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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 February 2003 and 4 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Nominal concentrations of benzyl salicylate were 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L.

Mean measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation of 0.00223 mg/L; control), 0.894, 1.64, 2.47, 4.02, and 6.23 mg/L. Final measured concentrations were 77 to 86% of the initial measured concentrations, indicating that once the aqueous solutions of benzyl salicylate were sealed into the test vessels with the daphnids, concentrations remained constant. Mean measured concentrations ranged from 78 to 91% of nominal concentrations.

- Sampling method:
Samples of dilution water collected daily during the definitive test had a hardness of 164 mg/L as CaCO3. A sample collected at the start of the test had an acidity of 160 mg/L as CaCO3 and an alkalinity of 62 mg/L as CaCO3. A sample collected at the end of the test had an alkalinity of 58 mg/L as CaCO3. A sample of this dilution water contained less than 10 mg/L particulate matter, 1.1 ppm total organic carbon, and less than 0.01 mg/L total residual chlorine.

- Sample storage conditions before analysis:
Samples were maintained at room temperature in sealed containers for 48 hours to determine the stability of the test substance in water under test conditions.
Vehicle:
yes
Details on test solutions:
An 8.0 mg/L stock solution was prepared by bringing 0.0080 g of test substance to 1,000 mL with dilution water in a Class A volumetric flask. The solution was mixed on a magnetic stirrer for approximately 30 minutes, sonicated for approximately 15 minutes, and then mixed on a magnetic stirrer for approximately four hours. A series of solutions was then prepared by bringing appropriate volumes of stock solution to 250 mL with dilution water in Class A volumetric flasks. Water quality measurements were made and each solution was subdivided into two clear glass 40 mL vials.

Nominal concentrations of benzyl salicylate were 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L.

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
No insoluble material was observed
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name:
daphnids

- Strain:
Daphnia magna

- Source:
They were produced from an in-house culture by adult daphnids that were maintained under test conditions at T.R. Wilbury Laboratories for 26 days. The original culture was obtained from Aquatic Biosystems Inc., Fort Collins, Colorado, on February 20, 2003.

- Age at study initiation (mean and range, SD):
less than 24 hours old

- Weight at study initiation (mean and range, SD):
0.47 mg

- Length at study initiation (length definition, mean, range and SD):
Not reported

- Valve height at study initiation, for shell deposition study (mean and range, SD):
Not reported

- Peripheral shell growth removed prior to test initiation:
Not specified

- Method of breeding:
Juvenile daphnids, Daphnia magna, less than 24 hours old, employed as test organisms were from a single source and were identified using an appropriate taxonomic key. They were produced from an in-house culture by adult daphnids that were maintained under test conditions at T.R. Wilbury Laboratories for 26 days. Prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions. The culture produced young before day 12 and a subsample of adults from this culture produced, on average, more than 3 young per day during the seven days before the start of the definitive toxicity test.

- Feeding during test
Daphnids were not fed during the test.

ACCLIMATION
- Acclimation period:
7 days

- Acclimation conditions (same as test or not):
Same as test

- Type and amount of food:
Test organisms were fed the freshwater algae, Selenastrum capricornutum, and a mixture of yeast and trout chow once each day before the test.

- Feeding frequency:
once each day before the test

- Health during acclimation (any mortality observed):
During the 48 hours prior to the beginning of the test, there was no mortality, and at the beginning of the test organisms were apparently free of disease, injuries, and abnormalities.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
160 to 180 mg/L as CaCO3
Test temperature:
20 ± 1°C
pH:
pH ranged from 7.0 to 7.2
Dissolved oxygen:
Dissolved oxygen concentration ranged from 7.8 to 9.3 mg/L (mean = 8.6 mg/L). The minimum dissolved oxygen concentration during the test was 86% saturation (saturation equals 9.1 mg/L at 20°C).
Salinity:
freshwater used.
Nominal and measured concentrations:
Nominal concentrations of benzyl salicylate were: 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L.

