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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:
2017-09-21 to 2019-10-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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 23 March 2006, Annex 5 corrected: 28 July 2011.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal test item loading rates of 100, 31.3, 9.77, 3.05 and 0.954 mg/L and control
- Sampling method: Analytical samples were taken and analysed from control and all test item loading rates at 0 hours (initial value) from fresh test solutions and after 72 hours from aged test solutions.
- Sample storage conditions before analysis: After receipt in the analytical laboratory the test medium samples (20 mL of each sample stabilized with 1 mL concentrated nitric acid) were stored at temperature between 1-10 °C until analysis. At the day of analysis, the samples were brought to room temperature.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Application. The following nominal test item loading rates were employed (spaced by a factor of 3.2): 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.
The test item was applied as water accommodated fraction (WAF). Stock solutions (S1, S2, S3) were prepared by directly weighing 100, 31.3 and 9.77 mg in 1000 mL test medium, respectively. These stock solutions were stirred in the dark at room temperature for 48 h (based on OECD Series on Testing and Assessment No. 23). Not the entire test item went into solution. Subsequently the undissolved test item was allowed to sediment and/or float for a period of 24 h until the phases had separated. After the settling the necessary volumes for the test were withdrawn via a Teflon tube from the medium level of the stock solution. Medium for the control was prepared in parallel and treated identically (stirred in the dark at room temperature for 48 h followed by a settling phase of 24 h). After the preparation the content of ortho-phosphate in the respective WAFs (control and all loading rates individually) was determined. lf the content of ortho-phosphate was reduced in the loading rates in comparison to the control, additional phosphate was dosed, and the ortho-phosphate content was determined again.
The test item solutions V1 and V2 were made by diluting S3 with test medium without algae to achieve the required test item loading rates. Algae were added to each solution individually, resulting in target cell densities of 0.5 >< 104 cells per mL in each solution. Approximately 50 mL of the prepared solutions were transferred to each test vessel.
- Eluate: N.a.
- Controls: AAP Medium only
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N.a.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): N.a.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
- Other relevant information: No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: semi-continuous

ACCLIMATION
- Acclimation period: Not necessary
- Culturing media and conditions (same as test or not): same as test; The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201 ). As the test item contains metal components, the content of NaZEDTA-2HZO in the employed AAP-medium was reduced to 0.100 mg/L (instead of 0.300 mg/L) as recommended by OCSPP 850.4500. The pH was adjusted to 7.5 i0.1 with NaOH and HCI.
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.4 – 23.2 °C
pH:
7.23 - 7.64
Nominal and measured concentrations:
Nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
Measured: Analytical Results: The measured initial concentrations of lutetium ranged from < LOQ (i.e. 3.5 mg lutetium/L) to 0.846 % of nominal. ln the aged samples the measured concentrations were between < LOQ and 0.520 % of nominal. Therefore, toxicological endpoints were evaluated using the nominal loading rates and the actual concentrations (based on the geometric mean of each measured concentration).
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL)
- Type (delete if not applicable): closed with aluminium caps, allowing exchange with air
- Material, size, headspace, fill volume: glass flask, 100 mL filling volume, filled with ca. 50 mL test solution
- Aeration: no; CO2 supply was ensured by continuous agitation: Test vessels were placed in an incubator on a pivoted bogie which turns around and induces shaking by regular sudden stops
- Type of flow-through (e.g. peristaltic or proportional diluter): static
- Renewal rate of test solution (frequency/flow rate): no
- Initial cells density: 5000 cells/mL
- Control end cells density: 641 600 cells/mL
- No. of organisms per vessel: N.a.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N.a.

GROWTH MEDIUM
- Standard medium used: yes; The medium used for the test was AAP-Medium (according to Annex 3 of OECD 201 ). As the test item contains metal components, the content of NaZEDTA-2HZO in the employed AAP-medium was reduced to 0.100 mg/L (instead of 0.300 mg/L) as recommended by OCSPP 850.4500.
- Detailed composition if non-standard medium was used: N.a.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: Yes, with NaOH and HCl at start of the test to 7.5 ± 0.1
- Photoperiod: continuously
- Light intensity and quality: 85.6 µEm-²s-1 (mean) at cell culture level.
- Salinity (for marine algae): N.a.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
At test start (0 hours) the number of cells in each control replicate was determined in duplicate. At defined dates (24, 48 and 72 hours), the number of cells in each replicate was determined in duplicate. The determination was performed by fluorescence measurement. The fluorescence measurements were performed with a fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0).
By the means of a calibration curve, where fluorescence signals (y axis) were plotted versus cell numbers (x axis), the cell numbers were derived from the fluorescence signals. To establish a calibration curve, the cell numbers were counted with a Neubauer chamber after preparation of a dilution series of a logarithmic growing Pseudokirchneriella subcapitata culture.
Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: N.a.
- Range finding study: Yes,
- Test concentrations: A limit test with a nominal test item loading rate of 100 mg/L was performed but was not conclusive due to statistically significant effects, thus a static test with nominal test item loading rates of 100, 31.3, 9.77, 3.05 and 0.954 mg/L and control was performed.
- Results used to determine the conditions for the definitive study: Yes


Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.189 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.002 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
other: for both: growth rate and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
other: for both: growth rate and yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
other: Yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.029 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
other: extrapolated from lutetium concentrations
Basis for effect:
other: Yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: The morphology of the algae cells was observed microscopically at test end. The cells were considered normal for the control and up to and including a nominal test item loading rate of 9.77 mg/L. No cells were observed at 31.3 and 100 mg/L nominal test item loading rate.
- Any stimulation of growth found in any treatment: no
- 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):
Significant inhibitory effects were determined for biomass integral and yield at test item concentrations of 0.800 mg/L (nominal) and above. Significant inhibitory effects were determined for growth rate at 2.00 mg/L (nominal) and above. The overall NOEC was therefore determined to be 0.320 mg/L (nominal), the corresponding LOEC was set at 0.800 mg/L (nominal).
The EC10-value for growth rate (ErC10) was determined to be 0.909 mg/L (nominal). The EC10-value for yield (EyC10) was 0.032 mg/L (nominal). The EC10-value for biomass integral (EbC10) was 0.634 mg/L (nominal).
The EC50-value for growth rate (ErC50) was determined to be 1.65 mg/L (nominal). The EC50-value for yield (EyC50) was 1.05 mg/L (nominal). The EC50-value for biomass integral (EbC50) was 1.07 mg/L (nominal).

Validity Criteria of the Study

Biomass Cell numbers, measured in the controls between 0 h and 72 hours, were found to increase by a factor of 125.80, which exceeds the threshold of 16. lt corresponds to a growth rate of 1.61336 per day

Coefficient of the mean coefficient of variation for the section-by-section specific growth: Variation rates (hours 0 - 24, 24 - 48 and 48 - 72) in the control cultures was 12 % (section by section) and did not exceed 35 %.

Coefficient of The coefficient of variation of average growth rate in replicate control: Variation cultures was 2.2 % and did not exceed 7 % for the whole test period.

 

Biological Results

Table 1: Average cell numbers for each sampling time and loading rate

Loading rate in mg/L

Average cell numbers [104/mL]

0 h

24 h

48 h

72 h

Control

0.51

2.23

13.92

64.16

0.954

0.51

2.16

13.63

62.64

3.05

0.51

1.80

9.42

44.17

9.77

0.51

1.53

6.65

31.07

31.3

0.51

1.33

4.89

20.60

100

0.51

0.89

0.59

0.32

 

Table 2: Percentage inhibition of growth rate

Loading rate in mg/L

% inhibition of growth rate

24 h

48 h

72 h

Control

0.0

0.0

0.0

0.954

2.7

0.9

0.8

3.05

14.8

12.0

7.8*

9.77

25.8

22.5

15.1*

31.3

35.5

32.6

25.0*

100

62.4

95.9

110.6*1)

1)Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

 

Table 3: Inhibition of Yield

Loading rate in mg/L

% inhibition of yield

24 h

48 h

72 h

Control

0.0

0.0

0.0

0.954

4.1

2.2

2.4 *

3.05

25.0

33.6

31.4*

9.77

40.7

54.2

52.0*

31.3

52.3

67.4

68.4*

100

77.9

99.4

100.3*1)

1)Value was set to 100 for EC10, 20, 50-calculation

* Statistically significant different to the control

 

Table 4: Individual cell numbers

Loading rate in mg/L

Cell numbers [104/mL]

Yield

0 h

24 h

 48 h

72 h

0-72 h

Control

0.54

2.33

14.29

62.41

61.87

0.50

2.23

14.59

61.96

61.46

0.53

2.29

13.79

60.66

60.13

0.46

2.20

13.53

67.24

66.78

0.56

2.14

13.62

66.97

66.41

0.46

2.18

13.72

65.72

65.26

Mean

0.511)

2.23

13.92

64.16

63.65

0.954

0.51

2.26

12.75

55.33

54.82

0.51

1.91

13.23

59.59

59.08

0.51

2.31

14.90

73.00

72.49

Mean

0.51

2.16

13.63

62.64

62.13

3.05

0.51

1.80

9.37

43.87

43.36

0.51

1.81

9.10

43.78

43.27

0.51

1.79

9.80

44.85

44.34

Mean

0.51

1.80

9.42

44.17

43.66

9.77

0.51

1.56

6.21

28.80

28.29

0.51

1.50

7.04

33.31

32.80

0.51

1.53

6.71

31.11

30.60

Mean

0.51

1.53

6.65

31.07

30.56

31.3

0.51

1.34

5.51

24.43

23.92

0.51

1.38

5.81

26.35

25.84

0.51

1.26

3.35

11.03

10.52

Mean

0.51

1.33

4.89

20.60

20.09

100

0.51

0.94

0.50

0.22

-0.29

0.51

0.85

0.58

0.31

-0.20

0.51

0.88

0.69

0.42

-0.09

Mean

0.51

0.89

0.59

0.32

-0.19

1)    The mean cell density of all control replicates is used as initial cell density for all treatment groups

