### Harcro s Chemical s Inc
.

520 0 Speake rRoa

d Kansa s City , K S 6610 6 

# cpp

f CBT C Telephone : 913/621-774

7 Fax : 913/621-774

6 E-mail : OrganicsCS@Harcros.co m 

# 20I 3 ;.rr: 2 6 6 A KI0 = 

## 2 1 Dat e 04/24/1 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 : Acut e Toxicit y t o Rainbo w Trou t 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 Acut e Toxicit y t o Rainbo w Trou t 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 Acut e Toxicit y t o Rainbo w Trou t 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 799.50870)
. I f an y furthe rinformatio n i s required , pleas e d o no t hesitat e t o contact m e a t (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 : Howar

d Stevenso n Jame s Grantha m Regards ,

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

# II

I II

I

CONTAINS N

O CB !

## REPOR T 

# harlai r

Sodiu m Glucoheptonate : Acute Toxicity To Rainbow Trout (Oncorhynchus mykiss)
Dat aRequirements/Tes

t Guidelines : • OEC D Guideline

s for Testin g o f Chemical s (1992 ) N o 203 , "Fish, Acut

e Toxicit y Test" . • Metho dC. l o f Commissio

n Regulatio

n (EC) No . 440/2008 . • U S EP A Draf t Ecologica l Effects Tes

t Guidelines OPPT

S 850.107 5 "Fis h Acut e Toxicit y Test , Freshwate

r an dMarine" . Stud y Director : S Harri s Tes t Facility : Harla n Laboratorie s Ltd . Shardlo w Busines s Par

k Shardlo w Derbyshir e DE7 2 2G D U K Sponsor : Harcro s Chemical s Inc . 3452 Corporat

e Driv e Dalto n G A 3072 0 UNITE D STATE S O F AMERIC

A

Harlan Stud

y Number : Stud y Completion Date : 4110469 4 0 8 Apri l 201 3

Pag e 1 of3 7p . 3

Harla n Stud y Number : 4110469 4 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 SIGNATUR

E OF CONTRIBUTIN G SCIENTIST S 6 SUMMAR Y 7 Introductio n 7 Method s 7 Results 7 GENERAL INFORMATIO N 8 Schedul e 8 Anima l Welfare 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 2. 1 Tes t

Ite m9 3 MATERIAL S AN D METHOD S 1 0 3. 1 Definition s an

d Abbreviation s 1 0 3. 2 Tes t

Species 1 0 3. 3 Tes t

Wate r 1 0 3. 4 Procedur

e 1 1 3.4. 1 Range-findin g Test s 1 1 3.4. 2 Definitiv e Test

1 2 4 RESULT S 1 4 4. 1 Range-findin g Test s 14 4. 2 Definitiv e Tes t

1 4 4.2. 1 Mortalit y Dat

a1 4 4.2. 2 Sub-Letha

l Effect s 1 5 4.2. 3 Observation s o n Test ItemSolubility 1 5 4.2. 4 Physico-Chemica l Measurement

s 1 5 4.2. 5 Verificatio n o f

Test Concentration s 1 5 5 CONCLUSIO N 1 6 6 REFERENCES 1 6 TABLE S 1 7 Table 1 Cumulativ e Mortality Dat ai n th e Range-findin g Tests 1 7 Table 2 Sub-letha

l Effect s o f

Exposur e i n th e Range-findin g Tests 1 8 Table 3 Cumulativ e Mortality Dat ai n th e Definitiv e Test

1 9 FIGURE S : 2 0 Figur e 1 Temperatur

e Measurement

s Recorde d Ove r th e 96-Hou r Tes t Perio d Usin g a Test o Temperatur

e Logge r 2 0 APPENDICE S 2 1 Appendix 1 Typical Wate r Qualit y Characteristic s 2 1 Appendi x 2 Analytica l Investigation s 2 3p . 4

Harla n Stud y Number : 4110469 4 Repor t Pag e 3

Appendix 3 Physico-Chemica l Measurement

s 3 6 Appendi x 4 Monitorin g Authority Statemen t o f GL P Complianc e 3 7p . 5

Harla n Stud y Number : 4110469 4 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 describedwas performe di n complianc e wit h U K GL P standards (Schedul

e 1 , Goo d Laborator y Practic e Regulation s 199 9 (S I 1999/3106 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 d1997 , ENV/MC/CHEM(98)17) . Thes e principle s ar

e compatibl e wit h Goo d Laborator

y Practic e regulation s specifie d b y regulator y authoritie s throughout

th e Europea n Community , th

e Unite dStates (EP

A an d FDA) , an dJapa n (MHLW , MAF F an

dMETI) .
Thi s repor

t full y an daccuratel

y reflect s th

e procedure s use

d an d dat agenerated . Ther

e wer e n o circumstance s consideredto hav e affecte d th

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

th e data . Stud y Director : S Harri s Harla n Stud y Number : Stud y Title : 4110469 4 Sodiu mGlucoheptonate : Acute Toxicity To Rainbow Trout {Oncorhynchus mykiss)



## Date : 0 8 AP

R 2013Harla n Stud y Number : 4110469 4 Repor t Pag e 5 

## QUALIT Y ASSURANC E STATEMEN T Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K Harla n Stud y Number : 4110469 4 Stud y Title : Sodiu mGlucoheptonate : Acute Toxicity To Rainbow Trout {Oncorhynchus mykiss) Th e genera l facilitie s an

d activitie s ar

e inspecte d a t least onc e ayea r an d th e result s ar

e reporte d to th e relevan t responsible perso

n an d management. Study-related procedure s conducte d a t th e tes t facilit y wer

e audited an

d inspected . Th

e detail s o f thes e audits an

d inspection s ar

e given below .
Date s an d Type s o f Q A Inspection

s Reporte d 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 Inspecte

d Repor

t Dat e 1 1 Octobe r 201 1 Genera

l Stud y Pla n Verificatio n NA 1 1 Octobe r 201 1 0 8 Januar y 201 3 Proces

s - base d Tes

t Ite mPreparatio

n 0 8 Januar y 201 3 10 Januar y 201 3 Proces

s - base d Tes

t Syste m Preparatio

n 1 0 Januar

y 201 3 0 7 Januar y 201 3 Proces

s - base d Exposur

e 0

7 Januar y 201 3 10 Januar y 201 3 Proces

s - base d Assessmen

t o f Respons e 10 Januar y 201 3 1 1 Januar y 201 3 Proces

s - base d Chemica

l Analysi s 1 1 Januar

y 201 3 0 2 Apri l 201 3 Repor t Audi t NA 0 2 Apri l 201 3 Th e complet e stud y plan (includin g stud

y specifi c supplement

) wa s verifie dfo r

GL P compliance a t repor t audit . Thi s statemen

t confirm s tha

t thi s repor

t reflect s th

e ra w dat aan d th e procedure s followed . Qualit y Assurance :

Date : ^ 

## 0 8 APR 20Kp . 7

Harla n Stud y Number : 4110469 4 Repor t Pag e 6


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



### Date : g AP

R 201 3p . 8



### Harla n Stud y Number : 4110469 4 Repor t Pag e 7


## SUMMAR

Y Introductio n A stud y wa s performe

d t o asses s th e acut e toxicit y o f th e tes t ite mt o rainbo w trou t {Oncorhynchu

s mykiss) . Th

e metho d followedwa

s designe d t o b e compatibl

e wit h th e OEC D Guideline s for Testin g o f Chemical s (1992 ) N o 203 , "Fish, Acut

e Toxicit y Test " reference d a s Metho dC. l o f Commissio

n Regulatio

n (EC ) No . 440/200

8 an d U S EP A Draf t Ecologica

l Effects Tes

t Guidelines OPPT

S 850.107 5 "Fis h Acut e Toxicit y Test , Freshwate

r an d Marine" . 

### Method s Followin g preliminar

y range-findin

g tests , fis h wer

e exposed , i n group s o f ten , t o a n aqueou s solutio n o f th e tes t ite move r arang e o f concentration

s o f 100 , 180 , 320 , 560, an

d 100 0 m g activ e ingredien

t (ai)/ L fo r aperio do f 9 6 hour s a t atemperatur

e o f 1 3 ° C t o 1 4 ° C unde r semi

stati c conditions . Th

e numbe r o f mortalitie s an

d an y sub-letha

l effects o f

exposur e i n eac h tes t an d contro l vesse l wer e determine

d 3 an d 6 hour s afte r th e star t o f exposur e an d the n dail y throughou t th e tes t unti l terminatio

n afte r 9 6 hours . Result s Th e 96-Hou r LC5 0 base d o n nomina l tes t concentration

s wa s greate r tha n 100 0 m g ai/L . Th e N o Observe d Effec t Concentratio

n wa s 100 0 m g ai/L . Analysi s o f

th e 100 0 m g ai/ L tes t preparatio n a t 0,24,48 , 7

2 an d 9 6 hour s showe d measure d tes t concentration

s t o rang e from91 % t

o 114 % o f nomina l an d s o th e result s ar e base d o n nomina l tes t concentration

s only . Th

e fresh tes t preparatio

n a t 4 8 hour s wa s measure d t o b e 184 % o f nominal , however

, a s th e correspondin

g old preparatio

n a t 7 2 hour s wa s nea r nomina l i t i s considere d tha t thi s resul t wa s anomalou

s an d ha s n o impac t o n th e outcom e o f th e test .

