# I CBIC Control Number 

# 357499

2013 DEC 

## 2lf Pi'll2• 

## 21

# 1111~11111~~ CHIMICA RER L'AGRICOLTURA

OPPT Document Control Office EPA East Building - Room E6428 U.S. Environmental Protection Agency 1201 Constitution Ave., NW Washington, DC 20004 Attention: Docket ID Number: EPA-HQOPPT-2009-0112 40 CFR 799.5089
Manerbio, 20 1 h December, 2013 Subj: 4-Ch/oro-a,a,a-trif/uoroto/uene (PCBTF): Submission of green algae toxicity study report
Dear Sir/Madam, please find here enclosed the study report N. CH-095/2013 "4-Chloro-a,a,atrifluorotoluene (PCBTF): toxicity to green algae Pseudokirchneriella subcapitata determined in a growth inhibition study" owned by Finchimica S.p.A., Via Lazio 13, Manerbio, Italy, which is submitted in support of KOWA American Corp., 55 East 59 1h Street, Suite 19A, New York, NY 10022. The right of reference to the mentioned study is granted by Finchimica S.p.A. with the purpose of allowing KOWA American Corp. to fulfil the regulatory requirements of U.S.EPA for the substance PBCTF. Yours faithfully,

?

Finchimica S.p.A.
-- Sede legale, Stabilimcnto: Via Lazio, 13
- 25025 Mancrbio (BS)
- Tel. 030 9389.01 Selezione Passante
- Tclefax 030 9389201 Capitalc Sociale Euro 2.250.000,00 int. vers., R.I.- Bs 00619040173 REA
- Bs n" 203922- C:F: 00619040173
- P. IVA 00573160983, M incomes n" Bs 021441 www.finchimica.it- info@finchimcia.it SOCIETA' SOGGETTA AL CONTROLLO E AL COORDINAMENTO DAPARTE Dl FINAGRO S.PA CON SEDEIN MILANO SISTEMA D1 GESTIONE Ai'.IENDJ\LE QUALITA. AMUIENTE. SICUREZZA. CON CERTIFIC:ATO Ill ECCELI..ENZA FINCHIMICA OTTElviPERJ\ ALD. Lgs 23 l/200 Ip . 2 

# Chem Service Controllt 0 ricorcho Fina l Repor t CH-095/201 3 

## GL P COMPLIANC E STATEMEN T 4-CHL0R0-a,a,a-TRIFLU0R0T0LUENE(PCBTF) :

TOXICIT YT O GREE N ALGA E Pseudokirchneriell a subcapitat a DETERMINE D I N AGROWT H INHIBITIO N STUD Y Thi s stud ywa s performe d b yth e Tes t Laborator

yo fChemServic e S.r.l . Controll i e Ricerch e unde rth e directio n o fFrancesc a No6 , i n complianc e wit h th e Stud yPla n CH-095/201 3 an d Goo d Laborator

yPractic e Standard s reporte d i n Italia n Legislativ e Decre e (D. LNo . 5 0 date d Marc h 2 n d , 2007 ) a s publishe d i n G.U . No . 8 6 o fApri l 13 th , 200 7 an d i n complianc e wit h OEC D Principle s o n GLP . Th e result s o fthi s repor tcompletel yan d faithfull yreflect th e RawDat a generate d byth e Study .

Dat e Stud yDirecto r

pag e 2 o f7 9

# Chem

Service CantrolU m riomronm SPONSO R



### Finchimica

S.p.a. Via Lazio, 13 25025 Manerbio

(BS) Italy 

# FINALREPORT
STUD YTITL E 

## 4-CHLORO-a,a s 

## a-TRIFLUOROTOLUENE(PCBTF) :

TOXICIT YT O GREE N ALGA E Pseudokirchneriell a subcapitat a DETERMINE D I N AGROWT H INHIBITIO N STUD Y

AUTHOR S Francesc a Noe , Rachel e Dini , Garagn a Desire e

TES TFACILITY ChemServic e S.r.l . Controll i e Ricerch e GL P Studie s Departmen t Vi a Fratell i Beltrami , 1 5 2002 6 Novat e Milanes e (Ml )

Ital y Ph:+3 9 (0) 2 356996 1 Fax : +3 9 (0 ) 2 3569965 0 G.L.P . Stud yNo . CH-095/201 3 CHEMSERVIC E S.r.l . CONTROLL I E RICERCH E SOCIETA ' SOGGETTAA LCONTROLL O E A LCOORDINAMENT O DAPART E D l FINAGR O S . P . A . CO N SED E I N MILAN O SED E LEGAL E E D OPERATIVA : 2002 6 NOVAT E MILANES E {Ml ) - VI AF.LL I BELTRAMI , 1 5 - TEL . 0 2 35 6 996. 1 - FAX0 2 38 2 01 4 4 6 RE A113284 7 - REGISTR O IMPRES E D l MILANO , CODIC E FISCAL E E P . IV A0702082015 0 - CAPITAL E SOCIAL E € 100.000,0 0 I.V . www.chemservice.it;

e-mail:info@chemservice.itp . 4



## o 

# Chem

Service

Stud yTitle :

Stud yNumber : Controill e rlcorcho Fina l Repor t CH-095/201 3 

## QUALIT YASSURANC E STATEMEN T 4-Choloro-a,a,a-trifluorotoluen e (PCBTF):Toxicityt o gree n alga e Speudokirchneriell a subcapitat a Determine d i n a Growt h Inhibitio n Stud y CH-095/201 3
I n accordanc e wit h ChemServic e S.r.l . Controll i e Ricerch e an d QualityAssuranc e procedure s fo r Goo d LaboratoryPractic e (GLP ) th e conduct o fth e stud yha s bee n inspecte d byQualit yAssuranc e an d th e result s ofinspection s wer e reporte d t o Stud yDirecto ran d Management . Stud yInspections : Date(s ) o fInspectio n Inspectio n Reportin g Date(s ) 2 1 March 201 3 StudyPlan 2 1 March 201 3 03 Ma y201 3 Draf tRepor tan d Ra wDat a 03 Ma y201 3 30 Ma y201 3 Fina l Repor t 30 Ma y201 3 I n additio n othe rproces s inspection s wer e periodicall ycarrie d ou t byQualityAssuranc e o n activitie s no tnecessaril yrelate d t o thi s stud yan d th e result s wer e reporte d t o Stud yDirecto ran d Tes t Facility Management . Th e mos trecen t proces s inspectio n i s identifie d below . Proces s Inspection : Date(s ) o fInspectio n Inspectio n Reportin g Date(s ) 03 Apri l 201 3 Acute Toxicit yto Gree n Algae 03 Apri l 201 3



## o Associate d laboratorie s an d suppor t function s wer e subjectt o regula rfacilit yinspection s i n accordanc e wit h QualityAssuranc e procedures . Th e result s i n thi s report completel yan d faithfull yreflectth e rawdat a generate d byth e study .
Debora Manera , Hea d o fQualit yAssurance , ChemServic e S.r.l . Controll i e Ricerch e Dat e

pag e 3 of7 9

# ChemService Controlli o rlooroho Fina l Repor t CH-095/201 3 

## STUD YIDENTIFICATIO N 4-CHL0R0-a,a,a-TRIFLU0R0T0LUENE(PCBTF) :

TOXICIT YT O GREE N ALGA E Pseudokirchneriell a subcapitat a DETERMINE D I N AGROWT H INHIBITIO N STUD Y
Stud yPla n Tes tite m Sponso r

Tes tFacilit y CH-095/201 3 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) Interna l sampl e code : 2911434-00 1 Batc h number : 11/201 2 Sampl e supplie d byth e Sponso r Finchimic a S.p.a . Vi a Lazio , 1 3 2502 5 Manerbi o (BS ) Ital y ChemServic e S.r.l . Controll i e Ricerch e GL P Studie s Departmen t Vi a Fratell i Beltrami , 1 5 2002 6 Novat e Milanes e (Ml ) Ital y Ph : +3 9 (0 ) 2 356996 1 Fax : +3 9 (0 ) 2 3569965 0 Stud yDirecto r Francesc a No e 

### Schedul e Initiatio n dat e Star to fexperimen t Star to fi n viv o experimen t Completio n o fi n viv o experimen t Star to fanalytica l phas e Completio n o fanalytica l phas e Completio n o fexperimen t Marc h 22,201 3 Ap r Ap r Ap r Ap r Ap r Ap r 102,201 3 102,201 3 I 05 , 201 3 102,201 3 I 16 , 201 3 I 23 , 201 3 pag e 4 o f7 9

# ChemService Controllio rtamrohmT Fina l Repor t CH-095/201 3 

## CONTENTS/ 1 pag e GL P Complianc e Statemen t2 QualityAssuranc e Statemen t3 Stud yIdentificatio n 4

Schedul e 4 Contents/ 1 5 Contents/

2 6 Lis to fAppendice s 6 KeyPersonne l 7 Summar y7 Introductio n 9 Material s an d method s 9 Tes tite m 9 Referenc e Ite m 1 1 Equipmen tfo rBiologica l Phas e 1 1 Definition s 1 1 Tes tsyste m 1 2 Experimenta l desig n 1 2 Dosag e an d concentration s 1 2 Experimenta l condition s 1 3 Evaluation s 1 5 Observation s an d determinatio n o fE y C X tE r C x an d th e NOE C 1 5 Calculation s 1 6 Measuremen t o fth e tes t concentration s 1 7 Calculatio n o fth e actua l tes t concentration s 1 7

pag e 5 o f7 9p . 7 

# ChemService Controlli im

rlcorohm Fina l Repor t CH-095/201 3 

## CONTENTS/

2 pag e Alga l strai n sensitivitychec k 1 7 Condition s fo rth e validityo fth e tes t1 8 Result s an d discussio n 1 8 Growt h rat e 2 5 Yiel d 2 7 Conclusion s 2 8 Reference s 2 9 Archivin g 2 9



## LIS TO F APPENDICE S pag e Appendi xA- Analytica l Phas e Repor t 3 0 Appendi xB - Tes tTemperatur e 5 8 Appendi xC - Statistica l Analysi s 6 0 Appendi xD - Cop yo fTes tIte m Certificat e o fAnalysi s 6 6 Appendi xE - Cop yo fReferenc e Ite m Certificat e o fAnalysi s 6 8 Appendi xF - Copyo fExtractio n Certificatio n o fAlga l Strai n 7 0 Appendi xG - Compositio n o fTes t Cultur e Mediu m (OEC D N 201 , 2011 ) 72 Appendi xH - Copyo fGL P Authorizatio n b yItalia n Healt h Departmen t74

pag e 6 o f7 9

## o p . 8



# ChemService Controlli 0 rtaorcho Fina l Repor t CH-095/201 3 

## KE YPERSONNE L o Stud yDirecto r Francesc a No e (f.noe@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e Stud yDirecto rdeput y Eric a Tedios i (e.tediosi@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e Tes tFacilit yManage r Giacom o Ravett a (giacomo.ravetta@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e Hea d o fQualit yAssuranc e Debora Manera (d.manera@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e Sponsor' s Representativ e Paol o Bett i (p.betti@finchimica.it ) Finchimic a S.p.a . Technicia n (biologica l phase ) Rachel e Din i (r.dini@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e Technicia n (analytica l phase ) Desire e Garagn a (d.garagna@chemservice.it ) ChemServic e S.r.l . Controll i e Ricerch e 

## SUMMAR Y Th e influenc e o fth e tes t ite m 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) o n th e growt h o fth e gree n alga l specie s Pseudokirchneriel/a subcapitata , formerl yknow n a s Selenastru m Capricornutum , wa s investigate d i n a 72-hou rful l tes taccordin g t o th e OEC D Guidelin e No . 201 , 2011 . Fo rthi s purpose , exponentiall ygrowin g tes t alga e wer e expose d fo r7 2 hours t o a n aqueou s tes tmediu m containin g th e tes t ite m a t fiv e differen t concentration s i n a geometri cseries , namel y7.9 , 11.9 , 17.8 , 26. 7 an d 40. 0 mg/ Lcorrecte d fo rit s purity(99. 3 % w/w) , unde rdefine d conditions . Beside s th e concentration , a negativ e contro l withou tth e tes tite m wa s als o prepare d t o chec k th e acceptabilit yo fth e alga l inoculum .

pag e 7 o f7 9p . 9 ) 

# ChemService ^^^^^^^^Z " Controti l 0 rtc&rcn * " 1 Fina l Repor

tCH-095/201 3 A tth e beginnin g o fth e test , th e p H valu e o fth e tes t mediu m range d betwee n 7.6 2 - 7.7 7 fo r tes tite m solution s an d wa s 7.9 1 fo rth e negativ e control . A tth e en d o fth e tes tth e p H value s o fnegativ e contro l range d betwee n 9.28 - 9.3 3 an d th e p H mea n value s o ftes t ite m solution s wer e i n th e rang e 9.3 1 - 9.60 . Th e incubatio n temperatur e wa s i n th e rang e 23. 3 ° C - 24. 1 °C . Ligh tintensitydurin g th e 72-hou rperio d wa s i n th e rang e 522 0 - 540 1 Lux . Th e actua l tes t concentratio n o fth e tes t ite m were analyticall ymeasure d a tth e beginnin g an d a t th e en d o fth e test . Additiona l sampling s fo ranalysi s a t2 4 hou rinterval s durin g th e exposur e perio d wil l b e take n i n orde rt o bette rdefin e los s o fth e tes titem . Th e analytica l recover

yo fth e concentration s o ftes t ite m i n th e freshl yprepare d solution s range d betwee n 72. 6 % an d 87. 1 % o fth e nomina l concentratio n o fth e tes titem . Afte r24 , 48 , an d 7 2 hours ofexposure , th e analytica l recover ywer e lowe rtha n th e limi t o f detectio n (L.O.D . = 100. 0 pg/L ) o fth e analytica l method , sinc e tes tite m evaporate d durin g th e 72-hou rperiod . Th e analytica l recoverie s wer e referre d t o th e tes t ite m conten t(correcte d fo rit s purity , 99. 3 %) . A s recommende d byOEC D Guidanc e Documen to n testin g o fdifficul t substance s No . 2 3 (2000) , th e biologica l result s wer e referre d t o th e geometri cmea n o ftes t ite m measure d concentrations , sinc e th e analytica l recoverie s oftes tite m wer e lowe rtha n th e rang e 8 0 % -12 0 % . Alga l cel l densitywa s measure d ever y2 4 hours takin g aliquot s fro m eac h tes tconcentratio n replicat e an d fro m negativ e control , dilutin g the m i n a NaC I 9 g/ Lsolutio n an d readin g wit h a n electroni cparticl e counter . A tth e tes ten d th e cel l densityi n th e negativ e contro l increase d o n averag e b ya facto ro f239 . Thi s valu e complie s wit h th e validitycriterio n o fth e test , accordin g t o th e mentione d guidelines , whic h indicate s a minimu m increas e facto ro f16 . Th e negativ e contro l als o metth e othe rvaliditycriterion , wit h a coefficien to fvariatio n o fdail y growt h rate s a t72 hours o f25. 6 % an d a coefficien to fvariatio n o faverag e growt h i n replicate s negativ e contro l culture s durin g th e tes t perio d o f1. 2 % . Fo rth e teste d concentrations , an ysignifican tbiologica l effectwa s showe d i n compariso n wit h negativ e contro l fo rbot h th e endpoints , yiel d an d growt h rate , afte r72 hours o fexposure .

pag e 8 o f7 9p . 1 0

Fina l Repor

t CH-095/201 3 

# ChemService

Controlli© rtomrchm

Th e 72-hou rEC 1 0 , EC 2 o , EC 5 0an d th e NOEC , calculate d i n term s o fgeometri cmea n o f measure d concentrations , fo rth e tw o end-points , ar e reporte d i n th e followin g table : Endpoin t 0-72hECi o 0 - 72 h EC 2 0 0 - 72 h EC 5 0 0 - 72 h NOE C Growt h rat e >0.4 1 >0.4 1 >0.4 1 0.4 1 Yiel d >0.4 1 >0.4 1 >0.4 1 0.4 1 

## INTRODUCTIO N Th e stud ywa s performe d t o evaluat e th e effecto fth e tes t ite m 4-chloro-a ) a,a-trifluorotoluen e (PCBTF ) o n th e growt h o fth e gree n alga l specie s Pseudokirchneriell a subcapitat a (Selenastru m capricornutum) . Th e Stud ywa s performe d i n complianc e wit h th e Stud yPla n CH 095/201 3 an d th e followin g guidelines : OEC D Guidelin e No . 201 , "Freshwate ralga e an d cyanobacteri a growt h inhibitio n test" , 2011 . OEC D Serie s o n Testin g an d Assessmen tN o 2 3 - Guidanc e Documen tO n Aquati cToxicit y Testin g O fDifficul tSubstance s An d Mixture s ENV/JM/MONO(2000)6 . Th e tes tmetho d an d th e tes tspecie s Pseudokirchneriell a subcapitat a ar e recommende d byth e internationa l tes tguidelines .



