*2.8. Statistical Analysis*

Student's *t*-test (*p* ≤ 0.05) was applied to find significant differences between values of heavy metals and genotoxicity parameters like mitotic inhibition (MI), physiological aberrations (PA), clastogenic aberrations (CA), and total aberration (TA) for untreated and treated effluents of the same industry. Chi-square test (*p* ≤ 0.05) was used to calculate the statistically significant differences between the values of genotoxicity parameters (MI, PA, CA, and TA) for effluents and the negative control. Statistical analysis was performed using Minitab version 14.0 (State College, PA, USA).

#### **3. Results and Discussion**

#### *3.1. Physico-Chemical Characteristics of Industrial Effluents and Soil*

Physico-chemical parameters of studied soil and industrial effluent samples are shown in Table 2. The mean pH of soil, that is, 8.02 was observed to be within the permissible limit of 6.5–8.5 and is alkaline in nature. Electrical conductivity (EC), which indicates the soil salinity, was also found to be within the prescribed limit. The studied soil sample had a sand content of 33.49%, silt content of 26.05%, and clay of 40.45%. The Ca, Mg and Na contents (mg/kg) of the soil sample were observed to be 120.24, 176.64, and 343.08, respectively. pH, the most significant parameter for the assessment of water quality, ranged from 6.67 to 8.90 and remained within the prescribed limits. Bulk density (BD) plays a vital role in the growth of plants as high BD can decrease the root penetration in soil. The mean bulk density (BD) of the studied soil sample was found to be 1.08. Organic matter content plays a chief role in the fertility of agricultural soils. Total organic carbon (TOC) in the present study was observed to be 2.22%. The pH of treated effluent of textile industry A (AT) was observed to be acidic while all other effluent samples showed basic pH. Dissolved calcium and magnesium in water are the two most common minerals that determine water hardness. Total hardness (mg/L) for AU, AT, BU, and BT was found to be 111.33, 151.33, 191.33, and 104.67, respectively. The calcium content in effluent samples ranged between 17.90–60.65 mg/L and magnesium content was seen in the range of 17.58–70.33mg/L. The order of chloride content (mg/L) was observed

to be: 232.41 (AU) > 142.47 (AT) > 114.07 (BU) > 66.74 (BT). The electrical conductivity varied from 511μS/cm to 1908.67 μS/cm. The value of total suspended solids (mg/L) was found to be minimum for AU (106.67) and maximum for AT (585) while the content of total dissolved solids (mg/L) was found to be minimum for AT (201.67) and maximum for BU (3666.67). The value of alkalinity (mg/L) varied from 356.67 to 656.67; sodium content (mg/L) from 141.08 (BU) to 333.63 (AU); and phosphate content (mg/L) from 1.48 (AT) to 2.08(BU). The value of total solids (mg/L) was found to be in the order of BU (3893.33) > AU (3473.33) > BT (2000) > AT (786.67).

According to Paul et al. [51], the basic nature of the pH of the industrial effluents was because of the usage of scouring and bleaching agents along with other various chemicals like caustic soda, hydrogen peroxide, and soap while pulping the waste. Similar results were also reported by Ramamurthy et al. [52]. Alkaline pH can have an adverse effect on soil permeability and soil microflora [53]. Total solid (TS) levels were higher in both AU and BU, which can lead to high turbidity of the water bodies into which these effluents are discharged. In the present study, higher levels of total dissolved solids (TDS) indicated high salt content in the effluents analyzed. Paul et al. [51] also reported higher values of TDS (2264–7072 mg/L) in textile effluents. The higher values of TDS are due to the addition of different chemicals during pulping and bleaching processes, which can have detrimental effects on aquatic flora and fauna.

The degree of hardness becomes higher as the calcium and magnesium content increases and is related to the concentration of multivalent cations dissolved in the water. The change in alkalinity depends on carbonates and bicarbonates, which in turn depends upon the release of CO2. The amount of total alkalinity in the effluents during the present study ranged from 356.667 to 656.667 mg/L. The hardness of water is mainly due to the presence of calcium and magnesium ions, and it is an important indicator of the toxic effect of poisonous elements [54].


