A complete version of the report is available at www.fluoridealert.org/NRC-fluoride.htm

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2.1.2.2 Fluoride-induced Effects on Agricultural and Forest Crops

Information on fluoride-induced injury to vegetation, which is often not directly applicable to the development of criteria, has appeared in numerous reports. This information is nevertheless important for an overall concept of fluoride phytotoxicity, and is therefore briefly reviewed.

Bennett and Hill (1973) exposed 4- to 8-week-old barley and alfalfa plants to hydrogen fluoride in fumigation chambers for a single 2-hour period, and measured carbon dioxide uptake as an index of photosynthesis. With this short exposure period, 50 ppb HF were required "before clearly measurable inhibition of the net carbon dioxide rates occurred". The percentage inhibition of apparent photosynthesis was linearly related to HF concentration throughout the range from 0 to 250 ppb, with no evidence of a no-effect threshold within the accuracy of the measurements. Poovaiah and Wiebe (1973) noted that fumigation of soybean plants with low (15 to 20 ppb) concentrations of HF for 1 to 4 hours caused stomatal closing, reduced transpiration, and increased leaf temperature. McLaughlin and Barnes (1975) report that trees which had accumulated 10 to 60 ug fluoride per g dry weight of leaf tissue (from sodium fluoride in an aqueous spray) had a reduced rate of photosynthesis and an increased rate of dark respiration.

Bale and Hart (1973a, b) exposed seedling roots of barley (Hordeum vulgare) to solutions containing 1 x 10-2, 1 x 10-4 and 1 x 10-6 M (190, 1.9 and 0.019 ppm) fluoride (as NaF or HF) for 12 to 72 hours and examined dividing cells for chromosomal aberrations. They concluded that "it is clear that each of the concentrations of sodium fluoride and hydrofluoric acid used in these experiments is capable of inducing chromosomal abnormalities and of producing mitotic inhibition in the meristematic region of roots".

Pack (1971a, b) grew beans from seedling to maturity in the presence of 0.58 to 10.5 ug fluoride/m3 air, then grew a second generation, without exposure to fluoride, from the seeds of these plants. Exposure of the first generation to as little as 2.1 ug F/m3 caused the development of less vigorous second-generation seedlings. Vins and Mrkva (1973) report a decrease of 30 to 70% in the diameter growth of pine trees at pollution levels that caused no otherwise-visible injury. These authors relate the decreased growth primarily to sulfur dioxide pollution, but there is an interesting relation between increasing fluoride emissions between 1960 and 1967 (their Fig. 1) and the rapid decline in annual diameter increments (their Fig. 5) during this same period. (NOTE: See also Carlson, C.E., and Hammar, W.P. 1976. Impact of fluorides and insects on radial growth of lodgepole pine. Proc. Montana Acad. Sci. 35: 39.)

Facteau et al. (1973) reported that the growth of pollen tubes in the styles of cherry blossoms was decreased by fumigation with hydrogen fluoride either before or after pollination. Pollen tube length, expressed as a percent of style length 72 hours after fumigation, decreased linearly as a function of the product of exposure-time and atmospheric fluoride level. A somewhat similar result was obtained in studies with apricot flowers (Facteau and Rowe 1977). Fluoride-induced reduction in pollen germination and tube growth has also been observed in tomato and cucumber plants (Sulzbach and Pack 1972) while inhibited seed production or fruiting has been reported, with soybean, bell-pepper, sweet corn, and cucumber being more susceptible than pea, grain sorghum, or wheat (Pack and Sulzbach 1976).

Conover and Poole (1971) found that cuttings of Cordyline terminalis var "Baby Doll", a horticultural foliage plant, suffered serious (approaching 50%) leaf necrosis when set for rooting in water containing 0.5 ppm fluoride.

"Soft-suture" of peaches is the "best known example of fluoride injury to fruit" (NAS 1971). Facteau and Rowe (1976) were able to induce this injury in Elberta peaches by spraying the trees at weekly intervals with 0.025% ammonium fluoride solution.