### p . 9

Harla n Stud y Number : 4110469 4 Repor t Pag e 8


## GENERA L INFORMATIO N Schedul e Experimenta

l Startin g Date : Experimenta

l Completio

n Date : 0

5 Marc h 201 2 0 8 Marc h 201 3 

## Anima l Welfar e Th e stud y wa s designe d an dconducte dt o caus e th e minimu msuffering o

r distres s t o th e animal s consisten t wit h th e scientifi c objectives an

di n accordanc

e wit h th e Harla n Laboratorie

s Ltd , Shardlow, U K

policy o n anima l welfar e an dth e requirement

s o f th e Unite dKingdom' s Animals (Scientific Procedures

) Act 1986 . Th e conduc t o f title stud y ma y b e retrospectivel

y reviewed

, a s par t o f th e Harla n Laboratories Ltd , Shardlow, U K Ethica l Revie w Process . Th e stud y wa s conducte

di n accordanc

e wit h th e UK Hom e Offic e Guidanc

e documen

t o n Regulator y Toxicolog

y an d Safet y Evaluatio

n Studie s an dth e OEC D guidanc e documen

t o n recognition , assessmen

t an d us e o f clinica l sign s a s human e endpoint s fo r experimenta

l animal s use d i n safet y evaluation . 

## Archivin g Unless instructe

d otherwise b

y th e Sponsor , al l origina

l dat aan dth e final repor

t wil l b e retaine d i n th e Harla n Laboratories Ltd , Shardlow, UK archive s fo r five year s afte r whic h instruction

s wil l b e sough t a s t o 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 t o th e Sponsor .

### p . 1 0

Harla n Stud y Number : 4110469 4 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 acute toxicity of the test item to rainbow trout

(Oncorhynchus mykiss). Rainbo w trou t i s afreshwate

r fish representativ

e o f awid e variet y o f natura l habitats , an

dca n therefor e b e considere

d 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 designe d t o b e compatibl

e wit h th e procedure

s indicate db y th e following internationall

y accepte d guidelines an

drecommendations :

• OEC D Guidelines fo r

Testin g o f Chemical s (1992 ) N o 20 3 "Fish , Acut

e Toxicit y Test" . • Metho dC. l o f Commissio

n Regulatio

n (EC ) No . 440/2008 . • U S EP A Draf t Ecologica l Effects Tes

t Guidelines OPPT

S 850.107 5 "Fis h Acut e Toxicit y Test , Freshwate

r an dMarine" . 

## 2 TES T ITE M 2.1 Tes

tIte m Th e integrit y o f th e supplie ddat arelatin g t o th e identity , purit

y an dstabilit y o f th e tes t ite mi s th e responsibilit

y o f th e Sponsor . Identification : Sodiu mGlucoheptonat

e Description : dar k brow n liqui d Batch : 92100010 0 Purity : 49.5 % w/ w Expir y / retes t date : 0 9 Februar y 201 5 Storag e conditions : roo mtemperatur

e i n th e dar k

### p . 1 1

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 0



## 3 MATERIAL S AN D METHOD S 3. 1 Definition s an d Abbreviation s LC50 : Th e calculate dconcentratio

n o f tes t ite mwhic h result s i n 50 % mortalit y withi n th e exposur e period . NOE C (N o Observe dEffec t Th e highes t tes t concentratio

n a t whic h n o significan

t toxi c Concentration) : effec t o n th e tes t organism s i s observe

dwithi n th

e exposur e period . Treatment: Comprise s tes t ite mtreatment

s an dcontrol (tes

t wate r only) . 3. 2 Tes tSpecie s Th e tes t wa s carrie dou t usin g juvenile rainbo

w trou t (Oncorhynchu

s mykiss) . Fis

h wer e obtaine dfromBrow Well Fisherie s Limited , Hebden

, nea r Skipton , Yorkshire , UK an d maintaine din-hous e sinc e 0 3 Decembe r 2012 . Fis

h wer e maintaine

di n aglass fibe

r tan k wit h a "singl e pass " wate

r renewa l system . Fis h wer e acclimatize

dt o tes t conditions from0

4 Januar y 201 3 t o 14 Januar

y 2013 . Th

e lighting cycl

e wa s controlle dt o giv e a1 6 hour

s ligh t an d8 hour s darknes s cycl e wit h 2 0 minut e daw n an ddus k transitio n periods . Th e wate r temperatur

e wa s controlle da t approximatel

y 1 4 ° C wit h adissolve

doxyge n conten t o f greate r tha n o r equa l t o 10. 3 m g O2/L. Thes

e parameter

s wer e recorde ddaily . Th

e stoc k fish wer e fe dcommercia

l trou t pellet s whic h wa s discontinue

dapproximatel

y 4 8 hour s prio r t o th e star t o f th e definitiv e test. Ther

e wa s zer o mortalit y i n th e 7 day s prio r t o th e star t o f th e tes t an d th e fish ha damea n standar d lengt h o f 4. 3 c m(s d= 0.2 ) an damea n weigh t o f 0.9 1 g (s d= 0.14 ) a t th e en do f th e definitiv e test. Base

d o n th e mea n weigh t valu e thi s gav e aloadin g rat e o f 0.4 6 g bodyweight/Iiter . Th e die t an ddiluen t wate r ar e considere

dno t t o contai n an y contaminan

t tha t woul daffect th e integrit y an doutcom e o f th e study . 3. 3 Tes tWate r Th e tes t wate r use d fo r bot h th e range-findin

g an ddefinitiv e test

s wa s th e sam e a s tha t use d t o maintai n th e stoc k fish. Laborator y ta p wate r wa s dechlorinate

db y passag e throug h a n activate dcarbo n filter (Purit e Serie s 500 ) an dpartl y softene d(ElgaNimbu

s 1248 D Duple x Wate r Softener ) givin g wate r wit h atota l hardnes s o f approximatel

y 14 0 mg/ L a s CaC03 . After

dechlorinatio

n an dsoftenin g th e wate r wa s passe d throug h aserie s o f compute r controlle dplat e hea t exchanger

s t o achiev e th e

### p . 1 2

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 1
require d temperature . Typica

l wate r qualit y characteristic s fo r

th e ta p wate r a s supplied , prio r t o dechlorinatio

n an dsoftening , ar

e give n i n Appendi

x 2 . A t th e star t o f th e definitiv e tes

t th e diluen t ha daparticulat

e matte r concentratio

n o f 0. 1 mg/L , tota l wate r hardness o f

14 4 mg/ L a s CaCOa , alkalinit y o f 101.2 mg/ L a s CaCCh , Tota l Organi c Carbo n conten t o f les s tha n th e limi t o f quantisation , determine

dt o b e 1, 0 m g C/L , chemica

l oxyge n deman do f 2.0 9 m g O2/ L an dconductivit

y o f 43 8 u-S/cm

. Th e chlorin e conten t range d fro m0.00 0 t o 0.09 1 m g CI2/L . Th

e wate r hardnes s an dalkalinit y wer e determine

d usin g th e method s describe d i n Fieldan

dOn-sit e Tes t Method s fo r th e Analysi s o f

Water s (British Standard s Institution , 1993) . 3. 4 Procedur e Al l tes t sampl e concentration

s wer e correcte dfo r awate r conten t o f 50.5% , an dar e reporte d a s milligrams o f

activ e ingredien

t (ai) pe r liter . 3.4. 1 Range-findin

g Test s Th e tes t concentration

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

t wer e determine

db y preliminar

y rangefindin g tests . In th e initia l range-findin

g tes t fis h wer e expose d t o asingl e tes t concentratio

n o f 100 0 m g ai/ L a s information fro mth

e Acut e Toxicit y t o Daphni aMagn astud y (Harla n Stud y Numbe r 41104695) suggeste

d tha t toxicit y wa s no t expecte da t thi s level , Th

e tes t ite mwa s dissolve d directl y i n dechlorinate

d ta p water . An amoun t o f tes t ite m(40.4 0 g) wa s dissolve di n dechlorinate

