Remediated marine sediment as growing medium for lettuce production: assessment of agronomic performance and food safety in a pilot experiment.

BACKGROUND: The use of reclaimed dredged sediments as growing media may offer a profitable alternative to their disposal as a waste and at the same time meets the need of peat-substitute substrates in horticulture. When sediments are reused to cultivate food crops, issues related to human health rise due to potential accumulation of contaminants in the product. This pilot study aimed at verifying the suitability of a reclaimed dredged port sediment, used pure or mixed with peat, as a growing medium for lettuce cultivation. RESULTS: The pure sediment caused a reduction in crop yield, probably due to its unsuitable physical properties, whereas the mixture sediment-peat and pure peat resulted in the same yield. Although the sediment contained potentially phytotoxic heavy metals and some organic pollutants, no symptoms of plant toxicity were noted. Besides, no organic contaminants were detected in lettuce heads, and heavy metals amounts were not hazardous for consumers. Conversely, plants grown in the sediment were particularly rich in minerals like Ca, Mg and Fe, and showed higher concentrations of organic acids and antioxidants. CONCLUSION: The use of the sediment as a growing medium for lettuce was shown to be safe for both inorganic and organic contaminants. Nevertheless, considering crop yield results, the mixture of the sediment with other materials is recommended in order to produce a substrate with more suitable physicochemical properties for vegetable cultivation. © 2019 Society of Chemical Industry.

Use of phytoremediated sediments dredged in maritime port as plant nursery growing media.

We evaluated the potential of a phytoremediated sediment (TR) dredged from maritime port as peat-free growth substrate for seven ornamental plants, in comparison with an untreated sediment (NT), in a greenhouse experiment. The studied plants were Quercus ilex, Photinia x fraseri, Viburnum tinus, Cistus albidus, Raphiolepis indica, Westringia fruticosa and Teucrium fruticans. Plant growth was monitored for ten months, and the changes in the physico-chemical properties, toxicity, microbial biomass and enzyme activities involved in the C, P and N cycles were also monitored during the plant growth period. The results showed that the studied ornamental plants could grow on both NT and TR sediments, but that the growth was higher on TR sediment. The plant growth induced changes in the sediment chemical functional groups, with clear separation between NT and TR sediments for each of the studied plant. Microbial biomass and enzyme activities significantly increased during the plant growth, more in TR than in NT sediment. Toxicity was detected in NT sediments during the plant growth whereas it was not observed in NT sediments during the whole growth period. We concluded that phytoremediation converted the dredged maritime sediments into suitable substrates for growing ornamental plants, and that the re-use by plant nursery industry can be a sustainable management and valorization for remediated sediments.

Agronomic Practices for Improving Gentle Remediation of Trace Element-Contaminated Soils.

The last few decades have seen the rise of Gentle soil Remediation Options (GRO), which notably include in situ contaminant stabilization ("inactivation") and plant-based (generally termed "phytoremediation") options. For trace element (TE)-contaminated sites, GRO aim to either decrease their labile pool and/or total content in the soil, thereby reducing related pollutant linkages. Much research has been dedicated to the screening and selection of TE-tolerant plant species and genotypes for application in GRO. However, the number of field trials demonstrating successful GRO remains well below the number of studies carried out at a greenhouse level. The move from greenhouse to field conditions requires incorporating agronomical knowledge into the remediation process and the ecological restoration of ecosystem services. This review summarizes agronomic practices against their demonstrated or potential positive effect on GRO performance, including plant selection, soil management practices, crop rotation, short rotation coppice, intercropping/row cropping, planting methods and plant densities, harvest and fertilization management, pest and weed control and irrigation management. Potentially negative effects of GRO, e.g., the introduction of potentially invasive species, are also discussed. Lessons learnt from long-term European field case sites are given for aiding the choice of appropriate management practices and plant species.

Uptake and distribution of perfluoroalkyl substances by grafted tomato plants cultivated in a contaminated site in northern Italy.

