SPONSO R

STUD Y TITL E Acut e Toxicity Scree nof Tinuvi n90 0 t o Scenedesmu s subspicatu s AUTHOR S

STUD Y COMPLETIO NDAT E Septembe r

28 , 199 3 PERFORMIN GLABORATOR Y

' PROJEC T I D Fina l Report Pag e 1 o f 6 7 This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is the property of the sponsor and may be used only for that purpose intended by the sponsor and as authorized under applicable law. Every other or additional use, exploitation, reproduction, publication or submission to other parties requires the written permission of the sponsor, except as otherwise required by applicable law (e.g., TSCA). The use of these studies/ documents for regulatory, registration or other commercial purposes [including but not limited to Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH)], without the express written permission of the sponsor is unauthorized and strictly prohibited.STUD Y NONCOMPUANC ESTATEMEN T

Study Noncomplianc e Statemen t fo r Fina l Report entitle d"Acut e Toxicit y Scree n o f

Tinuvi n 90 0 t o Scenedesmu s subspicatus " fo r I n accordanc e with inten t that a U aquati c toxicity test s conducte db y ou r facility follo w goo dlaboratory practices , study directo r fo r th e abov e tes t herei n confirm s tha t

th e study was conducte dinth e spiri t o f Goo dLaborator y Practic e standards , an dth e final report receive daLimite

drevie w b y Quality Assuranc e Unit . However , th e in-lif

e portio n o f

th e study wa s no t

audite db y Quality Assuranc e Unit . Th e dat aar e intende dfo r th e study sponsor' s interna l us e onl y an dar e no t suitabl e fo r

an y regulatory submission . N o analytica l confirmatio no f tes t concentration s wa s don e no r wa s th e stability o f

th e tes t substanc e unde r

tes t condition s investigated . A U origina l raw dat awer e provide dt o wit hth e final report

an dacop y wa s retaine dat 

### ^'^B-? 3 Dat e Study Directo rOUALIT Y ASSURANC ESTATEMEN T Quality Assuranc e Uni t

reviewe dStudy "Acut e Toxicity Scree n o f Tinuvi n 90 0 t o Scenedesmu s subspicatus, " fo r . Th e followin g audi t

wa s conducte do nthi s study . Dat e Reporte dt o Dat e Reporte dt o Dat e o f Inspectio n Phas e Inspecte dStudy Directo r Managemen t 09-21-9 3 Fma l Repor t an d09-21-9 3 09-22-9 3 Raw Dat a
Th e undersigne dconducte dth e final repor t audit . Th e audi t

indicates tha t

th e repor t

i s a n accurat e reflectio n o f

th e study a s i t

was conducte db y

Dat e Quality Assuranc e Officer nSIGNATUR EPAG E

Submitte dby :

Prepare dby :

Approve dby :

:na b 

### ^-;iS'f 5 Dat e 

### 9~ajy ) Dat e

yyy/9 j Dat e 

# ^A?A . ' 1 Dat eTABL EO FCONTENT S Pag e

SUMMAR Y 6 METHOD S AN D MATERIAL S 7 I . Tes t Specie s 7 n . Tes t Substanc e 7 m . Stati c Tes t

Procedur e 8 IV . Statistica l Analysi s 8 RESULT S 9 REFERENCE S 1 0 APPENDICE S - RAWDAT A 1 1 PROJEC T PERSONNE L1 2 APPENDI X I - BIOLOGICA LRAWDAT A 1 3 APPENDI X n - STATISTICA LANALYSI S DAT A 2 8 A . Studen t

r-tes t Dat a2 9 B . ANOV A an dDunnett' s Multipl e Mean s Tes t Dat a3 9 APPENDI X m- CORRESPONDENC EAN D PROTOCO L5 3SUMMAR Y

Subject : "Acut e Toxicity Scree n o f Tinuvi n90 0 t o Scenedesmu s subspicatus, " Fina l Report Sponsor : Testin g Facility :

Locatio n o f

th e Origina l Raw Dat aan dFina l Report :

Tes t Substance : Tinuvi n 90 0 Nomina l Tes t

Concentrations : Contro l (0.0) , Vehicl e Blank (DMF) , 1.0 , 10 , an d100 mg/ L Dilutio n Media : 7. 4 (pH ) Experimenta l Tes t

Dates : Initiatio n —Septembe r 3 , 199 3 Terminatio n—Septembe r 6 , 199 3 Lengtho f

Study : 7 2 hour s Results : 72-hou r

EC5 0 = > 10 0 mg/ L(95 % confidenc e limit s coul dno t b e determined ) 72-hou r no-observe deffec t

concentratio nwa s 100 mg/ L Tes t Species : Scenedesmu s subspicatu s Sourc e o f Organisms : Departmen t o f Botany , Cultur e Collectio no f Algae , University o f

