 1): the 10,000 Islands of south Florida (two subareas), the

Table 1. Summary of available data Museum Site sampled

Samples

Outer Islands Chokoloskee North Key Largo Northern Florida Keys Flamingo Long Pine Key Shark Valley Slough (SVS)

2 25 12 9 20 9 8

Total

85

Modern Yr. sampled

Samples

Yr. sampled

1957 1939 1948/49 1939/48 1948/49 1948/49 1947/48

25 25 17 – 21 11 19

2000 2000 2000 – 2000 2000 2000

118

Note: Indicates no samples taken or not applicable. Also, nine samples from Roelke et al. (1991) were analyzed making a grand total of 212 samples.

Retrospective Study of Mercury in Raccoons 209

Figure 1. Map showing the general sampling areas and locations of modern (Mod), museum (Mus) and Roelke et al. (R) samples of individual raccoons.

Shark Valley Slough, Long Pine Key, the north Florida Keys (two subareas), and the Flamingo area. Collections were directed at obtaining a number of raccoons equivalent to the number of museum samples to maximize the power and robustness of the statistical tests. Of the five regions, comparable data were obtained from four: (1) Flamingo located near the southern tip of Florida in a background area based on data from USEPA (Stober et al., 1996); (2) Long Pine Key located in Everglades National Park (ENP) just west of Miami in a former agricultural area; (3) North Key Largo south of the Miami urban area and considered a background site and (4) Shark Valley Slough in the middle of an area of high Hg in biota based on data from USEPA (1996): Shark Valley Slough provides the primary natural drainage pathway for the Everglades from Lake Okeechobee through ENP (Stober et al., 1995, 1996; Atkeson and Parks, 2001). The nine Roelke et al. (1991) samples were collected from Shark Valley Slough and immediate environs (n ¼ 6) and the background Flamingo area (n ¼ 3).

Raccoon hair was collected by cutting at the skin surface using a stainless steel scissors cleaned with isopropyl alcohol between specimens, or, later, by simply pulling out tufts of hair. The samples were then packaged, coded, and sent to the analytical laboratory where pre-selected analyses were performed. For the year 2000 samples, raccoons were collected using live traps (primarily Havahart #1079, Woodstream, Lititz, PA). For most animals, standard measurements were made of total length, tail length, ear, and hind foot. All animals were weighed, sexed and differentiated between immature and mature by condition of gonads, nipples and tooth wear. Raccoons were marked with two ear tags to avoid double counting through recaptures. To assess tissue relationships with hair MMHg, 11 sacrificed raccoons were obtained from the U.S. Fish & Wildlife Service and dissected samples to analyze Hg concentrations in blood, brain, hair, heart, kidney, liver, and muscle tissues. Also, the right canine or premolar 4 was extracted from the mandibles of these animals and age (usually ±0–1 year) was

210 Porcella et al. determined by cementum annuli analysis performed by Matson’s Laboratory in Milltown, MT. All samples were coded and chain of custody records assured sample anonymity until statistical analysis. Analytical procedures. Dr Lian Liang of CEBAM Analytical, Inc. in Seattle, WA analyzed hair, blood, and other tissue samples for MMHg, THg, selenium, and zinc. Methods used for analysis are taken from USEPA methods (1998, 1999), and are summarized as follows: Total mercury (THg): Samples were digested with HNO3/H2SO4 at 95 C for 3 h. The digestates were analyzed using a procedure in accordance with EPA Method 1631 for THg by oxidation, purge and trap, and cold vapor atomic fluorescence spectrometry. The method detection limit (MDL) is 0.5 ng/g. Methyl mercury (MMHg): Samples were digested with KOH/CH3OH at 75 C for 3 h. The digestates were analyzed using a procedure in accordance to EPA Method 1630 for MeHg by aqueous ethylation, purge, trap, GC separation, and cold vapor atomic fluorescence spectrometry. The MDL is 0.05 ng/g. Selenium (Se): Samples were digested with HNO3/HClO4 to HClO4 fume stage. The digestates were analyzed by hydride generation, Pd coated platform collection, and GFAAS detection (Liang, 1998). The MDL is 0.1 ng/g. Zinc (Zn): Samples were digested with HNO3, and analyzed using EPA 200.7 by ICP. The MDL is 0.1 lg/g. All analyses were accompanied by explicit quality assurance and quality control (QA/QC) protocols and procedures, which met full requirements of EPA methods. Two levels of THg and MMHg were analyzed in fish tissue from the International Atomic Energy Agency (IAEA) standard reference materials: IAEA142 – THg 0.127 lg/g and MMHg 0.047 lg/g; IAEA350 – THG 4.680 lg/g and MMHg 3.650 lg/g. All hair samples were ana-

