Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine and sodium polysulfide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-09 to 2019-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, Series On Testing And Assessment OECD No.23 (Second Edition)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/L (nominal)
- Sampling method: For determination of the test item concentration six replicate samples (25 mL per replicate) from the test concentration and six replicate samples (25 mL per replicate) from the control group were taken at the start and at the end of the test. The samples taken were sent to individual analysis. The analysis was performed by the analytical laboratory of TOXI-COOP ZRT. according to the results of the analytical method validation (Study number: 805-100-2122 and 805-100-4226) using UV/VIS spectrophotometric method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
The test item is poorly soluble in the test medium, therefore preparation of test solution was performed using the WAF method (according to OECD Series on Testing and Assessment No. 23). In order to achieve the maximum solubility level, the test solution was prepared as follows: a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving 0.1010 g test item amount into 1010 mL of the test medium (OECD medium) and handled in ultrasonic bath for approximately 10 minutes. This solution was mixed thoroughly for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 μm) to separate the possible non-dissolved test material. After the formulation procedure algal cells were immediately introduced into test solution (start of the experiment).

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding Conditions: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardized conditions according to the test guidelines.
- Method of cultivation: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days before this test.) The algal cultures used in this study did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.5 - 22.8°C (measured in the flask)
21.6 – 23.5°C (measured within the climate chamber)

pH:

7.59 – 9.09

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks filled with volumes of 100 mL algal suspension per replicate
- Type: Closed. The test flasks were covered with air-permeable stoppers.
- Initial cells density: 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity and quality: The light intensity was checked and recorded at the start of the test. The algal culture flasks were continuously illuminated. The average light intensity measured at the position occupied by algal culture flasks was 6576 lux (SD: 78 lux), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell concentration was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Other: The cell morphology was examined and recorded at 24, 48 and 72 hours.

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 the WAF method (according to OECD Series on Testing and Assessment No. 23) as follows:
For preparation of the test solution, a supersaturated solution (100 mg/L nominal loading) was prepared by dispersing/dissolving an amount of 0.0407 g test item in 407 mL OECD medium and handled in ultrasonic bath for 10 minutes. This solution was mixed for a period of 24 hours to achieve an equilibrated concentration and then filtrated through a membrane filter (0.45 μm) to separate the possible non-dissolved test material. Untreated control ran parallel in the test.
Algal cells were exposed to the treatment group plus a control, for 72 hours. The test was performed with three replicates in the treatment group and in the control.
The concentration levels used and results (72 h) of the preliminary range-finding test are summarised in table 1 in the section "Any other information on results including tables".
Based on the results of the non-GLP Preliminary Range-Finding Test (see above), the main test was a limit test performed using the WAF method (according to OECD Series on Testing and Assessment No. 23) including a loading rate of 100 mg/L and a concurrent control group.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and Yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

other: Growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Basis for effect:

other: Growth rate and Yield

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: No abnormal shape of algal cells was noticed in the control and at the test item concentration of 0.1 mg/L (=LOD; nominal 100 mg/L) during the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72h ErC50: 0.83 mg/L, (95 % confidence limits: could not be calculated)
- 72h EyC50: 0.57 mg/L, (95 % confidence limits: 0.45 – 0.72 mg/L)

Table 1: Results of the Preliminary Range-finding Test

Test Group	Untreated control	WAF* (100 mg/L nominal)
Average of cell number at 72 hours (x 10^4cells/mL)	66.8	65.4
Growth Rates (μ) [0-72h] % Inhibition of μ [0-72h]	0.0583 -	0.0581 0.5
Yield (Y) [0-72h] % Inhibition of Y [0-72h]	65.8 -	64.4 2.0

* Water accommodated fraction

Validity of the Test
- The cell density in the control cultures increased by a factor of 58.00 within 72 hours. This corresponds to a specific growth rate of 1.35 day-1.
- The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %
- CV for section-by-section growth rate day 0-1: 24.79 %
- CV for section-by-section growth rate day 1-2: 14.08 %
- CV for section-by-section growth rate day 2-3: 5.28 %
The mean coefficient of variation for section-by-section specific growth rates: 14.72 %.
- The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 3.57 %.
All validity criteria were met, therefore the study can be considered as valid.

