ÿWPCu –#c)@ÿ̯}C,/êußb>`Èv;ìç÷eù´¹=e©‘÷B íLÖ>¨MñÚÝ.ÃQ¨ûû”¬E„ê~-- ÿ ­ü™µ—Ù¿Ypʧ§Y¥j&úšvØ -_âaÏûZš3>ö?­Óðó³¹ÝGiý_é’æRÄwÂÌŸ1çvëWX·*qY0œ£¹ilª”’êOão»½ÅÜÜ+HCkãg)ç÷³ïÛ:z¬˜­ß 16vÂà»ÆXî}[ƒ^jëTu¬?q-ŒÔ5¯yŽV¹œãVø0Ö±'×§gkêIq.½¸]'<×D©Êi?OpMB˜ ãK®¾Eõ5K&ž6°þr ÙØÎW=J&øéOlbhæÓÎ+1vuH\sç5g9¢*`i_–œ¤¦Eÿ6E ´Q@g?䱚¬~]ÝWj+¾;CøÑuO:Hç‘]p0C°Ø•Ú5ðp:g{Á ›½¢a MæUê߆©„ðRâP–Þ]•;mƒKYávîÔf3÷¶ åöÖ0ÛGjŸÄ¾×2WNì¿E6 i}bÌ‹‡q¯Yö \ øyÔ9þ\rãqHÄá-'žŸ&(¯—xaú?!‡Á 0šU<µœñUN %ëñ 0(ùw@!4a<ujÿÿ:Default ParaÓ  ÓÔ€ôôôÔÔQ€ô\  `$Times NewRomanQÔÔ€ôôôÔÔQ€ô\  `$Times NewRomanQÔô\  `$Times NewRoman' WSU-ETCFSOP 100-01-01 4Dr. Barry Forsythe II 0 .   3|xP(ÖÃ9 Z ‹6Times New Roman RegularX($¡¡ GHÀIJEKLEMNOEPní$Ý ƒ!ÝÝ  ÝÑ  ÑÒܰÒÒܰÒÑX°ÑÓ  ÓÔ€X¡+XXXÔWSU„ETCF/SOP€600„04„01Ð X ÐPage€1€of€3ÌEffective€Date:€€10€October€1997ÌÌÓ  ÓTitle:€ò ò96€Hour€Acute€Toxicity€Test€For€òòChironomous€tentansóó.Ð ° Ð________________________________________________________________________ÌÌó óAuthor(s):à Œ àà ä à_______________________________à ô àDate:€€____________Ð Ðx Ðà 4 àà Œ àà ä àJim€EisenhauerÌÌSection€Leader:à ä à_______________________________à ô àDate:€€____________Ð ” <  Ðà 4 àà Œ àà ä àDr.€Michael€J.€LydyÌò ò________________________________________________________________________Ð l   Ðó óÌò ò1.0€€OBJECTIVEó óÐ Dì  ÐÌà 4 àTo€determine€the€ECòò50óó€value€for€a€compound€being€tested€on€òòChironomousÐ Ä Ѐ€€€€€€€€€€€€tentans.óóÐ ° ÐÌò ò2.0€€HEALTH€AND€SAFETYó óÐ àˆ ÐÌà 4 àLab€coat,€gloves€and€safety€glasses€must€be€worn€at€all€times.ÌÌò ò3.0€€PERSONNEL/TRAINING/RESPONSIBILITIESó óÐ 8 ÐÌà 4 àAny€WSU€employee/student€familiar€with€the€equipment,€laboratory€techniques,€Ìà 4 àand€trained€in€this€and€referenced€SOPððs€may€perform€this€procedure.ÌÌò ò4.0€€REQUIRED€AND€RECOMMENDED€MATERIALSó óÐ ,Ô ÐÌ€€ò ò4.1€€Required€Materialsó óÐ ¬ ÐÌà 4 à21€„€1€Liter€beakers€€€€€€€à ” àà ì à1€„€1Liter›€graduated€cylinderÐ àˆ  Ðà 4 àEnvironmental€Chamberà ” àà ì à40€L€of€MOD€Reconstituted€WaterÐ Ì t! Ðà 4 àConductivity€Meter€à < àà ” àà ì àDissolved€Oxygen€(DO)€MeterÐ ¸!`" Ðà 4 àpH€Meterà ä àà < àà ” àà ì à210€œ4òòthóó€Instar€Midge€Larvae›Ð ¤"L # Ðà 4 àœMicropipetter›€+€Tipsà < àà ” àà ì àAppropriate€Solvent€Ð #8!$ Ðà 4 àContaminant€or€StressorÌà 4 àÌò ò5.0€€PROCEDUREÐ T&ü#' ÐÌÓB.Ü'ž4Œ ä <”ìDœôL¤ü!°œXBÓ5.1€ó óAcquire€twenty€one,€1€L€glass€beakers€and€set€up€seven€sets€with€three€beakers€per€set.Ð ,(Ô%) ÐÌò ò5.2ó ó€Label€the€first€set€of€three€beakers€as€the€negative€controls,€and€the€next€set€of€threeÐ *°'+ Ðà ' à€€€€€beakers€as€the€solvent€controls.€€The€remaining€five€sets€of€three€beakers€Ìà ' à€€€€€should€be€labeled€with€the€appropriate€concentration,€and€the€name€ofÌà ' à€€€€€the€œstressor.€€Place€10€ml€of€sand€in€each€beaker€as€sediment€for€the€midges.›ÌÐ ¼-d+/ М›œ›‡à ' àà ž àà 4 àà Œ àà ä àà < àà ” àà ì àà D àà œ àà ô àà L à€€€€Page€2€of€œ3›Ìœ›Ìò ò5.3€ó óFill€each€beaker€with€1€L€of€aerated€moderately€hard€reconstituted€water€using€a€1€LÐ 0Ø Ѐ€€€€€graduated€cylinder.ÌÌò ò5.4ó ó€Remove€œòòC.€tentansóó›€from€the€culture€tank€by€œusing€the€appropriate€dip€net,€and€scoop€€€€Ð ø  Ѐ€€€€€up€some€of€the€flocculent€and€sediment€on€the€bottom€of€the€culture€tank.