**Table 2.** Physico-chemical characteristics (Mean ± S.E.) of collected samples (textile industrial effluents and soil) from Ludhiana, Punjab (India).

 (AU: Untreated effluent of textile industry A; AT: Treated effluent of textile industry A; BU: Untreated effluent of textile industry B; BT: Treated effluent of textile industry B; TOC: Total organic carbon). a BIS [55]; b Awashthi [56], \* Indicates statistically significant difference between values of parameters in untreated and treated effluents of the same industry. (Independent Student's *t*-test, *p* ≤ 0.05).

## *3.2. Heavy Metal Estimation*

#### 3.2.1. Heavy Metal Contents in Industrial Effluents

The results of a metal analysis of industrial effluents are given in Table 3. The contents (mg/L) of Cd, Cr, Co, Cu, Pb, and Zn observed for untreated effluents of textile industry A were 0.004, 0.06, 1.72, 0.02, 0.13, and 0.09, respectively, while the values (mg/L) of these metals for treated effluents were below detection limit (BDL), 0.05, 1.33, 0.02, 0.11, and 0.02, respectively. The heavy metal contents observed for untreated effluent of textile industry B were in the order Co (1.69) > Cd (1.33) > Pb (0.14) > Zn (0.13) > Cr (0.06) > Cu (0.03), while in the case of treated effluent, the order was Co (1.42) > Pb (0.11) > Zn (0.07) > Cr (0.06) > Cu (0.01) > Cd (0.001). Among the heavy metals analyzed, the contents of Co were found to be above the standard limits for the discharge of effluents from textile industries.

Among all metals, Co content was observed to be high in untreated and treated effluents of both textile industries A and B as compared to the prescribed limits. All other tested heavy metals were found to be within the permissible level. Metal contamination in textile effluents was reported to occur because of the wide usage of chemicals, colorants, mordants, and other additives like caustic soda, sodium carbonate, etc. during the manufacturing processes [57]. Adinew [58] also reported that different heavy metals such as cobalt, copper, and chromium in textile effluents were present within the dye chromophores. The presence of heavy metals in effluents produces several adverse effects on living organisms [59]. Metals like chromium, zinc, iron, mercury, and lead were reported to pose environmental challenges [60]. In the present study, there was a significant difference (*p* > 0.05) in Co, Pb, and Zn content between untreated and treated effluents of both textile industries.


(AU: Untreated effluent of textile industry A; AT: Treated effluent of textile industry A; BU: Untreated effluent of textile industry B; BT: Treated effluent of textile industry B). \* Indicates statistically significant difference between values of heavy metals in untreated and treated effluents of the same industry (Independent Student's *t*-test, *p* ≤ 0.05). a Awashthi [56]; b European Union Standards (EU) [61].

#### 3.2.2. Heavy Metal Contents in Soil

Table 3 shows the contents of various studied heavy metals in the soil of the agricultural field collected from the vicinity of textile industries. The contents (mg/kg) of Cd, Cr, Co, Cu, Pb, and Zn observed in samples were 1.33, 16.43, 214.60, 13.63, 57.33, and 92.52, respectively.

The chief sources of heavy metals in the roadside agricultural soils were documented to be the parent rock material, vehicular emissions, industrial activities, and agrochemicals like fertilizers and pesticides used for cultivation [18,62]. In the present study, soil samples were collected from agricultural fields in the vicinity of the textile industries. In the present study, three metals, that is, Cu, Cr, and Zn were observed to be low while Pb was higher in comparison to heavy metal contents reported from other parts of Punjab [63,64]. Also, Cd content was observed to be high in the present study in comparison to other parts of the world [65–70]. Cadmium is a toxic metal that causes serious health problems to humans, animals, and plants. Bhatti et al. [61] reported that the sources of the high levels of cadmium in the agricultural soils of Punjab were due to the usage of various agrochemicals like NPK (nitrogen, phosphate, potassium) fertilizers, pesticides, weedicides, etc. Industrial activities, lead mines, farmyard manure, and sewage sludge applications, etc. are reported to be the main sources of lead pollution in agriculture and plants [71]. Zinc pollution in roadside soils was caused by traffic-related activities such as vehicular emissions and weathering of crash barriers [18,72,73].

