标准编号:	GB/T 31270.11-2014
中文名称:	化学农药环境安全评价试验准则 第11部分：家蚕急性毒性试验
英文名称:	Test guidelines on environmental safety assessment for chemical pesticides. Part 11: Silkworm 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:	5000 字
翻译价格(元):	290.0
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 11 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 11: Silkworm 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 silkworm with leaf-dipping method and fumigation method. This part applies to silkworm acute toxicity tests with leaf-dipping method and fumigation method 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 toxicity test with leaf-dipping method, 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 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 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 3.1 Method overview The silkworm acute toxicity test is carried out by leaf-dipping method or fumigation method, and the relevant methods are selected according to the regulations of pesticide registration management and other regulations. 3.2 Leaf-dipping method The mulberry leaves are dipped into different concentrations of pesticide liquid, and dried to feed the silkworm. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed after 24h, 48h, 72h, and 96h; at the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 is calculated. 3.3 Fumigation method The test based on the pesticide application conditions in simulation chamber shall be carried out on the fumigation test device or fumigation chamber meeting the test requirements. The relevant parameters of fumigation test device or the fumigation chamber shall be designed according to the recommended dosage on the premise of meeting the test requirements. The test substance is quantitatively burned (or electrically heated) in the test device or the fumigation chamber. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 4 Test methods 4.1 Materials and conditions 4.1.1 Test organism The test silkworm (Bombyx mori) species are selected from Jingsong × Haoyue, Chunlei × Zhenzhu, Suju × Minghu, or other representative lines. The second-instar silkworm is used as materials for toxicity 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 silkworm may be used, and the amount thereof shall not exceed 0.1mL(g)/L. 4.1.3 Main apparatus and instruments Main apparatus and instruments are as follows: ——artificial climate chamber; ——electronic balance; ——culture dish; ——fumigation box, etc. 4.1.4 Test conditions Newly-hatched silkworm shall be fed at test temperature of (25±2)℃ and relative humidity of 70%～85%. 4.2 Test operations 4.2.1 Leaf-dipping method 4.2.1.1 Pre-test 3~5 concentration groups are set at a large difference according to the conditions of the formal test; the highest total survival concentration and the lowest total lethal concentration for the silkworm are calculated through pre-test. 4.2.1.2 Formal test 5~7 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, each group consists 20 silkworms and blank control is set; if solvent cosolvent is used, a solvent control group shall be added. 3 replicates are set for the control group and each concentration group. The second-instar silkworms are cultured in a culture dish, and the mulberry leaves are completely dipped into different concentrations of the pesticide liquid for 10s, and dried for use by the silkworms. The treated mulberry leaves are used for feeding throughout the test, then the toxicity symptoms and death conditions of test silkworms are observed at 24h, 48h, 72h, and 96h. At the end of the test, the data shall be subjected to statistic analysis and median lethal concentration LC50 and 95% confidence limit are calculated. If the insect growth regulator is adopted as test substance, and the death rate of silkworm increases by more than 10% between 72h and 96h, the observation time shall be extended until the death rate increases by less than 10% within 24h. 4.2.1.3 Limit test The upper concentration limit is set as 2000mg a.i./L, that is, if the silkworm does not die when the concentration of the test substance reaches 2000mg a.i./L, it is not necessary to continue the test. If the solubility of the test substance is less than 2000mg a.i./L, the maximum solubility is used as the upper concentration limit. 4.2.2 Fumigation method The fumigation test can be carried out in a fumigation box or other satisfactory test device. The test substance is quantitatively burned (or electrically heated) in the test device. From the beginning of fumigation, the toxicity symptoms of the silkworm in the fumigation test device are observed at 0.5h, 2h, 4h, 6h and 8h. After 8h, the silkworms in the test device are taken out, and the silkworm deaths at 24h and 48h are observed under the normal feeding conditions of the silkworms. 9 replicates are set for each treatment group and a blank control (3 replicates) is set at the same time. The food intake situation of silkworm (reduced or refusal), discomfort symptoms (escape, head-up, shaking head, headbang, twisting, struggling, emesis, etc.) and death conditions are observed and recorded. 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 silkworm in leaf-dipping method at each observation time (24h, 48h, 72h and 96h); data statistics software may also be adopted for analysis and calculation.

