Impact of fluoride on thyroid function and histopathology in cyprinus carpio: Implications for aquatic ecosystems.

Abstract

1. Introduction

Fluoride is an inorganic ion present in the natural environment in particulate and dissolved phases in the air, water, soil, and biota [1], [2]. Because of its positive effects when present in water at low concentrations, such as the prevention of tooth decay, its high concentrations in natural water bodies have caused severe concerns regarding adverse effects on human and ecological health [3]. Environmental fluoride pollution is on the rise with key human ventures, such as industrial discharge, agricultural waste, phosphate mining, and dumping high quantities of fluoride into water bodies [4]. In aquatic ecosystems, the presence of fluoride affects the physiology and health of fish in that habitat; fluoride is a sensitive bio-indicator because it comes into direct contact with contaminants [5].

Fluoride is a naturally occurring compound in water and its concentration depends on its location [6]. However, human influences, such as water pollutants from industrial effluents, poor disposal of waste, and the use of fluoride-containing fertilisers, have greatly influenced intensive fluoridation in many water sources [7]. Because fluoride ions penetrate water systems, they enter water and accumulate in the tissues of living organisms, disturbing their ability to correctly perform basic functions. Although fluoride has low toxicity at normal concentrations, it presents toxic effects at high concentrations or over long periods in a range of organisms, which are harmful to health through their interference with several important biological activities [8]. Signs of fluoride toxicity that have been reported by many researchers in fish include metabolic disorders, immunosuppression, stunted growth, and low fertility [9], [10].

Fluoride hazards are of immense concern in freshwater ecosystems, based on the diverse community of species inhabiting aquatic environments, and are sensitive to water quality shifts [11]. The toxicological impacts of fluorides are the same for all ecosystems, in addition to species-directed toxicities, with the effects manifesting through the organisms’ food webs and shifting distributions [12]. The presence of fluoride in freshwater habitats has considerable ecological and environmental impacts, which require further toxicological research.

It should also be noted that the endocrine system is among the most sensitive physiological systems to fluoride [13], [14]. In fish, the thyroid gland controls almost all major metabolic processes by releasing the end products of thyroxine T4 and T3. They have major functions in metabolic, growth, development, and reproductive processes in the body [15]. Interference with normal thyroid endocrine communication in water inhabitants results in dire ecological implications; they affect survivability and reproduction, and hence, the population and the balance thereof.

It causes antithyroid effects in several animal species, including fish, by inhibiting critical processes, such as the synthesis, secretion, and metabolism of thyroid hormones [16]. Jianjie et al. [17] indicated that changes in plasma concentrations of T4 and T3 were negatively affected by the inhibitory effects of fluoride in several fish species. Thompson et al. [18] posited that the primary metabolic process through which fluoride affects the thyroid is by reducing iodide uptake by the thyroid glands, impairing T4 to T3 conversion, and directly suppressing thyroid peroxidase, an enzyme required for thyroid hormone synthesis. These fluctuations in thyroid hormone levels have dire consequences on the physiology and overall welfare of aquatic species, including changes in their metabolism, retarded growth, and reproductive inefficiencies [16], [10].

In addition to the effects on thyroid hormone concentrations described above, the influence of fluoride on genes associated with thyroid hormone biosynthesis and action has also been identified [19]. For example, Sagliocchi et al. [20] used animal models to demonstrate that oxidative stress can influence the expression of enzymes such as deiodinase in the thyroid gland, and bioactive thyroid hormones such as T3 can be produced through the activation of T4 by these enzymes. Therefore, other agents, such as fluoride, which can cause oxidative stress, may even lead to upregulation of these enzymes, complicating the process of thyroid hormone homeostasis, which can elicit a chain of results throughout diverse biological processes and long-term impacts in the ecological system.

Thyroid hormones play a significant role in controlling fish metabolism, especially growth, development, and acclimatisation [21], [22]. This review provides an overview of various hormones and centres related to energy homeostasis in fish [23]. Most thyroid hormones in fish originate from the thyroid gland, although T4 dominates this gland. T4 is then converted to the more active form, T3, by deiodinase enzymes, because the body prefers using only T3 rather than storing it [24]. T3 controls significant physiological characteristics, including the metabolic rate of oxygen, rate of protein synthesis, and cardiovascular and nervous system activity [25]. Interference with this delicate hormonal balance by endocrine-disrupting chemicals such as fluoride affects fish well-being in ways that are not well understood, including poor body development, reduced fertility, and changes in stress [26].

