Presently, fish are increasingly exposed to various fertilizer components originating from fertilizer discharges from agriculture and chemical industry facilities or as emissions resulting from mining activities. In agriculture, the combined production of fish and plant, known as aquaponics, uses circulating nutrient-rich water and the addition of specific fertilizers to provide cultivated plants with all the essential nutrients, which, together, lead to better plant development and productivity of the system. Since the water used for this plant cultivation system usually originates from a closed aquaculture system and is therefore reused for plant growth, aquaponics is generally considered a highly sustainable method for crop production (Stathopoulou et al., 2021). However, as the single nutrient composition of the circulating water of an aquaculture system, as well as through supply by fish feed, is mostly insufficient (Sonneveld et al., 2009; Krishnasamy et al., 2012; Bittsanszky et al., 2016), the supplementation of fertilizers seems inevitable (Da Silva Cerozi, 2020; Stathopoulou et al., 2021). Similar to other plants, aquaponically grown plants have species-specific requirements of macro- and micronutrients. Since nitrogen (N) and phosphorus (P) are supplied by the fish in an aquaponic system, only the trace nutrients such as iron (Fe), copper (Cu), boron (B), molybdenum (Mo), manganese (Mn), zinc (Zn), and potassium (K) might be deficient to meet the plant’s requirements (Strauch et al., 2018). To balance these deficits in an aquaponic system, nutrients can either be added to the water as compounds of salts such as sulfates (SO₄ ⁻²), chlorides (Cl⁻), or nitrates (NO₃ ⁻); as part of chelated compounds such as ethylenediaminetetraacetic acid (EDTA), ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid) (EDDHA), or diethylenetriaminepentaacetic acid (DTPA); or as components of organic acids including fulvic acid or amino acids such as arginine, glycine, or histidine (Linnik and Ignatenko, 2015; Shuhaimi-Othman et al., 2015; Kasozi et al., 2019; Da Silva Cerozi, 2020; Neocleous et al., 2020; Yep and Zheng, 2020).

Not only for aquaponic plants, Zn is one of several elements necessary for development and growth by protein synthesis, carbohydrate metabolism, pollen formation, heat stress, protection from photo-oxidative damage, biological membrane preservation, or infection resistance (Alloway, 2008). Similar to other plants, aquaponic plants can react to a Zn deficiency situation with many symptoms, including browning of the leaves due to a specific crossing structure, stagnation of the growth process, and the formation of infertile buds (Goddek et al., 2019; Yang and Kim, 2019). In order to avoid the aforementioned deficits, Zn, as a component of different chemical compounds, is already supplemented at concentrations up to 0.45 mg/L in hydroponic systems (Krishnasamy et al., 2012; Neocleous et al., 2020). At present, in aquaponic cultivation systems, the targeted supplementation of Zn is processed in combination with an EDTA chelator, which provides resistance against oxidation or dissolution and enables a constant pH-dependent availability of Zn in the water to a plant (Rakocy, 2012).

As an essential nutrient for both plants and fish, Zn plays a fundamental role in cell metabolism. In finfish, the uptake, efflux, and compartmentalization of Zn ensure homeostasis and prevent its toxic overaccumulation (Zheng et al., 2010). Its main vectors into a fish body are the uptake through the gills or via the gastrointestinal tract (Watanabe et al., 1997; Coetzee et al., 2002; Mahboub et al., 2020). Zn²⁺ transport via a chelate complex (i.e., EDTA) has also not been excluded for aquatic species (Hockett and Mount, 1996; Karayakar et al. (2021). As an essential element for life, Zn is also present in several amino acids and enzymes and plays a central role in protein folding (Ezeonyejiaku et al., 2010, 2012). In addition, recent studies have underlined its importance in enhancing the reproduction, antioxidant response, and growth performance of fish (Onuegbu et al., 2018; Kaliky et al., 2019).

