標準編號:	GB/T 31270.12-2014
中文名稱:	化學農藥環境安全評價試驗準則 第12部分：魚類急性毒性試驗
英文名稱:	Test guidelines on environmental safety assessment for chemical pesticides. Part 12: Fish acute toxicity test
中標分類:	B13    肥料與土壤調理劑
行業分類:	GB    國家標準
ICS分類:	65.100 65.100    殺蟲劑和其他農用化工產品 65.100
發布機構:	中華人民共和國國家質量監督檢驗檢疫總局 中國國家標準化管理委員會
發布日期:	2014-10-10
實施日期:	2015-3-11
標準狀態:	valid
翻譯語言:	英文
文件格式:	PDF
中文字符數:	11000 字
翻譯價格(元):	400.0
交付時間:	付款后 1 個工作日

Codeofchina.com is in charge of this English translation. In case of any doubt about the English translation, the Chinese original shall be considered authoritative. GB/T 31270 consists of 21 parts under the general title Test guidelines on environmental safety assessment for chemical pesticides: ——Part 1: Transformation in soils; ——Part 2: Hydrolysis; ——Part 3: Phototransformation; ——Part 4: Adsorption/desorption; ——Part 5: Leaching in soil; ——Part 6: Volatility; ——Part 7: Bioconcentration test; ——Part 8: Degradation in water-sediment systems; ——Part 9: Avian acute toxicity test; ——Part 10: Honeybee acute toxicity test; ——Part 11: Silkworm acute toxicity test; ——Part 12: Fish acute toxicity test; ——Part 13: Daphnia sp.acute immobilisation test; ——Part 14: Alga growth inhibition test; ——Part 15: Earthworm acute toxicity test; ——Part 16: Soil microorganism toxicity test; ——Part 17: Trichogramma acute toxicity test; ——Part 18: Amphibian acute toxicity test; ——Part 19: Effects on non-target plants; ——Part 20: Livestock short-term dietary toxicity test; ——Part 21: Macro-crustacean toxicity test. This is Part 12 of GB/T 31270. This part is developed in accordance with the rules given in GB/T 1.1-2009. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. The issuing body of this document shall not be held responsible for identifying any or all such patent rights. This standard was proposed by and is under the jurisdiction of the Ministry of Agriculture of the People's Republic of China. Test guidelines on environmental safety assessment for chemical pesticides - Part 12: Fish acute toxicity test 1 Scope This part of GB/T 31270 specifies the basic requirements for materials, conditions, operation, quality control, data processing, test reports, etc. for acute toxicity test of chemical pesticides on fish. This part applies to fish acute toxicity test for the registration of chemical pesticides. It may be used as reference for other types of pesticides. This part does not apply to volatile and insoluble chemical pesticides. 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 2.1 median lethal concentration concentration of the test substance, at which 50% of the test organisms die in the acute toxicity test, expressed by LC50 Note: the unit is mg a.i./L. 2.2 test substance substance to be tested 2.3 chemical pesticide pesticide made of chemical substances through artificial synthesis, for some, the active substances in natural products are used as the mother body, and then subjected to imitation, structural transformation and innovation, they are bionic synthetic pesticides Synonym: synthetic organic pesticide. [Definition 2.3.1, NY/T 1667.1-2008] 2.4 technical material final product consisting of the active ingredients and impurities obtained during the manufacturing process, which shall not contain visible foreign substances or any additives, and if necessary, a small amount of stabilizer may be added [Definition 2.5.1, NY/T 1667.2-2008] 2.5 formulation product stable in use and made of technical material (or technical concentrate) of pesticides and auxiliaries [Definition 2.4, NY/T 1667.2-2008] 2.6 active ingredient; a.i. specific chemical ingredient that is biologically active in a pesticide product [Definition 3.1, NY/T 1667.2-2008] 2.7 static test test liquid is not replaced during the test 2.8 semi-static test test liquid is replaced at regular intervals (e.g. 24h) during the test to keep the concentration of the test liquid not lower than 80% of the initial concentration 2.9 flow-through test test liquid is continuously updated during the test 2.10 reference substances chemical substance or mixture used in a test to confirm or deny certain characteristics of a test substance or to judge the effectiveness of a test system 3 Test overview There are three methods for determining fish acute toxicity: static test, semi-static test and flow-through test. Appropriate method shall be selected depending on the nature of the test substance. Different concentrations of the test substance liquids are prepared separately. The toxicity symptoms and death count of test fish are observed and recorded every day during 96h test period to calculate LC50 and 95% confidence limits at 24h, 48h, 72h and 96h. 4 Test methods 4.1 Materials and conditions 4.1.1 Test organism Juvenile fishes of Brachydanio rerio, Cyprinus carpio, Oncothynchus mykiss, Oryzias latipes or Gobiocypris rarus are recommended as fish species; see Annex A for the specific full length and suitable water temperature. If other fishes are used as test materials, the corresponding pre-culture and test conditions meeting their physiological requirements shall be used and explanation shall be given. The test fish shall be healthy and disease-free and be uniform in size. Before the test, the fishes shall be pre-cultured for 7d ~ 14d under the same environmental conditions as the test. During pre-culture period, the fishes are fed once or twice a day, lit for 12h ~16h every day, and feces and food residues are removed in time. Feeding is stopped 24h before the test. 4.1.2 Test substance Pure pesticides, technical materials or formulation products shall be used as test substance. For insoluble pesticides, a small amount of organic solvents, emulsifiers or dispersing agents which are low in toxicity to fish may be used, and the amount thereof shall not exceed 0.1mL(g)/L. 4.1.3 Reference substance Potassium dichromate (superior to analytically pure). 