hexasodium 5-[(4-{3-[(8-benzamido-1-hydroxy-3,6-disulfonato-2-naphthyl)diazenyl]-4-sulfonatoanilino}-6-[(2-sulfonatoethyl)amino]-1,3,5-triazin-2-yl)amino]isophthalate

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:

The study was conducted between 24 October 2007 and 29 November 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

Magenta T-43

IUPAC Name:

Magenta T-43

Test material form:

solid: particulate/powder

Details on test material:

Sponsor's identification: MAGENTA T-43
Description: dark red solid
Storage conditions: room temperature, in the dark
Lot number: H186-5

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Verification of test concentrations:
Water samples were taken from the control and the 10, 18, 32, 56 and 100 mg/l test groups (replicates R1 - R2 pooled) at 0 and 48 hours for quantitative analysis. Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary. The method of analysis, stability, recovery and test preparation analyses are reported.

Test solutions

Vehicle:

yes

Details on test solutions:

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value. The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

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

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20°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 suspension of algae (Chlorella sp.). 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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Immobilisation or adverse reactions were recorded at 24 and 48 hours after the start of exposure.

Test conditions

Test temperature:

Water temperature was recorded daily throughout the test and was measured using a Hanna Instruments HI93510 digital thermometer. It was maintained at 21°C.

pH:

The pH was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.The pH range was between 7.9 to 8.0.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.1 0, 1.0, 10, 100 and 180 mg/l. The test material was dissolved directly in water.

Definitive test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Details on test conditions:

Procedure

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 0.1 0, 1.0, 10, 100 and 180 mg/l. The test material was dissolved directly in water.

An estimate of the predicted toxicity using Epiwin (version 3.12) indicated that the EC50 value for Daphna magna was in excess of 100 mg/l. Therefore a further test concentration of 180 mg/l was included in the range-finding test. An amount of test material (90 mg) was dissolved in water and the volume adjusted to 500 ml to give the 180 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 100, 10, 1.0 and 0.10 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 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded. The control group was maintained under identical conditions but not exposed to the test material.

Definitive test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Experimental preparation:
For the purpose of the definitive test the test material was dissolved directly in water. An amount of test material (200 mg) was dissolved in water and the volume adjusted to 2 litres to give the 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations of 56, 32, 18 and 10 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity. The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours.

Exposure conditions:
As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the test preparations. Duplicate test vessels were used for each test and control group. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light 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 material.

The test preparations were not renewed during the exposure period. Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

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:

mortality

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 54-69 (95% CL)

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Range-finding Test:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test were tabulated.
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However, immobilisation was observed at 100 and 180 mg/l.
Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive test.

Definitive Test
Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test were tabulated. The relationship between percentage immobilisation and concentration at 48 hours was plotted.

An estimate of the EC50 value at 24 hours was given by inspection of the immobilisation data. Analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations were determined and tabulated.

The No Observed Effect Concentrations after 24 and 48 hours exposure were 100 and 32 mg/l respectively. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curves at 24 and 48 hours.

The results obtained for the 100 mg/l test concentration in the definitive test showed a significant difference from those obtained for the 100 mg/l test concentration in the range-finding test. This was considered to be due to natural variation within the daphnia population.


The overall results indicate that the 48-Hour EC50 for the test material to Daphnia magna based on nominal test concentrations was 61 mg/l with 95% confidence limits of 54 - 69 mg/l. The No Observed Effect Concentration was 32 mg/l.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal so the results are based on nominal test concentrations only.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/l with 95% confidence limits of 0.43 - 0.53 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Results with reference substance (positive control):

Positive Control:
Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Safepharm Laboratories Project No: 0039/0971) during the positive control were provided. The relationship between percentage immobilisation and concentration at 24 and 48 hours were plotted.

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al. 1977) at 24 and 48 hours based on the nominal test concentrations were tabulated.

