OF- M TOXICWY OF HEXAVMEM Thai: for the Dog!” of Ph. D. MECHGAN STATE UNWERSITY Robert D. MacKonzio 1957 THESIS mcmcw STATE"U§~:1§‘€R31W EAST LANSHG, MlQHQAN LIBR .-1 .‘ Y Michigan State University "mm/nm / Ifm/I/I/I/WW/Wfi 11m 1/ " ' ' 3 1293 01088 0390 PLACE N RETURN BOX to roman this checkout from your «cord. TO AVOID FINES Mom on or baton date duo. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Adlai/Equal Opportunity IndMon Wanna-9.1 N-.._~—.—‘_——v—-————— ———- A STUDY OF THE TOXICITY OF HEKAVALEET AHD TRIVALENT CHROHIUH IN THE ALBINO RAT By Robert D. MacKenzie A THESIS Submitted to the College of Advanced Graduate Studies of Eichigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILUSQPHY Department of Chemistry 1957 manomeeemrrs The author wishes to express his sincere appreciation to Dr. Carl A. Hoppert, whose advice and interest tOgether with his patience and encouragement have greatly facilitated the completion of this uprk. He also wishes to thank Dr. Richard U. Byerrum for his helpful advice and interest in this work, as well as the other members of the Chemistry Department. Also special gratitude goes to er. Leo Klever, Foreman of Caretakers, Vitamin Assay Laboratory, for his assistance and helpful suggestions. In addition, the writer wishes to thank the United States Public Health Service for providing funds in support of this work. WM ii VITA The author was born August 18, 1928 in Chicago, Illinois. After receiving his elementary school education in Chicago, Detroit, and Cincinnati, attended Woodrow Wilson high school in washington, D. C. He was then in the United States Army for 18 months serving with the 82nd Airborne Division, after basic training. He entered the University of Cincinnati in September 19h8, and was graduated in June of 1952 with a Bachelor of Science Degree. During the summer of 1952 he worked at the William.S. herrell Company as an organic chemist. He enrolled in the Graduate School of Michigan State College in the fall of 1952, obtaining a Teaching Assistantship in December of that year. He re— mained at this position until receiving his Master of Science Degree in June 19Sh presenting as his thesis "The Use of Radioactive Ergosterol in the Study of’Lipid Absorption in the Albino Rat." He resmned his studies at z-aczragan State University in the Fall of 19524 as a Special Graduate Research Assistant under a United States Public Health Service Grant, which he held until the completion of his graduate program. He is married and has a son R. Bruce and two daughters Barbara and Catherine. A STUDY OF THE TOXICITY OF IIEIXAVALEWI' AND TEZIVALEZJ‘T CHECK-[HIM IN TEE ALBIEQO RAT 133' Robert D. hacKenzie AN ABSTRACT Submitted to the College of Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree or DOCTOR OF PHILOSOPHY Department of Chemistry Year 1957 Approved *9, Q- - j_j[i1:lij"%u __ L: J LETS-ACT T1 re mtemries imustri L1 use of c rzzo Stu-.1 emf the tisposal of afraid-tin wastes rarer suit :c in the cents dnatien sf rive: r an: well water supplies in certain areas of tide country. Lit tie is knots) ahcut the effects of the ingestion sf mull ain't-arts cf c3: ermine over a long; period. t was, therefore, the gar-1:335:24 of tE-is starry to eaterrdns at it at concentration in water harmful eifects rdght appear in albirw rate. It was heped that the results tar-132:1. lead to a more realistic (timer-rims stanc‘aard fer potable veater empires. The initial agrarian-ht with 35? day oid albino rats int-elven cone en- tratiens of chroxtzate ion ranging; {We 3 1 to 23;? parts per million in the water supply used in conjunction wit“; an adequate diet. Diced stur‘ies were eerie at ashtray inter vals seer a peridd of a year. its airfares-mes in the hem rlehin level or in the oil-ran .ntial u.‘ .it :3 cell cat-3t were ohnerved hetwem the experimental and corrtr cl mung-m. At the end of six mat a one rat of eac‘r s -.:. tats sacrificed to CU‘DQI'LBJI‘E t'r. e c2 we mid-n content of the livin‘, kidney 9- if f tar and to study tissue sections fer pretzels-{final cinemas. at the era} of the yes the rerrwiflm; animals were sacrificed and the spleen femur-Lax} in t: n arts-nineties tr tissues. There was no aria-canes of pat-relay in the tiesu- '25 star ied. The accumlatlen of c z’fl min-r1 in the tissues was fairly 810:! up to 5 ppm but increased rapidly at hijier D‘K‘n‘dtaau..0h3 emf: continued t1. .rou; vheut t .e period sf cheervatien. In OILS“: to caterpitina the 1.22.2? 21mm cf £62625. on the ate-:2- tic“ -.- of cur-2e 222% ion, rpm-pa cf starter? am nan-strayed rate were 'i-ven 12.22.: io~ active cit-re "5.2.2271 (-5312 ) by stew-cf: tube. The rate we re 2 '3. mac. 22.21 222-3t.22'=:.)<21- ism ca. 3223 an? aacrii‘i. 22d at ’3, 2..., 7'.., am) 1’23 hour a after lags-mien. The ad...“ t..3.' in ‘L: 2? 1:."231', iii-2:122:23“ blom‘, stair: 2.91:2?) -, int ‘Etvw 11:21:23 2.21:3. nHAI feces was Cirteflfs-nzsd. Aenroaszumly 5.3;}. (2.2:? t :a (es-3 me a?) arvwm by the eta-2'2 9:21am 22.2222. 0:2 '5: 2.573 by the non-suntan In moth-.2212 sari-:6 a corpa. "is-1.222 1.22223 via-tic in the :fi2sezgqtien of 1a- 2:). talent 3.22:2; tr‘rw 3.222.. Cit-.1153 (-r )2?" ' stare-ad :2sz 212:. 22-53): “e? rats, the Case (2:12:22): being given by atom 2722.- tuhe. Fee-:- 12:22 "a sitar 122;; action the rats we. - mutt 2. Liza hieoda 22.32- ext? 112202-22. the heart 9.22:?- aft-2r centrifuging, activity cmmta rams-i=3 on plane-2a .122. 92.x: calls. The remitts imicated that herrivalmt (2122202262123 1'1‘35 22528922131?- tea 2 2222 {22.22.2222 -. tar 9.: tent than trivalent Mid cemfimecl the greater 52325013.)an pram-25.02 rely aha-arm.- 111 the eta-wed rate. 22122:) Ct) 62:25:}- was-‘2‘ was the 0125-22 watt! .5221 t at 3-222:-t;m2al:2:2t text not titty: ant ail-21’22223-‘m22 can be 93mm: 2222:! hyt he 120:3- {21:22 3:. calls In :1 23.122223 522.2105 radioactive c?‘:r<::‘:(‘2.m:) 1:2 “- e fem of 5:255. chic-nets: , c.2- :.ranfi.c c.2larice, amid iii-€123 of egg-1:21 parts of car-2'2 was in jute-1 into ti‘zt': 8.15211 int 29.2.1 2-7.2. F3219 he 22:29 later 121(23):}. was 9:22....vr-x1 from the heart as bafare, cm‘rtL 2‘2 .21‘2;.;;2=.2‘ and t... 2e a2sti‘-.°;'-.ty of the pizza-m and ratio 222113 c 3t Jz'zfiin ad. The acti‘.‘-‘:-t'=f o'c'.)s-:~.21*J-=}2:3 $3532.32 the Laxatala‘mt f0; .21 was 32:3. ”202‘ Man ..at 10.1Fifii 123' 3112“?" aficctian, i:2:.‘.icatin;; that censidarable reduction to tie b.11- 9.1.2221. tit-.29 02.322.12.28 in tie tat-22:25:22.?“ "‘ 2- ‘ .‘ *"\‘ 52‘ "1 int .“fi ‘0‘ I «71" ‘4’: 1- {Lu '.‘| 2 " a T126 bloc-.1 of t-:,.'2"3 3.1mm a .22.... :3 222.20..- sier- .2" 4.2-2.21 .52 tan t at or be non-starved . A ‘7‘ Jtal co 23;».31'19011 Wes ram 83 0.3! 13M) 1.:2‘3119291-2- of? 1:337:12- :1;j;:2s;". “u".‘nsa'tltm of water (:oz‘xtaia'zing 32mm: ant 0:4. triv alazi c'. yes-2.3.2.12: engiz'alazzt to 213' {.3331 c. ratfii‘m. ‘t tin and; ofa “t1” :3 rats mar: 83': Mice: arr. the 11 my , spleen, kin‘nez‘a am? fzmr amines fc-r 0129073" .1112: an; swunm‘ for rd (.1‘98001’3133 6121163. The tissues 0f rate f~f3133*“"{1"“1 rt 0' ”21.; {'3 9 v. S: f". :3 >3 '5 “WNW 2:31; 3132' f; tin->8 {$720 arc'zm‘b 370323.321“ 2.31 the trivalarfi rm" * 3.) Li .. . Thur-c mm no my. draw of pairsflyg in mg of the 64:11: wig. It is 5132;103:2311; that. haze. ugh-.1. C‘ r0 '3. an 13 31):: 9:12:23 to a :mc’n greater extent. thm trival Mt. T313 remction of cia'o:xzta xmstes bcfarm tab-1‘ t‘itposal maul? C‘Wimmly grmfi gr rechzc‘a t -«1: 322.21: tra‘uial 32.33am 3 of um: 1‘ can't: tinA 210:1 cf ii .13 123373. TAJEE OF CONTENTS 'U SD ("2 0 IIV‘TROIJ'UCTIOI‘IOOOOI...QIOCOQOOOOOOOOOOOOOOOIOOOIOCQOUOCOIOOIOOC’OOOIO }ESTCELIC¢QIIOOOOOOOOOOOOOOOOCIOOOOOOOOOOICOODQOOOOIOQOO0.0.0.0000... occurrence Of Chromium....s....a.....a..o.......u...........¢.. Chromium.PoiBoning in Hana...oooooiooooocoooooooooi000000000000 Experimental Poisoning (Animal).....“anuuanuuuuus.5. Reaction With 3101031081 Haterials....