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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:
2 March 2017 - 8 April 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
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)
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours, from samples run alongside the test at 24 and 48 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
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: The culture medium used for both the range finding and definitive tests was the same as that used to maintain the stock culture. The media used in both the range finding and definitive tests was prepared with the addition of 250 mg/L of sodium bicarbonate to prevent inhibition of growth due to the restriction in gaseous exchange associated with testing in an enclosed system (Herman et al 1990).
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 : 7.7 - 8.3
t=72 : 8.3 - 10.1
Nominal and measured concentrations:
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) of the test item.

RANGE-FINDING TEST
The nominal loading rates of 10 and 100 mg/L were used.
The measured concentrations at 0 hours showed 2.6 and 13 mg/L, respectively.
No samples were taken at 72 hours in error, however, method development work showed the test item to be unstable, therefore it was considered appropriate to take additional samples at the 24 and 48 hours time points during the definitive test in order to better show the pattern of decline.

Because of the observed toxicity, a second range-finding test was conducted with nominal loading rates of 0.10 and 1.0 mg/L.

DEFINITIVE TEST
The nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were used.
The measured concentrations of the total peak area were: 0.16 -17 mg/L at 0 hours, 0.17-12 mg/L at 24 hours, 0.076-11 mg/L at 48 hours and 0.086-12 mg/L at 72 hours.
The measured concentrations of component 1 were: 0.052-1.0 mg/L at 0 hours, 0.032-0.80 mg/L at 24 hours, 0.026-0.62 mg/L at 48 hours and 0.018-0.70 mg/L at 72 hours.
Measured concentrations of component 2 could not be accurately obtained due to the changing chromatography of large unknown peaks present in the test samples.

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.

For the total peak area, the geometric means were: 0.22, 0.12, 0.95, 6.0 and 12 mg/L.
For the Component 1, the geometric means were: 0.058, 0.030, 0.22, 0.41 and 0.76 mg/L.
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)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (AAP-medium as described in OECD TG 201), with an addition of sodium bicarbonate (250 mg/L)

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) (Herman et al 1990)
- Intervals of water quality measurement: temperature - daily; pH - at t=0 and t=72 h
- Intervals of appearance of the test media assessment: daily.

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: Samples were taken at 23, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample.
- Chlorophyll measurement: no
- Other: All test and control cultures were inspected microscopically at 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations in the main study: 3.2
- Range finding study
* Test concentrations: 10 and 100 mg/L
* Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (November / December 2016)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
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: no
- Other:
- Any stimulation of growth found in any treatment: RF test: stimulation of growth found in the 0.1 and 1.0 mg/L solutions; main test: stimulation of growth found in some of the 1.0 and 3.2 mg/L 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: no
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 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 pH value of the control cultures (see Table 3) was observed to increase from pH 7.7 at 0 hours to pH 9.8 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 (see Figure 1) 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 EEC 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 Loading Rate

(mg/L)

pH

Cell Densities*(cells per mL)

pH

0 h

23 h

46 h

72 h

72 h

Control

R1

7.7

2.30E+04

1.01E+05

3.93E+05

9.8

R2

3.24E+04

1.46E+05

6.17E+05

R3

2.81E+04

1.56E+05

6.03E+05

R4

3.18E+04

1.62E+05

5.64E+05

R5

2.62E+04

1.19E+05

5.14E+05

R6

2.80E+04

1.48E+05

3.65E+05

Mean

2.83E+04

1.38E+05

5.09E+05

1.0

R1

7.8

2.64E+04

1.30E+05

4.64E+05

10.0

R2

2.45E+04

1.17E+05

5.34E+05

R3

3.11E+04

1.38E+05

5.79E+05

Mean

2.73E+04

1.28E+05

5.26E+05

3.2

R1

7.9

2.77E+04

1.11E+05

4.64E+05

10.1

R2

3.19E+04

1.42E+05

4.30E+05

R3

2.99E+04

1.41E+05

6.49E+05

Mean

2.98E+04

1.31E+05

5.14E+05

10

R1

7.9

2.11E+04

8.33E+04

3.70E+05

9.9

R2

2.12E+04

8.09E+04

3.94E+05

R3

1.28E+04

9.19E+04

4.62E+05

Mean

1.84E+04

8.54E+04

4.09E+05

32

R1

8.3

1.31E+04

3.32E+04

1.36E+05

9.5

R2

1.02E+04

3.40E+04

4.62E+05

R3

1.52E+04

4.08E+04

1.90E+05

Mean

1.28E+04

3.60E+04

2.63E+05

100

R1

8.0

7.95E+03

1.90E+04

2.07E+04

8.3

R2

8.07E+03

1.76E+04

1.67E+04

R3

1.27E+04

1.77E+04

2.10E+04

Mean

9.56E+03

1.81E+04

1.94E+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

Control

R1

0.066

0.064

0.052

R2

0.081

0.065

0.056

R3

0.075

0.075

0.052

R4

0.080

0.071

0.048

R5

0.072

0.066

0.056

R6

0.075

0.072

0.035

Mean

0.075

0.069

0.050


R1- R6= Replicates 1 to 6

Inhibition of growth rate and yield in the definitive test

Nominal Loading Rate
(mg/L)