dta p wate r an dth e volum e adjuste dt o 2 0 liters t o giv e th e 100 0 m g ai/ L tes t concentration . Mortalitie s wer e observe d a t thi s concentratio

n s o i t wa s considere

dappropriat

e t o conduc t asecon d rang e findin g test. I n th e secon d rang e findin g tes

t fis h wer e expose d t o aserie s o f tes t concentration

s o f 1.0 , 1 0 an d 10 0 m g ai/L . Th e tes t ite mwa s dissolve ddirectl y i n dechlorinate

dta p water. Amoun t o f tes t ite m(40.40 , 404.4

0 an d4040.4 0 mg ) wer e dissolve di n dechlorinate

dta p wate r an d th e volum e adjuste dt o 2 0 liter s t o giv e th e 1.0,1 0 an

d10 0 m g ai/ L tes t concentrations . In th e range-findin

g test s 3 fis h wer

e adde d t o eac h 2 0 lite r tes t an dcontro l vesse l an d maintaine da t 1 3 ° C t o 14 ° C i n atemperatur

e controlle

droo mwit h aphotoperio

do f 1 6 hour s ligh t an d8 hour s darkness wit

h 2 0 minut e daw n an ddus k transitio n period s fo r aperio do f u p t o 9 6 hour s unde r stati c tes t conditions . Th e contro l group s wer e maintaine

dunde r identica l conditions bu

t no t expose d t o th e tes t item .p . 1 3

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 2

Eac h vesse l wa s covered to reduc

e evaporation . Afte r

3 , 6 , 24 , 48 , 7 2 an

d 9 6 hour

s an y mortalitie s o

r sub-letha l effect s o f

exposur e wer e determined b y visual inspectio n o f th e tes t fish . 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 2 4 hours i n orde

r t o determin e th e stability o f

th e test ite munde

r tes t conditions . Al l samples wer e store d a t approximately -20 ° C prio r t o analysis . Only concentration s withi n th e rang e to b e use d fo r th e definitiv e tes t

wer e analyzed . 3.4. 2 Definitive Tes t Base d o n th e result s o f

the range-findin g test

s th e followin g tes t

concentration s wer

e assigne d t o th e definitiv e test : 100 , 180 , 320,56 0 an d 100 0 m g ai/L . 3.4.2. 1 Experimenta

l Preparation Fo r th e purpos e o f

the definitiv e test

th e tes t ite mwa s dissolve d directl y i n dechlorinated ta p water . Prio r

to weighing , the tes t

ite mwa s boile dto remov

e an y crystal s tha

t ha d formed . Amount s o f

tes t ite m(4040 , 7272 , 12930,2262 6 an d 4040 0 mg ) wer

e dissolve d i n dechlorinate d ta p wate r an d th e volume adjuste d t o 2

0 liter s to giv e th

e tes t concentration s o f

100 , 180 , 320 , 56 0 an d 100 0 m g ai/ L respectively . Th

e tes t medi awa

s stirre dwit h aflat

bladed mixe

r t o ensur e complet e dissolution . Prio r

to additio n o f

th e tes t animals , th

e p H o f th e tes t medi awa

s adjusted t o p H 7 . Th e concentratio n an

d stability o f

th e tes t ite mi n th

e tes t preparation s wer

e verifie db y chemica

l analysi s a t 0 (fres h media) , 24 , 48 , 7 2 (ol d an d fresh media

) an d 9 6 (fres h media

) hour s (se e Appendi x 3) , 3.4.2. 2 Exposur

e Condition s A s i n th e range-findin g tests , 2 0 lite r glas s exposur

e vessel s wer

e use d fo r eac h tes t concentration . A t

the star t o f th e test 1 0 fish wer

e placed i n eac

h tes t vesse l a t random , i n th

e test preparations . Th

e tes t vessel s wer

e the n covere d to reduce evaporatio n an d maintained a

t 1 3 ° C to 1 4 ° C i n atemperatur e controlle droo mwit h aphotoperio do f 1 6 hours ligh t

an d 8 hour s darknes s wit h 2 0 minute dawn an d dus k transitio n period s fo r

aperio d o f

9 6 hours . Th

e test vessels received n

o auxiliary aeration . Th

e fish wer e no t individuall y identifie dan d receive d n o foo ddurin g exposure . Th e contro l grou

p was maintaine d unde

r identica l condition s bu t

no t expose d to th e test item . A semi-static tes t

regime wa

s employed i n th

e test involvin g adail y renewa l o f th e tes t preparation s t

o ensur e tha t th e concentration s o f

th e tes t ite mremaine d nea

r nomina l an d t o preven t th e buil du p o f nitrogenou s wast

e products .

### p . 14

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 3

An y mortalitie s an

dsub-letha

l effect s o f exposur e wer e recorde da t 3 , 6 , 24 , 48 , 7

2 an d9 6 hour s afte r th e star t o f exposure . Th

e criteri ao f

deat h wer e take n t o b e th e absenc e o f bot h respirator

y movemen t an d respons e t o physica l stimulation . 3.4.2. 3 Physico-Chemica

l Measurement

s Th e wate r temperature, p H an

d dissolve doxyge n concentration

s wer e recorde ddail y throughou

t th e test. Th e measurement

s a t 0 hours , an

dafte r eac h tes t medi arenewa l a t 24,4 8 an d7 2 hours , represen t thos e o f th e freshl y prepare dtes t preparation

s while th e measurement

s take n prio r t o eac h tes t medi arenewal , an

d o n terminatio

n o f th e tes t afte r 9 6 hours , represen

t thos e o f th e use d o r 24-Hou r oldtes t preparations . Th

e pH an doxyge n wer e measure d usin g aHac h HQ30 dFlex i handhel dmete r an dth e temperatur

e wa s measure d usin g aHann aInstrument

s H I 9351 0 digita

l thermometer

. Th e temperatur

e wa s als o monitore

di n th e control hourl

y usin g aTest o temperatur e logger. 3.4.2.4 Verificatio n o f

Tes t Concentration

s Wate r sample s wer e take n fromth e contro l an dal l survivin

g tes t groups a t 0 (fresh media) , 24 , 48 , 7 2 (ol dan dfresh media

) an d9 6 (fresh media

) hour s fo r quantitativ

e analysis . Th

e sample s wer e store da t approximatel

y -2 0 °C prio r t o analysis . Duplicat e sample s wer e take n an dstore da t approximatel

y -2 0 ° C fo r furthe r analysi s i f necessary. Onl

y sample s o f th e NOE C wer e analyzed. Th e metho d o f analysis , recover

y an dtes t preparatio n analyse s ar e describe di n Appendi

x 3 . 3.4.2. 5 Evaluatio

n o f Dat a An estimat e o f th e LC5 0 value s wa s give n b y inspectio

n o f th e mortalit y data.p . 1 5

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 4



## 4 RESULT S 4. 1 Range-findin g Test s Cumulativ e mortality dat afro mth e exposur e o f rainbo w trou t

t o the tes t

ite mdurin g th

e rangefinding test

s ar e give n i n Tabl e 1 an

d sub-letha

l effect s o f

exposur e ar e give n i n Tabl

e 2 . Th e results showe

d no mortalitie s a

t th e tes t concentration s o f

1.0, 1 0 an

d 10 0 m g ai/L . However , mortalitie s wer e observe d a t 100 0 m g ai/L Afte r approximately 3 hours exposur e i n th e initia l rang

e finding tes t

3 out o f 3 fish a t 100 0 m g ai/L wer

e observe d t o exibi t alos s o f equilibrium . Du

e to anima

l welfare implication s (Animal s (Scientifi c Procedures ) Ac t 1986 ) these fish wer

e kille dan

d classe d a s mortalitie s fo r th e followin g observationa

l time point . Base d o n thi s informatio n tes t concentration s o f

100 , 180 , 320 , 56

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

the 1.0, 1 0 an

d 10 0 mg ai/L test

preparation s a

t 0 an d 2 4 hour s (se e Appendi x 2 ) snowe

d measure

d concentration s ranging fro m99 % to 126 % o f nomina l indicatin g tha t th e tes t ite mwa s stable unde

r test conditions . 4. 2 Definitiv e Tes t 4.2. 1 Mortalit

y Data Cumulativ e mortality dat afromth

e exposur

e o f rainbo w trou t

t o th e tes t ite mdurin g th

e definitiv e tes t

ar e give n i n Table 3 . Ther e wer e n o mortalitie s i n 1 0 fish expose d to aserie s o f

tes t concentration s o f

100 , 180 , 320 , 56 0 an d 100 0 mg ai/ L fo r

aperio d o f

9 6 hours . Inspectio n o f the mortality dat

agav e th e followin g results : 

