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Diss Factsheets

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
Study period:
06 March 2017- 20 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
April 13, 2004
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Octan-3-one
EC Number:
203-423-0
EC Name:
Octan-3-one
Cas Number:
106-68-3
Molecular formula:
C8H16O
IUPAC Name:
octan-3-one
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EM14089719
- Expiration date of the lot/batch: 01 August 2017
- Purity test date: 07/07/2016 ( certificate of analysis)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle:
Solubility in vehicle:
• Water 647.0 mg/L, at 20°C and pH= 6.3 (OECD 105)
• Other: …… Not indicated
Stability in vehicle:
• Water Not available
• Other: ….. Not indicated

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: n/a
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 10, 18, 32, 56 and 100 mg/L
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS:
Test facility test item number: 207812/A
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Octan-3-one
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Epiwin prediction vp of ~300 Pa
Specific gravity/density: Not available

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Single samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

- Sampling method:
Frequency: at t=0 h and t=48 h
Volume: 4.0 mL from the approximate centre of the test vessels

- Sample storage conditions before analysis:
Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility. Additionally, reserve samples of 4.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) 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:
- Method:
Preparation of test solutions started with the highest concentration of 100 mg/L applying a magnetic stirring period of 71 minutes (combined limit/range-finding test) or 33 minutes (final test) to accelerate the dissolving of the test item. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Differential loading: n/a
- Controls: Test medium without test item or other additives
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnia magna (Straus, 1820)
- Source: In-house laboratory culture with a known history.
- Age of study initiation (mean and range, SD): Test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old.
- 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
- Feeding during test: no

Other information:
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 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.
- 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 (RO-water, 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, macronutrients 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.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
closed system
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
180 mg/L as (CaCO3)
Test temperature:
The temperature continuously measured in a temperature control vessel varied between 19.9
and 20.5 °C during the test
pH:
Start of the test: 7.9-8.0
End of the test: 8.2-8.4
Dissolved oxygen:
Start of the test: 9.6-9.8 mg/L
End of the test: 8.4-8.6 mg/L
Nominal and measured concentrations:
Nominal: 10, 18, 32, 56 and 100 mg/L

Measured: 7.2, 14, 26, 48 and 89 mg/L

Details on test conditions:
TEST SYSTEM
Test duration: 48 hours
Test type: Static
Test vessels: 60 mL, all-glass,
Type: airtight closed with minimal headspace
Medium: Adjusted ISO medium
Number of daphnids: 20 per concentration
No. of organisms per vessel: 5 per vessel containing 60 mL of test solution
No. of vessels per concentration (replicates): 4
No. of vessels per control (replicates): 4
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnids: Within 41 minutes after preparation of the test solutions

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO 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 (RO-water, 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, macronutrients 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

- Culture medium different from test medium: no

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Daphnia were considered to be immobile if unable to swim within 15 seconds after gentle agitation of the test vessel.

RANGE-FINDING STUDY
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. No immobility was observed up to nominal concentrations of 10 mg/L, while immobility of 90% was observed at the highest tested concentration of 100 mg/L. Therefore, the expected EC50 was between 10 and 100 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
7.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence (17-24 mg/L)
Details on results:
In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discoloration or unusual behaviour such as trapping at the surface of the medium. The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Others:
Analysis of the samples taken at the start of the final test showed that measured concentrations were at 76 to 92% relative to nominal. During the 48-hour test period concentrations remained stable or decreased slightly. Based on these results, the average exposure concentrations were calculated to correspond with 7.2, 14, 26, 48 and 89 mg/L for the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively.

pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.
Results with reference substance (positive control):
The responses in this reference test with potassium dichromate 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).

The 24h-EC50 was 0.64 mg/L with a 95% confidence interval between 0.56 and 0.73 mg/L.
The 48h-EC50 was 0.48 mg/L with a 95% confidence interval between 0.43 and 0.53 mg/L.
Reported statistics and error estimates:
Calculation of EC50:
The 24 and 48h-EC50-value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (average measured) using the maximum likelihood estimation method. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Average Exposure Concentration Versus Nominal Concentration

Octan-3-one (ethyl amyl ketone)

Nominal concentration (mg/L)

Measured concentrations (mg/L)

Average exposure (mg/l)

T=0

T=48

10

7.63

6.71

7.2

18

13.7

14.3

14

32

27.2

24.9

26

56

47.5

48.0

48

100

91.7

86.5

89

Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

 

Average concentration Octan-3-one (ethyl amyl ketone) (mg/L)

Time(h)

Replicate

Control

7.2

14

26

48

89

0

 

 

A
B
C
D

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

 

Total introduced

20

20

20

20

20

20

24

A
B
C
D

0

0

0

0

0

0

0

0

0

0

0

0

5

2

3

4

5

5

4

4

3

5

5

5

 

Total immobilised

Effect (%)

0

0

0

0

0

0

14

70

18

90

18

90

48

A
B
C
D

0

0

0

0

0

0

0

0

0

2

0

0

5

4

4

4

5

5

5

4

5

5

5

5

 

Total immobilised

Effect (%)

0

0

0

0

2

10

17

85

19

95

20

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
see details on results
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to Octan-3-one (ethyl amyl ketone) was 21 mg/L based on average exposure concentrations (95% confidence interval between 17 and 24 mg/L).
Executive summary:

A study was performed to assess the acute toxicity of Octan-3-one (ethyl amyl ketone) on Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure. The study was conducted in accordance with the OECD Guideline for No. 202, 2004 and performed under GLP.  Twenty daphnids per group (5 per replicate, quadruplicate), less than 24 h old at the start of the test, were exposed to an untreated control and to nominal Octan-3-one (ethyl amyl ketone) concentrations of 10, 18, 32, 56 and 100 mg/L in closed systems. Analysis of the samples taken at the start of the final test showed that measured concentrations were at 76 to 92% relative to nominal. Based on these results, the average exposure concentrations were calculated to correspond with 7.2, 14, 26, 48 and 89 mg/L for the nominal concentrations of 10, 18, 32, 56 and 100 mg/L, respectively.

In conclusion, the 48h-EC50 for Daphnia magna exposed to Octan-3-one (ethyl amyl ketone) was 21 mg/L based on average exposure concentrations (95% confidence interval between 17 and 24 mg/L).