Pentyl salicylate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-11-2011 to 16-02-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study (OECD 202)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Amyl salicylate
- Physical state: liquid
- Purity test date: 99.9% (some of isomers)
- Lot/batch No.: PE00022143
- Expiration date of the lot/batch: 19 August 2012
- Storage condition of test material: Room temperature in the dark.

Analytical monitoring:

yes

Details on sampling:

Concentrations:
Nominal concentrations of amyl salicylate were 0 mg/L (control), 0.37, 0.67, 1.2, 2.2 and 3.7mg/L.

Mean measured concentrations of amyl salicylate were: Chemical analysis of the 0.37 and 3.7mg/l test preparations at 0 hours showed measured test concentrations of 0.31 and 1.6 mg/l were obtained (85% and 43% of nominal respectively) whilst analysis at 48 hours showed mean measured test concentrations of 0.23 and 1.8 mg/l were obtained (61 % and 50 % of nominal respectively). Given the decline in measured test concentration observed for the 0.37 mg/l test preparation it was considered appropriate to perform the definitive test using semi-static conditions in order to try and maintain near nominal test 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 the dark.

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under the test conditions. All samples were stored at approximately -20 °C prior to analysis.

Water samples were taken from the control and each test group (replicates R1 - R4 pooled) at 0 and 24 hours (fresh media) and at 24 and 48 h (old media) for quantitative analysis.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: HPLC using a Symmetry C18, 3.5µm (50 x 3 mm id) column; flow rate: 0.5ml/min; detector: UV/vis at 238nm; injection volume: 50µl
- Eluate: Methanol: water: formic acid (80: 20: 0.1 v/v/v)
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution): Approximately 550mg was dispersed in 11 litres of ELendt M7 media with the aid of propeller stirring for approximately 24 hours. After stirring the saturated solution was filtered. A series of dilutions was made from this saturated solution to give further nominal test concentrations of 2.2, 1.2, 0.67 and 0.37mg/l (equivalent to 1.6, 1.1, 0.57 and 0.39mg/l) based on the time weighted mean measured test concentrations.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnids
- Strain: magna

TEST ORGANISM
- Common name: Daphnids

- Strain: magna

- Source: 1st instar daphnia magna were derived from in-house stock. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 h old. These young were removed from the cultures and used for testing. Culture conditions ensured that reproduction was by parthenogenesis.

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

- Feeding during test: No

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The theoretical total water hardness of Elendt M7 medium is 250 mg/ L as CaCO3 and was therefore diluted in a ratio of 50:50 with deionised reverse osmosis water to reduce the total hardness.

Test temperature:

18 to 21 °C.

pH:

7.8 - 8.4

Dissolved oxygen:

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100 %. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was not considered to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Nominal and measured concentrations:

Definitive test nominal concentrations: 0.37, 0.67, 1.2, 2.2 and 3.7 mg/L, equivalent to 0.39, 0.57, 1.1 and 1.6 mg/L based on the time-weighted mean measured test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: Ground glass stoppered conical flasks
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: 250 mL, completely filled; control contained approximately 200 mL.
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Sami static with a daily renewal of the test preparations.
- Renewal rate of test solution (frequency/flow rate): Not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four
- No. of vessels per vehicle control (replicates): No data
- Biomass loading rate: No data

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Elendt M7 medium was used for culturing, range-finding and definitive tests.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobilisation

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: Nominal concentrations of 0.0037, 0.037, 0.37 and 3.7 mg/L.
- Results used to determine the conditions for the definitive study: No immobilisation was observed at the test concentrations of 0.0037, 0.037 and 0.37, however immobilisation was observed at 3.7 mg/L. Based on this information nominal test concentrations were selected for the definitive test. Chemical analysis of the 0.37 and 3.7 mg/ test preparations at 0 h showed measured test concentrations of 0.3 and 1.6 mg/L were obtained (85 % and 43 % of nominal respectively) whilst analysis at 48 h showed measured test concentrations of 0.23 and 1.8 mg/L were obtained (61 % and 50 % of nominal respectively). Given the decline in measured test concentration observed for the 0.37 mg/L test preparation, it was considered appropriate to perform the definitive test using semi-static conditions in order to try and maintain near nominal test concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence interval = 1.2 to 1.7 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.88 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval = 0.75 to 1.0 mg/L NOEL (48 hours)= 0.57mg/L

