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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was condcuted according to OECD TG 201 and in accordance with the Principles of Good Laboratory Practices
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-butoxyethyl benzoate
EC Number:
226-685-8
EC Name:
2-butoxyethyl benzoate
Cas Number:
5451-76-3
Molecular formula:
C13H18O3
IUPAC Name:
2-butoxyethyl benzoate
Test material form:
other: clear liquid
Details on test material:
- Name of test material (as cited in study report): 2-butoxyethyl benzoate (Butyl Cellosolve™ Benzoate)
- Physical state: clear liquid
- Analytical purity: 99.2%
- Lot/batch No.: 20130443-19
- Expiration date of the lot/batch: 02 April 2016
- Storage condition of test material: Ambient
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Target concentrations of 0 (Algal Assay Procedure Medium - AAP control), 0.625, 1.25, 2.50, 5.00, and 10.0 mg 2-Butoxyethyl benzoate/l. Test solutions were analyzed at test initiation and termination by HPLC/DAD. Mean measured concentrations were - Sampling method: The bulk dose solutions (AAP control, 0.625, 1.25,2.50,5.00 and 10.0 mg/l) were sampled for analytical confirmation on day 0 ofthe study following preparation. On day 3 (72 hrs), the replicate test solutions at each exposure level were pooled to provide composite algae containing samples for analytical confirmation, while test solutions containing no algae were sampled separately. Aliquots (~5 ml) were collected using an Eppendorf pipette and transferred to glass vials. The resulting sample was collected in autos ampler vials for analysis by high performance liquid chromatography with diode array detection (HPLC/DAD).

Test solutions

Vehicle:
yes
Details on test solutions:
Bulk test solutions were be prepared via direct addition of the test material (10.0 mg) to 1 L AAP to create a 10 mg/L primary stock (also used as the 10 mg/L bulk test solution). The primary stock was shaken until the solution appeared homogenous and no visibly undissolved test material remained. Subsequent bulk solutions were prepared via dilution of the primary stock with AAP to achieve nominal concentrations of 5.00, 2.50, 1.25, and 0.625 mg/L in volumetric flasks. The flasks were stoppered and shaken until the solutions appeared homogenous. Control bulk solution was AAP with no test material added. All solutions were clear and colorless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater alga
- Strain: subcapitata
- Source (laboratory, culture collection): in-house cultures initially obtained from the University of Texas at Austin Culture Collection (UTEX1648; lot # 111913)
- Age of inoculum (at test initiation): The algal inoculum for the test was prepared from a 3-day old stock culture
- Method of cultivation: under standard typical culture conditions, under continuous illumination of approximately 5200 ± 520 lux at a temperature of 23 ± 2ºC.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
not applicable
Test temperature:
23 ± 2ºC
pH:
pH for medium use on tests was adjusted to 7.5 ± 0.1 prior to solution preparation
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Target concentrations of 0 (Algal Assay Procedure Medium - AAP control), 0.625, 1.25, 2.50, 5.00, and 10.0 mg 2-Butoxyethyl benzoate/l. Test solutions were analyzed at test initiation and termination by HPLC/DAD. Mean measured concentrations were
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were sterilized 250-mL borosilicate Erlenmeyer flasks with foam stoppers each containing 50 mL test medium
- Initial cells density: A Coulter Multisizer 3 (Beckman Coulter, Brea, California) was used to determine the cell density of the stock culture. This evaluation determined that a 0.296 ml aliquot of the culture was required to inoculate each test vessel at an initial cell density of approximately 10000 cells/ml..
- Four replicate test vessels were prepared per test level and seven replicate test vessels were prepared at the medium control level. Each replicate contained 50 mL of the appropriate test solution. Three replicates in each test level and six replicates in the control level were inoculated with approximately 10,000 cells/mL. The additional replicate for each test and control level was not inoculated with algae and served as a counting blank. These blanks were used to correct the daily counts for the potential interference of the test material and to monitor pH without the algal biomass. At test initiation and following sampling for cell densities at 24 and 48 hours, the replicate test vessels were placed in a walk-in environmental chamber (Lab-Line
Environmental Chamber, Lab-Line Inc., Melrose, Illinois) on a shaker table (set at approximately 100 rpm) according to a computer-generated randomization.

