Ecotoxicology (2012) 21:1957–1964 DOI 10.1007/s10646-012-0930-3
Chronic toxicity of a laundry detergent to the freshwater flagellate Euglena gracilis Azizullah Azizullah • Peter Richter • Muhammad Jamil • Donat-Peter Ha¨der
Accepted: 10 May 2012 / Published online: 30 May 2012 Ó Springer Science+Business Media, LLC 2012
Abstract Chronic toxicity of the common laundry detergent Ariel on the freshwater alga Euglena gracilis was investigated by growing the alga in a medium containing the detergent for 7 days. Cell density, motility, swimming velocity, gravitactic orientation, cell shape, photosynthesis and concentration of light-harvesting pigments were used as end point parameters for the assessment of toxicity. Cell density was significantly reduced at a concentration of 1 mg l-1 or above. Among the other tested parameters, with the exception of cell shape, gravitaxis and chlorophyll b, all were adversely affected by the detergent at concentrations exceeding 1 mg l-1. It is concluded that long-term (7-days) exposure to the detergent caused significant toxicity to E. gracilis. Furthermore, long-term tests with E. gracilis can be used as sensitive indicator for the toxicity assessment of laundry detergents in aquatic environments. Keywords Euglena gracilis Ariel detergent Chlorophyll Motility Photosynthesis Toxicity
A. Azizullah (&) P. Richter Cell Biology Division, Department of Biology, Friedrich-Alexander University, Staudtstr. 5, 91058 Erlangen, Germany e-mail:
[email protected] A. Azizullah Department of Botany, Kohat University of Science and Technology (KUST), Kohat, Pakistan M. Jamil Department of Soil and Environmental Sciences, Faculty of Agriculture, Gomal University, DI Khan, Pakistan D.-P. Ha¨der Neue Str. 9, 91096 Mo¨hrendorf, Germany
Introduction Laundry and cleaning chemicals are commonly used substances in everyday life and are used in large quantities. For example, in Western Europe the annual consumption of detergent and softener products are 4,250 and 1,190 ktons, respectively (Pettersson et al. 2000). After usage, large quantities of detergents and their components reach aquatic and terrestrial environments (Liwarska-Bizukojc et al. 2005). The concentrations of surfactants in natural water vary from area to area. For example, Ghoochani et al. (2011) monitored detergent concentrations in different water channels in Iran and reported that concentrations of detergents in three channels, namely Firuz Abad, Sorkhe Hesar and Kan channels, ranged from 2.957 to 3.78, 0.875 to 1.986 and 0.1456 to 0.244 mg l-1, respectively. In Kolkata, India, the concentration of synthetic detergents in various water sources ranged from 0.084 to 0.425 mg l-1 (Ghose et al. 2009). Minareci et al. (2009) found that the concentration of anionic detergents in Gediz River, Turkey was in the range of 0.084–5.592 mg l-1. They also summarized concentrations of detergents in various rivers in different countries which reveal a high variation (for details see Minareci et al. (2009)). The synthetic detergents reaching the aquatic environment are of special concern as they can have potential adverse effects on aquatic organisms (Lal et al. 1983). The toxicity of the major components of commercial detergents (surfactants) to various aquatic organisms has been reported fairly well (Aizdaicher and Markina 2006; Chawla et al. 1987; Lal et al. 1983; Sanchez-Fortun et al. 2008; Warne and Schifko 1999; Yamane et al. 1984), but the effects of a detergent as a whole have rarely been considered (Markina and Aizdaicher 2007). Surfactants have been reported to adversely affect various groups of algae. For
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example, Pavlic et al. (2005) observed that commercially available different surfactants (anionic, amphoteric and non-ionic) inhibited the growth rate in the green algae Scenedesmus subspicatus and Pseudokirchneriella subcapitata as well as in the marine diatoms Skeletonema costatum and Phaeodactylum tricornutum. Similarly, sodium dodecyl sulfate was found to affect cell growth and other physiological activities in algal species, Dunaliella salina and Plagioselmis prolonga (Aizdaicher and Markina 2006; Markina 2010; Markina and Aizdaicher 2005). Some other studies also revealed that synthetic surfactants adversely affect different processes in algae (Chawla et al. 1987; Riess and Grimme 1993; Sanchez-Fortun et al. 2008; Yamane et al. 1984). Evaluating the effects of only the surfactants does not reflect the actual and net influence of the detergent as a whole on the aquatic environment, because most of the detergents used in every day life are