4.1.4 Main apparatus and instruments Main apparatus and instruments are as follows: ——dissolved oxygen meter; ——electronic balance; ——thermometer; ——acidometer; ——glass containers meeting the maximum carrying capacity; ——measuring cylinders, etc. 4.1.5 Test water Tap water that has been stored and dechlorinated for more than 24h (if necessary, treated with activated carbon) or diluted water marked with the formula is used as test water. The water hardness is between 10mg/L and 250mg/L (calculated as CaCO3), pH is between 6.0 and 8.5, and the dissolved oxygen is not less than 60% of the oxygen saturation value in air. See Annex A for the test water temperature (single test temperature is controlled at ±2℃). 4.2 Test operations 4.2.1 Selection of test method Static test, semi-static test or flow-through test may be selected according to the characteristics of the pesticide. If static or semi-static test is used, it shall be ensured that the concentration of the test substance in the test liquid during the test is not lower than 80% of the initial concentration. If the concentration of the test substance in the test liquid has a deviation of more than 20% during flow-through test, the actual concentration of the test substance in the test liquid shall be checked and the result calculated, or flow test is used to stabilize the concentration of the test substance in the test liquid. 4.2.2 Pre-test A number of concentrations at a large difference are set according to the formal test conditions. At least 5 fishes are used for treatment without replicate; the toxicity symptoms and death conditions of the test fish in 96h (or 48h) are observed and recorded. The highest total survival concentration and the lowest total lethal concentration for the test fish are determined by pre-test, and the concentration of the formal test is set within this range. 4.2.3 Formal test 5 to 7 concentration groups are set based on certain proportional difference (geometrical difference shall be controlled within 2.2 times) within a certain range of concentration determined by the pre-test, and a blank control group is also set. If solvent cosolvent is used, a solvent control group shall be added. At least 7 fishes are placed in each group without replicate, and it shall be ensured that the number of fish used in each group is the same. The toxicity symptoms and death counts of the test fishes are observed and recorded at any time within 6h after the start of the test and at 24h, 48h, 72h and 96h. If the fish has no visible movement by touching the fish tail with a glass rod, the fish is deemed to be dead and shall be removed in time. The test liquid temperature, pH and dissolved oxygen are measured and recorded daily. 4.2.4 Limit test The upper effective concentration limit is set as 100mg a.i./L, that is, if the test fish does not die when the concentration of the test substance reaches 100mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 100mg a.i./L, the maximum solubility is used as the upper concentration limit. 4.3 Data processing 4.3.1 Selection of statistical analysis method Karber method, linear interpolation method or probit graphical method may be adopted to calculate the median lethal concentration LC50 of pesticide toxicity on fish at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation. 4.3.2 Karber method The LC50 and 95% confidence limits of fish at 24h, 48h, 72h and 96h are obtained by Karber method. LC50 is calculated using Equation (1): logLC50＝Xm-i(∑P-0.5) (1) Where, Xm——the logarithm of the highest concentration; i——the logarithm of the ratio of two adjacent concentrations; ∑P——the sum of the death rate of each group (in decimal). 95% confidence limit is calculated using Equation (2): 95% confidence limit = logLC50±1.96SlogLC50 (2) Standard error is calculated using Equation (3): (3) Where, p——the death rate of one group; q——1-p; n——the number of fishes in each concentration group. 4.3.3 Linear interpolation method The linear scale coordinates are used to plot the curve for concentration of the test substance versus the death rate, and the LC50 at 50% death rate is calculated. 4.3.4 Probit graphical method A graph with the logarithm concentration as X-coordinate and the probit corresponding to the death rate as Y-coordinate is plotted in the semi-logarithmic paper. A straight line is drawn on the graph based on the measured values by visual method, and the logarithm concentration at 50% lethality is obtained from the straight line to estimate LC50.

