Toxicological & Environmental Chemistry

ISSN: 0277-2248 (Print) 1029-0486 (Online) Journal homepage: http://www.tandfonline.com/loi/gtec20

A comparitive study between the coal-biomass

briquette and raw coal in SO2 pollution and
adverse effects in rabbits

Cheng Shuqun , Zhou Yanrong , Wang Yi’nan , Wang Xun , Liu Yuanfu , Iwao
Uchiyama , Wang Qiangyue & Kazuhiki Sakamoto

To cite this article: Cheng Shuqun , Zhou Yanrong , Wang Yi’nan , Wang Xun , Liu Yuanfu , Iwao
Uchiyama , Wang Qiangyue & Kazuhiki Sakamoto (2006) A comparitive study between the coal-
biomass briquette and raw coal in SO2 pollution and adverse effects in rabbits, Toxicological &
Environmental Chemistry, 88:4, 665-672, DOI: 10.1080/02772240600856599

To link to this article: http://dx.doi.org/10.1080/02772240600856599

Published online: 01 Feb 2007.

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Download by: [University of Nebraska, Lincoln] Date: 13 June 2016, At: 18:34
 Toxicological & Environmental Chemistry, Oct.–Dec. 2006; 88(4): 665–672

A comparitive study between the coal-biomass

briquette and raw coal in SO2 pollution and
adverse effects in rabbits
Downloaded by [University of Nebraska, Lincoln] at 18:34 13 June 2016

CHENG SHUQUN1, ZHOU YANRONG1, WANG YI’NAN2,

WANG XUN1, LIU YUANFU1, IWAO UCHIYAMA3,
WANG QIANGYUE4, & KAZUHIKI SAKAMOTO4
1
School of Public Health, Chongqing University of Medical Sciences, Chongqing 400016,
P. R. China, 2Ear-nose-throat Department of Chongqing Daping Hospital, Chongqing 400014,
3
Department of Environmental Engineering, Graduate School of Engineering,
Kyoto University, Japan, and 4Faculty of Engineering, Department of Applied Chemistry,
Saitama University, Japan

(Received 30 May 2006; in final form 6 June 2006)

Abstract
This study was conducted to evaluate the adverse health effects on rabbits exposed to SO2 emitted
indoors from burning coals, and compare differences between coal-biomass briquette (B.B.) and
raw coal (R.C.). Thirty-six male rabbits were divided equally into three groups at random, and then
exposed to burning R.C., B.B., and the third without burning coal (Control) for 90 days.
Data showed that the average concentration of SO2 in 24 h in R.C. was 13.04 mg m
3, which was
5-fold greater than B.B. (2.68 mg m
3) and 31-fold higher than control (0.41 mg m
3). After 45 days,
the numbers of rabbits, with increased frequency of Comet cell was highest in R.C. After 90 days, the
% positive Comet cell was significant at 10.36% in R.C., 5.42% in B.B., and 1.73% in Control. There
was a nonlinear dose–effect relationship between % positive Comet cell and the concentration of SO2.
The incidence of interstitial pneumonia was 6/12 in R.C. and 4/12 in B.B. showing severe squamous
metaplasia with atypical hyperplasia in bronchial epithelia in R.C. animals. The results of study
indicate that use of B.B. reduced the emission of SO2; but the smoke emitted from burning coal still
produced DNA damage.

Keywords: Coal-biomass briquette, raw coal, rabbit, adverse effects

Correspondence: Cheng Shuqun, School of Public Health, Chongqing University of Medical Sciences, Chongqing 400016,
P. R. China. Fax: 86 23 68485008. E-mail: slow66@sina.com
ISSN 0277-2248 print/ISSN 1029-0486 online ß 2006 Taylor & Francis
DOI: 10.1080/02772240600856599
 666 C. Shuqun et al.

