Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

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:

The study was conducted between 5 September 2011 and 19 October 2011.

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 relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

see principles of method if other than guideline section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

see principles of method if other than guideline section

Principles of method if other than guideline:

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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description: off white powder
Purity: not applicable - complex mixture
Batch number: PS1 011 03
Date received: 11 July 2011
Expiry date: 18 November 2012
Storage conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Details on 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, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Pre-study work showed that a preparation period of 24 hours was sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, and following a 1-Hour settlement period the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expresed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

The reconstituted water used for both the range-finding and definitive test is defined below:
Stock Solutions
a) CaC12.2H20 - 11.76 g/l
b) MgS04.7H20 - 4.93 g/l
c) NaHC03 - 2.59 g/l
d) KCI - 0.23 g/l

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1 . The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Adult Daphnia were maintained in 150 ml glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 21 deg C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

Each culture was fed daily with a mixture of algal suspension and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any
contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

Test temperature:

Test temperature was between 21 and 22 deg C.

Some of the temperatures were measured to be slightly in excess of the 20 ± 1°C given in the study plan. This was considered not to affect the results of the test as no adverse effects of exposure were observed in the control daphnids throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter. After 48 hours the pH was measured to be between 7.9 and 8.2 for all samples.

Dissolved oxygen:

The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi Handheld meter. The dissolved oxygen concentrationw as measured to be between 8.4 and 8.8 mg O2/l after 48 hours. There were no treatment related differences for Oxygen concentration or pH.

Salinity:

Not applicable as fresh water

Nominal and measured concentrations:

Nominal - 100 mg/L
Measured: 0.022 mg/l

Details on test conditions:

TEST SYSTEM
An amount of test item (250 mg) was added to the surface of 2.5 litres of reconstituted water to give the 100 mg/I loading rate. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 ml discarded) to give the 100 mg/l loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.

As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 21°C to 22°C with a photoperiod of 16 hours light 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.

As a decline in measured test concentration was observed in the range-finding test over the 48-Hour test period a semi-static test regime was employed in the definitive test involving a daily renewal of the test preparations to ensure that the concentration of the test item remained as high as possible.

- Range finding study
- Test concentrations: 20 and 200 mg/l
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test a "Limit test" was conducted at a single loading rate of 100 mg/I to confirm that no immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (please see attachments section). No immobilisation was observed at 10 and 100 mg/iloading rate WAF.

Based on this information, a single loading rate of four replicates, of 100 mg/l, using a stirring period of 23 hours followed by a 1-Hour standing period, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilisation or adverse reactions to exposure were observed.

Chemical analysis of the 100 mg/l loading rate WAF test preparation at 0 hours (see Appendix 6) showed a measured test concentration of 0.022 mg/l was obtained. A decline in measured test concentration was observed at 48 hours to 0.0063 mg/l thereby indicating that the test item was unstable over the test duration.

Definitive test:
There was no immobilisation in 20 daphnids exposed to a 100 mg/l loading rate WAF for a period of 48 hours. Inspection of the immobilisation data gave the following results:
24 hours EL50 - 100 mg/L
48 hours EL50 - 100 mg/L

It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd., Project No: 41102080) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l. Exposure conditions for the positive control were similar to those in the definitive test. Analysis of the immobilisation data by the maximum-likelihood probit method (Finney 1971) at 24 and 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/I) 95% Confidence limits (mg/I)
24 1.5 1.3 - 1.8
48 0.99 0.85 - 1.1

The No Observed Effect Concentration after 24 and 48 hours was 0.56 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration. The results from the positive control with potassium dichromate were within the normal range for this reference item, and satisfied the conditions of this study.

Any other information on results incl. tables

Validation of Mixing Period:

Pre-study work indicated that there was no increase in the amount of total organic carbon by extending the preparation period for longer than 24 hours. Therefore, for the purposes of testing the test item was to be prepared using a 23-Hour stirring period followed by a 1-Hour settlement period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 (effective loading rate) value of greater than 100 mg/l loading rate WAF. The No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction. 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 DECO 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. Methods. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21°C to 22°C under semi-static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results. The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF. The No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

Chemical analysis of the test preparations at 0 and 24 hours (fresh media) showed measured test concentrations to range from 0.096 to 0.15 mg/L. A decline in measured test concentrations was observed at 24 and 48 hours (old media) in the range of 0.029 to 0.076 mg/L. This decline was considered to be due to the unstable nature of the test item.

Given that toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.