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Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa
Available online at www.sciencedirect.com
Agricultural Sciences in China
2007,6(3): 316-321
ScienceDirect
March 2001
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa W E Xia-li, YU Xiao-qing, LIU Yong-hong and DONG Yuan-yan College of Basic Science, Huazhong Agricultural Universify, Wuhan 430070, P.R.China
Abstract To study the growth effects of differing concentrations of bensulfuron-methyl on Chlorella pyrenoidosa and to evaluate the ecological risk, the effects of bensulfuron-methyl on the growth and the content change of chlorophyll and protein in Chlorella pyrenoidosu were studied through 96 h acute toxicity tests. Bensulfuron-methyl accelerated the growth of algae at lower concentrations (< 1 mg L-1) with content increase of chlorophyll or protein, and it inhibited the growth of algae at higher concentrations (> 5 mg L-1). The content of chlorophyll or protein in algae cells reduced with the increasing concentration of bensulfuron-methyl, exhibiting the good concentration-effect relationship. The 96 h-EC,, of bensulfuronmethyl upon the algae was 15.7 mg L-1. Bensulfuron-methyl has inhibiting effect on the growth of Chlorella pyrenoidosa and is low in toxicity.
Key words: Bensulfuron-methyl, Chlorella pyrenoidosa, growth, chlorophyll, protein
INTRODUCTION As a primary producer of aquatic ecosystem, algae plays an important role for the balance and stabilization of the ecosystem, which is an important index for monitoring and evaluating water environmental quality (Sun and Huang 2003; Sheng and Zhou 2002; Wang H Y et al. 2004). It is significant to study the growth effects of different concentrations of bensulfuron-methyl on Chlorella pyrenoidosa to show how bensulfuron-methyl induces toxicity in Chlorella pyrenoidosa and appraise ecological risk of agricultural chemicals. It can offer warranty for ecological index of pesticide pollution (Su et al. 2005; Ou et a l . 2003b; Ma et al. 2000). Bensulfuron-methyl which was developed in the 1970s is a pesticide of high efficiency and lower toxicity. At present, it is used widely in the growth of rice, maize, rape, wheat, and barley because of its high activity, small amount, better selectivity, and lower toxicity (Wei
et al. 1999; Fan et al. 1999). With the development and application of sulfonylurea herbicide, the residue of sulfonylurea herbicide in soil and its effects on the following crops prevents its development (Fan el al. 1999). The research has chosen Chlorella pyrenoidosa existing in nature in large quantity as its focus, with bensulfuron-methyl approach to toxicity test of Chlorella pyrenoidosa, studied the growth increment of algae, chlorophyll content, and protein content. The possibility for algae to degrade sulfonylurea herbicide was also discussed.
MATERIALS AND METHODS Reagent and instruments Bensulfuron-methyl is offered by the Shanghai Pesticide Research Institute (the purity is 99.1 %), China. The apparatus and equipment are as follows:
This paper is translated from its Chinese version in Scienriu Agriculturu Sinica YUE Xia-li, Ph D. Associate Professor, Tel: +86-27-87288247, E-mail: [email protected]
02007.CAAS. All rights resewed Publishedby Elsevierltd
317
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa
microscope, hemocytometer measurement, counter, sterilizing pot, conical flask, visible spectrophotometer, and high-speed freezing centrifuge.
Biology and its cultivation Chlorellapyrenoidosa is offered by Hydrobiont Research Institute of Wuhan, China. Cultivation of algae: In asepsis condition, well-growing Chlorella pyrenoidosa has been chosen and inoculated in a 250 mL conical flask with SE culture media. In 25 r2"C and 4 000 lx, atmosphere was acceded to algae liquid and was cultivated.
distilled at room temperature and darkness for 1-2 h. In 15 min, under 3 000-4 000 r/min, top solution of centrifugal extracting solution was calibrated to 10 mL with alcohol (90%). In contrast to alcohol (90%), absorbance data of extracting solution was measured by color comparison dish with a diameter of 1 cm at 652 nm in wavelength. Concentration of chlorophyll (mg L-') = A652/34.5x 1 000 Chlorophyll content in algae (mg L-I) = (Chlorophyll concentration x cubage of extracting solution x extension rate)/cubage of sample
Measurement of protein content Measurement of growth number in algae (Wang Y Y and Wang C H 2006; Lang and Zhao 2001) After 24,48, 72, 96, and 120 h, density of algae cells was measured by hemocytometer. Absorbance of algae suspension (A68o)was measured in 680 nm and linear relationship was based between different density of algae cells and absorbance. Biomass of algae was counted by the density of algae cells and its absorbance and their connection was proved by linear relationship.
