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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-10-01 to 2010-10-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
test concentrations for the definitive test were not properly chosen as the ErC50 falls outside the concentration range.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Name cited in study report: POPDA
- Lot/Batch number: LL100365
- Color: clear, colorless
- Purity: 94%
- Impurity: polypropylene glycol (approximately 6%)
- Date receipt: 2010-09-21
- Expiry date: 2011-12-31
- Storage: in the dark
Analytical monitoring:
yes
Details on sampling:
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 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 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 (4.6 ml) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/l.

The stock solution and each of the prepared concentrations was 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 ± 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.

Samples of the test preparations were taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. The 0-Hour samples were stored at approximately -20°C prior to analysis.
Vehicle:
no
Details on test solutions:
Definitive test

Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2, and 10 mg/l.

For the purpose of the definitive test, the test item was dissolved directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium and the volume adjusted to 500 ml to give a 100 mg/l stock solution from which a series of dilutions was made to give further stock solutions of 10, 3.2, 1.0, 0.32 and 0.10 mg/l. An aliquot (500 ml) of each of the 0.10, 0.32, 1.0, 3.2 and 10 mg/l stock solutions was separately inoculated with algal suspension (3.3 ml) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/l.

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

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

Verification of test concentrations

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, recovery and test preparation analyses are described in Appendix 4.

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 0.10 mg/l test cultures were observed to be pale green dispersions. The 0.32 and 1.0 mg/l test culture were observed to be very pale green dispersions, whilst the 3.2 and 10 mg/l test cultures were observed to be extremely pale green dispersions.


Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.2). 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 10E+03 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 10E+04 - 10E+05 cells/ml.

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.

Culture medium:
NaNO3 25.5 mg/l
MgCl2.6H2O 12.164 mg/l
CaCl2.2H2O 4.41 mg/l
MgSO4.7H2O 14.7 mg/l
K2HPO4 1.044 mg/l
NaHCO3 15.0 mg/l
H3BO3 0.1855 mg/l
MnCl2.4H2O 0.415 mg/l
ZnCl2 0.00327 mg/l
FeCl3.6H2O 0.159 mg/l
CoCl2.6H2O 0.00143 mg/l
Na2MoO4.2H2O 0.00726 mg/l
CuCl2.2H2O 0.000012 mg/l
Na2EDTA.2H2O 0.30 mg/l
Na2SeO3.5H2O 0.000010 mg/l

The culture medium was prepared using reverse osmosis purified deionised water (Elga Optima 15+) and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl.



Any deformed or abnormal cells observed:

All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures at 72 hours.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.
pH:
The pH values of each test and control flask are given in Table 2. Temperature was maintained at 24 ± 1ºC throughout the test.
The pH values of the control cultures (see Table 2) were observed to increase from pH 7.4 to 7.5 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.
Dissolved oxygen:
Not applicable
Salinity:
freshwater used
Nominal and measured concentrations:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

The results showed no effect on growth at the test concentrations of 0.10 mg/l. However, growth was observed to be reduced at 1.0, 10 and 100 mg/l
Chemical analysis of the 0.10, 1.0, 10 and 100 mg/l test samples at 0 and 72 Hours (see Appendix 4) showed measured test concentrations to range from 94% to 124% of nominal, thereby indicating that the test item was stable under test conditions for the test duration.

Based on this information test concentrations of 0.10, 0.32, 1.0, 3.2, and 10 mg/l were selected for the definitive test.
Details on test conditions:
TEST SYSTEM

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 characterised by a cell density of 6.15 x 10E+05 cells per ml. At initiation of the test the culture contained a nominal cell density of 4 x 10E+03 cells per ml. Inoculation of 500 ml of test medium with 3.3 ml of this algal suspension gave an initial nominal cell density of 4 x 10E+03 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.


- Control end cells density: algal cell density was approximately 4.08E+05 cells/ml

- No. of organisms per vessel:
initial cell density of approximately 4.00E+03 cells/ml.

- No. of vessels per concentration (replicates):
Three replicate flasks per concentration.

- No. of vessels per control (replicates):
Six replicate flasks.



OTHER TEST CONDITIONS
- Sterile test conditions: No

- Adjustment of pH:
No adjustments recorded

- Photoperiod:
Not applicable

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


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.
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). ECx values were then determined from the equation for the fitted line.

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). 


- Chlorophyll measurement:
Not recorded


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
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
15 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% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: not determined
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Not applicable
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
Yield
Remarks on result:
other: 95% confidence limits 1.6 - 2.8 mg/l
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: Not applicable
Details on results:
Exponential growth in the control (for algal test): yes

From the data given in Tables 2 and 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.