## MATERIAL S AN D METHOD S

Tes t ite m Asampl e o f4-chloro-a,a,a-trifluorotoluen e (PCBTF) , containe d i n a glas s bottle , wa s receive d byth e Tes tFacilityo n Februar y12 , 201 3 (interna l sampl e code : 2911434-001) .

pag e 9 o f7 9p . 1 1



# ChemService C'ontroifl 0 rtcorch* Fina l Repor t CH-095/201 3 Informatio n o n th e tes tite m i s a s follow s (th e tes tite m an d it s relevan t informatio n wer e provide d b

yth e Sponsor) : Tes tite m nam e Chemica l nam e CAS . numbe r EE C numbe r Molecula rweigh t Relativ e density(2 0 Empirica l formul a 'C ) 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) 4-chloro-a,a,a-trifluorotoluen e 98-56- 6 202-681- 1 180.5 5 g/mo l 1.336 1 g/m L(determine d i n n o GL P ChemServic e stud y numbe rCH-046/2013 ) CIC6H4CF

3

Structura l formul a

Purity Batc h numbe r Interna l numbe r Manufacturin g dat e Expir ydat e Storag e condition s

Stability 99.3 % (fro m Co Aan d TSD S supplie d bySponsor) 11/201 2 2911434-00 1 Januar y04 , 201 3 Januar y201 6 Th e tes t item , containe d i n th e origina l close d an d seale d bottle , i s store d a troo m temperatur e fa rawayfro m source s o fheat , i n accordanc e wit h Tes tSubstanc e Dat a Shee t(TSDS ) an d Materia l SafetyDat a Shee t(MSDS ) supplie d byth e Sponsor . n o therma l decompositio n whe n th e product i s store d an d handle d correctl y Th e tes tite m CoAi s annexe d i n Appendi xD t o thi s report .

pag e 1 0 of7 9

## o p . 1 2



# ChemService Controlli o rloorcho Fina l Repor t CH-095/201 3 

## Referenc e Ite m Referenc e ite m nam e 3,5-Dichloropheno l pestana l analytica l standar d CAS . numbe r591-35- 5 Batc h numbe r7262 X O Preparatio n dat e : Septembe r19,200 7

Expir ydat e Septembe r19 , 201 4 Chemica l formul a : C 6 H 4 Cl2 0 Molecula rweigh t163.0 0 g/mo l Storag e condition s Roo m temperature/wel l ventilate d roo m StabilityStabl e unde rstorag e condition s Th e referenc e ite m CoAi s annexe d i n Appendi xE t o thi s report .



## Equipmen t fo rBiologica l Phas e

- Shakin g apparatus , Falc , Interna l cod e No . 24 7

- p H meter , WT W315i , Interna l cod e No . 24 4

- Lamina rflo wbench , Interna l cod e No . 26 0

- Optica l microscop e N o 32 1

- Electroni cparticl e counter , Beckma n coulte rZ2 , Interna l cod e No . 55 2

- Magneti cstirre rtyp e AGE , Vel p Scientific , Interna l cod e No . 25 6

- Lux-meter , Test o 545 , Interna l cod e No . 25 2

- Micropipett e BIOHI Tprolin e (20-20 0 pL) No . 170/E 6

- Laborator

yWate rPurificatio n Systems , Sartoriu s Arium ® 611 , Interna l cod e No . 35 5

- Datalogge rTesto , T-174 , Interna l cod e No . 47 7 (precision : 0.48 ; correctio n factor , Cw0. 2 °C )

- Climati cchamber , KBW40 0 Binder , Interna l cod e N o 63 7

O Biomass :

Growth : Growt h rate : Yield : EYC X : E r C x : 

## Definition s

cell s numbe rpe rmillilitre . th e increas e i n cel l densityove rth e tes tperiod . th e increas e i n cel l densitype rtim e unit . th e biomas s a t th e en d o fth e exposur e perio d minu s th e biomas s a t th e starto fexposur e period . th e calculate d concentratio n o ftes tite m whic h result s i n a X % reductio n o f yiel d (Y ) compare d t o th e control . th e calculate d concentratio n o ftes tite m whic h result s i n a X % reductio n o f growt h rat e (r) compare d t o th e control . pag e 1 1 o f7 9p . 1 3 1



# ChemService

Controlli o rtomronm Fina l Repor t CH-095/201 3 NOEC : th e concentratio n o ftes t ite m whic h result s i n a not significan treductio n o f

yiel d (Y ) o rgrowt h rat e (r) compare d t o th e negativ e control .



## Tes t syste m Th e stud ywa s performe d wit h th e unicellular , fresh-wate rgree n alga e Pseudokirchneriell a subcapitat a strai n (formerl yknow n a s Selenastru m capricornutum) , culture d i n th e laboratorie s o fth e Tes t Facilityan d originall ypurchase d fro m th e Institut e o fPlan tPhysiolog yo fth e Universityo fGbttingen , Germany . Th e alga e wer e culture d i n a climati cchambe ra t2 4 ° C ±2 ° C unde rcontinuou s unifor m illuminatio n i n th e rang e 444 0 - 888 0 Lu xi n th e spectra l rang e 400-70 0 nm . Th e cultur e mediu m wa s th e sam e o fth e tes tmedium ; th e stoc k culture s wer e weekl ytransferre d t o fres h mediu m an d maintaine d i n continuou s shakin g t o ensure th e necessar yamoun to fC0 2an d t o kee p alga e i n suspension . Culture s containin g deforme d o rabnorma l cell s wer e discarded . Onl y exponentiall ygrowin g alga l culture s have bee n use d t o star tth e test . Th e cop yo fextractio n certificatio n i s attache d i n Appendi xF .



## Experimenta l Desig n Dosag e an d Concentration s Th e tes tconcentration s wer e chose n i n accordanc e wit h th e result s o fa pre-tes t(range-findin g test) , whic h wa s performe d no ti n complianc e wit h GL P Regulation s an d whos e Ra wDat a wer e archive d unde rth e cod e o fth e presen t study . Th e mai n tes tinclude d fiv e tes tite m concentration s i n a geometri cseries , namel y7.9 , 11.9 , 17.8 , 26. 7 an d 40. 0 mg/L , correcte d fo rit s purity(99. 3 % w/w) . Th e tes tmediu m wa s prepare d a s follows : prio rt o th e tes tstart , a 1 -litr e bottl e totall yfille d wit h 117 0 m Lo freconstitute d water , o fa 40. 0 mg/ L o fth e tes tite m solutio n wa s prepare d b ydosin g wit h a micropipett e 35.5. 0 \x Lo ftes t item , correspondin g t o 47. 4 m g o ftes tite m (considerin g it s density:1.336 1 g/m L, determine d i n n o GL P ChemServic e stud ynumbe rCH-046/2013 , an d it s purity99.3%) . Th e bottl e wa s tightl yclose d t o preven ttes tite m evaporation . Th e obtaine d solutio n appeare d transparen twit h oil ydroplet s o n th e botto m o fth e bottie , therefor e i twa s mixe d fo rtw o hours b yth e mea n o fa magneti cstirre ri n dar k condition s t o achiev e th e tota l solubilisatio n o ftes t item . Afte rstirring , th e solutio n appeare d homogenou s an d transparent . Afte rp H check , , th e tes tconcentration s wer e prepare d bytakin g adequat e aliquot s o fth e stoc k solutio n an d dilutin g the m int o reconstitute d wate ra s reporte d i n Tabl e 1 . Tes tflasks wer e inoculate d wit h alga l culture . Anegativ e contro l wit h alga l growt h mediu m onl ywa s als o tested . pag e 1 2 o f7 9

# o 

# ChemService Controlli « r/oaroh s
Tabl e 1 Tes t solution s preparation , Fina l Repor t CH-095/201 3



### O Teste d solution s Nomina l Tes t ite m

concentratio n (mg/L ) Fina l volum e o f

reconstitute d wate r (mL ) Adde d volum e o f

40. 0 mg/ Lsolutio n (mL ) 1 7. 9 25 0 49. 4 2 11. 9 25 0 74. 4 3 17. 8 25 0 111. 3 4 26. 7 25 0 166. 9 

## Experimenta l Condition s Th e treatmen tvessel s wer e identifie d b yexperimen tnumber , grou p an d replicat e number . Al l th e describe d procedure s wer e conducte d unde ra lamina rflo wbenc h t o ensur e th e sterility o fth e alga l cultures . Fo rth e tes tconcentration s an d fo rth e negativ e control , 10 0 m Lcapacit yconica l glas s flasks cappe d wit h ai rpermeabl e stopper s wer e used . Thre e replicate s fo rtes tite m concentration s an d si xfo rnegativ e contro l wer e prepared . A t tes t start , 5 0 m Lo ftes t solution s wer e poure d int o th e tes tflasks an d inoculate d wit h alga e t o obtai n a startin g cel l densityo fabou t10 4cells/mL . Th e alga l inoculu m wa s take n fro m a pre-culture , starte d 3 day s befor e th e beginnin g o fth e tes t i n orde rt o ensur e tha t th e assaywa s performe d wit h exponentiall ygrowin g alga l population . Th e cel l densityo fth e inoculu m wa s aroun d 10 6 cells/mL . Th e tes tflask s wer e incubate d i n a climati cchamber , unde rcontinuou s illuminatio n an d constan tshaking . Th e flasks wer e randoml ydistribute d unde rth e ligh tan d wer e replace d ever y daydurin g th e test . Temperature : Th e roo m temperatur e unde rth e ligh twa s continuousl ymonitore d durin g th e cours e o fth e stud ybyth e mea n o fa dat a logger . I t wa s i n th e rang e 23.3° C - 24. 1 ° C wit h a mea n valu e o f O 24. 0 ° C an d a standar d deviatio n o f0. 1 °C .

Th e temperatur e rang e provide d byOEC D guidelin e i s 2 4 ±2 C , thi s rang e wa s correcte d accordin g t o th e dat a logge rprecisio n (therefor e th e rang e t o b e considere d fo rth e tes t wa s 22. 5 - 25. 5 °C ) whil e temperatur e value s wer e correcte d accordin g t o correctio n facto r(Cw) . Measure d temperatur e value s are reporte d i n Figure 1 .

pag e 1 3 o f7 9

# ChemService Controlli o rlomrchm p . 1 5

Fina l Repor

tCH-095/201 3

C D a E .C D 27 26 25 24 23 22 2 1 20 1 9 

# r

^jS ^ Tim e (days ) Fig . 1 Temperatur e tren d durin g th e tes t perio d (blu e line) ; th e re d line s indicat e th e rang e

o facceptabl e temperature . Ligh tconditions : continuou s illumination ; ligh tintensitywa s range d betwee n 522 0 an d 540 1 Lux , withi n th e OEC D recommende d rang e (444 0 - 888 0 Lux) . Tes tduration : 72 hours Tes tmedium : Reconstitute d wate raccordin g t o OEC D Guidelin e 201 , 201 1 (se e Appendi xG) . Tes tmediu m p H wa s measure d i n th e tes t ite m solution s an d i n th e alga l cultur e mediu m a t th e beginnin g o fth e tes tan d i n eac h tes t flas k a t th e en d o fth e test . Mea n value s ove rth e replicate s ar e reported . Th e p H value s o ftes tmedi a wer e measure d wit h a portabl e pHmeter . p H value s wer e 7.9 1 fo r th e negativ e contro l an d range d betwee n 7.6 2 - 7.7 7 fo rth e tes t ite m solution s at th e beginnin g o fth e tes tperio d an d i n th e rang e 9.3 1 - 9.6 0 a tth e end . Th e mea n value s are reporte d i n Tabl e 2 . Th e p H i n th e negativ e contro l varie d b ya maximu m o f1.43 units , thi s valu e wa s considere d acceptabl e thoug h i twa s slightl yhighe rtha n th e recommende d limi tbyth e OEC D guidelin e statin g tha t th e p H value s i n th e negativ e contro l replicate s shoul d no tincreas e more tha n 1. 5 unit s durin g th e tes tperiod . Fo rfres h solution s th e p H value s wer e measure d samplin g a n aliquo timmediatel yafte rtes t solutio n preparation , befor e pourin g i ti n th e tes t flasks . Fo rspen t solution s (afte r7 2 hours) , p H wer e measure d i n eac h tes tflask . pag e 1 4 of7 9p . 1 6



# ChemService Controlit«rlaoroh* Fina l Repor t CH-095/201 3 Tabl e 2 p H value s i n th e tes tmedi a a t th e star t an d atth e en d o fth e test . Nomina l tes tite m concentration s (mg/L ) Star t( 0 hours ) p H En d (7 2 hours ) p H (mea n values ) 0. 0 (negativ e control ) 7.9 1 9.3 1 7. 9 7.7 7 9.3 1 11. 9 7.6 2 9.4 2 17. 8 7.7 7 9.5 2 26. 7 7.7 4 9.6 0 40. 0 7.6 4 9.5 0



