Exhaustive ecotoxicological evaluation of the product of malachite green biodegradation by Stenotrophomonas koreensis

doi:10.21203/rs.3.rs-4745352/v1

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Exhaustive ecotoxicological evaluation of the product of malachite green biodegradation by Stenotrophomonas koreensis

https://doi.org/10.21203/rs.3.rs-4745352/v1

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Stenotrophomonas koreensis effectively biodegrades malachite green, a mutagenic, carcinogenic, and teratogenic dye, raising the need to assess the safety of its degradation products before environmental release. This study addresses the gap in previous research by comprehensively evaluating the toxicity profile of malachite green degradation by S. koreensis. It incorporates a diverse range of organisms from terrestrial and aquatic ecosystems to assess the environmental impact of chemical introductions precisely. In-silico toxicological analysis was conducted using the online servers SwissADME, GUSAR, and vNN-ADMET, and ecotoxicological studies, following OECD guidelines. Aquatic toxicity was tested on cyanobacteria Anabaena flos-aquae, zooplankton Daphnia magna, aquatic macrophyte Lemna minor, and zebrafish Danio rerio. Terrestrial contamination risks were assessed with soil bacteria Bacillus subtilis, Bacillus pumilus, earthworm Eisenia fetida, important crop plants Triticum aestivum, Lens culinaris, and model organism Mus musculus. Genotoxicity was evaluated with Allium cepa root tip cells, and MTT assays were performed on HaCat cell line. Results indicate the degradation product's significantly reduced toxicity compared to malachite green. This study underscores the importance of thorough toxicity assessment in ensuring the safe disposal of dye degradation products into the environment.

Malachite green

biodegradation

Stenotrophomonas koreensis

ecotoxicology

The abundance of microorganisms in nature offers a wealth of untapped possibilities for human welfare. Only degrading any toxic dye does not fulfill the ecological requirement: a few studies suggest that the dye degradation products are more hazardous than the dyes themselves (Misal & Gawai, 2018; Saini et al., 2018). Toxicity assessment of the end products obtained after any degradation process is crucial to validate the process's safety. With the advancements in cheminformatics and data processing algorithms, in-silico approaches are often employed to support the experimental data (Furxhi et al., 2020). Studies on computational toxicology have highlighted its role in diverse industries including pharmaceutical, food, agricultural and cosmetic industry (Banerjee & Ulker, 2022; Idakwo et al., 2019). Integrating knowledge on statistics, programming languages, structural and chemical biology, several in-silico models are developed that exhibited good accordance with the toxicity experiments (for e.g., rat oral acute toxicity, toxicities to Tetrahymena pyriformis and Daphnia magna (Asadollahi-Baboli, 2012; Diaza et al., 2015; Golbamaki et al., 2014). These tools in the form of standalone software, online servers and platforms guide a researcher to elucidate the toxicity profile of the compounds of interest. Subsequently, using an array of bioassays with organisms from several ecological ranks allows for a more accurate assessment of the environmental impact of a toxic compound introduced into a given ecosystem. Photosynthetic bacteria, like the cyanobacteria, are believed to play a crucial role in the oxygenation of the earth’s atmosphere when the earliest traces of life appeared and has since formed the foundation of the aquatic ecosystem (Sánchez-Baracaldo & Cardona, 2020). The blue-green algae Anabaena flos-aque was chosen for our study. Zooplanktons feed on the aquatic algae and form the connecting link for energy transfer between the primary photosynthetic producer organisms, i.e., the algae and the larger vertebrates like fish (Lomartire et al., 2021; Sterner, 2009). Higher photosynthetic macrophytes like duckweeds (Lemna minor) also play significant roles in the water ecosystem. They provide a habitat for small amphibians and act as a food source for certain aquatic birds and fishes (Cao et al., 2018). Malachite green can pollute water and land (industrial run-offs often contaminate adjoining land areas). Bacteria in the soil are responsible for nutrient recycling and play crucial roles in carbon, nitrogen, sulfur, and phosphorous replenishment (Msimbira & Smith, 2020). They also produce plant growth hormones (Prasad et al., 2021). Earthworms (Eisenia fetida) have the potential to modify the physical and chemical properties of the soil, thereby influencing the life of other soil organisms (Bhadauria & Saxena, 2010). Chemical modification of soil includes better availability of essential nutrients like phosphorus and nitrogen to plants after being digested and excreted into the soil by earthworms in the form of “earthworm cast.” In terms of physical modification, earthworms dig up tightly packed soils to allow air circulation, which in turn leads to better interaction of oxygen with bacteria and promotes their growth (Lu et al., 2021). Dyes are known to have a negative impact on the germination of plants (Matpang et al., 2017; Rahman et al., 2018; Rajashekarappa et al., 2022). There can be significant and direct occupational exposure to malachite green by persons working in textiles, tanneries, or aquaculture farms (Okafoagu et al., 2017; Singh & Chadha, 2016). Effluents from these industries may run off into rivers or even into the urban water- supplies, thereby causing non-point-source pollution. Consumption of colored green vegetables and fish treated with malachite green for a prolonged period may also lead to direct exposure of the toxicant to human beings. Given the constantly expanding dye industries, determining the toxicity of textile dyes to the ecosystem is critical in determining the best course of action for future environmental safety concerns. The dye production industry has raised awareness about numerous environmental challenges, and products with negligible environmental impact are being manufactured and promoted. So, there is a need for the evaluation of the impacts of textile dyes and their corresponding degradation products on the ecosystem and based on the results an appropriate treatment technology could be designed in coming days keeping the users' interest in mind.

2.1 Dye and media

The dye Malachite green oxalate was purchased from Sisco Research Laboratories (India). Nutrient broth (Peptone 10 g/L; Beef extract 10 g/L; Sodium chloride 5 g/L; pH adjusted to 7) was obtained from Himedia Laboratories.

2.2 Preparation of the degradation product

A loop-full of S. koreensis was inoculated in 100 mL nutrient broth in a 250 mL flask and maintained overnight at 37°C under shaking conditions (200 rpm). Bacterial cells were removed during the late exponential growth phase after centrifuging the suspension at 10,000 rpm for 8 minutes at 4°C. After discarding the supernatant, the bacterial biomass was washed thrice with sterile Phosphate Buffer Saline (PBS). The biomass was re-suspended in sterile distilled water and adjusted to the final concentration Log₁₀CFU/mL = 6.2. Following the previous research on the industrial dye malachite green (Biswas et al., 2022), 5 mL of the bacterial suspension was added to 95 mL of malachite green oxalate solution and incubated at 37°C under static conditions for four hours to degrade the dye completely (S. koreensis degraded 98% dye in a span of 4 h without any media or supplementary nutrients). The decolorized product obtained after 4h was centrifuged at 10,000 rpm for 5 min to separate and discard the cell mass. The supernatant was collected, dried by lyophilization, and used for toxicity assays.

