## REPORT
CAS# 111-44-4: Algal Inhibition Test

Data Requirements/Test Guidelines: • OECD Guidelines for Testing ofChemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of CommissionRegulation (EC) No 761/2009 • US EPADraftEcological Effects Test Guideline OPPTS 850.5400 Study Director: TestFacility:

Sponsor:

Harlan Study Number: Study Completion Date: H Vryenhoef Harlan Laboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE722GD 

### UK Champion Technologies, Inc 3200 SouthwestFreeway Suite 2700 Houston Texas USA 77027 UNITED STATES OF AMERICA 41201182 24 July 2013

Page 1of44Harlan StudyNumber: 41201182 Report Page2 

## STATEMENT OFNO DATA CONFIDENTIALITYCLAIMS No claim ofconfidentiality, on any basis whatsoever, is made for any information contained in this document.

Submitter:

Date:Harlan StudyNumber: 41201182 Report 

## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK Harlan StudyNumber: Study Title: 41201182 CAS# 111-44-4: Algal Growth Inhibition Test Page 3

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/1O/EC.17). These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI).

This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affectedthe integrity ofthe study orthe validity ofthe data. Study Director: HVryenhoef



# ...!1£,.~~---······ Date. 

## 24 JUL 

## 2013Harlan StudyNumber: 41201182 Report Page4 

## CONTENTS STATEMENT OF NODATA CONFIDENTIALITY CLAIMS ...................................................2 STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE ....................................................3 CONTENTS ....................................................................................................................................4 QUALITY ASSURANCE STATEMENT......................................................................................6 SIGNATURE OF CONTRIBUTING SCIENTISTS ......................................................................7 SUMMARY ....................................................................................................................................8 Introduction............................................................................................................................8 Methods .................................................................................................................................8 Results .................................................................................................................................8 GENERAL INFORMATION .........................................................................................................9 Schedule .................................................................................................................................9 Archiving ...............................................................................................................................9 1 INTRODUCTIONAND PURPOSE ................................................................................... 1 0 1.1 Guidelines I Regulations ........................................................................................ 1 0 2 TEST ITEM ......................................................................................................................... 10 3 MATERIALS AND METHODS......................................................................................... 11 3.1 Test Species ........................................................................................................... 11 3.2 Culture Medium..................................................................................................... 12 3.3 Procedure ............................................................................................................... 12 3.3.1 Range-finding test.................................................................................... 12 3.3.2 Definitive test........................................................................................... 13 3.3.3 Evaluation ofdata.................................................................................... 14 3.4 Validation Criteria ................................................................................................. 16 4 RESULTS ............................................................................................................................ 17 4.1 Range-finding Test ................................................................................................ 17 4.2 Definitive Test ....................................................................................................... 17 4.2.1 Validation criteria.................................................................................... 17 4.2.2 Growth data ............................................................................................. 18 4.2.3 Observations oncultures.......................................................................... 19 4.2.4 Observations ontest item solubility ........................................................ 19 4.2.5 Physico-chemicalmeasurements ............................................................. 19 4.2.6 Verification oftest concentrations...........................................................20 5 CONCLUSION....................................................................................................................20 6 REFERENCES.....................................................................................................................20 TABLES ........................................................................................................................................21 Table 1 Cell Densities and Percentage Inhibition ofGrowth from the Range-finding Table 2 Table 3 Table 4 Table 5 Test.........................................................................................................................21 Cell Densities and pH Values inthe Definitive Test.............................................22 Daily Specific Growth Rates for the Control Cultures inthe Definitive Test.......23 Inhibition ofGrowth Rate, Yield and Biomass Integral inthe Definitive Test (0- 72 Hour)..........................................................................................................24 Inhibition ofGrowth Rate, Yield and Biomass Integral inthe Definitive Test (0- 96 Hour)..........................................................................................................25 FIGURES ......................................................................................................................................26 Figure 1 Figure 2 Mean Cell Densities v Time for the Definitive Test..............................................26 Temperature Measurements TakenThroughoutthe TestPeriod...........................27Harlan StudyNumber: 41201182 Report Page 5
APPENDICES ...............................................................................................................................28 Appendix 1 Appendix 2 Appendix 3 Appendix 4 Appendix 5 Certificate ofAnalysis ...........................................................................................28 Positive Control .....................................................................................................29 Culture Medium.....................................................................................................30 Analytical Investigations .......................................................................................31 Monitoring Authority Statement ofGLP Compliance ..........................................44Harlan StudyNumber: 41201182 Report 

## QUALITYASSURANCE STATEMENT
Harlan StudyNumber: Study Title: 41201182 CAS# 111-44-4: Algal Growth Inhibition Test Page 6

The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsible person and management. Study-related procedures conducted at the test facility were audited and inspected. The details of these audits and inspections are givenbelow. Reported to relevant Dates and Types ofQAInspections the StudyDirector and TestFacility

Management Date ofInspection Type ofInspection Phase Inspected Report Date 02 August 2011 General Study Plan N/A 02 August 2011 Verification 22 April 2013 Process

