Similarly, Ageratum conyzoides and Melia azedarach as medicinal plants promoted the growth and yield of rice and greatly reduced the growth of weed species . Batish et al.  also observed significant adverse effects of Tagetes minuta on the emergence and growth of the rice field weeds. Application of Anisomeles indica as an allelopathic plant for controlling weed species in wheat field inhibited the emergence and growth of weeds as same as chemical herbicide . Tinospora tuberculata , Malay name Batawali, belongs to the family Menispermaceae, and is a traditional medicinal plant . It is a plant that grows in primary rainforests in all parts of Malaysia, Thailand and Indonesia. There are a few studies that dealt with allelopathic activity of T. tuberculata . They observed the inhibitory activity of T. tuberculata against the germination and initial growth of crops and weed species. The current study was performed to  determine the allelopathic potential of T. tuberculata leaf methanol extract on the growth and development of the rice  and two common rice weeds, barnyard grass  and weedy rice , at three-leaf stage under hydroponic conditions; and  evaluate the role of phytotoxic compounds on the allelopathic activity of extract obtained from leaves of T. tuberculata.T. tuberculata plants growing in the Herbal Garden of University Putra Malaysia, Selangor, Malaysia, was used. T. tuberculata leaves were cleaned several times with tap water and air-dried for 3 weeks, then ground to a fine powder in a laboratory blender and stored in a refrigerator at 4°C until use.

Commercial seeds of barnyardgrass  and weedy rice  were purchased from Herbiseed company.Barnyardgrass seed coats were loosed by hand, one by one to improve the germination percentage. The seeds of rice  were manually collected in the rice field, Universiti Putra Malaysia. Rice and weedy rice seeds were surface sterilized in a 1:10  dilution of commercial hypochlorite bleach for 10 min and rinsed several times with distilled water. The rice seeds were cleaned, air-dried and all seeds stored in darkened,mobile vertical grow tables airtight containers at –18°C prior to their use. The germination of the seeds was randomly checked and was ~86–95%. Syringic acid , trans-ferulic Acid , trans-cinnamic acid , p-anisic acid , chlorogenic acid , vanillic acid , coumarin , gallic acid , caffeic acid  and benzoic acid  were supplied from Chemtron Biotechnology Sdn. Bhd . Vitexin, isovitexin , orientin  and isoorientin  were obtained from Sigma-Aldrich . Methanol and acetic acid of HPLC grade were obtained from Friendemann . Water was twice distilled and all chemicals used were HPLC grade.The experiment was conducted to determine the allelopathicpotential of the leaf methanol extract on the biomass and transpiration  of rice and rice weed seedlings  at the three-leave stage . Leaf methanol extract of T. tuberculata was prepared according to the methods described by Aslani et al. . Briefly, leaves of T. tuberculata were soaked in 1 000 mL of 80%  aqueous methanol, and shaken at room temperature  for 48 h. The solution was filtered through four layers of cheese cloth and then centrifuged at 3 000 r min–1 for 1 h. The solution was filtered again through a 0.2-µ, 15-mm syringe filters . The collected filtrate was evaporated to dryness under vacuum at 40°C using a rotary evaporator. Stock extract derived from leaves of T. tuberculata were diluted appropriately with 25% Hoagland’s solution  to obtain three different concentrations of hydroponic culture .

Seeds were added to moist germination paper and 7-dayold seedlings were subsequently added to 8 mL amber vials containing 7.5 mL of 25% Hoagland solution. The seedlings were placed in the glasshouse for 14 days. Five seedlings of uniform size per pot of each plant species were chosen and inserted carefully into holes in a styrofoam float. The seedlings were grown individually 2 cm apart and transplanted into a plastic pot  with 100 mL of 25% Hoagland’s solution amended with methanol leaf extract at 3.12, 6.25 and 12.5 g L–1. Hoagland’s solution without leaf extract was used as the control. The solution was replenished as needed with the appropriate solution to avoid exposure concentration changes. After 14 days, the plants were harvested; the root lengths and plant heights, biomass, transpiration volume and chlorophyll and carotenoid content were measured. The fresh leaves  of tested plants were taken, mixed and subsequently pulverized with liquid nitrogen to obtain a freeze dried mixture of old and young leaves. The freeze dried plant material of each sample  was extracted with aqueous 80% acetone and kept in the freezer at −10°C for 24 h. The suspension was filtered through a Whatman filter paper No. 1. Chlorophyll and carotenoid contents were determined spectrophotometrically using spectronic Genesys 20 spectrophotometer  at 3 wavelengths: 663 nm for chlorophyll a, 647 nm for chlorophyll b and 470 nm for carotenoids. Calculations were completed using Lichtenthaler’s equation  and expressed as mg g–1 fresh weight . The results of Experiment 1 showed that the amounts of tested index of rice, weedy rice and barnyardgrass seedlings grown under hydroponic culture were significantly affected when the leaf methanol extract of T. tuberculata in different concentrations  were added to the Hoagland’s solution. However, inhibition was more severe when these species were treated with higher concentrations.This suggested that allelopathic substances decreased stomatal conductance, leaf transpiration and net photosynthesis .

The concentrations required for 50% growth inhibition  of the root , shoot  and biomass  of target plants were calculated . It shows the leaf methanol extract had various degrees effects depending on target plant species and each tested index . The differences were evident from the rank values of each plant species and each tested index. Table 1 indicates that barnyardgrass was more sensitive followed by weedy rice and rice. Indeed, for half inhibition of biomass, root and shoot elongation of rice, the concentration of methanol extracts must be doubled to have the same effect registered on barnyardgrass. The present observations revealed that the photosynthetic pigments exhibited a significant decline in rice, barnyardgrass and weedy rice seedlings. Reduction of chlorophyll and carotenoid content in the presence of allelopathic plant extracts has been reported by Sodaeizadeh et al.  and Lokajová et al. . Zhou and Yu  opined that the inhibition of chlorophyll accumulation by allelochemicals might be due to the blockage of the biosynthetic pathway of chlorophyll or stimulation of chlorophyll degradation or both processes. A greater reduction was observed in chlorophyll a content compared to chlorophyll b in all target plants. These results are in accordance with the findings of Elisante et al.  and Lokajová et al. . Yang et al.  opined that probably two different enzymes located in the chloroplast of higher plants. Siddique and Ismail  demonstrated that chlorophyll content is closely related to plant dry matter production, reduction in leaf chlorophyll content would cause decreased photosynthesis and hence total plant growth.

Therefore, the present observations revealed that methanol extract may diminish the accumulation of dry matter in tested species through the decrease of chlorophyll levels and photosynthesis rate . Identification of the inhibitor components is important to determine the phytotoxic interference mechanism on receiver plants. The UFLC analyses showed T. tuberculata leaves are capable of producing numbers of compounds into the environment which may affect the growth of other plants in the vicinity. Verdeguer et al.  suggested that the suppressive effect of the extracts is dependent on the chemical composition. The chemical characterization of different fractions of Calamintha nepeta showed the magnitude of allelopathic activity depends on the presence of five phenolics  in varied amounts . Patterson  reported vanillic, ferulic, gallic and p-coumaric acids inhibited chlorophyll content, stomatal conductance, net photosynthetic rate and assimilation rate of soybean. The pure compound bioassay revealed that the components and their combination were biologically active by inhibiting germination and seedling growth of barnyardgrass. It shows that they are probably responsible for the allelopathic activity of T. tuberculata. Benzoic acid was found in the leaf methanol extract at the highest concentration , while the amount of chlorogenic acid was 427.8 mg kg–1 DW. On the other hand, benzoic acid and chlorogenic acid showed the highest and weakest allelopathic activities compared to the others . Therefore, the magnitude of inhibitory activity of the methanol leaf extract can be explained by the level of benzoic acid as most effective compound and orientin as one of the weakest allelopathic compounds. Therefore, based on the concentration and growth inhibitory effect of each tested compound, it seems that benzoic acid, among the other tested compounds, is the most effective in T. tuberculata allelopathic activity. Kato-Noguchi et al.  believed that benzoic acid could be a main contributor to allelopathic activity of Ginkgo biloba leaf extract. The mixture of all tested compounds was moderate inhibitory when compared with the allelopathic activity of individual compounds .

It may indicate that interactions of concentration and combination of compounds determine the allelopathic activity of a mixture. Biological activity of collective compounds depends on several factors such as: individual chemical structure, antagonistic, agonistic,mobile vertical farm additive, sequential, or synergistic effects . Both antagonism and synergism were observed when three flavonoids isolated from Derris urucu combined in pairs, applied on the seed germination and development of root and hypocotyl of Mimosa pudica . In contrast, Al Harun et al.  observed litter-mediated soil leachate of Chrysanthemoides monilifera had stronger phytotoxicity effects against Isotoma axillaris relative to individual phenolic compounds due to synergistic contributions of other unidentified compounds. Therefore, it might be the allelopathy activity of T. tuberculata leaves depending on the concentration, combination and inhibitory activity of its bioactive compounds. Phenolic and flavonoid compounds have been found in a wide range of plants and often exert allelopathic responses . Sludge produced during the municipal wastewater treatment process contains numerous nutrients such as nitrogen, phosphorus and small amounts of potassium , organic matter  and other trace elements. Therefore, this sludge could be used as a nutritional amendment to improve soil physical characteristics, fertility, microbial diversity and enzyme activity. It has been shown that the agricultural utilization of sludge is one of the most economical and effective options for sludge treatment . However, sludge is also rich in organic matter, pathogenic bacteria, parasitic eggs, heavy metals and other toxic and harmful substances that was difficult to degrade biologically . Such sludge could not be used in raw or dried form for agricultural application and composting, leading to the secondary environmental pollution caused by improper disposal of sludge . Ozone is a functional disinfectant, oxidizer, and reducer when used to treat sludge . Furthermore, the sludge mineralization process during ozone treatments could decompose organic matter into inorganic materials . Significant research efforts have been devoted to the application of ozone in sludge treatment , which promote cell cracking and reduce the number of excess sludge, thus reducing the waste disposal of activated sludge  and decreasing the chemical oxygen demand , chromaticity, and turbidity in conventional activated sludge process with ozone treating partial backflow sludge.

Ozone has also been shown to be a superior disinfectant compared to conventional disinfectants such as chlorine and its derivatives . When microorganisms were exposed to ozone, their cell walls were destroyed and internal organic matter was oxidized. The ozone could directly disrupt viral and bacterial metabolism and reproduction by breaking down macromolecule polymers. Most studies have focused on ozone treatments on sewage sludge derived from the municipal wastewater treatment plants where wastewater was collected from industrial and domestic sources . Very little research has been focused on the agricultural utilization of solely domestic sludge, which avoids the environmental pollution introduced by industrial sludge. The sludge was collected from the digester of the Jizhuangzi Wastewater Treatment Plant in Tianjin, China. The water content of the sludge was 99.5%. The sewage treatment plant only deals with domestic sewage, and the sludge is not doped with industrial sources. After testing, the selected experimental sludge met the specific requirements of the control standard of pollutants in agricultural utilization of sludge . The sludge contains numerous nutrients, meeting the plant growth to a certain extent. Through ozone treatment, the sludge organic pollutants and micro pollutants which were detrimental to both plants and human health were degraded . The sludge represented a viable alternative nutrient and water source for plants. Therefore, hydroponics was used in this experiment, which was a widely used technique for growing plants without soil, and could eliminate all the problems associated with soil culture.

When using blended fertilizers , Js were found to be lower for K+ and NO3− , but higher for NH4+ . That could be explained by a lower overall diffusivity due to the presence of two or more ions species in the DS, which also leads to lower reverse diffusion . It was also shown that the migration of NH4+ was favored compared to K+ as counter ion of NO3− . The smaller hydrated radius of NH4+ compared with K+  explains the higher diffusion of NH4+ when salts were tested together in DS . In addition, the reverse fluxes of the cations were facilitated by the negatively charged membrane surface, which enhances the cation diffusion to FS.This is because the DS concentration gradient drives the passage of both water and salts in opposite directions across the membrane, and higher fertilizer losses could be expected with higher water fluxes . However, it is hypothesized that if tests at 0.50 M would have been closer to osmotic equilibrium, as it happened with tests with 0.05 M, the corresponding losses would have been higher. This increase in losses is due to the longer time of contact between the solutions, which is required to achieve the targeted dilution rate but also enables more solute transport across the membrane. This issue is illustrated by the higher reverse salt fluxes in tests at 0.50 M than in the baseline tests at 0.05 M . These results highlight the importance of the setup conditions, as FO performance cannot only be valuated in terms of water and solute fluxes, but also in solute losses. Hence, it seems reasonable to look for conditions generating lower ion dilution instead of just considering the Js values. Since Js increases with DS concentrations , solute losses are expected to also be higher at higher mass dilution. For example, in tests with KNO3 alone, the initial DS concentration of 0.50 M lead to losses of around 37%, while 0.05 M KNO3 lead to losses of up to 70% for both potassium and nitrate to FS. However, looking at reverse fluxes, dutch bucket for tomatoes the Js was 1.5 mmol m− 2h− 1 for both K+ and NO3+ at 0.05 M, but 42.2 and 44.2 mmol m− 2h− 1 for K+ and NO3− respectively, for tests at 0.50 M of KNO3 in the initial DS .