Texa s at Austi n Ag e o f Cultur e a

t Study Initiation : 3 day s ol dMETHOD S AN D MATERIAL S

Th e study wa s performe dfollowin g th e procedure s outline di n a s approve db y o nFebruar y 25 , 1993 , an dth e study directo r o n Marc h10 , 1993 . I . Tes t

Specie s Th e paren t

stoc k o f Scenedesmu s subspicatu s (supplie r batc h#UTEX-2594 , cultur e 93-T-Sc ) use di nth e definitiv e tes t wa s obtaine dfro mth e Departmen t

o f Botany , Cultur e Collectio no f Algae , University o f

Texa s at Austin , Austin , Texas , o nAugus t 26 , 1993 . Th e paren t stoc k o f alga e wa s receive dgrowin g o n a

nagar slan t containe di na50-m Lcultur e tube . Th e alga e cultur e wa s identifie da s Scenedesmu s subspicatu s o nth e cultur e tub e label . Scraping s wer e take n fro mth e slan t an dplace di n250-m LErlenmeye r flasks containin g 100 m Lo f

steril e alga e nutrien t

media . Thes e flasks wer e incubate d i n an environmenta l chambe r at 2 4 + 2° C t o facihtat e alga e growth . Periodically , ne w Scenedesmu s subspicatu s culture s wer e initiate dusin g alo t o f th e paren t

stoc k o r clone dfro manexistin g cultur e derive dfromth e paren t stoc k i n 10 0 m Lo f

steril e cultur e medium . Culture s o f Scenedesmu s subspicatu s at AB C Laboratorie s wer e maintaine dunde r th e sam e environmenta l tes t condition s a s fo r th e definitiv e test . Th e alga l cultur e use dfo r thi s toxicity tes t

wa s 3 day s ol dat tes t initiation . n . Tes t

Substanc e Th e Tinuvi n 90 0 tes t

substanc e (batc h#E N306296.12 ) wa s receive dfro m Februar y 10 , 199 3 a s awhit e powder . Th e compoun dwa s assigne d referenc e #TS-631 3 an dwa s store dat roo mtemperature . Purit y o f th e tes t substanc e wa s reporte dt o b e > 9 9 % an dth e tes t concentration s wer e prepare dbase do ntota l produc t o f Tinuvi n900 . Fo r definitiv e testing , 1.0 , 10 , an d10 0 mg/ Lworkin g standard s wer e prepare d b y quantitativel y transferrin g 0.0005 , 0.0050 , an d0.050 0 g , respectively , o f th e tes t substanc e int o 500-m Lvolumetri c flasks, an deac hwa s brough t t o volum e withsteril e nutrien t

media . Afte r

eac hworkin g standar dwa s bough t t o volume , 0.05 0 m Lo f

DM Fwa s injecte dint o each . Eac hworkin g standar dwa s the n sonicate dfo r

3 0 minute s an dthe nmagneticall y stirre dfo r 3 0 minutes . Th e vehicl e blan k

wa s prepare db y injectin g a0.010-m Laliquo t o f DM Fint o eac h vehicl e blan k

replicat e containin g 10 0 m Lo f alga l nutrien t

media . Afte r preparatio n o f

tes t solutions , th e 1. 0 mg/ Lsolutio nwas clear , th e 1 0 mg/ L solutio n wa s clear

wit haprecipitate , an dth e 10 0 mg/ Lsolutio nwas cloud y with aprecipitate . Th e steril e alga l nutrien t mediu mwa s prepare da s describe di n th e protocol .m . Stati c Tes t Procedur e Th e stati c Scenedesmu s subspicatu s toxicity scree nwas conducte di n250-m L Erlenmeye r

flasks containin g 10 0 m Lo f alga e nutrien t

medi ao r

tes t solution . Eac htes t leve l an dth e control s wer e prepare di ntriplicat e an dreceive d1. 0 mL o f alga l inoculu mcontainin g approximatel y 1. 0 x 10 * cells/mL , resultin g in approximatel y 1. 0 x 10' ^ cells/m Lfo r

eac hflask. Initia l cel l count s o f th e contro l an dvehicl e blan k replicate s resulte di na

nactua l meancel l coun t o f 9. 3 X 10 ^ an d8. 5 x 10

^ ceUs/mL , respectively . Th e wate r quality parameter s o f temperatur e an dp H wer e measure di neac htes t solutio nat

tes t initiation . Th e temperatur e fo r

al l tes t solution s was 23° C an dth e p H range dfro m7. 4 t o 7.6 . After preparation , th e tes t

vessel s wer e positione drandomly , usin g acomputer generate drando mtable , an dincubate dfo r 7 2 hour s at

