Mitigation of Lead (Pb) Toxicity in Rice Cultivated with Ground Water and Waste Water by Application of Acidied Carbon

Toxicity induced by a higher concentration of lead (Pb) can signicantly decrease plants growth, gas exchange and yield attributes. It also caused cancer in humans. Use of organic amendments especially biochar has the capability to alleviate Pb toxicity in different crops. Application of biochar can decrease the uptake Pb by roots in plants. However, high pH of thermo pyrolyzed biochar makes it unt amendment for high pH soils. As Pb is an acute toxin and its uptake in rice is a major issue, current experiment was conducted with the aim to explore the ecacy of chemically produced acidied carbon (AC) for mitigation of Pb toxicity in rice. Leas has introduced articially at the rate of (0, 15 and 30 mg kg 1 soil) in combination with 0, 0.5 and 1 % AC underground water (GW) and waste water irrigation (WW) in rice. Results conrmed that addition of 1% AC signicantly improved plant height, number of tillers, spike length fresh and dry weights of spikes and straw, 1000 grains weight, photosynthetic rate, transpiration rate and stomatal conductance over 0 % AC under GW and WW irrigations at 30 mg Pb kg -1 soil (30Pb) toxicity. A signicant decrease in electrolyte leakage and plant Pb concentration by application of 0.5 and 1 % AC validated their effectiveness for mitigation of 30Pb toxicity in rice over 0 % AC under GW and WW. In conclusion, 1 % AC is effective amendment in the alleviation of Pb toxicity in rice irrigated with GW and WW at 30Pb.


Introduction
Toxicity induced by heavy metals, is a serious concern of soil and environmental health. These heavy metals entered in food chain when plants are cultivated in heavy metals contaminated soils 1,2 . Among different heavy metals, lead (Pb) is a potential pollutant and highly toxic for plants and humans. It has been observed that sul de ores melting and burning of fossil fuels played a signi cant role in the emission of Pb in environment 3 . Higher uptake of Pb can cause mutagenesis and cancer in humans 4,5 .
It also adversely affect the growth attributes, macronutrients uptake and chlorophyll contents of plants 5 . Low photosynthetic rate, transpiration rate and stomatal conductance are also some of negative effects of Pb which played signi cant role in making poor plants growth 6 .
On the other hand, limited availability of good quality irrigation water is a big hurdle for the achievement of optimum crops yield. On an average, the consumption of good quality water for domestic purposes and industries has tremendously increased. Such conditions resulted in generation of signi cant quantity (962,334 M gallons) of untreated waste water 7 . Most of farmer applied 30% of this untreated waste water as irrigation in urban and semi-urban agricultural lands (22 M ha land) to overcome the problem of irrigation water scarcity 7,8 . Readily available essential nutrients and low cost of waste water attracts the farming community for its utilization as irrigation 9 . However, they ignored the health hazards that are associated with the consumption of such untreated waste water irrigated crops 10 . It also induced detrimental effect on plants and soil 11 . Persistent organic pollutants and enrichment of heavy metals in waste water coming from sewage and industries make it un t. Microbes in soil remained unable to degrade the accumulated heavy metals which become the part of soil when waste water is irrigated 12 .
To deal with heavy metals induced toxicity in plants scientists have suggested many technologies 13 .
However, need of time is to adopt such methodologies and techniques which are cost-effective and remained actively bene cial for long duration. That's why, bio-sorbent have suggested for mitigation of heavy metals toxicity in plants. Activated carbon biochar is one of such amendment that has potential to immobilize heavy metals in soil [14][15][16] . Pyrolysis of organic waste compounds resulted in sequestration of carbon. It remained in soil for longer time period due to high resistant against decomposition 17 . Many essential nutrients that are integral part of biochar structure is also released in soil which also facilitate in the improvement of plants vegetative growth 18 . High porosity, surface charges stability and improved exchange sites played an imperative role in decreasing the uptake of these heavy metals in plants. When biochar is applied in soil, it not only contribute in better uptake of essential nutrients but also improve soil organic carbon pool which positively affect soil health 5,14,19−28 .
In Bangladesh, Vietnam and Indonesia, 60% calories are taken from rice 31 . That's why current experiment was conducted to check the in uence of acidi ed carbon in alleviation of Pb toxicity in rice plant. To the best of our knowledge, this is the rst kind of study where we use chemically produced acidi ed carbon in rice as an amendment for immobilization of Pb. We hypothesized that combined use of wastewater and chemically produced acidi ed carbon can be effective in minimizing Pb toxicity and enhancement growth, gas exchange attributes and yield of rice under Pd toxic conditions.

