University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange
Masters Theses Graduate School
8-1948
Effects of Various Fluorine Compounds on the Albino Rat
Robert Floyd Pevahouse University of Tennessee - Knoxville
Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes
Part of the Animal Sciences Commons
Recommended Citation Pevahouse, Robert Floyd, "Effects of Various Fluorine Compounds on the Albino Rat. " Master's Thesis, University of Tennessee, 1948. https://trace.tennessee.edu/utk_gradthes/3073
This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council:
I am submitting herewith a thesis written by Robert Floyd Pevahouse entitled "Effects of Various Fluorine Compounds on the Albino Rat." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Master of Science, with a major in Animal Science.
Charles S. Hobbs, Major Professor
We have read this thesis and recommend its acceptance:
Sam L. Hansard, Marshall C. Hervey, & Ollia E. Goff
Accepted for the Council: Carolyn R. Hodges
Vice Provost and Dean of the Graduate School
(Original signatures are on file with official studentecor r ds.) · June 17, 1948
To the Committee on Graduate Study :
I am submitting to you a thesis written by Robert Floyd Pevahouse entitle d "Effects of Various Fluorine Comp ounds on the Albino Rat ." I rec ommend that it be accepted for nine quarter hours credit in partial fulfillment of the requirements for the degree of Mas ter of Science, with a major in Animal Husbandry.
We have read this thesis and recommend its acceptance :
Accepted for the Committee
Dean oth�ra A THESIS Submitted to The Committee on Graduate Study of The University of Tennessee in Partial Fulfillment of th e Requirements for th e degree of ' Master of Science by Robert Floyd Pevahouse August 1948 ACKNOWLEDGMENT The author would like to acknowledge his in- debtedness to Dr . Charles S. Hobbs and Professo r Sam L. Hansard for th eir many helpful and nstrucco tive suggestions offered during the course of the work and in the preparation of this paper . He would also like to express his apprecia- tion to Dr. Marshall C. Hervey and Dr. Ollia E. Goff for many construc tive suggestions offered as the work progressed. Due acknowledgment is also expressed to the Depart- ment of Home Economi cs for supplying the experimental animals . , and to the Chemistry Department of the Agricultural Ex periment Station for doing the chemi cal analysis of the bones . R.F.P. 247328 FOREWORD No part of the information contained in this paper can be reproduc ed in any form without the written consent of the Head of the Department of Animal Husbandry of the University of Tennessee. TABLE OF CONTENTS PA GE INTRODUCTION. . . . • • . . • • . • • • • • • • • • • . . • • • • • • • • • • • • • • • • • • • • 1 LITERATURE REVI:&.'W ...... 3 EXPERIMENTAL ••••••...... 17 Feeding data...... 18 Weight records •••• ...... 19 Bone analysis •••• ...... 25 DISCUSSION OF RESULTS. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 32 SUMMARY AND CONCLUSIONS . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 45 BIBLIOGRAPHY. • • • . • • • • • • . • • • • • • • . • • • • • • . . . . • . • • • • • • • • • • • 46 LIST OF TABLES TABLE PA GE I. Concentration of Different Fluorine Comp ounds Used, Length of Time on Ration and Fluorine Analysis of Bones ...... 20 II . Average Group Weekly Gains and Total Gains for Four Week Period...... • ...... • . . . • 26 III. Effect of Time on Accumulation and Depletion of Fluorine in Bones of Rats Fed Graduated Levels of Sodium Fluorine ••••••••••••••, • • • • • • • 31 IN TRODUCTION At the beginning of the 19th century, an Italian chemist demons trated th e presence of fluo rine in the teeth of both man and animals. This discovery created con siderable int erest at that time and can probably be con sidered the initial step in stimulating research on the problem. In the light of our pres ent day knowledge , th ere is evidence th at the ingestion of small amounts of fluorine may be distinc tly benefi cial to livestock. However, the primary problem in animal nu trition is conc erned with definitely harmful affec ts resulting from an excessive in take. It was only a few ye ars ago that farmers we re using raw rock phosphate as a mineral supplement in rations for livestock. Although raw rock phosphate might supply the des ired mineral ingredients at a lower cost than they mi ght be obtained from other sources , it was learned that ro ck phosphate· produced abnormal calcification of ·the teeth and possibly other changes which greatly limited its us age. These changes were th e same as could be pro duced exp erimentally by us ing a chemi cally pure fluorine salt. 2 Observations in Europe, and in recent years in this country, show that cattle graz1ng ne�r industrial plants which give off fluorine in the smoke, such as aluminum or sup erphosphate plants, show excessive wear on the teeth, decrease in the rate of growth, and in some cases cause premature death . Since Tennessee is one of the larges t phosphate ' producing states, and since industrial plants have started a rather rap id program of expansion, there is a need for more informati�n on the problem of chronic fluorosis. The experiments reported in this thesis were de- signed primarily to study the relative toxicity of various , fluorine comp ounds in the albino rat. Experiments have been conducted to study the concentration of sodium fluoride and length of time of feeding necessary to produce fluorosis; to biologically test the fluorine concentration of samples of hay obt ained near industral plants known to give off fluorine substances; and to investigate substances in the ration which might offer promise of alleviating the toxic effects of fluorine in the albino rat . LITERATURE REVIEW Fluorine oc curs mos t abundantiy in nature in the mineral fluorspar (CaF2) and in cryolite, a double fluoride of aluminum and sodium. Mine rals cont aining small amounts of fluorine as biotlte, tourmaline , mu s covite, and apatite, distribut ed throughout the earth's crust , accounts for a part of the fluorine found in soils (24) . Jacobs and Reynolds (18) have investigated the fluorine content of various commerc ial grades and types of phosphate rock. Th e amount of fluorine found varied from 2.62 per cent in Tennessee phosphate "run of mine material" to 4.08 per cent for "carefully selected lump rock. " A fertilizer grade of the same rock contained 3.72 per cent fluorine , while a high grade washed and ground rock con tained 3.89 per cent . These values are similar to those obtained for samp les of phosphate rock occurring in North Africa, Idaho, Wyoming , and Florida . Gautier and Clausman (14) , on examination of 63 plant substanc es found an average of 0.00265 per cent fluorine in the fresh mat erial . Churchill, Boyland, and Martin (10) reported the fluorine content of alfalfa hay varied from 0.0015 per cent fluorine in the first cutting to 0.0037 per cent fluorine in the third cutting . A green sample of this third cutting cont ained 0.0053 per cent 4 fluorine. Further ·investigation by the same workers showed alfalfa leaves to contain 0.0046 per. cent fluorine while the alfalfa stems contained 0.0013 per cent fluorine . If a forage is exposed to gasses or smoke containing fluorine, the analysis for fluorine will increase greatly according to the work of Meyn and Viehl (26) and Bardelli and Nezani (2) . The fluorine content of so called "normal" bones varies greatly. This may be due partially to the method or analysis. Majumdar, Ray, and Sen (21) .observed that the fluorine content of bones and teeth increased with age. Thi s is especially true in the pelvis wh ere the concentra- f tion or fluorine per unit weight of bone is proportional to the age of the animal. The pelvis of a 3.5 year old bull contained 27 milligrams of fluorine (0. 