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Lead poisoning in cattle — transfer of lead to milk
The Science of the Total Environment, 111 (1992) 83-94 Elsevier Science Publishers B.V., Amsterdam
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Lead poisoning in c a t t l e - transfer of lead to milk Agneta Oskarsson ~'*, Lars Jorhem b, Johanna Sundberg ~, Nils-Gunnar Nilsson c and Lennart Albanus" ~'Toxicology Laboratory, hFood Research Laboratory, ~Food Production Division. at the National Food Administration, Box 622, S-751 26 Uppsala, Sweden (Received February I st, 1991; accepted February 18th, 1991 )
ABSTRACT The transfer of lead to milk in cattle in relation to blood lead levels and the uptake of lead in edible tissues was studied for an accidental exposure over 1 or 2 days to lead in excessive amounts from the licking of burnt storage batteries. The degree of exposure was monitored by determination of blood lead levels. Milk and blood samples were taken from eight cows, without acute symptoms of lead poisoning, during a period of 18 weeks. Two weeks after the accidental exposure, lead levels (mean + SD) in milk were 0.08 + 0.04mgkg -~ and in blood 0.36 + 0.04 mg kg ~in six of the cows. The relationship between lead concentration in blood and those in milk was found to be exponential and could be expressed by the equation: logy = 3.19x - 2.36 (r = 0.85, p < 0.001), where y and x are the lead concentrations in milk and blood, respectively. The lead level in milk was relatively constant up to a blood lead level of 0.2-0.3 mg kg ~, and increased sharply at higher blood levels. The biological half-life of lead in blood was shown to be ~ 9 weeks. In eight acutely sick cows, which were emergency slaughtered, the range of lead levels in edible muscle tissue was 0.23-0.50 mg kg ~wet weight. Very high concentrations were found in the kidneys, with a range of 70-330mgkg ~, and in the livers, with a range of 10-55 mg kg ~. Four of the cows were pregnant, in the first or second month of gestation, during the episode of exposure. The lead exposure was not found to disturb the gestation or development of the fetuses. INTRODUCTION
Lead is a common source of accidental poisoning in domestic animals (Allcroft and Blaxter, 1950; Hammond and Aronson, 1964; Aronson, 1971). The natural curiosity and licking habits of cattle make any available leadcontaining material a potential source of poisoning. Lead poisoning in cattle is usually the result of a single ingestion of a material containing a large quantity of lead. Sources of lead poisoning in cattle include lead-based paint, lead-containing lubricants and storage batteries. Poisoning in cattle can also result from long-term ingestion of crops or pasture forage contaminated by
* Author to whom correspondence and reprint requests should be addressed. 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
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lead from a local lead-emitting industry (Hammond and Aronson, 1964; Harbourne et al., 1968). Clinical manifestations of individual cases of lead poisoning have been described in the veterinary literature. However, much less information is available on the transfer of lead in lead-exposed cattle to milk and edible muscle tissues and organs consumed by man. The described episode of lead poisoning provided a unique opportunity to study the kinetics of lead in cows" milk and blood. Lead analysis of tissues and histological examination were performed. MATERIAL AND METHODS
Episode of lead e.x'posure On 5 July 1988, and for the following 3 days, two milking cows died and seven became acutely sick and were slaughtered. Poisoning was suspected and residues of burnt storage batteries (six to seven) were found on the pasture, where waste, including storage batteries, had been dumped and burnt on 4 July 1988. Another 11 milking cows, without any evident symptoms, had been grazing on the same pasture. Four of these cows were pregnant in the first or second month of gestation. After burning the dump, when presumably lead plates were made available from the storage batteries, the cows could have been exposed to lead for a maximum of 2 days. After that, poisoning was suspected, and the pasture was no longer used. The acutely sick cows are designated by Arabic numerals (1-8), and the cows without evident symptoms, examined for 18 weeks, are designated by Roman numerals (I-VIII).
Sample collection Milk and blood were collected from the surviving 11 cows 2 weeks after the lead exposure. One cow became acutely sick and was slaughtered after the first sampling. Two cows had lead levels below the detection limit in milk (0.005 mg kg ~) and constant low levels in blood at the first four samplings, after which no more samples were taken. The remaining eight cows (I-VIII) were examined for 18 weeks from 19 July: once a week for 3 weeks, once a fortnight for 6 weeks, and thereafter every fourth week for 8 weeks. In addition, blood was collected from three cows (II, III and VII1) at the time of slaughter 20 weeks after exposure. Blood and milk were also collected from two cows (I and V), as well as blood from their calves at delivery, 35 and 38 weeks, respectively, after the accident and on one additional occasion after that.
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TRANSFER OF LEAD TO MILK
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Milk and blood, l0 ml of each, were collected in acid-washed polyethylene tubes by the district veterinarian. At sample collection, precautions were taken to avoid contamination from the environment. Blood was collected with heparinized syringes, and the tubes contained 150 U of heparin. The level of lead in heparin and the lead released from the syringes and tubes used were analysed and found to be negligible. To avoid coagulation, the blood tubes were immediately inverted several times. The samples were mailed on the day of sampling to the National Food Administration and were frozen upon arrival on the day after collection. Tissue samples of muscle, liver and kidney were collected at slaughter from seven of the acutely sick cows (1-7) and from the cow (8) that became sick after 2 weeks for lead analysis and histological examination. Moreover, tissue samples were taken from three (lI, III and VIII) and two cows (I and V) slaughtered 20 and 62 weeks, respectively, after exposure. Two of these cows (II and III) were pregnant, approximately in the sixth month of gestation, and samples of liver, kidney and muscle were also taken from the fetuses for lead analysis.
Lead analysis All determinations were performed with a Perkin-Elmer 3030 graphite furnace atomic absorption spectrophotometer with a graphite furnace HGA-500 and an AS-40 autosampler. Deuterium background correction and L'vov graphite tubes were used. One milliliter of each blood sample was divided into two acid-washed Eppendorff tubes for duplicate analyses. Sample treatment was performed according to the method of Stoeppler et al. (1978) as modified by Elinder et al. (1983). Analytical accuracy was controlled for each sample run by means of external quality control samples, a gift from Birger Lind of the Department of Environmental Medicine, Karolinska Institute, Stockholm. The result of the quality control runs fell within the acceptance interval (Fig. 1) used in a global program for the biological monitoring of lead and cadmium in blood (Vahter, 1982). Lead levels in milk were determined directly by the method of standard addition after dilution of the milk 1"1 with 0.1 MHNO3. To control the analytical accuracy, a standard reference material, Non-fat Milk Powder (1549), from the US National Bureau of Standards, Washington, DC, was analysed for each run. The result was 0.016 4-_ 0 . 0 0 4 m g k g - ' (mean -I- SD, n = 4) as compared with the certified value of 0.019 + 0 . 0 0 3 m g k g - ' . Tissue samples were treated for lead analysis as described by Jorhem et al. (1984). The weighed tissue samples were ashed in a temperature-programmed furnace at a final temperature of 450°C. The ash was dissolved in 5ml
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8001 600] 3
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Fig. 1. Results for external quality control samples analysed together with the blood samples in the study. Solid line is the calculated regression line. Dotted lines indicate acceptance interval.
