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Major heavy metals contaminants of our environment are (Cd) cadmium, (As) arsenic, (Cr) chromium, (Hg) mercury, (Cu) copper, and (PB) lead. Accumulation of these heavy metals in soil and water are due to industrial and agricultural activities. Heavy metals are nonbiodegradable metallic elements so they accumulate in soil and water bodies with time. They have relatively high density. When present in soil and water above prescribed limits of concentrations then they are very toxic. Agricultural and industrial activities play a major role in the heavy metals’ accumulation in our environment, sugar mill involve both of these activities. In the production sugar cane, farmers consume large quantities of insecticides, herbicides, pesticides, chemical fertilizers and other medicines to enhance their crops production and as a result, sugar mill effluents have high amounts of heavy metals contaminants in it [1].

Impact of sugar mill effluent on soil and seed germination of tomato plants was studied by Pakistani researcher and found that average heavy metal (Cr, Pb, Cd, and Cu) concentrations in sugar mill effluent were greater than WHO standard. Rate of seed germination drops with increasing concentrations in irrigated water [2]. Bioremediation is process in which we use microorganisms and plants to degrade or remove toxic and hazardous pollutants from our environment [3]. Heavy metals presence in soil and water originates from anthropogenic and natural activities. When industrialization combined with urbanization then it enormously enhances heavy metals presence in our environment. These toxic contaminants accumulate in soil with time and as a result, they contaminate crops and water bodies [4].

Most toxic element known to mankind is mercury. Its exposure may be through inhalation and absorption. Elemental and inorganic mercury are methylated by bacterial action. It was found that 1gm mercury is enough to contaminate 20 acres surface area pond and making it unfit for fishing and drinking [5]. Heavy metals contaminants come in soil from different industrial discharges, ultimately contaminants come in crops like cane sugar, grains and vegetables. Automobile industry’s effluent contaminate our environment significantly by manufacturing unit, paints unit, wearing of parts, fluid leakage, wearing of tyres and wastes from batteries and radiators services units [6].Major heavy metals contaminants of our environment are (Cd) cadmium, (As) arsenic, (Cr) chromium, (Hg) mercury, (Cu) copper, and (PB) lead. Accumulation of these heavy metals in soil and water are due to industrial and agricultural activities. Heavy metals are nonbiodegradable metallic elements so they accumulate in soil and water bodies with time. They have relatively high density. When present in soil and water above prescribed limits of concentrations then they are very toxic. Agricultural and industrial activities play a major role in the heavy metals’ accumulation in our environment, sugar mill involve both of these activities. In the production sugar cane, farmers consume large quantities of insecticides, herbicides, pesticides, chemical fertilizers and other medicines to enhance their crops production and as a result, sugar mill effluents have high amounts of heavy metals contaminants in it [1].

Impact of sugar mill effluent on soil and seed germination of tomato plants was studied by Pakistani researcher and found that average heavy metal (Cr, Pb, Cd, and Cu) concentrations in sugar mill effluent were greater than WHO standard. Rate of seed germination drops with increasing concentrations in irrigated water [2]. Bioremediation is process in which we use microorganisms and plants to degrade or remove toxic and hazardous pollutants from our environment [3]. Heavy metals presence in soil and water originates from anthropogenic and natural activities. When industrialization combined with urbanization then it enormously enhances heavy metals presence in our environment. These toxic contaminants accumulate in soil with time and as a result, they contaminate crops and water bodies [4].

Most toxic element known to mankind is mercury. Its exposure may be through inhalation and absorption. Elemental and inorganic mercury are methylated by bacterial action. It was found that 1gm mercury is enough to contaminate 20 acres surface area pond and making it unfit for fishing and drinking [5]. Heavy metals contaminants come in soil from different industrial discharges, ultimately contaminants come in crops like cane sugar, grains and vegetables. Automobile industry’s effluent contaminate our environment significantly by manufacturing unit, paints unit, wearing of parts, fluid leakage, wearing of tyres and wastes from batteries and radiators services units [6].

