Pregnenolone

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Dec 2017 - 18 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

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 from the approximate centre of the test vessels, at t=0 h and t=48 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 were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to 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.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks
- Age at test initiation: <24 h
- Feeding during test: no feeding
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (as CaCO3)

Test temperature:

20°C

pH:

At t=0 h: 7.9-8.0
At t=48 h: 8.2-8.3

Dissolved oxygen:

At t=0 h: 8.6-8.8 mg/L
At t=48 h: 8.6-8.7 mg/L

Nominal and measured concentrations:

Nominal: Solutions containing 1.0, 10 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L
Measured: Only Control and 100% SS sample were measured. The 100% SS sample remained stable throughout the test period. The measured concentration was considered the maximum soluble concentration in test medium. therefore effect parameters were based on measured concentrations. See Table 1 in 'Any other information on results' for details on measured concentrations.

Details on test conditions:

The test was performed as Combined Limit/Range-Finding test.

TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4 for undiluted SS, 2 for 1.0 and 10% SS.
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 34 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis.
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH and dissolved oxygen: at the beginning and at the end of the test, for the control and highest test concentration. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 h and at 48 h

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Jan 2018)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.41 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

- No immobility was observed in the control and at any of the test concentrations throughout the test duration. In the control, 15% of the daphnids was trapped at the surface after 24 hours of exposure. Since the deviation was minimal (i.e. 15% instead of maximum 10%) and no effects (e.g. entrapment at the surface of the test solution; including immobility) were observed in the control and any test concentration after 48 hours of exposure, the test was considered valid.
- Mortality of control: none
- 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
- The 24h-EC50 was 0.87 mg/L with a 95% confidence interval between 0.80 and 0.95 mg/L.
- The 48h-EC50 was 0.54 mg/L with a 95% confidence interval between 0.49 and 0.61 mg/L.
- actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Any other information on results incl. tables

Table 1: Combined Limit/Range-finding Test: Test Samples

Time of sampling [hours]	Percentage of SS^# [%]	Analyzed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	100	0.410
48	0	n.d.	n.a.
	100	0.350	85

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

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

n.d. Not detected.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility

Time (h)	Replicate	Test substance; %SS prep. at a loading rate of 100 mg/L
		Control	1.0	10	100 [0.41]
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	20	20	20
24	A	0(1)	0(1)	0(1)	0
	B	0	0(1)	0	0
	C	0(1)			0
	D	0(1)			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total immobilised	0	0	0	0
	Effect %	0	0	0	0

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility,

[ ] between brackets: measured concentration given as mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 48h-EC50 for Daphnia magna exceeded the maximum solubility of the test item in test medium, i.e. exceeded a measured concentration of 0.41 mg/L.

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to Saturated Solutions of the test item prepared at a nominal loading rate of 100 mg/L (undiluted SS and untreated control: 4 replicates; 1.0 and 10% SS: 2 replicates). Measured concentration in the undiluted SS was 0.41 mg/L at t=0 h and remained stable throughout the test. Therefore, effect parameters were based on measured initial concentrations. No immobility was observed in the control and at any of the test concentrations. The 48h-EC50 for Daphnia magna exposed to the test item exceeded the maximum solubility of the test item in test medium, i.e. > 0.41 mg/L. The study is considered to be reliable without restrictions.