Chemosphere 120 (2015) 527–537
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Influence of taxa, trophic level, and location on bioaccumulation of toxic metals in bird’s feathers: A preliminary biomonitoring study using multiple bird species from Pakistan Naeem Akhtar Abbasi a, Veerle Leontina Bernard Jaspers b, Muhammad Jamshed Iqbal Chaudhry a,c, Sakhawat Ali d, Riffat Naseem Malik a,⇑ a
Environmental Biology and Ecotoxicology Laboratory, Department of Environmental Sciences, Faculty of Biological Sciences, Quaid-e-Azam University, Islamabad, 45320, Pakistan Department of Biology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway c WWF – Pakistan, Ferozpur Road, P O Box 5180, Lahore 54600, Pakistan d Department of Wildlife Management, PMAS Arid Agriculture University Rawalpindi, Pakistan b
h i g h l i g h t s The first study which screens toxic metals in multiple birds species of Pakistan. The study shows metals tendency for various taxonomic and trophic groups of birds. This study reports metals concentration snapshot along the stretch of Pakistan. It is the first endeavor which unveils the metals associated risks to local avifauna.
a r t i c l e
i n f o
Article history: Received 23 December 2013 Received in revised form 22 August 2014 Accepted 24 August 2014
Handling Editor: A. Gies Keywords: Toxic metals Bioaccumulation Taxonomic affiliation Trophic level Principal component analysis
a b s t r a c t Increasing concentrations of heavy metals in the environment and their effects on ecosystems and biota is still an imminent threat, particularly in developing parts of the globe. The aim of the present study was to screen the heavy metal concentrations in multiple bird species across Pakistan and to preliminary evaluate the influence of taxa, trophic level, and geographical location on heavy metal accumulation in various bird species. For this purpose, we measured the concentration of 9 heavy metals (Pb, Cd, Cr, Ni, Co, Cu, Fe, Zn and Mn) in feathers of 48 bird species from different localities in Pakistan. Species exhibited heterogeneous levels of heavy metals in feathers with marked inter and intra specific variations. Mean concentrations of studied metals in feathers followed the trend Fe > Zn > Cu > Pb > Mn > Cr > Ni > Co > Cd. Species belonging to closely related taxa (families) showed comparable metal concentrations in their feathers, inferring potential phylogenetic similarities in metal exposure or accumulation. In general, concentrations of metals were greatest in carnivorous species followed by omnivorous and insectivorous birds, and granivores showing minimal levels (p < 0.000). Furthermore, concentrations of metals varied significantly between locations (p < 0.000) exhibiting highest concentrations in Punjab province and Baluchistan, probably due to higher industrial and agricultural activity and runoff, respectively. With certain limitation, influence of trophic level, taxonomic affiliation and sampling location of birds on toxic metal accumulation was also statistically corroborated through principal component analysis (PCA). This study highlights that despite restricted emissions, heavy metals persist in the local environment and may pose elevated risks for the studied bird species in Pakistan. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Over the past few decades, excessive deposition of heavy metals in the environment and their negative impacts on ecosystems and ⇑ Corresponding author. Tel./fax: +92 5190643017. E-mail address: [email protected] (R.N. Malik). http://dx.doi.org/10.1016/j.chemosphere.2014.08.054 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
organisms has attracted the attention of ecotoxicologists throughout the globe (Frantz et al., 2012). Heavy metals are persistent elements and ubiquitous in the environment due to both naturally occurring geological cycles and anthropogenic activities. Terrestrial and aquatic ecosystems receive heavy metals for example through accidental discharge, waste dumping and/or atmospheric fallout. Subsequent exposure of biota results in accumulation of
528
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