Poly- and perfluoroalkyl substances (PFAS) are highly persistent and mobile pollutants raising alarming concerns due to their capability to accumulate in living organisms and exert toxic effects on human health. We studied the accumulation of different PFAS in the leaves and fruits of tomato plants grown on a PFAS-polluted soil in North-East Italy. Tomato plants were grafted with different rootstocks characterized by different vigor, and irrigated with PFAS-polluted groundwater. Leaves and fruits of the first and sixth truss were analyzed at full plant maturity. All tomato varieties accumulated PFAS in leaves and fruits, with the highest concentrations detected in the most vigorous rootstock and reflecting the PFAS concentration profile of the irrigation water. PFAS with a chain length from 4 to 8 C atoms and with carboxylic and sulfonic functional groups were detected in plant leaves, whereas only carboxylic C4, C5, and C6 PFAS were detected in tomato fruits. A general trend of decreasing PFAS concentrations in fruits upon increasing height of the plant trusses was observed. Calculation of the target hazard quotient (THQ) showed increasing values depending on the plant vigor. The hazard index (HI) values showed values slightly higher than 1 for the most vigorous plants, indicating potential risks to human health associated with the consumption of contaminated tomato fruits.

Design and experimental validation of an optimized microalgae-bacteria consortium for the bioremediation of glyphosate in continuous photobioreactors.

Glyphosate will be banned from Europe by the end of 2022, but its widespread use in the last decades and its persistence in the environment require the development of novel remediation processes. In this work, a bacterial consortium was designed de novo with the aim to remove glyphosate from polluted water, supported by the oxygen produced by a microalgal species. To this goal, bioinformatics tools were employed to identify the bacterial strains from contaminated sources (Pseudomonas stutzeri; Comamonas odontotermitis; Sinomonas atrocyanea) able to express enzymes for glyphosate degradation, while the microalga Chlorella protothecoides was chosen for its known performances in wastewater treatment. To follow a bioaugmentation approach, the designed consortium was cultivated in continuous photobioreactors at increasing glyphosate concentrations, from 5 to 50 mg L-1, to boost its acclimation to the presence of the herbicide and its capacity to remove it from water. C. protothecoides tolerance to glyphosate was verified through batch experiments. Remarkably, steady state conditions were reached and the consortium was able to live as a community in the reactor. The consortium activity was validated in both synthetic and real wastewater, where glyphosate concentration was reduced by about 53% and 79%, respectively, without the detection of aminomethylphosphonic acid formation.

Long-Term Effects of Soil Remediation with Willow Short Rotation Coppice on Biogeographic Pattern of Microbial Functional Genes.

Short rotation coppice (SRC) is increasingly being adopted for bioenergy production, pollution remediation and land restoration. However, its long-term effects on soil microbial communities are poorly characterized. Here, we studied soil microbial functional genes and their biogeographic pattern under SRC with willow trees as compared to those under permanent grassland (C). GeoChip analysis showed a lower functional gene diversity in SRC than in C soil, whereas microbial ATP and respiration did not change. The SRC soil had lower relative abundances of microbial genes encoding for metal(-oid) resistance, antibiotic resistance and stress-related proteins. This indicates a more benign habitat under SRC for microbial communities after relieving heavy metal stress, consistent with the lower phytoavailability of some metals (i.e., As, Cd, Ni and Zn) and higher total organic carbon, NO3--N and P concentrations. The microbial taxa-area relationship was valid in both soils, but the space turnover rate was higher under SRC within 0.125 m2, which was possibly linked to a more benign environment under SRC, whereas similar values were reached beyond thisarea. Overall, we concluded that SRC management can be considered as a phytotechnology that ameliorates the habitat for soil microorganisms, owing to TOC and nutrient enrichment on the long-term.

Unexpected enrichment of thallium and its geochemical behaviors in soils impacted by historically industrial activities using lead­zinc carbonate minerals.

Thallium is a trace metal with severe toxicity. Contamination of thallium (Tl) generated by steel and non-ferrous metals industry is gaining growing concern worldwide. However, little is known on Tl contamination owing to industrial activities using carbonate minerals. This study revealed abundant geochemical mobile/bioavailable Tl (> 65.7%, in average; mostly in oxidizable fraction) in soils from a carbonate-hosted PbZn ore utilizing area in China for the first time. Unexpected Tl enrichment was observed in soil accompanying with 3655, 7820, 100.1, 27.3 and 29.9 mg/kg (in average) of Pb, Zn, As, Cd and Sb, respectively. Characterization using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis further confirmed that historical industrial activities impose anthropogenic catastrophic effects on the local agricultural soil system. The ecological and health risk assessment of heavy metal(loid)s in soils proclaimed serious potential non-carcinogenic risks of Pb and V to adults, and Pb, Tl and As to children. Sequential extraction analysis showed that Tl, as well as Pb, Zn, Mn, Co, and Cd, mainly existed in the mobile fractions (exchangeable/acid-extractable, reducible and oxidizable), indicating an ecological risk of biological accumulation of multiple metal(loid)s in this area. These findings provide a theoretical basis for taking appropriate remediation measures in order to ensure safety of soils in such industrial areas likewise.

