Androst-4-ene-3,17-dione

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 to 21 Oct 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The substance was not completely soluble in test medium at the loading rates initially prepared. Preparation of test solutions started with loading rates of 10 and 100 mg/l. The loading rate of 100 mg/l was stirred for two days, whereas the loading rate of 10 mg/l was stirred for 1 hour. Both
were shortly treated with ultrasonic waves and then filtered through a 0.45 µm membrane filter. The clear and colourless filtrates were used as test solution. In addition, both filtered solutions were used to prepare lower test concentrations by subsequent dilution in test medium.

At the start of the test, the actual test concentrations were 0.85, 2.6, 8.3, 17 and 54 mg/L in samples taken from the solutions representing 10, 32, 100% of the 10 mg/L filtrate and 32 and 100% of the 100 mg/l filtrate. During the 72-hour test period the measured concentrations remained fairly stable with decreases less than 40%. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded to 0.79, 2.5, 8.0, 17 and 39 mg/L.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar; the suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C
- Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Stock culture medium: M1; according to the NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/ml. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use
- Pre-culture medium: M2; according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA) preventing precipitation
- Cell density: 10 E+4 cells/ml

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

No data

Test temperature:

21.3 - 22.9 °C, continuously measured

pH:

7.9 -8.2, measured at the beginning and at the end of the test.

Dissolved oxygen:

No data

Salinity:

n.a.

Nominal and measured concentrations:

At the start of the test, the actual test concentrations were 0.85, 2.6, 8.3, 17 and 54 mg/L in samples taken from the solutions representing 10, 32, 100% of the 10 mg/L filtrate and 32 and 100% of the 100 mg/L filtrate. During the 72-hour test period the measured concentrations remained fairly stable with decreases less than 40%.
Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded to 0.79, 2.5, 8.0, 17 and 39 mg/L.

Details on test conditions:

TEST CONDITIONS:
- Test vessels: 100 mL, all-glass, containing 50 mL of test solution
- Medium: M2
- Cell density: An initial cell density of 1 x 10E+4 cells/ml
- Illumination: Continuously using TLD-lamps of the type 'Cool-white' of 30 Watt, with a light intensity within the range of 77 to 88 µE.m-2.s-1
- Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned; During incubation the algal cells were kept in suspension by continuous shaking
- Test concentrations: 0.45 µm filtrates prepared at 10 and 100 mg/l and dilutions representing 32% of the 100 mg/L filtrate and 10 and 32% of
the 10 mg/L filtrate
- Controls: Test medium without test substance or other additives
- Replicates: 3 replicates of each test concentration; 6 replicates of the control; 1 extra replicate of each test group for sampling purposes; 2 replicates of the highest test concentration without algae

SAMPLING:
- Frequency: at t=0 h, t=24 h and t=72 h
- Volume: 2 mL
- Storage: Samples were stored in a freezer until analysis

MEASUREMENTS:
- pH: At the beginning and at the end of the test; the pH of the solutions should preferably not deviate by more than 1.5 units during the test
- Temperature of medium: Continuously in a temperature control vessel
- Appearance of the cells: At the end of the final test microscopic observations were performed on the test concentration closest to the EC50 to
observe for any abnormal appearance of the algae
- Recording of cell densities: Cells were counted using a microscope and a counting chamber

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.9 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 5.8 - 11 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 1.5 - 3.5 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

25 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 18 - 33 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 5.6 - 12 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

Algae were exposed for a period of 72 hours to K2Cr2O7 (Potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l and to a control.
The EC50 for growth rate reduction (ERC50: 0-72h) was 1.5 mg/l with a 95% confidence interval ranging from 1.1 to 2.1 mg/l. The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/l. Hence, the ErC50: 0-72h for the algal culture tested corresponds with this range.

Validity criteria for the measurement of the toxicity to algae:

Target condition according to guideline:	Actual condition according to the study:	Validity criteria met:
Exponentially growing test organisms are exposed to the test substance in batch cultures over a period of normally 72 hours.	Desmodesmus subspicatus was used as a test organisms. Exponential growth of the algae was confirmed. The test duration was 72 h.	Yes
For the final definitive test at least five concentrations, arranged in a geometric series with a factor not exceeding 3.2, should be selected.	Five concentrations with a spacing factor of below 3.2 were tested.	Yes
The cultures should be maintained at a temperature in the range of 21 to 24°C, controlled at ± 2°C.	The test temperature was 22 +/- 1°C.	Yes

Validity criteria fulfilled:

yes

Remarks:

cell density (control) increased > 16 within 2 d; variation for section-by-section specific growth rates (control) < 35%; variation of average specific growth rates during the whole test period in replicate control cultures < 7%.

Conclusions:

Regarding toxicity of Androstendion towards algae a 72 h-EC50 for growth rate reduction based on TWA concentrations of 25 mg/L was determined (95 % confidence interval 18 to 33 mg/L). The NOEC for growth rate reduction equalled a TWA concentration of 2.5 mg/L.

Executive summary:

The study procedures were based on the OECD guideline No. 201, 2006. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.3, 2008; Amended by EC No. 761/2009, the ISO International Standard 8692, 2004 and the OECD series on testing and assessment number 23, 2000. A final test was performed based on the results of a combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to a control, while three replicates were exposed to test groups representing 10, 32 and 100% of a 10 mg/L filtrate and 32 and 100% of a 100 mg/l filtrate. The total test period was 72 hours and the initial algal cell density was 10E+4 cells/mL. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

At the start of the test, the actual test concentrations were 0.85, 2.6, 8.3, 17 and 54 mg/L in samples taken from the solutions representing 10, 32, 100% of the 10 mg/l filtrate and 32 and 100% of the 100 mg/L filtrate. During the 72-hour test period the measured concentrations remained fairly stable with decreases less than 40%. Based on these results, the Time Weighted Average (TWA) exposure concentrations were calculated. The range tested based on TWA concentrations corresponded to 0.79, 2.5, 8.0, 17 and 39 mg/L. Androst-4-ene-3,17-dione reduced growth rate and inhibited the yield of the fresh water algae species significantly at a TWA concentration of 8.0 mg/L and higher.

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

The 72 h-EC50 for growth rate reduction based on TWA concentrations was 25 mg/L (95 % confidence interval 18 to 33 mg/L). The NOEC for growth rate reduction equalled a TWA concentration of 2.5 mg/L.

Description of key information

Regarding toxicity of Androstendion towards algae a 72 h-EC50 for growth rate reduction based on TWA concentrations of 25 mg/L was determined (95 % confidence interval 18 to 33 mg/L). The NOEC for growth rate reduction equalled a TWA concentration of 2.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

25 mg/L

EC10 or NOEC for freshwater algae:

2.5 mg/L