Natural Adsorbents as Therapeutic Candidates Against Necrotic Enteritis in Poultry: A Conceptual Review

Abstract

Necrotic enteritis (NE), primarily associated with Clostridium perfringens, remains a major enteric disease in poultry production, particularly under reduced-antibiotic and antibiotic-free systems. Natural adsorbents, including biochar, clay minerals, and graphite-based materials, have attracted interest because of their capacity to interact with toxins, microbial metabolites, pathogens, and the intestinal environment. This conceptual review synthesizes current evidence on the physicochemical and biological properties of these materials and evaluates their potential relevance to NE mitigation. Biochar and clay minerals have stronger poultry-related evidence, particularly for mycotoxin adsorption, gut microbial modulation, and performance responses, whereas graphite remains an emerging candidate supported mainly by in vitro, non-poultry, and graphite-derivative literature. Across all three adsorbent classes, direct evidence for NetB-specific adsorption is currently absent, making this a central research gap rather than an established mechanism. Therefore, this review proposes a structured evaluation pipeline integrating material characterization, in vitro toxin-binding and epithelial response assays, and in vivo poultry NE outcomes such as lesion scores, CP burden, barrier integrity, inflammation, oxidative stress, microbiome shifts, and growth performance. Overall, natural adsorbents should be viewed as promising but incompletely validated candidates requiring standardized, NE-specific testing before therapeutic or commercial application.

1. Introduction

Poultry farming is integral to global animal agriculture, supplying a vital source of economical protein for an expanding human population [1]. The industry makes up more than a third of the world’s meat production, and it is expected to grow to around half of all meat production, reaching 182 million tons in the next ten years [2]. Notwithstanding, the industry faces ongoing health challenges that jeopardize productivity and animal welfare, with necrotic enteritis (NE) remaining one of the most economically important poultry diseases and widely estimated to cost the global poultry sector approximately 2–6 billion U.S. dollars annually [3]. Beyond the economic burden, NE undermines gut integrity by inducing mucosal necrosis, barrier failure, malabsorption, diarrhea, anorexia, and dysbiosis, severely affecting poultry health and welfare [4]. NE is a re-emerging enteric disease in chicken caused by Clostridium perfringens (CP), especially in production systems where antibiotic growth promoters (AGPs) are restricted or banned [4,5]. Country-specific regulations such as the European Union (under Regulation EC No. 1831/2003 in 2006) and the United States Food and Drug Administration have placed restrictions on the use of AGP. The withdrawal of AGPs has left a therapeutic gap, exposing producers to additional economic losses and welfare issues from NE outbreaks. This expanding dilemma highlights the critical need for effective, antibiotic-free ways to manage NE, while maintaining poultry health and productivity.

Natural adsorbents (NAs) such as biochar [6,7], clay minerals [8], and graphite-based or graphene-derived materials [9] (Figure 1) have been employed in livestock production as mycotoxin binders to reduce the impact of aflatoxin, ochratoxin, and fumonisin contamination in feed. Beyond mycotoxin sequestration, these adsorbents may also have broader gut-health-related roles. Biochar has been reported to stabilize gut microbiota diversity [10] and clays to exhibit antimicrobial and immunomodulatory activities [11,12], while graphite derivatives such as graphene oxide demonstrate antibacterial and anti-biofilm properties [13]. Given these distinct mechanisms, the interaction between natural adsorbents and necrotic enteritis-associated protein toxins is expected to differ substantially, making it important to examine how these materials may function within the context of NE pathogenesis and toxin activity.

Despite these encouraging findings, the direct relevance of natural adsorbents to NE remains unclear. Most available studies have focused on general growth performance, mycotoxin mitigation, or broad gut-health outcomes rather than NE-specific mechanisms. In particular, limited attention has been given to how these materials may influence toxin adsorption, oxidative stress modulation, intestinal barrier protection, CP load, or NE-associated lesion severity. Therefore, a critical assessment is needed to determine which proposed mechanisms are supported by direct poultry evidence, which are based on indirect or non-poultry evidence, and which remain hypothetical. Accordingly, this review uses a conceptual framework to synthesize current mechanistic knowledge, evaluate translational relevance, and identify key gaps that must be addressed before natural adsorbents can be advanced as validated NE-control strategies.

Accordingly, this conceptual review addresses four guiding questions. First, which physicochemical properties of biochar, clay minerals, and graphite-based materials are most relevant to toxin adsorption, antimicrobial activity, oxidative stress modulation, and intestinal barrier protection in poultry? Second, what direct and indirect evidence supports the use of these adsorbents for NE mitigation, and where does the evidence remain extrapolative? Third, what gaps currently limit translation, particularly regarding NetB-specific adsorption, dose-response relationships, nutrient interactions, material standardization, and in vivo poultry validation? Fourth, how can a staged screening framework be used to prioritize adsorbents for future NE-focused testing? By addressing these questions, this review provides a critical conceptual framework rather than a definitive efficacy claim for natural adsorbents as NE therapeutics.

2. Literature Search Strategy and Evidence Synthesis

This article was developed as a structured conceptual review rather than a systematic review or meta-analysis. The purpose was to synthesize mechanistic and translational evidence on natural adsorbents as candidate non-antibiotic strategies for NE mitigation in poultry, with emphasis on biochar, clay minerals, graphite, and graphite-derived materials. Literature searches were conducted on PubMed, Web of Science, Scopus, Google Scholar, and relevant agricultural databases, including CAB Abstracts and AGRICOLA, where accessible. Searches covered publications from database inception to 15 April 2026. Because this review was conceptual rather than systematic, database hit counts were not used as exclusionary criteria; instead, source selection was based on relevance to the review objectives, primary-study status, mechanistic value, and translational relevance to poultry gastrointestinal health or NE pathogenesis. A total of 186 references were ultimately included in the manuscript after screening for relevance and removal of sources that were weakly aligned with the final scope.

Search terms were combined using Boolean operators and included: “necrotic enteritis” OR “Clostridium perfringens” OR “NetB” OR “poultry gut health” OR “broiler intestinal barrier” AND “biochar” OR “charcoal” OR “activated carbon” OR “clay mineral” OR “bentonite” OR “montmorillonite” OR “kaolinite” OR “zeolite” OR “graphite” OR “graphene oxide” OR “graphitic materials” AND “toxin adsorption” OR “mycotoxin binding” OR “antibacterial activity” OR “oxidative stress” OR “microbiota” OR “intestinal permeability” OR “short-chain fatty acids.” Additional primary studies were identified through backward and forward citation tracking of included articles.

Peer-reviewed primary research articles were prioritized. Studies were included if they addressed at least one of the following: physicochemical properties relevant to adsorption or gastrointestinal application; poultry or livestock studies involving biochar, clay minerals, graphite, graphene oxide, or related carbonaceous materials; in vitro or in vivo evidence for toxin adsorption, antimicrobial activity, oxidative stress modulation, epithelial protection, microbiome modulation, or nutrient interaction; and NE-relevant outcomes such as CP burden, intestinal lesions, epithelial integrity, inflammatory responses, oxidative stress markers, or growth performance. Studies were excluded if they were unrelated to animal gastrointestinal health, lacked relevance to adsorption or host-pathogen interaction, or could not be linked mechanistically to NE pathogenesis or poultry production.