§¢.......;.....;....3.... Analytical Chemistry Of ChromiumooooooboococoiooooDSQOococo-oi. Chromium in Tissuesooonoooiooooioooaoooooo‘oooobtéoéoooouooodoo EffeCt on Enzymes.....................................o........ a. EXPERIHEHTAL AND RESULT3000000.000.000.000IQOQOQIOOOIOQ00.00.0000. ExPeriment I -' Chronic TOXiCity'Study-o00000090000003.00903300 Rat Care and DietScoioio00300000000000noooooooiooiootooootoo BlOOd Studies-000900000000.00.00.00.05.00.060000030000303..- PathOIOfiyaoooooocoo-00003.300061030006506000000000.0.6.0.0.. CthfliflM;ADElESiSoooobocuoo000000606coo-ésoOQo-oooiooooooooo ReSUltBooooooo-coo-coo.o.00....0000..taco-000.000.00.00...to Experiment II - Absorption and Distribution Studies........... quaeriment IIa ”Distribution and Percent Absorption Study. PTOCSdurBoodoococooooooooioooiooooooooobooooic00000-00000 RCSUltsoccocooooooooooooooo0-00.00.ooooooooioaoooooéoooéé Experiment IIb ‘. Absorption Study Part I...o$oooooooooo.ooo Procedure..o............a.....a..a..........o.......o.... RGSUltSococooncocooaooooaoooococo-ooccooocooo00.000000... Experiment IIC .' Absorption.8tudy'Part IIc-¢Oooéooooooooooo ProcedurE....o..soo.oo....¢oo¢.oo.o......ho.o.......oo... ROSUltS.0.0.00.0...000.00.00.09...oooooooooo-ooooooooooon Experiment III - A Study of the Relative Toxicity of Homer valent and Trivalent Chromium.«150.000.63.00...0.00.60.00.00 Rat Care and Diets-o00.00.0030ooiooooooodioiooooiooooooié BlOOd Studies....aoo...........s...o........a....aa...... PathOlOF‘cocooooovooéoooooiéooooocooooooobSooo05060000090 Chromium AnalWBiSébSoioooooooooobo0.00000000000000000000' fiemfl‘tSOCOOOOCCOOOOOOCOIOOOOOOOOOOOCOIOIQOO‘COOOOOOOOOIO. DISCUSSICI‘:OD.OCOOOOO0.00.00...00.0.0000...OIOOOQOOOOIOIIOQO'OOODOO SUE” ’1 - - 0.0...IOOOOQIOOOOOIOQOOOIOOO0.0000,.OOOOOOIOIOOOOOOOOOOOOOO BImJIOG-R-APEHOOOOUQ0.000.000.0000...0.0.0...IOOOIIOOOOOOOOOOCOOOOO. viii Etta—442$: 1:“ H LIST OF TABLES TAKE Page I A'J'erage Initial weig‘itSooocooooooocooo.0000{cooQOOOQIOQOoo 15 II Composition Of the StOClC Dietownoo00-000....coco-0000.0... 1-6 III Average 33ng Weight Of 25:8.Ch GTOIIPcovoc-oooo‘o cocoon-coco... 2O . .o IV Average Food COI'ISUIKPtiOHoooooooto...oocooooooopooouoqooon. 2]- -v A o - v V Average water Intake of Bach Group..,.......,............. 22 VI Chromium Concentrations in Rat Tissues After Six MOnths Rq305ureuoocoocooogoooooooooococo-9000.0...ooooooooooooso. 23 VII Chromium Concentrations in.Rat Tissues After One Year..... 23 VIII Percent Retention in Tissues at Various Intervals........ 2o IX Average Activities in Red Cells and Plasma...............{ 30 X Conditions in Etcporiment 110.0...ooooooooaoooolooooocoozoo. 31 . v XI Average ACtiVitiOS in 323361 Cells and Phsz‘na. o a a 0 Q o o o o o o o o 0 o 33 XII Average Eater Intake 0f Each Group"....uunuuuon-u 37 o XIII Chromium Concentration in Rat Tissues..............;...... 38 LIST OF FIGURES FIGUL‘ZD Page ,4 I Average Tissue conC‘entratj-ORSIOOOOOOOOOOIIOOIOOOOO‘OQO00.0.0 2.) 1 ix INT?! ODU CTI ON INTRODUCTION Chromium is classified as a metal and occupies a position in Group VI of the periodic table. Though it is classified as a metal, it is an amphoteric substance depending on the environment and valence. Chromium can exist in three series of compounds 1) divalent chromium (basic), 2) trivalent chromium (weakly'basic and.amphoteric), and 3) hexavalent chromium (acid). Univalent and pentavalent chromium are also found in certain reactions. However, only the trivalent and hexavalent compounds are actually stable enough to be found in water supplies or in ores. Trivalent chromium is more stable than hexavalent in solution at a.pH below 7. Chromium is found mainly as chromite ore, also called chrome iron- stone or ferrous chromite (FeCr204 or FeO-Crzoa). Rarer ores such as chrome ochre (Craos) and chromitits (Fe203-2 Craoa) also occur. Chromite is mined in Rhodesia, the Transvaal, Cuba, Greece, Turkey, India, Russia, Yugoslavia, he Philippines and New Caledonia. In the United States it is found in fairly high concentrations in Arkansas in bauxite deposits and in serpentine soils in Maryland and California. Chromium is used in iron and steel alloys to increase harden- ability, strength at high temperatures, and resistance to abrasion, corrosion and oxidation. Chromium is an essential component of high- speed steel, and of many engineering steels, stainless steel, and a large preportion of other corrosion resistant alloys. Chromium compounds are used in the tanning of leather, for plating and anodizing metals, for the production of catalysts in gasoline and synthetic rubber manufacture, in the refractory industry, and in the manufacture of certain.pigments. In the refining of chrome ores, and in certain industries air contaminated with chromic acid mist from vats or dust of chromates is the principal type of exposure to chromium. Toxicity is usually due to inhalation or tepical contact. Hoxavalent chromium is generally considered the most toxic by these routes. Dermatitis and perforation of the nasal septum are frequently observed in workers exposed to chromate. When shin abrasions come in contact with chromate liquors, chrome ulcers or "Chrome holes" devoloP. Several cases of lung cancer were reported in Germany during the world war II in.plants where workers were exposed to zinc chromate dust. An analysis of the mortality data of the chromateeproducing industry in the United States, revealed that the death rate for cancer of the reSpiratory system was 21.8 percent of all deaths (16 times the expected incidence). It has been suggested that the monochromates may be the compounds responsible for lung cancer (2). The maximum allowable concentration of chromium as chromate dust or as chromic acid mist has been suggested as 0.1 milligrams of CrO3 per cubic meter of air. In recent years, chromium.has assumed increased importance as a contaminant of water supplies. Certain industries have disposed of chromate wastes by direct discharge into streams conveniently located. Le In other instances such wastes have been dumped into large holes in the ground resulting in contamination of ground water up to 25 ppm of ' chromium. Although the effects of acute chromium poisoning are well documented little has been done to study the hazards of prolonged ingestion of water containing small amounts of chromium. There is also a difference of opinion as to whether hexavalent chromium is more or less toxic than trivalent. . At present the allowable chromate concentration in.potable water supplies is 0.05 ppm expressed as chromium. This is assumed to be well below the harmful level. However, it is not always practical to maintain so lou'a concentration. Therefore more work needs to be done to estab- lish the limits of tolerance for chromium so that a more realistic and practical standard may be established. Therefore a study was made of the absorption and retention of chromium at various concentrations in the drinking water and certain tissues examined for pathological changes. HISTORI CAL HISTORICAL gpgurrencs of Chromium Chromium is found in wide areas all over the world. In the United States it is mined mainly in California and to lesser extent in myoming, Haryland and parts of Pennsylvania and Arkansas (1). In other areas it occurs in fairly high concentrations in soil. Davidson and Mitchell (6) reported from 150-350 ppm. of chromium in eigat Scottish soils. Two other soils contained as much as 1500-3000 ppm. In the United States concentrations of 150-300 ppm. have been found. According to Negas (7) inhibitors containing chromium are being sold to prevent corrosion of water pipes. When used as directed a con- centration of 0.31 ppm. of sodium chromate or 0.1 ppm. of chromium was found in the water. In one survey (8), the chromium content of twenty- four municipal water supplies in the United States ranged from 0.001-0.0h ppm. In the United States well water has been found to contain up to 25 ppm. of chromium (9). ghromiumIPoisoning_in_§an Chromium.may enter the body in three ways thus causing different effects. Poisoning may result by inhalation of chromic acid mists or