【商检行业标准(SN)】 出口贝类麻痹性贝类毒素检验方法

中华人民共和国进出口商品检验行业标准SN0352—95
出口贝类麻痹性贝类毒素检验方法Method for the determination of paralytic shellfisbpoison in shellfish for cxport1995-05-29发布
中华人民共和国国家进出口商品检验局1995-11-01实施
中华人民共和国进出口商品检验行业标准出口贝类麻痹性贝类毒素检验方法Metljod forhedeterminatinn nf paralytic shelltishpoiaon in shelfish far eiport1主预内穿与逐用范用
SN 0352-95
本标雅规定了出口贝类及其制品守麻游唑几类毒素检快的抽样，制单和生物测定太法，本际准活用非出门海产双壳类贝肉，以性和其他可供食用部分的麻冲性贝类牵系的检验。2术语和代号
2.1麻以类牵素(FSP)
化学绪树以方房站毒康（SaxItox:n）为代表的，握食后可产生麻游作用的在在成类体内的海洋物毒生物的化称.
2.2CFteomvr:rgiun f:rlr)
毒求转换察数。
2.3 DF(dilution facter)
释系数，
2.4 MU(nousc unic)
鼠单位。
2-5CMul(onrirrledmouseunit)
快正鼠单位：
2.6中位数
把变只值按大小次序排划，店于中位置的那个数值就是中位数。3抽样和糊样
3.1检骏批
必不过100力一检骏批，间一换批的商品成有相可站特征，好包装，标记，产地，垂克礼规格等
3.2抽样数
接各应验批的量、依下表拥这样：检整的重量、
11~·58
st--ton
如也做装的应均与持样。
抽样点件)效
中华人民共和国国家进出口商品检整局1995-05-29批准混仑样品数
1995-11-01实拖
3.3轴接方比
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安3.2规余的抽拌点（件)数，随机仙取样战。每5点件)拍取的始性只约一个源合样（即检验样出>，其重尽不少于2kg·装人消沾容器内，加时标记与，及时送交实验室检验，34分析价品的采
析样品要有充分药代表生.依受格贝美品种决定样品的采案量.收样个数不岁于12个贝类个体。即从2g湿个样品小排良好的贝声：去壳均成达20。对于全体过小的品种，以壳后肉成不少于2关决定采集个数，
新鲜其类不能皮时送性，接3.5.1上法将贝肉分高，将俩水后内203贝内放入100mL.IC1(0.18mol/.)中于4冷强保存（切例冷陈3.5试栏制资
3.5.1牡姆，蛤及筛贝
用错水将贝完外表彻底洗管，刘新壳肌开壳，用清水洗内部去除泥沙及其他外来物将闭壳肌和连接在胶合部的组织分片，竹细度出以内.切创州或内体，并光前不要加势或为麻醉剂。收策约2CC内没116烯子瓶水5m不要软内堆积？出率壳等杂物料贝肉质.3.5.2
取可食部分用准愉测，满下度均质迎程间品：1.35.3贝炎
将内所有内穿均（内及液体)例入闯质器充均质.妇果定人罐，将贝肉物水并收来游下的液伴，分别称重：格固心物和荡汁按亡例滤合，光分动质。35.4乐酸保存的！肉
诱去酸，分别存或贝均及峻液，将须十的贝函充分均质。3. 5.5冷你贝类
在室下，使冷冻蛇样品（带英双脱范的>呈半冷游状态：按3.3.1方法卡无，淋洗，取内，均质。35.6贝因下制品
十制可于HCI(0.16nvl/L>游液序料泡冷滤）,按3.5.4方达泌十，质。3.6试栏保存
上述5中经均质处理的偿品师不能改时检测：可取19口均质内加人1HC1(I.18rml/1,)等液，置于4℃:冷保存（尽亢能及时抢验,4测定方法
4.1方法提要
本克法买用报单位测定，对SP子以定重，拥单性定义为：对体电对21的小凹鼠胸腔纯射1则来提期激质，在m初时杀死心常院宽的战低声变。采用Saxito忙为声豪的标准品，将阅单化换算成每东的微克教。丧据小风注时贝类券取液的处亡时，李出鼠单拉，井按心显述耳，校出鼠单位，计节确会每1U其内内的PSP的微克效。所测定结果代表存在于贝内内务种化学结构PSP毒素的总量。
4.2试剂和材料
4.2.1盐能落使：3.13mal/L.将15ml.浓H用热增水烯择至11.4.2.2盐酸减：5Tol/E.格11.7L液HC1蒸罐永稀释至10mL：4-2-3氢致化钠溶液n.1tm./1.将4.6xa0H籍」11.俩水中.4.2.4瘀察性贝类年度(Sxitoxin)标准液：1rJg/mL，丝慢化，含有2n%为乙序指为保扩剂冷滋时.北限期段定
4.2.5体重19~21gICR品系的雄性随康小户鼠。2
4.3仪器和设备
4.3.1均质器。
4. 3.2天(满0. 5g)。
4.3.3两心机..
