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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:
10 January to 28 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 201 with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012 / signed on 30 November 2012)
Analytical monitoring:
yes
Details on sampling:
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 -20 ºC for further analysis if necessary.
- Given that chemical analysis of the range-finding test preparations had shown a decline in measured test concentration occurred over the test period, additional test samples were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.
- Sample storage conditions before analysis: All 0, 24 and 48 hours test samples were stored at approximately -20 °C prior to analysis at 72 hours.
Vehicle:
no
Details on test solutions:
PRE-STUDY SOLUBILITY
Information provided by the Sponsor indicated the water solubility of the test item to be 43.4 mg/L. Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.
A pre-study media preparation trial indicated that a dissolved test item concentration of approximately 48 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of test item (550 mg) was dispersed in 11 L 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 32, 10, 3.2 and 1.0% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with 8.8 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.
The concentration and stability of the test item in the test solutions were verified by chemical analysis at 0, 24, 48 and 72 hours.
- Controls: The control group was maintained under identical conditions but not exposed to the test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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 ± 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 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.
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
24 ± 1 °C throughout the test
pH:
pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 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:
No data
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
1.0, 3.2, 10, 32 and 100% v/v saturated solution
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Initial cells density: 5000 cells/mL
- Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 5.12 x 10^5 cells per mL. Inoculation of 900 mL of test medium with 8.8 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.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Culture medium according to OECD Guideline 201

OTHER TEST CONDITIONS
- 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.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Samples were taken at 0, 26, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Physico-chemical measurements: 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 WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

TEST CONCENTRATIONS
- Range finding study: 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. No effect on growth 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.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (Harlan Study Number 41300098)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Cl: 2.4-2.9 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.55 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.36 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% Cl: 0.81-1.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.55 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Inhibition of growth rate:
ErC10 (0 - 72 h): 1.5 mg/L
ErC20 (0 - 72 h): 1.9 mg/L
ErC50 (0 - 72 h): 2.6 mg/L; 95% confidence limits 2.4 – 2.9 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett 1955). There were no statistically significant differences (P≥0.05), between the control, 0.055 and 0.55 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 0.55 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 2.3 mg/L.

Inhibition of yield:
EyC10 (0 - 72 h): 0.36 mg/L
EyC20 (0 - 72 h): 0.52 mg/L
EyC50 (0 - 72 h): 1.0 mg/L; 95% confidence limits 0.81 – 1.3 mg/L
where EyCx is the test concentration that reduced yield by x%.
There were no statistically significant differences (P≥0.05), between the control, 0.055 and 0.55 mg/L test concentrations however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 0.55 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 2.3 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 0.055, 0.55 and 2.3 mg/L, however no intact cells and cell debris was observed to be present in the test cultures at 12 and 41 mg/L.

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, 0.055 and 0.55 mg/L test preparations were observed to be green dispersions. The 2.3 mg/L test preparations were observed to be pale green dispersions whilst the 12 and 41 mg/L test cultures were observed to be clear colorless solutions.
Results with reference substance (positive control):
ErC50 (0 – 72 h): 1.2 mg/L; 95% confidence limits 1.0 – 1.3 mg/L
EyC50 (0 – 72 h): 0.52 mg/L; 95% confidence limits 0.45 – 0.62 mg/L
No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
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).

Table 6.1.5/1: Inhibition of Growth Rate and Yield in the Definitive Test

 

Time-Weighted Mean Measured Test Concentration (mg/L)

Growth Rate

(cells/mL/hour)

Yield

(cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition *

Control

R1

0.076

-

1.19E+06

 

-

R2

0.072

9.16E+05

 

R3

0.072

8.95E+05

 

R4

0.070

7.76E+05

 

R5

0.066

5.68E+05

 

R6

0.066

5.67E+05

 

Mean

0.070

8.19E+05

 

SD

0.004

2.38E+05

 

0.055

R1

0.074

[6]

1.05E+06

 

[6]

R2

0.072

[3]

8.59E+05

 

R3

0.069

1

6.98E+05

 

Mean

0.072

[3]

8.71E+05

 

SD

0.003

 

1.79E+05

 

0.55

R1

0.069

1

7.42E+05

 

22

R2

0.066

6

5.62E+05

 

R3

0.067

4

6.14E+05

 

Mean

0.067

4

6.39E+05

 

SD

0.002

 

9.24E+04

 

2.3

R1

0.040

43

8.35E+04

 

85

R2

0.046

34

1.35E+05

 

R3

0.049

30

1.61E+05

 

Mean

0.045

36

1.26E+05

 

SD

0.005

 

3.95E+04

 

12

R1

0.008

89

5.92E+03

 

99

R2

0.004

94

4.45E+03

 

R3

0.000

100

2.46E+03

 

Mean

0.004

94

4.28E+03

 

SD

0.004

 

1.73E+03

 

41

R1

0.002

97

2.72E+03

 

100

R2

0.005

93

3.87E+03

 

R3

0.005

93

5.22E+03

 

Mean

0.004

94

3.94E+03

 

SD

0.002

 

1.25E+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]

 

Verification of test concentrations

Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.31, 1.2, 3.7, 13 and 42 mg/L were obtained. Given that chemical analysis of the range-finding test preparations had shown a decline in measured test concentration occurred over the test period, additional test samples were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

A concentration dependant decline in measured test concentration was observed at 24 and 48 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed (determined to be 0.051 mg/L) at 1.0% v/v saturated solution through to 42 mg/L at 100% v/v saturated solution. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ at 1.0, 3.2 and 10% v/v saturated solution to 36 mg/L at 100% v/v saturated solution.

