Ecotoxicological Evaluation of Wastewater Samples … · Ecotoxicological Evaluation of Wastewater...

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 5, 2011

© Copyright 2010 All rights reserved Integrated Publishing Association

Research article ISSN 0976 – 4402

Received on January, 2011 Published on February 2011 959

Ecotoxicological Evaluation of Wastewater Samples from Gadoon Amazai Industrial Estate (GAIE), Swabi, Pakistan

Azizullah Azizullah 1 , Peter Richter 1 , DonatPeter Häder 2 1 FriedrichAlexander University, Department of Biology, Cell Biology Division, Staudtstr.

5, 91058 Erlangen, Germany 2 Neue Str. 9, 91096 Möhrendorf, Germany

[email protected]

ABSTRACT

Industrial wastewater is a potential source of water pollution and a common threat to aquatic life. Chemical analysis of wastewater quantifies the concentrations of toxic substances but does not reflect their toxic effects on aquatic biota. Therefore, bioassessment is very necessary for monitoring of wastewaters quality. In the present study wastewater samples from Gadoon Amazai Industrial Estate (GAIE), Pakistan, were evaluated for their ecotoxicological effects using the automatic biotest ECOTOX and a Pulse Amplitude Modulated (PAM) fluorometer. Various physiological parameters of the freshwater flagellate Euglena gracilis like motility, swimming velocity, cell shape, gravitactic orientation and photosynthetic efficiency were used as end points. In addition, the water samples were analyzed for some common physicochemical properties. With some exceptions, most of the physicochemical properties of the tested samples were within the acceptable range of national environmental quality standards for municipal and industrial effluents (NEQS). However, all the water samples adversely affected different physiological parameters in Euglena gracilis. This study led to the conclusion that different toxic substances present in wastewater, even at low concentrations, adversely affect the physiology of aquatic biota. Gravitactic orientation and cell shape in Euglena gracilis were very sensitive to wastewater toxicity and can be used as reliable end points for wastewater bioassessment.

Keywords: ECOTOX, Euglena gracilis, Gravitaxis, Photosynthesis, Wastewater

1. Introduction

In the present age industrial wastewater is a potential threat to the quality of both surface and groundwater. An estimated 2 million tons of sewage and other effluents are discharged into the world’s waters every day. In developing countries the situation is worse where over 90% of raw sewage and 70% of untreated industrial wastes are dumped into surface water sources (Anonymous, 2010). The wastewaters of industries contain huge quantities of pollutants like nitrates, nitrites, cations and anions such as Ag + , Na + , K + , Mg 2+ , Ca 2+ Cl − , CO3

2 , HCO3 and

toxic metals like arsenic, iron, lead, mercury, chromium, cadmium, copper, nickel, zinc and cobalt (Sial et al., 2006; Ullah et al., 2009). In addition, wastewater also causes an increase in parameters like biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS), total suspended solids (TSS), and salinity and thus deteriorates the water quality.

In Pakistan, out of 6634 registered industries 1228 are considered to be highly polluting (Sial et al., 2006). Due to the high load of organic and toxic materials in their waste effluents, industries became a major source of water pollution in Pakistan (Nasrullah et al., 2006). It has


Azizullah Azizullah, Peter Richter, DonatPeter Häder

International Journal of Environmental Sciences Volume 1 No.5, 2011 960

been estimated that only 1% of industrial wastewaters in Pakistan is treated before being discharged (MOEPAK, 2005), and most of the industries dispose their waste effluents directly into the nearby drains, rivers, streams, ponds, ditches and open or agricultural land (Ullah et al., 2009). For example, river Kabul in Khyber Pakhtunkhwa, receives an estimated amount of 80,000 cubic meters of industrial effluents each day (MOEPAK, 2005).

Although several studies exist regarding the physicochemical analysis of industrial wastewaters in Pakistan, their harmful effects on aquatic organisms have hardly been analysed. In this study, in addition to some common physicochemical parameters, we evaluated the ecotoxic effects of wastewater samples from some selected industries of Gadoon Amazai Industrial Estate (GAIE), Swabi, Pakistan, using motility and photosynthetic parameters of Euglena gracilis as end points. The green flagellate E. gracilis has been reported in many studies as a reliable organism for bioassays. Due to its high sensitivity to heavy metals and other biologically active compounds, E. gracilis has proven to be a suitable organism for toxicity tests (Ahmed and Häder, 2010b; Danilov and Ekelund, 2001b; Ekelund, 1993; Tahedl and Häder, 1999).

Euglena gracilis orients itself in the water column by using external physical and chemical parameters such as light and gravity in search of a region optimal for growth and reproduction (Häder et al., 1999; Häder and Liu, 1990; Häder and Vogel, 1990; Lebert and Häder, 1999). To regulate its position in the water column, Euglena gracilis has developed precise mechanisms for phototactic and gravitactic orientation (Richter et al., 2003). Both responses are based on active physiological mechanisms (Häder, 1997; Lebert et al., 1996; Lebert et al., 1997; Richter et al., 2001). Motility and gravitactic orientation of E. gracilis were found to be sensitive parameters to various environmental stressors like organic compounds, heavy metals and sewage water (Tahedl and Häder, 1999), excessive visible and UV radiation, (Richter et al., 2007a), salinity (Richter et al., 2003) and herbicides (Pettersson and Ekelund, 2006). Photosynthesis parameters in Euglena, have also been reported as sensitive biomarkers for evaluating the toxicity of different substances (Ahmed and Häder, 2010b; GonzálesMoreno et al., 1997).

The main aim of the present study was to evaluate the selected wastewater samples from GAIE for their ecotoxic effects using various physiological parameters of the freshwater flagellate E. gracilis like motility, swimming velocity, cell shape, gravitactic orientation and photosynthesis as end points, and to evaluate the sensitivity and efficiency of ECOTOX and PAM fluorometer as instruments for water quality monitoring.

