Poultry Litter and Inorganic Fertilization: Effects on Biomass Yield, Metal and Nutrient Concentration of Three Mixed-Season Perennial Forages

Abstract

Poultry litter and fertilizers are normally added as soil amendments. The effects of poultry litter and inorganic fertilizers on three mixed-season perennial forages were studied for two years in the field to understand growth dynamics, metals, and nutrient uptake. The primary objective was to investigate the heavy metal and nutrient concentrations, biomass yield and forage potential of a cool-season forage, stinging nettle (Urtica dioica L.), relative to warm-season forages, bermudagrass (Cynodon dactylon (L.) Pers.) and switchgrass (Panicum virgatum L.). Forage cuttings and soil samples were analyzed for heavy metals and nutrients using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Total biomass yield was higher by 66% and 50% in switchgrass and bermudagrass, respectively, compared with stinging nettle for the first year. While the warm-season forages yielded more biomass over the cool-season forage, metal concentrations were significantly higher for all elements in the cool-season forage. Stinging nettle showed greater macro-nutrient uptake with 103.20 kg ha⁻¹, 0.87 kg ha⁻¹, 27.49 kg ha⁻¹ and 32.08 kg ha⁻¹ for Ca, Fe, Mg, and P, except for K with 223.51 kg ha⁻¹ compared with 267.29 kg ha⁻¹ and 283.96 kg ha⁻¹ for switchgrass and bermudagrass, respectively. Heavy metals were also higher in stinging nettle but were within the allowable limits for forages, indicating its potential as a resource for forages and nutrient cycling, particularly when double-cropped with warm-season forages.

1. Introduction

Poultry litter is an important source of organic fertilizer that is normally surface-applied on fields for the improvement of soil quality and increased yield in agricultural production. This is particularly common for pastures and hayfields, where different types of forages are cultivated for ruminant and non-ruminant animal nutrition [1,2,3]. Poultry litter contains valuable nutrients, including trace and non-trace metals, and its application can enhance soil productivity and improve soil quality by improving aggregate formation and stability. It improves water filtration capacity and supplies plant nutrients such as Ca, Mg, and K to soils [4,5,6]. However, poultry litter applied on fields to satisfy one nutrient requirement could result in oversupply and an excess of another [7]. One example is the excess of phosphorus that occurs when poultry litter is applied to provide adequate nitrogen [8,9,10]. This same situation could apply to short-term and long-term or repeated applications that cause the accumulation of heavy metals in the soil and the contamination of surface and groundwater. This is because the amount of poultry litter applied on the fields could exceed the nutrient requirements of those fields and the carrying capacity of the soil, even as the biomass yield of the forages is increased [11].

Heavy metals and other nutrients could also be taken up by forages due to increased poultry litter application and the subsequent concentration of metals in the biomass regardless of the seasonal preference of the forage type (warm or cool season). The heavy metal concentrations in the crop could vary and subsequently depend on the type of manure and the feeding practices of the animals [12]. A review by [13] found a wide variation in mean litter concentrations of Cu (32 to 593 mg Cu kg⁻¹) and Zn (125 to 496 mg Zn kg⁻¹) between various studies in 12 and 7 different experiments. In one study, [14], it was concluded that when dietary Zn content in poultry feed was decreased from 190 to 65 mg kg⁻¹, Zn concentration in broiler manure was reduced by 75%. Similarly, ref. [15] reported a strong correlation between the analyzed nutrients and metals in the feed and in their excreted waste. This was specific for the concentrations of As, Cu, Zn and P in soils that had received surface-applied poultry litter over a 14-year period; the concentrations of these metals in this soil were significantly greater than untreated soil at different depths and tillage conditions [2,16]. This trend was notable and consistent for many perennial forage grasses like bermudagrass (Cynodon dactylon (L.) Pers.) across various studies [17,18,19]. Similar results have also been seen in switchgrass (Panicum virgatum L.) [20]. More nutrient concentrations were also observed in switchgrass and perennial legumes like alfalfa (Medicago sativa L.) grown on poultry-litter-amended soils [21,22]. In comparison with inorganic fertilizers [23], the release and uptake of nutrients [24] as well as increased tissue Cu and Zn concentrations were recorded from lands with poultry litter applications. While soil amendments have been shown to increase heavy metals and affect soil health, amendments could immobilize the uptake of these metals while improving the yield of the crops [25], thereby reducing the accumulation, potential bioavailability, and the harmful effects of these heavy metals [26].

Stinging nettle (Urtica dioica L.) is an erect, herbaceous perennial forb and has been reported to have multiple uses. It is notable for fiber production [27,28,29] and food and medicinal purposes [30,31] and mostly compares with alfalfa in protein content [32,33]. Stinging nettle has been used as a supplement in dried and powdered forms, and is added to the feeds of various animals, including cows for increased milk production [34], horses and pigs for improved health and daily gains [35], as well as feeds for rabbits and captive gorillas [36,37]. When cut and dried, it was confirmed to be an excellent source of nutritious livestock feed [32].

However, agronomic research on its use as an alternative forage source is limited. Nevertheless, its biomass yield and quality depend on the availability of significant amounts of nitrogen (N), and it has been reported to have a tendency of accumulating heavy metals [38,39]. Additionally, studies on its biomass yield, trace metals (As, Cd, Cu, Mn, Zn) and nutrients (Ca, Mg, K, P) in response to poultry litter applications relative to other forages have not been done. Therefore, the objective of this study was to evaluate the forage potential of stinging nettle relative to bermudagrass and switchgrass, as well as relative biomass yield, nutrient, and heavy metal concentrations in mixed-season perennial forages planted in poultry litter- and inorganic fertilizer-applied fields.

2. Materials and Methods

2.1. Site Description, Experimental Design and Preparation

The experiment was conducted at Randolph Farm of Virginia State University located in the Tri-Cities area of Central Virginia (37.1° N; 77.3° W) at an elevation of 45 m above sea level. The soil type at the site is a Bourne series fine sandy loam (mixed, semiactive, thermic Typic Fragiudults). The research area was conventionally tilled and divided into 12 0.008-hectare plots with 3 m buffer stripes between plots. Each plot was treated as an experimental unit, and the forage treatments (switchgrass + poultry litter, bermudagrass + poultry litter, stinging nettle + poultry litter) were distributed in a completely randomized design (CRD) with four replicates per treatment, mostly due to the homogenous nature of the research area and the desired homogeneity of the experimental treatments. The perennials were planted in the spring (27 March) and emerged within the first 10 to 14 days after planting.

