Reaction mass of 2,6-Bis[(dimethylamino)methyl]-4-(1-{3-[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)phenol and 4-(1-{3,5-Bis[(dimethylamino)methyl]-4-hydroxyphenyl}-1-methylethyl)-2,6-bis[(dimethylamino)methyl]phenol

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))

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

Remarks:

test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations

Vehicle:

no

Details on test solutions:

Pre-treatment of test item and reference compound
- direct weighings were prepared
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours
- pH was measured and ranged between pH 8.8 - 10.6
- pH was adjusted to pH 7.2 - 7.4 with HCl

Pre-treatment of reference compound
- dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water
- pH was adjusted to pH 7 - 8 with HCl

Pre-treatment of test item with ATU
- direct weighings were prepared
- test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours
- pH was measured and ranged between pH 8.8 - 10.8 and was adjusted to pH 7.2 – 7.4 with HCl
- ATU solution: 2.32 g/L N-allylthiourea were weighed out and diluted with deionized water to 1 litre; 1.25 mL of the solution was given to all replicates for the determination of the heterotrophic oxidation before start of the incubation period

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: 2016-08-22
- Storage of sludge: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- pH of the suspension before application: 7.6
- Synthetic sewage feed : A synthetic waste water feed was prepared
- pH of the synthetic sewage feed : 7.5 ± 0.5

Pre-treatment of 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
- approx. 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

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20.4 - 21.0 °C

pH:

8.1 - 8.2, Pysico-chemical oxygen consumption control: 7.8

Nominal and measured concentrations:

test item: 10, 32, 100, 320, 1000 mg/L, 10, 32, 100, 320 and 1000 mg/L with ATU
3,5-Dichlorophenol : 2.5, 5, 10, 20 and 40 mg/L; 2.5, 5, 10, 20 and 40 mg/L with ATU
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.

Details on test conditions:

Exposure conditions
- Test item concentrations: 10, 32, 100, 320 and 1000 mg/L, 3 replicates; 10, 32, 100, 320 and 1000 mg/L with ATU, 3 replicates
- Control: 6 replicates, 6 replicates with ATU
- Test item concentration in physico-chemical oxygen consumption control: 1000 mg/L 1000 mg/L with ATU
- Concentration of reference compound 3,5-Dichlorophenol: 2.5, 5, 10, 20 and 40 mg/L 2.5, 5, 10, 20 and 40 mg/L with ATU
The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations.
- Test vessels: 300 mL glass Erlenmeyer flasks
- Test volume: 250 mL
- 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

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 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.:

332.4 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.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

916.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

604.8 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.:

98.1 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:

The effect values relate to a nominal concentration, since no analytical monitoring was performed.
All statistical evaluations to determine the NOEC and ECx values were calculated from the specific respiration rates at different test item concentrations using the statistics programme ToxRatPro Version 3.2.1 (released 2015-11-02).

Results with reference substance (positive control):

The EC50 of the reference compound 3,5-Dichlorophenol was 11.6 mg/L for total respiration, 20.5 mg/L for heterotrophic respiration and 4.1 mg/L for nitrification respiration.

Validity criteria fulfilled:

yes

Remarks:

see "Overall Remarks"

Conclusions:

The highest inhibition of activated sludge after 3 h of exposure to HPP 12879-1 was found for nitrification respiration: 3h-EC50: 604.8 mg/L, 3h-NOEC: 100 mg/L. Lower inhibition was found for heterotrophic and total respiration.

Executive summary:

A study was conducted to assess the toxicity of HPP 12879-1 to bacteria in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation)’ (adopted: 22 July 2010) and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02). 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).

A range finding test preceded the definitive test. This range finding test provided information about the range of concentrations to be used in the main test.

The activated sludge was exposed to HPP 12879-1 for 3 hours at different concentrations.

In the main test, 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 test was performed with different test item concentrations with 3 replicates. Each batch of activated sludge was checked using 5 concentrations in the range of 2.5 – 40 mg/L of 3,5-Dichlorophenol as a reference compound.

The respiration rate (oxygen consumption) is classified into two processes of oxidation, the oxidation of organic carbon (heterotrophic oxidation) and the ammonium oxidation (nitrification). By measuring the oxygen consumption rates in the presence and absence of a specific inhibitor of nitrification ATU (N-allylthiourea), total, heterotrophic and nitrification respiration rates were calculated.

Since some substances may consume oxygen by chemical reactivity, a physicochemical oxygen consumption control was carried out additionally for both sets. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

The respiration rate of each mixture was determined after aeration periods of 3 hours.

For HPP 12879-1 the following results were determined [mg/L]

Total respiration: EC 10: 332.4, EC 50: > 1000, NOEC (tα 0.05): 10

Heterotrophic respiration: EC 10: 916.2, EC 50: > 1000, NOEC (tα 0.05): 320

Nitrification respiration: EC 10: 98.1, EC 50: 604.8, NOEC (tα 0.05): 100

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

Description of key information

A study was conducted accroding to OECD Guideline 209 to assess the toxicity of HPP 12879 -1 to bacteria. The highest inhibition of activated sludge after 3 h of exposure was found for nitrification respiration: 3h-EC50: 604.8 mg/L, 3h-NOEC: 100 mg/L. Lower inhibition was found for heterotrophic and total respiration.

Key value for chemical safety assessment

EC50 for microorganisms:

604.8 mg/L

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

For HPP 12879-1 the following results were determined [mg/L]

Total respiration: EC 10: 332.4, EC 50: > 1000, NOEC (tα 0.05): 10

Heterotrophic respiration: EC 10: 916.2, EC 50: > 1000, NOEC (tα 0.05): 320

Nitrification respiration: EC 10: 98.1, EC 50: 604.8, NOEC (tα 0.05): 100

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