As a variety of fish species can live in the reused water of an aquaponic system, an imbalance in the essential plant nutrients such as Zn, which are metabolically active to the fish, can negatively affect the growth, health, and welfare of specific fish species (Karayakar et al., 2021). Thus, controlled supplementation is important. In particular, the concentration thresholds of these micronutrients should be well known and not exceeded in cultivated species. An overdose of micronutrients, which is usually species-specific, can ultimately reduce the growth, damage the internal organs, induce different types of diseases, increase mortality, and even change the behavior of fish (Karayakar et al., 2021). Various negative reactions of fish to Zn have recently been reported, including the increased mortality of Clarias gariepinus in LD₅₀ toxicity studies to zinc sulfate (ZnSO₄) at 71.0 and 75.3 mg/L supplementation (Ezeonyejiaku et al., 2010; Chidiebere, 2019); the inhibited growth of Oncorhynchus mykiss and Oreochromis niloticus (Eisler, 1993; Abdel-Tawwab et al., 2012); hypocalcemia in O. mykiss (Niyogi and Wood, 2006); the increased glucose, cortisol, and creatinine as a stress response in O. niloticus (Firat and Kargin, 2010; Abdel-Tawwab, 2016); behavioral alterations in C. gariepinus and O. niloticus (Ololade and Ogini, 2009; Duran and Erdem, 2014; Tunçsoy and Erdem, 2014; Chidiebere, 2019); changes in the physiological and biochemical properties of the blood or immune cells in Ictalurus punctatus and C. gariepinus (Lewis and Lewis, 1971; Gatlin et al., 1989; Coetzee et al., 2002; Ololade and Ogini, 2009); and the pathological alterations in the organs of Pimephales promelas, Pseudupeneus prayensis, and C. gariepinus (Brungs, 1969; Heath, 1995; Roméo et al., 1999; Coetzee et al., 2002; Shuhaimi-Othman et al., 2015), such as hemorrhage in the gills or fins and extensive body and gill mucus in Labeo rohita and O. mykiss (Bengari and Patil, 1986; Hogstrand et al., 2002; Wood, 2017). Furthermore, several accumulation ratios of Zn and Zn-EDTA in the gill and liver tissues of Cyprinus carpio had been described by Karayakar et al. (2021), which will be discussed in this context.

Over the last few years, tolerance tests for the compatibility of nitrate–phosphorus–potassium (NPK) fertilizers, trace potassium nutrients, and Fe-DTPA have been carried out for the African catfish (C. gariepinus; Burchell, 1822), confirming their beneficial potential in aquaponics (Ajima et al., 2015; Shuhaimi-Othman et al., 2015; Wenzel et al., 2021; Hildebrand et al., 2023). Aligning with this approach, the present study aimed to quantify the effects of Zn-EDTA on the health and welfare of African catfish fry and fingerlings in two separate experiments using concentrations relevant for aquaculture and aquaponics (approximately 0.5 mg/L). Therefore, the fry and juveniles of African catfish were treated with various concentrations of Zn-EDTA in two separate experiments, focusing on the growth performance, ethological indicators, blood and histopathological analyses, and genetic profiling of critical genes to assess the potential hazards of Zn fertilization to the selected fish species (Seibel et al., 2021). From this assessment, we expected Zn-EDTA to have no adverse effects on the health and welfare of this species at water concentrations equivalent to those recommended for plant growth in aquaponic systems (0.45 mg/L).