Introduction
China is a major consumer of coal comprising 75% of energy utilization. SO2 emitted
from burning coal is the main source of SO2 pollution in the atmosphere. In China 90% of
SO2 is derived from burning coal. With rapid economic development in China, emission
of SO2 is increasing annually. As Chongqing is a heavy industrial city, and coal produced
in this area mainly contains sulfur, the extensive use of coal in industries and homes
produces a large quantity of SO2, which reduces the quality of air and produces adverse
effects on human health [1]. SO2 is an irritant gas with corrosive properties resulting in
acid rain formation, producing building erosions, and is lethal to animals and plants.
Krasnowska et al. [2] demonstrated that SO2 increased the frequency of respiratory diseases
in rats. Several epidemiologic studies examined the correlation between SO2 exposure and
consequent mortality attributed to this emitted gas [3,4]. However, the effect of SO2 on
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health is usually slow in onset and not apparent until the organism is exposed to low
concentrations for a chronic period.
Coal-biomass briquette (B.B.) is made by adding some biological material as an
adsorbent to retain released sulfur. When briquettes burn, the emitted SO2 reacts with the
adsorbent and remains fixed in coal cinder. Thus, the emission of SO2 from burning
briquettes is reduced by 63% [5]. This study simulated the condition of using coal
at home by utilizing an animal model and eliminated the interfering role of confounding
factors which would occur in the natural environment. The aim of the study was to compare
the differences in SO2 emission between burning B.B. and burning raw coal (R.C.) indoors
with subsequent effects on mammalian health.

Materials and methods

Animal and experimental conditions
Thirty-six male rabbits (New Zealand, about 2 kg each, provided by Laboratory Animal
Center of Chongqing University of Medical Sciences) were randomly divided into three
groups, B.B., R.C., and blank group (Control), and were maintained for 90 days.

Laboratory room devices. The rooms for B.B. group and R.C. group were the same with size
and structure (3 m  2.5 m  2 m), installed an exhaust fan and a stove. The air of two
rooms was not convective to each other. The rabbits of Control group were maintained
at the Laboratory Animal Center. All the rabbits were subjected to standards used in
the Laboratory Animal Center. Rabbits that died from nonexperimental factors were
eliminated. During experiment the stoves were fired at a fixed time every day simulating
the common family cooking hours: 8:00–9:00 in the morning, 11:30–13:30 at noon,
and 17:00–19:00 in the evening. Coal was added at the same time in the two groups.

Experimental coal
All of the high sulfur R.C. (content of sulfur being higher than 3 kg/100 kg) came from
Chongqing Nantong Mine. The B.B. and R.C. were made to briquette with designed
formula and technological process by the factory belonging to Chengdu Research Institute
of Environment. Such formula and process were provided by Professor Kazuhiko Sakamoto
of Saitama University in Japan. An individual was especially assigned to supervise the
production and transportation of briquette. The composition of two kinds of briquette,
 A comparitive study between the coal-biomass briquette and raw in SO2 pollution 667

which include fixed carbon, gross sulfur and calcium sulfur ratio, and so on were analyzed
by the Southern Coal Examination Center of Geology and Mineral Ministry in China.

Environmental indices: SO2, CO

In each group, the concentrations of SO2 and CO were measured by TOXIC gas monitor
(Model PGM-35, made in the US) at the time of making and maintaining the fire in the
morning, noon, and evening daily. The measuring time is 10 min.

DNA damage of lymphocyte (Comet assay)

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Single cell gel electrophoresis (SCGE) assay was used to measure DNA damage of
lymphocytes according to the method of Rojas et al. [6]. Determinations were made
three times, (1) before the experiment, (2) 45 days later, and (3) at 90 day after the start.
One hundred cells from each sample were randomly selected randomly for the Comet assay.
The standard of classification of Comet tail is as follows: normal cell represents a round
fluorescence kernel, which is a Comet head without a tail. Damaged cells have a DNA
segment running out from kernel to anode and forms a bright head and tail. According to
the length of DNA from head to tail the damage is divided into three grades. Comet cell 1:
with visible DNA segment moving; Comet cell 2: with obvious DNA segment moving;
Comet cell 3: with long moving distance (Figure 1).

Pathological examination
The rabbits were sacrificed at the end of the experiment, bronchi and lung tissue excised,
fixed in 10% neutral formaldehyde, embedded with olefin and stained with hematoxylin
and eosin. The sections were observed and photographed with an optic microscope.

