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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
None
Analytical monitoring:
yes
Details on sampling:
No data
Vehicle:
not specified
Test organisms (species):
activated sludge
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastewater. The sludge was washed once 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 holding, the sludge was fed daily for two days with 50 ml synthetic wastewater* per liter. On the third day of holding immediately prior to use, the sludge was fed once again with 25 ml synthetic wastewater* per liter. During the entire holding period, the sludge was kept at room temperature under continuous aeration. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge was adjusted from 8.4 to 6.3 with a diluted sulfuric acid solution.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
21 °C
pH:
No data
Dissolved oxygen:
7.2 mg/l
Salinity:
No data
Nominal and measured concentrations:
The final nominal test item concentrations were 10, 32, 100, 320 and 1000 mg/l. Concentrations in excess of 1000 mg test item per liter were not tested.
Details on test conditions:
The test was performed in 1000-ml glass flasks. The test flasks were labeled with the RCC project number and all necessary additional information to assure unmistakable identification. At the start of the test 200 ml activated sludge inoculum with a sludge concentration of 2 g dry weight/I (corresponding to about 0.7 g dry material per liter test medium) was added. The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) first to a control, then to the test solutions of the reference item, then to the test suspensions of the test item, and finally to the second control. During the incubation period of exactly 3 hours all test media and the controls were continuously aerated with compressed air at a flow of approximately one liter per minute. The concentration of dissolved oxygen did not drop below 2.5 mg/l during the incubation period, and just before measurement of the respiration rates the dissolved oxygen concentrations were at least 7.2 mg/l. The temperature in the test media, measured in one control, was 21 °C at the start and at the end of the incubation period.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The 3-hour NOEC of FAT 60253/A to activated sludge microorganisms is at least at the limit of water solubility of FAT 60253/A under the present test conditions, or at least nominal 1000 mg/l. This value might be even higher, but concentrations in excess of nominal 1000 mg/l were not tested.
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 17.9 mg/l (the 95% confidence limits were not calculable). The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/l, confirming suitability of the used activated sludge.
Reported statistics and error estimates:
None

None

Validity criteria fulfilled:
yes
Conclusions:
The 3-hour NOEC of FAT 60253/A to activated sludge microorganisms is at least at the limit of water solubility of FAT 60253/A under the present test conditions, or at least nominal 1000 mg/l.
Executive summary:

The inhibitory effect of the test item FAT 60253/A on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the Commission Directive 87/302/EEC, and the OECD Guideline for Testing of Chemicals, No. 209. The following nominal concentrations of FAT 60253/A were tested: 10, 32, 100, 320 and 1000 mg/l. Concentrations in excess of nominal 1000 mg/l were not tested. 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 nominal 1000 mg/l the test item FAT 60253/A had no significant inhibitory effect (<15 %) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test suspensions, the saturation concentration (equilibrium) under the present test conditions was reached and resulted in less than 15 % inhibition after three hours contact time. Thus, the 3-hour EC50 is clearly higher than the limit of water solubility of FAT 60253/A under the present test conditions, or higher than nominal 1000 mg/l. The 3-hour NOEC of FAT 60253/A to activated sludge microorganisms is at least at the limit of water solubility of FAT 60253/A under the present test conditions, or at least nominal 1000 mg/l. This value might be even higher, but concentrations in excess of nominal 1000 mg/l were not tested. The 3-hour EC20 and EC80 could not be calculated, but are clearly higher than the limit of water solubility of FAT 60253/A, or higher than nominal 1000 mg/l.

Description of key information

The 3-hour NOEC of FAT 60253/A to activated sludge microorganisms is at least at the limit of water solubility of FAT 60253/A under the present test conditions, or at least nominal 1000 mg/l.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

In a key study, the inhibitory effect of the test item FAT 60253/A on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the Commission Directive 87/302/EEC, and the OECD Guideline for Testing of Chemicals, No. 209. The following nominal concentrations of FAT 60253/A were tested: 10, 32, 100, 320 and 1000 mg/l. Concentrations in excess of nominal 1000 mg/l were not tested. 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 nominal 1000 mg/l the test item FAT 60253/A had no significant inhibitory effect (<15 %) on the respiration rate of activated sludge after the incubation period of three hours. Based on the physical appearance of the test suspensions, the saturation concentration (equilibrium) under the present test conditions was reached and resulted in less than 15 % inhibition after three hours contact time. Thus, the 3-hour EC50 is clearly higher than the limit of water solubility of FAT 60253/A under the present test conditions, or higher than nominal 1000 mg/l. The 3-hour NOEC of FAT 60253/A to activated sludge microorganisms is at least at the limit of water solubility of FAT 60253/A under the present test conditions, or at least nominal 1000 mg/l. This value might be even higher, but concentrations in excess of nominal 1000 mg/l were not tested. The 3-hour EC20 and EC80 could not be calculated, but are clearly higher than the limit of water solubility of FAT 60253/A, or higher than nominal 1000 mg/l.