2.3 In-silico toxicity evaluation

Molecular and biological properties (such as drug-induced liver toxicity/hepatotoxicity, intestinal absorption, water solubility, blood-brain barrier permeation, AMES test, etc.) of all the by-products of degradation were analyzed using the online server of SwissADME (http://www.swissadme.ch), and vNN-ADMET (https://vnnadmet.bhsai.org/vnnadmet/) (Daina et al., 2017; Schyman et al., 2017). The acute (rat model) and environmental toxicity of the compounds were predicted from the online server of GUSAR (http://www.way2drug.com/) (Lagunin et al., 2011).

2.4 Ecotoxicological assessments

The ecotoxicological assessment of the biodegradation product of the toxic dye malachite green by S. koreensis was performed with a wide range of aquatic and terrestrial organisms, representing the critical levels of the ecosystem following previous researches (Amin et al., 2020; Gioia et al., 2018; Khan et al., 2020) while adhering to the OECD guidelines. To calculate an endpoint for quantitatively expressing the toxicity of the dye and the degradation product, a range of treatment solutions were prepared by two-fold serial dilutions, as mentioned by Gioia et al. (2018). Hence, 1/16, 1/8, 1/4, 1/2, and undiluted initial dye concentrations used in all previous experiments (translating to 6.25, 12.5, 25, 50, and 100 mg/L malachite green, respectively) along with their corresponding degradation products were tested.

2.4.1 Aquatic toxicity studies

Aquatic toxicity was measured using four indicative organisms of the four levels of the aquatic ecosystem: the primary producer, algae Anabaena flos-aque (algae growth inhibition test), aquatic plant Lemna minor (growth inhibition test), primary consumer crustacean Daphnia magna (acute immobilization) and secondary consumer zebra-fish Danio rerio (acute toxicity).

2.4.1.1 Algae growth inhibition test

The Algae growth inhibition test was conducted for a period of 96 h, maintaining the OECD 201 (2011) guidelines for toxicity testing (Hund-Rinke et al., 2022). The unicellular blue-green algae Anabaena flos-aque was used for this study. The cyanobacterial pure cultures were subcultured in BG11 medium [NaNO₃ 1.5 g, K₂HPO₄ 0.04 g, MgSO₄.7H₂O 0.075 g, CaCl₂.2H₂O 0.036 g, citric acid 0.006 g, ferric ammonium citrate 0.006 g, EDTA (disodium salt) 0.001 g, Na₂CO3 0.02 g, trace metal mix A5 1.0 mL, distilled water 1 L (pH 7.1 post-sterilization); trace metal mix A5 composition: H₃BO₃ 2.86 g, MnCl₂.4H₂O 1.81 g, ZnSO₄.7H₂O 0.222 g, NaMoO4.2H₂O 0.39 g, CuSO4.5H₂O 0.079 g, Co(NO3).6H₂O 49.4 mg, distilled water 1 L] and maintained under 16 h light and 8 h dark photoperiod at 25 ± 2ºC. Aliquots of algal culture were inoculated into BG 11 medium with malachite green and corresponding degradation product separately. The initial biomass density was maintained at 0.1 mg/mL for all the experiments. A. flos-aque cultures, including a dye-less control and the degradation product, were also set up similarly. These were incubated at 28 ºC under 16 h illumination in continuous shaking conditions for 96 h. Care was taken such that no other organic or inorganic contaminants could intervene in the system. After 96 h, 5 mL algal aliquots were filtered through Whatman GF/F 25 mm filter paper and dried at 60 ºC for 8 h. The average specific growth rate is calculated as the log of the biomass increase over a specific period and is calculated by Eq. 1 (Hund-Rinke et al., 2022):

$\:{\mu\:}_{i-j}\frac{(ln{X}_{j}-ln{X}_{i})}{({t}_{j}-\:{t}_{i})}$ Eq. 1

where:

µ _i-j is the average specific growth rate from time i to j;

X _i is the biomass at time i;

X _j is the biomass at time j;

t _i is the time (in days) of the i^th biomass measurement after beginning the exposure;

t _j is the time (in days) of the j^thbiomass measurement after beginning the exposure

The percentage inhibition of growth rate was calculated following Eq. 2 (Hund-Rinke et al., 2022):

$$\:\%{I}_{r}=\:\frac{{\mu\:}_{c}-\:{\mu\:}_{r}}{{\mu\:}_{c}}*100$$

Equation 2

where:

%I _r is the percentage inhibition in average specific growth rate;

µ _c is the average specific growth rate in the control group;

µ _r is the average specific growth rate for the treatment group

2.4.1.2 Lemna minor acute toxicity assay

The second level of aquatic toxicity bioassay was performed using the fresh-water macrophyte Lemna minor through experiments designed according to the OECD 221 (2006) guidelines for toxicity testing (Alkimin et al., 2019). The aquatic plants were collected during June from the Jadavpur University ponds and were thoroughly cleaned and then cultured in Hoagland’s medium for 8 weeks. The healthy plants were screened out for further work and maintained under laboratory conditions for another 7 days. Then, three colonies, each consisting of 4 fronds, were aseptically transferred into 250 mL borosilicate glass beakers containing 100 mL distilled water amended by the dye (6.25–100 mg/L), the corresponding degradation products, and a dye-less control. Each test solution, along with the control, was kept in triplicate at 25ºC for 16 h photoperiod. After an exposure of 96 h, the plant biomass for each treatment was recorded as a measure of toxicity. The specific growth rate and % growth inhibition were estimated from Equations 1 and 2, respectively. The total chlorophyll content was also measured for all the treatment groups. To estimate chlorophyll content, the plants were washed several times to remove any traces of dye, and the method described in a previous study was followed (Arnon, 1949). Total chlorophyll was extracted in 80% acetone (2 mL) in a pre-cooled mortar pestle and the extract was centrifuged at 3000 rpm for 15 min. The supernatant was used for the spectrophotometric measurement using Agilent Cary 60 UV-Vis spectrophotometer.