Maclean et ae. (1976) conclude that hydrogen fluoride (0, 5.0 and 9.7 ug F/m3 for 7 days) was more phytotoxic to tomato plants grown in magnesium-deficient media than to those grown in complete media. Similarly, Pack and Sulzback (1976) have demonstrated how calcium nutrition can influence the response of plants to airborne gaseous fluoride. Pilet and Bejaoui (1975) report that fluoride added to the culture medium for Rubus hispidus tissues markedly reduced oxygen absorption by the tissues, particularly in media deficient in calcium and magnesium. Increased levels of calcium and magnesium had a protective action, in that they lessened the degree of fluoride inhibition of oxygen absorption.

The concept that vegetation may be stressed by pollutants present at levels that induce relatively minor injury, or even at levels that do not induce detectable injury in normal healthy plants, requires further study. The importance of this concept relates to the possible summing of stresses from various sources, with the total stress inducing an injury that cannot be easily related to any one cause. Evidence of such stress-induced injuries resulting from multiple causes is difficult to establish experimentally, but the effect of magnesium deficiency discussed above (MacLean et al. 1976) is probably a dual-stress phenomenon. However, in foliage, there can also be an in situ effect of fluoride on magnesium. In a study of air pollution, Garrec et al. (1977) observed that fluoride accumulation led to a depletion in the magnesium content of pine needles; in addition, there was a similar depletion in foliar manganese content.

Probably the most striking example of multi-stress effects is to be found in studies of the relation between atmospheric pollution and insect infestations of forest species. The host-parasite relation is complex, and as noted by Heagle (1973), the presence of an atmospheric pollutant may act to either the advantage or disadvantage of the insect.

However, studies of forest species under field conditions have demonstrated that the stress placed on trees by pollutants can increase the degree of infestation by insects. Heagle (1973) states that "A common finding is that trees injured and weakened by pollutants are more likely to be attacked by insects that normally require weakened trees for successful reproduction". Jensen (1975) notes that "Some evidence has been provided that air-pollution stress can initiate and/or aggravate insect infestation and microbial infection of woody plants". Hay (1975) states that "Insects and mites have been implicated as a stress factor on trees being influenced by pollutant emissions".

Most of the above statements have been made relative to pollutants in general, but the work of Carlson et al. (1971, 1974) and of Carlson and Hammer (1974) shows that atmospheric fluoride induces an insect-favouring stress in forest trees. Failure to recognize this stress-factor led prior investigators to incorrectly diagnose a combined fluoride injury and insect attack in the "death-band area" near Kitimat, B.C. (Gordon 1976).

When pollutants act in combination, each exerts its own stress, and each can influence a different metabolic function. Thus, the effects of exposure to sulfur dioxide and gaseous fluoride mixtures induced additive effects on citrus species, but may have induced greater-than-additive effects on Zea mays and Hordium vutgare (Reinert et al. 1975).

In apricot orchards, trees that were stressed by competition from weeds showed more leaf damage from airborne fluoride than did trees from well-tended plots (Oelschlager and Moser 1969).

Fluoride-induced stresses undoubtedly affect vegetation in diverse ways, depending on the species and conditions. One possible mode of action in coniferous trees has been noted by Bligny et al. (1973) who report that exposure to fluoride delayed the formation of epicuticular waxes on the lower surfaces of Abus alba needles. This could increase water loss from the needles, and also increase their susceptibility to invasion by parasitic organisms. Keller and Schwager (1971) have attempted to relate fluoride-induced stress to an increased activity of an enzyme (peroxidase). Yee-Meiler (1974) has shown an increased phenolic content of Norway spruce subjected to "physiologischen Schadigungen" by fluoride (0.257 mg F/dm2 per 30 days).

Fluoride concentrations expressed in ug/dm2 per day or month refer to data collected by exposing lime-filter papers to ambient air for the started time...

2.1.3 Criteria for Crop Injury

Regression equations calculated from data presented in recent papers and which indicate mathematical relations between yield and airborne fluoride; or yield and foliar fluoride; or foliar fluoride, airborne fluoride and exposure time, are presented in Table 9. The yield vs airborne fluoride data are also presented in Fig. 2. Because of the small number of data-points available, some of these regressions do not achieve statistical significance. However, taken as a group, they reveal a consistent pattern of increasingly harmful effects with increasing exposure of vegetation to fluoride.

