Resinoid of Liquidambar styraciflua (Hamamelidaceae) obtained from exudate by organic solvents extraction

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:

From December 18th 2017 to January 18th 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test) were followed, with GLP compliance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for testing of chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006; Annex 5 corrected July 28, 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspection on 4, 5, and 6th of July, 2016, certificate signed on January 2017.

Specific details on test material used for the study:

No additional information

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test concentrations (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test.

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 22.5 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were filtered and added into test flasks containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. At the start of the test, the solution was observed to be clear and colourless.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Pseudokirchneriella subcapitata CCAP 278/4
- Source (laboratory, culture collection): Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
- Stock cultures : Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.
- Pre-culture : Pre-culture 2 to 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 1.104 cells.mL-1. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Test type:

static

Water media type:

other: the Original medium of OECD TG 201 was prepared in sterilised water.

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Test temperature:

23 +/- 2 °C

pH:

The pH of the medium was approximately in the range of 8.1 ± 0.2.
From 8.30 to 10.05.

Nominal and measured concentrations:

Loading rates (spaced by a factor of 2): 2, 4, 8, 16, and 32 mg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, cylindrical all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL
- Initial cells density: initial cell density of 5.103 cells.mL-1
- No. of vessels per control and concentration (replicates): 6 controls and 3 replicates of each loading rate for counting.

GROWTH MEDIUM
- Standard medium used: yes, original medium of OECD TG 201

TEST MEDIUM / WATER PARAMETERS
- Total organic carbon: TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025. Concentration of dissolved organic material in the control and the WAFs was checked by analysis of Total Organic Carbon (TOC) at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

OTHER TEST CONDITIONS
- pH : pH was measured at the start and at the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Photoperiod: Continuous illumination
- Light intensity and quality: 5238 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other: The criterion measured was the EL50(Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations : test solutions used in the definitive test were prepared to obtain the following loading rates (spaced by a factor of 2): 2, 4, 8, 16, and 32 mg.L-1.
- Range finding study : a range-finding-test was previously performed, the following nominal concentrations were tested : 1, 10, 32 and 100 mg/L
- Test concentrations: concentrations tested in the definitive test : 2, 4, 8, 16, and 32 mg.L-1.
- Results used to determine the conditions for the definitive study: 1.7 % inhibition at 1 mg/L after 72 hours, 47.1 % inhibition after 72 hours at 10 mg/L, 91.0 % inhibition after 72 hours at 32 mg/L and 92.9 % inhibition after 72 hours at 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate was used as reference substance

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.362 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 13.378 – 18.562

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

19.663 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 16.998 – 21.623

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.945 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 26.119 – 29.794

Details on results:

Based on the results of the range-finding test, the effect of the test item on Pseudokirchneriella subcapitata was tested in a definitive study using five loading rates.

Results with reference substance (positive control):

On August 21, 2017 (most recent test), the 72h-EC50 was 1.457 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (6) (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The software ToxRat® Professional was used to perform statistical analyses. Complementary statistical analyses were performed using another statistical spreadsheet (using Excel®) for the validity criteria of the study.

Table 6.1.5 / 2 : Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴).

	Replicate	Nominal concentration(mg test item.L^-1)*
		Control	2	4	8	16	32
t=24h	1	7.6	5.2	4.8	4.0	0.4	0.0
	2	7.2	4.8	5.5	4.4	2.0	0.0
	3	8.8	4.0	4.8	6.8	2.4	2.0
	4	4.4
	5	7.2
	6	6.4
	Mean	6.9	4.7	4.9	5.1	1.6	0.7
	Std. Dev.	1.47	0.61	0.23	1.51	1.06	1.15
	% Inh.	-	14.6	12.3	12.3	65.1	123.4
t=48h	1	32.4	22.8	21.2	16.4	10.0	0.4
	2	28.0	24.0	24.4	19.2	12.0	0.8
	3	28.8	26.4	23.6	21.2	12.8	0.8
	4	27.2
	5	24.8
	6	24.0
	Mean	27.5	24.4	23.1	18.9	11.6	0.7
	Std. Dev.	3.02	1.83	1.67	2.41	1.44	0.23
	% Inh.	-	2.9	4.3	9.4	21.6	94.0
t=72h	1	88.0	68.4	66.4	64.0	52.0	4.8
	2	81.2	64.4	72.0	73.2	50.8	2.0
	3	82.8	73.2	61.6	72.8	57.6	4.0
	4	78.8
	5	90.4
	6	84.8
	Mean	84.3	68.7	66.7	70.0	53.5	3.6
	Std. Dev.	4.32	4.41	5.21	5.20	3.63	1.44
	% Inh.	-	4.0	4.6	3.6	8.9	62.8