02 per cent) in a 100 gram sample . A 5.5 year old bull contained 73 .9 milligrams fluorine (0 .07 per cent) in the same size sample . McClure (24) summarized the results or several different investigators and set the average at from 0.01 to 0. 03 per cent fluorine in normal bones . Normal dental enamel contained 0.10 to 0.20 per cent fluorine . McClure (24) , in reviewing the work or Trebitsch, suggested that the ) hardest apatite, namely, fluor-apatite , 3 Ca3 (P04 2-CaF2 occurs in dental enamel and that the hardness of teeth depends on the presence of crystal masses of apatite. 5 There is evidence that the fluorine content of bones and teeth, as well as other body organs and tissues , may be increased by an increase in the fluorine intake . Bethke et al . (4} found that the fluorine content of the ash of femurs of pigs varied directly with the amount of fluorine in the ration. When pigs received 2 pounds of limestone or 2 pounds of steam bone meal as a mineral sup plement, the fluorine content of the bone ash was 409 and 231 parts per million respectively . However, when 100 grams of sodium fluoride was added to the 2 pounds limestone or wh en 2 pounds rock phosphate was fed, the bone ash con tained 11,077 and 10, 934 parts per million of fluorine respectively . McClure (24} in reviewing the work of Sonntag , report ed that, wh ereas normal teeth and bones contain about 0.3 per cent fluorine , the teeth of animals fed sodium fluoride contain 1.73 per cent fluorine and the bones con tained 1.29 per cent fluorine . Schul tz (30) reported that the ch emical analys is of the tibia, femur and humerus of several hundred rats have shown that the bones of rats fed moderate to large doses of fluorine contain increased per centages of fluorine and magnesium and decreas ed percentages of carbonates (carbon dioxide}. While these abnormalities in th e chemical composition of the bones of rats are not 6 strictly proportional to the amounts of fluorine in the rations, they do vary to a definite degree with the per centage of fluorine fed. Kick et al . (20) suggest that an increase in the fluorine content of the ration will cause an increase in the fluorine content of the bones. The increase was greater wh en sodium fluoride was fed than when calcium fluoride was fed. Meyn and Viehl (26) reported that the bones of cattle with chornic fluorine poisoning contained 7 to 9 times as much fluorine as was found in the bones of sound animals . This figure is based on their findings of 0.48 milligrams fluorine per gram in the lower jaw bone of normal cattle and 4. 68 milligrams per gram in infected animals . The rat is the laboratory animal most commonly used in fluorine investigations . For this reason there is more information available as to the toxic levels of the different fluorine compounds in the rat than in any other animals. Some of the most complete wo rk has been carried on by Schultz (30, 31 , 32), Bethke et al. (3, 4) , McClure and Mitchell (25), Smith and Leverton (35). Macroscopic examination of the incisor teeth for abnormal color and growth is the criterion mo st commonly use d to determine fluorosis in rats. Schultz (30) described the teeth of rats fed excessive amounts of fluorine as being 7 wh i te with an excessive overgrowth of the upper incisors and excess ive we aring or breaking off.of the lower incisors. Different investigators have required different amounts of fluorine to produce this effect. McClure and Mitchell (25) found that rats fed 0.0313 per cent fluorine in the form of sodium fluoride showed the abnormalities . Schul tz (30) obtained a loss of the pink na tural color of the teeth when as little as 0.01 per cent fluorine was fed in th e form of sodium fluoride but did not get an overgrowth of the incisors until 0.024 per cent was fed. This is in accord wi th Kick et al. {20 ) wh o reported normal shape of teeth when fluorine comb ined as sodium fluoride was fed at I a 0.01 per cent level or 0.05 per cent when combined as calcium fluoride. However, wh en the sodium fluori de was increas ed to 0.02 per cent , the mandibular incisors showed considerable wear , while the maxillary incisors became greatly elongated. Smi th and Lantz {33 ) reported that the feeding of sodium fluoride to ra ts at a 0.024 , 0.05 and 0.1 per cent of the ration had in each case interfered with the normal calcification of the teeth to an extent varying with the concentration. Other workers have reported approximately the same results . The amount of sodium fluoride ne ces s ary to produce th e characteristic abnormalities may be dependent to some 8 extent on the length of time the rats are on ration. Ac cording to Mayn ard (23), fluorine is a cumulative poison . At first the fluorine merely accumulates in the bones and teeth without evident harm and considerable time elapses before structur al injury becomes evident . In most of the toxicity studies of the different fluorine compounds , sodium fluoride has been used as a basis for comparison. Growth retardation , as well as calcification of the teeth , has been used to measure these· differences. Evans and Phillips (12} did a comp ar ative study with synthetic cryolite and sodium fluoride . A basal ration was supplemented to contain 0.08, 0.03 and 0.015 per cent fluorine from both carriers . The rats were kept on experiment for twelve weeks . The growth rate of the rats receiving 0.03 per cent sodium fluoride was similar to those receiving 0.08 per cent fluorine in the form of cryolite. Similar results were obtained with the 0.015 per cent sodium fluoride and 0.03 per cent cryolite. Ap parent normal growth occurred in the lower levels of fluorine reg ardless of the source . This does not agree with the work of Smith and Leverton (3 5} who required ten times as much fluorine combined as cryolite to produce the slightly stunted growth caused by 0.0228 per cent sodium fluoride. It should be noted however that Smith and Leverton (35} 9 us ed a natural cryolite wh ich is approximately half as soluble as synthetic cryolite . The work done at the University of Arizona by Smith and Leve rton (35) indicated that calcium fluoride was even less toxic than cryolite from the standpoint of growth interference in the rat . It required a concentration of 0.452 per cent fluorine in the form of calcium fluoride to produce the same slight stunting of growth obtained with 0�0226 per cent fluorine in the form of sodium fluoride . This is twice the concentration which interfered wi th the growth of the animals when the fluorine was in combination as cryolite and twenty times the amount necessary when it was combined as sodium fluoride . They attribute this partially to the fact that sodium fluoride is approximately one thousand times more soluble than calcium fluoride, and at leas t one hundred time s more soluble than· cryolite . Thi s comparison is rather high wh en comp ared to that of Kick et al. (20), who required 0.05 per cent fluorine in the form of calcium fluoride to produce the same toxic ef fects as 0.01 per cent as sodium fluoride, and Schul tz (31) and McClure and Mitchell (25) wh o