 

Table 5: Endpoints for the test item

 

Test item in mg/L

 

Growth rate

Nominal

Measured4)

ErL10, ErC101)

8.97

0.0249

95 % confidence limits

6.48 - 11.4

0.0150 – 0.0361

ErL50, ErC501)

35.0

0.189

95 % confidence limits

30.2 – 40.4

0.154 – 0.231

 

 

 

Yield

 

 

EyL10, EyC102)

1.45

0.00205

95 % confidence limits

0.985 – 1.96

0.00121 – 0.00310

EyL50, EyC502)

9.25

0.0285

95 % confidence limits

7.60 – 11.3

0.0217 – 0.0376

 

 

 

NOEC3)

0.954

0.00216

LOEC3)

3.05

0.00570

1) Probit analysis following Gompertz distribution

2) Probit analysis following normal distribution

3) Following Bonferroni-Holms corrected Welch test (left-sided, pS0.05) for growth rate and for yield

4) Based on geometric mean of each concentration level and extrapolated from lutetium concentrations

Validity criteria fulfilled:
yes
Conclusions:
Significant inhibitory effects were determined for growth rate and for yield at test item loading rates of 3.05 mg/L (nominal) and 0.00570 mg/L (actual) and above. The overall LOELR and LOEC were therefore determined to be 3.05 mg/L (nominal) and 0.00570 mg/L (actual), the corresponding NOELR and NOEC were set at 0.954 mg/L (nominal) and 0.00216 mg/L (actual).
The EL5O-value for growth rate (ErL50) was determined to be 35.0 mg/L (nominal), the EC50-value for growth rate (ErC50) was determined to be 0.189 mg/L (actual).
Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudokirchneriella subcapitata were exposed to LYSO at nominal concentrations of 0.954, 3.05, 9.77, 31.3 and 100 mg/L (measured: 0.00216, 0.00570, 0.0216, 0.203, 0.663 mg test item/L) under static conditions in accordance with OECD guideline 201.  The NOEC based on growth rate and yield equals a test item loading rate of 3.05 mg/L (nominal) and 0.00570 mg/L (actual). The EL50-value for growth rate (ErL50) was determined to be 35.0 mg/L (nominal), the EC50-value for growth rate (ErC50) was determined to be 0.189 mg/L (actual).  The % growth inhibition in the treated algal culture as compared to the control ranged from 0.8 to 110.6 %.

No further abnormalities were observed. 

This toxicity study is classified as acceptable and satisfies the guideline requirements for an aquatic algal growth inhibition toxicity study.

 

Results Synopsis

 

Test Organism: Pseudokirchneriella subcapitata

Test Type (Flowthrough, Static, Static Renewal): static

 

Table 5: Endpoints for the test item

 

Test item in mg/L

 

Growth rate

Nominal

Measured4)

ErL10, ErC101)

8.97

0.0249

95 % confidence limits

6.48 - 11.4

0.0150 – 0.0361

ErL50, ErC501)

35.0

0.189

95 % confidence limits

30.2 – 40.4

0.154 – 0.231

 

 

 

Yield

 

 

EyL10, EyC102)

1.45

0.00205

95 % confidence limits

0.985 – 1.96

0.00121 – 0.00310

EyL50, EyC502)

9.25

0.0285

95 % confidence limits

7.60 – 11.3

0.0217 – 0.0376

 

 

 

NOEC3)

0.954

0.00216

LOEC3)

3.05

0.00570

1) Probit analysis following Gompertz distribution

2) Probit analysis following normal distribution

3) Following Bonferroni-Holms corrected Welch test (left-sided, pS0.05) for growth rate and for yield

4) Based on geometric mean of each concentration level and extrapolated from lutetium concentrations

 

Endpoint(s) Effected: Yield, Growth rate

Description of key information

Lutetium-yttrium oxyorthosilicate, cerium doped was tested on the freshwater algae Pseudokirchneriella subcapitata according to OECD guideline No. 201 and under GLP.The tested concentrations were (nominal) 0.954, 3.05, 9.77, 31.3 and 100 mg/L (measured: 0.00216, 0.00570, 0.0216, 0.203, 0.663 mg test item/L). The result of the study was an ErC50 of 0.189 mg test item/L and a NOErC of 0.00216 mg test item/L based on the growth rate and measured test item concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:
189 µg/L
EC10 or NOEC for freshwater algae:
2.16 µg/L

Additional information

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