{"paper_id":"48bdb72d-e9df-41be-b07d-2e6ab6300b52","body_text":"Effects of dichlofluanid in tropical marine bivalves exposed to water and spiked sediments: an assessment of biomarker responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of dichlofluanid in tropical marine bivalves exposed to water and spiked sediments: an assessment of biomarker responses Ana Carolina Feitosa Cruz, Paloma Gusso-Choueri, Guacira de Figueiredo Eufrasio Pauly, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8593449/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Antifouling paints have been used to combat biofouling on submerged surfaces. They contain biocidal compounds that may be released into the environment and harm aquatic ecosystems; however, their effects on tropical organisms are little known. Dichlofluanid, or N-dichlorofluoromethythio-N′,N′-dimethyl-N-phenylsulfamide (C 9 H 11 Cl 2 FN 2 O 2 S 2 ), is a biocide present in antifouling paints which toxicity to tropical marine organisms is poorly understood. This study aimed to evaluate the effects of dichlofluanid at a cellular and biochemical level on bivalves exposed to seawater and sediment contaminated with this biocide, also considering sediments with two different concentrations of organic matter. Aqueous-phase tests were carried out with the mussel Perna perna , while tests with sediments used the clam Anomalocardia flexuosa. Then, cellular and biochemical biomarkers were analyzed. The neutral red retention time assay (NRRT), a cellular biomarker was assessed only in P. perna , while biochemical biomarkers (DNA damage, LPO, GSH, and activities of GST, GPx, EROD, and AChE) were analyzed in both organisms. Particle size, calcium carbonate content, and organic matter analyses were also conducted for the sediments. At higher concentrations, the NRRT assay showed effects in mussel hemocytes, denoting loss of lysosomal stability. Mussels also showed changes in biochemical biomarkers in the digestive glands and gills. In the clams exposed to sediment, adverse effects occurred in both organs and were more evident in animals exposed to sediments with higher levels of organic matter. Our study showed that dichlofluanid can affect marine bivalves at environmental concentrations and that organic matter may contribute to dichlofluanid exposure in clams. Dichlofluanid antifouling biomarkers ecotoxicology organic matter Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Dichlofluanid affected lysosomes of the mussel Perna perna from 10 µg/L Dichlofluanid affected gills and disgestive glands of P. perna from 1 µg/L Dichlofluanid affected gills and disgestive glands of Anomalocardia flexuosa from 1 ng/g Organically richer sediments induced worst effects in A. flexuosa Effects of dichlofluanid in bivalves occurred at environmental concentrations Introduction Biofouling can be defined as the settlement of organisms on natural and anthropic hard surfaces submerged in water. Manmade structures affected by biofouling include oil platforms, pipelines, dam gates, aquaculture facilities, and small and large boats. In commercial vessels, biofouling may increase fuel consumption by up to 40%. Biofouling accelerates hull corrosion, generating additional costs for naval transport. Furthermore, organisms attached to ship hulls favor bioinvasion mechanisms over commercial routes worldwide (Hilliam et al. 2024). The introduction of non-indigenous species (NIS) has been associated with a range of environmental, economic, and cultural impacts, including threats to human health (Molnar et al. 2008). Imbalances in food chains and the extinction of native species have been associated with a rapid increase in international trade in recent decades (Diagne et al. 2021). Therefore, strategies have been recommended by the International Maritime Organization (IMO) to avoid or reduce marine invasions, including those concerning combat biofouling. Hence, antifouling coatings have been developed and widely used to protect ships and boat hulls (Almeida et al. 2007). Most protective systems are currently based on paints containing one or a combination of biocides aimed at inhibiting biofilm formation and larval settlement on submerged surfaces (Thomas and Brooks 2010). Antifouling biocides are potentially harmful to aquatic ecosystems and include a range of organic and inorganic substances. The first commercial antifouling paints using copper and zinc oxides were replaced by those based on organotin compounds such as tributyltin (TBT) because of their low effectiveness (Fernandez and Pinheiro 2007). Since then, TBT-based antifouling paints have been extensively used around the world, mainly because of their high durability. However, they were banned by the Antifouling Systems Convention issued by the IMO due to their widespread toxic effects, including endocrine disruptors detected in ship and boat traffic areas (Dafforn et al. 2011; Evans et al. 2000). Thus, a new set of non-metallic and metallic compounds started to be used as active booster biocides in the current generation of antifouling paints, including compounds such as cuprous oxides, copper and zinc pyrithione, Zineb, DCOIT, Irgarol, Diuron, dichlofluanid, and chlorothalonil (Castro et al. 2011; Paz-Villarraga et al. 2022). Due to the extensive use of these compounds, areas under intense ship and boat traffic, such as ports, marinas, shipyards, and navigation channels, are exposed to biocide contamination leached from these coatings. In fact, reports of booster biocide residues reaching the water column and other environmental compartments, such as sediments and biota, are frequent (Albanis et al. 2002; Almeida et al. 2023; Campos et al. 2022a; Castro et al. 2012; Gatidou et al. 2007; Hamwijk et al. 2005; Vouvoulis et al. 2000), as well as the environmental risks caused by these substances (Abreu et al. 2021; Campos et al. 2022a). Dichlofluanid, or N-dichlorofluoromethythio-N′,N′-dimethyl-N-phenylsulfamide (C 9 H 11 Cl 2 FN 2 O 2 S 2 ), is a booster biocide currently used in antifouling paints (Paz-Villarraga et al. 2022). It is a non-metallic organochlorine compound with low water solubility and the potential to accumulate and associate with particulate matter (Kow = 3.7) (Dafforn et al. 2011; Evans et al. 2000). Dichlofluanid is also used as a fungicide and can induce mutagenic and carcinogenic effects in non-target marine invertebrates (Bellas 2006). However, studies on dichlofluanid toxicity in marine organisms are still scarce, particularly in polar and tropical organisms (Campos et al. 2022a). Thus, further investigations are needed to evaluate the biological effects of dichlofluanid on tropical aquatic organisms and provide information for its regulation at global and local levels. The use of biomarkers assessing biochemical, physiological or histological responses induced by dichlofluanid exposure, at the organism or “sub-organism” levels, may help to elucidate potential early impacts associated with this substance (Walker et al. 2005). Such approaches have traditionally used organisms such as fish and invertebrates as suitable biological models (Monteiro et al. 2024). Bivalve mollusks have great ecological and commercial importance and have been widely used to assess the bioaccumulation and effects of contaminants (Pereira et al. 2014; USEPA 1993). They are abundant and easy to collect, allowing the use of many individuals per experiment, and their sessile habits guarantee their exposure to contaminants. Moreover, bivalves are filter-feeding animals inhabiting both hard- and soft-bottom substrates directly exposed to water and/or sediment matrices (Pereira et al. 2014; USEPA 1993). In this sense, the organic matter present in sediments can influence the exposure of burrowing bivalves to dichlofluanid because organic compounds tend to have a higher affinity for organic carbon (Haitzer et al. 1998; Jones and De Voogt 1999). The sorption of dichlofluanid onto organic matter may reduce the fraction dissolved in the water but may favor the exposure of benthic organisms to this compound via feeding route. This study aimed to assess the cellular and biochemical effects of dichlofluanid on two species of marine bivalves exposed to water and sediment contaminated with this biocide. Additionally, we evaluated the influence of sediment organic matter (OM) enrichment on dichlofluanid toxicity by exposing the bivalves to sediments containing different OM concentrations. We hypothesized that dichlofluanid can induce cellular and biochemical alterations in marine bivalves, and that presence of OM in sediment would reduce dichlofluanid bioavailability and consequently its effects on A. flexuosa . Materials and Methods Water exposure To assess the biochemical and cellular effects of dichlofluanid dissolved in water, adult individuals of the brown mussel Perna perna (mean lengths = 6.69 ± 0.53 cm) were employed as biological models. Specimens were sourced from an aquaculture farm located at Cocanha Beach, Caraguatatuba, São Paulo (23°34'40.0\"S – 45°18'52.8\"W) and subsequently transported to the laboratory, where they were acclimatized under controlled experimental conditions. The experiment was conducted according to the protocols described by the USEPA (2002; 1993). The exposure treatments used five nominal dichlofluanid concentrations (0.01, 0.1, 1, 10.0, and 100 µg/L) in seawater. Negative controls containing only clean seawater (collected from a pristine coastal area) and acetone (cosolvent) diluted in seawater (concentration of 0.05%) were prepared. Three replicates were established in chambers conditions for 24h with gentle aeration, at 25 ± 2 °C, and under a controlled photoperiod (12 h light: 12 h dark). Fortification with dichlofluanid was then carried out, and twelve healthy P. perna individuals were introduced into each replicate. The exposure period was 96 hours. Every 24 h or whenever gamete release by the organisms was observed, the test solutions were renewed, and a new fortification was performed to ensure the maintenance of initial dichlofluanid concentrations and overall water quality. Numerous studies have shown that dichlofluanid tends to degrade rapidly in water, with a half-life of just a few hours, generating by-products, such as N-dimethyl-N-phenyl-sulphamide (DMSA) and dichloromethane aniline (Sakkas et al. 2001; Vouvoulis et al. 2000). After 48 h and 96 h of exposure, five individuals from each replicate of the respective treatment were taken and analyzed for the neutral red retention time assay (Lowe et al. 1995). Throughout the test, physicochemical parameters such as pH, salinity, temperature, dissolved oxygen, and ammonia were monitored and maintained within acceptable ranges to prevent any interference on the experimental results. The first four parameters were measured using appropriate electrodes, while ammonia concentrations were determined using a colorimetric method following the protocol described by Verdouw et al. (1978). At the end of the experiment, all organisms were euthanized, and their soft tissues (gills and digestive glands) were removed and stored at -80 °C for subsequent analysis of biochemical biomarkers. Sediment exposure The experiment using spiked sediments used two types of sediments, each characterized by its organic matter (OM) content. One sediment had a low OM content (~4%), whereas the other had a higher OM content (~10%). Both sediments were collected from distinct locations in the Cananéia-Iguape Peruíbe Estuarine Complex, a protected area located on the southern coast of São Paulo State. Low-OM sediment was collected from Arrozal (25°02.415' S, 47°55.540' W), and high-OM sediment was collected from Ariri (25°13.215' S, 48°02.492' W). Both sediments were spiked with dichlofluanid to assess potential differences in toxicity related to the organic matter content. As in the aqueous solution assays, negative controls (dichlofluanid-free) and analytical blanks (acetone-containing sediments of both types) were prepared. The dichlofluanid concentrations tested in the sediment were 1, 10, 100, 1000, and 10000 ng/g. For these experiments, the clam Anomalocardia flexuosa was used as the biological model. Adult individuals of A. flexuosa (mean lengths = 2.01 ± 0.21 cm) were collected in the Cananeia region, São Paulo (25°02.415' S, 47°55.540' W), transported to the laboratory, and acclimatized to the test conditions. The experiments were conducted as described by the USEPA (1993, 2002). Six replicates were prepared in glass chambers containing a sediment layer approximately 4 cm thick and 1.5 L filtered seawater. The test system was maintained under equilibrium conditions, with gentle aeration and at 25 ± 2 °C. After a 24-h acclimation period, seven healthy A. flexuosa individuals were introduced into each replicate. The tests were conducted under a controlled photoperiod (12 h light: 12 h dark), with continuous aeration and constant temperature (25 ± 2 °C) for 21 days. Throughout the experimental period, physicochemical parameters, including pH, salinity, temperature, dissolved oxygen, and ammonia, were monitored using the methodologies previously described for the aqueous solution tests. Sedimentological analyses The sediment grain size distribution was analyzed according to the protocol proposed by Mudroch and MacKnight (1994). Three aliquots (30 g) from each sample were dried in an oven at 60 °C for two days, washed through a 0.063 μm sieve to separate fine particles (silt and clay), and the sandy material retained on the sieve was dried again, sieved for 15 min in a RO-TAP shaker using sieves of different mesh sizes (Φ scale), and then weighed. The results were classified according to the Wentworth Scale (Wentworth, 1922). The calcium carbonate (CaCO₃) content in each sample was measured using the method described by Hirota and Szyper (1975), which involved sample digestion with 5N HCl for more than 24 h to remove CaCO 3 , followed by washing with distilled water and drying in an oven at 60 °C. The percentage of CaCO₃ present in the sample was calculated from the observed weight loss. The organic matter (OM) content was estimated using the ignition method described by Luczak et al. (1997), where aliquots of dry sediment were incinerated in a muffle furnace at 500 °C for 4 h. The percentage of OM was equal to the weight lost during ignition. Sediment spiking Sediment spiking was performed following the protocols described by ASTM (2008). Five dichlofluanid concentrations were used in the tests: 1, 10, 100, 1000, and 10000 μg/g. To prepare each, the respective amounts of dichlofluanid were added to the sediment based on the dry weight percentage. The moist sediment was partitioned into aliquots, and different concentrations of dichlofluanid were added accordingly. The analytical blank (clean sediment containing acetone) was subjected to the same procedure, while the control (clean) sediment consisted of natural sediments. All sediment samples were agitated for 15 min using a device that provided multidirectional rotation within a hermetically sealed glass container. After mixing, the contaminated sediments were stored in the dark at 4 °C for 72 h to allow equilibrium to be established between the contaminant, interstitial water, and the solid sediment phase. Subsequently, the sediments were distributed into test chambers for the exposure experiments. Comprehensive descriptions of the sediment spiking methodology, along with the analytical confirmation of dichlofluanid concentrations in the tested sediments, were provided by Campos et al. (2024). Dichlofluanid levels were determined using Gas Chromatography coupled with Mass Spectrometry (GC-MS). Biomarkers Analyses Neutral Red Retention Time (NRRT ) The analysis of Neutral Red Retention Time (NRRT) in hemocyte lysosomes followed the protocol described by Lowe et al. (1995). This biomarker was assessed only in experiments involving water exposure. Prior to the assay, the glass slides were pre-treated with a poly L-lysine solution to facilitate hemocyte adhesion. Hemolymph samples (0.5 mL) were extracted from the adductor muscle of the mussels using a hypodermic syringe containing a physiological solution (0.5 mL). After 20 min, 40 µL of the cell suspension was placed on each slide. Slides with hemocytes were kept in a dark and humid chamber for 15 min. Subsequently, 40 µL of the Neutral Red solution was added to each slide. After 15 min, the coverslips were placed over the samples. The slides were then examined under a microscope at 15-minute intervals during the first hour and subsequently up to a maximum of 120 min. The cells were analyzed to determine the retention time of neutral red by lysosomes. NRRT was obtained by estimating the proportion of cells in which lysosomal content leaked into the cytosol. When 50% or more of the cells on a glass slide exhibited such leakage, the respective organisms were considered physiologically stressed. Bio chemical Biomarkers The gills and digestive glands of both P. perna and A. flexuosa were kept on ice and homogenized in Tris-HCl buffer (50 mM TRIS; 1 mM EDTA; 1 mM DTT; 50 mM sucrose; 150 mM KCl; 100 mM PMSF; pH 7.6). Following homogenization, aliquots were separated for total protein (TP), DNA damage, and lipid peroxidation (LPO) analyses. The homogenates were then centrifuged at 12,000 × g for 20 min at 4 °C, and additional aliquots were collected for the analysis of TP, glutathione S-transferase (GST), and glutathione peroxidase (GPx) activity as well as for the quantification of non-protein thiols (GSH), acetylcholinesterase (AChE) activity, ethoxyresorufin-O-deethylase (EROD), and dibenzylfluorescein (DBF). All biomarker analyses were performed using a Biotek microplate reader, model Synergy™ HT. Total proteins were quantified using the Bradford method (Bradford, 1976), and this content was used to normalize all biomarkers data. DNA damage was assessed using the alkaline precipitation assay proposed by Olive (1988), with fluorometrically performed DNA quantification (λex 360 nm; λem 450 nm) (Gagné et al. 2006). The results were expressed as µg of DNA per mg of protein. LPO levels were determined by measuring thiobarbituric acid reactive substances (TBARS) using fluorescence detection (λex 532 nm; λem 556 nm) as described by Wills (1987). The concentration of peroxidized lipids was expressed as μM TBARS per mg of protein. AChE activity was measured at 412 nm using the colorimetric method (Ellman et al. 1961), and the results were expressed as µmol DTNB per min per mg of protein. GSH levels were determined according to the method described by Sedlak and Lindsay (1968). GSH levels were measured spectrophotometrically at 415 nm and expressed as nanomoles of glutathione per milligram of protein. GPx activity was determined spectrophotometrically at 340 nm, based on the method of Sies et al. (1979), and the results were expressed as nmol per minute per mg of protein. GST activity was measured at 340 nm and 25 °C using a microplate reader (Habig et al. 1974). The results were expressed as OD per minute per milligram of protein. EROD activity was evaluated using