heavy metals in their tissues and increase of metals accumulation in each succeeding feeding guild (Deng et al., 2007; Naccari et al., 2009). Presence of heavy metals in the environment poses a genuine threat to the quality and sustainability of ecosystems (Burger, 1993; Furness, 1993). A wide range of adverse effect due to heavy metals contamination in organisms, including endocrine and nervous disorders, genotoxicity, and certain physiological and behavioral abnormalities, has been observed in various studies (Burger and Gochfeld, 2000; Martin et al., 2003; Dauwe et al., 2004). Due to increasing public concern regarding heavy metal contamination, it is therefore necessary to assess, monitor, evaluate, manage and remediate ecological damage (Movalli, 2000; Naccari et al., 2009). In a living ecosystem, organisms vary in their level of metal accumulation depending upon their diet, trophic level, age, size, gender and various other internal or external parameters. Assessing the condition of an ecosystem by measuring the concentrations of contaminants in all compartments of the ecosystem is difficult, time consuming and labor intensive. So, for the sake of convenience various indicator (sentinel) species are commonly used which are exposed to the environment and could best reflect the state of that ecosystem in a wider range (Burger and Gochfeld, 2000; Scheifler et al., 2006; Brait and Antoniosi-Filho, 2011; Falq et al., 2011). Birds are useful sentinels for environmental contamination as they reside at the top of the food chain, are long lived, bioaccumulate a variety of contaminants, are widely distributed in the environment and sensitive to detect environmental change (Furness, 1993; Llabjani et al., 2012). Globally, birds are widely used for biomonitoring. Most of the recent literature has focused on birds of prey (Jager et al., 1996; Movalli, 2000; Battaglia et al., 2005; Burger and Gochfeld, 2009), water fowl and seabirds (Kim and Koo, 2007; Moreno et al., 2011), and small passerine birds (Dauwe et al., 2000; Jaspers et al., 2004; Leonzio et al., 2009). Raptor birds are particularly good bioindicators. These species, being at the top of the food pyramid can yield information over a large area around each sampling site, not only on bioavailability of contaminants but also their possible course of biomagnification in the food web (Battaglia et al., 2005). Some species have biological habits that increase the likelihood of exposure to contaminants and, in that way can produce relevant information that would be missed if only water or soil would be analyzed. Most recently, urbanelydwelled species like feral pigeon (Columba livia) have also been used for the environmental monitoring of urban environment (Brait and Antoniosi-Filho, 2011; Frantz et al., 2012). Measuring the pollutant concentrations directly in the internal tissues of birds is well in practice and proven to be the best indicator for bioaccumulative compounds. But recently, the need for alternative, noninvasive tissues for biomonitoring has arisen for various ethical, practical and conservationist reasons. Instead of sacrificing living birds, different non-invasive matrices such as eggs (Jaspers et al., 2005; Van den Steen et al., 2006; Eens et al., 2013) and feathers (Jaspers et al., 2004; Kim and Koo, 2007; Frantz et al., 2012) are commonly used for biomonitoring. Despite all its pros and cons, feathers offer many advantages as a useful non-destructive biomonitoring material and are often considered to be an excellent tool for monitoring bioaccumulativ compounds (Malik and Zeb, 2009; Garcia-Fernandez et al., 2013). Among the non-destructive matrices, feathers are most advantageous for bird biomonitoring because they are easy to collect, store and transport. Feathers can repeatedly be sampled from a living bird; moreover, samples can be collected without seriously affecting the fitness of the bird. Reproductive success of birds can also be monitored without interference (Spahn and Sherry, 1999). Furthermore body feathers can be removed from a bird without producing adverse effects on the bird so it is a much valued non-destructive biomonitoring tool for endangered species
(Behrooz et al., 2009; Jaspers et al., 2011). During the period of feather growth, heavy metals accumulate in feathers in proportion with the blood concentrations (Eens et al., 1999). Contrary to blood, which reflects short term exposure to metals (Furness, 1993), feathers exhibit a long term exposure because metal accumulation continues throughout feather growth until blood vessels atrophy from it. So feathers serve as an archive for food based bioaccumulative compounds (Burger and Gochfeld, 2000). Because of these specific properties and ease of utility, feathers have been used as a non-destructive biomonitoring tool for metals contamination for decades (Burger, 1993). Metal accumulation in birds is mostly affected by metal species, its bioavailability and exposure pathways (Burger et al., 2008). Moreover, in literature it is presumed and somehow proven that accumulation of