Unlocking the potential of biosynthesized zinc oxide nanoparticles for degradation of synthetic organic dyes as wastewater pollutants

The azo dyes released into water from different industries are accumulating in the water bodies and bioaccumulating within living systems thereby affecting environmental health. This is a major concern in developing countries where stringent regulations are not followed for the discharge of industrial waste into water bodies. This has led to the accumulation of various pollutants including dyes. As these developing countries also face acute water shortages and due to the lack of cost-effective systems to remove these pollutants, it is essential to remove these toxic dyes from water bodies, eradicate dyes, or generate fewer toxic derivatives. The photocatalysis mechanism of degradation of azo dyes has gained importance due to its eco-friendly and non-toxic roles in the environment. The zinc nanoparticles act as photocatalysts in combination with plant extracts. Plant-based nanoparticles over the years have shown the potential to degrade dyes efficiently. This is carried out by adjusting the dye and nanoparticle concentrations and combinations of nanoparticles. Our review article considers increasing the efficiency of degradation of dyes using zinc oxide (ZnO) nanoparticles and understanding the photocatalytic mechanisms in the degradation of dyes and the toxic effects of these dyes and nanoparticles in different tropic levels.


INTRODUCTION
The rapid increase of population affects the availability of pure and clean water. The pollutants and dyes become bioaccumulated in water bodies and the environment because of the release of pollutants into the nearby water bodies without any preprocessing and degradation. This has become a major concern for developing countries. The need to use safe and clean water leads us to approach methods of getting clean water (Brame et al. 2011;Jain et al. 2021).
Nanoparticles have many applications in the field of cosmetics, medicine, and wastewater management, etc. Nanoparticles can be synthesized from synthetic and biological sources with different elements such as silver, gold, copper, zinc, iron, etc. The chemical methods include two methods as: the bottom-up and top-down approaches which are costly and toxic. To cut down the expenses and reduce toxic effects, researchers have approached green synthesis methods (Agarwal et al. 2017). The biological sources for the generation of nanoparticles include the synthesis of nanoparticles from microorganisms, fungi, plants, etc. via phytochemicals. The phytochemicals consist of polyphenols, terpenoids, quinine, proteins, saponins, and alkaloids (Figure 1(a)). The phytochemicals are found in different parts of medicinal plants, fruits, nuts, cereals (Kurmukov 2013). These phytochemicals have many applications such as antibacterial, antiviral, catalytic properties, and many more. It also provides a route for the biosynthesis of nanoparticles through the green approach.

DEGRADATION OF DYES FROM ZnO NANOPARTICLES SYNTHESIZED BY MICROORGANISMS
Biodegradation of dyes using microorganisms is becoming increasingly popular due to the easy availability, cost-effective nature, and ease of growth under laboratory conditions. However, one of the significant reasons for the biological synthesis of ZnO nanoparticles is the existence of biologically active metabolites or enzymes that could be engaged as reducing agents during dye degradation (Khanna et al. 2019. Several dyes like methylene blue, methylene orange, have been degraded by ZnO nanoparticle synthesis using algae, bacteria, and fungi (Table 4).

