3β-hydroxyandrost-5-en-17-one acetate

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:

2017-05-17 to 2017-06-16

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: M16GB2547
- Expiration date of the lot/batch: 01 July 2018 (retest date)
- Purity test date: 26 August 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, weighed amounts were added to the dark brown test-bottles containing 200 mL Milli-RO water. tTe test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 9 to 18 minutes. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test item and test medium was ensured applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.
- Controls: blank controls: test medium without test item and treated in the same way as the test item solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: predominantly domestic sewage treatment plant, "Waterschap Aa en Maas", 's-Hertogenbosch, The Netherlands.
- Preparation of inoculum for exposure: coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ~105°C to determine the amount of suspended solids.
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium was added per L of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: 3.0 g/L of sludge

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

After 3 h of exposure, oxygen consumption was recorded for approximately 10 minutes.

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between 19 and 21°C during the test.

pH:

- Combined limit/range-finding test:
* The pH in all test vessels, before addition of sludge was 7.5.
* After the 3 hour exposure period the pH was between 7.1 and 7.9.
- Final test:
* The pH in all test vessels, before addition of sludge was between 7.4 and 7.5.
* After the 3 hour exposure period the pH was between 7.3 and 7.8.

Dissolved oxygen:

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

Salinity:

not applicable

Nominal and measured concentrations:

Loading rates for the test substance:
- Combined limit/range-finding test: 10, 100, 1000 mg/L
- Final test: 100, 180, 320, 560 and 1000 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: All dark brown open bottles/vessels
- Aeration: After 3-hour contact time, oxygen consumption was recorded for a period of approximately xx minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.
- Number of replicates: 5 (final test)
- Number of control replicates: 6
- Biomass loading rate: initial loading ca. 3.0 g dw/L

TEST MEDIUM / WATER PARAMETERS ADJUST IF DIFFERENT
- Source/preparation of dilution water:
Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3: 46.7 mg/L
KCl: 4.2 mg/L

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system. Oxygen consumption was monitored for approximately 10 min after the 3-h exposure period. Respiration rate was calculated from the measurements.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 1.8
- Range finding study: yes
- Test concentrations range finding study: loading rates of 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

other: ELR50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: NOELR

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data
- Blank controls oxygen uptake rate: 22 mg O2/g.h
- Coefficient of variation of oxygen uptake rate in control replicates: 12

Results with reference substance (positive control):

3-h EC50 = 2.6 mg/L (in the final test).
This is within the recommended range of 2-25mg/L confirming suitability of the activated sludge used.

Reported statistics and error estimates:

ECx and ELRx: For the reference item, calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using a non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For JNJ-83317-AAA (T008506) no ELR50-value could be calculated because effects were below 50% (ELR50 > 1000 mg/L).

* NOELR determination: An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

An activated sludge respiration inhibition test (according OECD guideline 209) with activated sludge from a predominantly domestic sewage treatment plant resulted in a 3-h ELR50 >1000 mg T008506/L. In conclusion,JNJ-83317-AAA (T008506) was not toxic to activated sludge at or below a loading rate of 320 mg/L. The results of the study can be considered reliable without restriction.

Description of key information

The study of Buitenweg (2017), investigating the toxicity of T008506 to microorganisms according to OECD guideline 209, was considered as the key study for endpoint coverage.

This study exposed activated sludge of a sewage treatment plant treating predominantly domestic sewage to a series of test substance concentrations (100, 180, 320, 560 and1000 mg/L) during 3 hours in a static test design, at a temperature of 20°C. The test item was not sufficiently soluble to allow the preparation of a 10 g/L stock solution in water. Therefore, the test item – Milli-RO water mixtures were magnetically stirred for a period of approximately 9 -18 minutes. The criteria for a valid test were fulfilled. The inhibitory effect of the test item T008506 on the respiration rate of aerobic waste water microorganisms of activated sludge resulted in a 3-h ELR50 >1000 mg/L. The 3-h NOELR was determined to be 320 mg/L.

T008506 was not toxic to activated sludge at or below a loading rate of 320 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

320 mg/L