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Item D Number
°2342
Author
Bonaccorsi, A.
Corporate Author
RdpOrt/ArtlClB TitlB Typescript: Study of the Bioavailability in the Rabbit of
the TCDD Present in Powdered Soil from Seveso Zone
A (Milan)
Journal/Book Title
Year
1983
Month/Day
June
Color
Number of Images
D
29
Descrlpton Notes
Friday, October 05, 2001
Page 2342 of 2422
�LSTISA^r 1383/19
Study of the bioavailability in the rabbit of the TCDD present
in •pondered soil from Seveso Zone A (Milan) '
1
o
*
o
1
A.Bonaccorsi , A.di Dorrtenico*", R.Fanelli', F.Merli , R.Motta ,
.
R.Vansati 2,f G.Zapponi. 2
1 Istitutq di Ricerche Farmacologiche' "Mario Mecri", Milan
2 Laboratory of Comparative Toxicology and Ecotoxicology,
Supreme Health Authority, Rome
Study conducted under tlis agreement befvesii the Special Departrier.t of Seveso {'/.ilan) of the Lombard-/ P.egio:-., tl.e "Mario
Hegri" Pharmacological Research Institute and the Istitxito
Superior® di Sanita (rLoma^ of 8 Jur.e 1321
�1
INTRODUCTION
TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is tiie most toxic of
the polychlorodibenzodioxins and is, as a rule, an unwanted microcontaminant of 2,4,5-trichlorophenol, produced on a large scale
by industry. TCDD has also been formed accidentally in relatively
high quantities in a few accidents at chemical plants, including
the one at the 1CMS3A chemical plant (Seveso, Milan) in July 1976.
This event led to diffuse TCDD contamination of the environment
whose gravity is still visible 5 years later (1).
Free TCDD has been the subject of numerous toxicological studies,
mostly designed to characterise its toxic properties rather than
to assess its absorption by the organism (2), However, exposure
to the poison in the environment is often due to the presence of
environmental substrates to which TCDD is in some way bound; more
rarely is the compound fouiid in the free form. Thus the present
study arose from the need to establish the bioavailability of TCDD
--hen the substance is administered to the rabbit on pondered soil
and not -in the free form. 3ir.ce previous work had shown that the
liver is the target organ in that species (3), accumulation of
TCDD in the liver was taker, as index of absorption. The various
forrrulations containing TCDD '"ere administered by gastric tube.
The extent of bioavailability may be of importance in ar. assessment of the toxicological risk.
2
METHODS OF ANALYSIS
The determination of TCDD in particulate soil was dons by mear.s
of a known GC-M5 method (4) adapted for small samples ;'~-2 c] and
v.sing t'/.e following sts^s: extraction in a Soxhlet ar^arav.is,
�chroma tography on a multilayer column (described later) and
chroinatography on a column of activated alumina.
The determination of TCDD in liver tissue was done by a GO-MS
method described in the literature ( 5 ) , using the following
steps: alkaline digestion, extraction with a solvent, chromatograp hy on an Extrelut column and chromatography on a column of
activated alumina.
As shown in Table 1, recovery by the method as a whole and by
two purification steps is on average always more than 9Q% in
the case of powdered soil samples. In the case of liver samples
recovery calculated with the addition of a tracer is B0%. For
further details see Appendix.
3. PREPARATION OF THE FORMULATIONS
TO \g of dry agricultural soil was removed from an area in Zone A
with a high level of contamination (mean TCDD >3COO ug/rr/) . The
stone-free material was spread on plastic sheets for drying in the
air (t =s 25-30°C) to constant weight (weight variations <2% over
24 h), crushed vigorously, remixed and sieved for collecting the
powder fraction with a particle si-ze of between 200 and ^00 mesh.
This fraction (-~0.5 -eg) was further mixed in a rotary evaporator
at a moderate speed for about 1C h to obtain a product with a
high degree of homogeneity. The results of tan analyses or. as
many portions of powder (on average ^2 g each) taken at random
revealed a mean contamination of Si - S pro (Table II). The
standard deviation value denotes a satisfactorily homogeneous
distribution of the contaminant. Portions of tl'.is powder (2.00 0.05 c sach) were administered to rabbits r see Treatment of the
rabbits) .
�TABLE I
Mean recoveries of TCDD for the methods of analysis used and
for two specific purification operations.
