Silybum marianum, 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:

05 - 07 March 2019

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Extract of Chardon marie without support
Batch/Lot Number: VDO06205A
Description: Light yellow brown cloudy oil
Purity: Considered as 100% No correction for purity of the test item was applied because this is an UVCB test item
Expiry date: 13 July 2019
Storage condition: Controlled room temperature (15-25 °C, ≤ 70 RH%)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Analytical monitoring:

yes

Details on sampling:

NPOC analysis
Alle glassware (sample tubes, beakers, flasks) were caregully washed with chromosulphuric acid and then by ultrapure water to remove organic contaminants.

Fore measurement of non-purgable organic organic content, the samples were acidified with 2% (v/v) amount from 1 M HCL. The CO2 released by acidification was removed by purging the solutions for 90 seconds with synthetic air (Sparge gas flow 80 ml/min).

50 ul of these acidified samples were injected for NPOC analysis. At least three injections were made from each solution.

Vehicle:

yes

Remarks:

ISO Medium

Details on test solutions:

Formulation:
Because the test item is poorly soluble in water, a test solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (ISO Medium) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solution.
Prior to treatment of each renewal period, test item solution was prepared by the method described above.

Untreated Control:
The dilution water (ISO-medium) was used without addition of the test item.


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.
The date of the last study (Study Code: 19/009-023DA) with reference item Potassium dichromate (batch no.: A0345704) is: 23 - 24 January 2019.
The 24h EC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.75 mg/L)

Dilution Water:
Reconstituted water (ISO medium, according to OECD 202) was used as dilution water for both the range finding and definitive tests. The same composition of reconstituted water was used for the tests and for breeding the test animals.

Test organisms (species):

Daphnia magna

Details on test organisms:

EXPERIMENTAL ANIMALS:
Species and strain: Daphnia magna
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Breeding The Daphnia are bred in Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
The health of the stock animals is continuously monitored by visual daily checking. Abnormal behaviour or significant decrease of population is recorded.
 
Justification of strain: Daphnia magna is the standard species of the acute immobilisation test.
Number of animals: There were 20 animals in test group and control group respectively, divided into 4 replicates (5 animals / replicate)
Age of the animals: They were less than 24 h old at the beginning of the test
Acclimatization: There was no acclimatization because the water used was similar to the culture water.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

The reconstituted water (ISO medium) had a total hardness of 246 mg/L (as CaCO3).

Test temperature:

The water temperature was measured at the start and at the end of the renewal periods in each test vessel. The test temperature was in the range of 20.1 – 20.7°C measured in the test vessels.
The additionally measured temperature in the climate chamber was between 19.9 – 21.0°C.

pH:

The pH of the test solution was not adjusted and not varied by more than 1.5 units in any one test. The pH was measured at the start and at the end of the renewal periods in each test vessel and was in the range of 7.22 – 7.68.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the renewal periods and was in the range of 6.7 – 8.6 mg/L.

Nominal and measured concentrations:

Prelminary range finding test
Nominal concentrations: 0.1, 1, 10, 100 mg/L nominal loading rates WAFs

Main test:
Nominal concentration: 100 mg/L nominal loading rates WAFs

Details on test conditions:

PERFORMANCE OF THE TEST:
The test duration was 48 hours. Twenty animals, divided into four groups (glass beaker with a volume of 50 mL) of five animals each (~40 mL test solution/flask; 8 mL test solution/animal) were used at the test concentration and for the control in a semi-static system. The animals were not fed during the test.
The test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours.
The choice of the test concentration was done on the basis of the results of a preliminary range-finding test.

Preliminary Range-Finding Test:
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Ten daphnids (divided into two replicates) in each test concentration and control were exposed for 48 hours under semi-static conditions (in absence of stability data).

The test solutions were prepared by appropriate dilution of the stock solution.

Test item concentrations in the Definitive Test:
Because no toxic response was observed during the preliminary concentration range-finding test, a Limit Test was carried out using only one test concentration 100 mg/L nominal loading rate (WAF) and one control group in a semi-static system.
The biological results are based on the test item nominal loading rate (WAF).

OBSERVATIONS:
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.
The number of immobilised animals and the percentage of immobility were determined at 24 and 48 hours.
The water temperature, the oxygen concentrations and pH of the control and the test solution were measured at the beginning and at the end of the renewal periods.

Reference substance (positive control):

not specified

Key result

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mortality

Key result

Duration:

48 h

Dose descriptor:

EL100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

nominal loading rate WAF

Basis for effect:

mobility

Details on results:

VALIDITY:
There was no immobilized animal in the control group and the dissolved oxygen concentration at the end of the test in control and test vessels was more than 3 mg/L.
All validity criteria were within acceptable limits and therefore the study can be considered as valid.

IMMOBILISATION:
The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour. In addition to immobility, no abnormal behaviour or appearance of test animals was detected.
Even if 5 % immobilization could be observed at the test item treated group, but this difference is negligible due to that the accepted control immobilization is 10 %.