2. Materials and Methods

2.1. Area of study

Gadoon Amazai Industrial Estate (GAIE) located in district Swabi is one of the main industrial estates in the province Khyber Pakhtunkhwa of Pakistan. The estate was established in 1988 on an area of about 1116 acres. At present, no treatment facilities exist in the estate and the waste effluents are discharged to the surrounding environment without any treatment. Different drains collect the wastewaters from various industries of the estate which ultimately end in the river Indus, the largest river of the country (Ahmad et al., 2002).

http://en.wikipedia.org/wiki/Pakistan




2.2. Water samples

Seven water samples were collected (Table 1) including one sample each from Sardar Chemicals Industries (W1), Saif Textile Mills (W2), Amin Soap and Oil Mills (W3), Syntron Limited (W4), Ink Chemicals (W5) and Uthman Ghee (W6). One sample was collected from the main drain which collects the wastewater from various industries of the estate (W7). All samples were collected at the discharge outlets of each industry in clean plastic bottles.

Table 1: Description of water samples collected from Gadoon Amazai Industrial Estate (GAIE).

Sample Code

Industry name and address Main products

W1 Sardar Chemicals Industries Plot # 29B, Gadoon Amazai Industrial Estate, Swabi

dyes and chemicals for leather, textile and paper industries

W2 Saif Textile Mills Plot #153,157,167, Gadoon Amazai Industrial Estate, Swabi

yarns

W3 Amin Soap and Oil Mills, Plot # 191/3L10 Gadoon Amazai Industrial Estate, Swabi

soap and detergents

W4 Syntron Limited, Phase # 1, Line # 1, Gadoon Amazai Industrial Estate, Swabi

woven polypropylene bags, fabrics, sheets / rolls

W5 Ink Chemicals Plot # 21 Gadoon Amazai Industrial Estate, Swabi

different products for paint, printing inks, textile printing, wood glue and shoe adhesives

W6 Uthman Ghee Plot #39, Gadoon Amazai Industrial Estate, Swabi

vanaspati Ghee (edible oil)

W7 Main drain, Gadoon Amazai Industrial Estate, Swabi

2.3. Physicochemical analysis

Water samples were analysed for some common physicochemical properties using standard methods. pH was measured according to DIN EN 27888 (1993) using a Metrohm 6.0253.100 glass electrode (Metrohm, Herisau, Switzerland) and electrical conductivity (EC) was determined with a WTW LF197 conductivity meter (WTW, Weilheim, Germany). Heavy metals were measured using ICPOES and open HNO3/H2O2 digestion according to DIN EN ISO 11885 (2009) using a Perkin Elmer, Optima 2000 ICP OES device (Perkin Elmer, Waltham, USA). Total phosphorus (TP) was determined photometrically (Lambda 20 UV/VIS spectrophotometer, Perkin Elmer, Waltham, USA) after H2SO4 digestion following the specifications of DIN EN ISO 6878 (2004). Total nitrogen (TN) was measured according to DIN 38409 H 27 : 199207 (1992) employing catalytic oxidation and chemoluminescence detection using a Mulit N/C 3100 TOC/TN analysator (Analytic Jena, Jena Germany). Total organic carbon (TOC) was determined according to DIN EN 1484 H3 (1997) by IRdetection after catalytic oxidation using a TOCOR 101 (Maihak, Bloomington, USA).




2.4. Cell culture and growth conditions

All experiments were performed with the freshwater flagellate Euglena gracilis KLEBS; strain Z, obtained from the algal culture collection at the University of Göttingen, Germany (Schlösser, 1994). The cells were grown in a mixture of mineral and organic medium (Checcucci et al., 1976; Starr, 1964) under continuous light of 20 W m 2 from mixed cool white and warm tone fluorescent lamps at a temperature of about 20 ºC. For the experiments with ECOTOX static cultures were employed while for PAM fluorometer experiments exponential phase cultures were used.

2.5. Analysis of motility and orientation

Motility, velocity, cell shape and orientation of Euglena gracilis were measured by using the automatic biotest, ECOTOX (Tahedl and Häder, 1999). The system operates in real time and tracks a virtually unlimited number of cells in parallel. The software uses the vectors of the tracks to calculate various parameters like percent motility, percentage of cells moving upwards, the mean velocity, cell compactness and rvalue. The motility parameter indicates the percentage of cells moving at a speed equal to or faster than the minimum velocity set in the program, and all other parameters are calculated only for the objects which fulfil this criterion. The parameter upward gives the percentage of cells which are moving towards the upper part of the cuvette (± 90° around the vertical direction upward). The rvalue is a statistic parameter which describes the precision of gravitactic orientation of the cells and ranges from 0 (when the cells are moving randomly) to 1 (when all the cells are moving in a single direction). The cell compactness (form factor) describes the shape of the cell and has the lowest value of 1 when the outline of the object is a circle and increases as the cell increases in length. The software plots the calculated parameters and compares the data with those of a previous control measurement, which was carried out immediately before the sample measurement using unpolluted water instead of the test sample. The results are expressed as percent inhibition for all parameters.

Measurements were made by the setting “automatically decreasing dilutions”. In this setting five different measurements with automatically decreasing dilutions (1:31, 1:15, 1:7, 1:3, and 1:1) and a control are taken. The filling time of the cuvette was 75 s and the inbetween rinsing time was 45 s. Time of tracking of the cells was 3 min. Minimum areas for objects to be included in vector analysis were set to 400 μm 2 and maximum at 2000 μm 2 . Minimum speed at which the cells were considered motile was set at 15 μm s −1 . Before the measurements were made, the cells were incubated in darkness for 30 min to avoid the effect of light.

