Open Veterinary Journal, (2023), Vol. 13(4): 451–465

Original Research

10.5455/OVJ.2023.v13.i4.7

Eichhornia crassipes from wastes to valuable products in water purification and influence on growth and impregnability in overwhelmed broiler chickens

Essam S. Soliman^1*, Rania A. Hassan², and Doaa S. Farid³

¹Department of Veterinary Public Health, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

²Animal Production Division, Department of Animal Wealth Development, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

³Department of Environmental Protection, Faculty of Environmental Agricultural Sciences, Arish University, El-Arish, Egypt

*Corresponding Author: Essam S. Soliman. Department of Veterinary Public Health, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. Email: soliman.essam [at] vet.suez.edu.eg

Submitted: 10/12/2022 Accepted: 19/03/2023 Published: 15/04/2023

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Abstract

Background: The implementation of green technologies is continually gaining attention worldwide and was considered for removing water pollutants and treating municipal water before its disposal.

Aim: Evaluation of the laboratory antimicrobial actions and chelating activities, and the field influence of Eichhornia crassipes on performance, biochemical and immunoglobulin concentrations, and intestinal microbiota in overwhelmed broiler chickens.

Methods: We assessed the laboratory antimicrobial actions of E. crassipes 1% suspension against bacterial (Escherichia coli O157: H7 and Salmonella Typhimurium) and fungal (Aspergillus niger and Candida albicans) microorganisms using a 96-well minimal inhibitory concentration, and the chelating activities of E. crassipes against calcium sulfate and copper sulfate. Also, we designed randomly four equal groups out of 200 1–day-old Ross^® 308 chicks on a deep litter system. Three groups (G1, G2, and G3) were supplied daily with E. crassipes suspension of 1% from the third day until the end of the experiment, while the fourth group (G4) received non-treated tap water. Broilers of G1–3 were challenged with calcium sulfate (75 mg.l^-1), copper sulfate (200 mg.l^-1), and S. Typhimurium (1.5 × 10⁶ CFU.ml^-1) polluted water respectively on the 7th, 14th, 21st, 28th, 35th days of age. We collected 1,914 samples by the end of the study, these samples included 90 E. crassipes pollutants and 480 E. crassipes microbial mixes, 192 sera, 192 intestinal swabs, and 960 tissues.

Results: Eichhornia crassipes treated water reveals highly significant (p < 0.01) improvements in water quality assessments and a highly significant (p < 0.01) increase in dissolved oxygen levels compared to tap water. Eichhornia crassipes 1% achieved a 100% adsorption capability for calcium and copper sulfate after 1-hour and 100% bactericidal (E. coli O157: H7 and S. Typhimurium) and fungicidal (A. niger and C. albicans) actions after 1, 2, 2, and 2-hours, respectively. Broilers treated with 1% E. crassipes revealed highly significant (p < 0.01) improvements in performance indices, carcasses characteristics, biochemical and immunological parameters, and highly significant (p < 0.01) decreases of cortisol hormone and bacteriological parameters in all treated broiler groups compared to the control.

Conclusion: Eichhornia crassipes 1% reveals a significant improvement in drinking water quality, as well as produces high adsorptive and antimicrobial actions. Eichhornia crassipes 1% improved performance traits, carcass quality, and intestinal microbiota in overwhelmed broilers.

Keywords: Antimicrobial, Broiler chickens, Eichhornia crassipes, Immunity, Performance.

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Introduction

Water pollution is a major worldwide reproducible problem with urbanization and industrial growth. Water quality has been used to point out the physicochemical characteristics to meet suitability for various uses (Li and Qian, 2018a). Water quality is influenced by numerous factors including source geology, meteorological conditions, topography, seasonal variations, and biological considerations (Li et al., 2017a). Clean and palatable water with freedom from disease-producing agents has been the main demand for livestock production (Tian and Wu, 2019). Water is negatively affected by populations' activities and becomes polluted with chemicals and infectious/zoonotic pathogens including bacteria (Soliman et al., 2009a) such as Escherichia coli, Salmonella, Leptospira, and Listeria, protozoa (Soliman et al., 2009b) such as Giardia and Eimeria, and parasitic agents such as Echinococcus, Schistosoma, and Faschiola. Biosecurity pillars have included water quality to provide birds with excellent/satisfactory water (Su et al., 2019).

Water purification has been performed using various methods such as photocatalysis (Raval et al., 2016), biochar (Palansooriya et al., 2020), sludge materials (Anastopoulos et al., 2017), ion exchange (Szczepanik, 2017), and eco-friendly byproducts as clay (Soliman et al., 2021d) and Oreochromis niloticus bone (Soliman et al., 2022). Developing eco-friendly, economical, and less destructive alternatives (Phytoremediation) for the handling of water pollutants is a worldwide interest (Shah, 2010). These eco-friendly means must possess higher absorption capabilities in short contact time and accumulation actions for longer periods (Mudgal et al., 2010). Aquatic plants such as Eichhornia crassipes are characterized by high invasiveness, wide spreading (tropical and sub-tropical countries), floating freely, and can grow up to 1 m above the water's surface (Pellegrini et al., 2018). It is known as a fast-growing runner plant and its seeds can remain viable for 28 years (Sarika et al., 2014).