Among the different metals analyzed, the content of Co was observed to be maximum in the soil sample analyzed during the present study. Cobalt is documented to occur naturally in soils following two main pathways, that is, weathering of rocks comprising of minerals and breakdown of organic matter. The major mechanism involved in cobalt content in soil includes the anthropogenic usage of cobalt salts. Smaller amounts of cobalt can also enter the soil from the airborne transport of particulate emissions and application of sewage sludge onto fields. Heavy metal mobility in soil was reported to be inversely related to the strength of adsorption by soil constituents. However, the adsorption of cobalt to soils was reported to be rapid by Kim et al. [74]. Cobalt is reported to be one of the beneficial elements for the growth of higher plants although there is no report available regarding its direct role in plant metabolism [75]. However, some studies showed that cobalt was required for nitrogen fixation by bacteria in the root nodules of plants belonging to the leguminous family [76,77]. Cobalt has also been documented to be necessary for the processes of stem growth, elongating the coleoptiles, and expanding leaf discs. Moreover, cobalt reduced the peroxidase activity resulting in the breakdown of Indole acetic acid (IAA). Application of cobalt through seed treatments improved the germination of a seed, stand establishment, growth, yield, and quality [78]. Yet, a higher concentration of cobalt was found to be toxic, causing chlorosis and necrosis and inhibited root growth by retarding cell division, hindering the uptake and translocation of nutrients and water [78,79].

Copper and zinc are considered essential elements for plant nutrition, however, these can also cause toxic effects, if their concentrations exceed the required limits [80]. Plants mainly absorb zinc as a divalent cation, which acts either as a metal component or enzymes or as a functional, structural, or regulatory co-factor of many enzymes [81]. Despite being a non-essential element, cadmium can also ge<sup>t</sup> highly accumulated in plants as reported by Nadian [82]. Pb being a toxic element decreases the biomass growth and disrupts the total chlorophyll content of plants [83]. Naureen et al. [84] observed that the concentration of heavy metals in plants varied from species to species. Another study reported that the accumulation of selected metals varied greatly among plant species and uptake of an element by a plant was primarily dependent on the plant species and the soil characteristics [85]. Similar observations were also made by Rattan et al. [86]. On the other hand, Muchuweti et al. [87] reported that the excessive accumulation of heavy metals in soils was due to elevated levels of heavy metals in wastewater used for irrigation that led to increased uptake of metals in crops.

#### 3.2.3. Heavy Metal Contents in Leaves of Plants

figfig:soilsystems-1285565-f001 shows the heavy metal contents in leaf samples of three plants *Cannabis sativa* L., *Chenopodium album* L., and *Ricinus communis* L. from the study area. The order of the heavy metals in the leaves of the three plants was observed to be Co > Zn > Pb > Cr > Cu > Cd. However, among three plant species, the order of heavy metal contents observed for Cd and Pb was *Cannabis sativa* L. > *Ricinus communis* L. > *Chenopodium album* L.; for Cr and Zn the order was *Chenopodium album* L. > *Ricinus communis* L. > *Cannabis sativa* L.; for Co the order was *Ricinus communis* L. > *Chenopodium album* L. > *Cannabis sativa* L.; and for Cu it was *Ricinus communis* L. > *Cannabis sativa* L. > *Chenopodium album* L.

**Figure 1.** Heavy metal contents in three plant species viz., *Cannabis sativa* L., *Ricinus communis* L., and *Chenopodium album* L. FAO/WHO, Adapted with permission from [56]. 2001, Indian standards: Awashthi.

In all the collected plant samples, the concentrations of different metals were observed to be above the European permissible limits like 50 mg/kg for cobalt, 0.2 mg/kg for cadmium, and 1 mg/kg for chromium. However, two metals and contents were determined to be lower than the Indian standards like Cd < 1.5 mg/kg and Cr < 20 mg/kg [88,89]. The concentration of copper in all three plant species was recorded to be less than both Indian standards of 30 mg/kg and European permissible limits of 73 mg/kg. Lead content was found to be higher than both Indian standards (2.5 mg/kg) and European permissible limits (0.43 mg/kg). The leaves of *Ricinus communis* L. and *Chenopodium album* L. had a higher zinc content than the Indian standards of 50 mg/kg and lower than that of European limits of 100 mg/kg. The content of zinc in *Cannabis sativa* L. was recorded to be less than both Indian standards and European permissible limits.

