Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

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:

19 July - 05 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13th April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 1996

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000

Version / remarks:

September 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

EC Number:

291-833-0

EC Name:

Phenol, 4-amino-, reaction products with 4-(2-naphthalenylamino)phenol and sodium sulfide (Na2(Sx))

Cas Number:

90480-94-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-aminophenol with 4-​(2-​naphthalenylamino)​phenol and sodium polysulfide

Test material form:

solid

Details on test material:

Test item: Leuco Sulphur Black 11
Appearance: black solid
CAS No: 90480-94-7
EC No: 291-833-0

Specific details on test material used for the study:

Date of production:26 October 2016
Expiry date: 26 October 2021

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

ANALYSIS OF THE SAMPLES
Four replicate samples were analysed from the test solutions at the start and at the end of the renewal
periods. Four replicate samples were analysed from the control as well.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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 0.0706 g test item to 706 mL test medium (ISO Medium) in the first renewal period and 0.0700 g test item in 700 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) to separate the possible non-dissolved test material.
- Controls: Negative control (ISO medium without addition of test item ), positive control (with the reference substance potassium dichromate)

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100-Gödöllő, Kotlán S. u. 3. Hungary
- Sex: Female
- Age of parental stock: Less than 24 h old at the beginning of the test
- Feeding during test: No
ACCLIMATION
- Acclimation period: 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.
- Acclimation conditions: Same as test
- Type and amount of food: The Daphnia were fed with concentrated algal suspension of Raphidocelis subcapitata during holding.
- Health during acclimation: Apparently healthy animals were used in this test with a known history

Study design

Test type:

semi-static

Water media type:

other: Reconstituted water (ISO Medium, according to OECD 202)

Limit test:

yes

Total exposure duration:

48 h

Remarks on exposure duration:

Animals were exposed to the test concentration for 48 hours. Vesseles contained 80 mL of test solution or control.

Post exposure observation period:

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 were considered to be immobile.

Test conditions

Hardness:

The measured hardness of the ISO Medium used in the study was 236.74 mg/L (as CaCO3).

Test temperature:

The test temperature was in the range of 20.2 - 20.9°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.4 – 21.0°C.

pH:

The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was in the range of 7.73 – 8.18 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 6.55 – 7.48 mg/L during the test.

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

The test vessels were kept in a climate chamber under controlled environmental conditions during the test and test solutions were not aerated. The water temperature, dissolved oxygen concentration and pH were measured in each test group at the start (before test solutions had been distributed into the test vessels) and in each test vessel at the end of each renewal period. Additionally, the ambient temperature was measured continuously using a min/max thermometer in the climate chamber.

Test type: Semi-static test. Based on the results obtained during analytical method validation (Study number: 805-100-3122) the test item is not stable for the duration of 48 hours in ISO Medium and not remains within 80 – 120% in comparison to that of the measured test item concentration at the start of the test throughout exposure. Therefore the test solution (as well control solution) was renewed once during the test (on day 1).
Temperature: The test temperature was in the range of 20.2 - 20.9°C measured in the test vessels during the test. The additionally measured ambient temperature within the climate chamber was between 19.4 – 21.0°C.
Oxygen Concentration: The dissolved oxygen concentration was in the range of 6.55 – 7.48 mg/L during the test.
pH: The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was in the range of 7.73 – 8.18 during the test.
Light-Dark Cycle: The experiment was carried out in complete darkness, as described by OECD 202.
Hardness: The measured hardness of the ISO Medium used in the study was 236.74 mg/L (as CaCO3).

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 0.41 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr. (dissolved fraction)

Basis for effect:

mobility

Details on results:

Validity of the Study

Immobilisation was 5.0 % (< 10 %) in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was in the range of 7.22 – 7.48 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 was analytically determined based on total product at the start and at the end of each 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 15 % of the lowest concentration of the calibration curve).
In the treated group the mean measured test item concentration was 0.68 mg/L at the start and 0.25 mg/L at the end of the first renewal period (corresponding to 36 % of the initial measured test concentration) while it was measured to be 0.78 mg/L at the start and 0.22 mg/L at the end of the second renewal period (corresponding to 32 % of the initial measured test concentration).
The exposure concentration was calculated as the geometric mean of the start and end values and determined to be 0.41 mg/L. This concentration was considered as the saturation concentration in the test medium, based on water-accommodated fractions of the test item (equivalent to 100 mg/L nominal concentration).

Biological Results and Performance of the Test

Testing units were kept in a climate chamber at a temperature of 19.4 – 21.0 °C, while temperature in the test glasses ranged between 20.2 - 20.9°C. The pH was measured as 7.73 – 8.18 and the dissolved oxygen concentration between 6.55 – 7.48 mg/L during the study (see Tables 4, 5 and 6 of Appendix I). The test was carried out in total darkness.
There was only one daphnid immobile (5.0 %) in twenty daphnids exposed in the control group and no immobilisation was observed in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Results with reference substance (positive control):

For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 392-202-3732) with reference item Potassium dichromate was: 28 February – 01 March 2018.
The 24h EC50 value was determined to be 1.41 mg/L (95% conf. limits: 1.19 – 1.68 mg/L), which falls within the range of 0.6 mg/L and 2.1 mg/L as given in the guideline.

