全 文 ：Safety Evaluation of Myricetin and Crude Extract
from Myrica rubra Leaves on Non-target Organisms
LI Qiao1，XU Jing2，ZHANG Shao-yong1，ZHANG Xu1，CHEN An-liang1*
1． College of Forestry and Biotechnology，Zhejiang Forestry University，Lin＇an 311300，China;2． Environmental Monitoring Station in Longyou
County of Quzhou City，Zhejiang Province，Quzhou 324400，China
Abstract ［Objective］The study aimed to supply important basis for developing environment-friendly pesticides with myricetin and crude extract of
Myrica rubra leaves as effective components． ［Method］According to Test guidelines for environmental safety evaluation on chemical pesticides，
the toxicity of myricetin and crude extract of M． rubra leaves on non-target organisms was determined and the safety evaluation was carried out．
［Result］Myricetin and crude extract of M． rubra leaves had low toxicity on non-target organisms，such as earthworm，silkworm and soil microbes．
Myricetin showed low toxicity and the crude extract of M． rubra leaves showed middle toxicity on tadpole． The high-concentration crude extract of
M． rubra leaves had some antifeedant effect on silkworm． ［Conclusion］Myricetin and crude extract of M． rubra leaves had low toxicity on non-tar-
get organisms in environment and they were relatively safe．
Key words Myricetin;Crude extract of M． rubra leaves;Non-target organisms;Safety evaluation
Received:May 15，2010 Accepted:July 8，2010
Supported by Major Program in Zhejiang Science and Technology De-
partment (2008C22G2100038)＆ Emerging Artists Project in Zhejiang
Province (21000037)．
* Corresponding author． E-mail:liqiaoxinong@163． com
Crude extract of M． rubra leaves has good inhibition effect
on many plant pathogens，which has the potential to become
agricultural fungicide［1 －3］． To investigate its effects on non-tar-
get organisms，the author measured the toxicity of crude ex-
tracts of M． rubra leaves and myricetin on the beneficial organ-
isms such as earthworms，tadpoles，silkworm and soil microor-
ganism，etc． according to the relevant test standard require-
ments of safety evaluation on chemical pesticide，and conduc-
ted preliminary evaluation on their environment safety，in order
to provide important basis for development of environmental
friendly pesticides，the effective ingredient of which were crude
extracts of M． rubra leaves and myricetin．
1 Materials and Methods
1． 1 Tested agents Crude extracts of M． rubra leaves:
M． rubra leaves were collected in the campus of Zhejiang For-
estry College in October 2006，which were air dried，parched
and crushed at 45 ℃，sieved through 40 mesh (pore size 0. 37
mm) ，and extracted with acetone for 3 times using infusion
method，extraction time was 72，48 and 24 h，filtrate was put
together and concentrated to a concentration of 2． 0 × 103 g /L
(dry weight)． The above dry samples were dissolved with ace-
tone，added with appropriate emulsifier，and made into EC with
the dry sample content of 2 g /ml for standby．
Myricetin was purchased from Hangzhou Hetian Biotech-
nology Co．，Ltd．，purity was 98% (HPLC grade) ，myricetin
was dissolved with acetone，added with appropriate emulsifier，
and made into EC with the dry sample content of 1 g /ml for
standby．
1．2 Tested organism Earthworm (Eisenia fetida)was pur-
chased from Lin＇an earthworm farms，healthy earthworm more
than 3 months old with the weight of about 350 mg was chosen
as test organism．
Tadpole (Rana limnochris)which were newly hatched for
10 d were collected in the campus of Zhejiang Forestry College，
after procuring for 1 d in laboratory，healthy tadpole with the
same size were selected，which did not feed throughout the trial
