Absolute of Dipteryx odorata (Fabaceae) obtained from beans by organic solvents treatment and subsequent ethanol 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:

2020

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)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Chemical analysis: 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.

Analytical instrument:
- Shimadzu TOC-VWP
- Calibration range: 0-5 mg/L
- Control solutions: LOQ: 0.3 mg/L, QC1: 1 mg/L, QC2: 4 mg/L
- Limit of quantification LOQ: 0.3 mg/L

Vehicle:

no

Details on test solutions:

Based on the results of range-finding tests, test solutions used in the definitive test were prepared to obtain the following nominal loading rates (spaced by a factor of 1.35): 30.0, 41.0, 55.0, 74.0 and 100.0 mg.L-1.
The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared 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 completely filled with test water (with a minimum headspace). After heating of the test item sample to approx. 80°C for about 1 hour, 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 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 enhance the extraction of 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 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 through an 11 μm filter to remove any suspended undissolved particle and added into test vessels. After filling and introduction of daphnids the test vessels were sealed immediately. No small bubble was observed in the test vessels. The test solutions in test vessels were observed to be clear and colourless.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Sexe: Female
- Age at study initiation (mean and range, SD): < 24 hours old
- 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.
- Breeding conditions: 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.
- Number of daphnids 20 per treatment group (including controls), divided into 4 groups of 5 daphnids
- Loading 5 daphnids per vessel each completely filled with test solution and without headspace

Test type:

semi-static

Water media type:

other: reconstituted water

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

No

Hardness:

No data.

Test temperature:

The temperature of the test medium was situated between 19.6 and 20.5°C throughout the test.

pH:

7.47- 8.66

Dissolved oxygen:

7.36 - 9.09 mg/L

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal loading rates (spaced by a factor of 1.35): 30.0, 41.0, 55.0, 74.0 and 100.0 mg test item.L-1

Details on test conditions:

TEST SYSTEM
- Test type Semi-static (24h renewals), in order to prevent the dissipation of the test item from test solutions because of its volatility and the expected degradation of some constituents, and to maintain dissolved O2 concentrations as far as possible; at each renewal, a new series of clean and sterile test vessels was used.
- Test vessels 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Test water Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202
- No. of organisms per vessel: 5 daphnids / unit
- No. of vessels per concentration (replicates): four replicate unites for each five treatment concentrations
- No. of vessels per control (replicates): 4 replicates for the water control and 4 replicates for the solvent control.
- Reference item treatment : Potassium dichromate was tested at three different concentrations and with four replicates per concentration.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

72.97 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95%CL: 63.13 - 89.29

Details on results:

After 48 hours of exposure, immobilisation rates were 0% at 30 mg.L-1, 20% at 41 mg.L-1, 30% at 55 mg.L-1, 45% at 74.0 mg.L-1 and 75% at 100.0 mg.L-1. Thus, the highest loading rate without effect (NOELR) was 30.0 mg.L-1.

Results with reference substance (positive control):

On November 7, 2019 (KD19-003; most recent test), the 24h-EC50 was 1.243 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.

Reported statistics and error estimates:

Statistical analyses were performed by the computer program ToxRat.

Validity criteria of the study

Controls: In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 3 mg.L-1 in controls and test vessels

Water quality parameters and environmental conditions throughout the test

The results of measurement of pH and oxygen concentrations remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen: ≥ 3 mg.L-1 at the end of the test).

Furthermore, the temperature of the test medium was situated between 19.6 and 20.5°C throughout the test, and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

pH-values during the final test 

		Nominal concentration*(mg test item.L^-1)
		Control	30.0	41.0	55.0	74.0	100.0
Start t=0 h		8.66	8.33	8.27	8.20	8.12	8.00
t=24 h	Old	8.45	7.80	7.71	7.67	7.57	7.49
	Fresh	8.50	8.01	7.97	7.82	7.80	7.67
End t=48 h		8.52	7.80	7.81	7.56	7.48	7.47

* WAF prepared at the given loading rate.

