p.1 

GREENWIC H CHEMICA L CONSULTING , INC . 

3300 PGA BOULEVARD • SUITE 635 n , , ,/ 

PALM BEACH GARDENS, FL 33410 M>" /" ^ 

TELEPHONE 561.578.81 10 

FACSIMILE 561.578.8120 

January 9, 2014 

CBIC Control Number 

357676 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

By UPS 

OPPT Document Control Office ^ O 

EPA East Building ~ ^g n i 

RoomE6428 o H m 

U.S. Environmental Protection Agency a*. S^" 

1201 Constitution Ave., NW 5 £ 3 

Washington, DC 20004 

CO 

Attention: Docket ID Number: EPA-HQ-OPPT-2009-0112 

40 CFR 799.5089 

Re: Study Plan Submission fot Algae Testing Conducted by the PCBTF Testing 

Consortium under the TSCA Section 4 Test Rule on the Testing of Certain 

High Production Volume Chemicals; Third Group 

Benzene, l-chloro-4-(trifluoromethyl)-

CAS RN 98-56-6 

(para-Chlorobenzotrifluoride (PCBTF)) 

Docket ID Number: EPA-HQ-OPPT-2009-0112 

40 CFR 799.5089 

Dear Sir/Madam: 

Through this letter, the PCBTF Testing Consortium submits to the U.S. Environmental 

Protection Agency (EPA or the Agency) a study plan for algae testing conducted on the 

chemical substance, benzene, l-chloro-4-(trifluoromethyl)- [CAS RN 98-56-6] which is 
p. 2 

CONTAINS NO CONFIDENTIA L 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

January 9, 2014 

Page 2 

currently subject to a Toxic Substances Control Act (TSCA) Section 4 test rule.1 For purposes 

of this letter, the chemical substance, benzene, l-chloro-4-(trifluoromethyl)-, is herein referred 

to as, PCBTF. The PCBTF Testing Consortium files this request in accordance with this test 

rule and the regulations set forth at 40 CFR 790.50 and 799.5089. 

L General Background 

Greenwich Chemical Consulting, Inc. (Greenwich Chemical Consulting) functions and 

operates as the panel manager for and authorized representative of the PCBTF Testing 

Consortium. Therefore, Greenwich Chemical Consulting submits this letter and the enclosed 

study plans on behalf of the PCBTF Testing Consortium's 13 member companies identified 

below:2 

• Brenntag North America, Inc. 

• Chemsynergy, Inc. 

• China Specialty Chemical Company, Ltd. 

• EMCO Chemical Distributors, Inc. 

• Greenchem Industries LLC 

• Kowa American Corporation 

• Makhteshim Agan of North America, Inc. 

• MC International, LLC3 

• Nexeo Solutions, LLC 

• Silver Fern Chemical, Inc. 

• Special Materials Company 

• Univar USA, Inc. 

• Valspar Corporation 

This final test rule addresses 15 high production volume (HPV) chemicals that went 

unsponsored under the Agency's voluntary HPV Chemical Challenge Program. One of the 15 

chemicals identified in the rule is PCBTF. The final rule identified the following two studies 

on PCBTF: 

• Aquatic Toxicity Testing: toxicity to plants (algae)) 

• Ready Biodegradation 

In its letter dated April 18, 2013, the PCBTF Testing Consortium timely submitted final test 

reports to EPA for these two studies. The Agency subsequently determined that the algae 

study was inadequate and required that the study be re-conducted for purposes of the TSCA 

Section 4 test rule.4 On November 6, 2013, the PCBTF Testing Consortium requested an 

1

 76 Eed, Reg, 65385; October 21, 2011. 

2

 See Attachment A. 

s

 MC International, LLC operates the entity, Miami Chemical, Inc., as a DBA. For purposes of U.S. Customs 

and TSCA, MC International, LLC is the importer. 

