Growth and physio-biochemical responses of Azolla liculoides subjected to short span of phenol exposure

Phenol and phenolic compounds are one of the major water pollutants increasing at an alarming rate due to uncontrolled anthropogenic activities. Aquatic plants are in the frontline to face the toxic effects of these pollutants. The present study attempts to study the effect of external phenols on a oating fern Azolla liculoides. In this context, the effect of four concentrations of crystalline phenol (0.5, 1, 2 and 3 mM) along with a control set (0 mM) were used to evaluate the changes in the responses of A. liculoides plants in terms of the major morphological, physiological and biochemical markers, after being exposed for 3 and 6 days. In this study, relative growth rate, relative frond number, relative frond surface area, relative water content, chlorophyll a and carotenoids decreased signicantly both with increasing phenol concentration and also with the duration of treatment. Frond necrotic area, electrolyte leakage and malondialdehyde content increased signicantly which demonstrated the negative effects of phenol. However, increase in proline content, total sugars, activities of antioxidative enzymes like superoxide dismutase, catalase and peroxidase indicated the presence of an inherent tolerance mechanism in this plant. Accumulation of H 2 O 2 also supported by the in situ localization of H 2 O 2 along with the changes in growth parameters and physio-biochemical indicators demonstrated that the extent of phenol toxicity increased with the increasing concentrations of phenol. The overall ndings also suggest that the A. liculoides plants can maintain their growth and physiology up to a concentration of 1 mM phenol. Azolla liculoides biomass to absorb 2, 4, 6-trichlorophenol from the aqueous solution. However, no studies have been conducted to assess the effect of phenol on the growth and physio-biochemical responses of Azolla liculoides. Therefore, this study attempts to provide an insight in to the responses of Azolla liculoides plants to different concentrations of phenol. For this purpose, the effect of phenol was evaluated in terms of morphological indicators such as relative growth rate, relative fond number (RFN), relative frond surface area (RFSA) and frond necrotic area (FNA). Physiological parameters like relative water content (RWC), electrolyte leakage (EL) and membrane lipid peroxidation were also observed. Most importantly, the effect of phenol exposure on the biochemical indicators of plants like the changes in osmolyte accumulation, photosynthetic pigments, enzymatic antioxidants and reactive oxygen species (ROS) were analyzed.


