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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Minimal transmission of zearalenone to milk of dairy cows a
b
D.B. Prelusky , P.M. Scott , H.L. Trenholm & G.A. Lawrence
a
b
a
Animal Research Centre , Agriculture Canada , Ottawa, Ontario, Canada , K1A 0C6 b
Bureau of Chemical Safety, Food Directorate, Health Protection Branch , Health and Welfare Canada , Ottawa, Ontario, Canada , KlA 0L2 Published online: 14 Nov 2008.
To cite this article: D.B. Prelusky , P.M. Scott , H.L. Trenholm & G.A. Lawrence (1990) Minimal transmission of zearalenone to milk of dairy cows , Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 25:1, 87-103 To link to this article: http://dx.doi.org/10.1080/03601239009372678
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J. ENVIRON. SCI. HEALTH, B25(l), 87-103 (1990)
MINIMAL TRANSMISSION OF ZEARALENONE TO MILK OF DAIRY COWS
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KEY WORDS:
zearalenone, dairy cow, milk, plasma, zearalenols, liquid chromatography, food safety, residues
1
2
1
D.B. Prelusky , P.M. Scott , H.L. Trenholm , and G.A. Lawrence 1
2
Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 and
2
Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, Canada KlA 0L2
ABSTRACT Milk and plasma levels of zearalenone (ZEN), α-zearalenol (αZEL),
(β-zearalenol
determined
after
(β-ZEL)
feeding
and
conjugated
lactating
cows
metabolites
with
ZEN.
instances where ZEN and α- and β-ZEL were detected
In
were those
in milk or
plasma, they occurred only as conjugates hydrolysable by treatment with
a
mixture
of
β-glucuronidase
and
aryl
sulfatase.
With
studies where 50 or 165 mg was fed daily to three cows for 21 day periods, neither dosage showed the presence of ZEN or metabolites in either milk or plasma (detection limits:
milk, 0.5 ng/ml, ZEN,
α-ZEL; 1.5 ng/ml, β-ZEL; plasma, 2-3 times higher). Contribution Number:
1628, Animal Research Centre 87
Copyright © 1990 by Marcel Dekker, Inc.
A dose of
88
PRELUSKY ET AL.
544.5 mg zearalenone per day given to a single cow for 21 days yielded maximum concentrations of only 2.5 ng ZEN/ml and 3.0 ng αZEL/ml
in
the milk.
In plasma, up
to
3 ng
ZEN/ml
detected during the initial 4 days of treatment.
could
be
At a dose of
1.8 g of zearalenone given over a one day feeding period, maximum
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milk levels of 4.0 ng ZEN/ml, 1.5 ng α-ZEL/ml, and 4.1 ng β-ZEL/ml were
observed
during
the
initial 2 days; corresponding
maximum
levels after a one day dose of 6.0 g zearalenone were 6.1, 4.0 and 6.6 ng/ml milk on days 2-3.
In plasma, peak ZEN concentrations (9 ,
and 13 ng/ml at the lower and higher one-day doses, respectively) occurred 12 hr after initial dosing, and declined to negligible levels by days 5-7.
Neither α- nor β-ZEL were detected in plasma.
Since measurable levels required very high oral doses of ZEN, milk would not normally pose
a human health hazard
as a result
of
feeding rations containing ZEN to lactating dairy cows.
INTRODUCTION Trans -zearalenone mycotoxin
produced
graminearum.
by
(ZEN)
is
several
an
estrogenic
species
of
and
carcinogenic
Fusarium.
notably
F.
Originally isolated as a result of investigations of
outbreaks of hyperestrogenism in pigs consuming contaminated corn (Kurtz and Mirocha,
1978),
ZEN has now been
found
in numerous
samples of agricultural commodities, not only feedstuffs but also grains used in food products (Kuiper-Goodman et al., 1987). There is some experimental evidence for transmission of ZEN, a-zearalenol
(a-ZEL)
and
/3-zearalenol
(£-ZEL)
into
milk
and
MINIMAL TRANSMISSION OF ZEARALENONE
89
tissues of domestic livestock following oral administration of ZEN (Palyusik et al., 1980; Vanyi et al., 1983; Mirocha et al., 1981, 1982; Hagler et al., 1980; James and Smith, 1982; Olsen et al., 1986), but only one report of natural occurrence of ZEN residues in animal products (Sandor, 1984).
Of particular importance when
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estimating human exposure to ZEN is a knowledge of carryover of ZEN and its metabolites into cow's milk.
Since ZEN does not seem
to be an important factor in the health of dairy cows at oral daily doses of up to 500 mg/cow (Weaver et al. , 1986a, 1986b), high levels of ZEN could be present in the feed without being noticed clinically.
Shreeve et al. (1979) could not detect ZEN in
milk of two cows fed concentrate ration containing 1.8 fig ZEN/g for 7 weeks.
Mirocha et al. (1981) found 481 ng/ml of total ZEN
(incuding conjugates) and similar concentrations of total a- and /9-ZEL in milk after feeding a cow grain containing 25 /Jg ZEN/g for 7 days; lower levels of total ZEN and a- and /J-ZEL were present in the milk when the concentration of ZEN in the grain was increased to 250 /ig/g, but for a shorter period of only 1 to 2 days.
After
a single 5-g dose fed to a cow by Hagler et al. (1980) there were only traces of ZEN and /3-ZEL, and then not until 5 days after dosing.
Further information on transmission of ZEN and metabo-
lites was obviously needed.
The present study was designed to
establish the minimum concentration of ZEN in dairy ration which would result in detectable amounts (low ng/ml) of toxin residues being transmitted into the milk.
90
PRELUSKY ET AL. EXPERIMENTAL
Feed Schedule: The three cows used in this study were Holstein cross-breeds weighing
480-580
kg.
Feed
intake
and
milk
production
were
monitored for 2 weeks prior to the ZEN treatment period to calcu-
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late the amount of dairy ration (Table 1) which should be fed, i.e.