4

 Letter from EPA to the PCBTF Testing Consortium dated September 26, 2013. 
p. 3 

CONTAINS N O CONFIDENTIAL 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

January 9, 2014 

Page 3 

extension for additional time to review and respond to EPA's comments. In that letter, the 

PCBTF Testing Consortium also requested that the study plan submission date be extended 

from November 12, 2013 to January 12, 2014. This extension, as well as the modified study 

plan due date, was formally granted by EPA in the Agency's letter of November 12, 2013. 

I I . Submission of the Algae Study Plan by the PCBTF Testing Consortium 

The PCBTF Testing Consortium, therefore, timely submits to EPA the study plan for a new 

algae test on the compound which was developed by the group's new contract lab, Harlan 

Laboratories Ltd. (Harlan Laboratories). As part of the development of this study plan, the 

PCBTF Testing Consortium provided to Harlan Laboratories the Agency's comments of 

September 26, 2013. Harlan Laboratories developed this study plan taking into account and 

incorporating these comments and also addressing the technical difficulties associated with 

testing PCBTF (Le., poor water solubility and high volatility). As with the first algae test and 

consistent with EPA's approval granted on February 9, 2012, this second test will also be 

conducted under OECD test guideline 201. 

The study plan submitted herein addresses all of EPA comments in its Notice of Inadequacy 

letter. It is noted though that in EPA's letter, the Agency discusses potential loss of the test 

material during sampling and, therefore, the use of additional replicates.5 The study plan at 

Section 3.7 addresses a modified use of additional replicates during both the limit test and full 

study. Harlan Laboratories provided additional comment on the proposed number of 

replicates with respect to the Agency's recommendation: 

The use of the additional replicates that EPA suggests would mean that in the case of a full 

test there would be 12 replicate vessels per test concentration (and control) i.e. a total of 72 

vessels. If the test was conducted as a limit test then the total number of vessels would still 

be 48. This would make the test too cumbersome and prone to error in the determination of 

cell densities as these would be monitored in individual replicate vessels on each cell 

determination point rather than from the same replicate vessels at 0, 24, 48 and 72 hours. As 

a compromise we've included additional vessels that will be sampled at 24 and 48 hours for 

analysis. These vessels will be treated in the same manner as those from which samples are 

taken for cell determination measurements such that measured concentrations in these vessels 

will mimic those in the test vessels that are incubated for the cell determinations. An additional 

replicate vessel will also be maintained fully closed for the 72-hour test period and analyzed at 

test termination to assess the effect that opening test vessels for the cell determinations 

has. This is a procedure that we've used on numerous occasions for volatile test items and has 

been accepted by various regulatory authorities. 

Therefore, the PCBTF Testing Consortium submits this algae study plan for the re-testing of 

the material under EPA's TSCA Section 4 test rule. Attached is a final draft study plan for 

review, comment, and approval by the Agency. 

5

 EPA's Notice of Inadequacy letter at Section C{2). 
p. 4 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

January 9,2014 

Page 4 

The PCBTF Testing Consortium thanks the Agency for its consideration in this matter. For 

all questions, comments, and determinations concerning this matter, please use the contact 

information given below. 

Mr. John V. Massingale 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

Telephone: (561) 578-8110 

Facsimile: (561) 578-8120 

E-mail: john@grcci.com 

Sincerely, 

John V. Massingale 

Panel Manager 

cc: Dr. J. Schaeffer-EPA 

PCBTF Testing Consortium 

Enclosure 
p. 5 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

U.S. Environmental Protection Agency 

January 9, 2014 

Page 5 

Attachment A 

Names and Addresses of Member Companies 

Companv Address 

Brenntag North America, Inc. 5083 Pottsville Pike 

Reading, PA 19605 

Chemsynergy, Inc. 1100 Burloak Drive 

Suite 101 

Burlington, ON L7L 6B2 

Canada 

China Specialty Chemical Company, Ltd. Unit A (Room 9) 3Ki Floor 

Cheong Sun Tower 

116-118 Wing Lok Street 

Sheung Wan 

Hong Kong 

EMCO Chemical Distributors, Inc. 2100 Commonwealth Avenue 

North Chicago, IL 60064 

Greenchem Industries, LLC 222 Clematis Street 

West Palm Beach, FL 33401 

Kowa American Corp. 55 East 59th

 Street 

19lh Floor 

New York, NY 10022 

Makhteshim Agan of North America, Inc. 171 Madison Avenue 

Room 1605 

New York, NY 10016 

MC International, LLC 2 NE 40th

 Street 

Suite 501 

Miami, FL 33137 

Nexeo Solutions, LLC 1400 Woodloch Forest Drive 

Suite 200 

The Woodlands, TX 77380 

Silver Fern Chemical Inc. 2226 Queen Anne Avenue N. 

