The use of physiological characteristics for comparison of organic compounds phytotoxicity

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The use of physiological characteristics for comparisonof organic compounds phytotoxicity

Marie Kummerova, Lucie Vanova *, Jana Krulova, Stepan Zezulka

Department of Plant Physiology and Anatomy, Institute of Experimental Biology, Faculty of Science, Masaryk University,

Kotlarska 2, 611 37 Brno, Czech Republic

Received 18 October 2007; received in revised form 21 January 2008; accepted 23 January 2008Available online 11 March 2008

Abstract

The influence of intact (FLT) and photomodified (phFLT) fluoranthene (0.05, 0.5 and 5 lmol l�1) and herbicide Basagran (5, 20, 35and 50 nmol l�1) on the germination, growth of seedlings and photosynthetic processes in pea plants (Pisum sativum L., cv. Garde) wasinvestigated. The germination was significantly inhibited already by the lowest concentration (0.05 lmol l�1) of FLT and phFLT, whileBasagran caused inhibition only in higher concentrations (35 and 50 nmol l�1). The growth of roots was significantly inhibited by higherconcentration 5 lmol l�1 of both FLT and phFLT and the shoot of seedlings was significantly influenced only by photomodified form. Thelength of root and shoot was inhibited already by concentration 5 nmol l�1 of Basagran. Organic compounds applied on chloroplasts sus-pension influenced primary photochemical processes of photosynthesis. In chlorophyll fluorescence parameters, the significant increase ofF0 values and the decrease of FV/FM and UII values by application of FLT (0.5 and 5 lmol l�1) and phFLT (0.05, 0.5 and 5 lmol l�1) wasrecorded. The maximum capacity of PSII (FV/FM) was influenced by the highest (50 nmol l�1) and the effective quantum yield of PSII (UII)already by the lowest (5 nmol l�1) concentration of Basagran. Hill reaction activity decreased and was significantly inhibited by higherconcentration (0.5 and 5 lmol l�1) of FLT and phFLT and already by the lowest concentration (5 nmol l�1) of Basagran.� 2008 Elsevier Ltd. All rights reserved.

Keywords: PAH; Herbicide; Pea plants; Phytotoxicity tests; Chlorophyll fluorescence; Hill reaction activity

1. Introduction

The development of human activities and industrializa-tion has led to an increased accumulation of organiccompounds in the environment. Polycyclic aromatic hydro-carbons (PAHs), which are by-products from the incom-plete combustion or pyrolysis of organic materials, areknown as priority pollutants (Thiele and Brummer,2002). Several of them are carcinogenic and/or mutagenicin animals, as well as phytotoxic (Meudec et al., 2006).Their fate is determined by their physico-chemical proper-ties; especially logKOW, solubility in water, adsorption

coefficients etc., responsible for their persistence in theenvironment (Meudec et al., 2006). The properties of PAHscan be changed either abiotically (e.g. photochemically) orby biological processes (e.g. oxidation supported by cyto-chrome P450). The essential role of the solar radiation inthe toxicity of polycyclic aromatic substances results notonly in photomodification but also in photosensitizationprocesses (Tukaj and Aksmann, 2007). During photomodi-fication, PAHs are structurally altered and form a complexmixture of photoproducts, which may be more toxic thanthe parent compounds most likely due to a combinationof increased solubility, bioavailability and reactivity(Huang et al., 1997b). Similarly to PAHs, herbicides, agroup of organic compounds, which are intentionallyintroduced into the environment, could influence not onlythe growth and development of the weed, but also thecrops due to the intensive use.

0045-6535/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2008.01.060

* Corresponding author. Tel.: +420 5 32 14 62 24; fax: +420 5 41 21 1214.

E-mail address: [email protected] (L. Vanova).