- based Test Item Preparation 22 April 2013 22 April 2013 Process

- based Test SystemPreparation 22 April 2013 22 April 2013 Process

- based Exposure 22 April 2013 25 April 2013 Process

- based Assessment ofResponse 25 April 2013 26 April 2013 Process

- based Chemical Analysis 26 April 2013 09 May 2013 ReportAudit N/A 09 May 2013 The complete study plan (including study specific supplement) was verified for GLP compliance at report audit. This statement confirms thatthis reportreflects the raw data and the procedures followed. Quality Assurance:



## 2 3JUL 2013Harlan StudyNumber: 41201182 Report Page 7 

## SIGNATURE OF CONTRIBUTING SCIENTISTS
Analytical Chemistry: D M Mullee Harlan Laboratories Ltd., ShardlowBusiness Park, London Road, Shardlow, Derbyshire, UK



# ················~············· Date: 

## 24 JUL 2013Harlan StudyNumber: 41201182 Report Page 8 

## SUMMARY Introduction A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing ofChemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 ofCommission Regulation (EC) No 761/2009 and the US EPA DraftEcological Effects Test Guideline OPPTS 850.5400. 

### Methods Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution ofthe test item at a concentration of 100 mg/L (six replicate flasks) for 96 hours, under constantillumination and shaking at atemperature of24 ± 1 °C. Samples ofthe algal populations were removed daily and cell concentrations determined for each control andtreatment group, using a Coulter® Multisizer Particle Counter. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture mediumto provide a source ofcarbon dioxide for algal growth. 

### Results Exposure ofPseudokirchneriella subcapitata to the test item gave EC50 values based on growth rate and yield ofgreaterthan 100 mg/L. TheNo Observed Effect Concentrationwas 100 mg/L. The range-finding test indicated that the test item was non-toxic at concentrations up to 100 mg/L. Therefore, the definitive testwas a limittestwith an exposure concentration of 100 mg/L. Analysis ofthe test preparations at 0 and 96 hours showed measured test concentrations to range from 99% to 100% ofnominal and so the results are based onnominal test concentrations only.Harlan StudyNumber: 41201182 Report 

## GENERALINFORMATION Schedule Experimental Starting Date: Experimental Completion Date: 

### Archiving 7March2013 19 

### April 2013 Page 9



### Unless instructed otherwise by the Sponsor, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as to further retention or disposal. Further retention or return of the data will be chargeable to the Sponsor.Harlan StudyNumber: 41201182 Report Page 10 

## 1 INTRODUCTIONAND PURPOSE This report contains a description ofthe methods used and results obtained during a study to investigate the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. Pseudokirchneriella subcapitata is a freshwater unicellular alga, representative of primary producers found in natural waters and can therefore be considered as an important non-target organism infreshwater ecosystems. 

### 1.1 Guidelines I

### Regulations This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
• OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 760/2009 • US EPA Draft Ecological Effects Test Guideline OPPTS 850.5400 

## 2 TESTITEM The integrity ofthe supplied data relating to the identity, purity and stability ofthe test item is the responsibility ofthe Sponsor. A Certificate ofAnalysis, provided by the Sponsor is provided in Appendix 1. Identification: Description: Batch: Purity: Expiry Date: Storage Conditions: CAS# 111-44-4 Clear colorless liquid 45296HKV 99.7% 7June 2013 Room temperature inthe darkHarlan StudyNumber: 41201182 Report Page 11 

## 3 MATERIALSAND METHODS Biomass: ECx:

Growth: Growth Rate (Average Specific Growth Rate): Yield:

LOEC (Lowest Observed Effect Concentration): NOEC (No Observed Effect Concentration): Treatment: Section-by-SectionGrowth Rate: AUC (AreaUnderthe Growth Curve): 

### 3.1 Test Species Measurement variable (fluorescence or algal cell density) as surrogate measure for algal biomass. The calculated concentration oftest item that results in an x% reduction of the respective growth parameter relative to the control (or solvent control, if applicable) within a stated exposure period. The increase inbiomass overthe testperiod. The logarithmic increase in biomass during the exposure period. The value of the measurement variable at the end of the exposure period minus the measurement variable's value at the start of the exposure period, calculated to express biomass increase during the test. The lowest test concentration at which a statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable). The highest test concentration at which no statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable). Comprises test item treatments, control (test medium only) and solvent control (ifapplicable). Specific growth rate for each day during the course ofthe test (days 0-1, 1-2, 2-3 and, 3-4 where appropriate). Biomass integral calculated as areaunderthe growth curve.

The test was carried out using Pseudokirchneriella subcapitata 

### strain CCAP 278/4. Liquid cultures ofPseudokirchneriella subcapitata 

### were obtained from the Culture Collection ofAlgae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and constantillumination at 21 ± 

### 1 °C. Prior to the start ofthe test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 

### cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100- 150 pm) and constant illumination at 24 ± 

### 1 °C until the algal cell density was approximately 104

- 

### 10 cells/mL.Harlan StudyNumber: 41201182 Report Page 12
A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item. Details of the positive control are given in Appendix 2. The positive control was conducted between 13 February 2012 and 17 February2012. 

### 3.2 Culture Medium The culture medium used for the range-finding and definitive test was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to provide a sufficient supply ofC02 and to counteract the increase in pH due to algal growth in an enclosed system (Herman et a/1990). The culture medium is defined inAppendix 3. 

