# Zebrafish (Danio rerio) Embryo–Larvae as a Biosensor for Water Quality Assessment

## TL;DR

Zebrafish embryos and larvae can detect harmful effects of wastewater, making them useful for assessing water quality and protecting public health.

## Contribution

This study demonstrates zebrafish as a practical and sensitive biosensor for evaluating water quality in wastewater treatment processes.

## Key findings

- Exposure to influent water caused significant malformations, reduced heart rate, and gene expression changes in zebrafish larvae.
- Effluent water had milder effects, which were further reduced when diluted, indicating safer discharge after treatment.
- Zebrafish embryos and larvae showed sensitivity to subtle phenotypic, behavioral, and molecular changes linked to water quality.

## Abstract

Wastewater treatment plants (WWTPs) are crucial for reducing pollutants and safeguarding ecosystems and human health. This study evaluated the quality of influent water (treated water before secondary (biological) treatment) and effluent water (discharged water after secondary treatment) from the León (Spain) WWTP. We used zebrafish (Danio rerio) embryos and larvae as sentinel organisms. Larvae were exposed to different concentrations of influent and effluent during their first 120 h of development, and multiple biological endpoints were analyzed, including survival, hatching, morphology, heart rate, behavior, regeneration, primordial germ cell migration, and gene expression. Exposure to 100% influent caused the strongest effects, including reduced survival, higher number of malformations, decreased heart rate, impaired regeneration, altered behavior and cell migration, and gene expression deregulation. Effluent exposure produced milder effects, decreased further when diluted, reflecting the natural mixing with river water. These results confirm that zebrafish embryos and larvae are sensitive biosensors capable of detecting subtle phenotypic, behavioral, regenerative, and molecular alterations linked to water quality. This research highlights zebrafish as a practical and cost-effective tool to complement current water quality monitoring of effluents released from treatment plants, enhancing the evaluation of effluent safety and contributing to improving environmental and public health protection.

Wastewater treatment plants (WWTPs) play a key role in the protection of the environment and public health by reducing the levels of pollutants released into the water. Here, we evaluate the quality of water obtained from two key points of the treatment process of a municipal WWTP (León, Spain) using zebrafish (Danio rerio) embryos and larvae as sentinels. Three experimental groups were established: (1) “Control” (CTRL) maintained in embryo medium, (2) “Influent” (I) exposed to influent water before the secondary (biological) treatment (concentrations: I-100% and I-75%), and (3) “Effluent” (E) exposed to effluent water from the secondary treatment (concentrations: E-100% and E-75%). Our results confirmed that survival was subtly affected in I-100% and E-100%, as well as the hatching rate in the effluent. Larvae exposed to both experimental conditions also presented a higher rate of malformations, affecting biometry and showing reduced embryo motility, with the exception of E-75%. The I-100% condition also caused reduced heartbeat, reduced fin regeneration, and a higher number of delocalized primordial germ cells. I-100%-exposed larvae showed dysregulation of four genes (foxm1l, cenpf3b, hoxc6a, and ddit3) out of the 19 studied. Effluent dilution mitigated the observed effects, and the model proved to be an effective additional test for wastewater treatment plants.