3β-hydroxyandrost-5-en-17-one acetate

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:

2017-06-09 to 2017-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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: 2018-07-01 (retest date)
- Purity test date: 2018-08-26 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not indicated

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples were taken before the start of the test and after 24 and 72 hours from each test medium and from the control. schedule below. In addition, the filter containing undissolved residue was kept for possible analysis. Additionally, reserve samples of 2.0 mL were taken for possible analysis. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Stored in a freezer until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an exprected low water solubility of the test item, a saturated solution (SS) was prepared at a loading rate of 100 mg/L applying a 3-day period of magnetic stirring at room temperature followed by filtration through a 0.45 µm membrane filter (RC55, Whatman). The filter was pre-conditioned with a small volume of test solution that was discardedThis SS was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. The final test solutions were all clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: yes
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22-23 °C

pH:

Final test:
t=0h: 8.1
t=72h: 7.9-8.0

Dissolved oxygen:

not applicable

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

Combined limit/range-finding test:
Samples from the control and the undiluted SS were analysed. The actual concentration in 100% SS was 0.95 mg/L at the start of the test, and decreased to 7.0% of the initial concentration at the end of the test. At the end of the test, test item was also detected in the control at a concentration higher than the concentration measured in the undiluted SS. Based on this finding, it could not be excluded that a possible toxic effect was masked and it was decided to perform a full test.

Final test:
nominal test concentrations: 10, 18, 32, 32 (without algae), 56 and 100% of the SS prepared at 100 mg/L
measured test concentration t = 0 h: 0.151, 0.205, 0.286, 0.372, 0.546, 0.896 mg/L
measured test concentration t = 24 h: 0.123, 0.191, 0.151, 0.308, 0.518, 0.627 mg/L
measured test concentration t = 72 h: 0.016*, 0.016*, 0.016*, 0.016*, 0.016*, 0.016* mg/L
TWA concentration: 0.075, 0.10, 0.10, 0.16, 0.24, 0.32 mg/L

* LOD x 0.5 = 0.016

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Initial cells density: 10,000 cells/mL
- Control end cells density: 358 x 10,000 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, according to the OECD 201 Guideline, formulated using Milli-RO water.
- Culture medium different from test medium: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: using TLD-lamps (with a light intensity within the range of 81 to 84 µE.m-2.s-1)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at680 nm using a spectrophotometer with immersion probe (pathlength = 20mm).
- Effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 1.8
- Range finding study: yes
- Test concentrations: 1.0 and 10% of the SS (setup as combined range finder/limit test)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100% v/v saturated solution, equivalent to a loading rate of 100 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 100% v/v saturated solution, equivalent to a loading rate of 100 mg/L Concentration based on the loading rate of the test substance.

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield) > 0.32 mg T008506/L corresponding with a LR of 100 mg/L
- 72-h EC10 (growth rate) > 0.32 mg T008506/L corresponding with a LR of 100 mg/L
- 72-h EC10 (yield) = 0.12 mg T008506/L (95% C.L. 0.073-0.19 mg/L)
- 72-h NOEC (yield) = 0.075 mg T008506/L (based on statistical significance)

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 72-h EC50 (growth rate) = 0.99 mg/L (95% C.L. 0.97 to 1.0 mg/L)
- 72-h EC50 (yield) = 0.37 mg/L (95% C.L. 0.36 to 0.37 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
No EC50-values could be calculated for growth inhibition because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).
The ECx values for yield inhibition were calculated with non-linear CDF (cumulative distribution fiction) regression model.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T008506 according to the OECD guideline 201 (GLP conditions). The EC50 for growth rate inhibition (72h-ErC50) was beyond the range tested, i.e. exceeded an average exposure concentration of 0.32 mg/L, being considered the maximum solubility of test item in a filtrate prepared at a loading rate of 100 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

The study of Tobor-Kaplon (2018), investigating the acute toxicity of T008506 to algae according to OECD guideline 201, was considered as the key study for endpoint coverage. The 72-h NOEC and 72-h EC50 for growth rate were determined to be ≥ 100 mg/L and > 100 mg/L respectively (test concentration based on loading rate). The average exposure concentration of 0.32 mg/L is being considered the maximum solubility of test item in a filtrate prepared at a loading rate of 100 mg/L The substance can be considered as not harmful to algae.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L