Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 16 January 2017. Experimental completion date: 02 May 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

EC Number:

233-316-4

EC Name:

Disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Cas Number:

10114-86-0

Molecular formula:

C27H22N6Na2O9S2

IUPAC Name:

disodium 3,3'-[carbonylbis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Test material form:

solid: particulate/powder

Details on test material:

Identification: CIM-42(Direct Yellow 132)
CAS No: 10114-86-0
Appearance/Physical state: Dark orange colored powder
Batch: 150909K
Purity: 90.3%
Expiry date: 01 October 2017
Storage conditions: Room temperature over silica gel, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of Test Concentrations
Samples were taken from the control and each test group from the freshly prepared bulk test preparation at 0 hours and from the old or expired pooled replicates at 48 hours for quantitative analysis. All samples were taken for analysis on the day of sampling. Duplicate samples were taken at and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Test Water:
Reconstituted water (Elendt M7) was used for the range-finding test. In the definitive test, due to test item precipitating out of solution in Elendt M7 in the range finding test, the media was diluted to a 50:50 ratio with deionized reverse osmosis water.

Media Preparation Trial:
In the range finding test it was observed that the test item precipitated out of solution in Elendt M7. Based on this and the results of the analytical investigations on the range finding test samples a media preparation trial was conducted (as below):

Solution Preparation:
Aliquots (500 mL) of Elendt M7 and deionized reverse osmosis water were combined to give 1 liter of a 50:50 ratio to be used as the test media.
A nominal amount of test item (50 mg) was dispersed, in 500 mL of the test media to produce a 100 mg/L test concentration. The appearance of the test concentration at 0, 24 and 48 hours was recorded and a sample taken for chemical analysis at 48 hours.

Results:
Visual Observations
Hours Observations:
0 Clear dark yellow solution
24 Clear dark yellow solution
48 Clear dark yellow solution

Chemical Analysis:
Time Point [Hours]: 48
Nominal Concentration of Test Item Cnom [mg/L]: 100
Sample Preparation Factor F: 1.0
Determined Concentration of Test Item C [mg/L]: 102
Percentage of Nominal Concentration [%]: 102

Discussion:
Based on this information for the definitive test the test item was prepared by dissolving 200 mg of test item into 2 liters of test media produced with a 50:50 ratio of Elendt M7 and deionized reverse osmosis water and to give a nominal test concentration of 100 mg/L.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

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

Adult daphnids were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

approximate hardness 125 mg/L as CaCO3

Test temperature:

Temperature was maintained at 21 °C to 22 °C throughout the test

pH:

7.5 - 7.2

Dissolved oxygen:

7.8 - 8.8 mg O2/L

Salinity:

not applicable freshwater test

Nominal and measured concentrations:

Range-finding Test: I
Nominal: 0.10, 1.0, 10 and 100 mg/L.

Definitive Test:
Nominal: 10, 18, 32, 56 and 100 mg/L.
Measured: range from 9.4 to 101 mg/L.

Details on test conditions:

Range-finding Test:
The test concentrations to be used in the definitive test were 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 further 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °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 group 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 and media preparation trial the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Aliquots (5 liters) of Elendt M7 and deionized reverse osmosis water were combined to give 10 liters of a 50:50 ratio to be used as the test media.

A nominal amount of test item (200 mg) was dissolved in test media and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 10, 18, 32 and 56 mg/L.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.

Exposure Conditions:
As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits: 54 - 120 mg/L

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test:
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. A sub lethal effect of exposure was seen in the 1.0 mg/L test concentration, one daphnid was seen to be trapped at the surface.

During the range finding test precipitation of the test item was observed at 24 hours (by visual inspection) in the 100 mg/L test concentration.

Chemical analysis of the 100 mg/L test preparation at 0 and 48 hours showed measured test concentration of 70 and 64 mg/L, respectively.

The measured test concentrations were significantly lower than expected during this test. The test item was seen to be precipitating out of solution at 24 hours and this was considered to be the cause of the lower than expected measured concentrations. The test item precipitation was considered to be due to the high hardness level in the media; as such a media preparation trial was conducted in order to determine if the hardness was the cause of the test item precipitation.

