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Biomarker responses in the earthworm, Dichogaster curgensis exposed to fly ash polluted soils
Ecotoxicology and Environmental Safety 118 (2015) 62–70
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Biomarker responses in the earthworm, Dichogaster curgensis exposed to fly ash polluted soils Vijaykumar L. Markad a, Tekchand C. Gaupale b, Shobha Bhargava b, Kisan M. Kodam a,n, Vikram S. Ghole a a b
Biochemistry Division, Department of Chemistry, Savitribai Phule Pune University, Pune 411007, India Department of Zoology, Savitribai Phule Pune University, Pune 411007, India
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 21 April 2015
Earthworms are globally accepted as a model organism in terrestrial ecotoxicology for assessment of environmental pollution. This study evaluated and compared effects of fly ash polluted soils collected from two geographically different thermal power plants on biomarker responses in the earthworm, Dichogaster curgensis. To evaluate relationship between distance sampling and biomarker responses in the earthworm D. curgensis, soil samples at 0.5, 1 and 3 km from thermal plant were analyzed for physico-chemical properties and metal concentrations. Biochemical alterations, lysosomal membrane stability, genotoxic effects, and histological changes were examined on 1, 7, and 14 d of exposure to fly ash contaminated soils collected from different thermal power plants. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were significantly increased, while glutathione reductase (GR) activity was found to be decreased in treated animals. Catalase (CAT) and glutathione-S- transferase (GST) activities were found to be increased initially up to 7 d exposure and further decreased on 14 d exposure. D. curgensis exposed to fly ash contaminated soils showed significant lysosomal membrane destabilization and DNA damage. Extensive histopathological changes were observed in the tissues of the body wall and intestinal tract of the exposed D. curgensis along with accumulation of heavy metals. These results demonstrate that soil pollution around thermal power plants has adverse biological effects of on the indicator organism D. curgensis and no correlation was found between distance and extent of biological biochemical responses. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biomarkers Dichogaster curgensis Fly ash Heavy metals Histopathology
1. Introduction A massive amount of fly ash (4750 million tons) is generated worldwide from coal-based thermal power plants (Yao et al., 2015). It has started globally receiving alarming attention due to its hazardous nature, widespread usage, and the manner of disposal; leading to severe environmental pollution (Maity et al., 2009; Markad et al., 2012). India generates higher amount of fly ash and utilizes lower percentage of fly ash compared to other countries. Therefore, major portion of it is disposed in ash ponds near the power plants occupying more than 65,000 acres of land (Pandey and Singh, 2010; Pandey et al., 2011). According to World Bank, by 2015 India will require 1000 km2 of land for the disposal of coal fly ash (Singh et al., 2010). These ash ponds became a potential source for contamination of soil and water streams (Mandal and Sengupta, 2006; Pandey et al., 2011, Dragović et al., 2013). n
Corresponding author. Fax: þ91 20 25691728. E-mail address: [email protected] (K.M. Kodam).
http://dx.doi.org/10.1016/j.ecoenv.2015.04.011 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Leaching and accumulation of organic and inorganic toxic compounds from fly ash is of major environmental concern and known to have severe adverse impact such as bioaccumulation of metals, oxidative stress, DNA damage, and reproduction on terrestrial and aquatic ecosystems (Ali et al., 2004; Chakraborty and Mukherjee, 2009; Grumiaux et al., 2010; Pandey and Singh, 2010). Fly ash contains heavy metals (Cu, Zn, Cd, Pb, Ni, Cr, etc.) and polyhalogenated compounds (Pandey and Singh, 2010; Pandey et al., 2011). Elevated trace metal concentrations around the thermal power plants have been reported earlier (Mehra et al., 1998; Basavaiah et al., 2012; Dragović et al., 2013). Basavaiah et al. (2012) reported the metal pollution of soils collected from Nashik thermal power station, Maharashtra, India using magnetic screening. However, biological effects of this environmental pollution remain to be investigated. Environmental quality monitoring programs recommended the integration of biological responses with chemical data for risk assessment of the contaminants. Earthworm, an important organism of soil ecosystem, is widely accepted as biological indicator of environmental pollution (Łaszczyca et al., 2004; Lukkari et al.,
V.L. Markad et al. / Ecotoxicology and Environmental Safety 118 (2015) 62–70
2004). Several biomarkers have been identified in the earthworms to assess the environmental impact of wide range of contaminants. An enhanced production of reactive oxygen species is a general pathway of toxicity induced by organic and inorganic toxic compounds (Livingstone et al., 1990; Stohs and Bagchi, 1995) leading to a condition of oxidative stress and subsequent alteration in antioxidant defense mechanism of the organisms. Therefore, in earthworms, biochemical responses (antioxidant defense system and lipid peroxidation) are regarded as fast, diagnostic, and prognostic indices of environmental pollution (Saint-Denis et al., 2001; Łaszczyca et al., 2004). The alkaline comet and neutral red retention (NRR) assays have been demonstrated as effective methods to evaluate the DNA damage and lysosomal membrane stability in earthworm coelomocytes, respectively (Weeks and Svendsen, 1996; Lourenco et al., 2011a; Markad et al., 2012). Histopathological changes in earthworms have been previously reported as valuable endpoint of metal toxicity (KilIç, 2011; Lourenco et al., 2011b). Therefore, the present study was designed to evaluate the biological effects of soil contamination around the thermal power plants by using earthworm as a model organism. Earthworms were exposed to fly ash polluted soils and various biomarker endpoints viz. DNA damage, lysosomal membrane integrity, biochemical responses and histological changes related to the effect of ROS were studied. Furthermore, bioaccumulation of heavy metals in earthworms was also assessed to elucidate cause–effect relationship.