As in other ectothermic animals, thyroid hormones are involved in osmoregulatory mechanisms, such as regular and orderly regulation and control of water and ionic concentrations, and thermoregulation of temperature adaptation [27]. Therefore, changes in thyroid activity severely influence the susceptibility of fish to death in contaminated water [28], [10]. In particular, it was found that due to the lowering of serum T4 and T3 concentrations by fluoride, the ability of fish to sustain the metabolic balance under the influence of other pollutants or climate change is reduced.

As thyroid hormones are essential for maintaining physiological homeostasis in fish, the negative effects of fluoride on thyroid activity are a crucial ecological issue [29], [30]. Fluoride pollution of the aquatic environment is universal, and the hazards of health impacts on fish may not only be confined to the level of the fish population but could also extend to the general population and the ecosystem [31]. In changes in the metabolic rate, growth, and reproductive activity of fish may have a vague impact on the population density of these species, thereby affecting the trophic structure and stability of freshwater ecosystems [25].

Cyprinus carpio was chosen as the model organism because of its ecological relevance, high physiological responsiveness, and utilisation in prior ecotoxicological investigations [32]. It is a bottom-dwelling fish that inhabits freshwater environments and is frequently affected by industrial and agricultural pollutants [33]. Therefore, it is a useful bottom-up bioassay for pollutants. The endocrine system is similar to that observed in other aquatic animals; hence, observations can be generalised more easily. Thus, Carassius carpio, as a model species, exhibits clear indices of physiological and histological changes to pollutants, such as thyroid hormones and glands, which are important for determining the effects of fluoride on aquatic environments [34]. Furthermore, its sound and highly specific control of the laboratory environment, as well as the large baseline data on its endocrinology and metabolic systems, guarantee positive and constant results.

This study also shows that fluoride pollution has a number of ecological implications that are not seen in other pollutants because it can only be episodic and stochastic; however, this distortion can greatly increase the level of contamination resulting from the discharge of industrial waste or agricultural chemicals. These characteristics make it critical to examine chronic, low-level fluoride exposure regimes for their combined, chronic impact on the life of fish species. Additionally, the results help explain the contradiction of the fact that fluorides are essential ions in large quantities in the human body, but in large concentrations, they are endocrine disruptors. The observed dose-dependent inhibitory effects on thyroid hormones and pathological alterations in the thyroid gland indicate the potential of fluoride to affect metabolic and reproductive processes in fish. This study focuses on the overall consequences of such disruptions in the food web structure and the stability of pelagic ecosystems. As one of the most popularly distributed and ecologically significant fish species, the present investigation offers important information for the development of new legislation to protect freshwater fish populations from fluoride toxicity.

Therefore, the present study was carried out to determine the impact of fluorides on thyroid hormones in Cyprinus carpio, which is known to have a higher tolerance to water pollutants and is mostly found in freshwater systems. Therefore, the present study aimed to determine the effect of fluoride on thyroid hormones in Cyprinus carpio, a preferred fish in ecotoxicity studies. Therefore, the findings of this study, which quantified blood plasma T4 and T3 concentrations in fish, will elucidate the interference effects of sublethal concentrations of fluoride in freshwater fish. The outcomes of this study may be of great interest to environmental managers and policymakers in developing improved strategies for the environmental management of the fluoride burden in drinking water and freshwater aquatic ecosystems.2.

Materials and methods

2.1. Study organism

This experiment was conducted on Cyprinus carpio, which is ranked as the most sensitive model fish species for ecotoxicological studies. The fish fingers were juvenile fish, 4–6cm in size, obtained from a farm in Ekurhuleni and held in the laboratory for 14 days. While acclimating fish were placed in 500L tanks used for holding fish throughout the experiment, aeration was used to provide adequate water circulation. The temperature inside the tanks was maintained at 27–28°C, pH was 7.0–7.5 and the dissolved oxygen level was above 5mg/L, which was important for the species. The tested fish were fed a standard commercial diet daily, and there were no mortalities during acclimatisation.

2.2. Control conditions and maintenance

To confirm the viability of the results, a control group consisting of Cyprinus carpio was kept in distilled, dechlorinated water free of fluoride, but with conditions similar to those obtained in the treatment groups where the fish were exposed to fluoride. Other critical factors such as water temperature (27–28°C), pH (7.0–7.5), and dissolved oxygen (>5mg/L) were maintained and regulated adequately. They provided the fish with a standard diet daily, and the tanks were cleaned to avoid accumulation of debris. The light/dark cycle was regulated at 12:12h to reduce stress, and all actions related to sampling were performed in the same manner for all groups. It also kept the ridge of the control group clear of contamination by extraneous variables or other treatments [35].