Agronomic performance and food safety of strawberry cultivated on a remediated sediment.

A marine sediment phytoremediated and homogenized by landfarming was tested for its potential recycle as growing media in horticulture. Two strawberry cultivars, Camarosa and Monterey, were grown on remediated sediment alone (TS100), commercial peat/pumice based growing medium (TS0) and a mixture 1:1 in volume of sediment and peat (TS50). Chemical fertility and strawberry production and safety of produced food were monitored for three consecutive productive seasons on the same growing media. During the first year of cultivation, plants grown on sediment-based media showed a significantly lower biomass production and fruit yield compared with peat, mainly due to the sediment low fertility. In the subsequent two years, the plant re-cultivation improved the sediment structure and N mineralization, and on the third cultivation year both strawberry cultivars showed higher fruit productivity and no accumulation of potentially toxic trace metals. The produced fruits did non accumulate high concentrations of trace metals, and risk assessment showed no risks for human health related to the consumption of strawberry produced on sediment-based growing media. We concluded that a phytoremediated sediment could be recycled as an ingredient of soilless growing media for reducing the environmental impact of plant nursery production and posing no risks for human health. These results show that reclaimed sediments could be reconsidered as a component material category in the new EU regulation on fertilizers.

Remediation of heavy metals and enhancement of fertilizing potential of a sewage sludge by the synergistic interaction of woodlice and earthworms.

Woodlice and the earthworm alone or in combination were used to improve physical properties, nutrient release, and heavy metals stabilization during composting of sewage sludge. Chemical properties of raw sludge (IS) were compared to those of composted sludge (CS), sludge + earthworms (VS), sludge + woodlice (WS), and sludge + earthworms + woodlice (VWS) after 50 and 100 days of composting. Physical properties and heavy metals accumulation by the studied fauna was determined after 100 days of composting. Highest proportions of fine particles, porosity and water holding capacity were in the VWS treatment, electrical conductivity, ash content, inorganic N and the total concentrations of P, K, Ca and Mg increased, whereas the pH value, the organic C, C:N ratio and humic acids content decreased in following order: VWS > VC > WS > CS > IS. Total concentrations of Pb, Cd and Ni in composted sludge were lower whereas concentrations of Zn, Cu and Mn were higher than in raw sludge, and woodlice showed higher bioconcentration factor (BCFs) than earthworm for all heavy metals in all treatments. We concluded that woodlice and earthworms synergistically improved the physicochemical properties of the compost and enhanced its potential use as amendment in agricultural soils.

Phytomanagement Reduces Metal Availability and Microbial Metal Resistance in a Metal Contaminated Soil.

Short rotation coppice (SRC) with metal tolerant plants may attenuate the pollution of excessive elements with potential toxicity in soils, while preserving soil resources and functionality. Here, we investigated effects of 6 years phytomanagement with willow SRC on properties including heavy metal levels, toxicity tested by BioTox, microbial biomass, enzyme activities, and functional gene abundances measured by GeoChip of soils contaminated by As, Cd, Pb and Zn, as compared to the same soils under non-managed mixed grassland representing no intervention treatment (Unt). Though metal total concentrations did not differ by SRC and Unt, SRC soils had lower metal availability and toxicity, higher organic carbon, microbial biomass, phosphatase, urease and protease activities, as compared to Unt soils. Significantly reduced abundances of genes encoding resistances to various metals and antibiotics were observed in SRC, likely attributed to reduced metal selective pressure based on less heavy metal availability and soil toxicity. SRC also significantly reduced abundances of genes involved in nitrogen, phosphorus, and sulfur cycles, possibly due to the willow induced selection. Overall, while the SRC phytomanagement did not reduce the total heavy metal concentrations in soils, it decreased the heavy metal availability and soil toxicity, which in turn led to less metal selective pressure on microbial communities. The SRC phytomanagement also reduced the abundances of nutrient cycling genes from microbial communities, possibly due to intense plant nutrient uptake that depleted soil nitrogen and phosphorus availability, and thus site-specific practices should be considered to improve the soil nutrient supply for phytomanagement plants.

