Acute Toxicity of the Insecticide Diazinon and Carbaryl to Calanoid and Cyclopoid Copepoda （Eodiaptomus, Mesocyclops and Thermocyclops）In Different Life Stages
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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 differ- ent 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 diazi- non 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 cope- pods 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 －133－ Acute toxicity of insecticide on Copepoda 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- －134－ TAKAHASHI et al 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- －135－ Acute toxicity of insecticide on Copepoda 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.