Chemosphere 120 (2015) 637–644

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Effect of malachite green toxicity on non target soil organisms R. Gopinathan a,1,3, J. Kanhere a,2,3, J. Banerjee b,⇑ a b

Biology Department, Indian Institute of Science Education and Research (IISER), Pune, Dr. Homi Bhabha Road, Pune 411 008, India Biology Department, 900 NCL Innovation Park, Indian Institute of Science Education and Research (IISER), Pune, Dr. Homi Bhabha Road, Pune 411 008, India

h i g h l i g h t s  Malachite green (MG) toxicity was tested on beneficial soil bacteria, fungi, earthworms and on seed germination of crop plants.  Genotoxicity, cytotoxicity and scanning electron microscopy assays concluded malachite green induced toxicity in soil microorganisms. 2 and 1.45 mg/kg respectively with evident morphological alterations.  Seed germination of Mung bean, Wheat and Mustard is unaffected in presence of MG upto 100 ppm.  MG negatively effects growth, physiology of tested soil borne micro organisms and earthworms raising concerns about its environmental hazard.

 Filter paper and artificial soil test on earthworms demonstrated a LC 50 of 2.6 mg/cm

a r t i c l e

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Article history: Received 24 February 2014 Received in revised form 8 September 2014 Accepted 12 September 2014

Handling Editor: Tamara S. Galloway Keywords: Malachite green Toxicity Microorganisms Earthworms Seed germination

a b s t r a c t Although malachite green (MG), is banned in Europe and US for its carcinogenic and teratogenic effect, the dye being cheap, is persistently used in various countries for fish farming, silk, dye, leather and textile industries. Current research, however, fails to elucidate adequate knowledge concerning the effects of MG in our ecosystem. In the present investigation, for the first time, an attempt has been made to study the effects of MG on soil biota by testing Bacillus subtilis, Azotobacter chroococcum, Saccharomyces cerevisiae, Penicillium roqueforti, Eisenia fetida and seeds of three crop plants of different families. Various tests were conducted for determining cytotoxicity, genotoxicity, acute toxicity, morphological and germination effect. Our data confirmed MG toxicity on fungi and bacteria (gram positive and gram negative organisms) showing elevated level of ROS. Genotoxicity caused in the microorganisms was detected by DNA polymorphism and fragmentation. Also, scanning electron microscopy data suggests that the inhibitory effect of MG to these beneficial microbes in the ecosystem might be due to pore formation in the cell and its eventual disruption. Filter paper and artificial soil test conducted on earthworms demonstrated a LC 50 of 2.6 mg cm2 and 1.45 mg kg1 respectively with severe morphological damage. However, seed germination of Mung bean, Wheat and Mustard was found to be unaffected in presence of MG up to 100 mg L1 concentration. Thus, understanding MG toxicity in non target soil organisms and emphasis on its toxicological effects would potentially explicate its role as an environmental contaminant. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Malachite green (MG), an N-methylated diaminotriphenylmethane basic dye is extensively used in aquaculture as it is highly efficient against fungal and protozoan infections (Sudova et al., 2007). It is also extensively used in food and textile industries. Due to its pervasive use and high solubility in water, MG can be ⇑ Corresponding author. Tel.: +91 (20) 25908001 E-mail address: [email protected] (J. Banerjee). Present address: 12 Atul Vihar, Lane 9, Dahanukar Colony, Kothrud, Pune 38, India. 2 Present address: Flat 6, Chinar Apartment, 34 Shilavihar Colony, Erandwane, Pune 38, India. 3 Equal contribution. 1

http://dx.doi.org/10.1016/j.chemosphere.2014.09.043 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

released into the environment by various sources. Studies have reported its carcinogenic (Lee et al., 2006), teratogenic (Culp et al., 1999) and reproductive abnormalities (Cha et al., 2001) spanning its effect from various fish to mammals (Srivastava et al., 2004). Moreover, it is also demonstrated that MG is highly persistent in the environment (Xie et al., 2012). Due to its health hazard concerns, MG is banned in Europe, United States and Canada (Mitrowska et al., 2007). Being extremely economic and highly potent against infections, it is still used steadily in various countries. In a recent report from Germany, malachite green was detected in suspended particulate matter from German rivers with concentrations ranging between LOD (1 ng g1 d.w.) and 543 ng g1 (Ricking et al., 2013). As MG can be resistant to natural biological degradation (El Qada et al., 2008), its residues might cause environmental pollution