Subject
Multilayer column
Alumina column
Soil 1 2
Soil 23
Soil 34
Hepatic tissLia"3
1
2
3
4
5
number of
data
71
22
20
16
Mean
recovery (°0
96-12
94- 5
92±15
31
9^i 8
94- 7
57
30-10
For further details and for the individual data on each subject see Table "Recoveries of TCDD" (Appendix).
Laboratory-contamined soil analysed \vithin £3 h of addition
of TCDD.
Laboratory-contaminated soil analysed after "aging" ( 1 month
37
Yield of TCDD (Cl ) added to uncoiltaminated ?articulate soil
and to Seveso1 particulate soil before extraction,
in the
range 10—1 CO ppb.
•3 7 •
/
Yield of TCDD (C1J/) added to the sample of hepatic tissv.e
before alkaline .digestion, in the ranee C.5-5 ppb.
�TABLE II
TCDD levels determined in ten samples of powdered soil from
Zone A after sieving and homogenisation.
„,__,„
,. _ Recovery2
Sample .,
g
1
2
3
4
5
6
7
8
9
10
2.15
1.80
2.06
2.11
1.92
2.32
2.00
1.89
2.00
2.00
TCDD found
*g
174
96
131
94
160
173
141
122
100
91
93.0
103.5
99.5
95.7
80.0
90.0
110.0
107.5
94.5
101.5
203
159
180
173
157
186
159
146
139
137
ppb
94.4
88.3
87.4
82.0
81,8
80.2
79.5
77.3
69.5
68.5
Mean TCDD3 concentration and its standard deviation:
8 1 - 8 ppb
1
Number of refluxings for the extraction in a Soxhlet
apparatus.
„„
2 Calculated on the quantity of tracer (I?.CDD(C1J/)) added
to the soil before extraction.
3 '.To correction "/as made for losses due to the method of
analysis because of the high recoveries (p>97%') .
�10 kg of soil presumably free from TCDD but morphologically
similar to that o£ Zone A was treated as described in the preceding section to get-^1 leg of ponder (200-^00 mesh). Analysis
of five samples («* 2 g each) revealed no TCDD at an analytical
sensitivity of 0.6 ppb. This powdered soil was administered to
rabbits as such in the tolerance tests (see Treatment of the
rabbits). One croup of accurately weighed portions (1.00-0,02 g
each) of the same powder was spiked with TCDD in an acetone solution (<3 ml/portion) at a level of 10.0-0.2 or 40-1 ppb); the
portions were left to "age" in the dark at 2C°C for over 1 month
before administration to the rabbits. A second group of portions
of pov/dered soil was contaminated (40-1 ppb) 48 h before administration to the rabbits, sufficient time for the solvent to evaporate. Each portion (or the combination of two) "/as the single
daily dose intended for each animal. Some samples of each group
were used for testing the recovery in the critical steps of the
analysis method.
The following were prepared: two solutions of TCDD in an acetonevegetable oil mixture (1:6) containing 1C.C-G.2 and 40.0-0.3 nc/ml
of poison respectively, and a'water-alcohol solution (l:l) containing 40.0-0.3 ng/ml. The single daily doses intended for each animal were 1 .00-0.01 and 2.0C-0.01 ml according to case.
4
T2EATKSITT OF TKS ?.A33ITS
Before treatment of the animals started, tests were done to find
out whether the repeated administration of riven quantities of
powdered soil could cause digestive tract disturbances that micht
affect the absorption of TCDD from the gut.. The tolerar.ee tests
�were done on six rabbits, each of which received a suspension
of powdered soil (2.0 g) in water (10 ml) by gastric tube daily
for 7 days. The animals presented no ill-effects, apart from a
brief interruption of weight gain on the first day of treatment.
3y the second day the bodyweight growth curve had already resumed its normal trend.
The previously described preparations were dosed by gastric tube
for 7 consecutive days to groups of 5-7 male white rabbits of
mean starting weight 2.4-0.2 kg (Fig. 1), caged singly. In all,
12 groups of animals were treated with the following preparations
(daily doses in brackets): a) powdered soil from Zone A, Soil S
(^80—160 ng daily); b) "aged" laboratory-contaminated soil, Soil
2 (20-40-80 ng daily); c) laboratory-contaminated powder, Soil 1
(40-80 ng daily); d) acetone-oil solution (20-40 ng daily); wateralcohol solution (40-80 ng daily). The daily dose (1.00 or 2.00)
of powdered soil was sxispended in 10 ml of water. The preparations
containing TCDD were given regularly at 09.00. On day 3 the animals
were killed and the livers removed and prepared for analysis.
