Introduction

In recent years, Pakistan has faced severe water scarcity due to an increasing population (currently about 225 million and projected to exceed 250 million by 2025). Such a situation is exerting immense pressure on national water resources; consequently, per-capita water availability has declined from nearly 5,000 m³ in 1951 to around 1,100 m³ in 2005, with forecasts indicating a further reduction to approximately 800 m³ by 20251,2. To cope with this shortage, untreated sewage water is increasingly used for irrigating crops and pastures. However, using this wastewater poses serious environmental and health concerns, as it can lead to poor water quality, heavy metal accumulation, and health risks for plants, animals, and humans1. Hussain et al.2 found that effluent samples from drains were unfit for irrigation due to high EC, SAR, and RSC, with Fe being the most abundant and Cd the least. A critical concern was that cadmium (Cd) levels exceeded permissible limits in effluents, soils, and edible vegetable parts, highlighting its potential threat to food safety and human health. Therefore, effective treatment of sewage effluent is essential before its use in irrigation to safeguard crop production and minimize health risks.

Soil biogeochemical processes can mobilize toxic metals, potentially contaminating water supplies and crops3. The metal solubility depends on various interactions between waste, soil properties, and specific metals. Plants absorb these metals through soil interactions, and the metals can then enter the food chain, raising concerns about long-term health impacts4. Studies have shown that irrigation with urban wastewater can reduce growth and yield indicators in plants, such as harvest index and seed weight, with crop sensitivity varying based on effluent concentration5.

Soils in Pakistan generally exhibit low natural fertility: nitrogen is universally deficient, phosphorus is inadequate in over 90% of cultivated soils, potassium shows deficiencies in up to 40% of the area, and organic matter is critically low—averaging around 0.5%, with approximately 84% of samples falling below this level6,7. Due to the high cost and scarcity of chemical fertilizers, poor farmers often use agricultural and municipal wastewater, including SE, as a cheaper alternative for nutrients and organic matter, despite associated risks8. Canola (Brassica napus L.), an oilseed crop with global and national importance, serves as both a source of edible oil and livestock feed due to its high energy content and suitability as forage9.

This research focuses on assessing the impact of SE on soil chemistry and the growth and yield characteristics of different canola (Brassica napus L.) varieties. Canola’s growth under sewage irrigation conditions is used to evaluate its ability to absorb essential minerals such as calcium, magnesium, and manganese, which are frequently monitored to manage land application systems for wastewater treatment.

Materials and methods

Experimental site

A pot experiment was conducted in the Botany Department at Bahauddin Zakariya University in Multan, Punjab, Pakistan (30°15’50.0"N, 71°30’36.4"E). To evaluate their physicochemical properties, soil samples from this location were air-dried and then sieved through a 2-mm mesh (Sultanpur soil series: coarse silty, mixed, calcareous, hyperthermic Fluventic Camborthids). The soil and water characteristics (Tables 1 and 2) were determined as follows:

Table 1 Physicochemical characteristics of experimental soil and sewage effluent.
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Table 2 Average heavy metal concentration in sewage effluent.
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Seed collection and sterilization

Four varieties of canola (CON III, AARI Canola, Punjab Canola, and Faisal Canola) were purchased from the Ayub Agricultural Research Institute, Faisalabad, Pakistan to prepare for the experiment. Seeds were surface-sterilized following a two-step procedure. Following the protocol of12 seeds were submerged in 70% ethanol for five minutes, followed by treatment with 5% sodium hypochlorite for 10 min. This combination ensures thorough sterilization, minimizing contamination risks and maintaining the integrity of the experiment. The seeds were thoroughly cleaned with purified water and soaked for 24 h.

Irrigation

A soil moisture meter was used to maintain 70% field capacity in each pot for both SE and normal irrigation water13.

