- Email: [email protected]
Fluoride accumulation and toxicity in wild small mammals
Environmental Pollution 85 (1994) 161-167 ~.~.~.~.:v'jj
F L U O R I D E A C C U M U L A T I O N A N D T O X I C I T Y IN W I L D SMALL MAMMALS I. C. Boulton, J. A. Cooke Ecology Centre, School of Environment, University of Sunderland, Sunderland, UK
& M. S. Johnson* Industrial Ecology Research Centre, Department of Environmental and Evolutionary Biology, University of Liverpool. Liverpool. UK (Received 12 November 1992; accepted 29 April 1993)
Abstract Populations of two species" of small mammal, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L.), inhabiting grasslands contaminated by industrial sources of fluoride were examined for fluoride concentrations in skeletal tissue and for morphological changes in the teeth. Concentrations of fluoride in teeth and bones were higher for C. glareolus than for M. agrestis at the chemical works and smelter sites. Severe dental lesions were recorded on the incisor and molar teeth of both species at the chemical works and smelter sites, with less marked damage at the mine tailings dam. This is attributed to inter-site differences in fluoride speciation and the consequent effects on the availability of fluoride in the diet for bioassimilation. INTRODUCTION The effects of environmental fluoride contamination on animals of economic importance to man are well documented, through studies of fluoride accumulation and its toxic effects in bones and teeth (Shupe et al., 1972, 1987; NAS, 1974). Other studies have described fluoride accumulation in wild animals such as ungulates (Kay et al., 1975; Shupe et al., 1984) and small mammals (Gordon, 1974; Wright et al., 1978; Walton, 1987a; Andrews et al., 1989); but relatively little attention has been given to the relationship between the chemical speciation of inorganic fluorides, their accumulation and consequent toxic effects. The use of small mammals as indicators of environmental contamination is important in the ecological assessment of polluted sites (McBee & Bickham, 1990; NRC, 1991; Talmage & Walton, 1991). However, such an approach needs to be based upon a sound understanding of the accumulation of the target pollutant and its toxicology. To be of practical value, specific * To whom correspondence should be addressed. Environ. Pollut. 0269-7491/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain
concentrations found in body tissues need to be related to any toxicological effects and also to variables such as diet, age and the source of pollution. This paper reports an investigation of two small mammals, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L. ), through a comparative study of three grassland sites contaminated by industrial sources of fluoride: a mine tailings dam; an aluminium smelter, and a fluorochemical works. The fluoride levels in the hard tissues of small mammals are described in relation to morphological changes in the teeth.
MATERIALS AND METHODS Site description and sampling methods The mine waste site comprises grassland established on the revegetated surface of a fluorspar tailings dam which contains residual amounts of fuoride, lead and zinc (Johnson, 1980). The smelter site comprises plantations and rough grassland surrounding an aluminium reduction plant on the north-east coast of England. The chemical works contains an area of rough grassland within an industrial complex manufacturing hydrofluoric acid and both inorganic and organic fluorides. The latter two sites emit a combination of hydrogen fluoride gas and fluoride-laden dusts that contaminate vegetation, mainly by dry deposition. A comparable area of uncontaminated grassland in rural Northumberland was used as a reference site. Vegetation and small mammals were collected at all sites in October, 1988 and July, 1990. Vegetation was cropped at ground level and sorted into standing live and dead material plus litter. Plant material was oven dried at 60°C for 48 h before being ground without prior washing in a Cyclotech mill to pass a 500 /~m sieve. Small mammals were caught live using baited Longworth-style plastic traps and were then killed, weighed and deep-frozen prior to dissection in the laboratory. The skull, jawbones and hind limbs were
162
I . C . Boulton, J. A. Cooke, M. S. Johnson
Table 1. Summary of Scoring system for classification of fluoride-induced lesions in the teeth of small mammals a
Score
Incisor
Molar
0 [Normal]
Enamel smooth, glossy orangeyellow colour; normal shape,
White enamel, solid creamy dentine on grinding face; solid enamel 'ridge'.
1 [Questionable]
Slight deviation from normal,
Slight deviation from normal.
2 [Slight]
Faint horizontal banding of enamel; mottling; chalky spots; slight erosion,
Slight increase in erosion of grinding face; dentine darkened.
3 [Moderate]
Whitened enamel; mottling; incisor banding; erosion of tips; staining,
Increased erosion of grinding face; dentinal cavities; enamel chipped.
4 [Marked]
Enamel hypoplasia, pitting, staining; heavy erosion of cutting tips; loss of enamel colour,
Grinding surface nearly worn flat; dentinal cavities; severe staining; abnormal wear.
5 [Severe]
Lesions much worse than 4; cutting tips splayed and eroded to blunt 'stubs'; total colour loss; abnormal curvature,
Grinding surface worn flat; heavily cavitated dentine; severe staining; shrunken profile deformation of tooth roots.
with 1 cm 3 of 20% nitric acid, the washings being added to the volumetric flask whose contents were then made up to 5 cm 3. A sample (1 cm 3) of this digest was removed and 4 cm 3 of 1.5 M trisodium citrate buffer added, before the fluoride concentration was determined with an ion-selective electrode. The citrate acts to adjust the final solution pH and total ionic strength. The analytical procedure produced >95% recovery of fluoride in reference materials certified previously through interlaboratory collaborative studies: these were Assam tea and fluorosed horse bone, with certified total fluoride concentrations of 100 and 1600 mg F kg 1, respectively. Samples of dried live vegetation from all four sites were incubated at 37°C with both deionised water and 0.2 M hydrochloric acid (pH 2), the latter in an attempt to estimate the amount of fluoride present in vegetation that might be extracted and absorbed from the gastrointestinal tract through the action of stomach acids (Walton, 1987b). The extracted solutions were assayed for fluoride using a modified citrate-buffer-selective electrode technique. Statistical presentation of data
Parametric (t-tests) or non-parametric (Mann-Whitney U-test) statistical tests (Sokal & Rohlf, 1981) were carried out on the data where appropriate, and are the source of the probability values quoted in the text.
