Fatemeh Shokri ¹ , Parisa Ziarati^*2 and Zahra Mousavi³

¹Pharmaceutical Sciences Research Center, Pharmaceutical Sciences Branch ,Islamic Azad university , Tehran-Iran (IAUPS ).

²Department of Medicinal Chemistry, Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS), Tehran, Iran.

³Department of Pharmacology and Toxicology, Pharmaceutical Sciences Branch, Islamic Azad University ,Tehran - Iran (IAUPS ).

Corresponding Author E-mail: ziarati.p@iaups.ac.ir

DOI : https://dx.doi.org/10.13005/bpj/993

Abstract

Due to vast probable contaminations from pharmaceutical effluent,   the main purposes of this study were to decrease the heavy metal toxic levels from waste water of pharmaceutical research and educational laboratories. The potential ability of A. Vera for cleaning up contaminated educational sites soil and the probable capability of this plant to phytoextract different metals (Chrome (III) and (VI) , Nickel, Copper, Lead and Cadmium) were studied. The initial concentration of heavy metals/metalloid in the plants and effluents were analyzed before introduction into the studied soil samples. After 10 days of treatment up to 60 days in every ten days, final concentration of heavy metals/metalloid in effluent samples and plants were analyzed using Atomic Absorption Spectroscopy. Results showed that A. Vera   transition factors for all heavy metals in treated soil were higher than one and A. Vera   can up-take lead and  Cadmium after 20 days (p<0.01) more than other studied metals. A highly significant, although low, positive correlation ( r= 0.52, p=0.01, n=200) was found between Lead and Cadmium and chrome (III)  of the greenhouse –grown  A. Vera , compared to a non-significant and much lower correlation between the three  other heavy metals: Nickel, Cr(VI)  and Copper . The phytoremediation of lead and Cadmium trend by this plant indicates that A.vera   cultivated in the soil can be consider as a suitable hyper-accumulator by its relatively large ratio of biomass concentration of the contaminant to soil concentration. The present investigation shows that the A. Vera is effective and inexpensive adsorbent for the removal of Pb ,Cd, Ni, Cu, Cr (III) and (VI) from contaminated soil by heavy metals.

Keywords

Phytoremediation; Heavy metals; Aloe Vera L; Pharmaceutical effluent

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Copy the following to cite this article: Shokri F, Ziarati P, Mousavi Z. Removal of Selected Heavy Metals from Pharmaceutical Effluent by Aloe Vera L. Biomed Pharmacol J 2016;9(2).

