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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 3 2014, pp. 47-61 ISSN 1997-0838 Original Text Copyright © 2014 by Gadallah and Sayed
ORIGINAL ARTICLE
Impacts of Different Water Pollution Sources on Antioxidant Defense Ability in Three Aquatic Macrophytes in Assiut Province, Egypt
Mohamed A.A. Gadallah, Suzan A. Sayed*
Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt.
Fax number: 002088234278 Tel. number: 00201098019725
*E-Mail: [email protected]
Received March 13, 2014
The present study was undertaken to evaluate the impacts of surface water pollution with wastes coming from sewage effluents (Site 2), agricultural runoff (Site 4) and oils and detergents factory (Site 3) on the stability of leaf membrane (measured as injury %), hydrogen peroxide (H2O2), ascorbic acid (Asc A), lipid peroxidation, chlorophyll (Chl) content, soluble sugars (SS), soluble proteins (SP) and total free amino acids (TAA) of Cyperus alopeucroides, Persicaria salicifolia and Echinochloa stagnina. Concentration of H2O2, MDA and TAA were higher in the three plants collected from polluted sites as compared with those of plants grown in control Nile site (Site1). The opposite was true for Asc A, SS and SP where their concentrations reduced significantly in response to water pollution. Leaf membrane was more damaged (high injury %) in plants exposed to wastes from different sources than in plants growing at control site. The results of this study indicated that water pollution reduced the oxidative defense abilities in the three plants through reduction of Asc A activities, enhancement of H2O2 production and increasing MDA accumulation. In addition it impaired the metabolic activity through lowering the SS and SP contents and enhancement of TAA accumulation and increase membrane injury. The over production of hydrogen peroxide by the studied aquatic plants under water pollution could be used as an oxygen source needed to oxidize the more resistant organic and inorganic pollutants and used for pollution control and municipal and industrial wastewater treatment.
Key words: Antioxidant, Industrial wastes, Hydrophytes, Membrane stability, Sewage.
ORIGINAL ARTICLE
Impacts of Different Water Pollution Sources on Antioxidant Defense Ability in Three Aquatic Macrophytes in Assiut Province, Egypt
Mohamed A.A. Gadallah, Suzan A. Sayed*
Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut, Egypt.
Fax number: 002088234278 Tel. number: 00201098019725
*E-Mail: [email protected]
Received March 13, 2014
The present study was undertaken to evaluate the impacts of surface water pollution with wastes coming from sewage effluents (Site 2), agricultural runoff (Site 4) and oils and detergents factory (Site 3) on the stability of leaf membrane (measured as injury %), hydrogen peroxide (H2O2), ascorbic acid (Asc A), lipid peroxidation, chlorophyll (Chl) content, soluble sugars (SS), soluble proteins (SP) and total free amino acids (TAA) of Cyperus alopeucroides, Persicaria salicifolia and Echinochloa stagnina. Concentration of H2O2, MDA and TAA were higher in the three plants collected from polluted sites as compared with those of plants grown in control Nile site (Site1). The opposite was true for Asc A, SS and SP where their concentrations reduced significantly in response to water pollution. Leaf membrane was more damaged (high injury %) in plants exposed to wastes from different sources than in plants growing at control site. The results of this study indicated that water pollution reduced the oxidative defense abilities in the three plants through reduction of Asc A activities, enhancement of H2O2 production and increasing MDA accumulation. In addition it impaired the metabolic activity through lowering the SS and SP contents and enhancement of TAA accumulation and increase membrane injury. The over production of hydrogen peroxide by the studied aquatic plants under water pollution could be used as an oxygen source needed to oxidize the more resistant organic and inorganic pollutants and used for pollution control and municipal and industrial wastewater treatment.
Key words: Antioxidant, Industrial wastes, Hydrophytes, Membrane stability, Sewage.
Abbreviation: Asc A = ascorbic acid, Chl = chlorophyll, H2O2 = hydrogen peroxide, LP = lipid peroxidation, MDA = malonydialdhyde, SS = soluble sugars, PEG = polyethylene glycol, ROS = reactive oxygen species, SP = soluble proteins, TAA = total free amino acids.