dusts, by topical contact with chromic acid solutions, or'by ingestion of chromium containing materials. dost of the work done has involved acute toxicity although there has been some evidence of chronic symptoms. In 1827 Cumming (ll) wrote about “chrome holes," ulcers which developed usually on the hands and arms of workers exposed to ohromate solutions. Papules appear first, then change to pustules and finally to deep and penetrating ulcers. Previous abrasion of the shin is essential for this type of damage. The ulcer is characteristically sluggish, the edges markedly'indurated and undermined, clear cut, look- ing as if punched out, hence the name "chrome hole." The center has a scab resting on the slough and the floor underneath is gray. A very common site for this ulcerative process is the septum of the nose. Other sites are the fingernails, the knuckles, and eyelids. Ulcers may also form on the edge of the nostrils, on the toes if the shoes become soaked with chrome water, and, rarely, in the throat. The effect is gradual, and usually not very'painful. Becourt and czwamer in 12351 (12), apparently without knowledge of Cumming's paper, noted what was to them a nev'disease of the skin, occurring in.sone chr me workers in.Paris. They wrote to physicians in other countries asking if such things had ever been seen elsewhere and received an answer from Isaac Tyson of Baltimore in 1852, confirming their observations and saying that in.haryland workmen.protected them— selves by tying a wet sponge over the nose and mouth. Ducatel (13) of the University of Naryland, added details as to the character of chrome ulcers and reported that Beer of Baltimore had seen 20 cases, caused by chrome steam, which healed only when the so h was given up. Laymann (1h) examined 722 workmen who were exposed to chromates and found ulcers and perforation of the septum in 253 or 35%, respiratory disease in 8.8%, and digestive disorders in 12.3%. The above description can be said to be that of acute toxicity. The occurrence of possible chronic toxicity in one of the chronate using industries in Gcrnuuv was reported by Gross (3). He found several cases of lung cancer resulting from :zposure to zinc cin'ornate dust during World War II. Emchle and Gregorius (LL), in a study of the mortality in the chromate producing industry in the United. States, reported that 21.8 percent of all deaths were due to cancer of the reSpiratory systm. This is sixteen times the suspected ratio. Poisoning by ingestion has not been reported to any great extent. Only in the case of the accidental ingestion of a large amount of a chromium compound or when water was found to contain chr mum have case histories been documented. In a case reported by Krieger (157) biOpsy showed parenclmnatous degeneration of the liver and on the 2nd day mild *e occurred. The urine had an or=z:.-1;,3-red color and contained I.) kidney dame. albmnn and leucocytes. The symtoms disappeared after three days. Sender and Camp (15) reported a case of poisoning in a 1‘4 month old infant. The infant ingested some chromite ore which was slozsfly released from the metro-intestinal tract. One week after ingestion convulsions, stupor, dilation of pupils and fever occurred. There were still. some symptoms six months later. David and Lieber (9) reported finc‘ing a fondly using water from a well containing 1.0 ppm of chromium. T‘s. used this water for times years tfith no apparent ill effects. Chromium was detected. by spectrograpi'ie analysis in the feces 031‘ one of the person' s checked. laggeriflental Poisoning (my) There have been no long term or chronic studies of chromium toxicity reported in the literature. However, there have been many experiments on acute toxicity. Plum (1?) reported distinct changes in serum bilirubin in rabbits three weeks after being given a daily intravenous dose of 0.001 grams of chromium per kilogram of body weight. Toxic effects reported were: hemorrhage of the msculture, gastro- enteritis with necrosis in the stomach and small intestine, leukemic changes in the blood, fatty endocarclitis, fatty degeneration of the liver and parenchynatous nephritis. In- a few cases of post-mortem exasfination of humans, shrivelled kidneys have also been observed. Gross and Heller (18) working with young rats reported that potassium chromate in drinking water caused toxic symptoms in two to three months when the water contained 1324, ppm of chromium or more. 39.6WJfiJ-h Biolocical flglg Chromium compounds are found to react with proteins and peptides to form a metalnorgano complex, also known as a chelation complex. I. P. Stralchov (19) reported that the cationic or trivalent chrorr‘ium will chelate with the carboxyl group and the hydroxyl group of hydroxy- proline or with serine whereas the aniorflc or chromate ion reacts with amino groups. There is no evidence that hexavalent chromium can be chelated. Trivalent chromium, however, is strongly chelated and is the form in which it is bound to the tissue proteins. Gray and Sterling (20) are of the opinion that when hexavalent is absorbed into the red blood cell it is reduced before combining with the globin moity of hemo- globin. In the tanning of leather Gustavson (22) has shown that even such small amounts of combined chromium as 0.5-1.5 grams per 100 grams collagen imparts a high degree of stability. Thus chromium influences the properties of the collagen and possibly other proteins ‘to a great extent. He reported that the reaction occurs best with basic chronic salts such as (05"9:$Cr) $04 and forms a modified type of internal complex salts (chelgts compounds, involving groups from different protein chains). According to this concept, the initial reaction is an ionic interaction of cationic chromium complexes, such as (CrQOSO,)nan* with the charged carboxyl groups of collagen, the sulfate ions being compen- sated by the ~NI-I; ions. The carbonyl groups, having a great tendency for complex formation and for direct attachment to chromium, penetrate into the coordination sphere, forming coordinate-covalent bonds. Since several chromium atoms are present in the large chromium complex, and in.view of the secondary aggregation of the fixed chrome complexes by further hydrolysis, possibilities exist for a multipoint interaction of one chainlike chromium complex with several carboxyl ions of the collagen lattice, resulting in the linking of adjacent protein chains by strong bonds, by means of the chrome bridge. Schuttleworth's (21) Conductivity data for gelatin solutions containing chronic salts points to the inactivation of carboxyl ions as the main reaction. .3. / Cr ..... 0H / ~. 3 \ / ‘ H0. “ NH 0 . c 'u ,,on / >c-(cz-12)2-coo .— ‘ca .. .. - - .. - ~- N - (CH2), - c\- H .r ‘. H L ’ H Iii ‘oH ' 3 ,9 ’ O \c s o / H-N / Hzouu/cr- x \ @Eical Cherigtry of Chromium Both colorimetric and spectrograptdc methods have been develOped for the detemfination of cl‘roxtium in tissues. The spectrographic method is quite complex and is generally better suited for qualitative analysis. For the colorimetric determination, chromium is oxidized. to chromate and treated with s-diphenylcarbazide. By this method as little as one part in twenty million may be determined. Urone and Anders (23) have reported that not or dry asking of tissue followed by oxidation with bromine in alkaline solution gives very good results. Saltzman (2‘4) reported an improved method. for the oxidation step. It consists of wet ashing of the tissue with sulmric and nitric acid until all orgardc matter is oxidized, removing the sulfuric acid in a muffle furnace at 550°C. and then washing the sides 01‘ the. Phillips beakers with aqua regia. After evaporating the solution to dryness on a steam bath, he residue is taken up in 0.3 N sulfuric acid and refluxed for 20 minutes with a slight excess of 0.1 1-! KIInO‘ so that a pink color persists. The excess permngante is decolorized. with a 5'55 solution of sodium azide, avoiding an excess of the latter. Since iron interferes