Adapted from Shupe et al. (1972). RESULTS removed and immersed in 10% papain enzyme at 60°C to enable the femur and teeth to be separated. The tissues were weighed and freeze-dried before further analysis. Examination of teeth for dental lesions Herbivorous mammals, including Microtus
agrestis,
have teeth that are adapted to the processing of fibrous and siliceous grasses prior to digestion: incisors which cut and trim grass stems and leaf blades, and molars which grind plant material to rupture fibres and cell walls (Oxberry, 1975). Excessive fluoride uptake and incorporation may result in morphological lesions that can be assessed and related to a scoring system (Shupe et al., 1972). The dental lesion scores given in the tables or text are modal values derived from an adaptation (Table 1) of the scoring system used originally to diagnose fluoride toxicosis in cattle (Shupe et al., 1972). Chemical analysis
After preparation, plant and animal materials were analysed for total fluoride using an acid extraction method adapted from the technique described by Andrews et al. (1989). Approximately 50 mg of tissue were treated with 1 cm 3 of 20% nitric acid (AnalaR) at 100°C for 1 h, in a digestion tube fitted with an air condenser in an aluminium block thermostat. After cooling, the digest was transferred to a 5 cm 3 volumetric flask and the tube and condenser rinsed twice
The total fluoride concentration in live vegetation from the tailings dam was four times higher than at the reference site. However, little of this fluoride was water soluble (7% of total) and the mean value of 5-8 mg kg (dry wt) was very similar to the equivalent of 3-6 mg kg ~ for the reference site; but 48% of the total fluoride in the railings dam vegetation was found to be soluble in HC1 (Table 2). Vegetation from the smelter site had over twice the total fluoride content of that at the tailings dam, and also showed higher percentage recoveries of both water- and HCl-extractable fluoride. Vegetation from the chemical works showed a higher total fluoride compared to the smelter site and, paradoxically, per-
Table 2. Total, water- and hydrochloric acid-extractable fluoride (mg kg-I dry wt) in composite live vegetation"
Site
n
Total
Reference
6
20 (0.9)
Tailings dam
6
80 (6.3)
15
187 (13)
6
549 (32)
Smelter Chemical works
Water
HC1
3.6 (0.3) [19] 5.8 (0.7) [7] 65 (5.4) [35] 144 (5.5) [26]
4.7 (0.2) [23] 38 (1.6) [481 111 (9.9) [59] 416 (40) [76]
" Mean values (+SE); [percentages of total fluoride extracted by method].
Fluoride accumulation and toxicity in wild small mammals
163
Table 3. Fluoride concentrations (mg kg -I dry wt) of femur, upper incisor and molar in the field vole (M. agrestis) and bank vole ( C. glareolus) ~
Site/species
n
Femur
Field vole (M. agrestis) Reference 19 291 (35) Tailings dam
19
1 169 (76)
Smelter
30
5 088 (301)
Chemical works
27
5 649 (325)
Incisor
Molar
289 (37) [0l 988 (120) [2] 2 530 (21) [31 3 466 (207) [5]
168 (18) [0] 716 (64) [1] 3 172 (351) [2] 4 060 (335) [5]
Bank vole ( C. glareolus) Reference 24 423 (21) Tailings dam Smelter Chemical works
2 24 7
210 (17) 378 (16) [0] [0] 1 556 (944) 234 (6) 641 (55) [21 [2] 8 011 (793) 4 387 (422) 6 397 (606) [41 [41 6 506 (1271) 5 271 (1080) 4 256 (994) [5] [41
Fig. 2. Smelter site: upper incisor of C. glareolus. higher in M. agrestis from the chemical works than from the smelter site, only in incisors was the difference significant (P < 0.01). The fluoride concentrations in C. glareolus from the smelter and chemical works were not significantly different (P > 0.05).
"Mean values (+SE); [modal dental lesion score].
Reference site
centage values for water- and HCl-extractable fluoride which were lower and higher, respectively, than at the smelter site. These results suggest that dietary fluoride assimilation by a consumer is in the following order:
Molars of M. agrestis from the reference site had significantly less fluoride than either the femur or incisor P < 0.01; Table 3). The fluoride concentration in the incisor of C. glareolus was significantly less than in the femur or molar (P < 0.0001). The incisors and molars of both species appeared normal.
reference < tailings dam < smelter < chemical works C o m p a r e d to the reference site, fluoride concentrations in teeth and bones were significantly higher in both Microtus agrestis and Clethrionomys glareolus from all three fluoride-contaminated sites (P < 0.0001; Table 3). The low fluoride concentrations in the tissues of animals from the reference site reflect the relatively low concentrations of dietary fluoride. Microtus agrestis from the tailings d a m had significantly lower fluoride concentrations than animals from the two atmosphericallycontaminated sites (P < 0.001), and correspondingly milder dental lesions. Although the mean fluoride concentrations for all hard tissues examined were
Tailings dam
At the tailings dam, the femur of M. agrest& contained more fluoride than the teeth, but only the concentration in the molar was significantly less than for the femur (P < 0-001; Table 3). Generally, only mild dental lesions were observed at this site: the incisors exhibited transverse banding just visible under the normal orangeyellow pigmentation (Fig. 1); the molars exhibited little change. Insufficient numbers of C. glareolus were caught to permit comparisons. Smelter
At the smelter site, femur fluoride concentrations in M.
iili~
Fig. 1. Tailing dam site: upper incisor of M. agrestis.
Fig. 3. Smelter site: molar of C. glareolus.
164
I.C. Boulton, J. A. Cooke, M. S. Johnson 10000
DO
o
Z=
0
°o Oo
v
oF
ov
o v
~v ~
v
~W
~
~'
,
[]
v
o.== =-% = E E~
v
~ •
•
1000
•
"'....."
o C.) 100 0
Fig. 4. Chemical works site: upper incisor of M. agrestis.
agrestis were significantly higher than for the teeth (P < 0.001) and, although concentrations were higher in molars compared to the incisors, the difference was not significant (P > 0.05). For C. glareolus there was significantly less fluoride in the incisors than either the femur or molar (P < O01; Table 3). In both species of vole, the incisors exhibited moderate to marked lesions, with erosion of cutting edges, colour loss, surface changes and enamel banding. The molars showed cavities (mainly in C. glareolus) and increased erosion of the grinding faces. Symptoms representative of those observed in both species are shown in Figs 2 and 3. Fluoride concentrations in the teeth of C. glareolus from the smelter site were significantly higher than in M. agrestis (P < 0.001), with higher modal lesion scores for the incisors but not molars, even though in C. glareolus the latter teeth had twice the concentration of fluoride. Chemical w o r k s At the chemical works the dental lesions recorded in both species were marked to severe, with incisors exhibiting total colour loss, scabbing and pitting of enamel, and gross erosion of the cutting tips to blunt stubs. The grinding surfaces of the molar teeth had worn flat, and cavities showed heavy staining. Symptoms representative of those observed in both species are shown in Figs 4 and 5. As with the smelter site, M.
I'0
r
j
20
30
i
j
40
Wet Body Weight (g)
Fig. 6. Relationship between body weight and log femur fluoride concentration in M. agrestis: ('k) reference site; (Q) tailings dam; (V) smelter; ([~) chemical works.
agrestis had femur fluoride levels significantly higher than in incisors or molars (P < 0.01), but the tissue fluoride concentrations in C. glareolus were not significantly different from each other (P > 0.05; Table 3). Mean fluoride concentrations in the teeth and femur of C. glareolus were higher than for M. agrestis, but these differences were not significant (P > 0-05). The lesion scores for both types of teeth were broadly similar. Analysis
of results
It appears that fluoride concentrations in both the femur and incisor do not increase with body weight in M. agrestis (Figs 6 and 7). Concentrations appear to reach an approximate equilibrium concentration which is sustained as body weight increases. There is some evidence that fluoride concentrations declined with increasing body weight at the reference site. The magnitude of the equilibrium concentration appears to be specific to the site of origin so, for the femur and incisor, this value is significantly higher in field voles from both the smelter and chemical works than in 10000
-
Q
o o~
OO O o
v v o
o
o:,, 1000
•ev
%. **% o O
100
L
0
I'0
20
L i
30
i
40
Wet Body Weight {g]
Fig. 5. Chemical works site: molar of M. agrestis.