Analytical Results

One test concentration at saturation (equivalent to 100 mg/L nominal concentration) and a concurrent control was tested in the main experiment. The concentration of the test item was analytically determined at the start and at the end of the test. Measured test item concentration was < LOD (Limit of Detection) over the test period of 72 hours. Therefore the biological results are based on the nominal and on the analytical method’s LOD, according to ECHA Guidance 2017, Chapter R.7b.

Table 2: Concentrations measured during the study

Nominal concentration (mg/L)	Start (May 14, 2019)		End (May 17, 2019)
	Mean of the measured concentrations (mg/L) (n=6)	Measured concentration in % of the nominal	Mean of the measured concentrations (mg/L) (n=6)	Measured concentration in % of the nominal
100	<LOD	-	<LOD	-
Control	<LOD	-	<LOD	-

LOD: 0.1 mg/L

Validity criteria fulfilled:

yes

Conclusions:

In this 72-hour growth inhibition study on algae (Raphidocelis subcapitata), the obtained results showed that the test item had not any significant toxic effects on the test system.
The 72-h NOEC was determined to be 0.1 mg/L (Limit of detection, nominal 100 mg/L).
The 72-h LOEC was determined to be > 0.1 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 2017.

Executive summary:

The effect of the test item Blue Sema on the growth of a unicellular green algal species was determined in a growth inhibition study according to OECD guideline 201 and EU Method C.3.
Exponentially growing cultures of Raphidocelis subcapitata were exposed to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration) in a limit test over a period of 72 hours. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. For determination of the test item concentration, samples were taken from the test item treated group and control at the start and end of the test. The test item concentration was determined using UV/VIS spectrophotometric method. Measured test item concentration was < LOD (Limit of Detection) over the test period of 72 hours, therefore the biological results are based on the nominal test concentration and the LOD, in line with ECHA 2017.
The results obtained in this study showed that the test item Blue Sema had not any significant toxic effects on the test system. The 72-h NOEC was determined to be 0.1 mg/L (Limit of detection, nominal 100 mg/L). The 72-h LOEC was determined to be > 0.1 mg/L (Limit of detection, nominal > 100 mg/L). All validity criteria were met.

Description of key information

In this 72-hour growth inhibition study on algae (Raphidocelis subcapitata), the obtained results showed that the test item had not any significant toxic effects on the test system.

The 72-h NOEC was determined to be 0.1 mg/L (Limit of detection, nominal 100 mg/L).

The 72-h LOEC was determined to be > 0.1 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 2017.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.1 mg/L

Additional information

The effect of the test item Blue Sema on the growth of a unicellular green algal species was determined in a growth inhibition study according to OECD guideline 201 and EU Method C.3.
Exponentially growing cultures of Raphidocelis subcapitata were exposed to the saturation concentration of the test item (equivalent to 100 mg/L nominal concentration) in a limit test over a period of 72 hours. Because of low solubility of the test item, test solution was prepared by utilizing the water accommodated fraction (WAF) approach. For determination of the test item concentration, samples were taken from the test item treated group and control at the start and end of the test. The test item concentration was determined using UV/VIS spectrophotometric method. Measured test item concentration was < LOD (Limit of Detection) over the test period of 72 hours, therefore the biological results are based on the nominal test concentration and the LOD, in line with ECHA 2017.
The results obtained in this study showed that the test item Blue Sema had not any significant toxic effects on the test system. The 72-h NOEC was determined to be 0.1 mg/L (Limit of detection, nominal 100 mg/L). The 72-h LOEC was determined to be > 0.1 mg/L (Limit of detection, nominal > 100 mg/L). All validity criteria were met.