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, 201

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 

591

h 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 I 
p. 2 

Chem Service Controllt 0 ricorcho 

Final Report CH-095/2013 

GLP COMPLIANCE STATEMENT 

4-CHL0R0-a,a,a-TRIFLU0R0T0LUENE(PCBTF): 

TOXICITY TO GREEN ALGAE 

Pseudokirchneriella subcapitata 

DETERMINED IN A GROWTH INHIBITION STUDY 

This study was performed by the Test Laboratory of ChemService S.r.l. Controlli e Ricerche 

under the direction of Francesca No6, in compliance with the Study Plan CH-095/2013 and 

Good Laboratory Practice Standards reported in Italian Legislative Decree (D.L No. 50 dated 

March 2n d, 2007) as published in G.U. No. 86 of April 13th, 2007 and in compliance with OECD 

Principles on GLP. 

The results of this report completely and faithfully reflect the Raw Data generated by the Study. 

Date 

Study Director 

page 2 of 79 
Chem Service 

CantrolU m riomronm 

SPONSOR 

Finchimica S.p.a. 

Via Lazio, 13 

25025 Manerbio (BS) 

Italy 

FINAL REPORT 

STUDY TITLE 

4-CHLORO-a,asa-TRIFLUOROTOLUENE(PCBTF): 

TOXICITY TO GREEN ALGAE 

Pseudokirchneriella subcapitata 

DETERMINED IN A GROWTH INHIBITION STUDY 

AUTHORS 

Francesca Noe, Rachele Dini, Garagna Desiree 

TEST FACILITY 

ChemService S.r.l. Controlli e Ricerche 

GLP Studies Department 

Via Fratelli Beltrami, 15 

20026 Novate Milanese (Ml) 

Italy 

Ph:+39 (0)2 3569961 

Fax: +39 (0) 2 35699650 

G.L.P. Study No. 

CH-095/2013 

CHEMSERVICE S.r.l. CONTROLLI E RICERCHE 

SOCIETA' SOGGETTA AL CONTROLLO E AL COORDINAMENTO DA PARTE Dl FINAGRO S. P. A. CON SEDE IN MILANO 

SEDE LEGALE ED OPERATIVA: 20026 NOVATE MILANESE {Ml) - VIA F.LLI BELTRAMI, 15 - TEL. 02 356 996.1 - FAX 02 382 014 46 

REA 1132847 - REGISTRO IMPRESE Dl MILANO, CODICE FISCALE E P. IVA 07020820150 - CAPITALE SOCIALE € 100.000,00 I.V. 

www.chemservice.it; e-mail:info@chemservice.it 
p. 4 

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Chem Service 

Study Title: 

Study Number: 

Controill e rlcorcho 

Final Report CH-095/2013 

QUALITY ASSURANCE STATEMENT 

4-Choloro-a,a,a-trifluorotoluene (PCBTF):Toxicity to green algae 

Speudokirchneriella subcapitata Determined in a Growth Inhibition 

Study 

CH-095/2013 

In accordance with ChemService S.r.l. Controlli e Ricerche and Quality Assurance procedures for 

Good Laboratory Practice (GLP) the conduct of the study has been inspected by Quality Assurance 

and the results of inspections were reported to Study Director and Management. 

Study Inspections: 

Date(s) of Inspection Inspection Reporting Date(s) 

21 March 2013 Study Plan 21 March 2013 

03 May 2013 Draft Report and Raw Data 03 May 2013 

30 May 2013 Final Report 30 May 2013 

In addition other process inspections were periodically carried out by Quality Assurance on activities 

not necessarily related to this study and the results were reported to Study Director and Test Facility 

Management. The most recent process inspection is identified below. 

Process Inspection: 

Date(s) of Inspection Inspection Reporting Date(s) 

03 April 2013 Acute Toxicity to Green Algae 03 April 2013 

o 

Associated laboratories and support functions were subject to regular facility inspections in 

accordance with Quality Assurance procedures. 

The results in this report completely and faithfully reflect the raw data generated by the study. 

Debora Manera, 

Head of Quality Assurance, 

ChemService S.r.l. Controlli e Ricerche 

Date 

page 3 of 79 
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Controlli o rlooroho 

Final Report CH-095/2013 

STUDY IDENTIFICATION 

4-CHL0R0-a,a,a-TRIFLU0R0T0LUENE(PCBTF): 

TOXICITY TO GREEN ALGAE 

Pseudokirchneriella subcapitata 

DETERMINED IN A GROWTH INHIBITION STUDY 

Study Plan 

Test item 

Sponsor 

Test Facility 

CH-095/2013 

4-chloro-a,a,a-trifluorotoluene (PCBTF) 

Internal sample code: 2911434-001 

Batch number: 11/2012 

Sample supplied by the Sponsor 

Finchimica S.p.a. 

Via Lazio, 13 

25025 Manerbio (BS) 

Italy 

ChemService S.r.l. Controlli e Ricerche 

GLP Studies Department 

Via Fratelli Beltrami, 15 

20026 Novate Milanese (Ml) 

Italy 

Ph: +39 (0) 2 3569961 

Fax: +39 (0) 2 35699650 

Study Director Francesca Noe 

Schedule 

Initiation date 

Start of experiment 

Start of in vivo experiment 

Completion of in vivo experiment 

Start of analytical phase 

Completion of analytical phase 

Completion of experiment 

March 22,2013 

Apr 

Apr 

Apr 

Apr 

Apr 

Apr 

102,2013 

102,2013 

I 05, 2013 

102,2013 

I 16, 2013 

I 23, 2013 

page 4 of 79 
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Final Report CH-095/2013 

CONTENTS/1 

page 

GLP Compliance Statement 2 

Quality Assurance Statement 3 

Study Identification 4 

Schedule 4 

Contents/1 5 

Contents/2 6 

List of Appendices 6 

Key Personnel 7 

Summary 7 

Introduction 9 

Materials and methods 9 

Test item 9 

Reference Item 11 

Equipment for Biological Phase 11 

Definitions 11 

Test system 12 

Experimental design 12 

Dosage and concentrations 12 

Experimental conditions 13 

Evaluations 15 

Observations and determination of EyCXt ErCxand the NOEC 15 

Calculations 16 

Measurement of the test concentrations 17 

Calculation of the actual test concentrations 17 

page 5 of 79 
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Final Report CH-095/2013 

CONTENTS/2 

page 

Algal strain sensitivity check 17 

Conditions for the validity of the test 18 

Results and discussion 18 

Growth rate 25 

Yield 27 

Conclusions 28 

References 29 

Archiving 29 

LIST OF APPENDICES 

page 

Appendix A - Analytical Phase Report 30 

Appendix B - Test Temperature 58 

Appendix C - Statistical Analysis 60 

Appendix D - Copy of Test Item Certificate of Analysis 66 

Appendix E - Copy of Reference Item Certificate of Analysis 68 

Appendix F - Copy of Extraction Certification of Algal Strain 70 

Appendix G - Composition of Test Culture Medium (OECD N 201, 2011) 72 

Appendix H - Copy of GLP Authorization by Italian Health Department 74 

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ChemService 

Controlli 0 rtaorcho 

Final Report CH-095/2013 

KEY PERSONNEL 

o 

Study Director Francesca Noe (f.noe@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

Study Director deputy Erica Tediosi (e.tediosi@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

Test Facility Manager Giacomo Ravetta (giacomo.ravetta@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

Head of Quality Assurance Debora Manera (d.manera@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

Sponsor's Representative Paolo Betti (p.betti@finchimica.it) 

Finchimica S.p.a. 

Technician 

(biological phase) 

Rachele Dini (r.dini@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

Technician 

(analytical phase) 

Desiree Garagna (d.garagna@chemservice.it) 

ChemService S.r.l. Controlli e Ricerche 

SUMMARY 

The influence of the test item 4-chloro-a,a,a-trifluorotoluene (PCBTF) on the growth of the green 

algal species Pseudokirchneriel/a subcapitata, formerly known as Selenastrum Capricornutum, was 

investigated in a 72-hour full test according to the OECD Guideline No. 201, 2011. 

For this purpose, exponentially growing test algae were exposed for 72 hours to an aqueous 

test medium containing the test item at five different concentrations in a geometric series, 

namely 7.9, 11.9, 17.8, 26.7 and 40.0 mg/L corrected for its purity (99.3 % w/w), under defined 

conditions. 

Besides the concentration, a negative control without the test item was also prepared to check 

the acceptability of the algal inoculum. 

page 7 of 79 
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ChemService 

^^^^^^^^Z " Controti l 0 rtc&rcn * " 1 

Final Report CH-095/2013 

At the beginning of the test, the pH value of the test medium ranged between 7.62 - 7.77 for 

test item solutions and was 7.91 for the negative control. 

At the end of the test the pH values of negative control ranged between 9.28- 9.33 and the pH 

mean values of test item solutions were in the range 9.31 - 9.60. 

The incubation temperature was in the range 23.3 °C - 24.1 °C. 

Light intensity during the 72-hour period was in the range 5220 - 5401 Lux. 

The actual test concentration of the test item were analytically measured at the beginning and at 

the end of the test. Additional samplings for analysis at 24 hour intervals during the exposure 

period will be taken in order to better define loss of the test item. 

The analytical recovery of the concentrations of test item in the freshly prepared solutions 

ranged between 72.6 % and 87.1 % of the nominal concentration of the test item. 

After 24, 48, and 72 hours of exposure, the analytical recovery were lower than the limit of 

detection (L.O.D. = 100.0 pg/L) of the analytical method, since test item evaporated during the 

72-hour period. 

The analytical recoveries were referred to the test item content (corrected for its purity, 99.3 %). 

As recommended by OECD Guidance Document on testing of difficult substances No. 23 

(2000), the biological results were referred to the geometric mean of test item measured 

concentrations, since the analytical recoveries of test item were lower than the range 80 % -120 

%. 

Algal cell density was measured every 24 hours taking aliquots from each test concentration 

replicate and from negative control, diluting them in a NaCI 9 g/L solution and reading with an 

electronic particle counter. 

At the test end the cell density in the negative control increased on average by a factor of 239. 

This value complies with the validity criterion of the test, according to the mentioned guidelines, 

which indicates a minimum increase factor of 16. 

The negative control also met the other validity criterion, with a coefficient of variation of daily 

growth rates at 72 hours of 25.6 % and a coefficient of variation of average growth in replicates 

negative control cultures during the test period of 1.2 %. 

For the tested concentrations, any significant biological effect was showed in comparison with 

negative control for both the endpoints, yield and growth rate, after 72 hours of exposure. 

page 8 of 79 
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Final Report CH-095/2013 

ChemService 

Controlli © rtomrchm 

The 72-hour EC1 0 , EC2o, EC5 0 and the NOEC, calculated in terms of geometric mean of 

measured concentrations, for the two end-points, are reported in the following table: 

Endpoint 0-72hECio 0 - 72 h EC2 0 0 - 72 h EC5 0 0 - 72 h NOEC 

Growth 

rate 

>0.41 >0.41 >0.41 0.41 

Yield >0.41 >0.41 >0.41 0.41 

INTRODUCTION 

The study was performed to evaluate the effect of the test item 4-chloro-a)a,a-trifluorotoluene 

(PCBTF) on the growth of the green algal species Pseudokirchneriella subcapitata 

(Selenastrum capricornutum). The Study was performed in compliance with the Study Plan CH095/2013 and the following guidelines: 

OECD Guideline No. 201, "Freshwater algae and cyanobacteria growth inhibition test", 

2011. 

OECD Series on Testing and Assessment No 23 - Guidance Document On Aquatic Toxicity 

Testing Of Difficult Substances And Mixtures ENV/JM/MONO(2000)6. 

The test method and the test species Pseudokirchneriella subcapitata are recommended by the 

international test guidelines. 

MATERIALS AND METHODS 

Test item 

A sample of 4-chloro-a,a,a-trifluorotoluene (PCBTF), contained in a glass bottle, was received 

by the Test Facility on February 12, 2013 (internal sample code: 2911434-001). 

page 9 of 79 
p. 11 

ChemService C'ontroifl 0 rtcorch* 

Final Report CH-095/2013 

Information on the test item is as follows (the test item and its relevant information were 

provided by the Sponsor): 

Test item name 

Chemical name 

CAS. number 

EEC number 

Molecular weight 

Relative density (20 

Empirical formula 

'C) 

4-chloro-a,a,a-trifluorotoluene (PCBTF) 

4-chloro-a,a,a-trifluorotoluene 

98-56-6 

202-681-1 

180.55 g/mol 

1.3361 g/mL (determined in no GLP ChemService study 

number CH-046/2013) 

CIC6H4CF3 

Structural formula 

Purity 

Batch number 

Internal number 

Manufacturing date 

Expiry date 

Storage conditions 

Stability 

99.3% 

(from CoA and TSDS supplied by Sponsor) 

11/2012 

2911434-001 

January 04, 2013 

January 2016 

The test item, contained in the original closed and sealed 

bottle, is stored at room temperature far away from 

sources of heat, in accordance with Test Substance Data 

Sheet (TSDS) and Material Safety Data Sheet (MSDS) 

supplied by the Sponsor. 

no thermal decomposition when the product is stored and 

handled correctly 

The test item CoA is annexed in Appendix D to this report. 

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Final Report CH-095/2013 

Reference Item 

Reference item name 3,5-Dichlorophenol pestanal analytical standard 

CAS. number 591-35-5 

Batch number 7262X 

O Preparation date : September 19,2007 

Expiry date September 19, 2014 

Chemical formula : C6H4Cl20 

Molecular weight 163.00 g/mol 

Storage conditions Room temperature/well ventilated room 

Stability Stable under storage conditions 

The reference item CoA is annexed in Appendix E to this report. 

Equipment for Biological Phase 

- Shaking apparatus, Falc, Internal code No. 247 

- pH meter, WTW 315i, Internal code No. 244 

- Laminar flow bench, Internal code No. 260 

- Optical microscope No 321 

- Electronic particle counter, Beckman coulter Z2, Internal code No. 552 

- Magnetic stirrer type AGE, Velp Scientific, Internal code No. 256 

- Lux-meter, Testo 545, Internal code No. 252 

- Micropipette BIOHIT proline (20-200 pL) No. 170/E6 

- Laboratory Water Purification Systems, Sartorius Arium® 611, Internal code No. 355 

- Datalogger Testo, T-174, Internal code No. 477 (precision: 0.48; correction factor, Cw 0.2 °C) 

- Climatic chamber, KBW400 Binder, Internal code No 637 

O 

Biomass: 

Growth: 

Growth rate: 

Yield: 

EYCX: 

ErCx: 

Definitions 

cells number per millilitre. 

the increase in cell density over the test period. 

the increase in cell density per time unit. 

the biomass at the end of the exposure period minus the biomass at the 

start of exposure period. 

the calculated concentration of test item which results in a X% reduction of 

yield (Y) compared to the control. 

the calculated concentration of test item which results in a X% reduction of 

growth rate (r) compared to the control. 

page 11 of 79 
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Final Report CH-095/2013 

NOEC: the concentration of test item which results in a not significant reduction of 

yield (Y) or growth rate (r) compared to the negative control. 

