(Z)-N-octadec-9-enylhexadecan-1-amide

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Analytical monitoring:

yes

Remarks:

HPLC-MS-MS

Details on sampling:

The test item (nominal 100 mg) was dissolved in propan-2-ol (100 mL) to prepare a stock
solution with a concentration of 1000 mg/L. This stock solution was further diluted with
(50:50 v/v) propan-2-ol:acetonitrile to produce a solution of 10 mg/L. Defined volumes of this
solution were then diluted with (90:10 v/v) acetonitrile:test medium to obtain calibration
standards in the range of 0.010 to 0.10 mg/L. A second series of calibration standards was
similarly prepared in the range of 0.025 to 0.15 mg/L. These solutions were used to determine
the recovery and test sample concentrations. These standards were used to evaluate the linearity of the
analytical system.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
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), 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 Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter) to give a saturated solution of the test item.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: bred at testing facility
- Age at study initiation: < 24 h


BREEDING
- Breeding conditions: same as in test
- Type and amount of food: algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension.
- Feeding frequency: daily

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L

Test temperature:

20 - 22 °C

pH:

7.6 - 7.9

Dissolved oxygen:

8.6 - 9.7 mg/L

Nominal and measured concentrations:

Nominal test substance concentration: 10, 18, 32,56 and 100% v/v saturated solution

Details on test conditions:

TEST SYSTEM
- Test vessel: glass jars (250 mL nominal volume)
- Fill volume: 200 mL
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 Medium
- Intervals of water quality measurement: at test start and test termination

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h day-night regime
- Light intensity: 648 - 660 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mobility after 24 and 48 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: Immobilization was observed at 100% v/v saturated solution

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: (% v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The test substance concentration was analysed for verification of test concentrations at 0 and 48 hours by HPLC-MS-MS (Perkin Elmer, API 3000; triple stage quadrupole mass spectrometer). The test concentrations were below the limit of quantitation (LOQ= 0.014 mg/L).
Limit of Quantitation (LOQ)
The limit of quantitation (LOQ) was determined by calculating the sample concentration that
gave a peak equivalent to ten times the baseline noise. Using this method the limit of
quantitation was determined to be 0.014 mg/L.

Results with reference substance (positive control):

EC50 (48 h): 0.45 mg/L

Cumulative Immobilization Data

Nominal concentration (% v/v saturated solution)	Immobilized Daphnia (mean)
	24 h	48 h
Control	0	0
10	0	0
18	0	0
32	0	0
56	0	0
100	0	2

(Initial population: 5 per replicate)

Whilst there was no immobilization in 20 daphnids exposed to test concentrations of 10, 18, 32 and 56% v/v saturated solution, 2 daphnia were observed to be immobilized in the 100% v/v saturated solution test concentration. However, as this is only 10% immobilization this is not thought to be a true toxic effect of this test item. Inspection of the immobilization data gave the following results:

Time (h) EC50 (% v/v saturated solution)

24 >100

48 >100

The No Observed Effect Concentration after 24 and 48 hours exposure was 100% v/v saturated solution.

Description of key information

No immobilisation or other negative effects were observed in any of the daphnids exposed to the test substances up to the highest attainable concentration tested. The substance is not harmful to aquatic invertebrates.

Additional information

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.The test substance is highly insoluble in water and pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high-shear mixing. A pre-study media preparation trial indicated that the use of a saturated solution method of preparation was most appropriate for this test item. The 48-Hour EC50 for the test item to Daphnia magna based on nominal test concentrations was greater than 100% v/v saturated solution. The No Observed Effect Concentration was 100% v/v saturated solution. Chemical analysis of the test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed were obtained which was determined to be 0.0141 mg/L.