Introduction
Water pollution directly or indirectly affects all life forms. Presently all water-bodies like ponds, lakes, rivers, oceans, and even groundwater are highly polluted or under extreme threat of pollution. A signi cant number of water pollutants either belongs to phenolic group of compounds or produces phenolic compounds upon degradation (Kulkarni and Kawar, 2013). Phenol is mostly a colorless, water-soluble molecule that consists of a benzene ring along with at least one functional hydroxyl group. US Environmental Protection Agency have enlisted about 11 types of phenolic pollutants viz. Phenol,2,4,2,2,2,4,and pentachlorophenol in their priority list of toxic pollutants (Kieth, 1980). Common sources of phenolic compounds in the aquatic ecosystem originates from industry, sewage, agricultural and also from household wastes (Anku et al., 2017;Stewart and Stewart, 2008).
There has been a lot of concerns regarding the presence of phenolic compounds in aquatic ecosystems off late. Zhong et al. (2018) have found the presence of about 50 phenolic compounds from 3 different rivers in Tianjin, China. They quanti ed the concentrations of phenols in surface water, suspended particulate matter and sediments in the range of 1.35 mg/L, 0.719 mg/Kg dry weight and 2.94 mg/Kg dry weight respectively. Another report by Busca et al. (2008) have suggested the presence of organic pollutants in wastewater comprising of phenols in the range of 2.8 to 6800 mg/L, which was mainly attributed to the e uents from different industries like manufacturing units, petrochemical re neries, coal processing units etc.
A large number of works have been conducted to assess the effect of phenolic compounds on aquatic fauna. However, very few studies have focused on the effect of phenolic compounds on aquatic plants. On the basis of these works, it has been found that the aquatic plants respond differently to varying concentrations of phenolic compounds but the basic mechanism to resist the negative impacts of these toxic compounds is more or less similar (Jagetiya et al., 2013;Wasi et al., 2013). Inhibition of plant multiplication and reduction of growth was observed in Lemna minor and Elodia canadensis subjected to concentrations above 10 − 3 moles/L, that exhibited the phytotoxic effect of phenols on these macrophytes (Stom and Roth, 1981). According to Scragg (2006), Chlorella VT-1 shows more tolerance against the phenolic compounds upto a concentration of 400 mg/L, when compared to Chlorella vulgaris which is relatively less tolerant. At 400 mg/L phenol, 44% growth inhibition was observed in Chlorella VT-1, whereas growth of Chlorella vulgaris at the same concentration was completely inhibited. Similarly, reduction in frond multiplication was observed in Lemna paucicostata as a consequence of increasing phenol concentration (Park et al., 2012). On the other hand, several plants have been known to maintain their normal growth and physiology at higher concentrations of phenol by uptaking and utilizing the excess amount of phenols from the surrounding. Chang et al. (2020) reported that the submerged macrophyte -Hydrilla verticillata was capable to absorb and degrade phenolic compounds when exposed to 100 mg/L concentration with minute physiological effects. Lee et al., (2017) calculated the rate of removal of phenols using Ipomoea aquatica that showed about 72.2% survival rate when exposed to 300 mg/L phenol. They have shown that the plant was capable to remove phenol at the rate of 21 mg/L/day. Similarly, enhancement in the biomass of Lemna minuta plants was observed owing to its ability to utilize the phenolic compounds (Paisio et al., 2018).
Azolla liculoides is a free-oating small aquatic fern, that exhibits the ability to grow rapidly, and double its biomass within 3-5 days (Arora and Saxena, 2005). The association of Azolla with the diazotroph Anabaena azollae also boost the growth of this plant where nitrogen availability is low (Sood et al., 2008). Azolla spp. has been known to accumulate heavy metals like Zn, Fe, and Mn from the surrounding (Sood et al., 2012). These attributes of Azolla has been valued for the removal of water pollutants in aquatic ecosystem (Mashkani and Ghazvini, 2009;Rai, 2008). A previous study by Ali et al. (2013) have reported the usage of dried Azolla liculoides biomass to absorb 2, 4, 6-trichlorophenol from the aqueous solution. However, no studies have been conducted to assess the effect of phenol on the growth and physio-biochemical responses of Azolla liculoides. Therefore, this study attempts to provide an insight in to the responses of Azolla liculoides plants to different concentrations of phenol. For this purpose, the effect of phenol was evaluated in terms of morphological indicators such as relative growth rate, relative fond number (RFN), relative frond surface area (RFSA) and frond necrotic area (FNA). Physiological parameters like relative water content (RWC), electrolyte leakage (EL) and membrane lipid peroxidation were also observed. Most importantly, the effect of phenol exposure on the biochemical indicators of plants like the changes in osmolyte accumulation, photosynthetic pigments, enzymatic antioxidants and reactive oxygen species (ROS) were analyzed.
2 Materials And Methods 2.1 Plant material and experimental setup A laboratory based experiment was carried out in the Plant Biochemistry Laboratory, Department of Botany, University of North Bengal during summer season, 2019. Azolla liculoides, a free-oating aquatic fern, a member of Azollaceae was chosen for the present experiment. The plant was collected from a pond, near the University of North Bengal (NBU) campus, Siliguri, India during the month of March, 2019. After collecting the plants, it was transferred to a water tank (200×60 cm 2 ) in the experimental garden of the department. The water tank was fertilized every 7 days with 1g/L solution of magnesium chloride, rock phosphate and potassium (2:3:1 w/w/w) to support the growth of the plants and thereafter the mature plants of same age were relocated to the laboratory, where they were transferred to beakers containing 0.1X Hoagland solution (Gamborg and Wetter, 1975). The plants were then allowed to acclimatise for a week in laboratory conditions. For the experimental setup, four different concentrations of crystalline phenol viz. 0.5, 1, 2, and 3 mM were prepared in 0.1X Hoagland solutions along with 0 mM solution as the control set. Equal number of plants were transferred to beakers containing the treatment solutions in a plant growth chamber (25 o -28 o C temperature and 70%-75% RH with 16:8 hours of light and dark photoperiod condition maintained). Two different duration of treatment viz. 3 days (shorter duration) and 6 days (longer duration) was selected to evaluate the responses of plants subjected to different phenol concentrations.

