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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:
28-08-2018 to 31-08-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2016 ; signature: 2017
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS at the start (0 hours), 24 hours and at the end of the exposure (72 hours).
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS from freshly prepared media after 0 hours (with algae), 24, 48 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae (and without algae, respectively). The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at room temperature, until analysis if necessary. Prepared samples were stored in an autosampler at room temperature until analysis.
Vehicle:
no
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDAK CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA, Scotland, UK
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.

ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water – OECD TG 201 medium
- Any deformed or abnormal cells observed: None reported.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 23.0 ; max 24.0 and mean 23.5 °C
pH:
0 hours: pH 8.1 ± 0.1 (and 8.14 in control); 72 hours: pH 8.21-9.36 (definitive test concentrations) and pH 9.32 (controls). pH did not vary more than 1.5 units.
Nominal and measured concentrations:
- Preliminary test: 0 (control), 1.0, 10.0 and 100% saturated solution (non-GLP range finding test)
- Final test: 0 (control), 0.842, 2.19, 5.69, 14.8, 38.5 and 100% saturated solution
- Equivalent measured time weighted average concentrations (without algae): 0 (control), 0.0643, 0.264, 0.672, 0.781, 1.78, 10.1 mg/L
- Measured concentrations of the test item were in the samples without algae in the range of 0.148 to 14.4 mg/L at the start of exposure (0 h) and in the range of 6 to 89% of the initially test item concentrations at the end of exposure (72 h). Samples with algae gave results of 0.153 to 14.4 mg/L at the start of exposure (0 h) and in the range of 3 to 56% of the initially test item concentrations at the end of exposure (72 h).
All effect values given were based on time weighted average test item concentration after 72 hours for samples.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 6442 cells/ml
- Control end cells density: Mean (of replicates after 72 hours) 519354 cells/ml (or ca. x81 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.

GROWTH MEDIUM
- Standard medium used: Yes. OECD TG 201 medium. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition).
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.

OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area
- Salinity (for marine algae): Not applicable.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the highest preliminary test concentration level of 100% saturated solution.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.6. In definitive test justified from the results of the range finding study.
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: Yes.
- Test concentrations: Three replicates per concentration were exposed to dilutions representing: 0 (control), 1.0, 10.0 and 100% saturated solution
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.811 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I.: 0.774 - 0.893 mg/L ; based on time weighted average concentrations
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.453 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.I.: 0.300 - 0.557 ; based on time weighted average concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.264 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.344 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% C.I.: 0.316 - 0.373 ; based on time weighted average concentrations
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.127 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% C.I.: 0.104 - 0.150 ; based on time weighted average concentrations
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.064 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels:
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: Low dose stimulation was observed in the definitive test at 0.0643 mg/L time weighted average concentration only.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: No.
Results with reference substance (positive control):
- Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.09 mg/L with a 95% confidence interval ranging from 0.842 to 1.31 mg/L with headspace and 0.244 mg/L with a 95% confidence interval ranging from 0.175 to 0.350 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.548 mg/L with a 95% confidence interval ranging from 0.439 to 0.660 mg/L with headspace and 0.236 mg/L with a 95% confidence interval ranging from 0.175 to 0.350 mg/L
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data.
Reported statistics and error estimates:
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using Williams Sequential t-test Procedure. A Shapiro-Wilk's Normality test and a Levene's Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value
(acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.01

Table 1. Results of the Preliminary Range Finding Test (non GLP, 0 - 72 hours)

Saturated solution
[%]

Growth Rate Inhibition
[%]

Yield Inhibition
[%]

100

94

100

 10.0

18

52

  1.00

10

34

  0.100

20

55

 

Table 2. Percentage reduction in growth rate and inhibition of yield in the definitive test (at 72 hours)

Time weighted average test item concentration

Replicate

Growth rate

Growth rate inhibition

Yield

Yield

Inhibition

[mg/L]

No.

[d-1]

[%]

[d-1]

[%]

10.1

1

n.a.

100

n.a.

100

2

n.a.

100

n.a.

100

3

n.a.

100

n.a.

100

Mean

(+)

n.a.

100

(+)

n.a.

