4,4'-methylenebis[N-sec-butylaniline]

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:

01 Mar 2018 - 16 Mar 2018

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:

2010

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the amber test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis). The test item – Milli-RO water mixtures were magnetically stirred for a short period and subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.
- Controls: Control without test item or reference item; Reference item control; Abiotic control (Combined Limit/Range-Finding test only)

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's -Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). The pH was 7.2 (combined limit range-finding test) and 6.5 (final test) on the day of testing. The batch of sludge was used one day after collection. Therefore, 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

19 - 22°C

pH:

At start (before addition of sludge): 7.3 - 7.5
After 3 h: 6.2 - 7.6

Nominal and measured concentrations:

Nominal: 100, 180, 320, 560 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all-glass open bottles, 500 mL fill volume
- Aeration: continuous aeration with clean, oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per reference control (replicates): 1 replicate per concentration (4 concentrations used)
- No. of vessels per abiotic control (replicates): 1 (Combined Limit/Range-Finding Test only)
- Sludge concentration (weight of dry solids per volume): 1.5 g/L suspended solids in the final test mixture.
- Nitrification inhibitor used: none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)
- Synthetic medium: according to OECD 209.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not specified.

EFFECT PARAMETERS MEASURED
After the 3-hour contact time the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls.
The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.
Oxygen recording: determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Combined Limit/Range-finding study test concentrations: 10 (1 replicate), 100 (1 replicate) and 1000 (triplicate) mg/L. Control (6 replicates) and abiotic control (1 replicate).
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

180 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.:

232 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% Confidence interval: 132 - 409 mg/L

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

Details on results:

- The combined limit/range-finding showed 2%, 6% and 11% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. The EC50 was above the highest concentration tested (1000 mg/L).Since the effect observed at 1000 mg/L was statistically significant the NOEC could not be determined.
- There was no oxygen uptake from abiotic processes.
- Final Test: No statistically significant inhibition of the respiration rate of the sludge was recorded at 180 mg/L. At higher concentrations the inhibitory effect of the test item on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 15% inhibition at 320 mg/L to 21% at 1000 mg/L.

Results with reference substance (positive control):

EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total respiration (5 mg/L in the final test).

Reported statistics and error estimates:

ECx values:
- Reference item: 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis.
- Test item: probit analysis using linear maximum likelihood regression.
NOEC determination: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1: Final Test – Overview of the Results

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)#
Control	0	37.90	25.27
T1	100	41.78	27.85	-10.22
T2	180	34.68	23.12	8.49
T3	320	32.28	21.52*	14.83
T4	560	31.71	21.14*	16.35
T5	1000	30.07	20.05*	20.67

# The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' section for details on validity criteria.

Conclusions:

The test item was not toxic to waste water bacteria (activated sludge) at or below a concentration of 180 mg/L (NOEC). The EC10 was 232 mg/L (95% confidence interval: 132 - 409 mg/L). The EC50 was above 1000 mg/L.

Executive summary:

In a 3-h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 100, 180, 320, 560 and 1000 mg/L. The test item was not toxic at or below a concentration of 180 mg/L (NOEC), and inhibited respiration up to 21% in the highest test concentration. The EC10 was 232 mg/L (95% confidence interval: 132 - 409 mg/L), the EC50 was >1000 mg/L. The study met all validity criteria and is considered reliable without restriction.

Description of key information

In a 3-h activated sludge respiration inhibition study conducted according to OECD Guideline 209 and according to GLP Principles, microorganisms in activated sludge were exposed to the test item at nominal concentrations of 100, 180, 320, 560 and 1000 mg/L. The test item was not toxic at or below a concentration of 180 mg/L (NOEC), and inhibited respiration up to 21% in the highest test concentration. The EC10 was 232 mg/L (95% confidence interval: 132 - 409 mg/L), the EC50 was >1000 mg/L. The study met all validity criteria and is considered reliable without restriction.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

232 mg/L