Experimental design and treatment plan

The treatments included four varieties of canola—CON III, AARI Canola, Punjab Canola, and Faisal Canola—subjected to tap water and 60% Sewage effluent (SE). The SE was diluted with tap water to achieve a final concentration of 60%. A completely randomized design (CRD) was used, with four replications per treatment. Each canola variety was tested under two irrigation treatments: (1) tap water and (2) 60% diluted SE (sewage effluent diluted with tap water in a 60:40 ratio). For the experiment, we used plastic pots with a total volume of approximately 1000 cm³, with two plants grown per pot.

Fertilizer

The recommended NPK rates (100-150-200 kg ha⁻¹) were applied. Fertilizer doses based on soil weight per pot (0.5 g, 0.75 g, and 1.0 g) were applied at the time of seed sowing. Fertilizers were mixed into the soil and in each pot 10 kg soil was filled. The nitrogen source was urea, while a single superphosphate met the potassium and phosphorus requirements.

Harvesting and data collection

Crops were harvested at physiological maturity. An analytical grade balance was used to calculate the plant’s morphological attribute data. Samples were oven-dried at 70 °C for 48 h to determine dry weight.

Chlorophyll contents and carotenoids

The study utilized Arnon’s method14 to determine the levels of chlorophyll in freshly harvested leaves. An 80% acetone solution was used for the extraction process. Absorbance readings were taken at 663 nm, 645 nm, and 480 nm for the final computation of chlorophyll contents14 and carotenoids15.

Antioxidant

Superoxide dismutase activity was measured using NBT reduction at 560 nm16. Peroxidase (POD) activity was determined using the guaiacol method at 420 nm, where the reaction mixture contained enzyme extract, phosphate buffer (50 mM, pH 7.0), 20 mM guaiacol, and 40 mM H₂O₂17. The decrease in absorbance at 240 nm due to H2O2 breakdown determined catalase activity18. Ascorbate peroxidase activity was measured at 290 nm19. Malondialdehyde was determined by reacting to the sample extract with thiobarbituric acid (TBA) to form a colored complex. Malondialdehyde concentration was calculated at 532 nm20. To determine ascorbic acid content, 10% trichloroacetic acid was used. The centrifugation was performed for 10 min at 12,000×g. Using a spectrophotometer, absorbance was taken at 525 nm as described for the final assessment of ascorbic acid21. Hydrogen peroxide (H2O2) was determined to use a spectrophotometer at 390 nm following standard protocol22.

Determination of total phenolic content

The Folin-Ciocalteu reagent method was employed alongside a standard curve for chlorogenic acid to quantify phenolic compounds in fresh shoots and root tissues. Absorbance readings were taken at 740 nm to determine total phenolic content23.

Free proline

Free proline content was determined using the procedure described by24. Sulfosalicylic acid was used to extract the components, and glacial acetic acid solutions and ninhydrin were added. After adding the toluene layer and heating the mixture to 100 °C, the absorbance of the layer was measured at 520 nm.

N, P, K, and Na in shoot and root

Initially, nitrogen digestion was carried out using sulfuric acid25 while phosphorus and potassium were digested using a di-acid mixture26. To assess nitrogen content using a modified micro-Kjeldahl method25. The phosphorus content was assessed at 420 nm using the protocol25. For the quantification of potassium content, a flame photometer was used.

Statistical analysis

The collected data was statistically analyzed using OriginPro software by applying an analysis of variance models. Normality was tested using the Shapiro-Wilk test and homogeneity of variance was assessed using Levene’s test before applying statistical analysis. The adjusted Tukey’s multiple comparison test was used to determine significant differences among the treatments at p < 0.05. OriginPro software was also used to perform convex hull analysis, hierarchical clustering, and Pearson correlation analysis27.

