Category: hydroponic growing

Ebb and Flow involves flooding the plant tray with the nutrient solution using a pump that is connected to the solution tank at given time intervals with the use of a timer. The solution is later drained back to the nutrient tank. Adoption of hydroponics in East African countries like: Uganda and Tanzania, where this technology might offer a profitable agri-business and food security solution for urban dwellers by tapping into the growing demand for local produce, is still very low.It is likely to be more complicated to provide sufficient food for the fast-growing population using traditional agriculture in future, therefore soil-less cultivation is the right substitute technology to adapt effectively.There has also been a lot of attention given to urban agriculture among researchers, scientists and the general public which calls for more attention into hydroponics as it is considered an urban farming technology. Based on the impasse of challenges presented by conventional farming practices, urbanization and the increasing urban population as well as the ability of hydroponics to tackle these challenges, this study focused on examining the status and perception of soilless farming in Central Uganda and Northern Tanzania as an alternative sustainable cropping system to increasing food security and agdribusiness opportunities around urbanand peri-urban areas. Focus was specifically put on a couple of influential factors majorly socio-economic and agricultural factors surrounding the urban and semi-urban farmers and farms practicing hydroponics in these countries. The study assessed and categorized the benefits,fodder sprouting system challenges and recommendations for enhancing the implementation of this technology. It focused specifically on vegetable production because research has shown vegetables to be one of the most easy-to-cultivate crops under hydroponics as earlier mentioned.

The study was carried out in the months of April-July 2021 in the urban and periurban areas of Meru district located in Northern Tanzania and Wakiso district located in Central Uganda. Tanzania and Uganda are both located in East Africa and experience tropical climate conditions. Tanzania has an estimated population of 58 million while Uganda has approximately 44 million people. Northern Tanzania was selected as study site because it is one of the vegetable growing hot spots in the country and also has a couple of large hydroponic farms in the country while the Central Uganda was selected because it has majority of the urban and periurban farmers engaging in soilless farming. A total of 150 farmers/firms/farms were identified using snowball sampling through farmers groups and recommendations from expert farmers and agricultural bodies. Only 51 participants who practice vegetable production soilless farming technology majorly hydroponics around urban and periurban areas took part in the study. These participants included both farm owners of the hydroponic vegetable farms that as well as managers of firms that produce vegetables using hydroponics for either seed production or vegetables for sale.The biggest challenge reported was the high investment costs required to set up this high end technology especially for the fully automated greenhouse farms . This was also noted by Nicole et al. who identified high startup costs as a challenge for adoption of hydroponic farming technology. These costs include: greenhouse construction, costs of fertilizers, electricity for system installation, hydroponic equipment such as: PVC pipes, hydroponic net cups, climate monitoring systems among others. Artificial lighting, for instance through use of Light Emitting Diodes lights is sometimes deemed necessary for steady production making energy costs a key factor . The dependency on electricity is one of the factors that make hydroponics expensive . As earlier noted, majority of the farmers interested in the farming system adopted it at a small scale under non-controlled environments to cut down on the high initial costs needed for setting up the hydroponic units.

The development of low cost and easy to use hydroponic units will not only increase adoption of technology but also help farmers produce high quality vegetables . 22% of the farmers still reported that hydroponic farming requires enough technical knowledge which also continues to deter farmers from adopting the technology. For example: knowledge on the right amount of nutrients required for a particular crop, how to mix them, in what proportions and recycling. Majority of the respondents reported having learn about hydroponic farming using internet which further corelates with the high number of educated participants of the study. Controlled environment hydroponics requires some knowledge on how to run the climate control system within the green house for factors such as: humidity, temperature etc.… The need for technical knowledge for hydroponics such as: maintainace of PH and EC maintainance is one of the challenges of hydroponics . 6% of the farm operators who practiced hydroponics using high end technology such as: climate control systems accordingly reported a hitch related to maintainace of EC, PH and temperature of the nutrient solution and damage to crops in case of system failure. A failure or mismanagement of hydroponics can cause crop damage as also noted by Specht et al. who indicated that it is not sustainable if not well handled. 12% of the farmers stated that lack of adequate ideas or innovations on use of alternative locally available resources for hydroponic farming is a setback for the adoption of the farming system. For example: replacement of PVC pipes with buckets or bottles for growing hydroponic vegetables. Lack of adequate options of organic fertilizers for hydroponics in agricultural shops was another drawback surrounding hydroponics mentioned by approximately 20% of the respondents.

Other challenges reported by 9% were: bias from the community for hydroponic produce who consider them to be non-organic products, lack of variety of organic fertilizer alternatives and the timeliness needed by the system to avoid crop or system failure. Fig. 6 shows the drawbacks of hydroponic farming in Tanzania and Uganda. Fig. 7 reviews the advantages and disadvantages of hydroponic farming among urban and semi-urban farmers in Uganda and Tanzania while Fig. 8 further summarizes the recommendations made by the respondents which can assist increase the adoption of the technology among the two countries and Africa at large.Fresh green vegetation or fodder is an essential input that plays a significant role in animal feed. Fodder in the form of sprouted grains such as barley, wheat, maize, alfalfa, oats, millets, rye, sunflower seeds, and lentils can be grown in an environmentally controlled system. Commercial hydroponic fodder companies report that about 6-10 kg of fresh fodder could be produced from 1.0 kg grain within 7-10 days in controlled spaces with hydroponic techniques by providing suitable temperature, humidity, and light in the growing rooms. Hydroponic fodder production is a primitive technique that started in the 1800s or earlier. The basic principle for the hydroponic fodder system is that cereal grains respond to water or nutrients rich solutions for germination as well as growth to produce green plants in the short time of 6-9 days. This system has no chance of soil-borne insects, pests, disease attacks, and weed infestation because nutrients are directly fed to the roots and plants placed in trays of different dimensions. The interest in controlled environment fodder production is being revived due to the shortage of green fodder for livestock production in extreme climates. Also, water scarcity and food production needs on arable land motivate farmers to produce fodder in a controlled environment . Most Middle Eastern and African countries have a severe shortage of food supplies for livestock due to repeated drought and lack of water for irrigation. A recent report from the Food and Agriculture Organization of the United Nations indicates that global food production needs to be increased by about 60-70 % from the current levels to meet the increased food demand in 2050.

The livestock sector has made significant contributions to ensuring global food security. Milk consumption and meat production from livestock are increasing worldwide due to expanding the world population and better living standards. Worldwide milk consumption is expected to grow from 664 million tons by 2006 to 1077 million tons by 2050, and meat production will increase about twofold from 258 to 455 million tons. According to FAO, about 30% of world croplands are used for livestock feed production. Over 63% of arable land in the European Union produces animal feed instead of food for people. Fig. 1 shows the breakdown of global agricultural land use in pasturing, animal feed production, and human food. More than 70% of agricultural land is used for some aspects of livestock production, which contributes 14.5% of human-induced greenhouse gas emissions from feed production and enteric fermentation from ruminants. Therefore, fodder production in agricultural land is a conflicting issue in countries with food shortages; cereal grains, rice, oil seeds, and pulses on arable land are critical to ensure the food security of the increased population. An innovative approach for fodder production could have significant potential in reducing the carbon footprint in livestock production. Recent research showed that hydroponic fodder production in a shipping container could reduce GHG emissions by 7.4% compared with conventional farming for barley fodder production. A higher reduction could be achieved with improved seed-to-fodder output. The open field fodder production for livestock is disturbed by the abrupt climate changes and consumes ample water. The dry matter content and nutritive value of fodder could be reduced due to the temperature rise and increased CO2 concentration in the atmosphere from climate changes. Schlenker and Roberts reported that maximum maize production could be achieved at 29.0◦C, but a further increase in temperature hinders the maize productivity. Lobell and Field reported that maize production could be decreased by 8.3% with every 1.0◦C rise in temperature.

Easterling et al. reported that every 4◦C rise in temperature could cause a 34% reduction in wheat production. Also,microgreen fodder system the livestock sector accounts for about 8% of global human water use; and the trend of global temperature rise could increase livestock water consumption by about two to three times. Hydroponic fodder production systems could save a significant amount of water and reduce reliance on traditional arable production. Some studies reported that the same amount of fodder could be produced with 35-45% lower costs using 0.5% of growing spaces. The water-saving with hydroponic systems could be up to 95% compared with open field production. Also, the nutrient compositions of hydroponic fodder are relatively better than grounded/open-field systems. Most importantly, fresh green fodder for livestock could be supplied 365 days a year, even in extreme conditions like high northern latitudes and arid regions . Therefore, fodder production in CE settings is becoming popular in recent years in developed countries like the USA, Australia, and Canada. Fig. 2 shows the different CE facilities used for green fodder production, like low-tech poly-house, small-scale shipping containers, and large-scale production in warehouses with precise control of indoor environments. Controlled environment agriculture facilities with optimal temperature, relative humidity , water, and lighting can produce green fodder within six to ten days. CEFP is an energy-intensive approach for maintaining suitable thermal environments with heating, ventilation, air-conditioning , and artificial lighting with highly automated systems. Although hydroponically grown fodder is a highly nutritious feed, the costs could be two to five times higher for the same DM from the original grain. These high operating costs are primarily associated with the energy use for HVAC, lighting, and high capital costs for the automated CE growing systems. Although the concept of hydroponic fodder production is relatively old, very few research works have been published. A few research studies have been done on feeding fodder to dairy animals in the United States; most fodder research is conducted in Australia, India, and some Gulf countries. Most research has been undertaken on nutritional aspects and operating costs regarding DM contents of fodder production compared with original grains. A study reviewed the nutritional elements of hydroponic fodder and their impact on productivity for milk and meat. The study also analyzed the water use efficiency in hydroponic production compared with open-field production. Other studies have reviewed the basic fundamental of hydroponic fodder production and rationality from water conservation, land use, and minimal use of chemicals and its benefits for animal production. Singh et al. reviewed the challenges, opportunities, and status of fodder production in India, mostly covering the open field fodder production.

Taken together, our findings of evolved isolates displaying altered biofilm formation and motility properties and the detection of mutations within genes related to biofilm formation and motility in single evolved isolates as well as across independent endpoint populations indicates that adaptation of B. subtilis to A. thaliana roots under the employed conditions is associated with alterations in these two bacterial traits. While we found that the phenotypic and genetic changes of Ev6.1 and Ev7.3 conferred a fitness advantage over the ancestor during root colonization, adaptation to one certain environment may be accompanied by a loss of fitness in other environments . This has been demonstrated for Escherichia coli which following adaptation to low temperature showed reduced fitness at high temperature . In the example of the evolution of hyper-swarmers of P. aeruginosa, the hypers warmer clones out competed the ancestor in swarming, but lost in biofilm competitions . In this study, we demonstrate that adaptation of B. subtilis to A. thaliana roots is accompanied by an evolutionary cost. When Ev6.1 and Ev7.3 each were competed against the ancestor in LB + xylan under shaking conditions, i.e. an environment where plant compounds are present but biofilm formation is not required for survival, both evolved isolates suffered a fitness disadvantage. The observation that two evolved isolates, from independent populations and with different phenotypes and genetic changes, both suffered a fitness disadvantage in a non-selective environment might suggest the generality of such an evolutionary cost accompanying adaptation to A. thaliana roots. In our EE approach, B. subtilis was adapted to plant roots in the absence of other microbes. In the rhizosphere environment under natural conditions, B. subtilis is far from being the sole microbial inhabitant.

Instead, it engages in cooperative and competitive interactions with other members of the rhizosphere microbiome. We tested whether the evolved isolate, Ev7.3, displaying increased root colonization in the selective environment relative to the ancestor, would also show improved establishment on the root under more ecologically complex conditions. We found that in the presence of a synthetic, soil-derived community, Ev7.3 displayed enhanced establishment on the root compared with the ancestor in two out of four inoculation ratios. This enhanced establishment on the root by Ev7.3 is not expected to be caused by altered antagonistic activities toward the community members. First,hydroponic bucket no major changes in the inhibition of the community members were observed in confrontation colony assays. Secondly, an increased number of Ev7.3 cells on the root did not cause a reduction in the co-colonizing community. Finally, Ev7.3 did not harbor mutations in genes directly related to secondary metabolite production. Instead, enhanced establishment on the root by Ev7.3 in the presence of the community is possibly enabled by robust biofilm formation facilitating stronger attachment to the root and enhanced utilization of plant compounds. Interestingly, a study by Molina-Santiago et al. showed that compared with a Dmatrix mutant, co-inoculation of B. subtilis WT with Pseudomonas chlororaphis on melon leaves enabled co-localization of the two species as well as the closer attachment of B. subtilis to the left surface . The robust biofilm formed on the root by Ev7.3 possibly facilitated by increased matrix production may thereby not exclude the community members on the root but could rather allow them to incorporate into the matrix. This could also explain why the enhanced establishment of B. subtilis Ev7.3 on the root did not cause a reduction in the number of community cells attached to the root. Alternatively, the community may not be majorly affected by any difference in the establishment on the root between the ancestor and Ev7.3 owing to the low abundance of B. subtilis relative to the community. Further work is needed to elucidate the interactions between B. subtilis and this synthetic community during root colonization. In summary, these findings suggest that even though B. subtilis was evolved on A. thaliana in the absence of other microbes, it became highly adapted to the plant root environment enabling better establishment on the root also when the ecological complexity increases.