reported calcium fluoride to be approximately half as toxic as sodium fluoride . Many reports have been published as to the effect the fluorine comp ounds have on the teeth of rats and 10 humans when incorporated in the ration at a very low level. Smith et al. (33, 34), Marcovitch (22), DeEds and Thomas (11) , and others, have found a concentration of approximately 0.0014 per cent fluorine in the feed will cause a slight striation or motteling of the teeth. Smith and Leverton (35) conclude that the amount of fluorine required to cause initial damage to the rat incisor was so small th at dif ference in solubility of the compounds were not a factor, and no difference in toxicity of fluorine from the various compounds studied could be noted . Since the various fluosilicates are used as a spray material , most of the work done with them has been in the low level just described. The work of Smith and Leverton stated , however , that there was no difference in the lethal concentration or the effects upon the teeth of the rat when the fluorine was in the form of sodium fluoride , potassium fluoride , barium fluosilicate , or sodium fluo silicate . Cattle , both beef and dairy , are affected by ex cessive intake of fluorine. Reed and Huffman (28) at the Michigan Agricultural Experiment Station , studied the ef fect of adding rock phosphate to the ration of mature dairy cows. They reported that the animals were thrown off feed when 1. 5 per cent of rock phosphate containing 3.5 per cent fluorine was added to the grain ration. Taylor (36) 11 obtained approximately the same results when rock phosphate containing approximately 3.0 per cent fluorine was fed at the same levels. The animals pres ented a general un thrifty appearance and their teeth showed an abnormal amount of wear. When calcium fluosilicate was fed at the same levels, similar results were obtained. Phillip s, Hart, and Bohstedt (27) required only 0.625 per cent raw rock phosphate in the grain ration to produce typical fluorine toxicosis. Thi s raw rock phosphate contained approximately 3.55 per cent fluorine. More time was required to produce th e symp toms of fluorine poisoning at this level of feeding than was required for the higher levels. Huffman and , Reed (17) made a comparative feeding test of long duration in which they compared a mineral mixture of bone flour, rock phosphate, limestone, and a complex mineral mixture at a level of 1.5 per cent of the grain ration. They undfo that the animals re ceiving rock phosphate which contained 3.5 per cent fluorine made slower gains, presented a mo re unhe althy appearance, produc ed less milk and showed more wear on the teeth than did the other animal s. When the cattle were approximately 2.5 years old, the teeth were worn to such an extent that they were sensitive to cold water. Some teeth were worn to such an extent that the pulp cavities were exposed. There did not appear to be any effect on reproduction. 12 Meyn and Viehl (26) described fluorine poisoning in cattle, wh ich was obtained from eating feed produced near an indus trial plant wh ich gave off smoke containing fluorine, as follows : Most of the animals were found to be undernourished to varying degrees. In about a third of the animals, deep hunger hollow, strongly protruding hip and seat bones, and distinctly separated ribs were th e sign of severe emaciation . The skin of the animals were dry and hard. In the region of the thorax the skin lay fairly taut over the ribs and it was difficult to make a fold in it. In most animals, the hair was dull and lifeless, in some it was coarse and rough . Many cows stood mo tionless, with bent heads, in front of filled cribs without any desire to eat . The appearance of some of them was really pitiable . A young milch cow with no udder trouble gave only 1 litter of milk a day . Six cows had pronounced pains in the joints. They disliked getting up and hardly dared to move from one sp ot. It took energetic driving to get them to move aside , stepping carefully, with shaking limbs , bent backs, and anxious exothphalmi c glances. These cows were in a particularly under nourished condition. Almost wi thout ex ception, these animals had serious tooth damage . The damage consisted mainly of : discoloration, with or without injured spots in the dental enamel; an unnatural, often considerable degree of wear of the incisors . Forbes and co-workers (13) were among the first to report poor results with swine when rock phosphate was used as the mineral supplement . They found it to be less efficient than limestone, steam bone me al, precipitated bone or calcium carbonate in improving bone formation and calcium and phospho rus retention of growing pigs. Enough 13 of each supplement was fed to supply 10 grams of calcium per he ad per day. This amounted to approximately 18 grams of the raw rock phosphate. The experiment of Be thke, et al . (3) showed that fluorine in the form of sodium fluoride, when added to the ration of growing swine in the am ount of 30 grams per 100 pounds of feed, lowered the feed consumpt ion and thereby de creased the daily gains . The decreased feed consumption in lactating sows caused them to loose weight.rapidly and they fai led to return to normal wei ght af ter the pigs were weaned . The teeth of sows kep t for two or more ye ars on a ration high in fluorine were frequently worn to the gums and the pulp cavities exposed. Kick et al . (20) conducted feeding experiments with swine on a long time basis. Varying amounts of sodium fluoride and rock phosphate was added to the mineral sup- plement . At the conclus ion of the exper imen t, the basal group which had received no fluorine supplement had femur s th at presented a normal yellowish color, with smooth ex teriors, a dense appearance, and a decided lus ter . The bones of animals in the other lots had a lus terles s ap - pearance, were wh ite in color, had many protuberances, and showed a decided thickening . The only gr oup that showed a significant difference in breaking strength was the high 14 level of phosphate, wh ich showed a cons iderable decreas e in strength over the control group. .Short time experiments with swine by the investigators showed the same condi tions but not as marked as over a long time period. Soft bones occurred in both pigs and rats Which had been fed on rations containing rock phosphate at the Arkansas Experiment Station (1). Bohstedt and co-workers (5) found no ill effects on growth and fat tening wh en pigs were fed rock phosphate at a level of 0.4 per cent. In some cas es 0.8 per cent of rock pho sphate ret arded growth, wh ile 1.6 per cent had a harmful effect on both rate and economy of gain . The fluorine content of this particular rock phosphate was not given . However , it was of the kind or grade known as lime phosphate . The experimental work to determine the toxic level of fluorine in the sheep is rather limi ted. However, Greenwood (15), in reviewing the wo rk of Pierce, des cribed . sheep fed various levels (60-170 mi crograms daily) of fluorine in the form of rock phosphate for a period of three years. All of the animal s app eared to be in good health for th e first year. Thereafter, some of them began to eat less and lose weight. The bones of the animal s wh ich received doses greater than 120 mi crograms of fluorine daily exhibited the bone changes des cribed by Ki ck et al. (20). No organs , ex cept the bones and teeth, appeared to be affected by the 15 treatment . There seems to be varying op inions as to the ability of th e chick to withs tand fluorine in the ration. Buckner, Martin, and Insko (7) report ed that ro ck phosphate inter fered with the growth of chicks for the first few weeks but di d permi t normal growth after that time . Kennard and Whi te (19) reported that steam bone me al and rock phosphate appeared to be equally efficient in mineral mi xtures fe d to pullets or cockerels . No difference was noted on growth or egg produc tion wh en mixtures cons is ting of equal parts of limestone, salt, and either steam bone meal or rock phosphate was fed . Buckner, Martin , and Peter (6) did re port, however, that mature birds receiving rock pho sphate we re troubled wi th diarrhea and egg production was somewhat reduced. Halpin and Lamb (16), after studying various levels of feeding rock phosphate containing 3.52 per cent fluorine, concluded that no harmful effects were indicated at a 1 per cent level; some depression of growth resulted at a 2 per cent level; and seriously harmful effects were observed at a 3 per cent level. The solubility of some of the more important fluorine comp ounds in water have been reported by Carter (8, 9) and Seidell ( 29 ) as follows : 16 Com;Eound Gr8li1SL lOO c c water Tem;eerature ° Potassium Fluoride 48 .70 20 c. Magnesium Fluosilicate 23.53 20° c. Calcium Fluosilicate 10.58 20° c. Sodium Fluoride 4.05 25° c. Sodium Fluosilicate 0.65 17.5°C. Potassium Fluosilicate 0.12 17.50C. Synthetic Cryolite 0.06 25° c. Natural Cryolite 0.04 25° c. Calcium Fluoride 0.004 250 c. EXPERIMENTAL The general plan of the experiment was to feed growing albino rats different fluo rine comp ounds , or in some cases varying levels of the same compound , over a period of time necessary to determine the level that would produce chronic fluorine poisoning . The rats were killed and the long bones and lower jaw bone removed and analyzed for fluorine content . Rats ranging in age from 22 to 30 days were obtained from the stock colony in the Department of Home Economics . Three ra ts were placed in each cage . They were sexed to the extent that each cage conta ined one male �d one female , the th ird being selected as the supply on hand permitted. The rats had access to a stock diet before weaning , and some were maintained on this diet up to seven days af ter we aning . This di et wa s very low in fluorine . All experimental animals we re confined in round wire cages approximately nine inches in diameter and ten inches high with an elevated bottom. The feces and urine were collected on paper placed under each cage . This paper was changed dai ly. The feed jars and wa ter bottles were cleaned as often as considered necessary for sanitary purposes. The animal s received distilled water ad lib . 18 The laborat ory was heated wi th a thermos tatically controlled electric heater and a temperature of 75° to 80°F. maintained at al l times . At the conclus ion of each exper iment the rats were killed by etherization and the meat removed from the long bones and lower jaw bone by gross dissection followe d by boiling 1.5 hours in three gallons of wa ter to wh ich had been added lOOcc of soap powder. The bones were allowed to stand over night to dry and th en delivered to chemistry department for fluorine analys is. Feeding Data A syntheti c basal ration was us ed wh ich consisted of the following ingredients : Per cent Starch------42 .0 Dehydrated Alfalfa Meal------30 .0 Casein------15 .0 Fat (Snowdrift ) ------ 7.0 Cod Liver Oil (5 USP uni ts of D per gram feed) G.B .I. Salt Mixture No . 2 ------3.0 NaCl (Iodiz ed )------1.0 Br ewers Yeast------2.0 This basal rati on was supplemented with th e dif- ferent fluorine compounds to make up the potency calculated for each ration. This was computed on the molecular con- centrat ion of each fluorine carrier us ed. 19 A general analysis of th e basal ration showed the following results: Per cent Calcium------ .581 Phosphorous------ .445 Protein------ 18.22 Fat ------ 10.18 Fiber------ 6.53 Fluorine------0.0015 The ration was not changed throughout the feeding period. The quantity of feed given was regulated according to the demands of the animal s. In cas es wh ere a hay was to be studied for its fluorine content, a finely ground portion of this hay was subst ituted for the dehy�rated alfalfa leaf mea� in the basal ration. The different fluorine compounds fed, along with the concentration and length of time on feed, are sh own in Table I. Weight Records The rats used in this experiment were placed in their respective cages and given a lot number. Each rat was individually marked wi th in each lot. All animal s were weighed for three consecutive days and the av erage of these weights us ed as the initial weight . The second day of the three day series was used as the starting date of th e TABLE I CONCENTRATION OF DIFFERENT FLUOR INE COMP OUNDS USED , LENGTH OF TIME ON RATION AND FLUORINE ANALYSIS OF BONES ., 'CIS:: D'J ..-1 §� rn OG> p., s:: M � �� 0 " D'J ..-1 E-ts:l OAI 't:l..-1 s:: � 0 o::S G)., 1 None 0.0015 0.026 18 34 . 2 a Sodium Fluoride 0.0150 0.463 4 34 2 b Sodium Fluoride 0.0300 0.706 4 34 3 a Potas sium Fluoride 0.0150 0.416 3 34 3 b Pot assium Fluoride 0.0300 0.735 3 34 4 a Calcium Fluoride 0.0300 0.202 6 34 - 4 b Calcium Fluoride 0.0600 0.222 6 34 5 a Ro ck Phosphate 0.0150 0.343 4 34 5 b Rock Phosphate 0.0300 0.614 4 34 5 c Rock Pho sphate 0.0600 0.716 4 34 6 a Sodium Fluosilicate 0.0150 0.662 3 32 6 b Sodium Fluosilicate 0.0300 0.875 3 32 ro 6 c Sodium Fluosilicate 0.0600 1.164 3 32 0 7 a Potassium Fluosilicate 0.0150 0.614 3 32 TABLE I ( Continued ) CONCENTRATION OF DIFFERENT FLUORINE COMPOUNDS USED, LENGTH OF TIME ON RATION AND FLUORINE ANALYSIS OF BONES .p 'OS:: ell ..-! §� IQ 0 ()) � Q) P.r-1 s:1 r-1 a J:!Pt 0 � Cll ..-! 'd..-1 s:: Q) E-fS:: 8� Q).j.) CONCENTRATION OF DIFFERENT FLUORINE COMPOUNDS USED, LENGTH OF TIME ON RATION AND FLUORINE ANALYSIS OF BONES .p 'd� l7l ..-! §� C1l OCII p., Q) s:: a �� 0 �Cil ..-! OP. 'd..-1 � Q) E-t� 0::3 Q),P CONCENTRATION OF DIFFERENT FLUORINE COMPOUNDS USED, LENGTH OF TIME ON RATION AND FLUORINE ANALYSIS OF BONES .p 'd� C1l ..... � � C1l 0 (I) � (I) � ri a �� 0 al C1l ..... O P. 'd..-i � Q) 8� 0 ;::$ CD.P �� 0 til .Pcd 0 r...t CH..-i Q) Gf M Per cent Per cent Days 14 a High Blount Hay 0.0021 0. 079 6 3-t 2 e Sodium Fluoride 0.0025 0. 072 3 34 15 a High Blount Hay plus Sodium Fluoride Defluorinated Rock 6 34 Phosphate (2%} - 0. 0050 0. 114 16 None 0. 0015 ** 3* 34 16 a Sodium Fluoride 0.0025 3* 34 *All groups from 16 through 16 g contained 3 rats for first 2 weeks and 2 each until the end of the experiment. **Bone analysis for groups 16 through 16 g reported in Table III . rv � 25 experiment . All animals were fed a stock diet on the firs t day and placed on the experimental diet the second day . The experimental animals were individually weighed at weekly intervals . Table II gives the average gain for each individual group together with the lot total gain throughout th e ex- periment. Bone Analysis Th e bon�s were analyz ed by the method of Willard and Winter (37) wi th modific ations as us ed by the Aluminum Comp any of America and the University of Tennes see. ' The bones from each rat used in the experiment were individually analyz ed, but only the av erage for each group are reported in thi s paper. The average fluorine content of the bones analyzed in each group together with the length of time on the ration and number of rats per group , are reported on Table I. TABLE II AVERAGE GROUP WEEKLY GA INS AND TOTAL GAINS FOR FOUR .WEEK PERIOD .!