complex mixtures of various compounds which can interact antagonistically, additively, and synergistically towards toxicity in the aquatic environment (Warne and Schifko 1999). Therefore, it is needed to evaluate the toxicity of the detergent mixture as a whole (Markina and Aizdaicher 2007). Because of their trophic level, ubiquitous occurrence, and high sensitivity to environmental pollutants algae are good indicators of environmental changes and the health of aquatic ecosystems (Danilov and Ekelund 2000). Euglena gracilis has been proven to be a reliable organism for the toxicity assessment of toxic substances like heavy metals and other biologically active compounds (Ahmed and Ha¨der 2010; Gajdosova and Reichrtova 1996; Tahedl and Ha¨der 1999). Motility, cell shape and orientation parameters in E. gracilis have been used as sensitive indicators of the adverse effects of environmental stressors like organic compounds, heavy metals and sewage water (Ahmed and Ha¨der 2010; Tahedl and Ha¨der 1999). Cell growth, photosynthesis and photosynthetic pigments in Euglena have also been reported as sensitive biomarkers for evaluating the adverse effects of different substances (Azizullah et al. 2012; Gajdosova and Reichrtova 1996; Gonza´les-Moreno et al. 1997). In a previous study it has been determined that Ariel detergent affected motility, gravitaxis, velocity, cell shape, photosynthesis and light-harvesting pigments in E. gracilis when cells were exposed to the detergent for a short time (Azizullah et al. 2011). It has been reported that long-term (usually 7 days) exposure to toxic substances causes more adverse effects as compared to short-term exposure (Danilov and Ekelund 1999). To improve the initial observations and to include long-term effects and therefore chronic toxicity, cultures of E. gracilis were grown in media containing different concentrations of the detergent and the effects on various parameters were investigated after 7 days of growth.
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Materials and methods Organism and growth conditions The experiments were performed using axenic cultures of E. gracilis KLEBS; strain Z, obtained from the algal culture collection at the University of Go¨ttingen, Germany (Schlo¨sser 1994). The cultures were grown in a mineral medium (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 °C. All cultures were grown in 100-ml flasks with 50 ml of the culture media with an initial cell density of &8 9 104 cells per ml. Cultures were grown in static condition without orbital shaking. Cells were fixed with 70 % ethanol and counted with the help of a Thomas chamber under the microscope. Detergent used The commonly used laundry detergent Ariel (Procter & Gamble GmbH, Germany), available from the local market was used in this study. This detergent is a complex mixture of various compounds including anionic surfactants, zeolites (15–30 %), cationic and nonionic surfactants, phosphonates, polycarboxylates, soap (\5 %) and other ingredients like enzymes, perfumes linalool, butylphenyl methylpropional as mentioned on the packing. The code present on the packing (98996289) can be used to obtain complete information on the ingredients of the detergent used (www.info-pg.com). The cultures of E. gracilis were grown in media at eight different concentrations of the detergent, i.e. 0.1, 0.5, 1, 5, 10, 25, 50 or 100 mg l-1. The control cultures were grown without adding any detergent. Motility and orientation analysis The motility and orientation parameters of E. gracilis cells were measured by using the automatic biotest, ECOTOX (Tahedl and Ha¨der 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 r value. The motility parameter gives the percentage of cells moving at a speed equal to or faster than the minimum velocity set in the program. The parameter velocity gives the speed (swimming velocity) of the cells in lm s-1. The cell compactness (form factor) describes the shape of the cell and has the lowest value of 1 when the cell has an absolutely round shape and increases as the cell increases in length. The parameter upward gives the percentage of cells which are moving towards the upper part of the cuvette (±90° around the vertical direction). The r value is
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at 4 °C for 1 h to extract the pigments. The mixtures were then centrifuged at 6,0009g for 10 min at 4 °C to pellet the cells debris and the extract was scanned from 400 to 750 nm using a spectrophotometer (UV-2550, Shimadzu). Chlorophyll a, b and total carotenoids were determined according to Lichtenthaler and Wellburn (1983).