Rapid industrialization and addition of toxic substances to the environment are responsible for altering the ecosystems (Oketola and Osibanjo, 2007). In various periods of the world history, scarcity and pollution of natural resources made societies aware of the fact that the economy depends on energy and ecological services provided by these resources (Ward, 2006 and Gurluk, 2009).
Water is undoubtedly the most precious natural source that exists on our planet. The lack of clean water has always been as issue of environmental concern of all over the world. The main sources of water pollution are industrial, municipal and agricultural. Pollutants, of all degrees of degradability, when discharge into aquatic environment accumulates primary in water and sediment and in time translocates in various portion on a much wide scale.
Under the influence of water pollution, the plants exhibit changes in parameters related to photosynthesis, respiration, enzyme activities, syntheses of lipids, proteins and other metabolites (Steinbacchova-Vojtiskova et al., 2006; Warrier and Saroja, 2008). Many plants are very sensitive to water pollution, and pollutants can damage their leaves, impair plant growth, and limit primary productivity. Visible injuries, significant decrease in growth and loss in vitality can be observed in plants when exposed to toxicant environment for either a long or a short period of time depending on the concentration of toxicants (Karatas et al., 2009).
It is well documented that various a biotic stresses lead to the overproduction of reactive oxygen species
(ROS) in plants which are highly reactive and toxic and ultimately results in oxidative stress. ROS or free radicals are generated extra or intra-cellularly, which can exert toxic effects to the cell. These species may affect cell membrane properties and caused oxidative damage to lipid and proteins (Gill and Tuteja, 2010). Among ROS species, H2O2 is also an important signaling molecule (Wood et al., 2003), which is produced by the chloroplast and involved in the response of the plant to different types of environmental stressors (Perez-Ruiz et al., 2006). The major water canals within Assiut area of Egypt suffer from uncontrolled disposal of increased amounts of wastes generated by various human and industrial activities.
Since plants provide not only a cheap but also an easy way to monitor the different sources of environmental pollution, their different parts were used as bioindicators of these contaminations. Aquatic macrophytes are considered useful tool in long-term studies to follow pollutants variation in some environment (Dantas and Locligiani, 2003; Calheiros et al., 2007; Maddisn et al., 2009). The information's available on the effects of water pollution on oxidative parameters in hydrophytes were very scare or negligible. The majority of studies focus on the capacity of hydrophytes to remove and accumulate metals and ions from polluted water. Accordingly, in our study, we wanted to provide information on oxidative defense ability of three aquatic macrophytes exposed to different liquid wastes coming from oils and detergents factory, sewage effluents and agricultural runoff. A further aspect considered in this
paper was the role of these wastes had on the stability of leaf membrane, chlorophyll content and some soluble carbon and nitrogen fraction found in plants.
MATERIALS AND METHODS
Plants and water sampling
Three emergent macrophytes included; Persicaria salicifolia (Brouss Ex Willd) Assenov, Echionochloa stagnina (Retz.0 p. Beauv.) and Cyperus alopecuroides Rottb were collected from four sites, one at the River Nile (site 1 unpolluted, control), two at irrigation canals (Sites 2 and 3) and one at drainage canal (Site 4). Surface water at three sites was suffered from wastes coming from sewage effluents (Site 2), oils and detergents factory (Site 3) and agricultural runoff (Site 4). Plant samples were collected in triplicates and were rinsed with tap and distilled water and subdivided into shoots and roots. The plant organs were dried in an aerated oven at 70° C to constant mass.
Water samples were brought to the laboratory, filtrated through a Whatman 3-mm filter paper, and stored at 4° C before assaying. Each water sample was assayed for pH, electric conductivity, and concentration of a number of soluble cations and anions. Sampling was done in December 2012.
Plant analysis Membrane stability index
The stability of leaf membranes was assessed by determining electrolyte leakage from leaf segments exposed to dehydration (40% PEG) and heat (51 °C) stresses (Blum and Ebercon, 1981). The degree of membrane injury (based on electrolyte leakage) was calculated according to the following formula:
% injury = 1- [1- (T1 / T2) / 1- (C1 - C2)] x 100 where T1 and T2 represent the first and second conductance measurements on the treatment sample and C1 and C2 the first and second measurements on the control.