### 3.3 Procedure 3.3.1 Range-finding test The test concentration to be used in the definitive test was determined by a preliminary rangefinding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 96 hours. The test was conductedin250 mL glass conical flasks each completely filled withtestpreparation and sealed with ground glass stoppers. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium. An amount oftest item (100 mg) was dissolved in culture medium and the volume adjusted to 1liter to give a 100 mg/L stock solution from which a series ofdilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (900 mL) ofeach ofthe stock solutions was separately inoculated with algal suspension (15.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L. The stock solutions and each ofthe prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group was maintained under identical conditions butnot exposed to the test item. At the start ofthe range-finding test a sample ofeach test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 -730nm) and constantly shaken at approximately 150 rpm for 96 hours. After 96 hours the cell density ofeach flask was determined using a Coulter® Multisizer Particle Counter.Harlan StudyNumber: 41201182 Report Page 13


### A sample ofeach test concentration was taken for chemical analysis at 0 and 96 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20

## oc priorto analysis. 3.3.2 Definitive test Based on the result ofthe range-finding test a "limit test" was conducted at a concentration of 100 mg/L. 3.3.2.1 Experimental Preparation For the purpose ofthe definitive test, the test item was dissolved directly in culture medium. An 

### amount oftest item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/L stock solution. This stock solution was inoculated with algal suspension (32.4 mL) to give the requiredtest concentration of 100 mg/L. The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity. The concentration and stability ofthe test item in the test preparations were verified by chemical analysis at 0 and 96 hours (see Appendix 4). 3.3.2.2 Exposure conditions As in the range-finding test 250 mL glass conical flasks were used. Six flasks were used for the control and the 100 mg/L treatment group. The control group was maintainedunder identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of6.18 x 105 

### cells per mL. Inoculation of2 litres oftest medium with 32.4 mL ofthis algal suspension gave an initial nominal cell density of 1 x 104 

### cells per mL and had no significant dilution effect onthe final test concentration. The flasks were incubated (INFORS Multitron® Version 2 incubator) at 24 ± 

### 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380

- 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. Samples were taken at 0, 24, 48, 72 and 96 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.Harlan StudyNumber: 41201182 Report Page 14
3.3.2.3 Physico-chemical measurements The pH ofthe control and 100 mg/L test preparation was determined at initiation ofthe test and after 96 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded hourly using the incubators' internal pt-100 temperature probe and Multicom Software Package (see Figure 2). 

### 3.3.3 Evaluation ofdata 3.3.3.1 Comparison ofgrowthrates The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from the equation:

where: average specific growth rate from time t1 to tn cell concentration att1 cell concentration attn time offirst measurement time ofnth measurement The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather thanthe measured starting value in orderto increase the precision ofthe calculation. In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant. Percentage inhibition ofgrowth rate for each replicate test item vessel was calculated using the following equation: I 

## = f.lc- f.lt X 100

r f.lc where: percentage inhibition ofaverage specific growthrate mean average specific growth rate for the control cultures average specific growthrate for the test cultureHarlan StudyNumber: 41201182 Report Page 15
3.3.3.2 Comparison ofYield Yield is calculated as the increase in biomass over the exposure period using the following equation:

where: y 

### No Nn yield cell concentration atthe start ofthe test cell concentration atthe end ofthe test For each test concentration and control the mean value for yield along withthe standard deviation was calculated. Percentage inhibition ofyield was calculatedusing the following equation:

where: percentage inhibition ofyield meanvalue for yield inthe control group mean value for yield for the treatment group 3.3.3.3 Comparison ofbiomass integral The biomass integral (areaunderthe growth curve) was calculatedusing the following equation:

where:
area nominal cell concentration at start oftest measured cell concentration att1 measured cell concentration attn time offirst measurement after beginning oftest time ofnth measurement afterbeginning oftest Percentage inhibition ofthe biomass integral for each replicate test item vessel was calculated using the following equation:Harlan StudyNumber: 41201182 Report


### where: percentage inhibition ofthe biomass integral meanbiomass integral for the control cultures biomass integral for the test culture 3.3.3.4 Determination ofECxvalues Page 16



### ECx values were determined by inspection ofthe growthrate, yield and biomass integral data after 96 hours. 3.3.3.5 Statistical analysis A Student's t-test incorporating Bartlett'stest for homogeneity ofvariance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS 1999- 2001). 

### 3.4 Validation Criteria The results ofthe test are considered valid ifthe following performance criteria are met:
• The cell concentration ofthe control cultures must increase by a factor ofat least 16 after 72 hours (OECD/EC Guidelines) and by a factor of at least 100 after 96 hours (EPA/OPPTS Guidelines).
• The mean ofthe coefficients ofvariation ofthe section by section specific growth rates in the control cultures during the course ofthe test (0-24, 24-48 and 48-72 hours) must not exceed 35% (OECD Guidelines).
• The coefficient ofvariation ofthe average specific growth rate inreplicate control cultures after 72 hours mustnot exceed 7% (OECD Guidelines).
• The coefficient ofvariation ofthe cell density values in replicate control cultures must not exceed 20% after 72 and 96 hours (EPA/OPPTS Guidelines).Harlan StudyNumber: 41201182 Report Page 17 

## 4 RESULTS 4.1 Range-finding Test The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriellasubcapitatato the test item during the range-findingtest are given in Table 1. The results showed no effect on growth atthe test concentrations of0.10, 1.0, 10 and 100 mg/L. Based on this information a single test concentration, six replicates of 1 00 mg/L, was selected for the definitive test. Chemical analysis ofthe 100 mg/L test preparation at 0 and 96 hours (see Appendix 4) showed a measured test concentration of 109% ofnominal was obtained indicating that the test item was stable overthe test duration. 