metals in birds is influenced by a variety of parameters such as trophic position, taxonomic group, genetic variability, age, gender, size of bird and feathers types etc. (Burger and Gochfeld, 2000; Dauwe et al., 2002; Burger et al., 2008; Burger and Gochfeld, 2009; Zolfaghari et al., 2009; Lucia et al., 2010; Castro et al., 2011). Many studies revealed that metal accumulation differs in birds with different feeding habits, feeding strategies and trophic levels on the food chain. Birds on the top of the food chain usually accumulate more metals in their tissues than those which are lower on the food chain (Burger and Gochfeld, 2000; Tsipoura et al., 2008). Van Straalen and Ernst (1991) also suggest that levels of metal accumulation in indicator species might increase with the age of the organism and its trophic position. Effects of taxonomic affiliations of birds species on metals have been reported in a limited number of studies (Ochoa-acuna et al., 2002; Zolfaghari et al., 2009). To the best of our knowledge no comprehensive study has investigated the combined effects of trophic level, taxonomic affiliation, and geographic location on metal accumulation in birds. In Pakistan, so far no baseline data for metals contamination in bird is available. It is the first study in which we investigated metal concentrations in 48 selected bird species from different localities in Pakistan and compared toxic metal concentrations among different taxonomic groups, feeding guilds (trophic level) and widely stretched geographical locations in Pakistan. The purpose was to preliminary study the inter and intra-specific variation of heavy metal concentrations in different bird species from Pakistan. We made a first attempt to study the obscure linkages between heavy metal accumulation in different taxonomic groups and trophic levels in birds from Pakistan. In addition, we performed a geographical comparison to compare and evaluate the contamination level at different locations in Pakistan.
2. Materials and methods 2.1. Feathers sampling Feathers (approximately 0.25 g for each individual; 3–5 birds per species) from a total of 48 species were collected and analyzed from different locations of Pakistan (details on species and sample sizes per location are presented in Table S.I. 2). Whole sampling was carried out from September 2011 to August 2012. No birds were captured for the sole purpose of this particular study; all the samples were taken from birds caught in other studies and some for commercial uses. Fig. 1 demonstrates different sampling sites and number of species collected from different sites. Samples from different individuals (n) of any single species were collected around the same location in the study area. No species was sampled from two or more locations. Sampling sites included remote rural areas, rivers, lakes, dams, cities and two coastal sites. Most of the samples were collected from Punjab province, which is the hub of industrial and agricultural activities. No industrial unit or
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
529
Fig. 1. Location map showing 21 different sites (S1–S21). Parenthesis [] represents No. of birds sampled from that site.
large scale agriculture has been recorded in the vicinity of the sampling location from Sindh, KPK (Khyber Pakhtoonkhwa) province and AJK (Azad Jammu and Kashmir),while the sites from Balochistan province were totally devoid of any agricultural or industrial activities except Gawadar (Fig. 1; S 20), which is an international sea port. Birds were captured in most of the cases through cages and mist nets and released after collecting feathers safely. Feathers were plucked from different parts of the body depending on the species. Body feathers were preferably collected as they are less affected by molting (Mansouri and Hoshyari, 2012), but for a few cases tail and fully grown wing feathers were collected depending upon the sampling availability of different feathers for different species Keeping in consideration the fitness and survival of birds, we only collected 1–2 tail or wing feather/s from live individuals in such cases. Because of the variation in available of feather types for different species, pooled analysis (using mix of different feathers types from each individual) were performed to estimate the overall metal burdens. After collection, feathers were put into plastic envelopes transported to the laboratory and stored in the refrigerator at 4C prior to analysis. 