lyzed for MMHg. Results of quality control measurements for MMHg are listed in Table 2, and show very high quality. Where available, duplicate measurements were averaged for data analysis. Other constituents, THg, Se, Zn, were analyzed on selected samples. In addition, an experiment to evaluate the effects of tanning was performed. Almost all of the 1939 museum samples were treated with a bark extract prepared according to standard procedures of that era (Personal Communication to J. Newman from Judith Chupasko, The Museum of Comparative Zoology, Harvard University, July 5, 2000). This extract behaved as a dilute organic acid. Consequently, an experiment was performed by treating 10 skin plus hair samples with dilute acid (pH ¼ 5, dilute H2SO4). Finally, all values are expressed as mass per fresh weight (lg/g). In addition, good agreement was obtained for split samples of hair analyzed by the Florida Department of Environmental Protection Central Laboratory (FDEP, Tallahassee FL) and CEBAM (Table 2). The matrix and spike additions performed by FDEP gave about 80% recovery, and this result can explain the somewhat lower MMHg concentrations obtained by FDEP. Statistical Analysis. StatView V. 5 (SAS Institute, 1998) was used for all data analyses. Prior to fieldwork, power analyses (beta ¼ 0.8, alpha ¼ 0.05, differences between means assumed to be 50%) were performed to estimate the optimal number of modern samples to test the hypotheses about temporal and spatial variation. To insure the most credible comparison, it was found that the number of modern samples should be equal to the number collected in the museum samples. T-tests were used for the comparison of museum and modern samples at the four sites where sufficient numbers of measurements were available

Table 2. Quality control variables Variable Standards (IAEA), recovery, percent Duplicate analyses, mean in percent Standard additions, recovery, percent Blanks, less than value Laboratory splits, CEBAM/FDEP THg MMHg

Number analyzed

Value

9 12 4 10

99.7% 98% 97% 0.1–0.6 ng/g

8 11

1.07 1.21

Retrospective Study of Mercury in Raccoons 211

Results – methodological issues Biologic variables

9

total weight, ln(g)

(Long Pine, North Key Largo, Flamingo, and Shark Slough). The Roelke et al. (1991) data were not grouped with the other data for the statistical analyses.

8.6 8.2 7.8 y = 2.9x - 4.1 R2 = 0.55

7.4

Museum and modern samples were compared to determine whether the populations showed any obvious differences (Table 3). Mean values were essentially identical for all the parameters measured. Although the animals sampled showed little variation in the various length measurements (coefficient of variation (CV) between 12% and 15%), the hair mercury content varied extensively (CV ¼ 108%). Modern samples plus six museum samples and three samples from Roelke et al. (1991) showed a fairly good approximation of the allometric law for raccoons (Fig. 2): lnðlive weight; gÞ ¼  4:1 þ 2:9  ln(total length, cm)

ð1Þ

Such a relationship, with r2 ¼ 0.55 (n ¼ 88), shows that the trapped specimens were not overtly unhealthy. The three samples (R65, R66, R72) from Roelke et al. (1991) were close to the line of best fit as were four of the museum samples. Two museum samples appeared to have greater weight per length than the other 86 samples. Summary statistics for the length measurements of the specimens with such measurements show there was no significant difference between modern and museum samples (Table 3). No significant difference between male and female animals was observed. Although aging was not possible under the sampling scheme (except for the 11 sacrificed ani-

7 4

4.1

4.2

4.3

4.4

4.5

Figure 2. Length–weight relationships for 88 samples of raccoons collected mostly (3 in 1990, 6 in 1956–1957) in the year 2000.

mals, which will be discussed separately), separation by examination of the gonads into immature and adult groups showed that age had some effect on raccoon hair Hg. Adult animals had significantly greater MMHg concentrations in hair than immature specimens (p ¼ 0.0023); adult means were 8.37 (sd ¼ 8.14, n ¼ 97) and immature were 6.14 lg/g ww (sd ¼ 9.56, n ¼ 30). Cumbie (1975a) obtained a similar result. Contamination with inorganic mercury One concern in this study was whether the museum samples might contain mercury used as a preservative. Despite assurances from museum directors that this was probably not the case, all samples were analyzed for MMHg, and the statistical treatments were based completely on these measurements. A subset of modern hair samples was analyzed for total mercury (THg), and these data are summarized in Table 4. For hair the MMHg levels constituted 90% or greater of the

Table 3. Biologic variables are equivalent

All samples All museum All modern Modern = museum CV, percent; all samples

4.6

total length, ln(cm)