How genetic adaptation to the plant root in the absence of other microbial species differs from adaptation to plant root environments with varying levels of ecological complexity is the scope of future studies.From an applied perspective, experimental evolution of B. subtilis on plant roots represents an unexplored approach for developing strains with improved root attachment abilities for agricultural use. However, a biofilm-motility trade off as observed here may be undesirable when developing biocontrol agents owing to the growing evidence of motility as an important trait for bacterial root colonization in soil systems . The phenotypes associated with the adaptation of B. subtilis to A. thaliana roots presented here as well as the accompanying evolutionary cost and the increased root colonization also in the presence of resident soil bacteria highlight the importance of considering the selective environment if evolving PGPR for biocontrol purposes.Aquaponics is an agricultural technique that is touted as a promising alternative to solve the food and environmental crisis that the world faces today. As it is a combination of aquaculture and hydroponics , this technique can have high water use efficiency, and reduce the dependency on pesticides and fertilizers which adds to the sustainability of the system. This system has been shown to consume 50–70% less water compared to traditional agricultural systems, owing to its recyclability. Moreover, this system requires less pest control and has proven to be less impacted by harsh weather conditions, leading to yield increase. As we are moving towards the era of digital agriculture, efforts have been made to design physical prototypes for smart commercial aquaponic systems. Lobanov et al. has focused on proposing a new system for reducing the carbon and nitrogen content from fish waste to produce liquid fertilizer which is added to facilitate the growth of lettuce in aquaponic set-ups .

Karimanzira et al. introduced the concept of building an intelligent aquaponics system incorporating predictive analysis, system optimization and anomaly detection for maximizing productivity in commercial aquaponics through early fault detection . Mahanta et al. formulated a laboratory set-up as a prototype to grow soybeans in hydroponic solution using plasma activated water to decrease the amount of heavy metal uptake and optimize yield . Rau et al. designed a smart IoT based sensing and actuation system for growing rice by controlling the concentration of magnesium and nitrogen in hydroponic solution along with monitoring the environmental parameters of the greenhouse . A similar design was proposed by Dhal et al. using a smart IoT system for real time sensing and regulation of nutrients in commercial aquaponic set-ups depending on the season in which the crops were grown . Timsina et al. proposed the use of Machine Learning models to regulate nutrients for growing cereals in farmlands, but efforts are yet to be made for growing them in aquaponic set-ups .There have been recent advancements in the field of Smart Aquaponics which involve monitoring environmental parameters as well as plant growth through different Machine Vision-Based approaches in an IoT environment . Arvind et al. implemented an AutoML model trained with an XGradient boost algorithm, with 10-fold cross-validation taking into account the different sensor values recorded in the greenhouse and the fish count in the aquaponic tank which was extracted using the mask R-CNN image segmentation. This algorithm was used to control the triggering of the actuators in the system that in turn control the environmental parameters in the greenhouse; ensuring significant improvements in yield and water conservation, when compared with the conventional methods. Languico et al. did a comparative study of three ML estimators: K-Nearest Neighbours , Logistic Regression , and Linear Support Vector Machine , on the visionfeature extracted images of lettuce in a smart aquaponics set-up to monitor diseases that the crop may incur in its lifetime. A similar kind of study was done by Maleki-Kakelar et al. where multiple ML algorithms like linear and quadratic regression models, fuzzy systems and genetic programming was used to conduct regression analysis for improving urease activity aimed at strengthening the behaviour of soils . A study on images of lettuce leaves was conducted by Concepcion II et al. to detect diseases, wherein different feature selection processes were used to select the top four attributes for training the ML models .

A study on smart nutrient regularization for replenishing the Nitrogen, Phosphorus and Potassium content of the soil has been done by Ahmed et al. using genetic algorithms to provide the optimal level of nutrients needed for high production level of crops . Hiram Ponce et al. used a combination of Convolutional Neural Network for extracting features from tomato leaves along with a combination of Artificial Hydrocarbon Network as the dense layer to predict deficiency of nutrients in tomato plants . A similar kind of Deep Neural Network was implemented by Yadav et al. for apple foliar disease classification using Plant Pathology image data-sets . Nevertheless, limited research has been conducted on monitoring and regulating nutrient concentrations in the aquaponic solution using ML-based approaches. The current work focuses on building a ML algorithm that monitors the nutrient status of hydroponic irrigation water and outputs a recommendation system for regulation of these parameters. The main motivation behind this entire approach is to select the most important nutrients that need to be regulated in aquaponic environments depending on the output of Machine Learning classifiers trained on small data-sets. The main issues which one may face while designing such a data-driven approach has been discussed in the next paragraph. One of the major challenges with automation in aquaponics is the lack of sufficient data and the vast number of predictors that have to be used for making any inferences. This could result in what is referred to as the “Curse of Dimensionality” leading to the available data becoming sparse . Thus, it becomes extremely important to reduce the dimensionality of the data-set without losing valuable information. For this, stackable planters feature selection techniques become more relevant. To state a few, Recursive Feature Elimination and ensemble techniques such as the Extra Trees Classifier have proven to be highly effective. It is also equally important to check for the separability of the classes in the data-set. Many data visualization techniques like Principal Component Analysis and Multi-Dimensional Scaling plots can be used to understand how linearly separable the data is and what classifiers would be best suited for the purpose. Another major drawback especially in the case of small data-sets is the problem of “over-fitting” the data . Traditional ML and Deep Learning algorithms have a high probability of performing poorly on small data-sets.

A solution to this problem was suggested by Shao et al. who proposed deep Reinforcement Learning algorithms named MONEADD and did a comparative study with Knapsack and Traveling Salesman problem to design neural networks for different combinatorial optimization problems without much feature engineering which showed better scalability when distributed on multiple GPUs . On a similar note, to address this problem of over-fitting, Braga-Neto et al. proposed Bolstered Error Estimation method, which uses the same data for both classifier design and error estimation . In this form of error estimation, the variance setting for the Bolstering kernel is determined in a non-parametric manner from the data. For all the linear classification rules, the integrals in the Bolstered error estimation are computed in the same way. For the non-linear classification rules, a small number of Monte-Carlo samples are generated. In this study, three types of Bolstered error estimators, namely the Gaussian Bolstered re-substitution error estimator, semi-Bolstered re-substitution error estimator, and the Gaussian Bolstered Leave-One-Out error estimator, have been used with linear classifiers like LDA and SVC, along with non-linear classifiers like CART and KNN, and their results have been compared to identify the best performing classifier in this case. Based on the performance of the classifier with utmost optimal performance, a set of recommendation rules were prescribed and a comparative study was done on how this proposed Machine Learning based approach resulted in more optimal yield as compared to the baseline model.The data-set which was used in this case was recorded from three commercial aquaponic facilities located in Caldwell, Bryan, and Grimes counties in Texas, USA which are large producers of lettuce and other greens.

A significant increase in both leaf and plant artemisinin was observed among plants that were maintained under water deficit compared to well-watered plants . However, Selmar and Kleinw€achter argued that since drought stress also reduces growth and biomass production in most plants, drought stress-related increase in natural product concentrations does not mean that the rate of biosynthesis of natural products in the plants has increased. Another important and well-studied factor that influences the morphological and physiological processes in plants is light . When plants are exposed to low light intensity, they tend to have elongated leaves, and increased leaf surface area and plant height.High light intensity can induce plants to produce high starch and carbohydrate contents, which contribute positively to their biomass . Baligar et al. reported that in legumes, growth, nutrient uptake and use-efficiency ratios were higher at higher Photosynthetic Photon Flux Density than at lower PPFD. However, when plants are simultaneously exposed to more than one stress factor, the responses are more complex. For instance, it was observed that shading alleviated the negative impact of drought on leaf traits and biomass characteristics of Acer buergerianum Miq . On the other hand, there was no interactive effect between light and water treatments on biomass accumulation in Quercus suber L. seedlings . Holmgren argued that shading could reduce the impact of drought by limiting loss of water in soil during evaporation.

Puertolas et al. and Quero et al. described three hypotheses to predict the possible responses of plants to the interactive effects of water stress and light availability. These are trade-off hypothesis — plants that are adapted to deep shade may adapt relatively poorly to drought than other plants growing under higher light levels, hydroponic nft system facilitation hypothesis — shade enhances survival and physiological status of plants by decreasing evaporative demands and radiation loads, and orthogonal hypothesis — the combined effects of shade and water-shortage are independent, and their impacts are orthogonal. Puertolas et al. further argued that interactive responses are influenced by plant species, the intensity of water stress, the range of light intensities, the traits considered and seedling age or environmental conditions. Currently, however, reports that address the interaction of light and water stresses on bioactivity of medicinal plant extracts are scarce. Medicinal plants are an important source and inspiration for discovery of new products for drug development . Consequently, many research activities have focused on the manipulation of these secondary metabolites in plants and yields of medicinal materials in order to meet the demands of the pharmaceutical industry, traditional healers and the cosmetics industry . In South Africa, T. violacea bulbs and leaves are traditionally used for treatments of gastrointestinal ailments, asthma, fever and tuberculosis; the leaves are used to treat cancer of the oesophagus . Previously, crude extracts from T. violacea showed good antimicrobial activities against bacterial strains . T. violacea has been shown to have similar antibacterial and anti-fungal activities as Allium sativum . T. violacea is rich in sulphur-containing compounds including thiosulfinate marasmicin which exhibit significant antimicrobial activities . Kubec et al. isolated R– S- cysteine-4-oxide from the rhizomes of T. violacea. The sulphur compounds in T. violacea are unstable and J€ager and Stafford reported that grinding the rhizome material in liquid nitrogen and extraction with ethanol yielded the best results and the sulphur compounds in the rhizomes decreased rapidly upon storage, after harvest.

Methyl alpha-D-glucopyranoside, a bio-active compound that can selectively kill cancer cells was successfully isolated from T. violacea using apoptosis-guided purification.Tulbaghia violacea is regularly harvested from the wild by traditional healers, a practice that may cause decline of the species’ populations in the wild . The persistent high demand might eventually place T. violacea at risk of extinction . Hence, there is a need to develop optimum cultivation protocols that will ensure improved crop yield and quality of medicinal materials. Plant growth parameters, such as dry and fresh weights, plant height, and anti-fungal activity of plant extracts are useful indicators of yield and quality of medicinal materials. Since ambient environmental conditions during cultivation can influence plant physiology, plant health and crop yield, a greenhouse is a perfect facility for manipulating most exogenous factors like humidity, light, temperature and water. Furthermore, growing plants in greenhouses could help circumvent many challenges, such as land availability, water availability, season, climate, pests and diseases, which are major concerns with conventional cultivation of indigenous plant species . The objective of this study was to assess the individual and interactive effects of light intensity and watering regime on plant growth, nutrient uptake and anti-fungal activity of extracts of T. violacea plants, grown hydroponically.Data of the different plant growth parameters were recorded at the end of the experiment. The height of the plant was recorded at two months post treatment using a measuring tape. The number of leaves was enumerated at two months post-treatment. At the end of the experiment, plants were harvested and fresh weight was immediately measured. In order to determine the dry weight, harvested plants were placed separately in paper bags and dried in a thermo-oven at 70 C, and the dried plant samples were weighed.The anti-fungal activity was evaluated using the minimum inhibitory concentration value obtained in a microdilution assay. Fusarium oxysporum f. sp. glycines strain obtained by courtesy of the Phytomedicine Programme, University of Pretoria, South Africa was used as the pathogenic agent in the bioassay. The F. oxysporum strain was subcultured from stock agar plates and grown into nutrient broth for 4 h.

The concentration of fungal spores in the nutrient broth was determined using a haemocytometer. One hundred microlitres of solution containing crude acetone extracts of plant roots was serially diluted with sterile distilled water in 96-well microplates . The fungal suspension was added to each well of a 96- well microplate . Forty micro litre of 0.2 mg ml-1 of piodonitrotetrazolium chloride  dissolved in sterile distilled water was added to each microplate well, sealed in a plastic bag and incubated at 37 C and 100% RH. Acetone was used as a negative control. The MIC values were recorded after 6, 12 and 18 h. There were three replicates per treatment and per watering interval. The MIC value and the weight of the extract obtained following acetone extraction were used to determine the Total Activity . The unit of TA is ml g-1 and it indicates the degree to which the active compounds in one g of plant materials can be diluted and still inhibit the growth of the tested microorganisms .The watering regime significantly affected the growth parameters as leaf number, fresh and dry weights, and plant height reduced with increasing watering intervals. Generally, the shorter watering interval had higher fresh and dry mean weights compared to plants exposed to the longer watering intervals. These results are consistent with the findings of Xego et al. , which showed that more abundant growth in Siphonochilus aethiopicus correlated with shorter watering intervals. In another study, water deficit due to long watering intervals had significant negative effects on plant height, leaf number, and induced a higher biomass of adventitious and tap roots of mango . Interestingly, the positive effects of a shortened watering interval on growth and biomass observed in this study correlated well with the increased tissue macro-nutrients contents. This observation provides a plausible explanation of the mechanism through which watering interval can influence growth of plants. Higher tissue nitrogen content increases plant growth rates and shifts plant biomass partitioning to above ground structures . On the other hand, a decrease in water availability can reduce nutrient uptake, transportation and availability . Nitrogen availability and the internal N status of plants correlate positively with shoot∶root ratios . Potassium and phosphorus are other essential macro-nutrients that affect physiological processes and influence plant growth and metabolism .

