Hospital wastewater treated with a novel bacterial consortium (Alcaligenes faecalis and Bacillus paramycoides spp.) for phytotoxicity reduction in Berseem clover and tomato crops

Hospital wastewaters are produced in large volumes in Pakistan (∼362–745 L/bed.day) and are discharged without proper treatment. They are widely used by farmers for crop irrigation and induce a phytotoxic effect on plant growth. The study was conducted to evaluate the effect of untreated and treated hospital wastewater on seed germination of a fodder crop Trifolium alexandrinum (Berseem clover) and a food crop Solanum lycopersicum (tomato). A bacterial consortium was formed with three bacterial strains, i.e., Alcaligenes faecalis and Bacillus paramycoides spp., which were individually proven efficient in previous studies. The concentrations of untreated and treated hospital wastewater (25, 50, 75 and 100%) were used to irrigate these crop seeds. To assess the efficiency of treatment, the germination percentage, delay index, germination index, stress tolerance indices, seedling vigour index and phytotoxicity index were calculated and were statistically proven significant. The seeds grown in treated wastewater concentrations showed negative values of phytotoxicity indices (tomato: 0.36, 0.47, 0.78 and 1.11; Berseem clover: 0.23) which indicate a stimulatory or non-toxic effect on seedling growth. Our work proposes that this bacterial consortium is efficient for hospital wastewater treatment before crop irrigation.


