Effect of industrial wastewater on wheat germination, growth, yield, nutrients and bioaccumulation of lead

Due to fresh water scarcity, farmers are using polluted water for irrigation. This research was conducted to study the bioaccumulation of Pb in wheat (Cv. Shafaq-2006). The experiment was comprised of seven treatments of lead i.e. 0–1,000 mg Pb/kg. The results revealed that lead severely reduces germination (− 30%), seedling fresh weight (− 74%), seedling dry weight (− 77%), vigor index (− 89%), tolerance index (− 84%), plant height (− 33%), number of leaves (− 41%), root fresh weight (− 50%), shoot fresh weight (− 62%), root dry weight (− 63%), shoot dry weight (− 71%), and root length (− 45%). The physiological parameters also respond negatively like stomatal conductance (− 82%), transpiration rate (− 72%) and photosynthetic rate (− 74%). Similarly, biochemical parameters also showed negative impacts, like carotenoids (− 41), total chlorophyll (− 43), chlorophyll a (− 42) and chlorophyll b (− 53). Yield parameters like the number of seed/plant, seed weight/plant, 1,000 seed weight and harvest index were reduced by 90%, 88%, 44% and 61%, respectively in T6. In addition, protein contents (− 81%), phosphorous (− 60%) and potassium (− 55%) were highly effected in the highest lead concentration (T6). Lead accumulation was extremely higher in seeds (119%) as compared to control plants. Lead bio-accumulation above threshold concentrations in crop parts is a serious human health concern.

Treatments were named T o , T 1 , T 2 , T 3 , T 4 , T 5 , T 6 having 0, 100, 200, 400, 600, 800 and 1,000 mg of Pb/L. All the petri dishes were arranged in completely randomized design (CRD). 2 ml of each treatment was added in respective petridish on daily bases. Germination was noted with the emergence of radical. Data was collected for germination %, seedling length, plumule length, radical length and fresh biomass. Seedling vigor index and tolerance index was estimated by multiplying seedling length and germination percentage 21 . The Metals Tolerance Index is a diagnostic metric used to identify samples with a high degree of organisms tolerant of metals.
Pot experiment. The pot experiment was set up in Botanical Garden (located at The Mall road) of Government College University in ambient condition. No additional instrument was used to control any abiotic factor (light, temperature, humidity, etc.). Each pot (earthen, 12 inch diameter) was washed, dried, lined with polythene bag and filled with 5.5 kg soil (1:6, humus: garden soil) mix. Lead nitrate was added in the soil in different concentration. Treatments were named T o , T 1 , T 2 , T 3 , T 4 , T 5 , T 6 having 0, 100, 200, 400, 600, 800 and 1,000 mg of Pb kg −1 . Pot's positions were changed on a weekly basis to keep climatic condition uniform. Data of weather conditions and other meteorological parameters is given in Table S1. All other agronomic practices were kept same for all treatments. All parameters were measured at harvest. After harvesting, plants were washed, dried and stored in polythene zipper bags. Biomass (fresh and dry) of root/shoot and length were measured at harvest using electric balance and measuring rod. For measuring dry weight, crop samples were dried (at 70 °C) for 24 h. Yield attributes were calculated by the protocols of Zadoks 22 . Physiological and biochemical attributes. IRGA, 'Infra-Red Gas Analyzer' (LCA4 Model) was used to measure stomatal conductance, transpiration and photosynthesis rate. Three leaves from each pot were selected and readings were noted at 75th and 130th day 23 . Chlorophyll contents were determined by following the protocols of Arnon 24 . Carotenoid extraction was carried out in dim light where 1 g of randomly selected fresh leaves was crushed in acetone. Homogenate was filtered and more acetone was added to make the final volume up to 50 ml. Carotenoids were estimated with the help of spectrophotometer at the wavelength of 440.5 nm 25 .
where: "V" is the volume used for analysis; "383" is the extinction coefficient of carotenoids; "As" is the absorbance of the sample; "Ab" is the cuvette error; "W" is the weight of the sample in gram.
Mineral contents and quality attributes. Potassium was estimated through flame photometer (PF Pt JENWWAY, England) 26 . For phosphorous quantification, 1 g of plant sample was dried, crushed, and placed at 650 °C in the furnace for 3 h 27 . Burned samples was added in 10 ml of H 2 SO 4 (0.7 N), stayed for about 1 h and later filtered (through Whatman-No-42) and the final volume was raised to 50 ml.
Over dried potassium phosphate (0.43 g) was dissolved in one liter of distilled water to make stoke solution. Different standards (ranging from 2 to 50 ppm) were prepared from this stock solution. Ammonium vanadomolybdate (10 ml) and the respective standard (5 ml) were mixed and stayed for 10 min. Absorbance was noted by spectrophotometer at 410 nm. The same protocol was used for samples. The standard graph was plotted using absorbance value of the standard and absorbance value of phosphorous.
Nitrogen was determined by using Kjeldahl method using the following formula: where ' A' is the HCl used, B is HCl used for blank; 14.01 is atomic mass of nitrogen. While protein was estimated by multiplying the nitrogen contents (%) with 6.25.
Lead accumulation. Lead accumulation in roots, shoots and leaves were determined by following protocol of Chandra 28 . Crop samples were 1st washed with double distilled water and later with calcium chloride (10 mM solution). Samples were dried and turned into ashes at 450 °C. Digestion of ash was carried out in HNO 3 (2%), www.nature.com/scientificreports/ which was later filtered with glass fiber filter 29 . Lead was quantified by "inductively coupled plasma spectrophotometer (Thermo Electron; Model IRIS Intrepid II XDL, USA)". Detection limit ranges from 8 to 80 ng L −1 .