Results

Shoot length, shoot dry weight, leaf per plant, and flowering branch per plant

Variety CON III, AARI Canola, Punjab Canola, and Faisal Canola showed a significant increase (p < 0.05) in shoot length (53.23%, 50.18%, 51.99%, and 55.76%), shoot dry weight (78.39%, 70.08%, 66.96%, and 89.45%), leaf per plant (89.30%, 96.79%, 47.55%, and 67.77%), and flowering branch per plant (72.02%, 79.82%, 55.34%, and 62.71%) with SE in comparison to their respective controls under tap water. Faisal Canola showed the highest and AARI Canola showed the lowest increase in shoot length, shoot dry weight, leaf no./plant, and flowering branch per plant under SE (Fig. 1A, B, C, and D).

Fig. 1
figure 1

Effect of sewage effluent (SE) on different varieties of canola shoot length (A), shoot dry weight (B), leaf per plant (C), and flowering branch per plant (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Silique no./plant, seed yield/plant, and seed oil

Under SE, variety CON III, AARI Canola, Punjab Canola, and Faisal Canola exhibited a significant increase (p < 0.05) in silique per plant (66.96%, 56.97%, and 64.51%, and 49.26%,), seed yield/plant (70.31%, 83.51%, 51.40%, and 55.48%), and seed oil (15.70%, 16.14%, 9.87%, and 13.37%) more than their respective controls under tap water (Fig. 2A, B, and C).

Fig. 2
figure 2

Effect of sewage effluent (SE) on different varieties of canola silique no./plant (A), seed yield/plant (B), and seed oil (C). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Physiological and biochemical measurements

Adding SE showed a significant increase (p < 0.05) in chlorophyll a (68.57%, 81.48%, 59.80%, and 57.72%), chlorophyll b (75.32%, 75.53%, 39.28%, and 56.51%), total chlorophyll (71.49%, 78.81%, 56.38%, and 57.19%), and carotenoids (99.61%, 90.43%, 59.58%, and 77.84%) in CON III, AARI Canola, Punjab Canola, and Faisal Canola above their respective controls under tap water (Fig. 3A-D).

Fig. 3
figure 3

Effect of sewage effluent (SE) on different varieties of canola chlorophyll a (A), chlorophyll b (B), total chlorophyll (C), and carotenoids (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Variety CON III, AARI Canola, Punjab Canola, and Faisal Canola showed a significant decrease (p < 0.05)in shoot proline (67.29%, 58.23%, 88.82%, and 80.62%), root proline (20.75%, 20.37%, 26.13%, and 18.57%), shoot ascorbic acid (26.75%, 28.11%, 41.94%, and 39.57%), and root ascorbic acid (45.13%, 36.51%, 72.03%, and 57.05%) with SE in comparison to their respective controls under tap water (Fig. 4A-D).

Fig. 4
figure 4

Effect of sewage effluent (SE) on different varieties of canola shoot proline (A), root proline (B), shoot ascorbic acid (C), and root ascorbic acid (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Under SE, variety CON III, AARI Canola, Punjab Canola, and Faisal Canola exhibited a significant decrease (p < 0.05) in shoot total soluble phenolics (34.49%, 31.29%, 37.86%, and 36.11%), root total soluble phenolics (40.71%, 37.34%, 50.07%, and 54.92%), shoot H2O2 (65.01%, 47.26%, 78.47%, and 74.12%), and root H2O2 (35.74%, 28.74%, 48.61%, and 41.69%) more than their respective controls under tap water (Fig. 5A-D).

Fig. 5
figure 5

Effect of sewage effluent (SE) on different varieties of canola shoot total soluble phenolics (A), root total soluble phenolics (B), shoot H2O2 (C), and root H2O2 (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Adding SE showed a significant decrease (p < 0.05) in shoot MDA (66.68%, 70.30%, 69.09%, and 71.30%), root MDA (67.38%, 58.04%, 92.72%, and 83.89%), shoot SOD (46.01%, 37.91%, 45.24%, and 47.47%), and root SOD (41.75%, 23.88%, 61.01%, and 52.80%) of CON III, AARI Canola, Punjab Canola, and Faisal Canola above their respective controls under tap water (Fig. 6A-D).