Eichhornia crassipes usually survive in an ambient temperature of 25°C–30°C (12°C: 35°C), pH of 5.0: 7.5, and salinity of low than 15%, which above contributes to chlorosis and death (Kong et al., 2014). It has been recognized as an ecological plague in Egypt from 1879 to 1892 and contributed to biomass suffocation, diminishing fish reservoirs, and negatively influencing the economy (Twongo, 2019). Eichhornia crassipes has high absorbing power and thus has been used in wastewater purification. Eichhornia crassipes have been used in numerous fields such as braiding material and fiber source in India, Thailand, and Vietnam, biogas production, sludge field, adsorbent (Abdel Shafy et al., 2016), bio-cleaning (Ansari et al., 2014), food source, small scale paper production, carotene-based table vegetable, dermatological medicinal applications in horses, sustainable source of oxygen, and bio-herbicidal agent through the production of hydrogen peroxide and inhibition of the soluble peroxidase activities (Chai et al., 2013).

Here, in this study, we evaluated the laboratory antimicrobial actions of the E. crassipes 1% suspension against bacterial (E. coli O1527: H7 1.7 × 10¹¹ CFU.ml^-1 and Salmonella Typhimurium 1.5 × 10⁶ CFU.ml^-1) and mycotic (Aspergillus niger 2.5 × 10⁸ CFU.ml^-1 and Candida albicans 2.5 × 10⁸ CFU. ml^-1) microorganisms, and the chelating activity against inorganic (calcium sulfate, 75 mg.l^-1) and heavy metals (Copper sulfate, 200 mg.l^-1) water pollutants. We also evaluated the field influence of the E. crassipes 1% suspension on growth traits, some biochemical parameters, immunoglobulin concentrations, some antioxidant enzymatic activity, cortisol hormone levels, and intestinal microbiota in broiler chickens challenged once weekly with polluted/contaminated (calcium sulfate; 75 mg.l^-1, copper sulfate; 200 mg.l^-1, and S. Typhimurium; 1.5 × 10⁶ CFU.ml^-1) water.

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Materials and Methods

Study location and duration

The laboratory (antimicrobial and chelating activities) trials were run in the Veterinary Public Health Department Laboratory from June to July 2022 and the field experiment was run in the broilers' units at Suez Canal University from August to September 2022. Water quality, production traits, carcass quality, and biochemical and bacteriological examinations were run in the Veterinary Public Health Department Laboratory. Water electrolytes, antioxidant enzymes, and cortisol and immunoglobulin concentrations were run in the University Hospital Laboratories. Heavy metal analysis was run in the Toxicology Laboratory at Suez Canal University.

Eichhornia crassipes preparation

The procedures were run after Sarkar et al. (2017) with some modifications. Eichhornia crassipes leaves were obtained from the Ismailia canal in polyethylene bags. The leaves were washed with de-ionized water four to five times, cut into pieces, sun-dried for 24–72 hours, and minced in a mortar (Granite-Rock). The minced powder was extra-dried in a hot air oven (Daihan^®, Indonesia) at 80°C/24–48 hours. The powder was stored in a dark bottle.

Pollutant suspensions preparation

Inorganic calcium sulfate dihydrate (Spectrum^®, United States) and organic copper sulfate (HiMedia^®, India) powders were purchased. Calcium sulfate was dissolved at a rate of 75 mg.l^-1 in distilled water (Total hardness=2,100 mg.l^-1 CaCO₃; MPL < 75 mg.l^-1 and calcium hardness=1,600 mg.l^-1 CaCO₃; MPL < 60 mg.l^-1). Copper sulfate was dissolved at a rate of 200 mg.l^-1 in distilled water (Copper=5 mg.l^-1; MPL=1.3 mg.l^-1).

Propagation of bacterial and fungal isolates

The bacterial isolates were propagated after Soliman et al. (2021b, 2021c). Escherichia coli O157: H7 suspension (2.0 × 10⁴ CFU.ml^-1) was propagated into Mac-Conkey broth (Oxoid™) at 44°C/24 hours, eosin methylene blue agar (Oxoid^TM) at 37°C/24 hours, and tryptone soya broth (Oxoid™) providing 1.7 × 10¹¹ CFU. ml^-1 suspension. Lyophilized S. Typhimurium (3.4 × 10² CFU) was propagated into Rappaport-Vassiliadis broth (Oxoid™) at 37°C/24 hours, xylose lysine deoxycholate agar (Oxoid™) at 37°C/24 hours, and tryptone soya broth providing 1.5 × 10⁶ CFU.ml^-1 suspension.

