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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March 2017 - 23 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

April 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

HPLC method with UV detection

Details on sampling:

- Sampling method: Four parallel samples were taken from the 100 mg/L concentration level and from the control sample. The samples were prepared immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test solution used in the test was prepared by mechanical dispersion. An appropriate amount of test item was dissolved in the dilution water (ISO Medium) in order to obtain the concentration of 100 mg/L. Untreated control was included parallel in the test. The test solution was prepared immediately before introduction of daphnids.
- Controls:
untreated control: The dilution water (ISO Medium without addition of the test item) was used as untreated control.
toxic reference control: For the evaluation of the quality of the Daphnia clone and the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary
- Breeding conditions: Daphnia were bred in the Laboratory of TOXI-COOP ZRT. under similar temperature and light conditions as in the test, and in reconstituted water of a similar quality regarding to pH, components of the main ions and total hardness as the test water used in the test. The test animals were fed with centrifuged green alga suspension.
- Age of parental stock: less than 24 hours old
- Feeding during test: none

ACCLIMATION
- Acclimation period: Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.
- Health during acclimation: no mortality observed

Test type:

static

Water media type:

freshwater

Remarks:

ISO Medium (according to OECD 202) was used as dilution water

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

249 mg/L (as CaCO3)

Test temperature:

19.8 - 20.2 °C

pH:

7.89 - 8.31

Dissolved oxygen:

8.29 - 8.72 mg/L

Nominal and measured concentrations:

Nominal concentration: 100 mg/L (limit concentration)
Measured concentration: 91.12 mg/L % at the start and 95.21 mg/L at the end of the test

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Material, size, headspace, fill volume: glass, approximately 40 mL test medium
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Volume/Organism: 2 mL

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 hours light (artificial illumination) and 8 hours darkness

EFFECT PARAMETERS MEASURED:
The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile.

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: A preliminary test was performed at the nominal concentation of 100 mg/L in order to check the toxicity of the test item on the daphnids up to at least this concentration. In the preliminary test no immobility or any sub-lethal effect was detected in the treated concentration and in the control group during the 48-h exposure period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

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

Results with reference substance (positive control):

24-h EC50: 1.41 mg/L (95 % conf. limits: 1.19 – 1.68 mg/L)

Reported statistics and error estimates:

A limit test was performed and toxic effects were not observed, therefore, statistical analysis was not performed. The NOEC, LOEC and ECx values of the test item were determined directly from the raw data.

Table1: Results from Preliminary Range-Finding Test

Nominal concentrations [mg/L]	Untreated control	100
Number of treated animals	10	10
Number of immobilized animals (at 48h)	0	0

Table 2: Summary of biological endpoints in the main test

Endpoints	Concentration [mg/L] based on nominal loading rate
48 h-EC10	>100
48 h-EC20	>100
48 h-EC50	>100
48 h-NOEC	100
48 h-LOEC	>100

Validity criteria fulfilled:

yes

Conclusions:

In a 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item had no significant toxic effects on the mobility of Daphnia. The 48-h NOEC was determined as 100 mg/L nominal concentration and the LOEC and the EC50 values were determined to be higher than 100 mg/L.

Executive summary:

The purpose of this study was to evaluate the influence of the test item on the mobility, respectively survival, of Daphnia magna. A limit test was performed in which the test animals were exposed to aqueous test media containing the test item for 48 hours at the nominal concentration of 100 mg/L in the test medium, plus a control. The test was run according to OECD TG 202, EU method C.2 and EPA OPPTS 850.1010.

Young Daphnia were exposed to aqueous test medium containing the test item at 100 mg/L nominal concentration for an exposure time of 48 hours in a static test system. The percentage of Daphnia being no longer capable of swimming at the end of the test or being dead was recorded for each test concentration.

There was no immobilisation observed in the twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Accordingly, the 48-h NOEC was determined as 100 mg/L nominal concentration and the LOEC and the EC50 values were determined to be higher than 100 mg/L.

Description of key information

In a 48-hour acute toxicity test on Daphnia magna, the obtained results showed that the test item had no significant toxic effects on the mobility of Daphnia. The 48-h NOEC was determined as 100 mg/L nominal concentration and the LOEC and the EC50 values were determined to be higher than 100 mg/L.

Additional information

The purpose of this study was to evaluate the influence of the test item on the mobility, respectively survival, of Daphnia magna. A limit test was performed in which the test animals were exposed to aqueous test media containing the test item for 48 hours at the nominal concentration of 100 mg/L in the test medium, plus a control. The test was run according to OECD TG 202, EU method C.2 and EPA OPPTS 850.1010.

Young Daphnia were exposed to aqueous test medium containing the test item at 100 mg/L nominal concentration for an exposure time of 48 hours in a static test system. The percentage of Daphnia being no longer capable of swimming at the end of the test or being dead was recorded for each test concentration.

There was no immobilisation observed in the twenty daphnids exposed to either test item treated or control group. In addition to immobility, no abnormal behaviour or appearance of test animals was detected. Accordingly, the 48-h NOEC was determined as 100 mg/L nominal concentration and the LOEC and the EC50 values were determined to be higher than 100 mg/L.