This fact is also clear when looking at the specific reverse salt fluxes , since they indicate the mass of each ion passing through the membrane to the FS per each liter of water recovered. The SRSF of all ions except for phosphate were much higher in tests with high DS concentration than in those with low DS concentration . Therefore, since reverse fluxes and consequent nutrient losses increased with increasing DS concentration, tests with higher initial concentration can be expected to result in higher losses if the process had continued to run until osmotic equilibrium was reached. To reach adequate NPK concentrations to nourish hydroponic systems, five KNO3/DAP mixes were tested  and compared to the individual fertilizers. All the mixes achieved the target draw dilution  with initial Jw between 5.5 and 8.7 L m− 2 .h− 1 and without reaching osmotic equilibrium , which attests the possibility of achieving a higher dilution rate and the benefits of using blended fertilizers instead of individual ones. However, the low average Jw  might limit the feasibility of further diluting the DS, resulting in very large filtration units when the system is operated near osmotic equilibrium, as already mentioned in the previous section. Similar nutrient losses were experienced for ammonium and potassium , and they varied analogously along the mixes, , due to the similar nature of both cations. In line with previous tests, phosphate losses were almost negligible , while nitrate losses were the highest with up to 47%. Such high losses have not been reported before in the literature. However, the other studies addressing this topic  were carried out using higher DS concentrations with much faster filtration kinetics. This may indicate that the migration of draw solutes through the FO membrane in the current study was promoted by the long filtration time due to the operation at low water flux.

Former studies probably underestimated potential losses of the FDFO. Nutrient losses observed in this study might be too large in some cases, depending on the composition of salts and their concentrations in the DS. Additionally, the presence of nitrate or phosphate in the FS could make its management and discharge more complex, as these elements can cause eutrophication . This issue could be mitigated by treating the FS prior to discharge, as proposed by Wang et al. , who added micro-algae in their FS that could benefit from the nutrient fluxes from DS to FS. However, we consider that this approach would increase the costs and make the process more complex. Zou et al.  pointed out the need of system optimization, membrane development, long term evaluation, as well as other cost-effective strategies to reduce the reverse salt fluxes in FO, which is crucial for a proper FO operation. In the same line, our results point out the need to evaluate nutrient losses when regard to DS dilution close to real conditions of the FDFO application. Designing the system to limit the filtration time like operating in counter-current mode, applying hydraulic pressure, or increasing membrane surface area may help to avoid such important losses of DS ions. In any case, it is essential to develop membranes with higher reverse flux selectivity, as well as finding suitable FS-DS combinations for a more efficient FDFO process. The tests also showed that using DAP individually as DS led to lower ammonium losses compared to using blended DAP and KNO3. On the contrary, KNO3 alone caused greater nitrate and potassium losses . Therefore, for practical applications, phosphate fertilizers are a promising DS to be used individually, due to their low Js, while nitrate fertilizers should be used in combination with other salts to reduce their nutrient losses. These results point out the importance of choosing not only the right salts for the FDFO process but also the right combination. Nitrogen, phosphorous and potassium  concentrations at the end of the tests are presented in Fig. 3. Values within the target NPK concentration ranges  were achieved with MIX 5  without the need for further dilution of DS or changes in nutrient concentrations.

Other mixes achieved the targeted concentrations for some of the nutrients. MIX 2 and MIX 4 achieved 117 and 158 mg.L− 1 of N and 179 and 315 mg.L− 1 for K, respectively, while MIX 3 achieved 60 mg.L− 1 of P. However, special attention must be paid to P concentration since values higher than 62 mg.L− 1 could be toxic for the plants . Thus MIX 2 and 4, although with acceptable concentrations of N and K, are not considered suitable nutrient solutions due to the concentrations of P well above the reported toxicity level. It should be noted that some of the tested mixes could still serve as nutrient solutions for some growing stages, despite having a N and K content below the previously mentioned target . For example, the standard Hoagland solution , which is commonly used in hydroponic experiments, has been applied at half strength in some studies . In contrast to our results, the final DS concentrations obtained in previous studies  ware way higher than those required for plant growth, so they pointed out the need for substantial dilution prior to application. Other studies showed the potential of FDFO systems to achieve an adequate DS dilution for direct application for plants, but assuming an unlimited FS volume , which implies not considering the salinity buildup in the FS; or by applying extra pressure in order to increase the nutrient dilution in DS . Results of our study indicate that solutions with an appropriate nutrient content for hydroponics can be achieved with FO , showing promising applications of FDFO. MIX 5 setup could be used in small-scale applications,such as homes or small buildings, considering that only 2 L of concentrated fertilizer solution in DS could extract up to 30 L of FS, to be applied directly in small hydroponic systems within the same building.

Extrapolating from the volume of water extracted in the experimental setup , 757 L could be extracted for kg of fertilizer using MIX 5 . This is a much higher volume than indicated by Phuntsho et al. , who tested the performance of FDFO with 9 fertilizer salts and estimated that 1 kg of fertilizer  could extract up to 29 L water from seawater . However, nutrient losses should be considered in the balances, and low water fluxes could compromise the process. As pointed out by Suwaileh et al.  in a recent review about FO, further work is required regarding membrane development in order to increase the efficiency of the FO process. This study confirms that more selective membranes are required to increase the efficiency of FDFO process, by lowering nutrient losses, especially for monovalent ions. To evaluate the impact of feed salinity, tests with 6.5 mM of saline solutions in the FS and with 0.05 M of blended or individual fertilizer salts in DS were carried out and compared with baseline tests  . The initial EC for FS ranged between 730 and 1448 μS cm− 1 and osmotic equilibrium was achieved in all tests . A good fit of the molar balance for anions and cations in both FS and DS was observed , blueberry grow pot showing that even with more ions present in the FS, ions were passing through the membrane to equilibrate the charges . Results show that all tests with salts in FS presented lower initial water fluxes compared with the tests with DI water in FS , which is in accordance with other studies . This is explained by the osmotic pressure present in FS at initial time, related to salt presence , which decreases the net osmotic pressure and thus Jw . Although Jw were similar along the tests with salts in FS, tests with NaCl in FS showed the best performance in terms of water fluxes and draw dilution .

A higher difference in osmotic pressures between FS and DS was observed when having NaCl in FS, as its osmotic pressure is lower than those of the other FS salts at the same molar concentration. The results from all tests with salts in FS were far from the target mass dilution and extracted liters and followed the trend of MIX 1>DAP > KNO3 , as it was observed in tests with only DI in FS. All tests performed with KNO3 alone  ended up with less DS volume than at the beginning , and with the FS having an even higher EC than the DS . These results show that the presence of salts in FS limits the FDFO performance and application, as the target draw dilution was not achieved in any case. Fig. 5 shows the forward solute fluxes  as well as the reverse solute fluxes  for tests with fertilizers alone and blended. For DAP ions, phosphorous reverse fluxes were not influenced by FS salinity and remained minimal in all tests, including tests with just DI water in FS . Ammonium Js were slightly higher for blended fertilizers compared to using DAP alone  and showed a similar trend as when using DI water in FS. Results in Fig. 5a and c also show that the presence of salts in the FS had a strong impact on favoring ammonium passage through the membrane, because of the resulting higher Js. Ammonium reverse fluxes were up to one order of magnitude higher with salts in FS than with just DI water, the highest being 3.1 mmol m− 2 .h− 1 for tests with Na2SO4 in FS. This effect was less strong in tests with Mg2+ due to its better rejection by the membrane . For KNO3 ions, nitrate and potassium reverse fluxes were similar for tests with and without salts in FS  when tested with KNO3 alone.

Although the experiments were conducted under hydroponic conditions without rice paddy soil which have thesorption capacities to 2,4-DBP and 2,4-DBA to limit their direct volatilization and bioavailability, it still could be concluded that phytovolatilization was an effective process for the exchange of 2,4-DBP and 2,4-DBA between contaminated sites and the air phase. Moreover, phytovolatilization as well as total volatilization of 2,4-DBP and 2,4- DBA in planted treatments were closely related to the exposure time, biomass and growing status of the rice plants. With regarded to the long term exposure of those two contaminants, the biomass of rice seedlings gradually increased with growth, and the phytovolatilization would be significantly enhanced and become more important in the long distance transportation of 2,4-DBP and 2,4-DBA.Methylation and demethylation metabolites of 2,4-DBP and 2,4- DBA were detected in their individual exposure systems. Xenobiotic contaminants are generally metabolized under the biocatalysis of plant enzymes after being taken up by plant. O-methyltransferase and demethyltransferase have been extensively observed and mediate the methylation and demethylation of contaminants in high plants. None of daughter 2,4-DBP and 2,4-DBA were detected in both planted blank controls without PUF and the unplanted treatments with PUF, con- fifirming that methylation of 2,4-DBP and demethylation of 2,4-DBA were all mediated by rice plants, not chemical reactions in exposure systems. Similar interconversion was also reported between OH-PCBs and MeO-PCBs within rice plants . For the 2,4-DBP exposure system without PUF, the methylation product was rapidly formed in rice roots after 6 h exposure , and then constantly decreased. The methylation product could also be detected in both leaf and leaf sheath.

The concentration was firstly increased and then decreased in leaf sheath  but always increased in rice leaf during the exposure . The total amount of methylation metabolite increased during 6–48 h,nft growing system and then decreased from 155.3 ng  to 97.78 ng . Majority of methylation metabolite was distributed in the culture solution, accounting for 68.6–83.8% of the total amount of metabolite. While for exposure of 2,4-DBA, as seen from Fig. 4B, no demethylation metabolite was detected in exposure solution. The demethylation product was extensively detected in various rice tissues. The concentrations of demethylation product in rice leaf, root and leaf sheath and the total concentration in whole rice plant were all constantly increased during the exposure period, and reached to 16.34, 42.66, 61.78 and 39.53 mg kg−1 at the end of exposure. A majority of the total demethylation metabolite was accumulated in rice roots before 72 h, while demethylation product became dominant in the leaf sheath and leaf at 120 h. Likewise, the formed metabolites, 2,4-DBP and 2,4-DBA, were phytovolatilized from rice seedlings into the headspace. And the methylation metabolite  which observed in hydroponic solution could also be directly volatilized into the headspace. Herein, all of metabolites in exposure solution, plant tissues and those volatilized into the headspace were included to evaluate the transformation ratio  between 2,4-DBP and 2,4-DBA in the sealed system. The total volatilized methylation and demethylation metabolites detected in air phases of exposure systems with PUF were 126.8 ng and 218.5 ng . Including these volatilized metabolites, the demethylation ratio of 2,4- DBA was 12.0%, that is 32.0 times higher than the methylation ratio of 2,4-DBP  after exposure for 120 h . The demethylation process was obviously faster than the methylation process. A similar phenomenon was previously reported on the interconversion of OH– and MeO-PCBs in rice seedlings, in which the demethylation process was 7.70–18.2 times greater than methylation . Results illustrate that bromoanisoles are readily demethylated within rice plants and subsequently released as bromophenols into environment. Unfortunately, bromophenols are precursors to form more lipophilic and bioactive products  in rice plants, such as hydroxylated polybrominated diphenyl ethers  and polybrominated dibenzo-p-dioxins/dibenzofurans.