period．
Soil organism was collected in the surface soil samples
with the depth of 5 －20 cm out of the east gate of Zhejiang For-
estry College，soil samples were air dried，and sieved through
2 mm mesh．
Silkworm，2nd instar was selected which was Baiyun × Xin-
hang variety．
1． 3 Test methods
1． 3． 1 Toxicity determination on tadpole． R． limnochris was
selected，and toxicity determination was conducted using quasi-
static method［4］． Tadpoles for test were indoor cultured for 1 d，
which did not feed during the test，test vessel was gold fish
tank，test water was running water which had been stored and
aeration treated for more than 24 h，pH value was 7 －8，DO
was more than 5． 0 mg/L，temperature was (22 ± 2)℃，L∶ D
= 12∶12． The concentration of crude extracts of M． rubra leav-
es were selected as 0． 5，1．0，1．5，2．0 and 2．5 mg/L accord-
ing to pre-test，the concentration of myricetin were 50，60，70，
80 and 100 mg/L，treatment without fungicide was selected as
control，each concentration had three repeats． Each repeat
had 10 tadpoles． The trial lasted for 4 d，fungicide liquid was
changed every 24 h，poisoning symptoms and death conditions
of tadpoles were observed and recorded，died tadpole were
taken out in time．
1． 3． 2 Toxicity determination on earthworm． Filter paper con-
Plant Diseases and Pests 2010，1(4) :46 －50
tact method and natural soil method was adopted［5］． The con-
centration range in the test was confirmed through pre-test，
then 5 concentrations in this range were set in proportion．
Filter paper contact method:five treatments of crude ex-
tracts of M． rubra leaves including 10，15，20，25 and 30 mg/L，
five treatments of myricetin including 100，200，400，800，
1 000 mg/L，clear water and solvent were set as control，each
concentration had three repeats with 10 earthworms in each re-
peat． Two layers of filter paper with the diameter of 12 cm were
inserted into petri dish with the diameter of 12 cm，10 ml corre-
sponding concentration of fungicide liquid were evenly sprayed
on the filter paper which were placed in a fume hood to make
solvent evaporate;earthworms which had been cleaned bowel
were washed out，dried moisture with filter paper，and inocula-
ted in petri dish． The filter paper which had been wetted with 5
ml water were inserted in the inner side of petri dish cover，then
the petri dish was covered and cultured in the artificial climate
chamber (T20 ℃，Φ(70% ±3%) ，L∶ D = 12∶ 12) ，poisoning
symptoms were observed every 24 h，mortality condition was cal-
culated，the observation was conducted continuously for 5 d．
Poisoned soil method:totally 11 treatments were set in the
test，including crude extracts of M． rubra leaves 100，200，
400，800 and 1 600 mg/L，myricetin 1 000，2 000，4 000，
6 000 and 8 000 mg/L，and water control． Each treatment had
three repeats． 50 ml fungicides with different concentrations
were mixed with 1． 0 kg soil，and the soil with fungicide was
prepared． The content of soil moisture should be controlled
round 60%，it was suitable when the soil was hold between the
fingers，there was water seeped，not dripped from fingers;pH
value was 6． 0 －8． 0．
The soil with fungicide was put into goldfish tank with 10
earthworms in each tank，nylon net was used to fasten the tank
mouth，and cultured in the intelligent artificial climate chamber
(22 ℃，Φ(65% ±3%) ) ，mortality rate was checked in the 1st，
3rd，7th and 14th days．
1． 3． 3 Toxicity determination on soil organism． Direct absorp-
tion of confined alkali method was used in the test［6］． 50 g sur-
face soil which had been air dried and sieved through 2 mm
mesh was taken out，added with 1 g glucose and mixed，
