2.5. Combustion analysis of fecal and extracted tissue residues Ž
. The method used was a modification by Sheppard and Rodegker 1962 . The dry
sample was pressed into a pellet and placed in a Parre oxygen bomb, which was charged with 30 atm oxygen, submerged in a water bath and electrically combusted. The
bomb was then cooled to condense the vapors, followed by slowly discharging the exhaust through a cold water trap. The bomb was then opened, flushed with absolute
ethanol and the rinsate collected for
3
H analysis. The percentage recovery was approxi- mately 75 3.0.
2.6. Statistical analysis Ž
. The data expressed in counts per minute CPM recovered from the urine, bile, GI
tract, and liver from the clay-fed and non-clay-fed fish were subjected to the Mann– Ž
. Whitney U-test Siegel, 1956 . CPM data from the fecal matter obtained from these two
Ž .
groups were subjected to evaluation by the Student’s t-distribution McGhee, 1985 .
3. Results
Ž
3
. Ž
3
. The recovery of tritium
H counts from the tritiated aflatoxin H-AFB
is reported
1
as CPM based on the CPM obtained as described in Section 2. The CPM present in 20
Fig. 1. CPM recovered in the urine each day for a period of 7 days of feeding diets with 20 mgrkg of mgrkg
3
Ž H-labeled AFB with and without 2 bentonite. ns 3 for each point on each curve sampled randomly from
1
. Ž
. the 20 fish , except day 7 ns 2 . The standard deviation range averages about 8 of the mean samples
without clay and about 10 for samples with clay. Day 2 through day 6 showed a significant difference Ž
. P - 0.05, Mann–Whitney U-test .
ng of the
3
H-AFB , which was the amount fed in one capsule each day, was 1.04 = 10
6 1
CPM. The CPM recovered in the urine increased daily from day 1 to day 6, with the CPM
Ž .
at day 6 in the trout fed clay averaging about 85 less than for fish not fed clay Fig. 1 . Ž
. Day 2 through day 6 showed a significant difference P 0.05, Mann–Whitney U-test .
About 63 less label accumulated in the head waters from fish with clay compared to Ž .
those not fed the clay. Only samples 4 for 1 day of head water collections were counted.
The fecal CPM recovered showed that those fish fed with clay averaged about 470 Ž
. more label excreted in the feces than the fish not fed clay Fig. 2 . This was a significant
Ž .
difference P - 0.001, Student’s t-test . The CPM recovered in the fecal material
represents the only finding in which the trout fed with clay had values exceeding those not fed clay.
Results of tissue and body fluid analysis for the
3
H-label all indicate a much greater uptake of
3
H-AFB in fish given the non-clay diet. Blood samples from fish given either
1
diet were the only samples yielding no measurable recovery of CPM. Fig. 2 shows the comparison of
3
H-CPM recovered from feces, bile, and the GI tract from fish given the
3
H-aflatoxin contaminated diet with and without clay. These data yielded a greater Ž
. variation between similar samples than the data obtained from tissues Fig. 3 . There
Ž .
Fig. 2. CPM recovered in the bile, GI tract, and feces including contents . Trout were fed diets with 20 mgrkg
3
H-AFB with and without 2 bentonite. Fecal samples from eight different fish fed with clay were
1
Ž .
significantly different from eight different fish fed without clay P - 0.001, Student’s t-distribution . The bile Ž
. Ž
and GI tract values from the two groups three in each group were also significantly different P - 0.05, .
Mann–Whitney U-test .
was some difficulty in collecting all the bile present in each fish for
3
H counting. Even with the large standard deviation among samples, there was a clear difference between
average values obtained from fish fed the clay and non-clay diets. The bile and GI tracts from fish given the non-clay diet yielded about a 7.4 and 3.5, respectively, greater CPM
Ž recovery than that from fish fed the clay diet significant difference P - 0.05, Mann–
.
3
Whitney U-test . The amount of H in the GI tract was probably mostly due to bile Ž
3
. products transformed from H-AFB
since about 90 of this activity could be rinsed
1
away in samples from fish with or without clay. Also about 88 and 96 of the total CPM obtained from the unrinsed GI tract from the clay and non-clay diet-fed fish,
respectively, appeared in the methanol fraction and the remainder in the chloroform fraction. The bile derivatives of AFB tended to be more methanol soluble, while AFB
1 1
Ž .
itself partitioned more extensively into the chloroform phase Wong and Hsieh, 1980 . Fig. 3 shows the comparison of label from
3
H-AFB accumulated in the kidney, liver,
1
and carcass after 7 days of feeding fish
3
H-AFB contaminated diets containing 2
1
bentonite and no bentonite. All tissues showed a greater accumulation of label in fish fed diets with no clay. The data are reported as the total CPM from the methanol and
chloroform fractions and the residual tissue following extraction. The liver samples were Ž
. significantly different P - 0.05, Mann–Whitney U-test .
Fig. 4 was constructed from the total CPM obtained from all the materials listed with each sample expressed as a percentage of the total. The only sample from the clay-fed
Fig. 3. CPM recovered in the kidney, carcass, and liver after sacrificing fish following the 7-day feeding period. Trout were fed diets with 20 mgrkg
3
H-AFB with and without 2 bentonite. ns 2 for each kidney
1
and carcass value for each dietary treatment and ns 3 for liver samples from each treatment. The liver Ž
samples from fish fed diets with and without clay were significantly different P - 0.05, Mann–Whitney
. U-test .
Ž .
Fig. 4. Percentage recovery of CPM for bile, urine, GI tract including contents , kidney, carcass, liver, and feces based on the total CPM recovered from these tissues and excreta, from fish fed non-clay and 2 clay
containing diets contaminated with 20 mgrkg H-AFB .
1
Ž .
trout containing a higher percentage and also more CPM than that from the non-clay-fed Ž
. trout was the fecal sample 85 vs. 23 .
4. Discussion
