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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Test Conditions
Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water adjacent to the test vials in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured at four locations around the perimeter of the shaker tables with a VWR Traceable light meter at 0 hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux.Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189 photometer and Model LI-190SA probe. Test vessels were randomly placed on the shaker table at test initiation based on computer-generated random numbers. Following each observation interval, the remaining test vials were assigned new random positions based on computer-generated random numbers. Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the additional replicates initiated for this purpose. At test termination, after cell counts were completed, the replicate solutions for each treatment and the control were respectively composited for pH and conductivity measurements. Test solution pH was measured with a Yellow Springs Instrument (YSI) Model pH100 pH meter, and conductivity was measured with a YSI Model No. 33 salinity-conductivity-temperature meter.

Analytical Measurements
At test initiation, 24 hours and test termination (72 hours), a single sample was removed from each test solution and control and analyzed for methyl isoamyl ketone (MIAK). Samples analyzed on day 0 were removed from the replicate vials established for this purpose. Samples analyzed at 24 and 72 hours of exposure were collected prior to performing cell counts from the replicate vials established for that purpose. Since the sampling intervals were not equally spaced (e.g., 0, 24 and 72 hours), a time-weighted mean was calculated for each treatment level. At 24 and 72 hours of exposure, a sample was removed from additional replicate vials of the 25 mg/L nominal test concentration which did not contain algae. The results of these analyses were compared with that obtained for the 24- and 72-hour analyses of the 25 mg/L solution containing algae to assess the impact that algae had on the test substance concentration. Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.

Vehicle:
no
Details on test solutions:
Based on the results of preliminary testing conducted at Springborn Smithers, and consultation with the Study Sponsor nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L were selected for the definitive exposure. A 100 mg/L stock solution was prepared prior to test initiation by placing 0.200 g of methyl isoamyl ketone (MIAK) in a 2000-mL volumetric flask and bringing it to volume with AAP medium and 300 mg/L of NaHCO3. The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. Test solutions were prepared from dilutions of the 100 mg/L stock solution. Replicate approximately 60-mL VOA vials, 11 for the 6.3, 13, 25 and 50 mg a.i./L treatment levels, 12 for the 100 mg a.i./L treatment level and 20 for the control were conditioned prior to use by rinsing with untreated AAP medium. Each vial was filled to approximately 90% of the final volume with algal medium. Exposure solutions were individually prepared by adding the appropriate amount of the 100 mg/L stock solution to achieve the desired nominal concentration. Additional untreated AAP medium was used for the control.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The alga used in this toxicity test was the freshwater green alga, Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum, strain 1648, Class Chlorophyceae. The alga was obtained from University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.
The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water. The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2 C and continuous illumination at the surface of the medium with an intensity range of 4500 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test with methyl isoamyl ketone was taken from a stock culture that had been transferred to fresh medium three days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23
pH:
The pH of the test and control solutions ranged from 7.8 to 7.9 at test initiation. At test termination, the test and control solution pH ranged from 9.2 to 9.5. The increase in pH during the exposure is common in static algal cultures and is due to photosynthesis by the algae.
Nominal and measured concentrations:
Nominal: 6.3, 13, 25, 50 and 100 mg/L
Measured: 5.8, 12, 24, 53 and 99 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
19 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 9.0-40
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
44 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 30-67
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
24 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
76 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 46-96
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
24 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Cells exposed to all treatment levels tested and the controls were observed to be normal throughout the exposure period. The 72-hour cell density in the control averaged 71.54 x 104 cells/mL, respectively. Cell density in the 5.8, 13, 24, 54 and 100 mg/L treatment levels averaged 75.33, 61.08, 69.25, 53.83 and 44.25 x 104 cells/mL, respectively. The 0- to 72-hour growth rate in the control averaged 1.43 days-1. The 0- to 72-hour growth rate in the 5.8, 13, 24, 54 and 100 mg/L treatment levels each averaged 1.45, 1.38, 1.42, 1.33 and 1.27 days-1, respectively.
Reported statistics and error estimates:
Based on the results of Shapiro-Wilks' and Bartlett's Tests, the biomass data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. Statistical analysis determined a significant reduction in yield for the 54 and 100 mg/L treatment levels compared to the control data. Based on Williams’ Test, the 72-hour NOEC for yield was determined to be 24 mg/L. The 72-hour EyC10 value was determined to be 19 mg/L with 95% confidence intervals of 9.0 to 40 mg/L. The 72-hour EyC20 value was determined to be 44 mg/L with 95% confidence intervals of 30 to 67 mg/L. Since no treatment level tested resulted in ≥ 50% reduction in yield, the EyC50 value was empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested.

