Disodium {sulfonylbis[4,1-phenylenediazene-2,1-diyl(1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-5,3-diyl)]}dimethanesulfonate

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:

25-Oct-2010 to 15-Nov-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH),

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic: Date of inspection: 05 to 09-Nov-2007 and 26 to 30-Nov-2007; Date of decision: 2008-04-30, Date of signature: 12-Nov-2008

Test material

Test material information

Constituent 1

Reference substance name:

Acid Yellow RN 2903

IUPAC Name:

Acid Yellow RN 2903

Constituent 2

Reference substance name:

Vers. Kilo 6, 26.05.2009

IUPAC Name:

Vers. Kilo 6, 26.05.2009

Details on test material:

The test item and the following information concerning the test item were provided by the Sponsor:

Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65%
Expiration Date: 01-Aug-2015
Storage Conditions: At room temperature at about 20 °C, in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start and end of each test medium renewal period. For sampling from the aged test media, the contents of the respective replicates were combined prior to sampling. All samples were stored deep-frozen (at about -20 °C) immediately after sampling.

The concentrations of the test item Acid Yellow RN 2903 were analyzed in the duplicate test media samples from the loading rate of 100 mg/L and the dilution 1:2 from all sampling times (0, 24 and 48 hours). The samples of the loading rates of 1:4, 1:8 and 1:16 were not analyzed since the concentrations were below the 48 hour NOEC determined in this test.

From the control, only one of the duplicate samples was analyzed per sampling time. The analytical procedure and results are described in Attachemnt I - Analytical Investigations.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before the test medium renewal by dispersing 100 mg of the test item (100.2 and 100.3 mg, respectively) in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 5 minutes and intense stirring on a magnetic stirrer for 15 minutes in order to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. After the stirring period, the dispersion was left incubated overnight at room temperature for for 24 hours in the dark, followed by filtration and subsequent dilutions for the tested concentrations. The resting period of 24 hours was chosen according to the results of a pre-experiment (without GLP) which showed that the solution equilibrium was reached after this time.
After the 24-hour resting period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= addition of daphnids).

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred at Harlan Laboratories in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see below).

During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).

At the start of the test, the organisms used in the test were 6 24 hours old and were not first brood progeny.
The test method and the test species are recommended by the international test guidelines.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

2.5 mmol/L

Test temperature:

The water temperature was between 20 22 °C during the test (see attached Table 5).

pH:

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 8.0.

Dissolved oxygen:

The dissolved oxygen concentrations in the test media and control were at least 8.0 mg/L (see attached Table 4).

Salinity:

according to OECD medium

Nominal and measured concentrations:

The following concentrations were tested: the undiluted filtrate with the loading rate of 100 mg/L and the dilutions 1:2, 1:4, 1:8 and 1:16. Additionally, a control (test water without test item) was tested in parallel.

Details on test conditions:

The test was performed in 100 mL glass beakers filled with 50 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.

The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20 to 22 °C.

A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used. Light intensity during the light period was approximately between 523 and 640 lux.

The daphnids were not fed during the test.

A semi-static test with test medium renewal after 24 hours was performed to keep the concentrations of the test item in the test media as constant as possible during the test period of 48 hours. After 24 hours, the test organisms were placed in clean test vessels with freshly prepared test medium of the corresponding concentration.

The selection of the test concentrations was based on the results of a range-finding test (non-GLP).

The following concentrations were tested: the undiluted filtrate with the loading rate of 100 mg/L and the dilutions 1:2, 1:4, 1:8 and 1:16. Additionally, a control (test water without test item) was tested in parallel.

Concentrations above the solubility limit of the test item in the test water were not tested, in accordance with the test guidelines.

For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnid was 10 mL. Thus, the requirement of the test guidelines for the minimum volume of 2 mL test medium per daphnid was fulfilled. The daphnids were randomly distributed to the test vessels at initiation of the test.