Variable effects of shading on growth parameters, such as plant height, leaf number and dry and fresh weights were recorded in this study. The plants grown under the 40% shade produced significantly higher mean height and lower fresh and dry weights of aerial parts than those grown in the 0% shade treatment and short watering intervals . However, under the longest watering interval and 40% shading, higher number of leaves and higher dry weights were recorded when compared to 0% shading. These results are in agreement with that of Zervoudakis et al. on Salvia officinalis L., which showed that dry mass, number of leaves and physiological parameters had a strong positive correlation with the light intensity, and plant’s height and leaf photosynthetic pigments were increased among low light treated plants. Fiorucci and Fankhauser postulated that low photosynthetically active radiation can induce pronounced phenotypic responses in some species, such as elongation of stem-like structures, elevation of leaves, as well as reduced branching and acceleration of flowering. Under a short-term low irradiance , chlorophyll b transiently increased in Brassica campestris, but extension of shading time to a 15-day period led to significant decreases in relative chlorophyll a and anthocyanin . There are few studies done on the interactive effects of low light intensity and limited water on plant growth and secondary metabolite synthesis . In the present study, plants subjected to both the 21-day watering interval and low light intensity produced more leaves than those grown under the higher light intensity and equivalent watering interval. Research done by Sack et al. reported that shading could mitigate the negative impact of water stress. Under limited light, nft channel plants may accumulate carbohydrates in leaves; these soluble sugars may reduce water loss through turgor maintenance and reduction of stomatal aperture . L€ of et al. reported an interaction between irradiance and water stress on biomass partitioning in Fagus sylvatica seedlings. Yang et al. argued that plants in shade invest more to produce shoots and leaves than biomass. Broadly, these studies corroborate our finding that watering regime and light intensity have intercative effects on plant growth parameters. Hazrati et al. reported that about 50% of total solar radiation and irrigation after depleting 40% of soil water content were the most efficient treatments for chlorophyll fluorescence and pigments of Aloe vera L. A key finding in this study is that shading alleviated the negative effects of water deficit stress on plant growth. This is in agreement with the report of Guo et al. , in which drought alleviated shading effects on Acer buergerianum Miq., and the above ground facilitation hypothesis .

Nevertheless, it is worth mentioning that the responses of plants to water and light stress also depend on species. For example, Liu and Su reported that under low light, Taxus yunnanensis produced larger leaves and a higher shoot axis length per unit dry mass under high light, whereas the leaf size and biomass yield of T. chinensis were not sensitive to light. Secondary metabolites play an important role in plant defence. They protect plants against pathogens and herbivory . Drought stress can induce plants to produce higher concentrations of secondary metabolites. In this study, although there were no significant differences in the MIC values between plants in low and high light intensities, acetone bulbous root extracts of the plants that were exposed to the longest watering interval and 40% shading yielded the highest total activity. The increase in total activity suggests that there was an interaction between watering interval and light intensity in relation to the yield of acetone extract during extraction. Hazrati et al. reported that conditions of irradiance of full sunlight and water deficit stress favoured increased anthocyanin production in Aloe vera. In conclusion, broadly, three trends occurred in the results. Firstly, the total weight of T. violacea increased with shorter watering intervals under high irradiance. Secondly, shading alleviated the negative effect of water deficit stress on plant growth. Thirdly, the longest watering interval plants had the highest total activity of bulbous root extracts. Furthermore, light intensity and watering interval had significant interactive effects on anti-fungal activity and plant growth. These results also suggested that nutrient supply and subsequent tissue nutrient levels might be modulating the responses, such as plant growth and biomass, and anti-fungal activity of plant extracts in relation to light intensity and watering regime. Future studies should investigate the interactive effects of water deficit and shading on production of bio-active compounds in T. violacea.

Many different classes of antioxidants are present in vegetables and it is hard to elucidate which ones are more associated with the benefits. Also, the synergistic effect and interaction of different antioxidants in one food leads to the fact that the level of a single antioxidant is not a good indicator of the total antioxidant capacity of the food item. Therefore, measuring the total antioxidant capacity, which is the cumulative capacity of food components to scavenge free radicals, has become an effective way to evaluate the potential benefits of various vegetables in preventing or managing chronic diseases. The average TAC of themicro-greens samples evaluated in this study ranged from 1.06 to 1.18 mg/g, which are comparable to previous results on micro-greens and mature vegetables and fruits. Since the TAC assay was conducted using the methanolic extract used for TPC analysis, it was not surprising that there was no significant difference in both values among samples, as phenolic compounds could be the main contributor to the TAC in this experiment.A total of 150 participants’ data was collected and analyzed in the sensory study. The descriptive information of participants is shown in Table 1. The participants were asked about their prior experience in purchasing and consuming micro-greens. Eighty-two percent had not purchased and 69% had not consumed micro-greens before participating in this study.According to the results of the sensory study , scores for the smell, appearance,taste, and overall liking of micro-greens were all significantly higher for those from the local farm as compared to those from commercial.

A higher average of scores was noted for soil-grown farm samples as compared to water-grown farm samples but did not reach statistical significance. The average scores for all the sensory attributes of farm micro-greens were in the range of 4.54–5.38 out of 7 , while those for the commercial micro-greens were in the range of 3.09–3.68 . Several factors may contribute to the better sensory quality of the micro-greens samples from the farm. Firstly, the greatly higher level of chlorophyll as aforementioned gave the farm samples a more vibrant color as compared to the commercial ones,flood tray and therefore contribute to the higher evaluation on the appearance. Appearance of a food product, especially fruits and vegetables, is the initial quality that attracts consumers, and affects their first time purchase intention. Secondly, samples from the local farm were delivered the same day of harvest and were used for the sensory test on the following3d.However,the exact harvest time of the for transportation and storage until they reached the consumers. The freshness of vegetables may significantly affect the evaluation on all the sensory attributes, including smell, taste, and appearance. Thirdly, it was reported that total sugar content is a factor that can greatly affect the sweetness, bitterness, and sourness, and thus the taste of vegetables. The higher level of chlorophyll in the farm samples as observed may result in a higher production of sugar owing to greater capability of photosynthesis, which may contribute to the higher scores on the taste of those samples as compared to the commercial ones. Future analysis can be conducted to measure the sugar contents of micro-greens samples from different sources. To explore the impact of smell, taste, and appearance on the overall liking for micro-greens, the correlations among sensory attributes were analyzed. It was found that the overall liking was most strongly correlated with the taste of micro-greens.

Scores of overall liking were also strongly correlated with the appearance and the smell of micro-greens . The results indicated that taste, smell, and appearance all contributed to consumers’ perception of micro-greens but taste may be the best predictor. This is consistent with previous findings that flavor-related characteristics best predicted consumer preferences for overall eating quality, although visual quality characteristics also contributed.The population is increasing but the farmland is reducing in China. The supply of fresh vegetables has become an urgent problem. Therefore, plant factories are being increasingly used to solve this problem. Plant factory is an efficient agricultural system which can realize the annual continuous production of crops through environmental control in facilities.Hydroponic lettuce, a vegetable with a short growth cycle and high yield, has been widely cultivated in the plant factories. Hydroponic lettuce is mainly planted on a planting board, and a deep flow technique, a hydroponic technique in which the plant roots grow in a deep and flowing nutrient layer, was used to cultivate lettuce. The hydroponic lettuce is pulled out from the planting board when it is mature, and the roots are cut as required. Then the lettuce is sold in packaging. At present, harvesting of hydroponic lettuce is carried out manually, with high labor costs and low efficiency. Therefore, an automatic harvester that can process leafy vegetables is required. Study of the physical and mechanical properties of vegetables plays a vital role in the development of automatic equipment.

Knowing the physical and mechanical properties of hydroponic lettuce will be useful for the development of harvesting machinery. One of the basic and most important experiments in the study of mechanical properties of lettuce is the tensile experiment. It can reflect some of the mechanical properties of the lettuce stem and whole lettuce. This paper will use similar vegetables as a reference to carry out research due to a lack of research on the mechanical properties of leafy vegetables. The researches on pulling force of similar vegetables were mainly focused on root crops such as radish , carrot , and garlic , etc. Several studies including Chen et al., Li et al. and, Fu et al. have used the tensile experiment to obtain the pulling force of different varieties of radishes. They concluded that the maximum pulling force to harvest green radishes and red radishes were 90 N and 110 N, respectively. Xin et al. also obtained that the main distribution range of garlic pulling force was from 24 to 27 N under the normal harvest condition. The cabbage harvester designed by Du et al. can pull cabbages out of the soil with a success rate of 86.7%, but it can only be used in the field environment. At present, there are many studies on the pulling force of vegetables in the field, but fewer studies on the pulling force of hydroponic leafy vegetables in plant factories. Therefore, the pulling force of hydroponic leafy vegetables needs to be studied to develop automatic machinery in plant factories. In addition to the tensile experiment, a shear experiment can be carried out to determine the root cutting force of hydroponic lettuce. Kanamitsu and Yamamoto and Li et al. have used shear experiments to obtain the root cutting force of cabbage. They found that the root cutting force can be affected by cutting position and cutting speed, while cutting position had a more significant impact on the root cutting force.

Gao et al.concluded the optimal parameters combination among cutting position, cutting mode, cutting speed, cutting angle, clamping position and clamping angle to harvest hydroponic lettuce through an orthogonal experiment. However, this study was only applicable to the way of removing the root harvest. Chen et al.and Wu et al. used orthogonal experiments to investigate the cutting forces of broccoli and rape, respectively. They both concluded that the cutting position was the key factor affecting the cutting force. Although the above-mentioned studies are not about lettuce, these studies provide support for the research of root cutting force of hydroponic lettuce. Presently, studies of hydroponic lettuce have generally been limited to the sensory attributes, general appearance, wilting, decay and physiological disorders conducted mostly during investigations on the packaging, processing and storage conditions,ebb and flow tray there are few studies on the root cutting force of hydroponic lettuce. However, the root cutting force is an important parameter in the design of the cutting device of hydroponic lettuce harvesting machinery. Therefore, the root cutting force should be investigated to develop the automatic harvester. This study is aimed to investigate some of the physical and mechanical properties of hydroponic lettuce. It is carried out by means of using a tensile experiment, shear experiment, and moisture content experiment on multiple samples. The pulling force, root cutting force, geometric characteristics, and moisture content were obtained. The moisture content and pulling force are summarized and analyzed by mathematical statistics method. Meanwhile, the response surface method and variance analysis are used to analyze the change rule of root cutting force with different cutting positions and cutting speeds.The hydroponic lettuces were randomly chosen from Yangling modern agricultural demonstration park in Shaanxi Province, China. A total of 100 samples were purchased for this study. The vegetables were ripe and defect-free.

The experiments were conducted at the College of Mechanical and Electronic Engineering, Northwest A&F University, China. Because experimenting with all samples at once was impossible, the unused vegetables were grown in homemade Petri dishes.The design requirements of leaf vegetable harvesting machinery and the planting mode of hydroponic lettuce were referenced. The physical parameters of hydroponic lettuce, such as crown diameter , overlap length , total height , plant height , total root length , main root length , stem diameter , maximum expansion diameter of the root , total weight and net weight were selected as the evaluation indexes of the geometric characteristics of hydroponic lettuce. The investigation was carried out using a digital vernier caliper , a digital ruler , and an electronic balance to measure the twelve parameters of hydroponic lettuce . The CD is the maximum diameter of the projection of hydroponic lettuce in the horizontal plane under the natural growth state. The OL is the maximum value of the overlapping part of leaves with two adjacent lettuces in nature growth. The MRL is the distance between the planting board and the maximum root diameter of less than 30 mm . The MED is the maximum diameter of the projection in the horizontal direction of the lettuce root after the root leaves nutrient solution. The net weight is the weight after cutting the root. The ruler was used to measure the PH, CD, and TRL while the digital vernier caliper was short for measuring. Thirty hydroponic lettuces were randomly selected from the 100 samples which were purchased to experiment with physical properties.The direct drying method was used in this experiment to calculate the moisture content, according to the standard of GB / T 5009.3-2010 .

