Influence of the Application of Zn and Mn Obtained from Black Mass on Young Citrus Plant Growth

Abstract

The reuse of industrial waste is essential to reduce environmental impact and move towards sustainable development through methods that do not depend on limited resources. To this end, a fertilizer was developed from recycled alkaline batteries, transformed into a useful product rich in zinc and manganese (black mass). The aim is to use industrial waste to create an environmentally safe fertilizer. An experiment was conducted on young citrus plants grafted onto Carrizo rootstock, grown in pots with coconut fiber under greenhouse conditions in Valencia (Spain) for one year (2023–2024). A total of 120 plants were arranged in randomized blocks with three replicates of 10 plants per treatment. Four nutrient solutions derived from the Hoagland formulation were evaluated: control solution without Zn or Mn (SoC), solution with Zn and Mn sulfates (SoH), solution with Zn and Mn sulfates extracted from black mass (BMS), and solution with Zn and Mn lignosulfonate derived from black mass (BMLS). Morphological, growth, physiological, and nutritional parameters were analyzed in March and October. While morphological traits showed no significant differences among treatments, some physiological (stomatal conductance, transpiration) and biochemical variables (chlorophyll, carotenoids, P, K, Mg, and S concentrations) differed significantly depending on the nutrient source. Nevertheless, all plants maintained healthy growth and nutrient levels within optimal ranges, and no signs of phytotoxicity or heavy metal accumulation were detected.

1. Introduction

In a world that requires change and demands solutions toward sustainability, innovation becomes an essential driver, and the reuse of industrial waste serves as the bridge that allows us to transform challenges into opportunities, promoting responsible use and proper management of resources, thereby reducing negative environmental impacts [1]. In this context, the circular economy operates on the concept of implementing closed cycles in which materials and products are continuously recycled, whereby by-products or derived waste are used as raw materials, contributing to the conservation of natural resources and waste reduction [2,3]. This strategy is crucial in facing the challenges arising from the need to produce food for a growing population. To achieve food security, adaptation to climate change and lower emission intensity per product are required, and this transformation must be accomplished without consuming more resources than can be regenerated [4]. Therefore, the reuse of industrial waste as nutrient sources enriches agricultural soils and, consequently, the nutritional quality of crops, which is fundamental for human and animal health. At the same time, maintaining a balance of essential nutrients is paramount for plant health. The scarcity or toxicity of a nutrient restricts plant function; growth and yield decrease, even if other elements are present in adequate amounts [5]. Thus, fertilization represents a high cost in citrus production and has a substantial impact on profitability [6]. However, the production of fertilizers consumes non-renewable resources, leading to a decrease in availability for future generations.

In this context, new methods are required that do not depend on the consumption of these increasingly scarce natural resources, which also entail high environmental costs. This creates the need to develop circular economy procedures through the project “Development of a new fertilizer from the recycling of alkaline batteries,” based on the reuse of micronutrients from alkaline batteries, rich in zinc (Zn) and manganese (Mn), to develop a fertilizer from this waste. Here, the raw material is utilized and transformed into a useful product with appreciable concentrations of these essential nutrients, known as “black mass”. Black mass obtained from the recycling of alkaline batteries generally contains Zn (20–25%), Mn (28–31%), K (3.4–7.25%), Fe (0.17–0.83%), Pb (0.005–0.02%), Cd (0.01 ± 0.003%), Hg (<0.015%) and graphite (38–56%) [7,8].

The production process incorporates raw materials derived from the circular economy, such as residual sulfuric acid and lignosulfonates. Usually, this proposal complies with the DNSH (Do No Significant Harm) principle, established in Regulation (EU) 2020/852 [9]. Furthermore, as waste-derived materials may contribute to the long-term accumulation of trace metals in soils and crops, the proposed application rates are designed to be compatible with sustainable nutrient management. In this context, the use of black mass from alkaline batteries as a source of micronutrients is limited by the EU pollutant limits for fertilizer products set out in Regulation (EU) 2019/1009 [10].

Globally, alkaline batteries represent a significant and still largely unmanaged waste stream. It is estimated that around 300,000 tonnes of alkaline batteries are consumed every year [11,12]. Recent assessments further indicate that the annual consumption of approximately nine billion alkaline cells generates substantial amounts of waste [13]. In the European Union, 244,000 tonnes of portable batteries were placed on the market in 2022, while only 111,000 tonnes of used batteries were collected for recycling [14]. The high consumption of alkaline batteries due to their very short lifespan generates large amounts of waste [15], managed under Directive 2006/66/EC [16], which requires the collection and recycling of portable batteries. The challenge is to recover valuable elements from this abundant waste to support a circular economy [17] and, with proper management, prevent environmental pollution [18]. Therefore, one of the uses of this waste is its transformation into fertilizers due to its high Zn and Mn content [19].

Both nutrients play a critical role in growth and development, exhibiting similar deficiency symptoms and associated physiological disorders in crops. In Citrus, Zn and Mn deficiencies are particularly prevalent in Mediterranean orchards and in other regions with calcareous or high-pH soils. The availability of these micronutrients is markedly reduced due to their low solubility at alkaline pH; Zn²⁺ tends to precipitate as hydroxides and carbonates, and Mn forms poorly soluble oxides, thus decreasing their concentration in the soil solution and limiting plant uptake [20]. Zn is involved in many enzymatic activities, important in tryptophan synthesis (a precursor of auxins), chlorophyll synthesis, and photosynthesis; it strengthens resistance to diseases and environmental stress and aids in fruit set. Its deficiency manifests as interveinal chlorosis in new leaves, reduced leaf size, necrosis on margins, and shortened internodes, causing a rosette effect; in advanced cases, defoliation may occur, affecting growth and, therefore, fruit yield and quality. Zn toxicity is uncommon but can cause smaller leaf size, stunted plant growth, and/or reduced root growth, chlorosis in new leaves, and young leaves with necrotic tips [21,22,23,24].