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(Jin et al., 2013). Furthermore, MG is speculated to be toxic to the food web and can disturb aquatic life (Chen et al., 2014; Kanhere et al., 2014). Contrary to several studies on fish, literature on effect of MG on non-target and beneficial soil organisms is extremely scanty (Sudova et al., 2007) in spite of all its hazardous information. Soil is one of the most important resources for food production. Its intricate structure and function depends on its physical properties as well as its biological components. This study investigates the ecotoxicity of MG on different non target soil organisms representing different trophic levels using a battery of test assays. Microorganisms in soil are essential players in carbon, nitrogen and phosphorous cycling including several elements indispensable to life. Organisms considered in this study were Bacillus subtilis (important as bio-fertilizers and bio-pesticides), Azotobacter chroococcum (nitrogen fixing bacteria), Saccharomyces cerevisiae (nutrient source for predatory soil yeast species and nematodes), Penicillium roqueforti (saprophytic fungi, producing various secondary metabolites and mycotoxins), Eisenia fetida (decomposers, maintaining soil structure and nutrient cycling) and seeds of three crop plants (plants, primary photosynthetic producers) from different families. Accumulation of chemical contaminants in the soil causes various problems such as alteration in physical and chemical properties, decrease in soil fertility, adverse effect on microbial fauna and flora and on soil invertebrates. Therefore, it is necessary to investigate the impact of toxic pollutants reaching soil ecosystem through various routes affecting soil biodiversity both qualitatively and quantitatively. Research on the potential environmental risks posed by unnecessary MG use is important, so as to make evidence-based policy decisions on the future management of its liberal use and to implement measures to protect environmental assets. Our study therefore, could potentially raise awareness of the long ignored ecotoxic effects of MG on soil quality and its environment. 2. Materials and methods 2.1. Chemicals and glass wares All the chemicals were obtained from Sigma–Aldrich, USA, unless otherwise mentioned. Malachite Green (CAT no-M6880), 2’7’-DCF diacetate (CAT no-35845), 4’,6-diamidino-2-phenylindole (CAT no-D9542) were used in the study. Growth media were acquired from HiMedia, India. Glass wares from BOROSIL were used throughout the study. 2.2. Strains used and maintenance 2.2.1. Microorganisms (bacteria and fungi) All micro-organisms (except for S. cerevisiae) were procured from National Collection of Industrial Microorganisms (NCIM), Pune, India. B. subtilis (NCIM no. 2063) was cultured and maintained in Nutrient Agar (catalogue no. M001) at 37 °C. A. chroococcum (NCIM 2452) was cultured and maintained on modified nitrogen-free Burks’ media (catalogue no. M707) amended with 0.1% NH4Cl at 28 °C. S. cerevisiae (W303d-kindly provided by Dr. M. Lahiri, IISER, Pune) was cultured and maintained on modified Yeast Peptic digest Dextrose (YPD) Agar (catalogue no. M670) supplemented with 0.1% Adenine at 30 °C. P. roqueforti (NCIM 710) strain was cultured and maintained on Potato Dextrose Agar (PDA) (catalogue no. M096) at 28 °C. 2.2.2. Invertebrates (earthworm) E. fetida were obtained from National Toxicity Centre (NTC) Pune, India. They were maintained according to Organization for Economic Co-operation and Development (OECD) guidelines (OECD guidelines for Testing of Chemicals, 207, 1984).

