Response of chemical and biochemical soil properties to the spreading of biochar-based treated olive mill wastewater.

Despite the polluting potential olive mill wastewater (OMW) can be a useful source of nutrients and organic compounds to improve soil properties. The aim of this paper was to verify if biochar-based treatment of OMW could be an efficient method to contrast the richness in phenolic compounds and phytotoxicity of OMW making it more suitable. for soil amendment. In this study poplar biochar (BP) was more effective than conifer biochar (BC) in terms of adsorbing phenols and reducing phytotoxicity at different biochar rates (5 and 10 %). In soil amendment BP-treated OMW induced an increase of organic carbon by approximately 15 % and notably BP10 treated OMW enhanced available phosphorous by 25 % after 30 days of incubation. In soil amended with 10 % BP-treated OMW microbial biomass and enzymatic activities were significantly enhanced after 30 and 90 days, with no effect on cress seed germination. Therefore, biochar based-treatment could be cost-effective and able to facilitate the long-term management of OMW in terms of storage and disposal.

Aerobic biodegradation of propylene glycol by soil bacteria.

Propylene glycol (PG) is a main component of aircraft deicing fluids and its extensive use in Northern airports is a source of soil and groundwater contamination. Bacterial consortia able to grow on PG as sole carbon and energy source were selected from soil samples taken along the runways of Oslo Airport Gardermoen site (Norway). DGGE analysis of enrichment cultures showed that PG-degrading populations were mainly composed by Pseudomonas species, although Bacteroidetes were found, as well. Nineteen bacterial strains, able to grow on PG as sole carbon and energy source, were isolated and identified as different Pseudomonas species. Maximum specific growth rate of mixed cultures in the absence of nutrient limitation was 0.014 h(-1) at 4 °C. Substrate C:N:P molar ratios calculated on the basis of measured growth yields are in good agreement with the suggested values for biostimulation reported in literature. Therefore, the addition of nutrients is suggested as a suitable technique to sustain PG aerobic degradation at the maximum rate by autochthonous microorganisms of unsaturated soil profile.

Response of chemical and biochemical soil properties to the spreading of biochar-based treated olive mill wastewater.

Olive mill wastewater (OMW) is the effluent derived from the oil extraction processes from olives. Despite the polluting potential OMW can be a useful source of nutrients and organic compounds to improve soil properties. OMW could negatively affect soil and water quality as this waste is rich in phenolic compounds and has high COD and BOD5. Biochar-based treatment could be an efficient method to remediate OMW. In this study poplar biochar (BP) was more effective than conifer biochar (BC) in terms of adsorbing phenols and reducing phytotoxicity at different biochar rates (5 and 10 %). BP-treated OMW was used in soil amendment and induced an increase in chemical properties, especially in organic carbon after 30 days of incubation. In soil amended with 10 % BP-treated OMW microbial biomass, enzymatic activities, and cress seed germination were significantly enhanced after 30 and 90 days.

Combined bioaugmentation and biostimulation techniques in bioremediation of pentachlorophenol contaminated forest soil.

Pentachlorophenol (PCP) is quite persistent in the environment and severely affects different ecosystems including forest soil. The main objective of this work was to study different bioremediation processes of artificially PCP (100 mg kg-1) contaminated forest soil (Sc). In fact, we used bioaugmentation by adding two different bacterial consortia B1 and B2, biostimulation procedures by amendments based on forest compost (FC), municipal solid waste compost (MC), sewage sludge (SS), and phosphate, and their combined treatments. Soil physical and chemical properties, residual PCP, soil microbial biomass carbon, soil respiration and some enzymatic activities at zero time and after 30 d of incubation, were evaluated. A net reduction of PCP, 71% of the initial concentration, after 30 d-incubation occurred in the sample Sc+B1+FC, as the best performance among all treatments, due to natural attenuation, immobilization of PCP molecules in the forest soil through organic amendments, and the action of the exogenous microbial consortium B1. The single application of FC or B1 led to a depletion of PCP concentration of 52% and 41%, respectively. Soil microbial biomass carbon decreased in PCP contaminated soil but it increased when organic amendment also in combination with microbial consortia was carried out as bioremediation action. Soil respiration underwent no changes in contaminated soil and increased under FC based bioremediation treatment. These results demonstrate that the combined treatments of biostimulation and bioaugmentation might be a promising process for remediation of PCP contaminated soil.