Test system 

The study was performed with the unicellular, fresh-water green algae Pseudokirchneriella 

subcapitata strain (formerly known as Selenastrum capricornutum), cultured in the laboratories 

of the Test Facility and originally purchased from the Institute of Plant Physiology of the 

University of Gbttingen, Germany. 

The algae were cultured in a climatic chamber at 24 °C ± 2 °C under continuous uniform 

illumination in the range 4440 - 8880 Lux in the spectral range 400-700 nm. The culture medium 

was the same of the test medium; the stock cultures were weekly transferred to fresh medium 

and maintained in continuous shaking to ensure the necessary amount of C02 and to keep 

algae in suspension. Cultures containing deformed or abnormal cells were discarded. Only 

exponentially growing algal cultures have been used to start the test. 

The copy of extraction certification is attached in Appendix F. 

Experimental Design 

Dosage and Concentrations 

The test concentrations were chosen in accordance with the results of a pre-test (range-finding 

test), which was performed not in compliance with GLP Regulations and whose Raw Data were 

archived under the code of the present study. 

The main test included five test item concentrations in a geometric series, namely 7.9, 11.9, 

17.8, 26.7 and 40.0 mg/L, corrected for its purity (99.3 % w/w). 

The test medium was prepared as follows: 

prior to the test start, a 1 -litre bottle totally filled with 1170 mL of reconstituted water, of a 40.0 mg/L 

of the test item solution was prepared by dosing with a micropipette 35.5.0 \xL of test item, 

corresponding to 47.4 mg of test item (considering its density:1.3361 g/mL , determined in no GLP 

ChemService study number CH-046/2013, and its purity 99.3%). The bottle was tightly closed to 

prevent test item evaporation. 

The obtained solution appeared transparent with oily droplets on the bottom of the bottie, 

therefore it was mixed for two hours by the mean of a magnetic stirrer in dark conditions to 

achieve the total solubilisation of test item. After stirring, the solution appeared homogenous 

and transparent. 

After pH check, , the test concentrations were prepared by taking adequate aliquots of the stock 

solution and diluting them into reconstituted water as reported in Table 1. 

Test flasks were inoculated with algal culture. 

A negative control with algal growth medium only was also tested. 

page 12 of 79 
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ChemService 

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Table 1 Test solutions preparation, 

Final Report CH-095/2013 

O 

Tested 

solutions 

Nominal Test item 

concentration 

(mg/L) 

Final volume of 

reconstituted water 

(mL) 

Added volume of 

40.0 mg/L solution 

(mL) 

1 7.9 250 49.4 

2 11.9 250 74.4 

3 17.8 250 111.3 

4 26.7 250 166.9 

Experimental Conditions 

The treatment vessels were identified by experiment number, group and replicate number. 

All the described procedures were conducted under a laminar flow bench to ensure the sterility 

of the algal cultures. 

For the test concentrations and for the negative control, 100 mL capacity conical glass flasks 

capped with air permeable stoppers were used. 

Three replicates for test item concentrations and six for negative control were prepared. At test 

start, 50 mL of test solutions were poured into the test flasks and inoculated with algae to obtain 

a starting cell density of about 104 cells/mL. 

The algal inoculum was taken from a pre-culture, started 3 days before the beginning of the test 

in order to ensure that the assay was performed with exponentially growing algal population. 

The cell density of the inoculum was around 106cells/mL. 

The test flasks were incubated in a climatic chamber, under continuous illumination and 

constant shaking. The flasks were randomly distributed under the light and were replaced every 

day during the test. 

Temperature: 

The room temperature under the light was continuously monitored during the course of the 

study by the mean of a data logger. It was in the range 23.3°C - 24.1 °C with a mean value of 

O 24.0 °C and a standard deviation of 0.1 °C. 

The temperature range provided by OECD guideline is 24 ± 2 C, this range was corrected 

according to the data logger precision (therefore the range to be considered for the test was 

22.5 - 25.5 °C) while temperature values were corrected according to correction factor (Cw). 

Measured temperature values are reported in Figure 1. 

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p. 15 

Final Report CH-095/2013 

CD 

a 

E 

.CD 

27 

26 

25 

24 

23 

22 

21 

20 

19 

r 

^jS^ Time (days) 

Fig. 1 Temperature trend during the test period (blue line); the red lines indicate the range 

of acceptable temperature. 

Light conditions: 

continuous illumination; light intensity was ranged between 5220 and 5401 Lux, within the 

OECD recommended range (4440 - 8880 Lux). 

Test duration: 

72 hours 

Test medium: 

Reconstituted water according to OECD Guideline 201, 2011 (see Appendix G). 

Test medium pH was measured in the test item solutions and in the algal culture medium at the 

beginning of the test and in each test flask at the end of the test. Mean values over the 

replicates are reported. 

The pH values of test media were measured with a portable pHmeter. pH values were 7.91 for 

the negative control and ranged between 7.62 - 7.77 for the test item solutions at the beginning 

of the test period and in the range 9.31 - 9.60 at the end. The mean values are reported in 

Table 2. 

The pH in the negative control varied by a maximum of 1.43 units, this value was considered 

acceptable though it was slightly higher than the recommended limit by the OECD guideline 

stating that the pH values in the negative control replicates should not increase more than 1.5 

units during the test period. 

For fresh solutions the pH values were measured sampling an aliquot immediately after test 

solution preparation, before pouring it in the test flasks. 

For spent solutions (after 72 hours), pH were measured in each test flask. 

page 14 of 79 
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Controlit« rlaoroh* 

Final Report CH-095/2013 

Table 2 pH values in the test media at the start and at the end of the test. 

Nominal test item 

concentrations 

(mg/L) 

Start (0 hours) 

pH 

End (72 hours) 

pH (mean values) 

0.0 (negative control) 7.91 9.31 

7.9 7.77 9.31 

11.9 7.62 9.42 

17.8 7.77 9.52 

26.7 7.74 9.60 

40.0 7.64 9.50 

Evaluations 

Observations and determination of E y C x , ErCx and the NOEC 

Algal cell density was measured every 24 hours taking aliquots from each replicate of negative 

control and test item concentration, diluting them in a NaCI 9 g/L solution and reading with an 

electronic particle counter. 

The measured values were used to calculate the percentage inhibition of cell growth as yield 

and as growth rate in comparison to the negative control. 

The CETIS V.1.026D software was used to carry out the statistical analysis: it automatically 

chooses the most appropriate analysis for the pool of data. 

The EyCx and the ErCx values were determined by a Linear Interpolation analysis. 

The NOEC value for yield and growth rate endpoint were determined by comparison with the 

control, by Bonferroni Adj t method. 

As input data, the geometric mean of measured concentrations of test item 4-chloro-a,a,atrifluorotoluene (PCBTF) were used. 

The negative control was considered also to check that the growth of the algal population used 

in the test meets the validity criteria. 

Details of the statistical analysis are reported in Appendix C. 

page 15 of 79 
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Final Report CH-095/2013 

Calculations 

Average growth rate 

The average growth rate was calculated according to the following formula: 

In Xj - In Xs 

tj-ti 

(day1) 

(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 

The percent inhibition of growth rate for each treatment replicate from equation was calculated 

according to the following formula: 

% / f = - X100 

jUC 

% l r percent inhibition in average specific growth rate; 

j ! c mean value for average specific growth rate (y) in the control group; 

p, T average specific growth rate for the treatment replicate 

Yield 

The percent inhibition in yield (%ly) was calculated as follows: 

(Yc - YT) 

% !y= X 100 

Yc 

% l y percent inhibition of yield; 

Yc mean value for yield in the control group; 

YT value for yield for the treatment replicate 

page 16 of 79 
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ChemService 

Final Report CH-095/2013 

Controlli * rtooronm 

Measurement of the test concentrations 

The concentration of test item in the test medium was analytically measured as described in the 

analytical phase report (Appendix A to the present document). 

10 mL samples of test item solutions and negative control were collected from freshly prepared 

solutions at time 0 and at the end of the test period (72 hours) after removing algal cells by 

centrifugation. 

Duplicates samples of the test item solutions and the negative control were taken at the 

indicated times from the test solutions, but only one sample was analysed, while the other were 

stored as retain samples to be analysed only if necessary. 

At time 24 and 48 hours were collected an aliquot of 14 mL (10 mL for analysis and 4 mL as 

retained sample) for each concentrations. 

Calculation of the actual test concentrations 

According to the guidance document on difficult substances (OECD No. 23, 2000), in a semistatic test, if the concentration of the test item does not remain within 80 % -120 % of the 

nominal concentrations, the effect concentrations should be determined and expressed in terms 

of geometric mean of the measured concentrations, in order to take into account both the initial 

and the final actual concentrations. 

Since the analytical recoveries of test item during the test period (in the fresh and spent test 

item solutions) were lower than the range 80 % -120 % (75.7 % in fresh solution and lower than 

the L.O.D. in the spent solution), the geometric mean of measured concentrations was 

calculated (see Table 4) and the toxic endpoints were calculated in terms of geometric mean of 

test item measured concentrations. 

Algal strain sensitivity check 

The results of the most recently performed test with a reference substance are reported and 

compared with literature data to demonstrate that the algal strain used has got an adequate 

level of sensitivity to that substance. In the current case the results refer to a test performed on 

March 2013 (CH-167/2013) with 3,5-Dichlorophenol (see Certificate of Analysis in Appendix E) 

as reference item (Table 3). 

Table 3 Effect of 3,5-dichlorophenol on the growth of Pseudokirchneriella subcapitata 

Reference 72 h ErC5 0 (mg/L) Confidence interval 

(95%, mg/L) 

Internal study CH-167/2013 2.57 2.51 -2.63 

Reference data 

(UNI EN ISO 8692/2004) 2.08 -4.68 

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Final Report CH-095/2013 

CONDITIONS FOR THE VALIDITY OF THE TEST 

• A test is not considered acceptable if the cell density in the control cultures has not increased 

by a factor of at least 16 within the test period. 

• The coefficient of variation of daily growth rates in the control cultures must not exceed 35%. 

• The coefficient of variation of average growth in replicates control cultures must not exceed 

7%. 

RESULTS AND DISCUSSION 

A full test was performed to evaluate the effect of the test item 4-chloro-a,a,a-trifluorotoluene 

(PCBTF)on Pseudokirchneriella subcapitata. 

The obtained cell densities for each replicate are reported in Table 6. 

As shown in Table 5, the analytical recoveries of the concentrations of test item in the freshly 

prepared solutions ranged between 68.5 % and 87.1 % of the nominal concentration of the test 

item. 

At the 24, 48 and 72 hours the analytical recoveries were lower than the limit of detection 

(L.O.D.) of the analytical method. 

The analytical recoveries were referred to the test item content (corrected for its purity, 99.3 %). 

As recommended by the OECD Guidance Document on testing of difficult substances N. 23 

(2000), since the analytical recoveries were not in the range 80 % - 120 % during the test 

period, the biological results referred to the geometric mean of measured concentrations of test 

item (Table 4). 

page 18 of 79 
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p. 20 

ChemService Controlli m rloorcht 

Final Report CH-095/2013 

Table 4 Geometric mean of measured concentrations of test item. 

C 

Nominal Test 

item 

concentration 

[mg/L] 

Measured concentrations [pg/L] Geometric 

mean 

[M9/L] 

Geometric 

mean 

[mg/L] 

Nominal Test 

item 

concentration 

[mg/L] t = 0h t=24h t = 48h t = 72h 

Geometric 

mean 

[M9/L] 

Geometric 

mean 

[mg/L] 

7.9 5734.9 100.0* 100.0* 100.0* 275.2 0.28 

11.9 10362.0 100.0* 100.0* 100.0* 319.1 0.32 

17.8 13633.5 100.0* 100.0* 100.0* 341.7 0.34 

26.7 19678.7 100.0* 100.0* 100.0* 374.5 0.37 

40.0 27394.0 100.0* 100.0* 100.0* 406.8 0.41 

* When the measured concentration is lower than the L.O.D. of the analytical method, the L.O.D. itself 

(100.0 pg/L) is used for geometric mean calculation, as stated in OECD No. 23 (2000). 

O 

page 19 of 79 
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ChemService 

Controll i o rfoervh s 

Final Report CH-095/2013 

Table 5 Analytical recovery of the measured test concentrations to the nominal ones 

Time (hours) 

Nominal Test 

item 

concentration 

fmg/Ll 

analytical 

recovery 

[%] 

Mean analytical 

recovery 

(%) 

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 

24 7.9 * 

24 11.9 _* 

24 17.8 * 

-

24 26.7 _* 

24 40.0 _* 

48 7.9 * 

48 11.9 _* 

48 17.8 _* -

48 26.7 _* 

48 40.0 _* 

72 7.9 _* 

72 11.9 * 

72 17.8 _* -

72 26.7 * 

72 40.0 * 

*the analytical recovery were not determined because the measured concentration is lower than the 

L.O.D. of the analytical method. 

page 20 of 79 
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p. 22 

ChemService Controlli o rlooroho 

Final Report CH-095/2013 

In Table 6 the effect of test item on cell density is shown. 

O 

C 

Nominal 

test item 

concentration [mg/L] 

replicate 

Cell density (n. cells x 104 Nomina /mL) l 

test item 

concentration [mg/L] 

replicate 

24 h 48 h 72 h 

A 3.8670 45.1680 314.8370 

B 3.6340 35.6480 287.6570 

0.0 

(negative control) 

C 3.8480 34.8280 294.2170 

0.0 

(negative control) D 

E 

3.7860 

3.8590 

32.6880 

40.9680 

260.6170 

311.1970 

F 4.2570 33.3280 286.4770 

Average value 3.8752 37.1047 292.5003 

A 4.8420 41.2870 329.9220 

7.9 

B 5.1160 48.2670 331.9020 

7.9 

C 3.4110 43.9870 334.0820 

Average value 4.4563 44.5137 331.9687 

D 4.3910 47.1620 386.6210 

11.9 

E 4.8670 59.7220 387.4410 

11.9 

F 4.8140 51.0020 393.7610 

Average value 4.6907 52.6287 389.2743 

D 4.9290 51.0380 367.0020 

17.8 

E 4.4340 51.2580 395.2220 

17.8 

F 4.5510 47.9980 371.1020 

Average value 4.6380 50.0980 377.7753 

D 4.3490 47.4470 341.9380 

26.7 

E 4.9760 48.9270 346.2780 

26.7 

F 3.7000 47.7070 338.8180 

Average value 4.3417 48.0270 342.3447 

D 4.7680 50.1780 380.8120 

40.0 

E 4.4470 50.4380 366.1520 

40.0 

F 4.9540 56.9580 389.4320 

Average value 4.7230 52.5247 378.7987 

page 21 of 79 
p. 23 

ChemService Controlli a rtooroho 

Final Report CH-095/2013 

At test end cell density in the negative control resulted increased on average by a factor of 293. 