2 4 + 2° C unde r continuou s cool-whit e fluorescent lightin g an dconstan t

rotary agitation . Ligh t intensity wa s maintaine dat 80 0 + 10 % ft-c (approximatel y 860 0 lux ) an dth e agitatio n rat e wa s 10 0 rpm . Temperature , ligh t intensity , an doscillatio nrat e wer e monitore dthroughou t

th e study . Alga l cel l count s wer e conducte dat 0 hou r inth e contro l an dvehicl e blank replicate s an di n all tes t

chamber s after

7 2 hours . Th e alga l cel l count s wer e ac complishe dusin g ahemacytomete r an danOlympu s Mode l BH-2 microscope . Th e hemacytomete r

ha s tw o chamber s eac hwithnin e squares , 1 m mo naside . Th e averag e numbe r

o f cell s pe r 1 mm ^ wa s designate da s Q . Th e cente r squar e wa s subdivide dint o twenty-fiv e 0.20-m msquares . Th e averag e numbe r o f cell s pe r 0.0 4 mm ^ wa s designate da s R . Th e cel l density (d ) fo r agive nsuspensio n o f alga l cell s coul db e calculate dfro meithe r o f th e followin g equations : A . d(cells/mL ) = IO'* x Q (averag e numbe r o f cells/ 1 mm^) B . d(cells/mL ) = 10 * x V4 R(averag e numbe r

of cells/0.0 4 mm^) I n general , equatio n B wa s onl y use dwithvery dens e cel l population s suc ha s thos e encountere dwitha4
- t o 7-day-oI dalga l cultur e typicall y use dt o prepar e a n alga l tes t

inoculum . Alga l cel l count s durin g th e definitiv e study use dequatio n A . Whe n th e averag e numbe r o f ceU s pe r I-m msquar e wa s les s than1 1 alga l cells , al l nin e 1-m

msquare s wer e counte dan ddivide db y 9 t o obtai nth e averag e numbe r o f cell s pe r

1-m msquar e (Q ) o r

th e replicat e meanvalue . Fo r a U suspension s wit

hat leas t 1 1 ceU s pe r

1-m msquare , th e fou r

come r 1-m msquare s coul db e counte dan daverage dt o obtai nQ (repUcate meanvalue) . Th e cell s pe r milliUter s fo r

eac hrepUcate wer e calculate dan drecorded . IV . Statistica l Analysi s Th e SA S progra m(1 ) print s out th e raw data , plots , charts ; compute s th e mean numbe r o f ceU s b y treatmen t

an dhour ; an dtest s fo r

significan t

difference s from th e contro l an dvehicl e blan k meanat eac htimepoint .A Studen t f-tes t was conducte dt o determin e i f th e contro l an dvehicl e blank ce U coun t values wer e significantl y differen t { p < 0.05 ) fro meac hother . Th e tes t indicate dthat

ther e wa s no t

asignifican t

differenc e afte r

7 2 hours . Th e contro l an dvehicl e blan k

values wer e the npoole dfo r a U furthe r statistical analyses . A one-way analysi s o f

varianc e (usin g PRO C GL M i nSAS ) wa s conducte dfo r eac htimepoin t

withaDunnett' s compariso nt o th e poole dcontrols . A one-taile d Dunnett' s tes t

wa s conducte dat

th e 0.0 5 leve l o f

significanc e wit

hth e alternat e hypothesi s bein g that

th e meannumbe r

o f ceU s ha s bee nreduce dincompariso n t o th e poole dcontrol s mean . Prio r t o th e Dunnett' s test , aLevene' s tes t (2 ) wa s conducte dt o tes t

fo r homogeneity o f

varianc e ove r

treatment s at

eac hhour . I f

th e p valu e fro mth e Levene' s tes t i s greate r

tha n0.01 , indicatin g insignifican t heterogeneity , the n th e analysi s i s performe do nth e ce U count s divide dby 10"*. I f th e p valu e i s les s tha n0.0 1 fo r som e hour(s) , the nth e cel l count s for eac h repUcate ar e transforme dusin g th e squar e roo t o f th e cel l count , th e recommende dtransformatio nfo r coun t dat a(3) . RESULT S A 72-hou r

scree n withScenedesmu s subspicatu s expose dt o Tinuvi n90 0 wa s conducte da t tes t concentration s o f

1.0 , 10 , an d10 0 mg/ Lalon g withacontro l an dvehicl e blan k (DMF) . Tes t concentration s wer e base do n th e solubiUt y o f Tinuvi n90 0 i nalga e nutrien t media . Logarithmi c phas e growthwa s confirme dat 7 2 hour s wit

hameancoun t

o f 6. 9 x 10

^ an d 6. 8 X 10 ^ ceUs/mLi n th e contro l an dvehicl e blan k repUcates , respectively , that wer e 74
- an d 80-fol dincreases , respectively , fro mth e initia l 9. 3 x 10 ^ an d8. 5 x 10

