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Article

Compost Mitigates Metal Toxicity and Human Health Risks and Improves the Growth and Physiology of Lettuce Grown in Acidic and Neutral Loam-Textured Soils Polluted with Copper and Zinc

by
Sana Ullah
1,
Marius Praspaliauskas
2,
Irena Vaskeviciene
2,
Ahmed Hosney
1 and
Karolina Barcauskaite
1,*
1
Lithuanian Research Centre for Agriculture and Forestry, Institute of Agriculture, Instituto Al. 1, LT-58344 Akademija, Kedainiai District, Lithuania
2
Laboratory of Heat-Equipment Research and Testing, Lithuanian Energy Institute, Breslaujos St. 3, LT-44403 Kaunas, Lithuania
*
Author to whom correspondence should be addressed.
Land 2025, 14(3), 478; https://doi.org/10.3390/land14030478
Submission received: 15 January 2025 / Revised: 20 February 2025 / Accepted: 21 February 2025 / Published: 25 February 2025
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Abstract

:
A pot study was conducted to assess the potential of green waste compost on soil properties, growth, physiology, and metal uptake of lettuce plants grown in acidic and neutral loam-textured soils irrigated with copper- and zinc-polluted wastewater (WW). The experiment consists of sixteen treatments involving two different soils with and without compost addition (compost and NoCompost) and irrigated with unpolluted WW, Cu-polluted WW, Zn-polluted WW, and Cu- plus Zn-polluted WW, arranged following factorial layout with three replications. The results illustrated that Cu- and Zn-polluted WW significantly reduced the growth, chlorophylls, and carotenoid pigments of lettuce plants in both soils under NoCompost conditions. However, the decline in these attributes was more pronounced in acidic soil (45–59%) than in neutral soil (30–38%). In the case of neutral soil, Zn-polluted WW did not negatively affect these attributes compared to control. All the metal-polluted treatments increased total polyphenols, polyphenolic acids, flavonoids, and antiradical activity in lettuce shoots. Alternatively, the compost application consistently increased (8–50%) the growth and physiological attributes of lettuce in both soils. Compost treatment decreased root and shoot metal (Cu, Zn) concentrations and uptake by 25–60% and 16–25%, respectively, in both soils. Likewise, compost decreased the metal health risk index (37%—2.7 folds) in both soils. Copper-polluted WW decreased the dehydrogenase activity of soils more than Zn-polluted WW, but compost significantly increased it in both soils, enhancing the organic matter contents of both soils. Conclusively, the addition of compost at the rate of 2% substantially alleviated the metal toxicity thereafter human health risks in both soils.

1. Introduction

This century is facing serious consequences of heavy metal pollution due to surging industrial activities [1,2]. Wastewater (WW) from our anthropogenic activities plays a major role in the pollution of our environment with toxic metals [3]. Tragically, freshwater scarcity compels farmers to use WW for the irrigation of crops [4]. Moreover, their toxic nature, non-biodegradability, and persistence in the environment make the heavy metals more dangerous for all living organisms [5]. Irrigation with polluted WW leads to harm to our food chain, ultimately posing potential ecological and health risks [6]. Nowadays copper (Cu) and zinc (Zn) are the metals of concern in harming living organisms, especially when they exceed their permissible limits; therefore, their observed potential ecological risk was greater than previously thought [7]. After irrigation with polluted WW, Cu and Zn availability and uptake by plants are affected by different factors including metal mobility and concentration, soil texture, pH, microbial activity, organic matter contents, and redox potential of the soil [8]. Furthermore, acidic soils (low pH) contain more available metal ions compared to neutral and alkaline soils (high pH), ultimately increasing metal uptake, which results in excessive accumulation of metals in crop plants [9]. The intake of food crops, especially vegetables contaminated with excessive levels of metals, has adverse effects on human health [10,11].
Lettuce is a commonly used vegetable due to its taste and high nutritional value, containing fiber, protein, carbohydrates, and antioxidants [12,13]. In fact, it is considered a model indicator plant for metal-polluted soils due to its significant metal bioaccumulation efficiency and apparent growth response [14]. Different approaches are practiced in reducing metal pollution in food crops. However, the use of green waste compost (GWC) is considered a green technique and an environmentally better option because of its various characteristics [15]. GWC is a rich source of primary nutrients and organic matter, improves soil structure, increases microbial activities, and reduces metal uptake [16]. Moreover, GWC has good potential to bind the metal ions, hence, reducing substantial uptake by plants and, ultimately, reducing health risks in the food chain [17].
The FAO allowable levels for Cu and Zn in irrigation water are 0.2 and 2 mg L−1, respectively [18]. The total concentrations of both metals in collected wastewater were detected within the safe limits (<0.02 mg L−1) (Table 1). However, based on the (a) higher levels of these metals recorded in the sewage sludge of wastewater [19] and (b) screening tests (Table 2), the collected wastewater was further polluted with these metals.
Literature exists about compost potential to improve lettuce growth under metal-polluted soils [17,20]. However, the comparative studies about compost potential to boost lettuce growth in acidic and neutral loam soils polluted with Cu and Zn are still unclear. Therefore, it was hypothesized that the application of compost relieves metal toxicity and health risks in lettuce by improving soil properties and reducing metal uptake in acidic and neutral loam-textured soils irrigated with Cu- and Zn-polluted wastewater. The objectives of the experiment include assessing the effect of green waste compost on the (i) organic matter contents and dehydrogenase activity of soils; (ii) growth, physiology, and biological compounds of Lactuca sativa; and (iii) metal concentration, metal uptake, and health risks associated with lettuce plants grown in acidic and neutral loam-textured soils irrigated with Cu- and Zn-polluted wastewater.