2. Materials and methods 2.1. Soils collection and analysis The fly ash polluted soils were collected from the fields near the ash disposal sites (receive continuously fly ash and its leachates from ash ponds) of the Nashik thermal power plant, Nashik, India and Bhusaval Thermal Power Plant, Jalgaon, India that generated 1.27 and 0.69 million tones of fly ash in 2012–2013 (CEA, 2014). For several decades, these activities have produced high concentrations of trace elements in surface soils (Basavaiah et al., 2012). For each study area, three soil samples from different sites were collected (Nashik area: S1–S3; Jalgaon area: S4–S6), while reference soil (R) was collected from the garden of Savitribai Phule Pune University, Pune, India, with no history of pollution. The soil samples of surface horizons (0–20 cm) were collected from three different points of a site to obtain a composite sample (Supplementary Fig. 1). The pH and electrical conductivity of the samples were measured in 0.01 M CaCl2. The organic matter and total metal content of all test soils were determined as described earlier (Markad et al., 2012). The soil samples were digested with concentrated nitric and perchloric acid and metal concentrations were estimated by using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, ARCOS, Spectro, Germany) (Markad et al., 2012). 2.2. Laboratory exposure A synchronized culture of earthworm, Dichogaster curgensis was maintained in laboratory conditions as described earlier (Markad et al., 2012). The organisms were acclimated for at least one week in a container with reference soil. Earthworms (n¼ 15) were exposed to the soils (1 kg) in polythene culture pots (20 cm 10 cm 8 cm). The organisms were fed once in a week with 10 g of defaunated cattle manure per set. The experiments were carried under controlled laboratory conditions of temperature (22 72 °C), moisture content (40%), and photoperiod (12 hL:
63
12 hD). Earthworms were removed from the experimental sets on 1, 7, and 14 d of exposure and used for bioaccumulation, cytotoxic and genotoxic, biochemical, and histopathological studies. For metal bioaccumulation, a pool of 4 earthworms from each group (depurated for 48 h, freeze killed) was acid digested and metal concentrations were estimated by using ICP-AES as described earlier (Maboeta et al., 2003; Markad et al., 2012). 2.3. Cytotoxic and genotoxic studies Three earthworms from each group were randomly selected on 1, 7, and 14 d of exposure and coelomocytes were harvested by simple, non-invasive technique described earlier (Manerikar et al., 2008). Coelomocytes obtained were further processed to assess the lysosomal membrane integrity and DNA damage by using NRR and alkaline comet assay, respectively as reported earlier (Markad et al., 2012). 2.4. Biochemical assays Three earthworms were removed from each group at an interval of 1, 7, and 14 d of exposure, rinsed with distilled water and kept for 48 h on moist filter paper in Petri dishes to depurate their gut contents. The earthworms were homogenized at 4 °C for 1 min in Tris–HCl buffer (100 mM, pH 7.5) using Potter-Elvehjem homogenizer and centrifuged at 12,000g for 20 min to obtain postmitochondrial fraction. The aliquots were stored at 80 °C until further use. The enzyme assays were performed using temperature-controlled dual beam UV–visible spectrophotometer (Jasco V-630, Japan). All biochemical estimations of Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S- transferase (GST) and lipid peroxidation (in terms of malondialdehyde, MDA) were assayed using methods as described in our earlier report (Markad et al., 2012). 2.5. Analysis of histological alterations In order to examine histological changes in different tissues, earthworms were removed from the soil on 14 d of exposure, depurated for 48 h on moist filter paper and fixed in Bouin's fixative for 24 h. Tissues were further dehydrated in ascending grades of alcohol, cleared in xylene, infiltrated in molten paraffin wax at 60 °C and embedded. The transverse sections (7 mm thick) of earthworms were mounted on the poly-L-lysine coated slides and stained with hematoxylin-eosin double staining method. Slides were mounted in DPX and observed under light microscope (Carl Zeiss, Axioskop 40) and images were captured (KilIç, 2011; Lourenco et al., 2011a, 2011b). 2.6. Statistical analysis Data were expressed as the mean 7S.E.M. Significant differences between the results of the different treatment groups were determined using one-way ANOVA and Dunnet multiple comparison post-hoc test. The level of significance was considered po 0.05. All statistical analyses were carried out using GraphPad Prism software (Version 5.0, USA).
3. Results and discussion 3.1. Soil analysis Physico-chemical analysis and metal concentrations of reference and contaminated soils are shown in Table 1. A significant
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Table 1 Physico-chemical properties and total metal concentrations of the soils used in experiments. Parameters
R (reference)
Distance from source (km) pH Conductivity (mS/cm) Organic matter (%) Si (g kg 1) Fe (g kg 1) Ca (g kg 1) K (g kg 1) P (g kg 1) Na (g kg 1) Al (g kg 1) Mg (g kg 1) Mn (mg kg 1) Pb (mg kg 1) Zn (mg kg 1) Ni (mg kg 1) Cd (mg kg 1) Cr (mg kg 1) Cu (mg kg 1)
5.7 7 0.1 11.7 7 0.1 7.7 7 0.1 1777 1.0 5.6 7 0.5 22 7 0.6 3.6 7 0.7 1.8 7 0.2 0.5 7 0.1 8.3 7 0.5 10.5 7 0.5 6337 47 3.6 7 0.5 169 7 5 25.8 7 0.3 0.5 7 0.1 28.17 1.6 517 0.6
Nashik Thermal Power Plant, Nashik
Bhusaval Thermal Power Plant, Jalgaon
S1 0.5
S2 1
S3 3
S4 0.5
S5 1
S6 3
6.6 7 0.1 12.7 7 0.1 7.7 7 0.2 2177 1.6 8.4 7 0.9 23.2 7 0.5 3.4 7 0.5 27 0.3 0.6 7 0.1 12.7 7 0.7 19.6 7 1.1 8717 55 127 0.5 284 7 3 697 2.4 5.0 7 1.4 95.2 7 2.3 1267 0.4
6.9 7 0.1 12.9 7 0.1 6.7 7 0.2 2177 2.1 8.8 7 1.2 247 2.8 4.5 7 0.8 1.8 7 0.3 0.6 7 0.2 12.9 7 1.2 13.8 7 0.5 887 7 5 177 0.6 294 7 4 62.5 7 3 4.0 7 0.2 877 3.4 1337 1.2
6.7 70.2 13.1 70.2 7.5 70.3 217 72.5 8.8 71.5 22.2 71.5 3.4 70.2 2.2 70.4 0.9 70.4 13.4 71.5 14.1 71.3 815 722 17 70.7 280 723 68 70.8 3.0 70.2 84 72 89 70.3
6.7 7 0.1 13.0 7 0.1 7.6 7 0.1 2187 1.5 7.2 7 0.9 22.8 7 1.3 3.6 7 0.4 1.9 7 0.4 0.9 7 0.3 14.3 7 0.9 18.2 7 0.6 805 7 48 177 0.2 2477 4 637 10.2 4.5 7 0.7 867 4.4 1187 0.5
6.7 7 0.2 12.8 7 0.1 7.17 0.1 2177 2.3 7.17 0.9 23.4 7 2.5 4.0 7 0.5 2.2 7 0.4 0.7 7 0.3 13.4 7 0.8 16.3 7 0.6 860 7 21 187 0.6 2467 7 677 1.4 3.0 7 0.57 847 1.5 1197 4.9
6.6 7 0.2 12.7 7 0.1 7.4 7 0.2 2177 1 6.4 7 1.2 21.2 7 2.6 4.0 7 0.3 1.8 7 0.4 0.7 7 0.2 10.7 7 2.5 13.9 7 0.5 845 7 32 167 0.5 258 7 8 637 5.4 3.0 7 0.7 86.6 7 3.3 86.2 7 3.6
increase in pH and conductivity (mS cm 1) of all test soils was observed. Soils samples from Nashik Thermal Power Plant showed increase in conductivity, Fe, Na, Al, with distance of sampling. This may be due to increased level of soluble inorganic constituents present in the fly ash (Jala and Goyal, 2006; Singh et al., 2010). On the contrary, Fe, Na, Al content of soils from Bhusawal Thermal power plant decreased slightly with increase in distance. This suggests differential mechanism in spreading of these metals and could be due to difference in soil properties at different geographical locations. Cd content was higher at 0.5 km distance and then decreased with increase in distance in both soils. The total metal concentrations of the contaminated soils were found to be depending on the collection site; higher concentrations were observed in the soil samples collected from the vicinity of thermal power plant (samples S1 and S4). Heavy metal concentrations viz. Pb, Zn, Ni, Cd, Cr, and Cu were significantly high in all tested soils when compared to reference soil. These results are in agreement with previous report that found increased trace metal profile around Nashik Thermal Power Plant (Basavaiah et al., 2012). Similar observations were noted in soils surrounding the thermal power plant in Delhi, India (Mehra et al., 1998) and Serbia (Dragović et al., 2013). The observed increased metal concentrations may be due to leaching and accumulation of these elements in soils from fly ash (Mandal and Sengupta, 2006), where these elements are abundantly present (Markad et al., 2012). Maximum enrichment of heavy metals was observed in the top soils when compared to the soils collected from different depth surrounding the ash ponds (Mandal and Sengupta, 2006). The earthworm survival was not affected throughout the exposure period. There were no significant changes observed in the metal body burdens of D. curgensis after 1 d exposure, while, significant bioaccumulation of Cd, Pb, Ni, and Cr was noted on 7 and 14 d of exposure to fly ash polluted soils, when compared with control earthworms (Supplementary Table 1). This may be due to direct exposure of the earthworms to metals present in soil through dermal contact or by ingestion of water, polluted food and/or soil particles (Hobbelen et al., 2006). Similar results were reported in Eisenia andrei exposed to a contaminated soil from an abandoned uranium mine (Lourenço et al., 2011a). Cadmium accumulation was found to be depending on the total metal concentration in the soil. However, accumulation of other metals (Pb, Cr and Ni) did not depend upon the total metal concentration in
