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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:
2015-07-21 to 2015-07-24
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
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 1.00, 3.16, 10.0, 31.6 and 100 mg/L
- Sampling method: All concentration levels and the control were analytically verified via GC-MS/MS after 0 hours (start of exposure) and 72 hours (end of exposure). Separate replicates for the test item analysis at the beginning of the exposure were prepared without algae. For the test item analysis after 72 hours, separate replicates of the concentration levels 1.0 to 10.0 mg/L and control were prepared and incubated under test conditions. Samples of the concentration levels 31.6 to 100 mg/L were taken out of the test replicates. No specific treatment for removal of algae cells from the samples was applied.
- Sample storage conditions before analysis:
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION:
A stock solution of 100 mg/L was freshly prepared with dilution water. Direct addition of the test item and shaking until the test item was completely dissolved.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Test organism: Pseudokirchneriella subcapitals HINDAK, SAG 61.81
- Source (laboratory, culture collection): University Göttingen (Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universitat Göttingen; Nikolausberger Weg 18, 37073 Göttingen, Germany)
- Age of inoculum: A four days old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 35-70 µE/m²/s for 24 hours per day.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
21 -24 °C, controlled at ± 2 °C
pH:
7.90 - 8.70
Dissolved oxygen:
No data
Salinity:
Not applicable
Conductivity:
No data
Nominal and measured concentrations:
Nominal: Control, 1.00, 3.16, 10.0, 31.6 and 100 mg/L
Measured concentrations: The measured concentrations of the test item in the fresh media (0 h) were in the range of 82 to 97 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 9 to 94 % of the nominal values of all concentrations. For details see table 2 in section "Any other information in results incl. tables".
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks (vol. 250 mL) with cotton wool plugs. A test volume of 100 mL was used.
Initial cell density: Nominal: approximately 5x10^3 10^5 cells/mL; Current: 6153 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to the OECD guideline 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: 24 hours/day light
- Light intensity and quality: 60 -120 µE/m²/s; Fluorescent tubes, Osram L 36 W/11-865, cool daylight

EFFECT PARAMETERS MEASURED:
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll a-fiuorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. No self-fluorescence was found in the preliminary range finding test at the concentration level of 100 mg/L.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of exposure was carried out. The ceils were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

RANGE FINDING STUDY:
- Concentrations: 1.00, 10.0 and 100 mg/L
- Results used to determine the conditions for the definitive study: The results of the Range Finding Test are shown in Table 1 in the section "Any other information on material and methods incl. tables"
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
31.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
99.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% conficence interval: 37.2 - >100
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
57.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% cinfidence interval: 33.5 - 98.5
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
71.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield inhibition
Remarks on result:
other: 95% confidence interval: 35.7 - 99.0
Details on results:
- Observation of abnormalities: Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities.
- Unusual cell shape: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- ErC50 (72 h): 0.613 (95% condidence interval: 0.589 - 0.638)
- EyC50 (72 h): 0.281 (95% condidence interval: 0.234 - 0.314)
Reported statistics and error estimates:
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and Dunnett's test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The α-value (acceptable probability of incorrectly concluding that there is a difference) is α=0.05.
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, Microsoft Corporation
• SigmaPlot, Spss Inc.
• GraphPad Prism, GraphPad Software, Inc.

Measured Exposure Concentrations during the Definitive Test

The concentrations of the test itm were determined in the fresh media (0 h) and old media (72 h) of all tested concentration levels and the control via GC-MS/MS. For results, see Table 2. The measured concentrations of the test item in the fresh media (0 h) were in the range of 82 to 97 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 h) were in the range of 9 to 94 % of the nominal values of all concentrations. Effects were found only at the highest concentration of 100 mg/L. Therefore, the nominal concentrations were used for calculations.

Table 2: Measured Concentrations and Percentage of the Nominal Concentration of the test item in Fresh Medium (0 h) and Old Medium (72 h)  

Sampling date

0 hours

Start of exposure

7 hours

End of exposure

Start of analysis

2015-07-21

2015-07-241)

Nominal test item concentration [mg/L]

Art. 278717

Meas. conc. [mg/L]

%

Meas. conc. [mg/L]

%

100

97.2

97

93.5

94

31.6

29.9

95

25.5

81

10.0

9.66

97

0.9472)

9

3.16

2.59

82

0.447*

14

1.00

0.881

88

0.163*

16

Control

>LOQ

>LOQ

Meas. conc: Measured concentration of the test item (preconcentration and dilution factor taken into account)
%: Percent of the nominal concentration of the test item
LOQ: Limit of Quantification (0.5 mg/L test item)
1): Reinjected on 2015-07-27
2): Reinjected on 2015-07-28

*: Measured concentration < LOQ, therefore only for information

 

Table 3: Validity criteria

Validity criteria

Required

This study

Increase of the cell growth in the
control cultures

Exponentially, > 16-fold
corresponding to a specific
growth rate of 0.92/day

 

278-fold
(specific growth rate 1.88/day)

 

Mean coefficients of variation for
section-by-section specific growth
rates (days 0-1, 1-2 and 2-3) in the
control cultures

≤ 35%

19.5 %

Coefficient of variation of average
specific growth rates during the
whole test period in replicate control cultures

≤ 7 %

1.52 %

Validity criteria fulfilled:
yes
Conclusions:
In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitals after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD TG 201. Psedokirchneriella subcapitata were exposed to nominal test item concentrations of 1.00, 3.16, 10.0, 31.6 and 100 mg/L and a control. The concentrations of the test item in all test concentrations and the control were analytically verified by GC-MS/MS at the start and the end of the exposure. The measured concentrations of the test item in the fresh media (0 hours) were in the range of 82 to 97 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 hours) were in the range of 81 to 94 % of the nominal values of the two highest concentrations (31.6 and 100 mg/L) and in the range of 9 to 16 % of the concentration levels 1.00 to 10.0 mg/L. Effects were only found at the highest test concentration of 100 mg/L. Therefore, all effect values given are based on the nominal test item concentrations of the test item.

Under the conditions of this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitals after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 99.5 mg/L (37.2 ->100) and 57.9 mg/L (33.5 - 98.5), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L

Description of key information

In an algae growth inhibition test according to OECD TG 201, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitals after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 99.5 mg/L (37.2 - 100) and 57.9 mg/L (33.5 - 98.5), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
99.5 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to OECD TG 201. Psedokirchneriella subcapitata were exposed to nominal test item concentrations of 1.00, 3.16, 10.0, 31.6 and 100 mg/L and a control. The concentrations of the test item in all test concentrations and the control were analytically verified by GC-MS/MS at the start and the end of the exposure. The measured concentrations of the test item in the fresh media (0 hours) were in the range of 82 to 97 % of the nominal values at all tested concentration levels. The measured concentrations in the old media (72 hours) were in the range of 81 to 94 % of the nominal values of the two highest concentrations (31.6 and 100 mg/L) and in the range of 9 to 16 % of the concentration levels 1.00 to 10.0 mg/L. Effects were only found at the highest test concentration of 100 mg/L. Therefore, all effect values given are based on the nominal test item concentrations of the test item.

Under the conditions of this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitals after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for both inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were > 100 mg/L. The EC10-values with 95 % confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 99.5 mg/L (37.2 ->100) and 57.9 mg/L (33.5 - 98.5), respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 31.6 mg/L (reference 6.1.5-1).