Leuco polysulfided 4-[(2,4-dinitrophenyl)amino]phenol

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:

2019-06-04 to 2019-06-06

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment No. 23, Paris; February 2019

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the determination of the test item concentration, samples were taken from the test item treatment and control group at the start (prior to distributing of the test solution to the test vessels) and at the end of each renewal period in duplicate. The first series of samples taken (4 x 50 mL) were sent to individual analysis. The further samples (4 x 50 mL) are kept separately as a reserve until the date of issuing the Final Report at a temperature below -15 °C. After issuing of the Final Report the frozen samples are discarded.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
As the test item is poorly soluble in deionized water as well in the test medium, for preparation of test solution the water-accommodated fraction (WAF) approach was taken, as described in OECD Guidance Document No. 23. A supersaturated solution (100 mg/L nominal loading) was prepared by adding an amount of 0.1003 g test item to 1003 mL test medium (ISO Medium) in the first renewal period and 0.1002 test item in 1002 mL test medium (ISO Medium) in the second renewal period one day before the start of each renewal period.
The test solution was handled by ultrasonic bath for 10 minutes, thereafter stirred for a period of 24 hours to achieve an equilibrated concentration. The test solution was then filtrated through a membrane filter (0.45 µm; Thermo Nalgene® membrane*) to separate the possible non-dissolved test material. The test solution was freshly prepared in the testing laboratory just before introduction of the test animals (start of each renewal period).

*Lot number: 1247803
Expiry date: 31 January 2024)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Species and Strain: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection), 2100 Gödöllö, Kotlán S. u. 3. Hungary
- Breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT.
- Age at study initiation: Less than 24 hours old
- Sex: Female
- Feeding during test : No

ACCLIMATION
- Acclimation period: The test animals were bred under similar (or the same) conditions as that used during the exposure period (holding water, temperature, background colour etc.), therefore additional acclimatisation before the test was not necessary. Brood daphnids were maintained in dilution water at the test temperature for at least 48 hours prior to the start of the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

236.74 - 249.2 mg/L (as CaCO3)

Test temperature:

20.4-21.1 °C

pH:

7.36 - 7.94

Dissolved oxygen:

6.89-8.57 mg/L

Salinity:

Not applicable

Conductivity:

No data

Nominal and measured concentrations:

Nominal: 100 mg/L
Measured: The measured test item concentration was below LOD (0.2 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: glass, 150 mL, 20 mL, 130 mL
- Aeration: No
- Renewal rate of test solution: Based on the results obtained during the analytical method validation (study number. 805-100-4517) the test item is not stable for the duration of 48 hours in ISO medium. Therefore the test solution (as well control solution) was renewed once during the test (on day 1)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to OECD TG 202

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Complete darkness

EFFECT PARAMETERS MEASURED: Daphnia were observed for immobility or mortality by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile. In addition to immobility, any abnormal behavior or appearance is reported.

RANGE-FINDING STUDY
In order to select appropriate test concentrations for use in the definitive test, a non GLP preliminary range-finding test was conducted to determine the approximate toxicity of the test item. Due to the test item's low solubility, preparation of test solution was performed using WAF method (according to OECD Series on Testing and Assessment No. 23) as follows:
In the semi-static preliminary range-finding test the nominal test concentration of 100 mg/L was prepared by suspending 0.0200 g test item amount in 200 mL ISO Medium in the first renewal period, 0.0202 g test item amount in 202 mL ISO Medium in the second renewal period and handled in ultrasonic bath for 10 minutes. This solution was mixed thoroughly for a period of 24 hours to achieve an equilibrated concentration and then filtered through a membrane filter (0.45 µm) to separate the possible non-dissolved test material. The test solution and control were renewed once during the test (on day 1). Untreated control ran parallel in the test. In the preliminary range-finding test ten (2 x 5) Daphnids in the test concentration and control were exposed for 48 hours. The results (48-h) of the preliminary range-finding test are summarized in table 1 in the section 'Any other information on materials and methods incl. tables'
Based on the results of the non-GLP Preliminary Range-Finding Test, the main test was a limit test performed using WAF method (according to OECD Series on Testing and Assessment No. 23) including only one test concentration at saturation (equivalent to 100 mg/L nominal concentration) and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

mortality

Remarks on result:

other: The test item had no toxic effect up to and including aquatic saturation (i.e. limit test concentration) on Daphnia magna

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: Concentration based on mean measured concentration of the water-accommodated fraction of the test item (=LOD)

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: Concentration based on mean measured concentration of the water-accommodated fraction of the test item (=LOD)

Details on results:

Validity of the study:
Immobilisation was 0.0% (<10%) in the control group and the dissolved oxygen concentration during the test in control and test vessels was in the range of 6.89 -8 .57 mg/L (more than 3 mg/L in all cases). All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Analytical Results:
The concentration of the test item in the test solutions was analytically determined based on total product at the start and at the end of the renewal period of the experiment.
The test item was not detected in the untreated control group (i.e. signal intensities measured for the control samples were less than LOD; limit of detection).
In the treated group the measured test item concentration was below LOD (limit of detection) during the study. Therefore, it was calculated as LOD of the test item (0.2 mg/L). This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fraction of the test item (equivalent to 100 mg/L nominal concentration).

