2-vinylpyridine

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 March 2002 - 21 May 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 211 (Daphnia magna Reproduction 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 of the test and weekly thereafter from the freshly prepared solutions. (Total 3 times)
At the first renewal and weekly thereafter from the 24-hour old solutions. (Total 3 times)
- Sampling:
New (freshly prepared test solution); Samples were taken from the each vessel.
Old (old test solution before renewal); Samples were taken from the each vessel, and removed feeds by centrifugation.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was weighed and dissolved with dilution water to produce a stock solution of 100 mg/L. Test solution was prepared in each test vessel by mixing the appropriate volume of the stock solution with dilution water. The test solution was divided into each test vessel.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone A
- Source: Young daphnids produced by 18 d-old parents which were cultured in the testing laboratory were used. Daphnids originally came from the University of Sheffield (Address: Sheffield S10 2UQ, United Kingdom).
- Age at study initiation: ≪ 24 h-old
- Feeding during test: Chlorella vulgaris 0.1 - 0.2 mgC (contained organic carbon)/animal/day



OTHER
- Confirming the reproducibility of the test conditions:
Reference substance: pottasium dichromate, reagent chemical, Wako Pure Chemical Industries, Ltd.
Results: 48h EC50: 0.262 mg/L (within the acceptable range* in this laboratory)
*mean �} 2S.D.: 0.0990 - 0.341 mg/L [mean�}S.D.: 0.220 �} 0.061 mg/L (n=33)]
- Reason for selection of species: Species recommended in the test guideline
- Allocation to the test groups: At random

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

36.0 - 41.6 mg/L (as CaCO3)

Test temperature:

20.2 - 20.7 (20±1)°C

pH:

7.8 - 8.0 (The test was performed without adjusting pH. The pH of the test solutions were kept within ± 1.5 during the exposure.)

Dissolved oxygen:

8.3 - 8.7 mg/L (≫ 60 % of the O2 saturation value ( ≫approx. 5mg/L))

Nominal and measured concentrations:

Nominal concentrations: 4.00, 2.00, 1.00, 0.500 and 0.250 mg/L (geometric series with a factor of 2.0, basis on the 48-h acute immobilisation test and the preliminary test)
Measured concentrations: 3.71, 1.80, 0.901, 0.452 and 0.223 mg/L (% of the nominal: 94.2 - 102 % (at freshly prepared test solution) and 78.5 - 87.7 % (before renewal of the test solution))

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Material: Glass
- Size: 100 mL
- Fill volume: 80 mL
- Thermostatic bath: Water bath with warming/cooling unit
- Renewal rate of test solution: Once a day
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water, aerated sufficiently and controlled temperature



OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h photoperiod daily
- Light intensity: At room light (≪1200 lux)


OBSERVATION AND MEASUREMENTS
- Living or dead parent daphnia: The numbers of the living and the dead parent daphnia were recorded daily during the exposure. An animal is recorded as dead when it is immobile, i.e. when it is not able to swim, or if there is no observed movement of appendages or postabdomen under the stereomicroscope.
- The number of offspring and appearance: The numbers of offspring produced per parent animal were recorded everyday. And the appearance of offspring was observed.
- Appearance of parent animal: Body length, immobility, abnormal behaviour, presence of male, presence of eggs in the brood pouch, presence of ephippia or aborted eggs were observed.
- Test solutions: Dissolved oxygen concentration, pH, water temperature and total hardness of the test solutions were measured during the exposure (once a week at fresh and old media × three times). At the fresh media, another solution obtained from the container for preparation was used for the measurement. At the old media, the measurement was carried out for one test vessel in each level.



RANGE-FINDING STUDY
48-h Acute immobilisation test (Refer to test number 92527):
- Results used to determine the conditions for the definitive study: 48h EC50: 9.48 mg/L, NOEC: 3.23 mg/L
Preliminary test:
- Test concentrations: 5.00, 0.500 and 0.0500 mg/L (geometric series with a factor of 10)
- No. of organisms per concentration: 5
- Duration: 14 or 18 days
- Results used to determine the conditions for the definitive study: LOEC: 5.00 mg/L (nominal), NOEC: 0.500 mg/L (nominal)

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

2.58 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL = 1.80-3.71 mg/L

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.933 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL = 0.648-1.35 mg/L

Effect concentrations 3

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

1.46 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL = 1.32-1.62 mg/L

Effect concentrations 4

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

1.06 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL = 0.939-1.19 mg/L

Effect concentrations 5

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 6

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.901 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals: The mortality of the parent animals in the control was 0% at the end of the test, which meets the criteria for validity of the test (i.e. not more than 20%). All parent animals were dead at 3.71 mg/L within 14 days. The mortality of the parent animals at 1.80 mg/L was 90% at the end of the test. The mortality of the parent animals were 30% and 10% at 0.901 mg/L and 0.223 mg/L, respectively. There was no siginificant differences between mortality of the control and these groups (0.901 mg/L and 0.223 mg/L). The cumulative number of dead parent animals and mortality of parent animals during the exposure were shown in Table 6.
- Time to first brood release or time to hatch: 8 days (control), 9.1 days (Mean, 3.71 mg/L), 8 days (1.80 - 0.223 mg/L). See Table 7.
- The mean cumulative number of juveniles produced per parent animal: 114 (control, meets the criteria for validity of the test (i.e. ≫60)), 0 (3.71 mg/L ), 31 (1.80 mg/L), 84.9 - 94.4 (0.901 - 0.223 mg/L). See Table 8 and 9.
- Body length and appearance of parent animals:
3.71 mg/L; growth inhibition within 5-11 days, discoloured body within 7-10 days, reduced activity within 9-11 days
1.80 mg/L; discoloured body after 10 days, reduced activity after 14 days, leathargic after 19 days
0.901 mg/L; discoloured body after 13 days, reduced activity at 21th day
0.452 mg/L; discoloured body after 15 days, leathargic after 19 days
0.223 mg/L; discoloured body after 15 days
Control; No abnormal appearance were observed
- Ephippia: None. A few aborted eggs and dead offsprings were observed at all exposure groups except for the group of 3.71 mg/L.

