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© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3056
DETERMINATION OF DIOXINS/FURANS IN HUMAN
GALLBLADDER STONES AND GALLBLADDER TISSUES
AS INDFICATOR FOR POLLUTION
Sharif Arar
1
, Samer Awaideh
2
, Mohammed H Kailani
1
, Mahmoud Alawi
1,*
1
School of Science, Department of Chemistry, University of Jordan, Amman, 11942, Jordan
2
Faculty of Science, Department of Chemistry, Al Isra University, Amman-11622, Jordan
ABSTRACT
The levels of 17 congeners of Polychlorinated
dibenzo-p-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) in human gallbladder tissue
and gallbladder stone samples gathered from pa-
tients of the “Jordan university hospital” in the pe-
riod between February 2016 and April 2016, were
analyzed. All samples were extracted, cleaned-up,
and analyzed using GC-EI/MS. The total mean
concentration of dioxins/furans in gallbladder tis-
sues and stone samples were 1.62 µg/kg and 2.30
µg/kg, respectively. In all samples most of diox-
in/furans congeners were not detected. Octachlo-
rodibenzo-p-dioxin was the most abundant conge-
ner with average concentrations of 1.19 µg/kg in
gallbladder tissue samples and of 1.64 µg/kg in
gallbladder stone samples. The TEQ values for
PCDDs/Fs in all samples were found in the range of
0.40 to 124.9 ng TEQ/kg for gallbladder tissue
samples and 15.7 - 135.9 ng TEQ/kg for gallbladder
stone samples. The distribution coefficient for diox-
ins/furans were calculated between gallbladder
stones and gallbladder tissue in each sample. The
distribution coefficients, (Kd = C
stone
/C
tissue
) for
dioxins/ furans were found in the range 0.88 to
2.06. The variations in Kd depend mainly on the
type of gallbladder stone and on the fat content of
the gallbladder tissue (obesity) of the patient. The
results of this study show that both gallbladder
stones and gallbladder tissues can be used as a good
indicator for body pollution especially if the stone
was of pure cholesterol type. The concentrations
we found in this study are much higher than what
Muto [29] found, nevertheless there was no deaths
among the donors of the 39 samples in our study. It
is also attention drawing that the congeners
1,2,3,6,7,8-hexachlorodibenzino-p-dioxin and
1,2,3,4,6,7,8-heptaclorodibenzo-p-dioxin are pre-
sent at high frequency, numerically 97.4% and
100% respectively.
KEYWORDS:
Dioxins/Furans, Gallbladder tissues, Gallbladder stones,
Pollution, Toxicity equivalency (TEQ), Jordan
INTRODUCTION
Dioxins and Furans. Polychlorinated diben-
zo-p-dioxins (PCDDs) and polychlorinated diben-
zofurans (PCDFs) are planar tricyclic compounds,
that exhibit comparable toxicities and properties [1-
4]. PCDDs and PCDFs are semi-volatile and toxi-
cologically significant trace organic contaminants
[5]. They are introduced into the environment
through various sources and accumulate in human
tissues [6]. The importance of studying the bioac-
cumulation of these compounds come from their
high toxicity, their transport pathway between air,
water and food [1, 5].
According to Fiedler [7], the number of
PCDDs congeners is 75 and those of PCDFs is 135
depending on the positions of chlorine atoms on the
aromatic rings, with wide variations in the physio-
chemical properties between the congeners. Gener-
ally, these compounds are insoluble in water and
lipophilic. Dioxins and furans are unintentionally
produced through high temperature processes. They
have no commercial applications and easily intro-
duced to the environment due to their lipophilic
behavior and its transportation ability.
The sources of dioxins and furans are chemi-
cal processes which associated with the production
of trace amounts of dioxins/furans as byproducts
including the manufacturing of PCBs and OCPs.
The other sources are uncompleted combustion
processes such as chemical waste, hospital waste,
coal and peat; landfill gas combustion, crematoria,
and cement manufacture [7].
From all dioxin and furan congeners, only 17
of them have shown significant potential toxicity,
and bio-accumulation ability [2, 8]. 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) has been re-
ported to be the most toxic dioxin congener [3, 8],
and the strongest carcinogenic agent [3, 9], where
2,3,7,8-TCDF is about 10% as toxic as TCDD.
These compounds have high ability to
transport for long distance, are highly lipophilic,
have high penetration ability, high resistance
against metabolism and easily deposited onto sur-
faces of water, soil and plants. So, they exist in all
environmental compartments.
Gallbladder and Gallbladder Stones. Ac-
cording to the Florida Medical Center “Gallbladder
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3057
is a small pear-shaped sac that sits in the upper ab-
domen on the right side, just under the ribcage [10].
It is about 7 cm in length and its main function is to
store and concentrate the bile, a yellow -brown di-
gestive enzyme produced by the liver [11].
Bile contains cholesterol and bile salts. After a
meal the gallbladder contracts and bile passes down
the bile duct to enter the duodenum, the beginning
of the small bowel. If too much cholesterol accu-
mulates in the bile it crystallizes out as stones, ei-
ther by itself, or mixed with bile pigments. This can
form a sludgy gravel or stones as big as chestnuts.
The only way out for the stones is to travel down
the bile duct into the bowel. If they are too big they
will get stuck and cause severe pain and a number
of other problems [11].
There are several factors that predispose to
gallstones. They tend to run in families and are
twice as common in women. Being overweight can
also contribute. As quoted from Gastroenterological
Society of Australia “Gallstones are a common
problem as we age, with an estimated prevalence of
25–30% in Australians over the age of 50 years”
[12]. Gallstones are a common clinical finding in
the Western populations. The prevalence rates in
European adults is 10–15%, while in African and
Asian populations prevalence is 3–5 % [13-14]. In
the United States, the prevalence rates range from
5% to 27%. In American Indians, gallstone disease
is found in 30% of male and 64% of female [14-
15]. The prevalence of gallstones is rising day by
day in Pakistani population [16-17]. Gallstones
types are classified according to the cholesterol
content to pure cholesterol stones which are consti-
tutes around 90% cholesterol, pigment stones con-
stitutes around 90% bilirubin and mixed composi-
tion stones, which are constitute of varying percent-
ages of cholesterol, bilirubin and other substances
such as calcium carbonate, calcium phosphate and
calcium palmitate [14, 18, 19].
Literature Review. Saqib, et al. [14], studied
the recognition of various gallstones in the patients
attending surgical OPD (Outpatient Department) by
biochemical analysis of gallstones. The study was
done in ISRA’ hospital in Hyderabad /Pakistan dur-
ing 18 months on sixty-nine patients. They found
that the frequencies in different age groups were as
follows: 11.60% for the age group 15-24 years,
24.64% for 25-34 years, 53.63% for 35-44 years,
8.69% for 45-54 years and 1.45% for 55-64 years.
The frequencies ratio between males to females was
1: 2.45 and the frequencies of stone types were
55.07% for cholesterol stones, 28.99% for pigment
stones and 15.94% for mixed stones.
