N,N'-(2-chloro-1,4-phenylene)bis(3-oxobutaneamide)

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-11-23 to 2000-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

1. Copper (II) sulphate pentahydrate instead of Dichlorophenol (unsuitable for routine lab use) 2. Sludge was washed only twice, was deemed sufficient 3. Lower sludge concentration of 3 g/L +- 20 % was deemed sufficient instead of 4 g/L.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-010-4

EC Name:

-

Cas Number:

53641-10-4

Molecular formula:

C14H15ClN2O4

IUPAC Name:

N-[3-chloro-4-(3-oxobutanamido)phenyl]-3-oxobutanamide

Test material form:

solid

Sampling and analysis

Analytical monitoring:

not required

Test solutions

Vehicle:

no

Details on test solutions:

Disperson treatment: 15 min. ultrasonic treatment at room temperature
Stock solution: None, directly weighing
Test concentrations (nominal): 1000 - 1800 - 3200 - 5800 - 10000 mg/L
Controls: 2 control samples without test item were included in every test series at the beginning and at the end of the test.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

The sludge was washed twice with autoclaved tap water and diluted 1:3.

Nutrient solution: Synthetic waste water according to OECD 209

Dry sludge concentration: 2.9 g/L

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

18 - 22 °C

pH:

of activated sludge: 7.21

Nominal and measured concentrations:

Nominal: 1000, 1800, 3200, 5800, 10000 mg/L

Details on test conditions:

Permanent ventilation

Test formulation: 200 mL inoculum + 16 mL synthetic waste water + 284 mL aqua demin. + directly weighing of the test item

1 replicate per concentration and 2 per control

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate (p.a. MERCK), CAS 7758-99-8, purity 99.0-100.5 % Test concentrations: 58, 100, 180 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

7 102 mg/L

95% CI:

5 452 - 9 251

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

1 178 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

In the reference test the EC50-value was determined by probit analysis. The EC50-value of the reference item is:

EC50= 84 mg/L

The EC50-value of the reference item copper(II)-sulphate-pentahydrate is in the
confidence range of 56 - 146 mg/L.

Reported statistics and error estimates:

The EC-values of the test and reference item were calculated with probit analysis using softeware SigmaPlot (Windows) rel. 3.02, 1995, JANDEL SCIENTIFIC. Probit values were taken from WEBER (1986). Confidence intervals were calculated according to a standard procedure (BREITIG & TÜMPLING 1982).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The quality criteria were fulfilled: - The two control respiration rates differ not more than 15 %. - The EC50(3 hours) of copper(I1)sulphate pentahydrate is in the accepted range of 56 - 146 mg/L

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 and GLP principles was performed about 3 h with the test item CL-DAEP (batch-no. 90003CCL) on November 30, 1999 at the test facility DR. U. NOACK-LABORATORIUM FÜR ANGEWANDTE BIOLOGIE.
Test system was activated sludge of the municipal treatment plant of D-31137Hildesheim. The test method was static. Nominal concentrations of the test item were as follows:

1000- 1800 - 3200 - 5800 - 10000 mg/L

The respiration rates of control, reference and test item were measured after a contact time of three hours, and the inhibitory effects of the test and reference concentrations were determined in comparison to the control respiration rates.

The EC20 and EC50 values were determinable in the tested concentration range . In order to check the activity of the test system and the test conditions a reference test is carried out once every 7 days with copper-(II)-sulphate-pentahydrate as reference item and the reference toxicity determined. The EC50 value for the reference item was 84 mg/L tested December 02, 1999. The quality criteria of the test guideline were fulfilled.

EC-values with confidence interval of Cl-DAEP:

EC20 = 1178 mg/L

EC50 = 7102 mg/L (95% CI = 5452 - 9251 mg/L)

EC80 = > 10000 mg/L

The test item CL-DAEP is not toxic in concentrations 5_ 1178 mg/L to activated sludge of a municipal sewage treatment plant.