The problem of water pollution acquires greater relevance in the context of a developing agrarian economy like Pakistan. Even though, the leather industry is a leading economic sector in Pakistan, there is an increasing environmental concern regarding tanneries because they produce large amounts of potentially toxic wastewaters containing both trivalent and hexavalent chromium, which are equally hazardous for human population, aquaculture and agricultural activities in the area. Therefore, we defined the scope of the present study as to employ different bioassays to determine the eco-toxic potential of tannery effluent wastewater (TW) and its chromium based components i.e., potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3. Particle-induced X-ray emission (PIXE) analysis of TW was carried out to determine the concentration of chromium in TW and then equal concentrations of hexavalent (K2Cr2O7) and trivalent chromium Cr2(SO4)3 were obtained for this study. Cytotoxicity assay, artemia bioassay and phytotoxicity assay were utilized to investigate the eco-toxicological potential of different concentrations of TW, K2Cr2O7 and Cr2(SO4)3. All the dilutions of TW, K2Cr2O7 and Cr2(SO4)3 presented concentration dependent cytotoxic effects in these assays. The data clearly represents that among all three tested materials, different dilutions of K2Cr2O7 caused significantly more damage (P < 0.001) to vero cell, brine shrimp and germination of maize seeds. Interestingly, the overall toxicity effects of TW treated groups were subsequent to K2Cr2O7 treated group. Based on biological evidences presented in this article, it is concluded that hexavalent chromium (K2Cr2O7) and TW has got significant eco-damaging potential clearly elaborating that environmental burden in district Kasur is numerous and high levels of chromium is posing a considerable risk to the human population, aquaculture and agricultural industry that can obliterate ecosystem surrounding the tanneries.
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Key words: Tannery effluent wastewater, Potassium dichromate, Chromium sulphate, Cytotoxicity assay, Artemia, Phytotoxicity
In developing countries, rapid industrialization has impacted dramatically resulting in heavy losses to economic welfare in terms of their toxicological consequences on human health and ecosystem through water and air pollution Reddy and Behera (2006). Man-made pollution is affecting the natural worldwide water resources to such an extant that reinstatement to immaculate conditions is realistically impossible Tornqvist et al. (2011). Water pollution is a critical confront of the age attributing serious impact on diverse economic activities of the world. Scientific community is playing their vital role in increasing awareness of water pollution and people are realizing the significance of delicate equilibrium connecting water pollution and global ecosystem.
Developing agrarian economies, like Pakistan, require greater consideration in the context of problems linked with water pollution. Albeit the fact that leather industry is a leading economic sector in Pakistan, there is an escalating ecological apprehension concerning tanneries due to the production of large amounts of potentially toxic wastewaters containing both trivalent and hexavalent chromium Szpyrkowicz et al. (2001). An example of rigorous water pollution induced by tanneries is in district Kasur, where half of the total numbers of tanneries (~ 300) in Pakistan are operational in close proximity to each other (Figure 1).
The tanning process incorporates the transformation of animal skin to leather. The skin is submitted to several procedures to eliminate hair, fat and meat in which different chemicals, such as ammonium salts, chlorides, chlorobenzene, enzymes, formic acid, kerosene, lime, tenso-active agents, potassium, sodium hypochlorite, sodium hydroxide, sulphates, and sulphuric acid are used (Figure 2).The obtained hide is then finally processed with mineral salts, colours and chromium (Cr) to obtain leather. The effluent thus generated contains large concentrations of Cr as potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3, which are highly toxic Alvarez-Bernal et al. (2006). This tannery effluent wastewater (TW) is then disposed off into the environment without any processing and treatment. Therefore, the discharge of TW and associated toxic compounds into aquatic systems represents an enduring environmental dilemma due to their potential impact on population in the receiving aquatic water and impending effects on human health Abbas Alkarkhi et al. (2008). Additionally, these toxic substances make their entry to the "surface and subsurface aquifers" resulting in pollution of irrigation and drinking water Mohan et al. (2010),Krishna et al. (2009). Throughout the past 21 years (1990-2011), tannery industry in district Kasur has caused noteworthy obliteration to the local environment and has recently been spotlighted due to recognition of the escalating environmental stress being placed on its water resources and of the resulting environmental degradation. Moreover, 50,000-60,000 local inhabitants are suffering from water borne diseases associated with water pollution e.g., gastroenteritis, hyperchloremic acidosis, hypertension, arteriosclerosis, cardiac arrest, retinal toxicity, hepatic fibrosis, hepatocellular cancer, diabetes, sperm damage, feto-maternal death, and impaired neurobehavioral functions Shakir et al. (2011).
