Resinoid of Canarium luzonicum (Burseraceae) obtained from the exudate by cyclohexane 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:

2017-09-11 to 2017-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. All validity criteria were fulfilled.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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:

Not applicable

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:

Samples for analysis were taken from the control, the solvent 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. 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:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 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). The loading rates of the test item were weighed in glass vials and resuspended in acetone. Then the loading rates of the test item/vehicle were vortexed to obtain homogeneous suspensions, and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg.L-1, as recommended in OECD guidance on testing and assessment No. 23) of each preparation was carefully added to the surface of test water contained in the mixing vessels that 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 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 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 (WAFs).

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:

no

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

between 22.9 - 23-8 °C throughout the test (average value: 23.7°C)

pH:

Start(t=0 h): 8.16 to 8.26
End (t=72 h): 8.8.40 to 9.74

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L

Details on test conditions:

TEST SYSTEM
- 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 controls, 6 solvent controls and 3 replicates of each loading rate for counting. Moreover, a replicate of each treatment without algae was 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, according to the study plan.

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

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- Other:
- pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Temperature of Medium: Measured continuously in the growth chamber, over the study period.
- Light Intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following loading rates: 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

34.352 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 28.110 - 39.763 mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

19.735 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 0.268 - 26.826

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

45.464 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 39.192 - 50.803 mg/L

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

24.977 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 2.082 - 31.604 mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

77.722 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits: 72.205 - 83.219 mg/L

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

39.197 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% conf. limits: 30.747 - 147.380

Details on results:

See above

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 evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

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

	Nominal concentration(mg test item.L^-1)*
	Control	Solvent Control	1.0	3.2	10.0	32.0	100.0
Start t=0h	8.21	8.23	8.16	8.26	8.18	8.23	8.23
End t=72h	9.80	9.65	9.63	9.74	9.53	9.42	8.40

 * 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(without solvent)	Control(with solvent)	1.0	3.2	10.0	32.0	100.0
t=24h	1	5.2	4.4	2.8	4.8	2.8	3.6	2.0
	2	5.6	4.4	2.4	4.0	3.2	2.8	2.4
	3	4.8	4.4	4.4	5.6	4.8	2.0	2.0
	4	6.4	4.0
	5	4.0	4.8
	6	4.4	4.4
	Mean	5.1	4.4	3.2	4.8	3.6	2.8	2.1
	Std. Dev.	0.86	0.25	1.06	0.80	1.06	0.80	0.23
	% Inh.	-	-	16.2	-3.6	10.4	22.0	33.4
t=48h	1	21.6	20.0	21.6	15.2	14.0	9.2	2.0
	2	25.2	18.8	16.4	16.0	15.6	13.6	1.6
	3	18.0	25.6	22.0	19.2	15.6	11.2	1.2
	4	19.2	16.0
	5	15.6	22.4
	6	18.8	25.6
	Mean	19.7	21.4	20.0	16.8	15.1	11.3	1.6
	Std. Dev.	3.30	3.85	3.12	2.12	0.92	2.20	0.40
	% Inh.	-	-	1.7	6.2	9.0	16.9	69.5
t=72h	1	91.6	87.2	76.8	83.2	74.4	58.4	4.0
	2	78.8	99.2	66.8	67.2	88.0	56.0	2.4
	3	76.4	90.0	89.6	68.0	85.6	59.2	2.8
	4	82.8	91.2
	5	72.0	78.0
	6	83.6	84.8
	Mean	80.9	88.4	77.7	72.8	82.7	57.9	3.1
	Std. Dev.	6.77	7.07	11.43	9.02	7.26	1.67	0.83
	% Inh.	-	-	2.6	3.8	1.3	8.1	65.4
	% Inh.	-	-	0.9	2.1	-0.4	6.5	64.8

* WAF prepared at the given loading rate.

At test start 5000 algal cells/mL were incubated; 6 replicates of the controls, 6 replicates of the solvent controls and 3 replicates of each test loading rate.

Std. Dev.: standard deviation.

 

Validity criteria of the study

- Cell density in Controls: 177-fold increase within 72 hours. The specific growth rate was determined at 1.72 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 25.0%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.5%.

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.

The analytical results of this test revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item, and that TOC content in WAFs was maintained around ± 30-40%. Nevertheless, the test item presence in WAFs was proportional to the tested loading rates given the algal growth inhibition observed in the final test and the analytical test results (TOC content) without solvent during the range-finding test. Thus, we consider that this point does not interfere with the conclusion of the study.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.Therefore, and since the test item was a UVCB substance, the results were based on the nominal loading rates.