0.33
produced. line
ZEN
kg
of
dairy
ration
per
day
for
every
kg
of
milk
Diets containing ZEN were prepared by mixing crystal(IMC Pitman-Moore, Inc., Terre Haute, IN) with
small
amounts of ground corn in a mortar and pestle followed by blending of this spiked corn with the dairy cow ration in a mixer. trations
of ZEN were
confirmed
by
liquid
Concen-
chromatographic
(LC)
analysis (Trenholm et al., 1984). The
experimental
protocol
outlining
the
several
feeding experiments carried out is shown in Table 2.
dosing/
During
Experiments 1 and 2, the three cows were fed first 50 mg ZEN/day for a 21 day period, followed by 165 mg ZEN/day for a 21 day period.
In Experiment 3, three treatments using one animal each
were included. In addition, the caws were fed a daily roughage allotment of dry hay, 3 kg; corn silage, 16 kg; and alfalfa haylage, 9 kg.
The
daily feeding schedule was as follows: 6:00
-
feed 1/2 of dairy ration including 1/2 of ZEN dose;
7:00
-
feed 1/3 of roughage allotment;
15:00
-
feed remaining 1/2 of dairy ration including ZEN dose;
16:00
-
feed remaining 2/3 of roughage allotment.
MINIMAL TRANSMISSION OF ZEARALENONE
91
TABLE 1
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Composition of Ottawa Lactating Dairy Cow Ration 1979
Ingredient
* of Diet
Shorts Ground corn Ground barley Soybean meal Limestone Dicalcium phosphate Trace minerals salt Vitamin premix Molasses
24 40 4 23 1 1.5 1.5 0.2 5
TABLE 2 Experiment Protocol
Diet No.
1
1
3
25 mg x 2
2
2
3
82.5 mg x '.2
3
3A 3B 3C
1(A) 1(B) 1(C)
1
2
3
No. Animals 2
Daily Dosing Regimen 3
Exp.1
272.25 mg x 2 900 mg x 2 3000 mg x 2
Total Daily Dose
Days
50 mg
21
165 mg
21
544.5 mg 1800 mg 6000 mg
21
1 1
Animals were given a 21-day "wash-out" period betwen experiments to allow elimination of potential residues from previous study. Animals in Exp. 3 were labeled as A, B or C to distinguish the individual dosing protocol. ZEN was administered in 2.5 kg dairy ration/day, fed at 6:00 and 15:00 hr, daily.
92
PRELUSKY ET AL.
The amount of roughage fed to the cows was adjusted in the week prior to ZEN administration and maintained constant during the treatment period. During the course of the study, milk samples were taken daily (morning and evening) and tail venous blood samples were
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twice weekly
(Exp. 1,2) or serial (Exp. 3 ) .
taken
Feed intake, body
weights, and milk production were monitored throughout the study. Milk samples were analyzed for fat (%), protein (%), lactose
(%)
and somatic cell count. Determination of ZEN and a- and g-ZEL in Milk and Plasma: Milk reversed basic
samples phase
were
liquid
acetonitrile,
analyzed
for
chromatography acidification,
ZEN
and
a-
following
and
£-ZEL
extraction
partition
into
by
with
methylene
chloride (or chloroform) and cleanup on an aminopropyl solid phase extraction column (Scott and Lawrence, 1988).
For determination
of total ZEN, a-ZEL and /3-ZEL (free plus conjugated metabolites), milk
samples
were
incubated
with
^-glucuronidase
(from
Helix
pomatia. containing aryl sulfatase) prior to extraction in order to hydrolyze any such conjugates (Scott and Lawrence, 1988).
Most
determinations were single analyses but replicate extractions were done in 9 cases and results averaged.
Separate (single) analyses
for the free (unconjugated) compounds were made without addition of enzymes to the milk. using
the
following
gradient
chromatography:
15-30%
over
(no.
7
minutes
The LC method was modified slightly by
5
reversed
phase
(CIB) f°r
acetonitrile
in methanol-water
concave
on
gradient
Altex
liquid (61+35) solvent
MINIMAL TRANSMISSION OF ZEARALENONE
93
programmer) at a flow rate of 1 ml/min, with a 15 minute hold at 30% acetonitrile and pre-run equilibration of 10 minutes at 15% acetonitrile.
Earlier analyses (Experiment 1) were isocratic with
a mobile phase of methanol-water (61+35) containing 17% acetonitrile.
Detection of ZEN, a-ZEL, and /3-ZEL was by
fluorescence
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with an excitation wavelength of 236 nm and an emission wavelength of 470 nra; measurement was by peak area, except at the lowest levels, where peak heights were used. Performance of the analytical method was checked periodically by measuring recoveries of ZEN (5 ng/ml), a-ZEL (5 ng/ml), and /?ZEL (25 ng/ml) added to milk, with and without enzyme addition. The identity of ZEN and a- and /J-ZEL in selected milk samples was confirmed to some extent by comparative (peak ratio) determination at a fluorescence excitation wavelength of 274 nm. of
the
higher
concentrated
level
extracts
samples, (equivalent
compounds to
8
ml
For 5
were
detected
in
milk
injected)
by
absorption at 236 nm using a diode array UV detector (1KB Rapid Spectral Detector); the UV spectrum was scanned and compared with that
of
an
authentic
standard
chromatographed
under
the
same
conditions. Analysis of ZEN and metabolites in 2 ml plasma samples, were carried out according to the method of Prelusky et al. (1989), in which a series of pH-controlled solvent extractions were used to isolate the compounds of interest for determination by reversed phase liquid chromatography with fluorescence detection excitation, 418 nm emission).
(238 nm
94
PRELUSKY ET'AL. RESULTS
Animals: While there were no marked differences in feed intake and weight gains during the intial two feeding experiments, there were two incidences of decreased feed consumption during Experiment 3.
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With cow A (Exp. 3) feed intake decreased 30% for the first 2 days on treatment.
Diarrhea was pronounced on the first day of ZEN
administration;
by
normal.
day
2
the
stools were
loose, but
appeared
By day 2, the vulva was inflamed and edematous.
The
uterus and ovaries were small with a small corpus luteum on the left ovary.
The swollen vulva responded to treatment with lavage
and 20 mL antihistamine
(1.0 H Vetastim).