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Pollution of water resources due to TW plays primary role for destroying ecosystems, therefore, characterization of TW was recently carried out in our lab using particle-induced X-ray emission (PIXE) to determine the concentration of different toxic elements in TW (Figure 3) Shakir et al. (2011). But chemical characterization of TW alone may not be sufficient to describe the adverse effects of the complex mixtures of chemicals present in TW. The potential utility of bioassays for monitoring both environmental quality and health of organisms inhabiting polluted ecosystems has received increasing attention during the recent years Minier et al. (2006),Ozmen et al. (2008).
Based on our recently published estimates of chemical characterization of TW and region's aquifers in the area of district Kasur, we defined the scope of the present study as to utilize different bioassays to ascertain the eco-toxic potential of TW and its Cr based components i.e., K2Cr2O7 and Cr2(SO4)3, which, to our knowledge, has never been systematically monitored for the area.
MATERIALS AND METHODS
Water sampling and particle-induced X-ray emission analysis
Collection and characterization of TW samples using PIXE analysis was carried out using the same method as described in our previous publication Shakir et al. (2011) . Briefly, TW samples were collected from the tannery area of district Kasur using sterile glass bottles. The concentration of Cr-VI in selected TW samples was quantified ~ 1.27 mg/ml by the PIXE spectrum. Furthermore, based on atomic weights, equal concentrations of K2Cr2O7 and Cr2(SO4)3 were obtained to determine the acute toxic index of different Cr salts produced during the tanning process. First ten solutions of the two fold dilutions of each sample were utilized in this study to investigate the eco-toxicological potential of different samples containing Cr (Table 1).
Vero Cell line was obtained from Veterinary Research Institute, Lahore. Cells were cultured from frozen stocks (maintained at -196 Â°C) and grown to confluency in a humidified 37Â°C incubator with 5% CO2. The cells were cultured in the growth medium containing GME media (12.6 g/L) supplemented with 8% fetal bovine serum and 3 ml Amphotericin B (250mg/ml) Dillon et al. (2000). When confluent, the adherent cells were detached with 0.25% Trypsin/EDTA and each well of 96 well plate was seeded with cell suspension at the rate of 104 cells in each well. First ten dilutions of TW, K2Cr2O7 and Cr2 (SO4)3 and control samples (prepared in GMEM media) were added in each well (100 Âµl/well) and incubated for 48hrs.
MTT assay was employed to determine the cell viability as described by Ermolli et al Ermolli et al. (2001). At the end of incubation period, all media was removed and cells were washed with PBS solution. Each well was then incubated with 100 Âµl of MTT solution (5mg/ml) and incubated for 5 hours. The media was then finally replaced with 100 Âµl freshly prepared DMSO (5%). After 2 hours of incubation, optical density of culture plate was determined by ELISA reader at 570 nm and percentage cell survival was quantified for each sample.
In order to determine the eco-toxic potential of TW and Cr salts on aquifer, artemia bioassay was utilized as described by other scientists Lagnika et al. (2011),Lumor et al. (2011). The cysts of commercially available brine shrimp (Artemia Franciscan) were purchased (Artemia International LLC, USA) and were hatched in sterile artificial seawater (3% [wt/vol] artificial sea salt in H2O) (Figure 5A). Artemia larvae (Figure 5B) were then transferred to 96 well plate (10-15/well) containing 100 Âµl of test dilutions (as described above) of TW, K2Cr2O7 and Cr2(SO4)3 prepared in artificial sea water. The numbers of dead shrimps were then recorded after incubation in photo-incubator at 250C for various exposure times (1hr, 24hr, 48hr and 72hr).
"Seed root elongation inhibition test" was utilized for the assessment of phytotoxicity of TW, K2Cr2O7 and Cr2(SO4)3. Maize seeds (Zea mays) were cultured in petridishes for five days in dark room at 22 Â± 2oC Arias-Barreiro et al. (2010),Boluda et al. (2011). Ten dilutions (as described above) of TW, K2Cr2O7 and Cr2(SO4)3 were prepared in fresh water, 10 ml of which was used during five days of incubation on regular basis. After five days of incubation with different test dilutions, the number of germinated seeds and length of roots were quantified and compared with control.
