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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 18 February to 01 March 2019
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:
OECD Guideline for testing of chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006; Annex 5 corrected July 28, 2011.
Deviations:
yes
Remarks:
Although the pH level in the control varied by more than 1.5 units at the end of the test (1.88 units of difference) this was not considered to have affected the integrity of the study.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Commission Regulation (EC) No. 440/2008 (amended by Commission Regulation (EC) No. 761/2009 and by Commission Regulation (EU) 2016/266 of 7 December 2015), Part C, C.3.: "Algal Inhibition Test", p. 464-472, Official Journal of the European Union, dated May 30, 2008, L 142:1-739.
Deviations:
yes
Remarks:
Although the pH level in the control varied by more than 1.5 units at the end of the test (1.88 units of difference) this was not considered to have affected the integrity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 29-30-31 May 2018 / Certificate signed on 15 November 2018
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
Single samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration with algae dedicated exclusively to chemical analyses) at the start and every day thereafter until the end of the test.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The mixing vessel was a cylindrical glass bottle sealed with a screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and approx. 1 L test water was added. Then an excess of the test item (approx. 500 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23.5 hours of gentle stirring in the dark at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the remaining stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary based on the measured concentration of the stock solution (≈ 140 mg.L-1) and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells.mL-1 per vessel. At the start of the test, the test solutions in test vessels were observed to be clear and colourless.
- Eluate: Original medium of OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 3 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 150 mg.L-1, according to the study plan.
- Differential loading: Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to test water and inoculum to obtain the following nominal concentrations (spaced by a factor of approx. 1.80): 1.0, 1.8, 3.2, 5.8, 10.5 and 18.9 mg.L-1.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): At the start of the test, the test solutions in test vessels were observed to be clear and colourless.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata, CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
The temperature in the incubator was situated between 22.0 and 22.8°C throughout the test (average value: 22.7°C),
pH:
From 7.79 to 9.92
See table in "Any other information on results incl. tables".
Salinity:
Not applicable
Nominal and measured concentrations:
- Nominal concentrations: 1.0, 1.8, 3.2, 5.8, 10.5 and 18.9 mg/L
- Geometric mean measured concentrations after 48h: 1.01, 1.70, 3.33, 6.17, 11.44 and 19.17 mg/L
- Geometric mean measured concentrations after 72h: 0.64, 1.32, 0.66, 3.01, 10.60 and 18.10 mg/L
See table in "Any other information on results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, filled with 50 mL of test solution. This volume was lower than the one used for the range-finding test, where test vessels were completely filled (with minimum headspace), in order to reduce the algal density and thus minimise test item losses by algal adsorption/metabolism especially at the (non-toxic) lowest concentrations.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: see above
- Initial cells density: 5000 algal cells.mL-1
- Control end cells density: See table in "Any other information on results incl. tables".
- 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: Original medium from OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous illumination
- Light intensity and quality: The mean light intensity was of 5993 lux (range: 5605-6527 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in one vessel per concentration and the control.
- Temperature of medium: Measured continuously in the growth chamber, over the study period.
- Light intensity: Light intensity was measured once (t=0h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 1.80
- Range finding study: A range-finding test was conducted to determine the range of concentrations for the definitive test. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated stock solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L test water was added. Then an excess of the test item (approx. 500 mg) was carefully added directly to the surface of the test water. The mixing vessel was thereafter closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22.5 hours of gentle stirring in the dark and at room temperature, the contents of the vessel was allowed to stand undisturbed for at least 1 hour before use. The first 100 mL were discarded via the drain port. Samples were taken from the saturated stock solution and chemically analysed. Then the stock solution was diluted with test water as necessary (based on the measured concentration of the stock solution: 117.78 mg.L-1) and a fixed amount of inoculum to obtain the required test concentrations in the test vessels and a cell density of 5.103 cells.mL-1 per vessel. The test vessels were completely filled (with minimum headspace) and were closed with ground glass stoppers. At the start of the test, test solutions in test vessels were observed to be clear and colourless at all test concentrations. The test was carried out without adjustment of the pH. In this range-finding test, algal cells were exposed to a series of nominal test concentrations of 0.5, 1.0, 10.0 and 50.0 mg test item.L-1, and to a control. The test system was maintained in a temperature controlled cabinet (23°C ± 2°C) with continuous illumination for a period of 72 hours. See results of the range-finding test in "Any other information on results incl. tables".
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
7.218 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 5.972-8.685 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.669 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 1.041-2.296 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.022 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 7.062-9.105 mg/L
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
4.511 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 3.203-5.415 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.33 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Specific analyses of samples taken from all test concentrations at the start and the end of the test revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.
Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour ErC10 and ErC50 values were determined to be 1.669 and 7.218 mg.L-1, respectively. Since the 72-hour NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined in order to have a complete set NOEC/EC10/EC50 (validity criteria of the study at t=48h were also respected). The 48-hour ErC10 and ErC50 values were determined to be 4.511 and 8.022 mg.L-1, respectively. The 48-hour NOEC value for growth rate was 3.330 mg.L-1.
Results with reference substance (positive control):
On December 18, 2018 (most recent test), the 72h-EC50 was 0.955 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (expected 72h-ErC50: 0.92 mg.L-1 to 1.46 mg.L-1).
Reported statistics and error estimates:
The software ToxRat® Professional version 3.3 was used to perform statistical analyses.