A composite soil sample formed by mixing 20 core samples collected from the plots to a depth of 15 cm was analyzed to characterize initial soil conditions. The poultry litter used was also analyzed, and, together with the initial soil conditions, used to determine and calculate plant-available nitrogen (PAN) and other nutrients for the experimental layout (

Table 1. Initial experimental plot analysis for soil and poultry litter.

Component	pH	EC	Ca	Cd	Cu	Fe	K	Mg	Mn	P	Pb	Zn
		µS cm−1	mg kg−1
Soil	6.06	50	238	nd	0.83	143	139	35.8	9.5	97	4.9	1.2
Poultry Litter	6.58	17,430	15,308	0.14	241	1704	25,327	53,792	512	11,884	0.83	318

). The NO₃-N and the NH₄-N of the initial soil analyzed were also shown to be 0.56 mg kg⁻¹ and 6.62 mg kg⁻¹, respectively, while the Cd content was not detectable.

In line with the objective, each experimental plot received a uniform manual application of poultry litter and inorganic fertilizer from urea (CO(NH₂)₂) in the spring (21 March) for the required rate of 136 kg N ha⁻¹ before the establishment of the study and before the perennials were planted. Subsequently, plots received a split-surface application of poultry litter to supply 68 kg N ha⁻¹ and inorganic fertilizer from sodium nitrate (NaNO₃) to supply 68 kg N ha⁻¹ in the spring (24 March) and early fall (30 August/2 September), respectively, for the total target rate of 136 kg N ha⁻¹ annually (

Table 2. Poultry litter (PL) and inorganic fertilizer (IF) rates and sequence of applications.

Application Sequence	Date	Total N (%)		Amount of PL Applied	Amount of IF Applied
		PL	IF	kg plot−1
First application	Spring-Year 1	1.70	46-0-0	32.0	1.18
Second application	Spring-Year 2	3.88	16-0-0	7.01	1.70
Third application	Fall-Year 2	2.49	16-0-0	10.93	1.70

). Watering was done as needed via drip irrigation, and weeds were controlled manually. Rainfall, temperature, and relative humidity data were recorded for the duration of the study (Figure 1). Necessary physiological parameters and agronomic data and traits were visually monitored throughout the study.

2.2. Initial Soil and Poultry Litter Analyses

The preliminary analysis of the composite soil samples used to determine PAN described the initial soil condition as well as the poultry litter (Table 1). The poultry litter was analyzed for total N before each application in order to calculate the required N for application. Urea was used as the first inorganic fertilizer to supplement poultry litter, and sodium nitrate was used after the plants were established to avoid the potential for burns that could result from urea fertilizer applications (Table 2).

2.3. Field Sampling and Analyses

Soil Samples

A total of 5 soil core samples were collected and composited from the 0–15 cm and 15–30 cm depths of each plot 90 days after the spring manure and fertilizer applications. The samples were air-dried and crushed to pass a 2-mm sieve. The soil pH was measured in a 1:2 (material: deionized water, v/v) suspension after equilibration using a glass electrode. Electrical conductivity (EC) was determined in a 1:2 (material: deionized water, v/v) suspension using an Orion conductivity meter [40,41]. Mehlich-3 (M3) extractable nutrients and metals (Ca, Cu, Fe, K, Mg, Mn, P, Pb and Zn) were determined as described by [42]. After extractions, samples were analyzed using inductively coupled plasma-optical emission spectroscopy (ICP-OES). All soil samples analyzed were processed with blanks and the in-house soil standard sample. The in-house standard was produced from a field soil which was rigorously and repeatedly analyzed for its chemical properties as well as its M3 extractable nutrients and metal contents. The soil standard sample is used with blanks in between all other soil samples to monitor the accuracy and reproducibility of soil analyses done in the laboratory. Our results were within the established percentage range of 80% to 100% for the in-house standard soil.

2.4. Forage/Plant Samples

Forage harvest was completed three times for the season on a specified cutting frequency. A prolonged harvest interval of 45 days was considered valuable for nutrient or metal removal and uptake through the biomass. Therefore, at 35 days after planting, and on a bimonthly basis of about 45 days, a 1-m quadrate was randomly placed in each plot, and the biomass was manually harvested to a 10–12 cm stubble. Each quadrate space provided the first, second and third harvest (cuttings) or regrowth where applicable for the forage samples. A complete set of sequential harvesting was not possible in switchgrass due to insufficient regrowth resulting from the cessation of seasonal growth. Fresh biomass weights were recorded, and subsamples were collected and oven-dried at 60 °C to constant weight for the determination of dry matter yield. Dried samples were ground in a cyclone mill to pass a 1-mm-screen prior to analyses. Elemental concentrations in plant tissues were determined using the dry ashing method, whereby 2 g of each sample was heated at 480 °C for 16 h, then digested using concentrated nitric acid (HNO₃) and 3 M hydrochloric acid (HCl). The digest was filtered through Whatman No. 40 filter paper, and the filtrate was brought to a volume of 25 mL with 0.1 M HCl. The concentration of Ca, Cu, Fe, K, Mg, Mn, P, Pb and Zn in each extract was analyzed using ICP-OES. For quality assurance, blanks and apple leaf standards from the National Institute of Standards and Technology (NIST), Gaithersburg, MD, were included for each analysis. The recovery percentage of the elements ranged from 100% to 77% for all the elements analyzed, except Fe, which was not listed with the apple leaf NIST. The recovery percentages for Ca, K, Cu, Mn, P, Mg, Pb and Zn were 100, 96, 93, 87, 85, 78, 77 and 77%, respectively.

2.5. Data Analyses

Analysis of variance was used to determine statistically significant differences among treatments using the PROC GLM, SAS version 9.4. [43]. Separation of means was performed using Duncan’s multiple range tests at p ≤ 0.05 [44].

