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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 202 and in accordance with the Principles of Good Laboratory Practices (GLP).

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Principles of method if other than guideline:
not applicable
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
2-butoxyethyl benzoate
EC Number:
EC Name:
2-butoxyethyl benzoate
Cas Number:
Molecular formula:
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:
Details on sampling:
The bulk dose solutions (laboratory dilution water - LDW control, 6.25, 12.5, 25.0, 50.0 and 100.0 mg/L) were sampled for analytical confirmation on day 0 and day 1 of the study following preparation. On day 1 and day 2, the spent test solutions at each dose level were pooled to provide compsoite samples for analytical confirmation. Aliquots (-5-rnl) were collected using an Eppendorf pipette and transferred to glass vials. Samples were diluted to an acceptable analysis range with ultrapure water. These samples were analyzed by high performance liquid chromatography with diode array detection (HPLC/DAD).

Test solutions

Details on test solutions:
The dispersal of the test material in the surrounding medium was considered to represent the most probable route of exposure in the environment. Because all solutions were used on the day of preparation, assessment of stability was not required.
Bulk test solutions were prepared via direct addition of the test material (100.0 mg) to 1 litre laboratory distilled water (LDW) to create a 100 mg/L primary stock (also used as the 100 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 LDWto achieve nominal concentrations of 50.0, 25.0, 12.5, and 6.25 mg/L in volumetric flasks. The flasks were stoppered and shaken until the solutions appeared homogenous. Control bulk solution was LDW with no test material added. All solutions were clear and colorless.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: water flea (freshwater cladoceran)
- Strain: magna
- Source: in-house cultures initially obtained from Aquatic BioSystems, Inc. Fort Collins, Colorado
- Age at study initiation (mean and range, SD): neonates < 24 hours old
- Method of breeding: Neonates were only used from daphnids which have had at least 3 broods. The day before neonates were needed for testing (<24 hours prior to test initiation), neonates within the culture vessels were removed and discarded leaving only adult daphnids in the vessels. On the following day, neonates produced overnight in these same culture vessels were again removed from the vessels and placed into a glass beaker containing LDW. These neonates (<24 hours old) were held in an incubator until used for the study.

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
48 h
Post exposure observation period:
not applicable

Test conditions

Hardness - 54 – 66 mg/L CaCO3
Test temperature:
temperatures ranged from 20 – 21°C
pH ranged from 7.3 – 7.7
Dissolved oxygen:
8.3 – 9.2 mg/L (93 – 103% oxygen saturation)
not applicable
Nominal and measured concentrations:
Target concentrations were 0 (water control), 6.25, 12.5, 25.0, 50.0, and 100 mg 2-Butoxyethyl benzoate/l while mean analytical concentrations were
Details on test conditions:
- Test vessel: 50 mL glass beakers each containing approximately 40 mL of control or test solution and covered to reduce evaporation
- Type: closed
- No. of organisms per vessel: 5 daphnids/replicate
- No. of vessels per concentration (replicates): four replicates

- Source/preparation of dilution water: The water used for daphnid testing and culturing (referred to as laboratory dilution water or LDW) was Lake Huron water supplied to The Dow Chemical Company by the City of Midland Water Treatment Plant. The water was obtained from the upper Saginaw Bay of Lake Huron off Whitestone Point and was limed and flocculated with ferric chloride. The water was pumped to the laboratory prior to municipal treatment for human consumption. Before use in the laboratory, the water was sand-filtered, pH-adjusted with gaseous CO2, carbon-filtered, and UV-irradiated

Control Water (LDW): 0 – 24 hr
Hardness (mg/L CaCO3) - 54 – 66
Alkalinity (mg/L CaCO3) - 42 – 46
Conductivity (µS/cm) - 188 – 192
High Test Level (91.9 mg 2-Butoxyethyl benzoate/L) 0 – 24 hr
Hardness (mg/L CaCO3) - 42 – 66
Alkalinity (mg/L CaCO3) - 46 – 54
Conductivity (µS/cm) - 144 – 166

- Photoperiod: 16* hours light/dark cycle
- Light intensity: 516 - 656 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Daphnids were observed for immobility (inability to swim within approximately 15 seconds after gentle agitation of the test vessel) at approximately 24 and 48 hours of exposure. Any sublethal effects (abnormal behavior or appearance) were recorded. After approximately 24 hours (i.e., after 24 hour observations), the daphnids were transferred to fresh test solutions. Any vessels with 100% immobility were not renewed at that time. Dissolved oxygen, pH, and temperature data were recorded from each bulk test solution
at test initiation and the 24-hour renewal as well as from all test vessels (spent test solutions) at the 24-hour renewal and test termination. Water temperature was continuously monitored with a minimum/maximum thermometer placed in a surrogate vessel. Light intensity was measured at test initiation. Water hardness, alkalinity, and conductivity were measured from the control water and the 100 mg/L test level solution at 0 and 24 hours (fresh test solutions only).

