Introduction

The colonization of ship hulls by aquatic organisms initiates biofouling, a sequential process that begins with biofilm development and advances to the establishment of macrofouling assemblages (Yebra et al., 2004;Bixler and Bhushan, 2012;Abed et al., 2019). Biofouling negatively affects vessel efficiency by raising hydrodynamic drag, which in turn elevates fuel usage, exhaust emissions, and operational maintenance expenses (Thomas et al., 2002;Schultz et al., 2011;Lindholdt et al., 2015). Moreover, it contributes to the spread of invasive species, causing significant threat to local biodiversity (Davidson et al., 2009;Soroldoni et al., 2017). These issues have led researchers to explore antifouling strategies to prevent the early formation of biofilms and limit the accumulation of macrofouling organisms. Tributyltin (TBT) [(C₄H₉)₃Sn⁺] is an organotin compound widely used in antifouling paints to prevent biofouling on ship hulls (Fent, 1996). Its application has extended to industrial and agricultural sectors as a biocide (Hoch, 2001;Konstantinou and Albanis, 2004).

Due to its persistence, hydrophobicity, bioaccumulation potential, and endocrine-disrupting properties, TBT has become a significant contaminant in marine and freshwater ecosystems, particularly in coastal sediments near harbors and shipyards (Antizar-Ladislao, 2008). It readily accumulates in fatty tissues of marine organisms (Cole et al., 2015), causing acute and chronic toxicity, including endocrine disruption, nervous system impairment, hepatotoxicity, and behavioral changes (Matthiessen and Gibbs, 1998;Park et al., 2016). Even at low concentrations (< 1 ng L⁻¹), TBT induces imposex in gastropods, bivalve malformations, and growth inhibition in various marine species (Alzieu, 2000;Sousa et al., 2009;Amara et al., 2018). Human exposure occurs through seafood consumption, drinking water, and contaminated meat, prompting the European Water Framework Directive 2000/60/CE (WFD) to classify TBT as a priority hazardous substance. Due to its widespread toxicity, the International Maritime Organization (IMO) implemented a global ban on organotin-based antifouling systems under the International Convention on the Control of Harmful Anti-fouling Systems on Ships in 2008 (IMO, 2001). Over 70 countries have agreed to prohibit TBT in aquatic applications, yet its use persists in developing nations outside the IMO framework (Antizar-Ladislao, 2008). Despite the ban, TBT remains detectable in harbor waters (200–400 ng L⁻¹) and marine sediments (1–10 μg g⁻¹) (Sousa et al., 2009;Radke et al., 2013;Briant et al., 2013). This is due to the high persistence of TBT in sediments caused by high K_ow, and Kim et al. (2014) also pointed out that the current TBT levels in the southern sea of Korea are higher than the global environmental quality standards established to protect marine organisms (Kim et al., 2014). While its ecotoxicity has been studied in aquatic animals (Fent, 1996;Hoch, 2001;Novelli et al., 2002;Park et al., 2016;Kim et al., 2018;Min et al., 2018), research on its specific effects on polychaete remains limited. To date, investigations into the effects of TBT on marine polychaetes remain limited, with only studies measuring acute and chronic responses in Hydroides elegans and TBT accumulation in Armandia brevis (Meador and Rice, 2001; Lau et al., 2007).

Perinereis aibuhitensis was selected as a non-target benthic indicator species to investigate the acute toxic effects of TBT exposure. Polychaetes are widely distributed in marine environments and are extensively used in ecotoxicology and environmental studies owing to their broad distribution, manageable size, ease of handling in both field and laboratory settings, deposit-feeding habits, and sensitivity to environmental contaminants (Reish & Gerlinger, 1997;Dean, 2008;Rhee et al., 2012). Among them, P. aibuhitensis is a dominant species in estuarine environments across many Asian coastal regions (Kang et al., 2011;He et al., 2024). In particular, this species is widely used in ecotoxicology studies due to its tolerance to a wide range of salinity and temperature conditions (Kang et al., 2011;Eom et al., 2019;He et al., 2024). This investigation evaluated mortality rates and cholinergic activity in TBT-exposed P. aibuhitensis. Additionally, biochemical indices associated with oxidative stress were measured and interpreted in the context of acute toxicological outcomes. These findings offer important insights into the physiological consequences of TBT exposure in non-target benthic annelids, particularly regarding oxidative stress, neurotoxic effects, and alterations in burrowing behavior.

