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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:
2016-12-15 to 2017-03-17
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:
2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- sampling method: analytical samples were taken in the main at 0h (initial value), after 24h, 48h and 72h from aged solutions
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
- Stock solution: 18.8 mg of test item was mixed with test AAP-medium and homogenised by shaking for 5 minutes of ultrasonication; Final volume is 1000mL
- Dilutions of stock solution: 3 dilutions were made from the stock solution with test AAP-medium, to reach a final volume of 500mL per dilution
- Controls: test water without addition of the test item
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source: available in study report
- Age of inoculum (at test initiation): 3 to 4 days old pre-culture
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Microscopic evaluation of the cells at the end of exposure (morphological appearance check)
Test temperature:
22.4 – 23.0 °C
pH:
pH range of control (0h-72h): 7.28 - 7.84
pH range of samples (0h-72h): 7.34 - 7.91
Nominal and measured concentrations:
Nominal concentrations tested (spaced by a factor of 2.5): 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and a control
Measured concentrations (geometric mean concentrations): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 100mL volume with aluminium caps filled with 50mL test solution
- Initial cells density: 0.5 x 10^4 cells/mL
- Control end cells density: 33.22 x 10^4 cells/mL
- No. of organisms per vessel: 0.5 x 10^4 cells/mL, employed for the individual replicates
- No. of treatment groups: 6 (5 dose rates of the test item and a control)
- No. of replicates: 3 for each test item concentration and 6 for the control

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

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD AAP-medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination from the side
- Light intensity: 88.8 µE/m2 s

EFFECT PARAMETERS MEASURED: influence of test item on yield and growth rate of organisms

RANGE-FINDING STUDY
- Test concentrations: Control, 100, 10.0, 1.00, 0.100 and 0.0100 mg/L.
- Results used to determine the conditions for the definitive study: after 72 h at termination of the test no inhibition of growth rate or yield was observed for concentrations up to and including 1.00 mg/L. For higher test item concentrations: inhibition of growth rate peaked (reached the highest point) at 142.4 % at a nominal concentration of 100 mg/L and the inhibition of yield peaked at 102.0 % at a nominal concentration of 100 mg/L.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate tested at least twice a year
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.56 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.:
0.841 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.981 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.636 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.04 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.746 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.373 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Growth rate and Yield inhibition
Details on results:
- Exponential growth in the control (for algal test): yes
- Morphology of algae cells: the cells were considered normal for the control and up to and including a nominal test item concentration of 3.01 mg/L. No cells were observed at 7.52 and 18.8 mg/L nominal test item concentration.
Results with reference substance (positive control):
- Results with reference substance valid? yes, the EC50 values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the test organism is sensitive.
- Test rates: static main test with nominal concentrations of 0.0512, 0.128, 0.320, 0.800 and 2 mg/L and control
- Conclusions: significant inhibitory effects were determined for all parameters at test item concentrations of 0.800 mg/L (nominal) and above
- EC50 (growth rate): 1.64 mg/L (nominal); EC50 (yield inhibition): 0.976 mg/L (nominal)
- EC10 (growth rate): 0.695 mg/L (nominal); EC10 (yield inhibition): 0.354 mg/L (nominal)

Results non-GLP dose range finding test:

Concentration (mg/L) % inhibition after 72h
Growth rate Yield
Control 0 0
0.01 -7.5 -29.3
0.1 -8.7 -37.1
1 -6.3 -23.8
10 123.5 101.5
100 142.4 102

Biological results main study:

Percentage inhibition of growth rate:

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

0.481

-1.8

-1.8

1.3

1.20

-10.3

-3.6

-0.2

3.01

14.7

16.7

20.3

7.52

36.4

79.9

83.8

18.8

107.9

109.9

132.3

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

0.52

1.50

6.34

33.22

0.481

0.52

1.49

6.55

31.43

1.20

0.52

1.62

6.94

33.74

3.01

0.52

1.25

4.18

14.35

7.52

0.52

1.00

0.87

1.04

18.8

0.52

0.48

0.41

0.14

Analytical results: determination of test item concentrations

Nominal concentration (mg/L) Sampling  Measured concentration (mg/L) % of nominal Geometric mean (mg/L)
Control 0h fresh n.d    
24h aged n.d
48h aged n.d
72h aged n.d
0.481 0h fresh 0.436 91 0.152
24h aged 0.215 45
48h aged 0.107 22
72h aged 0.0507 11
1.2 0h fresh 1.08 90 0.373
24h aged 0.538 45
48h aged 0.275 23
72h aged 0.122 10
3.01 0h fresh 2.89 96 1.03
24h aged 1.51 50
48h aged 0.723 24
72h aged 0.368 12
7.52 0h fresh 7.51 100 2.56
24h aged 3.72 49
48h aged 1.74 23
72h aged 0.879 12
18.8 0h fresh 19.3 103 6.54
24h aged 9.83 52
48h aged 4.67 25
72h aged 2.09 11
Validity criteria fulfilled:
yes
Conclusions:
After an exposure of 72 h, the test item was found to inhibit the growth of Pseudokirchneriella subcapitata. The reported EC50-values (based on measured concentrations, geom. mean) are 1.56 mg/L for growth rate inhibition and 0.981 mg/L when yield inhibition was studied. The 72-hour EC10-values (based on measured concentrations, geom. mean) for inhibition of growth rate and yield were 0.841 and 0.636 mg/L, respectively.
Executive summary:

For this endpoint, one study is available in which the effect of the test item on growth of freshwater green algae Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201. The organisms were exposed over a period of 72 hours to 5 concentrations of the test item (nominal concentrations of: 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and control; corresponding to measured concentrations (geom. mean): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.).

The test item was found to inhibit the growth of Pseudokirchneriella subcapitata. The 72-hour EC50-values (based on measured concentrations) for both inhibition of growth rate (ErC50) and yield (EyC50) were 1.56 and 0.981 mg/L. The EC10-values (based on measured concentrations) for inhibition of growth rate and yield after 72 hours were 0.841 and 0.636 mg/L, respectively.

Validity criteria were fulfilled with this test set-up.

Description of key information

For this endpoint, one study is available in which the effect of the test item on growth of freshwater green algae Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201.

The 72-hour EC50-values (geometric mean, measured concentrations) for both inhibition of growth rate (ErC50) and yield (EyC50) were 1.56 and 0.981 mg/L. The EC10-values (geometric mean, measured concentrations) for inhibition of growth rate and yield after 72 hours were 0.841 and 0.636 mg/L, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.56 mg/L
EC10 or NOEC for freshwater algae:
0.841 mg/L

Additional information

The organisms were exposed over a period of 72 hours to 5 concentrations of the test item (nominal concentrations of: 18.8, 7.52, 3.01, 1.20, 0.481 mg/L and control. This corresponds to measured concentrations (geom. mean): 6.54, 2.56, 1.03, 0.373, 0.152 mg/L and n.d.).

Validity criteria were fulfilled with this test set-up.

The test item was found to inhibit the growth of Pseudokirchneriella subcapitata.