Table 6.1.5/1: pH-values during the final test

 

Nominal concentration

and corresponding measured concentration (geometric mean)

after 48h and

after 72h

(mg test item.L-1)

Control

(0)

(0)

1.0

(1.01)

(0.64)

1.8

(1.70)

(1.32)

3.2

(3.33)

(0.66)

5.8

(6.17)

(3.01)

10.5

(11.44)

(10.60)

18.9

(19.17)

(18.10)

Start t=0h

8.04

8.03

8.17

8.17

8.11

7.99

8.00

End t=72h

9.92

9.80

9.74

9.48

8.92

7.91

7.79

Although the pH level in the control varied by more than 1.5 units at the end of the test (1.88 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the additional sodium bicarbonate.

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells.mL-1x104).

 

Replicate

Nominal concentration

and corresponding measured concentration (geometric mean)

after 48h and

after 72h

(mg test item.L-1)

Control

(0)

(0)

1.0

(1.01)

(0.64)

1.8

(1.70)

(1.32)

3.2

(3.33)

(0.66)

5.8

(6.17)

(3.01)

10.5

(11.44)

(10.60)

18.9

(19.17)

(18.10)

t=24h

1

2.8

1.6

3.2

1.2

2.8

0.4

1.6

2

4.4

5.6

4.8

2.8

1.6

0.8

1.2

3

4.4

2.4

1.6

1.6

1.6

2.0

1.6

4

2.4

 

 

 

 

 

 

5

3.2

 

 

 

 

 

 

6

2.8

 

 

 

 

 

 

Mean

3.3

3.2

3.2

1.9

2.0

1.1

1.5

Std. Dev.

0.86

2.12

1.60

0.83

0.69

0.83

0.23

t=48h

1

9.6

13.6

14.0

11.2

4.8

0.8

0.4

2

15.2

8.4

12.8

8.4

5.6

0.4

0.8

3

12.4

10.0

15.6

8.4

6.0

2.0

0.4

4

11.6

 

 

 

 

 

 

5

11.6

 

 

 

 

 

 

6

14.0

 

 

 

 

 

 

Mean

12.4

10.7

14.1

9.3

5.5

1.1

0.5

Std. Dev.

1.98

2.66

1.40

1.62

0.61

0.83

0.23

t=72h

1

37.2

29.2

31.2

24.0

16.4

3.2

0.8

2

41.6

25.2

31.2

19.2

14.8

2.8

1.2

3

42.8

31.2

30.0

20.8

17.2

3.6

0.8

4

36.0

 

 

 

 

 

 

5

36.8

 

 

 

 

 

 

6

40.8

 

 

 

 

 

 

Mean

39.2

28.5

30.8

21.3

16.1

3.2

0.9

Std. Dev.

2.87

3.06

0.69

2.44

1.22

0.40

0.23

At test start 5000 algal cells.mL-1were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

Std. Dev.: standard deviation.

Table 6.1.5/3: Mean specific growth rate in P. subcapitata during the final test.