Growth and morphological attributes
To determine relative growth rate (RGR), fresh weights of the plants were initially measured (W 0 ) before the start of phenol treatments. After 3 and 6 days of treatment, the weight (W t ) of the same plants were taken and the relative growth rate was measured with the following equation -Other indicators of morphological changes observed in the plants subjected to phenol treatments were determined in terms of relative frond number (RFN), relative frond surface area (RFSA) and frond necrotic area (FNA). All the parameters were analysed from the captured images of plants at the end of treatment regime using Fiji-Image J software.
To measure the RFN, frond number of plants were counted at 0 days (N 0 ), 3 days and 6 days (N t where t = 3 or 6 days). RFN was measured using following formula -For RFSA, initial frond surface area at 0 days (A 0 ) and nal frond surface area (A t where t = 3 or 6 days) at 3 and 6 days was measured. RFSA was calculated by the following formula -For FNA, necrotic area of all the sets were measured at 0, 3 and 6 days (NA t where t = 0 or 3 or 6 days) and determined in terms of percentage of the total frond surface area of the respective sets (A t where t = 3 or 6 days). FNA was calculated using the following equation -2.3 Relative water content (RWC), Electrolyte leakage (EL) and Malondialdehyde content (MDA) Relative water content (RWC) was measured according to the standard protocol described by Barr and Weatherley (1962). The whole plants were utilized for the experiment. Fresh weight (F.W), turgid weight (T.W) and dry weight (D.W) was taken as described. Relative Water Content was measured with the following equation -Electrolyte leakage was measured as described by Lutts et al. (1996). The control and treated plant samples were washed thoroughly with double distilled water. Then the plants along with 10 mL of deionized water were incubated in a rotary shaker for 24 h at 25°C. Then the initial electrical conductivity was measured (L 0 ), after that, it was autoclaved and the nal electrical conductivity was measured (L t ) after cooling. Electrolyte leakage was calculated as follows -Membrane lipid peroxidation (MDA content) was measured following the described method of Heath and Packer (class="CitationRef">1968) with slight modi cations. Plants were homogenized in 0.1% TCA and centrifuged at 10,000 rpm, then the supernatant was collected. 0.5% of TBA in 20% TCA and 1 mL of supernatant was heated at 95°C for 30 min, after that it was cooled on ice. Absorbance was nally taken at 532 nm and 600 nm. MDA content was calculated using an extinction co-e cient of 155 mM − 1 cm − 1 .

Extraction and estimation of photosynthetic pigments
Chlorophyll was estimated according to the method of Harborne (1973) with slight modi cations by homogenizing the plant materials (0.5 g) in 80% acetone and centrifuged at 8000 rpm for 10 min. The supernatants were collected and the absorbance was taken at 470 nm, 647 nm, and 663 nm in a UV-VIS spectrophotometer against a blank of 80% acetone to estimate chlorophylls and carotenoids using the formula as given by Lichtenthaler (1987 Extraction of proline from the plant material was done by the method of Bates et al. (1973) with slight changes. Plant tissue (1 g) was homogenized in 3% sulfosalicylic acid (10 mL) and centrifuged at 8000 rpm for 10 min at room temperature and the supernatant was collected for estimation. The reaction mixture absorbance was measured spectrophotometrically at 520 nm Proline content estimated using a standard curve of L-proline.
For the extraction of total sugar, Harborne's (1973) method was followed. The treated plants along with control set was taken for ethanolic extraction (95%) and estimation of total sugar was done by Anthrone reagent following the method of Plummer (1978). A standard curve of D-glucose was used for the quanti cation.

Extraction and estimation of antioxidative enzymes
The treated plants along with control plants were homogenized in 50 mM sodium phosphate buffer, pH 6.8 (for peroxidase and catalase enzymes; pH 7.2 for ascorbate peroxidase enzyme along with 1 mM of ascorbate) and 100 mM potassium phosphate buffer pH 7.6 (for superoxide dismutase and glutathione reductase) under ice-cold conditions. The homogenates were centrifuged at 10,000 rpm at 4°C for 15 min. Fresh supernatants were used as crude enzyme extracts and estimation of protein content was done in each case following the method of Bradford (1976).
The activity of superoxide dismutase (SOD) was determined following the method of Dhindsa et al. (1981) with some minor adjustments. The reaction mixture contained 1.5 mL of 0.1 M potassium phosphate buffer (pH 7.6), 0.5 mL of dH 2 O, 0.2 mL of 0.2 M methionine (freshly prepared), 0.2 mL of 2.25 mM NBT, 0.1 mL of 1.5 M Na 2 CO 3 , 0.1 mL of 3 mM EDTA (freshly prepared), 0.2 mL of 60 µM ribo avin, and 0.2 mL of enzyme extract. The reaction mixture were illuminated for 10 minutes. The absorbance was taken at the wavelength of 560 nm in a UV-VIS spectrophotometer. 50% reduction of NBT compared to the control set containing no enzyme extract was represented to have 1 Unit of enzyme activity (EU).
Glutathione reductase (GR) activity was determined by Lee and Lee (2000) by measuring the oxidation of NADPH (€ = 6.2 mmol-1 cm-1) with minor modi cations. The reaction mixture consisted of 1 mL of 0.1 M potassium buffer (pH 7.6), 0.2 mL of 100 mM EDTA, 0.2 mL of 0.1 mM NADPH and 0.2 mL of enzyme extract, along with 0.1 mL of 6 mM glutathione (freshly prepared). The immediate change in absorbance of the reaction mixture was measured after every 30 seconds up to 3 min using a UV-VIS spectrophotometer at 340 nm.
Ascorbate peroxidase (APX) activity was assayed following the method of Asada and Takahashi (1987) with slight modi cation. The reaction mixture consisted of 0.01 mL of freshly prepared ascorbic acid, 0.01 mL of H 2 O 2 , 2.97 mL of 50 mM sodium phosphate buffer (pH 7.2) and 0.01 mL of enzyme extract. Reduction of absorbance was measured at 290 nm at regular intervals up to 3 minutes in a UV-VIS spectrophotometer.
Peroxidase (POX) activity was determined by following the method of Chakraborty et al. (1993). The reaction mixture consisted of 1 mL of 100 mM sodium phosphate buffer (pH 6.8), 1.7 mL of dH 2 O, 0.1 mL of H 2 O 2 , 0.1 mL of o-dianisidine, and 0.1 mL enzyme extract. Changes in absorbance was taken at 460 nm at regular intervals up to 3 minutes in a UV-VIS spectrophotometer.
Catalase (CAT) activity was assayed using the method of Chance and Machly (1955). 100 µl of the enzyme extract was added in 2.9 mL of H 2 O 2 -phosphate buffer. Changes in absorbance were taken immediately at 240 nm in a UV-VIS spectrophotometer at regular intervals up to 3 minutes in a UV-VIS spectrophotometer.

