Evaluation of Rare Earth Element-Associated Hormetic Effects in Candidate Fertilizers and Livestock Feed Additives

Rare earth elements (REEs) are recognized as emerging contaminants with implications in human and environmental health. Apart from their adverse effects, REEs have been reported as having positive effects when amended to fertilizers and livestock feed additives, thus suggesting a hormetic trend, implying a concentration-related shift from stimulation to inhibition and toxicity, with analogous trends that have been assessed for a number of xenobiotics. In view of optimizing the success of REE mixtures in stimulating crop yield and/or livestock growth or egg production, one should foresee the comparative concentration-related effects of individual REEs (e.g., Ce and La) vs. their mixtures, which may display distinct trends. The results might prompt further explorations on the use of REE mixtures vs. single REEs aimed at optimizing the preparation of fertilizers and feed additives, in view of the potential recognition of their use in agronomy and zootechny.


Introduction
Rare earth elements (REEs) are a group of metals encompassing lanthanoids from lanthanum to lutetium, as well as yttrium and scandium, that have become indispensable in present-day life because of their critical role in many modern and cutting-edge technologies [1,2]. In recent decades, an extensive body of literature on REE-associated adverse effects in a number of biota and laboratory test models has given cause for concern that environmental REE exposures may have deleterious impacts on flora and fauna [3]. A growing body of literature on human REE exposures in mining areas, including facilities dedicated to REE extraction and manufacturing, increasingly points to REE bioaccumulation and excretion. These include environmental, non-occupational exposures among residents in REE mining areas [4,5], and point to the still many knowledge gaps on potential health risks in REE-exposed workers [6,7].
Apart from industrial applications, REEs have been extensively used in Chinese agriculture as fertilizers to increase crop yield, and in zootechny as feed additives aimed at increasing livestock growth and egg laying, with likely prospects of their utilization outside China [7][8][9][10].
The REE-associated adverse effects and their stimulatory actions in plant and animal growth may be regarded as one more case of the hormesis phenomenon, as reviewed by Calabrese [11] and by Calabrese and Agathokleous [12].
In view of a possible hormetic trend for REEs, just as for an extensive number of agents already reported in the literature, it is increasingly clear that testing the dose-response trends of individual REEs as well as their combinations is of growing importance to identify the concentration ranges and combinations which can give rise to hormetic or toxic effects [13]. Resolving the doses at which hormesis may occur, as well as the nature of the hormetic effects, are discussed in the present review, with a special focus on the present state of art, as yet confined to Chinese agriculture and zootechny and on the possible extension of REE utilization in the production of fertilizers and feed additives in other countries, by appropriate authorization from food safety agencies.

Materials and Methods
A detailed reference search of the literature was carried out using the PubMed, Scopus, and ScienceDirect databases by interfacing the following keywords:

REE-Associated Adverse Effects
After the pioneering studies by Drobkov [14] in 1941 on the effects of REEs on the development of peas, and by Jha and Singh [15,16] assessing the induction of cytogenetic damage by two REEs (praseodymium and neodymium) in mice and in broad bean (Vicia faba), a thriving literature over recent decades has provided established evidence for a number of REE-associated adverse effects in a number of test models, as summarized in Table 1. Studies of REE toxicity in plant models were carried out on several crop and native species, showing decreased seed germination, root elongation, and mitotic activity for REE levels ≤ 5.0 mg/L [17][18][19][20][21][22]. More extensive studies of REE-associated toxicity were conducted in several animal models including mammals (mice and rats), fish (Danio rerio), and sea urchins, providing evidence for a number of adverse effects, including oxidative damage, lung and kidney toxicity, and developmental and cytogenetic damage [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. Altogether, the available body of literature on the adverse effects of REE exposures raises environmental health concerns.