The variations of heavy metal contents in soil have a direct influence on the accumulation of heavy metals in plants. However, the heavy metal accumulation in plants also depends on the type of plant species, plant organelles, and traffic density. The plant species included in the present study were preferred because these were wildly grown along the boundaries of the agricultural field at the time of sampling. The contribution of human beings to metal concentrations in the terrestrial environment has arisen mainly from mining, smelting, and industrial activities [90].

#### *3.3. Metal Bioaccumulation Factor (BAF)*

BAF is one of the main indices that provide insight into the heavy metal uptake capacity of plant species. In the present study, BAF values were used to estimate and compare the extent of accumulation of various metals such as Cd, Co, Cr, Cu, Pb, and Zn in leaves of the three plants from the soil. Figure 2 presents the BAF values for different plant samples collected during the study. A BAF value above 1 was observed only for cobalt (1.042) in the leaves of *Ricinus communis* L, which indicates a high level of metal bioaccumulation in *Ricinus communis* L. The order of accumulation of heavy metals in the leaves of *Ricinus communis* L. was Co (1.04) > Zn (0.56) > Cu (0.48) > Cr (0.46) > Pb (0.26) > Cd (0.18); for *Chenopodium album* L. the order was Co (0.78) > Cr (0.66) > Zn (0.62) > Cu (0.46) > Pb (0.26) > Cd (0.09); and for *Cannabis sativa* L. it was Co (0.73) > Cu (0.47) > Cr (0.45) > Cd (0.31) > Pb (0.28).

**Figure 2.** Bioaccumulation factor of Soil to plant.

The heavy metal accumulation examination provides very important information about the phytoremediation potential of the plant species. Transport and accumulation of heavy metals from soil to edible parts act as the major pathway for the entry of heavy metals into the food chain, which ultimately leads to various harmful effects [91]. The exposure of heavy metals to human beings leads to various health problems such as nervous system disorder, skin ailments, stomach problems, kidney damage, bone, and lung diseases [92–94]. The BAF of heavy metals depends upon the bioavailability of metals, which in turn depends upon the concentration of metal in soil, its chemical forms, the difference in uptake capability for different metals, and the growth rate of different plant species [53,95]. Different metals are accumulated in plants at variable rates depending on various factors such as physiology, requirements, and the metal uptake mechanism of plants, the physico-chemical characteristics of soil such as soil texture, soil pH and

soil organic matter, as well as quantity of heavy metals present in the soil [94,96–98]. According to the guidelines given by Baker [99], BAF values greater than 1 indicated that the plant was an accumulator for the metal being analyzed and considered as harmful for plant health [53,100]. It is documented that *Ricinus communis* has good tolerance and phytoremediation potential for the removal of nickel (Ni) from contaminated land areas [101,102]. In the present study also, maximum BAF values for Co were observed in *Ricinus communis* L., which showed that this plant had higher metal bioaccumulation capacity than *Chenopodium album* L. and *Cannabis sativa* L. The mechanisms of cobalt accumulation are still not properly defined. However, there are some Cu accumulators which have the potential for Co accumulation as well due to similar mechanisms in the accumulation of different heavy metals [103]. Considering the Co toxicity on the targeted cellular system of plants that can hyperaccumulate, Co has been found to be evolved in regulating Fe homeostasis thus avoiding the accumulation of free ions that can induce oxidative stress [104]. Wong et al. [105] reported that heavy metals in carbonate-bounded form were more bioavailable than the presence of metal in any other fractions. Tamoutisidis et al. [106] revealed that heavy metals were transported passively from the root system to the shoot system through xylem vessels and were accumulated in the zones of high transpiration rates.