Biological nitrification inhibition in the rhizosphere: determining interactions and impact on microbially mediated processes and potential applications.

Nitrification is the microbial conversion of reduced forms of nitrogen (N) to nitrate (NO3-), and in fertilized soils it can lead to substantial N losses via NO3- leaching or nitrous oxide (N2O) production. To limit such problems, synthetic nitrification inhibitors have been applied but their performance differs between soils. In recent years, there has been an increasing interest in the occurrence of biological nitrification inhibition (BNI), a natural phenomenon according to which certain plants can inhibit nitrification through the release of active compounds in root exudates. Here, we synthesize the current state of research but also unravel knowledge gaps in the field. The nitrification process is discussed considering recent discoveries in genomics, biochemistry and ecology of nitrifiers. Secondly, we focus on the 'where' and 'how' of BNI. The N transformations and their interconnections as they occur in, and are affected by, the rhizosphere, are also discussed. The NH4+ and NO3- retention pathways alternative to BNI are reviewed as well. We also provide hypotheses on how plant compounds with putative BNI ability can reach their targets inside the cell and inhibit ammonia oxidation. Finally, we discuss a set of techniques that can be successfully applied to solve unresearched questions in BNI studies.

Long-term effects of aided phytostabilisation of trace elements on microbial biomass and activity, enzyme activities, and composition of microbial community in the Jales contaminated mine spoils.

We studied the effectiveness of remediation on microbial endpoints, namely microbial biomass and activity, microbial and plant species richness, of an As-contaminated mine spoil, amended with compost (C) alone and in combination with beringite (B) or zerovalent iron grit (Z), to increase organic matter content and reduce trace elements mobility, and to allow Holcus lanatus and Pinus pinaster growth. Untreated spoil showed the lowest microbial biomass and activity and hydrolase activities, and H. lanatus as sole plant species, whereas the presented aided phytostabilisation option, especially CBZ treatment, significantly increased microbial biomass and activity and allowed colonisation by several plant species, comparable to those of an uncontaminated sandy soil. Microbial species richness was only increased in spoils amended with C alone. No clear correlation occurred between trace element mobility and microbial parameters and plant species richness. Our results indicate that the choice of indicators of soil remediation practices is a bottleneck.

Nanoparticles and modified clays influenced distribution of heavy metals fractions in a light-textured soil amended with sewage sludges.

We comparatively assessed the effect of modified clays (MCs) (zeolite and bentonite) and nanoparticles (NPs) (ZnO and MgO) on the distribution of cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) fractions in a light-textured soil amended with five sewage sludges (SSs) differed in chemical properties during a 92-day incubation experiment in the laboratory. Results indicated that presence of adsorbents and incubation time were critical parameters in stabilizing metals in SS-amended soil. The adsorbents that significantly decreased the easily soluble and exchangeable of metals in SS-amended soil (rate 2%) were bentonite, ZnO and MgO for Cd and Cu, ZnO and MgO for Ni, and bentonite and MgO for Zn. The calculated mobility factor of the metals at five incubation times (5, 15, 29, 57 and 92days) was found in the range of 34.7-51.8 for Cd, 16.7-20.0 for Cu, 15.0-27.7 for Ni, and 35.3-40.7 for Zn indicating a higher mobility of Cd. The addition of MCs and NPs decreased the available fraction of metals, and among them, we suggest bentonite and MgO as novel, promising and feasible adsorbents with the unique characteristics in ameliorating the toxic effects of metals by converting their form from labile to non-labile phases in the SS-amended soil.

Functional activity and functional gene diversity of a Cu-contaminated soil remediated by aided phytostabilization using compost, dolomitic limestone and a mixed tree stand.