The roots, stems, and leaves of hydroponic lettuce were divided into three groups because experimenting on the whole vegetable was impossible. The roots were cut into a segment with 30 mm, the stems were cut into thin slices with 2 mm, and the leaves were cut into strips with 5–10 mm. Five samples were selected randomly from each group for the experiment. The experiment was performed under room temperature by using a drying oven , drying dish, and electronic balance. The weight of the drying dish and the total weight of drying dish with sample were measured, the weights were recorded as m3 and m1, respectively. Then the drying dish with sample was put into the drying oven at a temperature of 103℃±2℃. After 4 h, the drying dish with sample was taken out and weighed for the first time. The drying dish with sample should be weighed after cooling to room temperature, and tweezers were suggested to be used when moving the drying dish. After that, the weight of them was weighed every 1 h until the weight difference between two times was less than 2 mg. The maximum force to pull the hydroponic lettuce out of the planting board was called pulling force. The power consumption of the harvesting machinery and the design of the pulling device need a reference to the pulling force. A tension meter and puller of hydroponic lettuce with a length 200 mm, a width 50 mm, a height 300 mm, and a U-shaped groove with a width of 25 mm was developed to determine the pulling force of hydroponic lettuce. The stem of hydroponic lettuce was placed in the Ushaped groove of the puller of hydroponic lettuce.

Capability of crop production and translocation are two key factors in crop yield formation.Therefore, studying the characteristics of dry matter and photosynthetic production of rice has great significance in understanding rice yield formation and regulating high yields and stable yields. Regarding the characteristics of production and accumulation of rice photosynthetic substances, researchers have carried out extensive studies on  high-yield varieties,high-yield populations and groups that exhibit different production levels and have proposed many valuable theories . Yang et al.  reported that a greater material capacity in terms of leaf area during the middle and late stages of development and a relatively high population growth rate resulted in increased material production postanthesis and an increased transport ability of postanthesis substances under ecological conditions in Yunnan Province, China. Compared with high-yield populations, super-high-yield populations exhibited greater dry matter accumulation in the middle stages of development, a greater leaf area index  at the HS, a superior population quality, and a greater photosynthetic ability and perfected the coordination of both the output and transport of the stem sheath materials in the late stages of development. Zhang et al.  reported that greater material production is an important reason for improving the production potential of super hybrid rice compared with common hybrid rice and conventional rice. Seedling age is one of the key approaches in regulating rice growth and development.

Therefore, it is important to study the effects of seedling age on dry matter production to maximize grain yield from HLMS for different rice cultivars. The objective of this study was to clarify the growth process and dry matter and photosynthetic production characteristics of seedlings of different ages grown as HLMS. The corresponding results will provide a practical reference and lay a theoretical foundation for the large-scale application of HLMS.The dry matter accumulation of the population and its ratio to total dry matter were slightly but not significantly greater for young seedlings than for old seedlings for both cultivars from the TS to the JS in 2014 ; these findings were consistent for Wuyunjing 24 in 2015,mobile grow rack and the difference was significant. The interaction between year, cultivar and seedling age did not affect the dry matter accumulation of the population. The dry matter accumulation of the population and its ratio to total dry matter showed no obvious pattern of differences between the treatments from the JS to the HS. However, the dry matter accumulation of the population was significantly greater for young seedlings than for old seedlings for both cultivars and during both study years from the HS to the MS except for 6 Liangyou 9368 in 2014, and with the exception of 6 Liangyou 9368 in 2014, there was a consistent trend for the ratio to total dry matter. Regarding the harvest index, consistent variation was found: the harvest index decreased with increasing seedling age for Wuyunjing 24 in both years, while no significant difference was detected in 2015, and the opposite trend occurred for 6 Liangyou 9368.The leaf, stem and sheath to total plant dry weight ratios decreased as the rice plants grew;however, the panicle to total plant dry weight ratio increased as the rice plants grew.

There were no significant differences between the different treatments in terms of the leaf to total plant dry weight ratio at the middle and late growth stages, except for 6 Liangyou 9368 in 2015 at the HS. The stem and sheath to total plant dry weight ratios exhibited no obvious pattern of differences between the treatments at the JS, HS and MS, and the panicle to total plant dry weight ratio exhibited no obvious pattern of differences between the treatments at the HS; however, the panicle to total plant dry weight ratio decreased with increasing seedling age and was consistent for both cultivars at the MS. Compared with that of the 27-day-old seedlings, the panicle to total plant dry weight ratio of 13-day-old seedlings of Wuyunjing 24 and 6 Liangyou 9368 increased by 2.33 and 1.79%, respectively, in 2014 and by 5.08 and 5.77% in 2015, but there was no significant difference between the treatments. The interaction between cultivar and seedling age in terms of the panicle-tototal plant dry weight ratio was significant each year.Grain yield significantly decreased with increasing seedling age for both Wuyunjing 24 and 6 Liangyou 9368 . No significant differences in grain yield were detected between the 13- and 20-day-old seedlings at transplanting for either cultivar. Compared with that of 13-day-old seedlings, the grain yield of 27-day-old seedlings decreased by 14.5% in 2014 and by 10.7% in 2015 for Wuyunjing 24, and the corresponding values were 10.5% in 2014 and 9.8% in 2015 for 6 Liangyou 9368. The panicle number significantly decreased with increasing seedling age, except for Wuyunjing 24 in 2014. The spikelets per panicle of Wuyunjing 24 significantly decreased with increasing seedling age, but no significant difference was found for 6 Liangyou 9368.

Moreover, no significant difference was found in the seed setting rate for either cultivar or during either year, except for Wuyunjing 24 in 2014, and no significant difference in the grain weight was found for either cultivar or during either year, except for Wuyunjing 24 in 2015. The interaction between year, cultivar and seedling age did not affect the grain yield. However, the interaction between cultivar and seedling age in terms of spikelets per panicle was significant for each year, but no significant differences were detected in terms of panicle number, grain weight or seed setting rate.Growth process and performance are genetic properties of rice cultivars and are determined mainly by their photonasty, thermoperiodicity and basic vegetation growth phases . These properties are also affected by ST, TS , cultivation methods , and ecological conditions , among other factors. The transplantation of seedlings of different ages is usually accomplished by one of two cultural methods: seeding at different times while transplanting at the same time or seedling at the same time while transplanting at different times. Both of these methods lead to differences in growth characteristics and environmental conditions, which then affect the growth stages. For traditional rice seedling cultivation methods, previous reports have shown that the whole growth phase  differed by 19 days when the seedling age at transplanting differed by 18 days , and the WGP differed by 15 days when the seedling age at transplanting differed by 15 days for the cultural method of seeding at different times while transplanting at the same time .

The corresponding values were 5–8, 8 and 6–12 days when the seedling age at transplanting differed by 14, 15 and 20 days, respectively, for the cultural method of seedling at the same time while transplanting at different times . In this research, the WGP differed by 13–15 days when the seedling age at transplanting differed by 14 days under the method involving HLMS. The above results showed that the difference during the MS was much smaller than that during other growth stages, although the seedling age at transplanting was different. Further analysis revealed that the growth process was accelerated with the transplantation of young seedlings and that the whole phase was shortened. The acceleration occurred mainly during the vegetative growth phase , and there was little difference in the reproductive growth phase . A previous report suggested that a longer growth phase increases grain yields when rice plants head and mature normally . Nevertheless, Wang et al.  reported that the grain yield increased with the extension of the growth phase has a limit. In the present study, the grain yield did not increase significantly for the old seedlings and even declined, although their growth phase was longer than that of the young seedlings. In addition, the results showed that the optimum seedling age for maximizing grain yield from HLMS not only depends on the length of the growth period but also may be associated with seedling quality, transplantation shock, etc. These results were consistent with those of the manually transplanted rice reported by Lampayan et al. .

Rice grain yield is the product of total dry matter accumulation and the harvest index, whereas yield formation is the result of individual plant and population dry matter accumulation,ebb and flow table distribution, translocation, and transformation . The transplantation of seedlings of different ages leads to differences in the uptake and utilization of heat, light and resources by rice plants and further affects the growth of individual rice plants and the population. Many studies have consistently shown that grain yield is closely related to total dry matter accumulation at the MS and to dry mater accumulation from the HS to the MS and that there is no significant relationship with dry matter accumulation before the JS or the harvest index under conditions of high yield . Previous reports suggested that the LAI at the HS and Pn decreased with increasing seedling age at transplanting, resulting in decreased total dry matter accumulation . The DMWPS and DMWP are major signs of individual rice plant growth and population quality, and increasing the potential of an individual plant to accumulate dry matter is beneficial for improving population dry matter accumulation . Su et al.  suggested that a higher DMWPS leads to a slow reduction in leaf area, increased photosynthetic potential and net assimilation rates and, ultimately, high grain yields. In this research, the grain yield was significantly positively correlated with total dry matter accumulation at the MS  and with the dry mater accumulation from the HS to the MS , and the grain yield was not significantly correlated with the harvest index  for different seedling ages at transplanting for HLMS; these results were similar to those reported by Wu et al. . Grain yield was negatively correlated with dry mater accumulation from transplanting to the JS  and was quadratically related to dry matter accumulation at the HS; these results were similar to those reported by Gong et al. . Therefore, dry mater accumulation at early growth stages  should be controlled properly, and it proportionally increased during the middle growth stage  and increased the dry matter accumulation during the late growth stage  drastically.

The lower LAI at the HS and Pn for the old seedlings may explain the lower dry matter accumulation for the HLMS; these results are similar to those of Zhang and Gong  and Zhu et al. . The DMWP is determined by both the DMWPS and the stems and tillers of the population . In the present study, the DMWPS from transplanting young seedlings at the MS was greater than that from transplanting old seedlings, and there were more stems and tillers in the population of young seedlings than in that of old seedlings, which is mainly due to the robust seedling quality, better mechanical transplantation quality, lower transplantation shock and rapid tiller emergence of young seedlings transplanted compared with old transplanted seedlings . A portion of the grain-filling material of rice comes from the photosynthetic products after heading, and the other portion comes from the redistribution of stored materials from the leaves, stems and sheaths . Compared with low-yielding rice, high-yielding rice generally has greater amounts of stored photosynthetic products in the leaves, stems and sheaths at the early and middle stages of growth and has greater proportions of photosynthetic products distributed to the panicle, and greater amounts of materials stored in the leaves, stems and sheaths of the latter can be transported to the panicle . Li et al.  reported that the dry matter exportation and exportation rates of the leaves, culms and sheathes per shoot of 25-day-old seedlings were significantly greater than those of 40-day-old seedlings. In this study, the leaf-to-total plant dry weight ratio did not clearly differ at the JS, decreased slightly at the HS, and increased slightly at the MS with increasing seedling age. In addition, the panicle-to-total plant dry weight ratio at the MS decreased with increasing seedling age. All of these results showed that the young transplanted seedlings had better Pn s and population support systems, proper allocation of dry matter to the different organs and a high panicle-to-total dry matter ratio at the MS. In addition, the transplantation of young seedlings results in a strong growth advantage, which may be related to the absence of premature senescence, strong root activity, a long duration of photosynthetic function of the leaves, high physiological and biochemical activity, etc.; all these factors need to be further studied.

Similarly, Cu plays a role as a micronutrient at lower concentrations. On the contrary, high amounts of these two metals once overcome the biophysical barriers in plants become toxic to plants and negatively affect essential biological activities including inhibition of photosynthesis, nutrient absorption and overall plant growth . This could happen after mechanical damage or morphological alterations or indirectly via blocking of aquaporins. It is also suggested that toxic outcomes might be related to reduced syntheses of cell wall components and supplies of essential nutrients . Organ-wise, roots are likely to be most affected by NPs because are the first organ to encounter soil-borne contaminants . We found that Zn was highly toxic to maize plants growing in hydroponics and soil, and that exposed plants exhibited significant reductions  in biomass and chlorophyll, soluble protein, and P contents . In a previous study, ZnO-NPs at 800 mg kg− 1 reduced net photosynthesis by 12% and relative chlorophyll contents by 10% in maize grown in soil for 20 days . Physiological reduction in chlorophyll contents  leads to reduced biomass production . Furthermore, Cu accumulation interferes with the enzymes responsible for chlorophyll biosynthesis and alters the protein compositions of photosynthetic membranes . Reduced chlorophyll yield has been attributed to  reductions in iron content  reduced efficiencies of enzymes required for chlorophyll biosynthesis, and  the replacement of Mg2+ from the porphyrin ring of chlorophyll by metals . Interestingly, at 40 DAS, the inhibition caused by NPs and bulk was smaller than at 20 DAS. This can be related to metal extraction or hyperaccumulator potential of maize plants.

