N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 FEB 2004 to 13 FEB 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Testguideline 202 Daphnia sp., 14-day Reproduction Test (including an Acute Immobilisation Test) Adopted April 4, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Remarks:

According to German Chemical Law (ChemG), Annex 1, in the version of May 8, 2001 and 1999/11/EU of March 8, 1999

Analytical monitoring:

no

Details on sampling:

Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance in water is below 1 mg/L. The concentration data given in the present report are therefore based on nominal concentrations.

Vehicle:

no

Details on test solutions:

The test substance was weighed into a beaker, mixed with water for dilution and homogenized
with the Ultra-Turrax. The mixtures were then stirred for approximately 48
hours with a magnetic stirrer to ensure that the solubility limit of the test substance in
the test water was reached. To remove fine undissolved particles the test batches
were filtered through a cellulose filter, which was saturated with the test substance
prior to use.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna - Clone 5 (IRChA, France)
Origin Stock culture of Aventis Pharma Deutschland GmbH, ProTox
Breeding conditions 100 mL beakers (height 90 mm, diameter 50 mm), filled with 70 mL test water (according to Elendt, M4) served as culture vessels. One daphnid was used per culture vessel. The animals were used for breeding up to an age of 42 days. The reproduction rate and the state of health of the animals were monitored daily apart from weekends. From these cultures daphnids with an age of < 24 h are taken as test animals.
The animals were fed three times a week with monocellular green algae (Desmodesmus subspicatus). The supplied diet was based on the amount of organic carbon (C). The ration level is 0.2 mg carbon / day / animal.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The total hardness of the dilution water was at 2.1 mmol Ca(2+) + Mg(2+) / L during the study.

Test temperature:

Control: 20.8 - 21.1 deg C
Treatment: 21.0 - 21.1 deg C

pH:

Control: 7.7 - 8.0
Treatment: 7.8 - 8.0

Dissolved oxygen:

[mg/L]
Control: 8.4 - 8.6
Treatment: 8.3 - 8.5

Nominal and measured concentrations:

100 mg/L nominal concentration
Control: 0 mg/L

Details on test conditions:

Reconstituted fresh water was used for breeding and dilution. Demineralized water with a conductance of < 1µS / cm was used for preparing the
artificial fresh water (according to Elendt, M4). The test water was allowed to stand for at least a further 24 hours in order to stabilize and was aerated until saturation. The pH-value, oxygen content, conductivity and total hardness were measured and recorded once a week.
The study was conducted in a static system. As test chambers 250 mL beakers were used (height 110 mm, diameter 70 mm, water level about 75 mm), each filled with 200 mL test water. The room temperature was about 21 deg C. The room was illuminated by fluorescent tubes for 16 hours each day.
20 daphnids, divided into two parallel groups of 10 animals, were used for each concentration. The test concentrations, which were selected on the basis of range finding tests, were as follows:
0 mg/L (control)
100 mg/L nominal limit concentration (treatment)

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Details on results:

Neither in the treatment replicates nor in the control any immobilized daphnids could be detected.

Validity criteria fulfilled:

yes

Conclusions:

In the acute immobilization test on Daphnia magna with the substance neither in the treatment nor in the control groups any immobilized daphnids could be observed:
EC0 (24 and 48 h) = 100 mg/L
EC50 (24 and 48 h) > 100 mg/L

Executive summary:

The substance was tested in Daphnia magna (water flea) for 48 hours in a static system. The concentrations tested were 100 mg/L and 0 mg/L (control). Before insertion of the test organisms the chamber contents were stirred for approximately 48 hours with a magnetic stirrer to ensure that the solubility limit of the test substance in the test water was reached. To remove fine undissolved particles the test batches were filtered through a cellulose filter, which was saturated with the test substance prior to use. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance in water is below 1 mg/L. The concentration data given in the present report are therefore based on nominal concentrations. In this 48-hour acute toxicity study of the submission substance the following immobility values were determined:

	after 24 hours	after 48 hours
ECo(mg/L)	100	100
EC50(mg/L)	> 100	> 100
EC100(mg/L)	not determined	not determined

In the control group no immobility was observed. The 100 mg/L group showed no effect in comparison to the control.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 MAR 2005 to 02 MAR 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solution The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 micro-m (RC, SCHLEICHER & SCHUELL).
Limit concentration Saturated solution (100 mg/L)
Dilution water According to EC 92/69 L383A C.2. Annex
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (Clone 5)
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE*mE(-2)*sE(-1).
Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding of the culture At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL.
Age of test organisms 2 - 24 h old

