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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:
8 October 2007 to 26 October 2007
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 methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Date of inspection: 30 August 2005 Date of Signature: 21 November 2005)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
417-560-0
EC Name:
-
Cas Number:
67881-98-5
Molecular formula:
C11H22NO6P
IUPAC Name:
trimethyl[2-({2-[(2-methylprop-2-enoyl)oxy]ethyl phosphonato}oxy)ethyl]azanium
Details on test material:
- Name of test material (as cited in study report): MPC
- Substance type: White powder
- Physical state: Solid
- Lot/batch No.: 270103 (received on 14 September 2007)
- Storage condition of test material: Approximately 4ºC in the dark.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: From the control and the 100 mg/l test group.

- Sampling method: Water samples were taken from the control and the test group (six replicates pooled) at 0 and 72 hours for quantitative analysis. Additional samples were taken at 0 and 72 hours in order to confirm the correct dosing procedures were followed.
- Sample storage conditions before analysis: Duplicate samples were taken at 0 and 72 hours hours and stored at approximately -20ºC for further analysis if necessary.

Test solutions

Vehicle:
no
Details on test solutions:
For the purpose of the definitive test, the test material was dissolved directly in culture medium. An amount of test material (100 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 200 mg/l stock solution. This stock solutions was mixed with algal suspension (500 ml) to give the required test concentrations of 100 mg/l. The stock solutions and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity

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):
Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyl, 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 continuous illumination at 21 ± 1ºC.

- Culturing media and conditions (same as test or not):
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture. 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 1E+03 cells/ml. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illustration at 24 ± 1ºC until the algal cell density was approximately 1E+04 – 1E+05 cells/ml.

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 reported.
Test temperature:
24 ± 1ºC.
pH:
7.3 (at 0 h ), 7.7 (at 72 h) in test group.
Dissolved oxygen:
Not reporded.
Salinity:
Not reporded.
Nominal and measured concentrations:
(Range-finding test) 0.10, 1.0, 10 and 100 mg/l
(Definitive test) 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed.
- Material, size, headspace, fill volume: 250 ml glass conical flasks.
- Aeration: No.
- No. of organisms per vessel: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.80 x 1E+05 cells per ml. This suspension was diluted to a cell density of 8.46 x 1E+03 cells per ml prior to use. At initiation of the test the culture contained a nominal cell density of 4 X 1E+03 cells per ml.

- No. of vessels per concentration (replicates): 6.
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
Stock Solutions
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


OTHER TEST CONDITIONS
- Adjustment of pH: 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.
- Photoperiod: Continuous illumination for 72 hours.
- Light intensity and quality: intensity approximately 7000 lux provided by warm white lighting (380 – 730 nm).
- Others: Constantly shaken at approximately 150 rpm for 72 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisiser Particle Counter.


TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100 mg/l.

- Results used to determine the conditions for the definitive study: The results showed no effects on growth at any test concentrations. Based on this information a single test concentration of six replicates, of 100 mg/l was selected for the definitive test (limit test).
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Basis for effect:
biomass
Details on results:
Thr growth rate, yielf and biomass integral of Desmodesmus subspicatus were not affected by the presence of the test material at a nominal test concentration of 100 mg/l over the 72-Hour exposure period. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.

- Exponential growth in the control (for algal test): yes

- Observation of abnormalities (for algal test): There were no abnormalities detected.

- Any observations that might cause a difference between measured and nominal values: The pH values of the controls were observed to increase from pH 7.4 at 0 hours to pH 7.7 - 7.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours therefore was within the limits given in the Test Guidelines.

- Effect concentrations exceeding solubility of substance in test medium: 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: See table in 'Any other information on results incl. tables'. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 100% of nominal and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only. The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test material in test medium. A method of analysis was validated and proven to be suitable for use.
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal range for this reference material.
Reported statistics and error estimates:
Not reported.

Any other information on results incl. tables

Table Analytical results

Sample Nominal Concentration (mg/l) Concentration Found (mg/l)  Expressed as a Percent of the Nominal Concentration (%)
0 hours Control <LOQ -
100 R1 - R3 94.0 94
100 R4 - R6 92.0 92
72 hours Control <LOQ -
100 R1 - R3 96.1 96
100 R4 - R6 99.9 100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Desmodesmus subspicatus has been investigated and gave an EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l.
Executive summary:

Introduction. A study was performed to assess the effect of the test material on the growth of the green alga Desmodesmus subspicatus. The method 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 Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.Following preliminary range-finding test,Desmodesmus subspicatuswas exposed to an aqueous solution of the test material at concentration of 100 mg/l (six replicate flasks) 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.

 

A positive control conducted approximately every six months used potassium dichromate as the reference material. Desmodesmus subspicatus was exposed to an aqueous solution of the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l (three replicate flasks per concentration) 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.

 

Results. Exposure of Desmodesmus subspicatus to the reference material gave EC50 values of greater than 100 mg/l. Correspondingly the No Observed Effect Concentration was 100 mg/l. Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 92% to 100% of nominal and the results are based on nominal test concentrations only. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/l.