### Harcro s Chemical s Inc
.

520 0 Speake rRoa

d Kansa s City , K S 6610 6 RECEIVE H OPP T CEI O Telephone : 913/621-774

7 Fax : 913/621-774

6 E-mail : OrganicsCS@Harcros.co m 

## 2QI

3 AP R 2 U PMII : 1 0 Dat e 04/23/1 3 Documen t Contro l Offic e (DCO ) (7407M ) Offic e o f Pollutio n Contro l an d Toxic s (OPPT ) U.S
. Environmenta l Protectio n Agenc y (EPA ) 120 0 Pennsylvani a Ave
. N W Washington , D.C
. 2046 0 (202-564-8930 ) Attn : TSC A Sectio n 4 4 0 CF R § 799.508 7 (Docke t # EPA-HQ-OPPT-2007-0531 ) RE : Alga l Inhibitio n Tes t CASR N 31138-65- 5 T o Who m I t Ma

y Concern : I n accordanc e wit h EPA' s fina l rule , Testin g o f Certai n Hig h Productio n Volum e Chemicals ; Secon d Grou p o f Chemicals , publishe d i n th e Federa l Registe ro n Januar y 7 , 201 1 a t 7 6 fed
. Reg
. 1067 , pleas e fin d enclose d th e fina l repor t fo rth e Alga l Inhibitio n Tes t o n D-gluco-Heptoni c acid , monosodiu m salt , (2.xi.) - [CAS # 31138-65-5]
. Thi s stud y wa s conducte d t o fulfil l th e Toxicit y t o Alga e requirement s o f testin g categor y C a s se t fort h i n Tabl e 2 o f 4 0 CF R 799.5087(j)
. I f an y furthe rinformatio n i s required , pleas e d o no t hesitat e t o contact m e at (913 ) 621-780 2 o rat asavini@harcros.com
.

An n Mari e Savin i Harcro s Chemical s Inc
. Regulator y Specialis t cc : Howard Stevenso n Jame s Grantha m Regards ,

Rand y Buckne r Do n Meuschke Joh n Mile s Fil e 

# I II I 

# III !

CONTAIN

S N O CB IP . 2



## REPOR T 

# harla n

Sodiu m Glucoheptonate : Algal Inhibitio n Tes

t
Dat a Requirements/Tes

tGuidelines : • OEC D Guideline

s fo r Testin g o f Chemical s (2006 ) N o 201 , "Freshwate

r Alg a an d Cyanobacteria

, Growt h Inhibitio n Test

" reference

d a s Metho d C.3 o f Commissio

n Regulatio

n (EC ) N o 761/200 9 • U S EP A Draf t Ecologica l Effects Tes

t Guidelin e OPPT S 850.540 0 Stud y Director : H Vryenhoe f Tes tFacility : Harla n Laboratorie

s Ltd . Shardlo w Business Par

k Shardlo w Derbyshir e DE7 2 2G D U K Sponsor : Harcro s Chemical

s Inc . 345 2 Corporat e Drive DaltonG A 3072 0 UNITE D STATE S O F AMERIC

A Harla n Stud y Number: Stud y Completio

n Date : 4110469 6 8 Apri l 201 3
Pag e 1 of5 1



## CONTAINS

NO CBp . 3 Harla n Stud y Number : 4110469 6 Repor

t Pag e 2 

## CONTENT S CONTENT S 2 STUD Y DIRECTO RSTATEMEN T O F GL P COMPLIANC E 4 QUALIT Y ASSURANC E STATEMEN T 5 SIGNATURE OF CONTRIBUTIN G SCIENTIST S 6 SUMMAR Y 7 Introductio n 7 Method s 7 Results 7 GENERAL INFORMATIO N : 8 Schedul e 8 Archivin g 8 1 INTRODUCTIO N AN D PURPOS E 9 1. 1 Guideline s /

Regulation s 9 2 TES T ITE M 9 3 MATERIAL S AN D METHOD S 1 0 3. 1 Definition s an

d Abbreviation s 1 0 3. 2 Tes t

Specie s 1 0 3. 3 Cultur

e Mediu m1 1 3. 4 Procedur

e 1 1 3.4. 1 Range-findin g test 1 1 3.4. 2 Definitiv e tes t

1 2 3.4. 3 Evaluatio n o f

dat a1 3 3. 5 Validatio n Criteri a1 6 4 RESULT S 1 7 4. 1 Range-findin g Tes t 1 7 4. 2 Definitiv e Tes t

1 7 4.2. 1 Validatio n criteri a1 7 4.2. 2 Growt

h dat a1 8 4.2. 3 Observation s o n culture s 2 0 4.2. 4 Observation s o n tes t ite msolubility 2 0 4.2. 5 Physico-chemica l measurement

s 2 0 4.2. 6 Re-growt

h tes t 2 0 4.2. 7 Verificatio n o f

test concentration s 2 0 5 CONCLUSIO N 2 1 6 REFERENCE S 2 1 TABLE S 22 Table 1 Cel l Densitie s an d Percentag

e Inhibitio n o f

Growt h fro mth e Range-findin g Tes t 22 Table 2 Cel l Densitie s i n th e Definitiv e Test

2 3 Table 3 Dail y Specifi c Growt h Rates fo r

th e Contro l Culture s i n th

e Definitiv e Test

2 4 Table 4 Inhibitio n o f Growt h Rate , Yiel dan

d Biomas s Integra

l i n the Definitiv e Test (0-7 2 Hour ) 2 5 Table 5 Inhibitio n o f Growt h Rate , Yiel dan

d Biomas s Integra

l i n the Definitiv e Test (0-9 6 Hour ) 2 6 Table 6 p H value s i n th e Definitiv e Test

2 7 FIGURE S 2 8P . 4



### Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 Figur e 1 Mea

n Cel l Densitie s v Tim e fo r th e Definitiv e Tes

t 2 8 Figur e 2 Inhibitio n o f

Growt h Rat e ( 0 - 7 2 hours ) 2 9 Figur e 3 Inhibitio n o f

Yiel d( 0 - 7 2 hours ) 3 0 Figur e 4 Inhibitio n o f

Biomass Integra

l ( 0 - 7 2 hours) 3 1 Figur e 5 Inhibitio n o f

Growt h Rat e ( 0 - 9 6 hours ) 3 2 Figur e 6 Inhibitio n o f

Yiel d( 0 - 9 6 hours ) 33 Figur e 7 Inhibitio n o f

Biomass Integra

l ( 0 - 9 6 hours) 3 4 Figur e 8 Temperatur

e Measurement

s Take n Throughou

t th e Tes t Perio d 3

5 APPENDICE

S 3 6 Appendi x 1 Positiv

e Contro l 3 6 Appendi x 2 Cultur

e Mediu m3 7 Appendi x 3 Analytica

l Investigations 3 8 Appendi x 4 Monitoring Authorit

y Statemen

t o f GL P Complianc

e 5 1p . 5

Harla n Stud y Number : 4110469 6 Repor

t Pag e 4 

## STUD Y DIRECTO RSTATEMEN T O F GL P COMPLIANC E Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K

Th e stud y described was performe d i n complianc e wit h U K GL P standards (Schedul

e 1 , Good Laborator y Practic e 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 accordance wit h GL P standard

s publishe d a

s OEC D Principle s o

n Goo d Laborator y Practic e (revise d 1997 , ENV/MC/CHEM(98)17) .

Thi s repor

t full y an d accuratel

y reflect s th

e procedure s use

d an d dat agenerated . Ther

e wer e n o circumstance s considered to hav e affecte d th

e integrit y o f the stud y o r th e validit y o f

th e data . Stud y Director : H Vryenhoe f Harla n Stud y Number : Stud y Title : Sodiu mGlucoheptonat

e Alga l Inhibitio n Test 41104469 6



## 0 8 AP R 

## 201 3p . 6



### Harla n Stud y Number : 4110469 6 Repor

t Pag e 5 

## QUALIT Y ASSURANC E STATEMEN T Harla n Laboratories Ltd. , Shardlo w Business Park

, Shardlow, Derbyshire , DE7 2 2GD , UK Harla n Stud y Number: 41104469 6 Stud y Title : Sodiu mGlucoheptonat

e Alga l Inhibitio n Tes

t Th e genera l facilitie s an

d activitie s ar

e inspecte d a t leas t onc e ayea r an d th e result s ar e reporte d t o th e relevan t responsibl e perso n an dmanagement

. Study-relate

d procedure

s conducte d a t th e tes t facilit y wer e audite d an d inspected. Th

e details o f thes e audit s an d inspection

s ar e give n below .
Date s and Types o

f Q A Inspection

s Reported t o th e relevan tStud y Directo r an

d Tes t Facilit y Managemen

t Dat e o f Inspectio n Typ e o f

Inspectio n Phas

e Inspected Repor t Dat e 02 Augus t

201 1 1 4 Januar y 201 3 1 4 Januar y 201 3 1 4 Januar y 201 3 1 7 Januar y 201 3 2 2 Januar y 201 3 1 4 Marc h 201 3 Genera l Stud y Pla n Verificatio n Proces s - base

d Proces s - base

d Proces s - base

d Proces s - base

d Proces s - base

d Repor t Audi t N/ A

Test Ite mPreparation Test Syste mPreparation Exposur e Assessmen

t o f Respons e Chemica l Analysi s N/ A 02 Augus t 201 1 1 4 Januar y 201 3 1 4 Januar y 201 3 1 4 Januar y 201 3 1 7 Januar y 201 3 22 Januar y 201 3 1 4 Marc h 201 3 

### Th e complet e Stud y Pla n (includin

g Stud y Specifi c Supplement

) wa s verifie dfor

GL P complianc e a t repor t audit . Thi s statemen

t confirms tha

t thi s repor t reflect s th e ra w dat aan dth e procedure

s followed . Qualit y Assurance :


## Date : ( j \ AP R20fl 'p . 7

Harla n Stud y Number : 4110469 6 Repor

t Pag e 6 

## SIGNATUR

E O F CONTRIBUTIN G SCIENTIST S Analytica l Chemistry : D M Mulle e Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K

Date : 

## 0 » AP R 201 3p . 8 Harla n Stud y Number : 4110469 6 Repor

t Pag e 7 

## SUMMAR

Y Introductio n A stud y wa s performe d to asses

s th e effec t o f th e tes t ite mo n th

e growt h o f th e gree n alg a Pseudokirchneriell asubcapitata . Th e method followe d tha

t described i n th

e OEC D Guideline s fo r Testin g o f

Chemical s (2006 ) N o 201 , "Freshwate r Alg aan d Cyanobacteria , Growt

h Inhibitio n Test " reference d a

s Metho d C. 3 o f

Commission Regulatio n (EC ) N o 761/2009 an d th e US EP A Draf t Ecological Effect s Tes t

Guideline OPPT

S 850.5400 . 