Foreword i 1 Scope 2 Terms and definitions 3 Test overview 4 Test methods 5 Test report Annex A (Informative) Body length and suitable temperature conditions for test fish Annex B (Informative) Classification of toxicity levels of pesticides to fish Bibliography

ICS 65.100 B 13 中華人民共和國國家標準 GB/T 31270.12—2014 化學農藥環境安全評價試驗準則 第12部分：魚類急性毒性試驗 Test guidelines on environmental safety assessment for chemical pesticides—Part 12：Fish acute toxicity test 2014—10—10發布 2015—03—11實施 中華人民共和國國家質量監督檢驗檢疫總局 中國國家標準化管理委員會 發布 前言 GB/T 31270《化學農藥環境安全評價試驗準則》分為21個部分： ——第1部分：土壤降解試驗； ——第2部分：水解試驗； ——第3部分：光解試驗； ——第4部分：土壤吸附/解吸試驗； ——第5部分：土壤淋溶試驗； ——第6部分：揮發性試驗； ——第7部分：生物富集試驗； ——第8部分：水-沉積物系統降解試驗； ——第9部分：鳥類急性毒性試驗； ——第10部分：蜜蜂急性毒性試驗； ——第11部分：家蠶急性毒性試驗； ——第12部分：魚類急性毒性試驗； ——第13部分：溞類急性活動抑制試驗； ——第14部分：藻類生長抑制試驗； ——第15部分：蚯蚓急性毒性試驗； ——第16部分：土壤微生物毒性試驗； ——第17部分：天敵赤眼蜂急性毒性試驗； ——第18部分：天敵兩棲類急性毒性試驗； ——第19部分：非靶標植物影響試驗； ——第20部分：家畜短期飼喂毒性試驗； ——第21部分：大型甲殼類生物毒性試驗。 本部分是GB/T 31270的第12部分。 本部分按照GB/T 1.1—2009給出的規則起草。 請注意本文件的某些內容可能涉及專利。本文件的發布機構不承擔識別這些專利的責任。 本部分由中華人民共和國農業部提出并歸口。 化學農藥環境安全評價試驗準則 第12部分：魚類急性毒性試驗 1 范圍 GB/T 31270的本部分規定了化學農藥魚類急性毒性試驗的材料、條件、操作、質量控制、數據處理、試驗報告等的基本要求。 本部分適用于為化學農藥登記而進行的魚類急性毒性試驗，其他類型的農藥可參照使用。 本部分不適用于易揮發和難溶解的化學農藥。 2術語和定義 下列術語和定義適用于本文件。 2.1 半致死濃度median lethal concentration 在急性毒性試驗中，引起50％的供試生物死亡時的供試物濃度，用LC50表示。 注：單位是mg a.i./L。 2.2 供試物test substance 試驗中需要測試的物質。 2.3 化學農藥 chemical pesticide 利用化學物質人工合成的農藥。其中有些以天然產品中的活性物質為母體，進行仿制、結構改造，創新而成，為仿生合成農藥。 同義詞：有機合成農藥synthetic organic pesticide。 [NY/T 1667.1—2008，定義2.3.1] 2.4 原藥technical material 在制造過程中得到的有效成分及雜質組成的最終產品，不能含有可見的外來物質和任何添加物，必要時可加入少量的穩定劑。 [NY/T 1667.2—2008，定義2.5.1] 2.5 制劑 formulation 由農藥原藥(或母藥)和助劑制成使用狀態穩定的產品。 [NY/T 1667.2—2008，定義2.4] 2.6 有效成分 active ingredient；a.i. 