Fig. 6
figure 6

Effect of sewage effluent (SE) on different varieties of canola shoot MDA (A), root MDA (B), shoot SOD (C), and root SOD (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Variety CON III, AARI Canola, Punjab Canola, and Faisal Canola showed a significant decrease (p < 0.05) in shoot POD (15.80%, 12.42%, 23.67%, and 19.78%), root POD (26.43%, 19.04%, 29.35%, and 29.21%), shoot CAT (28.11%, 22.80%, 30.75%, and 30.00%), and root CAT (35.13%, 26.16%, 52.39%, and 43.72%) with SE in comparison to their respective controls under tap water (Fig. 7A-D).

Fig. 7
figure 7

Effect of sewage effluent (SE) on different varieties of canola shoot POD (A), root POD (B), shoot CAT (C), and root CAT (D). Significant differences were detected at (p < 0.05) using the Tukey test; different letters on the bars represent the means of four replicates with standard error.

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Shoot and root N, P, K, and Na

Adding SE showed a significant increase (p < 0.05) in shoot N (22.48%, 18.54%, 21.67%, and 20.98%), shoot P (40.61%, 43.72%, 29.82%, and 37.81%), shoot K (29.14%, 30.59%, 26.43%, and 27.90%), and caused decrease in shoot Na (33.85%, 32.84%, 67.42%, and 50.86%) in CON III, AARI Canola, Punjab Canola, and Faisal Canola above their respective controls under tap water (Table 3).

Table 3 Effect of sewage effluent on the concentration of shoot N, P, K, and Na of different Canola varieties.
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Significant differences were detected at (p < 0.05) using the Tukey test; values represent the means of four replicates with standard error.

Applying SE showed a significant increase (p < 0.05) in root N (29.07%, 28.72%, 23.27%, and 29.85%), root P (44.68%, 56.00%, 31.05%, and 35.40%), root K (20.37%, 21.19%, 16.56%, and 20.23%), and caused a decrease in root Na (50.20%, 51.77%, 97.75%, and 68.56%) in CON III, AARI Canola, Punjab Canola, and Faisal Canola above their respective controls under tap water (Table 4).

Table 4 Effect of sewage effluent on the concentration of root N, P, K, and Na of different Canola varieties.
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Significant differences were detected at (p < 0.05) using the Tukey test; values represent the means of four replicates with standard error.

Discussion

The application of sewage effluent (SE) significantly enhanced the growth and physiological responses of canola varieties, particularly CON III, AARI Canola, Punjab Canola, and Faisal Canola. This increase in shoot length, shoot dry weight, leaf number, and flowering branches per plant suggests that SE supplies essential nutrients that are rapidly absorbed and utilized for growth28. SE is rich in macronutrients like nitrogen (N), phosphorus (P), and potassium (K), which are critical for cell division, leaf expansion, and shoot elongation29. These nutrients play a crucial role in chlorophyll synthesis and protein production, thereby directly promoting photosynthesis, biomass accumulation, and overall plant growth30. Punjab Canola’s higher response in shoot length and dry weight highlights a possible superior nutrient uptake efficiency or greater adaptability to SE among the studied varieties31. The increase in nutrient uptake might be due to the buildup of the nutrient pool in the soil, as observed in the study by Masto et al.32. They reported an increase in organic carbon (0.86%), total nitrogen (2,713 kg ha⁻¹), available nitrogen (397 kg ha⁻¹), available phosphorus (128 kg ha⁻¹), available potassium (524 kg ha⁻¹), and available phosphorus (65.5 kg ha⁻¹) in the upper soil layer (0–15 cm). In another study, conducted by Tandi et al.33, an increase in total Zn (1,563.9 mg kg⁻¹) and Cu (133.3 mg kg⁻¹) was observed in the topsoil (0–20 cm depth) with the use of sewage effluent. The application of sewage effluent also led to increased Zn and Cu uptake in lettuce and mustard crops34. Coelho et al.34 found an increase in the yield and quality of Urochloa brizantha, along with improved soil health, without leading to heavy metal contamination. However, they did not observe a positive response of sewage effluent on soil porosity, water infiltration rate, soil particle aggregation, or organic carbon in the topsoil.