This value complies with the validity criterion of the test, according to the mentioned guidelines, 

which indicates a minimum increase factor of 16. 

The negative control also met the other validity criterion, with a coefficient of variation of daily 

growth rates of 25.6 % and a coefficient of variation of average growth in negative control 

replicates during the test period of 1.2 %. 

The growth curves for each replicate of the negative control are represented in Figure 2; the 

growth curves for each concentration of test item are represented in Figures from 3 to 7, while 

Figure 8 represents the comparison between the growth curves of the different concentrations 

and of the negative control calculated on the mean density values. 

3500000 

3000000 

j 2500000 

£ 2000000 

£ 1500000 

(A 

| 1000000 

500000 

0 * 

0 

-•— a 

-»- b 

c 

•*- d 

e 

-•—f 

20 40 

time (hours) 

60 80 

Fig. 2 Growth curves for each replicate (a-f) of the negative control 

4000000 

3500000 

3000000 

2500000 

2000000 

1500000 

1000000 

500000 

0 

20 40 

time (hours) 

60 80 

Fig. 3 Growth curves for each replicate (a-c) of the concentration, 7.9 mg/L. 

page 22 of 79 
ChemService 

Controlli o rtamrcn* 

p. 24 

Final Report CH-095/2013 

4500000 

4000000 

3500000 

3000000 

2500000 

2000000 

1500000 

1000000 

500000 

0 * 

20 40 60 

time (hours) 

80 

Fig. 4 Growth curves for each replicate (a-c) of the concentration, 11.9 mg/L. 

4500000 

4000000 

3500000 

3000000 

2500000 

2000000 

1500000 

1000000 

500000 

0 * 

20 40 

time (hours) 

60 80 

Fig. 5 Growth curves for each replicate (a-c) of the concentration, 17.8 mg/L. 

page 23 of 79 
p. 25 

ChemService Controlli a rfaoroho~ 

Final Report CH-095/2013 

4500000 

4000000 

3500000 

3000000 

2500000 

2000000 

1500000 

1000000 

500000 

o 

20 40 60 

time (hours) 

80 

Fig. 6 Growth curves for each replicate (a-c) of the concentration, 26.7 mg/L. 

4500000 

4000000 

Zi 3500000 

| 3000000 

£ 2500000 

£ 2000000 

| 1500000 

° 1000000 

500000 

0 A 

20 40 

time (h) 

60 80 

Fig. 7 Growth curves for each replicate (a-c) of the concentration, 40.0 mg/L. 

page 24 of 79 
p. 26 

Final Report CH-095/2013 

ChemService 

Controlli m rlamrcho 

4500000 

4000000 

3 3500000 

| 3000000 

g 2500000 

^ 2000000 

•| 1500000 

* 1000000 

500000 

0 * 

Fig. 8 Growth curves for the concentration and the negative control, mean density values 

Growth rate 

For all the tested concentrations, the algal growth rate (r) after 72 hours of exposure was slightly 

higher than the negative control (mean % inhibition: -1.0%). 

The percentages of growth inhibition for the tested concentrations are reported in Table 7. 

Table 7 Algal growth rate and inhibition caused by the test item over 72 hours of exposure in 

comparison with negative control. 

Nominal test item 

concentration 

(mg/L) 

0-24 h 0-4 i8 h 0-72 h Nominal test item 

concentration 

(mg/L) 

Mean 

growth 

rate 

Mean 

inhibition 

% 

Mean 

growth 

rate 

Mean 

inhibition 

% 

Mean 

growth 

rate 

Mean 

inhibition 

% 

0.0 

(negative control) 0.0564 - 0.0751 - 0.0788 -

7.9 0.0616 -9.3* 0.0790 -5.2* 0.0806 -2.3* 

11.9 0.0644 -14.1* 0.0825 -9.7* 0.0828 -5.1* 

17.8 0.0639 -13.3* 0.0815 -8.5* 0.0824 -4.5* 

26.7 0.0609 -7.9* 0.0807 -7.3* 0.0811 -2.8* 

40.0 0.0646 -14.6* 0.0825 -9.8* 0.0825 -4.6* 

* the algal growth rate was slightly higher than the negative control 

page 25 of 79 
p. 27 

1 

ChemService 

Controlli 0 rlaorcrio 

Final Report CH-095/2013 

The statistical analysis was carried out by the means of CETIS elaboration software v1.026D; at 

each observation time the ErCi 0 ErC2o ErC5o time were assessed by a Linear Interpolation 

analysis, while the NOEC was assessed by Bonferroni Adj t method. 

The ErCio, the ErC2o, the ErC5o values and the NOEC, in terms of geometric mean of measured 

concentration of test item, were as follows: 

Time ErC-io ErC2o ErCso NOEC 

0-24h >0.41 > 0.41 > 0.41 0.41 

0-48h >0.41 > 0.41 >0.41 0.41 

0-72 h >0.41 >0.41 > 0.41 0.41 

The concentration-response chart at 72 hours for growth rate end-point is shown in Figure 9. 

Concentration is expressed as geometric mean of measured concentrations. 

0.090i 

1 

s 

Pi 

0.080:; 

0.07(H 

0.06<H 

0.050^ 

0.04(H 

0.030^ 

0.020: 

0.010i 

0.000 

0.00 

1 1 i 1 

0.10 0.20 

Conc-mg/L 

0.30 0.40 0.50 

Fig. 9 Concentration-response chart after 72 h exposure to 4-chloro-a,a,a-trifluorotoluene 

(PCBTF) expressed as geometric mean of measured concentration of test item for 

growth rate end-point. 

page 26 of 79 
p. 28 

Final Report CH-095/2013 

ChemService 

Controlli o rloorchm 

Yield 

After 72 hours of exposure, the yield (Y), as difference of algal density between the end and the 

beginning of the test, for all the test item concentrations were higher than the control (mean % 

inhibition: -6.0%). 

The percentages of growth inhibition in terms of biomass yield for the tested concentrations are 

reported in Table 8. 

Table 8 Algal yield inhibition caused by the test item over 72 hours of exposure in 

comparison with negative control. 

Nominal test item 

concentration 

(mg/L) 

Mean biomass at 72h 

(cell/mL) 

Mean growth inhibition 

% 

0.0 

(negative control) 2915003 -

7.9 3309687 -13.5* 

11.9 3882743 -33.2* 

17.8 3767753 -29.3* 

26.7 3413447 -17.1* 

40.0 3777987 -29.6* 

* the yield was higher than the in the negative control 

The statistical analysis was carried out by the means of CETIS elaboration software v1.026D; at 

each observation time, the EyCx were assessed by a Linear Interpolation analysis, while the 

NOEC was assessed by Bonferroni Adj t method. 

The EyCio, the EyC2 0 , the EyC5o and the NOEC at 72 hours, in terms of geometric mean of 

measured of test item, were as follows 

Time EyClO EyC20 EyC50 NOEC 

72 h >0.41 >0.41 >0.41 0.41 

Details of statistical analysis are reported in Appendix C. 

page 27 of 79 
p. 29 

ChemService Controlli o rtaorctio 

Final Report CH-095/2013 

The concentration-response chart at 72 hours for yield end-point is shown in Figure 10. 

Concentration is expressed as geometric mean of measured concentrations. 

3.0BO6-

2.5B06: 

2.0B06: 

1.5B0&^ 

1.0B06 

5.0B05H 

0.0BO0 

0.00 0.10 0.20 0.30 

Conc-mg/L 

0.40 0.50 

Fig. 10 Concentration-response chart after 72h exposure to 4-chloro-a,a,a-trifluorotoluene 

(PCBTF) expressed as geometric mean of measured concentration of test item for 

yield end-point. 

CONCLUSIONS 

The toxicity of test item 4-chloro-a,a,a-trifluorotoluene (PCBTF) was tested on 

Pseudokirchneriella subcapitata. 

The 72-hour EC10, EC2o and EC5 0 and the NOEC, calculated , in terms of geometric mean of 

measured concentrations of test item and nominal test item concentration, for the two endpoints, are reported in the following table: 

Endpoint 0 - 72 h EC1 0 0 - 72 h EC2 0 0 - 72 h EC5 0 0 - 72 h NOEC 

Growth 

rate 

>0.41 >0.41 >0.41 0.41 

Yield >0.41 >0.41 >0.41 0.41 

page 28 of 79 
p. 30 

ChemService 

Controlli o rlaorcho 

Final Report CH-095/2013 

REFERENCES 

1 Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 

December 2006, concerning the chemical substance registration, estimation, authorization 

and restriction (REACh). 

2 Italian Legislative Decree (D.L. No. 50 dated March 2n d, 2007) as published in G.U. No. 86 

of April 13th, 2007; 

3 Organisation for Economic Co-operation and Development (OECD) Principles of Good 

Laboratory Practice (GLP) [C(97) 186 (Final)]. 

4 OECD Guideline No. 201, "Freshwater algae and cyanobacteria growth inhibition test", 

2011. 

5 OECD Series on Testing and Assessment No 23 - Guidance Document On Aquatic Toxicity 

Testing Of Difficult Substances And Mixtures ENV/JM/MONO(2000)6. 

ARCHIVING 

ChemService S.r.l. Controlli e Ricerche archives the Raw Data, the Study Plan as original and 

the Final Report as true copy of the original for 4 years. 

Thereafter, the Sponsor will be contacted for instructions regarding dispatch or disposal of the 

study documentation. 

Reference material used for analytical phase is stored until the expiry date whereas the test 

item is stored for at minimum six months starting from the approval of the Final Report. 

At the end of this period, the test item will be disposed of according to the environmental 

regulations in force, unless a written return request from the Sponsor is received. 

Water samples taken for the analytical phase will be stored until the approval of the Final 

Report. 

page 29 of 79 
p. 31 

ChemService Controill a rfcorchm 

Appendix A to Final Report CH-095/2013 

APPENDIX A 

ANALYTICAL PHASE REPORT 

Appendix A page 30 of 79 
c 

ChemService Controlli o rlaorohm " 

Appendix A to Final Report CH-095/2013 

CONTENTS 

c Page 

Introduction 33 

Materials 33 

Reference material 33 

Water Samples 34 

Equipment 36 

Reagents 36 

Analytical Method 36 

Experimental 40 

Results 40 

Conclusions 50 

Representative chromatograms (Figures 1-4) 51-57 

O 

Appendix A page 31 of 79 
ChemService Controlli o rlcorch< 

Appendix A to Final Report CH-095/2013 

INTRODUCTION 

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. 

For the determination of 4-chloro-a,a,a-trifluorotoluene (PCBTF) residues, all water samples 

were analysed following the internal Analytical Method No. 092/2013, validated in the internal 

Study No. CH-092/2013. 

MATERIALS 

Reference Item 

The test item, 4-chloro-a,a,a-trifluorotoluene (PCBTF), was used also as reference item, to 

perform water samples quantification. Details are reported at page 10. 

A copy of the Certificate of Analysis supplied by the sponsor annexed in Appendix D. 

Appendix A page 32 of 79 
o 

ChemService 

^^^^^^^ ^ C* o n fj^f/j T tf' rlaarcn m ' ' 1 

Appendix A to Final Report CH-095/2013 

Water Samples 

Samples description is reported in Table 1 and water composition is defined in OECD 

Guideline 201, 2011 and reported in Appendix G. 

Test solutions were sampled in duble. One sample was immediately analysed, the other 

O stored in the fridge at about 4°C. 

At time 0 hour and at time 72 hours, test solutions were sampled in duplicate (two vials of 10 

mL were totally filled), at time 24, 48 hours were collected an aliquot of 5 mL for each 

replicate (three replicate for every concentrations), 4 mL of these were keep into vials totally 

filled and replace into fridge at 4°C, 10 mL were centrifuged and analysed. 

Table 1 4-chloro-a,a,a-trifluorotoluene (PCBTF): water samples from the growth 

inhibition test 

(hours)

 Wate r Sam P| e s

 (*) sampling

 Dat e o f analvsi s

 Volume (mL) 

0 Control 02/04/2013 02/04/2013 2x 10 

0 7.9 mg/L 02/04/2013 02/04/2013 2x10 

O 

0 11.9 mg/L 02/04/2013 02/04/2013 2x10 

0 17.8 mg/L 02/04/2013 02/04/2013 2x10 

0 26.7 mg/L 02/04/2013 02/04/2013 2x 10 

0 40.0 mg/L 02/04/2013 02/04/2013 2x10 

24 Control 03/04/2013 03/04/2013 10+4 

24 7.9 mg/L 03/04/2013 03/04/2013 10 + 4 

24 11.9 mg/L 03/04/2013 03/04/2013 10 + 4 

24 17.8 mg/L 03/04/2013 03/04/2013 10 + 4 

24 26.7 mg/L 03/04/2013 03/04/2013 10 + 4 

24 40.0 mg/L 03/04/2013 03/04/2013 10 + 4 

48 Control 04/04/2013 04/04/2013 10+4 

48 7.9 mg/L 04/04/2013 04/04/2013 10 + 4 

Appendix A page 33 of 79 
p. 35 

) 

ChemService 

Controlli o rlooront 

Appendix A to Final Report CH-095/2013 

Time 

(hours) Water Samples (*) Date of sampling 

Date of analysis Volume (mL) 

48 11.9 mg/L 04/04/2013 04/04/2013 10 + 4 

48 17.8 mg/L 04/04/2013 04/04/2013 10 + 4 

48 26.7 mg/L 04/04/2013 04/04/2013 10 + 4 

48 40.0 mg/L 04/04/2013 04/04/2013 10 + 4 

72 Control 05/04/2013 05/04/2013 2x10 

72 7.9 mg/L 05/04/2013 05/04/2013 2x 10 

72 11.9 mg/L 05/04/2013 05/04/2013 2x10 

72 17.8 mg/L 05/04/2013 05/04/2013 2x10 

72 26.7 mg/L 05/04/2013 05/04/2013 2x10 

72 40.0 mg/L 05/04/2013 05/04/2013 2x10 

(*) Nominal concentrations are expressed as test item 4-chloro-aJa,a-trifluorotoluene (PCBTF). 

