Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

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

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:

Adopted: 22 July 2010

Deviations:

yes

Remarks:

Deviation to SOP 00144 V.1 the test was performed on base of the new OECD 209 (2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

Deviation to SOP 00144 V.1 the test was performed on base of the new OECD 209 (2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

not specified

Details on test solutions:

Pre-treatment of test item and reference compound without ATU
Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 6.4-6.9. The pH was adjusted to pH 7.3-7.4 with NaOH.
For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.

Pre-treatment of test item with ATU
Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured and ranged between pH 6.3-6.4. The pH was adjusted to pH 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

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Origin: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
Date of collection: 2015-08-31
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 by dissolving the following amounts of substance per 1 litre of water.
16.0 g peptone
11.0 g meat extract
3.0 g urea
0.7 g NaCl
0.4 g CaCl2 x 2H2O
0.2 g MgSO4 x 7H2O
2.8 g K2HPO4
pH of the synthetic sewage feed: 7.5 ± 0.5

Test type:

not specified

Water media type:

other: deionised water

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Incubation time: 3 hours with permanent aeration

Post exposure observation period:

Stirring period of the test item before start of incubation time : 17 hours

Test temperature:

20 ± 2°C

pH:

6 - 8

Nominal and measured concentrations:

Nominal test item concentration of 100 mg/L

Details on test conditions:

Exposure conditions

Test item concentration/s : 100 mg/L, 3 replicates
100 mg/L with ATU (N-allylthiourea), 2 replicates

Control : 6 replicates
4 replicates with ATU (N-allylthiourea)

Test item concentration in physico-chemical oxygen consumption control : 100 mg/L

Concentration of reference compound 3,5-Dichlorophenol : 2.5, 5, 10, 20 and 40 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

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of respiration due to nitrification

Details on results:

EGGE 2806-1 showed 2.787 % respiration inhibition of activated sludge at the highest test item concentration of 100 mg/L.
EGGE 2806-1 showed no statistically significant difference of respiration inhibition of activated sludge between control and a limit test item concentration of 100 mg/L.

Reported statistics and error estimates:

STUDENT-t test for Homogeneous Variances
There is no statistically significant difference between Control and 100.000 mg/L.

The physico-chemical oxygen consumption has been determined at a test item concentration of 100 mg/L. As no physico-chemical oxygen consumption was observed at that test item concentration this observation also holds true for the lower test item concentrations.

The oxygen consumption in the presence of N-allylthiourea was determined in three 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. The temperature during the incubation was between 20.3 °C and 20.9 °C.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria of the test method were met

Conclusions:

EGGE 2806-1 showed 2.787 % 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.

Executive summary:

This study was performed to assess the toxicity of EGGE 2806-1 to bacteria. It was conducted in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010). This test method is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008).

The activated sludge was exposed to EGGE 2806-1 at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours.

EGGE 2806-1 showed 2.787 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L.

The EC50 is higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L.

The effect value relates to a nominal concentration, since no analytical monitoring was performed.

Description of key information

This study was performed to assess the toxicity of EGGE 2806-1 to bacteria. It was conducted in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010). This test method is in most essential parts equal to Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008).

The activated sludge was exposed to EGGE 2806-1 at a limit test item concentration of 100 mg/L. The respiration rate of each mixture was determined after aeration periods of 3 hours.

EGGE 2806-1 showed 2.787 % respiration inhibition of activated sludge at a test item concentration of 100 mg/L.

The EC50 is higher than 100 mg/L. The NOEC is equal or higher than 100 mg/L.

The effect value relates to a nominal concentration, since no analytical monitoring was performed.

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.