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Chemosphere 71 (2008) 2050–2059


More than 80% of the Earth’s land surface is coveredwith vegetation, which is exposed to organic compoundsduring all the development stages – from germination toreproduction (Wild et al., 1992). Plants can take up theorganic compounds by roots (Gao and Zhu, 2004), butespecially by particle-phase deposition on the waxy leafcuticle or by their uptake in the gaseous phase through sto-mata (Meudec et al., 2006; Wild et al., 2006). The plant’sability of PAHs and herbicides uptake, translocation,transformation and accumulation is a limiting factor forphytotoxicity of these compounds and can affect quantita-tively and qualitatively several biochemical and physiolog-ical processes taking part in biomass production(Kummerova et al., 2006). Moreover plants standing atthe beginning of the food chain allow these xenobioticsto enter higher trophic levels.

With the increasing level of environmental loading byPAHs and herbicides a development and use of new meth-ods for early indication of stress or affection caused byorganic compounds in vegetation becomes more acute. Aclose attention is paid to molecular, biochemical and phys-iological indicators of the exposure to organic compounds,which could precede the visible injury of plants. One of themost investigated metabolic processes is the photosynthe-sis, which represents a central pathway in plants resultingin the production of energy-rich organic compounds neces-sary for growth.

Often referred target of herbicide action in the photo-synthetic apparatus is the photosystem I (PSI) (Wakabay-ashi and Boger, 2004), but intact and photomodifiedPAHs affect mainly PSII (Marwood et al., 1999). Bothgroups of organic compounds could exhibit similar mecha-nism of photosynthesis inhibition based on their competi-tion with quinone electron acceptors (Huang et al., 1996;Magne et al., 2006). Moreover, effect of herbicides onwater-splitting complex (Oxygen Evolving Centre – OEC,Hill reaction) is very often used for an evaluation of theirpossible toxicity (Szigeti and Lehoczki, 2003; Kummerovaet al., 2006). Photosystem II electron transport is one of themost sensitive indicators of damage in the photosynthetic

apparatus (Krause and Weis, 1991). Analysis of the chloro-phyll fluorescence induction curve, or Kautsky curve, iswell-known tool for monitoring the physiological statusof the photosynthetic apparatus in photosynthesis research(Abbaspoor et al., 2006). The studies using this method forevaluating the effect of herbicides are sporadic (Abbaspooret al., 2006). Chlorophyll fluorescence parameters (initialchlorophyll fluorescence – F0, potential yield of photo-chemical reactions in PSII – FV/FM and effective quantumyield of PSII – UII) might be used as indicators of stressaffecting photochemical pathway of utilization of absorbedlight energy (Krause and Weis, 1991; Mallakin et al., 2002).

The aim of this study was to evaluate the effect of fluor-anthene (FLT), photomodified fluoranthene (phFLT) andherbicide Basagran on the germination, growth of the seed-lings and primary photosynthetic processes in pea plants.Fluoranthene was selected from the PAHs family as oneof the most frequent polycyclic aromatic hydrocarbons inthe environment of the Czech Republic (Holoubek,2000). Herbicide Basagran is used for suppression of weedin pea plant fields. Applied concentrations of FLT simulatea low and high loading of the environment; applied concen-trations of Basagran represent very low loading. We sup-pose the use of a variety of physiological stress indicators(phytotoxicity tests, chlorophyll fluorescence, Hill reactionactivity) for an early indication of the effect of both groupsof organic compounds.

2. Materials and methods

2.1. Fluoranthene, photomodified fluoranthene and Basagran

preparation

Fluoranthene (FLT; Supelco, USA) was dissolved inacetone (Labscan, Ireland) and filter-purified (ultrafiltra-tion) water (FP-H2O) to a concentration of 50 lmol l�1.This FLT stock solution was then diluted with FP-H2Oto final concentrations of FLT ranging from 0.05 to 0.5and 5 lmol l�1 (0.01, 0.1 and 1 mg l�1). Preliminary exper-iments demonstrated that the concentration of dissolvent