3. Results and Discussion

3.1. Precipitation, Temperature and Forage Productivity

Monthly precipitation (mm) and temperature (°C) records of the three-year experimental region are shown in Figure 1. Precipitation at the site was lower in the first 2 years during the growing season with a range of 39–151 mm and 27–205 mm for 2011 and 2012. Precipitation then increased slightly in the third year to a range of 10–236 mm, constituting a total rainfall for the three years of 752 mm, 775 mm and 911 mm, respectively. The temperature ranged from −10 °C to about 40 °C, with a mean of 21 °C, 20 °C and 19 °C for the three years, respectively, as shown in Figure 1. The temperature was not limiting for the warm-season forage grasses. Furthermore, it is likely that precipitation was sufficient and did not result in drought for the forages based on the rainfall recorded for the period. This is because bermudagrass is known to survive in climates with an annual rainfall of 635–2540 mm [45], while switchgrass can tolerate conditions of drought to about 510 mm annually [46]. Similarly, stinging nettle needs 430–860 mm of precipitation annually [47] to reach its growth potential. Climatic factors, among others, affect crop growth, and, based on the research and conclusion for many warm-season forages crops, the presented levels are considered adequate precipitation [19,48,49] without the propensity to reduce yield. The annual precipitation was therefore supplemented with drip irrigation as needed.

3.2. Biomass Yield, Nutrient and Metal Concentrations

3.2.1. Year One Harvests/Cuttings

As shown in

Table 3. Nutrient and metal concentrations in switchgrass (SG), bermudagrass (BG) and stinging nettle (SN) cuttings for the first and second year.

Treatments	Cutting	Yield	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
		kg ha−1	mg kg−1
Year 1
SG	1	7221 ± 2912 a	2528 ± 201 b	15.90 ± 2.3 b	47.08 ± 11.4 b	18,116 ± 2637 b	1830 ± 358 c	51.63 ± 28 bc	2703 ± 180 b	2.00 ± −0.24 a	18.10 ± 3.6 c
BG	1	4807 ± 1490 b	2590 ± 134 b	13.07 ± 1.31 c	52.68 ± 8.8 b	22,103 ± 2746 b	2073 ± 236 b	74.03 ± 27 bc	2966 ± 438 b	1.32 ± −0.12 b	22.98 ± 2.3 b
SN	1	2425 ± 1000 c	26,169 ± 2508 a	18.71 ± 2.7 a	250.35 ± 108 a	36,312 ± 3367 a	6429 ± 1293 a	109 ± 75 ab	6599 ± 278 a	0.59 ± 0.23 c	29.23 ± 7.2 a
SG	2	8786 ± 1765 a	1943 ± 80 b	9.85± 1.5 b	51.68 ± 15.7 a	10,488 ± 2262 c	1531 ± 272 b	109 ± 50 b	1342 ± 93 c	1.58 ± 0.28 ab	9.08 ± 1.9 c
BG	2	7198 ± 635 ab	3318 ± 310 b	11.01 ± 0.7 a	28.10 ± 3.9 a	18,197 ± 2017 b	1650 ± 53 b	132 ± 64 ab	2170 ± 38 b	1.26 ± 0.19 b	18.18 ± 2.7 ab
SN	2	3364 ± 1149 c	16,846 ± 1334 a	7.65 ± 1.9 bc	60.05 ± 17.0 a	40,860 ± 3886 a	3482 ± 343 a	198 ± 122 a	4048 ± 560 a	0.53 ± 0.37 c	15.40 ± 2.8 ab
SG	3	2152 ± 1277 b	2478 ± 372 c	9.20 ± 1.26 b	33.98 ± 3.9 c	9673 ± 357 c	1561 ± 235 bc	120 ± 86 a	1459 ± 193 c	1.12 ± 0.27 a	10.15 ± 2.0 b
BG	3	8834 ± 1017 a	13,629 ± 1139 b	10.67 ± 0.74 ab	141.48 ± 8.5 b	20,861 ± 1831 b	1227 ± 92 c	61.9 ± 46 b	1962 ± 81 b	1.27 ± 0.07 a	19.03 ± 2.1 a
SN	3	851 ± 492 c	18,644 ± 2502 a	7.36 ± 0.69 c	472.15 ± 85 a	41,940 ± 3153 a	4184 ± 972 a	76.6 ± 44 b	4733 ± 208 a	0.43 ± 0.22 b	20.60 ± 2.0 a
BG	4	10,426 ± 2020 a	2576 ±329 b	11.89 ± 1.95 a	39.30 ± 15 b	13,813 ± 2268 b	1298 ± 263 b	139 ± 95 a	1785 ± 190 b	1.93 ± 0.46 a	14.70 ± 1.3 b
SN	4	4200 b ± 1174	22,584 ± 737 a	8.23 ± 0.72 b	416.73 ± 188 a	34,693 ± 3191 a	4799 ± 254 a	66 ± 24 b	5562 ± 303 a	0.33 ± 0.20 b	21.73 ± 2.0 a
BG	5	5703 ± 568 a	2805 ± 181 b	14.79 ± 2.3 a	38.75 ± 2.2 b	14,075 ± 907 b	1491 ± 270 b	175.5 ± 112 a	2012 ± 90 b	2.00 ± 0.30 a	17.68 ± 2.4 a
SN	5	1192 ± 600 b	29,036 ± 737 a	10.81 ± 0.48 ab	582.48 ± −282 a	41,775 ± 7719 a	5907 ± 366 a	39.7 ± 12 b	7463 ± 850 a	0.43 ± 0.18 b	18.83 ± 2.2 a
Year 2
SG	1	6777 ± 660 b	2224 ± 263 b	4.78 ± 0.58 b	72.3 ± 2.76 b	20,138 ± 2552 c	1619 ± 167 b	69.7 ± 26.8 ab	1567 ± 188 b	0.49 ± 0.09 ab	15.3 ± 3.5 b
BG	1	8057 ± 4366 a	2316 ± 144 b	6.25 ± 0.21 b	63.8 ± 3.34 c	22,057 ± 1904 c	1675 ± 144 b	40.3 ± 20.3 bc	1887 ± 144 b	0.25 ± 0.18 b	16.5 ± 0.9 b
SN	1	2925 ± 825 c	13,556 ± 1818 a	14.30 ± 1.63 a	89.0 ± 20.87 a	46,294 ± 4252 a	4066 ± 404 a	63.6 ± 10.6 c	5499 ± 320 a	0.20 ± 0.24 b	23.2 ± 1.3 a
SG	2	8575 ± 3919 a	1512 ± 140 bc	2.45 ± 0.17 b	58.0 ± 8.98 b	7940 ± 1360 c	952 ± 50 c	66.7± 34.8 a	840 ± 48 c	0.24 ± 0.16 b	7.3 ± 1.3 c
BG	2	7644 ± 4941 ab	2297 ± 480 b	4.20 ± 0.56 b	77.2 ± 14.77 a	15,779 ± 2763 b	1245 ± 220 b	47.1 ± 18.1 b	1540 ± 372 b	0.40 ± 0.07 a	14.8 ± 3.4 b
SN	2	2895 ± 731 c	12,603 ± 2323 a	11.47 ± 3.15 a	50.7 ± 7.79 b	44,981 ± 8877 a	3419 ± 599 a	62.8 ± 11.7 a	5217 ±638 a	0.15 ± 0.18 bc	19.2 ± 2.8 a
SG	3	6651 ± 757 a	1961 ± 365 b	3.20 ± 0.14 c	68.6 ± 33.76 a	9488 ± 938 b	1246 ± 220 b	43.8 ± 25.7 ab	1069 ± 188 c	0.40 ± 0.11 b	8.6 ± 2.1 c
BG	3	5259 ± 661 b	2911 ± 373 b	5.75 ± 1.16 b	72.2 ± 20.02 a	8865 ± 3533 b	1593 ± 342 b	63.3 ± 28.0 a	1523 ±286 b	0.42 ± 0.15 a	21.8 ± 3.4 a
SN	3	4620 ± 284 c	13,102 ± 8773 a	11.73 ± 2.16 a	71.2 ± 13.77 a	38,373 ± 13,612 a	4790 ± 3131 a	35.9 ± 22.4 c	5918 ± 3871 a	0.21 ± 0.03 c	16.3 ± 9.7 b
BG	4	1203 ± 529 a	3756 ± 709 b	7.62 ± 0.91 ab	99.9 ± 13.28 a	14,926 ± 4896 b	2355 ± 727 b	52.7 ± 24.9 b	2051 ± 419 b	0.60 ± 0.11 a	28.2 ± 3.3 a
SN	4	814 ± 131 b	14,808 ± 2198 a	9.52 ± 2.46 a	79.4 ± 16.41 ab	40,031 ± 3940 a	4837 ± 768 a	76.3 ± 15.2 a	5114 ± 459 a	0.53 ± 0.15 ab	20.0 ± 0.8 ab
BG	5	5016 ± 1876 a	3595 ± 3.5 b	7.40 ± 0.20 b	80.6 ± 6.35 ab	15,473 ± 2511 b	2383 ± 19 b	42.4 ± 16.1 a	3041 ± 72 b	0.50 ± 0.04 a	25.5 ± 0.9 a
SN	5	2566 ± 565 b	20,333 ± 5286 a	11.67 ± 1.72 a	96.9 ± 6.27 a	43,072 ± 2333 a	6202 ± 834 a	41.2 ± 9.5 a	6863 ± 315 a	0.01 ± 0.01 b	21.1 ± 1.9 ab

