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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 November 2008 to 07 July 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
methyl 4-[4-(4-methoxybenzamido)-3-nitrophenyl]-3-methyl-4-oxobutanoate
Details on test material:
- Name of test material (as cited in study report): Gamma Nap
- Substance type: monoconstituent
- Physical state: beige yellow powder
- Analytical purity: 99.9%
- Impurities: sum is 0.1%
- Purity test date: 24 April 2009
- Lot/batch No.: 07/004/2
- Expiration date of the lot/batch: April 2010
- Stability under test conditions: stable until the end of the test
- Storage condition of test material: room temperature and protected from light

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Details on test solutions:
- Method: As the solubility of the test item in water is 0.41 mg/L (information supplied by the Sponsor), test suspensions at 10, 31.6, 100, 316 and 1000 mg/L were prepared by direct addition of the test item in the test vessels and vigorous agitation (using a magnetic stirrer) of these preparations for a few minutes before the beginning of the test (i.e. just before addition of the inoculum, i.e. activated sludge).

- Controls: A reference stock solution was prepared by dissolving 500 mg of the reference item, 3,5-dichlorophenol, in 10 mL of NaOH at 1 N. The obtained preparation was then diluted to 30 mL with deionized water. Aliquots of H2SO4 at 1 N were added while stirring until the point of incipient precipitation was obtained (approximately 8 mL of H2SO4 at 1 N were required). The solution was made up to 1000 mL using deionized water.
The reference stock solution was agitated for 17 hours. The pH of this solution was 8.08 after agitation. The pH of this solution was then adjusted to 7.98 using HCL 1 N after agitation.
Reference item suspensions were prepared by further dilution of the stock solution with appropriate volumes of dechlorinated water and inoculum.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
After sampling, the inoculum was left to settle and the main part of the supernatant was rejected. It was washed once with dechlorinated water and then filtered (to remove the biggest particles). Four samples of 50 mL were taken to determine the content in suspended matter (dry weight/v). The concentration of sludge (8.50 g/L) was then adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water.
The inoculum was sampled one day before the test and maintained under agitation, aeration and protected from light before use. As the sludge was not used immediately, 50 mL of sewage feed were added, per liter, just before agitation and aeration

Study design

Test type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
The oxygen concentration was determined for a period of approximately 10 minutes.

Test conditions

280 +/- 20 mg/L as CaCO3.

Test temperature:
The temperature was not recorded.
The pH value of the test item suspension at the highest concentration (1000 mg/L) was 7.69 after addition of activated sludge.
Dissolved oxygen:
Air was bubbled through each suspension at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements.
No data.
Nominal and measured concentrations:
Nominal concentrations: 10, 31.6, 100, 316 and 1000 mg/L.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 1 000
Nominal / measured:
Conc. based on:
other: dissolved and undissolved fractions of the test substance
Basis for effect:
inhibition of total respiration
respiration rate
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 17.71 mg/L i.e. between 5 and 30 mg/L. The test was thus considered to be valid.

Any other information on results incl. tables

Respiration rates and percent inhibitions:

                                             Concentration             Respiratory rate                  Inhibition

                                                   (mg/L)                    (mg O2.L-1.h-1)                       (%)

Control 1                                       0                                 30                               NA

Control 2                                       0                                 32                               NA


Reference item                               4                                 33                                   0

                                                    12                                 16                                 48

                                                    36                                   9                                 71


Gamma Nap                                10                               ND                               ND

                                                    31.6                            ND                               ND

                                                  100                               ND                               ND

                                                  316                               ND                               ND

                                                1000                                 30                                   3

NA: not applicable

ND: not determined


Applicant's summary and conclusion

Validity criteria fulfilled:
Under the experimental conditions of the study, the 3-hour EC50 of the test item Gamma Nap for activated sludge respiration inhibition was > 1000 mg/L (including the dissolved and undissolved fractions of the item).
The highest test item concentration without significant effect (NOEC) on the respiration was ≥ 1000 mg/L.
The test item should be considered as non-toxic for the micro-organisms of a water treatment plant.
Executive summary:

The inhibition potential of Gamma Nap on the respiration rate of an activated sludge at 1.6 g/L in dechlorinated water was investigated using the following suspensions:

·        two control suspensions (activated sludge alone),

·        five test item suspensions: activated sludge and Gamma Nap at a concentration(including the dissolved and undissolved fractions of the item)of 10, 31.6, 100, 316 or 1000 mg/L,

·        three reference item (3,5-dichlorophenol) suspensions: activated sludge and the reference item at a concentration of 4, 12 and 36 mg/L.


The five test item concentrations were prepared by direct addition, of the required among to reach the targets concentrations, of the test item to the test flask.

The oxygen consumption (i.e.respiration rate) of the test item, reference item and control suspensions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours.

The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and that of the reference item were determined considering the mean oxygen consumption of the controls as 100%.

In the first assay:

As the EC50 of 3,5-dichlorophenol was 34.53 mg/L, out of the acceptable range 5 to 30 mg/L, the first assay was invalidated and a second assay was performed with a new batch of  3,5-dichlorophenol. Thereby, results of the first assay are not presented in this report.


In the second assay

As the validity criteria were met (the difference between the respiration rates of the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 17.71 mg/Li.e.between 5 and 30 mg/L), the test was considered valid.


The respiration rate of the test item suspension at the highestconcentration(1000 mg/L) was equivalent to the respiration rate of the first control (i.e.these rates werewithin 15% of each other). Therefore, the oxygen consumption rate of the four other test item suspensions (10 to 316 mg/L) was not determined.


Consequently, the highest test item concentration without significant effect (difference inferior or equal to 15%) on the respiration rate, relative to mean of the controls, was superior or equal to 1000 mg/L.