Potassium sodium amino-hydroxy-[(3-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]-[(2-sulfonato-4-{[2-(substituted)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-disulfonate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 04 November 2014 and 04 December 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Principles of method if other than guideline:

None

GLP compliance:

yes

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and 100 mg/L test group from the bulk test preparation at 0 hours (fresh) and from the pooled replicates at 48 hours (old) for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Vehicle:

yes

Details on test solutions:

Range-finding Test
A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0, and 10 mg/L.

Definitive Test
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.

The test preparation concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 21 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

approximately 21 °C

pH:

7.2 - 7.5

Dissolved oxygen:

Equal to or greater than 8.8 mg O2/L

Salinity:

No data

Nominal and measured concentrations:

Range-finding Test: Nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.
Definitive Test: Nominal test concentration of 100 mg/L.

Details on test conditions:

Test Water
Reconstituted water (ISO medium) was used for both the range-finding and definitive tests.

Procedure
Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

A nominal amount of test item (50 mg) was dissolved in test water and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give the required test concentrations of 0.10, 1.0, and 10 mg/L.

Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.

The control was maintained under identical conditions but not exposed to the test item.

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.


Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EC Test Guidelines no significant immobilization or adverse reactions to exposure were observed.


Experimental Preparation
A nominal amount of test item (100 mg) was dissolved in test water and the volume adjusted to 1 liter to give the 100 mg/L test concentration.

The test preparation concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test item in the test preparation were verified by chemical analysis at 0 and 48 hours.


Exposure Conditions
As in the range-finding test 150 mL glass jars containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (497 to 502 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No significant immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the maximum test concentration given in the OECD/EC Test Guidelines no immobilization or adverse reactions to exposure were observed.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value indicating that the test item was stable over the test duration.


Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.


Immobilization Data
There was no significant immobilization in 20 daphnids exposed to a test concentration of 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

The 24 h EC50 was > 100 mg/L
The 48 h EC50 was > 100 mg/L

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Results with reference substance (positive control):

A positive control (Harlan Study Number 41400711) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971*) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977 *) at 48 hours based on the nominal test concentrations gave the following results:

The 24 h EC50 was 0.87 mg/L with 95 % confidence limits of 0.75 - 1.0 mg/L.
The 48 h EC50 was 0.71 mg/L with 95 % confidence limits of 0.65 - 0.78 mg/L.
The NOEC was 0.56 mg/L.
The LOEC was 1.0 mg/L.

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Reported statistics and error estimates:

An estimate of the EC50 values was given by inspection of the immobilization data.

Validation Criteria

The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was ≥3 mg/L in the control and test vessels.

 

Water Quality Criteria

Temperature was maintained at approximately 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

 

Observations on Test Item Solubility

The test preparation remained as a dark blue opaque solution throughout the test.

Cumulative Immobilization Data in the Range-finding Test

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0*
1.0	0	0
10	0	1^**
100	0	0

*Only 9 daphnia were observed, of which none were immobilized.

^**Single immobilized daphnia considered not to be significant.

Cumulative Immobilization Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	1	1	5*	1	0	0	1	2	10^*
100	1	0	0	0	1	5^*	1	0	1	0	2	10^*

R₁– R₄= Replicates 1 to 4

*Equal to and less than 10% immobilization considered not to be significant.

Water Quality Measurements

Nominal Concentration (mg/L)		0 Hours			24 Hours	48 Hours
		pH	mg O2/L	T°C	T ºC	pH	mg O2/L	T °C
Control	R1	7.2	9.2	20	21	7.5	8.9	21
100	R1	7.4	9.0	20	21	7.5	8.8	21

R₁= Replicate 1

Validity criteria fulfilled:

yes

Conclusions:

The 48-Hour EC50 of the test item to freshwater aquatic invertebrates was determined to be >100 mg/L.

Executive summary:

A study according to OECD Guideline 202 and EU Method C.2, was performed to assess the acute toxicity of the test item to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so the results are based on nominal test concentrations only. 1 out of 10 daphnids were found to be immobilised in control as well as test group. As, equal to or less than 10% immobilization is considered not to be significant as per the OECD and EU Guidelines, the 48-Hour EC50 of the test item to freshwater aquatic invertebrates was determined to be >100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Description of key information

The 48-Hour EC50 of the test item to freshwater aquatic invertebrates >100 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A study according to OECD Guideline 202 and EU Method C.2, was performed to assess the acute toxicity of the test item to Daphnia magna. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a concentration of 100 mg/L for 48 hours at a temperature of approximately 21°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal value and so the results are based on nominal test concentrations only. 1 out of 10 daphnids were found to be immobilised in control as well as test group. As, equal to or less than 10% immobilization is considered not to be significant as per the OECD and EU Guidelines, the 48-Hour EC50 of the test item to freshwater aquatic invertebrates was determined to be >100 mg/L. The No Observed Effect Concentration was 100 mg/L.