### 4.2 Definitive Test Cell density values determined at each sampling time and pH values at 0 and 96 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. Growth rate, yield and biomass integral values for the control and test cultures after 96 hours and percentage inhibition values are given in Table 5. The mean cell densities versus time for the definitive test are presented in Figure 1. 

### 4.2.1 Validation criteria The following data showthatthe cell concentration ofthe control cultures increased by a factor of 83 after 72 hours and by a factor of 130 after 96 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours and the EPA/OPPTS Guidelines that states the enhancement must be at least by a factor of 100 after 96 hours. Mean cell density ofcontrol at 0 hours Mean cell density ofcontrol at 72 hours Mean cell density ofcontrol at 96 hours 1.04 x 104 cells per mL 8.69 x 105 cells per mL 1.35 x 106 cells per mL The mean coefficient of variation for section by section specific growth rate for the control cultures over the test period (0-72 hours) was 28% and hence satisfied the validation criterion given inthe OECD Guideline which states the mean must not exceed 35%. The coefficient ofvariation for average specific growth rate for the control cultures over the test period (0- 72 h) was 2% and hence satisfiedthe validation criterion given inthe OECD Guideline which states thatthis must not exceed 7%.Harlan StudyNumber: 41201182 Report Page 18
The coefficient ofvariation ofthe cell density values in replicate control cultures was 9% after 72 hours and 1% after 96 hours and hence satisfied the validation criterion which states that this must not exceed 20%. 4.2.2 Growth data From the data given inTables 2, 4 and 5, it is clear that the growth rate (r), yield (y) and biomass integral (b) ofPseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence ofthe testitem at a nominal test concentration of 100 mg/L overthe 96-Hourexposure period. Itwas consideredunnecessary and unrealistic to test at concentrations in excess of 1 00 mg/L. Accordingly the following results were determined from the data: 4.2.2.1 Inhibition ofgrowthrate ErCIO (0

- 72 h) ErC2o (0

- 72 h) ErCso (0

- 72 h) ErClO (0- 96 h) ErC2o (0- 96 h) ErCso (0

- 96 h) :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L where ErCx is the test concentrationthat reduced growthrate byx%. Statistical analysis ofthe growth rate data at 72 and 96 hours was carried out for the control and 100 mg/L test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant decreases in growth rate

**(P~0.05),** between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L. 4.2.2.2 Inhibition ofyield EyC10 (0

- 72 h) EyC2o (0

- 72 h) EyCso (0

- 72 h) EyCIO (0

- 96 h) EyC2o (0- 96 h) EyCso (0

- 96 h) :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L where EyCx is the test concentrationthatreduced yield by x%.Harlan StudyNumber: 41201182 Report Page 19
Statistical analysis of the yield data at 72 and 96 hours was carried out as in Section 4.2.2.1. There were no statistically significant decrease in yield

**(P~0.05),** between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 1 00 mg/L.
4.2.2.3 Inhibition ofbiomass integral EbC10 (0

- 72 h) EbC2o (0

- 72 h) EbCso (0

- 72 h) EbC10 (0

- 96 h) EbC2o (0- 96 h) EbCso (0

- 96 h) :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L :>100 mg/L where EbCx is the test concentration that reduced biomass integral (area under the growth curve) byx%. Statistical analysis of the biomass integral data at 72 and 96 hours was carried out as in Section4.2.2.1. There were no statistically significant decrease in biomass integral

**(P~0.05),** between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based onbiomass integral was 100 mg/L. 4.2.3 Observations on cultures All test and control cultures were inspected microscopically at 96 hours. There were no abnormalities detected in any ofthe control or test cultures. 4.2.4 Observations on test item solubility At the start ofthe test all control and test cultures were observed to be clear colorless solutions. After the 96-Hour test period all control and test cultures were observed to be bright green dispersions. 4.2.5 Physico-chemical measurements The pH values ofthe control and 100 mg/L test preparation are given in Table 2. Temperature was maintained at 24 ± 1 °C throughoutthe test. The pH value of the control cultures (see Table 2) was observed to increase from pH 7.5 at 0 hours to pH 10.5 at 96 hours. This increase was considered to be due to the amount ofcarbon dioxide required by the large number of algal cells in the log phase of growth (see Figure 1) exceeding the transfer rate ofC02from the gaseous phase to the aqueous phase. In this situation C02 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase inthe pH ofthe culture. The increase in pH after 96 hours was in excess ofHarlan StudyNumber: 41201182 Report Page 20


### that recommended in the Test Guidelines (1.5 pH units after 96 hours). This was considered to have had no adverse effect on the results ofthe study given that the increase in cell concentration inthe control cultures exceeded the validation criterion given inthe Test Guidelines. 4.2.6 Verification oftest concentrations 

### Analysis of the test preparations at 0 and 96 hours (see Appendix 4) showed measured test concentrations to range from 99% to 100% ofnominal and so the results are based on nominal test concentrations only. 

