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Ecotoxicological information

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:
2017-02-14 to 2017-03-16
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:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 mg/L and control
- Sampling method: Analytical samples were taken in the main test at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test concentrations and control. For each sampling also a retain sample was taken.
- Sample storage conditions before analysis: All samples were stored deep frozen until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The necessary amount of test item for preparing the stock solution S1 was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by shaking. Afterwards the solution was clear and transparent with a yellow colouring. Lower test solutions were prepared by dilution of appropriate solutions with test medium.
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 mg/L and control
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: SAG 61.81
- Source: commercial supplier
- Method of cultivation: The algae are 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. Stock cultures are ordered regularly from commercial supplier.

ACCLIMATION
- Acclimation period: 3 to 4 days
- Culturing media and conditions: same as the test; Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 – 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.6 – 22.8 °C
pH:
pH of control: 7.29 – 8.07
pH of test: 7.19 - 8.23
Nominal and measured concentrations:
Nominal concentrations: 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 mg/L
Measured concentrations (based on the geometric mean of each concentration level): 66.2, 6.85, 0.506, 0.0422, 0.00277 and 0.000276 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, headspace, fill volume: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Renewal rate of test solution: no renewal
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 4.188E+05 /mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP-medium

OTHER TEST CONDITIONS
- Photoperiod: Continuously from the side
- Light intensity and quality: 88.8 µEm-2s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence microplate reader, determined in duplicate at 24, 48 and 72 hours
- Other: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72 hour. The light intensity of all positions of the incubator is measured once a year and was confirmed for one representative position at test start. Analytical samples of all test item concentrations and control were analysed at test start (0 hours fresh), at 24 hours (aged), 48 hours (aged) and at test end (72 hours aged).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
Range finding study
- Test concentrations: 100, 10.0, 1.00, 0.100, 0.0100 mg/L and control
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a flat dose response relation was observed for the inhibition of growth rate and yield from nominal concentrations of 0.0100 to 100 mg/L. The inhibition of growth rate peaked in 44.6 % and the inhibition of yield peaked in 84.6 % at a nominal concentration of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 66.2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.167 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.:
0.506 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Biological Results: After 72 h at termination of the test, a dose response relation was observed for the inhibition of growth rate and yield from nominal concentrations of 0.0100 to 100 mg/L. The inhibition of growth rate peaked in 38.3 % and the inhibition of yield peaked in 80.9 % at a nominal concentration of 100 mg/L. The cells were considered normal for the control and up to and including a nominal test item concentration of 10.0 mg/L. No cells were observed at 100 mg/L nominal test item concentration.
- Analytical Results: The measured initial concentration of test item ranged from 62 % to 90 % of nominal. In the aged samples the measured content was between < LOD and 80 % of nominal.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: most recent test: yield 0.976 mg/L (nominal), growth rate 1.64 mg/L (nominal)
- Other: Significant inhibitory effects were determined for all parameters at test item concentrations of 0.800 mg/L and above.

Results of non-GLP range finding test:

loading rate (mg/L) % inhibition at test end (72h)
growth rate yield
Control 0 0
0.010 4.5 15.6
0.100 8.5 28.9
1.0 15.1 45.7
10.0 21.4 58.3
100.0 44.6 84.6

A flat dose-response curve was observed.

Biological results: main study:

Average cell number for each sampling time and concentration:

Conc.

Average cell numbers/mL[104/mL]

[mg/L]

0 h

24 h

48 h

72 h

Control

0.65

1.81

8.43

41.88

0.00100

0.65

1.71

7.95

36.64

0.0100

0.65

1.90

7.68

37.86

0.100

0.65

1.92

7.34

37.10

1.00

0.65

1.82

5.46

23.28

10.0

0.65

1.32

3.50

12.93

100

0.65

0.73

2.42

8.52

Again a flat dose-response was observed.

Percentage inhibition of growth rate:

Conc.