Scarletol antiseptic
was applied to the vulva and the tail was elevated overnight to prevent further irritation.
With cow C (Exp. 3 ) , roughage intake
decreased 47% on the second to fifth day after ZEN administration. This cow had very liquid diarrhea on day 2 and by day 3 seemed very docile, with the left side of the vulva swollen, edematous and inflamed.
By evening of day 3, the cow seemed more alert and
the vulvular edema was receding.
Follicles could not be palpated
on the small ovaries; the uterus seemed small and firm. In
Experiment
3
the
average
daily
milk
production
was
estimated to be 24, 15 and 15 kg for cows A, B and C, respectively.
No major differences were observed in the milk chemistry
parameters studied during any of the experiments. Transmission into Milk: The method used for determination of ZEN and a- and 0-ZEL in
MINIMAL TRANSMISSION OF ZEARALENONE
95
milk was essentially that published recently by Scott and Lawrence (1988).
Percent recoveries and standard deviations (n-6), in the
absence and presence of enzymes, respectively, were 84.9 + 6.7 and 86.5 ± 12.9 for ZEN, 91.5 + 7.8 and 90.2 ± 10.2 for a-ZEL, and 95.6 + 9.7 and 85.7 + 13.0 for ,8-ZEL.
Limits of detection were
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approximately 0.3-0.5 ng/ml for ZEN and a-ZEL and 1.5 ng/ml for /3ZEL.
Recoveries from plasma by the method of Prelusky et al.
(1989) were similar to those reported above, although detection limits were about 2-3 times higher. The two initial transmission experiments, in which each of three cows consumed 50 mg ZEN daily in their food for 21 days, then later 165 mg daily for 21 days, proved to be negative.
No
free ZEN, a-ZEL, or /3-ZEL was detected in milk collected from the three cows in each study, although a random interference for ZEN or a-ZEL was observed in some samples.
Nor was transmission of
these compounds detected on sampling enzyme-treated milk collected on the same days; however, in a few treated samples ZEN or /8-ZEL may have been present at' the limit of detection of the method. Monitoring of plasma toxin levels over the 3 week course of each feeding trial failed to identify the presence of either the free or conjugated metabolites. Evidence for transmission of ZEN, a-ZEL and 0-ZEL into milk was
only
obtained
when
the
dose
of
zearalenone
was
further
increased to 544.5 mg daily (Fig. 1) and with one-day doses of 1.8 g or
6.0
g
(Figs. 2 and
3, respectively).
Even then no
unconjugated metabolites were detected in any of these three
96
PRELUSKY ET A L .
4-1
D) C
3-
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2i iu
o o o
1-
1
2
3
4
5 DAYS
6
7
8
9
FIGURE 1
Concentrations of total (conjugated) ZEN (0 — ) and a-ZEL (+...) in milk of cow A on diet A (544.5 mg ZEN/day x 21 days) (/9-ZEL was at or below detection limit).
E
z o o o
Ul
FIGURE 2 Concentrations of total (conjugated) ZEN (0 — ) , a-ZEL (+...), and 0-ZEL (x---) in milk of cow B on diet B (1.8 g ZEN x 1 day).
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MINIMAL TRANSMISSION OF ZEARALENONE
97
FIGURE 3 Concentrations of total (conjugated) ZEN (0 — ) , a-ZEL (+...), and /3-ZEL (x---) in milk of cow C on diet C (6.0 g ZEN x 1 day).
highest dose experiments, with the possible exception of a-ZEL and ZEN at the detection limit in milk from cow C on the morning of day 2 (not shown). All data presented in Figs. 1-3 are for conjugated ZEN and aand /0-ZEL; results for /9-ZEL are not recorded for cow A receiving 544.5 mg ZEN/day on a continual basis, as it was not found except at the detection limit in the p.m. milk collection of day 1 (not shown).
The low levels of ZEN and a-ZEL found in milk from this
cow (A) support our earlier negative observations at 10.9 and 3.3 times lower feed levels of ZEN (Exp. 1 and 2, respectively).
Two
typical chromatograms of experimentally positive milk extracts are shown in Fig. 4.
98
PRELUSKY ET AL.
15ZEN
a-ZEL
ZEN a-ZEL
in in UJ tr
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a. UJ
o cc
o u
Ul
a.
0J
1
1 10
0
0
20
to
20
TIME(min)
FIGURE 4 Liquid chromatograms of extracts of enzyme-treated day 2 (p.m.) milk: (a) cow A, 0.84 ng a-ZEL/mL and 0.96 ng ZEN/ml milk; (b) cow C, 3.9 ng /3-ZEL/ml, 2.6 ng a-ZEL/ml, and 5.1 ng ZEN/ml milk. Equivalent to 1 ml milk injected.
Confirmation
of
ZEN
and
a-
and
0-ZEL by
diode
array
UV
detection showed in all cases examined an absorption maximum in the region of 235 nm.
At the low levels found in these milks, it
was not always possible to distinguish the secondary absorption maxima
at
274 nm
Tashiro, 1981).
and
315 nm
(Pathre
et al., 1979; Ueno
and
Agreement between determinations made at 236 nm
and 274 nm excitation wavelengths were satisfactory in most cases where this procedure was used for confirmation.
MINIMAL TRANSMISSION OF ZEARALENONE
99
Distribution into Plasma: Concentrations administration
of
of high
ZEN
metabolites
in
levels of ZEN were
plasma
following
similarly very
low.
Diets A, B and C resulted in only trace amounts of conjugated ZEN being detected (Fig. 5 ) . No free metabolites were found, nor were
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the conjugated alcohols (a- or j8-ZEL). A),
a maximum
plasma
(day
concentration
3) was
of only
measured,
At 544.5 mg ZEN/day (diet 2.8
declining
ng
conjugated
quickly
ZEN/ml
thereafter
to
negligible levels (Fig. 5A). Following the two high one-day doses (diets B and C ) , plasma ZEN (conjugated) peaked at 8.8 and 12.9 ng/ml, respectively, both at the 12 hr sampling. tions decreased
Again concentra-
rapidly, falling to below detectable
levels
(1
ng/ml) by days 5-6.
