Estimation of Biological Toxicity by Copper oxychloride on Pisum sativum L. and Vigna radiata L.

The present century has witnessed the dependence of society on the products of chemical industry to provide food and other necessities to uplift our quality of life. The overpopulation, limitation of agricultural fields, food shortage in whole world need to produce more crops in a limited area. But so many types of fungal diseases like Powdery mildew of Vigna radiata caused by Erysiphe polygoni  (Ganesha, 2000; Suryawanshi et al., 2009), Cercospora leaf spot of Vigna radiata caused by Cercospora canescene (Khunti et al., 2005) and fungal disease like wilt disease of Red gram (Cajanas cajan) caused by Fusarium oxysporum (Raju et al., 2008), which cause yield loss of pulse grains. Therefore commercially many types of fungicides have been formulated for management of fungus in the crop fields and home gardens. They act quickly to cure fungal disorders to increase the rate of production. The widespread use of these fungicides may cause environmental and food contaminations (Tort and Turkyilmaz, 2003; Fisun and Rasgele, 2009). Adoption of conservation agriculture was found to be climate smart agricultural technique (IARI, Annual Report 2017-18). The first landmark in the control of phytopathogens is the discovery of Bordeaux mixture by P. M. A. Millardet in 1885. After that, Copper based compounds emerged as the most successful fungicides. Copper is an essential nutrient for plant, it plays an irreplaceable role in the function of a large number of enzymes which catalyse oxidative reaction in a variety of metabolic pathways (Lolkema and Vooijs, 1986; Marschner, 1995). However when absorbed in excess, copper can lead to inhibition of plant growth (Reboredo and Henriques, 1991; Ouzounidou, 1994), inhibition of root elongation, disturbance of mitosis (Fiskesjo, 1988) and damage root epidermal cells and root cell membrane (Ouzounidou et al., 1995). Pesticide toxicity results in reduction of chlorophyll and protein contents, accompanied by decreased photosynthetic efficiency of plants (Sharma et al., 2019). One of the copper based fungicide is Copper oxychloride (commercial name Blitox) was used against Alterneria leaf and flower blight of Marigold (Jash et al., 2004).
       
Peas are used as a field crop in 3 main areas: human consumption, livestock fodder and as a source of hay (Afonin et al., 2008). Pea is highly susceptible to pre emergence damping off and after emergence root and foot rots caused by soil borne and seed borne fungal infection (McPhee, 2003). The Mature mung bean seeds and flour is used in variety of dishes and sometimes grown for fodder, green manure, cover crop (Mall, 2017).
       
The current study was designed due to nutritional importance of Pisum sativum and Vigna radiata to explore the effect of copper oxychloride (a blue labeled fungicide) on their germination, growth parameters, chromosomal abnormalities and biochemical changes on growing seeds. So Pisum sativum and Vigna radiata used as a bioassay indicator to ascertain toxicity of copper oxychloride.

Experimental materials and chemical
 
Healthy seeds of Pisum sativum L. and Vigna radiata L. and the fungicide copper oxychloride 50 were procured from Rathindranath Krishi Vigyan Kendra, Visva-Bharati, Santiniketan, West Bengal. The fungicide used in this work was copper oxychloride 50 (CAS No. 1332-40-7) (Blitox. CuCl₂.3CuH₂O₂).
 
Experimental set-up and record of observation data
 
The present work had been carried out in green house of Departmental Garden of Botany, Visva Bharati, Santiniketan, in the month of December to March of 2017. Equal number and same sized seeds of both P. sativum and V. radiata were surface sterilized first with 70% ethanol for 3 minutes followed by 2% sodium hypochlorite for 3 minutes and washed thoroughly with sterilized distilled water for 3-4 times and dried at room temperature. The seeds were directly imbibed in aqueous solution of copper oxychloride of different concentration (0.5%, 1%, 2%) for 6 hours along with untreated control at room temperature. After treatment with fungicide solution, seeds were thoroughly washed and transfer in earthen pots (1:1 ratio of sand and soil) in three replicates for each treatment. The pots were regularly watered to maintain proper moisture. This study was repeated two successive years (2018 and 2019) at the same period of the year for validation of observational data.
       
Morphologically the radical emergence of nearly 1 mm has been considered as germinated seeds. After radical emergence (nearly 24-28 hours later), germination rates were counted for each concentration along with control. The length of radical and plumule were measured on 4^th and 8^th day interval to calculate R/P ratio.
 
Estimation of biochemical parameters
 
Total soluble protein were estimated from progeny seeds of treated and control plants by the method of Bradford (1976). The amount of proline was estimated from the leaves of treated and control plants by the method of Sadasivam and Manickam (1992). For estimation of leaf chlorophyll, standard method of Arnon (1949) was followed.
 
Estimation of cytotoxic parameters
 
The slides were prepared by squashing technique following the method of Roy et al., (2014). Five slides replication wise per treatment were prepared and viewed under 400X magnification (atleast 10 fields) to calculate mitotic index and abnormality index. The Mitotic inhibition calculated by the following formula. Statistical analysis
 
The regression analyses was performed using Microsoft Office Excel version 2013. One-way ANOVA with post-hoc DMRT were used to test for statistically significant differences of means between each treatment and corresponding control at P<0.05 level. Box plot analysis was done to compare the range of variation through median using SPSS of version 20.

