Reaction mass of 3-[(2-aminoethyl)ammonio]propane-1-sulfonate and 3,3'-(ethane-1,2-diyldiammonio)dipropane-1-sulfonate and ethane-1,2-diamine

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:

March 2008-January 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Cas Number:

14235-54-2

Molecular formula:

C5H14N2O3S

IUPAC Name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Details on test material:

- Physical state: liquid
- Composition of test material, percentage of components:
48.5 % (w/w) 3-[(2-aminoethyl)ammonio]propane-1-sulfonate
51.5 % water
- Lot/batch No.: 06060423/101
- Stability under test conditions: stable
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Two forms of the test substance are available:
1) A ca. 50 % solution of EPS in water. Actually the concentration was 48.5 % w/w. This form was used for the test and is the form which is produced and registered.
2) The highly viscous to solid test substance, containing ca. 5 % residual water. This form was produced by evaporating the water of the ca. 50 % solution of EPS in a rotating evaporator. Further removal of water would damage the test substance.
This form of the test substance was used for the physical-chemical test, the studies on hydrolysis and adsorption coefficient.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Six different concentrations of the test substance between nominal 3.13 and 100.00 mg per L nutrient medium, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only).
- Sampling method: At the start and at the end of the incubation period samples of the test media were drawn and the concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae, and of one replicate of each of the test and control cultures.
- Sample storage conditions before analysis: The samples were deep frozen on the day of sampling and unfrozen on the day of analysis. One sample each was taken and then analysed in duplicate by HPLC.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal test substance concentration of 111 mg/L was prepared by dissolving 229.1 mg test substance in 1 L of nutrient medium by manual homogenisation. Aliquots of this stock solution were diluted with nutrient medium to obtain lower concentrations, nominally spaced by a factor of 2. The preparations were made freshly before the start of the exposure.
- Eluate: no.
- Differential loading: no.
- Controls: For the negative control group only nutrient medium was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): none.
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable.
- One blank, containing the highest test substance concentration, but no algae, was incubated under the same culture conditions for analysis of the test substance concentration and comparison with the algae cultures.
- The test was performed without adjustment of the pH of the test substance cultures.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum).
- Strain: ATCC (American Type Culture Collection) 22662.
- Source (laboratory, culture collection): LGC Promochem GmbH, Germany.
- Age of inoculum (at test initiation): /
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, were continuously maintained in 250 mL conical flasks. each containing 100 mL nutrient medium, at a temperature of 23 °C under permanent light with an intensity of at least 6000 lux. In about weekly intervals 1 mL of the stock culture was diluted 100-fold with nutrient medium for precultivation and incubation is continued.

ACCLIMATION
- Acclimation period: Precultures were prepared by incubating a new stock culture for four days.
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

/

Post exposure observation period:

Observation during the 72 h exposure period.

Test conditions

Hardness:

Not reported.

Test temperature:

23 +- 2 °C

pH:

The pH was between 7.5 and 8.1 at the start of the incubation in the test cultures and it was 7.3 in the control cultures.
After 72 hours of incubation the pH was between 9.4 and 9.9 in the test cultures and it was 7.8 in the control cultures.

Dissolved oxygen:

Not reported.

Salinity:

Fresh water was used.

Nominal and measured concentrations:

With algae:
Nominal concentrations: 0 - 3.13 - 6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L.
Actual concentrations at the start: 0 - 2.9 - 6.3 - 13.0 - 25.0 - 49.1 - 92.4 mg/L.
Actual concentrations after 72 h: 0 - 2.6 - 5.5 - 10.8 - 23.5 - 46.0 - 99.0 mg/L.

Blank without algae:
Nominal concentrations: 100 mg/L.
Actual concentrations at the start: 100.6 mg/L.
Actual concentrations after 72 h: 91.1 mg/L.

In the blank without algae the actual test substance concentration after 72 hours was 90.5 % of the actual starting concentration. The results give no indications for an uptake of the test substance by the algae or for adherence of the test substance to them.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL closed Erlenmeyer flasks filled with 100 mL medium .
- Initial cells density: 10 000 cells/mL
- Control end cells density: the cell density in the control cultures increased by a factor of about 82
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Sterile deionised water and sterile concentrated nutrient medium 9 + 1 was used.
- Conductivity of the deionised water: <5 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h.
- Light intensity and quality: at least 6000 lux. Wavelength of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic cell counter.
- Chlorophyll measurement: no
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: no

Reference substance (positive control):

no

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

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): During the 72 hours incubation period the cell density in the control cultures increased by a factor of ca. 82, corresponding to about 6.4 generations.
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: In all concentrations tested, the influence on algal growth was low and ranged from 12.4 % inhibition to 6.8 % enhancement.
- Any observations that might cause a difference between measured and nominal values: none.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

Not relevant.

Reported statistics and error estimates:

Not relevant.

Any other information on results incl. tables

The results give no indications for an uptake of the test substance by the algae or for adherence of the test substance to them.

The test substance concentrations have been satisfactorily maintained to within 80% of the initial concentrations throughout the duration of the test.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

NOEC >= 100 mg/L.
EC50 > 100 mg/L.

Executive summary:

A Pseudokirchneriella subcapitata growth inhibition test according to the directive 92/69/EEC Part C.3 was performed to determine the possible effects of "3-[(2-aminoethyl)ammonio]propane-1-sulfonate" on the growth of a unicellular green algal species. Six different concentrations of the test substance between nominal 3.13 and 100.00 mg per L nutrient medium, spaced by a factor of 2.0, were tested against one concurrent negative control (nutrient medium only). In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation.Possible test substance effects were determined by comparison of the areas under the growth curves and by comparison of the growth rates. At the start and at the end of the experiment, samples of the cultures exposed to the test substance, of the control cultures and also of the blank with the highest concentration of the test substance, but no algae, were taken and analysed by HPLC.

The test substance concentrations have been satisfactorily maintained to within 80 % of the initial concentrations throughout the duration of the test. In all concentrations tested, the influence on algal growth ranged from 12.4 % inhibition to 6.8 % enhancement. Two identical "no observed effect concentrations" (NOECs, greater or equal 100 mg/L) and two identical EC50 values (greater 100 mg/L) were derived.