p. 1 GREENWIC H CHEMICA L CONSULTING , INC .

330 0 PG A BOULEVAR D • SUIT E 63 5 n, , , / PALM BEACH

GARDENS, FL

33410M>" /" ^ TELEPHON E 561.578.8 1 1 0 FACSIMILE 561.578.812 0

Januar y 9 , 201 4 CBI C Contro l Numbe r 

# 35767 6

CONTAIN S N O CONFIDENTIA L BUSINES S INFORMATIO N B y UP S

OPP T Documen t

Contro l Offic e ^ O EP A Eas t

Buildin g ~ ^g n i RoomE642 8 o H m U.S . Environmenta l Protectio n Agenc y a* . S^ " 120 1 Constitutio n Ave. , N W 5 £ 3 Washington , D C 2000

4 C O Attention : Docke t

I D Number : EPA-HQ-OPPT-2009-011 2 4 0 CF R799.508 9

Re: Study Plan Submission fot Algae Testing Conducted by the PCBTFTesting Consortium underthe TSCA Section 4 Test Rule on the Testing of Certain High Production Volume Chemicals; Third Group Benzene , l-chloro-4-(trifluoromethyl) - CA S R N 98-56- 6 (para-Chlorobenzotrifluorid e (PCBTF) ) Docke tI D Number : EPA-HQ-OPPT-2009-011 2 4 0 CF R799.5089

Dea r Sir/Madam : Throug h thi s letter , th e PCBT F Testin g Consortiu msubmit s t o th e U.S . Environmenta l Protectio n Agenc y (EP A o r the Agency ) astud

y plan fo r

alga e testin g conducte do n the chemical substance , benzene , l-chloro-4-(trifluoromethyl)

- [CA S R N 98-56-6 ] whic h i sp . 2 CONTAIN S N O CONFIDENTIA L BUSINES S INFORMATIO N U.S . Environmenta l Protectio n Agenc y Januar y 9 , 201 4 Pag e 2 currentl y subjec t

t o aToxi c Substance s Contro l Ac t (TSCA ) Sectio n 4 tes t

rule. 1 Fo r

purpose s o f thi s letter , th e chemica

l substance , benzene , l-chloro-4-(trifluoromethyl)- , i s herei n referre d t o as , PCBTF . Th e PCBT F Testin g Consortiu mfile s thi s reques t i n accordanc e wit h thi s tes t rul e an dth e regulation s se t fort h a t 4 0 CFR790.5 0 and799.5089 . L Genera

l Backgroun

d Greenwic h Chemical Consulting , Inc . (Greenwic h Chemical Consulting ) function s an d operate s a

s th e pane l manage

r fo r an dauthorize drepresentativ e o f th e PCBT F Testin g Consortium . Therefore , Greenwic h Chemical Consultin g submit s thi s lette r an dth e enclose d stud y plan s o n behal f

o f the PCBT F Testin g Consortium' s 1 3 membe r companie s identifie d below: 2

• Brennta g Nort h America , Inc . • Chemsynergy , Inc . • Chin aSpecialt

y Chemica

l Company , Ltd . • EMC O Chemica

l Distributors , Inc . • Greenche mIndustrie s LL C • Kow aAmerica n Corporatio n • Makhteshi mAgan o f Nort h America , Inc . • M C International , LLC 3 • Nexe o Solutions , LL C • Silve r Fern Chemical , Inc . • Specia

l Material s Compan

y • Univar

USA , Inc . • Valspa

r Corporatio n Thi s fina l tes t

rul e addresse s 1 5 hig h productio n volum e (HPV ) chemical s that wen t unsponsore d unde

r th e Agency' s voluntary HP V Chemical Challeng e Program . On e o f th e 1 5 chemical s identifie di n the rul e i s PCBTF . Th e final rul e identifie dth e followin g tw o studie s o n PCBTF :

• Aquati c Toxicit y Testing : toxicit y t o plant s (algae) ) • Read y Biodegradatio n I n it s letter

date dApri l 18 , 2013 , th e PCBT F Testin g Consortiu mtimel y submitte dfina l tes t report s t o EP A fo r

thes e tw o studies . Th e Agenc y subsequentl y determine dtha t

th e alga e stud y was inadequat

e an d require dthat

the stud y b e re-conducte dfo r

purpose s o f

th e TSC A Sectio n 4 tes t

rule. 4 O n Novembe r

6 , 2013 , th e PCBT F Testin g Consortiu mrequeste d a n

1 7 6 Eed , Reg , 65385 ; Octobe r 21 , 2011 . 2Se e Attachmen t

A . s M C International , LL C operate s the entity , Miam i Chemical , Inc. , a s aDBA . Fo r

purpose s o f

U.S . Custom s an dTSCA , M C International , LL C i s th e importer . 4 Lette r fro mEP A t o th e PCBT F Testin g Consortiu mdate dSeptembe r 26 , 2013 .p . 3 CONTAIN S N O CONFIDENTIA L BUSINES S INFORMATIO N U.S . Environmenta l Protectio n Agenc y Januar y 9 , 201 4 Pag e 3 extensio n fo r

additiona l tim e t o revie w an d respon d t o EPA' s comments . I n that letter , the PCBT F Testin g Consortiu mals o requeste d that th e stud y pla n submissio n dat

e b e extende d fro mNovembe r

12 , 201 3 t o Januar

y 12 , 2014 . Thi s extension , a s wel l a s th e modifie dstud

y plan due date , wa

s formall y grante d b y EP A i n th e Agency' s letter o f Novembe r

12 , 2013 . I I . Submissio n o f

the Alga e Stud y Pla n b y th e PCBT F Testin g Consortiu m Th e PCBT F Testin g Consortium , therefore , timel y submit s t o EP A th e stud y plan fo r

anew alga e tes t o n th e compoun d whic h wa s develope d b y the group' s new contrac t lab , Harlan Laboratorie s Ltd . (Harlan Laboratories) . A s part o f the development

o f thi s stud y plan , the PCBT F Testin g Consortiu mprovide d t o Harla n Laboratorie s the Agency' s comment s o f Septembe r 26 , 2013 . Harla n Laboratorie s develope d thi s stud y plan takin g int o account an d incorporatin g thes e comment s an d als o addressing th e technical difficultie s associated wit h testin g PCBT F (Le. , poo r wate r solubility an d hig h volatility) . A s wit h the firs t alga e tes t an d consistent

wit h EPA' s approval grante d o n Februar y 9 , 2012 , thi s second tes t wil l als o be conducte d under