with he color development, it can be removed by adding NaOII 10 which precipitates the iron as rages-H20. After the diphenylcarbazide is added and one minute allowed for develOpment of color, a u M solu- tion.of NaHéPO‘ is added to stabilize the color. The Optical density is then determined in the Beckman model DU spectrometer. According to M. Bose (25) the color is due to a complex formed between.divalent chromium and diphenyloarbazone. These are formed from an oxidation-reduction reaction in.which hexavalent chromium oxidizes the carbazide to carbazone and is itself reduced to the divalent state. The divalent ion forms a stable chelate with the carbazone. /”'“‘¢ 3. /“'i“" C\-0H +Cr ----‘> C-O-fr H \N-N-¢ \N-N-o This method does not differentiate between trivalent and hexavalent chromium, because the oxidation conditions would convert trivalent chromium to the hexavalent state. A means of distinguishing‘between trivalent and hexavalent chromium was suggested by Gray.gtlgl. (20) who found that hexavalent but not trivalent chromium was absorbed'by red blood cells. The ready absorption of hexavalent chromium are its retention has been.used to determine the life span of the red blood cell as well as blood volume. This difference in permeability was used as basis for studying the relative absorption of hexavalent and trivalent chromium from the digestive tract. 19hromiumLin Tissues Not too much has been done on the determination of chromium in plant and animal tissues. Stockinger (27) in a study of 107 samples of human blood found values between 0.0 to 0.022 (median 0.012) mg. Cr per 100 gm. of‘blood. Sixtybone urine samples varied between 0.0 to 0.15 mg. per liter (median 0.001). In a comprehensive study by Grushho (28) the following amounts of chromium were found in human tissues expressed in mg. percent of wet tissue: hair 0.2, nails 0.12, bile 0.08, salivary'gland O-Oh, kidney 0.028 (0.027), diaphragm 0.016, large intestine 0.012 (0.063). heart 0.01, liver 0.001 (0.013). brain 0.002, thyroid gland 0.005 (0.001.), gastric wall 0.007, cartilage 0.001, hypophysis 0.0006, spleen 0.0005 (0.01), muscle 0.0002, suprarenal gland 0.0005, pancreas 0.0002, small intestine 0.0006, lung 0.0007, blood 0.0035 (0.012), bone (0.085). Grushko also reported values for the rabbit and sheep. For rabbits he reported the following values: stomach contents 0.108, large intestine 0.16, gastric wall 0.07, wall of small intestine 0.013, claws 0.10, bone 0.09, cartilage 0.0h, heart 0.0h3, lung 0.011, liver 0.002, kidney 1.02, spleen 0.01, muscle 0.03. He also analyzed a number of foods and found the following values expressed in mg. Percent: flour 0.185, bread 0.087, semolina 0.065, buckwheat 0.028, cabbage 0.031, beets 0.025, carrots 0.011, egg (powdered) 0.003, vernicilli 0.005, fish 0.002, A sample of soil contained 0.0016, water (underground) 0.0009- 0.002 mg. per liter, river water 0.0011 (Angers) and 0.0019 (Iskat). 12 All analyses were made spectrographically. Whether these values give an accurate picture of the normal distribution of chromium is not certain, since it represents the work of only one investigator. Viseklgt.§l. (29) did some eXperiments in rats with trivalent- chromium as sodium chromite and chronic chloride and hexavalent chromium as sodium chromate. He injected Crsl (radioactive) intravenously'by jugular catheter. Sodium chromite was picked up in extremely large quantities by the tissues of the reticuloendothelial system and the liver accumulated approximately 90% of the dose. The excretion values were correspondingly low. .Considering the small amount excreted, and the high liver and spleen values at 21 and h2 days after injection, it is apparent that most of the dose was retained for some weeks after injection. The tibial epiphysis showed a higher concentration of Cru than did the other skeletal tissues, and the activity was slow in leaving all the tissues except the blood and lungs. The liver and spleen at the end of h2 days contained 33% and 50% reapectively of the activity observed at h days. _In contrast the lung showed only 10% of the activity at this time. No detectable radioactivity was found in the blood after 21 days. Chronic chloride attained its highest concentration in the liver, spleen and bone marrow, although less was retained than in the case of the sodium chromite. Considerable variation between animals, was observed but in all cases the activity'was removed.very slowly. The value for the liver at h5 days was 35% of the value observed 2h hours after dosing. In contrast, the concentration in all of the other tissues with the exception of the blood and lungs remained practically constant throughout the as day period. The blood contained practically no activity after the seventh day and at 1;,” days the lungs contained about 15% of the maxizmm value which was observed at six hours following injection. The Spleen appeared to gain activity over the 12 day period. Chronic chloride was the only compound administered orally, intra- tracheally, and intraperitoneally. Orally administered CrmCla was almost totally excreted in the feces at the end of four days. The dosage was not given but it was reported that only about 0.5% of the dose was absorbed from the digestive tract. Intratracheal injection indicated that less than 5% was absorbed from the lungs. Intraperitoneal injection of Orr-.101a caused extensive necrosis at the site of injection due to the high activity of the solution. Tissue uptake was variable and extremely small. When sodium clu'omate was injected the major uptake was by the liver, but in much lower amounts than for the trivalent chromium. The excretion was correSpomiingly higher. Twenty-five per- cent of the injected dose was deposited in the liver within half an hour after administration and at the end of h2 days less than one percent was present. This shows a marked difference in behavior from both trivalent compounds. All of the other organs lost activity with time except the spleen which gained. The concentration in the blood at the end of 21 days was about one fifth of that found at thirty minutes but declined to practically zero at L12 days. The greater retention by the blood of hexavalent chromium was due to its being absorbed and bound by the erythrocytes . The influence of chromium on enzyme systems has not been exten- sively investigated. Shimizu (31) reported that trivalent chromium stimulated the activity of pancreatic lipase. The only metals that activate it more were calcium, aluminum and ferric ions. According to Curran (32) chromium appreciably'increased the hepatic synthesis of cholesterol and fatty acids from acetate (96% to 202% . He reported that hexavalent chromium had a much lower activity than trivalent, and suggested that the hexevalent chromium.was reduced to trivalent chromium and that only trivalent chromium was really active. .. Eff? earn 3.1%]: C71? TA'L 1%?» RESULTS EXPERIMENT I -- Chronic Toxicity Study get Care and giets Sprague-Dawley albino rats 3h days old were used for this experi- ment. They were assembled into six groups, a control group receiving distilled water (I), and Groups II to VI given water containing 1.0, 5.0, 10.0, 15.0 and 25.0 parts per million (ppm) of chromate ion reapectively. The different concentrations were made up by diluting a stock solution (1000 ppm chromate ion) with distilled water. The salt used as a source of chromate ion was potassium chromate (chro‘). Except for the control group, each contained 8 males and 8 females. The control group contained 10 rats of each sex. The average initial weigjats are given in Table I. TABLE I AVERAGE INITIAL WEIGHTS ========================::=:a:;_sr-wv- vw——;fi:tl w___i__ :::__ _r__i Group Numb er . MaleBOd w% I 85 83 II 81 82 III 86 88 IV 86 8 3 v 88 82; VI 8" 81; 1 i l i ‘ l ‘ ‘ l The retainere placed in ingividual raised cages. The room I 1 _ ”D * no 0 1 J. I a temperature was maintainec between 7; and 7y F. tnrougnout tne year. All rats received, ad-ligitum, the stock diet shown in Table II. TAHLE II COi-fl’OSITION OF THE STOCK DIET WW“W Constituents Percent by'weight Ground yellow corn meal 32.5 Ground whole wheat 25.0 Powdered whole mi k 22.5 Linseed oil meal 10.0 Alfalfa 6.0 Brewer's yeast 3.0 Sodium chloride 1.0 _‘ _____ ___.._. _ M __‘ ‘._—_a_ g... v”.