2.2. Heavy metals analysis/quality control and assurance (QC/QA) In the laboratory (Ecotoxicology and environmental biology QAU, Pakistan), feathers were washed vigorously with tap water, followed by deionized water alternated with acetone three times to remove the external contamination (Burger and Gochfeld, 2000). Washing of feathers with water removes the external air born particles and dust effectively while the surfactant solution (acetone here) wiped off the oil, lipid contents and other sticky substances. Treating feathers in aforementioned methods removes a substantial fraction of external contamination, although the contamination through preening likely cannot be removed completely (Jaspers et al., 2008). As feathers from different parts of the body reflect different exposure to metals, a pooled analysis has been
made for this study by making a combined extract of different feathers type for each individual separately. This has been done because the aim of the present study was to find mean concentration of different metals using feathers irrespective of their point of origin. After washing for 1 h, feathers were oven dried at 80C, ground into powder form and weighed 0.1 mg for digestion. Digestion consisted of two separate treatments in Teflon tubes. At first weighed samples were treated with Nitric acid (70%) and heated at 120 °C for 24 h and then 1 ml Hydrogen peroxide was added and kept for 24 h again. The final volume of the digested solution was measured to compute the metal concentration (Frantz et al., 2012). Concentration of elements (Pb, Cd, Co, Cr, Cu, Fe, Mn, Ni, and Zn) was measured using the Fast Sequential Atomic Absorption Spectrometer (VarianFS-AA-240). Blanks were prepared in a similar fashion without samples to verify contamination and instrumental precision. To avoid contamination all the glassware used for metal analysis was treated with diluted nitric acid (0.1% v/v) (Malik and Zeb, 2009). Analysis was carried out in batches with standard calibration curve and spiked specimens. For Quality control, standard reference material (National institute of science and technology 1648e), in addition to blanks and spiked samples was run after each 10 samples. Mean recoveries for samples ranged from 95% to 107% for all the metals which is an acceptable limit. Recovery for the certified samples was within the 10% of the certified values. 2.3. Statistical analysis All statistical tests (except PCA) were computed using statistical software STASTICA (99 Edition 5.5 A). The distribution of the whole dataset was normal so parametric tests were applied. To investigate whether metals accumulate differently, their mean concentrations among species, families and trophic level was compared separately through Analysis of variances (ANOVAs). Using adjusted P values multiple comparisons were made through post hoc fisherman LSD test. All the P values less than 0.05 were considered
530
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
as statistically significant. All the concentrations of metals were represented in arithmetic mean (lg g 1) ± S.E. Principal component analysis (PCA) was performed as described by Llabjani et al. (2012) and Frantz et al. (2012) to visualize the similarities and association of metals and associations of species, depending upon the concentrations of toxic metals in their feathers. Euclidean biplots were developed to observe species dispersion along the first two principal axes (explained 57.83% variance). The third and fourth axis were not considered because they explained negligible variation (eigen values
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Influence of taxa, trophic level, and location on bioaccumulation of toxic metals in bird’s feathers: A preliminary biomonitoring study using multiple bird species from Pakistan Naeem Akhtar Abbasi a, Veerle Leontina Bernard Jaspers b, Muhammad Jamshed Iqbal Chaudhry a,c, Sakhawat Ali d, Riffat Naseem Malik a,⇑ a
Environmental Biology and Ecotoxicology Laboratory, Department of Environmental Sciences, Faculty of Biological Sciences, Quaid-e-Azam University, Islamabad, 45320, Pakistan Department of Biology, Norwegian University of Science and Technology (NTNU), Trondheim, Norway c WWF – Pakistan, Ferozpur Road, P O Box 5180, Lahore 54600, Pakistan d Department of Wildlife Management, PMAS Arid Agriculture University Rawalpindi, Pakistan b
h i g h l i g h t s The first study which screens toxic metals in multiple birds species of Pakistan. The study shows metals tendency for various taxonomic and trophic groups of birds. This study reports metals concentration snapshot along the stretch of Pakistan. It is the first endeavor which unveils the metals associated risks to local avifauna.
