N1,N2-dimethyl-N1-{2-[methyl(propan-2-yl)amino]ethyl}-N2-(propan-2-yl)ethane-1,2-diamine

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms, other

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

Version / remarks:

July, 2020

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Source: Sponsor; Batch: 10/16/2019
- Purity, including information on contaminants, isomers, etc.: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability in aqueous solution: 24 hours

Analytical monitoring:

no

Details on inoculum:

Activated sludge from the municipal wastewater treatment plant Breisgauer Bucht was used
as test system. The treatment plant has a capacity of 600 000 inhabitant equivalents and clarifies predominantly domestic wastewater. Sampling date of activated sludge was 2 December 2020. The activated sludge was kept aerated until use at test temperature. Dry solid of the activated sludge was determined as 4.2 g/L by weight measurements before and after drying at 105°C for about three hours (mean of triplicate measurements). The activated sludge was washed twice by settling the sludge, decanting the supernatant and resuspending the sludge in chlorine-free tap water. Before using, the activated sludge was diluted to 3 g/L dry solids with chlorine-free tap water, in order to obtain a final concentration of 1.5 g/L dry solids in the test.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Nominal and measured concentrations:

Nominal concentrations: 1, 10, 30 mg/L

Details on test conditions:

The different vessels must be started separately with an interval of exactly 15 min in order to
be able to measure the respiration rates one by one while making sure that the aeration time
is 3 h in every vessel. At time "0", 16 ml synthetic sewage and the test item or reference
compound were added to the test vessel and made up to 250 mL with chlorine-free tap
water. After measurement and adjustment of pH, 250 mL of activated sludge (3 g/L dry
solids) were added consequently obtaining a total volume of 500 ml with 1.5 g/L dry solids.
For the controls the same procedure was made as with the test vessels, however without test
or reference substance. All vessels were stirred and aerated throughout the contact time with
room air to assure an oxygen saturation of 60-70% using a membrane pump. Before starting
the test the oxygen saturation rate was measured.
After 3 h the aeration of the first vessel was shut off and the respiration rate was measured in
a BOD-bottle with an oxygen electrode while mixing with a magnetic stirrer.
This determination was repeated for each vessel at 15-min intervals, ensuring that the
contact time in each vessel was 3 h.

Reference substance (positive control):

yes

Remarks:

Name: 3,5-dichlorophenol 97% Source: Sigma-Aldrich, Lot MKBS0425V Sum formula: Cl2C6H3OH CAS-No.: 591-35-5 Storage conditions: room temperature Durability: 30 June 2025 Solubility in water: soluble

Key result

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:

A range finder test with limit test design with the concentration of 10, 100 and 1000 mg/L was
performed. The pH in the test vessels was 7.5 – 9.9 and was adjusted to 7.2 – 7.6.
Appendix I: Table 1 shows the oxygen concentration used for the calculation of the respiration rate and table 2 shows the calculation of the respiration rate and the percentage
of inhibition.
There was no inhibition observed in any of the test vessels. In the highest test concentration
1000 mg/L, the consumption rate was 23.6 mg/g sludge per hour and therefore even slightly
higher than the one in the blank control vessels (23.2 mg/L).

Results with reference substance (positive control):

In the limit test the EC50 of 3,5-dichlorphenol was calculated as 8.22 mg/L (see background information attached), and was therefore within the acceptable range of 2 to 25 mg/l. The pH in the
reference vessels was 7.3.

Reported statistics and error estimates:

The respiration rate is calculated from oxygen measurements using the linear portion of the
oxygen-time-curves. From the oxygen consumption (differences of oxygen concentrations)
the respiration rates are calculated in mg O2/(L*h). The inhibitory effect of the test item at a
particular concentration is expressed as a percentage of the mean of the control respiration
rates. The EC50 calculation was performed with the ToxRat software by probit analysis using linear
max. likelihood regression (ToxRat Professional Version 3.2.1, © ToxRat Solutions GmbH).
The EC50 is calculated from the slope of the curve.

Validity criteria fulfilled:

yes

Conclusions:

An OECD 209 Activated sludge inhibition test (2010) was conducted with the test item. This
was to assess the inhibition effect of different concentrations of test item to micro-organisms
of activated sludge by measuring the respiration rate after adding a standard amount of
synthetic sewage feed. The test met all guideline validity criteria.
The results for the inhibition of the total respiration of activated sludge were:
EC50 > 1000 mg/L
LOEC > 1000 mg/L
NOEC ≥ 1000 mg/L

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L