Material And Methodology
Experimental site and design A pot study was done for assessing the effect of acidi ed carbon on rice growth and yield under Pb contaminated soil. The rice was irrigated with ground water and waste water. The study was conducted in research area of Department Soil Science, Bahauddin Zakariya University Multan, Pakistan. The research area is located in semi-arid region characterized with low average rainfall and high mean temperature ( Figure 1).

Preparation of acidi ed carbon (AC)
Waste syrup of sugar was used to manufacture acidi ed carbon as described by Sultan et al. 32  properly mixed and 7 kg soil was lled in pots for further application of AC treatments in control and Pb contaminated soil. AC was incorporated well in soil at 0.5 % and 1 % rate.

Irrigation
Sewage water and ground water was used as treatment for irrigation purpose. Biochemical and chemical properties of waste water is described in Table 2.

Harvesting of crop
Crop was harvested manually after full maturity in November, 2019. The harvesting was done manually. were weighed on weighing balance. After separation of kernels from spikes, thousand grains were separated and weighed for determining the quality of seed.

Electrolyte leakage
For estimating the electrolyte leakage (EL) of plants, 0.2 g of fresh plant sample was taken and chopped in to small pieces and immersed in 10 ml of distilled water 36 . Incubation was done at 25 °C for 24 h. The electrical conductivity (EC1) was examined using pre-calibrated EC meter. The second EC (EC2) was measured by heating the test tubes in a water bath at 120 °C for 20 min. The nal value of EL was calculated using the equation as follows; Chlorophyll contents Fresh leaves were taken and cut in to small pieces and 0.5 g leaf samples were immersed in 10 ml acetone for 24 hours. Extract of chlorophyll was measured and color intensity was determined at 645 nm and 663 nm by the help of spectrophotometer 37 . From intensity values, chlorophyll contents were determined by following formula: where, OD = Optical density (wavelength), V = Final volume made, W = Fresh leaf made (g) Photosynthetic rate and stomatal conductance of rice leaves were determined by using Infrared gas analyzer (CI-340 Photosynthesis system, CID, Inc. USA). On a sunny day, the readings were taken between 10:30 and 11:30 AM at saturating intensity of light 38 .

Plant Digestion
Plants shoot, leaves and grains samples were oven dried and grinded separately to form homogeneous powder. A mixture of di-acid was prepared by 10:1:4 HNO 3, H 2 SO 4 and HClO 4. Plant material was mixed with conc. H 2 SO 4 and kept overnight. Before heating, 10ml acid mixture was added in that suspension.
Mixture was heated till clearing the solution and white fumes emerge 39 . Mixture was than cooled and stored for further nutrient analysis. For Pb determination, sample was ltered and directly ran on atomic spectrophotometer.

Acidi ed Carbon and Soil Total Cadmium
Cadmium can be quanti ed by the soil di-acid wet digestion. For this one-gram air dried soil, 3 ml HNO 3 was added and heated on hot plate at 145 °C for an hour. After that, 4ml of per chloric acid (HClO 4 ) was added and hot plated temperature was raised to 240 °C for one hour again 40 . After clearing the solution, ask was removed and kept for cooling. When solution temperature reached at room temperature, 50 ml DI water was added in it and then ltered that solution. Sample was run with deionized water as blank at atomic absorption spectrophotometer (graphite furner; Agilant 200) for estimating the Pb concentration in soil.

Statistical analysis
Data computation was made on Microsoft Excel 2019 ® and statistical analysis one-way ANOVA was performed by Origin 2020b. Difference among treatment means was assessed by using the least square difference (LSD) at 0.5% probability level. Pearson correlation and principal component analysis was done 41 .

