Acute Toxicity of the Insecticide Diazinon and Carbaryl to Calanoid and Cyclopoid Copepoda （Eodiaptomus, Mesocyclops and Thermocyclops）In Different Life Stages

環境毒性学会誌（Jpn. J. Environ. Toxicol.），9（2），133-139，2006

Acute Toxicity of the Insecticide Diazinon and Carbaryl to Calanoid and Cyclopoid Copepoda （Eodiaptomus, Mesocyclops and Thermocyclops）in Different Life Stages

Hirokazu Takahashi, Kwang-Hyeon Chang and Takayuki Hanazato

Institute of Mountain Science, Shinshu University 5-2-4 Kogandori, Suwa 392-0027 Japan

ABSTRACT We conducted acute toxicity tests using a calanoid species（Eodiaptomus japonicus） and two cyclopoid species（Mesocyclops pehpeiensis, Thermocyclops taihokuensis）of different life stages（nauplius and adult）and assessed their sensitivities to the insecticide diazinon and carbaryl.

The EC50 values of nauplii of the three species ranged between 2.8 and 4.1 ppb for diazinon and between 9.9 and 19.7 ppb for carbaryl, which were in the similar range of the

EC50 values of neonates of the cladoceran Daphnia for the same insecticides. In contrast,

adults of the copepod species showed much higher tolerance to the chemicals; their EC50 values were in a range between 30.6-46.8 ppb for diazinon and between 785.7-1190.0 ppb for carbaryl, which were 10-70 times higher than the values of nauplii. The difference in the sensitivity to the chemicals between neonates and adults was much larger in the copepods than in Daphnia. This suggests that the copepod populations are controlled mostly by the mortality of nauplii if they are exposed to insecticides.

Key words: Diazinon; Carbaryl; Calanoid copepoda; Cyclopoid copepoda; Acute toxicity

1. Introduction assessed to conserve the communities and the Various freshwater bodies are contaminated aquatic ecosystems. Recently, the assessment has with anthropogenic toxic chemicals such as pesti- been intensively performed using the toxicity data cides, which may exert direct impacts on some obtained in the laboratory toxicity tests（Antunes organisms and control the organic communities et al., 2004; Coors et al., 2004; De Schamphelaere et there（Day et al., 1987; Dodson et al., 1995; al., 2004; Flaherty and Dodson, 2005; Hanazato, Goodrich and Leach, 1990; Helgen et al., 1988; 1998b; Hanazato and Hirokawa, 2001）． Hanazato, 1998a, 2001）．The impacts should be

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Zooplankton is an important group of organ- Suwa（36°2’N, 138°5’E）water was taken up isms in the lentic ecosystems by playing as herbi- with a column sampler during July to September vores or carnivores in the food chains（Mills and from 2004 to 2005. Then, adult females of Forney, 1988）．Their sensitivity to toxic chemicals Eodiaptomus japonicus with eggs were isolated from is being analyzed. However, most of the test ani- the zooplankton samples, which were collected by mals are the crustacean Cladocera, especially the filtering the lake water through a 40-μm mesh net. Daphniidae animals. This is probably because（1） They were put into 16.5-mL plastic multiwell cham- they are the major herbivores feeding effectively on bers（Asahi Techno Glass Co. Ltd, Chiba, Japan）， the phytoplankton and are preferred food items of each contained two adult individuals and 15mL of fish,（2）they are sensitive to toxic chemicals, and culture water with the green alga Chlorella vulgaris （3）they perform parthenogenesis and are, there- at the density of 5 x 105 cells/mL. The culture fore, easily cultured in clones in the laboratory. The water was the mixture in equal measures of aged toxicity data on zooplankton other than Cladocera tap water and the filtered lake water, which was are insufficient, and are thus needed. collected from Lake Suwa and filtered with The crustacean Copepoda is another important Whatman GF/C filter. The Chlorella was purchased group in zooplankton community（Gophen, 1977; from the Chlorella Industry Co. Ltd.（Fukuoka, Kerfoot, 1978; Li and Li, 1979; Plaβmänn, Maier Japan）．The chambers with copepods were kept in and Stich, 1997; Williamson, 1980, 1984, 1986; the laboratory for three days under a constant tem- Williamson et al., 1986; Stemberger, 1985; Yoshida perature（20±3℃）and 16-h light and 8-h dark et al., 2001）． It includes Calanoida and photocycle to acclimatize the animals to the experi- Cyclopoida, some of which are herbivorous, but oth- mental condition. The culture water with food was ers are carnivorous or omnivorous. The copepods renewed on the second day. have a life style different from Cladocera. They per- Adult females of Mesocyclops pehpeiensis and form metamorphosis between the last naupliar and Thermocyclops taihokuensis with eggs were also col- the first copepodite stages, and always reproduce lected from Lake Suwa during May to September sexually. from 2004 to 2005. They were cultured for three In the present study, we conducted acute toxici- days in the laboratory in the same way as used for ty tests using a Calanoid species（Eodiaptomus Eodiaptomus but fed with the mixture of Chlorella japonicus）and two Cyclopoid species（Mesocyclops and a zooplankton assemblage（including the small pehpeiensis, Thermocyclops taihokuensis）of different cladoceran Bosmina and rotifers）collected from life stages（nauplius and adult）and assessed their the same lake. sensitivities to the insecticide diazinon and car- During the acclimation, nauplius larvae baryl, which often contaminate freshwater bodies appeared in the chambers. Their appearance was （Shiraishi et al., 1988; Hatakeyama et al., 1990, checked everyday, and the larvae（<24h old）were 1991）and may have negative impacts on aquatic picked up if any and used for the toxicity tests. animals. Adults were also used for the tests after the 3-day- acclimation. 2. MATERIALS AND METHODS The chemicals tested were the organophospho- For sampling of the animals used, 12 L of Lake- rus insecticide diazinon and the carbamate insecti-