Reported statistics and error estimates:

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

Any other information on results incl. tables

Results of the Preliminary Range-finding Test

Nominal concentration (mg/L)	Untreated control	100 (WAF)
Number of treated animals	10	10
Number of immobilised animals(at 48h)	0	0

Summary of the Biological Endpoints

Endpoints	Concentration
48 h-NOEC	0.41 mg/L
48 h-LOEC	> 0.41 mg/L

pH-values Measured during the Experiment

Test Group	Replicate	pH
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	8.18	7.82		7.78	7.73
	2		7.89			7.86
	3		7.89			7.89
	4		7.89			7.91
Saturated test concentration (0.41 mg/L measured)	1	8.13	7.87		7.83	7.95
	2		7.88			7.95
	3		7.88			7.94
	4		7.90			7.95

 

Dissolved Oxygen Concentrations Measured during the Experiment

Test Group	Replicate	Dissolved Oxygen Concentration (mg/L)
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	7.33	7.16		7.31	7.48
	2		7.15			7.43
	3		7.22			7.40
	4		7.22			7.36
Saturated test concentration (0.41 mg/L measured)	1	7.03	7.12		6.55	7.22
	2		7.07			7.29
	3		7.06			7.23
	4		7.05			7.30

 

Temperatures Measured during the Experiment

Test Group	Replicate	Temperature (°C)
		1^strenewal period		2^ndrenewal period
		0 h	24 h		24 h	48 h
Control	1	20.2	20.3		20.5	20.6
	2		20.3			20.7
	3		20.5			20.7
	4		20.2			20.9
Saturated test concentration (0.41 mg/L measured)	1	20.3	20.2		20.6	20.9
	2		20.4			20.8
	3		20.4			20.8
	4		20.3			20.7

Immobilization of the Test Animals

Test Group	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	1	1
Saturated test concentration (0.41 mg/L measured)	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0

 

 

Validation Results

 

Results of the Method Validation (Study No.: 805-100-3122)

Measurement repeatability (7 parallels)	CV% < 2 %
Repeatability of the Preparation (n=5)	CV%< 3 %
Linear range	0.2 - 20 µg/mL
Correlation coefficients	> 0.999
Limit of Quantification	0.2 µg/mL
Limit of Detection	0.2 µg/mL
Recovery from ISO medium	102 %
Recovery from 20XAAPmedium	95 %
Recovery from ultra-pure water	101 %
Accuracy(samples used for calibration)	≤ 5 % (10 % at LOQ)
Precision	≤ 6 %
Stability of the test item in ISO medium at the temperature 18-22 °C for 2 days	< LOD at the test conditions
Stability of the test item in 20XAAP medium at the temperature 22-26 °C for 7 days	66 % at the test conditions
Stock solution stability (1 mg/ml), after dissolution, dye complexation and color development	for at least 7 days at 5±3 °C

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 48-hour semi-static test according to EU Method C.2, OECD Guideline 202 and EPA OPPTS 850.1010 the 48-hour LOEC of the test item was determined to be > 0.41 mg/L (geometric mean measured). The test item had no toxic effect to Daphnia magna up to and including aquatic saturation of the test item.

Executive summary:

The acute toxicity of the test item was assessed on Daphnia magna in a 48 -hour Acute Immobilisation Test.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 ofDaphniaup to at least the saturation concentration (equivalent to 100 mg/L nominal concentration, prepared as water-accommodated fraction (WAF).

The quantificaton of the test item was performed by a previously validatedanalytical method by the analytical laboratory of TOXI-COOP ZRT. 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. The mean determined exposure concentration was 0.41 mg/L (based on total product) which was calculated as the geometric mean of the measured start and end concentrations of both renewal periods.

Healthy, young female daphnids < 24 hours of age and with known origin, history and acclimatisation conditions were used. Twenty Daphnia (divided into 4 replicates) were tested each, exposed to the saturated test concentration and in the control. Each test vessel contained approximately 80 mL test medium.Fully characterized (content, physico-chemical characteristics) ISO Medium was used as test 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.

There was only one daphnid immobile (5.0 %) in twenty daphnids exposed in the control group and no immobilisation was observed in the test item treated group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

All validity criteria were met and therefore the study can be considered as valid . A limit test was performed and toxic effects were not observed, therefore NOEC and LOEC values of the test item were determined directly from the raw data

In conclusion, the test item Leuco Sulphur Black 11 had no toxic effect up to and including aquatic saturation (i.e.limit test concentration)onDaphnia magna; the 48h LOEC is higher than the solubility level of the test item in the test medium, which correspondsto the mean measured concentration of 0.41 mg/L(based on total product). The NOEC value is equal to the mean measured test item concentration of 0.41 mg/L(based on total product).