a r t i c l e
i n f o
Article history: Received 23 December 2013 Received in revised form 22 August 2014 Accepted 24 August 2014
Handling Editor: A. Gies Keywords: Toxic metals Bioaccumulation Taxonomic affiliation Trophic level Principal component analysis
a b s t r a c t Increasing concentrations of heavy metals in the environment and their effects on ecosystems and biota is still an imminent threat, particularly in developing parts of the globe. The aim of the present study was to screen the heavy metal concentrations in multiple bird species across Pakistan and to preliminary evaluate the influence of taxa, trophic level, and geographical location on heavy metal accumulation in various bird species. For this purpose, we measured the concentration of 9 heavy metals (Pb, Cd, Cr, Ni, Co, Cu, Fe, Zn and Mn) in feathers of 48 bird species from different localities in Pakistan. Species exhibited heterogeneous levels of heavy metals in feathers with marked inter and intra specific variations. Mean concentrations of studied metals in feathers followed the trend Fe > Zn > Cu > Pb > Mn > Cr > Ni > Co > Cd. Species belonging to closely related taxa (families) showed comparable metal concentrations in their feathers, inferring potential phylogenetic similarities in metal exposure or accumulation. In general, concentrations of metals were greatest in carnivorous species followed by omnivorous and insectivorous birds, and granivores showing minimal levels (p < 0.000). Furthermore, concentrations of metals varied significantly between locations (p < 0.000) exhibiting highest concentrations in Punjab province and Baluchistan, probably due to higher industrial and agricultural activity and runoff, respectively. With certain limitation, influence of trophic level, taxonomic affiliation and sampling location of birds on toxic metal accumulation was also statistically corroborated through principal component analysis (PCA). This study highlights that despite restricted emissions, heavy metals persist in the local environment and may pose elevated risks for the studied bird species in Pakistan. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Over the past few decades, excessive deposition of heavy metals in the environment and their negative impacts on ecosystems and ⇑ Corresponding author. Tel./fax: +92 5190643017. E-mail address: [email protected] (R.N. Malik). http://dx.doi.org/10.1016/j.chemosphere.2014.08.054 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
organisms has attracted the attention of ecotoxicologists throughout the globe (Frantz et al., 2012). Heavy metals are persistent elements and ubiquitous in the environment due to both naturally occurring geological cycles and anthropogenic activities. Terrestrial and aquatic ecosystems receive heavy metals for example through accidental discharge, waste dumping and/or atmospheric fallout. Subsequent exposure of biota results in accumulation of
528
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
heavy metals in their tissues and increase of metals accumulation in each succeeding feeding guild (Deng et al., 2007; Naccari et al., 2009). Presence of heavy metals in the environment poses a genuine threat to the quality and sustainability of ecosystems (Burger, 1993; Furness, 1993). A wide range of adverse effect due to heavy metals contamination in organisms, including endocrine and nervous disorders, genotoxicity, and certain physiological and behavioral abnormalities, has been observed in various studies (Burger and Gochfeld, 2000; Martin et al., 2003; Dauwe et al., 2004). Due to increasing public concern regarding heavy metal contamination, it is therefore necessary to assess, monitor, evaluate, manage and remediate ecological damage (Movalli, 2000; Naccari et al., 2009). In a living ecosystem, organisms vary in their level of metal accumulation depending upon their diet, trophic level, age, size, gender and various other internal or external parameters. Assessing the condition of an ecosystem by measuring the concentrations of contaminants in all compartments of the ecosystem is difficult, time consuming and labor intensive. So, for the sake of convenience various indicator (sentinel) species are commonly used which are exposed to the environment and could best reflect the state of that ecosystem in a wider range (Burger and Gochfeld, 2000; Scheifler et al., 2006; Brait and Antoniosi-Filho, 2011; Falq et al., 2011). Birds are useful sentinels for environmental contamination as they reside at the top of the food chain, are long lived, bioaccumulate a variety of contaminants, are widely distributed in the environment and sensitive to detect environmental change (Furness, 1993; Llabjani et al., 2012). Globally, birds are widely used for biomonitoring. Most of the recent literature has focused on birds of prey (Jager et al., 1996; Movalli, 2000; Battaglia et al., 2005; Burger and Gochfeld, 2009), water fowl and seabirds (Kim and Koo, 2007; Moreno et al., 2011), and small passerine birds (Dauwe et al., 2000; Jaspers et al., 2004; Leonzio et al., 2009). Raptor birds are particularly good bioindicators. These species, being at the top of the food pyramid can yield information over a large area around each sampling site, not only on bioavailability of contaminants but also their possible course of biomagnification in the food web (Battaglia et al., 2005). Some species have biological habits that increase the likelihood of exposure to contaminants and, in that way can produce relevant information that would be missed if only water or soil would be analyzed. Most recently, urbanelydwelled species like feral