2.3. Evaluations of morphological alterations

Stereological assessment of thyroid gland architecture was performed to supplement the histological findings. Light microscopy and improving software were used to determine follicular size and epithelial thickness between the control and fluoride-exposed groups. The follicle size was homogenised, with the mean diameter of the follicles measuring 60±5 m, and the measured epithelial layer thickness was 10 ± 2 m. Slightly larger changes were observed in the high fluoride group (10mg/L): the mean follicular diameter was 35± 4 µm (p <0.05). Epithelial hypertrophy was also observed, and the epithelial thickness increased to 18 ± 3µm in the high-fluoride group compared to that in the control group (p<0.05). The changes in the medium-fluoride group (5mg/L) were intermediate, the follicular diameter was 45±6µm, and the epithelial thickness was 14±3µm. These quantitative data also support the histopathological observations established in the present investigation, capable of presenting statistical confirmation of the dose-dependent changes in thyroid gland architecture induced by fluoride exposure in experimental animals. These improvements should provide a more reliable interpretation of histological alterations and their association with thyroid disturbance [36], [37].

2.4. Fluoride exposure

Fluoride exposure treatments were prepared using analytical-grade sodium fluoride (Sigma-Aldrich, USA). Fluoride levels were reported in an earlier study [26], suggesting that the chosen levels were sublethal, with three groups used for low, middle, and high FL exposure. These groups were exposed to the following fluoride concentrations: low, 1mg/L; medium, 5mg/L; high, 10mg/L. The respective control groups were maintained in nonfluoride water. All exposure tanks were filled with 500L of dechlorinated water, and the fluoride concentrations were checked every two days to support a stable level during the study using an ion-selective electrode (ISE) Orion 9609BN, USA, Thermo Fisher. Fluoride was resupplied when it was depleted, and water quality factors were analysed periodically.

2.5. Blood sampling and hormonal analysis

Blood samples from six fish in all treatment groups were collected at four intervals (7, 14, 21, and 35 days) according to the guidelines for the use of animals in teaching and research. Venous blood was collected from the caudal vein using 1ml sterile syringes and placed in heparinised tubes. Blood plasma was prepared by centrifuging blood samples at 3000 rpm for 10 min in a cool centrifuge at 4°C. The collected plasma was aliquoted and stored at 20°C until further use.Non-individualised thyroxine (T4) and triiodothyronine (T3) levels were analysed using enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific,India). ELISA was performed according to the manufacturer’s instructions, and the optical density of each sample was measured at 450 nm on a microplate reader (BioTek Instruments, India). Each sample was tested in triplicates to ensure high accuracy. The sensitivity of the assay for T4 was determined to be 0.05 ng/ml and that of T3 at 0.02 ng/ml.

2.6. Statistical analysis

Clinical data were analysed using one-way ANOVA to compare plasma T4 and T3 concentrations between the control and treatment groups. LD means were compared using Tukey’s HSD test at each time point to determine differences in fluoride concentrations. All statistical analyses were performed using the SPSS software (SPSS 23.0.). Hence, the significance level was set at p <0.05. Data are presented as mean ± standard error of the mean (SEM).

2.7. Ethical considerations and post-experimentation procedures

At the end of the experimental period, fish were anaesthetized with an overdose (tricaine methanesulfonate, Sigma-Aldrich, India), which is considered humane when used on aquatic animals. After euthanasia, the fish were placed in a tank containing the lethal concentration of the anaesthetic agent. Subsequently, all fish tissues were treated with appropriate precision and used for histological and biochemical examinations.

3. Results

3.1. Effects of fluoride on plasma thyroxine (T4) levels in Cyprinus carpio

3.2. Effects of fluoride on plasma triiodothyronine (T3) levels

Similarly, in Cyprinus carpio, plasma T3 levels were affected by fluoride exposure (Fig. 2). By the end of the study on the seventh day, the T3 levels in the low-fluoride group were similar to those in the control group. On day 14, T3 levels within the media and high fluoride groups were considerably decreased, and T3 levels in media with 5 ppm of high fluoride were significantly different from the control (by 50%; p <0.01) (Fig. 2). The decrease in T3 concentration was sustained in this study, whereby the high-fluoride group had the lowest concentration of T3 on days 21 and 35 (Fig. 2).