Results
Results showed that application of different levels of acidi ed carbon (AC) signi cantly affect the plant height of rice cultivated under 0, 15 (15Pb) and 30 mg Pb kg − 1 (30Pb) soil irrigated with waste water (WW) and ground water (GW) irrigations. No signi cant change was observed in plant height in 15Pb and 30Pb from control where WW was applied at 0%AAC. In 0%AAC, plant height was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for plant height over control (no Pb) under GW at 1%AAC. No signi cant change was also observed in plant height in 15Pb and 30Pb from control where WW was applied at 1%AAC. In 1%AAC, plant height was statistically alike in control + WW, 15Pb + WW and 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW ( Fig. 2A). Increasing level of Pb was nonsigni cant for plant height over control (no Pb) under GW at 1%AAC. Signi cant change was noted in plant height in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, plant height was signi cantly high in control + WW and 15Pb + WW over control + GW and 15Pb + GW. Chord diagram con rmed that 1%ACC gave maximum increase in plant height as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better plant height as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 2B).
Addition of different levels of acidi ed carbon (AC) signi cantly changed spike length of rice cultivated with waste water (WW) and ground water (GW) irrigations under 0, 15 (15Pb) and 30 mg Pb kg − 1 (30Pb) soil irrigated. In 0%AAC, spike length was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for spike length over control (no Pb) under GW at 1%AAC. No signi cant change was also observed in spike length in 15Pb and 30Pb from control where WW was applied at 1%AAC. In 1%AAC, spike length was statistically alike in control + WW, 15Pb + WW but differed signi cantly in 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW respectively (Fig. 3A). Increasing level of Pb was non-signi cant for spike length over control (no Pb) under GW at 1%AAC. Signi cant change was noted in spike length in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, spike length was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW. Chord diagram showed that 1%ACC gave maximum increase in spike length as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better spike length as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 3B).
Number of spikes did not differ signi cantly in 15Pb + GW over control + GW. No signi cant change was observed in number of spikes in 15Pb and 30Pb from control where WW was applied at 0%AAC. In 0%AAC, number of spikes was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for number of spikes over control (no Pb) under GW at 1%AAC. No signi cant change was also observed in number of spikes in 15Pb and 30Pb from control where WW was applied at 1%AAC (Fig. 4A). In 1%AAC, number of spikes was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Increasing level of Pb was non-signi cant for number of spikes over control (no Pb) under GW at 1%AAC. Signi cant change was noted in number of spikes in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, number of spikes was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW. Results also con rmed that 1%ACC gave maximum increase in number of spikes as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better number of spikes as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 4B).
Number of tillers remained non-signi cant in 15Pb + GW over control + GW. Signi cant decrease was observed in number of tillers in 30Pb from control where WW was applied at 0%AAC. In 0%AAC, number of tillers was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW as compared to control + GW, 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for number of tillers over control (no Pb) under GW at 1%AAC. No signi cant change was also observed in number of tillers in 15Pb and 30Pb from control where WW was applied at 1%AAC. In 1%AAC, number of tillers was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. 15Pb was non-signi cant but 30Pb different signi cantly for number of tillers over control (no Pb) under GW at 1%AAC (Fig. 5A). Signi cant change was noted in number of tillers in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, number of tillers was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW. Results also con rmed that 1%ACC gave maximum increase in number of tillers as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better number of tillers as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 5B).
In 0%AAC, fresh weight of straw was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for fresh weight of straw over control (no Pb) under GW and WW at 1%AAC. In 1%AAC, fresh weight of straw was signi cantly higher in control + WW and 15Pb + WW over control + GW and 15Pb + GW respectively. Treatments 15Pb and 30Pb were non-signi cant for fresh weight of straw over control (no Pb) under GW at 1%AAC (Fig. 6A). Signi cant change was noted in fresh weight of straw in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, fresh weight of straw was signi cantly high in control + WW and 15Pb + WW over control + GW and 15Pb + GW. Results also con rmed that 1%ACC gave maximum increase in fresh weight of straw as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better fresh weight of straw as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 6B).
Effects of different application rates of acidi ed activated carbon and lead (Pb) levels under waste water (WW) and ground water (GW) irrigation was signi cant for fresh weight of spike. It was noted that 15Pb did not decrease fresh weight of spike over control (no Pb) under GW and WW at 0%AAC. However, 30Pb signi cantly decreased fresh weight of spike from control in GW at 0%AAC. In 0%AAC, fresh weight of spike was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW.
Increasing level of Pb was non-signi cant for fresh weight of spike over control (no Pb) under GW and WW at 1%AAC. In 1%AAC, fresh weight of spike was non-signi cantly higher in control + WW and 15Pb + WW over control + GW and 15Pb + GW respectively (Fig. 7A). Treatments 30Pb were non-signi cant for fresh weight of spike over control (no Pb) under GW at 1%AAC. Signi cant change was noted in fresh weight of spike in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, fresh weight of spike was signi cantly high in control + WW and 15Pb + WW over control + GW and 15Pb + GW. Results also con rmed that 1%ACC gave maximum increase in fresh weight of spike as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better fresh weight of spike as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 7B).
No signi cant change in dry weight of straw was observed by increasing Pb levels in WW at 0%AAC. In 0%AAC, dry weight of straw was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for dry weight of straw over control (no Pb) under GW and WW at 1%AAC. In 1%AAC, dry weight of straw was statistically alike in control + WW and 15Pb + WW over control + GW and 15Pb + GW respectively. Treatments 30Pb was signi cant for dry weight of straw over control (no Pb) under GW at 1%AAC (Fig. 8A). Signi cant change was noted in dry weight of straw in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, dry weight of straw was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in dry weight of straw as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better dry weight of straw as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 8B).