A B

C D

Figure 1. Comet assay (A: normal cell, B: Comet cell 1, C: Comet cell 2, D: Comet cell 3).
 668 C. Shuqun et al.

Analysis of data
All the data were entered into a computer and analyzed by variance analysis, Ridit analysis,
the multiply analysis through SAS software package. The criterion for significance was
set at p50.05.

Results
The composition analysis of bio-mass briquette and raw coal
Table I shows that a though the composition of R.C. of two experimental coals was similar,
the technological process and adsorbent material was different. The content of sulfur and
calcium is different, and the ratio of sulfur to calcium in B.B. is 6-fold higher than that of R.C.
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Comparison of SO2 concentrations

Table II shows that the concentration of SO2 in R.C. is approximately 7 times higher
than B.B. when making fire, and the 24-h average value in R.C. (13.04 mg m
3) is 5-fold
greater than B.B. Group (2.68 mg m
3) and 32-fold greater than Control (0.41 mg m
3),
indicating that use of B.B. markedly reduces the concentration of SO2 indoors.

Concentration of CO
The average concentration of CO in B.B. is quantitatively higher than R.C. (Table III)
which indicates that the contribution of CO from two types of coals to atmospheric
pollution was negligible.

Table I. The comparison of coal composition in B.B. and R.C.

Category Humidity Ash content Volatile Fixed carbon Gross sulfur Calcium Calcium/sulfur ratio

Briquette 2.02 39.10 43.12 34.64 2.71 6.60 2.44

Coal 2.08 36.77 19.74 50.75 3.47 1.44 0.41

Table II. Concentration of SO2 in each laboratory room (mg m
3).

Making fire Keeping fire

Groups  s
X  s
X 24 h average value

B.B. 3.60  1.05 2.44  0.64 2.68

R.C. 24.65  9.75 10.01  3.13 13.04
Control – – 0.41

Table III. Concentration of CO in each laboratory room (mg m
3).

R.C. B.B.

Time n Md Xmax Xmin n Md Xmax Xmin

Making fire 57 33.14 332.55 6.86 57 37.2 545.14 10.29

Keeping fire 60 15.5 72.2 2.40 60 22.4 134.86 0.57
 A comparitive study between the coal-biomass briquette and raw in SO2 pollution 669

Table IV. Comparison of Comet assay at three time points in each group.

Before experiment 45 days after 90 days after

Groups Rabbits (n) Positive Rabbits (n) Positive Rabbits (n) Positive

R.C. 12 4 11 10 11 11
B.B. 12 5 12 6 12 11
Control 12 5 11 5 11 7
Likelihood 2 ¼ 0.236 p ¼ 0.889 2 ¼ 6.738 p ¼ 0.039 2 ¼ 7.09 p ¼ 0.024

Table V. Percentage comparison of Comet cell after 90 days.

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The degree of positive cells

Groups Total cells Comet assay cell Positive rate (%) (x  s) Degree 1 Degree 2 Degree 3

B.B. 1100 117 10.36  1.48 79 23 15

R.C. 1200 65 5.42  1.30 47 15 3
Control 1100 17 1.73  0.81 15 2 0
The Ridit analysis of data from each group: 2 ¼ 193.7, p50.05.

DNA damage of lymphocytes of rabbits

Prior to the experiment, there were a few rabbits with DNA damage in lymphocytes
and there was no significant difference of Comet cell % among the three groups
(Table IV). After 45 days of coal burning, Comet cells increased significantly in the
R.C. group. After 90-days of coal burning, the Comet cells in rabbits in the B.B.
group also rose (Table IV). However, the % and severity of Comet cell was different
between groups (Table V). The % of Comet cell was 10.36 in R.C., 5.42 in B.B., and
1.73 in Control were significantly different. Table VI shows that % Comet cell
increased gradually with time, and the difference between prior to and after 90-day
coal burning was 4.92 in B.B. and 9.94 in R.C. From Figure 2, it can be seen that the
% DNA damage in lymphocytes increased with the concentration of SO2 after 90-day
coal burning, and there was a nonlinear dose–effect relationship between SO2
concentration and % positive cell (R2 ¼ 0.784).

