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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:
09 February 2017 - 04 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
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) 761/2009
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test groups
- Sampling method: from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours, sampling volume: 100 mL
- Sample storage conditions before analysis: freezer (all samples)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of a saturated solution (direct addition to test medium) - A nominal amount of test item (1100 mg) was dispersed in 11 liters of test water 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 6.25, 12.5 , 25 and 50% (v/v) saturated solution. An aliquot (2 liters) of each of the stock solutions was separately inoculated with 13.2 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
- 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, 1.3, 2.8, 5.7 and 12 mg/L test cultures were observed to be green dispersions whilst the 23 mg/L test cultures were observed to be clear colorless solutions.
- Other: Prior to use the test medium was aerated until the dissolved oxygen concentration was approximately air-saturation value.
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): 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.
- Age of inoculum (at test initiation): not specified
- 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°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. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture (AAP-medium as described in OECDTG201).

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions (same as test or not): no (see 'test conditions')
- Any deformed or abnormal cells observed: after 72 hours, no abnormalities were detected in any of the control or test cultures
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
24 +/- 1°C
pH:
test start (t=0): 8.0-8.1
test end (t=72 h): 8.5-10.3
Nominal and measured concentrations:
Nominal test concentrations (definitive test): 6.25, 12.5, 25, 50 and 100% v/v of a saturated solution prepared at a loading rate of 100 mg/L.

Measured concentrations at t=0: 1.51, 3.07, 6.25, 12.4 and 23.8 mg/L in corresponding test solutions at 6.25, 12.5, 25, 50 and 100% v/v of a saturated solution, respectively
Measured concentrations at t=72: 1.20, 2.54, 5.12, 11.3 and 22.5 mg/L in corresponding test solutions at 6.25, 12.5, 25, 50 and 100% v/v of a saturated solution, respectively

Since a slight decline in measured test concentration was observed at 72 hours (79% to 95% of initial) it was considered appropriate to calculate the effect parameters based on the geometric mean measured test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks, completely filled with test preparation and sealed with ground glass stoppers to reduce evaporation
- Aeration: no
- Initial cells density: 5 x 10^3 cells per mL (nominal)
- Control end cells density: 4.77 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, as desribed in OECD TG 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water
- Culture medium different from test medium: yes; the culture medium was AAP-medium whereas 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
- 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)
- Agitation: constant shaking at ca. 150 rpm for 72 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities were determined using a Coulter® Multisizer Particle Counter (at t=24, 48 and 72 h), the nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density
- Chlorophyll measurement: no
- Other: All test and control cultures were inspected microscopically at 72 hours for abnormalities (appearance, shape and size).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L (measured concentration in 100% saturated solution was ca. 19 mg/L)
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (December 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13 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.:
5.7 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): no
- Aggregation of algal cells: Some large clumps of algal cells were observed to be visible in the 100% saturated solution test cultures.
- Other:
- Any stimulation of growth found in any treatment: slight increase in growth as compared to controls in the definite test in the 6.25% and 12.5% dilutions of the 100% saturated solution
- 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 (ErC50 similar as solubility limit in test medium)
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).

The pH value of the control cultures was observed to increase from pH 8.1 at 0 hours to pH 10.3 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.

Cell densities and pH values in the definitive test

Geometric Mean Measured Test Concentration (mg/L)

pH

Cell Densities* (cells per mL)

