Characterization of pharmaceuticals industrial effluent using GC–MS and FT-IR analyses and defining its toxicity
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The physicochemical analysis of collected effluent sample for different parameters shown results as pH (pH 5.6 ± 0.11) slightly acidic, high conductivity (1563.34 ± 176 μs cm−1), total dissolved solids (920.34 ± 137 mg L−1), high BOD (7253.34 ± 1022 mg L−1), and COD (756.67 ± 1124 mg L−1) in the effluent sample. The results of heavy metals concentration are viz. as [Cu (1.98–2.56), Co (0.26–0.53), Cd (0.10–0.50), Ni (0.04–0.07), Pb (0.58–1.2), Mn (0.58–1.05), Cr (1.47–1.51), Zn (2.61–3.5), Fe (1.72–2.13), As (0.05–0.09), and Hg (0.003–0.006)]. Results revealed the higher concentration of BOD, COD, TDS, and conductivity and also the concentration of lead. Results of GC–MS also confirmed the high levels of organic pollutants in effluent. Further the effluent toxicity was evaluated by employing genotoxocity assays with the use of Allium cepa L. (onion) root tip cells. Genotoxicity measured mitotic index (MI) and chromosomal aberrations (CAs) in root tip cells obtained after treatment with effluent of 6.25, 12.5, and 25% concentration (v/v). The results of root growth test showed that inhibition of root growth occurred at effluent concentration ≥ 50% (v/v). The lowest MI was recorded (MI = 9.6%) in 25% of effluent concentration, showing a significant reduction in mitotic index compared with control which MI = 64.1%. Further, the chromosomal aberration was investigated in root tip cell after treating with different concentration ranges of effluent exhibiting various CA, viz. c-mitosis, chromosome loss, chromosome break, micronucleated cells, etc. The result suggests that the effluent contained toxic constituents, which imposed cytotoxic and genotoxic hazard.
KeywordsPharmaceutical industrial effluent Allium cepa Mitotic index Chromosomal aberration Micronuclei cell
Increase in global demand for drugs has made pharmaceutical industry one of the major 26 polluters of solid wastes and effluent into the environment (Anyakora et al. 2011). According to estimates, about half of the global wastewater from pharmaceutical industries is released without any recommended preprocessing (Osaigbovo and Orhue 2006; Anetor et al. 1999).
In spite of the fact that the volume of untreated or entirely treated pharmaceutical industrial wastewater is little, it contains a high level of pollutants because of the presence of non-biodegradable organic matter such as antibiotics, other prescription drugs, non-prescription drugs, animal and plant steroids, reproductive hormones, betalactamides, anti-inflammatories, analgesics, lipid regulators, anti-depressants, cytostatic agents, personal care products, detergent metabolites, flame retardants, product of oil use and combustion, and other broadly used chemicals, i.e., spent solvents, reaction residues, used filter media, etc., heavy metals (such as lead, mercury, cadmium, nickel, and chromium), and other pollutants (Babaahmadi et al. 2017; Ramola and Singh 2013; Vuppala et al. 2012; Chelliapan et al. 2011). Pharmaceutical industrial wastewater contains toxic and hazardous substances, the majority of which can be unfavorable to human well-being.
The effects of pharmaceutical chemicals on public health and environment are critical due to their acute toxicity, including genotoxicity and mutagenic potential (Nadal et al. 2004, Akintonwa et al. 2009; Bakare et al. 2009; Adeoye et al. 2015). Drugs of various types, as active metabolites or unmetabolized sometime, especially in developing countries, are not well managed on expiration and subsequently discarded in large amount as environmental discharge in the surrounding ecosystems. While in the environment most of the pharmaceutical are discharge toxicants present in wastewater accumulates in aquatic bodies, soil, and other biological systems, and often exceeds the critical threshold levels (Cleuvers 2003; Hernandoa et al. 2006; Larsson et al. 2007). A number of studies have identified specific chemical components in pharmaceutical effluents and demonstrated their toxic effects on living organisms (Daughton and Ternes 1999; Jones et al. 2001; Larsson et al. 2007; Idris et al. 2013). Therefore, in recent years, great concern is shown to study the impacts of discharged pharmaceutical effluents on the ecosystem and its services, and human wellness tied to them.
Method to frame the prescription drugs to more advanced in form of lesser bioactivity, enhanced absorption, and reduced excretion of waste and carbon footprint.
Which types of mechanisms may be used to enhance the approval and regulation of present, but new redesigned, advanced medicines using green alternative synthesis methods should be incentivized, which are least hazardous and reduce the environmental impacts.
Way of procedure modification to reduce the waste discharged quantity and use of less toxic constituents in manufacturing and also reduction in the carbon footprint
Does little quantity of the non-medication chronic exposure to prescription drugs within the water have an impact on the usefulness of those prescription drugs to deal with treat conditions or illnesses? What practices can be employed to lessen the amount of pharmaceutical waste among health care companies, pharmacists, insurers, and agriculture, thereby enhancing source reduction and pollutants prevention?
