p. 1

The Dow Chemical Company

1803 BUILDING Midland, Michigan 48674

August 6, 2008 USA

fi

By overnight courier to :

OPPT Document Control Office

EPA East, R.-n . 6428

1201 Constitution Avenue, NW

Washington, DC

ATTN : 8(d) Health and Safety Reporting Rule (Notification/Reporting )

By U

Document Control Office (DCO) (7407M

.S. mail to: Cz

)

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-2005-0055

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

; ;

:emical Company s h o~~~ ~~~~ 0l li/i'~ Ill// liii! ~ I~ ~ I~I I The Dow Cl li ~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 studies
p. 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 Programs

Dichlormid

Dogs Aug-02

: 1 Yr. Oral Toxicity Study in US Environmental Protection Agenc y

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 Programs

Dichlormid: 90-Day Feeding Study in Rats MRID 44606406 Jan-88

Dichlormid: Multigeneration Reproduction Office of Pesticide Programs

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 1
p. 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 Programs

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 2
p. 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 Programs

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 3
p. 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 Programs

Salmonella Typhimurium MRID 41561404 Jan-87

Office of Pesticide Programs

14-Week Inhalation Study with R-25788 in US Environmental Protection Agenc

Rats Jan-93

y

Office of Pesticide Programs

The Acute Oral Toxicity (LD50) of MRID 4156140 1

Dichlormid to the Bobwhite Quail Jan-90

Page 4
p. 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 Programs

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 Programs

Determination of the Water Solubility of R- US Environmental Protection Agenc y

25788 at pH 5 and 9 Jun-89

Office of Pesticide Programs

R-25788 Physical Properties MRID 42773501 Jun-89

Office of Pesticide Programs

Dichlormid

Dogs Aug-02

: 1 year Oral Toxicity Study in US Environmental Protection Agenc y

Page 5
p. 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 6
p. 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 putida

Study completed : 23 November 1988

Performing laboratory : ICI Brixham Laboratory

Imperial Chemical Industries PLC

Freshwater Quarry

Brixham Devon TQ5 8BA

U K

Sponsor : ICI Agrochemicals

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 1989
p. 9

FT68(7) /88 BL/B/3471 page 2 of 11

BL/B/347 1

DICHLORMID : Determination of the toxicity to Pseudomonas putida
P. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . .11
p. 12

BL/B/3471 page 5 of 11

I SUMMARY

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 METHODS

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 substance

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 value

inhibition 1 - ) x 100

( Mean optical density Blank

( 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

231-241 -

. Water Research 14,

2 Slabbert J L (1986) Improved bacterial growth test for rapid

water toxicity screening . Bull Environ Contam Toxicol 37,

565-569
p. 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- medium

Flask 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 for

Flask Contents density uninoculated Izhinumber (nominal concentrations) at 600 nm solution bition

(4 cm cells )

1-3 Control 0.575 0.535

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 .040

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 .042
p. 18

BL/B/3471 page 11 of 11

FIGURE 1

Z

O

~

~

Z

I

3

O

LO

Z

ry O

li

H ~

U ~

J

2 ~

LL Z

O w

ry

Z U

O a Z ~--°O

F- U

cr: "o

L

ao

w

h- q

w

A a

r

Ln

~ w m

Cy0

J

2

U

► -+ ..

CD

OOT O8 09 0b OZ 0

NOIlISIHNI
P. 19

G4 .3/O 1

ZENECA Agrochemicals Report Senes

RJ 2668B

Jeabtt's Hill Research Station

Bracknell Berkshire UK

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 Pages
p. 20

Page 1

Report No : RJ2668B

ZENECA Ag Products

(ZENECA Inc .)

