11

Journal of Food Protection, Vol. 48, No. 1, Pages 11-15 (January 1985)
CopyrighMntemational Association of Milk, Food, and Environmental Sanitarians

Aflatoxin Conversion by Dairy Cattle

Consuming Naturally-Contaminated Whole Cottonseed1
RALPH L. PRICE, 2 * J. H. PAULSON, 3 OTIS G. LOUGH, 4
CONRAD GINGG 5 and ANDY G. KURTZ 6

Department of Nutrition and Food Science, The University of Arizona, Tucson, Arizona 85721, Office of the Arizona State Chemist, Mesa, Arizona,
Cooperative Extension Service, University of Arizona, Phoenix, Arizona 85040, Triple G Dairies, Tolleson, Arizona and United Dairymen of Arizona,
Mesa, Arizona and Arizona Farm Bureau, Phoenix, Arizona

(Received for publication April 2, 1984)

ABSTRACT in feedlots because of its low cost and ready availability.

Whole cottonseed determined to have aflatoxin B, (AFB,)
levels of 5, 31, 104, 280, and 560 jxg/kg was fed as 15% of
the total dairy ration to a commercial herd of 90 grade Holstein
dairy cattle for 70 d. Milk from the bulk tank was sampled
either daily or after each milking and analyzed for aflatoxin
M] (AFM,). The ratio for AFB, in the dairy ration to AFM,
in the milk averaged 75 to 1 under conditions and at levels
tested with no consistent relation to the level of AFBj in the
feed. Approximately 1.6% conversion occurred during the
steady state of consumption and secretion. The federal action
level of 0.5 p-g AFM^L of milk would be produced by cows
consuming a ration containing 15% whole cottonseed contami-
nated with approximately 250 p-g AFB]/kg.
Regulation of aflatoxin-contaminated cottonseed meal
and whole cottonseed used for livestock rations continues
to be under reviewed by state and federal agencies. Af-
latoxin, the carcinogenic mold metabolite, can be found
in cottonseed grown in Southwestern irrigated areas at
levels which exceed those presently permitted in dairy ra-
tions (9).
A considerable portion of the cottonseed remains
whole, to be used in ways other than crushing. At pre-
sent, dairymen throughout the Western states routinely
add both cottonseed and cottonseed meal to dairy rations.
Whole, lint-covered cottonseed has been recognized as a
valuable dairy supplement because of its relatively low
cost, high energy value, and its ability to maintain
milkfat levels in milk from high-producing dairy cows
consuming large amounts of concentrate. In addition, it
has been used for many years as a feed for beef cattle
Arizona Agriculture Experiment Station Journal Article Number 188.
2
University of Arizona, Tucson.
Office of Arizona State Chemist.
University of Arizona. Phoenix
'Triple G Dairies and United Dairymen of Arizona.
6
Arizona Farm Bureau.
Whole cottonseed may comprise as much as 15% or more
of the total diet, while cottonseed meal is rarely fed at
levels exceeding 5% (7).
Federal and state regulation of aflatoxin-contaminated
cottonseed has restricted its feeding to lactating animals
to that containing less than 20 |xg/kg (2(9). Since cotton-
seed meal comprises a small percentage of a ration, ani-
mals will ingest only a low level in the total ration. Re-
cently, the federal action level for aflatoxin-contaminated
cottonseed meal was raised to 300 |xg/kg when it is used
as a feed ingredient in rations of beef, swine, and poultry
(10). This change has no effect on regulation of the use
of whole cottonseed in the rations of any animal nor on
the feeding of dairy animals.
Because whole cottonseed may comprise a significant
portion of a livestock ration and because contaminated
whole cottonseed may inadvertently enter dairy rations,
a decision has not yet been made on any change in the
federal action level for this commodity. Inadvertent or
deliberate inclusion of cottonseed intended for livestock
only into rations intended for dairy cows may occur in
spite of rigorous regulation. To prevent production of
milk contaminated with AFMi at levels greater than 0.5
|xg/L, it is necessary to establish the feed-through ratio
of aflatoxin on a large number of high-producing cows
being fed naturally-contaminated whole cottonseed with
more accuracy than presently exists. Dairymen, cotton
growers, livestock feed producers and regulators would
benefit by being able to have a better idea about the
levels of aflatoxin-contaminated cottonseed which can be
permitted in the dairy ration.
Studies designed to establish the amount of an ingested
dose of aflatoxin B] (AFBj) that could be expected to
be excreted as its mono-hydroxylated derivative, aflatoxin
M, (AFM,) into milk found that the conversion varied
from 0.2 to 3.2% (19). In most previously published
studies, the number of animals was limited because of
the costs of the animals and of the contaminated milk,