% Inhibition ofgrowth rate

[mg/L]

0 h–24h

0h–48h

0h–72h

Control

0.0

0.0

0.0

0.00100

6.7

2.6

3.41)

0.0100

-3.5

3.9

2.6

0.100

-4.3

7.8

4.9

1.00

-0.2

17.5

14.7

10.0

32.0

34.5

28.4 *

100

88.9

49.2

38.3 *

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

* Statistically significant different to the control

Toxicological endpoints for the test item:

  Test item [mg/L]
nominal measured, geometric mean
ErC10(Growth rate) 0.364 0.167
95 % confidential limits 0.108 – 0.858 0.0443 – 0.424
ErC20  3.84 2.14
95 % confidential limits 1.76 – 8.26 0.914 – 4.88
ErC50 > 100 > 66.2
EyC10(Yield)  0.0191 0.00665
95 % confidential limits 0.000403 – 0.105 0.000104 – 0.0426
EyC20 0.113 0.0464
95 % confidential limits 0.00811 – 0.430 0.00269 – 0.198
EyC50 2.36 1.28
95 % confidential limits 0.670 – 9.77 0.328 – 5.81
NOEC 1 0.506
LOEC 10 6.85

 

As the maximum inhibition growth rate was less than 50 %, no reliable EC50-value for growth rate (ErC50) was determinable. Hence the ECr50 was not calculated but considered to be > 100 mg/L (nominal) or > 66.2 mg/L (measured). The EC50-value for yield (EyC50) was 2.36 mg/L (nominal) or 1.28 mg/L (measured).

Analytical results:

Determined concentration of test item:

Test item nominal concentration [mg/L] Sampling Test item measured concentration [mg/L] % of nominal Geometric mean [%] Geometric mean concentration [mg/L]
Control 0 h fresh n.d. - - -
24 h aged n.d. -
48 h aged n.d. -
72 h aged n.d. -
0.001 0 h fresh 0.000622 62 27.6 0.000276
24 h aged < LOQ
48 h aged < LOQ
72 h aged < LOD
0.01 0 h fresh 0.0075 75 27.7 0.00277
24 h aged 0.0065 65
48 h aged 0.00395 40
72 h aged < LOQ
0.1 0 h fresh 0.0823 82 42.2 0.0422
24 h aged 0.0655 66
48 h aged 0.0417 42
72 h aged 0.0135 14
1 0 h fresh 0.89 89 50.6 0.506
24 h aged 0.722 72
48 h aged 0.512 51
72 h aged 0.2 20
10 0 h fresh 9.02 90 68.5 6.85
24 h aged 7.96 80
48 h aged 6.84 68
72 h aged 4.49 45
100 0 h fresh 79.5 80 66.2 66.2
24 h aged 79.3 79
48 h aged 65.5 66
72 h aged 45.7 46
Validity criteria fulfilled:
yes
Conclusions:
The overall LOEC was therefore determined to be 10.0 mg/L (nominal) or 6.85 mg/L (measured) and the corresponding NOEC was set at 1.00 mg/L (nominal) or 0.506 mg/L (measured).

As the maximum inhibition growth rate was less than 50 %, no reliable EC50-value for growth rate (ErC50) was determinable. Hence the ECr50 was not calculated but considered to be > 100 mg/L (nominal) or > 66.2 mg/L (measured).
Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD Guideline 201. A static main test with nominal concentrations of 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 mg/Land control was performed. Six replicates were employed for the control and three for each test item concentration. Initial cell densities of 5E+03 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours.

Analytical samples were taken and analysed in the main testfrom control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and72 hours from aged solutions. The measured initial concentration of test item ranged from 62% to 90% of nominal. In the aged samples the measured content was between < LOD and 80% of nominal. The corresponding geometric mean test concentrations are: 0, 0.000276, 0.00277, 0.0422, 0.506, 6.85 and 66.2 mg/L.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 10.0 mg/L (nominal) or 6.85 mg/L (measured) and above. The overall LOEC was therefore determined to be 10.0 mg/L (nominal) or 6.85 mg/L (measured), the corresponding NOEC was set at 1.00 mg/L (nominal) and 0.506 mg/L (measured). The EC10-value for growth rate (ErC10) was determined to be 0.364 mg/L (nominal) or 0.167 mg/L (measured). The EC10-value for yield (EyC10) was 0.0191 mg/L (nominal) or 0.00665 mg/L (measured).