2.4.1.3 Daphnia magna acute immobilization assay

Daphnia magna is a microcrustacean, representing the lowest level of the aquatic zoological organism. For the third level of aquatic toxicity testing, these were used following the OECD 202 guidelines (OECD, 2004) (Baumann et al., 2014). Adult daphnids were procured from Neet Aquarium, Tamil Nadu, and were cultured in 2 L glass beakers having 1.6 L hard water (160 mg/L CaCO₃), each having 20 adult daphnids. The set-ups were maintained in the incubator at 20ºC with a 16 h photoperiod. The daphnids were transferred to fresh water each week and the neonates obtained from the third broods were used for further studies. For the acute immobilization tests, five neonates were carefully transferred in 100 mL glass beakers containing 80 mL test solutions (6.25–100 mg/L malachite green and the corresponding degradation products) using sterile glass pipettes, and the beakers were kept covered with watch glasses. No food was provided during this phase, and immobilization or death was recorded after 24 and 48 h.

2.4.1.4 Danio rerio acute toxicity assay

An acute toxicity test was performed using zebrafish (Danio rerio) to estimate the effect of malachite green (6.25–100 mg/L) and the corresponding degradation product following the OECD 203 (2019) guidelines with some modifications (Burden et al., 2020). Each set of ten zebrafish of average length 2.0 ± 1.0 cm and average weight 0.45 ± 0.05 g was exposed to malachite green, the degradation product, and a dye-less control. Experiments were conducted in glass aquaria holding 10 L water with a photoperiod of 16 h and water temperature of 25°C. Fish mortality and abnormal behavior or appearance was recorded at regular intervals. Histopathological analysis of tissue sections was also performed.

2.4.2 Toxicity assays with land organisms

2.4.2.1 Toxicity studies on beneficial soil microorganisms

Strains Bacillus subtilis (ATCC 21332) and Bacillus pumilus (ATCC 7061), which are commonly found in soil, were used to assess the impact of the dye (100 mg/L) and its degradation product. Logarithmically growing cultures (1 mL) having optical density 1 at 600 nm were inoculated in 100 mL nutrient broth. Growth was analyzed by incubating the bacteria in the dye, the degradation product, and a dye-less control. Then the cell density was checked periodically till the stationary phase was reached. The effect of Malachite Green and the degradation product on these beneficial soil bacteria were evaluated. The disc diffusion method was employed to assess the impact of MG and the degradation products, while sterile distilled water was taken as control. Diameters of zones of inhibitions were measured after overnight (18 h) incubation at 37°C.

2.4.2.2 Eisenia fetida acute toxicity assay (contact test)

Acute toxicity assessment (contact test) was performed using earthworm (Eisenia fetida), according to the OECD 207 (1984) guidelines (van Gestel & van Dis, 1988). The E. fetida were acquired from Aastha Earthworms, West Bengal. Sterile 30 mL borosilicate glass culture vials were lined with filter papers (suitably cut to fit within the vials) and soaked in test solutions. In each vial, one healthy adult earthworm was carefully placed. The test was conducted for 48 h. Five vials (carrying one earthworm each) were maintained for each test solution and the corresponding degradation products. Sterile distilled water was used as a control. No food was provided during the tenure of the study. Mortality rates were recorded at 24 h intervals.

2.4.3 Phytotoxicity

The toxicity of the Malachite Green and the degradation product was evaluated using two economically important crop plants: wheat and pulses. Seed germination index, root and shoot lengths were taken into account. Tests were performed by methods described by the previous reports (Biswas et al., 2018). Triticum aestivum and Lens culinaris were obtained locally. Ten healthy seeds of each crop were placed in separate Petri plates lined with filter papers soaked in equal volumes of test solutions (Malachite Green, the degradation product, and water as a control). Germination was continuously monitored on a day-to-day basis. The lengths of plumules of the germinated seeds were measured, and the number of seeds germinated was counted each day until further germination stopped. Seeds that showed the emergence of radical piercing through the seed coat were considered to be germinated. Data for germination were recorded on the 16th day.

2.4.4 Genotoxicity

Allium cepa bulbs of uniform size and weight (3-3.5g approximately) were obtained locally, and the outer loose, flaky scales were removed carefully with the help of a pair of forceps. The fresh root primordia that emerged as a result of the denudation step were immersed in the test solutions (6.25–100 mg/L Malachite Green, the corresponding degradation products, and a dye-less control). These set-ups were maintained at 25°C for 72h with a 16h photoperiod. Terminal 1cm portions of the actively growing root tips were excised, rinsed with distilled water, and fixed in Carnoy’s solution1 (1:3 Glacial acetic acid: ethanol). The fixed root tips were hydrolyzed in 1N HCl and stained with 2% aceto-orcein. Slides were prepared with a drop of acetic acid (45%) followed by squashing. Squashed root tips were then observed under Leica DM 750 microscope and photographed with Leica DFC295 camera.

2.4.5 Cytotoxicity

Immortalized human keratinocytes (HaCaT) were used for this study. MTT assay and DAPI imaging of the cells using various concentrations of Malachite Green (6.25, 12.5, 25, 50, and 100 mg/L) and the corresponding degradation products were performed. HaCaT cells were cultured at 37°C in Dulbecco's Modified Eagle Medium, having 10% Fetal Bovine Serum (FBS) with 1% antimycotic-antibiotic solutions and were maintained at 5% CO₂ and 95% humidity. The cell line was seeded in 96 well plates at a density of 1 x 10⁴ cells per well. The cells were incubated for 24 h and treated with varying concentrations of the dye and the degradation product to evaluate the toxicity following standard MTT cell proliferation assay. Furthermore, the effects of the test solutions on the cellular viability were assessed using DAPI fluorescent staining following the manufacturer’s protocol and visualized by fluorescence microscope (Olympus IX71).

2.4.6 Blood/serum toxicity and histopathology

The undiluted dye (100 mg/L) and the degradation product were tested for blood/serum toxicity and histopathology using mice (Mus musculus). All the animals were individually maintained with proper ventilation and 12 h light and dark cycles and provided with sterilized food pellets and water ad libitum. For evaluation of acute systemic toxicity, malachite green and the degradation product (2 g/ Kg body weight) were administered orally for 30 days. After completion of the study period, the mice were anesthetized using Diazepam (IP, 10 mg/kg body weight) and Xylazine (IP, 10 mg/kg body weight) subsequently. Under anesthesia, about 1 mL of blood was collected for serological analysis using a facial vein puncture. The control and test animals were euthanized and organs like kidneys, liver, and spleen were aseptically collected for histopathological analysis. All the organ samples were fixed using 10% Neutral Buffered Formalin and maintained at room temperature. The fixed tissue samples were subjected to hematoxylin and eosin (H&E) staining, followed by paraffin embedding and sectioning, respectively.

All studies performed with animals were conducted on approval of the Institute Animal Ethical Committee of the Indian Institute of Technology, Kharagpur.

