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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2002-10-29 - 2002-11-21

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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).

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Yellow 17 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Three instead of six treatment replicates

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

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.

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 (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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

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

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

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

Key result

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

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

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

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

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Yellow 17 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass). Both, the inhibition of Algal cell growth and the reduction of growth rate were not statistically different from the control values.
Hence, the NOEC equalled the maximum solubility and the EC50 for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium at a 0.45 pm filtered test solution prepared at 100 mg/I, the regulatory limit concentration.

Results with reference substance (positive control):

Selenastrum capricornutum, fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 352417).
The positive control experiment was performed analogous to the actual study.
Start of first exposure: May 21, 2002 Completion last exposure: May 24, 2002
The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.74 mg/L with a 95 % confidence interval ranging from 0.46 to 1.2 mg/I. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/L. Hence, the EBC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/L with a 95 % confidence interval ranging from 1.2 to 1.9 mg/L. The historical ranges for growth rate reduction are in the range of 0.82 and 2.3 mg/I. Hence, the ERC50: 0-72h for the present batch is within this range.

Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrationsof the test item. The respective growth curves are shown in Figure 1 (see the Appendix I for the calibration curve and extinctions and cell densities per replicate).

Table 1: Mean cell densities (x 10⁴cells/ml) during the limit test

Nominal conc.* test item (mg/1)	Exposure time (hours)
	0	24	48	72
Blank-control	1.0	5.2	30.0	103.1
100	1.0	6.2	27.8	84.1

 * 0.45 µm filtrate

Table 2: Percentage inhibition of cell growth during the final test.

Nominal conc.* test item (mg/1)	Cell growth (0-72 hrs)
	Mean area (A)	Inhibition(%)
Blank-control	2022.74
100	1765.66	12.7

* 0.45 µm filtrate

Table 3: Percentage reduction of growth rate at different time intervals during the final test.

Nominal conc.* test item (mg/L)	Mean growth rate
	µ (0-24 hrs)	Reduction (%)	µ (0-48 hrs)	Reduction (%)	µ (0-72 hrs)	Reduction(%)
Blank-control	0.06869		0.07073		0.06426
100	0.07542	-9.8	0.06915	2.2	0.06141	4.4

*0.45 µm filtrate

Validity criteria fulfilled:

yes

Remarks:

1.      In the controls, cell density increased by an average factor of > 16 within three days. 2.      Further, all test conditions (pH and temperature) remained within the ranges prescribed by the protocol.

Conclusions:

Limit study with nominal test substance concentration of 100 mg/L. Because of the poor substance solubility in water (below 10 µg/L) analytical detection or monitoring was not feasible.
EC50: > 100 mg/L (nominal)
NOEC: = 100 mg/L (nominal)

Executive summary:

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in the test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass) which were not statistically different from the control values.

The NOEC equalled the maximum solubility. The EC₅₀for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium prepared with a 0.45 µm filtered test solution (nominal concentration 100 mg/L).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-04-23 - 2002-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Remarks:

OECD-Principles of Good Laboratory Practice

Specific details on test material used for the study:

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

Analytical monitoring:

no

Details on sampling:

Analytical monitoring had not been feasible, as substance solubility (below 10 µg/l) was beyond analytical sensitivity.

Vehicle:

no

Details on test solutions:

The batch tested was a yellow powder with a purity of 94.5%. The test substance was insoluble in water based on information supplied by the sponsor. Analytical confirmation of the actually dissolved concentration in test medium was not possible as no analytical method was available or could be developed with the necessary sensitivity to measure the soluble fraction of the test item.
This was based on previous work on a similar compound, which showed that the water solubility was less than 10 µg/I (NOTOX Project 289979). The soluble fraction present in saturated solutions prepared during that project were not distinct from the background responses. All possible efforts were taken to improve the detection limit of the analytical method. However, these were not successful.
Preparation of a test solution at maximum saturation of the test item in test medium started with a nominal concentration of 100 mg/I. This solution was treated with ultrasonic waves and subsequently magnetically stirred for 4 days. The resulting dispersion was then pre-filtered through a paper filter (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5µm), followed by filtration through a 0.45 µm membrane filter. The final test solution was a clear and colourless solution. The blank-control received similar treatment to correct for possible treatment related effects on algal growth.
Note that the use of centrifugation to obtain the water-soluble fraction proved not to be successful as a consequence of the behaviour of the test substance in water, i.e. large amounts remained floating an the water surface. After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Test organism: Selenastrum capricornutum, strain: NIVA CHL 1
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light (4000-9000 lux) in a climate room at a temperature of 23 ± 2°C.
- Preculture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 2x10^4 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22.5°C at the start of the test. Temperature was maintained during the test between 21.5 nd 23°C.

pH:

The pH was determined at the beginning and the end of the incubation period:
Control: 8.4 and 8.2
Test sample: 8.3 and 8.4

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentration (limit test): 100 mg/L;
Due to the poor water solubility (less than 10 µg/L), no analytical determination of the concentration was possible as analytical sensitivity had been too low.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- 100 ml, all glass
- 50 ml
- The algal cells were kept in suspension by continous shaking
- Initial cells density: 10E4 Cells/mL
- Control end cells density: 103.1x10E4 cells/mL
- No. of vessels per concentration (replicates): 6 replicates for the control and the limit test (100 mg/L nominal), each

GROWTH MEDIUM
- Standard medium used: yes, M2-medium according to ISO-Standard Standard "Algal growth inhibition test" Nov. 1989

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water - Tap water purified by reverse osmosis and then passed over activated carbon and ion-exchange cartridges (Millipore Corp., Bedford, Mass., USA).



