9beta,11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Type: predominantly domestic sewage (content of solid particles: 3.48 g/L)
- Origin: well-operated municipal sewage treatment plant (Kläranlage Berlin-Ruhleben, Germany),
- Microbial inoculum: Upon arrival at the laboratory, the activated sludge was aerated and stirred for approx. 2 hours and for a further 15 minutes without aeration; then it was left to settle for approx. 2 hours
- 3300 mL were taken from the supernatant and mixed with 3300 mL water and 264 mL nutrient solution; the mixture was aerated and stirred for approx. 18 h

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

0.5 h

Hardness:

No data

Test temperature:

22.4 - 23.6 °C

pH:

No data

Dissolved oxygen:

No data

Salinity:

n.a.

Conductivity:

No data

Nominal and measured concentrations:

- Test item concentrations (nominal): 10, 20, 40 and 100 mg/L
- Reference substance concentrations (nominal): 5, 15 and 30 mg/L

Details on test conditions:

I. Preparation of the control, reference and test substance solutions
The test vessels were prepared as follows:
- Control: 200 mL of the pre-culture were filled into a 250 mL Erlenmeyer flask. Then 8 mL of the nutrient solution and 42 mL aqua demineralized were added
- Test compound: The same mixture as above was prepared. The compound was added directly in amounts of 2.5, 5, 10 and 25 mg. This resulted in an inhomogenous suspension
- Reference substance (3,5-dichlorophenol): 200 mL pre-culture were mixed with 8 mL nutrient solution. 2.5, 7.5 and 15 mL of an aqueous stock solution (500 mg/I) were added and filled up to 250 mL
- The test vessels were prepared and incubated at intervals of approx. 7 minutes. Each test concentration of the reference substance and the test compound was incubated in duplicate. For the control, two test vessels were used at the beginning and 3 test vessels at the end of the test series.

II. Incubation conditions:
- The test vessels were incubated under stirring and aerating (45 L/h) for 30 minutes at room temperature.
- The measured temperature after the incubation of each test vessel was between 22.4 and 23.6 °C

III. Measurement of oxygen consumption:
- After the incubation period of 30 minutes approximately 25 mL from the first vessel were introduced into a measuring apparatus by a plastic tube avoiding air bubbles.
- This apparatus consisted of a cylindrical glass container fitted with a polarographic oxygen electrode (Fa. WTW, OXI 92) connected to a recorder
- The test mixture was stirred using a magnetic stirrer; over a period of four minutes the oxygen consumption was measured and recorded
- This determination was repeated for the contents of each vessel at 7-minute intervals, with the result that the incubating time was 30 minutes in each case

IV. Calculation of the inhibition of the respiration rate:
- In order to calculate the inhibitory effect of a test substance at a particular concentration, the respiration rate is expressed as a percentage of the mean of the three control respiration rates
- The inhibition percentage was plotted against concentration and EC20, EC50, EC80 were calculated by the computer probit analysis "Schering, Rechenzentrum" with 95 per cent confidence limits

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (Aldrich)

Key result

Duration:

0.5 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

Details on results:

Since the solubility of the test compound in an aqueous solution was far exceeded - indicated by the inhomogenous turbidity - it can be concluded from the findings that a saturated aqueous solution of Epoxid Valerat has no effects on the microbial population in sewage sludge.

Results with reference substance (positive control):

The reference compound 3,5-dichlorophenol showed an EC50 of 16.2 mg/L

Validity criteria fulfilled:

yes

Conclusions:

A saturated aqueous solution of Epoxid Valerat had no effects on the microbial population in sewage sludge. The reference compound showed an EC50 of 16.2 mg/L, which was in the required range (5 - 30 mg/L) noted in the guidelines (OECD, ISO, EEC). The control variability did not exceed the required value of <15%.

Executive summary:

The study was conducted in accordance with OECD Guideline 209 ‘Activated Sludge, Respiration Inhibition Test, adopted 4. Apr. 1984. The test compound was incubated for 30 minutes as suspensions including nutrients with microorganisms from a municipal sewage treatment plant. Additionally a reference substance (3,5-dichlorophenol) was tested according to the same procedure in order to verify the sensitivity of the test organisms. The test compound was incubated in 4 concentrations 10, 20, 40 and 100 mg/L and the reference substance in three concentrations (5, 15 and 30 mg/L). The test item and reference compound concentrations were not confirmed by analytical methods, they were based on nominal concentrations. Furthermore, additional vessels without test or reference substance were used as controls. All test concentrations were incubated in duplicate except the control, which was incubated in triplicate.

As a parameter for the inhibition of the oxygen consumption, the respiration rate of each test vessel was measured in a closed apparatus over a period of 4 minutes after incubation. The reference compound showed an EC50 of 16.2 mg/L, which is in the required range (5-30 mg/L) noted in the guidelines (OECD, ISO, EEC). The control variability did not exceed the required value of <15%. The results of the respiration inhibition test with activated sludge demonstrate that the test compound exerted relatively low toxicity on the microorganisms of activated sludge. Since the solubility of the test compound in an aqueous solution was far exceeded - indicated by the inhomogenous turbidity - it can be concluded from the findings that a saturated solution of the test compound has no effects an the microbial population in sewage sludge. The observed inhibition of respiration was probably not due to the test compound, particularly since no clear exposure-effect relationship was observed. This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microorganisms.

Description of key information

A saturated aqueous solution of Epoxid Valerat had no effects on the microbial population in sewage sludge. The reference compound showed an EC50 of 16.2 mg/L, which was in the required range (5 -30 mg/L) noted in the guidelines (OECD, ISO, EEC).

Key value for chemical safety assessment

EC50 for microorganisms:

100 mg/L

Additional information

"Should read: EC 50: >100 mg/L"

Since the solubility of the test compound in an aqueous solution was far exceeded - indicated by the inhomogenous turbidity - it can be concluded from the findings that a saturated aqueous solution of Epoxid Valerat has no effects on the microbial population in sewage sludge.