Willow, Salix alba, ext.

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-06-24 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 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 28 July, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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 as well as testing toxicity towards algae with light absorbing compounds;

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the analysis, the samples were taken in triplicate from the test item solutions at the start. After 1, 2 and 3 days, one sample was taken from each of three replicate test vessels per concentration level. Control samples were taken in each day with one sample. The test samples were diluted into the calibrated range with diluent (methanol/water 1/1) to 5 fold in case of the two highest concentrations and with methanol to 2 fold in case of the lower concentration samples. The control samples were measured directly, without any dilution.

Vehicle:

no

Details on test solutions:

The test solutions with a loading concentration of 6.25, 12.5, 25, 50 and 100 mg/L were prepared individually with direct addition of the test item: the test item was mixed into the algal growth medium (OECD Medium), ultrasonicated for 3 minutes and stirred manually at room temperature until homogeneity. Any 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).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of Citoxlab Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no

Hardness:

OECD 201 test medium

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was between 22.4°C and 22.7°C measured in the flask and between 22.0 and 22.9°C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.26 – 8.36 during the experiment.

Dissolved oxygen:

not applicable

Salinity:

not applicable, fresh water

Conductivity:

no data

Nominal and measured concentrations:

Water accommodated fractions with nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L were prepared individually and used for the test. 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

Duration:

72 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:

initial concentrations analytically confirmed based on marker compound salicin

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL10

Remarks:

based on nominal loading rate of WAF

Effect conc.:

54.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

initial concentrations analytically confirmed based on marker compound salicin

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 40.9 - 73.1 mg/L

Duration:

72 h

Dose descriptor:

NOELR

Remarks:

based on nominal loading rate of WAF

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

initial concentrations analytically confirmed based on marker compound salicin

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

LOELR

Remarks:

based on nominal loading rate of WAF

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (total fraction)

Remarks:

initial concentrations analytically confirmed based on marker compound salicin

Basis for effect:

other: growth rate and yield

Details on results:

There were no observed morphological deviations of algae cells during the experiment.
At the highest loading rate of the test item of 100 mg/L, only 18.9% growth rate reduction (0 – 72 h) was observed. As such, the 72 h ErC50 was determined to be > 100 mg/L. An indicative value for ErC50 was determined by extrapolation of the very flat dose-response curve [by Probit analysis (TOXSTAT software)] as 395.06 mg/L with a very large confidence interval (95 % confidence limits: 172.19 – 906.39 mg/L), reflecting the high uncertainty of extrapolation.
For further details, see IUCLID section "Any other information on results incl. tables" below.

Results with reference substance (positive control):

The 72h ErC 50: 0.87 mg/L, (95 % confidence limits: 0.80 – 0.95 mg/L)
The 72h EbC 50: 0.62 mg/L, (95 % confidence limits: 0.57 – 0.68 mg/L)
The 72h EyC 50: 0.52 mg/L, (95 % confidence limits: 0.48 – 0.57 mg/L)
These values are within the range of laboratory ring test data (see ISO Guideline No. 8692).

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and William’s Test (alpha = 0.05) by TOXSTAT software. Before, normal distribution (Shapiro-Wilk's test) and variance homogeneity (Levene's test) were confirmed.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by William’s Test.

Analytical results

The measured chemical marker for the test item was salicin, the UVCB test item was only partially soluble. Therefore, water accommodated fractions prepared individually at 5 nominal loading rates (given in the analytical results table below) were used as treatments and analysed during the test.

The following analytical results (with 95% confidence interval) were obtained for the test item calculated based on analysed concentrations of the marker compound salicin:

Nominal concentration (mg/L)	Determined concentration at the start (mg/L)	Determined concentration after 1 day (mg/L)	Determined concentration after 2 days (mg/L)	Determined concentration at the end (mg/L)
Control	not detected	not detected	not detected	not detected
6.25	< 6.67 (6.16 ± 0.532)*	< 6.67 (5.73 ± 0.597)*	< 6.67 (3.24 ± 1.078)*	not detected
12.5	11.8 ± 0.38	10.9 ± 0.50	< 6.67 (6.64 ± 1.243)*	not detected
25	24.6 ± 0.51	23.6 ± 0.67	10.8 ± 3.61	not detected
50	51.3 ± 1.26	58.7 ± 4.97	26.9 ± 8.70	not detected
100	97.2 ± 2.90	118 ± 5.3	75.2 ± 6.51	not detected

*) The concentrations in bracket are the extrapolated values based on the calibration, because these values are below the calibrated range. However, the peaks were detectable and calculated.

