Sea Urchins and Spheroids: How Ethical Models Are Rewriting Pesticide Toxicity Testing

Every year, more than 3.5 million tons of pesticides are released into the environment, and among them the most heavily applied class is also one of the most insidious: the anti-cholinesterase insecticides. These neurotoxic compounds, dominated by two chemical families known as organophosphates (OPs) and carbamates (CBs), are designed to disable the nervous systems of insects, but they do not stop at the fence line of the farm. A new review published in Frontiers in Cell Death by researchers at Italy’s National Research Council and the University of Genova argues that the tools used to judge their safety are overdue for an upgrade, and that a menagerie of ethically chosen biological models, from human cell lines to sea urchin embryos, could transform how regulators predict the harm these chemicals cause to people and ecosystems.

The core problem these compounds pose is biochemical and brutally simple. Both OPs and CBs target acetylcholinesterase (AChE), the enzyme that clears the neurotransmitter acetylcholine from the synaptic cleft after a nerve fires. When the enzyme is blocked, acetylcholine floods receptor sites, overstimulating the nervous system. But the two families differ in a crucial way. Organophosphates phosphorylate the serine residue in the enzyme’s catalytic gorge and remain tightly bound, producing long-lasting inhibition. Carbamates, by contrast, carbamylate the same residue reversibly, allowing the enzyme to recover within hours. This difference in binding chemistry translates directly into differences in environmental persistence: OPs, which are larger molecules of roughly 300 daltons, leave persistent residues on crops, while CBs of about 200 daltons break down into small inorganic molecules such as carbon dioxide.

Persistence, however, is a slippery concept. Pesticides are classified as non-persistent when they degrade by half in under 30 days, moderately persistent between 30 and 100 days, and persistent beyond 100 days. Yet even nominally short-lived compounds can linger far longer than expected. Organophosphates with short half-lives can persist for up to a year in soils with low pH and temperature, where adsorption to soil particles shields them from microbial degradation. Carbamates show equally dramatic pH dependence: a compound with a seven-day half-life may persist for 560 days at pH 6.0. In seawater, microbial degradation of insecticides like diazinon and chlorpyrifos is inhibited, extending their residence in marine environments. Because both families are lipophilic, they cross cell membranes and biological barriers, including the blood-brain barrier and the placenta, and accumulate in body fat, plant oils, and even human milk.

The human health toll is substantial. Pesticide intoxication is one of the most common causes of poisoning worldwide, with approximately one million cases and several hundred thousand deaths reported annually. Acute exposure, whether from accidents or intentional ingestion, produces the classic cholinergic crisis: nicotinic symptoms such as paralysis and headache, and muscarinic effects including respiratory distress, cramps, and lacrimation, which can be fatal. A particularly treacherous feature of thiophosphate organophosphates like chlorpyrifos, diazinon, and parathion is that the liver converts them during Phase I metabolism, via cytochrome P450 oxidases, into oxon derivatives that are even more potent cholinesterase inhibitors than the parent compounds. Symptoms can therefore worsen after the initial exposure as the most toxic metabolites accumulate.

Chronic, low-dose exposure is harder to pin down but arguably more worrying, because residues persist in air and food and exposure is nearly universal. Epidemiological studies have linked organophosphate exposure to an increased incidence of Parkinsonism in areas of intensive pesticide use, impaired memory, reduced psychomotor speed, and mood disturbances in adults, and neurobehavioral deficits in newborns of exposed mothers. Elevated levels of DMAP, a metabolite of thionophosphates, have been detected in children and adolescents, supporting a possible relationship between OP exposure and attention deficit hyperactivity disorder. Large cohort studies report increased risks of hormonally related cancers among spouses of pesticide applicators and associations between parental exposure and childhood leukemia. Occupational poisoning rates have reached 18.2 cases per 100,000 agricultural workers in developed countries, and domestic exposures are frequently underdiagnosed because the symptoms are nonspecific.

Perhaps the most vulnerable stage of life is the earliest. Embryonic development is a multiphasic process driven by rapid ion fluctuations that are modulated by the cholinergic system, and molecules related to acetylcholine signaling are involved in nearly every developmental event, from sperm-egg interaction through early cleavages to tissue differentiation and morphogenesis. Neurotoxic pesticides can cross the placenta and interfere with these processes, and clinical reports describe severe congenital anomalies following occupational or accidental OP exposure during pregnancy. Some compounds, such as the organophosphate fenitrothion, even display antiandrogenic activity, impairing male reproductive development and reducing sperm production and fertilizing capability. Because inductive signals in early embryos determine future body structures, the earliest developmental stages are the most sensitive, and small anomalies introduced at these stages can be amplified as development proceeds.

