Something Fishy: Fluoride and Zebrafish
Modern developmental toxicology and the evidence for thyroid hormone disruption
June is Thyroid Awareness Month in Canada.
This is the third article in our series exploring the scientific literature on fluoride’s effects on thyroid hormone metabolism and activity, from early pharmacological bioassays to modern toxicology.
In the first two articles, we examined fluoride’s effects on thyroid activity through two established pharmacological tests: the Gudernatsch Tadpole Test and the Reid Hunt Reaction. Both tests were built around reproducible biological effects. In the Gudernatsch Tadpole Test, thyroid material induced metamorphosis in tadpoles. In the Reid Hunt Reaction, thyroid material increased resistance to acetonitrile poisoning. In both cases, fluoride interfered with a thyroid-dependent biological response. Thousands of patients suffering from iodine-induced hyperthyroidism were treated with fluoride compounds, spanning a period of at least 60 years.
These tests belonged to an earlier era of thyroid pharmacology, but the principle remains important. Thyroid hormone activity is not defined only by serum hormone levels. It is also revealed by biological response. A substance that interferes with thyroid hormone metabolism or action may therefore be detected through altered development, altered toxicant response, or disrupted tissue-level hormone activity.
That brings us to zebrafish.
Zebrafish - Danio Rerio
Zebrafish entered thyroid toxicology by a different route than tadpoles. Tadpoles were the classical model because thyroid hormone visibly controls metamorphosis. Zebrafish became important later, through developmental biology, genetics, toxicology, and pharmacology (Streisinger et al., 1981; Kimmel et al., 1995).
Their embryos develop outside the mother, grow rapidly, and remain transparent during early development. This allows researchers to observe developmental effects directly while also measuring changes in thyroid hormone synthesis, transport, metabolism, and signalling.
In 2002, Wendl and colleagues introduced zebrafish as a model for thyroid development, showing that thyroid follicle formation could be studied genetically in this small aquatic vertebrate (Wendl et al., 2002).
Since then, zebrafish have become widely used in thyroid toxicology, especially during early developmental windows that are difficult to examine in mammals (Mezzalira et al., 2026; Porazzi et al., 2009).
By 2017, almost all components of the zebrafish thyroid axis had been characterized and found to be structurally and functionally comparable with those of higher vertebrates (Marelli & Persani, 2017).
The zebrafish therefore belong to a modern toxicological tradition, but the underlying principle is familiar from older pharmacology. Like the older thyroid bioassays, they ask whether a chemical alters a biological response. The difference is that zebrafish allow that response to be measured at several levels at once: gene expression, hormone signalling, development, behaviour, and survival.
Fluoride and Zebrafish
Just as most of the early studies on fluoride effects using the Gudernatsch Tadpole Test and the Reid Hunt Reaction were conducted in Germany, much of the modern research on fluoride effects on the thyroid has been conducted in China.
This is not surprising. China has a major endemic fluorosis problem in 28 out of 31 provincial-level administrative divisions (PLADs), affecting over 70 million individuals. The problem extends to 7,100 villages, distributed over 1,000 counties in China (CDC China 2021; Zhao et al., 2024).
China also has a serious problem with high iodine intake, largely related to its mandatory salt iodization program, which began in the mid-1990s and contributed to excessive iodine intake in many areas that already had high iodine levels in drinking water or from coal pollution. China moved rapidly from iodine-deficiency control to periods of excessive and more-than-adequate iodine intake after universal salt iodization: national schoolchild median urinary iodine rose from 164.8 µg/L in 1995 to 330.2 µg/L in 1997 and 306.0 µg/L in 1999; later national values were mostly in the more-than-adequate range, while some provinces remained excessive (Li et al., 2011; Liu et al., 2020).
This helps explain why so much of the modern fluoride-thyroid research has come from China. China is not simply a country with endemic fluorosis. It is also a country where fluoride exposure has coincided with both iodine deficiency and iodine excess in real populations. That history helps explain why Chinese researchers have devoted so much attention to fluoride’s effects on iodine metabolism and thyroid function.
Although there are many studies on fluoride effects on zebrafish generally, all of the studies on fluoride’s effects on the thyroid in zebrafish were conducted by a Chinese group of researchers led by Jianjie Chen and Prof. Jinling Cao at Shanxi Agricultural University. Remarkably, no studies on this specific subject appear to have been conducted in other countries.
Chen et al., 2016 - Males
The first study by this team was published in 2016. This was the same year that the National Toxicology Program in the US started its fluoride/neurodevelopment review. The Chen study exposed 600 male zebrafish to different fluoride concentrations and followed them for 90 days. Effects were measured at the end of the exposure period, as well as halfway through, at 45 days.
The results showed clear disruption of the thyroid system. Fluoride-exposed fish had increased T3 levels throughout the exposure period, while T4 levels declined at the higher concentrations after 90 days. This pattern is consistent with altered thyroid hormone metabolism and disturbed deiodinase activity. Stronger evidence came from the gene-expression data. Fluoride changed the expression of multiple genes in the hypothalamic-pituitary-thyroid axis, including NIS, TG, DIO1, DIO2, TTR, UGT1ab, and thyroid hormone receptors.
The timing also mattered. After 45 days, many thyroid-related genes were up-regulated, suggesting an activated or compensatory thyroid response. After 90 days, some responses persisted, while others weakened or reversed. The clearest example was DIO2, the gene encoding the D2 deiodinase that converts T4 into active T3 within tissues. DIO2 expression increased after 45 days, but decreased after 90 days in the higher fluoride groups. This showed that fluoride’s effects depended on both exposure time and dose.