ORGANIC DYES PRESENT IN WATER BODIES AND MECHANISM OF TOXICITY
The azo dyes are synthetic organic dyes with an azo functional group. Azo dyes are grouped into different classes as reactive dyes, disperse dyes, acidic dyes, basic dyes, direct dyes, vat dyes, sulfur dyes, and solvent dyes (Saratale et al. 2011;Madhushika et al. 2020). The synthetic dyes consist of 70% azo dyes in the textile, food, cosmetics industries, etc. Azo dyes are toxic in nature, acting as carcinogens, genotoxins and mutagens (Ventura-camargo & Marin-morales 2013) (Table 5).
Textile dyes get mixed with industrial pollutants and are highly toxic and carcinogenic. These dyes are also toxic to the biological treatment units, thereby making the treatment of these dyes extremely complex (Tunçal & Kaygusuz 2014). The toxic dyes accumulate in sediments and soil, which then transport the water systems. The dyes assimilate in fish gills and accumulate in tissues. The azo dyes of chromium complexes damage the growth and development of plants (Lellis et al. 2019). The UV or chlorination process is efficient in degrading dyes from wastewater, but it has not been used in water bodies like ponds and lakes (Nikravesh et al. 2020). The toxic effects of dyes can be observed in aquatic animals like Xenopus laevis and Danio rerio (zebrafish) and can cause developmental stage and embryonic damage, respectively. Xenopus laevis tadpoles at the 46 th stage of development were exposed to six textile dyes, mainly Astrazon Red, Astrazon Blue, Remazol Red, Remazol Turquoise Blue, Cibacron Red, and Cibacron Blue FN-R for 168 h in static conditions. The dyes caused oxidative stress, and the presence of organic pollutants caused increased levels of the glutathione S-transferase (GST) enzyme. The exposure of fishes to Metanil yellow causes increased GST enzyme activity in the liver and intestinal tissues (Güngördü et al. 2013). Zebrafish were exposed to textile dyes such as Maxilon blue 5G and Reactive blue 203 for 96 h, which caused acute toxicity and embryonic damage. The dyes have induced DNA damage and deformities in fish like curved body axis, tail malformation, and reproductive damage. Limited studies have been done on the mechanism of DNA damage caused by textile dyes (Köktürk et al. 2021). The toxic effects of textile dyes (Optilan yellow, Drimarene blue, and Lanasyn

Pecan
Carya illinoinensis 10 g of leaves are cut into pieces, ground into a paste, and soaked in 100 mL of deionized water in a 250 mL glass beaker. The solution was heated at 70°C for 30 min using a magnetic stirrer until the color of the solution changed. The aqueous leaf extract was left to cool down at room temperature, filtered using Whatman No. 1 filter paper, and centrifuged at 7,000 rpm for 30 min.
Olive Olea europaea 10 g of O. europaea leaves were mixed with 100 ml of deionized water. The mixture was heated at 60°C for 30 min using a stirrer heater. The resulting product was filtered.

Moringa
Moringa oleifera 5 g of leaves were washed thoroughly with distilled water, and the surfaces of leaves were sterilized using alcohol. These leaves were heated for 40 min in 100 ml of distilled water at 50⁰ C. Then, the extract was filtered with Whatman No. 41 filter paper.

Aloe barbadensis miller
Small pieces of peel were cut and grounded with pestle mortar in distilled water to make an aqueous solution of peel extract. The aqueous solution was filtered with Whatman filter paper No. 1 to remove debris.
Tannins, saponins, flavonoids, Weldegebrieal (2020) Okra Abelmoschus 10 mL of leaves mixed in 0.01 mol zinc acetate dihydrate was hydrolyzed with the 0.01 mol sodium hydroxide with the leaf extract, pH is adjusted to the basic at 9-11, then cool at room temperature. Centrifuge at 7,000 rpm for 10 min. Alkaloids, flavonoids, saponins, reducing sugars Weldegebrieal (2020)

Coriander
Coriandrum sativum 10 g Coriander leaf powder was dissolved in 100 ml of distilled water and stirred at 100°C for 15 min. The solution was then filtered with a 1.5-micron Whatman filter paper No. 1. 20 g of fruit was added in 100 mL of distilled water and boiled for 5 min. after boiling, the color of the aqueous solution was dark brown, and the mixture was allowed to cool to room temperature.

Mangosteen
Garcinia mangostana 8 g of fruit pericarps in 100 mL water, heated at 70-80°C for 20 min and then filtered.
Red powder puff

Calliandra haematocephala
Air-dried leaves were mixed with water in a 1:20 weight proportion and were heated in a dry-bath at 80°C for 15 min, to yield a thin pale-yellow soup of the leaf extract Tectona grandis 20 g of leaves were collected, weighed, washed under tap water. Collected leaves were cut into fine fragments and placed into a round-bottomed flask with 100 ml of double deionized water. The whole reaction mixture was heated at 60°C for 1 h and filtrate was obtained employing Whatman No. 1 filter paper.

