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Ecotoxicological information

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:
13 July 2018 to 30 August 2018
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of Inspection: 18 July 2017 - 20 July 2017 Date of Issue: 28 November 2017
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control (0), 1,0, 3.2, 10, 32 and 100 mg/L
- Sampling method:
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. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
An additional sample of each test concentration containing no algal cells was incubated alongside the test to provide samples for uninoculated analysis at 72 hours.
- Sample storage conditions before analysis:
Samples were stored frozen prior to analysis.
Vehicle:
yes
Remarks:
culture medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Nominal amounts of test item (194 and 62 mg) were each separately dissolved in culture medium with the aid of ultrasonication for approximately 5 minutes, and the volume adjusted to 1 liter to give 100 and 32 mgi/L stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2 and 1.0 mg/L. An aliquot (500 mL) of each of the stock solutions was separately inoculated with algal suspension (2.9 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
All concentrations were corrected for a test item water content of 48.4% w/w.
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
Culture medium
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None recorded
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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): Not reported
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. ). The master cultures were maintained under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C.
pH:
7.5 - 8.8
Nominal and measured concentrations:
Nominal: 1.0, 3.2, 10, 32 and 100 mg/L.
Measured: 0.760/0.836*, 3.04, 7.26/8.65*, 37.0, and 121 mg/L
*duplicate samples stored frozen prior to analysis
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type : closed
- Material, size, headspace, fill volume:
250 mL glass conical flasks, plugged with foam bungs
- Aeration:
N/A
- Type of flow-through (e.g. peristaltic or proportional diluter):
N/A
- Renewal rate of test solution (frequency/flow rate):
N/A
- Initial cells density: 5.00E+03 cells per mL (nominal); 4.24E+04 cells per mL (measured)
- Control end cells density: 2.04E+06 cells per mL
- No. of vessels per concentration (replicates) 6
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis purified deionized water

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted to 7.5 with 0.1N NaOH or HCl
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux, provided by warm white lighting (380 to 730 nm)
- Other: constantly shaken at approximately 150 rpm for 72 hours


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter (measured at 24, 48, and 72 hours)
- Other: the shape and size of the algal cells was inspected microscopically and any abnormalities recorded

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approx 3.2
- Range finding study
- Test concentrations: of 0.10, 1.0, 10 and 100 mg/L. Due to a technical oversight, the concentrations employed were not corrected for the test item water content as was stated in the Study Plan. As the test item contains 48.4% w/w of water, the equivalent test item concentrations employed were 0.0516, 0.516, 5.16 and 51.6 mg/L. This oversight was considered to have had no adverse effect on the outcome of the range-finding test as it was still possible to assign dose levels for the definitive test from the data obtained.
- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at the test concentrations of 0.0516, 0.516 and 5.16 mg/L. However, growth was observed to be reduced at 51.6 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape:
None observed
- Colour differences:
At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72-hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were green dispersions, whilst the 32 and 100 mg/L test cultures were light green or pale green dispersions.
- Flocculation:
None observed
- Adherence to test vessels:
None observed
- Aggregation of algal cells:
None observed
- Other:
Some healthy cells and some cell debris were observed to be present in the test cultures at 100 mg/L
- Any stimulation of growth found in any treatment:
N/A
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
None observed
- Effect concentrations exceeding solubility of substance in test medium:
N/A
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
ErC50 (72 h): 1.6 mg/L, 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (72 h): 0.77 mg/L, 95% confidene limits 0.68 to 0.87 mg/L
Reported statistics and error estimates:
Statistical analysis of the growth rate and yield 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).
Growth Rate: There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg/L test concentrations, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 3.2 mg/L. Correspondingly the LOEC based on growth rate was 10 mg/L.
Yield: . There were no statistically significant differences (P≥0.05), between the control, 1.0 and 3.2 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 3.2 mg/L. Correspondingly the LOEC based on yield was 10 mg/L.