OEC D tes t

guidelin e 201 . Th e stud y pla n submitte d herei n addresse s al l o f EP A comment s i n it s Notic e o f Inadequacy letter . I t i s note d thoug h that

i n EPA' s letter , the Agenc y discusse s potentia l los s o f th e tes t material durin g samplin g and , therefore , th e use o f additiona l replicates. 5 Th e stud y plan a t Sectio n 3. 7 addresse s amodifie duse o f additiona l replicate s durin g bot h the limi t tes t an d ful l study . Harla n Laboratorie s provide d additiona l commen t o n th e propose d number o f replicate s wit h respec t

t o the Agency' s recommendation : Th e use o f

th e additiona l replicate s that EP A suggest

s woul dmea

n that i n th e cas

e o f aful l tes t there woul db e 1 2 replicate vessel s per tes t concentratio n (and control ) i.e . atota l o f 7 2 vessels . I f

th e tes t was conducte d a

s alimi t tes t the n th e tota l number

o f vessel s woul dstil l b e 48 . Thi s woul dmak e the tes t to o cumbersome an d pron e t o erro r i n th e determinatio n o f cel l densitie s a

s thes e woul dbe monitore d i n individua l replicate vessel s o n eac h cel l determinatio n poin t

rathe r tha n fro mth e sam

e replicate vessel s a

t 0 , 24 , 4 8 an d 7 2 hours . A s acompromis e we'v e include d additiona l vessel s that wil l be sample d a

t 24 an d 4 8 hour s fo r analysis . Thes e vessel s wil l b e treate d i n th e sam e manner a s thos e fro mwhic h sample s ar e take n fo r cel l determinatio n measurement s suc h that measure d concentration s i n thes e vessel s wil l mimi c thos e i n th e tes t vessel s that

ar e incubate d fo r

th e cel l determinations . A n additiona l replicate vessel wil l als o b e maintaine d full y close d fo r th e 72-hour

tes t perio d an d analyze d a

t tes t terminatio n t o asses s th e effec t that openin g tes t

vessel s fo r

the cel l determination s has . Thi s i s aprocedur e tha t we'v e used o n numerou s occasion s fo r volatil e tes t

item s an d has bee n accepte d b y variou s regulator y authorities . Therefore , th e PCBT F Testin g Consortiu msubmit s thi s alga e stud y plan fo r

th e re-testin g o f th e material under

EPA' s TSC A Sectio n 4 tes t rule . Attache d i s afina l draf t stud y pla n fo r review , comment , an d approval b y the Agency .

5 EPA' s Notic e o f

Inadequac y lette r

a t Sectio n C{2) .p . 4 CONTAIN S N O CONFIDENTIA L BUSINES S INFORMATIO N U.S . Environmenta

l Protection Agenc

y Januar y 9,201 4 Pag e 4

Th e PCBT F Testin g Consortiu mthank s th e Agenc y fo r it s consideratio n i n thi s matter . Fo r al l questions , comments , and determination s concernin g thi s matter , pleas e use th e contac t informatio n give n below . Mr . Joh n V . Massingale Greenwic h Chemica

l Consulting , Inc . 3300 PG A Boulevard , Suit e 63 5 Pal mBeach Gardens , F L 3341 0 Telephone : (561 ) 578-811 0 Facsimile : (561 ) 578-8120 E-mail : john@grcci.co m Sincerely , Joh n V . Massingale Panel Manage

r

cc : Dr . J . Schaeffer-EP A PCBT F Testin g Consortiu m

Enclosurep . 5 CONTAIN S N O CONFIDENTIA L BUSINES S INFORMATIO N U.S . Environmenta

l Protection Agenc

y Januar y 9 , 201 4 Pag e 5 

### Attachmen tA Names an

d Addresse

s o f Membe r Companie

s Compan v Addres s Brennta g Nort h America , Inc . 508 3 Pottsvill e Pik e Reading , P A 1960

5 Chemsynergy , Inc . 110 0 Burloa k Driv e Suit e 10 1 Burlington , O N L7 L 6B 2 Canad a Chin aSpecialt

y Chemica

l Company , Ltd . Uni t A (Roo m9 ) 3 K iFloor Cheon g Su

n Towe r 116-11 8 Win g Lo k

Stree t Sheung Wa n Hon g Kon g EMC O Chemical Distributors , Inc . 2100 Commonwealt h Avenu e Nort h Chicago , I L 6006 4 Greenche mIndustries , LL C 22 2 Clemati s Stree t Wes t Pal mBeach , F L 3340 1 Kow aAmerican Corp . 5 5 Eas t 59 t hStree

t 19 l hFloor Ne w York , N Y 10022 Makhteshi mAgan o f

Nort h America , Inc . 17 1 Madiso n Avenu e Roo m1605 Ne w York , N Y 1001 6 M C International , LL C 2 N E 40 t hStree t Suit e 50 1 Miami , F L 3313 7 Nexe o Solutions , LL C 140 0 Woodloc h Fores t

Driv e Suit e 20 0 Th e Woodlands , T X 7738 0 Silve r Fern Chemica

l Inc . 222 6 Queen Ann e Avenu e N . Seattle , W A 9810 9 Specia l Material s Compan

y 70West 40th Street 2 nd Floor New York, NY

10018 Univar US A Inc . 17425 N E Unio n Hil l Roa d Redmond , W A 9805 2 Valspa r Corporatio n 90 1 3r d Avenu e Sout h Minneapolis , M N 5540 2p . 6