-) 4) 1=1 1=1 s= Q) -MO J:! Mori Q) 0-l-) r-t ::ial Ill p. .!-) Average Weekly Gains M �� al per Grou 4) §t CH � p .0 ell 'dO � Q) CH ..... Q) .!-).!-) 0 .!¥I .!¥I .!ill .!ill cO ! 1=1 al 1=1 Q) Q) Q) Q) 0 ..... M Q) Q) 4) Q) M 3� Q) 1!!:: !!!: i!= � r-t §t 0 () 1=1 cO 0 :::; .-tO .!-) 'd '0 ..c:l .!-) r-t CISO � Ill 1=1 M .!-) 0 � IX! 0 z r-t (.\l tQ � E-f Per cent 1 None 0. 0015 18 26 29 26 28 109 2 a Sodium Fluoride 0. 0150 6 23 21 19 21 84 2 b Sodium Fluoride 0. 0300 6 15 20 14 19 . 68 3 a Potassium Fluoride 0.0150 3 25 22 25 23 95 3 b Potassium Fluoride 0.0300 3 22 20 20 21 83 4 a Calcium Fluoride 0. 0300 6 18 21 21 28 88 4 b Calcium Fluoride 0.0600 6 17 21 18 25 81 5 a Rock Phosphate O. Ql50 6 20 25 21 26 92 5 b Rock Pho sphate 0. 0300 6 19 22 17 24 82 5 c Rock Phosphate 0.0600 6 20 18 18 26 82 6 a Sodium Fluosilicate 0. 0150 3 24 20 21 34 99 6 b Sodium Fluosilicate 0. 0300 3 19 17 13 27 76 6 c Sodium Fluosilicate 0. 0600 3 14 15 2 15 46 7 a Potassium Fluosilicate 0.0150 3 18 23 17 35 93 {\) (j) TABLE II ( Continued ) AVERAGE GR OUP WEEKLY GA INS AND TOTAL GA INS FOR FOUR WEEK PERIOD .p • R RR C) 'riO a M'l"i G) O.P ...-t ::;$a! Ol Average Weekly Gains P. ...., M �� aj per Group G) §t � � C"/l 'dO R • � ori � • ..P+l 0 ..!14 ..!14 ..!14 ..!14 aj :z; s:l al R • Q) Q) Q) C!) ori M Q) G) Q) M 3� Q) � � � � ...-t §t 0 o R aj 0 ::;$ ...-tO ...., rd rd ...., M ...-t cdO Ol R M :5 0 C!) ll:t 0 ! ...-t C\l t() .qt 8 Per cent 7 b Potassium Fluosilicate 0.0300 3 21 19 16 26 82 7 c Potassium Fluosilicate o:o600 3 14 16 12 19 . 61 8 a Natural Cryolite 0.0150 3 15 17 21 29 82 8 b Natural Cryolite 0.0300 3 19 22 20 16 77 8 c Natural Cryolite 0.0600 3 15 21 20 21 77 9 a Magnesium Fluosilicate 0.0150 3 25 35 29 30 119 9 b Magnesium Fluosilicate 0.0300 3 32 35 34 27 128 9 c Magnesium Fluosilicate 0.0600 3 16 23 25 27 91 10 a Synthetic Cryolite 0.0300 3 30 32 25 26 113 10 b Synthetic Cryolite 0.0600 3 24 28 23 24 99 11 a Calcium Fluosilicate 0.0150 3 33 27 26 24 110 11 b Calcium Fluosilicate 0.0300 3 25 21 20 14 7280 11 c Calcium Fluosilicate 0.0600 3 16 19 25 22 (\l � TABLE II (Continued) AVERAGE GROUP WEEKLY GA INS AND TOTAL GA INS FOR FOUR V�K PERIOD .j.) 4) s:= s:l s:l ..-10 � J:.i..-1 Q) Q.j.) Average Weekly Gains M ;j cai U,l per Group At MIX: .j.) J:.i lit ci Q) � ct-t p:: 'dO s:: fll Q) ct-t ..-1 § Q) .j.).j.) 0 .!:1:1 .!:1:1 .!:1:1 al z s:l al s:l 4) Q) Q) Q) c ..-1 J:.i Q) Q) Q) Q) J:.i 3� Q) := � � := r-1 � 0 0 s:= al 0 ;j MO .j.) 'd 'd .j.) • ..cl U,l .j.) 0 J:.i M caiO § s:l J:.i • c lit .0 z r-1 C\1 tl) � E-t Per cent 12 a High Blount Hay and Sodium Fluoride 0.0100 3 26 35 28 25 '114 12 b High Blount Hay �nd Sodium Fluoride 0.0150 3 24 29 23 18 94 2 c Sodium Fluoride 0.0050 3 31 32 28 20 111 12 c High Blount Hay and Sodium Fluoride 0!.0050 6 23 34 24 25 106 12 d High Blount Hay and Sodium Fluoride 0.0075 6 25 30 26 19 100 2 d Sodium Fluoride 0.0150 3 32 34 29 24 119 13 a Sodium Fluoride Defluorinated Rock PhoaEhate �2�} 0.0150 3 27 29 28 30 11. !\) 00 TABLE II (Continued) AVERAGE GROUP WEEKLY GA INS AND TOTAL GA INS FOR FOUR WEEK PERIOD G) � s::= s::= G) .no a �'" Q) O.P r-1 :;j ell l1l Pt r-IP:: .p Average Weekly Gains ·� IX. al G) §t fH p:j E•r Grou:12 aJ 'dO R Q) fH -n § G) .p.p 0 � � � � .. z s::= QS s::= Q) ., Q) Q) � '" r-IQ) M Q) Q) Q) Q) � :;j.p C) :1: !5: !3: � r-1 §t 0 () s::= aS 0 :;j r-IO .p rd rd � .p � r-1 cxso � l1l s::= � .p 0 � IX. 0 z r-1 � 1:1) .qt E-t Per cent 13 b Sodium Fluoride Defluorinated Rock Phosphate (4� ) 0.0150 3 22 26 24 23 95 14 a High Blount Hay 0. 0021 6 19 25 18 20 82 2 e Sodium Fluoride 0. 0025 3 30 35 29 26 120 15 a High Blount Hay Defluorinated Rock Ph osphate (2%) o-.oo5o 6 24 32 23 20 99 16 ){one O.OOlS -3* 28 35 38 38 139 *All groups from 16 through 16 g contained 3 rats for first 2 weeks and 2 each until the end of the experiment . N) (0 TABLE II (Continued ) AVERAGE GROUP YffiEKLY GAINS AND TOTAL GAINS FOR FOUR W�K PERIOD .p e � � � Cl ..-iO a � .... Q) O.P r-1 ::::$ aS Ol P4 .p Average Weekly Gains � �� aS per Group Cl � � 1:'/l 'dO � s:= Q) � .... � G) .p.p 0 � � � � aS :z; � aS � G) (J) G) (J) 0 .... � Q) Q) Cl G) � �2 Q) iJ: � � � r-1 §t 0 C) !=: aS 0 ::::$ r-10 .p 'd 'd ..Q .p � r-1 IISO 1 11.1 � � .p 0 0 lit 0 z r-1 C\l tQ .qt � Per· c ..t 16 a Sodium Fluoride 0.0025 3* 30 40 23 31 124 16 b Sodimn Fluoride o:oo50 3* 23 37 36 31 .127 16 c Sodium Fluoride 0.0075 3* 21 44 26 33 124 16 d-1 Sodium Fluoride 0.0100 3* 24 38 35 33 130 16 d-2 Sodimn Fluoride 0.0100 3* 24 35 33 34 126 16 e Sodium Fluoride 0.0125 3* 27 41 36 34 138 16 f Sodium Fluoride 0.0150 3* 30 35 35 32 132 16 ei Sodium Fluoride 0.-0200 3* 25 37 26 29 117 *All groups from 16 through 16 g contained 3 rats for first 2 weeks and 2 each until the end of the experiment. VI 0 TABLE III EFFECT OF TIME ON ACCUMULATION AND DEPLET ION OF FLUORINE IN BONES OF RATS FED GRADUATED LEVELS OF SODIUM FLUORINE G» s:=s:= ..-tO ri � ..... �s:= .p.J:l .PR Gil 0 � l=l 0 � 0 � 0 � l1.l l1.l ;:j� Q) G» Q) 4) Q$ l1.l � riP:: �IIQ �IIQ �Ol,.Qill� � a! 1%-i J:l �E-t • Q) p:: ft-4 § � J:l § � s:= o GIS o 1=1 'dO OI=!O OAO Ol=!'d� ft-4 Q) ...... � 0 Q)IQ,P Q).qi� Q).ql � 'd � �.p � Q) z a!� � ri aS � tl) cal tl) � ..... p:: ..... p:: .....� � Q) 3:! H M � � �� 0 0 � C) � 0 Q) 0 Q) OG>..-t;:jft-4 0 riO ;:j.p ;:j.P ;:j .p.p � � � �0 rift-4 rift-4 rift-4CidCD 0 z 0 1%-i< 1%-i *One rat from each group wa s killed at each of the time s indicated . � ...... DISCUSSION OF RESULTS . This investigation was made primarily to study the relative toxicity of the different fluorine compounds when fed in the ration of the albino rat . The criteria used for study were.: calcification of. the teeth; total weight gain; and fluorine analysis of the bones . All rats that were maintained on the rations sup- plemented with fluorine throughout the feeding period showed signs of bleaching of the teeth . Some of the groups that were maintained on a low fluorine diet showed only a. slight change in teeth color and no irregularities as to shape and form of teeth. Other groups which we�e maintained on relatively high fluorine diets, showed not only a com- plete bleaching of both the mandibal and maxillary incisors but a great wearing or shortening of the lower incisors accompanied by an elongation of the uppers. This is in accord with the findings of Schultz (30) who reported a loss of the pink natural color of the teeth when 0.01 per cent fluorine was fed in the form of sodium fluorine but did not get an overgrowth of the incisors until 0.024 per cent was fed . In comparing groups of rats fed a ration with equal fluorine concentration of at least 0.03 per cent fluorine , calcium fluoride produced less change in the teeth than any other group studies, and sodium fluosilicate 33 produced the greatest change . This is in accord with Kick at al . {20), Schul tz {31), McClur.e and Mi tchell {25), and Smith and Leverton {35), wh o reported calcium fluoride to have 1/2 to 1/5 the toxicity of sodium fluoride . Smi th and Leverton (35) cons idered the effects on the teeth to be approximately the same wh en the fluorine was combined as sodium fluoride or sodium fluosilicate. On the average the first change in the color of the • teeth was observed after approximately 14 days on our ex perimental ration. There were variations from th is figure , depending upon the compound us ed as a supplement . White striations