Methyl 2-[(1,5-dihydro-3-methyl-5-oxo-1-phenyl-4H-pyrazol-4-ylidene)ethylidene]-1,3,3-trimethylindoline-5-carboxylate

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:

2017

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))

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

I. Pre-treatment of test item without ATU:
- Method of application: direct weighing
- Stirring period of the test item before start of incubation time: 16 hours
- pH was measured and ranged between pH 6.2 - 6.3; pH was adjusted to pH 7.2 -7.4 with NaOH

II. Pre-treatment of the reference compound:
- Method of application: stock solution (500 mg/L prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluted to 0.5 litre with deionised water)
- pH was adjusted to pH 7 - 8 with HCl

III. Pre-treatment of test item with ATU
- Method of application: direct weighing
- Stirring period of the test item before start of incubation time: 16 hours
- pH was measured and ranged between pH 6.4 - 6.5; pH was adjusted to pH 7.3 - 7.4 with NaOH

- For the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all
replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period

- To determine the heterotrophic oxidation four additional controls and two replicates with the test item concentration 100 mg/L, all containing 1.25
mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L, were prepared.

- Vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no
activated sludge.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Type: Mixed population of aquatic microorganisms (activated sludge)
- Origin: Aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Date of collection: 2017-02-06
- Microbial inoculum: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Storage of sludge: Aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.8
- Synthetic sewage feed: A synthetic waste water feed was prepared
- pH of the synthetic sewage feed: 7.5 ± 0.5

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

No data

Test temperature:

18.7 - 19.3 °C

pH:

8.2 - 8.4

Nominal and measured concentrations:

- Test item concentration (nominal): 100 mg/L
- reference substance concentrations (nominal): 2.5, 5, 10, 20 and 40 mg/L

Details on test conditions:

Exposure conditions:
- Test item concentration: 100 mg/L, 3 replicates; 100 mg/L with ATU (N-allylthiourea), 2 replicates
- Control: 6 replicates; 4 replicates with ATU
- Test item concentration in physico-chemical oxygen consumption control: 100 mg/L
- Test vessels: 300 mL glass Erlenmeyer flasks
- Method of application: direct weighing
- Test concentration of the activated sludge: 800 mg/L suspended solids
- Test temperature: 20 ± 2°C
- Stirring period of the test item before start of incubation time: 16 hours
- Incubation time: 3 hours with permanent aeration

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Purity: 99.9%; Acros Organics, batch no. A0357150)

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Macrolex Orange R showed 0.243 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L. Thus, Macrolex Orange R showed no statistical significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L.
As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50.

Results with reference substance (positive control):

In the present test the EC50 of the reference compound 3,5-Dichlorophenol was determined as 13.81 mg/L using Probit-analysis.

The oxygen consumption in the presence of N-allylthiourea was determined in four controls without test item and in two replicates of the test item concentration 100 mg/L. As no inhibition was observed for the total oxygen consumption at 100 mg/L no differences between the heterotrophic and the nitrification oxygen uptake rates have to be calculated.

Validity criteria fulfilled:

yes

Remarks:

oxygen uptake rate >=20 mg oxygen per gram of activated sludge in an hour; coefficient of variation of oxygen uptake in the control replicates <= 30 % at the end of the test; EC50 of reference compound between 2 – 25 mg/L for total respiration

Conclusions:

Macrolex Orange R showed 0.243 % respiration inhibition of activated sludge at the highest test concentration of 100 mg/L after 3 hours. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Executive summary:

According to OECD Guideline 209 the activated sludge was exposed to a limit concentration of Macrolex Orange R of 100 mg/L. After 3 hours of exposure the substance showed 0.243 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

To measure the oxygen consumption, 250 mL of sludge with the test item (or control or reference compound) was incubated for 3 h in 300 mL closed Erlenmeyer flasks (with air inlet and outlet) and aerated through a glass tube at 50-100 L/h with clean oil-free air. For the measurement, the content of the Erlenmeyer flasks was completely transferred to 250 mL BOD bottles and oxygen content was measured with an oxygen meter (redox electrode). Six controls without the test item were included in the test design, three at the start and the others at the end of the test series.

The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated. The oxygen uptake rates of four additional controls and two replicates of the test item concentration 100 mg/L, all in the presence of N-allylthiourea, were prepared and measured after the exposure period. These values represent the heterotrophic respiration. As no significant inhibitory effect was measured at a limit test item concentration of 100 mg/L no statistical analysis was required to determine the EC50. The No Observed Effect Concentration was calculated according to STUDENT-t test for Homogeneous Variances using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

The EC50 of the reference compound 3,5-Dichlorophenol was 13.81 mg/L.

Description of key information

According to OECD Guideline 209 the activated sludge was exposed to a limit concentration of Macrolex Orange R of 100 mg/L. After 3 hours of exposure the substance showed 0.243 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L. The EC50 is higher than 100 mg/L and the NOEC is equal or higher than 100 mg/L. The effect value relates to a nominal concentration, since no analytical monitoring was performed.

The EC50 of the reference compound 3,5-Dichlorophenol was 13.81 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

"Should read: > 100 mg/L (EC50), >= 100 mg/L (NOEC)"