Document 4QKQJqKLrL6LqqwRd73Lpwqmj
AR226-0%6
TOXICITY TO AQUATIC PLANTS (e.g., Algae) TEST SUBSTANCE
Identity:
ammonium salt, CAS # 3625-26-1) Perfluorooctanoic acid, ammonium salt; mayalso be referred to as.
PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC116, FC-126, FC-169, or FC-143. (Octanoic acid, pentadecafluoro-
Remarks: The 3M production lot number was 37. The test sample is FC-
143. It's purity was not sufficiently characterized, though current information
iannddicCastepseritfliusoaromiaxntaulroegoufe9c6o.m5p-o1un0d0s%.test substance and 0 -- 3.5% Cs, Cr,
METHOD:
M60e0t/h9o-7d8-F0o1l8l;owAeSdT:M-MEo-d3if5i.e2d3(DmroadftelNeod.)
after those 2; OECD;
described by A.G. Payne.
USEPA
--
Type (test type): Acute Static
GLP: No
YSpeeacrisetsu:dySepleernfaosrtmreudm:capr1i9c8o1muturn.
ASnoaulryctei:caUlSmEoPniAto--riEnRgL:,
Corv., Oregon (July 14, 1981), Algal cell counts (cells/mi), cell
dry
weights,
and
temperature.
EStxaptoisstuircael pMeertihodo:ds:4,7, 10E, Casndva1l4udeasyasnd 95% confidence limits were
Tcaelsctuloartgedamutiislimzsingl3abMorSaitxcourry,caulltiunreea:r rAelggreasesfironommoad7e-ld.ay-old stock culture
Algal Nutrient Medium: Sterile synthetic algal nutrient medium. This
nutrient medium served as the diluent for all algal operations. The pH of this
sSytnotchketiacndalTgaelstmeSdoliuutmiownasPraedpjaursatteidont:o 7A.5p0r.i1marpryiosrtotcokussoeluitniaosnswaayss..
pprriempaarryedstioncaklgsaollumteiodniwuamsatdialuctoendcweintthraatligoaln
mofed5igu/Lm.
toAfptrerepmairxeintgh,e
the six
test
concentraions.
Exposure vessels: Sterile 250 mL Erlenmeyer flasks containing 50 mL of
test solution and stoppered with autoclaved foam plugs.
ANguimtabteirono:f Croepnltiicnautoeuss: ptlhartefeorm shaking at 100 + 10 rpm
Initial algal cell loading: 1.0 x 10* cells/mL.
NNoummibnearl ocfonccoenncternattriaotniso:ns:Bksicxonptlrouls,n1e0g0a,ti1v8e0,co3n2t0ro,l560, 1000, and 1800
TmegsltL conditions:
Temperature: 232C (70-77F) Fluorescent illumination: 400 ft. candles + 10%
C3791
BRES SULTS
eee
eee
Algal Growth Response ECs values
Exposure
(CoDnatyasct)
CemllL-Dry(W9e5%igCh.1t,
mCgellLl-(C9o5u%ntC.@1)
a1
149 (57-340)
49 (28-75)
[ [17 iao1 | m7o4e@9 e(s1t5t9i6)s | | [2asrpo@aess)n0)|||
(TY culGtruorewfrlaesskps.onse parameter; oldryweight (mg/L), measured Tn plicated Ses of
(2) Gofrocuwltthurreefslpasoknsse parameter; celkcount (numbercalls / mL), measured in tripicated sets
Algal
Growth
Response EC10 and EC90 (numobfceelrls
/vmaLlu)es
based
on
Cell-Count
(CEoxnptoascutr)e
[ Days 4 95367 |
[71 334 |
[0
2915) |
[# 5@1 8 |
EC
8(CmgLlnLol(c9a5l%cCu.i1,ete)
283 (150-590) 386 (Cnot calculated) 307 (Clnotcalculated)
Element values based on nominal concentrations.
cCoOnNcCuLUsSiIoONnSs
0 000
Ammonium mg/L with a
p9e5r%flucoornofoicdteanncoeatineteexrvhailbiotfs
a 14
14-day EC to 81 mg/L.
