N,N'-ethylenedi(diacetamide)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-09

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Principles of method if other than guideline:

In contrast to the guideline, it was decided to use copper (II) suphate pentahydrate instead of the prescribed 3,5-dichlorophenol. The usage of dichlorophenol is unsuitable for routine lab use.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethylenedi(diacetamide)

EC Number:

212-811-9

EC Name:

N,N'-ethylenedi(diacetamide)

Cas Number:

871-78-3

Molecular formula:

C6H12N2O2

IUPAC Name:

N-(2-acetamidoethyl)acetamide

Constituent 2

Reference substance name:

Diacetylethylenediamine (DAED)

IUPAC Name:

Diacetylethylenediamine (DAED)

Details on test material:

- Name of test material (as cited in study report): N,N'-Diacetylethylenediamine (DAED)
- Physical state: Solid, powder
- Analytical purity: 99.9 %
- Lot/batch No.: 14-016
- Expiration date of the lot/batch: 2010-05-21
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1000 mg/L were weighed out, dispersion treatment was agitation
- Differential loading: 1000 mg/L
- Controls: 2 control samples without test substance were included

Test organisms

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at sarstedt (Municipal sewage treatment plant of 31157 Sarstedt, Germany)
- Pretreatment: Washed twice with autoclaved tap water and adjusted to a dry weight concentration of 4.0 g/L +/- 10 %
- Dry sludge concentration: 4.38 g/L

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

Hardness:

Not determined

Test temperature:

20.4 °C

pH:

6.64

Salinity:

Not determined

Nominal and measured concentrations:

1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Composition of test medium: 200 mL inoculum + 16 mL synthetic waste water (acc. to the guideline) + 284 mL autoclaved tap water + test substance concentration (directly weighing of 500 mg)



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Determination of respiration rates subsequently to an incubation period of 3 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
- Test concentrations: 10 - 100 - 1000 mg/L
- Results used to determine the conditions for the definitive study: Yes
Inhibitions of the Range-Finding Test

Concentration
[mg/L] Inhibition
[%]
10 3
100 1
1000 2

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 109 (CI 54 - 156) mg/L

Reported statistics and error estimates:

The EC50 of the reference item was calculated with probit analysis using software SIGMAPLOT (Windows), SPSS CORPORATION.
Probit values were taken from WEBER (1986).

Any other information on results incl. tables

From the oxygen depletion the respiration rates were calculated in mg O₂/L×h (Table 2).

The inhibition of the respiration rates from the test and reference item concentrations in % was calculated in comparison with the respiration rates from the control.

Respiration Rates and Inhibition of Respiration of Test and Reference Item Concentrations

	Concentration	Respiration rate	Inhibition
	[mg/L]	[mgO2/L×h]	[%]
Test item	1000	29.2	-1
Control 1	–	28.4	–
Control 2	–	29.2	–
Reference item	180	8.4	71
	100	14.8	49
	58	22.4	22

Difference of the control respiration rates: 3 %

Promotion of respiration is signed with minus (-)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item N,N’-Diacetylethylenediamine (DAED) is not toxic at concentrations
> 1000 mg/L to activated sludge of a municipal sewage treatment plant.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item N,N’-Diacetylethylenediamine (DAED) (batch-no. 14-016) on 2009-06-09 at Dr.U.Noack-Laboratorien, D-31157 Sarstedt. Test system was activated sludge of the municipal treatment plant of D-31157 Sarstedt. The test was carried out under static conditions with the limit concentration 1000 mg/L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. No inhibition was observed for the test item.

In order to check the activity of the test system and the test conditions a reference test was carried out with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity was determined. The EC₅₀- value for the reference item was 109 mg/L.

Validity criteria of the test guideline were fulfilled.