 

Replicate

Nominal concentration

and corresponding measured concentration (geometric mean)

after 48h and

after 72h

(mg test item.L-1)

Control

(0)

(0)

1.0

(1.01)

(0.64)

1.8

(1.70)

(1.32)

3.2

(3.33)

(0.66)

5.8

(6.17)

(3.01)

10.5

(11.44)

(10.60)

18.9

(19.17)

(18.10)

t=0h - t=24h

1

1.723

1.163

1.856

0.875

1.723

-0.233

1.163

2

2.175

2.416

2.262

1.723

1.163

0.470

0.875

3

2.175

1.569

1.163

1.163

1.163

1.386

1.163

4

1.569

 

 

 

 

 

 

5

1.856

 

 

 

 

 

 

6

1.723

 

 

 

 

 

 

Mean

1.870

1.716

1.760

1.254

1.350

0.544

1.067

Std. Dev.

0.2530

0.6392

0.5555

0.4309

0.3231

0.8073

0.1661

% Inh.

-

8.2

5.9

33.0

27.8

70.9

42.9

t=0h - t=48h

1

1.477

1.652

1.666

1.555

1.131

0.235

-0.112

2

1.707

1.411

1.621

1.411

1.208

-0.112

0.235

3

1.605

1.498

1.720

1.411

1.242

0.693

-0.112

4

1.572

 

 

 

 

 

 

5

1.572

 

 

 

 

 

 

6

1.666

 

 

 

 

 

 

Mean

1.600

1.520

1.669

1.459

1.194

0.272

0.004

Std. Dev.

0.0806

0.1220

0.0495

0.0830

0.0571

0.4036

0.2001

% Inh.

-

5.0

-4.3

8.8

25.4

83.0

99.8

t=0h - t=72h

1

1.436

1.356

1.378

1.290

1.163

0.619

0.157

2

1.474

1.307

1.378

1.216

1.129

0.574

0.292

3

1.483

1.378

1.365

1.243

1.179

0.658

0.157

4

1.426

 

 

 

 

 

 

5

1.433

 

 

 

 

 

 

6

1.467

 

 

 

 

 

 

Mean

1.453

1.347

1.373

1.250

1.157

0.617

0.202

Std. Dev.

0.0244

0.0364

0.0075

0.0377

0.0256

0.0419

0.0780

% Inh.

-

7.3

5.5

14.0

20.4

57.5

86.1

Values were extracted from the computer program ToxRat

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat

Table 6.1.5/4: Concentration of the test substance in test water - Final test

Nominal

concentration

(mg.L-1)

Measured concentration (mg.L-1)

Relative loss to initial value

(% initial)

Geometric mean measured concentrations

 

t=24h

t=48h

t=72h

t=0h

mg.L-1

%

nominal

 

 

(t=0h - t=48h)

(t=0h - t=72h)

(t=0h - t=48h)

(t=0h - t=72h)

(t=0h - t=48h)

(t=0h - t=72h)

 

Control

Abs.

Abs.

Abs.

Abs.

N.A.

N.A.

N.A.

N.A.

N.A.

N.A.

 

1.0

1.43

0.98

0.74

0.16

48

89

1.01

0.64

101

64

 

1.8

2.20

1.63

1.37

0.62

38

72

1.70

1.32

94

73

 

3.2

4.03

3.15

2.90

 Abs.*

28

100

3.33

0.66

104

21

 

5.8

7.60

6.17

5.02

0.35

34

95

6.17

3.01

106

52

 

10.5

13.80

10.33

10.50

8.44

24

39

11.44

10.60

109

101

 

18.9

21.42

19.02

17.28

15.23

19

29

19.17

18.10

101

96

 

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.02 mg.L-1) and the LOD (0.01 mg.L-1); “Abs.” values were considered to be the half of the LOD (=0.005 mg.L-1) for geometric means calculations.

Table 6.1.5/5: Algal cell densities during the range-ginding test (expressed as density of algal cells.mL-1x104).

 

Replicate

Nominal concentration

 (mg test item.L-1)

Control

0.5

1.0

10.0

50.0

t=24h

1

7.2

6.8

3.2

0.8

0

2

5.2

5.2

2.8

2.0

0.4

3

2.0

4.4

5.2

0.8

0

Mean

4.8

5.5

3.7

1.2

0.1

Std. Dev.