Wilmington

Delaware 19850

Study Title

Dichlormid : Toxicity of Technical Material to th e Earthworm Eisenia fetida in an Artificial Soil Test

Data Requirement

Supplemental

Authors

D Jackson and J M Coulson

Study Completed On

08 December 1998

Performing Laborato ry

ZENECA AGROCHEMICALS (ZENECA LIMITED)

JEALOTTS HILL RESEARCH STATION

BRACKNELL, BERKS, RG42 6E T

UK

Laboratory Project ID

98J H005

Page 1 of 16 Pages

1, 1
p. 21

Page 2

Report No : RJ26686

Study Number 98JH005

Report Tide : Dichlormid : Toxicity of Technical Material to the Earthworm Eisenia fetida

in an Artificial 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 Date
p. 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 Report

02 Feb 1998 Protocol 02 Feb 1998

16 Apr 1998 Study Conduct 16 Apr 1998

09 Nov 1998 Final Report 10 Nov 1998

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 1998

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

Qua Assurance Date ctmhe'y- l~8
p. 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 Preparation 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 Artificial Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

3 .4 Addition of Test Earthworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

3 .5 Test Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

3 .6 Assessment of Earthworms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

3 .7 Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

4 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

5 CONCLUSIONS . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

6 PERSONNEL AND TEST FACILITIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

7 STUDY DATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10

8 RETENTI 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 material 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 : 98JH005

Report Title : Dichlonrid : Toxicity of Technical Material to the Earthworm Eisenia fetida

in an Artificial Soil Test

Authors : D Jackson and J M Coulson

SUMMARY

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 LC50 value for 2-chioroacetamide, tested as a toxic standard, was 52 mg kg'' .
p. 26

.

•

Page 7

Report No : RJ2668B

1 INTRODUCTION

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 Chemicals

2 .1 .1 Dichlormid Technical Material

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 Animals

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 Soil

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 PROCEDURES

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

Report No : RJ2668B

3 .4 Addition of Test Earthworms

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 . The batches of ten worms were ranked into weight order and

then split into four g roups ranging from 'heaviesY, to "lightest" . One batch from 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 Conditions

Th e conditions throughout the tests were as follows :

Soil moisture contents were within the range 50 ± 10% throughout the test pe riod

(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 Earthworms

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 definitive 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

very 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 control

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 CONCLUSIONS

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 DATES

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 RECORDS

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 REFERENCES

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/CG

WINo :40771

Date : 10 November 1998
p. 31

Page 12

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 after

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

exposure to 2-chloroacetamide in a rtificial 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 February 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

Control 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 14
p. 33

-

Page 14

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 - 4.82

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 condition

c - Assumed all dead. Bodies difficult to allocate to individuals due to decomposition

d - Only rear segments of remaining worm found, which were moving
p. 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. Surviving
p. 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 zo

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

Brixham 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 1991
p. 37

THIS IS A TRUE COPY OF THE ORIGINAL

DOCUMENT AND MAY HAVE MISSING

PAGES OR MAY CONTAIN PAGES THAT

ARE HARD TO READ
p. 38

r 1 t4lyu BL3967/B page 2 of 26

BL3967B

DICHLORMID: Chronic toxicity to Daphnia magna

Brixham study number: T388/A
p. 39

FT74/90 BL3967B page 3 of 26

BL3967B

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 cf,
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 6 194 1

\ 6f3lRl D 6Dowe Date

Quality Assurance Unit
p. 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

Programme Manager ~j 5

Date
FT74/90 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 organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 litr e 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 magna

Source of organisms Continuous laboratory cultures

Stage of organisms Less than 24 hours old

at the initiation

of study
BL39b //t3 pagepti & lb

2 INTRODUCTION

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 certificate

of analysis for the batch of mate rial stated that its purity was 96 .3% w/w ( analytical

reference 11842-03, sample identification 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 organism

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 RESULTS

4.1 Chemical analysis

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 Reproduction

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.
BL3967/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 <0.08

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 94

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 97

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 mean 5 .5 98
BL3967/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 10 100

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 18 100 100

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 33 103 103

Overall mean 33 103

56 0- 3 56 55 100 98

5- 7 57 59 102 105

7- 10 58 58 104 104

Mean 57 57 102 102

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 .0
tsl,~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 concentration mg/1

test Control 1 .8 1 3 .2 5.6 10 18 32 56

Mean 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) 10

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 clearance
BL3967/B pagdMof 26

