c' ""H 1,...., 1 •.. Li 1

1 t.LJ r•t ,, ...... 0 r r' I (',:~~ 1 '(., ~ •• J ri .• ~· i ' .,/ 

GREENWICH CHEMICAL CONSULTING, IJ~fltM>;:~ 16 I~HIQ: 21 

3300 PGA BOULEVARD • SUITE 635 

PALM BEACH GARDENS, FL 33410 

TELEPHONE 561.578.8110 

FACSIMILE 561.578.8120 

CBIC Control Number 

359537 

COPY 

April 15, 2014 

)By Central Data Exchange 

OPPT Document Control Office 

U.S. Environmental Protection Agency 

EPA East Building 

RoomE6428 

1201 Constitution Ave., NW 

Washington, DC 20004 

Attn.: TSCA Section 4 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

Re: Study Plan Submission and Request for Modification of Test Standards for 

Testing Conducted by Univar USA under the TSCA Section 4 Test Rule on the 

Testing of Certain High Production Volume Chemicals: Second Group of 

Chemicals 

D-erythro-Hex-2-enonic acid, .gamma.-lactone, monosodium salt 

CAS RN 6381-77-7 

(Sodium erythorbate) 

Docket Identification No. EPA-HQ-OPPT-2007-0531; 40 CFR 799.5087 

Dear Sir/Madam: 

On behalf of its client, Univar USA Inc. (Univar or the Company), Greenwich Chemical 

Consulting, Inc. (Greenwich Chemical Consulting) submits to the U.S. Environmental 

Protection Agency (EPA or the Agency) study plans and a request for the modification of test 

standards conducted on the chemical substance, D-erythro-hex-2-enonic acid, .gamma.-

v ""-/ \~ 
U.S. Environmental Protection Agency 

April15, 2014 

Page 2 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

lactone, monosodium salt [CAS RN 6381-77-7] which is currently subject to a Toxic 

Substances Control Act (TSCA) Section 4 test rule.1 Univar ftles this request in accordance 

with this test rule and the regulations set forth at 40 CFR 790.50 and 790.55. 

I. General Background 

As a matter of background, this test rule addresses 19 high production volume (HPV) 

chemicals that went unsponsored under the Agency's voluntary HPV Chemical Challenge 

Program. One of the 19 chemicals identified in the rule is D-erythro-hex-2-enonic acid, 

.gamma.-lactone, monosodium salt which is identified herein as sodium erythorbate. The rule 

requires manufacturers and importers of these HPV chemicals, including sodium erythorbate, 

to conduct testing to obtain screening level data for human health effects, environmental 

effects, chemical fate, and physical/ chemical properties. 

The Agency has issued this test rule under its TSCA Section 4(a)(1)(B) authority based on its 

determination that HPV compounds, including sodium erythorbate, are "produced in 

substantial quantities; there is or may be substantial human exposure to them; existing data are 

insufficient to determine or predict their health and environmental effects; and testing is 

necessary to develop such data."2 The final rule identifies the following studies and test 

protocols on sodium erythorbate: 

• n-Octanol/Water Partition Coefficient [40 CFR 799.6755, ASTM E 1147-92, or 40 

CFR 799.6756] 

• Ready Biodegradation3 

Aquatic Toxicity Testing4

: 

Acute toxicity to fish [ASTM E 729-96] 

Acute toxicity to Daphnia [ASTM E 729-96] 

Toxicity to plants (algae) [ASTM E 1218-04et] 

Because Univar had not yet begun importing sodium erythorbate with the intent to sell it for 

TSCA purposes at the time of the rule's effective date, the specific dates, as set forth in the 

fmal rule, for the submission of the letter of intent and test reports were not applicable. Univar 

subsequently submitted its letter of intent to test on January 27, 2014.5 Based on this, EPA 

stated in its response letter dated February 11, 2014 that study plans would be due April15, 

2014 and fmal reports would be due February 11, 2015.6 

1 January 7, 2011; 76 Fed. Reg. 1067. 2 Id. at 1069. 

3 The test rule identifies six ISO protocols and one ASTM standard for biodegradation testing. The protocol 

selection depends on the test substance's physical and chemical properties. 

4 Because the n-octanol/water partition coefficient is expected to be significantly less than 4.2 based on the 

compound's high water solubility, Test Group 1 is expected to apply. 

s Upon EPA's receipt of its January 27, 2014 letter of intent to test, Univar planned to begin importing this 

substance for both TSCA purposes and for continuing exempt FDA purposes. 

6 It is noted that since the time this test rule was issued through today, no other companies have come forward 

indicating their intent to test sodj.um erythorbate under this rule. 
U.S. Environmental Protection Agency 

April15, 2014 

Page 3 

II. Study Plan Submission 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

As required under 40 CPR 790.50 and 799.5087, the Company is required to submit to the 

Agency study plans prior to the initiation of testing. Univar has selected Harlan Laboratories 

Ltd. (Harlan Laboratories) to conduct all of these tests. The laboratory will conduct these 

studies at a single location. Also, Greenwich Chemical Consulting is serving as the study 

monitor for this testing. 

The study plans included herein identify administrative information, the TSCA Section 4 test 

rule, the sodium erythorbate test material, primary laboratory personnel, study objectives, test 

guidelines, materials and methods, observance of Good Laboratory Practice standards, and 

other related testing and procedural information. 

III. Request for Modification of Test Standards for the Use of OECD Test Guidelines 

All of the studies require the use of an ASTM, CPR, or ISO standard. Because of the 

validation. widespread use. and broad acceptance of Organization for Economic Cooperation 

and Development (OECD) test guidelines, as well as the near equivalency among the OECD. 

ASTM, CPR, and ISO protocols. Univar requests that EPA permit Univar's lab. Harlan 

Laboratories, to perform all five studies under the corresponding OECD guidelines. 

Specifically, we request that the testing be performed under the OECD test guidelines listed 

below. 

n-Octanol/Water Partition Coefficient [OECD 107f 

• Ready Biodegradation [OECD 301B] 8 

• Aquatic Toxicity Testing: 

Acute toxicity to fish [OECD 20W 

Acute toxicity to Daphnia [OECD 202r0 

Toxicity to plants (algae) [OECD 201 ]

11 

One significant benefit in permitting Harlan Laboratories to utilize the OECD protocols for 

this testing is that it will allow for more straightforward and expedient development of the 

data. Because it has significant experience and familiarity with the OECD test guidelines, 

Harlan Laboratories can begin testing immediately. Alternatively, the required use of the 

ASTM standards would delay the development of the test data.12 

7 Harmonized with EPA's guidelines under OCSPP 830.7550. 

8 Harmonized with EPA's guidelines under OCSPP 835.3110. 

9 Harmonized with EPA's guidelines under OCSPP 850.1075. 

IO Harmonized with EPA's guidelines under OCSPP 850.1010. 

II Harmonized with EPA's guidelines under OCSPP 850.5400. 

12 The labs contacted by Univar for testing under another TSCA Section 4 testing obligation advised that because 

of the additional time involved, the costs of performing tests under the ASTM standards is expected to be 20% 

to 30% higher. 
U.S. Environmental Protection Agency 

April15, 2014 

Page 4 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 

In addition, Harlan Laboratories was not familiar with the ASTM standards that EPA 

identifies in this and other recent TSCA Section 4 HPV rulemakings. Indeed, the lab was not 

familiar with or had experience with the ASTM standards and was surprised to learn that EPA 

was requiring their use in lieu of EPA's own Harmonized Test Guidelines or OECD 

guidelines.13 Also, the use of familiar OECD protocols decreases the chances for errors due 

to inadvertent deviations from the guidelines. 

Presumably, the Agency intends to eventually submit these data as part of dossiers under the 

international OECD HPV SIDS Program. The development of data on sodium erythorbate, 

under the internationally adopted and accepted OECD guidelines, would facilitate the 

Agency's efforts at the OECD level. 

It is also noted that all of the above ASTM standards internally reference other ASTM standards 

which either must or should be observed. This adds to the complexity and duration of testing. 

For example, in the algae study alone, ASTM E 1218-04e1 references 11 other ASTM standards. 

Similarly, in the combined fish and Daphnia standard under ASTM E 729-96, nine other ASTM 

standards are cited. Conversely, it is notable that none of the ASTM standards reference either 

the OECD guidelines or EPA's Harmonized Test Guidelines. 

Even though most of the discussion above pertains to the ASTM standards, Univar is formally 

requesting that EPA also approve the use of equivalent OECD test guidelines for these tests 

for which EPA has cited ASTM, CPR, or ISO guidelines. 

* * * 

Univar 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 

13 See EPA's Harmonized Test Guidelines at: http://~'W.epa.gov/ocspp/pubs/frs/home/guidelin.htm. 
U.S. Environmental Protection Agency 

April15, 2014 

Page 5 

Sincerely, 

John V. Massingale 

Principal 

cc: Ms. K. Gilje- Univar 

Ms. S. Lilley - Univar 

Mr. J. Schaeffer - EPA 

CONTAINS NO CONFIDENTIAL 

BUSINESS INFORMATION 
c 

STUDY PLAN 

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Determination of Partition Coefficient (n-Octanol/Water) 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

J A Walker 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL 33410 

UNITED STATES OF AMERICA 

41401172 

Page 1 of 10 
Harlan Study Number: 41401172 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 

INTRODUCTION AND PURPOSE ..................................................................................... 7 

1.1 Guidelines I Regulations .......................................................................................... 7 

2 TEST AND REFERENCE ITEMS ....................................................................................... 8 

2.1 Test Item .................................................................................................................. 8 

2.2 Reference Item ......................................................................................................... 8 

3 TEST ...................................................................................................................................... 9 

3.1 Partition Coefficient (n-OctanoliWater) .................................................................. 9 

3 .1.1 Test Specific Guidelines I Regulations ...................................................... 9 

3.1.2 Test System ............................................................................................... 9 

3.1.3 PrincipleoftheTest. .................................................................................. 9 

3.1.4 Performance of the Test. ............................................................................ 9 

3.1.5 Calculation ............................................................................................... 10 
Harlan Study Number: 41401172 

SIGNATURES 

Study Director: 

Test Facility 

Management: 

For the Sponsor: 

Study Plan Page 3 

J A Walker 

Date: 

Date: 

John V. Massingdale 

Greenwich Chemical Consulting, Inc. 

Date: 
Harlan Study Number: 41401172 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)17); and are in accordance with, and 

implement, the requirements ofDirectives 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). 
Harlan Study Number: 41401172 

GENERAL INFORMATION 

Proposed Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

Distribution List 

Study Plan 

01 May 2014 

17 July 2014 

31 July 2014 

Page 5 

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 Monitor: John V. Massingdale 

Greenwich Chemical Consulting, Inc. 

Quality Assurance: Harlan Laboratories Ltd. 

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 Study Monitor. 

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 

A GLP compliant report will be presented. A draft report will be sent to the Study Monitor for 

review and comments before issue of the final report. 
Harlan Study Number: 41401172 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. 
Harlan Study Number: 41401172 Study Plan Page 7 

1 INTRODUCTION AND PURPOSE 

The purpose of this study is to evaluate the partition coefficient (n-octanol/water) of the test 

item. 

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) 

Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

1.1 Guidelines I Regulations 

The 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. 

Where testing is conducted to satisfy the requirements of specific guidelines and/or regulations, 

this will be documented and reflected in the final report. 
Harlan Study Number: 41401172 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 I retest date, storage conditions and safety 

precautions to be taken. 

Information as provided by the Sponsor. 

Identification: 

2.2 Reference Item 

D-erythro-hex-2-enonic acid, .gamma.-lactone, 

monosodium salt [CAS RN 6381-77-7] 

Reference items, if used, will be identified in the individual test sections. 
Harlan Study Number: 41401172 Study Plan Page 9 

3 TEST 

3.1 Partition Coefficient (n-OctanoVWater) 

The purpose of this test is to evaluate the partition coefficient (Pow) between n-octanol and water 

for the test item. 

As the test item is a salt, testing will be performed by the shake-flask method (as indicated by 

test guidance). 

3.1.1 Test Specific Guidelines I Regulations 

The procedure outlined in this document has been designed to be compatible with the following 

guidelines and recommendations: 

• OECD 107, 27 July 1995 

3.1.2 Test System 

The test system consists of immiscible aqueous and n-octanol phases in the same vessel with a 

defined amount of test item pre-dissolved in the appropriate phase. Partition of the test item 

between both phases will be achieved by inversion of the mixture. 

