Persistent Pharmaceutical Pollutants Linked to Neurotoxicity and Oxidative Stress, Study Finds

Everyday Medicines in Water Alter Zebrafish Social Behaviour and Brain Chemistry

Pharmaceutical residues that survive wastewater treatment may affect aquatic animals in ways that standard toxicity tests fail to detect, according to a new study in zebrafish. Researchers found that seven days of exposure to environmentally relevant concentrations of several persistent medicines changed social behaviour, aggression, light–dark preferences and antioxidant-related enzymes, even though the fish did not show a significant change in a commonly used test of anxiety. The findings suggest that pollution from medicines may produce subtle, compound-specific neurological effects rather than a single, easily measured pattern of toxicity. The study, published in Ecotoxicology, examined carbamazepine, metformin and TMP, using behavioural assays alongside biochemical measurements. Its results offer a detailed glimpse of how pharmaceuticals can influence fish physiology at concentrations intended to reflect those found in contaminated aquatic environments, while also underscoring how much remains unknown about the consequences of longer exposures.

Pharmaceutical pollution enters rivers and lakes when active drug compounds pass through the human body and reach sewage systems, or when medicines are discarded improperly. Conventional wastewater treatment is designed primarily to remove organic matter, nutrients and pathogens, not every biologically active molecule. Some compounds can therefore remain in treated effluent and move into surface waters. Carbamazepine, an anticonvulsant and mood-stabilising drug, is especially persistent because its chemical structure makes it difficult to break down during treatment. Metformin, widely prescribed for diabetes, is also frequently detected in wastewater streams, while TMP is among the pharmaceutical compounds investigated for potential effects on aquatic life. Although these substances are present at low concentrations compared with therapeutic doses, aquatic organisms may experience continuous or repeated exposure. Their biological activity is precisely what makes them useful as medicines—and potentially disruptive as environmental contaminants.

To assess behavioural effects, the researchers used zebrafish, a small freshwater species whose nervous system, sensory responses and genetic pathways make it valuable in toxicology. Zebrafish are not miniature humans, and results from them cannot be translated directly into human health risks. They are, however, vertebrates with measurable behavioural repertoires and conserved biological mechanisms, allowing scientists to study how chemicals affect movement, social interaction and stress responses under controlled conditions. The team exposed the fish for seven days and then evaluated them using a series of behavioural tests. The Novel Tank Behavior Test is designed to capture anxiety-related responses when fish are placed in an unfamiliar tank. The Social Interaction Test measures the tendency of fish to remain near or engage with conspecifics. The Mirror Biting Behavior Test uses a fish’s reflection as a social stimulus and can reveal changes in aggression. A separate light–dark assay probes exploration and avoidance in contrasting environments.

The first result was also one of the most important: the Novel Tank Behavior Test did not reveal significant differences in anxiety-related behaviour. That finding does not mean the pharmaceuticals had no effect on the nervous system. Instead, it shows that the response depended on the behavioural domain being measured. Toxic chemicals do not necessarily produce a generalized increase or decrease in activity; they may alter specific neural circuits, sensory processes or motivational states. A fish could therefore perform normally in an unfamiliar tank while behaving abnormally toward other fish or a perceived rival. The absence of a signal in one assay also illustrates why environmental neurotoxicity studies increasingly combine multiple tests. If researchers had measured only novel-tank behaviour, they might have concluded that the treatments caused little or no neurological disturbance. The broader behavioural battery revealed effects that a single endpoint would have missed.

Social interaction was substantially reduced, particularly in fish exposed to carbamazepine. Social behaviour in zebrafish depends on visual perception, locomotor coordination, attention, threat assessment and the neural processing of social cues. A reduction in interaction could therefore arise from several mechanisms, including altered motivation, impaired sensory communication or a disruption in the balance between exploration and avoidance. The study does not establish which mechanism is responsible, but the compound-specific result is notable. Carbamazepine acts on excitable cells in therapeutic settings, and its persistence in water means that non-target organisms may encounter a molecule capable of influencing nervous-system function. Reduced sociability could have ecological consequences if it affects schooling, predator detection, mating or access to food. These possible outcomes remain to be tested, but the behavioural observation provides a warning that low-level exposure may influence interactions that are central to survival.

