Willow, Salix alba, 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:

2019-06-25 to 2019-06-27 (experimental phase)

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:

adopted 13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending Regulation (EC) No 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals. OECD Series on Testing and Assessment No. 23

Version / remarks:

Paris 08 February 2019

Deviations:

no

Remarks:

With regard to WAF preparation, analysis and reporting of results

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test was performed in a semi-static design, with media exchange after 24 hours. Animals were devided into 4 replicates.
For test item analysis, four replicate samples were taken from the fresh test item solution (before splitting into four replicates) and one sample was taken from each test vessel at the end of renewal periods (after 24 hours). The test samples were diluted into the calibrated range with diluent (methanol/water 1/1) to 5 fold. The control samples were measured directly, without any dilution.

Vehicle:

no

Details on test solutions:

A water accommodated fraction with a loading rate of 100.0 mg/L was prepared with direct addition of the test item: the test item was mixed into the test medium (ISO Medium) and stirred manually at room temperature until homogeneity. The non-dissolved test material was removed by filtration through a fine (0.22 μm) pre-saturated polyethersulfone membrane filter (Supplier: Millipore, Lot No.: MP180108SA, Expiry date: January 2021). As a Limit test was carried out, further dilution of stock solution was not performed.

Test organisms (species):

Daphnia magna

Details on test organisms:

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.

Number of animals: There were 20 animals in the test group and the 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 of the same composition as the culture water.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

no

Hardness:

The reconstituted water (ISO medium) had a total hardness of 247 mg/L (as CaCO3).

Test temperature:

The water temperature was measured at the start of the experiment and in 24-hour intervals thereafter in each test vessel. The test temperature was in the range of 20.4 – 20.9°C measured in the test vessels. The additionally measured temperature in the climate chamber was between 20.1 – 21.0°C.

pH:

The pH of the test solution was not adjusted and did not vary by more than 1.5 units in any one test. The pH was measured at the start and at the end of the experiment in each test vessel and was in the range of 7.52 – 8.07.

Dissolved oxygen:

The dissolved oxygen concentration was measured in each test vessel at the start and at the end of the test and was in the range of 7.5 – 8.7 mg/L.

Nominal and measured concentrations:

The nominal limit concentration of the UVCB test item was a water accommodated fraction with a nominal loading rate of 100 mg/L.
For details on analytical verification of test item concentrations see IUCLID section "Any other information on results incl. tables" below.

Details on test conditions:

See IUCLID section "Any other information on materials and methods incl. tables" below.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Remarks:

based on nominal loading rate of WAF

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

analytically confirmed based on marker compound salicin

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL0

Remarks:

based on nominal loading rate of WAF

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

analytically confirmed based on marker compound salicin

Basis for effect:

mobility

Details on results:

See IUCLID section "Any other information on results incl. tables" below.

Results with reference substance (positive control):

Test performed with reference item potassium dichromate (batch no.: A0345704): 23 - 24 January 2019.
Result:
24h EC50: 0.71 mg/L, (95 % confidence limits: 0.66 – 0.75 mg/L)

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of any toxic effects.
The EC50, NOEC, LOEC and EC100 values were determined directly from the raw data.

Analytical results based on analysis of the marker compound salicin

The measured chemical marker for the test item was salicin, the supplied test item was only partially soluble. A water accommodated fraction prepared at a nominal loading rate of 100 mg/L was used as the limit concentration and analyzed during the test. The test concentration was analytically determined at the beginning and at the end of each renewal period. Measured concentrations of the test item based on the marker salicin were 99 mg/L at the start and 104 mg/L at the end of the first renewal period, 101 mg/L at the start and 102 mg/L at the end of the second renewal period. The corresponding measured geometric mean test item concentration was 101.48 mg/L.

According to the WAF concept, the concentration of the UVCB test item is expressed as Loading Rate: this is considered to be the valid approach for a UVCB chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials based on OECD guidance document No. 23.

IMMOBILISATION

The number of immobilised animals and the percentage of immobility were determined at the 24th and 48th hour and are given in the table below.

In addition to immobility, no abnormal behaviour or appearance of test animals was detected.

Test Group	Number of	Number of	immobilised	animals
	treated	24	hours	48	hours
	animals	number	percent	number	percent
Control	20	0	0	1	5
100.0 mg/L (loading rate)	20	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

From a semi-static limit test on acute toxicity towards aquatic invertebrates (OECD 202; GLP), the following effect concentrations based on nominal loading rate (WAF) were determined for the UVCB test item (only partially soluble at 100 mg/L):
EL50 (48 h; Daphnia magna; immobilization; nominal loading rate of WAF) > 100 mg/L;
EL0 (48 h; Daphnia magna; immobilization; nominal loading rate of WAF) >= 100 mg/L.

Executive summary:

Acute toxicity of White willow bark extract was assessed with the acute immobilisation test on Daphnia magna over an exposure period of 48 hours in a semi-static system (OECD 202; GLP). Based on preliminary results obtained from a range finding test a limit test was performed at 100 mg/L nominal loading rate: due to limited solubility of the UVCB test item, according to OECD guidance document No. 23 a water accommodated fraction was prepared and used as the only test item concentration in the test.

All validity criteria were met during this study.

Under the conditions of this study the following (no)effect concentrations were derived for the toxicity of White willow bark extract on Daphnia magna:

EL50 (48 h; Daphnia magna; immobilisation; nominal loading rate of WAF) > 100 mg/L;

EL0 (48 h; Daphnia magna; immobilisation; nominal loading rate of WAF) >= 100 mg/L.

Based on analytical results for the marker compound salicin, the test item was stable during the renewal period of 24 hours.

Description of key information

From a semi-static limit test on acute toxicity towards aquatic invertebrates (OECD 202; GLP), the following effect concentrations based on nominal loading rate (WAF) were determined for the UVCB test item (only partially soluble at 100 mg/L):

EL50 (48 h; Daphnia magna; immobilization; nominal loading rate of WAF) > 100 mg/L;

EL0 (48 h; Daphnia magna; immobilization; nominal loading rate of WAF) >= 100 mg/L.

Additional information

Acute toxicity of White willow bark extract was assessed with the acute immobilisation test on Daphnia magna over an exposure period of 48 hours in a semi-static system (OECD 202; GLP). Based on preliminary results obtained from a range finding test a limit test was performed at 100 mg/L nominal loading rate: due to limited solubility of the UVCB test item, according to OECD guidance document No. 23 a water accommodated fraction was prepared and used as the only test item concentration in the test.

All validity criteria were met during this study.

Under the conditions of this study the following (no)effect concentrations were derived for the toxicity of White willow bark extract on Daphnia magna:

EL50 (48 h; Daphnia magna; immobilisation; nominal loading rate of WAF) > 100 mg/L;

EL0 (48 h; Daphnia magna; immobilisation; nominal loading rate of WAF) >= 100 mg/L.

Based on analytical results for the marker compound salicin, the test item was stable during the renewal period of 24 hours.

Because no effects were determined at all up to the limit loading level of the WAF (100 mg/L), no key value is given for chemical safety assessment as no EC50 could be determined.