Materials and Methods

1. Marine polychaete

The polychaete culture followed the protocol established in our previous study (Lee et al., 2024). Marine polychaetes P. aibuhitensis (≈ 1.29 ± 0.34 g; 3 months post-fertilization) were obtained from a commercial aquaculture facility in Yeosu, South Korea, and the maintenance in an automated aquaculture system at Incheon National University was carried out according to established method as describe by Lee et al (2024). In brief, they were cultured in artificial seawater (ASW; TetraMarine Salt Pro, Tetra, Cincinnati, OH, USA) in opaque polyethylene tanks (700 L) containing 30 cm of sieved sediment (sand-to-mud ratio of 8:2). Each tank housed approximately 1,000 individuals. Prior to the experiment, P. aibuhitensis were acclimated for 10 days under controlled conditions (16 h:8 h light-dark cycle, 17 ± 0.6°C, 32 PSU, 7.05 ± 0.51 mg L⁻¹ dissolved oxygen, pH 8.0) with continuous aeration. Half of the overlying ASW was renewed every two days. The diet consisted of ground TetraMarin® (Tetra, Blacksburg, VA, USA; 0.1 g per individual) supplemented with microalgae (Dunaliella sp., Isochrysis sp., and Tetraselmis sp.; 1 × 10⁶ cells mL⁻ ¹), provided three times weekly. Water quality parameters, including dissolved oxygen, pH, and salinity, were monitored every 12 h.

2. Acute toxicity test

Tributyltin chloride (TBTCl) was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and was directly dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Inc.). To evaluate survival rates under different TBT concentrations, 30 P. aibuhitensis individuals were exposed per concentration, divided into three replicates of 10 individuals each. The solvent DMSO was used at a maximum concentration of 0.05% in the TBT treatments. The acute toxicity test was conducted in an opaque glass fiber tank (55 × 45 × 36 cm; n = 10 per tank; 21st-century HighTech®, Busan, South Korea) without sediment. P. aibuhitensis were exposed to ASW as a control and to TBT concentrations ranging from 0.01 to 500 μg L⁻¹ for 96 h. The exposure conditions were consistent with those used in P. aibuhitensis culture, including trickle-flow aeration. Survival rates were recorded after 96 h. No food was provided throughout the experiment, and no mortality was observed in the ASW treatment group.

3. Measurements of burrowing and acetylcholinesterase activity

Due to the high mortality observed in P. aibuhitensis exposed to 10 μg L⁻¹ of TBT, physiological and biochemical analyses were conducted using a lower concentration of 1 μg L⁻¹. This concentration was the highest concentration that caused no statistically significant mortality during the 96-hour toxicity test (NOEC) and was therefore selected as the experimental exposure level. The methodology for the burrowing assay followed established protocols from previous studies (Haque et al., 2020). Surviving P. aibuhitensis from the 96-h exposure experiment with 0.05% (v/v) of DMSO and 1 μg L⁻¹ of TBT were subjected to a burrowing ability test. For this assay, 30 surviving individuals from each treatment group were transferred to clean ASW. The test was conducted in 200 mL glass fiber containers filled with 100 mL of ASW and sieved artificial sediment (mean grain size: 0.25 mm; depth: 5 cm) obtained from a polychaete aquaculture supplier, identical to the sediment used during acclimation. Ten individuals of P. aibuhitensis were placed at the center of the sediment surface to monitor the burrowing behavior, and three replicates were conducted. Full burrowing events were recorded for 30 min, with individuals that only partially burrowed classified as unburrowed.