### Method s Followin g apreliminary range-findin g test , Pseudokirchneriell asubcapitatawa s expose d t o a n aqueous solutio n o f

th e test ite ma t concentration s o f

10 , 32 , 100 , 32

0 an d 100 0 m g activ e ingredien t (ai)/L (thre e replicat

e flask s pe

r concentration) fo r

9 6 hours , unde

r constan t illuminatio n an

d shaking a

t atemperatur

e o f 2 4 ± 1 °C . Samples o f

th e alga l population s wer

e removed dail y an

d cel l concentration s determine d fo r eac h contro l an d treatmen

t group , using aCoulter

® Multisize r

Particl e Counter . Result s Exposur e o f Pseudokirchneriell asubcapitat ato th e test ite mgav e th e followin g results :

Time Point (Hours ) Respons e

Variabl e EC i 0(m g ai/L ) 95 % Confidenc e Limit s (m g ai/L ) N o Observed Effec t Concentratio n (NOEC ) (m g ai/L ) Lowes t Observe d Effec t Concentratio n (LOEC ) (m g ai/L ) Growt h Rat e 51 0 * 3 2 10 0 7 2 Yiel d 15 0 12 0

- 19

0 3 2 10 0 Biomas s 16

0 14

0

- 19

0 3 2 10 0 Growt h Rat e 79 0 * 10 0 32

0 9 6 Yiel d 220 18 0

- 26 0 1 0 3 2 Biomas s 180 14 0

- 21 0 3 2 10 0 Analysi s o f

th e test preparation s a

t 0 an d 9 6 hour s showe d measure

d tes t concentration s to rang

e fro m93 % t o 105 % o f

nomina l an d s o th e results ar

e base d o n nomina

l tes t concentration s only . A re-growt

h tes t wa s performe d whic h showe d th e test ite mt o be algistati c i n effect .

* I t wa s no t possibl e t o calculat e 95 % confidenc

e limit s fo r th e E r C S 0 value s a s th e dat agenerate d di dno t fit th e model s availabl e fo r th e calculatio n o f confidenc e limits .p . 9

Harla n Stud y Number : 4110469 6 Repor

t Pag e 8 

## GENERA L INFORMATIO N Schedul e Experimenta

l Startin g Date : Experimenta

l Completio n Date : 2 7 Februar y 201 2 2 1 Novembe

r 201 2 Archivin g Unles s instructe d otherwis e b y th e Sponsor

, al l original dat

aan d the fina l repor

t wil l b e retained 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 to furthe r

retentio n o

r disposal . Furthe

r retentio n o

r retur n o f th e dat awil l b e chargeabl e to th e Sponsor .p . 1 0

Harla n Stud y Number : 4110469 6 Repor

t Pag e 9 

## 1 INTRODUCTIO N AN D PURPOS E This report

contains a description of the methods used

and results obtained

during a study to investigate the effect

of the test item on the growth of the green alga Pseudokirchneriella Pseudokirchneriell asubcapitatai s afreshwate r unicellula

r alga , representative o f primar y producers foun d i n natura l water s an d ca n therefor

e be considered a

s a n importan

t non-targe t organis mi n freshwate

r ecosystems . 

## 1. 1 Guideline

s / Regulation

s Thi s stud y wa s designed t

o b e compatible wit h th

e procedure s indicate d b y th e followin g internationall y accepte

d guideline s an

d recommendations :

• OECD Guideline s fo r Testing o f

Chemical s (2006 ) N o 201 , "Freshwate r Alg aan d Cyanobacteria , Growt

h Inhibitio n Test " referenced a s Metho d C. 3 o f

Commission Regulatio n (EC ) N o 761/200 9 • US EP

A Draf t Ecologica

l Effect s Tes

t Guidelin e OPPT

S 850.5400

Th e integrit y o f th e supplie d dat

arelatin g to th

e identity , purity an

d stabilit y o f th e tes t itemi s th e responsibilit

y o f th e Sponsor . subcapitata.



## 2 TES T ITE M

Identification : Description : Batch : Purity : Expiry / Retes t Date : Storag e Conditions : Sodiu mGlucoheptonat

e dar k brow n liqui d 92100010 0 49.5 % w/w 9 Februar y 201 5 roo mtemperatur

e i n the dar

k

### p . 1 1

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 0 

## MATERIAL S AN

D METHOD S 3. 1 Definition

s an d Abbreviation

s Biomass : ECx :
Growth : Growt h Rat e (Averag e Specifi c Growt h Rate) : Yield :
LOE C (Lowes t Observe d Effec t Concentration) : NOE C (N o Observe d Effec t Concentration) : Treatment: Section-by-Sectio

n Growt h Rate : AU C (Are aUnde r th e Growt h Curve) : Measuremen t variabl e (fluorescenc

e o r alga l cell density

) a s surrogat e measur e fo r alga l biomass . Th e calculate d concentratio

n o f tes t ite mtha t result s i n a n x % reductio n o f th e respectiv

e growt h paramete

r relativ e t o th e contro l (o r 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 increas e i n biomass durin

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

t variabl e 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 lowes t tes t concentratio

n a t whic h astatisticall

y significan

t 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) . Th e highes t tes t concentratio

n a t whic h n o statisticall

y significan t inhibition 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 (tes t mediu monly) an d solven t contro l (i f applicable) . 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 0-1,1-2,2- 3 and

, 3- 4 wher e appropriate) . Biomass integra

l calculate d a s are aunde r th e growt h curve .
3. 2 Tes tSpecie s Th e tes t wa s carrie d ou t usin g Pseudokirchneriell

asubcapitat

astrai n CCA P 278/4 . Liqui d cultures o f

Pseudokirchneriellasubcapitat awer e obtaine d fromth e Cultur e Collectio n o f Alga e an d Protozo a(CCAP) , SAM

S Researc h Service s Ltd , Scottis

h Marin e Institute , Oban

, Argyll , Scotland . Maste

r cultures wer

e maintaine

d i n th e laborator

y b y th e periodi c replenishmen

t o f cultur e mediu m(Sectio n 3.2) . Th

e maste r cultures wer

e maintaine

d i n th e laborator

y unde r constan t aeratio n an d constan t illumination a

t 2 1 ± 1 °C . Prio r t o th e star t o f th e tes t sufficien t maste r cultur e wa s adde d t o approximatel

y 10 0 m L volume s o f cultur e medi acontaine d i n conica

l flask s t o giv e a n initial cell densit

y o f approximatel

y 10 3 

### cells/mL . Th

e flask s wer e plugge d wit h polyurethan

e foa mstopper s an d kep t unde r constan t

### Harla n Stud y Number : 4110469 6 Repor

t p . 1 2 Pag e 1 1 agitatio n b y orbita l shake r (10 0 - 15 0 rpm ) an d constan t illumination a

t 2 4 ± 1 °C until th

e alga l cel l densit y wa s approximatel

y 10

4 

### -1 0 cells/mL . A positiv e control (Harla

n Stud y Number : 41200591 ) use

d zin c chlorid e a s th e referenc e item . Detail s o f th e positive contro

l ar e give n i n Appendi

x 1 . Th e positiv e contro l wa s conducte d betwee n 1 3 Februar

y 201 2 an d 1 7 Februar y 2012 . 3. 3 Cultur e Mediu m Th e cultur e mediu muse d fo r bot h th e range-findin

g an d definitiv e test

s wa s th e sam e a s tha t use d t o maintai n th e stoc k culture . Th

e cultur e mediu muse d i n th e definitiv e tes

t ha d ahardnes

s o f 1 8 mg/ L a s CaCC> 3 an d aTota l Organi c Carbo n (TOC ) conten t o f les s tha n 1. 0 m g C/L . Th e cultur e mediu mi s define

d i n Appendi

x 2 , 3. 4 Procedur e 3.4. 1 Range-findin

g tes t Th e tes t concentration

s t o b e use d i n th e definitiv e tes

t wer e determine

d b y apreliminar

y rangefindin g test . Th

e range-findin

g tes t wa s conducte d b y exposin g Pseudokirchneriell

asubcapitat

a cell s t o aserie s o f nomina l tes t concentration

s o f 1.0 , 10 , 10 0 an d 100 0 m g ai/ L fo r aperio d o f 9 6 hours . Al l tes

t concentration

s wer e correcte d fo r ates t ite mwate r conten t o f 50.5% . Th e tes t wa s conducte d i n 25 0 m L glas s conica l flask s eac h containin

g 10 0 m L o f tes t preparatio n an d plugge d wit h polyurethan

e foa mbung s t o reduc e evaporation . Tw

o replicat e flasks wer

e use d fo r eac h contro l an d tes t concentration . Th

e tes t ite mwa s dissolve d directl y i n cultur e medium . An amoun t o f tes t ite m(101 0 mg

) wa s dissolve d i n cultur e mediu man d th e volum e adjuste d t o 50 0 m L t o giv e a100 0 m g ai/ L stoc k solutio n fro mwhic

h aserie s o f dilution s wa

s mad e t o giv e furthe r stoc k solutions o f

100 , 10 an d 1. 0 m g ai/L . An aliquo

t (45 0 mL) o f

eac h o f th e stoc k solution s wa s separatel

y inoculate

d wit h alga l suspensio

n (9. 7 mL

) t o giv e th e require d tes t concentration

s o f 1.0 , 10 , 10 0 an d 100 0 m g ai/L . Th e stoc k solutions an

d eac h o f th e prepare d concentration

s wer e inverte d severa l time s t o ensur e adequat e mixing an

d homogeneity . Th e contro l grou p wa s maintaine

d unde r identica l conditions bu

t no t expose d t o th e tes t item . A t th e star t o f th e range-findin

g tes t asampl e o f eac h tes t an d control cultur

e wa s remove d an d th e cel l densit y determine

d usin g aCoulter

® Multisize

r Particl e Counter . Th e flask s wer e the n plugge d wit h polyurethan

e foa mbung s an d incubate d (INFOR S Multitron

® Versio n 2 incubator

) a t 2 4 ± 1 °C unde

r continuous illumination (intensit y approximatel

y 700 0 lux ) provide

d b y war m whit e lighting (38

0 - 730 nm

) an d constantl y shake n a t approximatel

y 15 0 rp mfor 9 6 hours .p . 1 3

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 2 Afte r 9 6 hours the cel l densit

y o f eac h flas k wa s determined usin g aCoulte r Multisizer

Particl e Counter . A sampl e o f eac h tes t concentration wa

s take n fo r chemica l analysi s a

t 0 an d 9 6 hours i n orde

r t o determin e th e stability o f

th e tes t ite munde

r test conditions . Al l samples wer e store d a t approximately -20 ° C prio r t o analysis . 3.4. 2 Definitiv e tes

t Base d o n th e result s o f

th e range-findin g test

th e followin g test

concentration s wer

e assigne d to th e definitiv e test : 10 , 32 , 100 , 32 0 an d 100 0 m g ai/L . 3.4.2. 1 Experimenta

l Preparation Fo r th e purpos e o f

th e definitiv e test , th

e tes t ite mwa s dissolve d directl y i n cultur e medium . Al l test concentration s wer