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Fig. 1. Measurements of total length (TL) of the adults of Eodiaptomus and Cyclopoida（Mesocyclops, Thermocyclops）， and of their nauplii.

cide carbaryl, both of which are commonly used in of the animal. Then, the individual was measured the paddy fields, orchards and forests in Japan. and soon returned to the subsample bottle. The Both chemicals of > 99% purity were purchased copepods were exposed in a static system in the 10- from Wako Pure Chemical Industries Ltd. Japan. In mL beakers to each of six-test concentrations with a order to use the chemicals in the toxicity tests, a constant dilution rate（1.4-1.8）of diazinon or car- stock solution（1000 ppm）of each chemical was baryl and a solvent（ethanol）control. The ethanol prepared by dissolving 10 mg of the chemical in concentration in each test vessel was made equal to 99% ethanol to a final volume of 10 mL, and was the ethanol concentration in the vessel with the stored in a refrigerator throughout the tests. highest chemical concentration（8.2μL/L－2700 The toxicity tests were conducted following the μL/L）．Twenty beakers, each of which contained OECD guidelines No.202 for testing chemicals 10mL of the test solution and a test individual, （OECD, 1984）with slight modification for 48-h were prepared for each insecticide concentration or bioassays. At start of the tests, total lengths of the control. individuals used（nauplii and adults）were meas- The exposure to each chemical lasted 48 h, ured under a binocular microscope to the nearest when the animals were not fed and the solutions 10μm（Fig. 1）．Each individual in each subsam- were not aerated. ple bottle was picked up by a pipet and placed on a At the end of exposure, copepods incapable of

glass slide with a drop of the rearing medium. The swimming were counted. The 48-h EC50 values were medium volume was reduced to restrain the moving then determined using the probit analysis （Eco-

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Tox Statics Release 2.5, Oita Univ., Japan）．The in the present study（10-70 times）than Daphnia test of each life stage of each copepod species for [< 5 times; 3.8 times for D. pulex with carbaryl each insecticide was performed in triplicate, and （Hanazato and Hirokawa, 2001), 2.1 times for D. mean value ± SE of EC50（n = 3）was calculated. magna with carbaryl（Takahashi and Hanazato, submitted), 1.8 times for D. galeata mendotae with 3. RESULTS AND DISCUSSION fenbalerate（Day and Kaushik, 1987），and 3.0 The sensitivities of nauplii of the three copepod times for D. magna with fenvalerate（Day and species tested were similar to one another; their Kaushik, 1987）］． This suggests that the copepod 2 8 4 1 EC50 values were in a range between . and . populations are controlled mostly by the mortality ppb for diazinon, and between 9.9 and 19.7 ppb for of nauplii if they are exposed to insecticides. carbaryl（Table1）．The values are similar to or a The results of the present toxicity tests have little bit higher than the EC50/LC50 values of daph- shown that diazinon is more toxic to copepods than nids（LC50 of the newly born individuals of D. carbaryl （Table 1）． The same phenomenon has magna; 0.8, 1.5 ppb, and of Ceriodaphnia dubia; 0.5 also been demonstrated in Daphnia magna; The 48- and 0.8 ppb; Ankley et at, 1991; Burkepile, 2000）． h LC50 values for diazinon are 0.8, 1.5ppb, while However, the copepods become very tolerant to the those for carbaryl are 10.0ppb（Anlkey et at, 1991; chemicals when grown up to adults. This was Burkepile, 2000; Hatakeyama and Sugaya, 1989）． inferred from the results that the EC50 values of the Cyclopoid copepoda are common invertebrate adults were in the range of 30.6-46.8 ppb for diazi- predators in lakes and preferably prey on small non and of 785.7-1190.0 ppb for carbaryl, which cladocerans and rotifers. The cladoceran Leptodora were nearly 10 times higher than the values of nau- is also an important predator on the small zoo- plii for diazinon and approximately 70 times higher plankton species in lakes. They inhabit Lake Suwa for carbaryl. The phenomenon that the juveniles are and show seasonal succession; cyclopoid copepods more sensitive to insecticides than adults is com- become abundant in spring and are replaced by monly observed in daphnids（Day and Kaushik, Leptodora in early summer（Chang and Hanazato, 1987; Hanazato and Hirokawa, 2001; Takahashi and 2003）．Sakamoto et al.（2005）determined the