(cell
count)
value
of
43
b0 DaAmTAauQUAAwLIrTy0 Y 0 00 00 Reliability: Kiimisch ranking = 2. This study meets the criteria for quality pstueursbitstitynogafnacttehtehinetestsoitlmuestiuiotbnsw.taasncceonadnudctaecdt.ualHomweeavseurr,etmheent`sstuodfy tlhaeckasmoinufnotrmoaftitoenston
RREEFFEERREENNCCEESS
OOOO
3AsMsaTeycThensitcaMletRhepoodr,tRSeupomrmtarNyu,mbMeulrt0i0-6P,haPsreojEexcptoNsuumrebe/rRe9c9o7v0e0r3y00A0lg0a,l M. T.
Elnabarawy, October 16, 1981
03792
oOwTHeERr
Submitter: 3M Paul, Minnesota,
Company, 55133
Environmental
Laboratory,
P.O.
Box
33331,
St.
Last changed: 5/24/00
93733
inertia
TECHNICAL REPORT SUMMARY
TO: TECHNICAL COMMUNICATIONS CENTER -- 201.208
Imp=o1reptrtsarinte ton both siodfpeapse,sn wocoi 0TCC.)
i/16/8
Environmental Laboratory - EE & PC - 2-3E
New Methods Develo; nt - FC-143, Lot 37
Multi-Phase Exposure, Recovery Algal Assay
D. Bacon/R. Bohon,
/A. Welter
M. T. Elnabarawy
Test
Method
0535 9970030000 006
46981
secmcoonmorsy:p ome Comme owoerrion | "AGHA
2 hemi Ror
TERRY
awplicatieterme)
| 1.
To evaluate
ggreeneenratailognase
at"shSeetlahelengyaaslmtaryugmrobecwtahpartifrececostrpenoduntsuebmyoefovvfperereusmsheewvaetoriearlihe
fluorochemical FC-143, lot 37.
E(EEnv&. PCL-aDbi)v. 2. eToxpodseuvreeloporarnecaolvgealrytpeesrtiopdrsototcoolthebatseedstoonmvmanriinagb:le
A ESTSTT AT 20250 To os ratio SR Te Sore
In the
these triplicated four-phase initial effects on organisms
exposure/recovery following varied
assays,
exposures to generations.
FC-143 were assessed over several Test results gave an indication of
the
possible assessed
qeufafnetcittsatoinveallygalin ptoeprumlsatioofnsc.ell-Adlrgyalwebiigohmtass(mwga/1s
and were
cell-count measured
(No./ml). The and calculated
following for:
biomass
) indicators
A. Verification of inhibitory effects
1. Meexdpioasnuregsr.owth response ECsg (mg/l) from varied
32.. NEoC-jegffaencdt EClgeovel(smg/l) for same exposure periods.
B. Verification of algicidal effects
1. Resumption or absence of logarithmic growth in subcultures during varied periods of recovery.
te /
i - : 03794
PFaCg-e14T3w/oMTE November 16, 1981
CONCLUSIONS 1) pcMloaantnsetirdiaeslrpseecdipeotsos.sebessisnlgighatnlyECspghytinototxheicratnogealgoafe1a0n-d100aqmuga/tlicare
2) pT(hedereiaotadhlsgoaflofaglrrgeoacwlotvhecrerlyelsspi)onndsiaectatcienodncamleilnntirsmauatblicounlastlugritecesisdtaefdlo.lelfofTwehiecntgsalvgaarlied trceeeslstlusspmeartneedcreoidvaelri.end fTarhneedshmreensduuitmarenidengtrloowmgteahdriiturhmemsipiconnsgterheow(taEhbCgsgew)nhceenwasof the fitohnrehmrieebrfiotrocerasyei,enftfpeehrcopttroestaysendtohpaepssoisbsea,dsictcoealllal-nygraiolnwgdtiihcc,iadtaialvnedefc9f8eelclat-n.diviIsniotnh,e acreell-rdedeuscterducwthieorneaasndancelall-gdiecaitdha.l effect causes direct