## STUDYPLA N 

# harla n

Benzene , l-chloro-4-(trifluoromethyl)- , CA S R N 98-56-6

, para-Chlorobenzotrifluorid e (PCBTF) : Alga l Growt h Inhibitio

n Tes t

Stud y Director : TestFacility : Hele n Vryenhoef Harlan Laboratories Ltd . Shardlo w Busines s Par

k Shardlo w Derbyshir e DE7 2 2G D U K Sponsor : PCBT F Testing Consortiu m c/ o Greenwic

h Chemica

l Consulting , Inc . Address : 330

0 PG A Boulevard , Suit

e 63 5 Pal mBeac h Gardens , F L 3341 0 US A Stud y Monitor : Mr . Joh n V . Massingal

e Greenwic h Chemica

l Consulting , Inc . 330 0 PG A Boulevard , Suit

e 63 5 Pal mBeac h Gardens , F

L 3341 0 US A Harla n Stud

y Number : 4130473 1

Pag e 1 o f 1 9p . 7 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 2



### CONTENTS CONTENT S 2 SIGNATURE S 3 GOO D LABORATOR Y PRACTICE 4 GENERA L INFORMATIO N 5 Propose d Schedul e 5 Distributio n Lis t

5 Stud y Pla n Amendment

s an d Deviation s 5 Qualit y Assuranc

e 5 Reporting 5 Archivin g 6 1 INTRODUCTIO N AN D PURPOSE 7 1. 1 Guideline s /

Regulation s 7 2 TES T AN D REFERENC E ITEM S 8 2. 1 Tes

t Item8 2. 2 Reference Ite

m8 3 MATERIAL S AN D METHOD S 9 3. 1 Definition s an

d Abbreviation s 9 3. 2 Tes

t Syste m9 3. 3 Tes

t Wate r 1 0 3.4 Dosag

e 1 0 3. 5 . Material s 1 0 3. 6 Experimenta

l Condition s 1 1 3. 7 Stud

y Desig n 1 1 3. 8 Evaluation s 1 2 3.8. 1 Tes

t Organis mObservation s 1 2 3.8. 2 Wate

r Qualit y Criteri a1 2 3.8. 3 Analysi s o f

Tes t ItemConcentration s 1 3 3. 9 Dat

aAnalysi s 1 4 3.9. 1 Plottin g Growt

h Curve s 14 3.9. 2 Calculation o f

Growt h Rate s 1 4 3.9. 3 Yiel d1 5 3.9. 4 Plottin g Concentration-Response Curves 1 6 3.9. 5 Statistica

l Analysi s 1 6 3.1 0 Validity Criteri a1 6 4 REFERENCE

S 1 8 APPENDICES 1 9p . 8 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 3 

## SIGNATURE S

Stud y Director : H Vryenhoef

Date :

Tes t Facilit y Management : C Mea d

Date :

Fo r th e Sponsor : Joh n V . Massingal

e Greenwic h Chemica

l Consulting , Inc .

Date :P . 9 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 4 

## GOO D LABORATOR Y PRACTIC E Thi s stud y wil l b e performed i n compliance with U K GL P standard

s (Schedul

e 1 , Goo d Laborator y Practice Regulation s 199 9 (S I 1999/310

6 a s amende d b y S I 2004/0994)) . Thes

e regulation s ar

e i n accordanc

e with GL

P standard

s published a

s OEC D Principle s o

n Goo d Laborator y Practice a

s revise d 199 7 (ENV/MC/CHEM(98))7) ; an d ar e i n accordanc

e with , an d implement, th

e requirement

s o f Directive s 2004/9/E

C an d 2004/10/EC . Thes e principle s ar

e compatibl

e with Goo

d Laborator

y Practice regulation s specifie d b y regulator y authoritie s throughou

t th e Europea n Community , th

e Unite dState

s (EP A an d FDA) , an d Japa n (MHLW , MAFFan

d METI) .p . 1 0 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 5 

## GENERA L INFORMATIO N Propose d Schedul e Experimenta

l Startin g Date : Experimenta

l Completio

n Date : Draf t Repor t Date : 2 8 Februar y 201 4 3 1 Marc h 201 4 3 0 Apri l 201 4 Distributio n Lis t Th e origina l Stud y pla n wil l b e retaine d i n th e stud y file. Copie

s o f th e stud y pla n alon g wit h an y amendment

s wil l b e distribute

da s follows :

Stud y Pla n Amendment s an d Deviation s An y intende d chang e t o th e stud y pla n wil l resul

t i n a n amendmen

t t o stud y pla n approve d b y th e Stud y Directo r an d als o signe d b y tes t facilit y managemen

t an d th e Sponsor . Amendment

s wil l b e distribute

dt o all recipient

s o f th e stud y plan . Deviation s (unplanne

d changes ) fromth e stud y pla n wil l b e documente

d an d acknowledge

d b y th e Stud y Director. Quality Assuranc e Qualit y Assuranc

e wil l audi t an d inspec t study-relate

d procedure

s an d wil l repor

t an y audi t an d inspectio n result s i n writing t

o th e Stud y Directo r an d Tes t Facilit y Management

. Reportin g Sponsor : PCBT F Consortiu

m Qualit y Assurance : Harla n Laboratorie

s Ltd . Shardlow , U K Stud y Monitor : Joh n V . Massingal

e Greenwic h Chemica l Consulting , Inc .

