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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:
February 25 2003 to September 11 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, 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
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were collected at approximately 0 and 96 hours to measure concentrations of the test substance. Samples at test initiation were collected from the individual batches of test solution prepared for each treatment and control group prior to the addition of the algae. At test termination, samples were collected fom the pooled three replicates from each treatment and control group. All samples were taken directly from each respective treatment vessel and were analyzed immediately without storage.
Vehicle:
yes
Details on test solutions:
A primary stock solution was prepared by dissolving Phosflex TXP in dimethylformamide (DMF) at a nominal concentration of 10 mg/ml. The stock solution was mixed by inversion and appeared clear and colourless. Secondary stock solutions at nominal concentrations of 0.63, 1.3, 2.5 and 5.0 mg/mL were prepared in DMF by serial dilution of the primary stock. Aliquots of the appropriate stock solutions then were diluted with freshwater algal medium to prepare test solutions at nominal concentrations of 63, 130, 250, 500 and 1000 µg/L. All treatment and control groups appeared clear and colourless. The solvent concentration in the solvent control and treatment groups was 0.1 mL/L.
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 mediumat 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. The culture was last transferred to fresh medium 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 algal cells are dividing at a constant rate, is indicated by the linear section of the growth curve.
Prior to test initiation, an inoculum of of the algal cells was prepared in freshwater algal medium at a concentration of approximately 1.0 x 10 to the power of 6 cells/mL. The concentration of algal cells was verified using a hemacytometer and microscope, and 0.1 mL of the inoculum was added to each test chamber to achieve a nominal concentration of approximately 10000 cells/mL at test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None.
Hardness:
Not reported.
Test temperature:
24 +/- 2 °C.
pH:
7.9 - 9.8 during test
Dissolved oxygen:
Not reported.
Salinity:
Not applicable.
Nominal and measured concentrations:
Nominal: 63, 130, 250, 500, 1000 µg/L

Details on test conditions:
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 labelled with the project number, concentration and replicate, and were indiscriminately positioned daily on mechanical shakers in an environmental chamber designed to maintain the desired test temperature throughout the test. The test flasks were shaken continuously at 100 rpm.
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 twice daily during the test using a liquid-in-glass thermometer.
The algae were held under 24 hours per day of cool-white fluorescent lighting at an intensity of 4300 +/- 10% lux. Light intensity was measured daily at five locations surrounding the test flas ks on the shaker table. 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 test termination using a Thermo Orion Model 525 Aplus pH 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 prepared for each treatment and the control group.

