标准编号:	GB/T 31270.12-2014
中文名称:	化学农药环境安全评价试验准则 第12部分：鱼类急性毒性试验
英文名称:	Test guidelines on environmental safety assessment for chemical pesticides. Part 12: Fish acute toxicity test
中标分类:	B13    肥料与土壤调理剂
行业分类:	GB    国家标准
ICS分类:	65.100 65.100    杀虫剂和其他农用化工产品 65.100
发布机构:	中华人民共和国国家质量监督检验检疫总局 中国国家标准化管理委员会
发布日期:	2014-10-10
实施日期:	2015-3-11
标准状态:	valid
翻译语言:	英文
文件格式:	PDF
中文字符数:	11000 字
翻译价格(元):	400.0
交付时间:	付款后 1 个工作日

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 中华人民共和国国家标准 GB/T 31270.12—2014 化学农药环境安全评价试验准则 第12部分：鱼类急性毒性试验 Test guidelines on environmental safety assessment for chemical pesticides—Part 12：Fish acute toxicity test 2014—10—10发布 2015—03—11实施 中华人民共和国国家质量监督检验检疫总局 中国国家标准化管理委员会 发布 前言 GB/T 31270《化学农药环境安全评价试验准则》分为21个部分： ——第1部分：土壤降解试验； ——第2部分：水解试验； ——第3部分：光解试验； ——第4部分：土壤吸附/解吸试验； ——第5部分：土壤淋溶试验； ——第6部分：挥发性试验； ——第7部分：生物富集试验； ——第8部分：水-沉积物系统降解试验； ——第9部分：鸟类急性毒性试验； ——第10部分：蜜蜂急性毒性试验； ——第11部分：家蚕急性毒性试验； ——第12部分：鱼类急性毒性试验； ——第13部分：溞类急性活动抑制试验； ——第14部分：藻类生长抑制试验； ——第15部分：蚯蚓急性毒性试验； ——第16部分：土壤微生物毒性试验； ——第17部分：天敌赤眼蜂急性毒性试验； ——第18部分：天敌两栖类急性毒性试验； ——第19部分：非靶标植物影响试验； ——第20部分：家畜短期饲喂毒性试验； ——第21部分：大型甲壳类生物毒性试验。 本部分是GB/T 31270的第12部分。 本部分按照GB/T 1.1—2009给出的规则起草。 请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。 本部分由中华人民共和国农业部提出并归口。 化学农药环境安全评价试验准则 第12部分：鱼类急性毒性试验 1 范围 GB/T 31270的本部分规定了化学农药鱼类急性毒性试验的材料、条件、操作、质量控制、数据处理、试验报告等的基本要求。 本部分适用于为化学农药登记而进行的鱼类急性毒性试验，其他类型的农药可参照使用。 本部分不适用于易挥发和难溶解的化学农药。 2术语和定义 下列术语和定义适用于本文件。 2.1 半致死浓度median lethal concentration 在急性毒性试验中，引起50％的供试生物死亡时的供试物浓度，用LC50表示。 注：单位是mg a.i./L。 2.2 供试物test substance 试验中需要测试的物质。 2.3 化学农药 chemical pesticide 利用化学物质人工合成的农药。其中有些以天然产品中的活性物质为母体，进行仿制、结构改造，创新而成，为仿生合成农药。 同义词：有机合成农药synthetic organic pesticide。 [NY/T 1667.1—2008，定义2.3.1] 2.4 原药technical material 在制造过程中得到的有效成分及杂质组成的最终产品，不能含有可见的外来物质和任何添加物，必要时可加入少量的稳定剂。 [NY/T 1667.2—2008，定义2.5.1] 2.5 制剂 formulation 由农药原药(或母药)和助剂制成使用状态稳定的产品。 [NY/T 1667.2—2008，定义2.4] 2.6 有效成分 active ingredient；a.i. 农药产品中具有生物活性的特定化学结构成分。 [NY/T 1667.2—2008，定义3.1] 2.7 静态试验法static test 试验期间不更换试验药液。 2.8 半静态试验法semi-static test 试验期间每隔一定时间(如24 h)更换一次药液，以保持试验药液的浓度不低于初始浓度的80％ 2.9 流水式试验法flow-through test 试验期间药液连续更新。 2.10 参比物质 reference substances 在测试中为证实或否定供试物的某种特性或判断测试系统有效性而使用的化学物质或混合物。 3试验概述 鱼类急性毒性测定方法有静态法、半静态法与流水式试验法三种。应按供试物的性质采用适宜的方法。分别配制不同浓度的供试物药液，于96 h的试验期间每天观察并记录试验用鱼的中毒症状和死亡数，并求出24 h、48 h、72 h和96 h的LC50值及95％置信限。 4试验方法 4.1材料和条件 4.1.1供试生物 推荐鱼种为斑马鱼(Brachydanio rerio)、鲤鱼(Cyprinus carpio)、虹鳟鱼(Oncothynchus mykiss)、青鳉(Oryzias latipes)或稀有鮈鲫(Gobiocypris rarus)等的幼鱼，具体全长和适宜水温参见附录A。如果选用其他鱼类作为试验材料，应采用能够满足其生理要求的相应预养和试验条件，并加以说明。 试验用鱼应健康无病，大小一致。试验前应在与试验时相同的环境条件下预养7 d～14 d，预养期间每天喂食1次～2次，每日光照12 h～16 h，及时清除粪便及食物残渣。试验前24 h停止喂食。 4.1.2供试物 供试物应使用农药纯品、原药或制剂。对难溶于水的农药，可用少量对鱼低毒的有机溶剂、乳化剂或分散剂助溶，其用量不得超过0.1 mL(g)/L。 4.1.3参比物质 重铬酸钾(分析纯以上)。 4.1.4主要仪器设备 主要仪器设备如下： ——溶解氧测定仪； ——电子天平； ——温度计； ——酸度计； ——满足最大承载量的玻璃容器； ——量筒等。 4.1.5试验用水 试验用水为存放并去氯处理24 h以上的自来水(必要时经活性炭处理)或能注明配方的稀释水。水质硬度在10 mg/L～250 mg/L之间(以CaCO3计)，pH在6.0～8.5之间，溶解氧不低于空气饱和值的60％。试验水温参见附录A(单次试验温度控制在±2℃)。 4.2试验操作 4.2.1方法的选择 按农药的特性选择静态试验法、半静态试验法或流水式试验法。如使用静态或半静态试验法，应确保试验期间试验药液中供试物浓度不低于初始浓度的80％。如果在流水式试验法试验期间试验药液中供试物浓度发生超过20％的偏离，则应检测试验药液中供试物的实际浓度并以此计算结果，或使用流动试验法进行试验，以稳定试验药液中供试物浓度。 4.2.2 预试验 按正式试验的条件，以较大的间距设若干组浓度。每处理至少用鱼5尾，可不设重复，观察并记录试验用鱼96 h(或48 h)的中毒症状和死亡情况。通过预试验求出试验用鱼的最高全存活浓度及最低全致死浓度，在此范围内设置正式试验的浓度。 4.2.3 正式试验 在预试验确定的浓度范围内按一定比例间距(几何级差应控制在2.2倍以内)设置5个～7个浓度组，并设一个空白对照组，若使用溶剂助溶应增设溶剂对照组，每组至少放入7尾鱼，可不设重复，并保证各组使用鱼数相同，试验开始后6 h内随时观察并记录试验用鱼的中毒症状及死亡数，其后于24 h、48 h、72 h和96 h观察并记录试验用鱼的中毒症状及死亡数，当用玻璃棒轻触鱼尾部，无可见运动即为死亡，并及时清除死鱼。