2.6. Analysis of photosynthetic parameters

The photosynthetic parameters were measured with the help of a portable Pulse Amplitude Modulated fluorometer (PAM 2000, Walz, Germany). The quantum yield of photosystem II (Fv/Fm) was measured by the single saturating pulse method. The relative electron transport rate (rETR) was measured by using the setting of the light curve, which involved determination of fluorescence at different intensities of actinic light (0, 86, 155, 236, 327, 466, 658, 966, 1461, 2177 and 3199 µmol m 2 s 1 ). Cells were exposed to five different dilutions of the water samples i.e. 16.66, 8.33, 4.76, 2.94, 2 dilution factor (corresponding to 6, 12, 21, 34 and 50%) and a control. The fluorescence parameters were measured after 24 hours of incubation. In order to keep the primary electron acceptors (QA) in an oxidized state, the cells of E. gracilis were incubated in the dark for 20 min before the measurements were made.




2.7. Data analysis

All experiments were performed in three replicates. Means and standard deviation were calculated for each treatment using Microsoft Word Excel. Student ttest was used to calculate the significance of differences among the treatments and the control. The lowest dilution (highest concentration), at which no significant effect was observed for a given parameter, was considered as Gvalue for that parameter. For motility and orientation parameters the effect was considered significant if the inhibition was above the threshold values of the ECOTOX system (11.4% for motility, 12.3% for rvalue, 3.1% for upward, 3.4% for compactness and 6.8% for velocity). The effect was considered significant only if all three replicates exceeded the threshold value of a given parameter.

3. Results

3.1. Physicochemical properties

The physicochemical properties of all the tested water samples are given in Table 2. The pH values of all samples were in the alkaline range except for Amin Soap and Oil industry. Electrical conductivity ranged from 245 to 1090 µS/cm with the lowest one for Uthman Ghee and the highest for Amin Soap and Oil Mills. Heavy metal concentrations varied between the different samples. Cadmium (Cd) was below 0.01 mg L 1 in all samples. Copper (Cu) was detected in a range from 0.03 to 0.29 mg L 1 , with the highest concentration in the sample from Ink Chemicals industry. Iron (Fe) was below 1 mg L 1 in all samples except Ink Chemicals and Amin Soap and Oil where it reached 3.7 and 14 mg L 1 , respectively. Nickel (Ni) was below 0.01 mg L 1 in all samples except Amin Soap and Oil where it reached 1.4 mg L 1 . The Amin Soap and Oil sample also had the highest chromium (Cr) concentration (0.03 mg L 1 ); the remaining samples had a Cr concentration of ≤ 0.01 mg L 1 .

Table 2: Physicochemical properties of water samples collected from GAIE. All concentrations are given in mg L 1 .

Parameter

Sardar Chemicals (W1)

Saif Textile Mills (W2)

Amin Soap and Oil (W3)

Syntron Limited (W4)

Ink Chemicals (W5)

Uthman Ghee (W6)

Main drain (W7)

pH 7.47 8.16 6.00 8.25 7.88 8.02 7.90 EC (20 o C) µS/cm 894 466 1090 264 392 245 322

Cd <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Cu 0.06 0.04 0.05 0.04 0.29 0.04 0.03 Fe 0.45 0.31 14 0.23 3.7 0.51 0.20 Ni <0.01 <0.01 1.4 <0.01 0.01 <0.01 <0.01 Cr <0.01 <0.01 0.03 0.01 0.01 <0.01 <0.01 Zn 0.07 0.11 0.20 0.12 1.5 0.28 0.01 Pb 0.03 0.03 0.05 0.06 0.06 0.03 0.03 TP 0.17 0.82 2.4 <0.1 0.19 0.19 0.10 TN 20 6.6 8.2 3.3 20 3.1 3.3 TOC 47 10 93 9.5 110 10 9.2




Zinc (Zn) was detected in the range from 0.01 to 1.5 mg L 1 , the minimum being in the Main drain sample and the maximum in the Ink Chemicals water sample. Lead (Pb) occurred in the range from 0.03 to 0.06 mg L 1 . The minimum concentration of total phosphate was detected in the Syntron industry sample (<0.1mg L 1 ) and the maximum in the Amin Soap and Oil Mills (2.4 mg L 1 ). Total nitrogen (TN) varied in samples from 3.1 to 20 mg L 1 . Total organic carbon (TOC) was detected in a wide range (9.2 to 110 mg L 1 ). The minimum concentration of TOC was in the Main drain and the maximum in the Ink Chemicals water samples.

3.2. Effects of wastewater on motility, velocity, compactness and orientation of cells

The ECOTOX instrument determines the effects of wastewater on several motility and orientation parameters of E. gracilis in parallel. Figures 1 (a – g) show the effects of water samples on motility, cell speed, cell shape and orientation of E. gracilis. The inhibitory effects on motility were not very pronounced. The Sardar Chemicals wastewater sample affected motility at dilutions below 33.33 but the effect was not significant due to large standard deviation. The Saif Textile sample caused a 10 – 15 % inhibition of motility at the lowest two dilutions; however, the effect was not significant. Similar results were found for the Amin Soap and Oil, Ink Chemicals and Main drain samples, i.e. all these samples caused a nonsignificant inhibition of motility at low dilutions (high concentrations). Wastewater samples from Uthman Ghee and Syntron industry did not affect motility. Velocity was significantly reduced only by the lowest dilution of the Ink Chemicals sample. The rest of the samples either did not affect velocity or caused slight positive effects. The compactness of cells (cell shape) was reduced by wastewaters (the cells treated with wastewater became rounder as compared to the control) and showed ≈ 5 18% decrease at the lowest dilutions of the samples. The sample from Sardar Chemicals significantly reduced cell compactness at all dilutions except the lowest one. The Saif Textile sample caused a decrease in cell compactness; however, this was not significant in many cases due to large standard deviation. The sample from Amin Soap and Oil Mills significantly reduced cell compactness at all dilutions below 33.33. The effect of the sample from Syntron industry on cell compactness was not very pronounced, and a significant decrease was observed only at 2.94 dilution factor. The Ink Chemicals sample caused a decrease in cell compactness at dilutions below 8.33. The least sensitivity of cell compactness was observed to the sample from Uthman ghee industry where only ≈ 5% decrease was observed at the lowest dilution (highest concentration). The water sample from the main drain caused a concentrationdependent decrease in cell compactness which was significant at the lowest two dilutions.

