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
Study period:
from 3rd of June 2013 to 6th of July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was conducted according to OECD guidelines and under GLP rules.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Range finding test :
the results on the range finding test showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. Chemical analysis of the 100mg/L test preparation at 0 and 72 hours showed measured test concentrations of 9.3 and 8.5 mg/L respectively were obtained indicating that the test item was stable over the test duration. Whilst the measured test concentrations were a factor of 10 less than the obtained during the pre-study media preparation trial, inspection of the data could find no cause for this.
Based on this information an initial experiment was conducted at a single test concentration of 100 mg/L. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

- Initial experiment:
An initial experiment conducted as a limit test at a nominal concentration of 100 mg/L was terminated after 48 hours exposure due to significant inhibition of growth being observed. It was teherefore considered likely that the initial stock solution prepared for use in the range finding test had been incorrectly dosed, accounting for the inconsistenly low measured test concentration observed. Given this information nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected for the definitive test.

- Definitive test:
Nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were selected.

- Verification of test concentrations: Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 72 hours for quantitative analysis. All 0-Hour samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored at approximately -20 °C for further analysis if necessary.
Chemical analysis of the test preparations at 0 hours (see appendix 4 attached) showed measured test concentrations to range from 5.3 to 91 mg/L. Measured concentrations in the range of 5.2 to 94 mg/L (94% to 105% of the 0-hour measured test concentration) were observed at 72 hours and hence it was considered appropriate to calculate the results based on the 0-hour measured concentrations.

- Sample storage conditions before analysis: the 0-hours samples were stored at approximately -20°C prior to analysis, whilst the 72 hour test samples were analyzed on the day of receipt.

- Preparation of the test samples: The 0-hours samples were thawed with the aid of sonication, whilst the 72-hour samples were analyzed on the day of receipt. The test samples were extracted directly with 15 mL of hexane in the same bottle. An aliquot of the hexane layer was taken for analysis. See table 1 on appendix 4 attached for more information on preparation volumes.

Test solutions

Vehicle:
no
Details on test solutions:
Preliminary solubility work conducted indicated that it was not possible to obtain a a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

Based on this information the test item was categorized as being a "difficult substance" as defined by the OECD guideline document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 200). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

The results obtained from the pre-study media preparation trial conducted showed that near nominal concentrations were obtained irrespective of method of pre-treatment suggesting that the test item had completely dissolved. Based on this information the test item was prepared using a prolonged stir method of preparation: An amount of test item (1100 mg) was dispersed in 11 liters 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 liter discarded in order to pre-condition the filter) to give a stock solution with a 0-Hour measured concentration of 91 mg/L. A series of dilutions was made from this stock solution to give further stock solutions of 42, 23, 11 and 5.3 mg/L. An aliquot (2 lietrs) of each of the stock solutions was separately inoculated with algal suspension (11.4 mL) to give the test concentrations of 5.3, 11, 23, 42 and 91 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item were verified by chemical analysis at 0 and 72 hours (see attached appendix 4).

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures of Pseudokirchnerella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, Oban, Argyll, Scotland.
- Method of cultivation: 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 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100-150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10000 - 100000 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
no data
Test temperature:
Temperature of the control and each the preparation was maintained at 24 ± 1 ºC
pH:
The pH values of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 9.6 at 72 hours. The pH values of the control and each test preparation are given in table 2 attached.
This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
see table 4 in appendix 4 attached
Details on test conditions:
- The test item was known to be volatile and hence testing was conducted in completely filled, stoppered vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

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

- The experiment was conducted under constant illumination (intensity approximately 7000 lux) provided by warm white lighting (380-730 nm) and constantly shaken at apprximately 150 rpm for 72 hours.

- Pre-culture conditions gave an algal suspension in log phase growth chracterized by a cell density of 8.78 x 10E5 cells/mL. Inoculation of 2 liters of test medium with 11.4 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells/mL and had no significant dilution effect on the final test concentration.

- Samples were taken at 0, 23, 47 and 72 hours and the cell densities determined using a Coulter® Multizer Particle Counter.

- See Appendix 2. Culture medium for description of the standard culture medium prepared. The culture medium pH was adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): all test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 5.3 and 11 mg/L. However, some enlarged cells were observed in the 23 mg/L test cultures, enlarged cells debris were observed in the 42 mg/L test cultures and no intact cells were observed to be present in the 91 mg/L test cultures.
- Observations on Test Item solubility: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 5.3 and 11 mg/L test cultures were observed to be green dispersions. The 23 mg/L test cultures were observed to be pale dispersions, the 42 mg/L test cultures were observed to be very pale green dispersions and the 91 mg/L test cultures were observed to be clear colorless solutions.

Results with reference substance (positive control):
- Results with reference substance valid? yes
- ErC50 (0-72h): 1.2 mg/L; 95% confidence limits 1.0 - 1.3 mg/L
- EyC50 (0-72h): 0.52 mg/L; 95% confidence limits 0.45 - 0.62 mg/L
- NOEC based on growth rate: 0.25 mg/L
- NOEC based on yield: 0.25 mg/L
- LOEC based on growth rate: 0.50 mg/L
- LOEC based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

- For results see attached "Tables and Figures.pdf"

- Validation Criteria:

In the control, the biomass increased by a factor of 146 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 after 72 hours was fulfilled.

Mean cell density of control at 0 hours: 4.06 x 10E3 cells/mL

Mean cell density of control at 72 hours: 5.92 x 10E5 cells/mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 25% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 -72 h) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

The pH values of the control cultures was observed to increase from pH 7.9 at 0 hours to pH 9.6 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion given in the Test Guidelines.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchnerella subcapitata has been investigated over a 72-Hour period and gave the following results based on the 0-Hour measured test concentration:
-Growth rate:
EC50: 30 mg/L (95% confidence limits: 27 - 33 mg/L).
NOEC: 5.3 mg/L
LOEC: 11 mg/L

-Yield:
EC50: 15 mg/L (95% confidence limits: 14 - 17 mg/L).
NOEC: 5.3 mg/L
LOEC: 11 mg/L
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchnerella subcapitata were exposed to the test item to assess the effect on its growth. The method followed that described in the OECD Guidelines for testing of chemicals (2006) No 201, referenced as Method C.3 of Comission Regulation (EC) No 761/2009.

Following a preliminary range-finding test and initial experiment, Pseudokirchnerella subcapitata was exposed ti aqueous solutions of the test item at 0 -Hour measured concentrations of 5.3, 11, 23, 42 and 91 mg/L (three replicate flasks for each) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 ºC. The test item was prepared by stirring 100 mg/L of test item in culture medium. After the stirring period any undissolved test item was removed by filtration (0.2 µm filter) to produce the 91 mg/L stock solution from which a series of dilutions was made to give the remainder of the test solutions.

Results Synopsis

 

Test Organism: of Pseudokirchnerella subcapitata

Test Type: Static

 

-Growth rate:

EC50: 30 mg/L (95% confidence limits: 27 - 33 mg/L).

NOEC: 5.3 mg/L

LOEC: 11 mg/L

-Yield:

EC50: 15 mg/L (95% confidence limits: 14 - 17 mg/L).

NOEC: 5.3 mg/L

LOEC: 11 mg/L