2.62

1.22

1.29

0.69

0.23

% Inh. GR

-

-11.4

7.4

63.6

153.8

% Red. Y

-

-15.5

24.8

83.7

107.0

t=48h

1

27.6

30.0

32.0

6.0

0.8

2

31.2

24.0

25.2

5.2

0.4

3

32.0

23.6

26.4

4.0

0.4

Mean

30.3

25.9

27.9

5.1

0.5

Std. Dev.

2.34

3.59

3.63

1.01

0.23

% Inh. GR

-

3.9

2.1

43.9

99.8

% Red. Y

-

14.8

8.1

84.7

99.9

t=72h

1

75.2

86.4

80.0

58.4

0

2

87.6

63.6

71.6

43.6

0.4

3

82.8

84.0

76.8

48.4

0.8

Mean

81.9

78.0

76.1

50.1

0.4

Std. Dev.

6.25

12.53

4.24

7.55

0.4

% Inh. GR

-

1.1

1.4

9.7

108.9

% Red. Y

-

4.8

7.0

39.0

100.1

At test start 5000 algal cells.mL-1were incubated; 3 replicates of the controls and 3 replicates of each testconcentration.

% Inh. GR: %Inhibition of growth rate relative to the control determined by ToxRat; % Red. Y: %Reduction in yield relative to the control determined by ToxRat; Std. Dev.: standard deviation.

Table 6.1.5/6: Concentrations of the test item in test water (biotic samples) - Range-finding test

Nominal

concentration

(mg.L-1)

Measured concentration(mg.L-1)

Relative loss to initial value

(t=0h - t=72h) (%)

Geometric mean measured concentrations

Start

(t=0h)

End

(t=72h)

mg.L-1

%

nominal

Control

Abs.

Abs.

N.A.

N.A.

N.A.

0.5

0.79

Abs.

100

0.06

12

1.0

1.54

Abs.

100

0.09

9

 

10.0

12.62

Abs.

100

0.25

3

 

50.0

73.75

53.19

28

62.63

125

 

N.A.: not applicable

% = Percent of expected nominal concentration in test item.

Abs.= Absence: concentrations below the LOQ (0.02 mg.L-1) and the LOD (0.01 mg.L-1); “Abs.” values were considered to be the half of the LOD (=0.005 mg.L-1) for geometric means calculations.

Validity criteria fulfilled:
yes
Remarks:
See "Overall remarks"
Conclusions:
Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour ErC10 and ErC50 values were determined to be 1.669 and 7.218 mg/L, respectively.
Executive summary:

A study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to an aqueous solutionof the test item over 72 hours at the required nominal test concentrations 1.0, 1.8, 3.2, 5.8, 10.5 and 18.9 mg/L. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Samples taken from the control and all test concentrations were analysed at the start and every day thereafter until the end of the test in order to determine if concentrations of the test item were maintained.

Specific analyses of samples taken from all test concentrations at the start and the end of the test revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations.