Kitamura, et al. [20], studied the concentration
of 20 dioxin/furans congeners in 27 autopsy cases.
They found a total TEQ values in the bile of 43.2 ±
30.9 pg TEQ/g lipid, where the concentration of
1,2,3,4,6,7,8-heptachloro-p-dibenzodioxin was the
highest.
Up till now there is no research work dealing
with dioxins/furans in gallbladder tissues and
gallbladder stones. This will be the first study on
this field.
Aims of the Present Work. This study aims
to determine the concentrations of Polychlorinated
dibenzo-p-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) in gallbladder tissues and
gallbladder stones in samples gathered from the
Jordan hospital in Amman-Jordan as indicator for
human body pollution and to calculate the toxicity
risk in (ng TEQ/ g sample) concentration for
PCCD/Fs in both gallbladder tissues and stones.
Then to make a comparison between the
PCDDs/Fs concentrations in gallbladder tissues and
their concentrations in the gallbladder stones to
determine which of them is the most favorable site
of bioaccumulation and to calculate the distribution
coefficient of PCDDsd/Fs congeners between
gallbladder tissues and gallbladder stones. Study if
these POPs can be one of the agents that contribute
to the formation of dysplasia in the human tissues
by studying its concentrations in the available sam-
ples that have dysplasia which are tow samples
only.
CHEMICALS AND APPARATUS
Chemicals and Solvents. Solvents used were
as follows: n-hexane of 97% purity, and toluene of
98% purity were purchased from Tedia (USA);
ethanol absolute of 99% purity, n-nonane of 98.7%
purity, and isooctane of 98.7% purity were pur-
chased from Riedel deHaën (Hannover/ Germany);
dichloromethane of 97% purity and petroleum ether
(40-60 ºC) of 95% purity was purchased from Car-
lo-Erba (Italy); acetone of 99% purity was purchase
from GCC (UK). Chemicals used for the extraction
and clean-up were purchased as follows: Silica gel
60, 0.063–0.200 mm (70–230 mesh) from Merck
(Germany), anhydrous sodium sulfate extra pure
was purchased from Fluka (Switzerland) and Flo-
risil® 0.15–0.25 mm (60– 100 mesh) from Merck
(Germany) [6].
Standards and Internal Standards. EPA
method 1613 stock solution of PCDDs/PCDFs and
internal standard for PCDDs/PCDFs (1,2,3,4-
tetrachloro [
13
C
12
] dibenzo-p-dioxin), 200 ng/mL,
were purchased from Willington Laboratory (Cana-
da) [6].
Apparatus. The instrument used for the de-
termination of PCDDs/PCDFs was as mentioned in
Alawi, et al. [6].
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3058
TABLE 1
Participants information
Participants
No.
Cancer patient
(Yes / No)
Sex Age
(year)
BMI
*
(kg m
-2
)
Address Smoking
S
-
01
No
F
46
18.40
Hai Nazzal
No
S
-
02
No
F
36
34.50
Irbid
No
S
-
03
No
F
31
30.62
Sweileh
No
S
-
04
No
F
24
34.50
Um Alheeran
No
S
-
05
No
M
55
19.95
Wadi Alsair
Yes
S
-
06
No
F
52
17.90
Hai Nazzal
No
S
-
07
No
F
33
17.87
Tabarbore
No
S
-
08
No
F
45
17.72
Sweileh
Yes
S
-
09
No
M
52
33.16
Ain Albasha
Yes
S
-
10
No
M
74
23.50
Tla’a Ali
Yes
S
-
11
No
F
54
23.00
Alunzhah
No
S
-
12
No
F
44
35.02
Aljwaidah
No
S
-
13
No
F
28
17.41
Albaqa’a
No
S
-
14
No
F
24
39.52
Alsalt
No
S
-
15
N
o
M
70
33.27
Hainazzal
Yes
S
-
16
No
F
47
17.39
Jarash
No
S
-
17
No
F
57
17.00
South Shounah
No
S
-
18
No
F
28
19.70
Marj AlHamam
No
S
-
19
Dysplasia
F
40
36.01
Jabal AlHusein
No
S
-
20
No
F
75
17.00
Marka
No
S
-
21
No
M
76
18.61
Alge
ezah
No
S
-
22
No
F
59
24.05
North Marka
No
S
-
23
No
F
39
23.50
Abu Alanda
Yes
S
-
24
No
F
60
22.90
Aljbaihah
No
S
-
25
No
F
49
21.00
Dahiat Alrasheed
No
S
-
26
No
F
40
21.72
Aqaba
Yes
S
-
27
No
F
52
18.66
Ain Albasha
No
S
-
28
No
F
29
35.00
Alzarqa
No
S
-
29
No
M
43
16.91
Ajloun
Yes
S
-
30
Dysplasia
M
61
37.62
Albaqa’a
No
S
-
31
No
F
44
16.97
Amman
-
Tariq
No
S
-
32
No
M
21
18.83
Madaba
No
S
-
33
No
M
21
16.50
Dabooq
No
S
-
34
No
F
24
19.50
Shafabadran
No
S
-
35
No
F
56
39.20
Ala’shrafiah
No
S
-
36
No
F
52
16.32
Marka
No
S
-
37
No
F
28
21.67
Al
-
abdal
i
No
S
-
38
No
F
31
15.88
Ajloun
Yes
S
-
39
No
F
77
16.50
Al Bnaiat
No
*Body Mass Index (BMI) = Body weight / (Body height)
2
SAMPLING AND SAMPLE PREPARATION
Sample Collection. A total of thirty nine
samples of each fresh gallbladder tissues and
gallbladder stones were collected with collaboration
with the Institutional Review Board (IRB) in the
Jordan University Hospital (JUH) - surgery depart-
ment and histology laboratory in the period from
February 2016 to April 2016.
All samples were stored at – 20
ᵒ
C until analy-
sis. Sample data sheets with questionnaire were
filled for each patient; the summary of information
for all samples is listed in Table 1.
Sample Extraction and Clean-up. One gram
of each homogenized gallbladder tissue sample or
gallbladder stone sample was weighed into a
Soxhlet thimble, spiked with 200 μL IS (100 ppb)
and then extracted in a Soxhlet apparatus with 200
mL toluene for 16 h. The extracts were reduced to
ca. 2 mL using the rotary evaporator at 30 °C and
140 mbar [6].
The cleanup of the extracts was con-
ducted according to Hagenmaier [21] method, on
three columns as follows:
Macro Alumina Column. A glass column
(25x1.6 cm) was filled with 20 g dry (500 °C, 6h)
alumina B (Super I) and 10 g Na
2
SO
4
then washed
with 100 ml of n-hexane. Sample residues from the
extraction step were dissolved in 10 ml n-hexane
and added to the column, using Pasteur pipette. The
column was then eluted with 80 ml n-hexane, 100
ml n-hexane/ dichloromethane (98:2) and 180 ml n-
hexane/dichloromethane (1:1), successively. The
third (last) fraction which contains PCDD/Fs was
collected and evaporated to few milliliters at 40 °C
and 280 mbar (the vacuum is to be reduced step-
wise).