## Evaluation s Observation s an d determinatio n ofE y 

## C x 

## , E r 

## C x 

## an d th e NOE C Alga l cel l densitywa s measure d ever y2 4 hours takin g aliquot s fro m eac h replicat e o fnegativ e contro l an d tes t ite m concentration , dilutin g the m i n a NaC I 9 g/ Lsolutio n an d readin g wit h a n electroni cparticl e counter . Th e measure d value s wer e use d t o calculat e th e percentag e inhibitio n o fcel l growt h a s yiel d an d a s growt h rat e i n compariso n t o th e negativ e control . Th e CETI S V.1.026 D softwar e wa s use d t o carr you tth e statistica l analysis : i t automaticall y choose s th e mos t appropriat e analysi s fo rth e poo l o fdata . Th e EyC xan d th e E r C xvalue s wer e determine d bya Linea rInterpolatio n analysis . Th e NOE C valu e fo ryiel d an d growt h rat e endpoin twer e determine d bycompariso n wit h th e control , byBonferron i Ad j t method . A s inpu tdata , th e geometri cmea n ofmeasure d concentration s o ftes tite m 4-chloro-a,a,a trifluorotoluen e (PCBTF ) wer e used . Th e negativ e contro l wa s considere d als o t o chec k tha tth e growt h o fth e alga l populatio n use d i n th e tes tmeet s th e validitycriteria . Detail s o fth e statistica l analysi s ar e reporte d i n Appendi xC .
pag e 1 5 o f7 9p . 1 7 

# ChemService Controll i a rlcorch o "~ ' ——— TM Fina l Repor

t CH-095/201 3 

## Calculation s Averag e growt h rat e Th e averag e growt h rat e wa s calculate d accordin g t o th e followin g formula : I n X j - I n X s 

### tj-ti (day 1 

### ) (j. i-j is the average specific growth rate from time i to j; Xi is the biomass

at time /; Xj is the biomass

at time j Th e percen tinhibitio n o fgrowt h rat e fo reac h treatmen t replicat e fro m equatio n wa s calculate d accordin g t o th e followin g formula : % / f = - X10 0

jUC % l rpercen tinhibitio n i n averag e specifi cgrowt h rate ; j ! cmea n valu e fo raverag e specifi cgrowt h rat e (y ) i n th e contro l group ; p, Taverag e specifi cgrowt h rat e fo rth e treatmen treplicat e Yiel d Th e percen tinhibitio n i n yiel d (%l y ) wa s calculate d a s follows : (Y c- YT ) % !y = X10 0 Y c % l ypercen tinhibitio n o fyield ; Y cmea n valu e fo ryiel d i n th e contro l group ; YTvalu e fo ryiel d fo rth e treatmen t replicat e

pag e 1 6 o f7 9p . 1 8



# ChemService Fina l Repor t CH-095/201 3 Controlli * rtooronm 

## Measuremen to fth e tes tconcentration s Th e concentratio n o ftes tite m i n th e tes t mediu m wa s analyticall ymeasure d a s describe d i n th e analytica l phas e report(Appendi xAt o th e presen tdocument) . 1 0 m Lsample s o ftes t ite m solution s an d negativ e contro l wer e collecte d fro m freshl yprepare d solution s a ttim e 0 an d a tth e en d o fth e tes tperio d (7 2 hours ) afte rremovin g alga l cell s by centrifugation . Duplicate s sample s o fth e tes t ite m solution s an d th e negativ e contro l wer e take n a t th e indicate d time s fro m th e tes tsolutions , bu tonl yon e sampl e wa s analysed , whil e th e othe rwer e store d a s retai n sample s t o b e analyse d onl yi fnecessary . A ttim e 2 4 an d 4 8 hours wer e collecte d a n aliquo t o f1 4 m L(1 0 m Lfo ranalysi s an d 4 m La s retaine d sample ) fo reac h concentrations . 

## Calculatio n o fth e actua l tes tconcentration s Accordin g t o th e guidanc e documen t o n difficul tsubstance s (OEC D No . 23 , 2000) , i n a semi stati ctest , i fth e concentratio n o fth e tes t ite m doe s no t remai n withi n 8 0 % -12 0 % o fth e nomina l concentrations , th e effect concentration s shoul d b e determine d an d expresse d i n term s o fgeometri cmea n o fth e measure d concentrations , i n orde rt o tak e int o accoun t bot h th e initia l an d th e fina l actua l concentrations . Sinc e th e analytica l recoverie s o ftes t ite m durin g th e tes tperio d (i n th e fres h an d spen t tes t ite m solutions ) wer e lowe rtha n th e rang e 8 0 % -12 0 % (75. 7 % i n fres h solutio n an d lowe rtha n th e L.O.D . i n th e spen tsolution) , th e geometri cmea n o fmeasure d concentration s wa s calculate d (se e Tabl e 4 ) an d th e toxi cendpoint s wer e calculate d i n term s o fgeometri cmea n o f tes tite m measure d concentrations .



## Alga l strai n sensitivit ychec k Th e result s o fth e mos trecentl yperforme d tes t wit h a referenc e substanc e ar e reporte d an d compare d wit h literatur e dat a t o demonstrat e tha tth e alga l strai n use d ha s go t a n adequat e leve l o fsensitivit yt o tha t substance . I n th e curren tcas e th e result s refe rt o a tes t performe d o n Marc h 201 3 (CH-167/2013 ) wit h 3,5-Dichloropheno l (se e Certificat e o fAnalysi s i n Appendi xE ) a s referenc e ite m (Tabl e 3) . Table 3 Effect

of 3,5-dichlorophenol

on the growth of Pseudokirchneriella

subcapitata Referenc e 7 2 h E r C 5 0(mg/L ) Confidenc e interva l (95% , mg/L ) Interna l stud yCH-167/201 3 2.5 7 2.5 1 -2.6 3 Referenc e dat a (UN I E N IS O 8692/2004 ) 2.0 8 -4.6 8 pag e 1 7 o f7 9p . 1 9



# ChemService Con trolli o rloortsho Fina l Repor tCH-095/201 3 

## CONDITION S FO R TH E VALIDIT YO F TH E TES T

•Ates ti s no tconsidere d acceptabl e i fth e cel l densityi n th e contro l culture s ha s no tincrease d bya facto ro fa tleas t 1 6 withi n th e tes tperiod . •Th e coefficien to fvariatio n o fdail ygrowt h rate s i n th e contro l culture s mus tnotexcee d 35% . •Th e coefficien to fvariatio n o faverag e growt h i n replicate s contro l culture s mus t no t excee d 7% . 

## RESULT S AN D DISCUSSIO N Afull test was performed

to evaluate

the effect of the test item 4-chloro-a,a,a-trifluorotoluene (PCBTF)on

Pseudokirchneriella

subcapitata. Th e obtaine d cel l densitie s fo reac h replicat e are reporte d i n Tabl e 6 . A s show n i n Tabl e 5 , th e analytica l recoverie s o fth e concentration s o ftes t ite m i n th e freshl y prepare d solution s range d betwee n 68. 5 % an d 87. 1 % o fth e nomina l concentratio n o fth e tes t item . A tth e 24 , 4 8 an d 7 2 hours th e analytica l recoverie s wer e lowe rtha n th e limi t o fdetectio n (L.O.D. ) o fth e analytica l method . Th e analytica l recoverie s wer e referre d t o th e tes tite m conten t (correcte d fo rit s purity , 99. 3 %) . A s recommende d byth e OEC D Guidanc e Documen t o n testin g o fdifficul t substance s N . 2 3 (2000) , sinc e th e analytica l recoverie s wer e noti n th e rang e 8 0 % - 12 0 % durin g th e tes t period , th e biologica l result s referre d t o th e geometri cmea n o fmeasure d concentration s o ftes t ite m (Tabl e 4) .

pag e 1 8 o f7 9

# o p . 2 0



# ChemService Controlli m

rloorcht Fina l Repor t CH-095/201 3 Tabl e 4 Geometri cmea n o fmeasure d concentration s o ftes titem . 

### C Nomina l Tes t ite m concentratio n [mg/L ] Measure d concentration s [pg/L ] Geometri c mea n [M9/L ] Geometri c mea n [mg/L ] Nomina l Tes t ite m concentratio n [mg/L ] t= 0 h t=24 h t= 48 h t = 72 h Geometri c mea n [M9/L ] Geometri c mea n [mg/L ] 7. 9 5734. 9 100.0 * 100.0 * 100.0 * 275. 2 0.2 8 11. 9 10362. 0 100.0 * 100.0 * 100.0 * 319. 1 0.3 2 17. 8 13633. 5 100.0 * 100.0 * 100.0 * 341. 7 0.3 4 26. 7 19678. 7 100.0 * 100.0 * 100.0 * 374. 5 0.3 7 40. 0 27394. 0 100.0 * 100.0 * 100.0 * 406. 8 0.4 1
* Whe n th e measured concentratio n i s lowe rtha n th e L.O.D . ofth e analytica l method , th e L.O.D . itsel f (100. 0 pg/L) i s used fo rgeometri cmean calculation , a s state d i n OEC D No . 2 3 (2000) .



### O

pag e 1 9 o f7 9p . 2 1



# ChemService

Controll i o rfoervh s Fina l Repor tCH-095/201 3 Tabl e 5 Analytica l recover yo fth e measure d tes tconcentration s t o th e nomina l one s Tim e (hours ) Nomina l Tes t

ite m concentratio n fmg/L l analytica l

recover y [% ] Mea n analytica l recover y (%) 0 7. 9 72. 6 0 11. 9 87. 1 0 17. 8 76. 6 75. 7 0 26. 7 73. 7 0 40. 0 68. 5 2 4 7. 9 * 2 4 11. 9

_ * 2 4 17. 8 *

- 2 4 26. 7

_ * 2 4 40. 0

_ * 4 8 7. 9 * 4 8 11. 9

_ * 4 8 17. 8

_ * - 4 8 26. 7

_ * 4 8 40. 0

_ * 72 7. 9

_ * 72 11. 9 * 7 2 17. 8

_ *
- 7 2 26. 7 * 7 2 40. 0 * *th e analytica l recover

ywer e no tdetermined because th e measured concentratio n i s lowe rtha n th e L.O.D . o fth e analytica l method .

pag e 2 0 o f7 9

# o p . 2 2



# ChemService Controlli o rlooroho Fina l Repor t CH-095/201 3 I n Tabl e 6 th e effecto ftes t ite m o n cel l densityi s shown .


### O

C Nomina l tes tite m concentratio n [mg/L ] replicat e Cel l density(n . cell s x10 4 /mL ) Nomina l tes tite m concentratio n [mg/L ] replicat e 2 4 h 4 8 h 7 2 h A 3.867 0 45.168 0 314.837 0 B 3.634 0 35.648 0 287.657 0 0. 0 (negativ e control ) C 3.848 0 34.828 0 294.217 0 0. 0 (negativ e control ) D E 3.786 0

3.859 0 32.688 0

40.968 0 260.617 0

311.197 0 F 4.257 0 33.328 0 286.477 0 Averag e valu e 3.875 2 37.104 7 292.500 3 A 4.842 0 41.287 0 329.922 0 7. 9 B 5.116 0 48.267 0 331.902 0 7. 9 C 3.411 0 43.987 0 334.082 0

Averag e valu e 4.456 3 44.513 7 331.968 7 D 4.391 0 47.162 0 386.621 0 11. 9 E 4.867 0 59.722 0 387.441 0 11. 9 F 4.814 0 51.002 0 393.761 0

Averag e valu e 4.690 7 52.628 7 389.274 3 D 4.929 0 51.038 0 367.002 0 17. 8 E 4.434 0 51.258 0 395.222 0 17. 8 F 4.551 0 47.998 0 371.102 0

Averag e valu e 4.638 0 50.098 0 377.775 3 D 4.349 0 47.447 0 341.938 0 26. 7 E 4.976 0 48.927 0 346.278 0 26. 7 F 3.700 0 47.707 0 338.818 0

Averag e valu e 4.341 7 48.027 0 342.344 7 D 4.768 0 50.178 0 380.812 0 40. 0 E 4.447 0 50.438 0 366.152 0 40. 0 F 4.954 0 56.958 0 389.432 0

Averag e valu e 4.723 0 52.524 7 378.798 7

pag e 2 1 o f7 9p . 2 3 

# ChemService Controlli a rtooroho Fina l Repor tCH-095/201 3 A ttes ten d cel l densit yi n th e negativ e contro l resulte d increase d o n averag e b ya facto ro f293 . Thi s valu e complie s wit h th e validit ycriterio n o fth e test , accordin g t o th e mentione d guidelines , whic h indicate s a minimu m increas e facto ro f16 . Th e negativ e contro l als o me tth e othe rvalidit ycriterion , wit h a coefficien to fvariatio n o fdail y growt h rate s o f25. 6 % an d a coefficien to fvariatio n o faverag e growt h i n negativ e contro l replicate s durin g th e tes tperio d o f1. 2 % . Th e growt h curve s fo reac h replicat e o fth e negativ e contro l ar e represente d i n Figur e 2 ; th e growt h curve s fo reac h concentratio n o ftes tite m ar e represente d i n Figure s fro m 3 t o 7 , whil e Figur e 8 represent s th e compariso n betwee n th e growt h curve s o fth e differen tconcentration s an d o fth e negativ e contro l calculate d o n th e mea n densit yvalues . 350000 0 300000 0 j 250000 0 £200000 0 £150000 0 ( A | 100000 0 50000 0 0 * 0 -• — a -»- b c •*- d e -•— f 2 0 4 0 tim e (hours ) 6 0 8 0 Fig . 2 Growt h curve s fo reac h replicat e (a-f ) o fth e negativ e contro l



### 400000 0 350000 0 300000 0 250000 0 200000 0 150000 0 100000 0 50000 0 0 2 0 4 0

tim e (hours ) 6 0 8 0 Fig . 3 Growt h curve s fo reac h replicat e (a-c ) o fth e concentration , 7. 9 mg/L .
pag e 2 2 o f7 9

# ChemService Controlli o rtamrcn* p . 2 4

Fina l Repor t CH-095/201 3

450000 0 400000 0 350000 0 300000 0 250000 0 200000 0 150000 0 100000 0 50000 0 0 * 2 0 4 0 6 0

tim e (hours ) 8 0 Fig . 4 Growt h curve s fo reac h replicat e (a-c ) o fth e concentration , 11. 9 mg/L .

450000 0 400000 0 350000 0 300000 0 250000 0 200000 0 150000 0 100000 0 50000 0 0 * 2 0 4 0

tim e (hours ) 6 0 80 Fig . 5 Growt h curve s fo reac h replicat e (a-c ) o fth e concentration , 17. 8 mg/L .

pag e 2 3 o f7 9p . 2 5 

# ChemService

Controllia rfaoroho~ Fina l Repor t CH-095/201 3

450000 0 400000 0 350000 0 300000 0 250000 0 200000 0 150000 0 100000 0 50000 0 

# o 2 0 4 0 6 0

tim e (hours ) 8 0 Fig . 6 Growt h curve s fo reac h replicat e (a-c ) o fth e concentration , 26. 7 mg/L .