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test. Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 72-hour ErC10 and ErC50 values were determined to be 1.669 and 7.218 mg/L, respectively. Since the 72-hour NOEC value for growth rate could not be determined, the endpoint values at t=48h were also determined in order to have a complete set NOEC/EC10/EC50. Indeed, according to OECD 201 section 11, it is possible to shorten the test period to 48 hours whenever unlimited, exponential growth during the test has been obtained. This corresponds to at least a 16-fold growth in controls. Since the cell density in the control cultures increased by a factor of 25 within 48 hours exposure period, the results can also be provided after 48h according to OECD 201, section 11 (other validity criteria of the study at t=48h were also respected). Under the experimental conditions and based on the geometric means of measured exposure concentrations, the 48-hour ErC10 and ErC50 values were determined to be 4.511 and 8.022 mg/L, respectively. The 48-hour NOEC value for growth rate was 3.330 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
From 22 June 2012 to 17 July 2012
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
This study was performed according to OECD Guideline 201, EU Method C3 with GLP statement. As the mean Coefficient of Variation (CV) for section by section daily growth rates in control cultures was 61.7% from 0 to 72 hours (which exceeded the validity criteria of 35%) and as the measured concentrations after 72 hours had decreased less than the limit of detection at the critical concentrations, only the counts to 48 hours have been used to determine the endpoint values. However, even if the mean CV for section by section daily growth rates in control cultures showed a lower value from 0 to 48 hours, the validity criteria was not fulfilled (53.4%, so also >35%). These substantial differences between the section by section growth rate and the average growth rate indicate deviation from constant exponential growth. In addition, a severe loss of substance was observed during the test due to lactone metabolization by the high algae population between 24 hours (where the concentration was maintained) and 48 hours (where it dropped dramatically). It's assumed that this one very low value at 48 hours dropped the geometric mean of the EC10/NOEC < 1 mg/L. Finally, to verify the endpoint values, the ECx/NOEC/LOEC values were recalculated by the assessor with ToxRat Professional v2.10 and different results were obtained.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
, the mean coefficient for daily growth rates in the control cultures exceed 35%.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
, see deviation for OECD Guideline.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
17-08-2011
Specific details on test material used for the study:
- Physical state: Transparent clear to pale yellow liquid
- Storage condition of test material: Room temperature in dark
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the start of the definitive test, two samples (20 mL) were taken for the freshly-prepared control and test media. After 24, 48 and 72 hours, aliquots were removed from the replicate flasks for each group were pooled and samples (20 mL) of the pooled medium were taken for analysis. Additionally at 24, 48 and 72 hours, samples were taken from flasks containing the test substance at 0.763 and 50 mg/L but without algal cells, in order to obtain information on the extent of adsorption/absorption of the test substance by algal cells. All samples were added to vials containing hexane (10 mL) to facilitate the extraction process.
- Sample storage conditions before analysis: On each occasion, one of the samples was analysed and the other was stored in a freezer in case further analysis was required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance (100 mg) were dissolved in culture medium (2 L) in a volumetric flask and stirred in the dark at 2 hours. The contents of the flask were either used directly as the test medium (50 mg/L) or aliquots were diluted in culture medium to provide the test medium at the lower concentrations of 15.6, 7.81, 2.44 and 0.763 mg/L.
- Eluate: Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Guideline 201.
- Controls: 1
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Axenic, uni-cellular, liquid slope cultures of algae were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland and arrived on 21 March 2012.
- Age of inoculum (at test initiation): not applicable
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25°C. Subsequently, appropriate volumes of these primary cultures were aseptically transferred to fresh sterile algal nutrient medium to prepare secondary liquid cultures; these cultures were incubated, as stated above, for a further three days to provide an inoculum in the log phase of growth, characterised by a cell density of 8.35 x 10^5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
50 mg/L as NaHCO3.
Test temperature:
Measured initial and final temperatures were between 21.6 and 22.0°C.
The temperature ofthe incubator ranged between 22.1 and 23.1°C.
pH:
During the 72h exposure period, the pH of the control culture varied between 7.76 and 8.11 and the pH of the test flasks varied between 7.72 and 8.10.
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
See table 6.1.5/1 in "Any other information on results incl. tables".
Based on a geometric mean (which excluded measured concentrations at 72 hours due to abnormalities in cell densities and the considerable loss of substance at the time point), the overall mean measured levels of the test substance were 0.144, 0.779, 5.23, 11.9 and 39.8 mg/L. These values have been used in the calculation of results.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks (250 mL)
- Material, size, headspace, fill volume: Test vessels containing 150 mL control or test cultures
- Aeration: yes (gaseous exchange)
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 1.0 x 10^4 cells/mL
- Control end cells density: See table 6.1.5/2 in "Any other information on results incl. tables".
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable
Before the start of the test, the required number of empty test vessels were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (150 mL), each flask was then loosely plugged with a foam bung.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Filtered, dechlorinated tap water which had been softened and treated by reverse osmosis, before microfiltration and purification (resistivity of 18 Megohm/cm).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination at temperatures maintained at 23 +/- 2°C.
- Light intensity and quality: illuminated orbital incubator (4440 to 8880 Lux). Illumination provided by 6 x 30 W "cool white" 1 meter flurorescent tubes. To minimise the impact of differences in light intensity across the test area on algal growth, control and test flasks were re-positioned in the test area each day during the definitive test.
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber (Coulter Z Series Particle Count and Size Analyser)
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 0.763, 2.44, 7.81, 15.6 and 50.0 mg/L (nominal)
- Range finding study: yes (at 0.1, 1.0, 10 and 100 mg/L)
- Results used to determine the conditions for the definitive study: After 72 hours, algal growth was inhibited by 17, 27 and 98% respectively at 0.1, 10 and 100 mg/L, no inhibition occurred at 1.0 mg/L.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
5.94 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value reported in the study report. 95% CL: 4.65-7.44 mg/L.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
6.418 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value recalculated with ToxRat Professional v2.10. 95% CL: 5.625-7.260 mg/L (see attached ToxRat report).
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.876 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value reported in the study report. 95% CL: 0.462-1.56 mg/L.
Key result
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value recalculated with ToxRat Professional v2.10. 95% CL: 0.680-1.318 mg/L (see attached ToxRat report).
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.779 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value reported in the study report and confirmed by the assessor with ToxRat Professional v2.10
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
5.23 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Endpoint value reported in the study report and confirmed by the assessor with ToxRat Professional v2.10
Details on results:
Although the study was conducted to 72 hours, only the counts to 48 hours have been used to determine EC10, EC50, NOEC and LOEC values. This was considered to be a more relevant assessment of the study. The 48-hour values were more representative due to abnormally inconsistent cell counts between nominal concentrations of 7.81 and 15.6 mg/L at 72 hours; where cell counts at 7.81 mg/L where lower than those at 15.6 mg/L. In addition, measured concentrations of the test material at 48 hours were still within detectable levels at the critical concentrations for the NOEC, LOEC and ECx values (nominal 2.44 to 7.81 mg/L).