^ ceUs/mL . After 7 2 hours , alga l ce

U count s fo r

th e I.0- , 10- , an d100-mg/Ltes t level s wer e 104 , 94 , an d94 % o f th e poole dcontro l population , respectively . A precipitat e wa s observe dinth e 1 0 an d100 mg/ Ltes t repUcate s at

7 2 hours . A 72-hou r

EC5 0 fo r Scenedesmu s subspicatu s expose dt o Tinuvi n90 0 wa s estimate dt o b e >1 0 mg/ Lsinc e 50 % inhibitio n wa s no t observe di nan y o f

th e tes t concentrations . Th e 72-hou r no-observe deffec t

concentratio nwa s 100 mg/L .REFERENCE S

(1 ) P C DO S SAS/STA T Releas e 6.0 4 Copyrigh t 1985 , 1987 , 198 8 b y SA S Institut e Inc. , Cary , Nort hCaroUna , 27512-800 0 USA . (2 ) MilUken , G.A . an dD.E . Johnson . Analysi s o f Mess y Data , 1984 , Vol . 1 , p . 22 . (3 ) Zar , J.H . Biostatistica l Analysis , 1984 , 2n dedition , p . 241 .

AB C LAB S #40858-1 0APPENDICE S —RAWDAT A

NOTE : Som e o f

th e record s tha t

appear i nthi s raw dat aappendi x hav e bee nprovide da s exac t copie s o f origina l record s o nfU

e at . Thi s ha s bee n don e b y necessity fo r

certai ndat athat

ar e use dcommonl y insevera l studie s a t Suc hrecord s may includ e analytica l standar dsolutio npreparatio n sheets , organis mcultur e logs , an dhardblende dwate r chemica l scree nresults . Th e foUowin g symbol s may appear o nth e raw dat apage s include dinthi s report . C Calculatio n erro r

RRecordin g eno r EEntry erro r

FFor mchang e S SpelUng eno r

T Transcriptio neno r



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### pgO O I 2APPENDI X I - BIOLOGICA LRAWDAT A

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## Date : f-^ n Date : f/J-^- / 

### PiOfii l ROBSERVATION S AND/O R REMARK S FOR M

Tes t Material : VARIOU S Protoco l No . VARIOU S Stud yDirector : VARIOU S Stud y# : VARIOU S 09/02/93 : ^ Avisibl e solubilit ytes t wa s performe d o n Tinuvi n 90 0 usin g th e solvents^^cetonerDM F (Dimethylformamide) , ancrfethanol . Thre e stoc k

solution s wer e prepare d b yweighing n .OOOO g o f Tinuvi n 90 0 i n 3 separat e lOm L volumetri cflasks . Eac h wa s brough t t o volum e o f lOm L wit h Acetone , DMF , an d Ethano l respectively . Al l thre e volumetric s w / 10 0 mg/m L solution s wer e sonicatecri n exces s o f 1/ 2 a n hour . I n th e volumetric s containin g Tinuvi n 90 0 an d Acetone , an d Tinuvi n 90 0 an d Ethano l a larg e amoun t o f th e compoun d wa s no t i n solution . Th e volumetri c containin g Tinuvi n 90 0 an d DM F see m t o dissolv e a littl e bette r tha n th e othe rsolvents , bu t ther e wa s stil l a significan t amoun t o f compoun d lef t undissolved . Nex t a 50mg/m L solutio n o f Tinuvi n 90 0 wa s prepare d b yweighin g 0.5000 g o f tes t materia l i n a lOm L volumetri can d bringin g i t t o volum e wit h DMF . Al l compoun d dissolved , bu t whe n 0. 1 Om L o f th e 50mg/m L solutio n wa s injecte d i n 500m l D l wate ra n exces s o f whit e precipitat e wa s evident . Eve n afte rsonicatin g thi s lOmg/ L solutio n th e whit e precipitat e wa s abundant .

tS ) f-vj- l i l 73/0/^ 3 * © BrAtiSt^T S S-i«, o -Ton.t^^o r /fljii^tji X atiifoi a lOjQ^-Hk ^ NOTE : Individua l entrie s mus t b e date d an d initialed . Reviewe d by : Date : 9-/3-9^ " Stud yDirector: . 