Pathological observations
There were rabbits appearing with pneumonia in three groups, predominantly acute
interstitial pneumonia. However, the rate in the two coal groups was notably higher than
control, 6/12 in R.C., 4/12 in B.B., and 1/12 in the Control group. The hyperplasia of
bronchial epithelia of rabbits in R.C. was marked. It is notable that the size and stain
intensity of bronchial epithelial cells of a rabbit were different showing a disorderly array
and occasional nuclear disintegration, severe squamous metaplasia with atypical hyperplasia
was also noted (Figure 3).
 670 C. Shuqun et al.

Table VI. Percentage comparison of a comet cell at different times in three groups (x  s).

Groups Before experiment 1 45d 2 90d 3 2–1 3–1

B.B. Group 0.42  0.31 2.09  0.61 10.36  1.48 1.67 9.94
R.C. Group 0.50  0.33 1.00  0.54 5.42  1.30 0.50 4.92
Control Group 0.42  0.26 0.64  0.52 1.73  0.81 0.22 1.31

12

10

8
Percent (%)
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0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
SO2 concentration (mg m−3)

Figure 2. The nonlinear dose–effect relationship of SO2 to Comet cell (R2 ¼ 0.784).

Figure 3. The result of pathological test of lung and bronchus. (A) Interstitial pneumonia HE  100;
(B) Severe squamous metaplasia with atypical in bronchial epithelia HE  400.
 A comparitive study between the coal-biomass briquette and raw in SO2 pollution 671

Discussion
The concentrations of SO2 were measured in the kitchen was 0.95 mg m
3 when making
fire and 0.45 mg m
3 when maintaining fire by using bio-briquette, and these values were
less than those using R.C., 5.71 and 3.16 mg m
3. Hence, the use of bio-briquette
significantly reduced the concentration of SO2 in kitchen. Data also showed that despite the
composition of R.C. of two experimental coals being the same, because of the difference
of adsorbent and technological processes, the ratio of calcium to sulfur of briquette
was 6-fold different. When the other conditions remain unchanged, the ratio of calcium to
sulfur was higher, indicating the effectiveness of the adsorbent [7]. In this experiment
the concentration of SO2 in the R.C. was 24.65 mg m
3 when making fire which
was 49 times greater than the maximum admissible concentration in China. As the use
of bio-briquette markedly reduced the SO2 concentration indoors, it is important to
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popularize the use of bio-briquette to reduce SO2 pollution. It is noteworthy that

according to national standard of China (GB3095-82) the MAC of SO2 is 0.5 mg m
3,
SO2 produced by briquette is still higher than the MAC, and thus, needs further
improvements.
There are many reports that the incidence of lung cancer for animals is higher when
there is both SO2 and benzo[a]pyrene (BaP) present compared to BaP alone [8,9].
SO2 thus promotes the development of cancer through a mechanism not defined as
yet. DNA damage is the first step in cancer induction. However, DNA damage can
be repaired as repair mechanisms exist in mammals, which reduces the occurrence
of cancer. If repair ability is modified, the damage to DNA cannot be repaired or
repaired wrongly, resulting in DNA mutation and tumor development. Comet assay or
the SCGE is a sensitive, reliable, rapid, simple, and new technology for testing the DNA
damage of individual cells [10]. Data showed that the DNA damage in lymphocytes rose
with the increase of concentration of SO2 indoors and there was nonlinear dose–effect
relationship. BaP is typical carcinogenic substance and is inadvertently emitted from
coal smoke accompanying SO2 [11]. However, the coal source for B.B. and R.C. came
from same place. Further the consumed quantity, the size of the stove and room space
were identical, indicating that the quantity of BaP in exposed animals in two groups
were almost identical, but the pathologic observation and the damage rate of DNA was
notably different. Thus evidence suggests that SO2 played a role in cell DNA damage.
Whether the SO2 plays a leading role in inducing tumors of the respiratory tract needs
further study.

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