Find out the methods of safe disposal of unused, what are the methods to make certain safe disposal of unused, objectionable, or expired prescription drugs and enhanced disposed elimination of those substances from discharged wastewater, and in the long run the potable water?
Allium cepa (onion) offers easier pathway to the assessment of chromosomal degradation and interference in mitotic cycles. This is possible because A. cepa cells have large chromosomes and in reduced numbers (Levan 1938). Thus, this test has been broadly used to study the genotoxicity of an extraordinary assortment of environmental pollutants, such as heavy metals, pesticides, aromatic hydrocarbons, and complex mixtures of pollutants (Leme and Marin-Morales 2009).
Objective of the present study is to identify physicochemical parameters (viz. pH, electrical conductivity (EC), total dissolved solids (TDS), BOD, COD, and heavy metals (Cu, Co, Cd, Ni, Pb, Mn, Cr, Zn, Fe, As, and Hg) and organic pollutants in pharmaceutical effluent. The organic constituents were characterized using gas chromatography–mass spectrophotometer (GC–MS) and Fourier-transform infrared spectroscopy analyses (FT-IR). The effluent toxicity was studied by genotoxicity assays using Allium cepa L. (onion) root tip cells. Genotoxicity studies included measuring mitotic index (MI) and chromosomal aberrations (CAs) in root tip cells following treatment with 6.25, 12.5, and 25% (v/v) of effluent.
Materials and methods
The pharmaceutical industry effluent samples were collected from Lucknow, Uttar Pradesh (India). Effluent samples were collected from industry outlet point in sterile plastic containers of 1 L capacity. Total three samples were collected from pharmaceutical industry at 10 days time period. Samples were brought to laboratory in icebox and stored at 4 °C and filtered with Whatman filter paper no. 42. pH of collected effluent was measured with a digital pH meter (Metrohm, USA). Electrical conductivity (EC) and total dissolved solids (TDS) were determined by a conductivity meter (Thermo Orion, model 162A, USA). Biochemical oxygen demand (BOD) and chemical oxygen demand (COD) were determined by the titration method (APHA 2005). Heavy metals (Cu, Co, Cd, Ni, Pb, Mn, Cr, Zn, Fe, As, and Hg) were determined with atomic absorption spectroscopy (AAS) (GBC, Avanta Sigma, Australia). The detection of As was performed on inductively coupled plasma-optical emission spectrometer (ICP-OES, VARIAN720-ES).
To analyze the presence of different organic constituents in the pharmaceutical effluent, 50 mL of effluent was treated thrice with an equal volume of hexane (Koning et al. 2009). Extraction and derivatization of effluent sample were done using hexane and trimethyl silyl (BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) TMCS). Silylated sample (1 µL) was injected in GC–MS (Ultra TSQ Quantum XLS Mass spectrometer, Thermo Scientific, USA). The injection step was operated in splitless injection mode with a column temperature of 250 °C and flow of carrier helium gas with the flow rate of 1.1 mL min−1. The compounds were identified based on retention time (RT in minutes) and mass spectra obtained from full scan mode (m/z 45–800). Furthermore, FT-IR was done to identify the various organic functional groups. The effluent sample (50 mL each) was evaporated and subsequently dried (100 °C). Finally, a solid mass was obtained for each sample by mixing them with potassium bromide (KBr) (AR, Merck) and pellet was prepared. The analyses were carried out on FT-IR spectrum scan of 500–4000 cm−1 (PerkinElmer Spectrum RXI).
For the cytotoxicity bioassay, onion bulbs were purchased from a local market of Lucknow, India. Six different concentrations (% v/v) of pharmaceutical effluent (each 50 mL), viz. 6.25, 12.5, 25, 50, 75, and 100% v/v, were prepared. Five onion bulbs of equal size were treated and incubated at 23 °C with each concentration type in separate 50-mL Falcon™ tubes for 5 days. Dechlorinated tap water was used as a negative control and 5 mM ethane methyl sulfonate (EMS) treatment as the positive control. The solutions were changed every 24 h until the end of treatment.