Chlorophyll determination
Chlorophyll a and b contents were measured spectrophotometrically (Wellburn, 1994). Determination of the malonydialdhyde (MDA)
The level of lipid peroxidation in plant tissues was
measured by determination of MDA (Madhava Rao
and Sresty, 2000). MDA content was determined with
thiobarbituric acid (TBA) reaction.
Determination of hydrogen peroxide and ascorbic acid contents
Hydrogen peroxide and ascorbic acid
concentrations in the leaves were assayed according
the method of Velikova et al. (2000) and Jagota and
Dani (1982), respectively.
Determination of Soluble carbon and nitrogen metabolites contents
In hot water plant extract the contents of soluble
sugar, soluble proteins and total free amino acids in
shoot and root systems were measured according to
Buysse and Merckx (1993), Lowry et al. (1951) and
Lee and Takahshi (1956), respectively.
Water analysis
The pH values of water samples were determined
by pH meter (model pH-206 Lutron). The specific
electrical conductance in water samples was
measured by means of conductivity meter (model
4310 JEN WAY).
The contents of Ca2+ and Mg2+ were determined volumetrically by EDTA titration method (Schwarzenbach and Biederman, 1948). Na+ and K+ were analyzed by using flame photometer type M7P. Chlorides were determined by the silver nitrate titration methods described by Jackson (1973).
The significance of differences between the plants in response to the presence of pollution sources were determined by analysis of variance (F value).Dunken test was used for comparison between means to evaluate the effects of different pollution sources on the parameters tested, using SPSS for Microsoft Windows (Ver. 10.0, SPSS Inc., USA).
RESULTS
Hydrogen peroxide (H2O2)
Data of Fig. 1A indicate that water pollution with wastes coming from sewage effluent, agricultural runoff and oils and detergents factory caused an increase in H2O2 concentration in shoots and roots of the studied plants. Industrial waste water (Site 3) was more effective in H2O2 elevation than the other pollution sources. The effect of wastes was more pronounced in roots than in shoots. For example, concentration of H2O2 in roots of P salicifolia collected from Site 3 was about 5-fold that of plants collected from control site (Site 1).
Malonydialdhyde (MDA)
Lipid peroxidation (Fig. 1C), measured as malonydialdhyde, increased significantly in shoots and roots of the studied plants growing at polluted sites in comparing with their analogous collected from the control ones (Site 1). Maximum increase (about 5fold) was recorded in roots of P salicifolia grown in
site exposed to oils and detergents factory waste water.
Ascorbic acid
Concentration of ascorbic acid (Fig. 1B) in shoots and roots of the three studied plants showed opposite response trend to that of H2O2 and MDA. Regardless to pollution sources, surface water pollution resulted in a significant decrease in the concentration of Asc A except at site 3 (roots) and site 4 (shoot) in C alopecuroides where the concentration showed slight increase in response to surface water pollution with industrial and agricultural wastes.
Stability of leaf membrane
Exposure of leaf discs excised from control plants (Fig. 2A) to heat stress (51°C) caused 31.05, 55.27 and 56.47 % injury in leaf membrane of C alopecuroides , P salicifolia and E stagnina, respectively. Water pollution with the different wastes significantly increase membrane injury in the three studied plants except in P salicifolia exposed to agricultural wastes.
Dehydration stress (40% PEG) caused 33.27 (P salicifolia), 9.53 (C alopecuroides) and 32.47 % (E stagnina) injury. Sewage waste water reduced membrane injury in P salicifolia and E stagnina but it increased the injury percent in leaf membrane of C alopecuroides. Water pollution with wastes coming from agricultural drainage increased the membrane injury in the three studied plants. The same was true for oils and detergents factory waste water for E stagnina plants.
Chlorophyll content
The response of Chl a and Chlb contents to water
pollution (Fig. 2B) varied according to pollution source differences as well as plant species. The three waste water caused significant decrease in Chl a and b content in P salicifolia. On the contrary, contents of Chl a and b were higher in C alopecuroides and E stagnina plants grown in polluted sites than those in plants collected from control site (Chl b in E stagnina at S2 was an exception).
Soluble sugars
Changes in soluble sugars contents (Fig. 3A) showed irregular response to water pollution according to plant and pollution source differences. In general, significant differences (P < 0.05) in SS of shoots and roots of the studied plants were recorded between plants growing in control (Site 1) and polluted (Site 2, Site 3, Site 4) sites. The three waste water caused significant increases in SS contents of C alopecuroides shoots and roots. Contrarily, the same pollution sources reduced the contents of SS in E stagnina and P salicifolia shoots and roots.
