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Ecotoxicological information

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:
5 April 2016 - 7 July 2016
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)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Individual WAF solutions were prepared by mixing a calculated amount of the test substance in freshwater AAP medium at nominal loading rates of 6.3, 13, 25, 50 and 100 mg/L.
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Test substance used in the WAF solution preparation was a fine powder, measured out directly from the original container or ground with a mortar and pestle prior to measurement. The required mass of EXP1505385 test substance for each solution was weighed in a tared glass beaker and rinsed into a 4000 mL glass aspirator bottle with freshwater AAP medium. WAF solutions were mixed by stirring with a Teflon-coated stirbar and magnetic stirplate for 48 hours. A 30% vortex was maintained throughout the mixing period. After stirring, the test solutions were allowed to settle for one hour prior to use. Test substance was observed in the water column and on the surface of the solutions during mixing at the end of the one hour settling period. After mixing and settling, test solutions at WAF loading rates of 13, 25, 50, and 100 mg/L had a cloudy appearance, which increased with increasing concentration. Negative control solution and test solution at the WAF loading rate of
6.3 mg/L were clear and colorless. The test solutions were decanted from mid-depth. Negative control solution consisted of freshwater AAP medium without test substance added.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The freshwater green alga (Pseudokirchneriella subcapitata) was selected as the test species for this study. The species is representative of an important group of freshwater algae, and was selected for use in the test based upon a past history of use, and ease of culturing in the laboratory. Original algal cultures were obtained from the University of Toronto Culture Collection, and had been maintained in culture medium at Wildlife International, Easton, Maryland. Algal cells used in this test were obtained from Wildlife International cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh media four days prior to test initiation. The negative control organisms were expected to exhibit exponential growth over the 72-hour exposure period.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Samples were collected at approximately 24, 48 and 72 hours after initiation of exposure.
Hardness:
No data
Test temperature:
The test temperature ranged from 23.75 - 24.00 °C
pH:
The pH ranged from 6.0-9.7
Dissolved oxygen:
No Data
Salinity:
No Data
Conductivity:
No Data
Nominal and measured concentrations:
Individual WAF solutions were prepared by mixing a calculated amount of the test substance in freshwater AAP medium at nominal loading rates of 6.3, 13, 25, 50 and 100 mg/L.
Details on test conditions:
Test chambers were held in an environmental chamber at a temperature of 24 ± 2°C. The temperature of a container of water adjacent to the test chambers in the environmental chamber was recorded continuously using an Amega Scientific Corporation centralized monitoring system. The algae were held under 24 hours of cool-white fluorescent lighting throughout the test. The target light intensity was 6,000 lux ± 10%. Light intensity was measured at five locations surrounding the test flasks on the shaker table at test initiation. Light intensity was measured using a SPER Scientific 840006C light meter. The pH of the medium in each treatment and control group was measured at test initiation and at test termination using a Thermo Orion Model 4 Star Plus pH/ISE meter. At test initiation, pH was measured in the individual batches of test solution prepared for each treatment and the control group. At test termination, pH was measured in pooled samples of test solution collected from each of the biological replicates in each treatment and control group.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
56 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
This test achieved all validity criteria specified in the study protocol which was based on the OECD 201 guideline.

After 72 hours of exposure, inhibition of cell density in the nominal 6.3, 13, 25, 50 and 100 mg/L treatment groups (based on nominal WAF loading rates) was -4, -8, 9, 95 and 99%, respectively, relative to the negative control. Inhibition of growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was -1, -1, 2, 53 and 74%, respectively, relative to the negative control. Inhibition of yield in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was -4, -8, 9, 96 and 99%, respectively, relative to the negative control. After 72 hours of exposure, mean cell density, mean growth rate, and mean yield were significantly reduced (Dunnett’s test; p < 0.05) in the 50 and 100 mg/L treatment groups when compared to the negative control group. Consequently, the 72-hour NOEL was determined to be 25 mg/L.

