10-undecenyl 2-cyano-3,3-diphenylpropenoate

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

24 August 2009 and 11 September 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 19th August 2008, Date of signature 4th March 2009

Test material

Test material information

Constituent 1

Reference substance name:

947753-66-4

Cas Number:

947753-66-4

IUPAC Name:

947753-66-4

Constituent 2

Reference substance name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl-, 2-ethylhexyl ester

IUPAC Name:

2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl-, 2-ethylhexyl ester

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
- Name of surrogate test material: Ethyl hexyl methoxycrylene
- Physical state: light amber colored viscous liquid
- Analytical purity: not stated
- Lot/batch No.: LLR 711
- Date received: 16 April 2009
- Storage condition of test material: room temperature in the dark
- Other: information related to the identity, purity and stability of the test material was the responsibility of the Sponsor

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the solvent control flasks (R1 to R6 pooled) and the 0.013 mg/l test group (R1-R3 and R4-R5 flasks, pooled) for quantitative analysis. Duplicate samples were taken at 0 hours and stored at -20 deg C for further analysis if necessary. Sample volumes required precluded the storage of duplicate samples at 72 hours. Samples (300 ml) were removed and sodium chloride (30 g/100 ml)) added. Samples (300 ml) were extracted with dichloromethane (3 x 50 ml). Extracts were filtered through anhydrous sodium sulphate and combined extracts evaporated to dryness. Residues were re-dissolved in 3 ml of methanol to give final theoretical concentrations of 0.90 mg/l.

Test solutions

Vehicle:

yes

Details on test solutions:

The test material (100 mg) was dissolved in tetrahydrofuran and the volume adjusted to 10 ml to give a 100 mg/10 ml solvent stock solution. An aliquot (300 microliters) of the solvent stock solution was dispersed in 3 liters of culture medium with the aid of magnetic stirring for approximately 10 minutes to give a 1.0 mg/l stock solution prior to removal of any undissolved test material by centrifugation at 40,000 x g for 30 minutes to five a 0.013 mg/l stock solution.

The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Name: Desmodesmus subspicatus
- Strain: CCAP 276/20
- Source (laboratory, culture collection): culture collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyl, Scotland
- Age of inoculum (at test initiation): No information
- Method of cultivation: master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 +/- 1 deg C. Prior to the start of the test, sufficient master culture was added to approximately 100 ml volumes of culture media in conical flasks to give an initial cell density of approximately 1000 cells/ml. Flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 +/- 1 deg C until the algal cell density was approximately 10,000 to 100,000 cells/ml.

The culture medium used for both the range-finding and definitive tests was that same as that used to maintain the stock culture.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

Not reported

Test temperature:

24 +/- 1 deg C

pH:

7.6 at 0 hours; 7.4-7.5 at 72 hours
The deviation in the control cultures was less than 1.5 pH units after 72 hours and was within test guideline limits.

Dissolved oxygen:

Not reported

Salinity:

Not reported.

Nominal and measured concentrations:

Analysis of test preparations at 0 hours showed the measured concentrations (0.012 and 0.013 mg/l) to be higher that that obtained in the pre-study media preparation trial (0.0090 mg/l). Differences were attributed to different media types (tap water vs. algal culture media).

Analysis of 72-hour test preparations showed a decline in measured test concentrations with values of 0.0099 and 0.010 mg/l being obtained (duplicate). Based on these results, the geometric mean measured test concentration was calculated to be 0.011 mg/l. This was considered to represent the “worst case” analysis of the data.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: 250 ml conical flasks, containing 100 ml of test preparation
- Initial cells density: 4 x 10^3 cells/ml (approx.)
- Control end cells density: 1.96 x 10^5 (mean)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes


OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: approx. 7000 lux (warm light, 380 - 730 nm)


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell densities were determined using a Coulter(R) Multisizer Particle Counter


TEST CONCENTRATIONS
- Limit test: 0.013 mg/l (nominal)
- Range finding study:
- Test concentrations: 0.00090 and 0.0090 mg/l
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate; study conducted between 26 May 2009 and 29 May 2009

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.011 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

increased yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

increased yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

increased yield

Details on results:

Range finding test:

The results indicated no effect on growth at test concentrations of 0.00090 and 0.0090 mg/l. The 0.0090 mg/l test concentration was selected for the definitive test.