e corrected fo r

ates t ite mwate

r conten t o f 50.5% . Amount s o f

tes t ite m(202 0 an

d 64 6 mg ) wer

e eac h separatel

y dissolve d i n cultur

e mediu man d th e volum e adjuste d t o 1 lite r

to giv e 100

0 an d 32 0 m g ai/ 1 stoc

k solution s respectively . A series o f dilution s wa

s mad e fro mthese stoc

k solution s to giv e furthe r stoc k solution s o f

100 , 3 2 an

d 1 0 m g ai/L . A n aliquo t

(90 0 mL ) o f

eac h o f th e stoc k solution s wa

s separatel

y inoculate d wit h alga l suspensio

n (13. 5 mL ) t o giv e th e require d test

concentration s o f

10 , 32 , 100 , 32

0 an d 100 0 mg ai/L . Th e stoc k solution s an

d eac h o f th e prepare d concentration s wer

e inverte d severa

l time s to ensur

e adequat e mixin g an

d homogeneity . Th e concentratio n an

d stability o f

th e tes t ite mi n th

e test preparation s wer

e verifie d b

y chemica

l analysi s a t 0 an d 9 6 hour s (se

e Appendix 3) . 3.4.2. 2 Exposur

e condition s A s i n th e range-findin g tes t

25 0 m L glas s conica

l flask s wer

e used . Si x flask s eac

h containin g 10 0 m L o f tes t preparatio n wer

e use d fo r th e contro l an

d three flask s eac

h containin g 10

0 mL wer e use d fo r eac h treatmen

t group . Th e contro l grou

p was maintaine d unde

r identica l condition s but

no t expose d t o th e test item . Pre-cultur e condition s gav

e a n alga l suspensio

n i n lo g phas

e growt h characterized b

y acel l densit y o f 6.6 8 x 10 5 cell s pe

r mL . Inoculatio n o f

90 0 mL o f test mediumwit h 13. 5 m

L o f thi s alga l suspensio

n gav e a n initia l nomina

l cel l densit

y o f 1 x 10 4 cell s pe

r m L an d ha d n o significant dilutio n effec t

o n th e final tes t

concentration . Th e flask s wer

e plugge d wit h polyurethan e foa mbung s an

d incubated (INFOR S Multitron ® Versio n 2 incubator

) a t 2 4 ± 1 ° C unde

r continuou s illuminatio n (intensit y approximatel

yHarla n Stud y Number : 4110469 6 Repor

t p . 1 4 Pag e 1 3
700 0 lux ) provide d b y war mwhite lightin g (38 0 - 73 0 nm) an d constantl

y shake n a t approximately 15 0 rp mfo r 9 6 hours . Samples wer

e taken a

t 0 , 24 , 48 , 7 2 an

d 9 6 hours an

d th e cel l densitie s determine d using a Coulter® Multisize r

Particl e Counter . 3.4.2. 3 Physico-chemica l measurement

s A t th e reques t o f the Sponsor

, a t th e star t o f th e test th e p H o f th e contro l an

d test preparation s wa s adjuste d to p H 7 . Th

e p H o f th e contro l an

d eac h test preparatio n wa

s determined a

t initiatio n o f th e test an d afte r bot h 4 8 an d 9 6 hour s exposure . Th

e p H wa s measure d usin g aWT W p H 32

0 p H meter . Th

e temperatur

e withi n th

e incubato

r wa s recorded hourl y using th e incubators ' interna l pt-10 0 temperatur

e probe an

d Multico mSoftwar

e Packag e (se e Figur e 8) . 3.4.2. 4 Re-growt

h tes t A re-growt

h tes t wa s performe d afte

r 9 6 hour s to determin

e th e algicidal o

r algistati c effec t

o f th e test item . Aliquot s wer

e remove d fro meac

h replicat e contro l an

d tes t cultur e (0.25 mL an

d 0.5 0 m L respectively ) an d th e replicate s pooled . Fres h steril e cultur

e mediu m(10 0 mL ) wa

s adde d to eac h to ensur

e tha t th e tes t concentration wa

s reduce d to belo w th

e inhibitin g level . Th e flask s wer

e plugge d wit h polyurethan e foa mbung s ar

e incubated (INFOR S Multitron ® Versio n 2 Incubator) a

t 2 4 ± 1 ° C unde

r continuou s illuminatio n (intensit y approximatel

y 700 0 lux ) an d constantly shake

n a t approximatel

y 15 0 rp mfo r

12 0 hours . 3.4. 3 Evaluatio n o

f dat a 3.4.3. 1 Compariso 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 increase i n biomas s fromthe equation : lnN n -ln N
where : N i N „ t i t n average specifi c growt h rat e fromtime t i to t ,

to cel l concentratio n a

t t j cel l concentratio n a

t t n tim e o f firs t measuremen

t tim e o f n t hmeasuremen

tp . 1 5

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 4
Th e averag e specifi c growt

h rat e ove r th e test duratio n wa

s calculate d fo r

eac h replicat e contro l an d tes t ite mvesse

l usin g th e nominall y inoculate d cel l concentration a s th e starting valu

e rathe r tha n th e measure d startin g valu e i n orde

r to increas e th

e precisio n o f

the calculation . I n additio n th

e sectio n b y sectio

n specifi c growt

h rat e (day s 0-1 , 1-2

, 2- 3 an d 3-4) wa s calculated fo r th e contro l culture s an

d th e result s examined i n orde r to determin

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

growt h rat e fo r eac h replicat e test ite mvesse

l wa s calculated using th

e followin g equation : I r =^xl0 0 where : I r = percentage inhibitio n o f averag e specifi c growt

h rat e H e = mea

n averag e specifi c growt

h rat e fo r th e contro l culture s M t = average specifi c growt h rat e fo r th e test cultur e 3.4.3. 2 Comparison o f

Yiel d Yiel di s calculate d a

s the increas e i n biomas s ove r th e exposur e perio d using th

e followin g equation : Y = N n - N 0 where : Y = yiel d N o = cel l concentratio n a t th e star t o f th e test N n—cel l concentratio n a t th e en d o f th e tes t Fo r eac h tes t concentratio n an

d contro l th

e mea n value fo r

yiel dalon g wit h th

e standar d deviatio n wa s calculated . Percentag

e inhibitio n o f

yiel dwa s calculated usin g the followin g equation : I y= x 10

0 where : I y = percentage inhibitio n o f yiel d Y c = mea

n valu e fo r

yiel di n th

e contro l grou

p Y t = mea

n valu e fo r

yiel dfo r th e treatmen

t grou pp . 1 6

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 5
3.4.3. 3 Compariso n o f

biomas s integra

l The biomass integral

(area under the growth curve) was calculated using the following equation:

A = N i - N £ x

^+ N, + N 2 -2N 0x(t 2 _ ti)+ N„, 1+ N„-2N 0x(tn _ ^ where : A = are

a N o = nominal cel l concentration a t star t o f tes t N i = measure

d cel l concentration a

t t i N n= measure

d cel l concentration a

t t n t i = tim e o f

first measuremen

t after beginnin g o f

tes t t n= tim e o f

n * measuremen

t after beginnin g o f

tes t Percentag e inhibitio n o f

th e biomas s integra

l fo r eac h replicat e test ite mvesse

l wa s calculated using the followin g equation : A £ Z A , xl0 0 A c where : I A = percentage inhibitio n o f th e biomas s integra

l A c = mea

n biomas s integra

l fo r th e contro l culture s A t = biomas s integra l fo r th e tes t cultur e 3.4.3. 4 Determinatio n o f EC Xvalue s Fo r eac h individua l tes t

vesse l (mea n value s fo r

yield) , percentag

e inhibitio n (arithmeti c axis ) wa s plotte d against

tes t concentratio n (logarithmi c axis ) an d alin e fittedb y computerised interpolatio n using th e Xlfi t softwar e packag e (IDBS) . EC Xvalue s wer e then determined fromth e equation fo r th e fitte dline . Wher e appropriat

e 95 % confidenc e limit s fo r th e EC5 0 value s wer

e calculated , using th

e simplifie d metho d o f

evaluatin g dose-effec t

experiments o f

Litchfiel dan

d Wilcoxo n (1949) . 3.4.3. 5 Statistica

l analysi s On e wa y analysi s o f

varianc e incorporatin g Bartlett' s tes t fo r homogeneit

y o f varianc e (Soka

l an d Rohl f 1981 ) an d Dunnett' s multipl e compariso n procedur

e fo r comparing severa

l treatment

s wit h acontro l (Dunnet

t 1955 ) wa

s carrie d ou

t o n th e growt h rate , yiel dan

d biomas s integra

l dat aafter 7 2 an d 9 6 hours fo r

the contro l an

d al l test concentration s to determin e an y statisticall y significant

### p . 1 7

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 6 difference s betwee

n th e tes t an d contro l groups . Al l statistica

l analyse s wer e performe

d usin g th e SA S compute r softwar e packag e (SA S 199 9 - 2001) . 3. 5 Validatio n Criteri a Th e result s o f th e tes t ar e considere

d vali di f th e followin g performanc

e criteri aar

e met:

• Th e cell concentratio

n o f th e contro l culture s mus t increas e b y afacto r o f a t leas t 1 6 afte r 7 2 hour s (OECD/E

C Guidelines) an

d b y afacto r o f a t leas t 10 0 afte r 9 6 hour s (EPA/OPPT

S Guidelines) .

• Th e mea n o f th e coefficients o f

variatio n o f th e sectio n b y sectio n specifi c growt h rate s i n th e control culture s durin

g th e cours e o f th e tes t (0-24 , 24-48 an

d 48-7 2 hours ) mus t no t excee d 35 % (OEC

D Guidelines) .

• Th e coefficien

t o f variatio n o f th e averag e specifi c growt h rat e i n replicat

e contro l culture s afte r 7 2 hour s mus t no t excee d 7 % (OEC D Guidelines) .

• Th e coefficien

t o f variatio n o f th e cel l densit y value s i n replicat

e contro l culture s mus t no t excee d 20 % afte r 7 2 an d 9 6 hour s (EPA/OPPT

S Guidelines) .

