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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:
From 2016-02-01 to 2016-02-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 201 Alga Growth Inhibition Test [S]. Paris: OECD, Adopted 27 2011
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Batch No.: 150209
Purity: 97.7 wt%
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 25, 50, 100, 200, 400 mg/L
- Sample time: at the start of the test of 0 h, 24 h, 48 h and 72 h
Vehicle:
no
Details on test solutions:
- Test solution preparation: According to the design concentration, package 100mL of each concentration WAF solution into the test flask and filled by the 1.00 mL alga solution to prepare the 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test solution with the final alga concentration of about 10E+4 cells/mL
- Control: The blank control was composed of 1.00 mL alga solution and 100 mL test medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Species: Pseudokirchneriella subcapitata- FACHB-271
- Selection reason: The test organisms species is recommended by "The Guidelines for the Testing of Chemicals".
- Source: Bought from FACHB-Collection and cultured in the laboratory
- Age: Logarithmic growth phase alga cells
- Pre-culture: The pre-culture was incubated under the same conditions as the test cultures when still exponentially growing after 2-3 times transit culture. The logarithmic growth phase Alga used for test when the Alga cultures contain no deformed or abnormal cells after checking.
- Temperature in incubator: 22.3 °C -23.2 °C
- Light condition: Continuous light, the mean light intensity was 5027 lx - 5107 lx
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.3°C -23.2°C
pH:
at the start of the test: 7.70 to 7.82
at the end of the test: 6.47 to 7.92
Nominal and measured concentrations:
five nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L water-accommodated fraction of test substance groups (the nominal loading rates of active constituent of 24.4 mg/L, 48.8 mg/L, 97.7 mg/L, 195 mg/L and 391 mg/L calculated by the 97.7% purity of test substance) were set.
Details on test conditions:
Exposure condition
- Test method: The test solutions of each group were placed at constant temperature in a light incubator and started the test. They were shaken at least 3-4 times that was good for CO2 exchange between air and test solution.
- Control: a blank control
- Replicates: 6 replicates / blank control, 3 replicates / test substance group
- The initial cell concentration: 104 cells/mL (±25%)
- Volume of test solution: 101 mL / test vessel

Test environmental condition
- Light condition: Continuous light, the mean light intensity was 5027 lx - 5107 lx

GROWTH MEDIUM
- Standard medium used: yes, as recommended in OECD Guideline 201

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Microscope

Range finding study
- Test concentrations: The nominal loading rate of 10 mg/L, 32 mg/L and 100 mg/L WAF test group and a blank control and a nominal loading rate of 100 mg/L WAF test group without alga
- Results used to determine the conditions for the definitive study: The test algae growth inhibition rate of 10 mg/L, 32 mg/L and 100 mg/L WAF test groups were -6.7%, 2.8% and 16.5%.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
316 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% limit: 302-329 mg/L
Key result
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
36.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% limit: 33.2-40.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
EL50
At the end of the test, the average specific growth rate of blank control and 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAE test groups were 1.33 /d, 1.26 /d, 1.12 /d, 0.96 /d, 0.83 /d and 0.54 /d respectively. Compare with blank control, the inhibition rate of the average specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups were 5.03%, 15.5%, 27.4%, 37.3% and 59.0% respectively.

Cell observations and LOEL
At the end of the test, there was no significant difference on shape and size of cell between each test group and blank control under microscope. The condition of cells in the blank control was not abnormal.
The mean coefficient of variation for section-by-section specific growth rate in the blank controls was 28%. The coefficient of variation of average specific growth rates during the whole test period in control cultures was 2%.
Based on the statistical analysis results (Dunnett's test), there was a significant difference (p<0.05) on the specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups compare with blank control’s, therefore, the 72h-LOErL was 25 mg/L (based on the nominal loading rate WAF), but the 72h-NOErL was not obtained.
Results with reference substance (positive control):
After domestication, the 72h-ErC50 of reference substance K2Cr2O7 was tested for the Pseudokirchneriella subcapitata. It was 1.34 mg/L, and the 95% confidence limit was between 1.10 mg/L-1.53 mg/L, which was within the range of 1.19 mg/L ± 0.27 mg/L.

The measured concentrations of Lithium (Li) in test solutions:

The measured concentrations of Li in blank control were 0.00324 mg/L, 0.00240 mg/L, 0.00184 mg/L and 0.00332 mg/L, respectively.

The measured concentrations of Lithium in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 0.824 mg/L to 12.0 mg/L, equivalent to 111 % -122% of the nominal concentration of Li after removing the measured concentration of Li of blank control; and the measured concentrations of Li at the start of the test of 72 h ranged from 0.894 mg/L to 12.9 mg/L, equivalent to 116% - 133% of the nominal concentration of Li after removing the measured concentration of Li of blank control. During 24 h intervals, the measured concentrations of Li is stable, but the measured concentrations of Li of lower concentration test groups (25 mg/L, 50 mg/L, 100 mg/L test group) is slightly higher than 120% of the nominal concentration of Li.

The measured concentrations of oxalic ion (C2O42-) in test solutions:

The measured concentrations of C2O42- in blank control were lower than the limit of detection (LOD = 0.006 mg/L) at 0 h, 24 h, 48 h and 72 h of the test.

The measured concentrations of C2O42- in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 8.66 mg/L to 163 mg/L, equivalent to 96% - 119% of the nominal concentration of C2O42-; and the measured concentrations of C2O42- at the start of the test of 72 h ranged from 12.7 mg/L to 141 mg/L, equivalent to 9% - 149% of the nominal concentration of C2O42-. During the test, the measured concentration of C2O42- cannot always maintained±20% of the nominal concentration of C2O42-.