Phytoremediation is found to be a promising technique to reduce heavy metals contamination from soil and water bodies. One plant is used to remediate particular heavy metals specifically. Soil is treated with biochar before phytoremediation and mycorrhizal assistance increase heavy metals (HM) absorption area [7].Conventional heavy metals cleaning techniques are adsorption, ion exchange, ultra-filtration, flocculation, electrochemical, precipitation, heating process, chemical process and coagulation methods. These techniques have many disadvantages because these are not feasible at large areas of polluted habitats, very costly, not eco-friendly, produce toxic by-products and large amount of sludge. Phytoremediation / Bioremediation techniques are best convincing methods to eliminate organic chemicals, heavy metals and other pollutants from soil  and water bodies, it is economical, eco-friendly, feasible for large areas, energy efficient technique and produce waste by-products less [8]. There are several phytoremediation methods which are used for soil decontamination such as rhizofiltration, phytoextraction, Phyto stabilization, phytovolatilization and phytodegradation [9].

Figure-1: Different types of phytoremediation processes

India produces second largest quantity of cane sugar in the world and it produces largest amount of sugar mill effluent[1,3].

Sugar industry is very significant agriculture-based industry in India. In India, there were about 571 sugar industries and produced 24.5 million tons sugar in 2010-11. Daily production of liquid and solid wastes by these sugar mills is about 500 tons in the form of boiler ash, press mud and waste water. Press mud was about 3% of total quantity of cane crushed. Environmental pollution is very detrimental problems of India and the world. Reason of this is rapid industrial growth which produce huge amount of liquid and solid wastes. Lead and mercury were found to be present in least amount insoil samples of sugar mill effluent. Sugar mill effluent and sludge are found to have slightly high levels of heavy metals.Thus, sugar mill effluent is not fit for irrigation and sludge is also not suitable for agricultural purpose without using proper remedial measures [10]. Chromium, lead, cadmium and copper concentrationsare found above prescribed limit in sugar mill effluent [11].

In Uttarakhand state,some farmers use paper mill effluent in irrigationof sugarcane crop and this is the reason that Cr and Ni are found in higher concentration in sugarcane than WHO,FAO and IS prescribed standard while other heavy metals (Cu, Pb, Cd) are also found in significant amount [12].Rajshahi university, Bangladesh researchers studied effects of sugar mill effluent on surrounding area of mill. Bangladesh and found that aquatic organisms and fish face difficulty in survival because of heavy metals pollution. Water and soil bodies are found to have higher concentration of lead than permissible limit [13].

Seed germination efficiency of African marigold (tagetes erecta L) was studied in the presence of different concentration of SME and seen that germination percentage, shoot length, root length, dry weight, fresh weight, seed vigour index and tolerance index decrease regularly with increased concentrations of sugar mill effluent from 10 % to 100% SME. Percentage of phytotoxicity increased regularly with concentration increase from 10 % to 100% sugar mill effluent. It explains the impact of toxicity of heavy metals of SME. Here, seed vigour index is (%) germination × length of seedling. Tolerance index is ratio of mean length of longest root in different SME (%) and mean length of longest root in simple water ×100 [14]. 

in a researchit is found that Sugar mill industry of 3000 MT/day sugarcane crushing capacity release 400-500 m3/day effluent and significant amount Cr, Pb, Cd and Cu heavy metals are found in this effluent [15].

Sugar industry operates about 120-200 days/year and it is seasonal. In India, sugar mills are backbone of agriculturaland socio-economic development in India. The sugar mill effluent generates from soda and acids wastes, boiling house, mill house, waste water from boiler and condenser cooling water of mills. In India, sugar millsgenerate about 0.16 – 0.76 m3 waste water per ton crushed sugar cane [3].