Table III gives (a) the weights of the animals at death (p = 2.5
-0.3 kg; see also Fig. 2), (b) the weights of the whole livers
(see also Fig. 3) and (c) of the portions of hepatic tissue used
for analysis, -(d) the individual TCDD levels found in the hepatic :
tissue: in the case of groups or. broadly homogeneous treatments
(as "/ill be noted frcm the lack of significant differences betweer.
the specific values of the means) the cumulative mean was calculated on several homogeneous groups. Table III contains three
such cases: treatments with TCDD in solution at the dose of 40 r.c
daily, treatments with laboratory-cor.taminsEi powdered soil at the
dose of 40 ng daily -and treatments with laboratory-contaminated
powdered soil at the dose of SO nc daily. The results are surjur.arisec in Table IV
�Table III
Levels of TCDD found in the hepatic tissue of rabbits
after seven days treatment
Treatment
BodyLiver weight.. (e_)
weight 1 Total Sample 2
(kg?
1.90
2.43
2.72
2.55
2.57
2.65
2.57
2.57
2.73
2.70
2.70
2.32
73.9
70.0
107
84.1
73.0
90.1
98.0
86.9
98.0
100
118
80.0
22.0
21.0
22.0
22.0
20.0
19.0
20.0
18.0
18.0
20.0
21.0
20.0
1.85
2.20
Oil-acetone
2.35
2.33
2.47
2.35
2.25
40 ngj/die
ALcoholj-watei 2.35
2.31
2.30
2.57
2.05
2.67
40 ng/die
2.80
Acohol-watei 2.75
2.95
72.8
82.9
79.2
61.5
66.9
96.5
92.4
96.9
81.4
77.7
100
62.9
72.0
88.0
86.5
108
18.9
22.0
23.3
23.6
24.8
23.5
22.5
23.6
23.3
22.8
25.7
21.0
18.1
17.0
21.7
20.2
20 ng/die
Oil-acetone
20 ng/die
Soil 2*
40 ng/die
40 ng/die
Soil 1
s
40 ng/die
Soil 2
2.57
2.40
2.30
2.30
2.51
2.99
2.69
2.35
2.65
2,71
2.20
2.70
2.35
TCDD Found 3 TCDD found
( n g ) ( p p b ) mean value
Cppb )
7.78
6.04
5.83
5.19
3.26
6.58
6.46
5.14
5.13
4.93
4.24
2.52
29.8
29.2
27.7
27.6
26.7
•18.0
29.3
25.6
.23.7
22.2
22.4
13.1
29.3
23.9
27.1
19.9
0.353
0.288
0.265
0.236
0.163
0.347
0.323
0.286
0.285
0.246
0.202
0.126
1.57
1.32
1.19
1.17
1.08
0.766
1.30
1.08
1.02
0.972
0.870
0.626
1.62
1.41
1.25
0.984
0.26 ± 0.07
0.26 ± 0.08
i lfl
igi8
4>
n Qft
u ys
•f 0 77
'
.
/-\
-^ w
2. ( ] • £ //
W /
i
\J » M M
, „, ± 0.27
1<J1
Mean value for the three groups
1.1 ± 0.5
45.6
68.5
1.60
28.5
31.7
41.9
1.32
79.2
16.7
71.4
21.2
0.786 n QQ j_ Pi . *J b
O
T f.