4.3.4pH 。
4.3.5秒表，
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4.3.6肢璃器血：滤、盘简.穿量稚.撤拌等。4. 3. 7射器[1 mL}.
4.4测定步强
4.4.1PSP标准工作液的配划
4.4.1.用移液管液1mL（100gSx10xin/mL)标准液100ml容瓶1加入月HCl载化至pH3的带筛冰并忘穿.该减1gSaxicoxin/mLpH在2.-4.1之间，在3~4C下需稳定数周。4.4.1.2分别司11、15、2.25和30ml.水稀释1CmL依度为1R/mL标准减.希释波的pH应为2-4.
4.4.2中位数死广时间的标准液选择将4.4.1.2的各种浓度的我税释滤各1L疫腔注射数只小良，避择中空数死亡时间为元-7il.的法度剂质。妇革施度稀释波已可达要求，还需以1mL水的培减基进行补充薪释试验。例加：用25mL水格释的11ml.标注液在5-7mis杀死小鼠-还需进行24f-10和26！1C的筛释度试龄以19个小鼠为一组，用中位数与亡时间在5~7mi范因内的二种（最好是三种)帮释度的核准液注射小鼠。记录只小自睦监注射完毕至停止呼受的所需死亡间。每只小鼠事完称重，精确到.5R记录注射率至死凸时间的量短问随为5即？按为5$8校正1（见谢求？。
4.4.3每前转换系教的计募
计原中各种橘样液所止射的1U只小鼠的中位数死亡时：名某种稀释注东的所有10只小眠止位数死时间小于成大于了mim，则弃去该组结果，若另种稀液注射的10只小刷中位数死广时叫在5~？mir，即使英紫小系死时间可能小于5惑人于7min，也要使开流能所有数据，出是某组稀籽波经注驻片，1C只小鼠中有3只以1.择活，则要再取1C只小点进行承复试验。般据所选带择注的1u只小以中位数死亡时间为5～7min之间的死亡时间，由对录分别壳出良单位（MU）.再1附录查出小鼠的里基校正图了，两老相燕求得每忘外稀释浪的性正闻单位(.ML，将所选稀释游每变升实际毒意的微克教（以Saiitosin计算）除以CMU值便孕到毒求转换多数(CF).CFg SAX-/CMU.