About 50 million people residing at Ganga-Meghna-Bramhaputra basin are suffering from toxicity of arsenic. The global tolerance value of arsenic is 10 mg/kg soil while maximum acceptable limit is 20 mg/kg for agricultural soil recommended by (EU) European Union. The arsenic contamination in five blocks under malda district of west Bengal, India was studied and it is found that As concentration in ground water is 0.41-1.01 mg/l, this is greater than WHO standard for drinking water (0.01 mg/l) and FAO standard for irrigation water (0.10 mg/l). Arsenic accumulation is found in potato (0.456 mg/kg), rice grain (0.429 mg/kg), cereals (0.121-0.429 mg/kg), pulses and oilseeds (0.076-0.168 mg/kg), tuber crops (0.243-0.456 mg/kg), spices (0.031-0.175 mg/kg), fruits (0.021-0.145 mg/kg) and in vegetables (0.032-0.411 mg/kg). Thus, immediate action is required to mitigate [16].

In China, the mustard and wheat grown areas are high in arsenic contamination. When As contamination increased from <60 mg/kg to 80 mg/kg level of experimental soil then wheat yield reduced. Shoot of wheat and leaf of mustard have highest As contamination as 8.31mg/kg and 3.63 mg/kg respectively. Arsenic in wheat and mustard grains concentration did not cross permissible limit for food stuff of (1.0 mg/kg) [17]. The suggested permissible limit for mercury as 72 mg/kg [18]. Heavy metals contaminants in soil, vegetables and waterwere studied in farms and around oil refinery in south of Tehran city, it was seen that contamination of V, Co and Ni were above WHO standard. Industrial waste water was found responsible for this [19].

Large number of Sugar mills and industries contribute devastating role in augmenting heavy metals pollutions of our environment.

Table-1: Heavy metals standard in soil / dried sludge

Heavy Metals	WHO standard in soil	Global accepted average	Standard of European Union	FAO/ International Standard	CPCB, India
Parameters
Lead, Pb mg/kg	85 [4,6]	-----	------	30-50 [20]	NI
Copper, Cu, mg/kg	36 [4,6]	-----	------	------	NI
Cadmium, Cd, mg/kg	0.8 [4,6]	------	------	0.9-3 [20]	NI
Arsenic,  As, mg/kg	NI	10  [16]	20  [16]	30 [19] 20 [20]	NI
Mercury, Hg, mg/kg	NI	-----=	-----	0.3 [19] 0.03-2  [20]	6.6 Residential & Agricultural [5]
Chromium, Cr, mg/kg	100 [4,6]	------	-----	------	NI

NI= Not Informed,  ------ = Not available.

Phytoremediation word isconsist of two Latin words “phyto” and “remedium”. Phyto means plant and remedium means restore. At present both natural as well as genetically modified (transgenic) plants are used for this purpose. Hyperaccumulator plants may be used for extraction, absorption, degradation of HMs[21].In avoidancemechanism, plants restrict the absorption of pollutants by root cells and retard the heavy metals movement in plant tissues. The mechanism involves root sorption, metal precipitation and exclusion [21,22].Exclusion barrier restrict HMs accessibility in plant from soil to roots [21,23]. Heavy metals embed in cell wall where pectin groups behave as cation exchanger and stop entry in the cell [21,24]. In tolerance mechanism, heavy metal ions enter into the cytosol where plants make chelate with these ions to inactivate[21,22]. HM-ligand complexes (chelates)is transferred from cytosolto inactive compartment (trichomes, vacuole, leaf petioles and leaf sheaths) here heavy metals stay without any toxicity[21,25].When there is high concentration of heavy metals in rhizosphere, above strategies may not work to clean polluted site, because heavy metals may generate reactive oxygen species (ROS) in the cytoplasm and reactive oxygen species (ROS) produce oxidative stress [26]. Many antioxidant enzymes are used as ROS scavenger, these are superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione peroxidase (GR) [26,27].

Phytoextractionmay be called as phytoaccumulation, Phyto absorption and Phyto sequestration. Transport of minerals and toxic heavy metals by the roots into shoots is called absorption. Toxins accumulate in to shoots and roots which are harvested [9]. Plants allow heavy metals’ movement from roots to shoots, leaves, stems and other parts in phytoextraction technique [28]. Hyperaccumulator metallophytes do not face any developmental and functioning effects on themselves when they store heavy metals 500 times more concentration in higher parts than other parts [29]. Mechanisms are in developmental stage of these processes [21,30].