Mn is vital for several processes, essential for photosynthesis, acts as an enzyme activator (notably nitrate reductase), is fundamental for root development, and helps plants combat oxidative stress. Its deficiency produces interveinal chlorosis in young leaves and/or necrotic spots, stunted growth, and root alterations, affecting yield and quality. Mn toxicity, less common, presents symptoms like chlorosis similar to deficiency but in older leaves, necrosis at tips, smaller new leaves, reduced growth, and root damage, affecting the absorption of other nutrients [21,22,25]. According to the foliar sufficiency ranges proposed by Kadyampakeni and Morgan (2020) [6] for Zn and Mn in young citrus leaves, concentrations are classified as deficient (<18 mg kg⁻¹), low (18–24 mg kg⁻¹), optimal (25–100 mg kg⁻¹), high (101–300 mg kg⁻¹), and excessive or potentially toxic (>300 mg kg⁻¹).

This project, within the framework of the circular economy, transforms pollutants into a valuable resource for agriculture, addressing waste reduction and avoiding the extraction of finite natural resources, thereby decreasing environmental impact. The general objective is to obtain new fertilizers that effectively supply Mn and Zn to citrus trees, using products derived from industrial waste as raw materials, providing products without phytotoxic effects on plants. Thus, the specific objectives of the project were as follows: (1) to study the influence of black mass as a source of Zn and Mn on morphological, growth, and physiological parameters in young Navelina variety plants; (2) to determine and compare the concentration of chlorophyll, carotenoids, and mineral nutrients in young Navelina plants; and (3) to evaluate the phytotoxic effect of black mass as a source of Zn and Mn in young Navelina plants.

2. Materials and Methods

2.1. Description of the Experiment

The study was conducted at the Polytechnic University of Valencia (Spain) in a Venlo-type greenhouse. The experimental period lasted from October 2023 to October 2024. Climatic data for temperature and relative humidity were monitored using a Testo 174H datalogger (Testo SE and Co. KGaA, Lenzkirch, Germany). Greenhouse air temperature (T), relative humidity (HR), Vapor Pressure Deficit (VPD), and radiation (Rs) were recorded continuously with the data logger, and the Daily reference evapotranspiration (ET_o) was calculated using the Penman–Monteith FAO 56 method [26] (Table 1).

Table 1. Average climatic characteristics from the datalogger in the greenhouse. The data presented are the means ± SE of the months.

Year	Month	T Mean (°C)	HR Mean (%)	DPV (kPa)	Rs (MJ m−2)	ETo (mm d−1)
2023	October	22.35 ± 0.35	78.84 ± 3.69	0.69 ± 0.10	4.66 ± 0.11	2.52 ± 0.05
	November	19.06 ± 0.37	65.85 ± 2.57	1.24 ± 0.05	4.54 ± 0.12	2.25 ± 0.06
	December	18.15 ± 0.36	61.31 ± 2.43	1.10 ± 0.04	3.20 ± 0.11	1.54 ± 0.05
2024	January	20.26 ± 0.16	56.00 ± 2.10	1.28 ± 0.07	3.08 ± 0.12	1.59 ± 0.06
	February	21.13 ± 0.13	53.00 ± 1.99	1.68 ± 0.08	5.22 ± 0.17	2.75 ± 0.10
	March	20.85 ± 0.20	62.87 ± 3.02	1.43 ± 0.10	7.04 ± 0.26	3.68 ± 0.14
	April	21.93 ± 0.23	62.61 ± 2.28	1.67 ± 0.10	10.22 ± 0.26	5.49 ± 0.16
	May	23.90 ± 0.16	69.79 ± 2.40	1.50 ± 0.08	11.06 ± 0.23	6.23 ± 0.14
	June	26.30 ± 0.29	79.45 ± 1.32	1.44 ± 0.08	11.67 ± 0.25	6.96 ± 0.17
	July	28.67 ± 0.16	86.59 ± 0.85	1.17 ± 0.05	10.67 ± 0.13	6.69 ± 0.08
	August	29.08 ± 0.12	87.01 ± 0.99	1.22 ± 0.06	9.72 ± 0.17	6.15 ± 0.12
	September	25.58 ± 0.28	78.24 ± 2.55	1.04 ± 0.08	7.15 ± 0.13	4.19 ± 0.08
	October	22.90 ± 0.26	82.43 ± 2.46	0.84 ± 0.08	5.46 ± 0.10	3.00 ± 0.06

The plant material used consisted of one-year-old sweet orange (Citrus sinensis cv. Navelina) plants, grafted onto Carrizo rootstock (Citrus sinensis x Poncirus trifoliata), which were transplanted in September 2023. The plants were grown in 7 L pots filled with coconut fiber substrate (Espafibrac S.L., Segorbe, Spain), with an electrical conductivity (EC 1:1.5) of <0.7 mS·cm⁻¹, a pH (1:1.5) of 5.7–6.5, a bulk density (ρ_a) of 75–78 kg·m⁻³, the cation exchange capacity (CEC) of 10–30 meq·100 g⁻¹ and a water retention capacity of 48–53%. An initial analysis of the substrate was carried out to determine its nutrient content, including Zn and Mn (Table A1). In October 2024, the final substrate analysis was conducted. For this purpose, samples were collected from the pots of four plants, one per treatment (Table A1). Analyses of the coconut fiber substrate were carried out to determine moisture content according to EN 13041:2011 [27], total organic matter according to EN 13039:2011 [28], and total nitrogen using the Kjeldahl method following EN 13654-1:2002 [29]. Macro- and micronutrient contents were quantified after aqua regia digestion according to EN 13650:2001 [30].

2.2. Experimental Design

Four treatments (SoC, SoH, BMS, BMLS) were studied, based on variations in the Hoagland nutrient solution [31] with different sources of Zn and Mn (Table 2). A total of 120 citrus plants were used in the experiment, distributed among four nutrient solution treatments. For each treatment, three replications of 10 plants were established, resulting in 30 plants per treatment and 120 plants overall. The experiment followed a randomized complete block design, with plants randomly assigned within each replication and arranged on a 12 m² greenhouse bench (1.20 × 10 m) located in the center of the greenhouse to minimize environmental gradients of temperature, humidity, and radiation. The block effect was tested during the statistical analysis and was found not to be statistically significant.

