2-vinylpyridine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2002 - 26 March 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

: Ministry of the Environment, Japan(2001)

Test material

Test material information

Constituent 1

Reference substance name:

2-vinylpyridine

EC Number:

202-879-8

EC Name:

2-vinylpyridine

Cas Number:

100-69-6

Molecular formula:

C7H7N

IUPAC Name:

2-ethenylpyridine

Constituent 2

Reference substance name:

2-ethenylpyridine

IUPAC Name:

2-ethenylpyridine

Details on test material:

- Physical state: Red-brown and transparent liquid
- Analytical purity: 99.2 % (Capillary Column GC)
- Impurities: No data
- Lot/batch No.: ELE1957
- Storage condition of test material: in cool and dark place
- Stability under the storage condition: The test substance was stable under the storage conditions, because IR spectra of the test substance before and after the study were identical.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Frequency: at the start and the end of the exposure
- Sampling:
At the start of the exposure: Samples were taken from the container for preparation in each exposure level
At the end of the exposure: Samples were taken from each of the three vessels per concentration, and removed algae by centrifugation.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was weighed and dissolved in medium to prepare a solution of 1000mg/L. The solution was filtered by pressure filtration with a membrane filter of 0.45 μm, and the filtrate was used as a stock solution.
Appropriate volume of the stock solution and the medium were mixed in the vessel to prepare the test solution. The test solution was divided into each test vessel.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC22662
- Source: Pseudokirchneriella subcapitata which originally came from the American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland 20852-1776 U.S.A) and have been cultured in the testing laboratory were used.

OTHER
- Confirming the reproducibility of the test conditions:
Reference substance: pottasium dichromate, reagent chemical, Wako Pure Chemical Industries, Ltd.
Results: 72h EbC50: 0.332 mg/L (within the acceptable range* to background data in this laboratory)
*: mean ± 2S.D.: 0.296 - 0.442 mg/L [mean± S.D.: 0.369 ± 0.036 mg/L (n=22)]
- Reason for selection of species: Species recommended in the test guideline

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

No data

Test temperature:

22.9 - 23.3 °C (23 ± 2 °C)

pH:

At the start of exposure: 7.7 - 7.9
At the end of exposure: 7.9 - 9.5

Dissolved oxygen:

No data

Nominal and measured concentrations:

Nominal concentrations: 100, 55.6, 30.9, 17.1, 9.53 mg/L (geometric series with a factor of 1.8, basis on the range finding study)
Measured concentrations: 87.4, 48.1, 27.2, 15.0, 8.25 mg/L ( % of the nominal: 98.2 -102 % (0h) and 72.7 -78.1 % (72h))

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: erlenmeyer flask with silicon rubber plug
- Size: 500 mL
- Fill volume: 100 mL
- Incubator: Incubator with temperature control, continuous shaking(approximately 100rpm) and continuous illumination, maintained the uniform light intensity
- Initial cells density: 1.0 × 10^4 cells/mL
- Control end cells density: 102 × 10^4 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 3

GROWTH MEDIUM
- Standard medium used: yes (Sterile OECD medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous
- Light intensity and quality: Fluorescence light (4,100 - 4,200 lux)

OBSERVATION
- Cell density: Counted with a particle counter at 24, 48, and 72 h
- Cell condition: Under microscope at the end of the exposure
- Test solutions: Appearance of the test solutions was observed before and after the exposure. The pH of the test solution was measured at the start and the end of the exposure. For the measurement of pH, another solution sampled from the vessel for preparation was used at the start of the exposure and one test vessel in each test level was used for the measurement at the end of the exposure. The temperature and light intensity in the incubator were measured once a day during the exposure.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

50.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Dose descriptor:

other: 24-48h EC50

Effect conc.:

64.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Dose descriptor:

other: 24-72h EC50

Effect conc.:

64.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 5

Dose descriptor:

other: 24-48h NOEC

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Dose descriptor:

other: 24-72h NOEC

Effect conc.:

30.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

EC50
EbC50 (0 - 72h): 50.8 mg/L
ErC50 (24 -48h): 64.3 mg/L
ErC50 (24 -72h): 64.4 mg/L

NOEC
NOECb (0 - 72h): 30.9 mg/L
NOECr (24 - 48h): 30.9 mg/L
NOECr (24 - 72h): 30.9 mg/L