Gravitactic orientation of cells was found to show a very pronounced response to wastewater toxicity. The rvalue was the most affected parameter and showed more than 40% impairment at the lowest dilutions of most of the samples. Based on the Gvalues (the lowest dilution at which no significant effect was observed) calculated for rvalue, samples from Saif Textile, Amin Oil and Soap and Ink Chemicals were found to be more toxic and impaired the orientation of the cells (rvalue) at dilutions below 33.33 (concentration above 3%). Samples from Sardar Chemicals, Uthman Ghee and the Main drain impaired the orientation of the cells at dilutions below 16.66 (concentration above 6%). The Syntron industry sample was the least toxic sample and disturbed the orientation in the cells at dilutions below 8.33. Upward swimming of cells was affected in the same pattern as rvalue. Samples from Sardar Chemicals and Ink Chemicals significantly inhibited upward swimming of cells at dilutions below 33.33. The Saif Textile and Amin Soap and Oil samples caused a decrease in upward




swimming of cells at dilutions below 16.66. The rest of samples inhibited upward swimming of cells at dilutions below 8.33.

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33.33 16.66 8.33 4.76 2.94 2

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Inhibition (%

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16.66 8.33 4.76 2.94 2


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33.33 16.66 8.33 4.76 2.94 2


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16.66 8.33 4.76 2.94 2


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Figures 1: (a – g). Effect of wastewater on motility ( ), velocity ( ), compactness ( ), upward swimming ( ), and rvalue ( ) of E. gracilis. The abscissa shows the dilutions of the water sample and ordinate % inhibition (compared to the control) of the given parameters. The rvalue is a statistic parameter, so standard error bars cannot be calculated. Asterisks (*) show that the inhibition is significant. (a) Sardar Chemicals W1, (b) Saif Textile Mills W2, (c) Amin Soap and Oil Mills W3, (d) Syntron Limited W4, (e) Ink Chemicals W5, (f) Uthman Ghee W6, (g) Main Drain W7.

3.3. Effects of Wastewater on Photosynthesis

The photosynthetic parameters like quantum yield (Fv/Fm) and rETR in E. gracilis were measured after 24 h of exposure to the wastewater samples. No water sample significantly affected the quantum yield (Fv/Fm) except Sardar Chemicals wastewater where Fv/Fm significantly (p< 0.05) increased at the lowest dilution (Figures 2 a g). With increasing irradiances, all water samples were shown to have a positive effect on the rETR and yield as compared to the control. Samples from Saif Textile and Ink Chemicals caused an increase in rETR at dilutions below 16.66.

The rest of the samples were shown to have significant positive effects on rETR even at the highest dilution (lowest concentration) tested. With increasing light intensity, the photosynthetic yield decreased and fell to nearly 0.1 at the highest intensity of light in all experiments. However, cells exposed to wastewater samples showed significantly higher yields than the control at the respective light intensities. Since all the samples showed almost the same effects on rETR and yield, the data are shown only for one sample (Sardar Chemicals) as representative graphs (Figures 3 a, b). The overall results of the fluorescence parameters demonstrate that water samples tested in this study did not inhibit photosynthesis in E. gracilis but rather had a positive effect.




0.1

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Cont 16.66 8.33 4.76 2.94 2


Fv/Fm

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Cont 16.66 8.33 4.76 2.94 2

Dilution factor (DF) Fv/Fm

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0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Cont 16.66 8.33 4.76 2.94 2


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Cont 16.66 8.33 4.76 2.94 2


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Cont 16.66 8.33 4.76 2.94 2


Fv/Fm

g

Figures 2: (a – g). Effect of wastewaters on quantum yield of photosystem II (Fv/Fm) in E. gracilis. Each bar represents the mean of three replicates and error bars represent standard deviations. (a) Sardar Chemicals W1, (b) Saif Textile Mills W2, (c) Amin Soap and Oil Mills W3, (d) Syntron Limited W4, (e) Ink Chemicals W5, (f) Uthman Ghee W6, (g) Main drain W7.




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Figure 3: Effect of wastewater from Sardar Chemicals industry on the electron transport rate (rETR) (a), and yield (b) in E. gracilis with increasing light intensity. The dilutions used were control ( ), 16.66 DF ( ), 8.33 DF ( ), 4.76 DF ( ), 2.94 DF ( ), and 2 DF ( ). rETR and yield of the treated cells were significantly higher than the control at the respective light intensities at all dilutions tested. Similar results were obtained for other samples.