— w—v—v—iw—w—W ——~ Stock diet contained one micrOgram chromate ion in 50 grams of feed. w weekly records were kept of'body weight, food and water consumption. At the end of six months, one male and one female from each group were sacrificed. Tissues were taken for a study of pathological changes and chromium analysis. After one year the rest of the rats were sacrificed. Rats that died during the experimental period were examined to determine th cause of death and in a few cases tissue sections were prepared. _Qlood Studies Blood analyses were made at monthly intervals on four males and females of each group given chromium and itve males and females from 17 the control group. Blood studies included deterrinations of total red and white cell counts, differential white cell count and heroSlobin concentration.by a modified Sanford method (33). “'1, l-methyl- 1 Pathology To sacrifice the rats, 0.25 ml. of Halatal (sodium-stay u- Samples of butyl, barbiturate) was injected into the thoracic carit*. adrenal glands, liver, kidney, the following tissues were taken: spleen, brain, heart, stomach, duodenum, ileum, colon, and cross-sections Fixatives used were 10% of bone marrow from the sternum.and femur. neutral saline-formalin (standard stain.procedure for fat), Carnqy's All the tissues were stained fluid for glycosen, and Zenker's solution. with hematoxylin and eosin. Small portions of the liver, kidney, and adrenal gland were stained with Best's Carmine for glycogen and Sudan IV for fat. Qhromiun A3§£I§£§ The tissues from the rats sacrificed at 6 months were liver, kidney Those from the rats sacrificed after one year were liver, and £5337le- The tissues were frozen in a deep freeze at kidney, Spleen and femur. -lS°C. until analyses were performed. A weighed sample of each tissue was wet ashed on hot plates in 250 ml. Phillips beakers covered with watch glasses using concentrated sulfuric acid to char and then concentrated nitric acid to oxidize the ———v 1 All pathological studies were performed by the Department of Animal Pathology, H. S. U., under the direction of Dr. Robert F. Langham. 18 organic material. When all org, rue material had been oxidized, ti';e beakors were placed in a muffle fi1rnace at 530°C. and the sulfuric acid renoved. Subs-eqaent 3*, 5 ml. acpia regia was added (2 H1103 : 1 £101) and heating continued for about S minutes. The watch glasses were then remved and he beakers placed on a steam bath until they just became or: . he residue was dissolved in about 10 ml. of 0.5 N H3804 and 6 23 NaGH was added to precipitate most of the iron. 'rl'lis was centrifuged and the supernatant just acidified. A few drops of 0.1 N B22110“ were then added and the solution refluxed for 15’ minutes. If at this stage the pink color disappeared, enough permanganate was added to maintain the color for at least 5 minutes. This solution was then decolorized by adding a few drops of 5'93 sodium azide solution. The solution and wasE-dngs were combined, centrifuged and the supernatant transferred to a 25 m1. volumetric flask. Ten milliliters of 0.5 N 112804 was added, and then 1.0 ml. of dipnenylcarbazide re gent (0.62:3 gm. S-Diphenylcarbazide, 10 gas. phthalic antg'dride in 250 ml. of 95% ethanol, See Saltzman (210). After letting the color develop for one minute 2.5 ml. of 14 M Nal’IBPO4 was added and the solution adjusted to _ the mark with double distilled water (all reagents were prepared with double distilled water, distilled from glass in the presence of K3104); The Optical density of each solution was determined about 30 :finutes after color dove].0pment, at 52.3.0 m in a Beckman model LU. A standard curve was prepared using known quantities of ci'zroz'aate ion. The steward obtained indicated that Beer's law held between 1.5 ug. mid 52 ug. (upper limit of readings) in 25 ml. of solution. 19 The average weights at various periods given in Table III show .that there were no significant differences between the groups. Values for the average food consumption (Table IV) and water consumption (Table V) also shou'no significant differences between the various groups. he blood analyses indicate that the experimental groups did not differ significantly from the control group. In the microscopic study of tissue sections, very little was noticed. The only changes were found at the level of 25 ppm chromate ion and involved the kidneys. The proximal convoluted tubules appeared to be slightly more vacuolated than normal. There was a slight increase in the loss of albumin in the tubules and formation of nyalin casts. However, the changes were so minor that they were considered to be of slight significance. The tissue analyses for animals sacrificed at 6 months are shown in Table VI. ‘here is an indication that at low concentrations (0-5 ppm chronate ion), relatively'little stays in the tissues. However, between 5 and 10 ppm of chronate ion an appreciable increase in the rate of accumulation occurs (Figure I). The tissue analysis for rats sacrificed at the end of a.year are shown in.Table VII. The data indicate a similar trend, an increase in accumulation rate above 5 ppm of chronate ion (Figure I). 20 mom 0% men mom SN in en «3 8a emu in mm. me He mmm mrm Nam mom as con emu 3m an” «mm is 8 ma e 9e new. mom 2m New 3.... an 83 SH omm me we on E 9....“ am new mom RN m3 mm mm en 9m 3 as m HHH mam ems New one new mam 3m mam SH one mm. 5 a H can mam . m2 «.5 8m emm 3mm 3% 02 Hmm mm mm o H Jimmwr n a Emit 1 “well 1% “Mel 1 sec 1 t is r we sews wager,“ no.3.” masses was: edges...“ mag...“ Smash e3.“ egg mg,” NEW cg Ivoso 935 mail a 1; 1. e m H asses $55... :3 25 5mm mg no BEN—”ma. Hnom megabd .HHH was 21 mam men emm Re Sm Rm. 18... NS «3 mm mu 5 can u... mom Em a3 0% mum fie, m5 Em ma > a? Sm ed... 3% 3e 8 amm one mmm mmm. 2.. B Ham Hams mmm mmm ma. ohm mum m3 93 owm m HHH Rm 3m 8m eon m3 Em mom m8 ems. mam a HH Sm I... 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Cr/gm.Tissue —+— Female -I Year -e— Female -6 Months ’,. -o-'- Male---l Year ,x” / -9- Male --— 6 Months //’ / / // ° / / / a I I I I I I I, /,e I / , / I ' / I / I / , / I / / I / I // I, / , / / ’ a / . , / a ,l / l // / / x/ / / /’x // / " /// a-flfie.” 5 IO . .15 ' 25 mamas II - AJBORPTION AND DISTRIBUTION STUDIES Eqperiment IIa ~- Distribution axzd Percent Absom tie on Stqu Procedure Thirty-two male albino rats of the Sprague-Dawley strain, weighing about 2-130 gms. (2345-271), were divided into two equal groups. One group was starved for 2h hours before injecting the chromium, whereas 0.01). tiie other group was fed ado; 1tum. The same diet was used as in Experiment I (See Table II). The starved animals did not receive feed until two hours after the ciL ronium was injected. Each rat was injected. by stomach tube with a so: ium c2 II‘OI iate solu- tion containing ra*u oactive Chromium (Crs 1). The injecting dose was 51 57 micrograms of chromium, wiich contained 22.8 microcuriss of Cr in a volume of 1.5 ml. The sodium chronate was prepared by aiidizing a: ' Sr 813 witn hyurOg en peroxide in 6H NaOH. he excess hydrOgen.peroxide was removed.by long simmering on a hot plate and the pH of the final solution adjusted to 7.5. After the rats were injected, they were placed in metabolism ca gee. Feces and uring,were collected separately. This was facilitated by Spraying he metabolism cages with an 8C17110 plastic and placing a similarly treated small mesh screen at the bottom to prevent the feces from falling into tlze bea‘cer used for collecting the urine. The feces were removed from the bottom of the cage daily to minimize contamination of the urine. The rats were sacrificed at intervals of 6, 2h, 72, and 168 hours. L’ver, kidney, stomach, intestine, ans blood, as wellm the urine and 27 feces were selected for analysis. The samples were prepared for count- ing by partial oxidation with concentrated nitric acid and some hydrOgen peroxide. A 2.0 ml. sanple representing all or a known portion of each sample was counted in a well-type scintillation tube containing a thallium ctivated sodium iodide crystal. The counting was done at 1500 volts and the normal.background was 188 counts per minute. Results The average activities, expressed in terms of percentage of the injected dose, are found in Table VIII. Since the total amount of chromium injected was only 57 micrograms, this corresponds approximately to what a rat would ingest daily by drinking water containing 2 ppm. The distribution would be comparable to that found at the beginning of a chronic experiment; It appears that considerably more chromium is absorbed in the starved animals than in the non-starved. This can'be seen readily‘by comparing the activities in the urines of both groups at each interval. It also shows that the greatest part of the injected dose is not absorbed, but is excreted in the feces. 