Soluble proteins
In general soluble proteins (Fig. 3B) were higher in shoots and roots of C alopecuroides collected from polluted sites than in those of control site. On the other hand SP were often lower in P salicifolia and E stagnina plants growing at polluted sites in comparing with those of control site. Both changes (enhancing or reduction) in SP contents were statistically significant. Total free amino acids
Total free amino acids (Fig. 3C) showed reversed trend in their response to water pollution to that noticed for soluble proteins. P salicifolia and E stagnina plants growing in polluted sites had higher
TAA in their shoots and roots than in plants collected from control site. The reverse held true in C alopecuroides where the three waste water caused a considerable reduction in the contents of TAA of shoots and roots (roots at Site 2 was an exception). The increase or decrease in TAA was significant (P < 0.05).
Water analysis
Water samples collected from the study sites were analyzed for their physico-chemical properties (Tab. 1). The pH values were fluctuated in neutral and slight alkaline range depending on type of water body (Nile, irrigation and drainage canals). The lowest pH value (7.17) was recorded at site 2 and the highest value (8.88) was at site 3 polluted with wastes of oil and detergents factory.
Conductivity yields a measure of a water capacity to convey an electric current. It is clear that EC of irrigation and drainage canals was generally higher than those recorded for water sampled from the Nile. The EC values ranged between 107 mScm-1 at site 1 and 1032 mScm-1 at site 4.
Generally Na+ contents were higher than those of K+ in water samples collected from different water bodies. The highest value of Na+ (49.03 mg L-1) was noticed at site 2 of irrigation canals and the lowest (27.67 mg L-1) was at site 3. The maximum value of K+ (0.34 mg L-1) was recorded at site 1 and the minimum one (0.13 mg L-1) was at site 3. Calcium fluctuated between 13.33 mg L-1at site 3 and 30.67 mg L-1 at site 1. Chlorides contents of different water bodies decreased in the order of drainage canals > irrigation canals > River Nile. Their concentration
varied from 0.11 mg L-1 at site 1 to 0.26 mg L-1 at site 4. Generally, changes in the above mentioned water
properties, as indicated by analysis of variance (F value), were statistically significant.
Persicaiia salicifolia
Figure 1. Changes in (A) Hydrogen peroxide, (B) ascorbic acid (Asc A), (C) malonyldialdehyde (MDA) contents (цітюі g-1FW) of shoots and roots of Cyperus alopecuroides, Persicaria salicifolia, Echinochloa stagnina plants sampled from different polluted sites. Vertical bars represent standard deviation. Different letters denote significant differences between different treatments at p < 0.05 (DMRT). Site1: Control Nile site, Site 2: Sewage effluents, Site 3: Oils and detergents factory, Site 4: Agricultural runoff.
Figure 2. Changes in (A) the stability of leaf membranes to both heat stress (51oC) and dehydration (40% PEG) measured as % of membrane injury and (B) chlorophyll (Chl a, Chl b) contents (mg g-1 FW of leaves) of Cyperus alopecuroides, Persicaria salicifolia, Echinochloa stagnina plants sampled from different polluted sites. Vertical bars represent standard deviation. Different letters denote significant differences between different treatments at p < 0.05 (DMRT). Site1: Control Nile site, Site 2: Sewage effluents, Site 3: Oils and detergents factory, Site 4: Agricultural runoff.
Figure 3. Changes in (A) soluble sugar (SS), (B) soluble protein (SP) and (C) total free amino acid (TAA) contents (mg g-1 DW) of shoots and roots of Cyperus alopecuroides, Persicaria salicifolia, Echinochloa stagnina plants sampled from different polluted sites. Vertical bars represent standard deviation. Different letters denote significant differences between different treatments at p < 0.05 (DMRT). Site1: Control Nile site, Site 2: Sewage effluents, Site 3: Oils and detergents factory, Site 4: Agricultural runoff.
Table 1 Physico-chemical properties and their F-values for water samples from different polluted sites. Data are means of three replicates (± standard error).