Significant correlations between airborne fluoride levels and foliar fluoride concentrations ("C" and 'T' in Table 9) are difficult to attain under field conditions. However, if close attention is paid to the location of sampling sites and to the selection of foliage of uniform age from specific species and varieties, such correlations can be achieved. The correlation coefficient for Linzon's (1971) data (Table 9) is 0.48. In controlled greenhouse studies, the concentration of fluoride and the age of foliage are usually controllable factors; under these conditions, the time of exposure can be included as a component of the regression equation [data of McCune and Hitchcock (1971) and MacLean and Schneider (1973) in Table 9].

The data on the yield of oranqes (Leonard and Graves 1970, 1972) shown in Table 9 and Fig. 2, were for trees exposed for 28 months in field shelters with low ambient levels of fluoride pollution (i.e. 0.1 to 0.4 ug/m3). Data for beans (Pack 1971a) were from a 70-day greenhouse study at high levels of airborne fluoride. Both indicate a severe loss of yield with increasing fluoride levels, i.e. approximately 19% per 0.1 ug/m3 for oranges, and 3% per ug/m 3 (approximately 1.2 ppb) for beans. The data on strawberries (Pack 1972) are from a 5-month greenhouse study, and indicate about a 5% loss in weight of individual fruits per ug/m3 increase in airborne fluoride. This loss in yield (fruit set does not appear to have been affected) was accompanied by a statistically significant decline in fruit quality, as indicated by the "development rating" assigned by the original author.

Yield of oranges in field shelters was also related to the fluoride content of the foliage (Leonard and Graves 1972), declining by about 5% for each 100 ppm increase in fluoride in 10-month-old leaves.

In field tests, Israel (1974b) observed a highly significant multiple regression between the fluoride content of forage and of air-plus-soil. This was expressed as:

F(foliage) = 11.4 F(air) + 0.0085 F(soil),

where fluoride content of foliage and soil is expressed in ppm and of air in ug/dm2 per day absorbed by lime-paper.

In a recent survey of a Newfoundland region in Canada, Sidhu (1977a) concluded that "The safe levels of fluoride in air for forest species appear to be between 0.17 to 0.23 ug/m3." This is very close to the lower limit given by the estimates of Marier and Rose (1971), i.e. "The average gaseous fluoride level in ambient air should be below 0.4 ug/m3 and might have to be as low as 0.2 ug/m3."

2.2 EFFECTS ON ANIMALS

2.2.1 Aquatic Species

The ecological significance of the exposure of aquatic animals to fluoride has been studied to a limited extent, but much more research is required before broad conclusions can be drawn. The following paragraphs summarize the available recent information.

A large number of species have been shown to suffer injury from exposure to fluoride (Groth 1975a). The response of fish to fluoride is influenced by a number of factors such as species and strain, concentration of calcium and chloride in the water, temperature, and the size or age of fish used in the study (Sigler and Neuhold 1972). The response of other species to fluoride is probably influenced by at least some of these factors, but few data are available.

Fish and other aquatic species tend to accumulate fluoride from the environment, primarily in the skeleton (including the gills) and exoskeleton. Groth (1975a) has tabulated the accumulation of fluoride by a number of species. Stewart et al. (1974) analyzed specimens from an uncontaminated estuarine-coastal area of New Zealand. They report fluoride levels from 509 to 2885 ppm (ash basis) in the skeleton, and from 31 to 209 ppm in the exoskeletons, of different species. Wright and Davison (1975) also report "background" fluoride levels for a number of species, but give the data only as ug/g fresh weight. These authors also report data from controlled experiments that clearly demonstrate accumulation of fluoride in the exoskeleton of shore crab (Carcinus maenas ). Blue crab (Callinectus sapiduz), exposed to 20 ppm fluoride in water, accumulated fluoride in the exoskeleton and suffered a 4.5% reduction in growth increment per molt (Moore 1971). Moore estimates that this would result in a 52% reduction in the final size of an average crab.

Wright (1977) reported a whole-body fluoride concentration of 10 ppm, wet weight basis, in fry of Brown trout exposed to 5 ppm fluoride in tap water for 200 hours. These fry suffered increased mortality, as compared to fry in a control tank, but mortality appeared to occur only in a susceptible portion of the total population.

