p . 1

The Dow C hemical Company 1803 BUILDING Midland, Michigan 48674 August 6, 2008 USA f i By overnight courier to : OPPT Document Control Office EPA East, R .-n . 6428 1201 Constitution Avenue, NW Washington, D C ATTN : 8(d) Health and Safety Reporting Rule (Notification/Reporting ) By U .S . mail to : Cz Document Control Office (DCO) (7407M ) Office of Pollution Prevention and Toxics (OPPT) Environmental Protection Agenc y 1200 Pennsylvania Avenue, NW Washington, DC 20460-000 1 ATTN : 8(d) Health and Safety Reporting Rule (Notification/Reporting) _- RE : EPA-IfO-OPPT-2005-005 5 Dear Sir or Madam: With this letler The Dow Chemical Company ("Dow") is submitting a list of 40 health and safety studies related to acetamide, 2,2-dichloro-N,N-di-2-propenyl-, CAS No . 37764-25-3, known as dichlormid . These studies were previously submitted to the Office of Pesticide Programs ("OPP") without claim of confidentiality . Dow is also hereby submitting copies of five studies on dichlormid which may not have been previously submitted to OPP

. Dichlormid was added to 40 CFR § 716 .120 by notice at 71 Fed . Reg . 47130 (Aug . 16, 2006)
. Prior to the submission deadline, Dow conducted an adequate file search for responsive dichlormid s :udies, as required by 40 CFR § 716 .25, but found none . Recently, Dow discovere&~' studies on di-,hlormid which had not been found in the course of the 2006 file search . This submission provides information on those newly-discovered studies

. Sincerely,

. . , , -~, Linda C . Bu)-gert ~ ~,~ 

# Il/u ~ , .~ EH&S Product Regulatory Management ;;l

# li/i'~

# Ill// liii! I The Dow Cl

:emical Company s h o~~~ ~~~~ 0 li ~I ~ I~ ~ I~I 1803 Building Midland, MI 48674 PH : 989-636-1011 FAX : 989-638-9933 E-MAIL : lb ur)zert@dow .com 

# j t Attachments : List of dichlormid studies previously submitted to OPP

Five additional dichlormid studiesp . 2

List of studies of Acetamide, 2,2-dichloro-N,N-di-2-propenyl-, CAS No . 37764-25-3, known as

dichlormid, previously submitted to EPA's Office of Pesticide Program s TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTED
Office of Pesticide Program s Dichlormid : 1 Yr. Oral Toxicity Study in US Environmental Protection Agenc y Dogs Aug-02

Dichlormid :Technical : Acute Neurotoxicity Office of Pesticide Program s Study in Rats MRID 46353805 Nov-02

Dichlormid : 90 day SubChronic Office of Pesticide Program s Neurotoxicity Study in Rats MRID 46353806 Dec-02

Dichlormid : 14-Day Repeat Oral Dosing Office of Pesticide Program s Study in the Rat MRID 46366001 Mar-01

Office of Pesticide Program s Dichlormid : 90-Day Feeding Study in Rats MRID 44606406 Jan-88

Dichlormid : Multigeneration Reproduction Office of Pesticide Program s Toxicity Study in Rats MRID 46353801 Jun-00

Dichlormid : an Evaluation in the In Vitro Office of Pesticide Program s C o enetic Assay in Human Lymphocytes MRID 41561407 Jan-89

Page 1p . 3

TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTED

Dichlormid : 4-Hour Acute Inhalation Office of Pesticide Program s Toxcity in the Rat MRID 44606403 Jan-91

Dichlormid : Teratogenicity Study in the Office of Pesticide Program s Rabbit MRID 44606407 Jan-89

Office of Pesticide Program s Dichlormid : Biotransformation in the Rat MRID 46353802 Jun-00

Dichlormid : Excretion and Tissu e Distribution of a Single Oral Dose (100 Office of Pesticide Program s mg/kg) in the Rat MRID 46353803 Jun-00

Dichlormid : 2-Year Dietary Toxicity and Office of Pesticide Program s Oncogenicity Study in Rats MRID 44751801 Jan-98

Dichlormid : 80-week Oncogenicity Study in Office of Pesticide Program s Mice MRID 44529401 Jan-98

Dichlormid : 90-Day Oral Dosing Study in Office of Pesticide Program s Dogs MRID 41419401 Mar-90

Dichlormid : (R25788) : Acute Dermal Office of Pesticide Program s Toxicity to the Rat MRID 44606402 Jan-90 Page 2p . 4

TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTE D

Office of Pesticide Program s Dichlormid : Skin Irritation to Rabbit MRID 42807902 Jan-90

Office of Pesticide Program s Dichlormid : Eye Irritation to the Rabbit MRID 42807901 Jan-90

Office of Pesticide Program s Dichlormid : Acute Oral Toxicity to the Rat MRID 44606401 Jan-90

Dichlormid : Skin Sensitization to the Office of Pesticide Program s Guinea Pig MRID 44606405 Jan-90

Office of Pesticide Program s Dichlormid : Terato enicit Study in the Rat MRID 44606408 Jan-89

Office of Pesticide Program s Ocular Irritation Test for R-2578 MRID 44606404 Jan-89
Evaluation of the Potential of Dichlormid t o Induce Unscheduled DNA Synthesis in th e In Vivo-In Vitro Hepatocyte DNA Repair Office of Pesticide Program s Assay in the F-344 Rat MRID 41561408 Jan-89
Office of Pesticide Program s R-25788 : Safety Evaluation by Deitary US Environmental Protection Agency Feeding to Rats for 13 Weeks Jan-93
Page 3p . 5

TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTE D
Office of Pesticide Programs R-25788 : Safety Evaluation by Deitary US Environmental Protection Agenc y Feeding to Dogs for 13 Weeks Jan-93

R-25788 : Mutagenicity Evaluation in Bone Office of Pesticide Program s Marrow Micronucleus MRID 41561403 Jan-87

R-25788 : Unscheduled DNA Synthesis in Office of Pesticide Programs Rat Primary He atoc es MRID 44606409 Dec-86
R-25788 : Mutagenicity Evaluation i n L5178Y Mouse Lymphoma Multipl e Endpoint Test Cytogenetic Assay, Sister Office of Pesticide Program s Chromatid Exchange MRID 41561406 Jan-87

R-25788 : Mutagenicity Evaluation i n L5178Y Mouse Lymphoma Multiple Office of Pesticide Program s Endpoint Test Forward Mutation Assay MRID 41561405 Jan-87

R-25788 : Mutagenicity Evaluation in Office of Pesticide Program s Salmonella Typhimurium MRID 41561404 Jan-87
Office of Pesticide Program s 14-Week Inhalation Study with R-25788 in US Environmental Protection Agenc y Rats Jan-93
Office of Pesticide Program s The Acute Oral Toxicity (LD50) of MRID 4156140 1 Dichlormid to the Bobwhite Quail Jan-90 Page 4p . 6

TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTED

The Dietary Toxicity (LC50) of Dichlormid Office of Pesticide Program s to the Bobwhite Quail MRID 41561402 Jan-90

Dichlormid Technical : Determination of Acute Toxicity to Rainbow Trout (Salmo Office of Pesticide Program s gairdneri) MRID 41561419 Jan-89

R25788 : an Investigation of the Toxicity to Office of Pesticide Program s First Instar Daphnia magna MRID 41561418 Jan-89

Office of Pesticide Program s R-25788-Photodegradation in Water MRID 41561410 Jan-89

Additional Studies : R-25788 Aerobic Solid Office of Pesticide Program s Metabolism MRID 41561412 Jan-90
Office of Pesticide Program s Determination of the Water Solubility of R- US Environmental Protection Agenc y 25788 at pH 5 and 9 Jun-89

Office of Pesticide Program s R-25788 Physical Properties MRID 42773501 Jun-89
Office of Pesticide Program s Dichlormid : 1 year Oral Toxicity Study in US Environmental Protection Agenc y Dogs Aug-02
Page 5p . 7

TITLE NAME AND ADDRESS TO WHOM SENT MO/YR SUBMITTED

Dichlormid : 14 Day Repeat Rat Dosing Office of Pesticide Program s Study in the Rat MRID 46366001 Mar-01

Page 6p . 8 BL/B/3471 pagt G7 .1/01 Performing Laboratory Project Identification : R654/A FT68 (

BL/B/3471 DICHLORMID : Determination of the toxicity to Pseudomonas putid a
Study completed : 23 November 1988

Performing laboratory : ICI Brixham Laboratory Imperial Chemical Industries PLC Freshwater Quarr y Brixham Devon TQ5 8BA UK Sponsor : ICI Agrochemical s Imperial Chemical Industries PLC Fernhurs t Haslemere Surrey GU27 3JE UK

Authors J I Mather J F Tapp Approved by B R H William s March 1989p . 9 FT68(7) /88 BL/B/3471 page 2 of 11

BL/B/347 1 DICHLORMID : Determination of the toxicity to Pseudomonas putidaP . 10 FT68(7)/88 BL/B/3471 page 3 of 1 1

BL/B/3471 DICHLORMID : Determination of the toxicity to Pseudomonas putida Brixham study number R654/ A AUTHENTICATION I, the undersigned, hereby declare that this study was performed under my direction according to the principles of Good Laboratory Practice, and that this report represents a true and accurate record of the results obtained .

The following personnel carried out work on this study :

The conduct of this study has been inspected/audited in accordance with Imperial Chemical Industries' policies and procedures for Good Laboratory Practice, as follows Date of Date Inspection/audit QA report 22/23 .11 .88 Study conduct 24 .11 .88 26 .02 .89 Draft report 27 .02 .8 9 Final report 10• 3-~~P . 11 FT68(7)/88 BL/B/3471 page 4 of 1 1

BL/B/3471 DICHLORMID : Determination of the toxicity to Pseudomonas putida

CONTENTS page TITLE PAGE 1 CONFIDENTIALITY STATEMENT 2 GOOD LABORATORY PRACTICE AUTHENTICATION STATEMENT 3 CONTENTS PAGE 4 I SUMMARY 5 2 INTRODUCTION 5 3 TEST SUBSTANCE AND REFERENCE SUBSTANCE 5 4 MATERIALS AND METHODS 6 5 RESULTS 8 REFERENCES 8 TABLES 1 AND 2 9-10 FIGURE 1 11 The last page of this report is . . . . . . . . . . . . . . . . . . . . . . . . . . .11p . 12 BL/B/3471 page 5 of 11

I SUMMAR Y The bacterial growth inhibition of dichlormid technical was determined by measuring the toxicity of the substance to the bacterium Pseudomonas putida . A range of concentrations was tested

. The ECIO and EC50 values were found to be 370 and 1180 mg/1 nominal respectively
. 2 INTRODUCTION At the request of ICI Agrochemicals, Imperial Chemical Industries PLC, Fernhurst, Haslemere, Surrey, GU27 3JE, UK, the toxicity of dichlormid technical to Pseudomonas putida was determined at ICI Brixham Laboratory . The work was carried out under Study Number R654/A during the period 9-23 November 1988 . All original data together with other records relating to the study are filed in the Brixham Laboratory archive

. 3 TEST SUBSTANCE AND REFERENCE SUBSTANCE 3 .1 Test substanc e The test material, dichlormid technical, was supplied by ICI Agrochemicals, Imperial Chemical Industries PLC

. IUPAC chemical name : N,N-diallyl-2,2-dichloroacetamide Chemical Abstracts name : 2,2-dichloro-N,N-di-2-propenylacetamide Molecular structure : C',CHCO. N(C H2CH= CH 2)2

The sample was received at Brixham on 25 July 1988 and assigned the Brixham test substance reference number R654 . The material was a golden coloured liquid and the certificate of analysis for the batch of material stated that its purity was 97 .3% w/w (sample request number SA-2115-UK, sample number R-149 dated 12 July 1988)
. The sample was stored in the dark at ambient temperature in the container in which it was received until required for testing when an appropriate subsample was provided for the test operator

.p . 13 BL/B/3471 page 6 of 1 1

3 .2 Reference substanc e The reference substance employed was 3,5 dichlorophenol (99%), supplied by Aldrich Chemical Co Ltd, Gillingham, Dorset, UK
.
4 MATERIALS AND METHOD S The toxicity was measured by a method based on that originally described by Bringman and Kuhn (Ref 1) which has recently been modified by Slabbert (Ref 2) . This procedure measures the degree of inhibition of growth of a pure culture of Pseudomonas putida during a 6 hour period when the cells are in the logarithmic growth phase . 4 .1 Pseudomonas putid a Pseudomonas putida , strain NCIB9494, was obtained as a freeze-dried culture from National Collections of Industrial and Marine Bacteria Ltd, Aberdeen, UK . This was stored at 4°C until use . The freeze-dried culture was rehydrated in 0 .5 ml of nutrient broth (Oxoid Ltd) . A loop of this suspension was streaked onto a nutrient agar (Oxoid Ltd) slope in a universal bottle . This was incubated at 25°C for 24 hours, and then stored at laboratory temperature, until use as the stock culture
. 4 .2 Preparation of stock solutions 4 .2 .1 Test substanc e

Deionised water was added to 4 g of dichlormid technical to give a total volume of 1 litre . This was stirred in the dark for 5 days and allowed to stand in the dark for a further 9 days . This gave a nominal 4 g/1 stock solution of dichlormid technical which was used for the test . 4 .2 .2 Reference substanc e A nominal 500 mg/1 stock solution of 3,5 dichlorophenol was prepared by taking 0 .125 g of 3,5 dichlorophenol, making up to 250 ml with deionised water and stirring overnight
. 4 .3 Growth medium A basal salt solution was prepared by dissolving 26 .25 g dipotassium hydrogen phosphate 11 .25 g potassium dihydrogen phosphat e 1 .175 g trisodium citrate dihydrate 2 .5 g ammonium sulphat e 0 .25 g magnesium sulphate heptahydrat e in deionised water and making up to I litre with deionised water
. This solution was autoclaved at 121°C for 15 minutes
.p . 14 BL/B/3471 page 7 of 1 1

A olucose solution was prepared by dissolving 6 .25 g of glucose in deionised water and making up to 1 litre with deionised water . This solution was autoclaved at 121°C for 15 minutes
. Equal volumes of the salt solution and the glucose solution were mixed together in a sterile flask to form a growth medium concentrate . This was stored at 4°C until use

. 4 .4 Preparation of test inoculum

18-20 hours before the start of the test 4 ml of test medium concentrate were added to 46 ml of deionised water in a sterile conical flask . A loop of Pseudomonas putida stock culture was added to this growth medium solution, and then incubated overnight at 25°C on an orbital shaker (150 rpm)
. After this period the cells were diluted by addition of fresh growth medium solution at 25°C to an optical density which gave an absorbance of 0 .8 (+/- 0 .05) at 600 nm (4 cm cells) on a Uvikon 860 spectrophotometer . This was used as the test inoculum
. 4 .5 Test procedur e

A range of nominal concentrations (10, 32, 100, 320, 1000, 1600 and 3200 mg/1) of dichlormid technical was set up under sterile conditions, in duplicate conical flasks containing the inoculated growth medium . Three further flasks containing the reference toxic suhatance, 3,5 dichlorophenol, at 18 mg/1 nominal and three control flasks were also set up . In addition a series of single flasks was also included, to run in parallel at the control and each of the test substance concentrations, but containing no inoculum . These were the blank and chemical controls used to compensate for any background colour or turbidity
. The experimental design is shown in Table 1 . Each flask contained 4 ml of growth medium concentrate, the quantity of test or reference substance as shown, 1 ml of inoculum (except in the blank and chemical control flasks), and deionised water to give a final flask contents volume of 50 ml . After shaking at 150 rpm for 6 hours at 25°C on an incubator shaker, the optical density of each flask content was measured at 600 nm on a Uvikon 860 spectrophotometer . 4 cm quartz optical cells wer e used, with 8 % v/v growth medium solution in the reference cell

.p . 15 BL/B/3471 page 8 of 1 1

5 RESULTS Where flask contents were set up in duplicate or triplicate, the mean of the optical density values obtained were calculated (Table 2) . For the control and test concentrations, the corresponding blank and chemical control optical density values were then subtracted . The degree of inhibition of growth compared with that of the controls was calculated as follows : Mean optical density Chemical ) ( value of test - contro l X ( flask contents valu e inhibition 1 - ) x 100

( Mean optical density Blan k ( value of control - control )

flask contents value ) Percentage inhibition was plotted against log10 concentration (Figure 1) and the EC10 and EC50 values were read directly from this graph . These were the concentrations of dichlormid technical which caused 10 and 50% inhibition of growth respectively . The EC10 and EC50 values were 370 and 1180 mg/1 (nominal concentrations) respectively . The nominal 18 mg/1 solution of the reference substance, 3,5 dichiorophenol, gave 92w inhibition of growth indicating that the Pseudomonas putida culture was responding normally to this known toxicant .
REFERENCES I Bringman G and Kuhn R (1980) Comparison of the toxicity thresholds of water pollutants to bacteria, algae and protozoa in the cell multiplication inhibition test . Water Research 14, 231-241 - 2 Slabbert J L (1986) Improved bacterial growth test for rapid water toxicity screening . Bull Environ Contam Toxicol 37, 565-569p . 16 BL/B/3471 page 9 of 11

TABLE 1

DICHLORMID : DETERMINATION OF THE TOXICITY TO Pseudomonas putida EXPERIMENTAL DESIGN Client : ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technica l Test organism : Pseudomonas putid a Test medium : Artificial culture medium

