Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

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:

2017-09-06 to 2017-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline; EU Method; GLP

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

EC Number:

274-366-7

EC Name:

Sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Cas Number:

70179-77-0

Molecular formula:

C28H30N2O8S.Na

IUPAC Name:

sodium 3-[[4-amino-9,10-dihydro-3-[2-(2-methoxyethoxy)ethoxy]-9,10-dioxo-1-anthryl]amino]-2,4,6-trimethylbenzenesulphonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the controls were taken at the start of the test and at day 1.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the aged test media of all test concentrations and the control were collected at day 1 (after 24 hours of exposure) and at the end of the test by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Test Concentrations: 131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/L) and a control.
corresponding to following Initial mean measured concentrations of the test item:
134, 43.3, 13.0, 4.39 and 1.38 mg test item/L, and a control. Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test medium of the highest test concentration of nominal 131 mg test item/L was prepared by dissolving 78.7 mg test item into 600.8 mL test water by intense stirring for 15 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
Appearance of the Test Item in Test Medium: In the freshly prepared and aged test medium of the highest concentration of 134 mg test item/L were observed to be dark violet. The lower the concentration, the lighter the test medium was coloured ranging from violet (4.39, 13.0 and 43.3 mg test item/L) to light violet (1.38 mg test item/L). Additionally, the test item was observed to lie at the bottom of the test beaker in the aged test media at test concentration of 13.0 to 134 mg test item/L.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 1.5 to 21.5 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

20.4 to 20.6 °C in the freshly prepared media;
20.0 to 20.4°C in the aged test media

pH:

8.2 to 8.4 in the freshly prepared media;
7.9 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

8.5 to 8.9 mg/L in the freshly prepared media;
8.2 to 8.7 mg/L in the aged test media

Nominal and measured concentrations:

131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/L) and a control.
corresponding to following Initial mean measured concentrations of the test item:
134, 43.3, 13.0, 4.39 and 1.38 mg test item/L, and a control.

Details on test conditions:

Test Environment: Controlled environment room
Measurement of pH, Dissolved
Oxygen and Water Temperature:
The water temperature, pH-values and the dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group.
Water Temperature: 20.4 to 20.6 °C in the freshly prepared media;
20.0 to 20.4°C in the aged test media
pH-Values: 8.2 to 8.4 in the freshly prepared media;
7.9 to 8.0 in the aged test media; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 8.5 to 8.9 mg/L in the freshly prepared media;
8.2 to 8.7 mg/L in the aged test media Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 310 to 620 lux (measured once during the test).

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 134 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 134 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 134 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the initial mean measured test item concentration of 1.38, 13.0, 43.3 and 134 mg test item/L. At the concentration of 4.39 mg test item/L, one animal was immobile.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate performed in January 2017 (study code 88297220) the EC50 after 24 hours was determined to be 1.48 mg test item/L, indicating that the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.2 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item Acid Violet 126 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 134 mg test item/L. The 48-hour LOEC was determined to be > 134 mg test item/L and the 48-hour EC50 value was determined to be > 134 mg test item/L.

Executive summary:

Title:

Acid Violet 126: Acute Toxicity to Daphnia magna in a Semi-Static 48-hour Immobilisation Test

Purpose:

The purpose of this study was to determine the influence of the test item Acid Violet 126 on the mobility of Daphnia magna. For this purpose, young daphnids (< 24 hours old) were exposed in a semi-static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines:

Commission Regulation (EC) No 440/2008, Annex, Part C, C.2.: "Daphnia sp. Acute Immobilisation Test", Official Journal of the European Union (EN), dated May 30, 2008

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Material and Methods

Test Item: Acid Violet 126; batch no.: PQD21/1609023; purity: 76.1 %, according to certificate of analysis

Test Species: Female Daphnia magna, clone 5; 1.5 to 21.5 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: 131, 42.0, 13.1, 4.20 and 1.31 mg test item/L (referring to 100, 32, 10, 3.2 and 1.0 mg active ingredient/L) and a control.

corresponding to following Initial mean measured concentrations of the test item:

134, 43.3, 13.0, 4.39 and 1.38 mg test item/L, and a control.

Test Conditions: Water temperature: 20.0 to 20.6 °C; pH value: 7.9 to 8.4; dissolved oxygen concentration: 8.2 to 8.9 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 310 to 620 lux; and thus were within the ranges requested by guideline OECD 202

Results

Biological test results: After 48 hours of exposure no immobilisation of the test animals was observed in the control and in the initial mean measured test item concentration of 1.38, 13.0, 43.3 and 134 mg test item/L. At the concentration of 4.39 mg test item/L, one animal was immobile.

Analytical test results:

The quantification of the test item Acid Violet 126 in the test samples was performed using liquid chromatography with UV detection. In the freshly prepared test media at the start of the test and at the renewal of the test media 103% of the nominal test concentrations were found (average of all test concentrations). In the aged test media after 24 and 48 test duration, 68% of the nominal value was determined (average of all test concentrations).

Conclusion:

The toxic effect of the test item Acid Violet 126 to Daphnia magna was assessed in a semi-static concentration-response test. The 48-hour NOEC was determined to be ≥ 134 mg test item/L. The 48-hour LOEC was determined to be > 134 mg test item/L and the 48-hour EC50 value was determined to be > 134 mg test item/L. The initial concentrations and the concentrations during the test were determined in the analytical part. The test item didn’t remain dissolved in the test medium during the renewal period over 24 hours which is seen from the test item observations and was therefore not bioavailable. The test item was tested up to its limit of solubility in the test medium. Therefore all reported results refer to initial mean concentrations. This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.