Results with reference substance (positive control):

Not applicable

 Number and percentage of immobilised animals

Test Group	Number of treated animals	Number of immobilised animals
		24 hours		48 hours
		number	percent	number	percent
Control	20	0	0	0	0
100 mg/L nominal loading rate WAF	20	1	5	1	5

  Temperature measured in the test vessels (°C)

Test group	Replicate	Measuring
		0 h (fresh media)	24 h (old media)	24 h (fresh media)	48 h (old media)
Control	1	20.4	20.1	20.7	20.3
	2	20.4	20.1	20.7	20.3
	3	20.4	20.1	20.7	20.3
	4	20.4	20.1	20.7	20.3
100 mg/L nominal loading rate WAF	1	20.4	20.1	20.7	20.3
	2	20.4	20.1	20.7	20.3
	3	20.4	20.1	20.7	20.3
	4	20.4	20.1	20.7	20.3

 

Oxygen concentration measured in the test vessels (mg/L)

Test group	Replicate	Measuring
		0 h (fresh media)	24 h (old media)	24 h (fresh media)	48 h (old media)
Control	1	8.4	8.3	8.4	8.6
	2	8.4	8.3	8.4	8.6
	3	8.4	8.5	8.4	8.4
	4	8.4	8.5	8.4	8.4
100 mg/L nominal loading rate WAF	1	6.7	8.5	6.8	8.4
	2	6.7	8.5	6.8	8.4
	3	6.7	8.5	6.8	8.4
	4	6.7	8.5	6.8	8.4

 

pH measured in the test vessels

Test group	Replicate	Measuring
		0 h (fresh media)	24 h (old media)	24 h (fresh media)	48 h (old media)
Control	1	7.22	7.66	7.50	7.30
	2	7.22	7.66	7.50	7.30
	3	7.22	7.68	7.50	7.34
	4	7.22	7.68	7.50	7.34
100 mg/L nominal loading rate WAF	1	7.27	7.67	7.66	7.38
	2	7.27	7.67	7.66	7.38
	3	7.27	7.66	7.66	7.40
	4	7.27	7.66	7.66	7.40

 

DATA OF IMMOBILISATION

Immobilisation of the test animals

Test group	Number of animals	Number of immobilised animals
		24 h	48 h
Control	5	0	0
	5	0	0
	5	0	0
	5	0	0
100 mg/L nominal loading rate WAF	5	1	1
	5	0	0
	5	0	0
	5	0	0

 

 

Validity criteria fulfilled:

yes

Conclusions:

Acute toxicity of Extract of Chardon marie without support was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Extract of Chardon marie without support were the followings:

The 24h and 48h EL50 value: > 100 mg/L nominal loading rate WAF
The 48h EL100 value: > 100 mg/L nominal loading rate WAF
The 48h No-Observed Effect Loading Rate (NOELR): 100 mg/L nominal loading rate WAF
The 48h Lowest Observed Effect Loading Rate (LOELR): > 100 mg/L nominal loading rate WAF

Executive summary:

Acute toxicity of Extract of Chardon marie without support on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried outusing onlyone test concentration at100 mg/L nominal loading rate WAFand one control groupin the definitive test.

The test concentration was analytically determined at the start and at the end of the renewal periods, but could not be measured due to limitation of the analytical method used. Therefore, biological results are expressed in nominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multi-constituent substances.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

All validity criteria were met during this study.

 

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Extract of Chardon marie without supportwere the followings:

The 24h and 48h EL₅₀value: >100 mg/L nominal loading rate WAF

The 48h EL₁₀₀value:  >100 mg/L nominal loading rate WAF

The 48h No-Observed Effect Loading Rate (NOELR): 100 mg/L nominal loading rate WAF

The 48h Lowest Observed Effect Loading Rate (LOELR): >100 mg/L nominal loading rate WAF

Description of key information

Acute toxicity of Extract of Chardon marie without support was assessed with Acute immobilisation test on Daphnia magna, over an exposure period of 48 hours in a semi-static system.

All validity criteria were met during this study.

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Extract of Chardon marie without support were the followings:

The 24h and 48h EL50 value: > 100 mg/L nominal loading rate WAF

The 48h EL100 value: > 100 mg/L nominal loading rate WAF

The 48h No-Observed Effect Loading Rate (NOELR):       100 mg/L nominal loading rate WAF

The 48h Lowest Observed Effect Loading Rate (LOELR): > 100 mg/L nominal loading rate WAF

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

Acute toxicity ofExtract of Chardon marie without support on Daphnia magna was assessed in an Acute immobilisation test, over an exposure period of 48 hours in a semi-static test system.

Because no toxic response was observed during the preliminary range-finding test, a Limit Test was carried out using onlyone test concentration at 100 mg/L nominal loading rate WAF and one control group in the definitive test.

The test concentration was analytically determined at the start and at the end of the renewal periods, but could not be measured due to limitation of the analytical method used. Therefore, biological results are expressed in nominal loading rates WAFs (water accommodated fraction) based on OECD TG NO. 23 on poorly soluble and multi-constituent substances.

Twenty animals, divided into four groups (glass beaker) of five animals each were used at the test concentration and for the control.

All validity criteria were met during this study.

 

Under the conditions of this Daphnia magna acute immobilisation study the observed endpoints for the effect of Extract of Chardon marie without supportwere the followings:

The 24h and 48h EL₅₀value:>100 mg/L nominal loading rate WAF

The 48h EL₁₀₀value:  >100 mg/L nominal loading rate WAF

The 48h No-Observed Effect Loading Rate (NOELR): 100 mg/L nominal loading rate WAF

The 48h Lowest Observed Effect Loading Rate (LOELR): >100 mg/L nominal loading rate WAF