Pharmaceuticals and personal care products (ppcp,s) and their metabolites in the environment has become growing area of research in environmental science. Pharmaceuticals are introduced into aquatic by largest inputs of human ingestion and excretion (Williams 2005) Upto 90% of a drug can be excreted in its original, active form (Halling Sorenson et al., 1998)The presence of pharmaceuticals and personal care products and their metabolites in the environment has become a growing area of research in environmental science. The continuous usage of Antibiotic and antimicrobial products is leading to antibiotic resistance in bacteria.
More than 3000 different substances used as medicines, painkillers, antibiotics, contraceptives, beta-blockers, lipid regulators, tranquilizers and impotence drugs (Ternes et al.2004).The trace levels of pharmaceuticals in the water supply remains unknown.(Strauch et al .,2011)and these compounds mainly introduced into environment, mainly through waste water effluent from municipal treatment plant ,hospital effluents and live stock activities (Halling-Sorenson et al.,1998; Ternes 1998; Herber,2002; Fent et al. 2006;Besse and Garric,2009 K??mmerer and AL Ahmad,2010; vulliet and cren-olive, 2011;Brausch and Rand 2011).
Pharmaceutical and personal care products in the environment have been detected in water and wastewater effluents around the world.(Daneshwar et al., 2010;Rosal et al., 2010;Grover et al., 2011; Sim et al., 2011; Seri et al., 2011).The removal efficiency of pharmaceuticals depends on the nature of the target compounds and the type of technology and environmental conditions(eg dilution of waste water effluent,rainfall,temperature and sunlight) including the adsorption capacity of sludge(N,Bolong et al., 2009)
Large amounts of unmetabolized or unused PPCP with low concentrations of nanogram/litre to microgram/litre levels enter into Waste water via city sewage pipelines. PPCP,s are detected frequently in the effluents of WWTP (Carballa et al 2004; Leung et al 2011; Martin et al., 2012; Santos et al., 2007)The common procedures that are using to carry out the determination of organic compounds in environmental matrices such as Solid Phase extraction SPE(Martinez et al., 2010) Solid Phase micro extraction (SPME) (Rodriguez et al 2006) followed by separation and determination using liquid(LC) or Gas Chromatography. Liquid-liquid extraction (LLE) (Gomez et al.,2009) followed by separation and determination using liquid(LC) or gas chromatography(GC),couple with mass spectrometry(LC-MS or GC-MS).Tandem mass offers higher performance that single-quadrupole instruments, in terms of sensitivity and selectivity.So,triple-quadrupole-ion trap systems are one of the most frequently chosen techniques for analysing organic compounds in complex matrices.LC-MS/MS has been the technique most referenced for the analysis.(Gros et al.,Martinez Bueno et al.,2007)
Current waste water treatment plants are not specifically designed for the removal of micro pollutants, many of these pollutants are able to pass through waste water treatment process. In addition precautions and monitoring actions for micro pollutants have not been well established in most Waste water treatment plants.(Bolong et al., 2009)
The emergence of antibiotic resistance is common in areas where antibiotics are used (Aarestrup F et al., 1996, Klare I et al., 1995).The bacteria can able to develop different mechanisms to render ineffective to the antibiotics used against them. Resistance to multidrugs was first detected among enteric bacteria namely E coli, shigella and salmonella in the late 1950s to early 1960s(subha A et al., 2003) The presence of antibiotics in environmentally relevant concentration levels has been associated to chronic toxicity and the prevalence of resistance to antibiotics in bacterial species (Schwartz et al., 2006; K??mmerer, 2009).
Use of antibiotics may also accelerate the development of antibiotic resistance gene (ARG) and bacteria which shade health risks to human and animals (kemper 2008), there is always a chance to bacteria to transmit into environment and humans either directly or indirectly and it is needed to develop molecular methods to investigate the fate and transport of environmental antibiotic resistance genes. Polymerase chain reaction (PCR) assays are used for environmental samples for the detection of specific antibiotic resistance genes.(Mohapatra et al.,2008)
Richardson BJ et al., 2005 compared Pharmaceuticals and personal care products (ppcp) in Asia with reference to southern china are not well known and this paper presents a general description of the issues and status of PPCP and it is suggested that adequate evaluation of PPCPs in environmental media from the south east Asia region that is critically needed.
Hedgespeth M.L et al., 2012 studied the base line of the presence of quantity and spatial and temporal distribution of PPCPs in waste water treatment plants and also in the surface water of Charleston Harbour.
