C-C1

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:

Definitive test between 16th April 2007 and 19th April 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 relevant results

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Remarks:

Date of inspection: 30th August 2005 Date of signature: 21st November 2011

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification :C-C1
Description : dark blue powder
Batch number : MB-1
Date received : 5 March 2007
Storage conditions :room temperature over silica gel in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Standard solutions of test material were prepared in water at a nominal concentration of 100 mg/l.

- Sampling method: The test material concentration in the test samples was determined by high performance liquid chromatography (HPLC) using an external standard. The test material gave a chromatographic profile consisting of a single peak.
The method was based on a procedure supplied by the Sponsor.

- Sample storage conditions before analysis: -20°C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Range-finding test
Pre-study solubility work conducted indicated that the test material formed a coloured solution. It was therefore considered appropriate to conduct the range-finding test using a modified algal inhibition test method with increased light intensity (10000 lux) and decreased test volume (25 ml) in order to minimise the effects of light absorption by the test material at the wavelengths required for photosynthetic growth.

The test was conducted in 250 ml glass conical flasks plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks each containing 25 ml of test preparation were used for each control and test concentration. 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 from which a series of dilutions was made to give further stock solutions of 20, 2.0 and 0.20 mg/l. An aliquot (25 ml) of each of the stock solutions was separately mixed with algal suspension (25 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l dispensed into two replicate test vessels per concentration.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test material.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1ºC under continuous illumination (intensity approximately 10000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Spectrophotometer measurements
Given that the test material formed coloured test solutions it was considered appropriate to conduct spectrophotometer measurements on the stock solutions in order to determine what concentration significant absorption of light at the wavelengths required for photosynthesis occurred (460 and 665 nm).

Definitive test
Eperimental Preparation
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 solution was mixed with algal suspension (500 ml) to give the required test concentration of 100 mg/l.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

Due to the coloured nature of the test solutions prepared and following the current advice available from the competent authorities the test was conducted using a modified algal inhibition test method with increased light intensity and decreased test volume in order to minimise the effects of light adsorption by the test material at the wavelengths required for photosynthetic growth.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 2).

- Differential loading: No

- 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): The unsonicated stability vessel showed no evidence of insolubility or adherence to glass.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Not applicable
- Source (laboratory, culture collection): Liquid cultures of Desmodesmus subspicatus were obtained from the 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: 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.

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

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:

Maintained at 24 +/- 1°C throughout the test.

pH:

7.7 - 7.8 measured using a pH meter.

Dissolved oxygen:

not stated in report

Salinity:

Not applicable as freshwater study

Nominal and measured concentrations:

Nominal 100 mg/l in definitive test

nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours for definitive test

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable):closed - plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: 250 ml glass conical flasks were used. Six flasks each containing 25 ml of test preparation were used for the control and 100 mg/l treatment group.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 10^3 cells/ml
- Control end cells density: 1.2 x 10^5 cells/ml
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- 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: yes
- Adjustment of pH: not required
- Photoperiod: continuous illumination
- Light intensity and quality: approx. 10,000 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable as a limit test was conducted
- Justification for using less concentrations than requested by guideline: based on the results of the range finding study.
- Range finding study
- Test concentrations: 0.1, 1 , 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: based on the results of the range finding study a limit test was conducted.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Basis for effect:

growth rate

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. There were no abnormalities detected in any of the control or test cultures.

- Unusual cell shape: No

- Colour differences: At the start of the test all control cultures were observed to be clear colourless solutions whilst the test cultures were observed to be bright blue solutions. After the 72-Hour test period all control cultures were observed to be green dispersions whilst the test cultures were observed to be bright blue 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:

- Effect concentrations exceeding solubility of substance in test medium:

Results with reference substance (positive control):

- Results with reference substance valid? Yes

ErC50 (0 - 72 h) : 0.58 mg/l; 95% confidence limits 0.47 - 0.71 mg/l
EyC50 (0 - 72 h) : 0.16 mg/l; 95% confidence limits 0.14 - 0.19 mg/l
EbC50 (0 - 72 h) : 0.20 mg/l; 95% confidence limits 0.17 - 0.24 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on yield : 0.0625 mg/l
No Observed Effect Concentration (NOEC) based on biomass integral : 0.0625 mg/l
Lowest Observed Effect Concentration (LOEC) based on growth rate : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on yield : 0.125 mg/l
Lowest Observed Effect Concentration (LOEC) based on biomass integral : 0.125 mg/l
The results from the positive control with potassium dichromate were within the normal range for this reference material.

Reported statistics and error estimates:

 Statistical analysis
A Student’s t-test incorporating's test for homogeneity of variance (Sokal and Rohlf 1981) was carried out on the growth rate, yield and biomass integral data after 72 hours for the control and the 100 mg/l 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).

Any other information on results incl. tables

Verification of test concentrations

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so it was considered justifiable to estimate the EC₅₀ values in terms of the nominal test concentrations only.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Desmodesmus subspicatus to the test material gave EC50 values of greater than 100 mg/l and 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) and further modified following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to coloured test substances.

Methods. Following a preliminary range-finding test, Desmodesmus subspicatus was exposed to an aqueous solution of the test material at a 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.

Due to the coloured nature of the test solutions prepared and following the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Test Substances and Mixtures with regards to coloured test substances the study was conducted using a modified algal inhibition test method with increased light intensity and decreased test volume in order to minimise the effects of light adsorption by the test material at the wavelengths required for photosynthetic growth.

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 test material gave EC₅₀ values of greater than 100 mg/l and 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 be near nominal and so 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.

Exposure of Desmodesmus subspicatus to the reference material, potassium dichromate, gave an E_rC₅₀(0 - 72 h) of 0.58 mg/l; 95% confidence limits 0.47 - 0.71 mg/l, an E_yC₅₀(0 - 72 h) of 0.16 mg/l; 95% confidence limits 0.14 - 0.19 mg/l, and an E_bC₅₀(0 - 72 h) of 0.20 mg/l; 95% confidence limits 0.17 - 0.24 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate, yield and biomass integral was 0.125 mg/l and the No Observed Effect Concentration was 0.0625 mg/l.