2.5 Statistical Analysis

All data were analyzed by one-way ANOVA with Tukey-Kramer multiple comparison test.

3.1 In-silico toxicological analysis

According to (Biswas et al. 2022), GCMS/MS analysis of the product obtained upon the degradation of malachite green by S. koreensis indicated the presence of Michler’s ketone, 4-N,N-dimethylaminophenol, Benzophenone, N, N-dimethylaniline, 4-(N-methylamino)-benzophenone, 4-aminobenzaldehyde, anionic canonical form of 4-aminobenzaldehyde, 4-(N,N-dimethylamino)benzoic acid, and 4-(N-methylamino) benzoic acid. All the compounds fall in the applicability domain of toxicity prediction models of GUSAR. Additionally, the results from SwissADME and vNNADMET are presented in Table 1. Except for benzophenone, all the by-products of degradation showed a lower value of bioaccumulation factor. Considering the in-silico toxicological data related to the fathead minnow, Daphnia magna, and Tetrahymena pyriformis, the lethal concentration and 50% growth inhibition concentration were observed to be the lowest in malachite green, indicating its higher environmental toxicity compared to the degraded products.

Table 1

Prediction of ADMET properties of the compounds obtained from the degradation of malachite green
Product	GI absorption	BBB permeant	Water Solubility (Log S)	DILI	hERG blocker	AMES	Rat acute toxicity				Environmental toxicity
Product	GI absorption	BBB permeant	Water Solubility (Log S)	DILI	hERG blocker	AMES	Rat IP LD50 (mg/kg)	Rat IV LD50 (mg/kg)	Rat Oral LD50 (mg/kg)	Rat SC LD50 (mg/kg)	Bioaccumulation factor (Log10(BCF))	Daphnia magna LC50 (-Log10(mol/L))	Fathead Minnow LC50 (Log10(mmol/L))	Tetrahymena pyriformis IGC50 (Log10(mol/L))
Michler’s ketone	High	Yes	-4.12	No	No	Yes	255	41.41	1,883	746.8	1.230	4.726	4.726	1.250
4-(Dimethylamino)phenol	High	Yes	-2.41	Yes	No	Yes	28.5	57.2	1026	481.6	0.671	3.971	-0.361	-0.009
Benzophenone	High	Yes	-3.48	Yes	No	No	1312	56.92	4518	630.8	1.818	4.291	-1.470	0.798
N, N-dimethylaniline	Low	No	-2.47	Yes	No	No	122.2	24.06	569.9	264.9	0.868	3.914	-0.369	-0.212
4-(Methylamino)benzophenone	High	Yes	-3.66	Yes	No	No	515.4	34.43	1,768	2,065	1.333	4.982	-1.624	0.958
4-aminobenzaldehyde	High	Yes	-1.52	No	No	No	779.6	45.15	1643	561.7	0.422	5.094	-0.501	-0.363
4-(Dimethylamino)benzoic acid	High	Yes	-1.91	No	No	No	424.2	207.100	2,090	549.6	0.057	3.082	-0.172	-0.090
4-(Methylamino)benzoic acid	High	Yes	-2.4	Yes	No	No	521.9	228.3	1649	1.005	-0.054	3.378	-0.157	-0.019
Malachite Green Oxalate	Low	No	--10.83	No	No	No	278.4	34.36	1627	614.1	1.740	1.740	-3.056	-1.855
*GI – gastrointestinal, BBB – blood-brain-barrier, DILI – drug induced liver toxicity, IP - intraperitoneal route of administration, IV - intravenous route of administration, SC- subcutaneous route of administration, hERG - human Ether-à-go-go-Related Gene

3.2 Aquatic toxicity

3.2.1 Algae acute toxicity assay

A gradual decrease in the average specific growth rate could be observed with the increase in dye concentration, giving a clear indication of toxicity to the algae (Fig. 1).

The undiluted degradation product showed 26% inhibition, while dye concentrations higher than 50 mg/L led to complete inhibition of growth (Fig. 2). The percentage inhibition of growth was also calculated from the specific growth rate. For 6.25 mg/L malachite green and its degradation product, the percentage inhibition of growth was 44.31% and 4.26%, respectively, indicating significantly lesser toxicity (p < 0.05) of the degradation product than the original dye.

Algae form the basic level of all aquatic ecosystems. The impacts of the toxic element that inhibit the growth of these primary producers will inevitably reach the ecosystem's upper layers. Only limited research has been performed to determine the toxic effects of textile dyes on microalgae compared to other toxicants like heavy metals (Gita et al., 2019). The effect of Indigo dye-containing effluent on Scenedesmus quadricauda was studied by Chia and Musa (2014). They observed a systematic decrease in cell number with increased effluent concentration. The specific growth rate of yeast continually decreased with increasing concentrations of Reactive Blue 172 and Direct Red 28 (Chia & Musa, 2014). Gita et al. (2019) studied the effect of Optilan yellow, Drimarene blue, and Lanasyn brown on Chlorella vulgaris. They observed 100% growth inhibition for 30 ppm dye concentrations for all three dyes (Gita et al., 2019). All of the above-mentioned literature supported the observations of the present study. Although there are a few articles on the toxicity of dyes on algae, this is the first report on the toxicity assessment of the degradation product of malachite green on any microalgae. Since there is a paucity of data on the toxic effects of malachite green and its degradation product on microalgae, this article will aid future investigations.

3.2.2 Lemna minor acute toxicity assay

The floating macrophyte Lemna minor is a common biological indicator for toxicity estimation. The specific growth rates of the plants steadily decreased with the increase in the dye concentration to 50 mg/L (Fig. 3 and Fig. 4).

The highest specific growth rate was recorded for the control group. The specific growth rates of the plants grown in all the concentrations of the degradation product were significantly higher (p < 0.01) than the corresponding concentrations of the dye. More than 73% growth inhibition was recorded for the undiluted malachite green, while only 18% inhibition was recorded for the undiluted degradation product. A similar trend could be noted in the case of the chlorophyll content of L. minor. The lowest total chlorophyll (0.3 mg/g fresh weight) was recorded in the plants grown with the highest concentration of the dye (Fig. 5). Lesser chlorophyll contents correspond to lower photosynthetic capability and result in lower biomass. The chlorophyll content of plants grown in the degradation product, although less than that of the control was significantly higher (p < 0.01) than that of the plants grown in the dye.

