- Email: [email protected]
The effect of PCB-contaminated sewage sludge and sediment on metabolism of cucumber plants (Cucumis sativus L.)
Ecohydrology & Hydrobiology 14 (2014) 75–82
Contents lists available at ScienceDirect
Ecohydrology & Hydrobiology journal homepage: www.elsevier.com/locate/ecohyd
Original Research Article
The effect of PCB-contaminated sewage sludge and sediment on metabolism of cucumber plants (Cucumis sativus L.) Anna Wyrwicka a,*, Steffani Steffani b, Magdalena Urbaniak b,c a
University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Plant Physiology and Biochemistry, Banacha 12/16, 90-237 Ło´dz´, Poland b University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Applied Ecology, Banacha 12/16, 90-237 Ło´dz´, Poland c European Regional Centre for Ecohydrology of the Polish Academy of Sciences, Tylna 3, 90-364 Ło´dz´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 October 2013 Received in revised form 1 January 2014 Accepted 13 January 2014 Available online 8 February 2014
Increasing amounts of sewage sludge are produced nowadays, which need to be disposed of in a safe and responsible manner. Likewise, bottom sediments from small urban water bodies undergo periodical dredging and need to be utilized. These deposits often contain Persistant Organic Pollutants (POPs) and other toxic substances. Plants can be used to reduce these pollutants during or before disposal to land (phytoremediation). Cucurbitaceae are known to accumulate high levels of POPs, including polychlorinated biphenyls (PCB), compared with other plant species but such accumulation may lead to secondary oxidative stress that may limit their value. This study examined the impact of sewage sludge and urban lake sediment on soil toxicity, measured as PCB concentration, and changes in the antioxidative system of cucumber plants grown in the soils. There was an average reduction of PCB by 38.63% and 27.38% in soil amended with sewage sludge and sediment, respectively after 5 weeks of cucumber plant cultivation. In the case of plants grown with sewage sludge, guaiacol peroxidase (POx) activity significantly decreased to 49% of the control at the highest dose given, while that of glutathione S-transferase (GST) increased to 172% of the control value in the same treatment. a-Tocopherol concentration was higher in the plants grown in the sewage sludge amended soil. ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Phytoremediation PCDD PCDF Antioxidants Enzyme assay a-Tocopherol
1. Introduction Implementation of the Urban Waste Water Treatment Directive (UWWTD) (91/271/EEC) in Poland led to a sharp increase in the production of sewage sludge from nearly 350 000 to 641 000 Mg of d.w./yr in the period from 2000 to 2010. In the year 2015 this amount may exceed 713 000 Mg
* Corresponding author at: University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Plant Physiology and Biochemistry, Banacha 12/16, 90-237 Ło´dz´, Poland. Tel.: +48 42 6354420; fax: +48 42 6354423. E-mail address: [email protected] (A. Wyrwicka).
of d.w./yr. Since Poland is a major sewage sludge producer in Europe, with the mean value of about 37 kg per capita per year (Przewrocki et al., 2004), this increase has posed problems with its utilization and disposal. On the one hand, sewage sludge contains nutrients such as nitrogen and phosphorus, that are useful for agriculture (Rulkens, 2008; US EPA, 1998), on the other hand, it also contains a variety of toxic compounds including heavy metals, Persistent Organic Pollutants (POPs), Polycyclic Aromatic Hydrocarbons (PAHs), as well as pathogens and other microbial pollutants, that pose hazard to human health. The situation is similar for sediments deposited at the bottom of urban reservoirs and sedimentation ponds as
1642-3593/$ – see front matter ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.ecohyd.2014.01.003
76
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
they contain a broad range of pollutants originating from urban catchments (Marsalek et al., 2006; Wagner et al., 2008) including heavy metals, plant fertilizers and pesticides from lawns and gardens, oil from leaky cars, pet waste and other pollutants such as POP and PAH (Luna, 1968; Tsihrintzis and Hamid, 1997; US EPA, 1999; Walsh et al., 2004; Wildi et al., 2004; Christensen et al., 2006; Wagner et al., 2007; Jartun and Pettersen, 2010; Urbaniak et al., 2010, 2012, 2013; Urbaniak, 2013). One way of coping with the growing amount of sewage sludge and urban lake sediments, when they have to be dredged from reservoirs, is to use phytoremediation, the use of plants to reduce pollutants so that they are metabolized or concentrated for easier disposal (US EPA, 1999; Macek et al., 2000; Urbaniak, 2013). Some species of cucumber family, Cucurbitaceae, are known to accumulate higher levels of POP such as polychlorinated dibenzo-pdioxins (PCDD), polychlorinated dibenzofurans (PCDF) (Hu¨lster et al., 1994; Inui et al., 2008; Zhang et al., 2009) and PCB (White et al., 2005) in their tissues, compared with other plant species. Transfer of POP from soil to plants is especially interesting because these substances are strongly absorbed on soil organic matter and are rarely transported into plant roots and then transferred into aboveground plant tissues. Their high hydrophobicity suggests that the major pathway by which POP enter plant tissues is absorption from soil vapour (Trapp and Matthies, 1997). Many plant species e.g., lettuce, potato, tomato, rice, garland chrysanthemum, Chinese cabbage, maize, and soybean, can absorb POP through their roots. However, the amounts of these substances translocated from roots to shoots are usually very small. In contrast, the Cucurbitaceae can accumulate POP from soil in their leaves and fruits (Inui et al., 2008; Zhang et al., 2009). Consequently they may show secondary oxidative stress, which is the overproduction of reactive oxygen species (ROS) in plant cells (Gabara et al., 2003; Wyrwicka and Skłodowska, 2006; Gajewska et al., 2013). Oxidative stress occurs when there is a serious imbalance between the production of ROS and their neutralization. The production of ROS is a natural phenomenon in plant tissues and accompanies photosynthesis and respiration. Endogenously overproduced ROS such as superoxide anion (O2), hydroxyl radicals (OH), and peroxyl radicals (ROO) as well as singlet oxygen (1O2) and hydrogen peroxide (H2O2) spontaneously damage membrane lipids and proteins, nucleic acids and other biologically important molecules (Mittler, 2002) and may reduce plant growth and development. Plant cells are equipped with a large and integrated system of enzymatic and non-enzymatic antioxidants that regulate ROS level. Enzymatic antioxidants include superoxide dismutase (SOD; EC 1.15.1.1) (Dhindsa et al., 1981), catalase (CAT; EC 1.11.1.6) (Willekens et al., 1995), ascorbate peroxidase (APx; EC 1.11.1.11) (Asada, 1992), phenolic peroxidases e.g., guaiacol peroxidase (POx; EC 1.11.1.7) (Tayefi-Nasrabadi et al., 2011) and glutathione Stransferase (GST; EC 2.5.1.18) (Edwards and Dixon, 2004) while non-enzymatic ones include e.g., ascorbic acid (Noctor and Foyer, 1998), glutathione (Tausz and Grill, 2000), carotenoids, and a-tocopherol (Schafer et al., 2002).
The aim of the present study was to assess the impact of sewage sludge and urban reservoir sediment application to soils on toxicity, measured as PCB concentration, and changes in selected elements of the Cucumis sativus L. (cucumber) antioxidative system when the plants were grown on these soils. In particular the content of atocopherol, the main lipophilic non-enzymatic antioxidant, was measured to assess its function in plant defence. Activities of antioxidative and detoxifying enzymes such as POx and GST were determined to estimate the efficiency of protective systems. 2. Materials and methods 2.1. Soil preparation Sewage sludge from Lodz Municipal Wastewater Treatment Plant (LM WWTP) and sediments from the Sokoło´wka Sequential Biofiltration System (SSBS) were collected. The Sokoło´wka Sequential Biofiltration System was constructed in the upper section of the Sokoło´wka River in order to remove sediments, suspended solids, particulate pollutants, petroleum hydrocarbons, heavy metals and nutrients from stormwater runoff through its sedimentation and filtration mechanisms. The system comprises three different zones: the zone of hydrodynamically intensified sedimentation, the zone of intensive biogeochemical processes and the zone of intensive biofiltration. The sediment samples used in the experiment were collected from the first zone wherein the accelerated sedimentation of suspended matter and pollutants associated with it occurs. The sewage sludge and sediment samples were dried at 70 8C for 72 h then homogenized into small particles using a mortar and used as fertilizer for the soil samples for cucumber planting. The vegetable potting soil (specified for cucumber growth) used in the experiment was collected from Hollas Sp. z o.o. Pasłe˛k. Four treatments were used: a control C in which no sludge or sediment was added, and three levels of addition, 1.8 g, 5.4 g and 10.8 g per flower pot. The first corresponds to the dose of 3 tonnes ha1, the allowed dosage per year by the Regulation of the Minister of Environment of 13 July 2010 on municipal sewage sludge (Dz.U. Nr 137/2010 r., poz. 924); the second is the permitted dose of 9 tonnes ha1 per 3 years applied on one occasion; and the third, 18 tonnes ha1 is above the permitted level. Treatments are designated by the numerical dose per pot and the abbreviations SS for sewage sludge and SED for sediment. 2.2. Plant material Cucumber seeds (C. sativus L.) cv ‘‘Cezar’’ were germinated in Petri dishes for 7 days and the seedlings were planted into the control and sewage sludge- or sediment-amended soil. They were grown in a growth chamber at of 23 0.5 8C with 16 h light/8 h dark cycle and with 150 mmol m2 s1 photon flux density during the light period, and 60% relative humidity. Five-week-old plants with five fully expanded leaves were used for subsequent analysis.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