6 M HC1, evaporated to dryness on a water-bath, and the residue dissolved in 20.0 ml 0.1 M HNO3. To control the analytical accuracy, a standard reference material, Bovine Liver (1577a), from the US National Bureau of Standards, Washington, DC, was analysed. The results were 0.132 _+ 0.008mgkg ' (mean _+ SD; n = 3) as compared with the certified value of 0.135 _+ 0.015 m g k g 1.
Histological preparation Tissue specimens of liver, kidney and brain were fixed in a 10% aqueous solution of formaldehyde. Sections were stained with hematoxylin-eosin. R E S U LTS
The seven acutely sick cows exhibited signs of blindness, crashing into objects. Lead poisoning was confirmed from the results of lead analyses of liver, kidney and muscle tissue (Table 1). Included in the table are data for a cow that was weak, showing signs of blindness, and that was slaughtered 2 weeks after the episode. The lead levels in kidneys ranged from 70 to 330 mg kg ' wet weight. Lower levels were found in the livers of the poisoned cows, ranging from 10 to 55 mg kg '. Tissue samples of muscle contained lead levels of from 0.23 to 0.50 mg kg-'. Normal lead levels in bovine kidney, liver and meat from Sweden have recently been reported to be 0.10 + 0.06 (n = 68), 0.05 ___ 0.03 (n = 33) and <0.005 ___ 0.001 (n = 3 4 ) m g k g ~, respectively (mean _+ SD) (Jorhem et al., 1991). The remaining 10 cows did not show any immediate symptoms of lead poisoning. However, one cow (V) showed temporary fainting symptoms with stiff legs and tremor of the front legs 2 weeks after the lead exposure episode.
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TABLE 1 Lead levels (mg kg-' wet weight) in tissues and body fluids of cattle shortly (0-2 weeks) after exposure to lead Cow
Sampling time after exposure
Kidney
Liver
Muscle
I 2 3 4 5 6 7 8~
1 day 3 days 4 days 4 days 4 days 5 days 5 days 2 weeks
70 100 124 112 92 129 330 214
26 10 31 24 18 34 55 13
0.42 0.14 0.26 0.36 0.33 0.50 0.25 0.23
~'Blood and milk levels of lead 2 weeks after exposure were 1.18 and 1.56 mg kg ', respectively.
The level of lead exposure was determined from lead analysis of blood and milk, starting 2 weeks after the episode. In two of the 10 cows, lead levels in milk were below the detection limit ( < 0.005 mg kg '), and the blood concentrations were at a constant low level in eight samples taken in the 2-6 week period after the episode (0.019 + 0.006mgkg-~; mean + SD). These cows were evidently only exposed to lead to a low extent and are not included in further analyses of data. The time course of lead levels in blood and milk of the remaining eight cows followed for 2-18 weeks after the excessive ingestion of lead are shown in Fig. 2A-C. Six of the cows had similar levels of lead in blood (Fig. 2A, B), and the arithmetic means of blood and milk levels in these cows are shown in Fig. 2C, together with the results for one cow with high, and one with low, levels of lead. Two weeks after the accidental exposure, lead levels (mean + SD) in milk were 0.08 + 0.04mgkg -~ and in blood 0.36 + 0.04mg kg-~ in six of the cows. The highest lead levels found in eight cows studied for 18 weeks were 0.22mgkg -~ in milk and 0 . 4 8 m g k g - ' in blood. From the mean levels in blood shown in Fig. 2C, the apparent half-life of lead in the blood of cows was determined to be 8.5 weeks. The half-life calculated for each individual of the eight cows was 9.0 + 1.8 weeks (mean + SD). The excretion of lead via the milk of cows seems to be more complicated. There was an initial rapid decrease in lead levels in milk during the first 6-8 weeks, after which the levels increased or remained at a similar level (Fig. 2A-C). In the 2-6 week period after exposure, the half-life of lead in milk calculated from the mean levels in Fig. 2C was ~ 1.5 weeks. The relation between lead in the blood and milk of cattle is shown in Fig. 3. An exponential, rather than linear, relationship was observed. At
88
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Fig. 2(A-C). Time course of lead levels in blood and milk of eight cows after accidental exposure to lead on a single occasion. In Fig. 2C, heavy lines show mean blood and milk levels ( + SD) of the six cows shown in Figs 2A and B.
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TRANSFER OF LEAD TO MILK
Pb in milk~ mg/kg 0.25
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Fig. 3. R e l a t i o n s h i p between lead levels in b l o o d and milk from cattle after accidental exposure to lead on a single occasion (n = 64): log y = 3.19x 2.36 (r = 0.85, p < 0.001).
blood lead levels < 0 . 2 5 m g k g -I, the transfer into milk was relatively constant, and with the exception of three samples the concentration of lead in milk was < 0.03mgkg -t. At higher blood lead levels, there was a rapid increase in the lead concentration in milk. The relationship could thus be expressed by the equation: log y = 3.19x - 2.36 (r = 0.85, p < 0.001), where y is the lead concentration in milk and x is the lead concentration in blood. The exponential relationship had a higher correlation coefficient than if expressed as a linear relationship (r = 0.79, p < 0.001). The relation between lead in blood and that in milk in the samples obtained during the first 6 weeks, i.e. the time period before the milk level had plateaued (Fig. 2A-C), showed the same exponential milk-blood relationship as shown in Fig. 3 (data not shown). Lead levels in blood of calves and cows and milk of cows that delivered normal calves after complete gestation are shown in Table 2. Elevated blood lead levels were still found in the cows as late as 38 weeks after exposure to lead. Lead levels in colostrum were increased compared with the levels in mature milk 18 weeks after exposure. Lead levels in milk decreased rapidly after delivery and were almost at the detection limit in the mature milk. The fetuses of two cows (II and III) that were slaughtered in the sixth month of gestation were examined and found normal in size and development. The cows were thin. The lead levels in tissues from the calves and their mothers are shown in Table 2. Similar levels of lead were found in the kidney and the liver of the adult cattle at this time. In the fetuses, however, the liver had 10-fold or higher levels of lead compared with the kidney. Lead levels in the kidneys and livers of adult cattle were still elevated 62 weeks after exposure compared with normal levels.