3) eE(xxehp.igob.s,iutree>d7/rsdealcyooswv)eerryirnedrcueocsevudelrtysgreriaanttdeeiscratiienndhtihtbehiattisounbecxutalnetdnudrecedosn.esxepqouseunrtelsy
summary
geArveafelonuuarta-elpghaaaeslega"leSxepglroeosnwuatrshetr/urrmeecsopcvoaenprsryeicooarflnguattlhuena"sfsroaevysehrwwaastseervdeervuaenlliocpgeeeldnleurltaaortions
rFasCe-s1pt4oh3ne.syesmaThydiusrbienmgualftffieopcuhrtaedsceonbsayelcgeauxltpiovaseusrseaeyxpteoovsautlrhueea/trfeeldcuoovarelogrcayhlemcigocrmaoblwi,tnhations:
pd4eu/rr10ia,otdiso7n/7d,eofcr1e0ea/xs4p,eods.uanrdeThp1e4eridcaooydmssb.iniendAcsredauisrseadtc,iloenawrhlioyfleeianrcdehiscpaetcetdi,ve threecovery
creeexslpploo-sndusrreye/wwraeesicgohmvetearsy(umrgpe/hd1a)seinanwdatsercmefslilxoe-fdcouaanntti14n(cNrdoea.ya/ssm.el).inAlbgDairloym-awgsersiogwahtsth and
acveelrla-gceosuntofretsruilptlsicautseedd fsoetrsthoef ctaelsctulcautlitounreofflaEsCksso.'s (ng/1) vere
c"IanSlectluhelenasaetsetdrturmiEpClCsigacparvtiaecldourensmuutl(utmmig"p/hla)asreefopmrruelfstreiengstehenwdeartaietnrioTgnarbeleaenlga1al.lgaaesTshaeysse, the
evxaplouseusreretporetsheenttetshte mmaetdeirainalgrofworth4,re7s,pon10s,e a(nEdCsg14mgd/alys). following
pTehreiovdasluelsistoefd EeCa1orliaenrd are presented in Table
wEeCrgeo 2.
c(amlac/lu)latfeodr
einachteromfs tohfe
ceexlplo-scuoruent
and
No test concentrations were defined as a no-effect level.
C03795
LL Fc-la3/mTE PNaogveemTbherree16, 1981
Tgesrotoawvbtelhriisfihnyedtahlegfircaoibmdsaeelnaccehefoftfercitptslh,eicatatenedsdt tmosaettaesrsoifeaslstessputobscscuiulbltltueurreersesfwuleamrspektsion of
croemsbpionnesde avtastheevaleundateodf 10, 7, and 4 days.
efaoclhloewixnpgosurreesppeecrtiiovde. recAolvgaelryrepceorivoedrsy
of
INTRODUCTION
asIqynusattteeimsctifnoegrnvietrvhoeanlmupeaontstis,nigbluetnhieecfeflielncuftllsuaernocfaelgcaohfeemiaccrhaeelmircseauclbossmtmaoennncdeeasdlgaoalns
athemodel growth
(aquatic primary producers), aquatic plants, and phytoplankton.
Tcstoeixlmiluc-ldaeitfvofiresyci,tosn b)adrueriinnhtgiebsittethdoeryotnesatng.dr/oowriTnhgaelsgeailcggiarldoawltc.hultruersepsonstehsat mauyndebersgoa)
MATERIAL AND EXPERIMENTAL DESIGN
Tpsheoerlfuobtrlemesetdisnounbwsatttaehnircseamta(taFemCrb-ii1ae4l3n,t evrlaootlouma3t7t)eedmpiesirtsaatupfroietn.eentwihTaihltee ablpigooawaldsesrat,yosxicity
and the data generated form the basis of this report.
TthheosetesdtespcrriobteodcolbyutUiSlEiPzAed2; OECD; A. G. Payne. (1),
f6o0r0/9t-hi7s8-0s1t8u;dyASwTasM-mEo-d3i5f.i2e3d DarfaftterNo. (2), (3), (4).