pH

0 h

24 h

46 h

72 h

72 h

Control

R1

8.1

2.94E+04

9.76E+04

4.61E+05

10.3

R2

2.87E+04

8.36E+04

4.79E+05

R3

2.85E+04

1.10E+05

4.96E+05

R4

3.07E+04

1.25E+05

4.84E+05

R5

3.21E+04

1.19E+05

4.94E+05

R6

2.86E+04

8.06E+04

4.47E+05

Mean

2.97E+04

1.03E+05

4.77E+05

1.3

R1

8.1

2.91E+04

8.43E+04

4.80E+05

10.3

R2

3.20E+04

1.19E+05

5.29E+05

R3

2.15E+04

7.97E+04

5.04E+05

Mean

2.76E+04

9.43E+04

5.04E+05

2.8

R1

8.1

2.58E+04

1.11E+05

4.76E+05

10.3

R2

2.27E+04

9.33E+04

4.86E+05

R3

2.95E+04

9.06E+04

5.10E+05

Mean

2.60E+04

9.83E+04

4.91E+05

5.7

R1

8.0

2.39E+04

1.02E+05

4.06E+05

10.2

R2

2.63E+04

6.93E+04

4.83E+05

R3

2.84E+04

7.23E+04

4.52E+05

Mean

2.62E+04

8.12E+04

4.47E+05

12

R1

8.1

1.95E+04

3.79E+04

3.12E+05

10.1

R2

2.40E+04

3.80E+04

3.69E+05

R3

2.09E+04

6.88E+04

3.52E+05

Mean

2.15E+04

4.82E+04

3.44E+05

23

R1

8.1

8.98E+03

1.40E+04

3.31E+04

8.5

R2

1.08E+04

1.74E+04

3.59E+04

R3

6.78E+03

1.68E+04

2.50E+04

Mean

8.86E+03

1.60E+04

3.13E+04

*    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

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

0.074

0.055

0.060

16%

0.063

R2

0.073

0.049

0.067

19%

0.063

R3

0.073

0.062

0.058

13%

0.064

R4

0.076

0.064

0.052

19%

0.064

R5

0.077

0.060

0.055

19%

0.064

R6

0.073

0.047

0.066

21%

0.062

Mean

0.074

0.056

0.060

18%

0.063

 

 

 

 

 

 

CV = 2%

R1- R6= Replicates 1 to 6

Inhibition of growth rate and yield in the definitive test

Geometric Mean Measured Test Concentration (mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.063

 

4.56E+05

 

R2

0.063

 

4.74E+05

 

R3

0.064

 

4.91E+05

 

R4

0.064

-

4.79E+05

-

R5

0.064

 

4.89E+05

 

R6

0.062

 

4.42E+05

 

Mean

0.063

 

4.72E+05

 

SD

0.001

 

1.94E+04

 

1.3

R1

0.063

0

4.75E+05

 

R2

0.065

[3]

5.24E+05

 

R3

0.064

[2]

4.99E+05

 

Mean

0.064

[2]

4.99E+05

[6]

SD

0.001

 

2.49E+04

 

2.8

R1

0.063

0

4.71E+05

 

R2

0.064

[2]

4.81E+05

 

R3

0.064

[2]

5.05E+05

 

Mean

0.064

[1]

4.86E+05

[3]

SD

0.001

 

1.76E+04

 

5.7

R1

0.061

3

4.01E+05

 

R2

0.063

0

4.78E+05

 

R3

0.063

0

4.47E+05

 

Mean

0.062

1

4.42E+05

6

SD

0.001

 

3.88E+04

 

12

R1

0.057

10

3.07E+05

 

R2

0.060

5

3.64E+05

 

R3

0.059

6

3.47E+05

 

Mean

0.059

7#

3.39E+05

28#

SD

0.002

 

2.91E+04

 

23

R1

0.026

59

2.81E+04

 

R2

0.027

57

3.09E+04

 

R3

0.022

65

2.00E+04

 

Mean

0.025

60#

2.63E+04

94#

SD

0.003

 

5.67E+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]

# statistically significantly different to controls (P<0.05)

Analytical results for test samples

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

6.25

0.10

1.51

 

12.5

0.10

3.07

 

25

0.10

6.25

 

50

0.10

12.4

 

100

0.20

23.8

72

Control

0.10

<LOQ

 

6.25

0.10

1.20

 

12.5

0.10

2.54

 

25

0.10

5.12

 

50

0.10

11.3

 