a modified method by Gagné and Blaise (1993), and the results were expressed as pmol per minute per mg of protein. Dibenzylfluorescein (DBF) activity was determined according to Gagné et al. (2007), using fluorescence (λex 485 nm and λem 516 nm), and results were expressed as nmol per minute per mg of protein. Statistical and Exploratory Analyses The results from the NRRT assay were first checked for normality and homoscedasticity and then analyzed by one-way ANOVA with Dunnett’s post hoc test to detect significant differences in NRRT between the dichlofluanid concentrations, the analytical blank (acetone), and the control. The results obtained for biomarkers were first assessed for normality using the Kolmogorov–Smirnov test, followed by one-way ANOVA with Dunnett’s post hoc test (or an equivalent non-parametric test when necessary) to determine significant differences among dichlofluanid concentrations, the blank (acetone), and the control in the water exposure tests. For the sediment exposure experiments using matrices with different organic matter contents, a two-way ANOVA with Bonferroni’s post hoc test (or PERMANOVA for non-parametric data) was conducted to assess the effects of dichlofluanid concentration and sediment type and potential interactions between them. The results from the subchronic exposure tests (biomarkers) were further integrated using Principal Component Analysis (PCA) to identify patterns and potential groupings based on exposure conditions. For the sediment dataset, PERMANOVA was performed using the studied biomarkers (GSH, GST, GPx, EROD, AChE, LPO, and DNA damage). Results and Discussion Water exposure – Perna perna The physicochemical parameters of seawater at the Perna perna cultivation site, as well as those recorded in the test solutions during the experiments, are presented in the Supplementary Material (Tables S1 and S2). During the tests the mortality rates remained below 20% of the total number of exposed organisms (Figure S1). Neutral Red Retention Time Figure 1 presents the Neutral Red Retention Times (NRRT) after 48 and 96 h of exposure to dichlofluanid. At the initial time point, the organisms exhibited a mean retention time of 40.5 minutes. After 96 h, NRRT was significantly reduced (p < 0.05) in mussels exposed to 10 µg/L (mean NRRT= 33 min) and 100 µg/L (mean NRRT = 15 min) compared to the acetone control group (mean NRRT = 97.5 min). No significant differences in the NRRT were observed after 48 h of exposure. Hagger et al. (2005) reported that for the mussel Mytilus edulis , increased cytotoxicity induced by TBT using the Neutral Red assay occurred at concentrations above 0.5 µg/L and at shorter retention times than those observed in the present study. These findings suggest that dichlofluanid may exhibit lower cytotoxicity than TBT, a compound that is used in antifouling coatings and has been banned in several countries. Lysosomes are organelles responsible for handling tissue nutrition and repair, encompassing the main cellular structure for sequestration and detoxification of organic compounds, as reported in previous studies (Lowe et al. 1995). Under chemical stress, lysosomal membranes are damaged and leakage of lysosomal contents into the cytoplasm often occurs concurrently. Neutral red retention is minimally affected by natural factors and predominantly influenced by contaminants (Ringwood et al. 1998). Thus, diclofluanid induced the cytotoxicity observed in the present study, as shown by Suzuki et al. (2004). In our study, the significant responses observed for NRRT at higher dichlofluanid concentrations are concerning, since lysosomal membrane stability and integrity have been widely used for the early detection of toxic compound effects (Repetto et al. 2008). NRRT serves as an important indicator of cellular health, and any impairment in this parameter signals early signs of more severe adverse effects across multiple levels of biological organization, ranging from subcellular processes (Dailianis et al. 2003) to effects observable at the population and community scales. Bio chemical Biomarkers Figure 2 presents the results for the exposure biomarkers. The induction of GST activity in the digestive glands was observed at the highest dichlofluanid concentrations, as well as increased levels of GSH and GPx activity in the gills at the same concentrations. This induction of the antioxidant defense system has been previously reported for biocides used in antifouling paints, such as TBT, Irgarol and Diuron (Park et al. 2016). Regarding the biomarker of effect, lipid peroxidation (LPO) levels in the digestive glands were lower than those observed in the control groups (Fig. 3). This suggests that activation of detoxification mechanisms minimizes oxidative damage to cellular membranes. Cells possess various mechanisms to cope with oxidative stress, either by repairing damage or by reducing its occurrence through enzymatic and non-enzymatic antioxidant pathways. Enzymatic antioxidants neutralize oxidative stress (Sutcu et al. 2007). Principal Component Analysis (PCA) The PCA results for the water-based exposure tests are shown in Tables 1 and S3 and Figure 4. The first two principal components (PCs) explained 64.54% of the total variance in the dataset (Table 1). The PCA revealed that, for the PC1, the seawater control, the acetone blank, the initial time point (T0), and the lowest dichlofluanid concentration (0.01 μg/L) clustered together, while the other tested concentrations (0.1, 1, 10, and 100 μg/L) formed a second group (Table 1, Figure 4). Regarding PC2, the control and analytical blank clustered with the two lowest dichlofluanid concentrations (0.01 and 0.1 μg/L), while the higher concentrations (1, 10, and 100 μg/L) separated. T0 appeared to be isolated from all other treatments (Table 1, Figure 4). For PC1, positive associations were observed between GPx and GSH in the gills, and between GPx and GST in the digestive glands, along with an inverse relationship between DBF and GSH in the digestive glands (Table 1). These findings suggest activation of detoxification mechanisms in both the gills and digestive glands, which is also reflected in the individual biomarker plots. Although no statistically significant differences were detected, the plots for the digestive glands showed a decreasing trend in GSH and DBF levels (Fig. 2d and 2j). Moreover, a relationship between the enzymatic responses and the neutral red retention assay was observed. These effects were associated with intermediate and high dichlofluanid concentrations, although they did not result in measurable biochemical damage (i.e., lipid peroxidation or DNA damage). However, membrane-destabilizing effects were evident at higher concentrations, as demonstrated by significant lysosomal membrane destabilization revealed by the NRRT assay. As this is a biomarker of effect, the overall data suggest that detoxification pathways were not fully effective, leading to lysosomal membrane damage, especially at higher concentrations. Regarding PC2, negative correlations were found between GST and DNA damage in the gills, while positive correlations were observed between GPx and EROD, as well as between DNA damage, EROD, lipid peroxidation (LPO), and AChE in the digestive glands. This axis appears to have grouped variables that, when analyzed individually, did not show significant effects and thus did not contribute to a clear overall response in the biomarker data. Tab 1. Results of Principal Components Analysis (PCA) integrating all biomarkers analyzed in Perna perna mussels exposed to dichlofluanid concentrations in water. Factorial Scores for the initial time, control, acetone control, and dichlofluanid concentrations at 96 h in the water exposures. G = Gills; DG = Digestive Gland; NR = Neutral Red. PC Eigenvalue % Variance Cumulative Variance PC1 6.12 35.97 35.97 PC2 4.86 28.58 64.55 Variable PC1 PC2 DNA.G -0.24 -0.75 LPO. G -0.49 0.30 AchE. G 0.29 0.90 Erod. G -0.61 0.38 DBF G 0.10 0.27 GPx. G -0.87 0.15 GSH. G -0.83 0.22 GST. G -0.42 -0.65 DNA.DG -0.46 0.69 LPO.DG 0.31 0.83 AchE.DG 0.46 0.56 Erod.DG 0.12 0.63 DBF DG 0.87 -0.36 GPx.DG -0.53 0.56 GSH.DG 0.91 -0.21 GST.DG -0.93 -0.11 NRRT 0.69 0.55 Treatment PC1 PC2 T0 2.31 -4.80 T96_Control 1.70 0.53 T96_Acetone 2.67 2.98 T96_0.01 2.38 1.01 T96_0.1 -1.56 0.78 T96_1 -1.90 -0.43 T96_10 -2.72 -0.02 T96_100 -2.88 -0.05 In the projection of variables relative to the two factors extracted by PCA, the treatments were grouped into three clusters plotted on the plane formed by PCs 1 and 2 (Fig. 4). T0 was isolated from other samples. One group consisted of the control, acetone blank, and the lowest concentration (0.01 μg/L), whereas the other contained intermediate and higher dichlofluanid concentrations (0.1, 1, 10, and 100 μg/L) (Fig. 4). These data suggest a metabolic effect in animals exposed to concentrations above 0.1 μg/L. The analyses of biomarkers, either analyzed individually or in multivariate analyses, showed that dichlofluanid may induce significant alterations in antioxidant processes, as observed in the biomarkers directly involved with glutathione (GSH, GST, GPx). This can be explained by the mechanisms of action of dichlofluanid and its primary metabolite, DMSA (N'-dimethyl-N-phenyl-sulfamide). It is a multi-site herbicide and fungicide, which primary mode of action involves reactions of dichlofluanid with thiols within the cells of target organisms (Hamwijk et al., 2005). Thus, dichlofluanid is a reactive compound with sulfhydryl groups present in glutathiones (Yamano and Morita 1995). This study demonstrated an increase in GPx activity and GSH production in the gills, the first organs to combat xenobiotics, as the primary exposure route in bivalves occurs via the gills. This increase in GPx activity and GSH levels may be directly related to the biotransformation processes of dichlofluanid or the potential reactive oxygen species generated in the presence of xenobiotics (Keppler and Ringwood 2001). Biocides, such as dichlofluanid and DMSA, have pro-oxidant properties that cause imbalances between GSH and GSSG, leading to several other cell effects, including an increase in reactive oxygen species (ROS), which are eliminated by antioxidant enzymes such as GPx (Lushchak et al. 2018). In the digestive glands, the results showed increased GST activity and LPO levels at higher dichlofluanid concentrations, suggesting that the antioxidant system action was limited and unable to avoid oxidative stress. The effects observed in the digestive glands suggest that dichlofluanid is incorporated by bivalves and transported to the glands where detoxification processes occur. Digestive glands have been shown to be highly sensitive to xenobiotics in some bivalve species (Ramos-Gómez et al. 2011; Zhang et al. 2010). Considering all the analyses performed when exposing bivalves to dichlofluanid via water, some effects were observed in both the gills and digestive glands, particularly at the higher concentrations tested. However, no DNA damage or neurotoxicity was observed, and the effects on lysosomal membranes of hemocytes were noted at higher concentrations, corroborating that the toxic effects of dichlofluanid are caused by oxidative stress, as lysosomal membranes have altered permeability due to ROS (Lowe et al. 1995). Thus, our results suggest that the effects of dichlofluanid are more related to membrane destabilization due to increased ROS levels. Studies have shown that bivalve hemocytes can be affected by biocides used as antifouling agents, such as DCOIT (Campos et al. 2022b) and chlorotalonil (Morais et al. 2023). Moreover, in ascidians exposed to dichlofluanid, hemocytes exhibited several types of alterations, such as apoptosis, cell shrinkage, reduced motility, and phagocytic activity (Cima and Varello 2020). Sediment exposure – Anomalocardia flexuosa Sedimentological analyzes Table 2 presents some sediment properties, including grain size distribution, calcium carbonate (CaCO₃) content, organic matter (OM) percentage, and dry weight. Both sediments used in the experiment consisted mainly of very fine sands mixed with mud and fine sands, as well as smaller quantities of other textural fractions, indicating that potential variations in toxicity are unlikely to be attributed to differences in sediment texture. The sediment collected from Ariri exhibited a higher OM content (10.4%) than that from Arrozal, which contained approximately 4% OM. As for CaCO₃ concentrations, no substantial differences were observed between the sediments, suggesting that this parameter likely did not play a significant role in influencing the outcomes of the toxicity assessments. Tab 2. Grain size distribution, organic matter (OM), calcium carbonate (CaCO₃) content, and wet weight percentages of the sediments collected in Cananéia (São Paulo, Brazil) and used in the dichlofluanid exposure assays with Anomalocardia flexuosa . Gravel Very Coarse Coarse Medium Sand Fine Sand Very Fine Sand Silt & Clay OM CaCO 3 Wet Weight High OM 0.37 0.33 2.11 5.21 18.22 55.63 17.10 10.38 7.38 57.18 Low OM 0.00 0.07 0.62 1.42 8.66 74.53 13.81 4.00 5.88 49.52 OM: Organic Matter; CaCO 3 : Calcium carbonate. Chemical analyses As previously reported by Campos et al. (2024) and summarized in Table 3, dichlofluanid was quantifiable only at a concentration of 1000 ng/g, where it exhibited degradation rates of 87% within 6 h and 99% after 24 h. This compound is recognized as a rapidly degrading biocide with a very short environmental half-life (Hamwijk et al. 2005). Nevertheless, given that dichlofluanid has been detected in concentrations exceeding established toxicity thresholds and that its degradation can lead to the formation of potentially toxic transformation products, investigation of its effects remains ecologically relevant. Tab 3. Measured concentrations of dichlofluanid at 0, 6, and 24 hours post-spiking under equilibrium conditions, as previously reported by Campos et al. (2024). Nominal concentration (ng.g -1 ) Time 0h 6h 24h Dichlofluanid (LD = 0.5; LQ= 1) Blank <QL <DL <QL 10 <DL <QL <DL 100 <QL <QL <QL 1000 1189.5 ± 460.4 157.7 ± 36 16.9 ±7.4 DL= Detection limit; QL – Quantification limit. Exposure of Anomalocardia flexuosa Among the organisms exposed to sediments enriched with high OM content, there was no mortality. On the other hand, clams exposed to low OM sediment exhibited some mortality; however, the rates remained below 30% (Figure S2). The physicochemical parameters of the overlying water within the test chambers during the test are presented in Table S4, and remained within suitable ranges for the species (Cruz et al. 2021). Biomarkers of exposure and effect Figures 5 and 6 present the biomarker responses measured in A. flexuosa following exposure to dichlofluanid-spiked sediments. In gill tissues, sediments with high OM content induced an increase in GST and EROD activities at the highest concentration (10000 ng/g), a reduction in GSH levels, and elevated GPx and AChE activities at the three highest concentrations (100, 1000, and 10000 ng/g). Additionally, increased LPO levels were observed at intermediate concentrations (1 and 10 ng/g). Exposure to low-OM sediment led to a reduction in EROD activity at the highest concentrations (1000 and 10000 ng/g). In the digestive glands, exposure to high-OM sediment led to enhanced GST activity at intermediate concentrations (10 and 100 ng/g), decreased GSH and AChE activity, increased EROD activity and LPO levels at 10000 ng/g, decreased GPx activity at 1000 ng/g, and increased DNA damage at 10 ng/g. For low-OM sediment, the results showed decreased EROD activity at 1000 and 10000 ng/g, increased AChE activity at 10000 ng/g, and reduced LPO levels at varying concentrations (1, 10, and 10000 ng/g). Overall, both the gills and digestive glands exhibited consistent evidence of the activation of defense mechanisms, such as alteration of biotransformation processes (EROD), stimulation of conjugation mechanisms (GST activity), alteration of antioxidant responses (GPx, GSH), together with associated cellular effects (DNA damage and LPO), and altered AChE activity, which may indicate potential neurotoxicity. Such effects tended to be more pronounced in animals exposed to higher concentrations of dichlofluanid and organically richer sediments (i.e., high OM). Principal Component Analysis (PCA) The results of the PCA performed using the results obtained from the exposure tests with sediments are presented in Tables 4 and S5, and figure 7. The first two principal components accounted for 63.93% of the total variance (Table 4). PCA revealed that along PC1, the water control, acetone blank, T0, and lower dichlofluanid concentrations (i.e., 1 and 10 ng/g) clustered together, whereas the higher concentrations (100, 1000, and 10000 ng/g) formed a separate group (Table 4, Figure 7). Along PC2, T0, water control, and the acetone blank were grouped together and separated from the 1 and 10 ng/g concentrations, while the remaining dichlofluanid concentrations formed a distinct cluster (Table 4). For PC1, in sediments with high organic matter content, gill tissues showed a positive association between GST and AChE and a negative association between GSH and DNA damage. In the digestive glands, EROD and lipid peroxidation (LPO) were positively associated, whereas GSH, GPx, and AChE showed inverse associations. In low organic matter sediments, gills exhibited associations among EROD, GPx, DNA damage, AChE, and LPO, yet most of these variables did not show significant variations according to the ANOVA (Figures 5 and 6). In the digestive glands, an inverse relationship was observed between GST, GSH, and AChE (Table 4). For PC2, in high organic matter sediments, gills showed an association between GPx and LPO, while in the digestive glands, GST and DNA damage were positively associated, and AChE was negatively correlated. In the projection of the variables along the two principal components extracted by PCA, the data were grouped into three clusters plotted on the plane defined by PCs 1 and 2 (Fig. 7), similarly to the pattern observed in the water exposure experiments. The T0 treatment appeared to be isolated from the other treatments. The second cluster comprised the control, analytical blank, and lower concentrations of dichlofluanid (1, 10, and 100 ng/g). The third group had the highest concentrations (1000 and 10000 ng/g). These results also suggest a more pronounced metabolic effect in organisms exposed to higher dichlofluanid concentrations. Overall, the PCA results supported the occurrence of adverse effects in A. flexuosa individuals exposed to dichlofluanid through sediments, particularly under conditions of elevated organic matter. These effects were more consistent at higher concentrations; however, biochemical alterations were detectable even at the lowest tested concentrations, indicating that the animals responded to minimal levels of dichlofluanid in the environment. Tab 4. Results of Principal Components Analysis (PCA) integrating all biomarkers analyzed in Anomalocardia flexuosa individuals exposed to