DISCUSSION In the two initial experiments there was no indication that ZEN was affecting the dairy cows.
Even at the very high doses
given in Experiment 3, decreases in feed intake were transient. However, the estrogenic effects of ZEN were notable in Experiment 3 with swollen inflamed vulvas.
Although one would not expect ZEN
intake to be as high as studied in this experiment in a dairy operation, it does raise the possibility that subtle changes in reproductive performance may occur in dairy herds exposed to ZEN contaminated feed. Our findings are consistent with two of the three published reports
on transmission of ZEN and its metabolites
into
cow's
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100
PREtUSKY ET AL.
FIGURE 5 Concentrations of conjugated ZEN in plasma of cows A, B and C receiving diets A, B and C, respectively (544.5 mg ZEN/day x 21 days, 1.8 g ZEN x 1 day, and 6.0 g ZEN x 1 day).
milk.
The level of ZEN in feed concentrate used by Shreeve et al.
(1979), who detected no residues, was equivalent to a dose of 19 mg/cow/day, which was far too low to expect any transmission based on our own observations.
The single 5 g dose of ZEN of Hagler et
al.
to
(1980),
comparable
our
highest
one-day
dose
of
6 g
(2 x 3 g ) , yielded only "traces" of total ZEN and 0-ZEL in cow's milk.
"Traces" in this case can be interpreted as no more than 1-
2 ng/ml, since these levels were apparently measurable in sheep milk in the same publication.
Thus, the concentrations in cow's
milk were marginally lower (but appeared later) than the levels we observed.
Hagler et al. (1980) did not detect a-ZEL.
Mirocha et
al. (1981) detected all three compounds, as we did, but they found that the free forms were present in concentrations of the same order of magnitude as the conjugates, except after 1 day of a diet
MINIMAL TRANSMISSION OF ZEARALENONE
101
containing 250 pg zearalenone/g grain (equivalent to 1 g/cow/day) when no conjugates at all were detected.
The quantitative aspects
of this study were also quite different from our data; in fact concentrations of total ZEN, a-ZEL and 0-ZEL were as high as 481, 508
and
370 ng/ml,
respectively, which
represented
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consumed dose of 200 mg ZEN/day for 7 days.
0.7%
of
a
By comparison we
found concentrations of the order of only 1-3 ng/ml at nearly 3 times this daily dose of ZEN (Figure 1 ) . The
capacity
of
rumen
fluid
to
degrade
ZEN
(Kallela
and
Vasenius, 1982; Kiessling et al. , 1984) is one variable factor that could account for the wide discrepancies in transmission of ZEN to cow's milk.
Kiessling et al. (1984) found that most of the
degradation of ZEN to ZEL (mainly a-) was due to rumen protozoa. Furthermore, while there appears to be no available about residual blood ZEN,
levels
in ruminants
following
information exposure, to
studies involving a very closely related compound, zeranol
(zearalanol, Ralgro R ) in steers, have shown that clearance from plasma occurs very rapidly (Chichila et al., 1988).
This informa-
tion, in combination with the fact that the rumen is a barrier to systemic
absorption, and other yet to be determined
biological
characteristics of the toxin (i.e. volume of distribution, tissue uptake, protein binding, etc.), may all contribute to extremely low blood residual levels. In conclusion, our study has shown that to achieve measurable levels of total ZEN and a- and /3-ZEL in milk required feeding very high doses of ZEN to a cow.
While these low ng/ml levels in milk
102
PRELUSKY ET AL.
would in theory constitute a significant intake by humans (KuiperGoodman
et
al. , 1987),
in practice
protein
rations would
not
normally contain sufficient ZEN to provide a cow with even as much as 50 or 165 mg daily, at which doses we could not measure transmission to the milk and consequently would not expect any human
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health hazard.
ACKNOWLEDGEMENTS We are grateful to IMC Pitman-Moore, Inc. for a generous gift of ZEN, S.N. Dixon for additional standards of ZEN, a-ZEL, and fiZEL, and to T. Kuiper-Goodman for helpful discussion.
The authors
also wish to acknowledge Dr. K. Hartin, W.A. Emond, and operational staff for care of animals, and R. Warner and N. Zabolotny for their technical assistance.
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Olsen, M. ,
Kuiper-Goodman, T., Scott, P.M. and Watanabe, H., Regul. Toxicol. Pharmacol., 7, 253-306 (1987). Kurtz, H.J. and Mirocha, C.J., In "Mycotoxin Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Handbook", Vol. 2, Wyllie,
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Olsen, M., Mirocha, C.J., Abbas, H.K. and Johansson, B. , Poult. Sci., 65, 1905-1910 (1986). Palyusik, M., Harrach, B., Mirocha, C.J. and Pathre, S.V., Vet. Acad. Sci. Hung., 28, 217-222 (1980).
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Pathre, S.V., Mirocha, C.J. and Fenton, S.W., J. Assoc. Off. Anal. Chem., 62, 1268-1273 (1979). Prelusky, D.B., Warner, R.M. and Trenholm, H.L., J. Biomed. Appl., in press.
Chromatogr.,
Sandor, G., Acta Vet. Hung., 32, 57-69 (1984). Scott, P.M. and Lawrence, G.A., J. Assoc. Off. Anal. Chem., 71. 1176-1179 (1988). Shreeve, B.J., Patterson, D.S.P. and Roberts, B.A., Food Cosmet. Toxicol., 17, 151-152 (1979). Trenholm, H.L., Warner, R.M. and Fitzpatrick, D.W., J. Assoc. Off. Anal. Chem., 67, 968-972 (1984). Ueno, Y. and Tashiro, F., J. Biochem., 89. 563-571 (1981). Ványi, Á., Bata, Á. and Sándor, G.S., In "Proceedings of the International Symposium on Mycotoxlns, September 6-8, 1981, Cairo, Egypt", Naguib, K., Naguib, M.M., Park, D.L., Pohland, A.E., Eds., National Research Centre, Cairo, Egypt (1983), pp. 311-315. Weaver, G.A., Kurtz, H.J., Behrens, J.C., Robison, T.S., Seguin, B.E., Bates, F.Y. and Mirocha, C.J., Amer. J. Vet. Res., 47, 1395-1397 (1986a). Weaver, G.A., Kurtz, H.J., Behrens, J.C., Robison, T.S., Seguin, B.E., Bates, F.Y. and Mirocha, C.J., Amer. J. Vet. Res., 47, 1826-1828 (1986b).