## 5 CONCLUSION The effect ofthe test item on the growth ofPseudokirchneriella subcapitata

### has been investigated and gave EC5o

### values based on growth rate and yield ofgreater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L. 

## 6 REFERENCES HERMAN et a! 

### (1990) Impact ofvolatile aromatic hydrocarbons, alone and in combination, on growth ofthe freshwater alga Selenastrum capricornutum.Aquatic Toxicology 18, 

### 87- 100 SAS/STAT Proprietary Software Release 8.02 (1999- 2001), SAS Institute Inc, Cary, NC, USA. SOKAL, R RandROHLF, F J 

### (1981) Biometry. NewYork: W H FREEMAN and Company.Harlan StudyNumber: 41201182 Report Page 21 

## TABLES Table 1 CellDensities and Percentage Inhibition ofGrowth from the Range-finding Test Cell Densities* (cells permL) InhibitionValues(%) Nominal Concentration (mg!L) 0 Hours 96 Hours Growth Rate Yield/Biomass Integral Control R, 1.02E+04 8.68E+05 R2 1.01E+04 9.65E+05 

## - - Mean 1.01E+04 9.16E+05 0.10 R, 8.58E+03 5.37E+05 R2 9.60E+03 1.25E+06 [2] 2 Mean 9.09E+03 8.93E+05 1.0 R, 9.22E+03 1.04E+06 R2 1.45E+04 1.31E+06 [2] [29] Mean 1.18E+04 1.18E+06 10 R, 8.40E+03 1.41E+06 R2 7.82E+03 1.73E+06 [17] [72] Mean 8.11E+03 1.57E+06 100 R, 5.98E+03 1.33E+06 R2 7.33E+03 1.21E+06 [17] [39] Mean 6.66E+03 1.27E+06

* Cell densities represent the mean number ofcells per mL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R 1 and R2= Replicates 1 and2 [Increase in growth comparedto controls]Harlan StudyNumber: 41201182 Report Page 22
Table2 CellDensities and pH Values in the Definitive Test
Nominal Concentration pH Cell Densities* (cells permL) pH (mg/L) Oh Oh 24h 48 h 72h 96h 96h Control R1 1.14£+04 6.00£+04 3.04£+05 8.93£+05 1.35£+06 Rz 1.08£+04 5.03£+04 2.67£+05 7.59£+05 1.35£+06 R3 1.03£+04 5.72£+04 3.19£+05 9.01£+05 1.38£+06

**~** 7.5 1.05£+04 6.27£+04 3.20£+05 7.96£+05 1.34£+06 10.5 Rs 1.02£+04 5.98£+04 3.29£+05 8.81£+05 1.35£+06

**~** 9.30£+03 6.73£+04 3.26£+05 9.83£+05 1.35£+06 Mean 1.04£+04 5.96£+04 3.11£+05 8.69£+05 1.35£+06 100 R1 1.16£+04 6.22£+04 3.95£+05 1.27£+06 1.23£+06 Rz 1.13£+04 5.80£+04 3.47£+05 1.09£+06 1.43£+06 R3 1.13£+04 6.29£+04 3.46£+05 1.20£+06 1.48£+06

**~** 7.7 1.07£+04 6.02£+04 3.52£+05 1.05£+06 1.12£+06 10.6 Rs 1.08£+04 6.00£+04 3.05£+05 1.01£+06 1.43£+06

**~** 1.05£+04 5.88£+04 3.33£+05 1.02£+06 1.14£+06 Mean 1.10£+04 6.04£+04 3.46£+05 1.11£+06 1.31£+06

* Cell densities represent the mean number ofcells permL calculated from the mean ofthe cell counts from 3 counts for each ofthe replicate flasks. R1
-

**~** = Replicates 1to 6Harlan StudyNumber: 41201182 Report Page 23


### Table 3 Daily Specific Growth Rates for the Control Cultures in the Definitive Test
Daily Specific Growth Rate (cells/mL!hour) Day0- 1 Day 1-2 Day2-3 Day 3-4 Rt 0.075 0.068 0.045 0.017 R2 0.067 0.070 0.043 0.024 R3 0.073 0.072 0.043 0.018 Control

**~** 0.077 0.068 0.038 0.022 Rs 0.075 0.071 0.041 0.018

**~** 0.079 0.066 0.046 0.013 Mean 0.074 0.069 0.043 0.019

R1 -

**~** = Replicates 1to 6Harlan StudyNumber: 41201182 Report Page 24


### Table 4 Inhibition ofGrowth Rate, Yield and Biomass Integral in the Definitive Test (0-72 

### Hour) Growth Rate Yield Nominal Concentration (cells/mL/hour) (cells/mL) (mg/L) 0-72h % % Inhibition 0-72h Inhibition* Control RI 0.062 8.81£+05 Rz 0.060 7.48E+05 R3 0.063 8.91E+05 Rt 0.061 

### - 7.85E+05 

### - Rs 0.062 8.71E+05

**~** 0.064 9.74E+05 Mean 0.062 8.58E+05

SD 0.001 8.09E+04 100 RI 0.067 [8] 1.26E+06 Rz 0.065 [5] 1.08E+06 R3 0.066 [6] 1.18E+06 Rt 0.065 [5] 1.04E+06 Rs 0.064 [3] l.OOE+06