Padhye L.P et al.,2013 studied the occurrence and removal of 30 representative pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs) in an urban drinking water plant for a period of one year. This results showed that removal efficiencies are varied greatly among different PPCPs and EDCs and ozonisation process showed high removal efficiency.
Rehman MSU et al., 2013 discussed about the global risk of pharmaceutical contamination from high populated developing countries.
Li W.C 2014 in his review paper he summarized the recent studies on the Source, Occurrence, Fate and its effects of most common pharmaceuticals and risk management for controlling the pharmaceuticals.
Kosma CI et al., 2010 studies four seasons over a year to investigate the residues of 11 pharmaceuticals and personal care products (PPCPs) in selected areas like municipal and hospital waste water treatment plants in Greece and by her studies it is observed that the removal efficiency is higher in municipal waste water treatment plant than in hospital waste water treatment plant.
Jeong-wook Kevon and Jose M.Rodriquez has studied the occurrence and removal of selected pharmaceuticals and personal care products in three waste water treatment plants during a period of one year, antibiotics like sulfamethoxazole showed higher concentrations with around 1,640ng/l and carbamazepine showed lower concentrations with around 132ng/l ,
They concluded that the discharges from waste water treatment plants are the major sources of Pharmaceutical and personal care products contamination into surface water.
Miege C et al., 2008 created a database in order to quantitatively assess the occurrence and removal efficiency of pharmaceuticals and personal care products in Waste water treatment plants from 117 publications.
M.J.Martinez Bueno M.J et al., 2012 studied the occurrence and persistence of emerging organic pollutants in between the concentrations of few ng/l to few hundred ng/l and metabolites in five sewage treatment plants of Spain in two year case study.
Schwartz T et al., 2002 studied the detection of ARB and its resistance genes in waste water, drinking water and Biofilms. In different types of waters they found resistance like VAN (vancomycin) A in biofilms and Mec (Methicillin) A in hospital waste water.
Lapara TM et al., 2011 studied the impact of tertiary treated municipal waste water on the quantity of several antibiotic resistance determinants in Duluth-Superior harbour. Quantitative PCR technique is used to target the genes encoding tetracycline and integrons. This study demonstrates that the discharge of exceptionally treated waste water have significant effect on the quantities of antibiotic resistance genes.
Diwan V et al., 2010 quantified the antibiotics in water that are associated with hospitals in Ujjain, India and they found that susceptibility of e coli found in hospital effluent.
Ahammad Z.S et al., 2014 focused on blaNDM-1 resistance gene abundances associated with
5 seasonal human pilgrimages to the Upper Ganges River with compared to New Delhi Metallo-??-lactamase-1.
Mutiyar, P.K., Mittal, A.K.2014 explained in their review on risk assesment studies of antibiotic residues in water matrices in Indian scenario, he quantified by using a hazard quotient(HQ).Here HQs were developed by using measured environmental concentration(MEC) that are reported in the previous literature.
' There is no scientific data on pharmaceuticals and personal care products in the selected area.
' There are no Comparitive studies noted before with the selected water treatment plants.
' It gives the baseline documentation for the water treatment plants in controlling the spread of emerging pollutants.
' There is no proven data and very limited studies are carried out in India about the resistance studies of antibiotics towards bacteria.
1) A general survey of commonly used pharmaceuticals and personal care products (PPCPs) in the selected study area.
2) To determine the occurrence of pharmaceuticals and personal care products (PPCPs) hospital effluents, municipal waste waters, WWTP and river water.
3) To compare the occurrence of pharmaceuticals in four waste water treatment plants with different designs.
4) To estimate the concentrations of the pharmaceuticals and personal care products from initial concentration effluents to the final concentration effluents in waste water treatment plants and determination of the removal efficiency in each compartment in four waste water treatment plants with different operations.
5)To study the impact and proliferation of antibiotic resistance on antibiotics like Vancomycin, Streptomycin, Methicillin, beta lactams towards e coli, staphylococcus and Klebsiella bacteria in four waste water treatment plants and in aquatic environment.
Importance of proposed research:
Enormous amount of PPCPs into the environment is a source of alarm and great concern. People are aware on negative effects of PPCP than compare to the positive effects. The identification and quantification of PPCPs in waste water treatment plants is very important for the establishment of reasonable treatment options .The studies on PPCPs in different waste water treatment plants with different designs helps to understand the efficiency of the treatment plant in removing the pharmaceuticals. It is needed to understand the role of antibiotics and spread of antibiotic resistance bacteria and their possible ways in which the bacteria contribute its growth towards antibiotic resistance genes. Inspite of the lack of information as well as studies in PPCPs and the antibiotic resistance the maximum contamination of their pollutants remains unknown.