dichlofluanid concentrations in spiked sediments with two matrices of differing organic matter content. Factor scores for the initial time point, control, acetone blank, and dichlofluanid concentrations in sediment exposures over 21 days (Concentrations in ng/g). G = Gills; DG = Digestive gland; H = High organic matter; L = Low organic matter. PC Eigenvalue % Variance Cumulative Variance PC1 12.03 42.96 42.96 PC2 5.87 20.97 63.97 PC1 PC2 DNA.G.H -0.70 0.03 DNA.G.L -0.83 0.45 LPO. G.H -0.21 -0.85 LPO. G.L -0.51 0.28 AchE. G.H 0.86 0.30 AchE. G.L -0.74 -0.21 EROD. G.H 0.45 0.06 EROD. G.L -0.85 -0.26 GPx. G.H 0.05 -0.82 GPx. G.L -0.73 -0.38 GSH. G.H - 0.73 -0.29 GSH. G.L -0.42 -0.28 GST. G.H 0.83 -0.38 GST. G.L 0.31 -0.28 DNA.DG.H -0.35 -0.66 DNA.DG.L -0.36 0.14 LPO. DG.H 0.97 0.003 LPO. DG.L -0.46 0.56 AchE. DG.H -0.81 0.51 AchE. DG.L 0.90 -0.25 EROD. DG.H 0.94 0.02 EROD. DG.L -0.42 0.69 GPx. DG.H -0.77 -0.46 GPx. DG.L -0.25 - 0.81 GSH. DG.H -0.85 -0.14 GSH. DG.L 0.65 -0.48 GST. DG.H -0.01 -0.86 GST. DG.L -0.76 -0.18 Treatment PC1 PC2 T0 -0.85 4.24 T21_Control -3.14 1.38 T21_Acetone -2.39 1.29 T21_1 -2.12 -1.89 T21_10 -1.73 -3.28 T21_100 0.03 -2.22 T21_1000 3.08 0.50 T21_10000 7.13 -0.02 The results of the PERMANOVA using data obtained for both gills and digestive glands are presented in the Supplementary Material (Figs S3 and S4, and Tabs S6-S9). In summary, these results showed a clear separation between the different types of sediments, with a grouping of the high-OM sediment with the highest concentrations of the low-OM sediment, whereas the remaining concentrations of the low-OM sediment formed a second group for the gills. These results also highlight that, for both the high and low organic matter sediments, significant differences in biomarkers were observed at the highest concentrations. Similar results were observed for digestive glands, indicating separation by sediment type (i.e., OM content), and only the highest dichlofluanid concentrations of the high-OM sediment were isolated. When comparing sediments with different organic matter concentrations, this analysis revealed significant differences in the controls and at concentrations of 1, 10, and 10000 ng/g. In general, biological responses were more pronounced in organisms exposed to sediments with a high quantity of organic matter. In such matrices, organic compounds tend to adsorb onto organic matter, establishing various chemical interactions that directly influence their bioavailability and toxicity to benthic organisms (Haitzer et al. 1998; Jones and De Voogt 1999). The stability of contaminants in sedimentary matrices is also related to the rate or degree of organic matter decomposition (Said-Pullicino et al., 2007). This fraction, retained in the sediment, can be directly transferred to benthic organisms, such as A. flexuosa in the present study. According to Lagreze et al. (2022), A. flexuosa is a filter feeding mollusk that ingests particles suspended in the water using their siphons; however, it also ingests large amounts of organic and inorganic material deposited on the sediments (Rodrigues et al. 2010) and is thus susceptible to contaminants present in the sedimentary environment. Other booster biocides used in antifouling paints and with solubilities comparable to that of dichlofluanid, such as diuron (log Kow = 2.7-2.8) and TBT (log Kow = 3.1-4.11), also appear to be strongly influenced by the concentration of organic matter, with higher OM levels enhancing their retention (Rocha et al., 2013; Furdek et al., 2016). In this sense, the increased retention of dichlofluanid in OM richer sediments likely favors the exposure of A. flexuosa and consequently could explain the negative effects recorded in our study, especially at higher concentrations. Biomarker analyses revealed a typical response pattern in both the gills and digestive glands: activation of EROD, involved in phase I biotransformation processes, followed by the induction of conjugation enzymes such as GST, accompanied by the consumption of GSH during detoxification. However, the antioxidant system was insufficient to completely metabolize or eliminate dichlofluanid, resulting in lipid peroxidation. This outcome is consistent with previous findings by Suzuki et al. (2004), who demonstrated that dichlofluanid induced lipid peroxidation and cytotoxicity at most of the tested concentrations above 25 μM. In addition to oxidative stress, signs of neurotoxicity were observed in both tissues. A key target of some pesticides, particularly organophosphates, is the enzyme acetylcholinesterase (AChE), which hydrolyzes the neurotransmitter acetylcholine, and increased AChE activity has been associated with cellular apoptosis (Zhang et al. 2002). In this investigation, an increase in AChE activity was reported for both the gills and digestive glands, associated with biomarkers related to oxidative stress, such as LPO and DNA damage. This can be explained by the mode of action of dichlofluanid, which involves increased production of ROS, which causes oxidative stress (Keppler and Ringwood 2001; Lushchak et al. 2018). Therefore, our results demonstrate that dichlofluanid can harm neotropical benthic species, such as A. flexuosa , especially at higher concentrations and in organically rich sediments. However, more studies using other taxonomic groups are necessary to detail the ecological risks caused by dichlofluanid to tropical species. Conclusion Based on the results, it can be inferred that dichlofluanid induces cellular-level effects in both target and non-target species, as in mussel effects were observed from 0.1 µg/L (against a maximum measured environmental concentration of 3.37 µg/L (Campos et al. 2022a ) whereas in clams the effects started to occur from 1 µg/g, for a maximum MEC of 0.8 µg/g. These effects were more pronounced and evident in sediment exposures, which may be attributed to the tendency of the compound to adsorb onto sediment particles. Furthermore, organisms exposed to sediments with high organic matter content exhibited more intense alterations than those exposed to low-organic-matter sediments. Considering the full set of biomarker responses, the importance of using an integrated biomarker approach is evident as it enables early warning signals for aquatic environmental monitoring. Declarations Funding: This study was supported by Financiadora de Estudos e Projetos - FINEP (Process 1111/13–01.14.0141.00), and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Grant #456372/2013–0). ACFC (Grants #2016/02029-4 and #2021/06167-0), BGC (Grant #2017/10211–0), and GFEP (Grant #2018/23279-4) were funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). FCP (Grant # 88887.144657/2017-00) was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação para a Ciência e a Tecnologia (FCT, grants UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020). DMSA (PQ #308533/2018–6 and #313420/2023-8) and IBG (PQ #304398/2021-7) are research fellows of CNPq. Competing interests : The authors have no relevant financial or non-financial interests to disclose. Author Contribution statement ACFC: Conceptualization, Formal analysis, Methodology, Investigation; Writing – original draft . PGC: Methodology, Investigation, Writing – review & editing. GFEP: Methodology, Investigation, Writing – review & editing. BGC: Methodology, Investigation. FCP: Methodology, Investigation IBC: Supervision, Writing – review & editing DMSA: Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Writing – original draft . Acknowledgements We thank the NEPEA laboratory staff and the UNESP-CLP employees for their support. We are also grateful to Prof. Gilberto Fillmann (FURG) for the assistance, and “Rede Nacional de Estudos de Anti-incrustantes” (RNEA) for the support. DMSA thanks “Instituto Nacional de Ciência e Tecnologia em Identificação, quantificação, dispersão, riscos ambientais e mitigação da poluição por contaminantes emergentes em ambientes marinhos e costeiros (INCT CEMAR)” for the support (CNPq 408782/2024-2). References Abreu FEL, Martins SE, Fillmann G (2021) Ecological risk assessment of booster biocides in sediments of the Brazilian coastal areas. Chemosphere 276: 130155, https://doi.org/10.1016/j.chemosphere.2021.130155 Albanis TA, Lambropoulou DA, Sakkas VA, Konstantinou IK (2002) Antifouling paint booster biocide contamination in Greek marine sediments. Chemosphere 48, 475–485. https://doi.org/10.1016/S0045-6535(02)00134-0 Almeida E, Diamantino TC, de Sousa O (2007) Marine paints: The particular case of antifouling paints. Prog Org Coat 59: 2–20. https://doi.org/10.1016/j.porgcoat.2007.01.017 Almeida JC, Castro ÍB, Nunes BZ, Zanardi-Lamardo E (2023) Antifouling booster biocides in Latin America and Caribbean: A 20-year review. Mar Pollut Bull 189: 114718. https://doi.org/10.1016/j.marpolbul.2023.114718 ASTM (2008). Standard guide for collection, storage, characterization, and manipulation of sediments for toxicological testing. Annual Book of ASTM Standards, Vol. 11.05. Bellas J (2006). Comparative toxicity of alternative antifouling biocides on embryos and larvae of marine invertebrates. Sci Total Environ 367: 573–585. https://doi.org/10.1016/j.scitotenv.2006.01.028 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. https://doi.org/10.1016/0003-2697(76)90527-3 Campos BG, Figueiredo J, Perina F, Abessa DMS, Loureiro S, Martins R (2022a) Occurrence, effects and environmental risk of antifouling biocides (EU PT21): are marine ecosystems threatened? Crit Rev Environ Sci Technol 52: 3179–3210. https://doi.org/10.1080/10643389.2021.1910003 Campos BG, Fontes MK, Gusso-Choueri PK, Marinsek GP, Nobre CR, Moreno BB, Abreu FEL, Fillmann G, Mari RB, Abessa DMS (2022b) A preliminary study on multi-level biomarkers response of the tropical oyster Crassostrea brasiliana to exposure to the antifouling biocide DCOIT. Mar Pollut Bull 174: 113241. https://doi.org/10.1016/j.marpolbul.2021.113241 Campos BG, Perina FC, Abreu FEL, Fillmann G, Abessa DMS (2024) Degradation kinetics of antifouling biocides in sediment during the spiking equilibrium phase. Ecotoxicol Environ Contamin 19: 39–49. https://doi.org/10.5132/eec.2024.01.05 Castro ÍB, Westphal E, Fillmann G (2011) Tintas anti-incrustantes de terceira geração: novos biocidas no ambiente aquático. Quim Nova 34: 1021–1031. https://doi.org/10.1590/S0100-40422011000600020 Castro ÍB, Perina FC, Fillmann G (2012) Organotin contamination in South American coastal areas. Environ Monit Assess 184: 1781–1799. https://doi.org/10.1007/s10661-011-2078-7 Cima F, Varello R (2020) Immunotoxicity in Ascidians: antifouling compounds alternative to organotins—V. the case of dichlofluanid. J Mar Sci Eng 8: 396. https://doi.org/10.3390/jmse8060396 Cruz ACF, Pauly GFE, Araújo GS, Gusso-Choueri PK, Fonseca TG, Campos BG, Santelli RE, Freire AS, Braz BF, Bosco-Santos A, Luiz-Silva W, Machado W, Abessa DMS (2021) Metal bioaccumulation by the neotropical clam Anomalocardia flexuosa to estimate the quality of estuarine sediments. Bull Environ Contamin Toxicol 107: 106-113. https://doi.org/10.1007/s00128-020-03062-x Dafforn KA, Lewis JA, Johnston EL (2011) Antifouling strategies: History and regulation, ecological impacts and mitigation. Mar Pollut Bull 62(3): 453–465. https://doi.org/10.1016/j.marpolbul.2011.01.012 Dailianis S, Domouhtsidou GP, Raftopoulou E, Kaloyianni M, Dimitriadis VK (2003) Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring. Mar Environ Res 56: 443–470. https://doi.org/10.1016/s0141-1136(03)00005-9 Diagne C, Leroy B, Vaissière A-C, Gozlan RE, Roiz D, Jarić I, Salles J-M, Bradshaw CJA, Courchamp F (2021) High and rising economic costs of biological invasions worldwide. Nature, 592: 571-576. https://doi.org/10.1038/s41586-021-03405-6 Ellman GL, Courtney KD, Andres Jr V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7: 88–95. https://doi.org/10.1016/0006-2952(61)90145-9 Evans SM, Birchenough AC, Brancato MS (2000) The TBT Ban: Out of the Frying Pan into the Fire? Mar Pollut Bull 40: 204–211. https://doi.org/https://doi.org/10.1016/S0025-326X(99)00248-9 Fernandez MA, Pinheiro F (2007) New approaches for monitoring the marine environment: the case of antifouling paints. Int J Environ Health 1: 427–448. https://doi.org/10.1504/IJENVH.2007.017875 Furdek M, Mikac N, Bueno M, Tessier E, Cavalheiro J, Monperrus M (2016) Organotin persistence in contaminated marine sediments and porewaters: In situ degradation study using species-specific stable isotopic tracers. J Hazard Mater 307: 263–273. https://doi.org/10.1016/j.jhazmat.2015.12.037 Gagné F, Blaise C (1993) Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout ( Oncorhynchus mykiss ) after acute exposure to pulp and paper mill effluents. Water Res 27: 1669–1682. https://doi.org/10.1016/0043-1354(93)90131-Z Gagné F, Blaise C, Pellerin J, Pelletier E, Strand J (2006). Health status of Mya arenaria bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period. Ecotoxicol Environ Saf 64: 348–361. https://doi.org/10.1016/j.ecoenv.2005.04.007 Gagné F, André C, Cejka P, Gagnon C, Blaise C (2007) Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels ( Elliptio complanata ). Comp Biochem Physiol Part C: Toxicol & Pharmacol 145: 542–552. https://doi.org/10.1016/j.cbpc.2007.01.019 Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 249: 7130–7139. https://doi.org/10.1016/S0021-9258(19)42083-8 Hagger JA, Depledge MH, Galloway TS (2005) Toxicity of tributyltin in the marine mollusc Mytilus edulis . Mar Pollut Bull 51: 811–816. https://doi.org/10.1016/j.marpolbul.2005.06.044 Haitzer M, Höss S, Traunspurger W, Steinberg C (1998) Effects of dissolved organic matter (DOM) on the bioconcentration of organic chemicals in aquatic organisms—a review—. Chemosphere 37: 1335–1362. https://doi.org/10.1016/S0045-6535(98)00117-9 Hamwijk C, Schouten A, Foekema EM, Ravensberg JC, Collombon MT, Schmidt K, Kugler M (2005) Monitoring of the booster biocide dichlofluanid in water and marine sediment of Greek marinas. Chemosphere 60: 1316–1324. https://doi.org/10.1016/j.chemosphere.2005.01.072 Hilliam K, Floerl O, Treml EA (2024) Priorities for improving predictions of vessel-mediated marine invasions. Sci Total Environ 921: 171162. https://doi.org/10.1016/j.scitotenv.2024.171162 Hirota J, Szyper JP (1975) Separation of total particulate carbon into inorganic and organic components. Limnol Oceanogr 20(5): 896–900. https://doi.org/10.4319/lo.1975.20.5.0896 Jones KC, De Voogt P (1999) Persistent organic pollutants (POPs): state of the science. Environ Pollut 100: 209–221. https://doi.org/10.1016/S0269-7491(99)00098-6 Keppler C, Ringwood AH (2001) Expression of P-glycoprotein in the gills of oysters, Crassostrea virginica : seasonal and pollutant related effects. Aquat Toxicol 54: 195–204. https://doi.org/10.1016/S0166-445X(01)00151-5 Lagreze FJS, Sühnel S, Ramos RJ, Miotto M, Albuquerque MCP, Vieira CRW, de Melo CMR (2022) Bioaccumulation and depuration of Escherichia coli in the tropical clam Anomalocardia brasiliana at different salinities. Arq Bras Med Vet Zootec 74(1): 101-110. http://dx.doi.org/10.1590/1678-4162-12230 Lowe DM, Fossato VU, Depledge MH (1995) Contaminant-induced lysosomal membrane damage in blood cells of mussels Mytilus galloprovincialis from the Venice Lagoon: an in vitro study. Mar Ecol Prog Ser 129: 189–196. https://doi.org/10.3354/meps129189 Lushchak VI, Matviishyn TM, Husak VV, Storey JM, Storey KB (2018) Pesticide toxicity: a mechanistic approach. Exp Clynic Sci J 17: 1101-1136. http://dx.doi.org/10.17179/excli2018-1710 Luczak C, Janquin M-A, Kupka A (1997) Simple standard procedure for the routine determination of organic matter in marine sediment. Hydrobiologia 345: 87–94. https://doi.org/10.1023/A:1002902626798 Molnar JL, Gamboa RL, Revenga C, Spalding MD (2008) Assessing the global threat of invasive species to marine biodiversity. Front Ecol Environ 6(9): 485-492. https://doi.org/10.1890/070064 Monteiro A, Rodrigues V, Picado A, Dias JM, Abrantes N, Ré A, Rosa M, Russo M, Barreirinha A, Potiris M, Aghito M, Hänninen R, Majamäki E, Grönholm T, Alyuz U, Sokhi R, Kukkonen J, Jalkanen J-P (2024) Holistic evaluation of the environmental impacts of shipping in the sensitive region of Ria de Aveiro. Sci Total Environ 946: 174314. https://doi.org/10.1016/j.scitotenv.2024.174314 Morais LG, Gusso-Choueri PK, Abreu FEL, Castro IB, Abessa DMS, Choueri RB (2023) Multilevel assessment of chlorothalonil sediment toxicity to Latin American estuarine biota: effects on biomarkers, reproduction and survival in different benthic organisms. Sci Total Environ 872: 162215, https://doi.org/10.1016/j.scitotenv.2023.162215 Mudroch A, MacKnight SD (1994) Handbook of techniques for aquatic sediments sampling. CRC press. Olive PL (1988) DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells. Environ Mol Mutagen 11: 487–495. https://doi.org/10.1002/em.2850110409 Park MS, Kim YD, Kim B-M, Kim Y-J, Kim JK, Rhee J-S (2016) Effects of antifouling biocides on molecular and biochemical defense system in the gill of the pacific oyster Crassostrea gigas . PLoS One Dec 22;11(12):e0168978. https://doi.org/10.1371/journal.pone.0168978 Paz-Villarraga CA, Castro ÍB, Fillmann G (2022) Biocides in antifouling paint formulations currently registered for use. Environ Sci Pollut Res 29: 30090–30101. https://doi.org/10.1007/s11356-021-17662-5 Pereira CDS, Abessa DMS, Choueri RB, Almagro-Pastor V, Cesar A, Maranho LA, Martín-Díaz ML, Torres RJ, Gusso-Choueri PK, Almeida JE, Cortez FS. Mozeto AA, Silbiger HLN, Sousa ECPM, DelValls TA, Bainy, ACD (2014) Ecological relevance of sentinels’ biomarker responses: a multi-level approach. Mar Environ Res 96: 118–126. https://doi.org/10.1016/j.marenvres.2013.11.002 Ramos-Gómez J, Coz A, Viguri JR, Luque Á, Martín-Díaz ML, DelValls TÁ (2011) Biomarker responsiveness in different tissues of caged Ruditapes philippinarum and its use within an integrated sediment quality assessment. Environ Pollut 159: 1914–1922. https://doi.org/10.1016/j.envpol.2011.03.030 Repetto G, Del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3: 1125–1131. https://doi.org/10.1038/nprot.2008.75 Ringwood AH, Conners DE, Hoguet J (1998) Effects of natural and anthropogenic stressors on lysosomal destabilization in oysters Crassostrea virginica . Mar Ecol Prog Ser 166: 163–171. https://doi.org/10.3354/meps166163 Rocha PRR, Faria AT, Borges L, Silva LOC, Silva AA, Ferreira EA (2013) Sorção e dessorção do diuron em quatro latossolos brasileiros. Planta Daninha 31: 231–238. https://doi.org/10.1590/S0100-83582013000100025 Rodrigues AML, Borges-Azevedo CM, Henry-Silva GG (2010) Aspectos da biologia e ecologia do molusco bivalve Anomalocardia brasiliana (Gmelin, 1791) (Bivalvia, Veneridae). R bras Bioci Porto Alegre 8(4): 377-383. https://seer.ufrgs.br/index.php/rbrasbioci/article/view/114960 Said-Pullicino D, Erriquens FG, Gigliotti G (2007) Changes in the chemical characteristics of water-extractable organic matter during composting and their influence on compost stability and maturity. Bioresour Technol 98: 1822–1831. https://doi.org/10.1016/j.biortech.2006.06.018 Sakkas VA, Konstantinou IK, Albanis