Assessment of the Suitability of Melilotus officinalis for Phytoremediation of Soil Contaminated with Petroleum Hydrocarbons (TPH and PAH), Zn, Pb and Cd Based on Toxicological Tests

Abstract

The article presents issues related to the possibility of using toxicological tests as a tool to monitor the progress of soil treatment contaminated with petroleum substances (TPH, PAH), Zn, Pb and Cd in bio-phytoremediation processes. In order to reduce the high content of petroleum pollutants (TPH = 56,371 mg kg^-1 dry mass, PAH = 139.3 mg kg^-1 dry mass), the technology of stepwise soil treatment was applied, including basic bioremediation and inoculation with biopreparations based of indigenous non-pathogenic species of bacteria, fungi and yeasts. As a result of basic bioremediation in laboratory conditions (ex-situ method), the reduction of petroleum pollutants TPH by 33.9% and PAH by 9.5% was achieved. The introduction of inoculation with biopraparation-1 prepared on the basis of non-pathogenic species of indigenous bacteria made it possible to reduce the TPH content by 86.3%, PAH by 40.3%. The use of a biopreparation-1 enriched with indigenous non-pathogenic species of fungi and yeasts in the third series of inoculation increased to an increase in the degree of biodegradation of aliphatic hydrocarbons with long carbon chains and PAH by a further 28.9%. In the next stage of soil treatment after biodegradation processes, which was characterized by an increased content of heavy metals (Zn, Pb, Cd) and naphthalene, chrysene, benzo(a)anthracene and benzo(ghi)perylene belonging to polycyclic aromatic hydrocarbons, phytoremediation with the use of Melilotus officinalis was applied. After the six-month phytoremediation process, the following was achieved: Zn content by 25.1%, Pb by 27.9%, Cd by 23.2% and TPH by 42.2% and PAH by 49.9%. The rate of removal of individual groups of hydrocarbons was in the decreasing order: C₁₂-C₁₈ > C₆-C₁₂ > C₁₈-C₂₅ > C₂₅-C₃₆. PAHs tended to be removed in the following order: chrysene > naphthalene > benzo(a)anthracene > benzo(ghi)perylene. The TF and BCF coefficients were calculated to assess the capacity of M. officinalis to accumulate metal in tissues, uptake from soil and transfer from roots to shoots. The values of TF translocation coefficients were, respectively, for Zn (0.44), Pb (0.12), Cd (0.40). The calculated BCF concentration factors (BCF_roots > BCF_shoots) show that heavy metals taken up by M. officinalis are mainly accumulated in the root tissues in the following order Zn > Pb > Cd, revealing a poor metal translocation from the root to the shoots. This process was carried out in laboratory conditions for a period of 6 months. The process of phytoremediation of contaminated soil using M. officinalis assisted with fertilization was monitored by means of toxicological tests: Microtox, Ostracodtoxkit F^TM, MARA and Phytotoxkit^TM. The performed phytotoxicity tests have indicated variable sensitivity of the tested plants on contaminants occurring in the studied soils, following the sequence: Lepidium sativum < Sorghum saccharatum < Sinapis alba. The sensitivity of toxicological tests was comparable and increased in the order: MARA < Ostracodtoxkit F^TM < Microtox. The results of the toxicological monitoring as a function of the time of soil treatment, together with chemical analyses determining the content of toxicants in soil and biomass M. officinalis, clearly confirmed the effectiveness of the applied concept of bioremediation of soils contaminated with zinc, lead and cadmium in the presence of petroleum hydrocarbons.

Keywords: Melilotus officinalis; biodegradation; heavy metals; phytoremediation; polycyclic aromatic hydrocarbons; soil; total petroleum hydrocarbons; toxicological tests.

Figure 1

Comparison of identified n-alkane contents in Soil B: (a) after basic bioremediation; (b) after successive series of inoculations in laboratory conditions—ex-situ method (repetition number n = 9–10 p < 0.05). (Soil B) raw soil; (Soil B1) soil after basic bioremediation; (Soil B2) soil after inoculation with I biopreparation (1st series); (Soil B3) soil after inoculation with I biopreparation (2nd series); (Soil B4) soil after inoculation with II biopreparation (3rd series).