### Tim e (h ) LC 5 

### o (m g ai/L ) 3 >100 0 6 >100 0 2 4 >100 0 4 8 >100 0 7 2 >100 0 9 6 >100 0

### p . 1 6

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 5

Th e result s o f th e definitiv e tes

t showe d th e highes t tes t concentratio

n resultin g i n 0 % mortalit y t o b e greate r tha n o r equa l t o 100 0 m g ai/L , th e lowes t tes t concentratio

n resultin g i n 100 % mortalit y t o b e greate r tha n 100 0 m g ai/L . Th e N o Observe d Effec t Concentratio

n (NOEC ) wa s 100 0 m g ai/L . In th e initia l rang e finding test

, mortalitie s wer

e observe d a t 100 0 m g ai/L . Du

e t o atechnica

l oversigh t th e p H o f th e tes t preparation

s wa s no t adjuste d t o withi n specification . Adjustment s t o pH wer e mad e durin g th e definitiv e tes

t an d henc e i t wa s considere

d likel y tha

t th e mortalitie s observe d i n th e initial rang

e finding tes

t wer e du e t o apH effec t

rathe r tha n atru e toxic effect . 4.2. 2 Sub-Letha

l Effect s Ther e wer e n o sub-letha

l effects o f

exposur e observe d i n 10 fish expose

d t o ates t concentratio

n o f 100 0 m g ai/ L fo r aperiodo f

9 6 hours . 4.2. 3 Observation

s o n Tes tIte m Solubilit

y Throughou

t th e test , th e 10 0 an d 18 0 m g ai/ L tes t preparation

s wer e observe d t o b e ligh t yello w solution s increasin

g i n colo r wit h concentration, an

d th e 320 , 56 0 an d 100 0 m g ai/ L tes t preparation

s wer e observe d t o b e dar k brow n solution s increasin g i n colo r wit h concentration . 4.2. 4 Physico-Chemica

l Measurement

s Th e result s o f th e physico-chemica

l measurement

s ar e give n i n Appendi

x 3 . Temperatur

e wa s maintaine d a t 1 3 ° C t o 14 ° C throughou

t th e test , while ther

e wer e n o treatmen

t relate d difference s fo r

oxyge n concentratio

n o r pH . Th e oxyge n concentratio

n i n som e o f th e tes t vessel s wa s observe d t o hav e a n ai r saturatio n valu e (ASV ) i n exces

s o f 100% . Thi s wa

s considere

d t o b e du e t o th e presenc e o f microscopi

c ai r bubble s i n th e medi asuper-saturatin

g th e diluen t an d wa s considere

d no t t o hav e ha d a n impac t o n th e outcom e o r integrit y o f th e tes t a s n o advers e effects wer

e observe d i n th e contro l group . Th e wate r temperatur

e whic h wa s als o recorde d i n th e contro l vesse l approximatel

y ever y hou r usin g aTest o temperatur

e logge r (se e Figur e 1 ) range d fro m1 2 °C t o 1 4 °C . 4.2. 5 Verificatio

n o f Tes tConcentration

s Analysi s o f

th e 100 0 m g ai/ L tes t preparatio n a t 0, 24 , 48 , 7

2 an d 96 hour s showe d measure d tes t concentration

s t o rang e fro m91 % t

o 114 % o f nomina l an d s o th e result s ar e base d o n nomina l tes t concentration

s only . Th

e fres h tes t preparatio n a t 4 8 hour s wa s measure d t o b e 184 % o fp . 1 7

Harla n Stud y Number : 4110469 4 Repor t Pag e 1 6

nominal , however , a

s th e correspondin

g ol d preparation a

t 7 2 hours wa

s nea r nomina l i t i s considere d tha t thi s result

i s anomalou s an

d ha s n o impact o n the outcom

e o f th e test . 

## 5 CONCLUSIO N Th e acut e toxicit y o f

th e tes t ite mto th

e freshwate

r fis h rainbo w trou t

(Oncorhynchu s mykiss ) ha s bee n investigate d an

d gav e a96-Hou

r LC5 0 o f greate r tha n 100 0 mg ai/L . Th

e N o Observe

d Effec t Concentratio n wa

s 100 0 m g ai/L . 

## 6 REFERENCE S BRITIS H STANDARD S INSTITUTIO N (1993 ) Determinatio n o f Alkalinity . In : Fiel dan

d On

sit e Test Method s fo r

the Analysi s o f

Waters , (B S 1427) , p

p 33-35 . BRITIS H STANDARD S INSTITUTIO N (1993 ) Determinatio n o f Calciu man

d Tota l Hardness . In : Fiel dan

d On-sit e Tes t Method s fo r

th e Analysi s o f

Waters , (B S 1427) , p

p 35-36 .Harla n Stud y Number: 4110469

4 Repor t p . 1 8 Pag e 1 7


## TABLE S Tabl e 1 Cumulativ e Mortalit y Dat a i n th e Range-findin g Test s Nomina l Concentratio n (m g ai/L ) Cumulativ e Mortalit y (Initia l Populatio n = 3 ) Nomina l Concentratio n (m g ai/L ) 3 Hour s 6 Hours 2 4 Hours 48 Hour s 7 2 Hour s 9

6 Hour s Control * 0 0 0 0 0 0 1. 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 100 0 3* * 3 3 3 3 3

* Th e result s observe d fo r th e controls durin

g bot h range-findin

g test s wer e observe d t o b e identica l an d therefor e wer e no t separatel y tabulated . * * Afte r 3 hour s al l o f th e fis h wer e observe d t o hav e alos s o f equilibrium . Du

e to approac

h o f th e substantia

l severit y limi t (Animal s (Scientifi c Procedures ) Ac t 1986 ) thes e fis h wer e kille dan dclasse d a s mortalities a

t th e 3 hou r tim e point .Harla n Stud y Number: 4110469

4 Repor t p . 1 9 Pag e 1 8

Tabl e 2 Sub-letha l Effect s of Exposur e i n th e Range-findin g Test s Nomina l Concentratio n (m g ai/L ) Sub-lethal Effects Tim e (Hours ) Nomina l Concentratio n (m g ai/L ) Sub-lethal Effects 3 6 24 48 7 2 9

6 Control * N o abnormalitie s detected 3/ 3 3/

3 3/

3 3/

3 3/

3 3/

3 1. 0 N o abnormalitie s detected 3/ 3 3/

3 3/

3 3/

3 3/

3 3/

3 1 0 N o abnormalitie s detected 3/ 3 3/

3 3/

3 3/

3 3/3 / 3/

3 10 0 N o abnormalitie s detected 3/ 3 3/

3 3/

3 3/

3 3/

3 3/

3 100 0 Los s o f

equilibriu m 3/3* * A/ D A/ D A/ D A/ D A/ D

* Th e result s observe dfo r th e control s durin

g bot h range-findin

g test s wer e observe dt o b e identica l an d therefor e wer e no t separatel y tabulated . * * After 3 hour s al l o f th e fis h wer e observe dt o hav e aloss o f equilibrium . Du

e t o approac h o f th e substantia

l severit y limi t (Animal s (Scientifi c Procedures ) Ac t 1986 ) thes e fis h wer e kille dan

dclasse d a s mortalities a

t th e 3 hou r tim e point . A/ D = Al l fish dea

dHarla n Stud y Number : 4110469 4 Repor t Pag e 1 9

Tabl e 3 Cumulativ e Mortalit y Dat a i n th e Definitive Tes t Nomina l Concentratio n (m g ai/L ) Cumulativ e Mortalit y (Initia l Populatio n = 10) % Mortalit y Nomina l Concentratio n (m g ai/L ) 3

Hour s 6

Hours 2

4 Hour s 4

8 Hour s 7

2 Hour s 9

6 Hour s 9

6 Hour s Contro l 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 180 0 0 0 0 0 0 0 320 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0Harla n Stud y Number : 4110469 4 Repor t p . 2 2 Pag e 2 1



## APPENDICE S Appendi x 1 Typica l Wate r Qualit y Characteristic s WATE R SUPPL Y ZON E ZDB1 3 ALVASTO N REPORTIN G PERIOD : 01/01/201 0 - 31/12/201 0 PARAMETE R ANALYSI S UNIT S NO
.

SAMPLE S TAKE N CONCENTRATIO N O R VALU E

(AL L SAMPLES ) PCV+ SAMPLE S CONTRAVENIN G PC

V PARAMETE R ANALYSI S UNIT S NO
.