Data analysis.
Results were evaluated through ANOVA and DMR using Costat version 3.03 30 .

Result and discussion
This study has given significant results regarding the Pb toxicity in the germination, growth, physiological, biochemical and yield attributes of wheat. Data regarding germination attributes revealed that Pb treatments significantly affected the germination percentage, fresh weight, dry weight, plumule length, radical length and seedling length of the wheat (Table 1 and Fig. 1). Increase Pb concentration results in negative impacts in wheat. The highest decline recorded was in germination i.e. 30%. Radical length was decreased by 45%, fresh weight of seedling by 74%, dry weight of seedlings by 77%), seedling vigor index by 89% and tolerance index by 74% in T 6 (1,000 mg Pb/L) compared to the control (Table 1 and Fig. 1). Chun 31 observed that at 0-0.5 mg Pb/kg non-significant adverse impact of lead on germination index and energy required during the process of germination. Likewise, growth rate of roots and shoots also remain unaffected at low level contamination of lead 32 . However, concentration above 4.5 mg/kg of soil is toxic to plant health, which decreases germination 33 .
Roots of plants are more affected by lead toxicity as compared to other plant parts 12,34 . Excessive Pb contamination (including Pb) results in the formation of 'reactive oxygen species' (ROS) in mitochondria, chloroplast and  www.nature.com/scientificreports/ cellular compartments 14,35 . The results of (seedling growth) present study are in line with that of Yadav 36 who stated that growth attributes were significantly affected by increasing the Pb concentration. Co-cropping of T. minuta and soybean enhanced the Pb accumulation in T. minuta without posing any health risk from grain consumption of soybean 9 . Data regarding the growth attributes revealed that Pb significantly (p ≤ 0.01; 0.05) affect the growth attributes ( Table 2). Minimum plant height (58.2 ± 0.32 cm), root length (13.5 ± 0.24 cm), number of leaves (21.67 ± 0.05) were noticed in T 6 . Dry weight and fresh weight was also affected negatively at higher lead concentration. Minimum fresh weight of roots (0.66 ± 0.05 g), shoot (2.53 ± 0.04 g), and dry weight of roots (1.61 ± 0.02 g), shoot (0.34 ± 0.05 g) were noticed in T 6 ( Table 2). Gopal and Rizvi 37 also observed that due to increase in lead concentration length and weight of root and shoot decreases. Adverse effects of lead were not apparent at early stage of the plant's life but detrimental effects increased with the increase in maturity level of plant. Metal uptake, translocation and bio-accumulation are age dependent. Antoniadis 38 also reported that root development in excessive Pb concentration was poor. The major reason behind the reduction in biomass of plants is reduction in the rate of photosynthesis and nitrogen metabolism 39 . Osma 40 reported that specie and Pb concentration significantly effect the fresh weight in brassica. Decrease in fresh weight of shoots among different species was observed in following order, B. juncea. < B. oleracea I < B. carinata. Plants may develop resistance and as a result may survive in lead contaminated soils 41 . Gill et al., reported the damage in leaf mesophyll, root tips and disruption of golgbodies, diffused cell wall due to chromium toxicity at 400 µM 14 .