This is where the review’s central proposal comes in. Under European Regulation 1107/2009 and Council Directive 86/609/EEC, animal testing should be minimized and vertebrate experiments treated as a last resort, in line with the 3R principle of Replacement, Reduction, and Refinement. The authors systematically surveyed the literature, retrieving 277 records on organophosphates and 125 on carbamates from the Web of Science database, to identify which non-mammalian and in vitro models can reliably stand in for vertebrate testing. The answer is a tiered panel spanning nearly every level of biological organization: stabilized cell lines such as NT2 neurons and C6 glioma cells, primary cultures of nervous cells, immune cells from invertebrates, three-dimensional neural spheroids, and whole organisms including protists, fruit flies, brine shrimp, and sea urchins.

The cellular data are striking. C6 glioma cells exposed to diazinon at concentrations as low as 200 nanograms per liter, levels comparable to those measured in the blood and cord samples of mothers and children living in agricultural areas, showed dose-dependent micronuclei induction and significant AChE inhibition, suggesting that doses non-toxic to intact organisms may still weaken brain defense mechanisms. The NTera2/D1 human neuronal line, which expresses a complete cholinergic toolkit, showed time-dependent enhancement of apoptosis at diazinon concentrations of 10^-4 to 10^-5 M, with a paradoxical viability boost at 10^-6 M that the authors attribute to a dynamic balance between activated and blocked acetylcholine receptors. Three-dimensional retinal spheroids from chick embryos exposed to diazinon at 20 to 120 micromolar showed reduced AChE activity, inhibited differentiation of inner plexiform layer areas, and enhanced apoptosis, demonstrating that 3D models can capture tissue-level architecture that flat cultures miss.

Whole-organism models add ecological realism. The fruit fly Drosophila melanogaster, itself a target species, is so sensitive that its AChE has been engineered into biosensors for rapid detection of paraoxon and malaoxon. Brine shrimp cysts rehydrated in water containing pesticides at concentrations from 10^-11 to 10^-5 M showed dose-dependent decreases in hatching speed and larval survival, with chlorpyrifos oxon and carbaryl causing the strongest effects. But the standout model is the sea urchin. Species such as Paracentrotus lividus develop synchronously in transparent envelopes, allowing researchers to watch anomalies unfold in real time, and they possess the full set of cholinergic molecules from gamete interaction onward. Their AChE serves as a biomarker of environmental contamination, and researchers have proposed integrated toxicity indices that combine the number of anomalous embryos with the severity of skeletal and developmental defects to assess the health of coastal waters. Notably, deficits in skeletal spicule mineralization after exposure to the organophosphate Basudin open possible lines of inquiry into osteoporosis, while long-term effects have been associated with cardiovascular disease.

The review’s conclusion is a call for a panel approach rather than a single-model fix. Because cholinesterase enzymes exist in multiple molecular forms whose responsiveness to pesticides varies among species, tissues, and even physiological states, no one model can capture the full spectrum of risk. Electric eel AChE, for instance, is far more resistant to chlorpyrifos than rat brain or fruit fly enzyme, with inhibitory concentrations differing by an order of magnitude. By combining models of graded complexity, from molecules to spheroids to embryos, and measuring a broad range of endpoints including AChE inhibition, apoptosis, micronuclei, synaptogenesis, and developmental anomalies, toxicologists can build a more protective and predictive risk assessment. In an era when pesticide residues are nearly ubiquitous in environmental compartments, the humble sea urchin embryo may prove to be one of the most powerful sentinels we have.

Subject of Research: Hazard identification of organophosphate and carbamate pesticides using bioethical alternative models in toxicology

Article Title: Hazard identification and the harmful effects of organophosphates and carbamates in bioethical models: a review

Article References: Gambardella, C., Soro, P., Paladino, O., Falugi, C., Misurale, F., & Faimali, M. (2026). Hazard identification and the harmful effects of organophosphates and carbamates in bioethical models: a review. Frontiers in Cell Death, 5, Article 1680195. https://doi.org/10.3389/fceld.2026.1680195

Image Credits: AI Generated

DOI: 10.3389/fceld.2026.1680195

Keywords: organophosphates, carbamates, acetylcholinesterase, pesticides, neurotoxicity, 3R principle, bioassays, sea urchin embryos, brine shrimp, neural spheroids, risk assessment, emerging contaminants