- Range finding study - A range-finding test was conducted under static conditions. Test concentrations included 0 (LDW control), 0.1, 1, 10, and 100 mg/L. Ten daphnids were exposed to each test concentration (2 replicates per test concentration each containing 5 daphnids each). Temperature, dissolved oxygen, and pH were monitored throughout the exposure. After 48 hours, 100% immobility was observed in the 100 mg/L dose level. No immobility or other sublethal effects were observed in any other dose level or the control. Therefore, definitive test concentrations were set as: 0 (LDW control), 6.25, 12.5, 25.0, 50.0, and 100 mg 2-Butoxyethyl benzoate/l.
Reference substance (positive control):

Results and discussion

Effect concentrationsopen allclose all
24 h
Dose descriptor:
Effect conc.:
26.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 22.5 - 31.1
48 h
Dose descriptor:
Effect conc.:
15.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: 95% CL could not be calculated
48 h
Dose descriptor:
Effect conc.:
10.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
Details on results:
Following 24 hours of exposure, immobility was observed in 0, 0, 30, 95, and 100 of the daphnids at the 5.53, 10.8, 22.1, 45.4, and 91.1 mg/L test levels, respectively. At test termination (48-hours of exposure), immobility was observed in 0, 0, 100, 100, and 100%, of the daphnids at the 5.53, 10.8,
22.1, 45.4, and 91.1 mg/L test levels, respectively. No immobility was observed at the LDW control test levels during the conduct of the study. No changes in daphnid behavior or appearance were observed 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


Applicant's summary and conclusion

Validity criteria fulfilled:
The acute toxicity values for Daphnia magna exposed to 2-Butoxyethyl benzoate, over a 48-hour renewal exposure period and based on mean measured concentrations, were as follows:
- The 24-hour EC50 value, calculated by the USEPA Trimmed Spearman-Karber Program, was 26.5 mg/L, with a 95% confidence interval of 22.5 – 31.1 mg/L; the Spearman Karber trim was 0%.
- The 48-hour EC50 value, calculated by the USEPA Trimmed Spearman-Karber Program, was 15.5 mg/L. The confidence interval could not be calculated. The
Spearman Karber trim was 0%.
- The 48-hour NOEC, calculated by a Fisher’s Exact Test, was 10.8 mg /L.
Executive summary:

The purpose of this study was to assess the effects of 2-Butoxyethyl benzoate to the freshwater cladoceran,Daphnia magna. The study was performed for 48 hours with a renewal of the exposure solutions at 24 hours. Target concentrations were 0 (water control), 6.25, 12.5, 25.0, 50.0, and 100 mg 2-Butoxyethyl benzoate/L. Test solutions were analyzed at test initiation and termination by high performance liquid chromatography with diode array detection (HPLC/DAD). None of the analyses of the water control exhibited a concentration exceeding the lowest level quantitated (LLQ) equivalent to 0.25 mg 2-Butoxyethyl benzoate/L. Mean measured concentrations were <LLQ, 5.53, 10.8, 22.1,

45.4, and 91.9 mg 2-Butoxyethyl benzoate/L. The data collected were used to calculate the 24- and 48-hour EC50 values (the concentrations estimated to result in 50% immobility of the test population after 24- and 48-hours of exposure, respectively) and a 48-hour no observed-effect concentration (NOEC).

The acute toxicity values for the daphnid (D. magna) exposed to 2-Butoxyethyl benzoate over a 48-hour static-renewal exposure period and based on mean measured concentrations were as follows

- 24-hour EC50 = 26.5 mg/l

- 48 -hour EC50 = 15.5 mg/l 

- 48 -hour NOEC = 10.8 mg/l (based on the highest concentration exhibiting no significant immobility or sublethal effects)