As the evidence spans poultry science, material science, microbiology, toxicology, and nanomedicine, studies were synthesized narratively and categorized by translational relevance as: direct poultry NE evidence, poultry non-NE evidence, non-poultry in vivo evidence, in vitro mechanistic evidence, physicochemical/adsorption evidence, or hypothetical extrapolation. This classification was used to distinguish established findings from plausible but untested mechanisms and to identify research gaps requiring NE-specific validation.

3. Pathophysiology of NE in Poultry

The pathogenesis of an infectious disease elucidates the mechanisms of disease manifestation, necessitating a comprehensive understanding of the host-pathogen interaction that precipitates the condition. The principal etiological agent of NE is CP, a Gram-positive, rod-shaped, anaerobic, encapsulated bacterium that resides commensally in the gut of healthy poultry [14]. Although CP is a typical resident of the digestive tract and environment, its pathogenicity is closely associated with its capacity to generate a diverse range of strong toxins [15]. CP can be detected in the guts of healthy birds and in poultry production environments, including excreta, litter, and hatchery-associated samples [16,17]. Nevertheless, its presence alone does not signify disease [17]. The pathogenic potential of this bacterium manifests when virulence factors are expressed, notably the NetB (Necrotic enteritis B) toxin [18] (Figure 2).

The pathogenesis of NE is intricate, encompassing both CP proliferation and host susceptibility. High-protein diets, coccidial infection, dysbiosis of intestinal microbiota, and diminished mucosal immunity are all contributing factors to bacterial overgrowth and toxin production [19]. Figure 2 illustrates that the disease induces mucosal necrosis, compromises epithelial integrity, and elicits excessive inflammatory responses in poultry, leading to suboptimal performance, elevated morbidity, and significant mortality [20]. Subclinical NE diminishes efficiency and growth, whereas the acute clinical variant can lead to mortality rates of up to 50% [19].

Central to NE pathogenesis is the interplay between CP toxins and host oxidative stress responses. Excess reactive oxygen species (ROS) generated by immune activation and bacterial toxins promote epithelial death via lipid peroxidation, mitochondrial dysfunction, and DNA damage, triggering apoptosis [21], as shown in Figure 2. Alpha-toxin, a zinc-dependent phospholipase C, disrupts membrane phospholipids, causing cytotoxicity and inflammation, though it is not considered the principal virulence factor, since α-toxin-deficient strains can still cause NE [22]. The NetB toxin, a β-pore-forming toxin, is regarded as pivotal, compromising enterocyte membranes and leading to cell lysis, villus blunting, mucosal necrosis, and pseudo-membrane formation, with damage starting at the basement membrane [18,23]. TpeL toxin modifies Ras proteins and disrupts signaling pathways, promoting apoptosis, although its role is debated since it is found only in a subset of NetB-positive strains [24].

4. Existing Strategies for Controlling NE

Despite decades of research, control of NE remains challenging. Historically, the use of AGPs effectively suppressed CP proliferation [25], but the global movement to restrict or ban AGPs due to concerns over antimicrobial resistance has reignited NE outbreaks [26]. Vaccination strategies show promise but are not yet commercially reliable, as antigenic diversity and inconsistent protection limit their practical utility [27]. Alternatives such as probiotics [28], prebiotics [29], organic acids [30], and phytogenics [31] contribute to gut health but often yield inconsistent results under field conditions. Also, the differences in gut microbiota composition, feed formulations, pathogen loads, and rearing conditions have caused contradictory results across trials [32,33]. This situation necessitates a comprehensive re-evaluation of existing methods, underscoring the urgent requirement for integrated approaches that reduce reliance on traditional antibiotics.

5. Natural Adsorbents-Relevance in Poultry

5.1. Biochar

Biochar is produced through incomplete pyrolysis (heating to ~550 °C under oxygen-limited conditions) of organic materials like wood, straw, manure, crop residues, and leaves [34,35]. Biochar, contingent upon feed material and pyrolysis conditions, comprises 40–80% carbon, 0.1–0.8% nitrogen, 1–2% potassium, and 5–6% calcium on a weight/dry weight basis, with a cation exchange capacity (CEC) ranging from 25 to 150 cmol+/kg [34,36]. Biochar has historically been used in soil remediation [37], and it has recently been investigated as a feed supplement in animal husbandry due to its adsorptive qualities, ability to bind toxins, and potential to alter gut microbes [38,39]. The integration of biochar into poultry feeds has demonstrated promising outcomes, including a decrease in pathogenic bacteria, such as Campylobacter jejuni and Gallibacterium anatis, due to their adsorption capabilities [10,40]. The porous structure may facilitate the binding of selected metabolites in the gastrointestinal tract, but its relevance to NE requires direct validation in standardized challenge models [41].

Furthermore, poultry supplemented with biochar have demonstrated a wide range of physiological benefits, including improved immunological responses, lower plasma triglycerides, and elevated lipid profiles [42,43]. These findings suggest that biochar may support gut health through adsorptive and microbiota-associated mechanisms, but direct evidence for NE prevention remains limited. The type of feedstock and the pyrolysis conditions affect the composition and efficiency of biochar, which is made from a variety of biomass sources like bamboo, woody green waste, and rice husks [10,44,45]. As a result of its adsorptive properties and compatibility with sustainable farming approaches, biochar remains a plausible candidate for further NE-focused evaluation.

Aside from pathogen reduction, biochar has shown adsorption capacity for selected mycotoxins, including aflatoxins, ochratoxin A, and zearalenone [6,7]. This is relevant to poultry gut health because mycotoxin exposure can compromise intestinal integrity and immune function, potentially increasing susceptibility to secondary enteric disorders. Evidence from intestinal epithelial, animal, and poultry challenge models further supports that different mycotoxins can alter gastrointestinal function, microbial balance, and host responses relevant to enteric disease susceptibility [46,47,48]. Teleb et al. [49] reported that 0.5% kaolin or activated charcoal reduced the adverse effects of low-level aflatoxin exposure in broilers, improving growth-related outcomes and reducing mortality. Considering that activated charcoal is not identical to biochar, this study should be interpreted as indirect evidence that adsorbent materials could mitigate toxin exposure, rather than as direct evidence for biochar efficacy. The direct use of biochar for NE remains poorly researched; therefore, NE-specific challenge studies are still required before biochar can be considered a validated preventive strategy.

Table 1. Reported applications of biochar and charcoal-based materials in poultry and other animal systems.

Biochar Effects	Condition Targeted	Animal Type	Reference(s)
Increases HDL, lowers LDL; improves omega-3/6 ratio	Enhances lipid profile, immune support	Poultry (Ducks)	[43]
Adsorbs gut pathogens; reduces Campylobacter jejuni and Gallibacterium anatis	Bacterial infections in the gut	Poultry	[10,40]
Reduces plasma blood triglycerides; improves immunity	Cardiovascular/metabolic health	Poultry	[42]
Removes uremic toxins (e.g., urea, creatinine)	Toxin accumulation/detoxification	Poultry and cattle	[50]
Adsorbs toxic substances; detoxifies biogenic amines	Gastrointestinal health	Poultry	[51]
Reduces enteric methane emissions	Methane mitigation	Cattle	[44,52]
Increases protein efficiency ratio (PER), specific growth rate (SGR)	Growth performance, nutritional health	Fish	[53,54]

summarizes reported applications of biochar and charcoal-based materials in poultry and other animal systems.