Seattle, WA 98109 

Special Materials Company 70 West 40th Street 

2

nd Floor 

New York, NY 10018 

Univar USA Inc. 17425 NE Union Hill Road 

Redmond, WA 98052 

Valspar Corporation 901 3rd Avenue South 

Minneapolis, MN 55402 
p. 6 

STUDYPLAN 

harlan 

Benzene, l-chloro-4-(trifluoromethyl)-, 

CAS RN 98-56-6, 

para-Chlorobenzotrifluoride (PCBTF): 

Algal Growth Inhibition Test 

Study Director: 

Test Facility: 

Helen Vryenhoef 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Sponsor: PCBTF Testing Consortium 

c/o Greenwich Chemical Consulting, Inc. 

Address: 3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

Study Monitor: Mr. John V. Massingale 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens, FL 33410 

USA 

Harlan Study Number: 41304731 

Page 1 of 19 
p. 7 

Harlan Study Number: 41304731 Study Plan Page 2 

CONTENTS 

CONTENTS 2 

SIGNATURES 3 

GOOD LABORATORY PRACTICE 4 

GENERAL INFORMATION 5 

Proposed Schedule 5 

Distribution List 5 

Study Plan Amendments and Deviations 5 

Quality Assurance 5 

Reporting 5 

Archiving 6 

1 INTRODUCTION AND PURPOSE 7 

1.1 Guidelines / Regulations 7 

2 TEST AND REFERENCE ITEMS 8 

2.1 Test Item 8 

2.2 Reference Item 8 

3 MATERIALS AND METHODS 9 

3.1 Definitions and Abbreviations 9 

3.2 Test System 9 

3.3 Test Water 10 

3.4 Dosage 10 

3.5 . Materials 10 

3.6 Experimental Conditions 11 

3.7 Study Design 11 

3.8 Evaluations 12 

3.8.1 Test Organism Observations 12 

3.8.2 Water Quality Criteria 12 

3.8.3 Analysis of Test Item Concentrations 13 

3.9 Data Analysis 14 

3.9.1 Plotting Growth Curves 14 

3.9.2 Calculation of Growth Rates 14 

3.9.3 Yield 15 

3.9.4 Plotting Concentration-Response Curves 16 

3.9.5 Statistical Analysis 16 

3.10 Validity Criteria 16 

4 REFERENCES 18 

APPENDICES 19 
p. 8 

Harlan Study Number: 41304731 Study Plan Page 3 

SIGNATURES 

Study Director: H Vryenhoef 

Date: 

Test Facility 

Management: C Mead 

Date: 

For the Sponsor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Date: 
P. 9 

Harlan Study Number: 41304731 Study Plan Page 4 

GOOD LABORATORY PRACTICE 

This study will be 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 as revised 1997 (ENV/MC/CHEM(98))7); and are in accordance with, and 

implement, the requirements of Directives 2004/9/EC and 2004/10/EC. 

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). 
p. 10 

Harlan Study Number: 41304731 Study Plan Page 5 

GENERAL INFORMATION 

Proposed Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

28 February 2014 

31 March 2014 

30 April 2014 

Distribution List 

The original Study plan will be retained in the study file. Copies of the study plan along with any 

amendments will be distributed as follows: 

Study Plan Amendments and Deviations 

Any intended change to the study plan will result in an amendment to study plan approved by the 

Study Director and also signed by test facility management and the Sponsor. Amendments will 

be distributed to all recipients of the study plan. 

Deviations (unplanned changes) from the study plan will be documented and acknowledged by 

the Study Director. 