The research on the growth effects of different concentrations of bensulfuron-methyl on
Chlorellapyrenoidosa Six concentration groups and one blank control were used to measure acute toxicity of bensulfuron-methyl to algae. Absorbance of algae suspension sample was measured at an interval of 24 h. Concentration cost (inhibitory rate was 50%) was calculated by inhibitory rate (I) - concentration opposite digital quantity (1gC) 1 = [1- A sample 1A3 ,,,.
As specification curve with bovine serum albumin, admissible protein content of Chlorellapyrenoidosa was measured with Coomassie Brilliant Blue. Under 4 000 r/min, a few algae suspension was centrifuged for about 15 min, and the top solution was discarded. Five mL of distilled water was added, and the algae were transferred into mortar for 10 min under 4000 r/min, which was then abraded and centrifuged. The top solution was put into a calibration cuvette (10 mL) and distilled water (2 mL) was added into the residue, which was centrifuged for 10 min after suspension and was calibrated to calibration with top solution amalgamated. Protein content was measured by the solution. Protein content (mg L-') =' [Protein content which was measured (mg L-') x total volume of extracting solution (mL)]/volume of algae suspension (mL)
RESULTS Research on methods of measuring algae
x 1 00%
Measurement of chlorophyll content Under 50 kpa, algae suspension was filtered through a microporous membrane of 0.45 ym. The membrane was then cut and put into a glass mortar, with 7-8 mL of alcohol (90%) lapped and distilled chlorophyll. It was then transferred to calibration cuvette, and calibrated to 10 mL with alcohol (90%). The liquor was
Cell counting method is a basic method in measuring algae and is an effective basis in determining other methods. Fig. 1 shows better linear relationship between absorbance which was measured by spectral photometer and algae cell density by microscopic counting. The equation of linear regression is: A = 1 . 2 7 10-7C-0.0038, ~ R2=0.9973 Cell counting method is easy, but it requires considerable amount of work. The repeatability is not good
62007,CAAS. All fights resewed. Publishedby Elsevier Ltd.
YUE Xia-li et al.
318
and there is great influence from people and certain errors. With spectral photometer, standing crop of Chlorella pyrenoidosa was measured easily and quickly with no destruction, good repeatability, and few errors. This method is suitable for continuous measurement, which is easy and effective. Therefore spectral photometer will be chosen to measure the current volume of Chlorella pyrenoidosa in the following parts.
The growth effects of different concentrations of bensulfuron-methylon Chlorellapyrenoidosa Fig.2 shows the growth effects of different concentrations of bensulfuron-methyl on Chlorella pyrenoidosa.
In comparison with the control group, bensulfuronmethyl with light concentrations (c1 mg L-l) evidently promotes the growth of Chlorella pyrenoidosa. However, the high concentration can restrain its growth. There exists positive correlation between inhibition degree and medicament content, which shows clear correlation of content and effect. As shown in Fig.3, linear relationship is exhibited well between inhibition rates and lgC, which is graphed with 96 h-inhibition-ratesand 1gC. The linear regression formula is as follows:
I = 0.58541gC - 0.2008, R2= 0.9738 96 h median lethal concentration (96 h - EC,,) is 15.7 mg L-I. Referring to standards concerned, bensulfuron-methyl is lightly poisonous to Chlorella pyrenoidosa.
0 40
Effects of bensulfuron-methyl on chlorophyll content of Chlorellapyrenoidosa
0.31 0.30 0.25 0.20 0.15 (1.10
0.05
n 10
0
30
20
30
<'ell density (10: cell rnL I )
Fig. 1 Relationship between algae cell density and absorbance at 680 nm.