Test Species:
aAclhglbaoaercotce(orCcihcala-olfrerosepehywcacseualeto)ub,rteaiSoenfleednftahresotmrfurmeU schawS part--ieEcrBoRrpP EnlsuatnAukmFtaoronefi,.cthOgerreegoeornnder au(lnFgieacblerlulcauurllytaur1r8,ecewl1al9ss8,1)s.thoarveiTdnhgeinttehsettheaapldpgaearaekranaartcee49noCfo.n-amonteiwlemoon. The Inoculum:
Agilvgeaea fsrtoamrtain7g-doapyt-iomludmstioncokculcuumltulreevelwerofe gurseedenasalgianeoculum to
1Stoe0l-ednaasyt-ru0m1dcasptroicckorncuutltuumre1 sufficient number of viable
xins1u0r4edceltlhse/mplr.esenTchee oufsea of a 7 algal cells in the exponential
gccerulollwtstu/hrmelp)wh.aasse.detTehreminienditiuaslingalgaalhemceolclytocmoeuntterin(2t7h7e,00s0tock
C03796
FPaog-e1a3Fo/umrre
November 16, 1981
Algal Nutrient Medium:
Culture Flasks: Manidnertaelsti(nignoarlggaaneic)wasstaprnedpaarrdednuatsrieonuttlimneeddiuminfAotrtaccuhlmteunrting#1.
This nutrient medium for algal growth and
provided all mineral nutrients also served as the diluent for
essential all algal
operations PH of this
including synthetic
the preparation of stock solutions. algal medium was adjusted to 7.5+.1
The
Prior to use in assays.
=
Free gaseous exchange" to' somae Each 250 ml
comprised a
Erlenmeyer containing 50 ml test flask. Culture flasks
of test and all
solution other
glassware were #2. Autoclaved
specially prepared foam plugs used as
as described in flask closures,
Attachment permitted
Range-Pinding Test (Exploratory):
s1e5v0e0ranlosiosrders of magnitude: 100, 350, Eee 200 1 00 and Ihe exploratory test consisted of determining: the 4-day algal
biomass in triplicated flasks medium plus the test material
containing standard nutrient at concentrations covering
Full-Scale Test (Definitive):
14/0 days. The purvose. of ch exposure' (sontacty Clase nds All definitive algal assays were carried out
using 250 ml Erlenmeyer flasks containing 50
in ml
triplicate of test
solution. The definitive assay consisted of four
simultaneous exposure/recovery tests (4/10, 7/7, 10/4, and
to verify inhibitory effects. stage evaluated the' viability
The recovery (subculture) of growth-inhibited algae and
verified algicidal effects.
Substance ves prepared in' the Sioos mateiicy oath The following PC-143 logarithmic concentrations: 100, 180,
eb3rx20ap,cokseu5tr6e0,thset1a0g04e0-.,daaynTdhEiCss1o8d00voaslemuge/rslangwpeerreedoficutsdeeiddluttfioroonmisnpirtweaisaltiemsienltaehrceyted to
exploratory testing. algal nutrient medium
Three flasks containing 100% fresh Plus algal cells comprised the
nontreated controls. A fresh stock solution of the test
immediately prior solution was 7.4.
to testing. The initial Procedure and steps for
pH of this stock preparation of
algal test flasks are outlined in Attachment #3.
Each recovery stage was initiated by in the absence of the test material.
subculturing Algal cells
(0.5/50 ml) from each
resuspended i triplicated set of culture flasks were combined and then
nto fresh nutrient medium. Subsitare Lemods05797 are briefly described in Attachment #4.
Fc-143/uTE NPoavgeembFeirve16, 1981
Test Conditions:
cAollndiatligoanls,opeinraotridoenrs
wtoereavociadrriceodntaomuitnautnidoenr
aseptic with bacteria
other algae. Algal cultures were maintained in an
and
ecnovnidritoinomnesn:tal chamber under the following standard growth
Temperature - 23 + 2C (70-779) Fluorescent illumination - 400 t.C. + 10% SFrheaekinggas10e0xch+an1g0erp-m.Continuous Platform Algal Biomass Monitoring:
Tuchsoeiunngtaslgeai(ltcheeglrrlos/wamtllhg)a.lrecsepBloonlts-hedrgywraowswetihagphmpteraais(sumergde/lm)eqnutaasnndt/wioetrraetibyvmealdcyeellwbiyth
asltgaagles.cultBuortehsparfotceerdueraecsh foofr tahlegalexpboisoumraessamndeasruerceomveenrtys briefly described in Attachment #5.
are
uBtCigloizvianlgues3ManSdIXC9U5R%, caonfTRiAdCencSeystleimnitfsorwreergerecsaslicounlamtoeddels experimental aata.