All biochemical analyses were carried out on the second, third and fourth leaves from the control and treated plants. The leaves were harvested in the middle of the 16 h light period. 2.3. PCB concentrations PCB concentrations were determined in the control and amended soils before cucumber planting (the first day of the experiment) and after 5 weeks of cucumber growth (the last day of the experiment). The collected soil samples were stored in glass tubes in the dark at 4 8C for further extraction using a PCB RaPID Assay Sample Extraction Kit purchased from Tigret Ltd., and PCB analyzed using Enzyme Linked Immunosorbent Assay (ELISA) – PCB RaPID Assay. The PCB RaPID Assay kit applies the principles of enzyme linked immunosorbent assay to the determination of PCB and related compounds. The sample to be tested is added, along with an enzyme conjugate, to a disposable test tube, followed by paramagnetic particles with antibodies specific to PCB attached. Both, PCB and the enzyme conjugate compete for the antibody binding sites on the magnetic particles. The presence of PCB is detected by adding the enzyme substrate (hydrogen peroxide) and chromogen (3,30 ,5,50 -tetramethylbenzidine). The enzyme conjugate analogue bound to the PCB antibody catalyzes the conversion of the substrate/ chromogene mixture to a coloured product. Since enzyme competes with PCB for the antibody sites, the colour developed is inversely proportional to the concentration of PCB in the sample. Prior to running the assay, PCB in the given soil sample have to be extracted. In order to extract PCB, 10 g of soil (dry matter) were extracted into 20 mL of methanol in extraction jars, shaken for 1 min and left at room temperature for 1–2 min to separate the soil and solvent layers. The solvent was filtered and 25 mL was diluted in 25 mL of PCB Extract Diluent. The obtained extract was used as sample for further PCB analysis using a PCB RaPID Assay kit. In order to analyze the PCB content using the PCB RaPID Assay kit, aliquots (200 mL) of calibration standard PCB (0, 0.5, 2 and 10 ppm), the extracted sample, and the positive control solution (6 ppm) were added to the test tubes together with aliquots of enzyme conjugate (250 mL). After this, an aliquot (500 mL) of antibody, coupled with magnetic particles in buffered saline containing preservative and stabilizers, was added, thoroughly mixed and incubated at room temperature for 15 min using a RaPID Magnetic Separator. After incubation, the contents of each vial were decanted to a waste container to remove the solution containing any unbound reagents. The vials were then washed twice with washing solution (1 mL/vial). Following washing, an aliquot (500 mL) of colour solution containing hydrogen peroxide and 3,30 ,5,50 -tetramethylobenzidine in an organic base was added to each vial, shaken and incubated for 20 min. At the end of the incubation period an aliquot (500 mL) of stopping solution, containing 2 M sulphuric acid was added to each vial. The absorbance of
77
the liquid in each vial was measured at 450 nm using a SDI Differential Spectrophotometer. 2.4. Preparation of enzyme extracts from leaf tissues The leaves of the cucumber plants were ground (1:10, w/v) in an ice-cold mortar using 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 M NaCl, 1 mM EDTA, and 1 mM sodium ascorbate. The slurry was filtered through two-layers of Micracloth. The filtrate of homogenized cucumber leaves was then centrifuged (15,000 g for 15 min). After centrifugation, the supernatant was collected and POx and GST activities and protein concentration were measured. 2.5. Protein content of the extracts The protein content was determined by Bradford’s method (1976) with standard curves prepared using bovine serum albumin. 2.6. Enzyme assay POx activity was assayed according to a method modified from Maehly and Chance (1954) with guaiacol as a substrate. The reaction mixture contained 49 mM sodium acetate buffer (pH 5.6) 5 mM guaiacol, 15 mM H2O2, and enzyme extract (15–25 mg protein). The linear increase in absorbance at 470 nm due to the formation of tetraguaiacol (the millimolar extinction coefficient of tetraguaiacol at 470 nm; e = 26.6 mM1 cm1) was monitored for 4 min at 25 8C. Enzyme activity was expressed in mmol tetraguaiacol min1 mg1 protein. The total GST activity was determined with 1-chloro2,4-dinitrobenzene (CDNB) by a method modified from Habig et al. (1974). GST catalyses the conjugation of Lglutathione (GSH) to CDNB through the thiol group of GSH. The product of CDNB conjugation with GSH, dinitrophenyl thioether absorbs at 340 nm (e = 9.6 mM1 cm1). The reaction solution contained 100 mM potassium phosphate buffer (pH 6.25), 0.75 mM CDNB, 30 mM GSH and the enzyme extract (50 mg protein). Enzyme activity was expressed in units representing formation of dinitrophenyl thioeter (nmol min1 mg1 protein). All assays were made spectrophotometrically (UNICAM UV 300 UV-visible spectrometer) at 25 8C. 2.7. Determination of a-tocopherol Whole leaves were homogenized (1:5, w/v) in an ice-cold mortar using 50 mM sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl, 1% polyvinylpyrrolidone and 1 mM EDTA. The crude homogenate obtained after filtration was assayed for a-tocopherol content by the method of Taylor and Tappel (1976). After saponification of the sample with KOH in the presence of ascorbic acid, a-tocopherol was extracted with n-hexane. Fluorescence of the organic layer was measured at 280 nm (excitation) and 310 nm (emission) wavelengths using a F-2500 Fluorescence Spectrophotometer (Hitachi, Limited, Tokyo Japan). The
78
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
concentration of a-tocopherol was expressed as mg g1 fresh mass of original plant tissue. 2.8. Statistical analysis The results of PCB analysis were tested using Wilcoxon matched pair test. Differences of P < 0.05 were considered significant. The results presented for POx, GST and atocopherol are the means of eight replicates in two independent experiments. Sample variability is given as the standard deviation (SD) of means. The significance of differences between mean value was determined by a Student t test. Differences with P < 0.05 were considered significant. 3. Results 3.1. PCB concentration in soil before and after C. sativus L. cultivation Addition of sewage sludge led to increase in the PCB concentration in the soil samples. The smallest PCB concentration was noted in the control soil and the highest in soil amended with the highest sewage sludge dose (10.8 SS) (Fig. 1a). The analysis conducted after 5 weeks of cucumber growth showed reductions of PCB concentration by 6.25% (from 0.512 ppm to 0.480 ppm) in the control soil, 41.28% (from 0.940 ppm to 0.552 ppm) in
Fig. 1. Changes of PCBs concentrations in the soil fertilized with: (a) LM WWTP sewage sludge and (b) SSBS sediments, before &/ and after / 5 weeks of Cucumis sativus L. cultivation.
Fig. 2. Influence of LM WWTP sewage sludge and SSBS sediments on POx activity in the cucumber leaves. Symbols (*), (**), and (***) indicate values that differ significantly from the control plant at P < 0.05, P < 0.01, and P < 0.001, respectively. Symbols (a), (aa), (aaa) indicate values for 5.4 SS/5.4 SED that differ significantly from 1.8 SS/1.8 SED at P < 0.05, P < 0.01, and P < 0.001 respectively. Symbols (b), (bb), (bbb) indicate values for 10.8 SS/10.8 SED that differ significantly from 1.8 SS/1.8 SED at P < 0.05, P < 0.01, and P < 0.001 respectively. Symbols (c), (cc), (ccc) indicate values for 10.8 g that differ significantly from 5.4 SS/5.4 SED at P < 0.05, P < 0.01, and P < 0.001 respectively.
1.8 SS, by 38.39% (from 1.120 ppm to 0.690 ppm) in 5.4 SS, and by 36.22% (from 1.154 ppm to 0.736 ppm) in 10.8 SS. The lower the sewage sludge dose the greater the reduction of PCB concentration was observed. Nevertheless, the obtained values showed no statistically significant differences (Wilcoxon matched pair test). Addition of sediments also increased the PCB concentration in the soil samples. The highest concentration was noted at the highest sediment dose (10.8 SED) (Fig. 1b). After 5 weeks of cucumber growth, PCB concentrations were reduced by 7.90% (from 0.508 ppm to 0.472 ppm) in control soil, by 25.24% (from 0.808 ppm to 0.604 ppm) in the case of 1.8 SED; by 27.08% (from 0.960 ppm to 0.700 ppm) in 5.4 SED, and by 29.82% (from 1.120 ppm to 0.786 ppm) in 10.8 SED (Fig. 1b). The results demonstrated an increase in PCB reduction with increasing amendment of sediment. Nevertheless, also in this case no statistically significant differences were noted.
Fig. 3. Influence of LM WWTP sewage sludge and SSBS sediments on GST activity in the cucumber leaves. For further explanation, see Fig. 2.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
Fig. 4. Influence of LM WWTP sewage sludge and SSBS sediments on a-tocopherol concentration in the cucumber leaves. For further explanation, see Fig. 2.