Low mechano-energetic efficiency is associated with future left ventricular systolic dysfunction in hypertensives.

AIMS: In a hypertensive population with optimal blood pressure control with a long-term follow-up, we aimed at analysing possible predictors of left ventricular (LV) ejection fraction (LVEF) reduction, including indexed mechano-energetic efficiency (MEEi), a well-recognized echo-derived parameter of LV performance. METHODS AND RESULTS: The study population included 5673 hypertensive patients from the Campania Salute Network with a long-term follow-up, normal baseline LVEF (≥50%), and no prevalent cardiovascular (CV) disease. Patients developing LVEF impairment (LVEF < 50% or a reduction of at least 10 percentage points compared with baseline) were compared with patients with persistently normal LVEF. Optimal blood pressure control was achieved in about 80% of patients. Patients who experienced LVEF reduction were 2.41% during a long-term follow-up (mean duration 5.6 ± 3.9 years). At baseline, they were older (59.46 ± 11.58 vs. 53.40 ± 11.41, P < 0.0001) and showed higher LV mass index (53.3 ± 12.83 vs. 47.56 ± 9.58, P < 0.0001), left atrial (LA) volume index (14.4 ± 4.2 vs. 13.1 ± 2.8, P < 0.0001) and carotid intima-media thickness (1.99 ± 0.86 vs. 1.61 ± 0.73, P < 0.0001), lower MEEi (0.32 ± 0.08 vs. 0.34 ± 0.07, P = 0.037), and higher prevalence of CV events during follow-up (13.9% vs. 3%, P < 0.0001) compared with patients with persistently normal LVEF. A logistic regression analysis, performed after running univariate analyses and selecting parameters significantly associated with LVEF reduction, showed that having a CV event [odds ratio (OR) 7.57, P < 0.0001], being in the lowest MEEi quartile (OR 2.43, P = 0.003), and having a larger LA volume index (OR 1.08, P = 0.028) were all parameters independently associated with the development of LV systolic dysfunction. A further logistic regression model, performed by excluding patients experiencing CV events, demonstrated that the lowest MEEi quartile was independently associated with the evolution towards LVEF reduction (OR 2.35, P = 0.004), despite significant impact of LA volume index (OR 1.08, P = 0.023) and antiplatelet therapy (OR 1.89, P < 0.01). Receiver operating characteristic curves showed that the model including MEEi had higher accuracy than the model without MEEi in predicting LVEF reduction (areas under the curve 0.68 vs. 0.63, P = 0.046). CONCLUSIONS: Lower values of MEEi at baseline identify hypertensive patients more liable to develop LVEF reduction. In hypertensive setting, MEEi evaluation improves risk stratification for development of LV systolic dysfunction during long-term follow-up.

Can Lunar and Martian Soils Support Food Plant Production? Effects of Horse/Swine Monogastric Manure Fertilisation on Regolith Simulants Enzymatic Activity, Nutrient Bioavailability, and Lettuce Growth.