In 0%AAC, dry weight of spike was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for dry weight of spike over control (no Pb) under GW and WW at 1%AAC. In 1%AAC, dry weight of spike was statistically alike in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for dry weight of spike over control (no Pb) under GW at 1%AAC (Fig. 9A). Signi cant change was noted in dry weight of spike in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, dry weight of spike was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in dry weight of spike as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better dry weight of spike as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 9B).
Results showed that 0%AAC, 1000 grains weight was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. Increasing level of Pb was non-signi cant for 1000 grains weight over control (no Pb) under GW and WW at 1%AAC. In 1%AAC, 1000 grains weight was statistically alike in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for 1000 grains weight over control (no Pb) under GW at 1%AAC. Signi cant change was noted in 1000 grains weight in control over 15Pb and 30Pb where WW was applied at 1%AAC (Fig. 10A). In 1%AAC, 1000 grains weight was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in 1000 grains weight as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better 1000 grains weight as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 10B).
No signi cant change was observed in chlorophyll content where WW was applied under increasing level of Pb. In 0%AAC, chlorophyll content was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly decreased chlorophyll content over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, chlorophyll content was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for chlorophyll content over control (no Pb) under GW at 1%AAC. Signi cant change was noted in chlorophyll content in control over 15Pb and 30Pb where WW was applied at 1%AAC (Fig. 11A). In 1%AAC, chlorophyll content was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in chlorophyll content as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better chlorophyll content as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 12B).
A signi cant decrease was observed in electrolyte leakage where WW was applied under increasing level of Pb. In 0%AAC, electrolyte leakage was signi cantly low in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly increased electrolyte leakage over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, electrolyte leakage was signi cantly low in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for electrolyte leakage over control (no Pb) under GW at 1%AAC. Signi cant change was noted in electrolyte leakage in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, electrolyte leakage was signi cantly low in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW (Fig. 12A). Results also con rmed that 1%ACC gave maximum decrease in electrolyte leakage as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly low electrolyte leakage as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 12B).
Effects of different application rates of acidi ed activated carbon and lead (Pb) levels under waste water (WW) and ground water (GW) irrigation was signi cant for photosynthetic rate. It was noted that 30Pb signi cantly decreased photosynthetic rate over control (no Pb) under GW at 0%AAC. A signi cant change was observed in photosynthetic rate where WW was applied under increasing level of Pb. In 0%AAC, photosynthetic rate was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly decreased photosynthetic rate over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, photosynthetic rate was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for photosynthetic rate over control (no Pb) under GW at 1%AAC (Fig. 13A). Signi cant change was noted in photosynthetic rate in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, photosynthetic rate was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Chord diagram con rmed that 1%ACC gave maximum increase in photosynthetic rate as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better photosynthetic rate as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 13B).
A signi cant change was observed in transpiration rate where WW was applied under increasing level of Pb. In 0%AAC, transpiration rate was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly decreased transpiration rate over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, transpiration rate was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for transpiration rate over control (no Pb) under GW at 1%AAC. Signi cant change was noted in transpiration rate in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, transpiration rate was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW (Fig. 14A). Results also con rmed that 1%ACC gave maximum increase in transpiration rate as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly better transpiration rate as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 14B).
Signi cant change was noted in stomatal conductance in control over 15Pb and 30Pb where WW was applied at 1%AAC. In 1%AAC, stomatal conductance was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in stomatal conductance as compared to 1%AAC and 0%AAC in GW and WW.
However, application of 1%AAC also gave signi cantly better stomatal conductance as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 15B).
A signi cant change was observed in Pb concentration of rice where WW was applied under increasing level of Pb. In 0%AAC, Pb concentration of rice was signi cantly higher in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly decreased Pb concentration of rice over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, Pb concentration of rice was signi cantly higher in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for Pb concentration of rice over control (no Pb) under GW at 1%AAC. Signi cant change was noted in Pb concentration of rice in control over 15Pb and 30Pb where WW was applied at 1%AAC (Fig. 16A). In 1%AAC, Pb concentration of rice was signi cantly low in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum decreased in Pb concentration of rice as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly low Pb concentration of rice as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 16B).
In 0%AAC, soil Pb concentration was signi cantly low in 15Pb + WW and 30Pb + WW as compared to 15Pb + GW and 30Pb + GW. 30Pb signi cantly increased soil Pb concentration over control (no Pb) and 15Pb under GW and WW at 1%AAC. In 1%AAC, soil Pb concentration was signi cantly low in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + GW respectively. Treatments 30Pb was signi cant for soil Pb concentration over control (no Pb) under GW at 1%AAC. Signi cant change was noted in soil Pb concentration in control over 15Pb and 30Pb where WW was applied at 1%AAC (Fig. 17A). In 1%AAC, soil Pb concentration was signi cantly high in control + WW, 15Pb + WW and 30Pb + WW over control + GW, 15Pb + GW and 30Pb + WW. Results also con rmed that 1%ACC gave maximum increase in soil Pb concentration as compared to 1%AAC and 0%AAC in GW and WW. However, application of 1%AAC also gave signi cantly high soil Pb concentration as compared to 0%AAC under different levels of Pb in GW and WW (Fig. 17B). Pearson correlation showed that electrolyte leakage and plant Pb concentration has signi cant negative correlation with all the growth, yield and gas exchange attributes. Signi cant positive correlation was noted between electrolyte leakage and plant Pb concentration (Fig. 18).

