2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-methoxyphenyl)-3-oxobutyramide]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-10-29 - 2002-11-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The test had been performed according to relevant guidelines and compliant to GLP. The results are well documented and plausible. However, as the test was conducted according to earlier versions of the current guideline, minor methodological deviations are evident (instead of six treatment replicates as required by the guideline for a limit test, only three treatment replicates had been tested; growth rates for control replicates are only reported as mean values).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Three instead of six treatment replicates

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Remarks:

as described under § 19a, German Chemical Law (ChemG), Annex 1, in the version of May 8, 2001

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

EC Number:

228-787-8

EC Name:

2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

Cas Number:

6358-85-6

IUPAC Name:

2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis(3-oxo-N-phenylbutanamide)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

The test item is of very poor solubility in water. Analytical monitoring of the test substance concentrations in the test medium was not conducted, since no relevant results could be expected.

Test solutions

Vehicle:

no

Details on test solutions:

The test concentration of 100 mg/L was prepared in three replicates.
The test substance was weighed into a 250 mL beaker, diluted with algal medium, transferred to a 500 mL Erlenmeyer flask and filled up to final volume of 500 mL. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was filtered through a membrane filter of 0.45 µm pore size. The preparation was completed by adding suitable aliquots of algal precultures.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Species : Monocellular freshwater alga Desmodesmus subspicatus SAG Strain No. 86.81
Origin "Albrecht-von-Haller-Institute for Plant Sciences, Department of Experimental Phycology and Collection of Algal Cultures, University of Göttingen, Germany
Cultivation of algae: Cultivation of algae is carried out in the laboratories of Aventis Pharma Deutschland GmbH, ProTox under standardized conditions in accordance with the relevant test guidelines.
Sensitivity of the test species: To demonstrate that under the laboratory test conditions the sensitivity of the test species has not changed significantly, potassium dichromate (p.A.) is tested as a reference substance at least twice a year.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

Standard algal culture medium had been used according to the guideline (OECD TG 201 medium).

Test temperature:

in °C:
0 hours: 23
24 hours: 23
48 hours: 23
72 hours: 23

pH:

Controls (mean of replicates): start pH 7.8, end pH 8.9
test items (mean of replicates): start pH 7.6, end pH 8.4

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

The test item is of very poor solubility in water. Analytical monitoring of the test substance concentrations in the test medium was not conducted, since no relevant results could be expected.
The limit test was conducted at a nominal concentration of 100 mg/L.

Details on test conditions:

Test procedure:
The test concentration of 100 mg/L was prepared in three replicates. Six controls without test substance were run.
Test cultures containing the required concentrations of test substance and the required quantity of algal inoculum were prepared by adding aliquots of stock solutions of the test substance to suitable amounts of algal pre-cultures. The initial cell density in the test cultures was approximately 10E(4) cells/mL. The culture flasks were shaken and placed in the culturing apparatus. The cell density in each culture flask was determined at 24, 48 and 72 hours after the start of the test, using a Casy Coulter Counter (Schärfe Systeme, Reutlingen Germany).

Test conditions:
The cultures were maintained at a temperature in the range of 21 to 25 °C, controlled at ± 2 °C. In the culturing apparatus, continuous illumination in the range of 400 nm to 700 nm was provided with a light intensity between 6000 to 10000 lux.
The algae were kept in suspension by stirring in order to improve gas exchange and to reduce pH fluctuations in the test solution.
The test was carried out without pH adjustment. The pH was measured at the beginning of the test and after 72 hours.

Culture medium:
Standard algal culture medium had been used according to the guideline (OECD TG 201 medium). Only distilled water or deionized water with a conductivity of less than 1 µScmE(-1) was used. The pH of the medium after equilibration with air was 7.8.

DATA ANALYSIS
The measured cell density in the test cultures and controls are tabulated together with the concentrations of the test substance and the times of measurements. Algae response was calculated from changes in the area under the growth curve (biomass) and growth rate.
Percent inhibition of yield and percent inhibition of growth rate had been calculated according to the guideline.
CALCULATION OF EC50-VALUES AND THE NOEC
For the limit test according to the guideline Directive 92/69/EEC, Annex Part C, C.3 no statistical evaluation of the EC50 and NOEC was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate at least twice a year with the test organism

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the poor solubility of the test item no analytical determination of actual substance concentration possible

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Due to the poor solubility of the test item no analytical determination of actual substance concentration possible

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to the poor solubility of the test item no analytical determination of actual substance concentration possible

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Due to the poor solubility of the test item no analytical determination of actual substance concentration possible

Details on results:

Microscopical Observations:
Algae were observed at the beginning and at the end of the study. No visible changes of the shape of the algae were noticed either in the control or the treatment groups.
In the 100 mg/L treatment the inhibition of the Biomass was 2 % and the growth rate reduction was 3 % compared to the control.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Any other information on results incl. tables

Calculation of growth (area under growth curve) and growth rate exposure

Nominal conc.[mg/L]	Vessel number	Area	Growth rate			Growth inhib. [%]	Growth rate reduct. [%]
		0-72 hrs	0-24 hrs	0-48 hrs	0-72 hrs	0-72 hrs	0-24 hrs	0-48 hrs	0-72 hrs
Control	1-6	7979907	0.39	1.12	1.30	-	-	-	-
100	7-9	7788347	1.07	1.15	1.26	2	-175	-3	3

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Remarks:

Cell density controls: increase by an average factor of 50 (16 required by OECD 201); however, as only the mean section by section specific growth rates over all control replicates given, validity citerion cannot be checked.

Conclusions:

The submission substance was tested for toxicity to aquatic plants in a growth inhibition test with Desmodesmus subspicatus. Due to the poor solubility of the test item a limit test at 100 mg/L nominal concentration had been conducted. The following values were determined:
EC50 (72 h) for both, growth rate and biomass was larger than 100 mg/L nominal concentration
NOEC (72 h) for both, growth rate and biomass was equal to 100 mg/L nominal concentration.

Executive summary:

The purpose of this study was to determine the effects of the test substance on the growth of a unicellular green algae species.

Exponentially growing cultures of Desmodesmus subspicatus were exposed to 100 mg/L of the test substance and a control (0 mg/L) over several generations under defined conditions.

The cell density in each solution was measured at least every 24 hours. The inhibition of growth in relation to a control culture was determined.

According to Directive 92/69/EEC, Annex Part C, C.3. Algal Inhibition Test, it is sufficient to conduct a limit test with a test substance concentration of 100 mg/L if the inhibition of the biomass and growth rate is < 25 % compared to the control.

The test substance was weighed into a 250 mL beaker, diluted with algal medium, transferred to a 500 mL Erlenmeyer flask and filled up to final volume of 500 mL. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was filtered through a membrane filter of 0.45 µm pore size. The resulting solution was visually clear. No particulate matter was observed.

As requested by the sponsor analytical monitoring of the test substance concentrations in the test medium was not conducted, since no relevant results could be expected.

Algae were observed microscopically at the beginning and at the end of the study. No visible changes of the shape of the algae were noticed in either the control or the treatment groups.

In the 100 mg/L treatment the inhibition of the biomass was 2 % and the growth rate reductionwas 3 % compared to the control.

This resulted in the following effect concentrations:

Biomass                    Growth rate

(mg/L)                           (mg/L)

EC50	(0-72h)	>100	> 100
NOEC	(0-72h)	100	100