Received: August 10, 1989
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Minimal transmission of zearalenone to milk of dairy cows a
b
D.B. Prelusky , P.M. Scott , H.L. Trenholm & G.A. Lawrence
a
b
a
Animal Research Centre , Agriculture Canada , Ottawa, Ontario, Canada , K1A 0C6 b
Bureau of Chemical Safety, Food Directorate, Health Protection Branch , Health and Welfare Canada , Ottawa, Ontario, Canada , KlA 0L2 Published online: 14 Nov 2008.
To cite this article: D.B. Prelusky , P.M. Scott , H.L. Trenholm & G.A. Lawrence (1990) Minimal transmission of zearalenone to milk of dairy cows , Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 25:1, 87-103 To link to this article: http://dx.doi.org/10.1080/03601239009372678
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J. ENVIRON. SCI. HEALTH, B25(l), 87-103 (1990)
MINIMAL TRANSMISSION OF ZEARALENONE TO MILK OF DAIRY COWS
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KEY WORDS:
zearalenone, dairy cow, milk, plasma, zearalenols, liquid chromatography, food safety, residues
1
2
1
D.B. Prelusky , P.M. Scott , H.L. Trenholm , and G.A. Lawrence 1
2
Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada K1A 0C6 and
2
Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, Canada KlA 0L2
ABSTRACT Milk and plasma levels of zearalenone (ZEN), α-zearalenol (αZEL),
(β-zearalenol
determined
after
(β-ZEL)
feeding
and
conjugated
lactating
cows
metabolites
with
ZEN.
instances where ZEN and α- and β-ZEL were detected
In
were those
in milk or
plasma, they occurred only as conjugates hydrolysable by treatment with
a
mixture
of
β-glucuronidase
and
aryl
sulfatase.
With
studies where 50 or 165 mg was fed daily to three cows for 21 day periods, neither dosage showed the presence of ZEN or metabolites in either milk or plasma (detection limits:
milk, 0.5 ng/ml, ZEN,
α-ZEL; 1.5 ng/ml, β-ZEL; plasma, 2-3 times higher). Contribution Number:
1628, Animal Research Centre 87
Copyright © 1990 by Marcel Dekker, Inc.
A dose of
88
PRELUSKY ET AL.
544.5 mg zearalenone per day given to a single cow for 21 days yielded maximum concentrations of only 2.5 ng ZEN/ml and 3.0 ng αZEL/ml
in
the milk.
In plasma, up
to
3 ng
ZEN/ml
detected during the initial 4 days of treatment.
could
be
At a dose of
1.8 g of zearalenone given over a one day feeding period, maximum
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milk levels of 4.0 ng ZEN/ml, 1.5 ng α-ZEL/ml, and 4.1 ng β-ZEL/ml were
observed
during
the
initial 2 days; corresponding
maximum
levels after a one day dose of 6.0 g zearalenone were 6.1, 4.0 and 6.6 ng/ml milk on days 2-3.
In plasma, peak ZEN concentrations (9 ,
and 13 ng/ml at the lower and higher one-day doses, respectively) occurred 12 hr after initial dosing, and declined to negligible levels by days 5-7.
Neither α- nor β-ZEL were detected in plasma.
Since measurable levels required very high oral doses of ZEN, milk would not normally pose
a human health hazard
as a result
of
feeding rations containing ZEN to lactating dairy cows.
INTRODUCTION Trans -zearalenone mycotoxin
produced
graminearum.
by
(ZEN)
is
several
an
estrogenic
species
of
and
carcinogenic
Fusarium.
notably
F.
Originally isolated as a result of investigations of
outbreaks of hyperestrogenism in pigs consuming contaminated corn (Kurtz and Mirocha,
1978),
ZEN has now been
found
in numerous
samples of agricultural commodities, not only feedstuffs but also grains used in food products (Kuiper-Goodman et al., 1987). There is some experimental evidence for transmission of ZEN, a-zearalenol
(a-ZEL)
and
/3-zearalenol
(£-ZEL)
into
milk
and
MINIMAL TRANSMISSION OF ZEARALENONE
89
tissues of domestic livestock following oral administration of ZEN (Palyusik et al., 1980; Vanyi et al., 1983; Mirocha et al., 1981, 1982; Hagler et al., 1980; James and Smith, 1982; Olsen et al., 1986), but only one report of natural occurrence of ZEN residues in animal products (Sandor, 1984).
Of particular importance when
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estimating human exposure to ZEN is a knowledge of carryover of ZEN and its metabolites into cow's milk.
Since ZEN does not seem
to be an important factor in the health of dairy cows at oral daily doses of up to 500 mg/cow (Weaver et al. , 1986a, 1986b), high levels of ZEN could be present in the feed without being noticed clinically.
Shreeve et al. (1979) could not detect ZEN in
milk of two cows fed concentrate ration containing 1.8 fig ZEN/g for 7 weeks.
Mirocha et al. (1981) found 481 ng/ml of total ZEN
(incuding conjugates) and similar concentrations of total a- and /9-ZEL in milk after feeding a cow grain containing 25 /Jg ZEN/g for 7 days; lower levels of total ZEN and a- and /J-ZEL were present in the milk when the concentration of ZEN in the grain was increased to 250 /ig/g, but for a shorter period of only 1 to 2 days.
After
a single 5-g dose fed to a cow by Hagler et al. (1980) there were only traces of ZEN and /3-ZEL, and then not until 5 days after dosing.
Further information on transmission of ZEN and metabo-
lites was obviously needed.
The present study was designed to
establish the minimum concentration of ZEN in dairy ration which would result in detectable amounts (low ng/ml) of toxin residues being transmitted into the milk.
90
PRELUSKY ET AL. EXPERIMENTAL
Feed Schedule: The three cows used in this study were Holstein cross-breeds weighing
480-580
kg.