20.4
91.2
.762~\ w « yo ± U J
26.8
O
28.2
19.8
0.701
93.0
113
56.4
25.6
0.701
27.6
24.3
80.0
0.880
0.807
19.3
81.4
23.9
0.652
19.4
12.6
73.6
16.1
0.586 0.66 ± 0.15
110
27.5
87.7
12.6
0.572
22.0
74.4
15.4
0.560
27.6
0.559
23.6
13.2
63.3
�Continuation of Table: "Levels of TCDD in the liver")
Mean value for the two .groups
0.81 ± 0.51
80 ng/die
2.80
Alcohol-water 2.40
2.85
2.90
2.80
80 ng/die
2.80
2.93
Soil 1
2.53
2.85
3.15
80 ng/die
2.60
2.85
Soil 2
14.4
17.2
12.6
16,2
20.1
16.0
18.0
18.5
24.0
21.8
18.7
21.0
21.0
17.0
19,0 •
2.72
2.55
^80 ng/die
s.oj.1 3s
2.65
2.90
2.84
2.75
3.18
2.90
2.70
Mean value for the last two groups
1.5 ± 0.2
21.6
27.9
85.0
1.29
21.8
103
26.6
1.22
19.3
111
18.2
0.942
0.814 0.88 ± 0.28
115
20.5
16.7
19.7
128
13.6
0.692
105
17.8
0.6"3
12.0
21.7
11.7
116
0.540
M.60 ng/die
1.92
2.50
T f O
2.58
2.22
2.43
2.75
2.27
75.0
80.1
A A r\
94.9
88.0
90.2
100
89. S
2.65
Soil S
20.0
20.0
20.0
22.0
21.0
21.0
25.0
1/"S
/™S
49.1
35.4
32.6
38.8
45.2
28.8
32.2
30.7
28.2
25.4
33.9
33.4
32.3
26.1
25.7
3.41
5.11
2.59
2.40
2.15
1.80
1.79
1.66
1.17
1.16
1.81
1.59
1.54
1.53
1.35
85.0
83.1
94.9
119
114
93.0
113
109
111
125
97.2
80.0
100
103
98.0
77.2
58.0
41.6
45.5
40.5
31.2
23.0
i i
r
5.86
2.90
2.08
2.07
1.93
1.48
0.922
1
2.73 ± 0.52
1.S2 ± 0.52
1.56 ± 0.16
*
/"\O
2.18 ± 0.96
1 At the end of treatment
2 Portion of fresh liver used for the determination
3 Values corrected for losses inherent in the method of
analysis used (last column of Table on "Recoveries")
^Laboratory-contaminated soil left to "age" ( 1 month)
before administration
5
Laboratory-contaminated soil administered with 48 h of
spiking with TCDD
5
Powdered soil from Seveso (mean contamination: 81 ppb)
�TA3LS IV
Mean TCDD levels in the liver of rabbits treated for seven- consecutive days Tith various formulations containing the poison.
Vehicle
TCDD
ng/day
4
Solvent
Soil 2
Solvent
Soil 1-27
o
Solvent
Soil 1-2
Soil S'2
Soil S
20
20
40
40
80
80
• 80
'I ww
Number of
specimens
TCDD 'opb) in the liver
x - 6?
T t QC ^
0/
^ - >, P y /«* /
5
7
16
13
0.26-0.07
0.26-0.08
0.12-0.40
0.15-0.37
1 .1 -0.3
0.81^0.31
0.94-1 .3
0.54-1 .1
5
10
7
2.7 ±0.5
1 .5 -0.2
0.33^0.28
1 .7 -3.8
1 .3 -1 .8
0.43-1 .3
7
2.2 -1 .0
0.34-3.5
*
1
For further details and for data on the individual experiments
see Table "Levels of TCDD found in hepatic tissue".
2 Mean values and standard deviations.
3 Confidence interval
4
Oil-acetone
5
Laboratory-contaminated soil left to "age" (> 1 month) before
administration.
6
Combination of the results for treatments -"ith alcohol-v/ater
solution and those of oil-acetone solution.
7
Combination of the results for treatments \7ith the tv/o formulations of soil prepared: Soil 1 and Soil 2, the first of vliici
'•/as dosed v/ithin 48 h of spilcing '"ith TCDD.
8
Alcohol-v/ater.
S'
?ov/dered soil from Seveso (mean contamination: 31 o^b) .
�CONCLUSIONS
Table V summarises the results of the statistical analysis
(ANOVA and Duncan's test) conducted on the data given in Tables
III and IV. Regarding the bioavailability of TCDD, our conclusions are as follows:
(a) at the lowest dose tested (20 ng daily) no statistically
significant difference in bioavailability emerged between
TCDD in solution and TCDD on laboratory-contaminated powder;
(b) at the doses of 40 and 80 ng daily the bioavailability of
TCDD distributed over powdered soil appears on average to be
29-and 44% respectively, lower than that of TCDD in solution;
the lower limits of the confidence intervals (99/0 give a
decrease in bioavailability of 5% and 19% respectively;
(c) The bioavailability of•TCDD present on the powder of soil
taken from Seveso, contaminated after the 1CMESA accident,
.is on average 68% less than that of TCDD administered in solution; here again, however, the lower limit of the corresponding confidence interval (99/0 supplies a much smaller
variation in bioavailability (-40%);
(d) statistical analysis of the results obtained with treatments
at 40 ng daily shows that there are no significant difference:
in TCDD absorption between recently contaminated oowdered sci:
(Soil 1) and TCDD in solution, in two cases out of three (see
Table III);
(e) there is a significant difference (-45% on average) between
the bioavailability of TCDD present in the soil from Sevesc
and that of TCDD on laboratcry-contami-^sd soil; ir. this case
the lower limit of the confidence interval '9S?0 supplies 2.;-.
estimated decrease in bioavailability of only 5%;
�10
TABLE V
Statistical analysis of tlie results obtained (previous table)
for tlie evaluation of variations in bioavailability.
