C.I. Reactive Yellow 2

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test material was stored in a dark storage place at room temperature.
- Stability under storage conditions: stable.
- Solubility in dimethylsulfoxide: ≤ 439 g/L
- Solubility in acetone: ≤ 0.03 g/L
- Water solubility: ≥ 10 g/L

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF STOCK SOLUTIONS
- Stock solution of the test material: the test material (1383 mg) was accurately weighed with an electronic analytical balance and dissolved in purified water to obtain a 10.0 g/L stock solution.
- Stock solution of reference material (3,5-dichlorophenol): The reference material (100 mg) was accurately weighed with an electronic analytical balance and dissolved in approximately 80 mL of purified water. The pH of this solution was adjusted to 7.5 with 1 mol/L sodium hydroxide solution. The solution was filled up to 100 mL with purified water to obtain a 1000 mg/L stock solution.

PREPARATION OF TEST SOLUTIONS
- The pH of all test solutions was measured and confirmed to be within 7.5 ± 0.5.
- Text mixture (3 replicates): the 10.0 g /L stock solution of the test material (25 mL), the synthetic sewage (8 mL), purified water (02 mL) and the activated sludge (125 mL) were added to each vessel.
- Reference mixture (1 replicate/exposure level): the 1000 mg/L stock solution of the reference material (1, 2, 4 and 8 mL), the synthetic sewage (8 mL), purified water (the volume resulting from subtracting the additive volume of the stock solution of the reference material from 117 mL) and the activated sludge (125 mL) were added to each test vessel.
- Blank control (6 replicates): the synthetic sewage (8 mL), purified water (117 mL) and the activated sludge (125 mL) were added to each test vessel.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Sewage treatment plant where inoculum was collected: the activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurume-shi, Fukuoka, Japan.
- Concentration of suspended solid: 3115 mg/L
- The activated sludge (3 L) was fed with synthetic sewage (150 mL) and cultivated for 21.5 hours at 20 ± 2 °C under aerobic conditions in order to acclimate the test temperature. The activated sludge was then used.
- Preparation of synthetic sewage: the synthetic sewage (500 mL) was prepared from the following reagents, dissolved in 1 L of purified water: peptone (16 g), meat extract (11 g), urea (3 g), sodium chloride (0.7 g), calcium chloride (0.4 g), magnesium sulphate heptahydrate (0.2 g), and dipotassium hydrogen phosphate (2.8 g). The pH was then adjusted to 7.5 with 1 mol/L sodium hydroxide solution. The synthetic sewage was prepared the day before the test and was stored in a refigerator until use.

Test type:

not specified

Water media type:

not specified

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.5

Nominal and measured concentrations:

Nominal concnetrationof the test mixture: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Aerator: air pump (controllable flow rate at 0.5-1 L/min).
- Test vessel: 300 mL glass vessel.
- Volume of test solution: 250 mL
- Aeration rate: 0.5-1 L/min
- Replicate: 3 replicates/exposure level and 6 replicates/control.

OTHER TEST CONDITIONS
- Adjustment of pH: the pH was adjusted to 7.5 with 1 mol/L sodium hydroxide solution.

EFFECT PARAMETER MEASURED:
- Measurement of dissolved oxygen concentration (using a dissolved oxygen analyser) to calculate oxygen consumption rate.

MEASUREMENT OF DISSOLVED OXYGEN CONCENTRATION
- Instrument: dissolved oxygen analyser (No. DO-016).
- Method: after 3 hours of exposure the measurement bottle was filled with the test solution promptly and softly to avoid foaming. The concentration of dissolved oxygen (DO) in the test solution was measured for 10 minutes with stirring. The test solutions (blank control) were measured in 3 replicates at the beginning and at the end of exposure, individually.

CALCULATION OF PERCENTAGE INHIBITION OF RESPIRATION
- The concentration of DO (mg O2/L) was plotted against the measuring time (h) for each test solution, and a regression formula was derived from the linear part in the graph. The oxygen consumption rate (mg O2/L/h) was obtained from the absolute value of the slope of the regression formula.
- The percentage inhibition was calculated using the following equation:
Percentage inhibition of respiration (%) = [1 – (RS/RC)] x 100
- Where RS is the oxygen consumption rate in test or reference mixture (mg O2/L/h) and RC is the average oxygen consumption rate in the blank control (mgO2/L/h).