Extraction and estimation of total phenol
For the extraction of phenols, Mahadevan and Sridhar's (1982) method was followed. For the extraction, plants were crushed in 80% ethanol in dark and the ltrate was collected. Estimation of total phenol content was done following the method of Bray and Thorpe (1954). Brie y, the ltrate was mixed with Folin Ciocalteu's reagent and 20% Na 2 CO 3 solution and the absorbance was recorded at 650 nm. The total phenol content was calculated using a standard curve of catechol.

Hydrogen peroxide (H 2 O 2 ) content and in situ localization
The extraction and estimation of H 2 O 2 were done by the method of Jana and Choudhuri (1981) with slight modi cations. 0.5 g of plant sample was homogenized in 10 mL of 50 mM phosphate buffer (pH 6.5). The homogenate was centrifuged at 6000 rpm for 25 minutes. After that 3 mL of the supernatant was mixed with 1 mL of 0.1% titanium sulphate and centrifuged at 6000 rpm for 15 minutes. Finally, the absorbance was measured at 410 nm in a UV-VIS spectrophotometer.
For the detection of H 2 O 2 inside the plant tissue, the method of Thordal-Christensen et al. (1997) with minor modi cations was followed. The plants were washed thoroughly with dH 2 O and then incubated in dark for 24 h in 1 mg/mL of 3, 3'-Diaminobenzidine (DAB) (pH 3.8). After 24 h the whole plant was transferred to a beaker containing ethanol, lactic acid and glycerol in the ratio 4:1:1 and boiled in dark until all the chlorophyll was removed from the fronds.
Visualization of the H 2 O 2 stained region was done over an illuminated platform.

Statistical analysis
All the experiments were conducted in replicates (N = 3). Values shown in the gures and tables represent the average values ± standard deviation (SD) for all the treatment sets. One-way ANOVA was performed using IBM SPSS version 22 and the post-hoc analysis of the results were performed through Duncan's test taking a signi cance level of p ≤ 0.05. Correlation analysis was performed between the concentrations of phenol and all the parameters after 3 days and 6 days separately using two tailed Pearson's correlation co-e cient analysis in IBM SPSS version 22.
3 Results 3.1 Effect of phenol exposure on the growth and morphological attributes (RGR, RFN, RFSA and FNA) The varying effect of the different concentrations of phenol on Azolla liculoides plants was evident through the morphological appearance of the plants after the treatment regimes. With the increasing concentrations of phenol, marked impact was clearly visualized on the fronds in the form of necrotic lesions.
Further inference on the effect of phenol on the plants were evaluated in terms of relative growth rate (RGR), relative frond number (RFN), Relative frond surface area (RFSA) and frond necrotic area (FNA).
After 3 days of treatment, RGR of the plants was found to be signi cantly reduced in comparison to the control subjected to treatments with higher concentrations of phenol (≥ 2 mM). However, after longer duration of treatment RGR reduced signi cantly at all the phenol concentrations (Fig. 1A). This decrease in RGR ranged from 15-257% in comparison to the control set. Similarly, after 3 and 6 days of treatment, both RFN and RFSA were also found to gradually decrease with the increase in phenol concentrations, except in case of the plants treated with 0.5 mM phenol for 3 days (Fig. 1B-C). Highest decrease in the range of 106-121% in case of RFN and 148-169% for RFSA were observed in the plants treated with 3 mM phenol for 3 and 6 days. However, with the increase in phenol concentration and duration of treatment, FNA was found to be signi cantly enhanced (Fig. 1D). The extent of necrotic lesions was maximum in case of 3 mM treated plants, where more than 80% of the tissue was found to be affected.