SAMPLE S TAKE N MINIMU M AVERAG E MAXIMU M PCV+ NUMBE R PERCEN T 1, 2 Dichloroethan e ug/1 4 0 <0.08 0 <0,08 0 <0.08 0 3 0 0,00 Aluminiu m 3 3 <5.00 0 <6.68 4 12.00 0 20 0 0 0.00 Amelry n ug/ l 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0,00 Antimon y \igl\ 8 0.18 0 0.53 1 1.20 0 5 0 0,00 Arseni c ug/ l 8 <0.37 0 <0.37 0 <0.37 0 1 0 0 0,00 Atrazln e ug/ l 8 <0.00 3 <0,00 3 <0.00 3 0. 1 0 0.00 Azlnpho s methy l ug/ l 8 <0.00 4 <0.00 4 <0.00 4 0, 1 0 0.00 Benzen e 4 0 <0.06 0 <0.06 0 <0.06 0 1 0 0,00 Benz o 3,4-Pyren e M9' l 8 <0.00 1 <0.00 1 O.00 1 0.0 1 0 0.00 Boro n mg/ l 4 0 <0.01 7 0.02 0 0.03 8 1 0 0.00 Bfomad l ug/ i 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0 00 Bromat e Maf l 8 <0.60 0 <0.60 0 <0.60 0 1 0 0 0.00 Cadmiu m 8 0.08 0 0,12 1 0.16 0 5 0 0.00 Carbenazf m ug/ l 8 <0.00 2 <0.00 2 <0.00 2 0. 1 0 0.00 Carbetamid e ug i 8 <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.00 Carbophenophlo n ugf l 8 <0.01 2 <0.01 2 <0.01 2 0, 1 0 0.00 Chlorfenvinpho s MS " 8 <0.00 5 <0.00 5 <0.00 5 0. 1 0 0,0 0 Chlorotoluro n Mg/ l 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0.0 0 Chlorpyripho s Ethy l pg/ l 8 <0.01 0 <0.01 0 <0.01 0 0. 1 0 0.0 0 Chlorpyrlpho s Methy l Mfl " 8 <0.01 5 <0.01 5 <0.01 5 0. 1 0 0.0 0 Chromiu m ug/ i 6 <0.70 0 <0.70 0 0.70 0 50 0 0.0 0 Colou r mg/ l Pt/C o 3 8 <0.40 0 <0.60 8 2.60 0 2 0 0 0.0 0 Coppe r mg/ l 8 <0.00 3 <0.032 0,13 0 2 0 0.0 0 Cyanazln e Mfl/I 8 <0.00 5 <0.00 5 O.00 5 0. 1 0 0.0 0 Cyanid e ug/ l 4 0 <0.30 0 0.74 0 1 60 0 5 0 0 0.0 0 Demeton-S-Methy l Mfl/I e <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.0 0 Dlazlno n Mfl/I 8 <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.0 0 Dlchlorvo s Mfl/ I 8 <0.00 8 <0.00 8 <0.00 8 0. 1 0 0.0 0 Dlflufenica n ug/ i 8 «=0.00 5 <0.005 <0.00 5 0. 1 0 0.0 0 Dimelhoat e Mfl/ I 8 <0,00 5 <0.00 5 <0.00 5 0. 1 0 0.0 0 Dluro n ug/ l 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.0 0 E.col l No./10 0 m l 204 0.00 0 0.000 0,00 0 0 0 0.0 0 Enterococc l No
. /10 0 m l 8 0.00 0 0.00 0 0.00 0 0 0 0,0 0 Ethofumesate Mfl/ I 8 <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.0 0 Fenpropidl n Mfl/I 8 <0.0D 4 <0.004 <0.00 4 0. 1 0 0.0 0 Fenpropimorp h Mfl/I 3 <0.00 2 <0.00 2 <0.00 2 0. 1 0 0.0 0 Fluorid e mg/ l 8 0.27 0 0.33 1 0.41 4 1. 5 0 0.0 0 Flutriafo l Mfl/I 8 <0.00 7 <0.00 7 <0.00 7 0. 1 0 0,0 0 Heptenopho s Mfl/I 8 <0.00 5 <0.00 5 <0.00 5 0, 1 0 0.00 Iro n Mfl/I 7 6 <10.00 0 <26.57 9 488.00 0 20 0 1 1.3 2 Isoproturo n Mfl/I 8 <0.00 2 <0.00 2 <0.00 2 0. 1 0 0.00 Lea d Mfl/I 8 <0.50 0 <2,28 8 10.00 0 2 5 0 0,00 Llnuro n Mfl/I 8 <0.00 5 <0.00 5 <0.00 5 0, 1 0 0.00 Malathio n Mfl/I 6 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0.00 Manganes e Mfl/I 3 8 <1.80 0 <1.98 7 5.60 0 50 0 0,00 Mercur y Mfl/I 4 0 <0.01 2 0.01 8 0.19 9 1 0 0.00 Metaldehyd e Mfl/I 3 2 0.004 0.00 9 0.02 7 0. 1 0 0.00 Metamltro n Mfl/I 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.00 Metazachlo r Mfl/I 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.00 Methabenzthlazuro n Mfl/I 8 <0.00 3 < 0 00 3 <0.00 3 0, 1 0 0.00 Mevinpho s MS " 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0,0 0 Monollnuro n Mfl/I 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0.0 0 +PCV Prescribe d concentratio n o rvalu e fo rmandator y parameter

s an d specifie d limi t o rvalu e fo rnon-mandator y parameter

s (Indicator

s an d disinfectants)
.Harla n Stud y Number: 4110469

4 Typica l Wate r Qualit y Characteristic s Repor t p . 2 3 Pag e 2 2

WATE R SUPPL Y ZON E ZDB1 3 ALVASTO N REPORTIN G PERIOD : 01/01/201 0 - 31/12/201 0 PARAMETE R ANALYSI S UNIT S NO
.

SAMPLE S TAKE N CONCENTRATIO N O R VALU E (AL L SAMPLES ) PPV + SAMPLE S CONTRAVENIN G PC V PARAMETE R ANALYSI S UNIT S NO
.

SAMPLE S TAKE N MINIMU M AVERAG E MAXIMU M NUMBE R PERCEN T Nicke l 8 2.30 0 3.28 8 5.80 0 2 0 0 0.0 0 Nitrat e mg/ l 8 9.94 0 13.10 5 16.70 0 5 0 0 0.0 0 Nitrit e mg/ l 8 <0.00 3 O.00 5 <0,00 9 0. 5 0 0.0 0 Nitrite/Nitrat e Calculate d - 1 8 0.20 0 0.27 6 0.37 0 < V 0 0.0 0 Odou r Dilutio n 4 2 0.00 0 0.02 4 1.00 0 - 2 1 2.3 8 Numbe r PA H yg/ I 8 0.00 0 0.00 1 0.00 2 0. 1 0 0.0 0 Parathio n w i 6 <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.0 0 Pendimethali n 8 <0.00 5 O.00 5 <0.00 5 0. 1 0 0.0 0 P H p H valu e 7 6 7.02 0 7.49 7 7.87 0 Ma x 9.5 , 0 0 0 0 Mi n 6. 5 Phorat e pg i 8 <0.00 9 <0.00 9 <0.00 9 0. 1 0 0.0 0 Phosalon e ug/ l 8 <0.00 7 <0.00 7 <0,00 7 0. 1 0 0.0 0 PWmlcarf t rafl 8 <0.00 3 <O.O0 3 <0.003 0. 1 0 0.0 0 Plrimlphos-Methy l Mg/ i 8 <0.00 9 <0.00 9 <0.00 9 0. 1 0 0.0 0 Prometry n M9/ 1 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.0 0 Propachlo r M9/ l 8 <0.00 5 <0.00 5 <0.00 5 0. 1 0 0.0 0 Propazin ^ ug/ l 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0.0 0 Propetampho s von 8 <0.00 7 <0.00 7 <0.00 7 0. 1 0 0.0 0 Propiconazol e MS/ l 8 <0.01 0 <0.01 0 <0.01 0 0. 1 0 0.0 0 Propyzamjd e U0/ J 8 <0.00 4 <0.00 4 <0.00 4 0. 1 0 0.0 0 Seleniu m Mfl l 8 <0.22 0 <0.54 6 0.74 0 1 0 0 0.0 0 Simazin e Mfl/ I 8 <0.00 7 <0.00 7 <0.00 7 0. 1 0 0.0 0 Sodiu m mg/ l 8 14.00 0 25.25 0 30.00 0 20 0 0 0.0 0 Sulphat e mg/ l 4 0 23.80 0 50.34 5 99.80 0 2 5 0 0.0 0 Tast e Dilutio n Numbe r 4 2 0.00 0 0.00 0 0.00 0 2 0 0.0 0 Terbutry n Mfl/ I 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.0 0 Tetrachloroethen e an d Trichloroatrien e Mfl/ I 8 0.00 0 0.00 0 0.00 0 1 0 0 0.0 0 TelracWoromethart e (jg d 8 <0.018 <0.02 1 <0.03 0 3 0 0.0 0 Tota l Pesticide s Mfl/ I 3 2 0.00 0 0.00 9 0.02 7 0. 5 0 0.0 0 Tota l Trihalomethane s Mfl/ I 8 19.44 0 25.44 9 31.58 0 10 0 0 0.0 0 Triadlmefo n Mfl/ I 8 <0.00 9 <0.00 9 <0.00 9 0. 1 0 0.0 0 Trlallat e Mfl/ I 7 <0.00 8 <0.01 4 <0.05 0 0. 1 0 0.0 0 Triazopho s Mfl/ I 8 <0.00 3 <0.00 3 <0.00 3 0. 1 0 0.0 0 Trletazin e Mfl/ I 8 <0.00 6 <0.00 6 <0.00 6 0. 1 0 0.0 0 Turbidit y NT U 7 6 <0.03 0 <0.13 5 3.40 0 4 0 0.0 0 Fre e Chlorin e mg/ l 20 4 0.01 0 0.20 7 0.63 0