After 1 day, an unknown peak started to appear on the chromatograms, which caused increasing interference with increasing test item concentrations. The resolution of the peaks started to decrease continuously. Therefore, higher concentrations were probably calculated for the two highest concentration levels due to the increasing interference at 24 hours.

All initially analysed concentrations (0 h) are nearly equal to the nominal loading of the test item used for preparation of the WAFs. Accordingly, at least the marker compound salicin was quantitatively in solution up to the highest nominal loading of the test item. At 48 h a significant decrease of the marker salicin is mirrored by the reduced calculated test item concentrations, and at the end of the test, salicin concentration was below the limit of detection. However, other components of the UVCB test item may have been much more stable during the test but could not be analytically assessed individually.

Therefore and according to the WAF concept, with regard to derivation of (no)effect concentrations, all concentration levels are expressed as nominal loading rates as used for preparation of water accommodated fractions: this is considered to be the valid approach for a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) based on OECD guidance document No. 23 (2019).

CELL NUMBERS

The cell number in each flask was determined at the 24th, 48th, 72nd hours. The results of determinations for treatments and control are given as a graphical representation in the attached illustration.

Loading rate		Number of cells (x 10^4 cell/mL)
[mg/L]		0 h	24 h	48 h	72 h
Control		1	4	29	108
		1	5	34	96
		1	5	33	104
		1	6	35	98
		1	5	30	102
		1	5	32	100
	Mean	1.00	5.00	32.17	101.33
	SD	0.0	0.6	2.3	4.3
6.25		1	5	30	96
		1	6	28	99
		1	5	34	102
	Mean	1.00	5.33	30.67	99.00
	SD	0.0	0.6	3.1	3.0
12.5		1	3	27	92
		1	5	30	97
		1	5	31	101
	Mean	1.00	4.33	29.33	96.67
	SD	0.0	1.2	2.1	4.5
25		1	4	28	94
		1	3	24	92
		1	3	25	92
	Mean	1.00	3.33	25.67	92.67
	SD	0.0	0.6	2.1	1.2
50		1	4	20	67
		1	3	19	65
		1	3	17	60
	Mean	1.00	3.33	18.67	64.00
	SD	0.0	0.6	1.5	3.6
100		1	3	10	42
		1	3	12	39
		1	3	10	46
	Mean	1.00	3.00	10.67	42.33
	SD	0.0	0.0	1.2	3.5

AVERAGE SPECIFIC GROWTH RATES

The following table gives determined average specific growth rates (µ) together with significance results from statistical testing using William’s Test (alpha= 0.05):

Concentration	Growth rate (µ) and % inhibition of µ
Loading Rate	0–24 h		0–48 h		0–72 h
[mg/L]	µ	%	µ	%	µ	%
Control	0.0668	0.0	0.0723	0.0	0.0641	0.0
6.25	0.0696	-4.2	0.0712	1.4	0.0638	0.5
12.5	0.0600	10.2	0.0704	2.6	0.0635	1.0
25	0.0498*	25.5	0.0676*	6.5	0.0629*	1.9
50	0.0498*	25.5	0.0609*	15.7	0.0577*	10.0
100	0.0458*	31.4	0.0492*	31.9	0.0520*	18.9

* ):  statistically significantly different compared to the control values (William’s Test; a = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

From a static test on toxicity towards green algae (OECD 201; GLP), the following effect concentrations based on nominal loading rates (WAFs) were determined for the UVCB test item (only partially soluble at the higher concentrations):
EL50 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) > 100 mg/L;
EL10 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) = 54.7 mg/L (95% CI: 40.9 - 73.1).

Executive summary:

Toxicity of White willow bark extract towards green algae was assessed according to OECD TG 201 in a static system compliant with GLP. Based on preliminary testing results significant impact of light absorption by the brown coloured test item solution on algae growth could be excluded. Due to limited solubility of the UVCB test item, according to OECD guidance document No. 23 water accommodated fractions were prepared individually at 5 nominal loading levels and used as the treatment concentrations.