Sageretia thea
The aqueous solution of leaves mixed with a zinc nitrate solution.
Fresh peels were washed, dried at 50°C in the oven, then 3 g in 40 mL DDW:20 mL EtOH solvent was heated at 80°C for 10 min and then filtered.
Polyphenols, flavonoids, alkaloids, tannins, saponins (EtOH extract) Weldegebrieal (2020) Golden shower Cassia fistula 1:10 proportion of the coarsely powdered plant material to water was taken in a round-bottomed flask, and the extraction was carried out at 100°C with are flux arrangement for 5 h with constant stirring. The extract was filtered and centrifuged. Terpenoids, phenolic acid, flavonoids, proteins Weldegebrieal (2020) Garlic Allium sativum Fresh and finely sliced bulbs were boiled at 70-80°C for 20 min and then filtered.
Ganesh et al. (2019) Onion Allium cepa 5 g of dry brown outer onion peel were washed with tap water, followed by rinsing with distilled water and soaked in 50 mL of double-distilled water. The solution was boiled at 70°C for 15 min. The peel broth was filtered through Whatman No. 1 paper.
Phenolic compounds, proteins, and amino acids Rajkumar et al. (2019) Parsley Petroselinum crispum 20 g of fresh leaves of parsley were extracted in100 mL ultrapure water by refluxing for 60 min.
Vitamins (beta-carotene, thiamin, riboflavin, and vitamins C and E), fatty acids, volatile oils Stan et al. (2015) Loquat Eriobotrya japonica 25 g of the seed powder was mixed with 100 mL deionized water. The mixture was then stirred on a magnetic hotplate stirrer at 40°C for 60 min. Then, the supernatant was collected by Whatman No. 1 filter paper.
Phenolics, alcohols, sugars, and proteins Shabaani et al. (2020) Malabar cardamom Amomum longiligulare 25 mg of powder were diluted in 100 ml of distilled water, and the suspension was autoclaved for 30 min at 100°C to obtain an aqueous solution of extract. The extracts were centrifuged at 5,000 rpm for 10 min and filtered using Whatman No. 1 filter paper.

MECHANISMS OF DYE REMOVAL USING ZnO NANOPARTICLES
There are different types of synthetic textile dyes which include azo dyes, basic dyes, acidic dyes, nitro dye, disperse dye, vat dyes, direct dyes, mordant dye, reactive dye, solvent dye, reactive and sulfur dyes. The photocatalytic degradation mechanism is the same for all synthetic dyes, but they differ in the degraded product released. The zinc nanoparticles synthesized using medicinal plants are the mechanism responsible for degradation of azo dyes with the help of biosynthesized ZnO NPs under sunlight and UV irradiation. ZnO is a photocatalyst that helps in the degradation of dyes present in wastewater. ZnO nanoparticles are cost-efficient, and highly photoactive in the UV region. The plant extracted nanoparticles affect the morphology, and concentration of oxygen vacancies. The involvement of phytochemicals in nanoparticle synthesis increases the efficiency of ZnO NPs (Weldegebrieal 2020). Recent studies however showed that neither visible LEDs nor UV C irradiation resulted in photocatalytic bisphenol-A degradation. The inability of the amalgam UV C lamp to promote photocatalytic reactions in the vertical position could be attributed to either spatial photon emission caused by mercury gas settlement or photon-nanoparticle interaction at an incorrect collision angle. The vertically positioned UV LED-based illumination system, however, achieved a high energy consumption efficiency. Bisphenol-A was removed in the same manner as in the horizontally positioned reactor configuration, although the energy efficiency was almost unchanged (Tunçal 2020). ZnO nanoparticles were dispersed in an azo dye mixture in water in a ratio of 1:10 (w/v pH ¼ 6) under sonication for 20 min. The mixture was irradiated by sunlight, and absorption can be measured at regular intervals to observe the degradation of dyes. When light passes through the solution, electron-hole pair generation takes place (Equations (1) and (2)). The oxidation and reduction process creates free radicals and superoxides (Equation (3)) (Fageria et al. 2014). The OH functional group present in nanoparticles interacts with dye particles to generate OH free radicals (Equation (4)). The detailed mechanism of the photolytic dye degradation mechanism is shown below: The azo dye solutions can be prepared by making a stock solution of the desired concentration. This dye mixture has varying concentrations of nanoparticles in normal pH and is exposed to light. The concentration reduction can be calculated using Equation (5): In the equation, A₀ depicts the initial concentration of the dye solution, and A depicts the final concentration of the dye solution after the photocatalytic process (Fageria et al. 2014).

