Registration Dossier

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:
14 March 2008 - 22 August 2008.
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:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations:
A range of standard solutions covering 0.57 to 5.7 mg/l (exceeding the range of the working sample concentrations) was analysed.

- Sampling method:
The 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 developed by the Department of Analytical Services, Safepharm Laboratories Limited.

- Sample storage conditions before analysis:

Sponsor's identification: U-donor
Description : clear colourless liquid
Batch number : 071107
Date received : 6 December 2007
Storage conditions: room temperature over silica gel under nitrogen in the dark

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
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 with the aid of ultrasonication and shaking by hand for approximately 40 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 100, 50, 25 and 12.5 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 6.25, 12.5, 25, 50 and 100 mg/l.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The test material rapidly degrades to diethylamine, ethanol and a water soluble silicon compound. The concentration and stability of the degradation product diethylamine in the test preparations were verified by chemical analysis at 0 and 72 hours.

- Eluate:
Not applicable

- Controls:
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

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. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C 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.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
None recorded

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name:
Desmodesmus subspicatus.
Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.

- Strain:
Strain CCAP 276/20

- Source:
Obtained from the Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland.

- Age of inoculum :
Not recorded

- Method of cultivation:
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 deg C until the algal cell density was approximately 104 - 105 cells/ml.

ACCLIMATION

- Acclimation period:
Not recorded.

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

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.

* Elga Optima 15+ or Elga Purelab Option R-15 BP

- Any deformed or abnormal cells observed:
None recorded.

Study design

Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
72 h
Post exposure observation period:
Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l, however large cells clumped together were observed to be present in the test cultures at 100 mg/l.

Observations on test material solubility
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 6.25 mg/l test cultures were observed to be green dispersions. The 12.5, 25 and 50 mg/l test cultures were observed to be pale green dispersions whilst the 100 mg/l test cultures were observed to be extremely pale green dispersions.

Test conditions

Hardness:
Not recorded.
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test.
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 pH values of the control cultures were observed to increase from pH 7.0 – 7.1 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 and therefore was within the limits given in the Test Guidelines.

At the start of the test the test material vessels showed an increase in pH with increasing test concentration. This was considered to be due to an intrinsic property of the test material and as such no attempts were made to alter the starting pH of the test cultures.
Dissolved oxygen:
Not recorded
Salinity:
Not recorded
Nominal and measured concentrations:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel:
250 ml glass conical flasks
- Type: closed
- Material, size, headspace, fill volume: Glass, 250ml, 150ml, 100ml
- Aeration:

- Type of flow-through (e.g. peristaltic or proportional diluter):

- Renewal rate of test solution (frequency/flow rate):

- Initial cells density:

- Control end cells density:

- No. of organisms per vessel:
At initiation of the test the culture contained a nominal cell density of 4 x 103 cells per ml.

- No. of vessels per concentration (replicates):
Three, each containing 100 ml of test preparation.

- No. of vessels per control (replicates):
Six, each containing 100 ml of test preparation.


GROWTH MEDIUM
- Standard medium used: yes
For the purpose of the definitive test, the test material was dissolved directly in culture medium.


TEST MEDIUM

An amount of test material (100 mg) was dissolved in culture medium with the aid of ultrasonication and shaking by hand for approximately 40 minutes and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 100, 50, 25 and 12.5 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 6.25, 12.5, 25, 50 and 100 mg/l.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The test material rapidly degrades to diethylamine, ethanol and a water soluble silicon compound. The concentration and stability of the degradation product diethylamine in the test preparations were verified by chemical analysis at 0 and 72 hours


OTHER TEST CONDITIONS
- Sterile test conditions: yes

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not recorded

- Light intensity and quality:
warm white lighting (380 – 730 nm)

EXPOSURE CONDITIONS :

Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 2.48 x 105 cells per ml. This suspension was diluted to a cell density of 8.22 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 plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C 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.
Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

EFFECT PARAMETERS MEASURED :
- Determination of cell concentrations:
Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

- Chlorophyll measurement:
Not recorded

TEST CONCENTRATIONS
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 6.25, 12.5, 25, 50 and 100 mg/l.

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

The method of analysis, stability, recovery and test preparation analyses for the degradation product diethylamine are described in Attachment 1.

- Range finding study:
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.
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 with the aid of ultrasonication and shaking by hand and the volume adjusted to 500 ml to give a 200 mg/l stock solution. A series of dilutions was made from this stock solution 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.

POSITIVE CONTROL:
A positive control (Safepharm Laboratories Project Number: 0039/0994) used potassium dichromate as the reference material. An amount of reference material (100 mg) was dissolved in culture medium and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 2.0, 1.0, 0.50, 0.25 and 0.125 mg/l. An aliquot (250 ml) of each of the 0.125, 0.25, 0.50, 1.0 and 2.0 mg/l stock solutions was separately mixed with algal suspension (250 ml) to give the required test concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/l.

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. Six replicate flasks were prepared for the control and three replicate flasks prepared for each test concentration.
The flasks were incubated (INFORS Multitron Version 2 incubator) at 24 ± 1°C 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.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.

VALIDATION:
The results of the test are considered valid if the following performance criteria are met:
* The cell concentration of the control cultures must increase by a factor of at least 16 over the test period.

* The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.

* The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, yield and biomass integral
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, yield and biomass integral
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate 95% CL as data generated did not fit the models available for calculation of CL
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
23 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL = 20 - 27 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% Cl = 24 - 36 mg/l
Details on results:
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2 (Attachment 2). Daily specific growth rates for the control cultures are given in Table 3 (Attachment 2). Growth rates, yield and biomass integral values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (Attachment 2).

