calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

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:

March from 7th to 18th, 2016

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:

adopted 17th July, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test item concentrations were determined at the beginning of the test and after 2, 24, 48, 72 and 96 h of exposure.

Vehicle:

no

Details on test solutions:

Since the test item is soluble, the test solution was prepared by directly dissolving the test item in natural water.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish.
- Source: Aquarium Stern, 4310 Rheinfelden.
- Length at study initiation: length of 1–3 cm

ACCLIMATION
- Acclimation period: the fish were gradually acclimatized for at least an additional 12 days prior to testing.
- Acclimation conditions: the fish were gradually acclimatized to the test conditions and were held in water of similar temperature (23 ± 2.0 °C) and quality used in the test.
- Culture conditions: 16 h a day; 21–25 °C, controlled by a thermostat at ± 2 °C; medium Continuously aerated natural water of the town of Schlieren.

QUARANTINE
- Duration: young fish are held under quarantine conditions in 40 l glass aquaria until use in the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

Control: 21.5 - 22 °C
Test vessel: 21.5 - 22 °C
Controlled by a thermostat at ± 2 °C

pH:

Control: 6.8 - 8.3
Test vessel: 6.5 - 8.3

Dissolved oxygen:

Control: 6.9 - 8.0 mg/l
Test vessel: 7.0 - 8.4 mg/l

Nominal and measured concentrations:

100 mg/l nominal concentration.

Details on test conditions:

A fine-mesh dip net was used to transfer fish, taking care to minimise possible stress due to handling. The transfer was performed in an impartial manner to eliminate bias (as regards weight and length of the fish). No fish were damaged or dropped during the transfer.

TEST SYSTEM
- Test vessel: glass aquaria filled with 1.0 liter of water.
- Aeration: continuously aerated natural water of the town of Schlieren.
- Renewal rate of test solution: fresh test solutions were prepared after 24, 48 and 72 h, into which the fish were transferred after each 24 h exposure period.
- No. of organisms per vessel: 7 fish per test concentration and control.
- No. of vessels per concentration: 1 replicate.
- No. of vessels per control: 1 replicate. Natural water, without addition of any kind of additive, was used for the blank control.
- Biomass loading rate: total loading ≤ 1 g fish weight/l.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural water supplying the laboratory.
- pH: 7.4 - 7.8
- Electric conductivity (room temperature): 477 - 536 μS/cm
- Total Hardness: 20.6 - 24.2 °f H; 2.06 - 2.42 mmol/l; 206 - 242 mg/l
- CO3 Hardness: 18.6 - 21.7 °f H; 186 - 217 mg/l
- Residual Hardness: 2.0 - 3.0 °f H; 20 - 30 mg/l
- Acid consumption: 3.72 - 4.34 mmol/l
- Colony counts (30°C): 0 - 1 per ml
- Escherichia coli: 0 per 100 ml
- Enterococcus: 0 per 100 ml
- Nitrate: 7.3 mg/l
- Chloride: 16.1 mg/l
- Sulfate: 18.6 mg/l

OTHER TEST CONDITIONS
- Adjustment of pH: yes, the pH was adjusted to pH 6.5 - 7.0.
- Photoperiod: 16 h photoperiod a day, supplied by overhead white fluorescent tubes, no transition period.

TEST CONCENTRATIONS and SYSTEM
- Justification for concentration choice: the concentration was chosen based on preceding studies tests with Alga and Daphnia, where the 72 h ErC50 value for Alga and the 48 h-EC50 value for Daphnia were >100 mg/l nominal concentration (information provided by the sponsor).
- Justification for test system choice: because the test item proved to be readily biodegradable (ca. 96.9 % biodegradation within one week; information provided by the sponsor), semi-static exposure conditions were applied.

EFFECT MEASURED
Fish are considered to be dead if they show no reaction after touching the caudal fin and there is no visible movement (e.g. gill movements).
The median lethal concentration (LC50 value) is the concentration killing 50 % of the fish after a certain period of exposure.

PARAMETERS MEASURED
- Oxygen, pH, temperature: measurements were made prior to the addition of the fish to the test solutions and after 2h of exposure as well as in the old and fresh test solutions after each 24 h period until the end of the test.
- Behaviour and mortality: observations of any potential abnormal behaviour of the fish were made after 2, 24, 48, 72 and 96 h of exposure. Mortality of the fish was recorded at the same time intervals. Any dead fish were removed immediately from the test media, and their respective weight and length were recorded.
- Fish length and weight: at the end of the test, length and weight of all surviving fish were also recorded.