FACTORS AFFECTING DYE DEGRADATION
The degradation process can be affected by many factors, like size, pH, temperature, aeration, catalysts, dopants, and oxidation. The surface area plays an important role in catalysis reaction, and a bigger particle size inhibits the reaction (Talebian et al. 2013). The photodegradation observed in nano-size particles was more efficient than micro-size particles because of the high surface area and more availability of active sites (Ateeq 2012). The effect of pH on photodegradation was measured in the ranges 4, 7, and 10. The methyl orange dye was removed efficiently at pH ¼ 7, and pollutants were efficiently removed at pH ¼ 4 (Abbasi & Hasanpour 2017). ZnO at higher pH efficiently degrades anionic dyes such as Congo red. Photodegradation was better at pH ¼ 10 than pH ¼ 7 due to the high concentration of hydroxyl ions (Adam et al. 2018). The temperature varies for each dye to be degraded. The degradation of direct red-23 dye at different annealing temperatures showed different efficiencies of degradation, which include temperatures in the range of 400°C, 500°C, 700°C, and 800°C, and photodegradation efficiencies were 88.48%, 95.49%, 92.63%, and 86.40%, respectively. It was observed that at 600°C annealing temperature, complete degradation was observed (Umar et al. 2015). The synthesis of ZnO in the low-temperature reaction was efficient in Congo red degradation (Ong et al. 2016). The Reactive green 19 dye showed 77% degradation under 7 h in the presence of aeration, but in the absence of aeration, it resulted in the extension of reaction time to 8 h and a decrease in degradation efficiency to 56% (Lee et al. 2017). The ZnO nanoparticles extracted from Prosopis juliflora leaf extract degraded methylene blue with an efficiency of 99% under UV illumination and continuous aeration (Sheik Mydeen et al. 2020). The efficiency of degradation was directly proportional to the catalyst concentration. The reaction could not be proceeded due to the low catalyst surface. The degradation efficiency of decomposing methyl orange gradually increased from 50, 200, and 1,000 nm ZnO photocatalysts at pH ¼ 10 (Wang et al. 2007). With increased catalyst concentration, it opens up more active sites for interaction with the dye solution. The methylene blue degradation was higher at a high concentration of catalyst (de Moraes et al. 2018). The concentration of dopants was inversely proportional to degradation efficiency. Metals like Ni, Co, and Ti act as doping metals and affect degradation efficiency (Mohseni-Salehi et al. 2018). Silver-doped (2%) ZnO nanoparticles used to degrade Brilliant green dye resulted in higher photocatalytic efficiency than alone (Gnanaprakasam et al. 2016). The photolytic degradation of Brilliant green was 99% in the presence of TiO₂ (Munusamy et al. 2013). The photocatalytic degradation of Acid orange 7 dye follows first-order kinetics under the presence of hydrogen peroxide and sodium periodate (Sadik 2007). The degradation efficiency of dye decreased with an increase in dye concentration, and the rate increased with C oxidant /C dye ratio (Madhavan et al. 2006). The behaviour of ZnO suspensions was investigated at pH values ranging from 3 to 11 in order to investigate pH variation, the effect of dissolution on the zeta potential, and aggregate size stability. For all three constituents, the most stable pH region achieved in 1 hour corresponds to an initial pH of 7.7 (Fatehah et al. 2014).

ADSORPTION
The adsorption of dyes is essential to the efficient degradation of dyes. Several workers suggested that there was no relationship between adsorption of dyes and degradation in which they have used both anionic and cationic dyes (Liu et al. 2013). However, ZnO nanospheres were prepared by using a hydrothermal method and used efficient azo dye (Bismarck brown) in which different parameters affected the adsorption of dye and degradation by ZnO (Zaidi et al. 2019). Malachite green, Alexa fluor, and Congo red adsorbed a maximum of 2,963, 3,307, and 1,554 mg/g, respectively, on ZnO nanoparticles. The temperature and pH play an essential role in the adsorption process. The adsorption process was maximized by chemical precipitation, electrostatic attraction, and hydrogen bonding between the ZnO nanoparticle and different dyes (Zhang et al. 2016). ZnO supported with activated carbon or brick grain particles using the simple co-precipitation method resulted in a higher adsorption capacity for Malachite green and Congo red dyes (Raizada et al. 2014).