- Appearance of the test solutions: clear and colorless (freshly prepared test solution), light green (old test solution before renewal)
- 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 time weighted mean of the measured concentration because measured concentrations were exceeded ± 20% of nominal concentrations during the exposure.

STATISTICS
- 14d LC50: Binominal method
- 21d LC50: Probit analysis
- 21d EC50: Moving average method
- LOEC and NOEC: Bartlett test, one-way analysis of variance (number of dead parent animals), Kruskal-Wallis rank sum test (first-brood release day), one-way analysis of variance and Scheffe multiple comparison (cumulative number of juveniles produced per parent animal); The groups of higher concentration than LC50 or EC50 were excluded from statistics.

Any other information on results incl. tables

Table 6. Cumulative number of dead parent animals and mortality of parent animals

Measured concentration (mg/L)	Exposure days
	0	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
0.223	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (10)	1 (10)	1 (10)	1 (10)	1 (10)
0.452	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	1 (10)	1 (10)
0.901	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)	3 (30)	3 (30)
1.80	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	5 (50)	8 (80)	8 (80)	8 (80)	9 (90)	9 (90)
3.71	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	2 (20)	8 (80)	9 (90)	9 (90)	10 (100)	10 (100)	10 (100)	10 (100)	10 (100)	10 (100)	10 (100)	10 (100)

Values in parenthesis are mortality (%) of parent animals

Table 7. Time to first brood release of parent animals

Measured concentration (mg/L)	First-brood release day (Exposure days)											Mean
	Vessel No.	1	2	3	4	5	6	7	8	9	10
Control		8	8	8	8	8	8	8	8	8	8	8.0
0.223		8	8	8	8	8	8	8	8	8	8	8.0
0.452		8	8	8	8	8	8	8	8	8	8	8.0
0.901		8	8	8	8	8	8	8	8	8	8	8.0
1.80		8	8	8	8	8	8	8	8	8	8	8.0
3.71		8	9	8	8	9	9	9	9	9	13	9.1

Table 8. Mean cumulative number of juveniles produced per surviving parent animals

Measured concentration (mg/L)	Exposure days
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Control	0	0	0	0	0	0	0	14.7	14.7	14.7	42.8	42.8	42.8	74.1	74.2	74.2	74.3	97.2	97.2	97.2	114
0.223	0	0	0	0	0	0	0	14.0	14.0	14.0	42.4	42.4	42.4	70.6	71.4	71.4	71.4	86.0	86.8	86.8	94.4
0.452	0	0	0	0	0	0	0	14.6	14.6	14.6	42.4	42.4	42.4	72.2	72.7	72.7	72.7	82.4	82.4	82.4	93.0
0.901	0	0	0	0	0	0	0	13.6	13.6	13.6	35.1	36.7	38.3	60.6	63.1	63.1	63.1	75.4	75.4	79.1	84.9
1.80	0	0	0	0	0	0	0	12.0	12.0	12.0	28.0	30.0	30.0	30.0	31.0	31.0	31.0	31.0	31.0	31.0	31.0
3.71	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-

Only surviving parent animals at the end of the test were presented.

-: All parent animals were dead

Table 9. Cumulative number of juveniles produced per surviving parent animals

Measured concentration (mg/L)	Exposure days
	Vessel No.	1	2	3	4	5	6	7	8	9	10	Mean	S.D.	Significant difference
Control		96	86	95	110	122	124	114	122	136	132	114	16.7	#
0.223		67	70	99	119	112	73	109	80	-	121	94.4	22.0	#
0.452		111	87	124	70	-	129	95	66	65	90	93.0	24.2	#
0.901		66	-	83	99	-	112	-	61	94	79	84.9	18.2	#
1.80		-	-	-	-	-	-	-	-	-	31	31.0	*	Excluded
3.71		-	-	-	-	-	-	-	-	-	-	*	*	Excluded

-: Parent animals were dead

*: Unable to calculate or no data

1.80 mg/L and 3.71 mg/L were excluded from statistics

NOEC: 0.901 mg/L

LOEC: 1.80 mg/L

#: Indicated no significant difference

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Mortality among parental controls was < 205 and average offspring numbered greater than 60/mating pair.

Conclusions:

The reproductive toxicity of 2-Vinylpyridine to Daphnia magna was investigated in an OECD 211 guideline protocol, and time weighted average estimates of dosage were established because the measured concentrations were more than 20% lower than nominal concentrations . After 21 days of exposure, the EC50 for reproduction was 1.06 mg/L, and the NOEC was 0.901 mg/L. For mortality at 21 days, the LC50 was 0.933 mg/L.