Nomenclature

DCIP 2,6-dichlorophenol-indophenolF0 basic chlorophyll fluorescenceFLT fluorantheneFP-H2O filter-purified waterFV/FM maximum capacity of photosystem IIGA gibberrelin acidGC-FID flame-ionisation detector gas chromatographHPLC high performance liquid chromatographLHC(s) light harvesting complex(es)OEC oxygen-evolving centrePAH(s) polycyclic aromatic hydrocarbon(s)

PAM pulse amplitude-modulated fluorometerPAR photosynthetic active radiationphFLT photomodified fluoranthenePSI photosystem IPSII photosystem IIQA primary electron acceptorQB secondary plastoquinone acceptorTyr Z tyrosine ZUV ultra-violetUII quantum yield of electron transport through

photosystem II

M. Kummerova et al. / Chemosphere 71 (2008) 2050–2059 2051


did not affect seed germination and growth of seedlings(Kummerova and Kmentova, 2004) but control seeds werealways exposed to the same concentration of acetone. Thechlorophyll fluorescence parameters and Hill reactionactivity were unaffected by this concentration of solvent.

Photomodified fluoranthene (phFLT) was generated byirradiation of the intact fluoranthene (FLT) solution in ablack container using an UV lamp (medium pressure mer-cury vapour lamp MPK Tesla 125W) and a water-cooledborosilicate glass filter to provide UV light at k P 290 nmin order to simulate the emission spectrum of sunlight. Thetime of incubation (120 min) was sufficient to photomodifyabout 90% of the starting material (Huang et al., 1993).The concentration of FLT in experimental solutions andthe extent of photomodification were determined chro-matographically. HPLC analyses were performed on HP1050 Series chromatograph consisting of quaternary pump,programmable autosampler, diode-array detector, HP1046A programmable fluorescence detector and HP Chem-Server with HPLC 2D ChemStation software. The assem-bly was completed with HP 1100 thermostatted columncompartment (all Hewlett Packard, Germany). Acetonitrileof HPLC gradient grade, acetone of analytical grade (J.T.Baker, The Netherlands) and water purified by Milli-Q 185Plus system (Millipore, USA) were used. Sample volume of3.6 ll was injected into Vydac 201TP52 column250 � 2.1 mm I.D. packed with 5 lm polymeric octadecylphase with carbon load of 15.5–16% (Vydac, USA). AnODS-Hypersil cartridge 20 � 2.1 mm I.D., 5 lm octadecylphase (Hewlett Packard, Germany) was used as a guardcolumn. Columns were thermostatted at 20 �C. Stepwisegradient separation from 50% v/v to 100% v/v acetonitrilewas performed at the flow rate of 0.43 ml min�1. A com-plex mixture of photomodified PAHs, produced from eachPAH, was analysed using gas chromatography (GC-MS).GC-MS analyses were performed on the Finnigan GCQinstrument (Finnigan Mat, USA) with the column ZebronZB-5 (Phenomenex, USA; 30 m � 0.25 mm � 0.25 lm)using following temperature program: initial temperature60 �C hold for 1 min, then gradient 4 �C min�1–180 �C,then gradient 10 �C min�1–275 �C and hold 5 min at thistemperature. Temperature of the transfer line was 275 �C,ion source 200 �C. Linear velocity of the carrier gas(helium) was 40 cm s�1. Full scan spectra in the range ofrelative mass m/z 50–450 Da were obtained. Using thismethod we were able to identify in photomodified FLTsolutions the following compounds: 9H-fluoren-9-one, 4-hydroxy-9H-fluoren-9-one, phenanthrene-9,10-dione andphenanthrene-9-ol. These four compounds and all otherphotoproducts are very unstable and it is impossible at thistime to estimate the molar concentrations of these break-down products. Therefore, toxicity assays were carriedout on the basis of the concentrations of intact fluoran-thene present prior to incubation in UV light atk P 290 nm (Huang et al., 1993). To control the potentialside effects of the acetone on the toxicity of phFLT, ace-tone in water was also treated with UV light for 120 min

and used as control solution in the corresponding toxicityexperiments.