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SPSS for windows (version 12, SPSS inc., Chicago, IL, USA) was used to process and compare the data of all bioassays with control. Results were illustrated as means Â± SD and value of (P < 0.05) was termed as significant.
Figure 4 spotlights the percentage survival rate of vero cells, which were subjected to ten dilutions of three tested substances. Among all TW dilutions, application of D1- D5 caused very highly significant (P < 0.001) reduction in the cell survival, while highly significant (P < 0.01) reduction in cell survival was observed in the cells incubated with D6, whereas application of D7-D9 caused non-significant effects on vero cells. The maximum (P < 0.001) damage to cells with Cr2(SO4)3 dilutions were observed in groups treated with D1-D3, whereas, the maximum survival rate was recorded in groups treated with D6-D10. Application of D1- D7 dilutions of K2Cr2O7 caused very highly significant (P < 0.001) reduction in the cell survival. Surprisingly, D9 caused more damage (P < 0.01) to cells than D8. The data clearly represents that among all three tested materials, different dilutions of K2Cr2O7 caused significantly more damage to vero cell and TW group was subsequent to this group. Interestingly, the maximum number of cells survived in groups treated with Cr2(SO4)3 group.
The data representative of hatching physiology and exposure pathology of artemia is spotlighted in Figure 5, whereas comprehensive data set of time scale mortality associated with treatment of three tested materials is illustrated in Figure 6. During the 1st hr of incubation, significant increase (P < 0.05) in the mortality percentage of artemia was recorded with D1 and D2 (Figure 5D) in the group treated with TW, whereas only 1st dilution (D2) of K2Cr2O7 caused significant damage to artemia. Subsequent dilutions of all three tested materials presented no mortality during this time point (Figure 6A).
After 24 hrs of incubation, the toxic potential of K2Cr2O7 samples with lower concentrations (D6-D8) was significant better than TW and Cr2(SO4)3, whereas early dilutions (D1-D5) of K2Cr2O7 and TW caused almost identical pattern of mortality of artemia. Additionally, significant mortality was observed only in samples treated with higher concentration (D1-D5) of Cr2(SO4)3, while Cr2(SO4)3 proved non lethal at lower concentrations (Figure 6B). In nutshell, the toxicity for both K2Cr2O7 and TW are practically of the same level except for a limited condition (24hr incubation at D6-D8) where K2Cr2O7 showed higher toxicity than TW.
During 48 hrs of incubation, all three tested materials behaved similarly causing very highly significant (P < 0.001) increase in mortality with D1-D6 (Figure 6C), whereas 100% mortality was observed at 72 hrs of incubation in all samples treated with different dilutions of TW (Figure 5E), K2Cr2O7 and Cr2(SO4)3 (Figure 6D). The data clearly elaborates that among all tested materials, different dilutions of K2Cr2O7 proved more toxic than TW and Cr2(SO4)3.
After 5 days of incubation, the root length of maize seeds was measured revealing complex differentiating branching pattern of roots in control group (Figure 7B). Higher concentrations (D1-D6) of TW, K2Cr2O7 and Cr2(SO4)3 proved highly lethal resulting no germination of seeds (Figure 7C-F). Despite the fact that increase in root length was observed in the germinating seeds treated with the lower concentration of TW and Cr2(SO4)3 (Figure 7G-H), application of any concentration of K2Cr2O7 was highly toxic for germinating seed resulting in very highly significant reduction (P < 0.001) in root size and pattern (Figure 7I), proving K2Cr2O7 the most toxic tested sample in plant bioassay (Figure 8).