REE-Associated Hormetic Trends
Analogous to a number of chemical and physical agents [11,38], REE dose-response trends have been associated with hormesis, a phenomenon leading to stimulate (Greek: hormào) biological activities at lower concentrations compared to inhibition, bioaccumulation, and toxicity at higher exposure concentrations [39]. As shown in Table 2, evidence for REE-associated hormetic trends were reported in a set of studies conducted in several biota including plants, fungi, microbiota, and animals.
In particular, plant models including rice, bean, cabbage, and orange were exposed to varying levels of La, Ce, and Sc by testing some key endpoints including growth, germination, chlorophyll content, and oxidative stress parameters. The results reported on concentration-related hormetic trends in REE-exposed plants [40][41][42][43][44][45][46][47][48]. de Oliveira et al. [43] tested La 3+ exposures (5 to 150 μM) in soybean plants, by measuring a set of endpoints at low REE concentrations as plant growth, nutritional characteristics, photosynthetic rate, chlorophyll content, mitotic index, modifications in the ultrastructure of roots and leaves, and La mapping in root and shoot tissues. When La was applied, it was noted that the levels of some essential nutrients (Ca, P, K, and Mn) increased. Low La concentrations enhanced the photosynthetic rate and total chlorophyll content and led to a higher incidence of binucleate cells, with a slight increase in root and shoot biomass. At higher La levels, soybean growth was reduced. Liu et al. [44] tested La 3+ (0.05 to 1.5 mM) in rice plants for effects on reactive oxygen species and antioxidant metabolism. The results indicated that ROS levels declined after treatment with 0.05 mM La 3+ , with hormetic effects on the antioxidant metabolism in rice roots. Further, d'Aquino et al. [49] tested Trichoderma fungi to REE exposures ranging from 0.003 to 900 mM, and found increased growth of fungal biomass at low REE concentrations. Extending this work to bacteria, E. coli or microbial communities were exposed to several REEs by Técher et al. [50] and to Y(III) by Su et al. [51], who found increased growth kinetics and ammonia-oxidizing bacteria at low (< 20 mg/L) Y(III) concentrations but were inhibited by higher (≥ 20 mg/L) Y(III) concentrations.
Several studies of REE-associated hormetic effects were conducted in animal models (Table 2). Jenkins et al. [52] tested human dermal fibroblasts for profibrotic injury when exposed to REEs and found increased proliferation by low concentrations of REEs (1 to 10 μM), which turned to inhibition at higher (50 to 100 μM) REE concentrations. Decreased inflammatory parameters were reported by Hirst et al. [53] in mice exposed to low concentrations of CeO 2 nanoparticles. More recently, Zhang et al. [54] tested the response of rats to Y 2 O 3 exposure for growth endpoints, which were found to increase at low concentrations (20 ppm) and decrease at higher Y 2 O 3 concentrations (320 ppm).

REEs in Fertilizers
The established use of REEs as fertilizer components in Chinese agriculture dates back to the 1980s and was reported in early reviews [7,55,56]. A few reports in the past decade  Zhang et al. [54] have focused on some molecular endpoints in plants exposed to REE-containing fertilizers. Xu and Wang [57] found increased phosphorus uptake in maize after application of REE (La and Ce)-containing fertilizer, with applications of less than 10 kg/ha reported as increasing crop yield. Cheng et al. [58] exposed navel orange (Citrus sinensis) plants to a REE mixture (38.6 to 546 mg/kg in soil) by measuring a set of fruit quality indicators, including titratable acidity, total soluble solids, and vitamin C. The outcome was improved internal fruit quality in REE-exposed navel orange. A recent report by Lian et al. [59] investigated the effects of La 3+ on growth, photosynthetic ability, and phosphorus-use efficiency (PUE) in various organs of adzuki bean Vigna angularis seedlings. Treatment of young seedlings with La 3+ at 150 mg/L improved PUE in roots, stems, and leaves via the regulation of root elongation and activation of root physiological responses to P deficiency. La 3+ increased the level of superoxide dismutase and peroxidase, while it significantly decreased malondialdehyde content. The negative effects of P-deficiency on net photosynthetic rate, transpiration rate, and chlorophyll content in leaves were alleviated by La 3+ treatment.