5.1.1. Physicochemical Properties of Biochar

The functional activity of biochar depends strongly on feedstock type and pyrolysis conditions. Higher pyrolysis temperatures generally increase aromatic carbon content, structural stability, and pore development, whereas lower temperatures preserve more oxygen-containing functional groups that contribute to surface charge and cation exchange behavior [55,56]. For example, aflatoxin B1 has been shown in pig and mouse models to impair intestinal barrier integrity by reducing tight-junction proteins and promoting epithelial apoptosis [57]. Although this evidence is not NE-specific and is not derived from broilers, it supports the rationale that toxin sequestration may indirectly protect intestinal barrier function under toxin-associated stress. These properties are relevant to gastrointestinal applications because surface area, porosity, pH, ash content, volatile matter, and functional groups influence the capacity of biochar to absorb organic compounds, microbial metabolites, and selected mycotoxins [58,59,60,61,62,63,64].

In poultry, biochar-related benefits have been reported for aflatoxin-challenged broilers, gut microbial modulation, and reduction in selected poultry pathogens [6,10,40]. However, these findings should be interpreted with caution because the studies differ in disease context, feedstock type, inclusion level, pyrolysis condition, bird age, and outcome measures. For example, aflatoxin studies mainly support toxin-sequestration potential, whereas pathogen-reduction studies support possible microbiome or antimicrobial effects; neither study type directly establishes efficacy against CP-induced NE or NetB-associated injury. A cautious connecting hypothesis is that biochar may be most useful when its pore structure, surface area, pH, and functional groups align with the target luminal toxin, microbial metabolite, or pathogen-related stressor, but this requires validation under standardized poultry NE challenge conditions. These differences emphasize the need to characterize biochar materials before comparing biological efficacy across studies (

Table 2. Physicochemical Properties of Biochar and Their Functional Relevance.

Property	Functional Relevance to NE Control	Influencing Factor	Observed Range/Value	Reference
Surface Area	High surface area may enhance adsorption of selected luminal compounds; NetB-specific binding remains untested	Pyrolysis temp., feedstock	200–300 m2/g	[60]
Porosity	Supports microbial niche, gut integrity, and nutrient interaction	Biomass anatomy, pyrolysis conditions	Pore network and porosity vary with biomass structure and pyrolysis conditions	[65]
pH	Buffers gut acidity; can inhibit CP growth	Feedstock ash, pyrolysis temp	6.48–11.32	[66]
CEC	Facilitates nutrient retention; binds inflammatory ions and toxins	Functional groups, oxidation	26.7–222.4 mmolc kg−1	[67]
Ash Content	Source of minerals; high ash may clog pores	Feedstock (esp. manure vs. wood)	24.0–68.4%	[68]
Carbon Content	High aromatic carbon stabilizes the structure and limits microbial degradation	Pyrolysis temp., feedstock lignin	24.7–90.9%	[61]
Volatile Matter	Lower VM = more stable and safer; high VM may leach tars, affect microbiota	Pyrolysis temperature	9.7–84.8%	[69]
Functional Groups (-OH, -COOH)	Critical for CEC, ROS scavenging, polar toxin binding, and immune modulation	Low-temp pyrolysis, surface aging	Varies (increases with age)	[64,70]

).

In the context of NE, biochar with a high cation exchange capacity may offer greater adsorptive potential because negatively charged surface functional groups can enhance interactions with cationic species in the gut lumen [63]. Accordingly, high porosity and large surface area may thus increase the overall adsorption capacity of biochar. As NetB is a key pore-forming toxin involved in avian necrotic enteritis [18,71], these adsorptive properties may be relevant to NE mitigation, although direct primary evidence for NetB adsorption by biochar is still needed. Together, these properties support further evaluation of biochar as a candidate material for NE mitigation, but direct mechanism-specific validation remains necessary.

5.1.2. Source of Biochar

The different sources of biochar and their suitability for NE-focused adsorbent evaluation are summarized in

Table 3. Different Sources of Biochar.

Type	Biochar Source	Application Context	Reference(s)
Various trees	Orchard grass, chestnut oak, yellow poplar, white pine	Feed additive; processed at high temperature	[73]
Fast-growing grass	Bamboo	Used in pigs and fish diets to enhance immunity and growth	[45,74]
Agricultural waste	Rice husks, water spinach, cassava	Widely used in ruminants	[44,75]
Plant biomass	Woody green waste	Used in poultry diets for growth and egg yield improvement	[10,76]
Forestry waste	Pine wood chips	Used in cattle for methane reduction	[77]
Forestry waste	Jarrah wood	Promote weight gain and meat taste in cattle	[78]
Agricultural waste	Corn stover	Poultry diet to improve body weight and FCR	[12]
Livestock waste	Commercial broiler litter	Poultry diet: improved weight gain and FCR	[79]
Mixed plant residues	Apple tree branch, oak tree, rice straw	Affect the porosity and surface area of biochar	[80]

and

Table 4. Comparison of Biochar Feedstock Sources and Their Suitability for Therapeutic Application in NE.

Feedstock Type	pH	Surface Area (BET)	Porosity	Ash Content	Volatile Matter (%)	Fixed Carbon	Nutrient Content	Stability in Gut	Toxin Adsorption Potential	Risks	References
Softwood Biochar	Moderately alkaline (7–9)	50–300 m2/g	Mesoporous	<5%	Low (~10–15%)	70–80%	Low, mainly C	High, resists breakdown	Good, depends on porosity	Minimal if clean feedstock	[60,72,81]
Hardwood Biochar	Alkaline (8–10)	200–500 m2/g	Microporous + mesoporous	5–10%	Moderate (~15–25%)	75–85%	Low, mainly C	Very high, resists breakdown	Excellent, high microporosity	Minimal if clean feedstock	[82,83,84]
Poultry Waste Biochar	Strongly alkaline (9–12)	10–50 m2/g	Pores clogged with minerals	30–50%	High (~30–40%)	30–50%	High N, P, K, Ca	Lower, soluble due to salts/ash	Poor-moderate, ash interferes	Heavy metals, ammonia risk	[85,86,87]

, respectively. Wood-derived biochar is a promising feedstock for adsorption-based applications, but different wood types have different advantages. Softwood-derived chars have been reported to reach as high as 326.0 m²/g, compared with 221.0 m²/g for a hardwood counterpart in the same study [60]. Wood biochars can also develop vesicle- and slit-like pore structures during pyrolysis, which contribute to adsorption capacity [72]. In addition, increasing pyrolysis temperature promotes the development of condensed aromatic structures and greater structural ordering in biochar [55]. In the context of NE mitigation, these characteristics support the use of wood-derived biochar as a candidate adsorbent, but direct primary evidence for differential adsorption of NetB or other NE-associated toxins by specific wood feedstocks is still needed.