Another technique is Rhizofiltration. In rhizofiltration, rhizosphere is aninterface between soil and root and here, root secretions and soil microorganisms interact. The microorganisms survive on sloughed off plant which is known as rhizodeposition. Proteins and sugars compounds released by roots are termed as root exudates. This symbiosis happens with complex interactions (Rhizosphere -Wikipedia, en.m.wikipedia.org).

Plant root exudates may change pH of the interface and facilitate heavy metals absorption. [9].

pH of rhizosphere and root exudates are important and it is used to settle heavy metals on root surface (in microbiome). The plants increase microbial activities and microorganisms reduce the heavy metals contamination. [21]. Terrestrial plants such as Brassica juncea and H. annus have hairy root system which exhibit high tendency to amass heavy metals [31]. Sunflower plants have significant ability to decontaminate lead-polluted soil. Indian mustard may eliminate high concentration of lead in soil (upto 500mg/l) [32]. In blastofiltration mechanism, it is foynd that young seedlings have high tendency to reduce heavy metals from water [33]. In Rhizodegradation, microbes of rhizosphere secrete specific enzymes which convert high toxic to less toxic form of pollutants and biodegradation of organic pollutants take place. Rhizodegradation process increase when microbes receive nutrients from plant roots which secrete these [34]. The phytostabilization / phytorestoration stops bio-magnification of heavy metals [35,12]. Organic pollutants such as herbicides, insecticides and chlorinated solvents affect the soil which are decontaminated by phytodegradation mechanism [36]. Sunflower (Helianthus annus) is found effective in the reduction of methyl benzotriazole [37].

Contaminants are transformed into less toxic and volatile compounds in phytovolatilization, Then, they are released in atmosphere via transpiration through the stomata [38]. The plants used in the phytoremediation areTriticum aestivum, Trifolium repens, Crinum Americanum, Arabidopsis thaliana,Bacopa monnieri and Nicotiana tabacum [39]. Volatile organic compounds (VOC) evaporate by the leaves and stems in atmosphere in direct volatilization. Plant root manages the interface rhizosphere region which facilitate the conversionto less hazardous volatile forms in indirect volatilization [40]. while Se and Hg can also be removed by phytovolatilization [41].

HMs = Heavy metals, SME = sugar mill effluent, WHO = World health organization, FAO = food and agriculture organization, IS = Indian standard, CPCB = Central pollution control board, MT = Million ton, TCE = Trichloroethylene, PCE = Perchloroethylene.

Toxic effects of heavy metals:

Toxic effects of heavy metals areas reduced plant growth like-smaller plant, stem and leaves thus less production [1]. Heavy metals are absorbed from soil through water and accumulate in the plants’ tissues where they become health hazards for man and animals [10].  Long term consumption of contaminated sugarcane by people will pose severe health issue because these HMs also accumulate in body with time. Heavy metals like Nickel and Chromium when present in higher concentration than WHO, FAO and IS permissible limits are carcinogenic [12].

Toxicity of heavy metals present in sugar mill effluent is such that fish production decreased by 93% in canal, rice and potato production are decreased by 69% and 84% respectively, skin disease enhanced by 18% and human health hazards increased by 69% [13]. Cadmium is non-essential transition metal, its exposure happens with food, water, cigarette smoking and inhalation. cadmium accumulates in animal’s and plant’s tissues where its half-life period is about 28 years,it may cause lung, pancreas and breast cancer [42]. It affects and damage nervous system and immune system,it may destroy DNA. It can enhance a cancer level in cells. It may be cause of smell sense loss, taste sense loss, respiratory disease, hypertension and cardiovascular disease [43]. Cr⁶⁺ is very toxic substance for humans specially for those, who works in steel and  textileindustries. It may be the reason for dermatitis, ulcer, lung cancer, shortness of breath, brain damage, premature death, liver necrosis and kidney diseases. Chromium is found to be mutagenic and carcinogenic in humans. [44].

