步改變水體的生態環境 (Wendt-Rasch et al., 2004)。也因此，儘管在本研究中所 選用的三種藥劑對於小球藻並不會有直接的毒性影響，但考量到在環境中的生態
45
表十、不同物種之急毒性試驗半致死濃度
Table 9 Lethal concentration 50 of acute toxicity tests in different species Pesticide (mg L-1) Species Thiophanate-methyl 17.47
(13.14-23.22)a
1.72 (0.76-3.89)a
>50
Carbendazim 0.10 (0.07-0.16)a
0.41 (0.3-0.56)a
>50
Imidacloprid 62.17 (37.15-104.03)a
0.053 (0.0356-0.0773)a
77
a: 95% confidence interval (95% CI)
46
圖五、三種農藥在水蚤與端足蟲的半致死濃度相關性比較
Fig. 3 Correlation plot with 1:1 line. Plots indicate lethal concentration 50 of D. pulex and H. azteca exposed to three pesticides.
47
表十一、水蚤毒性實驗值與文獻值比較
Table 10 Laboratory data compared to literature data
Pesticide (mg L-1) This research Literature
D. pulex D. magna
Thiophanate-methyl 17.47 20.2a 25.1b
Carbendazim 0.10 0.13-0.22a
0.23b
Imidacloprid 62.17 85a
39.387b a: Pesticide manual
b: US EPA
48
表十二、端足蟲毒性實驗值與文獻值比較
Table 11 Laboratory data compared to literature data
Pesticide (mg L-1) This research Literature
H. azteca G. fossarum G. pulex Thiophanate-methyl 1.72
Carbendazim 0.41 0.075a 0.051b
0.077
Imidacloprid 0.053 0.07c 0.8d
0.405e
a: EC50 (7d) b: LC50 (7d)
c: EC50 (E=effect, paralyzed)
d: LC50 (48h) e: LC50 (72h)
49
圖六、水蚤於甲基多保淨中之劑量與毒性效應
Fig. 4 Mortality after D. pulex exposed to thiophanate-methyl
圖七、水蚤於貝芬替中之劑量與毒性效應
Fig. 5 Mortality after D. pulex exposed to carbendazim
50
圖八、水蚤於益達胺中之劑量與毒性效應
Fig. 6 Mortality after D. pulex exposed to imidacloprid
51
圖九、端足蟲於甲基多保淨中之劑量與毒性效應
Fig. 7 Mortality after H. azteca exposed to thiophanate-methyl
圖十、端足蟲於貝芬替中之劑量與毒性效應
Fig. 8 Mortality after H. azteca exposed to carbendazim
52
圖十一、端足蟲於益達胺中之劑量與毒性效應 Fig. 9 Mortality after H. azteca exposed to imidacloprid
53
圖十二、小球藻受甲基多保淨抑制之生長速率圖形
Fig. 10 Growth inhibition after C. vulgaris exposed to thiophanate-methyl
圖十三、小球藻受貝芬替抑制之生長速率圖形
Fig. 11 Growth inhibition after C. vulgaris exposed to carbendazim
54
圖十四、小球藻受益達胺抑制之生長速率圖形
Fig. 12 Growth inhibition after C. vulgaris exposed to imidacloprid
55 混合的組合中，透過貝芬替與無觀察危害反應劑量 (No-observed-adverse-effect level, NOAEL) 的益達胺混合 (圖十六) 與貝芬替與最低觀察危害反應劑量 (Lowest-observed-adverse-effect level, LOAEL) 的益達胺混合暴露 (圖十七) ，與 控制組 (單獨暴露貝芬替) 相比，致死率並沒有顯著性的差異。顯示益達胺的存 合物會受到吸附作用 (adsorption) 、生物轉化作用 (biotransformation) 、流佈作
56 同效應是來自於代謝農藥的酵素反應，如單加氧脢 (monooxygenases, P450s) 、 榖胱甘肽 S 轉移脢 (glutathione-S-transferases, GST) 、酯脢 (esterase) 被另一化 物抑制或誘導。在已知的文獻研究之中，具有確定的協同作用的殺菌劑以二氯苯 胺類 (Dichloroaniline) 與咪唑系 (Imidazole) 誘導 P450 為主，殺蟲劑以有機氯
57
類 (Organochlorines) 誘導 P450、GST、esterase 為主，有機磷類 (Organophosphorus) 抑制前述三種酵素為主 (Cabras et al., 1995)。本研究中，對於免賴得系殺菌劑甲 基多保淨與新菸鹼類殺蟲劑益達胺的協同作用，是首次知悉兩種藥劑所造成的協 同作用，推測可能原因是其中一種藥劑影響到水蚤的代謝解毒機制，而使得另一
