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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-10-18 to 2005-06-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylphenol
EC Number:
201-958-4
EC Name:
2-ethylphenol
Cas Number:
90-00-6
Molecular formula:
C8H10O
IUPAC Name:
2-ethylphenol
Constituent 2
Chemical structure
Reference substance name:
4-ethylphenol
EC Number:
204-598-6
EC Name:
4-ethylphenol
Cas Number:
123-07-9
Molecular formula:
C8H10O
IUPAC Name:
4-ethylphenol
Constituent 3
Chemical structure
Reference substance name:
3-ethylphenol
EC Number:
210-627-3
EC Name:
3-ethylphenol
Cas Number:
620-17-7
Molecular formula:
C8H10O
IUPAC Name:
3-ethylphenol
Test material form:
not specified

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
At test initiation (0 hour) and test termination (72 hours), a single sample was removed from each test concentration and the controls and analyzed for ethyl phenols concentration. Samples analyzed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels. Samples analyzed at test termination were removed from individually composited replicate solutions of the treatment levels and controls.
At 72 hours of exposure, a sample was removed from the replicate flask (D) of the 6.3 mg/L test concentration, which did not contain algae. The result of this analysis was compared with that obtained for the 72-hour analysis of the 6.3 mg/L solution containing algae to assess the impact that algae had on the test substance concentration.
Three quality control (QC) samples were also prepared at each sampling interval at nominal ethyl phenols concentrations of 1.00, 10.0 and 50.0 mg/Land remained with the exposure solution samples throughout the analytical process. Analysis of the QC samples was used to judge the precision and quality control maintained during the analytical process.

Test solutions

Vehicle:
yes
Details on test solutions:
The AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium with the following exceptions: Since screw cap tops, which do not readily permit gas exchange, were used as closures for the test vessels, an additional carbon source for photosynthesis was required. Therefore, an additional 300 mg/L of sodium bicarbonate was added to the algal medium. The pH of the AAP medium was not adjusted prior to use to avoid loss of the additional bicarbonate. Several liters of AAP medium were prepared using sterile, deionized water and were equilibrated to test temperature.
Prior to test initiation, a 300 mg/mL stock solution was prepared by placing 7.4 mL (7.5332 g based on a specific gravity of 1.018 g/mL) of ethyl phenols in a 25-mL volumetric flask and diluting to volume with acetone (CAS No. 67-64-1 ). The resultant 300 mg/mL stock solution was observed to be clear and colorless with no visible sign of undissolved test substance. The 300 mg/mL stock was used to prepare secondary stock solutions (250, 130, 63, 31 and 16 mg/mL).
Nominal test concentrations were prepared from these secondary stock solutions. All resulting test solutions were observed to be clear and colorless with no visible undissolved test substance. A solvent control was prepared by bringing 0.10 mL of acetone to a final volume of 1000 mL with AAP medium. The concentration of acetone in the solvent control was equal to the concentration of acetone present in each test solution (0.10 mL/L). Untreated algal medium was used to prepare the control.