Understanding the Antibiotic gene transfer mechanisms in complex environments remains a difficult issue for the assesment of resistance that are spreading rapidly into the environment.
The data of environmental levels of pharmaceutical and personal care products and its resistance helps to understand the environmental risk not only to aquatic organisms but also to human beings. It helps to adopt the modern technologies to reduce the pollution.
A very little amount of information is available regarding the water treatment plants and the treatment process in removing PPCPs and its metabolites. It is important to study and monitor the waste water treatment plants and sewage treatment plants because very few studies are carried in developing countries. Analyses of contaminants which includes the seasonal variations helps to understand that at what periods the ecosystem is more sensitive and their risk for the exposure of pharmaceuticals. More comprehensive studies are required to understand the behaviour of the pharmaceuticals especially antibiotics under different waste water treatment plants for their removal.
Waste water treatment operations has to be considered seriously because these are the important factors in the global ecology of antibiotic resistance in which the treatment plants contains numerous amounts of wastes like hospital effluents, human faecal matter specifically known for the substantial concentrations of both antibiotic resistance genes and antibiotic resistance bacteria. This study helps to understand the overall proliferation of antibiotics and its resistance.
The proposed research helps in quantifying and the removal capacity of pharmaceuticals and personal care products from waste water treatment plants. The following database based on the research can be used to make decisions while designing the new methods in the treatment plants to reduce the pollutants on the weight of evidence.
The general survey has to be done from the medical prescriptions of major hospitals to distributors and the pharmacy shops the production data from the pharmacy industries both from human and animal drugs. The database has to create for the better understanding regarding pharmaceuticals and their action , major side effects occurred from the prescribed products.
Determination of Pharmaceuticals by using HPLC-MS/MS:
The water samples are collected by Grab sampling.200 to 250 ml of water samples to be collected for analysis.PP grade (poly propylene) bottles are used for the collection of samples. Samples are collected from hospital effluents, municipal waste water and waste water treatment plants from each filtration compartment and finally from downstream of the river. The samples have to collect in time series as well as seasonal variation. In time series samples has to collect in 3 time intervals starting from 9:00am, 1:00pm and 5:00pm and seasons like winter, spring, summer and fall.
The parameters like PH, temperature, conductivity, TDS (total dissolved solids) and DO (dissolved oxygen) will be measured in onsite using HACH probe which is connected to multiparameter analysis.
Once collected, the samples were kept at 4??c .Then the waters are vaccum filtered with 0.45??m nylon membrane filter (saritorious made) because of their lower amount of suspended particulate matter.After filtration approximate volume of aqueous solution of 5%Na2EDTA was added,)0.1ml of mixed standard was spiked for the blank and procedural recovery. For extraction process oasis HLB 60mg catridges are used, the catridges are pre conditioned before passing with 5ml methanol and 5 ml of Milli Q and has to load with filtered samples. After filtration the following samples are washed with Milli Q and centrifuged for 2 min to remove the excess water (Gros et al., 2009) .The final elution of target compounds are analysed with 4 ml of pure methanol and air dried under nitrogen steam and refilled with 1ml of methanol and water (25:75 V/V) and finally 10 ??l of 1ng/ml standard mixture containing internal standard added to standard calibration.
Analysis was performed by HPLC with quadrupole linear ion mass spectrometry with ion sources by C18 guard column with a mixture of 1:1v/v (acetonitrile-methanol) and 0.1% of formic acid with 0.2ml/min and each sample was injected to HPLC MS/MS.(Gros et al.,2009).
Detection of antibiotic resistance:
Antimicrobial susceptibility testing on water samples with antibiotics like vancomycin, methicillin, tetracycline and streptomycin has to evaluate with agar disk diffusion method on Mueller Hinton agar plates (CLSI) and the detection of gene resistance is carried out by PCR technique. The DNA extraction is carried out by thawing and incubated to lyses the cells by using DNA extraction kit the primers has to select for the amplification. The extracted DNA was added to final volume of PCR mixture containing master mix, pcr buffer, and magnesium chloride, dNTPs and taq DNA polymerase (ampicillin co., Denmark) and distilled water .The thermal cycling comprised of following steps like denaturation (94-96??c) and annealing 68??c and elongation (72??c).The amplified products are visualised by electrophoresis in 2%agarose gels with ethidium bromide as an indicator.