Extract of Boswellia serrata (Burseraceae) obtained from exudate by solvent extraction

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11-09-2017 to 5-07-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from the control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). 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:

The definitive test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rates of 11, 23, 52, 114 and 250 mg.L-1 and to a control. The WAFs (for fresh media at t=0h and t=24h) were prepared in the dark under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. 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 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 24 +/- 2 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 discarded via the drain port. Then the WAFs were filtered and added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless. The test was carried out without adjustment of the pH.
Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

The total reconstituted water hardness was approximately 250 mg.L-1 (as CaCO3)

Test temperature:

requirements: 20°C ± 2°C; should not vary by more than 1°C at the end of the test

pH:

requirements: between 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

requirements: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Loading rates: control, 11, 23, 52, 114 and 250 mg.L-1 .

Details on test conditions:

TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2.2
- Range finding study: Daphnids were exposed to the nominal loading rates 10, 50, 100 and 250 mg.L-1 and to a control.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 250 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Biological results: Details are given in the table 6.1.3/1 in the section "any other information on results incl. tables".
- Analytical results: Details are given in the table 6.1.3/2 in the section "any other information on results incl. tables".
- Water quality parameters and environmental conditions throughout the test: During the test the pH, oxygen concentrations and the temperature of the medium were within the optimal ranges for daphnids.

After 48 hours of exposure, no significant immobilisation of the test animals was observed at any of the tested loading rates, although daphnids swam slower than the ones from the control. Based on these results, the 24 and 48-hour EL50 values were therefore > 250 mg.L-1.

Results with reference substance (positive control):

On March 8, 2018 (KD18-001; most recent test), the 24h-EC50 was 1.31 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

None

Biological results

Table 1 : Acute immobilisation of daphnids after 24 and 48 hours in the final test.

Nominal concentration*(mg test item.L^-1)	Replicate	Number of daphnids exposed	Response at 24h		Response at 48h
			Number	Total %	Number	Total %
Control	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
11	1 2 3 4	5 5 5 5	0 0 0 0	0	1 0 0 0	5
23	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
52	1 2 3 4	5 5 5 5	0 0 0 0	0	0 0 0 0	0
114	1 2 3 4	5 5 5 5	0 0 0 0	0	1 0 0 0	5
250	1 2 3 4	5 5 5 5	0 0 0 0	0	1 0 0 0	5

* WAF prepared at the given loading rate.

Daphnids swam slower at all loading rates compared to daphnids from the control.

Analytical results

Table 2: Concentrations of the test item in test water - TOC analysis results

	TOC (mg.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0h)	t=24h Old	t=24h Fresh	End (t=48h)
Control	0.80	0.74	1.02	0.81
11	1.91	1.58	1.98	1.14
23	3.60	1.19	2.85	1.49
52	5.40	1.77	5.05	2.22
114	5.96	3.30	3.91	3.86
250	9.00	6.08	3.81	7.16

* WAF prepared at the given loading rate.

Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

Although every effort was made to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), TOC analyses indicate that few organic compounds were found in the WAFs, and that overall WAFs concentrations were not stable between the start and the end of each exposure period (t=0h-t24hOld and t=24hFresh-t=48h).

However, it was considered that test item concentrations were maintained as much as possible since test solutions were renewed at t=24h. Moreover, despite the analytical results, the study was considered as valid given the results from the range-finding test which are consistent with the final test outcome, in agreement with the Study Monitor. Therefore, due to the complex nature of the WAF and since the test item was a UVCB substance, the results were based on nominal loading rates.

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was > 250 mg.L-1.

Executive summary:

A study was performed to assess the acute toxicity of the test item BOSWELLIA SERRATA EXTRACT to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 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). The criterion measured was the EL₅₀ (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of test animals within a period of 48 hours.

Following a preliminary range-finding test, daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 11, 23, 52, 114 and 250 mg/L and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. 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).

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was > 250 mg/L.

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was > 100 mg.L-1.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L

Additional information

For that endpoint, an experimental study with the registered substance was available. The study was performed to assess the acute toxicity of the test item BOSWELLIA SERRATA EXTRACT to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended byCommission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). 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.

Following a preliminary range-finding test, daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 11, 23, 52, 114 and 250 mg.L^-1 and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. 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).

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 value was > 100 mg.L-1.