Means within columns and cutting with the same letters are not significant at p < 0.05 levels with ± Standard Deviation.

, which presents the dry biomass yield for the first cutting, yield was higher by 66% and 50% in switchgrass and bermudagrass, respectively, compared with stinging nettle, while switchgrass was 33% higher than bermudagrass. A similar trend was observed in the second cutting, but for the third cutting, bermudagrass, the warm-season forage, had a higher yield that was significantly different from switchgrass by 75% and stinging nettle by 90%. The bermudagrass as the warm-season forage had a significantly higher yield than stinging nettle by 59% and 79% for the fourth and fifth cuttings, respectively. The dry biomass yield reveals a higher percentage for the warm-season forages and grasses than the cool-season stinging nettle for the five cuttings. Studies have shown that forage yield is generally higher for the first cutting than other times of the year. In a 4-cut system, the first cutting usually makes up about 35–38% of the year’s total forage yield [50], particularly in a monoculture or with variants of one forage. Cutting height can also affect bermudagrass yield and quality. Results of a study on cutting height and frequency indicated that cutting height and frequency interact to affect the productivity, quality, and composition of bermudagrass-crabgrass mixtures [51]. However, with different forage types, like grasses and forbs, or monocots and dicots, and seasonal types of warm and cool seasons, there could be variations between cuttings.

The nutrient and metal concentrations of Ca, Cu, Fe, K, Mg, Mn, P, Pb and Zn in the plant samples shown in Table 3 for the first cutting reveal a significantly higher concentration of all the elements in the cool-season than in the warm-season forage samples, except for Pb. The differences were at percentages of 90%Ca, 30%Cu, 39%K, 68%Mg, 31%Mn, 55%P, and 21%Zn, with a high 300%Fe and a decrease in Pb being an exception. When the total uptake was considered, stinging nettle showed a greater nutrient uptake of macronutrients for the first cuttings of the two years over bermudagrass and switchgrass, with 103.20 kg ha⁻¹, 0.87 kg ha⁻¹, 27.49 kg ha⁻¹ and 32.08 kg ha⁻¹ for Ca, Fe, Mg, and P, respectively. An exception was seen for K, with 223.51 kg ha⁻¹ compared with 267.29 kg ha⁻¹ and 283.96 kg ha⁻¹ for switchgrass and bermudagrass, respectively. The uptake of micronutrients was higher for the warm-season grasses, particularly with switchgrass. A similar trend was observed for all the five cuttings, except for Cu, for which inconsistencies were present from the second cutting, and Mn, for which inconsistencies were observed during the fourth and fifth cuttings.

Table 4. Nutrient and metal concentrations averaged over forage cuttings for the first and second year.

