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

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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

Remarks:

Certificate signed on November 15, 2018

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and all test solutions (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test.
Cell numbers were counted daily by microscope using a counting chamber.

Vehicle:

no

Details on test solutions:

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 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 headspace). 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, followed by a sterile filtration (0.22 µm), and were added into test vessels containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum of headspace. All the preparation was performed under strict sterile conditions. At the start of the test, the test solutions in all test vessels were observed to be clear and colourless. The test was carried out without adjustment of the pH.
- Test item: Based on the results of a range-finding test presented in Appendix VI, test solutions used in the definitive test were prepared to obtain the following nominal loading rates (spaced by a factor of approximately 1.90): 8, 15, 28, 53, 100 and 190 mg.L-1.
- Control:Test water without test substance but treated in the same way as the test substance solutions.
- Replicates: 6 controls and 3 replicates of each test concentration for counting. Moreover, additional (abiotic) replicates of each treatment without algae were prepared for chemical analyses.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, CCAP 278/4
Origin: 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.

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 22.2 and 23.6°C throughout the test (average value: 23.5°C).
The temperatures complied with the study requirements (23°C ± 2°C)

pH:

Control: 8.45 at t=0h and 9.73 at t=72h
WAFs: min= 8.25, max= 9.82
The pH level remained within the prescribed conditions

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Nominal concentrations: 0, 8, 15, 28, 53, 100 and 190 mg test item.L-1 (Loading rates)

Details on test conditions:

Test water: Original medium from OECD TG 201. The mixture was made up to 1000 mL with sterilised water. The pH of this solution was approximately in the range of 8.1 ± 0.2. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water to insure a satisfactory CO2 supply for the algal growth (for all treatments and inoculum suspension): 7 mL of NaHCO3 were 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.

Light regime: Continuous illumination
Light intensity: 4 440-8 880 lux
Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in test water at a cell density of 10 000 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.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

55.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

growth rate

Remarks on result:

other: 95%-CL: 52.76 - 58.53 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

95.66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

growth rate

Remarks on result:

other: 95%-CL: 89.80 - 101.70 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

44.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

other: Yield

Remarks on result:

other: 95%-CL: 31-95 - 62.14 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

65.23 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

other: Yield

Remarks on result:

other: 95%-CL: 42.44 - 98.33 mg/L

Details on results:

Cell density in control: 119.6-fold increase within 72 hours.
Coefficient of variation: 1. The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 33.2% in 72 hours.
2. The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 5.0%.

Thus the validity criteria were respected in this study.

Results with reference substance (positive control):

On May 29, 2020 (KA20-002; most recent test), the 72h-EC50 was 0.928 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-ErC50: 0.65 mg.L-1 to 1.73 mg.L-1).

Reported statistics and error estimates:

The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values).

Physico-chemical parameter values throughout the test

The temperature in the incubator was situated between 22.2 and 23.6°C throughout the test (average value: 23.5°C). The mean light intensity was 4872 lux (range: 4731-5083 lux). Thus, all test conditions remained within the ranges prescribed by the study plan (pH: not varying by more than 1.5 units at the end of the test in the control; temperature: 23°C ± 2°C, constant within 2°C; light intensity: 4440-8880 lux and not varying more than ± 15% from the average light intensity over the incubation area).

pH-values during the final test

	Nominal concentration*(mg test item.L^-1)
	Control	8	15	28	53	100	190
Start t=0 h	8.45	8.36	8.34	8.29	8.30	8.25	8.25
End t=72 h	9.73	9.82	9.56	9.56	9.30	8.59	8.35

* WAF prepared at the given loading rate.

 

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.

TOC analysis showed that WAFs concentrations were stable between the start and the end of the test,within the ± 20% of the initial TOC concentrations values. It should be recalled that the study was carried out using WAFs of a natural complex substance made of several constituents with different stabilities, solubilities and behaviours in aqueous solutions during testing.Therefore, and since the test item was a UVCB substance, the results were based on nominal loading rates.

A first definitive test has been performed with another test item sample. Results were not validated due to the presence of microorganisms in test flasks from t=48 h. TOC analysis at t=0 h obtained during this test are presented in the Analytical Report for information. A comparison of TOC contents of the WAFs between the previous and the new definitive test shows that the 0.22 µm filtration step after WAFs preparation has a limited impact on final WAFs concentrations.

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)	Relative loss to initial value (t=0h - t=72h) (%)
Control	0.80	0.85	-6
8	4.53	4.52	0
15	8.26	7.95	4
28	13.9	14.2	-2
53	22.4	23.2	-4
100	47.9	49.2	-3
190	91.0	88.5	-3

* WAF prepared at the given loading rate.