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TABLE 2
Lead levels (rag kg ~wet weight) in tissues and milk of cattle including fetuses and calves after exposure to lead Cow
Sampling time
Kidney
Liver
Muscle
Blood
Milk
after exposure (weeks) Cow II 6 m o n t h s fetus
20 20
0.86 0.026
0.88 0.24
< 0.007 <0.007
0.052 0.016
Cow Ill 6 m o n t h s fetus
20 20
1.4 0.026
1.5 0.48
< 0.007 < 0.007
0.090 0.008
Cow VIII
20
0.171
Cow V C a l f at delivery
35 35
O. I00 0.067
0.034
Cow V
38
0.121
0.006
Cow I Calf at delivery
38 38
0.066 0.05 I
0.045
Cow I
39
0.752
0.78 ~'
0.33"
0.40"
0.008
"Sampling time 62 weeks after exposure.
No lesions were found at the histological examination of the livers, kidneys and brains of two cows (II and III) and their fetuses. Intranuclear inclusion bodies in the proximal tubule cells of the kidneys were looked for, but not found. In cow No. VIII, which was not pregnant, two focal perivascular cuffings were found, one in the cerebral cortex and one in the outer medulla (Fig. 4). The cells were of the histiocytic and lymphocytic type. No other lesions were observed. DISCUSSION
In the present investigation, an exponential relationship between lead in blood and milk was demonstrated in cattle heavily exposed to lead on a single occasion. The degree of exposure was monitored by determination of the blood lead levels. The lead level in milk was relatively constant up to a blood lead level of 0.2-0.3mgkg -I, and increased sharply at higher blood lead levels. There was a high blood lead level in the two cows at delivery, which suggests mobilization of lead in connection with late gestation and delivery. Mobilization of lead during pregnancy has been shown in rats by Buchet et al. (1977). The lead level in two samples of colostrum was relatively high, but rapidly decreased in mature milk. The high level of lead in colostrum can result from the higher blood lead levels as well as different cellular transport
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TRANSFER OF LEAD TO MILK
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Fig. 4. Cerebral m e d u l l a with a p e r i v a s c u l a r cuffing with l y m p h o - h i s t i o c y t i c cells ( × 250).
of lead to colostrum compared with mature milk. Higher concentration in colostrum than in mature milk has also been shown for zinc, copper and manganese (Neville et al., 1983). H a m m o n d and Aronson (1964) found that the concentration of lead in milk was linearily related to the concentration in red blood cells (range 0.5-6 mg 1packed blood cells). The level in milk (range 0.05-0.3 mg kg -I ) was ,-~ 4% of the level in red blood cells. Calculations using a haematocrit value of 37% for adult cattle, and with 94% of the lead in blood being in the erythrocyte fraction, gives a concentration of lead in whole blood approximately four-fold higher in the study of H a m m o n d and Aronson (1964) than in our study, while the lead level in milk was at a similar level in the two studies. A major difference between the two studies is the duration of exposure, which was 2-3 months in the study of H a m m o n d and Aronson and 1-2 days in our study. Also, a lack of quality control of the analysis in older studies complicates comparison between different studies. Most probably, the lead level in milk reflects the serum lead level. Manton and Cook (1984) reported that serum lead in human blood is constant up to a blood lead concentration of 0 . 4 m g l -~ and rises sharply thereafter. The exponential relationship between lead in blood and in milk shown in our investigation means that, as the blood lead level increases, the rise in the milk lead level becomes progressively greater. This may be due to an increase in the fraction of lead in serum with increasing blood lead levels. This might also explain why H a m m o n d and Aronson (1964) found a linear relationship between lead in milk and in erythrocytes, which may not have been the case
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if whole blood had been used. Telisman et al. (1990) reported exponential relationships betwen the activity of erythrocyte 3-aminolevulinic acid dehydratase (ALAD) and erythrocyte protoporphyrin (EP) with respect to the blood lead levels in cows from a lead-contaminated area. These effects are related to the concentration of lead in the bone marrow, which might be reflected by the levels of lead in serum rather than the levels in whole blood. The authors suggest the use of A L A D activity and EP as exposure indicators rather than the lead concentration in whole blood. The biological half-life of lead in blood of cows heavily exposed to lead on a single occasion was shown to be --~9 weeks. This can be compared with the half-life of lead in blood of man, reported to be ~ 4 weeks (Rabinowitz et al., 1977; Chamberlain et al., 1978; Schfitz et al., 1987). As in man, there is probably also in cattle a compartment, represented by lead content of the skeleton, with a half-life of several years (Schfitz et al., 1987). Cases with clinical signs of poisoning in cattle have been reported at blood lead levels from 0.35 to 2.4mgl -I ( H a m m o n d and Aronson, 1964; Zmudski et al., 1983). There is, however, great variation and often a lack of quality control of the analytical data. Blood lead levels > 1.7 mg kg ~and sometimes lower have resulted in death (Hammond and Aronson, 1964). In our study, one cow with delayed, but evident, clinical signs had a blood lead level of 1.2 mg kg-~. The remaining cows without any symptoms of le~ad poisoning had an average lead level in blood of 0.36 mg kg-t 2 weeks after exposure. The histological finding, a focal non-suppurative encephalitis involving histiocytes and lymphocytes, from one c o w is of a type that has not been described in cases of lead poisoning. This form of encephalitis could be a sign of viral infection. However, it could not be excluded that lead poisoning could be the cause. Kradel et al. (1965) reported eosinophilic meningoencephalitis in a case of lead poisoning in cattle. The contamination of edible products and tissues from cattle experimentally exposed to lead has been studied by Sharma et al. (1982). As found in our study, as well as in the study by Zmudski et al. (1983), there was a moderate uptake of lead in skeletal muscle, but a strong accumulation of lead in liver and especially in the kidney. The acutely sick cows in our study had extremely high lead levels in the kidneys. The kidney is known to be a target organ for lead toxicity (Goyer, 1982). Studies on human milk have reported "normal" lead levels of --~0.003 mg kg-I (Larsson et al., 1981; Rockway et al., 1984; Schramel et al., 1988), but also higher levels of -~ 0.02 mg kg-~ (Moore et al., 1982; Ryu et al., 1983). Moore et al. (1982) reported a linear relationship between lead in blood and milk at lead levels < 0 . 0 4 m g k g -1 in most milk samples and < 0 . 4 m g k g -I in most blood samples. The levels in milk were --, 10% of the levels in blood.
