3-methyl-5-propylcyclohex-2-enone

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:

May-July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, to GLP

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:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

celery ketone

IUPAC Name:

celery ketone

Constituent 2

Reference substance name:

3-methyl-5-propylcyclohex-2-enone

EC Number:

223-069-0

EC Name:

3-methyl-5-propylcyclohex-2-enone

Cas Number:

3720-16-9

Molecular formula:

C10H16O

IUPAC Name:

3-methyl-5-propylcyclohex-2-en-1-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 3-Methyl-5-propylcyclohex-2-enone
- Substance type: Pure active substance
- Physical state: Extremely pale yellow liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): Not specified
- Composition of test material, percentage of components: 3-Methyl-5-propylcyclohex-2-enone 99.9%
- Isomers composition: Not applicable
- Purity test date: Not available
- Lot/batch No.: SC00005427
- Expiration date of the lot/batch: 16 December 2014
- Storage condition of test material: Room temperature, protect from light

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range finding: 0.10, 1.0, 10, and 100 mg/L (nominal); Definitive test: 1.0, 3.2, 10, 32 and 100 mg/L (nominal).
- Sampling method: Extraction of 200 mL test sample and 60 g sodium chloride with 20 mL hexane, analysis of hexane layer
- Sample storage conditions before analysis: Stored frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test substance samples (~12.8 mg, corresponding to 30 mg/L in 428 mL of test volume) are weighed in small aluminium boats and added directly to the test flasks, already containing 400 mL of mineral medium.
- Eluate: no data.
- Differential loading: no data.
- Controls: no data.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): SAMA Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Not available
- Method of cultivation: periodic replenishment of culture medium

ACCLIMATION
- Acclimation period: Not available
- Culturing media and conditions (same as test or not): the culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture
- Any deformed or abnormal cells observed: Not available

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not available

Test temperature:

24 ± 1 deg C

pH:

7.4 - 7.7

Dissolved oxygen:

Not available

Salinity:

Not available

Nominal and measured concentrations:

Concentrations: Range finding: 0.10, 1.0, 10, and 100 mg/L (nominal); Definitive test: 1.0, 3.2, 10, 32 and 100 mg/L (nominal).

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL flasks containing 100 mL test solution
- Aeration: Not available
- Type of flow-through (e.g. peristaltic or proportional diluter): Not available
- Renewal rate of test solution (frequency/flow rate): Not available
- Initial cells density: 4.88 x 10^3 cells/ml
- Control end cells density: 5.74 x 10^5 cells/ml
- No. of organisms per vessel: 4.88 x 10^5
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not available
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: Not available
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: Not available
- Conductivity: Not available
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was determined at the start of the test and again after 72 hours exposure. Temperature within the incubator was recorded daily

OTHER TEST CONDITIONS
- Sterile test conditions: Not available
- Adjustment of pH: Not available
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux (approximately), warm white lighting (380-730 nm)
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter Multisizer Particle Counter
- Chlorophyll measurement: Not available

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125 (approximately)
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L were used in a range-finding study
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 19-28 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 8.6-12 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): cell debris present at 32 and 100 mg/L
- Unusual cell shape: Not available
- Colour differences: Not available
- Flocculation: Not available
- Adherence to test vessels: Not available
- Aggregation of algal cells: Not available
- Any stimulation of growth found in any treatment: Not available
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not available
- Effect concentrations exceeding solubility of substance in test medium: At the start of the test all control and test cultures were observed to be clear colourless solutions.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, within normal ranges
- ErC50: 1.1 mg/L (growth rate; 0-72 h)
- EyC50: 0.51 mg/L (yield; 0-72 h)

Reported statistics and error estimates:

95% Confidence Limit (ErC50): 0.91 - 1.2 mg/L
95% Confidence Limit (EyC50): 0.45 - 0.59 mg/L

Any other information on results incl. tables

The effect of the test item on algal growth over 72 hours (based on nominal test concentrations):

Response Variable	EC10 (mg/L)	EC50 (mg/L)	95% Confidence Limit (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth Rate	6.8	23	19 - 28	3.2	10
Yield	3.3	10	8.6 - 12	3.2	10

The test was considered to be valid given that the cell concentration of the culture cells increased by more than a factor of 16 after 72 hours (increased by a factor of 118). Additionally, the mean coefficient of variation for section by section specific growth rate for the control cultures did not exceed 35% (equalled 8%) and the coefficient of variation for average specific growth rate for the control cultures did not exceed 7% over the course of the test period (equalled 3%).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The preferred observational end point in this study is algal growth rate inhibition because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1) and classification shall be based on the ErC50 (EU CLP regulation No 1272/2008 and its adaption 286/2011). In terms of the NOEC and EC10 values, there is a preference for use of the EC10 value because it is derived from the dose response curve. In contrast the NOEC depends on the experimental design (e.g. the concentrations used in the test). Thus the 72-h EC50 and EC10 based on growth rate are used for classification purposes.

Celery Ketone was observed to have a 72-hr algae EC50 of 23 mg/L (based on growth rate). The corresponding ErC10 was 6.8 mg/L.

Executive summary:

In an OECD guideline study (TG 201), to GLP, the 72 hour the EC50 for Celery Ketone were determined to be 23 mg/L (95% CL = 19 - 28 mg/L) based on growth rate. The corresponding EC10 was 6.8mg/L. The results were based on the nominal test concentrations since the majority of measured test concentrations at 0 and 72 hours were within the acceptable range (80 -120%) of nominal. The test was considered to be valid given that the cell concentration of the culture cells increased by more than a factor of 16 after 72 hours (increased by a factor of 118). Additionally, the mean coefficient of variation for section by section specific growth rate for the control cultures did not exceed 35% (equalled 8%) and the coefficient of variation for average specific growth rate for the control cultures did not exceed 7% over the course of the test period (equalled 3%).