Therefore, the occurrence of bromophenols and bromoanisoles needs to be considered as a potential risk factor in consumption of food plants.The contributions of inter conversion processes to the volatilizationof parent contaminants were further quantitatively evaluated and summarized in Fig. 5C and D. According to the equimolar reaction between parent and daughter compounds, the volatilized methylation and demethylation metabolites were transformed from 0.48 and 0.87 nmol of parent 2,4-DBP and 2,4-DBA accounting for 0.07% and 0.13% of their initial amounts , respectively. These amounts enhanced 4.95% and 2.69% of total volatilization mass , and 12.1% and 36.9% of phytovolatilization mass of parent 2,4-DBP and 2,4-DBA, respectively . Obviously, methylation and demethylation processes served as important strategies to volatilize their corresponding parent chemicals out of intact plants. Furthermore, the volatilization of metabolites was also an important process to reduce bioaccumulation of parent chemical in plants. These observations indicate that interconversion provides another pathway to volatilize phenolic chemicals and methyl-phenols pollutants from contaminated sites into the air phase. After calculation the mass balance of 2,4-DBP and 2,4-DBA in the treatments and exposure systems with PUF , it was found that the total recovered 2,4-DBP and 2,4-DBA in the unplanted treatments with PUF were only 88.5% and 87.0%, respectively. The photodegradation of target chemicals was avoided in incubation system by wrapping aluminum foil outside the brown glass reactors, and microbial transformation  was minimized by pre-autoclaving the solutions and glass bottles before exposure experiment. Therefore, we inferred that those unrecovered target compounds were resulted from the incompletely sampling of the volatilized chemicals in air phase. Although PUFs were placed in the systems, the analytes detected on PUFs only represent a fraction of the volatilized amounts. Namely, the total volatilization and the contribution of the interconversion to the volatilization are all underestimated. In planted exposure systems with PUF, the recoveries of 2,4-DBP  and 2,4- DBA  were significantly lower than those of unplanted treatments with PUF. Though non-sterilized rice plants were used for exposure experiments, our former study has shown that the microbial transformation  of 2,2′,4,4′-Tetrabromodiphenyl Ether  was far lower than pumpkin plant in the similar cultivation and exposure conditions . Thus, the significant differences between unplanted and planted treatments were inferred with the occurrence of other metabolic pathways or bound residues in rice plants .Lettuce  is a rich source of antioxidants such as polyphenols, ascorbic acid and carotenoids .

However, antioxidant content in plants can be manipulated by growing conditions and agronomic practices . At the same time, pre-harvest factors such as fertiliser application play a major role in determining product quality and shelf life of fruits and vegetables . Shortage or excess of nitrogen can positively or negatively affect quality parameters and nutritional components of lettuce . Although information is available on the influence of nitrogen in soil  or hydroponic systems on quality, the information on post harvest quality and bioactive compounds is limited. Nitrogen application of 225 kg ha–1 showed the least postharvest decay in romaine lettuce and defects in iceberg lettuce.Higher N application rates have been reported to negatively affect post harvest quality in romaine lettuce . Furthermore, Luna et al. recommended moderate levels of N to obtain better postharvest quality in lettuce. Growing salad vegetables with a short growing cycle like lettuce in a hydroponic system is a popular practice, which has many advantages such as providing good quality and sanitary products without soil contaminants, while benefiting the environment by reducing water and nutrient usage.Weight loss, colour, texture and appearance greatly affect post harvest quality,consumer acceptance and the saleable price of fresh cut lettuce.The term appearance describes the size, shape, wholeness, presence of defects and consistency for fresh cut vegetables . Texture of vegetables is influenced by cellular turgor pressure, which determines the consistency or weight loss of the product . In current marketing practice, fresh cut lettuce are packed in modified atmosphere packaging  and maintained at low temperature storage to retain quality and nutritional components similar to the whole original product at harvest . Shelf life of fresh cut vegetables is limited due to enzymatic browning that alters the colour of the product due to the production of brown pigments . Browning in fresh cut lettuce is one of the primary causes of quality loss . Furthermore, browning of lettuce affects sensory and biochemical properties which affect consumer acceptance of the product . Phenylalanine ammonialyase activity, polyphenol content , polyphenol oxidase and peroxidase activities are involved in the production of o-quinones browning pigments . Although several chemical treatments have been recommended to control the phenolic metabolism associated with browning, there are concerns about chemical toxicity related to food safety with regards to the recommended anti-browning agents or treatments .

Agronomic practices such as nitrogen  application rates were demonstrated as a tool to manipulate the enhancement of phytochemicals . Furthermore, N application rates can influence the quality and shelf life of fresh cut products . Since N application influences cell size, nft hydroponic system number of leaves and fresh mass, N available during the growth phase of the plant can also influence post harvest quality and shelf life . It is also important to understand the impact of nitrogen application on storage loss and phytochemical properties in red and green loose leafy lettuce cultivars used for fresh cuts. Optimum N application rates to obtain desirable yields with higher bioactive compounds and quality attributes differ between varieties . However, in practice, limited standard protocols are available for lettuce breeding companies in relation to fresh cut processing on cultivar selection or recommended preharvest N application. Therefore, the aim of this study was to determine the influence of different N application rates on green and red lettuce varieties grown for fresh cuts, packed in standard MAP and held at 5°C up to 12 days on the retention of overall quality , browning enzymes PAL, polyphenol oxidase , peroxidase , browning substrates  and ascorbic acid content. Percentage weight loss increased with storage time irrespective of the rates of pre-harvest N application in Multigreen 1  and Multired 4 . However, in Multigreen 3, lower rates of N application at preharvest stage showed higher weight loss with the storage time increasing . When compared the three varieties of fresh cut lettuce, the red variety, Multired 4 revealed higher percentage of weight loss. Percentage weight loss in Multigreen 1 was lower than in Multired 4 with the storage time increasing. In green variety Multigreen 3, the weight loss  increased around 1 to 2% with preharvest application less than 90 mg L–1 N. Weight loss is a vital factor associated with the saleable weight during marketing and weight loss higher than 5 to 10% has been reported to reduce the saleable value of fresh produce due to wilting . Weight loss is associated with water loss due to transpiration  which can occur through damage of the barriers that protect against transpiration during fresh cut processing . Weight loss within the MAP is affected by transpiration and moisture condensation within the packaging . However, in this study, the response to preharvest N application rates during storage differed between the different varieties. Lower rates of N application influenced weight loss with the storage time increasing in fresh cut green lettuce Multigreen 3 .

From our findings, the percentage weight loss was higher in Multired 4 due to lower thickness of the cuticle. According to previous reports, the influence of N application rate was very low on weight loss in crisphead cultivars Marius and Saladin . Unfertilised Butterhead lettuce  planted in soil showed higher weight loss than lettuce fertilised with 100 kg ha–1 N during 12 days of storage .Leaf colour of fresh cuts of the different types of lettuce varieties varied in response to preharvest N application during storage. Colour value L* , was not significantly affected by the different N application rates in fresh cuts of variety Multigreen 1 . When the light intensity decreased, leaves became darker with the storage time increasing . Although Multigreen 3 fertilized with lower preharvest N application retained the lightness or glossiness of the leaf up to 3 days during postharvest storage , the light intensity decreased irrespective of lower or higher preharvest N application rates with the storage time increasing However, in red variety Multired 4, light intensity was not remarkably influenced by storage time or preharvest N application rates .

Thus, the node assessment is conducted for the initial and current state of the farms that utilize the open field and hydroponic system as a production method in order to analyze the water and energy consumption. Two scenarios are conducted in order to investigate the change within energy and water consumption for the four farms. Agrico Agricultural Development, Global Farm and Al-Safwa Farm represent the first scenario that analyzes the water and energy consumption using the hydroponic greenhouse to produce tomato and groundwater for their water requirements. The second scenario is Al- Sulaiteen Agricultural & Industrial Complex  that uses RO to desalinate and treat brackish water within the hydroponic greenhouse. Along with the available growing method, the effect of seasonality on tomato farms is also considered. All meteorological data are extracted from the worldwide weather database for 13 weather stations that are mapped on Qatar map using ArcGIS. The following sections describe the required models and equations used in assessing energy and water consumption of the farming industry with the assumption that they are tomato farms. All equations utilized in this case study are illustrated below in Table 4.To estimate the overall water consumed by a tomato farm, two growing methods along with evapotranspiration factor per tomato crop are considered in this case study. Where the ET factor describes the amount of water lost from plant leaves  to the atmosphere, in addition to the amount that evaporates mostly from the soil surface after rain or irrigation . The amount of water lost through evapotranspiration is calculated using the Penman-Monteith equation, as recommended by the FAO .

The Penman-Monteith equation identified a set of weather parameters, specifically temperature , relative humidity ,ebb flow table mean daily radiation , wind speed  and soil density , where it produces a reference evapotranspiration rate  that can be used to compare water loss between hydroponic greenhouse and open field agriculture for various crops in various season. The atmospheric parameters are obtained from a weather database. Yet, in the full Penman-Monteith equation, other variables are assumed to be either as constant or negligible. The term of mean net daily radiation  in the Penman-Monteith equation describes the light-diffusing properties of the greenhouse covering. To describe this light diffusion, Rn term in the open-air equation is set to 1, while in the greenhouse equation it is set to a light diffusion rate for a specific greenhouse that is covered by a Polyethylene film. Hence, greenhouse covers that are rated with 88% light diffusion will have a value of 0.88 for the Rn term . It is important to note that the Rn values for open fields and greenhouses do not fully elaborate the solar radiation component. Furthermore, when soil heat density  is compared to  it will be relatively small and can be approximated at zero when the ground is covered in vegetation . Once the water savings in the open field and hydroponic greenhouse are estimated using the Penman-Monteith equation, the effect of crop characteristics that distinguish a typical field crop from the grass reference is implemented.To calculate the energy consumed by the tomato farms, two growing methods in addition to the source of water requirement are considered, as it has an impact on the overall energy consumption within a farm. Table 5 illustrates the main energy factors that are compiled from Jadidi and Sabouhi  study, in order to represent the total energy consumed in open field production. However, in the case of hydroponic greenhouse production, four energy components are included in the energy analysis: energy consumed by reverse osmosis plant; the use of electricity in the pumping of groundwater ; energy used during supplemental lighting and cooling loads.

The first component in the hydroponic greenhouse production is the annual energy consumption of brackish desalination plants, which is calculated using mass balance equations along with the specific energy consumption , the capacity and availability of the desalination plants . The SEC of desalinated water is one of the most critical factors characterizing the performance of the water supply . The SEC of various desalination processes and technologies is established in previous literature indicating a value with a range of 0.5 – 3 kWh/m3 for brackish water reverse osmosis . In addition, studies demonstrated that when specific data is not available for plant availability , then Pa is set as 90% . The second energy component is the energy consumed by electricity when pumping groundwater for irrigation. The shallow and renewable groundwater system in Qatar is composed mainly of two major aquifers, the northern aquifer and the southern aquifer, where the northern groundwater aquifer is the most important to Qatar’s developing agricultural sector. It is shallow with a range of 10–40 m deep and covers approximately 19% of Qatar’s land area. The southern aquifer and other secondary basins are smaller in size with diminished water quality and higher salinity levels. The agricultural activities in Qatar are very limited and are concentrated in the northern parts of the country . In this study, all the farms are located in the Northern Basin . Hence, the maximum depth of northern groundwater aquifer is used to determine the total energy consumed by pumping irrigation water. An irrigation pumping plant has three main components: power unit, pump and pump drive. In which, the overall pumping plant efficiency is a combination of the efficiencies of each separate component. It is assumed that all farms require energy to pump irrigation water and that all of the pumps use electricity as a power source, which has an average of 72–77% pumping efficiency . Hence, the groundwater aquifer depth , and its mass  along with pumping efficiency are considered in the energy calculation. Then, the average water requirement for tomato crop per season is used in conjunction with the estimated yield values for a tomato to estimate the energy use related to pumping in units of kJ/kg/y.

The third energy component is related to the energy used in supplemental artificial lighting, or what is known as energy per mole of photons , which is determined by assuming a 24- h photoperiod and a recommended daily Photosynthetically Active Radiation  of 0.4–0.5 mol/m2/s of both natural and supplemental light for optimal tomato production . It is also assumed that half of the required radiation could be obtained from natural lighting, and a wavelength of 400 – 700 nm. The resulting value is then used in conjunction with the estimated yield values for hydroponic greenhouse growing tomato to calculate the energy demand from supplemental lighting in units of kJ/kg/y. The energy use related to cooling loads is estimated by calculating the design heat load of a greenhouse in Qatar. The standard heat transfer equation is used to determine the fourth energy component. For the purpose of estimations, 20 °C is used as the set point temperature of the greenhouse, as this is the optimal temperature for tomato cultivation . Polyethylene is one of the most common materials used in greenhouse construction, and the overall heat transfer coefficient for this material is used in the calculations. For comparison purposes, all greenhouses are assumed to be closet to Al Khor weather station, in the North of Qatar; therefore, the average monthly temperatures for this region are used to estimate the temperatures external to the greenhouse. The final energy estimates are used in conjunction with the estimated yield values for a tomato to create a metric, in units of kJ/kg/y. Together, the estimates of energy use related to the supplemental lighting, water pumps, and cooling loads are combined to produce an overall estimate of energy use for tomato production in Qatar in units of kJ/kg/y .The methodology presented in this study is applied to a case study considering open-field agriculture, conventional greenhouses, and hydroponic greenhouses in Qatar. Nine risk factors comprising of temperature, humidity, solar radiation, soil quality , groundwater depth, groundwater recharge rate, groundwater salinity, and groundwater pH are selected to perform the AHP method for open-field agriculture and conventional greenhouse.