4. Discussion

The waste effluents of industries are a potential threat to water quality all over the world. In developing countries like Pakistan, the situation is further aggravated where the industrial wastes are often directly disposed off without any treatment. Literature studies reveal large variations in the physicochemical properties of wastewaters from different industries and even the same type of industries located in different areas and/or analysed by different workers. This variation is due to various factors like different locations of industries, time and methods of sampling, different methods and protocol of analysis and the prevailing conditions (temperature, rain etc) in the area of study. In our study, the physicochemical properties of all water samples were in agreement with previous reports in the country (Bangash et al., 2006; Ghafoor et al., 1994; Nasrullah et al., 2006; Sial et al., 2006; Tariq et al., 2006) and with some exceptions, were within the acceptable range of the national environmental quality standard for municipal and industrial effluents (NEQS) (Govt. of Pakistan, 1997). However, the concentration of pollutants in wastewaters with in NEQS does not guarantee that there is no toxicity to aquatic organisms. The continuous inflow in water bodies can result in wide spread pollution and damage to aquatic biota. Surface water pollution due to such practices has been reported in many countries. Javed and Hayat (1999) and Saeed and Bahzad (2006) reported that industrial and municipal wastewater from Lahore significantly increased the heavy metal concentrations and their toxicity in river Ravi. High concentrations of heavy metals in the water and sediments of the Chao Phraya river estuary in Thailand due to the bulk disposal of domestic and industrial effluents have been reported (Polprasert, 1982). Manga (1983) found that the industrial effluent input to the tributary rivers and direct discharges into the river Lagan were the main sources of heavy metal contamination in tidal Lagan sediments. Recently, Oguzie and Okhagbuzo (2010) reported that effluent discharges resulted in elevated concentrations of heavy metals in Ikpoba River in Nigeria.

In the present study the effects of wastewater were observed on a wide range of physiological parameters in E. gracilis. Due to the position of Euglena as a producer in the aquatic ecosystem, the measured end points have ecological importance not only for Euglena itself




but also for the food web as well as for the whole ecosystem. Motility and swimming velocity of cells were not significantly affected by wastewaters. The gravitactic orientation (rvalue and upward swimming) of cells was found to be very sensitive to wastewater and strongly impaired with decreasing dilutions (increasing concentrations) of wastewater. Gravitaxis, which is an active physiological phenomenon, can be inverted by certain environmental stressors like UV, increased salinity and toxicants (Richter et al., 2003). Wastewaters have a large number of active pollutants like heavy metals and some other compounds. Studies reveal that heavy metals like nickel, copper, cadmium, silver, lead, chromium, and zinc invert the gravitactic orientation in E. gracilis (Ahmed and Häder, 2010b; Tahedl and Häder, 2001). The density difference between the cell body and the surrounding medium plays an important role in the gravitactic orientation of the organism (Lebert et al., 1999). The sedimentation of the cell body inside the cytoplasmic membrane applies a force to the lower membrane which activates mechanosensitive ion channels in the membrane. This activation of mechano sensitive ion channels changes the membrane potential which triggers reorientation of the flagellar movement. By using micromolar concentrations of specific inhibitors of mechano sensitive ion channels such as gadolinium, graviorientation was impaired. This shows that the mechanism of mechanosensitive ion channels is involved in gravitaxis (Franco et al., 1991). The toxic substances present in water can directly affect these channels and hence invert gravitactic orientation of the cell (Tahedl and Häder, 1999).

Results obtained in the present study demonstrate that gravitactic orientation in E. gracilis is more sensitive and is strongly impaired by wastewaters as compared to other parameters. This is in agreement with previous studies reporting that gravitactic orientation of Euglena is more sensitive than other parameters to various pollutants in water (Ahmed and Häder, 2010a; Tahedl and Häder, 1999). Euglena gracilis has mechanosensitive channels in the cell membrane acting as gravireceptors (Häder et al., 2009) which can be a possible reason for the higher sensitivity of gravitactic orientation in Euglena to wastewaters and other toxins (Ahmed and Häder, 2010a). Previous studies also proved that motility and orientation in E. gracilis are more sensitive than photosynthesis (fluorescence parameters) to different pollutants. For example, in shortterm toxicity tests, the motility and gravitactic parameters were found to be more sensitive than photosynthetic parameters to heavy metals such as cadmium and nickel (Ahmed and Häder, 2010b). Similarly, the motility factors of E. gracilis were found to be adversely affected by wood ash concentrations exceeding 1015 g L 1 due to the elevated pH (Aronsson and Ekelund, 2005) but the same concentrations of wood ash did not affect photosynthesis in E. gracilis (Ekelund and Aronsson, 2007). Based on our observations of the analysis of a large number of wastewater samples from different industries (unpublished data), we conclude that gravitactic orientation of E. gracilis is its most sensitive parameter towards wastewater toxicity during shortterm tests (immediately after exposure).

A decrease in cell compactness was observed with decreasing dilutions (increasing concentrations) of wastewater, i.e. the treated cells became more rounded. The cell shape in E. gracilis has been recommended as sensitive physiological parameter to various environmental stressors in earlier studies (Häder et al., 1997). It has been reported that the species of the genus Euglena change their cell shape in response to increasing concentrations of water pollutants and other physical or chemical stressors (Conforti, 1998; Murray, 1981; Takenaka et al., 1997). According to Pettersson and Ekelund (2006) this change in cell compactness impairs the cell’s capacity to exert the required force on the cytoplasmic membrane which causes inhibition of upward swimming of cells and graviorientation. However, gravitaxis and cell shape are not necessarily coupled (Richter et al., 2007b). Also in




the present study, the responses of gravitactic orientation and cell shape to wastewater toxicity were independent of each other and showed different Gvalues.

