ÿWPC© Î@åÛi³»ÑÃaÛ ‰ "z_@«ÖƒPEV•yNQòá;ƒ1  js¬ÝÔä©×TýÔˆM‘em³nEc<¦ÅÛa:ÀŽ/œßˆdÞ»ªwIëÄÎ5ž®.ß`¯íî`I5'EyMö›‚°æ±0 ‹¡'iãÞ²vEOØæ–+[†s*5TX¤ÜèØ*ÊS¨={}|Ð]û3¯·¬Ùw§"Õä`_ð@l†Ø¨’}ˆ]:ß×à:«~PˆQ3——©è:Â/F(`7ß`Ç+¤_ ‡*ßÒ]?c'?£‡ô¤¶NLø‘A ?Ë7‰œ$"…üQóqë xÈæ‰aª*¶ëH%â^Nn¶9dK-_ÕDSé¿HЙŽÜ~Ø$Ño+ò¢ÝFdÛ3Fý¤½ˆs4TŠ.é?kU¯±ÕW¬³Ñl``u?9¹‘‡Ñæ ,Ã{Ü"!<³vÖ“uE/vê@—]†¿©^¼Èïâ:Êú-üîÏCÔY†±½P,5SòC€%%^qJ ~G¥ú=NÀ ø>s×ݹŽ87õ®O{èÅÇÇd@jÕظ’å³ ø óSoŒ¸¯ƒ€ÍÅÊ5*à@Ë`}mŠÛikö(¡ç­èka–ÙpÓ\Ÿ†3öï3<# ÒUNÝ %+ 0(1LYU<¥w@á4!5ND^ FR mT>k˜HP LaserJet 6LHPWðò,..,ðò0!(ÖÃ9 Z ‹6Times New Roman RegularX($¡¡C:\OFFICE\WPWIN\TEMPLATE\STANDARD.WPTô\  `$Times NewRoman ‘{’“”•–—e˜e™ení$3|x™,«þ$‰ÿU‹ÿÀÀÀ  WSU ETCFfemale:maleÝ ƒ!ÝÝ  ÝÔ_ÔÔ‡XF XXXÔ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€WSU„ETCF/SOP€400„05„01Ð ° Ѐ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Page€1€of€2Ô#†XF XXXF +#ÔÔ‡XF XXXF ÔÐ œì Ѐ€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€€Effective€Date:€€15€August€1997ÌÔ#†XF XXXF [#ÔÔ‡XF XXXF ÔÌTitle:€ò òFATHEAD€MINNOW€CULTURING€PROCEDURESÐ `° Ð________________________________________________________________________ÌÌó óAuthor(s):à ` àà ¸ à_______________________________à È àDate:€€____________Ð ( x Ðà  àà ` àà ¸ àDr.€Barry€L.€Forsythe€IIÌÌSection€Leader:à ¸ à_______________________________à È àDate:€€____________Ð ì <  Ðà  àà ` àà ¸ àDr.€Michael€J.€LydyÌò ò________________________________________________________________________Ð Ä  Ðó óÌò ò1.0€€OBJECTIVEó óÐ œì  ÐÌà  àTo€describe€the€procedures€used€in€maintaining€a€reproducing€colony€of€fathead€minnows€Ï€€€€€€€€€€€€€(òòPimephales€promelasóó).Ð `° ÐÔ#†XF XXXF .#ÔÌò ò2.0€HEALTH€AND€SAFETYÐ 8ˆ ÐÌ3.0€PERSONNEL/TRAINING/RESPONSIBILITIESó óÐ ` ÐÌò òà  à3.1€ó óPersonnelÐ è8 ÐÌà0  àà0` (#(#àò ò3.1.1ó ó€Only€those€individuals€trained€and€having€the€documentation€of€suchÐ Ä Ðtraining€€€€€€€€€€€will€be€allowed€to€assume€the€responsibility€of€the€fish€colonyððsÏmaintenance.Ð ` (#` (# ÐÌà  àò ò3.2ó ó€TrainingÐ xÈ ÐÌò òà0  àà0` (#(#à3.2.1ó ó€All€personnel€should€read€and€understand€the€USEPA€methods€paper€€€€€€€€€€€€€Ð T!¤ Ѐ€€€€€€€€(EPA/505/8„89/002b)€as€well€as€view€the€supplemental€video€prior€to€taking€Ï€€€€€€€€€€on€the€responsibility€of€colony€maintenance.Ð ` (#` (# ÐÌò òà0  àà0` (#(#à3.2.2ó ó€All€personnel€should€complete€a€tour€and€a€procedure€training€seminar€€€€€€€€€€€Ð %X # Ѐ€€€€€€€€presented€by€the€ð ðcolony€managerðð.