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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:
06 June 2016 - 30 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
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. 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 under test conditions to provide samples for analysis at 24 and 48 hours.

Storage:
The samples were analyzed on the day of receipt.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 9.9 mg/L could be obtained using a saturated solution method of preparation.

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% v/v saturated solution for a period of 72 hours.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 10, 1.0 and 0.10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (9.5 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.

Definitive Test:
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100% v/v saturated solution.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give stock solutions of 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (1800 mL) of each of the stock solutions was separately inoculated with 18.3 mL of algal suspension to give the required test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: strain CCAP 278/4
- Source: 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.
- 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 ± 2 oC.

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 oC 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:
Temperature was maintained at 24 ± 1°C throughout the test.
pH:
t=0: 7.7 - 7.8
t=72 h: 8.1 - 9.7
Nominal and measured concentrations:
TEST CONCENTRATIONS
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% v/v saturated solution for a period of 72 hours.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.0089 mg/L, to 8.9 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the LOQ to 8.4 mg/L suggesting that the test item was possible unstable over the test duration.

The results showed no significant effect on growth rate at the test concentrations of 0.10 and 1.0% v/v saturated solution. However, growth was observed to be reduced at 10 and 100% v/v saturated solution.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution were selected for the definitive test.

Definitive Test:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.074 to 7.4 mg/L. A decline in measured test concentrations was observed at 24 hour and 48 hours in the range of 0.059 to 7.6 mg/L, and less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.0089 mg/L) to 6.7 mg/L respectively. A further decline in measured concentrations was observed at 72 hours in the range of less than the LOQ to 6.4 mg/L.
Results are based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. In cases where the measured concentration was less than the LOQ of the analytical method following current regulatory advice a value of half the LOQ (i.e. 0.0044 mg/L) was used to enable calculation of the geometric mean measured concentration. The geometric mean measured test concentrations determined are given under "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed, the flasks were sealed with ground glass stoppers
- Fill volume: flasks completely filled
- Renewal of test solution: no, static
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 4.92 x 10^5 cells per mL. Inoculation of 1800 mL of test medium with 18.3 mL of this algal suspension gave an initial nominal cell density of 5 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

- Mean cell density of control at 0 hours: 5.00 x 10^3 cells per mL
- Mean cell density of control at 72 hours: 5.14 x 10^5 cells per mL

- No. of organisms per vessel: The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

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. The media used in both the range-finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).
The culture medium is defined below:

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.
For the purposes of the range-finding and definitive tests, additional sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use.
* Elga Optima 15+ or Elga Purelab Option R-15 BP

- 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 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.

OTHER TEST CONDITIONS
- Photoperiod: The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron Version 2 incubator) at 24 +/- 1 °C under continuous illumination provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- Light intensity and quality: intensity approximately 7000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations:
Samples were taken at 24, 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 10^3 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.

Reference substance (positive control):
yes
Remarks:
potassium dichromate (conducted between 07 December and 10 December 2015)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.0 - 1.7 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.38 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.:
0.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control:
Validation criteria:
The cell concentration of the control cultures increased by a factor of 103 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.
The mean coefficient of variation for section by section specific growth rate for the control cultures was 19% 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%.

- Observation of abnormalities:
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 0.029, 0.11 and 0.45 mg/L, however no intact cells were observed to be present in the test cultures at 2.1 and 7.1 mg/L.

- Water Quality
Temperature was maintained at 24 ± 1 ºC throughout the test.
The pH value of the control cultures was observed to increase from pH 7.8 at 0 hours to pH 9.7 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 increase in pH after 72 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 72 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentration in the control cultures exceeded the validation criterion 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, 0.029 and 0.11 mg/L test cultures were observed to be green dispersions. The 0.45 mg/L test cultures were observed to be pale green dispersions whilst the 2.1 and 7.1 mg/L test cultures were observed to be clear colorless solutions.

- Inhibition of Growth Rate
ErC10 (0 - 72 h): 0.38 mg/L
ErC20 (0 - 72 h): 0.60 mg/L
ErC50 (0 - 72 h): 1.3 mg/L; 95% confidence limits 1.0 – 1.7 mg/L

- Inhibition of Yield
EyC10 (0 - 72 h): 0.20 mg/L
EyC20 (0 - 72 h): 0.29 mg/L
EyC50 (0 - 72 h): 0.56 mg/L; 95% confidence limits 0.45 – 0.70 mg/L
Results with reference substance (positive control):
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- ErC50: 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
- EyC50: 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L
Reported statistics and error estimates:
Inhibition of Growth rate:
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, 0.029 and 0.11 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 0.11 mg/L.

Inhibition of Yield:
Statistical analysis of the yield data was carried out as for Growth rate. There were no statistically significant decreases in yield between the control and 0.029 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 0.029 mg/L.