However, the soil factors are eliminated from the analysis in the case of the hydroponic greenhouse as it is a soil-less growing method. Based on the analysis, weather factors such as temperature, solar radiation and humidity have the highest impact open-field agriculture, where their importance percentages that contribute to increasing the risk are 18.527%, 16.860% and 15.785% respectively. These results indicate that using an open field to grow and produce various types of crops will have a high risk of losing the harvest because of harsh weather conditions that make it much more challenging in managing the risk of weaker yield, thus many countries have shifted to a conventional greenhouse. This would allow farmers to have more environmental control over their growing crops. Temperature, humidity, irrigation and lighting process will be efficiently managed; thus, the yield can be 10–12 times higher when compared to open field cultivation, making crops much healthier and reliable. In the case of a conventional greenhouse, since the weather factors are controlled, it would have the lowest impact in comparison to groundwater factors. The results indicate that the groundwater salinity, pH and depth are equally important with a percentage of 18.12%. Similarly, Table 6 illustrates the importance weights for seven risk factors that affect hydroponic greenhouses. Since the plants in hydroponic farming are grown in a nutrient rich liquid solution instead of soil, all soil factors are eliminated and water factors have the highest impact on the farming condition, with a relative weight of 0.27503, 0.22404 and 0.13965 for groundwater salinity, groundwater pH and solar radiation respectively. The detailed results generated from the AHP method specifying the relative weights for open-field agriculture and conventional greenhouse are demonstrated in Appendix B. To validate the assumptions that are made for the AHP method, the Consistency Index  for an open field, conventional greenhouse and hydroponic greenhouse is estimated to be 0.03875, 0.09107 and 0.05632. Hence, the Consistency Ratio  is 2.7%, 6.3% and 4.3% respectively. Based on the literature, hydroponic grow table the CR should be less than 10%, therefore all assumptions validated the literature and the inconsistency within the subjective judgments is acceptable. When overlaying the locations of the four farms on the three risk maps generated from ArcGIS as illustrated in Fig. 8, the following can be deduced; when using open field and conventional greenhouse, the three farms – SAIC, Global and Al Sawfa farms – are expected to be located in low-risk areas .

However, AGRICO is situated in a high-risk area . Although both cases lead to the same result, there remains some variation in the overall risk maps, demonstrating that the amount of risk varies according to the utilized growing method. This is especially demonstrated in the third case which is the hydroponic greenhouse. Fig. 1 indicates that by transitioning the growing method from conventional greenhouse to hydroponic greenhouse, three farms will be located in an approximately high-risk area. This could be due to the fact that the quality of groundwater in these areas is poor, and thus the greenhouses fully depend on the water resource. The results are summarized in Table 7. The variability in risk maps obtained from ArcGIS is also assessed for the different seasons. Fig. 9 illustrates that there is a very slight variation between summer and winter. This is due to protected farming in conventional greenhouses, in addition to the AHP, allocating smaller weights to external weather conditions, such as temperature and humidity.Finally, the node assessments are conducted to assess the performance of the four farming industries in Qatar in terms of water and energy consumption, and by considering that all farms utilize both open-field agriculture and hydroponic greenhouse as growing methods. In addition, it is assumed that these farms have technologically evolved over time from open fields to hydroponic greenhouses for tomato production. Fig. 10 is an example of average weather data in the summer. The same maps are created for the rest of the seasons. These maps illustrate the variation within weather data  in different locations per different seasons.

Struvite forced precipitation has gained attraction since the 90’s, not only to avoid infrastructure damage but also as a P recovery technique . This process has been studied and improved in the past years making it a more efficient precipitation process . Although the production of struvite is gaining popularity, its commercial production is still scarce. The potential of P delivery of a WWTP in the form of struvite in the system where this study is located has been previously quantified by Rufí- Salís et al. , demonstrating the potential of these widespread installations to provide this ill distributed resource. In terms of application, the properties of struvite as an effective source of nutrients  for plants and its low solubility in water  make it a slow-releasing valuable fertilizer that can reduce economic costs in agriculture . However, only limited literature has explored the application of struvite in agricultural facilities. For example, Antonini et al. , Uysal et al. , Gell et al.  and Liu et al. assessed the maize performance of struvite with different characteristics and origins in different soils. In a review made by Li et al. we can see that almost all struvite trials found that vegetables grown with struvite had the same -or even improved- performance compared to controls with conventional fertilizers. Creating a closed-loop, waste-to-resource system such as that of struvite recovery within the city limits and not applying it at this scale seems contradictory within the concept of urban metabolism. In this sense, the synergy between struvite precipitation in urban WWTPs and urban agriculture seems worth exploring considering the potential of the latter to blur the lines between waste and resource within urban areas . This article aims to assess the potential of struvite precipitated in a WWTP as a fertilizer within the framework of urban metabolism.

Based on experimental and analytical results performed on a Phaseolus vulgaris crop grown in a hydroponic rooftop greenhouse, we determine the implications of fertilization with struvite in terms of yield, vertical farming racks water flows and P balances and provide recommendations to further improve the performance of this waste-to-resource fertilizer.Struvite granules were obtained from Aarhusvand A/S company from Aarhus, Denmark. This company distributes fertilizer grade struvite under the name PhosphorCare™, recovered using the Phosphogreen™ technology . This technology is based on a fluidized bed reactor that creates the specific conditions to precipitate struvite through the addition of magnesium chloride, sodium hydroxide and air. The final struvite granules have a size range of 0.5–1.5 mm. Common bean plant was chosen as the crop for this study, planting nursery plants . To apply the struvite to the plants, we considered different possibilities. Mixing it with the nutrient solution was discarded because the system could not benefit from the slow-release characteristics of struvite. Thus, we choose to directly apply the granules to the plant roots. Considering this option, we designed a system that consisted on mixing perlite with struvite inside a low-density polyethylene perforated bag with holes of no more than 1 mm diameter . At the same time, this system allows the interaction between struvite granules and roots and avoids the loss of undissolved struvite into the leachates due to draining through the perlite bag. Two different experiments were carried out: the validation test and the determination test, both of them using double growing lines with 8 substrate bags each . The validation test served as a previous experimental set-up to determine if the proposed methodology was functional and correct possible influencing variables in the experiment, such as the use of the plastic bag to retain the struvite close to the plant rhizosphere or to scale the most suitable quantities of struvite for the determination test. On the other hand the determination test was designed with the previous experience of the validation test.All other mineral fertilizers were maintained.

The initial nutrient concentration of the substrate was verified to be negligible at the beginning of the experiment through atomic spectroscopy and elemental analysis. Samples of the fertilizer solution were collected directly from the drippers placed in the perlite bags.To determine the PNS and PLIX, the respective samples were collected three times per week and externally analysed using ICP-OES atomic spectroscopy . PSI was quantified summing the amount of perlite in a specific bag with the amount of struvite that was applied, considering weights obtained by drying two struvite samples and two perlite samples at 105 °C in a furnace until reaching constant weight . PSF was quantified differently in each test. In the validation test, all 4 samples for a specific treatment were homogenized after extracting the roots, using distilled water to separate the struvite granules from the roots. After this process, two random samples were dried at 105 °C in a furnace until reaching constant weight, then grounded and digested with concentrated HNO3 in a Single Reaction Chamber microwave and externally analysed using ICP-OES atomic spectroscopy. On the other hand, in the determination test, roots were shredded, homogenized and integrated within every individual substrate sample. Then, a fraction of these samples was dried and analysed using the same method as in the validation test. PLV, and PST were determined based on the nutrient content of every plant separately. Leaves and stem were separated, sorted into paper envelopes and dried in a furnace at 65 °C until reaching constant weight , grounded and digested with concentrated HNO3 in a Single Reaction Chamber microwave before analyzing externally the concentration of P through ICP-OES atomic spectroscopy. The same methodology was applied to determine the PBN, with randomly chosen 500-gram bean samples being processed for every treatment. The measured P content of beans was multiplied by the measured rates of biomass production to estimate the rate of P accumulation in crop biomass.From September 13th to December 3rd, 2018, 10 double growing lines were used , distributing the treatments as showed in Fig. SM2 of the Supplementary material.

The aim of this experiment was to validate and keep track of different parameters of the system, like for instance, make sure that the small, perforated bag did not have negative consequences on the crop development. To do so, we split the control lines into two different treatments, VCB and VC0, using standard nutrient solution with and without the bags, respectively. Secondly, to check the correct development of bean plants with struvite in a hydroponic system, we applied different struvite amounts per plant: 5, 10, 15, 20 and 25 g corresponding to the treatments tagged as V5, V10, V15, V20 and V25, respectively. Additionally, a treatment with no struvite was tagged as V0. These amounts of struvite were based on previous experiments done with the same crop species and variety in hydroponic cultivation that accounted for P uptake . One week after the first harvest, KPO4H2 was added in the nutrient solution of struvite treatments until the end of the harvest to ensure a good nutrition to the plants during the production period, which is highly demanding in P .The production results for the control treatments VCB and VC0 showed that the perforated bag did not have any effect on the correct crop development and yield , as the yields from the different lines do not differ between them . Even though treatment VC0_1 generated more yield , it could be attributed to the fact that it was an exterior cropping line facing the border and thus received more radiation. Similarly, VCB_2 also produced more yield than its replicate although no significant differences where determined by the end of the experiment. On the other hand, treatments with struvite exerted a similar yield than the control treatments at the end of the crop. The treatment with the highest quantity of struvite had the highest production median , while the treatment with the lowest quantity of struvite had the highest mean . On the other hand, the treatment without struvite produced a really low yield . The similarities in terms of yield between all struvite treatments at the end of the cycle may be related to the addition of KPO4H2 fertilizer during the production phase. Moreover, we can see that struvite treatments produced more than the control in the first 3 harvests. This effect is similarly observed for the phenological stages . For the parameters that were quantified in different dates , side shoots , open flowers and floral buttons, we can see that the treatments with struvite not only had a correct early stage development, but also develop plant organs earlier than in control treatments.

The V0 treatment doesn’t show P results in leaves for 54 and 78 DAP because no leaves remained in the plant at the sampling time. For this same reason, there is a lack of data in beans for 78 DAP. Finally, concentration in beans for struvite treatments was similar to the one observed in the control. For all plant organs, a pattern in the accumulation of P in the plant tissue can be observed. In the first sampling all treatments show a rather low accumulation with greater content for plants with greater struvite quantities, in some cases also for the control treatments. For the second sampling, a bigger content difference can be seen with an acute increase of the V25 P content, especially for the stems and leaves. Finally, at 78 DAT, these differences between treatments even out and only treatment V0 remains significantly reduced. This last part however, does not correspond to the undissolved P in the perlite, where the P content in the substrate directly responds to the amount of struvite given, being always higher for the V25. The control treatments receive the P through irrigation making the existing content in the substrate comparably small.Figs. 3  shows the results of the accumulated yield per number of harvests, being the sixth harvest the final one before uprooting the plants. Only treatments S1  and S2.5 had lower yields than the control treatment , the first being significantly lower. On the other hand, all other treatments with 5 g of struvite or above produced more than the control treatment, demonstrating the potential of struvite to produce similar or even higher yields than with mineral fertilizer, as reported by Li et al. . As we can see in Fig. 3, it was not until the second harvest  that great differences were observed between the S1 yield and the other struvite treatments, while a decrease in S2.5 yield was observed between the 4th and 5th harvest, 57 and 64 DAP, respectively. Regarding the control treatment, vertical rack system the first harvest produced lower yield  than the S5 struvite treatment being even similar to the treatments with the lowest struvite application S1 .