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). For hardware and more details about ECOTOX see Tahedl and Ha¨der (1999). The filling time of the cuvette was 100 s and the rinsing time was 45 s. Time of tracking of the cells was 3 min. Minimum area for objects to be included in vector analysis was set to 400 lm2 and maximum at 2000 lm2. Minimum speed at which the cells were considered motile was set at 15 lm s-1. In order to avoid the effect of light, the cells of E. gracilis were incubated in darkness for 30 min before making measurements.
Data analysis All tests were performed in three independent replicates. Data were analyzed by Microsoft Excel to calculate the means and SD. The significance of differences among the treatments was calculated using one-way ANOVA. The difference was considered significant if the p value was smaller than 0.05 (p \ 0.05).
Photosynthesis measurement A portable Pulse Amplitude Modulated (PAM) fluorometer (PAM 2000, Walz, Germany) was used for measuring the photosynthetic parameters of E. gracilis cultures. The single saturating pulse method was used for measuring the quantum yield of photosystem II (Fv/Fm). To observe the effects on photosynthetic efficiency with increasing irradiances, the light curve setting was used which involved determination of fluorescence parameters at different intensities of actinic light (0, 86, 155, 236, 327, 466, 658, 966, 1461, 2177 and 3199 lmol m-2 s-1).
Results The results obtained show that cell growth, motility, swimming velocity, photosynthesis and light-harvesting pigments in E. gracilis were adversely affected by the detergent (Figs. 1, 2, 3; Tables 1, 2). The cell density was the most sensitive parameter, and a significant decrease in cell density was shown above 0.5 mg l-1 of the detergent (Fig. 1). The detergent caused a significant decrease in cell motility at concentrations exceeding 1 mg l-1 (Table 1). At the highest concentration (100 mg l-1) only 42 % cells were motile as compared to 93 % in the control. The swimming velocity of the cells was affected in the same pattern as motility, i.e. a significant decrease in cell speed was observed above 1 mg l-1 (Table 1). The cell compactness was not significantly affected by the detergent. A slight positive effect was
Pigments extraction and determination Chlorophyll was extracted in 80 % acetone (Sumida et al. 2007). Aliquots of cultures were centrifuged at a speed of 6,0009g for 10 min at 4 °C. An appropriate volume of 80 % acetone was added to the precipitated cells and kept
900000
a
a
100
ab
700000
b
600000
500000
c
Decrease in cell density (%)
800000
Cell density ml-1
Fig. 1 Density of E. gracilis cells per ml of a culture after 7 days of growth at different concentrations of Ariel. Each bar represents the mean of three replicates and the error bars represent SD. Values marked with same letters are not significantly different from each other (p \ 0.05). In the inset, dose–response curve of cell density is shown
80
EC50 = 2.42 mg l-1
60 40 20 0 -20 0.1
400000
1
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Ariel (mg L-1)
d
300000
d
d
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25
d
200000 100000 0
0.1
0.5
1
5
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-1
Ariel (mg l )
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200
0.4
150
* * * *
100
Yield
rETR
0.3
0.2
50
*
0.1
* * ** 0 0
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4000
-2 -1
* 0 0
Irradiance (µmol m s )
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1000
1500
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Irradiance (µmol m -2 s-1)
Fig. 2 Electron transport rate of E. gracilis cultures after 7 days of growth at different concentrations of Ariel. Concentrations (mg l-1) used were control (filled diamond), 0.1 (filled square), 0.5 (filled triangle), 1 (solid line with cross), 5 (solid line with asterisk), 10 (open circle), 25 (vertical line), 50 (horizontal line), 100 (dashed line). Asterisks indicate significant differences as compared to the control (p \ 0.05)