種藥劑造成的毒性提高，造成更強的毒性效應。
58
圖十五、混合暴露於貝芬替與益達胺之水蚤毒性與劑量效應
Fig. 13 Mortality after D. pulex exposed to carbendazim and imidacloprid
圖十六、貝芬替加上無觀察危害反應劑量的益達胺對水蚤之劑量與毒性效應 Fig. 14 Mortality after D. pulex exposed to carbendazim with NOAEL imidacloprid
59
圖十七、貝芬替加上最低觀察危害反應劑量的益達胺對水蚤之劑量與毒性效應 Fig. 15 Mortality after D. pulex exposed to carbendazim with LOAEL imidacloprid
圖十八、等毒性混合貝芬替與益達胺對水蚤之劑量與毒性效應
Fig. 16 Mortality after D. pulex exposed to equitoxic mixture of carbendazim and imidacloprid (**: p<0.05)
**
60
圖十九、混合暴露於甲基多保淨與益達胺之水蚤毒性與劑量效應
Fig. 17 Mortality after D. pulex exposed to thiophanate-methyl and imidacloprid
圖二十、甲基多保淨加上無觀察危害反應劑量的益達胺對水蚤之劑量與毒性效應 Fig. 18 Mortality after D. pulex exposed to thiophanate-methyl with NOAEL
imidacloprid (**: P<0.05)
**
61
圖二十一、甲基多保淨加上最低觀察危害反應劑量的益達胺對水蚤之劑量與毒 性效應
Fig. 19 Mortality after D. pulex exposed to thiophanate-methyl with LOAEL imidacloprid (**: P<0.05)
圖二十二、等毒性混合甲基多保淨與益達胺對水蚤之劑量與毒性效應
Fig. 20 Mortality after D. pulex exposed to equitoxic mixture of thiophanate-methyl and imidacloprid (**: P<0.05)
**
**
**
**
**
**
62
表十三、水蚤混合暴露之半致死濃度 Table 12 LC50 of mixture pesticide in D. pulex
Treatment (mg L-1) LC50 (48h) TU
Thiophanate-methyl 17.47
Carbendazim 0.10
Imidacloprid 62.17
Carbendazim with 1.8 mg L-1 (NOAEL) imidacloprid 0.12 Carbendazim with 3.6 mg L-1 (LOAEL) imidacloprid 0.08
Equitoxic mixture (carbendazim+imidacloprid) 2.62a Thiophanate-methyl with 1.8 mg L-1 (NOAEL) imidacloprid 8.35
Thiophanate-methyl with 3.6 mg L-1 (NOAEL) imidacloprid 4.16
Equitoxic mixture (thiophanate-methyl+imidacloprid) 0.27b a: TU>1, antagonism
b: TU<1, synergism
63
64
圖二十三、等毒性混合貝芬替與益達胺對端足蟲之劑量與毒性效應
Fig. 21 Mortality after H. azteca exposed to eqitoxic mixture of cabendazim and imidacloprid
圖二十四、等毒性混合甲基多保淨與益達胺對端足蟲之劑量與毒性效應 Fig. 22 Mortality after H. azteca exposed to eqitoxic mixture of thiophanate-methyl and imidacloprid
65
表十四、端足蟲混合暴露之半致死濃度 Table 13 LC50 of mixture pesticide in H. azteca
Treatment (mg L-1) LC50 (72h) TU
Thiophanate-methyl 1.72
Carbendazim 0.41
Imidacloprid 0.053
Equitoxic mixture (carbendazim+imidacloprid) 0.62a Equitoxic mixture (thiophanate-methyl+imidacloprid) 0.22b a: TU<1, synergism
b: TU<1, synergism
66
圖二十五、混合暴露的對數模型圖形比較 (空心：甲基多保淨加益達胺；實心：
貝芬替加益達胺)
Fig. 23 Three-logistic model of mixture exposing tests (empty : thiophanate-methyl + imidacloprid ; solid : carbendazim + imidacloprid)
67
表一五、混合暴露之混合毒性作用 (等毒性)
Table 14 Mixture toxicity effect of mixture exposing (TU=1:1)
Treatment D. pulex H. azteca Thiophanate-methyl
+ Imidacloprid
Synergism Synergism
Carbendazim +
Imidacloprid
Antagonism Synergism
68
六、 結論
1. 在三種非目標生物上，所選用的三種農藥呈現不同的毒性趨勢。對水蚤 (D.