Appendix A page 34 of 79 
p. 36 

ChemService 

Controllt * rfoorcho 

Appendix A to Final Report CH-095/2013 

Equipment 

- High performance liquid chromatograph (HPLC) equipped either with Triple Quadrupole 

Mass detector and DAD detector, ESI interface, binary pump, auto sampler 

and software for instrument management and data re processing, internal code No.568. 

- Laboratory Water Purification Systems, Sartorius Ahum® 611, internal code No. 355 

- Refrigerator, internal code No. 388 

- Aspirated Safety Storage Cabinet No. 57A 

- Usual laboratory glassware 

- Volumetric glassware 

- Micropipettes, internal code No. 170/E1 

Reagents 

-Water, HPLC grade 

- Methanol, analytical grade (VWR International) 

- Acetonitrile, HPLC grade (VWR International) 

- Phosphoric acid 85% (H3P04 ), reagent grade (Merck) 

-Algal growth medium (OECD 201, 2011) 

ANALYTICAL METHOD 

All controls and fortified water samples were analysed following the internal Analytical 

Method No. 092/2013 validated in the internal Study CH-092/2013. 

The presence of 4-chloro-a,a,a-trifluorotoluene (PCBTF) in test water samples was detected 

by virtue of the HPLC coupled with a diode array detector. 

The principal features of the method validation study are reported here below. 

• The analytical method was shown to be specific for 4-chloro-a,a,a-trifluorotoluene 

(PCBTF) residues in water samples from ecotoxicological tests. 

• The range tested, from 200.0 u,g/L to 5000.0 ng/L, nominal concentrations, was found 

to fit a linear model (correlation coefficient > 0.99). 

• Repeatability (Precision) and Recovery (Accuracy) of the method were verified in 

reconstituted water (OECD 203, 1992 and OECD 202, 2004), algal growth medium 

(OECD 201, 2011). Results are summarised below: 

Appendix A page 35 of 79 
ChemService Coniroiiftt rioorchi 

Appendix A to Final Report CH-095/2013 

Fortification level Mean found RSD% % Mean 

nominal corrected* (Precision) (Accuracy) 

Reconstituted 250.0 Lig/L 252.2 Lig/L 231.4 \xglL 10.2% 91.7% 

water 25000.0 (ig/L 25222.2 (u.g/L 21657.4 iig/L 10.8% 85.9% 

Algal 250.0 Lig/L 252.2 HQ/L 192.3 ng/L 3.6% 76.2% 

growth 

250.0 Lig/L 

medium 25000.0 M-g/L 25222.2 ng/L 24383.9 \ig/L 7.0% 96.7% 

concentration corrected for density and 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). 

• The limit of detection (L.O.D.) of this method is defined as 50 % of the lowest 

calibration level, i.e. 100.9 jig/L (ppb) in water matrix samples (nominal 100.0 |Kj/L). 

If the residue amount is greater than 5000.0 j^g/L, the aqueous sample must be suitably 

diluted in volumetric flasks. 

Water samples storage and preparation 

Water samples were collected immediately, after water solutions preparation, in vials filled 

without air inside; an aliquot was transferred into a vial, filling the vial to the border and 

analyzed to HPLC/DAD. 

If samples are stored at 4°C, analysis can be performed within one hour. 

Appendix A page 36 of 79 
p. 38 

o 

ChemService 

Controll i • rfooroh o 

Appendix A to Final Report CH-095/2013 

Chromatographic conditions 

HPLC column 

Agilent or equivalent 

Detector 

Column temperature 

Eluent A 

Eluent B 

Eluent mixture 

Eluent flow 

Volume of injection 

PCBTF R.T. 

Total Analysis Time 

Internal code LCN 157 

Gemini C18, 150x3 mm 

DAD, X=220 nm 

room temperature 

acetonitrile 0.1% H3P04 

water 0.1% H3P04 

A:B 55:45 

0.8 mL/min 

100 LLL 

5.2 minutes 

10 minutes 

C 

Appendix A page 37 of 79 
ChemService 

Controlli o rleorohm 

Appendix A to Final Report CH-095/2013 

Calculations 

PCBTF residue content in the water samples is calculated by the following formula. 

PCBTF (ng/L) = C s x D s 

where: 

C s = PCBTF concentration in the sample solution (Lig/L) 

D s = Dilution or concentration of the sample solution (if any) 

The C s value is calculated from the A s data using the calibration curve obtained with Cstd 

and As t<j data from injections of working standard solutions. 

where: 

A s = PCBTF peak area in the sample solution 

c

s t d

 = PCBTF concentration in the working standard solutions (ng/L) 

A

s t d

 = PCBTF peak area in working standard solutions 

The percentage recovery is calculated as follows: 

[ig/L found 

Recovery (%) = x100 

fig/L added 

Appendix A page 38 of 79 
p. 40 

o 

ChemService 

Appendix A to Final Report CH-095/2013 

Controlli o rlotmrohm 

EXPERIMENTAL 

For the determination of 4-chloro-a,a,a-trifluorotoluene (PCBTF), each sample was analysed 

following the procedures described in the Analytical Method No. 092/2013. 

In order to evaluate the performance of the method during the analysis of ecotoxicological 

samples two procedural accuracy tests at 252.1 ng/L and 25222.2 Lig/L were performed. 

RESULTS 

Note. Data in the tables are rounded values taken from Excel spreadsheets which will be 

archived together with the raw data. The use of Excel spreadsheets for calculations produces 

more accurate endpoints. These endpoints may slightly differ from the values derived by 

replacing the rounded values in the formulae given in the methods section. 

Data and results of the water samples analysis are reported in Tables 2 (a-b-c-d) and 3 (a-bc-d). Table 4 (a-b-c-d) shows the analysis sequence. 

O 

Appendix A page 39 of 79 
ChemService 

Appendix A to Final Report CH-095/2013 

Table 2a 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 0 h analysis - calibration with 

working standard solutions. 

4-chloro-a,a,atrifluorotoluene 

(PCBTF) 

WSS 1 

201.7 ng/L 

(Area) 

WSS 3 

1008.3 rig/L 

(Area) 

WSS 5 

5041.6 pg/L 

(Area) 

1

s t

 series 145.04 860.13 4365.40 

2

n d

 series 183.39 770.12 4446.48 

Mean 164.22 815.13 4405.94 

Delta 38 90 81 

Delta % 23.35% 11.04% 1.84% 

Y = mx + q 

Parameter m 

(slope) 

Parameter q 

(intercept) 

Parameter R2 

(correlation) 

0.88 -40.55 0.99983 

Table 2b 4-chloro-aJa)a-trifluorotoluene (PCBTF): time 24 h analysis - calibration with 

working standard solutions. 

4-chloro-a,a,atrifluorotoluene 

(PCBTF) 

WSS 1 

201.7 Lig/L 

(Area) 

WSS 3 

1008.3 ^g/L 

(Area) 

WSS 5 

5041.6 pg/L 

(Area) 

1

s t

 series 178.34 801.86 4391.62 

2

n d

 series 169.48 781.96 5672.98 

Mean 173.91 791.91 5032.30 

Delta 9 20 1281 

Delta % 5.09% 2.51% 25.46% 

Y = mx + q 

Parameter m 

(slope) 

Parameter q 

(intercept) 

Parameter R2 

(correlation) Y = mx + q 

1.02 -124.39 0.99847 

Appendix A page 40 of 79 
p. 42 

ChemService 

Controlli o rlamroho 

Appendix A to Final Report CH-095/2013 

Table 2c 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 48 h analysis - calibration with 

working standard solutions. 

4-chloro-a,a,atrifluorotoluene 

(PCBTF) 

WSS 1 

201.7 ng/L 

(Area) 

WSS 3 

1008.3 (ig/L 

(Area) 

WSS 5 

5041.6 pg/L 

(Area) 

1

s t

 series 165.42 752.79 4053.55 

2

n d

 series 145.27 697.06 3525.73 

Mean 155.35 724.93 3789.64 

Delta 20 56 528 

Delta % 12.97% 7.69% 13.93% 

Y = mx + q 

Parameter m 

(slope) 

Parameter q 

(intercept) 

Parameter R2 

(correlation) 

0.75 -14.15 0.99990 

Table 2d 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 72 h analysis - calibration with 

working standard solutions. 

4-chloro-a,a,atrifluorotoluene 

(PCBTF) 

WSS 1 

201.7 \ig/l 

(Area) 

WSS 3 

1008.3 ng/L 

(Area) 

WSS 5 

5041.6 pg/L 

(Area) 

1

s t

 series 109.65 723.38 3799.76 

2

n d

 series 112.14 680.33 3738.68 

Mean 110.90 701.86 3769.22 

Delta 2 43 61 

Delta % 2.25% 6.13% 1.62% 

Y = mx + q 

Parameter m 

(slope) 

Parameter q 

(intercept) 

Parameter R2 

(correlation) 

0.76 -50.92 0.99997 

Appendix A page 41 of 79 
p. 43 

ChemService 

^^^^^^^ ^ Controlli ft rlaordhm 

Appendix A to Final Report CH-095/2013 

Table 3a 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 0 h analysis - 4-chloro-a,a,atrifluorotoiuene (PCBTF) content in water samples from the growth inhibition 

test. 

Vial Identification Area s 

(ng/L)* 

PCBTF found 

(^g/L) 

PCBTF add 

(^g/L) 

Recovery 

(%) 

Spike Low 244.79 300.6 1.0 300.6 252.1 119.3 

Spike High 1892.70 2194.7 10.0 21946.8 25208.2 87.1 

Control tO 0.00 0.0 1.0 0.0 0.0 -

7.9 mg/L tO 464.63 573.5 10.0 5734.9 7900.0 72.6 

11.9 mg/L tO 872.22 1036.2 10.0 10362.0 11900.0 87.1 

17.8 mg/L tO 1160.40 1363.4 10.0 13633.5 17800.0 76.6 

26.7 mg/L tO 826.18 983.9 20.0 19678.7 26700.0 73.7 

40.0 mg/L tO 1165.99 1369.7 20.0 27394.0 40000.0 68.5 

Area s : Sample peak area 

C s : Injected solution concentration 

D

s : Dilution applied to the sample 

(*) Spike Low was quantified with WSS1, Spike high and water samples were quantified 

with the calibration curve reported in Table 2a. 

n.d. not detected, lower than L.O.D. (100.9 pg/L) 

Appendix A page 42 of 79 
p. 44 

o 

ChemService 

Controlli o rlaorono 

Appendix A to Final Report CH-095/2013 

Table 3b 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 24 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) content in water samples from the growth inhibition 

test 

O Vial Identification Area s 

(pg/L)* Ds 

PCBTF found 

(pg/L) 

PCBTF add 

(pg/L) 

Recovery 

(%) 

Spike Ultra Low 207.98 241.2 1.0 241.2 252.1 95.7 

Spike High 2126.62 2208.7 10.0 22087.3 25208.2 87.6 

Control t24 0.00 0.0 1.0 n.d. 0.0 -

7.9 mg/L t24 0.00 0.0 1.0 n.d. 7900.0 -

11.9 mg/L t24 0.00 0.0 1.0 n.d. 11900.0 -

17.8 mg/L t24 0.00 0.0 1.0 n.d. 17800.0 -

26.7 mg/L t24 0.00 0.0 1.0 n.d. 26700.0 -

40.0 mg/L t24 0.00 0.0 1.0 n.d. 40000.0 -

Area s : Sample peak area 

c

 s : Injected solution concentration 

D

s : Dilution applied to the sample 

(*) Spike Low was quantified with WSS1, Spike high and water samples were quantified 

with the calibration curve reported in Table 2b. 

n.d. not detected, lower than L.O.D. (100.9 pg/L) 

O 

Appendix A page 43 of 79 
p. 45 

ChemService 

Confronto riadrohm 

Appendix A to Final Report CH-095/2013 

Table 3c 4-chloro-a,a,a-trifiuorotoluene (PCBTF): time 48 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) content in water samples from the growth inhibition 

test 

Vial Identification Area s 

(pg/i-)* 

PCBTF found 

(pg/L) 

PCBTF add 

(pg/L) 

Recovery 

(%) 

Spike Ultra Low 186.53 242.1 1.0 242.1 252.1 96.1 

Spike High 1453.88 1947.6 10.0 19475.6 25208.2 77.3 

Control t48 0.00 0.0 1.0 n.d. 0.0 -

7.9 mg/L t48 0.00 0.0 1.0 n.d. 7900.0 -

11.9 mg/L t48 0.00 0.0 1.0 n.d. 11900.0 -

17.8 mg/Lt48 0.00 0.0 1.0 n.d. 17800.0 -

26.7 mg/L t48 0.00 0.0 1.0 n.d. 26700.0 -

40.0 mg/Lt48 0.00 0.0 1.0 n.d. 40000.0 -

Area s ; Sample peak area 

^ s: Injected solution concentration 

D

s : Dilution applied to the sample 

(*) Spike Low was quantified with WSS1, Spike high and water samples were quantified 

with the calibration curve reported in Table 2c. 

n.d. not detected, lower than L.O.D. (100.9 pg/L) 

Appendix A page 44 of 79 
c 

p. 46 

ChemService 

Contrail/ o rloorcnm~ 

Appendix A to Final Report CH-095/2013 

O 

Table 3d 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 72 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) content in water samples from the growth inhibition 

test 

Vial Identification Area s 

(pg/U* 

PCBTF found 

(pg/L) 

PCBTF add 

(pg/L) 

Recovery 

(%) 

Spike Ultra Low 142.25 258.7 1.0 258.7 252.1 102.6 

Spike High 2014.79 2727.5 10.0 27275.2 25208.2 108.2 

Control t72 0.00 0.0 1.0 n.d. 0.0 -

7.9 mg/L t72 0.00 0.0 1.0 n.d. 7900.0 -

11.9 mg/L t72 0.00 0.0 1.0 n.d. 11900.0 -

17.8 mg/L t72 0.00 0.0 1.0 n.d. 17800.0 -

26.7 mg/L t72 0.00 0.0 1.0 n.d. 26700.0 -

40,0 mg/L t72 0.00 0.0 1.0 n.d. 40000.0 -

Area s : Sample peak area 

^ s : Injected solution concentration 

D

s : Dilution applied to the sample 

(*) Spike Low was quantified with WSS1, Spike high and water samples were quantified 

with the calibration curve reported in Table 2d. 

n.d. not detected, lower than L.O.D. (100.9 pg/L) 

G 

Appendix A page 45 of 79 
p. 47 

ChemService 

Controlli o rloorchm 

Appendix A to Final Report CH-095/2013 

Table 4a 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 0 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) analysis sequence. 

