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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 October 2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study according to test guideline OECD 209 (not performed according to GLP). The test report is with detailed documentation. Further details on the test item are lacking in the report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Final test concentrations were prepared with test medium by dilution of stock solutions of the test substance.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Name and location of sewage treatment plant where inoculum was collected: ARA Werdhölzli, CH-8048 Zürich (03 October 2001; 9.30 a.m.)
- Preparation of inoculum for exposure: The activated sludge was used immediately after sampling from the treatment plant without adaptation.
lf the sludge was used on the following day, 50 ml of the synthetic sewage concentrate were added per liter of activated sludge and the sludge aerated overnight. Prior to the test, the sludge was washed twice with tap water.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
22 +/- 0.5 °C
pH:
7.5 +/- 0.5
Nominal and measured concentrations:
Nominal: 0, 125, 250, 500, 1000, 2000 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: 1000 ml flask
- Material, size, headspace, fill volume: total volume of test suspension of 500 ml
- Aeration: aerated by shaking (120 r.p.m.)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per reference compound concentration (replicates): 1
- Sludge concentration (weight of dry solids per volume): 1.6 g/l dry matter in the final mixture
- Nutrients provided for bacteria:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic sewage feed prepared with tap water: 16 ml of 100-fold sewage concentrate per 500 ml of final test mixture

OTHER TEST CONDITIONS
- Adjustment of pH: The pH-value was checked and adjusted to pH 7.5 +/- 0.5 with NaOH or H2SO4, if necessary

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): oxygen consumption

TEST CONCENTRATIONS
- Test concentrations: 0, 125, 250, 500, 1000 and 2000 mg/l
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol, 3, 10 and 30 mg/l
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 2 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
At the applied test concentrations, the test substance showed no significant toxic effects (<15 %) to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.
Results with reference substance (positive control):
The EC50 value of the reference substance 3,5-dichlorophenol was estimated to be 3 mg/l which was low but in the range of 5 mg/l thus confirming suitability of activated sludge and test conditions.

 Nominal concentration (mg/l)  Code  O2 consumption rates (mgO2/L h)  Respiration inhibition (%)
 0  B1  85.1
 0 B2   90.4  -
 125  T1 86.0   2.0
 250  T2  95.0  -8.3
 500  T3  83.7  4.6
 1000  T5  88.0  -0.3
 2000  T5  77.7  11.5
 Reference 3 mg/l  R1  44.3  49.5
 Reference 10 mg/l  R2  23.4  73.3
 Reference 30 mg/l  R3  8.5  90.4
Validity criteria fulfilled:
yes
Remarks:
The EC50 value of the reference substance 3,5-dichlorophenol was estimated to be 3 mg/l which was low but in the range of 5 mg/l. The variation between the two controls was + 3.1 %. Therefore, the test was considered valid.
Conclusions:
Based on these data the EC20, EC50, EC80 values of the test material were estimated to be >2000 mg/l.
Executive summary:

The inhibitory effect of the test material on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions. The inhibitory effect may include the effect on respiration and nitrification. The exposure time of the test substance on the activated sludge was 3 hours.

The nominal concentrations of the test material were 125, 250, 500, 1000 and 2000 mg/l, respectively.

These test concentrations were prepared by corresponding dilution of stock solutions of the test substance.

No chemical analysis of test concentrations was conducted. For the calculation of the effective concentration leading to 20, 50, and 80 % respiration inhibition (EC20, EC50, EC80) the nominal content of the test substance was used, assuming the test compound to be stable in water over 3 h.

At the applied test concentrations, the test substance showed no significant toxic effects (<15 %) to the activated sludge. The respiration rates were in the same range as determined for the two blank controls.

Based on these data the EC20, EC50, EC80 values of the test material were estimated to be >2000 mg/l.

Description of key information

The inhibitory effect of p-Nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) (CAS no. 7394-38-9) on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated under aerobic static conditions. The inhibitory effect may include the effect on respiration and nitrification. The exposure time of the test substance on the activated sludge was 3 hours.

At the applied test concentrations, the test substance showed no significant toxic effects (<15 %) to the activated sludge.

Based on these data the EC20, EC50, EC80 values of p-Nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) were estimated to be >2000 mg/l.

Key value for chemical safety assessment

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