Assessment of polychlorinated biphenyls and organochlorine pesticides in water samples from the Yamuna River

Polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) are toxic, persistent and bioaccumulative long-range atmospheric transport pollutants. These are transported worldwide affecting remote regions far from their original sources, and can transfer into food webs with a wide range of acute and chronic health effects. India ratified the Stockholm Convention with the intention of reducing and eliminating persistent organic pollutants (POPs), and encouraged the support of research on POPs. Despite the ban and restriction on the use of these chemicals in India, their contamination of air, water, sediment, biota and humans has been reported. In this study, surface water samples were collected during January 2012 from the Yamuna River in Delhi, India, and analyzed for PCBs and organochlorine pesticides (OCPs). The concentrations of ∑PCBs and ∑OCPs ranged between 2-779 ng L–1 and from less than 0.1 to 618 ng L–1 (mean 99±38 ng L–1 and 221±50 ng L–1, respectively). The PCB homolog was dominated by 3-4 chlorinated biphenyls. In calculating the toxicity equivalent of dioxin-like PCBs (dl-PCBs)using World Health Organization toxic equivalency factors, dl-PCBs accounted for 10% of a total of 27 PCBs. The concentration of ∑HCH ranged between less than 0.1 and 285 ng L–1 (mean 151±32 ng L–1). However, ∑DDTs concentrations varied between less than 0.1 and 354 ng L–1 (mean 83±26 ng L–1). The concentrations were lower than the US guideline values; however, levels of lindane exceeded those recommended in guidelines. Further in-depth study is proposed to determine the bioaccumulation of these pollutants through aquatic biota to assess the risk of contaminants to human health. Introduction Polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) are ubiquitous chemicals which are persistent, toxic and bioaccumulative in nature and are found in all environments of the earth.1,2 In 2004, the Stockholm Convention listed DDTs and PCBs as persistent organic pollutants (POPs) and aHCH, b-HCH, and g-HCH (lindane) were added to the list in 2009.3 These compounds enter aquatic environments through, for example, effluent release, atmospheric deposition, and runoff. They can be transferred into food webs and, finally, accumulated in aquatic organisms.4 For the past decades, there has been growing concern about the presence of these POPs in the environment and their threat to biological life. These are long-range atmospheric transport pollutants affecting regions far from their original sources, even in regions of the Arctic5 and Antarctic.6,7 Persistent organic pollutants have a wide range of effects on human health, such as carcinogenicity, nervous and reproductive disorders, and are also suspected to be hormonal disruptors.8 India actively participated in the International Negotiation Committee meetings leading to the drafting and acceptance of the Stockholm Convention. The Convention was adopted in May 2001 and came into force on 17th May 2004. India ratified the Convention on 13th January 2006 and it came into force on 12th April 2006. As a party to the Convention, India is obliged to abide by the objectives of the treaty with the intention of reducing and ultimately eliminating POPs, and encouraged to conduct research on POPs. In earlier studies, PCBs, DDT and HCH contaminations with various matrices have been reported in India, including water,9-11 soils,2,12,13 sediments,9,14-17 atmospheric air,18-20 and biological samples, including those from humans.21,22 This study focused on assessment of HCHs, DDTs and PCBs, including dioxinlike PCBs (dl-PCBs) in water samples from the Delhi stretch of the Yamuna River, India. Materials and Methods Description of study area The River Yamuna, a major tributary of the River Ganges, originates from the Yamunotri glacier of the lower Himalayas and enters Delhi near the village of Palla after a journey of approximately 224 km. Delhi is the administrative capital city of India with a population of approximately 18 million and a total area of 1483 km2. It lies between 28° 36’ 36” N and 77° 13’ 48” E on the banks of the River Yamuna. There is a barrage at Wazirabad that supplies drinking water to Delhi accounting for more than 70% of overall water supply. Generally, no water is allowed to flow beyond the Wazirabad barrage in the dry season as the available water is not adequate to satisfy the demand for water to serve Delhi. Whatever water flows downstream of the Wazirabad barrage is the untreated or partially treated domestic and industrial wastewater that enters the river through several drains. Delhi alone generates 1900 million liters per day of sewage and available water treatment facilities are not adequate to remove all the pollutants. Consequently, untreated and partially treated sewage laden with the city’s biological and chemical wastes enters the river every day.23 The Okhla barrage lies twenty-two kilometers downstream of the Wazirabad barrage and, here again, water is not allowed to flow through this barrage during the dry season. The total catchment area of the Delhi Journal of Xenobiotics 2012; volume 2:e6 Correspondence: Bhupander Kumar, National Reference Trace Organics Laboratory Central Pollution Control Board East Arjun Nagar Delhi110032 India. Tel. +91.0114.3102.378. E-mail: bhupander_kumar@yahoo.com