Dissolved oxygen concentrations (mg.L^-1) during the final test

		Nominal concentration*(mg test item.L^-1)
		Control	30.0	41.0	55.0	74.0	100.0
Start t=0 h		8.99	8.78	8.75	8.74	8.74	8.68
t=24 h	Old	9.03	7.82	7.89	7.91	7.61	7.43
	Fresh	9.09	8.37	8.26	8.32	8.47	8.09
End t=48 h		8.80	7.73	7.50	6.80	6.94	7.36

* WAF prepared at the given loading rate.

All these dissolved-oxygen concentrations correspond to values > 60% of the air-saturation value.

 

Analytical results

Concentration of dissolved organic material in the controls 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).

TOC analyses showed that organic compounds in the loading rates were found to be relatively stable between the start and the end of first exposure period (t=0h-t24hOld), and a little less stable during the second part of the test (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. In any case, 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 and as Effective Loading Rate 50, according to the OECD No. 23.

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=0 h)	t=24 h Old	t=24 h Fresh	End (t=48 h)
Control	0.80	1.06	1.51	0.80
30.0	13.6	18.1	18.5	13.6
41.0	18.4	18.4	21.0	18.1
55.0	27.6	30.4	41.4	22.9
74.0	33.5	33.1	45.9	33.2
100.0	49.9	45.0	46.5	44.9

* WAF prepared at the given loading rate.

N.A.: not applicable

 

TOC contents coming from test item only (by subtracting from the one of the control) for each treatment and at each time of the test is presented next:

 

	TOC content from test item (mg.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0 h)	t=24 h Old	Relative loss to initial value (t=0 h - t=24 hold)(%)	t=24 h Fresh	End (t=48 h)	Relative loss to initial value (t=24 hfresh- t=48 h)(%)
30.0	12.80	17.04	-33	16.99	12.80	25
41.0	17.60	17.34	2	19.49	17.30	11
55.0	26.80	29.34	-9	39.89	22.10	45
74.0	32.70	32.04	2	44.39	32.40	27
100.0	49.10	43.94	11	44.99	44.10	2

* WAF prepared at the given loading rate.

 N.A.: not applicable

 

Validity criteria fulfilled:

yes

Conclusions:

The EL50 (48h) for the immobilization of Daphnia magna by the test substance is of 72.97 mg test item/L.
95%-CL : 63.13 - 89.29 mg test item/L

Executive summary:

A study was performed to assess the acute toxicity of test item TONKA BEAN ABSOLUTE 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 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the 48 h EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 30.0, 41.0, 55.0, 74.0 and 100.0 mg.L-1, and to a control. The immobilisation 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).

TOC analyses showed that organic compounds in the loading rates were overall stable during the test period. After 48 hours of exposure, immobilisation rates were 0% at 30 mg.L-1, 20% at 41 mg.L-1, 30% at 55 mg.L-1, 45% at 74.0 mg.L-1 and 75% at 100.0 mg.L-1.

The toxic effect of TONKA BEAN ABSOLUTE to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 72.97 mg.L-1.

Description of key information

A study was performed to assess the acute toxicity of test item TONKA BEAN ABSOLUTE 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 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23). The criterion measured was the 48 h EL50 (Median Effective Loading rate), a statistically derived loading rate which is expected to cause immobility in 50% of the daphnids within a period of 48 hours.

Based on the results of range-finding tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 30.0, 41.0, 55.0, 74.0 and 100.0 mg.L-1, and to a control. The immobilisation 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).

TOC analyses showed that organic compounds in the loading rates were overall stable during the test period. After 48 hours of exposure, immobilisation rates were 0% at 30 mg.L-1, 20% at 41 mg.L-1, 30% at 55 mg.L-1, 45% at 74.0 mg.L-1 and 75% at 100.0 mg.L-1.

The toxic effect of TONKA BEAN ABSOLUTE to the freshwater invertebrateDaphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. Under experimental conditions, and based upon nominal loading rates, the 48-hour EL50 value was estimated to be 72.97 mg.L-1.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

72.97 mg/L

Additional information

The influence of the test substance on immobilisation of Daphnia magna was investigated according to OECD Guideline 202, with GLP statement. Daphnids were exposed to the test item under static conditions, for 48 h.