全 文 ：文章编号 :100028551 (2002) 0320174205
BEHAVIORS OF 14 C2BUTACHLOR ,
14 C2CHLORPYRIFOS AND 14 C2DDT IN
Rana japonica japonica GUENTHER
ZHANG Yi2qiang1 　ZHANG Hai2qing2 　ZHONG Chuang2guang1
ZHAO Xiao2kui1 　CHEN Shun2hua1
(11Laboratory of Isotopes , School of Life Sciences , Zhongshan University , Guangzhou , Guangdong prov. 　510275
21 Panyu Institute of Environmental Sciences , Panyu , Guangdong prov . 　511400)
ABSTRACT : The research on the behaviors of 14 C2butachlor , 14 C2chlorpyrifos and 14 C2DDT in the frog
Rana japonica japonica Guenther was carried out. After administrated per os to the frogs in doses of
380 ,347 ,363 Bq/ g , 14 C2butachlor , 14 C2chlorpyrifos and 14 C2DDT , were distributed respectively to vari2
ous organs within 24 h with specific accumulating organs as gallbladder , intestine and intestine , relevan2
tly to the pesticides described. Compared to that in gallbladder and intestine , the radioactivity of many
organs was extremely low , and this might due to the characters of the pesticides. Analysis of the metab2
olites of 14 C2DDT in frog at 24th hr demonstrated that DDT was difficult to be degraded. Most 14 C2
butachlor , 14 C2chlorpyrifos 14 C2DDT in liver and fat or ovary of frog was extractable with acetone.
However , there were some differences between the pesticides , and the organs as well. And 14 C2buta2
chlor , 14 C2chlorpyrifos or 14 C2DDT were better bound in liver than in fat.
Key words : 14 C2butachlor ; 14 C2chlorpyrifos ; 14 C2DDT; distribution ; metabolism ; Rana japonica japonica
Guenther
收稿日期 :1999208224
基金项目 :Supported by Intemational Atomic Energy Agency( IAEA) 7933/ MC;
作者简介 :张奕强 (1963～) ,男 ,中山大学生物科学院助教
Pesticides is an integral part of modern agriculture system , contributing significantly to improve crop
yields and enhance food production[1 ] . However , it has been estimated that less than 1 % of the pesti2
cides applied to crops attain the targets. Excess agrochemical moves throughout the environment and may
enter ecosystem by a number of pathways. The spread of pesticide residues in the environment has created
several current or potential future problems. It is observed that the application of the chemicals has leaded
to the death or the crisis of death of many organisms , especially the animals. All through the world , the
amphibian , an important group , are becoming extinct and the extinct rate increased with time. For in2
stance , the number of frogs and their diversities in the field have decreased to a very low level during last
decades[2 ] . Pesticides directly injure the amphibian , especially their larva or eggs , or they affect the
physiology , growth and development , and behavior of the amphibian through the food link. There was
471 　核 农 学 报　2002 ,16 (3) :174～178Acta Agriculturae Nucleatae Sinica
closely correlation between the extinct of amphibian in the three protect parks north to the Eally Lake of
Canada and the contamination of organic chloride pesticides there[3 ] . DDT contamination will result in the
malformation of the upper jaw of amphibian[4 ] . Many studies have been done on the biological behaviors
of pesticides in shellfishes crab[5 ] , or fish[6～9 ] , and given us much comprehension of the toxicology of the
pesticides. However , few in vestigations have been conducted to estimate the toxicological effects of pesti2
cides on amphibious animals , say the frogs , and their larva or eggs[10～11 ] . Here we report the primary
study on the behaviors of 14 C2butachlor , 14 C2chlorpyriofs , and 14 C2DDT in frog R . japonica japonica
Guenther.
1 　Materials and Methods
111 　Materials
Specimens of frog R . japonica japonica Guenther were brought from a market in Guangzhou and
identified in our laboratory. After 3d of breading in the laboratory , the healthy ones weighing about 10 ±
4 g were selected for the experiments.