of
TEST RESULTS
TSheelenraesstulrtusm cofaprbiicoomarsnsutummeaussuerdemeinntstheosfe tshteudgireesenaraelgae
:
sCFaeCll-1cf4u-3le,axtpelldaontaEtC3o7s,rqyf(aomngld/llo)wairnevgaldueeetxsapiolsieundrdeiciapnteirtnihgoedsaaltgtoaaflch4e,gdro7,wdtaht10a,resshapeneodtnss1.e4 to
etdaaecyrshnsoaforef tpcherelelse-excnpotouesndutreiannpdTearabirloeedsp1.rleissetTnehdteedavabloiuvneesTawboelfreeECc2.1aglcaRuneldcatoevEdeCrgygindfaotra
asnudmmarreiszuemdptiionnTaofblelo3g.arithmic growth in subcultures are
aTOfroewihslahltouwsnhtarsaitnehaFpitpgheuenreeadsctii1nonantdhoef 2.etxepsotUstumiraletiezrieinxagplertihamenednEtCt,ogotgviovaxeliuceiastn,yovceurrvveisew toxicity curves were constructed against time on semilog plots. TpdhaeetrafodrsamhteeadetrsbeypocMro.tmepdrT.isheEelrenoiarnbigairanaraewly baadnastdead.R.onR.stuRdoibeisdeadue.veloApcecdompaandnying
C03798
. PEaCg-ela3S/ixMTE
November 16, 1981
It should be noted that calculated on the basis
all the reported EC values were of the initial concentrations of
FC-143,
lot 37, in test solutions at the beginning of the bioassay.
REFERENCES
(1)
Miller, W. E., J. C. Greene, and Selenastrum Spricornutun Printz
T. Shiroyama. 1978. The Algal Assay Bottle Test:
Experimental sign, application, and U.S. Environmental Protection Agency,
data interpretation. Corvallis, Oregon.
EPA-600/9-78-018. 125 p.
(2)
ASTM-E-35.23. 1981. Proposed
CAlognadeu.ctinDgrafTtoxNioc.ity2. Tests with
Standard Practice For Freshwater and Saltwater
(3) OEfEfCeDctGsuiodneliBnieosticforSysTteesmtsi,ng Toefst Ch2e01mic"aAllsgae(,198G1r)owtShectIinohnibi2,tion Test," Adopted May 12, 1981.
(4)
OAPflagyanNlee,w TACo.hxeimGc.iictayalnsda.ndR. AIStH.TsMHAapSlpTllP,ica61t697i7,o9.np.tAo 1M7te1ht-eh18oS0da.feftoyr
Measuring Assessment
03799
TABLE 1 AlgalFCG-r1o4w3t,h RLoetspo3n7se to
ECs0's (mg/1) (1)
(ECxopnotsacutr)e
Days
Cell-DryWeight(2)
Cell-Cou(n3t)
4
149(57-341) (4)
49(28-75)
7
70(34-118)
30(21-40)
10
49(15-96)
27(8-50)
1a
73(25-147)
43(14-81)
(1) MreetghroedssiofonECmsoodelcsalcouflaetxipoenr:imen3tMalSIXdCaUtRa,. a TRAC system for (2) Girnowttrhiplriecsaptonesde speatrsamoeftecru;ltucreell-fdlraysksw.eight (mg/l), measured (3) mGeraoswuthredresipnontsreipplaircaamteetders;etscelofl-ccouulntture(nof.lacskesl.ls/ml), (4) 95% Confidence Limits.
0as09
.
Exposure (CDoanytsact)
4 7 10 14
TABLE 2
Algal Growth Response to
EC10'sFCa-n14d3,ECgLoo'ts 37(mg/1)
Based on Cell-Count (no. cells/ml)
EC1g
5.3(3-7) 3.3(2-4) 2.9(1-5) 5(2-8)
ECqq
624(No Limits) 283(150-590) 386(No Limits) 307(60-No Limits)
/)
03801
A
TABLE 3
Algal cnnRec@quienrythy StCaeglelo/Dmalta(SMuEmAmNarVyALUES) (1)
Treatment TaInyitaial--7A'lgbDaalyysCeel1l0LBoaavgdsisn"g(--2r)FhirnaDaealyAsAllegaall CCDeeallylls-cCoouunnet(D(3a3)y)s
Control 2,017 2,430 2,453
380,000 256,000 136,000
1100 mg/1 643 580 587
372,000 226,000 48,000
2 180 mg/L 530 290 280
348,000 204,000 20,000
3320 mg/1 383 273 380
300,000 214,000 20,000
4560 mg/L 237 137 290
236,000 200,000 18;000
5 1,000 mg/1 57
60 103
310,000 174,000 18,000
61,800 ng/1 73
73 73
182,000 54,000 18,000
(1) tBarsiepdlicoante0d.5smetls/5o0fmlculstuubrceultfulrasekss ecsotmabbilniesdh.ed from each (2) Measured at the end of each exposure period. (3) Measured at the end of each recovery period.