Figure 2

Alternation in polycyclic aromatic hydrocarbons (PAH) contents after consecutive stages of soil B purification in laboratory conditions—ex-situ method. (Soil B) raw soil; (Soil B1) soil after basic bioremediation; (Soil B2) soil after inoculation with I biopreparation (1st series); (Soil B3) soil after inoculation with I biopreparation (2nd series); (Soil B4) soil after inoculation with II biopreparation (3rd series). Naphthalene (N), Anthracene (A), Chryzene (CH), Benzo(a)anthracene (BaA), Dibenzo(a,h)anthracene (DaA), Benzo(a)pyrene (BaP), Benzo(b)fluoranthene (BbF), Benzo(k)fluoranthene (BkF), Benzo(ghi)perylene (BghiP), Indeno(1,2,3-cd)pyrene (IndP).

Figure 3

Comparison of the results of toxicity tests (expressed in TU toxicity units) of soil during the successive stages of biodegradation of petroleum pollutants—ex-situ method (repetition number n = 3, p < 0.05). (Soil B) raw soil; (Soil B1) soil after basic bioremediation; (Soil B2) soil after inoculation with I biopreparation (1st series); (Soil B3) soil after inoculation with I biopreparation (2nd series); (Soil B4) soil after inoculation with II biopreparation (3rd series).

Figure 4

Comparison of reduction in the concentrations of Zn, Pb, Cd, petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAH) during phytoremediation of soils: raw (A, B4), after fertilization (AF, B4F) and after phytoremediation carried out with M. officinalis (AFPh, BFPh) (repetition number n = 5–6, p < 0.05).

Figure 5

Comparison of the reduction in the content of specified groups hydrocarbons of TPH during phytoremediation of soils: raw (A, B4), after fertilization (AF, B4F) and after phytoremediation carried out with M. officinalis (AFPh, B4FPh) (n = 5–6, p < 0.05).

Figure 6

Comparison of the reduction in the content of individual PAH during phytoremediation of soils: raw (A, B4), after fertilization (AF, B4F) and after phytoremediation carried out with M. officinalis (AFPh, B4FPh) (repetition number n = 5–6, p < 0.05). Naphthalene (N), Anthracene (A), Chryzene (CH), Benzo(a)anthracene (BaA), Dibenzo(a,h)anthracene (DaA), Benzo(a)pyrene (BaP), Benzo(b)fluoranthene (BbF), Benzo(k)fluoranthene (BkF), Benzo(ghi)perylene (BghiP), Indeno(1,2,3-cd)pyrene (IndP).

Figure 7

Concentration of Zn, Pb and Cd in the biomass (shoots and roots) after 6-month phytoremediation enhanced by fertilization of soils AFPh and B4FPh (repetition number n = 3, p < 0.05).

Figure 8

Comparison of root growth inhibition (Lepidium sativum, Sinapis alba, Sorghum saccharatum) in the Phytotoxkit^TM test during soil phytoremediation (repetitions number n = 3, p < 0.05).

Figure 9

Comparison of death rate and growth inhibition (Heterocypris incongruens) during phytoremediation carried out with Melilotus officinalis, enhanced by fertilization of soils A and B4 (repetitions number = 3, p < 0.05).

Figure 10

Environmental risk assessment test MARA for soils A and B4 during 6 months of phytoremediation augmented by soils fertilisation AF and B4F with microorganisms (1. Microbacterium spaciec, 2. Brevundimonas diminuta, 3. Citrobacter freudii, 4. Comamonas testosteroni, 5. Entrococcus casseliflavus, 6. Delftia acidovorans, 7. Kurthia gibsoni, 8. Staphylococcus warneri, 9. Pseudomonas aurantiaca, 10. Serriatia rudidaea, 11. Pichia anomala), (MTC) Microbial Toxic Concentration (repetitions number n = 3, p < 0.05).