Physico-chemical properties of soil and FA-
The physico-chemical properties of soil and FA samples (Table-1) revealed that pH of soil was significantly (P < 0.05) improved from 7.48 in the control (T1) to 8.17 in 100% FA (T7) as we increased the concentration of FA in soil. The upsurge in pH of soil may be attributed to the counteraction of H+ ions by basic salts and solubilization of alkali metallic oxides of FA in the amended soils (Skousen et al. 2013). It has also been reported that an upsurge in soil pH resulted in the condensation of soluble cations in the FA and soil amendments (Sheoran et al. 2014). EC value significantly (P < 0.05) improved from T1 (0.001) to T7 (0.0047) with respect to increased application of FA. The maximum EC (0.0047 dS /m) was found in T7 (100% FA) while in T1 (control), 0.0012 dS /m was reported. FA addition to soil improved the Electrical conductivity by enhancing the concentration of soluble major and minor inorganic components (James et al. 2014). The same drift was observed in WHC (%) indicating a significant (P < 0.05) improvement from 14.28% in T1 to 43.72% in T7 at higher FA application with respect to control. The present study represented a gradual increase in Water holding capacity from control to 100% FA that may be due to the variation in particle size distribution and increases porosity (Panda and Biswal 2018). The similar trend was also spotted in moisture content (%) as it is significantly (P < 0.05) improved with FA application from control (1.931%) to 100% FA (3.971%). The enrichment in moisture content might be attributable to the binding potential of the FA-soil mixture to carry out the cation exchange reaction and with application of compaction effort the voids are employed by more water (Dixit et al. 2016). TDS was noticed to be improved by 66.6%, 75.91%, 76.8%, 84.09%, 84.81% and 85.06% at 10%, 20%, 40%, 60%, 80% and 100% respectively as compared to control. The bulk density of control soil (T1) was maximum at 1.19 g/cm3 and declined significantly (P < 0.05) with the addition of FA and hence was lowest (0.86 g/cm3) at 100% FA (T9). The bulk density however decreased with the increase in FA concentration in soil which may be attributed to an increase in clay particles (Mishra et al. 2017). This indicates a significant boost in the porosity and improved the water retention capacity of the soil.
Table 1
represents Physico-chemical properties of soil and FA amended soil. The values of all the treatments are means with S.D. of all the replicate measurement (n = 3)
Treatments
|
pH
|
WHC (%)
|
Moisture Content (%)
|
EC (Ms/m)
|
TDS (PPM)
|
Bulk Density (g/ml)
|
T1 (Control)
|
7.483 ± 0.27a
|
48 ± 3.27a
|
1.931 ± 0.43a
|
0.1 ± 0.14a
|
34.7 ± 4.47a
|
1.19 ± 0.137b
|
T2 (10%)
|
7.549 ± 0.26b
|
56 ± 5.66a
|
2.223 ± 0.23a
|
0.19 ± 0.15a
|
104.1 ± 3.28b
|
1.18 ± 0.107b
|
T3 (20%)
|
7.559 ± 0.22b
|
58.6 ± 3.98a
|
2.452 ± 0.14b
|
0.23 ± 0.1a
|
144.1 ± 8.18b
|
1.16 ± 0.08b
|
T4 (40%)
|
7.709 ± 0.23c
|
63 ± 2.16b
|
2.531 ± 0.56b
|
0.3 ± 0.20b
|
149.6 ± 5.46b
|
1.08 ± 0.23b
|
T5 (60%)
|
7.785 ± 0.23c
|
64 ± 3.27b
|
2.865 ± 0.60b
|
0.35 ± 0.28b
|
218.2 ± 4.7c
|
1.03 ± 0.01b
|
T6 (80%)
|
8.115 ± 0.18d
|
68 ± 8.64b
|
3.875 ± 0.67c
|
0.39 ± 0.26b
|
228.5 ± 7.2c
|
0.99 ± 0.107a
|
T7 (100%)
|
8.171 ± 0.30d
|
85.3 ± 3.77c
|
3.971 ± 0.44c
|
0.47 ± 0.25c
|
232.4 ± 2.08c
|
0.86 ± 0.11a
|
Different letters represent significant differences at P < 0.05 from Duncan’s multiple range test.