pigeon (Columba livia) have also been used for the environmental monitoring of urban environment (Brait and Antoniosi-Filho, 2011; Frantz et al., 2012). Measuring the pollutant concentrations directly in the internal tissues of birds is well in practice and proven to be the best indicator for bioaccumulative compounds. But recently, the need for alternative, noninvasive tissues for biomonitoring has arisen for various ethical, practical and conservationist reasons. Instead of sacrificing living birds, different non-invasive matrices such as eggs (Jaspers et al., 2005; Van den Steen et al., 2006; Eens et al., 2013) and feathers (Jaspers et al., 2004; Kim and Koo, 2007; Frantz et al., 2012) are commonly used for biomonitoring. Despite all its pros and cons, feathers offer many advantages as a useful non-destructive biomonitoring material and are often considered to be an excellent tool for monitoring bioaccumulativ compounds (Malik and Zeb, 2009; Garcia-Fernandez et al., 2013). Among the non-destructive matrices, feathers are most advantageous for bird biomonitoring because they are easy to collect, store and transport. Feathers can repeatedly be sampled from a living bird; moreover, samples can be collected without seriously affecting the fitness of the bird. Reproductive success of birds can also be monitored without interference (Spahn and Sherry, 1999). Furthermore body feathers can be removed from a bird without producing adverse effects on the bird so it is a much valued non-destructive biomonitoring tool for endangered species
(Behrooz et al., 2009; Jaspers et al., 2011). During the period of feather growth, heavy metals accumulate in feathers in proportion with the blood concentrations (Eens et al., 1999). Contrary to blood, which reflects short term exposure to metals (Furness, 1993), feathers exhibit a long term exposure because metal accumulation continues throughout feather growth until blood vessels atrophy from it. So feathers serve as an archive for food based bioaccumulative compounds (Burger and Gochfeld, 2000). Because of these specific properties and ease of utility, feathers have been used as a non-destructive biomonitoring tool for metals contamination for decades (Burger, 1993). Metal accumulation in birds is mostly affected by metal species, its bioavailability and exposure pathways (Burger et al., 2008). Moreover, in literature it is presumed and somehow proven that accumulation of metals in birds is influenced by a variety of parameters such as trophic position, taxonomic group, genetic variability, age, gender, size of bird and feathers types etc. (Burger and Gochfeld, 2000; Dauwe et al., 2002; Burger et al., 2008; Burger and Gochfeld, 2009; Zolfaghari et al., 2009; Lucia et al., 2010; Castro et al., 2011). Many studies revealed that metal accumulation differs in birds with different feeding habits, feeding strategies and trophic levels on the food chain. Birds on the top of the food chain usually accumulate more metals in their tissues than those which are lower on the food chain (Burger and Gochfeld, 2000; Tsipoura et al., 2008). Van Straalen and Ernst (1991) also suggest that levels of metal accumulation in indicator species might increase with the age of the organism and its trophic position. Effects of taxonomic affiliations of birds species on metals have been reported in a limited number of studies (Ochoa-acuna et al., 2002; Zolfaghari et al., 2009). To the best of our knowledge no comprehensive study has investigated the combined effects of trophic level, taxonomic affiliation, and geographic location on metal accumulation in birds. In Pakistan, so far no baseline data for metals contamination in bird is available. It is the first study in which we investigated metal concentrations in 48 selected bird species from different localities in Pakistan and compared toxic metal concentrations among different taxonomic groups, feeding guilds (trophic level) and widely stretched geographical locations in Pakistan. The purpose was to preliminary study the inter and intra-specific variation of heavy metal concentrations in different bird species from Pakistan. We made a first attempt to study the obscure linkages between heavy metal accumulation in different taxonomic groups and trophic levels in birds from Pakistan. In addition, we performed a geographical comparison to compare and evaluate the contamination level at different locations in Pakistan.
2. Materials and methods 2.1. Feathers sampling Feathers (approximately 0.25 g for each individual; 3–5 birds per species) from a total of 48 species were collected and analyzed from different locations of Pakistan (details on species and sample sizes per location are presented in Table S.I. 2). Whole sampling was carried out from September 2011 to August 2012. No birds were captured for the sole purpose of this particular study; all the samples were taken from birds caught in other studies and some for commercial uses. Fig. 1 demonstrates different sampling sites and number of species collected from different sites. Samples from different individuals (n) of any single species were collected around the same location in the study area. No species was sampled from two or more locations. Sampling sites included remote rural areas, rivers, lakes, dams, cities and two coastal sites. Most of the samples were collected from Punjab province, which is the hub of industrial and agricultural activities. No industrial unit or
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
529
Fig. 1. Location map showing 21 different sites (S1–S21). Parenthesis [] represents No. of birds sampled from that site.