the soil. 3.2. Biochemical responses Biochemical responses of the earthworms to environmental stress were regarded as early warning system for soil pollution (Łaszczyca et al., 2004). Fig. 1 depicts biochemical responses of D. curgensis, exposed to fly ash polluted soils. In the present study, SOD activity was significantly increased (15–31%) in all experimental sets and at all time points when compared with control earthworms. In our earlier work (Markad et al., 2012) SOD activity was decreased on 14 d exposure compare to 7 d at all the doses of fly ash except 40% fly ash dose. On the contrary, in this study, no decrease in SOD activity was noted. This indicates heavy contamination of soil samples in the vicinity of both the thermal power plants. No correlation between SOD activities and distance of soil sample location was established on day 14 (Fig. 1A). CAT activity was significantly increased in the earthworms on 1 d (7– 19%) and 7 d (13–28%) exposure to all test soils. However, on 14 d, CAT activity was significantly decreased when compared with 7 d, and was analogous to control earthworms (Fig. 1B). This decrease in CAT may be due to inhibition of enzyme by high cellular stress or inactivation by singlet oxygen, peroxyl radicals, and superoxide radical (Kono and Fridovich, 1982; Escobar et al., 1996). Further analysis of CAT activity data suggested no any correlation with distance soil sampling. Glutathione reductase reduces oxidized glutathione and maintains the cellular antioxidant status, whereas glutathione peroxidase utilizes reduced glutathione to eliminate H2O2 (Saint-Denis et al., 2001; Markad et al., 2012). The GR activity was significantly decreased (15–27%) in all treatment groups and at all exposure durations. While GPx activity was significantly increased (19–33%) in the earthworms exposed to all tested soils on 14 d exposure (Fig. 1C and D). Decrease in GR activity may be due to depletion of cellular NADPH level and/or inactivation of GR by metal binding (Goering et al., 1987; Saint-Denis et al., 2001; Łaszczyca et al., 2004). Glutathione S-transferase protects the tissue from oxidative stress by conjugation of glutathione with xenobiotics and lipid peroxidation products (Frova, 2006). In the present study, significant increase (14–21%) in GST activity was observed in earthworms on 7 d exposure. However, on 14 d, GST activity was significantly decreased (p o0.05) when compared to 7 d exposure
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Fig. 1. Biochemical responses of earthworm D. curgensis exposed to fly ash polluted soils; A) superoxide dismutase, B) catalase, C) glutathione reductase, D) glutathione peroxidase, E) glutathione S-transferase, and F) malondialdehyde on 1, 7 and 14 d of exposure. Data expressed as mean ± SD. Significant differences with control, n(p 4 0.05), # (p 4 0.01), $(p 4 0.001) (one-way ANOVA with Dunnet multiple comparison test).
and become normal as was in control earthworms (Fig. 1E). This decrease in GST activity could be attributed to depletion in GSH levels; evidenced by decreased GR activity and increased GPx activity (Łaszczyca et al., 2004; Maity et al., 2008). Similar response of GST activity was reported in earthworms from metal contaminated soils (Łaszczyca et al., 2004; Lukkari et al., 2004). The activities of GR, GPx and GST did not show any correlation with distance soil sampling. Lipid peroxidation is a sensitive indicator of oxidative stress
and various contaminants are known to induce lipid peroxidation through the formation of ROS. MDA is the lipid peroxidation product, widely used to measure the level of oxidative stress in the organisms (Saint-Denis et al., 2001; Sandrini et al., 2006). In the present study, significant increase (17–46%) in MDA level was observed on 7 and 14 d exposure in the earthworms of all test groups (Fig. 1F). This may be attributed to time-dependent enhancement in ROS generation. It can be noted that the reduction in CAT activity may favor lipid peroxidation due to accumulation of
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1d
45
7d
14d
40 35
NRR time (min)
30
#
$
$ $
#
#
$
#
$
$
$
25 †
20 †
15
†
†
† †
†
10 5 0 R
S1
S2
S3
S4
S5
S6
Soil samples Fig. 2. Neutral red retention time (min) for lysosomal membrane stability of coelomocytes derived from D. curgensis exposed to fly ash polluted soils assessed on 1, 7, and 14 d. Data expressed as mean7 SD. Significant differences #(p4 0.01), $(p 40.001), †(p 40.0001) compared with control (one-way ANOVA with Dunnet multiple comparison test).
H2O2, a precursor of hydroxyl radical that triggers lipid peroxidation (Sandrini et al., 2006). Similar observations were reported in invertebrates exposed to heavy metals (Saint-Denis et al., 2001; Ferreira-Cravo et al., 2009; Markad et al., 2012). Therefore, in the present study elevated MDA level along with increase in SOD, GPx activity, biphasic response in CAT, GST activity and decreased GR activity were observed, which reflects severe oxidative stress experienced by the earthworms upon exposure to fly ash polluted soils from both thermal plants. 3.3. Cytotoxic and genotoxic studies Lysosome is a sensitive target organ and its membrane stability has been used as a potential biomarker of toxic effects of diverse environmental contaminants (Weeks and Svendsen, 1996; Maboeta et al., 2003; Rocco et al., 2011). The results of the present study revealed a significant reduction in NRR time in the coelomocytes of earthworms exposed to contaminated soils. The NRR time for the earthworms in control group was 40 min, which reduced in all test groups to 29–32, 18–24 and 12–15 min on 1, 7, and 14 d, respectively (Fig. 2). This reduction in NRR time was found to be depending on the total metal concentrations in the soil and duration of exposure, which is consistent with earlier reports (Weeks and Svendsen, 1996; Maboeta et al., 2003; Rocco et al., 2011; Markad et al., 2012). The decreased lysosomal stability could be attributed to the peroxidation of membrane lipids. The level of DNA strand breaks in the organisms has been regarded as a sensitive biomarker for genotoxic effects of xenobiotics and widely used for environmental biomonitoring and risk assessment (Reinecke and Reinecke, 2004; Lourenco et al., 2011a). It
was observed that, the level of DNA damage (olive tail moment, % tail DNA) in coelomocytes was significantly high (ANOVA, po 0.0001) in earthworms exposed to contaminated soils as compared to reference soil (Table 2). The % DNA in the comet tail was increased by 40% on 1 d exposure, which was increased further to 60% on 14 d of exposure, compared to control. Similar trend was observed in olive tail moment for control and exposed earthworms. The observed DNA damage could be correlated to duration of exposure in accordance with previous reports of DNA damage in earthworms coelomocytes (Reinecke and Reinecke, 2004; Lourenco et al., 2011a). Integrity of lysosomal membrane and DNA in the earthworm coelomocytes were severely affected by the exposure to contaminated soils, may lead to immune compromised cells rendering the organism vulnerable to pathogenic insults. Metals and other contaminant are known to affect immunological function in invertebrates (Galloway and Depledge, 2001; Homa et al., 2003) through surplus production and accumulation of ROS that affects various cellular organelles and their repair systems (Squibb and Fowler, 1981; Stohs and Bagchi, 1995; Vallyathan et al., 1998). Our results with respect to various antioxidant enzymes suggested that ROS production had occurred in the earthworms exposed to contaminated soils. 