TA (2001) Photodegradation study of the antifouling booster biocide dichlofluanid in aqueous media by gas chromatographic techniques. J Chromatogr A 930)1-2): 135–144. https://doi.org/10.1016/S0021-9673(01)01193-1 Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 25(1): 192–205. https://doi.org/10.1016/0003-2697(68)90092-4 Sies H, Koch OR, Martino E, Boveris A (1979) Increased biliary glutathione disulfide release in chronically ethanol‐treated rats. FEBS Lett 103(2): 287–290. https://doi.org/10.1016/0014-5793(79)81346-0 Sutcu R, Altuntas I, Buyukvanli B, Akturk O, Ozturk O, Koylu H, Delibas N (2007) The effects of diazinon on lipid peroxidation and antioxidant enzymes in rat erythrocytes: role of vitamins E and C. Toxicol Ind Health 23: 13–17. Suzuki T, Nojiri H, Isono H, Ochi T (2004) Oxidative damages in isolated rat hepatocytes treated with the organochlorine fungicides captan, dichlofluanid and chlorothalonil. Toxicol 204(2-3): 97–107. https://doi.org/10.1016/j.tox.2004.06.025 Thomas KV, Brooks S (2010) The environmental fate and effects of antifouling paint biocides. Biofouling 26(1): 73–88. https://doi.org/10.1080/08927010903216564 USEPA (2002) Technical Basis for the Derivation of Equilibrium Partitioning Sediment Guidelines (ESGs) for the Protection of Benthic Organisms: Nonionic Organics: Draft. USEPA. Washington DC 20460. USEPA (1993) Guidance Manual Bedded Sediment Bioaccumulation Tests. USEPA. Washington DC 20460. EPA/600/R-93/183. Verdouw H, Van Echteld CJA, Dekkers EMJ (1978) Ammonia determination based on indophenol formation with sodium salicylate. Water Res 12(6): 399–402. https://doi.org/10.1016/0043-1354(78)90107-0 Vouvoulis N, Scrimshaw MD, Lester JN (2000) Occurrence of four biocides utilized in antifouling paints, as alternatives to organotin compounds, in waters and sediments of a commercial estuary in the UK. Mar Pollut Bull 40(11): 938–946. https://doi.org/10.1016/S0025-326X(00)00034-5 Walker CH, Sibly RM, Peakall DB (2005) Principles of ecotoxicology. Taylor & Francis, CRC press. Boca Raton, FL, USA. Wentworth CK (1922) A scale of grade and class terms for clastic sediments. J Geol 30: 377–392. https://doi.org/10.1086/622910 Wills ED (1987) Evaluation of lipid peroxidation in lipids and biological membranes. Biochemical toxicology: a practical approach. In: Snell K, Mullock B (eds) Biochemical Toxicology: a Practical Approach. Oxford: IRL Press, pp. 138–140. Yamano T, Morita S (1995) Effects of pesticides on isolated rat hepatocytes, mitochondria, and microsomes II. Arch Environ Contam Toxicol 28: 1–7. https://doi.org/10.1007/bf00213961 Zhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF (2002) Induction of acetylcholinesterase expression during apoptosis in various cell types. Cell Death Differ 9: 790–800. https://doi.org/10.1038/sj.cdd.4401034 Zhang Y, Song J, Yuan H, Xu Y, He Z, Duan L (2010) Biomarker responses in the bivalve ( Chlamys farreri ) to exposure of the environmentally relevant concentrations of lead, mercury, copper. Environ Toxicol Pharmacol 30: 19–25. https://doi.org/10.1016/j.etap.2010.03.008 Supplementary Files GraphicalAbstarct.png SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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Letters indicate statistically significant differences compared to the acetone control (p \\u0026lt; 0.05). Error bars represent standard deviations. C = control, C* = acetone control 0.05%.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/8424dccb48b2a877c474a0cb.png\"},{\"id\":100571189,\"identity\":\"f2ad1cfc-0c4f-42fa-aec8-4703b95b0268\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:41:07\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":175940,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eExposure biomarker responses in the gills and digestive glands of the mussel \\u003cem\\u003ePerna perna \\u003c/em\\u003eexposed to Dichlofluanid through water. Data are presented as box plots, with the boxes indicating the 25th and 75th percentiles; the line within each box represents the median value. Asterisks (*) above the data indicate statistically significant differences compared to the acetone control (p ≤ 0.05). Fig. 2.a. ANOVA, p = 0.0103; Fig. 2.b. ANOVA, p = 0.0005; Fig. 2.c. ANOVA, p = 0.0002; Fig. 2.d. ANOVA, p = 0.0013; Fig. 2.e.Kruskal–Wallis, p \\u0026lt; 0.0001; Fig. 2.f. ANOVA, p = 0.2299; Fig. 2.g. ANOVA, p = 0.0028; Fig. 2.h.ANOVA, p = 0.7243; Fig. 2.i.ANOVA, p = 0.0005; Fig. 2.j. ANOVA, p = 0.0043.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/ef1e24fd46e2a1e6c5f7dbb5.png\"},{\"id\":100595949,\"identity\":\"5728afbb-598b-46df-8f72-e5ab0f8b31d1\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:49:50\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":271428,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffect biomarker responses in the gills and digestive glands of the mussel \\u003cem\\u003ePerna perna\\u003c/em\\u003e exposed to Dichlofluanid through water. Data are presented as box plots, with the boxes representing the 25th and 75th percentiles. The line within each box indicates the median value. Asterisks (*) above the data denote statistically significant differences compared to the acetone control (p ≤ 0.05). Fig. 3.a. ANOVA, p = 0.0027; Fig. 3.b. ANOVA, p = 0.3107; Fig. 3.c. ANOVA, p = 0.0296; Fig. 3.d. ANOVA, p = 0.1856; Fig. 3.e. ANOVA, p = 0.0002; Fig. 3.f. ANOVA, p = 0.0004.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/5ff9103f4cb1489c86ed8c71.png\"},{\"id\":100595300,\"identity\":\"18f6ce89-6210-4e59-bf6c-079b4f6a7da0\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:48:10\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":172434,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProjection of variables on a plane formed by factors 1 and 2 resulting of Principal Components Analysis (PCA) integrating all biomarkers analyzed in \\u003cem\\u003ePerna perna\\u003c/em\\u003e exposed to various dichlofluanid concentrations in water. Br = Gills; GD = Digestive Gland; VN = Neutral Red Retention Assay.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/ab349c16f72d997a9642f499.png\"},{\"id\":100595951,\"identity\":\"ff189826-ac5e-41a9-a9ab-bed45a4aeb19\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:49:51\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":219364,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBiomarker responses of exposure in gills and digestive glands of the clam \\u003cem\\u003eAnomalocardia flexuosa \\u003c/em\\u003eexposed to dichlofluanid through spiked sediments. Data are presented as box plots, with boundaries indicating the 25th and 75th percentiles; the line within each box marks the median value. An asterisk (*) above the data points indicates a statistically significant difference compared to the acetone control within a specific organic matter condition. Letters denote significant differences between organic matter levels at the same dichlofluanid concentration (p = 0.05). High OM = High organic matter; Low OM = Low organic matter. Fig. 5.a. ANOVA, p = 0.0035; Fig. 5.b. ANOVA, p \\u0026lt; 0.0001; Fig. 5.c. ANOVA, p \\u0026lt; 0.0001; Fig. 5.d. ANOVA, p = 0.0057; Fig. 5.e. PERMANOVA, p = 0.001; Fig. 5.f. ANOVA, p \\u0026lt; 0.0001; Fig. 5.g. PERMANOVA, p = 0.001; Fig. 5.h. PERMANOVA, p = 0.001; Fig. 5.i. ANOVA, p = 0.0089; Fig. 5.j. ANOVA, p = 0.3946.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/cd7316e9967ee4d0e63c97f4.png\"},{\"id\":100595298,\"identity\":\"3e6e025e-80b6-4451-a3fe-35207cb86022\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:48:09\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":302382,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBiomarker responses of effect in gills and digestive glands of the clam \\u003cem\\u003eAnomalocardia flexuosa \\u003c/em\\u003eexposed to dichlofluanid through spiked sediments. Data are presented as box plots, with boundaries indicating the 25th and 75th percentiles; the line within each box represents the median value. An asterisk (*) above the data indicates statistically significant differences compared to the acetone control within a given level of organic matter. Letters denote statistically significant differences between organic matter levels for the same dichlofluanid concentration (p = 0.05). High OM = High organic matter; Low OM = Low organic matter. Two-way ANOVA: Fig. 6.a. p \\u0026lt; 0.0001; Fig. 6.b. p \\u0026lt; 0.0001; Fig. 6.c. p = 0.5284; Fig. 6.d. p = 0.0170; Fig. 6.e. p \\u0026lt; 0.0001; Fig. 6.f. p \\u0026lt; 0.0001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/a555a7fd37b58e3e3b1662c5.png\"},{\"id\":100594985,\"identity\":\"adb5fe35-48ae-4059-8ece-4f73bafecd18\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 13:46:52\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":112722,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eProjection of variables on a plane formed by factors 1 and 2 resulting of Principal Components Analysis (PCA) integrating all biomarkers analyzed in \\u003cem\\u003eAnomalocardia flexuosa\\u003c/em\\u003e exposed to various dichlofluanid concentrations in spiked sediments. G = Gills; DG = Digestive Gland; H = High organic matter; L = Low organic matter.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/c93d6c718e7a79e137e9646e.png\"},{\"id\":101880365,\"identity\":\"f071960b-a661-4c33-8bdc-3ce55d3803eb\",\"added_by\":\"auto\",\"created_at\":\"2026-02-04 14:57:40\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2285691,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/ad36e235-fd3a-4e89-b240-c87538367879.pdf\"},{\"id\":100571192,\"identity\":\"4aec2c34-cd80-4f83-8b9e-ef7ffd18de1b\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:41:07\",\"extension\":\"png\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":408479,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"GraphicalAbstarct.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/0c18ae08e42ab957e11ab8a6.png\"},{\"id\":100571198,\"identity\":\"b5f3c679-b590-44a6-96f5-85d01419a88b\",\"added_by\":\"auto\",\"created_at\":\"2026-01-19 09:41:07\",\"extension\":\"docx\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":216869,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryMaterial.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8593449/v1/ace9bcc1204c5805241d3dbc.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Effects of dichlofluanid in tropical marine bivalves exposed to water and spiked sediments: an assessment of biomarker responses\",\"fulltext\":[{\"header\":\"Highlights\",\"content\":\"\\u003cp\\u003eDichlofluanid affected lysosomes of the mussel Perna perna from 10 \\u0026micro;g/L\\u003c/p\\u003e\\u003cp\\u003eDichlofluanid affected gills and disgestive glands of P. perna from 1 \\u0026micro;g/L\\u003c/p\\u003e\\u003cp\\u003eDichlofluanid affected gills and disgestive glands of Anomalocardia flexuosa from 1 ng/g\\u003c/p\\u003e\\u003cp\\u003eOrganically richer sediments induced worst effects in A. flexuosa\\u003c/p\\u003e\\u003cp\\u003eEffects of dichlofluanid in bivalves occurred at environmental concentrations\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eBiofouling can be defined as the settlement of organisms on natural and anthropic hard surfaces submerged in water. Manmade structures affected by biofouling include oil platforms, pipelines, dam gates, aquaculture facilities, and small and large boats. In commercial vessels, biofouling may increase fuel consumption by up to 40%. Biofouling accelerates hull corrosion, generating additional costs for naval transport. Furthermore, organisms attached to ship hulls favor bioinvasion mechanisms over commercial routes worldwide (Hilliam et al. 2024). The introduction of non-indigenous species (NIS) has been associated with a range of environmental, economic, and cultural impacts, including threats to human health (Molnar et al. 2008). Imbalances in food chains and the extinction of native species have been associated with a rapid increase in international trade in recent decades (Diagne et al. 2021). Therefore, strategies have been recommended by the International Maritime Organization (IMO) to avoid or reduce marine invasions, including those concerning combat biofouling. Hence, antifouling coatings have been developed and widely used to protect ships and boat hulls (Almeida et al. 2007). Most protective systems are currently based on paints containing one or a combination of biocides aimed at inhibiting biofilm formation and larval settlement on submerged surfaces (Thomas and Brooks 2010).\\u003c/p\\u003e\\n\\u003cp\\u003eAntifouling biocides are potentially harmful to aquatic ecosystems and include a range of organic and inorganic substances. The first commercial antifouling paints using copper and zinc oxides were replaced by those based on organotin compounds such as tributyltin (TBT) because of their low effectiveness (Fernandez and Pinheiro 2007). Since then, TBT-based antifouling paints have been extensively used around the world, mainly because of their high durability. However, they were banned by the Antifouling Systems Convention issued by the IMO due to their widespread toxic effects, including endocrine disruptors detected in ship and boat traffic areas (Dafforn et al. 2011; Evans et al. 2000). Thus, a new set of non-metallic and metallic compounds started to be used as active booster biocides in the current generation of antifouling paints, including compounds such as cuprous oxides, copper and zinc pyrithione, Zineb, DCOIT, Irgarol, Diuron, dichlofluanid, and chlorothalonil (Castro et al. 2011; Paz-Villarraga et al. 2022).\\u003c/p\\u003e\\n\\u003cp\\u003eDue to the extensive use of these compounds, areas under intense ship and boat traffic, such as ports, marinas, shipyards, and navigation channels, are exposed to biocide contamination leached from these coatings. In fact, reports of booster biocide residues reaching the water column and other environmental compartments, such as sediments and biota, are frequent (Albanis et al. 2002; Almeida et al. 2023; Campos et al. 2022a; Castro et al. 2012; Gatidou et al. 2007; Hamwijk et al. 2005; Vouvoulis et al. 2000), as well as the environmental risks caused by these substances (Abreu et al. 2021; Campos et al. 2022a).\\u003c/p\\u003e\\n\\u003cp\\u003eDichlofluanid, or N-dichlorofluoromethythio-N\\u0026prime;,N\\u0026prime;-dimethyl-N-phenylsulfamide (C\\u003csub\\u003e9\\u003c/sub\\u003eH\\u003csub\\u003e11\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003eFN\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003eS\\u003csub\\u003e2\\u003c/sub\\u003e), is a booster biocide currently used in antifouling paints (Paz-Villarraga et al. 2022). It is a non-metallic organochlorine compound with low water solubility and the potential to accumulate and associate with particulate matter (Kow = 3.7) (Dafforn et al. 2011; Evans et al. 2000). Dichlofluanid is also used as a fungicide and can induce mutagenic and carcinogenic effects in non-target marine invertebrates (Bellas 2006). However, studies on dichlofluanid toxicity in marine organisms are still scarce, particularly in polar and tropical organisms (Campos et al. 2022a). Thus, further investigations are needed to evaluate the biological effects of dichlofluanid on tropical aquatic organisms and provide information for its regulation at global and local levels.\\u003c/p\\u003e\\n\\u003cp\\u003eThe use of biomarkers assessing biochemical, physiological or histological responses induced by dichlofluanid exposure, at the organism or \\u0026ldquo;sub-organism\\u0026rdquo; levels, may help to elucidate potential early impacts associated with this substance (Walker et al. 2005). Such approaches have traditionally used organisms such as fish and invertebrates as suitable biological models (Monteiro et al. 2024). Bivalve mollusks have great ecological and commercial importance and have been widely used to assess the bioaccumulation and effects of contaminants (Pereira et al. 2014; USEPA 1993). They are abundant and easy to collect, allowing the use of many individuals per experiment, and their sessile habits guarantee their exposure to contaminants. Moreover, bivalves are filter-feeding animals inhabiting both hard- and soft-bottom substrates directly exposed to water and/or sediment matrices (Pereira et al. 2014; USEPA 1993). In this sense, the organic matter present in sediments can influence the exposure of burrowing bivalves to dichlofluanid because organic compounds tend to have a higher affinity for organic carbon (Haitzer et al. 1998; Jones and De Voogt 1999). The sorption of dichlofluanid onto organic matter may reduce the fraction dissolved in the water but may favor the exposure of benthic organisms to this compound via feeding route.\\u003c/p\\u003e\\n\\u003cp\\u003eThis study aimed to assess the cellular and biochemical effects of dichlofluanid on two species of marine bivalves exposed to water and sediment contaminated with this biocide. Additionally, we evaluated the influence of sediment organic matter (OM) enrichment on dichlofluanid toxicity by exposing the bivalves to sediments containing different OM concentrations. We hypothesized that dichlofluanid can induce cellular and biochemical alterations in marine bivalves, and that presence of OM in sediment would reduce dichlofluanid bioavailability and consequently its effects on \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003ch2\\u003e\\u003cem\\u003eWater exposure\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eTo assess the biochemical and cellular effects of dichlofluanid dissolved in water, adult individuals of the brown mussel \\u003cem\\u003ePerna perna\\u003c/em\\u003e (mean lengths = 6.69 \\u0026plusmn; 0.53 cm) were employed as biological models. Specimens were sourced from an aquaculture farm located at Cocanha Beach, Caraguatatuba, S\\u0026atilde;o Paulo (23\\u0026deg;34\\u0026apos;40.0\\u0026quot;S \\u0026ndash; 45\\u0026deg;18\\u0026apos;52.8\\u0026quot;W) and subsequently transported to the laboratory, where they were acclimatized under controlled experimental conditions.\\u003c/p\\u003e\\n\\u003cp\\u003eThe experiment was conducted according to the protocols described by the USEPA (2002; 1993). The exposure treatments used five nominal dichlofluanid concentrations (0.01, 0.1, 1, 10.0, and 100 \\u0026micro;g/L) in seawater. Negative controls containing only clean seawater (collected from a pristine coastal area) and acetone (cosolvent) diluted in seawater (concentration of 0.05%) were prepared. Three replicates were established in chambers conditions for 24h with gentle aeration, at 25 \\u0026plusmn; 2 \\u0026deg;C, and under a controlled photoperiod (12 h light: 12 h dark). Fortification with dichlofluanid was then carried out, and twelve healthy \\u003cem\\u003eP. perna\\u003c/em\\u003e individuals were introduced into each replicate. The exposure period was 96 hours. Every 24 h or whenever gamete release by the organisms was observed, the test solutions were renewed, and a new fortification was performed to ensure the maintenance of initial dichlofluanid concentrations and overall water quality. Numerous studies have shown that dichlofluanid tends to degrade rapidly in water, with a half-life of just a few hours, generating by-products, such as N-dimethyl-N-phenyl-sulphamide (DMSA) and dichloromethane aniline (Sakkas et al. 2001; Vouvoulis et al. 2000).