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Diss Factsheets

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-4-2007 - 25-4-2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate
EC Number:
EC Name:
Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate
Cas Number:
Molecular formula:
tetrasodium 2-[bis(carboxylatomethyl)amino]pentanedioate
Details on test material:
- Name of test material (as cited in study report): tetrasodium-N,N-bis(carboxylatomethyl)-L-glutamate
- Trade name: Dissolvine GL-PD
- Substance type: chelating agent
- Physical state: white powder
- Composition of test material, percentage of components:
- Substance (incl impurities) 91.9% w/w
- Water 7.5 % w/w
- Lot/batch No.: CFC-8098
- Stability under test conditions: stable under test conditions
- Solubility in water: highly soluble in water (>45%)
- Storage condition of test material: at room temperature in the dark

Sampling and analysis

Analytical monitoring:
not required

Test solutions


Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Pretreatment: Activated sludge from a treatment plant treating predominantly domestic wastewater was used. The Activated sludge was used on the day of collection and before use the activated sludge was homogenized with a syringe.
- The activated sludge is kept aerated during the day at 20 +/- 2 °C
- Before use the pH of the activated sludge was determined (pH 6.0 - 8.0)
- Initial biomass concentration: The dry weight of the activated sludge in the incubation vessels was 1.55 g/l.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
0.5 h

Test conditions

not relevant
Test temperature:
20 +/- 2 °C
approximately pH 7
Dissolved oxygen:
aerobic (respiration rate is calculated between approximately 6.5 mgO2/L and 2.5 mg O2/L)
fresh water conditions
Nominal and measured concentrations:
Tested nominal concentrations of active ingredient tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate:
25.75 mg/L, 51.5 mg/L, 103 mg/L, 206 mg/L, 412 mg/L
Details on test conditions:
-The test was performed in 250 to 300 mL Erlenmeyers.
-The homogenized activated sludge was incubated in a shaking water batch (100 rpm, 20 °C) for 30 minutes.
-Synthetic sewage was added to the activated sludge (1.6 mL on a total volume of 50 mL).
Synthetic sewage contained per liter: 16 g peptone
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
-Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions of respectively 0.5 g/l and 1.0 g/l.
-Tap-water was used to fill up the total test volume to 50 ml.
- The dry weight concentration of the activated sludge in the test was 1.55 g/l
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 2.475 mg/l, 4.95 mg/l, 9.9 mg/l, 19.8 mg/l, 39.6 mg/l and 79.2 mg/l
-The tested concentrations of acitve ingredient tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate where
25.75 mg/l, 51.5 mg/l, 103 mg/l, 206 mg/l, 412 mg/l
-Controls without test substance (or reference compound) are measured at the beginning and at the end of the test
-The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.
Reference substance (positive control):

Results and discussion

Effect concentrations
0.5 h
Dose descriptor:
Effect conc.:
25.75 - 412 mg/L
Nominal / measured:
Conc. based on:
act. ingr.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
pH of the reaction mixtures after the incubation period ranged from 6.6 - 7.3
Temperature varied from 20.2 to 20.8 °C
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol = 8.7 mg/L
Reported statistics and error estimates:
The control respiration rates were within 15% of each other

Any other information on results incl. tables

Respiration rates of the activated sludge and inhibition percentages at various concentrations of 3,5 -dichlorophenol

 concentration (mg/l) Activity (mg O2/g/min)  Inhibition (%) 
 control  0.37  
 control  0.34  
 2.475  0.26  27
 4.95  0.25  30
 9.9  0.18  49
 19.8  0.09  75
 39.6  0.06  83
 79.2  0.04  89

Respiration rates of the activated sludge and inhibition percentages at various concentrations of tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

 Concentration (mg/l)  Activity (mg O2/g/min) Inhibition (%) 
 control  0.34  
 control  0.35  
 25.75  0.31  10
 51.5  0.32  7
 103  0.34  1
 206  0.34  1
 412  0.32  7

Applicant's summary and conclusion

Validity criteria fulfilled:
the control respiration rates are within 15% of each other, the EC50 of the reference compound is 8.7 mg/l this is within the prescried range ( 5 to 30 mg/l).
No inhibition of the respiration of the activated sludge was measured at a concentration of 412 mg/L tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate. Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate is therefore considered harmless to activated sludge.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice. The test is valid as shown by the EC50 of the reference compound, 3,5-dichlorophenol (8.7 mg/L). The control respiration rates which are within 15% of each other. The toxicity of tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate to activated sludge was determined by measuring the respiration rate after a contact time of 30 minutes at various concentrations of the test substance. The inhibitory effect of tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate at a particular concentration is expressed as a percentage of the two controls. No inhibition of the respiration of the activated sludge was measured at a concentration of 412 mg/L. Tetrasodium N,N-bis(carboxylatomethyl)- L-glutamate is therefore considered harmless to activated sludge.