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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 2018-08-06 to 2018-09-10
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:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: The samples were measured at following concentrations: 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L.
- Sampling method: For determination of the test item concentrations, three replicate samples (5 mL per replicate) were taken from each test solution and from the control at the start and at the end of the test.
- Sample storage conditions before analysis: Room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test solutions used in the test were prepared by mechanical dispersion without the aid of any solubilising agents. The test solutions were freshly prepared at the beginning of the experiment. A stock solution of 100 mg/L (nominal concentration) was prepared in OECD medium. The further test solutions were prepared by appropriate dilution of this stock solution.
- Controls:
Untreated Control: OECD Medium (without test item) was inoculated and examined in parallel in the study.
Toxic Reference Control: For the evaluation of the quality of the algae and the experimental conditions, potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Breeding conditions: The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for four days at this test).

ACCLIMATION
- Acclimation conditions: Same as the test.
Test type:
static
Water media type:
freshwater
Remarks:
OECD Medium, according to OECD 201
Limit test:
yes
Total exposure duration:
72 h
Remarks on exposure duration:
None
Post exposure observation period:
no
Hardness:
NA
Test temperature:
22.6 – 23.3 °C measured in the flask and 21.9 – 24.0 °C measured within the climate chamber
pH:
7.85 – 8.85
Dissolved oxygen:
NA
Salinity:
NA
Conductivity:
NA
Nominal and measured concentrations:
- Nominal: 6.25, 12.5, 25, 50 and 100 mg/L. Untreated control ran parallel in the test.
- Measured concentrations: The corresponding calculated geometric mean concentrations were the followings: 4.5, 8.9, 18.8, 37.9 and 76.3 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks of ~250 mL volume, Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker in flasks for an exposure time of 72 hours
- Type: closed, flasks were covered with air-permeable stoppers
- Initial cells density: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (1E07 algal cells per mL) to 100 mL test item solution. The initial cell density was about 1E04 cells/ mL in each test flask.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted algal growth medium (OECD Medium, according to OECD 201) was used as dilution water in the experiment. Separate stock solutions were first prepared in deionised water. The growth medium was prepared by adding an appropriate volume of these different stock solutions to deionised water in order to achieve the final concentrations. Stock solution 1 (macro nutrients): NH4Cl 15.0 mg/L, MgCl2 (anhydrous) 5.6 mg/L, CaCl2 × 2 H2O 18.0 mg/L, MgSO4 × 7 H2O 15.0 mg/L, KH2PO4 1.6 mg/L
Stock solution 2 (iron): FeCl3 × 6 H2O 64.0 μg/L, Na2EDTA × 2H2O 100.0 μg/L
Stock solution 3 (trace elements): H3BO3 185.0 μg/L, MnCl2 × 4 H2O 415.0 μg/L, ZnCl2 3.0 μg/L,
CoCl2 × 6 H2O 1.5 μg/L, CuCl2 × 2 H2O 0.01 μg/L, Na2MoO4 × 2 H2O 7.0 μg/L
Stock solution 4 (bicarbonate): NaHCO3 50.0 mg/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The algal culture flasks were continuously illuminated.
- Light intensity and quality: The average light intensity measured at the position occupied by algal culture flasks during the test was 7998 lux, which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm). The differences in light intensity between the test vessels did not exceed ± 15 % and therefore provided equal conditions for each test vessel.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Other: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
In order to select appropriate test concentrations for use in the definitive test, a preliminary range-finding test was conducted to determine the approximate toxicity of the test item. A stock solution of 100 mg/L (nominal concentration) was prepared by dissolving the test item in OECD medium. The further test solutions were prepared by appropriate dilution of this stock solution. Untreated control ran parallel in the test. Algal cells were exposed to each concentration of the test item plus control, for 72 hours. The test was performed with two replicates in treatment groups and three replicates in the control.
Concentrations: 0 (control), 0.1, 1, 10 and 100 mg/L. In the preliminary test 1.1 % and 100 % inhibition was determined at 10 and 100 mg/L respectively. Based on a non-GLP Preliminary Range-Finding Test, five test concentrations in a geometric series (with a spacing factor of 2) were used in the main test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (K2Cr2O7)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
13.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
33.7 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.:
4.5 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.:
5.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): yes. The cell density in the control has increased from nominal N = 1 × 10 000 cells/mL at the start of the test (0 hours) to N = 72.83× 10 000 cells/mL (mean value) after 72 hours in the in the control. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
- Unusual cell shape: No morphological abnormalities were observed in the control and in the lowest concentrations of 4.5 mg/L (measured) during the test. In the higher concentrations (8.9, 18.8, 37.9 and 76.3 mg/L measured) mostly swollen cells were observed. Furthermore some thin cells and pinches on cells were noticed in the 37.9 mg/L (measured) concentration.
- Colour differences: No effect observed
- Flocculation: No effect observed
- Adherence to test vessels: No effect observed
- Aggregation of algal cells: No effect observed
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