A GL P complian

t repor t wil l b e presented

. A draf t repor t wil l b e sen t t o th e Sponso r for revie w an d comment

s befor e issu e o f th e final report.p . 1 1 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 6

Archivin g Unles s instructed otherwis e b y th e Sponsor

, th e stud y plan , al l ra

w dat a(pape r an d electronic ) an d th e fina l repor

t wil l b e retaine d i n th e Harla n Laboratorie s Ltd , Shardlow , U K archive s fo r fiv e year s afte r whic h instruction s wil l b e sough t a s t o further retention o

r disposal . Furthe

r retention o

r retur n o f th e dat awil l b e chargeabl

e t o th e Sponsor . N o dat awil l b e discarde d withou t contacting th

e Sponso r to obtai n thei r writte n consent .p . 1 2 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 7 

## 1 INTRODUCTIO N AN D PURPOS E Th e purpose o f

thi s stud y i s t o asses s th e acut e toxicity o f

th e tes t ite mt o afreshwate

r alga . Th e tes t endpoin t i s inhibitio n o f

growth , expresse

d a s th e logarithmi c increase i n alga l biomas s (averag e growt h rate) during th

e exposur e period . Th

e toxicit y wil l b e expresse

d a s th e concentration bringin g abou t aspecifie d x % inhibitio n o f growt h (e.g . 50% ) an

d expresse d a s th e Th e Lowes t Observe d Effec t Concentratio

n (LOEC ) an

d th e N o Observe

d Effec t Concentratio

n (NOEC ) wil l also b

e determine

d i f applicable . A n additiona

l respons e variabl e recommende

d b y th e Tes t Guideline s i s yield , whic h ma y b e required t o fulfi l specifi c regulator y requirement

s i n som e countries . Result

s wil l therefor

e als o b e expresse d i n term s o f inhibitio n o f

yiel d(EyC x ) . Th e stud y wil l us e Pseudokirchneriell asubcapitat awhic h i s arepresentativ e specie s o f awid e variet y o f natura l habitats , an

d ca n therefor e b e considere

d a s a n importan

t non-targe t organis m i n freshwate

r ecosystems . Th e dispersa l o f th e tes t ite mi n th e surroundin

g mediu mi s considere

d t o represen

t th e mos t probabl e rout e o f exposur e i n th e environment . 1. 1 Guideline s /

Regulation s Thi s test method s describe d ar e designe d t o b e compatibl

e with th e procedures indicated b

y th e followin g internationally accepte d guideline s an

d recommendations :

• OEC D Guideline s fo r

Testing o f

Chemical s (2006 ) N o 20 1 "Freshwate r Alg aan d Cyanobacteria, Growt

h Inhibitio n Test" 1

• Metho dC. 3 o f

Commissio

n Regulation (EC ) No . 761/200 9

1 Anne x 5 corrected

: 2 8 Jul y 201 1p . 1 3 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 8 

## 2 

## TES T AN

D REFERENC E ITEM S 2. 1 Tes tIte m Th e tes t ite mwil l b e supplie db y o r o n behal f o f th e Sponso r includin g th e following informatio

n i f known ; description, batch , purity , expir

y / retes t date , storag e condition

s an d safet y precaution s t o b e taken. Informatio n a s provide d b y th e Sponsor .

2. 2 Referenc e Ite m Fo r evaluatio n o f th e qualit y o f th e alga l cultur e an d th e experimenta

l conditions , potassiu

m dichromat e wil l b e teste d a s apositiv e contro l twic e ayea r t o demonstrat

e satisfactor

y condition

s o f th e test . Th e referenc e ite mwil l b e supplie d b y acommercia

l supplie r an d wil l b e store d a s specifie d b y th e supplier. Identification : Benzene , l-chloro-4-(trifluoromethyl)- , CA S R N 98-56-6, para-Chlorobenzotrifluorid

e (PCBTF )p . 1 4 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 9

3 MATERIAL S AN D METHOD S 3. 1 Definition s an d Abbreviation s Biomass : EC K :

Growth : Growt h Rat e (Average Specifi c Growt h Rate) : Yield :

LOE C (Lowes t

Observe d Effec t Concentration) : NOEC (N o Observe

d Effec t Concentration) : Treatment : Measuremen t variabl e (alga

l cel l density ) a

s surrogat e measur e fo r alga l biomass . Th e calculated concentratio

n o f tes t ite mtha t result s i n a n x % reduction o f

th e respectiv

e growt h paramete

r relativ e t

o th e contro l (or

solven t control , i f

applicable) withi n astate d exposur e period . Th e increas e i n biomas s ove

r th e tes t period . Th e logarithmi c increase i n biomas s durin g th e exposur

e period . Th e valu e o f th e measuremen

t variable a

t th e en d o f th e exposur e perio d minu s th

e measuremen

t variable' s valu

e a t th e star t o f th e exposur

e period , calculate

d t o expres s biomas s increas e durin g th

e test . Th e lowest tes t concentratio

n a t whic h astatistically significant inhibitio n o f

growt h i s determine

d relativ e t

o th e contro l (or solven t control , i f

applicable) . Th e highest tes t concentratio

n a t whic h n

o statistically significant

inhibitio n o f

growt h i s determine

d relativ e t

o th e contro l (o r solven t control , i f

applicable) . Comprise s tes

t ite mtreatments , contro l (test mediu monly ) an

d solven t contro l (i f

applicable) . Section-by-Sectio

n Growt h Rate : Specifi c growt h rat e fo r eac h da y durin g th

e cours e o f th e tes t (day s 0-1 , 1-

2 an d 2-3) . 3. 2 Tes tSyste m Th e stud y wil l b e performed using Pseudokirchneriell asubcapitata . Thi s freshwate r alg a species i s recommende

d b y th e relevan t tes t guideline .Harla n Stud y Number : 4130473 1 Stud y Pla n p . 1 5 Pag e 1 0

