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Frontiers in ToxicologyPharmacology & ToxicologyPeer-reviewed journal article

Visual - Acoustic Thigmotaxis in Zebrafish Larvae: A High throughput NAM for Neurotoxicity Assessment

Mónica Torres-RuíziD, María Muñoz PalenciaiD, Antonio De la ViejaiD, Ana CañasiD

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Abstract

The nervous system is highly vulnerable to chemical disruption, yet current regulatory guidelines do not include behavioral endpoints that capture changes in stress-related responses. Zebrafish larvae, up to 5 days old, have emerged as a promising model to bridge this gap, offering genetic and neurochemical similarity to humans together with high throughput potential. In this work, we have developed and evaluated a larval thigmotaxis assay as a new approach methodology (NAM) to detect behavioral alterations caused by neuroactive substances. Thigmotaxis, or edge-preference behavior, was studied in zebrafish larvae exposed to a range of model compounds and challenged with both visual (light/dark) and acoustic (sound/silence) stimuli. We compared 24 round well plates, commonly used in behavioral assays, with 96 square well plates to increase throughput. The two formats showed equivalent results, supporting the use of the higher-capacity system. Classical controls confirmed assay performance with caffeine increasing thigmotaxis, while diazepam decreased it. Additional neuroactive substances with diverse modes of action (chlorpyrifos, nicotine, dexamethasone, ethylenethiourea) produced stimulus-dependent responses, whereas negative controls (saccharin, amoxicillin) had little or no effect. Benchmark dose modeling showed that thigmotaxis was generally more sensitive than traditional locomotor activity endpoints. Overall, this multiplexed visual-acoustic thigmotaxis assay proved reproducible, scalable, and sensitive. In neurotoxicity testing this method could be used both as a stand-alone assay or as part of a broader behavioral NAM battery to assess potential effects on the vertebrate nervous system. This method provides a practical and ethical tool to improve chemical safety assessment both in ecotoxicology and human toxicology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/703464v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ceea53org.highwire.dtl.DTLVardef@17a2a8borg.highwire.dtl.DTLVardef@17f16b1org.highwire.dtl.DTLVardef@aac24f_HPS_FORMAT_FIGEXP M_FIG C_FIG

In-depth summary

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (4 Feb 2026) · CC BY 4.0

A 96-well square plate zebrafish larva thigmotaxis assay with visual and acoustic stimuli matched 24-well round plates and detected neuroactive chemicals more sensitively than locomotion.

OECD neurotoxicity guidelines (424 and 426) rely on rodents and omit anxiety-related endpoints. Published zebrafish thigmotaxis work is low throughput, uses inconsistent conditions and rarely tests acoustic cues; this work offers a standardized higher-throughput larval format with visual and acoustic stimulation.

Key results

  1. ShownControl larvae in 96 square wells (96S) and 24 round wells (24R) showed equivalent thigmotaxis. Reference compounds were mostly equivalent, with exceptions for diazepam.

    TOST, margin ±10 pp, 90% CI. Controls equivalent for visual and acoustic time % and distance cm; only acoustic % distance differed. Caffeine equivalent in all conditions; diazepam different for visual light and acoustic quiet. · 2.4. Data Analysis; 3.1. Plate format comparison under visual and…; 4. Discussion

  2. ShownIn 96S plates, caffeine and diazepam largely gave the expected anxiogenic and anxiolytic effects, but responses depended on the stimulus, and diazepam raised edge time under some conditions.

    Caffeine raised thigmotaxis 20-25% at the highest concentration in light periods. Diazepam lowered it 30-40% at the highest concentration, only in dark periods. Diazepam raised edge time at the two highest concentrations under acoustic stimulation. 12 larvae per condition. · 2.3.2. Acclimation and Toxicant Exposure; 3.1. Plate format comparison under visual and…

  3. ShownFor chlorpyrifos, thigmotaxis responded at lower doses than total distance moved under both visual and acoustic stimulation.

