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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:
23 February 2017 - 18 March 2017
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 (Annex V corrected on 28 July 2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
761/2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of issue: 28 Oct 2016
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and each test group
- Sampling method: from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours, sampling volume: 200 mL
- Sample storage conditions before analysis: samples were analyzed directly on the day of receipt. Only duplicate samples (taken at t=0 h and t=72 h) were stored frozen for further analysis if necessary).
- Other: Two additional samples of each test concentration were prepared at the start of the test and incubated alongside the test to provide samples for analysis at 24 and 48 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of a saturated solution (direct addition to test medium) - 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 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 algal suspension (11.9 mL) 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.
- Controls: test medium without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): 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 1.0% v/v saturated solution test preparations were observed to be green dispersions. The 3.2% v/v saturated solution test cultures were observed to be pale green dispersions whilst the 10, 32 and 100% v/v saturated solution test cultures were observed to be clear colorless solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): originally obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland and maintained in the laboratory by the periodic replenishment of culture medium (AAP medium according to OECD TG 201).
- Age of inoculum (at test initiation): not specified
- Preparation of cultures: 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.
- Method of cultivation: The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 2°C.

ACCLIMATION
- Acclimation period: not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 +/- 1°C
pH:
7.6 - 8.4 at 0 h
8.1 - 10.1 at 72 h
Nominal and measured concentrations:
Based on the results of the range-finding test, performed at nominal test concentrations of 0.10, 1.0, 10 and 100% (v/v) of a saturated solution prepared at a loading rate of 50 mg/L (measured concentration in 100% saturated solution was 41.0 mg/L) it was decided to perform the definitive test with nominal concentrations of 1.0, 3.2, 10, 32% (v/v) and 100% of a saturated solution prepared at a loading rate of 50 mg/L.
Measured concentrations in the test solution: see table 1. Because a decline was observed in the measured test concentrations after 24, 48 and 72 hours, the geometric mean was calculated.
The mean of the three individual geometric mean values (per time interval) were: 0.12, 0.38, 1.5, 5.8 and 22 mg/L corresponding to nominal concentrations of 1.0, 3.2, 10, 32% (v/v) and 100% saturated solution prepared at 50 mg/L, respectively. The mean values were in the range of 34 - 74% of initial measured concentrations at t=0 h.

Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks, completely filled with test solution and sealed with ground glass stoppers
- Aeration: no
- Initial cells density: 5 x 10^3 cells per mL (nominal)
- Control end cells density: 5.37 x 10^5 cells per mL (mean, n=6)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: yes; for the purposes of the definitive tests, sodium bicarbonate (250 mg/L) was added to the prepared culture medium prior to use to provide a source of carbon dioxide for algal growth (in order to prevent inhibition of growth due to the restriction of gaseous exchange)
- Intervals of water quality measurement: temperature - daily; pH - at t=0 and t=72 h

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: intensity approximately 7000 lux, provided by warm white lighting (380 – 730 nm)
- Flasks were constantly shaken during the test period at approximately 150 rpm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at 28, 48 and 72 hours cell densities were determined using a Coulter® Multisizer Particle Counter
- Chlorophyll measurement: no
- Other: All test and control cultures were inspected microscopically at 72 hours. To determine the potential effect of the test item on the appearance of algal cells. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study:
* Test concentrations: 0.10, 1.0, 10 and 100% (v/v) of a saturated solution prepared at a loading rate of 50 mg/L
* Results used to determine the conditions for the definitive study: yes
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.:
0.77 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CI 0.66-0.89 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in the control or test cultures at 1.0% v/v saturated solution. Whilst cell debris was observed in the 3.2% v/v saturated solution test cultures, no intact cells were observed to be present in the 10, 32 and 100% v/v saturated solution test cultures.
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Test conditions: the increase of pH in the controls from 7.6 at 0 hours to 10.0 at 72 hours 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. The same is true for the increase in pH in the lowest test concentration. 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.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Tested concentrations: 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L
- 72-h ErC50: 1.4 mg/L (95% C.I.: 1.2-1.5 mg/L)
- Other: 72-h NOErC = 0.25 mg/L
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 1 Measured test concentrations in the final test

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

0.1

<LOQ

1.0

0.1

0.285

 

3.2

0.1

1.12

 

10

0.1

3.37

 

32

0.1

11.1

 

100

0.5

29.7

24

Control

0.1

<LOQ

1.0

0.1

0.166

 

3.2

0.1

0.321

 

10

0.1

1.44

 

32

0.1

5.98

 

100

0.5

26.0

48

Control

0.1

<LOQ

 

1.0

0.1

0.0682

 

3.2

0.1

0.303

 

10

0.1

1.19

 

32

0.1

4.47

 

100

0.5

18.0

72

Control

0.1

<LOQ

 