The fungal isolates were propagated following Soliman et al. (2021b). Aspergillus niger and C. albicans clinical isolates were propagated into sabouraud dextrose broth (SDB; HIMEDIA^®) at 37°C/24–72 hours, sabouraud dextrose agar (SDA; Oxoid™) at 37°C/24 hours, identified with lactophenol cotton blue stain (Hardy Diagnostics^®), and resuspended in SDB broth, providing 2.5 × 10⁸ CFU.ml^-1 suspensions.

In-vitro antimicrobial activities of E. crassipes

A 96-well minimal inhibitory concentration technique was run to evaluate the antimicrobial efficacy of E. crassipes 1% on E. coli O157: H7 (1.7 × 10¹¹ CFU. ml^-1), S. Typhimurium (1.5 × 10⁶ CFU.ml^-1), A. niger (2.5 × 10⁸ CFU. ml^-1), and C. albicans (2.5 × 10⁸ CFU. ml^-1) after Elshikh et al. (2016) with a few modifications. The 96 wells were inoculated with 100 µl of E. crassipes 1% suspension. Wells were inoculated with 10 µl from microbial suspensions as follows: E. coli O157: H7 into C1–3 (n =24 wells), S. Typhimurium into C4–6 (n =24 wells), A. niger into C7–9 (n=24 wells), and C. albicans into C10–12 (n =24 wells).

After contact times (0.25, 0.5, 1.0, 2.0, and 4.0 hours), 10 µl of the mixes were added into 10 ml of buffered peptone water (Oxoid™). The tubes were mixed at 37°C/2 hours (Elshikh et al., 2016). 10 µl were dropped onto eosin methylene blue, XLD, and SDA agar at 37°C/24 hours. The specific colonies were counted (Quebec Darkfield Colony Counter). The antimicrobial efficacies (%) of E. crassipes 1% were calculated by proportionating the surviving colonies to the initial counts as follows:

$\frac{\mathrm{The}\mathrm{initial}\mathrm{microbial}\mathrm{count}\mathrm{–}\mathrm{Number}\mathrm{of}\mathrm{Surviving}\mathrm{organisms}}{\mathrm{The}\mathrm{initial}\mathrm{microbial}\mathrm{count}}\times 100$

In-vitro chelating activities of E. crassipes

1 ml of E. crassipes 1% suspension (1 g.l^-1) was dispensed into each of three triplets of conical flasks containing either 1 l of calcium sulfate or copper sulfate. The mixes were harvested after contact times (0.25, 0.5, 1.0, 2.0, and 4.0 hours) for determining total and calcium hardness levels and copper concentrations.

Total hardness levels were quantified using the titrimetric method against ethylene diamine tetra-acetic acid; EDTA (0.01 mol.l-1) with aerochrom black-T indicator (American Public Health Association, 2012), calcium hardness levels were quantified using titrimetric method against EDTA with murexide powder indicator (American Public Health Association, 2012), and copper concentrations were quantified using atomic absorption spectrophotometer (American Public Health Association, 2012). The chelating efficacies (%) of E. crassipes 1% were calculated by proportionating the final to the initial concentrations.

In-vivo effectiveness of E. crassipes

Experimental birds’ housing and biosecurity

We used 200 1-day-old Ross^®308 broiler chicks from El-Helal Company—Egypt. We weighed the chicks on their arrival (W₀) and randomly divided them into 4 groups (50 birds each, 5 replicates of 10 birds) into units with a hay-deep litter system after Soliman and Hassan (2020b). The units were optimized with biosecurity measures following Soliman and Abdallah (2020a). These measures were foot dips of crude carbolic acid 5%, fly-proof nets, wild bird-proof entrances, secured food storage areas, proper interior arrangements to facilitate daily observation; feeding; and watering act, wire baited traps to discourage rodents, dry cleaning using brooms, wet cleaning using glutaraldehyde, disinfection using povidone-iodine 7.5%; sodium hydroxide 5%; and formaldehyde spraying respectively.

The units were supplied after Soliman and Hassan (2019) with a continuous lighting program (23 hours L and 1 hour D) using a white light-emitting diode of 20 W. The ventilation act was served by negative cross-ventilation using side-wall V-shaped windows (air inlets), ceiling fans for proper air distribution, and side-wall suction fans across the rooms (air outlets). The units' floors were covered with a thin film of a chemical litter treatment (superphosphate) at a rate of 0.5 g.m^-2 after Soliman et al. (2018) to maintain litter abiotic conditions.

Experimental birds' microclimate and management

The room's microclimatic temperature was optimized at 35°C by turning on indirect heat sources (halogen and oil heaters) following Soliman et al. (2021a). This temperature was sufficient for brooding. Starting from the eighth day of the rearing trial, the temperature was reduced by 0.5°C daily (3.5°C/week) until achieving 25°C–28°C/third week of the trial. Chicks were given ad libitum access to the experimented treated or non-treated drinking water. The dietary requirements of the broilers were met by corn-soybean ration (El-Eman company, Egypt) following the National Research Council (1994), and Applegate and Angel (2014) modifications. The nutritive ingredients of the used starter (1–14 days) and the grower (15–38 days) rations were protein (22.5% and 21%), energy (2,930 and 3,150 kcal), crude fiber (3.79% and 3.35%), and fat (5.4% and 2.67%), respectively.