14 C2butachlor (N2butoxymethyl222chloro22’, 6’2diethylacetonilide) ,14 C2chlorpyrifos (O ,O2diethyl2
O23 ,5 ,62trichloro222pyridiil phosphorotiate) , and 14 C2DDT [ 1 , 1 , 12trichloro22 , 22bis (pchloropheny2
lethane) ] were provided by IAEA.
The standards of butachlor , DA , DDT , DDE , DDMU , DDD , dicofols , DDA , chlorphyrifos , and
TCP were supplied by Chem Service Company.
Aquarium :pure glass , size in 50cm ×50cm ×30cm.
Digest solution :HClO4 , H2O2 and octanol .
Scintillation cocktail :PPO 4g , Triton X2100 333ml , and toluene 667ml.
112 　Methods
The frogs in either sex were grouped and bred in the Aquarius for 12h before experiments. The pes2
ticides 14 C2butachlor , 14 C2chlorpyrifos , and 14 C2DDT were solved with acetone and then diluted into 1127
×105dpmΠml , 1167 ×105dpmΠml , and 1145 ×105dpmΠml , relevantly. The concentrations of acetone
plus solubizer Tritone2100 were less than 015 % in the solutions. With a perfusion apparatus , each 10
frogs in a group were administrated per os with 14 C2butachlor , 14 C2chlorpyrifos , and 14 C2DDT in doses of
380 ,347 ,363 BqΠg. respectively. The experiments were conducted at temperature of 28 ℃and in saturat2
ed humidity. On the 24th hour , the frogs were anaesthetized and dissected. The analysis of the distribu2
tion and metabolism of these pesticides was made as described previously[12 ] . The organs of small size and
tissue samples were separated and mineralized directly into glass vials. The 14 C2radioactivity was mea2
sured after addition of the liquid scintillation cocktail . The much larger samples of frogs’remainder and
part of liver , fat body or ovary were minced and weighed before being extracted with acetone repeatedly.
Acetone extracts were combined and concentrated to reduce the volume following the metabolite analysis
with co2chromatography. The organic residue obtained was digested with the acid/ H2O2 mixture and the
14 C2radioactivity determined with the Liquid Scintillation Analyzer , Parkard Trib CA2000/ LL.
571　3 期 Behaviors of 14C2butachlor , 14C2chlorpyrifos and 14C2DDT in Rana japonica japonica Guenther
2 　Results and Discussions
211 　Distribution of 14 C2butachlor , 14 C2chlorpyrifos and 14 C2DDT in frog
After the chemicals were administrated to the frogs , they distributed to different organs or tissues. It
is found that the frogs had specific organs of accumulation for 14 C2butachlor , 14 C2chlorpyrifos and 14 C2
DDT respectively , gallbladder for 14 C2butachlor and intestine for 14 C2chlorpyrifos or 14 C2DDT( Table 1) .
It is noted that the 14 C2radioactivity in liver (326 BqΠg) was not the most one ,and it was only 018 % of
that of gallbladder (41541 BqΠg) . In frogs administrated per os with 14 C2butachlor , the radioactivity in
gallbladder was 720 times of that in bone. We consider that be related to the chemical character of buta2
chlor ———an amide compound. Except for gallbladder , intestine was also active in accumulating 14 C2
butachlor , but it only had 14 C2radioactivity of 4034 BqΠg , about 917 % of that of gallbladder. 14 C2buta2
chlor in intestine might be secreted from the gallbladder since the 14 C2radioactvity of stoma was 616 % of
that of intestine.
The accumulation of 14 C2chlorpyrifos in frog was different . The ratio of the highest radioactivity of
the organ (intestine) to the lowest (muscle) was 337. Except for that of intestine , the radioactivity of oth2
er organs varied between 39 BqΠg and 634 BqΠg.