Effect of FA on heavy metal accumulation in Calendula-
Heavy metal concentration in different treatments of soil and FA and plant grown in the same treatments is represented in Table 2. The significant pattern for metal accumulation was found, when Calendula plants were grown on different treatments of FA amended soil. Metals concentration in the leaves of Calendula were found in the trend- Ni > Co > Fe > Mn > Zn > Cr > Cu, though, in fly ash treated soil the concentration was in the trend- Ni > Fe > Co > Mn > Zn > Cr > Cu. Surprisingly, the accumulation of heavy metals (Cu, Cr, Ni, Mn, Fe, Zn, and Co) was noticed higher in plants grown on FA amended soil (100% FA) than control soil. This increase in level of lethal heavy metals (As, Cd, Cr, Pb and Hg) was also reported in rice plant grown in FA amended soil (Padhy et al. 2016) rely upon the physio-chemical properties of soil mixture. The bioaccumulation factor (BAF) of soil to plant is presented in Fig. 1. The data indicated that BAF differs from one metal to another metal and also confirmed that the BAF factor of the metals (Fe, Cu, Ni and Co) from soil to plant was higher in FA mixed soils as compared to unamended garden soil except for Cr, Mn and Zn. The bioaccumulation of metals relies upon several environmental aspects such as solubility, salinity, mineral uptake, pH, texture, chemical composition of the metal ions. The decrease in nutrient uptake in grain rice might be consequent of reduced availability of nutrients (elements) at high fly ash application was observed (Singh et al. 2016).
Table 2
Heavy metal concentration in different treatments of soil and FA and in the leaves of the plants grown in these treatments
Treatment
|
Cr (ppm)
|
Mn (ppm)
|
Fe (ppm)
|
Cu (ppm)
|
Zn (ppm)
|
Ni (ppb)
|
Co (ppb)
|
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Soil
|
Leaf
|
Control
|
0.18
|
0.01
|
0.83
|
0.24
|
65.33
|
2.28
|
0.07
|
0.03
|
0.19
|
0.13
|
66.86
|
4.88
|
28.00
|
1.33
|
10%
|
0.21
|
0.01
|
1.12
|
0.42
|
78.37
|
4.48
|
0.07
|
0.03
|
0.22
|
0.13
|
75.28
|
4.02
|
31.18
|
2.88
|
20%
|
0.21
|
0.01
|
1.16
|
0.43
|
76.88
|
2.88
|
0.07
|
0.03
|
0.21
|
0.16
|
81.18
|
5.66
|
31.27
|
3.13
|
40%
|
0.22
|
0.02
|
1.58
|
0.50
|
89.45
|
5.54
|
0.07
|
0.03
|
0.23
|
0.18
|
95.75
|
6.95
|
39.87
|
4.87
|
60%
|
0.21
|
0.02
|
1.93
|
0.64
|
91.81
|
6.36
|
0.07
|
0.04
|
0.25
|
0.19
|
103.46
|
9.08
|
44.47
|
4.07
|
80%
|
0.26
|
0.02
|
1.82
|
0.55
|
99.42
|
6.50
|
0.08
|
0.04
|
0.28
|
0.20
|
110.74
|
7.64
|
45.38
|
4.81
|
100%
|
0.26
|
0.02
|
2.15
|
0.60
|
104.22
|
6.89
|
0.08
|
0.04
|
0.33
|
0.22
|
120.16
|
11.13
|
51.08
|
7.54
|
Biochemical Parameters-
Effect of FA on Pigment content- Chlorophyll is the essential component of photosynthesis and occurs as a green pigment in chloroplast of plant tissues (Rao 2015). Generally, photosynthetic pigment content depends upon the leaf area, stomatal response, and plant growth performance. Plant response to heavy metals in FA is shown in the results of pigment concentration viz chl a, chl b, total chl and carotenoids. Chlorophyll and carotenoids content exhibit environmental stress induced impairment to the photosystem (Pandey 2013). The data on