Months	Cutting	Yield	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
		kg ha−1	mg kg−1
Year 1
May	1	4818 c	10,426 c	15.90 a	116.7 c	25,551 ab	3.44 ab	78.2 b	4089 b	1.30 a	23.43 a
July	2	6449 b	7369 d	9.52 c	46.6 c	23,181 b	2.22 c	146.2 a	2521 d	1.12 ab	14.22 c
August	3	3946 d	11,582 bc	9.07 c	215.9 b	24,158 b	2.32 c	86.2 b	2718 d	0.94 b	16.60 b
Sep	4	7313 a	12,580 b	10.08 c	228.0 ab	24,253 b	3.05 b	102.8 ab	3673 c	1.13 ab	18.21 b
Oct	5	3447 e	15,921 a	12.80 b	310.6 a	27,925 a	3.70 a	107.6 ab	4737 a	1.21 a	18.25 b
Year 2
May	1	5919 ab	6032 b	8.37 a	75.0 bc	29,496 a	2454 b	57.9 ab	2984 bc	0.31 b	18.3 b
July	2	6371 a	5471 b	6.04 b	62.0 c	22,900 bc	1872 b	58.9 ab	2532 c	0.26 b	13.8 c
August	3	4481 c	5992 b	6.89 b	70.7 c	18,909 c	2543 b	47.7 ab	2837 bc	0.34 b	15.5 bc
Sept	4	1009 e	9282 a	8.57 a	89.6 a	27,478 ab	3596 a	64.5 a	3583 b	0.56 a	24.1 a
Oct	5	3791 d	11,964 a	9.54 a	88.8 ab	29,273 a	4293 a	41.8 b	4952 a	0.25 a	23.3 a

Means within columns and cutting with the same letters are not significant at p < 0.05 levels.

shows the average yield over the five months of cuttings. Significant differences were observed between the warm months and the cool months for both types of forage crops being considered. Nutrient and metal concentrations, however, indicated that the cool-season forage had significantly higher levels relative to the warm-season forages, except for Pb, for which there was no difference.

When the average yield of individual forages was considered, as shown in

Table 5. Nutrient and metal concentrations averaged over forage crops for the first and second year.

Crops	Yield	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
	kg ha−1	mg kg−1
Year 1
Switchgrass	6053 b	2314 c	11.65 ab	44.2 b	12,759 c	1641 b	101 a	1835 c	1.57 a	12.4 c
Bermudagrass	7393 a	4984 b	12.29 a	60.1 b	17,810 b	1548 b	112 a	2179 b	1.56 a	18.5 b
Stinging Nettle	2496 c	22,654 a	10.56 b	356 a	39,116 a	4960 a	98 a	5681 a	0.46 b	21.2 a
Year 2
Switchgrass	7334 a	1899 b	3.44 c	66.3 b	12,522 b	1273 b	60.1 a	1159 c	0.38 a	10.4 b
Bermudagrass	5436 b	2975 b	6.24 b	78.6 a	15,420 b	1851 b	49.1 a	2008 b	0.43 a	21.3 a
Stinging Nettle	2147 c	14,880 a	11.70 a	77.4 a	42,550 a	4663 a	56.0 a	5722 a	0.22 b	19.9 a

Means within columns with the same letters are not significant at p < 0.05 levels.

, the warm-season forages recorded significantly higher yield than the cool-season crop. The cool-season forage had a significantly higher concentrations of all elements except for Pb. The relative nutrient and metal concentration followed the same trend for both the warm- and cool-season forages. For the first year, the order of nutrient and metal concentration was K > Ca > P > Mg > Fe > Mn > Cu > Zn > Pb for stinging nettle and K > Ca > P > Mg > Mn > Fe > Zn > Cu > Pb for bermudagrass and switchgrass, with an expected indication that the concentrations of the macro elements were more than that of the micro-elements. These results might be related to the growth and development of the specific forage.

3.2.2. Year Two Harvests/Cuttings

Table 3, for year 2, shows the dry biomass yield and corresponding nutrient contents as well as metal concentrations of the five cuttings during the second year. The biomass yield for the first cutting was significantly higher for the warm-season than for the cool-season forage. The second and third cuttings followed the same trend, and since the fourth and fifth cuttings were between just the bermudagrass and stinging nettle, the bermudagrass had a higher yield than the stinging nettle. Overall, bermudagrass recorded a consistently higher yield compared to stinging nettle and switchgrass for the first cutting, while switchgrass had the highest yield for the second and third cuttings.

The nutrient and metal concentrations from the forage samples shown in Table 3, year 2, appear to follow trends reported for the first year, with the cool-season forage having significantly higher concentrations of the elements than the warm-season grasses. The exceptions in the second year for metal and nutrient concentration levels were Cu and Mn, which were lower, as well as Pb, for which the cool-season forage had the lowest values.

The average of all the cuttings over the months for the second year presented in Table 4 shows that forages from the warm-season cuttings had significantly higher yields than the cool-season cuttings. Nutrient and metal concentrations were higher in the second year, apart from Mn, which was slightly lower in the cool-season forage.

Considering the average yield of specific forages for the second year, Table 5 indicates that switchgrass had a higher yield than bermudagrass, which had more biomass than stinging nettle. The nutrient and metal concentrations were higher for all forages, but their contents in stinging nettle were significantly higher than in switchgrass and the bermudagrass except for Pb. The contents were in the order of K > Ca > P > Mg > Fe > Mn > Cu > Zn > Pb for stinging nettle, K > Ca > Mg > P > Fe > Mn > Zn > Cu > Pb for switchgrass, and K > Ca > P > Mg > Fe> Mn > Zn > Cu > Pb for bermudagrass. In

Table 6. F statistics and significance levels for treatments and cuttings for the first and second year.