Mean measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation of 0.00223 mg/L; control), 0.894, 1.64, 2.47, 4.02, and 6.23 mg/L. Final measured concentrations were 77 to 86% of the initial measured concentrations, indicating that once the aqueous solutions of benzyl salicylate were sealed into the test vessels with the daphnids, concentrations remained constant. Mean measured concentrations ranged from 78 to 91% of nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
40 ml vials

- Type (delete if not applicable):
closed

- Material, size, headspace, fill volume:
Vials, which were filled to capacity to eliminate any head space, were sealed with Teflon-lined caps

- Aeration:
Yes

- Renewal rate of test solution (frequency/flow rate):
Prior to testing, the daphnid culture was maintained in 100% dilution water under static, renewal conditions

- No. of organisms per vessel:
Ten daphnids were indiscriminately added to each vial

- No. of vessels per concentration (replicates):
Two

- No. of vessels per control (replicates):
One

- Biomass loading rate:
loading rate during the definitive toxicity test was approximately 0.1 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water used for the acclimation of test organisms and for all toxicity testing was carbon filtered, deionized water collected at T.R. Wilbury Laboratories in Marblehead, Massachusetts. Water was adjusted to a hardness of 160 to 180 mg/L as CaCO3 and stored in polyethylene tanks where it was aerated and recirculated through particle filters, activated carbon, and an ultraviolet sterilizer. Prior to use, the pH of the dilution water was adjusted to <8.0 with 5% H3PO4.

- Total organic carbon:
1.1 ppm total organic carbon

- Particulate matter:
dilution water contained less than 10 mg/L particulate matter

- Metals:
Yes.
Aluminum, Arsenic, Boron, Cadmium, Calcium, Chromium,Cobalt, Copper, Iron, Lead, Magnesium, Mercury, Nickel, Potassium,Silver,Sodium and Zinc

- Pesticides:
Yes

- Chlorine:
less than 0.01 mg/L total residual chlorine.

- Alkalinity:
A sample collected at the start of the test had an acidity of 160 mg/L as CaCO3 and an alkalinity of 62 mg/L as CaCO3. A sample collected at the end of the test had an alkalinity of 58 mg/L as CaCO3.

- Ca/mg ratio:
None

- Conductivity:
600 to 620 μmhos/cm (mean = 600 μmhos/cm)

- Culture medium different from test medium:
Same

- Intervals of water quality measurement:
Samples of dilution water collected daily during the definitive test. A sample collected at the start of the test and at the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH:
No

- Photoperiod:
A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white fluorescent lights.

- Light intensity:
Cool-white fluorescent lights that provided a light intensity of approximately 32 footcandles (4 μEin/m2sec).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
None

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
Not specified

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study
control and at 0.01, 0.1, and 1.0 mg/L

- Test concentrations:
Nominal concentrations of benzyl salicylate were 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
1.21 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% confidence interval = 0.894 to 1.64 mg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.16 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% confidence interval = 0.894 to 1.64 mg/L
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
4.34 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% confidence interval = 2.47 to 6.23 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.25 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
other: immobilisation
Remarks on result:
other: 95% confidence interval = 1.64 to 4.02 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.894 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
immobilisation
Basis for effect:
other:
Remarks on result:
other: 95% confidence interval not stated
Details on results:
Insoluble material was not observed during the definitive toxicity test. Nominal concentrations of benzyl salicylate were: 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L. Mean measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation of 0.00223 mg/L; control), 0.894, 1.64, 2.47, 4.02, and 6.23 mg/L. Final measured concentrations were 77 to 86% of the initial measured concentrations, indicating that once the aqueous solutions of benzyl salicylate were sealed into the test vessels with the daphnids, concentrations remained constant. Mean measured concentrations ranged from 78 to 91% of nominal concentrations.

Biological data generated by the acute toxicity test are presented in Table 3 and water quality data are presented in Appendix A. One hundred percent survival occurred in the control and no control sublethal effects were noted during the exposure period. Water quality was within the normal range throughout the toxicity test. During the definitive toxicity test the conductivity range was 600 to 620 μmhos/cm (mean = 600 μmhos/cm), the pH ranged from 7.0 to 7.1, the temperature ranged from 19.9 to 21.0°C (mean = 20.7°C), and the dissolved oxygen concentration ranged from 7.8 to 9.3 mg/L (mean = 8.6 mg/L). The minimum dissolved oxygen concentration during the test was 86% saturation (saturation equals 9.1 mg/L at 20°C).