Effect on water status and membrane stability (RWC, EL and MDA)
Relative water content (RWC) was found to decrease signi cantly in comparison to the control plants with increasing concentrations of phenol both after 3 and 6 days of treatment (Table 1). Approximately, 90% RWC was observed in the plants exposed to 0.5 and 1 mM phenol concentrations for 3 and 6 days.
Also, after longer duration of treatment, RWC was found to decrease in comparison to that of 3 days across all the concentrations. Status of membrane integrity was evaluated through electrolyte leakage and membrane lipid peroxidation (MDA content). The electrolyte leakage increased with increasing phenol concentration for short as well as longer duration of exposure. Highest leakage in the order of 725-895% was observed in case of plants treated with 3 mM phenol concentration for 3 and 6 days respectively when compared to the control set (Table 1). Similarly, after 3 and 6 days of treatment, MDA was generally found to increase at all the concentrations. (Table 1). At 3 mM phenol treated plants, this increase in MDA ranged from 185-322% in comparison to the control set.

Effect on the pigment compositions
The amount of chlorophyll a and chlorophyll b of the plants increased at lower concentrations, but signi cantly reduced at higher concentrations when compared to the control set after shorter duration of treatment ( Fig. 2A). At highest concentration (3mM), chlorophyll a and chlorophyll b decreased approximately by 22% and 59% respectively. Moreover, chlorophyll a content of the plants reduced by more than 50% exposed to highest phenol concentration for 6 days. In contrast, the amount of chlorophyll b was found to be enhanced (64-162%) at all the concentrations in comparison to the control set. Carotenoid content was generally observed to be reduced with increasing phenol concentration both after shorter and longer duration of treatments, but this decrease was more signi cant in the plants exposed for longer duration when compared to the control set (Fig. 2B). This decline in carotenoid content after 6 days of treatment ranged from 15-36% in comparison to control set.

Effect on osmolyte accumulation
Accumulation of proline was found to be highest in plants exposed to 1 mM phenol concentration (0.061 mg/g for 3 days and 0.162 mg/g for 6 days).
Moreover, the accumulation of proline was found to be increased after 6 days of treatment in comparison to that of the 3 day treated plants across all the concentrations (Table 1). For instance, maximum increase in the accumulation of proline (164%) was observed in plants treated with 1 mM phenol concentration for 6 days when compared to that of the 3 day set.
Total soluble sugar content was generally observed to increase with increasing phenol concentrations both after shorter and longer duration of treatments except in the plants exposed to 3 mM phenol for 6 days. But the increase was more signi cant in the plants exposed for shorter duration when compared to the control set. The extent of this increase in total sugar content ranged from 22-64% in the phenol treated plants in comparison to control set.

Status of the enzymatic antioxidants and total phenols
The activity of superoxide dismutase (SOD) was found to increase signi cantly in plants exposed to phenol concentrations ≥ 1 mM when compared to the control set after 3 days of treatment (Fig. 3A). Moreover, SOD activity of the phenol treated plants showed an increase up to 2 mM phenol treated plant after 6 days of exposure in comparison to the control sets. In this regard, the highest increase of 155% was observed in case of 2 mM treated plants than control.
A decrease in the activity of glutathione reductase (GR) was observed in comparison to the control, when the plants were exposed for shorter duration of treatments (Fig. 3B). This decline in enzyme activity was found in the order of 44-63%. However, after 6 days of treatment a sharp enhancement in GR activity was recorded in case of 3 mM treated plants when compared to the control and also with respect to that of the plants treated with the same phenol concentration for shorter duration (510% increase from the 3 day plants).
The ascorbate peroxidase (APX) activities were found to increase with increasing phenol concentrations (≥ 2 mM) after 3 days (Fig. 3C). At the highest concentration, APX activity increased by approximately 30% than the control. After longer duration of treatment, APX activity was increased except in 2 mM phenol treated plant. Highest ascorbate peroxidase activity increased 293% in 3 mM phenol concentration in compare with control.
The peroxidase (POX) activity enhanced with increasing concentrations of phenol in comparison to control after 3 days of treatments, except at the highest concentration (3mM) where a signi cant decline (33% from the control) was observed (Fig. 3D). Also, after 6 days of treatment, signi cant decline in POX activity (68% from the control) was recorded in case of 3 mM phenol treated plants. Moreover, POX activity declined in the plants exposed to 2 and 3 mM phenol for 6 days when compared to that of the 3 day treated plants.
A signi cant enhancement in catalase (CAT) activity was observed in case of plants treated with higher concentrations (≥ 2 mM phenol) for shorter duration.
Highest activity in this regard was observed in case of 2 mM phenol treated plants which was approximately 45% more than the control plants (Fig. 3E). However, after longer duration of treatment, CAT activity was found to increase initially but at higher concentrations, the activity was found to be reduced signi cantly than the control.
Total phenolics also considered as non-enzymatic antioxidants was generally found to be signi cantly decreased in comparison to the control plants subjected to shorter duration of exposure, except at 0.5 mM treated plants (Fig. 3F). At 2 mM phenol concentration, highest decrease of 38% from the control was observed. However, in the plants exposed for longer duration, total phenol content signi cantly increased up to 1 mM concentration, but at higher concentrations the amount of that total phenols signi cantly reduced. Moreover, total phenol content increased at all the concentrations (11-67%) after 6 days of treatment in comparison to that of the 3 day treated plants.