2.2.3. Crop plants (seeds) Seeds of Mung beans (Vigna radiata – Fabaceae), Mustard seeds (Brassica nigra – Brassicaceae) and Wheat grains (Triticum aestivum – Poaceae) were obtained from local market for germination assay. 2.3. Growth inhibition assay The growth of each organism was analyzed by monitoring the cell division in presence of varying concentration of the dye. Samples of cultures were incubated at optimum temperature and periodically measured for cell density till stationary phase was reached. The OD (Optical Density) value was measured by Eppendorf Biophotometer in UVette having path length of 10 mm. Controls were kept for each concentration tested and all tests were done in triplicates. 2.3.1. Microbial growth inhibition test Initial cell density used were of 0.2 OD600 for B. subtilis, 0.3 for A. chroococcum, 0.25 for S. cerevisiae. P. roqueforti, growth pattern in presence of MG was determined by using a 96-well flat bottom microtitre plate (Meletiadis et al., 2001) with initial spore count of approximately 15  104 spores ml1. Readings were taken after 24 h with absorbance at 405 nm on Scientific VarioSkan Flash Multimode reader. As most of the MG toxicity studies conducted earlier were from 0.2 to 2 mg L1 (Arnold et al., 2009; Kanhere et al., 2014), we have chosen a similar concentration gradient (0–5 uM) for various organisms in the present study. 2.3.2. Toxicity test in earthworm For E. fetida, filter contact test and artificial soil test were carried out according to OECD guidelines (OECD, 1984) to assess MG toxicity. Filter contact test till 48 h was conducted to determine concentration range in which 0–100% mortality of the earthworms was obtained. Ten replicates were used for each concentration. Artificial soil test for 14 d was conducted to assess acute toxicity. A range of concentrations, 0, 0.1, 1.0, 10, 100 and 1000 mg kg1 dry soil were used to determine a concentration range that resulted in 0–100% mortality. To obtain LC50, test concentrations of 1.2, 1.3, 1.4, 1.5 and 1.75 mg kg1 and a control were used. Determination of lethal values LC50 of MG was determined by probit analysis. For this purpose value of each concentration was plotted along X-axis and percent of mortality along Y-axis and the eye fitted curve was drawn. LC50 values were calculated from the obtained curve (Matsumura, 1975). 2.3.3. Seed germination assay For germination assay, fifty seeds of each plant were tested on filter paper soaked with 10–100 mg L1 of MG and water as control. Percent germination was noted after 48 h and root length in centimeter was measured after 96 h. All assays were performed in triplicates. 2.4. Cytotoxicity assays 2.4.1. Comparison of protein profiles Total protein was extracted from B. subtilis, S. cerevisiae and P. roqueforti using TCA and acetone method (Damerval et al., 1986) and was further precipitated (Link and LaBaer, 2011). Isolated protein concentration was estimated by Bradford assay (Bradford, 1976). Whole cell protein was extracted from A. chroococcum using SDS method (Bhaduri and Demchick, 1983). Extracted proteins were re-suspended in Laemmli sample buffer (Cold Spring Harb Protoc, 2006) and loaded on a SDS-PAGE gel (Laemmli, 1970) for analysis. All protein isolations were carried out when the cells were in log phase of their growth.