The reproductive metrics, such as silique number, seed yield, and seed oil content, also increased notably under SE35. The rise in seed yield (with Punjab Canola showing the highest increase) can be attributed to SE’s positive impact on photosynthetic capacity, as seen in the significantly higher chlorophyll and carotenoid contents36. Chlorophyll a and b levels under SE facilitate more efficient light absorption, boosting photosynthetic energy capture, particularly under environmental stress, which translates to increased reproductive output37. The increased chlorophyll content under SE is associated with enhanced carbon assimilation and energy production38, allowing plants to allocate more resources to reproductive structures, thereby improving both silique number and seed yield. Furthermore, SE application appears to reduce plant stress, as indicated by decreased levels of stress-related metabolites, such as proline, ascorbic acid, and total soluble phenolics in both shoots and roots39. Proline typically accumulates in response to osmotic stress and aids in cellular water retention; the reduction of proline levels suggests that SE provides sufficient moisture and osmotic balance, reducing the need for such osmoprotectants35. Similarly, lower ascorbic acid and phenolic content may imply decreased oxidative stress due to SE, as plants receiving adequate nutrients often exhibit lower stress responses30. Reduced hydrogen peroxide (H2O2) and malondialdehyde (MDA) levels were observed in SE-treated plants, indicating less lipid peroxidation and cellular damage. MDA is a marker for oxidative stress, and its reduction points to a decrease in reactive oxygen species (ROS) formation30. Additionally, the observed decrease in antioxidant enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), further suggests that SE reduces oxidative stress5. These enzymes typically scavenge ROS, protecting plant tissues from oxidative damage; lower enzyme activity under SE indicates that nutrient sufficiency from SE minimizes ROS formation, reducing the need for heightened antioxidant defenses35. However, this reduced activity of antioxidants could have potential implications for plant resilience under extreme environmental conditions, such as drought or heavy metal stress. Therefore, further research is needed to assess the long-term impact of sewage effluent on plant stress tolerance in varying environmental conditions. The SE treatment influenced ion uptake in the plants, leading to increased levels of beneficial nutrients (N, P, and K) and reduced sodium (Na) accumulation in both shoots and roots. This favorable ion balance is critical, as high Na levels can be toxic, disrupting enzyme function and osmotic balance. The decrease in Na observed in SE-treated plants could help alleviate salt stress, improving water uptake and nutrient absorption, which are crucial for metabolic processes36. This efficient ion regulation and nutrient absorption in SE-treated plants likely contribute to the observed growth and yield improvements, as well as enhanced stress tolerance across the canola varieties studied. The use of treated sewage also can result in lower soil heavy metal contamination as evident in various studies. Coellho et al.34 found use of treated sewage effluent does not lead to soil heavy metal contamination. Rattan et al. found no difference in Pb and Cd concentrations between sewage effluent and tap water. The metal concentrations in both were within permissible limits.

Conclusion

The current study evaluated the dilution of sewage effluent (SE) for cultivating different canola varieties (CON III, AARI Canola, Punjab Canola, and Faisal Canola). Being nutrient-enriched, SE at 60% dilution had a positive impact on canola production, with Punjab Canola showing the most prominent response among the cultivars. Sewage effluent significantly improved canola growth attributes, enhancing root development, photosynthesis, and overall biomass production compared to tap water. Nutrient enrichment, including nitrogen, phosphorus, potassium, magnesium, and calcium, significantly improved canola growth attributes by enhancing root development, photosynthesis, and biomass production, in contrast to tap water irrigation, which lacked these essential nutrients. This approach could serve as a cost-effective and environmentally sustainable alternative to conventional water sources, particularly in water-scarce regions. However, further research is needed to conduct long-term field trials to evaluate soil health, heavy metal accumulation, microbial dynamics, and plant stress tolerance over multiple growing seasons and varying environmental conditions.