Willow, Salix alba, ext.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-24 to 2019-06-28 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017

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.
Analytical measurements were performed from the control and at the applied test concentration level at the beginning and at the end of the first and of the last renewal periods.
For test item analysis, three replicate samples were taken from the fresh test item solutions and at the end of the renewal periods (after 24 hours). The test samples were diluted with diluent (methanol/water 1/1) to 5 fold into the calibrated range. 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 mg/L was prepared with direct addition of the test item: the test item was mixed into the test medium (aquarium water) and stirred at room temperature until homogeneity. The non-dissolved test materials were removed (except of Day 0) 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.
Based on the analytical method validation (Study Code: 18/372-316AN) the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to the treatments, at each of the renewal periods test item solution was prepared according to the method described above.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Brachydanio rerio)
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group;
Body length of animals: 1.9 – 2.4 cm
Food and Feeding: The fish were not fed during the test;
Acclimatisation: more than 12 days;
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test period; the fish were healthy. There was no mortality of the population for seven days before the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

no

Hardness:

171 mg/L (as CaCO3)

Test temperature:

between 20.2 and 22.0 °C

pH:

The pH was in the range of 7.80 – 8.52

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 72 – 100% of the air saturation value at the temperature used.

Salinity:

not applicable, fresh water

Conductivity:

no data

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):

no

Duration:

96 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:

mortality (fish)

Remarks:

and sublethal effects

Key result

Duration:

96 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:

mortality (fish)

Remarks:

and sublethal effects

Details on results:

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

Results with reference substance (positive control):

Not applicable - no reference test

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of any toxic effects.
The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

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 the first and last renewal period (24 h). Measured concentrations of the test item based on the marker salicin were 104 mg/L at the start and 104 mg/L at the end of the first renewal period, and 101 mg/L at the start and 106 mg/L at the end of the last renewal period. The corresponding measured geometric mean test item concentration was 103.7 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.

Mortality Data

Cumulative mortality data from the exposure of Zebrafish to the test material during the definitive test are given in the table below:

Test Group	Cumulative Mortality (number of dead fish) [initial population = 7 fish]
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
100 mg/L (loading rate)	0	0	0	0	0	0

No sublethal effect was observed.

Body weight

The body weight of 7 fish was weighed at the start of the test. The measured and calculated data are listed in the table below.

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	1.12	0.16	0.22
100 mg/L (loading rate)	1.24	0.18	0.25

There was no considerable difference observed concerning body weights between the groups.

Validity criteria fulfilled:

yes

Conclusions:

From a semi-static limit test on acute toxicity towards freshwater fish (OECD 203; Danio rerio; 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 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) > 100 mg/L;
EL0 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) >= 100 mg/L.

Executive summary:

The acute aquatic toxicity of White willow bark extract towards fresh water fish was assessed according to OECD 203 on Zebrafish (Brachydanio rerio) over an exposure period of 96 hours in a

semi-static system compliant with GLP.

Because no toxic response was observed during the preliminary range-finding test, a limit test was carried out using one test item concentration (nominal loading rate of 100 mg/L) and one

control group in the definitive study under semi-static conditions. Because of the limited solubility of the test item in water and its UVCB character, in accordance with OECD guidance

document No. 23, water accommodated fractions (WAF) were prepared at 100 mg/L nominal loading.

The measured chemical marker for the test item was salicin, the supplied test item was only partially soluble. The test concentration was analytically determined at the beginning and at the

end of the first and of the last renewal periods. Measured concentration of salicin was 104 mg/L at the start and at the end of the first renewal period, 101 mg/L at the start and 106 mg/L

at the end of the last renewal period. Consequently, the corresponding measured geometric mean test item concentration based on salicin was 103.73 mg/L.

According to the WAF concept, the test item concentration 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.

One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre of test solution.

All achievable validity criteria were met during this study.

The following biological results were obtained from the valid and reliable study:

EL50 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) > 100 mg/L;

EL0 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) >= 100 mg/L.

Description of key information

From a semi-static limit test on acute toxicity towards freshwater fish (OECD 203; Danio rerio; 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 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) > 100 mg/L;

EL0 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) >= 100 mg/L.

Additional information

The acute aquatic toxicity of White willow bark extract towards fresh water fish was assessed according to OECD 203 on Zebrafish (Brachydanio rerio) over an exposure period of 96 hours in a

semi-static system compliant with GLP.

Because no toxic response was observed during the preliminary range-finding test, a limit test was carried out using one test item concentration (nominal loading Rate of 100 mg/L) and one

control group in the definitive study under semi-static conditions. Because of the limited solubility of the test item in water and its UVCB character, in accordance with OECD guidance

document No. 23, water accommodated fractions (WAF) were prepared at 100 mg/L nominal loading.

The measured chemical marker for the test item was salicin, the supplied test item was only partially soluble. The test concentration was analytically determined at the beginning and at the

end of the first and of the last renewal periods. Measured concentration of salicin was 104 mg/L at the start and at the end of the first renewal period, 101 mg/L at the start and 106 mg/L

at the end of the last renewal period. Consequently, the corresponding measured geometric mean test item concentration based on salicin was 103.73 mg/L.

According to the WAF concept, the test item concentration 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.

One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre of test solution.

All achievable validity criteria were met during this study.

The following biological results were obtained from the valid and reliable study:

EL50 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) > 100 mg/L;

EL0 (96 h; Danio rerio; mortality & sublethal effects; nominal loading rate of WAF) >= 100 mg/L.

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 LC50 could be determined.