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2.4.2. Detection of reactive oxygen species (ROS) assay 20 70 dichlorodihydrofluorescein diacetate (20 70 DCF diacetate), an oxidant sensitive probe, was employed to detect generation and accumulation of intracellular levels of ROS in cells exposed to MG (Wang and Joseph, 1999). Overnight grown cultures were treated with MG for 30 min and retreated with 10 lM 20 70 -DCF diacetate for additional 30 min. Qualitative analysis was carried out using Zeiss Imager.Z1. with Filter Set 10 (Cy2/GFP). 2.4.3. Scanning electron microscopy imaging Both MG treated and untreated microorganisms were fixed in log phase of their growth (Yamada and Sakaguchi, 1982) and their cellular morphologies were studied by scanning electron microscopy (Zeiss Ultra Plus FESEM). 2.5. Genotoxicity assay 2.5.1. DNA isolation and random amplification of polymorphic DNA (RAPD) Microbial DNA was extracted using Dneasy Blood and Tissue kit (Qiagen, cat. no. 69504) for B. subtilis, S. cerevisiae, A. chroococcum while for P. roqueforti, DNA was isolated as described earlier (AlSamarrai and Schmid, 2000). All DNA isolations were carried out when the cells were in log phase of their growth. Isolated DNA was run on 2% TAE agarose gel, and stained by ethidium bromide and assessed for possible DNA damage. For RAPD data, PCR (Polymerase Chain reaction) amplification was tested with eleven (10base pairs) random primers with genomic DNA as template. The nucleotide sequences of the primers used were (5 ? 3): OPN 1CTC ACG TTG G; OPO 08-CCT CCA GTG T; OPA 08-GTG ACG TAG G; OPA 09-GGG TAA CGC C; OPA 10-GTG ATC GCA G; OPA 18CCT CCA GTG T; OPC 05-GAT GAC CGC C; OPC 07-GTC CCG ACG A; OPC 10-TGT CTG GGT G; OPC 14-TGC GTG CTT G. RAPD amplifications were performed in a 10 lL reaction mixture containing 50 ng DNA, 1XPCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5 lM primer, and 2.5U DNA Taq polymerase. Two programs were used for PCR amplification. The first program was used for all microorganisms except B. subtilis. It consist of an initial denaturing step at 94 °C for 5 min, followed by 44 cycles of 94 °C for 1 min (denaturing), 40 °C for 1 min (for annealing) followed by 72 °C for 2 min (for extension) with an additional extension period of 15 min at 72 °C. The 2nd program used for B. subtilis, consists of an initial denaturing step of 94 °C for 4 min followed by 40 cycles of 94 °C for 45 s (denaturing), 35 °C for 45 s (annealing) and 72 °C for 1 min 30 s (extension) followed by additional extension period of 72 °C for 15 s. Amplified PCR products were detected by 2% TAE agarose gel, and stained by ethidium bromide. 2.5.2. DAPI staining DAPI (40 ,6-diamidino-2-phenylindole), is a fluorescent dye that selectively stains the DNA. Cellular nucleus of actively growing cells (log phase) was stained with DAPI stain having final concentration of 0.1 lg ml1 (Zachleder and Cepák, 1987). The cells were incubated for 30 min at their respective optimum temperature and visualized by Zeiss Imager.Z1 with Filter Set 49 (DAPI). 3. Results 3.1. Effect of MG on growth of bacteria, fungi, earthworm and germination of seeds Microbial samples were collected at different times throughout the growth cycle to test their sensitivity to MG. Physiological growth analysis showed that the toxicity of MG to soil microorganisms was concentration dependent. We found that B. subtilis

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(Fig. 1A(a)) and S. cerevisiae (Fig. 1A(c)) exhibited a dose dependent detrimental effect to MG as evident from prolonged lag phase of all the concentration tested. With B. subtillis, cellular growth was visible after 4 h of culture in presence of 0.5 lM MG, whereas with all other higher concentrations, an increase in OD was observed after 6 h of incubation compared to control. However, cellular growth was almost negligible in S. cerevisiae throughout the study except in control and 0.25 lM MG. Marked growth inhibition was noted in A. chroococcum with the two higher concentrations of MG (0.2 and 0.25 lM) in culture (Fig. 1A(b)). Whereas a linear growth pattern was noticed with P. roqueforti (Fig. 1A(d)), the OD values were low in comparison to the untreated samples. In general, except with the highest concentration tested, all the organisms could however survive and grow slow till the end of experiment. E. fetida when exposed to MG (in filter contact test) for 48 h, confirmed contact toxicities (Fig. 1B(a)) showing LC 50 of 2.6 mg cm2. In this test, morphological alterations like tail constrictions, abnormal swellings in clitellar region, dehydration and degeneration of the treated earthworms were observed (Fig. 1C). Coiling of the earthworms was also found frequently in MG treated samples. In the artificial soil test, earthworms revealed a LC 50 of 1.45 mg kg1 (Fig. 1B(b)) and developed multiple lesions, swelling of the clitella, fragmentation, etc., similar to paper test followed by death. Dehydration of the treated earthworms accompanied with their weight loss was also common. Hundred percent seed germination was documented in Mung bean, Mustard and Wheat when treated with MG concentration up to 100 mg L1. No significant detrimental effect on seed germination (Fig. 1D(a)) and their subsequent root length (Fig. 1D(b)) was recorded in this study. 3.2. Genotoxic effect of MG in bacteria and fungi Evident DNA alteration was depicted by RAPD analysis, despite a minor change in MG treated microbial genomic DNA mobility shift (Fig. 2A). Among all the primers tested, OPC-10 showed several changes in DNA banding pattern mostly in terms of loss or shift when untreated B. subtilis (Fig. 2B(a)) samples were compared to the treated ones. Marked changes in A. chroococcum DNA PCR with OPA-8 primer was also observed. Several new DNA bands were observed with the MG treated samples. In case of S. cerevisiae amplification, OPC-2 primer also resulted, amplification of new DNA bands with MG treated samples. Similarly, in P. roqueforti with OPO-8 primer, several new bands were amplified with 1.5 lM MG in comparison to control. However, with 2.0 lM MG concentration, almost all the bands disappeared (Fig. 2B(b)–(d) respectively). Genotoxicity was retested by DAPI staining of all the samples. Treated microbial cells displayed altered and severely segmented nuclear morphology (Fig. 2C). In contrast, untreated cellular nuclei appeared as tight compact single structures. 3.3. Cytotoxic effect of MG in bacteria and fungi Effect of MG treatment on total protein content of microbial cells were analyzed by SDS-PAGE (Fig. 3A). Changes in both qualitative (presence or absence of bands) and quantitative (band intensity) protein profile were noted in all the MG treated micro organisms. In B. subtilis (Fig. 3A(a)), A. chroococcum (Fig. 3A(a)), S. cerevisiae (Fig. 3A(b)), in the region of 25–10 kD, protein banding patterns clearly varied in comparison with their controls. Low molecular weight proteins were in general found to be affected with MG treatment. Also, changes in band intensity between 50 and 75 kD is noticed. However, in P. roqueforti, variation in band intensity was mostly evident for bigger sized proteins. Elevated ROS production (fluorescence emission) was detected in all concentrations of MG treated cells of B. subtilis, A. chroococcum,

