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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 March 2012 to 16 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
The test item was completely dissolved directly in culture medium.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Range-finding study: at the start of the range-finding stusy a sample of each test and control culture was removed at 72 h and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then sealed with ground glass stoppers and incubated at 24 ± 1 °C under continuous illumination provided by warm white lighting and constantly shaken. After 72 h of the initial range-finding study the cell density of each flask was determined using a Coulter® Multisizer Particle Counter. After 72 h of the second range-finding study the cell density of each flask was determined using a haemocytometer and light microscope. A sample of each test concentration was taken for chemical analysis at 0 and 72 h in order to determine the stability of the test item under test conditions. A samples were stored at -20 °C prior to analysis.

Definitive study: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 h. Samples were taken at 0, 24, 48 and 72 h and the cell densities were determined using a Coulter® Multisizer Particle Counter. Samples were taken from the control and each test group at 0 and 72 h for quantitative analysis. All samples were stored at approximately -20 °C prior to analysis. Duplicate samples were taken at 0 and 72 h and stored at approximately -20 °C for further analysis if necessary. Only samples at the NOEC were provided for analysis. Given that chemical analysis of the 100 mg/L test samples taken from the initial range-finding test indicated that a decline in measured test concentration occurred it was considered possible that the test item may have been absorbing to the algal cells present. As such, during the definitive test additional test samples were prepared at the start of the test with the omission of algal cells. These additional samples were then incubated alongside the test to provide samples for uninoculated analyses at 72 h.
Vehicle:
no
Details on test solutions:
Range finding study: The study was conducted in 250 mL glass conical flasks each completely filled with test preparation and sealed with ground glass stoppers 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 was dissolved in culture medium with the aid of high shear mixing at approximately 7500 rpm for 5 min and the volume adjusted to 1 L to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot of each of the stock solutions was separately inoculated with algal suspension ( 19.1 mL and 5.6 mL in the first and second range-finding studies respectively) to give the required test concentrations. 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. Based on the results of the range-finding test, initial experiments were performed as limit tests at a single concentration of 100 mg/L. Significant inhibition of growth however was observed and therefore a second range-finding test was conducted in order to determine the test concentration range to be employed in the definitive test.

Definitive study: For the purpose of the definitive test, the test item was dissolved directly in the culture medium. An aliquot of test item was dissolved in culture medium with the aid of high shear mixing at approximately 7500 rpm for 10 min and the volume adjusted to 3 L to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot of each of the stock solutions was separately inoculated with algal suspension to give the required test concentrations. The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity. The control group as maintained under identical conditions but not exposed to the test item.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- 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
- Age of inoculum (at test initiation):
- 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 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.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): The culture medium used for the definitive test was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system.
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± °C. The temperature within the incubator was recorded daily.
pH:
The pH of the control and each test preparation was determined at initiation of the test and after 72 h exposure. The pH was measured using a WTW pH 320 pH meter.
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.1 at 72 hours.
Nominal and measured concentrations:
Range-finding study: nominal 0.10, 1.0. 10 and 100 mg/L
Definitive study: nominal 1.0, 3.2, 10, 32 and 100 mg/L
Measured (100 mg/L group): 0 hr 99.9 mg/L; 72 hr 58.3 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type (delete if not applicable): closed with ground glass stoppers to reduce evaporation
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 7.42 x 10^5 cells per mL. Inoculation of 1500 mL of test medium with 10.1 mL of this algal suspension gave an initial cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentrations.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
The culture medium used for the definitive test was the same as that used to maintain the stock culture with the addition of 500 mg/L of sodium bicarbonate to counteract the increase in pH due to algal growth in an enclosed system.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination provided by warm white lighting (380 - 730 nm)
- Light intensity and quality: 7000 lux
- Other: constantly shaken at approximately 150 rpm for 72 h
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
76 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
Range-finding study: the results of the initial range-finding test showed no significant effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L. However, growth was observed to be reduced at 100 mg/L in the second-range-finding study. Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive study. Chemical analysis of the 100 mg/L test preparations taken from the initial range-finding test showed a decline in measured test concentration from 95 % of nominal at 0 h to 37 % of nominal at 72 h suggesting that the test item was either adsorbing to the algal cells present or was unstable over the test duration.

Physico-chemical measurements:
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 9.1 at 72 hours. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. The pH deviation in the control cultures was equal to 1.5 units after 72 hours and was therefore within the limits given in the Test Guidelines.
Results with reference substance (positive control):
A positive control 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 to the reference item gave the following results:
ErC50 (0-72 h): 1.4 mg/L; 95 % confidence limits 1.2- 1.7 mg/L
EyC50 (0-72 h): 0.59 mg/L; 95 % confidence limits 0.53- 0.65 mg/L
NOEC based on growth rate: 0.25 mg/L
MOEC based on yield: 0.25 mg/L
LOEC based on growth rate: 0.50 mg/L
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.