Hair Hg

Length, cm

lg/g

Length

Tail

7.5 7.6 7.4 8.3 108

73.7 73.5 74.1 76.6 12

21.5 20.6 22.9 22.5 15

Ear 5.2 5.2 5.2 5.5 12

Hind foot 10.8 10.7 10.9 11.4 13

212 Porcella et al. Table 4. THg and MMHg concentrations in raccoon hair samples collected during the year 2000 THg Description Split samples CEBAM analyses FDEP analyses Other CEBAM analyses Split + other (CEBAM)

n

8 8 38 46

Percent MMHg

MMHg lg/g

9.4 8.8 11.8 11.3

total Hg. This is in agreement with most estimates of hair mercury in a variety of mammals (e.g., Cernichiaria et al., 1995a, b). Tanning Very low values of hair MMHg were observed in the 1939 museum samples collected from Chokoloskee Island in the Ten Thousand Island area. These skins had been tanned with a dilute organic acid. The results showed that 91% of THg and 99% of MMHg were extracted from the hair using dilute H2SO4 (pH ¼ 5). These extraction levels are consistent with the levels observed in the tanned, museum samples which have a mean MMHg concentration of 0.05 lg/g which is about 1–5% of the average of background modern raccoon hair samples. Consequently, all tanned specimens were excluded from our analyses, which also resulted in the elimination of the Ten Thousand Island area from statistical analysis. Other trace metals Hair samples were analyzed for Se and Zn to determine if these elements could explain some of the differences between MMHg concentrations. Hair MMHg was not expected to show a strong relationship with hair Se levels, and the variance of the molar ratios was quite high: mean of 5.37, range of 0.07–25.21. Molar ratios for THg to Se averaged 6.1 with a similar broad range of 0.13– 27 moles/mole. Molar ratios for THg to Zn averaged 0.2 and varied between 0.0002 and 0.7 moles/ mole. The molar ratios of Zn to Se averaged 0.22 with a narrower range of 0.013–0.031. These data do not appear to have an application, and such ratios in other tissues like the liver might be more useful (Wagemann et al., 2000).

n

11 11 28 36

lg/g

8.1 6.6 10.8 10.1

85 75 95 0.93

Hair weight All hair Hg levels are reported in lg/g fresh weight. Dry weight measurements for hair showed that the mean dry weight was 94.5% of the fresh weight of hair with a range of 93–96% (n ¼ 14 samples). To maintain consistency with other tissue measurements, the fresh weight measurements were not adjusted to dry weight. Also, any correction would have had a small magnitude and a very small range.

Results – testing hypotheses Museum and modern samples The frequency distribution for all museum and modern samples (172 samples) that are considered equivalent and without artifact are shown in Fig. 3a. Thus, 31 museum samples were eliminated from the original sample total because the skins had been tanned. Tanning causes substantial loss of MMHg from the hair (see above). The data from the Roelke et al. (1991) samples will be discussed separately. The results in Fig. 3a show that most of the samples (74%) were less than 10 ppm MMHg, and 50% were less than 5 ppm. Separating the 172 samples into two groups as museum (Fig. 3b) and modern (Fig. 3c) samples showed little effect in means or frequency distribution. Similarly, the modern samples had 74% and the museum samples had 76% under 10 ppm with means that were virtually identical: 7.6 ppm for modern and 7.4 ppm for museum samples. When only those modern samples collected from the sites (Flamingo, Long Pine Key, North Key Largo, Shark Valley Slough) where museum samples had sufficient n are compared (Fig. 3d) (n ¼ 68), a

Retrospective Study of Mercury in Raccoons 213

Figure 3. Frequency distributions for various subsets of MMHg in raccoon hair collected during the year 2000 from south Florida sampling areas: (a) all samples less the artifacts of tanned samples (n ¼ 172); (b) all modern samples (n ¼ 118); (c) all museum samples less the artifacts of tanned samples (n ¼ 54); (d) all modern samples from four areas commensurate with the museum samples of 3c (n ¼ 68).

slightly higher mean value of 8.3 ppm was obtained for this subset, yet 71% had MMHg at less than 10 ppm. For the four data sets shown in Fig. 3, the CV was high and similar, 108%, 102%, 122%, and 116% for the sample sizes of 172, 118, 54, and 68, respectively. Furthermore, the range and mean values for raccoon hair MMHg do not differ greatly from those presented previously (Cumbie and Jenkins, 1974; Davis, 1981; also, see Cumbie, 1975a; Wren, 1986). However, a higher mean hair Hg value of 28.94 ppm (n ¼ 31, range 0.77–65.42) has been reported for raccoons in the Okefenokee Swamp (Arnold, 2000). Sheffy & St. Amant (1982) reported a mean value of 3.79 ppm for Wisconsin raccoon hair in comparison to these values from the southeastern US.