Mixed Silica Gel Column. A glass column
(25x1.6 cm) was filled from bottom to top with 2.0
g of dry (220 °C, 6h) silica, 5 g basic silica gel
mixed with 1 M NaOH (30% w/w), 2.0 g dry silica
gel, 10 g acidic silica gel mixed with concentrated
H
2
SO
4
(40% w/w), 2.0 g dry silica gel and finally
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3059
10 g anhydrous Na
2
SO
4
. The column was washed
with 100 ml n-hexane. Sample residues from the
previous column cleanup step were dissolved in 10-
20 ml n-hexane and transferred to the column using
Pasteur pipette. The column was then eluted with
250 ml n-hexane. The eluates were evaporated to
few milliliters at 30 °C and 240 mbar.
Mini Alumina Column. A glass column
(25x0.8 cm) was filled with 4 g of basic alumina B
(Super I) and 2.5 g of anhydrous Na
2
SO
4
. The col-
umn was washed with 10 ml n-hexane. The resi-
dues from previous column cleanup step were dis-
solved in 2-2.5 ml n-hexane and transferred to the
column using Pasteur pipette. The column was elut-
ed with 25 ml n-hexane/ dichloromethane (98:2)
and 30 ml n-hexane/ dichloromethane (1:1), succes-
sively. The second (last) fraction was collected and
evaporated to few milliliters then transferred to a
pear shaped flask using n-hexane and re-evaporated
almost to dryness at 30 °C and 240 mbar. The last
few drops of the solvent were evaporated using a
gentle nitrogen stream. 200 μL n-nonane were add-
ed to re-dissolve the residue, and transferred to mi-
cro-insert which placed in a small vial and closed
until GC analysis.
GC/MS ANALYTICAL METHOD
GC and GC Conditions. As mentioned in
Alawi, et al. [6].
* GC model: Agilent 6890 series II, with auto-
sampler injector 7683 series
* Injector: 280 °C, 1 μL/splitless
* Columns: TRB-5MS, 30m× 0.25 mm i.d. ×
0.25 μm film thickness
* Carrier gas: helium grade 5.0 (99.999%), 8 psi
* Flow rate: 1.0 mL/min
* Temperature programs: 150 °C (1
min)→200 °C, 12 °C/min→235 °C, 3 °C/min (8
min) → 310°C (8 min).
Mass Detector (MSD) Conditions. As men-
tioned in Alawi, et al. [6].
- Model: Agilent 5973 N, quadruple at 150 °C,
electron impact ionization, 70 eV at 230 °C
- 280 °C (auxiliary)
- Tuning substance: perfluorotributylamine
(PFTBA)
-Tuning masses: 69, 219, and 502 m/z
-Acquisition mode: total ion chromatogram
(TIC)
-Data analysis: MSD Chemstation software
METHOD VALIDATION
Detection limits and limits of quantitation.
The results show that for all studied PCDDs/PCDFs
the LOD were between 0.02 and 0.03 μg/L; the
LOQ were between 0.08 and 0.10 μg/L; the MDL
were between 0.02 and 0.03 μg/kg fat and the
MLOQ were between 0.08 and 0.16 μg/kg fat.
Instrument precision. The precision of the
instrument was measured through the injection of
three standard solutions of different concentrations.
High concentration: (20 ng/ml) for tet-PCDDs/Fs,
(100 ng/ml) for penta, hexa, hepta-PCDDs/Fs and
(200 ng/ml) for oct-PCDDs/Fs; then medium con-
centration: (10 ng/ml) for tetra-PCDDs/Fs, (50
ng/ml) for penta, hexa, hepta-PCDDs/Fs and (100
ng/ml) for octa-PCDDs/Fs and low concentration:
(5 ng/ml) for tetra-PCDDs/Fs, (25 ng/ml) for penta,
hexa, hepta-PCDDs/Fs and (50 ng/ml) for octa-
PCDDs/Fs. Each solution was injected three times.
Tables 2, 3, and 4 show the calculated concentra-
tions for the three injections, the average, standard
deviation (SD) and the coefficients of variation
(CV). The coefficients of variation (CV) were
TABLE 2
Instrument precision using high concentrations of standard solution mixture of PCDDs/PCDFs
No. Congener R.T
(min)
Calculated concentration (ng mL
-1
)
Trial 1
Trial 2
Trial 3
Average
SD
CV
(%)
1
2,3,7,8
-
Tetrachlorodibenzofuran
18.06
21.6
24.5
22.6
22.9
1.48
6.45
2
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
18.3
78
22.0
23.4
26.6
24.0
2.35
9.81
3
1,2,3,7,8
-
Pentachlorodibenzofuran
19.98
118
116
118
117.3
1.15
0.98
4
2,3,4,7,8
-
Pentachlorodibenzofuran
20.41
117
115
117
116.3
1.15
0.99
5
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
20.62
115
119
115
116.3
2.31
1.99
6
1,2
,3,4,7,8
-
Hexachlorodibenzofuran
22.53
93
100
95
96.0
3.61
3.76
7
1,2,3,6,7,8
-
Hexachlorodibenzofuran
22.63
90
95
90
91.7
2.89
3.15
8
2,3,4,6,7,8
-
Hexachlorodibenzofuran
23.12
105
112
105
107.3
4.04
3.77
9
1,2,3,7,8,9
-
Hexachlorodibenzofuran
23.26
103
115
1
09
109.0
6.00
5.50
10
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.35
97
115
97
103.0
10.39
10.09
11
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.67
113
100
116
109.7
8.50
7.76
12
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
23.89
107
95
107
103.0
6.93
6.73
13
1,2
,3,4,6,7,8
-
Heptachlorodibenzofuran
25.84
98
103
98
99.7
2.89
2.90
14
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
27.27
93
112
93
99.3
10.97
11.04
15
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
27.85
111
109
115
111.67
3.06
2.74
16
Octachlorodibenzo
-
p
-
dioxin
32.