 

Results of the determination of TOC analysis (mg.L^-1) at t=0 h - Previous definitive test (invalidated), without sterile filtration step (0.22 µm) 

Nominal concentration* (mg test item.L^-1)	Start (t=0h)
Control	0.33
8	5.15
15	9.43
28	14.3
53	28.4
100	40.5
190	61.6

* WAF prepared at the given loading rate.

Biological results

Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴)

	Replicate	Nominal concentration*  (mg test item.L^-1)
		Control	8	15	28	53	100	190
t=24 h	1	2.0	2.4	4.0	2.4	3.2	1.6	0.8
	2	2.4	4.0	2.8	2.0	3.6	0.4	0.4
	3	3.6	2.0	1.6	2.8	2.0	2.0	0.4
	4	3.2
	5	4.0
	6	4.0
	Mean	3.2	2.8	2.8	2.4	2.9	1.3	0.5
	Std. Dev.	0.84	1.06	1.20	0.40	0.83	0.83	0.23
t=48 h	1	18.0	17.2	12.8	13.2	11.2	2.8	1.6
	2	20.8	17.6	14.0	12.8	14.4	1.6	0.8
	3	22.8	16.0	15.6	11.2	10.0	4.0	0.8
	4	19.6
	5	20.0
	6	20.4
	Mean	20.3	16.9	14.1	12.4	11.9	2.8	1.1
	Std. Dev.	1.57	0.83	1.40	1.06	2.27	1.20	0.46
t=72 h	1	66.0	53.6	49.6	45.2	35.2	4.0	1.2
	2	42.0	46.8	38.4	43.2	47.2	3.6	0.8
	3	78.0	49.2	47.2	37.2	36.0	4.0	0.4
	4	46.0
	5	60.0
	6	66.8
	Mean	59.8	49.9	45.1	41.9	39.5	3.9	0.8
	Std. Dev.	13.61	3.45	5.90	4.16	6.71	0.23	0.40

* WAF prepared at the given loading rate.

At test start 5000 algal cells.mL^-1 were incubated; 6 replicates of the controls and 3 replicates of each test concentration.

The mean andstandard deviationvalues were taken from the computer program ToxRat.

Std. Dev.: standard deviation.

Yield of algal cells during the final test

	Replicate	Nominal concentration*  (mg test item.L^-1)
		Control	8	15	28	53	100	190
t=24 h	1	1.5	1.9	3.5	1.9	2.7	1.1	0.3
	2	1.9	3.5	2.3	1.5	3.1	-0.1	-0.1
	3	3.1	1.5	1.1	2.3	1.5	1.5	-0.1
	4	2.7
	5	3.5
	6	3.5
	Mean	2.7	2.3	2.3	1.9	2.4	0.8	0.0
	Std. Dev.	0.84	1.06	1.20	0.40	0.83	0.83	0.23
	% Red.	-	14.8	14.8	29.6	9.9	69.1	98.8
t=48 h	1	17.5	16.7	12.3	12.7	10.7	2.3	1.1
	2	20.3	17.1	13.5	12.3	13.9	1.1	0.3
	3	22.3	15.5	15.1	10.7	9.5	3.5	0.3
	4	19.1
	5	19.5
	6	19.9
	Mean	19.8	16.4	13.6	11.9	11.4	2.3	0.6
	Std. Dev.	1.57	0.83	1.40	1.06	2.27	1.20	0.46
	% Red.	-	16.9	31.0	39.8	42.5	88.4	97.1
t=72 h	1	65.5	53.1	49.1	44.7	34.7	3.5	0.7
	2	41.5	46.3	37.9	42.7	46.7	3.1	0.3
	3	77.5	48.7	46.7	36.7	35.5	3.5	-0.1
	4	45.5
	5	59.5
	6	66.3
	Mean	59.3	49.4	44.6	41.4	39.0	3.4	0.3
	Std. Dev.	13.61	3.45	5.90	4.16	6.71	0.23	0.40
	% Red.	-	16.8	24.8	30.2	34.3	94.3	99.5

* WAF prepared at thegiven loading rate.

Values were extracted from the computer program ToxRat.

% Red.: %Reduction in yield relative to the control determined by ToxRat.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 5. Mean specific growth rate inP. subcapitataduring the final test.