photosynthetic pigment content (chl a, chl b, total chl and carotenoids) of Calendula leaves as influenced by FA and soil mixture are interpreted in Table 3. In this study, chl a, chl b, carotenoids and total chl content was noted to get enhanced under low doses of FA but reduced under high FA application. A significant (P <0.05) boost in pigment content was found from control to 40% and then reduced from 60% to 100% of FA in soil. The constant increase from control to 40% FA amended soil was noticed in flowering stage but declined as the FA concentration increased above 60%. In pre-flowering stage alike observations were recorded however in post flowering stage, all three photosynthetic pigments depicted a gradual decline in comparison to the pre-flowering as well as the flowering stage. The maximum increase (P < 0.05) in chlorophyll a (32.03%) was recorded with 40% FA in the flowering stage followed by pre-flowering stage (22.76%) and finally in the post flowering stage (15.9%) respectively. However, the maximum upsurge (P<0.05) for chlorophyll b was recorded for the flowering stage of T4 Treatment (50%) followed by the pre-flowering stage (46.66%) and lastly in the post-flowering stage (40%). The highest upsurge (P < 0.05) of total chlorophyll content was observed at 40% FA treatment during flowering stage (28.98%) then by pre-flowering (16.66%) and post flowering stages (9.99%). Similar observations were also reported on pigment content of Solanum nigrum L. and Solanum melongena grown on different FA doses (Robab et al. 2010 and Gond et al. 2013). Accumulation of heavy metals at high concentration of FA resulting in the decline of chlorophyll content (Qurratul et al. 2014). The disintegration of photosynthetic pigment leads to replacement of Mg2+ ions in chlorophyll molecules by certain metal ions like Cu2+, Zn2+, Cd2+, Pb2+ and Ni2+ (Qurratul et al. 2014).
Carotenoids are the commonly occurring antioxidants found in the plants which help in protecting the chlorophyll molecule in various environmental conditions (Praveena and Murthy 2014). The results demonstrated that the carotenoids content was significantly (P < 0.05) increased at 40% FA treatment in flowering stage (70.58%) then by pre-flowering (41.93%) and post-flowering stages (40.74%). The increased level of carotenoids at 40% FA amendment may ascribe to its role in the fortification of the plant cell against toxic effects of free radical species. Similar results were obtained on Chickpea and Helianthus annus grown in different concentrations of FA (Pandey et al. 2010 and Pani et al. 2015b).
Table 3
represents effect of FA on Chl a, Chl b, Total Chl and Carotenoids at different stages of growth of Calendula officinalis L. Data is Mean value ± S.D (n= 3).
PARAMETERS/
STAGES
|
T1 (Control)
|
T2 (10%)
|
T3 (20%)
|
T4 (40%)
|
T5 (60%)
|
T6 (80%)
|
T7 (100%)
|
0% FA
|
10% FA
|
20% FA
|
40% FA
|
60% FA
|
80% FA
|
100% FA
|
A) Chlorophyll a (mg/g of fresh weight)
|
Pre- Flowering
|
0.95 ± 0.004 a
|
0.98 ± 0.013 a
|
1.11 ± 0.007 b
|
1.23 ± 0.035 ba
|
1.15 ± 0.003 b
|
0.92 ± 0.002 a
|
0.88 ± 0.002 abc
|
Flowering
|
1.03 ± 0.016 a
|
1.17 ± 0.139 b
|
1.25 ± 0.026 b
|
1.36 ± 0.142 b