Parameter Site 1 Site 2 Site 3 Site 4 F-values
PH 7.52±0.265 7.17±0.395 8.88±0.099 7.22±0.087 1.80ns
E.C [mScm-1] 107.3±31.5 836.6±26.36 459.3±8.45 1032±53.56 72.54**
Cl- 0.11 ±0.01 0.18±0.01 0.143±0.02 0.26±0.01 24.87**
Ca2+ 30.67±2.33 18.33±0.88 13.33±0.33 20.67±1.86 21.71**
Mg2+ 0.11 ±0.021 0.20±0.013 0.19±0.006 0.40±0.036 5.81ns
Na+ 39.67±3.53 49.03±1.93 27.67±0.92 43.63±0.38 69.96**
K+ 0.34±0.018 0.31±0.003 0.13±0.003 0.31±0.003 111.17**
Explanations: EC - electric conductivity, Site1: Control Nile site, Site 2: Sewage effluents, Site 3: Oils and detergents factory, Site 4: Agricultural runoff, ns - nonsignificant, * - significant at 5% confidence level; ** - significant at 1% confidence level. Contents of ions were expressed in mg L-1.
DISCUSSION
Various a biotic stresses lead to the overproduction of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA which ultimately results in oxidative stress (Gill and Tuteja, 2010). Measurement of H2O2 in plant tissue under water pollution is important because it can be precursor of highly reactive oxygen species. Surface water pollution with wastes coming from sewage effluents, oils and detergents factory discharge and agricultural drainages resulted in an increase in hydrogen peroxide (H2O2) and lipid peroxidation (MDA) concentrations in shoots and roots of the three studied plants as compared with those sampled from unpolluted River Nile site (Site 1). Currently, research data show that H2O2 can play a dual role in cells. During oxidative stress, H2O2 is a strong toxic oxidant causing cell damage or even cell death. At the same time it serves conversely as a signaling molecule to activate a rescue/defense system for restoring the redox homeostasis in plant cells (Yang and Poovalah,
2002; Desikan et a/., 2003). Numerous studies have shown that the application of H2O2 at low concentrations could improve plant tolerance to salt stress (De Azevedo Neto et a/., 2005 ; Li et al. 2011) and heavy metal stress (Lin et al., 2004; Hu et al., 2009; Xu et al., 2011; Guzell and Terzi, 2013). According to Hu et al. (2009) the increased metal stress tolerance was attributed to induced antioxidant defense system after pretreatment with H2O2 in rice seedlings.
Hydrogen peroxide has been commonly used as an oxygen source because of the limited concentrations of oxygen that can be transferred into the groundwater using above-ground aeration (Zappi et al., 2000). Therefore it has been used for pollution control, municipal wastewater treatment and industrial waste/wastewater applications. It may be needed to oxidize the more resistant organic and inorganic pollutants which contribute to BOD and COD -catalytic and may also affect BOD/COD removal and to improve supply and oxidation rate of suspended and dissolved particles that cause pollution in such
water effluent (Chen et al., 1996 and Badmus et a/., 2007). In this study the overproduction of H2O2 under water pollution especially in roots of the studied aquatic macrophytes could be used as oxygen source and supply oxygen to oxidize the more resistant organic and inorganic pollutants and could be promotes aeranchyma formation in these plants where aerenchyma formation is mediated by reactive oxygen species (Steffens et al., 2011).
Under water pollution conditions, ascorbic acid concentration decreased in shoots and roots of the studied plants. Ascorbic acid is the most abundant, powerful and water soluble antioxidants acts to prevent or minimizing the damage caused by ROS in plants (Smirnoff, 2005; Ather et al., 2008). It serves as a cofactor for enzymes involved in photosynthesis, hormone biosynthesis, and the regeneration of antioxidants such as a-tocopherol. Ascorbic acid was also found to enhance the efficiency of antioxidant defense system in plants and their tolerance to oxidative stress (Galli, 2013)
Leaf membrane damage by either heat (51 °C) or dehydration (40% PEG) was generally higher in plants exposed to liquid wastes from the three pollution sources compared to membrane injury in plants growing at unpolluted River Nile site (control). Increasing membrane injury under water pollution could be due to lower ascorbic acid concentration in polluted plants (Fig. 1) which can provide protection to membrane by directly scavenge the O2^ and OH (Gill and Tuteja, 2010). Enhancement accumulation of H2O2 in polluted plants (Fig. 1) which may affect membrane properties could be another alternative
probability to increase membrane damage (high injury %) under water pollution condition.