14 C2DDT was mostly distributed to intestine and fat tissue after digested by frog. The 14 C2radioactivi2
ty in intestine and fat tissue were 136 and 102 times of that in body juice. And the radioactivity in liver
was 616 % of that in intestine. As DDT is a lipophilic compound , it is well distributed to the fat tissue.
However , it was found that bone accumulated so much of 14 C2DDT while muscle accumulated so little.
In the whole body , the 14 C2radioactivity of butachlor , chlorpyrifos and DDT were very low compared
to those of the most2accumulating organs.
Table 1 　Distribution of 14 C2butachlor ( BUT) , 14 C2chlorpyrifos( CHL) and 14 C2DDT in frog
(BqΠg - 1 )
body skin heart
gall
bladder liver lung stoma intestine spleen fat muscle bone
body
juice testis ovary
BUT 181 193 75 33187 326 135. 4 331. 4 6657. 8 110. 6 240 124 63 391 60 70
CHL 82 201 165 634 617 228 262 1459 202 283 39 71 276 100 69
DDT 297 262 223 1314 819 483 278 12364 326 9301 94 2000 91 703 329
212 　Degradation of 14 C2DDT in frog
Analysis of the metabolites of 14 C2DDT in frog of either sex showed that DDT was difficult to be de2
graded. After 24h , the percentage of DDT in liver of fat was higher , 5416 % and 8814 % , respectively.
Since liver is the digestive organ , the metabolites of DDT were higher than that in the fat tissue. Howev2
er , our previous researches on the metabolism of DDT in mud carp Cirrhina molitorella showed that those
parts on the application point of TLC (O point) and those unknown (except the standards) , were much
less than DDE.
671 核　农　学　报 16 卷
Table 2 　Percentages of metabolites of DDT in liver and fat tissue of frog
metabolites DDE DDMU DDT DDD Dicofols O2point Others
liver 219 3. 1 54. 6 17. 3 0. 2 14. 9 7. 0
fat 0. 7 1. 3 88. 4 4. 9 2. 6 0. 5 1. 5
213 　Extractable and bounded residues of 14 C2butachlor , 14 C2chlorpyrifos and 14 C2DDT in C2
molitorella
The results indicated that most of 14 C2butachlor , 14 C2chlorpyrifos and 14 C2DDT in liver , fat or ovary
was extractable with acetone , and there were some differences between the pesticides and between the or2
gans or tissues (Table 3) . It was found that the bound parts of these chemicals in liver were higher than
those in ovary or in fat tissue as liver is the organ metabolizing the pesticides. However , it was noted that
the bound 14 C2DDT in liver or in fat tissue was less than the bound 14 C2butachlor or 14 C2chlorpyrifos in
the same organ or tissue. The possible explanation is that DDT is more lipophilic than butachlor and
chlorpyrifos and it combined to the tissue with the weak bounds , as the percentage of 14 C2DDT in liver is
higher than that in fat tissue. This result is different from our study with mud carp . In mud carp , most
butachlor and chlorpyrifos in viscera was extractable with acetone[12 ] .