Quality Assurance 

Quality Assurance will audit and inspect study-related procedures and will report any audit and 

inspection results in writing to the Study Director and Test Facility Management. 

Reporting 

Sponsor: PCBTF Consortium 

Quality Assurance: Harlan Laboratories Ltd. 

Shardlow, UK 

Study Monitor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

A GLP compliant report will be presented. A draft report will be sent to the Sponsor for review 

and comments before issue of the final report. 
p. 11 

Harlan Study Number: 41304731 Study Plan Page 6 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan, all raw data (paper and electronic) 

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. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
p. 12 

Harlan Study Number: 41304731 Study Plan Page 7 

1 INTRODUCTION AND PURPOSE 

The purpose of this study is to assess the acute toxicity of the test item to a freshwater alga. The 

test endpoint is inhibition of growth, expressed as the logarithmic increase in algal biomass 

(average growth rate) during the exposure period. The toxicity will be expressed as the 

concentration bringing about a specified x% inhibition of growth (e.g. 50%) and expressed as the 

The Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration 

(NOEC) will also be determined if applicable. 

An additional response variable recommended by the Test Guidelines is yield, which may be 

required to fulfil specific regulatory requirements in some countries. Results will therefore also 

be expressed in terms of inhibition of yield (EyCx). 

The study will use Pseudokirchneriella subcapitata which is a representative species of a wide 

variety of natural habitats, and can therefore be considered as an important non-target organism 

in freshwater ecosystems. 

The dispersal of the test item in the surrounding medium is considered to represent the most 

probable route of exposure in the environment. 

1.1 Guidelines / Regulations 

This test methods described are 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"1 

• Method C.3 of Commission Regulation (EC) No. 761/2009 

1

 Annex 5 corrected: 28 July 2011 
p. 13 

Harlan Study Number: 41304731 Study Plan Page 8 

2 TEST AND REFERENCE ITEMS 

2.1 Test Item 

The test item will be supplied by or on behalf of the Sponsor including the following information 

if known; description, batch, purity, expiry / retest date, storage conditions and safety 

precautions to be taken. 

Information as provided by the Sponsor. 

2.2 Reference Item 

For evaluation of the quality of the algal culture and the experimental conditions, potassium 

dichromate will be tested as a positive control twice a year to demonstrate satisfactory conditions 

of the test. 

The reference item will be supplied by a commercial supplier and will be stored as specified by 

the supplier. 

Identification: Benzene, l-chloro-4-(trifluoromethyl)-, 

CAS RN 98-56-6, 

para-Chlorobenzotrifluoride (PCBTF) 
p. 14 

Harlan Study Number: 41304731 Study Plan Page 9 

3 MATERIAL S AN D METHOD S 

3.1 Definitions and Abbreviations 

Biomass: 

ECK: 

Growth: 

Growth Rate (Average Specific 

Growth Rate): 

Yield: 

LOEC (Lowest Observed 

Effect Concentration): 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

Measurement variable (algal cell density) as surrogate measure 

for algal biomass. 

The calculated concentration of test 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 in biomass over the test period. 

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, if applicable). 

The highest test concentration at which no statistically 

significant inhibition of growth is determined relative to the 

control (or solvent control, if applicable). 

Comprises test item treatments, control (test medium only) and 

solvent control (if applicable). 

Section-by-Section Growth 

Rate: Specific growth rate for each day during the course of the test 

(days 0-1, 1-2 and 2-3). 

3.2 Test System 

The study will be performed using Pseudokirchneriella subcapitata. This freshwater alga 

species is recommended by the relevant test guideline. 
Harlan Study Number: 41304731 Study Plan 

p. 15 

Page 10 

An inoculum culture of algae will be set up three to four days before the start of the test. The 

algae will be cultivated under the test conditions and will be kept in the exponential growth 

phase until inoculation of the test solutions. 

3.3 Test Water 

Culture medium as defined in Appendix 1. 

3.4 Dosage 

The test item will be dissolved in the test water once at the start of the test. 