2. Materials and Methods

2.1. Screening Tests

Petri dish (90 × 15 mm, Ratiolab GmbH, Dreieich-Buchschlag, Germany) screening tests were conducted to refine and select the level of each metal for the main pot experiment. These tests aimed to assess the first effective lethal concentration of metals that can cause ≥50% reduction in the tested parameters of lettuce seedlings. Ten seeds of Lactuca sativa L. (Grand Rapids, UAB Agrofirma, Lithuania) were sown on double filter paper in petri dishes. Different Cu (2.5, 5, 10, 20, and 40 mg L−1) and Zn (5, 10, 20, 40, 80, and 160 mg L−1) concentrations were prepared with standard salts of copper- and zinc-nitrate in distilled water. Each treatment including control was replicated four times to ensure reliability in the obtained results. All the petri dishes were placed in a controlled climate growth chamber having 16/8 h light period, 24/16 °C day/night temperature, and 75% relative humidity. Germination energy and germination percentage were measured after 72 h and 10 days, respectively, as given in the formula equations. Likewise, after ten days, seedling root length and shoot length in centimeters were determined using a scale ruler.
G e r m i n a t i o n   e n e r g y % = N o .   o f   l e t t u c e   s e e d s   g e r m i n a t e d   a f t e r   72   h o u r s T o t a l   n u m b e r s   o f   l e t t u c e   s e e d s × 100
G e r m i n a t i o n   p e r c e n t a g e % = N o .   o f   l e t t u c e   s e e d s   g e r m i n a t e d   a f t e r   10   d a y s T o t a l   n u m b e r s   o f   l e t t u c e   s e e d s × 100

2.2. Characterization of Soils, Wastewater, and Compost

Two loam soils: acidic and neutral, were collected from two different research sites: Vezaiciai Klaipeda District and Dotnuva Kedainai District of the Lithuanian Research Centre for Agriculture and Forestry. Both soils were characterized as loam texture and classified as Retisol (acidic soil) and Cambisol (neutral soil) by the World Reference Base (WRB) [21]. After preparation, the soils were analyzed for different physicochemical parameters (Table 3). The soil pH was measured using 1M KCl by pH meter (XS Instruments, Carpi (MO), Italy). The soil texture was determined by the granulometric method ISO [22]. The organic matter contents were determined using a muffle furnace method as described by Dean [23]. Organic carbon was determined by the dry combustion method using infrared spectroscopy as described earlier [24]. Mineral N (NO2−, NO3−, NH4+) was measured by the injection flow spectrometric method using a FIASTAR-5000 analyzer (FOSS GmbH, Hilleroed, Denmark) following Swify and coworkers [24]. The available phosphorous and potassium were analyzed with ammonium-lactate and acetic-acid extracts, respectively [25], using a spectrometer (Shimadzu UV-1800, Kyoto, Japan) and a flame-photometer (JENWAY-PFP7, Wagtech, UK), respectively. The available Cu and Zn concentrations were extracted using 1M HCl (1:10 w/v) and ammonium-acetate buffer solution (pH 4.8), respectively, then analyzed using an atomic absorption spectroscopy method spectrophotometer [26,27]. For the total Cu and Zn concentrations, soil samples were digested in a di-acid mixture (HNO3 and HCl) in a microwave digestion apparatus, then extractants were analyzed on an inductively coupled-plasma mass spectrometer (PerkinElmer Inc., Waltham, MA, USA). Soil textures were evaluated following the granulometry method [22] (Table 1).
The treated wastewater was collected from a Lithuanian company (JSC Silales Vandenys, Silale, Lithuania) and stored in plastic cans for irrigation. The wastewater was characterized by its pH (by directly dipping an electrode of pH meter in the sample), organic matter [28], nitrogen [29,30], phosphorous and potassium [31,32], and total metal concentrations [33] (Table 1). In compost, pH (as described for soil), dry matter, and organic carbon contents were analyzed following standards procedures [28,34,35]. Phosphorous and potassium were analyzed by spectrophotometry (UV Mini 1240, Kyoto, Japan) and flame-photometry techniques (JENWAY-PFP7, Wagtech, UK), respectively, following the ISO protocols [31,32]. Other parameters were determined following the same standard methods used for soil analysis (Table 1).

2.3. Pot Experiment

A controlled pot (closed bottom, height 11.5 cm, diameter at the top 13 cm, diameter of the bottom 8 cm) setup was employed for the experiment in the same climate chambers used for the screening tests at the Lithuanian Research Centre for Agriculture and Forestry. In the NoCompost treatments, each plastic pot was filled with 1.0 kg of each soil without any compost treatment. While compost-treated designated pots received 980 g of each soil plus 20 g of compost, so compost-treated pots received compost at the rate of 2% (w/w). A total of 8 seeds of lettuce were sown in each pot, then later, at the proper seedling stand, 4 were allowed to grow till the harvest of the experiment. Four metal treatments were prepared with wastewater (WW) using the same Cu and Zn standard salts as in the case of screening tests. Control, Cu-polluted WW (20 mg L−1), Zn-polluted WW (80 mg L−1), and CuZn-polluted WW (20 + 80 mg L−1). Finally, following a factorial layout experiment consisting of 16 treatments: acidic soil and neutral soil, with compost and without compost (NoCompost), and WW polluted irrigations (2 soils × 2 compost levels × 4 wastewater irrigations). Then each treatment combination was replicated three times, resulting in a total of 48 experimental units. Each pot received a total of 12 irrigations with 1.6 L of WW throughout the growth period of 7 weeks. Hence, each Cu- and Zn-polluted WW irrigated pot received 80 mg and 320 mg of Cu and Zn, respectively. The primary nutrients, P and K, were applied according to the suggested rates, using a standard salt of KH2PO4 [36].

