Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 8
Issue 2
Environmental Development
Available Online: http://www.ejpau.media.pl/volume8/issue2/art-17.html


Małgorzata Hawrot, Andrzej Nowak
Department of Microbiology and Environmental Biotechnology, Agriculture University of Szczecin, Poland



Results of bioassays performed in Department of Microbiology and Environmental Biotechnology as well as statistical analysis point out to significant interdependence among decreasing amounts of diesel fuel in a soil and a germination level of spring barley (Ortega cv.) seeds, length of above ground parts of seedlings and their root system length. Calculated values of resistance indices (RI) express high susceptibility of spring barley towards diesel fuel presence in a soil and potential possibility to utilize the plant as a bio-indicator of changes occurring during remediation of soil polluted with diesel fuel.

Key words: biodegradation, diesel fuel, bioindicators, microorganisms, soil.


Soil pollution with oil-derived substances can be evaluated by means of chemical analyses and indirectly due to estimation of the phytotoxicity and zootoxicity levels. Chemical analysis makes possible to precisely determine the amount and type of a pollutant, whereas the indirect methods show not only the pollution effects, but also the estimation of bioremediation progress [3,5,6]. First of all, assessment of germination ability as well as inhibition of root and above ground part growth is used in bioassays with plants [7].

The aim of paper was to evaluate the efficiency of applied bioassays for assessment the changes of diesel fuel contents in the soil during its biodegradation.


Bioassays were carried out within the field experiment where influence of various bioremediation operations on biodegradation rate of diesel fuel was assessed. Soil under study (dust light loamy sand), with pH 6.7 in 1 mol KCl.dm-3, contained 0.09% of total nitrogen determined by means of Kiejdahl´s method and 1% of total carbon determined by Tiurin´s method. The soil was polluted with 5% diesel fuel. In addition, some modifications were applied (stirring, biogen introduction, microbial inoculation). Study objects created in such a manner are presented in Table 1.

Table 1. List of experimental objects


Applied operations


non-polluted soil, control


polluted soil, not modified with operations


polluted soil, fertilized, stirred


polluted soil, fertilized, stirred, inoculed with tribe mixture

The field experiment was set in a form of plots (1x1m size) by means of completely randomized blocks in four replications; it lasted for 5 months. Stirring (soil digging) was done every 14 days; fertilization was done at the beginning of the experiment and after 3 months. Nitrogen as NH4NO3 (100 kg N.ha-1), phosphorus - P2O5 (80 kg P.ha-1) and potassium - K2O (100 kg K.ha-1) fertilization was applied. Mixture of three earlier selected bacterial cultures was used for inoculation. The strain, definite as BS 101 (Pseudomonas sp.), BS 126 and BS 135 (Bacillus sp.), were described earlier by Hawrot and Nowak [4]. It was introduced into the soil at the beginning of experiment. Non-polluted and not modified with operations soil was the control object.

Level of spring barley (Ortega cv.) seeds was evaluated in bioassays (95% of germination ability). Comprehensive soil sample was taken every 30 days from 0-15 cm layer of every study object and then placed in pots of 500 g capacity. Fifteen barley seeds were sown in each pot at the depth of 1.5 cm. The experiment was performed for 10 days counting the amount of germinating seeds every 2 days. After test completing, the length of above ground part of seedling and length of its root system was measured. Also spring barley´s susceptibility towards diesel fuel presence in the soil was determined in the experiment. Resistance index (RI) proposed by Marecik et al. [8] was used.

Resistance index was calculated according to the following formula:

Modification of the method presented by Marecik et al. [8] referred to the measurement of root system length in the soil and not in the solution containing toxic substance.

Results achieved were subjected to statistical working out applying variance analysis.


The quantity of diesel fuel in the soil affected not only the number of germinating spring barley´s seeds (Fig. 1), but also the length of above ground part of the seedling (Fig. 2), length of root system (Fig. 3), as well as the look of seedlings. All seeds germinated only in control object in the first date of measurement. Diesel fuel at 5% concentration introduced into the soil of remaining objects made impossible to germination of barley´s seeds (Phot. 1).

Fig. 1. Percentage of seed germination in investigation objects

Fig. 2. Mean length of sprout in following measurement dates in experiment

Fig. 3. Mean length of root in following measurement dates in experiment

At the second date (30th day of polluted soil remediation), seeds germinated in fertilized and stirred object (object I), as well as in object II (fertilized, stirred and inoculed). In object I, mean number of seeds that germinated after 10 days of test was slightly above 13% in relation to that in control object. Number of germinating seeds was 33% as compared to the control in object II. Seedlings were twisted and their leaf tops had no pigment.

At 60th day of bioremediation, seeds germinated in all objects polluted with diesel fuel, but the highest germination percentage was observed in object where besides fertilization and stirring, also bio-augmentation (46%) was applied. Mean length of above ground part and root system was the highest in the same object. Seedlings in object 0 were twisted and had no pigment in tops (Phot. 2).

Phot. 1. Results of bioassay using soil taken at the first day of diesel fuel biodegradation (from left side object K, 0, I, II)

Phot. 2. Results of bioassay using soil taken at the 60th day of diesel fuel biodegradation (from left side object K, 0, I, II)

Gradual increase of number of germinating seeds in the soil of particular objects was observed in following dates. Seedling dimensions and length of their root system also increased. At the last day of experiment (after 150 days), 100% of seeds germinated in fertilized, stirred and inoculed object, 80% - in object subjected only to fertilization and stirring, and 46% - in object where no operations helping the diesel fuel biodegradation were applied. At the same time, seedlings still sowed different structure in object 0 than in control one - they were twisted with slightly yellow leaf tops (Phot. 3).