Exposure of daphnids to benzyl salicylate resulted in a 24 hour EC50 of 1.21 mg/L (95% confidence interval = 0.894 to 1.64 mg/L) and a 48 hour EC50 of 1.16 mg/L (95% confidence interval = 0.894 to 1.64 mg/L). This exposure also resulted in a 24 hour LC50 of 4.34 mg/L (95% confidence interval = 2.47 to 6.23 mg/L) and a 48 hour LC50 of 2.25 mg/L (95% confidence interval = 1.64 to 4.02 mg/L). The slope of the dose response curve based on mortality was 7.4. The 48 hour no observed effect concentration (NOEC) was 0.894 mg/L benzyl salicylate.
Reported statistics and error estimates:
Results of the toxicity test were interpreted by standard statistical techniques. The LC50 and EC50 values were calculated using the binomial method (Stephan et al. 1978). The slopes of the concentration-response curve was calculated using mortality data and the probit method (Stephan et al. 1978). The no observed effect concentration (NOEC) is the highest tested concentration of test substance that allowed at least 90% survival without any sublethal effects.

Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and resulted in a 24 hour EC50 of 1.21 mg/L (95% confidence interval = 0.894 to 1.64 mg/L) and a 48 hour EC50 of 1.16 mg/L (95% confidence interval = 0.894 to 1.64 mg/L). This exposure also resulted in a 24 hour LC50 of 4.34 mg/L (95% confidence interval = 2.47 to 6.23 mg/L) and a 48 hour LC50 of 2.25 mg/L (95% confidence interval = 1.64 to 4.02 mg/L). The slope of the dose response curve based on mortality was 7.4. The 48 hour no observed effect concentration (NOEC) was 0.894 mg/L benzyl salicylate.
Executive summary:

Introduction:

The acute toxicity of benzyl salicylate to the daphnid, Daphnia Magna was assessed, under static conditions, according to the OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). This was accomplished by determining the 24 and 48 hour EC50 and LC50 values, and the 48 hour NOEC.

Method: The test was performed under static conditions in sealed vials with no head space to minimize the potential loss of the test substance from test solutions to the atmosphere. The test was run with five concentrations of test substance at a temperature of 20 ± 1°C. The dilution water was deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 160 to 180 mg/L as CaCO3 and a pH less than 8.0. Nominal concentrations of benzyl salicylate were: 0 mg/L (control), 1.0, 1.8, 2.9, 4.8, and 8.0 mg/L. Mean measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation of 0.00223 mg/L; control), 0.894, 1.64, 2.47, 4.02, and 6.23 mg/L. These mean measured concentrations, which ranged from 78 to 91% of nominal concentrations, were steady throughout the exposure period. Insoluble material was not observed at any tested concentration during the definitive test. Organisms used to initiate the toxicity test were less than 24 hours old. They were obtained from an in-house culture that was acclimated to test conditions for more than seven days at T.R. Wilbury Laboratories and they were in good condition at the beginning of the test. Water quality was within normal limits during the toxicity test. The minimum dissolved oxygen concentration was 7.8 mg/L, which is 86% saturation at 20°C. Results & Conclusion: Exposure of daphnids to benzyl salicylate resulted in a 48 hour median effective concentration (EC50) of 1.16 mg/L (95% confidence interval = 0.894 to 1.64 mg/L) and a 48 hour median lethal concentration (LC50) of 2.25 mg/L (95% confidence interval = 1.64 to 4.02 mg/L). The 48 hour no observed effect concentration (NOEC) was 0.894 mg/L benzyl salicylate.

Description of key information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and resulted in a 48 hour EC50 of 1.16 mg/L and a 48 hour no observed effect concentration (NOEC) was 0.894 mg/L benzyl salicylate.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.16 mg/L

Additional information

The test (OECD 202) was performed under static conditions in sealed vials with no head space to minimize the potential loss of the test substance from test solutions to the atmosphere. The test was run with five concentrations of test substance at a temperature of 20 ± 1°C. Mean measured concentrations of benzyl salicylate were: ND (none detected at or above the limit of quantitation of 0.00223 mg/L; control), 0.894, 1.64, 2.47, 4.02, and 6.23 mg/L. These mean measured concentrations, which ranged from 78 to 91% of nominal concentrations, were steady throughout the exposure period. Exposure of daphnids to benzyl salicylate resulted in a 48 hour median effective concentration (EC50) of 1.16 mg/L (95% confidence interval = 0.894 to 1.64 mg/L) and a 48 hour median lethal concentration (LC50) of 2.25 mg/L (95% confidence interval = 1.64 to 4.02 mg/L). The 48 hour no observed effect concentration (NOEC) was 0.894 mg/L benzyl salicylate.