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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 05 November 2015 to 06 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)
Version / remarks:
(2007)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, 1-Litre test bottles were filled with 200 mL of test item mixtures in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for 25-28 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test item and test organisms was ensured applying continuous aeration and stirring.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3 g/L of sludge, as used for the test). The pH was 7.8 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- The amount of suspended solids in the final test mixture was 1.5 g/L (250 mL of 3 g/L in 500 mL)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20 - 22°C
pH:
- Before addition of sludge: 7.4 - 7.5
- After the 3 hour exposure period: 7.2 - 8.4
Dissolved oxygen:
The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation
Nominal and measured concentrations:
10, 100 and 1000 mg/L (combined limit/range-finding test only)
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass open bottles/vessels
- Total volume: 500 mL
- Aeration: yes, during the exposure period
- No. of vessels (1000 mg/L) (replicates): 3
- No. of vessels (10 and 100 mg/L) (replicates): 1
- No. of vessels per control (replicates): 6
- No. of vessels per reference (replicates): 3
- No. of vessels per abiotic control (replicates): 1
- No. of vessels per nitrification control (replicates): 2
- No. of vessels (1000 mg/L test substance + nitrification inhibitor (11.6 mg/L N-allylthiourea)) (replicates): 3

- Sludge concentration: 250 mL/500 mL
- Synthetic medium concentration: 16 mL/500 mL

MEDIUMS
- ISO medium: adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCL: 4.2 mg/L
- Synthetic medium (=sewage feed): according to OECD 209 (2010)

OTHER TEST CONDITIONS
- Adjustment of pH: no

PERFORMANCE OF THE TEST
The synthetic medium (=sewage feed,16 mL) made up to 50 mL with Milli-RO water (tap-water purified by reverse osmosis (Millipore Corp., USA)) and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined in the vessels (with the exception of blank control vessel 3 till 6 and nitrification control vessel 2). Thereafter 250 mL activated sludge was added. This was the start of the test. After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer. The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for all test/reference item concentrations and controls. The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels were identically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

OXYGEN RECORDING
Determination of oxygen was performed with multiple oxygen sensors connected to a BlueBox (GOSystemelektronik GmbH, Germany), a multichannel measuring and controlling system.
Reference substance (positive control):
yes
Remarks:
(3,5-dichlorophenol)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Details on results:
- The combined limit/range-finding test showed no inhibition, 1 and an average of 45% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The inhibition observed at 1000 mg/L was statistically significant.
- Based on the available results, a NOEC/EC10 could not be derived
- There was no significant oxygen uptake from abiotic processes.
- The result at 1000 mg/L with a nitrification inhibitor showed that the heterotrophic inhibition of the respiration rate was not inhibited.
- Based on the results of the total and heterotrophic inhibition, nitrification was calculated to be completely inhibited. Since, the heterotrophic respiration was stimulated and nitrification is calculated using the total and heterotrophic respiration rate the inhibition of nitrification could be overestimated. Since, no distorted or biphasic dose-response curve for total respiration was observed the effect on nitrification was not required according to the guidelines and was therefore not further investigated.
Results with reference substance (positive control):
EC50: 5.589 mg/L

The respiration rate of nitrification control vessel 2 could not be determined. No linear part in the oxygen depletion curve was observed. Evaluation: No statistical analysis was performed to assess for nitrification processes. Instead, the results obtained with the remaining nitrification control 1 were used for further calculations. The nitrification inhibition is a non-essential parameter since effects on the total respiration rate followed a normal dose-response effect.

Validity criteria fulfilled:
yes
Conclusions:
In a study in accordance with OECD 209 (2010) and according to GLP principles, an 3h-EC50 value of >1000 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria.
Executive summary:

In accordance with OECD 209 (2010) and according to GLP principles, the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. A combined limit/range-finding test was performed, with loading rates of 10, 100 and 1000 mg/L. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. A statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L (average of 45% inhibition). The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria. Based on the results of the study, an 3h-EC50 value of >1000 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria. No EC10 or NOEC could be derived.

Description of key information

In a study in accordance with OECD 209 (2010) and according to GLP principles, an 3h-EC50 value of >1000 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

In accordance with OECD 209 (2010) and according to GLP principles, the influence of the substance on the respiration rate of activated sludge was investigated after a contact time of 3 hours. A combined limit/range-finding test was performed, with loading rates of 10, 100 and 1000 mg/L. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. A statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg/L (average of 45% inhibition). The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria. Based on the results of the study, an 3h-EC50 value of >1000 mg/L was determined for the inhibitory effect of the substance on activated sludge bacteria. No EC10 or NOEC could be derived.