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Introduction

Pharmaceuticals are a large and diverse group of compounds designed to prevent, cure, and treat disease, and improve health [1-2].  Significant fractions of the parent compound are excreted in un-metabolized form or as metabolites (active or inactive) into raw sewage and wastewater treatment systems. Municipal sewage treatment plant effluents are discharged to water bodies or reused for irrigation, and bio-solids produced are reused in agriculture as soil amendment or disposed to landfill. Pharmaceuticals enter the environment from a myriad of scattered points [3].  The main sources of contamination include pharmaceutical production plants, WWTPs, hospitals, landfills and even graveyards [4-5]. The most investigated route of entry of pharmaceuticals into the environment is that from municipal WWTPs. Human excretion of unchanged or slightly transformed Active pharmaceutical ingredients (APIs) conjugated to polar molecules such as glucoronide enters the WWTP where these conjugates may then be cleaved, releasing the original API into the environment [6]. Activated sludge WWTPs has received particular attention [7-8]. A limited number of studies also found pharmaceuticals in drinking water [9] and hospital wastewater [10]. Pharmaceutical wastewater streams containing heavy metals are produced from different laboratories such as Toxicology and Food Toxicology Labs. Some examinations and chemical test and processes generate significant quantities of wastewaters containing heavy metals (such as cadmium, zinc, lead, chromium, nickel, copper, silver and titanium) from a variety of analyses tests. Heavy metals cause serious health effects, including reduced growth and development, cancer, organ damage, nervous system damage, and in extreme cases, death. Exposure to some metals, such as mercury and lead, may also cause development of autoimmunity, in which a person’s immune system attacks its own cells [11] .The conventional processes for removing heavy metals from wastewater include many processes such as chemical precipitation, flotation, adsorption, ion exchange, and electrochemical deposition. Chemical precipitation is the most widely used for heavy metal removal from inorganic effluent. Lime and limestone are the most commonly employed precipitant agents due to their availability and low-cost in most countries [12-13]. Lime precipitation can be employed to effectively treat inorganic effluent with a metal concentration of higher than 1000 mg/L. Other advantages of using lime precipitation include the simplicity of the process, inexpensive equipment requirement, and convenient and safe operations. However, chemical precipitation requires a large amount of chemicals to reduce metals to an acceptable level for discharge. Other drawbacks are its excessive sludge production that requires further treatment, slow metal precipitation, poor settling, the aggregation of metal precipitates, and the long-term environmental impacts of sludge disposal [13]. Recently, adsorption has become one of the alternative treatment techniques for wastewater laden with heavy metals. The efficacy of various plants in eliminating different heavy metal contaminants, particularly Lead and Cadmium is a major concern nowadays due to the vast soil pollution in many countries around the world especially Iran [14-20].Basically, adsorption is a mass transfer process by which a substance is transferred from the liquid phase to the surface of a solid, and becomes bound by physical and/ or chemical interactions [21]. Various low-cost adsorbents, derived from agricultural waste, industrial by-product, natural material, or modified biopolymers, have been recently developed and applied for the removal of heavy metals from metal-contaminated wastewater. In general, there are three main steps involved in pollutant sorption onto solid sorbent: (i) the transport of the pollutant from the bulk solution to the sorbent surface; (ii) adsorption on the particle surface; and (iii) transport within the sorbent particle. Technical applicability and cost-effectiveness are the key factors that play major roles in the selection of the most suitable adsorbent to treat inorganic effluent. Phytoremediation is environmental-friendly, cost-effective and natural green biotechnology for the removing xenobiotic, including toxic metals, from the environment using some species of the plants. The land in Iran is very fertile, providing favorable conditions for various medicinal plants to grow. Since ancient times, local people in every part of Iran have grown Aloe Vera in their home gardens, using the leaves of A. Vera to aid in healing wounds. A. Vera is cultivated in many places in Iran but not in wide range. About 90% of the plants are estimated to come from wild harvest [17, 22-23]. A. Vera has been on this earth for millennia and it is a member of the Liliaceae family. The plant Aloe vera  Linne, also known as Aloe barbadensis Miller, and Aloe vulgaris Lamarck [24]. As A. Vera can easily grow in Iran climates and due to vast probable contaminations from pharmaceutical effluent ,   the  main purposes of this study were  to decrease  the heavy metal toxic  levels from waste water of pharmaceutical research and educational laboratories  determine the potential ability of A. Vera for cleaning up contaminated  educational  sites soil and the  probable capability  of this  plant  to phytoextract different metals (Chrome , Nickel, Copper, Lead and Cadmium) and also determine metal transfer factors from soil (TFS) of A. Vera  in order to ascertain its  phytoremediation potential.

Experimental Section

Effluent samples were collected between 1 April to  october 5th, 2015. Sample collection containers (1 L, amber glass) were washed in hot water, rinsed three times with distilled water, rinsed three times with acetone, and then baked in a heated oven at 250ºC for a minimum of four hours. A 24-hour composite sample (500 mL of effluent) was collected by WWTP operators from each WWTP, using their own equipment, and 100 2 mL of a solution containing 5.0 g/L of Na2EDTA and 25 mg/L of ascorbic acid was added at the time of collection. The samples were shipped overnight on wet ice, and stored at 4°C until extraction. Because of the large number of sampling sites and chemical analytes, it was logistically too difficult and expensive to collect and analyze field blanks as well as duplicates from each location. Field blanks were collected from 20% of the sampling sites, with the field blanks being prepared from laboratory distilled water that was transferred into sampling containers and preserved at the time of collection. Duplicates were collected and analyzed for 10% of the sample sites.