Ox
According to Fig.4, the chlorophyll content increases 24 h after bensulfuron-methyl is added. However, 48 h later, different contents of bensulfuron-methyl show different effects and function to Chlorella pyrenoidosa. Bensulfuron-methyl with low concentration induces the growth of chlorophyll and increases the content of the chlorophyll. However, bensulfuron-methyl with high concentration shows obvious restrained function and the content of chlorophyll decreases with increasing bensulfuron-methyl. This result is consistent with the findings in the experiment that algae suspension becomes more and more yellow and shows light yellow at the final stage.
-
e
"0
14
48
12
96
I20
Time ( h )
Fig. 2 Effect of bensulfuron-methyl on the growth of Chlorella pyrenoidosa.
0 ' 0
I
0.5
1
1.5
2
2.5
IgC
Fig. 3 Relationship between 96 h-inhibition-rates and IgC.
02037, CAAS. All rightsresewed.Publishedby Elsevierltd.
319
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa
Effects of bensulfuron-methylon soluble protein content of Chlorella pyrenoidosa Fig.5 shows the change in total protein content of Chlorella pyrenoidosa in 120 h. The change of protein content is not obvious in 48 h with the addition of the pesticide. Following this, clear relationship appears between the change in protein content and the amount of bensulfuron-methyl used. In contrast to the control group, increase in protein content of Chlorella pyrenoidosa will be brought with low concentration of bensulfuron-methyl; but reduction appears with high
1.6 1.2
0.8 0.4 0
24
0
4X
72
96
120
Time (h)
Fig. 4 Effect of bensulfuron-methyl on chlorophyll content of Chlorella pyrenoidosa.
0.0mgL-'
0
5.0mgL-'
-i- 0.50mg L-l
-6-
10.0 mg L '
+ 1.0 mg L-'
-+-
20.0mgL1
I
I
1
I
24
28
72
96
I
120
Time ( h )
Fig. 5 Effect of bensulfuron-methyl on protein content of ChloreZla pyrenoidosa.
concentration of bensulfuron-methyl. One of the reasons is probably that low concentration of bensulfuronmethyl boosts the growth of Chlorella pyrenoidosa and high concentration restrains it. Moreover, the protein content of algae cell is closely related to its growth state.
DISCUSSION The photosynthesis, breath, and cell division are involved in the algae's growth which would be counteracted if any of the three links is interfered or destroyed (Liu et al. 2006; Yan et al. 2002; Wang Y H et al. 2004). Thorough research on the toxicity mechanism of pesticide to the algae could be used more objectively to appraise the ecological risks of pesticide and the highly efficient and low toxic pesticide can be chosen conveniently. And this research is very significant to develop an environment-friendly pesticide (Nie et al. 2002; Li et al. 2005; Peng et al. 2001; Ou et al. 2003a). In the environment polluted by pesticide, algae adapts to the changed environment by changing its growth and pattern of synthesis of the protein (Qi 2005; Zhang et al. 2001). A small amount of bensulfuron-methylis added into the algae suspension and the addition inspires the growth of algae cell's chlorophyll, promotes photosynthesis, and makes pesticide become nutritious material to be absorbed and used to grow the algae and increase the content of the protein (Shen et al. 1999; Yan et al. 1999). However, when it exceeds the given range of concentration, which means beyond the range of self-regulation of algae, bensulfuron-methyl will gradually counteract the growth of algae along with the rising concentration of bensulfuron-methyl. These result in the destruction of the structure of the algae cells, algae suspension turns yellow, content of chlorophyll declines, and the synthesis of algae cell is restrained, which leads to the decrease in protein content. The results from the present research are the same with Ou et al. (2003a) and Nie et al. (2002). Therefore, the two functions, which are the toxicity to algae from the pesticide and degradation of algae to the pesticide, exist in the process of interaction between the algae and pesticide. When both their concentrations are in different ranges, their functions hold different dominant positions. Therefore, different effects and functions
02007,CAAS. All tights reserved. PublishedbyElsevier LM.
YUE Xia-li et al.
320
are exhibited (Liu et al. 1998). When the concentration of bensulfuron-methyl is lighter, the pesticide begins to degrade and is absorbed as the nutritious material by algae to promote algae’s growth; however, the poisonous effect of pesticide will take the leading role on algae, so that its growth is restrained. An economic and effective way to improve the ecological environment is provided by strengthening the research on mechanism of degradation of the algae and using the function of the degradation to pesticide.