450000 0 400000 0 Z i 350000 0 | 300000 0 £250000 0 £200000 0 | 1500000 ° 1000000 500000 0 A 2 0 4 0

tim e (h ) 6 0 8 0 Fig . 7 Growt h curve s fo reac h replicat e (a-c ) o fth e concentration , 40. 0 mg/L .

pag e 2 4 o f7 9p . 2 6

Fina l Repor

t CH-095/201 3 

# ChemService

Controllimrlamrcho

450000 0 400000 0 3 350000

0 | 300000

0 g 250000

0 ^ 200000

0 • | 150000

0 * 100000

0 50000 0 0 *
Fig . 8 Growt h curve s fo rth e concentratio n an d th e negativ e control , mea n densityvalue s 

## Growt h rate Fo ral l th e teste d concentrations , th e alga l growt h rat e (r) afte r7 2 hours o fexposur e wa s slightl y highe rtha n th e negativ e contro l (mea n % inhibition : -1.0%) . Th e percentage s o fgrowt h inhibitio n fo rth e teste d concentration s ar e reporte d i n Tabl e 7 . Tabl e 7 Alga l growt h rat e an d inhibitio n cause d byth e tes tite m ove r7 2 hours o fexposur e i n

compariso n wit h negativ e control . Nomina l tes tite m concentratio n (mg/L ) 0-2 4 h 0- 4 i 8 h 0-7 2 h Nomina l tes tite m concentratio n (mg/L ) Mea n

growt h rat e Mea n

inhibitio n % Mea n

growt h rat e Mea n

inhibitio n % Mea n

growt h rat e Mea n

inhibitio n % 0. 0 (negativ e control ) 0.056 4 - 0.075 1 - 0.078 8 - 7. 9 0.061 6 -9.3 * 0.079 0 -5.2 * 0.080 6 -2.3 * 11. 9 0.064 4 -14.1 * 0.082 5 -9.7 * 0.082 8 -5.1 * 17. 8 0.063 9 -13.3 * 0.081 5 -8.5 * 0.082 4 -4.5 * 26. 7 0.060 9 -7.9 * 0.080 7 -7.3 * 0.081 1 -2.8 * 40. 0 0.064 6 -14.6 * 0.082 5 -9.8 * 0.082 5 -4.6 * * th e alga l growt h rate wa s slightl yhighe rtha n th e negativ e contro l pag e 2 5 o f7 9p . 2 7 1



# ChemService

Controlli 0 rlaorcrio Fina l Repor tCH-095/201 3 Th e statistica l analysi s wa s carrie d ou tb yth e mean s o fCETI S elaboratio n softwar e v1.026D ; a t eac h observatio n tim e th e E r Ci 0E r C 2 o E r C 5 o tim e wer e assesse d b ya Linea rInterpolatio n analysis , whil e th e NOE C wa s assesse d b yBonferron i Ad j tmethod . Th e E r Cio , th e E r C 2 o , th e E r C 5 o value s an d th e NOEC , i n term s o fgeometri cmea n o fmeasure d concentratio n o ftes titem , wer e a s follows : Tim e E r

C-i o E r

C2o ErCs o NOE C 0-24 h >0.4 1 > 0.4 1 > 0.4 1 0.4 1 0-48 h >0.4 1 > 0.4 1 >0.4 1 0.4 1 0-7 2 h >0.4 1 >0.4 1 > 0.4 1 0.4 1
Th e concentration-respons e char ta t7 2 hour s fo rgrowt h rat e end-poin ti s show n i n Figur e 9 . Concentratio n i s expresse d a s geometri cmea n o fmeasure d concentrations . 0.090 i



## 1 s Pi 0.080: ; 0.07( H 0.06< H 0.050 ^ 0.04( H 0.030 ^ 0.020 : 0.010 i 0.00 0 0.0 0 1 1i 1 0.1 0 0.2 0 Conc-mg/ L 0.3 0 0.4 0 0.5 0

Fig . 9 Concentration-respons e char tafte r7 2 h exposur e t o 4-chloro-a,a,a-trifluorotoluen e

(PCBTF ) expresse d a s geometri cmea n o fmeasure d concentratio n o ftes tite m fo r growt h rat e end-point . pag e 2 6 o f7 9p . 2 8

Fina l Repor

t CH-095/201 3 

# ChemService Controlli o rloorchm 

## Yiel d Afte r72 hours o fexposure , th e yiel d (Y) , a s differenc e o falga l densitybetwee n th e en d an d th e beginnin g o fth e test , fo ral l th e tes tite m concentration s wer e highe rtha n th e contro l (mea n % inhibition : -6.0%) . Th e percentage s o fgrowt h inhibitio n i n term s o fbiomas s yiel d fo rth e teste d concentration s ar e reporte d i n Tabl e 8 . Tabl e 8 Alga l yiel d inhibitio n cause d byth e tes t ite m ove r72 hours o fexposur e i n

compariso n wit h negativ e control . Nomina l tes tite m concentratio n (mg/L ) Mea n biomas s a t72 h (cell/mL ) Mea n growt h inhibitio n % 0. 0 (negativ e control ) 291500 3 - 7. 9 330968 7 -13.5 * 11. 9 388274 3 -33.2 * 17. 8 376775 3 -29.3 * 26. 7 341344 7 -17.1 * 40. 0 377798 7 -29.6 * * th e yiel d was highe rtha n th e i n th e negativ e contro l Th e statistica l analysi s wa s carrie d ou t b yth e mean s o fCETI S elaboratio n softwar e v1.026D ; a t eac h observatio n time , th e E y C xwer e assesse d bya Linea rInterpolatio n analysis , whil e th e NOE C wa s assesse d byBonferron i Ad j t method . Th e EyCio , th e E y C 2 0 , th e E y C 5 o an d th e NOE C a t72 hours , i n term s o fgeometri cmea n o f measure d o ftes titem , wer e a s follow s Tim e EyCl

O EyC 2 0 EyC5

0 NOE C 7 2 h >0.4 1 >0.4 1 >0.4 1 0.4 1 Detail s o fstatistica l analysi s ar e reporte d i n Appendi xC .

pag e 2 7 o f7 9p . 2 9



# ChemService Controlli o rtaorctio Fina l Repor t CH-095/201 3 Th e concentration-respons e chartat 7 2 hours fo ryiel d end-poin ti s show n i n Figur e 10 . Concentratio n i s expresse d a s geometri cmea n o fmeasure d concentrations .

3.0BO6 - 2.5B06 : 2.0B06 : 1.5B0& ^ 1.0B0 6 5.0B05 H 0.0BO 0 0.00 0.1 0 0.2 0 0.30

Conc-mg/ L 0.4 0 0.50 Fig . 1 0 Concentration-respons e char tafte r72 h exposur e t o 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) expresse d a s geometri cmea n o fmeasure d concentratio n o ftes tite m fo r yiel d end-point . 

## CONCLUSION S The toxicity

of test item 4-chloro-a,a,a-trifluorotoluene (PCBTF)

was tested on Pseudokirchneriella

subcapitata. Th e 72-hou rEC10 , EC 2o an d EC 5 0an d th e NOEC , calculate d , i n term s o fgeometri cmea n o f measure d concentration s o ftes tite m an d nomina l tes tite m concentration , fo rth e tw o end points , ar e reporte d i n th e followin g table : Endpoin t 0 - 7 2 h EC 1 0 0 - 72 h EC 2 0 0 - 7 2 h EC 5 0 0 - 7 2 h NOE C Growt h rat e >0.4 1 >0.4 1 >0.4 1 0.4 1 Yiel d >0.4 1 >0.4 1 >0.4 1 0.4 1

pag e 2 8 of7 9p . 30



# ChemService Controlli o rlaorcho Fina l Repor t CH-095/201 3 

## REFERENCE S 1 Regulatio n (EC ) N o 1907/200 6 o fth e Europea n Parliamen tan d o fth e Counci l o f1 8 Decembe r2006 , concernin g th e chemica l substanc e registration , estimation , authorizatio n an d restrictio n (REACh) . 2 Italia n Legislativ e Decre e (D.L . No . 5 0 date d Marc h 2 n d , 2007 ) a s publishe d i n G.U . No . 8 6 o fApri l 13 th , 2007 ; 3 Organisatio n fo rEconomi cCo-operatio n an d Developmen t(OECD ) Principle s o fGoo d Laborator

yPractic e (GLP ) [C(97 ) 186 (Final)] . 4 OEC D Guidelin e No . 201 , "Freshwate ralga e an d cyanobacteri a growt h inhibitio n test" , 2011 . 5 OEC D Serie s o n Testin g an d Assessmen t N o 2 3 - Guidanc e Documen t O n Aquati cToxicit y Testin g O fDifficul tSubstance s An d Mixture s ENV/JM/MONO(2000)6 . 

## ARCHIVIN G ChemServic e S.r.l . Controll i e Ricerch e archive s th e RawData , th e Stud yPla n a s origina l an d th e Fina l Repor

ta s tru e cop yo fth e origina l fo r4 years . Thereafter , th e Sponso rwil l b e contacte d fo rinstruction s regardin g dispatc h o rdisposa l o fth e stud ydocumentation . Referenc e materia l use d fo ranalytica l phas e i s store d unti l th e expirydat e wherea s th e tes t ite m i s store d fo ra tminimu m si xmonth s startin g fro m th e approva l o fth e Fina l Report . A tth e en d o fthi s period , th e tes t ite m wil l b e dispose d o faccordin g t o th e environmenta l regulation s i n force , unles s a writte n retur n reques t fro m th e Sponso ri s received . Wate rsample s take n fo rth e analytica l phas e wil l b e store d unti l th e approva l o fth e Fina l Report .

pag e 2 9 o f7 9p . 3 1 

# ChemService

Controill a rfcorchm AppendixAt o Fina l Repor

t CH-095/201 3


# APPENDI XA
ANALYTICA

L PHASE REPOR

T

Appendi xA pag e 3 0 o f 7 9

# c 

# ChemService Controlli o rlaorohm

"

Appendi xAt o Fina l Repor t CH-095/201 3 

## CONTENT S

cPag e

Introductio n 3 3 Material s 3 3 Referenc e materia l 3 3 Wate rSample s 3 4 Equipmen t3 6 Reagent s 3 6 Analytica l Metho d 3 6 Experimenta l 4 0 Result s 4 0 Conclusion s 5 0 Representativ e chromatogram s (Figure s 1-4 ) 51-5 7



### O

Appendi xApag e 3 1 o f7 9

# ChemService Controlli o rlcorch< Appendi xAt o Fina l Repor t CH-095/201 3 

## INTRODUCTIO N The analytical

phase was conducted

to determine

4-chloro-a,a,a-trifluorotoluene (PCBTF) residues content in water samples

coming from the biological

phase of ecotoxicological

test on green algae Pseudokirchneriella

subcapitata. Fo rth e determinatio n o f4-chloro-a,a,a-trifluorotoluen e (PCBTF ) residues , al l wate rsample s wer e analyse d followin g th e interna l Analytica l Metho d No . 092/2013 , validate d i n th e interna l Stud yNo . CH-092/2013 . 

## MATERIAL S Referenc e Ite m Th e tes t item , 4-chloro-a,a,a-trifluorotoluen e (PCBTF) , wa s use d als o a s referenc e item , t o perfor m wate rsample s quantification . Detail s ar e reporte d at pag e 10 . Acop yo fth e Certificat e o fAnalysi s supplie d b yth e sponso rannexe d i n Appendi xD .

Appendi xApag e 32 o f7 9

# o 

# ChemService

^^^^^^^ ^

C* o n fj^f/j T tf ' rlaarcn m ' ' 1 Appendi xAt o Fina l Repor tCH-095/201 3 

## Wate rSample s Sample s descriptio n i s reporte d i n Tabl e 1 an d wate rcompositio n i s define d i n OEC D Guidelin e 201 , 201 1 an d reporte d i n Appendi xG . Tes tsolution s wer e sample d i n duble . On e sampl e wa s immediatel yanalysed , th e othe r O store d i n th e fridg e a tabou t4°C .

A ttim e 0 hou ran d a ttim e 72 hours , tes t solution s wer e sample d i n duplicat e (tw o vial s o f1 0 m Lwer e totall yfilled) , a ttim e 24 , 4 8 hours wer e collecte d a n aliquo t o f5 m Lfo reac h replicat e (thre e replicat e fo rever yconcentrations) , 4 m Lo fthes e wer e kee p int o vial s totall y fille d an d replac e int o fridg e a t 4°C , 1 0 m Lwer e centrifuge d an d analysed . Tabl e 1 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : wate rsample s fro m th e growt h

inhibitio n tes t (hours ) Wate rSam P | e s(* ) samplin g Dat e o fanalvsi sVolum e (mL ) 0 Contro l 02/04/201 3 02/04/201 3 2 x1 0 0 7. 9 mg/ L02/04/201 3 02/04/201 3 2x1 0



### O 0 11. 9 mg/ L 02/04/201 3 02/04/201 3 2x1 0 0 17. 8 mg/ L 02/04/201 3 02/04/201 3 2x1 0 0 26. 7 mg/ L 02/04/201 3 02/04/201 3 2 x1 0 0 40. 0 mg/ L 02/04/201 3 02/04/201 3 2x1 0 2 4 Contro l 03/04/201 3 03/04/201 3 10+ 4 2 4 7. 9 mg/ L 03/04/201 3 03/04/201 3 1 0 + 4 2 4 11. 9 mg/ L 03/04/201 3 03/04/201 3 1 0 + 4 2 4 17. 8 mg/ L 03/04/201 3 03/04/201 3 1 0 + 4 2 4 26. 7 mg/ L 03/04/201 3 03/04/201 3 1 0 + 4 2 4 40. 0 mg/ L 03/04/201 3 03/04/201 3 1 0 + 4 4 8 Contro l 04/04/201 3 04/04/201 3 10+ 4 4 8 7. 9 mg/ L 04/04/201 3 04/04/201 3 1 0 + 4 Appendi xApag e 3 3 o f7 9p . 35 )



# ChemService Controlli o rlooront Appendi xAt o Fina l Repor t CH-095/201 3 Tim e (hours ) Wate rSample s (* ) Dat e o f samplin g Dat e o fanalysi s Volum e (mL ) 4 8 11. 9 mg/ L 04/04/201 3 04/04/201 3 1 0 + 4 4 8 17. 8 mg/ L 04/04/201 3 04/04/201 3 1 0 + 4 4 8 26. 7 mg/ L 04/04/201 3 04/04/201 3 1 0 + 4 4 8 40. 0 mg/ L 04/04/201 3 04/04/201 3 1 0 + 4 72 Contro l 05/04/201 3 05/04/201 3 2x1 0 7 2 7. 9 mg/ L05/04/201 3 05/04/201 3 2 x1 0 7 2 11. 9 mg/ L 05/04/201 3 05/04/201 3 2x1 0 7 2 17. 8 mg/ L 05/04/201 3 05/04/201 3 2x1 0 7 2 26. 7 mg/ L 05/04/201 3 05/04/201 3 2x1 0 72 40. 0 mg/ L 05/04/201 3 05/04/201 3 2x1 0 (* ) Nomina l concentration s are expresse d a s tes tite m 4-chloro-a J a,a-trifluorotoluen e (PCBTF) .