\\u003c/p\\u003e\\n\\u003cp\\u003eAfter 48 h and 96 h of exposure, five individuals from each replicate of the respective treatment were taken and analyzed for the neutral red retention time assay (Lowe et al. 1995). Throughout the test, physicochemical parameters such as pH, salinity, temperature, dissolved oxygen, and ammonia were monitored and maintained within acceptable ranges to prevent any interference on the experimental results. The first four parameters were measured using appropriate electrodes, while ammonia concentrations were determined using a colorimetric method following the protocol described by Verdouw et al. (1978). At the end of the experiment, all organisms were euthanized, and their soft tissues (gills and digestive glands) were removed and stored at -80 \\u0026deg;C for subsequent analysis of biochemical biomarkers.\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cem\\u003eSediment exposure\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThe experiment using spiked sediments used two types of sediments, each characterized by its organic matter (OM) content. One sediment had a low OM content (~4%), whereas the other had a higher OM content (~10%). Both sediments were collected from distinct locations in the Canan\\u0026eacute;ia-Iguape Peru\\u0026iacute;be Estuarine Complex, a protected area located on the southern coast of S\\u0026atilde;o Paulo State. Low-OM sediment was collected from Arrozal (25\\u0026deg;02.415\\u0026apos; S, 47\\u0026deg;55.540\\u0026apos; W), and high-OM sediment was collected from Ariri (25\\u0026deg;13.215\\u0026apos; S, 48\\u0026deg;02.492\\u0026apos; W). Both sediments were spiked with dichlofluanid to assess potential differences in toxicity related to the organic matter content. As in the aqueous solution assays, negative controls (dichlofluanid-free) and analytical blanks (acetone-containing sediments of both types) were prepared. The dichlofluanid concentrations tested in the sediment were 1, 10, 100, 1000, and 10000 ng/g.\\u003c/p\\u003e\\n\\u003cp\\u003eFor these experiments, the clam \\u003cem\\u003eAnomalocardia flexuosa\\u003c/em\\u003e was used as the biological model. Adult individuals of \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e (mean lengths = 2.01 \\u0026plusmn; 0.21 cm) were collected in the Cananeia region, S\\u0026atilde;o Paulo (25\\u0026deg;02.415\\u0026apos; S, 47\\u0026deg;55.540\\u0026apos; W), transported to the laboratory, and acclimatized to the test conditions. The experiments were conducted as described by the USEPA (1993, 2002). Six replicates were prepared in glass chambers containing a sediment layer approximately 4 cm thick and 1.5 L filtered seawater. The test system was maintained under equilibrium conditions, with gentle aeration and at 25 \\u0026plusmn; 2 \\u0026deg;C. After a 24-h acclimation period, seven healthy \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e individuals were introduced into each replicate. The tests were conducted under a controlled photoperiod (12 h light: 12 h dark), with continuous aeration and constant temperature (25 \\u0026plusmn; 2 \\u0026deg;C) for 21 days. Throughout the experimental period, physicochemical parameters, including pH, salinity, temperature, dissolved oxygen, and ammonia, were monitored using the methodologies previously described for the aqueous solution tests.\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cem\\u003eSedimentological analyses\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThe sediment grain size distribution was analyzed according to the protocol proposed by Mudroch and MacKnight (1994). Three aliquots (30 g) from each sample were dried in an oven at 60 \\u0026deg;C for two days, washed through a 0.063 \\u0026mu;m sieve to separate fine particles (silt and clay), and the sandy material retained on the sieve was dried again, sieved for 15 min in a RO-TAP shaker using sieves of different mesh sizes (\\u0026Phi; scale), and then weighed. The results were classified according to the Wentworth Scale (Wentworth, 1922). The calcium carbonate (CaCO₃) content in each sample was measured using the method described by Hirota and Szyper (1975), which involved sample digestion with 5N HCl for more than 24 h to remove CaCO\\u003csub\\u003e3\\u003c/sub\\u003e, followed by washing with distilled water and drying in an oven at 60 \\u0026deg;C. The percentage of CaCO₃ present in the sample was calculated from the observed weight loss. The organic matter (OM) content was estimated using the ignition method described by Luczak et al. (1997), where aliquots of dry sediment were incinerated in a muffle furnace at 500 \\u0026deg;C for 4 h. The percentage of OM was equal to the weight lost during ignition.\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cem\\u003eSediment spiking\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eSediment spiking was performed following the protocols described by ASTM (2008). Five dichlofluanid concentrations were used in the tests: 1, 10, 100, 1000, and 10000 \\u0026mu;g/g. To prepare each, the respective amounts of dichlofluanid were added to the sediment based on the dry weight percentage. The moist sediment was partitioned into aliquots, and different concentrations of dichlofluanid were added accordingly. The analytical blank (clean sediment containing acetone) was subjected to the same procedure, while the control (clean) sediment consisted of natural sediments. All sediment samples were agitated for 15 min using a device that provided multidirectional rotation within a hermetically sealed glass container. After mixing, the contaminated sediments were stored in the dark at 4 \\u0026deg;C for 72 h to allow equilibrium to be established between the contaminant, interstitial water, and the solid sediment phase. Subsequently, the sediments were distributed into test chambers for the exposure experiments. Comprehensive descriptions of the sediment spiking methodology, along with the analytical confirmation of dichlofluanid concentrations in the tested sediments, were provided by Campos et al. (2024). Dichlofluanid levels were determined using Gas Chromatography coupled with Mass Spectrometry (GC-MS).\\u003c/p\\u003e\\n\\u003ch2\\u003e\\u003cem\\u003eBiomarkers Analyses\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003ch2\\u003e\\u003cem\\u003eNeutral Red Retention Time (NRRT\\u003c/em\\u003e\\u003cem\\u003e)\\u003c/em\\u003e\\u003c/h2\\u003e\\n\\u003cp\\u003eThe analysis of Neutral Red Retention Time (NRRT) in hemocyte lysosomes followed the protocol described by Lowe et al. (1995). This biomarker was assessed only in experiments involving water exposure. Prior to the assay, the glass slides were pre-treated with a poly L-lysine solution to facilitate hemocyte adhesion. Hemolymph samples (0.5 mL) were extracted from the adductor muscle of the mussels using a hypodermic syringe containing a physiological solution (0.5 mL). After 20 min, 40 \\u0026micro;L of the cell suspension was placed on each slide. Slides with hemocytes were kept in a dark and humid chamber for 15 min. Subsequently, 40 \\u0026micro;L of the Neutral Red solution was added to each slide. After 15 min, the coverslips were placed over the samples. The slides were then examined under a microscope at 15-minute intervals during the first hour and subsequently up to a maximum of 120 min. The cells were analyzed to determine the retention time of neutral red by lysosomes. NRRT was obtained by estimating the proportion of cells in which lysosomal content leaked into the cytosol. When 50% or more of the cells on a glass slide exhibited such leakage, the respective organisms were considered physiologically stressed.\\u003c/p\\u003e\\n\\u003ch3 id=\\\"_Toc407449359\\\"\\u003e\\u003cem\\u003eBio\\u003c/em\\u003e\\u003cem\\u003echemical Biomarkers\\u003c/em\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe gills and digestive glands of both \\u003cem\\u003eP. perna\\u003c/em\\u003e and \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e were kept on ice and homogenized in Tris-HCl buffer (50 mM TRIS; 1 mM EDTA; 1 mM DTT; 50 mM sucrose; 150 mM KCl; 100 mM PMSF; pH 7.6). Following homogenization, aliquots were separated for total protein (TP), DNA damage, and lipid peroxidation (LPO) analyses. The homogenates were then centrifuged at 12,000 \\u0026times; g for 20 min at 4 \\u0026deg;C, and additional aliquots were collected for the analysis of TP, glutathione S-transferase (GST), and glutathione peroxidase (GPx) activity as well as for the quantification of non-protein thiols (GSH), acetylcholinesterase (AChE) activity, ethoxyresorufin-O-deethylase (EROD), and dibenzylfluorescein (DBF). All biomarker analyses were performed using a Biotek microplate reader, model Synergy\\u0026trade; HT.\\u003c/p\\u003e\\n\\u003cp\\u003eTotal proteins were quantified using the Bradford method (Bradford, 1976), and this content was used to normalize all biomarkers data. DNA damage was assessed using the alkaline precipitation assay proposed by Olive (1988), with fluorometrically performed DNA quantification (\\u0026lambda;ex 360 nm; \\u0026lambda;em 450 nm) (Gagn\\u0026eacute; et al. 2006). The results were expressed as \\u0026micro;g of DNA per mg of protein. LPO levels were determined by measuring thiobarbituric acid reactive substances (TBARS) using fluorescence detection (\\u0026lambda;ex 532 nm; \\u0026lambda;em 556 nm) as described by Wills (1987). The concentration of peroxidized lipids was expressed as \\u0026mu;M TBARS per mg of protein. AChE activity was measured at 412 nm using the colorimetric method (Ellman et al. 1961), and the results were expressed as \\u0026micro;mol DTNB per min per mg of protein. GSH levels were determined according to the method described by Sedlak and Lindsay (1968). GSH levels were measured spectrophotometrically at 415 nm and expressed as nanomoles of glutathione per milligram of protein. GPx activity was determined spectrophotometrically at 340 nm, based on the method of Sies et al. (1979), and the results were expressed as nmol per minute per mg of protein. GST activity was measured at 340 nm and 25 \\u0026deg;C using a microplate reader (Habig et al. 1974). The results were expressed as OD per minute per milligram of protein. EROD activity was evaluated using a modified method by Gagn\\u0026eacute; and Blaise (1993), and the results were expressed as pmol per minute per mg of protein. Dibenzylfluorescein (DBF) activity was determined according to Gagn\\u0026eacute; et al. (2007), using fluorescence (\\u0026lambda;ex 485 nm and \\u0026lambda;em 516 nm), and results were expressed as nmol per minute per mg of protein.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cem\\u003eStatistical and Exploratory Analyses\\u003c/em\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe results from the NRRT assay were first checked for normality and homoscedasticity and then analyzed by one-way ANOVA with Dunnett\\u0026rsquo;s post hoc test to detect significant differences in NRRT between the dichlofluanid concentrations, the analytical blank (acetone), and the control. The results obtained for biomarkers were first assessed for normality using the Kolmogorov\\u0026ndash;Smirnov test, followed by one-way ANOVA with Dunnett\\u0026rsquo;s post hoc test (or an equivalent non-parametric test when necessary) to determine significant differences among dichlofluanid concentrations, the blank (acetone), and the control in the water exposure tests. For the sediment exposure experiments using matrices with different organic matter contents, a two-way ANOVA with Bonferroni\\u0026rsquo;s post hoc test (or PERMANOVA for non-parametric data) was conducted to assess the effects of dichlofluanid concentration and sediment type and potential interactions between them. The results from the subchronic exposure tests (biomarkers) were further integrated using Principal Component Analysis (PCA) to identify patterns and potential groupings based on exposure conditions. For the sediment dataset, PERMANOVA was performed using the studied biomarkers (GSH, GST, GPx, EROD, AChE, LPO, and DNA damage).\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eWater exposure \\u0026ndash; Perna perna\\u0026nbsp;\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe physicochemical parameters of seawater at the \\u003cem\\u003ePerna perna\\u003c/em\\u003e cultivation site, as well as those recorded in the test solutions during the experiments, are presented in the Supplementary Material (Tables S1 and S2). During the tests the mortality rates remained below 20% of the total number of exposed organisms (Figure S1).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eNeutral Red Retention Time\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFigure 1 presents the Neutral Red Retention Times (NRRT) after 48 and 96 h of exposure to dichlofluanid. At the initial time point, the organisms exhibited a mean retention time of 40.5 minutes. After 96 h, NRRT was significantly reduced (p \\u0026lt; 0.05) in mussels exposed to 10 \\u0026micro;g/L (mean NRRT= 33 min) and 100 \\u0026micro;g/L (mean NRRT = 15 min) compared to the acetone control group (mean NRRT = 97.5 min). No significant differences in the NRRT were observed after 48 h of exposure. Hagger et al. (2005) reported that for the mussel \\u003cem\\u003eMytilus edulis\\u003c/em\\u003e, increased cytotoxicity induced by TBT using the Neutral Red assay occurred at concentrations above 0.5 \\u0026micro;g/L and at shorter retention times than those observed in the present study. These findings suggest that dichlofluanid may exhibit lower cytotoxicity than TBT, a compound that is used in antifouling coatings and has been banned in several countries.\\u003c/p\\u003e\\n\\u003cp\\u003eLysosomes are organelles responsible for handling tissue nutrition and repair, encompassing the main cellular structure for sequestration and detoxification of organic compounds, as reported in previous studies (Lowe et al. 1995). Under chemical stress, lysosomal membranes are damaged and leakage of lysosomal contents into the cytoplasm often occurs concurrently. Neutral red retention is minimally affected by natural factors and predominantly influenced by contaminants (Ringwood et al. 1998). Thus, diclofluanid induced the cytotoxicity observed in the present study, as shown by Suzuki et al. (2004). In our study, the significant responses observed for NRRT at higher dichlofluanid concentrations are concerning, since lysosomal membrane stability and integrity have been widely used for the early detection of toxic compound effects (Repetto et al. 2008). NRRT serves as an important indicator of cellular health, and any impairment in this parameter signals early signs of more severe adverse effects across multiple levels of biological organization, ranging from subcellular processes (Dailianis et al. 2003) to effects observable at the population and community scales.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cem\\u003e\\u003cu\\u003eBio\\u003c/u\\u003e\\u003c/em\\u003e\\u003cem\\u003e\\u003cu\\u003echemical Biomarkers\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eFigure 2 presents the results for the exposure biomarkers. The induction of GST activity in the digestive glands was observed at the highest dichlofluanid concentrations, as well as increased levels of GSH and GPx activity in the gills at the same concentrations. This induction of the antioxidant defense system has been previously reported for biocides used in antifouling paints, such as TBT, Irgarol and Diuron (Park et al. 2016).\\u003c/p\\u003e\\n\\u003cp\\u003eRegarding the biomarker of effect, lipid peroxidation (LPO) levels in the digestive glands were lower than those observed in the control groups (Fig. 3). This suggests that activation of detoxification mechanisms minimizes oxidative damage to cellular membranes. Cells possess various mechanisms to cope with oxidative stress, either by repairing damage or by reducing its occurrence through enzymatic and non-enzymatic antioxidant pathways. Enzymatic antioxidants neutralize oxidative stress (Sutcu et al. 2007).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003ePrincipal Component Analysis (PCA)\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe PCA results for the water-based exposure tests are shown in Tables 1 and S3 and Figure 4. The first two principal components (PCs) explained 64.54% of the total variance in the dataset (Table 1). The PCA revealed that, for the PC1, the seawater control, the acetone blank, the initial time point (T0), and the lowest dichlofluanid concentration (0.01 \\u0026mu;g/L) clustered together, while the other tested concentrations (0.1, 1, 10, and 100 \\u0026mu;g/L) formed a second group (Table 1, Figure 4). Regarding PC2, the control and analytical blank clustered with the two lowest dichlofluanid concentrations (0.01 and 0.1 \\u0026mu;g/L), while the higher concentrations (1, 10, and 100 \\u0026mu;g/L) separated. T0 appeared to be isolated from all other treatments (Table 1, Figure 4).\\u003c/p\\u003e\\n\\u003cp\\u003eFor PC1, positive associations were observed between GPx and GSH in the gills, and between GPx and GST in the digestive glands, along with an inverse relationship between DBF and GSH in the digestive glands (Table 1). These findings suggest activation of detoxification mechanisms in both the gills and digestive glands, which is also reflected in the individual biomarker plots. Although no statistically significant differences were detected, the plots for the digestive glands showed a decreasing trend in GSH and DBF levels (Fig. 2d and 2j). Moreover, a relationship between the enzymatic responses and the neutral red retention assay was observed. These effects were associated with intermediate and high dichlofluanid concentrations, although they did not result in measurable biochemical damage (i.e., lipid peroxidation or DNA damage). However, membrane-destabilizing effects were evident at higher concentrations, as demonstrated by significant lysosomal membrane destabilization revealed by the NRRT assay. As this is a biomarker of effect, the overall data suggest that detoxification pathways were not fully effective, leading to lysosomal membrane damage, especially at higher concentrations. Regarding PC2, negative correlations were found between GST and DNA damage in the gills, while positive correlations were observed between GPx and EROD, as well as between DNA damage, EROD, lipid peroxidation (LPO), and AChE in the digestive glands. This axis appears to have grouped variables that, when analyzed individually, did not show significant effects and thus did not contribute to a clear overall response in the biomarker data.