OTHER TEST CONDITIONS

- Photoperiod: continuously
- Light intensity and quality: TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 60 to 120 µE/mE2/s, not varying by more than 20%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, cells were counted by microscope, using a counting chamber. Thereafter cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with immersion probe (pathlength =20 mm). Varian Nederland BV., Houten, The Netherlands. Algal medium was used as blank.

TEST CONCENTRATIONS
Limit test, nominal concentration of 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass). Both, the inhibition of Algal cell growth and the reduction of growth rate were not statistically different from the control values.
Hence, the NOEC equalled the maximum solubility and the EC50 for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium at a 0.45 pm filtered test solution prepared at 100 mg/I, the regulatory limit concentration.

Results with reference substance (positive control):

Selenastrum capricornutum, fresh water algal growth inhibition test with potassium dichromate (NOTOX Project 352417).
The positive control experiment was performed analogous to the actual study.
Start of first exposure: May 21, 2002 Completion last exposure: May 24, 2002
The EC50 for cell growth inhibition (EBC50: 0-72h) was 0.74 mg/L with a 95 % confidence interval ranging from 0.46 to 1.2 mg/I. The historical ranges of the 72h EC50 for growth inhibition lie between 0.49 and 1.4 mg/L. Hence, the EBC50: 0-72h for the present batch corresponds with this range.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/L with a 95 % confidence interval ranging from 1.2 to 1.9 mg/L. The historical ranges for growth rate reduction are in the range of 0.82 and 2.3 mg/I. Hence, the ERC50: 0-72h for the present batch is within this range.

Table 1 shows mean cell densities measured at 24-hour intervals at the different concentrationsof the test item. The respective growth curves are shown in Figure 1 (see the Appendix I for the calibration curve and extinctions and cell densities per replicate).

Table 1: Mean cell densities (x 10⁴cells/ml) during the limit test

Nominal conc.* test item (mg/1)	Exposure time (hours)
	0	24	48	72
Blank-control	1.0	5.2	30.0	103.1
100	1.0	6.2	27.8	84.1

 * 0.45 µm filtrate

Table 2: Percentage inhibition of cell growth during the final test.

Nominal conc.* test item (mg/1)	Cell growth (0-72 hrs)
	Mean area (A)	Inhibition(%)
Blank-control	2022.74
100	1765.66	12.7

* 0.45 µm filtrate

Table 3: Percentage reduction of growth rate at different time intervals during the final test.

Nominal conc.* test item (mg/L)	Mean growth rate
	µ (0-24 hrs)	Reduction (%)	µ (0-48 hrs)	Reduction (%)	µ (0-72 hrs)	Reduction(%)
Blank-control	0.06869		0.07073		0.06426
100	0.07542	-9.8	0.06915	2.2	0.06141	4.4

*0.45 µm filtrate

Validity criteria fulfilled:

yes

Remarks:

1.      In the controls, cell density increased by an average factor of > 16 within three days. 2.      Further, all test conditions (pH and temperature) remained within the ranges prescribed by the protocol.

Conclusions:

Limit study with nominal test substance concentration of 100 mg/L. Because of the poor substance solubility in water (below 10 µg/L) analytical detection or monitoring was not feasible.
EC50: > 100 mg/L (nominal)
NOEC: = 100 mg/L (nominal)

Executive summary:

Exposure of the fresh water algal species Selenastrum capricornutum to the maximum soluble fraction of the test item in the test medium resulted in 4% reduction in growth rate and 13% inhibition of total cell growth (biomass) which were not statistically different from the control values.

The NOEC equalled the maximum solubility. The EC₅₀for both algal growth inhibition and growth rate reduction exceeded the maximum solubility of the test item in test medium prepared with a 0.45 µm filtered test solution (nominal concentration 100 mg/L).

Reference 4

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:

guideline study with acceptable restrictions

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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

Three instead of six treatment replicates

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

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.

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 (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.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

not applicable

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

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

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

Key result

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

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

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

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

Description of key information

For the assessment of toxicity of  the registration substance and its strucutral analogues, the Diarylide Yellow Pigments, towards aquatic plants two reliable studies on Pigment Yellow 12 and on Pigment Yellow 83 had been performed. For all studies with nominal pigment concentrations far above the solubility limit no toxicity towards algae could be observed. Judging from these experimental results the Diarylide Yellow Pigments are to be regarded as nontoxic to aquatic plants / algae.

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.

Additional information

For the assessment of the  toxicity of the registration substance and its strucutral analogues, the Diarylide Yellow Pigments, towards aquatic plants two reliable studies on Pigment Yellow 12 and on Pigment Yellow 83 had been performed with the green algae Desmodesmus subspicatus (Pigment Yellow 12) and Selenastrum capricornutum (Pigment Yellow 83). For all studies with nominal pigment concentrations far above the solubility limit no toxicity towards algae could be observed. Judging from these experimental results the Diarylide Yellow Pigments are to be regarded as nontoxic to aquatic plants / algae.