C-M5

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:

between 26 March 2007 and 27 April 2007.

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 no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

refined for coloured test substances

Principles of method if other than guideline:

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, "Alga, 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 refined for coloured test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the coloured test substance, (Memmert et al 1994).

The modified test system developed by Memmert et al (1994) enables quantification of the pure light filter effect of a coloured test substance on algal growth, and the total growth inhibition by both light absorption and toxicity, by the use of two experimental methods set up in parallel. The differences in algal growth between the two experimental methods are interpreted as the real toxic effect of the dyestuff on algal cell growth.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
0.10, 1.0, 10 and 100 mg/l in range-finding test.
3.2, 10, 32, 100, 320 mg/l in main tests.
- Sampling method: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using either a Coulter® Multisizer Particle Counter or a haemocytometer and light microscope.

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 at what concentration significant absorption of light at the wavelengths required for photosynthesis occurred (460 and 665 nm).

Test solutions

Vehicle:

yes

Details on test solutions:

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 adsorption by the test material at the wavelengths required for photosynthetic growth.
The test was conducted in 250 ml glass conical flasks each containing 25 ml of test preparation and 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, 10, 10 and 100 mg/l.


Experimental Preparation – Experiment A
In order to determine the inhibition of algal growth due to a combination of the toxic effects of the test material and the reduction in light intensity, algae were exposed to the test material at concentrations of 3.2, 10, 32, 100 and 320 mg/l, with glass petri dishes containing culture medium alone being placed above the test vessels.
Amounts of test material (320 and 100 mg) were each separately dissolved in culture medium and the volumes adjusted to 500 ml to give 640 and 200 mg/l stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 64, 20 and 6.4 mg/l. An aliquot (250 ml) of each of the stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 3.2, 10, 32, 100 and 320 mg/l.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.


Experimental Preparation – Experiment B
In order to determine the inhibition of algal growth due to light intensity alone, the test vessels contained algal cells in culture medium alone, whilst separate glass petri dishes containing the test material solutions at concentrations of 3.2, 10, 32, 100 and 320 mg/l were placed above the test vessels.
Amounts of test material (320 and 100 mg) were each separately dissolved in culture medium and the volumes adjusted to 500 ml to give 640 and 200 mg/l stock solutions respectively from which a series of dilutions were made to give the required test concentrations of 320, 100, 32, 10 and 3.2 mg/l. An aliquot (80 ml) of each of the 3.2, 10, 32, 100 and 320 mg/l test concentrations was separately placed in a glass petri dish, the overall depth of which was approximately half the depth of the test solutions in the conical flasks below.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test material on the algal cells.

The concentration and stability of the test material in the test preparations (Experiment A only) were verified by chemical analysis at 0 and 72 hours

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

Liquid cultures of Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. 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 degC.
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 degC until the algal cell density was approximately 2 x 104 - 105 cells/ml.

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

Study design

Test type:

other: Desmodesmus subspicatus was exposed to an aqueous solution of the test material for 72 hours, under constant illumination and stirred continuously via magnetic stirrer at a temperature of 22 to 25ºC. The test was conducted using two experimental methods.

Water media type:

not specified

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group.

Test conditions

Hardness:

Not recorded.

Test temperature:

22 - 25 deg C

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. The temperature within the incubator was recorded daily.

pH= 7.4-7.6

Dissolved oxygen:

Not recorded.

Salinity:

Not recorded.

Nominal and measured concentrations:

Water samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) from Experiment A alone at 0 and 72 hours for quantitative analysis. Duplicate samples were taken at each occasion and stored at approximately -20ºC for further analysis if necessary.

Analysis of the test preparations from Experiment B was not performed, as this was not a requirement of the test guidelines.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 100% to 120% of nominal. Analysis of the test preparations at 72 hours showed measured concentrations to be near nominal with the exception of the 3.2 mg/l test sample which showed a measured concentration of 64% of nominal. Analysis of a duplicate sample which had been stored frozen prior to analysis gave a measured concentration of 86% of nominal indicting the original value to be erroneous.

