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:
Agust 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor ,ethodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

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)
Remarks:
Date of inspection: September 15th 2009 Date of signature: November 26th 2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification :T-1540N
Description : white powder
Purity : >99%
Batch number : 990140
Date received : 10 May 2010
Expiry date : 30 November 2010
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: control (replicates R1 - R6 pooled) and the 0.018 mg/l test group (replicates R1 - R3 and R4 - R6 pooled) at 0 and 72 hours for quantitative analysis.

- Sampling method:
Pre-study Media Preparation Trial
A C1 solid phase extraction (SPE) cartridge was sequentially pre-conditioned with methanol and water*. A volume of test sample (500 ml) was acidified with 20 drops of formic acid, eluted through the cartridge and the cartridge dried. The test item was eluted from the cartridge with acetonitrile and made to volume (5 ml). The samples were then further diluted 1 ml to 10 ml in acetonitrile to give a response within the range of the calibration standards.

- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored at approximately 20ºC for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Pre-study media preparation trial
Information provided by the Sponsor indicated that the test item was insoluble in water. Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing, nor could a solution be obtained by dissolving the test item in an auxiliary solvent.
Based on this information the test item was categorised as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
An amount of test item (550 mg) was dispersed, in duplicate, in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for periods of 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
 Filtration through a 0.2 µm Gelman Acrocap filter (approximately 500 ml discarded in order to pre-condition the filter)
 Filtration through a 0.2 µm Gelman Acrocap filter (approximately 1 litre discarded in order to pre-condition the filter)


- Eluate: same as culture media

- Differential loading: not in definitive test

- Controls: The control group was maintained under identical conditions but not exposed to the test material.

- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable

- Evidence of undissolved material (e.g. precipitate, surface film, etc): After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 ml discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a 0-Hour mean measured concentration of 0.018 mg/l.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not stated in report
- Method of cultivation: 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 103 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 10 E4 - 10 E55 cells/ml.

ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
Not stated in report
Test temperature:
The temperature within the incubator was recorded daily.
Temperature was maintained at 24 ± 1ºC throughout the test.
pH:
The pH values of the control cultures were observed to increase from pH 7.1 at 0 hours to pH 7.5 – 7.6 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
Dissolved oxygen:
Not stated in report
Salinity:
Not stated in report
Nominal and measured concentrations:
Range finding test: 0.00024, 0.0024 and 0.024 mg/l (nominal)
Definitive test: 0.024 mg/l (nominal)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type (delete if not applicable): closed - plugged with polyurethane foam bungs to reduce evaporation
- Material, size, headspace, fill volume: 250 ml glass conical flasks each containing 100 ml of test preparation
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable, as static test conditions
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 1 x E04 - 1 x E05 cells/ml
- No. of vessels per concentration (replicates): six
- No. of vessels per control (replicates): six
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l
The culture medium was prepared using reverse osmosis purified deionised water* and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not required
- Photoperiod: constant illumination
- Light intensity and quality: intensity approximately 7000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable, as only one dose level used in definitive study
- Justification for using less concentrations than requested by guideline: Based on the result of the pre-study media preparation trial and range-finding test a "limit test" was conducted at a concentration of 0.024 mg/l to confirm that at the highest attainable concentration no effect on algal growth was observed.

- Range finding study
- Test concentrations: nominal test concentrations of 0.00024, 0.0024 and 0.024 mg/l
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at the nominal test concentrations of 0.00024, 0.0024 and 0.024 mg/l.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrations
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.018 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no abnormalities detected in any of the control or test substances.
- Unusual cell shape: No
- Colour differences: At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-Hour test period all control cultures were observed to be pale green dispersions whilst the 0.018 mg/l test cultures were observed to be extremely pale green dispersions.
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No

- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes

- EC50:
ErC50 (0 – 72 h) :0.74 mg/l*
EyC50 (0 – 72 h) :0.37 mg/l, 95% confidence limits 0.34 – 0.41 mg/l

- Other:
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/l
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/l
Lowest Observed Effect Concentration (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.
Reported statistics and error estimates:
Statistical analysis of the growth rate data was carried out for the control and 0.018 mg/l test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981). There were no statistically significant differences (P>=0.05), between the control and 0.018 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was considered to be equal to or greater than 0.018 mg/l.

Statistical analysis of the yield data was carried out. There were no statistically significant differences (P0.05), between the control and 0.018 mg/l test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was considered to be equal to or greater than 0.018 mg/l.

Any other information on results incl. tables

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 30 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours        :   4.89 x 103cells per ml
Mean cell density of control at 72 hours      :   1.46 x 105cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 22% 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 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
This study showed that there were no toxic effects at saturation.
Executive summary:

Introduction.A study was perford to assess the effect of the test item on the growth of the green alga Desmodesmus subspicatus.  Thethod 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 item was insoluble in water. 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, nor could a solution be obtained by dissolving the test item in an auxiliary solvent.

A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 0.024 mg/l was obtained from a saturated solution method of preparation.

Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to a solution of the test item at a 0-Hour mean measured concentration of 0.018 mg/l (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. 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 at a temperature of approximately 21°C for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 ml discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a 0-Hour mean measured concentration of 0.018 mg/l.

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

ResultsExposure of Desmodesmus subspicatus to the test item gave EC50values of greater than 0.018 mg/l and correspondingly the No Observed Effect Concentration was considered to be equal to or greater than 0.018 mg/l.

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 0.017 to 0.019 mg/l (approximately 75% of the nominal test concentration). It was considered that the differences observed in the measured test concentration obtained during the media preparation trial and definitive test were due to slight variances in the stirring speed and temperature of the saturated solution. As no decline in measured concentration was observed over the 72-Hour test period it was considered appropriate to base the results on the 0-Hour mean measured test concentration only.

This study showed that there were no toxic effects at saturation.