€€Dump€theÌ€€€€€€flocculent€and€sediment›€in€1€L€of€œaerated€›€moderately€hard€reconstituted€water€in€œanÌ€€€€€›€uncontaminated€œcontainer.€€Shake€the€container,€and€the€Midge€Larvae€will€spread€outÌà ' à€€€€€in€the€container.€€Two€or€three€scoops€may€be€necessary€to€get€enough€4òòthóó€InstarÐ ¬ T  Ðà ' à€€€€€Midges€for€the€test.›ÌÌò ò5.5€ó óPlace€ten€œ4òòthóó€Instar€Midgesòò›€óóinto€each€beaker€by€transferring€them€from€œtheÐ p   Ѐ€€€€›€œuncontaminated€container›€to€œthe€beaker€by€carefully€lifting€them€with€a€pair€ofÌ€€€€€€forceps.›€€ÌÌò ò5.6ó ó€If€any€œòòC.€tentansóó›€are€swimming€on€top€of€the€water,€gently€push€them€under€the€waterÐ $Ì Ðà ' à€€€€€using€your€œforceps,›€so€that€they€are€totally€œsubmerged,€and€laying€on€the€sediment.›ÌÌò ò5.7€ó óIn€each€of€the€three€negative€control€beakers,€check€and€record€the€pH,€DO,€and€Ð ì” Ðà ' à€€€€conductivity€using€the€appropriate€meter.ÌÌò ò5.8€ó óInoculate€each€set€of€beakers€using€the€micropipet€man,€and€the€appropriate€treatment.Ð ´\ ÐÌà ' àà ž àA.)€€Negative€Control:€No€TreatmentÌà ' àà ž àB.)€€Solvent€Control:€0.1ml€of€solvent€onlyÌà ' àà ž àC.)€€Treatment€#1:€0.1€ml€of€solvent€+€appropriate€concentration€of€contaminant.Ìà ' àà ž àD.)€€Treatment€#2:€0.1€ml€of€solvent€+€appropriate€concentration€of€contaminant.Ìà ' àà ž à€E.)€€Treatment€#3:€0.1€ml€of€solvent€+€appropriate€concentration€of€contaminant.Ìà ' àà ž à€F.)€€Treatment€#4:€0.1€ml€of€solvent€+€appropriate€concentration€of€contaminant.Ìà ' àà ž à€G.)€Treatment€#5:€0.1€ml€of€solvent€+€appropriate€concentration€of€contaminant.ÌÌò ò5.9€ó óCover€each€beaker€with€a€piece€of€aluminum€foil,€and€transport€the€beakers€to€the€Ð ð˜  Ѐ€€€€€environmental€chamber.€€Conditions€in€the€environmental€chamber€should€be€set€at€aÌ€€€€€€temperature€of€œ25›€ð$ðC,€and€a€photoperiod€of€16L:8D.Ð Ì!t" ÐÌò ò5.10€ó óThe€test€will€run€for€96€hours€(four€days),€before€the€beakers€can€be€removed€fromÐ ¤#L!$ Ðà ' à€€€€€€€the€environmental€chamber€and€the€number€of€œMidges›€showing€an€effect€at€eachÌà ' à€€€€€€€concentration€can€be€determined.ÌÌò ò5.11€ó óThe€endpoint€to€look€for€in€œòòC.€Tentansóó,›€is€the€œinability€of€the€Midge€to€form€a€ð ðfigureÐ X'%( Ѐ€€€€€€€8"›€upon€gentle€prodding€with€œa›€œpair€of€forceps.›ÌÌò ò5.12ó ó€Pour€the€majority€of€the€Hòò2óóO€into€a€separate€container€for€contaminants,€and€pour€the€Ð  *È'+ Ѐ€€€€€€€remaining€contents€into€a€different€contaminant€container€and€determine€the€numberÌ€€€€€€€€of€œMidges›€showing€an€effect.€€Pour€the€left€over€contents€down€the€drain,€taking€Ìœ€€€€€€€€care€not€to€splash€the€water.ÌÐ Ô-|+/ Ðà ' àà ž àà 4 àà Œ àà ä àà < àà ” àà ì àà D àà œ àà ô àà L à€€€€Page€3€of€3Ì›à ' à€€€€€€€œÌò ò5.13€ó óGraph€this€data€on€log€probit€paper€first,€and€then€use€probit€analysis€to€determine€Ð 0Ø Ðà ' à€€€€€€€the€ECòò50óó€for€the€compound.€€Trimmed€Spearman„Karber€method€may€be€used€on€ñNñ€Ð  È Ѐ€€€€€€€ñNññOññNñ€ñNññOñnon„normally€distributed€data€to€determine€the€ECÔ€€ÐXX¡+Ô50Ô€X¡+X€ÐÔ.€€Report€the€ECÔ€€ÐXX¡+ÔñGñ50ñGññHñÔ€X¡+X€ÐÔñHññIñ€valuñIññJññIñseñIññJññKñes€with€ñKññPñÐ  ´ Ѐ€€€€€€€ñPññKñcorresñKññLññKñtñKññLññMñponding€95%€confidence€intervals.ñMñÌà ' à€€Ì€€€€€à ž àà 4 àà Œ àà ä àà < àà ” àà ì àà D àà œ àà ô àà L àà ¤ àÐ Ðx ÐÌ