GRAPHICAL ABSTRACT INTRODUCTION
Agriculture, a major contributing factor to the gross domestic product of Pakistan (∼21%), is considered as the foundation of the country's economy (GOP ). The majority of the country's population (∼63%) is rural and is directly or indirectly associated with the agriculture sector (TWB ). Almost 90% of all the country's agricultural food production is carried out using water from the Indus Basin irrigation system (Qureshi ), which is regarded as the world's largest natural and continuous irrigation system. It is used for various purposes (PCRWR ): irrigation (∼60%), drinking (∼90%) and industrial use (∼100%). However, the system is now becoming one of the world's most overstressed and quickly diminishing natural water systems (NASA ). Additionally, Pakistan is predicted to face a shortage of water in the near future (Roberts ), with the country listed as the third most water-deficit in the world (IMF ) and its groundwater table is predicted to disappear by 2025 (PCRWR ).
Due to this freshwater shortage, the farmers are irrigating their crops with raw wastewaters coming from domestic, hospital, industrial and other waste effluent sources (Qadir et al. ). In Pakistan, the municipal wastewater production (3.06 × 10 9 m 3 /year) is more than 70% of the total produced wastewater (4.37 × 10 9 m 3 /year) (Murtaza & Zia ). The remaining 30% wastewater (1.31 × 10 9 m 3 /year) is produced from industrial use. All of these wastewaters are discharged into combined sewers in Pakistan (Murtaza & Zia ) containing impurities, dyes, disinfectants, pharmaceuticals, heavy metals, solvents and toxic chemical compounds without proper treatment (Emmanuel et al. ). The hospital wastewater containing pharmaceutical contaminants, heavy metals and toxic chemical compounds is more than 10% of the municipal wastewater (Ashfaq et al. ). The rest of the municipal wastewater is produced from domestic and other sources. The hospital wastewater is produced in large volumes (∼362-745 L/bed.day) and is discharged without proper treatment despite hospital waste management rules issued by the Ministry of Environment, Pakistan since 2005 (GoP ). This enormous amount of unsafe hospital wastewater needs special consideration (Meo et al. ). Reusing this untreated hospital wastewater for crop irrigation is extremely harmful to plants (Hamilton et al. ; Dwivedi ), animals and humans (Qadir et al. ; Keraita et al. ; Qadir et al. ; Contreras et al. ). It also pollutes the aquatic environment, leading to fish kills etc. (Hernando ). The treatment of hospital wastewater before its discharge into combined sewers would help to reduce freshwater pollution and increase its availability for safe use in crop irrigation. Previously, the biological methods using bacteria have been recognised as efficient, eco-friendly and more cost-effective than physicochemical methods for the treatment of combined wastewaters (Phugare ). However, their role in treatment of hospital wastewater was not confirmed. In our previous study (Rashid et al. ), we have shown the capacity of three bacterial strains (Bacillus paramycoides spp. and Alcaligenes faecalis) isolated from domestic and pharmaceutical wastewaters for the treatment of hospital wastewater. The hospital wastewater under study was characterised with pH (7.4), electrical conductivity (EC) (444 μs/cm), salinity (0.2 ppt), turbidity (51 NTU), total suspended solids (TSS) (2300 mg/L), total dissolved solids (TDS) (296 mg/L), chemical oxygen demand (COD) (396 mg/L), biological oxygen demand (BOD) (246 mg/L), biodegradability index (0.62), chromium (1.8 mg/L), lead (0.17 mg/L) and nickel (1.8 mg/L) and it contained a mixture of emergent pharmaceutic contaminants (i.e., phenol, salicylic acid, caffeine, naproxen, octadecene and diazepam). Though most of the parameters for untreated hospital wastewater are beyond the National Environment Quality Standards (NEQS), we achieved high percentage decolourization (>93%) and degradation (100-43%) of pharmaceutic pollutants found in hospital wastewater. This work recommends the potential use of these strains as a consortium as it involves a combined mechanism of metabolism among the co-existing bacterial isolates. For this, the first step would be to demonstrate the safe use of this consortium for crop irrigation by performing a phytotoxicity analysis. Phytotoxicity is the induction of any toxic effect induced within plants due to pollutants that delay seed germination or affect plant growth parameters (length and weight) (WRAP ). Previously, a reduction in phytotoxicity was observed in Lactuca sativa (lettuce) seeds irrigated with consortia treated wastewaters (Ceretta ). It has also been observed that biologically treated textile wastewaters used for crop irrigation were capable of improving the growth of plants (Velayutham ). However, extended work is still required for a comprehensive evaluation of the phytotoxicity of untreated and treated hospital wastewaters for crop irrigation.
The present study aimed to assess the phytotoxicity reduction by a novel bacterial consortium (B. paramycoides spp. and A. faecalis) (Rashid et al. ) in hospital wastewaters which are highly toxic, to allow the wastewater to be used safely for the irrigation of the main fodder crop in Pakistan i.e. Trifolium alexandrinum (Berseem clover), and the most popular food crop in Pakistan i.e. Solanum lycopersicum (tomato). Berseem clover is a vital winter fodder crop that plays an essential role in improving the dairy industry in Pakistan. Comparatively, tomato is a rapid-growing vegetable crop, providing a higher yield which is economically important. The reason for selecting these crops is that they are both considered highly valuable for the country's economy and it is the first time that these plants have been tested for phytotoxicity after wastewater irrigation. This will be determined using a range of matrices to assess plant healthseed germination percentage, delay index (DI), germination index (GI), stress tolerance indices (STIs), seedling vigour index (SVI) and phytotoxicity index (PI). These indices are predictors of phytotoxicity reduction in crop plants after irrigating with treated hospital wastewater and are compared with the irrigation with untreated hospital wastewater. The potential application of this work is to determine if the biotreatment with this consortium is a feasible method to treat highly toxic hospital wastewater and allow its safe reuse to irrigate two important crop plants, and therefore be an attractive alternative to meet the increasing demand of freshwater.

Collection of hospital wastewater
The hospital wastewater sample (50 L) was collected from three different points of discharges from a drainage site of a local hospital in Lahore, Pakistan, according to the standard protocols (APHA ). The drainage site of the hospital had a combined disposal tank station containing homogenized waste from all wards. The geographical coordinates of Lahore city are 31 34 0 55.36″ north and 74 19 0 45.75″ east at an altitude of 217 m (712 ft). The sample was collected on 15 March 2019.

Characterization of hospital wastewater
The full characterization of hospital wastewaters is not part of present study. However, in order to assess the efficiency of biotreatment, the following parameters were investigated according to standard protocols (APHA ) before and after the biotreatment of hospital wastewater. These parameters were compared with the NEQS (NEQS ) (Table 1), i.e. physical components (colour, odour, pH, EC, TDS, TSS, salinity [ppt] and turbidity [NTU]); biological components (BOD and isolation and identification of bacterial isolates); and chemical components (COD, heavy metal estimationarsenic, cadmium, chromium, lead and nickel, and identification of pharmaceutic contaminants). The heavy metal estimation was carried out using an Atomic Absorption Spectrophotometer (AA 7,000 F with Autosampler and Hydride Vapour Generator, Shimadzu, Japan) to access the efficiency of biotreatment. Most of the parameters for untreated hospital wastewater were beyond the range of the NEQS. The presence of pharmaceutic contaminants also highlighted the necessity of an effective biotreatment.