TABLE 3

DICHLORMID: CHRONIC TOXICITY TO Daphnia magna

REPRODUCTION DAT A

Client: ICI Agrochemicals, Imperial Chemical Industries PLC

Test subst ance: 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

Mean 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

offsprin g

% 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 from 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 52

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

NSD * * * *

.68 4.63 4.60 4.58 4.18

NSD = No significant (P=0.05) decrease from control

* = Significant (P=0.05) decrease from control
p. 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 10 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 20.0
P. 56

BL3967/B page 20 of 26

• TABLE6

DICHLORMID : CHRONIC TOXICITY TO Daphnia magna

pH OF NEW SOLUTIONS

Client: ICI Agrochemicals, Imperial Chemical Industries PLC

Test substance: Dichlormid technical

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 8.47
t3L396'//I3 pagep2 Rot 26

TABLE 6 cont d

DICHLORVIID : CHRONIC TOXICITY TO Daphnia magna

pH OF OLD SOLUTIONS

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

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

Da

o Nominal concentration mg/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

Maximum 9

.1 7.4 7.8 7.9 7.3 8.0 8.0 7.7 8.2 .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 1990

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 1991
p. 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

BL3991B

DICHLORMID : The 28 day LC50 to rainbow trout

(Salmo gairdneri)

Brixham study number: T3881B
p. 62

FT75/S0 BL3991/B page 3 of 28

BL399 , B

DICHLORIMID: 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 and Development (OECD) principles 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 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 re flection of the raw data generated .

Date C (

7ate ~-

3

-g/
FT75/90 rsLSyy

p. 63

i/ts page 4 or 23

BL3991/B

DICHLORMID: The 28 day LC50 to rainbow trout

(Salmo gairdneri)

Brixham study number: T388/B

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

26 November 1990 Study conduct 26 November 1990

1 Draft report

Final report

f ~ ~ 5 3 •9\

J L Gilbert Date

Quality Assurance Unit
p. 64

FT75/90 BL3991/B page 5 of 28

BL3991B

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

Date

Report approved by !_ 3 _ b

Progamme Manager '~ 7l

Date
p. 65

FI75/90 BL3991/Bpage 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 . . . . . . . . . . . . . . . . . . . . .

4.3 Preparation of test solutions . . . . . . . . . .

. .

.

.

.

.

. .

. .

.

.

. .

.

.

. .

.

. 9

. . . . . . . . . . . . . . 10

4.4 Analytical method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

4.5 Physical/chemical parameters

. . . . . . . . 10 . . . . . . . . . . . . . . . . . . . . . . . .

4.6 Observations of mortalities and symptoms of toxicity . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
p. 66

BL3991/B page 7 of 28

I SUMMARY

Sponsor ICI Agrochemicals, Imperial Chemical Industries PLC, Fernhurst,

Haslemere, Surrey, GU27 HE, UK

Contact Dr B D Cavell, Inte rnational (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 nam e

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 angle

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 956

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 study
p. 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 Brixham 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 solubility 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 capacity of 54 litres and a working volume of 45 lit re s

were used to hold the test fish. The test solutions were renewed at a nominal rate of 250

ml/minute so that a 95% exchange 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 method

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 angle

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 28

The means and measured ranges of diluent flow rate and the r anges 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 means 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/cm

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. 73

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

21

22

Mean mg/I 140 100 41 22 13 <0

measure .84 d

concn of

dichlormid

technical nominal % of 88 109 103 129 130
13L:~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 organism: Rainbow trout (Salmo gairdneri)

Test water : Freshwater

Nominal Mean

concn of measured Cumulative percentage mortality observ ed

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 TD

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 feeding

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 28
p. 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

b S

J

160 140 0.70 0.14 TD - - -

92 100 0.81 0

*00

.91 0+9 1 . 40 41 0 .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 14-28
p. 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 mate rial: 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 surviving