The genotoxicity studies were carried out with the help of five onion bulbs per effluent concentration grown in tap water initially for 48 h. After that when root growth of 1–2 cm observed, it transferred into three different concentrations of effluents, viz. 6.25, 12.5, and 25% (v/v), plus tap water as control (Chauhan and Sundararaman 1990). At the end of the exposure, root tips were extracted and fixed in Carnoy’s solution [alcohol (98% purity) and acetic acid (glacial 100%) in 3:1 ratio] for 12 h at 4 °C. After treatment, the residual fixative was removed by washing with distilled water (2–3 times) and subsequently hydrolyzed with 1 N HCl at 60 °C for 5–7 min. Hydrolyzed root tips were again washed with distilled water (2–3 times) and cut into pieces of approximately 1–2 mm. Afterward, staining of root tips was done with hematoxylin (Fiskesjo 1985; Chauhan and Sundararaman 1990). Finally, all the slides were studied under microscope to calculate the mitotic index percent (MI) and the score of chromosomal aberrations (CAs) percentage. MI was estimated by the use of numbers of dividing cells in mitosis phaseout of total number of observed cells per concentration. In our study, it was based on 1000 cells count (Fiskesjo 1985). CA was calculated as total aberrant cells/total observed cells per concentrations.
The results of MI and CA were subjected to analysis of variance (ANOVA) test followed by Dunnett’s post hoc multiple comparison tests. Statistical analysis was performed using STATISTICA 7.0.
Results and discussion
Physicochemical characteristics of pharmaceutical effluent
Mean ± SE
Standard permissible value
5.6 ± 0.11
EC (µs cm−1)
1563.34 ± 176
TDS (mg L−1)
920.34 ± 137
BOD (mg L−1)
7253.34 ± 1022
COD (mg L−1)
756.67 ± 1124
Heavy metal (mg L−1)
2.21 ± 0.19
0.39 ± 0.08
0.27 ± 0.12
0.05 ± 0.01
0.85 ± 0.90
0.84 ± 0.14
1.50 ± 0.02
3.11 ± 0.27
1.90 ± 0.12
0.07 ± 0.02
0.004 ± 0.001
Inhibition in root growth and length of Allium cepa were observed in the present study following the treatment with pharmaceutical industrial wastewater which was in concurrence with previous studies conducted by other researchers (Akintonwa et al. 2009; Bakare et al. 2009).
Indices of chromosomal abnormalities observed in the root tip cells of onions grown in different concentrations of pharmaceutical effluent
Pharmaceutical effluent concentration (%)
Mitotic index (%)
64.1 ± 0.68
3.1 ± 0.90*
25.0 ± 0.70*
17.1 ± 0.70*
9.6 ± 0.51*
0.0 ± 0.0
0.0 ± 0.0
11.6 ± 0.9
20.6 ± 1.51
31.4 ± 0.19
0.0 ± 0.0
0.0 ± 0.0
7.2 ± 0.59
13.8 ± 1.07
25.4 ± 0.82
0.0 ± 0.0
0.0 ± 0.0
5.4 ± 0.51
12.2 ± 1.07
22.4 ± 0.88
0.0 ± 0.0
0.0 ± 0.0
4.4 ± 0.51
8.0 ± 0.71
17.4 ± 0.88
0.0 ± 0.0
0.0 ± 0.0
4.0 ± 0.45
5.0 ± 0.89
9.4 ± 0.82
0.0 ± 0.0
0.35 ± 0.2
0.0 ± 0.0
3.2 ± 0.67
4.6 ± 0.51
0.0 ± 0.0
0.6 ± 0.31
0.0 ± 0.0
1.6 ± 0.51
2.0 ± 0.32
0.0 ± 0.0
0.9 ± 0.41
0.0 ± 0.0
1.4 ± 0.51
2.8 ± 0.38
Aberrant cells (%)
0.0 ± 0.0
0.25 ± 0.2
6.0 ± 0.8*
12.0 ± 0.49*
19.5 ± 0.39*
The present study finds out that the different heavy metals, phenols, drugs, and other organic compounds are the constituents of pharmaceutical effluent which is discharged into the wastewater. These constituents of pharmaceutical effluent are toxic in nature and can harm the environment. The various types of the raw materials which are used should be either modified or should be replaced with other nontoxic substances, and the manufacturing design and source of toxic substances should be reduced by employing various types of technological methods. The new concepts of zero liquid waste discharged should be involved in the waste disposal methods. It is critical to develop an integrated multidisciplinary treatment system which could efficiently degrade the pharmaceutical ingredients in the effluent before their discharge in the surrounding environment. The rules and regulations and norms of the pharmaceuticals industries should be strict. The monitoring of the pharmaceuticals industries should be carried out by the regulatory authority to check the illegal discharge of unused waste materials and wastewater in the environment, which pose health hazards for biotas when mixed with the water bodies.
Authors are very thankful to Central Drug Research Institute-SAIF, Lucknow (India), for providing FT-IR analytical facility. Authors also acknowledge Director, Forest Research Institute, Dehradun, and Indian Institute of Toxicology Research, Lucknow (India), for providing laboratory facility for sample analyses. Authors are also thankful to University Grant Commission (UGC), New Delhi (India), for providing fellowship in the form of UGC JRF and extended SRF (UGC JRF/SRF Ref. No-3634 NET-December 2012).
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