A Reproducible Method to Evaluate Sublethal Acoustic Stress in Aquatic Invertebrates Using Oxidative Biomarkers.

Underwater noise is a growing source of anthropogenic pollution in aquatic environments. However, few studies have evaluated the impact of underwater noise on aquatic invertebrates. More importantly, studies involving early developmental stages have been poorly addressed. Significant limitations are due to the lack of standardized protocols for working in the laboratory. Particularly, the design of uniform procedures in the laboratory is important when working with species that inhabit short-term changing habitats, such as estuaries, which makes it difficult to carry out repeated experiments in the natural habitat. Besides, controlling for environmental variables is also important when assessing the effect of a stressor on the physiological parameters of individuals. This experimental protocol addresses that gap by offering an adaptable laboratory-based method to evaluate sublethal physiological responses to sound exposure under highly controlled conditions. Here, we present a reproducible and accessible laboratory protocol to expose crabs to recorded boat noise and evaluate physiological responses using oxidative stress biomarkers. The method is designed for ovigerous females, as we evaluated the effects on embryos and early life stages (i.e., larvae), but it can be readily adapted to different life stages of aquatic invertebrates. A key strength of this protocol is its simplicity and flexibility: animals are exposed to noise using submerged transducers under well-controlled laboratory conditions, ensuring consistency and repeatability. Following exposure, tissues or whole-body samples can be processed for a suite of oxidative stress biomarkers-glutathione-S-transferase (GST), catalase (CAT), lipid peroxidation (LPO), and protein oxidation. These biomarkers are highly responsive, cost-effective indicators that provide a sensitive and early readout of sublethal stress. Together, the exposure and analysis steps described in this protocol offer a powerful and scalable approach for investigating the physiological impacts of underwater noise in crustaceans and other aquatic invertebrates. Key features • Enables measurement of oxidative stress markers across different life stages-from embryos to larvae and adult tissues-offering a complete view of physiological impact. • Ensures consistent, reproducible conditions through standardized exposure and sampling, supporting reliable comparisons across experiments. • Flexible protocol adaptable to