placed in 100 ml dry small beaker，and added with 5 ml water
for wetting，the beaker was placed in 2． 5 L dried wide-mouth
bottle，closely sealed，and cultured in the incubator with con-
sistent temperature of 25 ℃ for 7 d． Soil samples were added
with crude extracts of M． rubra leaves and myricetin，and made
the concentration of them be 1，10 and 100 mg/L in the soil，
respectively． The beaker contained 40 ml 0． 6 mol /L NaOH so-
lution was placed in wide-mouth bottle． NaOH solution was
separately taken out in 2，5，7，10，12 and 15 d，and fresh
NaOH solution was replaced，and continued to train． NaOH so-
lution which had been taken out was added with two drops of
phenolphthalein indicator and 5 ml 20% BaCl2 solution，and ti-
trated with 0． 4 mol /L HCl solution． CO2 release quantity in per
g of dry soil was calculated according to the consumption of HCl
solution．
V =(Blank value-titration value)×HCl molar concentration ×22． 4Dry soil weight × titration interval days
Note:V was CO2 release quantity in per g of dry soil;blank
value was 40 ml;dry soil weight was 50 g;HCl molar concen-
tration was 0． 4 mol /L．
1． 3． 4 Toxicity determination on silkworm． Poisoned leaf feed-
ing method was adopted［7］． The concentration of crude extracts
of M． rubra leaves were 10，40，80，160 and 320 mg/L，the
concentration of myricetin were 100，200，400，800 and 1 600
mg/L，each treatment had three repeats，water and solvent
was set as control． Clean fresh mulberry leaves were selected，
hole puncher with the diameter of 1 cm was used to punched
leaf discs，leaf discs was immersed in the above tested fungi-
cides for 30 s，and placed in the clean counter for natural dr-
ying． Leaf discs was placed in the petri dish with filter paper，
each dish had 15 leaves． 2nd early instar silkworm larvae which
had been starved for 4 h were selected，placed in the petri
dish，added with leaf discs，and placed in artificial climate
chamber (T(25 ± 1)℃，Φ65%)for cultivation，each treat-
ment had 10 heads． Normal leaves were used to feed after 24
h， toxicity symptoms were observed， mortality rate was
checked after 24 and 48 h．
1． 4 Data process and analysis Excel was used to make
toxicity regression curve equation according to reference［8］，
LC50 and 95% confidence limit were calculated．
2 Results and Analysis
2． 1 Effect of crude extracts of M． rubra leaves and myr-
icetin on tadpole Acute toxicity results of crude extracts of
M． rubra leaves and myricetin on tadpole were shown in Table
1． When tadpoles were placed in the water which had been
sprayed fungicide，they rotated and moved forward along helix
line，but their action were obtuse and movement were slow，
their moving lost balance，showing the action such as side
crouching，floatation and segmental venter upwards． When
tadpoles died，the abdomen near the base turned white，viscer-
a was visible，two gills were swollen and black，skin markings
were slightly blurred than normal tadpoles，body abdomen was
swollen，black，legs were drooped，and tail was curled．
As shown in Table 1，LC50 of crude extracts of M． rubra
leaves at 24 and 48 h separately were 1． 78 and 1． 44 mg/L;
LC50 of myricetin at 24 and 48 h separately were 63． 46 and
57. 31 mg/L． According to the proposed standard in Test
guidelines for environmental safety evaluation on chemical pes-
ticides，LC50 ＜1 mg/L was high-toxic pesticide，1 －10 mg/L
was middle-toxic pesticide，＞ 10 mg/L was low toxicity pesti-
cide［9］． The acute toxicity of crude extracts of M． rubra leaves
on tadpoles showed middle toxicity，myricetin showed low toxic-
ity，the toxicity of crude extracts of M． rubra leaves and myrice-
tin on tadpoles increased as the contact time increase． Howev-