	Biomass	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
Source of Variation	F Values
Year 1
Treatment	9.06 ***	57.3 ***	7.09 ***	6.4 ***	5.44 ***	7.90 ***	2.65 *	19.6 ***	5.94 ***	2.45 *
Cuttings	12.6 ***	52.2 ***	29.5 ***	10.2 ***	2.87 *	17.5 ***	1.73	73.5 ***	3.18	14.9 ***
Crop	67.6 ***	1138 ***	4.79 *	47.8 ***	323 ***	259 ***	0.21	627 ***	115 ***	34.8 ***
Treatment * cutting * crop	6.05 ***	236 ***	14.2 ***	14.9 ***	57.6 ***	52.9 ***	1.94	139 ***	23.2 ***	12.0 ***
Year 2
Treatment	1.21	1.09	3.54 **	2.8 *	1.0	0.78	1.61	0.30	3.68 **	5.34 ***
Cutting	8.26 ***	7.76 ***	8.83 ***	5.8 ***	8.2 ***	9.3 ***	1.62	6.61 ***	7.68 ***	16.16 ***
Crop	16.1 ***	92.2 ***	121 ***	0.36	171 ***	51.0 ***	1.03	14.7 ***	15.7 ***	22.5 ***
Treatment * cutting * crop	20.0 ***	18.5 ***	24.7 ***	3.4 **	31.7 ***	12.0 ***	1.5	14.7 ***	7.0 ***	11.8 ***

*** Significant at the 0.0001 probability level; ** significant at the 0.001 probability level; * significant at the 0.05 probability level.

, the sources of variations, interactions and the significant levels of the variables are shown. Almost all the treatment effects were significant, as were the interactions. Mn did not show any significance except for the treatment for the first year, while the biomass, K, Ca, and P indicated a non-significance for treatment in the second year. Pb also demonstrated non-significance in the first year. The elements that were not significant did not show any level.

From the results of the analyses, the biomass yield depended on the maturity of the plant and when it was harvested. The biomass yield of these three forages exhibited both specific and general attributes because changes in the foliage appearance were observed over time as plants grew and matured. The stinging nettle exhibited a growth pattern where the foliage increased as the crop matured and declined substantially as the stem matured. This is because of the high proportion of the leaves to the stem in young plants. As the plant grows and matures, the proportion of the leave changes and the stem lengthens and becomes fibrous, increasing its total proportion in the biomass [52,53], which might reduce the nutrient and metal concentrations present in the leaves. However, the bermudagrass and switchgrass did not exhibit such growth patterns, so the total biomass yield decreased due to seasonal changes. In a study to determine accurate and precise measurements of plant maturity, switchgrass exhibited a sigmoidal development pattern, with phases that were related closely to temperature and rainfall patterns [54]. Therefore, the plant’s growth rate is closely tied to climate, but the timing of reproductive development is linked to photoperiod [55]. Much like the growth stages and biomass yield, the nutrient and elemental concentrations of forage crops also depend on the maturity and time of harvest. In a study to determine the effect of different harvesting times on the chemical composition of stinging nettle, it was found that stinging nettle harvested in April had the highest levels of P, K, Fe, Zn, samples collected in July contained the highest levels of Mn, and samples collected in September had the highest amounts of Ca and Mg [31]. These findings agree with the observations made in this study. Overall, at all stages of growth, the stinging nettle had higher levels of all the elements measured compared to switchgrass and bermudagrass, suggesting that it has great potential for use in nutrient removal or cycling as well as environmental sustainability. However, if the yield is low, the plants may not take up much of the metals on a mass basis.

Stinging nettle has been shown to be very nutritious. Stinging nettle hay contains 21 to 23% crude protein, 3 to 5% crude fats, 35 to 39% non-nitrogen extracts, 9 to 21% crude fiber, and 19 to 29% ash. Amino acids in dehydrated stinging nettle meal are nutritionally superior to those of dehydrated alfalfa (Medicago sativa L.) meal [56]. With good management and favorable weather, bermudagrass can produce crude protein levels ranging from 8–16% and total digestible nutrient content of 55% or higher. The nutrient of switchgrass is said to be as high as 16–17% crude protein if harvested correctly. In a study, the crude protein content of switchgrass ranged from a high of 17% in the early growing season to a low of 4% at forage maturity [57], while total digestible nutrients ranged from 63% in the early season to 55% at forage maturity.

Forage quality is associated with nutrients, protein, digestibility, fiber, mineral, and vitamins, among other factors, and it generally depends on the time of harvest and age of the plant or stage of maturity. This is because forages possess a mixture of chemical, physical, and structural characteristics that determine the quality of that pasture. Forage quality also varies, not only among different forage species but also among varieties of specific forage species. Furthermore, the forage quality of a specific variety can also vary due to field and management conditions.

3.3. Nutrient and Metal Concentrations in the Soil after Harvests

Results for the first-year analysis of composite soil samples after harvests are shown in

Table 7. Soil pH, EC, nutrient and metals from forage plots for the first and second year.

Crop	pH	EC	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
		µS cm−1	mg kg−1
Year 1
Bermudagrass	6.06 ab	72.3 a	227 b	1.84 a	347 ab	191 a	46.2 b	21.2 a	185 b	10.4 a	2.39 a
Switchgrass	6.22 a	90.1 a	301 a	1.86 a	383 a	202 a	62.2 a	24.0 a	254 a	11.7 a	2.92 a
Stinging Nettle	5.88 b	78.2 a	211 b	1.51 a	318 b	174 a	40.7 b	19.5 a	172 b	11.2 a	2.01 a
Year 2
Bermudagrass	5.81 ab	101 b	322 a	1.11 a	149 a	241 a	51.9 a	19.1 a	160 a	5.50 a	2.35 a
Switchgrass	5.53 b	118 ab	327 a	1.12 a	150 a	212 a	50.7 a	13.5 a	125 a	5.24 a	2.10 a
Stinging Nettle	5.91 a	193 a	295 a	1.20 a	129 a	210 a	45.8 a	13.6 a	122 a	6.30 a	1.98 a

Means within columns with the same letters are not significant at p < 0.05 levels.

. Soil pH and EC were not significantly different, with respective readings being 6.06, 6.22 and 5.88 in bermudagrass, switchgrass and stinging nettle plots for pH, and 72.3 µS cm⁻¹, 90.1 µS cm⁻¹, and 78.2 µS cm⁻¹ for EC, respectively. Similarly, no significant differences in the nutrient and metal contents were observed among treatment plots, but Ca, Mg, and P contents were found to be slightly higher in switchgrass plots.