THE COMBINED ACTION OF ADSORPTION AND PHOTOCATALYTIC ACTIVITY OF ZINC OXIDE NANOPARTICLES IN DYE DEGRADATION
Adsorption and photocatalytic activity of the ZnO nanoparticles showed a higher efficiency to degrade the dyes. ZnO/Agmontmorillonite nanoparticles with Urtica dioica leaf extract increased the discoloration of methylene blue from 38.95 to 3300 91.95% (Sohrabnezhad & Seifi 2016). ZnO-CuO thin films were prepared by carbothermal evaporation with ZnO and Cu, and photocatalysis of methyl orange and methylene blue was observed in visible and UV light (Kuriakose et al. 2015). ZnOgraphene nanocomposites using grape and Eichhornia crassipes leaf extract degraded Rhodamine B dye efficiently with 70.0% and 97.5% degradation rate (Ramanathan et al. 2019). ZnO nanospheres generated by the hydrothermal method followed the pseudo-first-order rate reaction, degraded Bismarck brown dye with an efficiency of 94% after 2 hours of exposure (Zaidi et al. 2019). ZnO-graphene obtained by the hydrothermal process degraded Azure B dye 99% within 20 minutes of exposure under UV illumination (Rabieh et al. 2016). Strobilanthes crispus (B.) leaf extract fabricated with La 2 CuO 4 -decorated ZnO were capable of degrading Malachite green dye following pseudo-first-order kinetics (Yulizar et al. 2020).

MITIGATION OF ZnO NANOPARTICLE TOXICITY
Zebrafish share 70% of their genes with human beings. ZnO nanoparticles damage neural and vascular systems. Dissolved Zn causes less damage to the nervous system than chemically synthesized ZnO particles. Zebrafish when exposed to dissolved oxygen matter (DOM) water with ZnO nanoparticles, it converted to zinc ions that are toxic. ZnO damages the hatching rates and degrades the DOM (Kteeba et al. 2018). The ZnO nanoparticles synthesized from plant extracts are more efficient and eco-friendlier than chemically synthesized nanoparticles. Plant extracts were used as a reducing and stabilizing agent, and zinc nitrate can be used as a zinc precursor. ZnO nanoparticles are exposed to sunlight and electrons become excited from the valence band to conduction band resulting in the formation of superoxides and hydrogen oxide radicals which are potent reducing agents that are capable of degrading dyes, resulting in a less toxic degradation products such as carbon dioxide and water (Sharma et al. 2021).

FATE AND TOXICITY OF ZINC OXIDE NANOPARTICLES
In today's world, we use nanoparticles in all fields that lead to the accumulation of ZnO nanoparticles. When nanoparticles reach the water bodies, it leads to aggregation of nanoparticles that in suspension form, converted to zinc ions that induce toxicity (Beegam et al. 2016). ZnO nanoparticles enter the soil, and are converted to Zn 2þ in soil and plants. The soluble Zn was more toxic than ZnO nanoparticles .
ZnO nanoparticles affect soil, water bodies, the environment, and human health. Zn deficiency in humans leads to severe anemia, weakening of the immune system, inflammation, and lung toxicity due to inhalation of ZnO nanoparticles (Beegam et al. 2016;Rajput et al. 2018). The toxicity of ZnO nanoparticles can be observed in mammalian cells, bacteria, and zebrafish. In bronchial epithelial cell lines such as BEAS-2B and A549 cells, ZnO nanoparticles induce cytotoxicity and mitochondrial dysfunction. ZnO had both short-term and long-term effects on mammalian cells, and short-term effects included apoptosis, whereas long-term effects included increased ROS generation, and decreased mitochondrial activity (Vandebriel & De Jong 2012). The nanoparticles are toxic to both Gram-positive and Gram-negative bacteria as nanoparticles are bactericidal at the log phase of bacterial growth, and cell viability/membrane integrity was lost after 15 h of exposure. (Reddy et al. 2007). When ZnO nanoparticles enter the marine environment this leads to ROS production, toxicity in zebrafish embryos, and damage in their hatching enzyme due to hypoxia caused by ZnO nanoparticles (Bai et al. 2010) (Yung et al. 2014).