Monitoring total chlorophyll concentration can be used as early warning for toxic effect of water pollution on plants (Calheiros et al., 2009). In this study, P salicifolia plants collected from polluted sites had lower chlorophyll contents than those sampled from control site (Site 1). Reduction in Chl content under the influence of water pollution has been reported (Gadallah, 1994). Chlorophyll reduction induced by waste water could be associated with high concentration of mineral and increased alkalinity of water (e.g. site 3 polluted with oils and detergents factory wastes). Some of possible reason for any decreases in Chl content may be due to: 1- formation of chlorophyllase which is responsible for chlorophyll degradation (Drazkiewice, 1994). 2- retardation of chlorophyll synthesis and 3- enhancement of chlorophyll loss due to increases endogenous abscisic acid (Cizkova, 1990). On the contrary, contents of chlorophyll in E stagnina and C alopecuroides plants growing at polluted sites were higher than in those of control site. Our results confirming the finding of Calherios et al. (2009) who reported an increase in Phragmites australis chlorophyll content under the industrial water pollution. Increased Chl content in polluted plants especially those exposed to agricultural drainage could be due to the high Mg 2+ (Tab. 1).
Soluble sugars accumulated in shoots and roots of the studied plants showed irregular response to changes in water habitats and presence of pollution sources. E stagnina and P salicifolia plants collected
from polluted sites were lower in soluble sugars than those at control site. This reduction in SS under water pollution might be result of decrease photosynthesis (Warrier and Saroja, 2008) through chlorophyll reduction (Fig. 2). On the other hand, C alopecuroides plants growing at polluted sites had higher contents of chlorophyll than the control plants. The higher accumulation of SS in polluted plants with corresponding higher chlorophyll content (Fig. 2) indicates that increases in SS was the result of higher photosynthesis and increasing metabolic activities of the tissues under water pollution (Steinbachova-Vojtisova et a/., 2006). Accumulation of SS could be an adaptive mechanism of water pollution where SS are able to protect the structural integrity of cell membrane which has been as a measure of adverse environmental stresses. Water pollution with wastes coming from different sources decreased the content of soluble proteins in shoots and roots of P salicifolia. Lower SP contents under water pollution might be due to oxidative damage to proteins through over production of H2O2 (Figure 3) which may inactivate enzymes (Gill and Tuteja, 2010), enhancement of proteolysis or inhibition of protein synthesis. On the contrary, the same pollution sources increased SP content in C alopecuroides could be probably due to the high rate of nitrogen metabolism. Our results were in consist results of Warrier and Sayoja (2008).
Regardless of pollution sources, total free amino acids were generally higher in plants growing at polluted sites than in those at control site. These results are in agreement with the result of Steinbachova-Vojtisova et a/., (2006). Accumulation
of TAA in polluted plants in comparison with unpolluted control plants could be an adaptive response to help plants to ovoid toxic concentration of metals in polluted water (Tomsett and Thurman, 1988). The total free amino acids (especially proline) accumulation could be used as an indicator to exposure to stress such as water pollution (Megateli et al., 2009).
In conclusion, water pollution with different wastes coming from different sources adversely affected the metabolic activities and stability of leaf membrane of the investigated plants. The three waste water reduced the antioxidant defense ability through: 1-reduction of ascorbic acid concentration which considered as most powerful ROS scavenger. 2-enhancement H2O2 which has been established that excess of H2O2 in plant cells leads to the occurrence of oxidative stress and 3- increasing lipid peroxidation intensity. The over production of hydrogen peroxide by the studied aquatic plants under water pollution could be used as an oxygen source and oxidize the more resistant organic and inorganic pollutants which contribute to BOD and COD catalytic. The effect of water pollution was not the same for the three plants studied. The same pollution source could give antagonistic effect on the same parameter of the different species (e.g. Chl, SS and SP contents). The investigated macrophytes play vital role in oxidizing more resistant organic and inorganic pollutants, and provide a cheap and easy way to monitor the different source of water pollution.
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