Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

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

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)

Version / remarks:

2004

Deviations:

yes

Remarks:

The pH was not recorded in the limit test concentration (100 mg test item/L) after 48 h due to human failure. This deviation is not considered to influence the outcome of the test or impact the integrity of this study.

Principles of method if other than guideline:

Deviations from the Study Plan or the Guideline
Deviating from the study plan and the guideline, the pH was not recorded in the limit test concentration (100 mg test item/L) after 48 h due to human failure. Since the pH was within the required range, since no immobilities were observed during the test at the limit treatment of 100 mg test item/L, and since analytical verification of the test item concentration indicated stability of the test concentration, this deviation is not considered to influence the outcome of the test or impact the integrity of this study.
Otherwise, the study was performed according to the guideline, the study plan and its amendment(s) without deviation.

GLP compliance:

yes

Specific details on test material used for the study:

Storage conditions: Cool, dry, well ventilated, protected from light; in tightly closed container; not below 0 °C (stored at ambient temperature)

Analytical monitoring:

yes

Details on sampling:

Storage conditions: Cool, dry, well ventilated, protected from light; in tightly closed container; not below 0 °C (stored at ambient temperature)

Sampling for Chemical Analysis
Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
Control and test solutions were sampled in duplicate. The duplicate samples were kept separately as a reserve. The nominal volume per sample was 10 mL. mixed with 10 mL of acetonitrile. The volume of each sample was recorded.
After sampling and before shipment, all samples were stored in amber glass bottles in the dark at a temperature of ≤ -18°C. A record was kept for each sample.
Samples of untreated test medium were provided to the analytical laboratory in order to determine the recovery of the analytical method with the specific test medium. Samples were transferred to the test site for chemical analysis under the required storage conditions. The dates of transfer of the samples from ECT to the laboratory for chemical analysis were recorded in the raw data.

Vehicle:

no

Details on test solutions:

Preparation of Stock and Test Solutions
A stock solution was prepared at a nominal loading rate of 100 mg/L as described below by adding 100.25 mg of the test item to 1000 mL of the test medium. This stock solution (S1) was stirred (900 rpm) for 20 minutes at ambient temperature. The stock solution was visually examined for undissolved/particulate matter in the free water column. Visual inspection of the flask showed a clear solution without Tyndall effect.
The stock solution S1 was prepared once to prepare the test solutions.
The volume of the stock solution was large enough to prepare the test concentrations and all analytical samples.

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
Origin: The organisms were originally supplied by KU Leuven, Belgium
Age of parental daphnids: less than 35 d
Age of the daphnids at the beginning of the test: less than 24 h

The health of the Daphnia culture and the overall quality of the test organisms is monitored regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test design in order to determine the sensitivity of the organisms from the culture and to demonstrate that the test conditions are appropriate.

The most recent reference test with potassium dichromate performed in a separate study resulted in an EC50(24 h) of 0.74 mg/L.

Holding Conditions
Material of stock vessel: Glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 2-5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: within required ranges (20 +/- 2°C)
Photoperiod (light/dark): 16/8 h
Light intensity: within required ranges (50 to 1000 lx)
Aeration: None
Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week
Age of exposed daphnids at start < 24 hours

Test type:

static

Water media type:

freshwater

Remarks:

Water Quality Measurements: temperature, pH, dissolved oxygen content in % of air saturation value (ASV) and in mg/L

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

18 - 22 °C

pH:

7.7

Dissolved oxygen:

90 - 93 % of air saturation; 7.7 - 8.4 mg/L

Nominal and measured concentrations:

100 mg test item/L (based on the results of a preliminary non-GLP range finding test)

Details on test conditions:

Material and methods:
Test system: Daphnia magna (Straus)
Age of exposed daphnids at start < 24 hours
Medium: Elendt Medium M4
Endpoint: ECx (e.g. EC50)
Biological parameters: Immobilisation
Test duration: 48 h
Test units: 100-mL glass beakers, closed by watch glass
Volume of test solution per test vessel: 50-60 mL
Number of daphnids per test vessel: 5
Target temperature: 18 - 22°C
Photoperiod: 16 h light / 8 h dark
Aeration: none
Feeding during exposure: none
Test concentrations (nominal): Control; 100 mg test item/L
Number of replicates per concentration: 4
Number of replicates in the controls: 4
Renewal of test solution during exposure: Static, no renewal
Chemical analysis of test concentrations: At start and at end of exposure
Data evaluation: Evaluation of immobility.

Holding Conditions
The culture conditions of Daphnia magna as summarised below are similar to the test conditions.
Material of stock vessel: Glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 2-5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: within required ranges (20  2°C)
Photoperiod (light/dark): 16/8 h
Light intensity: within required ranges (50 to 1000 lx)
Aeration: None
Food: algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week
The selected holding vessels designated for production of the neonates for the test were cleared of all mixed-aged young daphnids on the day before test start. These vessels contained parental daphnids aged less than 35 days. On the day of test start (day 0) all newly produced neonates of these vessels were < 24 hours old. These neonates were collected for the test.

Reference substance (positive control):

yes

Remarks:

reference toxicant potassium dichromate (K2Cr2O7) to monitor health and overall quality Daphnia culture

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The test item concentration was stable during the test period, with >85% of the nominal concentration found in the fresh and aged solution. The biological findings are therefore expressed based on nominal concentrations.
In this limit test no immobilities were observed in control or in the limit concentration of 100 mg test item/L. It is therefore concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L).

