Pregnenolone

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Dec 2017 - 19 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006 (Annex 5 corrected 28 July 2011)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pregnenolone

EC Number:

205-647-4

EC Name:

Pregnenolone

Cas Number:

145-13-1

Molecular formula:

C21H32O2

IUPAC Name:

3-hydroxypregn-5-en-20-one

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: white crystalline powder
- Storage conditions: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations
- Sampling method: 2.0 mL at t=0 h, t=24 h and t=72 h
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (i.e. 32% of the SS) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium.Test solutions were prepared with a nominal loading rate of 100 mg/L applying a 30-minute period of ultrasonic waves followed by a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- 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: Test medium without test item or other additives

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: 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.
- Pre-culture: Three 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 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”)
- Pre-culture medium and test medium: M2, according to OECD 201

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21 - 23°C

pH:

At t=0 h: 8.0-8.2
At t=72 h: 8.1-8.2

Nominal and measured concentrations:

Nominal: Solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L.
Measured: Samples from 32 and 100% of SS and the control were analysed. The initial concentrations were 0.11 and 0.37 mg/L, respectively. At the end of the test the measured concentration remained stable for the highest test concentration, but not in the 32% SS sample. The effect parameters were based on measured initial concentration in the undiluted SS. See 'Details on results' and 'Any other information on results' for more details on the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cell density: 1 x 10^4 cells/mL
- Control end density (mean): 207 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure
- 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap water purified by reverse osmosis
- Intervals of water quality measurement: pH: at the beginning and at the end of the test. Temperature: continuously in a temperature control vessel.
- Appearance of the cells: at the end of the final test, microscopic observations were performed on the highest test concentration to observe for any abnormal appearance of the algae compared to the control.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 86 to 88 µE/m2/s

EFFECT PARAMETERS MEASURED
- Cell density at t=24, 48 and 72 h
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Combined Limit/Range-finding study test concentrations: Control and Solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L.
- Results used to determine the conditions for the definitive study: yes; 2.5% inhibition was observed in the highest test concentration but due to issues with the measured concentrations, a final test was performed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jan 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- Measured concentrations: Samples taken from solutions containing 32 and 100% of the SS prepared at a loading rate of 100 mg/L and the control were analysed. The initial concentrations were 0.11 and 0.37 mg/L, respectively. At the end of the test the measured concentration remained stable for the highest test concentration. After 24 hours the measured concentration remained stable at the solution containing 32% of the SS. However, this concentration had increased to 191% of initial at the end of the test. Therefore, the corresponding reserve sample was analysed. However, a response of 182% of initial was determined in the reserve sample. It is unclear why this response was observed at the end of the test. The concentration measured in the samples without algae was stable throughout the test duration. Similar as in the combined limit/range-finding test, a response was measured in the control sample at the end of the final test. Subsequently, the reserve control sample was analysed but showed a comparable measured response. It is unclear why this response is observed.
- Exponential growth in control: yes
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the undiluted SS when compared to the control.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 for growth rate inhibition (72h-ErC50) was 0.86 mg/L with a 95% confidence interval ranging from 0.84 to 0.88 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. The observed 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical significance: Dunnett’s Multiple t-Test Procedure, α=0.05, one-sided, smaller
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 3          
Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis (a)	Percentage of SS (b) [%]	Analyzed concentration [mg/L]	Relative to initial [%]
0	08 Jan 2018	17 Jan 2018	0	n.d.
			32	0.110
			32 (c)	0.112
			100	0.369
24	09 Jan 2018	17 Jan 2018	0	n.d.	n.a.
			32	0.114	104
			32 (c)	0.114	101
			100	0.344	93
72	11 Jan 2018	17 Jan 2018	0	0.142	n.a.
		19 Jan 2018	0 (d)	0.153	n.a.
		17 Jan 2018	32	0.210	191
		19 Jan 2018	32 (d)	0.199	182
		17 Jan 2018	32 (c)	0.108	96
			100	0.350	95

(a)   Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(b)   Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

(c)   Without algae.

(d)   Reserve sample.

n.d.  Not detected.

Table 2: Growth Rate And Percentage Inhibition For The Total Test Period

Test substance (mg/L)  % SS prep. at 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.777	0.0237	6
10	1.765	0.0162	3	0.63
18	1.746	0.0200	3	1.7
32	1.771	0.0077	3	0.33
56	1.751	0.0050	3	1.5
100	1.794	0.0088	3	-0.98

Table 3: Growth Rate And Percentage Inhibition At Different Time Intervals

Test substance (mg/L)  % SS prep. at 100 mg/L	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.392	0.0	1.561	0.0	1.377	0.0
10	3	2.354	1.6	1.579	-1.1	1.364	0.90
18	3	2.383	0.39	1.502	3.8	1.354	1.7
32	3	2.365	1.1	1.571	-0.62	1.376	0.059
56	3	2.348	1.8	1.557	0.28	1.347	2.1
100	3	2.421	-1.2	1.629	-4.3	1.331	3.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The ErC50 and ErC10 exceeded a measured concentration of 0.37 mg/L, being considered the maximum soluble concentration in medium. The 72h-NOErC was 0.37 mg/L.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to Saturated Solutions of the test item prepared at nominal loading rate of 100 mg/L (10, 18, 32, 56 and 100%, 3 replicates per concentration) and an untreated control (6 replicates). The initial measured concentration was 0.37 mg/L in the undiluted SS, and this concentration remained stable throughout the test period. Therefore, effect parameters were expressed in terms of concentration initially measured in the undiluted SS. No dose-related effects were observed in this study. The EC50 for growth rate inhibition (72h-ErC50) and 72h-ErC10 were >0.37 mg/L, the maximum soluble concentration in medium. The 72h-NOErC was 0.37 mg/L. The study met all validity criteria, and is considered reliable without restriction.