Analytical results for Range-Finding Samples:

Time Point

Nominal Concentration of
Test Item in Range-Finding Sample

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Range-Finding Sample


c

[hours]

[% v/v Saturated Solution]

 

[mg/L]

0

0.10

0.10

<LOQ

 

1.0

0.10

<LOQ

 

10

0.10

0.743

 

100

0.10

8.92

72

0.10

0.10

<LOQ

 

1.0

0.10

<LOQ

 

10

0.10

<LOQ

 

100

0.10

8.38

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

Nominal Concentration

(% v/v Saturated Solution)

Cell Densities* (cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

5.75E+03

3.83E+05

-

-

R2

4.28E+03

3.48E+05

Mean

5.02E+03

3.65E+05

0.10

R1

4.90E+03

3.56E+05

2

[1]

R2

5.37E+03

3.85E+05

Mean

5.13E+03

3.71E+05

1.0

R1

3.87E+03

2.60E+05

5

33

R2

4.17E+03

2.33E+05

Mean

4.02E+03

2.46E+05

10

R1

4.69E+03

2.17E+05

8

38

R2

4.11E+03

2.37E+05

Mean

4.40E+03

2.27E+05

100

R1

6.31E+03

1.17E+04

87

99

R2

3.40E+03

5.47E+03

Mean

4.85E+03

8.61E+03

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

R1and R2= Replicates 1 and 2

[Increase in growth compared to controls]

In the table below the nominal and measured concentrations in the main test are presented.

Time Point

Nominal Concentration of
Test Item in Test Sample

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Test Sample

 


c

[hours]

[% v/v Saturated Solution]

 

[mg/L]

0

Control

0.10

<LOQ

1.0

0.10

0.0735

 

3.2

0.10

0.242

 

10

0.10

0.775

 

32

0.10

2.58

 

100

0.10

7.35

24

1.0

0.10

0.0585

 

3.2

0.10

0.217

 

10

0.10

0.687

 

32

0.10

2.24

 

100

0.10

7.59

48

1.0

0.10

<LOQ

 

3.2

0.10

0.0451

 

10

0.10

0.462

 

32

0.10

1.90

 

100

0.10

6.73

72

Control

0.10

<LOQ

 

1.0

0.10

<LOQ

 

3.2

0.10

<LOQ

 

10

0.10

<LOQ

 

32

0.10

1.50

 

100

0.10

6.42

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

1.0

0.029

3.2

0.11

10

0.45

32

2.1

100

7.1

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

0.064

 

4.88E+05

 

R2

0.065

 

5.28E+05

 

R3

0.065

 

5.28E+05

 

R4

0.064

-

4.97E+05

-

R5

0.065

 

5.19E+05

 

R6

0.064

 

4.91E+05

 

Mean

0.065

 

5.09E+05

 

SD

0.001

 

1.89E+04

 

0.029

R1

0.066

[2]

5.82E+05

 

R2

0.066

[2]

5.87E+05

 

R3

0.066

[2]

5.79E+05

 

Mean

0.066

[2]

5.83E+05

[15]

SD

0.000

 

4.05E+03

 

0.11

R1

0.063

3

4.61E+05

 

R2

0.063

3

4.66E+05

 

R3

0.062

5

4.43E+05

 

Mean

0.063

4

4.56E+05

10

SD

0.001

 

1.23E+04

 

0.45

R1

0.058

11

3.24E+05

 

R2

0.057

12

2.95E+05

 

R3

0.059

9

3.35E+05

 

Mean

0.058

11

3.18E+05

37

SD

0.001

 

2.07E+04

 

2.1

R1

0.021

68

1.70E+04

 

R2

0.015

77

1.01E+04

 

R3

0.017

74

1.18E+04

 

Mean

0.018

73

1.30E+04

97

SD

0.003

 

3.57E+03

 

7.1

R1

0.009

86

4.77E+03

 

R2

0.005

92

2.25E+03

 

R3

0.007

89

3.57E+03

 

Mean

0.007

89

3.53E+03

99

SD

0.002

 

1.26E+03

 

*    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

[Increase in growth as compared to controls]

Validity criteria fulfilled:
yes
Remarks:
Validity criteria are met.
Conclusions:
The ErC50, ErC10 and NOErC were 1.3, 0.38 and 0.11 mg/L, respectively.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata, according to OECD TG 201 and GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium. After the stirring period any undissolved test item was removed by filtration to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. 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. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.074 to 7.4 mg/L. A decline in measured test concentrations was observed at 24 hour and 48 hours in the range of 0.059 to 7.6 mg/L, and less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.0089 mg/L) to 6.7 mg/L respectively. A further decline in measured concentrations was observed at 72 hours in the range of less than the LOQ to 6.4 mg/L. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The validity criteria of the test were met: The cell concentration of the control cultures increased by a factor of 103 after 72 hours (> 16); The mean coefficient of variation for section by section specific growth rate for the control cultures was 19%  (< 35%); The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% (< 7%). The exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations for Growth Rate: EC50 = 1.3 mg/L, EC10 = 0.38 mg/L and NOEC = 0.11 mg/L.

Description of key information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata, according to OECD TG 201 and GLP. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium. After the stirring period any undissolved test item was removed by filtration to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. 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. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.074 to 7.4 mg/L. A decline in measured test concentrations was observed at 24 hour and 48 hours in the range of 0.059 to 7.6 mg/L, and less than the limit of quantification (LOQ) of the analytical method employed (determined to be 0.0089 mg/L) to 6.7 mg/L respectively. A further decline in measured concentrations was observed at 72 hours in the range of less than the LOQ to 6.4 mg/L. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration. The validity criteria of the test were met: The cell concentration of the control cultures increased by a factor of 103 after 72 hours (> 16); The mean coefficient of variation for section by section specific growth rate for the control cultures was 19%  (< 35%); The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 2% (< 7%). The exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations for Growth Rate: EC50 = 1.3 mg/L, EC10 = 0.38 mg/L and NOEC = 0.11 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.3 mg/L
EC10 or NOEC for freshwater algae:
0.38 mg/L

Additional information