Basagran 600 (BASF s.r.o., Czech Republic) was dilutedin the water (FP-H2O) to the final concentrations of 5, 20,35 and 50 nmol l�1. Bentazone (3-isopropyl-1H-2,1,3-ben-zothiadiazin-4(3H)-on-2,2-dioxide, 480 g l�1) is an effectivecompound of commercial herbicide Basagran 600. Theconcentrations of Bentazone in applied herbicide Basagranwere 1.2, 4.8, 8.1 and 12 lg l�1.

2.2. Germination and growth of seedlings

After 24 h of soaking in FP-H2O, seeds were placed inPetri dishes (10 seeds per dish) on filter paper with 8 mlof each experimental solution. The control and all treat-ments were repeated 10 times. The seeds were germinatedand seedlings grew in growth chamber under controlledconditions (mean air temperature 25 ± 1 �C, relative airhumidity 80%) and in darkness. The germination of seedsand the parameters of early growth (length of root andshoot) of seedlings of pea plants were evaluated after 3and 7 days respectively (Czech Standard CSN, 1983). Thevalue of the germination expresses the percentage of germi-nating seeds related to number of planted seeds.

2.3. Cultivation of plants

After 3 days of germination, seedlings of pea plants(Pisum sativum L., cv. Garde) were delivered to the disheswith granulated polyethylene and FP-H2O. After next 2days seedlings were then transplanted into plastic vesselswith 2.5 l of Reid–York nutrient solution (Reid and York,1958) and cultivated for 18 and 25 days. Cultivation wasdone under the natural light conditions (max. irradiance400 lmol m�2 s�1 of photosynthetic active radiation –PAR) in an air-conditioned glasshouse at average air tem-perature of 23 ± 2 �C and relative air humidity from 60%to 80%. The nutrient solution was renewed every 2 daysand its pH value was regularly adjusted to 6.5.

2.4. Isolation of chloroplasts

Photosynthetic active pea leaves (fresh mass 5 g; 3rdand 4th leaf after 18-day cultivation; 4th, 5th and/or6th leaf after 25 days) were quickly homogenized incooled mortar with sea sand in 12.5 ml of extractionmedium with pH 7 containing 0.4 M sucrose, 0.05 MNa4P2O7, 0.01 M NaCl, 0.005 M MgCl2, 0.001 M EDTA,0.05% cystein and 0.03 M ascorbic acid. The chloroplasthomogenate was filtered through fine nylone net. The fil-trate was centrifuged at 1370 rpm for 5 min at 4 �C andthe supernatant was centrifuged again at 3230 rpm for10 min at 4 �C. Sedimented chloroplasts suspension wasthen softly stirred up in 17.5 ml of the re-suspensioningmedium with pH 7 containing 0.4 M sucrose, 0.05 MNa4P2O7, 0.01 M NaCl, 0.005 M MgCl2, 0.001 M EDTA,0.05% cystein and 0.0086 M K3[Fe(CN)6] and stored in

2052 M. Kummerova et al. / Chemosphere 71 (2008) 2050–2059


ice-cooled glass beaker in the dark. Immediately after theisolation before the measurements of chlorophyll fluores-cence and Hill reaction activity assessment the suspensionwas enriched with tested concentrations of FLT, phFLTand Basagran.

2.5. Chlorophyll fluorescence measurement

Effects of FLT, phFLT and Basagran on photosyn-thetic apparatus of pea chloroplast suspension weredetermined from chlorophyll fluorescence. A set of chlo-rophyll fluorescence parameters was determined from ananalysis of slow (Kautsky) kinetics supplemented withsaturation pulses (Kummerova et al., 2007). The param-eters of chlorophyll fluorescence (F0, FV/FM and UII; fordefinition of the parameters see Rohacek and Bartak,

1999) were measured using a PAM 2000 fluorometer(Walz, Germany).