To discuss the biological responses associated with behavioral disturbances (burrowing), we aimed to identify the molecular responses by analyzing AChE, which functions to modulate ACh and thus affect to movement (Métais, et al., 2019). AChE activity in P. aibuhitensis exposed to 1 μg L⁻¹ of TBT was assessed using the Ellman method (Ellman et al., 1961). The exposed polychaetes were homogenized in a Teflon homogenizer (Thomas Scientific, Logan Township, NJ, USA) with 0.1 M phosphate buffer (pH 8.0) at a 1:5 (w/v) ratio and centrifuged at 3,000 g for 30 min at 4°C. The resulting supernatant (100 μL) was mixed with 1.3 mL of 0.1 M phosphate buffer (pH 8.0), 50 μL of 5,5′ -dithiobis(2-nitrobenzoic acid) (DTNB, 0.01 M), and 10 μL of acetylthiocholine iodide (ATCh, 0.075 M) in a 3 mL cuvette. Both ATCh and DTNB were purchased from Sigma-Aldrich (St. Louis, MO, USA). Blank samples without ATCh and without the supernatant were prepared as controls. The reaction was incubated for 5 min (25°C) and the absorbance at 412 nm were measured using a spectrophotometer (Thermo™ Varioskan Flash, Thermo Fisher Scientific, Tewksbury, MA, USA). AChE enzymatic activity was normalized to the total protein content in the supernatant.

4. Measurement of antioxidant response

Lipid peroxidation in P. aibuhitensis was calculated by malondialdehyde (MDA) levels. Polychaetes exposed to 1 μg L⁻¹ of TBT for 96 h were homogenized in a cold buffer (20 mM Tris, 150 mM NaCl, 10 mM β -mercaptoethanol, 20 μM leupeptin, 2 μM aprotinin, and 100 μM benzamidine), and after centrifugation, the supernatants were heated to denature proteins. Thiobarbituric acid reactive substances (TBARs) were quantified by measuring absorbance at 535 nm (Thermo Varioskan Flash spectrophotometer). For this, MDA bis(tetramethoxypropane) was used as the calibration. The concentration of lipid peroxidation products in polychaete was expressed as M concentration (nM) of MDA per gram of whole-body tissue.

Glutathione (GSH) concentration in TBT-exposed P. aibuhitensis was measured using the Glutathione Assay Kit (Sigma-Aldrich). Polychaetes exposed to 1 μg L⁻¹ of TBT for 96 h were washed in 0.9% NaCl and homogenized in trichloroacetic acid (1:20, w/v), followed by centrifugation at 3000 × g for 10 min at 4°C. The supernatant was used to determine GSH levels based on absorbance at 412 nm using a Thermo Varioskan Flash spectrophotometer. GSH concentrations were calculated from a standard curve of 0, 150, and 350 μM GSH and expressed as nmol GSH per mg protein.

Enzymatic activities of catalase (CAT) and superoxide dismutase (SOD) were measured using assay kits (Sigma-Aldrich). Glutathione peroxidase (GPx) and glutathione reductase (GR) activities were analyzed using dedicated assay kits for Glutathione Peroxidase Cellular (Sigma-Aldrich) and Glutathione Reductase (Sigma-Aldrich), respectively. The enzyme activities were normalized to the total protein concentration in the samples and are presented as units per milligram of protein.

5. Statistical analysis

The data are presented as mean ± standard deviation (S.D.) values and were analyzed using the SPSS software package (ver. 17.0, SPSS Inc., Chicago, IL, USA). Statistical significances of measured variables were determined using one-way ANOVA. Post hoc analyses were performed to examine pairwise differences across time and concentrations (Tukey Honestly Significant Diference). A type I error probability of P < 0.05 was considered statistically significant.

Results

1. Mortality

The 96-h survival rates (%) of the P. aibuhitensis exposed to different concentrations of TBT significantly decreased with the rise in its concentrations (Fig. 1). No significant mortality was observed in the control group. The 96-h LC50 values were recorded as 23.7 μg L⁻¹.