TCDD
Formulations
(ng:day) compared ^
20
40
80
80
80
1
2
3
4
5
5
7
3
Soil
Soil
Soil
Soil
Soil
Significance
level of the
variation
Mean reduction (%)
of bioavailability
x
CI (S9%)3
2/Solvent nonsignificant
1-2/Solvent5
p <.01
6
1-2/Solvent
p < .01
S/Solvent7
p < .01
29
44
68
S/Soil 1-28
43
p < .01
5.0 - 53
19 - 68
40
95
5
- 81
•
AN OVA and Duncan test
In each pair the TCDD of the second formulation (Solvent) was
assigned maximum bioavailability (100%). This was arbitrarily
attributed also to the TCDD of the second formulation of the
Pair Soil S/Soil 1-2.
Confidence interval
Laboratory-contaminated soil after "aging" versus solution in
.oil-acetone.
Laboratory-contaminated soil ("aged" and "not aged") versus
solution (in alcohol-v/ater and oil-acetone).
Laborator2/>-contamined soil ("aged" and not "aged") versus
solution in alcohol-water.
Povdered soil from Seveso versus solution in aicohol-v/ater.
Powdered soil from Seveso versus laboratory-contaminated soil
("aged" and not "aged").
�11
(f) two highly significant linear regressions may be derived,
from the data obtained, vis. from the treatments with TCDD
in solution and with TCDD venicled by the laboratory-contaminated powder (Fig. 4); the two regression lines permit
an estimate of the mean levels that might have been present
in the liver following treatment with the above formulations
at the. dose of 160 ng daily: in contrast to what happens in
the case of the laboratory-contaminated soil, the value extrapolated for treatments with TCDD in solution is significantly higher than the value determined experimentally for
the group of animals treated with powdered soil from Seveso,
Soil S, at the: same daily dose (V160 ng daily) .
In sum, the data obtained indicate that in the rabbit the absorption of TCDD from orally dosed preparations is clearly less when
the poison is in the soil than in. solution. This phenomenon be/
come-s apparent at the highest doses.
Acknowledcmen t
/'
The authors thank the Lombardy Region, Special Department of Seveso, for financing this survey.
References
(1) Pocchiari, F., di Domenico, A., Silano, V . , e Zapponi, G. (1981)
Environmental Impact of the Accidental Release of Tetrachlorodibenzo-p-dioxin (TCDD) at Seveso (Italy). Presentato al Workshop on Human Health Aspects to Accidental Chemical Exposure of
Dioxin - Strategy for Environmental Reclamation and Community
Protection, 4-7 Ottobre 1981, Bethesda (Maryland, USA).
i'2; Poiger, H., e Schlatter, Ch. (1980): Influence of Solvents and
Adsorbents on Dermal and Intestinal Absorption of TCDD. Food and
Cosmetic Toxicology 18, 477-481.
�(3) Panelli, R., Bertoni, M.P., Castelli, M.G., Chiabrando, C.,
telli, G.P., Noseda, A., Garattini, S., Binaghi, C., Marazza, V.,
e Pezza, F. (1980): 2,3,~,8-Tetrachlorodibenzo-p-dioxin Toxic
Effects and Tissue Levels in Animals from the Contaminated Area
of Seveso, Italy. Archives of Environmental Contamination and
Toxicology £, 569-7/T:
(4) di Domenico, A., Silano, V., Viviano, G., e Zapponi, G. (1980):
Accidental Release of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
at Seveso, Italy: I. Sensitivity and Specificity of .Analytical
Procedures Adopted for TCDD Assay. Ecotoxico1ogy and Environmental Safety 4, 283-297.
(5) Panelli, R., Bertoni, M.P., Bonfanti, M., Castelli, M.G., Chiabrando, C., Martelli, G.P., Nod, M.A., Noseda, A., e Sbarra, C.
