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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:
22 - 25 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
not specified
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Mitsuboshi Chemical Co, Ltd. Lot: YY00G7P285
- Expiration date of the lot/batch: Stated on certificate of analysis (dated May 30, 2016) as "for 2 years after shipment date"
- Purity test date: 99.8%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Not specified - "The test solutions were utilized on the same day as preparation; thus, assessment of
stability of the test solution was not required".
- Solubility and stability of the test substance in the solvent/vehicle: Not specified - "Based on preliminary methodology work and results of the range-finding test, the test material was expected to be completely soluble at the selected test concentrations for the study".
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 10 mg/L (nominal) bulk test solution, equal to the highest test concentration, was prepared via direct addition of the test material to AAP medium. An aliquot containing 10 mg K-Salt (no adjustment for purity) was added to a 1-L volumetric flask filled to volume with AAP medium. The flask was mixed until the solution apppeared homogenous. The remaining bulk test solutions were prepared as dilutions of the 10 mg/L bulk test solution.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 10 mg/L bulk test solution
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Not specified
- Sampling method: Samples were collected from the bulk test solutions at test initiation and from pooled replicate test vessels at exposure termination. Blank replicates were analyzed separately at exposure termination. To confirm test solution preparations and associated stability over the course of the exposure, the collected samples were analyzed for K-Salt by high pressure liquid chromatography/diode array detection (HPLC/DAD).
- Sample storage conditions before analysis: Not specified
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 10 mg/L (nominal) bulk test solution, equal to the highest test concentration, was prepared via direct addition of the test material to AAP medium. An aliquot containing 10 mg K-Salt (no adjustment for purity) was added to a 1-L volumetric flask filled to volume with AAP medium. The flask was mixed until the solution apppeared homogenous. The remaining bulk test solutions were prepared as dilutions of the 10 mg/L bulk test solution
- Eluate: n/a
- Differential loading: n/a
- Controls: AAP medium control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Not specified
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Not specified
- Source (laboratory, culture collection): Initially obtained from the University of Texas at Austin Culture Collection in June 2015, stock cultures of this organism were maintained axenically by periodic transfer into sterile medium. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2ºC.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Axenically by periodic transfer into sterile medium. Algae were cultured under continuous illumination of approximately 5,200 ± 520 lux at a temperature of 23 ± 2ºC.

ACCLIMATION
- Acclimation period: n/a
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: Not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
n/a
Hardness:
Not specified
Test temperature:
22 - 23 ºC
pH:
7.4 - 7.9
Dissolved oxygen:
Not specified
Salinity:
Not specified
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations = 0 (AAP medium control), 0.10, 0.31, 1.0, 3.1 and 10 mg/L
Mean measured concentrations =
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized 250-mL borosilicate Erlenmeyer flasks with foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL
- Aeration: Not specified
- Type of flow-through (e.g. peristaltic or proportional diluter): Not specified
- Renewal rate of test solution (frequency/flow rate): Not specified
- Initial cells density: 10,000 cells/mL
- Control end cells density: 354.7 x 10^4 cells/mL (mean of 6 control replicates)
- No. of organisms per vessel: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes (AAP growth medium)
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Source - Lake Huron water supplied by the City of Midland Water Treatment Plant.
- Total organic carbon: Not specified
- Particulate matter: Not specified
- Metals: Not specified
- Pesticides: Not specified
- Chlorine: Not specified
- Alkalinity: Not specified
- Ca/mg ratio: Not specified
- Conductivity: Not specified
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature continuously monitored for 72 hour exposure period. pH measured at 0 hours before exposure period and at 72 hours at termination. Light intensity was measured at each position where inoculated replicate vessels
were placed during the exposure.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: pH for medium use on tests was adjusted to 7.5 ± 0.1 prior to solution preparation
- Photoperiod: 24 hour light
- Light intensity and quality: 4460 - 5250 lux
- Salinity (for marine algae): n/a

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Electronic particle counter. Total cell counts (average of two cell counts per replicate test vessel) were determined at approximately 24, 48 and 72 hours (± 1 hour from exposure initiation).
- Chlorophyll measurement: No
- Other: Morphilogical observations at test termination observed under a microscope

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline: n/a

- Range finding study: Yes
- Test concentrations: 0 (AAP medium control), 0.1, 1.0, 10.0 and 100 mg/L test item
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 3.22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.288 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
ca. 0.119 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Details on results:
- Exponential growth in the control (for algal test): Not specified
- Observation of abnormalities (for algal test): At test termination, microscopic evaluation of algal cells for the AAP medium control and the 0.0250, 0.119, 0.288 and 1.51 mg/L test levels revealed no abnormal observations. Microscopic evaluation of algal cells for the 7.17 mg/L test level revealed no algal cells present.
- Unusual cell shape: Not specified
- Colour differences: Not specified
- Flocculation: Not specified
- Adherence to test vessels: Not specified
- Aggregation of algal cells: Not specified
- Other: n/a
- Any stimulation of growth found in any treatment: Not specified
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Not specified
- Effect concentrations exceeding solubility of substance in test medium: Not specified
Results with reference substance (positive control):
It was confirmed that the test condition was valid, because the degree of degradation of aniline on Day7 and 14 determined by BOD was 75% and 76%, respectively
Reported statistics and error estimates:
Cell yield = EC50 95% confidence interval 0.720 (0.543 - 0.897) mg/L test item.
Growth rate = EC50 95% confidence interval 3.22 (2.69 - 3.75) mg/L test item.

Concentration of suspended substance in activated sludge was 6,900 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity values for Pseudokirchneriella subcapitata exposed to K-Salt over a 72-hour static exposure period and based on mean measured concentrations were as follows:
Cell yield: 72-hour EyC50: 0.720 mg/L (95% C.I. = 0.543 – 0.897 mg/L); 72-hour NOEC: 0.119 mg/L.
Growth rate: 72-hour ErC50: 3.22 mg/L (95% C.I. = 2.69 – 3.75 mg/L); 72-hour NOEC: 0.288 mg/L
Executive summary:

The potential effects of K-Salt to the freshwater green algae, Pseudokirchneriella subcapitata, over a 72-hour static exposure period were assessed according to OECD 201. These data were used to calculate effective concentration (EC) and no-observed-effect concentration (NOEC) values for 72-hour cell yield and 72-hour average specific growth rate.

Based on the results of a preliminary study, nominal test item concentrations were 0 (AAP medium control), 0.10, 0.31, 1.0, 3.1 and 10 mg/L. Mean measured test item concentrations were < LLQ (AAP medium control), 0.0250, 0.119, 0.288, 1.51 and 7.17 mg/L. Initial cell density in all replicates per test item concentration was 10,000 cells/mL.

The acute toxicity values for Pseudokirchneriella subcapitata exposed to K-Salt over a 72-hour static exposure period and based on mean measured concentrations were as follows:

Cell yield: 72-hour EyC50: 0.720 mg/L (95% C.I. = 0.543 – 0.897 mg/L); 72-hour NOEC: 0.119 mg/L.

Growth rate: 72-hour ErC50: 3.22 mg/L (95% C.I. = 2.69 – 3.75 mg/L); 72-hour NOEC: 0.288 mg/L.

Description of key information

Growth rate: 72-hour ErC50: 3.22 mg/L (95% C.I. = 2.69 – 3.75 mg/L); 72-hour NOEC: 0.288 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
3.22 mg/L
EC10 or NOEC for freshwater algae:
0.288 mg/L

Additional information