Absolute of Nicotiana tabacum (Solanaceae) obtained from leaves by organic solvent 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:

From 2020-11-16 to 2020-11-27

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)

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Chemical analysis in the test solutions was performed at T=0h and T=72h.
At T=0h, solutions for analysis were taken direcly from starting solutions without algae. At T=72h, measurement swere performed in abiotic replicates.
The algae cells per unit volume were measured during the test at 24, 48 and 72 hours with a particle counter and/or under a microscope with a Malassez counting cell if need be.

Vehicle:

no

Details on test solutions:

According to the results of the preliminary test, we proposed to test five concentrations for this study: 10 – 32 – 102 – 328 and 1050 mg/L(loading rates).
The prepared samples were stirred 24 hours at 20 ± 2°C, darkness. After this step, the samples were filtrated on cellulose acetate filter (porosity: 0.45 µm) after a settling period of 10 minutes.
The solutions obtained were named WAF (Water Accommodated Fraction).

Test organisms (species):

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

Details on test organisms:

Freshwater unicellular green alga Pseudokirchneriella subcapitata of the chlorococcal order strain number CCAP 278.4 was used in this study.
The algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 to 24 °C

pH:

7.6 - 8

Nominal and measured concentrations:

0, 10, 32, 102, 328, 1050 mg test item /L (loading rates)

Details on test conditions:

A growing exponential phase inoculum of the freshwater unicellular alga Pseudokirchneriella subcapitata was added in order to have an initial concentration of 100 000 cells/mL at the start of the test:
- 100 mL of algae cultures were grown in 250 mL erlenmeyer flasks which were placed in a cooling incubator at a temperature in the range of 21 to 24°C controlled at ±2°C and under a constant shaking fixed at 120 rpm and a continuous illumination comprised between 4 440 and 8 880 lux.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

79 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

other: yield (migrated information)

Remarks on result:

other: CL: [47-136] mg/L

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

other: yield (migrated information)

Remarks on result:

other: CL: [0-24] mg/L

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

growth rate

Remarks on result:

other: CL: [21-44] mg/L

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

349 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

growth rate

Remarks on result:

other: CL: [260-498] mg/L

Results with reference substance (positive control):

Results obtained with the reference item potassium dichromate:
The last algal growth inhibition test performed with the reference item K2Cr2O7 (June 02, 2020) gave an ErC50(0-72h = 0.88 mg/L (0.82 – 0.95), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from Mai 2012 as mentioned in the OECD 201 Guideline) and in accordance with previous data obtained by the laboratory.

  pH values and observations

Nominal test item loading rate (mg/L)	pH at the start (biotic replicates)	pH after 72 hours (biotic replicates)
WAF Control	7.9	8.0
WAF 10	7.9	7.7
WAF 32	7.9	7.7
WAF 102	7.9	7.6
WAF 328	7.9	7.7
WAF 1050	8.0	7.8

 Algal growth inhibition

Nominal test item loading rate (mg/L)		[algae] *10^4 /mL at 24 hours			[algae] *10^4 /mL at 48 hours			[algae] *10^4 /mL at 72 hours
		Count.	Average	CV	Count.	Average	CV	Count.	Average	CV
WAF Control	a	2,6	2,8	12,1	15,6	16,1	7,6	122,7	128,8	4,6
	b	3,2			15,8			121,7
	c	3,2			14,7			128,7
	d	2,7			17,6			137,8
	e	2,4			15,3			129,8
	f	2,6			17,6			131,8
WAF 10	a	2,7	2,5	6,0	17,8	17,0	4,2	131,7	126,2	4,2
	b	2,4			16,8			125,8
	c	2,5			16,4			121,1
WAF 32	a	2,6	2,5	4,6	17,9	17,8	6,7	129,6	131,5	5,3
	b	2,6			19,0			139,2
	c	2,4			16,6			125,6
WAF 102	a	2,6	2,6	2,2	11,9	11,6	2,3	31,6	25,5	20,9
	b	2,6			11,5			22,8
	c	2,5			11,4			22,0
WAF 328	a	2,4	2,4	2,4	3,1	3,0	3,9	11,5	11,4	6,1
	b	2,4			2,9			10,7
	c	2,5			2,9			12,1
WAF 1050	a	2,3	2,2	2,6	2,4	2,4	0,0	5,9	5,7	4,6
	b	2,2			2,4			5,4
	c	2,2			2,4			5,8

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of Total Carbon in solutions containing the test item “TOBACCO ABSOLUTE”, the measured concentration are well maintained within ± 20%t of the initial measured Total Organic Carbon (- 0.4% to +15.5%).
Consequently, the test item “TOBACCO ABSOLUTE” can be considered as stable in the testing conditions after 72 hours of exposure.