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

control
p. 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

SD 0

.9 9.9 10.0 9.9 9.9 9.8

.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 9.8
p. 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 Agrochemicals, 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=---

SD 0

Mean 7.79 7.71 7. 7

.10 0.12 0. 1

Minimum 7.41 7 .42 7.4

3 Maximum 7 .79 7.86 7.80
p. 80

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 PLC

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

SD 0

.2 15.0 15.0 15.1 15.0 15.0

.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 15.0
p. 81

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. 82

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 10

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) Mean SD Range

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 Range

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 control
p. 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

Alkal- (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 mg/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 4
p.85

~-oFIGURE 1

CONCENTRATION RESPONSE RELATIONSHIP

RAIN80V TROUT

DICHLORMID TECHNICAL

STUDY NO . T388/ B

100

80

60

40

-~E- 2e Day LC50

(38 W/ 1 )

~E 21 Day LC50

N 20 (36 sp/1 )

~" --~f- 14 Day c ~ c3~ LC50

20/1)

" 10 -~-- 10 Day LC50

6 (5D ~y I)

w

=E

6 \ (B3

7 Day LC50

no/ 1)

H

4 -~E- 4 Day LC50

(120 ap/1)

2

1

10 50 100 500 1000

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 Limited

Brixham Devon TQ5 8BA

UK

Sponsor ZENECAAgrocheniicals

Fernhurst Haslemere

Surrey GU27 3JE

UK

Authors D V Smyth

S E Magor

N Shillabee r

Approved by N R Gore

July 1998
p. 87

THIS IS A TRUE COPY OF THE ORIGINAL

DOCUMENT AND MAY HAVE MISSING

PAGES OR MAY CONTAIN PAGES THAT

ARE HARD TO READ
p. 88

BL6292IB page 2 of 22

DICHLORMID : Toxicity to the green alg a

Selenastrum capricornutum

Brixham study number: AF00211B
p. 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.

Date
P. 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 1998

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 Unit
P. 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 .

Study Director 01 01 g

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

Date
p. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 12

6 REFERENCES. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

TABLES . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

FIGURE. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 19

APPENDICES .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

The last page of this report is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

il I
p. 93

BL6292/B page 7 of 22

1 SUMMARY

Sponsor ZENECA Agrochemicals, Fernhurst, Haslemere, Surrey, GU27

3JE, UK

Contact Dr A M Skidmore, telephone : inte rnational (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

22662

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 1998

Nominal 24 t 2°C

test temperature

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 INTRODUCTION

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 MATERIALS

3.1 Test substance

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 species

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 solutions

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 method

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 I
p. 97

BL6292/B page 11 of 22

5 RESULTS

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 test

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 test

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 test

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 j
P. 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 of

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 1-1
p. 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 47.4 252

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 41.4 217

3 .0 B 5.68 44.7 225

C 5.44 44.4 213

Mean 5 .49 43.5 218

A 4.94 37.1 184

6.0 B 6.05 50.3 231

C 5.44 44.5 189

Mean 5.48 44.0 201

A 4.86 33.6 182

12 B 5.69 44.3 193

C 5.12 36.8 165

Mean 5.22 38.2 180

A 4.12 30.6 153

23 B 5.18 36.0 172

C 5.19 34.8 184

Mean 4.83 33.8 170

A 4.36 26.2 132

45 B 4.68 30.9 139

C 5.24 31.4 148

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 0.835

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-1
p. 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 control
p. 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

contro l

(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 control
P. 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 Day3

(mg 1-,) Replicate A Replicate B

Culture medium 7.3 8.3 8.3

contro l

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 7.6
p. 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. .

.

o ~ ~ ©

. 12 mg r'

. .

. 23 rng r

45 m9 r'

;C /~><t`---11'- 80 mgr

180 mg

U

0

0 1 2 3

Days

NB Nominal test concentrations shown