Hemens and Warwick (1972) and Hemens, et al. (1975) studied the potential environmental effects of the "scrub water" from an aluminum smelter in South Africa. Brown mussels (Perma perma) were the most sensitive of the organisms tested. In this species, mortality occurred at fluoride levels from 1.4 to 7.2 mg/l in sea water after exposure for 15 days, but lack of food during the test-period may have enhanced toxicity. All species tested had accumulated fluoride extensively (wholebody fluoride to water-borne external fluoride ratios varied from 25:1 to 149:1) after exposure for 72 days at 52 ppm fluoride. The authors interpret some of their data as being indicative of a greater fluoride accumulation during deposition of new skeletal material.

Lubinski and Sparks (1975) attempted to assess the total toxicity to Bluegills of several pollutants present in the Illinois river by expressing the contribution of each pollutant as "Bluegill toxicity units". They found that fluoride was one of six major contributors to the total toxicity of the river water. Taft and Martin (1974) have reported the absence of all living organisms in a fluoride-polluted zone of Tampa Bay.

2.2.2 Insects

Data on the effects of exposure of insects to fluoride are limited. Lillie (1970) has reviewed the literature on the toxicity of fluoride to honeybees and concluded that 4 to 5 ug of accumulated fluoride per bee may be the lethal level. Assuming an average dry weight per bee of 30 m , this corresponds to 130 to 170 ppm (dry weight). Trautwein et al. (1972) reported that the average total-body fluoride content of winter-killed bees ranged from 0.63 to 4.81 ug per bee (21 to 160 ppm dry weight basis). The highest levels were found in bees from hives located near sources of fluoride pollution.

Carlson and Dewey (1971) report data from the analysis of a number of insect species captured in non-polluted and polluted areas of Montana (Table 10). All insects were presumably collected live, but the honeybees with an average fluoride content of 221 ppm probably would not overwinter successfully. Other insects, such as bumblebees at 406 ppm and sphinx moth at 394 ppm fluoride, must be considered endangered, even in the absence of further evidence.

Mohamed (1971) reported evidence that exposure to fluoride caused chromosome damage and mutagenesis in fruit flies (Drosophila metanogaster). In a continuation of these studies, Gerdes et al. (1971a, exposed four strains of fruit flies at airborne gaseous HF levels of 0, 1.3, 2.9, 4.2 and 5.5 ppm for periods of up to 6 weeks. All flies were killed within 3 days at 5.5 ppm. All strains suffered at least 25% mortality in 6 weeks, even at the lowest level (1.3 ppm) of exposure, but the relation between mortality and fluoride concentration was non-linear, especially for the two "wild type" strains. In these two strains, about 65% of the population appeared to be resistant to fluoride, even at the 4.2 ppm level.

The offspring of the surviving flies from the 0, 1.3, and 2.9 ppm fluoride levels of the above experiment were also studied (Gerdes et 1971b). Statistically significant declines in fecundity and egg hatchability with increasing parental exposures were observed. Gerdes et al. concluded that the exposure to fluoride caused genetic damage. The dose-response plots for vegetation (Section 2.l.3) and swine (see Section 2.2.4) do not appear to indicate the existence of a no-effect threshold for fluoride. If the genetic effects in insects respond to dose in a similar manner, the cumulative genetic, evolutionary, and ecological effects of exposure to low levels of environmental fluoride could become manifest with continued exposure of successive generations.

Stewart et al. (1974) have provided data on the "background" fluoride levels in bones of various species in New Zealand, Tibia or entire skeletons were analyzed; data are reported as ppm in bone ash. (NOTE: Bones contain 50 to 70% ash. Thus a rough conversion of "ppm, ash" to "ppm, dry fat-free" can be made by multiplying the former by 0.6.) The mean value for two opossums was 247 ppm, and that for a single rabbit was 184 ppm fluoride. These values thus agree with those reported for Montana.

When compared with these background levels, bone fluoride concentrations ranging up to and above 5000 ppm dry fat-free basis, (Kay et al. 1975b; Newman and Yu 1976; Harris 1974) are clearly indicative of environmental contamination by fluoride and its ingestion by wild animals. Gordon (1970a) recorded extreme values of 12,700 ppm fluoride (ash basis) in the femur of Mus musculus, and of 16,000 ppm in a rabbit femur. A relation between bone fluoride levels in small rodents and the distance from a fluoride source has been demonstrated (Gordon 1970a).

