DB43∕T 1968-2020 穗状狐尾藻生长抑制试验操作技术规程(湖南省)

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ICS65.020CCSB16DB43湖南省地方标准DB43/T9168—2020穗状狐尾藻生长抑制试验操作规程GuidelineforMyriophyllumVerticillatumL.GrowthInhibitionTest2020-12-29发布2021-03-29实施湖南省市场监督管理局发布

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2DB43/T1968—2020目次前言························································································································Ⅲ1范围·····················································································································12规范性引用文件······································································································13术语和定义············································································································13.1扦插···············································································································13.2预培养············································································································13.3植物长············································································································13.4植物鲜重·········································································································13.5植物干重·········································································································14试验条件···············································································································24.1温度···············································································································24.2光照···············································································································24.3曝气···············································································································24.4暴露方式·········································································································24.5试验周期·········································································································25材料准备··············································································································25.1沉积物的制备···································································································25.2培养基的制备···································································································25.3生物试材的预培养·····························································································26试验仪器设备·········································································································37试验方法···············································································································37.1试验准备·········································································································37.2预试验············································································································37.3正试验············································································································37.4限度试验·········································································································37.5参比试验·········································································································38观察记录···············································································································39浓度检测···············································································································410数据处理··············································································································411质量保证与质量控制······························································································5附录A（资料性）SMARTandBARKO培养基配制方法·························································6附录B（资料性）预培养土培法“沉积物-狐尾藻”系统图················································7附录C（资料性）试验“沉积物-狐尾藻”系统图·····························································8I

3DB43/T1968—2020II

4DB43/T1968—2020前言本文件按照GB/T1.1—2020给出的规则起草。本文件某些内容可能涉及专利。本文件发布机构不承担识别这些专利的责任。本文件由湖南省农业农村厅提出。本文件由湖南省农业标准化技术委员会归口。本文件起草单位：湖南省植物保护研究所,长沙艾格里生物科技有限公司。本文件主要起草人：刘勇、陈昂、张德咏、张战泓、蒋桂芳、罗杰、熊浩、陈武瑛、罗香文。III

5DB43/T1968—2020IV

6DB43/T1968—2020穗状狐尾藻生长抑制试验操作规程1范围本文件规定了穗状狐尾藻（MyriophyllumVerticillatumL.）生长抑制试验的试验条件、材料的准备、操作方法、观察记录、数据处理、质量保证与质量控制等。本文件适用于湖南省穗状狐尾藻生长抑制试验的试验操作。2规范性引用文件下列文件中的内容通过文中的规范性引用而构成本文件必不可少的条款。其中，注日期的引用文件，仅该日期对应的版本适用于本文件；不注日期的引用文件，其最新版本（包括所有的修改单）适用于本文件。3术语和定义下列术语和定义适用于本文件。3.1扦插Cuttage扦插也称插条，是一种培育植物的常用繁殖方法。可以剪取某些植物的茎、叶、根、芽等，插入土中、沙中，或浸泡在水中，等到生根后就可栽种，使之成为独立的新植株。3.2预培养Pre-Culture在与试验系统相同的环境下进行预备试验，目的是使试验植株能够提前适应试验环境，提高植株的成活率，从而保证试验的质量。3.3植物长PlantLength是指将穗花狐尾藻从花盆完整取出，包括水面上植物和沉积物中植物根系部分，将植物垂直从上到下的长度。3.4植物鲜重PlantFreshWeight轻轻的将狐尾藻从烧杯中取出，包括根部，洗去泥沙。如果有断裂的根部在泥土中，将断裂的根部一同取出，并洗净。将植物放在吸水纸上吸干植物的多余水分，然后称重。3.5植物干重PlantDryWeight轻轻的将穗状狐尾藻从烧杯中取出，包括根部，洗去泥沙。如果有断裂的根部在泥土中，将断裂的根部一同取出，并洗净。放在烘箱中，60℃烘干至恒重，然后测干重。1