Trace elements such as Zn are necessary to fertilize aquaponic systems and to make up for the lack of supply for an improved and healthy plant growth. To achieve a better understanding of their effect on fish, we examined the responses of a common aquaponic-related living fish species (C. gariepinus) to Zn-EDTA fertilization in two single life stage experiments. In terms of its water quality requirements, C. gariepinus can tolerate various parameters of water quality, including temperatures between 18°C and 32°C, with optimal temperatures for development and growth ranging from 27°C to 29°C (Păpuc et al., 2019; Xu et al., 2022; Baßmann et al., 2023). While a pH value between 5.0 and 9.0 corresponds to the profile requirements of this species (Păpuc et al., 2019; Xu et al., 2022; Baßmann et al., 2023), in aquaponics, the pH value is generally lower (approximately 6.5–7.0) to allow a more efficient bacterial activity and nutrient uptake by plants (Tyson et al., 2004). The recommended DO values range from 3.0 to 6.0 mg/L (Păpuc et al., 2019); however, higher values up to approximately 9.0 mg/L are also considered suitable (Baßmann et al., 2020; Knaus et al., 2020; Baßmann et al., 2023). The reported EC tolerance ranges from 100 to around 2,000 µS/cm (Hanika and Kramer, 2000; Tyson et al., 2004). According to Knaus and Palm (2017) and Homoki et al. (2020), an oxidative regime with a positive redox potential between +83.4 and +151.7 mV is recommended for bacterial activity in aquaponic systems and is well tolerated by the African catfish. Concentrations of NO₃, NO₂, and NH₄(NH₃) ranging up to 697.5, 0.6, and 20.46 mg/L, respectively (>0.34 mg/L) and of CO₃ from >10 to<500 mg/L have been shown to still be tolerable (Molokwu and Okpokwasili, 2002; Schram et al., 2010, 2014; Roques et al., 2015; Baßmann et al., 2017; Knaus et al., 2020). In this study, the measured water temperatures ranged between 26°C and 28°C, the pH values were between 6.0 and 8.0, and the DO values varied from 6 to 8 mg/L. The EC values were measured between approximately 515 and 648 µS/cm, while the redox potentials ranged from +69 to +148 mV. Subsequently, all relevant parameters fitted the optimal or at least the adequate range for African catfish. Across both experiments, as the concentration of the fertilizer increased, the redox potential decreased, but remained in a positive (oxidative) state. Since the decrease in the redox potential in Exp. B was not linear, it can be assumed that the deactivation of the redox-active metal ions (Zn²⁺) through the use of an EDTA chelator had a buffering effect on the redox potential (Reddy and Patrick, 1977; Tuncay et al., 2011). The Zn concentrations were measured continuously in short intervals through a monitoring system and were maintained at predefined levels between 0.125 and 0.5 mg/L in Exp. A and between 0.5 and 2.0 mg/L in Exp. B. Although, in some cases, the treatment groups exceeded the general Zn requirement of plants in an aquaponic system (0.45 mg/L), this was not found to have any serious effects on the fish. Further monitored water chemical parameters (i.e., O₂, pH, NH₄, NO₂, NO₃, and PO₄) remained unaffected by the different Zn-EDTA concentrations or appeared to be less influenced by the treatment. Although it is known that the toxic effect of heavy metals is influenced by the pH and the water hardness (Viljoen et al., 2003; Kiyani et al., 2013; Chidiebere, 2019), in the present study, values between 232 and 265 mg/L (CaCO₃) were detected, corresponding to medium-hard to hard water and contributing to a low toxic effect of the metal ions.

As the growth of two African catfish life stages was assessed as additional data in this study, it should be mentioned that both experiments were not aimed at achieving the most efficient growth performance possible for the respective fish. Both experiments were conducted in aquarium systems that allowed only a limited amount of feed (nutrients), ensuring the stability of the system. Consequently, the fish in all treatment groups showed only a few grams of growth. In addition, the mortality of the African catfish life stages was determined in this study. For the African catfish fry (1.15–1.63 g), typical mortality ratios were described, ranging from 4.3% to 20.0% (Adewolu et al., 2008; Marimuthu et al., 2011); for fingerlings (102–288 g), these varied from 2.0% to 4.8% (Van de Nieuwegiessen et al., 2009; Knaus et al., 2020). As the tested lethal concentration of elemental Zn by 50% in 96 h (LC₅₀) to the African catfish has been calculated by Ezeonyejiaku et al. (2011) to the value of 70.135 mg/L and by Chidiebere (2019) to 75.36 mg/L, the exposure concentrations of up to 2.0 mg/L in the current study showed low lethal influence on the investigated fish species. In the present study, there were low mortalities, as only one fish died inside the tank (A-ZnEDTA-0.5). A lethal effect of the various Zn concentrations could therefore not be determined since mostly fish, which died in the experiments, had escaped from their tanks.

