4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012-06-14 to 2012-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Orange 13, 34 and Pigment Red 38 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was weighed out directly in an Erlenmeyer flask, filled up with 200 mL demieralised water and stirred overnight. Afterwards the pH of the additional concentration was adjusted to 7.5 +/- 0.5.
- Eluate: Test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
- Differential loading: 560 - 750 - 1000 mg/L, additionally: 1000 mg/L (pH adjusted)
- Vehicle: none
- Replicates: triplicates
The test item was weighed out directly in an Erlenmeyer flask, filled up with 200 mL demineralized water and stirred overnight.
Afterwards the pH of the additional concentration was adjusted to 7.5 +/- 0.5.

Controls: Test medium without test or reference item.
- Replicates: Triplicates at the beginning of the test + triplicates at the end of the test

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)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 3 g/L +/- 10 %. The sludge was used within 24 h after stirring.
- Dry sludge concentration: 2.91 g/L corresponding to 1.46 g/L suspended solids in the test vessels

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.2 °C

pH:

7.45 (activated sludge)
7.44 (synthetic waste water)

Salinity:

None, freshwater

Nominal and measured concentrations:

560 - 750 - 1000 mg/L, additionally: 1000 mg/L (pH adjusted)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3 at the beginning, 3 at the end of the test
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL autoclaved tap water
Test or reference item concentration (directly weighed into each test vessel)


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
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item Concentration
[mg/L] Pretreatment pH(test item in water) pH(test item in medium) Inhibition

[%]
10 Stirring overnight - - -6
100 Stirring overnight - - 2
1000 Stirring overnight - - 38
1000 Stirring overnight (6.00) 7.85* 7.42 1
1000 Stirring overnight 6.04 7.42 2
1000 Stirring overnight 6.02 7.48 11
1000 Stirring overnight (5.95) 7.12* 7.53 4
1000 Stirring overnight (5.96) 7.83* 7.60 -1

* adjusted with 1 M NaOH

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 91.2 mg/L

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) test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

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

Test Item / Reference Item Concentration [mg/L]		Repl.	pH*	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control		1		25.2	17.3	–	–
		2		27.6	18.9
		3		26.4	18.1
		4		23.2	15.9
		5		23.6	16.2
		6		23.6	16.2
Test Item	560	1		27.6		-11	-5
		2		26.0		-4
		3		25.2		-1
	750	1		26.4		-6	-2
		2		26.0		-4
		3		23.6		5
	1000	1		21.6		13	1
		2		24.8		0
		3		27.2		-9
	1000 (pH adjusted)	1	7.74	28.0		-12	-6
		2	7.80	25.6		-3
		3	7.57	26.0		-4
Reference Item	58	1		18.0		28	31
		2		16.8		33
		3		16.8		33
	100	1		12.0		52	53
		2		12.0		52
		3		11.2		55
	180	1		5.6		78	79
		2		5.2		79
		3		4.8		81

Repl. = Replicate

* adjusted with 0.1 M NaOH

Mean specific respiration rate of the control: 17.1 mg O2/g•h

Coefficient of variation of oxygen uptake rates of the control: 7.1 %

Suspended solids in the test vessel: 1.46 g/L

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item is 1000 mg/L, the EC50 of the test item is > 1000 mg/L.
The test item is not toxic up to the concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out for the test item compliant to GLP (reliability category 1). Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with test item concentrations of 560 - 750 - 1000 mg/L in a geometrical series with a dilution factor of 1.33 and additionally with the highest test item concentration 1000 mg/L, adjusted to a pH value of 7.5 ± 0.5. 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 -6 % to 1 %.

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 91.2 mg/L.

NOEC-Value of Test Item

	Test Item Concentration [mg/L]
NOEC*	1000

*) no test item related statistically significant inhibition (p 0.05), ANOVA

The NOEC of the test item is 1000 mg/L, the EC50 of the test item is > 1000 mg/L. The test item is not toxic up to the concentration of 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Description of key information

One toxicity to microorganism studies of reliability 1 for Pigment Red 38 is available.
Under the conditions of this test the NOEC of the submission substance after 3 hours was 1000 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water (2.2 μg/L).

For all three pigments of the Diazopyrazolone group consistently no aquatic toxicity has been found up to nominal concentrations well above the solubility limit. Therefore it is justified to use the data for all members of the group.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L