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

From 2020-10-14 to 2020-11-06

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

Analytical monitoring:

yes

Details on sampling:

Analysis were performed by Total organic Carbon measurements at initial time (T=0h) and at final time (T=48h). At T0h, All the solutions were analysed taken directly from the starting solutions without daphnids. At T48h, all the solutions were sampled from test tubes containing daphnids.

Observations of the daphnid’s immobility and behaviour were done after 24 and 48 hours in Control and exposed groups.

Any sign of stress observed on the daphnids after 24 and 48 hours of exposure to the test item were reported.
pH and dissolved oxygen were measured at the start and at the end of the test (including Controls).

Details on test solutions:

Seven test solutions of required concentrations were prepared by direct addition (by weighing) from the test item with the daphnids medium.
The prepared samples were stirred during 24 hours at 20 ± 2°C, darkness. After this step, the samples were filtrated on cellulose acetate filter (porosity: 0.45 µm – batch A26689264).
The test solutions were then filtered through a membrane filter: solutions obtained are named WAF (Water Accommodated Fraction).

Each volume of the daphnids test tubes was approximately 10 mL.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna Straus, clone number 5, from at least the third generation (i.e. the third brood) obtained with a non-cyclic parthenogenesis reproduction.
The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.
Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 µm and 800 µm (corresponding to daphnids less than 24 hours old).

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

None

Post exposure observation period:

No

Test temperature:

20.0 - 20.2°C

pH:

The pH of the solutions did remain in the range 6 – 9, no pH adjustment was necessary.

Nominal and measured concentrations:

Loading rates: 0, 5, 10, 18, 32, 60, 105, 190 mg test item /L

Details on test conditions:

The protocol consisted of exposing young daphnids at seven test item concentrations over a period of 48 hours in static conditions. Immobilisation of daphnids was recorded at 24 hours and 48 hours and compared with Control values.
The test was carried out under static conditions, which means that the test solution were not renewed at 24h.
The test contained a minimum of 32 glass test tubes as follow:
• Control: 4 replicates
• Loading rate 5 mg/L: 4 replicates
• Loading rate 10 mg/L: 4 replicates
• Loading rate 18 mg/L: 4 replicates
• Loading rate 32 mg/L: 4 replicates
• Loading rate 60 mg/L: 4 replicates
• Loading rate 105 mg/L: 4 replicates
• Loading rate 190 mg/L: 4 replicates

The test was performed in the dark between 18 and 22°C but constant within ±1°C. Daphnids were not fed during the test period.
Observations of the daphnid’s immobility and behaviour were done after 24 and 48 hours in Control and exposed groups.

Any sign of stress observed on the daphnids after 24 and 48 hours of exposure to the test item were reported.
pH and dissolved oxygen were measured at the start and at the end of the test (including Controls).

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

135 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Loading rates

Basis for effect:

mobility

Remarks on result:

other: Cl : 105 -198

Results with reference substance (positive control):

The last toxic response obtained on the reference item K2Cr2O7 (November 12, 2020) indicates an EC50-24h of 1.43 mg/L (1.30 – 1.57), indicating that the daphnids sensitivity is correct (within the accepted interval of 0.6 – 2.1 mg/L fixed by the International Standard ISO 6341 December 2012 as mentioned in the OECD 202 Guideline) and in accordance with historical data obtained by the laboratory.

pH and dissolved oxygen values during the final test

Nominal test item loading rate (mg/L)	pH
	T0	T 48h
WAF Control	7.8	7.6
WAF 5	7.9	7.6
WAF 10	7.9	7.5
WAF 18	7.8	7.5
WAF 32	7.8	7.5
WAF 60	7.8	7.5
WAF 105	7.8	7.4
WAF 190	7.8	7.2

Nominal test item loading rate (mg/L)	O2 (mg/L)
	T0  (Air: 9.5 mg/L)	T48 h  (Air: 9.3 mg/L)
WAF Control	9.3	9.1
WAF 5	9.2	9.0
WAF 10	9.1	8.8
WAF 18	9.2	8.8
WAF 32	9.1	8.7
WAF 60	9.0	8.7
WAF 105	8.9	8.6
WAF 190	8.9	8.7

Validity criteria fulfilled:

yes

Conclusions:

The results obtained for the test item “TOBACCO ABSOLUTE” on the toxicological endpoint “immobilisation” of daphnids give an EL50-48h = 135 mg/L [105 - 198], based on test item loading rate.

Executive summary:

The aim of this study was to determine the acute toxicity of the test item “TOBACCO ABSOLUTE” on the daphnids Daphnia magna, according to the OECD guideline 202, in static-conditions.

This study was carried out in accordance with the study plan no.20/1044F/d dated November 19, 2020.

There was no deviation and no amendment during this study.

 

Following the results of preliminary tests (non GLP), a final test, at seven nominal test item concentrations was performed, in order to evaluate the concentration which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EL₅₀-24h and EL₅₀-48h, relative to tested loading rates.

The immobility of the daphnids was determined in a closed static 48-hours test by visual observation after 24 and 48 hours.

The concentration of “TOBACCO ABSOLUTE” in the Control and the solutions was checked by Carbon analyser.

Observation time	24h	48h
EL10	Between 105 and 190 mg/L	52 mg/L [34 - 68]
EL50	≥190 mg/L	135 mg/L [105 - 198]

Description of key information

The aim of this study was to determine the acute toxicity of the test item “TOBACCO ABSOLUTE” on the daphnids Daphnia magna, according to the OECD guideline 202, in static-conditions.

This study was carried out in accordance with the study plan no.20/1044F/d dated November 19, 2020.

There was no deviation and no amendment during this study.

 

Following the results of preliminary tests (non GLP), a final test, at seven nominal test item concentrations was performed, in order to evaluate the concentration which immobilise 50% of the daphnids exposed to the test item after 24 and 48 hours, reported as EL₅₀-24h and EL₅₀-48h, relative to tested loading rates.

The immobility of the daphnids was determined in a closed static 48-hours test by visual observation after 24 and 48 hours.

The concentration of “TOBACCO ABSOLUTE” in the Control and the solutions was checked by Carbon analyser.

Observation time	24h	48h
EL10	Between 105 and 190 mg/L	52 mg/L [34 - 68]
EL50	≥190 mg/L	135 mg/L [105 - 198]

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

135 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.