Table 8. Soil pH, EC, nutrient and metal concentrations on soil depth from switchgrass (SG), bermudagrass (BG) and stinging nettle (SN) plots for the first and second year.

Crops	Depth	pH	EC	Ca	Cu	Fe	K	Mg	Mn	P	Pb	Zn
	cm		µS cm−1	mg kg−1
Year 1
SG	0–15	6.47 ± 0.28 a	100 ± 26 a	357 ± 108 a	2.20 ± 0.61 a	381 ± 51 a	265 ± 69 a	74 ± 21 a	27.7 ± 8.6 a	304 ± 43 a	12.0 ± 2.7 a	4.16 ± 1.85 a
	15–30	5.97 ± 0.18 b	83 ± 15 b	245 ± 54 b	1.52 ± 0.35 a	386 ± 58 a	139 ± 52 a	50 ± 12 b	20.2 ± 3.7 ab	203 ± 43 b	11.4 ± 2.7 a	1.69 ± 0.53 b
BG	0–15	6.30 ± 0.48 a	85 ± 64 a	271 ± 80 a	2.11 ± 1.12 a	364 ± 63 a	231 ± 151 a	56 ± 26 a	22.7 ± 3.6 a	235 ± 98 a	11.0 ± 1.4 a	3.20 ± 2.60 a
	15–30	5.82 ± 0.36 b	55 ± 31 b	183 ± 52 b	1.57 ± 0.43 a	330 ± 86 a	152 ± 79 a	36 ± 16 b	19.6 ± 5.2 ab	134 ± 47 b	9.8 ± 1.4 a	1.61 ± 0.52 b
SN	0–15	6.13 ± 0.24 a	83 ± 26 a	242 ± 73 a	1.66 ± 0.55 a	311 ± 69 a	208 ± 65 a	48 ± 11 a	18.6 ± 4.3 a	201 ± 27 a	10.3 ± 1.0 a	2.48 ± 0.87 a
	15–30	5.62 ± 0.57 b	77 ± 36 a	179 ± 66 b	1.35 ± 0.22 a	324 ± 104 a	140 ± 53 b	34 ± 11 ab	20.4 ± 6.2 ab	144 ± 33 b	12.3 ± 3.2 a	1.54 ± 0.35 ab
Year 2
SG	0–15	5.67 ± 0.41 a	75 ± 26 a	304 ± 84 a	1.47 ± 0.66 a	154 ± 26 a	225 ± 47 a	48 ± 18 a	13.9 ± 7.4 a	154 ± 62 a	5.6 ± 2.0 a	2.47 ± 1.70 a
	15–30	5.44 ± 0.15 a	80 ± 48 a	350 ± 84 a	0.93 ± 0.12 a	147 ± 48 a	198 ± 5.5 ab	54 ± 21 a	13.0 ± 1.6 a	93 ± 72 ab	4.8 ± 0.6 a	1.72 ± 0.68 a
BG	0–15	5.59 ± 0.53 a	75 ± 40 a	367 ± 84 a	1.34 ± 0.49 a	155 ± 39 a	273 ± 107 a	65 ± 25 a	13.5 ± 4.4 a	176 ± 90 a	5.2 ± 1.3 a	3.25 ± 3.00 a
	15–30	5.28 ± 0.35 a	45 ± 17 a	277 ± 59 a	0.98 ± 0.33 a	149 ± 45 a	209 ± 18 ab	39 ± 8.0 a	24.6 ± 27.0 a	144 ± 73 a	5.8 ± 1.2 a	1.44 ± 0.60 b
SN	0–15	5.75 ± 0.41 a	67 ± 9 a	309 ± 75 a	1.44 ± 0.41 a	126 ± 13 a	219 ± 25 a	51 ± 12 a	12.1 ± 1.3 a	150 ± 27 a	5.9 ± 1.6 a	2.79 ± 0.70 a
	15–30	5.39 ± 0.74 a	57 ± 13 a	281 ± 107 a	1.10 ± 0.41 a	133 ± 35 a	202 ± 12 ab	41 ± 14 a	15.1 ± 9.1 a	94 ± 39 ab	6.7 ± 3.4 a	1.16 ± 0.27 a

Means within columns with the same letters are not significant at p < 0.05 levels with ± standard deviation.

shows the pH and EC values of the soil, as well as the nutrient and metal concentrations separated between the topsoil and subsoil for each forage crop. For the first year, the topsoil had a slightly higher pH and EC than the subsoil, but although the pH difference was significant for all forages, the EC showed significant differences between the top and subsoil only for switchgrass and bermudagrass. The same trend was found for nutrient and metal concentrations, with the topsoil being slightly higher than the subsoil for all elements except for Fe, Mn and Pb. While the analyses of the first year (Table 8) showed a slightly significant difference between the top and subsoil for the forage crops, except Cu and Fe, the analyses of the second year also indicated a slight increase in the topsoil but did not show any significant differences among all the forage crops. This difference between the topsoil and subsoil could be due to the stability of the roots and its underlying soil interactions in the second year of study as well as the seasonal poultry litter and inorganic fertilizer applications of the second year in addition to the split applications, which provided an even distribution for the nutrients and metals. Additionally, nutrients strongly cycled by plants, such as P and K, could be more concentrated in the topsoil (upper 20 cm) than nutrients that are usually less limiting for plants, indicating that the vertical distribution of a limiting nutrient would be shallower as the nutrient becomes more scarce [58].

Similar trends to the first year were observed for the second year as presented in Table 7, but pH values were slightly lower, at 5.81, 5.53 and 5.91, respectively, for bermudagrass, switchgrass and stinging nettle plots, while EC values were slightly higher. However, the nutrient and metal concentrations were lower for all the elements except Ca and K, as well as Mg in the switchgrass treatment (Table 7). Importantly, the heavy metal levels were all within the allowable limits (US EPA 2021, 2000) [59,60].

Based on the results of the topsoil and subsoil discussed for pH and EC in Table 8, as well as the much lower nutrient and metal concentrations except for Ca and K, analyses of the interactions done between each soil from the forage crop plot and the soil depth were also not significant for the two seasons (table not shown). Overall, the sources of variations and interactions of the variables for the soil analyses discussed for the first and second years did not show any significance and were not shown.

