Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2017 - 23 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

reverse phase HPLC method using UV detection

Details on sampling:

The test items content was determined in ISO Medium at the start and at the end of the test. Five parallel samples were taken from the 100 mg/L concentration level and from the control sample.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion without the aid of any solubilising agents. An amount of 0.5 g test item was dissolved in 5000 mL dilution water (ISO Medium) in order to obtain the concentration of 100 mg/L. The test solution was freshly prepared just before introduction of the test animals (start of the experiment).

- Controls: The dilution water (ISO Medium without addition of the test item) was used as untreated control.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: Akvárium Magazin Kft. 1222-Budapest, Dévény u. 36, Hungary
- Age at study initiation: juveniles
- Length at study initiation: The body length of each fish were measured at the end of the test in order to check their compliance with the size range recommended for the species by the test guideline (OECD No. 203). The measured values were within the range of 2.50-2.90 cm.
- Weight at study initiation: At the start of the test (on day 0) 10 fish were introduced into aquariums, respectively. The body weight of fish was measured and recorded: first the weight of a vessel filled with water was determined. Then the fish were selected randomly and placed into this vessel. The weight of the group of fish was registered and the average body weight was calculated for 1 fish. The loading of the test aquariums was also calculated and was 0.64 g/L in the control and 0.48 g/L in the test item treated group.
- Method of breeding: Fish are bred in a well-known fish farm, under disease- and parasite-controlled conditions.
ACCLIMATION
- Acclimation period: at least 12 days before test initiation
- Acclimation conditions: same as test
- Type and amount of food during acclimation: fish were fed with appropriate, commercial diet for fish
- Feeding frequency during acclimation: at least three times per week until one day before the test start
- Health during acclimation: No mortality occurred in seven days before the start of the experiment, therefore the batch was considered to be acceptable for testing.

FEEDING DURING TEST
- No feeding during the exposure period.

Test type:

static

Water media type:

freshwater

Remarks:

ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post exposure observations were performed.

Hardness:

249 mg/L (as CaCO3).

Test temperature:

21.7 - 22.4 °C

pH:

7.48 - 8.12

Dissolved oxygen:

62.9 – 94.9 % of the air saturation value

Nominal and measured concentrations:

Nominal concentration: 100 mg/L (limit concentration)
Measured concentration: 100.8 mg/L at the start and 98.9 mg/L at the end of the test

Details on test conditions:

TEST SYSTEM
- Test vessel: aquariums
- Material, size, headspace, fill volume: glass, with approx. 6 litres capacity
- Aeration: Test solutions were not aerated during the test.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The ISO medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water proportionally.
Stock solution 1: CaCl2 x 2 H2O 11.76 g/L
Stock solution 2: MgSO4 x 7H2O 4.93 g/L
Stock solution 3: NaHCO3 2.59 g/L
Stock solution 4: KCl 0.23 g/L

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light: 8 h dark

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Toxic effects were not observed during the preliminary test, therefore, a limit test was carried out with one treatment group at a concentration of 100 mg/L (limit concentration) and a concurrent control.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

-Behavioural abnormalities:
- Observations on body length and weight: none
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: none

Reported statistics and error estimates:

Since a limit test was performed (and mortality has not occurred), no statistical analysis was necessary. The LC0, LC50, NOEC, LOEC and the LC100 values were determined directly from the raw data.

Sublethal observations / clinical signs:

Table1: Results from Preliminary Range-Finding Test

Nominal concentrations [mg/L]	Untreated control	100
Number of treated animals	3	3
Number of dead animals (at 96h)	0	0

Table 2: Summary of the biological endpoints in the main test

Parameter	Nominal concentration [mg/L]
96-h LC50	> 100
96-h NOEC	100
96-h LC0	100
96-h LOEC	> 100
96-h LC100	> 100

Validity criteria fulfilled:

yes

Conclusions:

The test item had no toxic effect up to at least the limit concentration of 100 mg/L on Zebrafish. Accordingly, the 96-h LC50 value was determined to be > 100 mg/L. The 96-h NOEC was determined to be 100 mg/L.

Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Brachydanio rerio). A limit test was performed in which the test animals were exposed to aqueous test media containing the test item for 96 hours at only one concentration (100 mg/L) plus a control in order to demonstrate that the test item is not toxic to Zebrafish up to at least this test concentration (i.e. limit concentration). Based on the results obtained during analytical method validation the test item was expected to be stable for the duration of 96 hours in ISO Medium, therefore, the experiment was carried out under static conditions.

For this purpose, young fish were exposed in a static test to aqueous test media containing the test item for 96 hours. The test was run according to OECD TG 203, EU method C.1 and EPA OPPTS 950.1075. Mortality and any sub-lethal effects were not observed during the 96-h exposure period neither in the treated nor in the control group. Accordingly, the 96-h NOEC was determined as 100 mg/L and the LOEC and the LC50 values were determined to be higher than 100 mg/L.

Description of key information

The test item  had no toxic effect up to at least the limit concentration of 100 mg/L on Zebrafish. Accordingly, the 96-h LC50 value was determined to be > 100 mg/L. The 96-h NOEC was determined to be 100 mg/L.

Additional information

The purpose of this study was to evaluate the acute toxicity of the test item on Zebrafish (Brachydanio rerio). A limit test was performed in which the test animals were exposed to aqueous test media containing the test item for 96 hours at only one concentration (100 mg/L) plus a control in order to demonstrate that the test item is not toxic to Zebrafish up to at least this test concentration (i.e. limit concentration). Based on the results obtained during analytical method validation the test item was expected to be stable for the duration of 96 hours in ISO Medium, therefore, the experiment was carried out under static conditions.

For this purpose, young fish were exposed in a static test to aqueous test media containing the test item for 96 hours. The test was run according to OECD TG 203, EU method C.1 and EPA OPPTS 950.1075.Mortality and any sub-lethal effects were not observed during the 96-h exposure period neither in the treated nor in the control group. Accordingly, the 96-h NOEC was determined as 100 mg/L and the LOEC and the LC50 values were determined to be higher than 100 mg/L.