All the dilutions of TW, K2Cr2O7 and Cr2(SO4)3 presented concentration dependent cytotoxic effects on vero cell line (Figure 4). Application of earlier dilutions of TW, K2Cr2O7 resulted in more than 80% (P < 0.001) loss of vero cells. Even though, identical pattern of cell survival was revealed in the groups (D1-D3) treated with higher concentration of Cr2(SO4)3, maximum survival of vero cells was observed in this group making the group least toxic. Despite the fact, that lower concentrations of TW also caused considerable damage to vero cell line, least survival rate was recorded in group treated with K2Cr2O7, making K2Cr2O7 the most toxic Cr based substance for vero cells (Figure 4). Although, some studies have demonstrated the cytotoxic effects of TW and of trivalent Cr compounds [Cr2(SO4)3] using RTG-2 fish cell line Riva et al. (2005) and MG-63 osteoblast-like cells Fleury et al. (2006) respectively, there is hardly any study demonstrating the comparative toxic potential of TW, K2Cr2O7 and Cr2(SO4)3 using MTT assay, which is one of the most reliable assay to determine cell survival in diverse conditions. The data presented here strongly advocate that TW, as a whole, and hexavalent Cr compound (K2Cr2O7) found in TW are hazardous for different biological cells. Wise et al, and Chen et al., have recently demonstrated concentration dependent cytotoxic effects of hexavalent Cr compounds using lung, skin fibroblasts and testes cells of North Atlantic right whale, which is supporting the data presented in this study Wise et al. (2008),Chen et al. (2009). Likewise, it has recently been revealed that exposure of hexavalent Cr; even at low concentration, has potential to cause oxidative stress, which is toxic for pulmonary cell lines Caglieri et al. (2008).
Eventhough, artemia bioassay has been demonstrated by several researchers to investigate the acute toxic effects of different toxicants Beketov and Liess (2006),Verriopoulos et al. (1987),Sleet and Brendel (1982); we here present successful application of artemia bioassay to investigate the toxic potential of effluent from tannery industry. The data presented in figure 6 clearly demonstrate that all three tested materials have caused significant mortality during the 1st (1hr post incubation) and 2nd (24 hrs post incubation) phase of experiment. The growth of brine shrimp was more sensitive to the higher concentrations of TW than K2Cr2O7 during the early phases of incubation. This early mortality of artemia might be associated with the increase burden of diverse toxicants in TW Gomes et al. (2011),Mohammadi et al. (2009),Haydar and Aziz (2009),Munz et al. (2008),Santosa et al. (2008). During the 2nd phase, almost identical pattern (P < 0.001) of artemia vulnerability to the higher concentrations of TW and K2Cr2O7 was observed, whereas, toxic effects of K2Cr2O7 were still significant (P < 0.05) even at lower concentrations (Figure 6B; D6-D8; Figure 6C; D7) making K2Cr2O7 more toxic among all three tested materials.
The data presented in figure 7 and 8 evidently demonstrate slow germination of maize seeds in all treated groups, lowest in group treated with K2Cr2O7 followed by TW and Cr2(SO4)3. Even though, seeds started to germinate with D2 of Cr2(SO4)3 and D4 of TW treated group, application of K2Cr2O7 presented highly detrimental (P < 0.001) effects on seed germination even with D10. At lower concentration, the growth pattern of roots in group treated with Cr2(SO4)3 (Figure 7G) was comparable to control group; thin main branch with several secondary branches. The group treated with lower concentration of TW presented single thick root without any branch (Figure 7H), while hardly any root was observed in K2Cr2O7 group (Figure 7I). These findings are inline with the research work of Calheiros et al who have reported that highly concentrated TW (100, 70 and 50%) with poor "water treatment" caused complete inhibition of germination of T. pratense Calheiros et al. (2008). Irrigation of agriculture land with TW has been shown to cause significant impairment in soil productivity resulting in 25-100% inhibition in seed germination Tayyar and Yapici (2009),Nath et al. (2009),Tayyar et al. (2008),Shanker et al. (2005),Nath et al. (2005),Karunyal et al. (1994). Kumar et al has recently demonstrated that Cr based tannery industry is creating detrimental impacts leading to soil degradations, damaging the physiological seed germination, and as a result root development system is critically spoilt leading to reduced plant yield Kumar et al. (2010). It is also evident from the graphical presentation of phytotoxicity data (Figure 8) that there is remarkable difference of "seed germination and root length" between K2Cr2O7 and Cr2(SO4)3 offered groups, proving K2Cr2O7 more lethal to this physiological event. Recently, identical differences in toxicity for cultivars was observed between hexavalent and trivalent Cr by Soudek et al. Soudek et al. (2010). Our results are also in accordance with the findings of López-Luna et al, who have reported greater toxic effects for hexavalent Cr on wheat, oat and sorghum seedlings because it is more mobile in soil than trivalent Cr and tannery sludge Lopez-Luna et al. (2009).