This fact reinforces the idea that the application of struvite could be benefificial for early stage plant development, as the validation test showed better behaviour in struvite than in control in phenological variables. This fact could be related to the NH4+ supply by struvite, which could benefit the plant root balance when combined with nitrate supply . The fact that previous literature suggests that NH4+ supply to common bean could be harmful for plant development could be related to the amount of NH4+ supplied. Because struvite does not only enable a slow release of P but also of NH4+, reaching NH4+ accumulation to harmful levels seems improbable. In terms of distribution, yields show an asymptote behaviour among treatments, where S20 produces the highest yield and S1, the lowest . Fig. SM15 in the Supplementary material shows how treatment S10 was detected as the exception for this tendency in terms of mean production,probably related to bias parameters like shapes in the greenhouse or a nonhomogenic distribution of struvite in the perlite bag. However, boxplots represented in Fig. SM16 shows how the median of the final amount of yield harvested for S10  follows the tendency, while not presenting outliers in the distribution.Fig. SM17 of the Supplementary materials shows that the irrigated water in the control and the struvite treatments was the same , while Fig. 4 shows the accumulated P during the entire cycle in the different water streams.

Three mature L. terrestris per earthworm-present experiment  were added to each earthworm-present jar, and the experiments left for a further 25 days. Experiments were watered every 2–3 days for the duration of the experiment, and earthworms weighed before and after the experiments.Fluorescein diacetate hydrolysis  assays were carried out immediately following completion of the soil-based experiments, on fresh soil sieved to 2.8 mm using a protocol described by Shaw and Burns . The FDH assay colourimetrically measures the hydrolytic capacity of soil as a proxy for biological activity. Briefly, approximately 1 g soil was equilibrated at room temperature with sterile potassium phosphate buffer  and reacted with 0.1 mL of a 1000 μg/mL solution of fluorescein diacetate in acetone for 30 min. To terminate the reaction, the solution was vortexted with 15 mL of 2: 1 chloroform: methanol , before centrifugal separation of the layers. The top layer containing the residual fluorescein was removed, centrifuged at 16,500 g for 5 min before analysis using a PerkinElmer Lambda 25 UV–vis spectrophotometer at 490 nm. Biological activity was expressed as μg fluorescein/g dry soil/30 min, calculated using external calibration with solutions containing known concentrations of fluorescein.Earthworm-plant experimental data were analysed using SigmaPlot for Windows and Excel 2016. Outliers in the data were identified as being those values which were more than 1.5 times the interquartile range above the third quartile or below the first quartile and were removed prior to further statistical analysis. Plant biomass in the presence and absence of earthworms in both the hydroponic and soil-based experiments, and pH in the hydroponic experiments were normally distributed  and were compared using a ttest. The impacts of plants and earthworms on FDH activity in the soils and phytohormone concentration in the hydroponic experiments were determined using two-way analysis of variance  with the presence or absence of earthworms and plants as the two factors and a p value of ≤0.05 taken to indicate significance differences. Phytohormone concentration data from the hydroponic experiments were not normally distributed and could not be normalised using standard transformations. However, because we wished to determine interaction effects between earthworms and plants on FDH activity and the concentration of phytohormones, and because ANOVA is robust to violations of assumptions of normality and equal variance, we analysed our data using two-way ANOVA followed by Holm Sidak post hoc testing, rather than one-way ANOVA on ranks considering first the impact of earthworms and then the impact of plants.

Although the genome of S. alba is not well studied, comparative genome mapping has shown that much of its genome is conserved in the closely related Brassica species B. napus,stacking flower pot tower with many regions of conserved gene order in the well-studied Arabidopsis thaliana . In both A. thaliana and B. napus, a number of studies have identified genes upregulated in response to stress . Therefore, using the B. napus genome as a guide, DNA primer sequences were designed, and tested to see if these primers could amplify S. alba orthologues in both the plant shoots and roots from the hydroponic experiments. Gene expression studies were conducted using quantitative PCR  to quantify levels of expression from genes in the ABA synthesis pathway , and genes upregulated in response to abiotic stresses including NaCl, drought or cold . Full details of the genes, and primer sequences used, can be found in the Supplementary information. Prior to qPCR, primer sequences were blasted against L. terrestris and E. fetida genomes to ensure sequences were specific to S. alba.FT-ICR-MS analysis was used to survey the range of phytohormones in the hydroponic and soil samples. Typical spectra of Fractions 1 and 3 from soil extracts are shown in Fig. 2A and B; analysis of hydroponic solutions resulted in less complex spectra in which phytohormone signals could be easily identified. For Fraction 1, ABA was detected in most  of the hydroponic and soil samples analysed . IAA was detected in only very few samples, despite it being widely reported in similar analysis of plant tissues. IAA is reported to be unstable at certain light levels, pHs and temperatures ; therefore, to determine whether IAA survived our extraction procedure, a small amount of an authentic IAA standard was spiked onto soil and analysed after extraction. IAA was detected at only very low intensity, suggesting that it cannot be extracted and detected effectively using this procedure, despite the low extraction temperatures. Analysis of Fraction 3 yielded a complex spectrum, attributed to the variety of compounds present within soil. However, the high mass accuracy analysis allowed the identification of several compounds related to plant growth including Ade, iP and Z as well as benzyladenine  and other adenosine-related products . Although iP was often present at very low intensity, it was detected in all samples analysed using FT-ICR-MS. As a result of the data from the untargeted FT-ICR-MS analyses, it was decided to proceed to quantification by MRM of Z, iP, Ade and ABA, using labelled standards of each as an internal standard. Analysis of IAA was also attempted, but in most cases this compound was not detected.Addition of known amounts of isotopically labelled internal standards prior to extraction allowed the quantification of levels of the four target compounds . The amount present in each sample  was determined by comparison of the peak areas of the diagnostic product ion, with those of the relevant internal standards. This method accounts for losses during extraction and chromatography, as losses of the labelled standard are assumed to be equivalent to loss of the target compounds.

Concentrations of the target compounds were then corrected to give values per gram of dry soil or per mL of hydroponic solution.Linearity of response for all compounds was demonstrated by injecting solutions containing both isotopically labelled and authentic standards at concentrations ranging from 0.1 to 100 ng/mL. Linearity was above 0.98 for all compounds  across the concentration range except for IAA; this has been attributed to the potential decay of IAA during the extraction process. Results for IAA have thus been reported only as either detected or undetected . Having demonstrated linearity, relative response factors  were calculated for each target compound from the peak area ratio of labelled standard: target compound in a solution containing a known quantity of each. This provides a correction based on the response expected from the target compound compared to the internal standard. Repeatability was determined by three replicate injections of solutions prepared at 0.01 ng/mL and expressed as the % relative standard deviation . The repeatability varied between compounds, with that for iP being comparatively weak but with all other compounds being under 10%. Limits of detection  are defined as the lowest concentration of a compound that can be reliably detected, and limit of quantification  the lowest concentration at which the compound can be reliably quantified. Whilst several studies have reported the extraction and detection of a range of phytohormones from plants , similar analysis in other matrices, such as soil and other growth media, has been neglected. However, the ability to examine the range and concentrations of phytohormones present in soil could potentially open up routes to understanding the influences of below-ground organisms  and rhizobacteria on plant growth. In this study, it has been demonstrated that a range of phytohormones can be extracted from both soil and hydroponic growth solution and detected using untargeted FT-ICR mass spectrometry. Furthermore, three commonly occurring cytokinins  as well as ABA were then quantified by MRM. Limits of detection ranged between 0.08 ng/g fresh soil for Ade and 2 ng/g fresh soil for iP, with much lower concentrations detectable in the hydroponic solution, as a much larger volume of matrix could be processed for analysis. The LoD for soil could potentially be improved by using larger masses of soil, although this was experimentally limited by the need to rapidly remove, without heating, the larger volumes of solvent that would be used. In addition, larger volumes of soil would have increased animal usage in the experiments. The repeatability of analyses varied, with the data for iP being prone to high levels of error but those for ABA and zeatin showing particularly good repeatability and linearity . There is scope for further refining these methods; previous studies report the detection of phytohormones in plant tissue at the picomole per gram fresh weight level .

The fast analysis time  reported here offers the ability to expand the method to examine additional compounds that may be present in soil, such as gibberellins and other auxins. Although these compounds were not detected by FT-ICR-MS in our experiments, and therefore not targeted for quanification, this may not be the case for others using our approaches. Applications for the ability to detect phytohormones in plant growth media are far reaching, including developing understanding of interactions between soil microbes and plants, and understanding the potential uses of plant growth-promoting bacteria, for example in agriculture. Despite the obvious potential value in understanding these relationships in terms of advancing land management practices to maximise crop growth , little research has been previously conducted in this area.Applying the developed analytical methods, experiments were carried out aimed at investigating the impact of the presence of earthworms with and without plants on the concentrations of phytohormones within the growth media. Despite no measurable differences in plant biomass in the presence of earthworms, a significant increase in the presence of ABA was detected when earthworms were present in hydroponic solution together with plants. This suggests that there could be interactions between the earthworms and plants that cause ABA to be produced. The possibility that the presence of earthworms alters the regulation pathways of certain phytohormone-related genes was tested for by molecular biology methods. A search of the earthworm genome for genes related to ABA production revealed no matches, indicating that earthworms are unlikely to be able to directly produce ABA. We hypothesise instead that the increase in ABA we observed in our earthworm-present experiments in hydroponic solution was caused by indirect influence. Further research would need to be carried out in order to fully assess the mechanisms by which earthworms may be involved in ABA  regulation in plants. An increase in ABA production in the presence of earthworms could be attributed to a range of indirect factors including increased competition for nutrients, or the chemical modification of the solution by earthworms. As ABA is frequently associated with abiotic or biotic stress, this seems the most obvious explanation for its increased presence . Analysis of the pH of the solutions did not reveal significant differences, although this is only a very broad measure of the degree to which the earthworms may have altered the environment. It is also possible that the presence of earthworms induced changes in the expression of genes known to be involved in plant stress responses. For example, in addition to affecting plant roots through burrowing, and physiological activities such as excretions ,vertical grow rack there is some evidence that earthworms also feed on living plant root material .

A small-scale study was therefore conducted to see if genes known to be involved in stress responses, or in the biosynthesis of ABA, were upregulated in either the plant roots or plant shoots grown in hydroponic solution in the presence of earthworms. However, only a few genes were tested and in only one case  was a significant difference  seen between the presence/absence of earthworms. Whilst this may be related to the observed differences in ABA concentrations, the metabolic pathway of ABA production is complex and as such this requires further investigation . In particular, transcriptomic-based studies could be employed to assess the effect of earthworms on global plant metabolic pathways. Substantial evidence exists that earthworms benefit crop yields . However, the observations from our hydroponic experiment suggest that under some circumstances, for example in an already stressed system, earthworms may in fact cause greater levels of stress to plants, resulting in higher levels of ABA in the soil. To our knowledge, no study has effectively dismantled the effects of earthworms in systems with limited nutrient availability.

Higher genetic gain results were obtained in open-pollinated sycamore  families measured at age seven years . The estimated high values were perceived to be inflated because members of the open-pollinated families were probably more closely related than half-sibs. The genetic gain for the number of leaves at age four was recorded as zero, since this variable presented zero heritability. Therefore no gain can be accumulated concerning this variable at this age. At age five, combined selection yielded a higher value than family selection. Among all the traits, the highest genetic gain was recorded for volume index and the joint model used resulted in a more efficient selection strategy. Gain can be increased if superior genotypes are vegetatively propagated , a possibility that exists for elite mother trees ofGreek origin regarding this experimental plantation . This result indicated that the provenance-progeny experimental trial of Taxiarchis has ample amounts of genetic variation in the quantitative traits studied and sufficient gains can be foreseen. Forests in the Republic of Serbia occupy 2.252.400 ha, in percentages expressed 29% of the total area of the Republic. In the past, the woods of Serbia were be-tween 35% – 40%.As far as the oaks themselves are state-owned, the highest share is 9.7%, followed by a hybrid with 7.5%, sugar 3.6%, gluten with 2.7%, and a brass with 0.9% . Since this paper deals with the sandwich, it should be noted that the forests of sandblast  in the Republic of Serbia cover 32,238 ha . This is a solid percentage and significant area under the alkaline forests in the Republic of Serbia, which represents a great ecological and economic potential and therefore should be paid attention and adequate treatment.Concerning air study of the area, it is quite continental to moderately continental.