计每纠10只小自的半均CF复，再取其组间平均（\F值，并以此为标准作带规格测。4.4.4CF值定期检查
如PSP校测有隔时间较长，每次测觉时要用适当的标哺蒂释液注射5其小氧断测定（P值加果周有儿次检测则用中位数能亡时州57min的标准帮释减每用检-一次，制的CF使应在原划定CF平内值的士20%监距内：告结果不符，用同样的标群稀释液另注射5只小鼠，综合光前注射的5只小金结果：算出CF值：并用同样的标准稀释减注射第二组10只小鼠，将第一组求出的CF值势一组的CF进行半均，即为个新的CI值。重效经监的F值通带年原结果的士20%之者经常发现有较大通差：在选行帝规检测前成谢该方法中是合存在未控制戒未意识到的可定因素。4.4.5样品中PSP的报取
取1心已均质的择于称过重的8C0L烧补中，加100mlHCl游(0.18ml1.)或样品中证待的酸，充分搅择，始查pH.PH火2.U~:4.，若需要，可逐滴人HCI(ui/L.1或Na)H3
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(0.1mol/)境伴以防止同产减化速年京破坏。将滤台物加热·并除除点需5min，冷却室益，训专已冷却混今物的pH至2.3~4.0。将派个物移率量简中并帮释至200ml将准合物倒回烧杯，揽产至均质状，偿其沉照至上消波呈半透明状，直至析分离旺不产生大至塔垂注针头的臣体控为上，2要成将据合物或上清液以3r/min离心min，或道过滤组过缺，保留处以述行心案延射均的液德。4.4.6小鼠试龄
将小乱移非记录垂量：每一样品注射三只小鼠。刘将只试验小录双腔江射1提收液。注射要准确※练，芒有一满以上提取藏溢出就预将该只小鼠去弃-并重新注射只小民，记求让射完毕时间，仔细观察小段停正呼收时所在明的死时间部小献呼出最后口气），州纱表记录死：时有。若小鼠的中觉数死时间小上血n则要进行稀群再射组小自只以到7m的死时所，注射未烊的样后后：1或2只小鼠的中位数死亡时问大「7m则需注射至心3只小鼠以确定样品的力。加需稀择详品，要逐滴划人HC1G.：或0.[ao/T.>,谢节释释浓nH至2.0~4.。5 结果的计尊与判断
5.1FSP毒力内让第与判断
根小限的死亡对间，在附求查出拍应为每升时液的鼠单位数MU。验动物重小于9或大于1则根执时出重校正系数。重成校正系效英以该只小课的MU梗得到MU计该组离的中位整M将存活鼠的外广时问税人mn或相当十小产心MU）以中位效按下式计算妞每样品中PS毒案的年。gPSP/1CCg=中位数CMU/mL×CF×1F×0任大于日内的被认为是有害的，对人美食用天安全。5.2M11毒的算与划断
（1）
取得麻师性以炎本素标准品有用难的实验室可使同鼠单位M付检验筛采于以算与判断。技5.1求出该约小配的中惊CMU，然后求止10Cg贝肉的MU值，MLi_ g肉 =中位数 CMU/mL. × DF × 2仁：何★于400ML./100g均的值即被议为足有半药，对人禁度用人安全，让：均验诺系的仲规以5.1为准。2
注：）以办沙表示。
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麻痹性贝类死亡时间鼠单拉的关系(补件)
12: ch
. 1154
小肽虽e
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附录B
小鼠体重校正表
（￥件）
附加说明
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本标准由中华人民共和国巨家出出商品检验局差出。本标准山中华人民共和国所步山口商品检教局贷责起举，牛标准主要制单人苏人路、税伟良详顾华。主要参考文献
1.Faraytie Sinellfish PoinonHiusiagirul Melyd (ACAC,15th Ed.,1gso)2. Laboratory I'rocedures tor the Examinatinn nf Seawaler imml Shellfish (APHA,5th Ed, -185)Professlonal Standard of the People's Republic ofChina for Import and Export Commodity InspectionMethod for the deferaination of paralytic shellfishpoison in shellfish for export1Serpe and eld or application5N0352--95
Tlis zLandard sperifies the mcthodg uf ampling sauple prepuration and determinarion bs bioag-say prouedure of paralyiic asellfigl poispn ia shcllfish End ahe.lfish jμruducis for exucirt.Thig scendurd ss upliet.blu tu tlu etprrminmrign 此内容来自标准下载网
2. 1. Paralyric shellfish [eisuu(PSP]Psp mtiam all nasine inlogical toxic substencee is shellfisa which can cause paralysis afier in*rsred,the repraseutative at ita chemical giructure is Saxiroxir..2.2CF
coavcrsion fector.
Eilution fartor.
mauyc urits.
trnrecled riouse unics.