3.2. Enzyme activities POx activity in the leaves of cucumber plants cultivated with sewage sludge showed significant changes from the control plants and among treatments (Fig. 2). Significant decrease in POx activity was observed in each treatment reaching 70% (P < 0.001), 65% (P < 0.001), and 49% (P < 0.001) of the control plant value for 1.8 SS, 5.4 SS and 10.8 SS, respectively. Moreover, significant differences were also observed among treatments. In 10.8 SS, POx activity was 71% (P < 0.01) and 76% (P < 0.01) of those observed in 1.8 SS and 5.4 SS treatments, respectively. In sediment-supplemented cultures, POx activity changes were not statistically significant from each other or the control (Fig. 2). The activity of GST increased with sewage sludge addition (Fig. 3). There were significant increases in 10.8 SS to 172% (P < 0.001) of the control value and to 170% (P < 0.001) and 148% (P < 0.001) of the values in 1.8 SS and 5.4 SS, respectively. GST activity in 10.8 SED was 117% (P < 0.05) of that for 5.4 SED variant (Fig. 3) but other differences for sediment amendment treatments and control were not significant. 3.3. a-Tocopherol concentration
a-Tocopherol concentration in the cucumber leaves increased with sewage sludge addition, but significant changes were observed only for 10.8 SS compared with control (150%, P < 0.01) and for 5.4 SS compared with 1.8 SS (119%, P < 0.05). The highest a-tocopherol concentration was observed in 10.8 SS with 12.86 mg g1 fresh mass. Amendment with sediment had no significant effects (Fig. 4). 4. Discussion Plants have been frequently shown to be able to remove POP from soils. Siciliano et al. (2003) demonstrated reduction of organochlorine compounds by about 30% during 2 years of plant cultivation; in soil without plants, the reduction was half as much. Nedunuri et al. (2000) reported reduction of aromatic compounds by about 42%
79
and 50% with fibre flax (Lolium annua) and St. Augustine grass (Stenotaphrum secundatum), respectively, over 21 months. Vervaeke et al. (2003) showed 57% reduction of aromatic compounds and mineral oils during 1.5 years of willow (Salix viminalis) cultivation. Other examples, using a combination of grass and fertilizers, showed remediation of soil contaminated with crude oil (Robinson et al., 2002; Banks et al., 2003; White et al., 2006). Zhang et al. (2009) showed that Cucurbita roots were very effective in dioxin uptake and its subsequent translocation. Cucumber and towel gourd (Luffa cylindrica Roem.) accumulated 0.5–3.5 times higher concentrations of dioxins than the predicted gross vapour leakages, indicating that they can significantly translocate dioxins from roots to shoots. The present study demonstrated an average 38.63% and 27.38% reduction of PCB in sewage sludge and sediment amended soils after 5 weeks of the cucumber plant cultivation. The removal of PCBs from the sewage sludge-amended soil was greater than its reduction from the soil fertilized with sediments. However, in the case of sewage sludge-amended soil the relative reduction decreased with the dose of sludge. The opposite situation was noted for the soil fertilized with sediments, where increasing sediment dose increased removal efficiency, but since none of these differences were statistically significant, they remain only indicative and may simply be due to random chance. The decreasing rate of PCB removal in the case of sewage sludge amended soils may be related to the occurrence of a wide range of other pollutants in the sewage sludge. Heavy metals are among the most important and dangerous substances noted in the sewage sludge from the LM WWTP (Drobniewska, 2006, 2008). Their very high concentrations, which usually exceed the allowed doses (according to government regulations in Poland: Dz.U. Nr 137/2010 r., poz. 924) suggest that they can negatively affect soil and plant condition (Gabbrielli et al., 1999; Gajewska et al., 2013) and thus influence the ability of treated plants to effectively remove the studied PCB. The uptake of contaminants by plants may also depend on other environmental factors. Inui et al. (2008) reported that this process was affected by organic matter content in soil, moisture content, pH and bioavailability of nutrients as well as plant characteristics. Moreover, the ability of plants to absorb PCB and to remove them from soil may be correlated with the particular mixture of PCB compounds present and the individual properties of particular PCB congeners. According to Whitfield A˚slund et al. (2007), highly chlorinated PCB congeners are least mobile and least soluble and thus become progressively adsorbed to the hydrophobic components in the lower part of the plant stem. Many studies have provided evidence that peroxidases, part of the cellular H2O2 detoxifying system, participate in important physiological processes such as lignification, suberinization and auxin catabolism. Guaiacol peroxidase (POx) is a crucial enzyme found in the cytosol, cell wall, vacuole and extracellular spaces and is involved in a range of processes related to plant growth and development. POx consumes H2O2 to generate phenoxy compounds that are polymerized to produce cell wall components such as lignin (Reddy et al., 2005). Our results showed a decrease in
80
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
POx activity in cucumber plants with increasing addition of sewage sludge. This may suggest the presence of toxic compounds in the sludge and an inhibitory effect on POx activity. In this situation toxic H2O2, the substrate for POx, might build up in plant cells. In tobacco cells exposed to low concentration of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans PCDD/PCDF in a suspension culture, POx activity was significantly increased (Zhang et al., 2012). However, it decreased with increasing concentration of PCDD/PCDF. The authors suggest that low concentrations of toxic substances allow plant cells to augment POx activity and to counteract oxidative stress, but at high concentrations, a significant toxic effect was observed. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in xenobiotic metabolism in plants (Marrs, 1996). GST catalyze the conjugation of reduced glutathione (GSH) with electrophilic substrates to form GSH-substrate conjugates, which are more polar and less toxic. Although GST were first noted as herbicide detoxifying enzymes in plants, many authors have demonstrated their involvement in the detoxification of PAH, PCB and heavy metals. Our results showed that in the leaves of plants grown in sewage sludge-amended soil, GST activity increased significantly, especially in 10.8 SS. High activity of this effective detoxifying enzyme indicates increased toxicity of soil. According to Flury et al. (1996) genes for GST are induced by factors that cause oxidative damage. The collective changes in POx and GST activities could also be interpreted as a compensatory mechanism for the reduced POx activity. In cucumber plants, GST may be an important enzyme to overcome oxidative stress, because no increase in other antioxidative enzymes was observed. Tocopherol is the main nonenzymatic antioxidant of the lipophilic fraction of cells. This molecule is synthesized only by photosynthetic organisms. Tocopherol maintains stability of cell membranes, and, as an antioxidant, protects chloroplast membranes against photooxidation (Trebst, 2003). Moreover, it seems to play a role in plant growth, development and senescence (Horvath et al., 2006). It is thought that tocopherol affects carbohydrate metabolism and is involved in controlling photosynthesis. Its physiological significance has been confirmed by studies showing increase in tocopherol content in plant tissue under unfavourable environmental conditions. After exposure of Arabidopsis plants to high light, a-tocopherol concentration approximately doubled (Giacomelli et al., 2007). Moreover, concentration of this lipophilic antioxidant increased in tobacco plants with leaf age (Dertinger et al., 2003). Increased content of a-tocopherol in our cucumber plants grown in the sewage sludge-amended soil might be correlated with the intensity of oxidative stress and might suggest that this antioxidant molecule participated in protection of plant cells against accelerated senescence caused by exposure to toxic compounds present in soil. Our results revealed that plants grown in the soil with the largest dose of sewage sludge showed a (nonsignificant) decrease in efficiency of PCB removal from soil, but a significant inhibitory effect on POx activity and the largest GST activity were observed suggesting high efficiency of detoxification processes. Decrease in POx
activity may contribute to ROS accumulation in plant cells and to increase in the concentration of the substances formed as a result of oxidative damage. Both processes might increase GST activity since GST gene transcription is directly induced by H2O2 and endogenous substances resulting from oxidative damage, which are induced mainly by hydroxyl radicals (OH), and are highly toxic (Danielson et al., 1987). In the sediment treatments, on the contrary, there was no significant response in the production of these enzymes, although PCB were present in the sediments. It is possible that in sediments, toxic substances other than for PCB, are present at lower concentrations and do not have the inhibitory effect on plant metabolism found for sewage sludge. PCB may therefore not be responsible for the plant reactions. It is also possible that PCB congeners present in the sediments are different and may be less toxic from those present in the sewage sludge. It cannot either be excluded that the ratio of toxic substances to nutrients is lower and more favourable in sediments and that the plants may be responding to higher nutrient levels. In sewage sludge, potassium concentration is limited because during its purification, potassium is removed, which does not occur in sediments (Drobniewska, 2006, 2008). These initial data provide a start for further investigations, including effects of different PCB congener patterns in sewage sludge and in sediments. It is always difficult to attribute cause and effect when dealing with collections of pollutants, such as occur in complex mixtures like sludge and sediment, and precise attribution may never be possible. The data do support the contention, however, that cucumber plants remove PCB from contaminated soils, though this is only one of many processes that might affect their growth. Further research might focus on the mechanism of PCB action and activation of plant protective systems that might improve the phytoremediation process. Conflict of interest None declared. Financial disclosure This research has been carried out as part of the following projects: Erasmus Mundus Master of Science in Ecohydrology (ECOHYD, 159659-1-2009-1-PT-ERA MUNDUS-EMMC) and ‘Innovative resources and effective methods of safety improvement and durability of buildings and transport infrastructure in sustainable development’ financed by the European Union from the European Fund of Regional Development based on the Operational Program of the Innovative Economy, POIG.01.01.02-10-106/09. References Asada, K., 1992. Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants. Physiologia Plantarum 85, 235–241. Banks, M.K., Kulakow, P., Schwab, A.P., Chen, Z., Rathbone, K., 2003. Degradation of crude oil in the rhizosphere of Sorghum bicolor. International Journal of Phytoremediation 5, 225–234. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein– dye binding. Analytical Biochemistry 72, 248–254.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82 Christensen, A.M., Nakajima, F., Baun, A., 2006. Toxicity of water and sediment in a small urban river (Store Vejlea˚, Denmark). Environmental Pollution 144, 621–625. Danielson, U.H., Esterbauer, H., Mannervik, B., 1987. Structure–activity relationships of 4-hydroxyalkenals in the conjugation catalyzed by mammalian glutathione S-transferases. Biochemical Journal 247, 707–712. Dertinger, U., Schaz, U., Schulze, E.-D., 2003. Age-dependence of the antioxidative system in tobacco with enhanced glutathione reductase activity or senescence-induced production of cytokinins. Physiologia Plantarum 119, 19–29. Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany 32, 93–101. Drobniewska, A., 2006. Sewage water management for environment quality and positive socio-economic feedbackIn: Demonstration Activity Report, The City of Lodz, GOCE 0118530 SWITCH. Drobniewska, A., 2008. Optymalizacja wykorzystania osadu s´ciekowego do produkcji bioenergii na podstawie wielkoskalowych eksperymento´w terenowych oraz modelu matematycznego. (Optimization of the Use of Sewage Sludge for Bioenergy Production on the Basis of LargeScale Field Experiments and Mathematical Model). (Ph.D. thesis)University of Lodz. Edwards, R., Dixon, D.P., 2004. Metabolism of natural and xenobiotic substrates by the plant glutathione S-transferase superfamily. In: Sandermann, H. (Ed.), Ecological Studies. Molecular Ecotoxicology of Plants, vol. 170. Springer-Verlag, Berlin, Heidelberg, pp. 17–50. Flury, T., Wagner, E., Kreuz, K., 1996. An inducible glutathione S-transferase in soybean hypocotyl is localized in the apoplast. Plant Physiology 112, 1185–1190. Gabara, B., Skłodowska, M., Wyrwicka, A., Glin´ska, S., Gapin´ska, M., 2003. Changes in the ultrastructure of chloroplasts and mitochondria and antioxidant enzyme activity in Lycopersicon esculentum Mill. leaves sprayed with acid rain. Plant Science 164, 507–516. Gabbrielli, R., Pandolfini, T., Espen, L., Palandri, M.R., 1999. Growth, peroxidase activity and cytological modifications in Pisum sativum seedlings exposed to Ni2+ toxicity. Journal of Plant Physiology 155, 639–645. Gajewska, E., Głowacki, R., Mazur, J., Skłodowska, M., 2013. Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions. Plant Growth Regulation 71, 13–20. Giacomelli, L., Masi, A., Ripoll, D.R., Lee, M.J., van Wijk, K.J., 2007. Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low. Plant Molecular Biology 65, 627–644. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercaptane acid formation. Journal of Biological Chemistry 246, 7130–7139. Horvath, G., Wessjohann, L., Bigirimana, J., Jansen, M., Guisez, Y., Caubergs, R., Horemans, N., 2006. Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues. Phytochemistry 67, 1185–1195. Hu¨lster, A., Mueller, J.F., Marschner, H., 1994. Soil–plant transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans to vegetables of the cucumber family (Cucurbitaceae). Environmental Science and Technology 28, 1110–1115. Inui, H., Wakai, T., Gion, K., Kim, Y.-S., Eun, H., 2008. Differential uptake for dioxin-like compounds by zucchini subspecies. Chemosphere 73, 1602–1607. Jartun, M., Pettersen, A., 2010. Contaminants in urban runoff to Norwegian fjords. Journal of Soils and Sediments 10, 155–161. Luna, B.L., 1968. Hydrology for Urban Land Planning. A Guidebook on the Hydrologic Effects of Urban Land Use, vol. 554. Geological Survey Circular Publishing, Washington, USA, , pp. 18. Macek, T., Mackova´, M., Ka´sˇ, J., 2000. Exploitation of plants for the removal of organics in environmental remediation. Biotechnology Advances 18, 23–34. Maehly, A.C., Chance, B., 1954. The assay of catalases and peroxidases. In: Glick, D. (Ed.), Methods of Biochemical Analysis, vol. 1. Interscience Publishers Inc., New York, pp. 357–425. Marrs, K.A., 1996. The functions and regulation of glutathione S-transferases in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 127–158. Marsalek, J., Jimenez-Cisneros, B., Malmaquist, E., Karmazus, P.A., Goldenfum, J., Chocat, B., 2006. Technical Documents in Hydrology 78. UNESCO, Paris. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410.
