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Relationship between fluoride content in bones and the age in European elk (Alces alces L.)
Camp. Bio~hem. Ph,wiol. Vol. Copyright
Pergamon
Printed
I I I C. No. I. pp. I 17-120. 1995 ‘t’ 1995 Elsevier Saence Ltd
m Great
Britain.
All
0742.84
13195 $9.50 + 0.00
rights
reserved
0742-8413(95)00012-7
Relationship between fluoride content in bones and the age in European elk (Alces alces L.) Z. Machoy, E. Dabkowska, and Z. Gqbczynska
D. Samujlo,
T. Ogofiski, J. Raczyriski
Department of Biochemistry, Pomeranian Medical Academy, al. Powstancow 72. 70-l 11 Szczecin, Poland; and Department of Biology, Warsaw University Bialystok, ul. Swierkowa 20 B, 15-950 Bialystok, Poland
Wielkopolskich Branch
The relation between fluoride content in bones (jaws) and the age of elks living in Poland in areas of relatively low pollution by industrial emissions was studied. Multiple regression analysis was performed by making use of 11 mathematical models. The relationship between fluoride content in bones and the animals age is best described by square root and linear models. Key words: Air fluoride; Animal Lower jaw; Mineral composition. Camp. Biochem.
Physiol.
age; Bone; Environment;
European
elk; Fluoride
accumulation;
11 IC, I1 7-120, 1995.
Introduction These investigations concerned both man and animals. The rectilinear dependence was elaborated by Mohamedally (1984) on material comprising almost 300 individuals having died in Great Britain. Other researchers conducted similar investigations on bones of human foetuses (after abortions, accidents) but they failed to find any rectilinear dependence of fluoride content on the age (Mokrzynski 1992). More attention was devoted to animal bones due to the fact that the material was easily accessible. The exact determination of the age, particularly in animals living at large (deer) is possible on the basis that the age of a deer may be defined by taking into account the development and attrition degree of the mandibular dentition. In very young individuals it is of help to assess the pace of growth and time at which the deciduous teeth change for permanent ones. In older animals the age is indicated by the degree of teeth attrition. These changes are known to occur on the friction face of premolars and molars (Lochman, 1987; Nowicka, 1992; Janczewski, 1984). Finally, at the estimation of fluorine content in bones, the studies involving man and animals as a whole must cope with an additional problem, namely the state of pollution of the natural
The expansion of industrialization in the world is accompanied by an increase of pollutants in the natural environment. One example is fluorine. When appearing as a microelement in food and water, it plays a positive role, since it exerts a favourable influence on the increased crystallization of hydroxyapatite in bones and prevents dental caries. However, overoptimal amounts of fluorine have a negative effect on physicochemical properties of the bones in humans and animals. Then the bone becomes fragile, hard and breakable, which enhances the development of skeletal fluorosis. The question has arisen as to what is the physiological dependence between fluoride accumulation in bones persisting throughout one’s life and the subject’s age. Fluorine accumulation in living organisms has been known for a long time now, and many researchers had reported much earlier that a rectilinear dependence existed between the age and the bound fluoride in the bones (Dabkowska 1987; Mohamedally 1984; Wix and Mohamedally, 1980). 10: A. Machoy, Department of Biochemistry, Pomeranian Medical Academy, al. PowstaGw Wielkopolskich 72, 70-l 1I. Szczecin, Poland. Received 28 February 1994; revised 14 July 1994; accepted 20 December 1994. Correspondence
117
118
Z. Machoy
environment. A lot of industrial emissions stemming from the production of chemical fertilizers, aluminum works and even from burning low quality coal for household requirements, contain fluorine compounds (Machoy et al., 1991; Newman and Murphy 1979). Keeping the above findings in mind we became interested in fallow game inhabiting areas which are relatively uncontaminated by industrial emissions. For this purpose the European elk was chosen. The European elk (Alces &es L.) is an animal that migrates and whose habitat is the north-eastern regions of Poland. The regions are not polluted by industrial emissions, being mainly covered by large forests within the boundaries of the Bialowieia Primeval Forest, a part of which forms the National Park (Pawlus et al., 1993). The mentioned regions are under legal protection. The elk in Poland, apart from belonging to the group of animals that are encountered not too frequently, is also under species protection. They are known to live in larger numbers in Scandinavia, Russia and Canada. That may be the reason why in this country their population is far from numerous. The study material comprised the jaws of the periodically shot elks originating from areas not polluted by industrial emissions. The actual paper is addressed to investigators dealing with the accumulation of elements in bones, particularly that of fluorine, and it also points to the possibility of mathematically determining the relationship between the content of accumulated component and the individual’s age.
