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Administrative data

Endpoint:
toxicity to terrestrial plants: short-term
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
The non-GLP study followed no guideline protocol, the actual exposure is unclear, and uncommon endpoints were investigated. Thus the study is not conclusive with regard to environmental risk assessment requirements.

Data source

Reference
Reference Type:
publication
Title:
Influence of 2.2'-bipyridyl, a photodynamic herbicide, on chloroplast development and pigment content in etiolated pea seedlings
Author:
Motowska A & Siedlecka A
Year:
1995
Bibliographic source:
Acta Physiologiae Plantarum 17(1): 21-30

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Peas were placed in sand for germination in the dark. After 8 days the etiolated plants were sprayed with solutions of the test item. After 6 or 12 hours under white light the leaves of the test organisms were examined for ultrastructural changes of the mesophyll cells by electron microscopy and the chlorophyll and carotenoid contents were determined.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-bipyridyl
EC Number:
206-674-4
EC Name:
2,2'-bipyridyl
Cas Number:
366-18-7
Molecular formula:
C10H8N2
IUPAC Name:
2,2'-bipyridyl
Details on test material:
- Name of test material (as cited in study report): 2.2'-B or 2.2'-bipyridyl

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
- Concentrations: All concentrations were sampled, but not all materials could be investigated (no pigment determination at 5mM)
- Sampling method:
(1) Leave material was sampled for pigment determination.
(2) For the morphometric analysis 40-50 plastid cross sections of the second leaf above the cotyledons were examined.
- Sample storage conditions before analysis: No storage

Test substrate

Vehicle:
yes
Details on preparation and application of test substrate:
- Controls: Vehicle control only
- Chemical name of vehicle (emulsifier): Tween 20 (CAS 9005-64-5)
- Concentration of vehicle in test medium (final test solution): 0.05 %

Test organisms

Species:
Pisum sativum
Plant group:
Dicotyledonae (dicots)
Details on test organisms:
- Common name: Pea
- Plant family: Fabales
- Variety: Illowiecki
- Prior seed treatment/sterilization: The seeds were soaked in water during 12 hours before sowing

Study design

Test type:
other: pigment formation of etiolated seedlings
Study type:
laboratory study
Substrate type:
other: sand
Limit test:
no
Total exposure duration:
29 h
Remarks:
or 23 h

Test conditions

Test temperature:
25 °C
Details on test conditions:
TEST SYSTEM
- No. of plants: 10 per treatment and vehicle control

GROWTH CONDITIONS
- Light source: Fluorescent tubes
- Light intensity and quality: White light; 5 W/m² at the leaf height

EFFECT PARAMETERS MEASURED:
(1) The inhibition of greening of etiolated seedlings was assessed on the basis of pigment determination. Leave material was extracted with 80% acetone (5 cm³ for 1 g) and stored at -4 °C. The Chlorophyll A and B concentrations were determined spectrophotometrically at 663 nm and 646 nm respectively (Spectrophotometer Shimadzu UV - 160A).
(2) Photodynamic destruction was assessed by electron microscope examination of the mesophyll cells ultrastructure. In randomly chosen unit areas of about 1 µm² on each plastid section the total length of thylakoids were measured by a curvometer. Grana length was measured as well in case they were present. The probes were prepared for electron microscopy by fixation in 2.5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4, 2 h). Then the samples were washed in buffer and used for electron microscopy after overnight incubation in Osmium tetroxide (cold, 2%).

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.3 to 1.7
Nominal and measured concentrations:
Spray concentrations of 1, 1.5, 2, 3, and 5 mM were used. This corresponds to 156.2, 234.3, 312.4, 468.6 and 780.9 mg/L as the Molecular weight of the test item is 156.187 g/mol.
Four mL of these test solutions were applied three times to ten plants. Consequently one plant may have been contaminated with 0.19, 0.28, 0.37, 0.56, or 0.94 mg of the test item respectively.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Species:
Pisum sativum
Duration:
6 h
Dose descriptor:
NOEC
Effect conc.:
<= 0.28 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks on result:
other: Dose estimated from 1.5 mM spray solution; No change in toto, in mesophyll cell cytoplasm vesicles which were formed from dilated endoplasmatic reticulum cisternae, chlorophyll A and B content reduced
Species:
Pisum sativum
Duration:
12 h
Dose descriptor:
NOEC
Effect conc.:
<= 0.28 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks on result:
other: Dose estimated from 1.5 mM spray solution; Number of thylakoid in grana 3 versus 4-5 in control, but average length indicates no evidence of strong inhibition
Species:
Pisum sativum
Duration:
6 h
Dose descriptor:
LOEC
Effect conc.:
<= 0.37 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks:
- loss of turgor in the upper part of epicotyl
Remarks on result:
other: Dose estimated from 2 mM spray solution
Species:
Pisum sativum
Duration:
12 h
Dose descriptor:
LOEC
Effect conc.:
<= 0.37 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks:
- loss of turgor in the upper part of epicotyl
Remarks on result:
other: Dose estimated from 2 mM spray solution
Species:
Pisum sativum
Duration:
6 h
Dose descriptor:
EC100
Effect conc.:
<= 780.9 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks:
- completly inhibited grana formation
Remarks on result:
other: Dose estimated from 5 mM spray solution
Species:
Pisum sativum
Duration:
12 h
Dose descriptor:
EC100
Effect conc.:
<= 780.9 other: mg per seedling
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
phytotoxicity
Remarks:
- completly inhibited grana formation
Remarks on result:
other: Dose estimated from 5 mM spray solution
Details on results:
The Chlorophyll A and B content generally significantly lower than in control. After 6 hours of illumination treament with the lowest concentration of 1 mM the Chlorophylls were about 45% of the control and 7% if treated with 3 mM.
Treatment with 5 mM spray solutions resulted in strong fading compared to the control after 6 hours of illumination and grana formation was completely inhibited.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
Spray application of the test item effected significant reduction in chlorophyll content in all concentrations and adverse ultrastructural changes from 2 mM spray solution concentration on.
Executive summary:

Mostowska & Siedlecka (1995) present a study on the effects of 2,2'-bipyridine (CAS 366-18-7) on chlorophyll formation and leaf mesophyll cell ultrastructure during the greening of etiolated pea (Pisum sativum) seedlings. No guideline was followed in the non-GLP experiments. As spray application was used and no analytical dose verification was performed the actual exposure of the test organisms keeps unclear. Thus the study is considered not conclusive with regard to environmental risk assessment requirements.

The plants were placed 8 days in sand for germination in the dark. After 8 days the etiolated seedlings were sprayed with solutions of to the test item. For greening the test organisms were then paced for 6 or 12 hours under white light. For evaluation the leaves were examined for ultrastructural changes of the mesophyll cells by electron microscopy and the chlorophyll and carotenoid contents were determined.

The test item was applied in 5 different spray solution concentrations of 1, 1.5, 2, 3, and 5 mM. This corresponds to 156.2, 234.3, 312.4, 468.6 and 780.9 mg/L as the Molecular Weight of the test item is 156.187 g/mol. Four mL of these test solutions were applied three times to ten plants. Consequently one plant may have been contaminated with 0.19, 0.28, 0.37, 0.56, or 0.94 mg of the test item respectively. However every treatment effected significant reduction in chlorophyll content the NOEC was assigned on the bases of ultramorphological changes to the 1.5 mM solution application, which leads to the 2 mM as LOEC.

The ultrastructural changes observed in the mesophyll cells were grana formation inhibition, prolamellar body transformation delay, and endoplasmic reticulum cisternae dilation.

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