81
Nedunuri, K.V., Govindaraju, R.S., Banks, M.K., Schwab, A.P., Chen, Z., 2000. Evaluation of phytoremediation for field-scale degradation of total petroleum hydrocarbons. Journal of Environmental Engineering 126, 483–490. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279. Przewrocki, P., Kulczycka, J., Wzorek, Z., Kowalski, Z., Gorazda, K., Jodko, M., 2004. Risk analysis of sewage sludge – Poland and EU comparative approach. Polish Journal of Environmental Studies 13, 237–244. Reddy, A.M., Kumar, S.G., Jyothsnakumari, G., Thimmanaik, S., Sudhakar, C., 2005. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere 60, 97–104. Robinson, S.L., Novak, J.T., Widdowsen, M.A., Crosswell, S.B., Fetterolf, G.J., 2002. Field and laboratory evaluation of the impact of tall fescue on polyaromatic hydrocarbon degradation in aged creosotecontaminated surface oil. Journal of Environmental Engineering 129, 232–240. Rulkens, W., 2008. Sewage sludge as a biomass resource for the production of energy: overview and assessment of the various options. Energy & Fuels 22, 9–15. Schafer, R.Q., Wang, H.P., Kelley, E.E., Cueno, K.L., Martin, S.M., Buettner, G.R., 2002. Comparing b-carotene, vitamin E and nitric oxide as membrane antioxidants. Biological Chemistry 383, 671–681. Siciliano, S.D., Germida, J.J., Banks, K., Greer, C.W., 2003. Changes in microbial community composition and function during a polyaromatic hydrocarbon phytoremediation field trial. Applied and Environmental Microbiology 69, 483–489. Tausz, M., Grill, D., 2000. The role of glutathione in stress adaptation of plants. Phyton 40, 111–118. Tayefi-Nasrabadi, H., Dehghan, G., Daeihassani, B., Movafegi, A., Samadi, A., 2011. Some biochemical properties of guaiacol peroxidases as modified by salt stress in leaves of salt-tolerant and salt-sensitive safflower (Carthamus tinctorius L.cv.) cultivars. African Journal of Biotechnology 10, 751–763. Taylor, S.L., Tappel, A.L., 1976. Sensitive fluorometric methods for tissue tocopherol analysis. Lipids 11, 530–538. Trapp, S., Matthies, M., 1997. Modeling volatilization of PCDD/F from soil and uptake into vegetation. Environmental Science & Technology 31, 71–74. Trebst, A., 2003. Function of b-carotene and tocopherol in photosystem II. Zeitschrift fu¨r Naturforschung 58c, 609–620. Tsihrintzis, V.A., Hamid, R., 1997. Modeling and management of urban stormwater runoff quality: a review. Water Resources Management 11, 136–164. Urbaniak, M., 2013. Biodegradation of PCDDs/PCDFs and PCBs. In: Chamy, R., Rosenkranz, F. (Eds.), Biodegradation – Engineering and Technology. InTech Publishing, Croatia, ISBN: 978-953-51-1153-5. Urbaniak, M., Skowron, A., Fra˛tczak, W., Zielin´ski, M., Wesołowski, W., 2010. Transport of polychlorinated biphenyls in urban cascade reservoirs: levels, sources and correlation to environmental conditions. Polish Journal of Environmental Studies 19, 201–211. Urbaniak, M., Skowron, A., Zielinski, M., Zalewski, M., 2012. Hydrological and environment al conditions as key drivers for spatial and seasonal changes in PCDD/PCDF concentrations, transport and deposition along urban cascade reservoirs. Chemosphere 88, 1358–1367. Urbaniak, M., Zielinn´ski, M., Kaczkowski, Z., Zalewski, M., 2013. Spatial distribution of PCDDs, PCDFs and dl-PCBs along the cascade of urban reservoirs. Hydrology Research 44, 614–630. US EPA, 1998. A Citizen’s Guide to Phytoremediation. EPA 542-F-98-011. Washington, DC. US EPA, 1999. Storm Water O&M Fact Sheet Handling and Disposal of Residuals. EPA 832-F-99-015. Washington, DC. UWWTD (91/271/EEC), Council Directive of 21 May 1991 concerning Urban Waste-Water Treatment. Vervaeke, P., Luyssaert, S., Mertens, J., Meers, E., Tack, F.M., Lust, N., 2003. Phytoremediation prospects of willow stands on contaminated sediments: a field trial. Environmental Pollution 126, 27–282. Wagner, I., Izydorczyk, K., Drobniewska, A., Fratczak, W., Zalewski, M., 2007. Inclusion of ecohydrology concept as integral component of systemic urban water resources management. The city of Lodz, case study, Poland.In: Scientific Conference SWITCH in Birmingham and New Directions in IURWM. Paris, SWITCHGOCE 018530. Project Report. Wagner, I., Marsalek, J., Breil, P., 2008. Aquatic Habitats in Sustainable Urban Water Management. Science Policy and Practice. Urban Water Series – UNESCO-IHP UNESCO Taylor & Francis Publisher, pp. 272. Walsh, C.J., Leonard, A.W., Ladson, A.R., Fletcher, T.D., 2004. Urban Stormwater and the Ecology of Streams. Cooperative Research Centre for
82
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
Freshwater Ecology and Cooperative Research Centre for Catchment Hydrology, Canberra, pp. 44. White, J.C., Parrish, Z.D., Isleyen, M., Gent, M.P., Iannucci-Berger, W., Eitzer, B.D., Kelsey, J.W., Mattina, M.I., 2005. Influence of citric acid amendments on the availability of weathered PCBs to plant and earthworm species. International Journal of Phytoremediation 8, 63–79. White Jr., P.M., Wolf, D.C., Thoma, G.J., Reynolds, C.M., 2006. Phytoremediation of alkylated polycyclic aromatic hydrocarbons in a crude oilcontaminated soil. Water, Air and Soil Pollution 169, 207–220. Whitfield A˚slund, M.L., Zeeb, B.A., Rutter, A., Reimer, K.J., 2007. In situ phytoextraction of polychlorinated biphenyl – (PCB) contaminated soil. Science of the Total Environment 374, 1–12. Wildi, W., Dominik, J., Loizeau, J.L., Thomas, R.L., Favarger, P.Y., Haller, L., Perroud, A., Peytremann, C., 2004. River, reservoir and lake sediment
contamination by heavy metals downstream from urban areas of Switzerland. Lakes and Reservoirs: Research and Management 9, 75–87. Willekens, H., Inze´, D., Van Montagu, M., Van Camp, W., 1995. Catalases in plants. Molecular Breeding 1, 207–228. Wyrwicka, A., Skłodowska, M., 2006. Influence of repeated acid rain treatment on antioxidative enzyme activities and on lipid peroxidation in cucumber leaves. Environmental and Experimental Botany 56, 198–204. Zhang, B., Zhang, H., Jin, J., Ni, Y., Chen, J., 2012. PCDD/Fs-induced oxidative damage and antioxidant system responses in tobacco cell suspension cultures. Chemosphere 88, 798–805. Zhang, H., Chen, J., Ni, Y., Zhang, Q., Zhao, L., 2009. Uptake by roots and translocation to shoots of polychlorinated dibenzo-p-dioxins and dibenzofurans in typical crop plants. Chemosphere 76, 740–746.