2. Burrowing and AChE activities

The burrowing activities of P. aibuhitensis previously exposed to 1 μg L⁻¹ of TBT for 96 h were significantly affected by TBT exposure (Fig. 2A). The polychaetes disappeared into the sand within 15 minutes in the control and DMSO-treated groups, while the burrowing ability was delayed at every time point in the 1 μg L⁻¹ of TBT-exposed P. aibuhitensis. The enzymatic activity of AChE was significantly reduced at 96 h following exposure to 1 μg L⁻¹ of TBT in P. aibuhitensis (P < 0.05) (Fig. 2B). No significant change in AChE activity was observed in the control and DMSO-exposed groups (P > 0.05).

3. Measurements of oxidative stress and antioxidant response

Intracellular MDA levels showed a significant increase following exposure to 1 μg L⁻¹of TBT for 96 h in P. aibuhitensis (P < 0.05) (Fig. 3A). Significant depletion of GSH content was also observed under the same exposure conditions (P < 0.05) (Fig. 3B). No significant changes in MDA or GSH content were observed in control and DMSO-exposed groups (P > 0.05).

Furthermore, all antioxidant enzymes tested—GPx, GR, CAT, and SOD—demonstrated significantly reduced activities after exposure to 1 μg L⁻¹ of TBT for 96 h in P. aibuhitensis (P < 0.05) (Figs. 3C, 3D, 3E, and 3F). No significant changes in enzyme activity were noted in the control and DMSO-exposed groups (P > 0.05).

Discussion

The 96-h survival rates of P. aibuhitensis exposed to TBT concentrations ranging from 0.01 to 500 μg L⁻¹ revealed survival/mortality thresholds, with an LC50 value of 23.7 μg L⁻¹. These results indicate that even microgram levels of TBT can cause significant mortality in this species. To date, TBT has been shown to exert acute toxicity at nano- and microgram levels per liter in aquatic animals (Kim et al., 2018). However, very limited toxicity data are available for marine polychaetes. TBT toxicity studies on polychaetes have primarily with attention to the growth of juveniles (Meador and Rice, 2001; Lau et al., 2007). For example, in an experiment on juveniles of Armandia brevis, growth inhibition to TBT was shown to be dose-dependent, and analysis of residual concentrations in tissues suggested that juveniles were about three times more sensitive than adults (Meador and Rice, 2001). It was also reported that the 48-h LC50 values for the eggs, 2-cells, trochophores, juveniles and adults of the marine polychaete H. elegans were 0.18, 0.97, 2.36, 2.86, and 4.36 μg L⁻¹, respectively (Lau et al., 2007). When it comes to the mortality data, although exposure durations differed between the two marine polycheates, a comparison of the LC50 values suggest that H. elegans is more sensitive to TBT than P. aibuhitensis. These differences may be due to species-dependent resistance to toxicants. Indeed, many studies have reported that P. aibuhitensis is highly tolerant to pollutants, whereas H. elegans is sensitive to chemical exposure (Xie et al., 2005;Lau et al., 2007). Furthermore, habitat and lifestyle may also be responsible for these results, as H. elegans forms a tube and feeds by extending its feathery gills to capture particles such as planktons directly from the water column, indicating that it is waterborne feeders. In contrast, P. aibuhitensis resides in sediments, where it is exposed to numerous compounds, including pollutants. As a result, P. aibuhitensis may have developed a higher tolerance to contaminants compared to other polychaetes.

