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

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:

March from 03rd to 05th, 2009

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:

April 2004

GLP compliance:

yes

Remarks:

temporary certificate

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A solution of 100 mg/l was prepared by direct weighting into aerated reconstituted water. The obtained suspension was mixed by the means of a magnetic stirrer for 24 hours to achieve the maximum solubilisation of the test item, and then it was filtered by a cartridge filter (pore diameter:
0.45 µm) in order to obtain an clear solution.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: originally supplied by the Italian Health Department (lstituto Superiore di Sanita) in September 2004.
- Breeding: the clone has been bred in the testing laboratories in reconstituted water of the quality identical to the water quality used in the tests (regarding pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Age of parental stock: at the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.
- Food type: daily fed a fixed amount of the laboratory cultured green algae Pseudokirchnerie/Ia subcapitata (formerly Selenastrum caprcornutum) and of a suspension of the yeast Saccharomyces cerevisiae.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

The measured values ranged between 19.8 and 22.6 °C during the test period, these values slightly exceeded the OECD’s recommended range (20.0 ± 2.0 °C), however this excess did not affect the results of the study, as showed by the immobilization data in the control.

pH:

Control: 7.42 - 7.50
Test vessels: 7.33 - 7.48

Dissolved oxygen:

Control: 6.52 - 6.82 mg/l
Test vessels: 6.32 - 6.8 mg/l

Nominal and measured concentrations:

100.0 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 ml glass beakers filled with 40 ml of test medium. The test vessels were labelled, including all necessary additional information to ensure unmistakable identification.
- Type: beakers were covered with glass plates to reduce the loss of water and to avoid the entry of dust into the solutions.
- Aeration: the test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, test water was not aerated.
- No. of organisms per vessel: 5 daphnids. The daphnids were randomly distributed to the test vessels at initiation of the test.
- No. of vessels per concentration: four replicates.
- No. of vessels per control: four replicates. Negative control with reconstituted water only was tested.
- Biomass loading rate: daphnid’s loading rate was more than one per 2 ml of test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water; analytical grade salts were dissolved in purified water.
- CaCl2 x 2H2O: 2.0 mmol/l (= 294.0 mg/l)
- MgSO4 x 7H2O: 0.5 mmol/l (= 123.0 mg/l)
- NaHCO3: 0.75 mmol/l (= 65.0 mg/l)
- KCI: 0.075 mmol/l (= 5.8 mg/l)
- Water Hardness: 2.5 mmol/l (= 250.0 mg/l as CaCO3)
- Alkalinity: 0.8 mmol/l
- Ratio of Ca : Mg: 4 : 1, based on molarity
- Ratio of Na : K: 10 : 1, based on molarity

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness daily photoperiod.
- Light intensity: within the range of 200-700 Lux.

EFFECT MEASURED
The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker were considered to be immobile).
The NOEC and LOEC were determined directly from the raw data by statistical comparison with the control (Fisher’s Exact analysis). The CETIS v.1.026D software was used to carry out the statistical analysis. As input data nominal concentration was used.

PARAMETERS MEASURED
Dissolved oxygen concentrations and pH were determined in the test medium and the controls at the start and at the end of the test period.
The appearance of the test medium was visually recorded at the start of the test and after 24 and 48 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

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

Details on results:

NOEC (48h): 100 mg/l (nominal)
LOEC (48h) > 100 mg/l (nominal)
The NOEC (highest concentration tested without toxic effects after the exposure period) was determined was equal to the nominal tested concentration, 100 mg/l at both the observation times. Given the nature of limit test, the LOEC and the 48-h IC50 could not be determined, but, according to the results, were determined to be greater than the nominal concentration tested (i.e., lC50 > 100 mg/l).
No remarkable observations were made conceming the appearance of the test medium. The test medium was a clear solution throughout the whole test duration.

In the control 0 % immobilization was observed during the test period. This value complies with the validity criteria of the test, according to the mentioned guidelines.

Results with reference substance (positive control):

The results of the most recently performed test with a reference substance are reported and compared with published data to demonstrate that the daphnia clone used has got an adequate level of sensitivity to that substance. In the current case, the results refer to a test run on May 2008 with potassium dichromate as reference substance (CHE-I-004/2008). The comparison shows that the end-point of the present test fits in the interval for the 24 h IC50 indicated in the ISO guideline.
IC50 (24h): 1.09 mg/l (CL 95 %: 0.92 - 1.29 mg/l)
Reference data: IC50 (24 h): 0.6 – 2.1 mg/l

Lethal effect of test item to Daphnia magna

Nominal concentration (mg/l)	N. of organisms	N. of immobilized daphnids		% of immobilized daphnids
		24 h	48 h	24 h	48 h
Blank control	20	0	0	0	0
100	20	1	2	5	10

Validity criteria fulfilled:

yes

Conclusions:

No adverse effect were observed to Daphnids after 48-hours of exposure at the nominal concentration of 100 mg/l under static conditions.

Executive summary:

Method

The acute toxicity of the test item Set Retard Plus to Daphnia magna was determined in a 48‑hour static limit test according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

For this purpose, juvenile daphnids were exposed to an aqueous test medium containing the test item at the only nominal concentration of 100.0 mg/L under defined conditions. The exposed organisms were checked for immobilization 24 and 48 hours after test initiation.

Observations

The pH of the test medium after preparation was 7.33 for test item solution and 7.50 for control. Therefore, the pH of the test medium was not adjusted before the start of the test.The room temperature was in the range 19.8 – during the test period, these values slightly exceeded the OECD’s  recommended range (20.0 ± 2.0 °C), however this excess did not affect the results of the study, as showed by the immobilization data in the control.In the control 0 % mortality was observed during the test period of 48 hours. This value complies with the validity criteria of the test according to the mentioned guidelines that provide a maximum value of mortality in the control of 10 %. No significant mortality was observed in the test medium after 24 and 48 hours (Fisher's Exact test), therefore the NOEC (highest concentration tested without toxic effects after the exposure period) of Set Retard Plus to Daphnia magna was equal to the nominal tested concentration of 100.0 mg/l.

Results

Based on the observed effects, the LOEC and the 48-h EC₅₀ were determined to be greater than the tested concentration, 100.0 mg/l.

Description of key information

EC50 (48h) > 100 mg/l (nominal)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

The acute toxicity of the test item Set Retard Plus to Daphnia magna was determined in a 48‑hour static limit test (100 mg/l), according to the EU Commission Directive 92/69/EEC, Part C.2 (1992), and the OECD Guideline for Testing of Chemicals, No. 202 (2004). No significant mortality was observed in the test medium after 24 and 48 hours (Fisher's Exact test), therefore the NOEC (highest concentration tested without toxic effects after the exposure period) of test item to Daphnia magna was equal/higher than the nominal tested concentration of 100.0 mg/l. No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the whole test duration. The LOEC and the 48-h EC50 were determined to be greater than the tested concentration, 100.0 mg/l.