In this study, physiological changes from exposure to Zn-EDTA, such as the distribution of leucocytes in fish, were additionally assessed for C. gariepinus fingerlings. The typical distributed leukocytes of unsexed African catfish (weighing approximately 320 g) have been described as 55.0% lymphocytes, 3.2% monocytes, and 43.4% neutrophils (Gabriel et al., 2004). Furthermore, Bello et al. (2014) described lymphocytes at 69.0%, monocytes at 3.0%, neutrophils/heterophils at 25.0%, and eosinophils at 3.0%. This study found the monocyte cell counts to be generally higher in African catfish fingerlings, while the neutrophil counts (even without Zn-EDTA) were lower compared with those in other studies. A significantly higher number of mature neutrophils in the ZnEDTA-2.0 group at the end of the experiment (after 32 days) were found along with a significant decrease in the number of meta-granulocytes between the initiation and the end of the experiment (21 days). Furthermore, the number of monocytes tended to increase over time (p< 0.1). According to Clauss et al. (2008); Havixbeck and Barreda (2015), and Havixbeck et al. (2016), a lower number of mature neutrophils in the peripheral blood together with a slight tendency to monocytosis, as observed in the present study, is suggestive of an incipient inflammatory response (day 21). As the mature neutrophil numbers decrease due to increased phagocytic activity, the proliferation and the formation of large numbers of new neutrophil meso- and metagranulocytes suggest an ongoing immune response. Increased numbers of mature neutrophils indicate the resolution of an earlier immune response as all pathogens have been eliminated (day 32). A potential inflammatory response in the current study was probably the result of severe skin lesions due to increased agonistic interactions rather than a response to the Zn concentration in the water. A distinct effect of the added Zn-EDTA is therefore questionable. Regarding the lesions observed in the African catfish, the average number of skin lesions in individual fish has been reported between 1.4 and 2.4 (Tort, 2011; Baßmann et al., 2017). In the current study, the skin lesion counts ranged from 0.39 to 1.42, which correspond to a normal range and were not influenced by the Zn treatments.

In previous studies, a broad span of different behavioral patterns of teleost fish in response to Zn exposure had been observed. These behavioral patterns include hyperactivity swimming at the surface, lethargy, and uncoordinated movements (C. gariepinus: Chidiebere, 2019); acute lethality (Scyliorhinus canicula: Crespo and Blasch, 1980); and rapid and erratic swimming, stereotypical movements, and loss of balance and general weakness with delayed reactions to light and sound (C. gariepinus: Ololade and Ogini, 2009). In the current study, significance in the behavioral alterations was found for swimming activity and air breathing in Exp. A and Exp. B ( Table 4 ). Previous studies have described the swimming activity of aquacultured African catfish to be 74.6%–99.0% (Van de Nieuwegiessen et al., 2008, 2009) and 71.9%–91.9% (Wenzel et al., 2022); therefore, the results of the current study are in the range between lower and normal activity. The swimming activity of the B-ZnEDTA-2.0 group on day 28 was significantly lower than that on day 8, but did not differ significantly from the control fish, which were more active. Therefore, a behavioral response to Zn-EDTA exposure cannot be excluded; however, the sample size was quite small, and other factors such as the group composition may have also played a role. Studies on the influence of Zn on the respiratory system of fish have shown that Zn disrupts the Ca²⁺ uptake in the gills of fish and causes hypoxemia, hypercapnia, acidosis, and hypocalcemia, depending on the carbonate concentration in the water (Skidmore, 1970; Spry and Wood, 1984; Sorensen, 1991), as cited in Crafford and Avenant-Oldewage (2011) and Chidiebere (2019). Van de Nieuwegiessen et al. (2008) observed air-breathing activities (i.e., event per fish per hour) between 12.0 and 104.0 with different fish stocking densities from 10 g to around 100 g, as well as activities between 80.0 and 83.0 with fish stocking densities from 102 to around 288 g (Van de Nieuwegiessen et al., 2009). In this study, a significant increase in air-breathing activity was found in the A-ZnEDTA-0.5 group between the start and the end of the experiment. As the effect also appeared in the control group in Exp. A and no similar effects were observed in Exp. B, a possible influence of the Zn concentration on this behavior pattern is questionable.