Feed
intake
and
milk
production
were
monitored for 2 weeks prior to the ZEN treatment period to calcu-
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late the amount of dairy ration (Table 1) which should be fed, i.e.
0.33
produced. line
ZEN
kg
of
dairy
ration
per
day
for
every
kg
of
milk
Diets containing ZEN were prepared by mixing crystal(IMC Pitman-Moore, Inc., Terre Haute, IN) with
small
amounts of ground corn in a mortar and pestle followed by blending of this spiked corn with the dairy cow ration in a mixer. trations
of ZEN were
confirmed
by
liquid
Concen-
chromatographic
(LC)
analysis (Trenholm et al., 1984). The
experimental
protocol
outlining
the
several
feeding experiments carried out is shown in Table 2.
dosing/
During
Experiments 1 and 2, the three cows were fed first 50 mg ZEN/day for a 21 day period, followed by 165 mg ZEN/day for a 21 day period.
In Experiment 3, three treatments using one animal each
were included. In addition, the caws were fed a daily roughage allotment of dry hay, 3 kg; corn silage, 16 kg; and alfalfa haylage, 9 kg.
The
daily feeding schedule was as follows: 6:00
-
feed 1/2 of dairy ration including 1/2 of ZEN dose;
7:00
-
feed 1/3 of roughage allotment;
15:00
-
feed remaining 1/2 of dairy ration including ZEN dose;
16:00
-
feed remaining 2/3 of roughage allotment.
MINIMAL TRANSMISSION OF ZEARALENONE
91
TABLE 1
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Composition of Ottawa Lactating Dairy Cow Ration 1979
Ingredient
* of Diet
Shorts Ground corn Ground barley Soybean meal Limestone Dicalcium phosphate Trace minerals salt Vitamin premix Molasses
24 40 4 23 1 1.5 1.5 0.2 5
TABLE 2 Experiment Protocol
Diet No.
1
1
3
25 mg x 2
2
2
3
82.5 mg x '.2
3
3A 3B 3C
1(A) 1(B) 1(C)
1
2
3
No. Animals 2
Daily Dosing Regimen 3
Exp.1
272.25 mg x 2 900 mg x 2 3000 mg x 2
Total Daily Dose
Days
50 mg
21
165 mg
21
544.5 mg 1800 mg 6000 mg
21
1 1
Animals were given a 21-day "wash-out" period betwen experiments to allow elimination of potential residues from previous study. Animals in Exp. 3 were labeled as A, B or C to distinguish the individual dosing protocol. ZEN was administered in 2.5 kg dairy ration/day, fed at 6:00 and 15:00 hr, daily.
92
PRELUSKY ET AL.
The amount of roughage fed to the cows was adjusted in the week prior to ZEN administration and maintained constant during the treatment period. During the course of the study, milk samples were taken daily (morning and evening) and tail venous blood samples were
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twice weekly
(Exp. 1,2) or serial (Exp. 3 ) .
taken
Feed intake, body
weights, and milk production were monitored throughout the study. Milk samples were analyzed for fat (%), protein (%), lactose
(%)
and somatic cell count. Determination of ZEN and a- and g-ZEL in Milk and Plasma: Milk reversed basic
samples phase
were
liquid
acetonitrile,
analyzed
for
chromatography acidification,
ZEN
and
a-
following
and
£-ZEL
extraction
partition
into
by
with
methylene
chloride (or chloroform) and cleanup on an aminopropyl solid phase extraction column (Scott and Lawrence, 1988).
For determination
of total ZEN, a-ZEL and /3-ZEL (free plus conjugated metabolites), milk
samples
were
incubated
with
^-glucuronidase
(from
Helix
pomatia. containing aryl sulfatase) prior to extraction in order to hydrolyze any such conjugates (Scott and Lawrence, 1988).
Most
determinations were single analyses but replicate extractions were done in 9 cases and results averaged.
Separate (single) analyses
for the free (unconjugated) compounds were made without addition of enzymes to the milk. using
the
following
gradient
chromatography:
15-30%
over
(no.
7
minutes
The LC method was modified slightly by
5
reversed
phase
(CIB) f°r
acetonitrile
in methanol-water
concave
on
gradient
Altex
liquid (61+35) solvent
MINIMAL TRANSMISSION OF ZEARALENONE
93
programmer) at a flow rate of 1 ml/min, with a 15 minute hold at 30% acetonitrile and pre-run equilibration of 10 minutes at 15% acetonitrile.
Earlier analyses (Experiment 1) were isocratic with
a mobile phase of methanol-water (61+35) containing 17% acetonitrile.
Detection of ZEN, a-ZEL, and /3-ZEL was by
fluorescence
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with an excitation wavelength of 236 nm and an emission wavelength of 470 nra; measurement was by peak area, except at the lowest levels, where peak heights were used. Performance of the analytical method was checked periodically by measuring recoveries of ZEN (5 ng/ml), a-ZEL (5 ng/ml), and /?ZEL (25 ng/ml) added to milk, with and without enzyme addition. The identity of ZEN and a- and /J-ZEL in selected milk samples was confirmed to some extent by comparative (peak ratio) determination at a fluorescence excitation wavelength of 274 nm. of
the
higher
concentrated
level
extracts
samples, (equivalent
compounds to
8
ml
For 5
were
detected
in
milk
injected)
by
absorption at 236 nm using a diode array UV detector (1KB Rapid Spectral Detector); the UV spectrum was scanned and compared with that
of
an
authentic
standard
chromatographed
under
the
same
conditions. Analysis of ZEN and metabolites in 2 ml plasma samples, were carried out according to the method of Prelusky et al. (1989), in which a series of pH-controlled solvent extractions were used to isolate the compounds of interest for determination by reversed phase liquid chromatography with fluorescence detection excitation, 418 nm emission).
(238 nm
94
PRELUSKY ET'AL. RESULTS
Animals: While there were no marked differences in feed intake and weight gains during the intial two feeding experiments, there were two incidences of decreased feed consumption during Experiment 3.
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With cow A (Exp. 3) feed intake decreased 30% for the first 2 days on treatment.