Environmental Cr generally subsists in different forms, but trivalent and hexavalent Cr are more common types. In leather industry, trivalent Cr is extensively employed as tanning agent, which is potential source of environmental contamination Song et al. (2000). The trivalent Cr is comparatively immobile than hexavalent Cr and is considered less toxic, as shown in data. But the disadvantage of trivalent Cr used in tanning is that is radially oxidized to form hexavalent Cr, which is highly toxic and soluble in water Sirajuddin et al. (2007). Therefore, Cr used in tanning should not be termed as "safe" in any form because the inter-conversion is quite rapid, as is the case in district Kasur.
Farmers in district Kasur have been using TW to irrigate their agricultural land since the last few decades, which has not only resulted in soil pollution and destruction of the aquatic life but has also significantly affected the food crops in the area. It was therefore vital to determine the eco-toxicological potential of TW and its Cr based integral constituents. Based on biological evidences presented in this article, it is concluded that hexavalent Cr (K2Cr2O7) and TW has got significant eco-damaging potential clearly elaborating that the environmental burden in district Kasur in numerous and high levels of Cr is posing a considerable risk to the human population, aquaculture and agricultural industry that can obliterate the ecosystem surrounding the tanneries. The current study also established that tannery industry is playing a key catastrophic role in exceeding environmental standards and is linked with waste streams beyond water quality standards. Moreover, comprehensive studies to describe the mechanisms involved in different pathologies associated with the exposure of TW are ongoing in our lab, which will address all the key questions related to eco-damaging potential of Cr based tannery industry in district Kasur.
This work was supported by the "Indigenous PhD grant" from Higher Education Commission, Islamabad,Â Pakistan".
Table 1: Different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3 used during this study
Label of dilutions
Levels of two fold dilutions
Concentration of chromium-VI in TW
Concentration of chromium in K2 Cr2O7
Concentration of chromium in Cr (SO4)3
Legends of the figures
Figure 1: Illustration of the study area, showing geographical description of district Kasur and clusters of tanneries (â-¡) in close proximity to agricultural land (empty area in the map)(A). The tanneries in the area produce tones of effluent wastewater (B), which is directly polluting agriculture land in the area (C) and posing great environmental threats to ecosystem.
Figure 2: Schematic presentation of the typical processing routes for leather tanning and finishing.
Figure 3: Representative spectrum of proton induced x-ray emission (PIXE) describing the concentrations of chromium VI and different elements revealed by chemical characterization of tannery effluent wastewater in district Kasur.
Figure 4: Graphical presentation describing the percentage survival of Vero cell line after being treated with different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3. Note the maximum damage caused in group treated with K2Cr2O7.
Figure 5: Photomicrographs demonstrating the effects of different dilutions of tannery effluent wastewater (TW) on the survival of artemia. Commercially purchased cysts of brine shrimp (Artemia Franciscan) were hatched in sterile artificial seawater (3% [wt/vol] artificial sea salt in H2O) (A) and their larvae (B) were exposed to different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3. In comparison with control artemia, diminished growth and increase mortality of artemia was recorded 1hr post incubation with D2 of TW (C). Note the significant increase in size of artemia observed 72hr post incubation with D9 of TW (D).
Figure 6: Graphical presentation describing the effect of different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3 on the mortality of artemia. The data presented clearly demonstrate dose dependent affects of all three tested materials on the survival of artemia. During early phases (1st hr and 24 hrs post incubation), the growth of brine shrimp was more sensitive to the higher concentrations of TW and lower concentration of K2Cr2O7, while the pattern of mortality was almost identical during the late phases (48 hrs and 72 hrs) causing highly significant mortality of artemia.
Figure 7: Pictorial presentation on the effect of different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3 on the germination of seeds. The data presented here clearly demonstrate that no seed was germinated in groups treated with higher concentrations (D1) of TW (C), K2Cr2O7 (D) and Cr2(SO4)3 (E) and treated seed presented deformed morphology (F) in comparison with control group (A). In addition, application of K2Cr2O7 caused significant damage to this process and hardly any root (I) was observed in this group (D8), whereas single thick root without any branches was observed in the same group treated with TW (H). The least damage was seen in group treated with Cr2(SO4)3 where branching pattern (G) was comparable to control group (B).
Figure 8: Graphical presentation describing phyotoxicity potential of different concentrations of tannery effluent wastewater (TW), potassium dichromate (K2Cr2O7) and chromium sulphate Cr2(SO4)3. Note decreased root length in all dilutions of K2Cr2O7 making it highly toxic to this physiological event.