The data currently available are not sufficient to indicate the environmental significance of fluoride pollution for wildlife, but there are indications of serious effects. Lameness induced by fluorosis has been observed in wild ungulates by Kay et al. (1975b), who note that it appeared to be more severe than the lameness observed in cattle at similar bone fluoride levels. In a predator-prey situation, even a minor loss of mobility can lead to rapid elimination of the individual affected. An apparent population age-shift was also observed (Kay et al. 1975b), and this "suggests that fluorosis was so severe that older, most susceptible, deer had been removed from that (the Teakettle mountain) herd".

In view of the data discussed above, we feel obligated to disagree with the statement made by Suttie (1977), to the effect that "There seems to be no real basis for assuming that these animals (wildlife) are any more susceptible to the adverse effects of fluoride ingestion than other herbivores, and it is generally felt that if the most sensitive domestic species, cattle, are protected the area will be safe for wildlife". In Section 2.2.4 on "Livestock", we discuss a number of factors that influence the severity of skeletal fluorosis. In a comparison of domestic to wild animals, nearly all of these factors [e.g. nutritional status (particularly in winter), physical exertion, variability of fluoride exposure-level, age when exposure begins, degree of individual variability, etc.] can be unfavorable to the wild animal. Suttie's statement ignores all of these factors, and also ignores the increased vulnerability that even mild fluorosis can create in a predator-prey situation. Kay et al. (1975b) observed that, for a given level of fluoride in the bones, deer appear to suffer more severe lameness than cattle. This confirms that the factors listed above do influence the severity of fluorosis, and also increase the wild animals' susceptibility to fluoride toxicity.

Data on wild birds are very limited. Stewart et al. (1974) and Kay et al. (1975a) have reported some background data (Table 12). In general, short-lived, seed-eating birds had lower bone fluoride levels than the longer-lived omnivorous species.

The mobility of birds largely precludes sampling of individuals who have remained in a fluoride contaminated area for long periods. However, the high "background" fluoride levels observed in some members of the omnivorous species suggests that there may be some danger of developing skeletal fluorosis. [Note: Fluoride levels exceeding 4,500 to 5,500 ppm, dry fat-free basis in the long bones, are considered indicative of marginal fluorosis in cattle (NAS 1971)].

House martins (Dilichon virbica) may be sensitive to fluoride, as few nests were found in heavily polluted areas (Newman 1977).

2.2.4 Livestock

Aschbacher (1973) has stated that "Of all airborne pollutants which may affect farm animals, fluorine has caused the most serious and widespread damage". Research on fluorosis in livestock has been extensive and a number of reviews have been published (Shupe 1970; Obel 1971; Shupe et al. 1972; Trautwein et al. 1972; NAS 1974; Fleischer et al 1974; Suttie 1977).

In brief, studies on skeletal fluorosis in livestock have led to the following conclusions:

(1) Fluorosis results from chronic ingestion of fluoride at levels above those usually arising from natural sources over a prolonged period; thus, it is more commonly observed in older animals.

(2) If exposure occurs during the period of tooth formation, tooth damage may occur. This can increase tooth wear and contribute to a decline in the nutritional status and well-being of the animal.

(3) In severe cases, animals become intermittently or permanently lame, and bone exostoses become radiologically or even visually apparent, especially near the leg joints.

(4) The severity of the fluorosis is influenced by a number of factors in addition to total fluoride intake and duration of exposure. Absorption of ingested fluoride is influenced by the chemical form and solubility of the fluoride and by other components of the diet (e.g. calcium, aluminum, etc., NAS 1974). Fluoride toxicity is enhanced by a low nutritional status of the animal (Suttie and Faltin 1973). The schedule of exposure also influences fluoride toxicity, with alternating periods of high and low exposure being more harmful than uniform exposures (Suttie et at. 1972). Physical activity also tends to increase the severity of bone lesions caused by excessive fluoride (Shupe and Olson 1971; Shupe et at. 1972).

The age of the animal when exposure begins also affects the development of fluorosis. This is especially important as regards dental effects caused by exposure during the period of tooth formation, although age also influences the receptivity (i.e. affinity) of bone for fluoride. Evidence for a declining rate of fluoride accumulation with age does not seem to have been presented for large domestic species, but has been shown with rats, rabbits, and dogs (WHO 1970; NAS 1971). Because bone lesions appear to be related to bone fluoride levels (NAS 1971), exposure to fluoride from weaning onward may be more harmful than exposures later in life.