JOURNAL OF FOOD PROTECTION, VOL. 48, JANUARY 1985

12 PRICE ET AL.

and they were usually low producers because of their ad- Analysis of other feed ingredients
The dairy ration prepared on-site consisted of barley, hominy, molas-
vanced stages of lactation (5). It has been postulated that
ses, beet pulp, wheat bran, corn silage, alfalfa hay, a cottonseed meal-
high producing cows are more likely to secrete a greater based vitamin and mineral supplement and the whole cottonseed lots
amount of AFM, into their milk because of a more rapid later described. Samples of each ingredient were ground with a Bauer
metabolic rate (6,21). The one large-scale study which mill to pass through a 20-mesh screen and were sent to all three labora-
may have given information on feed-through of aflatoxin tories for analysis by their respective methods for cottonseed analysis.
from naturally-contaminated cottonseed at a commercial
Composition of the dairy herd
dairy, suffered because of difficulty in accurately deter-
Ninety grade Holstein dairy cows representing all ages from 2 to
mining AFBj levels in the dairy ration (77). 9 years and in all stages of lactation through 215 d post-partum were
The purpose of this study was to establish the extent selected at random from those at a dairy of 1200 cows. Cows were
to which AFM! is excreted into the milk of a large placed in a typical dry lot dairy corral for 10 d before the experiment
for in-herd adjustment. Medical records were available and the cows
number of cows which have been fed to ration containing
were observed routinely throughout the experiment by a veterinarian.
15% whole cottonseed which had been naturally-contami-
nated with AFB] at different levels, under conditions rep- Feeding schedule
resentative of commercial scale and practice. The entire dairy ration was placed in the feed bunker. This included
Because of the recurrent nature of the contamination the whole cottonseed which was incorporated in an amount equal to
and the problems associated with regulating the resultant 15% of the total dry matter of the ration. The daily ration for each
animal consisted of the following:
milk, meat and other animal by-products, this study was
commissioned and supported by state educational, health, Mineral and vitamin supplement
industrial and regulatory institutions to further explore the (cottonseed meal-based) 1.32 kg 2.91 lb
effects of aflatoxin as it relates to its pass-through charac- Cracked barley 2.66 5.86
teristics. Corn hominy 2.05 4.52
Molasses 1.1 2.4
Wheat bran 2.3 5
MATERIALS AND METHODS Dried beet pulp 1.8 4
Alfalfa hay 7.3 16
Cottonseed procurement and preparation Corn silage 7.3 16
Whole cottonseed was donated by an Arizona oilseed crushing com- Whole cottonseed 3.6 8
pany. An attmept to locate cottonseed contaminated with AFB, at ap- Total 29 kg 64 lb
proximately the desired levels was made by first using records of the
company's original analyses and segregation of the seed when it entered Since the concentrate portion and the cottonseed were completely
from the gin. Samples were taken from the selected piles using a Prob- consumed, cleaning of the feed bunkers was limited to every-other-day
A-Vac pneumatic grain sampler (Cargill, Minneapolis, MN) and sampl- removal of alfalfa stems.
ing techniques described in the State of Arizona Feed Regulation R3-3- After the 10-d adjustment period, cows were given successively
53 (4). higher then lower levels of aflatoxin in the ration, according to the
A minimum of 7.5 kg (30 lb) was dehulled using a Bauer disc mill following schedule:
set at about 3-mm clearance. The meats were separated from the hulls Days 1-10 5 u.g/kg in cottonseed, 0.8 ^g/kg n feed
and lint by passing the milled seed across a shaker table perforated Days 11 -20 31 u.g/kg in cottonseed, 5.6 jig/kg n feed
with 5-mm (diameter) holes. Both portions were weighed, and the Days 21-32 104 u.g/kg in cottonseed, 18 ^g/kg n feed
meats were ground with another Bauer mill to pass a 20-mesh (U.S. Days 33-42 280 u.g/kg in cottonseed, 48 jig/kg n feed
Standard) screen. The entire ground meat portion was mixed thoroughly Days 43-52 560 u.g/kg in cottonseed, 94 ^ig/kg n feed
and passed through a divider to be split into four equal portions for Days 53-62 157 u.g/kg in cottonseed, 27 jig/kg n feed
distribution to each of the laboratories doing the aflatoxin analyses; one Days 61-70 8 n-g/kg in cottonseed, 0.8 ^g/kg n feed
was retained for future reference.
After levels of aflatoxin were determined, 5-ton lots from the selected Dry matter of all feed ingredients with the exception of the corn
piles were transported to the dairy farm where the study was to be silage was calculated as 90%. Dry matter of the silage was calculated
done. They were stored on concrete under cover but with natural air as 40%. This gave a total of 21.8 kg (48 lb) of dry matter in the
circulation. Sampling, sample preparation and analysis were carried out ration. The aflatoxin levels in the total ration were calculated by deter-
as before on the lots after delivery to the dairy farm. mining the amount of AFB| in both the cottonseed and the supplement
Analysis of aflatoxin in cottonseed and calculating its equivalent in 21.8 kg.
Laboratory A. Analysis for aflatoxins was by the Association of Offi-
cial Analytical Chemists (AOAC) method 26.A01-26.A08 for TLC (2) Milk sampling
with the following modifications: aluminum chloride-zinc acetate was Cows were milked twice daily; the combined milk was cooled to
used in place of lead acetate; chloroform rather than methylene chloride 4°C in a refrigerated bulk tank in which it was stored until samples
was used for partitioning; the chloroform was passed directly through were taken. The milk was agitated for 10 min before sampling with
a chromatographic column containing 4% deactivated alumina and silica a 225-ml glass container. Samples were collected initially once daily
gel; and the thin layer plates were developed with a tank solvent of in the morning during the first 25 d, but problems developed with freez-
toluene:ethyl acetate:formic acid (6:3:1) (v:v:v). ing milk which was stored overnight, so separate samples were col-
Laboratory B. Analysis was by the AOAC method for aflatoxins in lected twice daily from the morning and evening milkings throughout
cottonseed, 26.A01-26.A04, using the TLC method of quantitation. The the remainder of the trial. For protection against microbial activity, 4
method was modified by inclusion of a hexane wash of the acetone ml of a solution of 15 mg of sodium azide/ml was added to each liter
extract to remove the fat before chloroform extraction. No column of milk for preservation of the sample which was sent to Laboratory
cleanup was done before TLC. Z. Evening samples were stored at 4° overnight. After morning samples
Laboratory C. Analysis for aflatoxins was by the AOAC method, were taken, both samples were placed on ice and transported im-
26.A01-26.A08, for HPLC with a Waters, Inc. HPLC using a 10-^M mediately to the laboratories for analysis. Transportation to Laboratories
Partisil column. X and Y required less than 1 h and to Laboratory Z about 6 h.

