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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:
2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the study was conducted according to OECD TG 201, OPPTS 850.4500, ISO 14442:2006 and in accordance with the Principles of Good Laboratory Practice (GLP), the difference between the measured analytical concentrations and nominal concentrations were in excess of ±25%.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Remarks:
Some minor exceptions were noted and these did not have any impact on the quality and integrity of the study
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
other: ISO 14442:2006
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
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:
- Concentrations: 0, 1.9, 6.1, 19.5, 62.5 and 200 mg test item/L
Vehicle:
no
Details on test solutions:
A 200 mg test item/liter stock solution was prepared prior to test initiation by dissolving 0.2003 grams of the test item with 1000 ml dilution water. An oily film was observed on the surface of the stock solution and a strong smell occurred. After dissolving the test item, the stock solution was mixed for approximately one hour using a magnetic stirrer.
All resulting test solutions were shaken by hand and again mixed for a couple of minutes using a magnetic stirrer prior to division into the replicate vessels. To minimize the potential for volatilization of the test item during test solution preparation, each volumetric flask was kept closed. All test solutions were observed to be clear and colorless with no visible precipitate. Six dilution water control vessels containing only algal medium were established and maintained under the same conditions as the treatment level solutions.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: Pseudokirchneriella subcapitata (syn. Selenastrum capricornutum)
- Source (laboratory, culture collection): The alga was originally obtained from Albrecht-v.-Haller-Institut, Göttingen, Germany, and was maintained in stock culture at Springborn Smithers Laboratories (Europe).
- Method of cultivation: Three and six sterile 100 ml glass serum bottles crimped sealed with aluminum seals containing Teflon-lined rubber septa were prepared per treatment level and for the control, respectively. An aliquot of 65 ml of the appropriate test solution was placed in each replicate glass bottle and 0.38 ml of the preculture of Pseudokirchneriella subcapitata, at a density of 129 x 10(4) cells/ml, was immediately inoculated aseptically before the bottles were sealed. The resulting slight dilution from the inoculum was considered to be negligible. These volumes of inocula provided the required cell density of 0.75 x 10(4) cells/ml.

ACCLIMATION
- Culturing media and conditions: same as test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
not measured
Test temperature:
22.1 - 24.7 °C
pH:
6.80 - 11.04
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Nominal test concentrations: Control, 1.9, 6.1, 19.5, 62.5 and 200 mg test item/l
Initial measured concentrations: Control, 1.4, 4.5, 13.0, 45.2 and 141 mg test item/l
Details on test conditions:
The culture medium used was a synthetic algal assay growth medium, prepared by adding appropriate amounts of nutrient stock solutions to sterile, deionized water. Representative samples of the dilution water source used in the preparation of the culture medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals conducted at Bachema Schlieren, and Interlabor Belp AG, Belp, Switzerland. None of these compounds were detected at concentrations that are considered toxic in any of the water samples analyzed.

Three days prior to test start, the culture was maintained within the following conditions: a shaking rate of 99 rpm, a temperature of 22 to 24ºC and continuous illumination of 4746 to 5085 lux. Lighting was supplied by fluorescent bulbs. Culture flasks were shaken continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test was taken from a stock culture that had been transferred to fresh medium three days before testing.

The algal medium used to prepare the exposure solutions was the same as the culture medium. An aliquot of 10 ml of a 30 g/l sodium bicarbonate solution was added per liter algal medium to maintain the inorganic carbon level within the closed-system exposure, resulting in a final concentration of 300 mg sodium bicarbonate/l dilution water. The batch of medium used for the definitive test was equilibrated to test temperature. The pH of the medium was 7.10.

The test was conducted in a temperature-controlled room to maintain the test conditions specified in the study plan: a temperature of 24 ± 2 °C and continuous light intensity of 3900 to 4700 lux. An orbital shaker table provided a shaking rate of approximately 100 rpm. Test flasks were randomly placed on the shaker table at test initiation. Following each observation interval the test flasks were assigned new random positions.