### p . 1 8

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 7 

## 4 RESULT S 4. 1 Range-findin g Tes t Th e cell densitie

s an d percentag

e inhibitio n o f

growt h value s fro mth

e exposur e o f Pseudokirchneriell

asubcapitat

at o th e tes t ite mdurin g th e range-findin

g tes t ar e give n i n Tabl e 1 . Th e result s showe d n o effec t o n growt h a t th e tes t concentration

s o f 1. 0 an d 1 0 m g ai/L . However

, growt h wa s observe d t o b e reduce d a t 10 0 an d 100 0 m g ai/ L Base d o n thi s information tes

t concentration

s o f 10 , 32 , 100 , 32 0 an d 100 0 m g ai/ L wer e selecte d fo r th e definitiv e test . Chemica l analysis o f

th e 10 , 10 0 an d 100 0 m g ai/ L tes t preparation

s a t 0 an d 9 6 hour s (se e Appendi x 3 ) showe

d measure d tes t concentration

s t o rang e from87% t

o 129 % o f nomina l indicatin g tha t th e tes t ite mwa s stabl e ove r th e tes t duration . 4. 2 Definitiv e Tes t Cel l densit y value s determine

d a t eac h samplin g tim e ar e give n i n Tabl e 2 . Dail y specifi c growt h rate s fo r th e control cultures ar

e give n i n Tabl e 3 . Growt

h rate , yiel dan

d biomass integra

l value s fo r th e contro l an d tes t cultures afte

r 7 2 hour s an d percentag

e inhibitio n value

s ar e give n i n Tabl e 4 . Growt h rate , yiel dan

d biomas s integra l value s fo r th e contro l an d tes t cultures afte

r 9 6 hour s an d percentag

e inhibitio n value

s ar e give n i n Tabl e 5 . pH value

s determine

d a t 0,4 8 an d 9 6 hour s ar e give n i n Tabl e 6 . Th e mea n cell densities versu

s tim e fo r th e definitiv e tes

t ar e presente d i n Figur e 1 . Percentag

e inhibitio n value

s ar e plotte d agains t tes t concentratio

n i n Figur e 2 , Figur e 3 , Figur e 4 , Figur e 5 , Figur e 6 an d Figur e 7 . 4.2. 1 Validatio

n criteri a Th e followin g dat

asho w tha t th e cel l concentratio

n o f th e contro l cultures increase

d b y afacto r o f 16 0 afte r 7 2 hour s an d b y afacto r o f 239 afte r 9 6 hours . Thi s increas

e wa s i n lin e wit h th e OEC D Guidelin e tha t state s th e enhancemen

t mus t b e a t leas t b y afacto r o f 1 6 afte r 7 2 hour s an d th e EPA/OPPT

S Guidelines tha

t state s th e enhancemen

t mus t b e a t leas t b y afacto r o f 10 0 afte r 9 6 hours . Mea n cell densit

y o f control a t 0 hour s : 1.0

5 x 10

4 

### cell s pe r m L Mea n cell densit

y o f control a t 7 2 hour s 1.6

8 x 10

6 

### cell s pe r m L Mea n cell densit

y o f control a t 9 6 hour s : 2.5 1 x 10 6 

### cell s pe r m L Th e mea n coefficien

t o f variatio n for sectio n b y sectio n specific growt

h rat e for th e contro l cultures ove

r th e tes t period (0-72 hours

) wa s 9 % an d henc e satisfie d th e validation criterio

n give n i n th e OEC D Guidelin

e whic h state s th e mea n mus t no t excee d 35% .p . 1 9 Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 8 Th e coefficien t

o f variatio n fo r

averag e specifi c growt

h rat e fo r th e contro l culture s ove

r th e test perio d ( 0 - 7 2 h ) wa

s 1 % an d henc e satisfie d th

e validatio n criterio n give n i n th e OEC D Guideline whic h state s tha

t thi s mus t no t excee d 7% . Th e coefficien t

o f variatio n o f

th e cel l densit

y value s i n replicat

e contro l culture s wa

s 12 % after 7 2 hours an

d 3 % afte r

9 6 hours an

d henc e satisfie d th

e validatio n criterio n whic h states tha t thi s mus t no t excee d 20% . 4.2. 2 Growt

h dat a Fro mth e dat agive n i n Table s 2 an

d 4 , i t i s clea r tha t th e growt h rat e (r) , yiel d(y ) an

d biomas s integra l (b ) o f Pseudokirchneriell asubcapitat a(CCA P 278/4 ) wer

e affecte d b y th

e presenc e o f th e tes t ite move r the 96-Hou

r exposur e period . Accordingl y the followin g results wer e determined fromth

e data : 4.2.2. 1 Inhibitio n o f

growt h rat e

wher e E r C xi s th

e tes t concentration tha

t reduce d growt h rat e b y x% . Statistica l analysi s o f

th e growt h rat e dat aa t 7 2 an d 9 6 hours wa

s carrie d out

fo r th e contro l an

d al l tes t concentration s usin g on e wa y analysi s o f

varianc e incorporatin g Bartletf s tes t fo r homogeneit

y o f varianc e (Soka

l an d Rohl f 1981 ) an

d Dunnett' s multipl e comparison procedur e fo r comparing severa

l treatments wit h acontro l (Dunnet t 1955) . Afte r

7 2 hours ther

e wer e n o statisticall y significan t

difference s betwee

n th e control , 1 0 an

d 3 2 m g ai/ L test

concentration s (P^0.05) , howeve

r al l othe r tes t concentration s wer

e significantl y differen t (PO.05 ) and , therefor e th e "No Observed Effec t

Concentration " (NOEC ) base d o n growt

h rat e wa s 3 2 m g ai/L . Correspondingl

y th e "Lowes t

Observe d Effec t Concentration " (LOEC ) base d o n growt

h rat e wa s 10 0 m g ai/L . Afte r

9 6 hours ther

e wer e n o statisticall y significan t

difference s betwee

n th e control , 10 , 3 2 an d 10 0 m g ai/ L tes t

concentration s (P>0.05) , howeve r al l othe r tes t concentration s wer

e significantl y differen t

(P<0.05 ) and , therefor

e th e "N o Observe

d Effec t Concentration " (NOEC ) base d o n growt

h rat e wa s 10 0 m g ai/L . Correspondingl y th e "Lowes t

Observe d Effec t Concentration " (LOEC ) base d o n growt h rat e wa s 32 0 mg ai/L , E r C I 0( 0 ErC 2 0( 0 E r C 5 0( 0 E r C 9 0( 0 E rCi o ( 0 E r C 2 0( 0 E r C 5 0(0 E r C 9 0( 0 7 2 h )

7 2 h ) 7 2 h ) 7 2 h ) 9 6 h ) 9 6 h ) 9 6 h ) 9 6 h ) 200 mg ai/

L 33 0 mg ai/

L 79 0 mg ai/L * >1000m g ai/L 13 0 m g ai/ L 21 0 m g ai/ L 51 0 m g ai/L * >1000 m g ai/L

* I t wa s no t possibl e t o calculat e 95 % confidenc

e limit s fo r th e EC 5

0valu e a s th e dat agenerate d di dno t fit th e model s availabl e fo r th e calculatio n o f confidenc e limits .p . 2 0

Harla n Stud y Number : 4110469 6 Repor

t Pag e 1 9 4.2.2. 2 Inhibitio n o f

yiel d EyCjo( 0 EyC 2O ( 0 EyC 30 ( 0 EyC9( ) ( 0 EyCio( 0 EyC 20 ( 0 EyC 50 ( 0 EyC9 Q ( 0 72 h ) 7 2 h ) 7 2 h ) 7 2 h ) 9 6 h ) 9 6 h ) 9 6 h ) 9 6 h ) 47 0 m

g ai/ L 54 mg ai/L 9 0 mg ai/L 220 m g ai/L ; 95 % confidenc e limit s 18 0 - 260 mg ai/L 86 0 m g ai/ L 5 0 mg ai/ L 7 5 m g ai/ L 150 mg ai/L ; 95 % confidenc e limit s 12 0 - 19 0 mg ai/L

wher e E y C xi s th

e tes t concentration tha

t reduce d yiel db y x% . Statistica l analysi s o f

th e yiel ddat

aa t 7 2 an d 9 6 hour s wa s carrie d out

a s i n Sectio

n 4.2.2.1 . Afte r 7 2 hours ther

e wer e no statisticall y significan t difference s betwee

n th e control , 1 0 an

d 3 2 m g ai/ L test concentration s (P>0.05) , howeve r al l othe r tes t concentration s wer

e significantl y differen t (P<0.05 ) and , therefor

e th e "N o Observe

d Effec t Concentration " (NOEC ) base d o n yiel dwa

s 3 2 m g ai/L . Correspondingly th e "Lowes t

Observe d Effec t Concentration " (LOEC ) base d o n yiel dwa s 10 0 mg ai/L . Afte r

96 hour s ther e wer e n o statisticall y significan t

difference s betwee

n th e contro l an

d 1 0 mg ai/ L test

concentration (P>0.05) , howeve r al l othe r tes t concentration s wer

e significantl y different

(P<0.05 ) and , therefor

e th e "N o Observe

d Effec t Concentration " (NOEC ) base d o n yiel dwa

s 1 0 mg ai/L . Correspondingl y th e "Lowes t

Observe d Effec t Concentration " (LOEC ) base

d o n yiel dwa

s 3 2 m g ai/L . 4.2.2. 3 Inhibitio n o f

biomas s integra

l E b Ci o (0-72h ) 5 1 m g ai/ L E b C 2 0 ( 0 - 72h ) 7 8 m g ai/ L E b C 5 0(0-72h ) 16 0 m g ai/L ; 95 % confidenc e limit s 14 0 - 19 0 m g ai/L E b C 9 0(0 -72h ) 50 0 m g ai/ L EbCi o (0-96h ) 4 9 m

g ai/ L E b C 2 0( 0 - 9 6 h ) 7 8 m g ai/ L E b C 5 0( 0 - 9 6 h ) 18 0 m g ai/L ; 95 % confidenc e limit s 14 0 - 21 0 m g ai/L E b C 9 0(0-96h ) 62 0 m g ai/ L wher e EbC xi s the tes t

concentration tha

t reduce d biomas s integra

l (areaunde

r th e growt h curve ) b y x% . Statistica l analysi s o f

th e biomas s integra

l dat aa t 7 2 an d 9 6 hours was carrie d ou t a s i n Sectio n 4.2.2.1 . Ther

e wer e n o statisticall y significant difference s betwee

n th e control , 1 0 an

d 3 2 m g ai/L tes t

concentration s (P>0.05) , howeve r al l othe r tes t concentration s wer

e significantl y differen t (P<0.05 ) and , therefor

e th e "N o Observe

d Effec t Concentration " (NOEC ) base d o n biomas s integral wa

s 3 2 mg ai/L . Correspondingl y th e "Lowes t

Observe d Effec t Concentration " (LOEC ) base

d o n biomas s integra

l wa s 10 0 mg ai/L .p . 2 1

Harla n Stud y Number : 4110469 6 Repor

t Pag e 2 0 4.2. 3 Observations o

n culture s Al l test an d contro l culture s wer

e inspecte

d microscopically a

t 9 6 hours . Afte r

9 6 hour s ther e wer e n o abnormalitie s detecte

d i n th e contro l o

r test culture s a

t 10 , 32 , 10

0 an d 32 0 m g ai/L , howeve r cel l debri s was observe

d t o b e presen t i n th e test culture s a

t 100 0 mg ai/L . 4.2. 4 Observations o

n tes tite m solubilit

y A t th e star t o f th e tes t al l contro l culture s wer

e observe d to be clea

r colorles s solutions . Th

e test culture s wer

e observed t o rang

e fromclea

r colorles s solution s a

t 1 0 m g ai/L through to ambe r colore d solution s a

t 100 0 m g ai/L . Afte r

the 96-Hou

r tes t perio d al l control , 10 , 32 , 10 0 an d 32 0 m g ai/ L tes t

culture s wer

e observe d to b e gree n dispersion s whils t

th e 100 0 m g ai/ L test culture s wer

e observed t o be amber/gree n dispersions . 4.2.5 Physico-chemica l measurement

s Th e p H value s o f

th e contro l an

d eac h test preparation ar

e give n i n Tabl e 6 . Temperatur e wa s maintaine d a

t 2 4 ± 1 ° C throughou

t th e test . Th e p H valu e o f

the contro l culture s (se

e Table 6 ) wa

s observe d to increas e fro mp H 7. 0 a t 0 hour s to p H 8. 1 a

t 4 8 hours an

d p H 8. 2 a

t 9 6 hours . Thi s increas e wa s considere

d t o b e du e t o th e amoun t o f carbo n dioxid e require d b y th e larg e numbe