Foreword i 1 Scope 2 Terms and definitions 3 Test overview 4 Test methods 5 Test report Annex A (Informative) Classification of toxicity levels of pesticides to silkworm Bibliography

ICS 65.100 B 13 中华人民共和国国家标准 GB/T 31270.11—2014 化学农药环境安全评价试验准则 第11部分：家蚕急性毒性试验 Test guidelines on environmental safety assessment for chemical pesticides—Part 11：Silkworm 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11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11部分：家蚕急性毒性试验 1 范围 GB/T 31270的本部分规定了浸叶法和熏蒸法测定化学农药对家蚕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家蚕浸叶法毒性试验和熏蒸法毒性试验，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 在浸叶法毒性试验中，引起50％供试生物死亡时的供试物浓度，用LC50表示。 注：单位为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 product 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] Z.7 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 3.1 方法概述 家蚕急性毒性试验包括浸叶法毒性试验方法和熏蒸法毒性试验方法，根据农药登记管理法规及其他规定选择相关方法进行试验。 3.2浸叶法毒性试验 采用不同浓度的药液浸渍桑叶，晾干后饲喂家蚕。整个试验期间饲喂处理桑叶，观察24 h、48 h、72 h、96 h后受试家蚕的中毒症状及死亡情况，试验结束后对数据进行统计分析，并计算半致死浓度LC50值。 3.3熏蒸法毒性试验 针对卫生用药模拟室内施药条件进行的试验，应在满足试验要求的熏蒸试验装置或熏蒸室内进行。熏蒸试验装置或熏蒸室应在满足试验要求的前提下，按照推荐用药量设计相关参数。供试物在试验装置或熏蒸室中定量燃烧(或电加热)，从熏蒸开始，按0.5 h、2 h、4 h、6 h、8 h观察记录熏蒸试验装置内家蚕的毒性反应症状，8 h后将试验装置内的家蚕取出，在家蚕常规饲养条件下继续观察24 h及48 h的家蚕死亡情况。 4试验方法 4.1材料和条件 4.1.1供试生物 试验用家蚕(Bombyx mori)品种选用菁松×皓月，春蕾×镇珠，苏菊×明虎，或其他有代表性的品系。以二龄起蚕为毒性试验材料。 4.1.2供试物 供试物应使用农药纯品、原药或制剂。难溶于水的可用少量对家蚕毒性小的有机溶剂、乳化剂或分散剂助溶，其用量不得超过0.1 mL(g)/L。 4.1.3主要仪器设备 主要仪器设备如下： ——人工气候室； ——电子天平； ——培养皿； ——熏蒸箱等。 4.1.4试验条件 蚁蚕饲养和试验温度为(25±2)℃，相对湿度为70％～85％。 4.2试验操作 4.2.1 浸叶法 4.2.1.1 预试验 按正式试验的条件，以较大的间距设置3个～5个浓度组，通过预试验求出家蚕最高全存活浓度和最低全致死浓度。 4.2.1.2正式试验 根据预试验确定的浓度范围按一定比例间距(几何级差应控制在2.2以内)设置5个～7个浓度组，每组20头蚕，并设空白对照，加溶剂助溶的还需设溶剂对照。对照组和每一浓度组均设3个重复。在培养皿内饲养二龄起蚕，用不同浓度的药液完全浸渍桑叶10 s，晾干后供蚕食用。整个试验期间饲喂处理桑叶，观察并记录24 h、48 h、72 h和96 h试验用家蚕的中毒症状及死亡情况。试验结束后对数据进行数理统计，计算半致死浓度LC50值及95％置信限。 若供试物为昆虫生长调节剂，且试验72 h～96 h之间家蚕的死亡率增加10％以上，应延长观察时间，直至24 h内死亡率增加小于10％。 4.2.1.3 限度试验 设置上限浓度2 000 mg a.i./L，即在供试物达2 000 mg a.i./L时仍未见家蚕死亡，则无需继续进行试验。若供试物溶解度小于2 000 mg a.i./L，则采用其最大溶解度作为上限浓度。 4.2.2 熏蒸法 熏蒸试验可在熏蒸箱或其他可满足试验要求的试验装置内进行。供试物在试验装置内定量燃烧(或电热片加热)，从熏蒸开始，按0.5 h、2 h、4 h、6 h、8 h观察记录熏蒸试验装置内家蚕的毒性反应症状，8 h后，将熏蒸试验装置内的家蚕取出，在家蚕常规饲养条件下继续观察24 h及48 h的家蚕死亡情况。每个处理组设置9个重复，同时设置空白对照(设3个重复)。观察记录家蚕摄食情况(减少或拒食)、不适症状(逃避、昂头、晃头、甩头、扭曲挣扎、吐水等)及死亡情况等。 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)的家蚕浸叶法毒性的半致死浓度LC50，也可采用数据统计软件进行分析和计算。 4.3.2 寇氏法 用寇氏法可求出家蚕在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——各浓度组家蚕的数量。 4.3.3直线内插法 采用线性刻度坐标，绘制死亡百分率对试验物质浓度的曲线，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： ——对照组死亡率不超过10％； ——实验室内应定期(蚕卵每批一次，同批蚕卵至少每两个月一次)进行参比物质试验，推荐参比物质为乐果。 5试验报告 试验报告应包括下列内容： ——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； ——供试生物的名称、来源、大小及饲养情况； ——试验条件，包括试验温度、试验方法等； ——试验药液的浓度，24 h、48 h、72 h及96 h的LC50值(浸叶法)或8 h、24 h、48 h的死亡率(熏蒸法)和95％置信限，并给出所采用的计算方法； ——对照组家蚕是否出现死亡及异常反应； ——观察到的毒性效应，如受试家蚕的任何不正常的行为、中毒症状等； ——对家蚕的毒性等级划分参见附录A。 附 录 A (资料性附录) 农药对家蚕毒性等级划分 按农药对家蚕急性浸叶法的毒性半致死浓度LC50(96 h)，将农药对家蚕的急性毒性分为四级，见表A.1。 表A.1 农药对家蚕的毒性等级划分 毒性等级 LC50(96 h)/(mg a.i./L) 剧毒 LC50≤0.5 高毒 0.5200 熏蒸试验主要是针对卫生用药模拟室内施药条件下进行的试验，如果家蚕的死亡率大于10％以上时，即视为对家蚕高风险。 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] OECD(1998a).Guideline 213：Honeybees，acute oral toxicity test，OECD Guidelines for the Testing of chemicals. [4] OECD(1998b).Guideline 214：Honeybees，acute contact toxicity test，OECD Guidelines for the Testing of chemicals. [5] OECD(1992).Guidelines.203：Fish，acute toxicity test，OECD Guidelines for the Testing of Chemicals. [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. [7] NY/T 1154.14—2008农药室内生物测定试验准则 杀虫剂 第14部分：浸叶法 [8]GB/T 13917.10—2009农药登记用卫生杀虫剂室内药效试验及评价 第10部分：模拟现场