Appendi xApag e 3 4 o f7 9p . 36



# ChemService Controllt * rfoorcho Appendi xAt o Fina l Repor t CH-095/201 3 

## Equipmen t

- Hig h performanc e liqui d chromatograp h (HPLC ) equippe d eithe rwit h Tripl e Quadrupol e Mas s detecto ran d DA D detector , ES I interface , binar ypump , aut o sample r an d softwar e fo rinstrumen tmanagemen t an d dat a r e processing , interna l cod e No.568 .

- Laborator

yWate rPurificatio n Systems , Sartoriu s Ahum ® 611 , interna l cod e No . 35 5

- Refrigerator , interna l cod e No . 38 8

- Aspirate d SafetyStorag e Cabine tNo . 57A

- Usua l laborator

yglasswar e

- Volumetri cglasswar e

- Micropipettes , interna l cod e No . 170/E 1 

## Reagent s -Water , HPL C grad e

- Methanol , analytica l grad e (VW R International )

- Acetonitrile , HPL C grad e (VW R International )

- Phosphori caci d 85 % (H 3 P0 4 ) , reagen tgrad e (Merck) -Alga l growt h mediu m (OEC D 201 , 2011 )



## ANALYTICA L METHO D Al l control s an d fortifie d wate rsample s wer e analyse d followin g th e interna l Analytica l Metho d No . 092/201 3 validate d i n th e interna l Stud yCH-092/2013 . Th e presenc e o f4-chloro-a,a,a-trifluorotoluen e (PCBTF ) i n tes twate rsample s wa s detecte d byvirtu e o fth e HPL C couple d wit h a diod e arraydetector . Th e principa l feature s o fth e metho d validatio n stud yar e reporte d here below . •Th e analytica l metho d wa s show n t o b e specifi cfo r4-chloro-a,a,a-trifluorotoluen e (PCBTF ) residue s i n wate rsample s fro m ecotoxicologica l tests . •Th e rang e tested , fro m 200. 0 u,g/ Lt o 5000. 0 ng/L , nomina l concentrations , wa s foun d t o fi ta linea rmode l (correlatio n coefficien t> 0.99) . •Repeatability(Precision ) an d Recover y(Accuracy ) o fth e metho d wer e verifie d i n reconstitute d wate r(OEC D 203 , 199 2 an d OEC D 202 , 2004) , alga l growt h mediu m (OEC D 201 , 2011) . Result s ar e summarise d below :

Appendi xApag e 3 5 o f7 9

# ChemService Coniroiiftt

rioorchi Appendi xAt o Fina l Repor tCH-095/201 3 Fortificatio n leve l Mea n foun d RSD % % Mea n nomina l corrected * (Precision ) (Accuracy ) Reconstitute d 250. 0 Lig/ L 252. 2 Lig/

L 231.4

\xglL 10.2 % 91.7 % wate r 25000. 0 (ig/ L 25222. 2 (u.g/ L 21657.4

iig/L 10.8 % 85.9 % Alga l 250. 0 Lig/

L 252.2

HQ/L 192. 3 ng/ L 3.6 % 76.2 % growt h 250. 0 Lig/ L mediu m 25000. 0 M-g/ L 25222.2

ng/L 24383.9

\ig/L 7.0 % 96.7 % concentratio n correcte d fo rdensit yan d purity .

• The limit of quantification

(L.O.Q.) of this method is defined as the lowest fortification level of 252.2 Lig/L(ppb)

in water matrix samples (nominal

250.0 \ig/L).

•Th e limi to fdetectio n (L.O.D. ) o fthi s metho d i s define d a s 5 0 % o fth e lowes t calibratio n level , i.e . 100. 9 jig/ L(ppb ) i n wate rmatri xsample s (nomina l 100. 0 |Kj/L) . I fth e residu e amoun ti s greate rtha n 5000. 0 j^g/L , th e aqueou s sampl e mus tb e suitabl y dilute d i n volumetri cflasks . Wate rsample s storag e an d preparatio n Wate rsample s wer e collecte d immediately , afte rwate rsolution s preparation , i n vial s fille d withou tai rinside ; a n aliquo twa s transferre d int o a vial , fillin g th e via l t o th e borde ran d analyze d t o HPLC/DAD . I fsample s ar e store d a t4°C , analysi s ca n b e performe d withi n on e hour .

Appendi xApag e 3 6 o f7 9p . 38 

# o 

# ChemService Controll i •rfooroh o Appendi xAt o Fina l Repor tCH-095/201 3 Chromatographi ccondition s HPL C colum n Agilen to requivalen t Detecto r Colum n temperatur e Eluen tA Eluen tB Eluen tmixtur e Eluen t flo w Volum e o finjectio n PCBT F R.T . Tota l Analysi s Tim e Interna l cod e LC N 15 7 Gemin i C18 , 150x3 m m DAD , X=22 0 n m roo m temperatur e acetonitril e 0.1 % H3P0 4 wate r0.1 % H3P0 4 A: B 55:4 5 0. 8 mL/mi n 100 LL L 5. 2 minute s 1 0 minute s



### C

Appendi xApag e 3 7 o f7 9

# ChemService Controlli o rleorohm Appendi xAt o Fina l Repor tCH-095/201 3 Calculation s PCBT F residu e conten ti n th e wate rsample s i s calculate d b yth e followin g formula . PCBT F (ng/L ) = C sxD s where : C s= PCBT F concentratio n i n th e sampl e solutio n (Lig/L ) D s= Dilutio n o rconcentratio n o fth e sampl e solutio n (i fany ) Th e C svalu e i s calculate d fro m th e A sdat a usin g th e calibratio n curv e obtaine d wit h C s t d an d A s t< j dat a fro m injection s o fworkin g standar d solutions . where : A s= PCBT F pea k are a i n th e sampl e solutio n

c s t d =PCBT F concentratio n i n th e workin g standar d solution s (ng/L ) A s t d =PCBT F pea k are a i n workin g standar d solution s Th e percentag e recover yi s calculate d a s follows : [ig/ Lfoun d Recover y(% ) = x10 0 fig/ Ladde d

Appendi xApag e 3 8 o f7 9p . 4 0 

# o 

# ChemService Appendi xAt o Fina l Repor t CH-095/201 3 Controlli o rlotmrohm


## EXPERIMENTA L Fo rth e determinatio n o f4-chloro-a,a,a-trifluorotoluen e (PCBTF) , eac h sampl e wa s analyse d followin g th e procedure s describe d i n th e Analytica l Metho d No . 092/2013 . I n orde rt o evaluat e th e performanc e o fth e metho d durin g th e analysi s o fecotoxicologica l sample s tw o procedura l accuracytest s at252. 1 ng/ Lan d 25222. 2 Lig/ Lwer e performed . 

## RESULT S Note . Dat a i n th e table s are rounde d value s take n fro m Exce l spreadsheet s whic h wil l b e archive d togethe rwit h th e rawdata . Th e us e o fExce l spreadsheet s fo rcalculation s produce s more accurat e endpoints . Thes e endpoint s ma yslightl ydiffe rfro m th e value s derive d by replacin g th e rounde d value s i n th e formula e give n i n th e method s section . Dat a an d result s o fth e wate rsample s analysi s are reporte d i n Table s 2 (a-b-c-d ) an d 3 (a-b c-d) . Tabl e 4 (a-b-c-d ) show s th e analysi s sequence .



### O

Appendi xApag e 3 9 o f7 9

# ChemService Appendi xAt o Fina l Repor t CH-095/201 3 Tabl e 2 a 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 0 h analysi s - calibratio n wit h

workin g standar d solutions . 4-chloro-a,a,a trifluorotoluen e (PCBTF ) WS S 1 201. 7 ng/ L (Area ) WS S 3

1008. 3 rig/ L (Area ) WS S 5 5041. 6 pg/ L (Area ) 1 s tserie s 145.0 4 860.1 3 4365.4 0 2 n dserie s 183.3 9 770.1 2 4446.4 8 Mea n 164.2 2 815.1 3 4405.9 4 Delt a 3 8 9 0 8 1 Delt a % 23.35% 11.04% 1.84% Y = mx + q Paramete rm

(slope ) Paramete rq (intercept ) Paramete rR 2 (correlation ) 0.8 8 -40.5 5 0.9998 3 Tabl e 2 b 4-chloro-a J a ) a-trifluorotoluen e (PCBTF) : tim e 2 4 h analysi s - calibratio n wit h

workin g standar d solutions . 4-chloro-a,a,a trifluorotoluen e (PCBTF ) WS S 1 201. 7 Lig/ L (Area ) WS S 3

1008. 3 ^g/ L (Area ) WS S 5 5041. 6 pg/ L (Area ) 1 s tserie s 178.3 4 801.8 6 4391.6 2 2 n dserie s 169.4 8 781.9 6 5672.9 8 Mea n 173.9 1 791.9 1 5032.3 0 Delt a 9 2 0 128 1 Delt a % 5.09% 2.51% 25.46% Y = mx + q Paramete rm

(slope ) Paramete rq (intercept ) Paramete rR 2 (correlation ) Y = mx + q 1.0 2 -124.3 9 0.9984 7

Appendi xApag e 4 0 o f7 9p . 42 

# ChemService

Controllio rlamroho Appendi xAt o Fina l Repor t CH-095/201 3 Tabl e 2 c4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 4 8 h analysi s - calibratio n wit h

workin g standar d solutions . 4-chloro-a,a,a trifluorotoluen e (PCBTF ) WS S 1

201. 7 ng/ L (Area ) WS S 3

1008. 3 (ig/ L (Area ) WS S 5

5041. 6 pg/ L (Area ) 1 s tserie s 165.4 2 752.7 9 4053.5 5 2 n dserie s 145.2 7 697.0 6 3525.7 3 Mea n 155.3 5 724.9 3 3789.6 4 Delt a 2 0 5 6 52 8 Delt a % 12.97% 7.69% 13.93% Y = mx + q Paramete rm

(slope ) Paramete rq

(intercept ) Paramete rR 2 (correlation ) 0.7 5 -14.1 5 0.9999 0 Tabl e 2 d 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 72 h analysi s - calibratio n wit h

workin g standar d solutions . 4-chloro-a,a,a trifluorotoluen e (PCBTF ) WS S 1

201. 7 \ig/ l (Area ) WS S 3

1008. 3 ng/ L (Area ) WS S 5

5041. 6 pg/ L (Area ) 1 s tserie s 109.6 5 723.3 8 3799.7 6 2 n dserie s 112.1 4 680.3 3 3738.6 8 Mea n 110.9 0 701.8 6 3769.2 2 Delt a 2 4 3 6 1 Delt a % 2.25% 6.13% 1.62% Y = mx + q Paramete rm

(slope ) Paramete rq

(intercept ) Paramete rR 2 (correlation ) 0.7 6 -50.9 2 0.9999 7

Appendi xApag e 4 1 o f7 9p . 4 3



# ChemService

^^^^^^^ ^

Controlli

ft rlaordhm Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 3 a 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 0 h analysi s - 4-chloro-a,a,a trifluorotoiuen e (PCBTF ) conten t i n wate rsample s fro m th e growt h inhibitio n test . Via l Identificatio n Are a s 

### (ng/L) * PCBT F foun d (^g/L ) PCBT F ad d

(^g/L ) Recover y

(% ) Spik e Lo w 244.7 9 300. 6 1. 0 300. 6 252. 1 119. 3 Spik e Hig h 1892.7 0 2194. 7 10. 0 21946. 8 25208. 2 87. 1 Contro l t O 0.0 0 0. 0 1. 0 0. 0 0. 0 - 7. 9 mg/ Lt

O 464.6 3 573. 5 10. 0 5734. 9 7900. 0 72. 6 11. 9 mg/ Lt

O 872.2 2 1036. 2 10. 0 10362. 0 11900. 0 87. 1 17. 8 mg/ Lt

O 1160.4 0 1363. 4 10. 0 13633. 5 17800. 0 76. 6 26. 7 mg/ Lt

O 826.1 8 983. 9 20. 0 19678. 7 26700. 0 73. 7 40. 0 mg/ Lt

O 1165.9 9 1369. 7 20. 0 27394. 0 40000. 0 68. 5 Are a s: Sampl

e pea k are a C s : Injecte

d solution concentration D s : Dilution applie

d t o the sampl

e (*) Spik e Lo wwa s quantifie d wit h WSS1 , Spike hig h an d wate rsample s wer e quantifie d wit h th e calibratio n curv e reporte d i n Tabl e 2a . n.d . no tdetected , lowe rtha n L.O.D . (100. 9 pg/L )

Appendi xApag e 4 2 o f7 9p . 4 4 

# o 

# ChemService Controllio rlaorono Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 3 b 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 2 4 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) conten ti n wate rsample s fro m th e growt h inhibitio n tes t 

### O Via l Identificatio n Are a s (pg/L) * D s PCBT F foun d (pg/L ) PCBT F ad d

(pg/L ) Recover y

(% ) Spik e Ultr a Lo w 207.9 8 241. 2 1. 0 241. 2 252. 1 95. 7 Spik e Hig h 2126.6 2 2208. 7 10. 0 22087. 3 25208. 2 87. 6 Contro l t2 4 0.0 0 0. 0 1. 0 n.d . 0. 0 - 7. 9 mg/ Lt2 4 0.0 0 0. 0 1. 0 n.d . 7900. 0 - 11. 9 mg/ Lt2 4 0.0 0 0. 0 1. 0 n.d . 11900. 0 - 17. 8 mg/ Lt2 4 0.0 0 0. 0 1. 0 n.d . 17800. 0 - 26. 7 mg/ Lt2 4 0.0 0 0. 0 1. 0 n.d . 26700. 0 - 40. 0 mg/ Lt2 4 0.0 0 0. 0 1. 0 n.d . 40000. 0 - Area s: Sampl e peak

area

cs : Injected solutio n concentratio n D s : Dilutio n applie d to th e sampl e (* ) Spik e Lo wwa s quantifie d wit h WSS1 , Spik e hig h an d wate rsample s wer e quantifie d wit h th e calibratio n curv e reporte d i n Tabl e 2b . n.d . no tdetected , lowe rtha n L.O.D . (100. 9 pg/L )


### O

Appendi xApag e 4 3 o f7 9p . 4 5



# ChemService

Confronto

riadrohm Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 3 c4-chloro-a,a,a-trifiuorotoluen e (PCBTF) : tim e 4 8 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) conten ti n wate rsample s fro m th e growt h inhibitio n tes t Via l Identificatio n Are a s (pg/i-) * PCBT F foun d (pg/L ) PCBT F ad d