Climatic conditions differ depending on the altitude, relief and exposure with the average annual temperature is 11.8˚C, the period without frost of between 180 – 215 days and the period of sunshine of 2100 hours per year. The maximum and minimum temperatures ranged in their absolute amount of 41.7˚C. It can be concluded that there are large fluctuations in temperature, potted blackberry plant where the hottest months are July and August, and the coldest January and February. Furthermore, average annual rainfall is 681.8 mm, and since the winds are the most northern squally wind. The snow and wind have negative effects and fractures in cultures are noticeable as it is well known that black pine has deep roots and resistant to rippings by the wind, which is not the case with heavy snowfalls and strong winds. In order to study floristic composition and phytosociological determination of belonging study stands, phytocoenologically captured at all sample plots obtained by the method of Braun-Blanquet’s. In all experimental fields is done the following: entire dendrometric diameter measured, diameter, height, drill punched the tree, for taking increment cores and computation of gain, by Presler method. On the basis of the data, the volume and growth were calculated and measurements and analysis of growth rings were performed in the laboratory, by special instruments and related software . Observing the action of both endogenous and exogenous factors was conducted, supporting on the state of the stand and directly, based on quantitative and qualitative characteristics of volume increment. Monitoring the situation stands, covers, mostly components that are related to stand structure and which, together with the analysis increments allow for a realistic assessment of the current situation and future forecast development.The investigated area of G.J. Rogot once turned to Velika Morava  and, in most part, turned towards Lepenica. The third and least part of this gaz unit is not affected by any rivers, creeks, etc. Oak forests are in the aforementioned rubber unit “Rogot”, about the same age: 80 years. This is quite enough for the age of the tree, for dendroclimate analysis, to make the research valid. In the investigated area, oak forests will be analyzed through field experiments, depending on the hydrological impact . Hydrological influence is what they differ, given the position of the field. They are also distinguished by their solitude. A clear correlation between hydrological impact and dendrological characteristics of oak is obtained.Lepenica is the largest and most important river of the investigated area. It springs near Gledić Mountains in the village Goločelu, and flows into VelikaMorava as a left tributary. It flows from its source from the south-west towards the north-east through the Kraagujevac basin.

Lepenica’s length is 49 km. Earlier length was 60 km. and it ran parallel with Morava. However, since the great flood in 1987, it has been regulated and now flows in the direction of the northeast. Lepenica yields close to 4.5 m3 of water to the Great Moray. However, in the case of large droughts, as in 2017, this quantity is falling to 2 m3 . Lepenica runs 40 liters per second. We need to mention the local river: on the explored area: Dobrovidačka river, then: Cvetojevski potok, Botunjski potok, and others.In order to arrive at the final results, we used computer programs for the processing of climatological data . Computer programs are based on IPCC climate models, and programs are from the software package from the web site: KNMI Climate Explorer, like COFECHA programs, and ARSTAN. These programs -climate models  represent the basic tool for dynamic scaling of the results of global climate models and assessment of future climate change caused by the increase in the concentration of greenhouse gases in the earth’s atmosphere . It is the basic method for analyzing climate change in a specific research area. The results of climate models have been verified so far and have therefore enhanced confidence in this approach. Regular IPCC reports are based on the results of these models . The associated regional climate model EBU-POM, which was used, was created by linking the regional model, the atmosphere and the ocean . The acronym of this model is derived from the name of the component EBU  and POM . The reason for including the ocean component in the system is due to the fact that for long periods of time such as climatic, atmospheric and ocean interactions have to play a decisive role in defining the state of both components individually . For all climate models that are useful for obtaining desired results, it is necessary, above all, to “register users and log in” to access the system, except for access to climate data  they allow us to correlate and regression analysis, calculate possible extrema, and many other climate information that are necessary for dendrokylmatological research. The above programs, all the data we make available to them freely, are processed and a single essence  is displayed, which is graphically displayed. As we got it for the first, we got both the second and third field of view. The results and statistical data processing will be displayed through items.Children are particularly prone to orbital injury because the developing orbital bone offers little resistance. Foreign bodies most often encountered in the orbit are metallic objects and glass fragments.

Ocular complications of intraorbital foreign body include optic nerve damage, extraocular muscle palsy secondary to direct muscle trauma or nerve damage, proptosis and orbital cellulitis. It is uncommon for an orbital foreign body to present with isolated eyelid involvement. We report a rare presentation of an intraorbital foreign body presenting as a lower lid entropion in a 7-year-old boy. A 7-year-old boy presented to our eye clinic with a complaint of inward turning of the left lower lid for 3 days duration. According to the mother, the child was playing alone in the garden a week ago when part of the roof of their home suddenly broke off. She was alerted by the noise, and when she stepped into the garden, she noted a few bits of broken tiles on the ground. The child appeared to be unharmed, but complained of left eye irritation. She immediately brought him to a general practitioner, who reassured them that the patient had a normal eye examination. The left eye irritation resolved spontaneously, and the vision was good, with no double vision. However, after three days, the mother noticed that the left lower lid appeared to be inverted. When this did not resolve spontaneously, she brought her son to the ophthalmology clinic. On examination, the visual acuity in both eyes was 6/6. Extraocular movements were full in both eyes, but there was pain in the left eye on upgaze position. There was a left lower lid entropion, and palpation of the left eye revealed a deeply embedded foreign body in the lower lid . The entropion was attributed to the mechanical effect of the foreign body. There were no signs of a conjunctival or corneal laceration wound . Both anterior segments and fundi were normal. The intraocular pressure was 14 mmHg in both eyes. Skull X-ray showed a left lower lid radio-opaque foreign body. Computed tomography  scan of the orbits revealed a well-defined foreign object with sharp edges in the lower orbit . The object was projecting in an infero-oblique direction, fracturing and traversing the anterosuperior wall of the left maxillary sinus . It measured 1.4 cm × 0.8 cm in size. The left eye foreign body was removed via a transconjunctival approach. The object was embedded in the inferior lower fornix,tall pot stand extending until the inferior orbital rim. It was identified as a stone, which was removed in one piece . It measured 1.3 cm × 0.7 cm.

The patient was assessed further by the oral-maxillary-facial team, who decided that no internal fixation was required for the inferior orbital rim fracture. The patient was started on a five-day-course of intravenous amoxicillin clavulanate and metronidazole, which was continued orally for another week. Topical ceftazidime was also instilled 4 hourly. On post-operative day one, there was slight oedema of the left lower lid region, which resolved on subsequent follow-up. He was last seen 2 weeks post op and his wound had completely healed. He was planned for an entropion repair after 6 weeks.Intraorbital foreign bodies usually occur secondary to high velocity injuries, such as in gunshot or industrial accidents. However, in cases where the trauma is deemed trivial, patients may not give a clear history of a penetrating injury. In this case, as the patient was playing unsupervised, the mother could not provide a clear history of trauma, and even the examination by a general medical practitioner failed to reveal the intraorbital foreign body. We postulate that the most likely mechanism of injury was that upon hearing the sound of the roof breaking, or perhaps alerted by bits of the falling debris, patient looked upwards, and a piece of the debris pierced the tarsal conjunctiva to bury itself in the inferior orbital rim. Early clues giving rise to a clinical suspicion of an intraorbital foreign body include eyeball displacement, persistent inflammation and conjunctival chemosis. In cases where there is an associated fracture of the orbital floor, limitation of ocular movement with diplopia usually occurs, mainly due to inferior rectus entrapment and rarely, from nerve injury or muscle contusion. In cases of retained organic foreign bodies, decay of the material over time with superimposed infection almost always results in abscess formation. In cases where the foreign object is relatively inert, such as in this case,patients may be completely asymptomatic. It is likely that the initial discomfort experienced by the patient stemmed from the associated conjunctival wound. The latter was likely a self-sealing wound, which partially explains how the diagnosis was missed at the first presentation. A recent review highlights the importance of detailed examination of the conjunctiva and fornices as intraorbital foreign bodies usually penetrate the conjunctiva without causing eyelid laceration. Imaging is invaluable in the evaluation of the globe and orbital integrity post trauma. Although X-ray is the initial imaging in cases of suspected intraorbital foreign bodies, computed tomography  scan is the investigation of choice when there is a definitive history of penetrating injury, as even in cases of a radio-opaque object clearly visualised on X-ray, CT assists in planning of the surgical approach. It also enables to estimate the integrity of the orbital tissue and bone surrounding the foreign body.

This has already been put forward in , without showing any evidence, however. As expected the main effects of PBZ and GA3 were observed in shortening and elongation of seedlings stems, respectively. The body of evidences leads to the conclusion that GA deficiency caused by PBZ  resulted in a pronounced stem growth decrease and hence as stem-root system dry mass did not vary to a large extent, assimilates are directed towards the roots. Unequivocally, in the present investigation, not at all affected by both GA3 and PBZ  were mean leaf and internode number, total plant dry mass, leaf dry mass per plant, and the stem-root system dry mass per plant as a whole. Tea  is a perennial shrub, which is grown commercially in about 30 countries. The major tea exporting countries of the world are Kenya, China, India, Indonesia and Sri Lanka. It is one of the most popular beverages in the world and is a rich source of some essential dietary metals and metal-binding polyphenols. Tea contains several minerals, trace elements, antioxidant and therefore considered good healthy beverages. The environmental temperature of 18˚C – 30˚C and soil temperature between 20˚C – 25˚C are optimal for the plant growth. High annual rain fall and air humidity are other important weather requirement for cultivation of tea crops. Tea is grown in a verity of soil type such as alluvial soils, drained peat, sedimenttary from gneiss and granite, and soils derived from volcanic ash. Its growth is favoured in acidic conditions, with pH values ranging from 4.5 to 6.0. Although it will grow in soil pH as low as 4.0, soil pH higher than 5.6 is considered unsuitable without pH adjustment of the soil.A various reports have discussed the potential health implication of metal in tea, particularly tea bushes is known to accumulate trace metals. Very recent research finding indicated the positive and negative effect of drinking tea on the health. It was pointed out that some of the beneficial effects of drinking tea are prevention of chronic and cardiovascular disease, cancer, antioxidative detoxification and removal of cadmium in administered rats. Tea contents both essential and toxic elements such as Ca, Na, K, Mg and Mn are present at mg/g level,fodder system for sale whereas elements such as Cr, Fe, Co, Ni, Cu, Zn and Cd are present at level of few µg/L.

It is known that the source of heavy metal contamination in tea could be cause by uptake of metal from soil. The soil is contaminated by different type and amount of chemical substances added for improving the tea production. As a result, soil pollution by heavy metals becoming conspicuous. Therefore, control of tea quality and soil management has become very imperative. Tea plants  can take up metals from the soil, and inevitably, proportion will be transported to the tea leaves, which are prepared one of the most popular beverages in the world. Therefore the level of heavy metal concentration in tea leaves play a major role to assess the quality of tea due to stringent regulatory limit set by the government agencies. Thus this study is, proposed to assess the uptake level of essential and toxic metals from soil to tea leaves and infusion of the tea garden plot that are grown in Institute farm.tal farm was selected for this investigation. The general information of plot of tea garden including area, age, was summarized in Figure 1. Nine sampling spots were chosen for collection of soil, fresh tea leaves in descending order. Soil samples at depth of 20 cm and corresponding tea bush leaves samples were collected. Tea leaves samples were dried at room temperature, and rolled to prepared made tea in institute tea factory. Soil samples were dry at room temperature and then passed through a 2 mm nylon sieve. For organic matter determination the soil sample was ground more finely and passed through 0.5 mm sieve. The collected soil samples were subjected to measurement of fundamental chemical properties  and heavy metal concentrations using Atomic Absorption Spectrophotometer. The pH  of soil showed acidic in nature, and it is mentioned that acidification is advanced in the tea garden soils. Soils with low pH contain high amounts of Fe and Al oxides. Lower content of organic matter may be due to less application of organic matter in the tea experimental farm. The nitrogen was the second most abundantly found in tea garden after potash and phosphorus. Fortunately, the concentration of toxic heavy metals Pb, Ni and Cd in tea leaves, made tea and its infusions were too low or not detected in this study. However, there are reports of the availability of these metals at lower levels in different blended tea leaves. Al-Qud in 2003 has reported level of Cd within a range of nil – 0.18 mg/kg. Shen et al. 2008 reported that cadmium content in the tea infusion were in the ranged of 0.005 – 0.006 mg/kg. On the other hand, level of Pb was reported within a range of 0.03 – 14.84 mg/kg. According to Lagerwerff Pb emitted in exhaust fumes of petrol combustion as minute particles of inorganic Pb compounds accounts for about 80% of the total lead in the atmosphere.