(in Chinese) Lang B, Zhao X L. 2001. Study on the assessment of algaecide. Industrial Water Treatment, 21, 21-29. (in Chinese) Li X Q, Xu L G, Ma J Y. 2005. Combination toxicity of prometryne and permeating agent on Chlorella pyrenoidosa. China Environmental Science, 25,432-436. (in Chinese) Liu Q, Zhang X F, Li T W, Su X R. 2006. Effects of light on growth rate, chlorophyll level and cell cycle in four algae species. Journal of Dalian Fisheries University, 21, 24-30. (in Chinese) Liu Z, Tian S Z , Wong J H. 1998. Studies on positive growth response of Chlorella pyrenoidosa to low concentration Bis
CONCLUSIONS Different concentrations of bensulfuron-methyl have different function and effects on Chlorella pyrenoidosa. Light concentration of the bensulfuron-methyl (< 1 mg L-I)is capable of accelerating the growth of Chlorella pyrenoidosu and increases the content of chlorophyll and protein in the algae’s cell. And high concentrations of the bensulfuron-methyl (> 5 mg L-’)restrain the Chlorella pjirenoidosa’s growth and decrease the content of chlorophyll and protein accordingly. There is obvious measurement-effect relationship between bensulfuronmethyl and the growth of Chlorellu pyrenoidosa. 96 h - EC,, to Chlorella pyrenoidosa is 15.7 mg L-I, which means low noxious. Algae, an important factor influencing soil fertility in the farmland and a kind of basic source for fish in water is widely distributed in nature (Yan et al. 1999). And it takes an important position in the aquatic ecosystem. Because of the wide use of pesticides in the world, the algae’s survival, growth, and breeding is inevitably affected. Therefore, the research on the pesticide has a vital significance to poisoning symptoms and toxicity effect of algae; meanwhile, it is an important indicator to evaluate the security of pesticide to the environment.
Acknowledgements This work was financially supported by Natural Science Foundation of Hubei Province, China (4006066062).
References Fan Z J. Liu F M, Qian C F. 1999. The present situation and development of sulfonylurea herbicides. Pesticides, 38,6-9.
(2-Ethylhexyl) phosphoric acid. Environmental Chemistiy, 17,120-126. (in Chinese) Ma J Y, Chen J, Chai W G, Li M Z, Wu S G. 2000. Study on microscreening method to evaluate herbicidal activity using Chlorella pyrenoidosa. Chinese Journal of Pesticide Science, 2, 29-34. (in Chinese) Nie X P, Lan C Y, Lin L, Huang M H. 2002. The effects of aroclor 1254 on the growth of Chlorella pyrenoidosa and Scenedesmus obliquus. ACTA Scientiarum Naturalium Universitatis Sunyatseni, 41,68-71. (in Chinese) Ou X M, Lei M X, Huang M Z, Wang Y L, Wang X G, Fan D F. 2003a. Effects of novel sulfonylurea herbicide HNPC-(29908 on growth of green algae Chlorella pyrenoidosa. Chinese Journal of Pesticide Science, 5, 16-23. (in Chinese) Ou X M, Lei M X, Wang X G, Fan D F. 2003b. Accumulation and degradation of novel oxime insecticide HNPC-A9908 by Chlorella pyrenoidosa. China Environmental Science, 23, 475-479. (in Chinese) Peng J L, Yan G A, Shen G X, Deng X J. 2001. Effects of anaphthol on the glutathione and its reductase in Chlorella. Chinu Environmental Science, 21, 140-143. (in Chinese) Qi Q X. 2005. The influence of the copper sulfate on the growth of marine micro-algae. Journal of Aquaculture, 26, 11-1 3. (in Chinese) Shen G X, Yan G A, Peng J L, Yan X. 1999. Study on ecotoxicology for pesticides to algae II : Toxic mechanism and accumulation, degradation. Advances in Environmental Science, 7 , 131-139. (in Chinese) Sheng H, Zhou P J. 2002. Advance in the studies on effect of environmental organic pollutants on the algae growth. Acta Hydrobiologica Sinica, 26,529-535. (in Chinese) Su D, Tai P D, Li P J, Ke X. 2005. Toxic effects of lanthanides on Chlorella autotrophica. Chinese Journal of Ecology, 24,382384. (in Chinese) Sun H W, Huang G L. 2003. Mutual effects of algae and organic pollutants. Environmental Chemistry, 22, 440-444. (in Chinese)
02037,CAAS. All rights resewed. Publishedby ElsevierLtd.