農藥產品中具有生物活性的特定化學結構成分。 [NY/T 1667.2—2008，定義3.1] 2.7 靜態試驗法static test 試驗期間不更換試驗藥液。 2.8 半靜態試驗法semi-static test 試驗期間每隔一定時間(如24 h)更換一次藥液，以保持試驗藥液的濃度不低于初始濃度的80％ 2.9 流水式試驗法flow-through test 試驗期間藥液連續更新。 2.10 參比物質 reference substances 在測試中為證實或否定供試物的某種特性或判斷測試系統有效性而使用的化學物質或混合物。 3試驗概述 魚類急性毒性測定方法有靜態法、半靜態法與流水式試驗法三種。應按供試物的性質采用適宜的方法。分別配制不同濃度的供試物藥液，于96 h的試驗期間每天觀察并記錄試驗用魚的中毒癥狀和死亡數，并求出24 h、48 h、72 h和96 h的LC50值及95％置信限。 4試驗方法 4.1材料和條件 4.1.1供試生物 推薦魚種為斑馬魚(Brachydanio rerio)、鯉魚(Cyprinus carpio)、虹鱒魚(Oncothynchus mykiss)、青鳉(Oryzias latipes)或稀有鮈鯽(Gobiocypris rarus)等的幼魚，具體全長和適宜水溫參見附錄A。如果選用其他魚類作為試驗材料，應采用能夠滿足其生理要求的相應預養和試驗條件，并加以說明。 試驗用魚應健康無病，大小一致。試驗前應在與試驗時相同的環境條件下預養7 d～14 d，預養期間每天喂食1次～2次，每日光照12 h～16 h，及時清除糞便及食物殘渣。試驗前24 h停止喂食。 4.1.2供試物 供試物應使用農藥純品、原藥或制劑。對難溶于水的農藥，可用少量對魚低毒的有機溶劑、乳化劑或分散劑助溶，其用量不得超過0.1 mL(g)/L。 4.1.3參比物質 重鉻酸鉀(分析純以上)。 4.1.4主要儀器設備 主要儀器設備如下： ——溶解氧測定儀； ——電子天平； ——溫度計； ——酸度計； ——滿足最大承載量的玻璃容器； ——量筒等。 4.1.5試驗用水 試驗用水為存放并去氯處理24 h以上的自來水(必要時經活性炭處理)或能注明配方的稀釋水。水質硬度在10 mg/L～250 mg/L之間(以CaCO3計)，pH在6.0～8.5之間，溶解氧不低于空氣飽和值的60％。試驗水溫參見附錄A(單次試驗溫度控制在±2℃)。 4.2試驗操作 4.2.1方法的選擇 按農藥的特性選擇靜態試驗法、半靜態試驗法或流水式試驗法。如使用靜態或半靜態試驗法，應確保試驗期間試驗藥液中供試物濃度不低于初始濃度的80％。如果在流水式試驗法試驗期間試驗藥液中供試物濃度發生超過20％的偏離，則應檢測試驗藥液中供試物的實際濃度并以此計算結果，或使用流動試驗法進行試驗，以穩定試驗藥液中供試物濃度。 4.2.2 預試驗 按正式試驗的條件，以較大的間距設若干組濃度。每處理至少用魚5尾，可不設重復，觀察并記錄試驗用魚96 h(或48 h)的中毒癥狀和死亡情況。通過預試驗求出試驗用魚的最高全存活濃度及最低全致死濃度，在此范圍內設置正式試驗的濃度。 4.2.3 正式試驗 在預試驗確定的濃度范圍內按一定比例間距(幾何級差應控制在2.2倍以內)設置5個～7個濃度組，并設一個空白對照組，若使用溶劑助溶應增設溶劑對照組，每組至少放入7尾魚，可不設重復，并保證各組使用魚數相同，試驗開始后6 h內隨時觀察并記錄試驗用魚的中毒癥狀及死亡數，其后于24 h、48 h、72 h和96 h觀察并記錄試驗用魚的中毒癥狀及死亡數，當用玻璃棒輕觸魚尾部，無可見運動即為死亡，并及時清除死魚。每天測定并記錄試驗藥液溫度、pH及溶解氧。 4.2.4限度試驗 設置上限有效濃度100 mg a.i./L，即試驗用魚在供試物濃度達100 mg a.i./L時未出現死亡，則無需繼續試驗。若供試物溶解度小于100 mg a.i./L，則采用其最大溶解度作為上限濃度。 