Material and Methods The study material at our disposal consisted of 63 jaws of European elks (Alces alces L.). The age of the animals varied from I to 13 years. Mandibular bones, taken from animals having been killed during hunting, were deflated in acetone. The mandibular bones were bored with a dental drilling machine 3 cm below incisors, 1-2 mm deep. The material to be analyzed was collected from the cortical part of mandibular bone, for it is known that fluoride content in trabecular parts differs from that in cortical ones (Czerwinski et al., 1981). The pulverized material, in amount of 100 mg was desiccated at the temperature of 60°C. In consequence it lost about 1% of mass determined at room temperature. The error at defining fluoride in bones did not exceed 5%. That was obtained in a series of experiments involving 100 mg samples of pulverized mandibular bone. Next, the samples were shaken up for 2 min, placed in plastic laboratory tubes and kept until analysis
et al.
(Dabkowska, 1987; Nowicka, 1992; Samujlo 1994). After dissolution of 10 mg of pulverized bone in perchloric acid, followed by neutralization with a TISAB (Total Ionic Strength Adjustment Buffer), fluoride content was determined by means of a selective fluoride electrode (Radelkis Co., Hungary) (Dabkowska, 1987). The results were evaluated statistically. Pearson’s correlation coefficient was used for studying the correlation between the features. The significance level of 0.01 was implemented to serve as a criterion for approving or discarding hypotheses for drawing the conclusion, relying on statistical analysis. et al.,
Results The established results pertaining to the fluorine content in the mandibular bones in the European elk (Alces &es L.) together with short descriptive statistics are depicted in Table 1. The obtained results were subjected to linear regression analysis with the use of 11 mathematical models described by the following formulas Y = A + B/l’ y=Jp y zz x3 Y = x4 y = .,p Y = sqrt(X) Y = m(X) Y = log(X) Y=e’ Y= 10‘ Y=l/X
(linear) (X-square) (X-cubed) (X to the fourth power) (X to the fifth power) (square root of X) (natural log of X) (log base 10 of X) (Euler [e] = 2.71 . . . to the power of X) (10 to the power of X) (inverse of X),
whereas X is the independent variable (age) and Y is the dependent variable (fluoride content). In our studies the relationship is described best by the linear model (Fig. 1) and the square root model (Fig. 2).
Discussion The diet of the European elk in north-eastern Poland is variable, ranging from grass, herbs, ferns, lichens, mushrooms and even algae to twigs and shrubs (Pawlus et al., 1993)Taking the above into account we have disclosed that the game from the regions where the air is relatively low polluted or free of fluorine compounds, would be appropriate for finding out whether or not the fluoride accumulation in bones depends on the age and if the dependence tends to be rectilinear. The age of animals is determined on the basis of the teeth attrition degree with the exactitude up to 1 year. As evident from our previous investigations,
Relationship
between
y = 46.356 500
fluoride
content
in bones and age in elks
119
Multiple linear regression + 28.428*x R-square = 0.77665865
r
AGE (years) Fig. I. Regression
analysis
various types of bones belonging to the same skeleton reveal diverse fluorine content (Machoy, 1991). Fluorine accumulation in bones goes into interaction of hydroxyapatite. The hydroxyapatite hydroxyl groups are substituted by fluorine to fluoroapatite forms. Fluorine also displays a strong affinity towards such elements as calcium, magnesium and phosphorus. The fluoride content rises with the animal’s age (Kay et al., 1976, Dgbkowska 1987; Dabkowska and Machoy 1989; Machoy 1990). Our task consisted of ascertaining whether the dependence was rectilinear or not. For this purpose, analysis of regression was performed by resorting to the 11 mathematical models. In our studies the revealed dependence of fluoride content in bones on the animals age was described best by square root and linear models, for which Rsquared is equal to 0.78965277 and 0.77665865, respectively. Hence, over 77% of our results
y = (-58.821
linear model
arranged themselves in accordance with the above straight lines. Quite an interesting analysis of fluoride dependence on age, based on the value of the r correlation coefficient was presented by Kierdorf et al. (1989). Kierdorf et al. (1989) furnished Y = 0.583 with a significance level of < 0.001. In our studies, Pearson’s r correlation coefficient between age and the fluorine content of the jaw yielded 0.881 with the significance level being P < 0.001. We did not specify the animal’s sex, but in agreement with other authors this omission is almost meaningless (Kierdorf et al., 1989). We had at our disposal rather a small quantity of bones from older individuals, over 7 years old. Just then, it would not be possible to verify if the old individuals really do accumulate in their bones proportionally less fluoride than the younger ones, as reported by Kierdorf et al.