A positive effect on the photosynthetic parameters of E. gracilis was observed when exposed to wastewater for 24 h. These observations are in agreement with those by Danilov and Ekelund (1999) who found that shorttime exposure to wastewater samples from pulp and paper industry had a positive effect on the photosynthetic efficiency of Euglena. In shortterm exposure they observed that increasing concentrations of the uncleaned sample had continuously stimulating effects on the photosynthetic efficiency and worked protectively against UVB radiation (Danilov and Ekelund, 1999). A similar increasing trend in rETR was observed upon exposure of E. gracilis to wood ash solution (Ekelund and Aronsson, 2007). The results from the wastewaters and wood ash are comparable in the sense that both have a mixture of available nutrients and various biologically active substances. This positive effect on photosynthesis can be explained by one or a combination of the following factors, (1) The nutrients present in wastewater were available to, and satisfied the nutritional requirement of Euglena as compared to the control (diluted with deionized water) which resulted in higher photosynthesis than in the control. (2) The positive effect was due to a possible stimulatory effect of low doses of toxins as observed on the growth and some other physiological activities in some unicellular algae (Danilov and Ekelund, 2001a; Jennings, 1979; Stebbing, 1982; Vocke et al., 1980). (3) The protective role of wastewater against strong radiation can also be a possible reason for higher photosynthetic efficiency at high light intensities. The photosynthetic parameters in this study gave almost the same responses to all different samples, i.e. an increasing trend with decreasing dilution of water samples was observed. No inhibitory effect was observed on either yield or rETR after 24 h of incubation. Therefore, from an ecotoxicological point of view, it is concluded that photosynthetic parameters in E. gracilis do not seem to be suitable parameters for assessing wastewater toxicity in shortterm experiments. Probably in longterm assessments (7day tests), the photosynthetic parameter may give valuable results as reported by Danilov and Ekelund (1999) for wastewater from paper and pulp industry. Experiments are in progress to determine the validity and reliability of photosynthetic parameters for wastewaters toxicity assessment in longterm exposures.

For monitoring wastewater quality chemical analysis is a common practice; however, the chemical analysis alone cannot give quantitative information regarding the toxic effects on living biota (Kelly et al., 2004; Streb et al., 2002; Wong et al., 1995). Even the low concentration of toxicants shown by chemical analysis can possibly affect living organisms (Danilov and Ekelund, 2000). Therefore, the usage of bioassays is necessary for determining the quality of wastewater (treated and/or untreated) before being discharged into natural water bodies. For toxicity assessment in the aquatic environments, different algal tests are being used with cell number, fresh or dry weight, protein and nucleic acid contents, chlorophyll a fluorescence, photosynthetic CO2 fixation, ATP production, morphology or vital stainability as endpoints (Rai et al., 1994). However, most of these tests need a long time, hours or days and sometimes are expensive. In comparison, ECOTOX has low costs and needs a very short time for measurement. One complete measurement of a sample and control cycle lasts 6 to 10 min, depending on the setting of the software. In addition, automating measurement and data analysis is another advantage of ECOTOX which reduces the chances of personal error. Most of the tests used in biomonitoring of water quality involve a single parameter as end point, while ECOTOX uses several parameters (motility, cell velocity, gravitactic orientation (upward swimming and rvalue), and cell shape) in parallel as end points which increases reliability of the results. All these endpoint parameters of ECOTOX are easy to measure and give reliable and sensitive responses to various water pollutants. The




sensitivity of ECOTOX was found to be comparable or even higher than other biotests like Daphnia (motility), fish (mortality), gene toxicity tests (mutant genes), algae test (growth rate) and MICROTOX (Ahmed and Häder, 2010a).

5. Conclusions

Industrial wastewater is a potential threat to water quality and adversely affects various physiological activities in aquatic organisms. In the present study most of the toxic pollutants, as revealed by chemical analysis, were within the acceptable range of NEQS but still affected the physiology of E. gracilis. The resultant effect may be the synergistic effects of different toxic substances in wastewaters. Exclusive chemical analysis for assessment of wastewater quality is not sufficient and bioassessment is necessary to quantify the effects of biological toxicity of wastewaters. Gravitactic orientation and cell compactness in E. gracilis were found to be very sensitive and can be used as reliable and sensitive parameters for the bioassessment of wastewaters. Photosynthetic parameters in E. gracilis (using fluorescence techniques) can not be recommended for the assessment of wastewater toxicity in shortterm exposure.

Acknowledgments

The authors are thankful to Dr. Martin Schmid, Bayerisches Landesamt für Umwelt, München for skilful support in the analytical work. The authors also acknowledge Kohat University of Science and Technology (KUST), Kohat, Pakistan for granting a scholarship to Azizullah.

6. References

1. Ahmad, B., Zakir, S., Nawab, B., Jehangir, A., Farhan, D., 2002. Monitoring of wastewater quality of Gadoon Amazai Industrial Estate NWFP Pakistan. Asian Journal of Plant Sciences, Vol. 1, pp. 7072.

2. Ahmed, H., Häder, D.P., 2010a. Monitoring of waste water samples using the ECOTOX biosystem and the flagellate alga Euglena gracilis. Water Air and Soil Pollution, Published online DOI 10.1007/s1127001005524.

3. Ahmed, H., Häder, D.P., 2010b. Rapid ecotoxicological bioassay of nickel and cadmium using motility and photosynthetic parameters of Euglena gracilis. Environmental and Experimental Botany, Vol. 69, pp. 6875.

4. Anonymous, 2010. World Water Day 22.03.2010. Retrieved from www.worldwaterday2010.info. United Nations.

5. Aronsson, K.A., Ekelund, N.G.A., 2005. Effects on motile factors and cell growth of Euglena gracilis after exposure to wood ash solution; assessment of toxicity, nutrient availability and pHdependency. Water, Air and Soil Pollution, Vol. 162, pp. 353368.

6. Bangash, F.K., Fida, M., Fazeelat, 2006. Appraisal of effluents of some selected industries of Hayatabad Industrial Estate, Peshawar. Journal Chemical Society of Pakistan, Vol. 28, pp. 1619.




7. Checcucci, A., Colombetti, G., Ferrara, R., Lenci, F., 1976. Action spectra for photoaccumulation of green and colorless Euglena: evidence for identification of receptor pigments. Photochemistry and Photobiology, Vol. 23, pp. 5154.

8. Conforti, V., 1998. Morphological changes of Euglenophyta in response to organic enrichment. Hydrobiologia, Vol. 369, pp. 277285.

9. Danilov, R., Ekelund, N., 2000. Applicability of growth rate, cell shape, and motility of Euglena gracilis as physiological parameters for bioassessment at lower concentrations of toxic substances: An experimental approach. Härnösand, Sweden: Mid Sweden University, John Wiley & Sons, Inc.

