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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP compliant guideline study, published in peer reviewed literature, fully adequate for assessment
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Test was adapted to use volatile chemicals
GLP compliance:
Analytical monitoring:
Details on test solutions:
Stock solution of trichloroethylene was made in distilled water.
Test organisms (species):
activated sludge
Details on inoculum:
Trichloroethylene was tested against a culture of activated sludge origin that was maintained in the laboratory in a continuous flow reactor receiving a complex, synthetic feed designed to mimic domestic wastewater. Approximately 20 minutes before use, the organisms were removed from the continuous culture and allowed to equilibrate to the test temperature. They were then diluted to the appropriate concentration.
Test type:
Water media type:
Limit test:
Total exposure duration:
3 h
Post exposure observation period:
Test temperature:
24-26 °C
Dissolved oxygen:
>40% saturation
Not applicable
Nominal and measured concentrations:
Substance tested at solubility limit or 1000 mg/l
Details on test conditions:
The procedure used was as follows:

Appropriate quantities of cell suspension and OECD substrate solution were added to two 150-mL beakers to give cell and substrate SCOD concentrations ofapproximately 75 mg/L and 375 mg/L, respectively, after addition of the test compound. These concentrations were chosen to ensure a linear oxygen uptake rate throughout the test and to minimize the need for reaeration. Beakers were stirred with magnetic stirrers and aerated using Pasteur pipets for approximately 7 minutes to ensure oxygen saturation and a stable exogenous oxygen consumption rate.
Stirring and aeration were then stopped, the pipets were removed, and the test compound (inhibitor) was added to the test reactor, while an equal quantity of water was added to the control reactor.
The polytetrafluoroethylene plugs, containing DO probes, were then inserted so that headspace was eliminated. Stirring was resumed, and strip chart recorders were started to record oxygen concentrations. Measurement was continued for 5 minutes and the average utilization rate in each vessel was determined from the strip chart plots.
The test vessel rate was then expressed as a percent of the rate in the control and labeled the immediate response to the test compound. Stirring was continued for 21 additional minutes while DO concentration monitoring continued. If the DO concentration dropped below 40% of saturation it was returned to saturation by inserting the pipet into the reactor (adjusting the plug as needed) and very gently aerating. The pipet was again removed and the plug was readjusted to eliminate any head space. Typically, only one reaeration was required.
The strip chart recorders were restarted 26 minutes after addition of the test compound, and oxygen concentrations in both vessels were recorded for 5 minutes. After inspection of the plots to ensure linearity, the rates were determined. The rate in the test vessel was expressed as a percent ofthe rate in the control and labeled the 30-minute response to the test compound.
Reference substance (positive control):
3 h
Dose descriptor:
Effect conc.:
260 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
30 minute value for trichloroethylene was 260 mg/l.
Results with reference substance (positive control):
Results with the reference substance were found valid, lying between 5 and 30 mg/L.

Description of key information

The most critical value was the 3-h EC50 of 260 mg/l in activated sludge conducted under modified OECD conditions.

Key value for chemical safety assessment

EC50 for microorganisms:
260 mg/L

Additional information

Various tests have been carried out to study the toxicity of trichloroethylene to microorganisms. One study with activated sludge was available, for this study a modified version of OECD 209 “Activated sludge respiration inhibition test” was used. This modified version used more dilute cell and substrate concentrations, and conducted tests in vessels sealed with PTFE plug to prevent loss by volatilization. The 3-h EC50 of 260 mg/l is used in the assessment. Lower EC50 values were found in other toxicity studies, however the endpoints were less relevant for micro-organisms in sewage treatment plants.