The 72-h EC10, EC20, EC50 for growth were determined to be 13.5, 18.5 and 33.7 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 5.6, 8.1 and 16.6 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 8.9 mg/L. The 72-h NOEC for growth and yield was determined to be 4.5 mg/L. All validity criteria were met. The results are based on measured geometric mean test item concentrations.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: The following results were obtained:
The 72h ErC50: 1.08 mg/L, (95 % confidence limits: 0.79 – 1.51 mg/L)
The 72h EyC50: 0.61 mg/L, (95 % confidence limits: 00.49 – 0.76 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 24 h and 48 h, and at the end of the test (72 hours after the start of the test) using Excel for Windows software.
Percentage inhibition of growth rate (μ) and yield (y) were calculated using Excel for Windows software.
The ECx values and their confidence limits for growth rate and yield were calculated using Probit analysis by SPSS software.
For the determination of the LOEC and NOEC, the calculated mean growth rates and yield at the test concentrations were tested on significant differences to the control values using analysis of variance (ANOVA) and Dunnett’s test (α = 0.05; 2-sided) by SPSS software.
Validity criteria fulfilled:
yes
Conclusions:
The influence of the test substance on the growth of the freshwater algae Raphidocelis subcapitata was assessed in a static dose response test. The 72-hour ErC50 was determined to be 33.7 mg test item/L, the 72-hour NOEC was determined to be 4.5 mg test item/L and the associated 72-hour EC10 was determined to be 13.5 mg test item/L. All values were based on geometric mean measured concentrations.
Executive summary:

A key growth Inhibition Test of the test substance was performed. The purpose of this study was to determine the effect of the test item on the growth of a unicellular green algal species Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata and Selenastrum capricornutum) according to OECD 201 (2011) under GLP conditions. Exponentially growing cultures of Raphidocelis subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test method of application and the test species Raphidocelis subcapitata are recommended by the test guidelines. Nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were investigated in the main study. Corresponding calculated geometric mean measured concentrations were: 4.5, 8.9, 18.8, 37.9 and 76.3 mg/L. Concurrent control (OECD Medium without the test item) was performed. The measured concentrations of the test solutions were in the range of 84 – 87 % of the nominal at the start and 61 – 68 % at the end of the exposure. Biological results are based on the measured geometric mean test item concentrations as the analytically measured test item concentrations were not within the range of ± 20 % of the nominal over the test period of 72 hours. Exponentially-growing cultures of Raphidocelis subcapitata were exposed to the test item under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and, thus, over several algal generations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10 000 cells/mL at the start of the test in all of the test cultures. Glass flasks with a total capacity of 250 mL were used as test vessels. The volume of the test liquid in the vessels was 100 mL. The alga cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. For determination of the test item concentrations, samples were taken from each concentration level and the control at the start and at the end of the test. Concentrations were determined using HPLC-UV method. For the determination of the LOEC and NOEC, ANOVA and Dunnett’s test (α= 0.05; 2-sided) was used and for the determination of the ECx values Probit analysis was used (by SPSS software). The 72-h EC10, EC20, EC50 for growth were determined to be 13.5, 18.5 and 33.7 mg/L, respectively. The 72-h EC10, EC20, EC50 for yield were determined to be 5.6, 8.1 and 16.6 mg/L, respectively. The LOEC values based on growth rate and yield were determined to be 8.9 mg/L. The 72-h NOEC for growth and yield was determined to be 4.5 mg/L. All validity criteria were met. The results are based on measured geometric mean test item concentrations.

Description of key information

In the key 72 -h algal growth inhibition test with Raphidocelis subcapitata, The 72-h EC10 and EC50 for growth rate were determined to be 13.5 and 33.7 mg/L, respectively. The 72-h NOEC for growth was determined to be 4.5 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
33.7 mg/L
EC10 or NOEC for freshwater algae:
13.5 mg/L

Additional information

The influence of the test substance on the growth of the freshwater algae Raphidocelis subcapitata was assessed in a static dose response test according to OECD 201 (2011) under GLP conditions. The 72-hour ErC50 was determined to be 33.7 mg test item/L, the 72-hour NOEC was determined to be 4.5 mg test item/L and the associated 72-hour EC10 was determined to be 13.5 mg test item/L. All values were based on geometric mean measured concentrations. The validit criteria of the test were met.