No. Vial Identification No. Vial Identification 

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 Wash 1 CH09513_40.0 mg/L 

1 CH09513_Spk Low 1 Wash 

1 CH09513_SpkHigh 1 WSS1_B 

1 Wash 1 WSS3_B 

1 CH09513_Control 1 WSS5_B 

1 CH09513_7.9 mg/L 1 

Appendix A page 46 of 79 
p. 48 

ChemService 

^j^^^^^/ 1 Controlli o rtamronm 

Appendix A to Final Report CH-095/2013 

Table 4b 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 24 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) analysis sequence. 

No. Vial Identification No. Vial Identification 

1 WSS1_A-T24 1 Wash-T24 

1 WSS3_A-T24 1 WSS1_B-T24 

1 WSS5_A-T24 1 WSS3_B-T24 

1 Wash-T24 1 WSS5_B-T24 

1 CH09513_Spkl_ow-T24 1 Wash-T24 

1 CH09513_SpkHigh-T24 1 CH09513_7.9 mg/L-T24 reinj TQ* 

1 Wash-T24 1 CH09513_11.9 mg/L-T24 reinj TQ* 

1 CH09513_Control-T24 1 CH09513J7.8 mg/L-T24 reinj TQ* 

1 CH09513_7.9mg/L-T24 1 CH09513_26.7 mg/L-T24 reinj TQ* 

1 CH09513J1.9 mg/L-T24 1 CH09513_40.0 mg/L-T24 reinj TQ* 

1 CH09513J7.8 mg/L-T24 1 Wash-T24* 

1 CH09513_26.7 mg/L-T24 1 WSS5_C-T24 conferma risposta* 

1 CH09513_40.0mg/L-T24 

*sample injection was repeated at the end of the sequence, without dilution of the sample, since with 

the first dilution factors, test item concentrations were smaller than the linearity range. 

Appendix A page 47 of 79 
p. 49 

ChemService Controlli * rloorohm 

Appendix A to Final Report CH-095/2013 

Table 4c 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 48 h analysis - 4-chloro-a,a,atrifluorotoluene (PCBTF) analysis sequence. 

No. Vial Identification No. Vial Identification 

1 WSS1_A-T48 CH09513J1.9 mg/L-T48 

1 WSS3_A-T48 CH09513J7.8 mg/L-T48 

1 WSS5_A-T48 CH09513_26.7 mg/L-T48 

1 Wash-T48 CH09513_40.0 mg/L-T48 

1 CH09513_Spk LOW-T48 Wash-T48 

1 CH09513_SpkHigh-T48 WSS1_B-T48 

1 Wash-T48 WSS3_B-T48 

1 CH09513_Control-T48 WSS5_B-T48 

1 CH09513_7.9mg/L-T48 Wash-T48 

Table 4d 4-chloro-a,a,a-trifluorotoluene (PCBTF): time 72 h analysis - 4-ch!oro-a,a,atrifluorotoluene (PCBTF) analysis sequence. 

No. Vial Identification No. Vial Identification 

1 WSS1_A-T72 CH09513J1.9 mg/L-T72 

1 WSS3_A-T72 CH09513_17.8mg/L-T72 

1 WSS5_A-T72 CH09513_26.7 mg/L-T72 

1 Wash-T72 CH09513_40.0mg/L-T72 

1 CH09513_Spk Low-T72 Wash-T72 

1 CH09513_SpkHigh-T72 WSS1_B-T72 

1 Wash-T72 WSS3_B-T72 

1 CH09513_Control-T72 WSS5_B-T72 

1 CH09513_7.9 mg/L-T72 Wash-T72 

Appendix A page 48 of 79 
o 

ChemService Control!/ o rlooroho 

Appendix A to Final Report CH-095/2013 

CONCLUSIONS 

4-chloro-a,a,a-trifluorotoluene (PCBTF) content in water samples coming from the aquatic 

ecotoxicological test performed on P.subcapitata is reported in Table 5. 

Table 5 4-chloro-a,a,a-trifluorotoluene (PCBTF): summary results 

O 

Time 

(hours) Water Samples (*) 4-chloro-a,a,a-trifluorotoluene 

(PCBTF) (pg/L) 

0 Control 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 

24 Control n.d. 

24 7.9 mg/L n.d. 

24 11.9 mg/L n.d. 

24 17.8 mg/L n.d. 

24 26.7 mg/L n.d. 

24 40.0 mg/L n.d. 

48 Control n.d. 

48 7.9 mg/L n.d. 

48 11.9 mg/L n.d. 

48 17.8 mg/L n.d. 

48 26.7 mg/L n.d. 

48 40.0 mg/L n.d. 

72 Control n.d. 

72 7.9 mg/L n.d. 

Appendix A page 49 of 79 
Appendix A to Final Report CH-095/2013 

Time 

(hours) Water Samples (*) 4-chloro-a,a,a-trifluorotoluene 

(PCBTF) (pg/L) 

72 11.9 mg/L n.d. 

72 17.8 mg/L n.d. 

72 26.7 mg/L n.d. 

72 40.0 mg/L n.d. 

(*) Nominal concentrations for ecotoxicological samples are expressed as test item 4-chloroa,a,a-trifluorotoluene (PCBTF). 

Appendix A page 50 of 79 
p. 52 

ChemService 

Controlli * rtaorohm~ 

Appendix A to Final Report CH-095/2013 

Figure 1 4-chloro-a,a(a-trifluorotoluene (PCBTF): Representative chromatogram of 4-

chloro-a,a,a-trifluorotoluene (PCBTF) working standard solution. 

Qualitative Analysis Report 

Data Filename 

Sample Type 

Instrument Name 

Acq Method 

IRM Calibration Status 

Comment 

Sample Group Info. 

User Chromatograms 

TD_02.d 

Sample 

Insmjment 1 

PCBTF_CH09213.ro 

Not Applicable 

Sample Name 

Position 

User Name 

Acquired Time 

DA Method 

WSS3A 

VTal25 

4/2/2013 3:43:38 PM 

resefp'iu.m 

t i Q2 

4 

3 

2 

1-1 

0 

DAD1 - AiSig=220,1Q T0_02.d 

Noise (PeakToPeah) = 0.33; SMR (5.189iron) = 193.0 

— i 

70.09! 

2:5 A 3l5 4 4!S S S\B 4 6!B 1 7.'& 8 fl"3 (5 

Response UnilB (%) vs. Acquisition Time (min) 

3.5 

Start fct End Height Area Area % Signal To Noise 

4.789 5.189 5.73 76.09 860.13 100 198 

End Of Report — 

jjt'f;- AiitttfltTechnaloniai Page 1 of 1 Printed at : 2:59 PM on ; 4/3/2013 

Appendix A page 51 of 79 
p. 53 

ChemService Cantrolll a rlat»rchi 

Appendix A to Final Report CH-095/2013 

Figure 2 4-chloro-a,a,a-trifluorotoluene (PCBTF): Representative chromatogram of the 

Control water sample. 

Qualitative Analysis Report 

Data Ffaname 

Sample Type 

Instrument Name 

Acq Method 

I R H Calibration 

Comment 

Sample Group 

T0_08.d Sample Name CH09513_Control 

Sample Position Vi<il44 

Instrument 1 User Name 

PCBTF_CH09213.m Acquired Time 4/2/2013 10:48)20 PM 

Not Applicable DA Method reserptna.m 

Info. 

User Chromatograms 

x102 

4-

a2-

1. 

ODAD1 -A:Sig=22O,1BT0_08,d 

Noise (PaakToPeak) » 054 

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

Response Units (%> vs. Acquisition Time <min) 

End Of Report-

-jjj-- A|itatTectHwk>glM Page 1 of 1 Printed at: 3:00 PM on: 4/3/2013 

Appendix A page 52 of 79 
p. 54 

ChemService 

Controlli 0 rtcorohm 

Appendix A to Final Report CH-095/2013 

Figure 3 4-chloro-a,a,a-trifluorotoluene (PCBTF): Representative chromatogram of 

Control water sample fortified at low level (252.1 pg/L). 

Qualitative Analysis Report 

Data Filename 

Sample Type 

Instrument Name 

Acq Method 

IRM Calibration Status 

Comment 

Sample Group Info. 

User Chromatoerams 

T0_05.d Sample Name CH09513_Spk Low 

Sample Position Vial 42 

Instrument 1 User Name 

PCSTF_CH09213.m Acquired Time 4/2/201310:12:37 PH 

Not Applicable DA Method reserpfna,m 

K 1 0 2 

4 

3 

2-

1 

0 

DADT - A:Stg=220.1BTQ_G5.d 

Malae (PesKToPeak) « 0.56; SNR (5.1Q7min) = 41.2 

I ! -

244.76! j 

1 I .'3 2 2.S 3 315 4 4:3 fi 5:5 o Sla 1 7,'B. ft 

Response Units (%> vs. Acquisition Time (mln) 

6 a.'3 

Start RT End Height Area Area^fe Signal To Noise 

4.787 5.107 S.57 23.01 244,79 100 41.2 

— End Of Reporte r AflilHtTecbiwfaiaie* Page 1 of 1 Printed at: 2:59 PM on: 473/2013 

Appendix A page 53 of 79 
p. 55 

ChemService Controlli « rtcorchm 

Appendix A to Final Report CH-095/2013 

Figure 4 4-chloro-a,a,a-trifluorotoluene (PCBTF): Representative chromatogram of time 

0 h water sample at 17.8 mg/L. 

Qualitative Analysis Report 

Data Filename 

Sample Type. 

Instrument Name 

Acq Method 

IBM Calibration Status 

Comment 

Sample Group Ink 

User Chromatograms 

TO_ll,d Sample Name CH09513_17.3 mg/L 

Sample Position Via) 47 

Instrument 1 User Name 

PCBTF_CH09213.m Acquired Time 4/2/201311:24:05 PM 

Not Applicable DA Method rtstrpin*,m 

x10Z 

1 

0.75 

0.5 

0.25 

0 

DADt - A:Stg=22O.10TO_11.tl 

Noise (PeakToPeak) = 0.53; SNR (5.1Q7min) = 220.5 

1W.2JJ 

olS ] l."5 2 2:5 3 3'5 -1 4'.3 S 5i5 * ftfc 1 7.'& 4 S.'S 

Ftaaponse Units {%) vs. Acquisition Time (mln) 9:5 

start RT Height Area Area °*> Signal To Noise 

4.953 5.107 5.42 117.22 1160.4 100 2203 

End Of Report-

^X" Aiitantleohneliipiei Page 1 of 1 PrMsd at: 3:01 PM on: 4/3/2013 

Appendix A page 54 of 79 
p. 56 

ChemService Controlli o rtoorcho 

Appendix A to Final Report CH-095/2013 

Figure 5 4-chloro-aAa-trifluorotoIuene (PCBTF): Representative chromatogram of time 

24 h water sample at 17.8 mg/L. 

Qualitative Analysis Report 

Data Filename 

Sample Type 

Acq Method 

1RM Calibration 

Comment 

Sample Group Info. 

User Chrornatosrams 

T24_2I.d 

Sample 

Instrument 1 

PCBTF_CH09253.fn 

Not Applicable 

reinj TQ 

Sample Name CH09513..17.8 mg/L-T24 

Position Vial 5 

User Name 

Acquired Time 4/4/2013 9:49:50 AM 

DA Method reserpirw.m 

x10 1 

3 

2. 

1 

0. 

-1 

-2 

DAD1 - A:Sla=220.l6T24_21.d 

::!:f:jT[;r —-* - --

I'll ! 

|!!i | • 

jMM H ' l|l f 

-T--)--(-- r j 

—-

- \~ - i - !- -

I L

 11

 r 1 . i.—i— n 

ill ! 

: l 1-1 .!.-, 

• . .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-

Besponse Units (%) vs. Acquisition Time (mln) 9.5 

End Of Report' 

• f a AiibntTeclinetotiifla Page 1 of 1 Printed at: 3:14 PM on: 4/4/2013 

Appendix A page 55 of 79 
p. 57 

ChemService Controlli 0 rtooroho 

Appendix A to Final Report CH-095/2013 

Figure 6 4-chloro-a,a,ct-trifluorotoluene (PCBTF): Representative chromatogram of time 

48 h water sample at 17.8 mg/L. 

Qualitative Analysis Report 

Data Filename 

Sample Type 

Instrument Name 

Acq Method 

I R K Calibration Status 

T48_ll.d 

Sample 

Instrument 1 

PC8TF_CH09213.m 

Not Applicable 

Position 

0*9513,17.8 mg/UT48 

VfetS2 

Acquired Time 4/4/2013 7:0Si04 PM 

DA Method reserpfna.m 

Sample Group Info. 

User Chromatograms 

xlO 1 

3 

2 

1-1 

0 

-1 

-a 

DAD1 - A:Sig=220.1BT<ia_11-d 

h _ __ 

. i.. i J 

w "i ! < 

! . 1 

! I ! ! 

Response Units (%> vs. Acquisition Time (min> 

End Of Report-

•ijt A|i(mtTechnolDBl«i Page 1 of 1 Printed at: 8:57 AM on : 4/5/2013 

Appendix A page 56 of 79 
p. 58 

ChemService 

Controlli o rloorohm 

Appendix A to Final Report CH-095/2013 

Figure 7 4-chloro-a,a,a-trifluorotolijene (PCBTF): Representative chromatogram of time 

72 h water sample at 17.8 mg/L. 

Qualitative Analysis Report 

Data Filename 

Sample Type 

Instrumefit Name 

Acq Method 

IRM Calibration Status 

Comment 

Sample Group Info. 

