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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-09 to 2018-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken and analysed from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged test solutions.

Test solutions

Vehicle:
no
Details on test solutions:
A stock solution of 100mg/L was prepared. Lower test solutions were prepared by dilution of the stock solution with test medium. Algae were added to each solution individually targeting nominal cell densities of 0.5 × 104 cells per mL in each solution.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Pseudokirchneriella subcapitata Hindák (Sphaeropleales: Selenastraceae)
- Strain: SAG 61.81
- Source (laboratory, culture collection): MBM Sciencebridge GmbH, Göttingen
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory.

ACCLIMATION
- Acclimation period: 3 to 4 days before start of the test, test medium was inoculated with the test organism and held under test conditions in order to produce a pre-culture in the state of exponential growth.
- Culturing media and conditions (same as test or not): yes

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22.6 – 23.2 °C
pH:
7.49 – 8.30 (control)
7.26 - 8.34 (test concentrations)
Nominal and measured concentrations:
nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
geometric mean measured: 0.208, 0.158, 0.119, 0.0906, 0.0677mg/L and a control
The measured initial concentrations of analyte ranged from 79.9 % to 94.5 % of nominal. In the aged samples (24, 48 and 72h) the measured concentrations were
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Aeration: continuous agitation
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Initial cells density: 0.5 × 10^4 cells/mL.
- Control end cells density: 39.31 × 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes AAP-Medium ((according to Annex 3 of OECD 201))

OTHER TEST CONDITIONS
- Photoperiod: Continuously
- Light intensity and quality: 88.3 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (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).)
Reference substance (positive control):
yes
Remarks:
Potassium Dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.135 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.116 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.119 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.158 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.106 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Reported statistics and error estimates:
Probit analysis following normal distribution for the determination of EC values.
Jonckheere Terpstra test (left-sided, p<0.05) for growth rate and for yield for the determination of NOEC and LOEC.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

nominal Conc.

Average cell numbers [104/mL]

[mg/L]

0 h

24 h

48 h

72 h

Control

0.49

1.53

8.05

39.31

0.954

0.49

2.09

10.33

48.66

3.05

0.49

2.11

9.96

42.66

9.77

0.49

1.89

5.57

21.04

31.3

0.49

0.93

0.65

0.70

100

0.49

0.83

0.85

0.92

Table 2: Percentage inhibition of growth rate

nominal Conc.

% Inhibition of growth rate

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.954

-31.5

-9.8

-5.31)

3.05

-32.4

-8.4

-2.12)

9.77

-22.1

12.6

14.0

31.3

43.3

90.6

92.0 *

100

52.7

80.3

85.7 *1)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 3: Percentage inhibition of yield

niminal Conc.

% Inhibition of yield

[mg/L]

0 h – 24 h

0 h – 48 h

0 h – 72 h

Control

0.0

0.0

0.0

0.954

-53.8

-30.2

-24.11)

3.05

-55.8

-25.3

-8.72)

9.77

-34.6

32.8

47.0

31.3

57.7

97.9

99.5 *

100

67.3

95.2

98.9 *1)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

* Statistically significant different to the control

Table 4: Determined concentration of the test item

Test item nominal [mg/L]

Sampling

Test item found

Actual test item conc. [mg/L]

[mg/L]

% of nominal

Geometric mean [%]

Control

0 h fresh

< LOD

-

-

-

24 h aged

< LOD

-

48 h aged

< LOD

-

72 h aged

< LOD

-

0.954

0 h fresh

0.900

94.3

7.10

0.0677

24 h aged

< LOD1)

3.00

48 h aged

< LOD1)

3.00

72 h aged

< LOD1)

3.00

3.05

0 h fresh

2.88

94.5

2.97

0.0906

24 h aged

< LOD1)

0.938

48 h aged

< LOD1)

0.938

72 h aged

< LOD1)

0.938

9.77

0 h fresh

8.55

87.5

1.22

0.119

24 h aged

< LOD1)

0.293

48 h aged

< LOD1)

0.293

72 h aged

< LOD1)

0.293

31.3

0 h fresh

26.7

85.2

0.505

0.158

24 h aged

< LOD1)

0.0914

48 h aged

< LOD1)

0.0914

72 h aged

< LOD1)

0.0914

100

0 h fresh

79.9

79.9

0.208

0.208

24 h aged

< LOD1)

0.0286

48 h aged

< LOD1)

0.0286

72 h aged

< LOD1)

0.0286

 - = not calculated;LOD = 0.0286 mg/L test item;LOQ = 0.0954 mg/L test item

1)Analyte was below LOD in the sample. Therefore the LOD value was used for calculation of geometric mean according to OECD Guidance document No. 23 (2000)


Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The toxicity of the test substance to the green freshwater algae Pseudokirchneriella subcapitata was determined according to OECD 201 and GLP. The results are as follows:
The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.135 mg/L and 0.120 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.116 mg/L and 0.106 mg/L, respectively. The NOEC for both inhibition of growth rate and yield after 72 hours were 0.119 mg/L. All results are based on geometric mean measured values.