Characterization and development of the bacterial consortium
The bacteria used in the consortium were isolated in our previous study (Rashid et al. ). The isolates were identified as B. paramycoides spp. and A. faecalis sp. (Figure 1).
Phylogenetic analysis of the strains was carried out using the top 20 BLAST hits for each isolate. This was achieved by aligning the sequences using Muscle v. 3.8.425 (Edgar ) and a phylogenetic tree assembled in Geneious Prime using the Tamura-Nei genetic distance method (Tamura & Nei ) and Neighbor-Joining tree building Nd, Not detected; NSAID, non-steroidal anti-inflammatory drug. Significance is indicated by *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. method (Saitou & Nei ). The phylogenetic tree was then imported in to the Newick file format and edited in Evolview (Zhang et al. ). Phylogenetic analysis was conducted to determine the similarity of these species to each other and their respective closely identified BLAST sequences. The three isolates were distinct from these BLAST matches. Both B. paramycoides spp. clustered together, demonstrating that these isolates were highly similar. The closest cluster for three of the species was identified as Paenalcaligenes suuwonensis and Paenalcaligenes hominis ( Figure 2). For consortium development, one colony was picked from each of the plates of the three bacterial isolates. These were inoculated in a test tube (20 mL) containing sterilized Lysogeny broth medium (10 mL) and incubated at 37 C in a shaking incubator. After 24 h of incubation, the consortium was ready to be used for the biotreatment experiment.

Biotreatment
One colony of the consortium was added to a test tube (20 mL) with deionized water (10 mL) to form a consortium suspension (optical density ¼ 1). The consortium suspension (10%) was added to a conical flask (250 mL) containing hospital wastewater (100 mL) and incubated at 37 C for 48 h in a shaking incubator for the biotreatment. This suspension was considered as treated wastewater after this period.

Seed selection and sterilization
The fodder crop (T. alexandrinum, Berseem clover) and food crop (S. lycopersicum, tomato) seeds were generously provided by the Seed Certification Department, Lahore, Pakistan. The names of the certified variety for the crop seed were Berseem clover seed (Anmol) and Seminis Hybrid Tomato ONYX (Ori-Thailand). The germination experiments were conducted within a randomized complete block design with three replications. Fifteen Berseem clover seeds were sterilized in a laminar fume hood with 4% (v/v) bleach solution. Then, 50 μL of Tween 20 detergent was added to the bleach solution to avoid any contamination during germination.

Preparation of wastewater concentrations and germination experiment
A broad selection of dilutions of untreated and treated hospital wastewater concentrations, i.e. 25, 50, 75 and 100%, were chosen to simulate different levels of contaminant concentrations ( Figure 3). These concentrations were diluted with deionized water to assess the differential extent of phytotoxicity (Ceretta ). The sterilized seeds were placed on cotton (1 g) placed in sterilized glass Petri dishes (dimensions 100 × 15 mm). The cotton was wetted with a similar volume of the different dilutions of untreated and treated hospital wastewater (20 mL). Tap water (TW, 20 mL) and deionized water (20 mL) were used for the control treatment, separately (Kaushik et al. ). In total, there were 30 Petri dishes, including controls. Seeds were germinated under continuous white fluorescent tube lights with the light intensity of 2,500-3,500 lx/m 2 /s inside a growth room at 25 C.

Measurements of length and weight
The observations of seeds and growth parameters were recorded daily up to 1 week and germination indices were calculated. A seed was considered as germinated when its root was visible and measurable (i.e. >0.5 mm); the root lengths of ungerminated seeds were considered as zero. The length of root and shoot of the germinated seeds were measured from each experimental set. The shoot length was measured from the base of the primary leaf to the base of the hypocotyl in centimetres. Root length was measured from the tip of the primary root to the base of the hypocotyl in centimetres. By adding the root length and shoot length, the seedling length was calculated and expressed in centimetres. Both shoots and roots were oven-dried at 65 C overnight and the dry weights were determined.

