Ecotoxicology and Environmental Safety 52, 57–61 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2146, available online at http://www/idealibrary.com on
Di¡erential Sensitivity of Two Green Algae, Scenedesmus obliqnus and Chlorella pyrenoidosa, to 12 Pesticides Jianyi Ma,*,1 Rongquan Zheng,w Ligen Xu,z and Shufeng Wangz *Department of Resource and Environment, Zhejiang Forestry College, Linan 311300, People’s Republic of China; wCollege of Life Sciences, Zhejiang Normal University, Jinhua 321004, People’s Republic of China; and zCollege of Life Sciences, Zhejiang University, Hangzhou 310012, People’s Republic of China Received June 28, 2001
the environment with the explicit intention of exerting toxic eﬀects on one or more target organisms. Unfortunately, their toxicity is usually not limited to the location where they are applied. They reach other locations and environmental compartments through various physical transport processes, adversely aﬀecting organisms that happen to be present (Deneer, 2000). Moreover, their increased usage has elicited extensive research into pesticide eﬀects on nontarget organisms. Pesticides such as ﬂungicides appear to have a great potential for control of a wide range of fungal pests. However, there is not published information regarding their eﬀects on pure cultures of algae. Little is known about the sensitivity of algae to toxicants. Algae composing the primary producer level are of initial importance in providing the energy that sustains invertebrates and ﬁsh in most aquatic ecosystems. The action of toxic substances on algae is therefore not only important for the organisms themselves, but also for other links in the food chain. Algal toxicity tests are increasingly being used in bioassay test batteries for environmental management of chemical discharges and it has been observed in several studies that for a large variety of chemical substance algal tests are relatively sensitive bioassay tools. However, there have been few attempts to evaluate the relative vulnerability of algae to toxic stress with respect to that of other aquatic organisms. Tests on single species of algae are therefore of limited applicability in assessing the eﬀects of environmental contaminants on algal communities that are composed of an array of species with diﬀerent sensitivities. Algae species vary widely in their response to toxic chemicals (Boyle, 1984). A few reports have been published on the comparative toxicity of herbicides toward various test organisms (Abou-Waly et al., 1991; Ma and Liang, 2001; Ma, 2001). Relatively few reports have been involved with diﬀerential response of various algal species to fungicides and acaricides (Faust
Growth-inhibiting tests were carried out for 12 pesticides (including 11 fungicides: fosetyl-aluminum, benomyl, metalaxyl, iprodione, dimetachlone, carbendazim, thiophanate-methyl, bismerthiazol, procymidone, zineb, chlorothalonil, and the acaricide abamectin) in the green algae Chlorella pyrenoidosa and Scenedesmus obliqnus and the diﬀerential sensitivities of the two green algae to those pesticides were compared. The results indicate that the acute toxicity of benomyl to C. pyrenoidosa and S. obliqnus is the highest among all of the pesticides tested and is close to that of the photosynthesis-inhibiting herbicides atrazine, simazine, and chlorotoluron. Meanwhile, algal species vary widely in their response to the pesticides. The results demonstrated that there was a diﬀerential response to various pesticides by the two species of algae and that the sensitivity of various species of algae exposed to chlorothalonil varied by nearly two orders of magnitude; sensitivity to thiophanatemethyl varied by more than one order. Investigations using diﬀerent algal species as test organisms have demonstrated that algae vary greatly in their response to chemicals. Diﬀerential sensitivity of green species to the compounds could induce species shifts within communities. # 2002 Elsevier Science (USA) Key Words: Fungicides; acaricides; ecotoxicity; aquatic toxicity; algal sensitivity; Chlorella pyrenoidosa; Scenedesmus obliqnus.
Today environmental problems are multiple and complex, especially those arising from the disposal of identiﬁcation and the assessment of the toxicity of such substances. Assessment of human exposure to toxicants through biological monitoring oﬀers one means to evaluate the magnitude of the potential health risk of these chemicals (Ma et al., 2001). Pesticides diﬀer from most industrial organic chemicals in that they are brought into 1
To whom correspondence should be addressed. E-mail: [email protected]
0147-6513/02 $35.00 r 2002 Elsevier Science (USA) All rights reserved.