Fig. 7. Relationship between body weight and log incisor fluoride concentration in M. agrestis: ('k) reference site; (0) tailings dam; (T) smelter; (U]) chemical works.
Fluoride accumulation and toxicity & wild small mammals 10000
-
Table 4. Changes in dental lesion score with fluoride eoncentratio# (rag kg -1 dry wt) in incisor and molar, and wet body weight (g) in the field vole (M. agrestis) •
'~ ~ ~
v
v
•''
1000
~
165
.
®~
•
,
o • eoe
,
..
Lesion score
n
Incisor fluoride
n
Molar fluoride
Body weight
0 1 2 3 4 5
2 14 29 19 19 8
291 (88) 323 (61) 1 172 (154) 2 484 (191) 3 474 (236) 4 192 (414)
15 30 20 17 8 3
295 (42) 975 (195) 2 394 (306) 4 368 (372) 5 058 (434) 6 299 (150)
12.0 (3.0) 22.0 (2.2) 19.6 (1.2) 24.4 (2.0) 19.5 (1.7) 18.3 (1.2)
o
o
a Mean values (+SE) of combined results from the four field sites: 1988 and 1990.
100 100
1000
10000
Log Femur Fluoride Concentration (mgF kg-1 dry wt)
Fig. 8. Relationship between log fluoride concentration in femur and incisor in M. agrestis: ('k) reference site; (O) tailings dam; (V) smelter; (IS]) chemical works. Regression equation; log), = 0-76 log x + 0-61; r = 0-90, P < 0.0001. those animals from the tailings dam or reference sites (P < 0.0001). The equilibrium concentration in tailings dam animals was significantly higher-than at the reference site (P < 0.01). A significant positive correlation was observed between the fluoride concentration in the femur and both the incisor and molar (P < 0-0001). Individual data points appear to separate out into clusters specific to the type of site sampled (Figs 8 and 9). An increase in the lesion score of both the incisor and molar was positively associated with an increase in the concentration of fluoride in the teeth of M. agrestis (Table 4). However, the severity of dental lesions was dependent not on the age of the animal, as approximated to by body weight (Table 4), but to the level of fluoride contamination in the habitat the animal came from. Thus, the significantly higher fluoride concentrations in the teeth of voles from the smelter and chemical 10000
o °q~=
~T •
g~ ~
D
vv
1000
~ c
~o 0'~
O Q
8
"/,i, "" 100 100
1000
10000
Log Femur Fluoride Concentration (mgF kg-t dry wt)
Fig. 9. Relationship between log fluoride concentration in femur and molar in M. agrestis: (W) reference site; (Q) tailings dam; (V) smelter; ([]) chemical works. Regression equation: log y = 0.95 log x -0.06; r = 0.91, P < 0.0001.
works sites corresponded to the highest lesion scores. Lower scores in the teeth of tailings dam animals were associated with lower environmental fluoride concentrations (Table 3). DISCUSSION Levels of fluoride in vegetation from the tailings dam were considerably lower than those reported some years ago (Cooke et al., 1976; Wright et al., 1978; Andrews et al., 1989). Comparisons show that there has been at least an eight-fold drop in the total fluoride concentration in live vegetation in the last decade. It has already been suggested that, since land reclamation in 1974, there has been a progressive reduction in the overall availability of fluoride in the surface layers of the tailings profile, due to a combination of leaching, organic enrichment, complexation of soil-borne fluorides and uptake by plants (Andrews et al., 1989). When the present study was undertaken, fluoride uptake by new plant growth was relatively low, in line with the minimal water solubility of fluoride in plant tissue at this site (Cooke et al., 1976). Even with hydrochloric acid to mimic extraction by stomach acids, the solubility is moderate and, by inference, so is its assimilation. Compared to the tailings dam, fluoride levels in vegetation from the two sites contaminated by atmosphericallyderived fluorides are considerably higher, as is, proportionally, the fluoride extraction by both water and hydrochloric acid. This indicates the importance of the source of contaminating fluorides, with uptake of HF gas by leaves through stomata (Davis•n, 1983), and the deposited material on leaf surfaces, contributing to the higher water- and acid-soluble vegetation fluoride at the atmospherically-contaminated sites compared to the tailings dam. These results suggest that absorption and assimilation of dietary fluoride would be significantly higher in small mammals inhabiting the atmosphericallycontaminated sites, relative to those from the tailings dam surface. This is reflected in the significantly higher tissue fluoride concentrations, and an increased severity of dental lesions, in Microtus agrestis and Clethrionomys glareolus from the smelter and chemical works. Tissue fluoride concentrations and dental lesion scores were broadly similar for both vole species at
166
I . C . Boulton, J. A. Cooke, M. S. Johnson
three of the four field sites. However, at the smelter, tissue fluoride levels were significantly higher in C. glareolus than in M. agrestis, as was the incisor lesion score. Since results from the other three sites suggest that both species appear to have broadly similar tissue fluoride concentrations and dental lesions, the differences seen at the smelter are difficult to explain. Because of its long biological half-life, fluoride accumulation in hard tissues often exhibits a non-linear relationship with age (NAS, 1971; WHO, 1984), In young animals a much higher proportion of fluoride intake from the diet is retained in bone and teeth than is the case for older individuals. Zipkin and McLure (1952) showed that fluoride accumulation in the teeth and bones of rats was comparatively rapid in juveniles, but was considerably diminished in mature animals, so that tissue fluoride concentrations were then maintained at relatively constant levels so long as the rate of dietary fluoride intake remained stable. Caution needs to be exercised in using body weight as an approximation to the age of small mammals (Morris, 1972; Thomas & Bellis, 1980), but it appears that a rapid increase in tissue fluoride concentration occurs in young, recently weaned field voles, with the attainment of equilibrium concentrations in animals that are still juvenile and immature. Of equal significance, however, is the fact that the concentration that corresponds to this equilibrium in wild animals depends on the type o f contamination at the site of origin, and upon the concentration and chemical speciation of fluoride in the vegetation. There is some evidence from the present study that extraction of vegetation with hydrochloric acid gives a better estimate of the potential to assimilate fluoride than does an analysis for total or water-soluble fluoride. In field studies, other dietary components may contribute to differences observed in animals according to the type of fluoride-contaminated habitat. Because of high levels of fluorspar (CaF2) in the substrate, tailings dam vegetation may have a calcium content high enough to significantly reduce the net absorption of fluoride from the gut (Shupe et al. 1962; Harrison et al., 1984). This would limit the proportion of fluoride available to be incorporated in teeth and bone. A possible disruption of calcium homeostasis in small mammals, caused by the uptake of large quantities of calcium during digestion of the tailings dam vegetation, could result in the suppression of the normal mineralisation o f teeth and bone and, indirectly, the fractional uptake of fluoride by the above tissues. This could be mediated by changes in the hormonal control of calcium uptake and mobilisation (Waterhouse et al., 1980). In animals from the smelter and chemical works sites, severe tooth lesions were observed as frequently at low body weights as they were at higher values. Therefore, accumulation of fluoride by the teeth to levels sufficient to cause severe damage to the enamel has already taken place in relatively young individuals. It is possible that this rapid accumulation of fluoride
by the tissues of young animals might be a consequence of newly-weaned juveniles transferring from an infant diet of maternal milk to adult foodstuffs comprising fluoride-contaminated vegetation. In mammals, the transfer of fluoride from mother to the foetus and preweaned infant, via the placenta and milk respectively, is comparatively low (Stoddard et al., 1963; Hudson et al., 1967). On weaning, the consumption of vegetation high in fluoride would be expected to produce a significant increase in its absorption from the gut, and hence uptake by the body tissues. The total fluoride concentration in vegetation and its assimilation potential determine how rapidly that increase occurs, the magnitude of the equilibrium fluoride concentration in the hard tissues, and the severity of the dental lesions produced as a consequence.