2.4. Growth, Physiology, and Metal Analysis

During the 5th week of the growth period, fresh leaves of the lettuce plants were collected to determine chlorophylls and carotenoid contents [37]. For biological compounds, lettuce shoot samples were air-dried at room temperature and crushed in a grinder to powder. Then, extracts were obtained using methanol (75%) with a 1:10 solid–liquid ratio. Thereafter, antiradical activity, total flavonoids, polyphenols, and polyphenolic acids were analyzed on a spectrophotometer as described [38].
After harvest, the lettuce plant roots and shoots were separated and gently washed under running tap water following rinsing with distilled water. The root length was recorded using a scale. Then root and shoot dry weight were recorded after obtaining constant dry weight at 65 °C for three days in the oven. Thereafter, the metal tolerance index was measured by the following equation.
M e t a l   t o l e r a n c e % = T o t a l   d r y   w e i g h t   o f   p l a n t s   g r o w n   i n   p o l l u t e d   s o i l T o t a l   d r y   w e i g h t   o f   p l a n t s   g r o w n   i n   u n p o l l u t e d   c o n t r o l   s o i l × 100
For the metal analysis, oven-dried roots and shoots parts were mixed with HNO3 (68%) and then digested into the MARS6 digestion machine (CEM Coorporation, Matthews, NC, USA). After digestion, a 2% HNO3 solution was used to make the volume of 50 mL in a volumetric flask. Finally, all the samples were examined for metal concentrations on an ICP-OES (PerkinElmer Inc., Waltham, MA, USA). The metal uptake and transfer factors were calculated by the mentioned equations as suggested earlier [38].
M e t a l   u p t a k e   µ g   p e r   p l a n t = m e t a l   c o n c e n t r a t i o n × d r y   w e i g h t
T r a n s f e r   f a c t o r = M e t a l   concentration   in   lettuce   shoots M e t a l   concentration   in   lettuce   roots

2.5. Metal Intake and Health Risk Assessment

The daily intake of metals (Cu, Zn) (DIM) and metal risk assessment related to human health (HRI) were analyzed by the following equations [39].
D I M = M e t a l   c o n c .   i n   e a t a b l e   p a r t × D a i l y   f o o d   i n t a k e A v g .   b o d y   w e i g h t
H R I = D a i l y   I n t a k e   o f   M e t a l   ( D I M ) O r a l   r e f e r e n c e   d o s e
The metal concentrations (Cu, Zn) in edible parts are the same as given for lettuce shoot parts in mg kg−1. The daily vegetable or food intake is 0.24 kg day−1 [40]. The average body weight for European adults is 70.8 kg [41]. The oral reference doses for Cu and Zn are 0.04 and 0.3 mg day−1, respectively [42].

2.6. Soil Dehydrogenase Activity

After harvesting the lettuce plants, the soil was taken from each treatment in triplicate and mixed homogenized in triphenyltetrazolium chloride to facilitate electron transfer. The resulting solution of triphenyl formazan was quantified using a spectrophotometer as suggested earlier [43].

2.7. Data Collection, Analysis, and Visualization

All the collected replicated data went under statistical analysis using the Statistics 8.1 software. A three-way ANOVA following Tukey’s HSD test (p ≤ 0.05) was used to signify the treatment’s means. Data visualization was carried out by preparing the graphs in Microsoft Excel (365 version 2403). The correlation analysis among soil properties, growth, physiology, and metal uptake attributes of lettuce plants was performed using Origin-Pro version 2025. Likewise, the principal component analysis among these parameters under the influence of sixteen studied treatments was also evaluated using Origin-Pro version 2025.

3. Results

3.1. Screening Tests

Before the main pot experiment, screening tests were conducted to refine and select the first effective lethal dose of Cu and Zn that can cause at least ≥50% reductions in the tested parameters. The results of the petri dish screening tests revealed that increasing Cu and Zn concentrations left linear negative effects on the germination percentage, germination energy, root length, and shoot length of lettuce seedlings (Table 2 and Table 3). The decrease in these parameters was recorded as 5–98% and 6–98% with Cu and Zn concentrations, respectively, compared to the control. However, the first effective lethal dose that caused ≥50% reduction in the tested parameters of lettuce seedlings was observed at Cu 20 mg L−1 and Zn 80 mg L−1.

3.2. Pot Experiment

3.2.1. Growth, Metal Tolerance Index, and Physiology of Lettuce

The results regarding growth, metal tolerance index (MTI), and physiology of lettuce are presented in Figure 1. The results revealed that irrigation with polluted wastewater (WW) significantly reduced the root length and total dry matter of lettuce plants in both soils. However, in neutral soil, Zn-polluted WW irrigation did not cause any significant reduction in these growth attributes. The reduction in root length and total dry matter with Cu, Zn, or CuZn-polluted WW was recorded more in acidic soil (45%) than neutral soil (30%) compared to their unpolluted control treatment under NoCompost treatment. Meanwhile, the compost treatment increased root length and total dry matter by 15–36% across all irrigation treatments, including the unpolluted control, compared to untreated NoCompost soils.
Irrigation with polluted WW significantly decreased the MTI of lettuce plants in both soils (Figure 1f). Lower values of MTI were recorded in acidic soil (58–59%) than in neutral soil (71–100%) compared to their unpolluted control treatment under NoCompost conditions. Meanwhile, compost treatment increased the MTI of lettuce plants when irrigated with Cu and CuZn-polluted WW only in neutral soil compared to untreated NoCompost soils.
Irrigation with polluted WW significantly reduced the chlorophylls (a, b) and carotenoid contents of lettuce plants in both soils (Figure 1c–e). However, in neutral soil, Zn-polluted WW irrigation did not cause a significant reduction in these attributes. The reduction in chlorophylls and carotenoid contents with Cu, Zn, or CuZn-polluted WW was recorded more in acidic soil (59%) than in neutral soil (38%) compared to their unpolluted control treatment under NoCompost conditions. Meanwhile, compost treatment increased chlorophylls and carotenoid contents by 15–50% across all irrigation treatments, including the unpolluted control.
Similarly, irrigation with polluted WW significantly increased total polyphenols, polyphenolic acids, flavonoids, and antiradical activity of lettuce plants in both soils (Table 4). The increase in these compounds was recorded by 4–52% across all polluted WW treatments compared to their unpolluted control treatment under NoCompost conditions. Furthermore, compost treatment increased them by 8–25% across all polluted WW treatments compared to NoCompost in both soils. However, these parameters were more pronounced with Zn-polluted WW compared to either Cu- or CuZn-polluted WW in both soils. The pattern of increase in these compounds was consistent in both soils.

