Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

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:

26-29 June, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was performed according to standard guidelines, under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

and EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

The macro-nutrient concentration in the test medium was doubled.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

EC Number:

257-573-7

EC Name:

Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

Cas Number:

51981-21-6

Molecular formula:

C9H9NO8Na4

IUPAC Name:

tetrasodium 2-[bis(carboxylatomethyl)amino]pentanedioate

Details on test material:

- Name of test material: GBS-5
- Physical state: White powder
- Composition of test material, percentage of components:
- Substance (incl impurities) 94.99% w/w
- Water 5.01 % w/w
- Lot/batch No.: 40/7-11 (see CoA in the report attached)
- Storage condition of test material: Room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: control and 100 mg/L
- Sampling method: samples were taken from the control and each test group (replicates 1 to 6 pooled) at 0 and 72 hours.
- Sample storage conditions before analysis: No information

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 100 mg of test material was dispersed in culture medium and the volume adjusted to 500 mL to give a 200 mg/L stock solution. This stock solutionw as mixed with 500 mL algal suspension to give the test concentration of 100 mg/L.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 276/20
- Source: Culture Centre for Algae and Protozoa, Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria
- Method of cultivation: The culture was maintained in the laboratory by transfer of algal cells to fresh culture medium apporx. once per week. The culture was maintained at 21 ± 1 °C under continuous illumination and constant aeration.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

24 °C

pH:

0 h: 8.0 - 8.4
72 h: 9.9 - 10.1
The pH increased more than 1.5 units in some replicates. This was consideredto be due to the large number of cells in the end of the test and did not have an impact on the outcome of the test.

Nominal and measured concentrations:

control and 100 mg/L
Chemical analysis showed that the test material concentration was stable and the measured concentrations were equal to the nominals.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: the flasks were covered with aluminium foil
- Material, size, headspace, fill volume: 250 mL glass conical flasks
- Aeration: no
- Initial cells density: 10,000 cells/mL
- Control end cells density: 5.14x10E5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes, OECD medium

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, but the macro-nutrient concentration was doubled because of the chelating effect of the test material.
NH4Cl 30 mg/L
MgCI2.6H2O 24 mg/L
CaCl2.2H2O 36 mg/L
MgSO4.7H2O 30 mg/L
KH2PO4 3.2 mg/L
FeCl3.6H2O 0.16 mg/L
Na2EDTA.2H2O 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCI2 3x10E-3 mg/L
CoCl2.6H2O 1.5x10E-3 mg/L
CuCl2.2H2O 1x10E-5 mg/L
Na2MoO4.2H2O 7x10E-3 mg/L
NaHCO3 50 mg/L
The pH of this medium after equilibration with air is approximately 8.
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometrically at 665 nm, every 24 hours. The cell densities of the controls were also determined by direct counting with the aid of a haemocytometer to confirm that the absorbance values were sifficiently well correlated with cell density values.

TEST CONCENTRATIONS
- Range finding study: Three range finding studies were conducted. One with standard OECD medium which showed effects at the tested concentrations. After this two new range finding test were performed, one with trace element concentrations multiplied by a factor of 10 and one with macro-nutrient concentrations multiplied by a factor of 2.
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: The first range finding test showed no effect at 0.10 and 1.0 mg/L and significant inhibition was observed at 10 and 100 mg/L. The test with modified trace elements showed significant inhibition at 100 mg/L, but no effect at the other concentrations. The test with modified macro-nutrients showed no effects at any of the test concentrations.

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 94.99 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

5.01% w/w water

Basis for effect:

other: growth and biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities observed
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Reported statistics and error estimates:

A Students t-test was carried out on the area under the growth curve data at 72 hours for the control and 100 mg/L to determine significant differences between the control and the tested concentration.

Any other information on results incl. tables

Chemical analyses

Samples	Nominal concentration (mg/L)	Measured concentration (mg/L)
0 h	control	< LOQ
	R1 -R3*	102
	R4 -R6*	103
72 h	Control	< LOQ
	R1 -R3*	100
	R4 -R6 *	102

* pooled replicates 1 to 3 and 4 to 6, respectively

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test material is not toxic to aquatic algae.

Executive summary:

A study was performed to assess the effect of the test material, GBS-5, on the growth of the green alga Scenedesmus subspicatus. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No. 201, “Alga, Growth Inhibition Test” referenced as Method C.3 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Procedure
Following preliminary range-finding studies, Scenedesmus subspicatus was exposed to an aqueous dispersion of the test material at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 °C.
At the request of the Sponsor the culture medium used in the study was modified by multiplying the macro-nutrient concentration by a factor of 2.
Samples of the algal populations were removed daily and absorbance values determined for each control and treatment group.

Results
Exposure of Scenedesmus subspicatus to the test material gave EC50 values of greater than 100 mg/L and correspondingly the No Observed Effect Concentration was greater than or equal to 100 mg/L.
It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
Analysis of the test solutions at 0 and 72 hours showed the measured test concentrations to be near nominal and so the results are based on nominal test concentrations alone.