MECHANISM OF TOXICITY OF ZnO NANOPARTICLES IN IN VITRO MODELS
The highly toxic deposition of dyes in water bodies stops the oxygenation capacity of water and blocks sunlight from penetrating inside, affecting the biological activity of aquatic life and the photosynthesis process of aquatic plants. The dyes keep on accumulating in the sediments, in fishes, or other marine organisms. Decomposition of dyes into water bodies are carcinogenic or mutagenic compounds that cause allergies, skin irritation, or different tissue changes. The toxicity was observed in mammalian cells, which mimics human cell conditions in which briefly the authors have reported that ZnO induces cytotoxicity but not Zn 2þ . To understand the mechanism, they performed in vitro studies on bronchial epithelial cell lines, dermal cell lines, colon cell lines, and immune cells (RAW264.7). The ZnO nanoparticles dissolved the extracellular membranes of the cells and were converted to Zn 2þ that leads to lysosome destabilization. As zinc ion concentration increases inside the cells, it leads to a decrease in enzyme and transcription factors. Proteins such as bovine serum albumin adsorb to the surface of ZnO. ZnO damages the cells, which leads to calcium flux, ROS generation, membrane damage, and mitochondrial dysfunction (Vandebriel & De Jong 2012). Nanoparticles induce toxicity in both Gram-positive and Gram-negative bacteria and caused cell proliferation in cancer cells. The ultra-sonication of HL60 cancerous cell lines induced lipid peroxidation, which leads to enhancement of the mechanism in the presence of ZnO nanoparticles (Premanathan et al. 2011). E. coli is the most common pathogen that can be used to see toxicity mechanisms. The ZnO nanoparticles dissolve in medium and release Zn ions. High concentrations of ZnO nanoparticles damage physiological features, decreases toxicity tolerance levels, and causes deformation in the cell membrane, and leaking out of intercellular substances. The release of zinc ions leads to maximum toxicity   (Figure 2).

CONCLUSION AND FUTURE OUTLOOK
Organic pollutants such as azo dyes are common environmental pollutants, and considered hazardous materials for human health. Photocatalytic activity plays a significant role in the degradation of organic derivatives by ZnO and nanoparticles. ZnO nanoparticles have high thermal conductivity that helps in the removal of azo dye pollutants. Researchers have efficiently degraded azo dyes using plant-based nanoparticles. The photocatalytic degradation is enhanced by electron-hole pair generation. The nanoparticles generated from plant extracts help to increase the efficiency of degradation of dyes. Recently, several investigations have shown that plant extract nanomaterials have highly improved the photocatalytic efficiency compared to nanoparticles alone. To make nanoparticles, an appropriate compound, and an exclusive photocatalyst, researchers have tried different phytochemical concentrations to optimize the size of particles and surface area. Research has been carried out on numerous kinds of plant extract on ZnO as a photocatalyst by several approaches and the application in photodegradation of organic pollutants. The mechanism of toxicity is understood because of the interaction between the ZnO nanoparticles and different surfaces like bacteria, aquatic animals, and human cells. The deposition of nanoparticles damages cells, resulting in oxidative stress and is lethal to the environment and living organisms. The exact mechanisms of toxicity have still not been explored. With these pollutants either dyes or nanoparticles accumulate in water bodies and animal systems and this could pose a severe threat to health, especially in developing countries where water is consumed without much analysis or testing. Hence future posts should address new challenges for the application of ZnO in various emerging fields as well as well the identification of effects of these pollutants at the DNA level and how mutagenic they can be. However, these ZnO nanoparticles have been approved by the US FDA and are generally recognized as safe (GRAS), these nanoparticles can have huge prospects in the future towards biomedical, agricultural, and environmental remediation fields.

DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information. Water 3302