To make feasible the crewed missions to the Moon or Mars, space research is focusing on the development of bioregenerative life support systems (BLSS) designed to produce food crops based on in situ resource utilisation (ISRU), allowing to reduce terrestrial input and to recycle organic wastes. In this regard, a major question concerns the suitability of native regoliths for plant growth and how their agronomic performance is affected by additions of organic matter from crew waste. We tested plant growth substrates consisting of MMS-1 (Mars) or LHS-1 (Lunar) simulants mixed with a commercial horse/swine monogastric manure (i.e., an analogue of crew excreta and crop residues) at varying rates (100:0, 90:10, 70:30, 50:50, w/w). Specifically, we measured: (i) lettuce (Lactuca sativa L. cultivar 'Grand Rapids') growth (at 30 days in open gas exchange climate chamber with no fertilisation), plant physiology, and nutrient uptake; as well as (ii) microbial biomass C and N, enzymatic activity, and nutrient bioavailability in the simulant/manure mixtures after plant growth. We discussed mechanisms of different plant yield, architecture, and physiology as a function of chemical, physico-hydraulic, and biological properties of different substrates. A better agronomic performance, in terms of plant growth and optically measured chlorophyll content, nutrient availability, and enzymatic activity, was provided by substrates containing MMS-1, in comparison to LHS-1-based ones, despite a lower volume of readily available water (likely due to the high-frequency low-volume irrigation strategy applied in our experiment and foreseen in space settings). Other physical and chemical properties, along with a different bioavailability of essential nutrients for plants and rhizosphere biota, alkalinity, and release of promptly bioavailable Na from substrates, were identified as the factors leading to the better ranking of MMS-1 in plant above and below-ground mass and physiology. Pure Mars (MMS-1) and Lunar (LHS-1) simulants were able to sustain plant growth even in absence of fertilisation, but the amendment with the monogastric manure significantly improved above- and below-ground plant biomass; moreover, the maximum lettuce leaf production, across combinations of simulants and amendment rates, was obtained in treatments resulting in a finer root system. Increasing rates of monogastric manure stimulated the growth of microbial biomass and enzymatic activities, such as dehydrogenase and alkaline phosphomonoesterase, which, in turn, fostered nutrient bioavailability. Consequently, nutrient uptake and translocation into lettuce leaves were enhanced with manure supply, with positive outcomes in the nutritional value of edible biomass for space crews. The best crop growth response was achieved with the 70:30 simulant/manure mixture due to good availability of nutrients and water compared to low amendment rates, and better-saturated hydraulic conductivity compared to high organic matter application. A 70:30 simulant/manure mixture is also a more sustainable option than a 50:50 mixture for a BLSS developed on ISRU strategy. Matching crop growth performance and (bio)chemical, mineralogical, and physico-hydraulic characteristics of possible plant growth media for space farming allows a better understanding of the processes and dynamics occurring in the experimental substrate/plant system, potentially suitable for an extra-terrestrial BLSS.

Organic Matter Characterization and Phytotoxic Potential Assessment of a Solid Anaerobic Digestate Following Chemical Stabilization by an Iron-Based Fenton Reaction.

Digestates, a byproduct of the anaerobic bioconversion of organic wastes for the production of biogas, are highly variable in chemical and biological properties, thus limiting their potential use in agriculture as soil amendment. Using a lab-scale glass reactor, we aimed to assess the feasibility to chemically stabilize the solid fraction of an anaerobic digestate by applying a Fenton reaction under constant pH (3.0), temperature (70 °C), reaction time (8 h), and various combinations of H2O2 and Fe2+. In Fenton-treated samples, the phytotoxic potential (determined on a test plant), total phenols, and the bad smell odor index markedly declined, whereas total C and N remained unaltered. Thermogravimetric (TG) analysis and Fourier transform infrared (FT-IR) spectroscopy revealed contrasting changes in extracted humic and fulvic fractions being increased or depleted, respectively, in aromatic substances. Process feasibility and optimum conditions for an effective biomass stabilization were achieved with a H2O2/Fe2+ ratio between 0.02 and 0.03.

Oxidative transformation of aqueous phenolic mixtures by birnessite-mediated catalysis.

The catalytic efficiency of birnessite in the removal of catechol, hydroxytyrosol, methylcatechol and m-tyrosol, four phenols commonly present in polluted wastewaters, was studied in mono-substrate solutions or in mixtures of two, three, and four substrates. In single phenolic solutions the transformation order of phenols was catechol>hydroxytyrosol>methylcatechol>m-tyrosol. With phenolic mixtures different responses were observed and the amount of each phenol transformed and the crossing effects among the various phenols depended on the type and number of phenols present in the mixture. In particular, general inhibitory effects were observed for hydroxytyrosol and m-tyrosol that were transformed less when present in combination with the other phenols. By contrast the effects by the presence of more than one phenol were basically annulled for catechol and methylcatechol at 24 h incubation in all the mixtures. A simultaneous, but often no stoichiometric, release of soluble Mn2+ in the reaction mixtures occurred. The multi-substrate systems were designed to mimic birnessite-mediated oxidative processes that could occur under field conditions. Therefore they could be of great interest to environmental and soil science. The use of birnessite as a potential tool for an effective detoxification and recovery of phenol-polluted systems could be also envisaged.

May humic acids or mineral fertilisation mitigate arsenic mobility and availability to carrot plants (Daucus carota L.) in a volcanic soil polluted by As from irrigation water?

