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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:
2 May 2011 - 10 June 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD 23 Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
Deviations:
no
Principles of method if other than guideline:
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.

The test item was known to rapidly hydrolyse in culture medium (half life approximately 119 minutes). As such in accordance with the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000) it was considered appropriate to expose the test organisms to the test item hydrolysis product. The test item was therefore prepared as a saturated solution at an initial loading rate of 50 mg parent test item/L and stirred for a period of 24 hours in order to ensure that complete hydrolysis had occurred.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations: The concentration and stability of the hydrolysis product in the test solutions were verified by chemical analysis at 0 and 72 hours.

Sampling Method: 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. All 0-Hour samples were stored at approximately -20°C prior to analysis.

Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
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.

The test item was known to rapidly hydrolyse in culture medium (half life approximately 119 minutes). As such in accordance with the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000) it was considered appropriate to expose the test organisms to the test item hydrolysis product. The test item was therefore prepared as a saturated solution at an initial loading rate of 50 mg parent test item/L and stirred for a period of 24 hours in order to ensure that complete hydrolysis had occurred.


Range-finding test:

The results obtained from the pre-study media preparation trial conducted indicated that a saturated solution method of preparation followed by the removal of any undissolved hydrolysis product by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded) was most appropriate for this test item.

An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved hydrolysis product was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (500 ml) of each of the stock solutions was separately inoculated with algal suspension (11.2 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.


Definitive test:

An amount of test item (550 mg) was dispersed in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved hydrolysis product was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 litre discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (1 litre) of this saturated solution was inoculated with algal suspension (8.9 mL) to give the required test concentration of 100% v/v saturated solution.
The stock solution and prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
-Common name: Pseudokirchneriella subcapitata
-Strain: CCAP 278/4
-Source (laboratory, culture collection): 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.
-Age of inoculum (at test initiation): NDA
-Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1°C.

ACCLIMATION
-Acclimation period: NDA
-Culturing media and conditions (same as test or not): The culture medium used for the studies was the same as that used to maintain the stock culture.
-Any deformed or abnormal cells observed: NDA

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable

Test conditions

Hardness:
NDA
Test temperature:
24 ± 1 °C
pH:
Test vessels: 7.4 - 7.9
The pH values of the control cultures were observed to increase from pH 7.4 at 0 hours to pH 8.0 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:
NDA
Salinity:
NDA
Nominal and measured concentrations:
The test item was known to rapidly hydrolyse in culture medium (half life approximately 119 minutes). As such in accordance with the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000) it was considered appropriate to expose the test organisms to the test item hydrolysis product. The test item was therefore prepared as a saturated solution at an initial loading rate of 50 mg parent test item/L and stirred for a period of 24 hours in order to ensure that complete hydrolysis had occurred.

As the test item was known to rapidly hydrolyse the test preparations were analysed for the presence of the hydrolysis product only.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no hydrolysis product was in solution, just that which was so, was at a concentration of less than the LOQ.
Details on test conditions:
TEST SYSTEM
- Test vessel: conincal flask
- Type (delete if not applicable): closed with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 mL glass conical flasks each containing 100 mL of test preparation
- Aeration: NDA
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: 4.57E+03 cells per mL
- Control end cells density: 4.10E+05 cells per ml
- No. of organisms per vessel: N/A
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: N/A

NaNO3 25.5 mg/L
MgCI2.6H20 12.164 mg/L
CaCI2.2H20 4.41 mg/L
MgSO4.7H20 14.7 mg/L
K2HP04 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.1855 mg/L
MnCI2.4H20 0.415 mg/L
ZnCI2 0.00327 mg/L
FeCI3.6H20 0.159 mg/L
CoCI2.6H20 0.00143 mg/L
Na2MoO4.2H20 0.00726 mg/L
CuCI2.2H20 0.000012 mg/L
Na2EDTA.2H20 0.30 mg/L
Na2SeO3.5H20 0.000010 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP) and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCI.
- Total organic carbon: NDA
- Particulate matter: NDA
- Metals: NDA
- Pesticides: NDA
- Chlorine: NDA
- Alkalinity: NDA
- Ca/mg ratio: NDA
- Conductivity: NDA
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of each control and test flask was determined at initiation of the study and after 72 h exposure.
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
RANGE FINDING TEST
The resuls from the range finding study showed no effect on growth at any of the tested concentrations (0.1, 1.0, 10 and 100% v/v saturated solution).

Based on this outcome, 100% v/v saturated solution was selected for the definitive test. The experimental design conforms to the principles of a "limit test", in order to confirm that the highest achieveable concentration had no effect on growth.

OBSERVATIONS ON CULTURES
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

OBERVATIONS ON TEST ITEM SOLUBILITY
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 and test cultures were observed to be pale green dispersions.

VERIFICATION OF TEST CONCENTRATIONS
The test item was known to rapidly hydrolyse in culture medium (half life approximately 119 minutes). As such in accordance with the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 23, 2000) it was considered appropriate to expose the test organisms to the test item hydrolysis product. The test item was therefore prepared as a saturated solution at an initial loading rate of 50 mg parent test item/L and stirred for a period of 24 hours in order to ensure that complete hydrolysis had occurred.

As the test item was known to rapidly hydrolyse the test preparations were analysed for the presence of the hydrolysis product only.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no hydrolysis product was in solution, just that which was so, was at a concentration of less than the LOQ.
Results with reference substance (positive control):
A positive control (Harlan Laboratories Ltd Project No: 41100094) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 - 72 h) 1.2 mg/L, 95% confidence limits 0.98 - 1.4 mg/L
EyC50 (0 - 72 h) 0.48 mg/L, 95% confidence limits 0.43 - 0.54 mg/L

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:
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 control and the 100% v/v saturated solution 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).

Statistical analysis of the growth rate data was carried out for the control and 100% v/v saturated solution 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 decreases in growth rate (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100% v/v saturated solution.

Statistical analysis of the yield data was carried out as above. There were no statistically significant decreases in yield (P≥0.05), between the control and 100% v/v saturated solution test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100% v/v saturated solution.

Any other information on results incl. tables

Validity Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 90 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 : 4.57 x 103 cells per mL

Mean cell density of control at 72 hours : 4.10 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 30% 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%.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See above
Conclusions:
Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution.

As the test item was known to rapidly hydrolyse the test preparations were analysed for the presence of the hydrolysis product only.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no hydrolysis product was in solution, just that which was so, was at a concentration of less than the LOQ.

The study showed no toxic effects at saturation.
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 761/2009.

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was determined to be 100% v/v saturated solution.

As the test item was known to rapidly hydrolyse the test preparations were analysed for the presence of the hydrolysis product only.

Chemical analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.11 mg/L. This does not infer that no hydrolysis product was in solution, just that any that was present, was at a concentration of less than the LOQ.

The study showed no toxic effects at saturation.