Nicotine

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:

2014-11-10 to 2014-11-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed in accordance with standard test protocols (OECD 201 resp. EU C.3) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1 - 100 mg/L

Vehicle:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular freshwater green alga
- Genus: Desmodesmus
Species subspicatus
SAG Strain Number 86.81
Taxonomic position Chlorophyta - Chlorophyceae
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen)
- Method of cultivation: permanent culture

ACCLIMATION
- Acclimation period: 72 h
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21.6 - 23.6 °C

pH:

8.3-8.4

Nominal and measured concentrations:

Nominal concentrations: 1.0 / 3.2 / 10 / 32 / 100 mg/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: closed glass flasks total volume 65 mL
- Initial cells density: 5390 cells/mL
- Control end cells density: 352865 cells/mL
- No. of vessels per concentration (replicates): three replicates for each treatment
- No. of vessels per control (replicates): six replicates for the control
- No. of vessels per vehicle control (replicates): not applicable

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: To avoid a strong pH increase and CO2 shortage in the closed system, additional 6 mL sodium hydrogen carbonate solution (50 g/L) was added to 900 mL nutrient medium. Then, pH was adjusted to 7.0 (± 0.2) and the solution was filled up to 1000 mL with nutrient medium
- Photoperiod:continuous lightening
- Light intensity and quality: 5800 Lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: photometric measurement of optical density every 24 h
- Chlorophyll measurement: no


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: no
- Test concentrations: 1.0, 3.2, 10, 32, 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS No. 7778-50-9)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 25 - 55

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 9.8 - 17

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 2.7 - 11

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 3.3 - 7.9

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

Parameter: 72h ErC50 Value: 0.99 mg/L 95%-confidence-interval: 0.94 – 1.1 mg/L Range of interlaboratory test (EU C.3):0.60 – 1.03 mg/L
Parameter: 72h EyC50 Value: 0.55 mg/L 95%-confidence-interval: 0.52 – 0.58 mg/L Range of interlaboratory test (EU C.3): none stated

Reported statistics and error estimates:

The values for the Yield and the growth rate were used for the assessment of observed effect levels.
In order to select a suitable test for significance, it was checked whether equality of variance was given.
Equality of variance was tested using the following equation:
F = s1^2 / s2^2
with s1 being the greater variance and s2 the smaller one.
The calculated value F was compared with the F-test table (level of significance 95%). If the calculated value was smaller than the tabular value, equality of variance was given.
If equality of variance was given, the t-test was used; else, the WEIR test was used.

Cell Numbers Main Study

Nom. Conc. in mg/L	Repl.	Cell Number/mL
		0 h	24 h	48 h	72 h
0	1	5390	25872	88396	323400
	2	5390	28028	91630	336336
	3	5390	31262	100254	369754
	4	5390	26950	99176	403172
	5	5390	28028	98098	315854
	6	5390	29106	93786	368676
	Mean	5390	28208	95223	352865
	SD	0	1857	4707	33398
1	1	5390	29106	95942	350350
	2	5390	29106	74382	194040
	3	5390	33418	86240	299684
	Mean	5390	30543	85521	281358
	SD	0	2490	10798	79750
3.2	1	5390	26950	97020	366520
	2	5390	28028	108878	413952
	3	5390	29106	98098	269500
	Mean	5390	28028	101332	349991
	SD	0	1078	6557	73631
10	1	5390	30184	116424	211288
	2	5390	33418	118580	215600
	3	5390	31262	111034	210210
	Mean	5390	31621	115346	212366
	SD	0	1647	3887	2852
32	1	--	--	--	--
	2	5390	21560	20482	25872
	3	5390	20482	19404	24794
	Mean	5390	21021	19943	25333
	SD	0	762	762	762
100	1	5390	16170	16170	17248
	2	5390	15092	16170	26950
	3	5390	18326	20482	20482
	Mean	5390	16529	17607	21560
	SD	0	1647	2490	4940

SD = Standard Deviation

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met.

Conclusions:

The study was performed using five concentrations of 1.0 / 3.2 / 10 / 32 / 100 mg/L nominal concentration. Significant inhibition of algal growth was observed at the following concentrations: 100 / 32 / 10 mg/L
Because in the two lowest concentrated treatments no significant inhibition of algal growth could be observed, the lowest test item concentration was not used for evaluation of the results.
At the start and at the end of the test, the content of the test item in the test solutions was determined using HPLC. The measured concentrations in the treatments 3.2 / 10 / 32 / 100 mg/L lay between 96% and 103% of the nominal concentration at the beginning of the test and between 88% and 98% of the nominal concentration at the end of the test.
Therefore, the determination of the results was based on the nominal concentrations.
The EC50 values of potassium dichromate were determined in a separate reference test. For the estimation of the EC50 values of the positive control, the fits showed sufficient statistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory.
All validity criteria were met.
No observations were made which might cause doubts concerning the validity of the study outcome.

Executive summary:

The study was performed using five test item concentrations of 1.0 / 3.2 / 10 / 32 / 100 mg/L (nominal concentration). Incubation time (test system Desmodesmus subspicatus) was 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the corresponding solution at 440 nm every 24 hours with a spectral photometer. The cell density of the cultures was calculated based on the correlation curve between the adsorption and the cell density of the cultures determined with a cell counter. Growth rate µ and yield[1] were determined from the cell densities at the respective observation times.

Significant inhibition of algal growth was observed at the following test item concentrations:

100 / 32 / 10 mg/L

Because in the two lowest concentrated treatments no significant inhibition of algal growth could be observed, the lowest test item concentration was not used for evaluation of the results.

At the start and at the end of the test, the content of the test item in the test solutions was determined by HPLC. The measured concentrations in the treatments 3.2 / 10 / 32 / 100 mg/L lay between 96% and 103% of the nominal concentration at the beginning of the test and between 88% and 98% of the nominal concentration at the end of the test.

Therefore, the determination of the results was based on the nominal concentrations.

The EC50 values of potassium dichromate were tested in a separate reference test. The values lay within the normal range of the laboratory.

The following results for the test item nicotine were determined:

Endpoint	NOEC	LOEC	EC10	EC50
Growth Rate	3.2 mg/L	10 mg/L	5.2 mg/L	37 mg/L
Yield	3.2 mg/L	10 mg/L	5.3 mg/L	11 mg/L

[1]Yield (according to OECD Guideline 201) is defined as the difference between the biomass at the end and the biomass at the start of the exposure period.

Description of key information

One experimental study available in Desmodesmus subspicatus. The overall quality of the study is considered very high (Klimisch 1). The study is GLP compliant and appropriate for risk assessment, therefore no further testing is needed.

Key value for chemical safety assessment

EC50 for freshwater algae:

11 mg/L

EC10 or NOEC for freshwater algae:

5.2 mg/L