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC50 value calculated from all positive controls was 0.79 mg/l (sd = 0.23).

Reported statistics and error estimates:

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al. 1977) at 24 and 48 hours based on the nominal test concentrations were tabulated. From the 48h timepoint (EC50) a 95% confidence limit of 54-69 mg/l was reported.

Any other information on results incl. tables

Physico-chemical measurements:

The results of the physico-chemical measurements were tabulated. Temperature was maintained at 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

Observations on test material solubility:

The control was observed to be a clear, colourless solution throughout the duration of the test whereas the test concentrations of 10 to 100 mg/l were observed to be clear red/pink coloured solutions which increased in colouration as concentration increased.

Verification of test concentrations:

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to range from 103% to 106% of nominal value and so it was considered justifiable to calculate the EC50 values in terms of the nominal test concentrations only.

Physico-chemical measurements 

Nominal Concentration (mg/l)		0 Hours				24 Hours	48 Hours
		pH	Mg O2/l	%ASV*	T°C	T°C	pH	Mg O2/l	%ASV*	T°C
Control	R1	7.9	8.8	99	21	21	8.0	8.9	100	21
	R2	8.0	8.9	100	21	21	8.0	8.9	100	21
10	R1	7.9	8.9	100	21	21	8.0	8.9	100	21
	R2	7.9	8.9	100	21	21	8.0	8.9	100	21
18	R1	7.9	8.8	99	21	21	8.0	8.9	100	21
	R2	7.9	8.9	100	21	21	8.0	8.8	99	21
32	R1	8.0	8.8	99	21	21	8.0	8.8	99	21
	R2	7.9	8.9	100	21	21	8.0	8.8	99	21
56	R1	8.0	8.8	99	21	21	8.0	8.8	99	21
	R2	8.0	8.8	99	21	21	8.0	8.8	99	21
100	R1	7.9	8.9	100	21	21	8.0	8.8	99	21
	R2	7.9	8.9	100	21	21	8.0	8.8	99	21

 

*ASV = Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R1 -R2=Replicates 1 and 2

 

Cumulative immobilisation data in the range-finding test

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	0	0
10	0	0
100	0	4
180	0	4

 

 Immobilisation data: EC₅₀ values at 24 and 48 hours

Time (h)	EC50(mg/l)	95% Confidence Limits (mg/l)
24	>100	-
48	61	54-69

 

Cumulative Immobilisation Data in the Definitive Test

 

Nominal Concentration (mg/ml)	Cumulative Immobilised Daphnia (Initial Population: 10 per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0
56	0	0	0	0	4	3	7	35
100	0	0	0	0	10	10	20	100

R₁-R₂= Replicates 1 and 2

Immobilisation Data: Postive control EC₅₀values at 3, 24 and 48 hours

Time (h)	EC50(mg/l)	95% Confidence Limits (mg/l)
3	>3.2*	-
24	0.65	0.58-0.72
48	0.47	0.43-0.53

Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/ml)	Cumulative Immobilised Daphnia (Initial Population:10 per Replicates)
	3 hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	2	3	5	25	9	7	16	80
1.0	0	0	0	0	10	10	20	100	10	10	20	100
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	1	1	2	10	10	10	20	100	10	10	20	100

R₁-R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 value of 61 mg/l with 95% confidence limits of 54 - 69 mg/l. The No Observed Effect Concentration at 48 hours was 32 mg/l.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at concentrations of 10, 18, 32, 56 and 100 mg/l for 48 hours at a temperature of 21 °C under static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours. A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results

The 48-Hour EC₅₀ for the test material to Daphnia magna based on nominal test concentrations was 61 mg/l with 95% confidence limits of 54 - 69 mg/l. The No Observed Effect Concentration was 32 mg/1.

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal so the results are based on nominal test concentrations only.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.47 mg/l with 95% confidence limits of 0.43 - 0.53 mg/l. The No Observed Effect Concentration was 0.32 mg/1.