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Ecotoxicological information

Toxicity to terrestrial plants

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Administrative data

toxicity to terrestrial plants
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented and scientifically acceptable, but lacking testing guidelines
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Principles of method if other than guideline:
No guideline specified, cf. "Any other information on material and methods" for details.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibutyl phthalate
EC Number:
EC Name:
Dibutyl phthalate
Cas Number:
Molecular formula:
dibutyl phthalate

Test organisms

other: Hordeum vulgare, Spinacea oleraceae
Details on test organisms:
Protoplasts were prepared from barley {Hordeum vulgare L. cv. Simba) according to Hampp and Ziegler (1980). Grains were soaked for 16 h in aerated running tap water, and grown in a mixture of peat and sand (Ryberg and Sundqvist 1976) in a climate chamber irradiated with white light (General Electric F72P617 CW, USA) at an intensity of 40 W m^-2 (16 h light/8 h dark, 16°C/14°C, relative humidity 85%/75%). Protoplasts were prepared from 7-day-old leaves, using leaf segments 3 cm long and taken 5 cm from the base of the leaves. The incubations with enzyme were made at 22°C instead of at 28°C in Hampp and Ziegler (1980).

Thylakoids were prepared from spinach (Spinacea oleraceae L.) grown in a climate chamber irradiated with white light (General Electric F72P617 CW, USA) at an light intensity of 40 W m^-2 (10 h light/14 h dark, 21°C/17°C, relative humidity 70%) for 5-8 weeks. Thylakoids were prepared according to Radosevich et al. (1979) with the modifications that the chloroplasts were osmotically disrupted in 0.1 M NaCl, and suspended in a medium containing 0.2 M sorbitol instead of 0.1 M. When IC50 was determined according to Tischer and Strotmann (1977), their method for preparing thylakoids lacking the coupling factor was used.

Results and discussion

Effect concentrationsopen allclose all
Hordeum vulgare
Dose descriptor:
other: IC50
Effect conc.:
0 other: M
Basis for effect:
other: CO2 reduction
other: Spinacea oleraceae
Dose descriptor:
other: IC50
Effect conc.:
0 other: M
Basis for effect:
other: electron transport
other: Spinacea oleraceae
Dose descriptor:
other: IC50
Effect conc.:
0 other: M
Basis for effect:
other: uncoupled electron transport

Any other information on results incl. tables

The effect of DBP on photosynthesis in protoplasts prepared from barley

Protoplasts were used because we wanted to compare photosynthesis in higher plant with that of an alga, without interference by the long range transport of metabolites typical for higher plants. Photosynthesis was measured either as 14CO2-reduction or as CO2-dependent oxygen evolution. The chlorophyll concentrations were different in the two types of experiments (Tab. 1). DBP inhibited photosynthesis at concentrations higher than lO^-5 M (Fig. 3).

The effect of DBP on electron transport in isolated thylakoids from spinach

As photosynthesis was inhibited by DBP in both algae and protoplasts, we investigated in what photosynthetic system(s) the effect was localized. By choosing isolated thylakoids as a test system and by comparing the results obtained on different organisational levels (cells, protoplasts, thylakoids) we should be able to assign the effect of DBP to a certain level.

The effects of DBP on coupled and NH4CI-uncoupled basal electron transport in photosystem II + I are shown in Fig. 4. Electron transport was inhibited at DBP concentrations higher than 10^-4 M, the uncoupled system being the most sensitive system with an IC50 value of 3 X 10^-4. With a photosystem I assay (methylviologen as electron acceptor and dichlorophenolindophenol/ascorbate as electron donor couple) no effect was observed with DBP concentrations as high as 10^-3 (results not shown). Reactions in

photosystem II are thus being affected by DBP. In order to determine a chlorophyll independent IC50 value the method of Tischer and Strotmann (1977) was used. Here the ferricyanide dependent oxygen evolution in thylakoids devoid of coupling factor is measured at different chlorophyll concentration. By extrapolating to zero chlorophyll concentration the IC50 value in DBP treated thylakoids was found to be 2.5 x 10^-5 M.

Applicant's summary and conclusion

The CO2-reduction in isolated protoplasts prepared from barley (Hordeum vulgare L. cv. Simba) was inhibited by phthalate. The IC50 value was 2 x 10^-4 M. The electron transport in isolated thylakoids prepared from spinach was inhibited with an IC50 value of 3 x 10^-4 M. The IC50 value for
uncoupled electron transport extrapolated to zero chlorophyll concentration was 2.5 X 10^-5 M. The effect of di-n-butyl phthalate was localized to reactions in photosystem II. Di-n-butyl phthalate could thus be a pollutant which affects growth and photosynthesis of plants.
Executive summary:

This is part of a study conducted on algae, barley and spinat. Only the effects on barley and spinach are documented here. Those on algae can be found under section 6.1.5.