TEST SOLUTION
Appearance:
At the preparation of the test solutions: colorless and clear
At the end of the exposure:
- 30.9 -9.53 mg/L: green due to the algae growth
- 55.6 mg/L: pale green due to the algae growth
- 100 mg/L: colorless and clear

CELL GROWTH
- Control group, 30.9 mg/L: Exponential growth: Increased to 101 or more times of initial cell density at the end of exposure, meets the validity of the test (at least 16 times growth)
- 9.53, 17.1 mg/L: A little more growth than control groups
- 55.6 mg/L: Exponential growth although inhibition has shown
- 100 mg/L: Significantly inhibited

CELL CONDITION
- 9.53, 17.1 and 30.9 mg/L: Same as control groups
- 55.6 mg/L: Swelling
- 100 mg/L: Swelling (in many cells)


There were no factors which might have affected the reliability of the test.

Reported statistics and error estimates:

The results of the study were estimated by nominal concentration because the measured concentrations were kept within ±20% of nominal concentrations during the exposure.

ESTIMATION OF EC50
The percentage inhibition in each group was plotted on semilogarithmic graph against the corresponding concentration.
Linear regression analysis (least square method) was carried out using the data within the range showing linearity to estimate the EC50, which was estimated from the intercept of the regression line with the parallel drawn to the abscissa at 50% of inhibition.
The EC50 was denoted as:
based on area under the growth curve: EbC50 (0 - 72h)
based on growth rate: ErC50 (24 - 48h) or (24 - 72h)

ESTIMATION OF NOEC
Homogeneity of variance for the data: Bartlett’s test
Estimation of the significant difference in comparison with the control:
- Area under the growth curve: Kruskal-Wallis rank-sum test and Dunnett’s multiple comparison test (nonparametric)
- Growth rate: One-Way ANOVA and Dunnett’s multiple comparison test

Higher concentrations than EC50 on respective parameter were excluded for each analysis.
NOEC was determined in consideration of the whole test results in addition to these results of statistical analysis.

Any other information on results incl. tables

Table 5. Percent growth inhibition during the 72 -hr exposure

Nominal Concentration (mg/L)	No.	Area under the growth curves (×10^4)	Inhibition (%)	Growth rate (24-48h)	Inhibition (%)	Growth rate (24-72h)	Inhibition (%)
Control	1	1960	-	0.0678	-	0.0614	-
	2	1960	-	0.0718	-	0.0632	-
	3	1880	-	0.0671	-	0.0626	-
	Mean	1940	-	0.0689	-	0.0624	-
9.53	1	2200	-13.6	0.0725	-5.25	0.0631	-1.14
	2	2280	-17.8	0.0757	-9.90	0.0668	-7.14
	3	2180	-12.4	0.0748	-8.61	0.0662	-6.14
	Mean	2220	-14.6	0.0743	-7.92	0.0654	-4.81
17.1	1	2210	-14.1	0.0711	-3.30	0.0651	-4.31
	2	2150	-11.2	0.0717	-4.05	0.0626	-0.399
	3	2220	-13.5	0.0749	-8.74	0.0648	-3.92
	Mean	2190	-12.9	0.0726	-5.36	0.0642	-2.88
30.9	1	2070	-6.92	0.0710	-3.07	0.0659	-5.67
	2	1700	12.3	0.0731	-6.15	0.0603	3.39
	3	1660	14.2	0.0670	2.77	0.0595	4.68
	Mean	1810	6.52	0.0703	-2.15	0.0619	0.799
55.6	1	626	67.7	0.0443	35.7	0.0426	31.7
	2	596	69.2	0.0514	25.3	0.0459	26.5
	3	667	65.6	0.0412	40.2	0.0440	29.4
	Mean	630	67.5	0.0456	33.7	0.0442	29.2
100	1	57.5	97.0	0.00940	86.4	0.00724	88.4
	2	60.9	96.9	0.00895	87.0	0.00743	88.1
	3	66.8	96.6	0.0109	84.1	0.00871	86.0
	Mean	61.7	96.8	0.00976	85.8	0.00779	87.5