3.4. Histopathological examination Environmental contaminants cause histological alterations in animal tissues, which in turn significantly affect their functions. Histopathological changes in earthworms have been reported as a valuable marker of soil contamination with organic matter and heavy metals (Amaral et al., 2006; Giovanetti et al., 2010; KilIç, 2011; Lourenço et al., 2011b). The present study has shown the extensive histological changes in the tissues (body wall and intestinal tract) of D. curgensis exposed to fly ash polluted soils. The damage in epidermis was characterized by increased intercellular spaces, proliferation of glandular cells and loss of structural integrity (Fig. 3A). The circular and longitudinal muscle fibers showed loss of cell shape and structural integrity. The integrity of longitudinal muscle was entirely altered due to atrophy and necrosis (Fig. 3B–G). Alteration in muscle architecture may be due to dermal uptake of metals (Saxe et al., 2001; Hobbelen et al., 2006). Circular and longitudinal muscles of earthworms are known to accumulate heavy metals leading to detrimental effects in earthworm body wall (KilIç, 2011). Similarly, chloragogenous tissue and intestinal epithelium of D. curgensis showed histopathological alterations in the exposed animals. The cells of chloragogenous tissue lost their shape due to atrophy and necrosis when compared with controls earthworms (Fig. 4A). Deformation and atrophy of intestinal epithelium and inter villous spaces was observed in the earthworms exposed to polluted soils (Fig. 4B–G). The extensive loss and fusion to thin layer of intestinal villi structure was observed in treated earthworm, as reported earlier in the earthworms exposed to metals
Table 2 DNA damage in coelomocytes of D. curgensis exposed to fly ash polluted soils for 1, 7, and 14 d. Comet parameters
Duration of exposure (d)
R (reference soil)
S1
S2
S3
S4
S5
S6
Tail DNA (%)
1 7 14 1 7 14
8.9 7 0.21 a 9.247 0.3 a 10.247 0.3 a 1.36 7 0.04 a 1.94 7 0.07 a 2.08 7 0.05 a
12.69 7 0.4 b 14.58 7 0.3 b 17.077 0.4 b 2.517 0.11 b 3.727 0.08 b 5.58 7 0.12 b
12.617 0.3 b 15.217 0.4 b 16.477 0.4 b 2.217 0.09 bc 3.647 0.13 b 5.55 7 0.10 b
12.02 70.4 b 14.84 70.4 b 16.81 70.5 b 2.17 70.11 bc 3.38 70.10 b 5.11 70.10 bc
13.08 70.4 b 15.69 70.4 b 16.94 70.4 b 2.59 70.16 b 3.6770.09 b 5.71 70.12 b
12.647 0.3 b 14.98 7 0.3 b 16.077 0.3 b 2.25 7 0.8 b 3.54 7 0.09 b 5.277 0.10 bc
12.447 0.3 b 14.687 0.3 b 15.717 0.3 b 2.08 7 0.5 c 3.137 0.07 b 4.96 7 0.08 c
Olive tail moment
Results are expressed as mean 7 SEM (nZ 6). Means were compared by ANOVA and Tukey's multiple comparisons test. Different superscript alphabets represent statistical difference between two groups; values with same alphabets in a row are not significantly different from each other.
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Fig. 3. Transverse sections of D. curgensis showing histopathological changes on 14 d exposure to fly ash polluted soils. Control earthworm showed no deformities in structure (A). Loss of cell shape and structural organization of circular muscles; drastic damages in the longitudinal muscle layer of the treated earthworm (B–G). Arrows indicates histological alterations. Abbreviations: EP, epidermis; CM, circular muscle: LM, longitudinal muscle. Scale bar, 40 mm.
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Fig. 4. Transverse sections of D. curgensis showing histopathological changes in intestine, exposed for 14 d to heavy metal polluted soils. Control earthworm showed no deformities in structure (A). Deformation, degradation and necrosis of the chloragogenous; reduction in the thickness of the intestinal epithelium of the treated earthworm (B–G). Abbreviations: IP, intestinal epithelium; CH, chloragogenous tissue. Scale bar, 40 mm.
and radionuclides contaminated soils (Lourenço et al., 2011b). This may be due to bioaccumulation of metals in the epithelium through intestinal uptake where chloragogenous tissue of the earthworm serves as a major metal depository (Morgan and
Morgan, 1998; Morgan et al., 2002; Amaral et al., 2006). Morphological alterations in the earthworm chloragogenous tissue may imply excessive extrusion of chloragocytes for elimination of heavy metals (Morgan et al., 2002). Higher apoptotic rates and
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lower radial thickness of chloragogenous tissue and intestinal epithelium were reported in the earthworms inhabiting metal polluted soils (Amaral and Rodrigues, 2005; Amaral et al., 2006). The histological changes observed usually depend on the organism's antioxidant system, ability to repair the damage, the level of contaminant exposure (Haschek and Rousseaux, 1998). The histopathology results observed, reinforces biochemical and cytogenotoxic effects in earthworms exposed to a contaminated soil, and assume that the antioxidant defense system of the organism was not sufficient to handle metal induced oxidative stress leading to serious deleterious effects such as peroxidation of biomolecules, especially membrane lipids and DNA damage in the exposed earthworm.
4. Conclusions Effects of fly ash polluted soils on earthworm D. curgensis were evaluated in the present study. The results demonstrate that soil pollution around thermal power plants has adverse biological effects such as oxidative stress, histological damage, and bioaccumulation of metals that leads to DNA damage and destabilization of lysosomal membrane on the indicator organism D. curgensis. The soils around thermal power plants and fly ash disposal sites were polluted with metals and metalloids that pose serious risks to the entire earthworm populations and hence environmental health.
Acknowledgments The authors wish to thank Department of Science and Technology (DST), New Delhi, India (Grant no. SP/S0/AS-50/2005/2) for financial support, Sophisticated Analytical Instrument Facility (SAIF), IIT, Mumbai for ICP-AES analysis. The author VLM thanks the University Grants Commission (UGC), New Delhi, India (F.4-1/ 2006(BSR)/5-82/2007; Research Fellowship in Science for Meritorious Students) for research fellowship.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04. 011.
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Lourenço, J.I., Pereira, R.O., Silva, A.C., Morgado, J.M., Carvalho, F.P., Oliveira, J.M., Malta, M.P., Paiva, A.A., Mendo, S.A., Gonçalves, F.J., 2011a. Genotoxic endpoints in the earthworms/ sub-lethal assay to evaluate natural soils contaminated by metals and radionuclides. J. Hazard. Mater. 186, 788–795. Lourenço, J., Silva, A., Carvalho, F., Oliveira, J., Malta, M., Mendo, S., Gonçalves, F., Pereira, R., 2011b. Histopathological changes in the earthworm Eisenia andrei associated with the exposure to metals and radionuclides. Chemosphere 85, 1630–1634. Lukkari, T., Taavitsainen, M., Soimasuo, M., Oikari, A., Haimi, J., 2004. Biomarker responses of the earthworm Aporrectodea tuberculata to copper and zinc exposure: differences between populations with and without earlier metal exposure. Environ. Pollut. 129, 377–386. Maboeta, M.S., Reinecke, S.A., Reinecke, A.J., 2003. Linking lysosomal biomarker and population responses in a field population of Aporrectodea caliginosa (Oligochaeta) exposed to the fungicide copper oxychloride. Ecotoxicol. Environ. Saf. 56, 411–418. Maity, S., Roy, S., Chaudhury, S., Bhattacharya, S., 2008. Antioxidant responses of the earthworm Lampito mauritii exposed to Pb and Zn contaminated soil. Environ. Pollut. 151, 1–7. Maity, S., Bhattacharya, S., Chaudhury, S., 2009. Metallothionein response in earthworms Lampito mauritii (Kinberg) exposed to fly ash. Chemosphere 77, 319–324. Mandal, A., Sengupta, D., 2006. An assessment of soil contamination due to heavy metals around a coal-fired thermal power plant in India. Environ. Geol. 51, 409–420. Manerikar, R.S., Apte, A.A., Ghole, V.S., 2008. In vitro genotoxicity of fly ash leachate in earthworm coelomocytes. Toxicol. Environ. Chem. 90, 293–300. Markad, V.L., Kodam, K.M., Ghole, V.S., 2012. Effect of fly ash on biochemical responses and DNA damage in earthworm Dichogaster curgensis. J. Hazard. Mater. 215–216, 191–198. Mehra, A., Farago, M.E., Banerjee, D.K., 1998. Impact of fly ash from coalfired power stations in Delhi, with particular reference to metal contamination. Environ. Monit. Assess. 50, 15–35. Morgan, A.J., Turner, M.P., Morgan, J.E., 2002. Morphological plasticity in metal sequestering earthworm chloragocytes: morphometric electron microscopy provides a biomarker of exposure in field populations. Environ. Toxicol. Chem. 21, 610–618. Morgan, J.E., Morgan, A.J., 1998. The distribution and intracellular compartmentation of metals in the endogeic earthworm Aporrectodea caliginosa sampled from an unpolluted and a metal-contaminated site. Environ. Pollut. 99, 167–175. Pandey, V.C., Singh, J.S., Singh, R.P., Singh, N., Yunus, M., 2011. Arsenic hazards in coal fly ash and its fate in Indian scenario. Resour. Conserv. Recycl. 55, 819–835.