Multiple fixed-nonlinear I) + 119,9405*sqrt(x)
regression R-square = 0.78965227
5000 0
400 -
6
8
AGE (years) Fig. 2. Regression
analysis
square
root model
Z. Mach oy et ul.
120
(1989). Therefore, an extension this respect is recommended.
of the studies in
Czerwinski E., Skolarczyk A., Klewska A., Kaifosz J. and Szewczyk S. (198 I) Fluoride concentration in cortex and in full thickness samples of the iliac crest. Fluoride 14, I, 10-13.
Dabkowska E. (1987) An attempt to evaluate the industrial emission hazard to game on the basis of mineral composition changes in the masticatory system of the deer from Western Pomerania region (Polish). M.D. thesis, Pomeranian Medical Academy, Szczecin. Dabkowska E. and Machoy Z. (1989) Effects of fluortde pollution on calcium and magnesium content of mandibles (lower jaws) of wild game. Fluoride 22, 29-32. Dilbkowska E., Machoy-Mokrzynska A., Straszko J., Machoy Z. and Samujio D. Temporal changes of fluoride levels in jaws of European deer in industrial regions of Western Pomerania. Poland. Emir. Grochem. Hllh. (submitted). Janczewski Z. (1981) Deer.s Antbrs. PWRiL Warszawa. Kay E., Tourengean P. C. and Gordon C. C. (1976) Populational variation of fluoride parameters in wild ungulates from the Western United States. Fluoride 2, 73-90.
Kierdorf U.. Kierdorf H., Erdelen M. and Korsch J. P. (1989) Mandibular fluoride concentration and its relation to age in roe deer (Capreolus capreolus L.). Camp. Biothem.
Phwiol.
94A.
783-785.
-J. (1987) ‘Animal
Pmisrwowe
Wyduttxictwo
age determination Rolnicze
Z. (1990) Fluoride (Polish). Zesz~t~
Wroclaw
References
Lochmann
Machoy man
i LeSnr.
255.
(Polish).
Warsxwa-Krakbw
and its effects on animals Naukowr PAN, 2, 57 -75.
and
Ossolineum
Machoy Z. (1991) Are the fluoride content and the phvsical load-of bones in man related? Fluoride 24, 3, 160.rl02. Machov Z., Dabkowska E. and Nowicka W. (1991) Increased fluoride content in mandibular bones of deer living in industrialised regions of Poland. Enrir. Geochem. HI/h
13(3),
161 163.
Mohamedally S. M. (1984) Studies of the relative fluoride content of normal and pathologicallv mineralised human tissues. Fluoride 17, 246-251. . Mokrzvriski S. (1992) Mineral content analvsis of human fetal- bone (Polishj. MD thesis, Pomeranian Medical Academy, Szczecin. Newman J. R. and Murphy J. J. (1979) Effects of industrial fluoride on black-tailed deer (preliminary report). Fluoride 12, 1299135. Nowicka W. (1992) Evaluation of the course of processes in formation of bones and antlers of fallow deer on the basis of mineral composition analysis. MD thesis, Pomeranian Medical Academy, Szczecin. Pawlus G.. Machoy Z., Tustanowski S., Raczytiski J and Gebczytiska Z. (1993) Damage to the Charnobyl atomic power station had no influence on the emission quantity of beta and gamma radiation determined of elk living in the north-east region of Poland. Polish. Bromar. Chem. Taxvkol.
26, 77-79.
Samujio D., Machoy-Mokrzyriska A, Dqbkowska E., Nowicka W. and Paterkowski W. (1994) Fluoride accumulation in European deer antiers. ‘Em+. Sci. 2, 1899194. Wix P. and Mohamedally S. M. (1980) The significance of age-dependent fluoride accumulation in bone in relation to daily intake of fluoride. Fluoride 13, 100~104.