Contents lists available at ScienceDirect
Ecohydrology & Hydrobiology journal homepage: www.elsevier.com/locate/ecohyd
Original Research Article
The effect of PCB-contaminated sewage sludge and sediment on metabolism of cucumber plants (Cucumis sativus L.) Anna Wyrwicka a,*, Steffani Steffani b, Magdalena Urbaniak b,c a
University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Plant Physiology and Biochemistry, Banacha 12/16, 90-237 Ło´dz´, Poland b University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Applied Ecology, Banacha 12/16, 90-237 Ło´dz´, Poland c European Regional Centre for Ecohydrology of the Polish Academy of Sciences, Tylna 3, 90-364 Ło´dz´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 October 2013 Received in revised form 1 January 2014 Accepted 13 January 2014 Available online 8 February 2014
Increasing amounts of sewage sludge are produced nowadays, which need to be disposed of in a safe and responsible manner. Likewise, bottom sediments from small urban water bodies undergo periodical dredging and need to be utilized. These deposits often contain Persistant Organic Pollutants (POPs) and other toxic substances. Plants can be used to reduce these pollutants during or before disposal to land (phytoremediation). Cucurbitaceae are known to accumulate high levels of POPs, including polychlorinated biphenyls (PCB), compared with other plant species but such accumulation may lead to secondary oxidative stress that may limit their value. This study examined the impact of sewage sludge and urban lake sediment on soil toxicity, measured as PCB concentration, and changes in the antioxidative system of cucumber plants grown in the soils. There was an average reduction of PCB by 38.63% and 27.38% in soil amended with sewage sludge and sediment, respectively after 5 weeks of cucumber plant cultivation. In the case of plants grown with sewage sludge, guaiacol peroxidase (POx) activity significantly decreased to 49% of the control at the highest dose given, while that of glutathione S-transferase (GST) increased to 172% of the control value in the same treatment. a-Tocopherol concentration was higher in the plants grown in the sewage sludge amended soil. ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Keywords: Phytoremediation PCDD PCDF Antioxidants Enzyme assay a-Tocopherol
1. Introduction Implementation of the Urban Waste Water Treatment Directive (UWWTD) (91/271/EEC) in Poland led to a sharp increase in the production of sewage sludge from nearly 350 000 to 641 000 Mg of d.w./yr in the period from 2000 to 2010. In the year 2015 this amount may exceed 713 000 Mg
* Corresponding author at: University of Ło´dz´, Faculty of Biology and Environmental Protection, Department of Plant Physiology and Biochemistry, Banacha 12/16, 90-237 Ło´dz´, Poland. Tel.: +48 42 6354420; fax: +48 42 6354423. E-mail address: [email protected] (A. Wyrwicka).
of d.w./yr. Since Poland is a major sewage sludge producer in Europe, with the mean value of about 37 kg per capita per year (Przewrocki et al., 2004), this increase has posed problems with its utilization and disposal. On the one hand, sewage sludge contains nutrients such as nitrogen and phosphorus, that are useful for agriculture (Rulkens, 2008; US EPA, 1998), on the other hand, it also contains a variety of toxic compounds including heavy metals, Persistent Organic Pollutants (POPs), Polycyclic Aromatic Hydrocarbons (PAHs), as well as pathogens and other microbial pollutants, that pose hazard to human health. The situation is similar for sediments deposited at the bottom of urban reservoirs and sedimentation ponds as
1642-3593/$ – see front matter ß 2014 European Regional Centre for Ecohydrology of Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.ecohyd.2014.01.003
76
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
they contain a broad range of pollutants originating from urban catchments (Marsalek et al., 2006; Wagner et al., 2008) including heavy metals, plant fertilizers and pesticides from lawns and gardens, oil from leaky cars, pet waste and other pollutants such as POP and PAH (Luna, 1968; Tsihrintzis and Hamid, 1997; US EPA, 1999; Walsh et al., 2004; Wildi et al., 2004; Christensen et al., 2006; Wagner et al., 2007; Jartun and Pettersen, 2010; Urbaniak et al., 2010, 2012, 2013; Urbaniak, 2013). One way of coping with the growing amount of sewage sludge and urban lake sediments, when they have to be dredged from reservoirs, is to use phytoremediation, the use of plants to reduce pollutants so that they are metabolized or concentrated for easier disposal (US EPA, 1999; Macek et al., 2000; Urbaniak, 2013). Some species of cucumber family, Cucurbitaceae, are known to accumulate higher levels of POP such as polychlorinated dibenzo-pdioxins (PCDD), polychlorinated dibenzofurans (PCDF) (Hu¨lster et al., 1994; Inui et al., 2008; Zhang et al., 2009) and PCB (White et al., 2005) in their tissues, compared with other plant species. Transfer of POP from soil to plants is especially interesting because these substances are strongly absorbed on soil organic matter and are rarely transported into plant roots and then transferred into aboveground plant tissues. Their high hydrophobicity suggests that the major pathway by which POP enter plant tissues is absorption from soil vapour (Trapp and Matthies, 1997). Many plant species e.g., lettuce, potato, tomato, rice, garland chrysanthemum, Chinese cabbage, maize, and soybean, can absorb POP through their roots. However, the amounts of these substances translocated from roots to shoots are usually very small. In contrast, the Cucurbitaceae can accumulate POP from soil in their leaves and fruits (Inui et al., 2008; Zhang et al., 2009). Consequently they may show secondary oxidative stress, which is the overproduction of reactive oxygen species (ROS) in plant cells (Gabara et al., 2003; Wyrwicka and Skłodowska, 2006; Gajewska et al., 2013). Oxidative stress occurs when there is a serious imbalance between the production of ROS and their neutralization. The production of ROS is a natural phenomenon in plant tissues and accompanies photosynthesis and respiration. Endogenously overproduced ROS such as superoxide anion (O2), hydroxyl radicals (OH), and peroxyl radicals (ROO) as well as singlet oxygen (1O2) and hydrogen peroxide (H2O2) spontaneously damage membrane lipids and proteins, nucleic acids and other biologically important molecules (Mittler, 2002) and may reduce plant growth and development. Plant cells are equipped with a large and integrated system of enzymatic and non-enzymatic antioxidants that regulate ROS level. Enzymatic antioxidants include superoxide dismutase (SOD; EC 1.15.1.1) (Dhindsa et al., 1981), catalase (CAT; EC 1.11.1.6) (Willekens et al., 1995), ascorbate peroxidase (APx; EC 1.11.1.11) (Asada, 1992), phenolic peroxidases e.g., guaiacol peroxidase (POx; EC 1.11.1.7) (Tayefi-Nasrabadi et al., 2011) and glutathione Stransferase (GST; EC 2.5.1.18) (Edwards and Dixon, 2004) while non-enzymatic ones include e.g., ascorbic acid (Noctor and Foyer, 1998), glutathione (Tausz and Grill, 2000), carotenoids, and a-tocopherol (Schafer et al., 2002).
The aim of the present study was to assess the impact of sewage sludge and urban reservoir sediment application to soils on toxicity, measured as PCB concentration, and changes in selected elements of the Cucumis sativus L. (cucumber) antioxidative system when the plants were grown on these soils. In particular the content of atocopherol, the main lipophilic non-enzymatic antioxidant, was measured to assess its function in plant defence. Activities of antioxidative and detoxifying enzymes such as POx and GST were determined to estimate the efficiency of protective systems. 2. Materials and methods 2.1. Soil preparation Sewage sludge from Lodz Municipal Wastewater Treatment Plant (LM WWTP) and sediments from the Sokoło´wka Sequential Biofiltration System (SSBS) were collected. The Sokoło´wka Sequential Biofiltration System was constructed in the upper section of the Sokoło´wka River in order to remove sediments, suspended solids, particulate pollutants, petroleum hydrocarbons, heavy metals and nutrients from stormwater runoff through its sedimentation and filtration mechanisms. The system comprises three different zones: the zone of hydrodynamically intensified sedimentation, the zone of intensive biogeochemical processes and the zone of intensive biofiltration. The sediment samples used in the experiment were collected from the first zone wherein the accelerated sedimentation of suspended matter and pollutants associated with it occurs. The sewage sludge and sediment samples were dried at 70 8C for 72 h then homogenized into small particles using a mortar and used as fertilizer for the soil samples for cucumber planting. The vegetable potting soil (specified for cucumber growth) used in the experiment was collected from Hollas Sp. z o.o. Pasłe˛k. Four treatments were used: a control C in which no sludge or sediment was added, and three levels of addition, 1.8 g, 5.4 g and 10.8 g per flower pot. The first corresponds to the dose of 3 tonnes ha1, the allowed dosage per year by the Regulation of the Minister of Environment of 13 July 2010 on municipal sewage sludge (Dz.U. Nr 137/2010 r., poz. 924); the second is the permitted dose of 9 tonnes ha1 per 3 years applied on one occasion; and the third, 18 tonnes ha1 is above the permitted level. Treatments are designated by the numerical dose per pot and the abbreviations SS for sewage sludge and SED for sediment. 2.2. Plant material Cucumber seeds (C. sativus L.) cv ‘‘Cezar’’ were germinated in Petri dishes for 7 days and the seedlings were planted into the control and sewage sludge- or sediment-amended soil. They were grown in a growth chamber at of 23 0.5 8C with 16 h light/8 h dark cycle and with 150 mmol m2 s1 photon flux density during the light period, and 60% relative humidity. Five-week-old plants with five fully expanded leaves were used for subsequent analysis.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