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T h e c o n c e n t r a t i o n o f lead in b l o o d is c o n s i d e r e d to be a g o o d biological i n d i c a t o r o f c u r r e n t lead e x p o s u r e in cattle. F r o m the present s t u d y it can be c o n c l u d e d t h a t the lead c o n c e n t r a t i o n in milk is not a g o o d i n d i c a t o r o f c u r r e n t lead e x p o s u r e at levels in milk o f < 0.03 m g kg i or b l o o d lead levels < 0.25 m g kg -~. H o w e v e r , if the c o n c e n t r a t i o n o f lead in milk instead reflects the c o n c e n t r a t i o n in serum, milk levels o f lead m i g h t be even a better i n d i c a t o r t h a n lead in b l o o d o f the actual level in a target organ. ACKNOWLEDGEMENT This study was s u p p o r t e d by a g r a n t f r o m the N a t i o n a l (Swedish) E n v i r o n m e n t P r o t e c t i o n B o a r d , Project E n v i r o n m e n t a l H e a l t h M o n i t o r i n g System Based o n Biological Indicators. T h e e l a b o r a t e sample collection by D V M G 6 r a n K r a n t z a n d the excellent technical assistance o f Christina A s t r a n d Yates is gratefully a c k n o w l e d g e d . T h e sampling o v e r a long period o f time was m a d e possible by the valuable c o l l a b o r a t i o n with Prof. Paul Holtenius. REFERENCES Allcroft, R. and K.L. Blaxter, 1950. Lead as a nutritional hazard to farm livestock. J. Comp. Pathol., 60: 209-218. Aronson, A.L., 1971. Biologic effects of lead in domestic animals. J. Wash. Acad. Sci., 61: 110-113. Buchet, J.P., R. Lauwerys, H. Roels and G. Hubermont, 1977. Mobilization of lead during pregnancy in rats. Int. Arch. Occup. Environ. Health, 40: 33-36. Chamberlain, A.C., M.J. Heard, P. Little, D. Newton, A.C. Wells and R.D. Wiffen, 1978. Investigations into lead from motor vehicles. Harwell Report AERE-R, 9198, HMSO, London. Elinder, C.-G., L. Friberg and M. Jawaid, 1983. Lead and cadmium levels in blood samples taken from persons in the general population of Sweden. Environ. Res., 30: 233-253. Goyer, R.A., 1982. The nephrotoxic effects of lead. Monogr. Appl. Toxicol., 1: 338-348. Hammond, P.B. and A.L. Aronson, 1964. Lead poisoning in cattle and horses in the vicinity of a smelter. Ann. N.Y. Acad. Sci., 111: 595-611. Harbourne, J.F., C.T. McCrea and J. Watkinson, 1968. An unusual outbreak of lead poisoning in calves. Vet. Rec., 83: 515-517. Jorhem, L., P. Mattsson and S. Slorach, 1984. Lead, cadmium, zinc and certain other metals in foods on the Swedish market. V~r F6da, 36 (Suppl. 3): 135-208. Jorhem, L., S. Slorach, B. Sundstr6m and B. Ohlin, 1991. Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88. Food Addit. Contam., 8: 201-212. Kradel, D.C., W.M. Adams and S.B. Guss, 1965. Lead poisoning and eosinophilic meningoencephalitis in cattle - - a case report. Vet. Med./Small Anim. Clin., 60: 1045-1050. Larsson, B., S.A. Slorach, U. Hagman and Y. Hofvander, 1981. WHO collaborative breast feeding study. II. Levels of lead and cadmium in Swedish humal milk. 1978-1979. Acta Pediatr. Scand., 70: 281-284. Manton, W.I. and J.D. Cook, 1984. High accuracy (stable isotope dilution) measurements of lead in serum and cerebrospinal fluid. Br. J. Ind. Med., 41: 313-319.
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Moore, M.R., A. Goldberg, S.J. Pocock, A. Meredith, I.M. Stewart, H. MacAnespie, R. Lees and A. Low, 1982. Some studies of maternal and infant lead exposure in Glasgow. Scott. Med. J., 27: 113-122. Neville, M.C., J.C. Allen and C. Watters, 1983. The mechanisms of milk secretion. In: M.C. Neville and M.R. Neifert (Eds), Lactation, Physiology, Nutrition and Breast-Feeding. Plenum Press, New York, pp. 49-102. Rabinowitz, M.B., G.W. Wetherill and J.D. Kopple, 1977. Kinetic analysis 0flead metabolism in healthy humans. J. Clin, Invest., 58: 260-270. Rockway, S.W., C.W. Weber, K.Y. Lei and S.R. Kemberling, 1984. Lead concentrations of milk, blood, and hair in lactating women. Int. Arch. Occup. Environ. Health, 53:181-187. Ryu, J.E., E.E. Ziegler, S.E. Nelson and S.J. Fomon, 1983. Dietary intake of lead and blood lead concentration in early infancy. Am. J. Dis. Child, 137: 886-891. Schramel, P., S. Hasse and J. Ovcar-Pavlu, 1988. Selenium, cadmium, lead, and mercury concentrations in human breast milk, in placenta, maternal blood and the blood of the newborn. Biol. Trace Elem. Res., 15:111-124. Schfitz, A., S. Skerfving, J. Ranstam and J.O. Christoffersson, 1987. Kinetics of lead in blood after the end of occupational exposure. Scand. J. Work Environ. Health, 13: 221-231. Sharma, R.P., J.C. Street, J.L. Shupe and D.R. Bourcier, 1982. Accumulation and depletion of cadmium and lead in tissues and milk of lactating cows fed small amounts of these metals. J. Dairy Sci., 65: 972-979. Stoeppler, M., K. Brandt and C.T. Rains, 1978. Contribution of automated trace analysis. Part II. Rapid method for the automated determination of lead in whole blood by electrothermal atomic-absorption spectrophotometry. Analyst, 103: 714-722. Telisman, S., D. Prpi6-Maji6 and A. Kersanc, 1990. Relationships between blood lead and indicators of effect in cows environmentally exposed to lead. Toxicol. Lett., 52: 347-356. Vahter, M., 1982. Assessment of Human Exposure to Lead and Cadmium Through Biological Monitoring. National Swedish Institute of Environmental Medicine and Department of Environmental Hygiene, Karolinska Institute, Stockholm. Zmudski, J., G.R. Bratton, C. Womac and L. Rowe, 1983. Lead poisoning in cattle: reassessmerit of the minimum toxic oral dose. Bull. Environ. Contain. Toxicol., 30: 435-441.