Broilers were massively immunized via dechlorinated drinking water vaccination act that was served after 2–4 hours of water deprivation. Birds were vaccinated against infectious bronchitis (7th day), infectious bursal disease (14th and 21st days), and Newcastle virus disease (16th and 26th days). Close observation was followed for the early detection of any abnormalities and mortalities. The experiment was designed to last for 38 days during which the daily prevailing microclimatic conditions were recorded.

Eichhornia crassipes treated water and challenges act

Broilers were supplied daily with E. crassipes (1 g.l^-1) treated water for 4–6 hours, starting from the third-day-old throughout the experiment (38 days). Meanwhile, polluted water was supplied to groups as follows: G1 with calcium sulfate (75 mg.l^-1) polluted water, G2 with copper sulfate (200 mg.l^-1) polluted water, G3 with S. Typhimurium (1.5 × 10⁶ CFU.ml^-1) contaminated water, and G4 with non-polluted tap water (control negative). Polluted/contaminated water was supplied to birds weekly on the 7th, 14th, 21st, 28th, and 35th days. The repetition of the challenge was designed to evaluate the effectiveness of the E. crassipes to neutralize the impact of overwhelming challenges in broiler farms. On the challenge day, water deprivation was run for 2–4 hours to ensure the weekly challenge act, and the E. crassipes treated water was supplied following the challenge.

Performance indices (PIs)

Representative simple random samples were harvested from broilers' groups (44 birds/ group) on their arrival and weekly for weighing the live body weight (LBW.g^-1) with a digital scale (WONHENG^®, China). We calculated the sample size at a 95% confidence level following Thrusfield and Christley (2018) as follows:

n=(Z_{1- α/2})².p (1-p)/d²

where (n) is the samples' number, (Z_1-α/2) is the normal standard, (p) is the probable population proportions, and (d) is the absolute precision.

The PIs were calculated following Soliman and Hassan (2017). The weight gains (WG.g^-1) are the differences between week-ending weights (Wx) to initial weights (W₀). Feed (FI.g^-1) and water (WI.ml^-1) intakes are the proportions between the amounts consumed in a group to the number of surviving birds in such a group. The feed conversion ratios (FCR) are the proportions of FI.g^-1 to WG.g^-1, while the PI is the proportion of LBW.kg^-1 to FCR.

Sampling

We collected 1,914 samples divided into 570 including 90 E. crassipes pollutants mixes (3 replicates × triplets × 2 pollutants × 5 sampling times) and 480 E. crassipes microbial mixes (96 well × 5 sampling times) from the in-vitro study, and 1,344 including 192 sera, 192 duodenal swabs, and 960 tissues (breast muscles, liver, spleen, heart, and bursa of Fabricius) from 5 bird groups after the in-vivo study. Nine ml buffered peptone water (Oxoid™) were used as a vehicle for the duodenal swabs.

We sacrificed 192 broilers (48/group) by the end of the clinical trial (38th day) via slaughtering with decapitation for harvesting blood samples on serum tubes (BD Vacutainer^®). Samples were warmed in a water bath (Thermo^®, Germany) at 28°C/20 minutes and centrifuged (Fisher^® CL10) at 3,500 rpm/15 minute. Sera were pipetted (Thermo Scientific™) into Eppendorfs (2.5 ml) and stored at -20°C (Soliman et al., 2017). The carcasses were weighed (CW.g^-1) after de-feathering and evisceration. Breast muscles were incised for the bacteriological assessment. Edible organs (liver and heart) and immune organs (spleen, and bursa of Fabricius) were harvested and weighed (g). Carcasses were disposed of by burial with lime.

Biochemical profile and immunoglobulin concentrations

We analyzed sera (n =192) calorimetrically using ROCHE^® COBAS Integra 400 Plus chemical analyzer for some biochemical parameters [total protein (TP); g. dl^-1, alanine aminotransferase (ALT); IU.l^-1, urea; mg.dl^-1, creatinine (CREAT); mg.dl^-1, Glucose (GLUCO); mg.dl^-1, total cholesterol (TC); mg.dl^-1, and triglycerides (TGs); mg.dl^-1] and some antioxidant enzymatic activity [total antioxidant capacity (TAC); mM.ml^-1, malondialdehyde (MDA); nmol.ml^-1, and superoxide dismutase (SOD); U.ml^-1]. We analyzed sera also using ROCHE^® ELECSYS 1010 Immunoassay Analyzer for hormonal and immunological profiles (Cortisol hormone; mcg.dl^-1 and immunoglobulin G and M; mg.dl^-1).