A n inoculu

mcultur e o f alga e wil l b e se t u p thre e t o fou r day s befor e th e star t o f th e test . Th e alga e wil l b e cultivate d unde

r th e tes t condition s an

d wil l b e kep t i n th e exponentia

l growt h phas e unti l inoculatio n o f

th e tes t solutions . 3. 3 Tes tWate r Cultur e mediu ma s define d in Appendix 1 . 3. 4 Dosag e Th e tes t ite mwil l b e dissolve d i n th e tes t wate r onc e a t th e star t o f th e test . Dat asupplied b

y th e Sponso r indicate s tha

t th e test ite mha s awate r solubilit y o f 29 - 33. 8 mg/L . Therefor e th e test itemfall s int

o th e categor y o f a"difficul t

substance " a

s define d b

y th e OEC D Guidanc e Documen

t (OECD , 2000) , henc

e th e metho d o f preparatio

n o f th e tes t mediawil l b

e base d o n th e recommendations o f

thi s document . Th e tes t ite msolutio n wil l b

e prepare d a s asaturate

d solutio n using a

n initia l test

ite mdispersion o f 10 0 mg/L . Thi s tes

t ite mdispersio

n wil l b e stirred using amagneti

c stirre r a t arat e suc h tha t a vorte x representin

g approximatel

y 5% o f th e tes t wate r colum n heigh t i s produced . To reduc

e th e ris k o f volatilizatio n during th

e stirrin g period , th

e stirrin g wil l b

e conducte

d i n acompletel

y filledvesse

l (i.e . n o headspace

) seale d wit h anon-permeabl

e stoppe r e.g . glas s stopper

, a t a temperatur

e o f approximatel

y 2 1 ° C fo r

aperio d o f

2 4 - 4 8 hours . Afte r th e stirrin g period , excess undissolved tes t ite mwil l b e remove d b y a n appropriat

e metho d e.g . filtratio n o

r centrifugatio n an

d th e tes t organism s expose d t o th e resulting saturate

d solution . Pre-experiment

s wil l b e performed t

o determin

e th e solubilit y an d stabilit y o f th e tes t ite mi n th e tes t water, th e stirrin g perio d required t o achiev e saturation an

d th e suitabilit

y o f eithe r filtration o r centrifugatio n t

o remov e excess undissolved test

item . Accordin g t

o th e finding s o f

thes e preexperiments , appropriat

e procedures wil l b

e chose n to prepar

e th e tes t media . I f th e test ite mi s photodegradable , th

e preparatio

n o f th e tes t medi awil l b e performed a

s fa r a s possibl e i n th e dark . Detail s o f

th e metho d o f preparatio

n wil l b e documente

d i n th e data . 3. 5 Material s Glas s tes t vessels containing a

t leas t 10 0 m L test mediu mwil l b e used . Th

e tes t ite mi s known t

o b e volatil e (Dat

asupplied b

y th e Sponsor ) henc e th e tes t vessel s wil l b e completel

y fille dan

d seale d using glas s stopper

s (i.e . tes t performed i n aclose

d syste mwith n o headspace) . Th

e siz e o f th e test vessels an

d th e volum e o f tes t solutio n wil l b

e documente

d i n th e data .Harla n Stud y Number : 4130473 1 Stud y Pla n p . 1 6 Pag e 1 1 

### Th e tes t vessel s wil l b e labele d wit h th e stud y numbe r an d all necessar

y informatio

n t o ensur e uniqu e identification . 3. 6 Experimenta l Condition s Th e environmenta

l parameter

s wil l b e a s use d t o maintai n th e stoc k organisms . Th e tes t flask s wil l b e incubate d unde r th e following conditions :

• INFOR

S Multitron® Versio

n 2 incubato

r unde r constan t illuminatio

n (betwee n 444 0 an d 888 0 lux ) provide d b y war mwhit e lightin g (38 0 - 73 0 nm ) a t 2 4 ± 1° C an d agitate d b y orbita l shake r (approximatel

y 15 0 rpm) . Temperature

, ligh t intensit y an d oscillatio

n rat e o f th e incubatio n chambe r wil l b e recorde d dail y throughou

t th e test . 3. 7 Stud y Design Th e tes t desig n wil l includ

e thre e replicate s pe r tes t concentration

, si x replicate s o f th e contro l and , i f appropriate, si

x replicat e solven t contro l flasks . A blan

k consistin g o f cultur e mediu m alon e wil l als o b e prepared . In orde r t o investigat

e possibl e losse s o f tes t ite mdu e t o volatilit y whe n th e tes t vessel s ar e opene d o n adail y basis , additiona

l replicat e vessel s a t eac h concentratio

n wil l b e prepare d a t 0 hour s an d incubate d alongsid e th e othe r exposur e vessel s for analysi s a t 2 4 an d 4 8 hours . Thes

e vessel s wil l b e opene d an d asub-sampl

e remove d i n th e sam e manne r a s thos e take n for alga l cel l determination

s befor e th e tes t vesse l i s sample d for chemica l analysis . A furthe r replicat e vesse l a t eac h concentratio

n wil l b e prepare d a t 0 hour s an d incubate d alongsid e th e othe r exposur e vessels . This fourt

h replicat e wil l remai

n unopene d until terminatio n o f th e tes t whe n i t wil l b e analyze d for tes t ite mconcentration . Th e following condition

s wil l b e met : Initia l cel l densit y (cells/mL

) 2- 5 x 1 0 Tes t duratio n (hours ) 7

2 A stati c tes t desig n wil l b e applied. A t leas t fiv e concentration

s of th e tes t ite marrange d i n ageometri

c serie s wil l b e tested. Th

e spacin g facto r betwee n th e tes t concentration

s wil l preferabl

y no t excee d 2.2 . Th e actua l tes t concentration

s ar e assigne d accordin

g t o preliminar

y range-findin

g test s o r availabl e toxicit y dat aan d wil l b e documente

d i n th e dat aprio r t o th e star t o f th e definitive test

. I f i n th e range -p . 1 7 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 2

findin g tes

t afla t concentration-effect

relationshi

p i s determine

d and, thus , alarg

e concentratio

n rang e wil l hav e to b e tested , aspacin

g factor exceedin g 2. 2 (e.g . 3.2) wil l b

e applied . Th e lowest test concentratio

n shoul d hav e n o significant

effec t o n th e growt h o f algae . Th

e concentration series shoul d preferabl

y cove r th e rang e causin g 5 t o 75 % inhibitio n o f

alga l growt h rats . I n accordanc

e wit h th e tes t guideline s (EC , 2002) , concentration s o f th e test ite m abov e th e limi t o f solubilit y o f th e tes t ite mi n th e tes t wate r wil l no t b e tested . I f n o effec t i s observe

d i n th e preliminar

y range-findin g tes

t a t th e limi t o f solubilit y o f th e test ite mi n th e test water, aLimi t