86
212
241
235
229.3
15.31
6.67
17
Octachlorodibenzofuran
32.99
225
214
232
223.7
9.07
4.06
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3060
TABLE 3
Instrument precision using medium concentrations of standard solution mixture of PCDDs/Fs
No. Congener R.T
(min)
Calculated concentration (ng mL
-1
)
Tr
ial 1
Trial 2
Trial 3
Average
SD
CV
(%)
1
2,3,7,8
-
Tetrachlorodibenzofuran
18.06
9.8
10.2
10.2
10.1
0.24
2.41
2
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
18.378
11.2
11.9
10.5
11.2
0.70
6.25
3
1,2,3,7,8
-
Pentachlorodibenzofuran
19.98
48
51
49
49.3
1.53
3.10
4
2,3,4,7,8
-
Pentachlorodibenzofuran
20.41
53
49
48
50.0
2.65
5.29
5
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
20.62
52
48
47
49.0
2.65
5.40
6
1,2,3,4,7,8
-
Hexachlorodibenzofuran
22.53
43
47
48
46.0
2.65
5.75
7
1,2,3,6,7,8
-
Hexachlorodibenzofuran
22.63
42
44
44
43.3
1.15
2.66
8
2,3,4,6,7,8
-
Hexachlorodibenzofuran
23.12
47
48
42
45.7
3.21
7.04
9
1,2,3,7,8,9
-
Hexachlorodibenzofuran
23.26
44
42
46
44.0
2.00
4.55
10
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.35
54
52
51
52.3
1.53
2.92
11
1,2,3,6,7,8
-
Hexachlorodiben
zo
-
p
-
dioxin
23.67
46
45
45
45.3
0.58
1.27
12
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
23.89
45
44
48
45.7
2.08
4.56
13
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
25.84
52
53
55
53.3
1.53
2.86
14
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
27.27
51
42
44
45.7
4.7
3
10.35
15
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
27.85
48
52
51
50.3
2.08
4.14
16
Octachlorodibenzo
-
p
-
dioxin
32.86
99
97
96
97.3
1.53
1.57
17
Octachlorodibenzofuran
32.99
109
115
105
109.7
5.03
4.59
TABLE 4
Instrument precision using low concentrations of standard solution mixture of PCDDs/PCDFs
No. Congener R.T
(min)
Calculated concentration (ng mL
-1
)
Trial 1
Trial 2
Trial 3
Average
SD
CV
(%)
1
2,3,7,8
-
Tetrachlorodibenzofuran
18.06
4.4
4.2
5.3
4.6
0.59
12.65
2
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxi
n
18.378
5.2
5.6
4.9
5.2
0.35
6.71
3
1,2,3,7,8
-
Pentachlorodibenzofuran
19.98
26
27
24
25.7
1.53
5.95
4
2,3,4,7,8
-
Pentachlorodibenzofuran
20.41
22
24
22
22.7
1.15
5.09
5
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
20.62
24
21
26
23.7
2.52
10.63
6
1,2,3,4,7,8
-
Hexachlorodibenzofuran
22.53
29
28
27
28.0
1.00
3.57
7
1,2,3,6,7,8
-
Hexachlorodibenzofuran
22.63
26
23
22
23.7
2.08
8.80
8
2,3,4,6,7,8
-
Hexachlorodibenzofuran
23.12
27
25
29
27.0
2.00
7.41
9
1,2,3,7,8,9
-
Hexachlorodibenzofuran
23.26
26
24
25
25.0
1.00
4.00
10
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.35
23
21
22
22.0
1.00
4.55
11
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.67
26
26
27
26.3
0.58
2.19
12
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
23.89
24
21
23
22.7
1.53
6.74
13
1,2,3,4,6,7,8
-
Heptachlorodibenzo
furan
25.84
22
22
21
21.7
0.58
2.66
14
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
27.27
24
25
21
23.3
2.08
8.92
15
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
27.85
26
25
23
24.7
1.53
6.19
16
Octachlorodibenzo
-
p
-
dioxin
32.86
55
54
51
53.3
2.08
3.90
17
Octachl
orodibenzofuran
32.99
56
52
51
53.0
2.65
4.99
TABLE 5
PCDDs/Fs recoveries and method precision (CV %) for gallbladder tissue blanks
spiked with high concentration standard solution mixture
No. Congener
Average Concentration
Found (N=3)
( ± s )
Average recovery % CV(%)
Spiked concentration: 20 ng g
-1
1.
2,3,7,8
-
Tetrachlorodibenzofuran
21.23 ± 0.60
106
2.84
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
21.57 ± 0.25
108
1.17
Spiked concentration: 100 ng g
-1
3.
1,2,3,7,8
-
Pentachlorodibenzofur
an
102.43 ± 1.91
102
1.87
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
101.77 ± 1.37
102
1.34
5.
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
102.43 ± 1.91
102
1.87
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
99.43 ± 1.25
99
1.26
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
100.1
0 ± 1.01
100
1.01
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
95.77 ± 4.20
96
4.39
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
97.77 ± 2.66
98
2.72
10.
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
93.77 ± 5.85
94
6.24
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
96.43 ± 3
.39
96
3.51
12.
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
95.10 ± 4.53
95
4.76
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
94.43 ± 5.1
94
5.41
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
97.10 ± 2.82
97
2.90
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
102.7
7 ± 2.36
103
2.30
Spiked concentration: 200 ng g
-1
16.
Octachlorodibenzo
-
p
-
dioxin
202.67 ± 3.51
101
1.73
17.
Octachlorodibenzofuran
203.00 ± 2.65
102
1.30
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3061
found to be less than the accepted limit value for
trace analysis (CV< 15%) [22].
Recoveries. Nine portions each of one gram
sheep gallbladder tissue were used as blank samples
representing the human gallbladder tissues and nine
portions each of one gram weight of a mixture of
sand mixed with cholesterol compound (sigma,
99%) were used as blank samples representing the
human gallbladder stones.
Each of the nine blank samples was spiked
with the one of the PCDDs/Fs standard solution
mixtures to give the concentrations in three levels
high, medium and low concentration.
The high concentration level was as follows:
(20 ng/ml) for tetra-PCDDs/Fs, (100 ng/ml) for
penta, hexa, hepta-PCDDs/Fs and (200 ng/ml) for
octa-PCDDs/Fs. The medium concentration level
TABLE 6
PCDDs/Fs recoveries and method precision (CV %) for gallbladder stone blanks spiked with high
concentration standard solution mixture
No. Congener
Average Concentration
Found (N=3)
(
± s )
Average recovery % CV(%)
Spiked concentration: 20 ng g
-1
1.
2,3,7,8
-
Tetrachlorodibenzofuran
20.63 ± 0.65
103
3.15
2.
2,3,7,8
-
Tetrachlorodib
enzeno
-
p
-
dioxin
20.97 ± 0.85
105
4.83
Spiked concentration: 100 ng g
-1
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
103.00 ± 1.00
103
0.97
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
102.33 ± 0.58
102
0.56
5.
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
103.00 ± 1.00
103
0.
97
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
100.00 ± 2.00
100
2.00
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
100.67 ± 1.53
101
1.52
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
96.33 ± 5.13
96
5.33
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
98.33 ± 3.51
98
3.57
10.
1,2,
3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
94.33 ± 6.81
94
7.22
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
97.00 ± 4.36
97
4.49
12.
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
95.67 ± 5.51
96
5.76
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
95.00 ± 6.08
95
6.40
14.
1
,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
97.67 ± 3.79
98
3.88
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
103.33 ± 1.53
103
1.48
Spiked concentration: 200 ng g
-1
16.
Octachlorodibenzo
-
p
-
dioxin
201.33 ± 2.08
101
1.03
17.