10. Danilov, R.A., Ekelund, N.G.A., 1999. Influence of waste water from the paper industry and UVB radiation on the photosynthetic efficiency of Euglena gracilis. Journal of Applied Phycology, Vol. 11, pp. 157163.

11. Danilov, R.A., Ekelund, N.G.A., 2001a. Effects of Cu 2+ , Ni 2+ , Pb 2+ , Zn 2+ and pentachlorophenol on photosynthesis and motility in Chlamydomonas reinhardtii in shortterm exposure experiments. BMC Ecology 1, 1.

12. Danilov, R.A., Ekelund, N.G.A., 2001b. Responses of photosynthetic efficiency, cell shape and motility in Euglena gracilis (Euglenophyceae) to shortterm exposure to heavy metals and pentachlorophenol. Water, Air and Soil Pollution, Vol. 132, pp. 61 73.

13. DIN 38409 H 27: 199207, 1992. German standard methods for the examination of water, waste water and sludge Parameters characterizing effects and substances (group H) Standard test method for total nitrogen in original sample. Deutsches Institut für Normung E.V.

14. DIN EN 1484 H3, 1997. Water analysis Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC); German version EN 14841997. Deutsches Institut für Normung E.V.

15. DIN EN 27888, 1993. Water quality; determination of electrical conductivity (ISO 7888:1985); German version EN 27888: 1993. Deutsches Institut für Normung E.V.

16. DIN EN ISO 11885, 2009. Water quality Determination of selected elements by inductively coupled plasma optical emission spectrometry (ICPOES) (ISO 11885: 2007); German version EN ISO 11885: 2009. Deutsches Institut für Normung E.V.

17. DIN EN ISO 6878, 2004. Water quality Determination of phosphorus Ammonium molybdate spectrometric method (ISO 6878: 2004); German version EN ISO 6878: 2004. Deutsches Institut für Normung E.V.

18. Ekelund, N.G.A., 1993. The effect of UVB radiation and humic substances on growth and motility of the flagellate, Euglena gracilis. Journal of Plankton Research, Vol. 15, pp. 715722.

19. Ekelund, N.G.A., Aronsson, K.A., 2007. Changes in chlorophyll a fluorescence in Euglena gracilis and Chlamydomonas reinhardtii after exposure to woodash. Environmental and Experimental Botany, Vol. 59, pp 9298.




20. Franco, A., Jr., Winegar, B.D., Lansman, J.B., 1991. Open channel block by gadolinium ion of the stretchinactivated ion channel in mdx myotubes. Biochemical Journal, Vol. 59, pp 11641170.

21. Ghafoor, A., Raur, A., Arif, M., Muzaffar, W., 1994. Chemical composition of effluents from different industries of the Faisalabad. Pakistan Journal of Agriculture Sciences, Vol. 31, pp 367370.

22. GonzálesMoreno, S., GómezBarrera, J., Perales, H., MorenoSánchez, R., 1997. Multiple effects of salinity on photosynthesis of the protist Euglena gracilis. Physiologia Plantarum, Vol. 101, pp 777786.

23. Govt. of Pakistan, 1997. Pakistan environmental legislation and the National Environmental Quality Standards (NEQS).

24. Häder, D.P., 1997. Gravitaxis and phototaxis in the flagellate Euglena studied on TEXUS missions. In Life Science Experiments Performed on Sounding Rockets (19851994). Cogoli, A., Friedrich, U., Mesland, D., Demets, R. (eds). ESTEC, Noordwijk, The Netherlands: ESA Publications Division, pp 7779.

25. Häder, D.P., Lebert, M., and Richter, P., 1999. Gravitaxis and graviperception in flagellates and ciliates. In Proceedings 14th ESA Symposium on European Rocket and Balloon Programmes and Related Research (ESA SP437), Potsdam, Germany. pp 479486.

26. Häder, D.P., Lebert, M., Tahedl, H., Richter, P., 1997. The Erlanger flagellate test (EFT): photosynthetic flagellates in biological dosimeters. Journal of Photochemistry and Photobiology B: Biology 40, pp 2328.

27. Häder, D.P., Liu, S.M., 1990. Motility and gravitactic orientation of the flagellate, Euglena gracilis, impaired by artificial and solar UVB radiation. Current Microbiology, Vol. 21, pp 161168.

28. Häder, D.P., Richter, P., Schuster, M., Daiker, V., Lebert, M., 2009. Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: involvement of a transient receptor potentiallike channel and a calmodulin. Advances in Space Research, Vol. 43, pp 11791184.

29. Häder, D.P. and Vogel, K., 1990. Graviorientation in photosynthetic flagellates. Proceedings of the Fourth European Symposium on Life Science Research in Space (ESA SP307). pp 521526.

30. Javed, M., Hayat, S., 1999. Heavy metal toxicity of river Ravi aquatic ecosystem. Pakistan Journal of Agriculture Sciences, Vol. 36, pp 8189.

31. Jennings, J.R., 1979. The effect of cadmium and lead on the growth of two species of marine phytoplankton with particular reference to the development of tolerance. Journal of Plankton Research, Vol. 1, pp 121136.

32. Kelly, C.J., Tumsaroj, N., Lajoie, C.A., 2004. Assessing wastewater metal toxicity with bacterial bioluminescence in a benchscale wastewater treatment system. Water Research, Vol. 38, pp 423431.




33. Lebert, M. and Häder, D.P., 1999. Aquarack: longterm growth facility for 'professional' gravisensing cells. Proceedings of the 2nd European Symposium on the Utilisation of the International Space Station, ESTEC, Noordwijk, The Netherlands. 1618 November 1998 (ESASP 433). pp 533537.