REFERENCES
Andrews, S. M., Cooke, J. A. & Johnson, M. S. (1989). Distribution of trace element pollutants in a contaminated ecosystem established on metalliferous fluorspar tailings. 3: Fluoride. Environ Pollut., 60, 165-79. Cooke, J. A., Johnson, M. S., Davison, A. W. & Bradshaw, A. D. (1976). Fluoride in plants colonizing fluorspar mine waste in the Peak District and Weardale. Environ. Pollut., 11,9 23. Davison, A. W. (1983). Uptake, translocation and accumulation of soil and airborne fluoride by vegetation. In Fluorides: Effects on Vegetation, Animals and Humans, ed. J. L. Shupe, H. B. Peterson & N. C. Leone. Paragon Press, Inc., Salt Lake City, UT, USA, pp. 61 82. Gordon, C. C. (1974). Environmental Effects of Fluoride: Glacier National Park and Vicinity. US Environmental Protection Agency, Denver, CO, USA. Harrison, J. E., Hitchman, A. J. W., Hasany, S. A., Hitchman, A. & Tam, C. S. (1984). The effect of diet calcium on fluoride toxicity in growing rats. Can. J. Physiol. Pharmacol., 62, 259 65. Hudson, J. T., Stookey, G. K. & Muhler, J. C. (1967). The placental transfer of fluoride in the guinea pig. Arch. Oral Biol., 12, 23741. Johnson, M. S. (1980). Revegetation and the development of wildlife interest on disused fluorspar tailings dams in Great Britain. Reclam. Rev., 3, 209-16. Kay, C. E., Tourangeau, P. C, & Gordon, C. C. (1975). Industrial fluorosis in wild mule and whitetail deer from western Montana. Fluoride, 8, 182 91. McBee, K. & Bickham, J. W. (1990). Mammals as bioindicators of environmental toxicity. In Current Mammalogy (Vol. 2), ed. H. H. Genoways. Plenum Press, New York, USA, pp. 37 7. Morris, P. (1972). A review of mammalian age determination methods. Mammal Rev., 2, 69-104. NAS (1971). Biologic Effects of Atmospheric Pollutants: Fluorides. National Academy of Sciences, Washington, DC, USA. NAS (1974). Effects of Fluorides in Animals. National Academy of Sciences, Washington, DC USA. NRC (1991). Animals as Sentinels of Environmental Health Hazards. National Research Council, National Academy Press, Washington, DC, USA. Oxberry, B. A. (1975). An anatomical, histochemical and autoradiographic study of the ever-growing molar dentition of Microtus with comments on the role of structure in growth and eruption. J. MorphoL, 147, 337-54. Shupe, J. L., Milner, M. L., Harris, L. E. & Greenwood, D. A. (1962). Relative effects of feeding hay atmospherically
Fluoride accumulation and toxicity in wild small m a m m a l s contaminated by fluoride residue, normal hay plus calcium fluoride, and normal hay plus sodium fluoride to dairy heifers. Am. J. Vet. Res., 23, 777-87. Shupe, J. L., Olson, A. E., & Sharma, R. P. (1972). Fluoride toxicity in domestic and wild animals. Clinical Toxicol., 5, 195 213. Shupe, J. L., Olson, A. E., Peterson, H. B. & Low, J. B. (1984). Fluoride toxicosis in wild ungulates. J. Am. Vet. Med. Assoc., 185, 1295 300. Shupe, J. L., Christofferson, P. V., Olson, A. E., Allred, E. S. & Hurst, R, L. (1987). Relationship of cheek tooth abrasion to fluoride-induced permanent incisor lesions in livestock. Am. J. Vet. Res., 48, 1498 503. Sokal, R. R. & Rohlf, F. J. (1981). Biometry. In The Principles and Practice of Statistics in Biological Research (2nd edn). Freeman, New York, USA. Stoddard, G. E., Bateman, G. Q, Harris, L. E., Shupe, J. L. & Greenwood, D. A. (1963). Effects of fluorine on dairy cattle. IV. Milk production. J. Dairy Sci., 46, 720. Talmage, S. S. & Walton, B. T. (1991). Small mammals as monitors of environmental contaminants. Rev. Environ. Contam. Toxicol., 119, 47 145.
167
Thomas, R. E. & Bellis, E. D. (1980). An eye-lens weight curve for determining age in Microtus pennsylvanieus. J. Mammol., 61, 561-3. Walton, K. C. (1987a). Fluoride in bones of small rodents living in areas with different pollution levels. Water, Air Soil Pollut., 32, 113-22. Walton, K. C. (1987b). Extraction of fluoride from soil with water, and with hydrochloric acid simulating predator gastric juices. Sci. Tot. Environ., 65, 247-56. Waterhouse, C., Taves, D. & Munzer, A. (1980). Serum inorganic fluoride, changes related to previous fluoride intake, renal function and bone resorption. Clinical Sei., 58, 145 52. WHO (1984). Fluorine and Fluorides (Environmental Health Criteria 36). World Health Organization, Geneva, Switzerland. Wright, D. A., Davison~ A. W. & Johnson, M. S. (1978). Fluoride accumulation by long-tailed field mice (Apodemus sylvaticus L.) and field voles (Microtus agrestis L.) from polluted environments. Environ. Pollut., 17, 303 10. Zipkin, I. & McLure, F. J. (1952). Deposition of fluorine in the bones and teeth of the growing rat. J. Nutrit., 84, 611-20.