Table 6. Cell densities during the 72-hr exposure

Nominal Concentration (mg/L)	No.	Cell densities (x 10^4cells/mL)
		0 hour	24 hours	48 hours	72 hours
Control	1	1.00	5.41	27.5	103
	2	1.00	4.97	27.8	103
	3	1.00	5.03	25.2	101
	Mean	1.00	5.14	26.8	102
	SD	0	0.238	1.45	0.958
9.53	1	1.00	5.53	31.5	114
	2	1.00	5.00	30.8	124
	3	1.00	4.90	29.5	118
	Mean	1.00	5.14	30.6	118
	SD	0	0.339	1.01	4.77
17.1	1	1.00	5.29	29.2	120
	2	1.00	5.53	30.9	112
	3	1.00	5.15	31.1	116
	Mean	1.00	5.32	30.4	116
	SD	0	0.191	1.05	4.22
30.9	1	1.00	4.84	26.6	115
	2	1.00	4.64	26.8	83.6
	3	1.00	4.89	24.4	84.9
	Mean	1.00	4.79	25.9	94.4
	SD	0	0.136	1.33	17.5
55.6	1	1.00	3.68	10.7	28.5
	2	1.00	3.05	10.5	27.6
	3	1.00	3.87	10.4	32.0
	Mean	1.00	3.54	10.5	29.4
	SD	0	0.428	0.134	2.35
100	1	1.00	1.65	2.07	2.34
	2	1.00	1.71	2.11	2.44
	3	1.00	1.73	2.24	2.62
	Mean	1.00	1.70	2.14	2.47
	SD	0	0.0378	0.0903	0.143

Table 7. Results of statistical analysis

Nominal concentration (mg/L)	Detection index
	Area under the growth curve	Growth rate (24-48h)	Growth rate (24-72h)
9.53	-	-	-
17.1	-	-	-
30.9	-	-	-
55.6	#	**	**
100	#	#	#
Statistics	Bartlett’s test Kruskal-Wallis rank-sum test Dunnett’s multiple comparison test (nonparametric)	Bartlett’s test One-Way ANOVA Dunnett’s multiple comparison test	Bartlett’s test One-Way ANOVA Dunnett’s multiple comparison test

**: p<0.01

-: No significant difference

#: Statistical analysis was not performed because of higer concentration than EC50.

Calculation of inhibition rates in measured parameters on growth:

The mean value of cell concentration for each test level was plotted against time to produce growth curves.

Using this curve, inhibition rates were calculated comparing with control values on area under the growth curve and growth rate.

The area under the growth curve (A) was calculated according to the following formula:

A = [(N₁ - N₀) / 2] × t₁ + [(N₁ + N₂ - 2N₀) / 2] × (t₂ - t₁) + ... + [(N_n-1 + N_n - 2N₀) / 2] × (t_n - t_n-1)

N₀ = cell density at the start of the exposure

N₁ = cell density at t₁

N_n = cell density at t_n

t₁ = Time for fist measurement of cell density during the exposure

t_n = Time for n-th measurement of cell density during the exposure

Percentage of inhitition (I_A) was calculated according to the following formula:

I_A = [(A_c - A_t)/A_c] × 100

A_c = area under the growth curve in control group

A_t = area under the growth cuve in each test group

The growth rate (μ) was calculated as the logarithmic increase in biomass according to the following formula:

μ = (lnN_n - lnN₁) / (t_n - t₁)

N₁= cell density at t₁

N_n= cell density at t_n

t₁= Time for fist measurement of cell density during the exposure

t_n= Time for n-th measurement of cell density during the exposure

Percentage of inhibition (Iμ) was calculated according to the following formula:

Iμ = [(μ_c - μ_t) / μ_c ] × 100

μ_c = growth rate of control group

μ_t = growth rate of each test grop

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell densities in control cultures increased over 16-fold, and the concentration of the test material did not fall below 80% of original during 72 h.

Conclusions:

The acute toxicity of 2-Vinylpyridine to Pseudokirchnerella subcapitata (reported as Selenastrum capricornutum) was investigated in a standard test protocol (OECD guideline 201). After 72 h of exposure, the EC50 based on growth rate was 64.4 mg/L and on biomass expansion was 50.8 mg/L. The NOEC for based on growth rate nad on biomass expansion was 30.9 mg/L .