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

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:

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.2 (Acute Toxicity for Daphnia)

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: Nominal test substance concentrations of 6.25, 12.5, 25, 50 and 100 mg test substance per litre reconstituted water according to ISO 6341 were tested.
- Sampling method: Samples of the test media for determination of the actual test substance concentrations were taken immediately before introduction of the Daphnia and after 48 hours. Actual concentrations of the test substance in the test medium were determined in the surplus of the test media at the beginning of the test and in the pooled test media of the replicates at the end of the test.
- 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 of nominal 100 mg/L was prepared by addition of actual 204.8 mg test substance to 1000 mL reconstituted water according to ISO 6341 and manual homogenisation. Aliquots of this stock solution were diluted with reconstituted water 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 reconstituted water according to ISO 6341 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.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Visual observations were made at the same time as the animal observation. The control medium and the test media were clear and colourless throughout the test period.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Neonates of Daphnia magna (Crustacea, Cladocera), were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM). .
Batch No. of the ephippia in the test: DM110208.
- Strain: /
- Source: Ephippia were supplied in the microbiotest "Daphtoxkit F TM " (Daphnia magna; Fa. Micro BioTests Inc; Kleimoer 15, B-9030 Mariakerke (Gent); BELGIUM).
- Age at study initiatio): less than 24 hours old.
- Weight at study initiation: /
- Length at study initiation: /
- Valve height at study initiation, for shell deposition study: not applicable.
- Peripheral shell growth removed prior to test initiation: not applicable.
- Method of breeding: Neonates were obtained by hatching ephippia. The ephippia were hatched in the laboratory in a petri dish containing reconstituted water in a temperature controlled room at approx. 20-22 °C under continuous illumination of approximately 6 000 Lux. According to MicroBioTests Inc. the largest hatching occurs between 72 and 80 hours and the Daphnia must be collected at the latest 90 hours after initiation of hatching. The neonates were collected for this test at approximately 85 hours after initiation of hatching.
- Feeding during test: none.
- Food type: not applicable.
- Amount: not applicable.
- Frequency: not applicable.


ACCLIMATION
- Acclimation period: <24 h.
- Acclimation conditions (same as test or not): same.
- Type and amount of food: Approximately 2 hours before the start of the test the neonates were fed with a suspension of Spirulina powder, as also in traditional Daphnia cultures the neonates have the opportunity to take up some feed particles.
- Feeding frequency: once.
- Health during acclimation: not applicable.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None.

Test conditions

Hardness:

250 mg CaCO3 / L.

Test temperature:

20 °C.

pH:

Between 7.6 and 7.9 in the relevant test substance media. Between 7.4 and 7.8 in the control media.

Dissolved oxygen:

At least 8.9 mg/L.

Salinity:

Not applicable.

Nominal and measured concentrations:

Nominal: 0.0, 6.3, 12.5, 25.0, 50.0 and 100 mg/L.
Actual, at time 0 h: 0.0, 5.5, 11.3, 23.3, 45.0 and 96.8 mg/L.
Actual, at time 48 h: 0.0, 6.0, 12.3, 24.7, 48.3 and 94.8 mg/L.
Geometric mean of the actual concentrations: 0.0, 5.7, 11.8, 24.0, 46.6 and 95.8 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: Disposable multiwell test plates with 30 test wells covered with a transparent lid, provided with the Daphtoxkit F. Each plate has four 10 mL wells (replicates A, B, C, D) for each of the control and the concentrations tested. Additionally, for each concentration the plates are provided with "rinsing wells" to prevent dilution of the test substance during the transfer of the Daphnia from the hatching petri dish to the test wells.
- Material, size, headspace, fill volume: 10 mL policarbonate wells (according to Microbiotest), filled with 10 mL medium.
- Aeration: No extra aeration, but slight circulation of air is still possible when the wells are closed with the transparent lid.
- Type of flow-through: /
- 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: /


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISOD191007). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
- Culture medium different from test medium: no.
- Intervals of water quality measurement: Temperature daily. Oxygen and pH at the start and the end of the test. Total hardness before the start.


OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 4 a.m. to 8 p.m.
- Light intensity: /


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
24 and 48 hours after the start of the exposure to the test substance the neonates were observed for their swimming behaviour. Animals not able to swim within approximately 15 s after gentle agitation of the test container were evaluated as immobilised. Animals trapped at the surface were counted, to evaluate a possible influence of the test substance to the water surface tension.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: nominal 2.
- Justification for using less concentrations than requested by guideline: not applicable.
- Range finding study
- Test concentrations: not applicable.
- Results used to determine the conditions for the definitive study: not applicable.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

ca. 25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none.
- Observations on body length and weight: not applicable.
- Other biological observations: no.
- Mortality of control: no immobilisation in the negative control group.
- Other adverse effects control: no.
- Abnormal responses: no.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.
- Effect concentrations exceeding solubility of substance in test medium: no.

Results with reference substance (positive control):

- Results with reference substance valid: yes.
- EC50 24h: 1.27 mg/L
- Other: The batch of ephippia is provided together with a specification sheet with batch number and date of expiry. Additionally for confirmation of
the sensitivity of the test animals of the used batch the results of a reference test with potassium dichromate are provided.
The acceptable range of the EC50 24h according to the OECD guideline 202 is 0.6 mg/L to 2.1 mg/L.

Reported statistics and error estimates:

not applicable.

Any other information on results incl. tables

Quality criteria:

·        Constant conditions were maintained (temperature, pH).

·        There was no immobilisation in the negative control group at the end of the test.
(guideline: maximum of 10 %).

·        There was no control Daphnia trapped at the water surface.

·        The dissolved oxygen concentration was higher than 3 mg/L at each determination.

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

- Immobilisation:

Group	Nominal test substance concentration (mg/L)	Immobilisation at 24 h	Immobilisation at 48 h
K (Control)	0.00	0 / 20	0 / 20
C1	6.25	1 / 20	2 / 20
C2	12.50	0 / 20	1 / 20
C3	25.00	0 / 20	2 / 20
C4	50.00	0 / 20	4 / 20
C5	100.00	1 / 20	10 / 20

- Actual test substance concentrations:

Group	Nominal test substance concentration (mg/L)	Actual concentration at 0 h (mg/L)	Actual concentration at 48 h (mg/L)	Difference (0-48 h) between the actual concentrations (%)	Geometric mean 0 - 48 h (mg/L)
K (Control)	0.00	0.0	0.0	-	-
C1	6.25	5.5	6.0	9.1	5.7
C2	12.50	11.3	12.3	8.8	11.8
C3	25.00	23.3	24.7	6.2	24.0
C4	50.00	45.0	48.3	7.2	46.6
C5	100.00	96.8	94.8	-2.1	95.8

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

EC50,48h 100 mg/L.
EC0,48h ca. 25 mg/L.
NOEC48h 25 mg/L.

Executive summary:

The acute toxicity of " 3-[(2-aminoethyl)ammonio]propane-1-sulfonate" to Daphnia magna was determined according to EC-methods.

Nominal test substance concentrations of 6.25, 12.5, 25, 50, and 100 mg test substance per litre were diluted from a stock solution of nominal 100 mg/L. The stock solution was prepared by addition of actual 204.8 mg of a 48.5 % solution of"3-[(2-aminoethyl)ammonio]propane-1-sulfonate"in water, to 1000 mL reconstituted water according to ISO 6341 and was manually homogenised. Reconstituted water only was used for the negative control group. The exposure of the Daphnia was for 48 h in a static test.

Results:

EC50,48h 100 mg/L

EC0,48h ca. 25 mg/L

NOEC48h 25 mg/L