were firs t observed near the base of the tooth, gradually spreading with a cons tant wh itening effect toward the end of the tooth until it was completely bleached. The upp er incisors showed little signs of change until after the bottom incisors were completely bleached. None of the rats fed calcium fluoride showed any changes greater than tho se jus·t described. Rats receiving the lowe r levels of calcium fluoride , 0.015 and 0.03 per cent fluorine , re latively few individuals showed complete bleaching of the upper teeth. In the case of the rats fed more soluble comp ounds , such as sodium fluosilicate, the lower teeth started to wear and the uppers showed excessive growth about the time or even before they were completely bleached. These teeth obtained a dull chalky white color and often showed spots of erosion It 34 in the enamel. This is the same condition as described in the literature by Schul tz (31), Kick et !l• (20) and many others . The other compounds that were studied varied in their effects on the teeth between these two extremes , when fed at the same conc entration in the ration . Potas s ium fluosilicate produced a little earlier and more severe effects than did calcium fluosilicate, magnes ium fluosili cate, potassium fluoride, or sodium fluoride , these latter comp ounds appearing to have approximately the same effect. This is not entirely in agreement with Smi th and Leverton · (35), who reported the effect on the teeth of the albino rat to be approximately the same wh en fluorine was combined as sodium fluoride , potassium fluoride, barrium fluosilicate and sodium fluosilicate . The effects on the teeth were approximately the same wh en the supplement was in the form of synthetic cryolite or rock phosphate. Both of th ese produced more pronounced abnormalities than did natural cryolite fed at th e same level. This work parallels to some extent the study by Evans and Ph illips (12) where th e feeding of twice as much synthetic cryolite was required as wh en sodium fluoride was fed to produce the same ab normalities in the teeth. Smi th and Leverton (35) required 10 times as much natural cryolite as was required of sodium fluoride to produce the same tooth changes when the natural 35 cryolite was fed at 0.0226 per cent of the ration. A study was made with a graduate d level of sodium fluoride (groups 16 through 16 g) to determine the con centration necessary to produce recognizable signs of fluorosis in the teeth in a 15 day period. At the end of this period a slight striation could be observed, wh en the teeth were studied with the aid of a hand lense, in the group that received 0.0025 per cent fluorine in the ration. The group that received 0.0050 per cent showed definite striations and a slight bleaching effect at the end of 15 - days. The visible symp toms of fluorosis increased with the concentration of the fluorine carrier for the rest of the groups in this study. Two groups were fed a ration con taining 0.010 per cent fluorine. The only difference being that one contained plain salt (NaCl} and the other iodized salt. The iodized salt seemed to retard the bleaching effect of the fluorine for 3 to 5 days, but after that time no difference could be observed. In the study of the animal s that were fed to study the toxic effects of hay that was obtained ne ar an aluminum plant, the symptoms, as shown by the teeth, appear to be approximately the same as those obtained by feeding the same level of sodium fluoride . A little more wear was ob served, however, in th ese animals than in thos e receiving 36 so dium fluoride at the same fluorine level. This wear resembl ed greatly that obtained by the. feeding of the fluos ilicates, but , probably due to the low concentration in the ration, was not as severe . The addition of 2 per cent and 4 per cent defluorinated phosphate (group 13 a and 13 b ) to an adequate ration con taining 0. 015 per cent fluorine in the form of sodium fluoride, appeared to produce more normal tooth develop- ments than did their cont rol group (2d) . The action of the fluorine on the teeth was not completely averted but ap peared greatly ret arded. The teeth of the animals in group 2d were completely bleached on both the upper and lower incisors and showed some wear at the end of the feeding period. Group l3b had complete bleaching of the bot tom teeth but the top incisors were a normal pink color and showed no visible wear . This was probably caus ed by the increased calcium and phosphorus cont ent of the ration. However, McClure and Mitchell (25) reported that the calcium balance of rats was not affected by sodium fluoride at levels of 0.0105 per cent and 0.0313 per cent fluorine in the ration. They did report however, that a level of 0.0623 per cent fluorine in the form of so dium fluoride lowered the per cent of calcium that would have otherwise been re tained by the experimental animals . 37 No record was maintaine d of the feed consumed by each group . It was no ted however , tha� in the higher levels , above 0.015 per cent fluorine , the feed wasted in creased with the fluorine concentration in the ration . In mo st of the comp ounds studied, an increas ed con centration of fluorine in the ration caus ed a decrease in the total gain as shown in Table II. The most noticeable exception to this was wi th the gr oups fe d magnesium fluosilicate . The week to week decreas e in gain wi th in one group was not consistent. It mus t be remembered however , that the we ekly gains were taken only over a four week interval , a period of time considerably shorter than mo st previous wo rkers have used. As an example of the time other investi gators have continued their work, Smi th and Leverton (35) fed their animal s for a 6 we eks period, wh ile Schul tz (31) continued his investigation for approximately 17 we eks. This could pr obably ac count , in part at least, for the almost equal gains made wh en some of our groups were fed different levels of th e same compound . Some of the gr oups had hay wh ich was known to con tain fluorine subs tituted in th e bas al ration for the de hydrated alfalfa leaf meal . When studying th e decreased total gains made by animals receiving th is ration, con sideration should be given to th e fact that it pr obably contained more fiber and lower T.D.N. than the me al wh ich 38 it replaced. The study made using graduated levels of sodium fluoride does not show a proportional decreas e in total gain with an increase of fluorine in the ra tion as presented in Table II . The highest level fed was probably significantly lower than the control group , but because of the small number of experimental animal s and the length of time on feed, mo re study is needed before definite conclus ions are drawn . Thi s is in accord with Smith and Leverton ( 35) who required a ration containing 0.0226 per cent sodium fluoride to produce a slight stunting of growth. The fluorine analys is of the bones of the experimental animals as presented in Table I are in line with the findings of earlier workers . McClure ( 24 ) reported the average of fluorine content of normal bones to be between 0.01 and 0.03 per cent . Th is range , according to Ma jumdar et al . (21), will vary with the age of the animal. The fluorine content of the bones from all the control animals in this experiment was 0.026 per cent . McClure (24), Schultz (30) , Ki ck et al . (20), and others have reported an increase in the fluorine content of the bones wh en this element was added to the ration . The findings from th is work are in complete agreement with the findings of the se investigators . 