5.1.3. Limitation of Biochar Use

Despite its purported antibacterial, antioxidant, and adsorptive properties, direct evidence supporting biochar specifically against NE remains limited. Most published poultry studies have instead focused on broader outcomes such as growth performance, gut microbial changes, or toxin-related applications outside NE-specific or NetB models. In addition, depending on feedstock and pyrolysis conditions, some biochars may contain residual contaminants such as polycyclic aromatic hydrocarbons and heavy metals, highlighting the need for careful characterization before biological use [88]. Although dietary biochar has shown promise in some poultry studies, high inclusion levels may adversely affect feed intake or performance in certain contexts, indicating that dose optimization is necessary [12,89]. These limitations highlight the need for standardized production criteria and targeted NE-specific in vivo trials.

Biochar and clay minerals differ substantially in their dominant adsorption mechanisms. Biochar is a carbon-rich, porous material whose activity is influenced by feedstock, pyrolysis temperature, surface area, aromaticity, and oxygen-containing functional groups [55,56,58,59,60,61,62,63,64]. In contrast, clay minerals are naturally occurring aluminosilicate materials with layered structures and surface reactivity that may support enteric adsorption and gut-health applications [90,91]. This distinction provides the basis for considering clay minerals separately from biochar in the following section.

5.2. Clay Mineral

The Joint Nomenclature Committees (JNC) define clay minerals as naturally occurring materials made mostly of fine-grained minerals [90]. These materials are usually plastic when they contain the right amount of water, and they will harden when dried or fired [90]. Clay minerals, such as bentonite, zeolite, and kaolinite, have unique properties that help reduce mycotoxin exposure in poultry (Table 5). The addition of clay minerals like montmorillonite, kaolinite, and palygorskite to poultry feed may improve gastrointestinal health, enhance performance, and reduce the harmful effects of pathogens in chickens [91].

Research by Ghazalah et al. [92] shows that bentonite, a type of clay mineral, can reduce the negative impacts of mycotoxins in broiler diets. This absorption reduces their uptake in the gastrointestinal tract and promotes better health and growth in poultry birds [10,11,92]. This highlights how clay minerals may enhance gut integrity, which is crucial for preventing conditions like NE that can arise from gut dysbiosis and pathogen overgrowth.

In addition, studies have found that some clay minerals have antibacterial properties. Research by Lafi and Al-Dulaimy [93] showed that certain mineral clays have antimicrobial properties. This could be particularly significant for managing NE, as reducing the presence of CP and other pathogens is vital for effective disease control. Nonetheless, more studies are needed to substantiate the dual capabilities of clay minerals. As shown in Table 5, both layered and non-layered clay minerals have potential in tackling various animal health issues, especially gastrointestinal infections.

Table 5. Application of Clay in the Treatment of Animal Diseases/Infections.

Clay Type	Disease Effect Treated	Mechanism of Action	Animal Species	Reference
Montmorillonite (MMT)	Diarrhea, bacterial infections (e.g., E. coli, Salmonella enteritidis)	Adsorbs bacteria and toxins; enriched forms (e.g., Cu-MMT) enhance antimicrobial activity	Poultry, pigs	[94]
Bentonite	Aflatoxicosis, general toxin adsorption	Binds aflatoxins and mycotoxins, reducing toxin transfer to organs or milk	Broilers, milk samples from cows, camels, sheep and goats	[92,95]
Clinoptilolite (Zeolite)	Mycotoxicosis (e.g., aflatoxins), intestinal hygiene	Molecular sieve structure traps mycotoxins, supports immune response	Poultry	[96,97,98]
Sepiolite	Pathogen-induced intestinal damage	Adsorbs bacterial toxins and supports gut lining integrity	Broilers	[99]
Kaolinite	Digestive disorders, diarrhea	Anti-toxic properties, reduce water content in droppings	Piglets	[100]
Natural Zeolite	Mycotoxin effects, gut flora imbalance	High ion exchange and binding capacity; reduces NH3 emissions	Broilers, layers	[101,102]
Attapulgite (Palygorskite)	Mycotoxicosis, intestinal pathogens	High surface area for adsorbing toxins and pathogens	Broilers, pigs	[103]

Table 5. Application of Clay in the Treatment of Animal Diseases/Infections.

Clay Type	Disease Effect Treated	Mechanism of Action	Animal Species	Reference
Montmorillonite (MMT)	Diarrhea, bacterial infections (e.g., E. coli, Salmonella enteritidis)	Adsorbs bacteria and toxins; enriched forms (e.g., Cu-MMT) enhance antimicrobial activity	Poultry, pigs	[94]
Bentonite	Aflatoxicosis, general toxin adsorption	Binds aflatoxins and mycotoxins, reducing toxin transfer to organs or milk	Broilers, milk samples from cows, camels, sheep and goats	[92,95]
Clinoptilolite (Zeolite)	Mycotoxicosis (e.g., aflatoxins), intestinal hygiene	Molecular sieve structure traps mycotoxins, supports immune response	Poultry	[96,97,98]
Sepiolite	Pathogen-induced intestinal damage	Adsorbs bacterial toxins and supports gut lining integrity	Broilers	[99]
Kaolinite	Digestive disorders, diarrhea	Anti-toxic properties, reduce water content in droppings	Piglets	[100]
Natural Zeolite	Mycotoxin effects, gut flora imbalance	High ion exchange and binding capacity; reduces NH3 emissions	Broilers, layers	[101,102]
Attapulgite (Palygorskite)	Mycotoxicosis, intestinal pathogens	High surface area for adsorbing toxins and pathogens	Broilers, pigs	[103]

5.2.1. Physicochemical Properties of Clay Minerals

Clay minerals are naturally occurring aluminosilicates whose layered structures and surface reactivity make them useful adsorbents in animal gastrointestinal applications. Their crystal architecture, including 1:1 and 2:1-layer arrangements, influences hydration behavior, interlayer accessibility, and adsorption performance [104,105]. In general, clay minerals differ markedly in specific surface area and CEC, two properties that strongly affect their ability to bind ions, toxins, and other dissolved compounds. In a comparative study of bentonite, illite, and kaolinite, bentonite showed substantially greater surface area and CEC than kaolinite, supporting the view that higher-charge, higher-surface-area clays have greater adsorption potential [106]. Likewise, studies on smectitic clays have shown that adsorption efficiency depends on physicochemical characteristics such as mineral origin, native sodium content, swelling behavior, and charge-related properties [105]. Experimental work in digestive-tract models also confirms that clay minerals can modify the solubility of bivalent cations under ruminal, abomasal, and duodenal conditions, demonstrating that these materials actively interact with the gut environment [104]. Collectively, these characteristics support the use of selected clay minerals as enteric adsorbents that may reduce luminal toxin exposure, help maintain ionic balance, and lessen pathogenic burden during necrotic enteritis and related enteric disorders.

In addition, recent molecular studies suggest that matching the molecular size and polarity of targeted toxins in this case NetB with the appropriate clay interlayer architecture, and not simply maximizing surface area or CEC, is essential for selective toxin binding. Deng et al. and Deng and Szczerba [107,108,109] showed that CEC plays little to no role in the adsorption of most toxins, because common mycotoxins are nonionic or negatively charged at neutral pH (e.g., fumonisin) and therefore do not participate in cation exchange processes with smectites or zeolites. Instead, aflatoxin B1 (AfB1) is one of the few well-studied nonionic mycotoxins that bind to smectite clays primarily through polarity and size matching between the toxin and the non-polar nanoscale interlayer domains of smectites, allowing the planar AfB1 molecule to intercalate into the interlayer space [108]. Within this confined environment, adsorption is driven by ion-dipole interactions and coordination between exchange cations and the carbonyl groups of AfB1, as well as hydrogen bonding between carbonyl oxygens and hydration-shell water at higher humidity [107,109].