Details on results:

Chemical analysis of the test preparations at 0 h showed measured test concentrations to range from 91 % to 01 % of nominal whilst measured concentrations in the range of 80 % to 96 % of nominal were obtained from the 24 h old media test samples. Analysis of the freshly prepared test media at 24 h showed measured test concentrations to range from 76 % to 128 % of nominal whilst analysis of the corresponding old media samples at 48 h showed measured test concentrations to range from 70 % to 97 % of nominal. Given that some of the measured test concentrations were outside the 80 - 20 % acceptance limits, it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations, which were determined to be 0.39, 0.57, 1.1, 1.6 and 3.4 mg/L.
The NOEC after 24 and 48 h exposure was 0.57 mg/L. The NOEC is based upon no significant immobilisation at this concentration.
The slopes and their standard errors of the response curves at 24 and 48 h were 5.3 (SE = 1.0) and 9.2 (SE = 1.8) respectively.

Results with reference substance (positive control):

The results from the positive control were within the normal range for this reference item.

Reported statistics and error estimates:

The EC50 values and associated confidence limits at 24 and 48 hours and the slope of the response curve and its standard error were calculated by the maximum-likelihood probit method using the ToxCalc computer software package. Probit analysis is used where two or more partial responses to exposure are shown.

Cumulative immobilisation data in the definitive test:

Mean-weighted mean measured test concentration (mg/L)	Cumulative immobilized Daphnia (initial population: 5 per replicate)
	24 h						48 h
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.39	0	0	0	0	0	0	0	0	0	0	0	0
0.57	1*	0	0	0	1	5	1*	0	0	0	1	5
1.1	2	1	1	1	5	25	4	4	5	3	16	80
1.6	2	3	4	3	12	60	5	5	5	5	20	100
3.4	5	5	5	5	20	100	5	5	5	5	20	100

R1 - R4 = Replicates 1 to 4

* Single immobilised daphnid considered to be due to natural causes rather than a true toxic effect, as no further immobilisation was observed to have occurred.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity to freshwater Daphnia magna based on the time-weighted mean measured test concentration was determined to be EC50(48h)= 0.88mg/L.

Executive summary:

A GLP study was performed to assess the acute toxicity of the Amyl salicylate to Daphnia magna in accordance with OECD Guideline 202.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at nominal test concentrations of 0.37, 0.67, 1.2, 2.2 and 3.7mg/L for 48 hours at a temperature of 20C to 22C under semi-static conditions. The test item solutions were prepared by stirring excess (50mg/L) of test item in Elendt M7 medium using a propeller stirrer at approximately 1500rpm for 24 hours. After the stirring period any un-dissolved test item was removed by filtration (0.2m Sartorius Sartopore filter, first approximate 2 litres discarded in order to pre-condition the filter) to produce a saturated solution of the test item with a nominal concentration of 3.7mg/L. This saturated solution was then further diluted as necessary to provide the required test conditions.

Immobilisation and adverse reactions to exposure were recorded after 24 and 48 hours.

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 91% to 101% of nominal whilst measured concentrations in the range of 80% to 96% of nominal were obtained from the 24 -Hour old media test samples. Analysis of the freshly prepared test media at 24 hours showed measured test concentrations to range from 76% to 128% of nominal whilst analysis of the corresponding old media samples at 48 hours showed measured test concentrations to range from 70% to 97% of nominal.

Given that some of the measured test concentrations were outside of the 80 -120% acceptance limits it was considered appropriate to calculate the results based on the time-weighted mean measured test concentrations which were determined to be 0.39, 0.57, 1.1, 1.6 and 3.4mg/L.

The 48 -Hour EC50 (Daphnia magna) for the test item based on the time-weighted mean measured test concentrations was 0.88mg/L; 95% confidence limits 0.75 - 1.0mg/L. The NOEL was 0.57mg/L.

 

Description of key information

EC50(96 hours)= 0.88 mg/L (mean measured TWA)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.88 mg/L

Additional information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated. The test was performed under semi-static conditions. The results were based on the time-weighted mean measured test concentrations.