Ð ` (#` (# ÐÌà  àò ò3.3€ó óResponsibilityÐ Ð' #& ÐÌò òà0  àà0` (#(#à3.3.1ó ó€The€primary€responsibility€of€maintaining€the€colony€belongs€to€the€ð ðcolony€€Ð ¬)ü$( Ѐ€€€€€€€€€€managerðð,€and€then€falls€to€someone€named€by€the€manager,€who€has€been€€Ï€€€€€€€€deemed€adequately€trained,€in€case€of€the€managerððs€absence.Ð ` (#` (# ÐÌÐ `-°(, ÐÐ `-°(, Їà  àà ` àà ¸ àà  àà h àà À àà  àà p àà È àà  àPage€2€of€5ÌÌò òÌ4.0€REQUIRED€AND€RECOMMENDED€MATERIALSó óÐ tÄ ÐÌà  àGlass€AquariaÌà  àBiological€filter€(optional)Ìà  àAir€pump€with€stoneÌà  à4"€Spawning€tiles€(PVC)Ìà  àSeparatory€funnelsÌà  à6"€fish€netsÌà  àCleaning€utensilsÌà  àBrine€shrimp€cystsÌà  àFrozen€brine€shrimpÌà  àTetraminðð€fish€foodÌà  àAssorted€ñ—ñpipettsñ—ññ—ñpipettesñ—ñÌà  àNalgeneðð€Autoclave€tubÌà  àDoor€SheetÌÌò ò5.0€PROCEDUREó óÐ 4„ ÐÌà  àò ò5.1ó ó€Culture€Water€PreparationÐ  \ ÐÌò òà0  àà0` (#(#à5.1.1ó ó€Natural€water,€drinking€water,€or€reconstituted€water€may€be€used€in€the€€€€€€€€Ð è8 Ѐ€€€€€€€€culturing€of€fathead€minnows€if€it€is€deemed€to€be€toxicant€free.Ð ` (#` (# ЀÌà  àò ò5.2ó ó€Natural€WatersÐ ° ÐÌò òà0  àà0` (#(#à5.2.1ó ó€Surface€waters€such€as€those€from€a€river,€lake,€well,€or€spring€may€be€used€if€Ð ŒÜ Ѐ€€€€€€€€passed€through€a€carbon€and/or€sand€filter€followed€by€a€fine€filter€(ð ð5ððm).€€Ð |Ì Ѐ€€€€€€€€If€resident€fish€populations€exist€in€the€source€then€some€measures€must€be€Ï€€€€€€€€€taken€to€remove€potential€fish€pathogens.Ð ` (#` (# ÐÌò òà  à5.3ó ó€Drinking€WatersÐ ,#|! ÐÌò òà0  àà0` (#(#à5.3.1ó ó€Tap€water€may€be€used€if€dechlorinated€first.€€Dechlorination€can€be€€€€€€€€€€€€€€€Ð %X # Ѐ€€€€€€€€accomplished€by€three€common€methods;€aeration,€passage€through€a€carbon€Ï€€€€€€€€€filter,€or€the€use€of€sodium€thiosulfate.€€Some€care€must€be€taken€if€using€Ï€€€€€€€€€sodium€thiosulfate€as€it€exhibits€some€toxicity€to€fathead€minnows.Ð ` (#` (# ÐÌà  àò ò5.4ó ó€Reconstituted€WaterÐ ¨)ø$( ÐÌò òà0  àà0` (#(#à5.4.1ó ó€Deionized€water€from€an€adequate€ultrafiltration€system€may€be€combined€€€€€€Ð „+Ô&* Ѐ€€€€€€€€with€reagent€grade€chemicals€in€a€recipe€manner€according€to€those€Ï€€€€€€€€€guidelines€set€forth€by€EPA€and€ASTM..Ð`-°(,` (#` (# Їà  àà ` àà ¸ àà  àà h àà À àà  àà p àà È àà  àPage€3€of€5ÌÌò òà0  àà0` (#(#à5.4.2ó ó€Commercial€mineral€water€may€be€mixed€with€ultrafiltered€water€(1:4)€as€a€€€€€Ð ˆØ Ѐ€€€€€€€€source€of€culture€water.