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Ecotoxicological information

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
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
1. A total number of 21 daphnids was exposed to the 10 mg/l loading rate in the final test. Evaluation: This was taken into account for the determination of the EC50 value. The study integrity was not adversely affected by this deviation.
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
equivalent or similar to
Guideline:
ISO 6341 15 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Qualifier:
equivalent or similar to
Guideline:
other: OECD Number 23, 2000
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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
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

Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the
schedule below. In addition, the filter used to prepare the highest test concentration was retained for
possible analysis of the residue. The method of analysis is described in the appended Analytical
Report (APPENDIX 3).
Frequency at t=0 h and t=48 h
Volume 2 ml from the approximate centre of the test vessels
Storage Samples were stored in a freezer until analysis.
At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 2 ml were taken for possible analysis. If not 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:
Medium M7, as prescribed by Dr. Elendt-Schneider
(Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An
ultrastructural approach to antennal damage in Daphnia
magna Straus. Protoplasma 154, 25-33).
Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (ROwater,
GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following
concentrations:
Trace elements: B 0.125 mg/l
Fe 0.05 mg/l
Mn 0.025 mg/l
Li, Rb and Sr 0.0125 mg/l
Mo 0.0063 mg/l
Br 0.0025 mg/l
Cu 0.0016 mg/l
Zn 0.0063 mg/l
Co and I 0.0025 mg/l
Se 0.0010 mg/l
V 0.0003 mg/l
Na2EDTA.2H2O 2.5 mg/l
Macro nutrients: Na2SiO3. 9H2O 10.0 mg/l
NaNO3 0.27 mg/l
KH2PO4 0.14 mg/

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at
least third generation, obtained by acyclical parthenogenesis
under specified breeding conditions

Source In-house laboratory culture with a known history.
Reason for selection This system has been selected as an internationally
accepted invertebrate species.
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.
Characteristics For the test selection of young daphnids with an age of < 24
hours, from parental daphnids of more than two weeks old.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Immobility (including mortality) At 24 hours and at 48 hours.
pH At the beginning and at the end of the test, for all
concentrations and the control.
Dissolved oxygen At the beginning and at the end of the test, for all
concentrations and the control. In addition after 24 hours,
immediately after counting the immobilized daphnids oxygen
levels were measured in the test container with the solution
of lowest concentration at which all the Daphnia magna were
immobilized.
Temperature of medium Continuously in a temperature control vessel, beginning at
the start of the test.

Test conditions

Test temperature:
18-22°C
Nominal and measured concentrations:
Δ6-nandrolon Filtered solutions prepared at loading rates of 10, 18, 32, 56
and 100 mg/l
Controls Test medium without test substance or other additives

Analysis of the samples taken at the start of the final test showed measured concentrations of 8.7, 17,
30, 51 and 54 mg/l at the loading rates of 10, 18, 32, 56 and 100 mg/l, respectively.
Details on test conditions:
Test duration 48 hours
Test type Static
Test vessels 100 ml, all-glass
Medium Adjusted ISO medium
Number of daphnids 20 per concentration
Loading 5 per vessel containing 80 ml of test solution
Light No illumination
Feeding No feeding
Aeration No aeration of the test solutions.
Introduction of daphnids Within 45 minutes after preparation of the test solutions.

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (ROwater,
GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
Macro salts: CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l
Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following
concentrations:
Trace elements: B 0.125 mg/l
Fe 0.05 mg/l
Mn 0.025 mg/l
Li, Rb and Sr 0.0125 mg/l
Mo 0.0063 mg/l
Br 0.0025 mg/l
Cu 0.0016 mg/l
Zn 0.0063 mg/l
Co and I 0.0025 mg/l
Se 0.0010 mg/l
V 0.0003 mg/l
Na2EDTA.2H2O 2.5 mg/l
Macro nutrients: Na2SiO3. 9H2O 10.0 mg/l
NaNO3 0.27 mg/l
KH2PO4 0.14 mg/l
K2HPO4 0.18 mg/l
Vitamins: Thiamine 75.0 μg/l
B12 1.0 μg/l
Biotin 0.75 μg/l
The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.

Results and discussion

Effect concentrations
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: (18, 24)
Details on results:
24h-EC50= 41 (39,43)
48h-EC50 =20 (18,24)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
1. In the control, no daphnids became immobilised. 2. The oxygen concentration at the end of the test was ≥ 3 mg/l in control and test vessels.
Conclusions:
Under the conditions of the present study Δ6-nandrolon did not induce acute immobilisation of
Daphnia magna at 8.7 mg/l after 48 hours of exposure (NOEC).
The 48h-EC50 was 20 mg/l (95% confidence interval between 18 and 24 mg/l).
Executive summary:

Acute Toxicity Study in Daphnia magna with Δ6-nandrolon. The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, the procedures were designed to meet the test methods of the Commission Regulation (EC) No 440/2008, Part C.2, 2008, the ISO International Standard 6341, 1996 and the OECD series on testing and assessment number 23, 2000. The batch of Δ6-nandrolon tested was a white crystalline powder of unknown purity and not completely soluble in test medium at the initial loading rates prepared. Weighing of test substance was performed under either yellow or dimmed light. Preparation of test concentrations was performed under dimmed light. A final test was performed based on the results of a preceding combined limit/range-finding test. Preparation of test solutions started with loading rates of 10, 18, 32, 56 and 100 mg/l applying two days of magnetic stirring to reach maximum solubility of the test substance in the test medium. The resulting mixtures were filtered through a 0.45 μm membrane filter where after the clear and colourless filtrates were used for testing. Twenty daphnids per concentration (four replicates, five daphnids per replicate) were exposed to a control and the filtered solutions prepared at loading rates of 10, 18, 32, 56 and 100 mg/l. The total test period was 48 hours and samples for analytical confirmation of actual exposure concentrations were taken at the start and the end of the test. Analysis of the samples taken at the start of the final test showed measured concentrations of 8.7, 17, 30, 51 and 54 mg/l at the loading rates of 10, 18, 32, 56 and 100 mg/l, respectively. These concentrations remained stable during the 48-hour test period (98-101% of initial). Given these results, the effect parameters were based on initial exposure concentrations. The results indicated that the solubility limit of Δ6-nandrolon in test medium approximated 50 mg/l. The study met the acceptability criteria prescribed by the protocol and was considered valid. Δ6-nandrolon did not induce acute immobilisation of Daphnia magna at 8.7 mg/l after 48 hours of exposure (NOEC). The 48h-EC50 was 20 mg/l (95% confidence interval between 18 and 24 mg/l, based on initial measured concentrations).