G. MILHAUD AND SMAIL MEHENNAOUI:

INDICATORS OF LEAD, ZINC AND CADMIUM EXPOSURE IN CATTLE/I. RESULTS IN A POLLUTED AREA.

VETERINARY AND HUMAN TOXICOLOGY, 1988, 30(6), 513-517

INDICATORS OF LEAD, ZINC AND CADMIUM EXPOSURE IN CATTLE;!. RESULTS IN A POLLUTED AREA

G. E. Milhaud, S. Mehennaoui

Reprinted from Veterinary and Human Toxicology, Vol. 30, No. 6, December 1988, pp. 513-517

Indicators of Lead, Zinc and Cadmium Exposure in Cattle:I. Results in a Polluted Area

Guy E H l l h a u d DVM and S m a l l M e h e n n a o u r DVM

P h a r m a c y a n d T o x i c o l o g y , N a t i o n a l V e t e r i n a r y Schoo l A l f o r t ,9*1704 Ha I s o n s - A I f o r t , F r a n c e

( R e c e i v e d F e b r u a r y 2 , 1988; R e v i s i o n Rece i ved June 21, 1988;A c c e p t e d June 2*1, 1988)

ABSTRACT. Dairy ca t t le on a farm located [n the v i c in i t y of a lead and zinc-ore processing factory werestudied over 21 mo and compared w i th cat t le on a control farm. Mean da i l y intakes or' lead from the dietwere 4.3 mg/kg body weight, w i th great var ia t ions; mean dai ly zinc in takes were 5-6 mg/kg body weight ; andmean daily cadmium intakes were O-06'i mg/kg body weight. The 3 major indicators of contamination were bloodlead concentrations, w i t h mean values of 50 Ug/IOO ml of blood, zinc protoporphyrin w i th mean values of 165•^g/100 ml blood, and lead concentrat ions in hair which averaged 10 ug/g. Blood zinc concentrat ions and zincconcentrations were not s ign i f i can t ly increased. One cow developed fatal post-partum paralysis. Liver, kid-ney and bone lead concentrations and kidney cadmium concentrat ions were good "po^t-rnortem" indicators ofexposure.

Since 1971, studies have been conductedon catt le in the vic ini ty of a lead andzinc-ore processing factory in northernFrance. Blood samples have been collectedfrom live animals to determine concentra-tions of lead, cadmium, calcium, magnesium,zinc protoporphyrin (ZPP) and hemoglobin.Earlier urine samplings to determine deltaaminolevulinic acid proved d i f f i c u l t tocarry out and were stopped^ Most of theaffected cows and sheep were s laughtered.Necropsies were performed and tissues wereanalysed for lead, zinc and cadmium; ana-tomopathological, bacteriological, andvirological analyses were performed. Thefirst results were published previously( 1 , 2 ) .

Data on lead storage in bovine liair (3)prompted new research. A 2-year s tudy wasconducted on the cattle of the most pol lu tedf a r m to ascertain whether metal storage intail hair was a s ign i f i can t indicator ofexposure. The purpose of this communicationis to report the results of this investi-gation. Further investigation was conductedon 4 animals to determine the relationshipbetween ingestion of contaminated feed andthe 3 main parameters of exposure -- bloodlead concentrations, zinc protoporphyrinand lead concentrations in hair -- and alsoto study the decrease of these parameterswhen feed was no longer con tamina ted .

MATHlUALS AND METHODS

Animals

The test farm was located 1 km 6 north-north east of a lead and zinc-ore processingfactory. The control animals belonged tothe National Agronomic Institute located inan area free of contamination. The testfarm herd consisted of 20 Friesan cows and2 heifers born on the farm. The adult cowswere bought at about 5 years of age. Theanimals were fed corn si.lage in winter andfresh natural grasses from pastures insummer (zero-grazing). Mineral salts andsupplemented feed were added. Corn andnatural grasses came from various siteslocated 0.5 km to 2 km from the factory.

Samjole Co 1_1 ecjtion

The test program was conducted fromOctober 1, 1984 to April 16, 1986. Leadconcentrations at each site were determinedwhen grasses were harvested, which permittedus to assess the amount of lead ingested bythe animals over the period of testing.Samples of blood and hair were collected 3times — on June 17, 1985, on November 29,1985 and on April 16, 1986.