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: Algal assay procedure medium (AAP) approximate composition
Nutrient Concnetration (mg/l)
NaNO3 25.5
MgCl2 * 6H2O 12.2
CaCl2 * 2H2O 4.4
MgSO4 * 7H2O 14.7
NaHCO3 15.0
K2HPO4 1.044
H3BO3 0.186
MnCl2 * 4H2O 0.417
ZnCL2 0.00332
NaMoO4 * 2H2O 0.00726
CoCl2 * 6H2O 0.00143
CuCl2 * 2H2O 0.000011
Na2EDTA * 2H2O 0.3
FeCl3 * 6H2O 0.16

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algae were cultured in freshwater algal nutrient medium (i.e., AAP medium), prepared with sterile deionized water and reagent grade chemicals. The water source for the deionized water system was municipal water produced by the City of Midland Water Treatment Plant. The base water used to prepare the medium is passed through a series of activated carbon, (two) deionization polymer (US Filter Mixed Bed, Type 1), and a final filtration unit, prior to collection and autoclaving in clean glass containers. Prior to treatment, the base water used to prepare the media is analyzed periodically to verify that no contaminants are present at levels that may interfere with the test results

OTHER TEST CONDITIONS
- Adjustment of pH:pH for medium use on tests was adjusted to 7.5 ± 0.1 prior to solution preparation
- Photoperiod: continuous (i.e. 24 hours light)
- Light intensity and quality: target light intensity 5,200 ± 780 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Algal cell densities of the initial inoculum and test solutions were determined by electronic particle counting using a Coulter Multisizer 3 (Beckman Coulter, Brea, California) fitted with a 100 μm aperture tube. Total cell counts were determined at approximately 24, 48, and 72 hours. Cells were cumulatively counted at a lower threshold equivalent spherical diameter of approximately 2.6 μm to a higher threshold equivalent spherical diameter of approximately 8.7 μm. Two cell count readings were made per replicate and averaged. The readings for the blank replicates were used to correct for background in daily calculations. The adjusted cell counts were converted to cells x 10,000/mL (cell density) for statistical analysis and reporting.
In addition, at test termination morphological observations were done on a composited sample of the inoculated replicates at each test concentration. The cells were observed under a microscope (Olympus BH Microscope (Olympus Corporation, Tokyo, Japan); 20x or 40x objective lens; WF10x eyepiece; 1.25x Dual Observation Deck).