Growth Rate (cells/mL/hour)

Yield (cells/mL)

0 – 72 h

% Inhibition

0 – 72 h

% Inhibition*

Control

R1

0.061

 

3.88E+05

 

R2

0.067

 

6.12E+05

 

R3

0.067

 

5.98E+05

 

R4

0.066

-

5.59E+05

-

R5

0.064

 

5.09E+05

 

R6

0.060

 

3.60E+05

 

Mean

0.064

 

5.04E+05

 

SD

0.003

 

1.07E+05

 

1.0

R1

0.063

2

4.59E+05

 

R2

0.065

[2]

5.29E+05

 

R3

0.066

[3]

5.74E+05

 

Mean

0.065

[1]

5.21E+05

[3]

SD

0.002

 

5.79E+04

 

3.2

R1

0.063

2

4.59E+05

 

R2

0.062

3

4.25E+05

 

R3

0.068

[6]

6.44E+05

 

Mean

0.064

[0]

5.09E+05

[1]

SD

0.003

 

1.17E+05

 

10

R1

0.060

6

3.65E+05

 

R2

0.061

5

3.89E+05

 

R3

0.063

2

4.57E+05

 

Mean

0.061

4

4.04E+05

20

SD

0.002

 

4.74E+04

 

32

R1

0.046

28

1.31E+05

 

R2

0.063

2

4.57E+05

 

R3

0.051

20

1.85E+05

 

Mean

0.053

17

2.58E+05

49

SD

0.009

 

1.75E+05

 

100

R1

0.020

69

1.57E+04

 

R2

0.017

73

1.17E+04

 

R3

0.020

69

1.60E+04

 

Mean

0.019

70

1.44E+04

97

SD

0.002

 

2.40E+03

 

*       In accordance with the OECD test guideline only the mean 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]

Analytical results for test samples

Test Item:

Time Point

Nominal Loading Rate

cnom

Sample Preparation Factor



F

Determined Concentration of Test Item in Test Sample

 


c

[Hours]

[mg/L]

 

[mg/L]

0

Control

0.2

<LOQ

1.0

0.2

0.273

 

3.2

0.2

0.155

 

10

0.2

1.37

 

32

0.2

8.45

 

100

0.2

16.5

24

Control

0.2

<LOQ

1.0

0.2

0.283

 

3.2

0.2

0.172

 

10

0.2

1.11

 

32

0.2

6.30

 

100

0.2

11.6

48

Control

0.2

<LOQ

 

1.0

0.2

0.165

 

3.2

0.2

0.0763

 

10

0.2

0.843

 

32

0.2

5.44

 

100

0.2

11.3

72

Control

0.2

<LOQ

 

1.0

0.2

0.130

 

3.2

0.2

0.0860

 

10

0.2

0.481

 

32

0.2

4.06

 

100

0.2

11.6

LOQ                      =            Limit of Quantification

Component 1:

Time Point

Calculated Nominal Concentration of
Component 1 in Test Sample

cnom

Sample Preparation Factor



F

Calculated Concentration of Component 1 in Test Sample

 


c

[Hours]

[mg/L]

 

[mg/L]

0

Control

0.2

<LOQ

0.506

0.2

0.0864

 

1.62

0.2

0.0518

 

5.06

0.2

0.326

 

16.2

0.2

0.615

 

50.6

0.2

1.03

24

Control

0.2

<LOQ

0.506

0.2

0.0644

 

1.62

0.2

0.0319

 

5.06

0.2

0.238

 

16.2

0.2

0.449

 

50.6

0.2

0.797

48

Control

0.2

<LOQ

 

0.506

0.2

0.0524

 

1.62

0.2

0.0262

 

5.06

0.2

0.188

 

16.2

0.2

0.333

 

50.6

0.2

0.617

72

Control

0.2

<LOQ

 

0.506

0.2

0.0334

 

1.62

0.2

0.0176

 

5.06

0.2

0.151

 

16.2

0.2

0.289

 

50.6

0.2

0.701

LOQ                      =            Limit of Quantification

Analytical results for spiked recovery samples

Test Item:

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]

[%]

[%]

 

 

0.2

0.372

77*

8.2

 

 

0.2

0.427

0.500

0.544

0.2

0.420

 

 

0.2

0.465

 

 

0.2

0.404

 

 

0.2

0.394

81

4.4

 

 

0.2

0.429

0.500

0.521

0.2

0.444

 

 

0.2

0.425

 

 

0.2

0.418

 Acceptance Target

 

 

80-120

<10

Component 1:

Calculated Nominal Concentra­tion of
Component 1


cnom

Calculated Fortified Concentra­tion of Component 1 in the Spiked Sample


cfort

Sample Preparation Factor



F

Calculated Concentra­tion of Component 1 in the Spiked Sample


c

Mean Analytical Recovery
Rate



R

Precision (Relative Standard Deviation of Recovery)

[mg/L]

[mg/L]

 

[mg/L]

[%]

[%]

 

 

0.2

0.147

63*

13.6*

 

 

0.2

0.182

0.253

0.275

0.2

0.173

 

 

0.2

0.210

 

 

0.2

0.161

 

 

0.2

0.145

58*

6.4

 

 

0.2

0.156

0.253

0.264

0.2

0.168

 

 

0.2

0.151

 

 

0.2

0.143

 Acceptance Target

 

 

80-120

<10

Component 2:

Calculated Nominal Concentra­tion of
Component 2


cnom

Calculated Fortified Concentra­tion of Component 2 in the Spiked Sample


cfort

Sample Preparation Factor



F

Calculated Concentra­tion of Component 2 in the Spiked Sample


c

Mean Analytical Recovery
Rate



R

Precision (Relative Standard Deviation of Recovery)

[mg/L]

[mg/L]

 

[mg/L]

[%]

[%]

 

 

0.2

0.162

100

10.6*

 

 

0.2

0.213

0.181

0.197

0.2

0.205

 

 

0.2

0.212

 

 

0.2

0.197

 

 

0.2

0.143**

95

2.0

 

 

0.2

0.175

0.181

0.188

0.2

0.176

 

 

0.2

0.183

 

 

0.2

0.178

 Acceptance Target

 

 

80-120

<10

Validity criteria fulfilled:
yes
Remarks:
in controls 1. cell concentration increased by a factor of >16 (actual: 102) after 72 hours; 2. mean CV for section by section specific growth rate was <35% (actual: 21%); 3. CV for average specific growth rate over the test period was <7% (actual: 5%)
Conclusions:
The 72h-ErC50 and EC10 of the substance towards Pseudokirchneriella subcapitata were 9.5 and 5.0 mg/L based on the geometric mean measured test concentrations. The No Observed Effect Concentration was 0.95 mg/L.
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 201 and GLP. Due to the low aqueous solubility and complex nature of the test item, it was prepared as a Water Accommodated Fraction. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to the test item over a range of nominal loading rates 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. Though test vessels cannot be fully closed as oxygen is needed in algae test, it was attempted to limit the volatility of the substance. Concentrations were confirmed with a validated GC-FID method in samples taken from all treatments at t = 0, 24, 48 and 72 h. As the toxicity could not be related to one or the other constituent of Rosemarel, the total peak area was used to calculate the concentration of the substance. In addition, measured concentrations of component 2 could not be accurately obtained due to the changing chromatography of large unknown peaks present in the test samples.

The measured concentrations of the total peak area were: 0.16 -17 mg/L at 0 hours, 0.17-12 mg/L at 24 hours, 0.076-11 mg/L at 48 hours and 0.086-12 mg/L at 72 hours. The measured concentrations of component 1 were: 0.052-1.0 mg/L at 0 hours, 0.032-0.80 mg/L at 24 hours, 0.026-0.62 mg/L at 48 hours and 0.018-0.70 mg/L at 72 hours. Since the test concentration did not remain stable during the test period the geometric mean measured concentrations were calculated and used to express the endpoint. These calculations gave the 72h-ErC50 and EC10 of 9.5 mg/L and 5.0 mg/L, respectively.

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 201 and GLP. Due to the low aqueous solubility and complex nature of the test item, it was prepared as a Water Accommodated Fraction. Following preliminary range-finding tests, Pseudokirchneriella subcapitata was exposed to the test item over a range of nominal loading rates 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. Though test vessels cannot be fully closed as oxygen is needed in algae test, it was attempted to limit the volatility of the substance. Concentrations were confirmed with a validated GC-FID method in samples taken from all treatments at t = 0, 24, 48 and 72 h. As the toxicity could not be related to one or the other constituent of Rosemarel, the total peak area was used to calculate the concentration of the substance. In addition, measured concentrations of component 2 could not be accurately obtained due to the changing chromatography of large unknown peaks present in the test samples.

The measured concentrations of the total peak area were: 0.16 -17 mg/L at 0 hours, 0.17-12 mg/L at 24 hours, 0.076-11 mg/L at 48 hours and 0.086-12 mg/L at 72 hours. The measured concentrations of component 1 were: 0.052-1.0 mg/L at 0 hours, 0.032-0.80 mg/L at 24 hours, 0.026-0.62 mg/L at 48 hours and 0.018-0.70 mg/L at 72 hours. Since the test concentration did not remain stable during the test period the geometric mean measured concentrations were calculated and used to express the endpoint. These calculations gave the 72h-ErC50 and EC10 of 9.5 mg/L and 5.0 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
9.5 mg/L
EC10 or NOEC for freshwater algae:
5 mg/L

Additional information