The test substance at 0.763 and 50.0 mg/L, which had been incubated without algal cells, had higher measured levels (except on a single occasion; 50 mg/L at 48 hours) when compared to samples at the same concentrations incubated in the presence of algal cells. This result indicated that the presence of agal cells may have affected the stability of the test substance.

The mean coefficient for daily growth rates were 61.7% including days 2-3 or 53.4% excluding days 2-3, which exceeded the value specified in the protocol (35%).
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

Table 6.1.5/1: Measured concentrations

Nominal concentrations (mg/L)

Measured test substance concentrations (mg/L)

Overall mean ** (mg/L)

0h

%N

24h

%N

48h

%N

%t0

72h*

%N

Control

0.763

0.763A

2.44

7.81

15.6

50.0

50.0A

nd

0.647

-

1.96

5.64

11.5

35.7

-

-

85

-

80

72

74

71

-

nd

0.464

0.679

1.80

5.88

13.2

38.4

43.9

-

61

89

74

75

85

77

88

nd

nd1

0.819

0.134

4.31

11.0

46.1

40.6

-

1

107

5

55

71

92

81

-

2

127

7

76

96

129

114

nd

nd

0.230

nd

0.747

2.19

26.3

35.4

-

1

30

0

10

14

53

71

nd

0.144

0.711

0.779

5.23

11.9

39.8

39.9

nd: none detected (less than the limit of detection of 0.02 mg/L).

%N: % of the nominal concentration.

%t0: % of the initial concentration.

A: culture medium incubated under test conditions without algal cells.

1: applied as 0.01 mg/L, half limit of detection, for geometric mean calculation.

*: 72 -hour measured concentrations have not been included in determination of the mean measured concentrations.

**: Geometric mean excluding 72 hour values.

Table 6.1.5/2: Cell densities

Measured Concentration (mg/L)

Cell densities (cells/mL)

0h

24h

48h

72h*

Control

R1

10000

60050

192567

324533

R2

68983

205167

356467

R3

65683

149867

277967

R4

68650

146867

342833

R5

67017

126500

237467

R6

70650

147700

253633

Mean

10000

66839

161444

298817

0.144

R1

10000

76383

237900

360900

R2

72617

228733

384600

R3

69750

231800

406533

Mean

10000

72917

232811

384011

0.779

R1

10000

48183

134767

225267

R2

44317

120167

174800

R3

58150

149400

283133

Mean

10000

50217

134778

227733

5.23

R1

10000

13683

48833

73367

R2

14450

38833

51700

R3

16083

56567

81867

Mean

10000

14739

48078

68978

11.9

R1

10000

15383

23367

139400

R2

13883

24167

96100

R3

12317

22467

54633

Mean

10000

13861

23333

96711

39.8

R1

10000

21050

14933

13800

R2

11317

15233

18933

R3

9583

14000

19100

Mean

10000

13983

14722

17278

R1 -R6: replicate number.

*: 72 -hour counts have not been included in the determination of the study endpoints.