All biochemical analyses were carried out on the second, third and fourth leaves from the control and treated plants. The leaves were harvested in the middle of the 16 h light period. 2.3. PCB concentrations PCB concentrations were determined in the control and amended soils before cucumber planting (the first day of the experiment) and after 5 weeks of cucumber growth (the last day of the experiment). The collected soil samples were stored in glass tubes in the dark at 4 8C for further extraction using a PCB RaPID Assay Sample Extraction Kit purchased from Tigret Ltd., and PCB analyzed using Enzyme Linked Immunosorbent Assay (ELISA) – PCB RaPID Assay. The PCB RaPID Assay kit applies the principles of enzyme linked immunosorbent assay to the determination of PCB and related compounds. The sample to be tested is added, along with an enzyme conjugate, to a disposable test tube, followed by paramagnetic particles with antibodies specific to PCB attached. Both, PCB and the enzyme conjugate compete for the antibody binding sites on the magnetic particles. The presence of PCB is detected by adding the enzyme substrate (hydrogen peroxide) and chromogen (3,30 ,5,50 -tetramethylbenzidine). The enzyme conjugate analogue bound to the PCB antibody catalyzes the conversion of the substrate/ chromogene mixture to a coloured product. Since enzyme competes with PCB for the antibody sites, the colour developed is inversely proportional to the concentration of PCB in the sample. Prior to running the assay, PCB in the given soil sample have to be extracted. In order to extract PCB, 10 g of soil (dry matter) were extracted into 20 mL of methanol in extraction jars, shaken for 1 min and left at room temperature for 1–2 min to separate the soil and solvent layers. The solvent was filtered and 25 mL was diluted in 25 mL of PCB Extract Diluent. The obtained extract was used as sample for further PCB analysis using a PCB RaPID Assay kit. In order to analyze the PCB content using the PCB RaPID Assay kit, aliquots (200 mL) of calibration standard PCB (0, 0.5, 2 and 10 ppm), the extracted sample, and the positive control solution (6 ppm) were added to the test tubes together with aliquots of enzyme conjugate (250 mL). After this, an aliquot (500 mL) of antibody, coupled with magnetic particles in buffered saline containing preservative and stabilizers, was added, thoroughly mixed and incubated at room temperature for 15 min using a RaPID Magnetic Separator. After incubation, the contents of each vial were decanted to a waste container to remove the solution containing any unbound reagents. The vials were then washed twice with washing solution (1 mL/vial). Following washing, an aliquot (500 mL) of colour solution containing hydrogen peroxide and 3,30 ,5,50 -tetramethylobenzidine in an organic base was added to each vial, shaken and incubated for 20 min. At the end of the incubation period an aliquot (500 mL) of stopping solution, containing 2 M sulphuric acid was added to each vial. The absorbance of
77
the liquid in each vial was measured at 450 nm using a SDI Differential Spectrophotometer. 2.4. Preparation of enzyme extracts from leaf tissues The leaves of the cucumber plants were ground (1:10, w/v) in an ice-cold mortar using 50 mM sodium phosphate buffer (pH 7.0) containing 0.5 M NaCl, 1 mM EDTA, and 1 mM sodium ascorbate. The slurry was filtered through two-layers of Micracloth. The filtrate of homogenized cucumber leaves was then centrifuged (15,000 g for 15 min). After centrifugation, the supernatant was collected and POx and GST activities and protein concentration were measured. 2.5. Protein content of the extracts The protein content was determined by Bradford’s method (1976) with standard curves prepared using bovine serum albumin. 2.6. Enzyme assay POx activity was assayed according to a method modified from Maehly and Chance (1954) with guaiacol as a substrate. The reaction mixture contained 49 mM sodium acetate buffer (pH 5.6) 5 mM guaiacol, 15 mM H2O2, and enzyme extract (15–25 mg protein). The linear increase in absorbance at 470 nm due to the formation of tetraguaiacol (the millimolar extinction coefficient of tetraguaiacol at 470 nm; e = 26.6 mM1 cm1) was monitored for 4 min at 25 8C. Enzyme activity was expressed in mmol tetraguaiacol min1 mg1 protein. The total GST activity was determined with 1-chloro2,4-dinitrobenzene (CDNB) by a method modified from Habig et al. (1974). GST catalyses the conjugation of Lglutathione (GSH) to CDNB through the thiol group of GSH. The product of CDNB conjugation with GSH, dinitrophenyl thioether absorbs at 340 nm (e = 9.6 mM1 cm1). The reaction solution contained 100 mM potassium phosphate buffer (pH 6.25), 0.75 mM CDNB, 30 mM GSH and the enzyme extract (50 mg protein). Enzyme activity was expressed in units representing formation of dinitrophenyl thioeter (nmol min1 mg1 protein). All assays were made spectrophotometrically (UNICAM UV 300 UV-visible spectrometer) at 25 8C. 2.7. Determination of a-tocopherol Whole leaves were homogenized (1:5, w/v) in an ice-cold mortar using 50 mM sodium phosphate buffer, pH 7.0, containing 0.5 M NaCl, 1% polyvinylpyrrolidone and 1 mM EDTA. The crude homogenate obtained after filtration was assayed for a-tocopherol content by the method of Taylor and Tappel (1976). After saponification of the sample with KOH in the presence of ascorbic acid, a-tocopherol was extracted with n-hexane. Fluorescence of the organic layer was measured at 280 nm (excitation) and 310 nm (emission) wavelengths using a F-2500 Fluorescence Spectrophotometer (Hitachi, Limited, Tokyo Japan). The
78
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
concentration of a-tocopherol was expressed as mg g1 fresh mass of original plant tissue. 2.8. Statistical analysis The results of PCB analysis were tested using Wilcoxon matched pair test. Differences of P < 0.05 were considered significant. The results presented for POx, GST and atocopherol are the means of eight replicates in two independent experiments. Sample variability is given as the standard deviation (SD) of means. The significance of differences between mean value was determined by a Student t test. Differences with P < 0.05 were considered significant. 3. Results 3.1. PCB concentration in soil before and after C. sativus L. cultivation Addition of sewage sludge led to increase in the PCB concentration in the soil samples. The smallest PCB concentration was noted in the control soil and the highest in soil amended with the highest sewage sludge dose (10.8 SS) (Fig. 1a). The analysis conducted after 5 weeks of cucumber growth showed reductions of PCB concentration by 6.25% (from 0.512 ppm to 0.480 ppm) in the control soil, 41.28% (from 0.940 ppm to 0.552 ppm) in
Fig. 1. Changes of PCBs concentrations in the soil fertilized with: (a) LM WWTP sewage sludge and (b) SSBS sediments, before &/ and after / 5 weeks of Cucumis sativus L. cultivation.
Fig. 2. Influence of LM WWTP sewage sludge and SSBS sediments on POx activity in the cucumber leaves. Symbols (*), (**), and (***) indicate values that differ significantly from the control plant at P < 0.05, P < 0.01, and P < 0.001, respectively. Symbols (a), (aa), (aaa) indicate values for 5.4 SS/5.4 SED that differ significantly from 1.8 SS/1.8 SED at P < 0.05, P < 0.01, and P < 0.001 respectively. Symbols (b), (bb), (bbb) indicate values for 10.8 SS/10.8 SED that differ significantly from 1.8 SS/1.8 SED at P < 0.05, P < 0.01, and P < 0.001 respectively. Symbols (c), (cc), (ccc) indicate values for 10.8 g that differ significantly from 5.4 SS/5.4 SED at P < 0.05, P < 0.01, and P < 0.001 respectively.
1.8 SS, by 38.39% (from 1.120 ppm to 0.690 ppm) in 5.4 SS, and by 36.22% (from 1.154 ppm to 0.736 ppm) in 10.8 SS. The lower the sewage sludge dose the greater the reduction of PCB concentration was observed. Nevertheless, the obtained values showed no statistically significant differences (Wilcoxon matched pair test). Addition of sediments also increased the PCB concentration in the soil samples. The highest concentration was noted at the highest sediment dose (10.8 SED) (Fig. 1b). After 5 weeks of cucumber growth, PCB concentrations were reduced by 7.90% (from 0.508 ppm to 0.472 ppm) in control soil, by 25.24% (from 0.808 ppm to 0.604 ppm) in the case of 1.8 SED; by 27.08% (from 0.960 ppm to 0.700 ppm) in 5.4 SED, and by 29.82% (from 1.120 ppm to 0.786 ppm) in 10.8 SED (Fig. 1b). The results demonstrated an increase in PCB reduction with increasing amendment of sediment. Nevertheless, also in this case no statistically significant differences were noted.
Fig. 3. Influence of LM WWTP sewage sludge and SSBS sediments on GST activity in the cucumber leaves. For further explanation, see Fig. 2.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
Fig. 4. Influence of LM WWTP sewage sludge and SSBS sediments on a-tocopherol concentration in the cucumber leaves. For further explanation, see Fig. 2.
