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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:
01 March 2017 to 23 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Verification of Test Concentrations:
Samples were taken from the control and the 100% v/v saturated solution test group from the bulk test preparation at 0 hours, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken and stored frozen for further analysis if necessary. At each time point, only one sample was analyzed.

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 0.15 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 (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 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 0.10, 1.0 and 10% v/v saturated solution. An aliquot (900 mL) of each of the stock solutions was separately inoculated with algal suspension (6.8 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 a "limit test" was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable test concentration no effect on algal growth was observed.

A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorious Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution.
An aliquot (2.5 liters) of the stock solution was inoculated with 26.6 mL of algal suspension to give the required test concentration of 100% v/v saturated solution.
The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.
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:
yes
Total exposure duration:
72 h
Test temperature:
Temperature was maintained at 24 ± 1°C throughout the test.
pH:
t=0: in control pH = 7.6; in 100% v/v saturated solution pH = 7.8
t=72 h: pH = 10.1 in both control and 100% v/v saturated solution.
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 that measured concentrations of between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.0090 mg/L, and 0.33 mg/L were obtained. Measured concentrations at 72 hours were observed to range from less than the LOQ to 0.18 mg/L, indicating that the test item was possibly unstable under test conditions. See "Any other information on materials and methods incl. tables".

The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution, see "Any other information on results incl. tables".

Definitive Test:
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the highest attainable test concentration no effect on growth was observed.

Analysis of the 100% v/v saturated solution test preparation at 0 hours showed that a measured test concentration of 0.49 mg/L was obtained. There was no significant change in the measured concentrations at 24, 48 and 72 hours (i.e. all concentrations were within ± 20% of the 0-Hour measured concentration) and so the results are based on the 0-Hour measured test concentration only. See "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 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 4.70 x 10^5 cells per mL. Inoculation of 2.5 liters of test medium with 26.6 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.

- Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
- Mean cell density of control at 72 hours: 4.05 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): 6
- 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 the 100% v/v saturated solution 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 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 the 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 28 November 2016 and 01 December 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.49 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 0.49 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.49 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Growth Data:
From the data presented under "Any other information on results incl. tables" it is clear that the growth rate (r) of Pseudokirchneriella subcapitata (CCAP 278/4) was not affected by the presence of the test item at a test concentration of 0.49 mg/L over the 72-Hour exposure period.

- Inhibition of Growth Rate:
ErC10 (0 - 72 h): >0.49 mg/L
ErC20 (0 - 72 h): >0.49 mg/L
ErC50 (0 - 72 h): >0.49 mg/L

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

- Validation criteria:
The following data show that the cell concentration of the control cultures increased by a factor of 81 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.
Nominal cell density of control at 0 hours: 5.00 x 10^3 cells per mL
Mean cell density of control at 72 hours : 4.05 x 10^5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% 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%.

- Observation of abnormalities:
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in the control or test cultures at 72 hours.

- Water Quality
The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 10.1 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 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 and test cultures were observed to be green dispersions.

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: 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
- EyC50: 0.60 mg/L; 95% confidence limits 0.52 – 0.69 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 the 0.49 mg/L test group using a Student’s t-test. Results showed that a statistically significant difference was observed, however this corresponded to only 4% inhibition of growth. Inspection of the data showed that there was only a slight variability in the control results, which was considered to be the cause of the significance seen. As such the "No Observed Effect Concentration" (NOEC) based on growth rate was considered to be >= 0.49 mg/L.

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

Control

R1

7.07E+03

2.50E+05

-

R2

6.45E+03

2.99E+05

Mean

6.76E+03

2.75E+05

0.10

R1

6.45E+03

3.61E+05

[12]

R2

5.63E+03

3.79E+05

Mean

6.04E+03

3.70E+05

1.0

R1

7.07E+03

2.67E+05

0

R2

5.22E+03

2.30E+05

Mean

6.15E+03

2.48E+05

10

R1

6.10E+03

2.69E+05

[2]

R2

5.78E+03

2.18E+05

Mean

5.94E+03

2.44E+05

100

R1

4.08E+03

2.38E+05

[8]

R2

5.10E+03

2.50E+05

Mean

4.59E+03

2.44E+05

*    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.

R1and R2= Replicates 1 and 2

[Increase in growth compared to controls]

The measured test concentrations in the definite test were determined to be:

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

 

100

0.10

0.487

24

Control

0.10

<LOQ

 

100

0.10

0.430

48

Control

0.10

<LOQ

 

100

0.10

0.405

72

Control

0.10

<LOQ

 

100

0.10

0.393

LOQ                      =             Limit of Quantification

Inhibition of Growth Rate in the Definitive Test:

Nominal Concentration
(% v/v Saturated Solution)

Growth Rate (cells/mL/hour)

0 – 72 h

% Inhibition

Control

R1

0.062

 

R2

0.062

 

R3

0.063

 

R4

0.062

-

R5

0.059

 

R6

0.058

 

Mean

0.061

 

SD

0.002

 

100

R1

0.057

7

R2

0.058

5

R3

0.057

7

R4

0.061

0

R8

0.058

5

R6

0.060

2

Mean

0.059

4

SD

0.002

 


R1– R6= Replicates 1 to 6

SD= Standard Deviation

Validity criteria fulfilled:
yes
Remarks:
See details on results.
Conclusions:
The ErC50, ErC10 and NOErC were >0.49, >0.49 and >=0.49 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. The method was performed in compliance with OECD TG 201 and GLP principles. The test item was a poorly soluble pure compound and as such prepared as a saturated solution. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. 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. Analysis of the 100% v/v saturated solution test preparation at 0 hours showed that a measured test concentration of 0.49 mg/L was obtained. There was no significant change in the measured concentrations at 24, 48 and 72 hours (i.e. all concentrations were within ± 20% of the 0-Hour measured concentration) and so the results are based on the 0-Hour measured test concentration only. Exposure of Pseudokirchneriella subcapitata to the test item gave EC10 and EC50 values based on growth rate and on the 0-Hour measured concentration of greater than 0.49 mg/L. The No Observed Effect Concentration based on growth rate was determined to be equal to or greater than 0.49 mg/L. This study showed that there were no toxic effects at saturation.

Description of key information

Algal Growth Inhibition Test (OECD TG 201): The ErC50, ErC10 and NOErC were >0.49, >0.49 and >=0.49 mg/L, respectively.

Key value for chemical safety assessment

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method was performed in compliance with OECD TG 201 and GLP principles. The test item was a poorly soluble pure compound and as such prepared as a saturated solution. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solution was prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. Testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. 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. Analysis of the 100% v/v saturated solution test preparation at 0 hours showed that a measured test concentration of 0.49 mg/L was obtained. There was no significant change in the measured concentrations at 24, 48 and 72 hours (i.e. all concentrations were within ± 20% of the 0-Hour measured concentration) and so the results are based on the 0-Hour measured test concentration only. Exposure of Pseudokirchneriella subcapitata to the test item gave EC10 and EC50 values based on growth rate and on the 0-Hour measured concentration of greater than 0.49 mg/L. The No Observed Effect Concentration based on growth rate was determined to be equal to or greater than 0.49 mg/L. This study showed that there were no toxic effects at saturation.