Germination percentage (GP)
The GP is the ratio between the total number of germinated seeds to the total number of viable seeds. This parameter is an indicator to assess the viability of seeds and to see whether the treatment condition is suitable for the seeds to grow or not. The range of desirable GP for seeds irrigated with treated hospital wastewater is between 80 and 100%; whereas, less than 75% GP indicates poor germination (Solomon ). The GP was calculated using Equation (1)

Delay index (DI)
DI is the ratio between delay in germination time over control to germination time for control. This parameter is an indicator to see the delay in seeds that are germinated in different dilutions of untreated and treated hospital wastewater compared to the time required by those seeds germinated in control (DW). The desirable DI for seeds irrigated with treated hospital wastewater should be the same as the control. The higher the DI, the poorer the ability of seeds to germinate. DI was calculated using Equation (2)

Germination index (GI)
The GI is the product of relative seed germination (RSG) and relative root growth (RRG). This is the parameter

Stress tolerance indices (STIs)
STI is a valuable parameter to determine the high yield and stress tolerance capability in crop plants. STIs are comprised of six indices, namely the root length stress tolerance index (RLSTI), shoot length stress tolerance index (SLSTI), root fresh weight stress tolerance index (RFSTI), shoot fresh weight stress tolerance index (SFSTI), root dry weight stress tolerance index (RDSTI) and shoot dry weight stress tolerance index (SDSTI). These values determine the difference between the stress tolerance potential in shoots and roots between plants irrigated with untreated and treated hospital wastewater in terms of their lengths and fresh and dry weights. The desirable range of all STIs for seeds irrigated with treated hospital wastewater is larger than the STI values for the control and seeds irrigated with untreated hospital wastewater. These were calculated using Equations (6)

Seedling vigour index (SVI)
This is the property of seed that determines the level of performance of seed to germinate and emerge as a seedling. It is a measurable parameter to describe the germination characteristics linked with the seed performance. The desirable SVI values for seeds irrigated with treated hospital wastewater are larger than the SVI values for control and seeds irrigated with untreated hospital wastewater (Amin et al. ). These were calculated according to Equation (12), where the higher the SVI, the more dynamic the growth (Amin et al. ): SVI ¼ germination percentage × seedling length (12)

Phytotoxicity index (PI)
The PI indicates any delay in seed germination, plant growth inhibition or any side effect on seedlings caused by toxic pollutants. It also specifies any danger happening to the plant growth. The possible range of PI values is between 0 and 1, in which a positive PI value indicates a toxic effect on seedlings, whereas a negative PI value demonstrates a stimulatory or non-toxic effect (Tiquia et al. ). It was calculated based on germination and root elongation according to Equation (13)

Statistical analysis
Experiments were statistically analyzed through one-way analysis of variance (ANOVA) in which the factor was the concentration of hospital wastewater from biotreatment and the effects were the indices. The obtained data were statistically analyzed using GraphPad Prism ® 2020. The results were presented as means ± standard deviation (SD). The data were compared using a t-test with Welch's correction and two-tailed p-value calculation using GraphPad Prism software. The comparisons included the control (DW) with each treatment (tap water, TW, R25, T25, R50, T50, R75, T75, R100 and T100) and the raw wastewater within each concentration level with the corresponding treated wastewater (e.g. R25 with T25). For all comparisons, differences were considered significant when the probability level was less than 0.05.

Characterization of the bacterial consortium
The bacteria used in the consortium were isolated from our previous study (Rashid et al. ). The isolates were identified as two B. paramycoides species and A. faecalis sp. Phylogenetic analysis was conducted to determine the similarity of these species to each other and their respective closely identified BLAST sequences. The three isolates were distinct from these BLAST matches. Both B. paramycoides spp. clustered together, demonstrating that these isolates were highly similar. The closest cluster for three of the species was identified as P. suuwonensis and P. hominis.

Germination percentage
According to Solomon (), seeds grown in the laboratory showing more than 90% GP (e.g. super germination) are expected to grow ex situ with a GP of 65% or higher. Similarly, seeds with 85% GP in the laboratory (e.g. good germination) are expected to possess more than 50% GP.
Finally, seeds with 75% or less GP in the laboratory (e.g. legal germination) are expected to have at least 15% practical GP in fields. Both Berseem clover and tomato seeds showed 60-80% growth in all untreated hospital wastewater concentrations (R25, R50, R75 and R100), while the seeds tested showed super germination (95-100%) in different concentrations of treated hospital wastewater concentrations (T25, T50, T75 and T100).