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)

Remarks on result:

other: 99% CL

Details on results:

The no-observed-effect concentration (NOEC) was 100 mg/l nominal concentration.
At the nominal concentration of 100 mg/l test item, none of a total of seven fish died after 96 h of exposure. No mortality was observed in the blank control.
Toxic effects other than mortality, such as loss of coordination, hypo- or hyperactivity and swimming on the back, were not observed.

TEST CONCENTRATIONS
The test concentration could satisfactorily be maintained over the 96 h test period. Since all measurements were within the 80-120 % range of the nominal concentration, the assessment of the toxicity was based on the nominal concentrations.

Sublethal observations / clinical signs:

Concentrations of test item, measured by HPLC (mg/l)

Nominal concentration (mg/l)	0 h	2 h	24 h		48 h		72 h		96 h
			old	new	old	new	old	new
100	101	101	102	99.4	103	101	103	100	104

Data of surviving fish at the end of the exposure (96 h)

Nominal concentration (mg/l)	Length (mm) and weight (mg) of the individual fish								Total fish loading (g/l medium)*
Blank control	Length	12	14	12	11	11	13	12	-
	Weight	16	25	15	12	9	17	16	0.11
100	Length	11	10	14	14	14	14	11	-
	Weight	15	9	30	23	32	24	13	0.15

* maximal total loading recommended by OECD TG 203: 1 g fish per liter medium

All fish were within the recommended size range of 1-3 cm.

Validity criteria fulfilled:

yes

Conclusions:

LD59 (96h) > 100 mg/l (nominal)

Executive summary:

Method

The acute lethal toxicity of test substance to Danio rerio (Zebrafish) was investigated under semi-static exposure conditions over a period of 96 h, according to the OECD Guideline 203.

The only applied nominal concentration was 100 mg/l. Therefore, this study represents a limit test. This concentration was chosen based on preceding studies tests with Alga and Daphnia, where the 72 h ErC₅₀value for Alga and the 48 h-EC₅₀value for Daphnia were >100 mg/l nominal concentration.

Since the test item proved to be readily biodegradable, semi-static exposure conditions were applied. Since the test item is highly soluble, the test solution was prepared by directly dissolving the test item in natural water.

The concentrations of test item were measured by HPLC at the beginning of the test and after 2, 24, 48, 72 and 96 h of exposure, in old and fresh medium. These analyses showed that the test concentration could satisfactorily be maintained over the whole 96 h exposure period. Since all measurements were within the 80-120 % range of the nominal concentration, the assessment of the toxicity was based on the nominal concentrations.

Observations

At the nominal concentration of 100 mg/l of test substance, none of a total of seven fish died after 96 h of exposure. No mortality was also observed in the blank control.

Toxic effects other than mortality, such as loss of coordination, hypo- or hyperactivity and swimming on the back, were not observed.

The no-observed-effect concentration (NOEC) was 100 mg/l nominal concentration.

Results

Based on the binominal theory, the absence of mortality in such a test design provides at least 99 % confidence that the median lethal concentration (LC₅₀) of substance to Danio rerio (Zebrafish) is greater than the applied nominal concentration of 100 mg/l.

Description of key information

LD50 (96h) > 100 mg/l (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The acute lethal toxicity of test substance to Danio rerio (Zebrafish) was investigated under semi-static exposure conditions over a period of 96 h, according to the OECD Guideline 203 by means of a limit test (100 mg/l, nominal).

The concentration of test item was measured by HPLC at the beginning of the test and after 2, 24, 48, 72 and 96 h of exposure, in old and fresh medium. These analyses showed that the test concentration could satisfactorily be maintained over the 96 h test period. Since all measurements were within the 80-120 % range of the nominal concentration, the assessment of the toxicity was based on the nominal concentrations.

At the nominal concentration of 100 mg/l of test substance, after 96 hours, no deaths occurred. Toxic effects other than mortality, such as loss of coordination, hypo- or hyperactivity and swimming on the back, were not observed.

The NOEC can be considered as equal/higher than 100 mg/l (nominal); the LC50 resulted greater than the applied nominal concentration of 100 mg/l.