The following data show that the cell concentration of the control cultures increased by a factor of 87 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 : 4.69E+03 cells per ml
Mean cell density of control at 72 hours : 4.08E+05 cells per ml

The mean coefficient of variation for section by section specific growth rate for the control cultures was 5% 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 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Accordingly the following results were determined from the data:

Inhibition of growth rate

ErC10 (0 - 72 h) : 1.4 mg/l
ErC20 (0 - 72 h) : 3.3 mg/l
ErC50 (0 - 72 h) : 15 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 between the control, 0.10 and 0.32 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 0.32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 1.0 mg/l.

Inhibition of yield

EyC10 (0 - 72 h) : 0.36 mg/l
EyC20 (0 - 72 h) : 0.69 mg/l
EyC50 (0 - 72 h) : 2.1 mg/l; 95% confidence limits 1. 6 -2.8 mg/l

where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as ifor the growth rate There were no statistically significant differences between the control, 0.10 and 0.32 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 0.32 mg/l. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 1.0 mg/l.
Results with reference substance (positive control):
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 - 72 h) : 0.82 mg/l, 95% confidence limits 0.73 - 0.93 mg/l
EyC50 (0 ¿ 72 h) : 0.47 mg/l, 95% confidence limits 0.43 ¿ 0.51 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
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 and yield 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).

Table1              Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration

(mg/l)

Cell Densities*(cells per ml)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

4.81E+03

5.55E+05

 

 

 

R2

4.51E+03

5.77E+05

-

-

 

Mean

4.66E+03

5.66E+05

 

 

0.10

R1

3.94E+03

4.70E+05

 

 

 

R2

4.21E+03

3.86E+05

3

24

 

Mean

4.08E+03

4.28E+05

 

 

1.0

R1

4.69E+03

2.52E+05

 

 

 

R2

4.22E+03

3.96E+05

10

43

 

Mean

4.45E+03

3.24E+05

 

 

10

R1

4.77E+03

1.26E+05

 

 

 

R2

4.03E+03

9.89E+04

33

81

 

Mean

4.40E+03

1.12E+05

 

 

100

R1

5.02E+03

7.62E+04

 

 

 

R2

3.77E+03

5.86E+04

43

89

 

Mean

4.40E+03

6.74E+04

 

 

 


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

R1and R2= Replicates 1 and 2

Table2              Cell Densities and pH Values in the DefinitiveTest

Nominal Concentration

(mg/l)

pH

Cell Densities*(cells per ml)

pH

0 h

0 h

24 h

48 h

72 h

72 h

Control

R1

7.4

5.38E+03

1.89E+04

8.90E+04

4.07E+05

7.7

 

R2

7.5

4.52E+03

1.88E+04

7.90E+04

4.08E+05

7.8

 

R3

7.5

4.56E+03

1.73E+04

8.87E+04

4.32E+05

7.7

 

R4

7.5

4.62E+03

1.76E+04

8.75E+04

4.19E+05

7.8

 

R5

7.5

4.62E+03

1.58E+04

6.84E+04

3.63E+05

7.8

 

R6

7.5

4.46E+03

1.82E+04

9.02E+04

4.22E+05

7.8

 

Mean

 

4.69E+03

1.78E+04

8.38E+04

4.08E+05

 

0.10

R1

7.4

4.43E+03

1.79E+04

8.65E+04

4.53E+05

7.9

 

R2

7.4

4.49E+03

1.71E+04

8.56E+04

3.95E+05

7.8

 

R3

7.4

4.86E+03

1.64E+04

8.90E+04

4.12E+05

7.8

 

Mean

 

4.59E+03

1.71E+04

8.70E+04

4.20E+05

 

0.32

R1

7.4

4.86E+03

1.76E+04

8.45E+04

3.84E+05

7.8

 

R2

7.4

4.77E+03

1.51E+04

7.02E+04

3.23E+05

7.9

 

R3

7.4

4.41E+03

1.79E+04

8.55E+04

3.85E+05

7.8

 

Mean

 

4.68E+03

1.69E+04

8.01E+04

3.64E+05

 

1.0

R1

7.5

5.03E+03

1.72E+04

8.54E+04

2.94E+05

7.8

 

R2

7.4

4.27E+03

1.36E+04

5.96E+04

2.94E+05

7.7

 

R3

7.4

4.65E+03

1.53E+04

6.46E+04

3.04E+05

7.7

 