The phytoextraction of maize has also been demonstrated for Zn and Cu . During phytoextraction, plants may undergo some metal induced physiological and/or morphological alterations. These may include compartmentalization of increasing metal concentration in root cell plasma membrane, metal sequestration in vacuoles, stacking pots loading of metals in xylem vessels followed by transportation to upper ground parts and sequestration of metals in leaf cell membrane and vacuoles. Additionally, low-methylesterified pectins of root cell walls can also sequestrate the metals . These many processes can restrict the interactions between bioaccumulated Zn and Cu and maize cellular environment up to certain extent in soil environment. IR revealed that treatments with the tested materials affected biomolecules in roots  more than in leaves . The literature supports our results and suggests such biomolecular alterations in food crops. For example, CuO-NPs reduced the areas of CH2 and CH3 IR bands of lipids , and protein  signal shifting. Rico et al.  suggested C–N–H in-plane bend and C–N stretch vibrations in maize protein after CeO2-NP treatment . The distribution of lipids, lignins, and carbohydrates in maize vascular tissues corresponds to protein distribution patterns , and thus, any change in protein structure by metal treatment might alter lipid and carbohydrate levels and types. Carbohydrate associated IR peaks at 1164–883 cm− 1  can be corelated with bands for pectin in wheat plants exposed to CuO-NPs that resulted in decreased molecular mass of pectin . The low methyl esterified homo-galacturonan fractions of pectin contain free -COOH groups, which are mainly involved in the binding of divalent metals like Cu . Moreover, dissolution of CuO-NPs can be induced by interactions with proteins and organic acids inside plant tissues. Hence, due to strong affinities between divalent metals  and -OH, -COOH, and –SH groups, metal ions strongly interact and modify cell wall polysaccharides . In line with our observations  of Zn and Cu movement through maize organs, Zn deposition in maize roots and shoots was found to be 12–24 times higher over non-treated plants when 500 mg ZnO-NPs kg− 1 was present in soil . In addition, Zn uptake by maize exposed to ZnO-NPs, even during germination, has been reported to be much higher than that of Zn2+ ions , which corroborates with our results of higher Zn uptake in maize organs treated with ZnO-NP compared to Zn2+ .

However, the opposite trend was observed for soil. These observations suggest that Zn uptake by maize occurs mainly by ZnO-NP uptake in soilless medium  but from Zn2+  in soil , though it may be that soil constituents have some effect by hindering the nanoparticle mobility. The higher concentration of Zn in tissues of maize plants grown in hydroponics and soil is also in-line with the results of a study on corn seedlings . Cu uptakes also differed in maize organs when plants were cultivated in different media. Less accumulation of NPs in soil grown plants could be due to the soil derived chemical or physical transformations such as soil weathering, heteroaggregation, binding of NPs with soil organic matter, formation of copper-sulfur complexes, ZnS formation, formation of Cu2O from soil applied CuO-NPs that may limit the uptake of NPs from soil . Cu2+ and Zn2+ caused more inhibition/damage than CuO- and ZnONPs to protein synthesis . Protein inhibition by metals is usually caused by disulfide bond disruption . In one study, Au-NPs severely downregulated 25 genes encoding many essential proteins, including proteins involved in Fe transport, Cu transport, and protease inhibitor/seed storage/lipid transfer, and cytochrome P-450, nicotinamide synthase, and aquaporin . Observed declines in TSP levels in maize after NP or ion treatment could be attributed to the uptakes and translocations of metals even within above-ground parts and disruption of the maize proteome. Phosphorus, a vital plant macronutrient, an integral part of ATP and NADPH  playing crucial roles in major metabolic processes was also found deficient . Shoots generally accumulate more P than roots possibly due to rapid translocation of P from roots to shoots during the vegetative growth phase . In line with our results, nanoparticle treatments have been reported to repress the transcriptions of P metabolism-associated genes, for example, two P-transporter maize genes, GRMZM2G009045 and GRMZM2G326707, were found to be down-regulated by ZnO-NPs .

NPs mediated toxic effects can be associated with oxidative stress . To counteract this, plants have evolved mechanisms to protect themselves from stressors. One such mechanism involves the increased proline production . Proline may be beneficial in maize  by acting as a singlet oxygen quencher and a scavenger of free radicals and other oxidative species  or  by maintaining osmotic balance and homeostasis . Furthermore, a recent metabolomic study revealed that enhanced Ce bio-uptake increased proline levels in beans . Intracellular oxidative stress  in maize induced by ZnO-NPs or CuO-NPs can also lead to apoptosis, recognized as deficit in DNA content  as evidenced by a sub-G1 peak during cell cycle analysis . Similarly, it has been reported that NiO-NPs  caused 65.7% of tomato root cells to undergo apoptosis or necrosis and increased caspase-3 like protease activity 2.14-fold , and in ZnO-NPs  treated wheat plants, NPs induced PI fluorescence in dead or membrane compromised root cells . We observed NPs triggered LPO and antioxidant production in maize plants, which highlights the stress-alleviating potential of maize when grown in polluted environments . Similarly, ZnONPs at 500 mg kg− 1 in soil significantly enhanced LPO and induced H2O2 production in green pea plants . In the present study, the production of formazan in NBT assay was found to be inversely related to nanoparticle concentrations suggesting higher concentrations result in greater dismutation of O2- by SOD enzymes. The O2- as a primary ROS is usually the first reactive species to be released in cells. Subsequently, it is reduced to other ROS  either directly or via metal- or enzyme-catalyzed reactions . Therefore, SOD enzymes rapidly convert O2- to relatively less toxic H2O2. In line with our results of surface and deep scanning by SEM-EDXmapping and TEM , the adsorption and uptake of Fe2O3–NPs in tomato vegetative tissues were reported , and as was observed for CuO-NP  aggregates , hematite and ferrihydrite NPs  were detected by confocal laser scanning microscopy as red spots in maize seedlings .

Entrapping of CuO-NPs and translocation across epidermal cell walls  suggest their uptake by cells via endocytosis-like structures in maize root cortical cells and transportation of NPs follows. For instance, Cu accumulation occurred in shoots of CuO-NP exposed plants but not in CuO-bulk or Cu2+ treated plants . It is worth noting that the Casparian strip  plays an important part in plant protection, but that at the root apex it is not fully developed . Therefore, we suggest that in the current study, NPs passed through root apices to maize steles and were then transported to shoots via xylem. Zn2+ and Cu2+ were more toxic than NPs, irrespective of growth conditions. This could be due to heavy influx of ions in the root apoplast through transporters and metal chelators and enhanced by the negatively charged cell wall due to the presence of cellulose, pectins, and glycoproteins acting as specific ion exchangers. On the other hand, NPs are taken up by plants majorly in nano-particulate form and sometimes compartmentalized in cells. Also, due to quick toxicity by ions, maize plants could not survive for longer. Similalry, grow lights more reduction in Cucumis sativus biomass was evident by Yb3+ compared to Yb2O3-NPs . Additionally, the impact of ions varies depending on the oxidation states. Our results also concur with the findings of Cui et al. , who observed cucumber plants were more sensitive to Ag+ than Ag-NPs at same concentrations. For better understanding the toxic outcomes of Zn and Cu types on maize growth, a comparative table summarizing differences in magnitude of maize growth inhibition by different metal types is presented . Panax ginseng Meyer is a famous traditional medicinal plant belonging to the Araliaceae family. The genus name Panax originates from the word panacea, which means “a remedy for all diseases.” The 4e6-year-old roots of this perennial herbaceous plant are mainly used for medicinal purposes. P. ginseng leaves are palmate, and the flflowers bloom in June.

Ginseng has primarily been cultivated in the forest areas of East Asia including Korea, China, Russia, and Japan. Traditionally, P. ginseng is cultivated in soil, and numerous pharmacological and phytochemical studies of the extracts or compounds from soil-grown plants were conducted. P. ginseng contains ginsenosides, polyacetylenes, sugars, and some essential oils used for enhancement of immunocompetence, nutritional fortification, improvement of liver function, and their anticancer, antioxidant, and antidiabetic effects. More than 70 kinds of saponins have been isolated from P. ginseng. There is a growing interest in using safe, high-quality agricultural products, leading to hydroponic cultivation of ginseng using high-tech culture facilities. Hydroponic cultivation of ginseng takes much less time than soil cultivation and is accomplished in just 3e4 months in a moisture, light, and temperature-controlled environment without pesticide treatment. Hydroponically cultivated ginseng is mainly used in fresh and high-quality ginseng products. The aerial parts of hydroponic P. ginseng are reported to contain higher contents of total ginsenosides than the roots. This study was initiated to isolate active metabolites from the aerial parts of hydroponic P. ginseng. Of note, glycosyl glycerides have never been isolated from hydroponic P. ginseng. Therefore, this study is designed to isolate and identify glycosyl glycerides as well to evaluate their potential for inhibition of NO production. Monogalactosyldiacylglycerol  and digalactosyldiacylglycerol  are commonly present in the chloroplast membrane of ginseng. The MGDG and DGDG constitute up to about 70% of chloroplast lipids.The galactolipids play roles in the photosynthesis and regulation of lipid biosynthesis during phosphate deprivation. Furthermore, glycosyl glycerides were reported to have antifilarial, anticancer, antitumor, and many antiinflammatory activities. Therefore, this study describes the procedure for isolation and identification of four glycosyl glycerides from the hydroponic P. ginseng, and evaluation of their anti-inflammatory activities on NO production in lipopolysaccharide -stimulated RAW264.7 macrophage cells.The root of Panax ginseng Meyer has been used as traditional medicine in East Asian countries for more than 2000 years. Various processed products from P. ginseng have been introduced globally. Panax ginseng has antioxidant and antiinflflammatory properties; thus, it is under investigation for its therapeutic effects on skin disorders, including atopic dermatitis. Intake of red ginseng  extract reportedly attenuated eczema, transepidermal water loss , and skin squamation in patients with AD. Also, an RG extract decreased 1-flfluoro-2,4-dinitrobenzene-induced ear thickness, TEWL, and levels of immunoglobulin E, thymic and activation-regulated chemokines , thymic stromal lymphopoietin, and tumor necrosis factor -a in mice. The beneficial effects of P. ginseng are attributable to ginsenosides, which are the main active compounds in its roots. However, phenolic compounds, including phenolic acids and flavonoids, have also been detected in the fruits, leaves, and roots of P. ginseng aged 3e6 years.

The controlled temperature in S2 results in a doubled yield with respect to on-field cultivation, but still half of the one of vertical hydroponic . Having a high production in a limited space is one of the main qualities of vertical hydroponic systems. Moreover, since no soil is needed, the cultivation can take place in many different spaces: on rooftops, indoor, on abandoned industrial sites, on walls. An additional positive characteristic of hydroponics is that the quality and eventual contamination of the soil does not represent a risk for the products – simply because the two compartments are not in direct contact. This advantage is shown in the agricultural land occupation of scenario S1: by being soil independent and vertical, this type of hydroponics requires from four to 20 times less agricultural land than conventional agriculture. However, this means that hydroponic production needs to be supported by an external input of fertilizers to satisfy the nutrient requirement of the plants. The use of NPK fertilizers in the three scenarios is reported in Table 2. Scenario S1 needs more nutrient input than greenhouse cultivation, but less than on-field production, where part of the applied fertilizers is lost due to leaching processes. The different NPK proportion indicates that hydroponics allows an optimization of nutrient supply to support the growth phases of the plants. These results do not completely explain the impacts on freshwater eutrophication: in scenarios S1 the production of electricity and fertilizers are the main contributing processes, and in scenario S2 the production of heat is the dominant process. Scenario S3 has a negligible use of these inputs, but needs more fertilizers, which determines the emission of nutrients into the water streams.

It is important to distinguish where the emissions of nutrients take place, since eutrophication is a local phenomenon. In this sense, in scenario S1 and S2 the major risk of eutrophication is in the areas where fuels, hydroponic nft metals and fertilizers are extracted, i.e. mining sites distant from the place of final use. On the contrary, in scenario S3 the release of nutrients following the application of fertilizers represents a noteworthy impact on local water quality. About marine eutrophication, the actors in play are the same of freshwater eutrophication , but since scenario S3 has a high release of nitrogen , its impact is higher than scenario S1. Regarding eutrophication, a merit of hydroponics is the efficient use of nutrients, that, by being recycled with the water, are not released through the soil but remain available for the plants. The impact on freshwater ecotoxicity is due to the heat consumption in scenario S2, whereas for the other two scenarios several processes impact with the same order of magnitude: electricity production, fertilizers production, equipment production, pesticide production and transport. The combination of equipment and energy requirements is responsible of the impact of scenario S1 on fossil depletion. Scenario S2, due to the necessity to heat the greenhouse, has a performance more than ten times worse, while on-field cultivation , that is relatively low-input, has half the impact of S1. A benefit of urban agriculture is the shortening of the supply chain and the reduction of losses during the transport of the products. Thanks to the proximity of the farm to the consumers, the vegetables are fresher and do not need to be cooled during the delivery. For these reasons, we assumed zero losses in the transport phase. For conventional agriculture, on the other hand, the reported distribution losses are approximately 12%.The quality of the data influences the results. The primary data collected for scenario S1 refer to four months of production. Even if the farmers considered the seasonal variability for a better estimation of the annual consumptions, e.g. the water consumption, we recognise that the annual estimations could eventually not correspond to the actual values. Moreover, scenarios S2 and S3 are based on LCI datasets representative of the Integrated Production in Switzerland. Even though the authors of the datasets affirm that, most probably, their data are representative for similar cultivations in industrialised countries, these values cannot capture the peculiarities of country-specific cultivation practices. In conclusion, we acknowledge that the data do not have the same level of precision, and this affects the quality of the results.