1.0

0.1

0.0271

 

3.2

0.1

0.181

 

10

0.1

0.982

 

32

0.1

3.80

 

100

0.5

14.2

A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.17 to 26 mg/L at 24 hours, 0.068 to 18 mg/L at 48 Hours and from 0.027 to 14 mg/L at 72 hours (Table 1). Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data (Table 2)

Table 2 Geometric mean measured test concentrations

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

Expressed as a Percentage of the 0-Hour Measured Test Concentration (%)

1.0

0.12

42

3.2

0.38

34

10

1.5

45

32

5.8

52

100

22

74

Table 3 Cell densities and pH values in the definitive test

Nominal Concentration

(% v/v Saturated Solution)

pH

Cell Densities* (cells per mL)

pH

0 h

28 h

48 h

72 h

72 h

Control

R1

7.6

3.38E+04

1.21E+05

5.51E+05

10.0

R2

3.31E+04

1.14E+05

5.32E+05

R3

3.57E+04

1.31E+05

4.89E+05

R4

3.70E+04

1.59E+05

5.30E+05

R5

3.97E+04

1.40E+05

5.97E+05

R6

3.77E+04

1.51E+05

5.20E+05

Mean

3.62E+04

1.36E+05

5.37E+05

1.0

R1

7.8

3.92E+04

1.42E+05

5.41E+05

10.1

R2

4.25E+04

1.47E+05

5.39E+05

R3

4.21E+04

1.60E+05

5.94E+05

Mean

4.13E+04

1.50E+05

5.58E+05

3.2

R1

7.9

3.38E+04

6.26E+04

2.25E+05

9.3

R2

3.39E+04

7.08E+04

2.67E+05

R3

3.12E+04

5.94E+04

2.21E+05

Mean

3.30E+04

6.43E+04

2.38E+05

10

R1

7.9

1.07E+04

1.17E+04

1.42E+04

8.2

R2

1.18E+04

9.91E+03

1.06E+04

R3

1.13E+04

8.71E+03

9.71E+03

Mean

1.13E+04

1.01E+04

1.15E+04

32

R1

7.9

8.01E+03

8.74E+03

9.68E+03

8.1

R2

9.15E+03

7.92E+03

1.03E+04

R3

8.95E+03

8.65E+03

9.98E+03

Mean

8.70E+03

8.44E+03

1.00E+04

100

R1

8.4

8.07E+03

7.51E+03

6.83E+03

8.3

R2

7.89E+03

7.01E+03

9.18E+03

R3

7.04E+03

7.36E+03

6.91E+03

Mean

7.67E+03

7.29E+03

7.64E+03

*    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.

R1- R6= Replicates 1 to 6

Table 4 Daily specific growth rates for the control cultures in the definitive test

 

Daily Specific Growth Rate (cells/mL/hour)

Day 0 - 1

Day 1 - 2

Day 2 - 3

Coefficient of Variation (Section by Section Daily Growth Rate)

Average Specific Growth Rate

(Day 0-3)

Control

R1

0.068

0.064

0.063

5%

0.065

R2

0.067

0.062

0.064

5%

0.065

R3

0.070

0.065

0.055

13%

0.064

R4

0.071

0.073

0.050

20%

0.065

R5

0.074

0.063

0.060

11%

0.066

R6

0.072

0.069

0.052

17%

0.065

Mean

0.070

0.066

0.057

12%

0.065

 

 

 

 

 

 

CV = 2%

R1- R6= Replicates 1 to 6

Table 5 Inhibition of growth rate and yield in the definitive test

Nominal Concentration
(% v/v Saturated Solution)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.065

 

5.46E+05

 

R2

0.065

 

5.27E+05

 

R3

0.064

 

4.84E+05

 

R4

0.065

-

5.25E+05

-

R5

0.066

 

5.92E+05

 

R6

0.065

 

5.15E+05

 

Mean

0.065

 

5.32E+05

 

SD

0.001

 

3.60E+04

 

1.0

R1

0.065

0

5.36E+05

 

R2

0.065

0

5.34E+05

 

R3

0.066

[2]

5.89E+05

 

Mean

0.065

[1]

5.53E+05

[4]

SD

0.001

 

3.14E+04

 

3.2

R1

0.053

18

2.20E+05

 

R2

0.055

15

2.62E+05

 

R3

0.053

18

2.16E+05

 

Mean

0.054

17

2.33E+05

56

SD

0.001

 

2.56E+04

 

10

R1

0.014

78

9.16E+03

 

R2

0.010

85

5.62E+03

 

R3

0.009

86

4.71E+03

 

Mean

0.011

83

6.50E+03

99

SD

0.003

 

2.35E+03

 

32

R1

0.009

86

4.68E+03

 

R2

0.010

85

5.34E+03

 