Copper is interconvertible between Cu(I) and Cu(II) states in redox reactions which is used by cuproenzymes. In this way, copper generate hydroxyl and superoxide radicals thus make it very reactive. It may cause Wilson disease in humans due to cellular damage [45]. Lead (Pb) works as significant cumulative body poison.  Lead may cause kidney disease, central nervous system disorder, gastrointestinal tract and mental retardationdisease. Children below  6 years age are at high impactrosk. It causes nausea, insomnia, memory loss, anorexia, reproduction  disability,  reduced heam synthesis and tumor disease in humans. Lead poisoning result in Encephalopathy disease which include coma, headache, vertigo convulsion and tremor[44].

 Mercury (Hg) is most toxic metal element found in the environment where methyl mercury is very dangerous compound for humans and it may cause deformities in children, it badly affects central nervous system. It may be cause of mental retardation, convulsions, breaking of chromosome in genetic defects, defects in pulmonary function, kidney anddyspnoea [44].

The mercury can cross the skin, blood, placenta and brain barrier and become reason for devastating health. At present, total global mercury emissions in the atmosphere is 5000 tonnes/year,  80% of this is due to anthropogenic activity, Chlor-alkali industry, fluorescent tube lamps, Neon lamps, thermometer, mercury cells.Mercury can damage central nervous system, skin, kidney and liverdiseases [46].

pH is found to affect the solubility of heavy metals in the water. Solubility of these heavy metals in alkaline conditions (pH above 7) is very less. Metal solubility enhance in slightly acidic conditions. At the same pH value, metal solubilities increasewith redox potential decrease. If one think to increase solubilities of HMs, pH is more significant than redox potential effects [47].

Study Area:

Several sugarindustries are situated in Meerut district of Uttar Pradesh state, India for exampleMawana sugar mill,Daurala sugar mill and Mohiuddinpur sugar mill etc. Meerut region has humid subtropical climate and monsoon season. Climate of Meerut region There is winter climate from November to March with very less rainfall and summer climate from March to October while there is sufficient rainfall from July to October in Meerut region. Average temperature (annual) is 24.1⁰C/75.3⁰ F and average rainfall (annual) is 886mm/34.9 inch per year [48]. Study area’s latitude and longitude position are latitude   28⁰59’4.7184” N and longitude 77⁰42’21.4416” E [49].            

Collection of Sample:

The soil / sludge samples are collected from different places of Sugar Mills effluent drain of District-Meerut, U P, India, stored in well washed and cleaned plastic containers and kept at room temperature. The semi solid sludge samples collected from sugar mill effluent drain which are dried in open buckets at room temperature for 1 month and this dried sludge / soil is used for plants to bioremediate pollution.

Phytoremediation process:This dried sludge was used to fill pots. 60 seeds of sunflower plant are sown in five pots and their germination was observed properly in first week of May-2022.Because of heavy metals contamination in the soil, only 2- seeds germinated out of 20 seeds sown (average) and only 1-plant could survive out of 2-plants germinated (average). The mean hight of the sunflower plants were near >12 inches above soil level. The plants died after completing their life span. After removing dried plants from pots, we again tested the soil for heavy metal concentrations in it. The data is shown in table-2.

Methodology Used:

The Atomic Absorption Spectroscopy (AAS method) based technique is used to obtain the heavy metal concentrations in the dried sludge samples of sugar mill effluent drain. AAS (atomic absorption Spectroscopy) is spectroscopical analytical technique where it is used to measures the concentrations of heavy metal elements quantitatively and qualitatively.  All the testing procedure steps and methods were adopted as per prescribed AAS method.    

