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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:
20 June 2016 - 29 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) No 761/2009
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD series on testing and assessment number 23
Version / remarks:
December 14, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 100% saturated solution
- 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. An aliquot (2500 mL) of this saturated solution was inoculated with algal suspension (16.3 mL) to give the required test concentration of 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 and test cultures were observed to be green dispersions.
- 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): 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
- Age of inoculum (at test initiation): not specified; 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
- Culturing media and conditions (same as test or not): no (culture medium = AAP-medium acc. to OECD 201, test medium = AAP-medium with addition of sodium bicarbonate)
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
no data
Test temperature:
24 +/- 1°C
pH:
t=0 : 7.8 - 7.9
t=72 : 9.9 - 10
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 100 mg/L (measured concentration in 100% saturated solution was ca. 3.3 mg/L) it was decided to perform the definitive test as a "limit test" at 100% of the saturated solution prepared at a loading rate of 100 mg/L.
Measured concentrations in the test solution: 0.74 mg/L at t=0 and 0.24 mg/L at t=72 h

Geometric mean measured test concentrations were used to calculate effect parameters, to account for the decline in measured test concentration during the exposure period and in order to give a “worst case” analysis of the data.
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 and losses through volatilization
- Aeration: no
- Initial cells density: 5 x 10^3 cells per mL (nominal)
- Control end cells density: 4.54 x 10^5 cells per mL (mean, n=6)
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: initially (at t=24 and 48 h) cell densities were determined using a Coulter® Multisizer Particle Counter, however cell counts conducted at 48 hours showed a significant decrease in cell numbers in the 100% v/v saturated solution test preparations. Visually however there was no notable difference in the appearance of the cultures compared to the control; both were observed to be pale green dispersions. Examination of the 100% v/v saturated solution test cultures under the microscope showed that there were numerous clumps of algal cells present which were perhaps not being detected by the Coulter Particle counter. As such, it was considered appropriate for the remainder of the test to determine cell numbers using a haemocytometer and light microscope in order to ensure that an accurate count of the numbers of algal cells present was obtained. Cell densities using the haemocytometer and light microscope were determined at 51 and 72 hours.
- Chlorophyll measurement: no
- Other: All test and control cultures were inspected microscopically at 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable - limit test
- Range finding study
* Test concentrations: 0.1, 1.0, 10 and 100% (v/v) of a saturated solution
* Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (December 2015)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.42 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.:
> 0.42 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: not during the definite test (RF test: stimulation of growth found in the 0.1 and 10 % (v/v) saturated solutions; stimulation of yield observed in 0.1, 1.0 and 10 % (v/v) of the saturated solutions)
- 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: yes
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: 1.5 mg/L (95% C.I.: 1.3-1.7 mg/L)
- Other: 72-h NOErC = 0.50 mg/L
Reported statistics and error estimates:
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100% v/v saturated solution test concentration 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 7.8 at 0 hours to pH 9.9 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

Nominal Concentration

(% v/v Saturated Solution)

pH

Cell Densities*(cells per mL)

pH

0 h

24 h

51 h

72 h

72 h

Control

R1

7.8

1.31E+04

9.00E+04

4.57E+05

9.9

R2

1.33E+04

8.33E+04

4.17E+05

R3

1.06E+04

9.33E+04

4.40E+05

R4

1.45E+04

7.33E+04

4.40E+05

R5

1.40E+04

8.67E+04

5.00E+05

R6

1.76E+04

1.00E+05

4.67E+05

Mean

1.39E+04

8.78E+04

4.54E+05

100

R1

7.9

9.86E+03

8.33E+04

4.23E+05

10.0

R2

9.09E+03

8.67E+04

4.60E+05

R3

8.04E+03

8.67E+04

4.23E+05

R4

8.59E+03

8.00E+04

4.77E+05

R5

8.27E+03

7.67E+04

4.40E+05

R6

1.56E+04

8.67E+04

4.57E+05

Mean

9.91E+03

8.34E+04

4.47E+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.

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

Control

R1

0.040

0.071

0.077

R2

0.041

0.068

0.077

R3

0.031

0.081

0.074

R4

0.045

0.060

0.085

R5

0.043

0.068

0.083

R6

0.053

0.064

0.073

Mean

0.042

0.069

0.078


R1- R6= Replicates 1 to 6

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

0.063

 

4.52E+05

 

R2

0.061

 

4.12E+05

 

R3

0.062

 

4.35E+05

 

R4

0.062

-

4.35E+05

-

R5

0.064

 

4.95E+05

 

R6

0.063

 

4.62E+05

 

Mean

0.063

 

4.49E+05

 

SD

0.001

 

2.85E+04

 

100

R1

0.062

2

4.18E+05

 

R2

0.063

0

4.55E+05

 

R3

0.062

2

4.18E+05

 

R4

0.063

0

4.72E+05

 

R5

0.062

2

4.35E+05

 

R6

0.063

0

4.52E+05

 

Mean

0.063

1

4.42E+05

2

SD

0.001

 

2.18E+04

 


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

R1– R6= Replicates 1 to 6

SD= Standard Deviation

The measured concentration obtained from the freshly prepared 100% saturated solution was significantly lower than that obtained during the preliminary media preparation trial. This was considered to be due to the reduced solubility of the test item in the test media due to the presence of inorganic salts. This was considered to have had no adverse effect on the outcome of the test.

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

0.1

<LOQ

 

100

0.1

0.744

72

Control

0.1

<LOQ

 

100

0.1

0.237

LOQ                      =            Limit of Quantification

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]

[%]

[%]

 

 

0.1

0.106

96

7.2

 

 

0.1

0.0990

0.1

0.102

0.1

0.0951

 

 

0.1

0.102

 

 

0.1

0.0876

 Acceptance Target

 

 

80-120

<10

Validity criteria fulfilled:
yes
Remarks:
in controls 1. cell concentration increased by a factor of >16 (actual: 91) after 72 hours; 2. mean CV for section by section specific growth rate was <35% (actual: 31%); 3. CV for average specific growth rate over the test period was <7% (actual: 2%)
Conclusions:
The 72h-ErC50 and EC10 of the substance towards Pseudokirchneriella subcapitata were both > 0.42 mg/L based on the geometric mean measured test concentrations. The No Observed Effect Concentration was above the water solubility limit.
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 and GLP. 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 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 only the 100% saturated solution was tested (limit test). Pseudokirchneriella subcapitata was exposed for 72 hours (six replicate flasks per control and limit test concentration) under constant illumination and shaking at a temperature of 23 - 24°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 concentration was 0.74 mg/L in the 100% saturated solution. At the end of the test, the measured test concentration was 0.24 mg/L. Since the test concentration did not remain stable during the test period the geometric mean measured concentration was calculated and used to express the endpoint. This study showed that there were no toxic effects at the water solubility and 72h-ErC50 and EC10 were > 0.42 mg/L, 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 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 100 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 only the 100% saturated solution was tested (limit test).Pseudokirchneriella subcapitatawas exposed for 72 hours (six replicate flasks per control and limit test concentration) under constant illumination and shaking at a temperature of 23 - 24°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 concentration was 0.74 mg/L in the 100% saturated solution. At the end of the test, the measured test concentration was 0.24 mg/L. Since the test concentration did not remain stable during the test period the geometric mean measured concentration was calculated and used to express the endpoint.This study showed that there were no toxic effects at the water solubility and72h-ErC50 and EC10 were > 0.42 mg/L, based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater algae:
0.42 mg/L

Additional information