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### 9 0 8 ^ Mea n Q V a ( Tes t Solutio n [volum e (mL ) o f inoculum \ es t vesse l (ml ) j

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s , 1973 , ed i :iet yo f Am e /lateria l #16 : TIO N

nar y 

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lu e = 9< \ volu r ) _sto c a in o Alok' -

_uA i a 1 te d Jan e rica , In c 3-384 ) a Date : _ Date : _ \/sci-<f^(n .

L i n prepare d oculu m Q X 10*'- ' quares ) " " 

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n e o f washe d k cultur e t o d d t o tes t culu m {mL )
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09-01-199 3

n 1 2 3 4 5 6 7 

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I D 4085 8 1 0 C 4085 7 0 . 1 0 B CONTRO L A 4085 7 0.1 0 C 4085 8 1. 0 B 4085 7 1. 0 C 4085 8 1 0 A 4085 7 VB A 4085 7 VB C 4085 8 VB B CONTRO L C 4085 7 1. 0 B 4085 8 10 0 A 4085 8 1. 0 C 4085 7 1 0 B 4085 7 VB B 4085 7 1 0 A 4085 7 0.1 0 A CONTRO L B 4085 8 10 0 C 4085 8 VB A 4085 7 1 0 C 4085 8 1. 0 A 4085 8 1 0 B 4085 8 10 0 B 4085 7 1. 0 A 4085 8 VB C

DAT AENTERE D BY s REVIEWE D BY : 

### STUDY DIRECTOR : ^'/6-9 3 

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i nu^ i r j '^O O SUPPLIE D INFORMATIO N Dat e Received : 0'^/jc>/^. 5 inggfl d I n By : K- h f ^ Re

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Stud yDirector : Dat e Prepared / V liter s o f ( 1 mLA. ) o f th e autoclave d f withgr wJM H p H adjustmen t the n store d i n a Macronutrlen t £ wate r

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mriou ^ \JaKiniJ < nitlal s ^'3^?/4t> y steril e nutrien t medi a wer e pre p nutrien t solution s liste d below . reagen t water . Th e solutio n v

_usin g a Cornin g 14 0 p H met e th e solutio n wa s passe d throug l 1 refrigerator . >toc k Solutions : Eac h prepare d
NaNO , NaHCO j MgS04«7H2 0 MgClj-eHj O CaCla^aHjO K,HP0 4 too k Solutions : Prepare d I n IOC
MnCl2»4H2 0 NajEDTA^aHj O HaBO , FeCI,«6Hj O Na2Mo04»2H, 0 ZnCl j CoClj-eHj O CuCl3»2H, 0 y i PREPARATIO N Protoco l No. : "Jano u Stud v# : \lat\ox \

>are d wit h nutrien t aliquot s Th e solutio n wa s prepare d v i/as p H adjuste d t o 7. 5 ±0. 1 r , LD . No.1714-175 . A \ \ 0.45-/m i Millipore * filter s a r i n 100 0 m L o f steril e reagen t Targe t Weigh t 25.50 0 g 15.00 0 g 14.70 0 g 12.16 4 g 4.41 0 g 1.04 4 g K ) m L o f steril e reagen t wat e Targe t j f ^ Weigh t [[ | ^ 415. 4 m g § j 1 30 0 m g | 5 185. 5 m g *

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## pg0 0 2 5yOBSERVATION S AND/O R REMARK S FOR M

Tes t Material : VARIOU S Protoco l No . VARIOU S Stud yDirector : VARIOU S Stud y# : VARIOU S 8/31/9 3 ^ O n Augus t 26 , 199 3 a slan t labele d UTE X 164 8 Selenastru m capricornutu m !ot # 93-1648-4) , a tes t tub e labele d UTE X LB230 8 Skeletonem a costatu m lot # 93-LB2308-4 ) containin g a liv e cultur e i n suspension , a slan t labele d UTE X 259 4 Scenedesmu s subspicatu s lot # 93-2594-4) , a slan t labele d UTE X 144 4 Anabaen a flos-aqua e lot # 93 - 1444-4) , an d a slan t labele d UTE X 66 1 Navicul a pelliculos a lot # 93-66 1 4 ) wer e received . Slant s an d suspensio n wer e store d a t roo m temperature .

' - ^ 8/31/93 : Scraping s o f th e liv e culture s wer e take n an d ascepticall yplace d i n steril e 25 0 m L E-flasks , eac h containin g 10 0 m l o f thei rrespectiv e alga l nutrien t media . Th e tw o Skeletonem a costatu m culture s wer e place d i n a n environ menta l chambe ra t 20° C ±2° C wit h a 16-hr . light/8-hr . dar k phot o period . Th e tw o Anabaen a flos-aqua e culture s wer e place d i n a n environmenta l chambe ra t 25° C ±1'* C wit h continuou s lighting . Tw o Selenastru m capricornutu m an d tw o Scenedesmu s subspicatu s culture s wer e place d i n a n environmenta l chambe ra t 24° C ±2° C wit h continuou s lightin g an d agitation . Preparatio n o f al l plug s wa s don e ascepticall yunde ra U.V , hood .