Algal growth measurements and observations
Test medium samples were collected from each replicate of the treatment and control group for the determination of algal cell densities. Samples were collected at approximately 24 hour intervals during the 96 hour exposure and were held for a maximum of four days under refridgerated conditions sufficient to inhibit growth until cell counts could be performed. Cell counts were performed using an electronic particle counter (Coulter electronics Inc.) Prior to conducting cell counts, the linearity of the instrument response was determined at settings previously established for Selenastrum capricornutum. A primary counting standard containing Selenastrum capricornutum cells was prepared, the density was verified using a hemacytometer and a microscope and the standard was subsequently diluted to provide a series of seven counting standards for the determination of instrument linearity. Theoretical densities were assigned to each secondary counting standard based upon the verified density of the primary counting standard and the dilution ratio. The cell densities of the counting standards were measured using the electronic particle counter and were compared to the theoretical densities by performing a least squares regression analysis. Cell counts for samples collected during the test were conducted once instrument linearity was demonstrated (i.e. the R-squared value obtained through the regression analysis was 1.0). A single aliquot of each sample collected during the test was diluted with an electrolyte solution (Isoton). Three 0.5 mL volumes of the diluted sample were counted and the resulting counts were averaged. The cell density of the sample was determined by adjusting the mean cell count (cell/mL) obtained using the particle counter, based upon the y intercept and slope calculated through the regression analysis and the dilution factor.
Samples of test solution were collected from each of the replicates per treatment and control group at the end of the test. These samples were pooled within their respective treatments and subsamples were removed and examined microscopically for atypical cell morphology (e.g. changes in cell shape, size or colour). Cells in the replicate test chambers were also assessed for aggregations or flocculations of cells, and adherence of cells to the test chamber.
The calculation of cell densities, area under the growth curve, growth reates and percent inhibition values, as well as all statistical analyses, was conducted using the "SAS System for Windows", Version 8.02.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 011 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
other: cell density, area under the growth curve and growth rate
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 1 011 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
other: cell density, area under the growth curve and growth rate
Key result
Duration:
72 h
Dose descriptor:
other: NOAEC
Effect conc.:
112 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
other: cell density and area under the growth curve (biomass)
Key result
Duration:
96 h
Dose descriptor:
other: NOAEC
Effect conc.:
112 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
dissolved
Basis for effect:
other: cell density and area under the growth curve (biomass)
Key result
Duration:
72 h
Dose descriptor:
other: NOAEC
Effect conc.:
233 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks:
growth rate
Key result
Duration:
96 h
Dose descriptor:
other: NOAEC
Effect conc.:
1 011 µg/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
growth rate
Details on results:
Nominal concentrations selected for use in this study were 63, 130, 250, 500 and 1000 µg/L. Samples collected on Day 0 had recoveries of 82, 86, 93, 98 and 101% of nominal concentrations, respectively. Samples collected on Day 4 had recoveries of 54, 57, 51, 63 and 62% of nominal concentrations, respectively. Due to the decline in the test substance concentrations during the test, Day 0 recoveries were used in the estimation of EC50 values.
Temperatures ranged from 23.3 to 24.7 °C and were within the 24 +/- °C range established for the test. The pH of the test solutions at test initiation was 7.9 and at test termination ranged from 8.9 to 9.8. The pH tended to increase relative to increases in algal densities, which is typical for tests conducted with Selenastrum capricornutum. The light intensity ranged from 3910 to 4710 lux, which was in the desired range of 4300 +/- 10% lux.
The toxicity of Phosflex TXP to Selenastrum capricornutum was determined by evalutaing changes in cell density over the 96 hour exposure period. Cell densities were used to calculate areas under the growth curve (biomass) and growth rates for each 24 hour interval of exposure. Changes in mean cell density in the negative control replicates over the 96-hour exposure period indicated that exponential growth of cells occured in those replicates.
After 72 hours of exposure, inhibition of cell density in the 52, 112, 233, 485 and 1011 µg treatment groups was 27, 6.1, 26, 42 and 45 %, respectively, relative to the pooled controls. Treatment related effects were apparent in the three highest test concentrations. After 72 hours of exposure, the difference between the pooled control and the 52, 233, 485 and 1011 µg/L treatment levels was statistically significant (Dunnett's test, p<0.05), but differences in the 112 µg/L treatment level was not statistically significant (Dunnett's test, p>0.05). Due to the lack of concentration dependant response, the effects noted in the 52 µg/L treatment level were not considered to be treatment related. Consequently, the 72-hour NOAEC for cell density was 112 µg/L. After 96 hours of exposure, inhibition of cell density in the 52, 112, 233, 485 and 1011 µg/L treatment groups was 15, 4.1, 18, 22 and 31%, respectively, relative to the pooled controls. Treatment related effects were apparent in the three highest test concentrations. Dunnett's test indicated that cell densities were significantly reduced(p<0.05) in the 233, 485 and 1011 µg/L treatment levels. Consequently, the 96 hour NOAEC for cell density was 112 µg/L.