As the EC10 -value for growth rate appeared to be below the NOEC determined, the NOEC is the value that is to be used further on for risk assessment purposes. The NOEC was associated with approximately 15% inhibition of the growth rate.

As the maximum inhibition growth rate was less than 50 %, no reliable EC50-value for growth rate (ErC50) was determinable. Hence the ECr50 was not calculated but considered to be > 100 mg/L (nominal) or > 66.2 mg/L (measured). The EC50-value for yield (EyC50) was 2.36 mg/L (nominal) or 1.28 mg/L (measured).

Description of key information

For this endpoint, one study is available in which the toxicity of the test item to algae was assessed according to OECD 201.

Significant inhibitory effects were determined for growth rate and yield of algae at test item concentrations of 10.0 mg/L (nominal), corresponding to 6.85 mg/L measured, and above. The overall LOEC was therefore determined to be 10.0 mg/L (nominal) or 6.85 mg/L (measured), the corresponding NOEC was set at 1.00 mg/L (nominal) or 0.506 mg/L (measured). The EC10-value for growth rate (ErC10) was determined to be 0.364 mg/L (nominal) or 0.167 mg/L (measured). The EC10-value for yield (EyC10) was 0.0191 mg/L (nominal) and 0.00665 mg/L (measured). As the EC10 -value for growth rate appeared to be below the NOEC determined, the NOEC is the value that is to be used further on for risk assessment purposes. The NOEC was associated with approximately 15% inhibition of the growth rate.

As the maximum inhibition growth rate was less than 50 %, no reliable EC50-value for growth rate (ErC50) was determinable. Hence the ECr50 was not calculated but considered to be > 100 mg/L (nominal) or > 66.2 mg/L (measured). The EC50-value for yield (EyC50) was 2.36 mg/L (nominal) or 1.28 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:
66.2 mg/L
EC10 or NOEC for freshwater algae:
0.506 mg/L

Additional information

For this endpoint only one test is available. 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 concentrations of 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration. Initial cell densities of 5E+03 cells/mL were employed for the individual replicates. The increase of cell numbers was assessed over a test period of 72 hours. The test was performed in 100 mL Erlenmeyer flasks each containing~ 50 mL test solution.

Analytical samples were taken and analysed in the main test 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 solutions. The measured initial concentration of test item ranged from 62% to 90% of nominal. In the aged samples the measured content was between< LOD and 80% of nominal.

Significant inhibitory effects were determined for growth rate and yield at test item concentrations of 10.0 mg/L (nominal) and 6.85 mg/L (measured) and above. The overall LOEC was therefore determined to be 10.0 mg/L (nominal) and 6.85 mg/L (measured), the corresponding NOEC was set at 1.00 mg/L (nominal) and 0.506 mg/L (measured). The EC10-value for growth rate (ErC10) was determined to be 0.364 mg/L (nominal) and 0.167 mg/L (measured). The EC10-value for yield (EyC10) was 0.0191 mg/L (nominal) and 0.00665 mg/L (measured). As the EC10 -value for growth rate appeared to be below the NOEC determined, the NOEC is the value that is to be used further on for risk assessment purposes. The NOEC was associated with approximately 15% inhibition of the growth rate.

As the maximum inhibition growth rate was less than 50 %, no reliable EC50-value for growth rate (ErC50) was determinable. Hence the ECr50 was not calculated but considered to be > 100 mg/L (nominal) or > 66.2 mg/L (measured). The EC50-value for yield (EyC50) was 2.36 mg/L (nominal) or 1.28 mg/L (measured).