3.2. Enzyme activities POx activity in the leaves of cucumber plants cultivated with sewage sludge showed significant changes from the control plants and among treatments (Fig. 2). Significant decrease in POx activity was observed in each treatment reaching 70% (P < 0.001), 65% (P < 0.001), and 49% (P < 0.001) of the control plant value for 1.8 SS, 5.4 SS and 10.8 SS, respectively. Moreover, significant differences were also observed among treatments. In 10.8 SS, POx activity was 71% (P < 0.01) and 76% (P < 0.01) of those observed in 1.8 SS and 5.4 SS treatments, respectively. In sediment-supplemented cultures, POx activity changes were not statistically significant from each other or the control (Fig. 2). The activity of GST increased with sewage sludge addition (Fig. 3). There were significant increases in 10.8 SS to 172% (P < 0.001) of the control value and to 170% (P < 0.001) and 148% (P < 0.001) of the values in 1.8 SS and 5.4 SS, respectively. GST activity in 10.8 SED was 117% (P < 0.05) of that for 5.4 SED variant (Fig. 3) but other differences for sediment amendment treatments and control were not significant. 3.3. a-Tocopherol concentration
a-Tocopherol concentration in the cucumber leaves increased with sewage sludge addition, but significant changes were observed only for 10.8 SS compared with control (150%, P < 0.01) and for 5.4 SS compared with 1.8 SS (119%, P < 0.05). The highest a-tocopherol concentration was observed in 10.8 SS with 12.86 mg g1 fresh mass. Amendment with sediment had no significant effects (Fig. 4). 4. Discussion Plants have been frequently shown to be able to remove POP from soils. Siciliano et al. (2003) demonstrated reduction of organochlorine compounds by about 30% during 2 years of plant cultivation; in soil without plants, the reduction was half as much. Nedunuri et al. (2000) reported reduction of aromatic compounds by about 42%
79
and 50% with fibre flax (Lolium annua) and St. Augustine grass (Stenotaphrum secundatum), respectively, over 21 months. Vervaeke et al. (2003) showed 57% reduction of aromatic compounds and mineral oils during 1.5 years of willow (Salix viminalis) cultivation. Other examples, using a combination of grass and fertilizers, showed remediation of soil contaminated with crude oil (Robinson et al., 2002; Banks et al., 2003; White et al., 2006). Zhang et al. (2009) showed that Cucurbita roots were very effective in dioxin uptake and its subsequent translocation. Cucumber and towel gourd (Luffa cylindrica Roem.) accumulated 0.5–3.5 times higher concentrations of dioxins than the predicted gross vapour leakages, indicating that they can significantly translocate dioxins from roots to shoots. The present study demonstrated an average 38.63% and 27.38% reduction of PCB in sewage sludge and sediment amended soils after 5 weeks of the cucumber plant cultivation. The removal of PCBs from the sewage sludge-amended soil was greater than its reduction from the soil fertilized with sediments. However, in the case of sewage sludge-amended soil the relative reduction decreased with the dose of sludge. The opposite situation was noted for the soil fertilized with sediments, where increasing sediment dose increased removal efficiency, but since none of these differences were statistically significant, they remain only indicative and may simply be due to random chance. The decreasing rate of PCB removal in the case of sewage sludge amended soils may be related to the occurrence of a wide range of other pollutants in the sewage sludge. Heavy metals are among the most important and dangerous substances noted in the sewage sludge from the LM WWTP (Drobniewska, 2006, 2008). Their very high concentrations, which usually exceed the allowed doses (according to government regulations in Poland: Dz.U. Nr 137/2010 r., poz. 924) suggest that they can negatively affect soil and plant condition (Gabbrielli et al., 1999; Gajewska et al., 2013) and thus influence the ability of treated plants to effectively remove the studied PCB. The uptake of contaminants by plants may also depend on other environmental factors. Inui et al. (2008) reported that this process was affected by organic matter content in soil, moisture content, pH and bioavailability of nutrients as well as plant characteristics. Moreover, the ability of plants to absorb PCB and to remove them from soil may be correlated with the particular mixture of PCB compounds present and the individual properties of particular PCB congeners. According to Whitfield A˚slund et al. (2007), highly chlorinated PCB congeners are least mobile and least soluble and thus become progressively adsorbed to the hydrophobic components in the lower part of the plant stem. Many studies have provided evidence that peroxidases, part of the cellular H2O2 detoxifying system, participate in important physiological processes such as lignification, suberinization and auxin catabolism. Guaiacol peroxidase (POx) is a crucial enzyme found in the cytosol, cell wall, vacuole and extracellular spaces and is involved in a range of processes related to plant growth and development. POx consumes H2O2 to generate phenoxy compounds that are polymerized to produce cell wall components such as lignin (Reddy et al., 2005). Our results showed a decrease in
80
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
POx activity in cucumber plants with increasing addition of sewage sludge. This may suggest the presence of toxic compounds in the sludge and an inhibitory effect on POx activity. In this situation toxic H2O2, the substrate for POx, might build up in plant cells. In tobacco cells exposed to low concentration of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans PCDD/PCDF in a suspension culture, POx activity was significantly increased (Zhang et al., 2012). However, it decreased with increasing concentration of PCDD/PCDF. The authors suggest that low concentrations of toxic substances allow plant cells to augment POx activity and to counteract oxidative stress, but at high concentrations, a significant toxic effect was observed. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in xenobiotic metabolism in plants (Marrs, 1996). GST catalyze the conjugation of reduced glutathione (GSH) with electrophilic substrates to form GSH-substrate conjugates, which are more polar and less toxic. Although GST were first noted as herbicide detoxifying enzymes in plants, many authors have demonstrated their involvement in the detoxification of PAH, PCB and heavy metals. Our results showed that in the leaves of plants grown in sewage sludge-amended soil, GST activity increased significantly, especially in 10.8 SS. High activity of this effective detoxifying enzyme indicates increased toxicity of soil. According to Flury et al. (1996) genes for GST are induced by factors that cause oxidative damage. The collective changes in POx and GST activities could also be interpreted as a compensatory mechanism for the reduced POx activity. In cucumber plants, GST may be an important enzyme to overcome oxidative stress, because no increase in other antioxidative enzymes was observed. Tocopherol is the main nonenzymatic antioxidant of the lipophilic fraction of cells. This molecule is synthesized only by photosynthetic organisms. Tocopherol maintains stability of cell membranes, and, as an antioxidant, protects chloroplast membranes against photooxidation (Trebst, 2003). Moreover, it seems to play a role in plant growth, development and senescence (Horvath et al., 2006). It is thought that tocopherol affects carbohydrate metabolism and is involved in controlling photosynthesis. Its physiological significance has been confirmed by studies showing increase in tocopherol content in plant tissue under unfavourable environmental conditions. After exposure of Arabidopsis plants to high light, a-tocopherol concentration approximately doubled (Giacomelli et al., 2007). Moreover, concentration of this lipophilic antioxidant increased in tobacco plants with leaf age (Dertinger et al., 2003). Increased content of a-tocopherol in our cucumber plants grown in the sewage sludge-amended soil might be correlated with the intensity of oxidative stress and might suggest that this antioxidant molecule participated in protection of plant cells against accelerated senescence caused by exposure to toxic compounds present in soil. Our results revealed that plants grown in the soil with the largest dose of sewage sludge showed a (nonsignificant) decrease in efficiency of PCB removal from soil, but a significant inhibitory effect on POx activity and the largest GST activity were observed suggesting high efficiency of detoxification processes. Decrease in POx
activity may contribute to ROS accumulation in plant cells and to increase in the concentration of the substances formed as a result of oxidative damage. Both processes might increase GST activity since GST gene transcription is directly induced by H2O2 and endogenous substances resulting from oxidative damage, which are induced mainly by hydroxyl radicals (OH), and are highly toxic (Danielson et al., 1987). In the sediment treatments, on the contrary, there was no significant response in the production of these enzymes, although PCB were present in the sediments. It is possible that in sediments, toxic substances other than for PCB, are present at lower concentrations and do not have the inhibitory effect on plant metabolism found for sewage sludge. PCB may therefore not be responsible for the plant reactions. It is also possible that PCB congeners present in the sediments are different and may be less toxic from those present in the sewage sludge. It cannot either be excluded that the ratio of toxic substances to nutrients is lower and more favourable in sediments and that the plants may be responding to higher nutrient levels. In sewage sludge, potassium concentration is limited because during its purification, potassium is removed, which does not occur in sediments (Drobniewska, 2006, 2008). These initial data provide a start for further investigations, including effects of different PCB congener patterns in sewage sludge and in sediments. It is always difficult to attribute cause and effect when dealing with collections of pollutants, such as occur in complex mixtures like sludge and sediment, and precise attribution may never be possible. The data do support the contention, however, that cucumber plants remove PCB from contaminated soils, though this is only one of many processes that might affect their growth. Further research might focus on the mechanism of PCB action and activation of plant protective systems that might improve the phytoremediation process. Conflict of interest None declared. Financial disclosure This research has been carried out as part of the following projects: Erasmus Mundus Master of Science in Ecohydrology (ECOHYD, 159659-1-2009-1-PT-ERA MUNDUS-EMMC) and ‘Innovative resources and effective methods of safety improvement and durability of buildings and transport infrastructure in sustainable development’ financed by the European Union from the European Fund of Regional Development based on the Operational Program of the Innovative Economy, POIG.01.01.02-10-106/09. References Asada, K., 1992. Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants. Physiologia Plantarum 85, 235–241. Banks, M.K., Kulakow, P., Schwab, A.P., Chen, Z., Rathbone, K., 2003. Degradation of crude oil in the rhizosphere of Sorghum bicolor. International Journal of Phytoremediation 5, 225–234. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein– dye binding. Analytical Biochemistry 72, 248–254.
