Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

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:

08-10 December 2015

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

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of “OF14 AT” in daphnids test solutions were performed in the test solutions.
In order to check the initial concentrations and maintenance of the exposure concentrations of the test item during the exposure period, chemical analysis of the “OF14 AT” in daphnids test solutions were analysed at the beginning and after 24 h (before renewal). Method for the chemical analysis of OF14 AT in samples from daphnids ecotoxicology testing by GC/FID:
- at the beginning of the test (T = 0 h)
- at the end of the test (T = 24 h before renewal)
- for the control (i.e. daphnids test medium filtered), the highest and the lowest concentrations.
These solutions were analysed taken directly from the starting solutions without daphnids.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item is soluble in water. Five solutions were then prepared in the test medium by weighing the test item (10.08, 22.01, 48.41, 106.48 and 234.35 mg test item were dissolved in 100 mL of test medium: 100, 220, 484, 1065 and 2343 mg/L, respectively).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna Straus, clone number 5
- Daphnia magna Straus, clone number 5, from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
- Age at study initiation: Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 and 800 μm (corresponding to daphnids less than 24 h old).
- Method of breeding: Daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4 (same conditions as for main test).
- Feeding during test: None

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

None

Hardness:

250 mg/L of CaCO3

Test temperature:

20 ± 1 °C (18-22 °C)

pH:

8.6

Dissolved oxygen:

9.1 mg/L

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentrations: 100, 220, 484, 1065 and 2343 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass test tube (approximately capacity 24 mL)
- Type: closed
- Each glass test tube (approximate. capacity 24 mL) 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 (frequency/flow rate): Test solutions were renewed after 24 h of exposure.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M4 ELENDT medium

OTHER TEST CONDITIONS
- Light intensity: Darkness

EFFECT PARAMETERS MEASURED
- Observations of the daphnids mobility were carried out after 24 and 48 h of exposure to the test item. Any immobile animals were removed from the tests vessels once they were observed.
- Any sign of stress (e.g. abnormal behaviour), determined by visual observation of the daphnids at 24 and 48 h of exposure to the test item were reported.
- pH and dissolved oxygen were measured in the test solutions before (old solutions) and after renewal (fresh solutions) for each concentration (including controls).

TEST CONCENTRATIONS
- Range finding study: 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, the following test concentrations 100, 220, 484, 1065 and 2343 mg/L were proposed for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 2 343 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2 343 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- No immobilization was observed at any of the concentration tested.
- No sign of stress was observed on the mobile daphnids after 48 h of exposition.

Results with reference substance (positive control):

The last toxic response obtained on the reference item potassium dichromate (December 02, 2015) showed an EC50-24 h of 1.21 mg/L (1.09 – 1.35), indicating that the daphnids sensitivity was 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 Guideline) and in accordance with historical data obtained by the laboratory.

Table 6.1.3/1: Daphnids immobilization

Nominal test item concentrations (mg/L)	Number of daphnids exposed at the start	Immobilised daphnids (%)
		24 h	48 h
Control	20	0 (0%)	0 (0%)
100	20	0 (0%)	0 (0%)
220	20	0 (0%)	0 (0%)
484	20	0 (0%)	0 (0%)
1065	20	0 (0%)	0 (0%)
2343	20	0 (0%)	0 (0%)

 

Table 6.1.3/2: Results of the chemical analyses in daphnids test solutions

Nominal test item concentration	mg “OF14 AT”/L in daphnids solutions		Variation between T0 and T24 hours (%)
	Results at T0 hour	Results at T 24 hours
Dilution water control evaporated	< 12.5	< 12.5	NA
Dilution water control Silylated	< 250	< 250	NA
100 mg/L	113.44	40.14	- 64.6 %
2343 mg/L	2333.33	1533.43	- 34.3 %

NA: not applicable

The test item concentrations were found to be unstable in the testing conditions (35.4 - 65.7 % of the initial concentrations at the end of the 24 h incubation periods).

 

Validity criteria:

In the control, not more than 10 % of the daphnids showed an immobilization.

The dissolved oxygen concentration was determined to be higher than 3 mg/L in the control and test vessels. The validity criteria were fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, 48 h EC50 of the test item “OF14 AT” was determined to be > 2343 mg/L expressed as nominal test item concentration.

Executive summary:

A study was performed to assess the acute toxicity of the test item, OF14 AT to the freshwater invertebrate Daphnia magna was investigated in a semi-static test according to OECD Guideline 202 with GLP compliance. 

 

The test was performed in sterilized vessels and sterile conditions with renewal at 24 h of the test solution. Groups of twenty Daphnia magna, less than 24 h old, were exposed for 48 h under semi-static exposure conditions to a control solution (Elendt M4 medium) and to test item “OF14 AT”, prepared in Elendt M4 medium. The test item concentrations were prepared under sterile conditions in order to obtain the required nominal concentration of 100, 220, 484, 1065 and 2343 mg/L. Observations of the Daphnia magna in each control and test vessel were performed at 24 and 48 h.

 

After 48 h, no immobilization was observed at any test concentration. After 24 and 48 h, the highest concentration at which no immobilisation occurred was 2343 mg/L (nominal).

 

The low and upper concentrations of the test item were determined by chemical analyses at start and end of 24 h incubation period before renewal. The test item concentrations were found to be unstable (35.4 - 65.7 % of the initial concentrations at the end of the 24 h incubation periods).

 

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

 

Under the test conditions, 48 h EC50 of the test item “OF14 AT” was determined to be > 2343 mg/L expressed as nominal test item concentration.

Description of key information

Under the test conditions, 48 h EC50 of the test item “OF14 AT” to the aquatic invertebrate Daphnia magna was determined to be > 2343 mg/L expressed as nominal test item concentration.