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 Laboratories culture facility.
The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.
If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide. Stock cultures were grown in 125-mL glass flasks each containing 50 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange.
The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 2 °C and continuous illumination at the surface of the medium with an intensity of approximately 7000 to 9100 lux (650 to 850 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 ethyl phenols was taken from a stock culture that had been transferred to fresh medium three days before testing.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
22 to 24 °C
pH:
at test start: 8.2 - 8.3
at test end: 9.4 - 9.9
Conductivity:
350 to 400 µmhos/cm
Nominal and measured concentrations:
nominal: 1.6, 3.1, 6.3, 13 and 25 mg/L
measured: 1.1, 2.0, 5.2, 16 and 22 mg/L
Details on test conditions:
Due to the volatile nature of the test substance, the following procedures were used to minimize the loss of test substance: 1.) the test substance was transferred volumetrically rather than weighed, and 2.) screw top exposure vessels were used rather than traditional caps which allow air exchange.
Three replicate, sterile 250-mL Erlenmeyer flasks per treatment level (A, Band C) were prepared for each treatment level and the controls. All flasks were conditioned prior to use by rinsing with the appropriate exposure solution. One hundred milliliters of the appropriate exposure solution was then placed in each replicate flask. The control and solvent control flasks were prepared and maintained under the same conditions as the treatment vessels but contained no ethyl phenols. All test vessels were fitted with screw top caps with Teflon® liners to minimize the potential for volatilization of the test substance.
After the test solutions were added to the test flasks ( 100 mL per flask), a 0.383-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 261 x 104 cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 104 cells/mL.
In order to estimate the impact that the presence of algal biomass had on the test substance concentration, an additional replicate flask (D) of the 6.3 mg/L (nominal) treatment level was prepared. This flask, which was not inoculated with algae, was analyzed after 72 hours of exposure for ethyl phenols concentration. The result of this analysis was compared with the result for the 6.3 mg/L solution containing algae.
The test was conducted in an environmental chamber designed to maintain the test conditions specified in the guideline: a temperature of 24 ± 1 °C, continuous light intensity of 7000 to 9100 lux (650 to 850 footcandles) provided by Premira Vitalux fluorescent bulbs. Two orbital shaker tables provided a shaking rate of 100 ± 10 rpm. All test vessels were fitted with screw top caps with Teflon-liners to minimize volatilization of the test substance.
At each subsequent 24-hour interval, cell counts were conducted on three replicate vessels (A, B and C) of the treatment solution and the controls using a hemacytometer (Neubauer Improved) and compound microscope. One sample was removed from each flask for counting. One or more hemacytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were also made and recorded at each 24-hour interval.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
5.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Since no concentration tested resulted in >=50% inhibition, the 72-hour E,C50 was empirically
estimated to be > 22 mg/L, the highest mean measured concentration tested. The 72-hour NOEC was determined to be 5.2 mg/L.
Reported statistics and error estimates:
A t-Test (Sokal and Rohlf, 1981) was used to compare the total biomass and average growth rate data of the control to that of the solvent control. Ifno significant difference was determined, control and solvent control data were pooled for further statistical analysis to determine treatment level effects. If a significant difference was detected, the treatment data were compared to the solvent control data.
A computer program, TOXSTAT® (Gully et al., 1996) was used to calculate the EC values and 95% confidence limits. If less than the required response was observed (i.e., <50% response), the EC value was empirically estimated to be greater than the highest concentration tested.
Based on the results of statistical analysis performed for 72-hour total biomass and average growth rate data, the No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p <= 0.05) for each parameter when compared to the appropriate control data, was determined. The data were first checked for normality using Shapiro-Wilks' Test (Weber et al., 1989) and for homogeneity of variance using Bartlett's Test (Horning and Weber. 1985). If the data sets passed the test for homogeneity and normality. Williams· Test (Williams, 1971, 1972) was used to detect treatment levels that were significantly different from the appropriate control data. lfthe data did not pass the tests for homogeneity and normality, then Kruskal-Wallis' Test was used to determine the NOEC. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99% level of certainty was applied. A computer program,
TOXSTA T® (Gully et al., 1996) was used to perform these calculations.

Any other information on results incl. tables

Table 2: Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to ethyl phenols. 

























































































































































































































  Cell density (x 10^4 cells/mL)
  Observation interval (hours)
Mean measured concentration (mg/L)244872
ControlA5.7522.75148.00
 B6.2534.50132.00
 5.5047.25153.67
 Mean (SD)5.83 (0.83)34.83 (12.25)144.56 (11.24)
Solvent controlA5.2532.00196.00
 B8.2527.75157.67
 6.5026.50146.67
 Mean (SD)6.67 (1.51)28.75 (2.88)166.78 (25.9)
1.1A7.5024.25174.00
 B8.2515.00260.50
 4.5027.25152.00
 Mean (SD)6.75 (1.98)22.17 (6.39)195.50 (57.36)
2.0A6.2527.75163.67
 B6.0028.25150.67
 7.5020.00144.00
 Mean (SD)6.58 (0.8)25.33 (4.63)152.78 (10)
5.2A5.2527.00123.75
 B6.0030.50187.67
 6.0021.25105.75
 Mean (SD)5.75 (0.43)26.25 (4.67)139.06 (43.05)
16A4.756.7582.50
 B5.0022.7590.00
 6.2529.0072.00
 Mean (SD)5.33 (0.8)19.50 (11.48)81.50 (9.04)
22A3.759.7564.50
 B5.5013.0060.75
 2.7515.5044.25
 Mean (SD)4.00 (1.93)12.75 (2.88)56.50 (10.77)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
During this study, the mean 72 hour control cell growth (144.56 x 104 cells/ml) exceeded the requirement of a 16-fold increase from the initial density of 1.0 x 104 cells/ml.
Conclusions:
Since no concentration tested resulted in >=50% inhibition, the 72h EC50 was empirically estimated to be > 22 mg/L, the highest mean measured concentration tested. The 72h NOEC was determined to be 5.2 mg/L.
Executive summary:

The objective of this study was to determine the effect of ethyl phenols on the growth of the freshwater green alga Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum.


Since no concentration tested resulted in >=50% inhibition, the 72h EC50 was empirically estimated to be > 22 mg/L, the highest mean measured concentration tested. The 72h NOEC was determined to be 5.2 mg/L.