Continued population growth and increased industrialization have caused the degeneration of ecosystems. In the case of the river and ocean water quality, the primary source of pollution is the discharge of industrial wastewater, which may be contaminated with dyestuffs and colorants from textiles, plastics, paper, food, cosmetics, and pharmaceuticals (Saratale et al., 2011). Over 100,000 commercially available dyes and more than 2.8x105 metric tons of dyestuffs are produced worldwide every year (Jin et al., 2007). However, 10–15% of the dye fails to bind with the fibers during textile processing (Supaka et al., 2004; Rai et al., 2005). The uncontrolled discharge contaminating with azo dyes to water bodies causes serious environmental problems, including eutrophication by reducing light penetration and production of different amines under anaerobic conditions (Khan et al., 2013).
Several studies have shown that the release of azo dyes into the environment from the effluents of dye-utilizing industries has become a major concern in wastewater treatment due to the toxic, mutagenic, and carcinogenic characteristics of these dyes and their biodegradation products, which can cause different damages to the dye-exposed organisms (Al-Sabti, 2000; Tsuboy et al., 2007; Caritá and Marin-Morales, 2008; Sompark et al., 2021). Some azobasic, acid, and direct dyes are classified to be toxic or very toxic to fish, crustaceans, algae, and bacteria. Mutagenic effects were observed from the textile azo dye (chlorotriazine reactive red 120) by the induction of micronuclei in the fish erythrocytes (Al-Sabti, 2000). Genotoxicity and mutagenicity to HepG2 mammalian cells have been reported for C.I. disperse blue 291 dye via the induction of DNA fragmentation, formation of cell-bearing micronuclei, and an increase in the index of apoptosis (Tsuboy et al., 2007). A similar effect on DNA damage is also found in higher plants. Industrial effluents stained by reactive dyes were demonstrated to lead micronucleus and chromosome aberration in Allium cepa test systems (Caritá and Marin-Morales, 2008).
Reactive red 141 (RR141) is a diazo reactive dye with bright red color, high molecular weight sulfonated diazo reactive dye substituted with aromatic amines (MW 1,774 g/mol, C52H26Cl2N14Na8O26S8, and λmax 543 nm) (Fig. 1) Both sulfonated and unsulfonated azo dyes have a negative effect on the wastewater as they are difficult to be naturally degraded. Besides, the dye has been widely used in different industries, it is a source of water contamination (Arni et al., 2021). Toxicity tests of RR141 were done in green algae (Chlorella sp.) and water fleas (Moina macrocopa) (Vinitnantharat et al., 2008). This revealed that algae could utilize dyestuffs as a carbon source, and the 96-h EC50 of RR141 to Chlorella sp. was 95.55 mg/L, while dye toxicity to water fleas, the 48-h LC50 was 18.26 mg/L. For the removal of RR141 dye from an aqueous solution, an alternatively low-cost adsorbent (corn stover with 3-aminopropyltrietoxysilane, CS-APTES) was developed and employed for capacitive adsorption (Carijo et al., 2019), as well as pyrrhotite ash (Mouldar et al., 2020), nanochitin particles (Boonurapeepinyo et al., 2011), and photodegradation by CdS nanomaterials (Senasu & Nanan, 2017). Various reactive textile dyes can also be decolorized by the bacteria isolated from the soil sample collected from contaminated sites, i.e., Pseudomonas sp. (Kalyani et al., 2008). A recombinant Escherichia coli strain containing genomic DNA fragments from an azo-reducing wild-type Pseudomonas luteola strain displayed a magnified reactive azo dye (reactive red 22) decolorization (Chang et al. 2000). Such synergistic decolorization by carbon adsorption and biodegradation of Pseudomonas luteola strain in biological activated carbon (BAC) process has been trial to treat azo-dye contaminants in textile wastewater (Lin and Leu, 2008). For the chemical degradation of RR141, the photo–Fenton reaction was demonstrated to be most effective by using CuFeO2 as a catalyst, together with Bacillus lentus BI377 (Arni et al., 2021).
Since higher plant offers a helpful system for screening and monitoring the effect of harmful or poisonous substances in the environment, the commonly used legume crop mung bean (Vigna radiata (L.) Wilczek) (a diploid, 2n = 22) is one of the optimal choices for physiological and molecular analyses in toxicology (Raj et al., 2014). Its seedling is a significant short-term assay for phytotoxicity assessment employing different parameters, i.e., seed germination and seedling vigor index, to consider the impacts of toxic substances on plant growth (Zeyad et al., 2019; Yadav et al., 2019). The mung bean test, a simple, fast, sensitive, and highly reproducible plant model, has been practically regarded for the toxicity evaluation of environmental contaminants presented in water, wastewater, and soil. For genotoxicity in the plant model, it has been commonly studied in the root tip cells of Allium cepa L. (Haq et al., 2016). Besides, other plants have also been used in phytotoxicity/genotoxicity testing with the seeds of sorghum (Sorghum vulgare) and urad bean (Phaseolus mungo) (Kalyani et al., 2008), kidney bean (Phaseolus vulgaris) (Enan, 2006; Cenkci et al., 2009), soybean (Glycine max L.) (Özkan & Aksoy, 2019), tomato (Lycopersicon esculentum, Mill) seed varieties (Jaja and Odoemena, 2004), oat plants (Avena sativa) (Swaileh et al., 2008), spinach (spinacia oleracea L.) (Lawal et al., 2011), and water hyacinth (Eichhornia crassipes) (Moghanm et al., 2020).
Recent advances in molecular biology have led to the development of selective and sensitive assays for DNA analysis in ecogenotoxicology. Various DNA-based techniques, i.e., RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), SSR (simple sequence repeats), ISSR (inter simple sequence repeats) and VNTR (variable number of tandem repeat), are generally used to evaluate variation in DNA sequence levels (Atienzar and Jha, 2006). Since the technique RAPD has been successfully applied to detect genotoxicity with clear revealed differences in the DNA fingerprints of organisms in conditions with the genotoxic agents, it has also been employed to detect genomic sequence changes in plants exposed to DNA damaging agents, including toxic metal ions, e.g., cadmium on barley (Hordeum vulgare), rice (Oryza sativa L.) and wheat (Triticum aestivum L.) (Liu et al., 2005; Liu et al., 2007; Azimi et al., 2013), mercury on Sesbania grandiflora L., duckweed (Lemna minor) (Malar et al., 2015; Zhang et al., 2017), arsenic on rice (Oryza sativa L.) (Ahmad et al., 2012), flusilazole on Allium cepa (Ozakca and Silah, 2013), and aluminium, nickel and cobalt on maize (Zea mays L.) (Erturk et al., 2013, Taspinar et al., 2018). Other reports with increase annealing temperature to reduce non-specific reaction during PCR reaction, such HAT-RAPD (high annealing temperature random amplified polymorphic DNA) techniques allow discrimination between closely related and morphologically indistinct species, providing greater polymorphism, reproducibility and resolution (Wongsawad and Wongsawad, 2010). The amount of DNA damage and mutations determined through genomic DNA by RAPD as a molecular marker technique has been shown as the genotoxic effect of toxic chemicals regarded to alter polymorphism and genomic template stability (GST) through the changes in the RAPD band profile (Cenkci et al., 2010).
This research aimed to study the phytotoxicity and genotoxicity of RR141 dye in mung bean seedlings. Phytotoxicity assessments included seed germination and seedling growth experiments in pot plants for short-term and long-term dye exposures. Genotoxicity measured by HAT-RAPD for estimating polymorphism and percentages of genomic template stability (GTS) was also evaluated.