Maximum reduction in all biochemical and physiological attributes was noticed in those pots whose soil was spiked with 1,000 mg Pb/kg. all the treatments differ significantly (p ≤ 0.01; 0.05). A linear decrease in the physiological and growth attributes was noted with increase in Pb concentration. Regarding the physiological attributes maximum reduction in photosynthetic rate (44%), stomatal conductance (82%) and transpiration rate (72%) was noticed in T 6 ( Table 3). Maximum reduction in chlorophyll a (44%), chlorophyll b (53%), carotenoids contents (42%) and total chlorophyll contents (43%) was noticed in those pots whose soil was spiked with the  www.nature.com/scientificreports/ 1,000 mg Pb/kg (Fig. 2). Similar results were observed by Zeng 42 that performed an experimental analysis by applying lead acetate at six different levels (0-900 mg/kg). This may be due to non-toxic effect of lead at low concentration. All the physiological and biochemical attributes of the wheat was reduced by increasing the Pb concentration that might be due to lead toxicity as it damages the chloroplast structure, decreases chlorophyll synthesis, restricted enzymatic and carotenoids activities. Lead is also responsible for obstructions in electron transport chain, deficiency of carbon dioxide (due to closure of stomata) and altered thylokoid membrane. Qadri 43 reported that metal presence reduce the chlorophyllase and results in low chlorophyll content. Studies have indicated that chlorophyll b contents is more disturbed then chlorophyll a 39 . Rafaqat et al., reported the decline in photosynthetic rate in Brassica napus L. due to the elevated stress of chromium 5 . All the Pb treatments significantly (p ≤ 0.01; 0.05) decrease the mineral contents and the quality attributes of the wheat (Table 4). Phosphorous content decreased from 11 to 58%, potassium contents from 14 to 34%, nitrogen contents from 38 to 82% and proteins contents from 18 to 81% in the T 6 as compared to the control. Range of all the yield attributes was as follows, phosphorous contents (18.85-7.51 ppm), potassium contents (409.20-269.57 ppm), nitrogen contents (1.54-0.28%) and protein contents (9.63-1.75%).
Result of Pot experiment were significantly changed by Pb treatments (Table 5). At higher concentration the yield decrease. Minimum number of seeds per plant (16.33 ± 0.58), seed weight per plant (0.85 ± 0.09 g), 1,000 seed's weight (30.03 ± 0.63 g), straw weight per plant (2.62 ± 0.12 g), and harvest index (263.39 ± 11.52%) was noticed in T 6 ( Table 5). In order to feed the growing population, farmers demand those varieties of crops which give more yields all the yield attributes were reduced by the increasing Pb concentrations. Scientists are  Table 4. Effect of various concentrations of lead on the mineral contents and quality attributes of wheat plant. T 0 = 0 mg Pb/kg; T 1 = 100 mg Pb/kg; T 2 = 200 mg Pb/kg; T 3 = 400 mg Pb/kg; T 4 = 600 mg Pb/kg; T 5 = 800 mg Pb/ kg; T 6 = 1,000 mg Pb/kg; Treatment means with different letters in the same column are significantly different from one another according to Duncan Multiple Range Test at (p ≤ 0.05); values represent the Means ± SE. www.nature.com/scientificreports/ also focused on developing new and improved varieties (with genetic engineering), to produce more yield. The current research investigate severe negative impacts of Pb on wheat yield. Xiong 44 reported that cadmium, lead and zinc accumulate in cabbage and spinach which were grown near smelter. Lead interferes with active sites of enzymes and results in low yield. Zheljazkov and Nielsen 45 revealed that a 400 m away source of lead (through air) affected corn mint yield by 16%. Similarly, 14% decrease in yield of essential oils was observed due to air pollution 46 . Gill et al ., reported increased size and number of starch grains due to chromium in oilseed rape cultivars 24 . The presence of heavy metals revealed elite molecular transporters (MTs) genes that were responsible for water transmembrane transporter activity 5 .