To compare the performance of the systems, we chose the environmental indicators considered the most representative for LCA analyses of agricultural systems. We recognise that the agricultural sector can deliver other positive and negative effects, such as potential contribution to biodiversity, social issues and economic development. Consequently, integrating the analysis with other methodologies could give a broader perspective on the impacts of agriculture on an environmental, economic and social level.Constructed wetlands are commonly applied secondary or tertiary wastewater treatment systems . CWs exploit natural processes to remove pollutants via substrata, vegetation and microorganisms . CWs were traditionally employed to remove traditional pollutants, but are recently being used to remove micro-pollutants from wastewater. Numerous studies reported the adverse effects of micro-pollutants on human and environmental health . Effects include feminization of fish, short and long-term toxicity to biota, and the development of antibiotic resistance among natural and anthropogenic microbiomes . Currently, phyiscochemical technologies, such as ozonation, and membrane and activated carbon filters are implemented as tertiary treatment to remove micro-pollutants . However, such high-tech, expensive methods are less suitable in many emerging and developing economies . CWs are an attractive alternative: CWs are cost effective, eco-friendly and easy to operate and maintain . However, a key limitation in application of CWs is their high land area requirements, typically termed a land footprint. The CW substratum plays the key role in determining the size of a CW. Substrata remove various pollutants directly by sorption, precipitation, filtration and biodegradation . In addition, the substratum supports plant and microbial growth. Initially, soil was used as substratum. Recently, sand and gravel have been selected for their high hydraulic conductivity, resulting in improved performance of CWs. However, the application of these materials has limitations due to low removal of nitrogen and phosphorous, and more recently for micro-pollutants . Considering the importance of substrata for CW performance, selection of proper substrata could both reduce CW footprint while also removing micro-pollutants. Hydroponic substrata are good candidates for use in CWs.

Developed for hydroponic plant growth in horticulture, hydroponic substrata have attractive properties, such as higher oxygen and water holding capacity, large surface area and retain nutrients . In this study, the use of four commonly used hydroponic substrata were compared: mineral wool, pumice, wood fibre and coconut fibre. To date, a limited number of studies were conducted on their usability in CWs and on their potential to remove micro-pollutants from wastewater . Yang et al.  reviewed the studies which focused on the use of emerged substrata  in CWs to remove conventional pollutants. However, suitability of the emerged substrata to remove micro–pollutants was not reported. Furthermore, Wang et al.  articulated that there is a lack of comprehensive studies available on the suitability of hydroponic CW substrata  to remove various pollutants. Therefore, there is an urgent need to study the potential of the four substrata to remove micro-pollutants in CWs. Generally, sorption is one of the initial removal processes of a pollutant in the filter bed of CW. Sorption is an umbrella term covering both absorption  and adsorption  processes, and is especially used when these two mechanisms cannot be distinguished . Isotherm models, such as Freundlich and Langmuir models can be used to unravel the sorption mechanisms from experimental data to distinguish surface related or absorption related mechanisms.These include physico-chemical properties of a sorbent and sorbate, such as acidity  and hydrophobicity  of a pollutant, and organic matter content of a substratum . These factors influence the affinity between a sorbent and sorbate and transport of the sorbates from bulk solution to sorption sites, influencing extent and kinetics of sorption. Thus, hydroponic channel understanding sorption mechanisms is crucial for successful applications of a substratum in CWs treating micro-pollutant-containing wastewater. This study aims to investigate the applicability of four hydroponic substrata for efficient removal of micro-pollutants from wastewater. We determine sorption affinities and kinetics, and interpret this data using isotherm models and physico-chemical properties of substrata and micro-pollutants.

These results are used to assess the suitability of the substrata in CWs, both for micro-pollutant removal and for reducing CW footprint.Sorption kinetics were studied using the sorption of the micro-pollutants into the substrata over the contact time . Fast initial sorption to organic substrata was observed for most of the studied micro-pollutants during the first 6 h, followed by a slower sorption phase. This suggests diffusion into internal substratum was limiting: rapid sorption to the outer layer of the substrata is followed by slower diffusion in an apparent first order process. Naproxen and ibuprofen exhibited distinctly slower kinetics during sorption to the organic substrata. For inorganic substrata, even slower kinetics were observed for the studied micro-pollutants . A thorough and further mechanistic interpretation of the sorption kinetic data using pseudo-first and second-order models was not possible due to insufficient R2 values resulting from fitting data with these models . The profile of the sorption kinetics  was used to identify equilibrium. The sorption curves levelled off between 24 and 72 h, indicating that sorption reached a status of equilibrium. Based on this, the concentration obtained at 72 h was taken as an estimate of the equilibrium concentration Ce.Organic substrata wood fibre and coconut fibre sorbed micro-pollutants more than the inorganic substrata mineral wool and pumice . The observed sorption affinity of the organic substrata with the micro-pollutants followed the order: trimethoprim>carbamazepine >caffeine>sulfamethoxazole>ibuprofen= naproxen . A similar order of sorption affinity for trimethoprim, carbamazepine and sulfamethoxazole was found for soils by Kodešová et al. . Ibuprofen and naproxen, containing -COOH groups, and sulfamethoxazole, containing -SO2-NH moieties, have acidic protons which can dissociate and form anionic species . Subsequently, a strong repulsion occurs between these anionic species and negatively charged surface of the organic substrata. Although sulfamethoxazole has a basic NH2 group , it has acidic protons due to the presence of an acidic -NHand an electron-withdrawing -SO2- in the vicinity. The presence of two basic NH2 groups in trimethoprim explains its highest sorption affinity. Carbamazepine has one amido group and the basicity of an amido group is lower than of an amino group.

This could explain the sorption order: trimethoprim is followed by carbamazepine. Caffeine has a lone pair of electrons at the non-methylated N site . Therefore, caffeine acts as a proton-acceptor, basic, so it is positively charged and attracted to the negatively charged surface. Caffeine is hydrophilic whereas carbamazepine is hydrophobic due to its dibenzoazepine structure . The hydrophobicity positively affects the sorption . Therefore, carbamazepine is followed by caffeine in the sorption order. The sorption of micro-pollutants on organic rich materials appears to be a trade-off between electrostatic interaction and hydrophobic interactions between the organic matrices and the micro-pollutants. This is further depicted in a four quadrant matrix . In the high pKa and high Log Kow quadrant, both electrostatic interactions and hydrophobicity positively affect the sorption, as indicated by high KF values for carbamazepine on the organic substrata. For the low pKa and high Log Kow quadrant, the positive effect of hydrophobicity of iburprofen and naproxen appears to be largely counteracted by strong electrostatic repulsion due to deprotonation . At the quadrant of high pKa and low Log Kow, an intermediate sorption on the organic substrata was observed for caffeine and sulfamethoxazole. In this study, no compounds falling into the low pKa and low Log Kow quadrant were investigated. We speculate based on our results, that even lower sorption is to be expected for compounds in this quadrant on our selected substrata. Inorganic substrata mineral wool and pumice generally exhibited much lower sorption and slower sorption kinetics towards the studied micro-pollutants. Mineral wool is a non-reactive neutral material. Therefore, unlike wood fibre and coconut fibre, mineral wool has no extensive net negative surface charge. Furthermore, mineral wool has the lowest surface area among all the substrata . These properties together explain the low sorption of the micro-pollutants.

FQs themselves, their synthesis by-products, and the metabolites of bacteria are raising increasing concern regarding their ecological risks . Such compounds exhibit toxicity and non-biodegradability and are expected to form complexes with metal ions and other unknown organic compounds, representing hazardous environmental and health impacts. The resistance capacity of microorganisms will also be inhibited . Constructed wetlands have been used internationally for over two decades in a variety of aquatic enhancement projects . CWs are an important and effective measure used to purify wastewater that can effectively remove antibiotics and pollutants . In the existing research, most of the studies on antibiotics in constructed wetlands address the effects of high concentrations  on the removal of antibiotics, the generation and removal of resistance genes, etc . The effect of antibiotics in the natural environment on the purification of pollutants in constructed wetlands and the influence mechanism are still not known. Moreover, temperature is the key factor affecting the water purification effect of constructed wetlands . The removal rate of selenium by cattail floating systems is 93–100% at 35 ◦C in summer and 51–100% at 5 ◦C in winter . Low-temperature stress has two distinct components: chilling and freezing. Usually, chilling is defined as occurring at temperatures that are lower than the normal growing temperature for a given species but higher than 0 ◦C, and freezing is defined as occurring at temperatures less than 0 ◦C.However, ebb and flow trays the potential threat and impact of antibiotics with respect to the environment cannot be ignored.

Whether different concentrations of antibiotics have an effect on pollutant removal under different temperature conditions and the difference in the influence mechanism are worth investigating. In this study, a hydroponic culture experiment was designed, and the reactors were placed in an artificial climate chamber at 4–8 ◦C and in a greenhouse at 25–28 ◦C . Different concentrations of LOFL were put into the reactors at two temperature conditions. The concentration of 0–10.0 µg/L may appear in the environment, and 100 µg/L is the concentration in emergencies, which can strengthen the impact of LOFL on the entire system . The objectives of the present study were  to evaluate the long-term effects of different LOFL levels on pollutant removal in CWs at low and normal temperatures;  to clarify the changes in the bacterial community structure of CWs after long-term exposure to LOFL at low and normal temperatures; and  to explore the correlation analysis of environmental factors and predict the functional changes at low and normal temperatures. The experiment was conducted in an open field at the China Research Academy of Environmental Sciences , Beijing, China . Plants were hydroponically cultured in white plastic buckets. Each incubator, 191 mm bottom diameter × 214 mm top diameter × 210 mm height, was filled with 4.5 L culture medium . Plants were fixed in planting baskets and incubated with tap water for 2 months in a greenhouse  before the start of the experiment. Hydroponic culture was used in this experiment, and the devices were placed in an artificial climate room  or greenhouse . Plants with similar growth were selected. Four Iris pseudacorus L. specimens were planted in each incubator and fixed with a guardrail. Specimens were cultivated for 81 days, from May 31 to August 20. Temperature was the main factor affecting the degradation of pollutants, and the concentration of LOFL was the secondary factor. The removal rate of pollutants increased with increasing temperature and contact time and decreased with increasing LOFL concentration. The removal rate of NH4+ was greatly affected by the environmental conditions , while the removal rates of TN and TP were less affected by environmental factors, and the removal rate of CODCr was the least affected. Normal-temperature conditions are more beneficial than low-temperature conditions to the degradation of pollutants.

Under the two temperature conditions, there was a significant difference in the removal rate of pollution indices between the control group  and the experimental groups  . The pollutant removal rate of the control group was lower than those of the experimental groups, which indicated that the plants played an important role in the system. At normal temperature, the removal rate of TN was the highest at 0 µg/L LOFL, with a value of 82.90%. Any concentration of LOFL has a certain inhibitory effect on the removal rate of TN. The degree of inhibition of TN removal at a concentration of 0.3 µg/L was lower than that at 0.1–0.2 µg/L, which indicated that the former concentration promoted the degradation of TN compared with the effect of 0.1–0.2 µg/L. Under the condition of low temperature, a LOFL concentration of 0.1–0.5 µg/L promoted the removal of TN by the system. When the concentration of LOFL was 0.2 µg/L, the promoting effect was the most significant, and the removal rate was 73.28%. The degradation of TN was inhibited when the concentration of LOFL was more than 1 µg/L, and when the concentration of LOFL was 100 µg/L, the removal rate decreased to 66.91%. There was a significant difference in ammonia nitrogen removal efficiency between 0 and 0.5 µg/L LOFL and 100 µg/L . Moreover, the difference between 10 µg/L and 100 µg/L was significant. At normal temperature, with increasing LOFL, the removal rate of ammonia nitrogen showed a continuous inverted “V” shape; the tip of the “V” appeared at 0, 0.3, and 10 µg/L, and the removal rates were 85.10%, 87.40% and 77.90%, respectively. The removal rate of ammonia nitrogen was the highest at a LOFL concentration of 0.3 µg/L, and the degradation of ammonia nitrogen was inhibited at other concentrations. When the LOFL concentration was 100 µg/L, the removal rate decreased to 59.99%. LOFL concentrations of 0.1–0.5 µg/L can promote the degradation of ammonia nitrogen, and the maximum removal rate was 45.03%  at low temperature. When the concentration of LOFL was higher than 1 µg/L, it showed an inhibitory effect, and the removal rate was 28.90–30.18%. This result showed that a high concentration of LOFL has a great effect on ammonia nitrogen and inhibits the degradation of ammonia nitrogen. Overall, the removal rate of ammonia nitrogen at low temperature was 17.67–40.96% lower than that at normal temperature. The removal rate of CODCr at normal temperature was 2.06–27.9% higher than that at low temperature.