JOURNAL OF FOOD PROTECTION, VOL. 48. JANUARY 1985

 AFLATOXIN CONVERSION BY DAIRY COWS
Aflatoxin Mi analysis
Laboratory X. When received, milk samples were placed into a re-
frigerator at 4°C. Analyses were performed on a weekly basis with
AOAC method 26.090-26.094 for AFM, using TLC visual comparison
and estimation (2).
Laboratory' Y. Milk was analyzed daily along with the cooperative's
quality control samples using AOAC method 26.A10-26.A14 for
HPLC. Analysis was done on 20-JJ.I portions of the sample extracts.
Percent recovery was determined by adding known amounts of AFM,
in acetonitrile:benzene (10:90) to aflatoxin-free milk.
Laboratory Z. Milk was stored overnight at 4°C and analyzed daily
except for weekends. AOAC method 26.A10-26.A14 was used. TLC
development was for 8 cm using ether:methanol:water (95:4:1) as a de-
veloping solvent. Confirmation of AFM, identity was by AOAC method
26.A16. Percent recovery was determined by spiking aflatoxin-free milk
with known amounts of AFM| in acetonitrile:benzene (10:90).
RESULTS AND DISCUSSION
Dairy herd
On days 11, 29, 41, and 43 it was necessary to remove
individual animals from the herd because of mastitis or
hardware disease. They were not replaced. Total feed ra-
tions to the herd were adjusted accordingly. Average
daily milk production of the entire period was 27.7 L (60
lb) per animal. The 8% decrease in milk production (av-
erage of 28.4 to 26.1 L) per animal over the 70 d of the
lactation period was considered to be normal according
to Dairy Herd Improvement Association (DHIA) records.
DHIA records kept on all individual cows showed no
aberrations from previous lactations.
Procurement of aflatoxin-contaminated cottonseed.
One of the major problems in feeding a naturally-con-
taminated feed has always been the difficulty in determin-
ing the exact amount of aflatoxin that is in the experi-
mental feed. Cottonseed has the same spotty distribution
of contamination that has been seen in peanuts and in
corn (27). Some investigators have attempted to avoid
this problem by feeding known amounts of pure aflato-
xin. This, we believe, is an artificial situation which does
not duplicate what will be seen under commercial condi-
tions. In this study, we attempted to determine, as far
as possible, the true levels of aflatoxin in all groups by
first selecting a portion of cottonseed, which had been
analyzed by the oil mill has having approximately the de-
sired AFB, concentration. After analysis on-site, trans-
portation to the dairy farm, resampling and reanalysis,
the cottonseed was used as a feed ingredient only if the
coefficient of variation of all analyses was less than 25%.
TABLE 1. Aflatoxin B] (\x,glkg) in lots of whole cottonseed included as 15% of the dairy ration.
Desired Control Lab A
levels number TLC
0 482 6 5
20 466 32
100 467 103
300 492 213
1000 496 539
300 709 157
Mineral mix 483 16
JOURNAL OF FOOD PROTECTION, VOL. 48, JANUARY 1985
13
The mean value of all analyses of a specific lot was then
used for calculation.
Analytical results of different lots are shown in Table
1. Some of the lots did not meet the specifications and
were discarded. The original thought was to feed cotton-
seed contaminated with AFB, at 0, 20, 100, 300, 1000,
300, and 0 (xg/kg as 15% of the dairy ration. It was ex-
pected that the stepwise increase of AFB! in the ration
would minimize the time to reach the constant conversion
rate at each level, and a 10-d feeding period at each level
would provide sufficient samples for statistical evalua-
tion. By comparing results near the end of the test with
those at the beginning of the test, we hoped to have an
indication of the effect of feeding high levels of AFB,
upon the rate of metabolic conversion. The average levels
of AFB, in the lots of cottonseed selected were 5, 31,
104, 280, 560, 157, and 5 (xg/kg.
The factor which was surprising is that the salt and
mineral mix had 15 (xg AFB,/kg. Cottonseed meal had
been added as filler. It was necessary to include this in
the total aflatoxin consumed during the experiment.
Milk analysis
Each of the laboratories which was asked to analyze
milk samples had several years of experience in AFM,
analysis. One was regulatory, one was commercial and
the other was research. Before this experiment, numerous
check samples consisting of naturally-contaminated milk
had been distributed regularly to each of the laboratories
as part of in-state quality control.
In this experiment, examination of the results of AFM,
analysis indicate that the results from laboratories Z and
Y have a correlation of 0.96 with each other and a corre-
lation of 0.98 with changes in levels of the AFB, fed.
The results from laboratory X have serious anomalies;
when levels would be expected to be highest, they are
only moderate; when they would be expected to be de-
creasing, they suddenly rise. Whereas laboratories Z and
Y analyzed daily, and laboratory Z had sodium azide
added as a preservative because of the 72-h holding
period over the weekends, laboratory X held samples
from several days to 2 weeks at times until personnel
were available from other analytical duties for these
analyses. In addition, in an international check sample
program involving more than 80 laboratories, the official
AOAC method used by laboratory X has recently been
shown to give the poorest reproducibility of all official
LabB LabC Average
TLC HPLC AFB,
5 5
28 34 31
100 108 104
265 361 280
705 435 560
160 153 157
15 Missing 16
14 PRICE ET AL.