MA ET AL.
et al., 2000; Ferrando et al., 1996). For purposes of comparing diﬀerential sensitivity among populations of green algae, set of acute toxicity tests have been devised. The work reported here was done to examine the eﬀects of 11 fungicides and 1 acaricide that have been widely used in China on the green algae Chlorella pyrenoidosa and Scenedesmus obliqnus and to compare diﬀerential sensitivity among populations of these two green algae to those pesticides. MATERIALS AND METHODS
Chemicals. All of the fungicides and insecticides tested were purchased in the people’s Republic of China and their chemical structures are given in Table 1. The pesticides tested were dissolved with a little acetone or distilled water. The concentration of solvent in medium was kept at a minimum due to tested pesticid’s solubility. The concentration of solvent in medium was less than 0.05%. The U.S. Environmental Protection Agency recommends maximum allowable limits of 0.05% solvent for acute tests and 0.01% for chronic tests (Jay, 1996), this level has no signiﬁcant eﬀect on the data of toxicity (Ma and Liang, 2001). Test organisms. The Chinese National Environment Protection Agency recommends the green algae Chlorella pyrenoidosa and Scenedesmus obliqnus for acute tests as ecological indicators because of their high sensitivity to the compounds (Chinese NEPA, 1990). The toxicity tests were
carried out with freshwater unicellular green algae C. pyrenoidosa and S. obliqnus obtained from the Institute of Wuhan Hydrobiology, Chinese Academic of Science. The algae were kept on agar slants at approximately 41C. Nutrient media. The medium for the algal growth inhibition test with C. pyrenoidosa or S. obliqnus was prepared in accordance with Chinese National Environmental Protection Agency Guidelines 201 (Chinese NEPA, 1990), using HB-4 medium composed of distilled water and the following chemical ingredients (mg/liter): (NH4)2SO4 (200), Ca(H2PO4)2?H2O+(CaSO4?H2O)(30), MgSO4? 7H2O,NaHCO3 (100), KCL(23), FeCl3(1.5), and A5 liquid (0.5 ml, chemical ingredients of A5 liquid are H3BO3 2.86, MnCl2?4H2O 1.81, ZnSO4 7H2O 0.222, Na2MoO4?2H2O 0.0391, CUSO4?5H2O 0.079 g/liter) (Ma et al., 2001). The culture medium was sterilized at 1211C, 1.05 kg cm2 for 30 min (Kong et al., 1999). Test methods. Single cells of algae were propagated photoautotrophically in a 250-ml Erlenmeyer ﬂask containing 100 ml liquid, kept on a rotator shaker (100 rpm) at 251C, and illuminated with cool-white ﬂuorescent lights at a continuous light intensity of 5000 lx. For cell experiments, 15-ml aliquots of the HB-4 medium containing single algal cells (initial spectrophotometric data: OD680 nm=0.050) were distributed to sterile 50-ml Erlenmeyer ﬂasks. The media for C. pyrenoidosa and S. obliqnus were then treated with various concentrations of pesticides ranging from zero to 150 mg/liter, and incubated for 96 h
TABLE 1 Selected Herbicides and Chemical Structures Fungicide
Fosetyl-aluminium 90% Benomyl 25% Thiophanate-methyl 70% Metalaxyl 25% Iprodione 95%
TC WP WP WP TC
Distilled water Distilled water Distilled water Distilled water Acetone o0.05%