(pg/L ) Recover y

(% ) Spik e Ultr a Lo w 186.5 3 242. 1 1. 0 242. 1 252. 1 96. 1 Spik e Hig h 1453.8 8 1947. 6 10. 0 19475. 6 25208. 2 77. 3 Contro l t4 8 0.0 0 0. 0 1. 0 n.d . 0. 0 - 7. 9 mg/ Lt4 8 0.0 0 0. 0 1. 0 n.d . 7900. 0 - 11. 9 mg/ Lt4 8 0.0 0 0. 0 1. 0 n.d . 11900. 0 - 17. 8 mg/Lt4 8 0.0 0 0. 0 1. 0 n.d . 17800. 0 - 26. 7 mg/ Lt4 8 0.0 0 0. 0 1. 0 n.d . 26700. 0 - 40. 0 mg/Lt4 8 0.0 0 0. 0 1. 0 n.d . 40000. 0 - Area s; Sampl e peak

area ^ s : Injected solutio n concentratio n D s : Dilutio n applied to th e sampl e (* ) Spik e Lo wwa s quantifie d wit h WSS1 , Spike hig h an d wate rsample s wer e quantifie d wit h th e calibratio n curv e reporte d i n Tabl e 2c . n.d . no tdetected , lowe rtha n L.O.D . (100. 9 pg/L )

Appendi xApag e 4 4 o f7 9

# c p . 4 6



# ChemService Contrail/ o rloorcnm~ Appendi xAt o Fina l Repor tCH-095/201 3



### O Tabl e 3 d 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 7 2 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) conten t i n wate rsample s fro m th e growt h inhibitio n tes t Via l Identificatio n Are a s (pg/U * PCBT F foun d (pg/L ) PCBT F ad d

(pg/L ) Recover y

(% ) Spik e Ultr a Lo w 142.2 5 258. 7 1. 0 258. 7 252. 1 102. 6 Spik e Hig h 2014.7 9 2727. 5 10. 0 27275. 2 25208. 2 108. 2 Contro l t7 2 0.0 0 0. 0 1. 0 n.d . 0. 0 - 7. 9 mg/ Lt7 2 0.0 0 0. 0 1. 0 n.d . 7900. 0 - 11. 9 mg/ Lt7 2 0.0 0 0. 0 1. 0 n.d . 11900. 0 - 17. 8 mg/ Lt7 2 0.0 0 0. 0 1. 0 n.d . 17800. 0 - 26. 7 mg/ Lt7 2 0.0 0 0. 0 1. 0 n.d . 26700. 0 - 40, 0 mg/ Lt7 2 0.0 0 0. 0 1. 0 n.d . 40000. 0 - Are a s : Sampl e pea k are a ^ s : Injecte d solution concentration D s : Dilution applie

d t o the sampl

e (* ) Spik e Lowwa s quantifie d wit h WSS1 , Spik e hig h an d wate rsample s wer e quantifie d wit h th e calibratio n curv e reporte d i n Tabl e 2d . n.d . no tdetected , lowe rtha n L.O.D . (100. 9 pg/L ) 

### G

Appendi xApag e 4 5 o f7 9p . 4 7



# ChemService

Controlli o rloorchm Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 4 a 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 0 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) analysi s sequence . No . Via l Identificatio n No . Via l Identificatio n 1 WSS1_ A 1 CH09513_11. 9 mg/ L 1 WSS3_ A 1 CH09513_17. 8 mg/ L 1 WSS5_ A 1 CH09513_26. 7 mg/ L 1 Was h 1 CH09513_40. 0 mg/ L 1 CH09513_Sp k Low 1 Was h 1 CH09513_SpkHig h 1 WSS1_ B 1 Was h 1 WSS3_ B 1 CH09513_Contro l 1 WSS5_ B 1 CH09513_7. 9 mg/ L 1

Appendi xApag e 4 6 o f7 9p . 4 8



# ChemService ^j^^^^^/ 1Controlli o rtamronm Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 4 b 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 2 4 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) analysi s sequence . No . Via l Identificatio n No . Via l Identificatio n 1 WSS1_A-T2 4 1 Wash-T2 4 1 WSS3_A-T2 4 1 WSS1_B-T2 4 1 WSS5_A-T2 4 1 WSS3_B-T2 4 1 Wash-T2 4 1 WSS5_B-T2 4 1 CH09513_Spkl_ow-T2 4 1 Wash-T2 4 1 CH09513_SpkHigh-T2 4 1 CH09513_7. 9 mg/L-T2 4 rein j TQ * 1 Wash-T2 4 1 CH09513_11. 9 mg/L-T2 4 rein j TQ * 1 CH09513_Control-T2 4 1 CH09513J7. 8 mg/L-T2 4 rein j TQ * 1 CH09513_7.9mg/L-T2 4 1 CH09513_26. 7 mg/L-T2 4 rein j TQ * 1 CH09513J1. 9 mg/L-T2 4 1 CH09513_40. 0 mg/L-T2 4 rein j TQ * 1 CH09513J7. 8 mg/L-T2 4 1 Wash-T24 * 1 CH09513_26. 7 mg/L-T2 4 1 WSS5_C-T2 4 conferm a risposta * 1 CH09513_40.0mg/L-T2 4 *sampl e injectio n wa

s repeated a

t th e en d o

fth e sequence , withou t dilutio n o fth e sample , since wit h th e firstdilutio n factors , testite m concentration s wer e smalle rtha n th e linearit

yrange .

Appendi xApag e 4 7 o f7 9p . 4 9



# ChemService

Controlli * rloorohm Appendi xAt o Fina l Repor tCH-095/201 3 Tabl e 4 c 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 4 8 h analysi s - 4-chloro-a,a,a trifluorotoluen e (PCBTF ) analysi s sequence . No . Via l Identificatio n No . Via l Identificatio n 1 WSS1_A-T4 8 CH09513J1. 9 mg/L-T4 8 1 WSS3_A-T4 8 CH09513J7. 8 mg/L-T4 8 1 WSS5_A-T4 8 CH09513_26. 7 mg/L-T4 8 1 Wash-T4 8 CH09513_40. 0 mg/L-T4 8 1 CH09513_Sp k LOW-T4 8 Wash-T4 8 1 CH09513_SpkHigh-T4 8 WSS1_B-T4 8 1 Wash-T4 8 WSS3_B-T4 8 1 CH09513_Control-T4 8 WSS5_B-T4 8 1 CH09513_7.9mg/L-T4 8 Wash-T4 8 Tabl e 4 d 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : tim e 72 h analysi s - 4-ch!oro-a,a,a trifluorotoluen e (PCBTF ) analysi s sequence . No . Via l Identificatio n No . Via l Identificatio n 1 WSS1_A-T7 2 CH09513J1. 9 mg/L-T7 2 1 WSS3_A-T7 2 CH09513_17.8mg/L-T7 2 1 WSS5_A-T7 2 CH09513_26. 7 mg/L-T7 2 1 Wash-T7 2 CH09513_40.0mg/L-T7 2 1 CH09513_Sp k Low-T7 2 Wash-T7 2 1 CH09513_SpkHigh-T7 2 WSS1_B-T7 2 1 Wash-T7 2 WSS3_B-T7 2 1 CH09513_Control-T7 2 WSS5_B-T7 2 1 CH09513_7. 9 mg/L-T7 2 Wash-T7 2

Appendi xApag e 4 8 o f7 9

## o 

# ChemService Control!/ o rlooroho Appendi xAt o Fina l Repor t CH-095/201 3 

## CONCLUSION S 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) conten t i n wate rsample s comin g fro m th e aquati c ecotoxicologica l tes t performe d o n P.subcapitat a i s reporte d i n Tabl e 5 . Tabl e 5 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : summar yresult s



### O Tim e (hours ) Wate rSample s (* ) 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) (pg/L ) 0 Contro l n.d . 0 7. 9 mg/ L 5734. 9 0 11. 9 mg/ L 10362. 0 0 17. 8 mg/ L 13633. 5 0 26. 7 mg/ L 19678. 7 0 40. 0 mg/ L 27394. 0 2 4 Contro l n.d . 2 4 7. 9 mg/ L n.d . 2 4 11. 9 mg/ L n.d . 2 4 17. 8 mg/ L n.d . 2 4 26. 7 mg/ L n.d . 2 4 40. 0 mg/ L n.d . 4 8 Contro l n.d . 4 8 7. 9 mg/ L n.d . 4 8 11. 9 mg/ L n.d . 4 8 17. 8 mg/ L n.d . 4 8 26. 7 mg/ L n.d . 4 8 40. 0 mg/ L n.d . 72 Contro l n.d . 7 2 7. 9 mg/ L n.d . Appendi xApag e 4 9 o f7 9Appendi xAt o Fina l Repor t CH-095/201 3 Tim e (hours ) Wate rSample s (* ) 4-chloro-a,a,a-trifluorotoluen e (PCBTF ) (pg/L ) 72 11. 9 mg/ L n.d . 7 2 17. 8 mg/ L n.d . 72 26. 7 mg/ L n.d . 7 2 40. 0 mg/ L n.d . (*) Nomina l concentration s fo recotoxicologica l sample s are expresse d a s tes tite m 4-chloro a,a,a-trifluorotoluen e (PCBTF) .

Appendi xApag e 5 0 o f7 9p . 52



# ChemService Controlli * rtaorohm~ Appendi xAt o Fina l Repor t CH-095/201 3 Figur e 1 4-chloro-a,a ( a-trifluorotoluen e (PCBTF) : Representativ e chromatogra m o f4 - chloro-a,a,a-trifluorotoluen e (PCBTF ) workin g standar d solution . Qualitativ e Analysi s Repor t Dat a Filenam e Sampl e Typ e Instrumen tNam e Ac q Metho d IR M Calibratio n Statu s Commen t Sampl e Grou p Info . Use rChromatogram s TD_02.d Sampl e Insmjmen

t 1 PCBTF_CH09213.r

o No t Applicabl

e Sampl e Nam e

Positio n Use rNam e Acquire d Tim e D AMetho d WSS3 A

VTal2 5 4/2/201 3 3:43:3 8 P

M resefp'iu. m

t i Q 2 4 3 2 1- 1 0 DAD 1 - AiSig=220,1 Q T0_02. d Nois e (PeakToPeah ) = 0.33 ; SM R (5.189iron ) = 193. 0

— i
70.09 !

2: 5 A3l 5 4 4! S S S\ B 4 6! B 1 7.' & 8 fl"3 ( 5 Respons e Unil B (% ) vs . Acquisitio n Tim e (min ) 3. 5 Star t fct En d Heigh t Are a Are a % Signa l T o Nois e 4.78 9 5.18 9 5.7 3 76.0 9 860.1 3 10 0 19 8
En d O fReport—

jjt'f; - AiitttfltTechnalonia i Pag e 1 o f1 Printe d at : 2:5 9 P M on ; 4/3/201 3
Appendi xApag e 5 1 o f7 9p . 5 3 

# ChemService Cantrolll a rlat»rchi Appendi xAt o Fina l Repor

t CH-095/201 3 Figur e 2 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : Representativ e chromatogra m o

fthe Contro l wate rsample .

Qualitativ e Analysi s Repor t Dat a Ffanam e Sampl e Typ e Instrumen t Nam e Ac q Metho d I R H Calibratio n Commen t Sampl e Grou p T0_08. d Sampl e Nam e CH09513_Contro l Sampl e Positio n Vi<il44 Instrumen t 1 Use rNam e PCBTF_CH09213. m Acquire d Tim e 4/2/201 3 10:48)2 0 PM No t Applicabl e D AMetho d reserptna. m Info . UserChromatogram

s x10 2 4 - a2 - 1 . ODAD 1 -A:Sig=22O,1BT0_08, d Nois e (PaakToPeak ) » 05 4

o: s i i. s S 2. s 4 i s 4 4' M 6 s!5 6 els 1 7.' & a £3 Respons e Unit s (%

> vs . Acquisitio n Tim e <min )
En d O fReport -

-jjj- - A|itatTectHwk>gl M Pag e 1 of1 Printe d at : 3:0 0 P M on : 4/3/201 3

Appendi xApag e 52 o

f7 9p . 5 4



# ChemService Controlli0rtcorohm Appendi xAt o Fina l Repor tCH-095/201 3 Figure 3 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : Representativ e chromatogra m o f

Contro l wate rsampl e fortifie d a tlo wleve l (252. 1 pg/L) .

Qualitativ e Analysi s Repor t Dat a Filenam e Sampl e Typ e Instrumen tNam e AcqMetho d IR M Calibratio nStatu s Comment Sampl e Grou p Info . Use rChromatoeram s T0_05.d Sampl e Nam e CH09513_Sp k Lo w Sampl e Positio n Via l 4 2 Instrumen

t 1 Use r Nam e PCSTF_CH09213.

mAcquire dTim e 4/2/201310:12:3 7 P H No tApplicabl e D AMetho d reserpfna, m

K 1 0 2 4 3 2 - 1 0 DAD T- A:Stg=220.1BTQ_G5. d Mala e (PesKToPeak ) « 0.56 ; SN R (5.1Q7min ) = 41. 2
I! - 244.76 ! j 1 I.' 3 2 2. S 3 31 5 4 4: 3 fi 5: 5 o Sl a1 7,'B . ft

Respons e Units (%> vs . AcquisitionTim e (mln ) 6 a.'

3 Star t R T En d Heigh t Are a Area^f e Signa l T o Nois e 4.78 7 5.10 7 S.5 7 23.0 1 244,7 9 10 0 41. 2

—En d O fReport e r AflilHtTecbiwfaiaie * Pag e 1 o f1 Printedat : 2:5 9 P M on : 473/201 3

Appendi xApag e 5 3 o f7 9p . 55



# ChemService Controlli « rtcorchm Appendi xAt o Fina l Repor t CH-095/201 3 Figure 4 4-chloro-a,a,a-trifluorotoluen e (PCBTF) : Representativ e chromatogra m o ftim e 0 h wate rsampl e a t 17. 8 mg/L . Qualitativ e Analysi s Repor t

Dat a Filenam e Sampl e Type . Instrumen tNam e Ac q Metho d IB M Calibratio n Statu s Commen t Sample Group Ink Use rChromatogram s TO_ll, d Sampl e Nam e CH09513_17. 3 mg/ L Sampl e Positio n Via ) 4 7 Instrumen t1 Use rNam e PCBTF_CH09213. m Acquire d Tim e 4/2/201311:24:0 5 P M Not Applicable

DAMethod rtstrpin*,m

x10 Z 1 0.75 0. 5 0.2 5 0 DAD t- A:Stg=22O.10TO_11.t l Nois e (PeakToPeak

) = 0.53 ; SN R (5.1Q7min) = 220. 5 1W.2J J

ol S ] l."5 2 2: 5 3 3' 5 - 1 4'. 3 S 5i 5 * ftfc 1 7.'& 4 S.' S Ftaapons e Unit s {% ) vs . Acquisitio n Tim e (mln ) 9: 5 star t R

T Heigh t Are a Area °*> Signa l T o Nois e 4.95 3 5.107 5.4

2 117.2

2 1160. 4 10 0 220 3
En d O fReport -

^X " Aiitantleohneliipie i Pag e 1 o f 1 PrMs d at : 3:0 1 P M on : 4/3/201 3

Appendi xApag e 5 4 o f7 9p . 5 6



# ChemService Controlli o rtoorcho Appendi xAt o Fina l Repor tCH-095/201 3 Figur e 5 4-chloro-aAa-trifluorotoIuen e (PCBTF) : Representativ e chromatogra m o ftim e

2 4 h wate rsampl e a t 17. 8 mg/L .