Table 2. Nutrient solutions formulated with different sources of Zn and Mn.

Treatment	Description
SoC (Control)	Hoagland nutrient solution without Zn and Mn supplementation
SoH (Hoagland)	Hoagland nutrient solution with Zn and Mn
BMS (Black Mass Sulfate)	Hoagland nutrient solution with Zn and Mn sources derived from black mass treated with sulfates
BMLS (Black Mass Lignosulfonate)	Hoagland nutrient solution with Zn and Mn sources derived from black mass treated with lignosulfonate

The SoH nutrient solution contained macronutrients (in mmol L⁻¹): 15 NO₃⁻, 1.0 H₂PO₄⁻, 2.0 SO₄²⁻, 6.0 K⁺, 4.0 Ca²⁺, and 2.0 Mg²⁺, and micronutrients: 46 µM B, 9.1 µM Mn²⁺, 0.77 µM Zn²⁺, 0.31 µM Cu²⁺, 0.12 µM MoO₄²⁻, and 36 µM Fe³⁺. The BMS nutrient (11.47% Zn; 14.58% Mn) contained the same macronutrients as SoH and the following micronutrients: 46 µM B, 1.16 µM Mn²⁺, 0.77 µM Zn²⁺, 0.31 µM Cu²⁺, 0.12 µM MoO₄²⁻, and 36 µM Fe³⁺. The BMLS nutrient (2.04% Zn; 2.89% Mn) also contained the standard macronutrients and the following micronutrients: 46 µM B, 1.29 µM Mn²⁺, 0.77 µM Zn²⁺, 0.31 µM Cu²⁺, 0.12 µM MoO₄²⁻, and 36 µM Fe³⁺. Finally, the SoC nutrient solution contained the same macronutrients as SoH, but the micronutrients were: 46 µM B, 0 µM Mn²⁺, 0 µM Zn²⁺, 0.31 µM Cu²⁺, 0.12 µM MoO₄²⁻, and 36 µM Fe³⁺. The nutrient solutions had an electrical conductivity (CE) of 2.6 dS m⁻¹ and a pH of 6.1.

Irrigation was carried out manually using graduated containers, with the irrigation volume adjusted according to the crop water requirements. These requirements were determined from crop evapotranspiration (ETc), estimated according to the methodology proposed by the FAO [26]. Over the entire experimental period, a total of 65 L of water was applied per plant. For each irrigation event, both the irrigation volume and the number of applications were scheduled to maintain drainage between 10% and 20%, occurred between 7 h and 8 h to minimize surface evaporation. Drip irrigation system was carried out with an emitter rate of 2.0 L h⁻¹.

BMS and BMLS were prepared from the material commonly known as black mass, which constitutes the main component of spent or discharged alkaline batteries after the removal of the casing and other structural plastic and metallic parts. The black mass represents the predominant fraction of the battery, accounting for approximately 60–75% of its total weight. This material mainly consists of zinc oxide (ZnO), the mixed manganese–zinc oxide compound ZnMn₂O₄ (heterolite), potassium chloride (KCl), and graphite (pure carbon). It may also contain residual metallic zinc. In addition, trace amounts of minor or non-critical metals such as indium (In) and lithium (Li) can be present. If the battery selection process is inadequate, contaminants such as cadmium (Cd), nickel (Ni), or even mercury (Hg) may occur. However, contaminated batches are eliminated through prior quality control, as fertilizer regulations regarding heavy metals are highly stringent. Finally, the concentrations of metals were: Cd (0.01%), Pb (0.01%), Hg (84.45 ppm), and Ni (0.22%). Black mass was subjected to acidic leaching with 14 M H₂SO₄ (solid-to-liquid ratio 1 kg·L⁻¹) to solubilize Zn and Mn as their corresponding sulfates. Although ZnO and ZnMn₂O₄ exhibit different solubility and dissolution kinetics under milder conditions, under these strongly acidic leaching conditions, both phases are fully converted into dissolved Zn²⁺ and Mn²⁺ sulfate species, so differences in the initial solid-phase speciation do not persist in the final fertilizer formulations or affect metal bioavailability. The dried sulfate mixture was subsequently reacted with ammonium lignosulfonate, added at 15 equivalents with respect to the total Zn and Mn content, to yield a solid Zn/Mn lignosulfonate complex. The resulting product was dried, cryo-milled under liquid N_2, and either granulated or dissolved in water (up to 400 g·L⁻¹) to obtain solid and liquid fertilizer formulations, which were characterized prior to agronomic trials.

Upon extraction from black mass, all Zn and Mn compounds are ideally converted into their respective sulfates, resulting in the BMS product. Subsequently, the BMS can react with ammonium lignosulfonate to produce the BMLS formulation.

Various parameters were monitored, and regular evaluations were conducted to assess the plant’s response to the different treatments.

2.3. Plant Growth and Morphology Parameters

Plant height was measured using a meter stick from the base of the stem to the tip of the highest shoot. Stem diameter was measured with a BRIXO 800723 digital caliper (Fraschetti S.p.A., Pofi, Italy) at a height of 3 cm from the base of the stem. The number of leaves per plant was obtained through direct counting. These measurements were taken at the beginning of the experiment and subsequently monthly for all plants in each treatment. Leaf area (LA) was determined using the ImageJ software 1.53e (National Institutes of Health, Bethesda, MD, USA). Digital images of the leaves were captured against a white background with a ruler as a calibration reference. The scale was adjusted, and the total leaf area was calculated. Subsequently, biomass was determined using the same plants utilized for leaf area measurements. Each plant was separated into leaves, stems, and roots, and each part was weighed fresh. The samples were then oven-dried at 110 °C for 24 h to obtain dry weight. Total biomass was calculated as the sum of the dry weights of the different plant parts. Both measurements were taken at the beginning of the experiment and subsequently every three months on one plant from each repetition.