39 The experimental animal s wh ose rations were sup plemented wi th fluorine combined as calcium fluo ri de stored less fluorine in the bones than any other group receiving the same concentration in the ration, as is presented in Table I. The groups showing the greatest storage of fluorine were the ones fed fluorine in the form of potassium fluosilicate and sodium fluosilicate . More fluorine was stored in the bones of the animals wh en sodium fluosilicate was fed at a level of 0.0150 per cent than when potassium fluosilicate was fed at the same level . However, at the 0.030 and 0.060 per cent level of feeding , mo re was stored in th e bones wh en combined as potas sium fluosilicate . In all groups fed comp ounds containing fluorine at th e higher levels of feeding--0.015 to 0.06 per cent fluorine--there was one group which received a ration con taining 0.030 per cent fluorine . Therefore, using this level of fluorine as a basis for comparison, the comp ounds resulting in the greatest storage of fluorine in the bones were in this order: potas sium fluosilicate, sodium fluosilicate, potas sium fluoride , sodium fluoride , ro ck phosphate, natural cryolite, synthetic cryolite, calcium fluosilicate, magne sium fluosilicate, and calcium fluoride . Th ere is some variation from this , however, wh en cons idering the fluorine comp ounds comb ined in the concentrations of 0.015 and 0.06 per cent of the ration as shown in Tabl e I. 40 Each of these groups were composed of three rats , and one bone sample was tested for each of th ,se rats , and these individual averages compiled in Table I. Consequently , a slight error in analysis or individual differences of the bon es analyzed could cause a distorted view of the whole group, because of the limited number of animals used in each group . The amount of fluorine deposited in the bon es from the different di etary compounds were not always in the same order as expected , using the wear and bleaching of the teeth as the criterion for judgment . Sodium fluosilicate and potassium fluosilicate on this basis would have been expected to store the greatest per cent of flu6rine, and calcium fluoride would have been expected to have stored the smaller percentage . This was true in all levels tested when th e fluorine made up more th an 0.015 per cent of the ration . Again, judging from the changes observed in the teeth , one would have expected calcium fluosilicate , magnesium fluosilicate , potassium fluoride , and sodium fluorid e to have deposited· approxim at ely the same amount of fluorine in the bones . Sodium fluorid e and potassium fluoride did deposit approximat�ly the same amount of fluorine in the bones when fed at a concentration of both 41 0.015 and 0.03 per cent of the ration . The amount de- posited was app roximat ely 60 per cent to 70 per cent of the amount deposited by sodium fluosilicate and potas sium fluosilicate . However, the chemical analys is of the bone s showed less fluorine stored wh en the rat ion was supplement ed wi th calcium fluosilicate and magnesium fluosilicate at 0.015 and 0.03 per cent of the ration than was found wh en the same concentration of natural cryolite, rock pho sphate, or synthetic cryolite was fed. Both of the se comp ounds resulted in a gre ater retention of fluorine wh en fed at 0.06 per cent of the ration than did synthetic cryolite fed at the same concentration . Magnesium fluosilicate f showed a gre ater storage of fluorine in the bones than did rock phosphate at th is high level of feeding as shown in Table I. The feeding of natural cryolite resulted in a higher fluorine content of the bones than rock ph osph ate when both we re fed in such an amount to supply 0.015 per cent fluorine in th e ration . However, wh en the ration contained 0.03 per cent fluorine , the ration supplemented with rock phosphate caus ed a greater retention of fluo rine in the bones than did either natural cryolite or synthetic cryolite . There was little difference in fluorine content of the bones of rats when the sba al ration was supplemented 42 with either rock phosphate or synthetic cryolite to make the total fluorine content up to D6 per cent . Natural cryolite caused a slightly higher storage at thi s level, however, than either rock phosphate or synthetic cryolite at th e same levels . This is not in agreement with earlier workers , Smith and Leverton (35), wh o have reported synthetic cryolite to be mo re soluable than natural cryolite and, probably for thi s reas on, more toxic . When the same comp ound was fed at different levels , the amount of fluorine stored in the bones was , in mo st cases , proportional to the concentration in the ration. Th ere are some exc eptions to this however, as observed in I Table I. Thi s is in agreement wi th the work of Schul tz (30) who reports that the feeding of fluorine to the albino rat will cause an increase in the fluorine content of the bones , but not always strictly in proportion to the amount in the ra tion . The study of the animals that received hay wh ich was known to contain a high per cent fluorine , substitut ed into the basal ration for th e dehydrated alfalfa leaf meal , present s an interesting picture . Group l2b, wh ich received a ration containing this hay and enough sodium fluoride to make a total fluorine content of 0.015 per cent of the ration, showed a greater storage of fluorine in the bones than did similar animals receiving only sodium fluoride at 43 the same per cent of the ration (Group 2a ). Th is would suggest that the fluorine present in the hay was comb ined in some form which was more toxic than sodium fluoride . The same condition was pres ented in group 2c and 12c as shown in Table I. The ration fed both groups wa s calculated to contain the same amount of fluorine , but the group re- ceiving the high fluorine hay showed a greater storage of fluorine in the bones . Group l4a received a ration con- taining no fluorine supplement added and was calculated to 1 contain 0.0021 per cent fluorine . Group 2e received a ration to wh ich sodium fluoride had been added to make the entire fluorine content of the ration 0.0025 per cent . I Group 14a showed a gre ater storage of fluorine than did group 2e even though the total fluo rine content of the ration was less . This would strengthen the previous statement th at the fluorine in th e hay was app arently comb ined in a form more toxic than sodium fluoride . When defluorinated ro ck phosphate was added to the ration containing fluorine , the amount of fluorine de- posited in the bones ap peared to be slightly retarded in most cases . Group s 2d, l3a, and l3b received a ra tion lThe hay that was substituted into the basal ration in the amount of · 30 per cent contained 0.0070 per cent fluorine . 