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Diss Factsheets

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:
This study was conducted between 10 February 2017 and 17 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
EC No. 761/2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylenepropane-1,3-diyl diacetate
EC Number:
223-225-8
EC Name:
2-methylenepropane-1,3-diyl diacetate
Cas Number:
3775-29-9
Molecular formula:
C8H12O4
IUPAC Name:
2-[(acetyloxy)methyl]prop-2-en-1-yl acetate
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification : 2-methylene-1, 3-propanediol diacetate (CAS: 3775-29-9)
Physical state/Appearance: Clear colorless liquid
Batch: 20160308 (- 88)
Purity: 99.7%
Expiry Date: 31 March 2019
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Range-Finding Test
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis

Definitive Test
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

Test solutions

Vehicle:
no
Details on test solutions:
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 Pseudokirchneriella subcapitata 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 item was dissolved directly in culture medium.
An amount of test item (50.6 mg) was dissolved in culture medium with the aid of ultrasonication for 5 minutes and the volume adjusted to 500 mL to give a nominal 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.0 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.

Definitive Test
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.
Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 25 minutes and the volume adjusted to 2 liters to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (9.0 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.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, 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 illumination at 21 ± 2”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 10^3 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 10^4 – 10^5 cells/mL.

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:

NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgSO4.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 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
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
Not reported
Test temperature:
24 ± 1 ”C
pH:
pH = 7.2 - 7.4
Dissolved oxygen:
Not reported
Salinity:
Not reported
Conductivity:
Not reported
Nominal and measured concentrations:
Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours showed measured test concentrations to range from 9.4 to 97 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.045 mg/L indicating that the test item was unstable under the conditions of the test.
Details on test conditions:
Experimental Design and Study Conduct
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 Pseudokirchneriella subcapitata 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 item was dissolved directly in culture medium.
An amount of test item (50.6 mg) was dissolved in culture medium with the aid of ultrasonication for 5 minutes and the volume adjusted to 500 mL to give a nominal 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (5.0 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 item.
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 ± 1 ºC 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.
A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

Definitive Test
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.
Experimental Preparation
A nominal amount of test item (200 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 25 minutes and the volume adjusted to 2 liters to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 50, 25, 12.5 and 6.25 mg/L. An aliquot (1 liter) of each of the stock solutions was separately inoculated with algal suspension (9.0 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 concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. 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.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.52 x 105 cells per mL. Inoculation of 1 liter of test medium with 9.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration.
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.

Assessments
Test Organism Observations
Samples were taken at 22, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
The appearance of the test media was recorded daily.

Verification of Test Concentrations
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Additional samples of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
Range-finding Test
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.
Chemical analysis of the 10 and 100 mg/L test preparations at 0 hours (see Annex 3) showed measured test concentrations to range from 9.4 to 97 mg/L. A decline in measured test concentrations was observed at 72 hours to less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.045 mg/L indicating that the test item was unstable under the conditions of the test.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 hours (see Table 1) showed measured test concentrations to range from 4.9 to 92 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 4.0 to 85 mg/L at 24 hours, 1.1 to 41 mg/L at 48 hours and 0.15 to 2.3 mg/L at 72 hours.
Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 2.3, 4.8, 11, 22 and 52 mg/L.

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.
From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly the following results were determined from the data:

Inhibition of growth rate
ErC10 (0 - 72 h) : 17 mg/L
ErC20 (0 - 72 h) : 26 mg/L
ErC50 (0 - 72 h) : 51 mg/L

where ErCx is 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 (P0.05), between the control, 2.3, 4.8 and 11 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 11 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 22 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 9.4 mg/L
EyC20 (0 - 72 h) : 13 mg/L
EyC50 (0 - 72 h) : 21 mg/L; 95% confidence limits 18 - 25 mg/L

Where:
EyCx is the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out as above. There were no statistically significant differences (P0.05), between the control, 2.3, 4.8 and 11 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 11 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 22 mg/L.

Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 69 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.
Nominal cell density of control at 0 hours : 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 3.45 x 105^ cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 17% 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%.

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 2.3, 4.8 and 11 mg/L. Some enlarged cells were observed to be present in the test cultures at 22 mg/L whilst some enlarged and misshapen cells were observed to be present in the 52 mg/L test cultures.

Water Quality Criteria
The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures (see Table 2) was observed to increase from pH 7.2 at 0 hours to pH 7.4 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.

Observations on Test Item Solubility
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 2.3, 4.8 and 11 mg/L test cultures were observed to be green dispersions. The 22 mg/L test cultures were observed to be pale green dispersions whilst the 52 mg/L test cultures were observed to be extremely pale green dispersions.
Results with reference substance (positive control):
Positive Control
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.

Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Table 2            Cell Densities and pH Values in the Definitive Test

Geometric Mean Measured Test Concentration

(mg/L)

pH

Cell Densities* (cells per mL)

pH

0 h

22 h

48 h

72 h

72 h

Control

R1

7.2

1.42E+04

6.83E+04

3.82E+05

7.4

R2

1.69E+04

6.46E+04

1.87E+05

R3

1.82E+04

9.34E+04

4.09E+05

R4

1.20E+04

6.59E+04

2.46E+05

R5

1.56E+04

6.83E+04

2.61E+05

R6

1.32E+04

1.02E+05

4.27E+05

Mean**

1.46E+04

7.96E+04

3.45E+05

2.3

R1

7.2

1.56E+04

8.27E+04

3.19E+05

7.4

R2

1.69E+04

8.94E+04

3.76E+05

R3

1.56E+04

7.63E+04

3.96E+05

Mean

1.60E+04

8.28E+04

3.63E+05

4.8

R1

7.1

1.45E+04

1.00E+05

4.48E+05

7.3

R2

1.44E+04

9.52E+04

3.21E+05

R3

1.68E+04

7.48E+04

4.88E+05

Mean

1.52E+04

9.01E+04

4.19E+05

11

R1

7.1

1.68E+04

5.55E+04

3.35E+05

7.2

R2

1.54E+04

4.86E+04

2.53E+05

R3

1.39E+04

3.45E+04

2.39E+05

Mean

1.54E+04

4.62E+04

2.75E+05

22

R1

7.1

1.44E+04

3.07E+04

1.99E+05

7.2

R2

1.32E+04

3.68E+04

1.32E+05

R3

1.38E+04

3.57E+04

1.86E+05

Mean

1.38E+04

3.44E+04

1.72E+05

52

R1

7.0

9.39E+03

1.79E+04

2.69E+04

7.2

R2

1.02E+04

1.48E+04

4.19E+04

R3

8.42E+03

1.79E+04

5.12E+04

Mean

9.35E+03

1.69E+04

4.00E+04

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1- R6= Replicates 1 to 6

**Mean values calculated excluding replicate R2.

Table 3            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

0.047

0.060

0.072

R2

0.055

0.052

0.044

R3

0.059

0.063

0.062

R4

0.040

0.065

0.055

R5

0.052

0.057

0.056

R6

0.044

0.079

0.060

Mean*

0.050

0.063

0.058

R1- R6= Replicates 1 to 6

*Mean values calculated excluding replicate R2.

Table 4            Inhibition of Growth Rate and Yield in the Definitive Test

Geometric Mean Measured Test Concentration
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.060

 

3.77E+05

 

R2

0.050

 

1.82E+05

 

R3

0.061

 

4.04E+05

 

R4

0.054

-

2.41E+05

-

R5

0.055

 

2.56E+05

 

R6

0.062

 

4.22E+05

 

Mean**

0.058

 

3.40E+05

 

SD**

0.004

 

8.52E+04

 

2.3

R1

0.058

0

3.14E+05

 

R2

0.060

[3]

3.71E+05

 

R3

0.061

[5]

3.91E+05

 

Mean

0.060

[3]

3.58E+05

[5]

SD

0.002

 

4.01E+04

 

4.8

R1

0.062

[7]

4.43E+05

 

R2

0.058

[0]

3.16E+05

 

R3

0.064

[10]

4.83E+05

 

Mean

0.061

[6]

4.14E+05

[22]

SD

0.003

 

8.70E+04

 

11

R1

0.058

[0]

3.30E+05

 

R2

0.054

7

2.48E+05

 

R3

0.054

7

2.34E+05

 

Mean

0.055

5

2.70E+05

21

SD

0.002

 

5.20E+04

 

22

R1

0.051

12

1.94E+05

 

R2

0.045

22

1.27E+05

 

R3

0.050

14

1.81E+05

 

Mean

0.049

16

1.67E+05

51

SD

0.003

 

3.54E+04

 

52

R1

0.023

60

2.19E+04

 

R2

0.030

48

3.69E+04

 

R3

0.032

45

4.62E+04

 

Mean

0.028

51

3.50E+04

90

SD

0.005

 

1.23E+04

 

*       In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1-R6= Replicates 1 to 6

SD= Standard Deviation

**Mean and SD values calculated excluding replicate R2.

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results: Please see 'Executive Summary'.
Executive summary:

A study was perford to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.


 


 Methods.


Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 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.


Analysis of the test preparations at 0 hours showed measured test concentrations to range from 4.9 to 92 mg/L. A decline in measured test concentrations was observed after each    24-Hour period in the range of 4.0 to 85 mg/L at 24 hours, 1.1 to 41 mg/L at 48 hours and 0.15 to 2.3 mg/L at 72 hours.


Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.  The geometric mean measured test concentrations were determined to be 2.3, 4.8, 11, 22 and 52 mg/L.


Exposure ofPseudokirchneriella subcapitatato the test item gave the following results based on the geometric mean measured test concentrations:


 































Response Variable



EC50(mg/L)



95% Confidence Limits (mg/L)



No Observed Effect Concentration (NOEC) (mg/L)



Lowest Observed Effect Concentration (LOEC) (mg/L)



Growth Rate



51



 



*



 



11



22



Yield



21



18



-



25



11



22






*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.







*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.







*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.