本土狹長介形蟲(Stenocypris major)底泥生物毒性試驗方法之優化條件與物種靈敏度探討

由於大型底棲無脊椎動物群落的種類和結構同周圍環境之間的關係密切，近年來常被作為生物監測，反映水域底部的污染現況及生態毒理學風險評估之指標物種。美國材料試驗協會(ASTM)、美國環保署(USEPA)及經濟合作暨發展組織(OECD)等機構，已建立一系列標準之生物毒性試驗方法，評估受試驗生物經長時間暴露所造成的影響，但試驗生物馴養維護耗費大量人力成本與費用，因此市面上有了快篩式的TOXKIT生物毒性試驗套組的誕生，不僅能降低實驗室馴養成本，也能於短時間內進行具靈敏且可重複結果之生物毒性試驗。本研究將持續優化本土底棲無脊椎生物狹長介形蟲(Stenocypris major)底泥毒性試驗，穩定幼蟲暴露之初始體長。S. major與環檢所公告之底泥生物毒性試驗之Hyalella azteca和水體生物毒性試驗之 Daphnia magna試驗生物進行環境樣品暴露以了解作為毒性試驗物種之合適性與靈敏度，以利於S. major生物毒性試驗套組之開發。S. major暴露於水體參考毒物NaCl試驗中，四種不同暴露時間(24hr、48hr、72hr及96hr)之LC50分別為5230、4574、3632及3239 mg/L，顯示S. major屬於中耐受性物種。S. major休眠卵最適孵化條件以添加10：0.4 mL(中硬度水：營養液B)，於25℃及24hr全光照環境下進行孵化，孵化時間為72-168hr。S. major底泥毒性試驗之流程為，於6孔盤中每孔添加1mL底泥、4：0.8 mL(中硬度水：營養液C)及10隻S. major進行暴露，暴露時間為144hr，於25℃及24hr全暗環境下進行。參考樣品配方為石英砂70.5%、高嶺土24%、纖維素5%、白雲石0.49%及腐植酸0.01%組成，試驗終點控制組存活率≧80%及成長率≧50%，樣品以存活率及生長抑制率進行評估。暴露底泥樣品研究顯示，S. major與H. azteca兩者生物體存活率相比呈現相似之毒性效應；S. major與D. magna暴露於水體樣品，於上覆水和孔隙水之存活率同樣呈現相似之毒性效應，甚至更為靈敏，不同之環境參數呈現不同之毒性效應，顯示S. major適合作為環境監測之毒性試驗物種。本研究試驗因理化水質參數影響導致無法反應底泥污染物對生物體之毒性效應，建議優化毒性試驗方法，以利於不同環境參數之探討，完備試驗套組以提供商業化之可行性。

關鍵字

底棲無脊椎動物；介形蟲； Stenocypris major ；生物毒性試驗

並列摘要

Macrobenthic invertebrate communities and indicator species are often used in biological monitoring and ecotoxicological risk assessment to reflect the environmental pollution status of the bottom of the water column because of their close relationship with the quality of their surrounding environment. Organizations such as the American Society for Testing and Materials (ASTM), the U.S. Environmental Protection Agency (USEPA), and the Organization for Economic Cooperation and Development (OECD) have established a series of standard biotoxicity test methods to evaluate the effects of long-term exposure to test organisms. However, the domestication and maintenance of test organisms consume a lot of labor and other expenses. Therefore, the rapid screening TOXKIT biotoxicity test kit was created to not only reduce the costs of laboratory domestication but also perform sensitive and repeatable tests in a short time period. This study will continue to optimize the sediment toxicity test designed for the native benthic invertebrate Stenocypris major and standardize the initial body length of exposed larvae. S. major and Hyalella azteca in the biological toxicity testing of sediments (announced by the Environmental Inspection Institute) and Daphnia magna in the biological toxicity testing of water were exposed to environmental samples to understand their suitability and sensitivity for use in the S. major Biotoxicity Test Kit. In the testing of S. major exposed to reference toxicant NaCl in solution, the LC50 values for four different exposure times (24hr, 48hr, 72hr, and 96hr) were 5230, 4574, 3632, and 3239 mg/L, respectively, indicating that S. major was moderately tolerant. The optimal incubation conditions for S. major dormant eggs were to add 10:0.4 mL of medium hardness water: nutrient solution B, and incubate to hatching at 25 °C and 24hr full light for 72-168hr. The S. major sediment toxicity test procedure was to add 1 mL of sediment, 4:0.8 mL medium-hardness water: nutrient solution C, and 10 S. major to each well of a 6-well plate for an exposure time of 144 hr at 25 °C and 24hr total darkness. The formulated reference sediment constituents were 70.5% quartz sand, 24% kaolin, 5% cellulose, 0.49% dolomite, and 0.01% humic acid. The survival rate of the control group at the end of the test was ≧80% and growth rate was ≧50%. Their exposure to sediment samples showed that S. major and H. azteca exhibited similar toxic effects on survival rates. S. major and D. magna exposed to water samples had the same survival rates in overlying water and pore water, showing similar toxicity effects but being even more sensitive. Different environmental parameters showed different toxicity effects, indicating that S. major is suitable as a toxicity test species for environmental monitoring. Due to the influence of physical and chemical water quality parameters, this research test cannot reflect the overall toxic effect of sediment pollutants on organisms. We recommend that the toxicity test method be optimized to facilitate the discussion of different environmental parameters, and complete the test set to inform the feasibility of commercialization.