|
1.28 ± 0.031 b
|
1.01 ± 0.003 a
|
0.92 ± 0.002 bc
|
Post- Flowering
|
0.88 ± 0.021 a
|
0.93 ± 0.002 a
|
0.95 ± 0.034 a
|
1.02 ± 0.066 b
|
0.98 ± 0.005 a
|
0.85 ± 0.002 ab
|
0.81 ± 0.045 ab
|
B) Chlorophyll b (mg/g of fresh weight)
|
Pre- Flowering
|
0.30 ± 0.021 a
|
0.34 ± 0.028 a
|
0.39 ± 0.032 b
|
0.47 ± 0.009 bc
|
0.43 ± 0.046 bc
|
0.26 ± 0.004 abc
|
0.21 ± 0.003abc
|
Flowering
|
0.36 ± 0.034 a
|
0.45 ± 0.042 a
|
0.49 ± 0.012 ab
|
0.54 ± 0.024 ab
|
0.51 ± 0.047 ab
|
0.34 ± 0.026 abc
|
0.28 ± 0.021 abc
|
Post- Flowering
|
0.23 ± 0.052 a
|
0.26 ± 0.003 a
|
0.31 ± 0.004 b
|
0.36 ± 0.035 b
|
0.33 ± 0.077 a
|
0.21 ± 0.005 ab
|
0.17 ± 0.002 ab
|
C) Total Chlorophyll (mg/g of fresh weight)
|
Pre- Flowering
|
1.26 ± 0.004 a
|
1.29 ± 0.052 a
|
1.32 ± 0.073 a
|
1.47 ± 0.054 b
|
1.38 ± 0.014 ab
|
1.25 ± 0.006 a
|
1.21 ± 0.005 abc
|
Flowering
|
1.38 ± 0.051 a
|
1.42 ± 0.072 a
|
1.68 ± 0.054 b
|
1.78 ± 0.074 b
|
1.70 ± 0.068 b
|
1.36 ± 0.043 a
|
1.25 ± 0.021 ab
|
Post- Flowering
|
1.21 ± 0.007 a
|
1.25 ± 0.002 a
|
1.27 ± 0.028 a
|
1.33 ± 0.068 a
|
1.29 ± 0.003 a
|
1.19 ± 0.007 ab
|
1.12 ± 0.003 ab
|
D) Carotenoids (mg/g of fresh weight)
|
Pre- Flowering
|
0.31 ± 0.008 a
|
0.35 ± 0.005 a
|
0.39 ± 0.032 a
|
0.44 ± 0.065 b
|
0.42 ± 0.005 b
|
0.28 ± 0.002 a
|
0.23 ± 0.004 ab
|
Flowering
|
0.34 ± 0.029 a
|
0.39 ± 0.043 a
|
0.47 ± 0.056 a
|
0.58 ± 0.041 b
|
0.51 ± 0.054 b
|
0.32 ± 0.023 a
|
0.29 ± 0.037 ab
|
Post- Flowering
|
0.27 ± 0.024 a
|
0.30 ± 0.005 a
|
0.34 ± 0.043 a
|
0.39 ± 0.038 b
|
0.36 ± 0.006 a
|
0.25 ± 0.041 ab
|
0.21 ± 0.002 ab
|
T = Treatment, Values signify mean ± SD where the sample size (n=10)
Values presented in various letters differ significantly (P < 0.05) from the control values for different stages of growth as per the Duncan’s Multiple Range Test (DMRT).
Effect of FA on sugar, protein, nitrate content and nitrate reductase activity-
Effect of fly ash on reducing sugar- The observations on reducing sugar content (Fig 2A) in the leaves of Calendula officinalis showed that in the flowering stage, the significant (P < 0.05) enhancing sugar content (78.94%) was observed in T4 (40% FA amendment) then pre-flowering (73.91%) and post flowering stages (72.58%). However, T7 (100% FA) showed the lowest reducing sugar content (26.31%) at flowering, (23.91%) at pre-flowering and (19.35%) at post flowering stage. The rise in sugar content was noticed with FA application up to 60% FA soil ratio, further application (80% and 100%) causes decline in the sugar content in leaves. The trend of decline in sugar content at high fly ash application was directly correlated with the decline in the photosynthetic pigments. Similar results were obtained in Helianthus annus grown in FA amended soil (Pani et al. 2015a). It was inferred that the harmful effect of heavy metals (Pb, Cd, Ni, Cu) at higher FA applications reduces the sugar content in leaves however at low FA application in soil, these metals are within threshold limits which leads to an upsurge in the sugar content. The decline in the sugar content in heavy metal stressed plant leaves was due to the inhibition of photosynthetic activities was also noticed (Dash and Sahoo 2017).