Biological Results
Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of the test animals was detected in any of the test groups.
The results are based on nominal test item concentration and on the LOD, in line with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint Specific Guidance Version 4.0 (ECHA), June 2017.
The 48-h NOEC was determined to be 0.2 mg/L (Limit of detection, nominal 100 mg/L total product).
The 48-h LOEC was determined to be >0.2 mg/L (Limit of detection, nominal >100 mg/L total product).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
24h EC50 = 1.58 mg/L (95% conf. limits: 1.34-1.85 mg/L)

Reported statistics and error estimates:

A limit test was performed and toxic effects were observed, therefore statistical analysis was not necessary. The NOEC and LOEC values of the test item were determined directly from the raw data.

Validity criteria fulfilled:

yes

Conclusions:

In this acute 48-hour Daphnia magna immobilisation study performed using WAF method (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L (total product), the obtained results showed that the test item had not any significant toxic effect on the test system. The 48-h NOEC was determined to be 0.2 mg/L (Limit of detection, nominal 100 mg/L). The 48-h LOEC was determined to be >0.2 mg/L (Limit of detection, nominal >100 mg/L). All validity criteria were met. The results are based on nominal test item concentration and the LOD, in line with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint Specific Guidance Version 4.0 (European Chemicals Agency), June 2017.

Executive summary:

Acute toxicity of the test item was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test according to OECD TG 202 and 23. Young Daphnia were exposed to aqueous test media containing the test item for 48 hours in a semi-static test. A limit test was performed in which, the test animals were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item in the test solutions was performed by a previously validated analytical method. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV-VIS spectrophotometric method. As the measured start and end test item concentrations of both renewal periods were below the limit of detection (LOD), the determined exposure concentration was 0.2 mg/L which was calculated as LOD (according to OECD 23).

Healthy, young female daphnids <24 hours of age were exposed to the saturated test concentration and a control (ISO medium). The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of the test animals was detected in any of the test groups. All validity criteria were met and therefore the study can be considered as valid.

In this acute 48-hour Daphnia magna immobilization study performed using WAF method (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L (total product), the obtained results showed that the test item had not any significant toxic effect on the test system. The 48-h NOEC was determined to be 0.2 mg/L (Limit of detection, nominal 100 mg/L). The 48-h LOEC was determined to be >0.2 mg/L (Limit of detection, nominal >100 mg/L). The results are based on nominal test item concentration and the LOD, in line with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint Specific Guidance Version 4.0 (European Chemicals Agency), June 2017.

Description of key information

In the acute 48-hour Daphnia magna immobilisation study performed with the WAF (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L (total product, i.e. Leuco Sulphur Blue 11 containing 36.5 % dyestuff compared to Leuco Sulphur Blue 11 containing 97.4 % dyestuff), the obtained results showed that the test item had not any significant toxic effect on the test system. The 48 -h NOEC was determined to be 0.2 mg/L (limit of detection, nominal 100 mg/L). The 48 -h LOEC was determined to be >0.2 mg/L (limit of detection, nominal >100 mg/L). All validity criteria were met. The results are based on nominal test item concentration and the LOD, in line with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint Specific Guidance Version 4.0 (European Chemicals Agency), June 2017.

Additional information

Acute toxicity of the test item was assessed on Daphnia magna in a 48-hour Acute Immobilisation Test according to OECD TG 202 and 23. Young Daphnia were exposed to aqueous test media containing the test item (Leuco Sulphur Blue 11 containing 36.5 % dyestuff compared to Leuco Sulphur Blue 11 containing 97.4 % dyestuff) for 48 hours in a semi-static test. A limit test was performed in which, the test animals were exposed to aqueous test media containing the test item for 48 hours at the limit of its solubility in the test medium (i.e. saturation) plus a control in order to demonstrate that the test item has no influence on the mobility of Daphnia up to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF)).

The quantification of the test item in the test solutions was performed by a previously validated analytical method. Samples were taken from the test concentration and the control at the start and at the end of each renewal period of the experiment and analysed by UV-VIS spectrophotometric method. As the measured start and end test item concentrations of both renewal periods were below the limit of detection (LOD), the determined exposure concentration was 0.2 mg/L which was calculated as LOD (according to OECD 23).

Healthy, young female daphnids <24 hours of age were exposed to the saturated test concentration and a control (ISO medium). The immobilisation of the test animals was recorded 24 and 48 hours after test start. Environmental conditions were recorded at the start and at the end of each renewal period. All measured values remained within the acceptable ranges.

Immobilisation was observed neither in the control group nor in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of the test animals was detected in any of the test groups. All validity criteria were met and therefore the study can be considered as valid.

In this acute 48-hour Daphnia magna immobilization study performed using WAF method (according to OECD Series on Testing and Assessment No. 23) including a nominal loading rate of 100 mg/L (total product), the obtained results showed that the test item had not any significant toxic effect on the test system. The 48-h NOEC was determined to be 0.2 mg/L (Limit of detection, nominal 100 mg/L). The 48-h LOEC was determined to be >0.2 mg/L (Limit of detection, nominal >100 mg/L). The results are based on nominal test item concentration and the LOD, in line with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7b: Endpoint Specific Guidance Version 4.0 (European Chemicals Agency), June 2017.