Octachlorodibenzofuran
201.67 ± 0.
58
101
0.29
TABLE 7
PCDDs/Fs recoveries and method precision (CV %) for gallbladder tissue blanks spiked with medium
concentration standard solution mixture
No. Congener Average Concentration
Found (N=3)
(
± s )
Average recovery
%
CV(%)
S
piked concentration: 10 ng/g
1.
2,3,7,8
-
Tetrachlorodibenzofuran
10.73 ± 0.15
107
1.42
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
10.80 ± 0.62
108
5.78
Spiked concentration: 50 ng/g
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
53.33 ± 1.15
107
2.17
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
52.00 ± 2.65
104
5.09
5.
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
48.33 ± 6.03
97
12.47
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
48.00 ± 6.00
96
12.50
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
52.00 ± 2.65
104
5.09
8.
2,3,4,6,7,8
-
Hexachlo
rodibenzofuran
49.00 ± 4.36
98
8.90
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
50.00 ± 3.61
100
7.21
10.
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
47.00 ± 6.56
94
13.95
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
47.33 ± 5.86
95
12.38
12.
1,2,3,7,8,9
-
Hexachloro
dibenzo
-
p
-
dioxin
47.67 ± 6.03
95
12.65
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
48.33 ± 4.93
97
10.21
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
47.00 ± 6.08
94
12.94
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
55.67 ± 2.08
111
3.74
Spiked concentrat
ion: 100 ng/g
16.
Octachlorodibenzo
-
p
-
dioxin
108.33 ± 3.06
108
2.82
17.
Octachlorodibenzofuran
101.67 ± 6.43
102
6.32
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3062
TABLE 8
PCDDs/Fs recoveries and method precision (CV %) for gallbladder stone blanks spiked with high
concentration standard solution mixture
No. Congener Average Concentration
Found (N=3) ( ± s ) Average recovery % CV(%)
Spiked concentration: 10 ng g
-1
1.
2,3,7,8
-
Tetrachlorodibenzofuran
10.70 ± 0.20
107
1.87
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
10.77 ± 0.64
108
5.97
Spiked concentration: 50 ng g
-1
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
52.33 ± 1.53
105
2.92
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
51.00 ± 2.00
102
3.92
5.
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
47.33 ± 4.73
95
9.98
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
4
7.00 ± 4.58
94
9.75
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
51.00 ± 2.00
102
3.92
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
48.00 ± 2.65
96
5.51
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
49.00 ± 2.00
98
4.08
10.
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
46.00 ± 5.0
0
92
10.87
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
46.33 ± 4.16
93
8.99
12.
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
46.67 ± 4.51
93
9.66
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
47.33 ± 3.21
95
6.79
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
46.0
0 ± 4.36
92
9.48
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
54.67 ± 3.51
109
6.42
Spiked concentration: 200 ng g
-1
16.
Octachlorodibenzo
-
p
-
dioxin
106.67 ± 3.79
107
3.55
17.
Octachlorodibenzofuran
100.00 ± 3.61
100
3.61
was as follows: (10 ng/ml) for tetra-PCDDs/Fs, (50
ng/ml) for penta, hexa, hepta-PCDDs/Fs and (100
ng/ml) for octa-PCDDs/Fs and the low concentra-
tion level was as follows: (5 ng/ml) for tetra-
PCDDs/Fs, (25 ng/ml) for penta, hexa, hepta-
PCDDs/Fs and (50 ng/ml) for octa-PCDDs/Fs. This
process was done for three samples. All spiked
samples were mixed thoroughly, extracted, cleaned-
up and analyzed according to the above mentioned
method. The recoveries were done in triplicate at
different time. The average recoveries were calcu-
lated. The results are shown in Tables 5, 6, 7, 8, 9
and 10. All recoveries were found to be within the
acceptable range for analysis of (80-120%) [22].
RESULTS
General. Thirty nine of each gallbladder tis-
sues and gallbladder stones have been investigated
for PCDDs/Fs). The average age for normal per-
sons was 48 years (range from 21 to 77). The body
mass index (BMI) was ranged from 15.88 to 39.52.
The samples were chosen by convenience, and
there was no statistically representative survey
design used to select sampled persons. As such, no
inference to the population of sick or healthy peo-
ple can be made with confidence at this stage.
TABLE 9
PCDDs/Fs recoveries and method precision (CV %) for gallbladder tissue blanks spiked with low
concentration standard solution mixture
No. Congener Average Concentration
Found (N=3) ( ± s ) Average recovery % CV(%)
Spiked concentration: 5 ng g
-1
1.
2,3,7,8
-
Tetrachlorodibenzofuran
4.67 ± 0.38
93
8.11
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
5.23 ± 0.15
105
2.92
Spiked
concentration: 25 ng g
-1
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
24.00 ± 2.65
96
11.02
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
22.00 ± 2.65
88
12.03
5.
1,2,3,7,8
-
Pentachlorodibenzo
-
p
-
dioxin
23.33 ± 1.53
93
6.55
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
23.67 ± 2
.31
95
9.76
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
24.00 ± 1.00
96
4.17
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
22.67 ± 2.52
91
11.10
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
23.67 ± 1.53
95
6.45
10.
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.67 ± 3.21
95
13.
58
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.33 ± 1.53
93
6.55
12.
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
24.00 ± 1.73
96
7.22
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
23.00 ± 2.00
92
8.70
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
24.67 ± 0.58
99
2.34
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
25.67 ± 1.15
103
4.50
Spiked concentration: 50 ng g
-1
16.
Octachlorodibenzo
-
p
-
dioxin
43.67 ± 1.53
87
3.50
17.
Octachlorodibenzofuran
48.33 ± 5.51
97
11.39
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3063
TABLE 10
PCDDs/Fs recoveries and method precision (CV %) for gallbladder stone blanks spiked with
high concentration standard solution mixture
No. Congener
Average Concentration
Found (N=3)
(
± s )
Average recovery %
CV(%)
Spiked concentration: 5 ng g
-1
1.
2,3,7,8
-
Tetrachlorodibenzo
furan
4.63 ± 0.32
93
6.94
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
5.20 ± 0.20
104
3.85
Spiked concentration: 25ng g
-1
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
23.67 ± 2.52
95
10.63
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
21.67 ± 2.08
87
9.61
5.
1,2,3,7,8
-
Pe
ntachlorodibenzo
-
p
-
dioxin
23.00 ± 1.00
92
4.35
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
23.33 ± 2.08
93
8.92
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
23.67 ± 0.58
95
2.44
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
22.33 ± 2.08
89
9.32
9.
1,2,3,7,8,9
-
Hexachlorodib
enzofuran
23.33 ± 1.15
93
4.95
10.
1,2,3,4,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.33 ± 3.06
93
13.09
11.
1,2,3,6,7,8
-
Hexachlorodibenzo
-
p
-
dioxin
23.00 ± 1.00
92
4.35
12.
