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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:
19 February 2018 - 30 March 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)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 0.010, 0.032, 0.10, 0.32, 1.0 mg/L
- Sample storage conditions before analysis: The samples were stored frozen prior to analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the need to test at relatively low test concentrations a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which a series of dilutions was made to give the remaining test concentrations.
A nominal amount of test item (20 mg) was added to the surface of 20 liters of culture medium to give the 1.0 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1-Hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. A series of dilutions was made from the WAF to give further stock solutions of 0.32, 0.10, 0.032 and 0.010 mg/L loading rate WAF.
An aliquot (900 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.3 mL) to give the required test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L loading rate WAF.
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium

ACCLIMATION
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ±1 ºC
pH:
7.3 - 7.4 at test start, 7.8 - 8.6 at the end of the observation period
Nominal and measured concentrations:
0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks containing 100 mL of test preparation
- Aeration: constantly shaken at approximately 150 rpm for 72 hours.
- Type (delete if not applicable): closed
- Initial cells density: 5.00E03 cells/mL
- Control end cells density: 1.63E06 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Culture medium different from test medium: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: no
- Other:
Samples were taken at 19, 42 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 (5.00E03 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.



TEST CONCENTRATIONS
- Spacing factor for test concentrations: sqrt(10)
- Range finding study
- Test concentrations: first range-finding test: 10 and 100 mg/L, second range-finding test: 0.010, 0.10 and 1.0 mg/L
- Results used to determine the conditions for the definitive study:
The results showed no effect on growth at 0.010 mg/L loading rate WAF. However, growth was observed to be reduced at 0.10, 1.0, 10 and 100 mg/L loading rate WAF. Based on this information loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.07 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
0.11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits
Remarks:
0.10 to 0.13 mg/L
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.087 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
0.092 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities detected in the control or test cultures at 0.010, 0.032 and 0.10 mg/L loading rate WAF; cell breakdown was observed in the test cultures at 0.32 and 1.0 mg/L loading rate WAF
- Effect concentrations exceeding solubility of substance in test medium:
Observations on the test media were carried out during the mixing and testing of the WAF.
At both the start and end of the mixing period and following a 1-Hour standing period, the 1.0 mg/L loading rate WAF was observed to have formed a clear colorless water column with a very small number of globules of test item dispersed throughout the water column. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present.
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, 0.010 and 0.032 mg/L loading rate WAF test cultures were observed to be green dispersions. The 0.10 mg/L loading rate WAF test cultures were observed to be pale green dispersions whilst the 0.32 and 1.0 mg/L loading rate WAF test cultures were observed to be clear colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? yes

Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration 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, 0.010, 0.032 and 0.10 mg/L loading rates 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). The 0.32 and 1.0 mg/L loading rates were not included in the analysis as visual inspection of the data showed a significant effect on growth.

Validity criteria:

The following data show that the cell concentration of the control cultures increased by a factor of 327 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    :          5.00E03 cells per mL
Mean cell density of control at 72 hours       :          1.63E06 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 23% 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 to 72 hour) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition

Control

R1

0.080

-

1.59E+06

-

R2

0.081

1.70E+06

R3

0.082

1.77E+06

R4

0.080

1.57E+06

R5

0.081

1.69E+06

R6

0.079

1.46E+06

Mean

0.081

1.63E+06

SD

0.001

1.13E+05

0.010

R1

0.082

[1]

1.86E+06

 

R2

0.084

[4]

2.17E+06

 

R3

0.082

[1]

1.83E+06

 

Mean

0.083

[2]

1.95E+06

[20]

SD

0.001

 

1.88E+05

 

0.032

R1

0.085

[5]

2.32E+06

 

R2

0.082

[1]

1.88E+06

 

R3

0.081

0

1.67E+06

 

Mean

0.083

[2]

1.96E+06

[20]

SD

0.002

 

3.34E+05

 

0.10

R1

0.057

30

2.88E+05

 

R2

0.051

37

1.95E+05

 

R3

0.047

42

1.47E+05

 

Mean

0.052

36

2.10E+05

87

SD

0.005

 

7.16E+04

 

0.32

R1

-0.002

102

-7.76E+02

 

R2

0.001

99

2.21E+02

 

R3

0.006

93

2.45E+03

 

Mean

0.002

98

6.32E+02

100

SD

0.004

 

1.65E+03

 

1.0

R1

-0.022

127

-4.00E+03

 

R2

-0.015

119

-3.27E+03

 

R3

-0.019

123

-3.71E+03

 

Mean

-0.019

123

-3.66E+03

100

SD

0.004

 