At each subsequent 24-hour interval, cell counts were conducted on each replicate vessel of the treatment levels and the control using a hemocytometer (Neubauer Improved) and a Leica DMLS microscope. For counting, one sample was removed from each bottle using a single-use syringe. To avoid the loss of test item from the test solutions, the syringes were stuck through the rubber septa of the sealed bottles. One or more hemocytometer field(s), each 0.1 cm x 0.1 cm in size and 0.01 cm deep, containing 0.0001 mL of culture, were examined for each sample until at least 400 cells or a total of four fields were counted. Observations of the health of the algal cells were also made at each 24-hour interval.

Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test bottles in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers were recorded daily. Light intensity was measured with a Pancontrol LX 1308 at hour 0 and at each 24-hour interval during the exposure period. Test bottles were randomly placed on the shaker table at test initiation. Following each observation interval the test bottles were assigned new random positions. The pH of the test solutions and control was measured at test initiation, after 72 hours of exposure and at test termination. Measurements at test initiation were conducted in the test solutions remaining in the mixing vessels after the individual test bottles had been filled. After 72 hours of exposure, pH measurements were performed in a composite sample of all replicates from each test concentration and control solution using a single-use syringe for sampling. At test termination (hour 96), after cell counts were completed, the replicate vessels for the test concentrations and the control were composited for pH measurements. Test solution pH was measured using a Metrohm 691 pH-meter.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
41.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL - 40.3-44.2
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
4.5 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
13 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
41.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 39.6 - 43.1
Details on results:
After 72 and 96 hours of exposure, cells exposed to all the treatment levels tested and the control were observed to be normal.

The 96-hour cell densities in the control and 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L treatment levels averaged 276, 256, 280, 147, 11.1 and 2.1 x 104 cells/mL, respectively.

The 72-hour yield in the control and 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L treatment levels averaged 179, 162, 167, 79.2, 8.0 and 0.2 x 104 cells/mL, respectively. Statistical analysis (William’s Test, p < 0.05) demonstrated a significant reduction in yield among algae exposed to 13.0, 45.2 and 141 mg test item/L treatment levels when compared to the control. The 96-hour yield in the control and 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L treatment levels averaged 275, 255, 280, 146, 10.3 and 1.3 x 104 cells/mL, respectively. Statistical analysis (William’s Test, p < 0.05) demonstrated a significant reduction in yield among algae exposed to 13.0, 45.2 and 141 mg test item/L treatment levels when compared to the control.

The 72-hour growth rate in the control and 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L averaged 1.84, 1.81, 1.82, 1.57, 0.82 and 0.06 day(-1),respectively. Statistical analysis (William’s Test, p < 0.05) demonstrated a significant reduction in growth rate among algae exposed to 13.0, 45.2 and 141 mg test item/L treatment levels when compared to the control. The 96-hour growth rate in the control and 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L averaged 1.49, 1.47, 1.49, 1.33, 0.68 and 0.21 day(-1), respectively. Statistical analysis (Mann-Whitney Test, p < 0.05) demonstrated a significant reduction in growth rate among algae exposed to 13.0, 45.2 and 141 mg test item/L treatment levels when compared to the control.

During this study, the 72-hour cell growth in the control was 179 x 10(4) cells/mL, which fulfills the above criterion of a 16 fold increase within 72 hours. The mean daily growth rate CV through 72 hours and the CV for the 0 to 72 hour average growth rate for the control were 23 and 1.3%, respectively, which also fulfill the criteria given for green algae.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Standard statistical methods were employed

Preliminary Test

A 96-hour preliminary range-finding exposure was conducted at Springborn Smithers Laboratories (Europe) at nominal UCAR n-Pentyl Propionate concentrations of 0.020, 0.20, 2.0, 20 and 200 mg test item/l and a control. Three 100 -ml glass serum bottles crimped sealed with aluminum seals containing Teflon-lined rubber septa were established for each concentration and the control. Following 96 hours of exposure, cells exposed to the 0.020, 0.20, 2.0 and 20 mg test item/l and the control were observed to be normal. In the highest treatment level ciliates were observed. Cell densities in the control and 0.020, 0.20, 2.0, 20 and 200 mg test item/l treatment levels averaged 277, 273, 278, 279, 108 and 0.8 x 104 cells/ml, respectively.