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cspan id=\\\"_Toc407569921\\\"\\u003eTab 1. Results of Principal Components Analysis (PCA) integrating all biomarkers analyzed in \\u003cem\\u003ePerna perna\\u003c/em\\u003e mussels exposed to dichlofluanid concentrations in water. Factorial Scores for the initial time, control, acetone control, and dichlofluanid concentrations at 96 h in the water exposures. G = Gills; DG = Digestive Gland; NR = Neutral Red.\\u0026nbsp;\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003cdiv align=\\\"\\\"\\u003e\\n \\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"454\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003ePC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003eEigenvalue\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e% Variance\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003eCumulative Variance\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e6.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e35.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e35.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e4.86\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e28.58\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e64.55\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eVariable\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eDNA.G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.75\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eLPO. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.49\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.30\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eAchE. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.90\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eErod. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.61\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eDBF G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.27\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGPx. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.87\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.15\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGSH. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGST. G\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.65\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eDNA.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.69\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eLPO.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eAchE.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.56\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eErod.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.63\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eDBF DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.87\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.36\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGPx.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.53\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.56\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGSH.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.91\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eGST.DG\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.93\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.11\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eNRRT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.69\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.55\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eTreatment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e2.31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-4.80\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_Control\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e1.70\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.53\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_Acetone\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e2.67\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e2.98\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e2.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e1.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_0.1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-1.56\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.78\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-1.90\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.43\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-2.72\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003eT96_100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-2.88\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.05\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eIn the projection of variables relative to the two factors extracted by PCA, the treatments were grouped into three clusters plotted on the plane formed by PCs 1 and 2 (Fig. 4). T0 was isolated from other samples. One group consisted of the control, acetone blank, and the lowest concentration (0.01 \\u0026mu;g/L), whereas the other contained intermediate and higher dichlofluanid concentrations (0.1, 1, 10, and 100 \\u0026mu;g/L) (Fig. 4). These data suggest a metabolic effect in animals exposed to concentrations above 0.1 \\u0026mu;g/L.\\u003c/p\\u003e\\n\\u003cp\\u003eThe analyses of biomarkers, either analyzed individually or in multivariate analyses, showed that dichlofluanid may induce significant alterations in antioxidant processes, as observed in the biomarkers directly involved with glutathione (GSH, GST, GPx). This can be explained by the mechanisms of action of dichlofluanid and its primary metabolite, DMSA (N\\u0026apos;-dimethyl-N-phenyl-sulfamide). It is a multi-site herbicide and fungicide, which primary mode of action involves reactions of dichlofluanid with thiols within the cells of target organisms (Hamwijk et al., 2005). Thus, dichlofluanid is a reactive compound with sulfhydryl groups present in glutathiones (Yamano and Morita 1995). This study demonstrated an increase in GPx activity and GSH production in the gills, the first organs to combat xenobiotics, as the primary exposure route in bivalves occurs via the gills. This increase in GPx activity and GSH levels may be directly related to the biotransformation processes of dichlofluanid or the potential reactive oxygen species generated in the presence of xenobiotics (Keppler and Ringwood 2001). Biocides, such as dichlofluanid and DMSA, have pro-oxidant properties that cause imbalances between GSH and GSSG, leading to several other cell effects, including an increase in reactive oxygen species (ROS), which are eliminated by antioxidant enzymes such as GPx (Lushchak et al. 2018). In the digestive glands, the results showed increased GST activity and LPO levels at higher dichlofluanid concentrations, suggesting that the antioxidant system action was limited and unable to avoid oxidative stress. The effects observed in the digestive glands suggest that dichlofluanid is incorporated by bivalves and transported to the glands where detoxification processes occur. Digestive glands have been shown to be highly sensitive to xenobiotics in some bivalve species (Ramos-G\\u0026oacute;mez et al. 2011; Zhang et al. 2010).\\u003c/p\\u003e\\n\\u003cp\\u003eConsidering all the analyses performed when exposing bivalves to dichlofluanid via water, some effects were observed in both the gills and digestive glands, particularly at the higher concentrations tested. However, no DNA damage or neurotoxicity was observed, and the effects on lysosomal membranes of hemocytes were noted at higher concentrations, corroborating that the toxic effects of dichlofluanid are caused by oxidative stress, as lysosomal membranes have altered permeability due to ROS (Lowe et al. 1995). Thus, our results suggest that the effects of dichlofluanid are more related to membrane destabilization due to increased ROS levels. Studies have shown that bivalve hemocytes can be affected by biocides used as antifouling agents, such as DCOIT (Campos et al. 2022b) and chlorotalonil (Morais et al. 2023). Moreover, in ascidians exposed to dichlofluanid, hemocytes exhibited several types of alterations, such as apoptosis, cell shrinkage, reduced motility, and phagocytic activity (Cima and Varello 2020).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eSediment exposure \\u0026ndash; Anomalocardia flexuosa\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cpre\\u003e\\u003cem\\u003e\\u003cu\\u003eSedimentological analyzes\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/pre\\u003e\\n\\u003cp\\u003eTable 2 presents some sediment properties, including grain size distribution, calcium carbonate (CaCO₃) content, organic matter (OM) percentage, and dry weight. Both sediments used in the experiment consisted mainly of very fine sands mixed with mud and fine sands, as well as smaller quantities of other textural fractions, indicating that potential variations in toxicity are unlikely to be attributed to differences in sediment texture. The sediment collected from Ariri exhibited a higher OM content (10.4%) than that from Arrozal, which contained approximately 4% OM. As for CaCO₃ concentrations, no substantial differences were observed between the sediments, suggesting that this parameter likely did not play a significant role in influencing the outcomes of the toxicity assessments.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eTab 2. Grain size distribution, organic matter (OM), calcium carbonate (CaCO₃) content, and wet weight percentages of the sediments collected in Canan\\u0026eacute;ia (S\\u0026atilde;o Paulo, Brazil) and used in the dichlofluanid exposure assays with \\u003cem\\u003eAnomalocardia flexuosa\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"706\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 78px;\\\"\\u003e\\n \\u003ch3\\u003e\\u0026nbsp;\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eGravel\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eVery Coarse\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eCoarse\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 74px;\\\"\\u003e\\n \\u003ch3\\u003eMedium Sand\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003eFine Sand\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eVery Fine Sand\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 59px;\\\"\\u003e\\n \\u003ch3\\u003eSilt \\u0026amp; Clay\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003eOM\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eCaCO\\u003csub\\u003e3\\u003c/sub\\u003e\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003eWet Weight\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 78px;\\\"\\u003e\\n \\u003ch3\\u003eHigh OM\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e0.37\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e0.33\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e2.11\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 74px;\\\"\\u003e\\n \\u003ch3\\u003e5.21\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003e18.22\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e55.63\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 59px;\\\"\\u003e\\n \\u003ch3\\u003e17.10\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003e10.38\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e7.38\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e57.18\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 78px;\\\"\\u003e\\n \\u003ch3\\u003eLow OM\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e0.00\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e0.07\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e0.62\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 74px;\\\"\\u003e\\n \\u003ch3\\u003e1.42\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003e8.66\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e74.53\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 59px;\\\"\\u003e\\n \\u003ch3\\u003e13.81\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 57px;\\\"\\u003e\\n \\u003ch3\\u003e4.00\\u0026nbsp;\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e5.88\\u0026nbsp;\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 64px;\\\"\\u003e\\n \\u003ch3\\u003e49.52\\u003c/h3\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003eOM: Organic Matter; CaCO\\u003csub\\u003e3\\u003c/sub\\u003e: Calcium carbonate.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eChemical analyses\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAs previously reported by Campos et al. (2024) and summarized in Table 3, dichlofluanid was quantifiable only at a concentration of 1000 ng/g, where it exhibited degradation rates of 87% within 6 h and 99% after 24 h. This compound is recognized as a rapidly degrading biocide with a very short environmental half-life (Hamwijk et al. 2005). Nevertheless, given that dichlofluanid has been detected in concentrations exceeding established toxicity thresholds and that its degradation can lead to the formation of potentially toxic transformation products, investigation of its effects remains ecologically relevant.\\u003c/p\\u003e\\n\\u003cp\\u003eTab 3. Measured concentrations of dichlofluanid at 0, 6, and 24 hours post-spiking under equilibrium conditions, as previously reported by Campos et al. (2024).\\u003c/p\\u003e\\n\\u003cdiv align=\\\"\\\"\\u003e\\n \\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"548\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 132px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd rowspan=\\\"2\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eNominal concentration (ng.g\\u003csup\\u003e-1\\u003c/sup\\u003e)\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"3\\\" style=\\\"width: 303px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eTime\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 132px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0h\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e6h\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e24h\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd rowspan=\\\"4\\\" style=\\\"width: 132px;\\\"\\u003e\\n \\u003cp\\u003eDichlofluanid\\u003c/p\\u003e\\n \\u003cp\\u003e(LD = 0.5; LQ= 1)\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003eBlank\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;DL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;DL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;DL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026lt;QL\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e1000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e1189.5 \\u0026plusmn; 460.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 104px;\\\"\\u003e\\n \\u003cp\\u003e157.7 \\u0026plusmn; 36\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 95px;\\\"\\u003e\\n \\u003cp\\u003e16.9 \\u0026plusmn;7.4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003ch3\\u003eDL= Detection limit; QL \\u0026ndash; Quantification limit.\\u003c/h3\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eExposure of Anomalocardia flexuosa\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAmong the organisms exposed to sediments enriched with high OM content, there was no mortality. On the other hand, clams exposed to low OM sediment exhibited some mortality; however, the rates remained below 30% (Figure S2). The physicochemical parameters of the overlying water within the test chambers during the test are presented in Table S4, and remained within suitable ranges for the species (Cruz et al. 2021).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003eBiomarkers of exposure and effect\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFigures 5 and 6 present the biomarker responses measured in \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e following exposure to dichlofluanid-spiked sediments. In gill tissues, sediments with high OM content induced an increase in GST and EROD activities at the highest concentration (10000 ng/g), a reduction in GSH levels, and elevated GPx and AChE activities at the three highest concentrations (100, 1000, and 10000 ng/g). Additionally, increased LPO levels were observed at intermediate concentrations (1 and 10 ng/g). Exposure to low-OM sediment led to a reduction in EROD activity at the highest concentrations (1000 and 10000 ng/g).\\u003c/p\\u003e\\n\\u003cp\\u003eIn the digestive glands, exposure to high-OM sediment led to enhanced GST activity at intermediate concentrations (10 and 100 ng/g), decreased GSH and AChE activity, increased EROD activity and LPO levels at 10000 ng/g, decreased GPx activity at 1000 ng/g, and increased DNA damage at 10 ng/g. For low-OM sediment, the results showed decreased EROD activity at 1000 and 10000 ng/g, increased AChE activity at 10000 ng/g, and reduced LPO levels at varying concentrations (1, 10, and 10000 ng/g).\\u003c/p\\u003e\\n\\u003cp\\u003eOverall, both the gills and digestive glands exhibited consistent evidence of the activation of defense mechanisms, such as alteration of biotransformation processes (EROD), stimulation of conjugation mechanisms (GST activity), alteration of antioxidant responses (GPx, GSH), together with associated cellular effects (DNA damage and LPO), and altered AChE activity, which may indicate potential neurotoxicity. Such effects tended to be more pronounced in animals exposed to higher concentrations of dichlofluanid and organically richer sediments (i.e., high OM).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u003cu\\u003ePrincipal Component Analysis (PCA)\\u003c/u\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe results of the PCA performed using the results obtained from the exposure tests with sediments are presented in Tables 4 and S5, and figure 7. The first two principal components accounted for 63.93% of the total variance (Table 4). PCA revealed that along PC1, the water control, acetone blank, T0, and lower dichlofluanid concentrations (i.e., 1 and 10 ng/g) clustered together, whereas the higher concentrations (100, 1000, and 10000 ng/g) formed a separate group (Table 4, Figure 7). Along PC2, T0, water control, and the acetone blank were grouped together and separated from the 1 and 10 ng/g concentrations, while the remaining dichlofluanid concentrations formed a distinct cluster (Table 4).\\u003c/p\\u003e\\n\\u003cp\\u003eFor PC1, in sediments with high organic matter content, gill tissues showed a positive association between GST and AChE and a negative association between GSH and DNA damage. In the digestive glands, EROD and lipid peroxidation (LPO) were positively associated, whereas GSH, GPx, and AChE showed inverse associations. In low organic matter sediments, gills exhibited associations among EROD, GPx, DNA damage, AChE, and LPO, yet most of these variables did not show significant variations according to the ANOVA (Figures 5 and 6). In the digestive glands, an inverse relationship was observed between GST, GSH, and AChE (Table 4). For PC2, in high organic matter sediments, gills showed an association between GPx and LPO, while in the digestive glands, GST and DNA damage were positively associated, and AChE was negatively correlated.