Waste water Effluent

Effluents from five educational and research laboratories in pharmaceutical sciences branch, Azad university in Tehran, including, Food Science and Technology research   (Effluent 1&2), Toxicology (Effluent 3,4), Analytical chemistry(Effluent 5) were used in this study. Effluent 1 and Effluent 2 were from the same laboratory but were collected on separate occasions with a 3 week time interval. Although Effluent 1 and Effluent 2 come from the same WWTP, they were treated as 2 different effluents due to the variability of their characteristics. This difference is attributed to the significant experiments which occurred following the first sampling event. After collection, the effluent was immediately transported to the research laboratory for analysis. Physico-chemical parameters such as pH, Electrical Conductivity, Total Solids, Total Dissolved Solids, Total hardness, Chloride, Sulphate, Dissolved oxygen, , Calcium, Sodium, Cadmium, Lead, Zinc, Copper, Chrome, Manganese ,Iron and Potassium were analyzed as per the standard methods [26-27].

The initial concentration of heavy metals/metalloid in the plants and effluents were analyzed before introduction into the studied soil samples. After 10days of treatment up to 60 days in every ten days, final concentration of heavy metals/metalloid in effluent samples and plants were analyzed using Atomic Absorption Spectroscopy. The samples were analyzed by an Atomic Absorption Spectrophotometer Model AA-6200 (Shimadzu, Japan) using an air-acetylene flame for heavy metals and using at least five standard solutions for each metal. All necessary precautions were taken to avoid any possible contamination of the sample as per the AOAC guidelines [28-30].  The efficiency of plant in accumulating heavy metals in their areal parts such as leaf, stem and also root were calculated using bio-concentration factor.

Analysis of Samples

Vera (one month old plants) were grown in a local nursery until transplant into the research study. For metal analysis 2g of shoots along with leaves and roots of 50 numbers of plants sample were taken separately in every ten day in this study during 60 days. 8ml of concentrated nitric acid (65%) and 1 ml of per chloric acid (70%) was added and 1 ml hydrogen peroxide (17, 26). Application of concentrated HNO₃ along with thirty percent hydrogen peroxide H₂O₂ (Merck) for mineralization of samples to the complete digestion of samples (15-16, 31) following   Environmental Protection Agency (EPA) Method 3052 was done. All glassware and plastic containers used were washed with liquid soap, rinsed with water, soaked in 10% volume/volume nitric acid at least overnight, and rinsed abundantly in deionized water and dried in such a manner to ensure that any contamination does not occur. Five-point calibration curves (five   standards and one blank) were constructed for each analyte. The calibration curve correlation coefficient was examined to ensure an r² ≥ 0.998 before the start of the sample analysis. The digested samples were diluted with 10% HNO₃ and brought up to 50 mL and analyzed by a graphite furnace atomic absorption spectrophotometry, (GFAAS). The measurements were performed using a Perkin Elmer   Pin A Acle 900 T atomic absorption (AA) spectrophotometer and using at least five standard solutions for each metal. All recoveries of the metals studied were greater than 95%Digestion followed by the measurement of total concentrations of Cd using Atomic Absorption Spectrophotometer using an air-acetylene flame for heavy metals: Pb, Cd, Co, Cu, Cr, Ni and Cu. All necessary precautions were taken to avoid any possible contamination of the sample as per the AOAC guidelines Physical and chemical properties and concentrations of heavy metals (Chrome, lead, Cadmium, Copper, Cobalt and Nickel) in soils before and after planting Vera   and also after the growth period (in every 10 days) measured (21-26). In order to assess amount of heavy metals transfer from soil to plant (shoot and root), translocation factor was determined by dividing metal concentration at shoot by its concentration at root (27-29).  Different parts of plant samples (roots and leaves) were separated and washed and digested by wet method according the standard protocol for measuring Cadmium and Lead. The uptake rate is given by the following equation [17, 32, 21].

U = (TSCF) (T) (C) (1)

Where U = uptake rate of contaminant, mg/day

TSCF = transpiration stream concentration factor, dimensionless

T = transpiration rate of vegetation, L/day

C = aqueous phase concentration in soil water or groundwater, mg/L.

The ratios were higher than one it was considered as suitability of plant at that condition for use in phytoremediation. The enrichment factor (EF) has been calculated to derive the degree of soil contamination and heavy metal accumulation in soil and in plants growing on contaminated site with respect to soil and plants growing on uncontaminated soil [12,14,17,31-32]. Metal contents were detected by Atomic Absorption Spectrophotometer by wet digestion method in Research Laboratory in Pharmaceutical Sciences Branch University.