T72_lt,d Sample Name ONBBajJX mg/L-T72 

Sample Position Vial 26 

Instrument 1 UserName 

PCBTF_CH09213.m Acquired rune 4/5/2013 4:57.27 PM 

Not Applicable DA Method reserpina.ni 

User Chromatograms 

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 OlS 1 7.5 a 9 9.5 

Response Units {%) vs. Acquisition Time (mln) 

End Of Report — 

~ $ AiftonilttcluHihtBii* Page 1 of 1 Printed at: 9:26 AH on: 4/8/2013 

Appendix A page 57 of 79 
Appendix B to Final Report CH-095/2013 

APPENDIX B 

TEST TEMPERATURE 

Appendix B page 58 of 79 
p. 60 

ChemService 

Con trolll a rlaorohm 

Ch*mSoMc*S/.l 

Appendix B to Final Report CH-095/2013 

T_dbampcr___ra CH-005/2919 

Corrected 

ttmptf. turt (*C> 

Corrected m in 

i * m pvature f*o 

ccnwleaiibax 

$06 12.00W » 1 : s i 225 S6.6 

607 13,0003 23.7 2 U S2JS 20.fi 

305 14.0000 230 24 22.1 20.6 

MO 16,0Q« 2)0 24.1 225 25.E 

BID 16.0000 239 241 22 .S zn.c 

at. 

1T.0OOO 230 24 1 225 25.5 

512 18.0000 239 241 225 28.8 

_13 le.oaorj 219 24:1 225 28.6 

SM 30.00 CO 239 24.1 225 ss.s 615 21.0000 239 24.1 225 20.E 

.10 22.0000 23.9 24-1 225 25.5 

517 02090013 23.0000 239 241 22.5 25,5 

me O.DOCO 29.9 24.5 22* 26.5 

_.9 i.oaoo 2 U 24.1 225 25.5 

s_o 3.0000 23.9 24.1 225 255 

621 3.00.00 23 9 24.1 225 28.8 

622 4.00 CO 239 24.1 225 25.5 

623 6,00 CO 23.9 24.1 22 S 2D.S 

924 fl.oaoo 23.9 24.1 225 2fl.fi 

525 7.0ft CO 23.9 24.1 225 25.5 

KB B.oaoo 239 24 229 2D.9 

627 8.00 CO 23.9 24 225 2fl.fi 

»a 10.0000 23.9 24 225 25 5 

529 ii.oaoo 23.8 24 229 26.5 

MO 12.03.CO 23.0 24 225 285 

531 13,0000 237 23.9 225 25,5 

532 14.IXL0O 23.8 24 225 205 

633 10.0000 216 24 225 255 

534 la.oaoo 23.8 24 225 2G.6 

535 1T.00.00 23.6 24 225 255 

538 18.0000 ».e 24 £2$ 255 

637 10,0000 23 8 24 226 265 

MB 20.03.00 23.6 24 229 285 

53V 21 .WOO 246 24 225 255 

(WD 22.00.00 23.6 24 223 259 

941 OJrtHffiOlS 23.00.00 23.6 24 225 255 

542 O.OOOO 33.6 24 225 255 

Ha l.flO.00 23.0 24 22.5 Z05 

544 2.0000 23.6 24 215 255 

545 a.oaoo 23,6 24 22.6 256 

846 400C0 23.6 24 22.6 25.6 

S47 5 WOO 33.6 24 225 255 

1MB fi.CO.00 23.6 24 22.5 256 

549 7.00.00 23.6 24 225 25.5 

550 6.00.00 23.6 24 22.8 255 

ESI BJOOLOO 23,6 24 22.5 255 

992 10.0000 23.6 24 22.5 25.4 

SS9 11.0000 23.6 24 22.5 255 

CM 12.0000 23,6 24 225 255 

MS 13.TO. CO 237 23.9 22.5 2E.5 

556 14.0000 23.8 24 22.6 255 

DO. 10.0000 23.6 24 22.5 255 

658 10.00.00 23.8 24 22.5 255 

.5. 17.W.00 33.6 24 22.5 255 

660 ia.oo.uo 23,6 24 22.5 25.5 

M l 1B.00.00 23,6 24 22.5 25.5 

562 20.0000 23,6 24 32.5 255 

«a 21.00.00 23,6 24 22.5 255 

564 22*000 23.6 24 22.5 255 

GOG 040412913 23.00.00 23,6 24 22,5 256 

m 0.00.00 23,6 24 £2.5 256 

H7 1.00.00 23,6 24 22.5 256 

MO 200.00 23.6 24 22.5 256 

560 300.00 23.6 24 22.8 256 

BTfl 4.00.C.Q 23,6 24 22.9 256 

5J1 fi.OO.C0 23.6 24 22.5 256 

Eja 640.00 23.6 24 22.5 255 

813 T.CO.DO 23,6 24 22.5 255 

6X4 6.00,00 23.6 24 22.5 255 

BTS fi.CO.flO 23,8 24 22.5 255 

6T« 10.W.OD 23.8 24 22,5 255 

577 11.WOO 23.6 24 22.fi 255 

07a MiMotm 12O0.00 23.8 24 22.5 ».B 

M l 0WWMekf477 

•externa. 24>t FfetlUoiti UI*C 

24,0 Cw:0J*C 

OA. 0,1 CWMM t •meenturan mB*:2U45J 

Ax 

Appendix B page 59 of 79 
ChemService 

Controlli 0 rtooroho 

Appendix C to Final Report CH-095/2013 

APPENDIX C 

STATISTICAL ANALYSIS 

Appendix C page 60 of 79 
p. 62 

ChemService 

Controll i o rtooroh m ' 

Appendix C to Final Report CH-095/2013 

Statistical analysis in terms of measured geometric mean of 4-chloro-a.a.atrifluorotoluene (PCBTF) for end-point growth rate: 

PagatoT i 

. . ftcportOffta: 1»apf-1310,15 XM CETIS Data Worksheet o^z^wsjcHoasfloia 

Satoiwtlnim Ofowtti Tt*t ChmMnrlea ftrX CONTROtU K RICERCHE 

San Data: KapMS spwha: SWensaiM* caprfcomulum sanptiCocM: 922933909 

ending Out: 05apr-13 Protocol: OECO20U2QU Smplitw*: *foruor 

Sampta Dad: Material: PCBTF SMpto Station; 

• • ! 'iiijui-'.ipffiimc j • ...li, i.LiM fFOMh "J 21* 

Q N 1 2 00684 (MffW 

0 N 2 3 90GU O.flW 

0 N 3 T 04561 0.074 0.07» 

D N 4 21 oottsa oar» O.OJW 

0 N 5 »e 90563 09773 0.07B7 

0 N « a 00004 oora O.W» 

o.a 1 12 009W Done 0O8O5 

0.2B 2 6 D.0W 09306 90606 

0.S8 3 a 00511 aorst 0.0B07 

4.32 1 is 0,0510 09tK» 0«2 T 

0.33 2 10 a « » 00BM 0.0329 

0.32 3 13 o.oaw OOCIP 0063 

0,34 1 11 00993 OOOtfl 0062 

0.34 2 ia O0S21 aou 0903 

es» 3 10 aoaat aaotx 00322 

0.37 % i t 0.0112 oow 9961 

037 2 » ft0W» D9B1 ttWtt 

0.3? 3 14 aowo 90009 owns 

0.41 f 1 0.0431 9*818 0.0925 

041 2 SO 0.0*22 OJOBIT OJ032 

041 3 4 0.0*87 9.C642 00636 

000-155-136*1 CETISTM 41.026O *i«twJ9(fttt3 RmrtSiWOByLifeC— 

€0 VlSiUW* 

Appendix C page 61 of 79 
p. 63 

ChemService Controlli o rtaoroht 

Appendix C to Final Report CH-095/2013 

CETIS Test Summary 

Pageier 2 

Report 0««: 16 «pM3 ID, 17 XM 

I** : fM-3W2-55a&,OHimG(2013 

$4krm*trvm Qmwth T#«l Own * trvicm S./.L CONTROLLI E RICERCHE 

Teat Hoe 1t-7715-96*S Teat Type: QKMtfhrete Deration: 72h 

Start Data: C2«pM3 PWtOGOl! oecD2otrzoii Spedea: Stiaoaotnim caprlpomuum 

EadhflOatr, C5apM3 OHWaMr, Algal Cuture Madia Soorce: iBbonMryoolDure 

Setup 0«l«: 02apr-1312.a& Brine: 

Comment* CHOW2013 

FlfllMl 

oeomfMoniBOfi 

Sample Hot 0O-22ft3-3Ufi Malarial: PCBTF Cienl: FkKrtrrJca B.pA 

Sempla Deter Codac 922S3385B Project Product aludy 

Receive Dtta- Aouraa: Bponaor 

&*mpla*4H:

 ri 'A Waflcwi: 

Coennenta: Batch number; 11/2012 

Internal number. 2S11434.001 

MMWMurinfl date: January W, 2013 

Exp*ydali: January 2016 

Comparlaon Summary 

Anelnti Er*a>olM NOEL UXL ChV MOP MettKMl 

04-2573-039D grawtft rsla 24h 041 >OA1 r*A 14*1 * Qonforronl Adjl 

06-3374-aW2 g^awnntarffltl ><M1 m, 4.67% BorthmxilA^I 

10-2603-4708 pmMh tftto 72h 041 >0*1 WA 1.39% BontarronlAdjt 

Pofcil Cattaiata Summaiy 

AftfttyaJt Efldpotftf % effect Conc^noO. mic t 9S%UCL Maffcod 

M-36W-67fl2 groWhmo*2*ti 6 >0.41 WA Lknoar rrMeipoUitiQO 

10 *0>I1 m WA 

15 >041 HEA WA 

20 »041 WW WA 

25 >0A^ m. NfA 

40 >041 UiA NfA 

60 *0J4 1 NCA NfA 

«0 >041 H!K WA 

W-7602-5875 {jrowlrt r<*9 <Bh 6 >04T NfA NfA Linear Intarpovwcn 

10 >04f NfA NfA 

15 >0.41 WA NfA 

20 >0.41 NfA NfA 

25 >0.41 K/A N7A 

46 >Mt WA WA 

60 > M t WA WA 

86 >041 HVA WA 

1244)08-2607 gwMh rale 72h 5 >0.41 WA NfA UwlntarpoiBilon 

ID >04I WA WA 

16 >041 WA WA 

20 

26 

>0L41 

>a4i 

WA 

WA 

WA 

t-UA 

40 

60 

BO 

>0L41 

>041 

>0.41 

WA 

WA 

WA 

HFA 

WA 

WA 

000*169-126.1 06718"' vt.owo ^gMf t Approval^ : 

Appendix C page 62 of 79 
p. 64 

ChemService Controlli o rlomroHm 

Appendix C to Final Report CH-095/2013 

CETIS Test Summary 

ReportDeea: 19apr-1319.17XM 

LMc O4-394M535CHfja!W013 

growth rati 

Cenc-ntglL 

24h Summary 

Control Type Rap* Ma an Minimum MmlnKiri r. S I SO cv 

0 NegatnwContr 0 005642 0.03390 006040 0.00080 0.00313 3*6% 

0.28 3 0.06160 0.06110 0.08800 0.00629 0,00917 

032 3 0.08433 006(60 0O85M 9.00137 0,00238 3*9 * 

0,34 3 0.08300 0.OSZ10 009659 9.00133 0.00231 3At% 

9JJ7 3 0.06067 0.05450 000690 0.00390 0.00021 1020% 

0-41 3 0.06467 0.06220 000670 0.00132 O.0O22S 3.61% 

growth rate 4 * Bonwnary 

Cflrw-ms/i, Cantrot'Typ* Rap* Mean Minimum Maximun a Si SO CV 

0 Negativw Con tr 6 0.07613 0.07260 0.07946 OLOOIOS 0.00267 3.96% 

02B 3 0.07003 0.07T60 0.08060 &O0096 0.00166 2.10% 

0.32 3 0.08247 0.06930 0.08520 000144 0.00260 3X13% 

034 3 0-M1S0 0.06060 0,08200 OJJ0045 0.0097S 0.96% 

0-37 3 ftOOGM OsOftMO 0.00109 acwoie 0.00033 CAO* 

041 3 0JM25D o.oaieo 0,08420 000065 0.00147 1.79% 

growth raw 72hSumanary 

Coiic-rngn. Control Type Rape Mean MMmum MaMlreun 1 « SO CV 

0 rtegeitvaConlr O 0.07803 0.O7T30 0.07&90 OOOOW 0.00093 t.19% 

an 3 OuWOBO 0.08050 0.09070 QJWOM 0.00010 0.12% 

0.32 3 000283 0.06270 0,06300 QJWKW 0.00015 0-16% 

a.a* 3 Q»24& O.06ZO0 0,06300 000931 O0Q063 0.84% 

0.37 0.M103 0.06090 0.09130 000009 0,00015 0.10% 

0.41 0J38243 006200 0.06380 000023 0.00040 040% 

twvftnia 24ft Data* 

Ccmeang/L Contra* Twe Ravi flap 2 Rap 3 Rep 4 Raps Rap 4 

0 HegolrrtiComr 005640 0.06360 O.0561O 0.Q5&M 005030 QO6O40 

0.25 000570 0.00000 0.06110 

0.32 ooeieo 0.06600 0,06650 

0.34 OJOOSSO 0.062IO 0.06310 

0.37 ooei20 0.06600 0.06460 

0.41 ooatfj 0.06220 9.06670 

growth r«ta 4th Detail 

C*ne-mOA CoetroiTyp* Repi Rap t Rep 3 Rap 4- KapS Rapt 

0 NagaUva Ccnlr 007640 0.07460 OO74O0 0.07200 0.07730 007300 

02a OA77BO 0.Q60U 0078*0 

032 0X16030 046620 0-06190 

0JJ4 OOfltOO 0.0A2OO OJ06060 

037 006040 0.00100 0J36OS0 

041 0O6160 0.0B170 0OM20 

growth rata T2h Detail 

Conc-ntB/l Cental Type Rap 1 R*p2 Rap 3 Rap 4 RapS Rapt 

0 Nopailva Conir 0,07000 0O7860 007090 0X17730 0.07970 0.07660 

028 0X16050 000000 049070 

0O2 0.08270 006299 004300 

034 0O8200 OJTOQO QJ09220 

037 o.oeioo 000120 OXWSO 

041 0.00250 908200 ooazao 

000*155-120-1 CETIS* vt O20D 

Appendix C page 63 of 79 
p. 65 

ChemService 

Control!/ » riot 

Appendix C to Final Report CH-095/2013 

iaaijg^^ * ~ --"-•wo „ 

CETIS Data Worksheet 

PftgaloM 

MpwlDfte 19apM310.0BXM 

S*i*rtMtrtfni OnwCh Tail 

02*pM3 

05«pf-t3 

SwSqMHIrun caprfcwmAum 

PCBTF 

SmvftCaat: 

3*mpiaSo««*: 

seia^etttttofv 

EOT£J MHM0WW* rttMiDMvlty 

o

1 H i 19 1D0W 314HM 

a « 2 4 10OM »rwro 

c N 9 1B KQ00 

« N 4 * 10SO» S0U1T0 

a N 9 2 1HK» ainera 

c n e » unto 3»<47TO 

OifJ i 1ft 10300 SW0W6 

a.a 2 I 10000 31HC20 

aa 3 11 WOW 3M0UO 

03S 1 » 10MO 

ON Z 10DH 3ST441D 

o » 3 » WW* 

034. 1 IS 10000 S6JDD30 

03H X fl 1QOM j»aj o 

a » 3 Ifl WHO WU0» 

OAT 1 « wcw 5*1*380 

M r 2 6 W0W 3*81710 

Cat1 3 IT *«)(» 93U1BD 

041 1 2t tocw M0M20 

0.41 a 3 100W 3H1S» 

041 3 to 1MM aawao 

M0-15&-126-1 CETISTM vl.0260 

Appendix C page 64 of 79 
p. 66 

ChemService 

Controlli o rlooroho 

Appendix C to Final Report CH-095/2013 

CETIS Test Summary 

Report Onto: 19 fipr-13 10.12 XM 

Unle 0<-35M2-5535O«fly2a 13 

SaJeneetnjfr* orawtn Te« Cheanw KVtee Iri . COHTROUJ B RICCRCHE 

Tt«l Mo: 11-7715^596 TeatType: YWd Duration: 72h 

Start Data: G2apr*13 Protocol; •0ECO2OW2O11 Speotae: Seferoalrum ^rimmubjm 

EflattnflM" 06ao--13 a t Wreter: A^CtftureModto Source: iBtcraiwy cortof* 

Setup Oat*: 02 spr-1312.00 Brine: 

CofflMtrRt: C*W85f20l3 

Fill test 

geomotriofnaan 

Sample Hoc «-22fl3-M5fl Material! PCBTF Cllanrt: Flnchliwca S.rxA. 