2.6. Hill reaction activity assessment

Hill reaction activity was measured spectrophotochemi-cally (630 nm) as the rate of DCIP (2,6-dichloro-indophe-nol) reduction (%) by the chloroplast suspension affectedby FLT, phFLT and Basagran during irradiation usingUV–VIS Spectrophotometer SPECORD 205 (AnalyticJena AG, Germany). For detail information about prepa-ration of DCIP and determination of the Hill reactionactivity see Kummerova et al. (2006).

2.7. Statistics

For a statistical evaluation of results, the softwareSTATISTICA 6 (StatSoft, Inc.�) was used. The obtainedresults are means of at least six repetitions of each assessedparameter. The significance of the differences of the aver-age values between the treatments was evaluated by theanalysis of variance of simple classification after precedingverification of normality and homogenity of the variance(ANOVA, P = 0.05) or by non-parametric Kruskal–Wallistest. The comparison of means was based on the method ofTukey test.

3. Results

3.1. Germination

The percentage of germinated seeds decreased with theincreasing concentration of organic compounds in the envi-ronment (Table 1). The significant decrease was causedalready by the application of 0.05 lmol l�1 FLT andphFLT. Basagran significantly inhibited the germinationin concentration 35 and 50 nmol l�1.

Table 1The effect of increasing concentration of FLT, phFLT and Basagran onthe germination of pea plants

Treatment Concentration Germination (%) Statistics

FLT (lmol l�1) 0 94.00 ± 1.52a a0.05 82.00 ± 2.00b bc0.5 79.00 ± 2.33b c5 63.00 ± 2.60c d

phFLT (lmol l�1) 0 94.00 ± 1.52a a0.05 79.00 ± 2.77b c0.5 75.00 ± 2.69b cd5 61.00 ± 2.77c d

Basagran (nmol l�1) 0 94.00 ± 1.52a a5 94.00 ± 4.00ab ab

20 94.00 ± 4.00ab ab35 80.00 ± 3.16bc bc50 76.00 ± 2.45c cd

Data represents as mean ± standard error (n = 100). Different letters bythe value show significant differences between effects of concentrations ofthe same compound (P = 0.05). Letters in column statistics mark generaldifferences.

Table 2The effect of increasing concentration of FLT, phFLT and Basagran on length of root and shoot of pea plants

Treatment Concentration Length of root (mm) Statistics Length of shoot (mm) Statistics

FLT (lmol l�1) 0 97.73 ± 5.83a a 33.14 ± 3.54a a0.05 91.28 ± 3.12ab abc 33.45 ± 1.78a a0.5 92.09 ± 4.94ab ab 32.81 ± 2.30a ab5 86.54 ± 4.55b bcd 32.48 ± 2.37a abc

phFLT (lmol l�1) 0 97.52 ± 8.25a a 32.55 ± 3.54a a0.05 95.03 ± 8.50ab ab 33.52 ± 3.37a a0.5 90.09 ± 6.24ab abc 30.56 ± 3.69ab abc5 76.56 ± 7.84c ef 27.50 ± 4.25bc bcd

Basagran (nmol l�1) 0 97.17 ± 5.33a a 33.21 ± 4.30a a5 88.48 ± 8.16b bcd 27.06 ± 3.18b cd

20 78.21 ± 5.83bc def 24.67 ± 4.10bc de35 67.94 ± 8.49c fg 20.30 ± 4.15c e50 60.83 ± 6.50c g 12.91 ± 4.54d f

Data represent mean ± standard deviation (n = 100). Different letters by the value show significant differences between effects of concentrations of thesame compound (P = 0.05). Letters in column statistics mark general differences.



3.2. Growth of seedlings

All concentrations of FLT and phFLT reduced thelength of roots and shoots (Table 2). Significant inhibitionof root growth was detected by the application of5 lmol l�1 FLT and phFLT. Only the concentration5 lmol l�1 phFLT significantly influenced the shoot lengthof seedlings. The lowest concentration of Basagran

(5 nmol l�1) significantly reduced both root and shootlength.