Besides surface charge and adsorption, clay minerals also possess hydration, swelling or pore structure, and ion exchange characteristics that contribute to their usefulness as natural adsorbents in poultry systems. Broiler studies with clinoptilolite and bentonite have shown improvements in gut-related parameters or mitigation of aflatoxin effects [110,111], while adsorption studies show that these performance differences depend on physicochemical traits such as charge, swelling behavior, and mineral origin [105]. Many natural aluminosilicate adsorbents used in feed and environmental systems have near-neutral point-of-zero-charge values; for example, natural zeolite has been reported at about pH 6.2, while bentonite-based adsorbents commonly fall near neutral to slightly alkaline values depending on treatment and composition [112,113]. Because pH at the point of zero charge influences whether the surface is net positive or net negative, it can affect electrostatic interactions with dissolved compounds in gut digesta [112,114].

Smectite-rich clays such as bentonite are expandable and take up substantial amounts of water, and swelling bentonites have been shown to perform well as aflatoxin adsorbents [105,115]. In contrast, kaolinite is a non-expanding clay with comparatively high structural stability, a property that limits excessive swelling relative to smectites [116,117]. Zeolites such as clinoptilolite are crystalline porous materials whose adsorption and ion exchange behavior are closely linked to their framework structure, and primary studies have demonstrated effective uptake of ammonia and heavy metals by natural or modified clinoptilolite [118,119]. Taken together, these complementary physicochemical properties support the therapeutic potential of selected clay minerals as relatively low-cost and physiologically compatible agents for reducing luminal toxin burden and improving gut health in poultry, especially in antibiotic-free production systems.

5.2.2. Potential Limitations and Safety Considerations Regarding Clay Minerals

Although clay minerals have detoxifying and antibacterial potential, their effects appear strongly shaped by mineral type, inclusion level, and dietary context. Smectites, zeolites, and bentonites can bind aflatoxins and improve selected gut-health or performance outcomes under toxin pressure, but their adsorption is not fully selective [120,121]. For example, copper-bearing montmorillonite at 1.5 g/kg diet improved growth performance, digestive enzyme activity, intestinal morphology, and microflora in broilers [94], whereas calcium bentonite showed dose-dependent responses, with 2% inclusion producing better growth responses than 5% inclusion and higher inclusion altering digesta moisture, intestinal viscosity, serum lipids, and meat fatty-acid composition [122]. These findings suggest that clay efficacy may follow a non-linear response, where moderate inclusion supports toxin binding or gut function, but excessive inclusion may reduce nutrient utilization or alter digesta properties. Because increased intestinal viscosity can depress nutrient utilization, especially lipid digestion, clay-based NE strategies should be evaluated not only for toxin-binding efficacy but also for unintended nutrient sequestration, viscosity changes, and raw material safety [122,123]. Safety assessment is also important because some natural clays can carry environmental contaminants if sourcing and processing are poorly controlled; for example, dioxins have been identified in mined clay products and in ball clay associated with poultry-feed contamination [124,125].

5.3. Graphite-An Emerging Natural Adsorbent

Of the three adsorbent classes reviewed, graphite has the weakest direct poultry evidence base. No published in vivo poultry study currently demonstrates that dietary graphite prevents or mitigates necrotic enteritis, and no primary study has directly shown adsorption or neutralization of NetB by graphite. Therefore, graphite is considered here as an emerging material rather than a validated NE therapeutic candidate. Nevertheless, observations from our laboratory (manuscript in preparation) support a biologically plausible mechanistic basis for its investigation, and the experimental gaps that must be addressed before its therapeutic potential can be fully evaluated are identified here.

Graphite is a crystalline carbon material composed of stacked hexagonal carbon sheets held together by weak interlayer forces [126,127,128]. Natural graphite is obtained from geological deposits, whereas synthetic graphite is produced by high-temperature graphitization of carbonaceous precursors [129,130]. Differences in purity, morphology, defect density, processing history, and precursor source may influence surface behavior and reproducibility [131,132,133,134]. These characteristics are relevant because batch-to-batch consistency and contaminant control would be essential before graphite could be evaluated as a feed-grade adsorbent.

The graphite-related evidence should be interpreted according to material class. Selected graphene-derivative studies have reported antibiofilm and stress-response effects in bacterial systems [135], while engineered graphitic nanozymes have shown pH-responsive antimicrobial activity in a non-poultry Helicobacter pylori model [136]. Bulk graphite and related graphene-family materials have also shown antibacterial activity in selected in vitro systems, although this evidence cannot be directly transferred to dietary graphite in poultry [137]. Additional graphene oxide studies in non-poultry infection, biofilm, and gut models are summarized in

Table 6. Reported biological activities of graphite- and graphene-derived materials in selected experimental models.

Material	Animal Model/Condition	Reported Mechanism or Biological Outcome	Reference
Graphite	Escherichia coli (in vitro)	Exhibits antimicrobial activity via membrane disruption and oxidative stress (glutathione oxidation); lower efficacy than GO/rGO, but still noteworthy	[137]
Graphene Oxide (GO)	Mouse, Klebsiella pneumoniae lung infection	Antimicrobial and anti-inflammatory blocks bacterial growth, boosts macrophage response, reduces tissue damage, and mortality	[138]
GO	Cow, Staphylococcus aureus mastitis	Disrupts biofilms, kills intracellular pathogens, enhances antibiotic susceptibility	[139]
GO-alginate hydrogel	Mouse, S. aureus skin infection	Eradicates biofilm, promotes tissue repair with minimal cytotoxicity	[140,141]
Oral GO	Mouse, gut microbiota and colon structure	Modulates gut microbiota, alters colon morphology, suggesting gut-barrier interaction	[142]

[138,139,140,141,142]. Evidence for graphene oxide and reduced graphene oxide is more extensive than evidence for bulk graphite, but it cannot be directly transferred to graphite because oxidation and exfoliation substantially increase surface area, hydrophilicity, oxygen-containing functional groups, and biological reactivity [143,144,145]. Therefore, graphene oxide and engineered graphitic materials should be treated as mechanistic analogues, not as direct evidence that dietary graphite is effective against poultry NE.

Available graphite-derivative studies suggest possible antibacterial, antibiofilm, oxidative stress-modulating, and microbiome-related effects, but most evidence comes from in vitro systems or non-poultry models [135,137,142]. Oral graphene oxide has also been shown to alter gut microbial composition and colon ultrastructure in mice, indicating that dose, material chemistry, and host context may strongly influence biological outcomes [142]. These findings support further hypothesis-driven investigation but do not establish graphite as a near-term therapeutic for NE. The most important gaps are the absence of poultry NE challenge studies, the lack of purified NetB adsorption or neutralization assays, limited information on safe dietary inclusion levels, and uncertainty about whether graphite would retain functional activity under gastrointestinal and feed-processing conditions. Accordingly, future work should first evaluate graphite using standardized physicochemical characterization, CP inhibition assays, NetB-binding assays, intestinal epithelial cell protection models, and controlled in vivo poultry NE studies before therapeutic claims are made. Table 6 provides an overview of graphite and graphene-derived materials and their reported biological activities in selected experimental models.