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Lead poisoning in c a t t l e - transfer of lead to milk Agneta Oskarsson ~'*, Lars Jorhem b, Johanna Sundberg ~, Nils-Gunnar Nilsson c and Lennart Albanus" ~'Toxicology Laboratory, hFood Research Laboratory, ~Food Production Division. at the National Food Administration, Box 622, S-751 26 Uppsala, Sweden (Received February I st, 1991; accepted February 18th, 1991 )
ABSTRACT The transfer of lead to milk in cattle in relation to blood lead levels and the uptake of lead in edible tissues was studied for an accidental exposure over 1 or 2 days to lead in excessive amounts from the licking of burnt storage batteries. The degree of exposure was monitored by determination of blood lead levels. Milk and blood samples were taken from eight cows, without acute symptoms of lead poisoning, during a period of 18 weeks. Two weeks after the accidental exposure, lead levels (mean + SD) in milk were 0.08 + 0.04mgkg -~ and in blood 0.36 + 0.04 mg kg ~in six of the cows. The relationship between lead concentration in blood and those in milk was found to be exponential and could be expressed by the equation: logy = 3.19x - 2.36 (r = 0.85, p < 0.001), where y and x are the lead concentrations in milk and blood, respectively. The lead level in milk was relatively constant up to a blood lead level of 0.2-0.3 mg kg ~, and increased sharply at higher blood levels. The biological half-life of lead in blood was shown to be ~ 9 weeks. In eight acutely sick cows, which were emergency slaughtered, the range of lead levels in edible muscle tissue was 0.23-0.50 mg kg ~wet weight. Very high concentrations were found in the kidneys, with a range of 70-330mgkg ~, and in the livers, with a range of 10-55 mg kg ~. Four of the cows were pregnant, in the first or second month of gestation, during the episode of exposure. The lead exposure was not found to disturb the gestation or development of the fetuses. INTRODUCTION
Lead is a common source of accidental poisoning in domestic animals (Allcroft and Blaxter, 1950; Hammond and Aronson, 1964; Aronson, 1971). The natural curiosity and licking habits of cattle make any available leadcontaining material a potential source of poisoning. Lead poisoning in cattle is usually the result of a single ingestion of a material containing a large quantity of lead. Sources of lead poisoning in cattle include lead-based paint, lead-containing lubricants and storage batteries. Poisoning in cattle can also result from long-term ingestion of crops or pasture forage contaminated by
* Author to whom correspondence and reprint requests should be addressed. 0048-9697/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved
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lead from a local lead-emitting industry (Hammond and Aronson, 1964; Harbourne et al., 1968). Clinical manifestations of individual cases of lead poisoning have been described in the veterinary literature. However, much less information is available on the transfer of lead in lead-exposed cattle to milk and edible muscle tissues and organs consumed by man. The described episode of lead poisoning provided a unique opportunity to study the kinetics of lead in cows" milk and blood. Lead analysis of tissues and histological examination were performed. MATERIAL AND METHODS
Episode of lead e.x'posure On 5 July 1988, and for the following 3 days, two milking cows died and seven became acutely sick and were slaughtered. Poisoning was suspected and residues of burnt storage batteries (six to seven) were found on the pasture, where waste, including storage batteries, had been dumped and burnt on 4 July 1988. Another 11 milking cows, without any evident symptoms, had been grazing on the same pasture. Four of these cows were pregnant in the first or second month of gestation. After burning the dump, when presumably lead plates were made available from the storage batteries, the cows could have been exposed to lead for a maximum of 2 days. After that, poisoning was suspected, and the pasture was no longer used. The acutely sick cows are designated by Arabic numerals (1-8), and the cows without evident symptoms, examined for 18 weeks, are designated by Roman numerals (I-VIII).
Sample collection Milk and blood were collected from the surviving 11 cows 2 weeks after the lead exposure. One cow became acutely sick and was slaughtered after the first sampling. Two cows had lead levels below the detection limit in milk (0.005 mg kg ~) and constant low levels in blood at the first four samplings, after which no more samples were taken. The remaining eight cows (I-VIII) were examined for 18 weeks from 19 July: once a week for 3 weeks, once a fortnight for 6 weeks, and thereafter every fourth week for 8 weeks. In addition, blood was collected from three cows (II, III and VII1) at the time of slaughter 20 weeks after exposure. Blood and milk were also collected from two cows (I and V), as well as blood from their calves at delivery, 35 and 38 weeks, respectively, after the accident and on one additional occasion after that.
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Milk and blood, l0 ml of each, were collected in acid-washed polyethylene tubes by the district veterinarian. At sample collection, precautions were taken to avoid contamination from the environment. Blood was collected with heparinized syringes, and the tubes contained 150 U of heparin. The level of lead in heparin and the lead released from the syringes and tubes used were analysed and found to be negligible. To avoid coagulation, the blood tubes were immediately inverted several times. The samples were mailed on the day of sampling to the National Food Administration and were frozen upon arrival on the day after collection. Tissue samples of muscle, liver and kidney were collected at slaughter from seven of the acutely sick cows (1-7) and from the cow (8) that became sick after 2 weeks for lead analysis and histological examination. Moreover, tissue samples were taken from three (lI, III and VIII) and two cows (I and V) slaughtered 20 and 62 weeks, respectively, after exposure. Two of these cows (II and III) were pregnant, approximately in the sixth month of gestation, and samples of liver, kidney and muscle were also taken from the fetuses for lead analysis.
Lead analysis All determinations were performed with a Perkin-Elmer 3030 graphite furnace atomic absorption spectrophotometer with a graphite furnace HGA-500 and an AS-40 autosampler. Deuterium background correction and L'vov graphite tubes were used. One milliliter of each blood sample was divided into two acid-washed Eppendorff tubes for duplicate analyses. Sample treatment was performed according to the method of Stoeppler et al. (1978) as modified by Elinder et al. (1983). Analytical accuracy was controlled for each sample run by means of external quality control samples, a gift from Birger Lind of the Department of Environmental Medicine, Karolinska Institute, Stockholm. The result of the quality control runs fell within the acceptance interval (Fig. 1) used in a global program for the biological monitoring of lead and cadmium in blood (Vahter, 1982). Lead levels in milk were determined directly by the method of standard addition after dilution of the milk 1"1 with 0.1 MHNO3. To control the analytical accuracy, a standard reference material, Non-fat Milk Powder (1549), from the US National Bureau of Standards, Washington, DC, was analysed for each run. The result was 0.016 4-_ 0 . 0 0 4 m g k g - ' (mean -I- SD, n = 4) as compared with the certified value of 0.019 + 0 . 0 0 3 m g k g - ' . Tissue samples were treated for lead analysis as described by Jorhem et al. (1984). The weighed tissue samples were ashed in a temperature-programmed furnace at a final temperature of 450°C. The ash was dissolved in 5ml
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8001 600] 3
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o
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Fig. 1. Results for external quality control samples analysed together with the blood samples in the study. Solid line is the calculated regression line. Dotted lines indicate acceptance interval.