About 50% of this falls somewhere within the region of 100 m from the road, rendering Pb concentration to be higher in roadside soils and in its vegetation. Some essential elements such as Mn, Zn, Fe and Cu were studied, which play vital to humans for growth, metabolism, and hormones balancing. The Mn content in the tea infusion was reported in the ranged of 0.56 – 7.9 mg/kg after 5 min extraction time. Low level of phosphorus plays significant role in the metal uptake of Mn, Cu and Ni especially. Several studies demonstrated that plants take up metal by the phosphate pathway due to their chemical similarity. Similar trend was observed in case of manganese it increases from location U1 to L1 as phosphorus decreases. Mn was predominant heavy metal found in the fresh tea leaves followed by the Fe. Similar findings have been reported that Mn was found in highest concentration followed by Fe in fresh leaves of tea. AL-Qud 2003 suggested the ability of the tea plant to accumulate heavy metals particularly Mn, Fe and Zn, to a lesser extent Cu and reported higher levels of Mn in the tea plant within the range of 390 – 900 mg/kg. At present, the main transaction mode in China is competitor deal. Higher transaction costs and lower efficiency, insufficient market information and unjust transaction price are competitor deal’s main weakness, which restrict the development of agricultural circulation and agricultural industrialization. Compared with competitor deal mode, the auction market can result in significant savings for both sellers and buyers. Savings are made by reducing transaction costs, increasing the circle of potential customers as well as by improving the search-andfind capabilities for all members concerned. Electronic auctioning in particular is a rapidly expanding application. The additional benefit of the auction is the matching of demand and supply at the ‘best price’ at one specific point in time. The advantages, however, must be weighed up against lower switching costs for auction participants. And with these good reasons, for the vast majority of floriculture business the auction is the World Trade Center: a better way to realize an assortment of flowers with as broad a scope as possible, produced across the world, and available for market price reflecting the supply and demand in a short time. The auction provides a hugely diverse selection of trade companies with equally diverse quantities of flowers and plants. The Netherlands is the international crossroads for commercial trade in flowers and plants. As the largest producers of flowers, plants and tree nursery products, the Netherlands processes more than fifty percent of the international trade. On 1 Jan. 2008, Bloemenveiling Aalsmeer and the former FloraHolland merged into one company, under the name FloraHolland. FloraHolland is the largest and successful flower auction in the world, through which approximately 44 million cut flowers and 4.8 million houseplants and garden plants are traded daily. FloraHolland is market leader and key player in the international floriculture sector.

China is one of the biggest countries for production and sales in flowers and plants. Yunnan is the largest center of greenhouse horticulture in china and provides more than fifty percent of the domestic production for China. In Yunnan province there are approximately 16000 growers and almost 6000 wholesales companies and exporters. Kunming International Flower Auction Market  is the only normally operational flower auction market in China, through which each dayapproximately 1.2 million cut flowers are sold to domestic and international market. For KIFA, Nov. 2007 was a month full of positive developments with new operational process, inner logistics and information system of the organization, fodder growing system which resulted in transaction capacity from 300 thousands to 4000 thousands daily. The KIFA continues to invest in strengthening the markets and increasing gains in efficiencies throughout the chain of production, supply and sales. However, some researchers have concluded that the auction doesn’t suit to Chinese fresh agricultural product’s trade because of unsuccessful operation. In despite of improving on operational performance, there are still huge gaps between FloralHolland and KIFA. In this paper we compare operational goals and performance of FloraHolland with KIFA, which are two typical flower auction markets in two countries. We also analyze some reasons for those gaps between these two auction markets. Finally, some crucial factors are presented for developing Chinese flower auction market in the future. The remainder of this article is structured as follows: First we briefly review the related literature on flower auction. Section 3 compares operational goals of FloraHolland with KIFA and analyzes some reasons for these gaps. In Section 4, we compare key figures 2007 of FloraHolland with 2008 of KIFA and analyze the differences of operational performances between two auction markets. In Section 5, we give five operational strategy’s emphasis for developing Chinese flower markets. Section 6 ends with conclusions and implications for research and practice. Prior research abroad on developing flower auction typically emphasized on its function and transaction price. Heezen and Baets discussed functions of the Dutch Flower Auction on the view of organizational structure. More researches on flower auction have discussed price discovery mechanisms. A central argument of these articles was that flower auction would improve communication searches, monitoring and information-sorting capabilities, reduce transaction costs and allow purchasers to take advantage of production economics available in markets. Bakos argued that auction marketplaces could reduce the search costs that buyers must incur to acquire information about seller prices and product offerings. The lowered search costs allow buyers to look at more product offerings and make it difficult for sellers to sustain high prices. Koppius et al. compared screen auctioning and physical auction.

They reckoned that screen auctioning entailed a decrease in information about flower quality compared to the physical product representation and might be a partial explanation for reduced prices in electronic markets. A central argument of Chinese research was considering adaptability of implementing agricultural product auction method in China from the macroeconomic perspective. Zhu Xin-Kai considered that the auction method doesn’t suit to Chinese practice in terms of government malfunction, market mechanism faultiness and lower-level agency institution. Kou Ping-Jun and Lu Feng-Jun argued that it must be careful to put agricultural product auction methods in practice because of some limitations of social condition and history. Li Da-Sheng and Zhang Guang-Hui discussed some technical economic issues about agricultural product auction and characterized that auction method was the direction about reform and development of agricultural product currency systems. However, it is the fact that almost all of Chinese fresh agricultural product auction markets had bad financial position over the past years. KIFA was the same status before Nov. 2007. After then new business model, which was designed by Qin Kai-Da, Yang Bao-Jian and et al., was successfully introduced into KIFA. In this paper, however, we emphasize on how to develop Chinese flower auction markets through comparative analysis. Having operated for over a hundred years, FloraHolland has developed a mature vision and mission: In a market that is characterized by upscaling and internationalization, FlorHolland wants to maintain and increase its strong market position.

The reason of chlorophyll reduction was deterioration of pigments in leaves from enzymatic conversion by chlorophyllase. Chlorophyllase continuously reacts during storage, causing chlorophyll in lettuces content to gradually decrease. However, there was no linear relationship found between the relative content of chlorophyll and root length, as shown in Table 1. To then, chlorophyll content loss was further analyzed. Changes in the loss rate of chlorophyll content with different root lengths is presented in Fig. 3. Gradual risings in the loss rate of chlorophyll content were found at all samples during storage. In the group of 3 cm, the loss rate of chlorophyll content was 27.3% at the end of storage, same as the group of the whole root length. The percentage of the loss amounted was maximum in the five groups. In the groups of 6 cm and 9 cm, the loss rates of chlorophyll content were 23% and 24%. In the group of 0 cm, the percentage of the loss amounted was minimal that was 20%. The reduction in chlorophyll is mainly related to chlorophyllase. After lettuce leaves the nutrient solution, retaining roots may not affect the activity or content of chlorophyllase. Therefore, whether to retain the lettuce roots will not affect the relative content of chlorophyll.Change of chromatic aberration with different root lengths at successive storage was shown in Fig. 4.Overall, gradual rises in chromatic aberrations were recorded throughout the storage in all cases. One of the reasons for the color change of the leaves is enzymatic browning. Generally, browning reaction is assumed to be a direct consequence of the action of oxidizing enzymes polyphenol oxidase  and peroxidase on phenolic substrates. O-quinones are produced by these reactions and can subsequently be converted to brown pigments by non enzyme-catalyzed reactions, which changes leaf color . Therefore, the increasing values of chromatic aberration due to the continuous browning reaction during storage. Fig. 4 showed that chromatic aberrations of the groups of retaining root were smaller than that of the group without root. In the group of 0 cm, the chromatic aberration was 8.7 NBS at the end of storage. It was maximum in the five groups. In the group of 3 cm and 6 cm, the chromatic aberrations were 7.8 NBS and 6.3 NBS, respectively. In the group of the whole root length, it was 6.9 NBS. In the group 9 cm, the chromatic aberration was 6.0 NBS that was minimal in the five groups. Retaining roots helps reduce the change of chromatic aberration. Ali et al. indicated that ascorbic acid  could inhibit PPO activity by reducing the degree of browning. Ascorbate peroxidase  can oxidize AA and inhibits the effects of AA.

The leaves and roots of lettuce have APX and PPO. But the APX activity of lettuce leaf was superior to that of root,hydroponic nft where produce more AA. Therefore, retaining root can increase AA content to inhibit PPO activity, which can reduce the degree of browning. The longer roots were retained, the more the AA will produce, and the greater the degree of browning will reduce. Therefore, the root length can be appropriately extended to reduce the color change of the hydroponic lettuce.Changes in the weight loss rate of lettuces are presented in Fig. 5. The weight loss rate of hydroponic lettuce increased with time increased. Transpiration and respiration of lettuce during storage will be conducted, which mainly leads to moisture loss. Therefore, the transpiration and respiration of lettuce made the rate of weightlessness continue to increase. Variations were found among the effects of different root lengths on the weight loss rate during the storage. In the group of 0 cm and 3 cm, the weight loss rates at the end of storage were all 1.8%, which were maximum in the five groups. In the group of 6 cm and 9 cm, the weight loss rates were 1.68% and 1.26%, respectively. In the group of the whole root length, the weight loss rate was 1.23% that was minimal in the five groups. The relationship between the weight loss rate of hydroponic lettuce and the root length was linear. The weight loss rate decreases as the length of root retention increases. Weight loss of leafy vegetables is mainly caused by moisture loss. Qiu et al.indicated that lettuce mainly absorbs water through roots. Samples in our study were stored in a high-humidity environment. Retaining roots makes lettuces absorb moisture from the high-humidity environment. The longer roots were retained, the more water will be absorbed, and the less weight will be lost. Therefore, longer roots were recommended to be retained during storage to reduce lettuce weight loss. Compared with hydroponic lettuce without root, the hydroponic lettuce which retained root has a better effect for retaining freshness. And it had the best effect of retaining freshness when the root length was 9 cm. Therefore, it was recommended to retained the root length of 9 cm when harvesting hydroponic lettuce. In this study, it was found that influenced indicators in others’ research may not be suitable for this experiment. For example, chlorophyll relative content could not reflect the effect of root length for retaining freshness. Therefore, it was necessary to choose influenced indicators that were suitable for this experiment. The packaging method may be different when harvesting with root. The future research direction is the packaging method of hydroponic lettuce which retained root.Species of lilies were used as ornamental plant for thousands of years. In the genera there are about 110 of wild species. Nevertheless the first hybrids derived from the 19th century. Classifications of lily species were set before several centenaries. DNA technology has helped to identify and distinguish the lily species and also detected their genetic diversity. In Lithuania, just naturally grows wild lily , other species are introduced. Identification of hybrids using DNA analysis was started at 20th century, nine decades. Natures of hybridswere confirmed analyzing DNA of parental forms and their offspring using RAPD method. Plant population genetics study systematic biology and conservation, is a big interest in the genetic diversity in natural populations. Reflecting on the evolutionary history of the species, genetic diversity in natural populations is very useful for those who study plant population genetics, systematic biology and conservation.

Plant species across the wider geographical region can be divided into different ecotypes; this division could be influenced by ecological factors, which are more important for the geographical variation of plants. Therefore, successful management, preservation and reestablishment of sustainable populations of rare, threatened, or endangered species depend on a complete understanding of the species, including levels and structure of genetic variation. In Lithuania, quite a lot various lily  species are introduced. However, not all species of our weather conditions are suitable. That’s why we have to do morphologic and genetic researches to learn out plants resources. It is also important to compare the results with the global genetic results and the plant of origin. In Lithuania there weren’t any lily genetic researches done. Molecular markers are recognized as a good method for the identification of plant species. Accordingly, these markers could be helpful in the breeding aimed at new cultivar development through early detection and screening of plants that have so far escaped cultivation. Also to improve plants taxonomy or in fine cultivars number in collections, genetic diversity of plants was detected using various methods. Such methods as restriction fragment length polymorphism , analysis of chloroplast DNA random amplified polymorphic DNA , simple sequence repeat  and intersimple sequence repeat  have enabled researchers to investigate genetic diversity among various plant species across natural populations. The efficiency of these methods has already been demonstrated in phylogenetic and population genetic studies. Local populations of traditional cultivars provide a valuable resource for plant breeding as well as for the preservation of genetic diversity. The exploration, evaluation, and conservation in situ and ex situ of genetic diversity in natural populations are imperative to guarantee sustainable development. Random amplified polymorphic DNA method has often been used for estimating genetic variation within and among plant populations, cultivars and species, and also was used for determining the hybridism of inter-specific hybrids in the genus Lilium. Recently, the usefulness of inter-simple sequence repeat  markers that amplify the genomic sequence between two simple sequence repeats  using anchored simple sequence repeat  primers has also been reported. To find out recourses of lily in Lithuanian, it is necessary to evaluate the variability using of genetic methods. It is also very important to evaluate and compare the genetic variation between species and wild populations of wild samples. There are some problems with naturally grown wild lily populations and introduced Lilium species, varieties and hybrids. There could be several origin possibilities; they could be sowed out from introduced lilies, hydroponic channel also there could be hybrids. After molecular DNA analysis to choose lily we can more efficiently and faster to make the biggest part of selection work processes. Also there would be a possibility during plant regeneration process to control proportions of variability and stability.This study offered the first detailed analysis of the genetic diversity and population structure for 3 different lily species 1 varieties and 1 hybrid grown in Lithuania based on ISSR molecular markers that have been proven to be valuable for the determination of genetic diversity.