Safety Considerations for Graphite and Graphene-Derived Materials

Although graphite and graphene-based materials have shown antioxidants, cytoprotective, and antibacterial effects in several experimental systems, direct evidence for their efficacy against necrotic enteritis or the NetB toxin remains very limited [146,147]. Most available data come from non-NE models, including an in vitro gut-liver injury model and an in vivo zebrafish microbiome model, so extrapolation to poultry should be made with caution [146,148]. Furthermore, oral exposure to graphene oxide has been shown to alter gut microbial composition and colon ultrastructure in mice, while microbiome-dependent immune effects have also been reported in zebrafish, indicating that dose and host context may strongly influence biological outcomes [142,148]. These findings highlight the need to define safe dietary inclusion limits and to conduct validation studies specifically in poultry before practical application can be considered.

6. Natural Adsorbent’s Mechanism(s) of Action

6.1. Biochar

Biochar appears to act in the poultry gut through a first-line adsorptive mechanism, as illustrated in Figure 3, where its porous carbon matrix and reactive surface sites trap harmful luminal compounds before they contact the intestinal mucosa. Primary studies have shown that biochar-based adsorbents can bind important mycotoxins, including aflatoxin B1, ochratoxin A, and zearalenone [6]. Other carbonaceous adsorbents have also shown adsorption of deoxynivalenol under appropriate conditions [149]. In poultry, this toxin-sequestering role is complemented by a microbiota-modulating effect. In layer chickens, dietary biochar altered intestinal microbial communities and reduced Campylobacter load without distorting overall microbial richness and diversity, supporting the mechanism in Figure 3 in which biochar suppresses pathogenic bacteria while preserving broader community structure [10]. A follow-up poultry study further showed that feed supplementation with biochar lowered pathogens such as Gallibacterium anatis and Campylobacters, with the most pronounced effects observed at 2% dietary inclusion [40].

The downstream consequence of these actions is likely a gut environment more favorable for beneficial microbial metabolism, epithelial stability, and immune protection, as depicted in Figure 3. As biochar reduces pathogen pressure while reshaping intestinal microbial composition, it may indirectly support fermentation patterns associated with short-chain fatty acids, especially butyrate-producing communities, although evidence for direct biochar-butyrate metabolism in poultry remains limited [40,150]. This matters because butyrate is a well-established mediator of gut barrier function: primary studies show that it enhances tight-junction assembly and improves epithelial barrier integrity [151,152]. In vivo poultry, evidence also shows that biochar can reduce systemic toxic stress. In broilers challenged with aflatoxin B1, poultry litter biochar improved growth-related outcomes and restored serum protein and albumin while lowering indicators of liver injury, indicating that luminal adsorption can translate into measurable physiological protection [6]. Together, these findings support the mechanism proposed in Figure 3: biochar first adsorbs toxins and limits pathogen abundance, then stabilizes the microbial ecosystem and indirectly promotes barrier integrity and gut immunity.

6.2. Clay Minerals

Clay minerals such as bentonite, montmorillonite, zeolite, and halloysite appear to improve poultry gastrointestinal health through a sequential mechanism that begins with ion exchange and luminal adsorption, as illustrated in Figure 4. Their layered aluminosilicate surfaces can bind aflatoxin B1 in the poultry gastrointestinal model, although the same materials may also sequester essential trace minerals, confirming both their detoxifying potential and their non-selective binding risk [120]. In broilers, calcium montmorillonite adsorbed aflatoxin B1 and, when added to feed at 0.5%, significantly reduced the adverse effects of aflatoxin exposure, which fits the Figure 4 step showing toxin capture in the gut lumen before absorption [153]. Clay supplementation has also been linked with improved digestive conditions; for example, Transcarpathian zeolite improved gut morphology and digestive enzyme activity in broilers, supporting the Figure 4 pathway connecting luminal stabilization with better digestive function [154].

Beyond toxin binding, clay minerals may also support gut health by reducing pathogen pressure, preserving mucosal integrity, and stabilizing the intestinal ecosystem, as depicted in the central and right-hand portions of Figure 4. Natural antibacterial clays can inhibit bacteria through surface-related geochemical mechanisms, although most direct membrane-disruption evidence comes from non-poultry systems and should therefore be extrapolated with caution [155]. In poultry, palygorskite supplementation improved immunity, oxidative status, intestinal integrity, and barrier function in broilers, while a later broiler study showed improved antioxidant capacity, immune status, intestinal barrier function, and altered cecal microbiota [156,157]. Halloysite has also been used successfully in broiler production, improving feed conversion and house hygiene parameters, which supports the broader Figure 4 concept that clay can contribute to a healthier gut environment [158]. Taken together, these findings support the mechanism proposed in Figure 4: clay first exchanges ions and sequesters harmful luminal compounds, then helps limit microbial stress while supporting digestion, barrier health, and microbial balance in the poultry gut.

6.3. Graphite

Graphite-derived materials have been reported to influence bacterial survival, oxidative stress, adsorption-related interactions, and host responses in selected experimental systems, as summarized in Figure 3. Sharp graphenic edges can physically damage bacterial membranes, leading to leakage of intracellular content and loss of viability [13,159]. Surface chemistry also shapes the response, because graphene oxide can exert contact-dependent antibacterial effects that vary with sheet size and oxidation state and may involve oxidative stress-related pathways [159,160]. In epithelial models, graphene oxide has reduced ROS and improved cell viability during gut-liver injury, indicating a cytoprotective antioxidant mechanism in addition to its antimicrobial activity [146]. Graphene oxide can also modify the intestinal microbial environment by increasing short-chain-fatty-acid-producing bacteria such as Clostridium cluster IV and Allobaculum spp., which suggests an additional microbiota-mediated mechanism [161]. Collectively, these findings suggest that graphite-derived materials may be relevant to NE through combined effects on bacterial survival, oxidative injury, and gut microbial balance, although direct evidence for NetB binding or in vivo NE control is still limited.

Understanding microbiota-host interactions is important for optimizing the therapeutic potential of graphene oxide while minimizing unintended biological effects. Oral GO has been shown to alter gut microbial composition in mice, including increasing short-chain-fatty-acid-producing bacteria such as Clostridium cluster IV and Allobaculum spp. [161]. Short-chain fatty acids are important mediators of gut health because they support epithelial barrier integrity, influence immune signaling, and serve as energy substrates for colonocytes [162,163]. However, prolonged or dose-dependent exposure to graphene-based materials can also disturb microbial composition and metabolism, with reported changes in gut community structure, metabolite profiles, and host physiological outcomes [164,165].