Nominal Vol Growth stock of De- mediumFlask Nominal soln stock ionised concen- Inocnumber Contents concn concn soln water trate ulu m (ml) (ml) (ml) (ml)
1-3 Control - - - 45 4 1 4-6 3,5 dichlorophenoi 18 mg/1 500 mg/1 1 .8 43 4 1 7,8 Dichlormid 3200 mg/1 4 g/1 40 5 4 1 9,10 Dichlormid 1600 mg/1 4 g/l 20 25 4 1 11,12 Dichlormid 1000 mg/1 4 g/1 12 .5 32 .5 4 1 13,14 Dichlormid 320 mg/1 4 g/1 4 41 4 1 15,16 Dichlormid 100 mg/1 4 g/1 1 .25 43 .8 4 1 17,18 Dichlormid 32 mg/1 4 g/l 0 .4 44 .6 4 I 19,20 Dichlormid 10 mg/1 4 g/1 0 .125 45 4 1 21 Blank - - - 46 4 - 22 Dichlormid 3200 mg/1 4 g/1 40 6 4 - 23 Dichlormid 1600 mg/1 4 g/1 20 26 4 - 24 Dichlormid 1000 mg/1 4 g/1 12 .5 33 .5 4 - 25 Dichlormid 320 mg/1 4 g/l 4 42 4 - 26 Dichlormid 100 mg/1 4 g/1 1 .25 44 .8 4 - 27 Dichlormid 32 mg/1 4 g/1 0 .4 45 .6 4 - 28 Dichlormid 10 mg/1 4 g/1 0 .125 46 4 -p . 17 BL/B/3471 page 10 of 11

TABLE 2

DICHLORMID : DETERMINATION OF THE TOXICITY TO Pseudomonas putida OPTICAL DENSITY RESULT S
Client : ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technica l Test organism : Pseudomonas putid a Test medium : Artificial culture medium

Mean of minus optical value fo rFlask Contents density uninoculated Izhinumber (nominal concentrations) at 600 nm solution bition (4 cm cells )
1-3 Control 0 .575 0 .53 5 4-6 18 mg/1 3,5 dichlorophenol 0 .044 92 7,8 3200 mg/1 dichlormid 0 .032 0 100 9,10 1600 mg/1 dichlormid 0 .211 0 .173 68 11,12 1000 mg/1 dichlormid 0 .353 0 .315 41 13,14 320 mg/1 dichlormid 0 .539 0 .502 6 15,16 100 mg/1 dichlormid 0 .557 0 .518 3 17,18 32 mg/1 dichlormid 0 .560 0 .522 2 19,20 10 mg/1 dichlormid 0 .602 0 .560 0 21 Blank uninoculated 0 .04 0 22 3200 mg/1 dichlormid uninoculated 0 .037 23 1600 mg/1 dichlormid uninoculated 0 .038 24 1000 mg/1 dichlormid uninoculated 0 .038 25 320 mg/I dichlormid uninoculated 0 .037 26 100 mg/I dichlormid uninoculated 0 .039 27 32 mg/1 dichlormid uninoculated 0 .038 28 10 mg/1 dichiormid uninoculated 0 .042p . 18 BL/B/3471 page 11 of 11

FIGURE 1

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# ENECA Agrochemicals Report Senes RJ 2668B 

## Jea

## btt 's Hill Research Statio n Bracknell Berkshire U K

Dichlormid Toxicity of Technical Material to the Earthworm Eisenia fetida in an

Artificial Soil Test
.

Authors : D Jackson and J M Coulson Research Department
Authorised : Dr P J Campbell Date of Issue : 17 December 1998

[ Page 1 of 16 Pagesp . 20

Page 1 Report No : RJ2668B ZENECA Ag Products (ZENECA Inc .) Wilmington Delaware 19850 Study Title Dichlormid : Toxicity of Technical Mate rial to th e Earthworm Eisenia fetida in an Artificial Soil Test
Data Requiremen t Supplemental

Authors D Jackson and J M Coulso n Study Completed On 08 December 1998
Performing Laborato ry ZENECA AGROCHEMICALS (ZENECA LIMITED) JEALOTTS HILL RESEARCH STATION BRACKNELL, BERKS, RG42 6E T

UK

Laborato ry Project ID 98J H005
Page 1 of 16 Pages

1, 1p . 21
Page 2 Report No : RJ26686 Study Number 98JH00 5 Report Tide : Dichlormid : Toxicity of Technical Mate rial to the Earthworm Eisenia fetida in an A rtificial Soil Test

- .p . 22
Page 3 Report No : RJ2668B Study Number : 98JH005 Report Title : Dichlormid : Toxicity of Technical Material to the Earthworm Eisenia fetida in an Artificial Soil Test
.

GOOD LABORATORY PRACTICE COMPLIANCE STATEMEN T I, the undersigned, declare that the objectives laid down in the protocol were achieved and that the data generated are valid. The report fully and accurately reflects the procedures used and the raw data generated in the above study
. The study was conducted in compliance with the United Kingdom Good Laboratory Practice Regulations 1997 . These regulations are in accordance with the Organisation for Economic Co-operation and Development Principles of Good Laboratory Practice
. These international standards are acceptable to the United States Environmental Protection Agency and this study, therefore, satisfies the requirements of 40 CFR Part 160
.

Date

Date

Submitter Datep . 23
Page 4 Report No : RJ2668B Study Number 98JH005 Report Title : Dichiormid : Toxicity of Technical Material to the Earthworm Eisenia fetida in

an Artificial Soil Test

. 

### Quality Assurance Statemen t In accordance with ZENECA Agrochemicals policy and procedures for Good Laboratory Practice, the conduct of this study has been inspected/audited by the Quality Assurance Section at JealotYs Hill Research Station, Bracknell, Berkshire, UK
. Date of Inspection Inspection/Audit Date of Inspection Repo rt 02 Feb 1998 Protocol 02 Feb 199 8 16 Apr 1998 Study Conduct 16 Apr 1998 09 Nov 1998 Final Report 10 Nov 199 8 In addition, the following facility and procedure inspections associated with this type of study have been carried out
. 16 Dec 1997 Laboratory Facilities 19 Jan 199 8 So far as can be reasonably established, the methods described and results incorporated in this report accurately reflect the raw data produced during the study

.

t J A Vickers 151 

# L ctmhe'y

# - l~8 Qua Assurance Datep . 24

Page 5 Report No : RJ2668B CONTENTS Page No TITLE PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 CONFIDENTIALITY STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 GLP COMPLIANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 QUALITY ASSURANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 SUMMARY . . . .. .. .. .. .. .. .. . . .. .. .. .. .. . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. . . . . . . .. .. .. ... . ... .. .. .. .. .. .. .. .. . .. . . .. .. .. . ... . . .. .. .. .. 6 1 INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 2 MATERIALS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 .1 Test Chemicals .. .. .. . .. . .. .. .. .. . . .. . .. .. .. .. .. . . . . . .. .. .. .. .. .. . . . . . .. .. . .. .. .. .. .. . . . .. . ... .. .. .. .. .. . . . . . .. .. .. .. .. .. . . .. .. .. . .7 2 .1 .1 Dichlormid Technical Material
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 2 .2 Test Animals .. .. .. .. .. .. . . . . .. .. . . . . . . . .. .. .. . . .. . . .. . .. .. .. .. .. .. . . .. . . . . . . .. .. . . .. .. ... . . .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. . . .. .. .. . . 7 2 .3 Test Soil
. . . . . . . . . . . . . . . . . .. .. . . .. .. . . . . .. . . .. . . . . . . . . . . . . .. .. .. .. . . .. . . .. . .. . . . .. .. .. .. . .. .. . . .. .. .. .. .. .. .. .. .. . . . . . . . . . . . . .. .. .. . .. . .8 3 EXPERIMENTAL PROCEDURES
. .. .. .. . . .. .. . . . . . . .. .. .. . . . . . . .. .. .. .. ... .. .. . . .. .. .. . . .. .. .. .. .. . .. . . ... .. .. .. . . .. .. .8 3 .1 P reparation of Test Solutions for Initial Test . . . .. .. . . . . .. .. .. .. . .. .. ... . .. .. .. .. .. .. .. .. .. .. .. . ... .. .. .. .. .. .. .8 3 .2 Preparation of Test Solutions for Definitive Tests
. . .. .. . . . . . .. .. . . . . . . .. .. . . .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .8 3 .3 Application and Incorporation of Test Solutions in A rtificial Soil . .. .. . .. .. .. .. .. .. .. .. .. .. . . . . .. .8 3 .4 Addition of Test Earthworms ... .. .. .. .. .. .. .. . . . .. .. .. . .. .. .. .. . . ... .. .. .. . .. .. .. .. .. .. .. .. .. .. . .. .. . . ... . .. ... . ... . . . .. .9 3 .5 Test Conditions . ... . ... . ... . .. .. .. . . . ... .. .. .. . . .. .. .. . .. .. .. .. . .. .. .. ... . ... . . .. .. ... .. .. .. . . . . .. .. .. .. .. .. .. .. .. . .. . . . . ... . . . .. .9 3 .6 Assessment of Ea rthworms
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 3 .7 Statistical Analyses
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 4 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 5 CONCLUSIONS
. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 PERSONNEL AND TEST FACILITIES
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 7 STUDY DATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 8 RETENT I ON OF RECORDS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 9 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Appendix I Table 1 : Initial Test - Number of dead and weight of live if
. fetfda after 14 days exposu re to Dichiormid technical mate ri al in artificial soil
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 Table 2 : Definitive Test Number of dead and weight of live if
. feiida after 14 days exposure to Dichlormid technical material In artificial soil . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14 Table 3 : Number of dead and weight of live if
. fetlda after 14 days exposu re to 2-

chloroacetamide in a rtificial soil . . ... .. .. .. ... .. .. .. .. .. .. .. . . . . . . ... .. .. ... .. . .. . . .. .. .. .. .. .. .. .. . ... .. .. .. ... . . . . .1 5 Table 4 : Treatment means of E
. fetida bodyweights after 14 days exposure to Dichiormid technical material in artificial soil. . . .. .. .. .. . .. .. .. .. .. .. .. .. . . ... . ... .. .. . .. .. . .. .. .. .. .. .. . . .. ... . .. .. . . .. .. .. ... .. .16

}p . 25
Page 6 Report No : RJ26688 Study Number : 98JH00 5 Report Title : Dichlonrid : Toxicity of Technical Material to the Earthworm Eisenia fetida in an Artificial Soil Test Authors : D Jackson and J M Coulson SUMMAR Y Eisenia fetida (clitellate adults) were exposed to concentrations of dichlormid as technical material in an artificial soil for 14 days at 20 ± 3°C . An initial and a definitive test were conducted

. The value for the 14 day LC ., was 391 mg ai kg -' soil with confidence limits of 125 and 500 mg ai kg-1 . At the 31 .25 mg ai kg- ' soil level, there were no deaths, behavioural effects, significant effects on external conditions or significant bodyweight changes (P=5 %) and thus the No Observed Effect Level (NOEL) was at least 31 .25 mg ai kg " soil
.
The LC 50 value for 2-chioroacetamide, tested as a toxic standard, was 52 mg kg''

.p . 26

. 

### • Page 7 Report No : RJ2668B 1 INTRODUCTIO N

The toxicity of dichlormid as technical material to the earthworm Eisenia fetida was determined in laboratory tests based on the OECD' and EEC2 guidelines . The earthworm was chosen as the test organism as a representative of invertebrates potentially at risk in the soil
. E. fetrda was selected as the test species as it is widely used in earthworm laboratory tests and is recommended by the above OECD and EEC guidelines . This study was carried out between February and April 1998

. 2-chioroacetamide is tested periodically to monitor the response of earthworms to a substance of known toxicity . The study with 2-chloroacetamide3 (RJ2395B) with earthworms from the same source as those used for the test with dichlormid, was conducted in July 1997 at ZENECA Agrochemicals, Jealotts Hill Research Station, Bracknell, Berkshire, UK
. 2 MATERIALS 2 .1 Test Chemical s 2 .1 .1 Dichlormid Technical Materia l

IUPAC Name : NN-diallyl-2,2-dichloroacetamide

. Technical dichlormid (R-25788 Tech . Lot No . GBL1601) was used . Analysis (analytical reference WRC 14107-31, March 1993) by ZENECA Ag Products, Western Research Center, showed it to contain 97 .9% w/w dichlormid

. The stability of the material was confirmed by analysis in September 1997 by Product Characterisation Sciences, ZENECA Agrochemicals, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK. Between this date and use, the dichlormid material was stored at ambient temperature and was presumed to be stable under these conditions . The dichlormid was in acetone for no more than 55 minutes prior to application to the soil in the definitive test and was assumed to be stable for this period . To ensure homogeneity of the test substance, each concentration was shaken prior to aliquot removal during the dilution process. Immediately before application to the soil, each concentration was thoroughly shaken

. 2 .2 Test Animal s

Eisenia fetida used were clitellate adults that had been cultured in bulb fibre compost at Jealott's Hill . The earthworms were supplied by Monkfield, The Aquatic Centre, Cambridge, U .K. The cultures arrived at Jealott's Hill in spring 1990 and have since been cultured in a controlled environment room

. The mean weight t standard deviation (SD) for E

. fetida used were 0 .50 t 0 .03 g and 0 .46 t 0 .03 g in the initial and definitive test, respectively

.p . 27
Page 8 Report No ;RJ2668B 2 .3 Test Soi l

The test soil was a mixture of the following ingredients in oven dry (approximately 105°C) weight proportions : 70% fine silica sand (Grade 110), supplied by British Industrial Sands, Redhill, Surrey, U
. K 20% kaolinite clay (GTY Powder), supplied by English China Clay International Ltd, John Keay House, St. Austell, Cornwall, PL25 4 13J, U .K
. 10% peat (ICI Growers Blocking Compost) organic matter 90 .5%, supplied by Munro Horticulture Ltd, Goodwood, Chichester, Sussex U .K
. Calcium carbonate was incorporated into the artificial soil at 5 g kg-' Ingredients, in their natural fresh weight form, were mixed in a rotary cement mixer

. 3 EXPERIMENTAL PROCEDURE S 3 .1 Preparation of Test Solutions for Initial Tes t

Dichlormid solutions were prepared in acetone and made up to 25 cm' for application to the artificial soil . 25 cm' of acetone was used in the preparation of the control soil

. The solutions were applied to 1 .25 kg (dry weight equivalents) of artificial soil . Soil concentrations were 0, 1, 10, 100 and 1,000 mg ai kg' artificial soil (dry weight)

. 3.2 Preparation of Test Solutions for Definitive Test s

The initial test indicated that the LC ,, was between 100 and 1,000 mg ai kg'' soil

. Dichlormid was tested at 31 .25, 62.5, 125, 250, 500 and 1,000 mg ai kg -' in the definitive test. The dichlormid solutions were prepared in acetone and made up to 50 cm3 for application to 2 .5 kg (dry weight equivalents) of artificial soil . The control comprised 50 cm3 of acetone per 2 .5 kg artificial soil
. 3 .3 Application and incorporation of Test Solutions in Artificial Soi l

The weighed amount of test soil was spread out evenly over a sheet of "Benchkote" in a fume cupboard. The test solution was applied evenly over the soil surface from a 50 cm' gas tight syringe fitted with a fan-jet . The acetone was allowed to evaporate for approximately 60 minutes before the soil was placed in a mixing bowl . The weighed artificial soil was then placed into a food mixer bowl . The soil was mixed for two minutes by a heavy duty food mixer to evenly incorporate the test substance into the soil . Deionised water was then added to bring the moisture content of the soil up to 50% and mixing continued for a further two minutes

. From the prepared treated soil, 750 g (fresh weight) aliquots were weighed into 1 litre glass beakers . For the initial test, E

. fetida were exposed in two aliquots (replicates) of treated soil and for the definitive tests four aliquots of soil per treatment

.p . 28
Page 9 Repo rt No : RJ2668B 3 .4 Addition of Test Ea rthworm s

Clitellate E. fetida, which had been preconditioned in untreated a rtificial soil overnight, were selected at random into groups of ten . Each batch of ten E. fetida was weighed so that any weight change at the end of the exposure pe riod could be determined . The mean weight and standard deviation of E . fetida used in the test was determined from these weighings . Th e batches of ten worms were ranked into weight order and then split into four g ro ups ranging from ' heaviesY, to "lightest" . One batch fro m each group was randomly allocated to each treatment. The E
. fetida were placed on the surface of the soil between 1 and 2 hours after chemical treatment, and cling film was placed over the top to prevent them from escaping . Six small holes were made in the cling film to allow ventilation
. 3 .5 Test Condition s

Th e conditions throughout the tests were as follows : Soil moisture contents were within the range 50 ± 10% throughout the test pe ri od (measured at the beginning and end of the tests) . Moisture contents were maintained by surface watering with deionised water. The pH of the soils remained within the range 6 .2 ± 0 .2 . E

. fetida in the test soil were held in a constant environment room at 20 t 3°C . A 24 hour light regime was maintained from six fluorescent white tubes . Light intensity was 600 ± 200 lux. 3 .6 Assessment of Ea rthworms

E . fetida were assessed for mortality and any obvious abnormal effects after 14 days in the initial test and after 7 and 14 days in the defi nitive tests . The soil was tipped onto a clean su rface and E . fetida were recorded as dead if there was no response to a mechanical stimulus of their front end . Any E. fetida not found after careful searching of the soil were assumed to be dead . Each batch of ten E. fetida was weighed at the beginning and end of each test to determine any change in bodyweight. 3 .7 Statistical Analyses

