Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 January 2013 to 04 February 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 mg/L
- Sampling method: 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.
- Sample storage conditions before analysis: The 0 hour samples were stored at approximately -20 °C prior to analysis, whilst the 72 hour samples were analysed on the day of receipt. Duplicate samples were stored at approximately -20 °C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
An amount of test material (1100 mg) was dissolved in 11 litres of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and as a precautionary measure any undissolved test material was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 100 mL discarded in order to pre-condition the filter) to give a stock solution with a nominal concentration of 100 mg/L. 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 (500 mL) of each of the stock solutions was separately inoculated with 5.7 mL of algal suspension 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 was unstable in light; as such all test material preparation was performed under laboratory safety lighting or shielded from the light.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata strain CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, 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 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 °C until the algal cell density was approximately 10^4 - 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
The pH value of the control was 7.8 when measured at 0 hours and 7.7 at 72 hours.
The pH values of the test solutions at nominal concentrations ranged from 7.5 to 7.7 when measured at 0 hours and from 7.6 to 7.7 at 72 hours.
Nominal and measured concentrations:
- Nominal concentrations: 0, 6.25, 12.5, 25, 50 and 100 mg/L
- Measured concentrations (0 hours): - Measured concentrations (72 hours):
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: Closed (plugged with polyurethane foam bungs to reduce evaporation)
- Material, size, headspace, fill volume: 100 mL fill volume
- Aeration: No; flasks were constantly shaken at approximately 150 rpm for 72 hours
- Initial cells density: 5 x 10³ cells/mL
- Control end cell density: 6.56 x 10^5 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes; AAP Culture medium. The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1 N NaOH or HCL. Components (mg/L): NaNO3 25.5, MgCl2.6H2O 12.16, CaCl2.2H2O 4.41, MgSO4.7H2O 14.6, K2HPO4 1.044, NaHCO3 15.0, H3BO3 0.186, MnCl2.4H2O 0.415, ZnCl2 0.00327, FeCl3.6H2O 0.160, CoCl2.6H2O 0.00143, Na2MoO4.2H2O 0.00726, CuCl2.2H2O 0.000012 and Na2EDTA.2H2O 0.30.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionised water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: Warm white lighting 380 to 730 nm and intensity approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Yes. The test was conducted in the same manner as the main test; two replicate flasks were used for each control and test concentration.
- Range-finding test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes. The results showed no effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % CL: 25 - 32 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % CL: 13 - 16 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Mean cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 1. Daily specific growth rates for the control cultures are given in Table 2. Mean growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 3.

GROWTH DATA
From the data given in Table 1 and Table 3, it is clear that the growth rate (r) and yield (y) were affected by the presence of the test material over the 72-hour exposure period. The results were determined from the data based on nominal test concentrations.

- Inhibition of Growth Rate
ErC10 (0 - 72 h): 16 mg/L
ErC20 (0 - 72 h): 20 mg/L
ErC50 (0 - 72 h): 28 mg/L; 95 % confidence limits 25 - 32 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 and Dunnett's multiple comparison procedure for comparing several treatments with a control. There were no statistically significant differences between the control and 6.25 mg/L test concentration (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 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): 7.0 mg/L
EyC20 (0 - 72 h): 9.0 mg/L
EyC50 (0 - 72 h): 14 mg/L; 95 % confidence limits 13 - 16 mg/L
Where EyCx is the test concentration that reduced yield by x %.
Statistical analysis of the yield data was carried out as previously. There were no statistically significant differences between the control and 6.25 mg/L test concentration (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 6.25 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 12.5 mg/L.

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 and 25 mg/L, however cell debris was observed to be present in the test cultures at 50 mg/L and no intact cells were observed to be present in the test cultures at 100 mg/L.

VERIFICATION OF TEST CONCENTRATIONS
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 93 to 95 % of nominal. A concentration dependant decline in measured test concentration was observed at 72 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed at 6.25 mg/L through to 94 % of nominal at 100 mg/L. This decline was considered to be due to adsorption of the test material to the algal cells present as greater losses were observed at the lower test concentrations. This effect was considered to be due to there being greater numbers of algal cells in the lower concentrations and hence greater surface area for adsorption to occur. It can be considered that the algal cells were exposed to nominal concentration throughout the test duration and as such the results are based on nominal test concentrations only

VALIDATION CRITERIA
The following data show that the cell concentration of the control cultures increased by a factor of 119 after 72 hours.
Mean cell density of control at 0 hours: 5.54 x 10³ cells per mL
Mean cell density of control at 72 hours: 6.56 x 10^5 cells per mL
The mean coefficient of variation for section by section specific growth rate for the control cultures was 10 % 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 3 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.
Results with reference substance (positive control):
A positive control used potassium dichromate as the reference material 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 material gave the following results:
ErC50 (0 - 72 h): 1.1 mg/L; 95 % confidence limits 1.0 - 1.3 mg/L
EyC50 (0 - 72 h): 0.70 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.50 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 1.0 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 material.
Reported statistics and error estimates:
- Determination of ECx Values
For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation using the Xlfit software package (IDBS). ECx values were then determined from the equation for the fitted line.
Where appropriate 95 % confidence limits for the EC50values were calculated, using the simplified method of evaluating dose-effect experiments (Litchfield and Wilcoxon, 1949).