Introduction
Polychlorinated biphenyls (PCBs), hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) are ubiquitous chemicals which are persistent, toxic and bioaccumulative in nature and are found in all environments of the earth. 1,2In 2004, the Stockholm Convention listed DDTs and PCBs as persistent organic pollutants (POPs) and a-HCH, b-HCH, and g-HCH (lindane) were added to the list in 2009. 3These compounds enter aquatic environments through, for example, effluent release, atmospheric deposition, and runoff.They can be transferred into food webs and, finally, accumulated in aquatic organisms. 4For the past decades, there has been growing concern about the presence of these POPs in the environment and their threat to biological life.These are long-range atmospheric transport pollutants affecting regions far from their original sources, even in regions of the Arctic 5 and Antarctic. 6,7Persistent organic pollutants have a wide range of effects on human health, such as carcinogenicity, nervous and reproductive disorders, and are also suspected to be hormonal disruptors. 8ndia actively participated in the International Negotiation Committee meetings leading to the drafting and acceptance of the Stockholm Convention.The Convention was adopted in May 2001 and came into force on 17 th May 2004.India ratified the Convention on 13 th January 2006 and it came into force on 12 th April 2006.As a party to the Convention, India is obliged to abide by the objectives of the treaty with the intention of reducing and ultimately eliminating POPs, and encouraged to conduct research on POPs.

Description of study area
The River Yamuna, a major tributary of the River Ganges, originates from the Yamunotri glacier of the lower Himalayas and enters Delhi near the village of Palla after a journey of approximately 224 km.Delhi is the administrative capital city of India with a population of approximately 18 million and a total area of 1483 km 2 .It lies between 28° 36' 36" N and 77°1 3' 48" E on the banks of the River Yamuna.There is a barrage at Wazirabad that supplies drinking water to Delhi accounting for more than 70% of overall water supply.Generally, no water is allowed to flow beyond the Wazirabad barrage in the dry season as the available water is not adequate to satisfy the demand for water to serve Delhi.Whatever water flows downstream of the Wazirabad barrage is the untreated or partially treated domestic and industrial wastewater that enters the river through several drains.Delhi alone generates 1900 million liters per day of sewage and available water treatment facilities are not adequate to remove all the pollutants.Consequently, untreated and partially treated sewage laden with the city's biological and chemical wastes enters the river every day. 23The Okhla barrage lies twenty-two kilometers downstream of the Wazirabad barrage and, here again, water is not allowed to flow through this barrage during the dry season.The total catchment area of the Delhi

Sample collection
Sampling was carried out at the 12 pre-determined sampling stations on the Yamuna River in Delhi extending between Palla and Okhla (Figure 1).Water samples in duplicates were collected in amber glass bottles during January 2012.Amber glass sampling bottles (1 L) were washed successively with detergent and tap water and then distilled water before sample collecting.Surface water samples were collected using a stainless steel bucket and transferred to sampling bottles.The glass bottles were filled to the top with the sample water to eliminate air bubbles.After appropriate labeling, the sample bottles were transported on ice to the laboratory and stored at 4°C followed by an extraction within seven days.