pulex) 而言，殺菌劑貝芬替毒性最高、甲基多保淨次之，殺蟲劑益達胺最低；
對端足蟲 (H. azteca) 而言，益達胺最高、貝芬替次之，甲基多保淨最低；對 小球藻 (C. vulgaris) 而言，因貝芬替與甲基多保淨到達最高溶解量時仍無法 達成生長速率半抑制，因此認為受此三者農藥毒害可能性較小。
2. 在貝芬替的毒性影響上，水蚤 (D. pulex) 較端足蟲 (H. azteca) 敏感；在益達 胺上，端足蟲 (H. azteca) 則是較水蚤 (D. pulex) 敏感兩個尺度。在新菸鹼類 殺蟲劑上，端足蟲 (H. azteca) 是較水蚤 (D. pulex) 具有較高的敏感性。
3. 在混合暴露試驗中，對水蚤 (D. pulex) 而言，貝芬替與益達胺混合的毒性效 應是拮抗作用，甲基多保淨與益達胺混合則是協同作用。對端足蟲 (H. azteca) 而言，兩種混合暴露皆是協同作用，其中以甲基多保淨與益達胺混合的協同 效應為最大。
由以上結果可得，在進行生態環境影響評估時，底棲生物、例如本研究所選 用的端足蟲 (H. azteca) 的毒性試驗結果是必須的，因其生活型態、與環境中毒 物接觸頻仍，也顯現出較常用的試驗生物高的敏感度。而混合暴露的毒性試驗結 果也顯現，殺菌劑與殺蟲劑同時存在會造成比原先預期來得大的毒性效應，以混 合形式存在的農藥毒性對於水體中的生物影響仍需持續關注。
69
Ashauer, R., Hintermeister, A., Potthoff, E., Escher, B.I., 2011. Acute toxicity of organic chemicals to Gammarus pulex correlates with sensitivity of Daphnia magna across most modes of action. Aquatic Toxicology 103, 38-45.
Bal, R., Naziroglu, M., Turk, G., Yilmaz, O., Kuloglu, T., Etem, E., Baydas, G., 2012.
Insecticide imidacloprid induces morphological and DNA damage through oxidative toxicity on the reproductive organs of developing male rats. Cell Biochemistry and Function 30, 492-499.
Becker, L., Scheringer, M., Schenker, U., Hungerbuehler, K., 2011. Assessment of the environmental persistence and long-range transport of endosulfan.
Environmental Pollution 159, 1737-1743.
Belden, J.B., Gilliom, R.J., Lydy, M.J., 2007. How well can we predict the toxicity of pesticide mixtures to aquatic life? Integrated Environmental Assessment and Management 3, 364-372.
Belden, J.B., Lydy, M.J., 2000. Impact of atrazine on organophosphate insecticide
70
toxicity. Environmental Toxicology and Chemistry 19, 2266-2274.
Berenzen, N., Lentzen-Godding, A., Probst, M., Schulz, H., Schulz, R., Liess, M., 2005. A comparison of predicted and measured levels of runoff-related pesticide concentrations in small lowland streams on a landscape level. Chemosphere 58, 683-691.
Bereswill, R., Golla, B., Streloke, M., Schulz, R., 2012. Entry and toxicity of organic pesticides and copper in vineyard streams: Erosion rills jeopardise the efficiency of riparian buffer strips. Agriculture, Ecosystems & Environment 146, 81-92.
Bereswill, R., Golla, B., Streloke, M., Schulz, R., 2013. Entry and toxicity of organic pesticides and copper in.vineyard streams: Erosion rills jeopardise the efficiency of riparian buffer strips. Agriculture, Ecosystems & Environment 172, 49-50.
Binelli, A., Provini, A., 2003. DDT is still a problem in developed countries: the heavy pollution of Lake Maggiore. Chemosphere 52, 717-723.
Bjorge, C., Brunborg, G., Wiger, R., Holme, J.A., Scholz, T., Dybing, E., Soderlund, E.J., 1996. A comparative study of chemically induced DNA damage in isolated human and rat testicular cells. Reproductive Toxicology 10, 509-519.
Bliss, C.I., 1939. The toxicity of poisons applied jointly. Annals of Applied Biology 26, 585-615.
Cabras, P., Garau, V.L., Angioni, A., Farris, G.A., Budroni, M., Spanedda, L., 1995.
Interactions during Fermentation between Pesticides and Enological Yeasts Producing H2S and SO2. Applied Microbiology and Biotechnology 43, 370-373.