er，all toxicity values were higher than the standard value，
which could consider to be basically safety to aquatic organisms
under the right concentration．
2． 2 Toxicity of crude extracts of M． rubra leaves and myr-
icetin on earthworm When earthworm contacted with the fil-
ter paper that had been sprayed with high concentrations of
crude extracts of M． rubra leaves，the body continued to distort
74LI Qiao et al． Safety Evaluation of Myricetin and Crude Extract from Myrica rubra Leaves on Non-target Organisms
and struggle，losing the ability of crawl and escaping，body sur-
face of some earthworms leaked some yellow fluid，the bodies
were longer，while earthworms in lower concentration group
had slight phenomenon． After contamination for 24 h at 20 ℃，
twist state of earthworms was more relax compared with the ini-
tial state，the bodies was stiffened and black，and showed
slight twist state，which had strong response to stimulation，
segment began to appear the phenomenon such as swelling
and erosion，some back produced small swollen bubble，when
back or small swollen bubble were stabbed，yellow pus-like fluid
flew out． Annular zone of some earthworms at 48 h appeared
swollen erosion，the other parts shrank，bloody pus-like fluid
appeared in the filter paper． After earthworm died，the body
sections were completely broken，large amount of yellow pus-
like liquid appeared in the filter paper，some decayed and emit-
ted stench． The symptoms in myricetin treatment were similar
but relatively slight． In the high concentration group of crude
extracts of M． rubra leaves，earthworm action was slow，which
did not drill into the soil immediately，the body of died earth-
worm ulcerated，showing bead like． The symptoms of myricetin
group was similar．
Table 1 Acute toxicity of crude extracts of M． rubra leaves and myricetin on tadpole
Fungicide Time∥h Regression equation Correlation coefficient LC50∥mg/L 95% confidence limit
Crude extracts of M． rubra leaves 24 Y = 2．83X + 4．29 0． 968 1． 78 1． 58 －2． 00
48 Y = 2．97X + 4．53 0． 961 1． 44 1． 31 －1． 59
Myricetin 24 Y = 6．89X － 7．41 0． 99 63． 46 60． 69 －66． 35
48 Y = 6．99X － 7．29 0． 98 57． 31 54． 18 －60． 62
As shown in Table 2，LC50 of crude extracts of M． rubra
leaves in filter paper contact test at 24 and 48 h separately were
17． 41 and 15．39 mg/L，while LC50 of myricetin at 24 and 48 h
fseparately were 364． 11 and 180． 54 mg/L;LC50 of crude ex-
tracts of M． rubra leaves in poisoned soil test at 7 and 14 d
separately were 2 129．88 and 476．69 mg/L，while LC50 of myr-
icetin at 7 and 14 d separately were 8 705． 32 and 5 561．19
mg/L． Current classification of pesticide toxicity on earthworms
adopted the proposed standard in Test guidelines for environ-
mental safety evaluation on chemical pesticides，LC50 ＜1 mg/
L was high-toxic pesticide，1 －10 mg/L was middle-toxic pesti-
cide，＞10 mg/L was low toxicity pesticide［9］． LC50 of crude ex-
tracts of M． rubra leaves and myricetin in petri dish and soil
were all greater than 10 mg/L． When the exposure time was
longer，LC50 was smaller，that was toxicity was greater． There-
fore，the main component of two new fungicides was safe to
earthworm，when they were developed for popularized applica-
tion in farmland．
Table 2 Toxicity of crude extracts of M． rubra leaves and myricetin on earthworm
Fungicide Method Time Regression equation Correlation coefficient LC50∥mg/L 95% confidence limit
Crude extracts of Filter paper contact method 24 h Y = 3． 52X + 0． 63 0． 99 17． 41 16． 10 －18． 81
M． rubra leaves 48 h Y = 3． 84X + 0． 44 0． 96 15． 39 14． 19 －16． 69
Poisoned soil method 7 d Y = 0． 93X + 1． 73 0． 98 2 129． 88 1 271． 87 －3 566． 72
