Reaction mass of Benzenepropanal, 4-ethyl-α,α-dimethyl- and 3-(2-ethylphenyl)-2,2-dimethylpropanal

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Jun 2016 to 01 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The pH did not remain within 7.5 ± 1.5 (raised up to 10.1), but this was considered not to have an adverse effect on the result of the study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours. Samples were taken from the solvent control and 2.0 mg/L test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary. Two additional samples of the 2.0 mg/L test concentration were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed (OECD, 2000). A nominal amount of test item (200 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 200 mg/10 mL solvent stock solution. An aliquot (300 µL) of this solvent stock solution was dispersed in 3 litres of culture medium with the aid of magnetic stirring for 10 minutes to give a 2.0 mg/l stock solution. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 2.0 mg/L stock solution An aliquot (2500 mL) of this stock solution was inoculated with algal suspension (27.8 mL) to give the required test concentration of 2.0 mg/L. The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2 °C.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

- Test start (0h): 7.9
- Test termination (72h): 10.1

Nominal and measured concentrations:

- Nominal concentration: 0 (control), 0 (solvent control) and 2.0 mg/L (based on a range-finding study); see 'Any other information on materials and methods incl. tables' for test concentration considerations.
- Measured concentration (t=0): - Measured concentration (t=72h):

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flasks sealed with ground glass stoppers
- Type: closed
- Fill volume: 250 mL; test vessels were completely filled.
- Initial cells density: 5.00E+03 cells per mL (nominal)
- Control end cells density: 5.66E+05 cells per mL (control); 5.95E+05 cells per mL (vehicle control)
- No. of organisms per vessel: 5.00E3 cells per mL
- 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, AAP-medium

TEST MEDIUM / WATER PARAMETERS
- Preparation: The culture medium was prepared using reverse osmosis purified deionized water
- Intervals of water quality measurement: At initiation of the test and after 72 hours exposure.
- Other: The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).

OTHER TEST CONDITIONS
- Adjustment of pH: the pH adjusted to 7.5 with 0.1N NaOH or HCl
- Light intensity: approximately 700 lux, continuous
- Light quality: warm white lighting (380-720 nm)
- Other: flasks were constantly shaked at approximately 150 rpm for 72 hours

EFFECT PARAMETERS MEASURED:
- Algal cell densities. Samples were taken at 27, 48 and 72 hours cell densities were determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00E+03 cells/mL) was taken as the starting cell density.
- Appearance (shape and size): To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- Other: The pH of the control, solvent control and 2.0 mg/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

RANGE FINDING STUDY
- Test concentrations: 0.020, 0.20 and 2.0 mg/L
- Results used to determine the conditions for the definitive study: The results showed no significant effect on growth rate. Based on this information a single test concentration of six replicates, of 2.0 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Toxicity: This study showed that there were no toxic effects at saturation.
- Observation of abnormalities: There were no abnormalities detected in any of the control or test cultures.
- Test item solubility: At the start of the test all control, solvent control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, solvent control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

The 72-h ErC50 and EbC50 for the reference substance were determined to be 1.5 (95% C.L. 1.3 - 1.7 mg/L) and 0.79 mg/L (95% C.L. 0.70 - 0.89 mg/L), respectively.

Reported statistics and error estimates:

A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the solvent control and the 2.0 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

The 72-h EC50 based on either growth rate or biomass are > 1.2 mg/L in freshwater green algae (P. subcapitata). The NOEC values based on growth rate/biomass are determined at >=1.2 mg/L.

Executive summary:

The toxicity towards freshwater algae was determined in a limit test according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 0 (vehicle control) and 2 mg/L for 72 hours under static conditions. Analysis of the test preparations at 0, 24, 48 and 72 hours showed a decline in measured concentrations. Therefore, it was therefore justifiable to base the results on the geometric mean measured test concentration in order to give a "worst case" analysis of the data. The cell density in each flask was determined at the start of the test and after 24, 48 and 72 hours exposure. The mean values were plotted against time to produce growth curves from which the cell growth rate was calculated. No toxic effects were observed at saturation. The ErC5 and EbC50 values based on the geometric mean measured test concentration were determined to be greater than 1.2 mg/L. The NOErC and NOEbC values were determined to be 1.2 mg/L (geometric mean of measured concentrations).

Description of key information

The toxicity towards freshwater algae was determined in a limit test according to OECD TG 201 and in compliance with GLP criteria. In this study, exponentially growing freshwater algae (Pseudokirchneriella subcapitata) were exposed to nominal test substance concentrations of 0 (control), 0 (vehicle control) and 2 mg/L for 72 hours under static conditions.Analysis of the test preparations at 0, 24, 48 and 72 hours showed a decline in measured concentrations. Therefore, it was therefore justifiable to base the results on the geometric mean measured test concentration in order to give a "worst case" analysis of the data. The cell density in each flask was determined at the start of the test and after 24, 48 and 72 hours exposure. The mean values were plotted against time to produce growth curves from which the cell growth rate was calculated. No toxic effects were observed at saturation. The ErC5 and EbC50 values based on the geometric mean measured test concentration were determined to be greater than 1.2 mg/L. The NOErC and NOEbC values were determined to be 1.2 mg/L (geometric mean of measured concentrations).