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Biomarker responses in the earthworm, Dichogaster curgensis exposed to fly ash polluted soils Vijaykumar L. Markad a, Tekchand C. Gaupale b, Shobha Bhargava b, Kisan M. Kodam a,n, Vikram S. Ghole a a b
Biochemistry Division, Department of Chemistry, Savitribai Phule Pune University, Pune 411007, India Department of Zoology, Savitribai Phule Pune University, Pune 411007, India
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2014 Received in revised form 9 April 2015 Accepted 10 April 2015 Available online 21 April 2015
Earthworms are globally accepted as a model organism in terrestrial ecotoxicology for assessment of environmental pollution. This study evaluated and compared effects of fly ash polluted soils collected from two geographically different thermal power plants on biomarker responses in the earthworm, Dichogaster curgensis. To evaluate relationship between distance sampling and biomarker responses in the earthworm D. curgensis, soil samples at 0.5, 1 and 3 km from thermal plant were analyzed for physico-chemical properties and metal concentrations. Biochemical alterations, lysosomal membrane stability, genotoxic effects, and histological changes were examined on 1, 7, and 14 d of exposure to fly ash contaminated soils collected from different thermal power plants. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were significantly increased, while glutathione reductase (GR) activity was found to be decreased in treated animals. Catalase (CAT) and glutathione-S- transferase (GST) activities were found to be increased initially up to 7 d exposure and further decreased on 14 d exposure. D. curgensis exposed to fly ash contaminated soils showed significant lysosomal membrane destabilization and DNA damage. Extensive histopathological changes were observed in the tissues of the body wall and intestinal tract of the exposed D. curgensis along with accumulation of heavy metals. These results demonstrate that soil pollution around thermal power plants has adverse biological effects of on the indicator organism D. curgensis and no correlation was found between distance and extent of biological biochemical responses. & 2015 Elsevier Inc. All rights reserved.
Keywords: Biomarkers Dichogaster curgensis Fly ash Heavy metals Histopathology
1. Introduction A massive amount of fly ash (4750 million tons) is generated worldwide from coal-based thermal power plants (Yao et al., 2015). It has started globally receiving alarming attention due to its hazardous nature, widespread usage, and the manner of disposal; leading to severe environmental pollution (Maity et al., 2009; Markad et al., 2012). India generates higher amount of fly ash and utilizes lower percentage of fly ash compared to other countries. Therefore, major portion of it is disposed in ash ponds near the power plants occupying more than 65,000 acres of land (Pandey and Singh, 2010; Pandey et al., 2011). According to World Bank, by 2015 India will require 1000 km2 of land for the disposal of coal fly ash (Singh et al., 2010). These ash ponds became a potential source for contamination of soil and water streams (Mandal and Sengupta, 2006; Pandey et al., 2011, Dragović et al., 2013). n
Corresponding author. Fax: þ91 20 25691728. E-mail address: [email protected] (K.M. Kodam).
http://dx.doi.org/10.1016/j.ecoenv.2015.04.011 0147-6513/& 2015 Elsevier Inc. All rights reserved.
Leaching and accumulation of organic and inorganic toxic compounds from fly ash is of major environmental concern and known to have severe adverse impact such as bioaccumulation of metals, oxidative stress, DNA damage, and reproduction on terrestrial and aquatic ecosystems (Ali et al., 2004; Chakraborty and Mukherjee, 2009; Grumiaux et al., 2010; Pandey and Singh, 2010). Fly ash contains heavy metals (Cu, Zn, Cd, Pb, Ni, Cr, etc.) and polyhalogenated compounds (Pandey and Singh, 2010; Pandey et al., 2011). Elevated trace metal concentrations around the thermal power plants have been reported earlier (Mehra et al., 1998; Basavaiah et al., 2012; Dragović et al., 2013). Basavaiah et al. (2012) reported the metal pollution of soils collected from Nashik thermal power station, Maharashtra, India using magnetic screening. However, biological effects of this environmental pollution remain to be investigated. Environmental quality monitoring programs recommended the integration of biological responses with chemical data for risk assessment of the contaminants. Earthworm, an important organism of soil ecosystem, is widely accepted as biological indicator of environmental pollution (Łaszczyca et al., 2004; Lukkari et al.,
V.L. Markad et al. / Ecotoxicology and Environmental Safety 118 (2015) 62–70
2004). Several biomarkers have been identified in the earthworms to assess the environmental impact of wide range of contaminants. An enhanced production of reactive oxygen species is a general pathway of toxicity induced by organic and inorganic toxic compounds (Livingstone et al., 1990; Stohs and Bagchi, 1995) leading to a condition of oxidative stress and subsequent alteration in antioxidant defense mechanism of the organisms. Therefore, in earthworms, biochemical responses (antioxidant defense system and lipid peroxidation) are regarded as fast, diagnostic, and prognostic indices of environmental pollution (Saint-Denis et al., 2001; Łaszczyca et al., 2004). The alkaline comet and neutral red retention (NRR) assays have been demonstrated as effective methods to evaluate the DNA damage and lysosomal membrane stability in earthworm coelomocytes, respectively (Weeks and Svendsen, 1996; Lourenco et al., 2011a; Markad et al., 2012). Histopathological changes in earthworms have been previously reported as valuable endpoint of metal toxicity (KilIç, 2011; Lourenco et al., 2011b). Therefore, the present study was designed to evaluate the biological effects of soil contamination around the thermal power plants by using earthworm as a model organism. Earthworms were exposed to fly ash polluted soils and various biomarker endpoints viz. DNA damage, lysosomal membrane integrity, biochemical responses and histological changes related to the effect of ROS were studied. Furthermore, bioaccumulation of heavy metals in earthworms was also assessed to elucidate cause–effect relationship.
