CGL 210

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Commission Directive 87/302/EEC, Part C, Activated Sludge Respiration Inhibition Test, 1988

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

- Nominal final test item concentrations: 6.3, 12.5, 25, 50 and 100 mg/L (concentrations exceeding 100 mg test item per liter were not tested).
According to the results of a pre-test, no stock solution could be prepared due to the low water solubility of the test substance.
- Test item amounts of 3.2, 6.7, 12.5, 25.1 and 49.9 mg were directly weighed (by means of an analytical balance) into the test flasks. Then 284 mL of tap water was added to each flask. The test item was mixed into the tap water by ultrasonic treatment over 15 minutes and intense stirring over 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 200 mL of the activated sludge inoculum were added.
The stirring period of 24 hours was chosen as a convenient period, since no information was available about the stability of the test item in water.
- Two controls (tap water, synthetic wastewater and activated sludge inoculum, without test item) were tested in parallel under otherwise identical test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Fullinsdorf, Switzeriand) 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 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 6.6 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:

20°C

pH:

Start of the test: pH = 7.9-8.1 for the control and pH = 8.0 for the test concentrations
End of the test: pH = 8.8 for the control and pH = 7.5-8.8 for the test concentrations

Dissolved oxygen:

Start of the test: 7.2-7.5 mg/L for the control and 7.5-7.8 mg/L for the test concentrations
End of the test: 7.3-8.4 mg/L for the control and 6.9-7.3 mg/L for the test concentrations

Salinity:

no data

Nominal and measured concentrations:

Nominal concentrations: 6.3, 12.5, 25, 50 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type closed
- Material, size, headspace, fill volume: 1000 mL glass flasks

At the start of the test 200 mL activated sludge inoculum with a sludge concentration of 2.0 g dry weight/L (corresponding to about 0.8 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 solutions 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.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- No. of vessels per positive control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Synthetic wastewater: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 x 2 H2O, 0.2 g MgSO4 x 7 H2O, 2.8 g K2HPO4, filled up to a final volume of 1 L with deionized water
- Culture medium different from test medium: yes
- Intervals of water quality measurement: The pH-values and the dissolved oxygen concentrations were determined in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and end of the incubation period and was continuously monitored in all test media and the controls during measurement of the respiration rate. Before the addition of activated sludge, the appearance of the test media was recorded.

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 0X1 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 6.3 mg/L the test item was not completely dissolved in the test media. Thus, all test concentrations were cleariy above the water solubility limit of the test substance under the present test conditions.
Up to and including the 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 3 hours.
The inhibition of the respiration rates of the activated sludge after the incubation period were in the range of -18.3 to 5.1% compared to the controls. No concentration effect relationship was determined.
Thus, the 3-hour NOEC (EC15) of the test substance to activated sludge microorganisms was at least 100 mg/L or higher. The 3-hour EC20, EC50 and EC80 could not be calculated, but were clearly higher than 100 mg/L.
The oxygen consumption rates of the two controls (run at the start respectively the end of the test) differed only by 0.6% (guideline-recommended maximum variation: 15%).

Results with reference substance (positive control):

The 3-hour EC50 ofthe reference item 3,5-dichlorophenol (positive control) was calculated to be 15 mg/L with 95% confidence limits from 11 to 20 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5-30 mg/L, confirming the suitability of the activated sludge used.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (3h) of the test substance is higher than 100 mg/L.

Description of key 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

Additional information

A study was performed according to OECD guideline 209 and GLP (RCC Ltd. 2002h). 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 (3h) is at least 100 mg/L and the EC50 (3h) is higher than 100 mg/L.