Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

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:

14 Novermber 2019

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:

The test sample was corrected for the purity of the test material.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTION
1204 mg of the test sample (1001 mg as the test material) was accurately weighed with an electronic analytical balance and dissolved in purified water in order to obtain 10.01 g/L stock solution of the test material (100 mL).

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Inoculum source: Activated sludge was obtained from an aeration tank of a sewage treatment plant treating predominantly domestic sewage in Kurumeshi, Fukuoka, Japan.
- Preparation of inoculum for exposure: The activated sludge (3 L) was fed with the synthetic sewage (150 mL) and cultivated for 18 hours at 20 ± 2 °C under aerobic condition in order to acclimate the test temperature. The activated sludge was then used. Acclimation to the test material was not performed.
- Concentration of suspended solids: 2480 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.5 ± 0.5

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel (ca. 300 mL)
- Fill voulme: 250 mL
- Aeration: Yes. Aerated with an air pump (controllable flow rate at 0.5–1 L/min)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 6

PREPARATION OF TEST SOLUTIONS
The test mixture was prepared by adding 25 mL of the test material solution (10.01 g/L) to 8 mL synthetic sewage, 92 mL purified water and 125 mL activated sludge in each test vessel.
The reference mixture was prepared by adding 1, 2, 4 or 8 mL of the reference substance solution (1000 mg/L) to 8 mL synthetic sewage, purified water (the volume subtracting the additive volume of the stock solution of 3,5-dichlorophenol from 117 mL) and 125 mL activated sludge in each test vessel.
The blank controls were prepared by adding 8 mL synthetic sewage to 117 mL purified water and 125 mL activated sludge in each vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: No. The pH of all the test solutions was measured and confirmed to be within 7.5 ± 0.5.

EFFECT PARAMETERS MEASURED
Dissolved oxygen concentration was measured using a dissolved oxygen analyser. After 3 hours of exposure, the measurement bottle was filled with the test solution promptly and softly with attention to avoid foaming. Afterwards, the concentration of dissolved oxygen (DO) in the test solution was measured continuously for 10 minutes with stirring. The test solutions (blank control) were measured in 3 replicates at the beginning and the end of the exposure, individually.

ESTIMATION OF EC50
The concentration of DO (mgO2/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 slope of the regression formula.
The percentage inhibition of respiration was then calculated by the following equation:
Percentage inhibition of respiration (%) = [ 1 - (RS / RC)] x 100
where:
RS : Oxygen consumption rate in test or reference mixture (mg O2/L/h)
RC : Average oxygen consumption rate in blank control (mg O2/L/h)

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes. The results suggested that EC50 of the test material was higher than 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 as nominal loading concentration.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (4.0, 8.0, 16 and 32 mg/L)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Preliminary Test
The percentages of respiration inhibition were -1%, 1% and 4% at test material concentrations of 10, 100 and 1000 mg/L, respectively.

Main Test
The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 4%. In the test mixture, the EC50 of the test material was estimated as " >1000 mg/L" because less than 50% of inhibition rate was obtained at the exposure level.

Results with reference substance (positive control):

In the reference mixture, the percentage inhibition in each exposure level was plotted on semi-logarithmic graph against the corresponding concentration. The EC50 of 3,5-dichlorophenol was calculated from the regression formula obtained by probit analysis. The EC50 was determined using computer program (running on Microsoft software "Excel") constructed by the Test Facility.
The EC50 of 3,5-dichlorophenol as reference item was 9.8 mg/L.

Summary of results on oxygen consumption rate and percentage inhibition of respiration after 3 hours of exposure

Test solution	Concentration (mg/L)	Oxygen consumption rate (mg O2/L/h)	Percentage inhibition of respiration (%)	Average percentage inhibition of respiration (%)
Test mixture	1000	55.3	7	4
	1000	56.8	5
	1000	59.3	1
Reference mixture	4.0	48.0	19	-
	8.0	36.1	39
	16	16.4	72
	32	7.07	88
Blank control	-	At the beginning: 57.1, 56.6, 58.2 At the end: 61.0, 62.2, 62.5	-	-

 Average oxygen consumption rate in blank control: 59.6 mg O2/L/h

Validity of the test

The test was valid because the test results met the following criteria:

a) The EC50 of 3,5-dichlorophenol as reference item was 9.8 mg/L, and this value met the criterion (2–25 mg/L).

b) The coefficient of variation of the oxygen consumption rate in the blank controls between at the beginning and the end of the exposure was 5.5%, and this value met the criterion (less than 30%).

c) The oxygen consumption rate per gram (dry weight) of activated sludge per hour in the blank controls was average 48.1 mgO2/g/h, and this value met the criterion (≥20 mg O2/g/h).

Validity criteria fulfilled:

yes

Conclusions:

The test material showed 4 % respiration inhibition of activated sludge at a test material concentration of 1000 mg/L. The 3-hour EC50 value was therefore determined to be > 1000 mg/L.

Executive summary:

The toxicity of the test material to aquatic mircoorganisms was investigated in a study which was conducted in accordance with the standardised guideline OECD 209 and under GLP conditions.

During the study, activated sludge from a sewage plant (treating predominantly domestic sewage) was exposed to the test material (1000 mg/L), blank control solution and solution with positive reference substance (3,5-dichlorophenol at 4.0, 8.0, 16 and 32 mg/L) for a period of 3 hours.

Dissolved oxygen was recorded and respiration rates were calculated.

Under the conditions of the study the test material showed 4 % respiration inhibition of activated sludge at a test material concentration of 1000 mg/L. The 3-hour EC50 value was therefore determined to be > 1000 mg/L. All of the validity criteria were fulfilled.

Description of key information

The test material showed 4 % respiration inhibition of activated sludge at a test material concentration of 1000 mg/L. The 3-hour EC50 value was therefore determined to be > 1000 mg/L.

Additional information

The toxicity of the test material to aquatic mircoorganisms was investigated in a study which was conducted in accordance with the standardised guideline OECD 209 and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, activated sludge from a sewage plant (treating predominantly domestic sewage) was exposed to the test material (1000 mg/L), blank control solution and solution with positive reference substance (3,5-dichlorophenol at 4.0, 8.0, 16 and 32 mg/L) for a period of 3 hours.

Dissolved oxygen was recorded and respiration rates were calculated.

Under the conditions of the study the test material showed 4 % respiration inhibition of activated sludge at a test material concentration of 1000 mg/L. The 3-hour EC50 value was therefore determined to be > 1000 mg/L. All of the validity criteria were fulfilled.