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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between November 2009 and February 2010

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

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

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:

Pellicer

IUPAC Name:

Pellicer

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: all concentration levels were tested
- Sampling method: The concentration of the test item was measured at each test concentration level, at the start (0h), at 24h and 48h and at the end (72h) of the test. Samples were transferred into HPLC vials and directly analysed. Samples were twofold diluted with water before the HPLC
analysis. 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:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 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 (OECD Medium) two days before the start of the study (on day -2). This solution was shaken for 24 hours at ~30 ºC, then was left settling for 24 hours at ~20 ºC (on day -1) and thereafter was filtrated by paper filter or by a 0.45 μm filter (on day 0). The test solutions was prepared just before introduction of the algae on day 0 (= start of the experiment).

Stock solution: 1000 mg/L nominal concentration

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green Algae
- Strain: Not stated in report
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal
Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): Not stated in report
- Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.
- Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.

ACCLIMATION
- Acclimation period: not stated in report
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

All test and control cultures were inspected microscopically at 72 hours.

Test conditions

Hardness:

Not stated in report

Test temperature:

Culture temperature was checked at the beginning of the study and every 24 hours in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) in a vessel established for this purpose only within the climate chamber. The cultures were maintained at a temperature in the range of 21 – 24°C.

pH:

The pH was measured in the control(s) and in the used test concentration in each test vessel at the start and at the end of the experiment. The range of the pH was 7.50 – 8.00 at the start and 7.49 – 9.13 at the end of the study

Dissolved oxygen:

Not stated in report

Salinity:

Not applicable as freshwater 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: 3.0; 5.3; 7.5; 14.9 and 30.1 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:250 mL conical flasks
- Material, size, headspace, fill volume: fill volume: 100 mL
- Aeration: not stated
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not renewed
- Initial cells density: 10E4 cells/mL
- Control end cells density: not stated
- No. of organisms per vessel: each containing approximately 10E4 alga cells/mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
The 4th and 5th replicates of the test item groups and the 7th and 8th replicates of the control group were used for analytical determination on days 1 and 2, because the test item was not stable during this 72 hours test. The results of these replicates were used for the calculation of the concentrations.

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Nominal concentrations
Macro-nutrients: NaHCO3 50.0 mg/L
CaCl2 x 2 H2O 18.0 mg/L
NH4Cl 15.0 mg/L
MgSO4 x 7 H2O 15.0 mg/L
MgCl2 x 6 H2O 12.0 mg/L
KH2PO4 1.6 mg/L
Trace Elements: MnCl2 x 4 H2O 415.0 mg/L
H3BO3 185.0 mg/L
FeCl3 x 6 H2O 64.0 mg/L
Na2EDTA x 2H2O 100.0 mg/L
Na2MoO4 x 2 H2O 7.0 mg/L
ZnCl2 3.0 mg/L
CoCl2 x 6 H2O 1.5 mg/L
CuCl2 x 2 H2O 0.01 mg/L

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not stated
- Photoperiod: continuously illuminated
- Light intensity and quality: 8512 lux
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
- Chlorophyll measurement: not stated

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
- Results used to determine the conditions for the definitive study: 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: 3.0; 5.3; 7.5; 14.9 and 30.1 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limits: 27.5 - 41.9

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

109.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence limits 50.2 - 237.9

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

33.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% Confidence limits: 27.1 - 42

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

30.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 8

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

14.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

As the deviation from the nominal concentrations was more than 20 percent at all concentration levels during the test; the results are based on the measured geometric mean test item concentrations. The corresponding geometric mean of the measured concentrations was: 3.0; 5.3; 7.5; 14.9 and 30.1 mg/L.

Results with reference substance (positive control):

The ErC 50 : 0.92 mg/L, (95 % confidence limits: 0.83 – 1.02 mg/L)
The EbC 50 : 0.55 mg/L, (95 % confidence limits: 0.50 – 0.61 mg/L)
The EyC 50 : 0.47 mg/L, (95 % confidence limits: 0.43 – 0.52 mg/L)

Reported statistics and error estimates:

The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”.

Statistical comparisons of biomass, average specific growth rates and yield in control and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (a = 0.05) by TOXSTAT software.

For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations will be tested on significant differences to the control values by Bonferroni t-Test.

Any other information on results incl. tables

The experiment is valid because the cell density in the control cultures increased by a factor of 69.50 within 72 hours. The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % during the course of the test (days 0-1; 1-2; 2-3). The section-by-section growth rates remained nearly constant indicating the exponential growth for the entire study period.

The coefficient of variation of average specific growth rates during the whole test period in the control cultures did not exceed 7 %.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met during this study.

Conclusions:

In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with PELLICER, the 72-h EC50 based on growth rate was determined as 109.3 mg/L. Yield was the most sensitive parameter with an 72-h EC50 of 33.7 mg/L. The overall NOEC was determined to be 7.5 mg/L

Executive summary:

The effect of Pellicer test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours. The guidelines followed are OECD No. 201, EPA Ecological Effects Test Guidelines OPPTS 850.5400 and Commission Directive 92/69/EEC Method C.3.

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: 3.0; 5.3; 7.5; 14.9 and 30.1 mg/L. At the start of the study the alga cell number was 104 cells/mL in all of the test cultures.

In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with Pellicer, the 72-h EC50 based on growth rate was determined as 109.3 mg/L. Yield was the most sensitive parameter with an 72-h EC50 of 33.7 mg/L. The overall NOEC was determined to be 7.5 mg/L.