Mean

 

4.65E+03

1.54E+04

6.98E+04

2.97E+05

 

3.2

R1

7.5

4.50E+03

9.52E+03

3.00E+04

1.47E+05

7.7

 

R2

7.5

4.42E+03

8.25E+03

2.48E+04

1.43E+05

7.7

 

R3

7.6

4.55E+03

1.41E+04

4.85E+04

1.54E+05

7.6

 

Mean

 

4.49E+03

1.06E+04

3.44E+04

1.48E+05

 

10

R1

8.6

3.69E+03

6.54E+03

2.06E+04

6.22E+04

7.6

 

R2

8.6

4.22E+03

7.24E+03

1.58E+04

5.49E+04

7.6

 

R3

8.6

4.19E+03

7.31E+03

2.07E+04

7.60E+04

7.6

 

Mean

 

4.03E+03

7.03E+03

1.90E+04

6.44E+04

 

 


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

R1- R6= Replicates 1 to 6

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

0.065

0.065

0.063

 

R2

0.065

0.060

0.068

 

R3

0.061

0.068

0.066

 

R4

0.062

0.067

0.065

 

R5

0.057

0.061

0.069

 

R6

0.063

0.067

0.064

 

Mean

0.062

0.065

0.066

 


R1- R6= Replicates 1 to 6

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

Nominal Concentration
(mg/l)

Growth Rate

(cells/ml/hour)

Yield

(cells/ml)

0 ¿ 72 h

% Inhibition

0 ¿ 72 h

% Inhibition*

Control

R1

0.064

 

4.02E+05

 

 

R2

0.064

 

4.04E+05

 

 

R3

0.065

 

4.27E+05

 

 

R4

0.065

-

4.15E+05

-

 

R5

0.063

 

3.58E+05

 

 

R6

0.065

 

4.17E+05

 

 

Mean

0.064

 

4.04E+05

 

 

SD

0.001

 

2.44E+04

 

0.10

R1

0.066

[3]

4.49E+05

 

 

R2

0.064

0

3.90E+05

 

 

R3

0.064

0

4.07E+05

 

 

Mean

0.065

[1]

4.15E+05

[3]

 

SD

0.001

 

3.03E+04

 

0.32

R1

0.063

2

3.79E+05

 

 

R2

0.061

5

3.18E+05

 

 

R3

0.063

2

3.81E+05

 

 

Mean

0.062

3

3.59E+05

11

 

SD

0.001

 

3.57E+04

 

1.0

R1

0.060

6

2.89E+05

 

 

R2

0.060

6

2.89E+05

 

 

R3

0.060

6

3.00E+05

 

 

Mean

0.060

6

2.93E+05

27

 

SD

0.000

 

5.82E+03

 

3.2

R1

0.050

22

1.43E+05

 

 

R2

0.050

22

1.39E+05

 

 

R3

0.051

20

1.50E+05

 

 

Mean

0.050

21

1.44E+05

64

 

SD

0.001

 

5.48E+03

 

10

R1

0.038

41

5.85E+04

 

 

R2

0.036

44

5.07E+04

 

 

R3

0.041

36

7.18E+04

 

 

Mean

0.038

40

6.03E+04

85

 

SD

0.003

 

1.07E+04

 

[ ]


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

R1¿ R6= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave an ErC50 (0 - 72 h) of 15 mg/l and an EyC50 (0 - 72 h) of 2.1 mg/l; 95% confidence limits 1.6 - 2.8 mg/l. The Lowest Observed Effect Concentration based on growth rate and yield was 1.0 mg/l and the No Observed Effect Concentration was 0.32 mg/l. The ErC10 was 1.4 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 0.10, 0.32, 1.0, 3.2 and 10 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.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

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

15*

 

**

 

0.32

1.0

Yield

2.1

1.6

-

2.8

0.32

1.0

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 87% to 103% of nominal and so the results are based on nominal test concentrations only.