Bacteriological examinations

Frozen breast muscle samples (n=192) were thawed, smashed in the stomacher (Seward^®, UK), and added to 9 ml of buffered peptone water. Breast muscles (n=192) and duodenal swabs (n=192) solutions were diluted up to (10^-8) after American Public Health Association (2017) to cover the contamination range. We analyzed the dilutions for aerobic total bacterial (TBC) onto standard plate count agar (Oxoid™), Enterobacteriaceae (TEC) onto eosin methylene blue agar, and Salmonella counts (TSC) onto CHROMagar^TM Salmonella (BD BBLei) using the drop plate (Kim and Lee, 2016) at 37°C/24–48 hours. Ideal colonies were counted on the Dark-field colony counter (Murray et al., 2015).

Statistical analysis

We analyzed the data using the Statistical Package for Social Sciences (SPSS version 27.0, IBM Corp, NY, USA) software package (SPSS, 2020). The multifactorial two-way analysis of variance was employed to determine the overall influence of the variables (water pollutants and broiler's age) and their interactions. The statistical model is as follows:

Y_ijk=µ + α_i + β_j + (αβ)_ij + Ɛ_ijk

where Y_ijk is the final measurement of the dependent variables; µ is the overall mean; α_i is the fixed effect of the pollutants, β_j is the fixed effect of the broiler's age, (αβ)_ij is the interactions, and Ɛ_ijk is the random error. We expressed microbial counts as logarithms (Log) using Microsoft Excel 2016. We expressed the results as highly significant at (p < 0.01), significant at (p ≤ 0.05), and non-significant at (p > 0.05).

Ethical approval

The laboratory and field clinical trials' designs and materials were approved by the Scientific Research Ethics Committee, Faculty of Veterinary Medicine, Suez Canal University–Ismailia, Egypt with approval number (2022025). Broilers were housed and approached in comfortable microclimatic conditions as much as possible to prevent the development of any overwhelming challenges other than the induced (Polluted and/or contaminated water). We minimized the number of birds as an understudy as possible and followed the laboratory animal rights (3Rs).

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Results

Water analysis

Eichhornia crassipes treated water in Table 1 revealed highly significant (p < 0.01) decreases in physicochemical such as electrical conductivity (df=17, F=442.2, and p=0.000), total dissolved solids (df=17, F=442.24, and p =0.000), total alkalinity (df=17, F=66.9, and p=0.000), sulfates (df=17, F=164.9, and p=0.000), nitrates (df=17, F=136.1, and p=0.000), and total hardness (df=17, F=138.4, and p=0.000), electrolyte such as sodium (df=17, F=17.9, and p =0.001), potassium (df=17, F=306.4, and p =0.000), calcium (df=17, F=12.0, and p =0.003), magnesium (df=17, F=20.7, and p=0.000), and chloride (df=17, F=42.8, and p=0.000), heavy metal such as iron (df=17, F=4.0, and p =0.063), copper (df=17, F=9.3, and p=0.007), and zinc (df=17, F=30.2, and p=0.000), and microbial assessments such as aerobic TBC (df=17, F=552.3, and p=0.000), TEC (df=17, F=110.5, and p=0.000), and TSC (df=17, F=10.8, and p=0.005) compared to tap water. Eichhornia crassipes treated water also revealed highly significant (p < 0.01) increases in the dissolved oxygen levels (df=17, F=43.0, and p =0.000). pH levels reveal no-significant differences (df=17, F=0.258, and p=0.619) in E. crassipes treated and tap water.

Table 1. Water analysis (Mean ± SE) in tap and E. crassipes treated water.

In-vitro adsorption and antimicrobial efficacies

Eichhornia crassipes 1% suspension reveals in Figure 1A 100% adsorption (chelating) activity (p < 0.01) for copper (Cu⁺², df=44, F=444.8, and p=0.000) and total (df=44, F=6,124.5, and p=0.000) and calcium (df=44, F=5,549.7, and p=0.000) hardness after 1-hour. Eichhornia crassipes 1% suspension also reveals in Figure 1B 100% killing (antimicrobial) efficacy (p < 0.01) against E. coli O157: H7 (df=119, F=13,843.9, and p =0.000), S. Typhimurium (df=119, F=2,595.2, and p =0.000), A. niger (df=119, F=7,738.1, and p=0.000), and C. albicans (df=119, F=8,773.4, and p=0.000) after 1, 2, 2, and 2-hour respectively.