2. Materials and methods 2.1. Soils collection and analysis The fly ash polluted soils were collected from the fields near the ash disposal sites (receive continuously fly ash and its leachates from ash ponds) of the Nashik thermal power plant, Nashik, India and Bhusaval Thermal Power Plant, Jalgaon, India that generated 1.27 and 0.69 million tones of fly ash in 2012–2013 (CEA, 2014). For several decades, these activities have produced high concentrations of trace elements in surface soils (Basavaiah et al., 2012). For each study area, three soil samples from different sites were collected (Nashik area: S1–S3; Jalgaon area: S4–S6), while reference soil (R) was collected from the garden of Savitribai Phule Pune University, Pune, India, with no history of pollution. The soil samples of surface horizons (0–20 cm) were collected from three different points of a site to obtain a composite sample (Supplementary Fig. 1). The pH and electrical conductivity of the samples were measured in 0.01 M CaCl2. The organic matter and total metal content of all test soils were determined as described earlier (Markad et al., 2012). The soil samples were digested with concentrated nitric and perchloric acid and metal concentrations were estimated by using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES, ARCOS, Spectro, Germany) (Markad et al., 2012). 2.2. Laboratory exposure A synchronized culture of earthworm, Dichogaster curgensis was maintained in laboratory conditions as described earlier (Markad et al., 2012). The organisms were acclimated for at least one week in a container with reference soil. Earthworms (n¼ 15) were exposed to the soils (1 kg) in polythene culture pots (20 cm 10 cm 8 cm). The organisms were fed once in a week with 10 g of defaunated cattle manure per set. The experiments were carried under controlled laboratory conditions of temperature (22 72 °C), moisture content (40%), and photoperiod (12 hL:
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12 hD). Earthworms were removed from the experimental sets on 1, 7, and 14 d of exposure and used for bioaccumulation, cytotoxic and genotoxic, biochemical, and histopathological studies. For metal bioaccumulation, a pool of 4 earthworms from each group (depurated for 48 h, freeze killed) was acid digested and metal concentrations were estimated by using ICP-AES as described earlier (Maboeta et al., 2003; Markad et al., 2012). 2.3. Cytotoxic and genotoxic studies Three earthworms from each group were randomly selected on 1, 7, and 14 d of exposure and coelomocytes were harvested by simple, non-invasive technique described earlier (Manerikar et al., 2008). Coelomocytes obtained were further processed to assess the lysosomal membrane integrity and DNA damage by using NRR and alkaline comet assay, respectively as reported earlier (Markad et al., 2012). 2.4. Biochemical assays Three earthworms were removed from each group at an interval of 1, 7, and 14 d of exposure, rinsed with distilled water and kept for 48 h on moist filter paper in Petri dishes to depurate their gut contents. The earthworms were homogenized at 4 °C for 1 min in Tris–HCl buffer (100 mM, pH 7.5) using Potter-Elvehjem homogenizer and centrifuged at 12,000g for 20 min to obtain postmitochondrial fraction. The aliquots were stored at 80 °C until further use. The enzyme assays were performed using temperature-controlled dual beam UV–visible spectrophotometer (Jasco V-630, Japan). All biochemical estimations of Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S- transferase (GST) and lipid peroxidation (in terms of malondialdehyde, MDA) were assayed using methods as described in our earlier report (Markad et al., 2012). 2.5. Analysis of histological alterations In order to examine histological changes in different tissues, earthworms were removed from the soil on 14 d of exposure, depurated for 48 h on moist filter paper and fixed in Bouin's fixative for 24 h. Tissues were further dehydrated in ascending grades of alcohol, cleared in xylene, infiltrated in molten paraffin wax at 60 °C and embedded. The transverse sections (7 mm thick) of earthworms were mounted on the poly-L-lysine coated slides and stained with hematoxylin-eosin double staining method. Slides were mounted in DPX and observed under light microscope (Carl Zeiss, Axioskop 40) and images were captured (KilIç, 2011; Lourenco et al., 2011a, 2011b). 2.6. Statistical analysis Data were expressed as the mean 7S.E.M. Significant differences between the results of the different treatment groups were determined using one-way ANOVA and Dunnet multiple comparison post-hoc test. The level of significance was considered po 0.05. All statistical analyses were carried out using GraphPad Prism software (Version 5.0, USA).
3. Results and discussion 3.1. Soil analysis Physico-chemical analysis and metal concentrations of reference and contaminated soils are shown in Table 1. A significant
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Table 1 Physico-chemical properties and total metal concentrations of the soils used in experiments. Parameters
R (reference)
Distance from source (km) pH Conductivity (mS/cm) Organic matter (%) Si (g kg 1) Fe (g kg 1) Ca (g kg 1) K (g kg 1) P (g kg 1) Na (g kg 1) Al (g kg 1) Mg (g kg 1) Mn (mg kg 1) Pb (mg kg 1) Zn (mg kg 1) Ni (mg kg 1) Cd (mg kg 1) Cr (mg kg 1) Cu (mg kg 1)
5.7 7 0.1 11.7 7 0.1 7.7 7 0.1 1777 1.0 5.6 7 0.5 22 7 0.6 3.6 7 0.7 1.8 7 0.2 0.5 7 0.1 8.3 7 0.5 10.5 7 0.5 6337 47 3.6 7 0.5 169 7 5 25.8 7 0.3 0.5 7 0.1 28.17 1.6 517 0.6
Nashik Thermal Power Plant, Nashik
Bhusaval Thermal Power Plant, Jalgaon
S1 0.5
S2 1
S3 3
S4 0.5
S5 1
S6 3
6.6 7 0.1 12.7 7 0.1 7.7 7 0.2 2177 1.6 8.4 7 0.9 23.2 7 0.5 3.4 7 0.5 27 0.3 0.6 7 0.1 12.7 7 0.7 19.6 7 1.1 8717 55 127 0.5 284 7 3 697 2.4 5.0 7 1.4 95.2 7 2.3 1267 0.4
6.9 7 0.1 12.9 7 0.1 6.7 7 0.2 2177 2.1 8.8 7 1.2 247 2.8 4.5 7 0.8 1.8 7 0.3 0.6 7 0.2 12.9 7 1.2 13.8 7 0.5 887 7 5 177 0.6 294 7 4 62.5 7 3 4.0 7 0.2 877 3.4 1337 1.2
6.7 70.2 13.1 70.2 7.5 70.3 217 72.5 8.8 71.5 22.2 71.5 3.4 70.2 2.2 70.4 0.9 70.4 13.4 71.5 14.1 71.3 815 722 17 70.7 280 723 68 70.8 3.0 70.2 84 72 89 70.3
6.7 7 0.1 13.0 7 0.1 7.6 7 0.1 2187 1.5 7.2 7 0.9 22.8 7 1.3 3.6 7 0.4 1.9 7 0.4 0.9 7 0.3 14.3 7 0.9 18.2 7 0.6 805 7 48 177 0.2 2477 4 637 10.2 4.5 7 0.7 867 4.4 1187 0.5
6.7 7 0.2 12.8 7 0.1 7.17 0.1 2177 2.3 7.17 0.9 23.4 7 2.5 4.0 7 0.5 2.2 7 0.4 0.7 7 0.3 13.4 7 0.8 16.3 7 0.6 860 7 21 187 0.6 2467 7 677 1.4 3.0 7 0.57 847 1.5 1197 4.9
6.6 7 0.2 12.7 7 0.1 7.4 7 0.2 2177 1 6.4 7 1.2 21.2 7 2.6 4.0 7 0.3 1.8 7 0.4 0.7 7 0.2 10.7 7 2.5 13.9 7 0.5 845 7 32 167 0.5 258 7 8 637 5.4 3.0 7 0.7 86.6 7 3.3 86.2 7 3.6