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Fig. 2. Effects of parameters in exposed to sub-lethal concentration of Fluoride Significant differences between experimental groups and the control group are revealed by different letters (p <0.05). Data are demonstrated as mean ± S.E.

3.3. Dose-response relationship between fluoride concentration and hormone levels

3.4. Observations of thyroid gland

The thyroid glands of the exposed fish were observed under a light microscope (Fig. 3). Normal thyroid follicles with well-preserved epithelial architecture were observed in the control group, suggesting normal thyroid function. However, in the high fluoride exposure group, the size of the thyroid follicles was significantly smaller, and epithelial cells of the thyroid gland showed hypertrophy and hyperplastic changes, indicating a functional disorder of the thyroid gland (Fig. 3). The medium fluoride group also showed similar changes, although slight, in comparison with those observed in the high fluoride group.

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Fig. 3. Schematic representation of Cyprinus carpio exposed Fluoride particle using in aquatic ecology.

4. Discussion

The findings of this study provide strong evidence that fluoride interferes with the endocrine system of thyroid hormones in Cyprinus carpio, a preferred ecotoxicological model organism. More specifically, this study revealed a significant decline in plasma thyroxine (T4) and triiodothyronine (T3) levels as well as thyroid histopathological changes in fish subjected to multiple fluoride concentrations. The information reported here is consistent with literature reviews and experimental data that illustrate the endocrine-disrupting efficacy of fluoride in fish species and contribute to the hypothesis documenting metabolic regulation dysfunction and overall reduced fish health.

4.1. Fluoride-induced disruption of thyroid function

The T4 and T3 recorded in the present study in response to increased fluoride concentrations in Cyprinus carpio agree with earlier related studies that have shown the same effect in fish species under sub-lethal concentrations of the chemical. In this study, water with 5 mg/L and 10 mg/L of fluorides significantly reduced T4 levels to approximately 42% and 50%, respectively, after 35 d of water exposure. In terms of functional changes, the high fluoride group also had their T3 concentrations reduced by half compared to the control. This dose-dependent suppression is in concordance with the study by Nouri et al. [38], who reported lower T4 and T3 concentrations in Oncorhynchus mykiss exposed to fluoride at 5 and 10mg/L. Therefore, the effect of fluoride on the thyroid appears to be species- and concentration-dependent.

The influence of fluoride disorganisation on the thyroid system can be caused by interference with the most significant thyroid processes, explained by the synthesis of thyroid hormones, as well as their secretion and metabolism. The reduction in T4 and T3 levels is likely due to several mechanisms. There are three main mechanisms by which goitrogens cause hypothyroidism: blocking iodide uptake by the thyroid gland, interference with thyroid peroxidase enzyme activity, and blocking the functioning of deiodinase enzymes that convert T4 to T3.

This is in concordance with Liu et al. [39], who opined that fluoride exposure in Carassius auratus (goldfish) brought about a similar suppression of thyroid hormones, causing metabolic breakdown. Interestingly, in the present investigation, a much higher concentration of fluoride (10 mg/L) reduced T4 and T3 to a greater extent, and thus, the thyroid dysfunction was severe at higher concentrations of fluoride. In this regard, the observation made here dovetails with the dose response, represented in Fig. 2 by Waugh et al. [43], concerning the effect of excessive fluoride levels on thyroid hormones in Salmo salar.

Notably, T3 levels in the present study were significantly lower than the baseline, a difference that was greater than the decrease in T4 levels. T3 decreased more significantly, which could be associated with the effect of fluoride on the action of the three deiodinase enzymes in the conversion of T4 to T3. The mechanism of action has been reviewed in various studies. For instance, Rodríguez et al. [26] established that fluoride exposure reduces deiodinase activity in Gambusia affinis, thereby limiting the transformation of exercised T4 to the active T3 form, which prescribes low circulating T3 levels. In our data are in line with this assumption that fluoride may influence the synthesis and rate of conversion required for thyroid hormone regulation., Rodríguez et al. [26] confirmed that the testes of Pimephales promelas had follicular atrophy and epithelial hyperplasia related to fluoride, and excess damage to testicular tissue at the cellular level by fluoride exposure. Therefore, the present study employed a similar exposed fish, Oryzias latipes, and referred to the study by Zhang et al. [40] to show thyroid follicle damage, which simultaneously explained the direct structural effect of fluoride on the thyroid gland.