6 M HC1, evaporated to dryness on a water-bath, and the residue dissolved in 20.0 ml 0.1 M HNO3. To control the analytical accuracy, a standard reference material, Bovine Liver (1577a), from the US National Bureau of Standards, Washington, DC, was analysed. The results were 0.132 _+ 0.008mgkg ' (mean _+ SD; n = 3) as compared with the certified value of 0.135 _+ 0.015 m g k g 1.
Histological preparation Tissue specimens of liver, kidney and brain were fixed in a 10% aqueous solution of formaldehyde. Sections were stained with hematoxylin-eosin. R E S U LTS
The seven acutely sick cows exhibited signs of blindness, crashing into objects. Lead poisoning was confirmed from the results of lead analyses of liver, kidney and muscle tissue (Table 1). Included in the table are data for a cow that was weak, showing signs of blindness, and that was slaughtered 2 weeks after the episode. The lead levels in kidneys ranged from 70 to 330 mg kg ' wet weight. Lower levels were found in the livers of the poisoned cows, ranging from 10 to 55 mg kg '. Tissue samples of muscle contained lead levels of from 0.23 to 0.50 mg kg-'. Normal lead levels in bovine kidney, liver and meat from Sweden have recently been reported to be 0.10 + 0.06 (n = 68), 0.05 ___ 0.03 (n = 33) and <0.005 ___ 0.001 (n = 3 4 ) m g k g ~, respectively (mean _+ SD) (Jorhem et al., 1991). The remaining 10 cows did not show any immediate symptoms of lead poisoning. However, one cow (V) showed temporary fainting symptoms with stiff legs and tremor of the front legs 2 weeks after the lead exposure episode.
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TABLE 1 Lead levels (mg kg-' wet weight) in tissues and body fluids of cattle shortly (0-2 weeks) after exposure to lead Cow
Sampling time after exposure
Kidney
Liver
Muscle
I 2 3 4 5 6 7 8~
1 day 3 days 4 days 4 days 4 days 5 days 5 days 2 weeks
70 100 124 112 92 129 330 214
26 10 31 24 18 34 55 13
0.42 0.14 0.26 0.36 0.33 0.50 0.25 0.23
~'Blood and milk levels of lead 2 weeks after exposure were 1.18 and 1.56 mg kg ', respectively.
The level of lead exposure was determined from lead analysis of blood and milk, starting 2 weeks after the episode. In two of the 10 cows, lead levels in milk were below the detection limit ( < 0.005 mg kg '), and the blood concentrations were at a constant low level in eight samples taken in the 2-6 week period after the episode (0.019 + 0.006mgkg-~; mean + SD). These cows were evidently only exposed to lead to a low extent and are not included in further analyses of data. The time course of lead levels in blood and milk of the remaining eight cows followed for 2-18 weeks after the excessive ingestion of lead are shown in Fig. 2A-C. Six of the cows had similar levels of lead in blood (Fig. 2A, B), and the arithmetic means of blood and milk levels in these cows are shown in Fig. 2C, together with the results for one cow with high, and one with low, levels of lead. Two weeks after the accidental exposure, lead levels (mean + SD) in milk were 0.08 + 0.04mgkg -~ and in blood 0.36 + 0.04mg kg-~ in six of the cows. The highest lead levels found in eight cows studied for 18 weeks were 0.22mgkg -~ in milk and 0 . 4 8 m g k g - ' in blood. From the mean levels in blood shown in Fig. 2C, the apparent half-life of lead in the blood of cows was determined to be 8.5 weeks. The half-life calculated for each individual of the eight cows was 9.0 + 1.8 weeks (mean + SD). The excretion of lead via the milk of cows seems to be more complicated. There was an initial rapid decrease in lead levels in milk during the first 6-8 weeks, after which the levels increased or remained at a similar level (Fig. 2A-C). In the 2-6 week period after exposure, the half-life of lead in milk calculated from the mean levels in Fig. 2C was ~ 1.5 weeks. The relation between lead in the blood and milk of cattle is shown in Fig. 3. An exponential, rather than linear, relationship was observed. At
88
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L E A D POISONING IN C A T T L E
89
TRANSFER OF LEAD TO MILK
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Fig. 3. R e l a t i o n s h i p between lead levels in b l o o d and milk from cattle after accidental exposure to lead on a single occasion (n = 64): log y = 3.19x 2.36 (r = 0.85, p < 0.001).
blood lead levels < 0 . 2 5 m g k g -I, the transfer into milk was relatively constant, and with the exception of three samples the concentration of lead in milk was < 0.03mgkg -t. At higher blood lead levels, there was a rapid increase in the lead concentration in milk. The relationship could thus be expressed by the equation: log y = 3.19x - 2.36 (r = 0.85, p < 0.001), where y is the lead concentration in milk and x is the lead concentration in blood. The exponential relationship had a higher correlation coefficient than if expressed as a linear relationship (r = 0.79, p < 0.001). The relation between lead in blood and that in milk in the samples obtained during the first 6 weeks, i.e. the time period before the milk level had plateaued (Fig. 2A-C), showed the same exponential milk-blood relationship as shown in Fig. 3 (data not shown). Lead levels in blood of calves and cows and milk of cows that delivered normal calves after complete gestation are shown in Table 2. Elevated blood lead levels were still found in the cows as late as 38 weeks after exposure to lead. Lead levels in colostrum were increased compared with the levels in mature milk 18 weeks after exposure. Lead levels in milk decreased rapidly after delivery and were almost at the detection limit in the mature milk. The fetuses of two cows (II and III) that were slaughtered in the sixth month of gestation were examined and found normal in size and development. The cows were thin. The lead levels in tissues from the calves and their mothers are shown in Table 2. Similar levels of lead were found in the kidney and the liver of the adult cattle at this time. In the fetuses, however, the liver had 10-fold or higher levels of lead compared with the kidney. Lead levels in the kidneys and livers of adult cattle were still elevated 62 weeks after exposure compared with normal levels.