Validity criteria fulfilled:
no
Remarks:
The mean coefficient of variation for section by section daily growth rates in control cultures was 53.4% at 48h and 61.7% at 72h. The other validity criteria were met.
Conclusions:
Although the study was conducted to 72 hours, only the counts to 48 hours have been used to determine endpoints values. After 48h of exposure to the test substance, the ErC10 and ErC50 values were determined at 0.990 mg/L and 6.418 mg/L, respectively (recalculated with ToxRat Professional v2.10). The NOEC for growth rate was 0.779 mg/L.
Executive summary:

This study was performed according to EU method C3 and OECD guideline 201 with GLP statement to assess the effect of the test substance on the growth of the unicellular green alga Pseudokirchneriella subcapitata.

Triplicate algal cultures, with an initial cell density of 1.0 x 10^4/mL were exposed to the test substance dissolved in algal nutrient medium at nominal concentrations of 0.763, 2.44, 7.81, 15.6 and 50 mg/L. The test media were prepared in OECD medium from an aqueous stock solution, which was made by the direct addition of the test substance to the dilution medium. The cultures were incubated in an orbital incubator under continuous illumination at temperatures ranging between 22.1 and 23.1°C for 72 hours.

At the start of the test, the measured levels of the test substance in samples of the test cultures ranged between 71 and 85% of their nominal values and were maintained fro the initial 24 hours of the test (between 72 and 115% of initial values). After 48 hours concentrations had decreased with the lowest dose being below the limit of detection of the analytical method, at the other concentrations, measure levels ranged between 5% and 92% of their nominal values with the greatest losses occurring at the lower levels. At 72 hours, measured concentrations had decreased further in all test groups, with values ranging between less than the limit of detection to 14% of their nominal values, except at 50.0 mg/L which was 53% of its nominal value.

Based on a geometric mean (which excluded measured concentrations at 72 hours due to abnormalities in cell densities and the considerable loss of substance at the time point), the overall mean measured levels of the test substance were 0.144, 0.779, 5.23, 11.9 and 39.8 mg/L. These values have been used in the calculation of results.

Cell numbers were counted daily to monitor growth.

All validity criteria were not fulfilled. For the control culture, the biomass increased by a factor of 16.1 and 29.9 respectively within the 48h and 72h test period, and the mean coefficient of variation (CV) for the average specific growth rates was 4.96% and 6.65% respectively calculated for 72h and 48h exposure periods. However, the mean CV for section by section daily growth rates in control cultures was 53.4% at 48 hours and 61.7% at 72 hours. As this latest value at 72 hours was unacceptable and as the measured concentrations after 72 hours had decreased less than the limit of detection at the critical concentrations, only the counts to 48 hours have been used to determine the endpoint values. However, even if the mean CV for section by section daily growth rates in control cultures showed a lower value from 0 to 48 hours, the validity criteria was not fulfilled (53.4%, so also >35%). These substantial differences between the section by section growth rate and the average growth rate indicate deviation from constant exponential growth. In addition, a severe loss of substance was observed during the test due to lactone metabolization by the high algae population between 24 hours (where the concentration was maintained) and 48 hours (where it dropped dramatically). It's assumed that this one very low value at 48 hours dropped the geometric mean of the EC10/NOEC < 1 mg/L. Finally, to verify the endpoint values, the ECx/NOEC/LOEC values were recalculated by the assessor with ToxRat Professional v2.10 and different results were obtained.

In conclusion, after 48h of exposure to the test substance, the recalculated ErC10 and ErC50 values were 0.990 mg/L and 6.418 mg/L, respectively. The NOEC for growth rate was 0.779 mg/L. For all reasons presented above, this study is considered invalid.

Description of key information

OECD Guideline 201, EU Method C.2, GLP, key study, validity 1:

72h-ErC10 (Pseudokirchneriella subcapitata) = 1.669 mg/L based on geometric mean measured concentrations.