Effect on the accumulation of H 2 O 2 and in situ localization
In the present study, the H 2 O 2 content increased signi cantly with increasing phenol concentrations both after 3 and 6 days of treatment (Fig. 4A). The accumulation H 2 O 2 was found to be highest at 3 mM phenol treated plants which ranged from 129-252% in comparison to the control set. Similar trends were observed with the DAB stained fronds to locate the accumulation of H 2 O 2 in the plant tissues. The presence of H 2 O 2 was observed as reddish-brown patches formed due to the complexation of H 2 O 2 and DAB (Fig. 4B). After 3 and 6 days of treatment, this reddish-brown patches gradually increased with the increasing phenol concentrations. Also, the reddish-brown patches were found to be comparatively more after 6 days in comparison to that of the 3 days treated plants.    (Chang et al., 2020, Lee et al., 2017, Zazouli et al., 2014, Stom and Roth, 1981. In the present study, the effect of different concentrations of phenol was evaluated on the performance of Azolla liculoides which is a commonly growing weed in the water bodies. The experimental results indicated that under the effect of phenol exposure, growth rate of A. liculoides plant decreased signi cantly after 6 days of treatment (longer duration of exposure), and this decrease in growth rate enhanced with the increasing concentrations of phenol (Fig. 1A). Also, the effect on the growth was maximum incase of 3 mM phenol treated plants. This decrease in growth rate could also be associated with the simultaneous decrease in the generation of new fronds with the increasing concentrations of phenol (Fig. 1B). This type of reduction in growth was previously reported in case of Azolla pinnata plants exposed to two different pesticides -chlorpyrifos and cypermethrin (Prasad et al., 2015). Also, Sood et al. (2011) reported that Azolla microphylla exposed to ≥ 20 µM paraquat concentration for 10 hours exhibited the maximum effect on plant growth. Moreover, in the present study the relative surface area of the plants were found to decrease under the effect of phenol concentrations ≥ 1 mM both after 3 and 6 days of treatment (Fig. 1C).