Trace elements (TEs) availability, biochemical activity and functional gene diversity was studied in a Cu-contaminated soil, revegetated for six years with a mixed stand of willow, black poplar, and false indigo-bush, and amended or not with compost plus dolomitic limestone (OMDL). The OMDL amendment significantly reduced Cu and As availability and soil toxicity, and increased the biochemical activity and microbial functional diversity assessed with the GEOCHIP technique, as compared to the unamended soil (Unt). The OMDL soil showed significantly higher abundance of 25 functional genes involved in decomposition organic compounds, and 11, 3 and 11 functional genes involved in the N, P and S biogeochemical cycles. Functional gene abundance was positively correlated with nutrient contents but negatively correlated with Cu availability and soil toxicity. The abundance of microbial functional genes encoding for resistance to various TEs also increased, possibly due to the microbial proliferation and lower Cu exposure in the presence of high total soil Cu concentration. Genes encoding for antibiotic resistance due to the co-occurrence of TEs and antibiotic resistant genes on genetic mobile elements. Overall, phytomanagement confirmed its potential to restore the biological fertility and diversity of a severely Cu-contaminated soil, but the increase of TEs and antibiotic resistant gene abundances deserve attention in future studies.

Phytoremediated marine sediments as suitable peat-free growing media for production of red robin photinia (Photinia x fraseri).

Sediments dredged by an industrial port, slightly contaminated by heavy metals and petroleoum hydrocarbons, were phytoremediated and used as peat-free growing media for the red robin photinia (Photinia x fraseri L.). Plants were grown on sediment only (S), sediment mixed with composted pruning residues (S + PR), sediment fertilized with controlled release fertilizers (S + F) and peat-based growing media as control (C). Plant elongation and dry weight, leaf contents of chlorophyll, malondialdehyde (MDA), macronutrients and heavy metals were determined at the end of one growing season. Environmental impact related to the use of sediment-based as compared to peat-based growing media was assessed by the Life Cycle Analysis (LCA). Sediment-based growing media presented significantly higher bulk density, pH and electrical conductivity values, lower C and N contents, and significantly higher total and available P. Red robin photinia grown on S + F growing media showed morphological and chemical parameters similar to those of control plants (C), whereas plants grown on S and S + PR showed lower growth. Leaf concentration of nutrients and heavy metals varied depending on the considered element and growing media, but were all within the common values for ornamental plants, whereas the highest MDA concentrations were found in plants grown on traditional growing media. The LCA indicated the use of sediments as growing media reduced the C footprint of ornamental plant production and the contribute of growing media to the environmental impact per produced plant. We concluded that sediments phytoremediation and use in plant nursery is a practical alternative re-use option for dredged sediments.

Evaluation of dredged sediment co-composted with green waste as plant growing media assessed by eco-toxicological tests, plant growth and microbial community structure.

Dredged sediments have currently no broad reuse options as compared to other wastes due to their peculiar physico-chemical properties, posing problems for the management of the large volumes of sediments dredged worldwide. In this study we evaluated the performance of sediment (S) co-composted with green waste (GW) as growing medium for ornamental plants. Analysis of the microbial community structure, eco-toxicological tests, were conducted on sediments at 1:1 and 3:1S:GW composting ratios. Sediment-based growing media were then reused to growth the ornamental plant Photina x fraseri in a pilot-scale experiment and plants' physiological and chemical parameters were measured. The results showed that co-composting with green waste increased the diversity of bacteria, fungi and archaea as compared to the untreated sediments, and that both the 1:1 and 3:1 S:GW composted sediments had no substantial eco-toxicological impacts, allowing an excellent plant growth. We concluded that co-composted of sediment with green waste produce a growing medium with suitable properties for growing ornamental plants, and represent a sustainable option for beneficial use of dredged sediments.

Biochemical parameters and bacterial species richness in soils contaminated by sludge-borne metals and remediated with inorganic soil amendments.

The effectiveness of two amendments for the in situ remediation of a Cd- and Ni-contaminated soil in the Louis Fargue long-term field experiment was assessed. In April 1995, one replicate plot (S1) was amended with 5% w/w of beringite (B), a coal fly ash (treatment S1+B), and a second plot with 1% w/w zerovalent-Fe iron grit (SS) (treatment S1+SS), with the aim of increasing metal sorption and attenuating metal impacts. Long-term responses of daily respiration rates, microbial biomass, bacterial species richness and the activities of key soil enzymes (acid and alkaline phosphatase, arylsulfatase, beta-glucosidase, urease and protease activities) were studied in relation to soil metal extractability. Seven years after initial amendments, the labile fractions of Cd and Ni in both the S1+B and S1+SS soils were reduced to various extents depending on the metal and fractions considered. The soil microbial biomass and respiration rate were not affected by metal contamination and amendments in the S1+B and S1+SS soils, whereas the activity of different soil enzymes was restored. The SS treatment was more effective in reducing labile pools of Cd and Ni and led to a greater recovery of soil enzyme activities than the B treatment. Bacterial species richness in the S1 soil did not alter with either treatment. It was concluded that monitoring of the composition and activity of the soil microbial community is important in evaluating the effectiveness of soil remediation practices.