The 11 largest values (>20 lg/g) from the main distribution shown in Fig. 3a are of considerable interest because they appear anomalous. For these so-called outliers, seven samples were included in the modern samples (about 10% of the total) and four in the museum samples (7%). All but one of these samples were collected from south of the Tamiami Trail (Hwy 41), an area in the northern sector of ENP known for high biotic Hg in south Florida (Lange et al., 1993; Stober et al., 1995; Frederick et al., 2002). Six of the seven modern sample outliers were from the Shark Valley Slough south of Hwy 41 with the seventh from north of Hwy 41 while three museum samples were from Long Pine Key and the fourth was from Shark Valley Slough (along Hwy 41). There are samples

214 Porcella et al. in the Shark Valley Slough and Long Pine Key areas that are quite low – for example, values of 1.55 and 8.45 ppm were found along with the six modern samples above 30 ppm in the area south of Hwy 41. This broad distribution suggested due to dominance in feeding by individuals that feeding behavior might account for differences in hair MMHg concentrations (for discussions of raccoon feeding behavior, Johnson, 1970; see Davis, 1981). Samples were grouped according to location and date of collection (museum and modern) (Table 5). These results show that the means vary widely with location of the samples, and account for the high CV’s. The means from North Key Largo were lowest for both museum and modern samples, while Long Pine Key had the highest mean for the museum samples. Modern samples from Shark Valley Slough had the highest means, and samples from south of Hwy 41 were the highest of all, averaging 27 ppm. The range of the mean values was comparable for both museum and modern samples: 19 for the museum samples (Long Pine Key to North Key Largo) and 18 for the modern (Shark Valley Slough samples from south of Hwy 41 to North Key Largo). Interestingly, six additional hair samples from animals in a study by Roelke et al. (1991) collected in spring 1990 from Shark Valley Slough (not included in Fig. 3) provide the highest MMHg levels of all the samples analyzed, averaging 72 ppm (range of 44– 124 ppm). Roelke et al. (1991) provided three control samples obtained from the Flamingo area

averaging 2.8 ppm, which is less than the museum and modern means for Flamingo in Table 5. There is no consistent pattern in the differences in mercury concentrations between the museum and modern samples. Rejection of samples because of tanning occurred primarily in the Ten Thousand Islands area (Chokoloskee and Outer Islands) where intermediate hair MMHg concentrations were obtained in modern samples (ave. ¼ 6.6 ppm). Only three untanned museum samples were available from the Ten Thousand Island area; these had hair MMHg levels of 10.6, 12.2, and 9.7 ppm (1939–1957). Looking at the remaining four areas with relatively equal sample sizes in the museum and modern samplings – Flamingo, Long Pine Key, North Key Largo, and Shark Valley Slough – gives inconsistent results. Overall, Shark Valley shows an insignificant increase for the modern samples (p ¼ 0.52). Long Pine Key (p ¼ 0.027) and Shark Valley north of Hwy 41 (p ¼ 0.05) show decreases for the modern samples while Flamingo (p ¼ 0.002), North Key Largo (not significant, p ¼ 0.18), and Shark Valley south of Hwy 41 show increases for the modern samples (p ¼ 0.05). The ratios of modern/museum were 1.88 for Flamingo, 1.51 for North Key Largo, 0.48 for Long Pine Key, and 1.33 for Shark Valley Slough (2.38 for south of Hwy 41 and 0.56 for north of Hwy 41). The overall ratio for the set of compared data (modern ¼ 8.4; museum ¼ 7.4) was 1.12. The processes that control MMHg delivery to raccoons

Table 5. Comparison of MMHg levels in modern and museum hair samples of raccoons* Number of samples

MMHg, lg/g fresh weight

Location

Museum

Modern

Museum

Modern

Range-museum

Range-modern

North Key Largo Flamingo Long Pine Key Shark Valley Slough SVS, so. of Hwy 41 SVS, no. of Hwy 41 TTI, Chokoloskee TTI, Outer Islands Sands Key Roelke (1991) 1990 Roelke (1991) 1990

15 20 9 8 (8) (8) 1 2 2

17 21 11 19 8 11 25 25 – 6 3

1.0 3.8 19.6 11.2 (11.2) (11.2) 10.6 11.0 4.5 – –

1.5 7.2 9.4 14.9 26.7 6.4 5.4 7.9 – 72.5 2.8

0.031–3.00 0.201–10.2 4.85–36.6 3.37–30.1 – – 10.6 9.69–12.2 4.44–4.60 – –

0.17–4.17 1.01–13.2 2.90–15.3 0.46–13.1 1.55–43.1 0.46–25.8 0.71–16.3 2.96–14.0 – 44.3–124.0 2.2–3.4

*Bold indicates significantly different at p < 0.05; – indicates not applicable. Shark Valley Slough museum samples were collected along US 41, and are compared to the areas north and south of US 41.