7DB43/T1968—20204试验条件4.1温度20～25℃。4.2光照采用LED灯光照。每天光照时间16h。光照强度约8000～10000Lux。4.3曝气试验开始前，每个容器内增氧曝气12h。试验期间，试验容器内不能曝气。4.4暴露方式静态法。4.5试验周期14天。5材料准备5.1沉积物的制备应准备两种人工土，无营养盐人工土和加入营养盐的人工土（表2）。为了狐尾藻根部可以吸收到营养盐，保证植物正常生长。含营养盐的人工土需要加入300mg/kg的Na3PO4•12H2O和150mg/kg的NH4Cl。详细配制方法见附录A的表A1、表A2。5.2培养基的制备试验选用SMARTandBARKO培养基，应在试验开始前配制好足够用量的培养基，确保配制用水不含氯，用水水质应符合NY5051的规定。SMARTandBARKO培养基配制方法参见附录A的A.3。5.3生物试材的预培养5.3.1预培养（水培）试验前14天，从保种藻中剪取新枝条，均长约5cm，在配有SMARTandBARKO培养基中进行预培养7天。5.3.2预培养（土培）试验前7天，从SMARTandBARKO培养基中选取藻枝条，剪取没有异种侵染，长势良好，没有侧枝的主枝条进行试验前的预培养。5.3.3沉积物预培养（土培系统的制备）于大玻璃缸中底部加入4cm已配制好的有营养的沉积物，再铺上1cm无营养的沉积物，最后铺上一层约3～5cm厚的石英砂，以防加入培养基时使沉积物移位。再加缓慢加入约25cm高的SMARTand2

8DB43/T1968—2020BARKO培养基。最后将水培好的藻枝条插入沉积物中进行预培养，7天的后用于试验。参见图附录B的B.1。6试验仪器设备游标卡尺、培养架、照度计、pH计、溶解氧仪、EC测定仪、2L烧杯、量筒、容量瓶、剪刀、移液器、色谱分析仪、质谱分析仪等。7操作方法7.1试验准备7.1.1狐尾藻枝条的剪切于预培养系统中挑取长约8cm的枝条用于试验。7.1.2试验沉积物系统的制备2L玻璃烧杯。底部加入350g已配制好的有营养的沉积物，再铺上200g无营养的沉积物，最后铺上一层约3～5cm厚的石英砂，以防加入培养基时使沉积物移位。再加缓慢加入约1.5LSMARTandBARKO培养基。一天后将9.1.1中剪切好的枝条扦插在OECD沉积物上。参见图附录C的C.1。7.2预试验按正试验的要求以较大间距设置4～5个浓度处理组，求出供试物使试验用藻生长受抑制的最低浓度和不受抑制的最高浓度，在此范围内设置正式试验，试验对照组设置5个重复，试验处理组设置3个重复，每重复扦插一根新枝。7.3正试验在预试验确定的浓度范围内以一定比例间距（几何级差控制在3.2倍以内）设置5～7个浓度组，并设置一个空白对照组，使用助溶剂的还应增设溶剂对照组，对照组设置10个重复，试验处理组设置10个重复，每重复扦插一根新枝。7.4限度试验设置上限浓度为100mga.i./L，即在供试物达到100mga.i./L时，未对藻产生影响。对照组10个重复，试验处理组10个重复，每重复扦插一根新枝。7.5参比试验为检验实验室的设备、条件、方法及供试生物的质量是否合乎要求，设置参比物质作方法学上的可靠性检验。推荐使用3,5-二氯苯酚作为参比物质对穗状狐尾藻生长抑制试验进行检测，EC50范围在4.7mg/L至6.1mg/L。8观察记录8.1在试验第0天，随机选取15棵试验所用狐尾藻，测定并记录其植物鲜重和植物干重。3