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82 Christensen, A.M., Nakajima, F., Baun, A., 2006. Toxicity of water and sediment in a small urban river (Store Vejlea˚, Denmark). Environmental Pollution 144, 621–625. Danielson, U.H., Esterbauer, H., Mannervik, B., 1987. Structure–activity relationships of 4-hydroxyalkenals in the conjugation catalyzed by mammalian glutathione S-transferases. Biochemical Journal 247, 707–712. Dertinger, U., Schaz, U., Schulze, E.-D., 2003. Age-dependence of the antioxidative system in tobacco with enhanced glutathione reductase activity or senescence-induced production of cytokinins. Physiologia Plantarum 119, 19–29. Dhindsa, R.S., Plumb-Dhindsa, P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. Journal of Experimental Botany 32, 93–101. Drobniewska, A., 2006. Sewage water management for environment quality and positive socio-economic feedbackIn: Demonstration Activity Report, The City of Lodz, GOCE 0118530 SWITCH. Drobniewska, A., 2008. Optymalizacja wykorzystania osadu s´ciekowego do produkcji bioenergii na podstawie wielkoskalowych eksperymento´w terenowych oraz modelu matematycznego. (Optimization of the Use of Sewage Sludge for Bioenergy Production on the Basis of LargeScale Field Experiments and Mathematical Model). (Ph.D. thesis)University of Lodz. Edwards, R., Dixon, D.P., 2004. Metabolism of natural and xenobiotic substrates by the plant glutathione S-transferase superfamily. In: Sandermann, H. (Ed.), Ecological Studies. Molecular Ecotoxicology of Plants, vol. 170. Springer-Verlag, Berlin, Heidelberg, pp. 17–50. Flury, T., Wagner, E., Kreuz, K., 1996. An inducible glutathione S-transferase in soybean hypocotyl is localized in the apoplast. Plant Physiology 112, 1185–1190. Gabara, B., Skłodowska, M., Wyrwicka, A., Glin´ska, S., Gapin´ska, M., 2003. Changes in the ultrastructure of chloroplasts and mitochondria and antioxidant enzyme activity in Lycopersicon esculentum Mill. leaves sprayed with acid rain. Plant Science 164, 507–516. Gabbrielli, R., Pandolfini, T., Espen, L., Palandri, M.R., 1999. Growth, peroxidase activity and cytological modifications in Pisum sativum seedlings exposed to Ni2+ toxicity. Journal of Plant Physiology 155, 639–645. Gajewska, E., Głowacki, R., Mazur, J., Skłodowska, M., 2013. Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions. Plant Growth Regulation 71, 13–20. Giacomelli, L., Masi, A., Ripoll, D.R., Lee, M.J., van Wijk, K.J., 2007. Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low. Plant Molecular Biology 65, 627–644. Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases. The first enzymatic step in mercaptane acid formation. Journal of Biological Chemistry 246, 7130–7139. Horvath, G., Wessjohann, L., Bigirimana, J., Jansen, M., Guisez, Y., Caubergs, R., Horemans, N., 2006. Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues. Phytochemistry 67, 1185–1195. Hu¨lster, A., Mueller, J.F., Marschner, H., 1994. Soil–plant transfer of polychlorinated dibenzo-p-dioxins and dibenzofurans to vegetables of the cucumber family (Cucurbitaceae). Environmental Science and Technology 28, 1110–1115. Inui, H., Wakai, T., Gion, K., Kim, Y.-S., Eun, H., 2008. Differential uptake for dioxin-like compounds by zucchini subspecies. Chemosphere 73, 1602–1607. Jartun, M., Pettersen, A., 2010. Contaminants in urban runoff to Norwegian fjords. Journal of Soils and Sediments 10, 155–161. Luna, B.L., 1968. Hydrology for Urban Land Planning. A Guidebook on the Hydrologic Effects of Urban Land Use, vol. 554. Geological Survey Circular Publishing, Washington, USA, , pp. 18. Macek, T., Mackova´, M., Ka´sˇ, J., 2000. Exploitation of plants for the removal of organics in environmental remediation. Biotechnology Advances 18, 23–34. Maehly, A.C., Chance, B., 1954. The assay of catalases and peroxidases. In: Glick, D. (Ed.), Methods of Biochemical Analysis, vol. 1. Interscience Publishers Inc., New York, pp. 357–425. Marrs, K.A., 1996. The functions and regulation of glutathione S-transferases in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 127–158. Marsalek, J., Jimenez-Cisneros, B., Malmaquist, E., Karmazus, P.A., Goldenfum, J., Chocat, B., 2006. Technical Documents in Hydrology 78. UNESCO, Paris. Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410.
81
Nedunuri, K.V., Govindaraju, R.S., Banks, M.K., Schwab, A.P., Chen, Z., 2000. Evaluation of phytoremediation for field-scale degradation of total petroleum hydrocarbons. Journal of Environmental Engineering 126, 483–490. Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279. Przewrocki, P., Kulczycka, J., Wzorek, Z., Kowalski, Z., Gorazda, K., Jodko, M., 2004. Risk analysis of sewage sludge – Poland and EU comparative approach. Polish Journal of Environmental Studies 13, 237–244. Reddy, A.M., Kumar, S.G., Jyothsnakumari, G., Thimmanaik, S., Sudhakar, C., 2005. Lead induced changes in antioxidant metabolism of horsegram (Macrotyloma uniflorum (Lam.) Verdc.) and bengalgram (Cicer arietinum L.). Chemosphere 60, 97–104. Robinson, S.L., Novak, J.T., Widdowsen, M.A., Crosswell, S.B., Fetterolf, G.J., 2002. Field and laboratory evaluation of the impact of tall fescue on polyaromatic hydrocarbon degradation in aged creosotecontaminated surface oil. Journal of Environmental Engineering 129, 232–240. Rulkens, W., 2008. Sewage sludge as a biomass resource for the production of energy: overview and assessment of the various options. Energy & Fuels 22, 9–15. Schafer, R.Q., Wang, H.P., Kelley, E.E., Cueno, K.L., Martin, S.M., Buettner, G.R., 2002. Comparing b-carotene, vitamin E and nitric oxide as membrane antioxidants. Biological Chemistry 383, 671–681. Siciliano, S.D., Germida, J.J., Banks, K., Greer, C.W., 2003. Changes in microbial community composition and function during a polyaromatic hydrocarbon phytoremediation field trial. Applied and Environmental Microbiology 69, 483–489. Tausz, M., Grill, D., 2000. The role of glutathione in stress adaptation of plants. Phyton 40, 111–118. Tayefi-Nasrabadi, H., Dehghan, G., Daeihassani, B., Movafegi, A., Samadi, A., 2011. Some biochemical properties of guaiacol peroxidases as modified by salt stress in leaves of salt-tolerant and salt-sensitive safflower (Carthamus tinctorius L.cv.) cultivars. African Journal of Biotechnology 10, 751–763. Taylor, S.L., Tappel, A.L., 1976. Sensitive fluorometric methods for tissue tocopherol analysis. Lipids 11, 530–538. Trapp, S., Matthies, M., 1997. Modeling volatilization of PCDD/F from soil and uptake into vegetation. Environmental Science & Technology 31, 71–74. Trebst, A., 2003. Function of b-carotene and tocopherol in photosystem II. Zeitschrift fu¨r Naturforschung 58c, 609–620. Tsihrintzis, V.A., Hamid, R., 1997. Modeling and management of urban stormwater runoff quality: a review. Water Resources Management 11, 136–164. Urbaniak, M., 2013. Biodegradation of PCDDs/PCDFs and PCBs. In: Chamy, R., Rosenkranz, F. (Eds.), Biodegradation – Engineering and Technology. InTech Publishing, Croatia, ISBN: 978-953-51-1153-5. Urbaniak, M., Skowron, A., Fra˛tczak, W., Zielin´ski, M., Wesołowski, W., 2010. Transport of polychlorinated biphenyls in urban cascade reservoirs: levels, sources and correlation to environmental conditions. Polish Journal of Environmental Studies 19, 201–211. Urbaniak, M., Skowron, A., Zielinski, M., Zalewski, M., 2012. Hydrological and environment al conditions as key drivers for spatial and seasonal changes in PCDD/PCDF concentrations, transport and deposition along urban cascade reservoirs. Chemosphere 88, 1358–1367. Urbaniak, M., Zielinn´ski, M., Kaczkowski, Z., Zalewski, M., 2013. Spatial distribution of PCDDs, PCDFs and dl-PCBs along the cascade of urban reservoirs. Hydrology Research 44, 614–630. US EPA, 1998. A Citizen’s Guide to Phytoremediation. EPA 542-F-98-011. Washington, DC. US EPA, 1999. Storm Water O&M Fact Sheet Handling and Disposal of Residuals. EPA 832-F-99-015. Washington, DC. UWWTD (91/271/EEC), Council Directive of 21 May 1991 concerning Urban Waste-Water Treatment. Vervaeke, P., Luyssaert, S., Mertens, J., Meers, E., Tack, F.M., Lust, N., 2003. Phytoremediation prospects of willow stands on contaminated sediments: a field trial. Environmental Pollution 126, 27–282. Wagner, I., Izydorczyk, K., Drobniewska, A., Fratczak, W., Zalewski, M., 2007. Inclusion of ecohydrology concept as integral component of systemic urban water resources management. The city of Lodz, case study, Poland.In: Scientific Conference SWITCH in Birmingham and New Directions in IURWM. Paris, SWITCHGOCE 018530. Project Report. Wagner, I., Marsalek, J., Breil, P., 2008. Aquatic Habitats in Sustainable Urban Water Management. Science Policy and Practice. Urban Water Series – UNESCO-IHP UNESCO Taylor & Francis Publisher, pp. 272. Walsh, C.J., Leonard, A.W., Ladson, A.R., Fletcher, T.D., 2004. Urban Stormwater and the Ecology of Streams. Cooperative Research Centre for
82
A. Wyrwicka et al. / Ecohydrology & Hydrobiology 14 (2014) 75–82
Freshwater Ecology and Cooperative Research Centre for Catchment Hydrology, Canberra, pp. 44. White, J.C., Parrish, Z.D., Isleyen, M., Gent, M.P., Iannucci-Berger, W., Eitzer, B.D., Kelsey, J.W., Mattina, M.I., 2005. Influence of citric acid amendments on the availability of weathered PCBs to plant and earthworm species. International Journal of Phytoremediation 8, 63–79. White Jr., P.M., Wolf, D.C., Thoma, G.J., Reynolds, C.M., 2006. Phytoremediation of alkylated polycyclic aromatic hydrocarbons in a crude oilcontaminated soil. Water, Air and Soil Pollution 169, 207–220. Whitfield A˚slund, M.L., Zeeb, B.A., Rutter, A., Reimer, K.J., 2007. In situ phytoextraction of polychlorinated biphenyl – (PCB) contaminated soil. Science of the Total Environment 374, 1–12. Wildi, W., Dominik, J., Loizeau, J.L., Thomas, R.L., Favarger, P.Y., Haller, L., Perroud, A., Peytremann, C., 2004. River, reservoir and lake sediment
contamination by heavy metals downstream from urban areas of Switzerland. Lakes and Reservoirs: Research and Management 9, 75–87. Willekens, H., Inze´, D., Van Montagu, M., Van Camp, W., 1995. Catalases in plants. Molecular Breeding 1, 207–228. Wyrwicka, A., Skłodowska, M., 2006. Influence of repeated acid rain treatment on antioxidative enzyme activities and on lipid peroxidation in cucumber leaves. Environmental and Experimental Botany 56, 198–204. Zhang, B., Zhang, H., Jin, J., Ni, Y., Chen, J., 2012. PCDD/Fs-induced oxidative damage and antioxidant system responses in tobacco cell suspension cultures. Chemosphere 88, 798–805. Zhang, H., Chen, J., Ni, Y., Zhang, Q., Zhao, L., 2009. Uptake by roots and translocation to shoots of polychlorinated dibenzo-p-dioxins and dibenzofurans in typical crop plants. Chemosphere 76, 740–746.