### B y "Thi s i s a n exac

t copyof Th e origina l document "



## .date-±2±2 ^
NOTE : Individua l entrie s mus t b e date d an d initialed . Reviewe d by : Stud yDirector : Date:__iilii3 _ Date : A// h



### p

g 0 0 2 b

## P i 0 0 ^ 1( ,

APPENDI X n- STATISTICA LANALYSI S DAT A A . Studen t

Mes t Dat a B . ANOV A an dDunnett' s Multipl e Mean s Tes t Dat a



## pgfl 0 2 8

### A . Studen t Mest DataPRINTOU T O F RA W DAT A FO R CONTROL S OU R 7 2 7 2 7 2 7 2 7 2 7 2 LEVE L

CO N CO N CO N VE H VE H VE H RE P

A B C A B C CELL_10 4 NO_CELL S

N= 6

ANALYSI S BY ; REVIEWE D BY ; DATE : 08SEP9 3 DATE : ^-1 3 -'7 3 

### pg0 0 3 0DISTRIBUTIO NO F CEL L COUNT S FO R EAC H HOU R

LOO K FO R UNUSUA L VALUE S WHIC H MA Y B E INCORREC T DAT A HOUR=7 2 FREQUENC Y O F CEL L 10 4

SYMBO L LEVE L C CO N SYMBO L LEVE L V VE H

ANALYSI S BY : REVIEWE D BY : DATE : 08SEP9 3 DATE : (/-/3
- f J 

### pg0 0 3 lMEA NCEL L COUNT S FO R CONTROL S B Y HOU R MEANO F CELL_10 4 B Y LEVE L GROUPE D B Y HOU R HOU R LEVE L CELL_104 FRE Q MEA N 7 2 CO N VE H ********************************** * 3 69.4166 7

********************************* * 3 68.1666 7 1 0 2 0 3 0 4 0 5 0 6 0 7 0

CEL L 10 4 MEAN

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : J 

## p g 0 0 3 2DESCRIPTIV E STATISTIC S (N , MIN , MAX , MEAN , STANDAR D DEVIATION , AN D CV ) FO R NUMBE R O F CELLS/M L DIVIDE D B Y 10** 4

LEVE L CO N VE H NO_REP S

3 3 MIN_C10 4

6 8 6 5 HOUR=7 2 -

MAX_C10 4 71.7 5 70.0 0 AVR_C10 4

69.416 7 68.166 7 STD_C10 4

2.0361 3 2.7537 9 CV_C10 4

2.9332 0 4.0397 8

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : DATE : ^-/i'9 3 

### pg 0 03 3DESCRIPTIV E STATISTIC S (N , MIN , MAX , MEAN , STANDAR D DEVIATION , AN D CV ) FO R NUMBE R O F CELLS/M L
LEVE L CO N VE H NO_REP S

3 3 MIN_CEL L

68000 0 65000 0 HOUR=7 2 -

MAX_CEL L 71750 0 70000 0 _—^^^^^^^——— ^

AVR_CEL L 694166.6 7 681666.6 7 STD_CEL L

20361.3 2 27537.8 5 CV_CEL L

2.9332 0 4.0397 8

ANALYSI S BY : DATE : 08SEP9 3 

### REVIEWE D BY : DATE : 9'/?"? ^ PgO 0 3 4REPEATE D MEAStJRE S ANALYSI S O F VARIANC E COMPARIN G CONTROL S AVERAGE D OVE R AL L TIM E POINT S FRO M 7 2 HOUR S I F PR> F FO R SOURCE=LEVE L (BOTTO M LINE ) I S LES S THAN.0 5 AN D I F DIFFERENC E I S BIOLOGICALL Y MEANINGFU L THE NUS E VEHICL E O R CARRIE R A S CONTRO L Genera l Linea r Model s Procedur e Clas s Leve l Informatio n Clas s LEVE L RE P HOU R Level s

2 3 1 Value s CO NVE H A B C 7 2 Numbe r o f observation s i n dat a se t = 6

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : DATE : 9-J^-9 3 

### ^ pg0 0 3 5REPEATE D MEASURE S ANALYSI S O F VARIANC E COMPARIN G CONTROL S AVERAGE D OVE R AL L TIM E POINT S FRO M 7 2 HOUR S I F PR> F FO R SOURCE=LEVE L (BOTTO M LINE ) I S LES S THA N.0 5 AN D I F DIFFERENC E I S BIOLOGICALL Y MEANINGFU L THE NUS E VEHICL E O R CARRIE R A S CONTRO L

