{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}{[(2,4 and 2,5 and 2,6)-dimethylphenyl and 2-ethylphenyl]amino}methaniminium 3-[(4-anilinophenyl)diazenyl]benzenesulfonate

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:

From 2017-04-18 to 2017-04-19

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:407940
- Expiration date of the lot/batch: 2024

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: rome temperature, protected from light in the tightly closed original container
- Stability under test conditions: stable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Pretreatment of test material prior to testing: stirring overnight

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition: the appropriate quantity of test item is weighed out in Erlenmeyer flasks containing synthetic waste water and demineralised water and stirred overnight
- Controls: test medium without test item or reference item.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant of 31137 Hildesheim, Germany.
- Reason for the selection: non-adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste
- Pretreatment: Sludge washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling (activated sludge received the 2017-04-08)

Test type:

static

Water media type:

other: demineralised water

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Test temperature = nominal 18-22°C (actual = 20.2°C).

pH:

pH-value of the activated sludge = 7.59
pH-value of the synthetic waste water = 7.31

Adjustment was not necessary.

Dissolved oxygen:

dissolved oxygen keep above 60-70% saturation

Nominal and measured concentrations:

Nominal concentration: geometrical series of 4, 16, 64, 250 and 1000 mg/L (dilution factor of 4)

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flasks, DIN 12380
- Material, size, headspace, fill volume: test vessel filled with 500 mL
- Aeration: permanent
- No. of vessels per concentration: 5 replicates
- No. of vessels per blanck control : 3 replicates at the beginning of the test and 3 replicated at the end of the test
- No. of vessels per positive control : 3 replicates
- Sludge concentration (weight of dry solids per volume): 3.12 g/L (i.e. 1.56 g/test vessel)
- Nutrients provided for bacteria: synthetic waster water according to OCDE Guideline 209

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: demineralized water.

OTHER TEST CONDITIONS
- Adjustment of pH: not necessary


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
TEST CONCENTRATIONS
- Spacing factor for test concentrations: factor of 4
- Range finding study: yes at 10 mg/L, 100 mg/L and 1000 mg/L of test item with 2 replicates per concentration. 2 additional replicates added to determine the inhibition of nitrification, and 2 other replicates were measured with and without allylthiourea (ATU)
- Test concentrations: 4 mg/L, 16 mg/L, 64 mg/L, 250 mg/L and 1000 mg/L of test item
- Results used to determine the conditions for the definitive study:
* 10 mg/L, repli. 1: inhibition of -6 %
* 10 mg/L, repli. 2: inhibition of 13%
* 100 mg/L, repli. 1: inhibition of 43%
* 100 mg/L, repli. 2: inhibition of 47%
* 1 000 mg/L, repli. 1: inhibition of 60%
* 1 000 mg/L, repli. 2: inhibition of 60%

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a. CAS 7758-99-8

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 115 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: confidence interval

Remarks:

103-127 mg/L

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: no statistically significant inhibition (P < 0.001)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 50.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: confidence interval

Remarks:

46.0-53.8 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

ca. 62.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: confidence interval

Remarks:

60.0-65.4 mg/L

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: confidence interval

Remarks:

-

Details on results:

- Blank controls oxygen uptake rate:
* replicate 1 = 35.2 mg/O2g.h
* replicate 2 = 34.0 mg/O2g.h
* replicate 3 = 34.8 mg/O2g.h
* replicate 4 = 34.4 mg/O2g.h
* replicate 5 = 34.8 mg/O2g.h
* replicate 6 = 34.8 mg/O2g.h
- Blank control of the mean specific oxygen uptake rate = 22.2 mg/O2g.h
- Coefficient of variation of oxygen uptake rate in control replicates: 1.2%

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50 = 109 mg/L (in the range of 53 - 115 mg/L).

Reported statistics and error estimates:

Normality test (Shapiro-Wilk) performed with α=0.05. P=0.62.
One Way ANOVA performed with α=0.05. P<0.001.
Multiple comparison versus control group (Dunnett’s method).
Values are statistically different between control and treatments at 64, 256 and 1000 mg/L of Yellow MGF (P<0.05)

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 16 mg/L and the EC50 is 115 mg/L

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Sepisolfast yellow MG-F (batch number:407940) from 2017-04-18 to 2017-04-19, with the definitive exposure phase on 2017-04-19 at the test facility.

The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 4 - 16 - 64 - 256 - 1000 mg/L.

The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -2 % to 57 %.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 109 mg/L.

Table 1 : NOEC and EC-Values with Confidence Interval of Sepisol fast yellow MG-F after 3 h exposure time, Inhibition of Total Respiration

	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	16	̶
EC10	50.2	46.0 – 53.8
EC20	62.8	60.0 – 65.4
EC50	115	103 – 127
EC80	> 1000	-

*) No statistically significant inhibition (P < 0.001), ANOVA, Dunnett`s Method

Conclusion: the NOEC of Sepisol fast yellow MG-F is 16 mg/L and the EC₅₀ is 115 mg/L.

Description of key information

Activated sludge, Respiration Inhibition test: EC₅₀ (3h): 115 mg/L corrected to 75 mg/L (water solubility) (OECD TG 209; static).

Key value for chemical safety assessment

EC50 for microorganisms:

75 mg/L

EC10 or NOEC for microorganisms:

16 mg/L

Additional information

The EC50 (3 hours) was 3 mg/L with 95% confidence limits of 103 -127 mg/L. The inhibition range from 2% to 57%. However, as the substance has a water solubility 75 mg/L, the EC50 for the test substance has been considered to be 75 mg/L.

Other data (non corrected):

The EC10 (3 hours) was 50.2 mg/L (measured concentrationx) with 95% confidence limites of 46 -53.8 mg/L.

The NOEC (3 hours) was 16 mg/L.