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa Wang H Y, Cai M Y, Guo S Y. 2004. Microalgae and environmental monitoring. Environmental Science and Technology, 27,98102. (in Chinese) Wang Y H, Yuan J H, Ding F Y, Zhang J P, Tang L, Chen W. 2004. Toxicity effect of styrenen on four algae species. Journal of Shanghai N o m l Universily (Natural Sciences), 33,97-101. (in Chinese) Wang Y Y, Wang C H. 2006. Culture condition optimization of Chlorella sp. Journal of Yantai Universiby (Natural Science and Engineering Edition), 19, 125-129. (in Chinese) Wei D B, Zhang A Q, Han S Q, Wang L S. 1999. Advance in sulfonylureasherbicides research.Advances in Environmental
321
Science, 7,34-42. (in Chinese)
Yan G A, Shen G X, Yan X, Peng J L. 1999.Study on ecotoxicology for pesticides to algae 11: toxic effect. Advances in Environmental Science, 7,96-106. (in Chinese) Yan X, Yang Y Q, Li Y K, Sheng G X, Yan G A. 2002. Accumulation and biodegradation of anthracene by Chlorella protothecoides under different trophic conditions. Chinese Journal ofApplied Ecology, 13, 145-150. (in Chinese) Zhang W, Yan H, Wu Z L. 2001. Toxic effects of copper on inhibition of the growths of unicellular green algae. China Environmental Science, 21,2-7. (in Chinese)
(Edited by WANG Lu-han)
Q2007,CAAS.All tights resewed. PublishedbyElsevierLtd.
Agricultural Sciences in China
2007,6(3): 316-321
ScienceDirect
March 2001
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa W E Xia-li, YU Xiao-qing, LIU Yong-hong and DONG Yuan-yan College of Basic Science, Huazhong Agricultural Universify, Wuhan 430070, P.R.China
Abstract To study the growth effects of differing concentrations of bensulfuron-methyl on Chlorella pyrenoidosa and to evaluate the ecological risk, the effects of bensulfuron-methyl on the growth and the content change of chlorophyll and protein in Chlorella pyrenoidosu were studied through 96 h acute toxicity tests. Bensulfuron-methyl accelerated the growth of algae at lower concentrations (< 1 mg L-1) with content increase of chlorophyll or protein, and it inhibited the growth of algae at higher concentrations (> 5 mg L-1). The content of chlorophyll or protein in algae cells reduced with the increasing concentration of bensulfuron-methyl, exhibiting the good concentration-effect relationship. The 96 h-EC,, of bensulfuronmethyl upon the algae was 15.7 mg L-1. Bensulfuron-methyl has inhibiting effect on the growth of Chlorella pyrenoidosa and is low in toxicity.
Key words: Bensulfuron-methyl, Chlorella pyrenoidosa, growth, chlorophyll, protein
INTRODUCTION As a primary producer of aquatic ecosystem, algae plays an important role for the balance and stabilization of the ecosystem, which is an important index for monitoring and evaluating water environmental quality (Sun and Huang 2003; Sheng and Zhou 2002; Wang H Y et al. 2004). It is significant to study the growth effects of different concentrations of bensulfuron-methyl on Chlorella pyrenoidosa to show how bensulfuron-methyl induces toxicity in Chlorella pyrenoidosa and appraise ecological risk of agricultural chemicals. It can offer warranty for ecological index of pesticide pollution (Su et al. 2005; Ou et a l . 2003b; Ma et al. 2000). Bensulfuron-methyl which was developed in the 1970s is a pesticide of high efficiency and lower toxicity. At present, it is used widely in the growth of rice, maize, rape, wheat, and barley because of its high activity, small amount, better selectivity, and lower toxicity (Wei
et al. 1999; Fan et al. 1999). With the development and application of sulfonylurea herbicide, the residue of sulfonylurea herbicide in soil and its effects on the following crops prevents its development (Fan el al. 1999). The research has chosen Chlorella pyrenoidosa existing in nature in large quantity as its focus, with bensulfuron-methyl approach to toxicity test of Chlorella pyrenoidosa, studied the growth increment of algae, chlorophyll content, and protein content. The possibility for algae to degrade sulfonylurea herbicide was also discussed.