	Replicate	Nominal concentration*  (mg test item.L^-1)
		Control	8	15	28	53	100	190
t=0 h - t=24 h	1	1.386	1.569	2.079	1.569	1.856	1.163	0.470
	2	1.569	2.079	1.723	1.386	1.974	-0.223	-0.223
	3	1.974	1.386	1.163	1.723	1.386	1.386	-0.223
	4	1.856
	5	2.079
	6	2.079
	Mean	1.824	1.678	1.655	1.559	1.739	0.775	0.008
	Std. Dev.	0.2867	0.3593	0.4619	0.1684	0.3110	0.8720	0.4002
	% Inh.	-	8.0	9.3	14.5	4.7	57.5	99.6
t=0 h - t=48 h	1	1.792	1.769	1.621	1.637	1.555	0.861	0.582
	2	1.864	1.781	1.666	1.621	1.680	0.582	0.235
	3	1.910	1.733	1.720	1.555	1.498	1.040	0.235
	4	1.834
	5	1.844
	6	1.854
	Mean	1.850	1.761	1.669	1.604	1.578	0.828	0.351
	Std. Dev.	0.0387	0.0249	0.0495	0.0437	0.0933	0.2309	0.2001
	% Inh.	-	4.8	9.8	13.3	14.7	55.3	81.1
t=0 h - t=72 h	1	1.628	1.558	1.532	1.501	1.418	0.693	0.292
	2	1.477	1.513	1.447	1.486	1.516	0.658	0.157
	3	1.683	1.530	1.516	1.436	1.426	0.693	-0.074
	4	1.507
	5	1.596
	6	1.632
	Mean	1.587	1.534	1.498	1.475	1.453	0.681	0.125
	Std. Dev.	0.0793	0.0229	0.0452	0.0340	0.0544	0.0203	0.1852
	% Inh.	-	3.4	5.6	7.1	8.4	57.1	92.1

* WAF prepared at the given loading rate.

Values were extracted from the computer program ToxRat.

% Inh.: %Inhibition of growth rate relative to the control determined by ToxRat.

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Under the experimental conditions and based upon nominal loading rates, the 72-hour EL50 for the parameters growth rate and yield were determined to be 95.660 mg.L-1 and 65.227 mg.L-1, respectively. The 72-hour EL10 for growth rate was 55.568 mg.L-1 and for yield was 44.559 mg.L-1. The 72-hour NOELR value for growth rate was 15.000 mg.L-1 and for yield was 8.000 mg.L-1.

Executive summary:

A study was performed to assess the test item TONKA BEAN ABSOLUTE 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, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (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 of8, 15, 28, 53, 100 and 190 mg.L^-1 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 analysis of Total Organic Carbon (TOC) at the start and the end of the test.

The analytical results of this test showed that WAF concentrations were stable between the start and the end of the test, within the ± 20% of the initial TOC concentrations values.

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based up on nominal loading rates, the 72 -hour EL₅₀ for the parameters growth rate and yield were determined to be 95.660 mg.L^-1and 65.227 mg.L^-1, respectively. The 72 -hour EL₁₀ for growth rate was 55.568 mg.L^-1and for yield was 44.559 mg.L^-1. The 72 -hour NOELR value for growth rate was 15.000 mg.L^-1and for yield was 8.000 mg.L^-1.

Description of key information

Under the experimental conditions andbased upon nominal loading rates,the 72 -hour EL₅₀for the parameters growth rate and yield were determined to be 95.660 mg.L^-1and 65.227 mg.L^-1, respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

95.66 mg/L

EC10 or NOEC for freshwater algae:

55.57 mg/L

Additional information

For that endpoint, a study on the registered substance was available.

A study was performed to assess the test item TONKA BEAN ABSOLUTE 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, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 and with the “Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (OECD No. 23).

Following a preliminary range-finding test, algal cells were exposed toWater Accommodated Fractions (WAFs)of the test itemover a range of nominal loading values of 8, 15, 28, 53, 100 and 190 mg.L^-1and 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 analysis of Total Organic Carbon (TOC)at the startand the end of the test.

The analytical results of this test showed that WAF concentrations were stablebetween the start and the end of the test,within the ± 20% of the initial TOC concentrations values.

The toxic effect of test item to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based upon nominal loading rates,the 72 -hour EL₅₀ for the parameters growth rate and yield were determined to be 95.660 mg.L^-1and 65.227 mg.L^-1, respectively. The 72 -hour EL₁₀for growth rate was 55.568 mg.L^-1and for yield was 44.559 mg.L^-1. The 72 -hour NOELR value for growth rate was 15.000 mg.L^-1and for yield was 8.000 mg.L^-1.