1,2,3,7,8,9
-
Hexachlorodibenzo
-
p
-
dioxin
23.67 ± 1.53
95
6.45
13.
1,2,3,4,6,7,8
-
Heptachlor
odibenzofuran
22.67 ±1.53
91
6.74
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzo
-
p
-
dioxin
24.33 ± 0.58
97
2.37
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
25.33 ± 1.53
101
6.03
Spiked concentration: 50 ng g
-1
16.
Octachlorodibenzo
-
p
-
dioxin
47.33 ± 2.08
95
4.40
17
.
Octachlorodibenzofuran
50.33 ± 2.08
101
4.14
Concentration of PCDDs/Fs in Gallbladder
tissue and Gallbladder Stone Samples. For each
sample three processes were performed: weighing,
Soxhlet extraction and clean-up. The final extract
was injected onto the GCMS column three times. A
standard solution mixture of the 17 PCDDs/Fs con-
geners was injected every batch of samples (8 – 10
samples) to produce a chromatogram used for the
calculation of the Relative Peak Area (RPA) for
each standard compound (RPA
standard
). In the same
way, RPA for each compound in the unknown
chromatogram (RPA
unknown
) was compared with
(RPA
standard
) to find the concentration of (X) com-
pound in the unknown sample, as shown in the fol-
lowing equation:
Conc. of (X) in unknown sample = Conc. of (X) in
standard x (RPAunknown / RPAstandard)
Table 11 shows the analytical results of the
gallbladder tissue samples presented as average ±
standard deviation ( ± s) for each compound.
Tables 12 shows the analytical results of the
gallbladder stone samples presented as average ±
standard deviation ( ± s) for each compound.
Toxicity equivalencies were calculated for all
samples by converting the concentrations to the ng
TEQ/kg samples as follows:
TEQ (ng TEQ/kg) = Conc. of unknown sample
μg/kg tissues TEF
The sum of toxicity equivalence concentra-
tions are shown in the bottom of Table 11 for
gallbladder tissue samples.
The sum of toxicity equivalence concentra-
tions are shown in the bottom of Table 12 for
gallbladder stone samples.
DISCUSSION
Octachlorodibenzo-p-dioxin has eight chlorine
atoms which make it highest lipophilic in compari-
son with other PCDDs/Fs and it has a high abun-
dance in the environment so it has the highest aver-
age concentrations in all samples in both tissues
and stones which are 1134.49 μg/kg and 1645.39
μg/kg respectively. Eight PCDDs/Fs congeners
have zero concentrations or less than the detection
limit.
The TEQ values for each PCDDs/Fs congener
in each gallbladder tissue and stone sample were
calculated and the sums are presented in Tables 11
and 12 for tissues and stones respectively and
summarized in Figures 1 and 2 for tissues samples
and Figures 3 and 4 for the stones samples. The
sum of TEQ of each PCDDs/Fs congener show that
highest toxic PCDDs/Fs congeners in gallbladder
tissues and stones were 1,2,3,7,8-
pentachlorodibenzo-p-dioxin, 1,2,3,6,7,8-
hexachlorodibenzo-p-dioxin and 1,2,3,7,8,9-
hexachlorodibenzo-p-dioxin due to their high con-
centration and high Toxicity Equivalency Factors
(TEF).
A comparison between the average concentra-
tion of each PCDDs/Fs in gallbladder tissue sam-
ples and gallbladder stone samples is presented in
Figure 5. Most of the detected PCDDs/Fs conge-
ners show an accumulation in the gallbladder stones
more than in the gallbladder tissue samples.
The equilibrium in concentrations of
PCDDs/Fs congeners between the gallbladder tis-
sues and gallbladder stones can be represented by
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3064
the distribution coefficient Kd which can be calcu-
lated by dividing the total concentration of
PCDDs/Fs congeners in all gallstone samples by
the total concentration in the gallbladder tissue
samples as shown in the following equation:
Kd = ∑concentration in stone / ∑ concentration in
gallbladder stones.
The results are presented in Table 13 and Fig-
ure 6 which shows that the Kd values ranged be-
tween 0.86 for 1,2,3,4,7,8-Hexachlorodibenzo-p-
dioxin and 2.96 for 1,2,3,4,6,7,8-
Heptachlorodibenzofuran This slight variation is
due to the chemical structure of each congener
which determine its lipophilic character and its abil-
ity to bind stronger to the fat or to the cholesterol.
The type of gallbladder stone which determine its
cholesterol content while the obesity which deter-
mine the percent of fat in the gallbladder tissue
sample.
TABLE 11
Concentration of PCDDs/Fs (µg kg
-1
tissue) in gallbladder tissue samples (G-1 - G-13)
Average Concentration (µg kg
-1
tissue ( ± s )
Congener
Nr.
*
(TEF)
*
G-1
G-2
G-3
G-4
G-5
G-6
G-7
G-8
G-9
G-10
G-11
G-12
G-13
1 (0.1)
<
MDL
*
**
87.2
±
5.2
<
MDL
45.7 ±
5.0
49.4 ±
6.7
<
MDL
44.3 ±
5.5
68.6 ±
8.2
<
MDL
35.4 ±
3.5
35.0 ±
1.7
46.4 ±
5.2
48.9
±
4.8
2 (1.0) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
3
(0.03)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
4 (0.3) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
5 (1.0) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
28.8
±1.7
<
MDL
<
MDL
<
MDL
49.7
± 3.4
6 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
29.5 ±
1.4
<
MD
L
<
MDL
<
MDL
<
MDL
7 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
8 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
9 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
10
(0.1)
<
MDL
42.7
±
4.7
<
MDL
<
MDL
<
MDL
35.7 ±
4.4
<
MDL
49.3 ±
1.7
26.9 ±
3.0
<
MDL
<
MDL
<
MDL
27.9
±
3.7
11
(0.1)
<
MDL
101.8
±
5.4
104.1
±
14.1
94.3 ±
5.3
304.5
±
35.8
80.9 ±
6.3
<
MDL
238.7
±
23.4
< MD
L
206.3
±
24.4
111.2
±
10.4
229.1
±
21.7
243.6
±
26.3
12
(0.1)
<
MDL
52.3
±
2.6
157.6
±
12.6
178.7
±
21.9
132.6
±
6.4
61.2 ±
3.6
79.8
±
10.7
30.9 ±
4.1
59.7 ±
5.9
48.5 ±
6.2
54.6 ±
7.7
<
MDL
50.1 ±
3.2
13
(0.01)
<
MDL
132.7
±
8.0
55.8
±
4.5
<
MDL
60.6 ±
7.9
120.7
±
5.4
83.6
±
7.6
113.7
±
10.3
35.4 ±
1.6
31.9
±
1.9
<
MDL
<
MDL
<
MDL
14
(0.01)
<
MDL
386.1
±
39.2
328.9
±
40.4
132.5
±
13.8
336.1
±
41.1
194.7
±
26.5
88.4 ±
6.1
120.4
±
9.1
76.6 ±
6.4
283.9
±
17.8
93.9 ±
8.8
341.0
±
30.5
113.3
±
13.3
15
(0.01
)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
16
(0.000
3)
289 ±
19.5
252..4
± 30.1
1428.