Fig. 1. In-vitro adsorption and antimicrobial efficacy (Reduction percentage Mean ± SE) of E. crassipes 1% suspension. (A) Chemical adsorption action. Initial levels of total hardness=2,100 mg.l^-1 CaCO₃, calcium hardness=1,600 mg.l^-1 CaCO₃, and copper=5 mg/l. (B) Microbial survival. Initial levels of E. coli O157: H7=1.7 × 10¹¹ CFU.ml^-1, Salmonella Typhimurium=1.5 × 10⁶ CFU.ml^-1, A. niger =2.5 × 10⁸ CFU.ml^-1, and C. albicans =2.5 × 10⁸ CFU.ml^-1

Growth traits

Eichhornia crassipes 1% treated water reveals in Table 2 highly significant (p < 0.01) improvements of WG (df=3, F=11.1, and p=0.000), PI (df=3, F=35.8, and p=0.000), WI interactions (df=12, F=22.8, and p =0.000), WI/FI ratio (df=3, F=48.9, and p=0.000), FCR (df=3, F=24.5, and p =0.000), and FI (df=3, F=47.1, and p=0.000) in all treated broiler groups compared to the control. The overall means concerning age reveal in Table 2 highly significant (p < 0.01) improvements of WG (df=4, F=987.3, and p =0.000), FCR (df=4, F=120.6, and p =0.000), and PI (df=4, F=843.2, and p=0.000) in the fourth week, an d WI (df=4, F=1,021.8, and p=0.000) in the second week, as well as, highly significant (p < 0.01) decreases in FI (df=4, F=4,914.4, and p =0.000) in the second week.

Live birds’ weights and carcasses traits

Eichhornia crassipes 1% treated water reveals in Table 3 highly significant (p < 0.01) increases in LBW (df=3, F=191.1, and p=0.000) in G3 (broilers challenged with S. Typhimurium) compared to other challenged groups and the control. Carcass weights (CW, Table 3) reveal highly significant (p < 0.01) increases (df=3, F=256.2, and p=0.000) in G1 (broilers challenged with calcium sulfate) and G2 (broilers challenged with copper sulfate) with no significant differences between the two groups. Liver (df=3, F=126.0, and p=0.000), heart (df=3, F=89.8, and p=0.000), and spleen (df=3, F=355.3, and p=0.000) weights reveal highly significant (p < 0.01) increases in G3 compared to all other groups (Table 3). Bursa's weights (Table 3) reveal highly significant (p < 0.01) increases (df=3, F=348.0, and p=0.000) in G3 and G2 with no significant differences between the two groups.

Table 2. PIs (Mean ± SE) in different groups supplied with E. crassipes treated water (1 g.l^-1).

Biochemical, immunological, and hormonal profiles

Eichhornia crassipes 1% treated water reveals highly significant (p < 0.01, Table 4) improvements of the measured biochemical such as TP (df=3, F=60.6, and p=0.000), ALT (df=3, F=4,329.5, and p =0.000), urea (df=3, F=142.4, and p =0.000), CREAT (df=3, F=762.1, and p=0.000), GLUCO (df=3, F=1,197.9, and p=0.000), TG (df=3, F=17,063.2, and p=0.000), and TC (df=3, F=557.1, and p=0.000), and antioxidant parameters such as TAC (df=3, F=4,491.8, and p=0.000), MDA (df=3, F=1,625.8, and p=0.000), and SOD (df=3, F=640.0, and p =0.000) in all treated broiler groups compared to the control. We reported that E. crassipes 1% treated water reveals in Table 5 highly significant (p < 0.01, Table 5) increases in immunoglobulins G (df=3, F=112.5, and p=0.000) and M (df=3, F=36.3, and p=0.000) of G3 compared to the other treated groups and the control. Meanwhile, the cortisol hormone (Table 5) reveals highly significant (p < 0.01) decreases (df=3, F=358.0, and p=0.000) in all treated broiler groups compared to the control.

Bacteriological assessments

Eichhornia crassipes 1% treated water in Figure 2 reveals highly significant (p < 0.01) decreases in the TBC of intestinal swabs (df=3, F=29.9, and p =0.000) and breast muscles (df=3, F=13.2, and p =0.000), TEC of intestinal swabs (df=3, F=24.0, and p=0.000) and breast muscles (df=3, F=7.6, and p=0.000), and TSC (duodenal swabs) (df=3, F=5.0, and p=0.002) in all treated broiler groups compared to the control.

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Discussion

Water quality in broiler farms contributes to the general conditions of production, performance, and reproduction (Maharjan et al., 2016). Broilers hold an important position among food animals, thus requiring a healthy environment and water of high quality to contribute to good health and production (Magor et al., 2013). Drinking water in broiler farms can be easily polluted/contaminated with a variety of micro-organisms through the birds themselves or the surrounding sources contributing to a potential risk to the broilers and humans (Van der et al., 2016). According to Maes et al. (2019), the arriving pollutants concerning some micro-organisms such as Stenotrophomonas maltophilia, Pseudomonas geniculata, and Pseudomonas aeruginosa contribute to a lowering in the water quality and characterization.

Table 3. Live, carcass, and organ weights (Mean ± SE) in different groups supplied with E. crassipes (1 g.l^-1) treated water.

Table 4. Biochemical profile (Mean ± SE) in different groups supplied with E. crassipes (1 g.l^-1) treated water.

Table 5. Immunoglobulin concentrations and stress markers (Mean ± SE) in different groups supplied with E. crassipes (1 g.l^-1) treated water.