Dimetachlone Bismerthiazol Carbendazim Procymidone
40% 20% 50% 50%
WP WP WP WP
Distilled Distilled Distilled Distilled
Zineb Chlorothalonil Abamectin
80% WP 30% SC 95% TC
water water water water
Distilled water Distilled water Acetone o0.05% o0.05%
Chemical structure Ethyl hydrogen phosphonate Methyl-l-(butylcarbamoyl)-2-benzimidazole carbamate Dimethyl 4,40 -(o-phenylente) bis(3-thioa(lophanate) Methyl-(2-methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate 3-(3,5-Dichlorophenyl)-N-isopropyl-2,4-dioxoimidazolidine-lcarboxamide N-(3,5-Dichlorophenyl)succinidide N,N0 -Methylene-di(2-amino-5-mercapto-1, 3,4-thiodiazole Methylbenzimidazol-zyl-carbamate N-(3,5-Dichlorophenyl)-1,2-dimethylcyclopropane-1,2dicarboximide Zinc ethylene bis(dithiocarbamate) Tetrachloroisophthalonitrile Mixtureb
CAS Registry No. 15845-66-6 17804–35–2 23564-05-8 57837-19-1 36734-19-7 24096-53-5 52316-55-9 32809-16-8 12122-67-7 1897-45-6 71751-41-2
TC, technical product; WP, wettable powder; SC, suspension concentrate. b(10E, 14E, 16E, 22Z)-( 1R, 4S, 50 S, 6S, 60 R, 8R, 12S, 13S, 20R, 21R, 24S)6 -[(S)-sec-butyl]-21,24-dihydroxy-50 ,11,13,22-tetramethyl-2-oxo-(3,7,19-trioxatetracyclo[126.96.36.199,8.020,24]pentacosa-10,14,16,22-tetraene)-6-spiro-20 (50 ,60 -dihydro-20 H-pyran)-12-yl 2,6-dideoxy-4-O-(2,6-dideoxy-3-O-methyl-a-L-arabino-hexopyranosyl)-3-O-methyl-a-L-arabino-hexopyranoside and (10E, 14E, 16E, 22Z)-(1R, 4S, 50 S, 6S, 60 R, 8R, 12S, 13S, 20R, 21R, 24S)-21,22-dihydroxy-60 -isopropyl-50 ,11,13,22-tetramethy1-2-oxo-(3,7,192,6-dideoxy-4-O-(2,6-dideoxy-3-O-methyltrioxatetracyclo[188.8.131.52,8.020,24]pentacosa-10,14,16,22-tetraene)-6-spiro-20 -(50 ,60 -dihydro-20 H-pyran)-12-yl a-L-arabino-hexopyranosyl)-3-O-methyl-a-L-arabino-hexopyranoside. 0
SENSITIVITY OF S. Oblignus AND C. Pyrenoidosa TO 12 PESTICIDES
on an orbital shaker (100 rpm) at 251C with a continuous light intensity of 5000 l (Wijk et al., 1998; Oliveira-Filho and Paumgartten, 2000). A wide range concentrations were developed in a previous test to determine the adequate range of toxicity for each pesticides. Then, similar test doses were tested according to the previous test (MorenoGarrido et al., 2000). Cell counts were correlated with absorbance over time for 96 h on a Shimadzu UV-2401PC spectrophotometer. The most suitable wavelength to use for monitoring culture growth was 680 nm; Kasai (1993) reported that cell numbers and OD680 nm were highly correlated. In our previous work (Ma et al., 2001; Ma and Liang, 2001), a strong conﬁrmed in the experiment, with coeﬃcient of correlation r values40.99 and signiﬁcance level Po0.01 for C. pyrenoidosa or S. obliqnus tested. Thus, growth of algal cells was calculated indirectly using spectrophotometric data in this work. Each pesticide concentration was tested in triplicate. Appropriate control systems containing no pesticide were included in each experiment. Control and treated cultures were grown under the same conditions of temperature, photoperiod, and shaking of the stock cultures. In each experiment, percentage inhibition values, relative to growth in control systems, were calculated using spectrophotometric data (Ma et al., 2001).