increase in pH and conductivity (mS cm 1) of all test soils was observed. Soils samples from Nashik Thermal Power Plant showed increase in conductivity, Fe, Na, Al, with distance of sampling. This may be due to increased level of soluble inorganic constituents present in the fly ash (Jala and Goyal, 2006; Singh et al., 2010). On the contrary, Fe, Na, Al content of soils from Bhusawal Thermal power plant decreased slightly with increase in distance. This suggests differential mechanism in spreading of these metals and could be due to difference in soil properties at different geographical locations. Cd content was higher at 0.5 km distance and then decreased with increase in distance in both soils. The total metal concentrations of the contaminated soils were found to be depending on the collection site; higher concentrations were observed in the soil samples collected from the vicinity of thermal power plant (samples S1 and S4). Heavy metal concentrations viz. Pb, Zn, Ni, Cd, Cr, and Cu were significantly high in all tested soils when compared to reference soil. These results are in agreement with previous report that found increased trace metal profile around Nashik Thermal Power Plant (Basavaiah et al., 2012). Similar observations were noted in soils surrounding the thermal power plant in Delhi, India (Mehra et al., 1998) and Serbia (Dragović et al., 2013). The observed increased metal concentrations may be due to leaching and accumulation of these elements in soils from fly ash (Mandal and Sengupta, 2006), where these elements are abundantly present (Markad et al., 2012). Maximum enrichment of heavy metals was observed in the top soils when compared to the soils collected from different depth surrounding the ash ponds (Mandal and Sengupta, 2006). The earthworm survival was not affected throughout the exposure period. There were no significant changes observed in the metal body burdens of D. curgensis after 1 d exposure, while, significant bioaccumulation of Cd, Pb, Ni, and Cr was noted on 7 and 14 d of exposure to fly ash polluted soils, when compared with control earthworms (Supplementary Table 1). This may be due to direct exposure of the earthworms to metals present in soil through dermal contact or by ingestion of water, polluted food and/or soil particles (Hobbelen et al., 2006). Similar results were reported in Eisenia andrei exposed to a contaminated soil from an abandoned uranium mine (Lourenço et al., 2011a). Cadmium accumulation was found to be depending on the total metal concentration in the soil. However, accumulation of other metals (Pb, Cr and Ni) did not depend upon the total metal concentration in
the soil. 3.2. Biochemical responses Biochemical responses of the earthworms to environmental stress were regarded as early warning system for soil pollution (Łaszczyca et al., 2004). Fig. 1 depicts biochemical responses of D. curgensis, exposed to fly ash polluted soils. In the present study, SOD activity was significantly increased (15–31%) in all experimental sets and at all time points when compared with control earthworms. In our earlier work (Markad et al., 2012) SOD activity was decreased on 14 d exposure compare to 7 d at all the doses of fly ash except 40% fly ash dose. On the contrary, in this study, no decrease in SOD activity was noted. This indicates heavy contamination of soil samples in the vicinity of both the thermal power plants. No correlation between SOD activities and distance of soil sample location was established on day 14 (Fig. 1A). CAT activity was significantly increased in the earthworms on 1 d (7– 19%) and 7 d (13–28%) exposure to all test soils. However, on 14 d, CAT activity was significantly decreased when compared with 7 d, and was analogous to control earthworms (Fig. 1B). This decrease in CAT may be due to inhibition of enzyme by high cellular stress or inactivation by singlet oxygen, peroxyl radicals, and superoxide radical (Kono and Fridovich, 1982; Escobar et al., 1996). Further analysis of CAT activity data suggested no any correlation with distance soil sampling. Glutathione reductase reduces oxidized glutathione and maintains the cellular antioxidant status, whereas glutathione peroxidase utilizes reduced glutathione to eliminate H2O2 (Saint-Denis et al., 2001; Markad et al., 2012). The GR activity was significantly decreased (15–27%) in all treatment groups and at all exposure durations. While GPx activity was significantly increased (19–33%) in the earthworms exposed to all tested soils on 14 d exposure (Fig. 1C and D). Decrease in GR activity may be due to depletion of cellular NADPH level and/or inactivation of GR by metal binding (Goering et al., 1987; Saint-Denis et al., 2001; Łaszczyca et al., 2004). Glutathione S-transferase protects the tissue from oxidative stress by conjugation of glutathione with xenobiotics and lipid peroxidation products (Frova, 2006). In the present study, significant increase (14–21%) in GST activity was observed in earthworms on 7 d exposure. However, on 14 d, GST activity was significantly decreased (p o0.05) when compared to 7 d exposure
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Fig. 1. Biochemical responses of earthworm D. curgensis exposed to fly ash polluted soils; A) superoxide dismutase, B) catalase, C) glutathione reductase, D) glutathione peroxidase, E) glutathione S-transferase, and F) malondialdehyde on 1, 7 and 14 d of exposure. Data expressed as mean ± SD. Significant differences with control, n(p 4 0.05), # (p 4 0.01), $(p 4 0.001) (one-way ANOVA with Dunnet multiple comparison test).
and become normal as was in control earthworms (Fig. 1E). This decrease in GST activity could be attributed to depletion in GSH levels; evidenced by decreased GR activity and increased GPx activity (Łaszczyca et al., 2004; Maity et al., 2008). Similar response of GST activity was reported in earthworms from metal contaminated soils (Łaszczyca et al., 2004; Lukkari et al., 2004). The activities of GR, GPx and GST did not show any correlation with distance soil sampling. Lipid peroxidation is a sensitive indicator of oxidative stress
and various contaminants are known to induce lipid peroxidation through the formation of ROS. MDA is the lipid peroxidation product, widely used to measure the level of oxidative stress in the organisms (Saint-Denis et al., 2001; Sandrini et al., 2006). In the present study, significant increase (17–46%) in MDA level was observed on 7 and 14 d exposure in the earthworms of all test groups (Fig. 1F). This may be attributed to time-dependent enhancement in ROS generation. It can be noted that the reduction in CAT activity may favor lipid peroxidation due to accumulation of
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1d
45
7d
14d
40 35
NRR time (min)
30
#
$
$ $
#
#
$
#
$
$
$
25 †
20 †
15
†
†
† †
†
10 5 0 R
S1
S2
S3
S4
S5
S6
Soil samples Fig. 2. Neutral red retention time (min) for lysosomal membrane stability of coelomocytes derived from D. curgensis exposed to fly ash polluted soils assessed on 1, 7, and 14 d. Data expressed as mean7 SD. Significant differences #(p4 0.01), $(p 40.001), †(p 40.0001) compared with control (one-way ANOVA with Dunnet multiple comparison test).