Data supplied by the Sponsor indicates that the test item has a water solubility of 29 - 33.8 mg/L. 

Therefore the test item falls into the category of a "difficult substance" as defined by the OECD 

Guidance Document (OECD, 2000), hence the method of preparation of the test media will be 

based on the recommendations of this document. 

The test item solution will be prepared as a saturated solution using an initial test item dispersion 

of 100 mg/L. This test item dispersion will be stirred using a magnetic stirrer at a rate such that a 

vortex representing approximately 5% of the test water column height is produced. To reduce 

the risk of volatilization during the stirring period, the stirring will be conducted in a completely 

filled vessel (i.e. no headspace) sealed with a non-permeable stopper e.g. glass stopper, at a 

temperature of approximately 21 °C for a period of 24 - 48 hours. 

After the stirring period, excess undissolved test item will be removed by an appropriate method 

e.g. filtration or centrifugation and the test organisms exposed to the resulting saturated solution. 

Pre-experiments will be performed to determine the solubility and stability of the test item in the 

test water, the stirring period required to achieve saturation and the suitability of either filtration 

or centrifugation to remove excess undissolved test item. According to the findings of these preexperiments, appropriate procedures will be chosen to prepare the test media. 

If the test item is photodegradable, the preparation of the test media will be performed as far as 

possible in the dark. Details of the method of preparation will be documented in the data. 

3.5 Materials 

Glass test vessels containing at least 100 mL test medium will be used. The test item is known to 

be volatile (Data supplied by the Sponsor) hence the test vessels will be completely filled and 

sealed using glass stoppers (i.e. test performed in a closed system with no headspace). The size 

of the test vessels and the volume of test solution will be documented in the data. 
Harlan Study Number: 41304731 Study Plan 

p. 16 

Page 11 

The test vessels will be labeled with the study number and all necessary information to ensure 

unique identification. 

3.6 Experimental Conditions 

The environmental parameters will be as used to maintain the stock organisms. 

The test flasks will be incubated under the following conditions: 

• INFORS Multitron® Version 2 incubator under constant illumination (between 4440 and 

8880 lux) provided by warm white lighting (380 - 730 nm) at 24 ± 1°C and agitated by 

orbital shaker (approximately 150 rpm). Temperature, light intensity and oscillation rate 

of the incubation chamber will be recorded daily throughout the test. 

3.7 Study Design 

The test design will include three replicates per test concentration, six replicates of the control 

and, if appropriate, six replicate solvent control flasks. A blank consisting of culture medium 

alone will also be prepared. 

In order to investigate possible losses of test item due to volatility when the test vessels are 

opened on a daily basis, additional replicate vessels at each concentration will be prepared at 0 

hours and incubated alongside the other exposure vessels for analysis at 24 and 48 hours. These 

vessels will be opened and a sub-sample removed in the same manner as those taken for algal 

cell determinations before the test vessel is sampled for chemical analysis. 

A further replicate vessel at each concentration will be prepared at 0 hours and incubated 

alongside the other exposure vessels. This fourth replicate will remain unopened until 

termination of the test when it will be analyzed for test item concentration. 

The following conditions will be met: 

Initial cell density (cells/mL) 2-5 x 10 

Test duration (hours) 72 

A static test design will be applied. 

At least five concentrations of the test item arranged in a geometric series will be tested. The 

spacing factor between the test concentrations will preferably not exceed 2.2. The actual test 

concentrations are assigned according to preliminary range-finding tests or available toxicity 

data and will be documented in the data prior to the start of the definitive test. If in the range-
p. 17 

Harlan Study Number: 41304731 Study Plan Page 12 

finding test a flat concentration-effect relationship is determined and, thus, a large concentration 

range will have to be tested, a spacing factor exceeding 2.2 (e.g. 3.2) will be applied. 

The lowest test concentration should have no significant effect on the growth of algae. The 

concentration series should preferably cover the range causing 5 to 75% inhibition of algal 

growth rats. In accordance with the test guidelines (EC, 2002), concentrations of the test item 

above the limit of solubility of the test item in the test water will not be tested. 

