Azarbre

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:

June-August 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 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Preliminary solubility work indicated that it was not possible to obtain a testable 100 mg/L stock solution using traditional methods of preparation e.g. ultrasonication and high shear mixing, and so the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000).

A media preparation trial determined that a prolonged stir method could produce a 100 mg/L solution.

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:

Not applicable

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 test water 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 discarded in order to pre-condition the filter) to give the 100 mg/L test concentration.
- Eluate: Deionised reverse osmosis water
- Differential loading: No
- Controls: Test water only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): undissolved test item was filtered off

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: no data
- Source: In-house laboratory cultures
- Age at study initiation (mean and range, SD): neonates <24 hours old (1st instar)
- Weight at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): no data
- Method of breeding: parthenogenesis
- Feeding during test: No
- Food type: no data
- Amount: no data
- Frequency: no data

ACCLIMATION
- Acclimation period: Not available
- Acclimation conditions: same as test
- Type and amount of food: mixture of Algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no data

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

250 mg/L as calcium carbonate

Test temperature:

20-21 deg C

pH:

7.7 - 8.0

Dissolved oxygen:

8.8 - 9.2 mg(O2)/L

Salinity:

Not applicable

Nominal and measured concentrations:

0.10, 1.0, 10 and 100 mg/L nominal test item concentrations (range finding test). Measured concentrations were in the range of 82 % to 90 % of the nominal concentrations.

10, 18, 32, 56 and 100 mg/L nominal test item concentrations (definitive test) . Measured concentrations were in the range of 81 to 88 % of the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass jars containing approximately 200 mL of test preparation
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not available
- Renewal rate of test solution (frequency/flow rate): The test preparations were not renewed during the exposure period
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium): CaCl2.2H2O (294 mg/L); MgSO4.7H2O (123 mg/L); NaHCO3 (64.75 mg/L); KCl (5.75 mg/L)
- Culture medium different from test medium: Yes, Elendt M7 was used as culture medium
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: If necessary with NaOH or HCl
- Photoperiod: 16 hours light, 8 hours darkness (20 minute dawn and dusk intervals)
- Light intensity: 502-521 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mobility at 24 and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 1.8
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: yes
- Test concentrations: 0.10, 1.0, 10, 100 mg/L in range finding study
- Results used to determine the conditions for the definitive study: Noimmobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However immobilisation was observed at 100 mg/L. Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were used for the definitive test

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Remarks on result:

other: 30-42 mg/L (95% CL)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Details on results:

No detailed results from the tested concentrations are presented in the report.
Other biological observations: No data

Results with reference substance (positive control):

- Results with reference substance valid? Yes, within normal range (0.6-2.1 mg/L; EC50-24 hour)
- Mortality: None stated
- EC50: 0.87 mg/L (24 hour); 0.71 mg/L (48 hour)

Any other information on results incl. tables

Cumulative immobilisation data in the definitive test

Nominal concentration (mg/L)	Cumulative immobilised Daphnia (Initiall population: 5 per replicate)
	24 hours						48 hours
	1	2	3	4	Total	%	1	2	3	4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	0	0	0	0	0	0
18	0	0	0	0	0	0	0	0	0	0	0	0
32	3	2	2	1	8	40	5	3	2	1	11	55
56	3	1	3	5	12	60	4	3	4	5	16	80
100	4	5	5	4	18	90	5	5	5	5	20	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Azarbre was observed to have a 48-hour Daphnid EC50 of 35 mg/L

Executive summary:

In an OECD guideline study (TG 202), to GLP, Azarbre was observed to have a 48-hour Daphnid EC50 of 35 mg/L (95% CL = 30 - 42 mg/L).

The results were based on nominal concentrations, as measured test concentrations at 0 h and 48 h were in a range of 80% to 92% of the nominal concentrations