Conventional water treatments (coagulation, sedimentation, flocculation, membrane filter, irradiation, and adsorption) influence the water's physicochemical characteristics as recorded by Robinson et al. (2001) to achieve water of good quality at a high cost, and the treatment process wastes require special treatment or disposal and thus increase the cost. On the other hand, Mishra and Maiti (2017) revealed that the adsorption and phytoremediation processes are waste-free and provide higher advantageous water conditions compared to other treatments as they use natural and biological products such as rice husk, bark, coal, wood dust, tree bark powder, lignin, seaweeds, orange peel, banana peel, coconut pulp, and E. crassipes. Eichhornia crassipes is advantageous by higher adsorption capabilities in shorter time and accumulation (leaves, roots, and bulb) for long periods against chemical and biological pollutants (Thapa et al., 2016), and can be used in biogas production, feed product for fish and animals, microbial metabolism because of its high carbon contents, vermicomposting field, and in medicinal uses (Sharma et al., 2016).

Fig. 2. Logarithmic bacterial counts (Mean) in different groups supplied with E. crassipes (1 g.l^-1) treated water. G1=Broilers challenged with calcium sulfate (75 mg.l^-1), G2=Broilers challenged with copper sulfate (200 mg.l^-1), G3=Broilers challenged Salmonella Typhimurium (1.5 × 10⁶ CFU.ml^-1), G4=control broilers. TBCi=Total bacterial counts in intestinal swabs, TBCm=Total bacterial counts in breast muscles, TECi=Total Enterobacteriaceae counts in intestinal swabs, TECm=Total Enterobacteriaceae counts in breast muscles, TSC=Total Salmonella count in intestinal swabs.

Eichhornia crassipes reveals some capabilities in different studies such as enhancing the water quality characteristics, minimizing the pollutants' (heavy metals, and dyes) concentrations (Priya and Selvan, 2017), minimizing the biological oxygen demands (Wei et al., 2019), extract palm oil from mill waste in Malaysia, bio-indication for heavy metal pollution of rivers in Pakistan (Srivastava et al., 2019), Phyto-purification of wastewater (Victor et al., 2016), reduction of chromium levels (99.5%) in wastewater 15 days post-treatment (Saha et al., 2017), and extreme reduction of nitrates in wastewater combined with eutrophication (Wenwei et al., 2016).

Eichhornia crassipes treated water in the current study reveals highly significant declines in physicochemical, electrolytes, heavy metals, and microbial assessments and a highly significant increase in dissolved oxygen levels compared to tap water. The results agreed with those of Wuijts et al. (2018) who recorded that E. crassipes can actively minimize heavy metals and coloring agent concentrations from the water through bio-sorption, precipitation, and accumulation. Hartmann et al. (2018) also reported the high capabilities of E. crassipes to absorb dyes and heavy metals using electrostatic forces and improve water quality characteristics. Mahmood et al. (2010) recorded that E. crassipes water treatment contributed to a significant reduction of the biological and chemical oxygen demand (70.0%), the total solids (50.6%), and pH to a level that counteracts microbial survival. Also in agreement with our results, Dar et al. (2011) evaluated the capability of E. crassipes to treat different dilutions of wastewater (25%, 50%, 75%, and 100%) and recorded significant improvements in the physicochemical and biological parameters with significant increases of the dissolved oxygen levels. They recommended E. crassipes as an adsorbent to treat wastewater from agricultural, industrial, and municipal sources.

Eichhornia crassipes 1% suspension in the current results achieved 100% reductions of cobber and total and calcium hardness after 1-hour and 100% antimicrobial efficacies against E. coli O157: H7, S. Typhimurium, A. niger, and C. albicans after 1, 2, 2, and 2-hours, respectively. The results were synchronized with those of Vishwakarma et al. (2018) who reported that E. crassipes contributed to a shift in the pH up to 8.6 which is unfavorable to microbial growth and survival, and a significant reduction in the levels of total dissolved solids, conductivity, total hardness, biological and chemical oxygen demands, nitrate-nitrogen, and ammonium nitrogen in the treated water. Our results were also synchronized with those of Long et al. (2018) who revealed the strong abilities of E. crassipes for minimizing copper, cadmium, lead, zinc, chromium, and iron up to 97.3%, 94.8%, 94.7%, 96.8%, 94.3%, and 93.5%, respectively. Deng et al. (2018) recorded in agreement with the current results the strong antimicrobial activity of E. crassipes against Mycobacterium phlei, Candida parapsilosis, and Rhizopus oryzae.

