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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:
From 2017-11-06 to 2017-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.
- Sampling method: taken at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test item concentrations and control. At each sampling time point a retention sample was taken.
- Sample storage conditions before analysis: stored deep frozen

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
stock solution S1: weighed necessary amount of test item to a volumetric flask and added test medium up to the bench mark, homogenised by shaking. Lower test solutions: dilution of stock solution S1 with test medium
- Evidence of undissolved material: the solution was clear and transparent.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák
- Strain: SAG 61.81
- Source: commercial supplier
- Age of inoculum (at test initiation):
- Method of cultivation: grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

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

Test conditions

Test temperature:
22.7 – 23.1 °C
pH:
7.70 – 8.15
Nominal and measured concentrations:
Nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps
- Type: closed
- Fill volume: filled up with ~ 50 mL test solution
- Aeration: CO2 supplied by continuous agitation
- Initial cells density: 0.5 E+04 cells/mL
- Control end cells density: 26.35 E+04 cells/mL
- 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: AAP-Medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.
- Photoperiod: Continuously
- Light intensity and quality: fluorescent tubes, 88.8 μEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence microplate reader (infinite 200Pro)
- Other: morphological appearance: observed microscopically at the end of the test; pH: Measured at t = 0 h and t = 72 h; temperature: measured continuously and recorded at hours 0, 24, 48 and 72; light intensity: measured once a year and was confirmed for one representative position at test start.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study
Reference substance (positive control):
yes

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 63.2 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.:
> 63.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
63.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Inhibition: After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for test item concentrations up to and including 100 mg/L. The inhibition of growth rate peaked in 6.6 % and the inhibition of yield peaked in 23.8 % at a nominal test item concentration of 31.3 mg/L.
- Cell morphology: normal for the control and all test item concentrations. At 31.3 and 100 mg/L bacteria were observed in the solutions.
- Analytical results: The measured initial concentration of active ingredient was 81 % of nominal at 100 mg/L. A degradation of the active ingredient was observed during the test with 29 % of nominal measured at test end. The measured initial concentration of active ingredient in the replicates containing the 100 mg/L test solution without algae was 74 % of nominal at 100 mg/L. A degradation of the active ingredient was observed during the test with 46 % of nominal measured at test end.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 1.23 mg/L for growth rate; 0.685 mg/L for yield; 0.639 mg/L for biomass intergral
Reported statistics and error estimates:
- LOEC and NOEC: Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield
- ECx: Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

Any other information on results incl. tables

Toxicological endpoints for the test item:

 

Test item [mg/L]

nominal

Actual (geometric mean)

ErC10 (Growth rate)

>100

>63.2

95% confidential limits

n.c

n.c

ErC20

>100

>63.2

95% confidential limits

n.c.

n.c.

ErC50

>100

>63.2

95% confidential limits

n.c.

n.c.

EyC10 (Yield)

n.d.

n.d.

95% confidential limits

n.c.

n.c.

EyC20

n.d.

n.d.

95% confidential limits

n.c

n.c

EyC50

>100

>63.2

95% confidential limits

n.c.

n.c.

NOEC

100

63.2

LOEC

-

-

n.d.: Not determined

n.c : Not caculated

Individual cell numbers:

Conc. [mg/L]

Cell numbers [1E+04/mL]

Yield

0h

24h

48h

72h

0h–72h

 

 

 

Control

0.63

1.29

5.63

25.52

24.89

0.51

1.32

6.05

28.78

28.27

0.52

1.17

5.02

17.18

16.66

0.54

1.63

6.07

26.49

25.95

0.54

1.77

7.57

32.39

31.85

0.51

1.68

6.73

27.75

27.24

Mean

0.541)

1.48

6.18

26.35

25.81

 

 

0.954

0.54

1.01

5.77

25.48

24.94

0.54

1.65

6.24

25.24

24.70

0.54

1.75

6.80

25.53

24.99

Mean

0.54

1.47

6.27

25.42

24.88

 

 

3.05

0.54

1.18

5.74

20.00

19.46

0.54

1.74

6.87

29.90

29.36

0.54

1.62

5.96

22.65

22.11

Mean

0.54

1.51

6.19

24.18

23.64

 

 

9.77

0.54

1.43

5.44

21.67

21.13

0.54

1.61

5.99

25.25

24.71

0.54

1.49

6.09

25.38

24.84

Mean

0.54

1.51

5.84

24.10

23.56

 

 

31.3

0.54

1.13

4.03

17.55

17.01

0.54

1.46

5.66

18.76

18.22

0.54

1.23

6.37

24.32

23.78

Mean

0.54

1.27

5.35

20.21

19.67

 

 

100

0.54

1.88

6.32

28.60

28.06

0.54

1.76

7.00

28.50

27.96

0.54

1.59

7.29

31.67

31.13

Mean

0.54

1.74

6.87

29.59

29.05

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) and 63.2 mg/L (actual) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal) and 63.2 mg/L (actual).
The EC10-, EC20- and EC50-value for growth rate and the EC50-value for yield were considered to be > 100 mg/L (nominal) and > 63.2 mg/L (actual).
Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD 201.

A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05 and 0.954 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration. One replicate with 100 mg/L test item concentration without algae was run in parallel which was used for the analytical sampling. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution.

Toxicological endpoints were evaluated using the nominal and actual concentrations (based on the geometric mean) of the test item.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) and 63.2 mg/L (actual) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal) and 63.2 mg/L (actual).

The EC10-, EC20- and EC50-value for growth rate and the EC50-value for yield were considered to be > 100 mg/L (nominal) and > 63.2 mg/L (actual). Due to an inhibition of yield below 50 % and a missing concentration response relation no reliable values were calculable and the EC10- and EC20-value for yield were not determined.