F L U O R I D E A C C U M U L A T I O N A N D T O X I C I T Y IN W I L D SMALL MAMMALS I. C. Boulton, J. A. Cooke Ecology Centre, School of Environment, University of Sunderland, Sunderland, UK
& M. S. Johnson* Industrial Ecology Research Centre, Department of Environmental and Evolutionary Biology, University of Liverpool. Liverpool. UK (Received 12 November 1992; accepted 29 April 1993)
Abstract Populations of two species" of small mammal, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L.), inhabiting grasslands contaminated by industrial sources of fluoride were examined for fluoride concentrations in skeletal tissue and for morphological changes in the teeth. Concentrations of fluoride in teeth and bones were higher for C. glareolus than for M. agrestis at the chemical works and smelter sites. Severe dental lesions were recorded on the incisor and molar teeth of both species at the chemical works and smelter sites, with less marked damage at the mine tailings dam. This is attributed to inter-site differences in fluoride speciation and the consequent effects on the availability of fluoride in the diet for bioassimilation. INTRODUCTION The effects of environmental fluoride contamination on animals of economic importance to man are well documented, through studies of fluoride accumulation and its toxic effects in bones and teeth (Shupe et al., 1972, 1987; NAS, 1974). Other studies have described fluoride accumulation in wild animals such as ungulates (Kay et al., 1975; Shupe et al., 1984) and small mammals (Gordon, 1974; Wright et al., 1978; Walton, 1987a; Andrews et al., 1989); but relatively little attention has been given to the relationship between the chemical speciation of inorganic fluorides, their accumulation and consequent toxic effects. The use of small mammals as indicators of environmental contamination is important in the ecological assessment of polluted sites (McBee & Bickham, 1990; NRC, 1991; Talmage & Walton, 1991). However, such an approach needs to be based upon a sound understanding of the accumulation of the target pollutant and its toxicology. To be of practical value, specific * To whom correspondence should be addressed. Environ. Pollut. 0269-7491/94/$07.00 © 1994 Elsevier Science Limited, England. Printed in Great Britain
concentrations found in body tissues need to be related to any toxicological effects and also to variables such as diet, age and the source of pollution. This paper reports an investigation of two small mammals, the field vole (Microtus agrestis L.) and the bank vole (Clethrionomys glareolus L. ), through a comparative study of three grassland sites contaminated by industrial sources of fluoride: a mine tailings dam; an aluminium smelter, and a fluorochemical works. The fluoride levels in the hard tissues of small mammals are described in relation to morphological changes in the teeth.
MATERIALS AND METHODS Site description and sampling methods The mine waste site comprises grassland established on the revegetated surface of a fluorspar tailings dam which contains residual amounts of fuoride, lead and zinc (Johnson, 1980). The smelter site comprises plantations and rough grassland surrounding an aluminium reduction plant on the north-east coast of England. The chemical works contains an area of rough grassland within an industrial complex manufacturing hydrofluoric acid and both inorganic and organic fluorides. The latter two sites emit a combination of hydrogen fluoride gas and fluoride-laden dusts that contaminate vegetation, mainly by dry deposition. A comparable area of uncontaminated grassland in rural Northumberland was used as a reference site. Vegetation and small mammals were collected at all sites in October, 1988 and July, 1990. Vegetation was cropped at ground level and sorted into standing live and dead material plus litter. Plant material was oven dried at 60°C for 48 h before being ground without prior washing in a Cyclotech mill to pass a 500 /~m sieve. Small mammals were caught live using baited Longworth-style plastic traps and were then killed, weighed and deep-frozen prior to dissection in the laboratory. The skull, jawbones and hind limbs were
162
I . C . Boulton, J. A. Cooke, M. S. Johnson
Table 1. Summary of Scoring system for classification of fluoride-induced lesions in the teeth of small mammals a
Score
Incisor
Molar
0 [Normal]
Enamel smooth, glossy orangeyellow colour; normal shape,
White enamel, solid creamy dentine on grinding face; solid enamel 'ridge'.
1 [Questionable]
Slight deviation from normal,
Slight deviation from normal.
2 [Slight]
Faint horizontal banding of enamel; mottling; chalky spots; slight erosion,
Slight increase in erosion of grinding face; dentine darkened.
3 [Moderate]
Whitened enamel; mottling; incisor banding; erosion of tips; staining,
Increased erosion of grinding face; dentinal cavities; enamel chipped.
4 [Marked]
Enamel hypoplasia, pitting, staining; heavy erosion of cutting tips; loss of enamel colour,
Grinding surface nearly worn flat; dentinal cavities; severe staining; abnormal wear.
5 [Severe]
Lesions much worse than 4; cutting tips splayed and eroded to blunt 'stubs'; total colour loss; abnormal curvature,
Grinding surface worn flat; heavily cavitated dentine; severe staining; shrunken profile deformation of tooth roots.
with 1 cm 3 of 20% nitric acid, the washings being added to the volumetric flask whose contents were then made up to 5 cm 3. A sample (1 cm 3) of this digest was removed and 4 cm 3 of 1.5 M trisodium citrate buffer added, before the fluoride concentration was determined with an ion-selective electrode. The citrate acts to adjust the final solution pH and total ionic strength. The analytical procedure produced >95% recovery of fluoride in reference materials certified previously through interlaboratory collaborative studies: these were Assam tea and fluorosed horse bone, with certified total fluoride concentrations of 100 and 1600 mg F kg 1, respectively. Samples of dried live vegetation from all four sites were incubated at 37°C with both deionised water and 0.2 M hydrochloric acid (pH 2), the latter in an attempt to estimate the amount of fluoride present in vegetation that might be extracted and absorbed from the gastrointestinal tract through the action of stomach acids (Walton, 1987b). The extracted solutions were assayed for fluoride using a modified citrate-buffer-selective electrode technique. Statistical presentation of data
Parametric (t-tests) or non-parametric (Mann-Whitney U-test) statistical tests (Sokal & Rohlf, 1981) were carried out on the data where appropriate, and are the source of the probability values quoted in the text.