**~** 0.064 [3] 1.01£+06 Mean 0.065 [5] 1.10E+06 [28]

SD 0.001 1.03E+05

*In accordance withthe OECD test guideline only the mean value for yield is calculated R1 -

**~** = Replicates 1to 6 SD = Standard Deviation [Increase in growth as comparedto controls] Biomass Integral (cells/mL/hour) % 0-72h Inhibition 1.89E+07 1.61£+07 1.92E+07 1.81E+07 

### - 1.93E+07 2.06E+07 1.87E+07 1.51£+06 2.56E+07 [37] 2.22E+07 [19] 2.36E+07 [26] 2.19E+07 [17] 2.03E+07 [9] 2.10E+07 [12] 2.24E+07 [20] 1.89E+06Harlan StudyNumber: 41201182 Report Page 25


### Table 5 Inhibition ofGrowth Rate, Yield and Biomass Integral in the Definitive Test (0- 96 Hour) GrowthRate Yield Nominal Concentration (cells/mL/hour) (cells/mL) (mg/L) % 0-96h % Inhibition 0-96 h Inhibition* Control RI 0.051 1.34£+06 Rz 0.051 1.34£+06 R3 0.051 1.37£+06 Rt 0.051 

### - 1.33£+06 

### - Rs 0.051 1.34£+06 R.<; 0.051 1.35£+06 Mean 0.051 1.34£+06

SD 0.000 1.30£+04 100 RI 0.050 2 1.22£+06 Rz 0.052 [2] 1.42£+06 R3 0.052 [2] 1.47£+06 Rt 0.049 4 1.11£+06 Rs 0.052 [2] 1.42£+06 R.<; 0.049 4 1.13£+06 Mean 0.051 1 1.29£+06 4

SD 0.002 1.59£+05

* In accordance withthe OECD test guideline onlythe mean value for yield is calculated R1 - R.<; = Replicates 1to 6 SD = StandardDeviation [Increase in growth as comparedto controls] Biomass Integral (cells/mL/hour) 0-96h % Inhibition 4.55£+07 4.12£+07 4.64£+07 4.35£+07


### - 4.59£+07 4.85£+07 4.52£+07 2.49£+06 5.53£+07 [23] 5.22£+07 [16] 5.54£+07 [23] 4.78£+07 [6] 4.94£+07 [9] 4.68£+07 [4] 5.11£+07 [14] 3.76£+06Harlan StudyNumber: 41201182 Report 

### Figure 2 Temperature Measurements Taken Throughout the TestPeriod



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**23.0+----------------.----------------.----------------.--------------~** 0 24 48 72 96 Time (Hours)Harlan StudyNumber: 41201182 

## APPENDICES Appendix 1 Certificate ofAnalysis


## SIGMA-ALDRICH'' Report

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..... Page 28Harlan StudyNumber: 41201182 Report Page 29 Appendix 2 Positive Control 

### A positive control (Harlan Study Number: 41200591) used zinc chloride as the reference item at concentrations of0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata 

### (CCAP 278/4) to the reference item gave the following results:
No Observed Lowest

Time Point Response EC50 (mg/L) 95% Confidence Effect ObservedEffect

(Hours) Variable Limits (mg/L) Concentration Concentration (NOEC) (mg/L) (LOEC) (mg/L) GrowthRate 0.31 0.28 

### - 0.35 0.10 0.32 72 Yield 0.26 

### * 0.10 0.32 Biomass 0.27 

### * 0.10 0.32 Growth Rate 0.35 0.30 

### - 0.40 0.10 0.32 96 Yield 0.27 

### * 0.10 0.32 Biomass 0.26 

### * 0.10 0.32 

### The results from the positive control with zinc chloride were within the normal ranges for this reference item.



### * It was not possible to calculate 95% confidence limits for these EC50 values as the data generated did not fit the models available for the calculation ofconfidence limits.Harlan StudyNumber: 41201182 Report Page 30 

### Appendix3 Culture Medium

NaN03 25.5 mg/L MgCb.6H20 12.16 mg/L CaCb.2H20 4.41 mg/L MgS04.7H20 14.6 mg/L K2HP04 1.044 mg/L NaHC03 15.0 mg/L H3B03 0.186 mg/L MnCbAH20 0.415 mg/L ZnCb 0.00327 mg/L FeCh.6H20 0.160 mg!L CoCb.6H20 0.00143 mg/L Na2Mo04.2H20 0.00726 mg/L CuCb.2H20 0.000012 mg/L Na2EDTA.2H20 0.30 mg/L The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 

### 0.1 with O.lNNaOH or HCl. For the purposes of the range-finding and definitive test, additional sodium bicarbonate (500 mg/L) was addedto the prepared culture medium priorto use.



### *Elga Optima 15+ orElga Purelab Option R-15 BPHarlan StudyNumber: 41201182 Report Page 31 

### Appendix 4 Analytical Investigations 

## 1 MATERIALSAND METHODS 1.1 Introduction The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard. The test item gave a chromatographic profile consisting ofa single peak. The method was developed by the Department ofAnalytical Services, Harlan Laboratories Ltd, Shardlow, UK. 