#### *3.4. Genotoxicity of Industrial Effluent*

The genotoxic potential of textile industrial effluents, before and after treatment was evaluated on the basis of percent aberrant cells. The results of genotoxic potential analysis of effluents and distilled water (negative control) using the *Allium cepa* test system are shown in Table 4. Among all physiological aberrations, delayed anaphases and stickiness were the most frequent type of aberrations while chromatin bridges dominated among clastogenic aberrations. Total chromosomal abnormalities in the meristematic cells of root tips of *Allium cepa* exposed to untreated effluents were significantly higher as compared to those exposed to treated effluents. The reduced mitotic index clearly indicates the cell division reduction in the root meristematic cells, which may be due to the collaborating effects of a complex mixture of cytotoxic chemicals like metals present in the textile industrial effluents. The total percentage aberration including both physiological (laggards, vagrants, stickiness, delayed anaphases, and c-mitosis) and clastogenic (chromatin bridges and chromosomal breaks) aberrations are shown in Figure 3. The total percent aberrant cells were observed to be 29.36% for AU, 27.48% for BU, 19.69% for AT, 16.52% for BT, and 3.84% for the negative control. The results obtained indicate the less toxic nature of the treated effluents of both textile industries (A and B) as compared to the untreated effluents, which can be due to the decrease in heavy metal contents.



(NC: Negative Control; (AU: Untreated effluent of textile industry A; AT: Treated effluent of textile industry A; BU: Untreated effluent of textile industry B; BT: Treated effluent of textile industry B); TDC: Total Dividing Cells; MI: Mitotic Index; PA: Physiological Aberration; CA: Clastogenic Aberration; TA: Total Aberration). \* Indicates statistically significant difference between values of genotoxicity parameters (MI, PA, CA, and TA) for untreated and treated effluents of same industry (Independent Student's *t*-test, *p* ≤ 0.05). # Indicates statistically significant difference between values of genotoxicity parameters (MI, PA, CA, and TA) for effluents and negative control (Chi square test, *p* ≤ 0.05).

**Figure 3.** Induction of physiological and clastogenic chromosomal aberrations in root tip cells of *Allium cepa* under exposure to industrial effluents and distilled water (Negative Control). Physiological aberrations (Cm: C-mitosis; Da: Delayed anaphase; Lg: Laggards; St: Stickiness; Vg: Vagrants; Aa: Abnormal anaphases; Am: Abnormal metaphases); Clastogenic aberrations (Cb: Chromatin bridges; Bk: Chromosomal breaks), Sample codes (AU: Untreated effluent of textile industry A; AT: Treated effluent of textile industry A; BU: Untreated effluent of textile industry B; BT: Treated effluent of textile industry B); NC: Negative control.

The results observed in the present study indicate the mitogenic, as well as the clastogenic effects of the textile effluents, which were evident from the low value of the mitotic index (MI) and higher values of the chromosomal aberrations assay when compared to the results obtained from treatment with negative control (distilled water). The statistical analysis (Chi square test) also revealed that there is a significant difference between the values of the genotoxicity parameters viz., physiological aberration (PA), clastogenic aberration (CA), and total aberration (TA) along with mitotic index (MI) for effluents of both textile industries (A and B) and negative control at *p* ≤ 0.5). Chromosomal aberration is an important indicator for assessing the genotoxicity of textile effluents [107]. The *Allium cepa* root chromosomal aberration assay has been widely used for cytotoxic as well as genotoxic mitotic studies [41,108–110]. The reduction in the values of mitotic index, in the present study, indicated the cytotoxic effects, whereas the induction of chromosomal and nuclear abnormalities showed genotoxic effects. Both cytotoxic and genotoxic effects were endorsed by various environmental pollutants [44].

The higher genotoxic response of root tip cells under exposure to untreated effluent of textile industry A as compared to untreated effluent of B industry can be attributed to the presence of high content of Cd, Co, Na, and Cl. Some authors have reported the cytotoxic and genotoxic effects using the *Allium cepa* test system following exposure to heavy metals [111,112]. Cd has been shown to reduce the mitotic index (MI) and enhance the induction of chromosomal aberrations, as well as micronuclei, in various studies [113–116]. Grover and Kaur [117] studied the genotoxic potential of textile and paper mill effluents and sewage water following the *Allium cepa* chromosomal aberration assay and reported that the industrial effluents induced the formation of micronuclei and chromosomal abnormalities in the root tip cells of *Allium cepa*. Genotoxicity of both untreated and treated textile industrial effluents was evaluated using the *Allium cepa* test system by Vijayalakshmidevi and Muthukumar, [118] and they observed a reduction in the mitotic index, as well as induction of various types of chromosomal aberrations in the root tips exposed to effluent. It is possible that some chemicals in the complex chemical mixtures could have stimulatory effects on the mitotic process while some others might have mito-depressive effects [43]. Similar results were also reported for genotoxicity of textile industrial effluents using the *Allium cepa* test system by other authors [42,108,119,120] and they demonstrated the induction of chromosomal abnormalities and decrease in the mitotic index in root tips cells treated with the effluent. Therefore, mitotic responses observed in this study could be due to the overall collaborative effects such as additive, antagonistic, and synergistic of the complex chemical mixtures in the effluents on the root meristematic cells.