LC ,, estimation on the mo rtality data was carri ed out by logit analysis . There were ve ry little data available with which to construct a dose-response curve . To overcome this, the 0% response at the 250 mg ai kg

-' rate was adjusted upwards by 2 .5% at each replicate in order to be able to fit a curve. The LCw was estimated a s 391 mg ai kg' . The confidence limits produced were 125 and 500 mg ai kg
- ' The highest two treatment rates were omi tted from the analysis of bodyweight because of insufficient data and because a clear effect on mo rtali ty existed at these concentrations . The remaining bodyweight data was analysed by analysis of variance appropriate to a randomised complete block design with the blocks determined by the initial bodyweights . The F-test for the overall effect of treatment was statistically significant (P=0.0002) and so, using the pooled estimate of error variance,p . 29
Page 1 0 Report No : RJ2668B comparisons were made between each treatment mean and that of the control using t-tests (two-sided)
. LC50 estimation was carried out using LOGITPC, version 1 .2' . The bodyweight data was analysed using SAS, version 6 .115
. 4 RESULTS

Mortality and weights of live E
. fetida at the beginning and end of the tests are presented in Table 1 for the dichlormid initial test and Table 2 for the definitive test

. Table 3 presents the data for 2-chioroacetamide from Reference 3
. The 14 day LC5, value for dichiormid was 391 mg ai kg-' with confidence limits of 125 - 500 mg ai kg''
. E . fetida bodyweights at the start and end of each test are presented alongside the mortality results (Tables 1 and 2) . Analysis of variance of final bodyweights is presented in Table 4 . The rates 250, 125 and 62 .5 mg ai kg-' resulted in statistically significant differences in bodyweight change relative to the contro l At the 31 .25 mg ai kg-' soil level, there were no deaths, behavioural effects, significant bodyweight changes (P = 5%) and thus the No Observed Effect Level (NOEL) was at least 31 .25 mg ai kg' soil
. The 14 day LC50 value for 2-chloroacetamide was 52 mg kg-' with confidence limits of 40 - 80 mg kg', showing that the test system was valid

. 5 CONCLUSION S

The 14 day LC50 value for dichlormid technical material was 391 mg ai kg-' soil . The 14 day No Observed Effect Level was at least 31 .25 mg ai kg-'
. 6 PERSONNEL AND TEST FACILITIE S

The tests were carried out by D Jackson, H J Gough and T Fleming in the Ecological Risk Assessment Section, JealotYs Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK
. Statistical analyses were carried out by E C Mclndoe in International Field Biology and Statistics Section, Jealotts Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK
.
7 STUDY DATE S

The experimental phase of the study was carried out between February and April 1998
.p . 30

~ . _ Page 1 1 Report No : RJ2668B 8 RETENTION OF RECORD S

The raw data for this study and a copy of this report are stored under Study Number 98JH005 in the ZENECA Agrochemicals GLP Archives, Jealott's Hill Research Station, Bracknell, Berkshire, RG42 6ET, UK . The data for 2-chioroacetamide are stored under Study Number 97JH240 at the same location

. 9 REFERENCE S

1 . OECD Guidelines for Testing of Chemicals, number 207 (April 1984)

. 2 . EEC Guideline C(L1)4 Toxicity for Earthworms, Artificial Soil Test, Revision 6 prepared for annex V of EEC Directive 791831 (Ref . DG XU128/82)
. 3. Jackson D and J M Coulson (1997) . 2-chloroacetamide : Toxicity to the Earthworm Eisenia fetida in an Artificial Soil Test . ZENECA Agrochemicals Report No

. RJ2395B
. 4 . Muhammad I(1988) . Small Projects Report : User Specification for Micro Logit Analysis Programme LOGITPC, version 1 .2 . ICI Agrochemcials Report No

. JS002A/2
. 5 . SAS Users Guide : Statistics, Version 6 .11 . SAS Institute Inc ., Cary, NC, USA
.

Filename : RJ2668B .doc Location : E :/GROUPMlP/RJ Reference : JMC/C G WINo :40771 Date : 10 November 1998p . 31
Page 1 2 Report No :RJ2668B

Appendix Table 1 : Initial Test - Number of dead and weight of live E . fetida after 14 days exposure to dichiormid technical material In artificial soil
. Table 2 : Definitive Test - Number of dead and weight of live E . fetida afte r 14 days exposu re to dichiormid technical material in artificial soil. Table 3 : Number of dead and weight of live E . fet►da after 14 days

exposu re to 2-chloroacetamide in a rt ificial soil . Table 4 : Treatment means of E. fetida bodyweights after 14 days

exposure to dichionnid technical material in a rtificial soil .p . 32
Page 1 3 Report No : RJ2668B Table 1 : Initial Test - Number of dead and weight of live E . fetida after 14 days exposure to dichlormid technical material in artificial soil
. TEST STARTED - 4 Februa ry 1998 Dichiormid Replicate Numbers Dead Weight of 10 Concentration in Soil out of 10 E .fetida (g) (mg ai kg
- ') Day 0 Day 14 % Change Cont ro l 1 0 5 .45 5 .85 +7 . 3

2 0 4 .88 5 .20 +6.6 1 1 0 5 .46 5 .83 +6 .8

2 0 4 .74 5 .30 +11

. 8 10 1 0 5 .02 5 .28 +5 .2

2 0 4 .70 5 .30 +12 .8 100 1 0 5 .10 5 .15 +1
. 0

2 0 4 .69 4 .89 +4 .3 1,000 1 10 5 .49 - -

2 10 4 .69 -= 100 % mortality at day 14p . 33

- Page 1 4 Report No : RJ2668B Table 2 : Definitive Test
- Number of dead and weight of live E
. fetida after 14 days

exposure to dichiormid technical material in artificial soil
. TEST STARTED - 1 April 1998 Dichlormid Replicate Numbers dead Weight of 10 E . fetida (g) Concentration in Soil out of 1 0 (mg ai kg'') 7 days 14 days pay 0 Day 14 % Change Control 1 0 0 5 .39 5 .15 -4 .5

2 0 0 4 .70 4 .90 +4 .3 3 0 0 4 .35 4 .35 0 4 0 0 4 .23 4 .31 +1
. 9 31 .25 1 0 0 5 .03 4 .77 -5 .2

2 0 0 4 .79 4 .60 -4 .0 3 0 0 4 .57 4 .10 -10 .3 4 0 0 4 .28 4 .18 -2

. 3 62 .5 1 0 0 4 .85 4 .54 -6 .4

2 0 0 4 .81 4 .57 -5 .0 3 0 0 4 .39 4 .22 -3 .9 4 0 0 4 .18 4 .09 -2
. 2 125 1 0 0 4 .65 4 .29 -7 .7

2 0 0 4 .66 4 .46 -4 .3 3 0 0 4 .42 4 .04 -8 .6 4 0 0 4 .04 3 .53 -12
. 6 250 1 0' 0 4 .97 4 .16 -16 .3

2 0' 0 4 .67 3 .55 -24 .0 3 0' 0 4 .51 3 .78 -16.2 4 on 0 4 .27 3 .55 -16
. 9 500 1 9° 10 4 .84 - -

2 10° - 4 .65 - - 3 9°0 9° 4 .57 - - 4 10` - 4 .24 - - 1,000 1 10 - 5 .15 - -

2 10 - 4 .8 2 3 10 - 4 .51 - - 4 10 - 4 .27 - - -= 100 % mortality atdays 7or1 4 a - All worms with behavioural effects, curled up when removed from soil b - Remaining live worm in poor conditio n c - Assumed all dead . Bodies difficult to allocate to individuals due to decomposition d - Only rear segments of remaining worm found, which were movingp . 34

Page 1 5 Report No : RJ26688 Table 3 : Number of dead and weight of live E . fetida after 14 days exposure to 2- chioroacetamide in artificial soil . Abstracted from Reference 3 . TEST STARTED - 15 July 1997 2-chloroacetamide Replicate Numbers Dead Weight of 10 Concentration in out of 10 E
. fetida (g) Soil (mg k9"') 7 14 Start Finish % days days Day 0 Day 14 Change Control 1 0 0 4 .91 4 .59 -6
. 5

2 0 0 5 .58 5 .07 -9 .1 3 0 0 5 .80 5 .33 -8 .1 4 0 0 5 .96 5 .56 -6
. 7 20 1 0 0 5 .50 5 .29 -3 .8

2 0 0 5 .63 5 .08 -9 .8 3 0 0 5 .83 5 .61 -3 .8 4 0 0 6 .00 5 .59 -6
. 8 40 1 0 0 5 .43 5 .26 -3.1

2 1 1 5 .62 5 .32b -5 .3 3 0 0 5 .74 5 .18 -9 .8 4 0 0' 5 .96 4 .70 -21

. 1 80 1 10 - 5 .51 - -

2 10 - 5 .60 - - 3 10 - 5 .68 - - 4 10 - 5 .86 - - = 100 % mortality at day 7 a - I womi affected, several rear segments missin g b Where mortality occurred, the end weight and the percentage weight change were adjusted to account for the dead earthworms using the following calculation : End weight for 10 worms (g) - Weight of remaining worms (g)x No . Exposed (10) No . Survivingp . 35
Page 1 6 Report No : RJ2668B Table 4 : Treatment means of E. fetida bodyweights after 14 days exposure to dichlormid

technical material in artificial soil . Dichlormid Definitive Test Concentration in Mean Final % Change soil (mg ai kg-1) Bodyweight (g) Relative t o Control 250 3 .76 ' -19 .6 125 4 .08 ' -12 .8 62 .5 4 .36 * -6 .9 31 .25 4 .41 -5 .7 Control 4 .68 N/ A ' Statistically significant (P=5%) from the control

.p . 36 nLJyoi/ts page i or z o 

### Performing laboratory project identification T388/A (FT74/90) G6 .2/01

y



### BL3967/B DICHLORMID : Chronic toxicity to Daphnia magna



### Data requirement OECD Guideline Number 202 Study completed 7 December 1990 Performing laboratory Imperial Chemical Industries PLC Group Environmental Laboratory Brix

### ham Devon TQ5 SBA UK Sponsor ICI Agrochemicals Imperial Chemical Industries PLC Fernhurst Haslemere Surrey GU27 3JE UK Authors K M Stewart J F Tapp S A Sankey T D Williams KWJLong Approved by B R H Williams March 1991p . 37



# THIS IS A TRUE COPY OF THE ORIGINAL DOCUMENT AND MAY HAVE MISSING PAGES OR MAY CONTAIN PAGES THAT

ARE HARD TO READp . 3 8 

### r 1 t4lyu BL3967/B page 2 of 26 BL3967B DICHLORMID : Chronic toxicity to Daphnia magna Brixham study number: T388/Ap . 39 FT74/90 BL3967B page 3 of 26 BL3967 B DICHI,oRIMID : Chronic toxicity to Daphnia magna
Brixham study number : T388/A



### GOOD LABORATORY PRACTICE COMPLIANCE STATEMEN T This study has been conducted in compliance with the principles of Good Laboratory Practice (GLP) laid down in the United Kingdom Department of Health Compliance Programme (1989) . These principles are themselves in accordance with the Organisation for Economic Cooperation and Development (OECD) principles of Good Laboratory Practice ISBN 9264 12367 9
. Under the Memorandum of Understanding, signed by both the United States and the United Kingdom, this study is considered to satisfy the requirements of 40 CFR Part 160
. I believe that this study is valid for the purpose for which it was conducted and that this report is a true reflection of the raw data generated
.


## Date y 

## i
Date ~! 31 c f,p . 40 F174/90 BI-3967B page 4 of 26 BL3967/B DICHLORIVIID : Chronic toxicity to Daphnia magna

Brixham study number: T388/A


### QUALITY ASSURANCE STATEMENT The conduct of this study has been inspected/audited in accordance with Impe rial Chemical Industries' policies and procedures for Good Laboratory Practice, as follows Date Inspection/audit Date of QA report 16 November 1990 Study conduct 16 November 1990 8 February 1991 Draft report 20 February 1991 6 MaruU, )9q1 Final report 6 1941



## \ 6f3lRl D 

### 6 Dowe Dat e Quality Assurance Unitp . 41 F174/90 BL3967/B page 5 of 26 BL3967/B DICHLORMID ; Chronic toxicity to Daphnia magna
Brixham study number : T388/A
AUTHENTICATION I, the undersigned, hereby declare that this study was performed under my direction according to the principles of Good Laboratory Practice and that this report represents a true and accurate record of results obtained.

Study Director Date The following personnel carried ou
Principal Investigator Bioassay Date
Principal Investigator Analysis ~ ~ . ~, . 

# 91 Date

Report approved by 5 Programme Manager ~j DateFT 74/90 p . 42 BL3967/B page 6 of 26 BL3967/B 

### DICHLORMID : Chronic toxicity to Daphnia magna CONTENTS page TITLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CONFIDENTIALITY STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT . . . . . . . . . . 3 QUALITY ASSURANCE STATEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 AUTHENTICATION STATEMENT

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 SUMMARY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 INTRODUCTION

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 METHODS AND MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .1 Test substance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 Preparation of test solutions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 .3 Test org anism
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 .4 Dilution water

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 .5 Test apparatus and conditions

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 .6 Experimental design

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3 .7 Test procedure
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3 .8 Feeding
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3 .9 Water quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3 .10 Chemical analysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4 .1 Chemical analysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.2 Observations of mortalities and symptoms of toxici ty
. . . . . . . . . . . . 11 4 .3 Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 .4 Length

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 .5 Water quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 REFERENCES

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 TABLES 1-7
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14-22 APPENDIX 1 (Reconstituted/dilution water)

. . . . . . . . . . . . . . . . . . . . . . . . 23 APPENDIX 2 (Dilution water parameters)

. . . . . . . . . . . . . . . . . . . . . . . . . . 24 APPENDIX 3 (Analytical method)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25-26

(Figure 1 ) The last page of this report is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

### BL3967/B page'74of 26 I SUMMARY Sponsor ICI Agrochemicals, Imperial Chemical Industries PLC, Feinhurst,

Haslemere, Surrey, GU27 HE, U K Contact Dr B D Cavell, telephone : International (44) 428 644061

Mr J F Tapp, telephone : International (44) 803 882882 Location of study, raw Imperial Chemical Industries PLC, Group Environmental Laboratory, data and final report Brixham, Devon, TQ5 8BA, UK Test material Dichlormid technical common name IUPAC Chemical 

### NN-diallyl-2,2-dichloroacetamide name Chemical Abstracts 

### 2,2-dichloro-N,N-di-2-propenylacetamide name Subject Chronic toxicity to Daphnia magna Test doses Control and nominal concentrations of 1 .8, 3 .2, 5 .6, 10, 18, 32 and 56 mg/1 Test dates 16 November to 7 December 1990 Length of test 21 days Nominal test 20 ± 1°C temperature Results based on 95 % mean measured confidence Calculation concentrations Time LC50 interval method as mg of dichlormid ' technical per litre 1-7 day >57 - - 14 day 35 30 - 43 Moving average angle 21 day 24 18 - 32 Moving average angle No observed effect concentration (NOEC) = 1 .7 mg/1 (mean measured) . Test species/strain Daphnia magn a Source of organisms Continuous laboratory cultures Stage of organisms Less than 24 hours old at the initiation of studyBL39b //t3 pagepti & lb 2 INTRODUCTIO N 

### At the request of ICI Agrochemicals, Imperial Chemical Industries PLC, the chronic toxicity of dichlormid technical to Daphnia magna was determined. The study was undertaken between 12 November and 7 December 1990 . The study number was T388/A and the Brixham sample number was T388 . All original data together with other records relevant to this study are filed in the ICI Group Environmental Laboratory archive at Brixham. 

### The methods were based on those described in the relevant OECD method (Ref 1) modified by individually separating the Daphnia. 3 METHODS AND MATERIALS 3.1 Test substance The test substance, dichlormid technical, was supplied by ICI Agrochemicals, Imperial Chemical Industries PLC. IUPAC Chemical name : N,N-diallyl-2,2-dichloroacetamide . Chemical Abstracts name : 2,2-dichloro-N,N-di-2-propenylacetamide . Molecular structure:



### C12CHC0•N(CH2CH=CH2)2

The sample was received at Brixham on 26 April 1990 and assigned the Brixham test substance reference number T388 . The material was a pale yellow liquid and the certi ficate of analysis for the batch of mate rial stated that its purity was 96 .3% w/w ( analytical reference 11842-03, sample identi fication 11489-41-01, analysis dated 10 January 1989 certificate dated 17 April 1990) . Dichlormid technical has a quoted solubili ty in water of 5000 mg/1 at 20°C (ICI Agrochemicals) . During the definitive run of the test 1 .8 g of test substance were used to make up the test solutions .

### BL39671B page"Sf 26
3 .2 Preparation of test solutions Each time new test solutions were prepared, 200 mg of test substance were dissolved in two litres of dilution water to give a stock solution containing 100 mg/l. This stock solution was stirred for 24 hours prior to use to obtain complete dissolution of the test substance
. New test solutions were prepared every Monday, Wednesday and Friday by the addition of appropriate volumes of the stock solution to dilution water . 