The justification for the selection of the test system and test method ts that they are 

recommended by international guidelines. 

3.1.3 Principle of the Test 

In principle, the log Pow can be determined by the shake flask method (log Pow between -2 and 4 ). 

Limitations of the method are described in the relevant guidelines. 

3.1.4 Performance of the Test 

A preliminary estimate of the partition coefficient of the test item will be generated. This may 

be obtained from the ratio of the approximate solubilities of the test item in n-octanol and water 

(by visual assessment) or calculated using appropriate modeling software. 

In order to determine a partition coefficient, equilibrium between all interacting components of 

the system must be achieved, and the equilibrium concentrations of the items dissolved in the 

two phases must be determined. 
Harlan Study Number: 41401172 Study Plan Page 10 

Before the partition coefficient can be determined, the two individual phases will be 

pre-saturated by shaking both n-octanol and water with a sufficient quantity of the opposite 

phase. A stock solution of the test item will be prepared in either n-octanol saturated water or 

water saturated n-octanol. The actual mass concentration of this stock solution will be 

determined using a suitable analytical method. 

Three ratios of the two phases will be carried out, each in duplicate, as detailed in the method 

guidelines. Ratio selection will be based on the characteristics of the test item and analytical 

methodology, but will contain both twice and half the n-octanol to water ratio of the middle 

ratio. 

The test solutions will be partitioned and phase separation performed by centrifugation before 

analysis to determine the concentration of the test item in the initial stock solution and in both 

phases. The total quantity of test item present in both phases will be calculated and compared 

with the quantity of the test item originally introduced (derived from the analyzed stock solution 

concentration). 

3.1.5 Calculation 

The partition coefficient (Pow) is defined as the ratio of the equilibrium concentrations (c) of a 

dissolved test item in a two-phase system consisting of two largely immiscible solvents. The 

partition coefficient therefore is the quotient of two concentrations (no dimension) and is usually 

given in the form of its logarithm to base ten (log pow). 

All observations which may be used to interpret the results of the tests will be documented. 

Where appropriate, mean values and standard deviation will be calculated and linear regression 

analysis (least square method) will be applied. 

Calculations performed will be presented in the study report. 
c 

STUDY SPECIFIC SUPPLEMENT 

To General Study Plan: 920-30 

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Assessment of Ready Biodegradability; C02 Evolution Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

Proposed Schedule: 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

NBest 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Univar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL 33410 

UNITED STATES OF AMERICA 

41401177 

31 May 2014 

30 June 2014 

31 July 2014 

Page 1 of4 
Harlan Study Number: 41401177 Study Specific Supplement Page 2 

Test Item 

Information as provided by the Sponsor. 

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7] 

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) 

Section 4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

Special Conditions 

Study specific parameters: 

GSP Section 1.1 Guidelines I Regulations: 

The study will be conducted according to the following internationally accepted guidelines and 

recommendations: 

• OECD Guidelines for Testing of Chemicals (1992) No 301B "Ready Biodegradability: C02 

Evolution Test" 

• Method C.4-C of Commission Regulation (EC) No. 440/2008 

• US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 (Paragraph 

(m)) 

GSP Section 3.2.1 Inoculum: 

The study will be conducted using aerobic activated sewage sludge from Loughborough 

sewage treatment works. 

GENERAL INFORMATION 

Distribution List 

The original study specific supplement will be retained in the study file. Copies of the study 

specific supplement and general study plan along with any amendments will be distributed as 

follows: 

For the Sponsor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK 
Harlan Study Number: 41401177 Study Specific Supplement Page 3 
Harlan Study Number: 41401177 

SIGNATURES 

Study Director: 

Test Facility 

Management: 

For the Sponsor: 

Study Specific Supplement Page4 

NBest 

Date: 

CMead 

Date: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Date: 

Note: These signatures also acknowledge the procedures described in the corresponding General 

Study Plan. 
GENERAL STUDY PLAN: 920~30 

harlan® 

... 

Assessment of Ready Biodegradability; C02 Evolution Test 

Test Facility: 

Signatures: 

Author: 

Test Facility Management: 

Quality Assurance: 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

N Clarke BSc 

.~ ......................... . Date: 2 9 AUG 2012 

J G Riley BSc (Hons) MRQA 

..... 2..~!.~ .................. . Date: 0 5 SEP 2012 

Page 1 of17 
General Study Plan: 920-30 Page2 

CONTENTS 

GOOD LABORATORY PRACTICE ............................................................................................. 3 

GENERAL INFORMATION ......................................................................................................... 4 

Study Plan Amendments and Deviations ............................................................................... 4 

Quality Assurance .................................................................................................................. 4 

Reporting ............................................................................................................................... 4 

Archiving ............................................................................................................................... 4 

1 INTRODUCTION AND PURPOSE ..................................................................................... 5 

1.1 Guidelines I Regulations .......................................................................................... 5 

2 TEST AND REFERENCE ITEMS ....................................................................................... 6 

2.1 Test Item .................................................................................................................. 6 

2.2 Reference Item ......................................................................................................... 6 

3 MATERIALS AND METHODS ........................................................................................... 6 

3.1 Definitions and Abbreviations ................................................................................. 6 

3.2 Test System .............................................................................................................. 7 

3.2.1 Inoculmn .................................................................................................... 7 

3.2.2 Justification ................................................................................................ 7 

3.2.3 Preparation ofinoculmn ............................................................................ 7 

3.3 Mineral Medimn ...................................................................................................... 8 

3.4 Dosage ..................................................................................................................... 8 

3 .4.1 Test Item Preparation ................................................................................ 8 

3 .4.2 Reference Item Preparation ....................................................................... 8 

3.5 Materials .................................................................................................................. 8 

3.6 Experimental Conditions ......................................................................................... 8 

3. 7 Study Design ............................................................................................................ 9 

3. 7.1 Test Item Concentration ............................................................................ 9 

3.7.2 Nmnber ofVessels .................................................................................. 10 

3.7.3 Procedure ................................................................................................. lO 

3.8 Evaluations ............................................................................................................ 11 

3 .8.1 Observations ............................................................................................ 11 

3 .8.2 IC Analysis .............................................................................................. 11 

3.8.3 IC/TC Ratio ............................................................................................. 11 

3.8.4 Analyzers ................................................................................................. 12 

3.9 Data Evaluation ..................................................................................................... 12 

3.9.1 Percentage Carbon Content ..................................................................... 12 

3.9.2 Percentage Biodegradation ...................................................................... 12 

3.9.3 Total C02 Evolution ................................................................................ 13 

3.10 Validity Criteria ..................................................................................................... 13 

4 REFERENCES .................................................................................................................... 14 

APPENDICES ............................................................................................................................... 15 

Appendix I Mineral Medium ............................................................................................................... 15 

Appendix 2 Methods ofDissolving!Dispersing Test Item in the Test System ..................................... l6 
General Study Plan: 920-30 Page 3 

GOOD LABORATORY PRACTICE 

The associated 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) 17); 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 (MHL W, MAFF and METI). 
General Study Plan: 920-30 Page4 

GENERAL INFORMATION 

Study Plan Amendments and Deviations 

Any intended change to the study plan (general study plan and study specific supplement) 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 (general study plan and study specific 

supplement) 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 

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. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), 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. 
General Study Plan: 920-30 PageS 

1 INTRODUCTION AND PURPOSE 

The purpose of this study is to determine the ready biodegradability of the test item in a 

biodegradation test by following carbon dioxide (C02) evolution of the test item by means of 

inorganic carbon analysis over a period of usually 28 days when exposed to sewage sludge 

micro-organisms under defined conditions. 

The results of the study are of value in the assessment of the persistence of the test item in the 

aquatic environment. 

1.1 Guidelines I Regulations 

The 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 (1992) No 301B "Ready Biodegradability: 

C02 Evolution Test" 

• Method C.4-C of Commission Regulation (EC) No. 440/2008 

• US EPA Fate, Transport and Transformation Test Guidelines OCSPP 835.3110 

(Paragraph (m)) 
General Study Plan: 920-30 Page6 

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 I retest date, storage conditions and safety 

precautions to be taken. 

Where the test item is administered in a vehicle, and the homogeneity, concentration or stability 

of the test item in that vehicle is not determined an impact assessment will be made by the Study 

Director, this will be reflected in the final report and an exception will be included in the GLP 

compliance statement. 

2.2 Reference Item 

Sodium benzoate, at a concentration of 10 to 20 mg organic carbon/L (equivalent to 

17.1 to 34.3 mg/L), will be included in the study. 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Readily biodegradable: 

1 0-Day window: 

Inorganic carbon (IC): 

Total carbon (TC): 

Total organic carbon (TOC): 

Dissolved organic carbon 

(DOC): 

Test items may be considered to be readily biodegradable if 

~ 60% biodegradation is attained in a 10-Day window within 

the 28 days of the test. 

The 10 days immediately following the attainment of 10% 

biodegradation. 

Carbon derived from inorganic sources e.g. carbon dioxide, 

carbonic acid, and carbonate. 

This is the sum of the organic carbon and the inorganic carbon 

present in a sample. 

This is the sum of the organic carbon in solution and in 

suspension. 

Dissolved organic carbon is the organic carbon present in 

solution or that which passes through a 0.45 J.Lm filter or 

remains in the supernatant after centrifuging at approximately 

4000 g for 15 minutes. 
General Study Plan: 920-30 Page 7 

3.2 Test System 

The test method and test system are consistent with the testing guidelines and should provide a 

rational basis to assess the ready biodegradability of the test item under aerobic conditions. 

3.2.1 Inoculum 

The study will be performed with aerobic activated sewage sludge from the aeration stage of a 

local wastewater treatment plant, which treats predominately domestic sewage. Details of the 

source of inoculum to be used in the study will be specified under the special conditions section 

of the study plan and documented in the data. 

3.2.2 Justification 

Sewage sludge/treatment micro-organisms have been selected following the recommendations of 

OECD Guideline No 301, Commission Regulation (EC) No. 440/2008 and US EPA Fate, 

Transport, and Transformation Test Guidelines OCSPP 835.3110. 

Pre-adaptation of the inoculum to the test item is not permitted. 

3.2.3 Preparation of Inoculum 

The aerobic activated sewage sludge will be washed by centrifugation or settlement and 

resuspension. The supernatant (liquid phase) will be decanted and the solid material 

resuspended in tap water or mineral medium. If necessary, this procedure will be repeated but 

the fmal wash will be in mineral medium. An aliquot of the fmal sludge suspension (this may be 

homogenized) will be weighed, thereafter dried and the concentration of suspended solids will be 

calculated. 

Based on this ratio, calculated amounts of wet sludge may be suspended in mineral medium to 

obtain a concentration equivalent to 4 g (±10%) dry material per liter. 

The inoculum is maintained on aeration prior to use and may be pre-conditioned to experimental 

conditions for up to 7 days by aeration at test temperature. Prior to use, the sludge may be 

diluted with mineral medium to a concentration of 1 g dry material per liter. Defined volumes of 

this diluted activated sludge will be added to each test vessel to obtain a final concentration of 

30 mg dry material per liter. 

Preparation and pre-conditioning of the inoculum may be performed with similar concurrent 

studies. However, the raw data, copies of the raw data or transcribed data of this work will be 

included in the study data file. 
General Study Plan: 920-30 Page 8 

3.3 Mineral Medium 

The test will be conducted using a defined mineral medium (see Appendix 1). The mineral 

medium is considered not to contain any contaminant that would affect the integrity or outcome 

ofthe study. 

3.4 Dosage 

3.4.1 Test Item Preparation 

If necessary, pre-experiments will be performed to determine the solubility/dispersibility of the 

test item in the test system. According to the fmdings of these pre-experiments, appropriate 

procedures will be chosen to prepare the test media (typical methods are summarized in 

Appendix 2). 

The test item will be dissolved/dispersed in a defined mineral medium once at the start of the test. 

If auxiliary solvents, surfactants or inert carriers are used to aid in the dispersion, they should not 

be toxic to bacteria and must not be biodegradable or cause foaming under the test conditions. 

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

3.4.2 Reference Item Preparation 

The reference item will be dissolved in the mineral medium once at the start of the test and 

added to the test vessels via a concentrated stock solution. 