Tes t ma y b e conducte

d usin g only thi s concentration an d th e correspondin

g control . I n thi s case

, th e numbe r o f tes t concentratio

n replicate s wil l b

e double d t o six . Th e contro l grou

p wil l b e expose d t o th e test wate r alone . Dat

afro mth e contro l grou

p ma y b e share d wit h simila

r concurren

t studies . Th e test wil l b e carried ou

t withou t adjustmen

t o f pH . I f th e p H fall s outsid

e th e rang e 6-9 , th e Sponso r wil l b e contacte

d fo r advice o n whethe r asecon d test should b

e performe d wit h adjustmen t o f p H t o tha t o f th e test water . However, p H adjustmen t wil l not b e carried ou

t i f th e tes t solution s ar

e change d to an y significant

exten t an d i f achemica

l reaction o

r precipitatio n i s caused . 3. 8 Evaluation s 3.8. 1 Tes tOrganis

m Observation

s A small volum

e o f th e alga l suspensio

n wil l b e withdraw n fro meac

h test flas k daily fo r th e measuremen

t o f alga l biomass , an

d wil l not b e replaced . Th

e alga l concentratio

n o f eac h sampl e i s determine

d b y direct counting using aCoulter ® Particle Counter . Thre e determination s ar

e mad e fo r eac h sample . Observation s wil l b

e mad e o n th e tes t ite mpreparation s dail

y during th

e exposur e period . To determin

e apotentia

l effec t o f th e tes t ite mo n th e appearanc

e o f alga l cells , asampl

e wil l b e take n fro mth e tes t an d contro l culture s a

t th e en d o f th e test . Th e shap e an d siz e o f th e alga l cell s wil l b e inspecte d microscopicall

y an d an y abnormalitie s recorded . 3.8. 2 Wate r Qualit

y Criteri a Th e p H o f th e tes t mediawil l b

e measure d i n al l treatment

s a t th e star t an d en d o f th e test . Th e p H o f th e control s shoul

d no t normally deviat

e b y mor e tha n 1. 5 p H unit s durin g th

e test . A n explanation wil l b

e provided in th

e repor t i f p H deviation s i n exces s o f 1. 5 units ar e observed .p . 1 8 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 3

Th e wate r temperatur

e wil l b e measure d daily i n a

n Erlenmeye

r flas k fille dwit h wate

r an d incubate d unde r th e sam e condition s a

s th e tes t flasks . Th e appearanc

e o f th e tes t medi awil l b e recorde d daily . 3.8, 3 Analysi s o

f TestIte m Concentration

s 3.8,3- 1 Samplin

g Sample s o f th e tes t mediawil l b

e take n a t initiatio n an

d terminatio n o f

bot h th e range-findin g an d definitiv e test

s fo r analysis . Durin g th

e definitiv e tes

t additiona l sample s a t 2 4 an d 48 hour s wil l b e analysed . Wher

e possibl e duplicat

e sample s wil l b e take n a t eac h occasion , thes

e sample s ma y als o b e analyze d shoul d th e nee d arise . Analysi s o f

sample s take n fro mth e range-findin g tes

t i s performed i n orde

r t o determin

e th e stabilit y o f th e tes t ite munde r tes t condition s ove

r th e exposur e period . Th

e replicat e test vessel s a t eac h concentration wil l b

e poole d to giv e on

e sample . Th

e tes t concentration s provided fo r analysi s wil l b

e documente

d b y th e Stud y Director

i n th e data . Fo r th e purposes o f

th e definitiv e test, th

e replicat e tes t an d contro l vessels wil l b

e poole d t o giv e on e sample . Shoul

d th e analysi s o f

th e range-findin g test

sample s sho w adeclin

e i n measure

d test concentration ove

r th e tes t duration the

n additiona

l tes t sample s ma y b e prepare d a t tes t initiatio n wit h th

e omission o f

alga l cell s an d incubate

d alongsid

e th e tes t fro mwhic h sample

s wil l b e take n fo r analysi s a

t tes t termination . I f tP e test mediu mvolum e fro mth e actua l tes t i s to o smal

l to perfor

mth e required analyses , requisit e volume s o f

th e freshly prepare

d tes t mediawil l b

e incubate

d separately , bu

t unde r identica l condition s a

s i n th e actua l test . Fin£l detail s o f

th e sample s t o b e take n an d th e sampl e size , etc , wil l b

e documente

d i n th e data . 3.8.3- 2 Storag

e an d sampl e preparatio

n Al l sample

s wil l b e analyze d immediatel

y afte r samplin g o r store d a t approximatel

y -20° C unti l analysis . I f th e test ite mha s bee n show n t o b e unstabl e unde r th e storag e conditions , th

e sample s wil l b e analyze d immediatel

y afte r samplin g without prio r storag e o r measure s wil l b e take n (e.g . p H adjustmen

t o r additio n o f

organi c solvents ) i n orde

r t o maintai n th e stabilit y o f th e tes t ite munti l analysis . 3.8.3. 3 Analysi s o f

sample s Th e tes t ite mformulation s wil l b

e analyze

d fo r concentratio

n an d stabilit y b y Harla n Laboratorie s Ltd , Shardlow , U K Analytical Services .p . 1 9 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 4