*Value determined from the equation for the fitted line as no concentration tested resulted in 50% inhibition of growth rate

**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.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-07-28 to 2005-11-16
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Conducted according to ISO 10253; No information given on test substance purity or stability (responsibility of the Sponsor). No analytical monitoring of the test concentrations.
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
yes
Remarks:
No data on purity or stability of test substance.
GLP compliance:
yes
Remarks:
Statement, no certificate
Specific details on test material used for the study:
- Name as cited in study report: Jeffamine D-230
- Substance type: Active
- Color: colorless
- Lot/Batch number: 4W512
- Date receipt: 2005-07-05 727.9g; 2005-07-13 750.6 g
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared by adding 0.4 g of test substance to 500 mL sterile diluent. Test concentrations were made by diluting the stock solution.
- Eluate: Sterile enriched seawater
- Differential loading:
- Controls: Sterile enriched seawater and a potassium dichromate postive control.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Skeletonema costatum
- Source (laboratory, culture collection): Stllmeadow Inc. in-house laboratory culture
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: Continuous laboratory mass culture

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not):Same as test
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
N/A
Test temperature:
20+/- 2 °C
pH:
Initial pH range-8.3-9.6 SU
Final pH range-7.9-8.9
Dissolved oxygen:
N/A- test flasks were gently aerated.
Salinity:
30 =/- 5 ppt
Nominal and measured concentrations:
Nominal (Definitive test)-25, 50, 100, 200 and 400 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Flasks
- Material, size, headspace, fill volume: 250 mL glass, with stoppers
- Aeration: constant; Flasks were hand-swirled twice daily.
- Initial cells density: 5000 cells/mL
- Control end cells density: 157 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Sterile seawater with addition of Guillard f/2 + silicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic seatwater enriched with Guillard f/2 + silicates
- Culture medium different from test medium: No
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuous light
- Light intensity and quality: 280-560 ft.c
- Salinity (for marine algae): 35 +/- 5 ppt

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: hemacytometer


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study:yes
- Test concentrations: 0.1, 1.0, 10, 100, 500 and 1000 mg/L (for range-finding)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (10 mg/L, JT Baker Lot# V11654)
Key result
Duration:
72 h
Dose descriptor:
IC50
Effect conc.:
141.72 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL was 89.89-173.04 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
33.34 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL was 0.00-52.02 mg/L
Details on results:
- Exponential growth in the control (for algal test): yes
Results with reference substance (positive control):
- Results with reference substance valid? Validity criteria not reported.
- Other: No cells alive at 24 h
Reported statistics and error estimates:
The IC50 was determined using the linear interpolation method.
Validity criteria fulfilled:
yes
Remarks:
Control density increased by >16 x in 72 h, and control pH did not vary by >1.0 SU.
Conclusions:
The 72 hr IC50 of the test substance to the alga Skeletonema costatum was determined to be 141.72 mg/L, with 95% confidence limits of 89.89-173.04 mg/L. The 72 hr EC 10 for growth rate was determined to be 33.34 mg/L.

Description of key information

The 72 hr EC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to the freshwater algae Pseudokirchneriella subcapitata based on growth rate was found to be 15 mg/L (Vryenhoef & Mullee, 2010). The 72 hr EC50 based on yield was found to be 2.1 mg/L.  The 72 hr ErC10 was determined to be 1.4 mg/L. The 72 hr IC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to the marine algae Skeletonema costatum was determined to be 141.72, with 95% confidence limits of 89.89-173.04 mg/L (Stillmeadow Inc., 2005). The 72 hr ErC10 was determined to be 33.34 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
15 mg/L
EC50 for marine water algae:
141.72 mg/L
EC10 or NOEC for freshwater algae:
1.4 mg/L
EC10 or NOEC for marine water algae:
33.34 mg/L

Additional information

A 72-h GLP and Guideline ISO 10253 study was conducted with the marine algae Skeletonema costatum. The test temperature was 20 deg C at 30 +/- 5 ppt salinity. Standard media plus silica was used. Test concentrations were 25, 50, 100, 200 and 400 mg/L plus a Control, 6 replicates each. The culture was 3 -4 days old, in exponential growth. The 72 -h IC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to the alga Skeletonema costatum was determined to be 141.72, with 95% confidence limits of 89.89-173.04 mg/L. The 72 -h ErC10 was determined to be 33.34 mg/L.

A second 72 h study was conducted with the freshwater algae Pseudokirchneriella subcapitata, strain CCAP 278/4. This study also followed GLP and OECD Guideline 201. The test culture was one week old. Test concentrations were Control, 0.10, 0.32, 1.0, 3.2 and 10 mg/L. Three replicates of each concentration were exposed, with 6 replicates for the Control. The 72 hr EC50 of Poly[oxy(methyl-1,2-ethanediyl)], alpha-(2-aminomethylethyl)-omega-(2-aminomethylethoxy)- to Pseudokirchneriella subcapitata based on growth rate was found to be 15 mg/L by extrapolation from the growth rate inhibition curve. The 72 -h EC50 based on yield was found to be 2.1 mg/L. The 72 hr ErC10 was determined to be 1.4 mg/L.