3.2.2. Metal Concentrations, Uptake, and Translocation

The results regarding metal concentrations, uptake, and translocation are given in Figure 2 and Figure 3. Irrigation with metal-polluted wastewater (WW) significantly increased the metal (Cu, Zn) concentrations and uptake of respective metals in both soils.
The increase in root and shoot metal (Cu, Zn) concentrations with Cu-, Zn-, or CuZn-polluted WW was more pronounced in acidic soil than neutral soil, compared to their unpolluted control treatment (Figure 2). In contrast, compost treatment significantly decreased these metal concentrations across all irrigation treatments compared to NoCompost soils. Compost treatment decreased root and shoot Cu concentrations and Zn concentrations by 33–64% and 25–50%, respectively, in respective metal-polluted WW treatments under NoCompost conditions in both soils. Similarly, Cu-, Zn-, or CuZn-polluted WW significantly increased the shoot metal (Cu, Zn) uptake in both soils, compared to their unpolluted control treatment, under NoCompost treatment (Figure 3). In contrast, compost treatment significantly decreased the shoot metal (Cu, Zn) uptake across all polluted WW irrigation treatments compared to NoCompost soils. Compost treatment decreased the shoot Cu uptake and Zn uptake by 22–59% and 16–25%, respectively, in respective metal-polluted WW treatments under NoCompost conditions in both soils.
The results regarding the root-to-shoot Cu transfer factor (TF) revealed that relatively higher TF values (>1) were observed with Cu- than CuZn-polluted WW in both soils under NoCompost conditions (Figure 3). However, an exception of higher TF value was also observed with Zn-polluted WW in neutral soil. Because in this treatment, Cu concentration was less compared to Cu- or CuZn-polluted WW, hence, it preferred to be transferred to above-ground parts by plants. In contrast, compost treatment decreased these values across all the irrigation treatments in both soils. However, compost treatment did not influence the Cu TF value in CuZn treatment in neutral soil. For Zn, lower TF values (<1) were observed either with Zn- or CuZn-polluted WW treatments under NoCompost conditions. Furthermore, compost treatment did not decrease these values in any of the Zn-polluted WW treatments in both soils.

3.2.3. Metal Intake and Health Risk Assessment

Results regarding the daily metal intake and health risk assessment are presented in Figure 4. Irrigation with polluted wastewater (WW) significantly increased the daily consumption of metals (Cu, Zn) and the health risk index in both soils. The increase in daily intake of Cu and Zn with Cu-, Zn-, or CuZn-polluted WW was more pronounced in acidic soil (200–320%) than neutral soil (100–230%), compared to their respective unpolluted control treatment, under the NoCompost condition (Figure 4). In contrast, the application of compost significantly decreased (37%—2.7 folds) the daily consumption of Cu and Zn in both soils, compared to unpolluted control under the NoCompost condition. Similarly, the increase in health risk indexes of Cu and Zn with Cu-, Zn-, or CuZn-polluted WW was more pronounced in acidic soil (2–32 folds) than neutral soil (1–23 folds) compared to their respective unpolluted control treatment under the NoCompost condition (Figure 4). In contrast, the application of compost significantly decreased (37%–2.7 folds) the health risk indexes of Cu and Zn in both soils compared to unpolluted control under the NoCompost condition.

3.2.4. Soil Properties

The results of soil properties are presented in Figure 5. All the polluted WW treatments decreased the dehydrogenase activity of both soils. The negative effect of Cu-polluted WW on microbial activity was more pronounced than Zn-polluted WW in both soils compared to their respective unpolluted control treatments. In contrast, the application of compost significantly increased this activity consistently in both soils compared to the NoCompost conditions. Similarly, compost amendment significantly increased the organic matter contents of both soils compared to the NoCompost conditions.

3.2.5. Correlation and Principal Component Analysis

The correlation analysis among soil properties, growth, physiology, and metal uptake attributes of lettuce plants is presented in Figure 6. The results stated that the growth and physiology (total dry weight, root length, metal tolerance index, chlorophylls, carotenoids) of lettuce plants depicted a strong positive relationship with the dehydrogenase activity of soils. In contrast, lettuce growth and physiology and dehydrogenase activity showed a negative relationship with root and shoot metals (Cu, Zn) concentrations and uptake. Notably, a strong negative relationship between lettuce growth and physiology was depicted with Cu concentrations and uptake. Similarly, the results of the PCA analysis illustrate PC1 and PC2 as principal components with a total of 77% of the variance in the analyzed data (Figure 7). The red dots showed the sixteen treatment combinations. The Zn concentrations, uptake, HRI, and biological compounds are grouped in the top left quadrant; copper concentrations, uptake, and HRI are grouped in the bottom left quadrant; while soil parameters, growth attributes, MTI, chlorophylls, and carotenoids are grouped on the right side.