\\u003c/p\\u003e\\n\\u003cp\\u003eIn the projection of the variables along the two principal components extracted by PCA, the data were grouped into three clusters plotted on the plane defined by PCs 1 and 2 (Fig. 7), similarly to the pattern observed in the water exposure experiments. The T0 treatment appeared to be isolated from the other treatments. The second cluster comprised the control, analytical blank, and lower concentrations of dichlofluanid (1, 10, and 100 ng/g). The third group had the highest concentrations (1000 and 10000 ng/g). These results also suggest a more pronounced metabolic effect in organisms exposed to higher dichlofluanid concentrations. Overall, the PCA results supported the occurrence of adverse effects in \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e individuals exposed to dichlofluanid through sediments, particularly under conditions of elevated organic matter. These effects were more consistent at higher concentrations; however, biochemical alterations were detectable even at the lowest tested concentrations, indicating that the animals responded to minimal levels of dichlofluanid in the environment.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp id=\\\"_Toc407569926\\\"\\u003eTab 4. Results of Principal Components Analysis (PCA) integrating all biomarkers analyzed in \\u003cem\\u003eAnomalocardia flexuosa\\u0026nbsp;\\u003c/em\\u003eindividuals exposed to dichlofluanid concentrations in spiked sediments with two matrices of differing organic matter content. Factor scores for the initial time point, control, acetone blank, and dichlofluanid concentrations in sediment exposures over 21 days (Concentrations in ng/g). G = Gills; DG = Digestive gland; H = High organic matter; L = Low organic matter.\\u003c/p\\u003e\\n\\u003cdiv align=\\\"\\\"\\u003e\\n \\u003ctable border=\\\"0\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 127px;\\\"\\u003e\\n \\u003cp\\u003ePC\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003eEigenvalue\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 102px;\\\"\\u003e\\n \\u003cp\\u003e% Variance\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 135px;\\\"\\u003e\\n \\u003cp\\u003eCumulative Variance\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 127px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e12.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 102px;\\\"\\u003e\\n \\u003cp\\u003e42.96\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 135px;\\\"\\u003e\\n \\u003cp\\u003e42.96\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 127px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 113px;\\\"\\u003e\\n \\u003cp\\u003e5.87\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 102px;\\\"\\u003e\\n \\u003cp\\u003e20.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\" style=\\\"width: 135px;\\\"\\u003e\\n \\u003cp\\u003e63.97\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eDNA.G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.70\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eDNA.G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eLPO. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.85\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eLPO. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.51\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eAchE. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.86\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.30\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eAchE. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.74\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.21\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eEROD. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.45\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.06\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eEROD. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.85\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.26\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGPx. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.05\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.82\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGPx. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.73\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGSH. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-\\u003cstrong\\u003e0.73\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGSH. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGST. G.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.83\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGST. G.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.31\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eDNA.DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.35\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.66\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eDNA.DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.36\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eLPO. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.97\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.003\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eLPO. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.56\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eAchE. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.81\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.51\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eAchE. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.90\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eEROD. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.94\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.02\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eEROD. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.42\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.69\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGPx. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.77\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.46\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGPx. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.25\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-\\u003cstrong\\u003e0.81\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGSH. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.85\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGSH. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e0.65\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.48\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGST. DG.H\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.01\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.86\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eGST. DG.L\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003e-0.76\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.18\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eTreatment\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003ePC1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003ePC2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-0.85\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e4.24\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_Control\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-3.14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e1.38\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_Acetone\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-2.39\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e1.29\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-2.12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-1.89\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_10\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e-1.73\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-3.28\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_100\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e0.03\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-2.22\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_1000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e3.08\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e0.50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 165px;\\\"\\u003e\\n \\u003cp\\u003eT21_10000\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 151px;\\\"\\u003e\\n \\u003cp\\u003e7.13\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd colspan=\\\"2\\\" valign=\\\"top\\\" style=\\\"width: 161px;\\\"\\u003e\\n \\u003cp\\u003e-0.02\\u0026nbsp;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n\\u003c/div\\u003e\\n\\u003cp\\u003eThe results of the PERMANOVA using data obtained for both gills and digestive glands are presented in the Supplementary Material (Figs S3 and S4, and Tabs S6-S9). In summary, these results showed a clear separation between the different types of sediments, with a grouping of the high-OM sediment with the highest concentrations of the low-OM sediment, whereas the remaining concentrations of the low-OM sediment formed a second group for the gills. These results also highlight that, for both the high and low organic matter sediments, significant differences in biomarkers were observed at the highest concentrations. Similar results were observed for digestive glands, indicating separation by sediment type (i.e., OM content), and only the highest dichlofluanid concentrations of the high-OM sediment were isolated. When comparing sediments with different organic matter concentrations, this analysis revealed significant differences in the controls and at concentrations of 1, 10, and 10000 ng/g.\\u003c/p\\u003e\\n\\u003cp\\u003eIn general, biological responses were more pronounced in organisms exposed to sediments with a high quantity of organic matter. In such matrices, organic compounds tend to adsorb onto organic matter, establishing various chemical interactions that directly influence their bioavailability and toxicity to benthic organisms (Haitzer et al. 1998; Jones and De Voogt 1999). The stability of contaminants in sedimentary matrices is also related to the rate or degree of organic matter decomposition (Said-Pullicino et al., 2007). This fraction, retained in the sediment, can be directly transferred to benthic organisms, such as \\u003cem\\u003eA. flexuosa\\u0026nbsp;\\u003c/em\\u003ein the present study. According to Lagreze et al. (2022), \\u003cem\\u003eA. flexuosa\\u0026nbsp;\\u003c/em\\u003eis a filter feeding mollusk that ingests particles suspended in the water using their siphons; however, it also ingests large amounts of organic and inorganic material deposited on the sediments (Rodrigues et al. 2010) and is thus susceptible to contaminants present in the sedimentary environment.\\u003c/p\\u003e\\n\\u003cp\\u003eOther booster biocides used in antifouling paints and with solubilities comparable to that of dichlofluanid, such as diuron (log Kow = 2.7-2.8) and TBT (log Kow = 3.1-4.11), also appear to be strongly influenced by the concentration of organic matter, with higher OM levels enhancing their retention (Rocha et al., 2013; Furdek et al., 2016). In this sense, the increased retention of dichlofluanid in OM richer sediments likely favors the exposure of \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e and consequently could explain the negative effects recorded in our study, especially at higher concentrations.\\u003c/p\\u003e\\n\\u003cp\\u003eBiomarker analyses revealed a typical response pattern in both the gills and digestive glands: activation of EROD, involved in phase I biotransformation processes, followed by the induction of conjugation enzymes such as GST, accompanied by the consumption of GSH during detoxification. However, the antioxidant system was insufficient to completely metabolize or eliminate dichlofluanid, resulting in lipid peroxidation. This outcome is consistent with previous findings by Suzuki et al. (2004), who demonstrated that dichlofluanid induced lipid peroxidation and cytotoxicity at most of the tested concentrations above 25 \\u0026mu;M. In addition to oxidative stress, signs of neurotoxicity were observed in both tissues. A key target of some pesticides, particularly organophosphates, is the enzyme acetylcholinesterase (AChE), which hydrolyzes the neurotransmitter acetylcholine, and increased AChE activity has been associated with cellular apoptosis (Zhang et al. 2002). In this investigation, an increase in AChE activity was reported for both the gills and digestive glands, associated with biomarkers related to oxidative stress, such as LPO and DNA damage. This can be explained by the mode of action of dichlofluanid, which involves increased production of ROS, which causes oxidative stress (Keppler and Ringwood 2001; Lushchak et al. 2018). Therefore, our results demonstrate that dichlofluanid can harm neotropical benthic species, such as \\u003cem\\u003eA. flexuosa\\u003c/em\\u003e, especially at higher concentrations and in organically rich sediments. However, more studies using other taxonomic groups are necessary to detail the ecological risks caused by dichlofluanid to tropical species.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eBased on the results, it can be inferred that dichlofluanid induces cellular-level effects in both target and non-target species, as in mussel effects were observed from 0.1 \\u0026micro;g/L (against a maximum measured environmental concentration of 3.37 \\u0026micro;g/L (Campos et al. \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2022a\\u003c/span\\u003e) whereas in clams the effects started to occur from 1 \\u0026micro;g/g, for a maximum MEC of 0.8 \\u0026micro;g/g. These effects were more pronounced and evident in sediment exposures, which may be attributed to the tendency of the compound to adsorb onto sediment particles. Furthermore, organisms exposed to sediments with high organic matter content exhibited more intense alterations than those exposed to low-organic-matter sediments. Considering the full set of biomarker responses, the importance of using an integrated biomarker approach is evident as it enables early warning signals for aquatic environmental monitoring.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u0026nbsp;\\u003c/strong\\u003eThis study was supported by Financiadora de Estudos e Projetos - FINEP (Process 1111/13\\u0026ndash;01.14.0141.00), and Conselho Nacional de Desenvolvimento Cient\\u0026iacute;fico e Tecnol\\u0026oacute;gico \\u0026ndash; CNPq (Grant #456372/2013\\u0026ndash;0). ACFC (Grants #2016/02029-4 and #2021/06167-0), BGC (Grant #2017/10211\\u0026ndash;0), and GFEP (Grant #2018/23279-4) were funded by the Funda\\u0026ccedil;\\u0026atilde;o de Amparo \\u0026agrave; Pesquisa do Estado de S\\u0026atilde;o Paulo (FAPESP). FCP (Grant # 88887.144657/2017-00) was funded by Coordena\\u0026ccedil;\\u0026atilde;o de Aperfei\\u0026ccedil;oamento de Pessoal de N\\u0026iacute;vel Superior (CAPES) and Funda\\u0026ccedil;\\u0026atilde;o para a Ci\\u0026ecirc;ncia e a Tecnologia (FCT, grants UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020). DMSA (PQ #308533/2018\\u0026ndash;6 and #313420/2023-8) and IBG (PQ #304398/2021-7) are research fellows of CNPq.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e: The authors have no relevant financial or non-financial interests to disclose.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contribution statement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eACFC: Conceptualization, Formal analysis, Methodology, Investigation; Writing \\u0026ndash; original draft\\u003cstrong\\u003e.\\u003c/strong\\u003e PGC: Methodology, Investigation, Writing \\u0026ndash; review \\u0026amp; editing. GFEP: Methodology, Investigation, Writing \\u0026ndash; review \\u0026amp; editing. BGC: Methodology, Investigation. FCP: Methodology, Investigation IBC: Supervision, Writing \\u0026ndash; review \\u0026amp; editing DMSA: Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Writing \\u0026ndash; original draft\\u003cstrong\\u003e.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank the NEPEA laboratory staff and the UNESP-CLP employees for their support. We are also grateful to Prof. Gilberto Fillmann (FURG) for the assistance, and \\u0026ldquo;Rede Nacional de Estudos de Anti-incrustantes\\u0026rdquo; (RNEA) for the support. DMSA thanks \\u0026ldquo;Instituto Nacional de Ci\\u0026ecirc;ncia e Tecnologia em Identifica\\u0026ccedil;\\u0026atilde;o, quantifica\\u0026ccedil;\\u0026atilde;o, dispers\\u0026atilde;o, riscos ambientais e mitiga\\u0026ccedil;\\u0026atilde;o da polui\\u0026ccedil;\\u0026atilde;o por contaminantes emergentes em ambientes marinhos e costeiros (INCT CEMAR)\\u0026rdquo; for the support (CNPq 408782/2024-2).\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eAbreu FEL, Martins SE, Fillmann G (2021) Ecological risk assessment of booster biocides in sediments of the Brazilian coastal areas. Chemosphere 276: 130155, https://doi.org/10.1016/j.chemosphere.2021.130155\\u003c/li\\u003e\\n \\u003cli\\u003eAlbanis TA, Lambropoulou DA, Sakkas VA, Konstantinou IK (2002) Antifouling paint booster biocide contamination in Greek marine sediments. Chemosphere 48, 475\\u0026ndash;485. https://doi.org/10.1016/S0045-6535(02)00134-0\\u003c/li\\u003e\\n \\u003cli\\u003eAlmeida E, Diamantino TC, de Sousa O (2007) Marine paints: The particular case of antifouling paints. Prog Org Coat 59: 2\\u0026ndash;20. https://doi.org/10.1016/j.porgcoat.2007.01.017\\u003c/li\\u003e\\n \\u003cli\\u003eAlmeida JC, Castro \\u0026Iacute;B, Nunes BZ, Zanardi-Lamardo E (2023) Antifouling booster biocides in Latin America and Caribbean: A 20-year review. Mar Pollut Bull 189: 114718. https://doi.org/10.1016/j.marpolbul.2023.114718\\u003c/li\\u003e\\n \\u003cli\\u003eASTM (2008). Standard guide for collection, storage, characterization, and manipulation of sediments for toxicological testing. Annual Book of ASTM Standards, Vol. 11.05.