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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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
Based on the results of a pre-experiment, no stock solution could be prepared due to the low water solubility of the test item under the present test conditions.
Therefore, test item amounts of 3.1, 6.2, 12.4, 25.1 and 50.3 mg were directly weighed (by means of an analytical balance) into the respective amber test flasks and mixed into 284 mL of tap water by ultrasonic treatment for ten minutes and intense stirring for 24 hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. Then, 16 mL of synthetic wastewater and immediately prior to incubation the activated sludge inoculum were added.
The final nominal test item concentrations were 6.25, 12.5, 25, 50 and 100 mg/L.
Concentrations exceeding 100 mg test item per liter were not tested. The test item and its dispersions were handled in a dark room under red light.

Additionally, two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) and the reference item 3,5-dichlorophenol (positive control) at nominal concentrations of 5, 16 and 50 mg/L were tested in parallel under otherwise identical test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf, Switzerland) treating predominantly domestic wastwater.
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 the holding of two days prior to use, the sludge was fed daily with 50 mL synthetic wastewater per liter and was kept at room temperature under continuous aeration until use. Immediately before use, the dry weight of the activated sludge was measured again in the inoculum used in the test. The pH of the activated sludge inoculum was adjusted from 8.6 to 7.0 with a diluted sulfuric acid solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
no data
Test temperature:
18°C at the start of the test and 19°C at the end of the test (measured in one control)
pH:
At the start of the test: 7.1-8.1 (control) and 7.9-8.1 (test concentrations)
At the end of the test: 8.8 (control) and 8.8 (test concentrations)
Dissolved oxygen:
At the start of the test: 8.3 mg/L (control) and 8.2-8.3 mg/L (test concentrations)
At the end of the test: 8.4-8.5 mg/L (control) and 7.4-8.6 mg/L (test concentrations)
Salinity:
no data
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50, 100 mg/L
Details on test conditions:
TEST SYSTEM
The test was performed in 1000mL amber glass flasks.
At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 3.0 g dry weight/L (corresponding to about 1.2 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.

TEST MEDIUM / WATER PARAMETERS
- 16 g peptone; 11 g meat extract; 3.0 g urea; 0.7 g NaCl; 0.4 g CaCl2 -2H2O; 0.2 g MgSO4-7H2O; 2.8 g K2HPO4 per liter dechlorinated drinking water.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Measurement of the respiration rates:
For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after exactly three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW OXI 539 meter, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim, Germany), and was continuously recorded for about ten to fifteen minutes. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2/L/minute) was determined from the linear part of the respiration curve in the range of 6.5-2.5 mg O2/L.
In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least 10 minutes
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
other: EC80
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Down to the lowest test concentration of 6.25 mg/L the test item was not homogeneously distributed in the test medium. Thus, all test concentrations were clearly above the water solubility limit of the test substance.
Up to and including the highest concentration of 100 mg/L the test item 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 <6.25 mg/L and resulted in less than 15% inhibition after three hours contact time. Thus, the 3-hour EC20, EC50 and EC8O were clearly higher than the limit of water solubility of the test substance under the present test conditions, or higher than 100 mg/L.The 3-hour NOEC of the test substance to activated sludge microorganisms was at least at the limit of water solubility of the test substance under the present test conditions or at least 100 mg/L. This value might be even higher, but concentrations exceeding 100 mg/L were not tested.
The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 14.9% (guideline-recommended maximum variation: 15%
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 12 mg/L with 95% confidence limits from 3 to 29 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming suitability of the activated sludge used.
Validity criteria fulfilled:
yes
Conclusions:
The EC50 (3h) of the test substance is higher than 100 mg/L (nominal concentration).
Executive summary:

In this guideline (OECD 209) study conducted to GLP standards, the 3 hour EC50 (EC 438-340-0) of the test material to sewage microorganisms was determined to be >100 mg/l (nominal). The test was conducted upon a washed activated sewage sludge taken from a municipal sewage treatment plant. The test material is not expected to inhibit sewerage microorganisms at low concentrations.

Description of key information

Study conducted to recognised testing guidelines with GLP.

Key value for chemical safety assessment

EC50 for microorganisms:
100 mg/L

Additional information

The inhibition of the degradation activity of activated sludge is not anticipated when introduced in appropriate low concentrations. No toxic effects occur at the range of solubility.The study was performed according to OECD guideline 209 and GLP (RCC Ltd. 2002k). Up to and including the concentration of 100 mg/L (nominal concentration), the test substance had no significant inhibitory effect (<15%) on the respiration rate of activated sludge after the incubation period of three hours. The NOEC is at least 100 mg/L and the EC50 (3h) is higher than 100 mg/L.