Qualitativ e Analysi s Repor t Dat a Filenam e Sampl e Typ e Ac q Metho d 1R M Calibratio n Commen t Sampl e Grou p Info . Use rChrornatosram s T24_2I. d Sampl e Instrumen t1 PCBTF_CH09253.f n No tApplicabl e rein j T Q Sampl e Nam e CH09513..17. 8 mg/L-T2 4 Positio n Vial 5 Use rNam e Acquire d Tim e 4/4/201 3 9:49:5 0 A M D AMetho d reserpirw. m

x10 1 3 2 . 1 0 .

- 1

- 2 DAD 1 - A:Sla=220.l6T24_21. d



# ::!:f:jT[; r —-

* - -

- I'll ! 

### |!!i | 

### • 

## jM M 

## H ' l|l f -T--)--(-- rj

— - - \ ~ - i - ! - - I L 1 1 r1 . i.—i— n ill ! 

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### . .M ! 

### J... L_!__ _

.. L

### J . I . L

' F 1 1 1 3. 5 4 4.S . 5 5, 5 6 6. 5 7 7.5 - Bespons e Unit s (% ) vs . Acquisitio n Tim e (mln ) 9. 5

En d O fReport '

• f a AiibntTeclinetotiifl a Page 1 o f1 Printe d at : 3:1 4 P M on : 4/4/201 3

Appendi xApag e 5 5 o f7 9p . 5 7



# ChemService Controlli 0 rtooroho Appendi xAt o Fina l Repor t CH-095/201 3 Figure 6 4-chloro-a,a,ct-trifluorotoluen e (PCBTF) : Representativ e chromatogra m o ftim e

4 8 h wate rsampl e a t 17. 8 mg/L .

Qualitativ e Analysi s Repor t Dat a Filenam e Sampl e Typ e Instrumen tNam e Ac q Metho d I R KCalibratio n Statu s T48_ll. d Sampl e Instrumen t1 PC8TF_CH09213. m No tApplicabl e Positio n 0*9513,17. 8 mg/UT4 8 VfetS 2 Acquire d Tim e 4/4/201 3 7:0Si0 4 P M D AMetho d reserpfna. m Sampl e Grou p Info . Use rChromatogram

s xl O 1 3 2 1- 1 0

- 1

- a DAD 1 - A:Sig=220.1BT<ia_11- d

h __ _

. i. . i J 

## w" i ! < ! . 1 ! I ! ! Respons e Unit s (%

> vs . Acquisitio n Tim e (min >
En d O fReport -

•ij tA|i(mtTechnolDBl« i Pag e 1 o f 1 Printe d at : 8:5 7 A M on : 4/5/201 3

Appendi xApag e 5 6 o f7 9p . 5 8



# ChemService Controlli o rloorohm Appendi xAt o Fina l Repor tCH-095/201 3 Figure 7 4-chloro-a,a,a-trifluorotolijen e (PCBTF) : Representativ e chromatogra m o ftim e

72 h wate rsampl e at 17. 8 mg/L .

Qualitativ e Analysi s Repor t Dat a Filenam e Sampl e Typ e Instrumefi tNam e Ac q Metho d IR M Calibratio n Statu s Commen t Sampl e Grou p Info . T72_lt, dSample Nam e ONBBajJ X mg/L-T72 Sampl e Positio nVia l 2 6 Instrumen

t1 UserName PCBTF_CH09213.

mAcquire drun e 4/5/201 3 4:57.2 7 P M No tApplicabl e D AMetho d reserpina.n i
Use rChromatogram s x10 l 4 - 3 - 2 - 1 - 0 -

- 1 -2 - ~&S~~ \l_

5 S 2. 5 4 3: 5 4 4'. 5 S 5' 5 6 Ol S 1 7. 5 a9 9. 5 Respons e Unit s {% ) vs . Acquisitio n Tim e (mln )

En d O fReport—

~ $ AiftonilttcluHihtBii * Pag e 1 o f1 Printe d at : 9:2 6 A H on : 4/8/201 3

Appendi xApag e 5 7 o f7 9Appendi xB t o Fina l Repor t CH-095/201 3 

# APPENDI XB
TES T TEMPERATUR

E

Appendi xB pag e 5 8 o f7 9p . 60



# ChemService Con trolll a rlaorohm

Ch*mSoMc*S/. l Appendi xB t o Fina l Repor

t CH-095/201 3 T_dbampcr___r

a CH-005/291 9 Corrected ttmptf . tur t(*C

> Correcte

d m i n i * m pvatur e f* o ccnwleaiiba

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## Ax Appendi xB pag e 5 9 o f7 9

# ChemService Controlli 0 rtooroho Appendi xC t o Fina l Repor tCH-095/201 3


## APPENDI XC
STATISTICA L ANALYSI S

Appendi xC pag e 6 0 o f7 9p . 62



# ChemService

Controll i o rtooroh m ' Appendi xC t o Fina l Repor t CH-095/201 3 Statistica l analysi s i n term s o fmeasure d geometri cmea n o f4-chloro-a.a.a trifluorotoluen e (PCBTF ) fo rend-poin tgrowt h rate :

Pagato Ti

. . ftcportOffta: 1»apf-1310,1 5 X M CETI S Dat

a Workshee

t o^z^wsjcHoasfloi a Satoiwtlni m Ofowtt i Tt* t ChmMnrle a ftrXCONTROtU KRICERCHE Sa n Data : KapM S spwha : SWensaiM* caprfcomulum sanptiCocM : 92293390 9 endin g Out : 05apr-1 3 Protocol : OECO20U2Q U Smplitw* : *foruo r Sampt a Dad : Material : PCBT F SMpt o Station ;

• • ! 'iiijui-'.ipffiimc j •...li , i.Li M fFOM h " J 21 * Q N 1 2 0068 4 (Mff W 0 N 2 3 90G U O.fl W 0 N 3 T 0456 1 0.07 4 0.07 » D N 4 2 1 ootts a oar » O.OJW 0 N 5 »

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# ChemService Controlli o rtaoroht Appendi xC t o Fina l Repor tCH-095/201 3

CETI S Tes

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Appendi xC pag e 6 2 o f7 9p . 6 4



# ChemService Controlli o rlomroHm Appendi xC t o Fina l Repor t CH-095/201 3

CETIS Tes

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Appendi xC pag e 6 3 o f7 9p . 6 5 

# ChemService

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Appendi xC pag e 6 4 o f7 9p . 66



# ChemService Controlli o

rlooroho Appendi xC t o Fina l Repor t CH-095/201 3

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Appendi xC pag e 6 5 o f7 9Controllio rlaorohm Appendi xD t o Fina l Repor

t CH-095/201 3



## APPENDI XD
COP YO F TES TITE M CERTIFICAT E O F ANALYSI S

Appendi xD pag e 6 6 o

f7 9p . 6 8



# ChemService

Controllim rlcorcho Appendi xD to Fina l Repor t CH-095/201 3



### CERTIFICATEOFANALYSIS PRODUCT: 4-ddoro-a^a-trifluortKolueo c (PCBTF ) MANUFACTURE

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Appendi xD pag e 6 7 of7 9p . 6 9 

# ChemService

Controllim rlcarchm Appendi xE t o Fina l Repor t CH-095/201 3



## APPENDI XE
COP YO F REFERENC E ITE M CERTIFICAT E O F ANALYSI S

Appendi xE pag e 6 8 o f7 9

# ChemService Controllimrlcorcho p . 7 0

Appendi xE to Fina l Repor t CH-095/201 3

SIGMA-ALDRIC H Slgma-Aldrlc hLaborchenikalie nQnb H 0-3091 8 SM1Z « Telefon : +4

9 5137 8238-15 0 Riedel-d e Hae n CERTIFICAT E O F ANALYSI S / INSPECTIO N CERTIFICAT E 3. 1 acc.t o DI N E N 1020 4 Saalie , 05.10.2007/844258/07/2422 5 Order-No. ; Custoner-So. : Order-Code : Quantity : Productio ndata : 19.Sap.200 7 lac . Ratas t

Data : 19.Sep.201 4 Article/Product : 4607 0 Batc h: 7262X 3,5-Dichlorophenol e PESTANAL ® 

### Referenc e Materia l (RM ) 1 . Genera l Information Formula : C6H4C12

0 CAS-No. : [591-35-5 ] Usag e : Metabolit e Mola r mass : 163.0 0 g/Mol e R e coram , storag e temp. : roomtetnp . Th e estimatedrelative error

of asingle measuremen

t of th e assa y ca nb e expectedto b

e ±0. 5 % (confidenc e leve

l 95% , n=6) . 2 . Batc hAnalysi s Identit y (NMR

) complyin g Assa y (GC

) 99. 9 are a% Wate r (Kar lFischer ) 0.08 6 % Meltin g rang e 65.4-66. 0 ° C 04.Oct.200 7 Dat e o fAnalysi s 3 . Advic e an d Remark

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# ChemService

Controlli m rlcore no Appendi xF t o Fina l Repor tCH-095/201 3



## APPENDI XF COP YO F EXTRACTIO N CERTIFICATIO N

O F ALGA L STRAI N

Appendi xF pag e 7 0 o f7 9

# o 

# ChemService Controllimrlomrono Appendi xF t o Fina l Repor

t CH-095/201 3



### O GEORG-AUGUST-UNIVERSITA TGOTTINGE N ALBRECHT-VON-HALLER-INSTITU T FO RPFLANZENWISSENSCI-IAPTE N Experimentell e Phykologi e un d S am mlun g vo nAlgenkulture n(SAG) Dr , M aik etoren z Nikolausberge

r We g 18,37073 Gottingen, Fax

. 0551/397871 , email ; epsag©gwdg.de Chemservic

e S.R.L MChiar aNer i viaF.II IBertram ! 15 1-2002 6 Novate-Milanese ITAL Y Gottingen , 5t ho f Jul y 201 0 Extractio ncertificatio n Thi s is t o certif y th e compan y Chemservic

e S.R.L . tha

t th e alga l cultur e wit hth e strai nnumbe r SA G61.8 1 pu

t a t you r disposa l contain s alga e o f th e species : Pseudokirchneriella subcapitata (formerlycalled: SelenastrumCapricorn utttm; Ankistrodesmus subcapitata; Ankistrodesmus bibraianus; Raphidocelis subcapitata) Th e strai nmentione

dabov e wa s las t rename db y u s int o Pseudokirchneriellasub
capitat abecaus e o f ne w researc hinsight s (Hindfik , F . (1990

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e Appendi xF pag e 7 1 o f79

# ChemService Controllim rlooroho Appendi xG t o Fina l Repor t CH-095/201 3



## APPENDI XG
COMPOSITIO N O F TES TCULTUR E MEDIU M

(OEC D N 201 , 2011 )

Appendi xG pag e 7 2 o f7 9

# ChemService Controtll m rlcorcho Appendi xG t o Fina l Repor tCH-095/201 3 Reconstitute d Water : I n deionise d wate r(conductivity< 5 pScm" 1 ) analytica l grad e salt s wil l b e adde d t o followin g fina l nomina l concentrations : O Macro-nutrients : NaHC0 350. 0 mg/ L CaCI 2 x2H 2 0 18. 0 mg/ L NH 4 C I 15. 0 mg/ L MgS0 4 x7H 2 0 15. 0 mg/ L MgCI 2 x6H 2 0 12. 0 mg/ L KH 2 P0 41. 6 mg/ L Trac e Elements : Na 2 EDTAx2 H 2 0 100. 0 pg/ L FeCI 3x6 H 2 0 64. 0 pg/ L MnCI 2 x4H 2 0 415. 0 pg/ L H3BO 3 185. 0 pg/ L Na 2 Mo0 4x2 H 2 0 7. 0 pg/ L ZnCI 23. 0 pg/ L CoCI 2 x6H 2 0 1. 5 pg/ L CuCI 2x2 H 2 Q 0.0 1 pg/ L p H 8. 1 ±0. 2



### O

Appendi xG pag e 7 3 o f7 9

# ChemService Appendi xH t o Fina l Repor tCH-095/201 3 Controlli m rlcoroho



## APPENDI XH Cop yofG.L.P . Authorizatio n b vItalia n Healt h Departmen t (date d Apri l 26th . 2012 )

Appendi xH pag e 7 4 o f7 9p . 7 6



# ChemService

Controllim rlamroHm Appendi xH t o Fina l Repor t CH-095/201 3

DIPARTIMENT O DELL ASANITA 1 PUBBLIC AE DELL1NN0VAZI0N E

DIREZION E GENERAL E DELL APREVENZION E

UFFICIOVI-EXDGPREV Biotecfwtogl

e e Buon a Pratlc a d l Laboratory

) Vi a Giorgi o Ribotla, 5 0014 4 Rurr w CER17FIC AT O O fCONFORMtTA ' ALC A BUON APRATIC AD

fLABORATOR Y {Decret o Legislativ o 02 marz o 2007 , n . S O - Direttiv a 2004/9/CE )
Roma , 26/04)201 2 Certificazion« : 014/201 2 A i sens ! deil'articol o 4 comm a 1 de l Decreto Legislative n . 5 0 de l 2 marzo 2007 , dell ' articol o 3 de l decreto ministerial e 4 lugli o 1997 e tenuto conto dell'esit o favorevol e deii'lspezion e effettuata i n data 14-15/02/201 2 S I CERTIFIC A ch e I I Centr

e d i Saggio : Chemservic e S.r.l , - Controll i e Ricerch e

Vi a F.ll l Beltram i 1 5 2002 6 Novat e Milanes e (Ml )

e idone o a d effeltuare ne l rfspelto dell e Buon e Pratich e d l Laboratori o d i cu i all'altegato I I de l citat o decreto legislative :

Appendi xH pag e 7 5 o f7 9

# ChemService Controlli m rlaorohi Appendi xH t o Fina l Repor

t CH-095/201 3

1 ) TeatRsteo-Chimtc l

umidtta ; Atfdlt a eatolfnita ; PH ; Punt o d i fusione ; Punt o d i Inflammability ; ResldU D a l setacdo ^ Sagnablllta ; Sospenslblllto ; Pes o specsflco apparente ; Sostani e Insolgbil t I n acetone ; Schilwi a persistente ; Densit a relativa l Tensloft cd l vapor e a divers e temperature ; Asgett a ( stnt o fislco , colore , odore e wmcentrazlon ] sogli a pe rl e sostanz e co n color e cgust o intense ) I n aequo) ) Spettr a d i assorbJmentb , (UV/VIS , IR , NM R e MS) , estron e molar e fl relativ e lungheza a d'onda ; Solubilit a I n acqua , compres l gt l effott l de l p H (d o 5 a 9 ) e delt a temperatur e sull e solubilita ; Solubilit a ne l solvent l organic! , compres i gi i effctt i dell e temperatur e suIl a solUblilta ; Coefficien t d l riparttzion e n-ottanoio/acqua rcompres i gl i effett l de l p H (d a 5 a 9 ) cdelt a temperature ; Degradazlon e ossldatlv a rotothlmica ; Stabiltt a dell e cmufaoni ; StabHft a accelerat e e a lung o terminc , idcntit o de l prodott l d l degradazlone ; Stabiltt a a bass e temperature ; Stabiltt a ne t solvents organic ! impieijat i noll e preparaaoni ; Stabilftaall e dilulztanl ; Infiamrnablflta ; Temperatur a d l autoaccenslon e Tension s superfidale ; Proprlet a ossldand ; peattMt o nt t wnfrOrttJ de l contenltorf ; VJscoslta ; Spontaneita ; Setacdartient o a d umido/a soceo ; Granulornstrla ; Contenut o i n potvcre ;

Appendi xH pag e 76 o f7 9p . 78



# ChemService

Controllim rlamrcho Appendi xH t o Fina l Repor tCH-095/201 3

Capsot a d i palverlzzaztane ; Capacste d i versamento j CompatlbHIta ; Fluids . 4 ) Stud i d ( towiclt a arnbiental e H I organltm l acquatic l e terreBtr f

Tossicit a acut a pes o - tes t stabco/semistatico/fluss o contlnuo ; Tossicit a acut a Daphni a - tes tstatlco/semistatico ; Iniblzionc delt

acrcsclt aalaale ; Tossidt a acut a lombridtl ; Tes td l inibizlon e deli o resplrwfon e d i fang N attivi ; Tossicit a prolungat o peso - tes t semi-stetlco ; Tossicit a su b letal e lombrico ; Tossicit a cronlc a lombrlco ; RJpraduiion e Oaphnla-bes tseml-statica . Tossicit a s u plant e acquatlch e Lemna ; Tossicit a s u microorganism ! de l suolo : tes t d i nitrificazion e e carbo n ificazionc . 5 ) Stud i s u comportamcnt o i n acqua,barro ( irla ; stud i d l blOBCttimulazlon e
> Stabilit yi n acqua , tass o d i idroltsi , degradazlon e fotoxhimica , quantitativ e e d identi cdel/ l prodotto/ ) d * d^rjrattazlrjr"? , C9rt?"t ? rtl rit«TM*i«?lrtni* J I divrus l p H (d a s a •Stabiiit a all'arla tdegradazlon e fatochlmlca Pidentit a del/de i prodotto/ l d i degradaztone .