Only 9 animals (7 cows and 2 heifers)were included in all the 3 sample-co11actionson account of herd renewals and ejcc.lu.sion of

Vet Hum Toxicol 30 (6j December 1988 513

cows about to calve. The first sample col-lected 18 animals (16 cows and 2 heifers);the second sampling had 19 animals (includ-ing 12 from the first group), and the thirdsampling comprised 17 animals (all from thesecond group). All the adult cows on thefarm were slaughtered, and liver, kidneyand bone samples were collected for toxi-cology and histopathology.

For each sample-collection 15 controldairy cows (Friesan cattle In the same agegroups as the test cows) were chosen atrandom among 132 animals. Blood and hairsamples were collected on January 10, 1986,June 28, 1986, and January 15, 1987. Tenanimals were submitted for all the 3 sam-plings. No control animals were slaughtered.

Blood samples were collected from the sub-caudal vein in 3 heparinlzed evacuated bloodcollection tubes (Becton Dickinson VacutainerSystemes, Europe BP 37 38241, Meylan, Cedex,France). One tube was centrifuged at highspeed to obtain nonhemolysed plasma. Hairwas clipped for 30 cm from the median partof the tail, above the end tuft, and placedin a polyethylene bag. The Hair had neverbeen clipped before. The second and thirdhair samplings were from hair which had grownagain.

Analyses^

Blood lead. Determination of lead wasby anoding stripping voltammetry (ESA 3010A, Esa Inc, 45 Wiggins Avenue, Bedford) ona 100 ill sample (detection limit was 2 ug/100 ml).

Zinc and copper. Determination of Znand Cu in plasma was by a flame absorptionspectrophotometer (Perkin Elmer 303) on a1 ml sample diluted 1:5 (4) (detection limitwas 10 pg/100 ml).

Cadmium. Determination of Cd was by anatomic absorption spectrophotometer equippedwith a graphite furnace (Perkin Elmer HGA72) after calcination and extraction by 5NUNO 3 (5) (detection limit was 0.1 ug/100 ml)

Calcium and magnesium. Colorimetry withBio-Merieux reactives (ref 61 041 and 61411, Marcy 1'Etoile 69290 Craponne, France)was used.

Zinc prptopprphyri^. Direct determinationwas performed on a blood drop with hemato-fluorometer AVIV (Biochemical Incorporation,Towbin Av 810, Box 994, Lakwoot NG).

Hemoglobin. Determination was with aCoultronics hemoglobinometer (29 avenueGeorges Pompidou, 95580 Margency, France).

Hair, liver, kidney, bone. The hair waswashed with 1% Triton X and demineralizedwater using a procedure derived from Ward(6). Proceeding in stages, the sampleswere mineralized at 450 C with sulphuricacid. The ashes were treated with concen-trated nitric acid (15 N), and the dried

extract was again mi nei:al i'/.ed ;il, -150 C.The white ashes werr dissolved :i n d i l u t e dnitric acid (5 N). ZJm; and copper wore .determined by flame absorption spectro-photometry, while lead and cadmium weredetermined by atomic absorption spectro-photometry with furnace. Detection limits(ppm/dry wt) were:

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Statistical Analysis

Statistical calculations were performedusing student's t-Test. Correlation anal-ysis was carried out using statisticalmethods of simple correlation between leadand ZPP levels in the blood and lead con-centrations in the'hair.

RESULTS

The estimated intake of lead throughfeeding in the polluted area was 6 mg/kg/day between October 1, 1984 and January 15,1985, and 4.8 nig/kg/day between January 16,1985 and May 4, 1985. It varied from 3.2mg/kg/day to 0.5 mg/kg/day between May 5,1985 and June 19, 1985 (when the firstsamples were collected) (Fig 1). Betweenthe first and the second sample-collections,the daily intake of lead was very variablewith intake ranging from 2.5 to 3 mg/kg/day,separated by very high peaks (22.5 mg/kg/day from June 20 to June 24, 24.5 mg/kg/dayfrom July 7 to July 12). After October 12(48 days prior to the second sample-col-lection), the daily intake stabilized at2.5 mg/kg/day and remained unchanged be-tween the second and the third sample-collections.

F i g'ure 1 . Lead i ntake through feed ing from October10, 8<l utit i I Apri i 16, 86.'