(1980): Routine Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin
in Biological Samples from the Contaminated Area of Seveso, Italy. Bulletin of Environmental Contamination and Toxicology 24,
818-SUI
(6) Baser, H.R., e Rappe, C. (1978): Identification of Substitution
Patterns in Polychlorinated Dibenzo-p-dioxins (PCDDs) by Mass
Spectrometry. Chemosphere 7, 199-211.
�13
APPENDIX
Methods of analysis
(a) For the oowdered soils:
The procedure of analysis for the samples of powdered soil
was one that has already been v/idely used for TCDD determinations in various environmental substrates at Seveso (4), adapted for the type of samples under study ar reported later on.
For a detailed discussion of the procedure see References.
.T
The three steps used (extraction, purification on a multilayer
column and purification on an alumina column) were subjected
to numerous preliminary tests in order to ensure practically
quantitative recoveries and a high degree of reproducibility.
riT,
Extraction, All the samples of powdered soil were extracted
in a Soxhlet apparatus for ~24 h (refluxing 4-6 times/ft.) with
dichloromethane (--75 rail, HPLC grade) as solvent. The'sample
was prepared for extraction by mixing intimately ^2 g of dry
powder with 8 g of anhydrous sodium sulfate. The extract "-as
left to evaporate slowly under a stream of nitrogen in order
to obtain a moist residue.
Par if i cat ion on multilayer column. This column (l.D. 1C mr.:
total length 250 mm) consisted (from bottom to 'top): glass
wool; anhydrous sodium sulfate 0.5 cm; activated silica gel
1.5 cm; mixture of anhydrous sodium sulfate and sodium bicarbonate (4:1 w/w) 2 cm; celite 545 1 cm; mixture of concentrated sulfuric acid and celite 5^-5 (1:1 w/w) 5 cm; anhydrous
sodium sulfate 1 cm. The secuence of the lasers is shown in
Fig. 1 . Before use, the multilayer column (one for each attraction) was washed -with three portions (i c ral each) of dichloromethane, followed by ~liree portions (10 ml) of :i-sen-cans
The residue of extraction was rale en \i^ with ± rn' o- ---^c.--^-- =
�14
(HPLC grade) and transferred to tlie top of the column before
the oentane washings had eluted cornsletelv; the operation -;as
J*
*•»
J.
-•
*
repeated another seven tines in the same "/ay to ensure quantitative transfer. Percolation 'vas by gravity. The eluate collected (^32 ml) \vas allowed to evaporate to -dryr.ess spontaneous ly.
Purification on column or ac'civatgdalumina. Alumina March
(neutral aluminium oxide 90, 170-230 mesh) vas Icept in an over,
at l30°ci5°C for 14 h and then stored in a desiccator on silica
gel ready for subsequent use. The columns (l.D. 10 mm, total
length/-*250 mm) -;;ere prepared by inserting first a piece of
glass ";ool, fcllov/ed by 5 cm of alumina (r-4 g) and 0.5 cm of anhydrous sodium sulfate. The residue from the multilayer column
'^as taken up quantitatively \vith five successive portions (2 ml
each) of n-hexane and transferred in succession on to the top of
the column.
There followed three washings (10 ml each) "/ith a
mixture of n-hexane and dichloronvethane (1/0 and four elutions
~'ith a total of 20 ml (5 ml per elution) of a mixture of r.-hexar.
and dichloromethane 20%. This eluate (-20 ml) ~;as carefully
collected in a bealcer and left to evaporate to a moist residua;
this was then transferred quantitatively tn a suitable container
evaporated to dryr.ess and diluted to a kncr/n volume for t2:e assa
by means of irGC/irMS according to the procedure describee in tl:
reference cited.
(b) For the samples of hepatic tissue:
The determinations on the liver samples ••/•ere cone by means of" a
procedure already used for the determination of TCDD in rabbit
• liver (5), as reported later. For a ds-ailec discussion of rlie
procedure see references.
�15
Alkaline digestion. About 20 g of liver <vas hydrolysed "/itl:
10 ml NaOK 10 N plus 20 ml ethanol at 90°C and refluxed- for
1 h.
Extraction. After cooling, the sample in the alkaline medium
was extracted twice with n-hexane (20 ml each time) .
Purification on column of Sxtrelut. A column of Sxtrelut was
pretreated with 20 ml concentrated sulfuric acid and left to
equilibrate for 4-14 h. The total hexane solution ( ~ 0 ml)
.4
'•was left to percolate through the column, which was subs ecu sr.tl;
washed with 20 ml n-hexane. The eluate was evaporated to dryness in a stream of nitrogen at room temperature.