### 3 .3 Test organis m The test organism was the freshwater crustacean, Daphnia magna Straus, obtained from continuous laboratory cultures at the ICI Group Environmental Laboratory . The stock cultures of Daphnia were maintained in a reconstituted water medium, identical to the test dilution water, at a temperature of 20 ± 2°C, and a photoperiod of 16 hours light :8 hours dark
. The cultures were fed a defined diet of algae (Chlorella vulgaris) and yeast
. Culture conditions were such that the Daphnia reproduction was by diploid parthenogenesis
. Daphnia <24 hours old, obtained from a single culture vessel, were used for testing . 

### 3 .4 Dilution wate r The reconstituted water medium used for testing (and maintenance of stock cultures) is described in Appendix 1 . The pH, hardness, alkalinity, conductivity and total organic carbon were determined for each batch of dilution water used in this study . Three batches of dilution water were analysed for a range of heavy metals . 

### The water was aerated for >2 hours before use
. 3 .5 Test apparatus and conditions Pyrex glass beakers were used as test vessels, with 10 replicates per exposure concentration, each containing 80 ml of test solution providing a depth of 48 mm . The beakers were covered with loose fitting lids
. The test solution temperature was maintained at 20 ± 1°C . A photoperiod of 16 hours light : 8 hours dark was provided at an average intensity of 746 Lux (cosine receptor, measured during the study) . The test solutions were not aerated
.

### BL3967B page q'l~of 26
3 .6 Experimental design Ten replicate vessels, each containing one Daphnia were employed for the control and each concentration of test substance . The nominal concentrations tested were as follows : Control, 1 .8, 3 .2, 5 .6, 10, 18, 32 and 56 mg/litre. The positions of the test treatments were randomly allocated within the test area
. 3 .7 Test procedure The test procedure used was based on the OECD Guideline No 202 (modified by individually separating the Daphnia) (Ref 1), and followed the ICI Group Environmental Laboratory standard operating procedure AQ108, version 03
. At the start of the test, one Daphnia was added to each test vessel in sequence down the rows . These Daphnia were termed the Po generation. Mortality of the Po generation was recorded daily for each test vessel. Mortality was defined as absence of any movement by the organism, when examined by eye, for a period of 15 seconds . Observations were made at the same time for any other symptoms of toxicity
. Observations were made daily from Day 6 for the presence of offspring (termed Fl generation) in each vessel . The test solutions were prepared on the day of use and were renewed every Monday, Wednesday and Friday. On each renewal day, the new test solutions were dispensed to a second set of test vessels . The surviving P. generation were transferred to the new solutions, using a wide bore pipette, minimising transfer of medium . The old solutions were sampled, as appropriate, for physical and chemical analysis (see Sections 3 .9 and 3 .10 below) and the F, generation removed from each vessel and counted . The numbers of live and dead FI were recorded
. At the end of the test the length (apex of helmet to base of spine) of each surviving Po Daphnia was measured, using a microscope with an eyepiece graticule previously calibrated with a stage micrometer
. The duration of the test was 21 days
. 3.8 Feeding The test Daphnia were fed daily with cultured algae (Chlorella vulgaris), strain CCAP 211/12 and yeast
.BL3967/B page .1417of 26 The algal cultures were harvested and centrifuged and the algal cells resuspended in Daphnia culture medium (reconstituted water) . The algal stocks were measured for cell density by Coulter counter, stored at approximately 4°C in the dark, and used within one week
. A yeast stock was prepared weekly by dispersing 0 .5 g dried bakers yeast in 50 ml of test dilution water, and was stored at approximately 4°C in the dark . A yeast suspension containing 1,000 mg/1 was prepared daily from this stock by making a 10 x dilution with test dilution water . 

### A mixture of the above suspensions was provided daily such that each vessel received 3 .0 x 10' algal cells and 0 .25 mg of dried yeast per day
. 3 .9 Water quality The dissolved oxygen concentration of the dilution water (excess control solution) was measured for each set of test solutions prepared
. The pH of each newly prepared test solution was measured using excess remaining after filling the test vessels . The pH and dissolved oxygen concentration of two of the replicates of the old test solutions were measured after transfer of the P . generation
. Temperatures were measured daily by thermometer in an additional replicate vessel (without Daphnia) for each treatment . Hourly temperature measurements were also recorded automatically in the additional control vessel
. 3 .10 Chemical analysis The analytical method used in this study was based on ICI Group Environmental Laboratory standard operating procedures AL200 version 01 and AL201 version 01 which are summarised in Appendix 3
. On six renewal occasions samples of each new test solution were analysed for the concentration of the test substance . Samples from the corresponding old solutions were analysed on the following renewal day
. 

### 4 RESULT S 4 .1 Chemical analysi s The measured concentrations for the new and old test solutions are given in Table I . The overall mean measured concentrations ranged from 94 to 103% of the nominal values and are referred to as the mean measured concentrations elsewhere in the text . Also reported are the separate mean results for the old and new test solutions

. 

### 4 .2 Observations of mortalities and symptoms of toxicity Mortalities of P
. Daphnia in each treatment on each test day are given in Table 2, together with other symptoms observed
.liL590//13 pagept Ar Lo There were no mortalities in the control and mean measured test concentrations up to and including 18 mg/1 after 21 days . After 21 days there was 100 % mortality in both the 33 and 57 mg/1 mean measured test concentrations . 

### The LC50 values were calculated at various time intervals by ICI Group Environmental Laboratory computer program "LC50" using Stephan's method (Ref 2) . The LC50 values are shown below as mg of technical material per litre based on mean measured concentration s

LC50 95% confidence intervals Calculation method 1-7 day >57 - - 14 day 35 30 - 43 Moving average angle 21 day 24 18 - 32 Moving average angle Reduced food clearance was observed in the 33 and 57 mg/1 mean measured test concentrations (Table 2) . 4 .3 Reproductio n The total number of offspring (F,) obtained from each replicate vessel are given in Table 3, which also shows the mean and standard deviation for each treatment and the control . At mean measured concentrations of 33 and 57 mg/1 (in which 100% mortality of P
. Daphnia occurred) reproduction was concluded to be affected without the need for statistical analysis, and the mean offspring values were not corrected for mortality . The remaining data did not meet the assumptions for analysis of variance . Therefore a nonparametric test, Steel's Many-One-Rank Test (Ref 3) was used to determine significant effects on reproduction. There was a significant (P = 0 .05) decrease in mean number of offspring (FL) produced at the 5 .5 and 18 mg/1 mean measured concentrations compared to the control. At mean measured concentrations of 1 .7, 3 .1 and 10 mg/1 mean reproduction was not significantly reduced
. In the control and test concentrations up to and including 55 mg/1, all individual Po Daphnia released the first offspring by day 9 . Therefore : No observed effect concentration (NOEC) for reproduction = 3 .1 mg/1 (mean measured)
. 4 .4 Length The length of surviving Daphnia (Po) on day 21 are given in Table 4, which also shows the mean and standard deviation for each treatment and the control
. The length data did not meet the assumptions for analysis of variance . Therefore a nonparametric test, Steel's Many-One-Rank Test (Ref 3) was used to determine significant effects on length . At mean measured concentrations of 3 .1 mg/1 and above, the mean length of surviving Daphnia was significantly (P = 0.05) less than that of the control . At a mean measured concentration of 1 .7 mg/1 mean length was not significantly reduced
.t31,390//ts page R -At Lb Therefore : No observed effect concentration (NOEC) for length = 1 .7 mg/1 (mean measured)
. 

### 4 .5 Water quality Temperatures, pH values and dissolved oxygen concentrations of the test solutions recorded during the study are given in Tables 5, 6 and 7 respectively . Water quality parameters for the dilution water batches used are given in Appendix 2
. The overall ranges of temperature, pH and dissolved oxygen were as follows : Temperature 19 .0 to 20.1° C pH (new) 8 .24 to 8 .51 (old) 7 .95 to 8 .82 Dissolved oxygen:- Dilution water (new) 9 .1 to 9 .5 mg/1 Test solutions (old) 7 .3 to 10.2 mg/i The overall range of the temperatures recorded automatically at hourly intervals over the 21 days was 18 .7 to 20 .5°C. Only 27 measurements were outside the range 20 ± 1 .0°C and it is considered this did not invalidate the results of the test . 

### REFERENCES 1 OECD (1984) Guidelines for Testing of Chemicals, Method 202, Part II Adopted 4 April 1984. 2 Stephan C E (1977) Methods for calculating an LC50 . Proceedings first annual symposium on aquatic toxicology . Aquatic Toxicology and Hazard Evaluation . Ed: Mayer F L Hamelinlc JL, ASTM STP 634 65-84
. 3 Horning W B and Weber C I(1985) . Short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organisms . USA EPA/600/4- 85/014
.BL39 67/B pagept45%f 26 TABLE 1 

### DICHLORMID : CHRONIC TOXICITY TO Daphnia magna CONCENTRATION OF DICHLORMID TECHNICAL DETERMINED BY

QUANTIFICATION OF DICHLORIvII D
Client : ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technica l Test organism : Daphnia magna Test water : Reconstituted water (see Appendix 1 )

Nominal Measured Percent concn concn (m g/1) nominal (mg/1) Days of test New soln Old soin New Old Control 0 - 3 <0 .08 <0 .0 8



### 5 - 7 <0 .08 <0 .08 7 - 10 <0 .08 <0 .08 12 - 14 <0 .08 <0 .08 14 - 17 <0 .08 <0 .08 19 - 21 <0 .08 <0.08 1 .8 0 - 3 1 .7 1 .6 94 89

5- 7 1 .7 1 .7 94 94 7- 10 1 .9 1 .7 106 94 12-14 1 .8 1 .7 100 94 14 - 17 1 .8 1 .7 100 94 19 - 21 1 .7 1 .7 94 94 Means 1 .8 1 .7 100 94 Overall mean 1 .7 9 4 3 .2 0 - 3 3 .1 3 .1-1 97 97

5-7 3 .1t 3 .2 97 100 7 - 10 3 .2 3 .1 ; 100 97 12 - 14 3 .1 i 3 .1 97 97 14- 17 3 .1 3 .0i 97 97 19 - 21 3 .1 t 3 .1 97 97 Means 3 .1 3 .1 97 97 Overall mean 3 .1 9 7 5 .6 0 - 3 5 .5 5 .3 98 95

5 - 7 5 .6 5.5 100 98 7 - 10 5 .6 5 .7 100 102 12- 14 5 .5 5 .5 98 98 14 - 17 5 .6 5 .4 100 96 19 - 21 5 .7 5 .6 102 100 Means 5 .6 5 .5 100 98 Overall me an 5 .5 98BL3967/B pageMlof 26 TABLE 1 contd 

### DICHLORMID : CHRONIC TOXICITY TO Daphnia magna CONCENTRATION OF DICHLORMID TECHNICAL DETERMINED BY

QUANTIFICATION OF DICHLORMI D Nominal Measured Percent concn concn (mg/1) nominal (mg/1) Days of test New soln Old soln New Old 

### 10 0- 3 10 10 100 100

5- 7 10 9 .9 100 99 7- 10 10 10 100 100 12-14 9 .9 10 99 100 14 - 17 10 9 .7 100 97 19-21 10 9 .9 100 99 Mean 10 9 .9 100 99 Overall mean 10 10 0 18 0- 3 18 17 100 94

5- 7 18 18 100 100 7- 10 18 18 100 100 12 - 14 18 18 100 100 14 - 17 19 18 106 100 19 - 21 18 18 100 100 Mean 18 18 100 10 0 Overall mean 18 100 

### 32 0- 3 32 32 100 100

5- 7 32 33 100 103 7- 10 33 33 103 103 12 - 14 33 33 103 103 14 - 17 33 32 103 100 Mean 33 33 103 10 3 Overall mean 33 10 3 56 0- 3 56 55 100 98

5- 7 57 59 102 105 7- 10 58 58 104 104 Mean 57 57 102 10 2 

### Overall mean 57 102 i Mean of triplicate analyses : New solutions : Old solutions : Day 5 3 .1, 3 .1, 3 .2 Day 3 3 .0, 3 .1, 3 .1 Day 12 3 .1, 3 .1, 3 .1 Day 10 3 .1, 3 .1, 3 .2 Day 19 3 .1, 3 .2, 3 .1 Day 17 3 .0, 3 .0, 3 .0tsl,~yoi~n ~a~t~l~2ui co TABLE 2 DICHLORMID : CHRONIC TOXICITY TO Daphnia magna MORTALITY AND OBSERVED EFFECT S 

### Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technical Test organism: Daphnia magn a Test water : Reconstituted water (see Appendix 1 )

Date Day Number dead (of 10 tested)

of Nominal concentra tion mg/1 test Control 1 .8 1 3 .2 5 .6 10 18 32 56 Me an measured concentration (ms/1 

### 1 .7 3 .1 5 .5 10 18 33 57 16-11-90 0 0 0 0 0 0 0 0 0 17-11-90 1 0 0 0 0 0 0 0 0 18-11-90 2 0 0 0 0 0 0 0 0 19-11-90 3 0 0 0 0 0 0 0 0 20-11-90 4 0 0 0 0 0 0 0 0 21-11-90 5 0 0 0 0 0 0 0 0 22-11-90 6 0 0 0 0 0 0 0 0 23-11-90 7 0 0 0 0 0 0 0 0 24-11-90 8 0 0 0 0 0 0 0 0 25-11-90 9 0 0 0 0 0 0 0 3 26-11-90 10 0 0 0 0 0 0 0 (a) 10 (a) 27-11-90 11 0 0 0 0 0 0 0 a 10 28-11-90 12 0 0 0 0 0 0 0 (a) 10 29-11-90 13, 0 0 0 0 0 0 0 (a) 10 30-11-90 14 0 0 0 0 0 0 3 (a) 1 0 1-12-90 15 0 0 0 0 0 0 6 (a) 10 2-12-90 16 0 0 0 0 0 0 8 a 10 3-12-90 17 0 0 0 0 0 0 8 (a) 10 4-12-90 18 0 0 0 0 0 0 10 10 5-12-90 19 0 0 0 0 0 0 10 10 6-12-90 20 0 0 0 0 0 0 10 10 7-12-90 21 0 0 0 0 0 0 10 10 Observed effects : a reduced food clearan ceBL3967/B pagdMof 26 TABLE 3 

### DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

REPRODUCTION DAT A Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technica l Test organism: Daphnia magn a Test water: Reconstituted water (see Appendix 1)

Total offspring per parent Replicate Nominal concentration number Control 1 .8 3 .2 5 .6 10 18 32 56 Me an measured concentration m g/1) 1 .7 3 .1 5 .5 10 18 33 57 1 207 199 207 203 209 181 (50) (7) 2 218 196 175 190 187 170 (40) (13) 3 197 201 183 179 191 177 (32) (6) 4 189 202 152 197 200 152 (28) (11) 5 204 203 211 155 192 135 (49) (14) 6 212 199 187 196 189 166 (32) (8) 7 202 207 180 183 188 150 (68) (10) 8 194 210 162 181 191 139 (35) (10) 9 204 199 208 177 200 165 (42) (4) 10 200 213 182 184 200 164 (30) (13 ) 

### Mean 203 203 185 185 195 160 41 10

NSD NSD * NSD * + + Std dev 8 .5 5 .4 19 .6 13 .5 7 .1 15 .4 12 .3 3 .3 Total 2027 2029 1847 1845 1947 1599 406 96 offspring % Dead 0 0 0 0 0 0 1 .5 0 + = All parent Daphnia died during the test period; reproduction considered

significantly lower than the control without the need for statistical analysis NSD = No significant ( P=0.05) decrease compared with control * = Significant (P =0 .05) decrease compared with contro l () = The values in parentheses denote offspring fr om a Po Daphnia which had died before Day 21 .n .54 BL3967/B page 18 of 26 TABLE 4 

### DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

LENGTH MEASUREMENTS Client : ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technica l Test organism : Daphnia magna Test water: Reconstituted water (see Appendix 1)
Replicate Length in scale divisions (Day 21) number Nominal concentration (m g/1 )

Control 1 .8 3 .2 5 .6 10 18 Mean measured concentration (m g/1 ) 1 .7 3 .1 5 .5 10 18 1 59 57 56 57 57 54 2 60 58 56 57 57 52 3 60 58 56 57 56 52 4 57 57 58 56 55 51 5 58 57 56 56 56 51 6 59 56 57 56 56 52 7 58 59 56 54 57 48 8 56 57 57 57 56 49 9 57 57 57 57 55 51 10 57 57 58 56 56 5 2 Mean 58 .1 57 .3 56 .7 56 .3 56 .1 51 .2 Std dev 1 .4 0 .8 0 .8 0 .9 0 .7 1 .7 Length in mm (1 mm = 12.24 scale divisions ) 

### Mean 4 .75 4 .68 4 .63 4 .60 4 .58 4 .18 NSD * * * * NSD = No significant (P=0.05) decrease from control * = Signi ficant (P=0.05) dec rease from controlp . 55 BL3967IB page 19 of 26 TABLE 5 DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

TEMPERATURE OF TEST SOLUTIONS
. 

### Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technical Test organism : Daphnia magna Test water : Reconstituted water (see Appendix 1)
Temperature OC Date Day Nominal concentration m



### Of Control 1 .8 3 .2 5 .6 1 0 18 32 56 test Mean measured concentration (m

1 .7 3 .1 5 .5 10 18 33 57 16-11-90 0 19 .9 19 .4 19 .5 19 .4 19 .4 19 .6 19 .5 19 .7 17-11-90 1 19 .9 19 .7 19 .7 19 .6 19 .6 19 .7 19 .9 20 .0 18-11-90 2 19 .8 19 .1 19 .1 19 .1 19 .0 19 .1 19 .4 19 .5 19-11-90 3 19 .9 19 .7 19 .7 19 .7 19 .6 19 .7 19 .7 19 .9 20-11-90 4 20 .0 19 .5 19 .3 19 .2 19 .1 19 .5 19 .7 19 .8 21-11-90 5 20 .0 19 .5 19 .5 19 .6 19 .5 19 .5 19 .7 19 .8 22-11-90 6 19 .8 19 .3 19 .3 19 .3 19 .3 19 .4 19 .6 19 .6 23-11-90 7 19 .6 19 .8 19 .3 19 .6 19 .5 19 .8 19 .9 19 .9 24-11-90 8 19 .9 19 .8 19 .8 19 .9 19 .9 19 .9 19 .8 19 .9 25-11-90 9 19 .9 20 .0 20 .0 20 .1 20 .0 20 .0 19 .9 19 .9 26-11-90 10 19 .8 19 .3 19 .3 19 .4 19 .0 19 .2 19 .4 - 27-11-90 11 19 .2 19 .9 20 .0 19 .9 19 .9 19 .8 19 .8 - 28-11-90 12 19 .5 20 .0 19 .9 19 .9 19 .9 19 .9 19 .9 - 29-11-90 13 19 .6 19 .7 19 .7 19 .7 19 .7 19 .8 19 .9 - 30-11-90 14 19 .6 19 .8 19 .9 19 .8 19 .8 19 .9 19 .7 - 1-12-90 15 19 .6 19 .8 19 .9 19 .9 19 .9 19 .9 19 .8 - 2-12-90 16 19 .0 19 .1 19 .1 19 .2 19 .1 19 .2 19 .2 - 3-12-90 17 19 .5 19 .6 19 .7 19 .6 19 .6 19 .7 19 .8 - 4-12-90 18 19 .6 19 .8 19 .9 20 .0 20 .0 19 .9 19 .7 - 5-12-90 19 19 .7 19 .8 19 .8 19 .6 19 .7 20 .0 - - 6-12-90 20 19 .4 19 .7 19 .8 19 .8 19 .8 19 .8 - - 7-12-90 21 19 .8 20 .0 20 .0 19 .9 19 .7 20 .0 - - Range : Minimum 19 .0 19 .1 19 .1 19 .1 19 .0 19 .1 19 .2 19 .5 Maximum 20 .0 20 .0 20 .0 20 .1 20 .0 20 .0 19 .9 20.0P
. 56 BL3967/B page 20 of 26 • TABLE 6 DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

pH OF NEW SOLUTION S

Client : ICI Ag rochemicals, Imperial Chemical Industries PLC Test substance: Dichlormid technica l 

### Test organism: Daphnia magna Test water: Reconstituted water (see Appendix 1)

pH Date Day Nominal concentration (mg/1 )



### of Control 1 .8 3 .2 5 .6 10 18 32 56 test Mean measured concentration (mg/1 )

1 .7 3 .1 5 .5 10 18 33 57 16-11-90 0 8 .45 8 .48 8 .47 8 .47 8 .46 8 .47 8 .45 8 .45 19-11-90 3 8 .47 8 .42 8 .46 8 .46 8 .44 8 .44 8 .47 8 .47 21-11-90 5 8 .50 8 .46 8 .49 8 .47 8 .47 8 .48 8 .47 8 .46 23-11-90 7 8 .44 8 .45 8 .43 8 .46 8 .46 8 .45 8 .44 8 .45 26-11-90 10 8 .49 8 .48 8 .49 8 .49 8 .51 8 .49 8 .47 - 28-11-90 12 8 .43 8 .38 8 .40 8 .40 8 .40 8 .39 8 .38 - 30-11-90 14 8 .45 8 .45 8 .47 8 .46 8 .45 8 .47 8 .43 - 3-12-90 17 8 .44 8 .44 8 .45 8 .45 8 .45 8 .45 8 .47 - 5-12-90 19 8 .46 8 .24 8 .44 8 .44 8 .47 8 .48 - - Range : 

### Minimum 8 .43 8 .24 8 .40 8 .40 8 .40 8 .39 8 .38 8 .45 Maximum 8 .50 8 .48 8 .49 8 .49 8 .51 8 .49 8 .47 8 .47t3L396'//I3 pagep2 Rot 26 TABLE 6 cont d 

### DICHLORVIID : CHRONIC TOXICITY TO Daphnia magna

pH OF OLD SOLUTION S pH Date Day Nominal concentration (mg/1 )

of Control 1 .8 3 .2 5 .6 10 18 32 56 test Me an measured concentration (mg/1)



### 1 .7 3 .1 5 .5 10 18 33 57 19-11-90 3 8 .66 8 .66 8 .67 8 .72 8 .71 8 .68 8 .68 8 .71

8 .62 8 .66 8 .67 8 .70 8 .70 8 .68 8 .68 8 .7 1 21-11-90 5 8 .58 8 .59 8 .60 8 .69 8 .65 8 .65 8 .81 8 .82

8 .59 8 .59 8 .66 8 .63 8 .64 8 .62 8 .67 8 .77 23-11-90 7 8 .37 8 .29 8 .23 8 .29 8 .29 8 .29 8 .31 8 .35

8 .28 8 .34 8 .29 8 .36 8 .30 8 .29 8 .31 8 .69 26-11-90 10 8 .40 8 .36 8 .32 8 .27 8 .34 8 .31 8 .27 8 .18

8 .31 8 .36 8 .33 8 .42 8 .49 8 .37 8 .20 8 .80 28-11-90 12 8 .28 8 .20 8 .23 8 .23 8 .16 8 .23 8 .21 -

8 .25 8 .21 8 .25 8 .18 8 .19 8 .21 8 .25 - 30-11-90 14 8 .25 8 .32 8 .28 8 .36 8 .14 8 .33 8 .33 -

8 .13 8 .30 8 .29 8 .13 8 .32 8 .26 8 .45 - 3-12-90 17 8 .12 8 .01 8 .28 8 .09 8 .08 8 .21 8 .14 -

7 .95 8 .02 8 .14 7 .99 8 .10 8 .14 8 .39 - 5-12-90 19 8 .17 8 .09 8 .22 8 .22 8 .23 8 .37 8 .39 -

8 .10 8 .12 8 .21 8 .21 8 .19 8 .25 8 .58 - 7-12-90 21 8 .18 8 .24 8 .20 8 .35 8 .29 8 .26 - - 8 .26 8 .20 8 .23 8 .33 8 .26 8 .29 - - Range : 

### Minimum 7 .95 8 .01 8 .14 7 .99 8 .71 8 .68 8 .14 8 .18 Maximum 8 .66 8 .66 8 .67 8 .72 8 .08 8 .14 8 .81 8 .82

### BL-' )967/B page 22of 26 TABLE 7 DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

DISSOLVED OXYGE N Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test substance : Dichlormid technical 

### Test organism : Daphnia magna Test water: Reconstituted water (see Appendix 1 )

Date D Dissolved oxygen ms/1

D a o Nominal concentration m g/1 test Dilution water Control 1 .8 3 .2 5 .6 10 18 32 56 Mean measured concentration m e/l) 

### 1 .7 3 .1 5 .5 10 18 33 57 16- 1 1-90 0 9 .2 - - - - - - - - 19-11-90 3 9 .3 9 .4 9 .8 9 .7 9 .7 9 .4 9 .6 9 .4 9
. 8 9 .4 9 .6 9 .4 9 .8 9 .6 9 .8 9 .8 10 .0 21-11-90 5 9 .3 9 .4 9 .0 9 .3 9 .2 9 .4 9 .3 9 .5 10 .0 9 .4 9 .2 9 .6 9 .4 9 .4 9 .2 9 .6 9 . 6 23-11-90 7 9 .4 8 .6 8 .4 8 .4 8 .5 8 .4 8 .4 8 .3 8 .4 8 .4 8 .6 8 .4 8 .5 8 .5 8 .4 8 .4 9 .6 26-11-90 10 9 .1 9 .0 8 .6 8 .6 8 .6 8 .8 8 .6 8 .4 8
.2 8 .6 8 .6 8 .6 9 .2 9 .4 8 .8 8 .0 10 .2 28-11-90 12 9 .4 7 .9 8 .4 8 .5 8 .3 8 .2 8 .2 8 .0 - 8 .6 8 .4 8 .4 8 .4 8 .3 8 .4 8 .3 - 30-11-90 14 9 .5 8 .4 8 .8 8 .3 8 .6 8 .8 8 .7 8 .4 - 8 .0 8 .6 8 .6 7 .8 8 .8 8 .4 8 .6 - 3-12-90 17 9 .3 8 .0 7 .8 8 .5 7 .9 8 .0 8 .2 7 .7 - 7 .4 7 .8 7 .9 7 .3 8 .1 8 .0 8 .6 - 5-12-90 19 9 .2 8 .7 8 .5 8 .7 8 .9 8 .6 9 .1 8 .8 - 8 .4 8 .3 8 .6 8 .8 8 .4 8 .6 8 .6 - 7-12-90 21 - 8 .5 9 .0 8 .6 9 .1 9 .0 9 .0 - - 9 .1 8 .8 8 .8 9 .0 8 .8 8 .9 - - Range : 

### Minimum 9 .1 7 .4 7 .8 7 .9 7 .3 8 .0 8 .0 7 .7 8 .2 Maximum 9 .5 9 .4 9 .8 9 .7 9 .8 9 .6 9 .8 9 .8 10 .2 * Dilution water refers to the dissolved oxygen concentration of the new solutions

. All others are for the old solutions
.p . 59 BL3991/B page 1 of 2 8 

### Performing laboratory project identification T388/B (FT75/90) G5 .1/02

BL3991B DICHLORMID : The 28 day LC50 to rainbow trout (Salmo gairdneri

)

Data requirements OECD Guideline Number 204 Study completed 17 December 199 0 Performing laboratory Imperial Chemical Industries PLC Group Environmental Laboratory Brixham Devon TQ5 SBA 

### UK Sponsor ICI Alp' ochemicals Imperial Chemical Industries PLC Fernhurst Haslemere Surrey GU27 3JE UK Authors J F Tapp S A Sankey J E Caunter R D Stanley Approved by B R H Williams March 1991p . 60



# THIS IS A TRUE COPY OF THE ORIGINAL DOCUMENT AND MAY HAVE MISSING PAGES OR MAY CONTAIN PAGES THAT

ARE HARD TO READ

### FI75/90 BL3991/B page Tot z8 BL3991 B DICHLORMID : The 28 day LC50 to rainbow trout (Salmo gairdneri) Brixham study number : T3881Bp . 62 FT75/S0 BL3991/B page 3 of 28 BL399 , B DICHLOR IMID : The 28 day LC50 to rainbow trout (Salmo gairdneri )

Brixham study number : T388/B

GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study has been conducted in compli ance with the principles of Good Laboratory Practice (GLP) laid down in the United Kingdom Depa rtment of Health Compli ance Programme (1989) . These principles are themselves in accordance with the Org anisation for Economic Coopetation an d Development (OECD) p rinciples of Good Laboratory Practice ISBN 9264 12367 9 . Under the Memorandum of Underst anding, signed by both the United States and the United Kingdom, this study is considered to satisfy the re quirements of 40 CFR Part 160 . I believe that this study is valid for the purpose for which it was conducted and that this report is a true re flection of the raw data generated .



### Date C (

7ate ~- 3 -g/p . 63 FT75/90 rsLSyy i/ts page 4 or 2 3 BL3991/B DICHLORM ID : The 28 day LC50 to rainbow trout (Salmo gairdneri)
Brixham study number : T388/B


### QUALITY ASSURANCE STATEMEN T The conduct of this study has been inspected/audited in accord ance with Impe rial Chemical Industries' policies and procedures for Good Laboratory Practice, as follows Date Inspection/audit Date of QA report 26 November 1990 Study conduct 26 November 1990

1 Draft repor t



### Final report



## f ~ ~ 5 3 •9\

J L Gilbert Date Quality Assurance Unitp . 64 FT75/90 BL3991/B page 5 of 28 BL3991 B DICHI,ORMID : The 28 day LC50 to rainbow trout (Salmo gairdneri)
Brixham study number : T388/B
AUTHENTICATION I, the undersigned, hereby declare that this study was performed under my direction according to the principles of Good Laboratory Practice and that this report represents a true and accurate record of nhtained,

Study Director j j- ?- C t Date The following personnel carried o
Principal Investigator Bioassay Date

Principal Investigator Analysis 9-~- 9 Dat e

Report approved by !_ 3 _ b Progamme Manager '~ 7l Datep . 65 FI75/90 BL3991/B page 6 of 28 BL3991/B DICHLORMID : The 28 day LC50 to rainbow trout (Salmo gairdneri) CONTENTS page TITLE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CONFIDENTIALITY STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT

. . . . . . . . 3 QUALITY ASSURANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 AUTHENTICATION STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 

### 1 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .1 Test species
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3 .2 Test material
. . . . . . . . . 9
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 TEST METHOD AND CONDITIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.1 Bioassay test method

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4 .2 Apparatus

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4 .3 Preparation of test solutions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4 .4 Analytical method
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4 .5 Physical/chemical parameters
. . . . . . . . 10 . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Observations of mortalities and symptoms of toxici ty
. . . . . . . . . . . . . . . 11 5 RESULTS 11 5 .1 Analytical results

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 .2 Mortalities and symptoms of toxicity

. . . . . 11 5 .3 Physical/chemical parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 REFERENCE
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 TABLES 1-11
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14-25 FIGURE 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 APPENDIX 1
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27-28 The last page of this report is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28p . 66 BL3991/B page 7 of 28 I SUMMAR Y 

### Sponsor ICI Agrochemicals, Imperial Chemical Industries PLC, Fernhurst,

Haslemere, Surrey, GU27 HE, U K Contact Dr B D Cavell, International (44) 428 644061 Mr J F Tapp, International (44) 803 88288 2 Location of study, raw Imperial Chemical Industries PLC, Group Environmental data and final repo rt Laboratory, Brixham, Devon, TQ5 8BA, U K Test material Dichlormid technical common nam e IUPAC N, N-diallyl-2,2-dichloroacetamide Chemical name Chemical 2,2-dichloro-N, N-di-2-propenylacetamide Abstracts name Subject Final report of 28 day LC50 to rainbow trout (Salmo gairdneri) Test dates 19 November to 17 December 199 0 Test doses Dilution water control and nominal concentrations of 10, 17, 40, 92 and 160 mg/1 Length of test 28 day dynamic (flowthrough) test, no aeration Nominal test 15 ± 1 .0° C temperature Results based on 95 % mean measured confidence Calculation concentrations as Time LC50 interval method mg of dichlormid 4 day 120 98 - 150 Moving average angle technical per 7 day 83 62 - 130 Moving average angle litre 10 day 50 40 - 63 Moving average angl e



### 14 day 39 30 - 50 Moving average angle 21 day 36 27 - 46 Moving average angle 28 day 36 27 - 46 Moving average angle No observed effect level (NOEL) = <13 mg/I Test species/strain Rainbow trout (Salmo gairdneri), Batch 95 6 Source of organisms Upwey Trout Farm, Upwey, Weymouth, Dorset, UK Weight/length of Weight range 0 .48 - 2 .03, mean 1 .33 g dilution water control Length range 33 - 51, mean 44 mm fish at end of exposure period Physical condition at All fish were in good condition at the initiation of the study initiation of studyp . 67 BL3991/B page 8 of 28
2 INTRODUCTIO N At the request of ICI Agrochemicals, Imperial Chemical Industries PLC, the 28 day LC50 (median lethal concentration) of dichlormid technical to rainbow trout (Salmo gairdneri) was determined in freshwater
. A dynamic (flowthrough) system was used to determine the 4, 7, 10, 14, 21 and 28 day LC50 values
. The definitive test was undertaken between 19 November 1990 and 17 December 1990

. The Brixham study number was T388B and the Brixham sample number was T828

. All original data together with other records relevant to this study are filed in the ICI Group Environmental Laboratory archive at Brixham
. 

### 3 MATERIALS 3 .1 Test species Rainbow trout (Salmo gairdneri, also known as Oncorhynchus mykiss) a freshwater species, was selected as the test organism
. The fish were obtained from Upwey Trout Farm, Upwey, Weymouth, Dorset, UK on 16 October 1990 . No mortalities were observed in the fish in the 6 days prior to the test

. The pretest diet used for the fish was BP Mainstream (batch ref FF123), a proprietary product. The batch of fish used for this study (batch ref 956) was held for 34 days at 15 .0 t 1 .0°C before the start of the test . The fish were held in fibre glass tanks under daylight and artificial lighting
. No medication was given to the fish prior to the test

. During the test the fish were fed Promin (batch ref FF122) a proprietary product, at a rate of 2% of the total body weight of fish present per day . The mean weight of the fish was determined by bulk weighing each test population on days 0, 14 and 28

. At the end of the exposure period all surviving fish were individually weighed and measured after excess moisture had been removed using tissue paper . The range in weight of the dilution water control fish was 0 .48 to 2 .03 g with a mean of 1 .33 g and the range in length was 31 to 51 mm with a mean of 44 mm . The loading of dilution water control fish was 0.30 g/l
. Ten rainbow trout were used in each test concentration and dilution water control . The fish were randomly assigned to the test vessels and the vessels were arranged in random order on the test rig
.p . 68 BL3991/B page 9 of 28 3 .2 Test material The test material, dichlormid technical, was supplied by ICI Agrochemicals, Impe rial Chemical Industries PLC . IUPAC chemical name : N, N-diallyl-2,2-dichloroacetamide Chemical Abstracts name : 2,2-dichloro-N, N-di-2-propenylacetamide Molecular structure:
C12CHC0•N(CH2CH=CH2)2

The sample was received at ICI Group Environmental Laboratory , Brixham, on 26 Ap ril 1990 and assigned the B rixham test substance reference number T828 . The substance was a viscous yellow liquid and the certificate of analysis for the batch of substance stated that its purity was 96 .3% w/w (sample identification : 11489-41-01, analytical reference 11842- 03, certificate dated 17 April 1990) . The solubili ty of the test substance in water is quoted at 5000 mg/1 at 20°C and the specific gravity at 1 .202 (ICI Agrochemicals) . The test substance was stored in the dark at room temperature in the container in which it was received until required for testing when approp riate sub-samples were provided for the test operators . 