514 Vet Hum Toxicol 30 (6) Uecembe'- 1988

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Zinc and cadmium concentrations were notmeasured in all the samples, but many anal-yses conducted in the area showed that theconcentrations of zinc in the forageamounted to about 130% of lead concentra-tions. The concentrations of cadmiumamounted to about 1.5% of the lead con-centrations .

The estimated intake of lead, zinc andcadmium by the animals on the control farmwas 0.025 mg/kg/day of lead, 0.002 mg/kg/day of cadmium, 1.2 mg/kg/day of zinc.

Table 1 shows the mean values obtainedin blood and hair from the 3 samplings onthe control farm and from each sampling inthe contaminated area, with the statisticalsignificance between the results on thecontrol farm and the contaminated farm(t-test). Table 2 shows the mean concen-trations of these elements in the liver,kidney and bone of the animals reared inthe contaminated area.

DISCUSSION

Figure 2 shows that 2 blood parameters,lead and ZPP levels were multiplied by afactor above 10. The same applied to leadconcentrations in hair. In spite of sig-nificant statistical differences, theother parameters did not show such vari-ations.

In spite of the high concentrations ofzinc contained in the diet, there were in-significant differences between blood zincconcentrations in test and control animalsin the first sampling. The concentrationswere 3 times higher in the third sampling

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of test animals, but those samplings wereof poor quality and hardly reliable. Inthe third sampling only the zinc concentra-tions in hair were significantly differentbetween test and control animals. The zincin kidney and liver were not above thereference values (7,8). This suggeststhat zinc absorption is decreased afterbody zinc status has been elevated bydietary means. This phenomenon has alsobeen described in the rat (9).

In the first sampling, blood cadmium con-centrations in test animals were lower thanin control animals. Hair concentrationswere higher but the difference was notsignificant on account of the high standarddeviation. The concentrations in kidneyand liver were markedly above what is con-sidered normal by Dorn et al (10) — 1.04and 0.24 ppm respectively — but were belowthe critical concentrations of cadmium inhuman renal cortex (210-260 ppm) reportedby Nomiyama (11).

Blood and hair copper concentrations inthe control animals were below the normallimits of deficiency suggested by Lamandet al (4), 70 ug/100 ml and 7 Mg/g respec-tively. Concentrations in the first sam-pling of test animals were not signifi-cantly increased compared with controls,which confirms the absence of contamina-tion by copper. The concentrations ob-tained in the other samplings were signifi-cantly different in various ways.

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Figure 2. Blood parameters in control cows (and test cows (f i rst sampl ing :— •)

Vet Mum Toxicul 30 (6) December 1988 515

Blood calcium concentrations in test andcontrol animals were slightly below thenormal values (97-124 mg/1) suggested byKaneko (12). However, magnesium and hemo-globin concentrations were within normallimits (18-23 mg/1, 8-14 g/100 ml, respec-tively ).

What can be inferred from the great vari-ance of the 3 parameters, lead and ZPP con-centrations in the blood and lead concentra-tions in the hair? Fig 3 shows the relation-ship between lead in blood and in hair foreach animal and in each series of samples.In the first sampling, blood lead concentra-tions were comparatively low in all animalsand unrelated to the lead in the hair, wheremean concentrations were the highest. Thecows that spent an average of 19.3 -f 9.6months (range 6 to 41 mo) in sheds, accumu-lated high amounts of lead in the hair(which can be considered an organ of ex-cretion) because lead intakes were highduring the several months before sample-collection. Reversely, in the weeks whichpreceded this sampling, lead intakes wereparticularly low (Fig 1) and blood leadconcentrations were only 34.3 + 12.7 ug/100ml, whereas they were 50.8 + 22.5 pg/100 ml6 mo before (December 7, 1984). At the timeof the second sampling, blood lead concentra-tions were higher and in very low correlationwith the lead in the hair (r=0.34). At thetime of the third sampling, mean blood leadconcentrations were not very different butthe correlation was much greater (r=0.89). .This closer correlation appeared due to thefact that lead intake remained unchanged(2.5 mg/kg/day) between October 12, 1985and April 16, 1986 (ie, during the 48 daysbefore the second sampling and over the 135days which separated the second ai.d thethird samplings) (Fig 1).

Fig 4 shows the relationship between leadand ZPP in the blood of each animal and ineach series of samples. Correlation isstrong in the results of the first sampling(r=0.92), and lower in the second (r=0.73)and third samplings (r=0.61). It is stat-istically significant at p < 0.01 in all 3cases. When an animal starts ingesting

Figure 3- Relationship between Blood and Hair leadlevels.