Purification on column of activated alumina.
The columns of
neutral alumina (45 x 5 mm), with a layer of anhydrous sodium
sulfate at the top (5 mm), were washed with 3 ml dichloromethar.;
and activated at 270°C for 12 h or at 4GQ°C 'for 4 h. The residue
from the -column of Sxtrelut was taken up with n-hsxane (total
volume-*3 nil) and transferred to the top of the column. This
was followed by an elution with 5 ml carbon tetrachloride followed by 4 ml dichloromethane. The latter fraction, containing
TCDD, was evaporated to dryness. The residue was taken up -*itl
dioxane (0.100 ml) and analysed by GC and mass fracmentograp'.v-.
*
N
In some cases samples from croups on like treatment "/ere pooled
and analysed by high resolution GC (25000 theoretical plates) aiid
MS•according to a method reported in the literature (6).
All the reacents and standards used in this study were of a hic'.i
degree of purity and were grade H?LC or R3, where possible. Tl'.e
TCDD used vas of the same purity as the environmental standards .
Table VI and Figs. 5-11 summarise all the results and informaticappropriate for defining the quality of the analysis methods used.
�18
Table VI
Rcoveries ("£) of TCDD for the methods of analysis used
and for two specific purification operations.
Multilayer
column
141. 0"
123. 5
120. 0
115. 5
114. 6
114. 5
114. 3
110. 5
110. 5
109. 0
108. 5
108.0
107. 5
105. 0
104, 5
104.0
104. 0
103. 8
103. 5
103. 1
103. 0
102. 6
102. 0
101. 5
101. 5
100. 9
100. 8
99. 9
99. S
99. 0
98. 1
98. 0
98. 0
97. 5
96. 0
96. 0
95. 0
95.0
95.0
94.5
94.0
94.0
93.5
91.5
91.0
91.0
90.0
90.0
89.0
89.0
88.9
88.0
88.0
88.0
87.0
86.5
85.5
84.0
84.0
84.0
84.0
84.0
83.5
83.5
82.4
82.0
81.5
81.0
78.0
76.0
73.8
65.1 +
57. 0'
Alumina
column
Soil 1
Soil 2
102.0
101.5
101.0
100.0
99.4
135. 8"
116. 7
109. 4
104. 6
102. 0
102. 0
99. 1
•99. 0
98. 0
97. 6
97. 6
95. 5
95. 0
94. 0
92. 0
87. 5
85. 5
69. 4
66. 6
66. 5
60. 3
106. 0
102. 6
98. 0
97. 5
97. 0
95. 5
94. 0
93. 5
93. 0
92. 5
92. 0
91.0
86. 0
81, 9
81. 4
77. 9
98.0
96.5
95.6
95.0
94.0
93.5
93.5
92.5
92.0
91.8
91.5
91.0
90.0
89.0
87.5
82.7
82.7
74.0"
47.0"
2
1
':
Soil 3'
110.0
107.5
105.5
103.5
101.5
99.5
99.5
99.5
99.0
98.0
97.5
97.5
97.0
95.7
94.5
94.5
• 93.0
93.0
92.5
90.0
90.0
88.5
88.5
88.0
88.0
87.5
86.5
86.0
86.0
85.0
80.0
Hepatic
tissue
108. 5"
99. 0
98. 2
96. 6
95. 2
94. 8
91. 0
89. 3
89. 0
88. 6
88. 3
88.0
88. 0
88. 0
88. 0
87. 0
86. 2
86. 0
86. 0
85. 0
85. 0
85. 0
84. 0
84. 0
84. 0
83. 3
83. 3
82. 4
82. 0
80.0
80.0
78.5
78.2
77.5
77.3
77.0
77.0
77.0
76.0
76.0
75.5
75.2
75.0
73.0
72.8
72.2
71.4
71.0
70.2
67.0
65.5
64.3
63.0
63.0
62.0
62.0
61.0
60.4
Means and standard deviations and number of data used in
the calculation:
-.
94 ± 5
80 ± 10
92 ± 15 92 ± 8
96 ± 12
94 ± 7
{20}
{16}
{31}
{71}
{5 7}
{22}
1
Laboratory-contaminated soil
Laboratory-contaminated soil left to "age"
3
Recoveries of TCDD (Cl ") added to samples of powdered
soil and of hepatic tissue before extraction
"Aberrant (according to Chauvenet)
2
3
�19
14
12
N
Media
Dev. St
Min
Max
3
4 .