REEs in Livestock Feed Additives
Analogous to their use in fertilizers, REEs have been used in Chinese zootechny as livestock feed additives, as reported by Wang and Xu [60] in their review of an extensive body of literature encompassing Chinese and Japanese papers dating back to the 1980s and the 1990s, and in a recent review by Abdelnour et al. [61]. Mechanistic and up-to-date reports are summarized in Table 3. He et al.
[62] tested diet supplementation of a REE mixture in piglets (300 mg/kg) and reported an increased body weight gain and feed conversion ratio. The same positive effects were found by Wang and Xu [60] who supplemented piglets with LaCl 3 (100 mg/kg BW). A recent study by Xiong et al. [63] evaluated the effects of a REE mixture (200 mg/kg BW) on sows and their offspring, observing improved antioxidant activity, immunity, reproduction of sows, and growth of piglets. Liu et al. [64] supplemented Simmental steers with LaCl 3 (400 to 1800 mg/day) and found improved rumen fermentation, urinary excretion, and feed digestibility. Renner et al. [65] supplemented fattening bulls with a mixture of REE citrates (100 to 300 mg/ kg dry matter) and found that REE supplementation affected dry matter intake, but not live weight gain, clinical chemical parameters, and ion concentrations significantly. Peripheral blood mononuclear cells were significantly increased in REE-supplemented bulls. He et al.
[62] fed Ross broiler chicks with either the chloride or citrate salts of REEs, and found improved growth performance of broilers without affecting carcass composition and health of the broilers. Cai et al. [66] fed broiler chickens with REE-enriched yeast (500 to 1500 mg/kg BW) and found improved growth performance. Durmuş and Bölükbaşı [67] supplemented laying hens with La 2 O 3 (100 to 400 mg/kg BW) and observed improved feed conversion ratio, egg production, and egg shell life. In further work, the same group [8] supplemented laying hens with CeO 2 , finding similar results as increased egg shell breaking strength and decreased oxidative stress parameters.
Beyond those experimental reports, it must be recognized that an official stamp of approval for the use of REE-based feed additives in a more widespread way globally is yet to be forthcoming, as reviewed by Squadrone et al. [68]. At least in one case, to our knowledge, a safety statement was provided by the EFSA Panel FEEDAP [69] for the feed additive Lancer®, a REE citrate mixture to be used in piglet diet. The EFSA Panel stated that uncertainty still remains on possible developmental neurotoxicity of Lancer® since it was unable to identify a no observed adverse effect level. However, the FEEDAP Panel considered that exposure to La and Ce from products of animals treated with Lancer® at 250 mg/kg feed would not add a significant contribution to the background exposure of these elements. The FEEDAP Panel concluded that the use of Lancer® in feed for weaned piglets according to the proposed conditions of use does not represent a safety concern for the consumer and for the environment.
Though there is currently little data available on the progress of other candidate feed additives, it is to be expected that increasing knowledge on the hormetic effects of REE-based materials will lead to further regulatory approval of REE-containing feed additives in the not-too-distant future.

Toward Production of REE Mixtures as Hormetic Agents
Under a historical perspective, the pioneering studies of hormesis by Stebbing [70] in 1982, revisiting the nineteenthcentury Arndt-Schulz Law, have now made hormesis a wellknown phenomenon in biological sciences, medicine, and pharmacology. In the more specific fields of agriculture and zootechny, and in the use of REEs as ingredients for fertilizers and feed additives, a persuasive body of evidence reports advantages to using REEs for increasing crop yield and livestock performance. Indeed, as well-theorized by Edward Calabrese and his group [11][12][13]38], REEs display hormetic dose-response trends, just as with a number of other chemical and physical agents, which are being underpinned with increasingly sophisticated theoretical frameworks [71][72][73][74]. However, it should be noted that REEs are rarely present individually but usually more likely as a mixture of REEs. For this reason, it is timely to begin considering the effect on biota of multiple REEs concomitantly present, particularly at very low concentrations and how hormetic effects might be modulated or negated. For example, as observed by Jacob et al. [75], when pharmaceuticals such as diazepam and simvastatin are individually present at concentrations below the no observable effect concentration, combinations of these at such concentrations indicate toxicity, e.g., to Aliivibrio fischeri. Hence, the need should be recognized for more studies involving mixtures, particularly at very low concentrations, since chemicals are subject to interactions and modifications which may result in antagonistic, additive, or synergistic effects.
This was the case, reported in our early studies [76,77], of a shift from stimulation to inhibition of sea urchin sperm fertilization rate by exposures to sub-micromolar levels of either cadmium or zinc, compared to their mixtures, respectively. Subsequent and recent investigations have further explored the concentration-related hormetic trends of several agents compared to their binary or multiple mixtures, such as antibiotics [78][79][80], industry wastewater [81], pharmaceuticals [82][83][84], and fungicides [85].
In view of likely developments in the production and use of REE-based fertilizers and feed additives, and in view of open questions persisting on the efficacy of using REE mixtures and their concentration-related trends, ad hoc investigations are required aimed at verifying the single vs. combined use of REEs in these production and use scenarios.
Funding Open access funding provided by Università degli Studi di Napoli Federico II within the CRUI-CARE Agreement.

Conflict of Interest The authors declare no competing interests.
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