3.5 Materials 

Glass test vessels (5 liters) containing 3 liters oftest preparation will be used. The test vessels 

will be labeled with the study number and all necessary information to ensure unique 

identification. 

3.6 Experimental Conditions 

Test duration: Normally 28 days. At the discretion of the Study Director the 

test may be terminated before Day 28 if three consecutive 

determinations confirm complete degradation. With the 

Sponsor's approval and depending upon the results obtained, 

the test may be extended for a further 2 to 3 weeks. In such a 

case the test item could not be classed as readily biodegradable. 

Any extension would be subject to an additional cost. The test 

dates will be documented in the data. 
General Study Plan: 920-30 

Light conditions: 

Test temperature: 

Aeration: 

COz absorption: 

pH: 

3. 7 Study Design 

Page9 

In darkness. 

The test will be conducted at a temperature of 22 ± 2°C. The 

room temperature will be recorded throughout the test at least 

on each working day. Additionally, the temperature may be 

checked on each sampling occasion in a separate vessel 

containing water and incubated under the same conditions as 

the test vessels. 

COz-free air is bubbled through the solution in each test vessel 

at a rate of 30-100 mL/min per vessel and stirred continuously 

by magnetic stirrers to prevent settlement of the sludge. COzfree air will be produced by passing compressed air through a 

glass column containing self-indicating soda lime granules 

(Carbosorb~ or the air will be passed through a bottle, 

containing about 750 mL of a 2 M NaOH solution to trap COz. 

One empty 500 mL Dreschel bottle is placed in the com~ressed 

air line prior to passage of the air through the Carbosorb to act 

as a moisture trap. 

The C02 produced by the culture vessels will be collected in 

2 x 500 mL Dreschel bottles containing a suitable volume of 

0.05 MNaOH. 

Prior to test start (Day 0), the pH will be measured in each test 

vessel after the addition of the test or reference item, and, if 

necessary, adjusted to pH 7.4 ± 0.2 using a diluted hydrochloric 

acid or sodium hydroxide solution. The pH will be measured 

again in each vessel at the end of incubation. 

3.7.1 Test Item Concentration 

10 to 20 mg organic carbon/L, in duplicate. The total organic carbon (TOC) content of the test 

item will be calculated based on the information (molecular formula or carbon content) provided 

by the Sponsor or specific measurement (e.g. DOC analysis). 

Lower test concentrations may be employed if the test item is toxic to sewage sludge microorganisms. The actual concentration used will be documented in the data. If a repeat test is 

required due to the test item toxicity, this will be subject to an additional cost. The test item can 

be assumed to be not inhibitory if in the toxicity control more than 25% degradation (based on 

total mg TOC added) occurs within 14 days. 
General Study Plan: 920-30 Page 10 

3.7.2 Number of Vessels 

The following vessels will be prepared: 

• 2 test item vessels, containing the test item (1 0 to 20 mg organic carbon/L) in 

inoculated mineral medium. 

• 2 inoculum control vessels, containing inoculated mineral medium alone (blank). 

• 2 procedure control vessels, containing the reference item, sodium benzoate 

(10 to 20 mg organic carbon/L), in inoculated mineral medium. 

• 1 toxicity control vessel, containing inoculated mineral medium, the reference item, 

sodium benzoate (10 to 20 mg organic carbon/L) and the test item (10 to 20 mg organic 

carbon/1). 

Experience has shown that an abiotic control is not required. The abiotic control is designed to 

show chemical degradation of the test item and it is considered that C02 would not be produced 

from such degradation and hence the abiotic control is omitted. 

Should an abiotic control be required a vessel will be prepared containing the test item in noninoculated mineral media and a sterilizing agent to prevent microbial growth (e.g. 0.1 giL 

sodium azide or 10 mg/L HgCh). This will only be performed at the request of the Sponsor and 

at an additional cost. 

If auxiliary solvents, surfactarits or inert carriers are used to aid in the dispersion of the test item, 

the control and reference item vessels will be prepared containing an identical concentration of 

auxiliary solvent, surfactant or inert carrier as in the test item vessels. 

Data from the control and reference group may be shared with similar concurrent studies. 

However, the raw data, copies of the raw data or transcribed data of this work will be included in 

the study data file. 

3. 7.3 Procedure 

Approximately 24 hours prior to the start of the study, to each 5 liter vessel, typically 2400 mL 

of mineral medium will be added and an appropriate volume of the activated sewage sludge 

inoculum to give 30 mg ss/1 in the final volume of 3 liters. The test media will be aerated 

overnight with co2..:free air to purge the system of carbon dioxide. 

On the following day (Day 0), the test item and/or reference item will be added to the relevant 

vessels and the volume in all flasks made up to 3 liters by the addition of mineral medium 

(which was overnight purged with C02-free air). The inoculum control (blank) will be prepared 

in a similar manner with the omission of test and reference items. Two absorber flasks, 

containing a suitable volume of 0.05 M NaOH which will be documented in the data, will be 

connected in series to the exit air line of each test flask. 
General Study Plan: 920-30 Page 11 

On Day 28 (after pH measurement), 1 mL of concentrated HCl will be added to each test flask. 

Then the flasks will be aerated overnight to drive off any residual/dissolved C02 present in the 

test suspensions. On Day 29, a sample of both absorber flasks will be withdrawn and analyzed 

for IC to determine residual C02 remaining in the 3 liter test suspensions. Samples of the second 

NaOH absorber flask will be analyzed to correct for any carryover of C02. 

3.8 Evaluations 

3.8.1 Observations 

The appearance of the test preparations will be observed on Day 0 and then periodically 

throughout the test and, if relevant, changes in the appearance will be recorded. 

3.8.2 IC Analysis 

Samples (the exact volume will be recorded in the data) are taken from the first C02 absorber 

vessel for IC analysis on Day 0 and typically every second or third day during the first 14 days 

(to cover the 10-day window) and on Days 21, 28 and 29 to give a total of nine sampling 

occasions. 

Additional sampling days may be included if required for identification of the 10-Day window. 

The volume of sample removed is recorded at each sampling time. On the 29th day the last 

analysis of evolved carbon dioxide is made. The second absorber vessel is sampled on Day 0 and 

Day 29 only in order to correct for any carryover of carbon dioxide. 

Each sample is subjected to a direct analysis of the evolved C02 in triplicate. Depending on the 

degradability of the test item, an additional sample from the second absorber flask may be 

withdrawn at an earlier time point during the exposure period. 

Samples are stored in glass containers and are either analyzed immediately or stored at 

approximately -20°C. 

3.8.3 IC/TC Ratio 

For soluble test items, in order to calculate the IC/TC ratio, samples (the exact volume will be 

recorded in the data) will be removed from the test item and inoculum control vessels after 

making up to the final volume of 3 liters. All samples will be either filtered through a 0.45 ~-tm 

filter (an initial volume discarded, details to be recorded in the data) or for those test items which 

show significant adsorption to glassware, fllters and/or activated sewage sludge, centrifuged 

(approximately 4000 g, 15 min), prior to analysis. 
General Study Plan: 920-30 Page 12 

In order to enable calculation of the IC/TC ratio in the test item preparations for insoluble test 

items, samples (the exact volume will be recorded in the data) will be removed from the test item 

prior to dosing and the inoculum control vessels after preparation. All samples will be either 

filtered through a 0.45 f.Lm filter (an initial volume discarded, details to be recorded in the data) 

or centrifuged (approximately 4000 g, 15 minutes), prior to analysis. The nominal TC 

contribution of the test item, e.g. 10 mg C/L, will be added to that obtained from TC analysis for 

the test item before calculation of the IC/TC ratio. 

3.8.4 Analyzers 

All samples will be analyzed using a TOC infrared gas analyzer equipped with an automatic 

sampler for use if required. For calibration of the TOC analyzer, a standard sample is measured 

at the start or at the end of each run to prove conformity with the calibration curve. Details of 

the analyzer used and the calibrations performed will be documented in the data. 

3.9 Data Evaluation 

3.9.1 Percentage Carbon Content 

The theoretical amount of carbon present in the test item is calculated as follows: 

No of C atoms x mol wt of C xlOO% 

mol wt of test item 

Where the molecular weight of the test item is not known, the TOC present as test chemical 

maybe derived from calculation or specific measurement. 

3.9.2 Percentage Biodegradation 

The amount of C02 produced, expressed as IC (mg), is given by multiplying the concentration of 

IC in the NaOH absorbent by the volume of the absorbent (this will be different on each 

sampling occasion due to the removal of sample). 

The amount of carbon dioxide produced must be corrected for endogenous carbon dioxide 

production and is, therefore, determined by the difference in carbon dioxide content between the 

experimental and control absorber vessels. 

The percentage degradation based on C02 evolution is calculated from: 

OJ' d d . * mg IC from test flask- mg lC from COntrOl flask } "o egra at10n = . x 00 

mg TOC added as test chemical 

• The conversion factor for carbon to carbon dioxide is 3.67 
General Study Plan: 920-30 Page 13 

The volume obtained on the day of measurement is a cumulative value and no additions are 

required. 

The amount of IC present in the second absorber vessels is also taken into consideration when 

calculating the degradation values for the vessels. 

3.9.3 Total C02 Evolution 

The total C02 evolution in the control vessels at the end of the test is calculated from the 

equation below. The inorganic carbon values for replicates R1 and R2 on Day 28 are meaned 

before substitution into the equation. 

Total C02 evolution=mgiC in control x 

100 

x--

1

-- %C of C02 test volume 

. 100 1 

= mgiC mcontrol x -- x27.29 3 

3.10 Validity Criteria 

Test items giving a result of ~60% yield of C02 (within 28 days) should be regarded as readily 

biodegradable. This level must be reached within 10 days of biodegradation exceeding 10%. 

The test is considered valid if the difference of the extremes of replicate values of production of 

C02 at the time the plateau is reached, at the end of the test or at the end of the 10-Day window, 

as appropriate, is less than 20%. 

The results of the degradation test are considered valid if in the same test the reference item 

yields~ 60% biodegradation (in a 10-Day window) by Day 14. 

The toxicity control should attain ~ 25% degradation by Day 14 for the test item to be considered 

as non-inhibitory. 

The total C02 evolution in the control vessels at the end of the test should not normally exceed 

40 mg/L medium(= 120 mg/3 liters, corresponding to 33 mg C per flask), however values up to 

70 mg/L are acceptable. Data from studies where values in excess of 70 mg/L are obtained 

should be critically examined. 

The IC content of the test item in the mineral medium at the beginning of the test should be <5% 

of the TC. 
·. 

General Study Plan: 920-30 Page 14 

4 REFERENCES 

HANDLEY, J. W., MEAD, C., RAUSINA, G. A., W AID, L. J., GEE, J. C. and HERRON, S. J. 

(2002) The Use of Inert Carriers in Regulatory Biodegradation Tests of Low Density Poorly 

Water-soluble Substances. Chemosphere, 48, pp. 529-34. 

INTERNATIONAL STANDARDS ORGANISATION (ISO) (1995) Standard 10634: Water 

quality - Guidance for the preparation and treatment of poorly water-soluble organic 

compounds for the subsequent evaluation of their biodegradability in an aqueous medium. 

Geneva: International Standards Institute. 
--"··--~··-··-- ... ------------···--···-------········--···----·-------·-· ··--·-------···-----·····----------·····-·····----·--····· ., 

General Study Plan: 920-30 Page 15 

APPENDICES 

Appendix 1 Mineral Medium 

The mineral medium will be prepared according to the testing guidelines using purified water*. 

Analytical grade salts will be dissolved in purified water to obtain the following stock solutions: 

i) Stock Solutions 

(a) KH2P04 8.50 giL 

KzHP04 21.75 g/L 

NazHP04.2H20 33.40 giL 

~Cl 0.50g/L 

The pH of this solution should be 7.4 

(b) CaCh 

or CaC1z.2H20 

27.50 giL 

36.40 giL 

22.50 giL 

(d) FeCh.6H20 0.25 giL 

(In order to avoid having to prepare solution (d) immediately before use add one drop 

of concentrated HCl per liter as a preservative. 

ii) Preparation 

To 1 liter (fmal volume) of purified water* are added the following volumes of solutions (a) 

to (d). 

10 ml of Solution (a) 

1 ml of Solution (b) 

1 ml of Solution (c) 

1 ml of Solution (d) 

iii) Storage 

Stock solutions (a) to (d) are stored at approximately 4°C and are stable for a period of 1 year. 