Retrospective Study of Mercury in Raccoons 215 appear to differ between sites where low exposures might occur (Flamingo and North Key Largo) while areas with higher concentrations appear to be dominated by MMHg production and bioaccumulation processes (Long Pine Key and Shark Valley Slough). The Roelke et al. (1991) samples – though taken from areas around our Shark Valley Slough samples (south of Hwy 41) – appear to represent a different seasonal time frame of exposure. Roelke et al. samples were collected in May– June, whereas both museum and modern Shark Valley Slough (North of Hwy 41) samples were collected in October, and modern Shark Valley Slough samples south of Hwy 41 were collected in March and October. The fact that raccoons are opportunistic feeders raises the question of whether the higher values seen in the Roelke et al. samples were a result of higher natural Hg uptake in seasonally available food rather than a real difference between the years of collection. Statistically significant differences in MMHg levels between the museum and modern hair samples existed at two locations, Flamingo and Long Pine Key. At the Flamingo location the MMHg concentrations in the modern and museum samples were 7.1 and 3.9 lg/g, respectively. At the Long Pine sampling location, the museum samples were significantly higher than the modern samples: 19.6 versus 9.4 lg/g. At the other two locations (North Key Largo and Shark Valley Slough) the measured concentrations in the modern samples were slightly elevated above the museum, but these differences were not statistically significant. Overall, the variability among the samples at each location and each sampling period was high. The coefficients of variation ranged between 50%

and greater than 100%. Power analyses conducted during the design phase of this study indicated that the collection of 20 samples at each location would provide the ability to detect minimum changes on the order of 30–50% between the mean MMHg concentrations in the hair of the raccoons. Thus, the a priori estimates were less than observed variation. Tissue mercury The distributions of Hg among the tissues studied indicate that the highest concentrations and burdens of mercury are in hair and liver (Table 6). Marked differences in speciation of mercury exist between these two compartments. The liver is almost all inorganic Hg (93%), while the hair is almost all MMHg (99%). The liver acts as the site of detoxification (Wren, 1986; Clarkson, 1994), changing MMHg to HgII where sequestration processes undoubtedly store the mercury (Davis, 1981). The kidney also may play a role in demethylation, accounting for the high inorganic Hg concentrations in the kidney (55%). High inorganic Hg in liver and kidney relative to other tissues also has been reported for river otter, and generally also for wild mink (Jernelov et al., 1976; Wren et al., 1980; Evans et al., 2000; Fortin et al., 2001). In marine mammals, one fraction of liver Hg has molar ratios of Hg to Se of 1:1, suggesting that this insoluble moiety sequesters Hg and prevents further toxicity (Wagemann et al., 2000). Comparison of relative MMHg levels in tissues (normalized to blood concentrations) showed that hair was highest, followed by liver, kidney, muscle, heart, brain, and blood in their respective ratios to

Table 6. Mean mercury concentrations in tissues of raccoon samples* Percent MMHg

Average ww, lg/g Tissue type Blood Brain Hair Heart Kidney Liver Muscle * Sample n = 11.

THg 0.126 0.286 10.600 0.457 1.490 16.200 0.570

Inorganic Hg 0.016 0.010 0.100 0.005 0.813 15.759 0.240

MMHg 0.110 0.276 10.500 0.452 0.677 0.441 0.546

0.87 0.96 0.99 0.99 0.45 0.03 0.96

216 Porcella et al. blood: 96:10:6:5:4:2.5:1. Although these values are in the same relative order, they were calculated differently from Arnold (2000) who used total Hg measurements for hair, liver, kidney, muscle, brain, respectively: 10:7:5:1:1. Hair serves as an efficient mechanism for eliminating MMHg directly (Clarkson, 1994). Davis (1981) used radioisotopes to estimate the biological half-life (B1/2) for raccoon hair THg, and for tissues with high percentages of MMHg; this approach provides a first approximation of its elimination. Hair B1/2 is apparently 229 days (Davis, 1981). The raccoon hair B1/2 is much higher than hair in humans and other primates (B1/2 ¼ 45 days) (Chernicciaria et al., 1995a, b). Other tissues that contained a high fraction of MMHg (>95% MMHg) were brain, heart and muscle with B1/2’s of 62, 41 and 35 days, respectively (Davis, 1981). Despite having a high fraction of inorganic Hg that is apparently sequestered, Davis (1981) estimated that the liver had a lower half-life than hair, i.e., B1/2 ¼ 108 days. The liver B1/2 of 108 days is considerably less than that of the marine mammal, the ring-necked seal, which has a Hg B1/2 of 28 years (Wagemann et al., 2000). A two-compartment model allows for calculation of a more reasonable estimate of the labile fraction of Hg in the whole body of marine mammals (B1/2 ¼ 50 days; Claude Joiris, Belgium; E-mail:

[email protected], personal communication, October 2001). One question was whether hair was a good predictor of Hg in other tissues. Using the coefficient of determination, r2, as a guide, hair does not appear to predict Hg levels in other tissues at a high level of precision (Table 7), with r2 values between 0.24 and 0.58. Slopes ranged between 0.01 and 6.55. The maximum r2 value showed that 58% of the variance in hair could be associated with blood Hg levels. Furthermore, unlike Cumbie (1975a) our results showed that hair MMHg or THg did not correlate well with either variable measured in liver or muscle. Logarithmic transformations did not improve these correlations. The longer B1/2 for raccoon hair suggests that correlation would not be that good between hair and other tissues. Furthermore, the slope values of Cumbie (1975a) do not appear to approach the ones shown in Table 7. Cumbie (1975a) measured THg only but had greater numbers of samples than this study. Using THg did not improve correlation in this study. One major difference in this study and that of Cumbie’s may account for the different result: The range of our values for hair MMHg was 5.1–14.0 lg/g (n ¼ 11) for most of our tissue comparisons, while, for example, Cumbie (1975a) had a greater n and greater range from 1 to 28 lg/g (n ¼ 60). A greater number of paired

Table 7. Regression slopes for selected variables* Dependent

Independent

Slope

r2

Blood MMHg Brain MMHg Heart MMHg Kidney MMHg Liver MMHg Muscle MMHg Brain MMHg Heart MMHg Kidney MMHg Liver MMHg Muscle MMHg Brain MMHg Kidney MMHg Muscle MMHg Kidney HgII

Hair MMHg Hair MMHg Hair MMHg Hair MMHg Hair MMHg Hair MMHg Blood MMHg Blood MMHg Blood MMHg Blood MMHg Blood MMHg Heart MMHg Heart MMHg Heart MMHg Liver HgII

6.546 0.030 0.044 0.090 0.085 0.056 1.450 2.360 3.430 6.880 2.240 0.617 1.260 1.300 0.024

0.58 0.51 0.46 0.47 0.24 0.39 0.51 0.56 0.29 0.66 0.26 0.92 0.38 0.86 0.71

* Samples n = 11 (except n = 24 for blood:hair regression); all based on lg/g, live (wet) weights; intercepts not shown because not different from zero.

Retrospective Study of Mercury in Raccoons 217 samples were available for our hair:blood comparison (n ¼ 24; range of hair MMHg ¼ 2.7–34.7), which likely accounts for the higher r2 value for blood compared to some of the other tissue comparisons. Arnold (2000) also reported a significant correlation between THg concentrations in raccoon hair with liver, kidney, muscle and brain (n ¼ 32; p < 0.01). However, their average hair THg values (28.94) were much greater than either Cumbie’s or this study with a correspondingly greater range (0.77–65.42). Liver and kidney inorganic mercury levels correlate well (r2 ¼ 0.71), suggesting that the liver is the source of HgII to the kidney or that the kidney demethylates MMHg as well. Note that blood MMHg correlates reasonably with brain, heart, and liver MMHg. Heart and muscle are highly correlated, as would be expected for these similar tissues, but the correlation between blood and muscle is poor despite the reasonable correlation between blood and heart. The highest correlation is between heart and brain MMHg (r2 ¼ 0.92), with a slope approaching 1.0 which suggests similar delivery processes in line with their similar B1/2. The muscles and brain tissues correlate well with reasonable r2 values and slopes approaching 1.0. Little or no relationship was found between age and most tissue Hg concentrations among the 11 animals from which multiple tissue samples were taken and age determined by tooth cementum annuli analysis. However, a weak correlation was found between age and THg in liver (r2 ¼ 0.39).

Discussion Two primary hypotheses were tested in this study: (1) that differences do not exist in the MMHg concentrations in the hair of raccoons between museum and modern samples and (2) that differences in the MMHg concentrations in the hair of raccoons do not exist between sampling sites. The first hypothesis that the two different sampling times did not show a difference could not be rejected. The second hypothesis that the four sampling areas were not different was rejected. Clearly, the individual sites vary in the temporal pattern of Hg in hair of raccoons. The two background sites (North Key Largo and Flamingo)

