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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:
From June 09, 2009 to June 09, 2010
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
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were collected at approximately 0, 72, and 96 hours to measure concentrations of the test substance. At test initiation samples were collected for each treatment and control group prior to distribution into test chambers. At 72 hours, samples were collected from individual analytical replicates. At test termination, the biological replicates from each respective treatment and control group were pooled and then sampled. All samples were collected in glass vials and processed immediately for analysis.
Vehicle:
no
Details on test solutions:
A primary stock solution was prepared at a nominal concentration of 30 mg test substance/L, by mixing 15 mg of test substance in algal medium, to achieve a final volume of 500 mL. The stock was inverted at least 20 times to mix, and appeared clear and colorless with foam on the surface. A secondary stock solution was prepared at a nominal concentration of 3.0 mg/L by mixing 100 ml of the primary stock in algal medium to a final volume of 1000 mL. The secondary stock solution, which also acted as the highest concentration test solution, was proportionally diluted with freshwater algal medium to prepare the six additional test solutions at nominal concentrations of 0.047, 0.094, 0.19, 0.38, 0.75, and 1.5 mg/L. Test solutions were mixed by inversion and appeared clear and colorless. The negative control was freshwater algal medium without test substance.
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, Ltd., Easton, Maryland. Algal cells used in this test were obtained from Wildlife International, Ltd. 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 three 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.

Test Apparatus: test chambers were sterile, 250-mL Erlenmeyer flasks plugged with foam stoppers and contained 100 mL of test or control medium.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24.8 -25.6 st. C
pH:
7.5-7.7 (test start); 7.9-8.9 (test termination)
Nominal and measured concentrations:
Nominal: Negative Control; 0.047 mg/L; 0.094 mg/L; 0.19 mg/L; 0.38 mg/L; 0.75mg/L; 1.5 mg/L; 3.0 mg/L
Mean measured: < LOQ; 0.039 mg/L; 0.068 mg/L; 0.16 mg/L; 0.29 mg/L; 0.64 mg/L; 1.3 mg/L; 2.7 mg/L
Details on test conditions:
The algal cells were cultured and tested in freshwater algal medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified Wildlife International, Ltd. well water. The test medium then was prepared by adding appropriate volumes of the stock nutrient solutions to purified well water. The pH was 7.4. The medium was then sterilized by filtration (0.22 µm) prior to use.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% Confidence limits
Remarks:
0.71 – 1.14 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
cell number
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.01 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence limits
Remarks:
1.91 – 2.11 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Conditions for the validity of the test:
1) The mean cell density in the control flasks increased by a factor greater than 16 within three days. The factor was 163
2) The coefficient of variation of average specific growth rate in the control replicates during the whole test period did not exceed 7%. It was 1.01%.
3) The mean percent coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) did not exceed 35%. It was 7.56%.
4) Algal growth in the controls reached logarithmic growth phase by 96 hours.

- After 72 hours of exposure, inhibition of cell density in the 0.039, 0.068, 0.16, 0.29, 0.64, 1.3, and 2.7 mg test substance/L treatment groups was 11, -2.3, -0.28, 10, 39, 66, and 97%, respectively, relative to the negative control.
- Percent inhibition of area under the growth curve in the 0.039, 0.068, 0.16, 0.29, 0.64, 1.3, and 2.7 mg test substance/L treatment groups was 12, -0.07, 2.0, 15, 34, 59, and 94%, respectively, relative to the negative control.
- Percent inhibition of growth rate in the 0.039, 0.068, 0.16, 0.29, 0.64, 1.3, and 2.7 mg test substance/L treatment groups was 2.3, -0.47, 1.2, 1.9, 9.7, 21, and 72%, respectively, relative to the negative control.
Dunnett’s test indicated that cell density, area under the growth curve, and growth rate were significantly reduced (p<0.05) in the 0.64, 1.3, and 2.7 mg test substance/L treatment levels relative to the negative control. EC20 values were calculated as a 72-hour NOAEC estimate and were 0.52, 0.56, and 1.27 mg/L for the cell density, biomass and growth rate, respectively. The 72-hour EC50, EbC50, and ErC50 values were 0.90, 1.02, and 2.01 mg test substance/L, respectively.
Reported statistics and error estimates:
The calculation of cell densities, areas under the growth curve, growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows”, Version 8.2.
Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, the 72 h EC50, based on cell density, was 0.90 mg/L with the 95% confidence intervals of 0.71 – 1.14 mg/L and 72 h NOEC was 0.52 mg/L. The 72 h EC50, based on growth rate, was 2.01 mg/L, with the 95% confidence intervals of 1.91 – 2.11mg/L and 72 h NOEC was 1.27 mg/L. The 72 h EC50, based on biomass , was 1.02 mg/L with the 95% confidence intervals of 0.80 – 1.29 mg/L and 72 h NOEC was 0.56 mg/L.
Executive summary:

A study was conducted to determine the toxicity of the test substance to the freshwater green alga, Pseudokirchneriella subcapitata, during a 96 h exposure period according to OECD Guideline 201, EU Method C.3 and EPA OPPTS Method 850.5400, in compliance with GLP. The algae were exposed to seven test concentrations and a negative control (culture medium) under static conditions for 96 h. Six replicate test chambers in the control group and three replicate test chambers in each treatment group were maintained. An additional replicate was included in each control and treatment group and served as an analytical replicate. Nominal test substance concentrations were based upon the results of an exploratory range finding toxicity test and were of: 0.047, 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L. Measured concentrations were determined from samples of medium collected from the stock solutions used to prepare each treatment level at the beginning of the test, from individual analytical replicates on Day 3, and from pooled replicates of each treatment and control group at test termination. At test start, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of  approximately 10,000 Pseudokirchneriella cells/mL. Samples were collected from each replicate test chamber at approximately 24 h intervals to determine cell densities, which were subsequently used to calculate areas under the growth curve and growth rates. Cell densities, areas under the growth curve and growth rates were used to calculate percent inhibition values relative to the control over the 96 h exposure period. EC50, EbC50 and ErC50 values were calculated, when possible, based upon cell density, area under the growth curve and growth rate, respectively. Under the study conditions, the 72 h EC50, based on cell density, was 0.90 mg/L with the 95% confidence intervals of 0.71 – 1.14 mg/L and 72 h NOEC was 0.52 mg/L. The 72 h EC50, based on growth rate, was 2.01 mg/L, with the 95% confidence intervals of 1.91 – 2.11mg/L and 72 h NOEC was 1.27 mg/L. The 72 h EC50, based on biomass, was 1.02 mg/L with the 95% confidence intervals of 0.80 – 1.29 mg/L and 72 h NOEC was 0.56 mg/L (Minderhout, 2010).

Description of key information

A study was conducted to determine the toxicity of the test substance to the freshwater green alga, Pseudokirchneriella subcapitata, during a 96 h exposure period according to OECD Guideline 201, EU Method C.3 and EPA OPPTS Method 850.5400, in compliance with GLP. The algae were exposed to seven test concentrations and a negative control (culture medium) under static conditions for 96 h. Six replicate test chambers in the control group and three replicate test chambers in each treatment group were maintained. An additional replicate was included in each control and treatment group and served as an analytical replicate. Nominal test substance concentrations were based upon the results of an exploratory range finding toxicity test and were of: 0.047, 0.094, 0.19, 0.38, 0.75, 1.5 and 3.0 mg/L. Measured concentrations were determined from samples of medium collected from the stock solutions used to prepare each treatment level at the beginning of the test, from individual analytical replicates on Day 3, and from pooled replicates of each treatment and control group at test termination. At test start, an inoculum of the algal cells was added to each test chamber to achieve a nominal concentration of  approximately 10,000 Pseudokirchneriella cells/mL. Samples were collected from each replicate test chamber at approximately 24 h intervals to determine cell densities, which were subsequently used to calculate areas under the growth curve and growth rates. Cell densities, areas under the growth curve and growth rates were used to calculate percent inhibition values relative to the control over the 96 h exposure period. EC50, EbC50 and ErC50 values were calculated, when possible, based upon cell density, area under the growth curve and growth rate, respectively. Under the study conditions, the 72 h EC50, based on cell density, was 0.90 mg/L with the 95% confidence intervals of 0.71 – 1.14 mg/L and 72 h NOEC was 0.52 mg/L. The 72 h EC50, based on growth rate, was 2.01 mg/L, with the 95% confidence intervals of 1.91 – 2.11mg/L and 72 h NOEC was 1.27 mg/L. The 72 h EC50, based on biomass, was 1.02 mg/L with the 95% confidence intervals of 0.80 – 1.29 mg/L and 72 h NOEC was 0.56 mg/L (Minderhout, 2010).

Key value for chemical safety assessment

EC50 for freshwater algae:
2.01 mg/L
EC10 or NOEC for freshwater algae:
1.27 mg/L

Additional information