MATERIALS AND METHODS Reagent and instruments Bensulfuron-methyl is offered by the Shanghai Pesticide Research Institute (the purity is 99.1 %), China. The apparatus and equipment are as follows:
This paper is translated from its Chinese version in Scienriu Agriculturu Sinica YUE Xia-li, Ph D. Associate Professor, Tel: +86-27-87288247, E-mail: [email protected]
02007.CAAS. All rights resewed Publishedby Elsevierltd
317
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa
microscope, hemocytometer measurement, counter, sterilizing pot, conical flask, visible spectrophotometer, and high-speed freezing centrifuge.
Biology and its cultivation Chlorellapyrenoidosa is offered by Hydrobiont Research Institute of Wuhan, China. Cultivation of algae: In asepsis condition, well-growing Chlorella pyrenoidosa has been chosen and inoculated in a 250 mL conical flask with SE culture media. In 25 r2"C and 4 000 lx, atmosphere was acceded to algae liquid and was cultivated.
distilled at room temperature and darkness for 1-2 h. In 15 min, under 3 000-4 000 r/min, top solution of centrifugal extracting solution was calibrated to 10 mL with alcohol (90%). In contrast to alcohol (90%), absorbance data of extracting solution was measured by color comparison dish with a diameter of 1 cm at 652 nm in wavelength. Concentration of chlorophyll (mg L-') = A652/34.5x 1 000 Chlorophyll content in algae (mg L-I) = (Chlorophyll concentration x cubage of extracting solution x extension rate)/cubage of sample
Measurement of protein content Measurement of growth number in algae (Wang Y Y and Wang C H 2006; Lang and Zhao 2001) After 24,48, 72, 96, and 120 h, density of algae cells was measured by hemocytometer. Absorbance of algae suspension (A68o)was measured in 680 nm and linear relationship was based between different density of algae cells and absorbance. Biomass of algae was counted by the density of algae cells and its absorbance and their connection was proved by linear relationship.
The research on the growth effects of different concentrations of bensulfuron-methyl on
Chlorellapyrenoidosa Six concentration groups and one blank control were used to measure acute toxicity of bensulfuron-methyl to algae. Absorbance of algae suspension sample was measured at an interval of 24 h. Concentration cost (inhibitory rate was 50%) was calculated by inhibitory rate (I) - concentration opposite digital quantity (1gC) 1 = [1- A sample 1A3 ,,,.
As specification curve with bovine serum albumin, admissible protein content of Chlorellapyrenoidosa was measured with Coomassie Brilliant Blue. Under 4 000 r/min, a few algae suspension was centrifuged for about 15 min, and the top solution was discarded. Five mL of distilled water was added, and the algae were transferred into mortar for 10 min under 4000 r/min, which was then abraded and centrifuged. The top solution was put into a calibration cuvette (10 mL) and distilled water (2 mL) was added into the residue, which was centrifuged for 10 min after suspension and was calibrated to calibration with top solution amalgamated. Protein content was measured by the solution. Protein content (mg L-') =' [Protein content which was measured (mg L-') x total volume of extracting solution (mL)]/volume of algae suspension (mL)
RESULTS Research on methods of measuring algae
x 1 00%
Measurement of chlorophyll content Under 50 kpa, algae suspension was filtered through a microporous membrane of 0.45 ym. The membrane was then cut and put into a glass mortar, with 7-8 mL of alcohol (90%) lapped and distilled chlorophyll. It was then transferred to calibration cuvette, and calibrated to 10 mL with alcohol (90%). The liquor was
Cell counting method is a basic method in measuring algae and is an effective basis in determining other methods. Fig. 1 shows better linear relationship between absorbance which was measured by spectral photometer and algae cell density by microscopic counting. The equation of linear regression is: A = 1 . 2 7 10-7C-0.0038, ~ R2=0.9973 Cell counting method is easy, but it requires considerable amount of work. The repeatability is not good
62007,CAAS. All fights resewed. Publishedby Elsevier Ltd.