Ð ` (#` (# ÐÌà  àà ` àò ò5.4.3ó ó€Shelf€life€is€approximately€14€days.Ð P   ÐÌà  àò ò5.5€ó óWater€DeliveryÐ , | ÐÌà  àà ` àò ò5.5.1ó ó€If€using€a€flow„through€system€turnover€should€be€at€least€3„4€times€per€day.Ð  X  ÐÌò òà0  àà0` (#(#à5.5.2ó ó€Static€tanks€should€have€either€an€under„the„gravel€filter€or€an€external€filter.€€Ð ä4  Ѐ€€€€€€€€Renew€the€water€by€replacing€25%,€every€2€or€3€days,€with€distilled€water.Ð ` (#` (# ÐÌà  àò ò5.6ó ó€FeedingÐ ¬ü  ÐÌà  àà ` àò ò5.6.1ó ó€Feeding€The€Larvae€Ð ˆØ ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#à5.6.1.1ó ó€From€hatching€to€the€age€of€30€days€the€larvae€should€be€fed€live,€€€€Ð d´ Ѐ€€€€€€€€€€€less€than€24€h€old,€brine€shrimp,€òòArtemia€spp.óóÐT¤¸ (#¸ (# Ѐ€€€€€€€Ìò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.1.2ó ó€Fish€should€be€fed€twice€daily,€Monday€„€Friday,€and€once€a€day€onÐ ,| Ðweekends.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.1.3ó ó€Observe€consumption€so€as€to€estimate€an€adequate€amount€of€€€€€€€€€Ð ôD Ðbrine€shrimp.Ð (#(# ÐÌà  àà ` àà ¸ àò ò5.6.1.4ó ó€Siphon€our€any€excess€food€each€day.Ð ¼  ÐÌò òà  àà ` à5.6.2ó ó€Feeding€Juveniles€and€AdultsÐ ˜è ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.2.1ó ó€Fish€older€than€30€days€and€less€than€90€days€should€be€fed€partiallyÐ t!Ä Ðfrozen,€adult€brine€shrimp.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.2.2ó ó€Fish€should€be€fed€twice€daily,€Monday€„€Friday,€and€once€a€day€onÐ <$Œ" Ðweekends.Ð (#(# ÐÌà  àà ` àà ¸ àò ò5.6.2.3ó ó€Feed€an€amount€ñ˜ñthañ˜ññ˜ñthatñ˜ñ€will€be€consumed€in€10„30€minutes.Ð 'T"% ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.2.4ó ó€Naturally€occurring€periphyton€in€tanks€serve€as€a€good€dietaryÐ à(0$' Ðsupplement.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.6.2.5ó ó€Commercial€flake€food,€such€as€Tetraminðð€may€be€used€to€feed€fishÐ ¨+ø&* Ðolder€than€90€days.Ð (#(# ÐÐ „-Ô(, Ðà  àà ` àà ¸ àà  àà h àà À àà  àà p àà È àà  àPage€4€of€5ÌÌà  àò ò5.7ó ó€CulturingÐ ˆØ ÐÌà  àà ` àò ò5.7.1ó ó€Initiating€CulturesÐ d´ ÐÌà  àà ` àà ¸ àò ò5.7.1.1ó ó€Embryos€may€be€obtained€from€a€reliable€research€laboratory.Ð @  ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.1.2ó ó€Once€acclimated€to€25ððC€the€embryos€will€hatch€in€4„5€days€andÐ  l Ðmay€be€treated€as€described€earlier.