After 72 hours of exposure, inhibition of ara under the growth curve in the 52, 112, 233, 485 and 1011µg/L treatment groups was 25, 6.2, 25, 39 and 40%, respectively, relative to the pooled controls. Treatment related effects were apparent in the three highest test concentrations. After 72 hours of exposure, the difference between the pooled control and the 52, 233, 485 and 1011 µg/L treatment levels was statistically significant (Dunnett's test, p<0.05), but differences in the 112 µg/L treatment level was not statistically significant (Dunnett's test, p>0.05). Due to the lack of concentration dependant response, the effects noted in the 52 µg/L treatment level were not considered to be treatment related. Consequently, the 72-hour NOAEC for area under the growth curve was 112 µg/L. After 96 hours of exposure, inhibition of area under the growth curve in the 52, 112, 233, 485 and 1011 µg/L treatment groups was 19, 5.0, 21, 30 and 36% respectively, relative to pooled controls. Treatment related effects were apparent in the three highest test concentrations. While Dunnet's test indicated that area under the growth curve was significantly reduced (p<0.05) in only the 485 and 1011 µg/L treatment levels, the 233 µg/L treatment group did show 21% inhibition when compared to the negative control. Although not significantly different a 21% inhibition is considered to be a treatment related effect. Consequently, the 96-hour NOAEC for area under the growth curve was 112µg/L.
Aftyer 72 hours of exposure, inhibition of growth rate in the 52, 112, 233, 485 and 1011 µg/L treatment groups was 7.1, 1.4, 6.4, 11 and 13%, respectively, relative to the pooled controls. Treatment related effects were apparent in the three highest test concentrations. After 72 hours of exposure, the difference between the pooled control and the 52, 233, 485 and 1011 µg/L treatment levels was statistically significant (Dunnett's test, p<0.05), but differences in the 112 µg/L treatment level was not statistically significant (Dunnett's test, p>0.05). Due to the lack of concentration dependant response, the effects noted in the 52 µg/L treatment level were not considered to be treatment related. Consequently, the 72-hour NOAEC for growth rate was 112 µg/L. After 96 hours of exposure, inhibition of growth rate in the 52, 112, 233, 485 and 1011 µg/L treatment groups was 2.8, 0.70, 3.4, 4.3 and 6.5%, respectively, relative to the pooled controls. While Dunnett's test indicated that growth rate was significantly reduced (p<0.05) in the 485 and 1011 µg/L treatment levels, the 4.3 and 6.5% inhibition from the control in 485 and 1011 µg/L treatment groups, respectively, was not considered to be treatment related, since this amount of inhibition was small (<10%) and considered to be within an acceptable limit for Selenastrum capricornutum. Consequently, the 96-hour NOEC for growth rate was 1011 µg/L.
After 96 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/flocculation of algae in the controls or in any treatment group. There were no noticable changes in cell morphology at concentrations at or below 112 µg/L when compared to the control. However, enlarged cells were noted in the 233, 485 and 1011 µg/L treatment groups at 96 hours.
Results with reference substance (positive control):
Not applicable.
Reported statistics and error estimates:
As above.
Validity criteria fulfilled:
yes
Conclusions:
Selenastrum capricornutum were exposed to five concentrations of Phosflex TXP and evaluated for effects on cell density, area under the growth curve and growth rate. The 72 and 96-hour EC50, based on cell density, area under the growth curve and growth rate, was > 1011 µg/L, the highest tested concentration. The 72 hour NOAEC, based on cell density and area under the growth curve (biomass) was 112 µg/L. The 96-hour NOAEC based on cell density was 112µg/L.
Executive summary:

Selenastrum capricornutum were exposed to five concentrations of Phosflex TXP and evaluated for effects on cell density, area under the growth curve and growth rate. The 72 and 96-hour EC50, based on cell density, area under the growth curve and growth rate, was > 1011 µg/L, the highest tested concentration. The 72 hour NOAEC, based on cell density and area under the growth curve (biomass) was 112 µg/L. The 96-hour NOAEC based on cell density was 112 µg/L.

No toxicity at the limit of solubility in water was therefore noted.

Description of key information

Short term toxicity to Algae

Key value for chemical safety assessment

EC50 for freshwater algae:
1 011 µg/L
EC10 or NOEC for freshwater algae:
112 µg/L

Additional information

Selenastrum capricornutum were exposed to five concentrations of Phosflex TXP and evaluated for effects on cell density, area under the growth curve and growth rate. The 72 and 96-hour EC50, based on cell density, area under the growth curve and growth rate, was > 1011 µg/L, the highest tested concentration. The 72 hour NOAEC, based on cell density and area under the growth curve (biomass) was 112 µg/L. The 96-hour NOAEC based on cell density was 112µg/L.

No toxicity at the limit of solubility in water was therefore noted.