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Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Harlan Laboratories Ltd.,Zelgliweg 1, 4452 Itingen, Switzerland

Test material

Details on test material:
Identity: Glyoxylic acid 50%
Batch No.: B191000111
Purity: 50.1%
Expiration Date (as provided by the Sponsor): January 2011
Expiration Date (as handled at Harlan Laboratories): 31-Jan-2011
Solubility in Water: Miscible in all proportions (20 °C)
Aggregate State / Physical Form at Room Temperature: Liquid
Color: Colorless to yellowish
Vapor Pressure: 10 hPa (25 °C)
Density: 1.33 – 1.34 g/cm3 (20 °C)
Storage Conditions: At room temperature at about 20 °C, away from direct sunlight.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
not required
Details on sampling:
Not applicable

Test solutions


Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
Based on this ratio, an aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test.

* Synthetic sewage feed:
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
filled up to a final volume of 1 liter with deionized water

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
Not applicable

Test conditions

Not applicable
Test temperature:
The temperature in the test media, measured in one control, was 20 °C at the start and at the end of the incubation period.
The pH of the activated sludge inoculum was 7.1.
The pH of the stock solution with a Glyoxylic acid 50% concentration of 10 g/L (used to prepare the five test concentrations) was adjusted from pH 1.8 to 7.4 with a 1M sodium hydroxide solution.
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates, the dissolved oxygen concentrations were at least 8.6 mg/L.
Not applicable
Nominal and measured concentrations:
The inoculum had a sludge concentration of 2.4 g/L dry weight (corresponding to about 1.0 g dry material per liter test medium).
Details on test conditions:
The test was performed in 2000-mL glass beakers. The test vessels were labeled with the necessary information to ensure unmistakable identification.
At the start of the test, synthetic sewage feed and activated sludge inoculum (see Sections 3.3 and 3.4.1) were added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, secondly to the test solutions of the reference item, thirdly to the test solutions of the test item, and finally to the second control.
During the incubation period of 3 hours, all test media and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.
Reference substance (positive control):

Results and discussion

Effect concentrations
3 h
Dose descriptor:
Effect conc.:
> 2 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Remarks on result:
other: Not applicable
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 10 mg/L (the 95 % confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Applicant's summary and conclusion

Executive summary:

The inhibitory effect of Glyoxylic Acid 50% on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test in accordance with OECD TG 209 and GLP. The following nominal concentrations of the test item Glyoxylic Acid 50% were tested: 20, 64, 200, 640 and 2000 mg/L (corresponding to 10, 32, 100, 320 and 1000 mg active ingredient per liter, respectively). In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the highest concentration of 2000 mg/L, the test item Glyoxylic Acid 50% had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of three hours. Thus, the 3-hour NOEC (EC10) of Glyoxylic acid 50% to activated sludge microorganisms was at least 2000 mg/L (equivalent to 1000 mg active ingredient per liter). This value might even be higher, but concentrations above 2000 mg/L were not tested. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 2000 mg/L.