Lemna minor is a bio-indicator plant that forms colonies in freshwater bodies and is commonly employed to evaluate environmental and toxicological concerns. As an example, the effect of the textile dye Gentian Violet was studied on the growth parameters of L. minor (Adomas et al., 2020). They reported that Gentian violet changed the putrescine (Put) production pathway, raised tyramine content, inhibited S-adenosyl methionine decarboxylase activity, and inhibited ornithine decarboxylase (ODC) activity. Micropollutants like caffeine, benzophenone, bisphenol, etc., exert inhibitory effects on the growth and chlorophyll production of Lemna minor (Fekete-Kertész et al., 2015). The presence of even a very nominal amount of dye in the growth media can cause distortions in the leaf and root anatomy. For example, 0.01 µg/L Congo Red causes severe shriveling of the upper and lower epidermis. At 0.04 µg/L Congo Red, reduction of mesophyll tissues and dissolution of aerenchyma occurs in L. minor, and leaves start falling at the bottom of the aquarium (Al Zurfi et al., 2020).

3.2.3 Daphnia magna immobilization assay

Daphnia magna, the aquatic microcrustacean, can survive in various water bodies ranging from freshwater ponds to acidic lakes (Ebert, 2005). The dye and the degradation product were tested for potential ecotoxicity on the representative zooplankton Daphnia magna. No deaths were observed in the lowest dye concentration (6.25 mg/L) in the first 24 h. While all of the daphnids died within 2 h of exposure in the highest dye concentration (100 mg/L), more than 86% viability was recorded for those treated with the undiluted degradation product. A notably elevated viability (p < 0.01) was observed among Daphnia neonates exposed to the degradation product compared to those exposed to the dye (Fig. 6).

The toxicity of textile dyes and their degradation products have been tested on D. magna by some authors. Decolorization of the toxic azo dyes Acid Red 88 and Acid Black 172 was done by immobilizing laccase enzyme of Trametes villosa (Gioia et al., 2018). In the D. magna acute toxicity test, the dye Acid Red 88 showed lesser toxicity than the degradation product. The non-genotoxic dye Direct Blue 218 has a 48 h LC50 of 3.6 mg/L for D. magna (Bae & Freeman, 2007). Previous study on the degradation product of the azo dye Reactive Black 5 was found out to be slightly more toxic than the original dye due to the production of aromatic amines during the degradation process (Cuervo Lumbaque et al., 2017). For azo dyes, in most cases, the degradation products were more toxic than the original dyes due to the production of copious amounts of aromatic amines. The 48 h EC50 value for Daphnia magna ranged from 0.29–0.77 mg/L for azo dye Reactive Black 5 (Kanhere et al., 2014; Muhammad Raziq Rahimi et al., 2017). However, for triphenylmethane dyes like malachite green, the observations are different. Except for algae and cyanobacteria, the superparamagnetic iron oxide nanoparticles had no harmful effect on Lemna minor or D. magna up to a concentration of 1 g/L (Plachtová et al., 2018). In the present study, it is observed that the product of malachite green degradation by S. koreensis is significantly less toxic than the original dye (p < 0.05).

3.2.4 Danio rerio acute toxicity assay

Zebrafish (Danio rerio) treated with malachite green showed loss of equilibrium, production of excessive mucus, and convulsion. The fish treated with the undiluted dye showed hemorrhagic lines anterior to the anal fin, and 100% fish mortality was recorded within 38 min for dye. For the lowest dose of malachite green (6.25 mg/L), all the fish died within 3 h (Table 2). The experiment was carried out for 7 days with the corresponding dosages of the degraded product, although no death or abnormal behavior was recorded for the degraded product.

Table 2

Percentage mortality of *Danio rerio* on exposure to malachite green (MG) and its degradation product (DP)
Two-fold serial dilutions	Percentage fish mortality
	40 min		80 min		160 min		320 min
	MG	DP	MG	DP	MG	DP	MG	DP
1/16 Dil	0	0	30	0	80	0	100	0
1/8 Dil	20	0	40	0	100	0	100	0
1/4 Dil	50	0	70	0	100	0	100	0
1/2 Dil	80	0	100	0	100	0	100	0
Undiluted	100	0	100	0	100	0	100	0

The liver of zebrafish is a model for studying liver diseases in humans. Bile improves lipid-soluble nutrient absorption while facilitating the excretion of cholesterol and toxic metabolites, especially bilirubin (Romano et al., 2020). Prominent dilations of the bile ducts were observed in the fish exposed to the dye and it progressively increased with the increase in the concentration of the dye. In contrast, the ones treated with the degradation product showed no significant morphological changes (Fig. 7).

Dilation of the bile ducts in case of alcoholic liver disease in zebrafish had been previously reported (Lin et al., 2015). Dilated bile duct may be a manifestation of cholestasis which disrupts bile flow, resulting in bile fluid accumulation in the liver. Significant morbidity and mortality may be caused by cholestasis due to pruritus, malnutrition, portal hypertension, biliary cirrhosis, etc. (Pham & Yin, 2019). Blocking bile flow at the level of extrahepatic biliary ducts is a significant mechanism of cholestasis, and retained bile leads to hepatotoxicity (Shah & John, 2018). Failure to transport these bile salts results in their accumulation in the liver. The bile salts' powerful detergent-like effect causes membrane injury and degradation of membrane function, reflected by the histopathological examinations of Danio liver tissues (Fig. 8).

Malachite green has already been reported to exert detrimental toxicity on the liver (Donya, 2012). But our study revealed that the product obtained after the biological degradation of malachite green by this bacterium renders it remarkably safe for Danio rerio. Thus, it can be stated that the degradation of the dye by the bacteria leads to mitigating the toxic effects that the untreated dye exerts on aquatic life.

3.3 Toxicity to beneficial soil bacteria

3.3.1 Microbial toxicity assay

Several bacteria that inhabit the soil add to the soil fertility by processes like nutrient mineralization, promotion of plant growth hormones, or playing roles as plant pest repellents. We chose two such strains B. subtilis and B. pumilus to see if the dye and its degradation product exert any toxic effect on them (Figs. 9 and 10). This was important because the dyes that come out through the effluents not only pollute the rivers and water bodies but also contaminate adjoining land areas.

The growth curves of the two bacterial strains were observed after exposing them to the undiluted dye (100 mg/L) and its degradation product. In the undiluted dye exposure, a prominent extension of the lag phase was observed. The lag phase is known to prepare the bacteria for the upcoming phases of the growth cycle. An extended lag phase, therefore, indicates that the bacteria require more time to prepare themselves for reproduction which, in turn, is a clear sign of toxicity. On the other hand, the bacteria grown in the presence of the undiluted degradation product followed a very similar timescale as the control. The removal of toxicity in the degradation product could thereby be claimed.