4. Discussion

4.1. Screening Tests

The lethal dose of Cu (20 mg L−1) and Zn (80 mg L−1) caused >50% reductions in the studied parameters of lettuce seedlings (Table 2 and Table 3). Toxic Cu and Zn concentrations negatively affect the normal functioning of enzymes involved in germination, plumule, and radicle growth [44,45]. Moreover, metal toxicity induces free radicals production and disrupts photosynthesis, ultimately impairing the seedling growth parameters, including root length and shoot length [46]. Based on the screening test results, Cu 20 mg L−1 and Zn 80 mg L−1 were considered to prepare polluted wastewater for the main pot experiment.

4.2. Pot Experiment

4.2.1. Growth, Metal Tolerance Index, and Physiology of Lettuce

Copper- and Zn-polluted wastewater (WW) significantly reduced the growth and chlorophyll pigment attributes of lettuce plants in both soils (Figure 1) because Cu and Zn toxicity induces oxidative stress, which, in turn, damages photosynthetic apparatus, chloroplast, and thylakoid membranes, ultimately reducing photosynthesis in plants [47]. Then, the decreased rate of photosynthesis led to reduced growth, including stunted growth and ultimately less dry matter and root length (Figure 1) [48]. However, the decline in these attributes was more pronounced in acidic soil than in neutral soil. This is because under an acidic pH, metal ions are easily available for uptake by the plants, hence, increasing metal concentrations inside the plant body [49]. It demonstrates the comparative concentration-dependent toxicity in plants between acidic and neutral soils as described (Figure 1 and Figure 2) [50]. Hence, the pronounced decline in total plant dry weight and root length is associated with the toxic levels of Cu (>21 mg kg−1 dry weight) and Zn (>21 mg kg−1 dry weight) in lettuce shoots (Figure 1 and Figure 6) as previously described [51]. Recently, it was also found that a decline in dry weight, photosynthetic pigments, and metal tolerance index (MTI) of lettuce plants was associated with higher shoot Cu concentrations and background applied Cu levels in the soil [52].
In the case of neutral soil, Cu- and CuZn-polluted WW negatively affected the growth and chlorophyll pigments compared to Zn-polluted WW (Figure 1). It might be due to the higher intensity of Cu to damage the plants about the lower concentration needed for plant growth in comparison to Zn (Figure 6 and Figure 7) [53]. Furthermore, increased radicles production and damage to the defense system under Cu stress make it more harmful than Zn [54]. Similarly, MTI scored higher with Zn- than Cu- or CuZn-polluted WW (Figure 1). It might be ascribed to better dehydrogenase activity, chlorophyll pigment production, root growth, dry biomass, and antiradical activity with Zn- than Cu- or CuZn-polluted WW (Figure 1 and Figure 6). Because these compounds play their role in the plant defense system under metal toxicity [55], hence, lettuce plants attained better survival with Zn- than Cu- or CuZn-polluted WW. Accordingly, in neutral soil, Zn-polluted WW did not cause any reduction in the growth and chlorophyll pigments of lettuce plants. It highlights the lettuce ability to counteract metal toxicity with better metal resilience or tolerance under Zn-polluted WW (Figure 1) [38]. Likewise, all the polluted WW treatments significantly increased the production of biological compounds including polyphenols, polyphenolic acids, flavonoids, and antiradical activity with a consistent trend in both soils (Table 4). Furthermore, lettuce plants achieved better production of these compounds and antiradical activity with Zn- than Cu- or CuZn-polluted WW. The correlation and PCA analysis also depicted a positive relationship between polyphenols (polyphenols, polyphenolic acids) and Zn uptake (Figure 6 and Figure 7). It showed biosynthesis and accumulation of phenolic compounds under Zn toxicity and, ultimately, their role in better survival under stressful environments [56]. These results are in line with previous experiments, which demonstrate that Zn is less toxic to the growth and physiology of lettuce plants with more production of biological compounds, antiradical activity, and MTI than Cu or CuZn [38,57].
The compost treatment increased all attributes of lettuce including dry weight, MTI, chlorophyll pigments, biological compounds, and anti-radical activity consistently in both soils compared to NoCompost conditions (Figure 1). Compost is rich in organic substances that favor microbial growth and ensure the availability of essential nutrients for plant growth under metal stress [58]. The humic substances in compost bind Cu and Zn to reduce uptake; hence, reduced metal uptake by plants relieved the burden of metal-induced oxidative stress [59]. Under metal stress, compost combats oxidative stress by inducing the production of antioxidants including polyphenols, polyphenolic acids, flavonoids, and antiradical activity (Table 4) [60]. These results are similar to the findings of [61], who depicted that a significant improvement in growth, physiology, and antioxidant enzyme activities was associated with reduced Cu and Zn uptake in pakchoi plants grown in a metal-contaminated and compost-amended soil.