2“." a? "b .L ‘ ub‘. —' WP“ Fxnerinont IIB - Absarption Sit _— In experiment Ila, it was found that there was a greater absorp- tion in the starved than in the non-starved animals. The presence of food in the digestive tract obviously decreases the absorption of chromium and it may do so by'changin; the valence. There is no 23 t I'Irlll till I'll il ti l ll. ‘1 1 IIIII 111 ‘11 1'1 ll4‘ 1 i 1‘ m.a0 ~.m0 0.00 5.40 0.00 p.h0 0.0m :.Hm anesooea smmueee 0.m m.0m m.00 ea.00 p.00 m.~0 m000.0 me.a moose 3.0 a.m mm.w m.m ~0.H 00.“ 00.0 Ha.0 means 0H0.0 . 000.0 0H0.0 00H.0 000.0 0H.0 ~H0.0 No.0 assene .0H0.0 . 0000.0 «No.0 HHH.0 000.0 0H.0 000.0 ma.0 “span ~00.0 m00.0 _ H0.0 040.0 «00.0 0a.0 , «no.0 N0H.0 eooam H00.0 . mH0.0 . m.H m0.0 ~.0H m.a~ m.0m H.H0 maaemspaH 0H0.0 .. ma0.0 aa.0 _ 0H.0 0.H 00.0 04.0 m.~ eoaaopm . enema. smatammnmmmnemsnspm emsaepmtqoe sosnsem _emeneemraoz eoenmpm emensemueoeylmo>ammm. soapaeeo0 0ea as gm 1‘0 Amasoav sea? II 1 iii {H immom memoranafl 9:3me 1? t 1111 i 1 i I‘ll!!! g i gambfi mDOHfidb 54 mumbdea 2H ZOHBZHBMm BZMOmmm HHHb was 2? satisfactory way to differentiate_the valence states of chromium in tissues. Gray and Sterling (20) have shown that erythrocytes will absorb chromate ions but not trivalent chrommn. The next two experi- ments use this difference in absorption by red cells as a means of distinguishing'between the two forms of chromium. Procedure Sixteen male albino rats weighing about 330 fine (308-375) were equally'dividod into four groups. Two groups received hexaralent chromium by stomach tube, four rats being starved for 2h hours before injection and the other four fec gragibitun. The other rats received trivalent chromium, four being starved and four non-starved. Each rat received by stomach tube 125 micro-curios in 1.0 ml. of solution containing 131 micrograms total chromium. The hexavalent chromium was given as the sodium salt (see Experiment IIA for prepara- tion) at pH 7.5. Chronic chloride at pH 5.0 was the source of trivalent chromium. Four hours after the rats were injected they were anesthetized with ether and blood removed from the heart before he rats died. The syringes were rinsed with heparin to prevent clotting. The blood was centrifuged and the red cells and plasma partially oxidized with concentrated nitric acid and hydrOgen peroxide. As in the previous experiment 2.0 ml. samples were then counted in the scintillation counter at 1500 volts. Results he average activities found in the red blood cells and plasma are shown in Table IX. Also shown for comparison are 'the ratios between counts in the red cells and plasma per unit volume. Although these are probably not absolute values, they do reflect the influences of valence state and alimentation on the amount and distribution of chromium. The results also indicate that even in the starved animals there may be some reduction of the hexavalent chromium before absorption can take place. TABLE IX VciLAGE ACTIETPIES III KILL CELLS AID FLASH. Scents per Linute Group “;"Nscr5104 '— Orgibl3 Edna Kon- Ccnditions Starved ‘Starved Starved Starved Avg. Count/m1. whole Blood 159 261 217 188 AVg. Count/ml. RBC (correctedf 313' 67 0* 0* Avg. Count/ml. Plasma (corrected? M90 592 In? 398 Ratio RBC:Plasma lxa.76 128.8 -~ - w __ ____._ _ __.‘.__— w‘ _— ww *— _— W WT *The corrections are based on the assumption that red cells absorbed no trivalent chromium. The actual counts found in red cells were lb and 10 for the starved and non-starved rats respectively and are probably due to incomplete removal of the plasma. - Experimentmllc -- absorption Studz;§art II In one valence state absorbed at a greater rate than the other or are both valence states absorbed at about the same rate? Why do the starved animals fed hexavalent chromium absorb more chromium than the 31 non-starved or those fed trivalent chromium? To answer these questions more completely the following experiment was made. Procedure Twentybfour male albino rats weighing 3&5 gms. (330-371) were used in this experiment. The conditions of this experiment were as follows: TABLE X CONDITIONS IN EXPERIMENT IIc Group Nuhber Condition ‘ ._._ _‘ __‘ A.— v—y W _v v.-..,-.- “—— v 1 NaZCrO‘ injected into starved rats 2 Naacro‘ injected into nonvstarved rats 3 ‘ rCla injected into starved rats h CrCl3 injected into non-starved rats 5 Equal mixture of both valence states injected into starved rats ' 6 Equal mixture of'both valence states injected into nonrstarved rats ‘ _ __A__. A fl‘“ ‘— __ w ————v “ ww—v— Four rats were used in each group. In this experiment radioactive chromium was injected directly into the intestine. The rats were anesthetized with ether and incisions of about l—l.5 inches made just below the lower rib in the center of the abdomen. The intestine and stomach were located and the chromium solution injected.about 1.5 inches below'the stomach. One milliliter of solution contained 375 micrograms of total chromium and an activity of 125 micro-curiae was injected into each rat. The solutions were prepared as in Experiments Ila and IIb. he equal mixture of both valence states was made by adding 5.0 ml. of the sodium chromate solution to 5.0 ml. of chronic chloride solution. The trivalent chromium formed a slight colloidal suspension. Since the particles were so small, a homagenous solution was easily maintained. The solutions were injected with a 2 ml. syringe equipped with a short 27 gauge needle. Four hours after the injections, the rats were bled from the heart with a syringe rinsed in heparin. The blood was centrifuged and the plasma carefully removed with a pipette. The red cells were washed three times with isotonic saline solution to remove most of the plasma from the red cells. The red cells and plasma were then partially oxidized with concentrated nitric acid and hydrogen peroxide and 2.0 ml. samples counted in the scintillation «zounter as previously described. Results The average activity in counts per minute for each group is shown in Table XI. The table includes average counts per minute per milli- liter of whole blood, average counts per minute per milliliter of plasma and average counts per minute per milliliter of red blood cells. The table also includes the ratios of red cell to plasma counts for each group and condition. Absorption was greatest in starved rats receiving hexavalent chromium and lowest in non-starved rats receiving trivalent chromium. 33 TABLE XI AVERAGE ACTIVITIES IN RED CELLS AND PLASMA Average Counts_per Minute Group fifififiirfifibod 'Plasma '“ Hffitfi. . Ratio Number For ml. Per ml. Per ml. RBCaPlasma "1" 1 2,500. 3,135. 1,886. 1:1.66 ’ 3 3‘ 2 1,098. 1,587. 500. 1: 3.17 e *1" 3 230 653 3 1: 217 .m 68* h 13h #33 6 1:72 52-7 5 1,19 7 37 328 1: 2.25 / 5m 6 285 1481 132 1: 3.6L; _.