2.4. Physiological Parameters

Gas exchange was measured using an LI-6400 portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). Measurements were conducted in June and October 2024 under steady-state conditions, with saturating light (1200 μmol m⁻² s⁻¹), a CO₂ concentration of 400 ppm, and an airflow rate of 500 µmol s⁻¹. During the measurements, environmental conditions were as follows: in June, the temperature ranged from 29 to 31 °C with relative humidity of 55–60%; in October, the temperature ranged from 22 to 24 °C with relative humidity also between 50 and 65%. Solar radiation in June ranged from 800 to 1200 μmol m⁻² s⁻¹ between 11:00 a.m. and 12:00 p.m., and in October between 600 and 1000 μmol m⁻² s⁻¹. Eight plants per treatment were measured, selecting fully expanded, healthy leaves free of mechanical damage or disease, specifically the third or fourth leaf from the apex of the shoot. The evaluated parameters were stomatal conductance (gsw), apparent transpiration rate (E_apparent), quantum efficiency of photosystem II (ΦPSII), and electron transport rate (ETR).

2.5. Leaf Chlorophyll Concentration

The content of chlorophyll (a, b, and total) and carotenoids was determined following the protocol described by Lichtenthaler (1987) [32]. This analysis was conducted at the beginning of the experiment and then quarterly. For each treatment group, 8–10 fully developed, expanded, and healthy leaves were randomly collected at a standardized time. Monthly, non-destructive measurements of chlorophyll content (SPAD values) were taken using a portable MC-100 chlorophyll concentration meter (Apogee Instruments, Logan, UT, USA). The SPAD index (Soil and Plant Analyzer Development) provides a relative estimate of leaf chlorophyll concentration. For these measurements, three fully developed and expanded leaves in good condition were selected, specifically the third or fourth fully expanded leaf from the apex. All measurements were taken under uniform light conditions.

2.6. Leaf Nutrient Concentration

To determine the concentrations of essential nutrients such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), sulfur (S), manganese (Mn), zinc (Zn), boron (B), sodium (Na), and chlorine (Cl), 20–25 fully developed, expanded, and healthy leaves were randomly collected per treatment group. Additionally, at two points during the experimental period (March and October 2024), foliar analyses were conducted to determine the presence of heavy metals such as nickel (Ni), lead (Pb), cadmium (Cd), chromium (Cr), cobalt (Co), and mercury (Hg). Nitrogen content was determined using the Kjeldahl method following AOAC Official Method 984.13 [33], including sample drying, grinding, acid digestion, distillation, and titration as prescribed. The remaining mineral nutrients were quantified according to AOAC Official Method 985.01 [33], which includes wet digestion of the powdered leaf material and subsequent elemental determination using atomic absorption spectroscopy (AAS), in accordance with the standardized analytical conditions described in the method.

2.7. Statistical Analysis

The statistical analysis was performed using Statgraphics Centurion 19 (Statgraphics Technologies, The Plains, VA, USA). First, the normality and homoscedasticity of the data were evaluated. Then, a one-way analysis of variance (ANOVA) was conducted to determine the statistical influence of the study factor (treatment with different sources of Zn and Mn) using the F-Snedecor statistical test (p < 0.05). Mean separations between the different levels of the study factor were performed, when appropriate, using Student’s statistical t-test with the Least Significant Difference (LSD) method at p < 0.05.

3. Results

In general terms, the results showed no significant differences between the nutrient solutions, which ensures the absence of harmful substances and, therefore, the health of the plants. Consequently, no toxic effects were found in the young citrus plants treated with nutrient solutions prepared with Mn and Zn obtained from black mass.

3.1. Initial Parameters

The results of the main morphological and growth parameters, as well as leaf nutrient concentrations one month after transplanting (October 2023), showed no statistically significant differences among treatments. Likewise, no statistically significant effects were observed in the concentration of the main mineral elements (Table 3 and Table 4). It is worth noting that sodium (Na) concentrations were below the detection limit (<5 mg/kg) in all treatments and are therefore not included in the table.

Table 3. Initial results of morphological, growth and physiological parameters (height; stem diameter; leaves; biomass; LA, Leaf Area and SPAD, Soil and Plant Analyzer Development), in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), one month after transplantation (October 2023).

Treatment	Height	Stem Diameter	Number of Leaves	Biomass	LA	SPAD
	(cm Plant−1)	(mm Plant−1)	(N° Plant−1)	(g Plant−1)	(cm2 Plant−1)
SoC	88.81 a	12.85	64.00	109.95	21.93	53.19
SoH	76.95 b	12.47	61.00	103.89	20.72	49.91
BMS	76.87 b	11.64	60.00	100.65	20.07	41.95
BMLS	88.74 a	14.11	61.00	109.29	21.80	54.19
Source (df)
Treatment (3)
SS	21.52	19.12	2.32	10.44	12.46	21.34
p-value	0.046	0.321	0.978	0.821	0.771	0.566

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

Table 4. Initial results of nutrient element concentration in leaves on a dry matter basis in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), one month after transplantation (October 2023).

Treatment	N	P	K	Ca	Mg	S	Mn	Zn	B
	(%)						(mg·kg−1)
SoC	2.85	0.45	4.15	2.31	0.63	0.58	19.74	38.96	100.03
SoH	2.69	0.43	3.93	2.19	0.60	0.55	18.65	36.81	94.51
BMS	2.61	0.42	3.80	2.12	0.58	0.53	18.07	35.66	91.57
BMLS	2.83	0.45	4.13	2.30	0.63	0.57	19.62	38.72	99.42
Source (df)
Treatment (3)
SS	91.22	82.46	81.46	94.16	72.46	77.55	88.89	79.51	75.98
p-value	0.541	0.632	0.441	0.571	0.673	0.885	0.892	0.567	0.789

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

3.2. Plant Growth and Morphology Parameters

The results obtained for plant height, stem diameter, and number of leaves (Table 4) showed no statistically significant differences (p > 0.05) attributable to the analyzed factor (treatment with different sources of Zn and Mn) at the two evaluation points (March and October 2024).

