Triazine derivative

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:

The range-finding test was conducted between 26 March 2007 and 29 March 2007 and the definitive test between 14 May 2007 and 17 May 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 not 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

method refined for coloured test substance (see prinicples of method section)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

as for OECD 201

Principles of method if other than guideline:

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)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Sponsor's identification: C-Y9
Description : yellow powder
Batch number : MB-1
Date received : 6 March 2007
Storage conditions: room temperature over silica gel in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Experiment A: 3.2, 10, 32, 100 and 320 mg/l

- Sampling method:
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 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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

RANGE-FINDING TEST:
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The 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.

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.

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:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 3.2, 10, 32, 100 and 320 mg/l.
For the purpose of the definitive test, the test material was dissolved directly in culture medium using two parallel experimental set ups.

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.

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 Desmodesmus subspicatus were obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, 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 constant illumination at 21 ± 1°C.
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 10E3 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°C until the algal cell density was approximately 2 x 10E4 - 10E5 cells/ml.

ACCLIMATION
- 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.

Study design

Test type:

static

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Samples were taken at 0, 26, 49 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

Test conditions

Test temperature:

The flasks were incubated (ETAD incubator) at 25 to 26ºC. 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.
Experiment A - The pH values of the control cultures were observed to increase from pH 7.5 at 0 hours to pH 8.3 – 8.4 at 72 hours.
Experiment B - The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.4 – 8.5 at 72 hours.

Nominal and measured concentrations:

Experiment A and B - nominal concentrations of 3.2, 10, 32, 100 and 320 mg/l.

Measured - Analysis of the test preparations from Experiment A showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Details on test conditions:

TEST SYSTEM
Exposure 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.

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 in the ETAD cabinet 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 1.97 x 10E5 cells per ml. This suspension was diluted to a cell density of 8.56 x 10E3 cells per ml prior to use. At initiation of the test the culture contained a nominal cell density of 4 x 10E3 cells per ml.

GROWTH MEDIUM
Culture Medium:
The culture medium is defined below -
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 (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.

OTHER TEST CONDITIONS
The flasks were incubated under continuous illumination (intensity approximately 7000 lux) and constantly stirred at approximately 100 rpm for 72 hours.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were dtermined using a Coulter Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Result from Experiment A

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: Result from Experiment A

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

130 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: Result from Experiment A

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: Result from Experiment A

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield and biomass

Remarks on result:

other: Result from Experiment A

Details on results:

Observations on cultures:
All test and control cultures for Experiment A and B were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.

Observations on test material solubility:
Experiment A:
At the start of the test all control cultures were observed to be clear colourless solutions. The test cultures were observed to range from pale yellow solutions at 3.2 mg/l through to dark yellow/orange solutions at 320 mg/l. After the 72-Hour test period all control cultures were observed to be pale green dispersions. The test cultures were observed to range from pale yellow/green dispersions at 3.2 mg/l through to dark yellow/orange dispersions at 320 mg/l.

Experiment B:
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, 3.2 and 10 mg/l test cultures were observed to be pale green dispersions whilst the 32, 100 and 320 mg/l test cultures were observed to be very pale green dispersions.

Results with reference substance (positive control):

The cell densities from exposure of Desmodesmus subspicatus (CCAP 276/20) to the reference material during the positive control are given in Table 9 (see attached background material). Daily specific growth rates for the control cultures are given in Table 10 (see attached background material) whilst growth rates, yield and biomass integral values are given in Table 11 (see attached background material).

Accordingly the following results were determined from the data:
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:

See any other information on results incl.tables section.

Any other information on results incl. tables

Range-finding Test

The cell counts and percentage inhibition of growth values from the exposure of Desmodesmus subspicatus to the test material during the range-finding test are given in Table 1 (see attached background material).

Whilst significant inhibition of growth was observed at 0.10 and 100 mg/l the results showed no effect on growth at the test concentrations of 1.0 and 10 mg/l. It was therefore considered that the inhibition of growth observed at 0.10 mg/l was due to possible contamination of the test vessels.