    Visual BMD 0.3-0.4 mg/L (thigmotaxis) vs 3-43 mg/L (distance). Acoustic BMD 1.8-2.5 mg/L (thigmotaxis). In quiet periods, 1.8 mg/L (thigmotaxis) vs 4.9 mg/L (distance). · 3.3. Neuroactive and negative substances visual…; 3.4. Neuroactive and negative substances…

  4. ShownNicotine, dexamethasone and ethylenethiourea also showed thigmotaxis as the more sensitive endpoint, with stimulus-dependent patterns.

    Nicotine dark-period BMD 0.15 mg/L (thigmotaxis) vs 0.8 mg/L (distance). Dexamethasone acoustic BMDs about 0.3 mg/L with no significant activity change. Ethylenethiourea visual distance BMD two orders of magnitude above thigmotaxis. · 3.3. Neuroactive and negative substances visual…; 3.4. Neuroactive and negative substances…; 4. Discussion

  5. SuggestedThe putative negative substances saccharin and amoxicillin had essentially no effect, apart from amoxicillin at the highest concentration in visual tests.

    Visual tests: no significant changes except amoxicillin at the highest concentration. Acoustic tests: no significant changes in thigmotaxis or distance up to 100 mg/L. · 3.3. Neuroactive and negative substances visual…; 3.4. Neuroactive and negative substances…

Methods, data and reproducibility

Zebrafish larvae (120 hpf) were exposed for 1 h in 24R or 96S plates and tracked under dark/light and tapping/silence regimes. Percent edge time and distance moved were analyzed with ANOVA, TOST equivalence and Hill-model BMD fits.

  • Control larvae compared across 24 round and 96 square well plates under visual and acoustic stimulation · Zebrafish (Danio rerio, AB strain) · n = 12 larvae per condition
  • Caffeine and diazepam concentration series in both plate formats · Zebrafish (Danio rerio, AB strain) · n = 12 larvae per condition
  • Chlorpyrifos, nicotine, dexamethasone and ethylenethiourea concentration series in 96S plates · Zebrafish (Danio rerio, AB strain) · n = 12 larvae per condition
  • Saccharin and amoxicillin as putative negatives, tested in 96S plates · Zebrafish (Danio rerio, AB strain) · n = 12 larvae per condition
Methods & tools
DanioVision platform with Observation Chamber (Noldus), EthoVision XT v. 16, GraphPad Prism 10.2.3, Two-way ANOVA with Sidak's multiple comparisons, EPA Benchmark Dose Online Tool (BMDS), Hill model, Two One-Sided Test (TOST) equivalence analysis, Microsoft Excel, Fixed-effects inverse-variance meta-analysis
Reagents & environment
EthoVision XT v. 16; GraphPad Prism 10.2.3; EPA BMDS Online; DanioVision observation chamber held at 28 °C.
Reproducibility gaps
Data available only on request; no code or analysis scripts reported; TOST was run in Excel.
Limitations (6)
  • Results at 120 hpf may not extrapolate to other larval stages or adults, and developmental variability in receptor expression may affect interpretation. · stated by the authors
  • Edge preference was not coupled to molecular endpoints such as cortisol or neurotransmitter levels, so the anxiety-like interpretation is behavioral only; overlapping neurotransmitter systems complicate mode-of-action reading. · stated by the authors
  • Inter-laboratory reproducibility and the domain of applicability are untested; multi-laboratory ring trials are still needed. · stated by the authors
  • In a handful of cases the BMDS tool could not fit a model, so no BMD was calculated, and the highest concentrations were sometimes excluded from BMD fits when responses were non-monotonic. · stated by the authors
  • Each condition used 12 larvae, and curation removed the highest and lowest value per group plus low-movement larvae. This may shape variance and BMD estimates. · noted in this summary
  • The ±10 pp equivalence margin is of similar size to the stated assay SD (approx. 10 to 30 pp), so equivalence conclusions may have limited power; only four neuroactive compounds support the sensitivity claim. · noted in this summary

Read this if you are setting up a zebrafish larval behavioral assay for neurotoxicity screening and want plate-format equivalence and benchmark dose comparisons.

Citations
3
FWCI
10.28
Field percentile
top 2%

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