Estimation of Heavy Metals in Effluents

The heavy metals/metalloid in the effluent samples such as Chrome, Nickel, Copper,  lead, cadmium and  Iron  were analyzed before and after treatment by AAS after digestion of plant materials by AOAC method [28-32].  The plant samples were washed in deionized water dried (24 hrs at 80°C) immediately to stabilize the tissue and stop enzymatic reactions. After drying, samples were ground to pass a 1.0mm screen using the appropriate Wiley Mill. After grinding, the sample were thoroughly mixed and a 5- to 8-g aliquot withdrawn for analyses and storage [33]. Weighed 0.5 to 1.0 g of dried (80°C) plant material that has been ground (0.5 to 1.0 mm) and thoroughly homogenized and place in a tall-form beaker or digestion tube. Added 5.0 ml concentrated HNO₃ (65 %) and cover beaker with watch glass or place a funnel in the mouth of digestion tube and allow to stand overnight or until frothing subsides. Place covered beaker on hot plate or digestion tube into block digester and heat at 125°C for 1 hour. Removed the digestion tube and allowed cooling. Added 1 to 2 ml 30% H₂O₂ and digest at the same temperature. Repeated heating and 30% H₂O₂ additions until digest is clear. Add additional HNO₃ as needed to maintain a wet digest. After sample digest is clear, removed watch glass and lowered temperature to 80°C. Continued heating until near dryness. Added dilute HNO₃ (10%), and deionized water to dissolve digest residue and bring sample to final volume.

Total dissolved solids (TDS)

The total solid concentration in waste effluent represents the colloidal form and dissolved species. The probable reason for the fluctuation of value of total solid and subsequent the

value of dissolved solids due to content collision of these colloidal particles. The rate of collision of aggregated process is also influenced by pH of these effluents.

Chemical oxygen demand (COD)

The chemical oxygen demand test (COD) determines, the oxygen required for chemical oxidation of organic matter with the help of strong chemical oxidant. The COD is a test which is used to measure pollution of domestic and industrial waste. The waste is measure in terms of equality of oxygen required for oxidation of organic matter to produce CO2 and water. It is a fact that all organic compounds with a few exceptions can be oxidizing agents under the acidic condition. COD test is useful in pinpointing toxic condition and presence of biological resistant substances. For COD determination samples were preserved using H2SO4 and processed for COD determination after the entire sampling operation was complete.

 Biochemical oxygen demand (BOD)

For BOD, 5 samples were immediately processed after Collection for the determination of initial oxygen and incubated at 20 °C for 5 days for the determination of BOD5.

Chlorides

Chlorides are generally present in natural water. The presence of chloride in the natural water can be attributed to dissolution of salts deposits discharged of effluent from chemical industries, oil well operations, sewage discharge of effluent from chemical industries, etc.

Sulphates

Sulphate in one of the major ions  occurring in natural water. Sulphate being a stable, highly oxidized, soluble form of sulphur and which is generally present in natural surface and ground waters. Sulphate itself has never been a limiting factor in aquatic systems. The normal levels of sulphate are more than adequate to meet plants need.

Bio-concentration Factor

The bio concentration factor is a measure of bioaccumulation of heavy metals. It can be calculated by dividing the trace element concentration in plant tissues (ppm) at harvest by initial concentration of the element in the external nutrient solution (ppm) [34-36].

 

 

Statistical Analysis

For testing statistical significance, student’s t-test (SPSS-20) was used. Independent sample t-test was used for finding the mean difference of each parameter control with plants.

Results and Discussion

Chemical extraction of the soil profile before and after  planting  A.Vera    samples during 60 days were studied  in pH= 5.6-7.1 ,  as plant availability of certain heavy metals depends on soil properties such as soil and contains exchange capacity and on the distribution of metals among several soil fractions . All the soil data are expressed on a dry basis.

Results in figures 1,2 and 3  showed significant differences in cadmium,  Cr (III) , Cr(VI) and lead      up taking by roots and leaves of plant. The best results for uptake of Nickel and copper was in the soil with pH=6.2 among different samples while for lead up taking was in 6.4 and for copper up taking was in pH=5.9. This range of pH had no affecting in zinc up taking. Chemical extractions of the soil profile before treatment of heavy metals by Aloe Vera L.  is shown in the table 1. Data is averages of the profiles.