6.4. NetB Adsorption as a Central Unproven Mechanism

A critical limitation across the natural adsorbent literature is the absence of direct evidence demonstrating adsorption or neutralization of NetB by biochar, clay minerals, graphite, or graphite-derived materials. NetB is a major pore-forming toxin associated with avian NE, and its structure and function have been experimentally characterized [18,71]. However, current support for adsorbent-NetB interaction is based on physicochemical plausibility rather than direct experimental validation. For biochar, the rationale is based on its porosity, surface area, and functional groups known to support adsorption of some organic compounds and mycotoxins [6,7,50]. For clay minerals, the rationale is based on their toxin-binding studies showing that adsorption depends on molecular size, polarity, interlayer accessibility, and exchangeable cations [105,107,108,109,120,153]. For graphite and graphene-derived materials, the rationale is derived from surface interactions, antimicrobial activity, and biomolecule adsorption reported in non-NE systems [137,143,144,145,159,160]. However, none of these findings confirms binding of NetB under poultry gastrointestinal conditions. Therefore, NetB adsorption should be treated as a testable hypothesis and a priority research gap. Future studies should directly evaluate purified NetB binding, residual toxin activity, epithelial cytotoxicity, and dose-dependent adsorbent performance under simulated poultry intestinal conditions before NetB neutralization is presented as a mechanism of NE control.

To clarify the strength and translational relevance of the evidence supporting each proposed mechanism,

Table 7. Evidence level and translational readiness of natural adsorbent mechanisms relevant to NE mitigation.

NAs	Proposed Mechanism	Main Evidence Base	Poultry Relevance	Current Confidence	Key NE-Specific Gap	References
Biochar	Mycotoxin adsorption	In vitro adsorption and poultry aflatoxin studies	Moderate	Moderate	NE challenge studies must separate mycotoxin protection from direct CP/NetB effects	[6,7]
Biochar	Microbiome/pathogen modulation	Poultry non-NE studies	Moderate	Moderate	Need standardized NE models measuring lesion scores, CP load, SCFAs, and microbiome shifts	[10,40,150]
Biochar	NetB adsorption	Physicochemical plausibility only	Conceptual	Low	Direct purified NetB binding and residual cytotoxicity assays are absent	[18,71]
Biochar	Feed-manufacturing compatibility and dose effects	Broiler feed-performance studies	High	Moderate	Need realistic inclusion rates that do not impair performance, viscosity, or digestibility	[79,89]
Clay minerals	Aflatoxin adsorption	In vitro and poultry studies	High	Moderate-high	Need comparison with NE-associated bacterial toxins and nutrient-binding risk	[105,107,108,109,120,153]
Clay minerals	Nutrient/mineral sequestration	In vitro poultry gastrointestinal model	High	Moderate	Need dose thresholds that preserve trace mineral bioavailability	[120]
Clay minerals	Antibacterial/gut-barrier support	Poultry and non-poultry studies	Moderate	Low-moderate	Need CP-specific and NE lesion studies	[94,110,111,156,157,158]
Graphite	Antibacterial contact/oxidative stress	In vitro graphite/graphene-family material studies	Low	Low	Need poultry-relevant CP, IEC, and in vivo NE validation	[137,159,160]
Graphene oxide	Microbiome modulation	Mouse/zebrafish/in vitro models	Indirect	Low	Effects may be beneficial or adverse depending on dose, host, and material chemistry	[142,161,164,165]
Graphitic nanozymes	pH-responsive antimicrobial activity	Non-poultry H. pylori model	Very indirect	Low	Mechanism depends on engineered nanozyme properties and cannot be assumed for dietary graphite	[136]
Graphite/GO	NetB adsorption	No direct evidence	Conceptual	Very low	Highest-priority gap requiring purified NetB binding and epithelial protection assays	[18,71,137]

summarizes whether the evidence is derived from poultry studies, non-poultry models, in vitro systems, physicochemical plausibility, or hypothetical extrapolation. The “current confidence” classification was assigned based on the proximity of the evidence to poultry NE application, the presence or absence of direct NE-relevant outcomes, and the degree of experimental validation. Mechanisms were considered moderate to high confidence when supported by poultry studies measuring relevant outcomes such as mycotoxin adsorption, gut-health responses, pathogen modulation, performance, or intestinal integrity. Mechanisms were classified as low or very low confidence when evidence was limited to non-poultry models, in vitro systems, engineered material platforms, or physicochemical plausibility without direct poultry NE validation. Because no study has directly demonstrated NetB sequestration, adsorption, or neutralization by biochar, clay minerals, graphite, or graphene-derived materials, NetB-related mechanisms were classified as conceptual or very low confidence. This classification also accounts for whether differences in feed composition, challenge model, bird age, material purity, inclusion level, or processing conditions limit direct comparison across studies.

In summary, the physicochemical mode of action of adsorbents differs fundamentally from other biological alternatives. Unlike probiotics or vaccines, which depend on microbial competition or immune priming, natural adsorbents exert direct physicochemical sequestration of toxins and microbial metabolites [166,167]. This mechanism is not influenced by antimicrobial resistance patterns and remains stable under feed-processing conditions. Furthermore, adsorbents may provide multifunctional benefits, including toxin binding, oxidative stress attenuation, and modulation of the luminal microenvironment, making them a feasible option in antibiotic-free production systems.

When compared with other non-antibiotic interventions used against NE, such as probiotics, phytogenic compounds, organic acids, and vaccination strategies, natural adsorbents offer several distinctive features (

Table 8. Comparison of Non-Antibiotic Strategies for NE Mitigation in Poultry.

Strategy	Primary Mechanism	Evidence in Poultry	Advantages	Key Limitations
NAs: clay, biochar, graphite	Physicochemical toxin sequestration, cation exchange, adsorption of microbial metabolites, and possible microbiome modulation	Clay minerals bind aflatoxin and can improve performance under mycotoxin challenge [111,153,173]; biochar modulates gut microbiota and may reduce selected poultry pathogens [10,40]; graphite evidence remains indirect and mainly from in vitro or non-poultry graphene-family studies [137,142,159,160,161]	Resistance-independent; potentially stable in feed; multifunctional effects; compatible with antibiotic-free systems	Inclusion levels must be optimized; possible nutrient or mineral sequestration; limited direct in vivo NE evidence; graphite evidence remains exploratory
Probiotics	Competitive exclusion, immune modulation, microbial balance	Some probiotic interventions reduce NE-associated lesion scores and improve gut integrity in broilers [174]	Supports microbiota and barrier function; industry accepted	Strain-specific effects; stability and efficacy vary across diets, environments, and challenge models
Phytogenics	Plant-derived antimicrobial, antioxidant, and anti-inflammatory effects	Phytogenic products can reduce NE-associated gut damage and performance loss in broilers [31,175]	Natural compounds; multi-target biological effects	Variable composition, dose response, and product consistency
Vaccines, including NetB-based vaccines	Immune priming against CP virulence factors	Recombinant NetB vaccination provides partial experimental protection against NE [176,177]	Mechanistically targeted; may reduce disease susceptibility	Variable field efficacy; delivery and cost challenges
Organic acids	Lower gut pH, inhibit pathogen growth, support barrier function and microbial metabolites	Organic acid blends improve intestinal integrity, SCFA profiles, and microbiota in NE-challenged broilers [30,178,179]	Broad antimicrobial and gut-health effects	Efficacy depends on acid type, coating, dose, diet, and farm conditions

). Probiotics and phytogenics primarily modulate microbial populations or host signaling pathways but may exhibit variability due to strain stability, storage conditions, or environmental stressors [168,169]. Vaccination strategies targeting NetB and other virulence factors have shown promise in experimental models; however, protective efficacy may vary depending on challenge strain and field conditions [18,170]. In contrast, adsorbents function through non-biological mechanisms, including electrostatic interactions, cation exchange, and surface adsorption, allowing immediate binding of toxins and microbial by-products in the gut lumen [166,171]. Nevertheless, inclusion levels must be carefully optimized, as excessive adsorption capacity may interfere with nutrient availability and mineral bio-accessibility [172]. Therefore, a balanced risk-benefit evaluation, including dose-response assessments, lesion scoring, gut integrity markers, and production performance metrics, is essential when positioning natural adsorbents as therapeutic candidates for NE mitigation.