Delay index
The Berseem clover plant was strongly influenced by irrigation with untreated wastewater at all concentrations as it showed a higher value of DI (2) for all untreated hospital wastewater concentrations. The tomato showed a high value of DI (1.25) at all concentrations of untreated hospital wastewaters. The order of DI among these two crop plants followed the trend: Berseem clover < tomato.

Measurements of length and weight
The effects of treated (T) and untreated (R) wastewater concentrations (25, 50, 75 and 100%) were compared for the seedling growth of Berseem clover and tomato. The associated statistical measurements indicated that the means of lengths and weights were statistically significant. The comparisons included the control (DW) with each treatment (TW, R25, T25, R50, T50, R75, T75, R100 and T100) and the raw wastewater within each concentration level with the corresponding treated wastewater (e.g. R25 with T25). It was observed that the seedling length of Berseem clover (shoot and root) was longer (shoot: 1.6-2.7 cm; root: 2.9-4.6 cm) in treated wastewater concentrations than in untreated wastewater concentrations (shoot: 1-1.8 cm; root: 1.2-2.9 cm). Likewise, the root and shoot weights of Berseem clover (fresh and dry) were found to be greater in treated wastewater concentrations than in untreated wastewater concentrations (Table 2). It was also observed that the seedling lengths of tomato (shoot and root) were longer (shoot: 3.7-4.4 cm; root: 4.9-7.6 cm) in treated wastewater concentrations than in untreated wastewater concentrations (shoot: 1.6-2.8 cm; root: 1.7-3.3 cm). Similarly, the root and shoot weights of tomato (fresh and dry) were found to be greater in treated wastewater concentrations than in untreated wastewater concentrations (Table 3).

Germination index
In our previous study, we showed the presence of organic pollutants (phenol, salicylic acid, caffeine, naproxen, octadecene and diazepam) in the raw/untreated hospital wastewater (Rashid et al. ). In this present study, the GI values for Berseem clover and tomato seeds irrigated with four concentrations of untreated hospital wastewater (R25, R50, R75 and R100) were increased to 60-150% and 189-278% in treated hospital wastewater (T25, T50, T75 and T100), respectively. Compared to seeds irrigated with tap water (as control), the GI values for tomato seeds irrigated with treated hospital wastewater were higher (Figure 5(a)). Although the GI values of Berseem clover seeds irrigated with treated hospital wastewater were lower than control (TW) they were higher than the GI values of seeds irrigated with raw hospital wastewater ( Figure 5(b)). This result also indicates the sensitivity of tomato seeds compared to the Berseem clover seeds.
Zucconi & De Bertoldi () previously mentioned that the 60% increase in GI values compared to distilled water is an indication of improved germination. Our results indicate 100% increase in GI values compared to distilled water. The low values of GI in both crop seeds irrigated with untreated wastewater concentrations reflect the presence of organic pollutant compounds that inhibited the RSG as well as the RRG. Our results agree well with the previously reported work by Tiquia et al. (). The increase in GI values in Berseem clover and tomato seeds irrigated with treated hospital wastewater concentrations suggest the efficiency of the treatment in removing or reducing the concentrations of organic pollutant compounds. The present study also confirms the presence of three heavy metals: nickel, chromium and lead (Figure 4(b)) in untreated hospital wastewater with low GI and high EC values ( Figure 5(a)). However, the treated hospital wastewater showed high GI and EC values due to the negligible amounts of heavy metals ( Figure 5(b)). This efficacy indicates the increase in RSG as well as the RRG (Selim et al. ). The high GI values in seeds irrigated with treated hospital wastewater also elucidate the decrease in phytotoxicity (Tiquia et al. ).
higher the tolerance index, the more tolerant the genotype. This higher percentage in STIs for Berseem clover seeds irrigated with treated wastewaters therefore indicates the high yield and stress tolerance capability in Berseem clover.
These high values determine the stress tolerance potential in plant shoots and roots in terms of lengths and fresh and dry weights. The values of the six STIs (RLSTI, SLSTI, RFSTI, SFSTI, RDSTI and SDSTI) for tomato seeds irrigated with untreated wastewater concentrations were also increased to 349, 177, 536, 675, 2,400 and 3,040%, respectively, in treated wastewaters (Table 5). This percentage increase is indicative of high yield and stress tolerance capacity in the tomato plant. It also supports the efficacy of hospital wastewater treatment that enhanced the extent of stress tolerance capability in both crop plants.