90
A OSKARSSONET AL.
TABLE 2
Lead levels (rag kg ~wet weight) in tissues and milk of cattle including fetuses and calves after exposure to lead Cow
Sampling time
Kidney
Liver
Muscle
Blood
Milk
after exposure (weeks) Cow II 6 m o n t h s fetus
20 20
0.86 0.026
0.88 0.24
< 0.007 <0.007
0.052 0.016
Cow Ill 6 m o n t h s fetus
20 20
1.4 0.026
1.5 0.48
< 0.007 < 0.007
0.090 0.008
Cow VIII
20
0.171
Cow V C a l f at delivery
35 35
O. I00 0.067
0.034
Cow V
38
0.121
0.006
Cow I Calf at delivery
38 38
0.066 0.05 I
0.045
Cow I
39
0.752
0.78 ~'
0.33"
0.40"
0.008
"Sampling time 62 weeks after exposure.
No lesions were found at the histological examination of the livers, kidneys and brains of two cows (II and III) and their fetuses. Intranuclear inclusion bodies in the proximal tubule cells of the kidneys were looked for, but not found. In cow No. VIII, which was not pregnant, two focal perivascular cuffings were found, one in the cerebral cortex and one in the outer medulla (Fig. 4). The cells were of the histiocytic and lymphocytic type. No other lesions were observed. DISCUSSION
In the present investigation, an exponential relationship between lead in blood and milk was demonstrated in cattle heavily exposed to lead on a single occasion. The degree of exposure was monitored by determination of the blood lead levels. The lead level in milk was relatively constant up to a blood lead level of 0.2-0.3mgkg -I, and increased sharply at higher blood lead levels. There was a high blood lead level in the two cows at delivery, which suggests mobilization of lead in connection with late gestation and delivery. Mobilization of lead during pregnancy has been shown in rats by Buchet et al. (1977). The lead level in two samples of colostrum was relatively high, but rapidly decreased in mature milk. The high level of lead in colostrum can result from the higher blood lead levels as well as different cellular transport
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TRANSFER OF LEAD TO MILK
91
Fig. 4. Cerebral m e d u l l a with a p e r i v a s c u l a r cuffing with l y m p h o - h i s t i o c y t i c cells ( × 250).
of lead to colostrum compared with mature milk. Higher concentration in colostrum than in mature milk has also been shown for zinc, copper and manganese (Neville et al., 1983). H a m m o n d and Aronson (1964) found that the concentration of lead in milk was linearily related to the concentration in red blood cells (range 0.5-6 mg 1packed blood cells). The level in milk (range 0.05-0.3 mg kg -I ) was ,-~ 4% of the level in red blood cells. Calculations using a haematocrit value of 37% for adult cattle, and with 94% of the lead in blood being in the erythrocyte fraction, gives a concentration of lead in whole blood approximately four-fold higher in the study of H a m m o n d and Aronson (1964) than in our study, while the lead level in milk was at a similar level in the two studies. A major difference between the two studies is the duration of exposure, which was 2-3 months in the study of H a m m o n d and Aronson and 1-2 days in our study. Also, a lack of quality control of the analysis in older studies complicates comparison between different studies. Most probably, the lead level in milk reflects the serum lead level. Manton and Cook (1984) reported that serum lead in human blood is constant up to a blood lead concentration of 0 . 4 m g l -~ and rises sharply thereafter. The exponential relationship between lead in blood and in milk shown in our investigation means that, as the blood lead level increases, the rise in the milk lead level becomes progressively greater. This may be due to an increase in the fraction of lead in serum with increasing blood lead levels. This might also explain why H a m m o n d and Aronson (1964) found a linear relationship between lead in milk and in erythrocytes, which may not have been the case
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if whole blood had been used. Telisman et al. (1990) reported exponential relationships betwen the activity of erythrocyte 3-aminolevulinic acid dehydratase (ALAD) and erythrocyte protoporphyrin (EP) with respect to the blood lead levels in cows from a lead-contaminated area. These effects are related to the concentration of lead in the bone marrow, which might be reflected by the levels of lead in serum rather than the levels in whole blood. The authors suggest the use of A L A D activity and EP as exposure indicators rather than the lead concentration in whole blood. The biological half-life of lead in blood of cows heavily exposed to lead on a single occasion was shown to be --~9 weeks. This can be compared with the half-life of lead in blood of man, reported to be ~ 4 weeks (Rabinowitz et al., 1977; Chamberlain et al., 1978; Schfitz et al., 1987). As in man, there is probably also in cattle a compartment, represented by lead content of the skeleton, with a half-life of several years (Schfitz et al., 1987). Cases with clinical signs of poisoning in cattle have been reported at blood lead levels from 0.35 to 2.4mgl -I ( H a m m o n d and Aronson, 1964; Zmudski et al., 1983). There is, however, great variation and often a lack of quality control of the analytical data. Blood lead levels > 1.7 mg kg ~and sometimes lower have resulted in death (Hammond and Aronson, 1964). In our study, one cow with delayed, but evident, clinical signs had a blood lead level of 1.2 mg kg-~. The remaining cows without any symptoms of le~ad poisoning had an average lead level in blood of 0.36 mg kg-t 2 weeks after exposure. The histological finding, a focal non-suppurative encephalitis involving histiocytes and lymphocytes, from one c o w is of a type that has not been described in cases of lead poisoning. This form of encephalitis could be a sign of viral infection. However, it could not be excluded that lead poisoning could be the cause. Kradel et al. (1965) reported eosinophilic meningoencephalitis in a case of lead poisoning in cattle. The contamination of edible products and tissues from cattle experimentally exposed to lead has been studied by Sharma et al. (1982). As found in our study, as well as in the study by Zmudski et al. (1983), there was a moderate uptake of lead in skeletal muscle, but a strong accumulation of lead in liver and especially in the kidney. The acutely sick cows in our study had extremely high lead levels in the kidneys. The kidney is known to be a target organ for lead toxicity (Goyer, 1982). Studies on human milk have reported "normal" lead levels of --~0.003 mg kg-I (Larsson et al., 1981; Rockway et al., 1984; Schramel et al., 1988), but also higher levels of -~ 0.02 mg kg-~ (Moore et al., 1982; Ryu et al., 1983). Moore et al. (1982) reported a linear relationship between lead in blood and milk at lead levels < 0 . 0 4 m g k g -1 in most milk samples and < 0 . 4 m g k g -I in most blood samples. The levels in milk were --, 10% of the levels in blood.