44 containing 0.0150 per cent fluorine in the form of sodium fluoride . This ration was supplemen�ed with defluo rinated ro ck phosphate as shown in Table I. At this level of feeding it required 4 per cent of the pho sphate to bring about a slight reduc tion in fluorine storage in the bone . However, when the fluorine content of the ration was only 0.005 per cent , as is the case in groups 2c and 15a, 2 per cent defluo rinated rock phosphate caus ed a slight de creas e in the amount of fluorine stored in the bones . This would suggest that the higher concentration of fluorine in the feed will require a relatively higher per cent of mineral in the form of defluorinated rock phosphate to partially alleviate the toxic effects . Becaus e of the small number of exp erimental animal s involved, there was very little definite information ob tained from the study of fluorine analysis of the bones of rats maintained for different lengths of time on the graduated level of sodium fluoride as presented in Table III. These animal s showed cons ide rable variance so that the differenc e be"tween lots is not graduat ed in direct proportion to the per cent of fluorine in the ration . However, the per cent of fluorine in the bones did decrease in all cases when the animals were taken off the supplemented ration and returned to the basal ration for a 15 day period. SUMMARY AND CONCLUS IONS The previously reported wo rk of many investigators that the supplementat ion of a ration with a fluorine com pound would cause abnormal calcification of the teeth, re tarded growth and an increase in the fluorine stored in the bones was supported by the evidence presented in this study . Evidence was presented to show that there was a dif ference in the toxic effects of fluorine, depending up on the substances with which it is comb ined. This toxic effect does not always vary directly with the solubility of the compound . Evidence of fluorine poisoning was observed in the teeth of young growing albino rats in 15 days when as little as 0.0050 per cent fluorine was present in the ration in th e form of sodium fluoride . It was indicated that the fluorine pres ent in legume hay grown near an aluminum plant was more toxic than sodium fluoride . Experimental evidence indicated that the addition of an excess amount of a mineral, such as defluorinated rock phosphate , would retard to some extent the toxicity of fluorine in the albino rat . BIBLIOGRAPHY 47 Bibliography 1. Arkans as Agricultural Experiment Station 28th Annual Report , 1926 . Bul . No . 21 5, pp . 23-27 . 2. Bardell!, P. and C. Menz ani . 1937-38 . Fluorosis Atti . 1s t Veneto Sci . Part II, 97 , pp . 623-647 . 3. Bethke , R. M. and C. H. Ki ck. 1933 . The effect of fluorine on reproduc tion and lactation in swine . Ohio Agr . Expt. Sta. , Bul . 516 :83 ( 51st Ann . Rpt .) 4. Be thke , R. M. , C. H. Kick, B. H. Edgington, and O. H. Wilder. 1929 . The effect of feeding sodium fluoride and rock phosphate on bone development in swine . Ame r. Soc . Animal Prod. Proc ., pp . 29-33 . 5. Bohstedt, G. , E. B. Hart , J. G. Halpin, A. R. Lamb and P. H. Phillips . 1931 . Study being made of safe limi ts for ro ck pho sphate as a mineral feed. Wi s. Agr. Exp t. Sta . Bu l . 421, 105-107 (Ann . Rpt . 1930- 1931 ). 6. Buckner, C. D. , J. H. Mart in and A. M. Peter. 1928 . The relative utiliz ation of different calcium com pounds by hens in the produc tion of eggs .· Jour . Agr . Res ., 36 : 263-268 . 7. Buckne � G. D., J. H. Martin and W. M. Insko, Jr . 1929 . The relative utilization of certain calcium com pounds for the growing chick. Poultry Sci . 9:1-5. 8. Carter, R. H. 1929 . Solubili ties of some inorgani c fluorides in water at 250C . Ind . Engin . Chem. 20 :1195 . 9. Carter, R. H. 1930 . Solubilities of fluosilicates in water. Ind . Engin . Cham . 22 :886-887 . 10 . Churchill , H. V. , J. R. Rowley, and L. H. Martin . 1948 . Fluorine content of certain vegetation in a wes tern Pennsylvania area. Analyt ical Chem . 20 , p. 69 . 11 . DeEds , F. and J. 0. Thomas . 1933 . Comparative chronic toxicities of fluorine comp ounds . Soc . Expt . Bi ol . and Med . 31 :824-825 . 48 12. Evans , R. J. and P. H. Phillips. 1939 . A study of the comparative toxicity of cryolite fluorine and sodium fluoride for the rat . Ame r. Jour . Physiol. 126 :713-719 . 13 . Fo rbes , E. B., J. 0. Halverson, L. T. Morgan, J. A. Schulz , E. B. Wells , C. H. Hunt , and A. R. Winters . 1921 . The utiliz ation of calcium comp ounds in animal nutrition . Ohio Agr . Exp t. Sta . Bul . 347 :1-99 . 14 . Gaut ier, A. , and P. Clausman. 1916 . Fluorine in the vegetabl e kingdom. Compt. Rend . Acad. Sci., 162, 105 . 15 . Greenwood, A. A. 1940 . Fluorine intoxi cation . Physiol . Revs . 20 : 582-616 . 16 . Halpin, J. G. , and A. R. Lamb . 1932 . Th e effect of ground phosphate rock fed at various levels on the growth of chicks and egg produc tion. Poultry Sci. 11 :5-13 . 17 . Huffman , C. F. , and 0. E. Re ed. 1930. Result s of a long- time mineral feeding experiment w�th dairy cattle . Mi ch . Agr . Expt. Sta . Cir. Bul . 129 :1-11 . 18 . Jac obs , D. D. , and D. s. Reynolds . 1928 . The fluorine content of ph osphate rock. As soc . Off . Agr . Chern. Jour . 11 :237-250. 19 . Kennard, D. c., and P. S. Whi te . 1922. Mineral mixtures for growth of chicks and egg production . Ohio Agr. Expt. Sta . Mo . Bul . 7:171-177. 20 . Kick, C. H. , R. M. Bethke , B. H. Edgington , 0. H• M • . Wilde r, P. R. Record, W. Wilder, T. J. Hill, and S. W. Chase. 1935 . Fluorine in animal nu trition . Ohio Agr. Expt. Sta. Bul . 558, pp . 1-77. 21 . Ma jumdar, B. N. , S. N. Ray and K. C. San . 1943. Fluorine intoxication of cattle in India. Indian Jour . of Ve terinarin Scienc e 10 :95-107 . 22. Marcovitch, S. 1928 . Studies on toxicity of fluorine comp ounds. Tenn . Agr. Expt . Sta. Bul . 139, pp . 1-47 . 23 . Maynard, Leonard A. 1947 . Animal Nutrition . McGraw Hill Book Company , 2nd ed. New York, pp . 161-166 . 49 24. McClure , F. J. 1933 . A review of fluorine and its physiological effects . Physiol . Revs . 13 :277-300. 25 . McClure, F. J. and H. H. Mitchell . 1931 . The effect of fluorine on the calcium metabolism of albino rats and the composition of the bones . Jour . Biol . Chem. , 90 : 297-320. 26. Meyn , A. and K. Viehl . 1941 . Chronic fluorine poisoning in cattle. Arch . wiss. prakt . Tierheilk. 76 :329-339. 27. Phillips, P. H., E. G. Hart , and G. Bohs tedt . 1934 . Chronic toxicosis in dairy cows due to ingestion of fluorine . Wi s. Agr. Expt . Sta. Res . Bul . 123 , pp . 1-30 . 28. Reed, 0. E. and c. F . Huffman . 1930. The results of a five year mineral feeding investigation with dairy cattle. Mich . Agr. Expt. Sta. Tech . Bul . 105, pp . 3- 63 . 29. Seidell, Athevton . 1940. Solubilities of inorgani c and me tal organic c0mp ounds . Third Edition Vol. I, D. Van Nastrand Co. New York . 30 . Schultz , J. A. Effects on the ingestion of fluorides upon the teeth, bones, blood, and tissues of albino rats . Iowa Agr . Expt . Sta. Rep . on Agr. Res. for 1936 :78-80. 31 . Schultz , J. A. 1938 . Fluorine toxicosis on the albino rat . Iowa Agr . Expt . Sta. Res . Bul . 247, pp . 160-242. 32 . Schultz , J. and A. R. Lamb . 1925 . The effect of fluorine as sodium fluoride on the growth and re produc tion of albino rats. Science 61 :93-94 . 33 . Smith, M. C. and E. L. Lantz . 1933 . Experimental production of mottled enamel . Ariz . Agr. Expt . Sta. Tech . Bul . 45 :327-359 . 34 . Smi th , M. C., E. L. Lantz , and H. V. Smi th . 1931 . The cause of mottled enamel, a defect in human teeth. Ariz . Agr. Expt . Sta . Tech. Bul . 32, pp . 253-281 . 35 . Smi th , Margaret Cammack and Ruth Leverton. 1934 . Comparative toxicity of rluorine comp ounds . Ind . Engin . Chem. 26 :791-797 . 50 36. Taylor, G. E. 1929 . The effect of fluorine in dairy cattle ratiorr. Mich . Agr. Exp t. Sta. Quart . Bul . 11, No . 3, pp. 101-104 . 37 . Willard, H. H. and 0. B. Winter. 1933. Volumetric method for determination of fluorine . Jour . Ind. Eng . Chem. Analyt . Ed . 5:7-10.