Extract of Boswellia serrata (Burseraceae) obtained from exudate by solvent 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 2017-08-07 to 2018-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline No. 201. The 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:

Duplicate samples for analysis were taken from the control and the limit test loading rate 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
- Method: 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 1 L cylindrical glass bottles sealed with screw caps. A magnetic stirring bar was placed in each mixing vessel and test water was added. The loading rates of the test item were weighed on weighing boats that afterwards were placed above the mixing vessel and rinsed with test water. The mixing vessels were then carefully and completely filled (with a minimum of headspace) with the remaining volume of test water, 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 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. Then the WAFs were filtered and 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):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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.
- Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
- 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 1.10^4 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:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

No data

Test temperature:

22.8-23.3 °C throughout the test (average value: 23.3 °C)
This complied with the requirements (23°C ± 2°C, constant within 2°C).

pH:

Start(t=0 h): 8.27 - 8.31
End (t=72 h): 10.12 - 10.20
Although the pH level in the control varied more than 1.5 units at the end of the test (1.85 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.

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

no data

Nominal and measured concentrations:

Nominal concentrations: 100 mg/L (limit test)

Details on test conditions:

TEST CONDITIONS
- 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.
Test environment: Controlled environment cabinet (23°C ± 2°C); vessels were distributed randomly in the incubator and redistributed over the test at t=24h and t=48h. During incubation, the algal cells were kept in suspension by continuous magnetic stirring.

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: 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.

RANGE-FINDING TEST
A preliminary test has been realised on the test item. The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg.L-1 and to a control. Two different conditions for the preparation of WAF have been tested: with and without solvent (acetone). Results with and without solvent showed no significant effect at any of the tested loading rates. Therefore, it was decided to perform a limit test at 100 mg.L-1 (loading) for the final study.

TEST CONCENTRATIONS
- Results used to determine the conditions for the definitive study: Based on the results of a range-finding test, a limit test was performed at 100 mg.L-1 (loading) in order to demonstrate that the test item had no significant effect on the test organism at this test concentration or that EL50 was higher than 100 mg.L-1.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth 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

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth 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

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 February 14, 2017 (most recent test), the 72h-EC50 was 1.438 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 software ToxRat® Professional was used to perform statistical analyses. No reported statistics and error estimates.

Table 1: pH-values during the final test

	Nominal concentration (mg test item.L^-1)*
	Control	100
Start t=0h	8.27	8.31
End t=72h	10.12	10.20

Table 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	4.8	8.8
	2	3.6	5.6
	3	10.4	7.2
	4	5.6	10.0
	5	6.4	4.8
	6	7.6	6.0
	Mean	6.4	7.1
	Std. Dev.	2.39	2.00
	% Inhib.	-	-4.9
t=48h	1	22.0	35.2
	2	26.4	26.4
	3	24.4	34.8
	4	28.0	25.6
	5	24.8	30.0
	6	26.8	32.4
	Mean	25.4	30.7
	Std. Dev.	2.13	4.12
	% Inhib.	-	-4.7
t=72h	1	98.0	105.2
	2	90.4	110.0
	3	93.6	103.2
	4	103.6	98.0
	5	96.0	113.2
	6	102.0	111.6
	Mean	97.3	106.9
	Std. Dev.	5.01	5.78
	% Inhib.	-	-1.8

* WAF prepared at the given loading rate.

% Inhib.: %Inhibition of growth rate relative to the control determinedby the computer program ToxRat (8).

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: 195-fold increase within 72 hours. The specific growth rate was determined at 1.76 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 16.0%.

2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 1.0%.

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.

Even if the TOCanalysisindicated that organic compounds were found at low value in the loading rates, analytical resultsshowed thatWAFs concentrations were overall stablebetween the start and the end of the exposure period.

It should be noted that aWAF 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 thenominaltest loading rates.

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	< 0.30	0.40
Control	< 0.30	0.39
100	3.91	4.44
100	4.42	4.89

* WAF prepared at the given loading rate.

 

 

Physico-chemical parameter values throughout the test

Although the pH level in the control varied more than 1.5 units at the end of the test (1.85 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 moresodium bicarbonate.

The mean light intensity was of 5212 lux (range: 4845-5719 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).

The temperature in the incubator was situated between 22.8 and 23.3°C throughout the test (average value: 23.3°C), and complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C).

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based on nominal concentrations, 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.

Executive summary:

A study was performed to assess the test item Boswellia serrata extract 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 and was performed under GLP conditions.

A limit test was performed following the results of a range-finding test. Algal cells were exposed toWater Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item.L^-1and 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.

Even if the TOCanalysisindicated that organic compounds were found at low value in the loading rates, analytical resultsshowed thatWAFs concentrations were overall stablebetween the start and the end of the exposure period. 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 concentrations, 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 concentrations, the 72-hour EL₅₀ and 72-hour EL₁₀ values for the parameters growth rate and yield were estimated to be higher than 100 mg.L^-1.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A reliable experimental study was available to assess the ability of the registered substance Boswellia serrata extract to generate toxic effects on Pseudokirchneriella subcapitata, under static conditions. This study was performed under GLP conditions and according to the OECD 201 guideline.

A limit test was performed following the results of a range-finding test. Algal cells were exposed toWater Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item.L^-1and 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.

Even if the TOC analysis indicated that organic compounds were found at low value in the loading rates, analytical results showed that WAFs concentrations were overall stable between the start and the end of the exposure period. The results are therefore based on nominal concentrations. After 24, 48 and 72 hours of exposure no significant inhibition of algal growth relative to control values was observed.

Based on the result of this study, the substance would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

Besides some pH values that did not meet the requirements (but this deviation did not affect the integrity of the study), all the validity criteria of the study as laid down in the guideline were respected. Therefore, this study was considered as valid for that endpoint.