Table 1: Heavy Metal concentration in studied Pharmaceutical Laboratories ‘ effluent before treating

Heavy Metal Content	Concentration (mg/kg DW ± SE*)
Lead	30.731 ± 0.106
Cadmium	9.879 ± 0.011
Nickel	30.98 ± 0.318
Chrome	19.043 ± 1.204
Copper	101.650 ± 10.006
Iron	42.089± 1.819

SE^* = Standard Error

Results showed that A. Vera   transition factors for all heavy metals in treated soil were higher than one and A. Vera can up-take lead and  Cadmium after 20 days (p<0.01) more than other studied metals. In figures 1 and 4, the phytoremediation of lead and Cadmium trend by this plant indicates that A.vera   cultivated in the soil can be consider as a suitable hyper-accumulator by its relatively large ratio of biomass concentration of the contaminant to soil concentration. Although A. Vera can uptake Cr (III) and (VI) after 10 days significantly (p < 0.05), but Cr (III) can be absorbed in a slightly manner up to 60 days (figures 2 and 3).

Figure 1: The mean content of Pb ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

Figure 2: The mean content of Cr(VI)  ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

Figure 3: The mean content of Cr (III) ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

Figure 4: The mean content of Cd   ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

The heavy metals uptake rate by this plant is significantly affected by number of plant cultivated as for lead uptake (p<0.005) while for Cd and chrome the p-value was less than 0.01. The percentage of copper uptake by leaves and roots of this plant has been demonstrated in figure 5, it has been indicated that plants had more potential to uptake Nickel and Copper after 10 days by root and the absorbing trend were decreased after 20 days. A highly significant, although low, positive correlation ( r= 0.52, p=0.01, n=200) was found between Lead and Cadmium and chrome (III)  of the greenhouse –grown  A. Vera , compared to a non-significant and much lower correlation between the three  other heavy metals: Nickel, Cr(VI)  and Copper .

Figure 5: The mean content of Cu   ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

Figure 6: The mean content of Ni   ( mg/kg ) in Aloe Vera L. leaves and root parts and studied soil samples after treatment during 60 days. Click here to View figure

 

Conclusion

Phytoremediation has been advanced in the last few years by increased understanding of the mechanisms of plant uptake and the various types of enzymatic metabolism that occur. Sorption and uptake constants such as the RCF and TSCF may help model plant uptake rates of various chemicals, allowing more accurate prediction of treatment times required for phytoremediation technology. Research into enzymatic transformation pathways will help determine the ultimate fate of chemicals in a plant remediation system. Our results for chrome translocation indicate that A. Vera has high potential for taking –up this toxic heavy metal and Translocation factor in all conditions were higher than one which  proves  that metal concentrations in shoots were  higher than roots and  the plant is suitable for phyto-remediation and the heavy metals uptake rate by this plant is significantly affected by time of growing plant as for lead uptake (p<0.01) while for Cd and Cr(III) the p-value was less than 0.03. The present investigation shows that the A. Vera plant is effective and inexpensive adsorbent for the removal of Pb, Cd, Ni, Cu, Cr (III) and Cr (VI) from contaminated soil by heavy metals. Aloe vera, is considering as a medicinal plant with diverse therapeutic applications, also used in many different countries. Through human trade and migration, this plant came to be known and was widely used to cure burns and wounds throughout ancient civilizations. Successful cultivation of this plant is economically attractive provided marketing is not a problem. The cultivation of Aloe vera has acquired great commercial importance for medicinal products and cosmetics processing. Aloe vera cultivation demands skill and it is also very labor intensive. Better management can results in much higher income and net profit .The nutrient make up of Aloe Vera is one of a kind and has amazing natural healing properties. Its uses are multiple and undoubtedly, the nature’s gift to humanity and it remains for us to introduce it to ourselves and thank the nature for its never-ending gift. This plant also could be used for treating contaminated soil and rescue the environment. This research conduct adsorption of heavy metals by its leaves and root  and proved that this friendly method should  gain more attention and research interest for the removal of heavy metals from contaminated soil  due to its surface area, adsorption capacity and plenty abundant in nature must be followed seriously. Also, this research suggests more investigations by other genera and families of cost-effective plants.

Acknowledgement

Financial Supports from Pharmaceutical Sciences Branch, Islamic Azad University (IAUPS) is gratefully acknowledged.

Conflicts of Interest

None of the authors have any conflicts of interest associated with this study.

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