Ion exchange and reverse osmosis water treated tap water (Elga Optima 15+, Elga Purelab Option R-15 or Elga 

Purelab Option R-15 BP) 
General Study Plan: 920-30 Page 16 

Appendix 2 Methods of Dissolving/Dispersing Test Item in the Test System 

Following the recommendations of the International Standards Organisation (ISO, 1995) and in 

the published literature (Handley et al, 2002) typical methods of preparation that may be 

employed in the biodegradation test are summarized below. 

Readily Water Soluble Items: A concentrated aqueous stock solution of the test item will be 

prepared in reverse osmosis purified deionized water or mineral medium and a suitable aliquot 

added to inoculated mineral medium to give the required test concentration. 

In order to prepare an aqueous stock solution, dissolution methods such as ultrasonication, high 

shear mixing and prolonged stirring/shaking may be used. 

Low Water Solubility Items: In order to aid dispersion of the test item in the test system and 

increase the potential for biodegradation to occur the following methods may be used: 

Ultrasonication: The required amount of test item will be dispersed in an aliquot of mineral 

medium with the aid of ultrasonication prior to dispersal in inoculated mineral medium to give 

the required test concentration. 

High Shear Mixing: The required amount of test item will be dispersed in an aliquot of mineral 

medium with the aid of high shear mixing prior to dispersal in inoculated mineral medium to 

give the required test concentration. 

Adsorption onto Silica Gel: The required amount of test item will be adsorbed onto granular 

silica gel. The test item/silica gel combination will then be dispersed in an aliquot of mineral 

medium prior to dispersal in inoculated mineral medium to give the required test concentration. 

When dispersing the test item/silica gel combination in the aliquot of mineral medium, shaking 

by hand, ultrasonication or high shear mixing may be used to aid dispersion. 

Adsorption onto Filter Paper: The required amount of test item will be adsorbed onto glassfibre 

filter paper. The test item/filter paper combination will then be added into inoculated mineral 

medium to give the required test concentration. When adsorbing the test item onto the glassfibre 

filter paper efforts should be made to spread the test item across the surface of the filter paper in 

order to maximize the surface area that will be exposed to the micro-organisms present in the test 

system. 

Addition onto Glass Slide: The required amount of test item will be weighed directly onto a glass 

microscope slide or coverslip. The slide/coverslip carrying the test item will then be added into 

inoculated minerale medium to give the required test concentration. When weighing the test 

item onto the slide/coverslip efforts should be made to spread the test item across the surface in 

order to maximize the surface area that will be exposed to the micro-organisms present in the test 

system. 
'· 

General Study Plan: 920-30 Page 17 

Preliminary Solution in a Volatile Solvent: A stock solution of the test item may be prepared in a 

suitable solvent e.g. acetone, diethyl ether, chloroform. A suitable aliquot of this solvent stock 

solution will then be dispensed onto a glassfibre filter paper and the solvent allowed to evaporate 

to dryness prior to addition of the filter paper containing the test item to inoculated mineral 

medium to give the required test concentration. As with the adsorption onto filter paper method, 

efforts should be made during addition of the solvent stock solution to the filter paper to spread 

the test item across the surface of the filter paper to maximize its surface area. 

Care should be taken when using this method of addition of the test item to ensure that all traces 

of solvent are removed prior to addition of the filter paper to the test vessel as trace amounts of 

solvent can cause inhibitory effects or affect percentage biodegradation values. 

Alternatively an aliquot of the solvent stock solution can be dispensed to an empty test vessel 

and the solvent allowed to evaporate to dryness prior to addition of inoculated mineral medium 

to give the required test concentrations. Again care should be taken to ensure that all traces of 

solvent are removed from the test vessel prior to addition of the inoculated mineral medium. 

A solvent stock solution of the test item can also be dispensed onto silica gel and the solvent 

allowed to evaporate to dryness by passing a steady stream of compressed air (0.5- 1.0 L /min) 

over the solvent stock solution/silica gel. In order to ensure the solvent has been removed, the 

test item coated silica gel can be placed in an oven over night (temperature of oven to be advised 

by Study Director). The test item coated silica gel may be stored in a labeled glass container 

prior to use in the study. 

Preliminary Solution in a Non-Volatile, Non-Degradable Solvent: A stock solution of the test 

item may be prepared in a suitable solvent e.g. silicone oil (Polydimethylsiloxane ). An aliquot 

of this solvent stock solution will then be dispensed into inoculated mineral medium to give the 

required test concentration. 
STUDY SPECIFIC SUPPLEMENT 

To General Study Plan: 902-21 

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Acute Toxicity to Rainbow Trout 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

Proposed Schedule: 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

D Sacker 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

U nivar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL 33410 

UNITED STATES OF AMERICA 

41401173 

30 April 2014 

31 May 2014 

30 June 2014 

Page 1 of 4 
Harlan Study Number: 41401173 Study Specific Supplement Page 2 

Test Item 

Information as provided by the Sponsor. 

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7] 

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 

4 Test Rule and the regulations set forth under 40 CPR 799.5087. 

Special Conditions 

Study specific parameters: 

GSP Section 1.1 Guidelines I Regulations: 

The study will be conducted according to the following internationally accepted guidelines and 

recommendations: 

• OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish Acute Toxicity Test" 

GSP Section 3.2 Test System: 

The study will be conducted using Rainbow Trout (Oncorhynchus my kiss) 

GSP Section 3.3 Test Water: 

The study will be conducted using reconstituted water 

GSP Section 3.7 Study Design: 

The study will be conducted using 7 fish per test concentration. 

Any pH adjustments of the exposure solutions will be conducted according to section 14 of 

OECD 203 details of which will be included in the final test report. 

If measured concentrations are ± 20% of the nominal concentrations, results will be based on 

nominal concentrations. If measured concentrations are not ± 20% of nominal concentrations, 

results will be based on geometric mean measured concentrations. 

The study report will provide a detailed description of the analytical method that will be 

developed prior to and utilized during the test. 

All elements described in item 23 of OECD 203 will be included in the final test report. 

Particulate matter concentration, chemical oxygen demand (COD), total organic carbon 
Harlan Study Number: 41401173 Study Specific Supplement Page 3 

(TOC), hardness, alkalinity and conductivity of the dilution water will be determined at the 

start of the study. 

GENERAL INFORMATION 

Distribution List 

The original study specific supplement will be retained in the study file. Copies of the study 

specific supplement and general study plan along with any amendments will be distributed as 

follows: 

For the Sponsor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK 
Harlan Study Number: 41401173 

SIGNATURES 

Study Director: 

Test Facility 

Management: 

For the Sponsor: 

Study Specific Supplement Page 4 

D Sacker 

Date: 

CMead 

Date: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Date: 

Note: These signatures also acknowledge the procedures described in the corresponding General 

Study Plan. 
GENERAL STUDY PLAN: 902-21 

Acute Toxicity to Fish 

Test Facility: 

Signatures: 

Author: 

Test Facility Management: 

Quality Assurance: 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shard! ow 

Derbyshire 

DE722GD 

UK 

H VryenhoefBSc 

o~~,Lzo,f········· 

~712 ........................ 

Date: 1 ·1· SEP 2012 

J G Riley BSc (Hons) MRQA 

Date: 1. 2 SEP 2012 

Page 1 of 16 
General Study Plan: 902-21 Page2 

CONTENTS 

GOOD LABORATORY PRACTICE ............................................................................................. 3 

GENERAL INFORMATION ......................................................................................................... 4 

Animal Welfare ...................................................................................................................... 4 

Study Plan Amendments and Deviations ............................................................................... 4 

Quality Assurance .................................................................................................................. 4 

Reporting ............................................................................................................................... 4 

Archiving ............................................................................................................................... 4 

1 INTRODUCTION AND PURPOSE ..................................................................................... 6 

1.1 Guidelines I Regulations .......................................................................................... 6 

2 TEST AND REFERENCE ITEMS ....................................................................................... 7 

2.1 Test Item .................................................................................................................. 7 

2.2 Reference Item ......................................................................................................... 7 

3 MATERIALS AND METHODS ........................................................................................... 7 

3.1 Definitions and Abbreviations ................................................................................. 7 

3.2 Test System .............................................................................................................. 7 

3.3 Test Water ................................................................................................................ 8 

3.4 Dosage ..................................................................................................................... 8 

3.5 Materials .................................................................................................................. 9 

3.6 Experimental Conditions ......................................................................................... 9 

3. 7 Study Design ............................................................................................................ 9 

3.8 Evaluations ............................................................................................................ 11 

3.8.1 Test organism observations ..................................................................... 11 

3.8.2 Water quality criteria ............................................................................... 11 

3.8.3 Analysis of Test Item Concentrations ..................................................... 12 

3.9 Statistical Analysis ................................................................................................ 13 

3.10 Validity Criteria ..................................................................................................... 13 

4 REFERENCES .................................................................................................................... 14 

APPENDICES ............................................................................................................................... IS 

Appendix 1 Recommended Fish Species .............................................................................................. 15 

Appendix 2 Test Water ......................................................................................................................... 16 
General Study Plan: 902-21 Page3 

GOOD LABORATORY PRACTICE 

The associated 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) 17); 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 (MHL W, MAFF and METI). 
General Study Plan: 902-21 Page4 

GENERAL INFORMATION 

Animal Welfare 

The study is designed and will be conducted to cause the minimum suffering or distress to the 

animals consistent with the scientific objectives and in accordance with the Harlan Laboratories 

Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's 

Animals (Scientific Procedure) Act 1986. This general study plan is subject to review and the 

conduct of the study may be included as part of the Harlan Laboratories Ltd, Shardlow, UK 

Ethical Review Process. 

The associated study will also be conducted in accordance with the UK Home Office Guidance 

document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance 

document on recognition, assessment and use of clinical signs as humane endpoints for 

experimental animals used in safety evaluation. 

Study Plan Amendments and Deviations 

Any intended change to the study plan (general study plan and study specific supplement) 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 (general study plan and study specific 

supplement) 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 

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

and comments before issue of the fmal report. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), 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. 
General Study Plan: 902-21 Page 5 

·---~-·----------------------

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. 
General Study Plan: 902-21 Page6 

----------------------------.. --.. --.. ---.. --..... ----

1 INTRODUCTION AND PURPOSE 

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

toxicity will be expressed as the median lethal concentration (LC50) or median lethal loading rate 

(LLso). 

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) 

and the Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL) will also be determined if applicable. 

The species used will be a freshwater species representative 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 I Regulations 

The test methods described are designed to be compatible with the procedures indicated by the 

following internationally accepted guidelines and recommendations: 

• The OECD Guidelines for Testing of Chemicals (1992) No 203 "Fish, Acute Toxicity 

Test" 

• Method C.1 of Commission Regulation (EC) No. 440/2008 

• JMAFF Test Guidelines 2-7-1-1 (2000) 
General Study Plan: 902-21 Page7 

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 I retest date, storage conditions and safety 

precautions to be taken. 

Where the test item is administered in a vehicle, and the homogeneity, concentration or stability 

of the test item in that vehicle is not determined an impact assessment will be made by the Study 

Director, this will be reflected in the final report and an exception will be included in the GLP 

compliance statement. 

2.2 Reference Item 

In the interest of animal welfare, a positive control using a reference item will not be performed. 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

LCsoiLLso: 

LOEC (Lowest Observed 

Effect Concentration) or LOEL 

(Lowest Observed Effect 

Loading rate): 

NOEC (No Observed Effect 

Concentration) or NOEL (No 

Observed Effect Loading rate): 

Treatment: 

3.2 Test System 

The calculated concentration or loading rate of test item which 

results in 50% mortality within the exposure period. 

The lowest test concentration or loading rate at which a 

significant toxic effect on the test organisms is observed within 

the exposure period. 

The highest test concentration or loading rate at which no 

significant toxic effect on the test organisms is observed within 

the exposure period. 

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

solvent control (if applicable). 

The study will be performed using juvenile fish supplied by an accredited live fish supplier, 

details of which will be documented in the data, or from in-house breeding cultures. The species 

of fish used will be selected depending on the test guideline to be followed and will be 

documented on the study specific supplement. Recommended species, along with recommended 

temperature range and test length range for each species, are given in Appendix 1. 
General Study Plan: 902-21 Page 8 

Following a 48-Hour settling in period, mortalities are recorded and the following rejection 

criteria applied. 