72h-ErC50 (Pseudokirchneriella subcapitata) = 7.218 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
7.218 mg/L
EC10 or NOEC for freshwater algae:
1.669 mg/L

Additional information

One valid key study, performed in 2019, is available to assess the toxicity of the registered substance to the growth of the unicellular algae species Pseudokirchneriella subcapitata, according to OECD Guideline 201 and EU Method C.3 with GLP compliance (LPL, 2019). Following a preliminary range-finding test, algal cells (with an initial cell density of 5000 algal cells/mL) were exposed to an aqueous solution of the registered substance over 72 hours at the required nominal test concentrations of 1.0, 1.8, 3.2, 5.8, 10.5 and 18.9 mg/L, in closed and static conditions. In addition, according to the results obtained in the prevous study (HLS, 2012), presented below, and the preliminary range-finding test in the present study, the test conditions were adapted in order to reduce the algal density and thus minimise test item losses by algal adsorption/metabolism especially at the (non-toxc) lowest concentrations. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Specific analyses of samples taken from all test concentrations at the start and the end of the test revealed unavoidable test item losses. These decreases were inversely proportional to the tested concentrations, suggesting potential adsorption/metabolism effects by algae at the (non-toxic) lowest concentrations, as expected. Since the deviation of the exposure concentrations of the test substance was greater than ± 20% of the initial concentrations, the results were based on the geometric means of measured exposure concentrations, corresponding to 0.64, 1.32, 0.66, 3.01, 10.60 and 18.10 mg/L, respectively, after 72 hours. All validity criteria were fulfilled. According to the results of this test, the 72-hour ErC10 and ErC50 values were determined to be 1.669 and 7.218 mg/L, respectively.

Another study is available (HLS, 2012). This study, performed in 2012, was also conducted on the registered substance according to OECD Guideline 201 and EU Method C.3 with GLP compliance to assess the effect of the substance on the growth of the unicellular green alga Pseudokirchneriella subcapitata. In this study, algal cells (with an initial cell density of 10000 algal cells/mL) were exposed to an aqueous solution of the registered substance over 72 hours at the required nominal test concentrations of 0.763, 2.44, 7.81, 15.6 and 50 mg/L, in open and static conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. At the start of the test, the measured levels of the test substance in samples of the test cultures ranged between 71 and 85% of their nominal values and were maintained for the initial 24 hours of the test (between 72 and 115% of initial values). After 48 hours of exposure, concentrations had decreased with the lowest dose being below the limit of detection of the analytical method, at the other concentrations, measure levels ranged between 5% and 92% of their nominal values with the greatest losses occurring at the lower levels. At 72 hours of exposure, measured concentrations had decreased further in all test groups, with values ranging between less than the limit of detection to 14% of their nominal values, except at 50.0 mg/L which was 53% of its nominal value. Based on a geometric mean (which excluded measured concentrations at 72 hours due to abnormalities in cell densities and the considerable loss of substance at the time point), the overall mean measured levels of the test substance were 0.144, 0.779, 5.23, 11.9 and 39.8 mg/L. These values have been used in the calculation of results. All validity criteria were not fulfilled. For the control cultures, the biomass increased by a factor of 16.1 and 29.9 respectively within the 48h and 72h test period, and the mean coefficient of variation (CV) for the average specific growth rates was 4.96% and 6.65% respectively calculated for 72h and 48h exposure periods. However, the mean CV for section by section daily growth rates in control cultures was 53.4% at 48 hours and 61.7% at 72 hours. As this latest value at 72 hours was unacceptable and as the measured concentrations after 72 hours had decreased less than the limit of detection at the critical concentrations, only the counts to 48 hours have been used to determine the endpoint values. However, even if the mean CV for section by section daily growth rates in control cultures showed a lower value from 0 to 48 hours, the validity criteria was not fulfilled (53.4%, so also >35%). These substantial differences between the section by section growth rate and the average growth rate indicate deviation from constant exponential growth. In addition, a severe loss of substance was observed during the test due to lactone metabolization by the high algae population between 24 hours (where the concentration was maintained) and 48 hours (where it dropped dramatically). It's assumed that this one very low value at 48 hours dropped the geometric mean of the EC10 < 1 mg/L. Indeed, after 48 hours of exposure, the ErC10 and ErC50 values were determined in the study report to be 0.876 mg/L and 5.94 mg/L, respectively. The endpoint values were recalculated by the assessor with ToxRat Professional v2.10 and different results were obtained: 48h-ErC10 = 0.990 mg/L (so close to the critical value 1.0 mg/L for classification); 48h-ErC50 = 6.418 mg/L. For all reasons presented above, this study is considered invalid.

In conclusion, according to the valid key study (LPL, 2019), the 72-hour ErC10 and ErC50 values, retained as key values, were 1.669 and 7.218 mg/L, respectively.