2.6Mcria death line
After arenginn the dearth tinu varubls frurm largr I:t nrnl.taemedian deatl: time is -he oncwbich is at dlu- rrillle. peition.3Samplingandseunplepreparpiion3. 1Inapectan lot
Tar: yu:ility :if ian inspmriicn, lo1 should nnt be more than 1oo tonnes. The charucierisics of t'aeTargr within the same inspecticn lot such as packing park ,origin,naason and spccification ,shosld bethezamt.
3.2Quatity of aamples tuker
Appruved by the Stae Adminiktration nfImpart ind Expart Commotlly lispectlon orthe People's Repnbllc or China an May29.1995Inplemented from Nov.1.1995
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Nuraber ol tonner in eaahNurnbcr u!xaples in be takenirwpeclionJnt
less thur. IG
11 --50
51—100
Sainpler shouid be tekerl wsifernly if te cargo Bre ir bulk.Nurober of mixed satmples
3.3 Sumplirg prneshlit:
A inneier of samples arecified in 3.2 arc tascn at randunt. One mixil utapleci.*. sample for de.prntinat:on )censisis of [ioe samplcs. Tinc tutal wriglit sif rach mixed rample shouid no: be less than2 kg, 'The: Pat in:o clean runrainrra,wlich shall be sealed,labeled and zent to jaboraory ia 1:me3. 4 tolicecion tf amlytital ramplesT'ee airialytical saroples should be teprerentacive. The size uf thu suripk is guvernrrl ly lhe rliall[ieh apecies being exanined. Mre than 12 suunr! shllish are cnllecled fram 2 kg pl mixed sample lorshuckiug, Far mast spieius,thin nirnple siae will yield approximately 200 g ol mea1 ard ahell liqucr.Wnen ahclifish uf al xmullr size are beirg czamined.collel more shellfinh ta produce gou g chucked.ats
[Jnlese fresh ghellfish ern be anslyzed [rurr:ly ,xrperate the slellish nteatt.see 3. :. 1) and prc-serve 2ri g of the dreined meal in IGo ml. nf HClco. R mal/L) according to section 3. 5. 1. Refrigeratear 4'c: until enuiysix ix xtaried.3.5Prrnaratica of est sampie3 5.1 Clame oysterx-and musseiaThoroughly elcan uulsidu: af phellfizh wnh freslh watr.Cut adductor muselee and open. Kinae ingidc with frusl, war 'is reraove the sand and other loreign Taaterial. Shuek the mest fro shel' car.ful.ly ,msl in tut oy daage budy of mallesk. Colleer approximutely 2ui g af -irat on an Nn. lo sieve witlnur layering,ure. et drain for 5 min, Pieb uul iml linurd khell pieces, Griad the draiaed meat in ablerder until homogenuus.
3. 5.2Seallo11s
Sipuratln -lilsin Fnrtion for analysis. Itam and grind as in 3. 5. J.3.5.3 Canned shellfisk
Transfer the eartrn runeenin sf lhe ean(meat and liquid) into blender and blead enil homogeenus.For lurgr cunnul-ain the mueat and cullec: sll liguid.Ielernsine the weighes of meat and volumuf linjuid. Recambire the jortioas of each in proportiorate qupncitics. Bleul retwrnbiuwl mrlcws aniillonngeneous
3. 5, 4Acid preserved skellfish Trut.ixDrainl thu: atid xl.liru finni chellfish and keep both Iractions, Bdend the drained mcat until homo-getun
3. 5.5 Frozen shellish
Make the frazen sumptus(aheliri-l sr tutaig) aeui-Erozen at rnkom temperature.then open,tinge,sauek enel blund ux in 3. 5. l.3. 5. 6Prnduets nf dried sheltish weauImretre the dred ahellfish pruducts in HCl<0. 18 mil/l.Janl.antinn,drain and grind as in 3. 5. 1.3 6Srorage uf tes: gunple
If the. lurug+uiamrt mears treated in 3. 5 can not be analyzed in time,precrve 1oc g of the honog9
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caized meat in HCl(o, I5 mol/L),refrigerate at 4 until analysis is startcd.4Methnd nfdetermlnatlon
4.1 Principle
P'S is quantified by bioaasey uaing che mouse unit(MU) s the unic uf meusurement.The MU isdefised rs the minimum amount ul juisull reyuired lu kill : mu,une wrighing 2ti in Is sin whrnI. U mL uf shellfixh wxllard is injar led iniraperitoneallytIP). The bioaseay μrocedure has been modifiedby Jsin purified saxitoxin as a relererice siandard and convercing manae units zo microgrRms(μr? afcoxin. Fhis bioassay procedure and the exprcasian of results in terms of toxin concctulruiott is prescert-:ep:estml tnlal laxiciy from lwo or mare:ox.nscd here,but ic is reeuglizyl th: i licusaay rekulix may4.2 Reagents and marcriels
4. 2. 1 Hydrochlorie acid,G. 18 raal/L,dilute 1e mL uf cuncenrarion HCl tu 1 L wiuh rlistiletl watur-4. 2. 2 Hydrochlorie azid ,5 nwl/l.ililutr 41. 7 nal, nf enncenlralinn HC: tn 1GC mL, rith: distilled wa-.et.