(5) There are distinct differences among domestic species in their tolerance to fluoride. Cattle seem to be the most sensitive of the common North American domestic species, whereas swine are less sensitive and poultry are comparatively resistant.

Although there is general agreement on the above points throughout the industrialized countries, there is a diversity of opinion as to the levels of fluoride that can be permitted in animal forages and feeds. There are a number of reasons for this diversity, not the least of which has been the emphasis placed on osteosclerosis by many researchers in the field, and the difficulty of quantitatively expressing the degree of osteofluorotic injury. Particular attention should be given to the more insidious forms of osteofluorosis, such as the marked arthritic changes observed in dairy cattle fed fluoride-contaminated phosphate supplements (Griffith-Jones 1977).

Three indices of fluoride exposure have been proposed for use with livestock. The most widely accepted index in the U.S. is the fluoride content of fodder (and of feed supplements); however the fluoride content of bone is a more useful diagnostic index, and the fluoride content of urine may also have some diagnostic value.

Suttie (1969a) has proposed that standards for the fluoride content of forage should be set at:

not over 40 ppm, dry weight basis, as a yearly average;
not over 60 ppm, for more than 2 consecutive months;
not over 80 ppm, for more than one month.

Various U.S. State regulations make it unlawful for an industry to emit fluoride at a level that will cause the fluoride content of locally-grown forage to exceed 30 (Kay 1971) or 40 (Gordon and Tourangeau 1977) ppm, dry weight basis. These levels have been selected largely on the basis of data obtained in controlled animal studies (Suttie 1969a) which are not always relevant to actual farm conditions.

In controlled tests, conditions are selected to minimize the effects of many of the factors, discussed on p. 46, that are known to influence the severity of fluorosis. For example, the exposure level is kept constant or varied on a simple controlled schedule; animals of uniform age are selected; adequate nutrition is provided; physical exertion is restricted; and individual responses are largely eliminated by randomization and averaging. Obviously, the severity of fluorosis observed in such tests will be less than those to be expected in some individual animals in a range herd. In general, it can be concluded that the toxicity of a substance having a crippling effect will be underestimated by studies done on penned animals (i.e., those having restricted mobility) rather than on grazing animals whose nutritional needs cannot be met without mobility.

Bourbon et al. (1971) and Gordon and Tourangeau (1977) have suggested a single standard of 20 ppm fluoride, air-dried basis, for all fodder. However, it must be noted that soils and fertilizers also contribute to the fluoride content of fodders. Suttie (1969b) reported that "some rather high fluoride forages (112 ppm) can be found in areas with no known source of industrial fluorides ..." Thus, regulations that attempt to control the level of fluoride in fodders by restricting airborne industrial emissions may prove inadequate.

Standards controlling the fluoride content of fodders also fail to provide protection against high fluoride levels in mineral supplements and other types of feed (Suttie 1969b; Marier 1971; Obel 197 Griffith-Jones 1977; Hillman 1977).

The fluoride content of bone has also been suggested as a quantitative index of exposure to fluoride (NAS 1971). For monitoring of live animals, this would require an inconvenient biopsy; but, in the case of farm herds, post-mortem samples from slaughtered animals are often available. Results of feeding trials at the University of Wisconsin (summarized in NAS 1971) indicated that bone fluoride levels in "the range of 4,500 to 5,500 ppm (dry fat-free basis in long bones such as metacarpal or metatarsal) might be considered as the marginal zone of toxicosis, and that lower concentrations were not indicative of damage". This conclusion, however, appears to be specific to the experimental conditions used. Another NAS (1974) report has stated:

"Cancellous bone such as the frontal ribs, vertebrae, and those of the pelvis, have a higher fluoride content than the more compact metatarsal and metacarpal bones ... There is also a marked variation in the fluoride content of such different anatomical areas (within) bone (such) as the metatarsal or metacarpal; the diaphyseal portion has a lower fluoride content than the metaphyseal portion"

Obel and Erne (1971) observed serious fluorosis in calves with 500 to 2,400 ppm, and in cows with 900 to 2,800 ppm fluoride in metacarpal bone ash (assuming 60% ash, these figures correspond to 300, 1,440, 540 and 1,680 ppm, dry fat-free basis, respectively). Obel and Erne suggest that a phosphate deficiency may have contributed to the severity of fluorosis in some of the cattle examined. Zumpt (1975) observed fluorosis in sheep at femur bone fluoride levels of 2,400 to 3,200 ppm dry fat-free basis.