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Fig. 1. Inhibition growth assays in presence of MG: (A) Growth dynamics of B. subtilis (a), A. chroococcum (b), S. cerevisiae (c), and P. roqueforti (d). Legend at the bottom represents range of MG concentrations studied. (B) Toxicity of MG assessed on E. fetida by filter contact test after 48 h (a) and artificial soil test after 14 d (b). (C) Morphological alterations observed in filter contact test for 8 mg cm2. Arrow indicates dehydration, swelling at the clitellar region, tail constriction and body curling. (D) Percent germination of seeds after 48 h of exposure to MG (a) and effect on root length after 72 h on germination in presence of MG (b).

S. cerevisiae and P. roqueforti compared to untreated cells (Fig. 3B). Apart from this, possible alterations in structural morphology were observed under scanning electron microscopy. SEM images revealed that in B. subtilis, there is noticeable decrease in the individual cell size (Fig. 3C(b) and (c)). On an average, untreated cells measured around 9 lm in length, while MG treated cells measured 3–4 lm only. Along with this, higher concentration of MG (0.1 lm) exhibited cell surface alterations (Fig. 3C(a)). Although, A. chroococcum showed no cell size reduction, multiple dents were visible in comparison to the untreated cells. Treated S. cerevisiae developed several irregular bud scars without preferential position at the polar ends. Surface alteration with random folds were found in MG treated yeast cells in contrast to smooth surface and single polar bud scar of untreated cells (Fig. 3C(c)). In our study, treated P. roqueforti showed irregular and distorted flattened surface along with multiple pores in contrast to untreated smooth surfaced cells (Fig. 3C(d)).

4. Discussions Despite a ban on MG usage by several countries in edible fish culture, the dye is persistently used in various other industries including ornamental fish culture all over the world (Sudova et al., 2007). MG being extremely soluble in water, poses a great concern to soil and aquatic organisms. There are several reports of detection of MG in areas where it was banned earlier (Ricking et al., 2013). Many of these investigations are pertaining to fishes only. Recent data from Kanhere et al. (2014) showed toxic effects of MG to the aquatic food chain. To our knowledge, there was no previous information of MG’s effect on beneficial soil organisms and its implication. Our findings on MG revealed that it can cause genotoxic and cytotoxic effects in different soil microorganisms. These toxic effects as observed in our study could be due to increased ROS pro-