Carrot (Daucus carota L.) is a widely consumed root vegetable, whose growth and safety might be threatened by growing-medium arsenic (As) contamination. By this work, we evaluated the effects of humic acids from Leonardite and NPK mineral fertilisation on As mobility and availability to carrot plants grown for 60 days in a volcanic soil irrigated with As-contaminated water - representing the most common scenario occurring in As-affected Italian areas. As expected, the irrigation with As-contaminated water caused a serious toxic effect on plant growth and photosynthetic rate; the highest rate of As also inhibited soil enzymatic activity. In contrast, the organic and mineral fertilisation alleviated, at least partially, the toxicity of As, essentially by stimulating plant growth and promoting nutrient uptake. The mobility of As in the volcanic soil and thus its phytoavailability were differently affected by the organic and mineral fertilisers; the application of humic acids mitigated the availability of the contaminant, likely by its partial immobilisation on humic acid sorption sites - thus raising up the intrinsic anionic sorption capacity of the volcanic soil; the mineral fertilisation enhanced the mobility of As in soil, probably due to competition of P for the anionic sorption sites of the soil variable-charge minerals, very affine to available P. These findings hence suggest that a proper soil management of As-polluted volcanic soils and amendment by stable organic matter might mitigate the environmental risk of these soils, thus minimising the availability of As to biota.

Biochar based remediation of water and soil contaminated by phenanthrene and pentachlorophenol.

Phenanthrene (Phe) and pentachlorophenol (PCP) are classified as persistent organic pollutants and represent serious concern for the environment as they are toxic and ubiquitous. Biochar based remediation is an emerging technology used in water and soil contamination. In this study we used poplar (BP) and conifer (BC) biochars to remediate water and soil contaminated by Phe and PCP. BP and BC were able to remove completely either Phe or PCP from contaminated water within one to three days. When biochar was confined in a porous membrane, BC and BP maintained their sorption efficiency for several remediation cycles. However, in these conditions BC allowed faster Phe removal. In soil remediation experiments, addition of two biochar rates, i.e. 2.5 and 5 mg g-1, strongly reduced Phe extractability (up to 2.7% of the initially added Phe with the larger BC dose). This was similar to the behavior observed when compost was applied in order to verify the role of soil organic matter in the fate of both contaminants. PCP extractability was reduced only up to 75% (in average) in all samples including those with compost amendment. Only larger amount of biochar (20 and 50 mg g-1) allowed reduction of the extractable PCP and nullified phytotoxicity of the contaminant.

Mitigation of olive mill wastewater toxicity.

The toxicity of olive mill wastewaters (OMW) is commonly attributed to monomeric phenols. OMW were treated in an aerated, stirred reactor containing agricultural soil, where the oxidative polymerization of phenols took place. In 24 h, OMW monomeric phenols decreased by >90%. This resulted in a corresponding reduction in phytotoxicity, as measured by germination tests with tomato and English cress seeds, and in microbial toxicity, as measured by lag phase duration in Bacillus cereus batch growth. Soil germination capability after irrigation with OMW was assessed in long-term pot experiments. The relative germination percentage of tomato was higher when the soil was irrigated with treated OMW rather than with untreated ones, although it was lower than the control (e.g., soil irrigated with distilled water). At longer incubation times, a complete recovery of the soil germination capability was achieved with treated, but not with untreated, OMW.

Assessing the effectiveness of Byssochlamys nivea and Scopulariopsis brumptii in pentachlorophenol removal and biological control of two Phytophthora species.

Bioremediation and biological-control by fungi have made tremendous strides in numerous biotechnology applications. The aim of this study was to test Byssochlamys nivea and Scopulariopsis brumptii in sensitivity and degradation to pentachlorophenol (PCP) and in biological-control of Phytophthora cinnamomi and Phytophthora cambivora. B. nivea and S. brumptii were tested in PCP sensitivity and degradation in microbiological media while the experiments of biological-control were carried out in microbiological media and soil. The fungal strains showed low PCP sensitivity at 12.5 and 25 mg PCP L(-1) although the hyphal size, fungal mat, patulin, and spore production decreased with increasing PCP concentrations. B. nivea and S. brumptii depleted completely 12.5 and 25 mg PCP L(-1) in liquid culture after 28 d of incubation at 28 °C. Electrolyte leakage assays showed that both fungi have low sensitivity to 25 mg PCP L(-1) and produced no toxic compounds for the plant. B. nivea and S. brumptii were able to inhibit the growth of the two plant pathogens in laboratory studies and reduce the mortality of chestnut plants caused by two Phytophthorae in greenhouse experiments. The two fungal strains did not produce volatile organic compounds able to reduce the growth of two plant pathogens tested.