> 10% in 7 days: 

5-10% in 7 days: 

< 5% in 7 days: 

rejection of entire batch 

continue acclimatisation for an additional 7 days 

acceptance of batch 

The fish will be acclimatised for a minimum period of 12 days at Harlan Laboratories Ltd, with 

at least 7 days acclimatisation prior to test start in water of the quality and temperature to be used 

in the test. 

The fish will be fed commercial fish food, details of which will be documented in the data, 

regularly on demand. Feeding will be discontinued 24 hours prior to testing. The diet and 

diluent water are considered not to contain any contaminant that would affect the integrity or 

outcome of the study. 

Animals will be maintained in glass or fibreglass tanks at a loading rate not exceeding 0.8 g body 

weight per liter in static systems. This loading rate may be exceeded in flow-through systems. 

Aeration will be supplied to provide ;;:: 80% Air Saturation Value (ASV). 

3.3 Test Water 

The animals will be maintained in either reconstituted test water, local non chlorinated well 

water of drinking quality or dechlorinated laboratory tap water (see Appendix 2). 

3.4 Dosage 

If necessary, pre-experiments will be performed to determine the solubility and stability of the 

test item in the test water. According to the findings of these pre-experiments, appropriate 

procedures will be chosen to prepare the test media. 

The test item will be dissolved or dispersed in the test water once at the start of the test (static 

test), daily (semi-static test) or continuously (dynamic test) depending on the stability of the test 

item in the test water. 

Methods such as prolonged mixing, ultrasonication, high shear force stirring or the production of 

Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the 

nature of the test item. If auxiliary solvents or surfactants are used to aid in the dispersion, they 

will be used at a maximum concentration of 100 ~LIL (fmal volume). If the test item falls into 

the category of a "difficult substance" as defined by the OECD Guidance Document No.23 

(OECD, 2000), the method of preparation of the test media will be based on the 

recommendations of this document. 
General Study Plan: 902-21 Page9 

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 an appropriate amount of test medium. The test vessels will be 

covered to reduce evaporation and to avoid entry of dust into the solutions. Volatile chemicals 

will be tested in sealed vessels using a volume of test media to provide a minimal headspace (i.e. 

test performed in a closed system). The size of the test vessels and the volume of test solution 

will depend on the species used and will be documented in the data. 

The test vessels will be labelled 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 animals. Auxiliary aeration 

will be supplied to the test vessels via narrow bore glass tubes to give ;:::: 60% Air Saturation 

Value (ASV) unless evidence of instability ofthe test item under aeration is shown. For dynamic, 

continuous flow tests the diluent water supply only will be aerated. For tests conducted to 

OPPTS guidelines no auxiliary aeration will be supplied to the test vessels except for static tests 

where aeration may be used if the oxygen consumption is expected to drop below 60% Air 

Saturation Value (ASV) due to the chemical characteristics of the test item. The use of aeration 

will be documented in the data. 

The test will be performed under temperature-controlled conditions. The temperature will 

depend on the test species used (see Appendix 1) with a maximum deviation of± 1 oc during the 

test. 

A 16-hour light to 8-hour dark cycle will be applied with a 20- 30 minute transition period. If 

the test item is light sensitive, the test will be performed as far as possible in the dark or under 

reduced light conditions to avoid photolytic degradation of the test item. 

3. 7 Study Design 

Seven fish will be used at each test concentration and in the control(s). 

The loading rate should not be greater than 1 g bodyweight per liter of test medium for static and 

semi-static tests. This loading may be exceeded in dynamic, continuous flow tests. 

Animals will be selected at random from the total number collected. The animals will not be 

individually identified. The animals will not be fed during the test. 
General Study Plan: 902-21 Page 10 

Dependent on the test item stability, the test will be conducted with no medium renewal (static 

test), with daily medium renewal (semi-static test) or continuous medium renewal (dynamic test). 

The choice of test media renewal regimes is dependent upon the known characteristics of the test 

item and the results of the pre-test stability analysis. The actual method of test media renewal 

will be documented in the data. 

For static or semi-static tests, the test will be started by addition of the test organisms to the test 

vessels within 30 minutes of the test media being added to the test vessels. For tests conducted 

using continuous medium renewal (dynamic tests), the test will be started after the concentration 

of test item has attained a stable plateau. Alternatively the test item may be added to the test 

vessels at the required concentration prior to starting the dosing apparatus, in such cases the test 

organisms will be added to the test vessels within 30 minutes of the start of the dosing apparatus. 

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 rangefinding 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 chosen test concentrations should permit the determination of the 96-Hour LCso (or LLso) 

and, as far as possible, span the range of 0 to 100% mortality. In accordance with the test 

guidelines (EC, 2002), concentrations of the test item higher than 100 mg/L (1 000 mg/L for 

agrochemical formulations tested according to JMAFF guidelines), or above the limit of 

solubility of the test item in the test water (whichever is the lower) will usually not be tested. 

If no effect is observed in the preliminary range-finding test at the highest concentration 

recommended in the relevant test guideline, or the limit of solubility of the test item in the test 

water (whichever is the lower), a Limit Test may be conducted using only this concentration and 

the corresponding control(s). In this case, the test will be conducted using a single test vessel 

together with a control and solvent control vessel, if appropriate. 

Where appropriate a threshold test will be used. The lowest ECso/ELso value from an Algal 

Growth Inhibition test or Acute Toxicity to Daphnia magna study is set as the threshold 

concentration. A "Limit Test" will then be carried out as detailed previously at this threshold 

concentration. If no mortalities are observed this indicates that fish is not the most sensitive 

species and that the LC50 is greater than the threshold concentration. If one or more fish from the 

test group die, the test will be terminated and a test using a range of test concentrations will be 

conducted. 

The control group will be exposed to the test water alone. In the interests of animal welfare, data 

from the control group may be shared with similar concurrent studies. 
General Study Plan: 902-21 Page 11 

Where auxiliary solvents/surfactants are used, a second control will be employed. The test 

organisms will be exposed to a concentration of solvent equal to that employed in the test series, 

this solvent concentration will not exceed 100 JlLIL. In the interests of animal welfare, data 

from the solvent control group may be shared with similar concurrent studies. 

In the interests of animal welfare, ifthe pH falls outside the range 6-9, the pH will be adjusted to 

that of the test water prior to addition of the test organisms to the test preparations. In such cases, 

the adjustment of pH will be discussed with the Sponsor prior to proceeding with the test. 

3.8 Evaluations 

3.8.1 Test organism observations 

The fish are inspected after 3, 6, 24, 48, 72 and 96 hours and at intermediate times when 

appropriate. Mortalities are recorded and any abnormalities in appearance or behaviour shown 

by surviving fish are summarised. The criteria of death are absence of respiratory movement and 

the lack of response to physical stimulation. Dead fish are removed when observed. 

During the test, the principles mentioned in the OECD Humane Endpoints Guidance Document 

No. 19 (OECD, 2002) will be taken into consideration. Test fish, which are found in moribund 

condition, will be humanely killed before test end due to reasons of animal welfare. For the 

calculation of the LCso value the sacrificed fish will be taken into account as dead fish. 

The length and weight of a representative batch of fish will be determined e.g. fish from the 

stock tank prior to the start of the test or the control fish at the end of the test 

3.8.2 Water quality criteria 

Measurements of pH, dissolved oxygen and temperature in each surviving test vessel (prior to 

and post test media renewal for semi-static tests) are recorded daily. 

The appearance of the test media will be recorded daily. 
General Study Plan: 902-21 Page 12 

3.8.3 Analysis of Test Item Concentrations 

3.8.3.1 Sampling 

Samples of the test media will be taken at least at initiation, termination and an intermediate time 

point of the definitive test for analysis. In the range-finding test, samples of the test media will 

be taken at 0 hours and after 24 or 96 hours. Where possible duplicate samples will be taken at 

each occasion, these samples may also be analyzed should the need arise. 

Samples will be taken from the control (or solvent control where appropriate) and each surviving 

test group or at the observation point when all fish are dead in a certain test concentration. 

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 test concentrations 

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

Final 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. 

It may be necessary for some chemicals which form emulsions or dispersions to filter or 

centrifuge some samples as part of the pre-test validation process to determine the proportion of 

test item in the dissolved form. 

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. 

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 96-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. 

If the test item is a formulation, test item concentrations will be determined by the measured 

concentrations of the active ingredient(s). WAFs will be characterized by analysis of the main 

test item component(s). 
., 

General Study Plan: 902-21 Page 13 

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. 

Analytical pre-experiments may be performed within a different Harlan Laboratories study. 

3.9 Statistical Analysis 

The LCso (or LLso) values and associated 95% confidence limits for all appropriate observation 

times will be calculated using an appropriate statistical method which will be documented in the 

data. All LCso (or LLso) values will be rounded to two significant figures. 

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is 

the highest test concentration (or loading rate) at which no significant toxic effect on the test 

organisms is observed within the exposure period. 

The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL) is the lowest test concentration (or loading rate) at which a significant toxic effect on the 

test organisms is observed within the exposure period. If the test has been conducted as a limit 

test then it is not possible to determine a LOEC or LOEL. 

3.10 Validity Criteria 

For the test to be valid, the following criteria should be fulfilled: 

In the (solvent) control, not more than one of the fish should die or show signs of stress during 

the 96 hours. 

The dissolved oxygen concentration at the end of the test should be ~60% of Air Saturation 

Values (ASV) in the control and test vessels. 
General Study Plan: 902-21 Page 14 

4 REFERENCES 

OECD (2002) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as 

Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Series on 

Testing and Assessment, No. 19. OECD Publishing. 

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 911414/EEC. 
General Study Plan: 902-21 Page 15 

APPENDICES 

Appendix 1 Recommended Fish Species 

The following species are recommended for use according to the OECD, EU, and JMAFF 

guidelines. 

Species Recommended Test Recommended Total 

Temperature Range ec) Length of Test Fish (em) 

Brachydanio rerio 21-25 OECD 2.0 ± 1.0 

Zebra fish EU 3.0± 0.5 

JMAFF 2.0 ± 1.0 

Cyprinus carpio 20-24 OECD 3.0 ± 1.0 

Common carp EU6.0±2.0 

JMAFF 5.0 ± 1.0 

Lepomis macrochirus 21-25 OECD 2.0 ± 1.0 

Bluegill sunfish EU 5.0±2.0 

JMAFF 3.0 ± 1.0 

Oncorhynchus mykiss 13- 17 OECD 5.0 ± 1.0 

Rainbow Trout EU 6.0±2.0 

JMAFF 5.0 ± 1.0 

Pimephales promelas 21-25 OECD 2.0 ± 1.0 

Fathead minnow EU 5.0±2.0 

JMAFF 2.0 ± 1.0 
General Study Plan: 902-21 Page 16 

Appendix 2 Test Water 

Reconstituted Water 

The reconstituted water will consist of analytical grade salts dissolved in purified water to obtain 

the following nominal concentrations· 

Ingredients I Concentration 

Properties mmoJJL mWL 

CaC1z.2H20 1.0 147 

MgS04.?H20 0.25 61.5 

NaHC03 0.38 32.5 

KCl 0.038 2.9 

Water Hardness 1.25 125 as CaC03 

Alkalinity 0.4 - The ratio ofCa:Mg and Na:K will be 4:1 and 10:1 respectively based on molarity. 

The reconstituted water will be aerated until oxygen saturation is reached. The pH of the test 

water after equilibration will be in the range of7.0 to 8.5. 

Local Tap Water 

Local water (non chlorinated well water of drinking quality) will be total hardness reduced by 

ion exchange. The total hardness will be approximately 160 to 250 mg/L as CaC03. The water 

will be aerated until oxygen saturation is reached. The pH of the test water after equilibration 

will be in the range of7.8 to 8.8. 

Dechlorinated Laboratory Tap Water 

Laboratory tap water will be dechlorinated by passage through an activated carbon filter (Purite 

Series 500) and a proportion of the incoming water softened (Elga Nimbus 1248D Duplex water 

softener) and then remixed with the main supply to give a water hardness of approximately 

140 mg/L as CaC03 and pH 6.0- 8.5. After dechlorination and softening the water will then be 

passed through a series of computer controlled heat exchangers to achieve the required 

temperature. When a "single pass" system is not possible, the water will be recirculated via a 

suitable purification filter. 
STUDY SPECIFIC SUPPLEMENT 

To General Study Plan: 900-22 

c 

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Daphnia sp., 48-Hour Acute Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

Proposed Schedule: 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

D Sacker 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

U nivar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL 33410 

UNITED STATES OF AMERICA 

41401174 

30 April 2014 

31 May 2014 

30 June 2014 

Page 1 of4 
Harlan Study Number: 4140 117 4 Study Specific Supplement Page 2 

Test Item 

Information as provided by the Sponsor. 