Chemicals and solvents
Chemicals and solvents were purchased from Merck, India.Silica gel 60 (0.063-0.100 mm) was from Sigma-Aldrich (St. Louis, MO, USA).Prior to use, silica gel and anhydrous sodium sulphate was cleaned separately with methanol dichloromethane and acetone in a Soxhlet extractor for 8 h each and stored air tight at 130°C.Certified reference standards were available for organochlorine pesticides (OCPs) and PCBs purchased from Sigma-Aldrich and from Dr. Ehrenstorfer (GmbH, Augsburg, Germany), respectively.

Sample extraction and cleanup
Samples were extracted, purified and analyzed according to the methods established by the US Environmental Protection Agency. 24riefly, a 1 L water sample was extracted three times with dichloromethane.The dichloromethane extract (lower layer) was drained into a funnel containing 5 cm of anhydrous sodium sulphate.The dichloromethane extract was then evaporated to 2-3 mL on the rotary vacuum evaporator (Eyela, Japan).Extracts were cleaned using silica gel column chromatography consisting of 2 cm of anhydrous sodium sulphate (approx.2.0 g) overlaid with 10 cm of activated silica gel (approx.10.0 g) and topped with another 2 cm of anhydrous sodium sulphate.Once the column was prerinsed with 30 mL of hexane, the sample was added and then a 50 mL mixture of hexane and dichloromethane (1:1 v/v).The eluted extract was concentrated using a rotatory vacuum evaporator and TurboVap (Caliper, Hopkinton, MA, USA) under a gentle stream of pure nitrogen and solvent-exchanged into hexane to 1.0 mL.The extract was transferred to auto sampler vial for quantification by gas chromatograph equipped with an electron capture detector (GC-ECD).

Instrumental analysis
In the present study, 4 DDT isomers, 4 HCH isomers and 27 congeners of PCBs were analyzed.Separation and quantification of pesticides was carried out using a gas chromatograph (Perkin Elmer, Clarus 500, Waltham, MA, USA) equipped with an Electron Capture Detector (ECD, 63 Ni), on a fused silica capillary column 25 m¥0.20 mm i.d.0.33 mm film (Elite -1 ).The column oven temperature was initially maintained at 170°C and programmed to 220°C (7 o C min -1 ) and again taken to 250°C at 5°C min -1 and held for 6.86 min.The injector and detector temperature were maintained at 250°C and 350 o C, respectively.Purified nitrogen gas was used as carrier at the flow rate of 1.0 mL min -1 .The separation and quantification of PCBs was performed by GC-ECD (Shimadzu 2010, Japan), on fused silica capillary column (HP-5MS, Agilent) 60 m¥0.25 mm¥0.25 mm film.The temperature program of the column oven was set at 170°C for 1 min then increased by 3°C min -1 to 270°C, kept for 1 min, then further increased by 10°C min -1 to 290°C, and maintained for 3 min.The injector and detector temperatures were maintained at 225°C and 300°C, respectively.Purified nitrogen gas was used as carrier at a flow rate of 1.0 mL min -1 .