The absorption spectrum of chlorophyll a, the principal photoreceptor in the chloroplasts of green plants, shows that it has a double peak (Bidwell, 1979) with maximum absorption occurring at approximately 460 nm and 665 nm. As such if coloured solutions formed by the test material absorbed light at either of these wavelengths it would reduce the light intensity available for photosynthetic growth to occur.

Spectrophotometer measurements performed on test samples at concentration of 0.20, 2.0, 20 and 200 mg/l (see Table 2 - attached background material) indicated that whilst no significant adsorption occurred at 665 nm, a concentration dependent increase in adsorption values occurred at 460 nm. Based on this information and given that inhibition was observed despite the use of a modified test method it was considered justifiable to conduct the definitive test using the modified method developed by Memmert et al (1994) in order to confirm whether the inhibition of algal growth observed was due to true test material toxicity or a result of reduction in light available for photosynthetic growth.

Based on this information and in order to ensure that both EC50 and NOEC values were obtained test concentrations of 3.2, 10, 32, 100 and 320 mg/l were selected for the definitive test.

Definitive Test

Experiment A

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3 (see attached background material). Daily specific growth rates for the control cultures are given in Table 4 (see attached background material). Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 5 (see attached background material).

Experiment B

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 6 (see attached background material). Daily specific growth rates for the control cultures are given in Table 7 (see attached background material). Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 8 (see attached background material).

Validation criteria

Experiment A

The following data show that the cell concentration of the control cultures increased by a factor of 86 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:   4.33 x 10³cells per ml
Mean cell density of control at 72 hours:   3.71 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 27% 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 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Experiment B

The following data show that the cell concentration of the control cultures increased by a factor of 83 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:   4.04 x 10³cells per ml
Mean cell density of control at 72 hours:   3.34 x 10⁵cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 28% 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 5% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data

Experiment A

From the data given in Tables 3 and 5, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate

E_rC₁₀(0 - 72 h)             : 52 mg/l
E_rC₂₀(0 - 72 h)             : 160 mg/l
E_rC₅₀(0 - 72 h)             : > 320 mg/l*

where E_rC_xis the test concentration that reduced growth rate by x%.

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

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, 3.2, 10 and 32 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 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 100 mg/l.

Inhibition of yield

E_yC₁₀(0 - 72 h)            : 11 mg/l
E_yC₂₀(0 - 72 h)            : 19 mg/l
E_yC₅₀(0 - 72 h)            : 100 mg/l

where E_yC_xis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as for growth rate. There were no statistically significant differences between the control, 3.2, 10 and 32 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 yield was 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 100 mg/l.

Inhibition of biomass integral

E_bC₁₀(0 - 72 h)            : 13 mg/l
E_bC₂₀(0 - 72 h)            : 31 mg/l
E_bC₅₀(0 - 72 h)            : 130 mg/l

where E_bC_xis the test concentration that reduced biomass integral (area under the growth curve) by x%.

Statistical analysis of the biomass integral data was carried out as for growth rate. There were no statistically significant differences between the control, 3.2, 10 and 32 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 biomass integral was 32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 100 mg/l.

Experiment B

From the data given in Tables 6 and 8, it is clear that the growth rate (r), yield (y) and biomass integral (b) of Desmodesmus subspicatus (CCAP 276/20) were affected by the presence of the test material over the 72-Hour exposure period.

Accordingly the following results were determined from the data:

Inhibition of growth rate

E_rC₁₀(0 - 72 h)             : 43 mg/l
E_rC₂₀(0 - 72 h)             : 150 mg/l
E_rC₅₀(0 - 72 h)             : > 320 mg/l*

where E_rC_xis the test concentration that reduced growth rate by x%.

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, 3.2 and 10 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 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/l.