If no effect is observed in the preliminary range-finding test at the limit of solubility of the test 

item in the test water, a Limit Test may be conducted using only this concentration and the 

corresponding control. In this case, the number of test concentration replicates will be doubled to 

six. 

The control group will be exposed to the test water alone. Data from the control group may be 

shared with similar concurrent studies. 

The test will be carried out without adjustment of pH. If the pH falls outside the range 6-9, the 

Sponsor will be contacted for advice on whether a second test should be performed with 

adjustment of pH to that of the test water. However, pH adjustment will not be carried out if the 

test solutions are changed to any significant extent and if a chemical reaction or precipitation is 

caused. 

3.8 Evaluations 

3.8.1 Test Organism Observations 

A small volume of the algal suspension will be withdrawn from each test flask daily for the 

measurement of algal biomass, and will not be replaced. The algal concentration of each sample 

is determined by direct counting using a Coulter® Particle Counter. Three determinations are 

made for each sample. 

Observations will be made on the test item preparations daily during the exposure period. 

To determine a potential effect of the test item on the appearance of algal cells, a sample will be 

taken from the test and control cultures at the end of the test. The shape and size of the algal cells 

will be inspected microscopically and any abnormalities recorded. 

3.8.2 Water Quality Criteria 

The pH of the test media will be measured in all treatments at the start and end of the test. The 

pH of the controls should not normally deviate by more than 1.5 pH units during the test. An 

explanation will be provided in the report if pH deviations in excess of 1.5 units are observed. 
p. 18 

Harlan Study Number: 41304731 Study Plan Page 13 

The water temperature will be measured daily in an Erlenmeyer flask filled with water and 

incubated under the same conditions as the test flasks. 

The appearance of the test media will be recorded daily. 

3.8,3 Analysis of Test Item Concentrations 

3.8,3-1 Sampling 

Samples of the test media will be taken at initiation and termination of both the range-finding 

and definitive tests for analysis. During the definitive test additional samples at 24 and 48 hours 

will be analysed. Where possible duplicate samples will be taken at each occasion, these 

samples may also be analyzed should the need arise. 

Analysis of samples taken from the range-finding test is performed in order to determine the 

stability of the test item under test conditions over the exposure period. The replicate test vessels 

at each concentration will be pooled to give one sample. The test concentrations provided for 

analysis will be documented by the Study Director in the data. 

For the purposes of the definitive test, the replicate test and control vessels will be pooled to give 

one sample. Should the analysis of the range-finding test samples show a decline in measured 

test concentration over the test duration then additional test samples may be prepared at test 

initiation with the omission of algal cells and incubated alongside the test from which samples 

will be taken for analysis at test termination. 

If tPe test medium volume from the actual test is too small to perform the required analyses, 

requisite volumes of the freshly prepared test media will be incubated separately, but under 

identical conditions as in the actual test. 

Fin£l details of the samples to be taken and the sample size, etc, will be documented in the data. 

3.8.3-2 Storage and sample preparation 

All samples will be analyzed immediately after sampling or stored at approximately -20°C until 

analysis. 

If the test item has been shown to be unstable under the storage conditions, the samples will be 

analyzed immediately after sampling without prior storage or measures will be taken (e.g. pH 

adjustment or addition of organic solvents) in order to maintain the stability of the test item until 

analysis. 

3.8.3.3 Analysis of samples 

The test item formulations will be analyzed for concentration and stability by Harlan 

Laboratories Ltd, Shardlow, UK Analytical Services. 
p. 19 

Harlan Study Number: 41304731 Study Plan Page 14 

The test item concentrations will be measured in at least one of the duplicate test medium 

samples. However, the samples from test concentrations below the 72-Hour NOEC will not 

normally be analyzed. The remaining samples may be analyzed, if necessary. 

Stock solutions may also be analyzed, if necessary, for example if the test concentrations are 

below the limit of quantitation of the analytical method or for reasons of stability. 