large scale agriculture has been recorded in the vicinity of the sampling location from Sindh, KPK (Khyber Pakhtoonkhwa) province and AJK (Azad Jammu and Kashmir),while the sites from Balochistan province were totally devoid of any agricultural or industrial activities except Gawadar (Fig. 1; S 20), which is an international sea port. Birds were captured in most of the cases through cages and mist nets and released after collecting feathers safely. Feathers were plucked from different parts of the body depending on the species. Body feathers were preferably collected as they are less affected by molting (Mansouri and Hoshyari, 2012), but for a few cases tail and fully grown wing feathers were collected depending upon the sampling availability of different feathers for different species Keeping in consideration the fitness and survival of birds, we only collected 1–2 tail or wing feather/s from live individuals in such cases. Because of the variation in available of feather types for different species, pooled analysis (using mix of different feathers types from each individual) were performed to estimate the overall metal burdens. After collection, feathers were put into plastic envelopes transported to the laboratory and stored in the refrigerator at 4C prior to analysis. 2.2. Heavy metals analysis/quality control and assurance (QC/QA) In the laboratory (Ecotoxicology and environmental biology QAU, Pakistan), feathers were washed vigorously with tap water, followed by deionized water alternated with acetone three times to remove the external contamination (Burger and Gochfeld, 2000). Washing of feathers with water removes the external air born particles and dust effectively while the surfactant solution (acetone here) wiped off the oil, lipid contents and other sticky substances. Treating feathers in aforementioned methods removes a substantial fraction of external contamination, although the contamination through preening likely cannot be removed completely (Jaspers et al., 2008). As feathers from different parts of the body reflect different exposure to metals, a pooled analysis has been
made for this study by making a combined extract of different feathers type for each individual separately. This has been done because the aim of the present study was to find mean concentration of different metals using feathers irrespective of their point of origin. After washing for 1 h, feathers were oven dried at 80C, ground into powder form and weighed 0.1 mg for digestion. Digestion consisted of two separate treatments in Teflon tubes. At first weighed samples were treated with Nitric acid (70%) and heated at 120 °C for 24 h and then 1 ml Hydrogen peroxide was added and kept for 24 h again. The final volume of the digested solution was measured to compute the metal concentration (Frantz et al., 2012). Concentration of elements (Pb, Cd, Co, Cr, Cu, Fe, Mn, Ni, and Zn) was measured using the Fast Sequential Atomic Absorption Spectrometer (VarianFS-AA-240). Blanks were prepared in a similar fashion without samples to verify contamination and instrumental precision. To avoid contamination all the glassware used for metal analysis was treated with diluted nitric acid (0.1% v/v) (Malik and Zeb, 2009). Analysis was carried out in batches with standard calibration curve and spiked specimens. For Quality control, standard reference material (National institute of science and technology 1648e), in addition to blanks and spiked samples was run after each 10 samples. Mean recoveries for samples ranged from 95% to 107% for all the metals which is an acceptable limit. Recovery for the certified samples was within the 10% of the certified values. 2.3. Statistical analysis All statistical tests (except PCA) were computed using statistical software STASTICA (99 Edition 5.5 A). The distribution of the whole dataset was normal so parametric tests were applied. To investigate whether metals accumulate differently, their mean concentrations among species, families and trophic level was compared separately through Analysis of variances (ANOVAs). Using adjusted P values multiple comparisons were made through post hoc fisherman LSD test. All the P values less than 0.05 were considered
530
N.A. Abbasi et al. / Chemosphere 120 (2015) 527–537
as statistically significant. All the concentrations of metals were represented in arithmetic mean (lg g 1) ± S.E. Principal component analysis (PCA) was performed as described by Llabjani et al. (2012) and Frantz et al. (2012) to visualize the similarities and association of metals and associations of species, depending upon the concentrations of toxic metals in their feathers. Euclidean biplots were developed to observe species dispersion along the first two principal axes (explained 57.83% variance). The third and fourth axis were not considered because they explained negligible variation (eigen values