Statistical evaluation. For growth inhibition tests with the green algae, EC50 values (pesticide concentration required to cause 50% reduction in growth) were computed. EC50 values were calculated using linear regression analysis of transformed pesticide concentration as natural logarithm data versus percentage inhibtion (Ma et al., 2001). All correlation coeﬃcients were 40.90. RESULTS
Acute toxicity of 12 fungicides and acaricides to the green algae C. pyrenoidosa and S. obliqnus is summarized in Table 2. The 96-h EC50 values of fosetyl-aluminum varied around 6–35 mg/liter (105 M), metalaxyl varied around 70–22 mg/liter (105 M), iprodione varied around 6–42 mg/ liter (104–105 M), dimetachlone varied around 6–52 mg/ liter (104–105 M), and cerbendazim varied around 19– 35 mg/liter (104–105 M). The average acute toxicity of those fungicides to the green algae at the midpoint was of the pesticides tested. The 96-h EC50 values of thiophanatemethyl varied around 5–138 mg/liter (104–105 M); the acute toxicity of thiophanate-methyl was the lowest among the pesticides tested. The 96 h–EC50 values of bismerthiazol varied around 0.8–4.1 mg/liter (105–106 M), procymidone varied around 0.6–0.8 mg/liter (106 M),
TABLE 2 Diﬀerential Sensitivity of Two Green Algae, Scenedesmus obliqnus (2) and Chlorella pyrenoidosa (1), to 12 Fungicides and Insecticides Fungicide Fosetyl-aluminum Benomyl Thiophanate methyl Metalaxyl Iprodione Dimetachlone Bismerthiazol Carbendazim Procymidone Zineb Chlorothalonil Abamectin a
Regression equationa (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (1) (2) (2) (2)
Y=26.2940+12.3719X Y=6.9494+12.1860X Y=88.1610+12.9480X Y=70.7940+13.8946X Y=11.4870+39.1324X Y=6.3081+11.4318X Y=19.3286+9.6308X Y=27.7789+28.7152X Y=14.1084+19.9400X Y=58.4818+29.0284X Y=78.3519+32.6434X Y=13.1417+33.0481X Y=51.9517+18.4372X Y=32.9035+12.2211X Y=44.7152+26.7141X Y=11.5613+13.0416X Y=56.0813+21.1271X Y=55.4460+14.6678X Y=67.4166+27.2055X Y=-59.1567+13.5943X Y=106.2007+24.4384X Y=16.0494+16.2593X Y=19.9857+30.5436X Y=13.0025+18.5993X
Y percentage inhibition; X=natural logarithm of herbicide concentration. SL, signiﬁcance level. c CC, correlator coeﬃcient b
SLb 0.0296 0.0050 0.0097 0.0416 0.0804 0.0010 0.0317 0.0325 0.0030 0.0028 0.0575 0.0306 0.0411 0.0307 0.0274 0.0711 0.0191 0/0143 0.0337 0.0091 0.0042 0.0255 0.0070 0.0330
CCc 0.9704 0.9742 0.9903 0.9584 0.9196 0.9909 0.9683 0.9089 0.9971 0.9824 0.9425 0.9694 0.9589 0.9693 0.9726 0.9289 0.9809 0.9477 0.9663 0.9612 0.9769 0.9227 0.9930 0.9670
EC50 (mg/L) 6.7945 34.2194 0.0525 0.1994 5.7554 137.768 21.1605 7.4793 6.0495 41.9757 51.0085 6.7571 0.8996 4.0509 34.6575 19.0562 0.7499 0.6898 0.5272 0.5099 0.1003 8.0693 9.8882 7.3096
1.92 10 9.66 105 1.81 107 6.88 107 1.68 105 4.03 104 7.58 105 2.68 105 1.83 105 1.27 104 2.09 104 2.77 105 3.19 106 1.44 105 1.20 104 6.59 105 2.66 106 2.45 106 1.91 106 1.85 106 3.77 107 3.03 105 1.13 105 8.38 106
5.0360 3.7981 23.9372 0.3534 6.9387 0.1325 4.5030 0.5498 0.9198 0.9672 80.4516 0.7392
MA ET AL.