The dissected organs for histological microscopy analysis of C. gariepinus fingerlings revealed small alterations upon exposure to Zn-EDTA. Studies that examined the toxic effects of various concentrations of elemental Zn in different salts (ZnSO₄ or ZnCl₂) on O. mykiss, Tilapia sparmanii, or C. gariepinus revealed alterations in the epithelium (Lloyd, 1960; Skidmore, 1970; Hogstrand et al., 2002) and in the functionality of the gills (Burton et al., 1972; Mallatt, 1985; Van Rensbrug, 1989; Wood, 2017), as well as accumulations in different organs such as the gill, liver, muscle, and skin (Awoyemi et al., 2014; Duran et al., 2015). Later, using ICP-MS analysis, the combined effects and influence of Zn and Zn-EDTA (in concentrations from 4.0 to 8.0 mg/L) on C. carpio were investigated simultaneously by Karayakar et al. (2021) for more than 7 days of exposure. Their results revealed accumulations of Zn and Zn-EDTA in the tissues of the liver and gill at different uptake ratios. Moreover, they found a decreased ratio of Zn accumulation in tissue when the EDTA chelating agent was present in the treatment. In carp, the highest concentrations of Zn and Zn-EDTA were accordingly detected in the gills, as this organ is the most exposed to the environment of a fish (Chavan and Muley, 2014). Other studies investigated the toxic effects of Zn-EDTA on Palaemon elegans (Nugegoda and Rainbow, 1988). The authors described low Zn uptake in the presence of EDTA, as the chelator had a 1:1 molar binding ratio to Zn²⁺ and therefore reduced the amount of bioavailable Zn²⁺ ions (Nugegoda and Rainbow, 1988). Accordingly, a higher regulation ability of the Zn household was concluded. As metal components are taken up primarily by the fish liver, invading metal ions are therefore transformed into metallothionein proteins (Cherian and Nordberg, 1983; Shukla et al., 2007; Rose et al., 2014). These protein–metal complexes may be water-insoluble and become sequestrated in lipid or fatty tissues, where they accumulate (Montaser et al., 2010; Awoyemi et al., 2014). The polar property of the chelator and its ligands, in order to shift through a membrane and become a metal–protein complex, appears to play a key role in the accumulation process. Hydrophilic metal complexes (Zn-EDTA) do not appear to be able to cross the hydrophobic cell membrane, as it is negatively charged and lipophobic (Nugegoda and Rainbow, 1988). Unlike lipophobic complexes, lipophilic complexes (such as Fe-DTPA) are able to cross these cell membranes and start to accumulate (Asik et al., 2020; Hildebrand et al., 2023). Our data documented low effects of the Zn-EDTA treatment in the liver and moderate effects in the gill tissues based on the staining and microscopy results. As only a light coloration was observed in the liver tissue at a concentration of 2.0 mg/L Zn-EDTA after 32 days of exposure, the accumulation process of Zn-EDTA in the liver tissue of the investigated species could not be verified at a quantitative level using the staining method. Therefore, further investigations on the liver and gill tissues of C. gariepinus are recommend using advanced methods such as atomic absorption spectrophotometry (AAS) or inductively coupled plasma (ICP) analysis in order to verify and assess the accumulation process in this context more precisely. However, the present results are consistent with the findings and expectations of Nugegoda and Rainbow (1988) and Karayakar et al. (2021), who assumed and found a lower accumulation potential of Zn alone and in combination with EDTA chelating agents. In addition, this study emphasized the low toxicity of Zn-EDTA to fish internal organs, which would be expected to be much higher when using elemental Zn chloride or sulfate compounds for exposure (Awoyemi et al., 2014; Duran et al., 2015; Karayakar et al., 2021). As the major effects on the investigated organs in this study were primarily observed in the gill tissues, the present results of the grading system used (G0–G2, unremarkable to mild severity) were therefore based on the morphological alterations of the gill epithelium, which had been previously described by Lloyd (1960), Skidmore (1970), Zheng et al. (2011), and Srivatava and Prakash (2019), but did not have a significant effect on the respiratory system or the behavior.

