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The exponential growth of the human population requires an increasing application of nitrogen (N) fertilizers, causing environmental pollution. Biochar (B) amended soil has been suggested as a sustainable agricultural practice to improve crop yield and mitigate agricultural pollutants’ contamination. Evaluating the effect of fertilization on Brassica crops, in combination with spectral analysis, may specify changes in the chemical composition of the vegetable as a result of N fertilization. This study characterized cauliflower tissues treated with N fertilizer and biochar, employing Fourier Transform Infrared spectroscopy. The experiment was conducted in cauliflower mesocosms treated with two doses of N fertilizer (130 and 260 kg N ha−1) with or without B. Attenuated total reflectance fractions were used to characterize fractions of curds, leaves, stems, and roots in the infrared using a Fourier transform. Principal component analysis was performed to classify the main differences among cauliflower tissues concerning treatments. FTIR spectra of Brassica oleracea L. var. botrytis tissues were related to nitrogen-based agricultural practices. The specific molecules associated with functional groups in cauliflower tissues were phenols, amides, proteins, amines, and glucosinolates. Biochar amended soil resulted in higher peaks that correspond to the stretching of phenols and proteins. The application of sustainable nitrogen fertilizers might influence the absorption bands characteristic of cauliflower’s typical metabolites. The research allows the identification of Brassicaceae’s functional molecules with a potential agronomic application.

Aquifer represents an oligotrophic environment that sustains a relatively small amount of microbial cells, mostly non-culturable. Due to this dominance of unculturable microorganisms in natural ecosystems, studying microbial communities and their functionality should include culture-independent approaches based on molecular techniques using DNA analysis (Purswani et al. 2011). For practical reasons, aquifer routine analyses focus on groundwater samples, while solid aquifer samples are typically not included (Ritalahti et al. 2010). The amount of groundwater collected, together with the types and concentrations of inhibitory compounds if present, determine the abundance of the target biomarker(s) available for subsequent analyses. Hence, filtering large volumes of groundwater seems beneficial, but for practical purposes, 0.5–2 L of water are typically collected, depending on groundwater characteristics (Ritalahti et al. 2010). In this work, environmental DNA (eDNA) was extracted from groundwater samples, filtering three different initial volumes (1000 ml, 500 ml, and 250 ml) of water samples, using 0.22 µm membranes. Also, two DNA extraction commercial kits were tested, DNeasy PowerWater Kit and DNeasy PowerSoil kit (Qiagen, Germantown, MD) specific for water samples and solid matrix, respectively. For DNeasy PowerWater Kit the standard protocol was carried out, whereas a modified protocol equipped with the Inhibitor Removal Technology® (IRT) was selected for testing DNeasy PowerSoil kit. In order to minimize operator bias, both the protocols were made semi-automated by using a QIAcube provided by Qiagen for genomic DNA extraction. Additional steps to the PowerSoil IRT protocol were performed for optimizing chemical and mechanical cell lysis processes and facilitating the sample dispersion into the buffer solutions. eDNA was verified via electrophoresis and quantified fluorometrically. eDNA extracted from 250 ml of groundwater sample using the DNeasy PowerSoil kit with modified IRT protocol was also tested in downstream applications, including Polymerase Chain Reactions (PCRs) with specific primer pairs for the identification of microbial targets. Results suggest that the PowerSoil modified IRT protocol was the best performing one, allowing a higher eDNA yield from all the water sample volumes tested. In addition, plotting on a graph eDNA concentration values against sample volumes filtered, the yield of PowerSoil modified IRT protocol appeared more similar to an ideal direct proportionality than the yield of PowerWater standard protocol. eDNA quality was suitable for PCR analyses and the identification of bacterial targets, including bacterial subgroups (α and β-proteobacteria) and single species of interest, such as Shewanella oneidensis capable of hexavalent chromium reduction (Tumolo et al. 2020). Under these good performances, the PowerSoil optimized IRT protocol was also applied in a further experiment about bioremediation to extract eDNA from 50 ml of water spiked with 1000 µg/l of hexavalent chromium. The resulting genomic material was successfully used in quantitative PCR (qPCR) assays for monitoring the relative abundance of Shewanella oneidensis during the bioremediation process, allowing to highlight the hexavalent chromium inhibitor effect on the selected microbial target (Ancona et al. 2020). Downstream applications of eDNA obtained using DNeasy PowerSoil kit resulted in positive outcomes for both the experiments previously described. In light of this, it is possible to conclude that this kit combined with the protocol adjustments proposed in this work, can be a performing tool for eDNA extraction, also from small amounts of water sample collected from oligotrophic or degraded environments. Further investigations will be oriented to optimize eDNA extraction from the aquifer solid portion characterized by few nutrients and microbial cells to better understand how microbial populations can distribute themselves between solid and aqueous phases.