4.2.3Socium hydruxide,0.1 mol/Lidissotve 4.6 g af AsOH in 1 1. of distilled warer4. 2.4 PsP Standard Solution(Saxitoxin),1U0 μR/mL;Ecidilied with HCl,with 20% ethyl aleuhul u: pregervativc,bciaa stabie indefinitely in a tuol plece4.2.5Heelihy mle mit of ihe [CR suran,weighing 19 to 21 g.d.3Apparatusand cquipmen:
4.3.1Homogeaizer.
A.3. 2Torsion belane:.U. 5 g rsilivity.4. 3-3Cen, rilirgr.
4.3.4pJ meter.
4.3.5Suop-wa1ch.
4.3.BGlaasware;buukrrs.grulni.inil eylindar.volumeriir flaek,ulasa slirriag rods elc.4. 3-7 Syringe(_ mf.).
4.4 Frocedore
4.4.1Prepravion af pspP stndard salution4. 4. 1. 1 Prepere PSP referenee suluian (l g si.xitixin/nt.Jhy pipe1lirg 1.n) m1. nd sland:rd aalulium (lo μg Saxiinkin/nml.) into a :o mL volomccric flask and nakr up to volume with distilled wa-ter acicificd wich HCl(to about pH3). Jhia solutian is gtable for sevcrel wccks if atored ar 3 to C: andaf ihe final pH is betreea 2. J nd 4. 0.4. 4. 1.2 Dilute 1U mL [pustiorus uf 1 μg/ml. rrferrnr snlusiell will It.15.20.2,ant 3n ml. nd dieIill.d wul+r,rtsprt:lively-maincein pl nf dilutians a- between 2. G and 4, U.4. 4.2 Selection of standerd eclution fur median death tirnelnject 1 niL of each ecncertration as in 4. 4.1.2 into a few rest mierrchuuse the zuneeniratiunswhich caugc a medien dueth tiwr uf s l ? nin. H rerlain rrmrenllalinl twn gel :le result reeired,leelunlitiuul ililu:irs n I ml, :nrremente of dietilled water ;e. K- sif :5 ml. cf relcsence solutioa dilutedwith 25 mL distilled water kills mice in 5 tu 7 nuin.thea tesl the dilutions ol l0 ml, reference solutianlus 24 ml distilled wate: and of 10 mL refercnce solution [plus 26 ml dis=illeci weter.Oac gruug cosists uf lo nicmd inmrtmmil moum will I mtof 1w'or paefe.ailyhee,referrurr tilulinux thal cause mediaa desth tine of : to 7 min. Rccord thr death time which is the elapsed10
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limt: beiweell nnupletinn nf the iniection and the last gasying breath af the mousc.Weigh and revord the weigtn of each mouse ta nearegt 0. 5 g helorehanid. Reco-d injerrr enedeath time to thc ncercst 5 s intervel;thus 7 : is rownded as 5 9,und s s us 1u s(sct appendix A).4. 4.3Calruliuinens of ranveisinn [arlarCale:late Tedian death timc for cach group of lo mice by using tach dilutikn, Discard the resultsfor any group ul 10 mice giving & median death rime lena than5 pr more than 7 min.If any group of 10mise gives s median deuth timu belweeti unutl ' ttin,inelude all grouus of Lo triue sedl fur hat diln-liutt in nilxripurml talkulations even hrugh sme desth tinier Eall outside the dexired ranpe. If nr:tlan s mice ir. a roup of lo nice survive the injection ot a purticulur di.utior. ol the reterencv solutiun.Fepai ihe iajeetion with a nr #roup of c misc.Lsing the ceata tirne for uech tiousi, in arb gtjup