Effect of copper oxychloride on seed germination and radical-plumule ratio
 
The rate of seed germination has recorded more increased value in V. radiata (33.48%) than P. sativum (20.44%) at higher (2%) concentration of the fungicide, in compare to untreated control (Fig 1). The seed germination frequency has increased progressively with the increased concentration (0.5%, 1%, 2%) of copper oxychloride in both tester plants. The R/P ratio of both Pisum sativum and Vigna radiata were decreased with the increased concentration of copper oxychloride show that higher concentrations of fungicide has a reducing effect on the ratio of embryonic root and shoot length (Fig 2 a,b). The 2% concentration recorded lowest value of root/shoot ratio. Linear regression analysis has shown that germination frequency and ratio of radical-plumule length on 4^th and 8^th day were significantly dependent on the increase concentration of the applied fungicide compare to control (Fig 3a,b; Fig 4a-d). The seed germination percentage is positively correlated with increasing concentration indicates significant effect of copper oxychloride where copper act as an essential micronutrient for seed germination at the initial growth stage (Verma et al., 2011).
 

 

 

 

 
Effect of copper oxychloride on total leaf chlorophyll, seed protein and leaf proline content
 
The amount of total leaf chlorophyll in control plants of V. radiata recorded less than P. sativum. However treatment of seeds with copper oxychloride significantly decreased the amount of total leaf chlorophyll in both plants with increasing concentrations (Fig 5a). V. radiata has shown drastic decreased value of total leaf chlorophyll than P. sativum at 2% concentration than control, 0.5 and 1%. Stress usually hampers stomatal functioning (Poschenrieder et al., 2004) and retardation of chlorophyll biosynthesis (Singh et al., 2003). There was marked increase in total seed protein (mg/gm) of both P. sativum and V. radiata with increasing concentration of copper oxychloride solution. Although, at the highest concentration (2%) total seed protein decrease suddenly which is significant in V. radiata (Fig 5b). Similar result was also observed by Verma et al., (2011), in protein content of shoot and root of Vigna radiata treated with copper sulphate. In case of total seed protein of both Pisum sativum and Vigna radiata, increased protein with increase concentration of copper oxychloride than control may be due to the plant defense mechanism (Maksymiec, 1997). Regression curve (Fig 6 a-d) show total leaf chlorophyll is negatively correlated and total seed protein is almost positively correlated with increasing concentration except 2% in V. radiata. The total leaf proline estimation show interesting result (Fig 5c). Proline is an amino acid, protects the plants from various stresses (Hayat et al., 2012) The total leaf proline show negative correlation with increasing concentration of copper oxychloride in P. sativum (Fig 6e). On the other hand, totally opposite result was observed in  V. radiata where at highest concentration (2%), the amount of total leaf proline has increased abruptly (15.03%) than control (Fig 6f). Chen et al., (2001) indicated that proline accumulation in detached rice leaves upon exposure to excess Cu was due to proteolysis and increased activities enzymes of proline metabolism. Higher accumulation of endogenous proline in V. radiata may be due to same reason at higher concentration. Linear regression analysis has explained clearly that, the variation of chlorophyll, protein and proline content of both the plants are significantly depending on the concentration of applied fungicide.  
 

 

 
Box plot analysis (Fig 7a,b) figured out that copper oxychloride has pronounced effect on R/P ratio as observed on 4^th day and 8^th day, followed by biochemical characters in both experimental plants. But the overall degree of fungicidal effect was comparatively more drastic in Vigna radiata.
 

 
Cytotoxic effect of copper oxychloride on root tip cells
 
It was observed that increasing concentration of fungicide resulted decreased mitotic index with increased percentage of chromosomal abnormality and mitotic inhibition in roots of P. sativum and V. radiata (Fig 8a,b). The copper oxychloride induced various types of chromosomal aberrations like stickiness, laggard, clumping, c-mitosis, fragmentation, multipolarity, diagonal anaphase with bridge (Fig 9, 10). The linear regression analysis confirmed that abnormality index and mitotic inhibition exhibited significant positive correlation with increasing concentration (Fig 11c-f). The cytological study confirmed that copper oxychloride acted as mitotic cell division depressor and chromosome aberration inducer on plant cells, when absorbed in high dosage (Fig 11a,b) because the inhibition of cell division occurred in cell cycle. The inhibition of spindle formation leads to abnormality such as stickiness, laggard chromosome and multipolarity. The mitotic inhibition in both plants increased with increasing concentration of copper oxychloride. The Copper-containing polyphenol oxidase (PPO is a tetramer that contains four atoms of copper per molecule and binding sites for two aromatic compounds and oxygen) seems to function in defense mechanism of cells. After fungal or bacterial infection of the cells, they produce hydroxyphenols and quinines having fungicidal or bacteriocidal properties (Vaughn et al., 1988).
 

 

 

 

The bioindicator plants chosen for study, Pisum sativum and Vigna radiata recorded very low percentage of morphological, biochemical and cytological damages at lower concentration of copper oxychloride. At lower concentration copper oxychloride may be act as essential micronutrient and plant growth stimulator but higher concentration resulted accumulation and inactivation of proteins resulting increased in total seed protein. Therefore it may infer from this study that copper oxychloride act as a safe fungicide for protection of P. sativum and V. radiata at lower concentration.

The first and second authors express their acknowledgement to Dr. Tustu Mandal for his kind help rendered during the experiment.