Estra-4,6-dien-3-one, 17-hydroxy-, (17.beta.)-

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:

23 Jan 2012 to 23 Feb 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to internationally accepted guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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:

(1S,3aS,3bR,9aR,9bS,11aS)-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

EC Number:

700-803-2

Cas Number:

14531-84-1

Molecular formula:

C18H24O2

IUPAC Name:

(1S,3aS,3bR,9aR,9bS,11aS)-1-hydroxy-11a-methyl-1H,2H,3H,3aH,3bH,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-one

Constituent 2

Reference substance name:

Δ6-nandrolon

IUPAC Name:

Δ6-nandrolon

Details on test material:

Identification Δ6-nandrolon

Molecular formula C18H24O2
Molecular weight 272.39
CAS Number 14531-84-1
Description White crystalline powder (determined at NOTOX)
Batch PPEMC0010 (taken from label)
Purity Not indicated by the sponsor; treated as 100% pure
Test substance storage At room temperature protected from light
Stability under storage conditions Stable
Expiry date 30 April 2012
6.1.2. Study specific test substance information
Volatile No
Test substance handling
Use amber-coloured glassware or wrap container in tinfoil.
Stability in water Not indicated
Solubility in water Not indicated

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the
schedule below. The method of analysis is described in the appended Analytical Report
(APPENDIX 6).
Frequency at t=0 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 and the end of the test period.
Additionally, 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

Details on test solutions:

Δ6-nandrolon Filtered solutions prepared at loading rates of 1.0, 3.2, 10,
32 and 100 mg/l
Controls Test medium without test substance or other additives
Replicates 3 replicates of each test concentration;
6 replicates of the control;
1 replicate of the highest concentration without algae.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is an unicellular algal species sensitive to toxic
substances in the aquatic ecosystem and has been selected
as an internationally accepted species.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Details on test conditions:

Test duration 72 hours
Test type Static
Test vessels 100 ml, all-glass, containing 50 ml of test solution
Medium M2
Cell density An initial cell density of 1 x 104 cells/ml.
Illumination Continuously using TLD-lamps of the type „Cool-white‟ of
30 Watt, with a light intensity within the range of 74 to
86 E.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.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

4

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72

Dose descriptor:

EC50

Effect conc.:

15

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72

Dose descriptor:

NOEC

Effect conc.:

2.8

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72

Dose descriptor:

EC10

Effect conc.:

2.1

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72

Dose descriptor:

EC50

Effect conc.:

6.6

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The mean cell densities measured during the combined limit/range-finding test are presented in Table
1. Table 2 presents the percentages growth rate reduction and yield inhibition per concentration. Note
that from 24 hours of exposure onwards a test substance precipitate and a floating layer were
observed at the highest test concentration. Based on the results samples taken from the filtrates
prepared at 1.0 and 100 mg/l were analysed. The initial concentrations were 0.97 and 53 mg/l. These
concentrations remained stable during the test period (81-101% of initial, see also Table 2 of the
appended Analytical Report). The expected EC50 for growth rate reduction was between
concentrations obtained in filtrates prepared at 10 and 100 mg/l. The expected EC50 for yield inhibition
was between concentrations obtained in filtrates prepared at loading rates of 1.0 and 10 mg/l.
All test conditions were maintained within the limits prescribed by the protocol.

Any other information on results incl. tables

Mean cell densities (x 104 cells/ml) during the final test

Loading rate1 Δ6-nandrolon (mg/l)	Exposure time (hours)
	0	24	48	72
control	1.0	4.0	17.6	77.0
10% of 1.0	1.0	4.3	17.3	79.8
1.0	1.0	4.5	24.9	96.4
10	1.0	3.6	6.4	30.8
100	1.0	1.5	1.3	1.0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The EC50 for growth rate reduction (ERC50: 0-72h) was 15 mg/l with a 95% confidence interval ranging
from 9.8 to 24 mg/l.
The EC50 for yield inhibition (EYC50: 0-72h) was 6.6 mg/l with a 95% confidence interval ranging from
4.2 to 10 mg/l.

Executive summary:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Δ6-nandrolon reduced growth rate and inhibited the yield of this fresh water algae species significantly at 9.6 mg/l and higher. The EC50 for growth rate reduction (ERC50: 0-72h) was 15 mg/l with a 95% confidence interval ranging from 9.8 to 24 mg/l. The EC50 for yield inhibition (EYC50: 0-72h) was 6.6 mg/l with a 95% confidence interval ranging from 4.2 to 10 mg/l. The NOEC for both growth rate reduction and yield inhibition was 2.8 mg/l.