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 fish

Administrative data

Endpoint:

short-term toxicity to fish

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 meeting the requirements of the OECD Principles of Good Laboratory Practice

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.1 (Acute Toxicity for Fish)

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

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
A limit test with a nominal test substance concentration of 100 mg per litre dilution water was performed
- Sampling method: Samples of the test medium for determination of the actual test substance concentration were taken immediately before introduction of the fish and approximately after 4, 24, 48, 72 and 96 hours. One sample each was taken and then analysed in duplicate by HPLC.
When the test media were changed after 24, 48 and 72 hours of exposure, samples were taken from the old as well as from the new media.
- Sample storage conditions before analysis: The samples were deep frozen on the day of sampling and unfrozen for the analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A solution with a nominal test substance concentration of 100 mg/L was prepared by dissolving 1031 mg test substance (solution as obtained by the sponsor) in 5 litres
dilution water and manually homogenization. In order to maintain the actual test substance concentration above 80 % of the actual starting
concentration new test media were prepared for test media change at 24, 48 and 72 hours after the start of the test.
- Controls: Control animals were holded in dilution water.
- Evidence of undissolved material: No: Visual observations were made at the same time as the animal observation and the media changes.
The control medium and the test media were clear and colourless throughout the test period.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish (Danio rerio).
- Strain: not applicable
- Source: Fa. Amazonas Aquarium, A-2840 Grimmenstein (preliminary test) and Fa. Tomy`s Zoo GmbH, Joseph Steblgasse 6, 2345 Brunn am Gebirge (main study)
- Feeding during test: none
- Observations on body length and weight: body length 3.31 +/- 0.12 cm, weight 0.66 +/- 0.12 g at the end of the study

ACCLIMATION
- Acclimation period: Approx. 6 weeks.
- Acclimation conditions (same as test or not):
Dilution water for holding and exposure
Continuous aeration by water recirculation using an EHEIM filter pump (Exposure: no aeration),
Holding: full glass vessel (47 cm x 29 cm x 31 cm), each filled with ca. 40 L and covered with a transparent plastic lid. Water was purified by recirculation (power filter pump EHEIM) through filter wool (Exposure: full glass vessels (29 cm x 20 cm x 24 cm), filled with 5 L of control and test medium
and covered with transparent plastic lids)
Artificial light from 4 a.m. to 8 p.m. during holding and exposure
- Type and amount of food: TetraMin; Tetra Werke, Dr.rer.nat. Ulrich Baensch GmbH, D-49304 Melle, feeding ad libitum
- Feeding frequency: daily (during exposure: no feeding)
- Health during acclimation: no mortality or any sign of disease was observed

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Test conditions

Hardness:

200 mg CaCO3/L

Test temperature:

20° C

pH:

7.3 - 7.8 in the control medium, 7.7 - 8.0 in the test substance medium

Dissolved oxygen:

At least 79 % of the saturation value at the corresponding temperature throughout the test period.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentrations:
at time 0 h: 100,75 mg/L,
at time 4 h: 101,90 mg/L,
at time 24 h: before medium change: 101,30 mg/L, after medium change: 101,30 mg/L
at time 48 h: before medium change 101,30 mg/L, after medium change: 100,40 mg/L
at time 72 h: before medium change 102,00 mg/L, after medium change: 100,00 mg/L
at time 96 h: 99,60 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material, size, fill volume: full glass vessels (29 cm x 20 cm x 24 cm), filled with 5 L of control and test medium and covered with transparent
plastic lids.
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate):1x/d
- No. of organisms per vessel: 7 fish
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: Maximum loading of 1.0 g fish/litre


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was local tap water, mixed 1:1 with deionised water, to achieve the
appropriate water hardness as required by the guideline.
- Intervals of water quality measurement: pH, temperature and dissolved oxygen was measured at 0h and at 24 h, 48 h and 72 in each case before
and after the media change, and at 96 h. Total hardness was measured before the start of the study.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Artificial light from 4 a.m. to 8 p.m


EFFECT PARAMETERS MEASURED
All animals were observed for alterations and behavioural changes at introduction in the test vessels and approximately 2, 4, 24, 48, 72 and 96
hours afterwards.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable, a limit test with one concentration was conducted
- Range finding study
- Test concentrations: Nominal 10 and 100 mg/L
- Results used to determine the conditions for the definitive study:
In a preliminary test with 2 test substance concentrations of nominal 10 and 100 mg/L, one negative control and 3 fish per concentration, no fish
in any concentration died within 96 hours.
According to the EC directive, a limit test may be performed at 100 mg/L in order to demonstrate that the LC50 is higher than this concentration.
Therefore it was decided to perform a limit test with only one concentration (100 mg/L).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 5

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 6

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 7

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: none
- Observations on body length and weight: body length 3.31 +/- 0.12 cm, weight 0.66 +/- 0.12 g
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

- Results with reference substance valid? not applicable, no reference substance

Reported statistics and error estimates:

Not applicable

Any other information on results incl. tables

Sublethal observations / clinical signs:

Quality criteria:

• The actual test substance concentration of the test medium has been satisfactorily maintained to within 80% of the initial concentration throughout the duration of the test.
•  Constant conditions were maintained (temperature, pH).
• No mortality occurred in the negative control group.
• The dissolved oxygen concentration was higher than 60% of the air saturation value throughout the test.

Mortality

Group	Nominal test substance concentration	Total number of dead fish after the start of exposure
	(mg/L)	0 hours	24 hours	48 hours	72 hours	96 hours
Control group	0	0	0	0	0	0
Test substance	100	0	0	0	0	0

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

LC50 96 h: > 100 mg/L
LC0 96 h: >= 100 mg/L
NOEC 96 h: 100 mg/L

Executive summary:

A limit-test with a concentration of nominal 100 mg test substance per L dilution water was performed.

7 Zebrafish(Danio rerio) were exposed in the test substance group and 7 in the control group. Their length was between 3.1 and 3.5 cm.

A ca. 50 % solution of test substance in water was used. According to the sponsor the actual concentration of the test substance was 48.5 % w/w. A nominal test substance concentration of 100 mg/L was therefore prepared by dissolving 1031mg test substance in 5 litres dilution water and manual homogenization. The dilution water was local tap water, mixed 1:1 with deionised water, to achieve the appropriate water hardness as required by the guideline.

This dilution water was also used as the negative control substance and for fish holding prior the test.

The exposure of the Zebrafish was for 96 h in a semi-static test with renewal of test and control media after 24, 48 and 72 hours.

The results of the study revealed a LC50 after 96 h of exposure of more than 100 mg/L, a NOEC of 100 mg/L and a LC0 after 96 hours greater or equal 100 mg/L.