Adapted from Shupe et al. (1972). RESULTS removed and immersed in 10% papain enzyme at 60°C to enable the femur and teeth to be separated. The tissues were weighed and freeze-dried before further analysis. Examination of teeth for dental lesions Herbivorous mammals, including Microtus
agrestis,
have teeth that are adapted to the processing of fibrous and siliceous grasses prior to digestion: incisors which cut and trim grass stems and leaf blades, and molars which grind plant material to rupture fibres and cell walls (Oxberry, 1975). Excessive fluoride uptake and incorporation may result in morphological lesions that can be assessed and related to a scoring system (Shupe et al., 1972). The dental lesion scores given in the tables or text are modal values derived from an adaptation (Table 1) of the scoring system used originally to diagnose fluoride toxicosis in cattle (Shupe et al., 1972). Chemical analysis
After preparation, plant and animal materials were analysed for total fluoride using an acid extraction method adapted from the technique described by Andrews et al. (1989). Approximately 50 mg of tissue were treated with 1 cm 3 of 20% nitric acid (AnalaR) at 100°C for 1 h, in a digestion tube fitted with an air condenser in an aluminium block thermostat. After cooling, the digest was transferred to a 5 cm 3 volumetric flask and the tube and condenser rinsed twice
The total fluoride concentration in live vegetation from the tailings dam was four times higher than at the reference site. However, little of this fluoride was water soluble (7% of total) and the mean value of 5-8 mg kg (dry wt) was very similar to the equivalent of 3-6 mg kg ~ for the reference site; but 48% of the total fluoride in the railings dam vegetation was found to be soluble in HC1 (Table 2). Vegetation from the smelter site had over twice the total fluoride content of that at the tailings dam, and also showed higher percentage recoveries of both water- and HCl-extractable fluoride. Vegetation from the chemical works showed a higher total fluoride compared to the smelter site and, paradoxically, per-
Table 2. Total, water- and hydrochloric acid-extractable fluoride (mg kg-I dry wt) in composite live vegetation"
Site
n
Total
Reference
6
20 (0.9)
Tailings dam
6
80 (6.3)
15
187 (13)
6
549 (32)
Smelter Chemical works
Water
HC1
3.6 (0.3) [19] 5.8 (0.7) [7] 65 (5.4) [35] 144 (5.5) [26]
4.7 (0.2) [23] 38 (1.6) [481 111 (9.9) [59] 416 (40) [76]
" Mean values (+SE); [percentages of total fluoride extracted by method].
Fluoride accumulation and toxicity in wild small mammals
163
Table 3. Fluoride concentrations (mg kg -I dry wt) of femur, upper incisor and molar in the field vole (M. agrestis) and bank vole ( C. glareolus) ~
Site/species
n
Femur
Field vole (M. agrestis) Reference 19 291 (35) Tailings dam
19
1 169 (76)
Smelter
30
5 088 (301)
Chemical works
27
5 649 (325)
Incisor
Molar
289 (37) [0l 988 (120) [2] 2 530 (21) [31 3 466 (207) [5]
168 (18) [0] 716 (64) [1] 3 172 (351) [2] 4 060 (335) [5]
Bank vole ( C. glareolus) Reference 24 423 (21) Tailings dam Smelter Chemical works
2 24 7
210 (17) 378 (16) [0] [0] 1 556 (944) 234 (6) 641 (55) [21 [2] 8 011 (793) 4 387 (422) 6 397 (606) [41 [41 6 506 (1271) 5 271 (1080) 4 256 (994) [5] [41
Fig. 2. Smelter site: upper incisor of C. glareolus. higher in M. agrestis from the chemical works than from the smelter site, only in incisors was the difference significant (P < 0.01). The fluoride concentrations in C. glareolus from the smelter and chemical works were not significantly different (P > 0.05).
"Mean values (+SE); [modal dental lesion score].
Reference site
centage values for water- and HCl-extractable fluoride which were lower and higher, respectively, than at the smelter site. These results suggest that dietary fluoride assimilation by a consumer is in the following order:
Molars of M. agrestis from the reference site had significantly less fluoride than either the femur or incisor P < 0.01; Table 3). The fluoride concentration in the incisor of C. glareolus was significantly less than in the femur or molar (P < 0.0001). The incisors and molars of both species appeared normal.
reference < tailings dam < smelter < chemical works C o m p a r e d to the reference site, fluoride concentrations in teeth and bones were significantly higher in both Microtus agrestis and Clethrionomys glareolus from all three fluoride-contaminated sites (P < 0.0001; Table 3). The low fluoride concentrations in the tissues of animals from the reference site reflect the relatively low concentrations of dietary fluoride. Microtus agrestis from the tailings d a m had significantly lower fluoride concentrations than animals from the two atmosphericallycontaminated sites (P < 0.001), and correspondingly milder dental lesions. Although the mean fluoride concentrations for all hard tissues examined were
Tailings dam
At the tailings dam, the femur of M. agrest& contained more fluoride than the teeth, but only the concentration in the molar was significantly less than for the femur (P < 0-001; Table 3). Generally, only mild dental lesions were observed at this site: the incisors exhibited transverse banding just visible under the normal orangeyellow pigmentation (Fig. 1); the molars exhibited little change. Insufficient numbers of C. glareolus were caught to permit comparisons. Smelter
At the smelter site, femur fluoride concentrations in M.
iili~
Fig. 1. Tailing dam site: upper incisor of M. agrestis.
Fig. 3. Smelter site: molar of C. glareolus.
164
I.C. Boulton, J. A. Cooke, M. S. Johnson 10000
DO
o
Z=
0
°o Oo
v
oF
ov
o v
~v ~
v
~W
~
~'
,
[]
v
o.== =-% = E E~
v
~ •
•
1000
•
"'....."
o C.) 100 0
Fig. 4. Chemical works site: upper incisor of M. agrestis.
agrestis were significantly higher than for the teeth (P < 0.001) and, although concentrations were higher in molars compared to the incisors, the difference was not significant (P > 0.05). For C. glareolus there was significantly less fluoride in the incisors than either the femur or molar (P < O01; Table 3). In both species of vole, the incisors exhibited moderate to marked lesions, with erosion of cutting edges, colour loss, surface changes and enamel banding. The molars showed cavities (mainly in C. glareolus) and increased erosion of the grinding faces. Symptoms representative of those observed in both species are shown in Figs 2 and 3. Fluoride concentrations in the teeth of C. glareolus from the smelter site were significantly higher than in M. agrestis (P < 0.001), with higher modal lesion scores for the incisors but not molars, even though in C. glareolus the latter teeth had twice the concentration of fluoride. Chemical w o r k s At the chemical works the dental lesions recorded in both species were marked to severe, with incisors exhibiting total colour loss, scabbing and pitting of enamel, and gross erosion of the cutting tips to blunt stubs. The grinding surfaces of the molar teeth had worn flat, and cavities showed heavy staining. Symptoms representative of those observed in both species are shown in Figs 4 and 5. As with the smelter site, M.
I'0
r
j
20
30
i
j
40
Wet Body Weight (g)
Fig. 6. Relationship between body weight and log femur fluoride concentration in M. agrestis: ('k) reference site; (Q) tailings dam; (V) smelter; ([~) chemical works.
agrestis had femur fluoride levels significantly higher than in incisors or molars (P < 0.01), but the tissue fluoride concentrations in C. glareolus were not significantly different from each other (P > 0.05; Table 3). Mean fluoride concentrations in the teeth and femur of C. glareolus were higher than for M. agrestis, but these differences were not significant (P > 0-05). The lesion scores for both types of teeth were broadly similar. Analysis
of results
It appears that fluoride concentrations in both the femur and incisor do not increase with body weight in M. agrestis (Figs 6 and 7). Concentrations appear to reach an approximate equilibrium concentration which is sustained as body weight increases. There is some evidence that fluoride concentrations declined with increasing body weight at the reference site. The magnitude of the equilibrium concentration appears to be specific to the site of origin so, for the femur and incisor, this value is significantly higher in field voles from both the smelter and chemical works than in 10000
-
Q
o o~
OO O o
v v o
o
o:,, 1000
•ev
%. **% o O
100
L
0
I'0
20
L i
30
i
40
Wet Body Weight {g]
Fig. 5. Chemical works site: molar of M. agrestis.