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Fig. 2. Comparison of MG induced genotoxicity: (A) Effect of MG on genomic DNA of B. subtilis (a), A. chroococcum (b), S. cerevisiae (c), and P. roqueforti (d). (B) RAPD profiles with primer OPA-18. Arrows indicate differential banding pattern. (a) B. subtilis lane 1 – untreated, lane 2, 3–0.075 and 0.1 lM MG, lane 4 – I kb DNA ladder (b) A. chroococcum. Lane 1 – 1 kb DNA ladder, lane 2 – untreated, lane 3, 4–0.175 and 0.25 lM MG (c) S. cerevisiae. Lane 1 – 1 kb DNA ladder, lane 2 – untreated, lane 3, 4–0.5 and 1.0 lM MG (d), P. roqueforti. Lane 1 – I kb DNA ladder, lane 2 – untreated, lane 3, 4–1.5 and 2.0 lM MG (d). (C) DAPI staining of chromosomal DNA in untreated and treated cells of (a) B. subtilis, (b) A. chroococcum (c), S. cerevisiae, and (d) P. roqueforti scale bar represents 1 lm.

duction because of MG stress. Several detrimental environmental conditions as well as stress can lead to enhanced ROS activity, which is usually produced in cells as metabolic byproducts and can affect organism’s growth (Qian et al., 2009). In this study, MG mediated stress in cells was demonstrated by activation of the ROS specific probe 20 70 DCF diacetate (Fig. 3B). Our data suggests that microorganisms in contact with MG, generated higher ROS, indicating onset of stress and disturbance of intracellular redox homeostasis. This reduces growth of the microorganisms which ultimately might also lead to death of the samples (Trachootham et al., 2008). After a phase of initial retarded growth with MG treatment, we observed that these microbes can resist the damage to a certain extent and can resume growth. This could be due to up regulation of the gene expressions whose products function to either protect cells from the effects of ROS or repair any resulting damage (Tu et al., 2012). However, being reactive in nature, ROS is known to cause damage to DNA and proteins, etc. (Halliwell and Gutteridge, 2007). As protein synthesis is vital for

proper cell growth and proliferation (Watkins and Norbury, 2002), we analyzed the total protein lysate of the tested organisms through SDS-PAGE (Gibbins, 2004) to visualize preliminary changes of polypeptide patterns. Our SDS-PAGE analysis revealed elimination of several protein bands in MG treated samples compared to the control (Fig. 3A). The disappearance of proteins might indicate that there could be direct relationship between MG stress and protein regulation. These proteins might be responsible for overall integrity, protection against oxidative stress, and change in osmotic pressure (Bahramppour et al., 2013). Protein band intensity (decrease and increase) change as observed here, possibly suggests up and down regulation of some specific proteins, as and when needed by the microbes for their better survivability. Dyavaiah et al. (2011) earlier reported that protein profile change is closely related to DNA alterations. Our genotoxicity studies further confirmed this notion. Genomic DNA analysis of treated and untreated samples showed a negligible mobility shift (Fig. 2A). This might be because of considerable heterogeneity within a sin-

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Fig. 3. Comparison of cytotoxicity induced by MG: (A) Comparison of protein profiles (a) Lane 1 – untreated B. subtilis, lane 2, 3 – B. subtilis – with 0.075, 0.1 lM MG respectively. Lane 5 – untreated A. chroococcum, lane 6, 7 – A. chroococcum with 0.175, 0.25 lM MG. Lane 4 – biorad all blue protein ladder, (b) Lane 1 – untreated S. cerevisiae, Lane 2, 3 – S. cerevisiae with 0.5, 1.0 lM MG respectively. Lane 5 – untreated P. roqueforti, lane 6, 7 – P. roqueforti with 1.5, 2.0 lM MG. Lane 4 – biorad all blue protein ladder. (B) Generation of reactive oxygen species (ROS) in presence of MG in B. subtilis (a), A. chroococcum (b), S. cerevisiae (c), and P. roqueforti (d). (C) Scanning electron microscopy of B. subtilis (a), A. chroococcum (b), S. cerevisiae (c), and P. roqueforti (d) in presence of MG.