The mean cell densities versus time for the definitive test are presented in Figure 1 (Attachment 3). Percentage inhibition values are plotted against test concentration in Figure 2, Figure 3 and Figure 4 (Attachment 3).

Verification of test concentrations:
The test material rapidly degrades to diethylamine, ethanol and a water soluble silicon compound. The test concentrations were monitored by analysis of the test samples for diethylamine only.

Analysis of the test preparations at 0 and 72 hours (see Attachment 1) showed measured concentrations of diethylamine to range from 84% to 94% of nominal.

Given that the test organisms were exposed to a mixture of degradents, and that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole the results are based on nominal test concentrations only.


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

Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Daily Specific Growth Rate (cells/ml/hour)
Day 0 - 1 Day 1 - 2 Day 2 - 3
Control R1 0.054 0.051 0.088
R2 0.044 0.059 0.088
R3 0.047 0.059 0.086
R4 0.052 0.053 0.088
R5 0.047 0.056 0.090
R6 0.055 0.057 0.081
Mean 0.050 0.056 0.087

Observations on cultures
All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 6.25, 12.5, 25 and 50 mg/l, however large cells clumped together were observed to be present in the test cultures at 100 mg/l.

Observations on test material solubility
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 6.25 mg/l test cultures were observed to be green dispersions. The 12.5, 25 and 50 mg/l test cultures were observed to be pale green dispersions whilst the 100 mg/l test cultures were observed to be extremely pale green dispersions.

- Any stimulation of growth found in any treatment:
None

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None recorded
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 (Safepharm Laboratories Project No: 0039/0994) are given in Table 5 and Figure 5. Daily specific growth rates for the control cultures are given in Table 6 (Attachment 2) whilst growth rate, yield and biomass integral values are given in Table 7 (Attachment 2). Percentage inhibition values are plotted against test concentration in Figure 6, Figure 7 and Figure 8 (Attachment 3).

Accordingly the following results were determined from the data:
ErC50 (0 - 72 h) : 0.57 mg/l; 95% confidence limits 0.48 - 0.66 mg/l
EyC50 (0 - 72 h) : 0.32 mg/l; 95% confidence limits 0.29 - 0.35 mg/l
EbC50 (0 - 72 h) : 0.31 mg/l; 95% confidence limits 0.28 - 0.35 mg/l
No Observed Effect Concentration (NOEC) based on growth rate : 0.25 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 : 0.50 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:
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 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

 Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure ofDesmodesmus subspicatusto the test material during the range-finding test are given in Table 1 (Attachment 2).

The results showed no effect on growth at the test concentrations of 0.10, 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/l.

Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100 mg/l were selected for the definitive test.

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 96 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                            :   4.24 x 103cells per ml
Mean cell density of control at 72 hours                          :   4.09 x 105cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 32% 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 0% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth data

From the data given in Tables 2 and 4 (Attachment 2), it is clear that the growth rate (r), yield (y) and biomass integral (b) ofDesmodesmus 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

ErC10(0 - 72 h)             : 16 mg/l
ErC20(0 - 72 h)             : 32 mg/l
ErC50(0 - 72 h)             : 100 mg/l*

where ErCxis 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 and 6.25 mg/l test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 6.25 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 12.5 mg/l.

Inhibition of yield

EyC10(0 - 72 h)            : 6.0 mg/l
EyC20(0 - 72 h)            : 9.9 mg/l
EyC50(0 - 72 h)            : 23 mg/l; 95% confidence limits 20 - 27 mg/l

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

There were no statistically significant differences between the control and 6.25 mg/l test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 6.25 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 12.5 mg/l.

 Inhibition of biomass integral

EbC10(0 - 72 h)            : 6.0 mg/l
EbC20(0 - 72 h)            : 11 mg/l
EbC50(0 - 72 h)            : 30 mg/l; 95% confidence limits 24 - 36 mg/l

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

There were no statistically significant differences between the control and 6.25 mg/l test concentration (P³0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on biomass integral was 6.25 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on biomass integral was 12.5 mg/l.


*It was not possible to calculate 95% confidence limits for the ErC50value as the data generated did not fit the models available for the calculation of confidence limits.

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 an ErC50 (0 - 72 h) of 100 mg/l*, an EyC50 (0 - 72 h) of 23 mg/l; 95% confidence limits 20 - 27 mg/l, and an EbC50 (0 - 72 h) of 30 mg/l; 95% confidence limits 24 - 36 mg/l. The Lowest Observed Effect Concentration based on growth rate, yield and biomass integral was 12.5 mg/l, and the No Observed Effect Concentration was 6.25 mg/l.

* It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Executive summary:

In a 72 hour acute toxicity study, the cultures ofDesmodesmus subspicatus, Strain CCAP 276/20 were exposed to U-donor at nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/l, under renewal conditions in accordance with the EU Method C.3 (Algal Inhibition test).  The NOEC and LOEC value was based on growth rate, yield and biomass integral, were 6.25 and 12.5 Mg/L, respectively. 

 

The following abnormalities were noted:  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 6.25 mg/l test cultures were observed to be green dispersions. The 12.5, 25 and 50 mg/l test cultures were observed to be pale green dispersions whilst the 100 mg/l test cultures were observed to be extremely pale green dispersions. 

This toxicity study is classified as R53 May cause long-term adverse effects in the aquatic environment and satisfies the guideline requirements for Algal growth inhibition toxicity study.

Results Synopsis 

Test Organism: Algae

Test Type Static Renewal: 

72 .hr EC50:  100 mg/L

72 hr NOEC:  6.25mg/L

Endpoint Effected:  growth