03802
TFr e2. Te oiurEe Cm unve fran,
ResponseTo FC 143 Lor 2
le
FER
HER LTT]
EET
|
iii == H ihe H 0HH mi
IH
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nieii Tmh i ab
TT
"Tm m fHLGHI HEHE
Wl 5 e H | L Il T Lfi . Ee Hibs Hi
EERE HE PEE ee
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i SN 3i A
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ere ----
BfiY po m--n -- - I i
mn nm
hil BDa Dave
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. Faure 2, Tox\uTyY CURVE Aue STREET 31
OO
FTFERRE | eer To [EPEAT
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J4l iA i| E 4 I i AIlT Ah il v gq [IH re
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ee
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Lo
ATTAGHNENT 1
NUTRENTHED UNFoRERESHATERALGAE
A. BArCeROaNrUTeRdIEsNTeSpaSrTaOCK aSTOLULTOIONNeS., (CONCE(NCOTNCRENATRTATEEDD )
1) 25.500 gm NaNO3 in 1 liter DI water. 2) 1.044 gm KPO, in 1 liter DI water.
3) 12.159 gn HCl; 6Hy0 in 1 liter DI water. 4) 14.700 gn MgSO, 7H;0 In 1 Miter DI water.
5) 4.410 gn CaCly 2H;0 in 1 liter DI water.
6) 15.000 gnNatCoy In liter DI water.
> MEsT un(cowS cormareny
1) 185.5 mg Hypo
2) 415.6 mg mci, bo 3) 3.27 mg znc1,
1) 1.43 ng cacly 60 5) 0.01 mg cucl, 6) 7.26 ng Naghodys 2430 7) 9 mg Fecly
8) 300 mg NapEDTA-2H,0
: PREPARATION OF SYNTHETIC ALGAL NUTRIEN MEDIUM
=
Add
one ml of each micronutrients
macronutrient stock mix per
stock solution plus one ml 1 liter of deionizedwater;
= sonlcate and then filter through 0.22 ym membrane;
= adjust pH to 7.5 & 0.1;
~ store in the dark at 4%.
:
03505
i.
ATTACHMENT 2
PREPARATION OF ALGAL CULTURE FLASKS
All fbloarsokssiluisceadteIngmlaaissnta(iKnIiMnAXg).and testing algae were made of = Flasks were brushed Inside with a stiff bristle brush; - washewdithwitthopnwoant-eprh;osphate detergent (MICRO) and rinsed 3 tines - rinsed with a 10% HC solution; = rinsed 3 times with tap water and 3 times with 0.1. water; = dried in an oven at 70C for 2 hours (placed Inverted); - and autoclaved with foan plugs Inserted at 121 for 20 minutes.
03s06
i
-
ATTACHMENT 3
TY
conTroL
N8.2 * None
1100 (Sugfsonf] N12
+ Low
# 18ougl (3) Hoy C18
3Zoudl(Wd | N50 450wil (28)| Np,
* B52 * Sw
slocougl (50)| 3g 2, + lowd
6 Boowdl (30)
7
20.2
+ 18m
.
+ A a morml
L8
- 50
+ 1.8
-
50
+ 18
$0
+18
- 50
+ 18
+ Lg
- 30
-
50
+ L8
.
-
50
- 50
1)
2)
Determine
Adjust pH
Initial algal cell count
of algal nutrint medium
In
to
the
7.5
stock
+ 0.1
culture.
mi.
3) Make up stock solutions with algal nutrient medium as desired.