SamptoOatv: Code: B228336S9 Project Product atudy 

RwcetwDel* Souroa: aaonew 

giwapH Age: MiA Station: 

Comment*: Bwcft number: 1U2012 

mtorrvt wrnbon 291*434-001 

Msnutocturtngi date: January 04,2013 

Eipiiydate: January 2010 

Cwwpert**'1 * 

AnalyaJa 

lunwwy 

Eiwajwinl NOEL4 LOi l CMf MBDp Maeiod 

0>24«M)r43 VMtl 041 >041 WA 6.53* BonranwiAdjt 

Petrrt KMrneM Summery 

Anatyala Endpetnt %CH*c* CwwittfL OftKUCL Mettled 

11-2346-0465 vtatt 5 >041 WA WA Linear inierpofaflon 

10 >041 WA WA 

15 >041 WA WA 

20 >04>1 WA WA 

29 *04.t WA WA 

40 >041 WA WA 

50 >041 WA WA 

SO >041 WA WA 

V 

Cotw*mgrl Control Type Rape Iftaen MMeww MMIMIU n 8C W CV 

0 N40*Bw CortT 0 2A2E*6 2jeOG«« 3.UE+6 •J02E+4 t-JBBE+S 6.74% 

029 3 331Eta 3ME** 3,336*6 • •20E+4 2J08E+4 063% 

032 3 3ABE*6 3.90E+6 2.26E*4 3.91 E+4 1J01H 

0.34 3 3.7JE*6 3JWE+6 3,84£*C 6.60E*4 1J2E+5 409% 

£L37 3 3.41E+S 3.3BEt6 3.4541*0 3.76E*4 1.10% 

0.41 3 3.78E40 3j65Etfl 3.eee*s 6.B0E+4 1.1BE** 3,t3% 

VMctOeM 

ConcmgJL Cental Type Rapi Rep 2 Rap 3 Rap 4 Reps Rapft 

0 N*oalN«C<inlr 3.14E+6 2J7£-*« 293E*6 3«0E*« 3.10E+8 2.6fiE«6 

0.2B 3.20E+6 3.31E46 3.ME*6 

0.32 3.95E*« 3ME46 3.93E*6 

0.34 3flfl6« a94E-** 3.70E+6 

o.&r 341E+6 34<SE44 3.ME+6 

0.41 J,69E*6 3J6RE+* 3.BBE+6 

000-155.126-1 CETtS"* vlJ020I> 

Appendix C page 65 of 79 
Controlli o rlaorohm 

Appendix D to Final Report CH-095/2013 

APPENDIX D 

COPY OF TEST ITEM 

CERTIFICATE OF ANALYSIS 

Appendix D page 66 of 79 
p. 68 

ChemService 

Controlli m rlcorcho 

Appendix D to Final Report CH-095/2013 

CERTIFICATE OF ANALYSIS 

PRODUCT: 4-ddoro-a^a-trifluortKolueoc (PCBTF) 

MANUFACTURER :FiKhlmiaiS,p.A. 

LOT: Li/3012 

I FINCHIMICA 

MOLECULAR FORMULA C1C6H4CF3 

MOLECULAR WEIGHT |«0J3 

N°CAS W-56-6 

PARAMETERS FINCHIMICA 

Lot 11/2012 

ASSAY % 99,3% 

0.1 J « 

>clo«>4)erizotrtfkioridc 0.55% 

DicWoroderi smites <J101 % 

AcidUy <1 nwlAg 

Waicr 76m$rlig 

Manufacturing dace: January 2013 

Expiry daw January 2016 

MfjMtbta, January 6,2013 

Thhi Ctftlfloato of Anah/tfs is a computer printout tl Is vsllcl without signature. 

RrwWraica S.p.A. 

OC department 

ViaLuio, 13 

33025 Manerbio-BS 

ITALY 

Appendix D page 67 of 79 
p. 69 

ChemService 

Controlli m rlcarchm 

Appendix E to Final Report CH-095/2013 

APPENDIX E 

COPY OF REFERENCE ITEM 

CERTIFICATE OF ANALYSIS 

Appendix E page 68 of 79 
ChemService 

Controlli m rlcorcho 

p. 70 

Appendix E to Final Report CH-095/2013 

SIGMA-ALDRIC H 

Slgma-Aldrlch Laborchenikalien QnbH 0-30918 SM1Z« 

Telefon: +49 5137 8238-150 

Riedel-de Haen 

CERTIFICATE OF ANALYSIS / 

INSPECTION CERTIFICATE 3.1 acc.to DIN EN 10204 

Saalie, 05.10.2007/844258/07/24225 

Order-No.; 

Custoner-So.: 

Order-Code: 

Quantity: 

Production data: 19.Sap.2007 

lac. Ratast Data: 19.Sep.2014 

Article/Product: 46070 Batch : 7262X 

3,5-Dichlorophenole PESTANAL® 

Reference Material (RM) 

1. General Information 

Formula: C6H4C120 

CAS-No.: [591-35-5] 

Usage : Metabolite 

Molar mass: 163.00 g/Mole 

Re coram, storage temp.: roomtetnp. 

The estimated relative error of a single measurement of the assay can be expected to be ± 0.5 % 

(confidence level 95%, n=6). 

2. Batch Analysis 

Identit y (NMR) complying 

Assay (GC) 99.9 area % 

Water (Karl Fischer) 0.086 % 

Melting range 65.4-66.0 °C 

Date of Analysi s 04.Oct.2007 

3. Advice and Remarks 

• Tha nlniiram ahelf Uf a ia based on tha entrant knowledge and holds only for propar storage conditions In tha 

originally closed flasks/ packages. 

• Nhanavar tha contalnar la opened for renoval of aliqout portion* of tha aubatanca, tha parion handling tha 

aubatanca muet assure, that tha Integrity of tha aubatanca ia maintained and propar records of al l it a 

handling* are leapt. Special can ha* to be takan to avoid any contamination or adulteration of the substance. 

• He herewith confirm that the delivery is effected according to tha technical delivery conditions agreed. 

• A eaaple of the above nantloned batch showed the listed analytical values. 

• Particular properties of the products or the suitability for a particular area of application are not assured. 

• Ha guarantee a proper quality within our General Condition! of Sales. 

Slgma-Aldrich Laborchemikallen GmbH 

Quality Assurance r(Dr. Gaede) 

Works Inspector 

Appendix E page 69 of 79 
p. 71 

ChemService 

Controlli m rlcore no 

Appendix F to Final Report CH-095/2013 

APPENDIX F 

COPY OF EXTRACTION CERTIFICATION 

OF ALGAL STRAIN 

Appendix F page 70 of 79 
o 

ChemService Controlli m rlomrono 

Appendix F to Final Report CH-095/2013 

O 

GEORG-AUGUST-UNIVERSITA T GOTTINGE N 

ALBRECHT-VON-HALLER-INSTITUT 

FOR PFLANZENWISSENSCI-IAPTEN 

Experimentelle Phykologie und 

S a mm lung von Algenkulturen (SAG) 

Dr, Ma ike torenz 

Nikolausberger Weg 18,37073 Gottingen, Fax. 0551/397871, email; epsag©gwdg.de 

Chemservice S.R.L 

M Chiara Neri 

via F.III Bertram! 15 

1-20026 Novate-Milanese 

ITALY 

Gottingen, 5th of July 2010 

Extraction certification 

This is to certify the company Chemservice S.R.L. that the algal culture with the 

strain number SAG 61.81 put at your disposal contains algae of the species: 

Pseudokirchneriella subcapitata 

(formerly called: Selenastrum Capricorn utttm; Ankistrodesmus subcapitata; 

Ankistrodesmus bibraianus; Raphidocelis subcapitata) 

The strain mentioned above was last renamed by us into Pseudokirchneriella subcapitata because of new research insights (Hindfik, F. (1990) Biologicke" Pace 35: 

209). Responsible for the numerous names in the past is the constantly developing 

taxonomic research on algae. A future renaming of this strain is not impossible. 

Only the strain number, here SAG 61.81, testifies the originality of the algal material. Duplicates of this algal strain also exist in other algae culture collections 

(CCAP, UTEX), some of these arej^ll kept as Selenastrum capricornutum. 

SAG Curator ^^^S ^ 

Visit and order through our homepage: 

www.cpsag.uni-goetlingen.de 

Appendix F page 71 of 79 
ChemService 

Controlli m rlooroho 

Appendix G to Final Report CH-095/2013 

APPENDIX G 

COMPOSITION OF TEST CULTURE MEDIUM 

(OECD N 201, 2011) 

Appendix G page 72 of 79 
ChemService Controtll m rlcorcho 

Appendix G to Final Report CH-095/2013 

Reconstituted Water: In deionised water (conductivity < 5 pScm"1) analytical 

grade salts will be added to following final nominal 

concentrations: 

O Macro-nutrients: NaHC03 50.0 mg/L 

CaCI 2 x2H2 0 18.0 mg/L 

NH4CI 15.0 mg/L 

MgS0 4 x7H2 0 15.0 mg/L 

MgCI 2 x6H2 0 12.0 mg/L 

KH2P04 1.6 mg/L 

Trace Elements: Na2EDTA x 2 H2 0 100.0 pg/L 

FeCI3 x 6 H2 0 64.0 pg/L 

MnCI 2 x4H2 0 415.0 pg/L 

H3BO3 185.0 pg/L 

Na2Mo04 x 2 H2 0 7.0 pg/L 

ZnCI2 3.0 pg/L 

CoCI 2 x6H2 0 1.5 pg/L 

CuCI2 x 2 H2Q 0.01 pg/L 

pH 8.1 ±0.2 

O 

Appendix G page 73 of 79 
ChemService 

Appendix H to Final Report CH-095/2013 

Controlli m rlcoroho 

APPENDIX H 

Copy of G.L.P. Authorization bv Italian Health Department 

(dated April 26th. 2012) 

Appendix H page 74 of 79 
p. 76 

ChemService Controlli m rlamroHm 

Appendix H to Final Report CH-095/2013 

DIPARTIMENTO DELLA SANITA1 PUBBLICA E DELL1NN0VAZI0NE 

DIREZIONE GENERALE DELLA PREVENZIONE 

UFFICIOVI-EXDGPREV 

Biotecfwtogle e Buona Pratlca dl Laboratory) 

Via Giorgio Ribotla, 5 

00144 Rurrw 

CER17FICA TO Of CONFORMtTA' ALCA 

BUONA PRATICA Df LABORATORY 

{Decreto Legislativo 02 marzo 2007, n. SO - Direttiva 2004/9/CE) 

Roma, 26/04)2012 Certificazion«: 014/2012 

Ai sens! deil'articolo 4 comma 1 del Decreto Legislative n. 50 del 2 marzo 2007, 

dell' articolo 3 del decreto ministeriale 4 luglio 1997 e tenuto conto dell'esito favorevole 

deii'lspezione effettuata in data 14-15/02/2012 

SI CERTIFICA 

che II Centre di Saggio: 

Chemservice S.r.l, - Controlli e Ricerche 

Via F.lll Beltrami 15 

20026 Novate Milanese (Ml) 

e idoneo ad effeltuare nel rfspelto delle Buone Pratiche dl Laboratorio di cui all'altegato II 

del citato decreto legislative: 

Appendix H page 75 of 79 
ChemService Controlli m rlaorohi 

Appendix H to Final Report CH-095/2013 

1) TeatRsteo-Chimtcl 

umidtta; 

Atfdlta eatolfnita; 

PH; 

Punto di fusione; 

Punto di Inflammability; 

ResldUD al setacdo^ 

Sagnablllta; 

Sospenslblllto; 

Peso specsflco apparente; 

Sostanie Insolgbilt In acetone; 

Schilwia persistente; 

Densita relatival 

Tensloftc dl vapore a diverse temperature; 

Asgetta ( stnto fislco, colore, odore e wmcentrazlon] soglia per le sostanze con colore c gusto 

intense) In aequo)) 

Spettra di assorbJmentb, (UV/VIS, IR, NMR e MS), estron e molare fl relative lunghezaa d'onda; 

Solubilita In acqua, compresl gtl effottl del pH (do 5 a 9) e delta temperature sulle solubilita; 

Solubilita nel solventl organic!, compresi gii effctti delle temperature suIla solUblilta; 

Coefficient dl riparttzione n-ottanoio/acquar compresi gli effettl del pH (da 5 a 9) c delta 

temperature; 

Degradazlone ossldatlva rotothlmica; 

Stabiltta delle cmufaoni; 

StabHfta accelerate e a lungo terminc, idcntito del prodottl dl degradazlone; 

Stabiltta a basse temperature; 

Stabiltta net solvents organic! impieijati nolle preparaaoni; 

Stabilftaalle dilulztanl; 

Infiamrnablflta; 

Temperatura dl autoaccenslone 

Tensions superfidale; 

Proprleta ossldand; 

peattMto ntt wnfrOrttJ del contenltorf; 

VJscoslta; 

Spontaneita; 

Setacdartiento ad umido/a soceo; 

Granulornstrla; 

Contenuto in potvcre; 

Appendix H page 76 of 79 
p. 78 

ChemService 

Controlli m rlamrcho 

Appendix H to Final Report CH-095/2013 

Capsota di palverlzzaztane; 

Capacste di versamentoj 

CompatlbHIta; 

Fluids. 