Pergamon
Printed
I I I C. No. I. pp. I 17-120. 1995 ‘t’ 1995 Elsevier Saence Ltd
m Great
Britain.
All
0742.84
13195 $9.50 + 0.00
rights
reserved
0742-8413(95)00012-7
Relationship between fluoride content in bones and the age in European elk (Alces alces L.) Z. Machoy, E. Dabkowska, and Z. Gqbczynska
D. Samujlo,
T. Ogofiski, J. Raczyriski
Department of Biochemistry, Pomeranian Medical Academy, al. Powstancow 72. 70-l 11 Szczecin, Poland; and Department of Biology, Warsaw University Bialystok, ul. Swierkowa 20 B, 15-950 Bialystok, Poland
Wielkopolskich Branch
The relation between fluoride content in bones (jaws) and the age of elks living in Poland in areas of relatively low pollution by industrial emissions was studied. Multiple regression analysis was performed by making use of 11 mathematical models. The relationship between fluoride content in bones and the animals age is best described by square root and linear models. Key words: Air fluoride; Animal Lower jaw; Mineral composition. Camp. Biochem.
Physiol.
age; Bone; Environment;
European
elk; Fluoride
accumulation;
11 IC, I1 7-120, 1995.
Introduction These investigations concerned both man and animals. The rectilinear dependence was elaborated by Mohamedally (1984) on material comprising almost 300 individuals having died in Great Britain. Other researchers conducted similar investigations on bones of human foetuses (after abortions, accidents) but they failed to find any rectilinear dependence of fluoride content on the age (Mokrzynski 1992). More attention was devoted to animal bones due to the fact that the material was easily accessible. The exact determination of the age, particularly in animals living at large (deer) is possible on the basis that the age of a deer may be defined by taking into account the development and attrition degree of the mandibular dentition. In very young individuals it is of help to assess the pace of growth and time at which the deciduous teeth change for permanent ones. In older animals the age is indicated by the degree of teeth attrition. These changes are known to occur on the friction face of premolars and molars (Lochman, 1987; Nowicka, 1992; Janczewski, 1984). Finally, at the estimation of fluorine content in bones, the studies involving man and animals as a whole must cope with an additional problem, namely the state of pollution of the natural
The expansion of industrialization in the world is accompanied by an increase of pollutants in the natural environment. One example is fluorine. When appearing as a microelement in food and water, it plays a positive role, since it exerts a favourable influence on the increased crystallization of hydroxyapatite in bones and prevents dental caries. However, overoptimal amounts of fluorine have a negative effect on physicochemical properties of the bones in humans and animals. Then the bone becomes fragile, hard and breakable, which enhances the development of skeletal fluorosis. The question has arisen as to what is the physiological dependence between fluoride accumulation in bones persisting throughout one’s life and the subject’s age. Fluorine accumulation in living organisms has been known for a long time now, and many researchers had reported much earlier that a rectilinear dependence existed between the age and the bound fluoride in the bones (Dabkowska 1987; Mohamedally 1984; Wix and Mohamedally, 1980). 10: A. Machoy, Department of Biochemistry, Pomeranian Medical Academy, al. PowstaGw Wielkopolskich 72, 70-l 1I. Szczecin, Poland. Received 28 February 1994; revised 14 July 1994; accepted 20 December 1994. Correspondence
117
118
Z. Machoy
environment. A lot of industrial emissions stemming from the production of chemical fertilizers, aluminum works and even from burning low quality coal for household requirements, contain fluorine compounds (Machoy et al., 1991; Newman and Murphy 1979). Keeping the above findings in mind we became interested in fallow game inhabiting areas which are relatively uncontaminated by industrial emissions. For this purpose the European elk was chosen. The European elk (Alces &es L.) is an animal that migrates and whose habitat is the north-eastern regions of Poland. The regions are not polluted by industrial emissions, being mainly covered by large forests within the boundaries of the Bialowieia Primeval Forest, a part of which forms the National Park (Pawlus et al., 1993). The mentioned regions are under legal protection. The elk in Poland, apart from belonging to the group of animals that are encountered not too frequently, is also under species protection. They are known to live in larger numbers in Scandinavia, Russia and Canada. That may be the reason why in this country their population is far from numerous. The study material comprised the jaws of the periodically shot elks originating from areas not polluted by industrial emissions. The actual paper is addressed to investigators dealing with the accumulation of elements in bones, particularly that of fluorine, and it also points to the possibility of mathematically determining the relationship between the content of accumulated component and the individual’s age.