Th e tes t ite mconcentration s wil l b

e measure d i n a t leas t on e o f th e duplicat

e tes t mediu m samples . However, th

e sample s fro mtest

concentration s belo

w th e 72-Hou r NOE C wil l no t normall y b e analyzed . Th

e remainin

g sample s ma y b e analyzed , i f

necessary . Stoc k solution s ma

y also b e analyzed , i f

necessary , fo r

exampl e i f th e tes t concentration s ar

e belo w th e limi t o f quantitation o f

th e analytica

l metho d o r fo r reason s o f stability . Th e analytica

l metho d (e g HPLC , LC-MS , GC , AA S o r photospectrometry) wil l eithe r b e provide db

y th e Sponso r an d adapted, i f

necessary , t

o th e analytica

l condition s a

t Harla n Laboratorie s Lt d o

r develope d b y Harla n Laboratorie s Ltd . Adaptatio n o r developmen

t o f th e analytica l metho d wil l no t b e performed unde

r GLP . Detail s abou

t th e analytica

l metho d use d wil l b e given i n a

n Appendi

x o f th e fina l report . Th

e accurac y o f th e analytica

l method wil l b

e verifie db y recoverie s spannin

g th e concentratio

n rang e to b e analyze d withi n thi s study . 3. 9 Dat a Analysi s 3.9. 1 Plottin

g Growt h Curves Th e cell densit

y i n th e contro l an

d test culture s togethe

r with th e tes t ite mconcentration s an

d time s o f measurement

, recorde d t o th e neares t whole hour, ar

e tabulate d an d growt h curve s ar

e constructe d b y plottin g cel l densit

y (logarithmi c axis ) agains t tim e (arithmeti c axis) . Al l dat apoints ar

e criticall y examine

d an d i f i t is obviou s tha

t procedura

l mistakes ca

n b e identifie dand/o

r considere d highl y likely , th

e specifi c dat

apoin t i s marke d a s a n outlier an d no t includedi n subsequen

t statistica l analyses . Reason

s fo r rejectio n o f

adat apoin t a s a n outlie r wil l b e clearl y state d i n th e fina l report . Acceptabl e reason s ar e onl y procedura

l mistakes an

d no t jus t ba d precision . Outliers (marke d a s such ) wil l b e retaine d amongs t th e dat apoints show

n i n th e fina l report . 3.9. 2 Calculatio n o

f Growt h Rate s Th e averag e specifi c growt

h rat e fo r aspecifie d perio d i s calculate d a s th e logarithmi c increas

e i n biomas s fro mth

e equation : lnN n-In N t B - t , where : N i N n t i averag

e specifi c growt

h rat e fro mtim

e t i t o t n . cel l concentratio

n a t ti . cel l concentratio

n a t t n . tim e o f firs t measurement

.p . 2 0 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 5

t n= tim

e o f nt h measurement

. Th e averag e specifi c growt

h rat e fo r eac h replicat e contro l an

d tes t ite mvesse l i s calculated fo r th e perio d 0-7 2 hour

s usin g th e nominally inoculated cel l concentratio n a s th e starting valu

e rathe r tha n th e measure d starting valu

e i n orde r t o increase th

e precision o f

th e calculation . I n additio n th

e section-by-sectio

n growt h rat e ( 0 - 24,2 4 - 4 8 an

d 4 8 - 7 2 hours ) i s calculated fo r th e contro l culture s an

d th e result s examine

d i n orde r t o determin

e whethe r th e growt h rat e remains constant . Percentag e inhibitio n o f

growt h rat e fo r eac h replicat e test ite mvesse l i s calculate

d using th

e followin g equation : I r =^^-xl0 0 where : I r = percentag

e inhibitio n o f

averag e specifi c growt

h rate . H e = mea

n averag e specifi c growt

h rat e fo r th e contro l cultures , p t = averag

e specifi c growt

h rat e fo r th e test culture . Wher e auxiliar y solvents/surfactant

s ar e used, percentag

e inhibitio n i n th

e test culture s i s calculated b

y compariso

n t o th e solven t contro l cultures . 3.9. 3 Yiel d Yiel di s calculated a

s th e increas e i n biomas s ove

r aspecifie d perio d using th e followin g equation : Y = N n-N t where : Y = Yield . N t - cel l concentratio n a t th e beginning o f

th e specifie d period . N n= cel l concentratio n a t th e en d o f th e en d o f th e specifie d period . Fo r eac h test concentratio

n an d control , th

e mea n valu e fo r yield , along with th

e standar d deviatio n i s calculated fo r th e perio d 0 - 72 hours . Percentag e inhibitio n o f

yiel di s calculated using th e followin g equation :p . 2 1 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 6

x 10 0 Wher e 

## h Y c Y t percentag

e inhibitio n o f

yiel d mea n valu e fo r yiel di n th

e contro l grou

p mea n valu e fo r yiel dfo r th e treatmen

t grou p 3.9. 4 Plottin

g Concentration-Respons e Curves Fo r eac h individua

l tes t vessel , percentag

e inhibitio n (arithmeti c axis ) i s plotte d agains t tes t concentration (logarithmi c axis ) an d aline fittedb

y computerise

d interpolatio n e.g . Xlfit . Any dat apoints identifie da

s outlier s i n th

e first phas e o f dat aevaluation ar

e disregarde

d whe n fitting th e concentration-response curve . Th e ECm , EC20 , EC5 0 values ar

e rea d fro mth e graph . Wher e appropriat

e 95 % confidenc e limit s fo r th e EC5 0 wil l b e calculated , using th

e simplifie d method o f

evaluating dose-effect

experiment

s o f Litchfiel dan

d Wilcoxo n (1949) . Al l EC5 0 value s an d 95 % confidenc e limit s wil l b e rounde d t o tw o significant