Phot. 3. Results of bioassay using soil taken at the 150th day of diesel fuel biodegradation (from left side object K, 0, I, II)

Statistical analysis revealed highly significant correlation between diesel fuel content in the soil (biodegradation percent) and parameters evaluated in bioassay (Table 2). Changes of diesel fuel content in the soil were performed in another experiment according to norm PN-75/C-04573/10 for study on substances extractable with organic solvents.

Table 2. Matrix of correlation coefficients among parameters studied

Studied parameters

Correlation coefficients





Diesel fuel content in soil(A)





Germination percentage (B)





Length of above ground part (C)





Length of below ground part (D)





** - highly significant correlation coefficients

Comparing mean length of root system in objects polluted with diesel fuel with that in control one, resistance indices were achieved that inform about the susceptibility of spring barley towards diesel fuel presence in the soil. These indices are presented in Table 3.

Table 3. Resistance indices determined in following days of biodegradation


Following days of biodegradation




























Statistical analysis revealed highly significant correlation between measurement date and resistance index value (r = 0.5731). Higher diesel fuel contents decrease were determined in every following measurement date corresponding to the following days of polluted soil bioremediation. Modifications applied in the soil of particular objects favored diesel fuel decomposition process thus its toxic influence on plant´s development and growth diminished. Increasing values of RI, particularly for object II (fertilized, stirred and inoculed), can be the proof. Determined correlation coefficient between diesel fuel content in the soil and resistance index was highly significant; its value was also high (r = 0.8678).


Loss of the diesel fuel in soil, as a result of biodegradation, is determined by means of proper chemical analyses. Bioassays can also be a form of evaluation, because plant´s reactions can reflect the level of environmental pollution with toxic substance [1,7,8]. Significant dependence between decreasing content of diesel fuel in the soil (biodegradation percentage) and spring barley (Ortega cv.) seed germination was found in present study - similarly results received Adam and Duncan [2]. Maliszewska-Kordybach [7] also found highly significant interaction between parameters investigated in study upon the influence of polycyclic aromatic hydrocarbons (PAH) on chosen monocotyledonous and dicotyledonous plants; the lower PAH content, the longer root system and above ground part.

Resistance index (RI) determined for spring barley points out to its high susceptibility associated with the presence of diesel fuel in the soil. Therefore, the plant could be utilized as an indicator of changes during diesel fuel biodegradation in the soil. Furthermore, highly significant correlation between diesel fuel loss in the soil and resistance index value (r = 0.8678), draws to supposition that the determination of this parameter (similarly as germination percentage assessment) can also be a form of monitoring the bioremediation course of soils polluted with oil-derived products.

Study performed point out to wider, than it suggested by Marecik et al. [8], possibility for utilizing the bioassays - not only as a method for selection the plants with phytoremediative properties, but also as a way for finding potential bio-indicators of oil-derived substances presence in the environment, as well as progress of polluted soil bioremediation.


  1. Adam G., Duncan H. J., 1999. Effect of diesel fuel on growth of selected plant species. Environ. Geochem. Health 21: 353-357.

  2. Adam G., Duncan H., 2002. Influence of diesel fuel on seed germination. Environ. Pollut. 120: 363-370.

  3. Błażejewski M., Jankowiak J., Jaskowski J., 1988. Rekultywacja pól uprawnych skażonych substancjami ropopochodnymi. [Reclamation of cultivable fiels contaminated with petroleum substances]. Człow. Śr. 12 (3): 325-344 [in Polish].

  4. Hawrot M., Nowak A., 2003. Evaluation of microorganisms activity in a process of diesel fuel biodegradation during culturing under laboratory conditions. Pol. J. Natur. Sci. 15 (3): 619-628.

  5. Kiepas-Kokot A., 1999. Reakcje dżdżownic Eisenia fetida i rzepiku ozimego na zanieczyszczenie gleb substancjami ropopochodnymi. [Earthworms' reactions Eisenia fetida and winter agrimony on pollution soils with petroleum substances]. VI Ogólnopol. Symp. Nauk.-Tech. "Biotechnologia Środowiskowa", Wrocław, 47-54 [in Polish].

  6. Łebkowska M., 1992. Stan i perspektywy rozwoju metod badań toksyczności i biodegradacji. [Status and perspective of development of methods investigations toxicity and biodegradation]. Biotechnologia 1 (16): 39-46 [in Polish].

  7. Maliszewska-Kordybach B., 1998. Biodegradacja i oddziaływanie WWA w środowisku glebowym, wpływ na rośliny w początkowym stadium ich rozwoju. [Biodegradation and influence PAHs in soil environment, influence on plant in initial stage their development]. Ogólnopol. Symp. Nauk.-Tech. "Bioremediacja gruntów", Wisła-Bukowa, 171-176 [in Polish].

  8. Marecik R., Grajek W., Olejnik A., 1999: Testy korzeniowe jako metoda selekcji roślin o potencjalnych zdolnościach fitoremediacyjnych. [The radicular tests as method of the plants' selection about potential phytoremediation abilities]. VI Ogólnopol. Symp. Nauk.-Tech. "Biotechnologia Środowiskowa", w ramach I Krajowego Kongresu Biotechnologii, Wrocław, 291-298 [in Polish].

Małgorzata Hawrot
Department of Microbiology and Environmental Biotechnology,
Agriculture University of Szczecin, Poland
Słowackiego 17, 71-434 Szczecin, Poland

Andrzej Nowak
Department of Microbiology and Environmental Biotechnology,
Agriculture University of Szczecin, Poland
Słowackiego 17, 71-434 Szczecin, Poland
Phone: +48 91 42 50 234
email: anowak@agro.ar.szczecin.pl

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.