Reference substance (positive control):

yes

Remarks:

tested twice a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

In the analyzed test medium samples from the start and end of the two test medium renewal periods the average concentrations found in the non-aged treatment samples were 2.67 and 5.33 mg/L (day 0) and 3.29 and 6.73 mg/L (day 1), whereas the concentrations found in the aged treatment samples were 2.53 and 4.98 mg/L (day 1) and 3.28 and 6.43 mg/L (day 2). Consequently, the test water renewal ensured nearly constant concentration of the test item during the test. The

The biological results are listed in the attached Table 1.

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the highest loading rate of 100 mg/L. Thus, the 24-hour EC50 was clearly higher than the loading rate of 100 mg/L.

The 24-hour EC0 was 100 mg/L. The 24-hour EC100 was >100 mg/L.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the dilution 1:2. At the highest loading rate of 100 mg/L, two test organisms were found to be immobile. Thus, the 48-hour EC50 was clearly higher than the loading rate of 100 mg/L. The 48-hour EC0 was determined to be at the dilution of 1:2, while the NOEC of Acid Yellow RN 2903 was determined to be at the loading rate of >100 mg/L. Due to the low toxicity of the test item, the 48-hour EC100 was determined to be clearly higher than the solubility limit of the test item in the test water. However, loading rates above 100 mg/L were not tested, in accordance with the test guidelines.

All test media were colored by the test item throughout the entire test duration (see attached Table 2).

At the beginning and end of the test medium renewal periods, the pH values of the test media were between 7.8 and 8.0. The dissolved oxygen concentrations in the test media and control were at least 8.0 mg/L (see attached Table 4), and water temperature was between 20 22 °C during the test (see attached Table 5).

The test is considered to be valid, as in the control no daphnids showed immobilization or other signs of disease or stress (e.g., discoloration or unusual behavior such as trapping at the surface water). Furthermore, the dissolved oxygen concentration at the end of the test was >=3 mg/L in the control and test vessels.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in October 2010 (48-hour EC50: 0.56 mg/L, study C97643) indicated that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2010: 0.43 to 1.1 mg/L).

Reported statistics and error estimates:

The NOEC, EC0 and EC100 were determined directly from the raw data.
The 24 and 48-hour EC50 of the test item could not be calculated, because none of the responses exceeded 50%.

Any other information on results incl. tables

Please find attached:

Attachment 1: Appendix I - Analytical Investigations

Tables 1 to 5:

Table 1: Effect ofAcid Yellow RN 2903on the Mobility of Daphnia magna

Table 2: Appearance of the Test Media during the Test Period

Table 3: pH Values in the Treatments

Table 4: Dissolved Oxygen Concentrations (mg/L) in the Treatments

Table 5: Temperature (°C) in the Treatments

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48-hour EC50 was clearly higher than the loading rate of 100 mg/L. The 48-hour EC0 was determined to be at the dilution of 1:2, while the NOEC of Acid Yellow RN 2903 was determined to be at the loading rate of >100 mg/L.

Executive summary:

The acute toxicity of the test item Acid Yellow RN 2903 to Daphnia magna was determined in a 48‑hour semi-static test according to the EU Commission Directive 92/69/EEC, Part C.2, the Commission Regulation (EC) No. 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was stirred for 15 min and left incubated overnight at room temperature for a period of 24 hours in the dark. Then, the dispersion was filtered. Theundiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and1:16were used as test media. Additionally, a control was tested in parallel.

 

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

In the analyzed test medium samples from the start and end of the two test medium renewal periodsthe average concentrations found in the non-aged treatment samples were 2.67 and 5.33 mg/L (day 0) and 3.29 and 6.73 mg/L (day 1), whereas the concentrations found in the aged treatment samples were 2.53 and 4.98 mg/L (day 1) and 3.28 and 6.43 mg/L (day 2), respectively.

 The biological test results (based on the loading rates) were as follows:

 

– 24-hour EC50:		>100	mg/L
	(95% confidence limits could not be determined)
– 24-hour EC0:		100	mg/L
– 24-hour EC100:		>100	mg/L
– 48-hour EC50:		>100	mg/L
	(95% confidence limits could not be determined)
– 48-hour EC0:		50	mg/L
48-hour NOEC:		>100	mg/L
– 48-hour EC100:		>100	mg/L