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL50-(0-72h) = 349 mg/L [260 – 498], value expressed in nominal test item loading rate.

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL10-(0-72h) =28 mg/L [21 – 44], value expressed in nominal test item loading rate.

Executive summary:

The aim of this study was to determine the effects of the test item “TOBACCO ABSOLUTE” on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The algae were exposed to Water Accommodated Fractions (WAFs) of the test item, following the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

There was no deviation and no amendment during this study.

Following the results of preliminary tests (non-GLP), a final test at five nominal test item loading rates was performed, in order to evaluate the significant effect on the test organism. The effects on the algal growth were determined by cell counting daily for 72 hours.

The concentration of “TOBACCO ABSOLUTE” in the control and the solutions was checked from WAF algal solutions by Carbon Analyzer during the study at the start (T=0h) and at the end of the test (T=72h).

 

Test item: TOBACCO ABSOLUTE

Test organisms: Pseudokirchneriella subcapitata

Exposure duration: 72 hours

Test concentrations: Control – 10 – 32 – 102 – 328 and 1050 mg/L

Incubator: Temperature 22.8 °C –  23.2°C ; shaking at 120 rpm; light intensity: 5928 Lux

Measurement of cell growth: Beckman Coulter Z2 and Malassez counting cell

Validity criteria: In the control, the increase of the biomass during 72 hours was determined to be higher than a factor of 16.The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.

 

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL50-(0-72h) = 349 mg/L [260 – 498], value expressed in nominal test item loading rate.

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL10-(0-72h) =28 mg/L [21 – 44], value expressed in nominal test item loading rate.

Description of key information

The aim of this study was to determine the effects of the test item “TOBACCO ABSOLUTE” on the growth of the unicellular green alga Pseudokirchneriella subcapitata, according to the OECD 201 guideline (March 2006, corrected July 2011).

The algae were exposed to Water Accommodated Fractions (WAFs) of the test item, following the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

There was no deviation and no amendment during this study.

Following the results of preliminary tests (non-GLP), a final test at five nominal test item loading rates was performed, in order to evaluate the significant effect on the test organism. The effects on the algal growth were determined by cell counting daily for 72 hours.

The concentration of “TOBACCO ABSOLUTE” in the control and the solutions was checked from WAF algal solutions by Carbon Analyzer during the study at the start (T=0h) and at the end of the test (T=72h).

 

Test item: TOBACCO ABSOLUTE

Test organisms: Pseudokirchneriella subcapitata

Exposure duration: 72 hours

Test concentrations: Control – 10 – 32 – 102 – 328 and 1050 mg/L

Incubator: Temperature 22.8 °C –  23.2°C ; shaking at 120 rpm; light intensity: 5928 Lux

Measurement of cell growth: Beckman Coulter Z2 and Malassez counting cell

Validity criteria: In the control, the increase of the biomass during 72 hours was determined to be higher than a factor of 16.The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35% and 7% in the control.

The validity criteria were successfully fulfilled.

 

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL50-(0-72h) = 349 mg/L [260 – 498], value expressed in nominal test item loading rate.

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “inhibition of growth rate” indicate an ErL10-(0-72h) =28 mg/L [21 – 44], value expressed in nominal test item loading rate.

Key value for chemical safety assessment

EC50 for freshwater algae:

349 mg/L

EC10 or NOEC for freshwater algae:

28 mg/L

Additional information

An experimental study, conducted in accordance with conditions recommended in OECD guideline and under GLP, is on-going. It should be considered as a key study. The result should be retained as key data.