### 1.2 TestItem The test item is described inthe biological part ofthis study. 

### 1.3 Analytical Standard The test item described inthe biological part ofthis study was also used as the analytical standard. 

### 1.4 Analytical Procedure 1.4.1 Storage 

### The 0 hour samples were stored at approximately -20 

## oc whilst the 96 hour samples were analyzed onthe day ofreceipt. 1.4.2 Reagents and Solvents 

### Test medium: as described inthe biological part ofthis study. Solvents: methyl tert-butyl 

### ether, Fisher Scientific, HPLC grade. MethanolHarlan StudyNumber: 41201182 Report Page 32 Analytical Investigations 1.4.3 Preparation ofCalibration Standards The test item (nominal 100 mg) was dissolved in methyl tert-butyl ether (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. This stock solution was further diluted with methyl tert-butyl ether to obtain a nominal 100 mg/L calibration standard. A duplicate calibration standard was similarly prepared at 100 mg/L. These duplicate calibration standards were usedto determine the recovery and test sample concentrations. 1.4.4 Preparation ofLinearity Standards The test item (nominal 100 mg) was dissolved in methyl tert-butyl ether (100 mL) to prepare a stock solution with a nominal concentration of 1000 mg/L. Defined volumes of this stock solution were diluted with methyl tert-butyl ether to obtain standards in the range of 1.0 to 150 mg/L. A second standard was similarlyprepared at a nominal concentration of 100 mg/L. These standards were usedto evaluate the linearity ofthe analytical system. 1.4.5 Preparation ofSpiked Recovery Samples To demonstrate the validity ofthe analytical procedure, volumes oftest medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in methanol to prepare a stock solution with a concentration of 10000 mg/L. A defined volume ofthis stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 100 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition ofthe test item (synthetic control) was also analyzed. 1.4.6 Preparation ofTest Samples The 0 hour test samples were thawed with the aid of sonication whilst the 96 hour test samples were analyzed on the day ofreceipt. The test samples were extracted directly with 100 mL of methyl tert-butyl ether in the same bottle as shown in Table 1. An aliquot ofthe methyl tert-butyl ether layer was taken for analysis.Harlan StudyNumber: 41201182 Report Analytical Investigations 

### 1.4.7 Instrumental Parameters Gas chromatograph: Detector:
Column:

Inletliner: Carrier gas: Pressure: Oventemperature Program:
Injection mode:
Inletpurgetime: Detectortemperature: Retentiontime: Agilent 6850, with autosampler FID
Rxi-1 ms (Restek) (15mx 0.25 mmi.d.; 1 J..Lm film thickness)
4 mm emptyliner nitrogen 15 psi injector: 250

## oc oven:40

## oc for 1minute with 20 °C/minuteto 150

## oc for 1minute splitless injection volume: 1 J..LL
onat 1minute 250°C approximately 4.6 minutes Page 33Harlan StudyNumber: 41201182 Report Page 34 Analytical Investigations 

### 1.4.8 Calculations A linearresponse was obtained during the validation ofthe analytical method, so for the definitive test samples a single-point calibration was performed using duplicate calibration standards. The mean peak areas ofeach calibration standard were corrected to a nominal concentration and the meanvalue taken. The concentration "x" of the test item in an injected sample was calculated by the following equation:
X 

## = Rspi·N Real std where x: Rspl: Rca! std: N: (1) concentration ofthe testitem in injected sample [mg/L] meanpeak arearesponse ofthe testitemininjected sample [counts] meanpeakarearesponse for the calibration standard, correctedto nominal concentration [counts] nominal calibration standard concentration [ mg/L]
The concentration ofthe testitem inthe test samples was calculated using the following equation:

c = X ·F 

### R/100 where c: x: F: R: (2) concentrationofthe test item inthe test sample [ mg/L] concentrationofthe testiteminthe injected sample solution [mg/L] samplepreparationfactor recoveryrate [%] (Ifnot correcting for recoverythenR is taken as 1 

### 00) The recovery rate (R) ofthe spikedrecovery samples was calculatedusing the following equation: 

### R =-c-·100 cfort (3) where R: recoveryrate [%] c: determined concentrationofthe testiteminthe spikedrecovery sample [mg/L] Cfort: fortified concentration ofthe testitem inthe spikedrecovery sample [mg/L] The percentage ofnominal concentration ofthe test item in the test samples was calculated using the following equation:Harlan StudyNumber: 41201182 Report Page 35 Analytical Investigations
%nominal

## =_c_ ·100 cnom (4) where Cnom: nominal concentrationinthe test sample [mg/L]Harlan StudyNumber: 41201182 Report Page 36 Analytical Investigations 

## 2 RESULTS 2.1 Range-Finding Test The analytical methodology was not validated prior to the analysis ofrange-finding samples; full validation was later conducted specifically at the test concentration to be employed in the definitive test. The results for the range-finding test samples are summarized in Table 3. 