r o f alga l cell s i n th

e lo g phas

e o f growt h (se e Figur e 1 ) exceedin

g th e transfe r rat e o f CO 2 fromth e gaseou s phas e to th e aqueou s phase . I n thi s situatio n CO 2 require d fo r

photosynthesi s an

d growt h woul d b

e derive d fro m bicarbonat

e i n solutio n whic h results i n a n increase i n th

e p H o f th e culture . Th

e increas e i n p H after 9 6 hours wa

s i n exces s o f

1 p H uni t

after 9 6 hours . Thi s wa

s considered to hav

e ha d n o advers e effec t o n the result s o f

th e stud y given tha

t th e increas e i n cel l concentratio n i n th e contro l culture s exceede

d the validatio n criterio n given i n th e Test Guidelines . 4.2.6 Re-growt

h tes t Re-growt h wa s observed to hav

e occurred i n th

e control , 10 , 32,10 0 an

d 32 0 mg ai/L test

culture s afte r 4 8 hours an

d i n the 100

0 m g ai/L test cultur e after 12 0 hours . Thes

e result s indicat

e tha t th e test ite mwas algistati c i n effect . 4.2. 7 Verificatio n o f

tes t concentration s Analysi s o f

the tes t preparation s a

t 0 an d 9 6 hours (se

e Appendi x 3 ) showe

d measure

d test concentration s to range fro m93 % to 105 % o f nomina l an d s o th e result s ar

e base d o n nomina

l test concentration s onl yp . 2 2

Harla n Stud y Number : 4110469 6 Repor

t Pag e 2 1 

## 5 CONCLUSIO N Th e effec t o f the tes t ite mo n th

e growt h o f Pseudokirchneriell asubcapitataha s bee n investigated ove r a96-Hou

r perio d an

d gav e th e followin g results :
Tim e Poin t (Hours) Respons

e Variabl e EC 5 0(m

g ai/L ) 95 % Confidenc

e Limit s (m g ai/L) N

o Observe d Effec t Concentratio

n (NOEC ) (m g ai/L ) Lowes

t Observe d Effec t Concentratio

n (LOEC ) (m g ai/L) Growt h Rat e 51 0 * 3 2 10 0 7 2 Yiel d 15 0 12

0

- 19

0 3 2 10 0 Biomass 16

0 14

0

- 19

0 3 2 10 0 Growt h Rat e 79

0 * 10 0 32

0 9 6 Yiel d 22 0 18

0

- 26

0 10 3 2 Biomas s 18

0 14

0

- 21 0 3 2 10 0 A re-growt

h tes t was performe d whic h showe d th e test ite mto be algistati c i n effect . 

## 6 REFERENCE S DUNNETT , C W (1955 ) A Multipl e Compariso n Procedur

e fo r Comparing Severa

l Treatment

s Wit h aControl . J A

mSta t Asso c 50,1096-1121 . LITCHFIELD , J T and WILCOXON , F (1949 ) A Simplifie d Metho d o f Evaluatin g Dose-Effec t Experiments . J Pharmaco l Ex p The r 96 , 99-113 . SAS/STAT Proprietar

y Softwar

e Releas e 8.0 2 (199 9 - 2001) , SA

S Institute Inc , Cary , NC , USA . SOKAL , RRan

d ROHLF , F J (1981 ) Biometry . Ne w York : W H FREEMA N an d Company . Xlfit , I D Busines s Solution s Ltd .

* I t wa s no t possibl e t o calculat e 95 % confidenc

e limit s fo r th e Er C 5 0 value

s a s th e dat agenerate d di dno t fit th e model s availabl e fo r th e calculatio n o f confidenc e limits .p . 2 3
Harla n Stud y Number: 4110469

6 Repor t Pag e 2 2 

## TABLE S Tabl e 1 Cel l Densitie s an d Percentag e Inhibitio n o f Growt h fro m th e Range-findin g Tes t Cel l Densities * (cell s pe

r mL ) Inhibitio n Value s (% ) Nomina l Concentratio n (m g ai/L ) 0 Hour s 9 6 Hour s Growt h Rat e YielcVBiomas s

Integra l Contro l R i

R 2 Mea n 1.14E+0

4 1.16E+0 4 1.I5E+04 2.22E+0 6

2.25E+0 6 2.23E+0 6 - - 1. 0 R i 1.07E+0 4 2.27E+0 6 R 2 1.04E+0

4 2.04E+0 6 0 3 Mea n 1.05E+0

4 2.16E+0 6 1 0 R i 1.03E+0 4 2.09E+0 6 R 2 1.00E+0

4 1.84E+06 0 1 2 Mea n 1.02E+0

4 1.97E+06 10 0 R i 1.04E+0 4 1.67E+06 R 2 1.07E+0

4 1.81E+06 4 2 2 Mea n 1.06E+0

4 1.74E+06 100 0 R i 1.06E+0 4 1.59E+0

5 R 2 1.26E+0

4 1.78E+0

5 4 9 9 3 Mea n 1.16E+0

4 1.68E+0

5

* Cel l densitie s represen

t th e mea n numbe r o f cell s pe r m L calculate

d fromth e mea n o f th e cell count

s fro m3 count

s fo r eac h o f th e replicat e flasks . K i an d R 2 = Replicate

s 1 an d 2p . 2 4 Harla n Stud y Number : 41104696 Repor t Pag e 2 3
Tabl e 2 Cel l Densitie s i n th e Definitiv e Tes t Nomina l Concentratio n Cel l Densities * (cell s pe r mL ) (m g ai/L ) O h 2 4 h 4 8 h 7 2 h 9 6 h Contro l R i 1.09E+0 4 5.43E+0

4 2.80E+0 5 1.41E+0 6 2.44E+0 6 R z 1.00E+0

4 5.09E+0

4 3.58E+0 5 1.77E+0 6 2.45E+0 6 R 3 1.00E+0

4 6.18E+0

4 3.75E+05 1.85E+0 6 2.47E+0 6 R 4 1.09X+04 6.35E+0 4 3.59E+0 5 1.62E+0 6 2.55E+0 6 R 5 1.06E-KJ

4 6.10E+0

4 3.60E+0 5 1.50E-KJ 6 2.61E+0 6 R * 1.06E-KJ

4 6.16E+0

4 4.14E+0 5 1.90E-KJ 6 2.50E+0 6 Mea n 1.05E+0

4 5.88E+0

4 3.58E+0 5 1.68E+0 6 2.51E+0 6 1 0 R i 1.13E-H) 4 4.86E+0

4 3.13E+05 1.37E+0 6 2.74E+0 6 R 2 1.03E+0

4 6.35E+0

4 3.81E+05 1.79E+06 2.80E+0 6 R 3 1.31E+0

4 5.41E+0

4 2.89E+0 5 1.57E+0 6 2.57E+0 6 Mea n 1.16E+0

4 5.54E+0

4 3.28E+05 1.58E+06 2.70E+0 6 3 2 R ] 1.27E+0 4 5.80E+0

4 2.59E+0 5 1.43E+06 2.24E+0 6 R 2 1.03E-KJ

4 5.36E+0

4 3.14E+05 1.74E+06 2.19E+0 6 R 3 1.15E+0

4 5.28E+0

4 3.57E+05 1.50E+0 6 2.14E+0 6 Mea n 1.15E+04 5.4SE-K M 3.10E+0 5 1.55E+06 2.19E+0 6 10 0 R i 1.05E+0 4 4.87E+0

4 2.60E+0 5 1.24E+06 2.06E+0 6 R 2 1.1OE+0

4 4.37E-KJ

4 2.93E+0 5 1.16E+06 2.14E+0 6 R 3 1.15E+0

4 4.88E+0

4 3.00E+05 1.16E+06 1.70E+06 Mea n 1.10E+0

4 4.71E+0

4 2.84E+0 5 1.19E+06 1.97E+06 320 R i 1.15E+0 4 3.20E+0

4 5.82E+0

4 3.13E+0 5 9.14E+05 R 2 1.07E+0

4 3.59E+0

4 7.49E+0

4 2.90E+0 5 8.75E+05 R 3 1.04E+0

4 3.55E+0

4 7.26E+0

4 3.39E+0 5 1.01E+06 Mea n 1.09E+0

4 3.45E-KJ

4 6.86E+0

4 3.14E+0 5 9.31E+05 100 0 R i 1.07E+0 4 1.91E-K)

4 2.02E+0

4 4.49E+04 1.29E-KJ 5 R 2 1.09E+0

4 2.03E+0

4 2.11E+0

4 2.60E+04 8.90E+0 4 R 3 1.13E+0

4 2.21E+0

4 2.06E+0

4 4.34E+04 7.08E+0 4 Mea n 1.10E+0

4 2.05E+0

4 2.06E+0

4 3.81E+0

4 9.64E+0

4

* Cel l densities represen

t th e mea n numbe r o f cell s pe r m L calculate

d fromth e mea n o f th e cel l count s from3 count

s fo r eac h o f th e replicat e flasks . R ] - Rf i = Replicate

s 1 t o 6p . 2 5

Harla n Stud y Number : 4110469 6 Repor

t Pag e 2 4
Tabl e 3 Dail y Specifi c Growt h Rates fo r th

e Contro l Culture s i n th e Definitive Test Dail y Specifi c Growt

h Rat e (cells/mL/hour ) Da y 0 - 1 Da

y 1 - 2 Day2- 3 Da y 3 - 4

Contro l R i 0.07 0 0.06 8 0.067 0.023

Contro l R 2 0.06 8 0.08 1 0.067 0.01 3 Contro l R 3 0.07 6 0.07 5 0.06 6 0.01 2 Contro l R

4 0.07 7 0.07 2 0.06 3 0.01 9 R 3 0.07 5 0.07 4 0.06 0 0.023 Contro l
R

5 0.07 6 0.07 9 0.064 0.01 1 Contro l

Mea

n 0.07 4 0.07 5 0.06 5 0.01 7

R i - R « = Replicate

s 1 t o 6p . 2 6
Harla n Stud y Number: 41104696 Repor t Pag e 2 5

Tabl e 4 Inhibitio n o f Growt h Rate , Yiel d an d Biomas s Integra l i n th e Definitiv e Tes t ( 0 - 7 2 Hour ) Growt h Rat e Yiel d Biomas s Integra l Nomina l Concentratio n (cells/mL/hour) (cells/mL ) (cells/mL/hour) (m g ai/L ) 0-72 h % Inhibitio n 0-72 h % Inhibition * 0-7 2 h % Inhibitio n Contro l R i