100

0.20

22.5

LOQ                      =            Limit of Quantification (0.019 mg/L, based on calculating the sample concentration that gave a peak equivalent to ten times the baseline noise)

Geometric mean measured test concentrations

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

6.25

1.3

12.5

2.8

25

5.7

50

12

100

23

Analytical results for spiked recovery samples

Nominal Concentra­tion of
Test Item


cnom

Fortified Concentra­tion of Test Item in the Spiked Sample


cfort

Sample Preparation Factor



F

Determined Concentra­tion of Test Item in the Spiked Sample


c

Mean Analytical Recovery
Rate


R

Precision (Relative Standard Deviation of Recovery)

[mg/L]

[mg/L]

 

[mg/L]

[%]

[%]

 

 

0.10

1.07

107

0.66

 

 

0.10

1.06

1.00

0.995

0.10

1.07

 

 

0.10

1.06

 

 

0.10

1.07

 Acceptance Target

 

 

80-120

<10

Validity criteria fulfilled:
yes
Remarks:
in controls 1. cell concentration increased by a factor of >16 (actual: 95) after 72 hours; 2. mean CV for section by section specific growth rate was <35% (actual: 18%); 3. CV for average specific growth rate over the test period was <7% (actual: 2%)
Conclusions:
The 72h-ErC50, ErC10 and NOErC of the substance towards Pseudokirchneriella subcapitata were 21.0, 13.0 and 5.7 mg/L, respectively, based on geometric mean measured test concentrations.
Executive summary:

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with OECD TG No 201 andGLP. A saturated solution was prepared at a loading rate of 100 mg/L applying a 24-hour period of propeller stirring at approx. 1500 rpm followed by removal of any undissolved test item by filtration.In the definitive test an untreated control and dilutions of the 100% saturated solution (6.25, 12.5, 25 and 50% (v/v), respectively) as well as the 100% saturated solution were tested. The final test solutions were all clear and colourless. Pseudokirchneriella subcapitata was exposed for 72 hours (six replicate flasks per control and three replicates per test concentration) under constant illumination and 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 and 72 h. At the start of the test, the measured test concentrations ranged from 1.5 to 23.8 mg/L. At the end of the test, the measured test concentrations ranged from 1.2 to 22.5 mg/L.Since the test concentration slightly decreased during the test period (79 -95% of initial measured concentrations) the geometric mean measured concentrations were calculated and used to express the endpoint. This study showed that the substance caused statistically significant differences between the control and test concentrations >5.7 mg/L (P<0.05). The 72h-ErC50, 72h-ErC10 and 72h-NOEC were 21.0, 13.0 and 5.7 mg/L, respectively, based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the effect of the substance on the growth of the green alga Pseudokirchneriella subcapitata. The study was conducted in accordance with OECD TG No 201 andGLP. A saturated solution was prepared at a loading rate of 100 mg/L applying a 24-hour period of propeller stirring at approx. 1500 rpm followed by removal of any undissolved test item by filtration.In the definitive test an untreated control and dilutions of the 100% saturated solution (6.25, 12.5, 25 and 50% (v/v), respectively) as well as the 100% saturated solution were tested. The final test solutions were all clear and colourless. Pseudokirchneriella subcapitata was exposed for 72 hours (six replicate flasks per control and three replicates per test concentration) under constant illumination and 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 and 72 h. At the start of the test, the measured test concentrations ranged from 1.5 to 23.8 mg/L. At the end of the test, the measured test concentrations ranged from 1.2 to 22.5 mg/L.Since the test concentration slightly decreased during the test period (79 -95% of initial measured concentrations) the geometric mean measured concentrations were calculated and used to express the endpoint. This study showed that the substance caused statistically significant differences between the control and test concentrations >5.7 mg/L (P<0.05). The 72h-ErC50, 72h-ErC10 and 72h-NOEC were 21.0, 13.0 and 5.7 mg/L, respectively, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
21 mg/L
EC10 or NOEC for freshwater algae:
13 mg/L

Additional information