[No public or meaningful name is available]

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:

25 July 2008 and 5 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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 (incl. QA statement)

Remarks:

The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Test material information

Constituent 1

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: An aliquot (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.10, 1.0, 10 and 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.
- Sample storage conditions before analysis:

Sponsor's identification : NDS-Li
Description : white crystalline solid
Batch number : 7001
Date received : 12 May 2008
Storage conditions : room temperature in the dark

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For the purpose of the definitive test, the test material was dissolved directly in culture medium.
- Eluate: Same as culture media
- Controls: A positive control (Harlan Laboratories Ltd Project No: 0039/1066) used potassium dichromate as the reference material at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
- 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):None

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- 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 available
- Method of cultivation: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. The prepared media was sterilised by 0.2 µm membrane filtration.

ACCLIMATION
- Acclimation period:Not available
- 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.

Culture Medium
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.

- Any deformed or abnormal cells observed: None

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not available

Test temperature:

24 ± 1°C. The temperature within the incubator was recorded daily.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter

Dissolved oxygen:

Not available

Salinity:

Not available

Nominal and measured concentrations:

Range finding test:The initial range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
Definitive test:Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:250 ml glass conical flasks
- Type (delete if not applicable):closed,plugged with polyurethane foam stoppers to reduce evaporation.
- Material, size, headspace, fill volume: The test was conducted in 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
- Renewal rate of test solution (frequency/flow rate):Not applicable
- Initial cells density: At initiation of the test the culture contained a nominal cell density of 4 x 103 cells per ml.
- Control end cells density: 5.68 x 10^5 cells per ml
- No. of organisms per vessel:Not applicable
- 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
- Detailed composition if non-standard medium was used: For the purpose of the definitive test, the test material was dissolved directly in culture medium.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:No
- Photoperiod:continuous illumination
- Light intensity and quality:(intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm)


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:Not applicable
- Other:


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: None
- Range finding study
- Test concentrations: The test concentrations to be used in the definitive test were determined by preliminary range-finding tests. The initial range-finding test was conducted by exposing Desmodesmus subspicatus cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
The test was conducted in 250 ml glass conical flasks each containing 100 ml of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks 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 (100 ml) of each of the stock solutions was separately mixed with algal suspension (100 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.
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 7000 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.
The results of the initial range-finding test showed a relatively flat response in terms of inhibition of growth rate and so a second range-finding test was conducted in an identical manner to the first by exposing Desmodesmus subspicatus cells to nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l for a period of 72 hours.
Exposure conditions in the second range-finding test were the same as those in the initial test.

- Results used to determine the conditions for the definitive study:Based on the results of the range-finding tests the following test concentrations were assigned to the definitive test: 10, 20, 40, 80 and 160 mg/l.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL not stated

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL not stated

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL not stated

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CL not stated

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL not stated

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL not stated

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

not specified

Remarks:

yield

Remarks on result:

other: 95 % CL not stated

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

40 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL not stated

Details on results:

- Exponential growth in the control (for algal test): yes.
- Any stimulation of growth found in any treatment: There were no abnormalities detected in any of the control or test cultures at 72 hours.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: There were no abnormalities detected in any of the control or test cultures.
- 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, 10 and 20 mg/l test cultures were observed to be pale green dispersions whilst the 40, 80 and 160 mg/l test cultures were observed to be very pale green dispersions.

Results with reference substance (positive control):

- Results with reference substance valid?Yes
- EC50:
- Other:

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences between the control, 10 and 20 mg/l test concentrations (P0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 20 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 40 mg/l

Any other information on results incl. tables

The following data show that the cell concentration of the control cultures increased by a factor of 34 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                      :   4.09 x 10³cells per ml
Mean cell density of control at 72 hours                   :   1.40 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 29% 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:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave the following results:
Response Variable EC50 (mg/l) No Observed Effect Concentration (NOEC) (mg/) Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate > 160* 20 40
Yield 57 20 40
Biomass 92 20 40

Executive summary:

Introduction.A study was perford 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 Council Regulation (EC) No 440/2008.

Methods. Following preliminary range-finding tests,Desmodesmus subspicatus was exposed to an aqueous solution of the test material at concentrations of 10, 20, 40, 80 and 160 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 ofDesmodesmus subspicatusto the test material gave the following results:

Response Variable	EC50(mg/l)	No Observed Effect Concentration (NOEC) (mg/)	Lowest Observed Effect Concentration (LOEC) (mg/l)
Growth Rate	> 160*	20	40
Yield	57	20	40
Biomass	92	20	40

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 86% to 126% of nominal and so the results are based on nominal test concentrations only.

*It was not possible to calculate an E_rC₅₀value as no concentration tested resulted in greater than 50% inhibition of growth rate.