Effect of fly ash on soluble protein- Data on protein concentration in the Calendula leaves as influenced by FA and soil is depicted in Fig-2B. The significant (P < 0.05) upsurge in protein content of Calendula officinalis leaves was observed in 40% FA amended soil at flowering stage (31.08%) then pre-flowering (28.38%) and post-flowering stages (22.22%) in comparison with control in all growth stages of plant. Further high FA application (80% and 100%) causes a significant (P < 0.05) drop in protein content in leaves in a growth-dependent manner. It has been observed that the high protein content occurs due to the synthesis of additional stress shock proteins and stimulating biochemical changes in plant cells because of increased heavy metal stress (Sharma and Singh 2019). But this study revealed that at high application of FA in soil, the protein level was found to be decreased in the calendula leaves (Fig-2B). The decline in the amount of protein might be due to the increase in the protease activity and further catabolic enzymes (Pani et al. 2015a). Similar observations were also reported in Pisum sativum and Brassica juncea respectively by Sharma et al. 2010 and Gautam et al. 2012. It was also noticed that FA contains high amount of heavy metals which induces the production of ROS which may further harm the photosynthetic system and damage the proteins (Shahid et al. 2014).
Effect of fly ash on nitrate content- Nitrate content in Calendula leaves is graphically determined in Fig 2C. The nitrate content was found significantly (P < 0.05) increased at 40% FA amended soil ratio in pre-flowering (78.23%), flowering (87.11%) and post-flowering stages (50.63%) with respect to control. The post-flowering stage shows a gradual decline in all concentrations compared to pre-flowering and flowering stages. Nitrate content was noticed as lowest in flowering stage (44.27%) then pre-flowering (32.15%) and post flowering stage (29.44%) of T7 treatment. Nitrate content has a strong correlation with protein content and photosynthetic pigment rate as plant uses nitrates as a source of nitrogen which is required for protein building which is an important criterion for healthy growth of plants (Hikosaka and Osone 2009). As nitrate content decreases, the pigment concentration in the leaves of the plant decreases significantly (Chaturvedi et al. 2013). In this study, the decrease nitrate content was observed (Fig-2C) at higher FA application as FA alone does not contain nitrogen which is responsible for the reduced plant growth.
Effect of fly ash on Nitrate Reductase (NR) Activity- Results of NR activity (Fig 2D) demonstrated a significant increase (P < 0.05) at T4 (40% FA amendment) in comparison to all treatments in all stages of plant growth. The maximum upsurge was observed for the flowering stage (91.6%) then pre-flowering (51.31%) and post-flowering stages (48.50%). Higher level of FA amendments (80% and 100%) showed reduced NR activity in a growth-dependent manner at all stages of plant growth. Nitrate reductase serves as the first enzyme catalyzing the assimilation pathway and is involved in the reduction of nitrate to nitrite (Karwat et al. 2019). In this study, the NR activity (Fig- 2D) was found to be significantly (P < 0.05) reduced at high FA application, possible due to the disintegration of the enzyme molecules (Gupta et al. 2009). The decrease in Nitrate reductase activity may be due to either the decline in the protein content, or because of the availability of the substrate. Similar results on NR activity inhibition were also reported in Prosopis juliflora L. at high FA application (Rai et al. 2004). As FA encompasses heavy metals, the inhibition of activity of Nitrate reductase might be attributed to the constraining ability of the heavy metals as they can bind to the SH- groups of certain enzymes (Sahay et al. 2015).
In the present study, all the biochemical parameters were found significant (P < 0.05) at flowering stage, pre-flowering stage and post-flowering stage for all FA-soil treatments. The sequence of the results indicates that due to aging of plant at post-flowering stage, it has poorer metabolic events that resulted in lessened biochemical activity compared to younger leaves in pre-flowering stage however maximum biochemical activity was reported in the leaves of the Calendula plant for the flowering stage suggesting the highest rate of metabolic activities (Parween et al. 2011).