- Statistical Analysis
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control 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).

Table 1: Mean Cell Densities and pH Values in the Definitive Test

Nominal Concentration

(mg/L)

pH (0 h)

Cell Densities (cells per mL)

pH (72 h)

0 h

24 h

48 h

72 h

Control

7.8

5.54E+03

2.21E+04

1.21E+05

6.56E+05

7.7

6.25

7.7

6.03E+03

1.68E+04

9.40E+04

6.42E+05

7.7

12.5

7.6

5.42E+03

1.81E+04

7.81E+04

3.70E+05

7.7

25

7.5

5.66E+03

1.32E+04

3.93E+04

1.22E+05

7.7

50

7.5

5.44E+03

7.15E+03

7.70E+03

7.07E+03

7.6

100

7.5

4.75E+03

4.43E+03

4.85E+03

3.69E+03

7.6

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.

 

Table 2: Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Replicate

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

1

0.064

0.064

0.075

2

0.059

0.066

0.077

3

0.067

0.075

0.070

4

0.057

0.070

0.073

5

0.061

0.071

0.065

6

0.063

0.076

0.064

Mean

0.062

0.070

0.071

 

Table 3: Mean Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration

(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 h

% Inhibition

0 to 72 h

% Inhibition

Control

0.068

-

6.51E+05

-

6.25

0.067

1

6.36E+05

2

12.5

0.059

13

3.65E+05

44

25

0.044

35

1.16E+05

82

50

0.005

93

1.63E+03

100

100

-0.004

106

-1.06E+03

100

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of this study, the 72 h ErC50 was determined to be 28 mg/L (95 % CL 25 to 32 mg/L); the NOEC and LOEC values based on the growth rate were determined to be 6.25 and 12.5 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the test material on the growth of the unicellular green alga Pseudokirchneriella subcapitata in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at nominal concentrations of 0, 6.25, 12.5, 25, 50 and 100 mg/L (six replicate flasks for the control, three replicate flasks per concentration) for 72 hours at 24 ± 1 °C. Cultures were subjected to continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and were constantly shaken at approximately 150 rpm.

The test material solution was prepared by dissolving 1100 mg of test material in 11 litres of culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure, any undissolved test material was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre-condition the filter) to give a 100 mg/L stock solution from which a series of dilutions was made to give the remainder of the test concentrations.

Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

The growth rate (r) and yield (y) were affected by the presence of the test material over the 72-hour exposure period. The results were determined from the data based on nominal test concentrations.

Under the conditions of this study, the 72 h ErC50 was determined to be 28 mg/L (95 % CL 25 to 32 mg/L); the NOEC and LOEC values based on the growth rate were determined to be 6.25 and 12.5 mg/L, respectively. The 72 h EyC50 was determined to be 14 mg/L (95 % CL 13 to 16 mg/L); the NOEC and LOEC values based on the yield were determined to be 6.25 and 12.5 mg/L, respectively.

Description of key information

The 72 h ErC50 was determined to be 28 mg/L (95 % CL 25 to 32 mg/L); the NOEC and LOEC values based on the growth rate were determined to be 6.25 and 12.5 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
28 mg/L
EC10 or NOEC for freshwater algae:
6.25 mg/L

Additional information

A study was performed to assess the effect of the test material on the growth of the unicellular green alga Pseudokirchneriella subcapitata in accordance with the standardised guidelines OECD 201 and EU Method C.3 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test material at nominal concentrations of 0, 6.25, 12.5, 25, 50 and 100 mg/L (six replicate flasks for the control, three replicate flasks per concentration) for 72 hours at 24 ± 1 °C. Cultures were subjected to continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 - 730 nm) and were constantly shaken at approximately 150 rpm.

The test material solution was prepared by dissolving 1100 mg of test material in 11 litres of culture medium using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure, any undissolved test material was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 100 mL discarded in order to pre-condition the filter) to give a 100 mg/L stock solution from which a series of dilutions was made to give the remainder of the test concentrations.

Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

The growth rate (r) and yield (y) were affected by the presence of the test material over the 72-hour exposure period. The results were determined from the data based on nominal test concentrations.

Under the conditions of this study, the 72 h ErC50 was determined to be 28 mg/L (95 % CL 25 to 32 mg/L); the NOEC and LOEC values based on the growth rate were determined to be 6.25 and 12.5 mg/L, respectively. The 72 h EyC50 was determined to be 14 mg/L (95 % CL 13 to 16 mg/L); the NOEC and LOEC values based on the yield were determined to be 6.25 and 12.5 mg/L, respectively.