methods tested (77). Laboratory X also reported values
less than 0.1 jxg/L as less than 0.1 ji-g/L rather than try
to estimate the value. This is a satisfactory system for
regulation but not for statistical evaluation.
Because of the difficulty in trying to assign values to
those reported as "less than" a certain value, and be-
cause of the reported variations in the analytical methods
used, the results from laboratory X were excluded from
estimation of the level of AFM, in the milk. Figure 1
shows the levels of AFB, in the cottonseed and the
AFM, found in the milk over the course of the experi-
ment.
Calculation of AFB, infeed-AFM, in milk ratio
Two different methods may be used to calculate the
ratio of AFB, in the feed to AFM, in the milk. Both
are useful for different purposes.
To calculate the percentage of AFB, that was con-
verted to AFM, and excreted in the milk, the total
amount of AFM, produced each day by all cows was
divided by the total AFB, consumed daily and a regres-
sion equation of these two factors was calculated. To
allow for biological equilibrium to occur, the average of
AFM | values obtained from only days 3-10 of each feed-
ing period were used. The resulting regression equation
was Y=.0055 + .0159X, r2 = 0.962, n = 55 where Y
would be the p.g of AFM, excreted daily in the milk
and X would be the jxg of AFB, consumed daily by the
animal. If values from the initial and final 10-d periods
(where analysis was close to detection limits for both
AFB, and AFM,) were excluded from the calculations,
the regression equation (Y = -.0025 + .0162X,
r2 = 0.955, n = 41) was the result. If values from day 3
were excluded (to allow more time for equilibrium to
occur), the equation was Y=.0069 + .0162, ^ = . 9 6 4 ,
n = 36. The slopes and intercepts of these regression equ-
ations were not significantly different. The high correla-
tion coefficients support the results of earlier research
showing a linear relationship between AFB, consumption
and AFM, excretion (14). In this instance, however, be-
cause the data were sequentially obtained, the day-to-day
regression observations may be somewhat inflated (75).
An attempt to verify autocorrelation gave inconclusive re-
sults (8).
The slopes and intercepts of these equations are differ-
ent from those of Mertens et al. (14) who used data of
all previous tests where steady state of excretion was at-
tained. The present study would predict significantly
higher levels of AFM, excretion than would either their
average regression equation, Y = -515 + .0091X,
r ^ 0.976, n = 20, or that which they called the higher

1.30

1.20

1.10

1.00
5 ppb in 31 ppb in 104 ppb in 2 8 0 ppb in 560 ppb in 157 ppb in 8 ppb in
.90 - cottonseed cottonseed cottonseed cottonseed cottonseed cottonseed cottonseed