Acclimation period: approx. 2h

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

total hardness of 160 to 180 mg CaCO3/L

Test temperature:

18 - 22 °C, ± 1 °C constant

pH:

Control: 7.8 - 7.94
Treatment: 7.57 - 7.81

Dissolved oxygen:

[mg/L]
Control: 7.67 - 8.69
Treatment: 8.05 - 8.69

Nominal and measured concentrations:

Nominal limit concentation: 100 mg/L
Control: 0 mg/L

Details on test conditions:

Illumination Max. 20 µE*mE(-2)*sE(-1)
Photoperiod 16 / 8 hours light / dark cycle
Exposure vessel type Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
Number of study organisms 20 per concentration and control divided into 4 parallel samples
Feeding during the study The daphnids were not fed during the study.
Endpoints assessed Immobilisation of daphnids

A static preliminary range finding test was conducted as limit test with the saturated solution (NON-GLP-state).

Measurements Prior to test start pH-value, dissolved oxygen concentration,conductivity, total hardness and temperature of the dilution water were measured. At the beginning of the test water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control groups. After 48 h the water parameters were measured in all replicates per concentration and control. The room temperature was recorded throughout the test with a thermohygrograph.
Age of test organisms: 2-24 h old

2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2 for the reference item) groups with 5 (10 for the reference item) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure.
The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Details on results:

Neither in the control nor in the treatment replicates were any immobilzed daphnids observed.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in mg/L:
EC10 : 1.51
EC50 : 1.84 (CI 1.74 - 1.95)
EC100 : 3.20
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30

Reported statistics and error estimates:

The effects of the limit concentration were determined directly from the test results.
EC50- and EC10-values were only calculated for the reference item via probit analysis according to WEBER (1986). Calculation of the confidence intervals was carried out using standard procedures according to BREITIG G. & TÜMPLING W. (1982).

Software
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: SigmaPlot rel. 2000 (2000), SPSS CORPORATION

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.
EC0 (24 and 48 h) = 100 mg/L nominal concentration
EC50 (24 and 48 h) > 100 mg/L nominal concentration

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L; the maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6) as limit concentration of the test item was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.84 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1: Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

Test Concentration	IMMOBILISATION [%]
	24 h Replicates					48 h Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
Saturated solution (100 mg/L)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 APR 2005 to 08 APR 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solution The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 µm (RC, SCHLEICHER & SCHUELL).
Limit concentration Saturated solution (100 mg/L)
Dilution water According to EC 92/69 L383A C.2. Annex
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (Clone 5)
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE*mE(-2)*sE(-1).
Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding of the culture At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL.
Age of test organisms 2 - 24 h old

Acclimation period: approx. 2h

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

total hardness of 160 to 180 mg CaCO3/L

Test temperature:

18 - 22 °C, ± 1 °C constant

pH:

Control: 7.69 - 7.82
Treatment: 7.73 - 7.85

Dissolved oxygen:

[mg/L]
Control: 7.86 - 8.18
Treatment: 8.17 - 8.23

Nominal and measured concentrations:

Nominal limit concentation: 100 mg/L
Control: 0 mg/L

Details on test conditions:

Illumination Max. 20 µE*mE(-2)*sE(-1)
Photoperiod 16 / 8 hours light / dark cycle
Exposure vessel type Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
Number of study organisms 20 per concentration and control divided into 4 parallel samples
Feeding during the study The daphnids were not fed during the study.
Endpoints assessed Immobilisation of daphnids

Measurements Prior to test start pH-value, dissolved oxygen concentration,conductivity, total hardness and temperature of the dilution water were measured. At the beginning of the test water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control groups. After 48 h the water parameters were measured in all replicates per concentration and control. The room temperature was recorded throughout the test with a thermohygrograph.
Age of test organisms: 2-24 h old

2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2 for the reference item) groups with 5 (10 for the reference item) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure.
The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Details on results:

Neither in the control nor in the treatment replicates were any immobilzed daphnids observed.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in mg/L:
EC10 : 1.48
EC50 : 1.80 (CI 1.68 - 1.93)
EC100 : 3.20
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30

Reported statistics and error estimates:

The effects of the limit concentration were determined directly from the test results.
EC50- and EC10-values were only calculated for the reference item via probit analysis according to WEBER (1986). Calculation of the confidence intervals was carried out using standard procedures according to BREITIG G. & TÜMPLING W. (1982).