Table 6.1.5 / 3 : pH-values during the final test

 

	Nominal concentration(mg test item.L^-1)*
	Control	2	4	8	16	32
Start t=0h	8.34	8.32	8.30	8.36	8.32	8.41
End t=72h	9.95	10.01	10.03	10.05	9.86	8.02

* WAF prepared at the given loading rate.

Table 6.1.5 / 4 : Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L^-1) - Final test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)	Relative loss to initial value (t=0h - t=72h)(%)
Control	1.15	1.49	-30
2	1.64	0.99	40
4	3.24	1.57	52
8	2.31	1.41	39
16	4.21	2.27	46
32	8.1	3.68	55

* WAF prepared at given loading rate

Table 6.1.5 / 5 : Concentrations of the test item in test water (abiotic samples) - TOC analysis results (range-finding test).

	TOC (mg.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.53	0.65
1	0.65	0.66
10	3.06	1.84
32	7.59	6.12
100	12.30	8.79

* WAF prepared at the given loading rate.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria have been fulfilled in the present study.

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for growth rate was determined to be 27.945 mg.L-1. The 72 -hour EL10 for growth rate was 16.362 mg L-1.

Executive summary:

One experimental key study is available to assess the toxicity of the registered substance to algae.

In this study (LPL, 2018), the toxicity of the registered substance to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined in a closed static test of 72 hours, according to OECD Guideline 201 and EU Method C.3 with GLP compliance. Growth rate and yield parameters were recorded.

Following a preliminary range-finding test, algae were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2, 4, 8, 16, and 32mg.L^-1 and to a control.

Cell numbers were counted daily by microscope. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs.

TOC analyses indicated that few organic compounds were found throughout the test in the WAFs, maintained close to ± 50% of the initial concentration between the start and the end of the test.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for growth rate was determined to be 27.945 mg/L and the 72 -hour EL10 for growth rate was 16.362 mg/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 1:

72h-ErC50 (Pseudokircheriella subcapitata) = 27.945 mg/L (95% CL: 26.119 - 29.794 mg/L), based on nominal loading rates

72h-ErC10 (Pseudokircheriella subcapitata) = 16.362 mg/L (95% CL: 13.378 - 18.562 mg/L), based on nominal loading rates

Key value for chemical safety assessment

EC50 for freshwater algae:

27.945 mg/L

EC10 or NOEC for freshwater algae:

16.362 mg/L

Additional information

One experimental key study is available to assess the toxicity of the registered substance to algae.

In this study (LPL, 2018), the toxicity of the registered substance to the unicellular freshwater green alga Pseudokirchneriella subcapitatawas determined in a closed static test of 72 hours,according to OECD Guideline 201 and EU Method C.3 with GLP compliance. Growth rate and yield parameters were recorded.

Following a preliminary range-finding test, algae were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2, 4, 8, 16, and 32 mg.L^-1and to a control.

Cell numbers were counted daily by microscope. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs.

TOC analyses indicated that few organic compounds were found throughout the test in the WAFs,maintained close to ± 50% of the initial concentration between the start and the end of the test.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 for growth rate was determined to be 27.945 mg/L and the 72 -hour EL10 for growth rate was 16.362 mg/L.