_ » . - Tota l Chlorin e mg/ l 20 4 0.04 0 0.27 0 0.71 0 3 - - Ammoniu m mg/ l 3 8 <0.00 9 <0.01 2 0,04 9 0. 5 0 0.0 0 Chlorid e mg/ l 4 0 9.66 0 21.05 2 50,80 0 25 0 0 0.0 0 Clostridiu m perfringen s No
. /10 0 m l 3 8 0.00 0 0.00 0 0.00 0 0 0 0.0 0 Colifor m bacteri a No
. /10 0 m l 20 4 0.00 0 0.00 0 0,00 0 0 0 0.0 0 Colon y Coun t afte r4 8 hour s a t 37' C No./10 0 m l 7 6 0.00 0 0.78 9 31.00 0 Jt
- - Colon y Coun t afte r7 2 hour s a t 22' C No./10 0 m l 7 6 0.00 0 2.03 9 85.00 0 _A
- - Conductivit y uS/c m a t 20" C 3 3 315.00 0 419.00 0 486.00 0 250 0 0 0.0 0 Gros s Alph a Activit y Bq/ I 8 0.03 2 0.06 9 0.09 0 0.1 s Gros s Bet a Activit y Bq/ I 8 O.00 1 <0.03 6 0.05 3 1 s Tota l Organi c Carbo n mg/ l 4 0 <0.13 0 0.92 0 2.02 0

_ 4 - - +PC V Prescribe d concentratio n o rvalu e tormandator y parameter

s an d specifie d limi t o rvalu e fo rnon-mandator y parameter

s (indicator

s an d disinfectants)
. 1 Th e formul a fo rth e calculate d Nitrate/Nitrit e leve l I s [Nltrate]/5 0 + [Nitrite]/3
. Thi s numbe rha s n o units
. 2 Acceptabl e t o customer s an d n o abnorma l change
. 3 N o numerica l limi t applies
. 4 N o abnorma l change
. 5 Guidance level s only
. Monitore d fo rth e calculatio n o f Tota l Indicative Dos e (TID) , whic h i s th e regulator y parameter . N o sample s exceede d th e TI D I n 2010
.p . 2 4

Harla n Stud y Number : 4110469 4 Repor

t Pag e 2 3
Appendi x 2 Analytica l Investigation s 

## Analytica l Investigation

s CONTENT S CONTENT S 2 3 1 MATERIAL S AN D METHOD S 2 4 1. 1 Introductio n 2 4 1. 2 Tes t

Ite m2 4 1. 3 Analytica l Standar d 2 4 1. 4 Analytica l Procedur e 2 4 1.4. 1 Storag

e 2 4 1.4. 2 Reagent

s an d Solvent s 2 4 1.4. 3 Preparation o f

Calibratio n Standard

s 2 5 1.4.4 Preparation o f

Linearit y Standard

s 2 5 1.4. 5 Preparation o f

SpikedRecover

y Samples 2 5 1.4. 6 Preparation o f

Tes t Sample s 2 5 1.4. 7 Instrumenta

l Parameter

s 26 1.4. 8 Calculation s 26 2 RESULT S 2 8 2. 1 Range-Finding Test 2 8 2. 2 Definitiv e Test

2 8 2.2. 1 Validatio n o f

th e Analytical Metho d2 8 2.2. 2 Tes t

Samples 2 8 3 DISCUSSIO N 29 TABLE S 3 0 Table 1 Preparation o f Test Samples 3 0 Table 2 Calibratio n Standar d Dat a3 0 Table 3 Results fo r Range-Finding Sample

s 3 1 Table 4 Result s fo r SpikedRecover

y Sample s 3 1 Table 5 Results fo r Tes t Sample s 3 2 FIGURE S 3 3 Figur e 1 Calibratio n Plo t 3 3 Figur e 2 Chromatogra

mo f Calibratio n Solutio n (1. 0 m g ai/L ) 3 4 Figur e 3 Chromatogra

mo f Biological Contro l Sampl e 3 4 Figur e 4 Chromatogra

mo f Test Sampl e (100 0 m g ai/L ) 3 5p . 2 5

Harla n Stud y Number : 4110469 4 Repor

t Pag e 2 4 Analytica l Investigation

s 

## 1 MATERIAL S AND METHOD S 1. 1 Introductio n Th e tes t ite mconcentratio n i n th

e tes t sample s wa s determined b y direc t

injectio n int o th

e mass spectromete

r (HPLC-MS ) usin g a

n externa l standar d to calibrat

e th e system . Al l test sampl e concentration s wer

e corrected fo r

awate r conten t o f 50.5% , an

d ar e reporte da s milligram s o f

activ e ingredient

(ai ) pe r liter . Th e metho dwa

s developedb 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 describedi n th

e biological par

t o f thi s study . 1. 3 Analytica l Standar d Th e tes t ite mdescribedi 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 samples wer

e storeda t approximatel

y -2 0 ° C prio r to analysis . 1.4. 2 Reagent s an dSolvent s Water : prepare

dusin g a n ELG A Pur

e La b Optio n R- l 5 wate

r purificatio n system . Test medium : a

s describedi n th

e biological par

t o f thi s study . Solvents : methanol , Fishe r Scientific , HPL

C grade . Reagents : ammonia , Fishe r Scientific , Analytical reagent .Harla n Stud y Number : 4110469 4 Analytica l Investigation

s Repor t p . 2 6 Pag e 2 5

1.4. 3 Preparatio n o

f Calibratio n Standards The test item (nominal

200mg) was dissolved

in diluent*(100mL) to prepare a stock solution with a concentration of

1000mg ai/L. This stock solution was further

diluted with diluent*to produce a solution of

10mg/L. Defined

volumes of

this solution were then diluted with diluent* to obtain calibration standards in the range of 0.10to 1.0mg ai/L. A second series of calibration standards was similarly prepared in the range of

0.25to 1.5mg ai/L. These solutions were used to determine the recovery and test sample concentrations. 1.4. 4 Preparatio n o

f Linearity Standards Th e tes t ite m(nominal 200 mg ) wa s dissolvedi 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 dwit h diluent * to produce asolutio n o f

1 0 mg/L . Define dvolume s o f thi s solutio n wer

e then dilute dwit h diluent * to obtai n calibratio n standards i n th e rang e o f 0.1 0 to 1.

0 mg ai/L . A secon

d serie s o f

calibratio n standard s wa s similarl y preparedi n th e rang e o f 0.2 5 t o 1. 5 m g ai/L . Thes

e standard

s wer e use d t o evaluat e th e linearity o f

th e analytica

l system . 1,4. 5 Preparatio n o f

Spike dRecover

y Sample s To demonstrat

e the validit y o f

th e analytica

l procedure , volume s o f

tes t mediumwer

e spiked wit h th e tes t ite man dit s recover

y wa s assessed. Th

e tes t ite m(nominal 200 mg ) wa s initiall y dissolvedi n tes t

mediumto prepar

e astoc k solutio n wit h aconcentratio n o f 100 0 m g ai/L . Define dvolume s o f

thi s stoc k solutio n wer

e dilute dwit h tes t

mediumto obtai n spikedrecover y sample s a t concentration s o f

10 0 m g ai/L . Fiv e replicate s a t eac h concentratio n level wer e prepare dan dsubjecte dto th

e sam e treatmen

t a s th e tes t samples . I n addition , tes t mediu m withou t th e additio n o f

the tes t item(syntheti c control ) wa s als o analyzed . 1.4. 6 Preparatio n o

f Tes tSample

s Th e tes t samples wer

e thawe dwit h th

e ai do f sonication . The

y wer e furthe r dilute dint

o th e calibratio n rang

e initiall y i n tes t

mediu man dthen furthe r

dilute dwit h diluent * a

s show n i n Table 1 , an

dthen analyze ddirectl y using th e instrumenta

l procedure .