R 2 R J R * R 5 R e Mea n S D 0.06 9

0.07 2 0.07 2 0.07 1 0.07 0 0.07 3 0.07 1 0.00 1 - 1.40E+06 1.76E+06 1.84E+06 1.61E+06 1.49E+06 1.89E+06 1.67E+06 1.97E+0 5 - 2.44E+07

3.05E+07 3.21E+07 2.90E+07 2.75E+07 3.37E+07 2.95E+07 3.33E+0 6 -

1 0 R i 0.06 8 4 1.36E+06 2.45E+07 1 7

R 2 0.07 2 [1 ] 1.78E+06 3.16E+0 7 

# m R 3 0.07 0 1 1.56E+06 2.65E+07 1 0 Mea n 0.07 0 1 1.57E+06 6 2.75E+07 7 S D 0.00 2 2.12E+0 5 3.65E+0 6 3 2 R i 0.06 9 3 1.41E+06 2.41E+0 7 1 8

R 2 0.07 2 [1 ] 1.73E+06 2.91E+07 2 R 3 0.07 0 1 1.49E+06 2.72E+07 8 Mea n 0.07 0 1 1.54E+06 7 2.68E+07 9 S D 0.00 2 1.64E+0 5 2.49E+0 6 10 0 R i 0.067 6 1.23E+06 2.17E+07 2 6

R 2 0.06 6 7 1.15E+06 2.14E+07 2 7 R 3 0.06 6 7 1.15E+06 2.17E-HJ 7 2 6 Mea n 0.06 6 7 1.18E+06 29 2.16E+07 2 6 S D 0.00 1 4.79E+0 4 1.77E+0 5 320 R i 0.04 8 3 2 3.01E+05 5.32E+0 6 8 2

R 2 0.04 7 3 4 2.80E+0 5 5.54E+0 6 8 1 R 3 0.04 9 3 1 3.28E+05 6.06E+0 6 7 9 Mea n 0.04 8 3 2 3.03E+05 8 2 5.64E+0 6 8 1 S D 0.00 1 2.43E+0 4 3.81E+0 5 100 0 R . 0.02 1 7 0 3.42E+0 4 8.82E+05 9 7 R 2 0.01 3 8 2 1.51E+0 4 7.06E+0 5 9 8 R 3 0.020 7 2 3.20E+0 4 9.43E+0 5 9 7 Mea n 0.01 8 7 5 2.71E+0 4 9 8 8.44E+05 9 7 S D 0.00

4 1.04E+0 4 I.23E+0 5

* I n accordanc

e wit h th e OEC D tes t guidelin e onl y th e mea n valu e fo r yiel d fo r eac h tes t concentratio

n i s calculate

d R i - R e - Replicate

s 1 t o 6 S D = Standar

d Deviatio

n [Increas e in growt

h a s compare

d t o controls ]p . 2 7

Harla n Stud y Number : 41104696 Repor t Pag e 2 6

Tabl e 5 Inhibitio n o f Growt h Rate , Yiel d an d Biomas s Integra l i n th e Definitiv e Tes t ( 0 - 9 6 Hour ) Growt h Rat e Yiel d Biomas s Integra l Nomina l Concentratio n (cells/mL/hour) (cells/mL ) (cells/mL/hour) (m g ai/L ) 0-9 6 h % Inhibitio n 0-9 6 h % Inhibition * 0-96 h % Inhibitio n Contro l R i

R 2 R 3 R 4 R 5 R $ Mea n S D 0.05 7

0.05 7 0.05 7 0.05 8 0.05 8 0.05 8 0.05 8 0.00 1 - 2.43E+0 6 2.44E+0 6 2.46E+0 6 2.54E+0 6 2.60E+0 6 2.49E+0 6 2.49E+0 6 6.65E+0 4 - 7.04E+07

8.09E+0 7 8.36E-KJ 7 7.87E+07 7.67E+07 8.63E+0 7 7.95E+07 5.61E+0 6 -

1 0 R i 0.05 8 0 2.73E+0 6 7.36E+07 7

R 2 0.05 9 [2 ] 2.79E+0 6 8.65E+0 7 [9 ] R 3 0.05 8 0 2.55E+0 6 7.59E+0 7 4 Mea n 0.05 8 [1 ] 2.69E+0 6 [8 ] 7.87E+07 1 S D 0.00 1 1.26E+0 5 6.89E+0 6 3 2 R i 0.05 6 3 2.23E+0 6 6.79E+07 1 5

R 2 0.05 6 3 2.18E+0 6 7.59E+07 4 R 3 0.05 6 3 2.13E+0 6 7.06E+07 1 1 Mea n 0.05 6 3 2.18E-+0 6 1 3 7.15E+07 1 0 S D 0.000 5.13E+0 4 4.09E+0 6 10 0 R i 0.05 6 3 2.05E+0 6 6.12E+0 7 23 R 2 0.05 6 3 2.13E-KJ 6 6.08E+0 7 2

4 R 3 0.05 4 7 1.69E+06 5.58E+0 7 3 0 Mea n 0.05 5 4 1.96E+06 2 2 5.93E+0 7 2 6 S D 0.00 1 2.34E+0 5 2.97E+0 6 32 0 R i 0.047 1 9 9.02E+05 1.98E+0 7 7 5 R 2 0.047 1 9 8.64E+05 1.93E+0 7 7 6 R 3 0.04 8 1 7 9.95E+05 2.20E+07 7 2 Mean 0.047 1 8 9.20E+05 63 2.03E+07 7 4 S D 0.00 1 6.75E+0 4 1.41E+06 100 0 R i 0.02 7 5 3 1.19E+0 5 2.73E+0 6 9 7 R 2 0.02 3 6 0 7.81E+0

4 1.85E+06 9 8 R 3 0.02 0 6 6 5.95E+0 4 2.07E+0 6 9 7 Mea n 0.02 3 60 8.54E+0 4 9 7 2.22E+0 6 9 7 S D 0.00

4 3.02E+0 4 4.60E+0 5

* In accordanc

e wit h th e OEC D tes t guidelin e onl y th e mea n valu e fo r yiel d fo r eac h tes t concentratio

n i s calculate

d Ri-R e = Replicate

s 1 t o 6 S D = Standar

d Deviatio

n [Increas e i n growt

h a s compare

d t o controls ]p . 2 8

Harla n Stud y Number : 4110469 6 Repor

t Pag e 2 7
Tabl e 6 p H values i n th e Definitive Tes t
Nomina l Concentratio n (m g ai/L ) p H Nomina l Concentratio n (m g ai/L ) 0 Hours 4 8 Hour s 9 6 Hours Nomina l Concentratio n (m g ai/L ) Pre-adjustmen t Post-adjustmen

t 4 8 Hour s 9 6 Hours Contro l 7. 7 7. 0 8. 1 8. 2 1 0 7. 2 7. 0 8. 1 8. 2 3 2 7. 4 7. 0 8. 0 8. 1 10 0 8. 7 7. 0 7. 9 8. 1 32 0 9. 3 7. 0 7. 8 8. 0 100 0 9. 6 7. 0 7. 7 8. 0p . 2 9 Harlan Study Number: 41104696 Repor t Pag e 2 8 

## FIGURE S Figur e 1 Mea

n Cel l Densitie

s v Tim e fo r th

e Definitive Tes t

I.O0E-+O7 1

1.00E+06- "

o 1.00E-H35

-

1.00E+0 4

1.00E-H33 4 8 Tim e (Hours )p . 3 0
Harla n Stud y Number: 41104696 Repor t Pag e 2 9 Figur e 2 Inhibitio n o f Growt h Rat e ( 0 - 72 hours ) 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0

-1 0 Growt

h Rat e i ! i 

## : L ; • i i/'. 

### i 

## ' \i i | 

# i j 

## I! i i i i | 

## 1 | i

I ! | ! ;



### • i i i i i . t .. . i 1 1 0 10 0 Concentratio n (m g ai/L ) 100 0p . 3 1

Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 0Harla n Stud y Number: 41104696 Repor t Pag e 3 1p . 3 3
Harla n Stud y Number: 41104696 Repor t Pag e 3 2 Figur e 5 Inhibitio n o f Growt h Rat e ( 0 - 9 6 hours ) 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0

-1 0 Growt

h Rat e i i

I1 ! ! i ; 

# | I [ j 

### + i i i i

- — 1 1 0 10 0 Concentratio n (m g ai/L ) 100 0Harla n Stud y Number: 41104696 Repor t Pag e 3 3p . 3 5
Harla n Stud y Number: 41104696 Repor t Pag e 3 4 Figur e 7 Inhibitio n o f Biomas s Integra l ( 0 - 9 6 hours ) 10 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 Biomass Integra

l j j

] | i j j i 

## j! / i i / v.xX/.. 

# Xk + \ \

i j
I— i i i i 1 0 10 0 Concentratio

n (m g ai/L ) 100 0p . 3 6

Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 5 Figur e 8 Temperature Measurement s Take n Throughou

tth e Tes tPerio d
26. 0

Tim e (Hours )p . 3 7

Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 6 

## APPENDICE S Appendi x 1 Positive Contro l A positiv e contro l (Harla n Stud y Number : 41200591 ) use d zin c chloride a

s th e referenc e ite ma

t concentration s o f

0.010 , 0.032 , 0.10 , 0.3 2 an d 1. 0 mg/L . Exposur e condition s an

d dat aevaluatio n fo r

th e positiv e contro l wer

e simila r to those i n th

e definitiv e test . Exposur e o f Pseudokirchneriell asubcapitata(CCA P 278/4 ) to th

e referenc e ite mgav

e th e followin g results :

Time Point (Hours ) Respons e

Variabl e EC 5 0(mg/L ) 95 % Confidenc e Limit s (mg/L ) N o Observed Effec t Concentratio n (NOEC ) (mg/L ) Lowes t

Observe d Effec t Concentratio n (LOEC ) (mg/L )

7 2 Growth Rat

e 0.3 1 0.2 8 - 0.3 5 0.1 0 0.3 2 7 2 Yiel d 0.2 6 * 0.1 0 0.3 2 7 2 Biomas s 0.27 * 0.1 0 0.3 2
9 6 Growth Rat

e 0.3 5 0.3 0 - 0.40 0.1 0 0.3 2 9 6 Yiel d 0.27 * 0.1 0 0.3 2 9 6 Biomas s 0.2 6 0.1 0 0.3 2 Th e results fro mth

e positiv e contro l wit h zinc chloride wer e withi n th

e norma l ranges fo r

thi s referenc e item .

