Registration Dossier

Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sucrose di(acetate) hexaisobutyrate
EC Number:
204-771-6
EC Name:
Sucrose di(acetate) hexaisobutyrate
Cas Number:
126-13-6
IUPAC Name:
Sucrose Acetate Isobutyrate
Details on test material:
Identification:Sucrose acetate isobutyrate
Description:light amber colored extremely viscous liquid
Batch: A525 00620

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
The pH of the control and 100% v/v saturated solution test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily. Samples were taken from the control and the 100% v/v saturated solution test group (replicates R1 - R6 pooled) at 0 and 72 hours for quantitative analysis of the test concentrations. Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

Test solutions

Vehicle:
no
Details on test solutions:
Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution. An amount of test item (550 mg) was dispersed in 11 liters of culture medium (50 mg/L loading rate) with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 um Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. An aliquot (1 liter) of the 100% v/v stock solution was inoculated with 5.5 mL algal suspension to give the required test concentration of 100% v/v saturated solution.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. 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 ± 1 °C. Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of
approximately 10e3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100- 150 7m) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10e4 - 10e5 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Test temperature:
Temperature was maintained at 24 ± 1 °C throughout the test.
pH:
The pH value of the control cultures was observed to range from pH 8.1 at 0 hours to pH 7.8 at 72 hours.
The pH value of the 100% saturated solution was observed to range from pH 8.0 at 0 hours to pH 7.9 at 72 hours.
Nominal and measured concentrations:
Chemical analysis of the 100% v/v saturated solution (50 mg/L nominal loading rate) at 0 hours showed a measured test concentration of 0.12 mg/L was obtained. Analysis of the test preparation at 72 hours showed a decline in measured test concentration to 0.044 mg/L. The geometric mean measured test concentration was determined to be 0.073 mg/L.
Details on test conditions:
250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100% v/v saturated solution treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 9.07 x 10e5 cells per mL. Inoculation of 1liter of test medium with 5.5 mL of this algal suspension gave an initial nominal cell density of 5 x 10e3 cells per mL. The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF loading rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: WAF loading rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: WAF loading rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: WAF loading rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 100% saturated solution
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 100% saturated solution
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 100% saturated solution
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.073 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 100% saturated solution
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and 50 mg/L test group using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There were no statistically significant differences (P≥0.05), between the control and 50 mg/L test group and therefore the "No Observed Effect Loading Rate" (NOELR) based on growth rate was 50 mg/L. Statistical analysis of the yield data was carried out in a similar manner. There were no statistically significant differences (P≥0.05), between the control and 50 mg/L test group and therefore the NOELR based on yield was also 50 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with 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 76112009. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing. A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 0.12 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a nominal 50 mg/L WAF solution. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration to produce a 100% v/v saturated solution of the test item with a 0-Hour measured concentration of 0.12 mg/L.

Six replicate flasks were exposed 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. Exposure of Pseudokirchneriella subcapitata to the test item, based on the the WAF loading rate gave EL50 values of greater than 50 mg/L. The No Observed Effect Loading Rate (NOELR) was also 50 mg/L. Exposure of Pseudokirchneriella subcapitata to the test item, based on the geometric mean measured test concentration gave EC50 values of greater than 0.073 mg/L. The No Observed Effect Concentration was 0.073 mg/L. This study showed that there were no toxic effects at saturation.