### During the definitive run of the test 1 .1 litres ( 1 .3 kg) of test substance were used to supply the test solutions
. 4 TEST METHOD AND CONDITIONS 4 .1 Bioassay test method The bioassay test method used in this study was based on OECD Guideline Number 204 using ICI Group Environmental Laboratory standard operating procedure BA92, version 07
. 

### 4.2 Apparatus The apparatus used in this study was a dynamic, continuous flowthrough system . The test vessels, dosing lines, mixing chambers and stock vessels were all constructed of glass . A minimum quantity of silicone rubber tubing was used to connect the components together. Glass vessels with a maximum capaci ty of 54 litres and a working volume of 45 lit res were used to hold the test fish. The test solutions were renewed at a nominal rate of 250 ml/minute so that a 95% exch ange of the test solutions was calculated to occur within 9 hours.p . 69 BL3991/B page 10 of 2 8 The stock solutions (of undiluted dichlormid technical) were fed to the glass mixing chambers by a series of Watson-Marlow peristaltic pumps and the dilution water was supplied using a capillary flow control system . The nominal toxicant flow rates required for nominal concentrations of 160, 80, 40, 20 and 10 mg/1 were 0 .04, 0.02, 0 .01, 0 .005 and 0 .0025 g/min respectively . Toxicant flow rates were controlled by using silicone rubber tubing of specific diameter. The nominal toxicant flow rates achieved, determined by measurements prior to the start of the test, were 0 .040, 0 .023, 0 .01, 0 .023 and 0 .0043 g/min. Therefore, the calculated nominal concentrations used for this study were 160, 92, 40, 17 and 10 mg/1 . These are referred to as the nominal concentrations elsewhere in the text . Magnetic stirrers in the mixing chambers were used to ensure mixing . The test solutions were then passed through 9 litre settlement tanks, to collect any undissolved test material, before they passed into the fish exposure vessels. Due to varied toxicant flow rates used, the nominal dilution ratios of the toxicant stock to dilution water ranged from 6250 to 100,000
. The dilution water was dechlorinated tap water supplied from a 100 m' reservoir with an average retention time of 24 hours . It was passed through activated carbon, filtered to remove particulate material and dechlorinated with sodium thiosulphate . The treated water was then held in a secondary reservoir with a capacity of 36 m' and an average retention time of 8 hours . The water was then passed through an ultra violet steriliser to a third storage tank with capacity of 13 .5 m3 . It was delivered via a ring circuit to a temperature controlled header tank in the test laboratory (Room 354) set to a nominal temperature of 15°C
. Calibration of the dilution water and toxicant flow rates was undertaken daily for the first three days and then 3 times per week. The dilution water flow rates were measured by collecting for 1 minute prior to input to the test vessels . The toxicant stock flow rates were calculated after weighing the toxicant stocks over a known period of time

. 

### 4 .3 Preparation of test solutions The nominal test exposure concentrations used in this study were 160, 92, 40, 17 and 10 mg of dichlormid technical per litre, together with a dilution water and solvent control

. Each test concentration was run unreplicated
. The toxicant stocks were made with undiluted dichlormid technical . The stock jars used had a maximum capacity of 500 ml . For the nominal exposure concertrations of 160 and 92 mg/1, the jars were filled to capacity and topped up every 7 and 14 days respectively

. The stock jars for the nominal exposure concentrations of 40, 17 and 10 mg/1 were filled with 500, 250 and 125 ml of dichlormid technical respectively . These toxicant stocks were topped up as required
. 4 .4 Analytical metho d The analytical method employed to determine the levels of dichlormid technical in this study by quantification of dichlormid was based on ICI Group Environmental Laboratory standard operating procedures AL200 version 01 and AL201 version 01 . This is a high performance liquid chromatographic method which is outlined in Appendix 1 . Samples of the test solutions were taken for analysis on days 1, 2, 3, 8, 10, 15, 17, 22, 24 and 28

. The samples were taken from the middle of each test vessel

.p . 70 ti L.SYY 1/li page 1 t 

### or L2S 

### 4 .5 Physical/chemical parameters The dissolved oxygen, pH and temperature were measured in each test vessel daily for the first 3 days and then 3 times per week until the end of the study . In addition, the water hardness, conductivity and the free and residual chlorine content of the dilution water were determined daily (Monday to Friday) . Measurements of pH were carried out with a Corning Model 240 pH meter. Dissolved oxygen values were measured with a Yellow Springs Instruments dissolved oxygen meter Model 51B . Temperature values were determined for all vessels by a mercury-in-glass thermometer calibrated to 0 .1°C . A continuous record of the temperature in one vessel was also made using a thermistor probe linked to a chart recorder . Conductivity was measured with a Jenway 4010 conductivity meter. Hardness was determined by titration with standard EDTA solution using BDH total hardness indicator and alkalinity was measured by an electrometric method

. The photoperiod in this study was 16 hours light and 8 hours darkness with a 10 minute transition period at 06 :00 and 22 :00 hours
. The available free and total residual chlorine were determined separately by a colorimetric method using a lovibond comparator based upon the reaction between the various chlorine species and N,N-diethyl-p-phenylene diamine (DPD) to form a stable pink complex

. Interference from metal ions was suppressed using EDTA as a chelating agent . The combined available residual chlorine was calculated by subtracting the free available from the total available residual chlorine
. 

### 4 .6 Observations of mortalities and symptoms of toxicit y Observations of mortalities and symptoms of toxicity were made daily. Mortalities were recorded daily and symptoms of toxicity were recorded on days 4, 7, 10, 14, 21 and 28

. The LC50 value was calculated at various time intervals by the ICI Group Environmental Laboratory computer program "LC50" using Stephan's method (Ref 1) . The results calculated by the moving average angle method were selected and the dose response curve was plotted (Fig 1) using a Hewlett-Packard plotter
. 

### 5 RESULTS 5.1 Analytical results The analytical results for the levels of dichlormid technical are reported in Table 1 . The mean measured values ranged from 88 to 130% of nominal values in the test vessels . It was considered that the relatively high mean measured values were due to the toxicant pumping rates being too high for short periods of time . Representative chromatograms are shown in Appendix 1
. 5.2 Mortalities and symptoms of toxicit y Observations of mortalities are shown in Table 2, and observations of the symptoms of toxicity are reported in Table 3
. The general symptoms of toxicity noted in this study were laboured, slow, irregular and rapid respiration, quiescence, dark discolouration, loss of balance, coughing, no feeding response, reduced feeding response and ceased swimming

.p . 71 BL3991/B page 12 of 2 8 

### The LC50 values and 95% confidence intervals are shown below as mg of dichlormid technical per litre based on mean measured concentrations
. 95% confidence Calculation Time LC50 interval metho d 4 day 120 98 - 150 Moving average angle 7 day 83 62 - 130 Moving average angle 10 day 50 40- 63 Moving average angle 14 day 39 30 - 50 Moving average angle 21 day 36 27- 46 Moving average angle 28 day 36 27 - 46 Moving average angl e There were no mortalities in the dilution water control or in the mean measured concentration of 13 mg/l
. A graph has been plotted (Fig 1) of the derived LC50 values and their respective 95% confidence intervals . The confidence intervals are shown as horizontal bars
. The no observed effect level (NOEL) of dichlormid technical (based on mortalities and symptoms of toxicity) was found to be <13 mg/1 (mean measured)
. The threshold level of lethal effect was determined at 22 mg/1 and the threshold level of observed effect was determined at 13 mg/1 (mean measured concentrations)
. The lowest mean measured concentration at which there was 100% mortality was 100 mg/1
. The mean weight of fish measured by bulk weighing on days 0, 14 and 28 are reported in Table 4. The mean weight of fish in the dilution water control increased by 102% over the exposure period. The mean weight of surviving fish in the nominal exposure concentrations of 40, 17 and 10 mg/1 increased by 16 to 42% . It was concluded that dichlormid technical affected fish weight at all exposure concentrations
. The mean lengths and weights of individually measured fish from each test vessel at the end of the exposure period are reported in Table 5
. 

### 5 .3 Physical/chemical parameters During the test the means and ranges of oxygen concentration, pH, and temperature in the exposure vessels (including the controls) were :

Overall mean Range Dissolved oxygen : 9 .9 mg/1 9 .4 - 10 .2 mg/1 pH : 7 .70 7 .38 - 7 .86 Temperature : 15 .1 °C 14 .6 - 15 .2 ° C The continuous record of temperature made in the dilution water control tank noted the range to have stayed within 15 ± 1 .0°C
.p . 72 BL3991/B page 13 of 2 8 

### The means and measured ranges of diluent flow rate and the ranges of toxicant flow and the dilution ratios were :

Overall mean Range Diluent flow rate : 250 ml/minute 245 - 260 ml/minute Toxicant flow rate : N/A 0 .0018 - 0 .0463 ml/minute Dilution ratio : N/A 5292 - 141667 The nominal toxicant flow rate and consequently the dilution ratio varied with concentration . The me ans and measured ranges of total hardness and conductivity of the dilution water were : Overall mean Range 

### Total hardness 36 .0 mg/1 as CaCO3 31 .3 - 40.7 mg/1 as CaCO3 Conductivity 126 pS/cm 120 - 133 }1S/c m Full results are given in Tables 6-11
.
REFERENCE 1 Stephan C E (1977) . Methods for calculating an LC50 In : Aquatic Toxicology and Hazard Evaluation . Mayer F L, Hamelink J L, Editors
. Proceedings 1st Annual Symposium on Aquatic Toxicology, ASTM, 1977, STP 634 pp65-84

.p . 7 3 li L.Sy`J 1/li page 14 

### of 2 8 

### TABLE 1 DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT CONCENTRATIONS OF DICHLORM ID TECHNICAL IN TEST VESSELS DETERMINED BY QUANTIFICATION OF DICHLORMI D Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water : Freshwater
Date of Sample Nominal concn (mg/1) Dilution sample day water 160 92 40 17 10 control 

### 20.11 .90 1 65 130 44 15 9 .2 <0 .04 15 16 21 .11 .90 2 220 100 44 21 11 <0 .07 22 .11 .90 3 TD 96 40 17 11 <0 .07 27 .11 .90 8 - 97 42 24 16 <0 .07 24 24 29 .11 .90 10 - 96 41 25 11 <0 .07 4.12 .90 15 - TD 46 24 15 <0 .07 25 24 6 .12 .90 17 - - 39 21 18 <0 .07 11 .12 .90 22 - - 56 26 12 <0 .84 25 26 13 .12 .90 24 - - 29 21 11 <0 .82 17 .12 .90 28 28 22 13 <0 .80 2 1 22 Mean mg/I 140 100 41 22 13 <0 .84 measured concn of dichlormid % of 88 109 103 129 13 0 technical nominal13L :~yy I/li page 15pot 48 TABLE 2 

### DICHLORIVIID : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF PERCENTAGE MORTALITIES DURING TEST Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test org anism : Rainbow trout (Salmo gairdneri) Test water : Freshwater

Nominal Mean concn of measured Cumulative percentage mortality observed dichlormid concn of technical dichiormid

technical 4 days 7 days 10 days 14 days 21 days 28 days mg/1 mg/I (23 .11 .90) (26 .11 .90) (29.11 .90) (3 .12.90) (10 .12.90) (17 .12 .90) 160 140 100 TD

- 92 100 0 30 100 T D 40 41 0 0 10 60 70 70 17 22 0 0 0 10 20 20 10 13 0 0 0 0 0 0 Dilution water <0 .84 0 0 10 10 10 10 control Ten fish were exposed in each test concentration and in the dilution water control .p . 75 BL3991/B page 16 of 28 TABLE 3 

### DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT

SYMPTOMS OF TOXICITY OBSERVED Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material: Dichlormid technical Test organism : Rainbow trout (Sabnw gairdneri) Test water : Freshwater
Day no Day Nominal concn of dichlormid technical mg/1 160 92 40 17 10 4 23 .11 .90 C i C/abcdei B/bfj C/j A 7 26 .11 .90 TD C/abcdefhi C/abcdhjl C/j C/j km 10 29 .11 .90 - Ct C/acdghi C/adj C/j 14 3 .12 .90 - TD C/afj C/acdj C/j 21 10 .12 .90 - - C/abj C/adj C/j 28 17 .12 .90 - - C/ac C/abcd C/d Definition of symbols A = No significant effect. 10% or less of test population dead or exhibiting

symptoms of toxicity
. B = Partial effect. Between 11% and 30% of test population dead or exhibiting

symptoms of toxicity
. C = Significant effect . More than 30% of test population dead or exhibiting

symptoms of toxicity
. TD = Terminated i = 100% mortality g = Irregular respiration a = Quiescent h = Coughing b = Sounding i = Not feedin g c = Weak j = Reduced feeding d = Dark discoloured k = Ceased swimming e = Slow respiration 1 = Rapid respiration f = Loss of balance m = Laboured respiration The fish were not fed on day 28p . 76 BL3991/B page 17 of 28 TABLE 4 

### DICHI .ORMID : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF MEAN FISH WEIGHTS (DETERMINED BY BULK WEIGHING) AND CONSEQUENT FEEDING RATE S
Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water: Freshwater Nominal Mean Day 0 Day 14 Day 28 concn of measured (19 .11 .90) (3 .12 .90) (17 .12 .90) dichlormid concn of technical dichlormid Mean Nominal Mean Nominal % Mean %

technical fish food fish food change fish change

weight weight/ weight weight/ in fish weight in fis h



### day day weight weight from from day 0 day 0 rng/1 mg/I g g Q g J b S 160 140 0 .70 0 .14 TD - - - 92 100 0 .81 0

*00 . 40 41 0 .91 0+9 1 .15 +26 17 22 0 .74 0+22 0 .86 +16 10 13 0 .71 0 .+39 1 .01 +42 Dilution water <0 .84 0 .75 0 .+47 1 .52 +102 

# L control TD = Terminated NOTE Nominal food weight calculated from total fish weight on day 0 fed to fish on days 0-13 Nominal food weight calculated from total fish weight on day 14 fed to fish on days 14-28p . 77 BL3991/B page 18 of 28 TABLE 5 

### DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROU T FISH WEIGHT AND LENGTH RANGE AND MEANS AT END OF EXPOSURE PERIOD Client: ICI Agrochemicals, Impe rial Chemical Industries PL C Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water: Freshwater

Nominal Mean Number Weight Mean Length Mean concn of measured of fish range weight range length dichlormid concn of survivin g technical dichlot7nid

technical mg/1 mg/1 g ~ mm mm 160 140 0 - - - - 92 100 0 - - - - 40 41 3 0 .58-0 .78 0 .65 36-39 38 17 22 8 0 .45 - 1 .21 0 .77 34 - 43 39 10 13 10 0 .30-1 .40 0 .91 31-46 39 Dilution water <0 .84 9 0 .48-2 .03 1 .33 33 - 51 44 controlp . 78 tSL .Syyl/ti page 1 9 of 28 TABLE 6 

### DICHLORMID TECHNICAL : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF DISSOLVED OXYGEN CONCENTRATIONS IN EXPOSURE VESSELS Client : ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water : Freshwater

Date Day Dissolved oxygen (mg/1)

Dilution Nominal concentration (mg/1) water control 10 17 40 92 160 19 .11 .90 0 9 .8 9 .8 9 .8 9 .8 9 .8 9 .8 20 .11 .90 1 9 .8 9 .8 9 .8 9 .8 9 .8 9 .8 21 .11 .90 2 9 .6 9 .6 9 .8 9 .8 9 .6 9 .8 23 .11 .90 4 9 .8 10 .0 10 .0 9 .8 10 .0 - 26 .11 .90 7 9 .8 9 .8 9 .8 9 .6 9 .8 - 28 .11 .90 9 10 .0 9 .8 10 .0 10 .0 10 .0 - 30 .11 .90 11 10 .0 10 .0 10 .0 10 .2 10 .2 - 3 .12 .90 14 9 .8 9 .4 9 .8 9 .8 - - 5 .12 .90 16 10 .0 10 .0 10 .0 10 .2 - - 7 .12 .90 18 10 .0 10 .0 10 .0 10 .0 - - 10 .12 .90 21 10 .2 10 .2 10 .2 10 .2 - - 1 2 .12 .90 23 10 .0 10 .0 10 .0 9 .8 - - 14 .12 .90 25 10 .0 10 .0 10 .2 10 .0 - - 17 .12 .90 28 10 .2 9 .8 10 .2 10 .2 - - Mean 9 .9 9 .9 10 .0 9 .9 9 .9 9 .8 SD 0 .17 0 .20 0 .15 0 .20 0 .20 - Minimum 9 .6 9 .4 9 .8 9 .6 9 .6 9 .8 Maximum 10 .2 10 .2 10 .2 10 .2 10 .2 9 .8p . 79 BL3991/B page 20 of 28 TABLE 7 

### DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF pH MEASUREMENTS IN EXPOSURE VESSELS Client : ICI A grochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salrno gairdneri) Test water: Freshwater

pH Date Day Dilution Nominal concentration (mg/1)

water control 10 17 40 92 160 i9 .11 .90 0 7 .78 7 .79 7 .80 7 .79 7 .81 7 .81 20.11 .90 1 7 .79 7 .78 7 .80 7 .81 7 .76 7 .74 21 .11 .90 2 7 .64 7 .72 7 .77 7 .62 7 .68 7 .76 23 .11 .90 4 7 .77 7 .85 7 .73 7 .82 7 .86 - 26 .11 .90 7 7 .77 7 .82 7 .80 7 .80 7 .86 - 28 .11 .90 9 7 .63 7 .86 7 .64 7 .63 7 .60 - 30 .11 .90 11 7 .78 7 .74 7 .78 7 .78 7 .67 - 3 .12 .90 14 7 .41 7 .42 7 .43 7 .38 - - 5 .12 .90 16 7 .67 7 .59 7 .62 7 .66 - - 7 .12 .90 18 7 .74 7 .69 7 .70 7 .81 - - 10 .12 .90 21 7 .74 7 .73 7 .72 7 .55 - - 12 .12 .90 23 7 .70 7 .65 7 .70 7 .74 - - 14 .12 .90 25 7 .64 7 .59 7 .60 7 .64 - - 17 .12 .90 28 7 .69 7 .71 7.7 =--- Mean 7 .79 7 .71 7

. 7 SD 0 .10 0 .12 0
. 1 Minimum 7 .41 7 .42 7 .43 Maximum 7 .79 7 .86 7 .80p . 8 0 17LJ771/L kla~t, . :,1 ut LO 

### TABLE 8 DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF TEMPERATURE MEASUREMENTS IN EXPOSURE VESSELS
Client : ICI Agrochemicals, Imperial Chemical Industries PL C Test material : Dichiormid technical Test organism : Rainbow trout (Salnio gairdneri) Test water : Freshwater

Temperature ° C Date Day Dilution Nominal concentration (mg/1) water control 10 17 40 92 160 19 .11 .90 0 15 .2 14 .9 15 .1 15 .1 15 .0 15 .0 20 .11 .90 1 15 .2 15 .0 15 .1 15 .2 15 .0 15 .0 21 .11 .90 2 15 .2 15 .0 15 .1 15 .2 15 .0 15 .0 23 .11 .90 4 15 .2 15 .0 15 .1 15 .2 15 .0 - 26.11 .90 7 15 .2 15 .0 15 .1 15 .2 15 .0 - 28 .11 .90 9 15 .1 15 .0 15 .0 15 .1 14 .9 - 30 .11 .90 11 15 .2 15 .0 15 .1 15 .1 15 .0 - 3 .12 .90 14 15 .2 15 .0 15 .0 15 .1 - - 5 .12 .90 16 15 .2 15 .0 15 .1 15 .2 - - 7 .12 .90 18 15 .2 15 .0 15 .1 15 .1 - - 10 .12 .90 21 15 .2 15 .0 15 .0 15 .1 - - 12 .12 .90 23 15 .2 15 .0 15 .1 15 .2 - - 14.12 .90 25 15 .2 14 .9 15 .0 15 .1 - - 17 .12 .90 28 15 .2 15 .0 15 .1 15 .1 - - Mean 15 .2 15 .0 15 .0 15 .1 15 .0 15 .0 SD 0 .03 0 .04 0 .05 0 .05 0 .04 - Minimum 15 .1 14 .9 15 .0 15 .1 14 .9 15 .0 Maximum 15 .2 15 .0 15 .1 15 .2 15 .0 15 .0p . 8 1 a.r~ . .ii
. .i .r l . . .bv ... . . v. ...~ TABLE 9 DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT

FLOW DATA FOR THE TEST VESSEL S 

### Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water : Freshwater

Flow data (mUmin ) Date Day Dilution Nominal concentration (mg/1) water control 10 17 40 92 160 19 .11 .90 0 Diluent 245 250 255 245 245 245

Toxicant - 0 .0031 0 .0043 0 .0081 0 .0210 0 .0355 Dilution ratio - 80645 59302 30247 11667 690 1 20 .11 .90 1 Diluent 245 250 255 245 245 245

Toxicant - 0 .0029 0.0048 0 .0093 0 .0258 0 .0463 Dilution ratio - 86207 53125 26344 9496 5292 21 .11 .90 2 Diluent 245 250 255 245 245 245

Toxicant - 0 .0026 0 .0046 0 .0084 0 .0237 0 .0418 Dilution ratio - 96154 55435 29167 10338 586 1 23 .11 .90 4 Diluent 245 250 255 245 245 -

Toxicant - 0 .0023 0.0040 0 .0084 0 .0198 - Dilution ratio - 108696 63750 29167 12374 - 26 .11 .90 7 Diluent 245 250 255 245 245 -

Toxicant 0 .0028 0 .0044 0 .0090 0 .0209 - Dilution ratio - 89286 57955 27222 11722 - 28 .11 .90 9 Diluent 250 255 255 245 260 -

Toxicant - 0 .0027 0.0045 0 .0085 0 .0200 - Dilution ratio - 94444 56667 28824 13000 - 30 .11 .90 11 Diluent 245 255 255 245 245 -

Toxicant - 0 .0026 0 .0047 0 .0085 0 .0198 - Dilution ratio - 98077 54255 28824 12374 - 3 .12 .90 14 Diluent 250 250 260 245 - -

Toxicant - 0 .0027 0 .0044 0 .0084 - Dilution ratio - 92593 59091 2916 7 5 .12 .90 16 Diluent 245 250 255 245 - -

Toxicant - 0 .0032 0 .0044 0 .0081 - - Dilution ratio - 78125 57955 30247 _p . 8 2 DLJ771/D hilb'C

. :.J Ut LO TABLE 9 contd DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT

FLOW DATA FOR THE TEST VESSEL S

Flow data (ml/min) Date Day Dilution Nominal concentration (mg/1) water control 10 17 40 92 160 7 .12 .90 18 Diluent 250 250 260 245 - -

Toxicant - 0 .0036 0 .0046 0 .0067 - - Dilution ratio - 69444 56522 36567 - - 10 .12 .90 21 Diluent 250 255 260 245 - -

Toxicant - 0 .0018 0 .0052 0 .0077 - - Dilution ratio - 141667 50000 31818 - - 12.12 .90 23 Diluent 245 260 260 250 - -

Toxicant - 0 .0034 0 .0065 0 .0077 - - Dilution ratio - 76471 40000 32468 - - 14 .12 .90 25 Diluent 260 250 260 255 - -

Toxicant - 0 .0027 0 .0047 0.0058 - Dilution ratio - 92593 55319 43966 - - 17.12 .90 28 Diluent 245 250 255 250 - -

Toxicant - 0 .0029 0 .0049 0 .0057 - - Dilution ratio - 86207 52041 43860 - -p . 83

--- . ~ TABLE 1 0 DICHLORMID : THE 28 DAY LC50 TO RAINBOW TROUT SUMMARY OF FLOW DATA FOR THE TEST VESSELS
Client ICI Agrochemicals, Imperial Chemical Industries PLC Test material : Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water : Freshwater
Nominal Diluent flow (ml/min) No of concentration samples (mgn) Me an SD R ange DWC 248 4 .3 245 - 260 14 10 252 3 .2 250 - 260 14 17 257 2 .5 255 - 260 14 40 246 3 .1 245 - 255 14 92 247 5 .7 245 - 260 7 160 245 - 245 - 245 3
Nominal Toxicant flow (ml/min) No of concentration samples (mg/1) Mean SD R ange 

### DWC - - - 0 10 0 .0028 0 .00045 0.0018 - 0 .0036 14 17 0 .0047 0 .00059 0 .0040 - 0 .0065 14 40 0 .0079 0 .00109 0 .0057 - 0 .0093 14 92 0 .0216 0 .00231 0 .0198 - 0 .0258 7 160 0 .0412 - 0 .0355 - 0 .0463 3
Nominal Dilution ratio No of concentration samples (mg/1) Mean SD Range DWC - - - 0 10 92186 17435 .0 69444 - 141667 14 17 55101 5528 .5 40000 - 63750 14 40 31992 5617 .0 26344 - 43966 14 92 11567 1237 .6 9496 -13000 7 160 6018 - 5292 - 6910 3 DWC = Dilution water controlp . 84 tsWyy l/ts page :J or LS TABLE 1 1 

### DICHLOItIvIID : THE 28 DAY LC50 TO RAINBOW TROUT DETERMINATION OF WATER QUALITY OF DILUTION WATER Client: ICI Agrochemicals, Imperial Chemical Industries PLC Test material: Dichlormid technical Test organism : Rainbow trout (Salmo gairdneri) Test water : Freshwater

Chlorine Total Total A lkal- (pg/l Cl,) ammonia filterConduct- inity Hardness (NH3-N) able Test ivity m8/t as mg/1 as Free Combined mg/1 solids Date day pH pS/cm CaCO, CaCO3 resid residual m g/l 19 .11 .90 0 7 .88 127 21 .0 35 .3 <4 <4 20 .11 .90 1 7 .79 128 34 .7 <4 4 <0 .05 21 .11 .90 2 7 .80 127 36 .7 <4 < 4 22 .11 .90 3 7 .61 122 19 .6 313 <4 < 4 23 .11 .90 4 7 .72 121 33 .0 <4 <4 <0 .05 0 .9 26.11 .90 7 7 .48 120 20 .0 33 .0 <4 < 4 27 .11 .90 8 7 .48 127 32 .7 <4 <4 0 .09 28 .11 .90 9 7 .42 126 333 <4 < 4 29 .11 .90 10 7 .28 129 20 .4 33 .0 <4 < 4 30 .11 .90 11 7 .37 124 36 .7 <4 <4 0 .07 0 .5 3 .12 .90 14 7 .77 127 23 .2 38 .7 <4 <4 0 .05 4 .12 .90 15 7 .71 126 39 .3 <4 1 4 5 .12 .90 16 7 .60 127 40 .7 <4 <4 6.12 .90 17 7 .70 123 23 .4 38 .7 <4 4 7 .12 .90 18 7 .65 128 32 .3 <4 <4 <0 .05 10 .12 .90 21 7 .75 126 19 .6 37 .0 <4 < 4 11 .12 .90 22 7 .84 132 36 .3 <4 <4 <0 .05 12.12 .90 23 7 .83 131 40 .0 <4 < 4 13 .12 .90 24 7 .75 128 23 .6 39 .0 <4 <4 14.12 .90 25 7 .76 133 36 .0 <4 <4 17
. 1 2.90 

# f 28 7 .93 121 19 .8 37 .7 <4 4p .85~-o

### FIGURE 1

CONCENTRATION RESPONSE RELATIONSHIP RAIN80V TROUT DICHLORMID TECHNICAL STUDY NO . T388/ B

100 80 60 40 -~E- 2e Day LC5 0 (38 W/ 1 ) ~E 21 Day LC50 N 20 (36 sp/ 1 ) ~" --~f- 14 Day LC50 

## c ~ c3~ 20/1) " 10 -~-- 10 Day LC50 6 (5D ~y I) w =E 7 Day LC50 6 \ (B3 no/ 1) H 4 -~E- 4 Day LC50 (120 ap/1)
2

1 10 50 100 500 100 0



## MEASURED CONCENTRATION (mg/I)

Version 4 .31 ( 19/071 90)p . 86 BL6292/B page t of 22 Performing laboratory project identification AF00211B G8 .1/02 Sponsor work identification number (WINO) 4016 4

BL62921B Copy number DICHLORMID : Toxicity to the green alga Selenastrum capricornutum

{ Data requirements OECD Guideline 201 "Alga, Growth Inhibition Test" Study completed 21 February 1998 Performing laboratory Brixham Environmental Laboratory ZENECA Limite d Brixham Devon TQ5 8BA UK Sponsor ZENECA Agrocheniicals Fernhurst Haslemere Surrey GU27 3JE UK Authors D V Smyth S E Magor N Shillabee r Approved by N R Gore July 1998p . 87



# THIS IS A TRUE COPY OF THE ORIGINAL DOCUMENT AND MAY HAVE MISSING PAGES OR MAY CONTAIN PAGES THAT

ARE HARD TO READp . 88 BL6292IB page 2 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum capricornutum Brixham study number : AF00211Bp . 89 BL6292/B page 3 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum capricornutum Brixham study number : AF0021/B 

### GOOD LABORATORY PRACTICE COMPLIANCE STATEMEN T
This study was conducted in compliance with the UK principles of Good Laboratory Practice (United Kingdom GLP Regulations 1997) . These principles are in accordance with the OECD Principles of Good Laboratory Practice 1981 (OECD Environment Monograph No. 45) . These intemational standards are acceptable to the United States Environmental Protection Agency and this study, therefore, satisfies the requirements of 40 CFR Part 160
. This study is valid for the purpose for which it was conducted and this report is a true reflection of the raw data generated
.

DateP
. 90 BL6292/B page 4 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum capricornutum Brixham study number : AF0021/B



### QUALITY ASSURANCE STATEMEN T
This study has been inspected/audited in accordance with ZENECA Limited's policies and procedures for Good Laboratory Practice, as follow s Date Inspection/audit Date of QA report 16 July 1998 Draft report 16 July 199 8 Ack r~ qqCVg' Final report ~ JUI~ A9oty

Facilities and procedures associated with this type of study are periodically inspected in accordance with QA Standard Operating Procedures
. So far as can be established, the methods described and the results incorporated in the final report accurately reflect the raw data produced during the study
.


# ~' aa R H Swarbrick Date J Quality Assurance UnitP
. 91 BL6292/B page 5 of 22 DICHLORMID : Toxicity to the green alg a Selenastrwn capricornutum Brixham study number : AF0021 B 

### AUTHENTICATION STATEMENT
I, the undersigned, hereby declare that this study was performed under my direction according to the principles of Good Laboratory Practice and that this report represents a true and accurate record of results obtained
.

g Study Director 01 01 Date

The following personnel carried out work on this study :
Principal Scientist, ~998' Regulatory Toxicology Date
Principal Scientist, Zcy 5'„4.y Regulatory Analysis Date

Report approved by Business Manager Datep . 92 BL6292B page 6 of 22 DICHLORMID : Toxicity to the green alga Selenastrum capricornutum Brixham study number : AF0021/B 

### CONTENTS page TITLE PAGE . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 CONFIDENTIALITY STATEMENT . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
. . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . 3 QUALITY ASSURANCE STATEMENT
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . AUTHENTICATION STATEMENT . 5 CONTENTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 SUMMARY
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . 7 2 INTRODUCTION
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... .. ... .. ... .. . .... .... ... .. .. .. .. .. . .. ... .. ... .. ... . .. ... .... ... . ... 8

. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . ... ... .. .. 3 MATERIALS 8 3 .1 Test substance
. . . . . . . . . . . . . ... . .. .. ... . ... .. .... .. .. ... .. ... .. .... ... .. .. .. .... . .. .. .. ... .. .. .. ... . .. ... ..... .. .. . 8 3 .2 Test species
. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 .3 Culture medium
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4 TEST METHODS AND CONDITIONS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.1 Test procedure and apparatus . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 4.2 Preparation of test solutions
. . . . . . . . . . . . . . .. . 1 0
. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Analytical method
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 4 .4 Physical and chemical parameters
. . . .. . . . . . . . . .. . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 5 RESULTS
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 .1 Analytical data
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 .2 Biological data
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 11 5 .2.1 Areas under the growth curve
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 5 .2.2 Growth rates
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . 12 5 .3 Physical and chemical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 1 2 6 REFERENCES
. . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 TABLES
. . .. . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 FIGURE
. . . . . . . . . . . . . . . . . .. .. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 19 APPENDICES
.. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 20 The last page of this report is
. . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . .22

il Ip . 93 BL6292/B page 7 of 22 1 SUMMARY Sponsor ZENECA Agrochemicals, Fernhurst, Haslemere, Surrey, GU27

3JE, UK Contact Dr A M Skidmore, telephone : international (44) 1428 644061 Mr N Shillabeer, telephone : international (44) 1803 882882 Location of study, raw Brixham Environmental Laboratory, ZENECA Limited, Brixham, data and final report Devon, TQ5 8BA, UK Test substance Dichlormid common name Active ingredient N,N-diallyl-2,2-dichloroacetamide [UPAC chemical name Active ingredient 2,2-dichloro-N,N-di-2-propenylacetamide Chemical Abstracts name Subject Dichlormid : Toxicity to the green alga Selenastrum capricornutum Guideline followed OECD guideline 201, Alga, Growth Inhibition Test (Ref 1), als o

satisfies requirements of the relevant EU guideline (Ref 2) Test species Green alga Selenastrum capricornutu m Source of org anisms Brixham Environmental Laboratory culture from strain ATCC

2266 2 Test concentrations Culture medium control and nominal dichlormid technical concentrations of 1 .5, 3 .0, 6 .0, 12, 23, 45, 90 and 180 mg 1" 1 Length of test 72 hours static, no aeration, shaken Test dates 18 to 21 February 199 8 Nominal 24 t 2°C test temperatu re Results based on Based on areas under the growth curve (Ref 1) the results obtained nominal concentrations as mg were : of dichtormid per litre No observed effect (P=0 .05) concentration (NOEC) = 6 .0 mg 1"'

Lowest observed effect (P=0 .05) concentration (LOEC) = 12 mg 1" Median effective concentration, biomass (EbC50) = 33 mg 1-1 95% confidence limits = 29 to 39 mg 1 Based on logarithmic growth rate over the test period, the results obtained were : No observed effect (P=0 .05) concentration (NOEC) = 23 mg 1-1 Lowest observed effect (P=0 .05) concentration (LOEC) = 45 mg 1"1 Median effective concentration, growth rate (E,C50) = 80 mg 1"1 95% confidence limits = 70 to 94 mg 1- ~p . 94 BL6292/B page 8 of 22
2 INTRODUCTIO N At the request of ZENECA Agrochemicals, Fernhurst, Haslemere, Surrey, GU27 3JE, the toxicity of the herbicide safener dichlormid to the green alga Selenastrum capricornutwn was determined
. The definitive test was undertaken between 18 and 21 February 1998 . The study number was AF0021/B and the Brixham sample number was AF0021 . The ZENECA Agrochemicals reference number for the study was WINO 40164 . All original data together with other records relevant to the study are filed in the Brixham Environmental Laboratory archive
. 