Canton, J.H., 1976. The toxicity of benomyl, thiophanate-methyl, and BCM to four freshwater organisms. Bulletin of Environmental Contamination and Toxicology 16, 214-224.
Carter, S.D., Hein, J.F., Rehnberg, G.L., Laskey, J.W., 1984. Effect of benomyl on the reproductive development of male-rats. Journal of Toxicology and
71
Environmental Health 13, 53-68.
Cedergreen, N., Kamper, A., Streibig, J.C., 2006. Is prochloraz a potent synergist across aquatic species? A study on bacteria, daphnia, algae and higher plants.
Aquatic Toxicology 78, 243-252.
Chen, W.C., Yen, J.H., Chang, C.S., Wang, Y.S., 2009. Effects of herbicide butachlor on soil microorganisms and on nitrogen-fixing abilities in paddy soil.
Ecotoxicology and Environmental Safety 72, 120-127.
Chiba, M., Singh, R.P., 1986. High-performance liquid-chromatographic method for simultaneous determination of benomyl and carbendazim in aqueous-media.
Journal of Agricultural and Food Chemistry 34, 108-112.
Cuppen, J.G.M., Van den Brink, P.J., Camps, E., Uil, K.F., Brock, T.C.M., 2000.
Impact of the fungicide carbendazim in freshwater microcosms. I. Water quality, breakdown of particulate organic matter and responses of macroinvertebrates.
Aquatic Toxicology 48, 233-250.
Drescher, K., Boedeker, W., 1995. Assessment of the combined effects of substances - the relationship between concentration addition and independent action.
Biometrics 51, 716-730.
Hess, R.A., Nakai, M., 2000. Histopathology of the male reproductive system induced by the fungicide benomyl. Histology and Histopathology 15, 207-224.
Holtman, M.A., Kobayashi, D.Y., 1997. Identification of Rhodococccus erythropolis isolates capable of degrading the fungicide carbendazim. Applied Microbiology and Biotechnology 47, 578-582.
Iwasa, T., Motoyama, N., Ambrose, J.T., Roe, R.M., 2004. Mechanism for the
differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera.
Crop Protection 23, 371-378.
Kapoor, U., Srivastava, M.K., Srivastava, L.P., 2011. Toxicological impact of
72
technical imidacloprid on ovarian morphology, hormones and antioxidant enzymes in female rats. Food and Chemical Toxicology 49, 3086-3089.
Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: a national reconnaissance.
Environmental Science & Technology 36, 1202-1211.
Krueger, D.A., Dodson, S.I., 1981. Embryological induction and predation ecology in Daphnia pulex. Association for the Sciences of Limnology and Oceanography 26, 219-223.
Li, Y., Yang, H.-, Zhang, X., Yu, Y., Li, J., 2007. Characteristics of adsorption and desorption of lead on live Chlorella vulgaris. Biotechnology 6, 029.
Lim, J.H., Miller, M.G., 1997. The role of the benomyl metabolite carbendazim in benomyl-induced testicular toxicity. Toxicology and Applied Pharmacology 142, 401-410.
Loewe, S., 1953. The problem of synergism and antagonism of combined drugs.
Arzneimittel Forschung 3, 285-290.
Lukancic, S., Zibrat, U., Mezek, T., Jerebic, A., Simcic, T., Brancelj, A., 2010. Effects of exposing two non-target crustacean species, Asellus aquaticus L., and
Gammarus fossarum Koch., to atrazine and imidacloprid. Bulletin of Environmental Contamination and Toxicology 84, 85-90.
Lydy, M.J., Austin, K.R., 2004. Toxicity assessment of pesticide mixtures typical of the Sacramento-San Joaquin Delta using Chironomus tentans. Archives of Environmental Contamination and Toxicology 48, 49-55.
Ma, J.Y., Xu, L.G., Wang, S.F., Zheng, R.Q., Jin, S.H., Huang, S.Q., Huang, Y.J., 2002a. Toxicity of 40 herbicides to the green alga Chlorella vulgaris.
Ecotoxicology and Environmental Safety 51, 128-132.
73
Ma, J.Y., Zheng, R.Q., Xu, L.G., Wang, S.F., 2002b. Differential sensitivity of two green algae, Scenedesmus obliqnus and Chlorella pyrenoidosa, to 12 pesticides.
Ecotoxicology and Environmental Safety 52, 57-61.
Malev, O., Klobucar, R.S., Fabbretti, E., Trebse, P., 2012. Comparative toxicity of imidacloprid and its transformation product 6-chloronicotinic acid to non-target aquatic organisms: Microalgae Desmodesmus subspicatus and amphipod Gammarus fossarum. Pesticide Biochemistry and Physiology 104, 178-186.