The analytical method (eg HPLC, LC-MS, GC, AAS or photospectrometry) will either be 

provided by the Sponsor and adapted, if necessary, to the analytical conditions at Harlan 

Laboratories Ltd or developed by Harlan Laboratories Ltd. Adaptation or development of the 

analytical method will not be performed under GLP. Details about the analytical method used 

will be given in an Appendix of the final report. The accuracy of the analytical method will be 

verified by recoveries spanning the concentration range to be analyzed within this study. 

3.9 Data Analysis 

3.9.1 Plotting Growth Curves 

The cell density in the control and test cultures together with the test item concentrations and 

times of measurement, recorded to the nearest whole hour, are tabulated and growth curves are 

constructed by plotting cell density (logarithmic axis) against time (arithmetic axis). 

All data points are critically examined and if it is obvious that procedural mistakes can be 

identified and/or considered highly likely, the specific data point is marked as an outlier and not 

included in subsequent statistical analyses. Reasons for rejection of a data point as an outlier 

will be clearly stated in the final report. Acceptable reasons are only procedural mistakes and 

not just bad precision. Outliers (marked as such) will be retained amongst the data points shown 

in the final report. 

3.9.2 Calculation of Growth Rates 

The average specific growth rate for a specified period is calculated as the logarithmic increase 

in biomass from the equation: 

lnNn -In N 

t B - t , 

where: 

Ni 

N n 

ti 

average specific growth rate from time ti to t n. 

cell concentration at ti. 

cell concentration at tn. 

time of first measurement. 
p. 20 

Harlan Study Number: 41304731 Study Plan Page 15 

t n = time of nth measurement. 

The average specific growth rate for each replicate control and test item vessel is calculated for 

the period 0-72 hours using the nominally inoculated cell concentration as the starting value 

rather than the measured starting value in order to increase the precision of the calculation. 

In addition the section-by-section growth rate (0 - 24,24 - 48 and 48 - 72 hours) is calculated for 

the control cultures and the results examined in order to determine whether the growth rate 

remains constant. 

Percentage inhibition of growth rate for each replicate test item vessel is calculated using the 

following equation: 

I r =^^-xl0 0 

where: 

I r = percentage inhibition of average specific growth rate. 

He = mean average specific growth rate for the control cultures, 

p t = average specific growth rate for the test culture. 

Where auxiliary solvents/surfactants are used, percentage inhibition in the test cultures is 

calculated by comparison to the solvent control cultures. 

3.9.3 Yield 

Yield is calculated as the increase in biomass over a specified period using the following 

equation: 

Y = N n -N t 

where: 

Y = Yield. 

Nt - cell concentration at the beginning of the specified period. 

Nn = cell concentration at the end of the end of the specified period. 

For each test concentration and control, the mean value for yield, along with the standard 

deviation is calculated for the period 0 - 72 hours. Percentage inhibition of yield is calculated 

using the following equation: 
p. 21 

Harlan Study Number: 41304731 Study Plan Page 16 

x 100 

Where 

h 

Yc 

Yt 

percentage inhibition of yield 

mean value for yield in the control group 

mean value for yield for the treatment group 

3.9.4 Plotting Concentration-Response Curves 

For each individual test vessel, percentage inhibition (arithmetic axis) is plotted against test 

concentration (logarithmic axis) and a line fitted by computerised interpolation e.g. Xlfit. Any 

data points identified as outliers in the first phase of data evaluation are disregarded when fitting 

the concentration-response curve. The ECm, EC20, EC50 values are read from the graph. 

Where appropriate 95% confidence limits for the EC50 will be calculated, using the simplified 

method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949). All EC50 

values and 95% confidence limits will be rounded to two significant figures. 

3.9.5 Statistical Analysis 

The Lowest Observed Effect Concentration (LOEC) is the lowest concentration tested at which 

significant (P < 0.05) inhibition of growth relative to the control is observed within a given 

exposure time. However, all test concentrations above the LOEC must have an effect equal to or 

greater than those observed at the LOEC. When these two conditions cannot be satisfied, a full 

explanation of how the LOEC has been selected will be given in the test report. 