Correlation analysis between the concentration of phenols and the other parameters
The decrease in growth rate and surface area of the plants could be correlated with the extent of phenol injury that were evident in the form of black necrotic lesions on the fronds which increased with the increase in phenol concentrations (Fig. 1D). Similar type of necrotic lesions have been observed in Salvinia auriculata when the plants were exposed to cadmium for 10 days (Wolff et al., 2012). These ndings clearly indicate that the A. liculoides plants are sensitive to increasing concentrations of phenols which affect the plant growth signi cantly.
In the present study, the extent of decrease in relative water content (RWC) was maximum in plants subjected to higher concentrations (≥ 2 mM) of phenol (Table 1). Due to the decrease in RWC, plants are subject to osmotic stress which affects the growth rate, by impairing the physiological processes such as decreasing stomatal conductance and CO 2 assimilation (Lawlor, 2002). Decrease in RWC also destabilizes the photosynthetic machinery and directly impacts the plant productivity . Exposure to increasing phenol concentrations also elevated the MDA contents in A. liculoides, which may be due to the increase in the peroxidation of membrane lipids (Table 1). Moreover, the impact of phenol on the membrane integrity could be understood in terms of increasing electrolyte leakage with the increase in the concentrations of phenol. Under stressed conditions plants generates free radicals in signi cant amount, which get stuck into membrane lipids and generates lipid hydroperoxide. The peroxidized membranes are affected by elevated electrolyte leakage and reduction of membrane-uidity (Kapoor et al., 2016). Similar enhancements in the MDA content of Azolla microphylla plants subjected to increasing concentrations of an insecticide -endosulfan have been shown to be related with the potential of this toxic compound to disturb the membrane stability (Raja et al., 2012a). In an aquatic species Chang et al. (2020) observed that Hydrilla verticillata exposed to phenol shows an increased amount MDA content. Therefore, it is quite evident from our results that the phenolic compounds directly affect the membrane integrity of A. liculoides plants, especially at higher concentrations.
Chlorophyll and carotenoids are essential photosynthetic pigments and the status of these pigments in plants under stressed environment holds the key for their survival. A. liculoides plants under the exposure of external phenol triggers a considerable change in the pigment composition. After 3 days of treatment, chlorophyll a and b increased at lower concentrations ( Fig. 2A) which may be due its ability to resist low concentrations of phenol toxicity. However, at higher concentrations, both after 3 and 6 days of treatment chlorophyll a decreased signi cantly. This decline may result due to the disruption of photosystem as reported by Sánchez-Viveros et al. (2011). They have also reported while working on the effect of arsenic on A. liculoides, that the chlorophyll a content remained almost unchanged up to a concentration of ≤ 30 µg/mL of the heavy metal, while it decreased at higher concentrations (≥ 60 µg/mL). Carotenoids are the accessory pigments and also confer some protective mechanism to cope up with oxidative stress (Roberts et al., 2014). In our study, with shorter duration of exposure to phenol, carotenoid content does not show any signi cant changes with increasing concentrations of phenol, but it was found to decrease along with the increase in duration of treatment (Fig. 2B). A similar report of reduction in carotenoid content has been observed in another species of Azolla, when the plants were exposed to lead. This decline in carotenoid content has been attributed to the degradation of pigments due to an increased accumulation of ROS (Roberts et al., 2014).
Accumulation of an enhanced levels of osmolytes in plant tissues can help the plants to cope up with the negative impacts of osmotic stress (Samarina et al., 2020). Osmolytes such as soluble sugars and proline increased at all the concentrations of phenol after shorter duration of exposure. After longer duration of treatment, this enhancement was observed only in case of plants exposed to lower concentrations (≤ 1 mM) ( Table 1). A positive correlation between abiotic stress tolerance and free proline accumulation has been reported by Martinez et al. (2016). In our study, the enhanced accumulation of proline was observed in 6 day treated plants in comparison to the 3 day treated plants across all the concentrations of phenol. This may be due to the presence of tolerance mechanism in plants up to a certain extent, which may assist the plants to survive in stressed condition. Abscisic acid dependent proline biosynthesis from glutamic acid, inhibition of proline degradation, reduction of protein biosynthesis may contribute to the elevated accumulation of free proline (Cao et al., 2020;Rana et al., 2017;Ren et al., 2018). In a similar experiment depicting the effect of an insecticide -monocrotrophos on Azolla microphylla, proline content was found to increase signi cantly (Raja et al., 2012b). However, the decrease of proline and total soluble sugars in Azolla liculoides at highest phenol concentration (3 mM) after 6 days pointed towards the inability of the macrophyte to tolerate such high concentrations of phenol (Table 1). This ndings are also in accordance with the changes in the growth pattern of the plants observed morphologically as well as through the other indicators.
Under oxidative stress extensive increase in internal phenolic compounds, which also functions as non-enzymatic antioxidants plays a key role in cellular homeostasis. In shorter duration of treatments, the phenol content of plants decreased. This may be due to the elicitation of oxidative stress and osmotic imbalance inside the plants under the effect of external phenol treatment. However, in our results, we have observed an increase in the tissue phenol content across all the concentrations with the longer duration of treatment, which indicates a certain level of tolerance mechanism in A. liculoides (Fig. 3F). Similar ndings have been reported an increase in the tissue phenolics of Azolla imbricata plants with the duration of treatment subjected to different concentrations of cadmium (Dai et al., 2006). Among the different types of ROS, H 2 O 2 is the prime representative with relative stability and longer half-life which can determine the cellular stability. From our results, H 2 O 2 content and in situ localization gives a clear view of the impact of increasing toxicity with the increasing concentrations of phenol on A. liculoides (Fig. 4A-B). The enhanced levels of H 2 O 2 promotes oxidative stress as well as activates the antioxidative machinery by actively regulating the cellular signaling to protect the plants (Mittler, 2002, Prasad andSingh, 2011).