Hanazato, submitted）．However, the difference in 24-h LC50 of Leptodora for carbaryl as 3.48 ppb, sug- the tolerance to insecticides between neonates and gesting that this animal species is very sensitive to adults is much larger in the copepod species used insecticides in comparison with copepods. Because

TABLE 1. Total lengths and 48-h EC50 values（mean ± SE; n=3）for diazinon and carbaryl to nauplii and adult females of Eodiaputomus japonicus, Mesocyclops pehpeiensis and Thermocyclops taihokuensis.

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Japanese lakes are often contaminated with insecti- two Bosmina species（Cladocera, Crustacea） cides runoff from paddy fields in spring （Shiraishi co-exsisting in a lake: their relationship with et al., 1988），the insecticide contamination could invertebrate predators. J. Plankton Res., 25, be a factor inducing the dominance of copepods in 141-150 the spring community of invertebrate predators in Day, K.E. and Kaushik, N.K.（1987）．Short-term Lake Suwa. exposure of zooplankton to the synthetic Ecosystem is composed of huge number of pyrethroid, fenvalerate, and its effects on rates species, all of which are related complicatedly from of filtration and assimilation of the alga, one another. Therefore, a direct and negative Chlamydomonas reinhardii. Arch. Environ. impact given by a toxic chemical on a species could Contam. Toxicol. 16, 423-432. be exerted to whole communities through biological Day, K.E., Kaushik, N.K. and Solomon, K.R. interactions（Hanazato, 2001）．However, the （1987）．Impact of fenvalerate on enclosed direct chemical impact should differ depending on freshwater planktonic communities and on in species and life stages as shown in the present situ rates on enclosed freshwater planktonic study. Thus, we should increase toxicity data of the communities and on in situ rates of filtration of chemicals contaminating natural environment for zooplankton. Can. J. Fish. Aquat. Sci., 44, various organic species of different life stages to 1714-1728. evaluate the chemical impact on ecosystems. De Schamphelaere, K.A.C., Canli, M., Van Lierde, V., Forrez, I., Vanhaecke F. and Janssen C.R. REFERENCES （2004）Reproductive toxicity of dietary zinc to Antunes, S.C., Castro, B.B., Gonc,alves, F.（2004）． Daphnia magna. Aquat. Toxicol., 70, 233-244. Effect of food level on the acute and chronic Dodson, S.I., Hanazato, T. and Gorski, P.R.（ 1995）． responses of daphnids to lindane. Environ. Behavioral responses of Daphnia pulex exposed Pollut., 127, 367-375. to carbaryl and Chaoborus kairomone. Environ. Ankley, G.T., Dierkes, J.R., Jensen, D.A., and Toxicol. Chem., 14, 43-50. Peterson, G.S.（1991）Piperonyl Butoxide as a Gophen, M.（1977）Food and feeding habits of Tool in Aquatic Toxicological Research with Mesocyclops leuckarti （ Claus） in Lake Organophosphate Insecticides. Ecotoxicol Kinneret（Israel）．Freshwater Biol., 7, 513- .Environ. Saf., 21, 266-274 518. Burkepile, D.E., Moore, M.T. and Holland, M.M. Goodrich, M.S. and Leach, J.J.（1990）． A behav- （2000）Susceptibility of Five Nontarget ioral screening assay for Daphnia magna: a Organisms to Aqueous Diazinon Exposure. Bull. method to assess the effects of xenobiotics on Environ. Contam. Toxicol., 64, 114-121 spatial orientation. Environ. Toxicol. Chem., 9, Coors, A., Hammers-Wirtz, M. and Ratte, H.T. 21-30. （2004）． Adaptation to environmental stress Flaherty, C.M. and Dodson, S.I.（2005）Effects of in Daphnia magna simultaneously exposed to a pharmaceuticals on Daphnia survival, growth, xenobiotic. Chemosphere, 56, 395-404. and reproduction. Chemosphere, 61, 200-207. Chang, K.H. and Hanazato, T.（2003）．Seasonal Kerfoot W.C.（1978）．Combat between predatory and reciprocal succession and cyclomorphosis of copepods and their prey: Cyclops, Epischura,

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（受付：2006年10月10日；受理：2006年11月21日）

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