A) Control flasksPreparations:
1)
2)
Add
Add
inoculum (yolune predetermined); Initial
aalugtaolclauvterdiafnotunmeplduigosn. to bring volume to
coll
Soni;
loading of 1 x
swirl flasks,
10cells/ml;
and place
B) Test flasks:
1) Add algal nutrientmedium; 2) Add test material (dissolved In algal medium if required); 3) swirl flasks;
4) Add algal inoculum;
* S5t) oSwi sowmon: rl flasks, and pl 54/8 (5, ace autoclaved 000m/ foam plugs.1060mA),
RACH Iw = Swag.
03807
Kg
ATTACHMEN4T
SUBCULTURE MET5 HOmDi7I5N0AmLGAL ASSAY:
OBJECTIVE: To determine algal recovery response in absence of test substance.
PROCEDUR:E = TranCsofrenrin0g.=5imS!mlfrsotmereialceh ocefnttrhefutgreipltiucbeast.e culture flasks into
- Centrifuge at 10,000 rpm for 5 minutes.
E "= Decent supernatant 11quid and save theprecipitated algal cells.
= Usinfguttrhieenstammeedcieunmtritfougaefitxuebed,vorleusmues:penId5aal.lgal calls in algal
=
ix
astnuhdsopreotnursgaihnolsnyfearinndttohdeeacracelnmetaainntih2ne5g0tmvolpolEu1rm0lemeln(mo5ef1y)esrlgoafclulatlcguaarllel
cseuls)pension, flask.
= Usintgnouttbrhrieiennsgtamtmeoetcdaeilnutmvroiilfnuutmgoeetthtoeub5se0bs,omvlet.rEarnlsefnemreySemrlcuofltufrreeshflaaslhg,al
03808
CLL
ATTACHMEN5T
ALGAL BIOHASS MONITORING
A. GRAVIMETRIC CELL DRY WEIGHT (1)
The fpiolrteersirzeec.omended is Millipore type BD with an 0.6 micrometer
The method Is as fol lows:
|
= Dry filters for two hours at 70C in an oven;
=
Cool filters in a desiccator containing least two hours before weighing;
desiccant,
for at
= Flitevracauumsuoirtabplreesmsueraesurnoetd taloieqxucoetedof8ptshie; culture under a = Rinse the filter funnel with D.1. water; = Dry dtehesicfcialttoerr ftoorcotnwsotahnotursweiagndhtweaitgh7.0C, cool in a Basic instrument used: Analytical Balance; Mettler ME 30.
B. SPECTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL a (2)
Basic instrument used: Stectrophotameter; Bausch & Lamb, Spectronic 20. . IHMPIRCORVIEIDENTEEURBA(UUEsRed0.1FommebDEeEuProH4E1MOaCeYvTOeMrEToEFR COUToNaTIiNGr CHFLAUBERAMBERAANNDD OOPPTTIICCAALL
Mm.
Hiller, w. ., J. C. Greene, and capricornutum Printz Algal
T. Shiroyama. 1978. selenastrum Assay Bottle Test: Experimenta
RDeess.ign`,SerAipepsliEcPaAt-i6o0n0,/9-a7n8d-0D1a8t.a ICnotrevraplrleitsa,tioOnregPorno.tocoplp.. 27E.col.
(2). APHA-AWWaMtAe-rWPCaFnd (W1a97s5t)e.wat`eSrt,and(aH,rdFrMaentshoond,s mfaonragt.heedE.x)a,minlaittihoendiotfion, pp. 1030-1031.
3). Brite-Line (8), American Optical Corporation, Buffalo, New York.
03809
= CiEiAS C mSY BREE
foBoEdRS
R4e d LERENEeR TER ge
: Heth
:
c ns oonf iiALGALBiRASoSAwY:eTBOATLTLELsToEmSaTta(nAyA:BT) we
7
i
fr hdr
* ALGAL GROWTH RESPONSE TO
ECgy's
%
EXPOSURE (1)
RECOVERY (2)
: ws
CELL-DRY WEIGHT
~(mg/1.
*
0.28 ~ 2H]
A
9
NG
CELL COUNT np. cells/ml'
28
2000-39,
[| +]
CELL-DRY WEIGHT 9/1.
CELL COUNT no. cells/ml:
UENTIAL C! TI
XPOSURE/RECOVERY PER}
Ys):
~. b+ 10 747 10+h waeo
(1) Prodruescptoinvsiet.y is based on percent growth . reductionivalues indicate inhibitory (2) Prodruecstpiovnsiet.y is based on parcent growth reduction;values indicate recovery
03810