Ð (#(# Ðà  àÌà  àò òà ` à5.7.2ó ó€DevelopmentÐ ä4  ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.2.1ó ó€Fish€30€days€old€may€be€used€for€acute€toxicity€testing€or€grownÐ À  Ðour€as€brood€stock.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.2.2ó ó€Decreased€stocking€densities€and€the€addition€of€spawning€tiles€willÐ ˆØ Ðspeed€up€maturation.Ð (#(# Ðà  àà ` àÌà  àà ` àà ¸ àò ò5.7.2.3ó ó€Signs€of€maturity€begin€to€appear€at€3„4€months.Ð P  ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.2.3.1ó ó€Males€will€develop€a€wide€head€with€tubercles€along€with€€Ð ,| Ѐ€€black€coloration€on€their€sides.Ð h(#h(# Ðà  àÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.2.3.2ó ó€Females€will€be€smaller€with€olivaceous€color;€and€will€€€€€€€Ð ôD Ѐ€€exhibit€an€ovipositor€when€mature€enough€to€spawn.Ð h(#h(# Ðà  àÌà  àà ` àò ò5.7.3ó ó€SpawningÐ ¼  ÐÌà  àà ` àà ¸ àò ò5.7.3.1ó ó€There€are€two€options€for€separating€adults€for€breeding.Ð ˜è ÐÌò òà  àà ` àà ¸ àà  à5.7.3.1.1ó ó€Spawning€pairs€may€be€set€up€with€one€tile.Ð t!Ä ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.3.1.2ó ó€Place€a€group€of€mature€fish€in€a€tank€with€up€to€4€€€€€€€€€€€€€Ð P# ! Ѐ€€spawning€tiles,€resulting€in€a€female:male€ratio€of€8:3.Ð h(#h(# Ðà  àÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.3.2ó ó€Females€may€release€100€„€400€eggs€on€the€underside€of€theÐ &h!$ Ðspawning€tiles€whereupon€they€are€fertilized€by€the€male.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.3.3ó ó€Fathead€minnows€spawn€approximately€every€4€„€5€days€in€theÐ à(0$' Ðlaboratory.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.3.3.1ó ó€If€no€embryos€are€produced€in€a€3€week€period,€replace€€€€€Ð ¨+ø&* Ѐ€€the€pair€or€the€entire€group,€unless€the€fish€are€young€Ï€€€adults.Є-Ô(,h(#h(# Ðà  àà ` àà ¸ àà  àà h àà À àà  àà p àà È àPage€5€of€5ÌÌà  àà ` àà ¸ àò ò5.7.3.4ó ó€Collecting€EmbryosÐ ˆØ ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.3.4.1ó ó€Tanks€should€be€checked€mid„morning€each€day€for€€€€€€€€€€€Ð d´ Ѐ€€embryos.Ð h(#h(# Ðà  àà ` àÌò òà  àà ` àà ¸ àà  à5.7.3.4.2ó ó€Remove€spawning€tiles€with€tongs.Ð , | ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.3.4.3ó ó€If€embryos€are€to€be€used€in€toxicity€testing€then€they€may€Ð  X  Ѐ€€be€removed€from€the€tile€with€a€gentle,€circular,€rubbing€Ï€€€motion€while€keeping€the€tile€submerged.