To assess the exact amount of decrease in toxicity, a conventional agar-cup assay was performed. No inhibition zone was observed in the case of the degradation product. At the same time, a clear halo of 53 mm and 62 mm was recorded for the malachite green for B. subtilis and B. pumilus respectively (Fig. 11 and Table 3). Many authors have used microbial toxicity studies to test the ecotoxicity of dyes and their degradation products. Staphylococcus aureus was subjected to malachite green and its degradation by Bacillus vietnamensis.

Figure 11 Agar cup assay of malachite green and the degradation product with (a) Bacillus subtilis and (b) Bacillus pumilus

Table 3

Agar cup assay of *B. subtilis* and *B. pumilus* with dye and degradation product
Bacterial strains	Control	Malachite Green	Degradation product
Bacterial strains	Diameter of inhibition zone (cm)
Bacillus subtilis	No inhibition	0.53 ± 0.015	No inhibition
Bacillus pumilus	No inhibition	0.62 ± 0.025	No inhibition

Values are expressed as Mean ± SD of three replicates

Compared to the malachite green (50 mg/L), the degradation product showed no inhibition zone, indicating its lesser toxicity to S. aureus (Kabeer et al., 2019). Malachite green (100 mg/L) was more toxic than its degradation products on Escherichia coli (Chen et al., 2010). A study employed the bacterium Ochrobactrum pseudogrignonense to degrade malachite green (Chaturvedi & Verma, 2015). Agar cup assays with S. aureus indicated that the degradation products were significantly safer than the undegraded malachite green. Enterobacter asburiae degraded malachite green and its degradation products were non-toxic to Micrococcus luteus, Klebsiella pneumonie, and Aspergillus sp (Mukherjee & Das, 2014). It was observed that malachite green extended the lag phase of non-target soil bacteria and fungi like Bacillus subtilis, Azotobacter sp. Saccharomyces cerevisiea etc. (Gopinathan et al., 2015). The observations of the present study were consistent with these reports.

3.3.2 Eisenia fetida acute toxicity assay

Like beneficial soil bacteria, earthworm Eisenia fetida also contributes to soil fertility. They are also known as “natural tillers of the earth” and form an integral part of vermicomposting. These organisms are important for organic farming, which is now regarded as one of the most ecologically sustainable techniques for increasing soil fertility, rather than using chemical fertilizers. Earthworms respire through their skin and the chemicals to which they are exposed are directly absorbed inside their body through the skin. A contact toxicity test was performed by exposing the earthworms to dye and the degradation product. In 48h, only a slight decrease in viability was observed (93.4%) for earthworms treated with degradation product compared to the control (100% viability). E. fetida exposed to the undiluted malachite green showed severe damage leading to their decomposition. In contrast, the ones exposed to the undiluted degradation product showed no significant alteration in their appearance (Figs. 12 and 13). Significantly reduced toxicity in terms of the organism’s viability was exhibited by the degradation product compared to dye concentrations ¼^th dilution onwards (p < 0.01).

Several authors have reported bloody lesions, depigmentation, and detachment of the posterior part of the body under the effect of toxic pesticides in earthworms (Chakra Reddy & Venkateswara Rao, 2008; Rao & Kavitha, 2004; Yasmin & D’Souza, 2010). Similarly, toxic textile dyes can be lethally ecotoxic to E. fetida. For example, the textile dye Acid Red causes DNA damage and death in coelomates, including E. fetida (Sathya et al., 2009). (Gopinathan et al., 2015) studied the effect of malachite green on some non-target soil organisms, including E. fetida. The estimated 48 h LC₅₀ was 2.6 mg/cm². The azo dye Red BS and Methyl Red were tested on Pheretima posthuma. The LD₅₀ of Red BS and Methyl Red dye solution was 120.22 mg/L and 218.77 mg/L, respectively. Initially, sluggish movements were observed, followed by reduced pigmentation, the development of multiple lesions, and death. When the degradation product was evaluated in P. posthuma, the toxicity was much lower: no deaths were reported after 48 hours of application of the degradation product of the 100 mg/L Red BS (Gupte et al., 2013). Observations of the present study are consistent with the above reports as explained in the previous paragraph.

3.4 Phytotoxicity

Wheat and pulses are the major cereal crops and form the staple diet of the Indian subcontinent. Germination of seeds is a fairly common approach to determine the phytotoxicity of the chemicals to the crops. Of the two crops, Triticum aestivum seemed to be more affected by the toxicity of the dye, where germination was completely inhibited on exposure to the undiluted dye (100 mg/L), while the corresponding degradation product led to 58.3% germination. In T. aestivum, the germination percentage in the 1/16 dilution of the degradation product surpasses that in the control, while the germination percentage for the 1/8th dilution is nearly equivalent to the control. In Lens culinaris, more than 85% germination was recorded for the 1/16th dilution of the degradation product, which is nearly comparable to water (89.35%). In a 16 day study, significantly higher germination rates were recorded for crops grown in the degradation product than those grown in the dye (Fig. 14).

Phytotoxicity is the inhibitory effect exerted by a toxic element on plant germination and overall growth. If utilized for irrigation in agricultural fields, untreated industrial effluents contaminated with dyes and other hazardous chemicals may be detrimental. When water is scarce, however, sufficiently treated and recycled wastewater can be utilized instead. In this scenario, phytotoxicity testing is critical. Several plant species have been used for the assessment of phytotoxicity. Lucerne seeds grown in malachite degradation products showed germination rates similar to water (Du et al., 2011). Nicotiana tabacum and Lactuca sativa seeds showed similar results (Yang et al., 2016). (Amin et al., 2020) studied both aquatic and terrestrial phytotoxicity of Direct Red-81 and its degradation product. The dye inhibited Lemna minor growth, while its degradation product was significantly less toxic. Phytotoxicity tests with Vigna radiata, Raphanus sativus, and Abelmoschus esculentus showed similar results where the untreated dye was more toxic than the degraded product. The degradation of Congo Red by recombinant enzymes expressed by Pichia pastoris yielded degradation products that were less toxic than the original dye (S. Liu et al., 2020). In our study, phytotoxicity experiments indicated that biodegradation of malachite green by S. koreensis generated significantly safer and much less toxic compounds.