Seedling vigour index
The SVI values of Berseem clover seeds irrigated with untreated hospital wastewater concentrations (R25, R50, R75 and R100) were increased to 27, 54, 145 and 40% in treated wastewater concentrations (T25, T50, T75 and T100), respectively. Similarly, the SVI value of tomato seeds irrigated with untreated wastewater concentrations (R25, R50, R75 and R100) were increased to 82, 173, 339 and 206% in treated wastewater concentrations (T25, T50, T75 and T100), respectively. These increased SVI values of Berseem clover and tomato plants germinated at different concentrations of treated hospital wastewater demonstrate that the growth is more dynamic in these treated wastewaters. Previously, Rusan et al. () showed that the SVI  The associated statistical measurements indicated are calculated from one-way ANOVA and Sidak multiple comparison testing. *Denotes the p-value *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 within different treatments. The F-value is the ratio of two mean square values. A large F ratio means that the variation among group means is higher than expected by chance. Our results agree well with these as we obtained much higher SVI values. This indicates the level of performance of these seeds, which have been irrigated with treated hospital wastewater, to germinate and emerge as a seedlings.

Phytotoxicity index
In the present study, the PI values in seeds irrigated with treated hospital wastewater were statistically proven significant compared to the PI values of seeds irrigated with untreated hospital wastewater for both crop plants. For tomato seeds grown in tap water (TW) and untreated hospital wastewater concentrations (R25, R50, R75 and R100), the PI values were positive (0.15, 0.31, 0.53, 0.39 and 0.08), which indicates an extremely toxic effect on seedling growth (Figure 6(a)). In contrast, the seeds irrigated with all treated hospital wastewater concentrations (T25, T50, T75 and T100) showed negative PI values (À0.36, À0.47, À0.78 and À1.11), which indicates a strong stimulatory or non-toxic effect on seedling growth. For Berseem clover seeds grown in untreated hospital wastewater concentrations (R25, R50, R75 and R100), we observed positive values of PI (0.60, 0.27, 0.42 and 0.42) (Figure 6(b)). This highlights a highly toxic effect induced by the raw wastewater even when diluted. However, the seeds irrigated with the T75 treated hospital wastewater and tap water showed negative PI value (À0.23 and À0.51), which indicates a stimulatory non-toxic effect on seedling growth. Referring to the previous literature, the low PI value in seeds irrigated with tap water is attributed to the presence of higher concentrations of nitrogen, phosphorous and potassium (Rusan et al. ). After irrigation with treated hospital wastewater, it may be considered that the reduction in the phytotoxicity value is due to the reduction in heavy metal concentration, phenolic and other toxic organic compounds, and stress tolerance (

CONCLUSIONS
In this present study, we used a novel consortium made of three bacterial strains (two B. paramycoides spp. and one Alcaligenes faecalis) for irrigating Berseem clover and tomato that were individually proven efficient in our previous studies (Rashid et al. ). The germination  percentages for both crop plants irrigated with treated hospital wastewater concentrations (T25, T50, T75 and T100) were between 95% and 100%, which is considered as super germination. The seedling lengths and weights of Berseem clover and tomato (shoots and roots) were higher in treated wastewater concentrations than in the untreated wastewater concentrations. The values of six STIs (RLSTI, SLSTI, RFSTI, SFSTI, RDSTI and SDSTI) for Berseem clover and tomato seeds irrigated with treated wastewater concentrations were increased from 170% to 14,800%. The seeds grown in all treated wastewater concentrations (T25, T50, T75 and T100) showed negative values for phytotoxicity indices (tomato: À0.36, À0.47, À0.78 and À1.11; Berseem clover: À0.23), which indicates a strong stimulatory or non-toxic effect on seedling growth. Our work endorses that the biotreatment with this consortium is a feasible method to treat hospital wastewater before irrigation of tomato and Berseem clover crop plants and therefore is an attractive alternative to meeting the increasing demand for freshwater. However, if fruits and nuts are produced by plants irrigated by treated hospital wastewater, the authors recommend that these produces should be tested to make sure they are safe for consumption.