YUE Xia-li et al.
318
and there is great influence from people and certain errors. With spectral photometer, standing crop of Chlorella pyrenoidosa was measured easily and quickly with no destruction, good repeatability, and few errors. This method is suitable for continuous measurement, which is easy and effective. Therefore spectral photometer will be chosen to measure the current volume of Chlorella pyrenoidosa in the following parts.
The growth effects of different concentrations of bensulfuron-methylon Chlorellapyrenoidosa Fig.2 shows the growth effects of different concentrations of bensulfuron-methyl on Chlorella pyrenoidosa.
In comparison with the control group, bensulfuronmethyl with light concentrations (c1 mg L-l) evidently promotes the growth of Chlorella pyrenoidosa. However, the high concentration can restrain its growth. There exists positive correlation between inhibition degree and medicament content, which shows clear correlation of content and effect. As shown in Fig.3, linear relationship is exhibited well between inhibition rates and lgC, which is graphed with 96 h-inhibition-ratesand 1gC. The linear regression formula is as follows:
I = 0.58541gC - 0.2008, R2= 0.9738 96 h median lethal concentration (96 h - EC,,) is 15.7 mg L-I. Referring to standards concerned, bensulfuron-methyl is lightly poisonous to Chlorella pyrenoidosa.
0 40
Effects of bensulfuron-methyl on chlorophyll content of Chlorellapyrenoidosa
0.31 0.30 0.25 0.20 0.15 (1.10
0.05
n 10
0
30
20
30
<'ell density (10: cell rnL I )
Fig. 1 Relationship between algae cell density and absorbance at 680 nm.
Ox
According to Fig.4, the chlorophyll content increases 24 h after bensulfuron-methyl is added. However, 48 h later, different contents of bensulfuron-methyl show different effects and function to Chlorella pyrenoidosa. Bensulfuron-methyl with low concentration induces the growth of chlorophyll and increases the content of the chlorophyll. However, bensulfuron-methyl with high concentration shows obvious restrained function and the content of chlorophyll decreases with increasing bensulfuron-methyl. This result is consistent with the findings in the experiment that algae suspension becomes more and more yellow and shows light yellow at the final stage.
-
e
"0
14
48
12
96
I20
Time ( h )
Fig. 2 Effect of bensulfuron-methyl on the growth of Chlorella pyrenoidosa.
0 ' 0
I
0.5
1
1.5
2
2.5
IgC
Fig. 3 Relationship between 96 h-inhibition-rates and IgC.
02037, CAAS. All rightsresewed.Publishedby Elsevierltd.
319
Effect of Bensulfuron-Methyl on Growth of Chlorella pyrenoidosa
Effects of bensulfuron-methylon soluble protein content of Chlorella pyrenoidosa Fig.5 shows the change in total protein content of Chlorella pyrenoidosa in 120 h. The change of protein content is not obvious in 48 h with the addition of the pesticide. Following this, clear relationship appears between the change in protein content and the amount of bensulfuron-methyl used. In contrast to the control group, increase in protein content of Chlorella pyrenoidosa will be brought with low concentration of bensulfuron-methyl; but reduction appears with high
1.6 1.2
0.8 0.4 0
24
0
4X
72
96
120
Time (h)
Fig. 4 Effect of bensulfuron-methyl on chlorophyll content of Chlorella pyrenoidosa.
0.0mgL-'
0
5.0mgL-'
-i- 0.50mg L-l
-6-
10.0 mg L '
+ 1.0 mg L-'
-+-
20.0mgL1
I
I
1
I
24
28
72
96
I
120
Time ( h )
Fig. 5 Effect of bensulfuron-methyl on protein content of ChloreZla pyrenoidosa.
concentration of bensulfuron-methyl. One of the reasons is probably that low concentration of bensulfuronmethyl boosts the growth of Chlorella pyrenoidosa and high concentration restrains it. Moreover, the protein content of algae cell is closely related to its growth state.