Definitive test:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 1. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4. (The tables have been provided in the attached background material section).

Validation criteria:
Control cultures increased by a factor of 44 after 72 hours and the cell concentration of the solvent control cultures increased by a factor of 36 after 72 hours. This satisfies the OECD Guideline that states enhancement must be at least a factor of 16 after 72 hours.
- Mean cell density of control at 0 hours: 4.47 x 10^3 cells/ml
- Mean cell density of control at 72 hours: 1.96 x 10^5 cells/ml
- Mean cell density of solvent control at 0 hours: 4.34 x 10^3 cells/ml
- Mean cell density of solvent control at 72 hours: 1.58 x 10^5 cells/ml

The mean coefficients of variation for section by section specific growth rate for the control and solvent control cultures were 34% and 32% respectively, satisfying the validation criteria (OECD Guideline) which states the mean must not exceed 35%.

The coefficients of variation for average specific growth rate for the control and solvent control over the test period were 4f% and 6% respectively and hence satisfied the validation criterion given in the OECD guideline stating that this value must not exceed 7%.

Results with reference substance (positive control):

Exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material gave the following:
ErC50 (0-72 hours): 0.79 mg/l
EyC50 (0-72 hours): 0.30 mg/l

The results from the positive control were within the nominal ranges for this material.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the solvent control and 0.013 mg/l test group using a student's t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There was no statistically significant decrease in growth rate (P> 0.05), between the solvent control and 0.013 mg/l test group and therefore the NO Observed Effect Concentration (NOEC) based on growth rate was 0.013 mg/l.

Any other information on results incl. tables

Analysis of the test preparations at 0 hours showed the measured concentrations (0.012 and 0.013 mg/l) to be higher than that obtained in the pre study media preparation trial (0.0090 mg/l). This difference was considered to be due to the different types of media used, dechlorinated tap water in the media preparation trial and algal culture culture medium in this test.

Analysis of the test preparations at 72 hours showed a decline in measured test concentrations with values of 0.0099 and 0.010 mg/l being obtained. This decline in measured concentration was in line with that observed in the stability analyses conducted.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentration in order to give a worst case analysis of data. The geometric mean measured test concentration was calculated to be 0.011 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test material on the growth of Desmodesmus subspicatus gave a No Observed Effect Concentration of 0.013 mg/l (nominal).

Based on the geometric mean measured test concentrations the EC50 values were estimated to be greater than 0.011 mg/l. The corresponding No Observed Effect Concentration was 0.011 mg/l.

Executive summary:

Introduction. 

A study was perford to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Methods.

Information provided by the Sponsor indicated that the test material was insoluble in water. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing. A pre-study media preparation trial conducted indicated that a dissolved test material concentration of approximately 0.0090 mg/l could be obtained using a solvent spike method of preparation indicating this to be the limit of water solubility of this material under test conditions.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a 0-Hour mean measured concentration of 0.013 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

The difference observed between the measured concentrations obtained from the solvent spike preparation during the pre-study media preparation trial and definitive test was considered to be due to the different types of diluent used.

Results. 

Exposure of Desmodesmus subspicatus to the test material gave EC₅₀values of greater than 0.013 mg/l. The No Observed Effect Concentration was determined to be 0.013 mg/l.

The test concentration of 0.013 mg/l was the highest attainable test concentration that could be prepared due to the limited solubility of the test material in water and auxiliary solvent, and having due regard to the amount of auxiliary solvent permitted in the test under the OECD Guidelines.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.012 to 0.013 mg/l. A slight decline in measured concentrations was observed at 72 hours in the range of 0.0099 to 0.010 mg/l. This decline was in line with the preliminary stability analysis conducted which also showed a decline in concentration over a 72-Hour period.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geotricanasured test concentrations in order to give a "worst case" analysis of the data.

Exposure of Desmodesmus subspicatus to the test material gave EC₅₀ values based on the geometric mean measured test concentrations of greater than 0.011 mg/l. The No Observed Effect Concentration was determined to be 0.011 mg/l.