3.3. Induced chlorophyll fluorescence

Significant increase of F0 was recorded in pea chloro-plast suspension exposed to the highest concentrations5 lmol l�1 FLT and phFLT and 50 nmol l�1 Basagran

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Fig. 1. The effect of increasing concentration of fluoranthene (FLT) on the chlorophyll fluorescence parameters measured in chloroplast suspension of peaplants after 18 and 25 days of cultivation; F0 – initial chlorophyll fluorescence; FV/FM – ratio of variable to maximal chlorophyll fluorescence and UII –effective quantum yield of photochemical energy conversion in PSII. Different letters above the box plots show statistically significant differences betweenvalues. The point inside the box represents mean value, borders of the box indicate standard error and bars represent standard deviation (P = 0.05).



(Figs. 1–3). With the increasing concentration (0.05, 0.5and 5 lmol l�1) of FLT and phFLT and the age of plants(18 and 25 days) FV/FM value significantly decreased. Onlythe concentration 50 nmol l�1 of Basagran caused signifi-cant decrease of this parameter. The significant decreaseof UII was recorded by the application 0.5 and 5 lmol l�1

FLT and phFLT, and by 0.05 lmol l�1 phFLT in chloro-plast suspension from older plants (25 days). Already5 nmol l�1 of Basagran significantly decreased UII. The rec-ognized changes of chlorophyll fluorescence parameterswere already found in chloroplast suspension isolated from18-day-old plants.

3.4. Hill reaction activity

The results (Fig. 4) showed that the Hill reaction activityof isolated chloroplast suspension of 18-day-old pea plantswas significantly affected by addition of FLT (0.5 and5 lmol l�1), phFLT (0.5 and 5 lmol l�1) and Basagran (5,20, 35 and 50 nmol l�1).

4. Discussion

A yield of crops could be influenced among different bio-tic and abiotic factors also by organic compounds like bothPAHs and herbicides. In natural conditions all develop-mental stages of plants could be affected not only by theintact fluoranthene but also by the products of its pho-tomodification arising in the atmosphere and even in planttissues due to the effect of solar radiation. Although, mostof the herbicides, like Basagran used in our work, are selec-tive herbicides, a part of them could be taken up after theirapplication by roots of not only weeds but also of the cul-tivated plants. Basagran is used for the suppression of weedin pea plant fields. In our experiment pea plants were cul-tivated in controlled laboratory conditions in Reid–Yorknutrient solution in order to eliminate the possible effectof heterogeneous conditions of the soil environment. Peaplant belongs to protein-rich crops with low portion of lig-nin and low content of organic acids, tannins and phenoliccompounds in tissues (Kummerova et al., 2006). This factenabled us to homogenize pea plants easily and to extractintact cell compartments, especially chloroplasts.

For an assessment of the influence of selected organiccompounds on the early developmental stages a germina-tion test and root elongation test were used. Our resultsdemonstrate that low concentrations of Basagran have noeffect on the germination of pea seeds unlike the lowestapplied concentrations of intact (FLT) and photomodified(phFLT) fluoranthene (Table 1). Significant reduction ofgermination and the growth of plants in other plant speciesby the effect of FLT and phFLT was recorded by Huanget al. (1997b), McConkey et al., (1997) and Kummerovaand Kmentova (2004). According to these authors, theaffection of seed germination could be related both to thekind of endosperm and to the changes in gibberellin acid(GA) activity inducing many hydrolytic enzymes. Inhibi-

tion of GA activity induced by FLT and phFLT could beone of the reasons of lower germination. The differencein the efficiency of PAH and the herbicide could be con-nected with the fact that Basagran causes firstly the inhibi-tion of the photosynthetic apparatus. Other mechanisms bywhich herbicides influence the growth include inhibition ofmitosis, inhibition or stimulation of tissue enlargement andelongation and alteration of patterns of tissue differentia-tion patterns (Cartwright, 1976; Wakabayashi and Boger,2004). Since the growth of pea plant seedlings proceededin darkness (Czech Standard CSN, 1983), we suppose, thatBasagran could even affect another processes than photo-synthesis. This could be proved by the significant reductionof the root and shoot length recorded in our experiment in7-day-old seedlings exposed already to the lowest appliedconcentration of Basagran (5 nmol l�1) (Table 2).