Guo et al., 2019 - Females and Sex Differences
The group’s second study was a follow-up using the same basic exposure design, but this time in adult female zebrafish (Guo et al., 2019). Female fish were exposed to 0, 20, 40, and 80 mg/L of sodium fluoride for 45 and 90 days. As in the male study, fluoride impaired growth, damaged thyroid tissue, altered T3 and T4 levels, and changed expression of HPT-axis genes.
However, there were great differences in the direction of response. In the male study, fluoride increased all measured HPT-axis genes after 45 days. In females, fluoride decreased most measured HPT-axis genes at 45 days, especially at 40 and 80 mg/L. By 90 days, TG, DIO1, and DIO2 were increased at the higher concentrations, while UGT1AB was decreased at 80 mg/L.
Taken together, the two studies show that fluoride’s thyroid effects in zebrafish were dose-dependent, time-dependent, and sex-dependent. Male and female zebrafish both showed thyroid disruption, but the pattern was not the same.
Lu et al., 2022 - Lead & Fluoride
The team’s third study was published in 2022, and involved the effects of both lead and fluoride together (Lu et al., 2022). Again, the zebrafish were evaluated after 45 and 90 days.
The main finding was that both fluoride and lead damaged the zebrafish thyroid system. Thyroid sections showed loss of colloid/glia and follicular epithelial hyperplasia, indicating structural thyroid injury. Fluoride and lead also disturbed oxidative stress balance in the thyroid, altered T3 and T4 levels, and changed expression of thyroid endocrine-related genes.
The combined exposure was more damaging than either chemical alone. The authors reported that F + Pb aggravated thyroid hormone disruption, produced stronger oxidative stress effects, and had mainly additive effects, with some synergistic effects on thyroid gland damage.
Again, it was found that males were more sensitive than females to fluoride or lead thyroid toxicity. That lead and fluoride may have additive and synergistic effects has been shown in other studies from around the world.
Chen et al., 2023 - Embryos
In 2023, Chen’s team investigated the effects of fluoride on zebrafish embryos and larvae, again using sodium fluoride solutions of 0, 20, 40, and 80 mg/L (Chen et al., 2023).
This fourth zebrafish study shifted the focus from adult thyroid disruption to early-life developmental toxicity. Instead of exposing adult male or female fish for 45 and 90 days, the researchers exposed zebrafish embryos and larvae for 9 days. They measured hatching rate, malformation rate, mortality, heart rate, oxidative stress markers, T3 and T4, and expression of HPT-axis genes.
The developmental toxicity was clear. Fluoride decreased hatching rate and heart rate, while increasing malformation rate and mortality. Reported abnormalities included developmental delay, spinal curvature, tail deformity, pericardial edema, and yolk edema. These effects generally worsened as fluoride concentration and exposure time increased.
The thyroid hormone response was biphasic over time. At 3 days, fluoride reduced T3 and T4 at the higher concentrations. By 6 and 9 days, the response had reversed, with T3 and T4 increasing in several exposed groups, especially at higher concentrations.
The gene-expression results also changed over time. At 3 days, CRH and NIS increased, while TSH, TRα, TRβ, and NKX2.1a decreased. At 6 days, CRH, TRα, and TRβ increased, while TSH and NIS decreased. At 9 days, TSH, TRα, TRβ, and NKX2.1a increased, while NIS and UGT1ab decreased; TG increased at 40 and 80 mg/L.
Taken together, these studies form a coherent zebrafish record. Fluoride disrupted thyroid hormones, thyroid tissue, and HPT-axis gene expression, with downstream effects on oxidative stress, development, and survival. The effects depended on dose, exposure duration, sex, and developmental stage.
It therefore came as no surprise when later studies by this team showed that fluoride induced neurotoxicity in zebrafish (Xu et al., 2024; Chen et al., 2025).
The NTP and SEAZIT
By 2016, zebrafish had become an established model for studying developmental toxicity and thyroid disruption. That same year, the NTP began SEAZIT, the Systematic Evaluation of the Application of Zebrafish in Toxicology, to improve consistency between laboratories.
As mentioned above, 2016 was also the year the NTP began its investigation into fluoride’s effects on neurodevelopment.
That timing is hard to ignore.
Zebrafish studies were directly relevant because they measured thyroid hormones, HPT-axis gene expression, tissue effects, and development.
The NTP was supposed to “assess effects on thyroid function to help evaluate potential mechanisms of impaired neurological function.”
Yet the NTP made no meaningful effort to investigate these mechanisms. It did not evaluate the zebrafish fluoride thyroid literature. It did not conduct zebrafish fluoride assays of its own. Nor did it examine the older pharmacological evidence from the Gudernatsch Tadpole Test, the Reid Hunt Reaction, or the research on fluoride treatment in iodine-induced hyperthyroidism.
An oversight?
The earlier pharmacological record showed fluoride interfering with thyroid-dependent biological responses. The modern zebrafish studies showed fluoride altering thyroid hormones, thyroid tissue, and HPT-axis genes.
Instead, the NTP relied heavily on a highly questionable rodent study (McPherson et al., 2018) and changed its own protocol regarding iodine as a key confounder (PFPC, 2023), despite decades of research showing that fluoride and iodine interacted biologically.
Experiments using the Gudernatsch Tadpole Test had shown that the iodine-to-fluoride ratio measured in blood was approximately the same as the ratio required for fluoride to antagonize T4 activity in tadpoles, about 1 part iodine to 7 parts fluoride.
Later studies reached the same conclusion: fluoride toxicity was not isolated, but depended on iodine status, thyroid function, and the balance between iodine and fluoride.
If iodine was prevalent, iodine toxicity dominated; if fluoride was prevalent, fluoride toxicity dominated.
For a review charged with exploring mechanistic evidence, ignoring both the zebrafish evidence and the older pharmacological record is hard to defend.
Very fishy, indeed.
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