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7] 

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 

4 Test Rule and the regulations set forth under 40 CFR 799.5087. 

Special Conditions 

Study specific parameters: 

GSP Section 1.1 Guidelines I Regulations: 

The study will be conducted according to the following internationally accepted guidelines and 

recommendations: 

•The OECD Guidelines for Testing of Chemicals (2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

GSP Section 3.2 Test system 

Daphnia used in the test should be from a healthy stock (i.e., showing no signs of stress such as 

high mortality, presence of males and ephippia, delay in the production of the first brood, 

discolored animals, etc.). 

Any pH adjustments of the exposure solutions will be conducted according to section 13 of 

OECD 202 details of which will be included in the final test report. 

GSP Section 3.8.2 Water quality criteria 

Temperature, pH and dissolved oxygen measurements will be taken from replicate R1 only at 

the start and end of the test, and prior to and post test medium renewal if applicable. 

If measured concentrations are ± 20% of the nominal concentrations, results will be based on 

nominal concentrations. If initial measured concentrations are not ± 20% of the nominal 

concentrations but measured concentrations of the old exposure solution are ± 20% of the 

initial measured concentrations, results will be based on initial measured concentrations. If 

measured concentrations are not ± 20% of nominal concentrations, results will be based on 

geometric mean measured concentrations. 

The study report will provide a detailed description of the analytical method that will be 

developed prior to and utilized during the test. 
Harlan Study Number: 41401174 Study Specific Supplement Page 3 

All elements described in item 27 of OECD 202 will be included in the final test report. 

GENERAL INFORMATION 

Distribution List 

The original study specific supplement will be retained in the study file. Copies of the study 

specific supplement and general study plan along with any amendments will be distributed as 

follows: 

For the Sponsor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK 
Harlan Study Number: 41401174 

SIGNATURES 

Study Director: 

Test Facility 

Management: 

For the Sponsor: 

Study Specific Supplement Page4 

D Sacker 

Date: 

CMead 

Date: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Date: 

Note: These signatures also acknowledge the procedures described in the corresponding General 

Study Plan. 
GENERAL STUDY PLAN: 900-22 

Daphnia sp., 48-Hour Acute Immobilization Test 

Test Facility: 

Signatures: 

Author: 

Test Facility Management: 

Quality Assurance: 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

H VryenhoefBSc 

J G Riley BSc (Hons) MRQA 

........................ ~&.~ ~ ...... ~ ................ . Date: 1 2 SEP 2012 

Page 1 of 15 
General Study Plan: 900-22 Page2 

CONTENTS 

GOOD LABORATORY PRACTICE ............................................................................................. 3 

GENERAL INFORMATION ......................................................................................................... 4 

Study Plan Amendments and Deviations ............................................................................... 4 

Quality Assurance .................................................................................................................. 4 

Reporting ............................................................................................................................... 4 

Archiving ............................................................................................................................... 4 

1 INTRODUCTION AND PURPOSE ..................................................................................... 5 

1.1 , Guidelines I Regulations .......................................................................................... 5 

2 TEST AND REFERENCE ITEMS ....................................................................................... 6 

2.1 Test Item .................................................................................................................. 6 

2.2 Reference Item ......................................................................................................... 6 

3 MATERIALS AND METHODS ........................................................................................... 6 

3 .1 Definitions and Abbreviations ................................................................................. 6 

3.2 Test System .............................................................................................................. 7 

3.3 Test Water ................................................................................................................ 7 

3.4 Dosage ..................................................................................................................... 7 

3. 5 Materials .................................................................................................................. 8 

3.6 Experimental Conditions ......................................................................................... 8 

3. 7 Study design ............................................................................................................ 8 

3.8 Evaluations ............................................................................................................ 10 

3.8.1 Test organism observations ..................................................................... 10 

3.8.2 Water quality criteria ............................................................................... tO 

3.8.3 Analysis ofTest Item Concentrations ..................................................... 10 

3.9 Statistical Analysis ................................................................................................ 11 

3.10 Validity Criteria ..................................................................................................... 12 

4 REFERENCES .................................................................................................................... 13 

APPENDICES ............................................................................................................................... 14 

Appendix 1 Reconstituted Water - Elendt M7 Medium ...................................................................... 14 

Appendix 2 Reconstituted Water- Test Water .................................................................................... 15 
General Study Plan: 900-22 Page3 

GOOD LABORATORY PRACTICE 

The associated 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) 17); and are in accordance with, and 

implement, the requirements ofDirectives 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 (MHL W, MAFF and METI). 
General Study Plan: 900-22 Page4 

GENERAL INFORMATION 

Study Plan Amendments and Deviations 

Any intended change to the study plan (general study plan and study specific supplement) 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 (general study plan and study specific 

supplement) 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 

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

and comments before issue of the fmal report. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), all raw data (paper and electronic) and the fmal 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. 
General Study Plan: 900-22 PageS 

1 INTRODUCTION AND PURPOSE 

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

invertebrate Daphnia magna. The toxicity will be expressed as the median effect concentration 

(ECso) or median effect loading rate (ELso) for immobilization. 

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) 

and the Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL) will also be determined if applicable. 

Daphnia magna is a freshwater invertebrate representative 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 I Regulations 

The test methods described are designed to be compatible with the procedures indicated by the 

following internationally accepted guidelines and recommendations: 

• The OECD Guidelines for Testing of Chemicals (2004) No 202 "Daphnia sp., Acute 

Immobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 

• JMAFF Test Guidelines 2-7-2-1 (200 1) 
General Study Plan: 900-22 Page6 

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 I retest date, storage conditions and safety 

precautions to be taken. 

Where the test item is administered in a vehicle, and the homogeneity, concentration or stability 

of the test item in that vehicle is not determined an impact assessment will be made by the Study 

Director, this will be reflected in the final report and an exception will be included in the GLP 

compliance statement. 

2.2 Reference Item 

For evaluation of the quality of the Daphnia magna clone 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. 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Immobilization: 

ECso/ELso: 

LOEC (Lowest Observed 

Effect Concentration) or LOEL 

(Lowest Observed Effect 

Loading rate): 

NOEC (No Observed Effect 

Concentration) or NOEL (No 

Observed Effect Loading rate): 

Treatment: 

Those organisms which are not able to swim within 15 seconds 

after gentle agitation of the test container are considered to be 

immobile. 

The calculated concentration or loading rate of test item which 

results in 50% immobilization within the exposure period. 

The lowest test concentration or loading rate at which a 

significant toxic effect on the test organisms is observed within 

the exposure period. 

The highest test concentration or loading rate at which no 

significant toxic effect on the test organisms is observed within 

the exposure period. 

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

solvent control (if applicable). 
General Study Plan: 900-22 Page7 

3.2 Test System 

The study will be performed with young daphnids of the species Daphnia magna Straus, which 

is the preferred test species recommended by the test guideline. A clone of this species was 

originally supplied by the University of Sheffield/UK. The clone (defined by the supplier as 

clone 5) is successfully bred at Harlan Laboratories Ltd under temperature and light conditions 

identical to those of the test. 

During the culturing period, the parental daphnids will be fed an algal suspension and a Tetramin 

suspension. Details will be documented in the culturing records. Culture conditions ensure that 

reproduction will be by parthenogenesis. 

The animals will be single housed in 150 ml glass beakers containing Elendt M7 medium (see 

Appendix 1 ). 

To obtain young daphnids for the test which are equal to or less than 24 hours old, any young 

daphnids will be removed from the culture one day prior to initiation of the test such that the 

young daphnids produced overnight are at the required age. 

The daphnids will not be fed during the test. 

3.3 Test Water 

The test water parameters are defined in Appendix 2. The parental daphnids are cultivated in 

water of the same quality (in respect to pH, main ions, and total hardness). · 

3.4 Dosage 

If necessary, pre-experiments will be performed to determine the solubility and stability of the 

test item in the test water. According to the findings of these pre-experiments, appropriate 

procedures will be chosen to prepare the test media. 

The test item will be dissolved or dispersed in the test water once at the start of the test (static 

test), daily (semi-static test) or continuously (dynamic test) depending on the stability of the test 

item in the test water. 

Methods such as prolonged mixing, ultrasonication, high shear force stirring or the production of 

Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the 

nature of the test item. If auxiliary solvents or surfactants are used to aid in the dispersion, they 

will be used at a maximum concentration of 100 J..I.LIL (final volume). If the test item falls into 

the category of a "difficult substance" as defined by the OECD Guidance Document (OECD, 

2000), the method of preparation of the test media will be based on the recommendations of this 

document. 
General Study Plan: 900-22 Page 8 

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 50 mL of test medium will be used. The test vessels will be 

covered to reduce evaporation and to avoid entry of dust into the solutions. Volatile chemicals 

will be tested in completely filled and stoppered test vessels (i.e. test performed in a closed 

system). The size of the test vessels and the volume of test solution will be documented in the 

data. 

The test vessels will be labelled 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 will be performed in a temperature-controlled room. The water temperature will be 

maintained at 18- 22°C with a maximum deviation of ±1 oc during the test. 

A 16-hour light to 8-hour dark cycle will be applied with a 20 - 30 minute transition period. The 

light intensity during the light period will be between 200 and 1200 Lux and will be documented 

in the raw data. If the test item is light sensitive, the test will be performed as far as possible in 

the dark or under reduced light conditions to avoid photolytic degradation of the test item. 

3. 7 Study design 

A total of 20 daphnids per treatment will be used and divided into four replicates. The following 

conditions will be met: 

Total volume per replicate 

Volume I daphnid 

Loading rate I L 

2::10 mL 

2::2m.L 

:::;500 daphnids 

The daphnids will be randomly distributed to the test vessels at initiation of the test. 

For static or semi-static tests, the test will be started by addition of the test organisms to the test 

vessels within 30 minutes of the test media being added to the test vessels. For tests conducted 

using continuous medium renewal (dynamic tests), the test will be started after the concentration 

of test item has attained a stable plateau. Alternatively the test item may be added to the test 
General Study Plan: 900-22 Page9 

vessels at the required concentration prior to starting the dosing apparatus, in such cases the test 

organisms will be added to the test vessels within 30 minutes of the start of the dosing apparatus. 

The duration of the test will be 48 hours. 

Dependent on the test item stability, the test will be conducted with no medium renewal (static 

test), with daily medium renewal (semi-static test) or continuous medium renewal (dynamic test). 

In a semi-static test, the Daphnia will be transferred to the fresh media by wide-bore pipette. 

The choice of test media renewal regimes is dependent upon the known characteristics of the test 

item and the results of the pre-test stability analysis. The actual method of test media renewal 

will be documented in the data. 

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-fmding tests or available toxicity 

data and will be documented in the data prior to the start of the definitive test. If in the rangefinding 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 chosen test concentrations should preferably cover the range causing 0 to 100% immobility. 

In accordance with the test guidelines (EC, 2002), concentrations of the test item higher than 

100 mg/L (1000 mg/L for agrochemical formulations tested according to JMAFF guidelines), or 

above the limit of solubility of the test item in the test water (whichever is the lower) will usually 

not be tested. 

If no effect is observed in the preliminary range-finding test at the highest concentration 

recommended in the relevant test guideline, or the limit of solubility of the test item in the test 

water (whichever is the lower), a Limit Test may be conducted using only this concentration and 

the corresponding control(s). 

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

shared with similar concurrent studies. 

Where auxiliary solvents/surfactants are used, a second control will be employed. The test 

organisms will be exposed to a concentration of solvent equal to that employed in the highest 

concentration in the test series, this solvent concentration will not exceed 100 J..tLIL. Data from 

the solvent 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. 
General Study Plan: 900-22 Page 10 

3.8 Evaluations 

3.8.1 Test organism observations 

The nwnber of immobilised Daphnia in each vessel will be recorded after 24 and 48 hours. 

Those organisms not able to swim within 15 seconds after gentle agitation of the test beaker will 

be considered to be immobile. 