Plant height ranged from about 90 to 98 cm in March and from 118 to 131 cm in October, with BMS and BMLS showing the lowest and highest values, respectively, but these differences were not statistically significant (Table 5). Stem diameter also varied within a narrow range among treatments (approximately 13–17 mm on both dates), indicating no significant effect. The number of leaves per plant ranged from roughly 90 to 161 leaves, with SoC tending to produce more leaves in March and BMS in October. Similarly, biomass per plant did not differ significantly between treatments during the evaluated period (p > 0.05). Average biomass increased from around 110–123 g plant⁻¹ in March to about 298–318 g plant⁻¹ in October, with BMLS generally showing the highest values and SoH the lowest. In March, LA values with SoH tended to be the greatest leaf area and SoC the lowest, whereas in October, all treatments converged to similar LA values (≈26–31 cm² plant⁻¹), confirming the absence of a clear treatment effect.

Table 5. Temporal evolution of morphological and growth parameters in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), six and twelve months after transplantation (March and October 2024).

Treatment	Height		Diameter		Number of Leaves		Biomass		LA
	(cm Plant−1)		(mm Plant−1)		(N° Plant−1)		(g Plant−1)		(cm2 Plant−1)
	Mar	Oct	Mar	Oct	Mar	Oct	Mar	Oct	Mar	Oct
SoC	97.00	124.40	13.43	15.29	113.00	158.00	118.69	312.07	24.78	27.89
SoH	91.86	130.60	12.89	16.94	91.00	147.00	108.87	297.58	31.65	30.61
BMS	90.43	118.00	12.78	16.07	96.00	161.00	113.62	311.63	26.98	28.94
BMLS	98.36	127.40	14.25	16.30	102.00	139.00	122.82	317.82	25.62	26.89
Source (df)
Treatment (3)
SS	9.42	6.87	8.25	13.53	10.6	10.73	18.69	10.90	50.63	24.37
p-value	0.489	0.759	0.550	0.495	0.610	0.600	0.333	0.593	0.088	0.203

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

3.3. Physiological Parameters

Regarding the physiological parameters (Table 6), it is worth noting that the statistical analysis of the factor evaluated, both in June and October 2024, showed no significant effect on the efficiency of photosystem II (PhiPS2), whereas a statistically significant influence was observed for the remaining parameters (p < 0.05).

Table 6. Results of physiological parameters (stomatal conductance, g_sw; apparent transpiration rate, E_apparent; quantum efficiency of photosystem II, ΦPSII; and electron transport rate, ETR; in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), nine and twelve months after transplantation (June and October 2024).

Treatment	gsw	E_Apparent	PhiPS2	ETR
	(mol m−2 s−1)	(μmol m−2 s−1)
	Jun
SoC	0.10 ab	1.57 a	0.69	41.40 b
SoH	0.12 a	2.33 a	0.67	46.14 a
BMS	0.09 b	1.60 b	0.68	46.10 a
BMLS	0.08 c	1.42 b	0.68	43.31 b
Source (df)
Treatment (3)
SS	9.7	19.4	3.45	5.18
p-value	0.012	0.011	0.736	0.044
	Oct
SoC	0.06 d	0.94 c	0.71	38.47 b
SoH	0.32 a	5.04 a	0.69	42.70 a
BMS	0.26 b	4.47 a	0.69	41.49 ab
BMLS	0.19 c	3.30 b	0.69	39.31 b
Source (df)
Treatment (3)
SS	67.48	70.26	0.99	2.40
p-value	0.000	0.000	0.811	0.053

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

In June, stomatal conductance (g_sw) values ranged from 0.08 mol m⁻² s⁻¹ (BMLS) to 0.12 mol m⁻² s⁻¹ (SoH), and apparent transpiration varied between 1.42 mmol m⁻² s⁻¹ (BMLS) and 2.33 mmol m⁻² s⁻¹ (SoH). The photosynthetic efficiency (PhiPS2) remained stable among treatments (0.67–0.69), with no significant differences. However, electron transport rate (ETR) did show statistically significant differences (p = 0.044), with values ranging from 41.40 µmol m⁻² s⁻¹ (SoC) to 46.14 µmol m⁻² s⁻¹ (SoH).

In October, the SoH treatment again showed the highest values of gsw (0.32 mol m⁻² s⁻¹) and transpiration (5.04 mmol m⁻² s⁻¹), while SoC recorded the lowest in both parameters (0.06 mol m⁻² s⁻¹ and 0.94 mmol m⁻² s⁻¹, respectively). PhiPS₂ remained stable, with no statistically significant differences (0.66–0.71), as did ETR (38.47–42.70).

3.4. Leaf Chlorophyll Concentration

Chlorophyll concentration (Table 7) showed statistically significant differences (p < 0.05) in both March and October. On both dates, the SoH treatment exhibited the highest values (0.41 and 0.31 mg g⁻¹ FW, respectively), with statistically significant differences compared to the other treatments. This trend was supported by the SPAD parameter, which also reached its maximum values under SoH on both dates (62.86 in March and 66.69 in October)

Table 7. Temporal evolution of chlorophyll and carotenoid concentration in leaves on a dry matter basis and the SPAD index in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), six and twelve months after transplantation (March and October 2024).

Treatment	Chlorophyll T		Carotenoids		SPAD
	(mg g−1 FW)
	Mar	Oct	Mar	Oct	Mar	Oct
SoC	0.19 c	0.18 c	0.05 c	0.05 c	44.81 b	65.73
SoH	0.41 a	0.31 a	0.11 a	0.08 a	62.86 a	66.69
BMS	0.23 b	0.18 c	0.07 b	0.05 c	44.06 bc	62.90
BMLS	0.22 bc	0.26 b	0.06 b	0.07 b	38.62 c	69.30
Source (df)
Treatment (3)
SS	91.53	95.32	89.82	94.84	84.66	21.94
p-value	0.000	0.000	0.000	0.000	0.000	0.253

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

3.5. Leaf Nutrient Concentration

The leaf nutrient concentration of the three main macronutrients in plants (N, P, K) showed a statistically significant influence (p < 0.05) throughout the period analyzed, depending on the sampling dates. However, no clear behavioral patterns were observed between the elements and the different sources evaluated (Table 8).