R3

0.010

85

4.98E+03

 

Mean

0.010

85

5.00E+03

99

SD

0.001

 

3.28E+02

 

100

R1

0.004

94

1.83E+03

 

R2

0.008

88

4.18E+03

 

R3

0.004

94

1.91E+03

 

Mean

0.005

92

2.64E+03

100

SD

0.002

 

1.34E+03

 

*    In accordance with the OECD test guideline only thean 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:
In controls: cell concentration increased by a factor of >16 (e.g. 107.4) after 72 hours, mean CV for section by section specific growth rate was <35% (e.g. 12%) and CV for average specific growth rate over the test period was <7% (e.g. 2%).
Conclusions:
The 72h-ErC50, -ErC10 and -NOEC were 0.77, 0.28 and 0.12 mg/L, respectively, based on geometric mean measured test concentrations.
Executive summary:

A study was performed to assess the effect of the test substance on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with OECD TG No 201 and GLP. A saturated solution was prepared at a loading rate of 50 mg/L applying a 24-hour period of propeller stirring at approx. 1500 rpm followed removal of any undissolved test item by filtration. The final test solutions were all clear and colourless. Based on the results of a range-finding test (at 0.1, 1.0, 10 and 100% (v/v) of the saturated solution), in the definitive test nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v of the saturated solution prepared at a loading rate of 50 mg/L were used. Pseudokirchneriella subcapitata was exposed for 72 hours (six replicate flasks per control and three flasks per test concentration) under constant illumination and gentle shaking at a temperature of 24 +/- 1°C. Test concentrations were confirmed with a validated GC-FID method in samples taken from all treatments at t = 0 h and t=72 h and additionaly at t=24 h and t=48 h in samples taken from vessels without algae.

Results: The test concentrations did not remain stable during the test period: at 0, 1, 3.2, 10. 32 and 100 mg/l saturated solution at 0h the concentrations were about a third of these saturated concentrations: 0.29, 1.12, 3.37, 11.1 and 29.7 mg/l, respectively. After 72 hours these were: 0.027, 0.18, 0.982, 3.8 and 14.2 mg/l, respectively. Based on this decrease, the geometric mean measured concentrations were calculated for each 24 -hour interval and the arithmetic mean of the three values was used to express the endpoint. Geometric mean measured concentrations were: 0.12, 0.38, 1.5, 5.8 and 22 mg/L for nominal concentrations of 1.0, 3.2, 10, 32 and 100% of the saturated solution, respectively. The 72h-ErC50, -ErC10 and -NOEC were 0.77, 0.28 and 0.12 mg/L, respectively, based on geometric mean measured test concentrations.All acceptability criteria were met and the study was considered to be valid.

Description of key information

A study was performed to assess the effect of the test substance on the growth of the green algaPseudokirchneriella subcapitata. The study was conducted in accordance with OECD TG No 201 and GLP.A saturated solution was prepared at a loading rate of 50 mg/L applying a 24-hour period of propeller stirring at approx. 1500 rpm followed removal ofany undissolved test item by filtration. The final test solutions were all clear and colourless.Based on the results of a range-finding test (at 0.1, 1.0, 10 and 100% (v/v) of the saturated solution), in the definitive test nominal concentrations of 1.0, 3.2, 10, 32 and 100% v/v of the saturated solution prepared at a loading rate of 50 mg/L were used.Pseudokirchneriella subcapitatawas exposed for 72 hours (six replicate flasks per control and three flasks per test concentration) under constant illumination and gentle shaking at a temperature of 24 +/- 1°C.Test concentrations were confirmed with a validated GC-FID method in samples taken from all treatments at t = 0 h and t=72 h and additionaly at t=24 h and t=48 h in samples taken from vessels without algae.

Results: The test concentrationsdid not remain stable during the test period: at 0, 1, 3.2, 10. 32 and 100 mg/l saturated solution at 0h the concentrations were about a third of these saturated concentrations: 0.29, 1.12, 3.37, 11.1 and 29.7 mg/l, respectively. After 72 hours these were: 0.027, 0.18, 0.982, 3.8 and 14.2 mg/l, respectively. Based on this decrease, the geometric mean measured concentrations were calculated for each 24 -hour interval and the arithmetic mean of the three values was used to express the endpoint. Geometric mean measured concentrations were:0.12, 0.38, 1.5, 5.8 and 22 mg/L for nominal concentrations of 1.0, 3.2, 10, 32 and 100% of the saturated solution, respectively. The 72h-ErC50, -ErC10 and -NOEC were 0.77, 0.28 and 0.12 mg/L, respectively, based on geometric mean measured test concentrations.All acceptability criteria were met and the study was considered to be valid.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.77 mg/L
EC10 or NOEC for freshwater algae:
0.28 mg/L

Additional information