Fig. 3 Quantum yield of photosystem II with increasing intensity of actinic light after 7 days of growth of E. gracilis cultures at different concentrations of Ariel. Concentrations (mg l-1) used were control (filled diamond), 0.1 (filled square), 0.5 (filled triangle), 1 (solid line with cross), 5 (solid line with asterisk), 10 (open circle), 25 (vertical line), 50 (horizontal line), 100 (dashed line). Asterisks indicate significant differences as compared to the control (p \ 0.05)
Table 1 Motility, swimming velocity, gravitactic orientation (upward swimming of cells and r value) and cell compactness (shape) of E. gracilis after 7 days of growth at different concentrations of Ariel Ariel (mg l-1)
Motility (%)
Velocity (lm s-1)
Upward swimming cells (%)
r value
Cell compactness
0
93.1 ± 2.3a
82.6 ± 2.7a
67.1 ± 6.4a
0.35a
6.3 ± 0.03a
a
a
a
a
6.4 ± 0.39a
0.1
94.0 ± 0.4
0.5 1
92.4 ± 1.1a 92.5 ± 0.8a
5
75.3 ± 7.0ab
10 25
83.1 ± 2.2
c
41.7 ± 21.9
c
44.9 ± 10.4b c
50
41.8 ± 8.2
100
42.6 ± 19.7c
66.7 ± 6.2
0.35
81.1 ± 3.4ab 83.7 ± 1.8a
65.7 ± 2.3a 70.6 ± 7.3a
0.34a 0.42a
6.6 ± 0.24a 7.3 ± 0.44a
74.6 ± 2.2bc
74.0 ± 0.7a
0.44a
5.9 ± 0.62a
71.1 ± 2.9
a
0.38
a
5.8 ± 1.05a
72.6 ± 9.0
a
0.44
a
6.6 ± 1.02a
75.3 ± 1.6
a
0.47
a
6.4 ± 0.34a
0.50a
6.5 ± 0.23a
70.8 ± 4.4
cd
67.9 ± 9.2
cd
64.9 ± 1.2
d
62.8 ± 7.9d
76.9 ± 9.5a
Values given are means ± SD of three replicates. Values with similar letters indicate no significant differences from each others (p \ 0.05, oneway ANOVA)
observed on gravitaxis as evident from upward swimming cells and r value (Table 1). For example, at 100 mg l-1 of detergent &77 % cells were swimming upward as compared to 67 % in the control. Similarly, the mean r value of the cultures grown at 100 mg l-1 was 0.50 as compared to 0.35 in the control culture. It was observed that chlorophyll a and total carotenoids in E. gracilis were sensitive to detergent stress and decreased significantly at a concentration above 1 mg l-1 (Table 2). As compared to chlorophyll a and carotenoids,
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chlorophyll b was more resistant to detergent stress, and only a slight (non-significant) decrease was shown in this pigment (Table 2). Due to this smaller effect on chlorophyll b as compared to chlorophyll a, a decrease in the chlorophyll a/b ratio was observed with increasing concentration of the detergent (Table 2). The quantum yield of photosystem II (Fv/Fm) was adversely affected by the detergent at a concentration of 5 mg l-1 and above (Table 2). Similar effects were observed on rETR (relative electron transport rate) and quantum yield with increasing
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Table 2 Concentrations of chlorophyll a, b and total carotenoids, chlorophyll a/b ratio and quantum yield of photosystem II (Fv/Fm) in E. gracilis cultures after 7 days of growth at different concentrations of Ariel Ariel (mg l-1)
Chlorophyll a (lg ml-1)
Chlorophyll b (lg ml-1)
Total carotenoids (lg ml-1)
Chlorophyll a/b ratio
Fv/Fm
0
5.9 ± 0.4a
0.91 ± 0.07a
2.1 ± 0.1a
6.5
0.38 ± 0.01a
a
a
a
6.6
0.39 ± 0.01a
a
0.1
5.8 ± 0.1
0.87 ± 0.04
5.9 ± 0.5 5.6 ± 0.4ab
0.92 ± 0.08 0.85 ± 0.08a
2.0 ± 0.2 1.9 ± 0.3a
6.4 6.6
0.38 ± 0.02a 0.39 ± 0.02ab
5
4.6 ± 0.3bc
0.79 ± 0.06a
1.7 ± 0.1ab
25
c
3.5 ± 0.7
c
3.9 ± 0.2
c
a
2.1 ± 0.2
0.5 1 10
a
0.78 ± 0.07
a
0.79 ± 0.05
a a
50
4.3 ± 0.2
0.88 ± 0.03
100
3.8 ± 0.3c
0.84 ± 0.04a
5.8
0.29 ± 0.04bc
1.2 ± 0.2
c
4.5
0.16 ± 0.02c
1.4 ± 0.1
bc
4.9
0.31 ± 0.03c
1.3 ± 0.0
bc
4.9
0.27 ± 0.06c
4.6
0.32 ± 0.01c
1.2 ± 0.1c
Values given are means ± SD of three replicates. Values with similar letters indicate no significant differences from each others (p \ 0.05, oneway ANOVA)
light intensity, i.e. a significant decrease was shown at a concentration of 5 mg l-1 and above (Figs. 2, 3). It is evident from the overall data of this study (Tables 1, 2; Figs. 1, 2, 3) that the Ariel detergent caused a maximum inhibition of various parameters in E. gracilis at a concentration of 10 mg l-1 and with further increase in concentration there was either no increase in the inhibitory effect or it was even smaller as compared to 10 mg l-1 (although significant as compared to the control). This effect was more apparent in the case of photosynthetic parameters (Fv/Fm and ETR).