Table 8. Temporal evolution of N, P, and K element concentration in leaves on a dry matter basis in the four treatments six and twelve months after transplantation (March and October 2024).

Treatment	N		P		K
	(%)
	Mar	Oct	Mar	Oct	Mar	Oct
SoC	4.71	3.59 a	0.41 a	0.24 c	4.11	5.63 a
SoH	4.7	3.66 a	0.33 b	0.26 b	4.33	4.70 b
BMS	4.86	3.82 a	0.37 ab	0.29 a	4.04	4.66 b
BMLS	4.69	3.86 a	0.39 a	0.31 a	4.13	5.46 a
Source (df)
Treatment (3)
SS	1.95	19.02	40.08	69.95	7.04	57.46
p-value	0.955	0.324	0.038	0.000	0.752	0.003

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

With respect to foliar nitrogen concentration, no significant differences were detected at any of the sampling dates (p > 0.05). Values ranged from 4.73% (SoH) to 4.69% (BMLS) in March, and from 4.10% (SoH) to 3.45% (SoC) in October.

In contrast, foliar phosphorus concentration was significantly influenced by the factor under study (p < 0.05) at both sampling dates. In March, the SoC treatment showed the highest p value (0.41%), while SoH presented the lowest (0.33%). In October, the highest p concentration was recorded in BMLS (0.31%), whereas SoC showed the lowest value (0.24%).

Finally, foliar potassium concentration was not statistically affected by the treatment in March, with similar values across treatments (ranging from 4.04% to 4.33%). However, in October, K concentration increased in BMLS (5.46%) and SoC (5.63%), in contrast to SoH, which had the lowest value (4.70%).

The secondary macro nutrient concentration (Ca, Mg, S) was significant influenced (p < 0.05) by the studied factor on certain sampling dates (Table 9).

Table 9. Temporal evolution of Ca, Mg, and S element concentration in leaves on a dry matter basis in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), six and twelve months after transplantation (March and October 2024).

Treatment	Ca		Mg		S
	(%)
	Mar	Oct	Mar	Oct	Mar	Oct
SoC	1.63 b	1.36 b	0.41	0.29 b	0.38 ab	0.30 b
SoH	1.88 a	1.52 a	0.34	0.35 a	0.43 a	0.35 a
BMS	1.88 a	1.06 c	0.37	0.30 b	0.36 b	0.33 a
BMLS	1.60 b	1.07 c	0.38	0.30 b	0.34 b	0.33 ab
Source (df)
Treatment (3)
SS	39.34	89.7	33.13	63.6	42.99	44.94
p-value	0.041	0.000	0.085	0.001	0.026	0.020

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

For Ca, significant differences between treatments were detected in March (p = 0.041), with the SoH and BMS treatments showing the highest values (1.88%) compared to BMLS (1.60%). In October, however, no statistically significant differences were observed (p = 0.460), with similar values recorded across treatments (1.59–1.74%).

With respect to Mg, a significant treatment effect was recorded in October (p = 0.015), with BMLS (0.39%) and SoC (0.38%) showing the highest concentrations, while SoH had the lowest value (0.33%). In March, although differences were not statistically significant (p = 0.085), the SoC treatment exhibited the highest concentration (0.41%) compared to the lowest in SoH (0.34%).

On the other hand, S content in plants showed a significant difference in March (p = 0.026), with SoH presenting the highest concentration (0.43%) and BMLS the lowest (0.34%). In October, differences were not statistically significant (p = 0.207), with values ranging between 0.35% and 0.39%.

The study on Zn and Mn concentrations in the leaves of young “Navelina” plants showed varying results regarding the statistically significant influence of the Zn and Mn source and the sampling times (Table 10). Specifically, for Mn, significant differences were observed (p < 0.05). On both dates, SoH presented the highest concentrations (41.12 and 46.12 mg·kg⁻¹ plant⁻¹, respectively), while BMS showed the lowest values (28.97 and 33.61 mg·kg⁻¹ plant⁻¹).

Table 10. Temporal evolution of Mn and Zn element concentration in leaves on a dry matter basis in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), six and twelve months after transplantation (March and October 2024).

Treatment	Mn		Zn		B
	(mg·kg−1)
	Mar	Oct	Mar	Oct	Mar	Oct
SoC	30.79 b	38.19 b	23.12	15.02 bc	37.07	43.76 b
SoH	41.12 a	46.12 a	26.43	17.41 a	32.35	54.37 a
BMS	28.97 b	33.61 c	21.75	14.63 c	38.79	44.78 b
BMLS	30.10 b	37.16 b	24.08	15.98 b	36.82	47.74 b
Source (df)
Treatment (3)
SS	70.76	82.75	36.38	59.23	36.59	70.06
p-value	0.000	0.000	0.059	0.002	0.058	0.000

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

Regarding Zn, the treatment effect was not statistically significant in March (p = 0.059), although SoH recorded the highest numerical concentration (26.43 mg·kg⁻¹ plant⁻¹) and BMS the lowest (21.75 mg·kg⁻¹ plant⁻¹). In contrast, in October, a statistically significant effect was observed (p = 0.002). The highest Zn concentration was again found in SoH (17.41 mg·kg⁻¹ plant⁻¹), higher than in BMS (14.63 mg·kg⁻¹ plant⁻¹) and BMLS (15.98 mg·kg⁻¹ plant⁻¹), while SoC showed an intermediate value (15.02 mg·kg⁻¹ plant⁻¹).

Concerning the potential toxicity caused by critical elements such as B, Cl, and Na, the results showed variable responses depending on the elements and the sampling date (Table 10). In the case of B, statistically significant differences between treatments were observed in October (p = 0.000), with the SoH treatment showing the highest concentration (54.37 mg·kg⁻¹ plant⁻¹), significantly higher than those recorded in SoC (43.76 mg·kg⁻¹ plant⁻¹), BMS (44.78 mg·kg⁻¹ plant⁻¹), and BMLS (47.74 mg·kg⁻¹ plant⁻¹). In June, although no significant differences were detected (p = 0.058), BMS presented the highest value (38.79 mg·kg⁻¹ plant⁻¹), while SoH showed the lowest concentration (32.35 mg·kg⁻¹ plant⁻¹).