Range-finding Test

The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

Table 1: Cell Densities and Percentage Inhibition of Growth from the Range‑finding Test

Nominal Concentration
(mg/L)

Cell Densities*(cells per mL)

Inhibition Values (%)

0 Hours

72 Hours

Growth Rate

Yield

Control

R1

6.37E+03

6.64E+05

-

-

R2

6.28E+03

1.24E+06

Mean

6.32E+03

9.50E+05

0.0516

R1

6.28E+03

1.25E+06

[4]

[28]

R2

6.37E+03

1.19E+06

Mean

6.32E+03

1.22E+06

0.516

R1

6.13E+03

1.08E+06

[1]

[9]

R2

6.48E+03

9.97E+05

Mean

6.31E+03

1.04E+06

5.16

R1

6.57E+03

8.48E+05

4

7

R2

7.30E+03

9.22E+05

Mean

6.94E+03

8.85E+05

51.6

R1

7.45E+03

3.78E+05

21

64

R2

5.81E+03

3.21E+05

Mean

6.63E+03

3.50E+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

R  = Replicate

-   = Not applicable

[ ]= Increase in growth compared to controls

The results showed no effect on growth at the test concentrations of 0.0516, 0.516 and 5.16 mg/L. However, growth was observed to be reduced at 51.6 mg/L.

Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 0.516, 5.16 and 51.6 mg/L test preparations at 0 hours showed measured test concentrations to range from 87% to 99% of nominal. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of 66% to 99% of nominal. This was considered to be due to possible adsorption of the test item to the algal cells present.

Definitive Test

Verification of Test Concentrations

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 121% of nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 61% to 118% of nominal. Analysis of additional samples prepared at the start of the test with the omission of algal cells showed measured test concentrations at 72 hours to range from 88% to 137% of nominal suggesting that the losses observed in those samples where algae were present was due to adsorption of the test item to the algal cells present rather than instability under the conditions of the test.

Given this it was considered appropriate to calculate the results based on the nominal test concentrations only as this gives a more accurate measure of the levels of test item to which the algae were exposed.

Growth Data

Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

Table 2            Cell Densities and pH Values in the Definitive Test

Nominal Concentration
(mg/L)

pH

Cell Densities* (cells per mL)

pH

0 Hours

24 Hours

48 Hours

72 Hours

72 Hours

Control

R1

7.8

4.16E+04

2.66E+05

2.18E+06

8.6

R2

4.33E+04

2.54E+05

1.99E+06

R3

4.43E+04

2.62E+05

2.04E+06

R4

4.28E+04

2.58E+05

2.13E+06

R5

4.05E+04

2.43E+05

1.96E+06

R6

4.21E+04

2.40E+05

1.92E+06

Mean

4.24E+04

2.54E+05

2.04E+06

1.0

R1

7.7

4.48E+04

2.14E+05

1.36E+06

8.6

R2

4.50E+04

2.77E+05

2.18E+06

R3

4.22E+04

2.53E+05

1.82E+06

Mean

4.40E+04

2.48E+05

1.79E+06

3.2

R1

7.6

4.07E+04

2.72E+05

1.80E+06

8.8

R2

4.34E+04

2.98E+05

1.84E+06

R3

4.17E+04

2.72E+05

1.82E+06

Mean

4.19E+04

2.81E+05

1.82E+06

10

R1

7.6

3.66E+04

2.43E+05

1.46E+06

8.6

R2

3.68E+04

2.54E+05

1.51E+06

R3

3.68E+04

2.41E+05

1.34E+06

Mean

3.67E+04

2.46E+05

1.43E+06

32

R1

7.5

2.39E+04

1.38E+05

7.40E+05

8.3

R2

2.15E+04

1.38E+05

7.93E+05

R3

2.17E+04

1.13E+05

6.35E+05

Mean

2.24E+04

1.29E+05

7.22E+05

100

R1

7.5

1.44E+04

5.29E+04

2.36E+05

7.8

R2

1.38E+04

4.76E+04

1.98E+05

R3

1.42E+04

5.05E+04

2.17E+05

Mean

1.41E+04

5.03E+04

2.17E+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

R  = Replicate

Table3            Daily Specific Growth Rates for the Control Cultures in the Definitive Test