14 d Y = 1． 38X + 1． 31 0． 98 476． 69 390． 54 －581． 86
Myricetin Filter paper contact method 24 h Y = 1． 67X + 0． 73 0． 99 364． 11 309． 47 －428． 41
48 h Y = 2． 58X － 0． 814 0． 96 180． 54 154． 28 －211． 26
Poisoned soil method 7 d Y = 1． 45X － 0． 71 0． 99 8 705． 32 6 278． 40 －12 070． 37
14 d Y = 1． 58X － 0． 93 0． 98 5 561． 19 4 494． 39 －6 882． 75
2． 3 Toxicity of crude extracts of M． rubra leaves and myr-
icetin on soil organism As shown in Fig． 1 and Fig． 2，
throughout the determination period，CO2 release quantity in
the soil containing crude extracts of M． rubra leaves and myr-
icetin were greater than control． CO2 release quantity was rela-
tively greater in the second day adding with fungicide，subse-
quently decreased，which began to increase in the fifth day，
and reached the maximum in the 7th day． CO2 release quantity
significantly reduced from the 7th day，which reached relative
lower value and became stable in the 10th day，CO2 release
quantity declined again in the 12th day，reaching the lowest val-
ue during the whole determination period． Variance analysis
was conducted between the different treatments of crude ex-
tracts of M． rubra leaves and myricetin，the results showed that
CO2 quantity exhaled by soil organisms in the different treat-
ments of crude extracts of M． rubra leaves and myricetin had
no significant difference，CO2 quantity exhaled by organisms in
the different treatments of crude extracts of M． rubra leaves
and myricetin also had no significant difference，while crude ex-
tracts of M． rubra leaves had stronger activation effect on soil
organisms． The results showed that crude extracts of M． rubra
leaves and myricetin had no inhibition effect on soil organism，
at the same time，they had certain activation effect，activation
effect had no significant difference in the concentration range of
1 －100 mg/L，indicating that different concentrations of crude
extracts of M． rubra leaves and myricetin had no adverse
effects on the normal respiration of soil microorganism．
2． 4 Toxicity of crude extracts of M． rubra leaves and myr-
icetin on silkworm When leaf discs with high concentrations
84 Plant Diseases and Pests 2010
Fig． 1 Effects of crude extracts of M． rubra leaves on the
respiration of soil microbe
Fig． 2 Effects of Myrecetin on the respiration of soil mi-
crobe
were added，silkworm immediately appeared head rising phe-
nomenon，while the symptoms were not obvious in the low con-
centration group． Silkworm showed certain antifeedant phe-
nomena to the treated leaf discs with high concentrations after
48 h，antifeedant degree showed direct ratio with fungicide con-
centration． No individual died，all could normally molt．
Table 3 Toxicity determination results of crude extracts of
M． rubra leaves and myricetin on silkworm(48 h)
Fungicide
Treatment
concentration
mg/L
48 h eating leaf
area∥cm2
Antifeedant
percentage
%
Crude extracts of 10 35． 33 0 e
M． rubra leaves 40 33． 76 4． 4 d
80 28． 65 18． 9 c
160 23． 16 34． 4 b
320 19． 63 44． 4 a
Myricetin 100 34． 54 2． 2 d
200 33． 91 4． 0 c
400 33． 76 4． 4 bc
800 33． 36 5． 6 b
1 600 32． 19 8． 9 a
Ck 35． 33 －
Note:Ducan method was adopted to analyze the antifeedant percent-
age，the same letters stood for no difference among treatments．
Results showed that no death appeared when silkworm
consumed the mulberry leaves for 48 h which had been im-
mersed with different concentrations of myricetin，indicating that
crude extracts of M． rubra leaves and myricetin had no toxicity
activation on silkworm． However，higher concentrations of liq-
uid had certain antifeedant effect on silkworm，which enhanced