Ð h(#h(# ÐÌà  àà ` àò ò5.7.4ó ó€HatchingÐ ¼  ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.4.1ó ó€There€are€two€methods€plausible€for€the€hatching€of€fatheadÐ ˜è  Ðminnow€embryos.Ð (#(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.4.1.1ó ó€Roll€the€embryos€off€the€tiles€into€a€culture€dish€€€€€€€€€€€€€€€€€Ð `° Ѐ€€containing€culture€water€and€pipet€them€into€a€separatory€€Ï€€€funnel€filled€with€aerating€culture€water.€€After€two€days€€Ï€€€empty€the€separatory€funnel€into€a€pan€(preferably€white),€Ï€€€at€a€temperature€of€25ððC.€€The€embryos€will€hatch€in€2€„€3€Ï€€€days.Ð h(#h(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà0h(#(#àà  à5.7.4.1.2ó ó€Place€the€entire€spawning€tile€into€a€pan€fulled€with€€€€€€€€€€€€Ð Ø( Ѐ€€aerating€culture€water,€at€25ððC,€and€the€embryos€will€€Ï€€€hatch€in€4€„€5€days.€€Arrange€the€tiles€so€ñ™ñthañ™ññ™ñthatñ™ñ€the€bubbles€€€€Ï€from€the€air€stone€flow€over€the€eggs€so€as€to€keep€algal€€€€€Ïand€fungal€colonies€from€forming.€€These€can€entrap€the€€€€€Ïlarvae€as€they€hatch.€€Check€the€tiles€on€days€1€and€2€for€€€€€Ïfungus€or€lack€of€viability€and€remove€any€such€with€€€€€Ïforceps.€€Minimize€disturbances€on€days€3€„€5,€as€it€may€€€€Ïcause€early€hatching.€€If€50%€of€the€embryos€from€any€one€€€€Ïtile€have€to€be€removed€because€of€lack€of€viability,€then€€€€Ïdiscard€all€remaining€eggs.Ð h(#h(# ÐÌò òà0  àà0` (#(#àà0¸ ` (#` (#àà0¸ (#¸ (#àà ¸ à5.7.4.2ó ó€Disinfect€all€used€tiles€by€soaking€in€a€chlorine€bath€(1€mL/L)€for€1Ð ì&<"% Ðhour,€followed€by€rinsing€with€tap€water.€€Neutralize€any€residualÏchlorine€with€sodium€thiosulfate€(100€mg/L)€for€10€minutes,€andÏfinally€rinse€with€culture€water€and€allow€to€air€dry.Ð (#(# ÐÌò ò6.0€QUALITY€CONTROL€CHECKS€AND€ACCEPTANCEÐ Œ+Ü&* ÐÌó óà0  àThe€procedures€in€this€Standard€Operating€Procedure€will€be€reviewed€periodically.Ðd-´(,(#(# Їò òà  àà ` àà ¸ àà  àà h àà À àà  àà p àà È àà  àó óPage€6€of€6ò òÐ ° ÐÌ7.0€LITERATURE€CITEDó óÐ ˆØ ÐÌU.S.€Environmental€Protection€Agency,€1989.€€Culturing€of€fathead€minnows€(òòPimmephalesÐ `° Ðpromelasóó)ññ:ñññ‘ñññ€ñññ‘ñññ€Sñññ’ñupplemental€Report€for€video€training€tape.€€EPA/505/8„89/002b.Ð L œ ÐÌ„„„„„„„„„„€1989.€€Culturing€of€fathead€minnows€ñ’ññ“ñ(òòPimmephalesóóñ“ññ”ñ€ñ”ññ•ñòòpromelasóó):ñ•ññ–ñ€Video€training€tape.ñ–ñ