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T h e c o n c e n t r a t i o n o f lead in b l o o d is c o n s i d e r e d to be a g o o d biological i n d i c a t o r o f c u r r e n t lead e x p o s u r e in cattle. F r o m the present s t u d y it can be c o n c l u d e d t h a t the lead c o n c e n t r a t i o n in milk is not a g o o d i n d i c a t o r o f c u r r e n t lead e x p o s u r e at levels in milk o f < 0.03 m g kg i or b l o o d lead levels < 0.25 m g kg -~. H o w e v e r , if the c o n c e n t r a t i o n o f lead in milk instead reflects the c o n c e n t r a t i o n in serum, milk levels o f lead m i g h t be even a better i n d i c a t o r t h a n lead in b l o o d o f the actual level in a target organ. ACKNOWLEDGEMENT This study was s u p p o r t e d by a g r a n t f r o m the N a t i o n a l (Swedish) E n v i r o n m e n t P r o t e c t i o n B o a r d , Project E n v i r o n m e n t a l H e a l t h M o n i t o r i n g System Based o n Biological Indicators. T h e e l a b o r a t e sample collection by D V M G 6 r a n K r a n t z a n d the excellent technical assistance o f Christina A s t r a n d Yates is gratefully a c k n o w l e d g e d . T h e sampling o v e r a long period o f time was m a d e possible by the valuable c o l l a b o r a t i o n with Prof. Paul Holtenius. REFERENCES Allcroft, R. and K.L. Blaxter, 1950. Lead as a nutritional hazard to farm livestock. J. Comp. Pathol., 60: 209-218. Aronson, A.L., 1971. Biologic effects of lead in domestic animals. J. Wash. Acad. Sci., 61: 110-113. Buchet, J.P., R. Lauwerys, H. Roels and G. Hubermont, 1977. Mobilization of lead during pregnancy in rats. Int. Arch. Occup. Environ. Health, 40: 33-36. Chamberlain, A.C., M.J. Heard, P. Little, D. Newton, A.C. Wells and R.D. Wiffen, 1978. Investigations into lead from motor vehicles. Harwell Report AERE-R, 9198, HMSO, London. Elinder, C.-G., L. Friberg and M. Jawaid, 1983. Lead and cadmium levels in blood samples taken from persons in the general population of Sweden. Environ. Res., 30: 233-253. Goyer, R.A., 1982. The nephrotoxic effects of lead. Monogr. Appl. Toxicol., 1: 338-348. Hammond, P.B. and A.L. Aronson, 1964. Lead poisoning in cattle and horses in the vicinity of a smelter. Ann. N.Y. Acad. Sci., 111: 595-611. Harbourne, J.F., C.T. McCrea and J. Watkinson, 1968. An unusual outbreak of lead poisoning in calves. Vet. Rec., 83: 515-517. Jorhem, L., P. Mattsson and S. Slorach, 1984. Lead, cadmium, zinc and certain other metals in foods on the Swedish market. V~r F6da, 36 (Suppl. 3): 135-208. Jorhem, L., S. Slorach, B. Sundstr6m and B. Ohlin, 1991. Lead, cadmium, arsenic and mercury in meat, liver and kidney of Swedish pigs and cattle in 1984-88. Food Addit. Contam., 8: 201-212. Kradel, D.C., W.M. Adams and S.B. Guss, 1965. Lead poisoning and eosinophilic meningoencephalitis in cattle - - a case report. Vet. Med./Small Anim. Clin., 60: 1045-1050. Larsson, B., S.A. Slorach, U. Hagman and Y. Hofvander, 1981. WHO collaborative breast feeding study. II. Levels of lead and cadmium in Swedish humal milk. 1978-1979. Acta Pediatr. Scand., 70: 281-284. Manton, W.I. and J.D. Cook, 1984. High accuracy (stable isotope dilution) measurements of lead in serum and cerebrospinal fluid. Br. J. Ind. Med., 41: 313-319.
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Moore, M.R., A. Goldberg, S.J. Pocock, A. Meredith, I.M. Stewart, H. MacAnespie, R. Lees and A. Low, 1982. Some studies of maternal and infant lead exposure in Glasgow. Scott. Med. J., 27: 113-122. Neville, M.C., J.C. Allen and C. Watters, 1983. The mechanisms of milk secretion. In: M.C. Neville and M.R. Neifert (Eds), Lactation, Physiology, Nutrition and Breast-Feeding. Plenum Press, New York, pp. 49-102. Rabinowitz, M.B., G.W. Wetherill and J.D. Kopple, 1977. Kinetic analysis 0flead metabolism in healthy humans. J. Clin, Invest., 58: 260-270. Rockway, S.W., C.W. Weber, K.Y. Lei and S.R. Kemberling, 1984. Lead concentrations of milk, blood, and hair in lactating women. Int. Arch. Occup. Environ. Health, 53:181-187. Ryu, J.E., E.E. Ziegler, S.E. Nelson and S.J. Fomon, 1983. Dietary intake of lead and blood lead concentration in early infancy. Am. J. Dis. Child, 137: 886-891. Schramel, P., S. Hasse and J. Ovcar-Pavlu, 1988. Selenium, cadmium, lead, and mercury concentrations in human breast milk, in placenta, maternal blood and the blood of the newborn. Biol. Trace Elem. Res., 15:111-124. Schfitz, A., S. Skerfving, J. Ranstam and J.O. Christoffersson, 1987. Kinetics of lead in blood after the end of occupational exposure. Scand. J. Work Environ. Health, 13: 221-231. Sharma, R.P., J.C. Street, J.L. Shupe and D.R. Bourcier, 1982. Accumulation and depletion of cadmium and lead in tissues and milk of lactating cows fed small amounts of these metals. J. Dairy Sci., 65: 972-979. Stoeppler, M., K. Brandt and C.T. Rains, 1978. Contribution of automated trace analysis. Part II. Rapid method for the automated determination of lead in whole blood by electrothermal atomic-absorption spectrophotometry. Analyst, 103: 714-722. Telisman, S., D. Prpi6-Maji6 and A. Kersanc, 1990. Relationships between blood lead and indicators of effect in cows environmentally exposed to lead. Toxicol. Lett., 52: 347-356. Vahter, M., 1982. Assessment of Human Exposure to Lead and Cadmium Through Biological Monitoring. National Swedish Institute of Environmental Medicine and Department of Environmental Hygiene, Karolinska Institute, Stockholm. Zmudski, J., G.R. Bratton, C. Womac and L. Rowe, 1983. Lead poisoning in cattle: reassessmerit of the minimum toxic oral dose. Bull. Environ. Contain. Toxicol., 30: 435-441.