Software
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: SigmaPlot rel. 2000 (2000), SPSS CORPORATION

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.
EC0 (24 and 48 h) = 100 mg/L nominal concentration
EC50 (24 and 48 h) > 100 mg/L nominal concentration

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L; the maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6) as limit concentration of the test item was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.80 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1: Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

Test Concentration	IMMOBILISATION [%]
	24 h Replicates					48 h Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
Saturated solution (100 mg/L)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

From 01 MAR 2005 to 02 MAR 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Justification for type of information:

Justification for type of information: see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solution The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 micro-m (RC, SCHLEICHER & SCHUELL).
Limit concentration Saturated solution (100 mg/L)
Dilution water According to EC 92/69 L383A C.2. Annex
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (Clone 5)
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE*mE(-2)*sE(-1).
Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding of the culture At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL.
Age of test organisms 2 - 24 h old

Acclimation period: approx. 2h

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

total hardness of 160 to 180 mg CaCO3/L

Test temperature:

18 - 22 °C, ± 1 °C constant

pH:

Control: 7.8 - 7.94
Treatment: 7.57 - 7.81

Dissolved oxygen:

[mg/L]
Control: 7.67 - 8.69
Treatment: 8.05 - 8.69

Nominal and measured concentrations:

Nominal limit concentation: 100 mg/L
Control: 0 mg/L

Details on test conditions:

Illumination Max. 20 µE*mE(-2)*sE(-1)
Photoperiod 16 / 8 hours light / dark cycle
Exposure vessel type Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
Number of study organisms 20 per concentration and control divided into 4 parallel samples
Feeding during the study The daphnids were not fed during the study.
Endpoints assessed Immobilisation of daphnids

A static preliminary range finding test was conducted as limit test with the saturated solution (NON-GLP-state).

Measurements Prior to test start pH-value, dissolved oxygen concentration,conductivity, total hardness and temperature of the dilution water were measured. At the beginning of the test water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control groups. After 48 h the water parameters were measured in all replicates per concentration and control. The room temperature was recorded throughout the test with a thermohygrograph.
Age of test organisms: 2-24 h old

2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2 for the reference item) groups with 5 (10 for the reference item) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure.
The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Details on results:

Neither in the control nor in the treatment replicates were any immobilzed daphnids observed.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in mg/L:
EC10 : 1.51
EC50 : 1.84 (CI 1.74 - 1.95)
EC100 : 3.20
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30

Reported statistics and error estimates:

The effects of the limit concentration were determined directly from the test results.
EC50- and EC10-values were only calculated for the reference item via probit analysis according to WEBER (1986). Calculation of the confidence intervals was carried out using standard procedures according to BREITIG G. & TÜMPLING W. (1982).

Software
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: SigmaPlot rel. 2000 (2000), SPSS CORPORATION

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.
EC0 (24 and 48 h) = 100 mg/L nominal concentration
EC50 (24 and 48 h) > 100 mg/L nominal concentration

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L; the maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6) as limit concentration of the test item was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.84 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1: Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

Test Concentration	IMMOBILISATION [%]
	24 h Replicates					48 h Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
Saturated solution (100 mg/L)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Reference 5

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

From 11 FEB 2004 to 13 FEB 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Justification for type of information:

Justification for type of information: see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Testguideline 202 Daphnia sp., 14-day Reproduction Test (including an Acute Immobilisation Test) Adopted April 4, 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Remarks:

According to German Chemical Law (ChemG), Annex 1, in the version of May 8, 2001 and 1999/11/EU of March 8, 1999

Analytical monitoring:

no

Details on sampling:

Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance in water is below 1 mg/L. The concentration data given in the present report are therefore based on nominal concentrations.

Vehicle:

no

Details on test solutions:

The test substance was weighed into a beaker, mixed with water for dilution and homogenized
with the Ultra-Turrax. The mixtures were then stirred for approximately 48
hours with a magnetic stirrer to ensure that the solubility limit of the test substance in
the test water was reached. To remove fine undissolved particles the test batches
were filtered through a cellulose filter, which was saturated with the test substance
prior to use.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species Daphnia magna - Clone 5 (IRChA, France)
Origin Stock culture of Aventis Pharma Deutschland GmbH, ProTox
Breeding conditions 100 mL beakers (height 90 mm, diameter 50 mm), filled with 70 mL test water (according to Elendt, M4) served as culture vessels. One daphnid was used per culture vessel. The animals were used for breeding up to an age of 42 days. The reproduction rate and the state of health of the animals were monitored daily apart from weekends. From these cultures daphnids with an age of < 24 h are taken as test animals.
The animals were fed three times a week with monocellular green algae (Desmodesmus subspicatus). The supplied diet was based on the amount of organic carbon (C). The ration level is 0.2 mg carbon / day / animal.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The total hardness of the dilution water was at 2.1 mmol Ca(2+) + Mg(2+) / L during the study.