The heavy metals Pb, Cu,  Cd, As, Hg and Cr concentrations in the dried sludge samples of SME  are Pb (35.66 mg/kg), Cu (03.33 mg/kg), Cd (30 mg/kg), As (62.66 mg/kg), Hg (45 mg/kg)  and Cr (12.33 mg/kg), and pH of soil is 7.77 before phytoremediation. Because of heavy metals contamination in the soil, only 2- seeds germinated out of 20 seeds sown (average) and only 1-plant could survive out of 2-plants germinated (average). The mean hight of the sunflower plants were near >12 inches above soil level. The plants died after completing their life span. After removing dried plants from pots, we again tested the soil for heavy metal concentrations in it. The data is shown in table-2. The data after phytoremediation process are  Pb (32 mg/kg), Cu (03.03 mg/kg),  Cd (27.66 mg/kg) and As (44 mg/kg),  Hg (34.66 mg/kg) Cr (11.1 mg/kg) by plants and pH of the soil was 8.5. The data is given in the table-2. The concentrations of heavy metals are measured in mg per kg soil. Sunflower plant (Helianthus annus L) is significantly effective in reducing concentrations of Lead (Pb) from 35.66 mg/kg to 32 mg/kg. The dried sludge of SME has low level of Lead concentration already than permissible limit of 50 to 85 mg/kg soil according to data. Sunflower plant reduces copper concentration from 03.33 mg/kg to 03.03 mg/kg in dried sludge of SME, this data show that Copper is reduced very less in this process because its concentration was already very less in comparison of standard data of copper. The dried sludge of SME has low level of copper concentration than permissible limit of 36 mg/kg soil. Sunflower plant reduces the Cadmium concentration from 30 mg/kg to 27.66  mg/kg in dried sludge of SME. The dried SME sludge has very high level of  Cadmium concentration than permissible limit of 0.8 to 3 mg/kg soil. Sunflower plant reduce the Arsenic concentration from 62.66 mg/kg to 44 mg/kg in dried SME sludge. The dried SME sludge has high level of Arsenic concentration than permissible limit of 20 mg/kg soil. Sunflower plant reduce the Mercury concentration from 45 mg/kg to 34.66 mg/kg in dried SME sludge. The dried SME has high level of Mercury concentration than maximum permissible limit of 6.6 mg/kg soil. Sunflower plant reduce the Chromium concentration from 12.33 mg/kg to 11.1 mg/kg in dried sludge of SME. The dried SME sludge has very low level of Chromium concentration than permissible limit of 100 mg/kg soil. 

Thus, this dried sludge of sugar mill effluent has high level of Cd, As and Hg heavy metals. According to the literature of toxicity of heavy metals available, the result is that this sludge must not be allowed to spread in the nearby area because of its very high heavy metals contamination. This sludge is not contaminated with Pb, Cu and Cr heavy metals because their concentrations are below permissible limits in soil.

Table-2: Heavy metals in the dried sludge / soil of  SME drain of Meerut region, U P , India before and after phytoremediation with sun flower plants.

Heavy metals	Heavy metal in dried sludge /soil before phytoremediation Test Results				Heavy metal in dried sludge /soil after phytoremediation. Test Results				Test Method
Parameters	T.R-1	T R-2	T R-3	Average	T R-1	T R-2	T R-3	Average
PH	7.76	7.78	7.78	7.77	8.50	8.52	8.48	8.5	2720 (p-26)
Lead, mg/kg	36	36	35	35.66	32	30	34	32	AAS method
Copper, mg/kg	03	04	03	3.33	2.9	3.0	3.2	3.03	AAS method
Cadmium, mg/kg	30	31	29	30	30	26	27	27.66	AAS method
Total arsenic, mg/kg	62	64	62	62.66	42	46	44	44	AAS method
Mercury, mg/kg	44	46	45	45	34	36	34	34.66	AAS method
Chromium, mg/kg	12	12	13	12.33	10.5	11.2	11.6	11.10	AAS method

T R=Test Result

Statistical Analysis:

Graph-1: Heavy metals concentrations in dried SME sludge of Meerut region, UP, India before phytoremediation and after phytoremediation with permissible limits of heavy metals in soil. 

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