100

 1.78

1

-0.016

100

-298

100

2

-0.014

100

-263

100

3

 

-0.027

100

-501

100

Mean

(+)

-0.019

100

(+)

-354

100

 0.781

1

0.934

36

99752

81

2

0.803

45

65245

87

3

0.660

55

40212

92

Mean

(+)

0.799

45

(+)

68403

87

 0.672

1

1.02

30

131826

74

2

1.01

31

126126

75

3

1.02

30

130922

74

Mean

(+)

1.02

31

(+)

129625

75

 0.264

1

1.30

11

307274

40

2

1.31

10

324726

37

3

1.31

10

325123

37

Mean

(-)

1.31

11

(+)

319041

38

 0.0643

1

1.47

-1

524369

-2

2

1.47

-1

525610

-2

3

1.46

1

499717

3

Mean

(-)

1.47

0

(-)

516565

-1

Control

1

1.47

 

 

523847

 

2

1.45

 

 

487851

 

3

1.47

 

 

521911

 

4

1.46

 

 

511236

 

5

1.46

 

 

511435

 

6

1.47

 

 

521191

 

Mean

1.46

 

 

512912

 

Negative inhibition = increase in growth

n.a. = data not determinable

Validity criteria fulfilled:
yes
Conclusions:
The test item 72h-EC50 (growth rate reduction) was 0.811 (C.I. 0.774 – 0.893) mg/L based on time weighted average concentrations. The corresponding EC10 was 0.453 (C.I. 0.300 – 0.557) mg/L and the NOEC was 0.264 mg/L.
Executive summary:

The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL.A saturated solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water one day prior to the start of exposure. The saturated solution was stirred for 24 ± 1 hour (1100 rpm, room temperature). After completion of stirring, the test item solution was allowed to stand for 1 hour for separation of the phases. Thereafter, the saturated solution was removed by siphoning from the approximate bottom of the glass flask. The saturated solution was used as highest concentration level and for preparation of further dilution levels. Glass flasks without headspace were used to reduce losses of the test item. No presence of undissolved test item during preparation and during the test was detected. Six concentrations were tested in a geometrical series with a dilution factor of 2.6 (nominal): 0.842, 2.19, 5.69, 14.8, 38.5 and 100 % of the saturated solution, corresponding to the time weighted average test item concentrations after 72 hours of 0.0643, 0.264, 0.672, 0.781, 1.78, 10.1 mg/L, respectively. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS at test start, after 24 and 48 hours and the end of exposure. After 24 and 48 hours, the measured concentration were between 59 to 95 % and 15 to 67% of the initially measured concentrations, respectively. At test end the test item concentrations were between 3 to 56% of the initially measured concentrations. Time weighted average concentrations with algae were calculated to show the worst-case scenario. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) 0.811 (C.I. 0.774 – 0.893) mg/L based on time weighted average concentrations. The corresponding EC10 was 0.453 (C.I. 0.300 – 0.557) mg/L and the NOEC was 0.264 mg/L.

Description of key information

ErC50 (algae; growth rate) = 0.811 (C.I. 0.774 – 0.893) mg/L TWA concentration, 72-hour, freshwater, OECD TG 201, 2018

ErC50 (algae; growth rate) = 0.453 (C.I. 0.300 – 0.557) mg/L TWA concentration, 72-hour, freshwater, OECD TG 201, 2018

NOEC (algae; growth rate) = 0.264 mg/L TWA concentration, 72-hour, freshwater, OECD TG 201, 2018

Key value for chemical safety assessment

EC50 for freshwater algae:
0.811 mg/L
EC10 or NOEC for freshwater algae:
0.264 mg/L

Additional information

Key study: OECD TG 202, 2018 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 6442 cells/mL.A saturated solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water one day prior to the start of exposure. The saturated solution was stirred for 24 ± 1 hour (1100 rpm, room temperature). After completion of stirring, the test item solution was allowed to stand for 1 hour for separation of the phases. Thereafter, the saturated solution was removed by siphoning from the approximate bottom of the glass flask. The saturated solution was used as highest concentration level and for preparation of further dilution levels. Glass flasks without headspace were used to reduce losses of the test item. No presence of undissolved test item during preparation and during the test was detected. Six concentrations were tested in a geometrical series with a dilution factor of 2.6 (nominal): 0.842, 2.19, 5.69, 14.8, 38.5 and 100 % of the saturated solution, corresponding to the time weighted average test item concentrations after 72 hours of 0.0643, 0.264, 0.672, 0.781, 1.78, 10.1 mg/L, respectively. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS at test start, after 24 and 48 hours and the end of exposure. After 24 and 48 hours, the measured concentration were between 59 to 95 % and 15 to 67% of the initially measured concentrations, respectively. At test end the test item concentrations were between 3 to 56% of the initially measured concentrations. Time weighted average concentrations with algae were calculated to show the worst-case scenario. The validity criteria of the test guideline were fulfilled. The EC50 for growth rate reduction (72h-ErC50) 0.811 (C.I. 0.774 – 0.893) mg/L based on time weighted average concentrations. The corresponding EC10 was 0.453 (C.I. 0.300 – 0.557) mg/L and the NOEC was 0.264 mg/L.