H2O2, a precursor of hydroxyl radical that triggers lipid peroxidation (Sandrini et al., 2006). Similar observations were reported in invertebrates exposed to heavy metals (Saint-Denis et al., 2001; Ferreira-Cravo et al., 2009; Markad et al., 2012). Therefore, in the present study elevated MDA level along with increase in SOD, GPx activity, biphasic response in CAT, GST activity and decreased GR activity were observed, which reflects severe oxidative stress experienced by the earthworms upon exposure to fly ash polluted soils from both thermal plants. 3.3. Cytotoxic and genotoxic studies Lysosome is a sensitive target organ and its membrane stability has been used as a potential biomarker of toxic effects of diverse environmental contaminants (Weeks and Svendsen, 1996; Maboeta et al., 2003; Rocco et al., 2011). The results of the present study revealed a significant reduction in NRR time in the coelomocytes of earthworms exposed to contaminated soils. The NRR time for the earthworms in control group was 40 min, which reduced in all test groups to 29–32, 18–24 and 12–15 min on 1, 7, and 14 d, respectively (Fig. 2). This reduction in NRR time was found to be depending on the total metal concentrations in the soil and duration of exposure, which is consistent with earlier reports (Weeks and Svendsen, 1996; Maboeta et al., 2003; Rocco et al., 2011; Markad et al., 2012). The decreased lysosomal stability could be attributed to the peroxidation of membrane lipids. The level of DNA strand breaks in the organisms has been regarded as a sensitive biomarker for genotoxic effects of xenobiotics and widely used for environmental biomonitoring and risk assessment (Reinecke and Reinecke, 2004; Lourenco et al., 2011a). It
was observed that, the level of DNA damage (olive tail moment, % tail DNA) in coelomocytes was significantly high (ANOVA, po 0.0001) in earthworms exposed to contaminated soils as compared to reference soil (Table 2). The % DNA in the comet tail was increased by 40% on 1 d exposure, which was increased further to 60% on 14 d of exposure, compared to control. Similar trend was observed in olive tail moment for control and exposed earthworms. The observed DNA damage could be correlated to duration of exposure in accordance with previous reports of DNA damage in earthworms coelomocytes (Reinecke and Reinecke, 2004; Lourenco et al., 2011a). Integrity of lysosomal membrane and DNA in the earthworm coelomocytes were severely affected by the exposure to contaminated soils, may lead to immune compromised cells rendering the organism vulnerable to pathogenic insults. Metals and other contaminant are known to affect immunological function in invertebrates (Galloway and Depledge, 2001; Homa et al., 2003) through surplus production and accumulation of ROS that affects various cellular organelles and their repair systems (Squibb and Fowler, 1981; Stohs and Bagchi, 1995; Vallyathan et al., 1998). Our results with respect to various antioxidant enzymes suggested that ROS production had occurred in the earthworms exposed to contaminated soils. 3.4. Histopathological examination Environmental contaminants cause histological alterations in animal tissues, which in turn significantly affect their functions. Histopathological changes in earthworms have been reported as a valuable marker of soil contamination with organic matter and heavy metals (Amaral et al., 2006; Giovanetti et al., 2010; KilIç, 2011; Lourenço et al., 2011b). The present study has shown the extensive histological changes in the tissues (body wall and intestinal tract) of D. curgensis exposed to fly ash polluted soils. The damage in epidermis was characterized by increased intercellular spaces, proliferation of glandular cells and loss of structural integrity (Fig. 3A). The circular and longitudinal muscle fibers showed loss of cell shape and structural integrity. The integrity of longitudinal muscle was entirely altered due to atrophy and necrosis (Fig. 3B–G). Alteration in muscle architecture may be due to dermal uptake of metals (Saxe et al., 2001; Hobbelen et al., 2006). Circular and longitudinal muscles of earthworms are known to accumulate heavy metals leading to detrimental effects in earthworm body wall (KilIç, 2011). Similarly, chloragogenous tissue and intestinal epithelium of D. curgensis showed histopathological alterations in the exposed animals. The cells of chloragogenous tissue lost their shape due to atrophy and necrosis when compared with controls earthworms (Fig. 4A). Deformation and atrophy of intestinal epithelium and inter villous spaces was observed in the earthworms exposed to polluted soils (Fig. 4B–G). The extensive loss and fusion to thin layer of intestinal villi structure was observed in treated earthworm, as reported earlier in the earthworms exposed to metals
Table 2 DNA damage in coelomocytes of D. curgensis exposed to fly ash polluted soils for 1, 7, and 14 d. Comet parameters
Duration of exposure (d)
R (reference soil)
S1
S2
S3
S4
S5
S6
Tail DNA (%)
1 7 14 1 7 14
8.9 7 0.21 a 9.247 0.3 a 10.247 0.3 a 1.36 7 0.04 a 1.94 7 0.07 a 2.08 7 0.05 a
12.69 7 0.4 b 14.58 7 0.3 b 17.077 0.4 b 2.517 0.11 b 3.727 0.08 b 5.58 7 0.12 b
12.617 0.3 b 15.217 0.4 b 16.477 0.4 b 2.217 0.09 bc 3.647 0.13 b 5.55 7 0.10 b
12.02 70.4 b 14.84 70.4 b 16.81 70.5 b 2.17 70.11 bc 3.38 70.10 b 5.11 70.10 bc
13.08 70.4 b 15.69 70.4 b 16.94 70.4 b 2.59 70.16 b 3.6770.09 b 5.71 70.12 b
12.647 0.3 b 14.98 7 0.3 b 16.077 0.3 b 2.25 7 0.8 b 3.54 7 0.09 b 5.277 0.10 bc
12.447 0.3 b 14.687 0.3 b 15.717 0.3 b 2.08 7 0.5 c 3.137 0.07 b 4.96 7 0.08 c
Olive tail moment
Results are expressed as mean 7 SEM (nZ 6). Means were compared by ANOVA and Tukey's multiple comparisons test. Different superscript alphabets represent statistical difference between two groups; values with same alphabets in a row are not significantly different from each other.
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Fig. 3. Transverse sections of D. curgensis showing histopathological changes on 14 d exposure to fly ash polluted soils. Control earthworm showed no deformities in structure (A). Loss of cell shape and structural organization of circular muscles; drastic damages in the longitudinal muscle layer of the treated earthworm (B–G). Arrows indicates histological alterations. Abbreviations: EP, epidermis; CM, circular muscle: LM, longitudinal muscle. Scale bar, 40 mm.
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Fig. 4. Transverse sections of D. curgensis showing histopathological changes in intestine, exposed for 14 d to heavy metal polluted soils. Control earthworm showed no deformities in structure (A). Deformation, degradation and necrosis of the chloragogenous; reduction in the thickness of the intestinal epithelium of the treated earthworm (B–G). Abbreviations: IP, intestinal epithelium; CH, chloragogenous tissue. Scale bar, 40 mm.
and radionuclides contaminated soils (Lourenço et al., 2011b). This may be due to bioaccumulation of metals in the epithelium through intestinal uptake where chloragogenous tissue of the earthworm serves as a major metal depository (Morgan and
Morgan, 1998; Morgan et al., 2002; Amaral et al., 2006). Morphological alterations in the earthworm chloragogenous tissue may imply excessive extrusion of chloragocytes for elimination of heavy metals (Morgan et al., 2002). Higher apoptotic rates and
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lower radial thickness of chloragogenous tissue and intestinal epithelium were reported in the earthworms inhabiting metal polluted soils (Amaral and Rodrigues, 2005; Amaral et al., 2006). The histological changes observed usually depend on the organism's antioxidant system, ability to repair the damage, the level of contaminant exposure (Haschek and Rousseaux, 1998). The histopathology results observed, reinforces biochemical and cytogenotoxic effects in earthworms exposed to a contaminated soil, and assume that the antioxidant defense system of the organism was not sufficient to handle metal induced oxidative stress leading to serious deleterious effects such as peroxidation of biomolecules, especially membrane lipids and DNA damage in the exposed earthworm.
4. Conclusions Effects of fly ash polluted soils on earthworm D. curgensis were evaluated in the present study. The results demonstrate that soil pollution around thermal power plants has adverse biological effects such as oxidative stress, histological damage, and bioaccumulation of metals that leads to DNA damage and destabilization of lysosomal membrane on the indicator organism D. curgensis. The soils around thermal power plants and fly ash disposal sites were polluted with metals and metalloids that pose serious risks to the entire earthworm populations and hence environmental health.
Acknowledgments The authors wish to thank Department of Science and Technology (DST), New Delhi, India (Grant no. SP/S0/AS-50/2005/2) for financial support, Sophisticated Analytical Instrument Facility (SAIF), IIT, Mumbai for ICP-AES analysis. The author VLM thanks the University Grants Commission (UGC), New Delhi, India (F.4-1/ 2006(BSR)/5-82/2007; Research Fellowship in Science for Meritorious Students) for research fellowship.
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.04. 011.
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