5 ±
108.9
3294.
3 ±
200.3
1224.
9 ±
166.2
292.5
±
33.0
144.1
±
18.8
489.8
±
24.9
1896.
2 ±
207.5
1607.
2 ±
179.4
1720.
5 ±
185.6
1884.
0 ±
246.2
462.8
±
23.7
17
(0.000
3)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
∑PC
DDs/
Fs
289.0 1046.
2
2074.
8
3745.
6
2135.
5 785.6 440.2 1111.
3
2153.
0
2213.
2
2015.
1
2500.
5
1000.
3
∑TE
Qs
ng
TEQ/
kg
0.4 33.6 30.4 34.2 55.1 21.0 14.2 41.2 42.1 32.7 21.5 31.5 88.4
* Congener numbers as in Table 2; ** TEF = Toxicity Equivalency Factor *** MDL = method detection limit
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3065
TABLE 12
Concentration of PCDDs/Fs (µg kg
-1
tissue) in gallbladder stone samples (S-1 - S-13)
Average Concentration (µg kg
-
1
tissue ( ± s )
Congener
Nr.
*
(TEF
**
)
S-1
S-2
S-3
S-4
S-5
S-6
S-7
S-8
S-9
S-10
S-11
S-12
S-13
1 (0.1) 67.3 ±
5.4
53.5 ±
5.6
26.2 ±
1.4
60.2 ±
2.1
<
MDL
35.7 ±
4.4
28.4 ±
1.3
<
MDL
84.8
±
4.7
33.25
±
1.4
32.1 ±
1.6
14.9 ±
0.5
35.5 ±
4.4
2 (1.0)
<
MDL
*
**
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
3
(0.03)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
4 (0.3) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
5 (1.0) <
MDL
65.7
±5.0
<
MDL
<
MDL
59.3 ±
6.8
60.0
±
3.9
47.8 ±
5.7
<
MDL
<
MDL
<
MDL
<
MDL
51.8 ±
6.0
<
MDL
6 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
38.8 ±
1.3
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
7
(0.1)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
8 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
9 (0.1) <
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
10
(0.1)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
28.8 ±
3.3
<
MDL
54.82
±
1.8
<
MDL
<
MDL
<
MDL
11
(0.1)
54.1 ±
11.5
108.7
±
8.9
282.7
±
28.3
215.1
±
20.1
42.5 ±
4.9
64. 8±
6.3
<
MDL
33.0 ±
1.6
207.1
±
18.5
99.8 ±
6.6 < MD <
MDL
46.8 ±
3.1
12
(0.1)
58.6 ±
6.4
258.7
±
22.8
66.7 ±
7.9
99.6 ±
8.4
196.8
±
8.9
182.6
±
20.1
281.4
±
33.3
244.9
±
14.2
74.2
±
2.3
61.13
±
5.1
114.7
±
13.7
278.4
±
18.2
40.2 ±
3.6
13
(0.01)
109.4
±
8.1
232.3
±
25.5
217.9
±
24.8
151.2
±
15.7
90.1 ±
8.9
132.2
±
10.0
140.8
±
15.8
241.1
±
24.5
78.5
±
4.7
193.8
±
6.8
321.9
±
22.9
104.1
±
10.2
74.7 ±
6.2
14
(0.01)
107.4
±
10.0
295.4
±
11.0
327.3
±
36.7
127.9
±
10.3
180.8
±
18.1
104.9
±
7.9
166.7
±
6.6
106.9
±
9.9
472.6
±
30.2
260.4
±
19.4
54.0 ±
6.5
213.2
±
21.3
238.6
±
17.2
15
(0.01)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
16
(0.000
3)
1652.
0 ±
133.0
2354.
8 ±
215.5
1332.
2 ±
150.3
1039.
5 ±
54.4
1574.
5±
93.2
1057.
1 ±
38.4
1827.
3 ±
139.8
663.9
±
75.6
1628.
7 ±
69.6
1333.
6 ±
79.1
1603.
9 ±
55.8
2485.
9 ±
333.9
1107.
0 ±
17
(0.000
3)
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
<
MDL
∑PC
DDs/
Fs
2048.
8
3369.
0
2253.
6
1693.
5
2143.
9
1676.
3
2492.
4
1318.
6
2617.
3
2036.
7
2126.
4
3133.
4
1542.
8
∑TE
Qs
ng
TEQ/
kg
20.7 113.7 18.0 21.2 86.4 94.9 82.4 34.4 42.6 29.8 18.9 85.0 15.7
* Congener numbers as in Table 2; ** TEF = Toxicity Equivalency Factor; *** MDL = method detection limit
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3066
FIGURE 1
TEQ of PCDDs/Fs (ng TEQ kg
-1
) in gallbladder tissues for samples (G-1 to G-20)
FIGURE 2
TEQ of PCDDs/Fs (ng TEQ kg
-1
) in gallbladder tissues for samples (G-20 to G-39)
FIGURE 3
TEQ of PCDDs/Fs (ng TEQ kg
-1
) in gallbladder stone for samples (S-1 to S -20)
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3067
FIGURE 4
TEQ of PCDDs/Fs (ng TEQ kg
-1
) in gallbladder stone for samples (S-20 to S-39)
FIGURE 5
Comparison of the average concentration of each PCDDs/Fs (μg kg
-1
) in the gallbladder tissues (blue) and
gallbladder stones (red).
TABLE 13
Distribution coefficient (Kd) of PCDDs/Fs in gallbladders tissue and stone
No.
PCDDs/Fs congener
∑ PCDDs/Fs in gallbladder stone
∑ PCDDs/Fs in gallbladder tissues
Kd
1.
2,3,7,8
-
Tetrachlorodibenzofuran
2270.88
1417.40
1.60
2.
2,3,7,8
-
Tetrachlorodibenzeno
-
p
-
dioxin
0.00
0.00
0.00
3.
1,2,3,7,8
-
Pentachlorodibenzofuran
0.00
0.01
0.00
4.
2,3,4,7,8
-
Pentachlorodibenzofuran
0.00
0.00
0.00
5.
1,2,3,7,8
-
Pentachlorodibenzin
o
-
p
-
dioxin
520.23
231.61
2.25
6.
1,2,3,4,7,8
-
Hexachlorodibenzofuran
38.83
456.27
0.09
7.
1,2,3,6,7,8
-
Hexachlorodibenzofuran
0.00
0.00
0.00
8.
2,3,4,6,7,8
-
Hexachlorodibenzofuran
0.00
0.00
0.00
9.
1,2,3,7,8,9
-
Hexachlorodibenzofuran
0.00
0.00
0.00
10.
1,
2,3,4,7,8
-
Hexachlorodibenzino
-
p
-
dioxin
365.52
423.80
0.86
11.