每天测定并记录试验药液温度、pH及溶解氧。 4.2.4限度试验 设置上限有效浓度100 mg a.i./L，即试验用鱼在供试物浓度达100 mg a.i./L时未出现死亡，则无需继续试验。若供试物溶解度小于100 mg a.i./L，则采用其最大溶解度作为上限浓度。 4.3数据处理 4.3.1 统计分析方法的选择 可采用寇氏法、直线内插法或概率单位图解法计算每一观察时间(24 h、48 h、72 h、96 h)的鱼类急性毒性的半致死浓度LC50，也可采用数据统计软件进行分析和计算。 4.3.2 寇氏法 用寇氏法可求出鱼类在24 h、48 h、72 h和96 h的LC50值及95％置信限。LC50的计算见式(1)： logLC50＝Xm-i(∑P-0.5) (1) 式中： Xm——最高浓度的对数； i——相邻浓度比值的对数； ∑P——各组死亡率的总和(以小数表示)。 95％置信限的计算见式(2)： 95％置信限＝logLC50±1.96SlogLC50 (2) 标准误的计算见式(3)： (3) 式中： p——1个组的死亡率； q——1-p； n——各浓度组鱼类的数量。 4.3.3直线内插法 采用线性刻度坐标，绘制试验物质浓度对死亡百分率的曲线，求出50％死亡时的LC50值。 4.3.4概率单位图解法 用半对数纸，以浓度对数为横坐标、死亡百分率对应的概率单位为纵坐标绘图。将各实测值在图上用目测法画一条相关直线，从直线中读出致死50％的浓度对数，估算出LC50值。 4.4质量控制 质量控制条件包括： ——试验用鱼预养期间死亡率不得超过5％，对照组死亡率不超过10％，若鱼的数量少于10条，则最多允许死亡1条； ——试验期间，试验溶液的溶解氧含量应不低于空气饱和值的60％； ——实验室内用重铬酸钾定期(每批1次或者至少1年两次)进行参比物质试验，对于斑马鱼，LC50(24 h)应处于200 mg/L～400 mg/L之间； ——静态试验法和半静态试验法的最大承载量为1.0 g鱼/L水，流水式试验系统最大承载量可高一些。 5试验报告 试验报告至少应包括下列内容： ——供试物的信息，包括供试农药的通用名、化学名称、结构式、CAS号、纯度、基本理化性质、来源等； ——供试生物名称、来源、大小及预养情况； ——试验条件，包括试验温度、光照等，试验用水的温度、溶解氧浓度及pH等； ——试验药液的浓度及24 h、48 h、72 h和96 h的LC50值和95％置信限，并给出所采用的计算方法； ——对照组试验用鱼的死亡率、行为反应异常； ——试验质量控制条件描述； ——鱼的毒性等级划分参见附录B。 附 录 A (资料性附录) 试验用鱼体长要求及适宜温度条件 根据供试鱼的种类选择适宜全长的幼鱼及合适的水温进行试验(见表A.1)。 表A.1 试验用鱼的全长和适宜水温 鱼 种 全长/cm 适宜水温/℃ 斑马鱼 2.0±1.0 21～25 虹鳟鱼 5.0±1.0 13～17 青鳉 2.0±1.0 21～25 鲤鱼 3.0±1.0 20～24 稀有鮈鲫 3.0±1.0 21～25 附 录 B (资料性附录) 农药对鱼类毒性等级划分 按鱼类半致死浓度LC50(96 h)值，将农药对鱼类毒性分为四个等级，见表B.1。 表B.1 农药对鱼类的毒性等级划分 毒性等级 LC50(96 h)/(mg a.i./L) 剧毒 LC50≤0.1 高毒 0.110 参考文献 [1]NY/T 1667.1—2008农药登记管理术语 第1部分：基本术语 [2]NY/T 1667.2—2008农药登记管理术语 第2部分：产品化学 [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.农药环境毒理学研究.北京：中国环境科学出版社.1999.