Discussion
A signi cant decrease in growth and yield attributes of rice by increasing Pb concentration was due to toxicity induced by Pb in plants. High exposure of Pb to plants signi cantly enhanced the production of reactive oxygen species (ROS). These ROS induced oxidative stress in plants that adversely affect the growth attributes 42 . Scientists have observed that ROS disturb the electron transport chain and metabolism of different nutrients elements in plants 43,44 . Increase in uptake of Pb also restrict the activity of enzymes which regulate the mineral nutrients in plants. It also decrease the permeability of membrane and react with sulfhydryl groups at cellular level 45,46 . As an abiotic stress, heavy metals also stimulate endogenous production of stress generating ethylene. This ethylene react with cell membrane and activates chlase gene which played an imperative role in decreasing the chlorophyll contents in plants leaves 47 . Higher accumulation of stress ethylene in plant roots, play an imperative role in decreasing the root elongation. It promotes the thickness of plant roots via accumulation of dead cell in the root cortex. Such accumulation of death cells in root cortex results in the formation of lysigenous aerenchyma. In hypocotyls region, stress ethylene also decreases the cell divison. Low cell divison eventually results in poor elongation of roots and shoot in plants 48 .
Results of current study showed that application of waste water signi cantly increased the growth and 1000 grains weight of rice over ground water as irrigation under different levels of Pb (0, 15 and 30Pb). This increase was due to high N, P and K contents of waste water over ground water. Hamid et al. 49 also reported similar kind of results. They argued that waste water is a rich source of nutrition for the plants. It not only provide irrigation water for crops cultivation but also has potential to decrease the application rates of inorganic macronutrients fertilizers 49 .
Furthermore, addition of 0.5 and 1% AAC performed signi cantly better over control (0%AAC) for the increase in growth and 1000 grains weight in rice under 15 and 30Pb. The improvement in growth, chlorophyll contents and 1000 grains weight were due to less uptake of Pb as compared to control.
However, activated carbon ability to sorp nutrients also reduced the losses of N and improved its uptake in plants 50,51 . According to Chan et al. 52 the high surface area of activated carbon is basic reason for improved cation exchange sites in soil which resulted in better bioavailibility of nutrients. In addition, geometry, size, distribution and number of microspores in activated carbon play an e cacious role in the sorption of nutrients and water. Application of activated carbon in soil also makes rapid cycling of nutrients. Higher retention of nutrients and diversity of rhizobacteria improve the fertility level of soil and nutrients availability to the plants 53 . It also releases a signi cant amount of nutrients in soil solution that become part of the activated carbon structure during pyrolysis The concentration of nutrients present in activated carbon is dependent on the type of waste feedstock which is used to develop the activated carbon 54 .
Younis et al. 50 regarding uptake of P by addition of cotton sticks activated carbon. According to Singh et al. 55 it is the higher amount of K in activated carbon ash that contributes in the improvement of K concentration in plants. Better uptake of K might have maintained the turgor of cells and stomatal conductance by osmoregulation 56,57 . Polar and dispersive surface of biochar along with solid surface enerygy is directly associated with the retension of water molecules. The negative zeta potential of biochar in majority cases shows the prsecence of negative charges at biochar surface. Electrostatic force of attraction between negtively charged biochar surface and cations in soil solution facilitates the adsorption of nutrients at biochar surface 18 . Progressive degradation of cellulose and lignin in waste feedstock make the amorphous surface of biochar. This amorphous surface of biochar has micropores. Emission of volatile compound during pyrolysis creats the spaces which plays a role in absorption of water when biochar is applied in soil as an amendment 58 .