Treatments Phosphorous contents (ppm) Potassium contents (ppm) Nitrogen contents (%) Protein contents (%)
The results of the Pb accumulation in wheat plant including root, shoot and seed were depicted in the ( Table 6). Significant variation in the treatment means was revealed. The extent of increase in lead concentration was quite alarming compared to control treatments. For instance, roots showed 4,600% enhance in Pb quantity in T 6 . Likewise, shoots and seeds showed an increase of 9,800% and 118% in Pb concentration, respectively. Lead concentration decreases in parts of the wheat in the following order root > shoot > seeds. The considerable increase in lead concentration was noticed in T 6 as compared to the control (Table 6). Lead uptake by roots is directly proportional to the lead concentration. Other factors which contribute towards Pb adsorption include: type of fertilizer, amount of fertilizer, microbial activity, soil pH, precipitates of carbonate/phosphate and concentration of extractable lead in soil 47 . Antoniadis 38 reported lead translocation decreases significantly as the metal moves away from root, which implies that roots accumulate more lead then seeds and shoots. Different studies revealed high accumulation rate of lead in roots; Kenaf root may retain upto 85% of total Pb present in plant body 48 , Thlaspi praecox can accumulate up to 80% Pb 49 and Indian mustard (Brassica junce) has the tendency to accumulate upto 95% of total Pb 50 . Zhang 51 conclude that rice cultivars different variable in metal uptake and translocation. Lead absorption and translocation to leaves depends on number of factors and these factors and variable among different crops. Shoot-accumulators store more lead in shoots of the plant while root-accumulator tends to store large amount of lead in roots and allow very small concentration of lead to be transported to above ground parts of crop. Another study revealed that uptake of lead by plants depends upon Table 5. Effect of various concentrations of lead on yield attributes of wheat. T 0 = 0 mg Pb/kg; T 1 = 100 mg Pb/kg; T 2 = 200 mg Pb/kg; T 3 = 400 mg Pb/kg; T 4 = 600 mg Pb/kg; T 5 = 800 mg Pb/kg; T 6 = 1,000 mg Pb/ kg; Treatment means with different letters in the same column are significantly different from one another according to Duncan Multiple Range Test at (p ≤ 0.05); values represent the Means ± SE.

Treatments
Number of seed/plant Seed weight/plant (g) 1,000 seed weight (g) www.nature.com/scientificreports/ the surface area of roots 52 . Heavy metal effects root tip cells more as enlarged vacuole, disrupted cell membrane, plasmolysis and damaged mitochondrial thylakoid is more prominent in root cells 17 . Presence of different forms of lead in plants is also responsible for difference in its translocation rate. Sharma 53 concluded that ions and lower molecular complexes are more mobile then other forms. Roots may absorb more lead in such plants but high molecular weight and complexes restrict its translocation and distribution to aerial crop part 54 . In an experiment conducted by Basharat et al., the lead induce reduction in Brassica napus L. biomass, reduced macronutrients in shoot, increased ROS and MDA 6 . Ali et al., reported the use of plant growth regulators (5-aminolevulinic acid, ALA) can cpounter the injurious effect of heavy metals in crops 15 .

Conclusion
The present study revealed that lead imparts number of negative effects on germination, physiological, biochemical, yield, mineral, and growth and quality attributes of wheat crop. It was observed that all adverse impacts of lead were due to reduced photosynthesis rate and related phenomenon like decreased rate of transpiration and stomatal conductance. Accumulation rate of lead in wheat parts (shoots, seeds and roots) was also above threshold level. Lead contamination in wheat is alarming as it is the staple food in Pakistan. Slow bioaccumulation of lead ultimately leads towards serious disorders and illness. And certain remedial techniques should be adopted to restrict the entry of lead from contaminated soils to wheat. Maintaining supply of lead free wheat is the need of an hour.