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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:
June - August 2013
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes
Specific details on test material used for the study:
Identifier: EXP1200078
Appearance: Very dark brown (almost black) viscous liquid
Batch: E00275-350
Sample Expiration Date: end-2013
Purity:100%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
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. The required mass of EXP1200078 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 47 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. The test solutions were decanted from mid-depth and vacuum filtered through a 0.45 µm alpha cellulose disc prior to distribution into the replicate test vessels. All test solutions appeared clear and colorless following filtering as all particulate matter was removed.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
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 96-hour exposure period. Exponential growth phase, defined as the period of growth where the algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve on a logarithmic scale.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
not stated
Test temperature:
24 ± 2C.
pH:
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 replicates in each
treatment and control group.

pH values at 0h = 7.3 - 8.3
pH values at 96h = 8.7 - 9.2
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
Nominal / loading: negative control, 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 ± 2C

The algae were held under continuous cool-white fluorescent lighting throughout the test. The target light intensity was
6,000 lux ± 10%.

Test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and contained 100 mL of test or control
medium. The test flasks were shaken continuously at 100 rpm.

Initial cell density: The density of the culture was 4.35 x 106 cells/mL and 0.23 mL of the culture was added to each test
chamber to achieve a nominal concentration of approximately 10,000 cells/mL at test initiation.

Replicates per concentration (including control): 3

Growt medium: AAP; US EPA
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Area Under Growth Curve (mg/L)
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Area Under Growth Curve (mg/L)
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Area Under Groth Curve
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Area Under Growth Curve (mg/L)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
96 h
Dose descriptor:
NOELR
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:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
mg/L
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
mg/L
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
mg/L
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks:
mg/L
Details on results:
After 72 hours of exposure, inhibition of area under the growth curve in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 14, 13, 20, 12 and 25%, respectively, relative to the negative control. Inhibition of growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 3, 3, 4, 1 and 5%, respectively, relative to the negative control. Inhibition of yield in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 13, 13, 20, 6 and 23%, respectively, relative to the negative control. After 72 hours of exposure, mean area under the growth curve, mean growth rate and mean yield were significantly reduced (Dunnett’s test; p < 0.05) in the 25 and 100 mg/L treatment groups when compared to the negative control group (Table 4, attached). The 72-hour NOEL was determined to be 13 mg/L.

After 96 hours of exposure, inhibition of area under the growth curve in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 12, 10, 14, 13 and 26%, respectively, relative to the negative control. Inhibition of growth rate in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 2, 1, 2, 3 and 5%, respectively, relative to the negative control. Inhibition of yield in the 6.3, 13, 25, 50 and 100 mg/L treatment groups was 11, 7, 9, 16 and 27%, respectively, relative to the negative control. After 96 hours of exposure, 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. Mean area under the growth curve was significantly reduced in the 25 and 100 mg/L treatment groups (Dunnett’s test; p < 0.05) when compared to the negative control (Table 5, attached). The 96-hour NOEL was determined to be 13 mg/L, based on statistically significant reductions in area under the growth curve.
Validity criteria fulfilled:
yes
Remarks:
This test achieved all validity criteria specified in the study protocol which was based on the OECD 201 guideline.
Conclusions:
Pseudokirchneriella subcapitata were exposed to five nominal loading rates of 6.3 to
100 mg/L and evaluated for effects on area under the growth curve, growth rate and yield. EL50
values could not be calculated since less than 50% inhibition was observed in all treatment groups for
each of the parameters examined. EL50 values for all three endpoints were empirically estimated to
be greater than the highest loading rate tested, 100 mg/L. The 72 and 96-hour NOEL values
were determined to be 13 mg/L, at both exposure intervals.
Executive summary:

Exposure of Pseudokirchneriella subcapitata to test article gave EL50 values greater than 100 mg/L which was the highest loading rate WAF.

Description of key information

A well conducted study using Pseudokirchneriella subcapitata (OECD 201)were exposed to five nominal loading rates of 6.3 to 100 mg/L and evaluated for effects on area under the growth curve, growth rate and yield. EL50 values could not be calculated since less than 50% inhibition was observed in all treatment groups for each of the parameters examined. EL50 values for all three endpoints were empirically estimated to be greater than the highest loading rate tested, 100 mg/L. The 72 and 96-hour NOEL values were determined to be 13 mg/L, at both exposure intervals.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
100 mg/L

Additional information

Pseudokirchneriella subcapitata exposed to test article resulted in EL50 values greater than 100 mg/L which was the
highest loading rate WAF.