* Diluent : methanol:water:ammonia:tes

t mediu m(50:40:0.10:1 0 v/v/v/v )Harla n Stud y Number : 4110469 4 Analytica l Investigation

s Repor t p . 2 7 Page 26

1.4. 7 Instrumenta

l Parameter

s Autosampler : Pump : Detector : Column : Mobil e phase : Agilen t 120 0 Series Agilen t 120 0 Series Agilen t 120 0 Series MS D none , direc t

injectio n water : methanol : ammonia(500:500:1 , v/v/v ) Flo w rate : Injectio n volume : Colum n temperature : Ionizatio n mode : Polarity : Capillar y voltage : Ga s temperature : Nebulize r pressure : Fragmento

r voltage : Dryin g ga s flow: 0. 5 mL/minut e 1 0 u L
roo mtemperatur

e ES I negative 400 0 V 35 0 ° C 4 0 ps i 10 0 V 1 2 L/mi n Data acquisition mode: Single Ion Monitoring (SIM) SIM ions: 225m/z

1.4. 8 Calculation s A calibratio n curv e wa

s prepare dusing aquadratic functio n o f th e for mshown i n equatio

n (1) . Th e calibratio n dat

ai s presente

d i n Tabl e 2 an d Figur e 1 . Fro mth

e calibratio n curve , th

e concentration "x " o f

the tes t itemi n a

n injecte d sampl

e wa s calculate d usin g th

e followin g equation : -b + ^jb 2 -4-a-(c-y) (1)
X ~ 2-a wher e x : concentratio n o f th e test ite mi n injecte dsampl

e [m g ai/L ] y : average pea

k are ao f th e test ite mi n injecte dsampl

e [counts ]Harla n Stud y Number ; 4110469 4 Analytica l Investigation

s Repor t p . 2 8 Pag e 2 7

a , b , c : constant

s Th e concentratio n o f

th e tes t itemi n th

e test samples wa

s calculate d using th

e followin g equation :

c - R/10 0 wher e c x F 

### R (2 )

concentratio n 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 ite mi n th e injecte dsampl

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 the n Ri 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= •'for

t TO O (3 ) wher e R : c : recover

y rat e [% ] determine dconcentratio

n o f th e tes t itemi n th e spiked recover

y sampl e [m g ai/L ] c fort : fortifie dconcentration o f th e tes t ite mi n th

e spike d recover

y sampl e [m g ai/L ] Th e percentag

e o f nominal concentration o f th e tes t ite mi n th

e tes t sample s wa

s calculated using th e followin g equation : % nomina l = •100 (4 ) wher e nomina

l concentration i n th

e test sampl e [m g ai/L ]Harla n Stud y Number : 4110469 4 Analytica l Investigation

s Repor t p . 2 9 Pag e 2 8


## 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 conductedspecificall y a t th e tes t concentration s employe d i n th e definitiv e test . Th e result s fo r

th e range-findin g test

sample s ar e summarize

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

f th e Analytical Metho

d Specificit y Th e biological contro l sample s an d a n analyze

d analytica

l blan k showe d n o significant interferin g response a

t the retentio n time o f

th e tes t item . The standar

d solution s containe

d a pea k specifi c fo r

the tes t ite mwhose are

achange d accordingly wit h know n concentration , henc e th e specificit

y o f the metho db y retentio n time wa s confirmed . Linearit y Th e calibration dat

afo r th e calibratio n standard

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

e 2 an d Figur e 1 . Th e dat awa s foun dto hav

e aquadratic correlatio n withi n th e calibratio n range o f

0 t o 1. 7 mg/L . Th e R 2fi t o f th e calibratio n curv e t

o th e dat awa s 0.9973 , an

d wa s considered to b

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

y fortifie da

t arelevan

t concentratio n o f

test itemwa s prepare dfive-fol dan

danalyzed . Th

e result s obtained fo r

th e concentratio n o f

th e test itemi n th e recover y samples ar

e presented i n Table 4 . Th e methodwa

s consideredto be sufficientl y accurat e an dprecis e fo r

th e purpose s o f

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

s determined b y calculatin g te n time s th e standar d deviatio n o f th e response see

n i n fiv e controls . Usin g thi s metho d th e limi t o f quantitatio n wa

s determine

d t o b e 0.05 6 mg/L . 2.2. 2 Tes tSample

s Th e results obtaine dfo r

the concentration o f

th e test ite mfoun di n th

e tes t samples ar

e presente d i n Tabl e 5 . Typica l chromatograms ar e shown i n Figure s 2 to 4 .p . 3 0

Harla n Stud y Number : 4110469 4 Repor

t Pag e 2 9 Analytica l Investigation

s 

## 3 DISCUSSIO N Th e detectio n syste

mwa s foun dt o hav e acceptabl

e correlatio n to th

e fi t o f aquadratic equation . Th e analytica

l procedur

e wa s foun dt o hav e acceptabl

e recoverie s o f

tes t ite mi n tes t

medium . Th e metho d o f

analysi s wa

s validate dan

d prove n to b e suitable fo r

use .Harla n Stud y Number : 4110469 4 Analytica l Investigation

s Repor t p . 3 1 Pag e 3 0


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

s Nomina l Concentration o

f Tes tItem Sampl e Volum e Fina l Volum

e Sample Preparatio n Facto r Fina l Nomina l Concentration o

f Test Ite m [m g ai/L ] [mL ] [mL ] F [m g ai/L ] Contro l 1 100 , the n 5 - » 5 0 100

0

- 100 0 1 100 , the n 5 - > 5 0 100

0 1.

0 No t Applicabl e Tabl e 2 Calibratio n Standard Dat a Concentratio n o

f Tes tIte m Are a [m g ai/L ] [counts] 0 8.45 3 x 10 3 0.11 9 2.00 1 x 10 4 0.28 8 3.08 0 x 10 4 0.59 3 5.21 2 xlO 4 0.86 4 6.423 x 10 4 1.1 9 8.57 2 x 10 4 1.7 3 1.02 4 x10 sp . 3 2 Harla n Stud y Number: 4110469

4 Repor t Pag e 3 1 Analytica l Investigations 

### Tabl e 3 Result

s fo rRange-Findin

g Sample s Nomina l Concentratio n o f Tes t Ite mi n Range-Findin g Sampl e Measure d

Concentratio n Sampl e

Preparatio n Determine d

Concentratio n Tim e Poin t Nomina l

Concentratio n o f Tes t Ite mi n Range-Findin g Sampl e o f

Tes t Ite mi n Sampl e Via l Facto r o f Tes t Ite mi n Range-Findin g Sampl e % o f

Nomina l Concentratio n
X F c [hours ] [m g ai/L ] [m g ai/L ] [m g ai/L ] [% I 0 1.

0 0.12 3 1 0 1.2 3 12

3 1 0 1.1 3 1 0 11. 3 11 3 10 0 1.1 0 10 0 11 0 11 0 24 1.