Based on the results of Shapiro-Wilks’ and Bartlett’s Tests, the growth rate data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects. Statistical analysis determined a significant reduction in growth rate for the 54 and 100 mg/L treatment levels tested compared to the control data. Therefore, the 72-hour NOEC for growth rate was determined to be 24 mg/L. The 72-hour ErC10 value was 76 mg a.i./L with 95% confidence intervals of 46 to 96 mg/L. Since no treatment level tested resulted in ≥ 20% reduction in growth rate, the 72-hour ErC20 and ErC50 values were empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested.
Validity criteria fulfilled:
yes
Executive summary:

The 72 hour acute toxicity of methyl isoamyl ketone to the freshwater green alga Pseudokirchneriella subcapitata was conducted in a closed system following OECD Guideline 201. The test was conducted at nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L in the definitive exposure. Analytical determinations of the test solutions were made at test initiation, 24 hours and test termination (72 hours) and a time-weighted mean was calculated for each treatment level. The mean measured concentrations (as time weighted average) ranged from 92 to 110% of nominal concentrations and defined the treatment levels tested as 5.8, 12, 24, 53 and 99 mg/L. The 72-hour EyC10 value was determined to be 19 mg/L with 95% confidence intervals of 9.0 to 40 mg/L. The 72-hour EyC20 value was determined to be 44 mg/L with 95% confidence intervals of 30 to 67 mg/L. Since no treatment level tested resulted in ≥ 50% reduction in yield, the EyC50 value was empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested. The 72-hour NOEC for growth rate was determined to be 24 mg/L. The 72-hour ErC10 value was 76 mg a.i./L with 95% confidence intervals of 46 to 96 mg/L. Since no treatment level tested resulted in ≥ 20% reduction in growth rate, the 72-hour ErC20 and ErC50 values were empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested.

Description of key information

Key value for chemical safety assessment

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

Additional information

The 72 hour acute toxicity of methyl isoamyl ketone to the freshwater green algaPseudokirchneriella subcapitatawas conducted in a closed system following OECD Guideline 201. The test was conducted at nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L in the definitive exposure. Analytical determinations of the test solutions were made at test initiation, 24 hours and test termination (72 hours) and a time-weighted mean was calculated for each treatment level. The mean measured concentrations (as time weighted average) ranged from 92 to 110% of nominal concentrations and defined the treatment levels tested as 5.8, 12, 24, 53 and 99 mg/L. The 72-hour EyC10 value was determined to be 19 mg/L with 95% confidence intervals of 9.0 to 40 mg/L. The 72-hour EyC20 value was determined to be 44 mg/L with 95% confidence intervals of 30 to 67 mg/L. Since no treatment level tested resulted in ≥ 50% reduction in yield, the EyC50 value was empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested. The 72-hour NOEC for growth rate was determined to be 24 mg/L. The 72-hour ErC10 value was 76 mg a.i./L with 95% confidence intervals of 46 to 96 mg/L. Since no treatment level tested resulted in ≥ 20% reduction in growth rate, the 72-hour ErC20 and ErC50 values were empirically estimated to be > 100 mg/L, the highest time weighted average concentration tested.