 

Table 6.1.5/3: 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)
Control	0.52	0.93
Solvent Control	20.00	14.30
1.0	22.20	16.30
3.2	22.00	15.90
10.0	21.50	15.40
32.0	19.30	12.00
100.0	20.40	14.90

* WAF prepared at the given loading rate.

Physico-chemical parameter values throughout the test

- The results of measurement of pH are presented in Table 6.1.5/1. Although the pH level in the control varied more than 1.5 units at the end of the test (1.59 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the addition of more sodium bicarbonate.

- The temperature in the incubator was situated between 22.9 and 23.8°C throughout the test (average value: 23.7°C), and complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C).

- The mean light intensity was of 5037 lux (range: 4731-5491 lux), which remained within the ranges prescribed by the study plan (range: 4440-8880 lux and shall not vary 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 concentrations, the 72-hour EL50 for the parameters growth rate and yield were determined to be 77.722 mg.L-1 and 39.197 mg.L-1, respectively. The 72-hour EL10 for growth rate was 34.352 mg L-1 and for yield was 19.735 mg.L-1.

Executive summary:

A study was performed to assess the test item ELEMI RESINOID for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266(2) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L and to a control. The 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 controls and the WAFs was checked by TOC analysis at the start and the end of the test.

 

The analytical results of this test revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item, and that TOC content in WAFs was maintained around ± 30-40%. Nevertheless, the test item presence in WAFs was proportional to the tested loading rates given the algal growth inhibition observed in the final test and the analytical test results (TOC content) without solvent during the range-finding. Therefore, we consider that this point does not interfere with the conclusion of the study.

After 72 hours of exposure, the E_rL_x and E_yL_x values were as follows:

Parameter	Growth rate (mg.L^-1)	Yield (mg.L^-1)
72-hour EL10	34.352	19.735
95% conf. limits	28.110 – 39.763	0.268 – 26.828
72-hour EL20	45.464	24.977
95% conf. limits	39.192 – 50.803	2.082 – 31.604
72-hour EL50	77.722	39.197
95% conf. limits	72.205 – 83.219	30.747 – 147.380

 

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameters growth rate and yield were determined to be 77.722 mg.L-1 and 39.197 mg.L-1, respectively. The 72-hour EL10 for growth rate was 34.352 mg L-1 and for yield was 19.735 mg.L-1.

Description of key information

Under the experimental conditions and based on nominal concentrations, the 72-hour EL50 for the parameter growth rate was determined to be 77.722 mg/L (95% CL = 72.205 - 83.219) and the 72-hour EL10 for the parameter growth rate was determined to be 34.352 mg/L (95% CL = 28.110 - 39.763).

Key value for chemical safety assessment

EC50 for freshwater algae:

77.722 mg/L

EC10 or NOEC for freshwater algae:

34.352 mg/L

Additional information

For that endpoint, a reliable GLP experimental study performed on the registered substance was available. This study intended to assess the test item ELEMI RESINOID 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. 201 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

The study was carried out using WAFs (Water Accommodated Fractions). Different methods for the preparation of the WAFs have been tried in the preliminary test. Based on this pre-test, algal growth innibition was more pronounced following a chemical solubilisation (i.e use of solvent), thus the final test was performed using acetone (at the limit concentration of 100 mg solvent/L as recommended by the guideline OECD 23) for the preparation of Water Accommodated Fractions. Based on the results of the pre-experiment, test solutions were prepared to obtain the following loading rates for the final test: 1.0, 3.2, 10.0, 32.0 and 100.0 mg/L.

After 72 hours of exposure, the E_rL_x and E_yL_x values were as follows:

Parameter	Growth rate (mg.L^-1)	Yield (mg.L^-1)
72-hour EL10	34.352	19.735
95% conf. limits	28.110 – 39.763	0.268 – 26.828
72-hour EL20	45.464	24.977
95% conf. limits	39.192 – 50.803	2.082 – 31.604
72-hour EL50	77.722	39.197
95% conf. limits	72.205 – 83.219	30.747 – 147.380

Statistical analyses were performed by the computer program ToxRat.

This toxicity study complied with the requirements of the guideline, besides one minor deviation concerning some pH-value. This did not affect the integrity of the study. Thus, the study was considered acceptable for that endpoint.