Lupine, L. albus, ext.

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:

04-11 July 2016

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 January 2016

Analytical monitoring:

yes

Details on sampling:

In order to verify the initial concentrations and maintenance of the exposure concentrations of the test item during the exposure period, chemical analysis of the test item in daphnids test solutions were performed at the beginning and after 24 h (before renewal). Method for the chemical analysis of Total Carbon (TC) in samples containing the test item from daphnids ecotoxicology solutions by Total Organic Carbon Analyzer (described in the study 15/1256F/a) and the following protocol:
- in fresh solutions at the beginning of the test (T=0h)
- in old solutions (T=24h)
- for the control (i.e. daphnids test medium filtered) and concentration 100 mg/L
Schedule analysis at initial time:
-Filtered control Abiotic: 1 replicate
-100 mg/L Abiotic: 2 replicates
These solutions were analysed taken directly from the starting solutions without daphnids.
Schedule analysis at final time (before renewal: 24h):
-Filtered control Biotic: 1 replicate
-100 mg/L Biotic: 2 replicates

Vehicle:

no

Details on test solutions:

The test item is soluble and directly dispersed in test medium. The test medium and the solution (100 mg test item/L) were filtered through a membrane filter (cellulose acetate) with a mesh size of 0.22 um to sterilizing and all the manipulations were done under sterile conditions to prevent contamination.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna Straus, clone number 5
- Source: from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthogenesis reproduction.
- Age at study initiation: less than 24 hours old.
- Method of bredding: the daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.
- Feeding during test: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

250 mg/L of CaCO3

Test temperature:

19.9 - 20.1°C

pH:

8.1

Dissolved oxygen:

8.8 mg/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: 101.91 mg/L (at initial time) and 97.7 mg/L (after 24 hours of exposure)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass test tube
- Type: closed
- Each glass test tube (sterilized by autoclave) contained 10 mL of test solution and five daphnids; they were covered in order to avoid evaporation and dust deposition.
- Renewal rate of test solution: Renewal of test solutions at 24h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

OTHER TEST CONDITIONS
- Light intensity: Darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Observations of the daphnid's immobility and behaviour were carried out after 24 and 48 h in control and exposed groups.
- Any sign of stress observed on the daphnids after 48 hours of exposure to the test item were reported.
- pH and dissolved oxygen were measured before and after renewal of solutions (including the controls).

RANGE-FINDING STUDY
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No immobilization was observed at any of the concentration tested. Based on the results of the preliminary test, it has been proposed to perform a limit test at 100 mg/L for the study. This study was conducted on one concentration levels: 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

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:

- No immobilization was observed at 24 h and 48 h (including controls).
- No sign of stress was observed on the mobile daphnids after 48 h of exposure

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (June 27, 2016) gives an EC50-24h of 1.24 mg/L (1.13 - 1.37), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6 - 2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

Table 6.1.3/1: Daphnids immobilisation

Nominal test item concentration in mg/L	Number of daphnids exposed at the start	Immobilised daphnids (%)
		24 h	48h
Dilution water control	30	0 (0%)	0 (0%)
100.0	30	0 (0%)	0 (0%)

Table 6.1.3/2: Chemical analysis of daphnids solutions

Nominal test item concentration	100 mg/L
	At initial time	After 24 h of exposure	Variation (%)
Test item 100 mg/L	101.91	97.70	- 4.1 %

At the nominal concentration of 100 mg/L, the test item is considered as stable under test conditions because the measured concentrations of the "test item did not vary more than +/- 20% of initial measured or nominal concentration during the test

Table 6.1.3/3: Validity criteria conformity

	Values for OECD 202	Validity criteria for the test
Immobilised daphnids in control	=< 10 %	Ok (0%)
Dissolved Oxygen concentration	>= 3 mg/L	Ok (8.3 mg/L)

All the validity criteria were successful.

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item on the toxicological endpoint "immobilisation" give an EC50-24h and an EC50-48h > 100 mg/L, based on nominal test item concentration.

Executive summary:

The aim of this study was to assess the acute toxicity of the test item on the freshwater invertebrate Daphnia magna, according to the OECD 202 guideline. The study was performed at 100 mg/L, limit test, under semi-static conditions, with GLP compliance.

The test was performed in sterilized vessels and sterile conditions with renewal at 24 h of the test solution. Groups of thirty Daphnia magna, less than 24 h old, were exposed for 48 h to a control solution (Elendt M4 medium) and to the test item, prepared in Elendt M4 medium. The test item concentration was prepared under sterile conditions in order to obtain the required nominal concentration of 100 mg/L Elendt M4 medium.

The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC50 -24h and EC50 -48h were evaluated, relative to the tested concentration: 100 mg/L. After 48 h, no immobilization was observed in any test tubes.

In order to verify the initial concentrations and maintenance of the exposure concentrations of the test item during the ecotoxicological testing, a chemical analysis of the test item in the test solutions was performed. At the nominal concentration of 100 mg/L, the test item is considered as stable in the testing conditions because the measured concentrations of the test item did not vary more than +/- 20% of initial measured concentration during the test.

In the control, not more than 10 % of the daphnids showed an immobilization. In addition, the dissolved oxygen concentration was determined to be higher than 3 mg/L in the control and test vessels. Thus, the validity criteria were respected.

The results obtained for the test item on the toxicological endpoint "immobilisation" give an EC50-24h and an EC50-48h > 100 mg/L, based on nominal test item concentration.

Description of key information

Under the experimental conditions of a reliable study, the 48h - EC50 of the test substance to the aquatic invertebrate Daphnia magna was > 100 mg/L, the limit test concentration.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

A reliable study was available to assess the acute toxicity of the test item on the freshwater invertebrate Daphnia magna, according to the OECD 202 guideline. The study was performed under semi-static conditions, with GLP compliance.

The concentrations which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EC50 -24h and EC50 -48h were evaluated, relative to the tested concentration: 100 mg/L. After 48 h, no immobilization was observed in any test tubes.

At the nominal concentration of 100 mg/L, the test item was considered stable in the testing conditions. In addition, all the validity criteria were respected.

The registered substance exhibits an EC50-48h > 100 mg/L, based on nominal test item concentration.