4.2.2. Metal Concentrations, Uptake, and Translocation

All metal-polluted wastewater (WW) treatments significantly increased the root and shoot metal concentrations and shoot metal uptake in both soils (Figure 2 and Figure 3). It is a general phenomenon related to the concentration-dependent availability of metals in the soil solution. The increased Cu and Zn pollution in soil increased the metal uptake by roots and their accumulation in above-ground shoots parts (Figure 2 and Figure 3) [62]. However, lettuce roots and shoots retained more Cu and Zn concentrations in acidic soil than in neutral soil. Under acidic pH conditions, Cu2+ and Zn2+ ions are mobile and readily available in the soil solution; hence, they are actively taken up by the plant roots (Figure 2) [63]. Recently, ref. [64] reported a more pronounced release of Cu and Zn in acidic soil than in alkaline soil. Moreover, other recent studies also affirm the Cu and Zn dominance in acidic soils, hence, their risk of toxicity under such conditions [65,66].
Conversely, compost treatment decreased the Cu and Zn concentrations and uptake by lettuce plants in both soils (Figure 2 and Figure 3). Because compost acts as a pH neutralizer i.e., in the case of acidic soil, an increase in pH by carbonation tends to reduce the mobility of Cu2+ and Zn2+, reducing uptake by plant roots [67]. Secondly, compost contains diverse organic functional groups that provide surfaces for the adsorption of Cu2+ and Zn2+, ultimately reducing their available portion in the soil solution [68]. Thereafter, compost favors microbial diversity such as dehydrogenase activity due to enriched organic matter (Figure 5). In return, microbes transform these metals into less toxic and less available passive forms, ultimately decreasing metal uptake (Figure 3) [69]. The findings of Atav and Yüksel (2024) [70] showed that soil–plant transfer of Cu and Zn was recorded more in acidic soil than in neutral soil (Figure 2 and Figure 3). Likewise, these results are in line with different studies that demonstrated the potential role of compost in reducing Cu and Zn uptake by plants [71].
Regarding the metal transfer factor (TF), Cu-polluted WW gave TF values >1 in both soils compared to CuZn or unpolluted control treatment. It showed a higher transfer rate of Cu from roots to shoots in lettuce plants, ultimately higher Cu concentrations led to metal toxicity in both soils. Alternatively, compost treatment lowered (<1) the Cu TF; hence, lower Cu concentrations caused less damage to lettuce plants in both soils (Figure 1). In the case of Zn TF, the root-to-shoot transfer was less than the treatments where no Zn was applied. It showed that lettuce plants cannot efficiently transfer the Zn from root to shoot parts under Zn pollution.

4.2.3. Metal Intake and Health Risk Assessment

Regarding the daily intake of metals, Cu and Zn scored higher across the Cu-, Zn-, or CuZn-polluted WW treatments in both soils. These intake values for both metals (Cu 0.8–1.68 and Zn 1.45–4.30 mg kg−1 body weight day−1) (Figure 4) were found to be greater than the recommended limits (Cu 0.5 and Zn 1.0 mg kg−1 bw day−1) [72,73]. However, a lower safe value for Cu intake (0.46 mg kg−1 bw day−1) was observed with CuZn-polluted WW in neutral soil. Alternatively, compost application significantly lowered these values for Cu intake to a safe limit under all polluted WW treatments in both soils except Cu-polluted WW treatment in acidic soil. For Zn intake, compost application lowered the value to a safe limit (0.97 mg kg−1 bw day−1) only under CuZn-polluted WW treatment in neutral soil (Figure 4). This shows that lettuce grown on metal-polluted acidic soil can cause hazardous impacts on consumer health. Health risk indexes (HRI) of both metals with all polluted WW treatments were more than 1 in both soils under compost or NoCompost conditions. These findings suggest that lettuce grown on these soils is unsafe for consumers. Furthermore, the greater values of HRI in acidic soils are due to more availability and uptake of metals (Cu, Zn) by lettuce edible shoot parts (Figure 3 and Figure 4) [64]. Therefore, lettuce grown on acidic metal-polluted soil is more prone to pose health risks to consumers than on neutral metal-polluted soil. However, applying compost at the rate of 2% is not enough to bring HRI under the safe limit (HRI ≤ 1). Still, the compost application substantially reduced the HRI indexes by reducing metal concentration, uptake, and transfer to the edible parts (Figure 1, Figure 2, Figure 3 and Figure 4). Compost lowers these indexes and dilutes health risks by immobilizing the metals in soils and modifying pH and microbial activity [68,69]. These results are in support of those researchers who depicted a substantial reduction in health risk values for different vegetables under the application of compost [74,75].

4.2.4. Soil Properties

All the polluted WW treatments decreased the dehydrogenase activity (Figure 5) because exceeded concentrations of metals in soil negatively affect the microbes, such as by inhibiting nutrient availability and inducing oxidative stress, resulting in membrane damage. Hence, these metals, including Cu and Zn, inhibit enzyme synthesis by binding to active sites of enzymes, resulting in the deactivation of these enzymes [76]. However, due to the various roles in microbial metabolic processes, Zn may cause less damage than Cu (Figure 6 and Figure 7). However, increasing Zn concentrations can negatively affect microbial enzymatic activities as stated earlier [77]. In contrast, compost application substantially improved the dehydrogenase microbial activity in both soils. Compost improves soil health by increasing organic matter (Figure 5), essential nutrients, water and air transport, texture, and structure of soil [78]. All these factors, including organic matter, favor microbial growth under stressful environments, hence, increasing the dehydrogenase activity of soil (Figure 5). Organic matter provides food (carbon and energy) to increase microbial growth and population and, hence, improve dehydrogenase activity [79]. The findings affirmed the potential of compost to improve the dehydrogenase activity of Cu- and Zn-polluted soils [80].

5. Conclusions

The findings of the experiment demonstrated that polluted acidic soil has more detrimental effects on lettuce growth and physiology. Likewise, Cu-polluted (20 mg L−1) wastewater has more detrimental effects on soil properties and lettuce plant growth and physiology than Zn-polluted (80 mg L−1) wastewater. Furthermore, the negative effects of both Cu (20 mg L−1) and Zn (20 mg L−1) polluted wastewater irrigations on soil and lettuce attributes highlight the adverse impact on sustainable agriculture. Similarly, Cu (20 mg L−1) and Zn (20 mg L−1) polluted wastewater irrigations increased the Cu and Zn intake and health risk index. Alternatively, compost amendment improved all the soil and lettuce plant attributes in both soils following a similar trend. Application of compost at the rate of 2% substantially reduced daily intake and associated health risks in lettuce crop grown in soil irrigated with Cu (20 mg L−1) and Zn (80 mg L−1) polluted wastewater. So, this study underscores the compost application as a practical strategy to manage metal-contaminated acidic and neutral soils for resilient agricultural productivity.