Results with reference substance (positive control):

Test with potassium dichromate resulted in an EC50(24 h) of 0.74 mg/L.
According to the OECD guideline No. 202 EC50(24 h)-values for potassium dichromate obtained from different laboratories should range between 0.6 mg/L and 2.1 mg/L. The toxicity of the reference item is within this range. Therefore the results of this reference test are acceptable and the test conditions are reliable and the sensitivity of the test system could be demonstrated.

Reported statistics and error estimates:

In this limit test no immobilities were observed in the control or in limit concentration of 100 mg test item/L. A statistical evaluation for differences between treatment and control was therefore not required and was not performed.

Biological Results

Cumulative number of immobilised Daphnia magna after 24 and 48 h.

Nominal concentration	Total number of introduced daphnids	Total number of immobile daphnids	Immobile daphnids (% of introduced)	Total number of immobilie daphnids	Immobile daphnids (% of introduced)
[mg/L]		[24 h]	[24 h]	[48 h]	[48 h]
Control	20	0	0.0	0	0.0
100	20	0	0.0	0	0.0

Sublethal effects on the mobile daphnids were not observed.

Analytical Results

The test item concentration was stable during the test period, with >85% of the nominal concentration found in the fresh and aged solutions.

Summary of measured concentrations during the test.

Nominal concentration [mg test item/L]	Test period [h]	Age of test solution [h]	Measured concentration [mg/L]	% of nominal concentration
0 (control)	0	0	< 36.506	--
100	0	0	88.103	88.1
0 (control)	48	48	< 36.506	--
100	48	48	87.833	87.8

Analysis of control samples of test medium yielded no residues at or above the LOQ.

Since the measured concentrations were >80% of nominal, the biological endpoints are expressed based on nominal concentrations (mg/L).

Validity criteria fulfilled:

yes

Remarks:

immobilised daphnids in the control: 0% (<=10%); dissolved oxygen concentration in control and test vessels at the end of the test: >= 7.7 mg/L (3 mg/L); Signs of disease or stress did not exceed 10 per cent of the daphnids in the controls.

Conclusions:

In a limit test no immobilities were observed in control or in the limit concentration of 100 mg of Direct Yellow 86 to the water flea Daphnia magna under static conditions. Therefore the EC50 of the test item is higher than the limit concentration tested (EC50 > 100 mg test item/L).

Executive summary:

The aim of the study was to determine the acute toxicity of Direct Yellow 86 to the water flea Daphnia magna. To achieve this, a limit concentration of the test item in aqueous solution was prepared, and the test organisms were exposed to this concentration as well as to controls without the test item for a period of 48 hours under static conditions. During the test period the test solutions were not aerated. The daphnids were not fed. Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile.

Four replicates were used for the test item concentration and the control. Each replicate vessel contained five daphnids.

As there was no significant immobilisation compared to the control, the data obtained were analysed in order to demonstrate that the EC50 is higher than the limit concentration.

To verify the nominally applied concentrations, samples were taken from the test solutions and analytically measured. The samples designated for chemical analysis were shipped to the analytical test site and analysed under the responsibility of the principal investigator for analysis.

In this limit test no immobilities were observed in control or in the limit concentration of 100 mg test item/L. It is therefore concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L).

The requirement of the study is based on but not necessarily limited to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006.

Deviating from the study plan and the guideline, the pH was not recorded in the limit test concentration (100 mg test item/L) after 48 h due to human failure. Since the pH was within the required range, since no immobilities were observed during the test at the limit treatment of 100 mg test item/L, and since analytical verification of the test item concentration indicated stability of the test concentration, this deviation is not considered to influence the outcome of the test or impact the integrity of this study.

Description of key information

The aim of the study was to determine the acute toxicity of Direct Yellow 86 to the water flea Daphnia magna. To achieve this, a limit concentration of the test item in aqueous solution was prepared, and the test organisms were exposed to this concentration as well as to controls without the test item for a period of 48 hours under static conditions. During the test period the test solutions were not aerated. The daphnids were not fed. Those animals not able to swim within 15 seconds after gentle agitation of the test vessel were considered to be immobile.

Four replicates were used for the test item concentration and the control. Each replicate vessel contained five daphnids.

As there was no significant immobilisation compared to the control, the data obtained were analysed in order to demonstrate that the EC50 is higher than the limit concentration.

To verify the nominally applied concentrations, samples were taken from the test solutions and analytically measured. The samples designated for chemical analysis were shipped to the analytical test site and analysed under the responsibility of the principal investigator for analysis.

In this limit test no immobilities were observed in control or in the limit concentration of 100 mg test item/L. It is therefore concluded that the EC50 of the test item is higher than the limit concentration tested (nominal: 100 mg test item/L).

The requirement of the study is based on but not necessarily limited to Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December 2006.

Deviating from the study plan and the guideline, the pH was not recorded in the limit test concentration (100 mg test item/L) after 48 h due to human failure. Since the pH was within the required range, since no immobilities were observed during the test at the limit treatment of 100 mg test item/L, and since analytical verification of the test item concentration indicated stability of the test concentration, this deviation is not considered to influence the outcome of the test or impact the integrity of this study.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Should read "EC50 > 100 mg/L"