Depletion of pentachlorophenol in soil microcosms with Byssochlamys nivea and Scopulariopsis brumptii as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Fungal bioremediation is an emerging approach to rehabilitating areas fouled by recalcitrant xenobiotics. In the present study, we isolated two fungal strains from an artificially PCP-contaminated soil during a long-term bioremediation study and evaluated their potential as bioremediation agents in depletion and detoxification of PCP in soil microcosms. The two fungal strains were identified as: Byssochlamys nivea (Westling, 1909) and Scopulariopsis brumptii (Salvanet-Duval, 1935). PCP removal and toxicity were examined during 28 days of incubation. Bioaugmented microcosms revealed a 60% and 62% PCP removal by B. nivea and S. brumptii, respectively. Co-inoculation of B. nivea and S. brumptii showed a synergetic effect on PCP removal resulting in 95% and 80% PCP decrease when initial concentrations were 12.5 and 25 mg kg-1, respectively. Detoxification in bioaugmented soil and the efficient role of fungi in the rehabilitation of PCP contaminated soil were experimentally proven by toxicity assays. A decrease in inhibition of bioluminescence of Escherichia coli HB101 pUCD607 and an increase of germination index of mustard (Brassica alba) seeds were observed in the decontaminated soils.

Vertical and horizontal distributions of microbial abundances and enzymatic activities in propylene-glycol-affected soils.

The natural microbial activity in the unsaturated soil is vital for protecting groundwater in areas where high loads of biodegradable contaminants are supplied to the surface, which usually is the case for airports using aircraft de-icing fluids (ADF) in the cold season. Horizontal and vertical distributions of microbial abundance were assessed along the western runway of Oslo Airport (Gardermoen, Norway) to monitor the effect of ADF dispersion with special reference to the component with the highest chemical oxygen demand (COD), propylene glycol (PG). Microbial abundance was evaluated by several biondicators: colony-forming units (CFU) of some physiological groups (aerobic and anaerobic heterotrophs and microscopic fungi), most probable numbers (MPN) of PG degraders, selected catabolic enzymatic activities (fluorescein diacetate (FDA) hydrolase, dehydrogenase, and ß-glucosidase). High correlations were found between the enzymatic activities and microbial counts in vertical soil profiles. All microbial abundance indicators showed a steep drop in the first meter of soil depth. The vertical distribution of microbial abundance can be correlated by a decreasing exponential function of depth. The horizontal trend of microbial abundance (evaluated as total aerobic CFU, MPN of PG-degraders, and FDA hydrolase activity) assessed in the surface soil at an increasing distance from the runway is correlated negatively with the PG and COD loads, suggesting the relevance of other chemicals in the modulation of microbial growth. The possible role of potassium formate, component of runway de-icers, has been tested in the laboratory by using mixed cultures of Pseudomonas spp., obtained by enrichment with a selective PG medium from soil samples taken at the most contaminated area near the runway. The inhibitory effect of formate on the growth of PG degraders is proven by the reduction of biomass yield on PG in the presence of formate.

Formation and properties of organo-phosphatase complexes by abiotic and biotic polymerization of pyrogallol-phosphatase mixtures.

In this paper, the catalytic efficacy of peroxidase and manganese oxide, both commonly present in soil, to catalyze the formation of pyrogallol-phosphatase complexes was compared. The influence of several factors (e.g., the concentration of pyrogallol, the amount of catalysts, the nature of manganese oxide, birnessite, or pyrolusite, the incubation time, and the pH) on the transformation of pyrogallol and the characteristics and properties of the pyrogallol-phosphatase interaction products were investigated. The pyrogallol transformation mediated by both catalysts was very fast and increased by increasing the catalyst concentration. The nature of the catalyst also influenced the size and the molecular mass of the formed complexes. When polymerization of pyrogallol occurred with high intensity, a loss of phosphatase activity occurred, and it strongly depended on the pH at which the process was carried out and the catalyst. In particular, with peroxidase, the phosphatase activity was much lower in either suspensions or supernatants and not measurable in the insoluble complexes as compared to that measured in the presence of manganese oxides.