- Stabilit yne l suolo ,

- Stud i d l bioaccumulazJon e ne l slstem a d i saggi o pesce . 6 ) Studi su l resldul Analls l de l rcsklu i I n matricfvegetal* , acqua , terren l e fangm , tessut l animal! , latt e e prodctt l casearl , ol fe aria .

Appendi xH pag e 7 7 o f7 9p . 79



# ChemService

Controllim rlaorchm Appendi xH t o Fina l Repor t CH-095/201 3

8 ) Chlmtc a Cllnlc a e Analitic a

Chimica anaHtfca

•Svilupp o e valldazion e <J I metodich e anallfclch e mon o e multiresidual i I n matrie i vefletafi , acqua , terren l cfans In , tcssut l anlmall , latt e e prodott l casearl , Dil i e aria , •Messa a punt o o validazlon e d l metod l analttic i pe rf'ldentlficazion e e l a quantificaztcin e d l prindp t atUvf . •Mess a a punt o e validazlon e d l rnetod l analJtlc l pe rf'ldentlftcazlon e e fa quamificazion e dell e Impurezz e sfgniflcatrv e e rlfevanti . •Mess a a punt o e validazlon e d l metod l analitic l pe rl a detcrmlnazion e d l prlndp i attlv l e lor o metabotlt i i n fluid ! blologl d e d omogenat i d i tessut i onimalt . •Analis l d i prinefp i attSvi e impurezz e significativ e e CHevatitt .

Stud i d i metabolism o e famtacccfnetScd Biodegradabilib a pront a - test manometne o Blodegradabilit a ultim a * close tbottl e tes t Biodegradabilrt a ultim a - evoluzion e d i CG2 . Sogg fd i ottlvit a batterIcld a e funglcld a s u dlslnfettant i chimlc l e d antisettlci , I n sospension e e s u superfl d no n porose , usat l i n comp o allmentare , industriale , domestic o e nell e comunlta .

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### emission©. Appendi xH pag e 7 8 o f7 9p . 8 0



# ChemService

Controlli m rloorcno Appendi xH t o Fina l Repor tCH-095/201 3

L a present s certrficazion e no n verr a rinnovata, a l lermin e dell a scadenz a naturale , qualor a no n si a presen ta

t a , a l sens ! de

tcomm a 2 deli'arlicol o 4 de l D.M . 4 lugli o 1997 (pubblicat o nell a G.U , n . 22 9 de l 1/10/1937) , e a l plt t tard l 9 0 glorn i prim a dell a scadenza , domand a d i rinnovo dell a stess a completa dell a sched a riassunliva aggiornata .

I LDIRETTOR E DELL'UFFICI O VI 0

Dr.ssa Grazi a Ballacc i

Appendi xH pag e 7 9 o f7 9p . 8 1

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51AG4J99D5/1A9 E Dop o aver e stampat o quest a Letter a d i Vettura : 1 . Utilizzat e i l pulsant e "Stampare " i n quest a pagin a pe rstampar e l a Letter a d i Vettur a co n l a vostr a stampant e lase ro a gett o d'inchiostro . 2 . Regat e l a pagin a stampat a lung o l a line a orizzontale . 3 . Inserit e l a Letter a d i Vettur a nell'apposit a bust a autoadesiv a e incollatel a sull a spedizion e i n mod o ch e l a part e co n i l codic e a barr e poss a esser e lett a co n l o scanner . Awertenza : Pe rl a spedizione , utilizzat e esclusivament e l a Lettera d i Vettur a stampat a originate . Utilizzar e pe rl a spedizion e un a fotocopi a dell a Letter a d i Vettur a e illegal e e potrebb e da rluog o a oner i supplementa l d i fatturazione , insiem e ali a cancellazion e de l vostr o Codic e Ciient e FedEx . TERMIN I LEGAL I E CONDIZION I DELL E DEFINIZION I D l SPEDIZION E FEDEX . I n quest a Lettera d l Vettura , "noi" , "hostro" , e TedEx"s l riferiscono a Federa l Egres s Corporation , l e su e filial ! e cortsoclat e e I rispettiv i dipendenti , agent i eappaltator i indlpendent! . Co n "voi' e "rostra"s i f a riferimento a l mittente , suo i dlpendenti , commlttent i e agenti . Qualora l a spedizion e abbl a origin s a( d l fuor i degl i Stat i Unitl , i l contratt o d l vettura s i intend e stipulat o co n l a consoclata , l a filial e o I'appaltatore indipendent e FedE xch e perprim o h a accettat o l a rostra spedizione . I I termin e "collo"lndic a qualsias i contenltor e o bust a accettat i d a no i pe rl a consegna , inclus i quell i d a roi presentat i a FedE xutilizzand o i nostr i sistem i automatici , misurazioni , manifest ! o Letters d i Vettura . I I termin e "spedizione " indic a tutt i i coll i ch e son o presentat i a FedEx e accettat i d a FedExs u urVunica Lettera d i Vettura . NOT ASU LTRASPORT O AEREO . Aqualsias i spedizion e intemazional e pe rvi a aere a s i applican o l a Convenzion e d i Varsavi a e i relativ i emendamenti . Dunqu e l a Convenzion e d i Varsavia , co n i relativ i emendamenti , disc i p i in a e i n molt i cas i limit a l a re s pon s abilit a d i FedE xpe rperdite , ritard i o dann i subit i dall a vostra spedizione . La Convenzion e d i Varsavia , com e emendata , limit a l a responsabilit a d i FedEx . Pe resempl o negl i Stat i Unit l l a responsabillt a e limitat a a 9,0 7 dollar i statunitens i pe rllbbr a (2 0 dollar ! pe rchilo) , a men o ch e no n si a stat o dichiarato u n valore superior e a i fin i dell a responsabillt a de l vettore , com e spiegat o qu i d i seguito , e no n abbiat e pagat o tutt e l e spes e supplementa l appllcablll . Llnterpretazion e e I'applicazfon e de i limlt l d l rssponsabllit a dell a Convenzion e d l Varsavi a posson o varfare a second a de l paesl . No n v i son o specifich e locallt a d'arrest o ufflclall , e FedExs ! riserva i l dlritt o d i fartransltar e l e spedizion i i n qualsias i mod o gl i sBmbr i pi u appropriato . NOT AA LTRASPORT O S U STRADA . L e spedizion i effettuat e unicament e s u strad a d a o vers o u n paes e ch e h a firmat o l a Convenzion e d i Varsavi a o i l Contratt o pe ri l trasport o intemazional e d i merc i s u strad a (i l "CMR" ) son o soggett e a i termin i e all e condizion i de l CMR , a presclnder e d a qualsias i altr a disposizion e contrari a dell a present e Lettera d l Vettura . Pe rquant a riguarda l e spedizion i effettuat e unicament e s u strada , i n cas o d i disaccord o tra l e disposizion i dell a CM R e l a present e Lettera d i Vettura , prevalgon o i termin i dell a CMR . LIMTAZION E D l RESPONSABILITA . S e no n dlversament e previst o dall a Convenzion e d i Varsavia , i t CM R o altr i trattat l intemazlonall , leggi , regolament l govematlvl , ordln ! o requlslti , l a massim a responsabillt a d i FedE x perdanno , perdita , ritardo , carenze , errat a o mancat a consegna , errat a o mancat a comunicazlon a d i Informazion i I n rapport o ali a vostra spedizion e e limitat a da l present e AccordD , second o i termin i e condizion i stlpulat ! ne l contratt o d l trasporto . Invitlam o a consultar e i l contratt o d l trasport o stabllit o nell a Guid a a l serviz l FedE xI n vigore , o i l su o equlvalente , pe rdeterminan t l e llmltazion i contrattuall . FedE x declin a ogn i responsabillt a su l caric o o asslcurazlon e a copertur a globale , m a e posslbll e pagare u n supplement s perogn i 10 0 US D supplementa l d i valor e dichiarat o a i fin i dell a responsabillt a de l vettore ( o ('equivalent s nell a valut a local e de l paes e d'ongine) . Qualora si a stat o dichiarato u n valore superior e a i fin i dell a responsabilit a de l vettore e si a stat o pagat o i l supplement , l a massim a responsabilit a d i FedE xsar a pa n alllmport o ch e risulta infenor e tra i l valore dichiarato e i read dann l subiti . ESONER O D l RESPONSABILITA . I N NESSU N CAS O FEDE XSAR ARESPONSABIL E PE R EVENTUAL I DANNI , SIAN O ESS I DIRETTI , INDIRETTI , ACCIDENTALI , PARTICOLAR I O EMERGENTI , PE R U N VALOR E SUPERIOR E AQUELL O DICHIARAT O A l FIN I DELLARESPONSABILITADE LVETTOR E (INCLUSA , m SENZALIMTAZION E ALCUNA , L APERDIT AD l ENTRAT E O GUADAGNI ) OPPUR E PE R I LREAL E VALOR E DELLASPEDIZIONE , S E INFERIORE , INDIPENDENTEMENT E DA LFATT O CH E FEDE X FOSS E O N O ACONOSCENZ ADELL'EVENTUALIT ACH E TAL I DANN I POTESSEROVERIFICARSI . FedE xno n e responsabil e peratt i o omission ! de l mittente , Inclus i m a senz a llmitazion e alcuna : u n errat a dlchiarazion e d i carico , imballaggi , misur e d i sicurezza , contrassegn l o indirizz o Insufficient ! o Inadeguatl ; oppur e peratt i o omission i de tdestinatarl o o d i chiunqu e altr o ch e abbl a u n Interess e nell a spedizione ; oppure perviolazion i de i termin i de l present e accord o d a part e d i un o de i contraenti . FedE xno n e responsabil e pereventual i danni , ritardi , carenze , errat a o mancat a consegna , errata o mancat a comunicazion e d i informazion i i n relazion e a spedizion i d i contanti , valut a o altr i articol i praibiti , oppure i n presenz a d i situazion i a l d i fuor i de l nostr a controllo , com e caus e d i forz a maggiore , pericol i dell'aria , condizion i meteoralogiche , ritard i d i natur a meccanica , att i d i nemlc i pubblici , guerra , sciopero , guerra civile , oppure peratt i o d omission i d i autorit a pubblich e (tr a cu i funzionar i doganal i e sanitari ) co n autorit a real e o apparente . NESSUN AGARANZIA . No n diam o alcun a garanzia , n e espliclt a n e Impllcita . RECLA M PE R PERDITE , DANN I O RITARDI . TUTT I I RECLA M DEVON O ESSER E FATT I PE R SCRITTO , ENTRO I TERMN I PRESCRITTI . PE R I DETTAGLI , VIINVITIAM O ACONSULTAR E L E TARIFFE , L AGUIDAA l SERVIZ I FEDE XI N VIGOR E OPPUR E L E CONDIZION I STANDAR D D l TRASPORTO . La Convenzion e d i Varsavi a preved e particolar l procedur e scritt e perl e richiest e d l risarclment o pe rdannl , ritard i o l a mancat a consegn a dell a spedizione . Inottre , llnterpretazion e e I'azion e dell e disposizion i d i rectam o dell a Convenzion e d i Varsavi a posson o va n are d a u n paes e all'altro . Pe rconoscer e i termin i entro cu i inoltrar e l a richlest a relativ a ali a rostra spedizione , s i preg a d l fa r riferimento ali a Convenzione . I I diritt o ali a richiest a d i risarcimento d i dann i ne i nostr i confront i s i intender a scadut o s e entro du e ann i no n e stat a intentat a causa , second o quant o stabilit o dall a Convenzione . FedE xno n h a alcu n obblig o d i agir e ne i confront i d i un a richiest a fin o a l complet o pagament o d i tutt e l e spes e d i spedizione . L'ammontare dell a richiest a d i risarciment o no n e deducibil e dall e spes e d i trasporto . S e i l destlnatari o accett a l a spedizion e senz a annotar e alcu n dann o sull a bollett a d l consegna , FedE xpresum e ch e l a spedizion e e stat a consegnat a i n buon e condizioni . Afflnch e u n reclam o pe rdann l poss a esser e pres o i n considerazione , i l contenuto , i l carton e d i spedizion e origlnal e e I'lmballaggi o devon o esser e tenut i a nostr a disposizion e pe ri l controllo . LEGISLAZION E COMPETENTE . Nell a mlsur a i n cu i un a disposizion e contenut a o a cu i s i f a riferimento nell a present e Lettera d l Vettur a d i trasport o vi a aere a e contrari a a trattat l Intemazlonal i i n vigore , leggl , regolamentl govemativi , ordln i o requisiti , ess a rest a valld a I n quant o facent e part e de l nostr a accordo , purch e no n si a dirimente . La nullit a o l a no n applicabilit yd i qualsias i disposizion e no n h a alcu n effett o sull e altr e part i dell a present e Lettera d i Vettur a d i trasport o vi a aerea . Salv o altriment i indicato , FEDERA LEXPRES S CORPORATION , 200 5 Corporat e Aronue , Memphis , T N 38132 , USA , B i l prim o vettore d i quest a spedizione . Indirizz o e-mai l indicat o i n www.fedex.com .