The LOEC for growth rate and yield at 72 hours will be determined using analysis of variance 

(ANOVA) incorporating a statistical test for homogeneity of variance and an adequate post hoc 

test (e.g. Dunnett test). 

The No Observed Effect Concentration (NOEC) is the test concentration immediately below the 

LOEC which, when compared to the control, has no statistically significant effect (P > 0.05), 

within a given exposure time. 

Where a limit test has been conducted, it is not possible to determine a LOEC, and the NOEC 

will be the limit test concentration. 

3.10 Validity Criteria 

For the test to be valid, the following performance criteria should be met: 
p. 22 

Harlan Study Number: 41304731 Study Plan Page 17 

The cell concentration of the control cultures must increase by a factor of at least 16 after 

72 hours. 

The mean of the coefficients of variation of the daily section-by-section specific growth rate in 

the control cultures after 72 hours (0 - 24, 24 - 48 and 48 - 72 hours) must not exceed 35%. 

The coefficient variation of the average specific growth rate in replicate control cultures after 

72 hours must not exceed 7%. 
Harlan Study Number: 41304731 Study Plan 

p. 23 

Page 18 

4 REFERENCES 

HERMAN, D. C, INNISS, W. E. and MAYFIELD, C. I. (1990) Impact of volatile aromatic 

hydrocarbons, alone and in combination, on growth of the freshwater alga Selenastrum 

capricornutum. Aquatic Toxicology, 18, pp. 87 - 100. 

LITCHFIELD, J. T. and WILCOXON, F. (1949) A Simplified Method of Evaluating DoseEffect Experiments. J. Pharmacol. Exp. Ther., 96, pp. 99-113. 

MAYER, P., NYHOLM, N., VERBRUGGEN, E. M. J., HERMENS, J. L. M. and TOLLS, J. 

(2000) Algal growth inhibition test in filled, closed bottles for volatile and sorptive materials. 

Environmental Toxicology and Chemistry, 19, pp. 2551 -2556. 

OECD (2000) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and 

Mixtures. OECD Series on Testing and Assessment, No. 23. OECD Publishing. 

EUROPEAN COMMISSION (2002) Sanco/3268/2001 rev. 4 (final) Guidance Document on 

Aquatic Ecotoxicology in the context of Directive 91/414/EEC. 

Xlfit, ID Business Solutions Ltd. 
p. 24 

Harlan Study Number: 41304731 Study Plan Page 19 

APPENDICES 

Appendix 1 Culture Medium 

Reconstituted test water (AAP/ASTM medium) prepared according to OECD Test Guideline 

No 201 will be used for algal cultivation and testing. Analytical grade salts will be dissolved in 

purified water to obtain the following final concentrations: 

Ingredients Concentration 

Macro-nutrient Macro-nutrients NaHC03 15.0 mg/L 

K2HP04 1.044 mg/L 

Macro-nutrients 

MgS04.7H2 0 14.6 mg/L 

Macro-nutrients 

MgCl 2.6H2 0 12.16 mg/L 

Macro-nutrients 

CaCl2.2H20 4.41 mg/L 

Macro-nutrients 

NaN03 25.5 mg/L 

Trace element elements H3BO3 186.0 ug/L 

MnCl 2.4H2 0 415.0 ug/L 

Trace elements 

ZnCl2 3.27 Mg/L 

Trace elements 

CoCl2.6H20 1.43 ug/L 

Trace elements 

CuCl2.2H20 0.012 ug/L 

Trace elements 

Na 2MoO„.2H2 0 7.26 ug/L 

Trace elements 

FeCl3.6H20 160.0 ug/L 

Trace elements 

Na2EDTA.2H20 300.0 ug/L 

The pH will be adjusted to 7.5. The water hardness (calculated) of the test water will be 

0.15 mmol/L (= 15 mg/L as CaC03). 

For the purposes of the test, additional sodium bicarbonate (up to 500 mg/L final concentration) 

will be added to the prepared culture medium as defined by Herman et al (1990) and Mayer et al 

(2000) to counter act CO2 depletion and the change in pH due to algal growth in sealed test 

vessels.