Diarrhea was pronounced on the first day of ZEN
administration;
by
normal.
day
2
the
stools were
loose, but
appeared
By day 2, the vulva was inflamed and edematous.
The
uterus and ovaries were small with a small corpus luteum on the left ovary.
The swollen vulva responded to treatment with lavage
and 20 mL antihistamine
(1.0 H Vetastim).
Scarletol antiseptic
was applied to the vulva and the tail was elevated overnight to prevent further irritation.
With cow C (Exp. 3 ) , roughage intake
decreased 47% on the second to fifth day after ZEN administration. This cow had very liquid diarrhea on day 2 and by day 3 seemed very docile, with the left side of the vulva swollen, edematous and inflamed.
By evening of day 3, the cow seemed more alert and
the vulvular edema was receding.
Follicles could not be palpated
on the small ovaries; the uterus seemed small and firm. In
Experiment
3
the
average
daily
milk
production
was
estimated to be 24, 15 and 15 kg for cows A, B and C, respectively.
No major differences were observed in the milk chemistry
parameters studied during any of the experiments. Transmission into Milk: The method used for determination of ZEN and a- and 0-ZEL in
MINIMAL TRANSMISSION OF ZEARALENONE
95
milk was essentially that published recently by Scott and Lawrence (1988).
Percent recoveries and standard deviations (n-6), in the
absence and presence of enzymes, respectively, were 84.9 + 6.7 and 86.5 ± 12.9 for ZEN, 91.5 + 7.8 and 90.2 ± 10.2 for a-ZEL, and 95.6 + 9.7 and 85.7 + 13.0 for ,8-ZEL.
Limits of detection were
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approximately 0.3-0.5 ng/ml for ZEN and a-ZEL and 1.5 ng/ml for /3ZEL.
Recoveries from plasma by the method of Prelusky et al.
(1989) were similar to those reported above, although detection limits were about 2-3 times higher. The two initial transmission experiments, in which each of three cows consumed 50 mg ZEN daily in their food for 21 days, then later 165 mg daily for 21 days, proved to be negative.
No
free ZEN, a-ZEL, or /3-ZEL was detected in milk collected from the three cows in each study, although a random interference for ZEN or a-ZEL was observed in some samples.
Nor was transmission of
these compounds detected on sampling enzyme-treated milk collected on the same days; however, in a few treated samples ZEN or /8-ZEL may have been present at' the limit of detection of the method. Monitoring of plasma toxin levels over the 3 week course of each feeding trial failed to identify the presence of either the free or conjugated metabolites. Evidence for transmission of ZEN, a-ZEL and 0-ZEL into milk was
only
obtained
when
the
dose
of
zearalenone
was
further
increased to 544.5 mg daily (Fig. 1) and with one-day doses of 1.8 g or
6.0
g
(Figs. 2 and
3, respectively).
Even then no
unconjugated metabolites were detected in any of these three
96
PRELUSKY ET A L .
4-1
D) C
3-
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2i iu
o o o
1-
1
2
3
4
5 DAYS
6
7
8
9
FIGURE 1
Concentrations of total (conjugated) ZEN (0 — ) and a-ZEL (+...) in milk of cow A on diet A (544.5 mg ZEN/day x 21 days) (/9-ZEL was at or below detection limit).
E
z o o o
Ul
FIGURE 2 Concentrations of total (conjugated) ZEN (0 — ) , a-ZEL (+...), and 0-ZEL (x---) in milk of cow B on diet B (1.8 g ZEN x 1 day).
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MINIMAL TRANSMISSION OF ZEARALENONE
97
FIGURE 3 Concentrations of total (conjugated) ZEN (0 — ) , a-ZEL (+...), and /3-ZEL (x---) in milk of cow C on diet C (6.0 g ZEN x 1 day).
highest dose experiments, with the possible exception of a-ZEL and ZEN at the detection limit in milk from cow C on the morning of day 2 (not shown). All data presented in Figs. 1-3 are for conjugated ZEN and aand /0-ZEL; results for /9-ZEL are not recorded for cow A receiving 544.5 mg ZEN/day on a continual basis, as it was not found except at the detection limit in the p.m. milk collection of day 1 (not shown).
The low levels of ZEN and a-ZEL found in milk from this
cow (A) support our earlier negative observations at 10.9 and 3.3 times lower feed levels of ZEN (Exp. 1 and 2, respectively).
Two
typical chromatograms of experimentally positive milk extracts are shown in Fig. 4.
98
PRELUSKY ET AL.
15ZEN
a-ZEL
ZEN a-ZEL
in in UJ tr
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a. UJ
o cc
o u
Ul
a.
0J
1
1 10
0
0
20
to
20
TIME(min)
FIGURE 4 Liquid chromatograms of extracts of enzyme-treated day 2 (p.m.) milk: (a) cow A, 0.84 ng a-ZEL/mL and 0.96 ng ZEN/ml milk; (b) cow C, 3.9 ng /3-ZEL/ml, 2.6 ng a-ZEL/ml, and 5.1 ng ZEN/ml milk. Equivalent to 1 ml milk injected.
Confirmation
of
ZEN
and
a-
and
0-ZEL by
diode
array
UV
detection showed in all cases examined an absorption maximum in the region of 235 nm.
At the low levels found in these milks, it
was not always possible to distinguish the secondary absorption maxima
at
274 nm
Tashiro, 1981).
and
315 nm
(Pathre
et al., 1979; Ueno
and
Agreement between determinations made at 236 nm
and 274 nm excitation wavelengths were satisfactory in most cases where this procedure was used for confirmation.
MINIMAL TRANSMISSION OF ZEARALENONE
99
Distribution into Plasma: Concentrations administration
of
of high
ZEN
metabolites
in
levels of ZEN were
plasma
following
similarly very
low.