zineb varied around 0.5–0.6 mg/liter (106 M), chlorothalonil varied around 0.1–8.1 mg/liter (105–107 M), and acaricides (abamectin) varied around 7–10 mg/liter (105– 106 M). The average acute toxicity of abamectin, bismerthiazol, procymidone, zineb, and chlorothalonil to C. pyrenoidosa and S. obliqnus was higher than that of fosetyl-aluminum, metalaxyl, iprodione, dimetachlone, carbendazim, and thiophanate-methyl. The 96-h EC50 values of benomyl varied around 0.05–0.2 mg/liter (10–7 M); its acute toxicity to the C. pyrenoidosa and S. obliqnus was the highest among all of the pesticides tested and was close to those of the photosynthesis-inhibiting herbicidesFAtrazine (0.12–0.15 mg/liter), slmazine (0.08–0.25 mg/ liter), and chlorotoluron (0.08–1.49 mg/liter) (Ma et al., 2001). In this work, wide variations occurred in response to fungicides and insecticides among individual species of the green algae. Scenedesmus proved to be more tolerant genera, whereas Chlorella were more sensitive to pesticides. Among 12 pesticides, C. pyrenoidosa was more sensitive to 6 pesticidesFfosetyl-aluminum, benomyl, thiophanatemethyl, iprodione, bismerthiazol, and chlorothalonilF than S. obliqnus; only 3 pesticidesFmetalaxyl, dimetachlone, and carbendazimFwere less sensitive than S. obliqnus and 3 pesticidesFprocymidone, zineb, and abamectinFwere close to S. obliqnus. Similar results were obtained using the 42 herbicides used as test compounds (Ma et al., 2001; Ma and Liang, 2001; Ma, 2001). Meanwhile, algal species vary widely in their response to toxic chemicals; the results demonstrated that there was a diﬀerential response to various pesticides among two species of algae and that the sensitivity of various species of algae exposed to chlorothalonil varied by nearly two orders of magnitude and that to thiophanate-methyl by more than one order. Investigations using diﬀerent algal species as test organisms have shown that algae vary greatly in their response to chemicals. Diﬀerential sensitivity of green algae to the compounds could induce species shifts within communities (Boyle 1984; Tadros et al., 1994). DISCUSSION
Little is known about the sensitivity of algae to pesticides, especially fungicides, which are associated with the use of small test volumes and which have poor solubility. Methods for conducting aquatic toxicity tests as described in various guidelines, including those of the OECD, EPA, EU, and ISO, are typically designed for substances that are essentially pure, readily water soluble, and chemically stable. As a consequence, when such methods are applied to unstable or complex substances, or sparingly soluble substances that do not display toxicity at the solubility limit, diﬃculties in conducting and interpreting toxicity tests are encountered (Ruﬂi et al.,
1998). Most fungicides are poorly water soluble and low solvent soluble; therefore, most of the substances tested were used in various formulations in this work. The pesticides tested were dissolved with a little acentone or distilled water. The concentration of solvent in medium was kept at a minimum. Most were the lower maximum allowable limit of 0.05% solvent for acute tests, which is recommended by the U.S. Environmental Protection Agency; this level was not signiﬁcant with respect to toxicity (Jay, 1996). Sensitivity to toxicants is an important criterion determining the suitability of a test for adoption into chemical regulations. Sensitive tests are more likely to yield EOx values that aﬀord protection to species and communities (Girling et al., 2000). Sensitive varies not only among toxicants, but also among taxonomic groups and species within texa. Hughes and Erb (1989) examined the relative sensitivity of four species of algae and one species of duckweed to 13 diﬀerent pesticides; they reported that no species could be identiﬁed as ‘‘always being the most sensitive or always the least sensitive’’ (Wang and Freemark, 1995). The same results have also been obtained in this test and using the 42 herbicides as test compounds in our works (Ma and Liang, 2001; Ma, 2001).
1. The decreasing order of average acute toxicity to two green algae, Chlorella pyrenoidosa and Scenedesmus obliqnus, of 12 pesticides was as follows: benomyl4zineb4 procymidone 4 bismerthiazol 4 chlorothalonil 4 abamectin 4metalxyl4fosetyl-alminum4iprodione4carbendazim4 dimetachlone4thiophanate-methyl. 2. The acute toxicity of benomyl to C. pyrenoidosa and S. obliqnus was the highest among all of the pesticides tested and was close to that of the photosynthesis-inhibiting herbicides. 3. Compared with the algal species tested, there are no species that are always the most sensitive or always the least sensitive, but in general, Chlorella was more sensitive to most of the pesticides than Scenedesmus. 4. The algal species varied widely in their response to 9 of the 12 pesticides tested. The sensitivity of various species of algae exposed to chlorothalonil varied by nearly two orders of magnitude, that to thiophanate-methyl varied by more than one order, and that to dimethachlone by nearly one order.
ACKNOWLEDGMENTS Financial assistance was provided by the Chinese National Pesticides R&D South Center and the Nature Science Foundation of Zhejiang Provience, China.
SENSITIVITY OF S. Oblignus AND C. Pyrenoidosa TO 12 PESTICIDES
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