As for Cl, concentrations remained below the detection limit (<0.1 mg·kg⁻¹ plant⁻¹) in all treatments. Similarly, Na levels stayed below the detection threshold (<0.05 mg·kg⁻¹ plant⁻¹) across all sampling dates and treatments.

Regarding the accumulation of heavy metals in leaves (Table 11), the values obtained for the different treatments were very low for all analyzed elements (Ni, Pb, Cd, Cr, Co, and Hg), both in June and October. With regard to mercury (Hg), all samples tested showed values below the detection limit of the analytical method used, indicating the absence of this metal in the system.

Table 11. Foliar concentration of heavy elements on a dry matter basis in the four treatments (SoC: control solution; SoH: Hoagland solution; BMS: solution with Zn and Mn sulfates (black mass); BMLS: solution with Zn and Mn lignosulfonates (black mass)), at two time points of the trial six and twelve months after transplantation (March and October 2024).

Treatment	Ni	Pb	Cd	Cr	Co
	(mg·kg−1)
	Mar
SoC	0.285 b	0.129 b	0.017 c	0.233 c	0.013 c
SoH	0.205 c	0.232 a	0.007 d	0.312 b	0.021 b
BMS	0.331 ab	0.135 b	0.021 b	0.402 a	0.000 d
BMLS	0.338 a	0.123 b	0.027 a	0.330 b	0.041 a
Source (df)
Treatment (3)
SS	80.29	92.96	95.20	82.32	98.29
p-value	0.000	0.000	0.000	0.000	0.000
	Oct
SoC	0.181 a	0.000 c	0.000	0.300 a	0.015 b
SoH	0.160 b	0.034 a	0.000	0.213 b	0.011 c
BMS	0.170 ab	0.018 b	0.000	0.157 c	0.000 d
BMLS	0.158 b	0.019 b	0.000	0.167 c	0.048 a
Source (df)
Treatment (3)
SS	40.25	99.24		96.54	99.72
p-value	0.037	0.000		0.000	0.000

Different letters in the same column indicate statistically significant differences according to the LSD test at p < 0.05. df: degrees of freedom. SS: sum of squares.

Overall, the results support the conclusion that none of the treatments evaluated induced phytotoxic accumulation of heavy metals in young citrus plants, and that the Zn and Mn sources applied (especially BMS and BMLS) did not lead to hazardous concentrations of metallic contaminants in the foliar tissues analyzed.

4. Discussion

Zinc and manganese deficiencies in plants can cause a significant loss of leaves, as well as chlorosis accompanied by necrotic spots and generalized leaf malformations, ultimately affecting vegetative growth and, in some cases, leading to plant death [21,34]. Plant morphology and growth kinetics may also be affected by zinc toxicity, resulting in a significant reduction in biomass [35]. Likewise, manganese toxicity affects plant growth by significantly interfering with primary metabolism [36]. Moreover, high concentrations of Mn and/or Zn have been reported to negatively affect fine root length density, turnover rate, and hence the leaf area [37]. According to the results, no effects were observed on morphological or growth parameters that would indicate either Zn or Mn deficiency or toxicity. In our trial, there were no significant differences in plant height, stem diameter, leaf number, biomass, and leaf area among SoC, SoH, BMS, and BMLS at either sampling date. These findings indicate that no clear negative effects of the different Zn and Mn sources on vegetative growth were observed.

Electron transport in photosynthesis and net photosynthesis can be significantly reduced by high concentrations of Mn, which may cause phytotoxicity in the plant [38,39]. Under these conditions, the photosynthetic efficiency of photosystem II (PhiPS2) can also be significantly affected [40]. According to the scientific literature, no toxic effects related to Mn excess were observed from the nutrient solutions applied in this study. Photosynthetic efficiency (PhiPS2) remained stable across all treatments and at both measurement times, falling within the optimal range for healthy plants [41]. Stomatal conductance may be negatively affected by high Zn concentrations in leaves when present at levels that could cause phytotoxicity [42,43]. In the present study, stomatal conductance was not negatively influenced by the Zn and Mn sources applied, and therefore, no toxic effects on the plants were observed. In particular, PhiPS2, stomatal conductance, and transpiration showed only minor, non-consistent differences among treatments and always remained within physiological ranges typical of well-functioning citrus plants, even when Zn and Mn were supplied from black mass.

With respect to chlorophyll content, the observed effects could be related to the higher availability of Mn in the SoH treatment. This micronutrient plays a key role as an enzymatic activator in chlorophyll biosynthesis and other metabolic processes associated with photosynthesis [21], which could explain the increased accumulation of photosynthetic pigments observed. Black. Mass-derived sources provide sufficient photosynthetic activity in BMs and BMLS plants, as evidenced by the maintenance of pigment levels compared to control plants. SoH consistently showed the highest levels of chlorophyll and carotenoids during both sampling dates.

Regarding the analyzed nutrient elements, a general assessment of the three most essential macronutrients (N, P, K) highlights the variability in the influence of the different Zn and Mn sources on their concentrations. No consistent pattern of influence was observed from the Zn and Mn sources on these parameters. The concentrations of N, P, and K remained within levels considered adequate, with relatively high values for all three elements, in line with the recommendations of [6,20]. At no point was a depressive effect of the Zn and Mn sources on these nutrient concentrations observed, indicating that the BMS and BMLS treatments did not exert any negative influence on the concentration of the three key macronutrients. Therefore, it can be considered that these sources did not produce any toxic effect that might reduce Zn or Mn concentrations in plant tissues. Our foliar analyses confirmed that N, P, and K concentrations were statistically similar among SoC, SoH, BMS, and BMLS in both March and October, demonstrating that the recycled micronutrient formulations did not disrupt macronutrient uptake or balance.