Quality assurance/quality control
Certified reference standards from Dr. Ehrenstorfer and Sigma-Aldrich were used for the instrument calibration and quantification of PCBs and pesticides, respectively.The target analytes were identified in the sample extract by comparing the accurate retention time from the standard mixture, and quantified using the response factors from five level calibration curves of the standards.Appropriate quality assurance quality control analysis was performed including analysis of procedural blanks (analyte concentrations were <MDL, method detection limit), random duplicate samples (standard deviation <5), and calibration curves with the r 2 value of 0.999.
The instrument detection limits were established by using 3:1 signal to noise ratio to determine a peak as a valid quantifiable peak.Each sample was analyzed in duplicate and the average was used in analytical calculations.Calculated concentrations were reported as less than the limit of detection if the peak area did not exceed the specified threshold (three times the noise).Concentrations below the limit of detection were assigned zero values for the statistical analysis.Method detection limits were established by processing eight aliquots of a sample spiked with a quantity sufficient to produce a detectable response (s/n >3) and multiplying the standard deviation by 3 (the t students value for eight replicates).MDL for PCBs and organochlorine pesticides were 1
PCBs are not used as single compounds but as technical mixtures of different congeners.Trichlorobiphenyl was primarily used in power capacitors and transformers while high chlorinated biphenyls were mainly used as additive. 25PCB homolog patterns observed in this study are presented in Table 2 and Figure 2. The PCB homolog was dominated with 3-4 chlorinated biphenyls and pattern was in order of; tetra-CBs (35%) > tri-CBs (25%) > hepta-CBs (22%) > hexa-CBs (9%) > penta-CBs (8%) (Table 2).The homolog patterns show that the lighter-weighted molecular PCBs were much higher than those higher-molecular weight PCBs, and accounted for 68% and 31%, respectively.However, sampling stations 10, 11 and 12 show dominance of high chlorinated biphenyls (heptachlorobiphenyls).Water quality at these locations is influenced by two major drains from which municipal and industrial wastewater enter the River.In an aquatic environment, the PCBs in water may come from industrial and municipal wastewater discharges and air deposition.These are usually adsorbed on the particles in water and settle in the sediment that could naturally become a sink of PCBs as PCBs are non-ionic compounds and the octanol/water partition coefficient (K OW ) is in the range of 10 4 -10 8 .
TEQ concentrations of PCBs with established dioxin-like activity, especially non-and monoortho substituted PCBs, in water samples from the Yamuna River were calculated by multiply-ing the concentration of each dioxin-like PCB congener by its 2, 3, 7, 8-TCDD substituted TEF (Table 3).The TEQ values ranged between less than 1-1600 pg WHO 2005 TEQ L -1 with a mean value of 221±143 pg WHO 2005 TEQ L -1 .
Differences in composition of HCH or DDT isomers and their ratios in the environment could indicate different contamination sources. 26The ratios of HCH and DDT isomers obtained from this study are presented in Table 5. Technical HCH consists principally of four isomers, a-HCH (60-70%), b-HCH (5-12%), g-HCH (10-15%), d-HCH (6-10%), while lindane contains more than 99% of g-HCH. 27The average concentration of a-HCH, b-HCH, g-HCH and d-HCH in this study was 13%, 35%, 14% and less than 0.01%, respectively.The a/g-HCH ratio has been used to identify the possible HCH source; the higher ratio (>3) indicates input of technical HCH and long-range transport.However, a smaller ratio (<1) is characteristic of lindane sources. 28The ratio of a-HCH/g-HCH in this study varied between 0.23 and 1.67 (mean 1.11).
The ratios between the parent compound of DDT and its metabolites [DDD dichlorodiphenyldichloroethane (DDD) and dichlorodiphenyldichloroethene (DDE)] can be used to identify the possible sources in the environment. 29In the present study, occurrence order of DDT isomers was DDT > DDE > DDD.It is reported that DDTs can volatilize to an ambient environment in a few days. 1 After the DDT applications, much of the DDT slowly converted to DDE and DDD under aerobic and anaerobic conditions, respectively, 30 hence the ratios between the DDT and DDE and DDD is often used as an indication of age (recent or historic) and biotransformation of the DDT.A ratio (DDT/DDD+DDE) much greater than 1 indicates fresh use of DDT; however, a small ratio indicates historic DDT applications.In the present study, the ratios of DDT/(DDD+DDE) were in the range of less than 0.1 to 2.08 (mean 0.86), indicating that the DDT contaminations are from historic usage and biotransformation of DDT in this area.The ratio of p,p'-DDT and p,p'-DDE can be used to estimate the presence of technical DDT in recent inputs.A ratio of less than 1 is considered to be a very old or aged mixture, while a relatively high (>1) ratio indicates DDT use in the last five years. 31,32In our study the pooled mean ratio of p,p'-DDT/p,p'-DDE was between 2.08 and 2.94 (mean 2.51) indicating input of DDT in recent years.The p,p'-DDT/∑DDT ratio for technical DDTs was reported to be less than 1. 33 The mean ratio of p,p'-DDT to ∑DDT in the present study was 0.50, which indicates the presence of technical DDT.Furthermore, the ratio of DDD/DDE can reveal the degradation pathways of DDT, since DDE and DDD are the aerobic and anaerobic degradation products of DDT, respectively.A DDD/DDE ratio of less than 1 (<1) shows aerobic degradation and more than one (>1) indicates anaerobic degradation. 26As shown in Table 5, the DDD/DDE ratios ranged between less than 0.1 and 2.44 (average 1.22).This indicates that degradation of parent DDT was prevalent under anaerobic conditions.