Fig. 7. Relationship between body weight and log incisor fluoride concentration in M. agrestis: ('k) reference site; (0) tailings dam; (T) smelter; (U]) chemical works.
Fluoride accumulation and toxicity & wild small mammals 10000
-
Table 4. Changes in dental lesion score with fluoride eoncentratio# (rag kg -1 dry wt) in incisor and molar, and wet body weight (g) in the field vole (M. agrestis) •
'~ ~ ~
v
v
•''
1000
~
165
.
®~
•
,
o • eoe
,
..
Lesion score
n
Incisor fluoride
n
Molar fluoride
Body weight
0 1 2 3 4 5
2 14 29 19 19 8
291 (88) 323 (61) 1 172 (154) 2 484 (191) 3 474 (236) 4 192 (414)
15 30 20 17 8 3
295 (42) 975 (195) 2 394 (306) 4 368 (372) 5 058 (434) 6 299 (150)
12.0 (3.0) 22.0 (2.2) 19.6 (1.2) 24.4 (2.0) 19.5 (1.7) 18.3 (1.2)
o
o
a Mean values (+SE) of combined results from the four field sites: 1988 and 1990.
100 100
1000
10000
Log Femur Fluoride Concentration (mgF kg-1 dry wt)
Fig. 8. Relationship between log fluoride concentration in femur and incisor in M. agrestis: ('k) reference site; (O) tailings dam; (V) smelter; (IS]) chemical works. Regression equation; log), = 0-76 log x + 0-61; r = 0-90, P < 0.0001. those animals from the tailings dam or reference sites (P < 0.0001). The equilibrium concentration in tailings dam animals was significantly higher-than at the reference site (P < 0.01). A significant positive correlation was observed between the fluoride concentration in the femur and both the incisor and molar (P < 0-0001). Individual data points appear to separate out into clusters specific to the type of site sampled (Figs 8 and 9). An increase in the lesion score of both the incisor and molar was positively associated with an increase in the concentration of fluoride in the teeth of M. agrestis (Table 4). However, the severity of dental lesions was dependent not on the age of the animal, as approximated to by body weight (Table 4), but to the level of fluoride contamination in the habitat the animal came from. Thus, the significantly higher fluoride concentrations in the teeth of voles from the smelter and chemical 10000
o °q~=
~T •
g~ ~
D
vv
1000
~ c
~o 0'~
O Q
8
"/,i, "" 100 100
1000
10000
Log Femur Fluoride Concentration (mgF kg-t dry wt)
Fig. 9. Relationship between log fluoride concentration in femur and molar in M. agrestis: (W) reference site; (Q) tailings dam; (V) smelter; ([]) chemical works. Regression equation: log y = 0.95 log x -0.06; r = 0.91, P < 0.0001.
works sites corresponded to the highest lesion scores. Lower scores in the teeth of tailings dam animals were associated with lower environmental fluoride concentrations (Table 3). DISCUSSION Levels of fluoride in vegetation from the tailings dam were considerably lower than those reported some years ago (Cooke et al., 1976; Wright et al., 1978; Andrews et al., 1989). Comparisons show that there has been at least an eight-fold drop in the total fluoride concentration in live vegetation in the last decade. It has already been suggested that, since land reclamation in 1974, there has been a progressive reduction in the overall availability of fluoride in the surface layers of the tailings profile, due to a combination of leaching, organic enrichment, complexation of soil-borne fluorides and uptake by plants (Andrews et al., 1989). When the present study was undertaken, fluoride uptake by new plant growth was relatively low, in line with the minimal water solubility of fluoride in plant tissue at this site (Cooke et al., 1976). Even with hydrochloric acid to mimic extraction by stomach acids, the solubility is moderate and, by inference, so is its assimilation. Compared to the tailings dam, fluoride levels in vegetation from the two sites contaminated by atmosphericallyderived fluorides are considerably higher, as is, proportionally, the fluoride extraction by both water and hydrochloric acid. This indicates the importance of the source of contaminating fluorides, with uptake of HF gas by leaves through stomata (Davis•n, 1983), and the deposited material on leaf surfaces, contributing to the higher water- and acid-soluble vegetation fluoride at the atmospherically-contaminated sites compared to the tailings dam. These results suggest that absorption and assimilation of dietary fluoride would be significantly higher in small mammals inhabiting the atmosphericallycontaminated sites, relative to those from the tailings dam surface. This is reflected in the significantly higher tissue fluoride concentrations, and an increased severity of dental lesions, in Microtus agrestis and Clethrionomys glareolus from the smelter and chemical works. Tissue fluoride concentrations and dental lesion scores were broadly similar for both vole species at
166
I . C . Boulton, J. A. Cooke, M. S. Johnson
three of the four field sites. However, at the smelter, tissue fluoride levels were significantly higher in C. glareolus than in M. agrestis, as was the incisor lesion score. Since results from the other three sites suggest that both species appear to have broadly similar tissue fluoride concentrations and dental lesions, the differences seen at the smelter are difficult to explain. Because of its long biological half-life, fluoride accumulation in hard tissues often exhibits a non-linear relationship with age (NAS, 1971; WHO, 1984), In young animals a much higher proportion of fluoride intake from the diet is retained in bone and teeth than is the case for older individuals. Zipkin and McLure (1952) showed that fluoride accumulation in the teeth and bones of rats was comparatively rapid in juveniles, but was considerably diminished in mature animals, so that tissue fluoride concentrations were then maintained at relatively constant levels so long as the rate of dietary fluoride intake remained stable. Caution needs to be exercised in using body weight as an approximation to the age of small mammals (Morris, 1972; Thomas & Bellis, 1980), but it appears that a rapid increase in tissue fluoride concentration occurs in young, recently weaned field voles, with the attainment of equilibrium concentrations in animals that are still juvenile and immature. Of equal significance, however, is the fact that the concentration that corresponds to this equilibrium in wild animals depends on the type o f contamination at the site of origin, and upon the concentration and chemical speciation of fluoride in the vegetation. There is some evidence from the present study that extraction of vegetation with hydrochloric acid gives a better estimate of the potential to assimilate fluoride than does an analysis for total or water-soluble fluoride. In field studies, other dietary components may contribute to differences observed in animals according to the type of fluoride-contaminated habitat. Because of high levels of fluorspar (CaF2) in the substrate, tailings dam vegetation may have a calcium content high enough to significantly reduce the net absorption of fluoride from the gut (Shupe et al. 1962; Harrison et al., 1984). This would limit the proportion of fluoride available to be incorporated in teeth and bone. A possible disruption of calcium homeostasis in small mammals, caused by the uptake of large quantities of calcium during digestion of the tailings dam vegetation, could result in the suppression of the normal mineralisation o f teeth and bone and, indirectly, the fractional uptake of fluoride by the above tissues. This could be mediated by changes in the hormonal control of calcium uptake and mobilisation (Waterhouse et al., 1980). In animals from the smelter and chemical works sites, severe tooth lesions were observed as frequently at low body weights as they were at higher values. Therefore, accumulation of fluoride by the teeth to levels sufficient to cause severe damage to the enamel has already taken place in relatively young individuals. It is possible that this rapid accumulation of fluoride
by the tissues of young animals might be a consequence of newly-weaned juveniles transferring from an infant diet of maternal milk to adult foodstuffs comprising fluoride-contaminated vegetation. In mammals, the transfer of fluoride from mother to the foetus and preweaned infant, via the placenta and milk respectively, is comparatively low (Stoddard et al., 1963; Hudson et al., 1967). On weaning, the consumption of vegetation high in fluoride would be expected to produce a significant increase in its absorption from the gut, and hence uptake by the body tissues. The total fluoride concentration in vegetation and its assimilation potential determine how rapidly that increase occurs, the magnitude of the equilibrium fluoride concentration in the hard tissues, and the severity of the dental lesions produced as a consequence.
