[No public or meaningful name is available]

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

Study period:

Experiment start date - 24 January 2005; Experiment completion date - 27 January 2005; Study completion date - 03 May 2005.

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:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Ltd, Environmental Chemistry & Pharmanalytics, CH-4452 Itingen, Switzerland

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

505.5 mg of the test item was directly weighed by means of an analytical balance and transferred to the test flask with 284 mL of tap water. The test item was mixed into the tap water by intense stirring for ten minutes at room temperature to dissolve a maximum amount of the test item, and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used. After the stirring period, 16 mL of synthetic wastewater and 200 mL of the activated sludge inoculum were added. Additionally, two controls containing only tap water, synthetic wastewater and inoculum were tested in parallel to the limit test concentration of the test item, 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, FüIIinsdorf, Switzerland) treating predominantly domestic wastewater. To eliminate possible inhibitory material, the sludge was washed twice 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 period of three 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. 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 8.9 to 7.1 with a diluted sulfuric acid solution.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19-20 °C

pH:

7.9-8.6

Dissolved oxygen:

7.8 - 8.8 mg/L

Nominal and measured concentrations:

- Nominal: 1000 mg/L.
- Measured: no analysis performed.

Details on test conditions:

The test was performed in 2000-mL glass beakers. The test vessels were labeled with the necessary information to ensure unmistakable identification. At the start of the test, synthetic wastewater and activated sludge inoculum were added. The inoculum had a sludge concentration of 3.1 g/L dry weight (corresponding to about 1.2 g dry material per liter test medium). The sludge was added in time intervals of 15 minutes (an arbitrary but convenient interval) in the following order: control, test solutions of the reference item, test solutions of the test item, and second control.

During the incubation period of exactly 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers.

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 TriOxmatic 300 and an oxygen meter WTW Oxi 539, 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 per 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 ten minutes.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

The test item had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours at the limit test concentration of 1000 mg/L. Thus, the 3-hour NOEC (EC15) of FAT 40819/A to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher, but concentrations above 1000 mg/L were not tested. The 3-h EC50 could not be calculated, but was clearly higher than 1000 mg/L. The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 2 % (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 21 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 the suitability of the activated sludge used.

Validity criteria fulfilled:

yes

Conclusions:

The 3-h EC50 value is >1000 mg/L in microorganisms.

Executive summary:

In a GLP-compliant 3-hour respiration inhibition test, performed according to OECD Guideline 209, the inhibitory effect of the test substance on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. One nominal concentration of 1000 mg/L was 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 the suitability of the activated sludge and the method used. The test substance had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. Thus, the 3-h NOEC (EC15) was at least 1000 mg/L. This value might even be higher, but concentrations above 1000 mg/L were not tested. Due to a lack of effects, the 3-h EC50 could not be calculated, but was clearly higher than 1000 mg/L.

Description of key information

The 3-h EC50 value is >1000 mg/L in micro organisms.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

In a GLPcompliant 3-hour respiration inhibition test, performed according to OECD Guideline 209, the inhibitory effect of the test substance on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. One nominal concentration of 1000 mg/L was 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 the suitability of the activated sludge and the method used. The test substance had no significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours. Thus, the 3-h NOEC (EC15) was at least 1000 mg/L. This value might even be higher, but concentrations above 1000 mg/L were not tested. Due to a lack of effects, the 3-h EC50 could not be calculated, but was clearly higher than 1000 mg/L.