Polychlorinated biphenyls
Significant levels of PCBs in different environmental matrices from Delhi and adjoining areas have been reported. 11,12,13,15,19The results of this study were compared with reports from Europe, America and other tropical countries (Table 6).Similar to our study, PCB distribution in water has been reported from Poland (average 60-440 ng L -1 ), 34 northern China (19.46 to 131.62 ng L -1 ). 35Higher levels of PCBs have been reported from the Pearl River, China (91-1353 ng L -1 ), 36 northern Nigeria (6721 ng L -1 ), 37 the Warri River (350-1300 ng L -1 ), the Ethiope River (1500 ng L -1 ) and the Benin River (30-2930 ng L -1 ) in Nigeria, 38,39 Konya, Turkey (505-2377 ng L -1 ), 40 and the Minjiang River, southeast China (204-2473 ng L -1 ). 41However, lower concentrations of PCBs than those found in our study have been reported from the Jiangsu section of the Yangtze River, China (<0.21-44.4ng L -1 ), 42 Kunming, China (13 to 72 ng L -1 ), 43 southern Moravia, Czech Republic (5.2 to 190.8 ng L -1 ), 44 and from the Yangtze River, China (1.23 to 16.6 ng L -1 ). 4 Organochlorine pesticides HCH and DDT isomers have been identified as contaminants worldwide (Table 6).DDT and g-HCH concentrations of 39 ng L -1 and 285 ng L -1 , respectively, have been reported in water samples from northern Nigeria. 37Total OCPs of 0.94-231.8ng L -1 have been reported from river water from Okinawa, Japan. 45There N o n -c o m m e r c i a l u s e o n l y have been reports of 3.13-10.60ng L -1 of ∑HCHs and 4.05-20.59ng L -1 of ∑DDTs in surface water from the Baiyangdian Lake, northern China 35 and of 8-239 ng L -1 DDTs and 6-234 ng L -1 HCHs in water samples from Egypt. 46Ghose et al. 47 reported lindane (10-430 ng L -1 ) and DDT (30-650 ng L -1 ) concentrations for ground water in greater Kolkata, India.Even higher values of chlorinated pesticides have been reported in the literature such as ∑OCPs (631-1540x10 3 ng L -1 ) for the Jukskei River in Gauteng, South Africa, 48 HCH (76-100 ng L -1 ) and DDT (116-848 ng L -1 ) in drinking water from Haryana, India, 49 661x10 3 -1434x10 3 ng L -1 of ∑OCPs reported from Ghana 50 and ∑OCPs (214.4-1819ng L -1 ) from the Minjiang River estuary, southeast China. 41he ratio of a-HCH/g-HCH obtained from this study reflects the usage of technical HCH as well as lindane (g-HCH) in this area.Other studies also anticipated the use of HCH mixture and lindane in this region. 12,13,15,16,19The ratios between the parent compound of DDT and its metabolites (DDD and DDE) suggest that local input of DDT cannot be ruled out, and this may be caused by observation of significant DDT concentrations in this area 12,13,16,19,49,51,52 coupled with the higher longrange atmospheric transport tendency of DDT under tropical climate conditions.The possible sources of DDTs are the combined effect of past and ongoing use in vector control.