Markelewicz, R.J., Hall, S.J., Boekelheide, K., 2004. 2,5-hexanedione and
carbendazim coexposure synergistically disrupts rat spermatogenesis despite opposing molecular effects on microtubules. Toxicological Sciences 80, 92-100.
Marking, L., 1985. Toxicity of chemical mixtures. Fundamentals of Aquatic Toxicology: Methods and Applications. Hemisphere Publishing Corporation Washington DC. 1985. p 164-176, 2 fig, 3 tab, 67 ref.
Mazellier, P., Leroy, E., Legube, B., 2002. Photochemical behavior of the fungicide carbendazim in dilute aqueous solution. Journal of Photochemistry and
Photobiology a-Chemistry 153, 221-227.
Nakai, M., Hess, R.A., Moore, B.J., Guttroff, R.F., Strader, L.F., Linder, R.E., 1992.
Acute and long-term effects of a single dose of the fungicide carbendazim (methyl 2-benzimidazole carbamate) on the male reproductive-system in the rat.
Journal of Andrology 13, 507-518.
Nakai, M., Moore, B.J., Hess, R.A., 1993. Epithelial reorganization and irregular growth following carbendazim-induced injury of the efferent ductules of the rat testis. Anatomical Record 235, 51-60.
Nauen, R., Ebbinghaus-Kintscher, U., Schmuck, R., 2001. Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera : Apidae). Pest Management Science 57, 577-586.
74
Norgaard, K.B., Cedergreen, N., 2010. Pesticide cocktails can interact synergistically on aquatic crustaceans. Environmental Science and Pollution Research 17, 957-967.
Ralston-Hooper, K., Hardy, J., Hahn, L., Ochoa-Acuna, H., Lee, L.S., Mollenhauer, R., Sepulveda, M.S., 2009. Acute and chronic toxicity of atrazine and its metabolites deethylatrazine and deisopropylatrazine on aquatic organisms. Ecotoxicology 18, 899-905.
Rehnberg, G.L., Cooper, R.L., Goldman, J.M., Gray, L.E., Hein, J.F., McElroy, W.K., 1989. Serum and testicular testosterone and androgen binding-protein profiles following subchronic treatment with carbendazim. Toxicology and Applied Pharmacology 101, 55-61.
Ritz, C., Streibig, J.C., 2005. Bioassay analysis using R. Journal of Statistical Software 12, 1-22.
Roast, S.D., Widdows, J., Jones, M.B., 2000. Disruption of swimming in the hyperbenthic mysid Neomysis integer (Peracarida : Mysidacea) by the organophosphate pesticide chlorpyrifos. Aquatic Toxicology 47, 227-241.
Rosenberg, D.M., Resh, V.H., 1993. Introduction To Freshwater Biomonitoring And Benthic Macroinvertebrates.
Schaefer, R.B., von der Ohe, P.C., Rasmussen, J., Kefford, B.J., Beketov, M.A., Schulz, R., Liess, M., 2012. Thresholds for the Effects of Pesticides on Invertebrate Communities and Leaf Breakdown in Stream Ecosystems.
Environmental Science & Technology 46, 5134-5142.
Strong, D.R., 1972. Life-history variation among populations of an amphipod (Hyalella azteca). Ecology 53, 1103-1111.
Tomizawa, M., Casida, J.E., 2003. Selective toxicity of neonicotinoids attributable to specificity of insect and mammalian nicotinic receptors. Annual Review of
75
Entomology 48, 339-364.
van Wijngaarden, R.P.A., Crum, S.J.H., Decraene, K., Hattink, J., van Kammen, A., 1998. Toxicity of Derosal (active ingredient carbendazim) to aquatic
invertebrates. Chemosphere 37, 673-683.
Wendt-Rasch, L., Van den Brink, P.J., Crum, S.J.H., Woin, P., 2004. The effects of a pesticide mixture on aquatic ecosystems differing in trophic status: responses of the macrophyte Myriophyllum spicatum and the periphytic algal community.
Ecotoxicology and Environmental Safety 57, 383-398.
Woods, M., Kumar, A., Correll, R., 2002. Acute toxicity of mixtures of chlorpyrifos, profenofos, and endosulfan to Ceriodaphnia dubia. Bulletin of Environmental Contamination and Toxicology 68, 801-808.
Zubrod, J.P., Baudy, P., Schulz, R., Bundschuh, M., 2014. Effects of current-use fungicides and their mixtures on the feeding and survival of the key shredder Gammarus fossarum. Aquatic Toxicology 150, 133-143.