Any abnormal behaviour or abnormal appearance of the daphnids (sub lethal effects other than 

immobility) will also be recorded at 24 and 48 hours. 

3.8.2 Water quality criteria 

For static and dynamic flow tests the temperature is recorded daily and pH and dissolved oxygen 

measurements are made for each test vessel at the start and end of the test. For semi-static tests, 

pH, dissolved oxygen and temperature are measured in each test vessel at the start of the test, 

prior to and post test mediwn renewal and at the end of the test. 

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, before and after the test mediwn renewal (if 

applicable), and termination of both the range-finding and definitive tests for analysis. 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 test concentrations 

provided for analysis will be docwnented by the Study Director in the data. 

For the purposes of the definitive test, the replicate test and control (or solvent control) vessels 

will be pooled to give one sample. 

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

requisite volwnes of the freshly prepared test media will be incubated separately (without 

daphnids), but under identical conditions as in the actual test. 

Final details of the samples to be taken and the sample size, etc, will be docwnented in the data. 
General Study Plan: 900-22 Page 11 

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. 

It may be necessary for some chemicals which form emulsions or dispersions to filter or 

centrifuge some samples as part of the pre-test validation process to determine the proportion of 

test item in the dissolved form. 

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. 

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 48-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. 

If the test item is a formulation, test item concentrations will be determined by the measured 

concentrations of the active ingredient(s). WAFs will be characterized by analysis of the main 

test item component(s). 

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. 

Analytical pre-experiments may be performed within a different Harlan Laboratories study. 

3.9 Statistical Analysis 

The 24 and 48-Hour EC50 (or EL50) values and associated 95% confidence limits will be 

calculated using an appropriate statistical method which will be documented in the data. All ECso 

(or EL50) values will be rounded to two significant figures. 
General Study Plan: 900-22 Page 12 

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is 

the highest test concentration (or loading rate) at which no significant toxic effect on the test 

organisms is observed within the exposure period. 

The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL) is the lowest test concentration (or loading rate) at which a significant toxic effect on the 

test organisms is observed within the exposure period. If the test has been conducted as a limit 

test then it is not possible to determine a LOEC or LOEL. 

3.10 Validity Criteria 

For the test to be valid, the following criteria should be fulfilled: 

In the (solvent) control, not more than 10% of the daphnids should show immobilization or other 

signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface 

water). 

The dissolved oxygen concentration at the end of the test should be ?:3 mg/L in the control and 

test vessels. 
'• 

General Study Plan: 900-22 Page 13 

4 REFERENCES 

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

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

INTERNATIONAL STANDARDS ORGANISATION (ISO) (1996) Standard 6341: Water 

quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, 

Crustacea) - Acute toxicity test. Geneva: International Standards Institute. 

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

Mixtures. OECD Series on Testing and Assessment, No. 23. OECD Publishing. 
General Study Plan: 900-22 Page 14 

APPENDICES 

Appendix 1 Reconstituted Water- Elendt M7 Medium 

Elendt M7 medium will be prepared by dissolving the following analytical grade salts and 

additives in deionised or purified water at the following concentrations: 

Main Compounds Trace Elements• 

mg/L 

CaCh.2H20 

MgS04.1HzO 

KC1 

NaHC03 

Na2Si03.9H20 

NaN03 

KH2P04 

KzHP04 

Water Hardness: 

Alkalinity: 

.~ 294 B H3B03 

123 Fe FeS04.7H20 2 

5.8 Mn MnChAH20 

65 Li LiC1 

10 Rb RbC1 

0.27 Sr SrCh.6H20 

0.14 Br NaBr 

0.18 Mo Na2Mo04.2H20 

Co CuC}z.2H20 

Zn ZnCh 

Co CoCh.6H20 

I KI 

Se Na2Se03 

v ~vo3 

EDTA Na2EDTA.2H20 

2.5 mmol/L (= 250 mg/L as CaC03) 

0.9mmol/L 

Vitamins 

p.g/L 

125 Thiamine HC1 75 

50 Cyanocoba1amine (B12) 1.0 

25 Biotin (B6) 0.75 

12.5 

12.5 

12.5 

3.1 

6.3 

1.6 

6.3 

2.5 

2.5 

1.0 

0.3 

625 

The pH of the prepared media will be in the range of 7.9 ± 0.3 and will be stored at 

approximately 21 °C. If necessary the pH may be adjusted with NaOH or HCl. 

1 Concentrations of the target elements in bold; the source compounds are given in the next column 2 Stock solutions ofFeS04.7H20 and Na2EDTA.2H20 are combined and autoclaved immediately 
General Study Plan: 900-22 Page 15 

Appendix 2 Reconstituted Water- Test Water 

Prior to the start of the study, the test water (specifications see below) will be aerated until 

oxygen saturation is reached. Aeration will not be performed during the experiment. The pH of 

the test water after equilibration will be in the range of 7.6- 8.0. If necessary the pH may be 

adjusted with NaOH or HCl. The pH will normally not vary by more than 1.5 during the test. 

The ratio ofCa:Mg and Na:K will be 4:1 and 10:1, respectively, based on molarity. 

Reconstituted test water according to ISO 6341 (ISO, 1996) will be used in the study. It will 

consist of analytical grade salts dissolved in purified water with a conductivity of< 5 J.tS cm-1 to 

obtain the following nominal concentrations: 

Ingredients I Concentration 

Properties mmoVL mg/L 

CaCh.2H20 2.0 294 

MgS04.1H20 0.5 123 

NaHC03 0.75 65 

KCl 0.075 5.8 

Water Hardness 2.5 250 as CaC03 

Alkalinity 0.8 -
STUDY SPECIFIC SUPPLEMENT 

To General Study Plan: 908-25 

D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium 

salt [CAS RN 6381-77-7]: 

Algal Growth Inhibition Test 

Study Director: 

Test Facility: 

Sponsor: 

Study Monitor: 

Harlan Study Number: 

Proposed Schedule: 

Experimental Starting Date: 

Experimental Completion Date: 

Draft Report Date: 

H Vryenhoef 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

U nivar USA Inc. 

17425 NE Union Hill Road 

Redmond 

WA 98052 

UNITED STATES OF AMERICA 

Greenwich Chemical Consulting, Inc. 

3300 PGA Boulevard, Suite 635 

Palm Beach Gardens 

FL 33410 

UNITED STATES OF AMERICA 

41401175 

30 April2014 

31 May 2014 

30 June 2014 

Page 1 of4 
Harlan Study Number: 4140 117 5 Study Specific Supplement Page 2 

Test Item 

Information as provided by the Sponsor. 

Identification: D-erythro-hex-2-enonic acid, .gamma.-lactone, monosodium salt 

[CAS RN 6381-77-7] 

This study is being performed based on the U.S. Toxic Substances Control Act (TSCA) Section 

4 Test Rule and the regulations set forth under 40 CPR 799.5087. 

Special Conditions 

Study specific parameters: 

GSP Section 1.1 Guidelines I Regulations: 

The study will be conducted according to the following internationally accepted guidelines and 

recommendations: 

• OECD Guidelines for Testing of Chemicals (2006) No 201 * "Freshwater Alga and 

Cyanobacteria, Growth Inhibition Test" 

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

GSP Section 3.2 Test System: 

The study will be conducted using Pseudokirchneriella subcapitata 

GSP Section 3.6 Experimental Conditions: 

Incubation of the test vessels will be in an INFORS Multitron® Version 2 incubator 

If measured concentrations are ± 20% of the nominal concentrations, results will be based on 

nominal concentrations. If measured concentrations are not ± 20% of nominal concentrations, 

results will be based on geometric mean measured concentrations. 

The study report will provide a detailed description of the analytical method that will be 

developed prior to and utilized during the test. 

All elements described in item 61 ofOECD 201 will be included in the final test report. 

• Annex 5 corrected 28 July 2011 
Harlan Study Number: 41401175 Study Specific Supplement Page 3 

GENERAL INFORMATION 

Distribution List 

The original study specific supplement will be retained in the study file. Copies of the study 

specific supplement and general study plan along with any amendments will be distributed as 

follows: 

For the Sponsor: John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK 
Harlan Study Number: 4140 117 5 

SIGNATURES 

Study Director: 

Test Facility 

Management: 

For the Sponsor: 

Study Specific Supplement Page 4 

H Vryenhoef 

Date: 

CMead 

Date: 

John V. Massingale 

Greenwich Chemical Consulting, Inc. 

Date: 

Note: These signatures also acknowledge the procedures described in the corresponding General 

Study Plan. 
GENERAL STUDY PLAN: 908-25 

Algal Growth Inhibition Test 

Test Facility: 

Signatures: 

Author: 

Test Facility Management: 

Quality Assurance: 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

H VryenhoefBSc 

Date: 1 1 SEP 2012 

J G Riley BSc (Hons) MRQA 

••••••••••••••••••••• ~ •••• 10 ........ ~ ••••••••••••••• 

Date: 1 2 SEP 201! 

Page 1 of19 
General Study Plan: 908-25 Page2 

CONTENTS 

GOOD LABORATORY PRACTICE ............................................................................................. 3 

GENERAL INFORMATION ......................................................................................................... 4 

Study Plan Amendments and Deviations ............................................................................... 4 

Quality Assurance .................................................................................................................. 4 

Reporting ............................................................................................................................... 4 

Archiving ............................................................................................................................... 4 

1 INTRODUCTION AND PURPOSE ..................................................................................... 5 

1.1 Guidelines I Regulations .......................................................................................... 5 

2 TEST AND REFERENCE ITEMS ....................................................................................... 6 

2.1 Test Item .................................................................................................................. 6 

2.2 Reference Item ......................................................................................................... 6 

3 MATERIALS AND METHODS ........................................................................................... 6 

3.1 Definitions and Abbreviations ................................................................................. 6 

3.2 Test System .............................................................................................................. 7 

3.3 Test Water ................................................................................................................ 7 

3.4 Dosage ..................................................................................................................... 7 

3.5 Materials .................................................................................................................. 8 

3.6 Experimental Conditions ......................................................................................... 8 

3. 7 Study Design ............................................................................................................ 8 

3.8 Re-growth Experiment .......................................................................................... 10 

3.9 Colored Test Items ................................................................................................. 10 

3.10 Evaluations ............................................................................................................ 11 

3.10.1 Test Organism Observations ................................................................... 11 

3.10.2 Water Quality Criteria ............................................................................. 11 

3.10.3 Analysis ofTest Item Concentrations ..................................................... 11 

3.11 Data Analysis ......................................................................................................... 13 

3.11.1 Plotting Growth Curves ........................................................................... 13 

3 .11.2 Calculation of Growth Rates ................................................................... 13 

3 .11.3 Yield ........................................................................................................ 14 

3.11.4 Plotting Concentration-Response Curves ................................................ 15 

3.11.5 Statistical Analysis .................................................................................. 15 

3 .12 Validity Criteria ..................................................................................................... 15 

4 REFERENCES .................................................................................................................... 17 

APPENDICES ............................................................................................................................... 18 

Appendix 1 Culture Medium ................................................................................................................ 18 

Appendix 2 Testing ofVolatile Test ltems ........................................................................................... l9 
General Study Plan: 908-25 Page3 

GOOD LABORATORY PRACTICE 

The associated 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)17); 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 (MHL W, MAFF and ·MET!). 
General Study Plan: 908-25 Page4 

GENERAL INFORMATION 

Study Plan Amendments and Deviations 

Any intended change to the study plan (general study plan and study specific supplement) 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 (general study plan and study specific 

supplement) 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 

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

and comments before issue of the fmal report. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan (general study plan and study specific 

supplement), 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. 
General Study Plan: 908-25 Page 5 

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 (or loading rate) bringing about a specified x% inhibition of growth (e.g. 50%) and 

expressed as the ErCx (or ErLx). 

The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL), and the No Observed Effect Concentration (NOEC) or No Observed Effect Loading 

rate (NOEL) 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 may therefore also 

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

The study will use one of the following species of freshwater alga which are representative of a 

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

organism in freshwater ecosystems: 

• Pseudokirchneriella subcapitata 

• Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) 

• Anabaena jlos-aquae 

• Navicula pelliculosa 

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 I Regulations 

The 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 

• JMAFF Test Guidelines 2-7-7 

1 Annex 5 corrected: 28 July 2011 
General Study Plan: 908-25 Page6 

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 I retest date, storage conditions and safety 

precautions to be taken. 