At test initiation algal cells appeared normal. While counting cells in the samples collected on days 2 and 3, abnormal cells were observed in the 50 and 100 mg/L treatment groups when compared to cells in the negative control. Cells in the 6.3, 13 and 25 mg/L treatment groups appeared normal when compared to the control. There was also no abnormal flocculation or aggregation of cells or adherence of algal cells to the test chambers in any of the experimental groups.
Reported statistics and error estimates:
Not applicable; <50% immobility precluded statistical calculation of an EL50 value.

See attached background documents

Validity criteria fulfilled:
yes
Conclusions:
The freshwater green alga, Pseudokirchneriella subcapitata, was exposed to five nominal loading rates of the Susbtance ranging from 6.3 to 100 mg/L. Effects were evaluated based on cell density, yield and growth rate using nominal loading rates. The 72 hour EL50 and EyL50, values were determined to be 32 mg/L, while the ErL50 value was 56 mg/L. The 72-hour NOEL was determined to be 25 mg/L.
Executive summary:

The objective of this study was to determine the toxicity of the test substance to the freshwater green alga (Pseudokirchneriella subcapitata) during a 72-hour exposure period.

 

The green alga (Pseudokirchneriella subcapitata) was exposed to five nominal WAF loading rates and a negative control (culture medium) under static conditions for 72 hours. Six replicate test chambers in the control group and three replicate test chambers in each treatment group were maintained. Due to the low solubility of the test substance, test solutions were prepared as water accommodated fractions (WAFs) and test concentrations are based upon the loading rate for each test solution. Nominal WAF loading rates selected for use in this study were 6.3, 13, 25, 50 and 100 mg/L. Particulates were observed in the 25, 50 and 100 mg/L test solutions throughout the 72-hour exposure. 

 

The toxicity of Substance to green alga (Pseudokirchneriella subcapitata) was determined by evaluating changes in cell density over a 72-hour exposure period. Cell densities were used to calculate growth rates at 24-hour interval of exposure and yield at 72 hours of exposure.

 

This test achieved all validity criteria specified in the study protocol which was based on the OECD 201 guideline.

 

After 72 hours of exposure, inhibition of cell density in the nominal 6.3, 13, 25, 50 and 100 mg/L treatment groups (based on nominal WAF loading rates) was -4, -8, 9, 95 and 99%, respectively, relative to the negative control. Inhibition of growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was -1, -1, 2, 53 and 74%, respectively, relative to the negative control. Inhibition of yield in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was -4, -8, 9, 96 and 99%, respectively, relative to the negative control. After 72 hours of exposure, mean cell density, mean growth rate, and mean yield were significantly reduced (Dunnett’s test; p< 0.05) in the 50 and 100 mg/L treatment groups when compared to the negative control group. Consequently, the 72-hour NOEL was determined to be 25 mg/L.

 

At test initiation algal cells appeared normal. While counting cells in the samples collected on days 2 and 3, abnormal cells were observed in the 50 and 100 mg/L treatment groups when compared to cells in the negative control. Cells in the 6.3, 13 and 25 mg/L treatment groups appeared normal when compared to the control. There was also no abnormal flocculation or aggregation of cells or adherence of algal cells to the test chambers in any of the experimental groups.

 

The freshwater green alga (Pseudokirchneriella subcapitata) was exposed to five nominal loading rates of the Substance ranging from 6.3 to 100 mg/L. Effects were evaluated based on cell density, yield and growth rate using nominal loading rates. The 72 hour EL50 and EyL50, values were determined to be 32 mg/L, while the ErL50 value was 56 mg/L. The 72-hour NOEL was determined to be 25 mg/L.

Description of key information

Pseudokirchneriella subcapitata, ErL50 (72h) = 56 mg/L (Growth rate); NOEL (72h) = 0.25 mg/L.

Key value for chemical safety assessment

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

Additional information