Cell densities and pH values in the definitive test

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

7.7

5.22E+03

1.53E+04

7.30E+04

3.77E+04

9.1

R2

5.90E+03

1.33E+04

8.17E+04

3.50E+05

R3

5.25E+03

1.39E+04

8.45E+04

4.08E+05

R4

5.06E+03

1.60E+04

8.14E+04

3.44E+05

R5

5.72E+03

1.45E+04

7.67E+04

3.79E+05

R6

5.34E+03

1.37E+04

8.20E+04

3.84E+05

Mean

5.50E+03

1.44E+04

7.99E+04

3.74E+05

1.0

R1

7.7

5.05E+03

1.87E04

1.22E+05

4.31E+05

9.3

R2

4.93E+03

1.94E+04

1.24E+05

5.19E+05

R3

5.02E+03

1.88E+04

1.26E+05

5.21E+05

Mean

5.00E+05

1.90E+04

1.24E+05

4.90E+05

3.2

R1

7.7

5.37E+03

2.94E+04

1.34E+05

3.97E+05

9.2

R2

5.43E+03

2.85E+04

1.30E+05

4.09E+05

R3

5.05E+03

2.61E+04

1.44E+05

4.18E+05

Mean

5.28E+03

2.80E+04

1.36E+05

4.08E+05

10

R1

7.7

5.46E+03

2.48E+04

1.41E+05

4.18E+05

9.2

R2

5.07E+03

2.67E+04

1.40E+05

4.27E+05

R3

6.48E+03

2.55E+04

1.30E+05

4.49E+05

Mean

5.67E+03

2.56E+04

1.37E+05

4.32E+05

32

R1

7.8

5.51E+03

2.75E+04

1.61E+05

6.50E+05

9.3

R2

5.66E+03

2.34E+04

1.38E+05

6.32E+05

R3

5.25E+03

2.19E+04

1.20E+05

6.13E+05

Mean

5.48E+03

2.43E+04

1.40E+05

6.32E+05

100

R1

8.0

5.49E+03

1.86E+04

1.19E+05

5.47E+05

9.0

R2

6.39E+03

2.12E+04

1.04E+05

5.60E+05

R3

5.60E+03

2.13E+04

9.13E+04

5.22E+05

Mean

5.83E+03

2.04E+04

1.05E+05

5.43E+05

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

R1- R6= Replicates 1 to 6

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

0.065

0.068

R2

0.041

0.076

0.061

R3

0.043

0.075

0.066

R4

0.048

0.068

0.060

R5

0.045

0.069

0.067

R6

0.042

0.075

0.064

Mean

0.044

0.071

0.064

 

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

0.060

 

3.72E+05

 

R2

0.059

 

3.44E05

 

R3

0.061

 

4.03E+05

 

R4

0.059

-

3.38E+05

-

R5

0.060

 

3.73E+05

 

R6

0.060

 

3.78E+05

 

Mean

0.060

 

3.68E+05

 

SD

0.001

 

2.36E+04

 

1.0

R1

0.062

[3]

4.26E+05

 

R2

0.064

[7]

5.14E+05

 

R3

0.065

[8]

5.16E+05

 

Mean

0.064

[6]

4.85E+05

[32]

SD

0.002

 

5.14E+04

 

3.2

R1

0.061

[2]

3.92E+05

 

R2

0.061

[2]

4.03E+05

 

R3

0.061

[2]

4.13E+05

 

Mean

0.061

[2]

4.03E+05

[9]

SD

0.000

 

1.06E+04

 

10

R1

0.061

[2]

4.13E+05

 

R2

0.062

[3]

4.22E+05

 

R3

0.062

[3]

4.43E+05

 

Mean

0.062

[3]

4.26E+05

[16]

SD

0.001

 

1.55E+04

 

32

R1

0.068

[13]

6.45E+05

 

R2

0.067

[12]

6.25E+05

 

R3

0.067

[12]

6.07E+05

 

Mean

0.067

[12]

6.26E+05

[70]

SD

0.001

 

1.88E+04

 

100

R1

0.065

[8]

5.41E+05

 

R2

0.066

[10]

5.53E+05

 

R3

0.065

[8]

5.17E+05

 

Mean

0.065

[9]

5.37E+05

[46]

SD

0.001

 

1.86E+04

 

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

R1-R6= Replicated 1 to 6

SD= Standard deviation

[Increase in growth as compared to controls]

Validation criteria

The cell concentration of the control cultures increased by a factor of 68 after 72 h. this increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 h.

Mean cell density of control at 0 h: 5.50 x 103cells per mL

Mean cell density of control at 72 h: 3.74 x 105cells per mL

The mean coefficient of variation for section specific growth rate for the control cultures was 24 % 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 % hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.