Profiling of the genetic primers used for the African catfish, which have been established in a previous experiment (Hildebrand et al., 2023) through the catfish head kidney ( Table 1 ), showed the significantly different expression of KMT2A dependent on the exposure time and the Zn-EDTA concentration. KMT2A encodes lysine methyltransferase 2A, which is involved in the early phase of hematopoiesis and in wound healing (Huang et al., 2015; Zhang et al., 2017; Davis et al., 2019), as well as in the development of granulocytes (Fujiki et al., 2011; Ichikawa et al., 2021). Based on the above-mentioned observations, we assumed that the increased granulocyte counts that occurred at later time points in our experiments correlate with the observed elevated KMT2A transcript levels. Furthermore, inconspicuous genes such as MTF1 were similarly expressed through the treatments. As MTF1 is involved in the production of metal-binding metallothionein (Wimmer et al., 2005; Günther et al., 2012), its homogeneous expression across the various Zn-EDTA concentration groups indicates matching Zn families in the examined individuals and a protective effect of Zn-EDTA on the fish organs.

In the aquaculture of African catfish, the process water can reach Zn concentrations between 0.334 and 0.413 mg/L (Strauch et al., 2018). These concentrations are close to those used in hydroponic plant production (0.45 mg/L) (Bittsanszky et al., 2016). In addition to the fact that the Zn in the process water is derived from compounds (such as ZnO, ZnCl, and ZnSO₄) in the fish diet, it is not clear whether the Zn compounds in the recirculating water could cause problems for the fish at higher concentrations (Duran et al., 2015) or are harmless to their health. Moreover, it is not known whether additional Zn-EDTA in the process water positively affects the Zn family in the fish or the dissociation equilibrium of the Zn compounds in the systems when the percentage feed intake is higher than that of industry standards. In the present study, the tested Zn-EDTA concentrations of up to 0.5 mg/L did not have any severe effects either to the fry or to the fingerlings at feed intakes between 0.7 and 3.0% bodyweight per day. Furthermore, the tested Zn-EDTA concentrations were most likely not intense enough to trigger the induction of the various relevant genes selected for the current study, although the possibility that the expression of several other genes not included as target genes in the present analysis may well respond to Zn-EDTA exposure could not be ruled out. Only the 2.0 mg/L concentration revealed negative effects, including the Zn accumulation in the liver tissue in the fingerling group, demonstrating a potential overdose. Consequently, the fertilizer ratio for younger fish should be adjusted according to the supplementation process in order to not exceed the maximum Zn concentration in the system. Since the tested concentration of 0.5 mg/L Zn-EDTA in our study did not harm the fish, we support the supplementation of Zn-EDTA to African catfish up to this concentration level. This also allows the supplementation in coupled aquaponics.

The growth and mortality of African catfish were not affected by Zn-EDTA at the tested concentrations up to 2.0 mg/L in C. gariepinus fry and fingerlings. The ethological analysis also showed no behavioral dependence on Zn-EDTA treatment. Since only minor histomorphological changes in the gill tissue occurred, especially in treatments with 0.5 and 2.0 mg/L Zn-EDTA, and the fertilization recommendation for Zn in hydroponics is 0.45 mg/L, we do not recommend an addition to aquaponics with African catfish above 0.5 mg/L. Furthermore, long-term studies should clarify whether there can also be a stronger influence on or damage to the animals in the case of a prolonged exposure. Our study revealed new opportunities for the careful use of fertilizers such as Zn-EDTA for a more effective, sustainable water management for entrepreneurs with aquaponic systems. Accordingly, we can now confirm, thus far, the relatively harmless potential of Zn-EDTA as a necessary trace element for plants and its toxic potential to the African catfish (C. gariepinus) to ensure better plant growth without harming the cultivated fish species at the intended concentration of 0.45 mg/L.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The animal study was approved by Landesamt für Landwirtschaft, Lebensmittelsicherheitund Fischerei Mecklenburg-Vorpommern - Veterinärdienste und Landwirtschaft. The study was conducted in accordance with the local legislation and institutional requirements.

M-CH: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. AR: Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. TG: Investigation, Writing – original draft. HP: Funding acquisition, Supervision, Writing – review & editing. BB: Conceptualization, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.