in whi.l, thu umralig drath sie Eall, bctwenl5 anl y nin.ilrlerininir the ent:espnnding Mtt Tam Append.x A. Tisiny Lhe weight af eah mansr,determine the wcight purrection tactur frum Appendix B, Multiply the MU by the weight cor:ectiun fac-tor 1o derermine the valeea far correeced rucusc unics(CMU) per m:ll:li-cr of the aclected dilurinns. D1-vide the calcelted pg poison/mL iri the Nilicled alilutium, by lle eeniar CMU .u ultain envirsiut!fn-lrir (CF) valur (SAX/CMT:),s, e. CF=ag SAX/CMU.Calculate -he averape of individt:al CF va.ues and ure the restlrant alue tu check ruuntine aszays.4.4. 4 Perjodic checking af CFvaiueIf ahellish Bre assyrd ml Juniger intrrvals,tleirnine CF valur an waef al: y hy injrting 5 (ricewi: n uaprptiule rlilulinn rd workinx standard. I assaye ase. made an aeveral days durink 1he. wee'k,eheck anly onee per wcr's wis a diluted standz.rd uch that the mediaa dcath tine falls betvcen t: and? rxn. The Cr'hs retermined ahnu.d agrce within ±2o of the Predetermiaed standard ['F. J: :e-gulta do not sgrce,byse the CF check un : lct ruusu group furinkl by injecting 3 nluilioial aicu withihe wirnr eliluuinn nf stanilarid PsP sniulion and incltf.ng 1he reanliy with thsse ra, :ne \:iyin-. 3mice. Injcet a second group of lu ire. The average CF value ubtzined from the turt groups ut lu tnit?tepreaenrs e new t'F value.
Rcpcated cheeks ot th CF valut ureinutily μruulueu tunnistrtt tusal:s wi:nint + 2uvo. I: widervari:icin are [euml freguenily ,investigale the pors hil.1y of L.neanlrnllet: o mmierrgnixal wnrialsies i:sthe method befare precerding with rountine assaye.4.4.5Extractionofps
Weigh 1oU g of heugunived ncul inu a arl 8iNi mnL luuker.Arr: I(O mL cf HCE(a. 18 rul/L?Crr ihe drained axid sn.uticr From sample chat had beer Freserved'.Sti: tharoughly .and clierk pll,pTTsaoald be between 2.0 z.nd 4. 0. If nesessury.adjosl FH by eddiug HCl(5 aul/L) or NzOH(r.. ! cl/L) dropwise and siir tn prevent lacal slkal:nization and desrrueron nf the pousor. Hnil ra.xture gentlyfer 5 .in end let ruul to room remjuriture, Wanent euul,eaeck jH aad udjunyt to 2. ty 1. if nessary.Trtus-fer thr mixlure I: grrclualel rylintier ancl dilut- [r: 2nl nl. with ilisiille.d wilr-.Raturun tle mixture co the caker-stir to honuogencity.and let eectle until the portioa of suptrnat:is itanslucet1 and car he deralured free of solid pksticias large emoigh To block 2s-gage hspocerrtt:ncedle, If aceessary centifugt mixturr ur st.pernate min at 3,u rpm ur Filetr tarough papar, (lnlyungh I'rpir ler perlnrht sirasrty is ueinnsary.4.4.6Mousc tcxt
Weigh and record weighl o: tkree rmce for each sample co be analyzer. lnz:l iniraoeriicre.:llycach uf three micr with I aL of uuntrifuged exiret. lietiun must be aueurute Euid shilled. R:jcut ny1
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