Test temperature:

Control: 20.8 - 21.1 deg C
Treatment: 21.0 - 21.1 deg C

pH:

Control: 7.7 - 8.0
Treatment: 7.8 - 8.0

Dissolved oxygen:

[mg/L]
Control: 8.4 - 8.6
Treatment: 8.3 - 8.5

Nominal and measured concentrations:

100 mg/L nominal concentration
Control: 0 mg/L

Details on test conditions:

Reconstituted fresh water was used for breeding and dilution. Demineralized water with a conductance of < 1µS / cm was used for preparing the
artificial fresh water (according to Elendt, M4). The test water was allowed to stand for at least a further 24 hours in order to stabilize and was aerated until saturation. The pH-value, oxygen content, conductivity and total hardness were measured and recorded once a week.
The study was conducted in a static system. As test chambers 250 mL beakers were used (height 110 mm, diameter 70 mm, water level about 75 mm), each filled with 200 mL test water. The room temperature was about 21 deg C. The room was illuminated by fluorescent tubes for 16 hours each day.
20 daphnids, divided into two parallel groups of 10 animals, were used for each concentration. The test concentrations, which were selected on the basis of range finding tests, were as follows:
0 mg/L (control)
100 mg/L nominal limit concentration (treatment)

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Due to the poor solubility of the test item, a saturated solution had been prepared at the nominal concentration and used after filtration

Details on results:

Neither in the treatment replicates nor in the control any immobilized daphnids could be detected.

Validity criteria fulfilled:

yes

Conclusions:

In the acute immobilization test on Daphnia magna with the submission substance neither in the treatment nor in the control groups any immobilized daphnids could be observed:
EC0 (24 and 48 h) = 100 mg/L
EC50 (24 and 48 h) > 100 mg/L

Executive summary:

The submission substance was tested in Daphnia magna (water flea) for 48 hours in a static system. The concentrations tested were 100 mg/L and 0 mg/L (control). Before insertion of the test organisms the chamber contents were stirred for approximately 48 hours with a magnetic stirrer to ensure that the solubility limit of the test substance in the test water was reached. To remove fine undissolved particles the test batches were filtered through a cellulose filter, which was saturated with the test substance prior to use. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance in water is below 1 mg/L. The concentration data given in the present report are therefore based on nominal concentrations. In this 48-hour acute toxicity study of the submission substance the following immobility values were determined:

	after 24 hours	after 48 hours
ECo(mg/L)	100	100
EC50(mg/L)	> 100	> 100
EC100(mg/L)	not determined	not determined

In the control group no immobility was observed. The 100 mg/L group showed no effect in comparison to the control.

Reference 6

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

From 06 APR 2005 to 08 APR 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

Justification for type of information:

Justification for type of information: see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Acetolone Pigments – Nanoforms and Bulk Forms (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Stock solution The solution (100 mg/L test item was weighed out) was prepared with dilution water one day prior to application.
Dispersion treatment The stock solution was shaken at room temperature with 20 rpm for 24 h (rotating shaker 3040, GFL) and membrane filtrated with 0.45 µm (RC, SCHLEICHER & SCHUELL).
Limit concentration Saturated solution (100 mg/L)
Dilution water According to EC 92/69 L383A C.2. Annex
Saturated solution: The maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna STRAUS (Clone 5)
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu)
Breeder DR.U.NOACK-LABORATORIEN, 31157 Sarstedt, Germany

Culture In 2-3 L glass vessels with approximatively 1.8 L culture medium, at 21 °C (temperatures of 20 - 25 °C are tolerated), in an incubator, 16 h illumination, illumination strength max. 20 µE*mE(-2)*sE(-1).
Culture medium Elendt M4, according to Elendt (1990), modified to a total hardness of 160 to 180 mg CaCO3/L.
Feeding of the culture At least 5x weekly ad libitum with a mix of Desmodesmus subspicatus and Chlorella vulgaris, with an algae cell density of > 10E(6) cells/mL.
Age of test organisms 2 - 24 h old

Acclimation period: approx. 2h

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

total hardness of 160 to 180 mg CaCO3/L

Test temperature:

18 - 22 °C, ± 1 °C constant

pH:

Control: 7.69 - 7.82
Treatment: 7.73 - 7.85

Dissolved oxygen:

[mg/L]
Control: 7.86 - 8.18
Treatment: 8.17 - 8.23

Nominal and measured concentrations:

Nominal limit concentation: 100 mg/L
Control: 0 mg/L

Details on test conditions:

Illumination Max. 20 µE*mE(-2)*sE(-1)
Photoperiod 16 / 8 hours light / dark cycle
Exposure vessel type Glass beakers (5 cm ID x 8 cm H), 50 mL volume were used.
Number of study organisms 20 per concentration and control divided into 4 parallel samples
Feeding during the study The daphnids were not fed during the study.
Endpoints assessed Immobilisation of daphnids

Measurements Prior to test start pH-value, dissolved oxygen concentration,conductivity, total hardness and temperature of the dilution water were measured. At the beginning of the test water parameters (pH value, oxygen concentration) were measured in one additional replicate per concentration and control groups. After 48 h the water parameters were measured in all replicates per concentration and control. The room temperature was recorded throughout the test with a thermohygrograph.
Age of test organisms: 2-24 h old

2 to 24 h old daphnids were used for the definitive study. They were obtained by removing the mother animals twice within 22 h.
Control replicates with dilution water (test medium without test item) were tested under the same conditions as the test groups.
20 daphnids divided into 4 (2 for the reference item) groups with 5 (10 for the reference item) animals per test vessel were used.
In the definitive study the immobilisation of the daphnids was observed after 24 and 48 hours of exposure.
The reference item was tested in five concentrations within the concentration range 0.58 to 5.8 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: As the test item is highly insoluble in water, a saturated solution was prepared at the nominal concentration and filtrated

Details on results:

Neither in the control nor in the treatment replicates were any immobilzed daphnids observed.

Results with reference substance (positive control):

The percentage immobility for the reference item was determined after 24 h. The EC100-value was determined directly from the test results. The EC10- and EC50- with 95 % confidence interval (CI) was determined in a probability network by interpolation according to standard procedures.
EC-values after 24 h of the reference item in mg/L:
EC10 : 1.48
EC50 : 1.80 (CI 1.68 - 1.93)
EC100 : 3.20
The EC50-value of the reference item potassium dichromate after 24 h is within the prescribed concentration range of 1.0 - 2.5 mg/L of quality criteria according to AQS: DIN Guideline 38412 L 30

Reported statistics and error estimates:

The effects of the limit concentration were determined directly from the test results.
EC50- and EC10-values were only calculated for the reference item via probit analysis according to WEBER (1986). Calculation of the confidence intervals was carried out using standard procedures according to BREITIG G. & TÜMPLING W. (1982).

Software
All data were computer generated and rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures. Calculations were carried out using software: SigmaPlot rel. 2000 (2000), SPSS CORPORATION

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.
EC0 (24 and 48 h) = 100 mg/L nominal concentration
EC50 (24 and 48 h) > 100 mg/L nominal concentration

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS) the effect of the saturated solution (100 mg/L; the maximum dissolved concentration of the test item that can be achieved under the test conditions in the test medium, acc. to OECD Series, No. 23 (2000)6) as limit concentration of the test item was determined according to OECD 202 (2004) and EC Directive 92/69/EC Method C.2 (1992). The limit test was conducted under static conditions over 48 h. 20 test organisms were exposed to the limit concentration and control. A reference test was carried out with potassium dichromate to determine the toxicity of the reference item. The EC50-value of the reference item of 1.80 mg/L after 24 h was within the prescribed concentration range of 1.0 to 2.5 mg/L according to AQS, DIN Guideline 38412 L 30. Water quality parameters pH-value and dissolved oxygen concentration, measured at 0 and 48 h, were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

Table 1: Percentage of Daphnids Incapable of Swimming after 24 and 48 h of Exposure (n = 20)

Test Concentration	IMMOBILISATION [%]
	24 h Replicates					48 h Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
Saturated solution (100 mg/L)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

 

Description of key information

PY 175 (Nano)

In the acute immobilization test on Daphnia magna with the submission substance neither in the treatment nor in the control groups any immobilized daphnids could be observed:

EC0 (24 and 48 h) = 100 mg/L

EC50 (24 and 48 h) > 100 mg/L

PY180 (Bulk)

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.

EC0 (24 and 48 h) = 100 mg/L nominal concentration

EC50 (24 and 48 h) > 100 mg/L nominal concentration

 

PY154 (Bulk)

At the limit concentration of 100 mg/L of the submission substance no effects on mobility to Daphnia magna were observed. Neither in the control nor in the treatment replicates were any daphnids immobilized.

EC0 (24 and 48 h) = 100 mg/L nominal concentration

EC50 (24 and 48 h) > 100 mg/L nominal concentration