The field clinical trial reveals highly significant improvements in PIs, live weights, carcasses quality, biochemical parameters, immunoglobulin concentrations, cortisol hormone, and bacteriological assessments in broilers supplemented with E. crassipes treated water compared to the control group. The results agreed with those of Che et al. (2020) who recorded similar capabilities of Eichhornia purpurea (100, 200, and 600 mg.l^-1) treated water which enhanced weight acquires and carcasses yield and they recommended E. purpurea (200 mg.l^-1) as a natural effective eco-friendly water treatment. Abdel Shafy et al. (2016) reported that E. crassipes collected from the Nile River in Egypt contain some nutritive ingredients such as protein (10.7%), fat (4.94%), crude fiber (17.9%), nitrogen (44.3%), calcium (1.42%), and phosphorus (0.58%) making it suitable as a feed supplement with enhanced weight gains and improved feed conversions, and potential adsorbent of heavy metals polluting the drinking water.

Jianbo et al. (2008) found an increased performance, FI, and egg production in ducks supplied with E. crassipes treated wastewater, and they attributed these improvements to the higher crude protein contents of the supplied E. crassipes. Rufchaei et al. (2021) documented an improvement in the marketing weights, weight gains, and growth rates in fish fed with 1.5% of E. crassipes than those fed 0%, 0.5%, and 1% of E. crassipes leaves powder. Dumaup et al. (2020) from another perspective revealed no significant effect on the broiler's performance except for FI when supplied with E. crassipes. Abdulganiyu et al. (2013) also recorded no significant differences in initial and final body weights, FIs, and FCR among day-old Isa Brown pullets that received E. crassipes (0%, 5%, 10%, and 15%) for 8 weeks.

The high recorded improvements in broilers supplemented with E. crassipes treated water in our study might be attributed to the highly digestible protein contents. Wimalarathane and Perera (2019) recommended the use of E. crassipes as a dietary supplement in animals and poultry for its nutritive value and low cost. Indulekha et al. (2019) also recommended the use of E. crassipes as a dietary supplement to increase the production in animals and poultry. The recorded prominent immune-stimulant, antioxidant activities, and the biochemical enhancements of E. crassipes treated water in our study could be attributed to its chemical composition that exhibits anti-inflammatory, antifungal, antibacterial, and anticancer functions. The results were synchronized with those of Guna et al. (2017) who showed an increase in DNA inhibition and DPPH radical scavenging properties of E. crassipes.

Eichhornia crassipes in our study could act as a phytoremediation agent and sanitize water from all the chemical pollutants and microbial contaminants used (challenges) contributing to the production of high-quality water, enhanced production, performance, and immunity. The same concept was concluded by Maharjan (2016) who reported that water entering the broiler farms usually is contaminated with a microbial load of up to 300 CFU.ml^-1, but hence the breeders disinfect water using chemical or eco-friendly alternatives contributing to enhanced performance. Jacobs et al. (2020) also reported that sanitized and/or treated water enhanced significant growth traits.

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Conclusion

Eichhornia crassipes 1% contributed to significant enhancements in the water's physicochemical characteristics such as declines of conductivity, total dissolved solids, total alkalinity, hardness, sulfates, nitrates, electrolyte (sodium, potassium, calcium, magnesium, and chloride), heavy metals (iron, copper, and zinc), and microbial counts (TBC, TEC, and TEC). Eichhornia crassipes 1% was also able to significantly increase the levels of dissolved oxygen. Eichhornia crassipes 1% suspension exhibited 100% adsorption activities for copper (5 mg.l^-1) and total (2,100 mg.l^-1 CaCO₃) and calcium (1,600 mg.l^-1 CaCO₃) hardness after 1-hour, and 100% antimicrobial activities against E. coli O157: H7 (1.7 × 10¹¹ CFU.ml^-1), S. Typhimurium (1.5 × 10⁶ CFU.ml^-1), A. niger (2.5 × 10⁸ CFU.ml^-1), and C. albicans (2.5 × 10⁸ CFU.ml^-1) after 1, 2, 2, and 2-hours respectively.

The clinical trial showed that E. crassipes 1% suspension significantly improved PIs (LBW, WG, FI, WI, FCR, and PI), carcasses quality, biochemical parameters, antioxidant enzymatic activities, immunoglobulin concentrations, cortisol hormone, and intestinal microbiota in all treated broilers. Thus, E. crassipes 1% could be used with great potential as an eco-friendly alternative for the water and wastewater treatment processes to reduce waste and treatment costs and provide broilers with water of good quality.

Authors’ contributions

ESS deliberated and executed the laboratory and field trails' design, run water physicochemical analysis, water, and broiler samples' microbial examination, participated in sera samples analysis for biochemical and antioxidant profiles, engaged in writing the manuscript, and reviewed the submitted version. RHA calculated and judged the growth traits, determined carcass characteristics, analyzed sera for biochemical and antioxidant profiles, and collaborated in the manuscript writing. DSF took a part in the water physicochemical analysis, biochemical analysis, and microbial assessments, and collaborated in the manuscript writing.

Acknowledgment

We would like to thank Prof. MA. Sobieh for his directions during the study and OF Mohamed for her help in the laboratory examinations.

Funding

The authors declare that the study was self-funded without receiving financial aid from the affiliating institute or any funding agency.

Conflict of interest

The authors declare that they have no competing interests or personal relationships that could influence the work reported in this manuscript.

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