Table 3 　Extractable and bounded 14 C2butachlor ( BUT) , 14 C2chlorpyrifos ( CHL)
and 14 C2DDT in liver , fat tissue and ovary of frog
liver fat tissue ovary
extractable bounded extractable bounded extractable bounded
BUT 28. 1 71. 9 74. 4 25. 6 53. 5 46. 5
CHL 17. 6 82. 4 82. 0 18. 0 68. 3 31. 7
DDT 67. 6 32. 4 99. 6 0. 4
References :
[ 1 ] 　Alexandratos N. World agricultur towards 2000 , FAO. London :Rome and Belhaven Press , 1998
[ 2 ] 　Pan Daoyi . Apply the pesticides rationally and protect the frogs , Agricultural Sciences and Technology Communication 1986 , (6) :27
[ 3 ] 　Russell R W Hecnar S J . The ghost of pesticides past . Froglog. 1996 , (19) :1
[ 4 ] 　Hallidaay T. DAP wymposium , Born. Froglog. 1995 ; (14) :1
[ 5 ] 　Zhong Chuangguang , Zhao Xiaokui , Chen Shunhua , et al , Accumulation and metabolism of four 14C2labelled pesticides by some ma2
rine animals ; Tropical Oceanology 1998 ; 17 (4) :51～56
[ 6 ] 　Chen Shunhua , Zhong Chuangguang , Zhao Xiaokui ; Absorption , distribution dynamics of 14 C2chloropyrifos in several kinds of ani2
mals and plants in fresh water ; Acta Agriculturae Nucleatae Sinca 1998 ; 12 (5) :286～292
[ 7 ] 　Zhong Chuangguang , Carvalho F P. Zhang Yiqiang , et al . ; Accumulation and metabolism of 14 C2paration , 14 C2chloropyrifos and
14C2butachlor in Tilapia sp. ; Tropical Oceanology 1998 ; 17 (suppl) :11～31
[ 8 ] 　Zhong Chuangguang , Carvalho F P. Chen Shunhua , et al . ; Studies on the accumulation , distribution , and degradation of 14C2DDT
in marine Tilapia sp. ; Acta Scientiarum Naturalium Universitatis Sunyatseni 1997 ; 36 (suppl 3) :52～57
[ 9 ] 　Deneer J W. Uptake and elimination of chloropyrifos in the guppy at sub2lethal and lethal aquenous conditions ; Chomosphere 1993 :
26 (9) :1607～1617
[10 ] 　Zhang Fuying and Li Xingfu , Toxicology of HCH to the eggs and larva of rana and fish , Chinese Environmental Sciences 1998 , 7 (7) :
771　3 期 Behaviors of 14C2butachlor , 14C2chlorpyrifos and 14C2DDT in Rana japonica japonica Guenther
2[11 ] 　Pan Daoyi , Toxicology of herbicides to tadpole of bog frog Rana limnocharis , J Zoology , 1990 , 25 (1) :32～34
[12 ] 　Zhong Chuangguang , Zhang Yiqiang , Carvalho F. P. , et al . , Accumulation and metabolism of 14C2butachlor , 14C2chlorpyrifos and
14C2DDT in Cirrhina molitorella ; Acta Scientiarum Naturalium Universitatis Sunyatseni 1999. (in press)
14 C2丁草胺、14 C2毒死蜱和14 C2DDT在日本
林蛙中的生物学行为
张奕强1 　张海清2 　钟创光1 　赵小奎1 　陈舜华1
(1 广州市中山大学生命科学学院同位素研究室　广东 广州　510275 ;
2 广东省番禺市环境科学研究所 , 广东 番禺　511400)
摘要 :用同位素示踪技术研究了14 C2丁草胺、14 C2毒死蜱和14 C2DDT 在日本林蛙 ( Rana
japonica japonica Guenther) 中的生物学行为。结果发现 ,14 C2丁草胺、14 C2毒死蜱和14 C2
DDT在 24h 后分布到青蛙的各个器官组织 ,并分别以胆囊、小肠、小肠为它们的特异
性浓集器官。与胆囊或小肠的14 C放射性活度比较 ,其它器官组织中的要小得多。14
C2DDT在日本林蛙中较难降解 ,24h 后 DDT母体在肝和脂肪组织中占 DDT代谢物的
5416 %和 8814 %。青蛙中的14 C2丁草胺、14 C2毒死蜱和14 C2DDT 可被丙酮提取 ,但三者
之间以及在青蛙的器官之间有差异。
关键词 :14 C2丁草胺 ;14 C2毒死蜱 ;14 C2DDT;分布 ;代谢 ;日本林蛙 ( Rana japonica japonica
Guenther)
871 Acta Agriculturae Nucleatae Sinica
2002 ,16 (3) :174～178