TEST CONCENTRATIONS
- Range finding study: Test solutions were prepared similarly as that of the definitive test. Two flasks per dose level (6 for control) were inoculated with a predetermined aliquot of algal inoculum to achieve 10,000 cells/mL. An uninoculated replicate (counting blank) was prepared at each dose level and control.
Cell counts were taken after approximately 72 hours of exposure. Results for the 0 (AAP control), 0.1, 1, 10, and 100 mg/L levels were 249.4, 237.4, 229.1, -2.816 (i.e., 0), and -5.256 (i.e., 0) cells x 104/mL. Based on these results, the EyC50 was estimated at between 1 and 10 mg/L. Definitive concentrations were set 0 (AAP control), 0.625, 1.25, 2.50, 5.00, and 10.0 mg/L.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
3.79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: cell yield
Remarks on result:
other: EyC50
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.982 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: cell yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.98 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth inhibition
Remarks on result:
other: ErC50
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.982 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth inhibition
Details on results:
Temperatures during the exposure period ranged from 23 – 24 ºC. The pH for all exposure concentrations and the control was 6.8 at test initiation, ranged from 7.0 – 7.5 in replicates with algae at test termination, and was 6.7 in replicates without algae at test termination. Light intensity ranged from 4430 – 5660 lux.
Cell Yield: All cell yield data were normally distributed and homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p > 0.01). Mean yields at 0-72 hours were 278.4, 283.9, 274.3, 214.9, 124.2, and 5.363 (x104) cells/ml for the control, 0.480, 0.982, 2.01, 4.16, and 8.62 mg 2-Butoxyethyl benzoate/L test levels, respectively. Between 0 and 72 hours, the mean inhibition response relative to the control ranged from -2 (i.e., no inhibition) to 98% inhibition of yield. At 0-72 hours, yields in the 2.01, 4.16, and 8.62 mg 2-Butoxyethyl benzoate/L test levels were significantly different from the control. Thus, the 0-72 hour NOEC was 0.982 mg 2-Butoxyethyl benzoate/L.For cell yield at 0-72 hours, the calculated EyC50 (95% confidence intervals) was 3.79 (3.47 – 4.13) mg 2-Butoxyethyl benzoate/L.
Growth rate: All growth rate data were normally distributed and but not homogeneous (Shapiro-Wilk Test, p > 0.01 and Levene’s Test, p < 0.01). Mean specific growth rates between 0 and 72 hours were 1.877, 1.884, 1.873, 1.790, 1.609, and 0.6111 (day-1) for the control, 0.480, 0.982, 2.01, 4.16, and 8.62 mg 2-Butoxyethyl benzoate/L test levels, respectively. From 0 to 72 hours, mean inhibition response relative to the control ranged from 0 to 67% of the mean specific growth rate. Mean specific growth rates in the 2.01, 4.16, and 8.62 mg 2-Butoxyethyl benzoate/L treatment levels at 0-72 hours were significantly different from the control. Thus, the 0-72 hour NOEC was 0.982 mg 2-Butoxyethyl benzoate/L. Between 0 and 72 hours, the calculated ErC50 (95% confidence intervals) was 6.98 (6.73 – 7.23) mg 2-Butoxyethyl benzoate/L
Morphological observations: Microscopic evaluation of cells at each test concentration and the control at test termination revealed no abnormal observations at any test level.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
standard statistical methods were employed

Any other information on results incl. tables

not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity values for Pseudokirchneriella subcapitata exposed to 2-Butoxyethyl benzoate over a 72-hour static exposure period and based on mean measured concentrations were as follows:
0-72-hour cell yield: EyC50 = 3.79 mg/L, NOEC = 0.982 mg/L
0-72 hour growth inhibition: ErC50 = 6.98 mg/L, NOEC = 0.982 mg/L
Executive summary:

The purpose of this study was to assess the effects of 2-Butoxyethyl benzoate to the freshwater green alga, Pseudokirchneriella subcapitata. The study was performed for 72 hours with target concentrations of 0 (AAP control), 0.625, 1.25, 2.50, 5.00, and 10.0 mg 2 -Butoxyethyl benzoate/L. Test solutions were analyzed at test initiation and termination by HPLC/DAD. None of the analyses of the media control exhibited a concentration exceeding the lowest level quantitated (LLQ) equivalent to 0.25 mg 2-Butoxyethyl benzoate/L. Mean measured concentrations were <LLQ, 0.480, 0.982, 2.01, 4.16, and 8.62 mg 2-Butoxyethyl benzoate/L. The data collected were used to determine EC50 (the concentration causing 50% inhibition) values for 72-hour cell density, 0-72-hour cell yield, and 0-72-hour average specific growth rate. No-observable-effect concentrations (NOEC) were determined for each endpoint based on the highest concentration with algal growth not significantly different from the control.

The acute toxicity values for Pseudokirchneriella subcapitata exposed to 2-Butoxyethyl benzoate over a 72-hour static exposure period and based on mean measured concentrations were as follows:

0-72-hour cell yield: EyC50 = 3.79 mg/L, NOEC = 0.982 mg/L

0-72 hour growth inhibition: ErC50 = 6.98 mg/L, NOEC = 0.982 mg/L