ANALYSI S BY : REVIEWE D BY : DATE ; 08SEP9 3 DATE : 9'13-9 3 

## pg0 0 3 bREPEATE D MEAStTRE S ANALYSI S O F VARIANC E COMPARIN G CONTROL S AVERAGE D OVE R AL L TIM E POINT S FRO M 7 2 HOUR S I F PR> F FO R SOURCE=LEVE L (BOTTO M LINE ) I S LES S THA N.0 5 AN D I F DIFFERENC E I S BIOLOGICALL Y MEANINGFU L THE NUS E VEHICL E O R CARRIE R A S CONTRO L

ANALYSI S BY : DATE : 08SEP9 3 

### REVIEWE D BY : DATE : 9'/3'9 3 

## pg0 0 3: i9
STUDEN T T-TES T COMPARIN G CONTROL S A T EAC H TIM E POIN T STARTIN G A T 7 2 HOUR S I F PR0B>|T | FO R EQUA L VARIANCE S I S LES S THA N.0 5 AN D I F DIFFERENC E I S BIOLOGICALL Y MEANINGFU L THE NUS E VEHICL E O R CARRIE R A S CONTRO L

ANALYSI S BY : REVIEWE D BY : DATE : 08SEP9 3 

### DATE : 9-13-^ 3 

### pg0fl 3 8

### B . ANOV A andDunnett' s Multipl e Mean s Tes t Data



### pg 

### 0 0 3 9ANALYSI S BY :



### REVIEWED BY : DATE : 08SEP9 3 'T 3 

## TgOfl

4 0ANALYSI S BY :

REVIEWE D BY : DATE : 08SEP9 3 r/3-f J 

## TgOOl t IANALYSI S BY :

REVIEWE D BY : DATE : 08SEP9 3 9'/3-'? 3 

### pg004 2ANALYSI S BY : DATE : 08SEP9 3



### REVIEWE D BY : DATE : ^'/3-9 ^ Pi004 3ANALYSI S BY : DATE : 08SEP9 3

REVIEWE D BY : 

### DATE : 9-/3-'^ 3 

### Pg0044 ^ANALYSI S BY : DATE : 08SEP9 3



### REVIEWE D BY : DATE : 9'l3-9 3 Pg0 0 4 5ANALYSI S BY : DATE : 08SEP9 3



### REVIEWE D BY : DATE : 9-/3-9 J 

## : Pg0fl4 b8

CONT_MS G CONT_LE V POOLE D CONTRO L USE D FO R COMPARISON S T O CONTRO L WIT H DUNNETT' S TES T POOLE D

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : DATE : '?'/3-9 3 

### ^ Pg 0 0 4 1RESULT S O F LEVENE' S TES T FO R HOMOGENEIT Y O F VARIANC E ACROS S TREATMENT S FO R EAC H HOU R I F P I S LES S THAN.0 1 THE NTH E ANALYSI S I S PERFORME D O NTH E TRANSFORME D VALU E (SQUAR E ROO T TRANSFORMATION ) OB S HOU R NUM_D F DEN_D F F P 1 7 2 3 1 1 1.7967 0 0.2059 4

ANALYSI S BY : DATE : 08SEP9 3 

### REVIEWE D BY : DATE : 9-/3-9 3 

## pg 0 0 4 81 0
ANOVAAN D DUNNETT' S MULTIPL E MEAN S COMPARISO N O NSQR_CEL L = SQRT(NO . O F CELLS/M L + 0.5 ) O R NUMBE R O F CELLS/M L DIVIDE D B Y 10** 4 DEPENDIN G O NRESULT S O F LEVENE' S TES T

FO R THA T HOU R HOUR=7 2 Genera l Linea r Model s Procedur e Clas s Leve l Informatio n Clas s Level s Value s TREATMN T 4 DUNNETT/CONTRO L LEVEL- 1 LEVEL- 2 LEVEL- 3

Numbe r o f observation s i n bygrou p = 1 5

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : DATE ; 9-/3-9 3 

### Pg0 0 4 9ANOVAAN D DUNNETT' S MULTIPL E MEAN S COMPARISO N O NSQR_CEL L = SQRT(NO . O F CELLS/M L +0.5 ) O R NUMBE R O F CELLS/M L DIVIDE D B Y 10** 4 DEPENDIN G O NRESULT S O F LEVENE' S TES T

FO R THA T HOU R 1 1

HOUR=7 2

ANALYSI S BY : 