0 0.12 6 1 0 1.2 6 12

6 1 0 0.99 3 1 0 9.9 3 99 10 0 1.0

5 10

0 10

5 10

5


### Tabl e 4 Result

s fo r Spiked Recover

y Sample s Nomina l Concentratio n o f Tes t Ite m Fortifie d

Concentratio n o f Tes t Ite mi n th e Spike d Sampl e Cfor t Measure d Concentratio n o f Tes t Ite mi n th e Sampl e Via l X Sampl e

Preparatio n Facto r

F Determine d Concentratio n o f Tes t Ite mi n th e Spike d Sampl e c Analytica l

Recovery Rat e

R Precisio n

(Relativ e Standar d Deviatio n o f Recovery ) [m g ai/L ] [m g ai/L ] [m g ai/L ] [m g ai/L ] [% ] [% 1 10 0 10

5 1.0

8 10

0 108 10 5 1.0

3 10

0 10

3 10 5 1.0

7 10

0 10

7 10

0 4. 5 10 5 1.0

9 10

0 10

9 10 5 0.97 5 10 0 97. 5 Acceptanc e Target 80-12 0 <1 0 Fo r reportin g purposes

, x ha s bee n calculate

d fro mc retrospectively

.Harla n Stud y Number: 4110469

4 Analytica l Investigations Repor t Pag e 3 2

Tabl e 5 Result s fo r Tes tSample s

Tim e Point Nominal

Concentratio n of Tes tIte m i n Tes tSampl e Measured

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

Preparatio n Facto r Determined

Concentratio n of Tes tIte m i n Tes tSampl e % o f Nominal Concentratio n
*-no m X F c [hours ] [mg/L ] [mg/L ] [mg/L ] [% ] 0 (fresh ) Contro l

100 0 <LO Q

0.557/0.992 * 100 0 100 0 <LO Q

557/992 * 57/99 * 24 (old ) Contro l

100 0 <LO Q

0.714/1.14 * 100 0 100 0 <LO Q

714/1140 * 71/114 * 24 (fresh ) Contro l

100 0 <LO Q

0.95 6 / 0.907 * 100

0 100 0 <LO Q

94 6 / 907 95/91 * 4 8 (old ) Contro l

100 0 <LO Q

0.659/0.932 * 100 0 100 0 <LO Q

65 9 / 932 * 66/93 * 4 8 (fresh ) Contro l

100 0 <LO Q

0.681 / 1.84 * 100

0 100 0 <LO Q

68 1 / 1840 * 68 /

184 * 7 2 (old ) Contro l

100 0 <LO Q

0.629 / 0.925 * 100

0 100 0 <LO Q

629/925 * 63 / 93 * 7 2 (fresh ) Contro l

100 0 <LO Q

0.809/1.11 * 100 0 100 0 <LO Q

809 / 1110 * 81/111 * 9 6 (old ) Contro l

100 0 <LO Q

0.791/0.929 * 100 0 100 0 <LO Q

791/929 * 79/93 * LO Q = Limi t o f Quantitatio

n no t applicabl e * = duplicat e sampl e store d a t approximatel

y -2 0 °C prio r t o analysi s Fo r reportin g purposes

, x ha s bee n calculate

d fro mc retrospectively

.Harla n Stud y Number : 4110469 4 Analytica l Investigation

s 

## FIGURE S Figure 1 Calibratio n Plo t Repor t Pag e 3 3Harla n Stud y Number: 4110469

4 Analytica l Investigations Repor t p . 3 5 Pag e 3 4
Figur e 2 Chromatogra m o f Calibratio n Solutio n (1. 0 m g ai/L ) MSD1TK^MSH[e{SVmS7^10me^L57SO2^2901I9 B*fl,StMR8 S 10 0

£00 0

400 0

200 0

\2 —T — 1. 6 Figur e 3 Chromatogra m o f Biologica l Contro l Sampl e MSO I TIC , fv 6 Rl e (SW*EB*^11OMWB2^5O0M)6O 1 D ) E&W.SlMFraf f 10 0
soooaxo7X0

- 6000

- 5000

- 4X0

- 3D00

- 2D0 0 1000

: 0 6 0 8 u 1 5

Respons e see n in tes t medi acontrol subtracte

d fromcontrols an

dtes t sample sp . 3 6

Harla n Stud y Number : 4110469 4 Repor

t Pag e 3 5 Analytica l Investigation

s Figure 4 Chromatogra m o f Tes tSampl

e (100 0 m g ai/L )

~1— ' ' • 1— ' 1 —'—1— i '— ' 1— 1 — 1 1 —I— 1 — ' ' 1— ' ' '— I '— • '— I '—•—'—1— ' ' " 1 ~ 0 2 O

A 06 Q 8 1 1 2 U 1 5 nf np . 3 7 Harla n Stud y Number; 4110469

4 Repor t Pag e 36

Appendi x 3 Physico-Chemica l Measurement s Nomina l Concentration (m g ai/L ) Tim e (Hours ) Nomina l Concentration (m g ai/L ) 0 Hours (Fres h Media ) 2

4 Hour s (Ol dMedia) 2 4 Hour s (Fres h Media ) Nomina l Concentration (m g ai/L ) p H m g 0 2 / L % ASV * T° C p H m g 0 2 / L % ASV * T° C p H m g Oj/ L % AS V T° C Contro l 7. 0 11. 0 10 5 1 3 7. 0 9. 1 8 8 1 4 7. 0 11. 2 10 9 1 4 10 0 7. 1 11. 0 10 5 1 3 7. 1 9. 2 89 1 4 7. 1 11. 4 11 1 1 4 180 7. 1 11. 0 10 5 1 3 7. 1 9. 3 9 0 1 4 7. 0 11. 4 11 1 1 4 320 7. 0 10. 8 10 3 1 3 6. 9 8. 9 8 6 1 4 7, 1 11, 1 10 8 1 4 56 0 7. 0 10. 5 10 0 1 3 7. 1 8. 7 84 1 4 7. 1 11. 1 108 1 4 100 0 7. 1 10. 3 9 8 1 3 7. 1 8. 4 8 2 1 4 7. 1 10. 7 10 4 1 4 Nomina l Concentration (m g ai/L ) Time (Hours) Nomina l Concentration (m g ai/L ) 4 8 Hours (Ol dMedia ) 48 Hours (Fres h Media) 7 2 Hour s (Ol dMedia

) Nomina l Concentration (m g ai/L ) p H m g Oj/ L % ASV * T° C P

H m g Oi/ L % ASV * T° C p H m g 0 2 / L % ASV * T° C Contro l 7. 4 9. 2 8 9 1 4 7. 0 11. 4 11 1 1 4 7. 2 9. 3 9 0 1 4 10 0 7. 3 9. 1 8 8 1 4 7. 1 11. 4 11 1 1 4 7. 2 9. 3 9 0 1 4 18 0 7. 3 9. 1 8 8 1 4 7. 1 11. 3 11 0 1 4 7. 2 9. 3 9 0 1 4 320 7. 3 8. 7 8 4 1 4 7. 0 11. 2 10 9 1 4 7. 1 9. 0 8 7 1 4 56 0 7. 3 8. 6 83 1 4 7. 1 U . l 

## n o 1 4 7. 2 8, 8 8 5 1 4 100 0 7. 3 8. 3 8 1 1 4 7. 1 10. 8 10 5 1 4 7. 2 8. 6 83 1 4 Nomina l Concentration (m g ai/L ) Tim e (Hours ) Nomina l Concentration (m g ai/L ) 7 2 Hours (Fres h Media ) 9

6 Hour s (Ol dMedia

) Nomina l Concentration (m g ai/L ) p H m g Oj/ L % ASV * T° C p H m g Oj/ L % ASV * T° C Contro l 7. 0 11. 0 10 5 1 3 7. 4 9. 2 89 1 4 10 0 7. 0 11. 3 11 0 1 4 7. 4 9. 2 8 9 1 4 18 0 7. 1 11. 2 10 7 1 3 7. 4 9. 2 89 1 4 320 7. 0 11. 1 10 8 1 4 7. 4 8. 8 8 5 1 4 56 0 7. 2 11. 2 10 7 1 3 7. 4 8. 7 84 1 4 100 0 7. 0 10. 8 10 3 1 3 7. 4 8. 6 8 3 1 4

*AS V = Dissolve

d oxyge n concentratio

n expresse

d a s apercentag

e o f Ai r Saturatio n Valu eHarla n Stud y Number : 4110469 4 Repor

t Pag e Appendi x 4 Monitorin g Authorit y Statemen

to f GL P Compliance 

### (DHj Department V_ / 

### of Health
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 Ltd Analvticai/Cfinira i Shardlo w Busines s Par k rhSuJ TM Londo n Roa d
. , - c 1 h f m Shardlo w Environmenta l Toxicit y Derb y Environmenta l Fat e DE7 2 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 Laborator y Practic e wa s carrie d ou t a t th e above tes t facilit y as 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 Laborator y Practic e Monitorin g Authorit y were 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 Laborator y Practice
. Thi s statemen t constitute s a Goo d Laborator y Practice Instrumen t (a s define d I n th e U KGoo d Laborator

y Practic e Regulation s 1999)
.

Dr
. Andre w J
. Gra

y Head , U KGL P Monitorin g Authorit y ^^^ ^ORIGI NID : KCK

A(913 ) 621-773 3 JAK E BOORE M Hf)RCRO S CHEMICAL

S IN C 520 0 SPEAKE

R R D KANSA S CITY . K

S 6610 6 UNITE D STATE S U S SHI P DATE : 25APR1 3 ACTWGT : O. S L

B MA N CAD : O970415^CAFE26O

B BIL L THIR

D PART Y TO 

## (DCO)(7407M)(OPPT

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