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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:
04 Feb - 12 Feb 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:
adopted March 2006, corrected July 28, 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all test item concentrations and from the control
- Sampling method: Duplicate samples were taken from the freshly prepared test media at test start. At test end the contents of test beakers of each treatment were poured together and duplicate samples were taken.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: Defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for 24 h in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (1 h) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration. Each concentration was prepared separately.
- Controls: Yes, medium without algae
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Göttingen, Germany.
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: pre-culture

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.24 mmol/L (= 24 mg/L) as CaCO3
Test temperature:
21.8 to 22.7 °C
pH:
8.1 to 10.1 (Control)
7.8 to 10.0 (Test item concentrations)
Nominal and measured concentrations:
100, 31.6, 10.0, 3.2 and 1.0 mg/L (nominal test item concentrations)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open (loosely covered)
- Material, size, headspace, fill volume: Erlenmeyer flasks of 50 mL volume with nominal 50 mL of test medium
- Initial cells density: 0.5 x 10E04 algal cells per mL test medium
- Control end cells density: 121.6 x 10E04 algal cells per mL test medium
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were added at the nominal concentrations in deionised water
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 6048 lux (range: 5820 to 6320 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell density on each observation time was determined by spectrophotometric measurement.The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae).
Based on the counted cell densities and the absorption from an algal suspension and its dilutions, a linear regression was performed for the calculation of the cell densities of the replicates during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range-finding test: Range-finding test was performed under non-GLP conditions
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL20
Effect conc.:
43.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
22.8 mg/L
Nominal / measured:
nominal
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 remarkable observations, clear test medium
Results with reference substance (positive control):
For the evaluation of the quality of the algae and the experimental conditions the reference item potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions.
EC50 (72 h) for growth rate: 1.11 mg/L
Reported statistics and error estimates:
Based on the calculated cell densities, the 72 hour ErC50 and the 72 hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by the three parametric normal concentration distribution function (CDF).
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Dunnett's t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Table 1: Cell numbers

Nominal loading rate

Flask No.

Density of algal cells [10000/mL] after

[mg test item/L]

 

24 h

48 h

72h

Control

1

3.917

25.443

110.325

 

2

3.736

24.320

120.327

 

3

4.099

27.877

130.746

 

4

3.917

25.069

123.036

 

5

3.917

23.945

118.452

 

6

3.736

25.630

126.787

 

M

3.887

25.381

121.612

 

S

0.137

1.384

7.089

1.0

1

3.917

24.507

117.410

 

2

3.554

23.009

110.325

 

3

4.099

23.945

110.950

 

M

3.857

23.821

112.895

 

S

0.278

0.757

3.922

3.2

1

4.645

23.197

109.700

 

2

4.463

20.763

96.781

 

3

4.281

22.073

107.825

 

M

4.463

22.011

104.769

 

S

0.182

1.218

6.981

10.0

1

3.008

18.516

90.113

 

2

3.554

18.142

90.946

 

3

3.554

19.078

93.447

 

M

3.372

18.579

91.502

 

S

0.315

0.471

1.735

31.6

1

2.644

10.841

54.481

 

2

2.826

9.905

53.022

 

3

2.664

11.777

57.190

 

M

2.705

10.841

54.898

 

S

0.105

0.936

2.115

100

1

3.190

2.791

13.431

 

2

1.917

2.042

14.473

 

3

2.281

2.604

12.806

 

M

2.463

2.479

13.570

 

S

0.656

0.390

0.842

m: mean value; s: standard deviation; At test start nominal 5000 algal cells/mL were incubated

Table 2: Growth Rates µ and Percentage Inhibition of µ during the Test Period

Nominal loading rate

Growth rate µ [1/day] and % inhibition of µ

[mg test item/L]

0 - 24 hours

0 - 48 hours

0 – 72 hours

 

µ

%

µ

%

µ

%

Control

2.050

-

1.963

-

1.831

-

1.0

2.041

0.4

1.932

1.6

1.806

1.3

3.2

2.188

-6.7

1.892

3.6 (*)

1.781

2.7 (*)

10.0

1.906

7.1

1.807

7.9 (*)

1.736

5.2 (*)

31.6

1.688

17.7 *

1.537

21.7 *

1.566

14.5 *

100

1.572

23.3 *

 0.796

59.4 *

1.100

39.9 *

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control;

* mean value significantly different from the control(tested with Dunnett's t-test (24, 48 and 72h),a = 0.05, one-sided)
(*) statistically significant nut not considered to be scientifically relevant

 

Table 3: Summary of Analytical Results

Sample Description

%of nominal 1)

 

Loading rate

fresh (0 h)

aged (72 h)

 

[%]

[%]

Control (untreated)

n.a.

n.a.

Dilution Solvent (analytical performance)

n.a.

n.a.

Control

n.a.

n.a.

1 mg/L Loading Rate

97

50

3.2 mg/L Loading Rate

89

58

10.0 mg/L Loading Rate

90

70

31.6 mg/L Loading Rate

61

56

100 mg/L Loading Rate

37

36

1) mean value of all measured samples per treatment group; RSD: relative standard deviation per treatment group; n.a.: not applicable

Table 4: Validity criteria for OECD 201.

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

243.2

Yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

13.5 %

Yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

1.1 %

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

ErL50 (72 h) > 100 mg/L (Growth rate, OECD 201, Pseudokirchneriella subcapitata, nominal)

ErL10 (72 h): 22.8 mg/L (Growth rate, OECD 201, Pseudokirchneriella subcapitata, nominal)

Key value for chemical safety assessment

Additional information

There is one experimental study available, in which the toxicity of the substance was assessed according to the OECD guideline 201 and GLP.In a static test, Pseudokirchneriella subcapitata was exposed to the five nominal loading rates of 1.0, 3.2, 10.0, 31.6, and 100 mg/L test item for 72 h (initial cell density: 0.5 * 10E+04 cells/mL). Since the substance is a UVCB, the test solution was applied as water accommodated fraction (WAF). The actual test item concentrations were analytically verified via quantification of one component of the UVCB for fresh and aged samples by HPLC- UV detection. After 72 h, a clear dose-response curve was obtained for inhibition of growth rate and yield. Based on the quantified UVCB component the test concentrations were found to decrease over the course of the 72 h test in all test concentrations, except for 100 mg test item/L. The initial measured concentrations ranged between 37 and 97% of nominal and the aged samples between 36 and 70% of nominal. Since the pH increased up to 10 during the experimental phase degradation via hydrolysis is potentially expected but due to a higher degradation for the fish study with a lower pH this reason for degradation via hydrolysis is not presumably. Biological significant effects were determined and resulted in EL10 (72 h) of 22.8 mg/L (nominal) and EL50 (72 h) of > 100 mg/L (nominal) based on growth rate. For yield statistically significant effects resulted in an EL10 (72 h) of 4.63 mg/L and EL50 (72h) of 25.4 mg/L. Since the test was dosed via the water accommodated fraction (WAF) approach, all reported results refer to loading rate.