ESTIMATION OF EC50 IN THE REFERENCE MIXTURE
- The percentage inhibition in each exposure level was plotted on a semi-logarithmic graph against the corresponding concentration. The EC50 of 3,5-dicholophenol was calculated from the regression formula obtained by probit analysis. The EC50 was determined using a computer programme constructed by the laboratory.

TEST CONCENTRATIONS
- Range finding study yes (in the preliminary test, three test concentration solutions of the test material at 10, 100 and 1000 mg/L were prepared. The percentages of inhibition were -2 %, -2 % and 3 % respectively. The results suggested that the EC50 of the test material was > 1000 mg/L therefore the test mixture was prepared as a limit test and the test concentration of the test material was carried out at 1000 mg/L)
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5% therefore the EC50 of the test material was estimated as > 1000 mg/L.

Results with reference substance (positive control):

The EC50 of the reference material (3,5-dichlorophenol) was 7.8 mg/L and therefore met the criterion of 2-25 mg/L.

Oxygen consumption rate and percentage inhibition of respiration after 3 hours of exposure

Test solution	Concentration (mg/L)	Oxygen consumption rate (mgO2/L/h)	Percentage inhibition of respiration (%)	Average percentage inhibition of respiration (%)
Test mixture	1000	53.4	4	5
	1000	54.6	1
	1000	50.7	8
Reference mixture	4.0	39.1	29	-
	8.0	28.5	49	-
	16	13.5	76	-
	32	8.08	85	-
Blank control	-	At the beginning: 55.1, 54.5, 54.5. At the end: 55.8, 56.2, 56.0.	-	-

Average oxygen consumption rate in the blank control: 55.4 mg O2/L/h

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study the EC50 of the test material was estimated as > 1000 mg/L.

Executive summary:

The 3-hour median effective concentration, EC50, of the test material was determined by conducting an activated sludge respiration inhibition test. This was investigated in accordance with the standardised guidelines OECD 209, under GLP conditions.

The activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage. In the preliminary test, three test concentration solutions of the test material at 10, 100 and 1000 mg/L were prepared. The percentages of inhibition were -2 %, -2 % and 3 % respectively. The results suggested that the EC50 of the test material was > 1000 mg/L therefore the test mixture was prepared as a limit test and the test concentration of the test material was carried out at 1000 mg/L. The test solutions were prepared, 3 replicates per exposure level and 6 replicates per control. The volume of the test solution was 250 mL and the test temperature was 20 ± 2 °C. The dissolved oxygen concentration was measured by a dissolved oxygen analyser. After 3 hours of exposure the measurement bottle was filled with the test solution promptly and softly to avoid foaming. Afterwards the concentration of dissolved oxygen in the test solution was measured for 10 minutes with stirring. The test solutions (blank control) were measured in 3 replicates at the beginning and at the end of the exposure, individually. The test was valid as the test results met the criteria. The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5%. Therefore, under the conditions of the study the EC50 of the test material was estimated as > 1000 mg/L.

Description of key information

Under the conditions of the study the EC50 of the test material was estimated as > 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The 3-hour median effective concentration, EC50, of the test material was determined by conducting an activated sludge respiration inhibition test. This was investigated in accordance with the standardised guidelines OECD 209, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage. In the preliminary test, three test concentration solutions of the test material at 10, 100 and 1000 mg/L were prepared. The percentages of inhibition were -2 %, -2 % and 3 % respectively. The results suggested that the EC50 of the test material was > 1000 mg/L therefore the test mixture was prepared as a limit test and the test concentration of the test material was carried out at 1000 mg/L. The test solutions were prepared, 3 replicates per exposure level and 6 replicates per control. The volume of the test solution was 250 mL and the test temperature was 20 ± 2 °C. The dissolved oxygen concentration was measured by a dissolved oxygen analyser. After 3 hours of exposure the measurement bottle was filled with the test solution promptly and softly to avoid foaming. Afterwards the concentration of dissolved oxygen in the test solution was measured for 10 minutes with stirring. The test solutions (blank control) were measured in 3 replicates at the beginning and at the end of the exposure, individually. The test was valid as the test results met the criteria. The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5%. Therefore, under the conditions of the study the EC50 of the test material was estimated as > 1000 mg/L.