with the concentration increase (Table 3)． Compared with
crude extracts of M． rubra leaves，myricetin had no significant
antifeedant effect on silkworm，while the antifeedant effect be-
tween different concentrations was not significant． Therefore，it
could be considered that crude extracts of M． rubra leaves and
myricetin were safe to silkworm．
3 Conclusions and Discussion
Acute toxicity of crude extracts of M． rubra leaves and
myricetin on environmental organisms such as tadpoles，earth-
worms，soil microbes and silkworm were determined using in-
door research method，and the result showed that acute toxicity
of crude extracts of M． rubra leaves on tadpole was middle tox-
icity，acute toxicity of myricetin on tadpole was low toxicity，
crude extracts of M． rubra leaves had certain toxicity on aquatic
organism． Crude extracts of M． rubra leaves and myricetin had
low toxicity on earthworms，which had no inhibition effect on the
respiration of soil microorganisms，instead certain activation
effect existed，indicating that tested compounds could be uti-
lized as carbon and nitrogen sources by soil microorganism af-
ter being added to the soil，and accelerated the growth of mi-
croorganisms． Crude extracts of M． rubra leaves and myricetin
had extremely low toxicity on silkworm． The results showed that
crude extracts of M． rubra leaves had certain toxicity on aquatic
organism，crude extracts of M． rubra leaves and myricetin had
low or no real toxicity on the other environmental organisms．
Crude extracts of M． rubra leaves showed high activity on the
various pathogens， being safety to environmental organ-
isms［1 －2，10 －12］． Crude extracts of M． rubra leaves could be a-
dopted as the main component to develop new types of agricul-
tural fungicide varieties with good environmental compatibility．
Myricetin is the main active ingredient in crude extracts of
M． rubra leaves，in which the content is about 6%［7］． Com-
pared with the toxicity data of crude extracts of M． rubra leaves
and myricetin on tadpole and earthworm，LC50 value of crude
extracts of M． rubra leaves was significantly lower than LC50
value of myricetin，indicating that there was compound with
high activity in crude extracts of M． rubra leaves．
Bio-pesticide products generally appear in the form of mix-
ture，in the evaluation of its environmental safety，the mixture
should be taken as the primary research material，while envi-
ronmental safety of main active ingredient could not instead of
product safety evaluation．
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杨梅素及杨梅叶粗提物对非靶标生物的安全性评价
李 桥1，徐 静2，张绍勇1，张 旭1，陈安良1*
(1．浙江林学院林业与生物技术学院，浙江临安 311300;2．浙江省衢州市龙游县环境监测站，浙江衢州 324400)
摘要 ［目的］为开发以杨梅素及杨梅叶粗提物为有效成分的环境友好型农药提供重要依据。［方法］参照“化学农药环境安全评价试验准则”，
测定杨梅素及杨梅叶粗提物对非靶标生物的毒性，并进行安全性评价。［结果］杨梅素和杨梅叶粗提物对蚯蚓、家蚕和土壤微生物等非靶标生
物均为低毒;杨梅素对蝌蚪表现为低毒，杨梅叶粗提物则表现为中毒，高浓度的杨梅叶粗提物对家蚕有一定拒食作用。［结论］杨梅素及杨梅叶
粗提物对环境非靶标生物毒性低，较为安全。
关键词 杨梅素;杨梅叶粗提物;非靶标生物;安全性评价
基金项目 浙江省科技厅面上项目(2008C22G2100038);浙江省新苗计划项目(21000037)。
作者简介 李桥(1985 － )，男，宁夏西吉人，硕士研究生，研究方向:生物农药及农药的环境毒理学。* 通讯作者。
收稿日期 2010-05-15 修回日期
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2010-07-08
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春小麦蚜虫空间分布型及抽样技术研究
任月萍，贺达汉 (宁夏大学农学院，宁夏银川 750021)
摘要 ［目的］研究春小麦蚜虫种群的空间分布型和田间抽样方法。［方法］统计试验地小麦上的蚜虫数量，采用聚集度指标法计算小麦蚜虫
的田间分布型，分析聚集的原因，确定田间理论抽样数。［结果］小麦蚜虫为聚集分布并呈负二项分布型，小麦蚜虫聚集分布是由本身的习性和
环境因素共同作用引起的;小麦蚜虫在田间的理论抽样数和样本方差、允许误差有关;样本方差(S2)愈小，允许误差(d)愈小，无放回理论抽样
数愈多;虫口基数不同，理论抽样数不同;当允许误差设定后，样本方差(S2)越大，理论抽样数越大。［结论］该研究为小麦蚜虫的预测预报和田
间防治提供了科学依据。
关键词 小麦蚜虫;空间分布型;理论抽样数
基金项目 国家自然科学基金项目(30660017)。
作者简介 任月萍(1958 － )，女，宁夏中宁人，教授，从事植物保护研究。
收稿日期 2010-05-01 修回日期 2010-08-09
05 Plant Diseases and Pests 2010