appear to show an increase in modern raccoons (year 2000) that indicate that MMHg levels have increased by 50–90% since 1948–1949. In Long Pine Key a decrease of about 50% was observed. The possibility was considered that MMHg fungicides may have been used in the agricultural area near the points of collection during the middle parts of the century, and that its subsequent ban during the early 1970’s may have led to the observed decrease. As late as 1960, seed treatment by mercury compounds, principally mercuric chloride, was recommended (although considered dangerous) for tomatoes, and cited as necessary for squash (Chupp and Sherf, 1960). Both were common crops grown in the Long Pine Key area (Cornwell and Atkins, 1975). However, no direct evidence to support this possibility was found. Indeed, while chemical analysis of 1974 Everglades soil samples in and near Long Pine Key showed moderately high mercury values, Hg levels in never-farmed samples were as high as those from farmed lands (Cornwell and Atkins, 1975). Mercury levels in soil cores from the Taylor Slough, just east of Long Pine Key, showed higher values occurring 20–180 years ago (Gough et al., 2000), unlike other studies showing higher Hg concentrations near the sediment surface in central Everglades cores (Delfino et al., 1993; Rood et al., 1995). The patterns in Shark Valley Slough are quite different depending on whether a raccoon sample was collected north of Hwy 41 (lower mean values that show a 150% increase) or south of Hwy 41 (higher mean values that show a 45% decrease). With both subareas lumped together in Shark Valley Slough, MMHg levels in hair increased 33%. If all four sites are lumped together, museum and modern samples differ only by 12%. Roelke et al. (1991) collected raccoon hair in the same Shark Valley Slough area, and our analyses of those samples suggest a pattern where MMHg concentrations in hair increased by a factor of 6.5 between 1947–1948 and 1990, and then decreased by a similar factor (nearly 5) between 1990 and 2000. Other factors were considered that may control Hg uptake. A review of dietary composition shows that raccoons are opportunistic omnivores with the proportion of animal to plant food consumed depending on location and season (USEPA, 1993). A habitat-specific analysis of raccoon stomach

218 Porcella et al. contents in north-central Florida revealed that in hammocks and sandhills, acorns were the most important food consumed, whereas in wetland habitats insects were the most abundantly consumed foods (Caldwell, 1963). Johnson (1970) discussed variation in raccoon diet for Alabama raccoons in agricultural and rural areas, and indicated that raccoons focus primarily on fruits and acorns in the late summer and fall when the animals accumulate the most fat. When these foods are depleted, the raccoons lose fat, and ingest more animal prey (largely crustaceans and insects) up to 20% of the total gut contents. Based on observation, Johnson argued that this is a low estimate because they could see the raccoons eating clams, but could not identify clams in the gut contents. Davis (1981) made similar comments about raccoons in north Florida and Georgia noting that some raccoons became dominant and would control the prime feeding areas. These observations probably account for the broad distribution of MMHg in hair among raccoons within any given area. It was noteworthy that similar distributions of hair Hg in raccoons have occurred in these older samplings (Cumbie, 1975a; Davis, 1981). In fact feathers collected by Monteiro and Furness (1997) show similar broad distributions of MMHg concentrations. Unfortunately, similar feeding studies have not occurred in south Florida where the ecosystems differ markedly from the more northern climates of the southeast – Alabama, Georgia, and north Florida. Nevertheless, similar food groups that dominate seasonal foraging of raccoons in other areas are also available in south Florida and considerable data are available on uptake of Hg in these groups. Measurements of food groups in the Shark Valley Slough area show increasing concentrations of THg across taxa from plants to fish. Maximum THg concentrations based on wet weight were 57.8 ng/g in aquatic vegetation, 74.4 ng/g in segmented worms, 496 ng/g in aquatic insects, and 1160 ng/g in fish (Loftus et al., 1998). Apple snails, very common throughout the Everglades, averaged 67 ng/g in the Shark Slough (Eisemann et al., 1997). Crayfish THg levels ranged from 32 ng/g in tissue to 81 ng/g in exoskeleton in the same area (D. G. Rumbold, South Florida Water Management District, personal communication, February 8, 2002).

The seasonal difference found in hair MMHg in Long Pine Key may reflect the apparent dominance of berries from Brazilian Pepper trees (Schinus terebinthifolius) in the raccoon diet during the period of availability. Based on observations of scat during this study, Schinus berries made up an important if not dominant part of the raccoon diet in Long Pine Key and the Shark Slough during the relatively short ripening season of December through February in this area (Ewel et al., 1982). No analysis of berries has been found but young leaves were found to contain 1–5 ng Hg/g (dry) (Guentzel et al., 1998). Raccoon hair samples taken in January (during berry season) contained an average of 6.8 lg/g (n ¼ 7), whereas hair samples taken in July–August (when animal prey are relatively more available) contained an average of 13.8 ng/g (n ¼ 4). The Brazilian pepper became dominant after 1961 based on Loope and Dunevitz (1981), and its availability could have caused the reduction in hair Hg observed for the raccoons in Long Pine Key between the museum (1948–1949) and modern samples (2000). Given the assumption that feeding patterns control the accumulation of MMHg in raccoons, high-end raccoons became the focus because they possibly represent similar feeding habits at the highest exposure levels. Thus, a better comparison in Shark Valley Slough might be the means of the high-end outliers: thus, high-end museum samples from Shark Valley Slough would have 17 ppm (n ¼ 2), Roelke’s samples have 72 ppm (n ¼ 6), and the modern samples would have 34 ppm (n ¼ 6, all from south of Hwy 41). In this scenario, the increase between 1947–1948 to 1990 would be a factor of four, while the decrease since 1990 to 2000 would be a factor of 2. Evaluation of the effects of feeding behavior is highly speculative because there are few data available for south Florida raccoons. Roelke et al. (1991) reported a much more extensive analysis of Hg in raccoons than represented by the relatively few hair samples analyzed in this study. Since Hg levels in Roelke’s study were reported only for muscle and liver samples, they are not directly comparable to our hair samples and were not included in our above analysis. Their data set, however, provides a good comparison of Hg loadings in raccoons across most of the southern Florida peninsula (nine