* I t wa s no t possibl e t o calculat e 95 % confidenc

e limit s fo r thes e EC 5

0 value s a s th e dat agenerate d di dno t fit th e model s availabl e fo r th e calculatio n o f confidenc e limits .p . 3 8 Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 7 Appendi x 2 Cultur e Mediu

m NaN0 3 25. 5 mg/ L MgCl 2 .6H 2 0 12.16 4 mg/

L CaCl 2 .2H 2 0 4.4 1 mg/ L MgS0 4 .7H 2 0 14. 7 mg/ L K 2 HP0 4 1.04 4 mg/

L NaHC0 3 15. 0 mg/ L H3BO 3 0.185 5 mg/ L MnCl 2 .4H 2 0 0.41 5 mg/ L ZnCl 2 0.0032 7 mg/

L FeCl 3 .6H 2 0 0.15 9 mg/ L CoCl 2 .6H 2 0 0.00143 mg/ L Na 2 Mo0 4 -2H 2 0 0.0072 6 mg/ L CuCl 2 .2H 2 0 0.00001 2 mg/ L Na 2 EDTA.2H 2 0 0.3 0 mg/ L Th e cultur e mediumwa

s prepare d using revers e osmosi s purifie d deionize d water * an d th e p H adjuste d to 7. 5 ± 0. 1 wit h O.lNNaO H o r HCL

* Elg aOptim a15 + o r Elg aPurela b Optio n R-1 5 B Pp . 3 9

Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 8 Appendi x 3 Analytica l Investigation s 

## Analytica l Investigation

s
CONTENT S CONTENT S 3 8 1 MATERIAL S AN D METHOD S 3 9 1. 1 Introductio n 3 9 1. 2 Test

Ite m3 9 1. 3 Analytica l Standar d 3 9 1. 4 Analytica l Procedur e 3 9 1.4. 1 Storag

e 3 9 1.4. 2 Reagent

s an d Solvent s 3 9 1.4. 3 Preparation o f

Calibratio n Standard

s 4 0 1.4.4 Preparation o f

Linearit y Standard

s 4 0 1.4. 5 Preparation o f

Spiked Recover

y Samples 4 0 1.4. 6 Preparation o f

Test Sample s 4 0 1.4.7 Instrumenta

l Parameter

s 4 1 1.4. 8 Calculation s 4 1 2 RESULT S 43 2. 1 Range-Finding Tes t 43 2. 2 Definitiv e Test

43 2.2. 1 Validatio n o f

th e Analytica l Metho d 43 2.2. 2 Tes t

Sample s 4 3 3 DISCUSSIO N 4 4 TABLE S 4 5 Table 1 Preparatio n o f Tes t Samples 4 5 Tabl e 2 Calibratio n Standar d Dat a4 5 Table 3 Result s fo r Range-Finding Sample

s 4 6 Table 4 Results fo r Spike d Recover

y Sample s 4 6 Table 5 Results fo r Tes t Samples 4 7 FIGURE S 4 8 Figur e 1 Linearity 4 8 Figur e 2 Chromatogra

mo f Calibratio n Solutio n (1. 5 m g ai/L ) 49 Figur e 3 Chromatogra

mo f Biologica l Contro l Sampl e 49 Figur e 4 Chromatogra

mo f Tes t Sampl e (1000 m

g ai/L ) 5 0p . 4 0

Harla n Stud y Number : 4110469 6 Repor

t Pag e 3 9 Analytica l Investigation

s 

## 1 MATERIAL S AN

D METHOD S 1. 1 Introductio n Th e tes t ite mconcentratio n i n th e test samples wa

s determined b y direc t

injectio n into th

e mass spectromete

r (HPLC-MS ) usin g a

n externa l standar d to calibrat

e th e system . Al l tes t sampl e concentration s wer

e corrected fo r

awate r conten t o f 50.5% , an

d ar e reported a

s milligram s o f

activ e ingredien

t (ai ) pe r litre . Th e metho d wa

s developed b y th

e Departmen

t o f Analytica l Services , Harla n Laboratorie s Ltd , Shardlow , UK . 1. 2 Tes tIte m Th e test ite mi s described i n th

e biological par

t o f thi s study . 1. 3 Analytica l Standar d Th e tes t ite mdescribed i n th

e biological par

t o f thi s stud y wa s als o use d a s th e analytica

l standard . 

## 1. 4 Analytica

l Procedur

e 1.4. 1 Storag e Th e sample s wer

e stored a t approximatel

y -2 0 ° C prio r

to analysis . 1.4. 2 Reagent

s an d Solvent

s Water : prepared usin g a n ELG A Pur

e La b Optio n R- l 5 wate

r purificatio n system . Test medium : a

s described i n th

e biological par

t o f thi s study . Solvents : methanol , Fishe r Scientific , LC-M S grade . Reagents : ammoni asolution , Fishe r Scientific , A Rgrade .p . 4 1

Harla n Stud y Number : 4110469 6 Repor

t Pag e 4 0 Analytica l Investigation

s 1.4. 3 Preparatio n o

f Calibratio n Standards Th e tes t ite m(nominal 10

0 mg ai ) wa

s dissolve d i n diluent * (100 mL ) to prepar e astoc k solutio n wit h aconcentratio n o f

100 0 m g ai/L . Thi s stoc

k solutio n wa

s furthe r dilute d wit h diluent * to produc e asolutio n o f

10 0 mg ai/L . Define d volume s o f thi s solutio n wer

e then dilute d wit h diluent * t o obtai n calibratio n standard s i n th e rang e o f 0.1 0 to 1.2

5 mg ai/L . A secon

d serie s o f calibratio n standards wa

s similarl y prepare

d i n th e rang e o f 0.2 5 to 1. 5 mg ai/L . Thes

e solution s wer e use d to determin

e th e recover y an d tes t sampl e concentrations . 1.4. 4 Preparatio n o

f Linearity Standards Th e tes t ite m(nominal 10

0 mg ai ) wa

s dissolved i n diluent * (100 mL ) to prepar e astoc k solutio n wit h aconcentratio n o f

100 0 m g ai/L . Thi s stoc

k solutio n wa

s furthe r dilute d wit h diluent * to produc e asolutio n o f

10 0 mg ai/L . Define d volume s o f thi s solutio n wer

e then dilute d wit h diluent " to obtai n calibratio n standard s i n th e rang e o f 0.1 0 to 1.2

5 mg ai/L . A secon

d series o f calibratio n standards wa

s similarl y prepare

d i n th e rang e o f 0.2 5 to 1. 5 m g ai/L . Thes

e standard

s wer e use d to evaluat

e th e linearit y o f th e analytica

l system . 1.4. 5 Preparatio n o

f Spike d Recover

y Sample s T o demonstrat

e th e validit y o f

th e analytica

l procedure , volume s o f

test mediumwer

e spiked wit h th e tes t ite man d it s recover

y wa s assessed. Th

e test ite m(nomina

l 10 0 m g ai ) wa s initiall y dissolve d i n diluent * t o prepar e astoc k solutio n wit h aconcentration o f 100 0 m g ai/L . Thi s stoc

k solutio n wa

s furthe r dilute d wit h diluent * to produc e asolutio n o f 1 0 mg ai/L . Define d volume s o f thi s stoc

k solutio n wer

e dilute d wit h test

mediu mto obtai n spike d recover y sample s a t concentration s o f

1. 0 mg ai/L . Fiv e replicate s a t eac h concentratio n leve l wer e prepare d an d subjected t

o th e sam e treatmen

t a s th e test samples . I n addition , test mediumwithou t

th e additio n o f th e tes t ite m(syntheti c control ) wa s als o analyzed . 1.4. 6 Preparatio n o

f Tes tSample

s Th e tes t sample s wer

e thawe d wit h th

e ai d o f sonication . The

y wer e furthe r dilute d into th

e calibratio n range wit h solvent/mixtur e a s show n i n Table 1 .

* Diluent:

- methanol:water:testmedia:ammoni a(50:40:10:0. 1 v/v/v/v) .p . 4 2

Harla n Stud y Number : 4110469 6 Analytica l Investigation

s Repor t Pag e 4 1

1.4. 7 Instrumental Parameter s Autosampler : Pump : Detector : Column : Mobil e phase : Agilen t 110 0 Agilen t 110 0 Agilen t 110 0 Serie s MS D none , direc t

injectio n methanol:water:ammoni asolutio n (500:500:1 , v/v/v ) Flo w rate : Injectio n volume : 0. 5 mL/minut e 1 0 u L Ionizatio n mode : Polarity : Capillar y voltage : Ga s temperature : Nebulize r pressure : Fragmento

r voltage : Dryin g ga s flow : Dat aacquisitio n mode : SI M ions : ES I negativ e 400 0 V 35 0 ° C 4 0 ps i 10 0 V 1 2 L/mi n Single Io n Monitorin g (SIM ) 22 5 m/ z 1.4. 8 Calculation s A calibratio n plo t

was prepare

d using alinea

r plo t o f th e for my = m

x + a. Th e concentratio n "x " o f

th e test ite mi n a

n injecte d sampl

e wa s calculate d b y th

e followin g equation :

x = m wher e x : y : m : a : (1 )

concentration o f

th e test ite mi n th e injecte d sampl

e [m g ai/L ] mea n pea k are aresponse o f

th e test ite mi n th

e injecte d sampl

e [counts ] slop e o f th e calibratio n plo t

[counts pe

r m g ai/L ] y-axi s intercept

[counts ]p . 4 3

Harla n Stud y Number : 4110469 6 Repor

t Pag e 4 2 Analytica l Investigation

s Th e concentratio n o f

th e test itemi n th e test sample s wa s calculate d usin g the followin g equation :

c 

## =dr^* F (2 ) R/10 0 wher e c x F R concentration o f th e tes t ite mi n th

e tes t sampl e [m g ai/L ] concentratio

n o f th e tes t itemi n th e injecte d sampl

e solutio n [m

g ai/L ] sampl e preparation facto r recover y rat e [% ] (I f no t correctin g fo r

recover y then R i s take

n a s 100 ) Th e recover y rat e (R ) o f th e spiked recover

y samples wa

s calculate d usin g th

e followin g equation : R= —-10

0 (3 )

C for t wher e R : recover

y rat e [% ] c : determine

d concentratio

n o f th e tes t itemi n th e spike d recover y sampl e [mg/L ] Cfort : fortifie dconcentration o f th e test ite mi n th

e spike d recover

y sampl e [m g ai/L ] Th e percentage o f

nominal concentration o f th e test ite mi n th

e tes t sample s wa

s calculated using th e followin g equation : % nomina l = • 10

0 (4 ) wher e Cnom : nomina

l concentration i n th

e test sampl e [m g ai/L ]p . 4 4

Harla n Stud y Number : 4110469 6 Repor

t Pag e 4 3 Analytica l Investigation

s 2 RESULT S 2. 1 Range-Findin g Tes t Th e analytica

l methodolog y wa

s no t validate d prio r

t o th e analysi s o f

range-findin g samples ; ful l validatio n wa

s late r conducted specificall y ove r th e tes t concentratio n range to b

e employed i n th

e definitiv e test . The result s fo r th e range-findin g test

sample s ar

e summarize

d i n Table 3 . 2. 2 Definitiv e Tes t 2.2. 1 Validatio n o

f th e Analytical Metho

d Specificit y Th e biologica l contro l sample s an d analytica

l blan k showe d asignifican t

interferin g respons

e a t th e mas s specifi c to the tes t item ; howeve

r thi s wa s take n int o consideratio n whe n calculatin g th

e standards , sample s an

d LOQ . Th

e standar d respons

e change d accordingly wit h know n concentration , hence th

e specificit

y wa s confirmed . Linearity Th e calibratio n dat

afo r th e calibration standard

s o f th e tes t itemi s given i n Tabl

e 2 an d Figur e 1 , Th e dat awa s foun d to hav

e alinea r correlatio n withi n th

e calibratio n rang

e o f 0 to 1. 5 m g ai/L . Th

e R 2 fi t o f th e calibratio n curv e t o th

e dat awa s 0.9993 , an

d wa s considered to b

e acceptable . Accurac y (Recovery ) and Precisio n A se t o f recover y sample s accuratel

y fortifie da

t arelevan t concentratio n o f

tes t ite mwa s prepare d five-fol dan

d analyzed . Th

e results obtained fo r

th e concentration o f

the tes t ite mi n th

e recover y sample s ar

e presente

d i n Tabl e 4 . Th e metho d wa

s considered to b

e sufficientl y accurat

e an d precis e fo r

th e purpose s o f

thi s test . Th e tes t sampl e result s wer

e no t corrected fo r

recovery . Limi to f Quantitatio n (LOQ ) Th e limi t o f quantitatio n (LOQ ) wa

s determined b y calculatin g thre e time s th

e concentration o f th e response see

n i n th e blank . Usin g thi s method th e limi t o f quantitatio n wa

s determined t

o b e 2. 3 m g ai/L . 2.2. 2 Tes tSample

s Th e result s obtaine d fo r

the concentration o f

th e test itemfoun d i n th

e tes t samples ar

e presente d i n Tabl e 5 . Typical chromatogram s ar e show n i n Figure s 2 to 4 .p . 4 5