DISCUSSION The photosynthesis, breath, and cell division are involved in the algae's growth which would be counteracted if any of the three links is interfered or destroyed (Liu et al. 2006; Yan et al. 2002; Wang Y H et al. 2004). Thorough research on the toxicity mechanism of pesticide to the algae could be used more objectively to appraise the ecological risks of pesticide and the highly efficient and low toxic pesticide can be chosen conveniently. And this research is very significant to develop an environment-friendly pesticide (Nie et al. 2002; Li et al. 2005; Peng et al. 2001; Ou et al. 2003a). In the environment polluted by pesticide, algae adapts to the changed environment by changing its growth and pattern of synthesis of the protein (Qi 2005; Zhang et al. 2001). A small amount of bensulfuron-methylis added into the algae suspension and the addition inspires the growth of algae cell's chlorophyll, promotes photosynthesis, and makes pesticide become nutritious material to be absorbed and used to grow the algae and increase the content of the protein (Shen et al. 1999; Yan et al. 1999). However, when it exceeds the given range of concentration, which means beyond the range of self-regulation of algae, bensulfuron-methyl will gradually counteract the growth of algae along with the rising concentration of bensulfuron-methyl. These result in the destruction of the structure of the algae cells, algae suspension turns yellow, content of chlorophyll declines, and the synthesis of algae cell is restrained, which leads to the decrease in protein content. The results from the present research are the same with Ou et al. (2003a) and Nie et al. (2002). Therefore, the two functions, which are the toxicity to algae from the pesticide and degradation of algae to the pesticide, exist in the process of interaction between the algae and pesticide. When both their concentrations are in different ranges, their functions hold different dominant positions. Therefore, different effects and functions
02007,CAAS. All tights reserved. PublishedbyElsevier LM.
YUE Xia-li et al.
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are exhibited (Liu et al. 1998). When the concentration of bensulfuron-methyl is lighter, the pesticide begins to degrade and is absorbed as the nutritious material by algae to promote algae’s growth; however, the poisonous effect of pesticide will take the leading role on algae, so that its growth is restrained. An economic and effective way to improve the ecological environment is provided by strengthening the research on mechanism of degradation of the algae and using the function of the degradation to pesticide.
(in Chinese) Lang B, Zhao X L. 2001. Study on the assessment of algaecide. Industrial Water Treatment, 21, 21-29. (in Chinese) Li X Q, Xu L G, Ma J Y. 2005. Combination toxicity of prometryne and permeating agent on Chlorella pyrenoidosa. China Environmental Science, 25,432-436. (in Chinese) Liu Q, Zhang X F, Li T W, Su X R. 2006. Effects of light on growth rate, chlorophyll level and cell cycle in four algae species. Journal of Dalian Fisheries University, 21, 24-30. (in Chinese) Liu Z, Tian S Z , Wong J H. 1998. Studies on positive growth response of Chlorella pyrenoidosa to low concentration Bis
CONCLUSIONS Different concentrations of bensulfuron-methyl have different function and effects on Chlorella pyrenoidosa. Light concentration of the bensulfuron-methyl (< 1 mg L-I)is capable of accelerating the growth of Chlorella pyrenoidosu and increases the content of chlorophyll and protein in the algae’s cell. And high concentrations of the bensulfuron-methyl (> 5 mg L-’)restrain the Chlorella pjirenoidosa’s growth and decrease the content of chlorophyll and protein accordingly. There is obvious measurement-effect relationship between bensulfuronmethyl and the growth of Chlorellu pyrenoidosa. 96 h - EC,, to Chlorella pyrenoidosa is 15.7 mg L-I, which means low noxious. Algae, an important factor influencing soil fertility in the farmland and a kind of basic source for fish in water is widely distributed in nature (Yan et al. 1999). And it takes an important position in the aquatic ecosystem. Because of the wide use of pesticides in the world, the algae’s survival, growth, and breeding is inevitably affected. Therefore, the research on the pesticide has a vital significance to poisoning symptoms and toxicity effect of algae; meanwhile, it is an important indicator to evaluate the security of pesticide to the environment.
Acknowledgements This work was financially supported by Natural Science Foundation of Hubei Province, China (4006066062).
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