The histological changes noted in the current study may also be attributed to the toxicity of fluoride on the gland during the manufacture and control of Thyroxin and Triiodothyronine. Previous studies have demonstrated that the use of fluorides leads to necrosis of thyroid tissue and directly influences hormone secretion [10]. Thus, the key position of the thyroid gland as a final link in metabolic processes is emphasised, and the effects of fluorides on organs trigger a series of endocrine disorders [17], [20].

The alterations in thyroid function due to fluoride in Cyprinus carpio reflect other studies on endocrine-disrupting chemicals (EDCs) in aquatic ecosystems. Some of the EDCs are industrial chemicals, pesticides, and pharmaceutical products that impact the hormonal way of action and result in negative effects on growth, reproduction, and metabolic processes in living aquatic species. Similar to fluoride, chemicals such as bisphenol A (BPA) and polychlorinated biphenyls (PCBs) disrupt endocrine system stability, affecting aquatic life as well. Therefore, these results show the importance of a holistic approach to the regulation of EDCs, since individual or combined impacts may increase or interact to worsen endocrine disruption and ecosystem impacts.

4.2. Ecological implications of fluoride-induced endocrine disruption

4.3. Comparison with findings in other fish species

4.4. Reversal of thyroid dysfunctions

In this case, it is also relevant to investigate whether thyroid disturbances in Cyprinus carpio are reversible after the elimination of fluoride exposure in aquatic systems [26], [45]. Earlier research on other species including rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) have shown that even after removal from water containing fluoride or any other EDCs, hormone levels may only improve partially [26], [45]; Nouri Recovery generally depends on such parameters as time and concentration of exposure, reserves of thyroid tissues and general reactivity of the organism [46]. In the current study, the observed depleted T4 and T3 levels, together with histopathological changes such as follicular atrophy and hypertrophy of the epithelial layer, suggested that fuller recovery could be a problem when exposed to high concentrations for longer periods of time. Nevertheless, thyroid tissue regeneration ability revealed by other scholars indicates that if the cause of the fluoride impact is removed, then regenerative ability is possible if the damage is not end-stage irreversible.

5. The limitations and future directions

The present study aimed to investigate the effect of fluoride on thyroid hormone levels and histopathological changes in Cyprinus carpio based on the concentration of fluoride. However, it could not explore molecular mechanisms or the outcomes of their mitigation-recovery potential, mainly because of constraints in funding and resources. Further research should involve other modes of action, such as iodide uptake inhibition and transgenerational effects, to gain a better understanding of the effects of fluoride on endocrine disruptions.

6. Conclusion

The present study demonstrates the impact of non-toxic fluoride concentrations on the thyroid hormone system in Cyprinus carpio, a model organism in ecotoxicological studies. A systematic examination was conducted to establish a strong negative correlation between F exposure and plasma T4 and T3 levels as well as histopathological changes in the thyroid gland. These disruptions were dose related, and the most significant effect was observed when the tap water fluoride concentration was 10 mg/L, resulting in the highest observed decrease in T4 and T3 levels.Therefore, these findings support other studies showing that graded levels of fluoride affect the endocrine systems of fish and other aquatic organisms. Similar thyroid hormone alterations in animals such as Oncorhynchus mykiss, Danio rerio, and Pimephales promelas have indicated that fluoride exposure decreases thyroid hormone levels, which affects thyroid histopathology. The present study builds on these findings by demonstrating that fluoride treatment results in thyroid dysfunction in Cyprinus carpio, and that the effect is dependent on fluoride concentration.Disturbance of thyroid hormones, which are essential for homeostatic processes including developmental, metabolic, and reproductive functions, is of significance in terms of ecological impact. Therefore, the long-term cumulative effects of fluoride on fish development could threaten the stability of aquatic environments, fish stocks, and fish growth. Fluoride is a common water pollutant owing to its industrial and agricultural effects on freshwater supplies; thus, higher standards should be imposed on fluoride concentration and intensively supervised to minimise ecological effects.In conclusion, this study was partially funded to increase the archives of research on fluoride bioaccumulation, its toxic impact on aquatic ecosystems, and its association with endocrine disruptions. Further experimental work is needed to ascertain the chronic toxic effects of fluoride on fish stocks and other life forms in aquatic systems. Furthermore, studies focused on elucidating the possible pathways through which fluoride disrupts thyroid function and its effects on other types of organisms will be useful in designing efficient mechanisms to protect organisms from fluoride toxicity.

Declaration of Competing Interest

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