Under normal-temperature conditions, LOFL at concentrations of 0.1–0.5 µg/L promoted the removal of CODCr, and the promoting effect of 0.2 µg/L was the largest: the removal rate of CODCr increased from 54.63% to 64.78% compared with that of the control group. When the concentration of LOFL was higher than that of 1 µg/L, it inhibited the degradation of CODCr. At 100 µg/L, the removal rate of CODCr was 50.64%. At low temperature, LOFL concentrations of 0.1–100 µg/L promoted the degradation of CODCr. Under the conditions of low and normal temperature, the removal rate of TP in the control group increased from 71.28% to 86.34% and from 83.49% to 92.19%, respectively. Plants played a key role in the removal of total phosphorus. Under normal-temperature conditions, concentrations of 0.1–0.5 µg/L can promote the removal of TP. The maximum removal rate appeared at 0.3 µg/L, and the removal rate was 95.79%. When the concentration of LOFL was higher than 1 µg/L, the removal rate of total phosphorus began to be inhibited. When the concentration of LOFL increased to 100 µg/L, the removal rate of TP decreased to 86.98%. At low temperature, concentrations of 0.1–1 µg/L can promote TP, and when the concentration of LOFL was higher than 1 µg/L, the removal efficiency was obviously inhibited. When the concentration of LOFL increased to 100 µg/L, the removal rate of TP was 80.20%. In this study, the average removal rate of NH4+ at normal temperature was 12.84–55.89% higher than that at low temperature. The average removal rate of TN was 14.71–40.96% higher than that at low temperature. The removal rate of TP was 5.22–12.21% higher than that at low temperature. Studies by relevant scholars have shown that the removal efficiencies of ammonia nitrogen , total nitrogen  and total phosphorus  at low temperatures were reduced by 15%, 45% and 16%, respectively, compared to those observed at the optimal temperature  . Those experimental results are similar to those of this study. At the same concentration of LOFL, the removal rate of pollutants in the system at room temperature was significantly higher than that at low temperature. In this study, the purification of pollutants was mainly due to plant absorption and bacterial degradation . Plants play an important role in hydroponic wastewater treatment systems and directly affect the quality of wastewater . The nutrient absorption mechanism for plant utilization involves plant extraction, 4×8 flood tray plant transformation, plant filtration and plant degradation . Through these processes, plants release exudates from their roots, which can stabilize, fix and bind organic pollutants, thereby reducing biodiversity . This process can further improve water treatment and potentially minimize the discharge of nutrients absorbed by plants.

Existing studies have shown that hydroponic systems can not only purify nutrients but also remove pollutants such as antibiotics . TN removal in hydroponic culture systems is mainly realized by physical precipitation, nitrification/denitrification and plant absorption. However, plant tissue may release and decompose pollutants . Therefore, the mechanism by which plants absorb pollutants and decompose plant tissues is a dynamic process . This study does not conduct an in-depth analysis of this problem. In the low-temperature environment, the removal rate of pollutants in the system was reduced mainly for two reasons. First, plant growth, development, and productivity are negatively affected by low temperatures . Two direct effects at the molecular level would be caused by the reduction in temperature. Enzyme activity and membrane flexibility are reduced with decreasing temperature . When plants suffer from cold temperature, although the photosynthetic light reaction is relatively stable, the activity of dark reactive enzymes is reduced. Therefore, photoinhibition of photosystem I and sometimes II occurs . Membrane elasticity also decreases with decreasing temperature. This decrease leads to membrane damage, which leads to increased electrolyte leakage . The supply of oxygen to Iris pseudacorus L. is restricted at low temperature, and the root activity decreases. These processes lead to the narrowing of voids connected with substrates and air, which affects radial oxygen loss. As a result, reoxygenation is reduced, and the assimilation of nitrogen pollutants is decreased . Therefore, in the low-temperature environment, the nutrient absorption and transformation ability of the plants decreased, resulting in a reduction in the removal rate. The second reason is that the bacterial activity in the system decreased at low temperature. The pollutant transformation processes and mass transfer in CWs were restrained when bacterial activity was affected by low temperatures. Therefore, the efficiency of the ecosystem function limits the removal efficiency of CWs .

The activity of anaerobic ammonia oxidation decreases at 10 ◦C, resulting in the deterioration of nitrogen removal performance . The bacterial activity and metabolic rate in the CW systems were reduced at low temperatures. This reduction impedes heterotrophic bacteria from decomposing organic pollutants . The nitrification reaction efficiency drops rapidly at temperatures of 20–30 ◦C, and the reaction almost stops at 5 ◦C . Denitrification is the most effective way to denitrify  CWs, and the reaction rate decreases at 15 ◦C . With increasing temperature in CWs, denitrification and dissimilatory nitrate reduction to ammonium  increased. After 81 days of culture, compared with the values at 6 days of culture, ace and sobs were larger, and Chao, coverage, Shannon and Simpson were smaller. The higher the concentration of LOFL is, the smaller ace and sobs are and the larger Chao, coverage, Shannon and Simpson are. The ACE and Chao indices indicate bacterial community richness, while the Shannon and Simpson indices reflect bacterial community diversity . Sobs is the actual observation of richness. Therefore, compared with the results at low temperature, the bacterial community richness was higher and the diversity of bacterial communities was lower at normal temperature.This difference might be because the microorganisms were more active in the high-temperature environment and the dominant flora inhibited the reproduction of the inferior flora. In the low-temperature environment, most of the bacteria were inhibited.

Wastewater, groundwater, and surface waters were collected from established monitoring locations in an agricultural watershed containing the City of Jacksonville’s Municipal Wastewater Land Treatment Site , a 2139-ha forest land treatment system in coastal North Carolina, U.S.A, that has treated wastewater since 1998 . The average 30-year annual rainfall is 1379 mm for the watershed . Municipal wastewater land application occurs over 30 km2 of the watershed to irrigate wastewater volumes of 1244 to 1590 mm per year over 890 ha of a mixed hardwood/pine forest. In July 2019, A one-day sampling campaign collected 4-L and 1-L water samples at established watershed monitoring locations for wastewater, surface water, and groundwater; these water samples served as “irrigation sources.” Municipal tap water that was deionized  was amended with Ionic Grow ™ and served as a reference source for hydroponic soybeans.These locations were used for recent multi-campaign targeted , non-targeted , and hydrologic modeling studies . Groundwater and surface water samples were collected using USGS National Field Manual for the Collection of Water Quality Data as described in Hedgespeth et al. and transported on ice prior to storage or processing. All water samples were collected on the same day within four hours of each other in pre-cleaned amber bottles that were triple-rinsed with deionized water and baked for 24 h at 60 °C, then rinsed again with methanol using a modified USGS protocol . One-liter water bottles were extracted within 24 h by solid phase extraction while four-L bottles were stored at 4 °C for hydroponic studies.

Details of representative field sample collection are provided in supplemental documents.Soybean seeds, G. max, flood table were purchased from Johnny’s Selected Seeds  and germinated at 30 °C for four days prior to start of the hydroponic study. Healthy seedlings were randomized and placed in pre-cleaned and methanol-rinsed 250 mL amber wide mouth jars that had been filled with one of the six different irrigation sources. Each jar was covered entirely with aluminum foil and crimped around the plant stem to prevent photo degradation of chemicals in the water . There were three replicates per irrigation source for a total of 18 hydroponic jars and two separate hydroponic studies. One study grew 18 soybean plants for two weeks per USEPA OCSPP 850.4800 method . A second study grew 18 soybean plants for eight weeks until plants produced bean pods and plant leaves began to yellow. The amount of irrigation source water used was recorded while plants grew. At harvest, plants were removed and separated into roots, shoots, and bean pods and weighed as separate entities for wet biomass. Plant tissues were placed on clean aluminum foil and frozen at −20 °C then freeze dried and ground prior to solvent extraction for HRMS analyses.Plant tissues  were freeze dried and cut with clean surgical scissors for solvent extraction. Plant tissue and CIPS samples were fortified with a 100 μL surrogate internal standard  solution prior to extraction . Approximately 1 g of root tissue and 3 g of shoot tissue was extracted by shaking with dichloromethane/acetone  for two hours with a moisture absorbing polymer and then filtered through 0.45 μm PTFE syringe filters. CIPS were first rinsed with deionized water then extracted with the same solvent mixtures by shaking. All samples were processed through gel permeation chromatography  clean up using an Envirogel GPC column  and methylene chloride at a flow rate of 3 mL/ min.

After GPC, plant samples with heavy chlorophyll content were further processed through a Florisil SPE Cartridge  following US EPA method SW-846/3620C  and then eluted with dichloromethane prior to HRMS analysis.We expected that municipal wastewater would have more chemical features, TICs, and ToxCast chemicals overall, but onsite groundwater, surface water downstream of the land treatment site, and tap water had more chemical features for grab samples extracted after 24 h. Mean totals of chemical features in CIPS  were more variable for wastewater and upstream surface water but not significantly different from other irrigation sources . Mean totals of TICs and ToxCast chemicals were similar across all irrigation sources for grab samples and CIPS. Box plot distributions of total NTA features in plant tissues and CIPS show an upward trend from roots to shoots to bean for each irrigation source except surface water downstream of the land treatment site . Soybean roots from off-site groundwater had the lowest mean total of chemical features . Mean totals of chemical features were similar for beans in wastewater, downstream surface water, and tap water and significantly higher than upstream surface water . The mean totals of chemical features in CIPS were not different across all irrigation sources, and mean totals of chemicals features were not different between CIPS and extracted beans for each irrigation source. Mean total counts of tentatively-identified chemicals  and abundance were greater for extracted from roots, shoots, beans, and CIPS than irrigation sources extracted after 24 h . Total counts of TIC numbers and chemical class distributions in roots were most similar to extracted irrigation sources. Roots did contain TICs categorized as cosmetics-personal care products-disinfectants-tobacco . This new class was also present in extracted shoots along with TICs categorized as natural products-toxins, food additives-preservatives, and amino acids-fatty acids-essential oilsvitamins. Not surprisingly, amino acids-fatty acids-essential oils-vitamins and food additives-preservatives were more apparent in shoot tissues than roots, beans, or CIPS.

Chemical class categories for beans were consistent with chemical classes in irrigation sources, but the number and abundance of TICs were two to three times greater in beans than irrigation sources. TICs in beans were different in class distribution and abundance across irrigation sources . Specifically, the number of TICs extracted from beans from plants exposed to downstream surface water and tap water sources were double the TICs extracted from other irrigation sources. TICs in beans from plants exposed to wastewater and upstream surface water remained the same in number and abundance to TICs extracted from each irrigation source. The mean number of TICs in CIPS were more similar to TICs extracted from water, but CIPS had more diverse chemical categories for all irrigation sources than roots, shoot, or beans. TICs for musks-fragrances, hormonespharmaceuticals-drugs, and insect repellents were identified in CIPS across all irrigation sources although these TICS were not detected in irrigation sources, roots, shoots, or beans . There were more ToxCast chemicals  extracted from roots, shoots, and CIPS than extracted from irrigation sources, and more ToxCast chemicals were extracted from municipal wastewater and LTS surface- and ground waters sources than other irrigation sources . However, total count of ToxCast chemicals were not different between CIPS and plant tissues for each irrigation source . Although a qualitative metric and surrogate for concentration, total abundance of ToxCast chemicals in plant tissues were two to four times greater than ToxCast chemical abundances in irrigation sources or CIPS. Surprisingly, mean total counts of ToxCast chemicals detected in plant tissues exposed to municipal wastewater was fewer than mean total counts of ToxCast chemicals detected in soybean tissues from other irrigation sources. For roots, shoot, beans, and CIPS, most ToxCast chemicals were assigned to the Multiple Chemical Use Category across all irrigation sources .

Chemical use categories for CIPS were more diverse than categories for extracted plant tissues and irrigation sources. CIPS had ToxCast chemicals assigned to musks-fragrances, insect repellants, and cosmetic-PCPs disinfectants-tobacco as well as multiple classes, hormones-PPCPs, and industrial plasticizers-fire retardants-surfactants-dyes .We evaluated distributions of TICs and ToxCast chemicals between extracted irrigation sources, plant tissues, and CIPS as a function of log Kow and abundance . At high abundance, irrigation sources and beans had TICs and ToxCast chemicals with log Kow values of 3 to 8 while CIPS had TICs and ToxCast chemicals with log Kow values of 1 to 3. For TICs chemicals at low abundance, log Kow values ranged from 1 to 8 with most TICs above log Kow values of 4 associated almost exclusively with plant tissues or irrigation sources. Likewise, ToxCast chemicals with log Kow values greater than 3.5 were associated almost exclusively with plant tissues or irrigation sources while CIPS had ToxCast chemicals with log Kow values between 1 and 4. CIPS detected more unique ToxCast chemicals from irrigation sources than solid phase extraction of irrigation sources or solvent extraction of plant tissues . There were 11 unique ToxCast chemicals detected in irrigation sources with 1-chloro-3-methyl benzene detected in wastewater, groundwater, and downstream surface waters . There were 13 unique ToxCast chemicals detected in beans with benzofuran detected in beans grown in upstream surface water and tap water and 2,6-dimethoxy phenol detected in beans grown in municipal wastewater and downstream surface water. CIPS detected over 30 unique ToxCast chemicals from irrigation sources . These ToxCast chemicals were frequently detected in multiple irrigation sources and represented more diverse chemical use categories. Only two ToxCast chemicals, hexadecane and 1,3-benzenediol, were detected in an irrigation source, CIPS, and extracted beans, and only 16 ToxCast chemicals were detected in either irrigation source-CIPS, CIPSbean, or irrigation source-bean pairings . CIPS and irrigation sources had more similar ToxCast chemicals for multi-use, industrial,rolling benches insectrepellents, cosmetic, and pesticide chemical use categories with log Kow ranges of 1.3 to 6.2. Extracted beans shared 6 ToxCast chemicals in common with extracted irrigation sources and only 4 ToxCast chemicals in common with CIPS.