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 to 1

Day 1 to 2

Day 2 to 3

Control

R1

0.088

0.077

0.088

R2

0.090

0.074

0.086

R3

0.091

0.074

0.085

R4

0.089

0.075

0.088

R5

0.087

0.075

0.087

R6

0.089

0.073

0.087

Mean

0.089

0.075

0.087

 

R  = Replicate

Table 4            Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.084

-

2.18E+06

-

R2

0.083

1.99E+06

R3

0.083

2.03E+06

R4

0.084

2.12E+06

R5

0.083

1.96E+06

R6

0.083

1.91E+06

Mean

0.083

2.03E+06

SD

0.001

1.01E+05

1.0

R1

0.078

6

1.36E+06

 

R2

0.084

[1]

2.17E+06

 

R3

0.082

1

1.82E+06

 

Mean

0.081

2

1.78E+06

12

SD

0.003

 

4.08E+05

 

3.2

R1

0.082

1

1.79E+06

 

R2

0.082

1

1.84E+06

 

R3

0.082

1

1.82E+06

 

Mean

0.082

1

1.82E+06

11

SD

0.000

 

2.36E+04

 

10

R1

0.079

5

1.45E+06

 

R2

0.079

5

1.50E+06

 

R3

0.078

6

1.33E+06

 

Mean

0.079

5

1.43E+06

30

SD

0.001

 

8.75E+04

 

32

R1

0.069

17

7.35E+05

 

R2

0.070

16

7.88E+05

 

R3

0.067

19

6.30E+05

 

Mean

0.069

17

7.17E+05

65

SD

0.002

 

8.06E+04

 

100

R1

0.054

35

2.31E+05

 

R2

0.051

39

1.93E+05

 

R3

0.052

37

2.12E+05

 

Mean

0.052

37

2.12E+05

90

SD

0.002

 

1.88E+04

 

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

R  = Replicate

SD  =Standard Deviation

[ ]  =Increase in growth compared to controls

-   = Not applicable

Validity criteria fulfilled:
yes
Conclusions:
It was not possible to calculate an ErC50 value as no more than 39% inhibition of growth rate occurred at the maximum concentration employed of 100 mg/L. It was therefore also not possible to determine 95% confidence limits for growth rate. The ErC10 was estimated to be 18 mg/L.

The EyC50 value was determined to be 19 mg/L, 95% confidence limits of 14-25 mg/L. The EyC10 was estimate dto be 2.9 mg/L.
Executive summary:

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriellasubcapitata. 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.

Following a preliminary range-finding test, 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 (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.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 84% to 121% of nominal. A concentration dependent decline in measured concentrations was observed at 72 hours in the range of 61% to 118% of nominal. Analysis of additional samples prepared at the start of the test with the omission of algal cells showed measured test concentrations at 72 hours to range from 88% to 137% of nominal suggesting that the losses observed in those samples where algae were present was due to adsorption of the test item to the algal cells present rather than instability under the conditions of the test.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the nominal test concentrations:

Response Variable

EC50
(mg/L)

 

EC10
(mg/L)

95% Confidence Limits (mgL)

No Observed Effect Concentration (NOEC)
(mg/L)

Lowest Observed Effect Concentration (LOEC)
(mg/L)

Growth Rate

>100*

18

Not determined due to nature of data

3.2

10

Yield

19

2.9

14

-

25

3.2

10

*It was not possible to calculate an ErC50value as no more than 39% inhibition of growth rate occurred at the maximum concentration employed of 100 mg/L.

Description of key information

72h ErC50 = > 100 mg/L, 72h ErC10 = 18 mg/L; OECD 201; Vryenhoef, 2018

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
18 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriellasubcapitata. 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.

It was not possible to calculate an EC50 based on inhibition of growth rate as no more than 39% inhibition of growth rate occurred at the maximum concentration employed. Therefore only the EC10 is reported.