34. Lebert, M., Porst, M., Richter, P., Häder, D.P., 1999. Physical characterization of gravitaxis in Euglena gracilis. Journal of Plant Physiology, Vol. 155, pp 338343.

35. Lebert, M., Richter, P., Häder, D.P., 1997. Signal perception and transduction of gravitaxis in the flagellate Euglena gracilis. Journal of Plant Physiology, Vol. 150, pp 685690.

36. Lebert, M., Richter, P., Porst, M., and Häder, D.P., 1996. Mechanism of gravitaxis in the flagellate Euglena gracilis. Bräucker, R. Proceedings of the 12th C.E.B.A.S.Workshops. Annual Issue 1996. pp 225234.

37. Manga, N., 1983. Heavy metal concentrations in the sediments of the tidal section of the river Lagan. Irish Journal of Environmental Sciences, Vol. 2, pp 6064.

38. MOEPAK, 2005. 'State of the Environment Report 2005 (Draft)'. Islamabad, Pakistan: Ministry of Environment, Government of Pakistan.

39. Murray, J.M., 1981. Control of cell shape by calcium in the Euglenophyceae. Journal Cell Science, Vol. 49, pp 99117.

40. Nasrullah, Naz, R., Bibi, H., Iqbal, M., Durrani, M.I., 2006. Pollution load in industrial effluent and ground water of Gadoon Amazai Induatrial Estate (GAIE) Swabi, NWFP. Journal of Agriculture and Biological Science, Vol. 1, pp 1824.

41. Oguzie, F.A., Okhagbuzo, G.A., 2010. Concentrations of heavy metals in effluent discharges downstream of Ikpoba river in Benin City, Nigeria. African Journal of Biotechnology, Vol. 9, pp 319325.

42. Pettersson, M., Ekelund, N.G.A., 2006. Effects of the herbicides Roundup and Avans on Euglena gracilis. Archives of Environmental Contamination and Toxicology, Vol. 50, pp 175181.

43. Polprasert, C., 1982. Heavy metal pollution in the Chao Phraya River estuary, Thailand. Water Research, Vol. 16, pp 775784.

44. Rai, L.C., Gaur, J.P., Soeder, C.J., 1994. Algae and Water Pollution. Stuttgart: E. Schweizerbart'sche Verlagsbuchhandlung.

45. Richter, P., Börnig, A., Streb, C., Ntefidou, M., Lebert, M., Häder, D.P., 2003. Effects of increased salinity on gravitaxis in Euglena gracilis. Journal of Plant Physiology, Vol. 160, pp 651656.

46. Richter, P., Helbling, W., Streb, C., Häder, D.P., 2007a. PAR and UV effects on vertical migration and photosynthesis in Euglena gracilis. Photochemistry and Photobiology, Vol. 83, pp 818823.




47. Richter, P., Lebert, M., Korn, R., Häder, D.P., 2001. Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis. Journal of Plant Physiology, Vol. 158, pp 3539.

48. Richter, P.R., Schuster, M., Lebert, M., Streb, C., Häder, D.P., 2007b. Gravitaxis of Euglena gracilis depends only partially on passive buoyancy. Advances in Space Research, Vol. 39, pp 12181224.

49. Saeed, M.M., Bahzad, A., 2006. Simulation of contaminant transport to mitigate environmental effects of wastewater in river Ravi. Pakistan Journal of Water Resources, Vol. 10, pp 4352.

50. Schlösser, U.G., 1994. SAGSammlung von Algenkulturen at the University of Göttingen. Botanica Acta, Vol. 107, pp 113186.

51. Sial, R.A., Chaudhary, M.F., Abbas, S.T., Latif, M.I., Khan, A.G., 2006. Quality of effluents from Hattar Industrial Estate. Journal of Zhejiang UniversityScience B, Vol. 7, pp 974980.

52. Starr, R.C., 1964. The culture collection of algae at Indiana University. American Journal of Botany, Vol. 51, pp 10131044.

53. Stebbing, A.R.D., 1982. Hormesisthe stimulation of growth by low levels of inhibitors. Science of the Total Environment, Vol. 22, pp 213234.

54. Streb, C., Richter, P., Sakashita, T., Häder, D.P., 2002. The use of bioassays for studying toxicology in ecosystems. Current Topics in Plant Biology, Vol. 3, pp 131 142.

55. Tahedl, H., Häder, D.P., 1999. Fast examination of water quality using the automatic biotest ECOTOX based on the movement behavior of a freshwater flagellate. Water Research, Vol. 33, pp 426432.

56. Tahedl, H., Häder, D.P., 2001. Automated biomonitoring using real time movement analysis of Euglena gracilis. Ecotoxicology and Environmental Safety, Vol. 48, pp 161169.

57. Takenaka, S., Kondo, T., Nazeri, S., Tamura, Y., Tokunaga, M., Tsuyama, S. et al., 1997. Accumulation of trehalose as a compatible solute under osmotic stress in Euglena gracilis Z. Journal of Eukaryotic Microbiology, Vol. 44, pp 609613.

58. Tariq, M., Ali, M., Shah, Z., 2006. Characteristics of industrial effluents and their possible impacts on quality of underground water. Soil and Environ., Vol. 25, pp 64 69.

59. Ullah, R., Malik, R.N., Qadir, A., 2009. Assessment of groundwater contamination in an industrial city, Sialkot, Pakistan. African Journal of Environmental Science and Technology, Vol. 3, pp 429446.

60. Vocke, R.W., Sears, K.L., O'Toole, J.J., Wildman, R.B., 1980. Growth responses of selected freshwater algae to trace elements and scrubber ash slurry generated by coal fired power plants. Water Research, Vol. 14, pp 141150.




61. Wong, S.L., Wainwright, J.F., Pimenta, J., 1995. Quantification of total and metal toxicity in wastewater using algal bioassays. Aquatic Toxicology, Vol. 31, pp 5775.

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