6-O-palmitoylascorbic acid

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-08 until 2015-08-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-O-palmitoylascorbic acid

EC Number:

205-305-4

EC Name:

6-O-palmitoylascorbic acid

Cas Number:

137-66-6

Molecular formula:

C22H38O7

IUPAC Name:

6-O-palmitoylascorbic acid

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC/UV

Details on sampling:

Samples were taken from the control and 100% v/v saturated solution test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours.
Samples were stored frozen prior to analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000; for references see original study report), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (50 mg/L) of test item in culture medium for a period of 24 hours prior to removing any undissolved test item present by filtration (0.2 µm Sartorius Sartopore, first approximate 1 liter discarded in order to pre-condition the filter) to give a saturated solution of the test item.

Range-finding test concentrations were 0.10, 1.0, 10 and 100% v/v saturated solution.

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100% v/v saturated solution.

A nominal amount of test item (550 mg) was dispersed in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Sartorius Sartopore filter (first approximate 1 liter discarded in order to pre-condition the filter) to give the required test concentration of 100% v/v saturated solution.

The prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Study design

Test type:

static

Water media type:

other: ISO medium

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

250 mg/L expressed as CaCO3

Test temperature:

21-22 °C

pH:

At test start: 7.8
At test end: 7.7 - 7.8

Dissolved oxygen:

At test start: 8.4 - 8.8 mg O2/L
At test end: 8.6 - 8.7 mg O2/L

Salinity:

Not particularly stated, but detailed description of test medium composition given in the original study report.

Nominal and measured concentrations:

Nominal concentration: 100% v/v saturated solution
Measured at sampling time 0 hours: < LOQ (0.10 mg/L)
Measured at sampling time 48 hours: < LOQ (0.10 mg/L)

Details on test conditions:

- Test vessels: open 250 mL glass jars containing approximately 200 mL of test preparation
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

The test vessels were covered to reduce evaporation and maintained in a temperature controlled room at approximately 22 °C with a photoperiod of 16 hours light (not exceeding 1500 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.

The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

yes

Remarks:

potassium dichromate used in separate study

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % v/v saturated solution

Nominal / measured:

nominal

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: % v/v saturated solution

Nominal / measured:

nominal

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 50 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 50 mg/L

Nominal / measured:

nominal

Conc. based on:

other: loading rate

Basis for effect:

mobility

Details on results:

VERIFICATION OF TEST CONCENTRATIONS
Analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, which was determined to be 0.10 mg/L. This does not infer that no test item was in solution, just that any dissolved test item present was at a concentration of less than the LOQ.

IMMOBILIZATION DATA
There was no immobilization in 20 daphnids exposed to a test concentration of 100% v/v saturated solution for a period of 48 hours.

SUB-LETHAL EFFECTS
No sub-lethal effects of exposure were observed throughout the test.

Results with reference substance (positive control):

Potassium dichromate was used as reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.
The 48-hour EC50 was 0.75 mg/L with 95% confidence limits of 0.69 to 0.82 mg/L, the NOEC was 0.56 mg/L. The results were within the normal range for this reference item.

Reported statistics and error estimates:

not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Ascorbyl palmitate induced no immobility in Daphnia magna exposed to a 100% v/v saturated solution (NOEC), prepared by filtration (ca. 0.2 µm) of a test preparation with a loading rate of nominal 50 mg/L.
Owing to the extremely low solubility of ascorbyl palmitate in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h-EC50 for Daphnia magna exceeded the maximum solubility of ascorbyl palmitate in water.

Executive summary:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

A preliminary media preparation trial indicated that a saturated solution method of preparation was appropriate for this test item. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 100% v/v saturated solution for 48 hours at a temperature of approximately 22 °C under static test conditions. The test item solution was prepared by stirring an excess (50 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any undissolved test item was removed by filtration (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item.

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

Exposure of Daphnia magna to the test item gave EC50 values of greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution. This study showed that there were no toxic effects at saturation.