* .80
_J
^ .70
Z
— .60
0.5 PPB-FDA ACTION LEVEL
.50
<
.40
oa
D_
D_ .30

.20 -

.10 -

.01 111 Jjlll

10 15 20 25 10 15 20 25 30 I 5 10
FEBRUARY MARCH APRIL

DATE

Figure 1. Aflatoxin M, in milk of cows fed a dairy ration containing 15% whole cottonseed naturally-contaminated with differ
levels of aflatoxin Bh

JOURNAL OF FOOD PROTECTION, VOL. 48, JANUARY 1985

 AFLATOXIN CONVERSION BY DAIRY COWS
excretion rate equation, Y = -6.11 = .0102, r 2 =.999.
Under the conditions of our study, more AFB, was con-
verted to AFM, than would have been expected (1.6%
vs 0.91%).
One of the explanations for these differences may be
that in naturally-contaminated products, more aflatoxin is
absorbed through the digestive system. However, our re-
sults are between the average of 0.69 and 2.2% found
by investigators feeding naturally-contaminated peanut
meal (1,16).
A more acceptable explanation is that high-producing
cows appear to convert more AFB, to AFM! through de-
toxification mechanisms to be secreted into the milk (72).
The average milk production per animal in this study
(27.7 L), exceeded by 50-200% the average production
of animals in all other studies in which milk production
was reported with the exception of one animal in the
study of Masri et al. (13).
The other method of calculation is probably more use-
ful for regulatory or commercial estimation of the level
of AFM, which might be expected in milk if a ration
is fed which contains a known amount of AFB, in cot-
tonseed comprising a known portion of the ration.
When the average level of AFB, in the total dairy ra-
tion during the steady state of any period (days 3-10) was
compared with the average levels of AFM, during the
same period, the average ratio was approximately 70 to
1. A slight tendency to lower values was seen at the
lower feeding levels. When the averages for the initial
and the final periods were removed from calculations, the
ratio was 78 to 1. This would indicate that the federal
action level for AFM, in milk would be reached by feed-
ing these high-producing cows a ration approximately 40
(jug AFB,/kg. These values compare favorably with some
previous experiments although the average ratio was
lower (AFM, levels were higher ) than the approximately
300 to 1 cited by a review of most previously published
experiments (78).
Since the federal action level of 0.5 |xg AFM,/L would
be produced in the milk of high-producing cows consum-
ing a ration containing 15% whole cottonseed contami-
nated with approximately 250 |xg AFB,/kg, it would be
inadvisable to raise the federal action level on whole cot-
tonseed to 300 (xg/kg because of the possibility of inad-
vertent use of this cottonseed for dairy rations. A raising
of the action level from the present 20 to 100 |xg/kg
would probably not present any regulatory problem other
than those involved with a multi-tiered regulation system
such as the system which has been used successfully in
Arizona for several years. It would, however, result in
occasional AFM, positive test results in milk. This may
be a problem for individuals, cooperatives, or states
which have the goal of producing milk with no detectable
levels of AFM,.
ACKNOWLEDGMENTS
Research supported in part by Western Regional Research Project W-
122, the Arizona Farm Bureau, Vegetable Growers of America, Arizona
Cotton Growers Association, Cotton City Gin, Pinal County Farm Bu-
JOURNAL OF FOOD PROTECTION, VOL. 48, JANUARY 1985
15
reau, Anderson Clayton and Co., Producers Cotton Oil, Acme Gin
Company, Arizona Cotton Growers Association, Pima County Farm Bu-
reau, Chandler Ginning Company, United Dairymen of Arizona, Triple
G Dairies, Office of the Arizona State Chemist, Arizona State Agricul-
tural Laboratories, Arizona Department of Health Services, and the
Arizona State Dairy Commissioner. Appreciation goes to Dr. Thomas
Russell, Karen Jorgensen, Michael Billotte, Janet Bessey, Mary Keefer,
and Robert Magnenat for sample collection, preparation and analysis
and to Drs. Robert Keuhl and B. L. Reid for statistical interpretations.
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