Where the test item is administered in a vehicle, and the homogeneity, concentration or stability 

of the test item in that vehicle is not determined an impact assessment will be made by the Study 

Director, this will be reflected in the fmal report and an exception will be included in the GLP 

compliance statement. 

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. 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Biomass: 

Growth: 

Growth Rate (Average Specific 

Growth Rate): 

Yield: 

Measurement variable (fluorescence or algal cell density) as 

surrogate measure for algal biomass. 

The calculated concentration or loading rate 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. 
General Study Plan: 908-25 

LOEC (Lowest Observed 

Effect Concentration) or LOEL 

(Lowest Observed Effect 

Loading rate): 

NOEC (No Observed Effect 

Concentration) or NOEL (No 

Observed Effect Loading rate): 

Treatment: 

Section-by-Section Growth 

Rate: 

3.2 Test System 

Page7 

The lowest test concentration or loading rate at which a 

statistically significant inhibition of growth is determined 

relative to the control (or solvent control, if applicable). 

The highest test concentration or loading rate 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). 

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

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

The study will be performed using either Pseudokirchneriella subcapitata, Desmodesmus 

subspicatus (formerly known as Scenedesmus subspicatus), Anabaena jlos-aquae or Navicula 

pelliculosa. These freshwater alga species are recommended by the relevant test guideline. The 

test species used will be identified in the study-specific supplement of the corresponding study. 

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 

If necessary, pre-experiments will be performed to determine the solubility and stability of the 

test item in the test water. According to the findings of these pre-experiments, appropriate 

procedures will be chosen to prepare the test media. 

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

Methods such as prolonged mixing, ultrasonication, high shear force stirring or the production of 

Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the 

nature of the test item. If auxiliary solvents or surfactants are used to aid in the dispersion, they 
General Study Plan: 908-25 Page 8 

will be used at a maximum concentration of 100 J!L/L (final volume). If the test item falls into 

the category of a "difficult substance" as defined by the OECD Guidance Document (OECD, 

2000), the method of preparation of the test media will be based on the recommendations of this 

document. 

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 15 mL test medium will be used. The test vessels will be 

covered/plugged to reduce evaporation and to avoid entry of dust into the solutions. Volatile 

chemicals will be tested in completely filled and stoppered test vessels (i.e. test performed in a 

closed system). The size of the test vessels and the volume of test solution will be documented 

in the data. 

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 one of 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 oc 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. 

• Temperature-controlled water bath under constant illumination (between 4440 and 

8880 lux) provided by fluorescent tubes installed above the test flasks. The light intensity 

will not vary by more than ±15% from the average light intensity over the incubation area. 

The temperature will be maintained at between 21 and 24°C and maintained at± 2°C. 

The test solutions will be continuously stirred by magnetic stirrers. 

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. 

If the presence of particles in the test media could interfere with the determination of the algal 

biomass, eg if using a Coulter® Particle Counter, one additional replicate per test concentration 
General Study Plan: 908-25 Page9 

---------------------------··~·············--····----

containing the test media without algae will be prepared in the definitive test as particle controls. 

These additional replicates will be incubated under the same conditions as the test media with 

algae and will be used for the correction of the algal biomass determination. In cases where 

preliminary work indicates no counts due to dispersed test item, the blank will consist of culture 

medium alone. 

Depending on the specific requirements of the relevant test guidelines, the following conditions 

will be met: 

Initial cell density ( cells/mL) 

Test duration (hours) 

A static test design will be applied. 

OECD/EC 

2-5 X 103 

72 

JMAFF 

5 X 1 03 - 1 X 1 04 

72 

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 rangefinding 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 rate. In accordance with the test guidelines (EC, 2002), concentrations of the test item 

higher than 100 mg/L (1000 mg/L for agrochemical formulations tested according to JMAFF 

guidelines), or above the limit of solubility of the test item in the test water (whichever is the 

lower) will usually not be tested. 

If no effect is observed in the preliminary range-finding test at the highest concentration 

recommended in the relevant test guideline, or the limit of solubility of the test item in the test 

water (whichever is the lower), a Limit Test may be conducted using only this concentration and 

the corresponding control(s). 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. 

Where auxiliary solvents/surfactants are used, a second control will be employed. The test 

organisms will be exposed to a concentration of solvent equal to that employed in the test series, 

this solvent concentration will not exceed 100 JJLIL. Data from the solvent 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 
General Study Plan: 908-25 Page 10 

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 Re-growth Experiment 

For JMAFF requirements, a re-growth experiment will be conducted as above using the control 

(or solvent control) cultures and a single test group that has shown significant inhibition of 

growth. The samples will be incubated for a further 10 days (maximum) under test conditions 

with samples of the cultures being taken at 0 hours and periodically thereafter for the 

measurement of algal biomass to determine when/if re-growth occurs. 

The re-growth experiment will not be carried out if a "limit test" has been conducted. 

3.9 Colored Test Items 

Colored test items may absorb biologically relevant wavelengths and thereby may impair algal 

growth due to a reduction in the light available for photosynthetic growth. Using the standard 

algal test method it is not possible to differentiate between reduced algal growth by light 

absorption of the colored test solutions or an inherent toxic effect of the test item. 

The absorption spectra of chlorophyll a, the principal photoreceptor in the chloroplasts of green 

plants, shows a double peak (Bidwell, 1979) with maximum absorption occurring at 

approximately 460 nm and 665 nm. As such, if colored solutions formed by the test item show 

significant absorbance at either of these wavelengths it would reduce the light intensity available 

for photosynthetic growth to occur. 

In cases where the t~st solutions are shown to have distinct absorbance at 460 nm or 665 nm, the 

range-fmding test and the definitive test will therefore be carried out according to the 

modification to the standard methodology currently recommended by various competent 

authorities. This includes reducing the test volume in the range of 5- 25 mL in order to shorten 

the light path through the test medium and increasing the light intensity to approximately 

8880 lux (±15%) in order to reduce as much as possible the light absorbance effect ofthe colored 

test solutions. 

If, after conducting a range-finding test using the modified method, a reduction in algal growth is 

observed, current regulatory advice is to change the test method to a Lemna Growth Inhibition 

Test (OECD, 2006) as Lemna are a floating plant species and hence colouration of the test 

solutions does not affect photosynthetic growth. 
General Study Plan: 908-25 Page 11 

----------------------------·····--···-············--

3.10 Evaluations 

3.10.1 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, haemocytometer and light 

microscope or by fluorescence measurement. 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. 

3.10.2 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. 

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. 

3.10.3 Analysis of Test Item Concentrations 

3.10.3.1 Sampling 

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

and definitive tests for analysis. 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 (or solvent 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. 
General Study Plan: 908-25 Page 12 

If required, in order to investigate possible losses of test item due to volatility when the test item 

vessels are opened on a daily basis, a fourth replicate vessel may be prepared at 0 hours and 

incubated alongside the other exposure vessels. This additional replicate will remain unopened 

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

If the 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. 

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

3.1 0.3.2 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. 

It may be necessary for some chemicals which form emulsions or dispersions to filter or 

centrifuge some samples as part of the pre-test validation process to determine the proportion of 

test item in the dissolved form. 

3.10.3.3 Analysis of samples 

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

Laboratories Ltd, Shardlow, UK Analytical Services. 

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. 

If the test item is a formulation, test item concentrations will be determined by the measured 

concentrations of the active ingredient(s). WAFs will be characterized by analysis of the main 

test item component(s). 

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 
General Study Plan: 908-25 Page 13 

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. 

Analytical pre-experiments may be performed within a different Harlan Laboratories study. 

3.11 Data Analysis 

3.11.1 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. 

3.11.2 Calculation of Growth Rates 

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

in biomass from the equation: 

where: 

= 

= 

= 

average specific growth rate from time t1 to tn. 

cell concentration at t1. 

cell concentration at tn. 

time of first measurement. 

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. 
General Study Plan: 908-25 Page 14 

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

following equation: 

I = J.lc- J.lt X 100 r 

J.lc 

where: 

= 

= 

percentage inhibition of average specific growth rate. 

mean average specific growth rate for the control cultures. 

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. 

3.11.3 Yield 

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

equation: 

where: 

= Yield. 

cell concentration at the beginning of the specified period. 

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: 

Where 

= 

= 

= 

percentage inhibition of yield 

mean value for yield in the control group 

mean value for yield for the treatment group 
General Study Plan: 908-25 Page 15 

3.11.4 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 or 

ToxRat. Any data points identified as outliers in the first phase of data evaluation are 

disregarded when fitting the concentration-response curve. The EC10, EC2o, ECso (or ELto, EL2o, 

ELso) values are read from the graph. 

Where appropriate 95% confidence limits for the EC5o (or ELso) will be calculated, using either 

the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949) 

or by Fieller's Theorem (1944). All EC50 (or EL50) values and 95% confidence limits will be 

rounded to two significant figures. 

3.11.5 Statistical Analysis 

The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate 

(LOEL) is the lowest concentration (or loading rate) 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 (or LOEL) 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 (or LOEL) for growth rate and yield at 72 hours will be determined using analysis of 

variance (ANOV A) incorporating a statistical test for homogeneity of variance and an adequate 

post hoc test (e.g. Dunnett test). 

The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is 

the test concentration (or loading rate) immediately below the LOEC (or LOEL) 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 (or LOEL), and 

the NOEC (or NOEL) will be the limit test concentration. 

3.12 Validity Criteria 

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

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%. 
., 

General Study Plan: 908-25 Page 16 

------------------·········-··--·-··-··-·-·· ·-------------

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

72 hours must not exceed 7%. 
'I 

General Study Plan: 908-25 Page 17 

4 REFERENCES 

BIDWELL, R. G. S. (1979) Plant Physiology. 2nd ed. London: Collier Macmillan. 

FIELLER, E. C. (1944) A fundamental formula in the statistics of biological assay, and some 

applications. Quarterly Journal of Pharmacy and Pharmacology, 17, pp. 117-123. 

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 

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

INTERNATIONAL STANDARDS ORGANISATION (ISO) (1999) Standard 14442: Water 

quality - Guidelines for algal growth inhibition tests with poorly soluble materials, volatile 

compounds, metals and waste water. Geneva: International Standards Institute. 

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. 

OECD (2006) Test No. 221: Lemna sp. Growth Inhibition Test. OECD Guidelines for the 

Testing of Chemicals, Section 2: Effects on Biotic Systems. OECD Publishing. 

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

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

ToxRat Professional, ToxRat® Solutions GmbH 

Xlfit, ID Business Solutions Ltd. 
General Study Plan: 908-25 Page 18 

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-nutrients NaHC03 15.0mg/L 

K2HP04 1.044 mg/L 

MgS04.1H20 14.6 mg/L 

MgCh.6HzO 12.16 mg/L 

CaCh.2HzO 4.41 m£"!11 

NaN03 25.5 mg/L 

Trace elements H3B03 186.0 ug/L 

MnCbAH20 415.0 ug/L 

ZnCh 3.27 ug/L 

CoClz.6H20 1.43 ug!L 

CuCb.2H20 O.Ol2ug/L 

Na2Mo04.2H20 7.26 ug/L 

FeCh.6H20 160.0 ug/L 

Na2EDTA.2HzO 300.0 uwL 

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). 

In tests conducted using Navicula pelliculosa the culture medium will be supplemented with 

Na2Si03.9H20 at a final concentration of 1.4 mg Si/L 
General Study Plan: 908-25 Page 19 

Appendix 2 Testing of Volatile Test Items 

In cases where the test item is known (or suspected) to be volatile, the following amendments to 

the study design may be made in order to minimize possible losses of test item over the test 

period: 

• The test vessels used will be glass conical flasks completely filled with test preparation 

and sealed with ground glass stoppers. 

• Additional sodium bicarbonate (up to 500 mg/L final concentration) may be added to the 

prepared culture medium as defined by Herman et al (1990) and Mayer et al (2000) to 

counter act the change in pH due to algal growth. The pH of the culture medium will be 

adjusted to 7.5 ± 0.1 with O.lN NaOH or HCl prior to use. 

• HEPES buffer (1430 mg/L) may be added to the prepared culture medium to counter act 

.the change in pH due to algal growth (ISO, 1999) 

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

are opened on a daily basis a fourth replicate 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. 

Details of these amendments to the study design will be documented in the data.