### 2.2 Definitive Test 2.2.1 Validation ofthe Analytical Method Specificity The biological control samples and an analyzed analytical blank showed no significant interfering response at the retention time ofthe test item. The standard solutions contained a peak specific for the test item whose area changed accordingly with known concentration, hence the specificity ofthe method by retentiontime was confirmed. Linearity The calibration data forthe calibration standards ofthe testitemis given inTable 2 and Figure 1. The data was found to have a linear correlation within the calibration range of0 to 169 mg/L. The R2 fit ofthe calibration curve to the data was 0.9999, and was considered to be acceptable. Accuracy (Recovery) and Precision A set ofrecovery samples accurately fortified at a relevant concentration oftest item was prepared five-fold and analyzed. The results obtained for the concentration ofthe test item in the recovery samples are presented inTable 4. The method was considered to be sufficiently accurate and precise for the purposes ofthis test. The test sample results were not corrected for recovery. Limit ofQuantitation (LOQ) The limit ofquantitation (LOQ) was determined by calculating the sample concentrationthat gave a peak equivalentto tentimes the baseline noise. Using this method the limit ofquantitation was determined to be 0.099 mg/L. 2.2.2 Test Samples The results obtained for the concentration ofthe test item found in the test samples are presented in Table 5. Typical chromatograms are shown in Figures 2 to 4.Harlan StudyNumber: 41201182 Report Page 37 Analytical Investigations 

## 3 DISCUSSION The detection system was found to have acceptable linearity. The analytical procedure was found to have acceptable recoveries oftest item in test medium. The method ofanalysis was validated andproven to be suitable for use.Harlan StudyNumber: 41201182 Report Page 38 Analytical Investigations 

## TABLES Table 1 Preparation ofTest Samples
Nominal Sample Sample FinalNominal FinalVolume Preparation Concentration ofTest Concentration of Volume Factor Item TestItem

[mg/L] [mL] [mL] F [mg/L] Control 100 100 1 100 100 100 100 1 100



### Table2 Linearity Data
Concentration ofTestItem Area [mg/L] [counts] 0 0 1.124 5.660 X 104 2.810 1.385 X 105 5.620 2.749 X 105 11.240 5.460 X 105 28.100 1.364 X 106 56.200 2.696 X 106 112.400 5.426x 106 115.200 5.616 X 106 134.880 6.521 X 106 168.600 8.035 X 106Harlan StudyNumber: 41201182 Report Page 39 Analytical Investigations Table 3 Results for Range-Finding Samples Nominal Measured Sample Determined Concentration of Concentration Preparation Concentration TesiIiem in ofTestItem in Factor ofTestItem in % ofNominal Time point Range-Finding Sample Vial Range-Finding Concentration Sample Sample
X F c Cnom [hours] [mg/L] [mg!L] [mg/L] [%] 0 100 109 1 109 109 96 100 109 1 109 109

Table 4 Results for SpikedRecovery Samples Nominal Fortified Measured Sample Determined Analytical Concentration Concentration Concentration Preparation Concentration Recovery Precision of ofTest Item in ofTestItem in Factor ofTestItem in Rate (Relative Test Item the Spiked the Sample the Spiked Standard Sample Vial Sample Deviation of Recovery) Cnom Cfort X F c R [mg/L] [mg/L] [mg/L] [mg!L] [%] [%]

100 115 126 1 126 126 1 126 127 1 127 110 0.78 126 1 126 128 1 128 Acceptance Target 80-120 <10
Forreporting purposes, x has been calculated from c retrospectively.Harlan StudyNumber: 41201182 Report Analytical Investigations Table 5 Results for Test Samples

Time point

[hours] 0 96 LOQ Nominal Measured Concentration Concentration of ofTestItem in TestItem in SampleVial Test Sample
Cnom X [mg/L] [mg!L] Control <LOQ

100 100 Control <LOQ

100 98.6 Limit ofQuantitation not applicable Sample Preparation Factor

F
1 I 1 I

Forreporting purposes, x has been calculated from c retrospectively. Page 40

Determined Concentration ofTest Item in %ofNominal

Test Sample Concentration

c [mg!L] [%] <LOQ 

## -

100 100 <LOQ


### -

98.6 99Harlan StudyNumber: 41201182 Analytical Investigations 

## FIGURES Figure 1 Linearity 

### 2 9000000 ·;:: 2- 8000000 co !!! <( 7000000 ..><: co Ql a.. 6000000 5000000 4000000 3000000 2000000 1000000 0 0 Report

y =48062x + 8914.5 R

2 =0.9999

50 100 Page 41

150 200

Concentration (mg/L)Harlan StudyNumber: 41201182 Report Page 42 Analytical Investigations Figure2 Chromatogram ofCalibration Solution (100 mg/L)

600

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**~** 300Harlan StudyNumber: 41201182 Report Page 43 Analytical Investigations 

### Figure4 Chromatogram ofTest Sample (100 mg/L)

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# _______,v_, 200Harlan StudyNumber: 41201182 Report 

### Appendix 5 MonitoringAuthority StatementofGLP Compliance 

# i

# oii) Department

**~** 

### · ofHealth
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC TEST FACILITY Harlan Laboratories Ltd Shardlow Business Park London Road Shardlow Derby DE72 2GD

DATE OF INSPECTION 10 July2012 TEST TYPE(S) Analytical/Clinical Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys/Chem. Tests Toxicology

An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999).



# ~~~ -:30

# /u /tz.... Page 44

Dr. Andrew J. Gray - Head, UK GLP Monitoring Authority • •