L-Lysine, N2,N6-bis[N-(1-oxododecyl)glutamyl]-, sodium salt (1:?)

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:

Between 25th November 2009 and 27th November 2009.

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 did not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13 - 22 October 2008, this GLP certificate is valid for 2 years

Test material

Test material information

Constituent 1

Reference substance name:

Pellicier

IUPAC Name:

Pellicier

Details on test material:

Name: Pellicer
Lot No.: 01-09-17
Chemical name: L-Lysine, N2, N6-bis[N-(1-oxododecyl)-L-α (or γ)-glutamyl]-, sodium salt (1;?)
Purity: 91 %
Appearance: White powder
Storage: At room temperature in the dark, protected from humidity
Expiry date: 17 January 2011

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 6.3, 12.5, 25.0, 50.0 and 100.0 mg/L
- Sampling method: The concentration of the test item was measured at the applied test concentrations, at the beginning and end of the water renewal periods. Samples were transferred into HPLC vials and directly analysed.
- Sample storage conditions before analysis: All samples will be analysed directly after sampling. If analysis directly after sampling is not possible, the samples will be stored in a freezer immediately after sampling, and will be kept stored until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

Because the test item is non soluble in the media used in this aquatic toxicity test the stock solution was prepared according to the WAF method (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, “Direct addition”. OECD Series on Testing and Assessment No. 23, Paris September 2000).


A supersaturated test item stock solution (nominally more than 100 mg/L) was prepared by dispersing/dissolving the test item amount without the use of any organic solvent into the Test Medium (ISO Medium) two days before the start of the renewal periods (on day -2 and -1). This solution was shaken for 24 hours at 30 ºC, then was left settling for 24 hours at 20 ºC (on day -1 and 0) and thereafter was filtrated by a 0.45 μm fine filter. The test medium was prepared just before introduction of the Daphnia on day 0 and 1 (= start of the renewal periods).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain: not stated
- Source: National Institute of Public Health, 1097 Budapest Gyáli u. 2-6. Hungary
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): not stated in report
- Length at study initiation (length definition, mean, range and SD): not stated in report
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: The Daphnia were bred in Ecotoxicological Laboratory of LAB Research Ltd.
- Feeding during test: no feeding during the test
The Daphnia were fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding.

ACCLIMATION
There was no acclimatization because the water used during the test was similar to the culture water

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

The number of mobile and immobilised test animals was observed and recorded 24 and 48 hours after the start of the test.

Test conditions

Hardness:

159 mg/L

Test temperature:

The test temperature was between 18 and 22 °C. Temperature (in the test vessels): 20.2 – 20.7 °C

pH:

The pH of the test solution was not adjusted and the deviation was less than 1.5 units in any one test. The pH ranged from 7.57 to 7.90.

Dissolved oxygen:

The dissolved oxygen concentration was greater than 3 mg/L during the test. Oxygen concentration: 6.7 – 6.8 mg/L

Salinity:

Not applicable as fresh water was used.

Nominal and measured concentrations:

The following nominal concentrations were used in the main study: 6.3, 12.5, 25.0, 50.0 and 100.0 mg/L.
The corresponding geometric mean of the measured concentrations was: 2.5; 8.7; 19.4; 39.5 and 89.3 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: labortory glass beakers: 40 ml
- Material, size, headspace, fill volume: Fill volume: not stated in report
- Aeration: not stated
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not stated

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The ISO medium (according to OECD 202) will be prepared by adding 25 ml each of
the following stock solutions to deionised water.
CaCl2 x 2 H2O 11.76 g/L
MgSO4 x 7H2O 4.93 g/L
NaHCO3 2.59 g/L
KCl 0.23 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: not adjusted
- Photoperiod: Artificial illumination, 16 hours light and 8 hours darkness.
- Light intensity: not stated in report

EFFECT PARAMETERS MEASURED
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0, 0.01, 0.1, 1, 10, 100 (mg/l)
- Results used to determine the conditions for the definitive study: A significant toxic response was observed during the 2nd preliminary range-finding test, therefore five test concentrations arranged in a geometric series (with a factor 2.0) and an untreated control group was tested in the main study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

47.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% Confidence Limits: 42.0 - 53.4 mg test item

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

8.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

19.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The following results are based on the geometric mean of the measured concentrations.
48h EC0 (NOEC) value: 8.7 mg test item/L
48h LOEC: 19.4 mg test item/L
48h EC100 value: >89.3 mg test item/L

Results with reference substance (positive control):

The 24h EC50: 0.84 mg/L, (95 % confidence limits: 0.72 – 0.98 mg/L) for Potassium dichromate

Reported statistics and error estimates:

The EC50 values w calculated by Probit analysis with 95 % confidence limits using TOXSTAT software.
The EC0 (NOEC), LOEC and the EC100 were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met during this study.

Conclusions:

The 48 hour EC 50 value has been determined to be 47.4 mg/L, the NOEC has been determined to be 8.7 mg/L.

Executive summary:

Acute toxicity of Pellicer was assessed with Acute Immobilisation Test on Daphnia magna, over an exposure period of 48 hours in a semi-static system according to EU method C2 and OECD Guideline 202.

The following nominal concentrations were used in the main study: 6.3, 12.5, 25.0, 50.0 and 100.0 mg/L. The corresponding geometric mean of the measured concentrations was: 2.5; 8.7; 19.4; 39.5 and 89.3 mg/L.

Four vessels were used in test groups and in the control group. Each group comprised twenty Daphnia, five in each of the four replicate vessels, each containing app. 40 ml test solution.

Under the conditions of this Daphnia magna acute immobilization study the calculated and observed endpoints for the effect of Pellicer were the followings:

The 48h EC50 value: 47.4 mg test item/L (95 % confidence limits (lower-upper)): 42.0 – 53.4 mg test item/L

48h (NOEC) value: 8.7 mg test item/L

48h LOEC: 19.4 mg test item/L