Reaction products of 4,4'-methylenebis(cyclohexylamine) and 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

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))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system Micro-organisms in activated sludge.
Source Municipal sewage treatment plant: 'Waterschap Aa en Maas',
's- Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.
Preparation of the sludge 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 7.8 on the day of
testing. The batch of sludge was used one day after collection;
therefore, 50 mL of synthetic medium 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

Hardness:

Adjusted ISO-medium, formulated using RO water (tap water
purified by reverse osmosis; GEON Waterbehandeling, Berkel-
Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Performance of the test1 The synthetic medium (16 mL) made up to 50 mL with
Milli- RO and 200 mL test item solution were mixed (total
volume 250 mL). The pH was determined. Thereafter 250 mL
activated sludge was added. This was the start of the test.
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 vessel. The temperature control vessel 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
probes connected to a BlueBox (GO-Systemelektronik GmbH,
Germany), a multichannel measuring and controlling system.

Test temperature:

20-22 C

Details on test conditions:

Contact time 3 hours, during which aeration and stirring took place.
Vessels All glass open bottles/vessels.
Milli-RO / Milli-RO water Tap water purified by reverse osmosis (Millipore Corp.,
Bedford, Mass., USA).
Synthetic medium 16 g peptone
(=sewage feed) 11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered.
The pH was within 7.5 ± 0.5.
Air supply Clean, oil-free air.
Aeration 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.

Reference substance (positive control):

yes

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

3 h

Dose descriptor:

other: ELR10

Effect conc.:

7.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

3 h

Dose descriptor:

other: ELR20

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

149 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Combined limit/range-finding test
The combined limit/range-finding test showed 17, 62 and 84% inhibition of the respiration
rate at a loading rate of 10, 100 and 1000 mg/L, respectively. Therefore, the expected ELR50
was between a loading rate of 10 and 100 mg/L. There was no oxygen uptake from abiotic
processes.
All test conditions and acceptability criteria prescribed by the study plan were met.

Final test
Inhibition of the respiration rate
The effects observed were in agreement with what was expected based on the results of the
combined limit/range-finding test. The results of this test allowed for reliable determination
of a NOELR and ELRx values.
No statistically significant inhibition of the respiration rate of the sludge was recorded at or
below a loading rate of 3.2 mg PACM BADGE adduct per litre
At higher loading rates the inhibitory effect of PACM BADGE
adduct on aerobic waste water (activated sludge) bacteria increased with increasing loading
rate, ranging from 16% inhibition at 10 mg/L to 77% at 1000 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test PACM BADGE adduct was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 3.2 mg/L (3-hour NOELR).
The 3-hour ELR10 was at a loading rate of 7.3 mg/L (95% confidence interval: 1.7 – 16 mg/L).
The 3-hour ELR20 was at a loading rate of 21 mg/L (95% confidence interval: 7.5 - 37 mg/L).
The 3-hour ELR50 was at a loading rate of 149 mg/L (95% confidence interval: 93 – 254 mg/L).

Executive summary:

Under the conditions of this present test PACM BADGE adduct was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 3.2 mg/L (3-hour NOELR).

The 3-hour ELR10 was at a loading rate of 7.3 mg/L (95% confidence interval: 1.7 – 16 mg/L).

The 3-hour ELR20 was at a loading rate of 21 mg/L (95% confidence interval: 7.5 - 37 mg/L).

The 3-hour ELR50 was at a loading rate of 149 mg/L (95% confidence interval: 93 – 254 mg/L).

Description of key information

Under the conditions of this present test PACM BADGE adduct was not toxic to waste water bacteria (activated sludge) at or below a loading rate of 3.2 mg/L (3-hour NOELR).

The 3-hour ELR10 was at a loading rate of 7.3 mg/L (95% confidence interval: 1.7 – 16 mg/L).

The 3-hour ELR20 was at a loading rate of 21 mg/L (95% confidence interval: 7.5 - 37 mg/L).

The 3-hour ELR50 was at a loading rate of 149 mg/L (95% confidence interval: 93 – 254 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

149 mg/L