Researchers at the University of Santiago de Compostela in Spain, led by Luis Rodríguez-Santos and Manuel Botana, carried out a 28-day repeated-dose oral toxicity study in mice, comparing the three principal diarrhetic shellfish toxins: okadaic acid, dinophysistoxin-1 and dinophysistoxin-2. The work, published in Current Research in Food Science, was designed to answer a question that regulatory agencies, including the World Health Organization and the Food and Agriculture Organization, have explicitly flagged as unresolved: what happens to the body when these toxins are ingested day after day at low doses, rather than in a single large serving?

The experimental design is notable for its attempt to mimic real human dietary exposure. Rather than forcing toxin solutions into the animals by gavage, the standard but stressful technique, the team delivered the toxins inside small pieces of bread that the mice voluntarily ate, a welfare-friendly approach that avoids confounding the results with handling stress. The daily dose of 90 micrograms per kilogram of body weight was calibrated to model a high-intake human dietary scenario derived from the European regulatory limit of 160 micrograms of okadaic acid equivalents per kilogram of shellfish meat. That calculation assumes a large 400-gram portion of contaminated bivalves for a 70-kilogram adult, then applies the standard 100-fold uncertainty factor used in toxicology to bridge the gap between animal and human exposure.

Over four weeks, the mice showed a pattern of persistent, cumulative toxicity that single-dose studies simply cannot capture. Dinophysistoxin-1 emerged as the most troublesome analogue, producing the greatest number of non-specific clinical signs and the most severe diarrhea, with significant effects in both sexes throughout the first three weeks of the study. Okadaic acid also caused significant clinical signs and diarrhea, particularly in females, whose fecal consistency scores became significantly worse than controls from the second week onward. Dinophysistoxin-2, by contrast, behaved remarkably differently: it produced almost no significant diarrhea at any point, reinforcing its reputation as the mildest of the three toxins.

That difference in clinical severity tracks closely with the toxins’ behavior inside the body. Using ultra-performance liquid chromatography coupled with tandem mass spectrometry, the researchers measured toxin concentrations in feces, intestinal contents and organs. Dinophysistoxin-2 was excreted rapidly and abundantly in feces while remaining barely detectable in tissues, suggesting poor gastrointestinal absorption and fast clearance that limits its systemic bioavailability. Dinophysistoxin-1 showed the opposite profile: the lowest fecal excretion but proportionally greater retention in the intestinal wall, indicating prolonged contact between the toxin and the gut lining. Okadaic acid fell somewhere in between, with high initial fecal excretion that declined over the study period. The highest tissue concentrations of all three toxins were found in the small and large intestines, precisely where the diarrheic damage occurs.

The microscopic evidence added another layer of concern. After 28 days of exposure, mice receiving okadaic acid and dinophysistoxin-1 showed significant thinning of the gastric mucosa, the protective lining of the stomach, a change the authors interpret as chronic epithelial alteration. Kidney sections revealed vascular congestion, tubular damage and an enlarged Bowman’s space, the cup-like structure where blood filtration begins, in animals treated with okadaic acid or dinophysistoxin-1, while dinophysistoxin-2-treated animals showed essentially preserved renal morphology. In the liver, dinophysistoxin-1 caused mild vascular changes, including dilated sinusoids and red blood cell extravasation, though hepatocyte architecture remained largely intact. Male mice given okadaic acid also showed a significant increase in relative heart weight, an intriguing systemic signal that warrants follow-up.

Perhaps the most striking findings involved sex. Female mice reduced their water intake significantly from day 14 onward, particularly those receiving okadaic acid, and showed trends toward lower urine output and higher urine protein-to-creatinine ratios, hints of subtle renal stress that the authors caution are qualitative rather than definitive proof of kidney dysfunction. Males, meanwhile, displayed more non-specific clinical symptoms overall, while females suffered more pronounced gastrointestinal effects. This sexual dimorphism in toxin response is a critical consideration for risk assessment, since most toxicological reference values are derived without fully accounting for sex-specific vulnerability.

The team also pioneered a novel biomarker approach, examining cyclophilin A and cyclophilin C, evolutionarily conserved proteins released into the extracellular space during inflammatory responses, in the intestinal contents of exposed mice. Detectable cyclophilin A and C appeared exclusively in the small intestinal contents of dinophysistoxin-1-treated animals, consistent with that toxin’s longer residence time in the gut and its harsher clinical profile. Cyclophilin C was absent from the intestinal contents of dinophysistoxin-2-treated animals across all replicates, mirroring that toxin’s milder effects. Because luminal contents lack stable housekeeping proteins for normalization, the authors present these results as descriptive qualitative support rather than quantified proof of inflammation, but the correlation with the toxicokinetic and clinical data is compelling.

The implications for the seafood consumer are nuanced. The authors are careful to note that their findings do not demonstrate that the current European regulatory limit is inadequate, nor do they establish a tolerable daily intake for these toxins. What the study does provide is the first comparative assessment of all three diarrhetic shellfish toxins under subchronic conditions using a voluntary feeding model that closely replicates natural dietary exposure. The data suggest that current toxicity equivalency factors, which assign okadaic acid and dinophysistoxin-1 a potency of 1 and dinophysistoxin-2 a potency of 0.6 based on older in vitro and acute intraperitoneal data, may need revision in light of oral toxicity evidence showing dinophysistoxin-1 to be substantially more harmful than its legal classification implies.

With climate change intensifying harmful algal blooms worldwide and extending the periods during which shellfish carry low but persistent toxin loads, the scenario modeled in this study becomes increasingly plausible for regular seafood consumers. The researchers acknowledge limitations, including the modest sample size of five animals per sex per group, the single exposure level tested, and the 28-day subchronic window, which falls short of the 90-day or lifetime studies needed to establish formal no-observed-adverse-effect levels. Still, by demonstrating that repeated low-dose exposure produces cumulative gastrointestinal, renal and hepatic effects with distinct toxin-specific and sex-specific profiles, the study delivers exactly the kind of harmonized comparative evidence that international expert panels have demanded, and it makes a strong case that the safety of the world’s favorite shellfish deserves evaluation through a longer, more chronic lens.

Subject of Research: Subchronic oral toxicity of diarrhetic shellfish toxins (okadaic acid, DTX1 and DTX2) in mice under repeated low-dose dietary exposure

Article Title: Regular exposure to low dose of diarrheic shellfish toxins: Implications for the seafood consumer

Article References: Rodríguez-Santos, L., Louzao, M. C., Cagide, E., Alvarez, M., Vale, C., Cifuentes, J. M., Vilariño, N., Graña, A., Carrera, C., Alvariño, R., Vieytes, M. R., Lolo, M., & Botana, L. M. (2026). Regular exposure to low dose of diarrheic shellfish toxins: Implications for the seafood consumer. Current Research in Food Science, 13, Article 101574. https://doi.org/10.1016/j.crfs.2026.101574

Image Credits: AI Generated

DOI: 10.1016/j.crfs.2026.101574

Keywords: diarrhetic shellfish toxins, okadaic acid, dinophysistoxin-1, dinophysistoxin-2, harmful algal blooms, shellfish safety, subchronic toxicity, food toxicology, marine biotoxins, toxicity equivalency factors, seafood consumption, cyclophilins