Given this decline in measured test concentrations it was considered justifiable to base the results on the time-weighted 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 was used for calculation purposes. The time-weighted mean measured test concentrations were determined to be:

 

Nominal Test Concentration

(% v/v Saturated Solution)

0 hour Measured Test Concentration (mg/L)

 

Time-Weighted Mean Measured Test Concentration (mg/L)

 

Expressed as a % of the 0 hour Measured Test Concentration

 

1.0

0.31

0.055

18

3.2

1.2

0.55

46

10

3.7

2.3

62

32

13

12

92

100

42

41

98

 

 

Validation criteria

The following data show that the cell concentration of the control cultures increased by a factor of 179 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: 4.59 x 103 cells per mL

Mean cell density of control at 72 hours: 8.24 x 105 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 6% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions and based on the time-weighted mean measured test concentrations, the EC10, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 1.5, 2.6, 0.55 and 2.3 mg/L, respectively (based on growth rate). EC10, EC50, NOEC and LOEC were 0.36, 1.0, 0.55 and 2.3 mg/L, respectively (based on yield).
Executive summary:

In a algal growth inhibition study performed according to OECD Guideline 201/ EU method C.3 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item at the nominal concentrations of1.0, 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. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.31, 1.2, 3.7, 13 and 42 mg/L were obtained. Given that chemical analysis of the range-finding test preparations had shown a decline in measured test concentration occurred over the test period, additional test samples were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

A concentration dependant decline in measured test concentration was observed at 24 and 48 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed (determined to be 0.051 mg/L) at 1.0% v/v saturated solution through to 42 mg/L at 100% v/v saturated solution. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ at 1.0, 3.2 and 10% v/v saturated solution to 36 mg/L at 100% v/v saturated solution.

Given the decline in measured test concentration over the 72-Hour test period it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations which were determined to be 0.055, 0.55, 2.3, 12 and 41 mg/L for the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test preparation respectively.

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, 0.055 and 0.55 mg/L test preparations were observed to be green dispersions. The 2.3 mg/L test preparations were observed to be pale green dispersions whilst the 12 and 41 mg/L test cultures were observed to be clear colorless solutions. After 72 hours there were no abnormalities detected in the control or test cultures at 0.055, 0.55 and 2.3 mg/L, however no intact cells and cell debris was observed to be present in the test cultures at 12 and 41 mg/L.

Under the test conditions and based on the time-weighted mean measured test concentrations, the EC10, EC50, NOEC and LOEC of the test item to Pseudokirchneriella subcapitata were 1.5, 2.6, 0.55 and 2.3 mg/L, respectively (based on growth rate). EC10, EC50, NOEC and LOEC were 0.36, 1.0, 0.55 and 2.3 mg/L, respectively (based on yield).

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokirchneriella subcapitata) =2.6 mg/L based on time-weighted mean measured test concentrations

72h-ErC10 (Pseudokirchneriella subcapitata) = 1.5 mg/L (95% CI: 2.4 -2.9 mg/L) based on time-weighted mean measured test concentrations

72h-NOECr (Pseudokirchneriella subcapitata) = 0.55 mg/L based on time-weighted mean measured test concentrations

Key value for chemical safety assessment

EC50 for freshwater algae:
2.6 mg/L
EC10 or NOEC for freshwater algae:
1.5 mg/L

Additional information

One key study is available (Harlan 2014) to assess the influence of the registered substance on the growth of the freshwater green algae species Pseudokirchneriella subcapitata under static conditions, for 72 h according to OECD Guideline 201 with GLP statement. The

The green algae were exposed to test item at the 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. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter®Multisizer Particle Counter. 

Chemical analysis of the test preparations at 0 hours showed measured test concentrations of 0.31, 1.2, 3.7, 13 and 42 mg/L were obtained. Given that chemical analysis of the range-finding test preparations had shown a decline in measured test concentration occurred over the test period, additional test samples were prepared at 0 hours and incubated alongside the test to provide samples for analysis at 24 and 48 hours.

A concentration dependant decline in measured test concentration was observed at 24 and 48 hours in the range of less than the limit of quantitation (LOQ) of the analytical method employed (determined to be 0.051 mg/L) at 1.0% v/v saturated solution through to 42 mg/L at 100% v/v saturated solution. Analysis of the test preparations at 72 hours showed measured test concentrations to range from less than the LOQ at 1.0, 3.2 and 10% v/v saturated solution to 36 mg/L at 100% v/v saturated solution.

Given the decline in measured test concentration over the 72-Hour test period it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations which were determined to be 0.055, 0.55, 2.3, 12 and 41 mg/L for the 1.0, 3.2, 10, 32 and 100% v/v saturated solution test preparation respectively.

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, 0.055 and 0.55 mg/L test preparations were observed to be green dispersions. The 2.3 mg/L test preparations were observed to be pale green dispersions whilst the 12 and 41 mg/L test cultures were observed to be clear colorless solutions. After 72 hours there were no abnormalities detected in the control or test cultures at 0.055, 0.55 and 2.3 mg/L, however no intact cells and cell debris was observed to be present in the test cultures at 12 and 41 mg/L.

Under the test conditions and based on the time-weighted mean measured test concentrations, the EC10, EC50, NOEC and LOEC of the test item toPseudokirchneriella subcapitatawere 1.5, 2.6, 0.55 and 2.3 mg/L, respectively (based on growth rate).