Harla n Stud y Number : 4110469 6 Repor

t Analytica l Investigation

s 

## 3 DISCUSSIO N Pag e 4 4

Th e detectio n syste

mwa s foun d to hav

e acceptabl

e linearity . Th

e analytica

l procedur

e wa s foun d to hav e acceptabl

e recoverie s o f

tes t ite mi n test

medium . Th

e metho d o f

analysi s wa

s validate d an d proven t o be suitabl e fo r use .

/p . 4 6

Harla n Stud y Number : 4110469 6 Repor

t Pag e 4 5 Analytica l Investigation

s 

## TABLE S Tabl e 1 Preparatio n o f Tes tSample

s Nomina l Concentratio n o

f Tes tItem [m g ai/L ] Sampl e Volum e [mL ] Fina l Volum

e [mL ] Sample

Preparatio n Facto r F Fina l Nomina l Concentration o

f Tes t Ite m [m g ai/L ] Contro l 5 5 0 1 0

- 1 0 5 5 0 1 0 1. 0 3 2 2

5 100 , the n 1 - > 1 0 4 0 0.8 0 10 0 5 50 , then l - > 1 0 10

0 1.

0 32 0 5 200 the n 1 - > 1 0 400 0.80 1000 1 100 , the n 1 - » 1 0 100

0 1.

0

no t applicabl e Tabl e 2 Calibratio n Standard Dat a Concentration o

f Tes tIte m Are a [m g ai/L ] [counts ] 0 3.33 5 x 10 4 0.10 4 6.81 2 xlO 4 0.25 7 1.14 0 x10 s 0.52 2 1.76 5 x 10 5 0.77 0 2.38 5 x 10 5 1.0 4 3.17 7 x 10 5 1.0 3 3.09 7 x 10 5 1,3 1 3.81 4 x 10 5 1.5 4 4.48 1 x 10 5p . 4 7

Harla n Stud y Number: 41104696 Repor t Pag e 4 6 Analytica l Investigations 

### Tabl e 3 Result

s fo r Range-Findin

g Sample s

Tim e poin

t Nomina

l Concentratio n o f Tes tIte m i n Range-Findin g Sampl e Measure d Concentratio n of Tes tIte m i n Sample Via l Sample

Preparatio n Facto r Determined

Concentratio n of Tes tIte m i n Range-Findin g Sampl e % o

f Nomina l Concentratio n

*-no m X F c [hours ] [m

g ai/L ] [m

g ai/L ] [m g ai/L ] [%

] 0 1 0 0.86 8 1 0 8.6 8 8 7 10 0 0.90 1 10 0 90. 1 9 0 100 0 0.99 8 100 0 99 8 10 0 9 6 1 0 1.2 9 1 0 12. 9 12 9 10 0 0.98 1 10 0 98. 1 9 8 100 0 1.0

2 100

0 101 6 10 2


### Tabl e 4 Result

s fo r Spike

d Recover y Sample s Nomina l Fortifie d Measured Sample Determined Analytica l Concentratio n Concentratio n Concentratio n Preparatio n Concentratio n Recover y Precisio n of o f

Tes tIte m i n of

Tes tIte m i n Facto r of

Tes tIte m i n Rate (Relative Tes tIte m th

e Spike d th

e Sample th e Spike d Standard Sampl e Via l Sampl e Deviation o f Recovery )

c no m Cfor l X F c R [m g ai/L ] [m

g ai/L ] [m

g ai/L ] [m g ai/L ] [%

] [%

] 100 0 101 1 0.99 1 100 0 99 1 0.89 0 100

0 89 0 1.0 2 100

0 101 8 9 8 6. 0 1.0 2 

## woo 102 1 1.0 4 100

0 103 7 Acceptanc e Target 80-120 

### <i o Fo r reportin g purposes

, x ha s bee n calculate

d fro mc retrospectively.p . 4 8
Harla n Stud y Number : 41104696 Repor t Pag e 4 7 Analytica l Investigations Tabl e 5 Result s fo r Tes tSample s

Tim e poin

t

[hours ] Nomina l Concentratio n of Tes tIte m i n Tes tSampl e [m g ai/L ] Measured

Concentratio n o f Tes tIte m i n Sample Via l

X [m g ai/L ] Sample

Preparatio n Facto r

F Determined

Concentratio n o f Tes tIte m i n Tes tSampl e

c [m g ai/L ] % o

f Nomina l Concentration
[% ] 0 Contro l <LO Q 1 0 <LO Q
-

1 0 0.93 9 1 0 9.3 9 9 4 3 2 0.78 0 4 0 31. 2 9 7 10 0 0.97 5 10 0 97. 5 9 8 320 0.81 8 400 327 10 2 100 0 1.0

2 100

0 1023 10 2 9 6 Contro l

1 0 3 2 10 0 320 100 0 <LO Q

0.92 9 0.80 3 1.0 1 0.83 5 1.0 4 1 0

1 0 4 0 10 0 400 100 0 <LO Q

9.2 9 32. 1 10 1 33 4 103 5 9 3

10 0 10 1 10 5 10 4 LO Q = Limi t o f Quantitatio

n no t applicabl e Fo r reportin g purposes

, x ha s bee n calculate d fro mc retrospectively

.p . 4 9
Harla n Stud y Number ; 41104696 Analytica l Investigations Repor t Pag e 4 8 

## FIGURE S Figur e 1 Linearit y

60000 0 • 45000 0 - 40000 0 - 35000 0 - 30000 0 • 25000 0 - 20000 0 • 15000 0 - 10000 0 • 5000 0 - 0 0. 0 y = 264456x+ 3887 7 R* = 0.999 3

0. 5 1.0 1, 5 2.

0 Concentfation{mgai/L

)p . 5 0 Harla n Stud y Number : 4110469 6 Analytica l Investigation

s Repor t Pag e 4 9
Figur e 2 Chromatogra m o f Calibratio n Solutio

n (1. 5 m g ai/L )

MSD 1 TIC , M S Flt a (S:\ANASERVSW1104696\ECAL146\ECAL146\024-2401.D } API-ES , SIM
. Frafl ' 10 0

ft

0 2 0. 4 0. 6 0. 8 1 1 2 1 4 1. 6 1. 8 ml n Figur e 3 Chromatogra m o f Biologica

l Contro l Sampl

e

MSDI TIC, IMS Rte(S.lANASERVSWf13488ffieCAL1'ieteCAL!JSl008-3801.Oi API-ES, SIM, Frag-

100

0 2 0 4 0. 6 0 5 1 1.2 1. 4 1. 6 1 8 ml n

Respons e see n i n tes t medi acontro l subtracte d fromcontrol

s an d tes t sample sp . 5 1

Harla n Stud y Number : 4110469 6 Analytica l Investigation

s Repor t Pag e 5 0
Figur e 4 Chromatogra m o f Tes tSampl

e (100 0 m g ai/L ) MSD 1 TIC , M S Fll s (S:\ANASERVSVM1C4696\ECAL146\ECAL146\018-1801.D ) API-ES , SIM , Frag : 10 0

- | r— . , 1 - n—•— 1 —•—r 0. 2 0 4 0 6 0 8 1 1. 2 1. 4 1 6 1. 8 ml nHarla n Stud y Number : 4110469 6 Repor

t p . 5 2 Pag e 5 1 Appendi x 3 Monitorin g Authorit y Statemen

to f GL P Compliance 

### (DH1 Department V_ 'ofHealth TH E DEPARTMEN T O F HEALT H O F TH E GOVERNMEN T

O F TH E UNITE D KINGDO M GOO D LABORATOR Y PRACTIC E STATEMEN T O F COMPLIANC E I N ACCORDANC E WIT H DIRECTIV E 2004/9/E C TES T FACILIT Y TES T TYPE(S ) Harla n Laboratorie s Lt d Analytlcaf/Cllnfca l Shardlo w Busines s Par k * rhaml „.„ , Londo n Roa d Chemistr y Shardlo w Environmenta l Toxicit y Derb y Environmenta l Fat e DE72 2G D Mutagenicit y Phys/Chem
. Test s Toxicolog y

DAT E O F INSPECTIO N 1 0 Jul y 201 2 A n inspectio n fo rcomplianc e wit h th e Principle s o f Goo d Laboratory Practic e wa s carrie d ou t a t th e abov e tes t facilit y a s par t o f th e U KGoo d Laborator y Practic e Complianc e Monitorin g Programme
. Thi s statemen t confirm s that , o n th e dat e o f issue , th e U KGoo d Laboratory Practic e Monitorin g Authorit y wer e satisfie d tha t th e abov e tes t facilit y wa s operatin g i n complianc e wit h th e OEC D Principle s o f Goo d Laboratory Practice
. Thi s statemen t constitute s a Goo d Laborator y Practic e Instrumen t (a s define d i n th e U KGoo d Laboratory Practic e Regulation s 1999)
.

Dr
. Andre w J
. Gray ^^^^^^^ ^ Head , U KGL P Monitorin g Authorit yORIGI NID : KCK

AC9I3 > 621-773 3 SHI P DATE : 23APR1 3 JAK E BDORE H ACTWGT : 0. 7 L B (IA N HARCRO S CHEMICAL

S IN C CAD : 0978415/CAFE260 8 520 0 SPEAKE

R R D KANSA S CITY . K

S SS10 G BIL L THIR D PART Y UNITE D STATE S U S TO 

## (DCO)(7407M)(OPPT

) U S EP A TSC A SECTIO N 4 120 0 PENNSYLVANI

A AV E N W WASHINGTO

N D C 2046 0 (202)664-693 0 REF : POSTAG

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