Diets A, B and C resulted in only trace amounts of conjugated ZEN being detected (Fig. 5 ) . No free metabolites were found, nor were
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the conjugated alcohols (a- or j8-ZEL). A),
a maximum
plasma
(day
concentration
3) was
of only
measured,
At 544.5 mg ZEN/day (diet 2.8
declining
ng
conjugated
quickly
ZEN/ml
thereafter
to
negligible levels (Fig. 5A). Following the two high one-day doses (diets B and C ) , plasma ZEN (conjugated) peaked at 8.8 and 12.9 ng/ml, respectively, both at the 12 hr sampling. tions decreased
Again concentra-
rapidly, falling to below detectable
levels
(1
ng/ml) by days 5-6.
DISCUSSION In the two initial experiments there was no indication that ZEN was affecting the dairy cows.
Even at the very high doses
given in Experiment 3, decreases in feed intake were transient. However, the estrogenic effects of ZEN were notable in Experiment 3 with swollen inflamed vulvas.
Although one would not expect ZEN
intake to be as high as studied in this experiment in a dairy operation, it does raise the possibility that subtle changes in reproductive performance may occur in dairy herds exposed to ZEN contaminated feed. Our findings are consistent with two of the three published reports
on transmission of ZEN and its metabolites
into
cow's
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100
PREtUSKY ET AL.
FIGURE 5 Concentrations of conjugated ZEN in plasma of cows A, B and C receiving diets A, B and C, respectively (544.5 mg ZEN/day x 21 days, 1.8 g ZEN x 1 day, and 6.0 g ZEN x 1 day).
milk.
The level of ZEN in feed concentrate used by Shreeve et al.
(1979), who detected no residues, was equivalent to a dose of 19 mg/cow/day, which was far too low to expect any transmission based on our own observations.
The single 5 g dose of ZEN of Hagler et
al.
to
(1980),
comparable
our
highest
one-day
dose
of
6 g
(2 x 3 g ) , yielded only "traces" of total ZEN and 0-ZEL in cow's milk.
"Traces" in this case can be interpreted as no more than 1-
2 ng/ml, since these levels were apparently measurable in sheep milk in the same publication.
Thus, the concentrations in cow's
milk were marginally lower (but appeared later) than the levels we observed.
Hagler et al. (1980) did not detect a-ZEL.
Mirocha et
al. (1981) detected all three compounds, as we did, but they found that the free forms were present in concentrations of the same order of magnitude as the conjugates, except after 1 day of a diet
MINIMAL TRANSMISSION OF ZEARALENONE
101
containing 250 pg zearalenone/g grain (equivalent to 1 g/cow/day) when no conjugates at all were detected.
The quantitative aspects
of this study were also quite different from our data; in fact concentrations of total ZEN, a-ZEL and 0-ZEL were as high as 481, 508
and
370 ng/ml,
respectively, which
represented
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consumed dose of 200 mg ZEN/day for 7 days.
0.7%
of
a
By comparison we
found concentrations of the order of only 1-3 ng/ml at nearly 3 times this daily dose of ZEN (Figure 1 ) . The
capacity
of
rumen
fluid
to
degrade
ZEN
(Kallela
and
Vasenius, 1982; Kiessling et al. , 1984) is one variable factor that could account for the wide discrepancies in transmission of ZEN to cow's milk.
Kiessling et al. (1984) found that most of the
degradation of ZEN to ZEL (mainly a-) was due to rumen protozoa. Furthermore, while there appears to be no available about residual blood ZEN,
levels
in ruminants
following
information exposure, to
studies involving a very closely related compound, zeranol
(zearalanol, Ralgro R ) in steers, have shown that clearance from plasma occurs very rapidly (Chichila et al., 1988).
This informa-
tion, in combination with the fact that the rumen is a barrier to systemic
absorption, and other yet to be determined
biological
characteristics of the toxin (i.e. volume of distribution, tissue uptake, protein binding, etc.), may all contribute to extremely low blood residual levels. In conclusion, our study has shown that to achieve measurable levels of total ZEN and a- and /3-ZEL in milk required feeding very high doses of ZEN to a cow.
While these low ng/ml levels in milk
102
PRELUSKY ET AL.
would in theory constitute a significant intake by humans (KuiperGoodman
et
al. , 1987),
in practice
protein
rations would
not
normally contain sufficient ZEN to provide a cow with even as much as 50 or 165 mg daily, at which doses we could not measure transmission to the milk and consequently would not expect any human
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health hazard.
ACKNOWLEDGEMENTS We are grateful to IMC Pitman-Moore, Inc. for a generous gift of ZEN, S.N. Dixon for additional standards of ZEN, a-ZEL, and fiZEL, and to T. Kuiper-Goodman for helpful discussion.
The authors
also wish to acknowledge Dr. K. Hartin, W.A. Emond, and operational staff for care of animals, and R. Warner and N. Zabolotny for their technical assistance.
REFERENCES Chichila, T.M.P., Silvestre, D., Covey, T.R. and Henion, J.D., J. Anal. Toxicol., 1 2 , 310-318 (1988). Hagler, M. , Dankó, G. , Horváth, L. , Palyusik, M. and Mirocha, C.J., Acta Vet. Acad. Sci. Hung., 28, 209-216 (1980). James, L.J. and Smith, T.K. , J. Anim. Sci., 55, 110-118 (1982). Kallela, K. and Vasenius, L., Nord. Veterinaermed., 34, 336-339 (1982). Kiessling, H.-K., Pettersson, H. , Sandholm, K. and Appl. Environ. Microbiol., 47, 1070-1073 (1984).
Olsen, M. ,
Kuiper-Goodman, T., Scott, P.M. and Watanabe, H., Regul. Toxicol. Pharmacol., 7, 253-306 (1987). Kurtz, H.J. and Mirocha, C.J., In "Mycotoxin Fungi, Mycotoxins, Mycotoxicoses. An Encyclopedic Handbook", Vol. 2, Wyllie,
MINIMAL TRANSMISSION OF ZEARALENONE T.D., Morehouse, L.G., (1978), pp. 256-268.
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Eds., Marcel Dekker, Inc., New York
Mirocha, C.J., Pathre, S.V. and Toxicol., 19, 25-30 (1981).
Robison,
T.S.,
Mirocha, C.J., Robison, T.S., Pawlosky, R.J. and Toxicol. Appl. Pharmacol., 66, 77-87 (1982).
Food
Cosmet.
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Received: August 10, 1989