Based on the two seasons, the slight decrease in pH over time was not considered large enough to affect availability. This is because soil pH plays a critical role in the availability of nutrients to plants or uptake from the soil [61]. Conversely, the EC increased by 28%, 24% and 59% for bermudagrass, switchgrass and stinging nettle plots, respectively. The EC also decreased significantly with increasing depth for all treatments over the seasons. Though it is an important indicator of soil health, affecting crop yield and influencing key soil processes, soil EC is highly variable. The EC is not consistent enough to accurately predict the behavior of the nutrients and elements [62,63]. This is because, in a humid environment, EC is temporal, depends on past management of the field and is typically elevated in the spring/summer shortly after application of soluble fertilizer and reduced in late winter/early spring following winter leaching. The soil analyses also indicated that the recovery of the nutrients, particularly P, was lower in bermudagrass plots than switchgrass and stinging nettle plots. The total P uptake for bermudagrass for the first cuttings of the 2 years was shown to be 29.46 kg ha⁻¹, while switchgrass had 30.14 kg ha⁻¹, and uptake for stinging nettle was 32.08 kg ha⁻¹. Similar findings were reported in a previous study on bermudagrass [64]. This might be associated with the influence of the properties of the soil, such as soil texture, which is a sandy loam in this study, so adsorption was not anticipated. Other studies on bermudagrass fertilized with broiler litter indicated that the buildup of soil P is influenced by soil physical and chemical properties. [19,64].

Generally, the analyses of the various components in this study could have a direct relationship with soil health. Sustainable agronomic production depends on healthy soil, which is affected by the physical, chemical, and biological properties of the soil and their combined components, including soil structure, pH and soil biology or organic matter. The concentrations of heavy metals in the soil have a tendency of altering these properties, and when they are out of balance, the soil health deteriorates. Soil biology and micro-organisms play key roles in soil fertility and soil health through organic matter dynamics and nutrient cycling [65,66]. When the soil is exposed to stressful situations, like extreme temperatures, drastic changes in pH or toxins from anthropogenic activities and mining, these organisms could be affected. Microbial viability in the soil decreases with increasing levels of heavy metal content from inorganic fertilization and some pesticides applied on agricultural lands [67,68], thus leading to substantial losses in crop productivity. However, for many of these heavy metals, uptake is influenced by soil characteristics like pH, cation exchange capacity (CEC), soil texture and organic carbon content. Moreover, metal uptake is plant-species dependent and is influenced by interactions of the elements in the soil solution [69,70]. A study of grasses and stinging nettle showed that the most important soil property that influenced metal concentrations in stinging nettle was the clay content, while organic matter (OM) and pH affected the concentrations for grasses [71]. Another study showed that the distribution of heavy metals in the alfalfa cultivated in three areas did not coincide with the average of these metals in the soils [72], suggesting a regional difference in the effect of heavy metals on the plants and their potential uptake. Similarly, research on alfalfa yield and soil properties which utilized flue gas desulfurization gypsum, biochar, bed ash, broiler litter ash and their combinations concluded that the amendments could be effective in reducing the accumulation and potential bioavailability as well as the harmful effects of heavy metals [26], thus preserving the health of the soil.

Experimental results have shown that the intensity of heavy metal accumulation in plants depends on the type of the soil, the species of plants, the physicochemical properties of heavy metals and their content in the soil [73]. This is because the toxicity and tolerance of metals were found to vary with crops and growth stages. While a high concentration of heavy metals could be detrimental to soil health, proper agronomic practices and management of farmlands, as well as the kinds of crops cultivated, will reduce the impact of heavy metals on the soil [74]. Seeking to improve soil health, increase forage productivity, improve forage nutritive value, and net farm profit in a hay production system, a study on soil microbial biomass under different seeding proportions and cropping systems of two forage grasses and one legume concluded that the total aboveground plant biomass was higher in a 50–50% mixture of grass and alfalfa than monoculture alfalfa and monoculture grass and can be used for improving soil health and forage productivity [75]. Similarly, intercropping of pasture ryegrass and forage alfalfa was found to increase the resistance of plants to heavy metals through the reduction of plant oxidative damage and increased antioxidant activity, which can increase biomass, reducing the absorption of Pb in forage plants [76].

The aforementioned scenarios and cropping systems confirm some of the strategies of our study towards a recommendation of double-cropping bermudagrass with a cool-season annual forage which has the potential to remediate high P soil [64]. This points to the potential of our approach for the recovery and recycling of nutrients following a poultry litter/fertilizer application on the soil.

4. Conclusions

This two-year field study compared the agronomic performance of two warm-season forage grasses (bermudagrass and switchgrass) with a potential cool-season forage crop (stinging nettle). While bermudagrass and switchgrass provided a significantly higher biomass yield than stinging nettle, stinging nettle tissue samples contained a higher heavy metal and nutrient concentration than bermudagrass and switchgrass, including a greater uptake of macronutrients over the warm season grasses. Nutrient and metal concentrations were lowest in bermudagrass, and there was no indication of any nutrient overload stemming from the application of poultry litter in the experimental field. Though a high concentration of heavy metals could be detrimental to soil health, employing proper management and cropping systems will sustain the health of the soil. Since the heavy metals and nutrient concentrations ebbed in the grasses and increased in the forbs, we propose mixed cropping of cool- and warm-season forages to mitigate the potential loss of P from surface-applied poultry litter and similar organic materials.

Furthermore, understanding the normal regional levels from agencies like the Environmental Protection Agency (US EPA) or World Health Organization (WHO) and their allowable limits for health concerns, as well as monitoring these levels or limits, would enable producers to maintain soil health and benefit from using manures or inorganic fertilizers for agricultural and environmental sustainability. The general result does not indicate that the concentration of heavy metals would be a problem in the field used for this study nor the crops as forages. Our results also suggest that stinging nettle could be a valuable forage crop comparable to traditional forage crops and could be a viable candidate for use in nutrients and bioremediation of high nutrients in the soil.