\\u003c/li\\u003e\\n \\u003cli\\u003eBellas J (2006). Comparative toxicity of alternative antifouling biocides on embryos and larvae of marine invertebrates. Sci Total Environ 367: 573\\u0026ndash;585. https://doi.org/10.1016/j.scitotenv.2006.01.028\\u003c/li\\u003e\\n \\u003cli\\u003eBradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248\\u0026ndash;254. https://doi.org/10.1016/0003-2697(76)90527-3\\u003c/li\\u003e\\n \\u003cli\\u003eCampos BG, Figueiredo J, Perina F, Abessa DMS, Loureiro S, Martins R (2022a) Occurrence, effects and environmental risk of antifouling biocides (EU PT21): are marine ecosystems threatened? Crit Rev Environ Sci Technol 52: 3179\\u0026ndash;3210. https://doi.org/10.1080/10643389.2021.1910003\\u003c/li\\u003e\\n \\u003cli\\u003eCampos BG, Fontes MK, Gusso-Choueri PK, Marinsek GP, Nobre CR, Moreno BB, Abreu FEL, Fillmann G, Mari RB, Abessa DMS (2022b) A preliminary study on multi-level biomarkers response of the tropical oyster \\u003cem\\u003eCrassostrea brasiliana\\u003c/em\\u003e to exposure to the antifouling biocide DCOIT. Mar Pollut Bull 174: 113241. https://doi.org/10.1016/j.marpolbul.2021.113241\\u003c/li\\u003e\\n \\u003cli\\u003eCampos BG, Perina FC, Abreu FEL, Fillmann G, Abessa DMS (2024) Degradation kinetics of antifouling biocides in sediment during the spiking equilibrium phase. Ecotoxicol Environ Contamin 19: 39\\u0026ndash;49. https://doi.org/10.5132/eec.2024.01.05\\u003c/li\\u003e\\n \\u003cli\\u003eCastro \\u0026Iacute;B, Westphal E, Fillmann G (2011) Tintas anti-incrustantes de terceira gera\\u0026ccedil;\\u0026atilde;o: novos biocidas no ambiente aqu\\u0026aacute;tico. Quim Nova 34: 1021\\u0026ndash;1031. https://doi.org/10.1590/S0100-40422011000600020\\u003c/li\\u003e\\n \\u003cli\\u003eCastro \\u0026Iacute;B, Perina FC, Fillmann G (2012) Organotin contamination in South American coastal areas. Environ Monit Assess 184: 1781\\u0026ndash;1799. https://doi.org/10.1007/s10661-011-2078-7\\u003c/li\\u003e\\n \\u003cli\\u003eCima F, Varello R (2020) Immunotoxicity in Ascidians: antifouling compounds alternative to organotins\\u0026mdash;V. the case of dichlofluanid. J Mar Sci Eng 8: 396. https://doi.org/10.3390/jmse8060396\\u003c/li\\u003e\\n \\u003cli\\u003eCruz ACF, Pauly GFE, Ara\\u0026uacute;jo GS, Gusso-Choueri PK, Fonseca TG, Campos BG, Santelli RE, Freire AS, Braz BF, Bosco-Santos A, Luiz-Silva W, Machado W, Abessa DMS (2021) Metal bioaccumulation by the neotropical clam \\u003cem\\u003eAnomalocardia flexuosa\\u003c/em\\u003e to estimate the quality of estuarine sediments. Bull Environ Contamin Toxicol 107: 106-113. https://doi.org/10.1007/s00128-020-03062-x\\u003c/li\\u003e\\n \\u003cli\\u003eDafforn KA, Lewis JA, Johnston EL (2011) Antifouling strategies: History and regulation, ecological impacts and mitigation. Mar Pollut Bull 62(3): 453\\u0026ndash;465. https://doi.org/10.1016/j.marpolbul.2011.01.012\\u003c/li\\u003e\\n \\u003cli\\u003eDailianis S, Domouhtsidou GP, Raftopoulou E, Kaloyianni M, Dimitriadis VK (2003) Evaluation of neutral red retention assay, micronucleus test, acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of \\u003cem\\u003eMytilus galloprovincialis\\u003c/em\\u003e (L.), in pollution monitoring. Mar Environ Res 56: 443\\u0026ndash;470. https://doi.org/10.1016/s0141-1136(03)00005-9\\u003c/li\\u003e\\n \\u003cli\\u003eDiagne C, Leroy B, Vaissi\\u0026egrave;re A-C, Gozlan RE, Roiz D, Jarić I, Salles J-M, Bradshaw CJA, Courchamp F (2021) High and rising economic costs of biological invasions worldwide. Nature, 592: 571-576. https://doi.org/10.1038/s41586-021-03405-6\\u003c/li\\u003e\\n \\u003cli\\u003eEllman GL, Courtney KD, Andres Jr V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7: 88\\u0026ndash;95. https://doi.org/10.1016/0006-2952(61)90145-9\\u003c/li\\u003e\\n \\u003cli\\u003eEvans SM, Birchenough AC, Brancato MS (2000) The TBT Ban: Out of the Frying Pan into the Fire? Mar Pollut Bull 40: 204\\u0026ndash;211. https://doi.org/https://doi.org/10.1016/S0025-326X(99)00248-9\\u003c/li\\u003e\\n \\u003cli\\u003eFernandez MA, Pinheiro F (2007) New approaches for monitoring the marine environment: the case of antifouling paints. Int J Environ Health 1: 427\\u0026ndash;448. https://doi.org/10.1504/IJENVH.2007.017875\\u003c/li\\u003e\\n \\u003cli\\u003eFurdek M, Mikac N, Bueno M, Tessier E, Cavalheiro J, Monperrus M (2016) Organotin persistence in contaminated marine sediments and porewaters: In situ degradation study using species-specific stable isotopic tracers. J Hazard Mater 307: 263\\u0026ndash;273. https://doi.org/10.1016/j.jhazmat.2015.12.037\\u003c/li\\u003e\\n \\u003cli\\u003eGagn\\u0026eacute; F, Blaise C (1993) Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout (\\u003cem\\u003eOncorhynchus mykiss\\u003c/em\\u003e) after acute exposure to pulp and paper mill effluents. Water Res 27: 1669\\u0026ndash;1682. https://doi.org/10.1016/0043-1354(93)90131-Z\\u003c/li\\u003e\\n \\u003cli\\u003eGagn\\u0026eacute; F, Blaise C, Pellerin J, Pelletier E, Strand J (2006). Health status of \\u003cem\\u003eMya arenaria\\u003c/em\\u003e bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period. Ecotoxicol Environ Saf 64: 348\\u0026ndash;361. https://doi.org/10.1016/j.ecoenv.2005.04.007\\u003c/li\\u003e\\n \\u003cli\\u003eGagn\\u0026eacute; F, Andr\\u0026eacute; C, Cejka P, Gagnon C, Blaise C (2007) Toxicological effects of primary-treated urban wastewaters, before and after ozone treatment, on freshwater mussels (\\u003cem\\u003eElliptio complanata\\u003c/em\\u003e). Comp Biochem Physiol Part C: Toxicol \\u0026amp; Pharmacol 145: 542\\u0026ndash;552. https://doi.org/10.1016/j.cbpc.2007.01.019\\u003c/li\\u003e\\n \\u003cli\\u003eHabig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J Biol Chem 249: 7130\\u0026ndash;7139. https://doi.org/10.1016/S0021-9258(19)42083-8\\u003c/li\\u003e\\n \\u003cli\\u003eHagger JA, Depledge MH, Galloway TS (2005) Toxicity of tributyltin in the marine mollusc \\u003cem\\u003eMytilus edulis\\u003c/em\\u003e. Mar Pollut Bull 51: 811\\u0026ndash;816. https://doi.org/10.1016/j.marpolbul.2005.06.044\\u003c/li\\u003e\\n \\u003cli\\u003eHaitzer M, H\\u0026ouml;ss S, Traunspurger W, Steinberg C (1998) Effects of dissolved organic matter (DOM) on the bioconcentration of organic chemicals in aquatic organisms\\u0026mdash;a review\\u0026mdash;. Chemosphere 37: 1335\\u0026ndash;1362. https://doi.org/10.1016/S0045-6535(98)00117-9\\u003c/li\\u003e\\n \\u003cli\\u003eHamwijk C, Schouten A, Foekema EM, Ravensberg JC, Collombon MT, Schmidt K, Kugler M (2005) Monitoring of the booster biocide dichlofluanid in water and marine sediment of Greek marinas. Chemosphere 60: 1316\\u0026ndash;1324. https://doi.org/10.1016/j.chemosphere.2005.01.072\\u003c/li\\u003e\\n \\u003cli\\u003eHilliam K, Floerl O, Treml EA (2024) Priorities for improving predictions of vessel-mediated marine invasions. Sci Total Environ 921: 171162. https://doi.org/10.1016/j.scitotenv.2024.171162\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eHirota J, Szyper JP (1975) Separation of total particulate carbon into inorganic and organic components. Limnol Oceanogr 20(5): 896\\u0026ndash;900. https://doi.org/10.4319/lo.1975.20.5.0896\\u003c/li\\u003e\\n \\u003cli\\u003eJones KC, De Voogt P (1999) Persistent organic pollutants (POPs): state of the science. Environ Pollut 100: 209\\u0026ndash;221. https://doi.org/10.1016/S0269-7491(99)00098-6\\u003c/li\\u003e\\n \\u003cli\\u003eKeppler C, Ringwood AH (2001) Expression of P-glycoprotein in the gills of oysters, \\u003cem\\u003eCrassostrea virginica\\u003c/em\\u003e: seasonal and pollutant related effects. Aquat Toxicol 54: 195\\u0026ndash;204. https://doi.org/10.1016/S0166-445X(01)00151-5\\u003c/li\\u003e\\n \\u003cli\\u003eLagreze FJS, S\\u0026uuml;hnel S, Ramos RJ, Miotto M, Albuquerque MCP, Vieira CRW, de Melo CMR (2022) Bioaccumulation and depuration of Escherichia coli in the tropical clam \\u003cem\\u003eAnomalocardia brasiliana\\u003c/em\\u003e at different salinities. Arq Bras Med Vet Zootec 74(1): 101-110. http://dx.doi.org/10.1590/1678-4162-12230\\u003c/li\\u003e\\n \\u003cli\\u003eLowe DM, Fossato VU, Depledge MH (1995) Contaminant-induced lysosomal membrane damage in blood cells of mussels \\u003cem\\u003eMytilus galloprovincialis\\u003c/em\\u003e from the Venice Lagoon: an in vitro study. Mar Ecol Prog Ser 129: 189\\u0026ndash;196. https://doi.org/10.3354/meps129189\\u003c/li\\u003e\\n \\u003cli\\u003eLushchak VI, Matviishyn TM, Husak VV, Storey JM, Storey KB (2018) Pesticide toxicity: a mechanistic approach. Exp Clynic Sci J 17: 1101-1136. http://dx.doi.org/10.17179/excli2018-1710\\u003c/li\\u003e\\n \\u003cli\\u003eLuczak C, Janquin M-A, Kupka A (1997) Simple standard procedure for the routine determination of organic matter in marine sediment. Hydrobiologia 345: 87\\u0026ndash;94. https://doi.org/10.1023/A:1002902626798\\u003c/li\\u003e\\n \\u003cli\\u003eMolnar JL, Gamboa RL, Revenga C, Spalding MD (2008) Assessing the global threat of invasive species to marine biodiversity. Front Ecol Environ 6(9): 485-492. https://doi.org/10.1890/070064\\u003c/li\\u003e\\n \\u003cli\\u003eMonteiro A, Rodrigues V, Picado A, Dias JM, Abrantes N, R\\u0026eacute; A, Rosa M, Russo M, Barreirinha A, Potiris M, Aghito M, H\\u0026auml;nninen R, Majam\\u0026auml;ki E, Gr\\u0026ouml;nholm T, Alyuz U, Sokhi R, Kukkonen J, Jalkanen J-P (2024) Holistic evaluation of the environmental impacts of shipping in the sensitive region of Ria de Aveiro. Sci Total Environ 946: 174314. https://doi.org/10.1016/j.scitotenv.2024.174314\\u003c/li\\u003e\\n \\u003cli\\u003eMorais LG, Gusso-Choueri PK, Abreu FEL, Castro IB, Abessa DMS, Choueri RB (2023) Multilevel assessment of chlorothalonil sediment toxicity to Latin American estuarine biota: effects on biomarkers, reproduction and survival in different benthic organisms. Sci Total Environ 872: 162215, https://doi.org/10.1016/j.scitotenv.2023.162215\\u003c/li\\u003e\\n \\u003cli\\u003eMudroch A, MacKnight SD (1994) Handbook of techniques for aquatic sediments sampling. CRC press.\\u003c/li\\u003e\\n \\u003cli\\u003eOlive PL (1988) DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells. Environ Mol Mutagen 11: 487\\u0026ndash;495. https://doi.org/10.1002/em.2850110409\\u003c/li\\u003e\\n \\u003cli\\u003ePark MS, Kim YD, Kim B-M, Kim Y-J, Kim JK, Rhee J-S (2016) Effects of antifouling biocides on molecular and biochemical defense system in the gill of the pacific oyster \\u003cem\\u003eCrassostrea gigas\\u003c/em\\u003e. PLoS One Dec 22;11(12):e0168978. https://doi.org/10.1371/journal.pone.0168978\\u003c/li\\u003e\\n \\u003cli\\u003ePaz-Villarraga CA, Castro \\u0026Iacute;B, Fillmann G (2022) Biocides in antifouling paint formulations currently registered for use. Environ Sci Pollut Res 29: 30090\\u0026ndash;30101. https://doi.org/10.1007/s11356-021-17662-5\\u003c/li\\u003e\\n \\u003cli\\u003ePereira CDS, Abessa DMS, Choueri RB, Almagro-Pastor V, Cesar A, Maranho LA, Mart\\u0026iacute;n-D\\u0026iacute;az ML, Torres RJ, Gusso-Choueri PK, Almeida JE, Cortez FS. Mozeto AA, Silbiger HLN, Sousa ECPM, DelValls TA, Bainy, ACD (2014) Ecological relevance of sentinels\\u0026rsquo; biomarker responses: a multi-level approach. Mar Environ Res 96: 118\\u0026ndash;126. https://doi.org/10.1016/j.marenvres.2013.11.002\\u003c/li\\u003e\\n \\u003cli\\u003eRamos-G\\u0026oacute;mez J, Coz A, Viguri JR, Luque \\u0026Aacute;, Mart\\u0026iacute;n-D\\u0026iacute;az ML, DelValls T\\u0026Aacute; (2011) Biomarker responsiveness in different tissues of caged \\u003cem\\u003eRuditapes philippinarum\\u0026nbsp;\\u003c/em\\u003eand its use within an integrated sediment quality assessment. Environ Pollut 159: 1914\\u0026ndash;1922. https://doi.org/10.1016/j.envpol.2011.03.030\\u003c/li\\u003e\\n \\u003cli\\u003eRepetto G, Del Peso A, Zurita JL (2008) Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 3: 1125\\u0026ndash;1131. https://doi.org/10.1038/nprot.2008.75\\u003c/li\\u003e\\n \\u003cli\\u003eRingwood AH, Conners DE, Hoguet J (1998) Effects of natural and anthropogenic stressors on lysosomal destabilization in oysters \\u003cem\\u003eCrassostrea virginica\\u003c/em\\u003e. Mar Ecol Prog Ser 166: 163\\u0026ndash;171. https://doi.org/10.3354/meps166163\\u003c/li\\u003e\\n \\u003cli\\u003eRocha PRR, Faria AT, Borges L, Silva LOC, Silva AA, Ferreira EA (2013) Sor\\u0026ccedil;\\u0026atilde;o e dessor\\u0026ccedil;\\u0026atilde;o do diuron em quatro latossolos brasileiros. Planta Daninha 31: 231\\u0026ndash;238. https://doi.org/10.1590/S0100-83582013000100025\\u003c/li\\u003e\\n \\u003cli\\u003eRodrigues AML, Borges-Azevedo CM, Henry-Silva GG (2010) Aspectos da biologia e ecologia do molusco bivalve \\u003cem\\u003eAnomalocardia brasiliana\\u003c/em\\u003e (Gmelin, 1791) (Bivalvia, Veneridae). R bras Bioci Porto Alegre 8(4): 377-383. https://seer.ufrgs.br/index.php/rbrasbioci/article/view/114960\\u003c/li\\u003e\\n \\u003cli\\u003eSaid-Pullicino D, Erriquens FG, Gigliotti G (2007) Changes in the chemical characteristics of water-extractable organic matter during composting and their influence on compost stability and maturity. Bioresour Technol 98: 1822\\u0026ndash;1831. https://doi.org/10.1016/j.biortech.2006.06.018\\u003c/li\\u003e\\n \\u003cli\\u003eSakkas VA, Konstantinou IK, Albanis TA (2001) Photodegradation study of the antifouling booster biocide dichlofluanid in aqueous media by gas chromatographic techniques. J Chromatogr A 930)1-2): 135\\u0026ndash;144. https://doi.org/10.1016/S0021-9673(01)01193-1\\u003c/li\\u003e\\n \\u003cli\\u003eSedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman\\u0026rsquo;s reagent. Anal Biochem 25(1): 192\\u0026ndash;205. https://doi.org/10.1016/0003-2697(68)90092-4\\u003c/li\\u003e\\n \\u003cli\\u003eSies H, Koch OR, Martino E, Boveris A (1979) Increased biliary glutathione disulfide release in chronically ethanol‐treated rats. FEBS Lett 103(2): 287\\u0026ndash;290. https://doi.org/10.1016/0014-5793(79)81346-0\\u003c/li\\u003e\\n \\u003cli\\u003eSutcu R, Altuntas I, Buyukvanli B, Akturk O, Ozturk O, Koylu H, Delibas N (2007) The effects of diazinon on lipid peroxidation and antioxidant enzymes in rat erythrocytes: role of vitamins E and C. Toxicol Ind Health 23: 13\\u0026ndash;17.\\u003c/li\\u003e\\n \\u003cli\\u003eSuzuki T, Nojiri H, Isono H, Ochi T (2004) Oxidative damages in isolated rat hepatocytes treated with the organochlorine fungicides captan, dichlofluanid and chlorothalonil. Toxicol 204(2-3): 97\\u0026ndash;107. https://doi.org/10.1016/j.tox.2004.06.025\\u003c/li\\u003e\\n \\u003cli\\u003eThomas KV, Brooks S (2010) The environmental fate and effects of antifouling paint biocides. Biofouling 26(1): 73\\u0026ndash;88. https://doi.org/10.1080/08927010903216564\\u003c/li\\u003e\\n \\u003cli\\u003eUSEPA (2002) Technical Basis for the Derivation of Equilibrium Partitioning Sediment Guidelines (ESGs) for the Protection of Benthic Organisms: Nonionic Organics: Draft. USEPA. Washington DC 20460.\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eUSEPA (1993) Guidance Manual Bedded Sediment Bioaccumulation Tests. USEPA. Washington DC 20460. EPA/600/R-93/183.\\u003c/li\\u003e\\n \\u003cli\\u003eVerdouw H, Van Echteld CJA, Dekkers EMJ (1978) Ammonia determination based on indophenol formation with sodium salicylate. Water Res 12(6): 399\\u0026ndash;402. https://doi.org/10.1016/0043-1354(78)90107-0\\u003c/li\\u003e\\n \\u003cli\\u003eVouvoulis N, Scrimshaw MD, Lester JN (2000) Occurrence of four biocides utilized in antifouling paints, as alternatives to organotin compounds, in waters and sediments of a commercial estuary in the UK. Mar Pollut Bull 40(11): 938\\u0026ndash;946. https://doi.org/10.1016/S0025-326X(00)00034-5\\u003c/li\\u003e\\n \\u003cli\\u003eWalker CH, Sibly RM, Peakall DB (2005) Principles of ecotoxicology. Taylor \\u0026amp; Francis, CRC press. Boca Raton, FL, USA.\\u003c/li\\u003e\\n \\u003cli\\u003eWentworth CK (1922) A scale of grade and class terms for clastic sediments. J Geol 30: 377\\u0026ndash;392. https://doi.org/10.1086/622910\\u003c/li\\u003e\\n \\u003cli\\u003eWills ED (1987) Evaluation of lipid peroxidation in lipids and biological membranes. Biochemical toxicology: a practical approach. In: Snell K, Mullock B (eds) Biochemical Toxicology: a Practical Approach. Oxford: IRL Press, pp. 138\\u0026ndash;140.\\u003c/li\\u003e\\n \\u003cli\\u003eYamano T, Morita S (1995) Effects of pesticides on isolated rat hepatocytes, mitochondria, and microsomes II. Arch Environ Contam Toxicol 28: 1\\u0026ndash;7. https://doi.org/10.1007/bf00213961\\u003c/li\\u003e\\n \\u003cli\\u003eZhang XJ, Yang L, Zhao Q, Caen JP, He HY, Jin QH, Guo LH, Alemany M, Zhang LY, Shi YF (2002) Induction of acetylcholinesterase expression during apoptosis in various cell types. Cell Death Differ 9: 790\\u0026ndash;800. https://doi.org/10.1038/sj.cdd.4401034\\u003c/li\\u003e\\n \\u003cli\\u003eZhang Y, Song J, Yuan H, Xu Y, He Z, Duan L (2010) Biomarker responses in the bivalve (\\u003cem\\u003eChlamys farreri\\u003c/em\\u003e) to exposure of the environmentally relevant concentrations of lead, mercury, copper. Environ Toxicol Pharmacol 30: 19\\u0026ndash;25. https://doi.org/10.1016/j.etap.2010.03.008\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Dichlofluanid, antifouling, biomarkers, ecotoxicology, organic matter\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8593449/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8593449/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eAntifouling paints have been used to combat biofouling on submerged surfaces. They contain biocidal compounds that may be released into the environment and harm aquatic ecosystems; however, their effects on tropical organisms are little known. Dichlofluanid, or N-dichlorofluoromethythio-N\\u0026prime;,N\\u0026prime;-dimethyl-N-phenylsulfamide (C\\u003csub\\u003e9\\u003c/sub\\u003eH\\u003csub\\u003e11\\u003c/sub\\u003eCl\\u003csub\\u003e2\\u003c/sub\\u003eFN\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003eS\\u003csub\\u003e2\\u003c/sub\\u003e), is a biocide present in antifouling paints which toxicity to tropical marine organisms is poorly understood. This study aimed to evaluate the effects of dichlofluanid at a cellular and biochemical level on bivalves exposed to seawater and sediment contaminated with this biocide, also considering sediments with two different concentrations of organic matter. Aqueous-phase tests were carried out with the mussel \\u003cem\\u003ePerna perna\\u003c/em\\u003e, while tests with sediments used the clam \\u003cem\\u003eAnomalocardia flexuosa.\\u003c/em\\u003e Then, cellular and biochemical biomarkers were analyzed. The neutral red retention time assay (NRRT), a cellular biomarker was assessed only in \\u003cem\\u003eP. perna\\u003c/em\\u003e, while biochemical biomarkers (DNA damage, LPO, GSH, and activities of GST, GPx, EROD, and AChE) were analyzed in both organisms. Particle size, calcium carbonate content, and organic matter analyses were also conducted for the sediments. At higher concentrations, the NRRT assay showed effects in mussel hemocytes, denoting loss of lysosomal stability. Mussels also showed changes in biochemical biomarkers in the digestive glands and gills. In the clams exposed to sediment, adverse effects occurred in both organs and were more evident in animals exposed to sediments with higher levels of organic matter. Our study showed that dichlofluanid can affect marine bivalves at environmental concentrations and that organic matter may contribute to dichlofluanid exposure in clams.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e\",\"manuscriptTitle\":\"Effects of dichlofluanid in tropical marine bivalves exposed to water and spiked sediments: an assessment of biomarker responses\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-01-19 09:41:02\",\"doi\":\"10.21203/rs.3.rs-8593449/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"198a4dae-6c3b-4ae1-8872-70010d347e7b\",\"owner\":[],\"postedDate\":\"January 19th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-01-19T09:41:02+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-01-19 09:41:02\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8593449\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8593449\",\"identity\":\"rs-8593449\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}