Azarbre

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:

June-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:

Diethyldimethylcyclohex-2-en-1-one

EC Number:

272-462-3

EC Name:

Diethyldimethylcyclohex-2-en-1-one

Cas Number:

68845-36-3

Molecular formula:

C12H20O

IUPAC Name:

2,3-diethyl-4,4-dimethylcyclohex-2-en-1-one

Test material form:

other: liquid

Details on test material:

Please note that although the substance is stated as the mono-constituent substance diethyldimethylcyclohex-2-en-1-one in the report, analytical data on the same batch shows that the test substance is in fact the multi-constituent substance Reaction mass of 3,5-diethyl-2,5-dimethyl-cyclohex-2-en-1-one and 3,5-diethyl-5,6-dimethylcyclohex-2-en-1-one .

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: Nominal test item concentrations: 0.10, 1.0, 10, 100 mg/L (range-finding study), 1.75, 4.81, 13.2, 36.4 and 100 mg/L (definitive study).
- Sampling method: Extraction of 200 mL sample 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: A nominal amount of test item (1100 mg) was dissolved in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure, any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre was discarded in order to precondition the filter) to give a 100 mg/L stock solution.
- Eluate: deionised reverse osmosis water
- Differential loading: No
- Controls: Not available
- 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): undissolved test material was filtered off

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): SAMS Research Services Ltd; Scottish marine Institute; Oban; Argyll; Scotland; Culture Collection of Algae and Protozoa
- 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 - 7.8

Dissolved oxygen:

Not available

Salinity:

Not available

Nominal and measured concentrations:

Nominal concentrations: 1.75, 4.81, 13.2, 36.4 and 100 mg/L (definitive study)
Time-weight mean measured concentrations: 0.56, 1.5, 4.4, 12 and 83 mg/L (definitive study)

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: 5.06 x 10^3 cells/mL
- Control end cells density: 4.49 x 10^5 cells/mL
- No. of organisms per vessel: 5.06 x 10^5 cells/mL
- 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)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water: NaNO3 (25.5 mg/L); MgCl2.6H2O (12.16 mg/L); CaCl2.2H2O (4.41 mg/L); MgSO4.7H2O (14.6 mg/L); K2HPO4 (1.044 mg/L); NaHCO3 (15.0 mg/L); H3BO3 (0.186 mg/L); MnCl2.4H2O (0.415 mg/L); ZnCl2 (0.00327 mg/L); FeCl3.62O (0.160 mg/L); CoCl2.6H2O (0.00143 mg/L); Na2MoO4.2H2O (0.00726 mg/L); CuCl2.2H2O (0.000012 mg/L); Na2EDTA.2H2O (0.30 mg/L)
- 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: 2.75 (approximately)
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (nominal) 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: 1.75, 4.81, 13.2, 36.4 and 100 mg/L (nominal).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.1 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.6 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.3 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

other: yield

Remarks on result:

other: 4.6-6.2% (95% CL)

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

4.4 mg/L

Nominal / measured:

meas. (TWA)

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): swollen and deformed cells were observed in the test cultures at 12 and 83 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
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

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 time-weighted mean measured test concentrations):

Response Variable	EC10 (mg/L)	EC50 (mg/L)	95% Confidence Limit (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth Rate	3.1	12	*	1.5	4.4
Yield	2.6	5.3	4.6 - 6.2	1.5	4.4

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

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 89). Additionally, the mean coefficient of variation for section by section specific growth rate for the control cultures did not exceed 35% (equalled 32%) 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:

Azarbre was observed to have a 72-hr algae EC50 of 12 mg/L (based on growth rate) and 5.3 mg/L (based on yield).

Executive summary:

In an OECD guideline study (TG 201), to GLP, 72 hour EC50s for Azarbre were determined to be 12 mg/L and 5.3 mg/L (95% CL = 4.6 - 6.2 mg/L) for growth rate and yield effects respectively. The results were based on time-weighted mean of the measured test concentrations. 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 89). Additionally, the mean coefficient of variation for section by section specific growth rate for the control cultures did not exceed 35% (equalled 32%) 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%).