4.3數據處理 4.3.1 統計分析方法的選擇 可采用寇氏法、直線內插法或概率單位圖解法計算每一觀察時間(24 h、48 h、72 h、96 h)的魚類急性毒性的半致死濃度LC50，也可采用數據統計軟件進行分析和計算。 4.3.2 寇氏法 用寇氏法可求出魚類在24 h、48 h、72 h和96 h的LC50值及95％置信限。LC50的計算見式(1)： logLC50＝Xm-i(∑P-0.5) (1) 式中： Xm——最高濃度的對數； i——相鄰濃度比值的對數； ∑P——各組死亡率的總和(以小數表示)。 95％置信限的計算見式(2)： 95％置信限＝logLC50±1.96SlogLC50 (2) 標準誤的計算見式(3)： (3) 式中： p——1個組的死亡率； q——1-p； n——各濃度組魚類的數量。 4.3.3直線內插法 采用線性刻度坐標，繪制試驗物質濃度對死亡百分率的曲線，求出50％死亡時的LC50值。 4.3.4概率單位圖解法 用半對數紙，以濃度對數為橫坐標、死亡百分率對應的概率單位為縱坐標繪圖。將各實測值在圖上用目測法畫一條相關直線，從直線中讀出致死50％的濃度對數，估算出LC50值。 4.4質量控制 質量控制條件包括： ——試驗用魚預養期間死亡率不得超過5％，對照組死亡率不超過10％，若魚的數量少于10條，則最多允許死亡1條； ——試驗期間，試驗溶液的溶解氧含量應不低于空氣飽和值的60％； ——實驗室內用重鉻酸鉀定期(每批1次或者至少1年兩次)進行參比物質試驗，對于斑馬魚，LC50(24 h)應處于200 mg/L～400 mg/L之間； ——靜態試驗法和半靜態試驗法的最大承載量為1.0 g魚/L水，流水式試驗系統最大承載量可高一些。 5試驗報告 試驗報告至少應包括下列內容： ——供試物的信息，包括供試農藥的通用名、化學名稱、結構式、CAS號、純度、基本理化性質、來源等； ——供試生物名稱、來源、大小及預養情況； ——試驗條件，包括試驗溫度、光照等，試驗用水的溫度、溶解氧濃度及pH等； ——試驗藥液的濃度及24 h、48 h、72 h和96 h的LC50值和95％置信限，并給出所采用的計算方法； ——對照組試驗用魚的死亡率、行為反應異常； ——試驗質量控制條件描述； ——魚的毒性等級劃分參見附錄B。 附 錄 A (資料性附錄) 試驗用魚體長要求及適宜溫度條件 根據供試魚的種類選擇適宜全長的幼魚及合適的水溫進行試驗(見表A.1)。 表A.1 試驗用魚的全長和適宜水溫 魚 種 全長/cm 適宜水溫/℃ 斑馬魚 2.0±1.0 21～25 虹鱒魚 5.0±1.0 13～17 青鳉 2.0±1.0 21～25 鯉魚 3.0±1.0 20～24 稀有鮈鯽 3.0±1.0 21～25 附 錄 B (資料性附錄) 農藥對魚類毒性等級劃分 按魚類半致死濃度LC50(96 h)值，將農藥對魚類毒性分為四個等級，見表B.1。 表B.1 農藥對魚類的毒性等級劃分 毒性等級 LC50(96 h)/(mg a.i./L) 劇毒 LC50≤0.1 高毒 0.110 參考文獻 [1]NY/T 1667.1—2008農藥登記管理術語 第1部分：基本術語 [2]NY/T 1667.2—2008農藥登記管理術語 第2部分：產品化學 [3]FAO(1989).Guidelines on environmental criteria for the registration of pesticides. Food and Agriculture Organization of the United Nations. [4]OECD(1992).Guidelines 203：Fish，acute toxicity test，OECD Guidelines for the Testing of Chemicals. [5]USEPA(1985).Part II，Toxic substances control act test Guidelines，Final Rules，Federal register. [6] 蔡道基.農藥環境毒理學研究.北京：中國環境科學出版社.1999.