Material and Methods The study material at our disposal consisted of 63 jaws of European elks (Alces alces L.). The age of the animals varied from I to 13 years. Mandibular bones, taken from animals having been killed during hunting, were deflated in acetone. The mandibular bones were bored with a dental drilling machine 3 cm below incisors, 1-2 mm deep. The material to be analyzed was collected from the cortical part of mandibular bone, for it is known that fluoride content in trabecular parts differs from that in cortical ones (Czerwinski et al., 1981). The pulverized material, in amount of 100 mg was desiccated at the temperature of 60°C. In consequence it lost about 1% of mass determined at room temperature. The error at defining fluoride in bones did not exceed 5%. That was obtained in a series of experiments involving 100 mg samples of pulverized mandibular bone. Next, the samples were shaken up for 2 min, placed in plastic laboratory tubes and kept until analysis
et al.
(Dabkowska, 1987; Nowicka, 1992; Samujlo 1994). After dissolution of 10 mg of pulverized bone in perchloric acid, followed by neutralization with a TISAB (Total Ionic Strength Adjustment Buffer), fluoride content was determined by means of a selective fluoride electrode (Radelkis Co., Hungary) (Dabkowska, 1987). The results were evaluated statistically. Pearson’s correlation coefficient was used for studying the correlation between the features. The significance level of 0.01 was implemented to serve as a criterion for approving or discarding hypotheses for drawing the conclusion, relying on statistical analysis. et al.,
Results The established results pertaining to the fluorine content in the mandibular bones in the European elk (Alces &es L.) together with short descriptive statistics are depicted in Table 1. The obtained results were subjected to linear regression analysis with the use of 11 mathematical models described by the following formulas Y = A + B/l’ y=Jp y zz x3 Y = x4 y = .,p Y = sqrt(X) Y = m(X) Y = log(X) Y=e’ Y= 10‘ Y=l/X
(linear) (X-square) (X-cubed) (X to the fourth power) (X to the fifth power) (square root of X) (natural log of X) (log base 10 of X) (Euler [e] = 2.71 . . . to the power of X) (10 to the power of X) (inverse of X),
whereas X is the independent variable (age) and Y is the dependent variable (fluoride content). In our studies the relationship is described best by the linear model (Fig. 1) and the square root model (Fig. 2).
Discussion The diet of the European elk in north-eastern Poland is variable, ranging from grass, herbs, ferns, lichens, mushrooms and even algae to twigs and shrubs (Pawlus et al., 1993)Taking the above into account we have disclosed that the game from the regions where the air is relatively low polluted or free of fluorine compounds, would be appropriate for finding out whether or not the fluoride accumulation in bones depends on the age and if the dependence tends to be rectilinear. The age of animals is determined on the basis of the teeth attrition degree with the exactitude up to 1 year. As evident from our previous investigations,
Relationship
between
y = 46.356 500
fluoride
content
in bones and age in elks
119
Multiple linear regression + 28.428*x R-square = 0.77665865
r
AGE (years) Fig. I. Regression
analysis
various types of bones belonging to the same skeleton reveal diverse fluorine content (Machoy, 1991). Fluorine accumulation in bones goes into interaction of hydroxyapatite. The hydroxyapatite hydroxyl groups are substituted by fluorine to fluoroapatite forms. Fluorine also displays a strong affinity towards such elements as calcium, magnesium and phosphorus. The fluoride content rises with the animal’s age (Kay et al., 1976, Dgbkowska 1987; Dabkowska and Machoy 1989; Machoy 1990). Our task consisted of ascertaining whether the dependence was rectilinear or not. For this purpose, analysis of regression was performed by resorting to the 11 mathematical models. In our studies the revealed dependence of fluoride content in bones on the animals age was described best by square root and linear models, for which Rsquared is equal to 0.78965277 and 0.77665865, respectively. Hence, over 77% of our results
y = (-58.821
linear model
arranged themselves in accordance with the above straight lines. Quite an interesting analysis of fluoride dependence on age, based on the value of the r correlation coefficient was presented by Kierdorf et al. (1989). Kierdorf et al. (1989) furnished Y = 0.583 with a significance level of < 0.001. In our studies, Pearson’s r correlation coefficient between age and the fluorine content of the jaw yielded 0.881 with the significance level being P < 0.001. We did not specify the animal’s sex, but in agreement with other authors this omission is almost meaningless (Kierdorf et al., 1989). We had at our disposal rather a small quantity of bones from older individuals, over 7 years old. Just then, it would not be possible to verify if the old individuals really do accumulate in their bones proportionally less fluoride than the younger ones, as reported by Kierdorf et al.