gle culture when cells are undergoing apoptosis as reported by Collins et al. (1997). Alternatively, it could be because of MG’S negative role in DNA intercalation (Chan et al., 2004). To confirm MG toxicity, we used RAPD analysis in our investigations. Since DNA fingerprinting can be an important sign of genotoxicity (Danylchenko and Sorochinsky, 2005), RAPD is routinely used to evaluate the toxicity of the environmental pollutants (Liu et al., 2012). Our RAPD analysis demonstrated polymorphisms in the form of the disappearance of bands, appearances of new bands in MG treated sample profile (Fig. 2B), suggesting DNA alterations similar to the previous report (Atienzar et al., 2002). In our study, MG treated cells showed amplification of several new fragments when compared to untreated cells (Fig. 2C). This can be explained by genomic template instability and availability of new priming sites after DNA breaks, transpositions and deletions (Bouteau et al., 2011), upon MG exposure. To reconfirm DNA damage, DAPI staining test was carried out. Our observations of nuclear features of treated samples revealed fragmented DNA in the nucleus indicating obvious DNA degradation which is a common apoptotic marker (Chen and Dickman, 2005). Considering RAPD analysis, which corroborated well with our DAPI staining results, it can be concluded that MG has a detrimental effect on genome of the tested organisms. At elevated concentrations, ROS has been shown to exert various deleterious effects on normal cellular pathways (Azad et al., 2014). Here, we tested the morphological damage by SEM study. Cytotoxicity in structural level in presence of MG is evident in the form of deformation, surface destruction and size reductions of microbial cells (Fig. 3C). These multiple pores and

deformities on the cell membrane might cause eventual lysis or cell bursting. Integrity of cell membrane is essential for cell survival and a toxic substance usually causes membrane alterations by binding and deactivating membrane proteins or by dissolving the lipid matrix. Similar formation of holes in the membrane through the action of chemicals was also reported earlier (Walum and Forsby, 1995). Additionally, they may also interact directly with functional proteins and stimulate toxic effects inside the cell. In our study, increased number of randomized bud scars in S. cerevisiae cell wall might be an indication of MG induced cellular stress which affects the normal cell division process in such a way that a cell makes multiple attempts to divide (Nollin and Borgers, 1975) for its survivability. In an earlier study, randomized budding pattern in yeast is reported to be associated with alterations in the yeast cytoskeleton (Walther et al., 1996). Likewise, yeast showing randomized bud scar in our study might be because of interaction of MG and proteins responsible for maintaining cytoskeleton structure. Being a significant part of soil biomass, earthworms are routinely used for various soil pollution assays (Lin et al., 2012). E. fetida when exposed to MG, developed multiple detrimental effects in our study. Degeneration of earthworms as noted, may indicate complete utilization of reserved body energy followed by autolysis of its own tissues (Mccosh and Getliff, 2003) in an unfavorable condition. Similar observations were also reported earlier with organ phosphorous pesticide (Reddy and Rao, 2008). As witnessed in the present study, weight loss accompanying coiling, could be due to muscular alterations rendering difficulties in their

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movement and in turn affecting their feeding behavior (Faheem and Khan, 2010). All these behavior might indicate the survival strategy of the earthworms in the stressed environment. Malachite green is commonly used for seed treatment in agriculture industry for promotional effect on seed health and vigor (Tonapi et al., 2006). However, our analysis did not reflect any detrimental or promotional effect on MG at concentrations up to 100 mg L1 in germination of seeds. Additional investigations would be necessary to comment on the effect of the dye at cellular level in the crop plants.

5. Conclusion Diverse soil processes are carried out by communities of organisms at different trophic levels. Micro-organisms and soil invertebrates especially earthworms play an indispensable role in maintaining the soil ecosystem. Our study suggest that MG is ecotoxic and is thus of great concern to soil health. Genotoxicity was detected in all bacteria (both gram positive and gram negative) and fungi cultured in presence of MG. Elevated ROS levels and alterations in protein profiles also concluded MG induced cytotoxicity. SEM observations confirmed concentration dependent morphological alterations. MG also had capability of inducing morphological alterations in E. fetida. However, no significant change was observed in germination of seeds and their subsequent root elongation. In conclusion, this study indicates that key components of soil ecosystem are adversely affected by MG. Consequently, MG confers a major concern to the soil environment and possible eventual ecosystem disruption.

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