Author Contributions

S.U. and K.B. conceptualized and designed the experiments. S.U. performed experiments. S.U. and K.B. wrote the initial draft of the manuscript. S.U., M.P. and I.V. participated in plant, soil, and data analyses. K.B. managed the resources and reviewed and edited the original draft. S.U., K.B. and A.H. performed data analysis. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data that support the findings of this study are openly available on request.

Acknowledgments

The authors are thankful to Taisa Vashkevich for supporting experimental analysis at the Agrobiology Lab. LAMMC.

Conflicts of Interest

The authors declare no conflicts of interest.

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Land 14 00478 g001
Figure 1. Response of growth and physiological attributes of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. (a) Total dry weight; (b) Root length; (c) Chl. a; (d) Chl. b; (e) Carotenoids; (f) Metal tolerance Index.
Figure 1. Response of growth and physiological attributes of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. (a) Total dry weight; (b) Root length; (c) Chl. a; (d) Chl. b; (e) Carotenoids; (f) Metal tolerance Index.
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Figure 2. Response of metal concentrations in roots and shoots of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Root Concentration; (b) Zn Root Concentration; (c) Cu Shoot Concentration; (d) Zn Shoot Concentration.
Figure 2. Response of metal concentrations in roots and shoots of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Root Concentration; (b) Zn Root Concentration; (c) Cu Shoot Concentration; (d) Zn Shoot Concentration.
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Figure 3. Response of shoot metal uptake and transfer factor of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Shoot Uptake; (b) Zn Shoot Uptake; (c) Cu Transfer factor; (d) Zn Transfer factor.
Figure 3. Response of shoot metal uptake and transfer factor of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD, and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Shoot Uptake; (b) Zn Shoot Uptake; (c) Cu Transfer factor; (d) Zn Transfer factor.
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Figure 4. Response of metal intake and risk index of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Intake; (b) Cu risk index; (c) Zn Intake; (d) Zn risk index.
Figure 4. Response of metal intake and risk index of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu- and Zn-polluted wastewater. Data are the mean of three replicates ±SD and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Cu Intake; (b) Cu risk index; (c) Zn Intake; (d) Zn risk index.
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Figure 5. Response of organic matter contents and dehydrogenase activity of acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater. Data are the mean of three replicates ±SD and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Organic matter; (b) Dehydrogenase activity.
Figure 5. Response of organic matter contents and dehydrogenase activity of acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater. Data are the mean of three replicates ±SD and the same letters on the bars are non-significant to each other statistically according to Tukey’s HSD tests. Ck = Control. (a) Organic matter; (b) Dehydrogenase activity.
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Figure 6. Correlation analysis among various soil properties and growth, physiology, and metal uptake attributes of lettuce plants. Blue and pink circles show positive and negative values, respectively, which increase with the size.
Figure 6. Correlation analysis among various soil properties and growth, physiology, and metal uptake attributes of lettuce plants. Blue and pink circles show positive and negative values, respectively, which increase with the size.
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Figure 7. Principal component analysis among various soil properties and growth, physiology, and metal uptake attributes of lettuce plants under sixteen treatment combinations. In treatment combinations; S1 acidic soil, S2 neutral soil, Ck control, C0 NoCompost, C1 Compost, Cu Copper, Zn Zinc.
Figure 7. Principal component analysis among various soil properties and growth, physiology, and metal uptake attributes of lettuce plants under sixteen treatment combinations. In treatment combinations; S1 acidic soil, S2 neutral soil, Ck control, C0 NoCompost, C1 Compost, Cu Copper, Zn Zinc.
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Table 1. Properties of soils, compost, and wastewater used in the experiment.
Table 1. Properties of soils, compost, and wastewater used in the experiment.
ParameterUnitAcidic SoilNeutral SoilWastewaterCompost
pHKCl-3.996.717.446.54
Dry matter%---52
Organic matter%4.132.5718-
Organic C%1.20.86-16
NH4+-Nmg kg−18.71.1-10
NO3−-Nmg kg−1---225
NO3−-N + NO2−-Nmg kg−19.42.956-
Kjeldahl Nmg kg−1--2.8-
Total Nmg kg−118.104.058.8235
PO4-Pmg kg−1 0.30
Available Pmg kg−19840--
Total Pmg kg−1---1819
Available Kmg kg−1174131--
Total Kmg kg−1--196558
Available Cumg kg−11.52.6 4.6
Total Cumg kg−16.07.1<0.0236
Available Znmg kg−11.20.3 11
Total Znmg kg−13433<0.02254