The ISSR-PCR identified relatively high genetic diversity , considerable genetic differentiation among species  and the gene flow  at the species level. Compared with other Lilium species, polymorphism of analyzed groups is higher than that of 13 Lilium species and of 16 interspecific hybrids, and that of the Asiatic hybrid lily-Montreux and Connecticut King, and also that of the Lilium tsingtauence and higher than in the Lilium maculatum var. bukosanense. The effective gene flow per generation  of all analyzed groups is higher than one successful migrant per generation , possibly to overcome the diversifying effect of random drift. Lilium is a long-lived perennial grassy species, which should enable the species to maintain a high level of genetic diversity. Furthermore, its high genetic diversity could come from seedling establishment in the early period. Our observations suggest that events that potentially reduce genetic diversity and increase genetic differentiation among populations such as genetic drift or inbreeding might have not occurred yet with the restricted distribution. Based on Nei’s gene diversity and AMOVA analyses for 3 lily species, 1 varieties and 1 hybrid genetic differentiation among species  and fixation index  also signify a genetic structure. The non-random temporal or spatial distribution of genetic variations of alleles and genotypes is often referred to as the genetic structure of a population. The level and distribution of genetic variability among and within groups can be shaped with a great number of factors including pollen and seed dispersal, successional stages, geographic distribution range, adult density,mating system, colonization history, and natural selection. We hope that the genetic diversity and structure of analyzed five different lily groups reported here offer important information for this endangered species .The Cucurbita genus, of American origin, is one of the most variable genera within the Cucurbitaceae family. The main economic value of the species resides in the consumption of its immature fruits as vegetables, commonly known as summer squashes. Summer squashes of the Zucchini type rank among the highest-valued vegetables worldwide, whereas the “winter squash” types  of Cucurbita pepo and related Cucurbita spp. are food staples and rich sources of fat and vitamins in developing countries. It has various medicinal effects comprising antidiabetic, antihypertensive, antitumor, antimutagenic, immunomodulating, antibacterial, anti-hypercholesterolemic, intestinal antiparasitic, antalgic, and anti inflammation effects, and utilization possibilities of various Cucurbitaceae species have been reported. A wide range of pathogens hamper the cultivation of C. pepo, with more than eight different viruses infecting the crop. Among these viruses, Papaya rings pot virus  and Zucchini yellow mosaic virus  are the most important due to their prevalence and the degree of damage caused, which reduces crop and fruit quality. The control of these viruses is difficult and conventional breeding has not succeeded in producing new varieties with resistance to more than one virus species.

The proportional fraction of ammonia released in each treatment, determined through direct measurement in the leachates, was considered when calculating the emission factor as well as the nitrogen given in the irrigation. For the same reason, TA  was also chosen to reflect the direct emissions due to the application of ammonia as well as other acidifying agents generated during transportation and manufacturing of fertilizers . FE  and ME  have been regarded as the most relevant impact categories when analyzing fertilization methodologies, especially considering nitrogen and phosphorous . These impact categories are especially relevant in this study since slow struvite dissolution can provide insight into the possible reduction of P leaching into fresh and marine waterbodies, and again, the addition of N through struvite can also be reflected in the leachate quantities. FRS  was added as a relevant impact category to reflect fossil energy-related emissions that could arise due to struvite precipitation and transport compared to mineral P. Finally, MRS  was chosen to reflect the extraction of finite mineral resources, especially focused on phosphate rock extraction versus the recycling and reuse of phosphorous in the form of struvite. The resulting productions for the three lettuce cycles can be observed in Table 1. Here, we can appreciate the average yields obtained for all three harvests and all treatments for their fresh and dry weights. Further information on the specific production within the marked pots  can be seen in Table 2 in the supplementary information. We identified a general decrease in yield during the third harvest, most likely due to a remarkable decrease in the overall temperature during 54 DAT and 81 DAT in contrast to the previous crop cycles . This variation in the climatic conditions can be observed in Fig. 1 in the supplementary information with the recordings of humidity, radiation and temperature during all three cycles. While temperatures still ranged between 20 ◦C and 25 ◦C, the sudden change in comparison to the previous two crop cycles may have caused a delay in lettuce growth.

While no great differences can be seen in the overall yield of our lettuce cycles, the close monitoring of our pots can give us the variability of the obtained yield for the lettuces grown with the same initial struvite. This finding means that from the same pot, we can monitor the obtained yield in all three cycles. Table 2 in the supplementary information provides us with such information showing a general decline in production,grow table with the most acute decrease in yield in the 5LE treatment with a − 11% difference between the first harvest and the second. On the other hand, the decline for treatments 10LE and 20LE was less pronounced, with − 2% for both. In the case of pepper plant growth and production, Tables 2 and 3 provide insight into the differences spotted between treatments. Table 2 provides the main measurements made of the pepper plants at the end of the experiment . While no significant differences were seen for the stem weight, an increase in the fresh and dry weight was observed with the increasing amount of struvite applied. The same increase was noted for the leaf weight, number, and LAI, showing significant differences in all but the latter. The yield produced by the pepper plants  showed a greater total weight for the 20P treatment. While the total number of fruits was also higher for the 20P treatment, the weight per fruit did not differ greatly from that of the other treatments. The results shown in Fig. 2 depict the P content in the lettuce crop after 27 days of growing in the greenhouse for all treatments. The amount of P found in the lettuce biomass is directly related to the amount of struvite given, being lowest for the 5LE treatment followed by the 10 L and finally 20LE treatments. The amounts of P found in the 5LE and 10LE treatments decrease noticeably over time in the second and third cycles, while the 20LE treatment does not experience a great reduction during the second cycle but rather on the third cycle. It is important to point out that the results found for the second crop cycle show a much greater variability than the first and third ones. The remaining struvite content in the perlite and therefore P remaining in the substrate were analyzed and plotted in Fig. 2. Here, we can appreciate a great difference between the struvite fertilization treatments and the control, since the nutrient content in the perlite slowly increases over time for the latter, while the P content in the struvite treatments fluctuates and slowly decreases due to its dissolution. Here, again, a much greater variability in the results was observed for the second cycle. Fig. 3 depicts the P content in pepper biomass, fruit and perlite, showing great variation between struvite fertilization treatments and the control. While our treatments showed a low P content of 1.2 mg/g in leaves and 0.7–0.8 mg/g in the plant stem, giving ranges of 0.02 to 0.03 g of P in the total dry biomass, the control treatment showed values within adequate ranges of 2.1 mg/g . The amount of P in the harvested fruits reveals the differences between treatments based on the great mobility within the plant. Fruits are an ultimate sink of the phosphorous content in plants, and this result is reflected with a very clear relation to the struvite treatment. The great variability seen in these results derives from the great difference found between harvests within the same treatment, while the first pepper fruit harvest contained greater P concentrations, the third suffered a great reduction for all treatments, even the control . Finally, the amount of P found in perlite responds to the initially given struvite.

The resulting phosphorous concentrations found in the leachates were calculated for the total outgoing water weekly per plant,generating the patterns found in Fig. 4. The accumulation of P in the leachates for the lettuce and pepper crops can be seen in supplementary information Fig. 6. The results for the lettuce crop show the discharge of phosphorous during all three cycles, recognizing a clear pattern before and after each harvest. This pattern was highly noticeable for the CLE treatment, where the phosphorous content in the leachates decreased with the growth of the plant and rose once the plant was harvested and replaced with a seedling. This same pattern can be observed for all struvite fertilization treatments for lettuce, finding greater amounts for 20LE and less for 10LE and 5LE. The phosphorous content in water, on the other hand, differs greatly when observing the CP and the 5P, 10P and 20P treatments. The biomass growth, climatic conditions and subsequent irrigation amount define the loss of phosphorous in the CP treatments, showing an overall decrease in the concentration with a peak at approximately Day 37 after transplanting. All treatments with struvite showed very low concentrations in the leachates, especially after 20 DAT. The results obtained in the previous sections enable us to generate the nutrient balance for P during these cycles for all treatments. This understanding helps us estimate the P flows into the plant, substrate and water. These nutrient balances were calculated for the P flows in the lettuce and pepper crops  and averaged to obtain data for one plant. In addition, the water balances per plant are given in Figures 9 and 10 in the supplementary information. The nutrient balance is subjected to potential inaccuracies given through the sampling of substrate, water and biomass and the generation of mean values for all samples generating approximate values close to 100%. The balance for lettuce gives an overall picture of the obtained results of the phosphorous flows into the plant biomass as well as leachates. Compared to the control treatment, the phosphorous flow into the outgoing water was approximately 10 to 14 times lower for the 10LE and 5LE treatments, respectively, while the flow into the plant biomass remained similar. The remaining phosphorous in perlite remained high in the 5LE and 10LE treatments, while more than half was reduced in the 20LE treatment. We also appreciate an accumulation of P in the perlite of the CLE treatment. For pepper,ebb flow table the biomass flows were divided between the fruits produced and the generated biomass on the day the plants were cut and weighed. Here, we can appreciate the great quantity of phosphorous found in the pepper fruits, which equals the total phosphorous found in the plant leaves and stem. The total biomass showed a great difference between the CP treatment and the struvite fertilization treatments, revealing a much greater P content in the control.

Due to the greater irrigation needs of pepper plants compared to lettuce plants, the CP treatment received an overall greater amount of P through irrigation compared to the CLE treatments. Therefore, although the P in the perlite and leachates is lower in CP than in the CLE treatments in terms of percentage, the absolute amounts are greater. In the case of the pepper plants fertilized with struvite, the P in the leached water was similar and even smaller than the amount found in the lettuce crop. The outgoing P in the leachates of the pepper plants was 10, 19 and even 35 times lower than that in the control treatment  for the 20P, 10P and 5P treatments, respectively. The calculated dissolution rates for the applied struvite in lettuce and pepper are shown in Fig. 5. The struvite dissolution was estimated by the P contained in the water leachates as well as P in the plant biomass. This dissolution has a direct impact on the P uptake by the plant that was estimated as the P contained in the P biomass. The results for lettuce show greater dissolution with a greater initial amount of struvite. The dissolution of the struvite was also higher during the first lettuce cycle , showing smaller differences between the second and third cycles . The dissolution rate found in the pepper crop was smaller than that in the lettuce crop but followed the same pattern as seen before, with greater dissolution with higher amounts of struvite. Fig. 6 shows the results for lettuce, and Fig. 7 shows the results for the environmental assessment of the fertilization treatments. Since only the fertilization of the crops was considered for the analysis , all differences will be related to the use of struvite instead of monopotassium phosphate  in the form of KPO4H2, leaving out the laboratory infrastructure and auxiliary equipment, as well as the end-of-life processes. The obtained results for six out of seven impact categories show that fertilization with struvite has lower impacts than the control, and for the cases of ET, MRS, FRS and GW, impacts are also reduced as we increase the amount of struvite applied. In terms of eutrophication, FE, which is directly related to the emissions to water, had the greatest impact on the control irrigated with mineral P, followed by 20LE, which was the treatment with the highest quantity of struvite per plant. ME, although related to the emissions to water, also does not decrease substantially for the struvite-treated crops due to its relation to nitrogen emissions, which are sustained for all treatments. Furthermore, we can observe that although a reduction of the impacts is occurring for the 20LE treatment, this reduction is most likely not a consequence of a reduced N emission to water but due to greater yields obtained; on the other hand, treatment 5LE is overshadowed by the lower yields generated and a proportionally greater N emission due to the smaller plant growth. The results obtained for the pepper crop indicate a considerably abrupt decrease in the emissions in all impact categories for treatments 10P and 20P in comparison to the CP treatment. In comparison to the lettuce crop, the ME was severely reduced for these two treatments. The 5P treatment with lower production rates and therefore lower FU experiences much greater values for all impact categories except FE and MRS, which are slightly below the control treatment CP in the latter.