3.5 Genotoxicity

The genotoxic effect of malachite green and its degradation product was analyzed by considering the MI (mitotic index) and the frequency of chromosomal aberrations in onion root tip cells. Allium cepa was used as a model plant because it has many advantages as research material, such as ease of handling, sensitivity, quick analysis, low cost, and the ability to be used in correlation with other models that use mammalian cells (Chaparro et al., 2010). The MI is defined as the number of cells undergoing mitosis divided by the total number of cells where all stages of mitosis are included in the count. Higher or lower MI with respect to the control can be indicative of an alteration of mitosis mechanisms (Leme & Marin-Morales, 2009). The MI was scored for each concentration of malachite green and the degradation product by analyzing approximately 5,000 cells. The control showed an MI of 8.18%. There is a clear reduction of MI corresponding with the increase in the dye concentration. The minimum MI was obtained (4.37%) for the undiluted malachite green, whereas for the undiluted degraded product, the MI was 5.28%. This observation suggests that although the toxicity of the dye was reduced from its original form. Besides scoring MI, the evaluation of chromosomal abnormalities in dividing cells was studied to estimate the toxicity of the dye and the degradation product. All the results indicated that the degradation product was less toxic than the dye but more toxic than the control (water). The study of chromosomal aberrations provides important information and may be considered an efficient method to investigate the genotoxic potential of various textile dyes and effluent (Kalyani et al., 2012). The chromosomal aberrations are shown in Fig. 15.

Several chromosomal aberrations were observed, such as c-metaphase, laggard chromosomes, anaphase bridges, sticky chromosomes, and telophase bridges. Increasing the concentration of the dye led to an increase in the rate of aberration (Table 4).

Table 4

Genotoxicity analysis of malachite green and the degradation product using *A. cepa*
Analysis	Control	Malachite Green			Degradation product
		Two-fold serial dilutions			Two-fold serial dilutions
		1/16 Dil	1/8 Dil	Undiluted	1/16 Dil	1/8 Dil	Undiluted
Mitotic Index (MI)	8.18 ± 0.11	6.42 ± 0.07	5.25 ± 0.02	4.37 ± 0.05	6.92 ± 0.06	5.73 ± 0.06	5.28 ± 0.02
Percentage of aberration	0.00	15.82 ± 0.36	24.49 ± 0.85	57.75 ± 0.72	11.27 ± 0.18	16.56 ± 0.7	23.51 ± 0.35
C-metaphase	0.00	17.33 ± 0.88	22.67 ± 0.88	29.67 ± 1.2	12.00 ± 0.58	16.33 ± 0.3	21.00 ± 1.15
Anaphase bridge	0.00	5.67 ± 0.33	13.33 ± 0.88	20.33 ± 0.88	3.67 ± 0.33	7.67 ± 0.33	12.33 ± 0.33
Sticky Chromosome	0.00	0.33 ± 0.33	6.67 ± 0.33	10.67 ± 0.88	0.00	2.67 ± 0.58	7.67 ± 0.88
Laggard	0.00	1.67 ± 0.67	3.33 ± 0.88	5.67 ± 1.45	0.00	1.33 ± 0.58	4.00 ± 0.92
Telophase bridge	0.00	3.98 ± 0.14	9.36 ± 0.38	14.34 ± 0.44	1.68 ± 0.33	3.33 ± 0.33	8.1 ± 0.36

Among all the chromosomal abnormalities, C-metaphase, chromosome bridge, and sticky chromosomes were observed more frequently. Stickiness in the chromosome may arise due to DNA depolymerization, nucleoprotein dissolution, breakage and exchange in basic folding fiber units of chromatids, and stripping of protein covering of the DNA in chromosomes (Mercykutty & Stephen, 1980). It was established that a sticky chromosome has an irreversible toxic effect that may lead to cell cycle cessation (Friskesjo, 1985). The chromosome bridges indicate the clastogenic activity of the agent (Leme & Marin-Morales, 2009). The laggard chromosomes are formed due to the degradation or depolymerization of the chromosomal DNA (Liu et al., 2004). In our study, although the biodegraded malachite green also showed a reduction in MI and some chromosomal aberrations, they were at a significantly lower rate than that of the original dye. Chromosomal aberrations are 50% lesser in degradation product than in the dye itself.

3.6 Cytotoxicity assays with human cell line

To test the toxicity of the dye and the degradation product on human cell lines, the growth of HaCat cell line was used. The viability of the cell line was significantly higher for the cells treated with the degradation product than those exposed to the dye, which was evident through the MTT results (p < 0.01). The results reflect that the bacterial treatment of the dye reduced its toxicity to a considerable extent (Fig. 16).

From the DAPI staining, it can be inferred that the degradation product was less toxic to the cell line when compared to the dye (100 mg/L). An increase in dye concentration leads to increased cell death. Nuclear fragmentation is prominently visible in much higher densities in the cells exposed to the dye than those exposed to the undiluted degradation product obtained after the bacterial treatment (Fig. 17).

3.7 Serum toxicity in mice

It was observed that the serum albumin values of the dye-treated (100 mg/L) animals increased when compared to the degradation product treated (100 mg/L) animals. Serum albumin has been reported to correlate with hypothyroidism positively (Koga et al., 2009). Increased serum levels may be attributed to its impaired metabolism due to the malachite green treatment. The albumin and cholesterol values of the animals exposed to the undiluted degradation product were significantly lower than the dye-treated animals. Similarly, the increase in serum cholesterol levels may be due to the effect of thyroid hormone on cholesterol synthesis and metabolism. The elevated amount of both HDL and LDL cholesterol in blood serum could be due to the effect of hypothyroidism that ultimately resulted in the increased values of cholesterol. Most of the circulating enzymes and transporters of the mammalian system can be regulated by the macromolecular concentration of metabolites. It was reported that the incidence of hypothyroidism might be associated with Non-Alcoholic Fatty Liver Disease (NAFLD) (Singh & Chadha, 2016). The increased values of total protein and uric acid of the malachite green-treated animals were similar as observed in case of NAFLD. These might be due to the stress experienced by the endoplasmic reticulum of liver cells that may have led to oxidative stress and insulin resistance in the dye-treated animals. The onset of these effects might have induced hyperuricemia and higher total protein concentration in the serum of the dye-treated animals compared to the control. It is well known that malachite green could directly impart toxic effects on the liver (Culp et al., 2006; Mittelstaedt et al., 2004). In this current study, the malachite green treatment seems to have exerted toxicity to the animals similar to NAFLD. The degradation product treated animals’ serum values for total protein; uric acid was significantly low when compared with dye-treated animals. It can be inferred from these observations that malachite green degradation by the S. koreensis showed reduced toxicity levels in animals when compared with the toxicity levels exerted by the original dye. However, the triglyceride values have not increased in the dye-treated animals. This might be due to the short course of the dye treatment to the animals. Figure 18 depicts the serum toxicity analysis where all the results are compared with the control group.