Sand %45.550.2--
Silt %38.233.1--
Clay %16.316.7--
Textural class-LoamLoam--
Table 2. Effect of increasing concentrations of copper on germination energy, germination percentage, root length, and shoot length of lettuce seedlings grown in petri dishes.
Table 2. Effect of increasing concentrations of copper on germination energy, germination percentage, root length, and shoot length of lettuce seedlings grown in petri dishes.
Copper Concentration (mg L−1)Germination
Energy (%)		Germination
Percentage (%)		Root Length (cm)Shoot Length (cm)
Control47.5 (5) a87.5 (5) a3.9 (0.3) a6.33 (0.1) a
2.545 (5.8) a82.5 (5) ab3.55 (0.1) a5.73 (0.2) b
542.5 (5) a72.5 (5) bc3 (0.2) b4.78 (0.2) c
1027.5 (5) b62.5 (5) c2.45 (0.2) c3.50 (0.2) d
2012.5 (5) c12.5 (5) d0.10 (0.0) d0.10 (0.0) e
4010 (0.0) c10 (0.0) d0.10 (0.0) d0.10 (0.0) e
HSD value at p ≤ 0.0510.5910.250.400.34
The data are the average of four independent petri dish replications ±SD values in the bracket. The same letter in the column shows a non-significant difference according to Tukey’s HSD test.
Table 3. Effect of increasing concentrations of zinc on germination energy, germination percentage, root length, and shoot length of lettuce seedlings grown in petri dishes.
Table 3. Effect of increasing concentrations of zinc on germination energy, germination percentage, root length, and shoot length of lettuce seedlings grown in petri dishes.
Zinc Concentration (mg L−1)Germination
Energy (%)		Germination
Percentage (%)		Root Length (cm)Shoot Length (cm)
Control45 (5.8) a87.5 (5) a4.63 (0.2) a5.98 (0.5) a
542.5 (5) ab82.5 (5) ab4.33 (0.2) a5.50 (0.2) a
1035 (5.8) a−c72.5 (5) bc3.65 (0.1) b4.75 (0.1) b
2032.5 (5) bc65 (5.8) c3.30 (0.1) b3.98 (0.2) c
4025 (5.8) c52.5 (5) d2.48 (0.3) c3.30 (0.1) d
8010 (0.0) d10 (0.0) e0.10 (0.0) d0.10 (0.0) e
16010 (0.0) d10 (0.0) e0.10 (0.0) d0.10 (0.0) e
HSD value at p ≤ 0.0510.610.030.350.49
The data are the average of four independent petri dish replications ±SD values in the bracket. The same letter in the column shows a non-significant difference according to Tukey’s HSD test.
Table 4. Response of biologically active compounds of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater.
Table 4. Response of biologically active compounds of lettuce plants grown in acidic and neutral loam-textured soils under compost and NoCompost conditions and irrigated with Cu and Zn polluted wastewater.
Soil CompostMetalTotal Polyphenols
(RUE g−1 DW)		Total Flavonoids
(RUE g−1 DW)		Total Polyphenolic Acids
(CA g−1 DW)		Anit Radical Activity (RUE g−1 DW)
Acidic soilNo CompostControl1.21 (0.02) f1.01 (0.01) e0.43 (0.003) f1.08 (0.02) a
Cu1.53 (0.01) d1.12 (0.01) d0.54 (0.01) de1.14 (0.01) d
Zn1.78 (0.01) b1.25 (0.01) c0.65 (0.003) b1.23 (0.01) a
CuZn1.52 (0.02) d1.12 (0.01) d0.54 (0.003) de1.15 (0.01) c
CompostControl1.37 (0.02) e1.12 (0.01) d0.53 (0.003) de1.23 (0.01) c
Cu1.66 (0.02) c1.31 (0.01) b0.62 (0.003) c1.33 (0.02) b
Zn2.08 (0.05) a1.40 (0.01) a0.74 (0.003) a1.42 (0.01) a
CuZn1.64 (0.01) c1.31 (0.01) b0.62 (0.001) c1.31 (0.01) b
Neutral soilNo CompostControl1.22 (0.02) f1.01 (0.004) e0.43 (0.002) f1.10 (0.01) a
Cu1.54 (0.02) d1.12 (0.002) d0.54 (0.003) de1.15 (0.01) a
Zn1.79 (0.01) b1.25 (0.002) c0.65 (0.001) b1.22 (0.01) c
CuZn1.53 (0.01) d1.12 (0.01) d0.54 (0.002) d1.14 (0.01) a
CompostControl1.38 (0.01) e1.12 (0.005) d0.53 (0.001) d1.23 (0.01) a
Cu1.67 (0.01) c1.31 (0.002) b0.63 (0.001) c1.33 (0.01) b
Zn2.08 (0.01) a1.40 (0.002) a0.74 (0.002) a1.43 (0.02) a
CuZn1.64 (0.02) c1.31 (0.01) b0.62 (0.01) c1.30 (0.02) b
HSD value at p ≤ 0.050.05570.01900.0010.0378
Data are the mean of three replicates ±SD in the brackets, and the same superscript statistical letters on the bracket in the column are non-significant to each other statistically according to Tukey’s HSD tests.
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Ullah, S.; Praspaliauskas, M.; Vaskeviciene, I.; Hosney, A.; Barcauskaite, K. Compost Mitigates Metal Toxicity and Human Health Risks and Improves the Growth and Physiology of Lettuce Grown in Acidic and Neutral Loam-Textured Soils Polluted with Copper and Zinc. Land 2025, 14, 478. https://doi.org/10.3390/land14030478

AMA Style

Ullah S, Praspaliauskas M, Vaskeviciene I, Hosney A, Barcauskaite K. Compost Mitigates Metal Toxicity and Human Health Risks and Improves the Growth and Physiology of Lettuce Grown in Acidic and Neutral Loam-Textured Soils Polluted with Copper and Zinc. Land. 2025; 14(3):478. https://doi.org/10.3390/land14030478

Chicago/Turabian Style

Ullah, Sana, Marius Praspaliauskas, Irena Vaskeviciene, Ahmed Hosney, and Karolina Barcauskaite. 2025. "Compost Mitigates Metal Toxicity and Human Health Risks and Improves the Growth and Physiology of Lettuce Grown in Acidic and Neutral Loam-Textured Soils Polluted with Copper and Zinc" Land 14, no. 3: 478. https://doi.org/10.3390/land14030478

APA Style

Ullah, S., Praspaliauskas, M., Vaskeviciene, I., Hosney, A., & Barcauskaite, K. (2025). Compost Mitigates Metal Toxicity and Human Health Risks and Improves the Growth and Physiology of Lettuce Grown in Acidic and Neutral Loam-Textured Soils Polluted with Copper and Zinc. Land, 14(3), 478. https://doi.org/10.3390/land14030478

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