figures. 3.9.5 Statistica

l Analysi s Th e Lowes t Observe d Effec t Concentratio

n (LOEC ) i s th

e lowest concentratio

n teste d a t whic h significant

( P < 0.05 ) inhibitio n o f

growt h relativ e t

o th e contro l i s observe

d withi n agive

n exposur e time . However, al l tes

t concentration s abov

e th e LOE C mus t hav e a n effec t equa l t o o r greate r tha n those observe

d a t th e LOEC . Whe

n thes e tw o condition s canno

t b e satisfied , aful l explanation o f

ho w th e LOE C ha s bee n selecte d wil l b e give n i n th e tes t report . Th e LOEC fo r

growt h rat e an d yiel da t 72 hour s wil l b e determine

d using analysi s o f

variance (ANOVA ) incorporatin g astatistica l test fo r homogeneit

y o f variance an

d a n adequat e pos t ho c tes t (e.g . Dunnet

t test) . Th e N o Observe

d Effec t Concentratio

n (NOEC ) is th

e tes t concentratio

n immediatel

y belo w th e LOE C which , whe

n compare

d to th e control , ha

s n o statistically significant

effec t ( P > 0.05) , withi n agiven exposur

e time . Wher e alimi t test ha s bee n conducted, i t

i s not possibl e to determin

e aLOEC , an

d th e NOE C wil l b e th e limi t test concentration . 3.1 0 Validit y Criteri a Fo r th e tes t t o b e valid , th e followin g performance criteri ashoul d b e met :p . 2 2 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 17

Th e cel l concentration o f

th e contro l culture s mus

t increas e b y afactor

o f a t leas t 1 6 afte r 7 2 hours . Th e mea n o f th e coefficient s o f

variatio n o f

th e daily section-by-sectio

n specifi c growt

h rat e i n th e contro l culture s afte

r 7 2 hour s (0 - 24 , 2

4 - 4 8 an d 4 8 - 72 hours ) mus

t no t excee d 35% . Th e coefficien t

variatio n o f

th e averag e specifi c growt

h rat e i n replicat

e contro l culture s after 72 hour s mus t no t excee d 7% .Harla n Stud y Number : 4130473 1 Stud y Pla n p . 2 3 Pag e 1 8 

## 4 REFERENCE S HERMAN , D . C , INNISS , W . E . an dMAYFIELD , C . I . (1990 ) Impac t o f volatil e aromati

c hydrocarbons , alon

e an di n combination , o

n growt h o f th e freshwate

r alg aSelenastru

m capricornutum . Aquati c Toxicology , 18 , pp . 8 7 - 100 . LITCHFIELD , J . T . an

dWILCOXON , F . (1949 ) A Simplifie dMetho do f Evaluating Dose

Effec t Experiments . J . Pharmacol . Exp . Ther. , 96 , pp . 99-113 . MAYER , P. , NYHOLM , N. , VERBRUGGEN , E . M . J. , HERMENS , J . L . M . an dTOLLS , J . (2000 ) Alga l growt

h inhibitio n tes

t i n filled, close

d bottle s fo r

volatil e an

dsorptive materials . Environmenta

l Toxicolog

y andChemistry , 19 , pp . 255 1 -2556 . OEC D (2000 ) Guidanc

e Documen

t o n Aquati c Toxicit

y Testin g o f Difficul t Substances an d Mixtures . OEC

D Serie s o n Testin g an dAssessment

, No . 23 . OEC

D Publishing . EUROPEAN

COMMISSION

(2002) Sanco/3268/2001 rev. 4 (final) Guidance Document on Aquatic Ecotoxicology in the context of Directive 91/414/EEC. Xlfit , I D Busines s Solution s Ltd .p . 2 4 Harla n Stud y Number : 4130473 1 Stud y Pla n Pag e 1 9 

## APPENDICE S Appendi x 1 Cultur

e Mediu m Reconstitute

dtes t wate r (AAP/AST

M medium

) prepare daccordin

g t o OEC D Tes t Guidelin e N o 20 1 wil l b

e use d for alga l cultivatio n an dtesting . Analytica

l grad e salt s wil l b e dissolve d i n purifiedwate

r t o obtai n th e following fina

l concentrations : Ingredient

s Concentratio n Macro-nutrient s NaHC0 3 15. 0 mg/L Macro-nutrient K 2 HP0 4 1.04 4 mg/ L Macro-nutrient s MgS0 4 .7H 2 0 14. 6 mg/L Macro-nutrient s MgCl 2 .6H 2 0 12.1 6 mg/ L Macro-nutrient s

CaCl 2 .2H 2 0 4.4 1 mg/L Macro-nutrient s

NaN0 3 25. 5 mg/L Trac e element

s H3BO

3 186. 0 ug/ L element MnCl 2 .4H 2 0 415. 0 ug/ L Trac e element

s ZnCl 2 3.27 Mg/ L Trac e element

s CoCl 2 .6H 2 0 1.4 3 ug/ L Trac e element

s

CuCl 2 .2H 2 0 0.01 2 ug/L Trac e element

s

Na 2 MoO„.2H 2 0 7.2 6 ug/L Trac e element

s

FeCl 3 .6H 2 0 160. 0 ug/ L Trac e element

s

Na 2 EDTA.2H 2 0 300. 0 ug/ L 

### Th e p H wil l b e adjuste dt o 7.5 . Th e wate r hardnes s (calculated

) o f th e tes t wate r wil l b e 0.1 5 mmol/

L ( = 1 5 mg/ L a s CaC03 

### ) . Fo r th e purpose s o f th e test , additiona l sodiu mbicarbonat

e (u p t o 50 0 mg/ L fina l concentration

) wil l b e adde d t o th e prepare d cultur e mediu ma s define d b y Herma n e t a l (1990 ) an d Maye r e t a l (2000 ) t o counte r ac t CO 2 depletio n an d th e chang e i n p H du e t o alga l growt h i n seale d tes t vessels .