Concentrations of White willow bark extract in the test solutions were determined at the start and then daily from all three replicates and the control. Considering that salicin is one of the active ingredients from the test item (15%), it was used as marker for the test item. Salicin content in aqueous samples was determined by an HPLC-UV method. As a result, all initially analysed concentrations (0 h) were nearly equal to the nominal loading of the test item used for preparation of the WAFs. Accordingly, at least the marker compound salicin was quantitatively in solution up to the highest nominal loading of the test item. At 48 h, the calculated test item concentrations were significantly reduced compared to initial, equivalent to a significant decrease of the marker salicin; and at the end of the test, salicin concentration was below the limit of detection. However, other components of the UVCB test item may have been much more stable during the test but could not be analytically assessed individually. Therefore and according to the WAF concept, with regard to derivation of (no)effect concentrations, all concentration levels are expressed as nominal loading rates: this is considered to be the valid approach for a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) based on OECD guidance document No. 23 (2019).

All validity criteria of the OECD TG 201 were met during this study.

The following (no)effect concentrations were derived for the toxicity of White willow bark extract towards algae (Pseudokirchneriella subcapitata):

Parameter (0-72 h)	Growth rate (r) Loading Rate [mg/L]	Yield (y) Loading Rate [mg/L]	Biomass (b) Loading Rate [mg/L]
	Calculation based on the loading rates
EC10	54.7	19.7	15.7
95 % conf. limits	40.9 – 73.1	15.8 – 24.4	12.4 – 19.9
EC50	> 100 mg/L	79.0	71.25
95 % conf. limits	not applicable	65.02 – 95.98	58.46 – 86.84
NOEC	12.5	12.5	6.25
LOEC	25.0	25.0	12.5

Description of key information

From a static test on toxicity towards green algae (OECD 201; GLP), the following effect concentrations based on nominal loading rates (WAFs) were determined for the UVCB test item (only partially soluble at the higher concentrations):

EL50 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) > 100 mg/L;

EL10 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) = 54.7 mg/L (95% CI: 40.9 - 73.1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

54.7 mg/L

Additional information

Toxicity of White willow bark extract towards green algae was assessed according to OECD TG 201 in a static system compliant with GLP. Based on preliminary testing results significant impact of light absorption by the brown coloured test item solution on algae growth could be excluded. Due to limited solubility of the UVCB test item, according to OECD guidance document No. 23 water accommodated fractions were prepared individually at 5 nominal loading levels and used as the treatment concentrations.

Concentrations of White willow bark extract in the test solutions were determined at the start and then daily from all three replicates and the control. Considering that salicin is one of the active ingredients from the test item (15%), it was used as marker for the test item. Salicin content in aqueous samples was determined by an HPLC-UV method. As a result, all initially analysed concentrations (0 h) were nearly equal to the nominal loading of the test item used for preparation of the WAFs. Accordingly, at least the marker compound salicin was quantitatively in solution up to the highest nominal loading of the test item. At 48 h, the calculated test item concentrations were significantly reduced compared to initial, equivalent to a significant decrease of the marker salicin; and at the end of the test, salicin concentration was below the limit of detection. However, other components of the UVCB test item may have been much more stable during the test but could not be analytically assessed individually. Therefore and according to the WAF concept, with regard to derivation of (no)effect concentrations, all concentration levels are expressed as nominal loading rates: this is considered to be the valid approach for a chemical substance of Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB) based on OECD guidance document No. 23 (2019).

All validity criteria of the OECD TG 201 were met during this study.

The following (no)effect concentrations were derived for the toxicity of White willow bark extract towards algae:

EL50 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) > 100 mg/L;

EL10 (72 h; Pseudokirchneriella subcapitata; growth rate reduction; nominal loading rates of WAFs) = 54.7 mg/L (95% CI: 40.9 - 73.1).

At the highest loading rate of the test item of 100 mg/L, only 18.9% growth rate reduction (0 – 72 h) was observed. As such, the 72 h ErC50 was determined to be > 100 mg/L.