7. Proposed Screening Framework for Evaluating Natural Adsorbents for NE Control in Poultry

The evaluation of natural adsorbents as candidate NE-control strategies requires a systematic framework that connects material characterization with poultry health outcomes [120]. Future research should follow a defined testing process that begins with a comprehensive analysis of adsorbent properties, including surface area, porosity, charge characteristics, and ash content [120,131]. The evaluation process should include NetB-binding or residual toxicity assays, antibacterial MIC/MBC assays, agar well diffusion assays, cytotoxicity testing, ROS modulation, and epithelial barrier integrity assessment (Figure 5). While in vitro IEC models provide valuable mechanistic insight and align with animal-welfare priorities, they cannot fully replicate the complex interactions between microbiota, immune function, and nutrient dynamics in the live bird [180,181]. Therefore, adsorbents that demonstrate strong in vitro activity should progress to standardized in vivo models assessing intestinal lesion scores, CP enumeration, serum cytokines (IL-1β, IL-6, IFN-γ), villus morphology, tight-junction integrity, microbiome shifts, oxidative stress markers, growth performance, and feed conversion ratio [182,183,184].

7.1. Dose-Response and Go/No-Go Decision Criteria

Progression between screening tiers should be based on predefined go/no-go criteria. At the physicochemical stage, candidate adsorbents should proceed only if they show acceptable batch consistency, feed-grade safety, low contaminant burden, stable pH behavior, and reproducible surface properties relevant to adsorption [61,68,88,120]. Key screening parameters should include specific surface area, pore structure, CEC, ash content, volatile matter, pH, surface functional groups, and contaminant burden. For biochar, materials with excessive ash, residual volatile matter, heavy metals, or polycyclic aromatic hydrocarbons should be excluded because these properties vary by feedstock and pyrolysis condition and may affect biological safety [61,68,88]. For clay minerals, progression should require toxin-binding activity without excessive sequestration of essential trace minerals, because aluminosilicates can bind both aflatoxin B1 and essential minerals in poultry gastrointestinal models [120].

At the in vitro stage, progression should require the absence of cytotoxicity at biologically relevant intestinal exposure levels, reproducible reduction in CP growth or toxin-associated epithelial injury, measurable protection against oxidative stress or barrier disruption, and minimal non-selective binding of essential nutrients or minerals [120,137,146,159,160]. For NE-focused screening, candidate materials should show measurable activity in at least one disease-relevant assay, such as CP inhibition, NetB-binding or residual toxicity assays, epithelial-cell viability, ROS reduction, or preservation of epithelial barrier integrity. Materials should not progress solely because they show high adsorption capacity if they also show cytotoxicity, strong nutrient sequestration, or adverse epithelial effects.

At the in vivo stage, candidate materials should be advanced only if they reduce NE lesion severity or pathogen/toxin-associated injury without impairing feed intake, body weight gain, feed conversion, nutrient utilization, or bird welfare [182,183,184]. Where a 0–4 intestinal lesion scoring system is used, an effective candidate should reduce lesion scores toward the none-to-mild range, preferably 0–1, and show a statistically meaningful reduction compared with the challenged untreated control. Additional practical benchmarks should include lower CP burden, improved villus morphology or tight-junction integrity, reduced inflammatory or oxidative stress markers, and no negative effect on growth performance or feed conversion.

Dose-response considerations and synergistic interactions are also important in adsorbent evaluation. Current poultry studies suggest that moderate dietary inclusion levels, commonly around 0.5–2% depending on material type and experimental context, may support toxin binding, antimicrobial activity, or gut-barrier protection, whereas excessive inclusion may reduce nutrient digestibility or alter digesta viscosity [89,153,185,186]. Therefore, the ideal biological response is likely to be non-linear, with moderate inclusion improving adsorptive or gut-protective effects while maintaining feed efficiency. Combinations such as montmorillonite with charcoal or clay with phytogenics may provide complementary benefits through toxin sequestration, pH buffering, and microbial modulation [180]. However, combinations may also create antagonistic effects through nutrient competition or excessive micronutrient adsorption, highlighting the need for systematic dose optimization within realistic dietary inclusion limits [120,172]. This multi-tiered validation pipeline ensures both mechanistic rigor and practical relevance for NE mitigation.

7.2. Translational and Commercial Considerations

Translation of natural adsorbents into poultry production requires consideration beyond biological efficacy. Commercial deployment will depend on scalable sourcing, batch-to-batch standardization, contaminant screening, cost-effectiveness, feed-manufacturing compatibility, pelleting stability, and regulatory acceptance as feed additives or therapeutic adjuncts. Biochar presents standardization challenges because feedstock type, pyrolysis temperature, ash content, residual volatile matter, heavy metals, and polycyclic aromatic hydrocarbons can differ across products [61,68,79,88,89]. Clay minerals require similar scrutiny because mineral origin, swelling capacity, CEC, interlayer chemistry, and environmental contamination can influence both efficacy and safety [105,120,124,125]. Graphite-based materials may face additional safety and regulatory barriers because graphite, graphene oxide, and engineered graphitic nanomaterials differ substantially in particle size, surface chemistry, bioavailability, and biological reactivity [137,142,143,144,145,159,160,161]. Finally, all adsorbents should be evaluated for unintended interactions with vitamins, trace minerals, medications, vaccines, enzymes, organic acids, probiotics, and phytogenic additives, because non-selective adsorption may reduce the efficacy of other dietary interventions [120,172,180].

8. Conclusions

Natural adsorbents, including biochar, clay minerals, and graphite-based materials, represent promising but unevenly validated candidates for supporting gut health and reducing enteric disease pressure in poultry. Biochar and clay minerals currently have stronger poultry-related evidence, particularly for toxin adsorption, gut environmental modulation, and selected performance or barrier-related outcomes. In contrast, graphite remains exploratory because most available evidence comes from graphite-derived materials, in vitro systems, or non-poultry models. Importantly, direct evidence showing that any of these adsorbents bind or neutralize NetB is still absent; therefore, NetB adsorption should be viewed as a priority hypothesis rather than an established mechanism. Future research should focus on standardized physicochemical characterization, dose optimization, nutrient-interaction studies, NetB-specific adsorption assays, and controlled in vivo NE challenge trials. Such studies are essential before natural adsorbents can be confidently advanced from conceptual promise to practical therapeutic use in poultry production.