REFERENCES
Andrews, S. M., Cooke, J. A. & Johnson, M. S. (1989). Distribution of trace element pollutants in a contaminated ecosystem established on metalliferous fluorspar tailings. 3: Fluoride. Environ Pollut., 60, 165-79. Cooke, J. A., Johnson, M. S., Davison, A. W. & Bradshaw, A. D. (1976). Fluoride in plants colonizing fluorspar mine waste in the Peak District and Weardale. Environ. Pollut., 11,9 23. Davison, A. W. (1983). Uptake, translocation and accumulation of soil and airborne fluoride by vegetation. In Fluorides: Effects on Vegetation, Animals and Humans, ed. J. L. Shupe, H. B. Peterson & N. C. Leone. Paragon Press, Inc., Salt Lake City, UT, USA, pp. 61 82. Gordon, C. C. (1974). Environmental Effects of Fluoride: Glacier National Park and Vicinity. US Environmental Protection Agency, Denver, CO, USA. Harrison, J. E., Hitchman, A. J. W., Hasany, S. A., Hitchman, A. & Tam, C. S. (1984). The effect of diet calcium on fluoride toxicity in growing rats. Can. J. Physiol. Pharmacol., 62, 259 65. Hudson, J. T., Stookey, G. K. & Muhler, J. C. (1967). The placental transfer of fluoride in the guinea pig. Arch. Oral Biol., 12, 23741. Johnson, M. S. (1980). Revegetation and the development of wildlife interest on disused fluorspar tailings dams in Great Britain. Reclam. Rev., 3, 209-16. Kay, C. E., Tourangeau, P. C, & Gordon, C. C. (1975). Industrial fluorosis in wild mule and whitetail deer from western Montana. Fluoride, 8, 182 91. McBee, K. & Bickham, J. W. (1990). Mammals as bioindicators of environmental toxicity. In Current Mammalogy (Vol. 2), ed. H. H. Genoways. Plenum Press, New York, USA, pp. 37 7. Morris, P. (1972). A review of mammalian age determination methods. Mammal Rev., 2, 69-104. NAS (1971). Biologic Effects of Atmospheric Pollutants: Fluorides. National Academy of Sciences, Washington, DC, USA. NAS (1974). Effects of Fluorides in Animals. National Academy of Sciences, Washington, DC USA. NRC (1991). Animals as Sentinels of Environmental Health Hazards. National Research Council, National Academy Press, Washington, DC, USA. Oxberry, B. A. (1975). An anatomical, histochemical and autoradiographic study of the ever-growing molar dentition of Microtus with comments on the role of structure in growth and eruption. J. MorphoL, 147, 337-54. Shupe, J. L., Milner, M. L., Harris, L. E. & Greenwood, D. A. (1962). Relative effects of feeding hay atmospherically
Fluoride accumulation and toxicity in wild small m a m m a l s contaminated by fluoride residue, normal hay plus calcium fluoride, and normal hay plus sodium fluoride to dairy heifers. Am. J. Vet. Res., 23, 777-87. Shupe, J. L., Olson, A. E., & Sharma, R. P. (1972). Fluoride toxicity in domestic and wild animals. Clinical Toxicol., 5, 195 213. Shupe, J. L., Olson, A. E., Peterson, H. B. & Low, J. B. (1984). Fluoride toxicosis in wild ungulates. J. Am. Vet. Med. Assoc., 185, 1295 300. Shupe, J. L., Christofferson, P. V., Olson, A. E., Allred, E. S. & Hurst, R, L. (1987). Relationship of cheek tooth abrasion to fluoride-induced permanent incisor lesions in livestock. Am. J. Vet. Res., 48, 1498 503. Sokal, R. R. & Rohlf, F. J. (1981). Biometry. In The Principles and Practice of Statistics in Biological Research (2nd edn). Freeman, New York, USA. Stoddard, G. E., Bateman, G. Q, Harris, L. E., Shupe, J. L. & Greenwood, D. A. (1963). Effects of fluorine on dairy cattle. IV. Milk production. J. Dairy Sci., 46, 720. Talmage, S. S. & Walton, B. T. (1991). Small mammals as monitors of environmental contaminants. Rev. Environ. Contam. Toxicol., 119, 47 145.
167
Thomas, R. E. & Bellis, E. D. (1980). An eye-lens weight curve for determining age in Microtus pennsylvanieus. J. Mammol., 61, 561-3. Walton, K. C. (1987a). Fluoride in bones of small rodents living in areas with different pollution levels. Water, Air Soil Pollut., 32, 113-22. Walton, K. C. (1987b). Extraction of fluoride from soil with water, and with hydrochloric acid simulating predator gastric juices. Sci. Tot. Environ., 65, 247-56. Waterhouse, C., Taves, D. & Munzer, A. (1980). Serum inorganic fluoride, changes related to previous fluoride intake, renal function and bone resorption. Clinical Sei., 58, 145 52. WHO (1984). Fluorine and Fluorides (Environmental Health Criteria 36). World Health Organization, Geneva, Switzerland. Wright, D. A., Davison~ A. W. & Johnson, M. S. (1978). Fluoride accumulation by long-tailed field mice (Apodemus sylvaticus L.) and field voles (Microtus agrestis L.) from polluted environments. Environ. Pollut., 17, 303 10. Zipkin, I. & McLure, F. J. (1952). Deposition of fluorine in the bones and teeth of the growing rat. J. Nutrit., 84, 611-20.