Assessment of zerovalent iron for stabilization of chromium, copper, and arsenic in soil.

Stabilization of soil contaminated with trace elements is a remediation practice that does not reduce the total content of contaminants, but lowers the amounts of mobile and bioavailable fractions. This study evaluated the efficiency of Fe(0) to reduce the mobility and bioavailability of Cr, Cu, As and Zn in a chromated copper arsenate (CCA)-contaminated soil using chemical, biochemical and biotoxicity tests. Contaminated soil was stabilized with 1% iron grit. This treatment decreased As and Cr concentrations in leachates (by 98% and 45%, respectively), in soil pore water (by 99% and 94%, respectively) and in plant shoots (by 84% and 95%, respectively). The stabilization technique also restored most of analyzed soil enzyme activities and reduced microbial toxicity, as evaluated by the BioTox test. After stabilization, exchangeable and bioaccessible fractions of Cu remained high, causing some residual toxicity in the treated soil.

Assessment of a particle size fractionation as a technology for reducing heavy metal, salinity and impurities from compost produced by municipal solid waste.

A physical fractionation of a compost obtained by municipal solid wastes (MSW) was conducted by dry-sieving process, to quantify coarse impurities and assess the distribution of nutrients, heavy metals and salinity values in particle size fractions of 2, 1.2-2, 0.8-1.2, 0.4-0.8, 0.2-0.4, 0.1-0.2 and <0.1mm diameter. The whole unfractionated compost and all physical fractions were analyzed for the same chemical parameters. The results showed that the studied compost was of a low grade due to high salinity and heavy metal concentrations, and the presence of coarse impurities, mainly glass. The physical fractionation analysis showed that heavy metal and base cations concentrations, and salinity values significantly increased with decreasing of particle size, whereas macro nutrients such as C, N and P were more evenly distributed among the different particle size fractions. Overall, our results showed that the removal of selected particle size fractions <0.8mm and coarse impurities (e.g. glass impurity >2mm) could significantly improve the compost quality without reduce its fertilization potential. We concluded that particle size fractionation is a feasible and sustainable approach to improve composted MSW materials for their safe recycle in agriculture.

Selecting chemical and ecotoxicological test batteries for risk assessment of trace element-contaminated soils (phyto)managed by gentle remediation options (GRO).

During the past decades a number of field trials with gentle remediation options (GRO) have been established on trace element (TE) contaminated sites throughout Europe. Each research group selects different methods to assess the remediation success making it difficult to compare efficacy between various sites and treatments. This study aimed at selecting a minimum risk assessment battery combining chemical and ecotoxicological assays for assessing and comparing the effectiveness of GRO implemented in seven European case studies. Two test batteries were pre-selected; a chemical one for quantifying TE exposure in untreated soils and GRO-managed soils and a biological one for characterizing soil functionality and ecotoxicity. Soil samples from field studies representing one of the main GROs (phytoextraction in Belgium, Sweden, Germany and Switzerland, aided phytoextraction in France, and aided phytostabilization or in situ stabilization/phytoexclusion in Poland, France and Austria) were collected and assessed using the selected test batteries. The best correlations were obtained between NH4NO3-extractable, followed by NaNO3-extractable TE and the ecotoxicological responses. Biometrical parameters and biomarkers of dwarf beans were the most responsive indicators for the soil treatments and changes in soil TE exposures. Plant growth was inhibited at the higher extractable TE concentrations, while plant stress enzyme activities increased with the higher TE extractability. Based on these results, a minimum risk assessment battery to compare/biomonitor the sites phytomanaged by GROs might consist of the NH4NO3 extraction and the bean Plantox test including the stress enzyme activities.