1.80
2.22
2.64
3.06
Fig. 1. Distribution by weight group of 44 male white
rabbits before treatment
�20
28
N
24
Medi a
Dev. St.
Win
Max
7Q
2.6
0.3
I .85
3.18
20
12
8
1 .75
2.25
2.75
3.25
Fig. 2. Distribution by weight group of 70 male white
rabbits on day 8 (sacrifice) of treatment.
Note (see Fig.l) that the animals presented an
average increase in weight in the week of
treatment; in some cases there was a slight
weight loss unrelated to any specific factor.
�20
N
Media
Dev. St.
Min
Max
=
=
=
=
=
70
92
16
61.5
128.0
12
8
56
80
104
128
grammi
Fig. 5. Distribution by weight group of the livers removed.
from the rabbits after treatment with various
formulations containing TCDD.
�3.5 .
3.0
y = 0.041x - 0.51
y = O.OZlx - 0.076
2.5
Q.
Q.
O
2.0
4-)
cn
0)
4-
1.5
O
O
O
1—
1.0
n s
80
120
160
ng/die
Fig. 4. Regression lines obtained by using the set of data
for treatments with TCDD in solution (Q) and the
set of data for treatments with laboratory-contaminated powdered soil ( 0 ) « The significance level
of the regressions is F ^ „ = 172.35, P< .001;
F \ 8 = 106.67, P< .001 respectively. To highlight
the results of treatments with Seveso soil, two
points have been plotted ( u) corresponding to
the mean values of the TCDD levels determined analytically in the two groups treated. The standard
deviation is shown above and below the value of
each mean.
�23
anidro
1 em
5 em
Celite -t- H2S04 (1:1 p/p)
t em
Calita
2 cm
NaHC03 +• Na2SC
1 . 5 em
Gel di Silice
1 cm
Ma.SO, anidro
(1:4)
Cotone
\
Fig. 5.
Multilayer column for the first stage of
purification of the extracts. Description
in the text.
�Z4
28 r
24 L
Media
Dev. St.
Min
Max
20 I
16 U
12 h
8 L
4 L
60
Fig. 6.
84
108
recovery
132
Block distribution of percentage recoveries
obtained from the multilayer columns (see
Table VI first column)
�25
12
N
Me<i1a
10
=
22
= 94
Dev. St. = 5
M1n
* 82.7
Max
« 102.0
o
76
Fig. 7. Block distribution of percentage recoveries
obtained from the alumina columns (see Table
VI, second column)
�2b
8
-
6
-
4
N
-
Media
Dev. St.
Min
Max
2 .
30
64
98
132
recovery
.8* Block distribution of percentage recoveries
obtained on applying the method of analysis to
the determination of TCDD added to uncontaminated powdered soil, within 48 h of addition
(Soil 1: see Table VI, third column).
�6
.
4 .
2 .
70
Fig. 9.
80
90
100
recovery
no
Block distribution of percentage recoveries
obtained on applying the method of analysis
to the determination of TCDD added to uncontaminated powdered soil left to "age"
(Soil 2: see Table VI, four-th column)
�N
8 -
Media
Dev. St.
Min
Max
6 -
4 -
2 .
70
114.8
Fig. 10. Block distribution of percentage recoveries
obtained on applying the method of analysis
to the determination of labeled TCDD added
to powdered soil of different types (laboratory-contaminated or contaminated following
the ICMESA accident) before extraction (see
Table VI, column five)
�29
14
N
12
Media
Dev. St.
Min
Max
10
8
45
57
69
81
93
105
% recovery
Fig. 11.
Block distribution of percentage recoveries
obtained on applying the method of analysis
to the determination of labeled TCDD added to
hepatic tissue before alkaline digestion
(see Table VI, sixth column)
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
094
Folder
The folder containing the original item.
2342
Series
The series number of the original item.
Series IV Subseries IV
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
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Bonaccorsi, A.
A. di Domenico
R. Fanelli
F. Merli
R. Motta
R. Vanzati
G. Zapponi
Date
A point or period of time associated with an event in the lifecycle of the resource
1983-06-01
Title
A name given to the resource
Typescript: Study of the Bioavailability in the Rabbit of the TCDD Present in Powdered Soil from Seveso Zone A (Milan)
Subject
The topic of the resource
dioxin
animal testing
soil testing