Biodegradation of phenanthrene and analysis of degrading cultures in the presence of a model organo-mineral matrix and of a simulated NAPL phase.

Two mixed bacterial cultures (C(B-BT) and C(I-AT)) degraded phenanthrene when it was: (i) in the presence of either hexadecane as a non aqueous phase liquid or a montmorillonite-Al(OH)x-humic acid complex as a model organo-mineral matrix; (ii) sorbed to the complex, either alone or in the presence of hexadecane. The cultures had different kinetic behaviours towards phenanthrene with or without hexadecane. The degradation of Phe alone as well as that of Phe in hexadecane ended in 8 and 15 days with C(B-BT) and C(I-AT) cultures, respectively. Hexadecane increased Phe bioavailability for C(I-AT) bacteria which degraded Phe according to first-order kinetics. The same effect was observed for C(B-BT) bacteria, but with an initial 2 days lag phase and in accordance with zero-order kinetics. The presence of hexadecane did not affect the degradation of phenanthrene sorbed and aged on the complex by C(I-AT )culture. This capability was exhibited also after experimental aging of 30 days. The dynamics of the bacterial community composition was investigated through PCR-DGGE (denaturing gradient gel electrophoresis) of 16S rRNA gene fragments. Individual bands changed their intensity during the incubation time, implying that particular microbe's relative abundance changed according to the culture conditions. Isolation of phenanthrene and/or hexadecane degraders was in accord with cultivation-independent data. Growth-dependent changes in the cell surface hydrophobicity of the two cultures and of the isolates suggested that modulation of cell surface hydrophobicity probably played an important role for an efficient phenanthrene assimilation/uptake.

Bioavailability of phenanthrene in the presence of birnessite-mediated catechol polymers.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants and contribute to the pollution of aquatic and terrestrial environments. In soil, their fate may be affected by interactions with the soil biological community and soil colloids. This study was conducted to investigate the fate of phenanthrene (Phe), selected as a representative PAH, in simplified model systems, which simulate processes naturally occurring in soil. Phe was interacted with catechol (Cat), an orthodiphenol, and common intermediate in the microbial degradation of PAHs, and birnessite (Bir), an abiotic strong oxidative catalyst abundant in soil. Two experimental conditions were investigated: Cat (5 mM)+Bir (1 mg ml(-1))+Phe (0.05 mg ml(-1)) mixed at the same time and incubated for 24 h at 25 degrees C (Cat-Bir-Phe) and Cat+Bir incubated for 24 h at 25 degrees C before Phe addition and then incubated for a further 24 h (Cat-Bir+Phe). After incubation, the systems were analysed for residual Cat and Phe, supplied with a selected Phe-degrading mixed bacterial culture, and then the microbial degradation of Phe and the growth of cells were monitored. Complex phenomena simultaneously occurred. Cat was completely removed after a 24-h incubation with Bir, and no interference by Phe in the Bir-mediated transformation of Cat was observed. Elemental analysis and UV-Vis and Fourier transfer infrared spectra showed that Cat transformation by Bir produced soluble and insoluble polymeric aggregates involving Phe. The hydrocarbon also interacted with the surfaces of Bir either previously coated (Cat-Bir+Phe sample) or not by Cat polymers. When a Phe-degrading bacterial culture was added to the systems after Bir-mediated Cat polymerisation, a different behaviour was observed in terms of Phe consumption and bacterial growth, thus suggesting differentiated availability of Phe to the microbial cells. The hydrocarbon was completely transformed in the presence of Bir and/or Bir covered by Cat polymers. By contrast a reduced degradation was measured when the Phe was involved in the polymerisation of Cat and entrapped in the Cat polymers (Cat-Bir-Phe). Although Cat showed a toxic, lethal effect on the bacterial cells, microbial growth was observed in the presence of Cat and Cat polymers, as the only C source. The mechanism leading to the different availability of Phe in the presence of Cat and Bir is still not clear. Further investigations are requested to provide more insight into such a complex phenomenon.