Background

Underwater noise pollution from motorboats and other human activities is increasingly recognized as a significant stressor in coastal and estuarine ecosystems [1]. Benthic crustaceans are especially vulnerable to acoustic disturbance due to their lifestyle, having restricted locomotion behaviors to escape from the noise source, compared to fish or marine mammals. While many previous studies have explored the behavioral and physiological effects of anthropogenic noise on marine vertebrates (see reviews for marine mammals [2] and fish [3]), standardized protocols for assessing oxidative stress responses across multiple developmental stages in invertebrates remain very limited (see review [4]). The semiterrestrial crab Neohelice granulata is a widely distributed and ecologically relevant species due to its role as an ecosystem engineer, serving as a valuable model for investigating the effects of underwater noise on its physiology [5]. This species is frequently exposed to fluctuating environmental conditions since it inhabits the intertidal zone of estuaries, salt marshes, and mangroves of the southwestern Atlantic Ocean, which implies a difficulty in carrying out experiments in the natural environment. This protocol addresses this gap by providing a controlled laboratory method to reproduce boat noise exposure and to measure oxidative stress biomarkers in embryos and larvae. Moreover, it could be extrapolated to studies involving adult tissues. Its reproducibility and adaptability make it a valuable tool for advancing research in marine ecotoxicology and understanding the sublethal effects of underwater noise on aquatic crustaceans.

Noise produced by motorized vessels is one of the most prevalent forms of underwater acoustic pollution. These vessels predominantly emit low-frequency sounds (10–500 Hz), which travel faster and farther underwater than in air, sometimes covering hundreds to thousands of kilometers and potentially affecting organisms across multiple ecological levels [6,7]. In benthic invertebrates—especially during early developmental stages—exposure to anthropogenic noise can disrupt physiological homeostasis, delay or accelerate developmental timing, and dysregulate stress-response pathways. For example, chronic boat-noise playback shortened embryonic development in N. granulata and produced physiological and biochemical changes in offspring (e.g., altered heart rate and elevated lipid peroxidation [8]). Similarly, repeated exposure to boat noise reduced embryo survival by approximately 20% in a marine gastropod, with corresponding impairments in larval viability [9]. Broad reviews of anthropogenic noise effects on marine biota also report that invertebrates exhibit changes in oxidative stress biomarkers, energy metabolism, and immune function when exposed to low-frequency noise [10]. For species like N. granulata, which inhabits shallow estuarine areas with frequent boat traffic, understanding the effects of this type of stressor is essential. The ability to evaluate oxidative stress responses in embryos and larvae under controlled conditions provides a robust framework for identifying early markers of sublethal stress and for examining how environmental challenges may influence different life stages. Conducting these experiments under controlled laboratory conditions is particularly important for estuarine species like N. granulata, as natural environments are highly dynamic, with substantial variability in temperature, salinity, oxygen levels, and other stressors that can fluctuate markedly within and between a few days. Laboratory-based approaches help isolate the effects of sound exposure from these confounding factors, enabling more accurate and reproducible assessment of the organism’s responses.

Despite the current growing evidence of noise impacts on aquatic invertebrates, there still remains a notable shortage of standardized laboratory protocols designed to investigate how such mechanical disturbances influence early life stages under controlled laboratory conditions [8,11]. This protocol was developed to help fill that gap. It offers a reproducible and accessible laboratory approach for exposing ovigerous females of N. granulata (and thus, subsequently embryos and larvae) to recorded boat noise. The setup uses submerged transducers to deliver sound in a controlled aquatic laboratory environment, ensuring consistency in exposure conditions across experiments. The method is straightforward and adaptable, allowing researchers to adjust exposure time, acoustic parameters, and sample type depending on the question at hand or the species involved. A major strength of this protocol lies in its integration with biochemical tools for evaluating organismal responses. Oxidative stress biomarkers—glutathione-S-transferase (GST), catalase (CAT), lipid peroxidation (LPO), and protein oxidation—provide early and sensitive indicators of physiological stress [12]. These markers are well-established in ecotoxicology: for instance, in octopus (Octopus vulgaris), coordinated increases in CAT, GST, and LPO were observed in digestive gland and arm tissues in response to metal exposure, validating their use as reliable biomarkers [13]. Similarly, studies in fish have documented that GST and CAT activities rise significantly under environmental stress (e.g., heavy metals or pharmaceuticals), preceding visible damage and correlating with pollutant levels [14]. Recent studies on sound exposure in N. granulata show that LPO, protein oxidation, and GST and CAT activities are biomarkers of oxidative stress under these conditions [5,8,15]. However, it is important to mention that the inclusion of other enzymes is also relevant, given that not all enzymes necessarily increase under stress. Moreover, these assays are relatively cost-effective, technically straightforward, and suitable for moderate-throughput screening. By combining acoustic exposure with these robust biochemical endpoints, this protocol delivers a versatile and scalable framework for investigating how anthropogenic noise affects aquatic invertebrates across life stages. Beyond its immediate application in physiology and toxicology, the protocol can be readily expanded to include behavioral assessments, multi-stressor designs (e.g., noise combined with temperature, hypoxia, or pollutants), and conservation-oriented questions. Its flexibility makes it suitable not only for laboratory-based mechanistic studies but also as a foundational tool for understanding broader ecological consequences of sound pollution in coastal and estuarine ecosystems.