Media Preparation Trial:
A test concentration of 100 mg/L was obtained using test media produced by a 50:50 ratio of Elendt M7 and deionized reverse osmosis water. After 48 hours no precipitation of the test item was seen.

Definitive Test:
Verification of Test Concentrations:
Analysis of the test preparations at 0-Hours showed measured test concentrations to range from 9.4 to 101 mg/L. Analysis of the test preparations at 48-Hours showed no significant change in the test item concentrations and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations.

Immobilization Data:
Visual inspection of the immobilization data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression 48 hours based on the nominal test concentrations gave the following results:
24 h EC50: >100 mg/L
48 h EC50: 74 mg/L

The No Observed Effect Concentration after 24 and 48 hours exposure was greater 100 and 18 mg/L, respectively. The Lowest Observed Effect Concentration after 48 hours exposure was 32 mg/L.
The slopes and their standard errors of the response curves at 24 and 48 hours were 0.14 (SE = 43) and 2.4 (SE = 0.079) respectively.

Sub-Lethal Effects:
Sub-lethal effects of exposure were observed in the 18, 56 and 100 mg/L test concentrations. These responses were pale, covered in debris and reduced mobility

Validation Criteria:
The test was considered to be valid given that none 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 equal to or greater than 3 mg/L in the control and test vessels.

Water Quality Criteria:
Temperature was maintained at 21 °C to 22 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
Throughout the test the light intensity was observed to be in the range 275 to 480 Lux.

Observations on Test Item Solubility
At the start and throughout the test the control solutions were observed to be clear colorless solutions. The 10 and 18 mg/L test solutions were observed to be pale yellow clear solutions. The 32 and 56 mg/L test solutions were observed to be yellow clear solutions and the 100 mg/L test solutions were observed to be dark yellow clear solutions.

Results with reference substance (positive control):

A positive control ( 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 was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

24 h:
EC50: 1.3 mg/L
NOEC: 1.0 mg/L
LOEC: 1.8 mg/L

48 h:
EC50: 1.2 mg/L
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

The No Observed Effect Concentration is based upon equal to or 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.

Any other information on results incl. tables

Table: Cumulative Immobilization Data and Observations in the Range-finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 10 Per Replicate)
	24 Hours		48 Hours
	Cumulative Immobilized Daphnia	Observations	Cumulative Immobilized Daphnia	Observations
Control	0	10 N	0	10 N
0.10	0	10 N	0	10 N
1.0	0	1 T 9 N	0	1 T 9 N
10	0	10 N	0	10 N
100	0	10 N	0	10 N

N = No sub-lethal effects observed

T = Trapped at surface

Table: Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	0	0	0	0	0	5 N	5 N	1 P+C 4 N	5 N
32	0	0	0	0	0	0	5 N	5 N	5 N	5 N
56	0	1	0	0	1	5	5 N	4 N	5 N	5 N
100	0	0	0	0	0	0	5 N	5 N	5 N	5 N

Nominal Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4
Control	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
18	0	1	0	0	1	5	5 N	4 N	1 R 4 N	5 N
32	2	0	2	0	4	20	3 N	5 N	3 N	5 N
56	2	2	4	3	11	55	3 N	3 N	1 R	2 N
100	1	2	4	3	10	50	4 R	3 R	1 R	2 R

R₁– R₄= Replicates 1 to 4

N = No sub-lethal effects observed

P = pale in color

C = Covered in debris

R = Reduced mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results for the 48 hour time point:
EC50 (mg/L): 74
95% Confidence Limits (mg/L): 54 - 120
No Observed Effect Concentration (NOEC) (mg/L): 18
Lowest Observed Effect Concentration (LOEC) (mg/L): 32

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods

Following a preliminary range-finding test and a media preparation trial, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 21 °C to 22 °C under static test conditions. The number of immobilized daphnia were recorded after 24 and 48 hours.

Results

Analysis of the test preparations at 0-Hours showed measured test concentrations to range from 9.4 to 101 mg/L. Analysis of the test preparations at 48-Hours showed no significant change in the test item concentrations and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations.

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
48	74	55  -  120	18	32