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

Inhibition of yield

E_yC₁₀(0 - 72 h)            : 7.4 mg/l
E_yC₂₀(0 - 72 h)            : 18 mg/l
E_yC₅₀(0 - 72 h)            : 84 mg/l

where E_yC_xis the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as for growth rate. There were no statistically significant differences between the control, 3.2 and 10 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 yield was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg/l.

Inhibition of biomass integral

E_bC₁₀(0 - 72 h)            : 6.7 mg/l
E_bC₂₀(0 - 72 h)            : 17 mg/l
E_bC₅₀(0 - 72 h)            : 81 mg/l

where E_bC_xis the test concentration that reduced biomass integral (area under the growth curve) by x%.

Statistical analysis of the biomass integral data was carried out as for growth rate. There were no statistically significant differences between the control, 3.2 and 10 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 biomass integral was 10 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 32 mg/l.

Disscusion

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.

Physico-chemical measurements

 Experiment A

The pH values of each test and control flask are given in Table 3. The pH values of the control cultures (see Table 3 - attached background material) were observed to increase from pH 7.5 at 0 hours to pH 8.3 – 8.4 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.

Experiment B

The pH values of each test and control flask are given in Table 6 (see attached background material). The pH value of the control cultures was observed to increase from pH 7.5 at 0 hours to pH 8.4 – 8.5 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. 

On two occasions the temperature of the incubator was in excess of that recommended in the test guideline (24 ± 1ºC) Given that all growth criteria were met and that no abnormalities were detected in the control cultures during microscopic examination this was considered to have had no adverse effect.

Verification of test concentrations

Analysis of the test preparations at 0 and 72 hours (see table below) showed measured test concentrations to be near nominal and so it was considered justifiable to calculate the EC₅₀values in terms of nominal test concentrations only.

Sample	Nominal Concentration (mg/l)	Concentration Found (mg/l)	Expressed as a Percent of the Nominal Concentration (%)
0 Hours	Control	<LOQ	-
	3.2	3.58	112
	10	10.1	101
	32	32.7	102
	100	100	100
	320	318	99
72 Hours	Control	<LOQ	-
	3.2	3.47	108
	10	10.5	105
	32	34.6	108
	100	107	107
	320	336	105

LOQ = Limit of quantitation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Experiment A:

The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 320 mg/l, an EyC50 (0 - 72 h) of 100 mg/l and an EbC50 (0 - 72 h) of 130 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 100 mg/l, and the No Observed Effect Concentration was 32 mg/l.

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

Analysis of the test preparations from Experiment A showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

Experiment B:
The effect of the test material on the growth of Desmodesmus subspicatus has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of greater than 320 mg/l, an EyC50 (0 - 72 h) of 84 mg/l and an EbC50 (0 - 72 h) of 81 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 32 mg/l, and the No Observed Effect Concentration was 10 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.

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, (Memmert et al 1994).

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 25 to 26ºC. 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.

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.

Experiment A

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀(0 - 72 h) value of greater than 320 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 terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀ (0 - 72 h) value of 100 mg/l. The Lowest Observed Effect Concentration based on yield was 100 mg/l and the No Observed Effect Concentration was 32 mg/l.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀ (0 - 72 h) value of 130 mg/l. The Lowest Observed Effect Concentration based on inhibition of biomass integral was 100 mg/l and the No Observed Effect Concentration was 32 mg/l.

Experiment B

In terms of growth rate, exposure of Desmodesmus subspicatus to the test material gave an E_rC₅₀ (0 - 72 h) value of greater than 320 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.

In terms of yield, exposure of Desmodesmus subspicatus to the test material gave an E_yC₅₀ (0 - 72 h) value of 84 mg/l. The Lowest Observed Effect Concentration based on yield was 32 mg/l and the No Observed Effect Concentration was 10 mg/l.

In terms of biomass integral (area under growth curve), exposure of Desmodesmus subspicatus to the test material gave an E_bC₅₀ (0 - 72 h) value of 81 mg/l. The Lowest Observed Effect Concentration based on inhibition of biomass integral 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.

Analysis of the test preparations from Experiment A showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

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.

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.