本土狹長介形蟲(Stenocypris major)生物毒性試驗方法休眠卵保存及孵化條件之優化

近年來透過生物進行環境評估已成主要趨勢，底棲大型無脊椎動物為受歡迎的指標物種，以反映水域底部的污染現況，還可用在生態評估，因而建立了許多底棲無脊椎動物毒性試驗。美國環保署(USEPA)及經濟合作暨發展組織(OECD)等機構，已建立公告多種標準的生物毒性試驗流程，但因維持生物馴養需要耗費大量人力成本，因此近年來快篩式的TOXKIT生物試驗套組（例如，DaphtoxkitTM和AlgaltoxkitTM等）亦廣受探討，主要優點除了實驗室降低馴養費用，亦能在短時間內透過套組進行生物毒性試驗。本研究藉由了解S. major當作毒性試驗物種的適宜性及靈敏度，嘗試優化經刺激後可孵化之S. major休眠卵的保存及孵化條件，以利未來生物毒性試驗套組之開發。由S. major曝露於水體參考毒物NaCl試驗中顯示S. major屬中耐受性物種，四種不同暴露時間(24hr、48hr、72hr及96hr)之LC50分別為6727、5080、3986及3720 mg/L。S. major底泥毒性試驗之流程為，於6孔盤中每孔添加1mL底泥及4mL中低硬度曝氣水(SFW)，其中含0.1mL之營養液及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。最適人工控制組底泥配方為石英砂重量比(75-90%)與高嶺土重量比(10-25%)所組成；枋山溪口樣站底泥屬砂質壤土為最適野外控制組。S. major暴露於添加不同濃度毒物CuSO4之參考底泥時，實測真實吸附濃度依序為96.22 mg/L、81.80 mg/L 、233.87 mg/L、及208.11 mg/L，生物暴露6天後，各樣品存活率均為0%，顯示S. major對Cu耐受性低。休眠卵之最佳保存條件為，於4℃及24hr全暗環境下覆水保存；最佳孵化條件為水分乾燥4hr內，需加蓋以石臘膜封住培養皿，可於168hr內添加10mL中低硬度曝氣水(SFW)及0.2mL營養液，於25℃及24hr全光照環境下進行孵化(光照強度約為5000-8000lux)，上述條件證實休眠卵最少可保存6個月，其平均孵化率可至少達80%。本研究初步研判S. major適合作為毒性試驗物種，且具開發毒性試驗套組之潛力，但關於S. major亞致死終點之穩定性，則仍需加入光照條件及溫度等影響因子的細部研究，來完備試驗套組以提供未來商業化之可能性。

關鍵字

底棲無脊椎生物；介形蟲；毒性試驗套組；狹長介形蟲

並列摘要

Aquatic toxicity testing protocols for watersheds are applied in regulatory settings worldwide since decades. Benthic m3ion that are suitable for environmental and ecological assessments. Therefore, several benthic invertebrate toxicity models have been developed. The United States Environmental Protection Agency (USEPA) and the Organization for Economic Cooperation and Development (OECD) have established a series of standardized procedures using different ecologically representative organisms for toxicity testing. However, labor intensity and generally high costs to maintain the organisms have become a problem for their applications. The screening TOXKIT biological tests (such as DaphtoxkitTM and AlgaltoxkitTM, etc.) were widely applied over the last years due to their greater economic feasibility. Nevertheless, there is a demand for even more sensitive toxkits using benthic invertebrates for fast screening. To clarify the suitability and sensitivity of the freshwater ostracod Stenocypris major as a toxicity test species. This study attempts to optimize the storage and incubation conditions of S. major resting eggs. For the development of a test kit such resting eggs should be able to get hatched after stimulation. The exposure of S. major to the water toxicant reference NaCl showed that S. major is an intermediate tolerant species. The LC50 obtained at four different exposure times (24 h, 48 h, 72 h and 96 h) were 6727, 5080, 3986 and 3720 mg/L. The procedure and protocol of the S. major sediment toxicity test was to mix 1 mL sediment and 4 mL medium-low hardness aeration water (SFW) with 0.1 mL of nutrient solution to wells in a 6-well plate, together with ten 3-7 days old S. major larvae placed for exposure. The exposure time was 144 h, at 25 ⁰C in a completely dark incubator. The constituent of the control group sediment formula was composed of a quartz sand weight ratio of (75-90%) and a kaolin weight ratio (10-25%); the sediment consisted of sandy loam of Fangshan estuary samples serving as the field reference site as survival of S. major resting eggs reaching up to 95%. When S. major was exposed to the above sediment spiked with different concentrations, 96.22 mg/L, 81.80 mg/L, 233.87 mg/L, and 208.11 mg/L, of CuSO4. Survival at all Cu concentrations were 0% after six days exposure, indicating a substantial sensitivity of S. major to sediment pollutants. Suitable storage conditions for resting eggs in a dark environment turned out to be 4°C and no longer than 24hr. The best incubation conditions were with 10 mL overlying water in the petri dish covered with parafilm. Low-hardness aerated water (SFW) and 0.2 mL nutrient solution were co-incubated at 25 °C and 24 h full light environment (light intensity was about 5000-8000 lux). The above conditions confirmed that resting eggs could be stored for at least 6 months. The average hatching rate can be as high as 80%. This study showed that S. major is suitable as a toxicity test species and has the potential to be used in a toxicity test kit. However, to achieve stable sublethal end points of S. major, details about confounding factors such as light conditions and temperature still have to be elaborated in the future.

並列關鍵字

Benthic invertebrate ； Ostracod ； Toxkit ； Stenocypris major

參考文獻

Almheiri, A., Mahajan, R., Maldacena, J., Zhao, Y., 2020. The Page curve of Hawking radiation from semiclassical geometry. Journal of High Energy Physics 2020, 1-24.

Google Scholar

Ankley, G., Schubauer-Berigan, M., Monson, P., 1995. Influence of pH and hardness on toxicity of ammonia to the amphipod Hyalella azteca. Canadian Journal of Fisheries and Aquatic Sciences 52, 2078-2083.

Google Scholar

Batley, G.E., Simpson, S., 2013. Sediment Quality Guidelines. in: Férard, J.-F., Blaise, C. (Eds.). Encyclopedia of Aquatic Ecotoxicology. Springer Netherlands, Dordrecht, 1015-1024.

Google Scholar

Berry, W.J., Boothman, W.S., Serbst, J.R., Edwards, P.A., 2004. Predicting the toxicity of chromium in sediments. Environmental Toxicology and Chemistry: An International Journal 23, 2981-2992.

Google Scholar

Besser, J.M., Brumbaugh, W.G., Kemble, N.E., Ivey, C.D., Kunz, J.L., Ingersoll, C.G., Rudel, D., 2011. Toxicity of nickel-spiked freshwater sediments to benthic invertebrates—Spiking methodology, species sensitivity, and nickel bioavailability. US Geological Survey.

Google Scholar

主題瀏覽