1,2,3,6,7,8
-
Hexachlorodibenzino
-
p
-
dioxin
5612.77
5887.61
0.95
12.
1,2,3,7,8,9
-
Hexachlorodibenzino
-
p
-
dioxin
5709.82
1992.58
2.87
13.
1,2,3,4,6,7,8
-
Heptachlorodibenzofuran
4785.65
1617.93
2.96
14.
1,2,3,4,6,7,8
-
Heptachlorodibenzino
-
p
-
dioxin
6278.03
6803.77
0.92
15.
1,2,3,4,7,8,9
-
Heptachlorodibenzofuran
0.00
0.00
0.00
16.
Octachlorodibenzino
-
p
-
dioxin
64170.04
44245.23
1.45
17.
Octachlorodibenzofuran
0.00
0.00
0.00
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3068
FIGURE 6
Distribution coefficient (K
d
) values of PCDDs/Fs between gallbladder stones and gallbladder tissues
TABLE 14
Classification of samples according BMI and the concentrations of PCBs in each class
Group of
BMI
(kg m
-2
)
Symbol Samples in
each class
Average
Concentration
in gallbladder
Tissue samples
(µ
g kg
-1
)
Average
Concentration
in gallbladder
Stone samples
(µ
g kg
-1
)
Average
Concentration
in all samples
(µg kg
-1
)
0 - 18.5 L
(low)
1,6,7,8,13,16,17,20,21,29,31,
33,36,38,39
1838 1730 3568
18.5 -25 M
(medium)
5,10,11,18,22,23,24,25,26,27,
32,34,37
1786 2192 3978
> 25 H
(high)
2,3,4,9,12,14,15,19,28,30,35 2391 3111 5502
To study the factors which affect the concen-
trations of PCDDs/Fs in the 39 samples of gallblad-
der tissues and gallbladder stones, many correla-
tions trails between the concentrations and many
factors were done and showed that there was no
significant correlation between the concentration
with age, sex, living place, smoking and healthy
state. But it shows a significant correlation with
obesity which is represented by the body mass in-
dex (BMI).
The samples were classified to three groups
depending on their BMI as shown in Table 14. The
average concentrations of PCDDs/Fs in gallbladder
tissues, stones and in both tissues and stones (total)
FIGURE 7
Average concentration of PCDDs/Fs in gallbladder stones, tissues and for both versus BMI groups
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3069
FIGURE 8
Total concentrations (ng/kg) in both gallbladder tissues and stones of all PCDDs/Fs congeners in each
sample.
TABLE 15
Comparison between the results (ng kg
-1
tissue) of this study and the findings of others [25]
Congeners
found
Gallbladder Tissue
Gallbladder Stones
Results of Muto
et al.,
1991
Mean
Range
Frequency
Mean
Range
Frequency
Mean
Range
Cause of
Death
1,2,3,7,8-
Pentachlorodi-
benzo
-
p
-
dioxin
46.3 28.8 – 82.7 12% 52.3 29.2 – 65.7 20.5% 11.0
4.6
1.8 – 41.8
-
--
Hepatoma
1,2,3,6,7,8-
Hexachlorodi-
benzo
-
p
-
dioxin
203.7 31.0 – 892.9 93.3% 157 31.0 – 473.5 93.3% 1.5
2.3
0 – 1.5
-
- -
Hepatoma
1,2,3,4,6,7,8-
Heptachlorodi-
benzo-p-dioxin
178.3 30.1 - 533 97.4 252 29.0 – 2262.4 84.6%
99.6
90.3
79.5
- -
20.7 – 108
- -
Hepatoma
Cancer
Goiter
Hepatoma
Octachlorodi-
benzo-p-dioxin 1075.1 117.5 – 1896.2 100% 1646.9 424.6 – 4229.5 100%
132
81.8
101
225
24.8 – 225
- -
- -
- -
Cancer
Goiter
Hepatoma
Hepatoma
Cancer
Goiter
for each group were calculated, and the correlation
with BMI is shown in Figures 7.
The total concentrations of all PCDDs/Fs in
gallbladder tissues and stones in each sample were
calculated and shown in Figure 8 which shows
high concentrations in the samples 14, 19, 30 and
35. Of these, samples 19 and 30 were diagnosed as
a dysplasia cases (the presence of cells of an ab-
normal type within a tissue, which may signify a
stage preceding the development of cancer), so
these compounds can affect the cells and change
their nature and then can be carcinogenic [23-24].
In the literature, there is very limited number
of publications deals with PCDDs/Fs in gallbladder
tissues, and nothing about gallbladder stones. Muto
et al. [25]
studied the tissue distribution of 2,3,7,8-
chlorine substituted dibenzo-p-dioxins in 11 pa-
tients who died of cancer. The concentrations of
some PCDDs/Fs he found in gallbladder tissues are
summarized in Table 15 and compared to the find-
ings of this study. Table 15 shows that the concen-
trations found in this study are much higher than
what Muto and his research group [29]
found, nev-
ertheless there was no deaths among the donors of
the 39 samples. It is also attention drawing that the
congeners 1,2,3,6,7,8-hexachlorodibenzino-p-
dioxin and 1,2,3,4,6,7,8-heptachlorodibenzo-p-
dioxin are present at high frequency, numerically
97.4% and 100% respectively.
CONCLUSIONS
[1] All PCDDs/Fs that exist in the gallbladder tis-
sue can exist in the gallbladder stone too which
mean both gallbladder stones and gallbladder
tissues can be good indicator for PCDDs/Fs
exposure.
[2] Distribution coefficients of PCCDs/Fs between
gallbladder tissues and gallbladder stones are
close to one, and its deviation from one depend
on the amounts of cholesterol in stone and the
amounts of fat in tissue.
© by PSP Volume 29 – No. 04A/2020 pages 3056-3071 Fresenius Environmental Bulletin
3070
[3] Significant correlation between body mass in-
dex (BMI) and concentration of PCDDs/Fs
take place in both gallbladder tissues and
gallbladder stones, so that as BMI increase the
concentrations of PCDDs/Fs also increase due
to the increasing in the bioaccumulation ten-
dency.
[4] Dysplasia correlated to the concentrations of
PCDDs/Fs, the higher the concentration, the
higher is the probability to have dysplasia
which means these pollutants can be one of the
most factors that induce the formation of dys-
plasia or cancer.
ACKNOWLEDGEMENTS
The authors would like to thank the Deanship
for academic research at the University of Jordan
for the financial support.
All procedures performed in studies involving
human participants were in accordance with the
ethical standards of the institutional and/or national
research committee and with the 1964 Helsinki
declaration and its later amendments or comparable
ethical standards.
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Received: 30.11.2019
Accepted: 26.01.2020
CORRESPONDING AUTHOR
Mahmoud Alawi
School of Science,
Department of Chemistry,
University of Jordan,
Amman, 11942 – Jordan
E-mail: alawima@ju.edu.jo