___ ____ ‘_ AA w— v—w‘w f It may be noted that the average count per minute per milliliter of whole blood in the starved group given the 50-50 mixture is about the same as that of the starved group receiving an equivalent amount of chromium as hexavalent by stomach tube in experiment IIb. This indicates a definite reduction in the stomach even when devoid of food. EXPERIflENT III - A STUDY OF THE RELATIVE TOXICITY OF HEXAVALENT AND TRIVALEIIT CHROZCEUM Rat Care and Diets Albino rats of the Sprague-Lawley strain were about 35 days old when the experiment was started. They were separated into three groups each composed of 9.females and 12 males. More males than females were used because of the greater mortality.rate of males in Experiment I from respiratory infection. Group I was the control group, Group II was given water containing 25 ppm of chromium as potassium chromate and Group III was given 25 pmeOf chromium in the form of chronic chloride solution. The rats were placed in individual cages. The room temperature was maintained.between 75° and 79°F. throughout the year. All rats received the stock diet previously used. weekly records were kept of body weight, food and water consumption. At the end of one year the rats were sacrificed. Tissues were taken for a study of pathological changes and for chromium analysis as in Experiment I. 1.3.1095 tadias Two weeks before the rats were sacrificed blood samples were taken from two males and two females of each group for the determination of hemoglobin concentration and differential cell count. Patholo g The rats were sacrificed by the same procedure as in Experiment I. Samples were taken from the following tissues: adrenal glands, liver, b) U1 kidneys, spleen, brain, heart, stomach, duodenum, ileum, colon, and cross-section of bone marrow from the sternum and femur. Fixatives used were Zenker's, neutral 10% saline-formalin and Carnqy's fluid. All the tissues were stained with hematoxylin and eosin as in Experiment I. Small portions of the liver, kidney and adrenal gland were stained with Bower's Carmine for glycogen and Sudan IV for fat. ghromiumLAnalygig The tissues analyzed for chromium were kidney, femur, liver and spleen. The procedure was that of Saltzman (2h) described in Experiment I, but with one modification. The 2.0 ml. of h M sodium dihydrogen.phosphate (NaflgPO‘) was added to the sample solution before the diphenylcarbazide reagent. This would bind any iron.present and thus prevent its interference in the chromium complex development. A standard curve was made by this modified procedure. Results When the weight gains and food consumption were averaged at the end of the year no differences were found between the groups of females. The high incidence of disease and mortality in the males makes any comparison between the male groups meaningless. Nevertheless, it is possible to conclude that the consumption of water containing as much as 25 ppm of chromium in either hexavalent or trivalent form produced no gross changes within.a year. The water intake, however, did show a slight variation (Table XII). Rats given hexavalent chromium.drank less than the controls or those \a (3‘ given trivalent Chromium. The females consumed only 77% as much water as the controls, while the average consumption of the males was 8h% that of the controls. There was no significant difference between the water consumption of the control and trivalent groups. The blood analysis showed no differences in blood cell morphology or hemoglobin concentration in any of the groups. MicroscOpic study of tissue sections revealed no evidence of pathology due to the in- gestion of either hexavalent or trivalent chromium. The concentration of chromium in each tissue in the different groups is shown in Table XIII. The data indicate a considerable dif- forence in chromium concentration between Group II and III. Group II, that given chromate, averaged about 5 times the concentration in Group III (trivalent chromium). The values in the tissues of Group III (25 ppm Cr., equivalent to 55.5 ppm chromate ion) correspond to values found in tissues in Experiment I at levels of 5, 10, 15 ppm chromate ion depending upon the tissue compared. mm 9... R an mm Hm an R em 3 . e E um mm AN AN mm NM mm Om. mm mm +0 HH mm mm mm R mm mm mm an mm mm H828 H L } secbfihs ewehfiha seehflha emekgha ghee?“ pagans QEWm leg.” . .. _ 038%. 0.3,” edges: eds.“ 356% odwm @595 2 m 1 , n i H A3982 ed mfie Li 'Il' Il'bllll 1111 1‘ 11.11 I'll-III I 1 “Hand modw mo @295” $9.55 m0< HHN mam. [I i. ‘I’I’l SA. mm.m mmd mmd math mo.H Rim Q...” .H HH 1.” O mmém. mm.m~ mH.m m.m gum. No.3. 33m...” 5.0..” +93 HH Rd omwo 36 mod 2.6 2.5 mmd mad H9350 H 3.98% mama “338,.“ ads.“ madam...“ e3.” 3.98m 03,“ 950.5 odmmms em mammgiembno$os 03mg nmfiaby.mos seed. m 1 198m! unmaefim 1 936an I‘ll..- 1 Ilqv 1 All I“; 11 ‘14! 1 ‘1 ammmHa add EH ZOHgEfivzoo wSHMSGB HHHN mend“. DISCUSSION DIS CUSSIOI' The main.purpose of this study was to determine the minimum concentration of chromium ingested in mater that would cause patho- logical changes in the rat. No changes were evident after one year at any of the levels used (Experiment I). A study of the concentration of chromium in the tissues indicated that at low concentrations (O-S ppm of chromate ion) little accumulation occurred. Between 5 and 10 ppm chromate ion there was an appreciable increase in chromium found in the tissues (Figure I). his change in retention was observed both at the end of 6 months and at the end of one year. The results of this study show that there was rclativer little retention of chromium when the water contained up to 5 ppm of chromate ion. This indicates that small amounts of ingested chromium are efficiently eliminated. The increased retention at higher intakes means that there is a physiological limit in the elimination of chromium. On the basis of these results it would not be unwarranted to consider 5 ppm of chromate ion as a maximum allowh able concentration for a healthy) normal albino rat. Whether this would apply to other animals including man can not be said. Diseases of the kidney might seriously impair the capacity to eliminate chromium and lead to harmful accumulations. Other diseases might make a person much more sensitive to chromium. The establishing of a higher allowable concentration than the present 0.05 ppm of chromium will have to be post— poned until further work on the metabolism of chromium has been done. ho Experiments I and III indicate the presence of very low concen- trations of chromium in the control animals. This can be expected because small amounts of chromium are found in food and water supplies. In the control groups of these experiments the largest concentration of chromium was found in the femur bone. This may suggest a method of controlling the concentration of chromium in the system. The bone may take it up and then slowly eliminate it. This may work efficiently only at very low intakes as chromium is not generally'known as a.bone seeker. The increase in soft tissues and the bone were similar. Above 5 ppm of chromate ion the soft tissues increased at a greater rate than the bone and at 25 ppm surpassed it in concentration (Cr./gm. tissue). At this level the rate of accumulation in.bone decreased. Experiment II indicated only S.§% maximum absorption of chromium occurred in the form of hexavalent chromium. Since the hexavalent chromium is apparently reduced to a.variable degree in the digestive tract, it is difficult to determine to what extent pure chronate ions would be absorbed if none was reduced. The chromate ion is obviously more readily absorbed than trivalent chromium. However, considerable amount of reduction occurs even in the stomach of the starved animals. Experiment IIc indicates reduction of chromate ion not only in the acid environment of the stomach, but also in the intestine and in the blood stream. Experiment III shows that about 5 times more hexavalent than tri- valent chromium was absorbed. The concentration found in the tissues in of the trivalent chromium group receiving water containing the equivalent of 55.5 ppm chromate ion, correspond to the amounts found in the tissues of the groups given S, 10, or 15 ppm of chromate ion in Experiment I depending on.the tissue. It is evident from these results that tri- valent chromium shows a much lower absorption than hexavalent. It is, therefore, suggested that chronate wastes be treated with a reducing agent before they are disposed of. The conclusion of this study is that the occurrence of hexavalent chromium in water is potentially more hazardous than trivalent chromium, because it is more readily absorbed into the body. No toxic symptoms have been observed at any of the intakes over a period of one year, although quite high concentrations were fOund in the tissues. Apparently tissues can accumulate considerable quantities of chromium without caus- ing damage. SUiIE ENRY 1. 