Figure *t. Relationship between blood lead and ZPPlevels.

lead daily, blood lead increases more rapid-ly than ZPP (13). In the present study,when the first samples were collected theanimals had been on the farm long enoughfor ZPP to reach fairly elevated and stablevalues. In the 9 animals that were sub-mitted to all the 3 samplings, the mean ZPPconcentrations were slightly lower at thesecond sampling but markedly higher at thethird (respectively, 177 + 131, 165 + 91,and 258 + 159 ug ZPP/100 ml). In 4 animals,progression was constant, but in the other5, the concentrations were lower at thesecond sampling. These findings are diffi-cult to interpret.

When the animals were slaughtered afteran average of 28.1 + 15 mo on the farm, thelead concentrations in kidney, liver andbone were moderate (Table 2). In liver andkidney, they were higher than normal, butthey were within limits which do not mani-fest clinical signs.

The individual results show that when thesample and criteria are considered in someanimals, concentrations are always abovemean, whereas in others they are alwaysbelow. One cow that had been 42 mo on thefarm calved in the morning of April 9, 1986and was found paralyzed and died immediatelyafter a magnesium and calcium perfusion.Exposure indicators were relatively high —60 ug/100 ml lead in the blood in December1984 and 46 ug/100 ml on November 29, 1985;ZPP concentrations were 63 ug/100 ml inDecember 84 and 160 ug on November 29, 1985;lead in the hair was 12.2 ug/g on June 19,1985 and 7,8 on November 29, 1985. Necropsyrevealed abundant percarditic liquid con-taining blood and a mild diffuse hepaticsteatosis. Lead concentrations were 4.2ppm (wet) in the liver, 5.5 ppm (wet) inthe kidney and 50 ppm (dry) in the bone.The latter f :i gure is surpr Is ing, given thetime of exposure and the blood lead con-centrations -

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As the 3;: indicators of lead exposure didnot have the same significance and did notvary in exactly the same way, it was interest-

516 Vet Hum Toxicol 30 (6) December 3988

ing to use them to evaluate risks. M o n i t o r i n gthe time the animals spend in a con tamina tedarea and the amounts of lead ingested daily |were useful to interpret these 3 parameters, fOn the other hand , the concentration of zincand cadmium in the blood and in the hair wasof l i t t le interest. In the liver and kidneys ,lead and cadmium concentrations were goodindicators of exposure; zinc concentrationwas a fair indicator; and the bone was avery good indicator of lead exposure.

ACKNOWLEDGEMENTS

We thank la Soclete Penarroya, 1'lnstitut Nationalde la Recherche Agronomique (INRA), 1'InstltutNational Agronomique de Paris-Grignon, les servicesd'Anatomie-Pathologique, d'Alimentation et depathologie du Betail de 1'Ecole Nationale Veterinaired'Alfort , H. JP Bertrand, le Docteur Hathot, lesDocteurs-Veterinaires Caron et Barras, Madame Pupin,for their help in conducting this research. Theassistance and cooperation of Mademoiselle EllaneCharles, Assistant Ingenieur Inra au Laboratoire dePharmacie et Toxicologie de 1'Ecole NationaleVeterinaire d 'Alfor t , are also gratefully acknowledged.

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2. Hithaud G, Pinault L: Le Saturn isme animal.Symbloses 9:1*5-55, 1977-

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I*. Lamand H: Les ol igoelaments . Dal foz edi t ions,78 pages, 1978.

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9. Smith KT, Cousins RJ, Si tbon 8L, Fai l la ML:Z inc absorption and metabol ism by isolated,vasculary perfused rat intestine. Journal ofNutr i t ion 108:309-3", 1978.

10. Dorn CR, Pierce JO, Chase GR, Phi l ips PE:Cadmium, copper, lead and zinc In blood, milkmuscles and other t issues of ca t t le from anarea of mul t ip le source contamination. InProc 7th Annual Conf Tract Subst Environ Health,Hemph ill DO, ed. Uni vers i ty of M i ssourI ,Columbia, pp 171-202, 197<t.

11. Nomiyama K: Renal e f fec ts of cadmium. InCadmi urn in the Envi ronment. Part II: HealthEffects, Nriagu JO, ed. 6*13-689, 1981.

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