### 3 MATERIAL S
3 .1 Test substanc e The test substance, dichlormid, was supplied by ZENECA Agrochemicals, Jealott's Hill Research Station, Nr Bracknell, Berkshire RG42 6ET
. IUPAC chemical name : N,N-diallyl-2,2-dichloroacetamide Chemical Abstracts name : 2,2-dichloro-N,N-di-2-propenylacetamide CAS RN: 37764-25- 3 Molecular structure :



### C 1


### C1 I
.

The batch of dichlormid was received at Brixham Environmental Laboratory on 1 4 January 1998 and assigned the Brixham test substance number AF0021 . The substance was an amber liquid and the certificate of analysis for the batch of material stated that its purity was 97 .9% '/W (WRC certificate Analytical Ref number 14107-31, dated 23 February 1994) . The stability of dichlormid was last confirmed on 15 September 1997 (Calibrant Ref. ASW-1219-R, Data ref. TSC Dichlormid P12 File/1 + 2) . The solubility is stated as 5000 mg 1- 1 at 20°C (Zeneca Agrochemicals)
.p . 95 BL6292JB page 9 of 2 2 During the definitive run of the test 0 .3617 g of dichlormid were used to prepare the test solutions
. The sample of dichlormid was stored in the dark, at ambient temperature, in the container in which it was received until required for testing, when appropriate subsamples were provided for the test operators
. 3 .2 Test specie s The test species was the unicellular green alga Selenastrum capricornutum (Strain ATCC 22662) from laboratory cultures maintained under axenic conditions . A 2 day old culture of the alga in the exponential growth phase was used as inoculum for the test . The culture was grown in the medium, and under the environmental conditions, described for the test
. 3 .3 Culture medium The culture medium used for the test, and for the maintenance of cultures of the alga used as inoculum for the test, is described in Appendix 1 and was that described by Miller et al (Ref 3)
. 4 TEST METHODS AND CONDITIONS
4 .1 Test procedure and apparatus The procedures employed were based on the relevant OECD guideline (Ref 1) . The study also complies with the relevant EU guideline (Ref 2)
. The test vessels were borosilicate glass conical flasks of 250 ml nominal capacity closed with polyurethane foam bungs . Each flask contained 100 ml of test solution . The cultures were incubated at 24 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination, with orbital shaking at approximately 160 rpm, in a Gallenkamp type INR-401 orbital incubator
. Six replicate cultures of the culture medium control, and triplicate cultures of each concentration of dichlormid were employed . The positions of the test vessels in the incubator were randomised by rows, and re-randomised daily. One blank vessel (without algal inoculum) for the culture medium control and each test concentration was incubated concurrently
. The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter model ZB, counting at a lower threshold equivalent spherical diameter of approximately 2 .3 pm
. Each replicate test vessel was inoculated with 1 .116 ml of the inoculum culture to give a nominal cell density of 1 .00 x 104 cells ml-1 . Three 100 ml volumes of Coulter electrolyte, inoculated in the same manner, had a mean measured cell density of 0 .991 x 104 cells ml" . The latter value was used for growth calculations
.p . 96 BL6292/B page 10 of 22 After 1, 2 and 3 days (24, 48 and 72 hours), samples were removed from each test and blank vessel . The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density
. 4 .2 Preparation of test solution s This study was run with a culture medium control together with nominal concentrations of 1 .5, 3 .0, 6 .0, 12, 23, 45, 90 and 180 mg 1- 1 . Dichlormid was soluble in water at a higher concentration than the test concentration selected . Therefore the test solutions were prepared without the use of an organic solvent
. 1000 ml volumes of the nominal 45, 90 and 180 mg I " test concentrations and a 2000 ml volume of the nominal 23 mg I " concentration were prepared by the direct addition of dichlormid to sterile culture medium . The lower test concentrations were prepared, using sterile culture medium, by the addition of aliquots of the nominal 23 mg 1" 1 test solution, to final volumes of 1000 ml . The control consisted of culture medium only . In all cases the final solutions contained nutrients as specified in Appendix 1
. After preparation, 1 visual assessment of the test solutions showed the control and nominal 1 .5, 3 .0, 6 .0, 12 and 23 mg I" test concentrations to be clear and colourless . The nominal 45, 90 and 180 mg I" ' test concentrations were seen to contain a faint, very fine white homogeneous suspension, which increased in density with nominal concentration and persisted despite vigorous stirring by magnetic follower and ultrasonication
. Using aseptic techniques, 100 ml volumes of the appropriate test solution were dispensed to each test and blank vessel, with the remainder of the test solutions being used for physical and chemical analyses
. 4.3 Analytical metho d The concentrations of dichlormid in the test solutions were measured using the method outlined in Appendix 2 . This is a high performance liquid chromatography method . At the start of the test, samples were taken of each test solution, using the excess remaining after filling the test vessels . At the end of the test each blank solution was sampled and analysed in the same manner . As visual assessments on the higher nominal test concentrations had shown the presence of suspended material, all samples were analysed pre and post centrifugation to determine exposure concentrations
. 4 .4 Physical and chemical parameters The pH of each test solution was measured at the start of the test, using the excess remaining after filling the test vessels . At the end of the test the pH of two of the replicate test solutions (containing algae) from the culture medium control and each test concentration was determined . The measurements were made using a Coming Model 240 pH meter . The temperature of the incubator was measured daily by a thermometer calibrated to 0 .1 °C, and conforming to BS593, and was continuously monitored, with hourly recording of values, using an automatic recording system linked to a thermistor
. The light intensity was measured once during the study, using Skye Instruments photometers reading in lux and quantum units
.

~ I Ip . 97 BL6292/B page 11 of 22 5 RESULT S
5 .1 Analytical data The concentrations of dichlormid in the test solutions at the start and end of the test are reported in Table 1 . The limit of determination of dichlormid in this study was 0 .34 mg I- ' . Typical chromatograms are repo rted in Appendix 2, Figure 1 . All analytical values are quoted to two significant figures and percentages to the nearest integer. It was considered that the measured values of dichlormid in both the p re and post centrifugation samples were all close to nominal values, suggesting that the dichlormid was in solution . The mean measured concentrations determined in the pre centrifuged samples ranged from 98 to 100 % of the nominal values. On the basis of these analyses the nominal dichlormid concentrations were used for the calculation and reporting of all results . 5 .2 Biological data The algal cell densities measured at each time pe riod are given in Table 2 . The means of these values are also shown in Table 2 and are plotted as growth curves in Figure 1
. 5 .2 .1 Areas under the growth curv e The area under the growth curve (days 0 to 3) was calculated for each replicate culture, according to the formula given in the OECD Guideline (Ref I) . Area = (No + N~ ) - 2No x t, + (Ni + N~ ) - 2No x (tZ - ti ) + ~°-i + NI) ) - 2No X (t„ - t„_' )
where No = Cell density at start of tes t Ni = Cell density at tj N„ = Cell density at t „ tj = Time (days) of first measurement after start of test t2 = Time (days) of second me asurement after start of test t„ = Time (days) of nth measurement after sta rt of tes t These areas were examined by one-way analysis of variance, and Dunnett's procedure (Ref 4) was used to identify significant differences (P=0.05) from the culture medium control . The mean areas under the growth cu rve, and the significant differences identified, are given in Table 3, together with the areas expressed as percentages of the culture medium control . The percentages were transformed to a probit scale and the median effective concentration (based on biomass curve area, EbC50) and its 95% confidence limits was calculated using the probit method (Ref 5)
. The results obtained from these statistical analyses, based on the nominal test concentrations, were as follows :p . 98 BL6292/B page 12 of 2 2 No observed effect (P=0 .05) concentration (NOEC) = 6.0 mg 1" 1 Lowest observed effect (P=0 .05) concentration (LOEC) = 12 mg I " Median effective concentration, biomass (EbC50) = 33 mg 1 " 95% confidence limits = 29 to 39 mg I- 1 5 .2.2 Growth rates The mean (days 0 to 3) growth rate of each replicate culture was calculated according to the formula: Growth rate = L°g"(Ni / N d t where Ni = Cell density at start of tes t N2 = Cell density at end of test t = Time interval = 3 days These data were analysed as described for the area method . The mean growth rates, and the significant differences identified, are given in Table 4, together with the rates expressed as percentages of the culture medium control . The results obtained from these growth rate analyses, based on the nominal test concentrations, were as follows : No observed effect (P=0 .05) concentration (NOEC) = 23 mg 1" 1 Lowest observed effect (P=0 .05) concentration (LOEC) = 45 mg 1" 1 Median effective concentration, growth rate (E~C50) = 80 mg 1" 1 95% confidence limits = 70 to 94 mg 1" 1 Because the growth rate at each concentration was relatively constant throughout the test period (see Figure 1) it was not considered necessary to analyse growth rates over shorter intervals
. 5 .3 Physical and chemical data At the start of the test the pH ranged from 7 .3 to 7 .4 and at the end of the test the range was from 7 .6 to 9 .9 (Table 5) . A maximum increase of 2 .6 pH units was observed over the test duration . This pH shift was considered to be a function of the very high growth factors observed (mean culture medium control 72 hour cell density = 237 x the 0 hour initial inoculum density) . The pH shift occurred despite a high orbital shaking rate of 160 rpm to improve mass transfer of carbon dioxide into the test solutions . At the test concentrations closest to both EC50 values (sections 5 .2 .1 and 5 .2 .2) the pH shift was less than 1 .0 unit
. The daily temperature measurements recorded, by thermometer, in the incubator ranged from 24 .0 to 24 .1 °C . The hourly temperature measurements, recorded automatically, remained within 24 ± 2°C
. The light intensity, measured once during the study, was 7330 lux (by cosine receptor)
. This was also measured in terms of quantum response and was 86 .9 μ einsteins m-' s"'
.

I jP
. 99 BL6292/B page 13 of 22 6 REFERENCES 1 OECD (1984) . OECD Guidelines for Testing of Chemicals 201 . Alga, Growth Inhibition Test . Adopted 7 June 1984
. 2 Official Journal of the European Communities, L 383 A, Part C .3, Algal inhibition test . 29 December 1992
. 3 Miller W E, Greene J C and Shiroyama T (1978)
. Selenastrum capricornutum Printz . Algal Assay Bottle Test: Experimental Design, Application and Data Interpretation Protocol . EPA-600/9-78-018, Corvallis, OR
. 4 Dunnett C W (1964) . New Tables for Multiple Comparisons with a Control
. Biometrics, 20, 482-491 . 5 Finney D J (1977) . Probit Analysis . Cambridge University Press .p . 100 BL6292/B page 14 of 22 DICHLORMID : Toxicity to the green alga Selenastrwn capricornutum TABLE I MEASURED CONCENTRATIONS OF DICHLORMID IN THE TEST SOLUTIONS Nominal Measured concentration of Mean Percentage concn of dichlormid measured of nominal dichlormid concn o f dichlormid (mg 1"1 ) 0 hour 72 hour (mg 1' 1 ) Pre Post Pre Post Pre (mg 1-1) centrifugation centrifugation centrifugation centrifugation centrifugation Culture medium <0 .24 <0 .24 <0 .34 <0 .34 <0 .34 - control 1 .5 1 .4 1 .5 1 .5 1 .5 1 .5 100 3 .0 3 .0 3 .0 3 .0 3 .0 3 .0 100 6 .0 5 .9 5 .9 5 .8 5 .8 5 .9 98 12 12 12 12 12 12 100 23 23 23 22 22 23 100 45 44 44 43 43 44 98 90 90 91 89 88 90 100 180 180 180 180 180 180 100 The limit of determination of dichlormid in this study was 0 .34 mg 1-1p . 101 BL6292/B page 15 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum capricornutum


### TABLE 2
ALGAL CELL DENSITIES Nominal Replicate Algal cell density concn of (x 10° cells ml-1) dichlormid (mg 1 " ) Day I Day 2 Day 3 Culture A 5 .34 35 .9 202 medium B 5 .73 41 .3 233 control C 5 .99 47 .4 25 2

D 5 .77 42 .4 224 E 5 .07 41 .4 253 F 7 .20 46 .9 247 Mean 5 .85 42 .6 235 A 5 .81 45 .5 224 1 .5 B 4 .95 43 .0 204

C 5 .82 44 .4 216 Mean 5 .53 44 .3 215 A 5 .35 41 .4 21 7 3 .0 B 5 .68 44 .7 225

C 5 .44 44 .4 213 Me an 5 .49 43 .5 21 8 A 4 .94 37 .1 184 6 .0 B 6 .05 50 .3 231

C 5 .44 44 .5 18 9 Mean 5 .48 44 .0 201 A 4 .86 33 .6 182 12 B 5 .69 44 .3 19 3

C 5 .12 36 .8 165 Mean 5 .22 38 .2 180 A 4 .12 30 .6 15 3 23 B 5 .18 36 .0 172

C 5 .19 34 .8 184 Mean 4 .83 33 .8 17 0 A 4 .36 26 .2 132 45 B 4 .68 30 .9 139

C 5 .24 31 .4 14 8 Mean 4 .76 29 .5 140 A 2 .76 6 .08 20 .4 90 B 2 .77 3 .01 25
. 8

C 2 .07 3 .73 11 .0 Mean 2 .53 5 .94 19 .1 A 1 .38 0 .600 0 .83 5 180 B 1 .70 1 .23 0 .470

C 0 .970 0 .640 0 .486 Mean 1 .35 0 .823 0 .597 Inoculum (Day 0) cell density = 0 .991 x 104 cells ml-1p . 102 BL6292/B page 16 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum capricornutum
TABLE 3 

### MEAN AREAS UNDER THE GROWTH CURVE, INDICATING STATISTICALLY SIGNIFICANT DIFFERENCES TOGETHER WITH PERCENTAGES OF THE

CULTURE MEDIUM CONTRO L Nominal concn of Mean area Percentage of dichlormid under growth culture medium curve control (mg 1" 1 ) (0-3 days ) Culture medium 163 .5 - control 1 .5 154 .7 95 3 .0 155 .7 95 6 .0 147 .6 90 12 131 .0* 80 23 121 .0* 74 45 101 .6* 62 90 15 .5* 9 180 0 .0* 0 * Significant difference (P=0 .05) from the culture medium controlp . 103 BL6292JB page 17 of 22 DICHLORMID : Toxicity to the green alga Selenastrum capricornutum
TABLE 4 

### MEAN GROWTH RATES OVER THE TEST PERIOD, INDICATING STATISTICALLY SIGNIFICANT DIFFERENCES TOGETHER WITH PERCENTAGES OF THE CULTURE MEDIUM CONTRO L Nominal concn of Mean growth Percentage of dichlormid rate culture medium control (mg 1"(0-3 days ) Culture medium 1 .823 - control 1 .5 1 .793 98 3 .0 1 .798 99 6 .0 1 .771 97 12 1 .734 95 23 1 .714 94 45 1 .649* 90 90 0 .986* 54 180 -0 .169* -9 * Significant difference (P=0 .05) from the culture medium controlP
. 1 BL6292JB page 18 of 22 DICHLORMID : Toxicity to the green alg a Selenastrum cap ricornutum
TABLE 5 TEST SOLUTION PH MEASUREMENTS Nominal concn of pH dichlormid 

### Day 0 Day 3 (mg 1-, ) Replicate A Replicate B Culture medium 7 .3 8 .3 8 .3 control 1 .5 7 .3 8 .2 9 .9 3 .0 7 .3 9 .0 9 .6 6 .0 7 .4 9 .1 8 .6 12 7 .4 8 .9 8 .8 23 7 .4 8 .3 8 .2 45 7 .4 8 .2 8 .1 90 7 .3 8 .0 7 .9 180 7 .3 7 .6 7 .6p . 104

• BL6292B page 19 of 22

DICHLORMID : Toxicity to the green alga Selenastrum capricornutum
FIGURE 1 

### EFFECTS ON GROWTH OF THE GREEN ALGA Selenastrum capricornutum AFTER 72 HOURS

0

~ KEY ~' -- CONTROL 4 1 .5 mg r' 3 .0 my r' X 8.0 mg f'

.._..Y . .. . 12 mg r' o ~ ~ ©. . . 23 rng r 45 m9 r' ;C /~><t`---11'- 80 mg r 180 mg U

0

0 1 2 3 Days

NB Nominal test concentrations shown