9DB43/T1968—20208.2在试验第0、7、14天，详细记录试验各处理每颗狐尾藻的植物根长、总茎长（包括侧枝长度）、植物总长。8.3在试验第0、7、14天，记录pH值、水温、光照强度、溶解氧。8.4在试验第14天，测定并记录试验各处理组狐尾藻的鲜重和干重。8.5在试验第0、7、14天，观察主茎的发育状况，是否有畸形，是否发黄、萎蔫；细菌滋生或藻类污染；生长状态异常，如发育不良、节间距变化、茎/叶扭曲、侧枝增殖、叶组织受损、松弛现象、茎节片断化等。根的状态（试验结束时），观察与对照组组相比，是否出现下列情况：无根；少根；生长情况一般；生长状况良好，与对照组相似。9浓度检测在试验第0、7、14天，采集试验溶液样本进行真实浓度检测。10数据处理10.1评估参数植物根长的总增长率，植物根长的总生物量；总茎长的总增长率，总茎长的总生物量；植物总长的总增长率，植物总长的总生物量；植株鲜重增长率，鲜重生物量；干重增长率，干重总生物量。10.2统计分析所有数据运用适当的统计软件如SAS、SPSS、DPS进行方差分析。确定供试物对供试生物的LOEC和NOEC值。选取处理组和对照组中穗状狐尾藻的茎、根、整株植物的长度、鲜重和干重分别作为计算平均比生长率抑制百分率和生物量增长量抑制百分率的响应变量。10.2.1平均比生长率抑制百分率穗状狐尾藻平均比生长率的抑制百分率（Ir），其计算按式（1）：μcμtIr100%………………………………………………（1）μc式中：Ir——植物平均比生长率抑制百分率（％）；μc——空白对照组植物生长速率平均值；μt——处理组植物生长速率平均值。生长速率是指植株长度、鲜重和干重的对数随时间的变化。处理组和对照组中的每一个重复计算得出一个生长速率值，每个处理组和对照组得到一个平均植株生长速率值和方差值，计算公式按式（2）：lnNjlnNiμi-j……………………………………………………（2）t式中：μi-j——从试验开始时间i到结束时间j的平均比生长率；Ni——在时间i时处理组或对照组的测量变量；Nj——在时间j时处理组或对照组的测量变量；t——从i到j的时间。4

10DB43/T1968—202010.2.2生物量增长量抑制百分率平均生物量增长量抑制百分率（Iy）的计算按式（3）：bcbtIy100%………………………………………………（3）bc式中：Iy——平均生物量增长量抑制百分率（％）；bc——对照组最终生物量与初始生物量之差；bt——处理组最终生物量与初始生物量之差。11质量保证与质量控制11.1用于试验的新枝条长度规格应尽量一致，不能超过20％误差。11.2供试新枝条必须没有其他异种的侵染，如绿藻等。11.3试验过程中空白对照组的pH变化不能超过1.5个单位。试验区域光照强度偏差不能超过15％。5

11DB43/T1968—2020附录A（资料性）SMARTandBARKO培养基配制方法表A.1人工土的配方成分试剂用量备注泥炭藓4-5％pH尽量接近5.5-6.0；泥炭是粉末状的，粒径＜1mm，干燥的。高岭土20％高岭石含量最好超过30％。石英砂75-76％50％以上细沙粒径在50um和200um之间。(0.05mm-0.2mm)湿度20-30％加入去离子水，湿度控制在20-30％。pH7±0.5加入CaCO3调pH。表A.2沉积物配比OECD沉积物泥炭藓（kg）高岭土（kg）石英砂（kg）去离子水（kg）总重（kg）含营养物6*5％=0.36*20％=1.26*75％=4.51L含(Na3PO41.8g；NH4Cl0.9g)6不含营养物3*5％=0.153*20％=0.63*75％=2.250.5L3注：1）计算的土壤含水率为14％。（不是最终的含水率）2）将Na3PO4和NH4Cl溶于去离子水，用配制好的溶液与土混匀。A3SMARTandBARKO培养基的配制表A.3SMARTandBARKO培养基配制表化学成分浓度(mg/L)CaCl2*2H2O91.7MgSO4*7H2O69NaHCO358.4KHCO315.4pH7.5-86

12DB43/T1968—2020附录B（资料性）预培养土培法“沉积物-狐尾藻”系统图图B.1预培养土培法“沉积物-狐尾藻”系统图7

13DB43/T1968—2020附录C（资料性）试验“沉积物-狐尾藻”系统图图C.1试验“沉积物-狐尾藻”系统图8