The fluoride content of urine has also been suggested (Burns 1970) as an index of fluoride ingestion by cattle. This index might be advantageous because of the ease of sample collection, but the relation between fluoride intake and urinary fluoride is not well established.

Although Burns (1970) reports a reasonably close relation between urinary fluoride and the fluoride concentration in samples from the pasture vegetation, Huber and Schurch (1970) report much less agreement. Israel (1974b) reports a correlation coefficient of 0.87 between annual average urinary fluoride levels from cattle and annual average feed and forage samples. Annual averages of urinary fluoride were based on 3 samples per year from each of 10 to 13 animals per herd. Thus, under practical conditions, it appears that extensive sampling is required for urine analyses to provide a reliable indication of fluoride ingestion. Burns (1970) suggests that 10 ppm would be "a suitable figure to use as a threshold level" for urinary fluoride. Based on the equation given by Israel (1974b), this would correspond to a fluoride content in the fodder of less than 20 ppm.

There are continuing difficulties in answering the question of whether or not fluorine is an essential element of diet (NAS 1974). The criteria for essentiality and the difficulties of proving it by animal experimentation have been discussed (Underwood 1962; NAS 1971). One of the greatest difficulties is that practically every natural water supply and foodstuff contains traces of fluoride and it is almost impossible to prepare fluorine-free (e.g. < 0.005 ppm) control diets adequate in other respects (NAS 1974). In view of the conflicting results and conclusions from experiments with mice (Underwood 1977) it is not yet possible to assign an essential role to traces of dietary fluoride.

We have found only one set of research data from which a mathematical relation between fluoride intake and the response of a livestock species can be calculated. Forsyth et al. (1972c) fed diets containing 0, 30, 150 and 450 ppm fluoride, as sodium fluoride, to young swine, and recorded average daily weight gains for up to 18 weeks. No data are given on the fluoride content of the basal diet. The data (reproduced in Fig. 3) indicate a linear decline in growth rate with increasing dietary fluoride. In the 18-week experiment, the values at 150 and 450 ppm fluoride are significantly (p < 0.01) different from the control values. The regression equations (our calculations) indicate a loss of about 4% in the average daily weight gain, over the 18-week period, for each 100 ppm increment in dietary fluoride. Said et al. (1974) have reported that "retarded liveweight gain was the first significant sign of fluorosis" in a 25-month study of Wether sheep fed from 53 to 70 ppm fluoride in the total ration.

In the absence of additional quantitative criteria, and in view of the fact that the indices of fluorosis discussed above (i.e. bone and urine fluoride concentrations) do not appear to be satisfactory relative to actual farm experience, and do not appear to give adequate protection to wild species, the present authors cannot suggest criteria that would be of use in setting or revising Canadian standards for exposure of animals to fluoride. However, two suggestions can be made.

1. It should be emphasized that total fluoride intake is the only reliable index of chronic exposure for fluoride. The use of maximum "safe-levels" of fluoride in fodders is based on the assumption that intake of fluoride from all other sources, including water, will be low and relatively constant. Reported instances in which fluoride from sources other than fodder detrimentally affected the health of animals (Obel 1971; Griffith-Jones 1972, 1977; Parsonson et al. 1975; Hillman 1977) testify to the need to assess the contribution from all sources. Oelschlager (1974) has stated that "there appears to be a lack of full appreciation of the extraordinary amounts of fluoride which reach the feed rations through mineral supplement mixtures..."

2. Further research aimed at developing criteria relating fluoride intake, preferably in mg/kg body weight per day, to tissue fluoride contents and injury should be stressed. This research should include studies on animals at less-than-optimum nutritional status. Attention should be paid to the less obvious effects of fluoride, such as the reduced growth of swine (and sheep) discussed above, rather than to osteofluorosis. Blood plasma F- should be assessed, as a possible indicator of fluoride exposure and the likelihood of fluoride intoxication. Nutritional factors are of extreme importance in chronic fluoride intake (see Section 5.6). Chronic dietary deficiencies can aggravate the effects of a given fluoride dosage, and such factors should be considered in the assessment of dose:response interrelations.

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