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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:
2016-03-29 to 2016-04-01
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
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 mg/L
- Dilution: acetonitril:HPLC water (40:60)
- Sample storage conditions before analysis: stored at 6 +/- 2 °C. Prepared samples were stored at room temperature.
Vehicle:
not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÄK, SAG 61.81
- Source: available in study report
- Method of cultivation: Nutrient medium Z according to Lüttge et ai. (1994); Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE m-2 s-1 for 24 hours per day.
- Age of inoculum (at test initiation): 3 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 start and the end of exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.
Test temperature:
21-24 °C (controlled at +/- 2°C)
pH:
ranging from 7.87 to 9.47
Nominal and measured concentrations:
Nominal concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
Measured concentrations (0 h): 6.56 - 13.0 - 25.1 - 48.6 - 97.6 mg/L
Measured concentrations (72 h): 6.37 - 12.9 - 25.5 - 49.1 - 97.9 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: flask
- Type: flasks cotton plug, with headspace
- Initial cells density: nominal -> approximately 5 x 103- 104cells/mL; current: 6305 cells/mL
- Control end cells density:
- No. of organisms per vessel: 6305 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Agitation: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.

GROWTH MEDIUM
- Standard medium used: yes


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


OTHER TEST CONDITIONS
- Photoperiod: light 24 h/day
- Light intensity and quality: Approximately 60 - 120 µE m-2 s-1; light homogeneity within 15 % over incubation area


EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: yes, excitation at 436 nm, emission at 685 nm
- Frequency: daily measurements

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: 100 % inhibition at 100 mg/L and 4 and 20 % inhibition at 10 mg/L for growth and yield, respectively

Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval: 36.6-39.6 mg/L
Remarks:
Nominal concentrations verified by measurements
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence interval: 17.7 - 20.8 mg/L
Remarks:
Nominal concentrations verified by measurements
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
Nominal concentrations verified by measurements
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
20.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: 95 % confidence interval: 18.5-22 mg/L
Remarks:
Nominal concentrations verified by measurements
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other: 95 % confidence interval: 11.9-15.2 mg/L
Remarks:
Nominal concentrations verified by measurements
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Yield inhibition
Remarks on result:
other:
Remarks:
Nominal concentrations verified by measurements
Details on results:
- Exponential growth in the control (for algal test): yes
- Unusual cell shape: no morphological abnormalities
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.564 mg/L growth rate inhibition (valid range: 0.797 +/- 0.428 mg/) and 0.322 mg/L yield inhibition (valid range: 0.415 +/- 0.275 mg/L).
Reported statistics and error estimates:
ECx-values of growth rate and yield inhibition with confidence intervals after 72 hours were calculated by sigmoidal dose- response regression with GraphPad Prism.

The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using Williams Sequential t-test Procedure. A Shapiro-Wilk's Normality test and a Levene’s Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

The mean cell densities measured are reported here:

Nominal test item concentration (mg/L) no. of replicates Mean cell density (cells/mL)
0h 24h 48h 72h
100 3 6305 11209 8165 3784
50 3 6305 13720 14431 35496
25 3 6305 19666 89488 646958
12.5 3 6305 27685 331112 2078334
6.25 3 6305 29255 398052 2453092
control 6 6305 25220 346667 2199062

This translates to following growth rates, yield and inhibition:

Nominal test item concentration (mg/L) no. of replicates Mean growth rate (d-1) Mean inhibtion of growth rate (%) Mean yield (cells/mL) Mean inhibtion of yield (%)
100 3 -0.174 100 -2521 100
50 3 0.575 71 29191 99
25 3 1.53 21 640653 71
12.5 3 1.93 1 2072029 6
6.25 3 1.99 -2 2446787 -12
control 6 1.95   2192757  

The section-by-section and average growth rates of the control group are as follows:

replicate 0-24h 24-48h 48-72h Mean (0-72h) SD (+/-) CV (%) Mean CV (%)
1 1.41 2.46 1.77 1.88 0.533 28.4 32.3
2 1.34 2.7 1.77 1.94 0.698 36
3 1.52 2.63 1.78 1.98 0.582 29.4
4 1.43 2.45 1.99 1.96 0.51 26.1
5 1.2 2.79 1.92 1.97 0.797 40.5
6 1.39 2.69 1.86 1.98 0.657 33.2
Mean 1.95
SD (+/-) 0.04
CV (%) 1.91

Measured exposure concentrations during the Definitive Test:

Sampling date

2016-03-29

Fresh medium

(0 hours)

2016-04-01

End of the exposure

(72 hours)

Start of analysis

2016-03-29

2016-04-01

Nominal conc. (mg/L)

Measured conc. (mg/L)

%

Measured conc. (mg/L)

%

100

97.6

98

97.9

98

50.0

48.6

97

49.1

98

25.0

25.1

100

25.5

102

12.5

13.0

104

12.9

103

6.25

6.56

105

6.37

102

Control

< LOQ

 

< LOQ

 

 

Validity criteria fulfilled:
yes
Conclusions:
After an exposure of 72 h, the test item was found to inhibit the growht of Pseudokirchneriella subcapitata. The reported EC50-values are 38.1 mg/L for growth rate inhibition and 20.2 mg/L when yield inhibition was studied. The 72-hour EC10-values for inhibition of growth rate and yield were 19.2 and 13.4 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 alga Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201. The nominal concentrations tested were 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Validity criteria were fulfilled with this test set-up. The test item was found to inhibit the growth of Pseudokirchneriella subcapitata. The 72-hour EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) were 38.1 (36.6 - 39.6) and 20.2 (18.5 - 22.0) mg/L. The EC10-values for inhibition of growth rate and yield after 72 hours were 19.2 and 13.4 mg/L, respectively.

Description of key information

OECD 201 (Pseudokirchneriella subcapitata): ErC50 = 38.1 mg/L; ErC10 = 19.2 mg/L (nominal concentrations)

Key value for chemical safety assessment

EC50 for freshwater algae:
38.1 mg/L
EC10 or NOEC for freshwater algae:
19.2 mg/L

Additional information

For this endpoint, one study is available in which the effect of the test item on growth of freshwater green alga Pseudokirchneriella subcapitata after 72 hours exposure was assessed according to OECD 201. The nominal concentrations tested were 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L. Validity criteria were fulfilled with this test set-up. The test item was found to inhibit the growth ofPseudokirchneriella subcapitata. The 72-hour EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) were 38.1 (36.6 - 39.6) and 20.2 (18.5 - 22.0) mg/L. The EC10-values for inhibition of growth rate and yield after 72 hours were 19.2 and 13.4 mg/L, respectively.