## *3.5. Pollution Assessment*

The Igeo, *CF*, Cdeg, mCdeg, PI, PLI, ERi and RI of the agricultural soil in the present study were calculated based on the heavy metals content in the studied soil sample. Table 5 indicates that the *CF* value of heavy metals in studied area was ranked as: Co (21.46) > Cd (13.57) > Pb (2.87) > Zn (1.30) > Cu (0.55) > Cr (0.47) and the Igeo value ranked as Co (3.84) > Cd (3.18) > Pb (0.93). Pollution levels of contamination factor (*CF*) were classified as: low contamination (*CF* < 1), moderate contamination (*CF* value in the range of 1–3), considerable contamination (*CF* value in the range of 3–6), and very high contamination (*CF* > 6) by Taylor and Mclennan [121] and Hakanson [122]. The *CF* value indicated that the soil is extremely polluted by Cd and Co, moderately polluted by Pb and Zn whereas unpolluted by Cu and Cr. The result of Igeo showed that soil is heavily contaminated by Cd and Co whereas uncontaminated by Cr, Cu, and Zn on the basis of the classification given by Muller [123] and Taylor and Mclennan [121]. Considering the Cdeg values > 32, PI values > 3, and PLI > 3, the studied soil was found to be extremely polluted with heavy metals whereas mCdeg (6.70) value in the present study indicated that soil has a high degree of contamination. ERi values for Cr, Cu, Pb, and Zn were observed to be below 40, indicating low potential ecological risk from these metals whereas Cd showed considerable potential ecological risk and Co exhibited very high potential ecological risk. In the present work, ERi values showed that Co is the major pollutant in the area which indicates that agricultural managemen<sup>t</sup> is a probable cause of heavy metals accretion. The potential ecological risk index (RI) demonstrated that the study area had considerable ecological risk considering the RI value in the range of 300–600.


**Table 5.** Metal pollution indices for collected soil samples from Ludhiana, Punjab (India).

Igeo: geoaccumulation index; CF: contamination factor; Cdeg: degree of contamination; mCdeg: modified degree of contamination; PI: Numerow's pollution index; PLI: pollution load index; ERi: potential ecological risk factor; RI: potential ecological risk index.

#### *3.6. Human Health Risk Assessment*

The non-carcinogenic, hazard quotient (*HQ*), and hazard index (*HI*) of analyzed heavy metals (Cd, Cr, Co, Cu, Pb, and Zn) through three exposure pathways, that is, ingestion, dermal contact, and inhalation for adults and children were calculated and results were shown in Table 6. The values of *HQ*ingestion, *HQ*dermal, and *HQ*inhalation for all studied heavy metals were found to be lower than 1 for both adults and children and thus indicated that there is no obvious risk to the population. The total carcinogenic risks (TCR) were calculated only for Cr as cancer slope factors (*SF*) for all three exposure pathways (ingestion, dermal contact, and inhalation) are not available for other heavy metals. The total carcinogenic risk value was found to be in the range of the permissible limit of 1 × 10−<sup>6</sup> to 1 × <sup>10</sup>−4, as provided by USEPA [28]. The results of cancer risk were shown in Figure 4.


**Table 6.** Exposure values for non-carcinogenic risks for adults and children from different exposure pathways in the study area.

> (ADI: Average daily intake; HQ: Hazard quotient; HI: Hazard index).

**Figure 4.** Distribution of carcinogenic risk of chromium (Cr) for adults and children in the study area. (CR; carcinogenic risk; TCR: total carcinogenic risk).