Details on test conditions:

As in the range-finding test 250 ml glass conical flasks were used.
For Experiment A six flasks each containing 100 ml of test preparation were used for the control and three flasks each containing 100 ml were used for each treatment group. Glass petri dishes containing 80 ml of culture medium were placed above each of the conical flasks.
For Experiment B three flasks containing 100 ml of algal suspension were prepared for the control and each treatment group. Glass petri dishes containing 80 ml of each test preparation were placed above each of the conical flasks (see Appendix 1).
The depth of preparation (15 mm) in the petri dishes for both Experiment A and B was half the depth of the preparation in the conical flasks (30 mm)
The control groups for both Experiment A and B were maintained under identical conditions but not exposed to the test material.
Due to the limited space available only three replicate control vessels were prepared for Experiment B.
Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.06 x 105 cells per ml. This suspension was diluted to a cell density of 8.12 x 103 cells per ml prior to use. At initiation of the test the culture contained a nominal cell density of 4 x 103 cells per ml.
The flasks were incubated (ETAD incubator) at 22 to 25ºC under continuous illumination (intensity approximately 7000 lux) and constantly stirred at approximately 100 rpm for 72 hours.
Samples were taken at 0, 24 and 51 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Coulter counts conducted at 51 hours showed inconsistent results and it was therefore considered appropriate to determine cell densities at 51 and 72 hours using a haemocytometer and light microscope in order to obtain a more accurate count of the number of algal cells present.

Culture Medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

150 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Result from Experiment A. Not possible to calculate 95% confidence limits

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment A

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

260 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Result from Experiment B. Not possible to calculate 95% confidence limits

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Experiment B

Details on results:

Experiment A:
These results indicate the combined toxic nature of the test material and the reduction in light intensity.

Experiment B:
In Experiment B, reduction in light intensity resulted in reduction of algal growth.

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material. Therefore, for classification purposes the results determined from Experiment A should be used.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.58 mg/l (growth rate)

Reported statistics and error estimates:

Determination of ECx values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS).
Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

Statistical analysis
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) was carried out on the growth rate, yield and biomass integral data for Experiments A and B after 72 hours for the control and all test concentrations 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

  Experiment A

Inhibition of growth rate

E_rC₁₀(0 - 72 h)           : 18 mg/l
E_rC₂₀(0 - 72 h)           : 39 mg/l
E_rC₅₀(0 - 72 h)           : 150 mg/l

 Experiment B

Inhibition of growth rate

E_rC₁₀(0 - 72 h)           : 69 mg/l
E_rC₂₀(0 - 72 h)           : 110 mg/l
E_rC₅₀(0 - 72 h)           : 260 mg/l

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

It was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material. Therefore, for classification purposes the results determined from Experiment A should be used.
Experiment A: EC50: 150 mg/l.
Not classifed as harmful to quatic organsims.

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, "Alga, 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 refined for coloured test substances, to differentiate between a reduced growth of algae due to a true toxic effect of the chemical or due to an indirect effect, a reduction in growth by light absorption of the coloured test substance, (Memmertet al1994).

Methods.Following a preliminary range-finding test,Desmodesmus subspicatus was exposed to an aqueous solution of the test material for 72 hours, under constant illumination and stirred continuously via magnetic stirrer at a temperature of 22 to 25ºC. The test was conducted using two experimental methods performed in parallel.

Experiment A

In Experiment A the algae were exposed to test material concentrations of 3.2, 10, 32, 100 and 320 mg/l (three replicate vessels per concentration). Glass petri dishes above the test vessels contained culture medium alone. Therefore, inhibition of algal growth in these test vessels was due to a combination of both the toxic effects of the test material and reduction in light intensity.

Experiment B

In Experiment B the glass petri dishes above the test vessels contained test material solutions at concentrations of 3.2, 10, 32, 100 and 320 mg/l.  The test vessels (three replicate vessels per concentration) contained algal cells in culture medium alone. Therefore inhibition of algal growth was due to a reduction in light intensity alone.

The difference between the inhibition values obtained in Experiment A and B can be interpreted as the true toxic effect of the test material on the algal cells.

Results.

Experiment A

In terms of growth rate, exposure ofDesmodesmus subspicatusto the test material gave an E_rC₅₀(0 - 72 h) value of 150 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 32 mg/l and the No Observed Effect Concentration was 10 mg/l.

These results indicate the combined toxic nature of the test material and the reduction in light intensity.

Experiment B

In terms of growth rate, exposure ofDesmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of 260 mg/l. The Lowest Observed Effect Concentration based on inhibition of growth rate was 100 mg/l and the No Observed Effect Concentration was 32 mg/l.

In Experiment B, reduction in light intensity resulted in reduction of algal growth.

Given that significant differences (greater than 10%) in the inhibition values between Experiments A and B were observed, it was considered that the effect of the test material on algal growth was not only due to a reduction in light intensity, but also due to the intrinsic toxic properties of the test material. Therefore, for classification purposes the results determined from Experiment A should be used.

Under the conditions of the test the test material is not classified as harmful to aquatic organisms.