Retrospective Study of Mercury in Raccoons 219 locations). If a first order estimate of hair Hg is estimated from Roelke et al. liver data (using Cumbie’s, 1975b regression equation; ln (10  hair Hg) ¼ 0.825  [ln (100  liver Hg)] ) 0.385, R ¼ 0.96), the 1990–1991 Hg burden can be compared with our historic (1947–1958) and modern (2000) hair samples in several additional locations. In general, Roelke et al. found relatively high levels across the central portion from the Big Cypress Swamp through Long Pine Key, with levels falling off precipitously at locations further to the west and east. The overall mean of hair Hg estimates for all five central Florida locations was 31.7 ppm for 1990–1991 (n ¼ 26), compared with measured hair Hg means of 15.4 ppm for 1947– 1949 (two locations, n ¼ 17), and 14.2 ppm for year 2000 (three locations, n ¼ 30). In this comparison, the increase between 1947–1949 and 1990–1991 would be a factor of only 2, while the decrease since 1990–1991 to 2000 also would be a factor of 2. At the background location of Flamingo, the 1990–1991 estimated hair Hg levels were comparable to the 1948–1949 levels but only about half that of modern hair Hg levels. As noted above, however, seasonal differences in feeding behavior may affect differences observed between years. In addition to feeding behavior, other factors must be invoked to explain differences in site average raccoon hair MMHg. Given that deposition (95% of input of Hg to the south Florida environment) is relatively uniform, how can a factor of nearly 20 difference in means from different sites be explained? This discrepancy must be caused by the differences in methylation rate and bioaccumulation through the food web caused by site-specific conditions (e.g., see Cleckner et al., 1998; Gilmour et al., 1998).

Conclusions • The hypothesis that MMHg in raccoon hair had remained the same during the last 50 years in south Florida could not be rejected. Thus, there is only speculation that raccoon hair mercury might have increased. • Different areas of south Florida have markedly different MMHg in raccoon hair by rejecting the hypothesis that the means were the same.

• Feeding and other behaviors likely account for the observed broad distributions of hair MMHg among different habitats of raccoons. Land-use differences (agriculture versus parklands) and hydrologic differences (canalization and drought) are likely to be important determinants of feeding behaviors. • Large increases in raccoon hair MMHg occurred in high biotic MMHg areas of south Florida as inferred by measurements in 1990– 1991 (note that apparent differences between periods may reflect different seasonal collection times). Thereafter, there is the inference that a subsequent decrease resulted in levels about the same as the mid-century levels, although in one high biotic MMHg area – Long Pine Key – natural mid-century levels appear higher than current levels. • Contrary to conclusions of other studies, hair MMHg in raccoons does not appear to be a very precise predictor of MMHg in other tissues (other studies showing high coefficients of correlation were based on total Hg and larger sample size). • Speciation of Hg in tissues is essential to evaluating Hg levels in other tissues. For example, liver Hg is virtually all inorganic except for a few percent MMHg. • In trying to assign causes for the observations, deposition seems least important. The production of MMHg plus the effects of feeding behavior have more to do with the MMHg in raccoon hair than deposition. Influence factors that affect methylation as well as availability of inorganic mercuric ion substrate control the methylation process. • Based on a priori power analyses, substantial sampling effort occurred to estimate historic Hg patterns. Until more is known about factors influencing Hg uptake in raccoons, such as the relative importance of feeding behaviors versus biogeochemical factors influencing local rates of methylation, raccoons appear to have somewhat limited value as an archive for studying historic Hg patterns; nevertheless, the number of uncompromised raccoon specimens available in museums comprise the most representative of all historical biotic archives for Hg in south Florida. Hg accumulation in raccoons can be a useful tool for prospective studies of Hg patterns

220 Porcella et al. and trends. However, feeding information is required to understand Hg accumulation.

Acknowledgments We are indebted to E. R. Rich and W. B. Robertson, Jr. for their contributions to obtaining the museum samples. T. D. Atkeson and D. M. Axelrad provided access to the FDEP Central Laboratory for QC purposes, and T. M. Chandrasekhar analyzed split samples. T. J. Doyle provided raccoon carcasses from the US Fish & Wildlife Service’s Ten Thousand Islands sea turtle management program. Major funding of the study came from Florida Power and Light Company.

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