4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-10-27 to 2016-10-28

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1000 mg/L was weighed out directly in Erlenmeyer flasks and stirred overnight.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1000 mg/L
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8., Triplicates

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2016-10-27
- Pretreatment: The sludge was 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.
- Dry sludge concentration: 2.92 g/L, corresponding to 1.46 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.8 °C

pH:

pH-value of the activated sludge 7.36
pH-value of the synthetic waste water 7.33

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 3
- 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Shaking aeration, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.
- Pretreatment: Stirring overnight

- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8,at the end of the test)
- Composition of test medium:

Test concentration [mg/L] 1000

Test Item [mg] 500

Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250



Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flat bed recorder.

TEST CONCENTRATIONS
Inhibition of Respiration (Preliminary Tests)

Inhibitions in the Preliminary Tests
A preliminary test (non-GLP) was carried out with the following test item concentrations of Disperse Orange 29:
1000 mg/L with two replicates. The pH was not adjusted.


Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH

(test item in medium) Inhibition


[%]
1st preliminary test - without pH adjustment, direct aeration
1000 7.62 1
- -1


Based on the results of the preliminary test it can be concluded that the heterotrophic respiration as well as the nitrification are not inhibited. Therefore, the definitive study was carried out with the concentration of 1000 mg/L in 3 replicates (limit test to determine the NOEC). The concentration was weighed out directly in the Erlenmeyer flasks.

Triplicates of the control without test item were included at the beginning and at the end of the test.

A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.

The reference item was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 948 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The P-value (acceptable probability of incorrectly concluding that there is a difference) isP=0.05.

The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h.

The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration of the Control and Test Item and Reference Item Concentrations

Test and Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	31.2	-	-
	2	31.6
	3	31.6
	4	28.8
	5	30.0
	6	30.8
Test item 1000	1	29.2	5	1
	2	32.0	-4
	3	30.4	1
Reference item 58	1	24.0	22	23
	2	22.8	26
	3	24.0	22
100	1	18.0	41	43
	2	17.6	43
	3	17.2	44
180	1	8.0	74	74
	2	8.0	74
	3	8.4	73

Repl. = Replicate

Coefficient of variation of oxygen uptake rates of the control: 3.6%

Suspended solids in the test vessel: 1.46 g/L

Specific Respiration Rates of the Control Replicates

		Repl.	Specific Respiration Rate Rs [mg O2/g×h]
	Control	1	21.4
		2	21.6
		3	21.6
		4	19.7
		5	20.5
		6	21.1
Mean value			21.0

Repl. = Replicate

Suspended solids in the test vessel: 1.46 g/L

 

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the nominal 3 h NOEC of the test substance was ≥1000 mg/L (equivalent to ≥948 mg a.i./L).

Executive summary:

A respiration inhibition test with activated sludge was conducted with the test substance (in the form of a dark brown solid of 94.8% purity) according to OECD Guideline 209. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The study was carried out under static conditions at a single limit concentration of 1000 mg/L for a duration of 3 h. The respiration rates of the control, reference and test substance 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 substance replicates was 1%. No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test. 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 110 mg/L. The validity criteria of the test guideline were fulfilled. Under the study conditions, the nominal 3 h NOEC of the test substance was ≥1000 mg/L (equivalent to ≥948 mg a.i./L) (Maischak, 2016).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

not specified

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture:
- Name and location of sewage treatment plant where inoculum was collected: BS wastewater treament plant Sindlingen, Germany
- Initial biomass concentration: 2 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

22.1 - 23.8

pH:

6.9-7.1

Nominal and measured concentrations:

Nominal: 0, 250, 500 and 1000 mg/L, equivalent to 0, 109, 219 and 437 mg a.i./L

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

793.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

346.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Details on results:

The EC10 was evaluated using the function "Y = ae EXP bx". Evaluation using exponential regression.

Results with reference substance (positive control):

Not reported.

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the nominal 3 h EC10was 793.7 mg/L, i.e. 346.8 mg a.i./L.

Executive summary:

A study was conducted to determine the toxicity of the test substance (in the form of a water soluble dispersion of 43.7% purity) to wastewater sludge bacteria according to OECD Guideline 209 and EU Method C.11. Sludge at the equivalent of 2 g dry mass/L was exposed to nominal concentrations of 0, 250, 500 and 1000 mg/L, equivalent to 0, 109, 219 and 437 mg a.i./L for 3 h. The positive reference 3,5 -dichlorophenol was run in parallel at 5 and 30 ppm. Temperature, pH and oxygen inhibition were recorded at test end. Under the study conditions, the nominal 3 h EC10 was 793.7 mg/L, i.e. 346.8 mg a.i./L (Voelskow, 1989).

Description of key information

Value used for CSA:

EC10, LC10 or NOEC for microorganisms: ≥948 mg a.i./L

Additional information

A respiration inhibition test with activated sludge was conducted with the test substance (in the form of a dark brown solid of 94.8% purity) according to OECD Guideline 209. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The study was carried out under static conditions at a single limit concentration of 1000 mg/L for a duration of 3 h. The respiration rates of the control, reference and test substance 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 substance replicates was 1%. No nitrification inhibition was observed at 1000 mg/L in the preliminary test and this endpoint was therefore not evaluated in the definitive test. 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 110 mg/L. The validity criteria of the test guideline were fulfilled. Under the study conditions, the nominal 3 h NOEC of the test substance was ≥1000 mg/L (equivalent to ≥948 mg a.i./L) (Maischak, 2016).

A study was conducted to determine the toxicity of the test substance (in the form of a water soluble dispersion of 43.7% purity) to wastewater sludge bacteria according to OECD Guideline 209 and EU Method C.11. Sludge at the equivalent of 2 g dry mass/L was exposed to nominal concentrations of 0, 250, 500 and 1000 mg/L, equivalent to 0, 109, 219 and 437 mg a.i./L for 3 h. The positive reference 3,5 -dichlorophenol was run in parallel at 5 and 30 ppm. Temperature, pH and oxygen inhibition were recorded at test end. Under the study conditions, the nominal 3 h EC10 was 793.7 mg/L, i.e. 346.8 mg a.i./L (Voelskow, 1989).

For risk assessment purposes, the results from the more recent study using purer test substance (Maischak, 2016) are taken into account.