Phenol, 4-(phenylamino)-, sulfurized, leuco deriv.

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

Study period:

01 June 2012 to 10 August 2012

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

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), C2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

swissmedic, decision: 19-November-2010

Analytical monitoring:

no

Details on sampling:

On request of the Sponsor no analytical work was performed.

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 each test medium renewal by dispersing 100 mg of the test item (dosing range: 100.0-100.3 mg) in 1000 mL of test water.

This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 96 hours at room temperature in the dark to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used. The pH of the test medium was 7.8.

After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.2 µm).

The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for the preparation of the test media of 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) and prior to the test medium renewal.

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 (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 test.

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 to 24 hours old and were not first brood progeny.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

not applicable

Hardness:

The Water Hardness was 2.5 mmol/L.

Test temperature:

The water temperature was 21 °C during the test.

pH:

At the beginning and end of the test medium renewal periods, the pH values of the test media were 7.8 (see attached Table 3).

Dissolved oxygen:

The dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L (see attached Table 4).

Salinity:

according to OECD guideline.

Nominal and measured concentrations:

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 ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. This corresponds to theoretical concentrations of 100, 50, 25, 12.5 and 6.3 mg/L. Additionally, a control was tested in parallel.

Details on test conditions:

Reconstituted test water was used in the study. For further details on test water please see section "Any other information on materials and methods incl. tables" below.

The test was performed in 100-mL glass beakers filled with 60 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 21 °C. The test was performed as far as possible in the dark to avoid a photolytic degradation of the test item. 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 transferred in clean test vessels with freshly prepared test medium of the corresponding concentration by wide-bore pipette.

The following loading rate/dilutions of C.I. Leuco Sulphur Brown 96 were tested: undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L. This corresponds to theoretical concentrations of 100, 50, 25, 12.5 and 6.3 mg/L. Additionally, a control (test water without test item) was tested in parallel. Loading rates exceeding 100 mg/L were not tested in accordance with the test guidelines.

The selection of the test concentrations was based on the results of a range-finding test.

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 12 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.

The test duration was 48 hours.

Reference substance (positive control):

yes

Remarks:

tested twice a year

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The biological results based on the theoretical concentrations 100, 50, 25, 12.5 and 6.3 mg/L are listed in the attached Table 1. The calculations are based on these theoretical concentrations as no analytical determination could be conducted.

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including 50 mg/L (1:2 dilution).

At the loading rate of 100 mg/L, 20% of the organisms were found to be immobile after 24 hours.

The 24-hour EC50 of the test item could not be calculated because none of the responses exceeded 50% and, thus, was determined to be > 100 mg/L. The 24-hour EC0 was 50 mg/L. The 24-hour EC100 was > 100 mg/L. The values are based on the theoretical concentrations as no analytical determination could be conducted.

After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including 50 mg/L (1:2 dilution).

At the loading rate of 100 mg/L, 30% of the organisms were found to be immobile after 48 hours.

The 48-hour EC50 of the test item could not be calculated because none of the responses exceeded 50% and, thus, was determined to be > 100 mg/L. The 48-hour EC0 and NOEC of C.I. Leuco Sulphur Brown 96 were both 50 mg/L, since no toxic effect was observed up to and including this nominal test concentration. The 48-hour EC100 could not be determined due to the low toxicity of the test item. However, concentrations above 100 mg/L were not tested, in accordance with the test guidelines.

Throughout the entire test duration, all test media were clear solutions (slightly colored by the test item at the two highest nominal test concentrations of 50 mg/L and 100 mg/L) (see attached Table 2).

At the beginning and end of the test medium renewal periods, the pH values of the test media were 7.8 (see attached Table 3). The dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L (Table 4), and the water temperature was 21 °C during the test.

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 February 2012 (48-hour EC50: 0.63 mg/L, Harlan Laboratories Study D50585) showed that the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2012: 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-hour and the 48-hour EC50 of the test item could not be calculated, because none of the responses exceeded 50%. The EC50 was, therefore, also determined directly from the raw data.

The biological test results (based on the theoretical concentrations) were as follows:

 

– 24-hour EC50:	> 100	mg/L
– 24-hour EC0:	50	mg/L
– 24-hour EC100:	> 100	mg/L
– 48-hour EC50:	> 100	mg/L
– 48-hour EC0and
48-hour NOEC:	50	mg/L
– 48-hour EC100:	> 100	mg/L

Validity criteria fulfilled:

yes

Conclusions:

The test item had moderate acute toxic effects on Daphnia magna. The 48-hour EC50 was determined to be > 100 mg/L. The value is based on the theoretical concentration as no analytical determination could be conducted.

Executive summary:

The acute toxicity of the test item C.I. Leuco Sulphur Brown 96 to Daphnia magna was determined in a 48‑hour semi-static test according to the EU Commission Directive 92/69/, 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 ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. This corresponds to theoretical concentrations of 100, 50, 25, 12.5 and 6.3 mg/L. 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).

 

No analytical work was performed on request of the Sponsor due to the specific properties of the test item (low solubility in water).

 

The biological test results (based on the theoretical concentrations) were as follows:

 

– 24-hour EC50:	> 100	mg/L
– 24-hour EC0:	50	mg/L
– 24-hour EC100:	> 100	mg/L
– 48-hour EC50:	> 100	mg/L
– 48-hour EC0and
48-hour NOEC:	50	mg/L
– 48-hour EC100:	> 100	mg/L

Description of key information

In a 48-hour semi-static test with Daphnia magna the 48-hour EC50 was calculated to be >100 mg/L.

Additional information

The acute toxicity of the test item C.I. Leuco Sulphur Brown 96 to Daphnia magna was determined in a 48‑hour semi-static test according to the EU Commission Directive 92/69/, 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 ultrasonic treated for 15 minutes and continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the loading rate of 100 mg/L and dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. This corresponds to theoretical concentrations of 100, 50, 25, 12.5 and 6.3 mg/L. 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). No analytical work was performed on request of the Sponsor due to the specific properties of the test item (low solubility in water).

The 48-hour EC50 was calculated to be >100 mg/L and the 48-hour NOEC was determined to be 50 mg/L.