Resinoid of Styrax tonkinensis (Styracaceae) obtained from exudate by ethanol 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 2017-10-23 to 2017-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD 201 guideline. All validity criteria were successful and there was no deviation

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on January 10, 2017

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the limit test loading rate (from a replicate of each treatment without algae dedicated exclusively to chemical analyses) at the start and the end of the test. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs. 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.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring. After having tried different methods for the preparation of the WAFs in the preliminary study, the protocol described hereafter has been selected for the final test.
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). After heating of the test item sample to approx. 60°C for about 2 hours, 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 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test flasks containing a fixed amount of inoculum (5.10^3 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.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 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 culture: 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: 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 x 10^4 cells/mL. 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:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

between 23.0 and 23.9°C throughout the test (average value: 23.8°C).
The temperatures complied with the study requirements (23°C ± 2°C)

pH:

Control: 8.84 at t=0h and 9.91 at t=72h
WAF 100 mg/L: 8.79 at t=0h and 10.16 at t=72h
The pH level remained within the prescribed conditions

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Limit test: 100 mg/L (loading rate of a WAF)

Details on test conditions:

TEST DETAILS
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 replicates for the control and the loading rate at 100 mg.L-1. Moreover, replicates of each treatment without algae were prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: yes: Original medium of OECD TG 201, Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: light intensity of 4,440-8,880 lux, did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED :
Cell numbers were counted daily by microscope using a counting chamber.

TEST CONCENTRATIONS
Based on the results of a range-finding test, a limit test was performed at 100 mg.L-1 (loading).
- Range finding study: Algal cells were exposed to the nominal loading rates (in triplicate) 1.0, 10.0, 32.0 and 100.0 mg.L-1 and to a control, according to two different methods of preparation:
- heating/sonication;
- use of solvent (acetone).

The WAFs were prepared in the dark under closed conditions and by slow-stirring.
- Results used to determine the conditions for the definitive study: After the range-finding study, it has been decided to do a limit tests with a loading rate of 100 mg test substance/L for the final test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: loading rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: loading rate

Details on results:

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed, confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no ErLx and EyLx values could be determined due to the absence of effect at the tested loading rate.

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 (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values).

Table 6.1.5/1: pH-values during the final test

	Nominal concentration
	(mg test item.L^-1)*
	Control	100
Start t=0h	8.84	8.79
End t=72h	9.91	10.16

* WAF prepared at the given loading rate.

Table 6.1.5/2: Algal cell densities during the final test (expressed as density of algal cells/mL x 10⁴)

	Replicate	Nominal concentration (mg test item.L^-1)*
		Control	100
t=24h	1	6.0	6.0
	2	6.0	4.4
	3	6.4	4.8
	4	5.2	5.2
	5	4.8	4.8
	6	4.4	7.6
	Mean	5.5	5.5
	Std. Dev.	0.79	1.18
	% Inhib.	-	0.4
t=48h	1	21.6	20.0
	2	16.0	18.8
	3	20.8	21.2
	4	16.8	22.8
	5	15.2	20.8
	6	16.4	25.6
	Mean	17.8	21.5
	Std. Dev.	2.70	2.39
	% Inhib.	-	-5.5
t=72h	1	60.4	64.0
	2	44.0	73.2
	3	60.0	72.4
	4	50.4	60.8
	5	53.2	68.4
	6	48.4	59.2
	Mean	52.7	66.3
	Std. Dev.	6.52	5.92
	% Inhib.	-	-5.0

* WAF prepared at the given loading rate.

% Inhib.: %Inhibition of growth rate relative to the control determinedby the computer program ToxRat.

At test start 5000algal cells.mL^-1were incubated; 6 replicates of the controls and 6 replicates of the limit test loading rate.

Std. Dev.: standard deviation.

Validity criteria of the study

Cell density in controls: 105-fold increase within 72 hours. The specific growth rate was determined at 1.55 day^-1, so greater than 0.92 day^-1.

Coefficient of variation:

1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 8%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.7%.

Thus the validity criteria were respected in this study.

Analytical results

Concentration of dissolved organic material in the controls and the WAFs was checked by TOC analysis at the start and at the end of the test.

Although every effort was made in a first time to extract and solubilise the soluble fraction of the test item in test water (heating, sonication, mixing without headspace…) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOC analysis indicated that organic compounds were found at low value in the WAFs at 100 mg/L, maintained close to ± 40% of the initial concentration between the start and the end of the test. However, it should be noted that the study was carried out using WAFs of a natural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing. Therefore, due to the complex nature of the WAF and since the test item was an UVCB substance, the results were based on the nominal test loading rates.

Table 6.1.5/3: Results of the determination of TOC analysis (mg/L) - Final (limit) test.

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	0.64	0.80
Control	0.62	0.45
100	3.89	2.30
100	4.33	2.40

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

All test conditions remained within the ranges prescribed by the study plan and the test guideline (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23°C ± 2°C, constant within 2°C; light intensity: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were considered to be higher than 100 mg.L-1.

Executive summary:

A study was performed to assess the test item for its ability to generate toxic effects in the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysisat the startand the end of the test.

Although every effort was made in a first time to extract and solubilise thesoluble fraction of the test item in test water (heating, sonication, mixing without headspace…) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOC analysis indicated that organic compounds were found at low value in the WAFs at 100 mg/L, maintained close to ± 40% of the initial concentration between the start and the end of the test.

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed,confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no E_rL_x and E_yL_x values could be determined due to the absence of effect at the tested loading rate.

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were therefore considered to be higher than 100 mg.L-1.

Description of key information

Under the experimental conditions and based on nominal loading rates, the 72-hour EL50 and 72-hour EL10 values for the parameters growth rate and yield were considered to be higher than 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

For that endpoint, a study on the registered substance was available. This study was performed to assess the test item for its ability to generate toxic effects in Pseudokirchneriella subcapitata, under static conditions and during 72 hours. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

A limit test was performed following the results of a range-finding test. Algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item/L and to a control. The potential inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. Concentration of dissolved organic material in the control and the limit test loading rate was checked by TOC analysis at the start and the end of the test. Although every effort was made in a first time to extract and solubilise thesoluble fraction of the test item in test water (heating, sonication, mixingwithout headspace…) and secondly to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOCanalysisindicated that organic compounds were found at low value in the WAFs at 100 mg/L,maintained close to ± 40% of the initial concentration between the start and the end of the test. However, it should be noted that the study was carried out using WAFs of anatural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing. Therefore, due to the complex nature of the WAF and since the test item was an UVCB substance, the results were based onthe nominaltest loading rates.

After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed,confirming the observations of the range-finding test where results showed no effect at any of the tested loading rates. Based on these results, no E_rL_x and E_yL_x values could be determined due to the absence of effect at the tested loading rate.

All validity criteria were fulfilled and the study comply with the requirements of the guideline. This study is therefore considered acceptable for that endpoint.