3,20-bis(ethylenedioxy)pregna-5,7-diene

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:

25 February to 28 February, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Done under GLP and OECD methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

other: crystalline powder

Details on test material:

Slightly yellow crystalline powder
Stored at room temperature in the dark.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours and at the end of the 72-hour exposure period.

Chemical analysis:
Singular samples for possible analysis were taken from all test concentrations and the control
according to the schedule below. In addition, the filter used for preparation of test solutions was
retained for possible analysis. The method of analysis is described in the appended Analytical
Report.

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

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 demonstrated by running a test vessel at the highest substance concentration 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.
Additionally, singular reserve samples of 2 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

A limit test combined with a range-finding test was performed. Six replicates of exponentially
growing algal cultures per group were exposed to an untreated control and a 0.45 μm filtered
solution prepared at 100 mg/L in the limit test. In the combined range-finding test, three
replicates per test group were exposed to solutions containing 1.0 and 10% of the filtrate
prepared at a loading rate of 100 mg/L. The test solutions were prepared in M2 medium. In
addition, one or two replicates of each concentration without algae, as a turbidity control or for
sampling, and one replicate of each test group for sampling purposes were also incubated.

All glass and capped, 100 mL erlenmeyers, containing 50 mL of test solutions were used.

The illumination was continuously by using TLD-lamps of the type ‘Cool-white’ (30 Watt), with a
light intensity within the range of 68 to 77 E.m-2.s-1. The capped vessels were distributed at
random in the incubator and were daily repositioned. During incubation the algal cells were kept
in suspension by continuous shaking. The total test period was 72 hours and the initial algal cell
density was 104 cells/mL.

Test organisms

Test organisms (species):

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

Details on test organisms:

As a test species the fresh water algae Pseudokirchneriella subcapitata, strain: NIVA CHL 1 of
the in house laboratory culture were used.

The 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. The illumination was 60 to 120 µE/m2
/s when measured in
the photosynthetically effective wavelength range of 400 to 700 nm. The stock culture was
cultured on M1 medium.
From the stock culture the pre-culture was started 3 days before the start of the experiment being
M2 medium inoculated 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 in the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

At the beginning of the test, cells were counted using a microscope and a counting chamber.
Thereafter cell densities were determined after 24, 48 and 72 hours of exposure by
spectrophotometric measurement of samples at 720 nm using a spectrophotometer with
immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates
without algae as background for the treated solutions.
At the end of the test microscopic observations were performed on the undiluted filtrate to
observe for any abnormal appearance of the algae
The pH was measured at the beginning and at the end of the test in the control and the undiluted
filtrate. The pH of the solutions should preferably not deviate by more than 1.5 units during the
test.
The temperature of the medium was continuously measured in a temperature control vessel,
beginning at the start of the test.

Test conditions

Hardness:

0.24 mmol/L(24 mg CaCO3/L)

Test temperature:

21-24°C

pH:

pH at 0 hrs. = 8.3
pH at 72 hrs. = 8.2

Nominal and measured concentrations:

Nominal test concentrations of 0.18, 0.32, 0.56, 1.0, 1.8, and 3.2 mg/L

Analysis of these additional test preparations showed measured concentrations 1.0, 10, and 100

Details on test conditions:

The 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. The illumination was 60 to 120 µE/m2
/s when measured in
the photosynthetically effective wavelength range of 400 to 700 nm. The stock culture was
cultured on M1 medium.
From the stock culture the pre-culture was started 3 days before the start of the experiment being
M2 medium inoculated 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 in the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Half of the LOD (LOD=15 μg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Half of the LOD (LOD=15 μg/L)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Half of the LOD (LOD=15 μg/L)

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

8 µg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Half of the LOD (LOD=15 μg/L)

Details on results:

Analysis of the samples taken from the filtrate prepared at 100 mg/L showed that the measured
concentration was below the Limit of Detection of the analytical method (LOD=15 μg/L) at both
the start and at the end of the test. On the filter used to prepare the test concentrations the test
substance was identified, which is indicative of the use and removal of Proketal during the test
solution preparation. Therefore, effect parameters were expressed as half of the LOD, namely
8 μ/L.

No significant differences were recorded between the values for both growth rate or yield at any
of the test concentrations when compared to the control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the
cells exposed to the highest concentration when compared to the control.

Reported statistics and error estimates:

Since the measured concentrations were below the Limit of Detection (LOD), the final exposure
concentration(s) were taken as a factor of 2 below the applicable limit. This procedure is based
on the OECD “Guidance document on the use of the harmonised system for the classification of
chemicals which are hazardous for the aquatic environment”.

Statistical analysis of the data was not needed as the effects recorded were not significant(<10%). Therefore, the NOEC was considered to be the highest concentration tested.

No EC50 could be calculated because the test substance proved to be non-toxic (EC >maximum soluble concentration).

Any other information on results incl. tables

Acceptability of the test 1. In the controls, cell density increased by an average factor of >16 within two days. 2. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35%. 3. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Protocol deviations

For the analysis of the test substance container A (batch: 0385) was used instead of container B (batch: HX-121088-716).

Evaluation: Since, both batches have comparable composition this has no effect on the study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, Proketal did not induce growth rate reduction or yield inhibition of
Pseudokirchneriella subcapitata at 8 μg/L after 72 hours of exposure (NOEC). This is the
measured concentration of the undiluted filtrate which is expressed as half of the LOD and
considered the maximum soluble concentration in the test medium.

The EC50 for both growth rate reduction (72h-ERC50) and yield inhibition (72h-EYC50) was
beyond the range tested, i.e. exceeded a concentration of 8 μg/L. This is the measured
concentration in the undiluted filtrate and expressed as half the LOD.

Due to the very low solubility of Proketal in the test medium, concentration levels that might be
toxic to algae could not be reached.