Growth data

The growth rate (r) and yield (y) of the algae were not affected by the presence of the test item over the 72-h exposure period. It was considered that the inhibition of growth observed at 100 mg/L during the second range-finding test was due to possible contamination from an unknown source. Accordingly the following results were determined from the data:

ErC10(0-72 h): >100 mg/L

ErC20(0-72 h): >100 mg/L

ErC50(0-72 h): >100 mg/L

Where ErCxis the test concentration that reduced growth rate by x %.

There were no statistically significant decreases in growth rate between the control and all test concentrations (P≥ 0.05) and therefore the NOEC based on growth rate was 100 mg/L.

EyC10(0-72 h): >100 mg/L

EyC20(0-72 h): >100 mg/L

EyC50(0-72 h): >100 mg/L

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

There was no significant decreases in yield (P≥ 0.05), between the control and all test groups and therefore the NOEC based on yield was 100 mg/L.

Physico-chemical measurements

The pH value of the control cultures was observed to increase from pH 7.7 at 0 h to pH 9.1 at 72 h. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth exceeding the transfer rate of CO2 from the gaseous phase to the aqueous phase. In this situation CO2 required for photosynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of culture. The pH deviation in the control cultures was equal to 1.5 pH units after 72 h and therefore was within the limits given in the Test Guidelines.

Verification of test concentrations

Analysis of the 100 mg/L test preparation at 0 h showed a measured test concentration of 100 % of nominal was obtained. A decline in measured test concentration was observed at 72 h to 58 % of nominal. Analysis of 100 mg/L test sample which had been prepared at the start of the test with omission of algal cells and incubated under test conditions showed a measured test concentration of 67 % of nominal was obtained. This result indicated that the decline in measured concentration observed was due to instability rather than adsorption of the test item to the algal cells present.

Results were based in the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be:

Nominal test concentration (mg/L)

Geometric mean measured test concentration (mg/L)

Expressed as a % of the nominal test concentration

100

76

76

Accordingly the following results were determined from the data based on the geometic mean measured test concentration:

Growth rate:

ErC10(0-72 h): >76 mg/L

ErC20(0-72 h): >76 mg/L

ErC50(0-72 h): >76 mg/L

NOEC: 76 mg/L

Yield:

EyC10(0-72 h): >76 mg/L

EyC20(0-72 h): >76 mg/L

EyC50(0-72 h): >76 mg/L

NOEC: 76 mg/L

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated accoriding to OECD Guideline 201 and gave EC50 values of > 100 mg/L. The NOEC was 100 mg/L. Based on the geometric mean measured test concentration the EC50 values were estimated to be > 76 mg/L. The NOEC was 76 mg/L.
Executive summary:

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (3 replicate flasks per concentration) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test item was potentially volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Hermanet al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

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

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Analysis of the 100 mg/L test preparation at 0 h showed a measured concentration of nominal was obtained. A decline in measured test concentration was observed at 72 h to 58 % of nominal. Analysis of a 100 mg/L test sample which had been prepared at the start of the test with the omission of algal cells and incubated under test conditions showed a measured test concentration of 67 % of nominal was obtained. This result indicated that the decline in measured concentration observed was due to instability rather than adsorption of the test item to the algal cells present.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a "worst case" analysis of the data.

Exposure to Pseudokirchneriella subcapitata to the test item gave EC50 values, based on the geometric mean measured test concentration, of greater than 76 mg/L. The No Observed Effect Concentration was 76 mg/L.

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated according to OECD Guideline 201 and gave EC50 values of > 100 mg/L. The NOEC was 100 mg/L. Based on the geometric mean measured test concentration the EC50 values were estimated to be > 76 mg/L. The NOEC was 76 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
76 mg/L
EC10 or NOEC for freshwater algae:
76 mg/L

Additional information

Introduction

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. 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 preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (3 replicate flasks per concentration) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1 °C.

The test item was potentially volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimise possible losses due to volatilisation. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth.

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

Exposure of Pseudokirchneriella subcapitata to the test item gave EC50values of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.

Analysis of the 100 mg/L test preparation at 0 h showed a measured concentration of nominal was obtained. A decline in measured test concentration was observed at 72 h to 58 % of nominal. Analysis of a 100 mg/L test sample which had been prepared at the start of the test with the omission of algal cells and incubated under test conditions showed a measured test concentration of 67 % of nominal was obtained. This result indicated that the decline in measured concentration observed was due to instability rather than adsorption of the test item to the algal cells present.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data.

Exposure of Pseudokirchneriella subcapitata to the test item give EC50values, based on the geometric mean measured test concentration, of greater than 76 mg/L. The NOEC was 76 mg/L.