Discussion Long-term exposure to Ariel detergent severely affected cell growth, motility parameters, light-harvesting pigments and photosynthetic efficiency in E. gracilis. The surfactants present in the detergent have been shown to adversely affect living cells in different ways. They may damage cell membranes (Chawla et al. 1987; Mikolajczyk and Diehn 1978), bind to various proteins in the cell and affect physiological and biochemical processes in the cell (Markina and Aizdaicher 2007) as well as interact with lipids and consequently change their fatty acid compositions (Nyberg and Koskimies-Soininen 1984). The observed decrease in cell growth of E. gracilis is probably the collective effect of several adverse effects of the detergent on different metabolic processes in the cell. The detergent caused a significant decrease in motility parameters like percentage of motile cells and swimming speed of the cells. It has been reported by Mikolajczyk and Diehn (1978) that detergents damage the plasmalemma of the flagellum and disorganize the flagellar microtubules in E. gracilis and hence impair cell motility. In addition,
detergents can also impair motility due to depletion of ATP resources in the cell, blockage of ATP synthesis as well as changes in ionic homeostasis of the cell (Aizdaicher and Markina 2006). A slight positive effect was observed on gravitaxis as evident from upward swimming cells and r value. In the control cultures relatively low numbers (67 %) of cells were moving upward. A possible reason for this is that cultures were relatively young (7 days) and young cultures do not show a precise negative gravitaxis (Ha¨der et al. 1998). In older cultures of E. gracilis usually most of the cells swim upward, i.e. cells show a precise negative gravitaxis. A partial explanation for the slight positive effect on gravitaxis can be that due to inhibition of cell growth by the detergent the cultures became static sooner than the control and hence showed a relatively precise gravitactic orientation. For a good vitality and balanced energetic status of phototrophic organisms an optimal quantity of the lightharvesting pigments in their thylakoid membrane is necessary (Fodorpataki et al. 2001; Kolber and Falkowski 1993). In the present study the detergent caused a decrease in chlorophyll a and total carotenoids in E. gracilis cultures. Detergents can affect the composition of the lightharvesting pigments by inhibition of their biosynthesis through disturbance of protein synthesis (Chawla et al. 1987) as well as by degradation of the pre-existing pigment molecules as they damage pigment-protein complexes and enhance degradation of pigments through photo-oxidation (Dekker et al. 2002; Ruban et al. 1999). Chlorophyll b was found to be more resistant than chlorophyll a and carotenoids to detergent stress. These results are in agreement with those reported by Ruban et al. (1999) who showed that chlorophyll b is held by the proteins more tightly than chlorophyll a and carotenoids. This stronger binding of chlorophyll b to proteins can be a possible reason for
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higher tolerance of chlorophyll b than other pigments to detergents. A decrease in the chlorophyll a/b ratio was observed with increasing concentration of the detergent. Such a decrease in the chlorophyll a/b ratio has been observed in E. gracilis upon exposure to various stresses like increased salinity and elevated concentrations of the fertilizers DAP (diammonium phosphate) and urea (Azizullah et al. 2012; Gonza´les-Moreno et al. 1997). These observations suggest that the decrease in chlorophyll a/b ratio in E. gracilis might be a common phenomenon under stress conditions. The results obtained for fluorescence parameters show that photosynthesis in E. gracilis was impaired by the detergent (Figs. 2, 3; Table 2). The adverse effects on photosynthesis can be the effect of the surfactants present in the detergent as reported in different species of algae (Chattopadhyay and Konar 1985; Sanchez-Fortun et al. 2008). However, the mechanism of inhibition of photosynthesis by detergent is not fully known and may follow a complex mode of action. Sukenik et al. (1989) reported that the detergent Triton X-100 disrupts the energy transfer from chlorophyll b to chlorophyll a in the light-harvesting complex II of Dunaliella tertiolecta without pigment extraction. Another study revealed that the detergent Triton X-100 caused disruption of energy transfer from the accessory chlorophyll a to the active pheophytin a due to proteinpigment structural perturbations (Tang et