The European Community (EC) Nitrates Directive (ND) (Council Directive 1991/676/EEC) aims to avoid water pollution by nitrates from poor farming practices. Under the ND, Nitrate Vulnerable Zones (NVZs) have been defined as areas where the concentration of agricultural nitrates in surface or groundwater exceeds 50 mg/L. Groundwater is one of the main sources of drinking water in Europe, so ensuring its quality is of significant importance. Monitoring of water parameters, the identification of pollution, the development of good agricultural practice codes is included in ND as measures and action programs. ND provides for the attribution of areas affected by nitrate pollution to the NVZ in which farmers must comply with measures necessary to reduce nitrogen in excess and protect the quality of environmental matrices. However, in some cases the quality of water bodies remains poor, suggesting that some action measures are ineffective or need improvements. Nevertheless, nitrates in excess in groundwater might be related to industrial inputs, untreated waste discharge, and sewage spilling. Due to this, identifying non-agricultural contributions to nitrate groundwater pollution can be of great interest. With this aim, the employment of biomarkers such as microbes associated with the intestinal tract of a specific host is under study for identifying fecal pollution. This strategy is known, in scientific literature, as Microbial Source Tracking (MST)(Furtula et al. 2011)The amplification of 16S rRNA genes of host-specific Bacteroidales allows discriminating against human and livestock fecal sources in samples from nitrate polluted environment. The aim of this work is to evaluate the sensitivity and specificity of these intestinal biomarkers in groundwater samples collected along with the Apulian Region (Southern Italy) in order to assess the applicability of the MST in the employ of ND. A preliminary study was carried out to validate the performance of seven markers for MST using two different approaches: Polimerase Chain Reaction (PCR) and quantitative Real-time PCR (qPCR) assays. For both PCR and qPCR experiments selected primer sets were checked using fecal samples of known origin as positive controls. PCR assay was used for human (HF74 and Enterococcus faecalis) (Bernhard and Field 2000, Jackson et al. 2004), cattle (CF123) (Bernhard and Field 2000), equine (HoF597) (Dick et al. 2005), and pig (Pig163) (Dick et al. 2005) markers screening. Two additional markers were tested through Real-time PCR: human (Human- Bacteroides) (Seurinck et al. 2005) and zootechnical (BacPre I) (Kobayashi et al. 2012). The results indicate that biomarkers can be considered reliable in distinguishing human from animal pollution. Nevertheless, our studies show that the tests conducted with the human Enterococcus faecalis biomarker do not discriminate the zootechnical source from the human one. In some cases, PCR-tested biomarkers cannot determine the source of contamination in environmental matrices due to the detection limit. Animal and human fecal markers were widely detected on eleven groundwater samples through Real Time PCR, highlighting the prevalent source of contamination in the environmental matrix. This research provides evidence that MST technology is a valid tool for local authorities to identify the source of nitrate contamination and review uncertainties during the NVZs definition and the action program development required by the European Nitrate Directive.

Remediation interventions based on the native bacteria’s capability to reduce Cr(VI) represent a valid strategy in terms of economic and environmental sustainability. In this study, a bioremediation test was carried out using viable microcosms set with groundwater and deep soil (4:1), collected from the saturated zone of an industrial site in Southern Italy that was polluted by ~130 µg L−1 of Cr(VI). Conditions simulating the potential natural attenuation were compared to the enhanced natural attenuation induced by supplying yeast extract or polyhydroxybutyrate. Sterile controls were set up to study the possible Cr(VI) abiotic reduction. No pollution attenuation was detected in the unamended viable reactors, whereas yeast extract provided the complete Cr(VI) removal in 7 days, and polyhydroxybutyrate allowed ~70% pollutant removal after 21 days. The incomplete abiotic removal of Cr(VI) was observed in sterile reactors amended with yeast extract, thus suggesting the essential role of native bacteria in Cr(VI) remediation. This was in accordance with the results of Pearson’s coefficient test, which revealed that Cr(VI) removal was positively correlated with microbial proliferation (n = 0.724), and also negatively correlated with pH (n = −0.646), dissolved oxygen (n = −0.828) and nitrate (n = −0.940). The relationships between the Cr(VI) removal and other monitored parameters were investigated by principal component analysis, which explained 76.71% of the total variance.

Chromium is a potentially toxic metal occurring in water and groundwater as a result of natural and anthropogenic sources. Microbial interaction with mafic and ultramafic rocks together with geogenic processes release Cr (VI) in natural environment by chromite oxidation. Moreover, Cr (VI) pollution is largely related to several Cr (VI) industrial applications in the field of energy production, manufacturing of metals and chemicals, and subsequent waste and wastewater management. Chromium discharge in European Union (EU) waters is subjected to nationwide recommendations, which vary depending on the type of industry and receiving water body. Once in water, chromium mainly occurs in two oxidation states Cr (III) and Cr (VI) and related ion forms depending on pH values, redox potential, and presence of natural reducing agents. Public concerns with chromium are primarily related to hexavalent compounds owing to their toxic effects on humans, animals, plants, and microorganisms. Risks for human health range from skin irritation to DNA damages and cancer development, depending on dose, exposure level, and duration. Remediation strategies commonly used for Cr (VI) removal include physico-chemical and biological methods. This work critically presents their advantages and disadvantages, suggesting a site-specific and accurate evaluation for choosing the best available recovering technology.

In this review, we examined the European legislative context on water protection concerning pesticide residues monitoring and the relevant National Action Plans and strategies that were undertaken in European countries to better identify and manage the problem in water bodies. Furthermore, we illustrated the development of analytical methodologies and criticalities connected to determine pesticide residues in water matrices, including sampling, sample preparation approaches, instrumental analyses and specific applications emphasising those works published after 2015. Details about sampling strategies, analytical feasibility, official methods, degradation products and behaviour in the environment for a subset of 160 pesticides are also provided.

Nitrogen (N) is a fundamental nutrient for plant growth and for the performance of biological functions. In agroecosystems, nitrogen fertilization is aimed at providing a suitable N dose for crop growth, avoiding the impoverishment or the improper enrichment of nitrogen compounds in soil. The high application of nitrogen fertilizers is the main cause of the increase in nitrate leaching and loss of the quality of natural resources (groundwater and soil). In the last decades, new sustainable technological approaches have been developed and applied on laboratory and field scales to reduce the impacts of nitrogen pollution on the environmental matrices and to improve the sustainability of agricultural management. This review highlights the results of the implementation of sustainable remediation new strategies to reduce pollution from a main agricultural contaminant (nitrate) and describes the benefits obtained from the use of these solutions in agroecosystems.