3. h. DZ Sin-em! Five groups of rats were administered concentrations of chromium, between 1.0 and 25.0 parts per :nillion as chromate ion, in the drinking water. During a one year period there were no differences between these groups and the controls as to water intake, food con- sumption or weight gain. An.analysis of blood at regular intervals for one year showed no significant differences between any of the groups given chromium and the control group. No significant microscopic changes were found in any of the tissues examined. Kidney, liver and femur were analyzed for chromium at the end of six months and the spleen included at the end of one year. The chromium content of the tissues increased from quite low to rela- tively large amounts. There was an abrupt rise in tissue retention at water concentrations above 5 ppm of dhromate ion. Starved and nonrstarved rats were injected by stomach tube with S7 micrOgrams of radioactive chromium in the form of chromate having an activity of 22.8 nicrocuries. They were sacrificed at intervals of 6, 2h, 72, and 168 hours. Approximately'5.5% of the injected dose was absorbed from the starved animals, while only 2.5% was absorbed from the non-starved animals. Blood, stomach, intestine, liver, kidney, urine and feces were analysed. The highest activity in If U.) the blood occurred at 6 hours or before, whereas the highest activity in the liver and.kidney occurred at about 2h hours, slowly decreasing thereafter. There was more activity in the tissues of the starved animals than in the non-starved. The rest was eliminated in the feces in about a week. Starved and non-starved rats were given radioactive sodium chronate or chronic chloride solution equal to 131 micrograms of chromium with an activity of 125 nicrocuries by stomach tube and blood samples taken h hours later. The activity of the plasma and red cells was determined separately; The starved animals receiving sodium chromate solution absorbed the most chromium while the non-starved group given chronic chloride absorbed the least. There was considerable re- duction of the chromate in the digestive tract. 6. Radioactive sodium chromate, chronic chloride or a mixture of equal parts of each were injected into the intestines of three groups of .rats. The amount in ected in each case was 375 microrrams with an 0 activity of 125 microcuries. Blood sarples were taken h hours after injection. The plasma and red cells were counted separately. The absorption was greatest in the starved animals given chronate and lowest in the nonrstarved group given chronic chloride. The fact that much higher blood values were obtained than in the previous experiment indicates that a considerable amount of reduction of chromate occurs in the stomach. .I a 7. Two groups of rats were administered 25 ppm of chromium either as hexavalent or trivalent chrommn. During a one year period, no toxic smaptoms occurred in either group. However, tissue concen- trations were much higher in the group given hexavalent chromium. This indicates a much greater absorption of chromate ion than tri- valent chromimu BIEEIOGRAPHY l. {a BIELI OGE'lAP HY Partington, J . R., “General and Inorganic Chedstry," pg. 738, I-Ecliillan and Co. Limited London (19m). Fairhall, L. T., "Industrial 'l‘oxicity,‘I pg. Sh, he Williams and ‘ Wilkins 00., Baltimore (19u9). 3. h. 9. 10. 12. Gross, E. and Aluens, U., "Lung; Cancer from Work in Chrome Industries ," Ber. f}, intern. kongr. Unfallmed. u. Berui‘sl-zrazmh. 'g, 966 (1939). Z'Eachle, w. and Gregorius, F., "Cancer of the Respiratory System in the United States Chromate-producing Industry,“ USPILS, Pub. Health Repts. _62, 111b, (1934-8). "Allowable Concentrations of Chronic Acid and Chromates," ASA Code 237.7 - 19'43 mazerican Stamiards Assoc. , New York, 1943. Davidson, A. H. Id. and iit-chell, R. L., "The Spectrographic DeterI-rdnation of Trace Elements in Soil II. The Variable Internal Standard Iiethod, Applied to the Detersdnation Chromium in the Cathode Layer Aron" J. Soc. Chem. Ind. ‘7, 213, 232 (1910). {c313 , S. 5., "The PhysiolOgical Aspects of IZineral Salts in Public Water Supplies ," J. Am. 1%ter E’Jorizs Assoc. g2, 2542-261; (1938). Braidech, M. EL, and Emery, F. H., "The Spectrographic Deters-un- ation of minor Chemical Constituents in Various Tx—Jater Supplies in the United States,’I J. in. Water Works Assoc. _21, 557-580 (1933). David, H. and Lieber, IL, "Underground Water Contmdnation by Chromimn Wastes ," Water and Dermga Works 23', 528-31; (1951). Hamilton, 1., ”InchIstrial Poisons in the United States ," pg, 319 , The i-ifacmillan Co. New York 1929. Cmmrdng, Ecinburpfiq tied. and Surg. Jour., 325;, 1311 (1827). Hamilton, 11., “Industrial Poisons in the United States ," pg. 319. Becourt and Chevallier, Ann. d'lnrg. publ. 3-2, 83 (18:33). I'Zanilton, A., ”Inchzstrial Poisons in the United States ," pg. 320. 1:6 13. Modern. Art. 0.. d. mun Gesundheit-lit 328 (1897); ‘ Mites, 1., 'W him: in the Uni State-f pg. 320. 3.1;. Luv-m. loom, J. WNW“ tutti-cut, !. 1!. Law 913, 55. Bullion, L, “W Pollen- in the mm BW,‘ pg. 320. '6”:um ,‘Dcut.u£.w. 8932 (i931 ). 0.1.15.752633m .92: 16. m. J. I. am ,0. D. 'chruin Poi-Inn; in Mm' n. a. m. 3.1.3.2, 3.2.. man-a (i939). ’ 1.7 Plus, 0.)! . 'Limcndiplmhmmn II. Inn-tintin- thehhnuecotamotmathcrmdm Picture, 3mm Edna; to the Erythrocyte mm: the Injuries; m Arch MW 18. m, U. 35 “m" ‘4'; G. .W “Whmmf 19.8%, 1.!" '33muufiuuonorwm- minibus-o Wt turret-in, .L cm.nsag,m~?.159-6h(1951. o.1.§§.10611(1952. 3.3., unassuming, 'mmuuucauwm ”muhcfltgnmnim gmaeamso). a. Simmons-tn, s. 0.. J. In. Lathe anus»- um. 1g, 669 (new). 22. W, I. 11., 'Advmu in Protein W) _791. 7, Edited b, m, H. LI “ m. J. to, acadélllic‘ PiGSé Inc. NoYo 3Sh‘h2l (l9h9). 23. firms, P. 1., “More, 1!. L, “MM 0: a-hl1 We 2: gr)1n m m. m» and firm," Ann. M3, 1317 19 0 . 21:. mm, 3. 3., mum at cm with mm by 3mm». Oxidation, m. on». 31,, 1016 (1952). 25. Bose, IL, mammuwuth W. I 0:132;I Ann. Chin. an. ig. 201-221 (1951.). 26. Sterling, L, all any, 8. J., "W at the cum; Bud 0911 Von-I hflmtyW,‘ J. Olin. Invest. Q, lam-16:9 (1950) 27. mm W. m. n. W 7W9» 28. 29‘. 30.. 32. 33. 3b.. W. Grusmco, I. 11., "Chromium as a Bioelement, " Bio‘ chimiya 13 , 122-6 (19L8). CA L2, 83021. Visek, N. J. et a1., I'2hatefoolism of Cr6 J‘by Azfirxals as Influenced by Chemical State," Proc. Soc. Exptl. Biol. Med. 8’ , 610-615 (1953). Shimizu, M., ”Pancreatic Lipase. I. Active Centers," J. Japan Biochem. Soc. 233,, 205-9 (1952-3). CA 51, 125.5% (1953). Shimizu,M ., "Pancreatic Lipase. II. Effects of Various Ions on Pancreatic Lipase,” J. Japan Biochem. Soc. _Q, 209-11 (1952-3). CA _1, 12h5ub (1953) Curran, G. 0., "Effects of Certain Transition Group Elements on Hepatic Synthesis of Cholesterol in the Rat," J. Biol. Chem. 210, 765’ (1951.). ”"“ Sanford, A. 11., gt._ 8.1., "The Photometer; and Its Use in the Clinical Laboratory,“ Am. J." Clin. Pat! cl. 2, 1.05 (1933). Ihelberger, C. W., "Possible Hazards from Chezrdoal Contamination in Water Supplies ," J. Am. Water Works Assoc. '2, 1027-32. (1950). MICHIGAN STATE UNIVERSITY 'oF AGRICULILJEE ALL"; war) suzmcs DEFARTIMCZ. .T Cw: CHJEESTRY EAST LANSING, MICHIGAN \MY ‘2 a JUL 1 011 JUL 2 2 '57 "F5"?