al. 1991). Chawla et al. (1987) showed that the surfactant alkylbenzene sulphonate adversely affected photolysis in Scenedesmus quadricauda which led to inhibition of photosynthesis. The disturbance of photolysis by surfactants can be the reason for a decrease in photosynthetically produced oxygen as reported in Attheya ussurensis and S. quadricauda (Chawla et al. 1987; Markina and Aizdaicher 2007). Since photosynthesis (light reaction) is an important process for producing energycarrying molecules (NADPH and ATP), the inhibition of photosynthesis by detergents can possibly cause ATP blockage which can be a possible reason for the high toxicity of detergents. The maximum inhibitory effect on various parameters of E. gracilis was shown at a concentration of 10 mg l-1 of the detergent, and no increase in the inhibitory effect was observed with further increase in concentration of the detergent. These observations were confirmed by repeating the experiments. However, the exact reason for these unexpected effects is not known and needs further investigations. The World Health Organization set the maximum allowable concentration of anionic detergents in drinking water to be 0.2 mg l-1 (Minareci et al. 2009). Some countries also defined maximum allowable concentrations of surfactants in surface water. For example, in Poland the maximum permissible concentrations for anionic and
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non-ionic surfactants in surface water has been set to 1 and 2 mg l-1, respectively (Pastewski and Medrzycka 2003). Similarly, in Croatia the maximum allowed concentration for both anionic and non-anionic surfactants in surface water is 1 mg l-1 while for cationic surfactants it is 0.2 mg l-1 (Ivankovi and Hrenovi 2010). The present results indicate that the detergent at a concentration of 1 mg l-1 or above adversely affects various parameters in E. gracilis. A survey of literature reveals that different detergents and their products have been reported in a wide concentration range reaching a maximum of above 5 mg l-1 in surface water (Ghoochani et al. 2011; Minareci et al. 2009). This shows that detergents in the range of the reported concentrations in such areas can be a possible threat to aquatic organisms and need to be properly monitored. In addition, the maximum allowed concentration of detergent in water may be revised and set below 1 mg l-1. To the best of our knowledge this is the first long-term study of the toxicity of Ariel detergent to freshwater algae. The only reports found are those on its toxicity to marine microalgae published by Markina and Aizdaicher (2007, 2010). Markina and Aizdaicher (2007) observed that after 14 days exposure Ariel detergent at a concentration of 1 mg l-1 had weak inhibitory effects on the growth rate of the marine microalga A. ussurensis but at a concentration of 10 mg l-1 it strongly inhibited cell growth and the cell number was reduced to 0.7 % of the control. The authors also found that Ariel caused damage to the light-harvesting pigments in A. ussurensis. The same was also found on P. prolonga and D. salina by Markina and Aizdaycher (2010). By comparing the two species they observed that P. prolonga was more sensitive to the influence of the detergent as compared to D. salina. The present results show that the sensitivity of E. gracilis to 7-days exposure to the detergent is of the same order of magnitude as for A. ussurensis, P. prolonga and D. salina and can be used as an indicator for the detergent pollution in freshwater aquatic environments. It can be concluded from these results that Ariel detergent adversely affects various physiological and biochemical processes in E. gracilis. Since this detergent is a complex mixture and its various constituents may interact antagonistically, synergistically or additively toward toxicity (Warne and Schifko 1999), the observed effect should be considered as the net effect of the detergent as a whole. However, the results with the single algal species can not be generalized to other algae in aquatic environments, since previous studies concluded that the toxicity of the detergents is species specific and the most toxic surfactant or detergent for one alga is not always the most toxic for other algae (Lewis 1990; Yamane et al. 1984).
Chronic toxicity of a laundry detergent Acknowledgments The authors acknowledge Kohat University of Science and Technology (KUST) Kohat, Khyber Pakhtunkhwa, Pakistan for granting a scholarship to Azizullah. Conflict of interest
The authors have no conflict of interest.
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