Different physico-chemical properties of FLT andphFLT and the higher reactivity of phFLT reflected ingrowth responses of plant seedlings. Inhibition of shootgrowth in seedlings caused by higher concentrations ofphFLT proves its increased toxicity. Seedling growth wasa far more sensitive endpoint than seed emergence for allsubstances (Sverdrup et al., 2003).

The results of phytotoxicity tests on pea plants cannotbe generalized to any other plant species in other develop-mental stages. The knowledge on the organic compoundseffect on different biochemical and physiological processesin higher plants is still insufficient. An inhibition of photo-synthetic processes is very often a key mechanism of toxiceffects of many noxious substances. The photosyntheticactivity can be assessed on the level of chloroplast, singleleaves, whole plants or even vegetation cover. It is wellknown, that chloroplasts are the first target of different abi-otic stress factors (Alscher et al., 1998).

An extent of changes in primary photochemical pro-cesses of chloroplast isolated from pea plants was deter-mined from the chlorophyll fluorescence measurements.Probable mechanism responsible for basal chlorophyllfluorescence value (F0) increase in organics-treated (0.5and 5 lmol l�1 intact and photomodified FLT and 20, 35and 50 nmol l�1 Basagran; Figs. 1–3) pea plants might bephosphorylation and detachment of light harvesting com-plexes (LHC) from the core of photosystem II (PS II). Suchchanges obviously lead to a decrease in efficiency of energytransfer from LHCs to reaction centers of PSII and thus anincrease in basal chlorophyll fluorescence (Krause andWeis, 1991). The effect of organics on thylakoid membranemight cause reversible inactivation of PSII (Huang et al.,1997a; Mallakin et al., 2002) which leads to F0 increase.

The maximum capacity of PSII (FV/FM) is a widely usedindicator of stress responses (Huang et al., 1997a) in pho-tosynthetic apparatus of plants. Our results showed thatthe largest decrease in this commonly presented parameterwas caused by 5 lmol l�1 phFLT (reduction by 42%) inchloroplast suspension from 25-day-old pea plants(Fig. 2). It can be assumed that the larger impact of thephotomodified FLT is connected with the redox state of



the primary electron acceptor QA. Quinones, the mainproducts of photomodification of PAHs (Delphin et al.,1998), are able to block the electron transport in the loca-tion where plastoquinone acts as an electron acceptor ordonor (Huang et al., 1997a). Significant effect of Basagran(50 nmol l�1) on the decrease of FV/FM proves the affectionof photosystem II, too.

The value of effective quantum yield of PSII (UII) in peachloroplasts suspension also decreased under the influenceof increasing intact, photomodified FLT and Basagranconcentration. Since the UII value corresponds to the elec-tron transport in thylakoid membrane, we might attributeorganics-induced decrease of UII to an inhibition of photo-chemical processes of photosynthesis related to electron

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Fig. 2. The effect of increasing concentration of photomodified fluoranthene (phFLT) on the chlorophyll fluorescence parameters measured in chloroplastsuspension of pea plants after 18 and 25 days of cultivation; F0 – initial chlorophyll fluorescence; FV/FM – ratio of variable to maximal chlorophyllfluorescence and UII – effective quantum yield of photochemical energy conversion in PSII. Data evaluation was the same as given in Fig. 1.



transport chain in thylakoid membrane. It could beassumed that products of PAHs transformation, especiallyquinones, could affect PSII and electron transport to pho-tosystem I. Marwood et al. (1999) found that intact andphotomodified anthracene inhibits electron transportdirectly by blocking photosystem II (PSII), or betweenPSII and PSI at cytochrome-b/f. Similarly, herbicides could

competitively substitute an oxidized form of plastoquinoneQB (Ikeda et al., 2003; Abbaspoor et al., 2006).

