3,20-bis(ethylenedioxy)pregna-5,7-diene

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2013 to 18 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In accordance with OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

solid: crystalline

Details on test material:

Slightly yellow crystalline powder

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Batch: HX-1212088-716
Purity/Composition: 95.6%
Expiry date: 23 September 2013 (Retest date)

Sampling and analysis

Analytical monitoring:

no

Test solutions

Details on test solutions:

Since the water solubility of Proketal was very low (<0.01 mg/L), nominal loading rates were prepared for the test. 1-Litre test bottles were filled with 200 mL of test substance mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with initial loading rates of 2.5 times the final loading rate. The test substance was transferred into the test medium after a 10 minute treatment with ultrasonic waves to pre dissolve the test substance in 10-20 mL of Milli-RO. These mixtures were stirred in closed dark brown bottles for approximately 24 hours except for the abiotic control which was stirred for approximately 27 hours. Subsequently, 16 mL synthetic medium, 250 mL sludge and Milli-RO water up to 500 mL were added resulting in the required loading rates. Optimal contact between the test substance and test organisms was ensured applying continuous aeration and stirring.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Micro-organisms in activated sludge from a Municipal sewage treatment plant: 'Waterschap Aaen Maas', Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage.

The number of micro-organisms was determined as the amount of Mixed Liquor Suspended
Solids (MLSS) per litre test medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Post exposure observation period:

After the 3-hour contact time, the oxygen consumption was recorded for a period of 10-15 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer with the exception of a short period

Test conditions

Test temperature:

The test was conducted under normal laboratory lighting in a temperature controlled room at 20 ± 2 ºC.
18.0-20.1 ºC.

pH:

The pH in all test series, before the addition of sludge was between 7.3 and 7.6

Dissolved oxygen:

The dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- Type (delete if not applicable): open
- Aeration: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, which showed normal sensitivity.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Effect concentrations 2

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:

In the combined limit/range-finding test no inhibition of the respiration rate of the sludge was recorded in two replicates at a loading rate of 1000 mg per litre. Since, the inhibition of the respiration rate in one replicate at a loading rate of 1000 mg/L could not be reliably determined, an additional limit test was performed.

Furthermore, there was no oxygen uptake from abiotic processes and 1000 mg/L with a nitrification inhibitor showed no heterotrophic inhibition of the respiration rate. Therefore, N-allylthiourea was not used in the limit test.

Results with reference substance (positive control):

The batch of activated sludge was checked for sensitivity by testing the reference substance 3,5-dichlorophenol, which showed normal sensitivity.

Reported statistics and error estimates:

In the additional limit test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg Proketal / litre (Bonferroni t-Test: α=0.05 Toxstat). The effects observed were in agreement with the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC.

Any other information on results incl. tables

The study met the acceptability criteria prescribed by the protocol and was considered valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test Proketal was not toxic to waste water (activated sludge) bacteria at or below a nominal loading rate of 1000 mg/L (NOEC). The EC50 exceeded a nominal loading rate of 1000 mg/L. This loading rate was far above the very low
water solubility of Proketal (<0.01 mg/L).

This loading rate was far above the very low water solubility of Proketal (<0.01 mg/L).