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal loading rate of test substance, the median effective loading rate (ErL50), the loading rate causing 10% effective (ErL10), lowest observable effect loading rate (LOErL) of alga were respectively 316 mg/L, 36.5 mg/L and 25 mg/L, and no observable effect loading rate (NOErL) of alga was less than 25 mg/L.
Executive summary:

The aim of this study is to determine the effects of the test substance on the growth of alga (Pseudokirchneriella subcapitata) according to OECD Guideline 201 Alga Growth Inhibition Test [S]. Paris: OECD, Adopted 27 2011.

Algal cultures of three replicates were exposed to the substance at nominal concentrations 25, 50, 100, 200, 400 mg/L and incubated under continuous illumination for 72 hours. Six replicate control cultures were also incubated concurrently with the test samples.

At the end of the test, the average specific growth rate of blank control and 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAE test groups were 1.33/d, 1.26/d, 1.12/d, 0.96/d, 0.83/d and 0.54/d respectively. Compare with blank control, the inhibition rate of the average specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups were 5.03%, 15.5%, 27.4%, 37.3% and 59.0% respectively.

At the end of the test, there was no significant difference on shape and size of cell between each test group and blank control under microscope. The condition of cells in the blank control was not abnormal.

The mean coefficient of variation for section-by-section specific growth rate in the blank controls was 28%. The coefficient of variation of average specific growth rates during the whole test period in control cultures was 2%.

Based on the statistical analysis results (Dunnett's test), there was a significant difference (p<0.05) on the specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups compare with blank control's.

The measured concentrations of Li in blank control were 0.00324 mg/L, 0.00240 mg/L, 0.00184 mg/L and 0.00332 mg/L, respectively. The measured concentrations of Lithium in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 0.824 mg/L to 12.0 mg/L, and the measured concentrations of Li at the start of the test of 72 h ranged from 0.894 mg/L to 12.9 mg/L. The measured concentrations of C2O42- in blank control were lower than the limit of detection (LOD = 0.006mg/L) at 0 h, 24 h, 48 h and 72 h of the test. The measured concentrations of C2O42- in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 8.66 mg/L to 163 mg/L, and the measured concentrations of C2O42- at the start of the test of 72 h ranged from 12.7 mg/L to 141 mg/L.

Based on the nominal loading rate of test substance, the median effective loading rate (ErL50), the loading rate causing 10% effective (ErL10), lowest observable effect loading rate (LOErL) of alga were respectively 316 mg/L, 36.5 mg/L and 25 mg/L, and no observable effect loading rate (NOErL) of alga was less than 25 mg/L.

Description of key information

Based on the nominal loading rate of test substance, the median effective loading rate (ErL50), the loading rate causing 10% effective (ErL10), lowest observable effect loading rate (LOErL) of alga were respectively 316 mg/L, 36.5 mg/L and 25 mg/L, and no observable effect loading rate (NOErL) of alga was less than 25 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
316 mg/L
EC10 or NOEC for freshwater algae:
25 mg/L

Additional information

The aim of this study is to determine the effects of the test substance on the growth of alga (Pseudokirchneriella subcapitata) according to OECD Guideline 201 Alga Growth Inhibition Test [S]. Paris: OECD, Adopted 27 2011.

Algal cultures of three replicates were exposed to the substance at nominal concentrations 25, 50, 100, 200, 400 mg/L and incubated under continuous illumination for 72 hours. Six replicate control cultures were also incubated concurrently with the test samples.

At the end of the test, the average specific growth rate of blank control and 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAE test groups were 1.33/d, 1.26/d, 1.12/d, 0.96/d, 0.83/d and 0.54/d respectively. Compare with blank control, the inhibition rate of the average specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups were 5.03%, 15.5%, 27.4%, 37.3% and 59.0% respectively.

At the end of the test, there was no significant difference on shape and size of cell between each test group and blank control under microscope. The condition of cells in the blank control was not abnormal.

The mean coefficient of variation for section-by-section specific growth rate in the blank controls was 28%. The coefficient of variation of average specific growth rates during the whole test period in control cultures was 2%.

Based on the statistical analysis results (Dunnett's test), there was a significant difference (p<0.05) on the specific growth rate of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF test groups compare with blank control's.

The measured concentrations of Li in blank control were 0.00324 mg/L, 0.00240 mg/L, 0.00184 mg/L and 0.00332 mg/L, respectively. The measured concentrations of Lithium in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 0.824 mg/L to 12.0 mg/L, and the measured concentrations of Li at the start of the test of 72 h ranged from 0.894 mg/L to 12.9 mg/L. The measured concentrations of C2O42- in blank control were lower than the limit of detection (LOD = 0.006mg/L) at 0 h, 24 h, 48 h and 72 h of the test. The measured concentrations of C2O42- in the nominal loading rates of 25 mg/L, 50 mg/L, 100 mg/L, 200 mg/L and 400 mg/L WAF solution test groups at the start of the test of 0 h ranged from 8.66 mg/L to 163 mg/L, and the measured concentrations of C2O42- at the start of the test of 72 h ranged from 12.7 mg/L to 141 mg/L.

Based on the nominal loading rate of test substance, the median effective loading rate (ErL50), the loading rate causing 10% effective (ErL10), lowest observable effect loading rate (LOErL) of alga were respectively 316 mg/L, 36.5 mg/L and 25 mg/L, and no observable effect loading rate (NOErL) of alga was less than 25 mg/L.

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