The difference of F0, FV/FM, and UII values in chloro-plast suspension treated by organic compounds againstthe control was deepening with the age of plant (25 days).More noticeable changes in chlorophyll fluorescenceparameters were recorded in samples exposed to the

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Basagran 5

Basagran 20

Basagran 35

Basagran 50 control

Basagran 5

Basagran 20

Basagran 35

Basagran 50

IIΦ

Fig. 3. The effect of increasing concentration of Basagran on the chlorophyll fluorescence parameters measured in chloroplast suspension of pea plantsafter 18 and 25 days of cultivation; F0 – initial chlorophyll fluorescence; FV/FM – ratio of variable to maximal chlorophyll fluorescence and UII – effectivequantum yield of photochemical energy conversion in PSII. Data evaluation was the same as given in Fig. 1.



photomodified form of FLT, which is related to physico-chemical properties of photoproducts arising from the pho-tomodification of FLT (Kummerova and Kmentova,2004).

In many studies for the assessment of vegetation con-tamination by PAHs the induced chlorophyll fluorescenceis utilized (Huang et al., 1996; Mallakin et al., 2002). Theevaluation of the effects of another group of organic com-pounds, herbicides, is based on the assessment of Hill reac-tion activity reduction (Baron et al., 1986). In this study

both approaches were applied for the evaluation of theeffect of both FLT and Basagran.

The lipophilic PAHs and their derivatives can disturbthe structure and function of biomembranes. Some organ-ics disrupt cell membranes indirectly through the formationof free radicals, inhibition of lipid synthesis, or changes inthe types of lipids synthesized (Huang et al., 1996; Tukajand Aksmann, 2007).

The mode of negative action of a large number of thecommercial herbicides is based on the inhibition of thephotosynthetic electron flow in the photosystem II complex(Barber and Andersson, 1992; Soskic et al., 1997; Hernan-dez-Terrones et al., 2003). In Hill reaction activity assess-ment considerable difference in the amount of reducedsynthetic electron acceptor DCIP was recorded after bothFLT concentration treatment (0.5 and 5 lmol l�1)(Fig. 4). Fluoranthene and especially products of its pho-tomodification could injure the primary electron donorTyr Z (Dudekula et al., 2005). Greater influence of herbi-cide Basagran on Hill reaction activity is proved by the sig-nificant decrease of DCIP reduction already by 5 nmol l�1

of Basagran. Significant effect of the herbicide not only onthe weed but also on cultivated plants is shown by the sig-nificant percentage reduction of the Hill reaction activityby its presence in concentrations of an order of magnitudelower than applied in the agriculture. Applied concentra-tions were selected with the respect to their direct applica-tion into the suspension of chloroplasts and therefore thepossibility of direct affection of photosynthetic processes.

The results of our study proved, that the effect of bothgroups of organic compounds is related to their concentra-tion, the length of exposure period and on the developmen-tal stage of the plants, too. Moreover, the effect of PAHscould be influenced by abiotic environmental factors likeUV radiation. Our results proved that selected physiologi-cal characteristics (germination test, root elongation test,induced chlorophyll fluorescence, Hill reaction activity)are suitable for the evaluation of the toxicity of intactand photomodified fluoranthene and Basagran. Theyenable to predict partially possible consequences of theenvironmental loading in the subsequent stages of thedevelopment. Nevertheless in the natural environment thevegetation is exposed to the global impact of various fac-tors and therefore the maximal carefulness is necessaryfor the verification of the results in the natural conditions.

Acknowledgement

This work was financed by the Ministry of Education,Youth and Sports of the Czech Republic (MSM0021622412).

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The use of physiological characteristics for comparison of organic compounds phytotoxicity