Eco-toxicological risk assessment
The PCB and OCP contamination levels in water from the Yamuna River in Delhi were compared with guideline values stipulated by the US National Oceanic and Atmospheric Administration (NOAA) 53 and the New Jersey Department of the Environment. 54The NOAA has recommended 2000 ng L -1 for total PCBs as criteria maximum concentration in ambient freshwater.The observed concentrations of PCBs in this study were lower than the criteria maximum concentration (CMC) value, indicating no adverse toxicity effects to the aquatic biota and other users.NOAA's recommended criteria maximum concentration for DDD, DDE and DDT are 600 ng L -1 , 1050¥10 3 ng L -1 and 550 ng L -1 , respectively.Therefore, concentrations of these pollutants in the Yamuna River were far below the recommended CMC.The New Jersey Department of the Environment has recommended 12.4¥10 3 ng L -1 , 495 ng L -1 and 26 ng L -1 as criteria continuous concentration for a-HCH, b-HCH and g-HCH, respectively.It is clear from Table 4 that g-HCH exceeds the recommended guideline concentration.As mentioned earlier, lindane formulation is registered for use in public health practices to control vector borne diseases.Eco-toxicological risk may not be ruled out since the level of g-   HCH exceeded the guideline values and might be a cause of concern for the river ecosystem.
Comparatively high organochlorine concentrations were detected in water samples collected from locations receiving wastewater through municipal drains (i.e.location ns. 4, 6, 9, 10, 11 and 12; Figure 3).This indicates that these pollutants have been used in this area and the chemicals have found their way to the river ecosystem.DDT and HCH have been used for public health purposes in India and contamination of PCBs in the local environment is restricted to transformer oil rather than technical mixtures used for industries and electrical appliances.India's production of electronic waste (e-waste) is growing at an exponential rate generating approximately 150,000 t/year, much of which is stockpiled or poorly managed.PCBs, which have been widely used in industrial production, may also be present in the electronic waste stream.Most of the ewaste generated in the country ends up in New Delhi for recycling purposes, though procedures are not regulated creating a possible source for PCBs.A statistically significant (P<0.05)positive linear correlation has been observed between the amount of e-waste generated in 2005 and the PCB concentration in the atmosphere of Indian cities. 20 The other sources of PCBs in the Delhi region were probably from open biomass burning and of emission depositions from wood processing, paint and dying chemicals, electrical and electronic waste recycling, and polyvinylchloride manufacturing units.

Conclusions
The concentration of PCBs, HCH and DDT in Yamuna River water are comparable with other tropical regions of the world.The lower chlorinated PCBs dominated the total PCBs and dl-PCBs accounted for only 10%.The concentrations of HCH and DDT were lower than US guidelines; however, levels of lindane were higher than the CMC indicating possible toxicological effects to aquatic biota.Wastewater coming from drains should undergo complete treatment for organic contaminants before entering the river.Further work is required to determine the bioaccumulation through tissues of aquatic biota (e.g.fish) to determine the contaminant levels in these living bodies.

Figure 1 .
Figure 1.Map showing sampling locations on the Yamuna River in Delhi, India.

Figure 2 .
Figure 2. Station-wise distribution of total % of polychlorinated biphenyl homolog in Yamuna River water.
1 ng/L, respectively.PCB congeners are denoted by their International Union of Pure and Applied Chemistry numbers.The dioxin-like PCBs (dl-PCBs) are assigned with the toxic equivalent factors (TEFs) based on the relative toxicity with 2, 3, 7, 8-tetrachloro dibenzo-p-dioxin (TCDD). 8Toxic equivalent quantities (TEQ) of dl-PCBs were calculated by multiplying the concentration of individual dl-PCB congener with the corresponding World Health Organization (WHO) TEFs.The results were reported as ng L -1 and pg WHO 2005 -TEQ L -1 .

Table 2 . Concentration of polychlorinated biphenyl homolog and their percentage in Yamuna River water.
PCB, polychlorinated biphenyl.

Table 3 . Toxic equivalency of dioxin-like polychlorinated biphenyls in Yamuna River water (pg World Health Organization-toxic equivalent quantities L -1 ).
dl-PCBs, dioxin-like polychlorinated biphenyls; SE, standard error.