Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

EC Number:

948-256-0

IUPAC Name:

Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction

Specific details on test material used for the study:

Name: Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extraction
EC number: 948-256-0
Batch number: 06/1234
Appearance: Amber crystal
Purity: 100% UVCB
Expiry date: 31 May 2020
Storage conditions: Refrigerated (2-8°C), protected from light and humidity (store in a tightly closed container)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken at the test concentration as well as from the control, at the beginning and at the end of the experiment.

Test solutions

Vehicle:

yes

Details on test solutions:

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.
OECD medium was constitued of stock solution 1 (macro nutrients), stock solution 2 (iron), stock solution 3 (trace elements) and stock solution 4 (bicarbonate).

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 Charles River Laboratories Hungary Kft.
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.
Breeding 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, after an incubation period of three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Test conditions

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 in the range of 22.6 – 22.8 °C measured in the flask and between 21.9 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 each test vessels. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.05 – 8.51 during the experiment.

Nominal and measured concentrations:

The concentration levels used (72 h) of the Preliminary Range-Finding Test are summarised below:
Nominal concentrations (mg/L nominal loading rate WAF): untreated control, 0.1, 1.0, 10.0, 100.0.


Because significant inhibition was not observed at the highest concentration level during the preliminary range-finding test, only one test concentration in the test medium (100 mg/L nominal loading rate WAF) and one control group were tested in a Limit Test.

Details on test conditions:

Light intensity. The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 7421 lux (equivalent to ~100 µE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm). The differences in light intensity between the test vessels did not exceed +/-15 % and therefore provided equal conditions for each test vessel.

PERFORMANCE OF THE TEST
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.

The test was performed with six replicates per test concentration and six replicates in the control group. Volumes of 100 mL test solution per replicate in 250 mL sterile Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
All procedures were performed under sterile conditions during the test.


Preliminary Range-Finding Test

A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.
During the formulation procedure the stock solution was prepared as described below :
Because the test item is a UVCB (Chemical Substances of Unknown or Variable Composition, Complex Reaction Products and Biological Materials) and poorly soluble in water, test item stock solution was prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C.
The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give the appropriate WAF solution. Although there is no stability data, the very low level of any organic substance in the water phase necessitates a maximal approach to generating the highest extraction into media that is practicable.

The concentration levels used (72 h) of the Preliminary Range-Finding Test are summarised below:
Nominal concentrations (mg/L nominal loading rate WAF): untreated control, 0.1, 1.0, 10.0, 100.0.


Because significant inhibition was not observed at the highest concentration level during the preliminary range-finding test, only one test concentration in the test medium (100 mg/L nominal loading rate WAF) and one control group were tested in a Limit Test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

ca. 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Results with reference substance (positive control):

The cell density in the control cultures increased by the factor of 70.17 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 11.71 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.83 %.
All validity criteria were met; therefore, the study can be considered as valid.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The effect of Resinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extractionwas assessed on algal growth using the unicellular green algaPseudokirchneriella subcapitata(formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.

 

Under the conditions of thisalgal growth inhibition testthe calculated endpoints for the effect ofResinoid of Boswellia serrata (Burseraceae) obtained from exudate by hexane extractionwere the following:

 

Summary of endpoints

Parameter	72 h ErL50 [mg/L]	72 h EbL50 [mg/L]	72h EyL50 [mg/L]	72h NOELR [mg/L]	72h LOELR [mg/L]
nominal loading rate	> 100.0 mg/L	> 100.0 mg/L	> 100.0 mg/L	100.0 mg/L	> 100.0 mg/L