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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-09 to 2012-11-29
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
equivalent or similar to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)

Test material

Test material form:
liquid: viscous
Details on test material:
Name as cited in the study report: Additin E 459
Other name: Triethylene glycol dimaleate
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
At the time of the testing the CAS Number that was used for Triethylene glycol dimaleate was 66790-57-6
In the course of the project the CAS Number 109828-01-5 was assigned, as this number describes better the structure of the main compound

Sampling and analysis

Analytical monitoring:
Details on sampling:
No details available

Test solutions

Details on test solutions:
Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8.
8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C.
Control vessels (inoculated sample without test item) were prepared the same way.

Test organisms

Test organisms (species):
activated sludge, domestic
Details on inoculum:
The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 4500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
No details available

Test conditions

No details available
Test temperature:
20 +/- 2°C
7.5 before application
7.8 / 7.9 / 7.8 during exposure (10 / 100 / 1000 mg/L)
7.8 (control)
Dissolved oxygen:
Test item 10 mg/L : 5.1 (start) 2.8 (end) mg O2/L
Test item 100 mg/L: 5.5 (start) 3.0 (end) mg O2/L
Test item 1000 mg/L: 4.7 (start) 2.9 (end) mg O2/L
Control 1: 4.7 (start) 2.6 (end) mg O2/L
Control 2: 5.2 (start) 2.6 (end) mg O2/L
A synthetic waste water feed was prepared by dissolving the following amounts of substance per 1 litre of water.
16.0 g peptone
11.0 g meat extract
3.0 g urea
0.7 g NaCl
0.4 g CaCl2 x 2H2O
0.2 g MgSO4 x 7H2O
2.8 g K2HPO4
Nominal and measured concentrations:
Nominal 10, 100 and 1000 mg/L
No measured concentrations
Details on test conditions:
Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 16 hours. The pH was measured and ranged between pH 2.3 and pH 4.0 and was adjusted to pH 7.2 – 7.4.
For the reference compound a stock solution at a concentration of 500 mg/L was prepared by dissolving 250 mg 3,5-Dichlorophenol in 5 mL of 1 N NaOH and diluting to 0.5 litre with deionised water. The pH was adjusted to pH 7 ± 0.5 with HCl.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
Additin E 459 showed 13.88 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L.
Results with reference substance (positive control):
EC50 of the reference compound 3,5-Dichlorophenol is in the range 5 – 30 mg/L
Reported statistics and error estimates:
respiratory rates of the 2 controls differ less than 15 % from each other

Any other information on results incl. tables

Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%- and 99%-confidence limits (according to Fieller`s theorem).

 Parameter  EC10  EC20  EC50  EC80
 Value [mg/l]  507.464  2277.062  n.d. n.d.
 lower 95%-cl  n.d.  n.d.  n.d.  n.d.
 upper 95%-cl  n.d.  n.d.  n.d.  n.d.
 lower 99%-cl  n.d.  n.d.  n.d.  n.d.
 upper 99%-cl  n.d.  n.d.  n.d.  n.d.

n.d.: not determined due to mathematical reasons or inappropriate data

Applicant's summary and conclusion

Validity criteria fulfilled:
All validity criteria of the test method were met.
The EC50 is higher than 1000 mg/L
Executive summary:

A study was performed to assess the toxicity of Additin E 459 to bacteria.

The study was conducted in accordance with Council Regulation (EC) No 440/2008, Method C.11 ”Biodegradation: Activated Sludge Respiration Inhibition Test” (2008). This test method is equal to OECD Guideline 209 (1984).

The activated sludge was exposed to Additin E 459 at different concentrations. The respiration rate of each mixture was determined after aeration periods of 3 hours.

Additin E 459 showed 13.88 % respiration inhibition of activated sludge at a test item concentration of 1000 mg/L.

The EC50 is higher than 1000 mg/L.

The effect value relates to a nominal concentration, since no analytical monitoring was performed.