Alfalfa, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 May to 05 July 2018

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:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at approximately 4ºC in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item. A 23-Hour stirring period followed by a 1-Hour standing period was considered sufficient to ensure equilibration between the test item and aqueous phase.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF test groups
- Sampling method: not available
- Sample storage conditions before analysis: not available

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).
- Eluate: not specified
- Differential loading: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 10 and 100 mg/L for a period of 72 hours. Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L
- Controls: control group: reconstituted water (Elendt M7), positive control: potassium dichromate at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): not specified
- Method of cultivation: not specified
ACCLIMATION
- Acclimation period: not specified
- Culturing media and conditions (same as test or not): Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 1000 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1°C until the algal cell density was approximately 10'000 to 100'000 cells/mL.
- Any deformed or abnormal cells observed: not specified

Test type:

other: shaken at 150 rpm

Water media type:

other: culture media

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1°C

pH:

culture medium: 7.5
definitive test: 8.3-8.6

Nominal and measured concentrations:

Range finding test: 1.0, 10, 100 mg/L nominal loading rate
Definitive test: 1.0, 3.2, 10, 32 and 100 mg/L nominal loading rate

Details on test conditions:

TEST SYSTEM
- Test vessel: range-finding test: conical flasks
- Material, size, headspace, fill volume: glass, 250 mL
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): none
- Renewal rate of test solution (frequency/flow rate): none
- No. of organisms per vessel: 5.00 x 1000 cells per mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): none
- Biomass loading rate: not available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: culture medium
- Total organic carbon: - Particulate matter: none
- Metals: not available
- Pesticides: not available
- Chlorine: not available
- Alkalinity: 7.5
- Ca/Mg ratio: not available
- Conductivity: not available
- Salinity: not available
- Culture medium different from test medium: not applicable
- Intervals of water quality measurement: not applicable

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continuous illumination provided by warm white lighting (380-730 nm)
- Light intensity: 7000 lux
EFFECT PARAMETERS MEASURED (with observation intervals if applicable): inhibition of algal growth

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L nominal loading rate
- Results used to determine the conditions for the definitive study: effects on algal growth

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

other: EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

other: EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: NOEL

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

other: LOEL

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): there were no abnormalities detected in any of the control or test cultures.
- Unusual cell shape: not specified
- Colour differences: not specified
- Flocculation: not specified
- Adherence to test vessels: not specified
- Aggregation of algal cells: none
- Other: none
- Any stimulation of growth found in any treatment: yes, at 3.2 and 32 mg/L loading rate WAF
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50:
ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
- Other:
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package.

See attached additional information on results.

Validity criteria fulfilled:

yes

Conclusions:

In algal growth inhibition study performed on Pseudokirchneriella subcapitata, and conducted according to OECD test guideline 201 and to GLP, the No Observed Effect Level values for growth rate and yield were determined at 100 and 32 mg/L loading rate as Water Accommodated Fraction after 72 hours of exposure to Alfalfa, ext., respectively. The effective loading causing 50% response (EL50) was determined at >100 mg/L loading rate WAF for growth rate and yield.

Executive summary:

A test conducted on Pseudokirchneriella subcapitata according to OECD test guideline 201 and to GLP was performed to assess the potential of Alfalfa, ext. to inhibit algal growth.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test and initial experiment, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32, and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Total Organic Carbon (TOC) analysis of the test preparations at 0 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), considered to be 1.0 mg/L, to 22 mg/L. A slight decline in carbon concentrations was observed at 72 hours in the range of less than the LOQ to 15 mg/L.

There were no abnormalities detected in any of the control or test cultures.

The NOEL values for growth rate and yield were determined at 100 and 32 mg/L loading rate as WAF after 72 hours of exposure to Alfalfa, ext., respectively. The effective loading causing 50% response (EL50) was determined at >100 mg/L loading rate WAF for growth rate and yield.

Description of key information

Growth rate at 72 hours:

NOEL: 100 mg/L loading rate WAF

EL50: >100 mg/L loading rate WAF

Yield at 72 hours:

NOEL: 32 mg/L loading rate WAF

LOEL: 100 mg/L loading rate WAF

EL50: >100 mg/L loading rate WAF

Additional information

A test conducted on Pseudokirchneriella subcapitata according to OECD test guideline 201 and to GLP was performed to assess the potential of Alfalfa, ext. to inhibit algal growth.

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to WAFs of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32, and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Total Organic Carbon (TOC) analysis of the test items at 0 hours showed measured carbon concentrations to range from less than the limit of quantification (LOQ), considered to be 1.0 mg/L, to 22 mg/L. A slight decline in carbon concentrations was observed at 72 hours in the range of less than the LOQ to 15 mg/L.

There were no abnormalities detected in any of the control or test cultures.

The NOEL values for growth rate and yield were determined at 100 and 32 mg/L loading rate as WAF after 72 hours of exposure to Alfalfa, ext., respectively. The effective loading causing 50% response (EL50) was determined at >100 mg/L loading rate WAF for growth rate and yield.