Based on these results, nominal concentrations of 1.9, 6.1, 19.5, 62.5 and 200 mg test item/l and a control were selected for the definitive exposure.

The pH of the test and control solutions ranged from 7.36 to 7.47 at test initiation. After 72 and 96 hours of exposure, pH measured in the composited test solutions of the treatment levels and the control ranged from 7.10 to 10.13 and from 6.80 to 11.04, respectively. The low pH value measured in the highest test concentration at test completion was most likely due to no cell growth and/or the degradation of the test item over the 96 hour test duration. Continuous temperature monitoring established a temperature range from 22 to 25°C during the test period. Light intensity of the test area ranged from 3921 to 4543 lux. The shaking rate was maintained throughout the exposure at 100 rpm.

 

After preparation of the test solution at day 0, the percentage recoveries ranged from 66.5 to 74.7% of the nominal concentrations. After 96 hours of exposure, recoveries of below the limit of quantification (LOQ) of 1.72 mg test item/L were found for all treatment levels. The 96-hour recovery of the 19.5 mg test item/L solutions without algae (abiotic control) was also below the LOQ. Based on this, it can be concluded that the reduction in concentration at hour 72 was not caused (only) by the presence of algae cell.

 

Based on these results, initial measured concentrations of 1.4, 4.5, 13.0, 45.2 and 141 mg test item/L were used for the evaluation of the biological data.

 

Analysis of the QC samples prepared in dilution water resulted in measured concentrations ranging from 90.7 to 114% (N = 6) of nominal concentration at hour 0 and 96. The results of the QC samples established that the appropriate quality control was maintained during the analyses of the exposure solutions.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96-hour (growth rate) EC50 of UCAR n-pentyl propionate to Pseudokirchneriella subcapitata (syn. Selenastrum
capricornutum) under static conditions in a closed system was 41.7 (initial measured concentration) mg/l with 95 % confidence limits ranging between 40.3 - 44.2 mg/l and the NOEC and the LOEC values being 4.5 and 13.0 mg/l.
Executive summary:

The purpose of this study was to determine the effects of UCAR (n-Pentyl Propionate) on the growth of Pseudokirchneriella subcapitata under static conditions in a closed-bottle system. The results are reported as the 72- and 96-hour No-Observed-Effect Concentration (NOEC) and Lowest-Observed-Effect Concentration (LOEC) values for yield and growth rate. Additionally, the 72- and 96-hour EC10, EC20, EC50 values for both endpoints, and the 24- and 48-hour EC50 values for yield were determined. The nominal test concentrations were - Control, 1.9, 6.1, 19.5, 62.5 and 200 mg test item/l (initial measured concentrations were - Control, 1.4, 4.5, 13.0, 45.2 and 141 mg test item/l).

Based on initial measured concentrations (mg test item/L), the 72- and 96-hour (growth rate) ErC50 values for n-pentyl propionate to Pseudokirchneriella subcapitata (syn. Selenastrum capricornutum) under static conditions in a closed system were 41.1 and 41.7 (initial measured concentration) mg/l with 95 % confidence limits ranging between 39.6 - 43.1 and 40.3 - 44.2 mg/l, respectively. The 72 and 96 -hour NOEC values were both 4.5 mg/L.

Description of key information

In a GLP study conducted according to  OECD TG 201, the 72-hour ErC50 (growth rate) of pentyl propionate to Pseudokirchneriella subcapitata was 41.1 mg/l with 95% fiducial limits ranging between 39.6 - 43.1 mg/l and the 72-hour NOEC was 4.5 mg/l

Key value for chemical safety assessment

EC50 for freshwater algae:
41.1 mg/L
EC10 or NOEC for freshwater algae:
4.5 mg/L

Additional information

In a GLP study conducted according to OECD TG 201, the 72 -hour ErC50 (growth rate) of pentyl propionate to Pseudokirchneriella subcapitata was 41.1 mg/l with 95% confidence limits ranging between 39.6 -43.1 mg/l, while the 96 -hour ErC50 (growth rate) was 41.7 (95% CL 39.6 - 43.1). Both the 72 -hour and 96 -hour NOEC values were 4.5 mg/L.