The mirror-biting test produced a contrasting pattern. Carbamazepine increased aggressive behaviour, whereas metformin reduced aggression. The opposing effects show that pharmaceutical pollutants cannot be treated as a single class with predictable biological consequences. Different drugs act on different molecular targets, are absorbed and eliminated at different rates, and may influence distinct pathways in the brain and endocrine system. Mirror biting is not identical to aggression in the wild: the fish is responding to its reflection rather than to a living opponent. Nevertheless, the assay is widely used as an indicator of social reactivity and competitive behaviour. A rise in mirror-directed attacks may reflect heightened arousal or altered threat processing, while a decline may indicate reduced motivation, impaired responsiveness or a shift in energy allocation. Without additional physiological and behavioural measurements, the study cannot distinguish among these possibilities, but the result demonstrates that even closely related endpoints can move in opposite directions after exposure to different medicines.

The light–dark test added another layer of complexity. Carbamazepine-treated fish spent significantly more time in both the light and dark zones, suggesting increased movement or exploration across the apparatus rather than a simple preference for one environment. Metformin-treated fish, by contrast, showed a preference for the dark zone. In zebrafish, light–dark behaviour is influenced by visual processing, arousal, stress reactivity and locomotor activity, so interpreting the assay requires care. A preference for darkness can indicate avoidance of bright light, while increased occupancy of both zones may signal hyperactivity, disinhibition or disrupted transitions between behavioural states. The researchers’ observations are therefore best understood as changes in environmental response, not as straightforward evidence of “anxiety” or its absence. The contrasting profiles again point to chemical-specific effects and suggest that pharmaceutical mixtures in natural waters could create behavioural outcomes that are difficult to predict from single-compound studies.

The biochemical results indicated that behavioural changes were accompanied by altered enzyme activity, with important differences between males and females. Acetylcholinesterase, or AChE, terminates signalling at cholinergic synapses by breaking down the neurotransmitter acetylcholine. Changes in AChE activity can modify the duration and intensity of nerve-to-muscle and nerve-to-nerve communication, making the enzyme a common biomarker of neurotoxicity. Males showed increased AChE activity across all treatment groups. Female fish responded differently: TMP- and metformin-treated females also showed increases, while carbamazepine-treated females showed a decrease. Such sex-specific patterns may reflect differences in metabolism, hormone regulation, receptor expression or the way each sex allocates energy during chemical stress. The findings do not prove that enzyme changes caused the observed behaviours, but their alignment with behavioural disruption strengthens the case for examining molecular and physiological endpoints together.

The investigators also measured superoxide dismutase, catalase and glutathione S-transferase, enzymes involved in the response to oxidative stress. Normal metabolism generates reactive oxygen species, chemically reactive molecules that can damage proteins, lipids and DNA when their production exceeds the organism’s capacity to neutralize them. Superoxide dismutase converts superoxide radicals into hydrogen peroxide, catalase then breaks hydrogen peroxide down into water and oxygen, and glutathione S-transferase helps attach glutathione to reactive compounds so they can be detoxified or removed. The study found intricate treatment- and sex-dependent changes in the activities of these enzymes. Rather than a uniform increase or decrease, the antioxidant and detoxification systems appeared to respond differently to the individual pharmaceuticals and in male and female fish. This complexity matters because enzyme activity can represent either an adaptive defence or evidence that the system is under strain. A short-term rise may indicate compensation, whereas a later decline could signal depletion or impairment.

The study’s seven-day duration and environmentally relevant concentrations make the findings useful for identifying acute responses, but they also define the limits of the conclusions. The work does not show whether the fish would recover after exposure ended, whether the changes would intensify during months of contamination, or how the compounds would interact when present together in real waterways. Persistent pharmaceuticals may be encountered repeatedly, and organisms in polluted habitats can face simultaneous stress from temperature changes, low oxygen, pathogens and other contaminants. The authors therefore call for longer-duration studies to investigate chronic consequences in aquatic ecosystems. Future experiments could connect enzyme changes to gene expression, brain structure, reproduction, predator avoidance and population-level outcomes, while also examining how mixtures behave. For now, the results deliver a clear message: a clean-looking waterway can still contain biologically active pollutants, and the first signs of harm may appear not as mass mortality but as altered social lives, aggression and the chemistry of the nervous system.