In relation to the secondary elements (Ca, Mg, S), Ca and Mg concentrations remained at low and optimal levels, respectively [6]. In the case of S, concentrations were elevated [20], a result consistent with its close relationship to N concentration, as reported by [21]. At no point was a negative influence of the Zn and Mn sources observed on the reduction in Ca, Mg, or S concentrations due to toxic effects, particularly in the case of the black-mass-derived treatments (BMS and BMLS). Mg concentration was not negatively affected by the Zn source applied. Therefore, no toxic effect of the element source was confirmed, since under these conditions, Zn competes with Mg, disrupting ionic balance [44]. Similarly, excess Mn can reduce Ca and Mg concentrations because of oxidative stress [45,46]. The slight differences detected in Ca, Mg, and S levels among treatments were not associated with any visual deficiency or toxicity symptoms, indicating that BMS and BMLS preserved an adequate secondary-nutrient status comparable to that of the conventional Hoagland solution.

About the study of the micronutrients Zn and Mn, and despite the variability observed in the results, Mn concentrations remained within optimal ranges across all treatments evaluated, according to the interpretation criteria of [6,20]. In contrast, Zn concentrations were considered low at all four fertilization levels studied [6,20]. The toxic effect of Zn generally occurs when concentrations exceed 300 mg·kg⁻¹, although in some species, toxicity may appear at levels below 100 mg·kg⁻¹ [25,47]. For Mn, [21,48] indicate that normal concentrations in plants can range from 30 to 500 mg·kg⁻¹, depending on the species. In the case of Zn, concentrations typically range from 25 to 150 ppm, with deficiency symptoms appearing at levels between 10 and 20 ppm [21]. Moreover, it has been reported that excessive levels of Mn or P may inhibit Zn uptake [21]; however, no adverse effect of Mn sources on Zn absorption was observed in the present study. Likewise, an excess of Zn may reduce Mn uptake [21], but such an effect was not confirmed in any of the treatments evaluated.

Additionally, high levels of Ca and Mg can induce Mn deficiency [24]; nevertheless, based on the results of this experiment, no adverse effects attributable to the sources used were observed on Mn concentrations. According to the results obtained in this study, none of the Zn and Mn sources used showed a depressive effect on the foliar concentrations of these elements. Throughout the 12-month experiment, the concentrations of Mn and Zn in the leaves remained within the expected sufficiency or low-sufficiency ranges for young citrus in all treatments. It indicates that inputs derived from black mass supplied these micronutrients without causing antagonistic interactions with other elements.

Concerning the potential toxicity caused by the three critical elements (B, Cl, and Na), it is worth noting that Cl and Na concentrations were consistently very low and below detection limits, indicating the absence of phytotoxicity. B concentrations did not reach critical thresholds either, remaining within normal ranges [6,20], thereby ruling out any phytotoxic effect of the Zn and Mn sources evaluated in this study. Even when treatments included recycled sources (BMS and BMLS), foliar B, Cl, and Na contents remained within safe margins. It indicates that the use of black mass did not introduce additional salinity or boron stress into the system.

Regarding heavy metals, there are currently no established reference values specifically for foliar tissue in citrus plants; however, an approximate comparison can be made based on existing legislation for fruits intended for human consumption. Considering that the results from this study are expressed on a dry weight basis, and that the average dry matter content in citrus fruits such as ‘Navelina’ ranges between 10% and 15%, the corresponding values on a fresh weight basis would be substantially lower than those reported, ruling out any risk of hazardous accumulation of these metals in the analyzed tissues, according to Regulation (EU) 2023/915 [49]. In the case of nickel (Ni), Regulation (EU) 2024/1987 [50]—which amends Regulation (EU) 2023/915—establishes for the first time a maximum limit of 0.40 mg·kg⁻¹ (fresh weight) for fruits, including citrus, applicable from 1 July 2025. Although the present study reports data on a dry weight basis, the concentrations of Ni observed in leaves remained low and posed no apparent toxicity risk. As for chromium (Cr) and cobalt (Co), there are currently no specific maximum limits established for these elements in fruits under EU food safety legislation. Nevertheless, the concentrations recorded in this trial were very low, with no indication of excessive accumulation in the foliar tissue analyzed. Although our measurements were limited to leaves from young, non-fruiting citrus plants, foliar concentrations of Cd, Pb, Ni, Cr, and Co were clearly below the maximum levels established for fruits in EU food safety legislation. Using fruit-based limits is therefore conservative, as several studies report that heavy metals in citrus trees tend to accumulate more in leaves and peel than in the edible pulp, where the lowest concentrations are usually found [51]. Overall, the results support the conclusion that none of the treatments evaluated induced phytotoxic accumulation of heavy metals in young citrus plants, and that the Zn and Mn sources applied (especially BMS and BMLS) did not lead to hazardous concentrations of metallic contaminants in the foliar tissues analyzed.

A field trial with the same citrus variety has been established, based on the greenhouse results. This ongoing study will be conducted over the following years to validate the results obtained from young plants and to evaluate their applicability under field conditions. Subsequent research could be supported by the uniformly low foliar concentrations of Ni, Pb, Cd, Cr, and Co observed across SoC, SoH, BMS, and BMLS, indicating that repeated fertigation with black-mass-derived fertilizers did not promote heavy-metal accumulation in young citrus plants, and thus justifies scaling the evaluation to field conditions.

5. Conclusions

The analyzed treatments of Zn and Mn sources did not generally affect the morphological and physiological parameters. However, some variability in leaf pigment concentration was observed among treatments. In particular, the Hoagland solution yielded the most balanced chlorophyll levels, suggesting a more stable response in pigment metabolism.

In general, the treatments did not significantly affect the concentration of the main mineral nutrients in the plants. All elements remained within adequate levels based on optimal reference ranges, and no signs of phytotoxicity or deficiencies were observed due to the use of alternative Zn and Mn sources derived from black mass. Furthermore, the concentrations of heavy metals in the leaf tissues remained below the detection limits of the analytical methods used, indicating no risk of contamination under the experimental conditions.

Overall, over the season of this 12-month study, no clearly depressive effects were detected on plant growth, key physiological indicators, or heavy metals accumulation resulting from the Zn and Mn sources derived from black mass. The results of this work could promote the integration of recycled micronutrient sources into fertilization programs as a practical step towards a circular economy in agriculture.