3.69E+02

 

 

R  = Replicate

SD=Standarddeviation

-   = Not applicable

[ ] = Increase in growth as compared to the control

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Loading Rate
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 to 72 Hour

% Inhibition

0 to 72 Hour

% Inhibition*

Control

R1

0.080

-

1.59E+06

-

R2

0.081

1.70E+06

R3

0.082

1.77E+06

R4

0.080

1.57E+06

R5

0.081

1.69E+06

R6

0.079

1.46E+06

Mean

0.081

1.63E+06

SD

0.001

1.13E+05

0.010

R1

0.082

[1]

1.86E+06

 

R2

0.084

[4]

2.17E+06

 

R3

0.082

[1]

1.83E+06

 

Mean

0.083

[2]

1.95E+06

[20]

SD

0.001

 

1.88E+05

 

0.032

R1

0.085

[5]

2.32E+06

 

R2

0.082

[1]

1.88E+06

 

R3

0.081

0

1.67E+06

 

Mean

0.083

[2]

1.96E+06

[20]

SD

0.002

 

3.34E+05

 

0.10

R1

0.057

30

2.88E+05

 

R2

0.051

37

1.95E+05

 

R3

0.047

42

1.47E+05

 

Mean

0.052

36

2.10E+05

87

SD

0.005

 

7.16E+04

 

0.32

R1

-0.002

102

-7.76E+02

 

R2

0.001

99

2.21E+02

 

R3

0.006

93

2.45E+03

 

Mean

0.002

98

6.32E+02

100

SD

0.004

 

1.65E+03

 

1.0

R1

-0.022

127

-4.00E+03

 

R2

-0.015

119

-3.27E+03

 

R3

-0.019

123

-3.71E+03

 

Mean

-0.019

123

-3.66E+03

100

SD

0.004

 

3.69E+02

 

 

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

R  = Replicate

SD=Standarddeviation

-   = Not applicable

[ ] = Increase in growth as compared to the control

Validity criteria fulfilled:
yes
Conclusions:
ErL10 (0 to 72 hour) : 0.070 mg/L loading rate WAF
ErL20 (0 to 72 hour) : 0.083 mg/L loading rate WAF
ErL50 (0 to 72 hour) : 0.11 mg/L loading rate WAF; 95% confidence limits
0.10 to 0.13 mg/L loading rate WAF

EyL10 (0 to 72 hour) : 0.087 mg/L loading rate WAF
EyL20 (0 to 72 hour) : 0.088 mg/L loading rate WAF
EyL50 (0 to 72 hour) : 0.092 mg/L loading rate WAF*
(* It was not possible to calculate 95% confidence limits for the EyL50 value as the data generated did not fit the models available for the calculation of confidence limits.)
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 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) 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 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 0.013 to 1.3 mg/L. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of 0.0086 to 1.3 mg/L (67% to 104% of the 0-Hour measured test concentrations).

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results:

 

Response Variable

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

EL10
(mg/L Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/L)

Lowest Observed Loading Rate (LOEL) (mg/L)

Growth Rate

0.11

0.10

-

0.13

0.070

0.032

0.10

Yield

0.092

 

*

 

0.087

0.032

0.10

 

(*It was not possible to calculate 95% confidence limits for the EyL50value as the data generated did not fit the models available for the calculation of confidence limits.

Description of key information

72 h ErL10: 0.070 mg/L loading rate WAF

72 h ErL50: 0.11 mg/L loading rate WAF; 95% confidence limits: 0.10 to 0.13 mg/L loading rate WAF

(OECD TG 201; Pseudokirchneriella subcapitata; WAF)

Key value for chemical safety assessment

EC50 for freshwater algae:
0.11 mg/L
EC10 or NOEC for freshwater algae:
0.07 mg/L

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 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) 761/2009.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 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 0.013 to 1.3 mg/L. A concentration dependent decline in measured test concentrations was observed at 72 hours in the range of 0.0086 to 1.3 mg/L (67% to 104% of the 0-Hour measured test concentrations).

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

 

Response Variable

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits (mg/L Loading Rate WAF)

EL10
(mg/L Loading Rate WAF)

No Observed Effect Loading Rate (NOEL) (mg/L)

Lowest Observed Loading Rate (LOEL) (mg/L)

Growth Rate

0.11

0.10

-

0.13

0.070

0.032

0.10

Yield

0.092

 

*

 

0.087

0.032

0.10

 

(*It was not possible to calculate 95% confidence limits for the EyL50value as the data generated did not fit the models available for the calculation of confidence limits.