The greater number and diversity of chemical use categories for TICs and ToxCast chemicals extracted from CIPS provide more comprehensive representations of chemical use categories for plant exposure and uptake than solid phase extractions of irrigation sources.Historically, agricultural producers have used wastewater to provide water and nutrients inputs to cultivate food crops with positive  and negative  responses of plant productivity due to nutrients, microbiome, and/or phytotoxicity of wastewater . In this study, groundwater irrigation sources yielded poor soybean biomass and no flowering while wastewater and surface waters yielded good growth, flowers, and fruits. Chemical parameters, such as conductivity  and chloride concentrations did not vary greatly for groundwater and wastewater  from prior sampling events. On-site and off-site groundwater did often have low redox conditions, acidic pH, and elevated dissolved iron, manganese, and sulfides that may negatively impacted soybean growth and fruit production . For wastewater and surface water irrigation sources, observed increases in soybean biomass and fruit production did not correlate with mean counts of chemical features or TICs. In fact, beans from plants grown in municipal wastewater had fewer identified TICs and ToxCast chemicals and lower abundances than beans produced from plants grown in municipal tap water. Beans grown in wastewater had fewer TICs and similar ToxCast numbers to upstream surface water, tap water, and surface water downstream of the Jacksonville wastewater site. Downstream surface water and tap water had similar TICs and ToxCast COC numbers, abundances, and types of chemical use categories. The municipal tap water comes from several drinking water plants located in watersheds with significant agriculture, septic systems, and industry. For the study watershed, prior targeted and non-targeted comparisons of organic chemicals for wastewater, surface water, and groundwater provide unique insight to the dilution effect of land treatment systems versus conventional wastewater treatment with dedicated surface water outfalls. Multi-campaign sampling assessments have demonstrated that concentrations of pharmaceuticals in wastewater are often 1 to 2 orders of magnitude greater than concentrations quantified in groundwater and surface water samples during below normal rainfall periods . Pharmaceutical concentrations in on-site groundwater and surface waters are only half-fold greater than upstream surface water or off-site groundwater during rainfall deficits and 1 to 2-fold lesser than concentrations detected downstream of a similar-sized, conventional wastewater treatment outfall . Because the land treatment system stores wastewater in large open reservoirs for 10–14 days and these reservoirs receive rainfall, consistent rainfall events can dilute the number of chemical features in wastewater to equivalent numbers detected in upstream surface waters . Aggregated ToxPI scores of TICs from multi-sampling campaigns were similar for chemicals of greatest abundance between upstream surface water samples and wastewater samples . After a catastrophic hurricane in 2018, the number of chemical features and total abundance in upstream surface water and off-site groundwater samples far exceeded those numbers determined for on-site and downstream surface waters and groundwater at two week and three months beyond the hurricane event .

For the analysis of PFAAs in soil, the soil was dried in an oven at 40  C until no further weight loss was recorded. After homogenization, 1 g of dried soil was weighed in a 15 mL PP tube and spiked with internal standards. The soil was then extracted with 10 mL MeOH by vortex mixing for 1 min and sonication for 10 min. Phase separation was achieved by centrifugation . The supernatant was transferred to a new 15 mL PP tube and concentrated in the Rapidvap. The extraction was repeated twice with 5 mL MeOH. The extracts were combined and concentrated in the Rapidvap to a final volume of 1 mL. For pore water analysis 20 g of the soil was put in a 50 mL centrifugation filter tube with a 0.2 mm nylon filter. After 20 min of centrifugation at 2000 RPM, 0.5 mL of pore water was transferred to a vial. The internal standards and MeOH were added to achieve a final volume of 1 mL. All final extracts were passed through an Acrodisc LC 13 GHP Pall nylon filter into 2 mL PP vials and stored at 4  C until analysis.An HPLC system  coupled with a tandem mass spectrometer was used to analyze the samples for PFAAs. A pre-column prior to the injection valve was used to remove potential background contamination from the LC system. Separation of the analytes was achieved using an ACE 3 C18-300 column  maintained at 30  C with a mobile phase gradient consisting of two eluents A and B , both containing 2 mM ammonium acetate. The gradient used for separation and the mass transitions as well as other mass spectrometer settings can be found in the Supporting Information. The mass spectrometer was equipped with an electrospray ionization interface operating in the negative ionization mode, berry pots and it was run in a scheduled MRM-mode. The purified extracts were diluted 1:1 with water prior to analysis to match the injection conditions of the HPLC. A volume of 20 ml was injected. Raw data were processed with the Analyst 1.5 software .Each sample was extracted three times and each extract was injected in duplicate.

The relative standard deviation of the concentrations derived from these six injections was <10% for all analytes in all samples. Concentrations were quantified using a twelve-point calibration with fitted correlation lines that had r2 values of >0.99 for all analytes; no weighting was applied. Further information on quality assurance and quality control is provided in our previous studies . Recoveries were determined by comparison with a matrix free solution spiked with internal standard immediately prior to injection. Average recoveries of the internal standards in the samples were between 22%  and 112% . Since mass labeled internal standards were used for quantifying the analytes, no correction for recovery was necessary. See Table S4 in the Supporting Information for detailed information on recoveries. Limits of quantification were calculated on the basis of the lowest validated calibration standard. They were derived from the amount injected back calculated to an extract volume of 1 mL and divided by the average extracted sample quantities. Method blanks were prepared repeatedly with the same extraction procedure as the samples, but showed no quantifiable contamination. Solvent blanks were injected every ten injections to check for contamination of the LC system and for memory effects, but no contamination or memory effects were observed during the study. All PFAA concentrations from the non-spiked lysimeters were subtracted from the concentrations in the spiked lysimeters. Any resulting concentrations below the LoQ were neglected. Since PFOS is the only compound for which branched isomers were included in the standards used for the calibration curve, branched isomers could only be quantified for PFOS.

All reported PFOS concentrations are sum concentrations of non-branched and branched isomers.The soil concentrations at the time of planting were generally within the intended concentration range . The soil concentrations at the harvest date show that the shortest chain PFAAs, the C4eC6 PFCAs and PFBS, were depleted. Less than 3% of the initial mass was left in the soil . Depletion occurred in both the upper and lower soil layers . In contrast, some 80e90% of the longer chain PFCAs dosed were still present in the soil at the harvest date. We analyzed the behavior of the PFAAs in the lysimeter soil in another paper in which we include data from 12 other lysimeters prepared in the same manner but planted with different crops . That work showed that the depletion of the shorter chained PFAAs was due to leaching, and that the leaching was greater than anticipated due to interactions between the PFAAs. This accelerated leaching increased with the initial PFAA contamination level of the soil. Lower precipitation towards the end of the growth period contributed to reduced leaching and more stable conditions; two weeks before harvest the lysimeters had already received 91% of the water input for the whole growth period . Hence, although the lettuce was exposed to changing PFAA concentrations in soil, the evidence indicates that the concentrations were more stable towards the end of the growth period when the plants were largest and transpiring  most.To evaluate the plant uptake of the PFAAs, the PFAA concentrations in the plant tissues were compared with the PFAA concentrations in the sampled exposure media, soil and pore water, using uptake factors. Concentrations in soil were only available for the start of the experiment and at the time of harvest, and concentrations in pore water were only available at harvest. We chose to use the concentrations in exposure media measured at harvest because a much larger portion of the plant growth and transpiration occurred during the latter part of the growth period and because soil concentrations were judged to be more stable .

It is nevertheless possible that the uptake factors for the shortest chain PFAAs are somewhat overestimated due to the depletion of these chemicals in the soil over the course of the experiment.Downy mildew  is one of the most severe and devastating diseases for grapevines . Although there are various plant protection products   that can solve this issue, viticulture has traditionally used copper-based products as a fungicide, which is the most efficient way controlling it, and has become an indispensable product for a large number of grape growers worldwide . The reliance on this product is even higher for organic farming since there are no synthetical alternatives accepted to be used and copper-based pesticides are indispensable for organic vine cultivation . Therefore, the continued use of these kinds of products in viticulture has triggered the accumulation of this metal in several vineyard area soils around Europe and worldwide  which may even affect the productivity of food crops . Although Cu is fixed by organic and clayey matter content in the soil, Sonoda et al. observed copper mobility due to organic matter degradation , suggesting possible groundwater contamination. The high potential ecotoxicity of copper in freshwater and its accumulation in European soils have raised concerns in the European Union, leading to a tightening of the legislation on the use of copper-based products, limiting the use to 28 kg/ha of copper for seven years . Even though other PPP such as synthetic organic fungicides could be considered to replace copper in conventional vineyard production, their use is also not exempt from environmental risks such as soil accumulation and surface or groundwater pollution depending on their composition and the site characteristics. For example, hydroponic grow system a recent publication study  observed that synthetic organic fungicides used for grapevine cultivation in northern Italy were present in 80% of monitored water wells, exceeding the Environmental Quality Standards  limits for groundwater in 30% of them. The toxicity of dimethomorph has been observed in several living organisms, such as fish and invertebrates, other soil organisms , soil and water microflora  and aquatic plants . Concerning zoxamide, low risk was observed for non-target arthropods, soil microorganisms, and non-target terrestrial plants .

The increase of organic vine cultivation during recent years has increased the application of other kinds of organic fungicides, those named natural or biopesticides, produced from a natural source  and with minimum adverse effects on the physiological processes of plants , as is the case of laminarin, which is considered to have low environmental toxicity by EFSA . However, although some ecological products are considered good candidates for reducing copper dependency in vineyards, there is no treatment as effective as copper for controlling grapevine downy mildew . The replacement of copper-based products can be done with the use of plant resistance-inducers  or biological control agents,which have no environmental hazards. Henceforth, the use of PPPs such as copper-based and organic fungicides in agriculture can involve a human threat due to the intake of crops cultivated under those conditions  and a negative impact on other non-target receptors such as water, other plants, and animals. In addition, the sustainable use of PPPs in agriculture must include an application rate according to the characteristics of the vegetation and adjusted spraying, in order to reduce product losses due to drift.Despite this, it is worth knowing that active substances of pesticides are washed away by rainfall, causing the deposition of pesticides on the soil , which means continuing to seek solutions for preventing groundwater contamination from agricultural activity. Bare soil, commonly used in low rainfall areas, allows the rainfall water to drag with it the PPPs losses remaining on the topsoil without any obstacles and can even be simulated and modelled . Moreover, erosion and nutrient loss in bare soil would be more pronounced due to torrential rainfall events that are expected to be more recurrent due to climate change. On the contrary, cover crops in vineyards have been proven to be effective in protecting the soil from erosion and nutrient loss, improving soil fertility, structure, soil microbial functional diversity, and balancing the productive, and vegetative parts of the vine.Although the phytoextraction effect of covers in viticulture is not sufficient to eliminate the entire volume of copper annually applied as a phytosanitary product against mildew , cover crops are suggested to reduce the amounts of pesticide leached and, consequently, the risk of groundwater contamination .

Despite this, there has been no research on the effect of covers on the soil leaching of the commonly used organic fungicides in vineyards  . The main objective of this work is to demonstrate the capability of using cover crops in vineyards for the mitigation and prevention of soil and groundwater pollution caused by the application of fungicides. Also, to study if the biodiversity of this cover crops has an effect on it. Copper, 3 conventional/synthetic organic fungicides , and 1 ecological fungicide  were selected. The specific objectives of this work were as follows: i) assessment of the soil type on fungicides leaching, ii) quantification of pollution reduction due to the use of cover crops, and iii) analysis of the vegetation effect on fungicides degradation. Five different PPPs  were selected based on previous trials carried out at OPTIMA project  according to fungicide efficacy against downy mildew in vineyards and lower environmental impact . Table 1 shows the commercial name, register number, the active ingredient, the molecule structure, the octanol-water partition coefficient  and the environmental risk for each of the fungicides studied. Codimur 50 , which is a copper-based product used as preventive treatments, is used as a control/reference treatment due to its preferential use in viticulture against mildew. Forum and Zorvec Vinabel  are novel synthetic organic fungicides, with a systemic action, with dual preventive and curative effect whereas Bion MX  is a synthetic inducer and activator of plant self-defense mechanisms. Finally, Vacciplant is an ecological organic fungicide product. All experimental studies were performed by using commercial fungicides supplied by the manufacturer’s brands or purchased from Agrogava.During the spray application process carried out for crop protection, there is an inevitable loss of fungicides to the ground. According to Gil , the amount of ground losses due to the spraying application process in a vineyard was calculated as 4.62 μg cm− 2 for an application of 1 kg Cu⋅ha− 1 under the worst conditions .