Level of antioxidant enzyme activity-
The leaf samples of Calendula officinalis grown in different FA and soil treatments were analysed for enzyme activity by using certain antioxidant enzymes (SOD, CAT, APX, and Peroxidase) as shown in Fig. 3. In this study, the antioxidant enzyme activities of Calendula officinalis displayed significant changes under various treatments of FA and soil mixture compared to control. From ANOVA results, various antioxidative enzymes (SOD, APX, CAT and Peroxidase) were significantly (P < 0.05) improved at high fly ash application. At 100% fly ash application, these activities were found to be highest in comparison with control (garden soil). The percent increase in SOD, CAT, APX and Peroxidase activity was 202.9%, 310.4%, 432.1% and 153.74% respectively, in 100% FA treatment compared to the control. Results indicated that low FA application in soil did not trigger oxidative stress whereas higher level of metals in FA amended soil causes disturbance in the cell membrane, cell disruption, interrupt various metabolic processes and causes inhibition in plant growth (Panda et al. 2018). Plant tolerance to heavy metal stress is directly associated with an upsurge in antioxidant enzymes to detoxify reactive oxygen species (Bisoi et al. 2017). This elevation in enzymatic activity might be due to the plant defense against ROS species generated by metal ions present in FA. The same drift was observed in Chickpea grown in various treatments of FA with soil and reported an upsurge in antioxidant enzyme activities (POD, CAT and MDA) in the roots, shoots and leaves of the plant at high FA concentration in soil. Similar observations were also recorded in lemongrass where an increase in antioxidant enzyme activity (APX, SOD, and GPX) was noticed to be improved under all FA treatments over control. In addition, the growth morphology viz shoot, root, total biomass and metal tolerance index were amplified at low FA (25%) as compared to control plants followed by decrease at higher concentration of FA (50%, and above) (Panda et al. 2018). Alteration in antioxidant enzyme activity rely upon different levels of stress, plant species, period of stress acquaintance, tolerance degree and predominant ecological conditions (Zouari et al. 2016).
Correlation between antioxidant enzymes, photosynthetic pigments and growth parameters-
The relationship between photosynthetic pigments, growth parameters and antioxidant enzymes of leaves of calendula grown in different fly ash soil treatments is presented in Table 4. Results in this study show positive correlations between photosynthetic pigments and growth parameters whereas negative correlations were observed amongst antioxidant enzyme activities (SOD, CAT, APX and Peroxidase) with growth, photosynthetic and biochemical parameters in calendula leaves. These results indicated that the antioxidant enzyme activities of calendula are incapable to detoxify the ROS species triggered by metal ions due to increased FA concentration in soil which negatively affects growth and biochemical parameters of plant.
Table 4
Correlation analysis among biochemical parameters and antioxidant enzyme responses of Calendula officinalis
|
Protein
|
Chl a
|
Chl b
|
Total Chl
|
Carotenoids
|
Sugar
|
Nitrate
|
NR
|
SOD
|
CAT
|
APX
|
Peroxidase
|
Protein
|
1
|
|
|
|
|
|
|
|
|
|
|
|
Chl a
|
.909**
|
1
|
|
|
|
|
|
|
|
|
|
|
Chl b
|
.936**
|
.984**
|
1
|
|
|
|
|
|
|
|
|
|
Total Chl
|
.878**
|
.922**
|
.928**
|
1
|
|
|
|
|
|
|
|
|
Carotenoids
|
.909**
|
.943**
|
.966**
|
.938**
|
1
|
|
|
|
|
|
|
|
Sugar
|
.870**
|
.905**
|
.928**
|
.971**
|
.906**
|
1
|
|
|
|
|
|
|
Nitrate
|
.903**
|
.960**
|
.951**
|
.977**
|
.934**
|
.954**
|
1
|
|
|
|
|
|
NR
|
.931**
|
.897**
|
.924**
|
.925**
|
.961**
|
.890**
|
.915**
|
1
|
|
|
|
|
SOD
|
− .489*
|
-0.147
|
-0.195
|
-0.169
|
-0.202
|
-0.142
|
-0.181
|
-0.365
|
1
|
|
|
|
CAT
|
− .552**
|
-0.282
|
-0.324
|
-0.198
|
-0.29
|
-0.206
|
-0.264
|
-0.389
|
.896**
|
1
|
|
|
APX
|
− .645**
|
-0.422
|
− .452*
|
-0.338
|
− .456*
|
-0.288
|
-0.37
|
− .564**
|
.821**
|
.779**
|
1
|
|
Peroxidase
|
− .553**
|
-0.315
|
-0.318
|
-0.218
|
-0.288
|
-0.155
|
-0.275
|
-0.379
|
.823**
|
.804**
|
.838**
|
1
|
Chl a- Chlorophyll a; Chl b- Chlorophyll b; Total Chl- Total Chlorophyll; NR- Nitrate Reductase; SOD- Superoxide dismutase; APX- Ascorbate Peroxidase; CAT- Catalase
**. Correlation is significant at the 0.01 level (2-tailed).
*. Correlation is significant at the 0.05 level (2-tailed).