Multiple fixed-nonlinear I) + 119,9405*sqrt(x)
regression R-square = 0.78965227
5000 0
400 -
6
8
AGE (years) Fig. 2. Regression
analysis
square
root model
Z. Mach oy et ul.
120
(1989). Therefore, an extension this respect is recommended.
of the studies in
Czerwinski E., Skolarczyk A., Klewska A., Kaifosz J. and Szewczyk S. (198 I) Fluoride concentration in cortex and in full thickness samples of the iliac crest. Fluoride 14, I, 10-13.
Dabkowska E. (1987) An attempt to evaluate the industrial emission hazard to game on the basis of mineral composition changes in the masticatory system of the deer from Western Pomerania region (Polish). M.D. thesis, Pomeranian Medical Academy, Szczecin. Dabkowska E. and Machoy Z. (1989) Effects of fluortde pollution on calcium and magnesium content of mandibles (lower jaws) of wild game. Fluoride 22, 29-32. Dilbkowska E., Machoy-Mokrzynska A., Straszko J., Machoy Z. and Samujio D. Temporal changes of fluoride levels in jaws of European deer in industrial regions of Western Pomerania. Poland. Emir. Grochem. Hllh. (submitted). Janczewski Z. (1981) Deer.s Antbrs. PWRiL Warszawa. Kay E., Tourengean P. C. and Gordon C. C. (1976) Populational variation of fluoride parameters in wild ungulates from the Western United States. Fluoride 2, 73-90.
Kierdorf U.. Kierdorf H., Erdelen M. and Korsch J. P. (1989) Mandibular fluoride concentration and its relation to age in roe deer (Capreolus capreolus L.). Camp. Biothem.
Phwiol.
94A.
783-785.
-J. (1987) ‘Animal
Pmisrwowe
Wyduttxictwo
age determination Rolnicze
Z. (1990) Fluoride (Polish). Zesz~t~
Wroclaw
References
Lochmann
Machoy man
i LeSnr.
255.
(Polish).
Warsxwa-Krakbw
and its effects on animals Naukowr PAN, 2, 57 -75.
and
Ossolineum
Machoy Z. (1991) Are the fluoride content and the phvsical load-of bones in man related? Fluoride 24, 3, 160.rl02. Machov Z., Dabkowska E. and Nowicka W. (1991) Increased fluoride content in mandibular bones of deer living in industrialised regions of Poland. Enrir. Geochem. HI/h
13(3),
161 163.
Mohamedally S. M. (1984) Studies of the relative fluoride content of normal and pathologicallv mineralised human tissues. Fluoride 17, 246-251. . Mokrzvriski S. (1992) Mineral content analvsis of human fetal- bone (Polishj. MD thesis, Pomeranian Medical Academy, Szczecin. Newman J. R. and Murphy J. J. (1979) Effects of industrial fluoride on black-tailed deer (preliminary report). Fluoride 12, 1299135. Nowicka W. (1992) Evaluation of the course of processes in formation of bones and antlers of fallow deer on the basis of mineral composition analysis. MD thesis, Pomeranian Medical Academy, Szczecin. Pawlus G.. Machoy Z., Tustanowski S., Raczytiski J and Gebczytiska Z. (1993) Damage to the Charnobyl atomic power station had no influence on the emission quantity of beta and gamma radiation determined of elk living in the north-east region of Poland. Polish. Bromar. Chem. Taxvkol.
26, 77-79.
Samujio D., Machoy-Mokrzyriska A, Dqbkowska E., Nowicka W. and Paterkowski W. (1994) Fluoride accumulation in European deer antiers. ‘Em+. Sci. 2, 1899194. Wix P. and Mohamedally S. M. (1980) The significance of age-dependent fluoride accumulation in bone in relation to daily intake of fluoride. Fluoride 13, 100~104.