4) Studi d( towiclta arnbientale H I organltml acquaticl e terreBtrf 

Tossicita acuta peso - test stabco/semistatico/flusso contlnuo; 

Tossicita acuta Daphnia - test statlco/semistatico; 

Iniblzionc delta crcsclta alaale; 

Tossidta acuta lombridtl; 

Test dl inibizlone delio resplrwfone di fangN attivi; 

Tossicita prolungato peso- test semi-stetlco; 

Tossicita sub letale lombrico; 

Tossicita cronlca lombrlco; 

RJpraduiione Oaphnla-best seml-statica. 

Tossicita su plante acquatlche Lemna; Tossicita su microorganism! del suolo: test di nitrificazione 

e carbon ificazionc. 

5) Studi su comportamcnto in acqua,barro( irla; studi dl blOBCttimulazlone 

> Stability in acqua, tasso di idroltsi, degradazlone fotoxhimica, quantitative ed identic del/l 

prodotto/) d* d^rjrattazlrjr"?, C9rt?"t? rtl rit«TM*i«?lrtni* JI divrusl pH (da s a 

• Stabiiita all'arlat degradazlone fatochlmlcaP identita del/dei prodotto/l di degradaztone. 

- Stability nel suolo, 

- Studi dl bioaccumulazJone nel slstema di saggio pesce. 

6) Studi sul resldul 

Anallsl del rcsklui In matricf vegetal*, acqua, terrenl e fangm, tessutl animal!, latte e prodcttl 

casearl, olf e aria. 

Appendix H page 77 of 79 
p. 79 

ChemService 

Controlli m rlaorchm 

Appendix H to Final Report CH-095/2013 

8) Chlmtca Cllnlca e Analitica 

Chimica anaHtfca 

• Sviluppo e valldazione <JI metodiche anallfclche mono e multiresiduali In matriei vefletafi, acqua, 

terrenl c fans In, tcssutl anlmall, latte e prodottl casearl, Dili e aria, 

• Messa a punto o validazlone dl metodl analttici per f'ldentlficazione e la quantificaztcine dl prindpt 

atUvf. 

• Messa a punto e validazlone dl rnetodl analJtlcl per f'ldentlftcazlone e fa quamificazione delle 

Impurezze sfgniflcatrve e rlfevanti. 

• Messa a punto e validazlone dl metodl analiticl per la detcrmlnazione dl prlndpi attlvl e loro 

metabotlti in fluid! blologld ed omogenati di tessuti onimalt. 

• Analisl di prinefpi attSvie impurezze significative e CHevatitt. 

Studi di metabolismo e famtacccfnetScd 

Biodegradabiliba pronta - test manometneo 

Blodegradabilita ultima * closet bottle test 

Biodegradabilrta ultima - evoluzione di CG2. 

Soggf di ottlvita batterIclda e funglclda su dlslnfettanti chimlcl ed antisettlci, In sospensione e su 

superfld non porose, usatl in compo allmentare, industriale, domestico e nelle comunlta. 

II presente certificate) ha la valldfta di meal 24 dalla data dl 

9} Altrl 

emission©. 

Appendix H page 78 of 79 
p. 80 

ChemService 

Controlli m rloorcno 

Appendix H to Final Report CH-095/2013 

La presents certrficazione non verra rinnovata, al lermine della scadenza naturale, 

qualora non sia present at a, al sens! det comma 2 deli'arlicolo 4 del D.M. 4 luglio 1997 

(pubblicato nella G.U, n. 229 del 1/10/1937), e al pltt tardl 90 glorni prima della 

scadenza, domanda di rinnovo della stessa completa della scheda riassunliva aggiornata. 

IL DIRETTORE DELL'UFFICIO VI 0 

Dr.ssa Grazia Ballacci 

Appendix H page 79 of 79 
p. 81 

From: 023569961 

Giovanna Attardi 

Chemservice s.r.l. 

via F.lli Beltrami, 15 

Novate Milanese, 20026 

ITALY 

Origin ID: QCMA FecOb: 

Express 

J1320130S2S0426 

SHIP TO: (202)564-8930 BILL SENDER 

EPA-HQ-OPPT-2009-0112 40CFR7995089 

US Environmental ProtectionAgency 

EPA East Building, room E6428 

OPPT Document Control Office, 

WASHINGTON, DC 20004 

us 

The Warsaw Convention may apply and will govern and in most cases limit the liability of Federal 

Express for loss or delay of or damage to your shipment. Subject to the conditions of the contract 

CONSIGNEE COPY - PLEASE PLACE IN POUCH 

Ship Date: 23DEC13 

ActWgt 0.5 KG 

CAD: 1008622567INET3430 

ex-EORI:07020820150 

REF: 

DESC-1: Correspondence/No Customs Value 

DESC-2: 

DESC-3: 

DESC-4: 

COUNTRY MFG: IT 

CARRIAGE VALUE: 0.00 EUR 

CUSTOMS VALUE: 0.00 EUR 

T/C: S 242490886 LVT: S 242490886 

SIGN: Giovanna Attardi 

EIN/VAT: 

PKG TYPE: PAK 

TM# 7974 6931 6220 

0430 

XC RDVA 

10:30A 

INTL PRIORITY 

ISR 

20004 

DC-US 

IAD 

51AG4J99D5/1A9E 

Dopo avere stampato questa Lettera di Vettura: 

1. Utilizzate il pulsante "Stampare" in questa pagina per stampare la Lettera di Vettura con la vostra stampante laser o a getto d'inchiostro. 

2. Regate la pagina stampata lungo la linea orizzontale. 

3. Inserite la Lettera di Vettura nell'apposita busta autoadesiva e incollatela sulla spedizione in modo che la parte con il codice a barre 

possa essere letta con lo scanner. 

Awertenza: Per la spedizione, utilizzate esclusivamente la Lettera di Vettura stampata originate. Utilizzare per la spedizione una 

fotocopia della Lettera di Vettura e illegale e potrebbe dar luogo a oneri supplemental di fatturazione, insieme alia cancellazione del vostro 

Codice Ciiente FedEx. 

TERMINI LEGALI E CONDIZIONI DELLE DEFINIZIONI Dl SPEDIZIONE FEDEX. In questa Lettera dl Vettura, "noi", "hostro", e TedEx"sl riferiscono a Federal Egress Corporation, le sue filial! e cortsoclate e I 

rispettivi dipendenti, agenti eappaltatori indlpendent!. Con "voi'e "rostra"si fa riferimento al mittente, suoi dlpendenti, commlttenti e agenti. Qualora la spedizione abbla origins a( dl fuori degli Stati Unitl, il 

contratto dl vettura si intende stipulato con la consoclata, la filiale o I'appaltatore indipendente FedEx che perprimo ha accettato la rostra spedizione. II termine "collo"lndica qualsiasi contenltore o busta 

accettati da noi per la consegna, inclusi quelli da roi presentati a FedEx utilizzando i nostri sistemi automatici, misurazioni, manifest! o Letters di Vettura. II termine "spedizione" indica tutti i colli che sono 

presentati a FedExe accettati da FedExsu urVunica Lettera di Vettura. NOTA SUL TRASPORTO AEREO. A qualsiasi spedizione intemazionale per via aerea si applicano la Convenzione di Varsavia e i 

relativi emendamenti. Dunque la Convenzione di Varsavia, con i relativi emendamenti, disci pi ina e in molti casi limita la res pons abilita di FedEx per perdite, ritardi o danni subiti dalla vostra spedizione. La 

Convenzione di Varsavia, come emendata, limita la responsabilita di FedEx. Per esemplo negli Stati Unitl la responsabillta e limitata a 9,07 dollari statunitensi per llbbra (20 dollar! per chilo), a meno che 

non sia stato dichiarato un valore superiore ai fini della responsabillta del vettore, come spiegato qui di seguito, e non abbiate pagato tutte le spese supplemental appllcablll. Llnterpretazione e 

I'applicazfone dei limltl dl rssponsabllita della Convenzione dl Varsavia possono varfare a seconda del paesl. Non vi sono specifiche locallta d'arresto ufflclall, e FedExs! riserva il dlritto di fartransltare le 

spedizioni in qualsiasi modo gli sBmbri piu appropriato. NOTA AL TRASPORTO SU STRADA. Le spedizioni effettuate unicamente su strada da o verso un paese che ha firmato la Convenzione di Varsavia o 

il Contratto per il trasporto intemazionale di merci su strada (il "CMR") sono soggette ai termini e alle condizioni del CMR, a presclndere da qualsiasi altra disposizione contraria della presente Lettera dl 

Vettura. Per quanta riguarda le spedizioni effettuate unicamente su strada, in caso di disaccordo tra le disposizioni della CMR e la presente Lettera di Vettura, prevalgono i termini della CMR. LIMTAZIONE 

Dl RESPONSABILITA. Se non dlversamente previsto dalla Convenzione di Varsavia, it CMR o altri trattatl intemazlonall, leggi, regolamentl govematlvl, ordln! o requlslti, la massima responsabillta di FedEx 

perdanno, perdita, ritardo, carenze, errata o mancata consegna, errata o mancata comunicazlona di Informazioni In rapporto alia vostra spedizione e limitata dal presente AccordD, secondo i termini e 

condizioni stlpulat! nel contratto dl trasporto. Invitlamo a consultare il contratto dl trasporto stabllito nella Guida al servizl FedEx In vigore, o il suo equlvalente, per determinant le llmltazioni contrattuall. FedEx 

declina ogni responsabillta sul carico o asslcurazlone a copertura globale, ma e posslblle pagare un supplements perogni 100 USD supplemental di valore dichiarato ai fini della responsabillta del vettore 

(o ('equivalents nella valuta locale del paese d'ongine). Qualora sia stato dichiarato un valore superiore ai fini della responsabilita del vettore e sia stato pagato il supplement, la massima responsabilita di 

FedEx sara pan alllmporto che risulta infenore tra il valore dichiarato e i read dannl subiti. ESONERO Dl RESPONSABILITA. IN NESSUN CASO FEDEX SARA RESPONSABILE PER EVENTUALI DANNI, 

SIANO ESSI DIRETTI, INDIRETTI, ACCIDENTALI, PARTICOLARI O EMERGENTI, PER UN VALORE SUPERIORE A QUELLO DICHIARATO Al FINI DELLA RESPONSABILITA DEL VETTORE (INCLUSA, 

m SENZA LIMTAZIONE ALCUNA, LA PERDITA Dl ENTRATE O GUADAGNI) OPPURE PER IL REALE VALORE DELLA SPEDIZIONE, SE INFERIORE, INDIPENDENTEMENTE DAL FATTO CHE FEDEX 

FOSSE O NO A CONOSCENZA DELL'EVENTUALITA CHE TALI DANNI POTESSEROVERIFICARSI. FedEx non e responsabile peratti o omission! del mittente, Inclusi ma senza llmitazione alcuna: 

un errata dlchiarazione di carico, imballaggi, misure di sicurezza, contrassegnl o indirizzo Insufficient! o Inadeguatl; oppure peratti o omissioni det destinatarlo o di chiunque altro che abbla un Interesse 

nella spedizione; oppure perviolazioni dei termini del presente accordo da parte di uno dei contraenti. FedEx non e responsabile pereventuali danni, ritardi, carenze, errata o mancata consegna, errata o 

mancata comunicazione di informazioni in relazione a spedizioni di contanti, valuta o altri articoli praibiti, oppure in presenza di situazioni al di fuori del nostra controllo, come cause di forza maggiore, 

pericoli dell'aria, condizioni meteoralogiche, ritardi di natura meccanica, atti di nemlci pubblici, guerra, sciopero, guerra civile, oppure peratti od omissioni di autorita pubbliche (tra cui funzionari doganali e 

sanitari) con autorita reale o apparente. NESSUNA GARANZIA. Non diamo alcuna garanzia, ne espliclta ne Impllcita. RECLAM PER PERDITE, DANNI O RITARDI. TUTTI I RECLAM DEVONO ESSERE 

FATTI PER SCRITTO, ENTROI TERMNI PRESCRITTI. PER I DETTAGLI, VIINVITIAMO A CONSULTARE LE TARIFFE, LA GUIDA Al SERVIZI FEDEX IN VIGORE OPPURE LE CONDIZIONI STANDARD 

Dl TRASPORTO. La Convenzione di Varsavia prevede particolarl procedure scritte perle richieste dl risarclmento per dannl, ritardi o la mancata consegna della spedizione. Inottre, llnterpretazione e I'azione 

delle disposizioni di rectamo della Convenzione di Varsavia possono van are da un paese all'altro. Per conoscere i termini entro cui inoltrare la richlesta relativa alia rostra spedizione, si prega dl far 

riferimento alia Convenzione. II diritto alia richiesta di risarcimento di danni nei nostri confronti si intendera scaduto se entro due anni non e stata intentata causa, secondo quanto stabilito dalla 

Convenzione. FedEx non ha alcun obbligo di agire nei confronti di una richiesta fino al completo pagamento di tutte le spese di spedizione. L'ammontare della richiesta di risarcimento non e deducibile dalle 

spese di trasporto. Se il destlnatario accetta la spedizione senza annotare alcun danno sulla bolletta dl consegna, FedEx presume che la spedizione e stata consegnata in buone condizioni. Afflnche un 

reclamo per dannl possa essere preso in considerazione, il contenuto, il cartone di spedizione origlnale e I'lmballaggio devono essere tenuti a nostra disposizione per il controllo. LEGISLAZIONE 

COMPETENTE. Nella mlsura in cui una disposizione contenuta o a cui si fa riferimento nella presente Lettera dl Vettura di trasporto via aerea e contraria a trattatl Intemazlonali in vigore, leggl, regolamentl 

govemativi, ordlni o requisiti, essa resta vallda In quanto facente parte del nostra accordo, purche non sia dirimente. La nullita o la non applicability di qualsiasi disposizione non ha alcun effetto sulle altre 

parti della presente Lettera di Vettura di trasporto via aerea. Salvo altrimenti indicato, FEDERAL EXPRESS CORPORATION, 2005 Corporate Aronue, Memphis, TN 38132, USA, B il primo vettore di questa 

spedizione. Indirizzo e-mail indicato in www.fedex.com.