### REVIEWE D BY : DATE : 08SEP9 3 DATE : 9'/3-9 j 

### pgOOS O1 2
ANOVAAN D DUNNETT' S MULTIPL E MEAN S COMPARISO N O NSQR_CEL L = SQRT(NO . O F CELLS/M L + 0.5 ) O R NUMBE R O F CELLS/M L DIVIDE D B Y 10** 4 DEPENDIN G O NRESULT S O F LEVENE' S TES T

FO R THA T HOU R HOUR=7 2

ANALYSI S BY : DATE : 08SEP9 3 REVIEWE D BY : DATE : fVJ-f J 

### PgOOS i1 3
ANOVAAN D DUNNETT' S MULTIPL E MEAN S COMPARISO N O NSQR_CEL L = SQRT(NO . O F CELLS/M L +0.5 ) O R NUMBE R O F CELLS/M L DIVIDE D B Y 10** 4 DEPENDIN G O NRESULT S O F LEVENE' S TES T

FO R THA T HOU R HOUR=7 2

ANALYSI S BY : DATE : 08SEP9 3 

### REVIEWE D BY : DATE : 4-/3-9 3 Pg 0 0 5 2

### ADDENDUM T O ANALYTICA LBIO-CHEMISTRY' S REPOR T NO .

Th e followin g correction s wer e identifie dfo r th e abov e cite dstudy : Pag e 9 , RESULTS . Secon dParagraph , Firs t Sentenc e Replace d" > 1 0 mg/L " with" > 10 0 mg/L" . Thi s correctio nreflect s th e actua l result s o f

th e study . W e ar e issuin g thi s addendu mt o repor t t o provid e th e correcte dpag e fo r th e abov e cite dfina l report

an ddoe s not

chang e an y reporte dresults . Thi s addendu mwil l b e attache d t o th e final report

fo r th e abov e cite dstudy .

TeamLeader , Aquati c Toxicolog yA Studen t ^tes t wa s conducte dt o determin e i f th e contro l an dvehicl e blan k cel l coun t values wer e significantl y differen t { p < 0.05 ) fro meac hother . Th e tes t indicate dthat

ther e wa s not

asignifican t

differenc e after

7 2 hours . Th e contro l an dvehicl e blan k

values wer e the npoole dfo r al l furthe r

statistica l analyses . A one-way analysi s o f

varianc e (usin g PRO C GL M i nSAS ) was conducte dfo r eac htimepoin t

withaDunnett' s compariso nt o th e poole dcontrols . A one-taile d Dunnett' s tes t

wa s conducte dat

th e 0.0 5 leve l o f

significanc e withth e alternat e hypothesi s bein g that

th e meannumbe r

o f cell s ha s bee nreduce di ncompariso n t o th e poole dcontrol s mean . Prio r t o th e Dunnett' s test , aLevene' s tes t (2 ) was conducte dt o tes t

fo r homogeneity o f

varianc e ove r

treatment s a t

eac hhour . I f

th e p valu e fro mth e Levene' s tes t i s greate r

tha n0.01 , indicatin g insignifican t heterogeneity , the n th e analysi s i s performe do nth e ce U count s divide db y IO'* . I f th e p valu e i s les s tha n0.0 1 fo r som e hour(s) , the nth e cel l count s fo r eac h replicat e ar e transforme dusin g th e squar e roo t o f th e cel l count , th e recommende dtransformatio nfo r coun t dat a(3) . RESULT S A 72-hou r

scree n withScenedesmu s subspicatu s expose dt o Tinuvi n90 0 was conducte dat tes t concentration s o f

1.0 , 10 , an d10 0 mg/ Lalon g withacontro l an dvehicl e blan k (DMF) . Tes t concentration s wer e base do n th e solubility o f Tinuvi n90 0 i nalga e nutrien t media . Logarithmi c phas e growthwa s confirme da t 7 2 hour s withameancoun t o f 6. 9 x 10

^ an d 6. 8 X 10 ^ cells/m Li n th e contro l an dvehicl e blan k replicates , respectively , tha t wer e 74
- an d 80-fol dincreases , respectively , fro mth e initia l 9. 3 x 10 ^ an d8. 5 X 10

^ cells/mL . Afte r 7 2 hours , alga l cel l count s fo r th e 1.0- , 10- , an d100-mg/Ltes t level s wer e 104 , 94 , an d94 % o f th e poole dcontro l population , respectively . A precipitat e wa s observe di nth e 1 0 an d 10 0 mg/ Ltes t

replicate s at

7 2 hours . A 72-hou r

EC50 fo r

Scenedesmu s subspicatu s expose dt o Tinuvi n90 0 was estimate dt o b e >10 0 mg/ Lsinc e 50 % inhibitio n wa s not observe di nan y o f

th e tes t concentrations . Th e 72-hou r no-observe deffec t

concentratio nwas 10 0 mg/L .