3.8 Histopathology with mice

The liver tissue of the animals fed with Malachite green dye showed the presence of degenerated hepatocyte cells when compared with the ones treated with the degradation product group and the control group (Fig. 19) after a 30 days treatment period.

The hepatocytes of the animals fed with malachite green were strewn in the hepatic parenchyma with the deeply stained nucleus. Degeneration of hepatocytes of the dye-fed animals was observed in various spots. The hepatic cells of the malachite green group animals did not show any prominent nucleus, which might be due to the ductal hyperplasia induced by the Malachite green dye. Hepatic blood vessels of the MG-treated animals were observed to be dilated when compared with the degradation product and control group. No toxicological symptoms were observed in the histopathological sections of the kidney and spleen of the animals. The medullary and cortical regions of the kidney of all groups of animals were prominent without any evidence of morphological disorientation. The reticular framework of the spleen cells treated with the dye, its degradation product, or the control group animals did not show any change in their orientation.

Ecotoxicity of dyes and their corresponding degradation products have been previously studied by many authors but were restricted to only a few species for each work (Amin et al., 2020; Gupte et al., 2013). The novelty of the present work lies in the fact that the entire range of representative organisms from the land and aquatic ecosystem was considered. Using a battery of bioassays with organisms from several taxonomic groups allows for a more accurate assessment of the environmental impact of a chemical molecule or mixture introduced into a given ecosystem (Gioia et al., 2018). Biological experiments using a variety of processes and organisms have been used to assess the toxicity of dyes and the products generated from various decolorization treatments (Bafana et al., 2011). Ecotoxicity results might be difficult to compare to other reported data since they depend on various aspects, such as the chemical nature of the dyes, the approach used in the bioassay, the biological model, the research question, and the expression of outcomes (Gioia et al., 2018).

Advanced Oxidation Processes (AOP) can generate potent oxidizing hydroxyl radicals (˙OH) and sulfate radicals (SO2−˙) through catalytic reactions under various conditions. The traditional Fenton’s reaction produces iron sludge (Fe (OH)3) precipitation, leading to secondary pollution concerns (Zhu & Zou, 2022). Physical adsorption techniques involve the use of adsorbent materials to remove malachite green from contaminated water or soil. While effective to some extent, many of the adsorbents used, such as activated carbon, can generate waste products and require disposal measures, contributing to environmental concerns. Additionally, the adsorption process does not involve complete degradation of the malachite green. Photocatalytic degradation utilizes catalysts such as titanium dioxide (TiO2) activated by UV light to break down malachite green molecules. While this method can be effective, it relies on energy-intensive UV light sources and may produce reactive oxygen species as byproducts, which can be harmful to the environment and aquatic life (Fujishima & Zhang, 2006). These ROS are highly reactive and can pose environmental and health risks (D’Souza et al., 2023). They can cause damage to cellular components, initiate chain reactions leading to the formation of other harmful compounds, and contribute to the production of smog and secondary pollutants in the atmosphere (Chen et al., 2021).

The product of Malachite green degradation by Stenotrophomonas koreensis was subjected to toxicological testing according to the OECD guidelines for testing of chemicals. We started with the preliminary in-silico toxicological prediction, and moved on to a wide variety of organisms belonging to the land and aquatic ecosystems, ranging from the prokaryotic unicellular bacteria to higher animals like mice and even human cell lines. From the elaborate ecotoxicological studies performed with the representative organisms of the key levels of the ecosystem, it can be ascertained that the final products of malachite green decolorization by this strain was significantly safe for release in the environment. Hence, overall, S. koreensis is an extremely promising bioresource which can be utilized to bioremediate malachite green efficiently. Unlike chemical or physical methods that often involve the use of harsh reagents or energy-intensive processes, biodegradation occurs naturally and typically does not introduce additional pollutants or consume large amounts of energy. Degradation of malachite green by S. koreensis aligns with principles of sustainability by utilizing the inherent capacity of microbial communities to degrade pollutants over time. It promotes a balanced ecosystem where microorganisms play a crucial role in maintaining environmental health, and offers the potential for long-term stability in pollutant removal. Incorporating S. koreensis into biological reactors presents a sustainable approach in managing dye-contaminated wastewater in textile industry treatment plants. Its capacity to accelerate dye degradation while yielding environmentally safe end products highlights its potential as an invaluable tool in addressing environmental concerns related to textile dyeing processes. In this quest, it was re-established that bacteria can be an ideal solution for combating the dye pollution globally, replacing the expensive, and hazardous conventional treatment procedures.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

This work was supported by the University Grants Commission, Government of India.

Author Contribution

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Piyali Basak, Shreya Biswas, Tathagata Adhikari, Bhuvaneshwaran Subramanian, Tuhin Kahali, Suproteem Mukherjee, Anwesha Mukherjee, and Debasmita Chakraborty. The first draft of the manuscript was written by Shreya Biswas, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgement

University Grants Commission, Government of India

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Exhaustive ecotoxicological evaluation of the product of malachite green biodegradation by Stenotrophomonas koreensis

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Dye and media

2.2 Preparation of the degradation product

2.3 In-silico toxicity evaluation

2.4 Ecotoxicological assessments

2.4.1 Aquatic toxicity studies

2.4.1.1 Algae growth inhibition test

2.4.1.2 Lemna minor acute toxicity assay

2.4.1.3 Daphnia magna acute immobilization assay

2.4.1.4 Danio rerio acute toxicity assay

2.4.2 Toxicity assays with land organisms

2.4.2.1 Toxicity studies on beneficial soil microorganisms

2.4.2.2 Eisenia fetida acute toxicity assay (contact test)

2.4.3 Phytotoxicity

2.4.4 Genotoxicity

2.4.5 Cytotoxicity

2.4.6 Blood/serum toxicity and histopathology

2.5 Statistical Analysis

3. Results and discussion

3.1 In-silico toxicological analysis

3.2 Aquatic toxicity

3.2.1 Algae acute toxicity assay

3.2.2 Lemna minor acute toxicity assay

3.2.3 Daphnia magna immobilization assay

3.2.4 Danio rerio acute toxicity assay

3.3 Toxicity to beneficial soil bacteria

3.3.1 Microbial toxicity assay

3.3.2 Eisenia fetida acute toxicity assay

3.4 Phytotoxicity

3.5 Genotoxicity

3.6 Cytotoxicity assays with human cell line

3.7 Serum toxicity in mice

3.8 Histopathology with mice

4. Conclusion

Declarations

Competing Interests

Funding

Author Contribution

Acknowledgement

References

Additional Declarations

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