The phenol induced toxicity creates imbalance by generation of free radicals of oxygen, which triggers the cellular antioxidative defense mechanism in plants. The major players in this protective mechanism are antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX), glutathione reductase (GR) and several other non-enzymatic antioxidants. Enhanced activity of these antioxidative enzymes helps plants to counteract the oxidative stress (Soares et al., 2019). As a primary response to oxidative stress, SOD converts the highly unstable and reactive superoxide radicals to comparatively less harmful H 2 O 2 to protect major cellular organelles. In the present study, remarkable increase in SOD activity at higher phenol concentrations subjected to longer duration of treatment reveals that the A. liculocoides plant possess the capability to catalyze the increased amount of superoxide radicals (Fig. 3A). A similar observation has been reported in a different macrophyte -Salvinia natans, where on exposure to copper and glyphosate separately or in combined form led to the enhanced activity of SOD (Liu et al., 2019). The enhancement in the generation of H 2 O 2 by the action of SOD triggers a set of antioxidative enzymes to eliminate H 2 O 2 . Although H 2 O 2 is an important cellular signaling molecule, it can also hamper several metabolic pathways and lead to destruction of cell organelles. Therefore, to eliminate excessive H 2 O 2 accumulated in plant tissues, a set of antioxidative enzymes like catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), etc. play their essential part in the scavenging mechanism (Saxena et al., 2016). In our study, enhanced CAT activity was observed in plants exposed to phenol for shorter duration, however, the CAT activity was found to decline after being subjected to longer duration of treatment (Fig. 3E). Devi and Prasad (1998) have also reported an enhancement in the activity of CAT in a free oating macrophte -Ceratophyllum demersum subjected to copper toxicity. The increased activity of CAT effectively assist the plants to cope up with oxidative stress imparted by the toxic water pollutants. But at higher concentrations of these pollutants, this enzyme do not provide much protection to the plants, which is also observed in case of the present study. The peroxidase isozymes (POX and APX) also plays a remarkable part in the scavenging of excessive H 2 O 2 . In our study, the APX and POX activities were found to be elevated at lower phenolic treatments, after which a considerable reduction was observed at higher concentrations of phenol (Fig. 3C-D). This indicates towards the ine ciency of the antioxidative machinery of the plants to provide su cient protection at higher concentrations. The enhanced POX and APX activities evidently enables the plant to employ a system for the coordinated elimination of peroxides and bypass the cellular damage, which has been previously observed on experiments with Salvinia molesta in response to arsenite toxicity (da Silva, 2018). Also, in the present study, the increased activity of POX and APX helped the A. liculoides to perform better at lower phenol concentration. Along with the scavenging of H 2 O 2 , for the detoxi cation of xenobiotics, and for controlling the equilibrium between GSSG and GSH ascorbate-glutathione cycle GR plays an important role during stressed condition (Vilvert, 2017). Apart from APX, GR is an essential component of ascorbate-glutathione cycle and effectively regulates the accumulation of H 2 O 2 . In the present study, in response to increasing phenol concentrations, noticeable reduction in the activity of GR was observed (Fig. 3B). Also the, GR activity in plants subjected to lower concentrations of phenol did not show any signi cant increase from the 3 day treated plants.
Similarly, in response to the exposure to oxitetracyline -a toxic water pollutant, Egria densa also shows a decline in the activity of GR which clearly suggests the relatively lesser importance of this enzyme in the detoxi cation of H 2 O 2 when compared to the other antioxidative enzymes (Vilvert, 2017).

Conclusion
The growth parameters, pigment contents and enhanced activities of antioxidative enzymes provides clear evidence that A. liculoides can maintain its normal growth and physiology up to a considerable level at lower phenol concentrations. Our results indicated that the A. liculocoides plants show tolerance upto a concentration of 1 mM phenol under laboratory conditions and above this concentration signi cant damages could be observed morphologically, physiologically and biochemically. The extent of tissue injury and accumulation of ROS like H 2 O 2 manifested by the plants exposed to higher concentrations of phenol for longer durations have clearly indicated the sensitivity of the plants. This sensitivity was also well re ected by the tissue damage in the form of necrotic lesions on the fronds and cessation in the growth of newer fronds. Though we have used crystalline phenol as a representative phenol compound to assess its toxicity on Azolla liculoides for the present study, still the results clearly indicate the potential of this plant to be used as a biological indicator for the rapid detection of other phenolic pollutants in water bodies. In this connection, further studies are required to be conducted to understand the molecular mechanism of the impact of phenolic pollutants on this plant. Moreover, the potential of this macrophyte should be evaluated for its ability to accumulate phenolic compounds and the probable strategies for phenol degradation in the aquatic ecosystem.