Given these habitat characteristics, the results of burrowing behavior and bioturbation activities of polychaete are a very important. Although AChE activity was not measured prior to 96 hours, making temporal alignment between AChE levels and burrowing impairment difficult, exposure to 1 μg L⁻¹ of TBT led to both a reduction in burrowing activity and inhibition of AChE. Similar results were observed in a study by Métais et al. (2019), where reduced burrowing activity in Hediste diversicolor was along with significant inhibition of AChE activity, supporting the link between neurotoxicity and behavioral impairments in polychaetes (Métais et al., 2019). The acute toxic effects of TBT on physiological and biochemical response have been widely reported in various aquatic animals (Fent, 1996;Hoch, 2001;Novelli et al., 2002;Antizar-Ladislao, 2008;Park et al., 2016;Kim et al., 2018;Min et al., 2018). Such toxic effects have the potential to disrupt metabolism, diminish detoxification capacity, and deplete energy reserves required for homeostatic maintenance, ultimately resulting in compromised burrowing activity. In P. aibuhitensis, delayed burrowing behavior observed in TBT exposed individuals implies potential neurotoxic impacts. The substantial energy demands associated with detoxification and metabolism may induce physiological stress and energy redistribution, thereby adversely affecting energetically intensive behaviors (Won et al., 2022). The impairment of movement such as burrowing inhibition observed in polychaetes is directly associated with significant inhibition of AChE activity in individuals exposed to TBT. Similar adverse effect of TBT have been documented in the Pacific oyster, Crassostrea gigas, including reduced ache mRNA expression and enzymatic activity (Park et al., 2016). Inhibition of AChE disrupt normal neurotransmission (Fulton and Key, 2001;Nunes, 2011), which may manifest as behavior alterations in polychaetes, including P. aibuhitensis (Eom et al., 2019;Haque et al., 2020;Lee et al., 2024).

Despite the environmental risks of TBT, few studies have examined its sub-lethal effects on the antioxidant defense system in marine organisms. To assess oxidative stress and antioxidant responses, P. aibuhitensis were exposed to 1 μg L⁻¹ of TBT for 96 h. In general, oxidative stress arises from an imbalance between ROS generation and removal, disrupting prooxidant-antioxidant equilibrium (Winston and Di Giulio, 1991;Valavanidis et al., 2006). Aquatic organisms rely on antioxidant defenses to mitigate ROS-induced damage, but excessive stress can impair these mechanisms (Lushchak, 2011). TBT exposure induced lipid peroxidation in P. aibuhitensis, evidenced by increased MDA levels, highlighting oxidative damage and reduced antioxidant capacity in this species.

GSH plays a crucial role in combating oxidative stress by detoxifying reactive intermediates, maintaining redox balance, and protecting cellular components (Lesser, 2006;Regoli and Giuliani, 2014). The decrease in total GSH levels in P. aibuhitensis measured after 96 h of exposure to TBT 1 μg L⁻¹ suggests that this organism utilizes GSH for ROS scavenging and has difficulty counteracting oxidative stress when GSH is depleted. Since GSH also inhibits lipid peroxidation by reacting with lipid peroxides and recycling antioxidants (Lushchak, 2011), its depletion may impair the organism’s ability to mitigate the elevated MDA levels induced by TBT exposure, exacerbating oxidative damage.

Assessing various antioxidant responses including SOD, CAT, GR, and GPx to environmental pollutants can provide a further understanding of the state of organism under oxidative stress. These enzymes have also proven valuable for evaluating homeostatic balance in polychaetes (Won et al., 2012, 2014;Eom et al., 2019;Haque et al., 2020;Lee et al., 2024). For instance, the collaborative action of CAT and SOD is critical for the conversion of ROS into harmless H₂O and O₂, thereby constituting primary defense mechanisms against oxidative damage (Lushchak, 2011;Regoli and Giuliani, 2014). The remarkable reduction in CAT and SOD activities in P. aibuhitensis indicates a decrease antioxidant capacity and increased venerability to oxidative stress. Furthermore, the observed decreases in GPx and GR activities in P. aibuhitensis imply a impaired capacity to mitigate oxidative stress, given the dependence of these enzymes on GSH for ROS detoxification (Winston and Di Giulio, 1991;Lushchak, 2011). Reduced levels of GSH, together with the inhibition of GPx and GR, indicate a disturbance in the antioxidant system. These alterations confirm that TBT acts as a trigger for oxidative stress, promoting lipid peroxidation and undermining antioxidant mechanisms in P. aibuhitensis. Our results establish a direct link between acute TBT exposure and multi-systemic dysfunction in P. aibuhitensis. These outcomes raise concerns about the long-term ecological consequences of sublethal TBT concentrations, which may undermine the sustainable of ecosystem and resilience of benthic populations.