Comparative Sublethal Toxicity of Sodium Dodecyl Sulfate and Mercuric Chloride in the Freshwater Planarian Girardia tigrina

Comparative Sublethal Toxicity of Sodium Dodecyl Sulfate and Mercuric Chloride in the Freshwater Planarian Girardia tigrina | 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 Comparative Sublethal Toxicity of Sodium Dodecyl Sulfate and Mercuric Chloride in the Freshwater Planarian Girardia tigrina Victor Eduardo Souza-Aguiar, Welligton Luciano Braguini This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7457039/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 Synthetic chemicals with high environmental persistence, including anionic surfactants and heavy metals, pose significant risks to freshwater ecosystems. This study evaluated the sublethal toxicity of sodium dodecyl sulfate (SDS) and mercuric chloride (HgCl₂) on the freshwater planarian Girardia tigrina , a widely used ecotoxicological model. Median lethal concentrations (LC₅₀) were determined over 96 hours, revealing a time- and dose-dependent toxicity, with SDS (LC 50 = 45.27 µg/L) exhibiting higher acute toxicity than HgCl₂ (LC 50 = 229.16 µg/L). Sublethal concentrations (5, 10, 25 µg/L) were used to assess locomotor behavior, reproduction, regeneration, and acetylcholinesterase (AChE) activity. Both SDS and HgCl₂ impaired locomotion in a concentration- and time-dependent manner, with partial recovery after prolonged exposure, indicating potential neurophysiological compensation. Reproductive performance was severely affected, with reduced fecundity and fertility, prolonged cocoon hatching times, and complete inhibition of viable cocoons at higher concentrations. Regeneration assays revealed that SDS delayed head and eye regeneration primarily after day 3, whereas HgCl₂ induced rapid and sustained inhibition from day 2 onward. AChE activity exhibited compound-specific alterations: HgCl₂ induced a biphasic response (initial stimulation followed by suppression), whereas SDS caused sustained activation. These multifaceted effects indicate disruption of neurochemical balance, reproductive physiology, and stem cell-mediated regeneration. Collectively, the findings highlight the ecological risks of SDS and HgCl₂, emphasizing the sensitivity of planarians as sentinel species for detecting sublethal toxicological effects in freshwater environments. This study underscores the importance of evaluating behavioral, reproductive, regenerative, and biochemical endpoints to support comprehensive environmental risk assessments and inform regulatory policies on emerging pollutants. Sodium dodecyl sulfate Mercuric chloride Sublethal toxicity Freshwater planarian Girardia tigrina Behavioral toxicity Regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Synthetic contaminants with long environmental half-lives, particularly anionic surfactants and heavy metals, have raised increasing concern due to their ubiquity in aquatic ecosystems and their ability to produce both acute and chronic biological effects. Among them, sodium dodecyl sulfate (SDS) is one of the most widely used surfactants, present in detergents, cosmetics, pharmaceuticals, and industrial formulations. In aquatic organisms, SDS can compromise membrane integrity, interfere with enzymatic pathways, and promote oxidative imbalance (Cruz-de-Carvalho et al., 2022 ; Gibson et al., 2016 ). Likewise, mercuric chloride (HgCl₂), a soluble inorganic mercury compound, is highly toxic and readily enters food webs, where it may accumulate and exert effects ranging from reproductive failure to neurotoxicity and oxidative damage (Saturday, 2018 ; Urbano et al., 2022 ; Vaidya and Mehendale, 2014 ). While SDS is predominantly introduced into surface waters through wastewater effluents, HgCl₂ inputs are more often associated with industrial and mining activities (Banaee, 2024 ; Ge et al., 2022 ). Despite their different chemical properties, both substances are capable of producing severe impacts on aquatic invertebrates. However, their comparative and combined effects on sensitive model species such as planarians remain poorly investigated. The large-scale production of synthetic surfactants, currently exceeding 15 million tons annually, reflects their economic importance but also their potential ecological burden, as most are derived from fossil-based feedstocks (Fung et al., 2023 ). Their amphiphilic structure, consisting of hydrophilic and hydrophobic moieties, allows them to associate with biological membranes, disturbing cell homeostasis. At relatively low concentrations, SDS (C₁₂H₂₅OSO₃⁻ Na⁺; sodium lauryl sulfate) can increase membrane permeability by interacting with proteins and lipids. With rising concentrations, progressive extraction of phospholipids destabilizes membranes, ultimately leading to structural collapse, impaired function, and cell death (Qiao et al., 2020 ; Stewart et al., 2017 ; Thomas and White, 1989 ). These properties, together with its strong foaming capacity, complicate removal during wastewater treatment and account for its frequent detection as a pollutant with ecotoxicological relevance (Najim et al., 2022 ; Ying, 2006 ). Global demand for synthetic surfactants continues to rise, with market projections indicating growth from nearly USD 45 billion in 2021 to more than USD 80 billion by 2030 (Zargar and Srivastava, 2024 ). Because most of these compounds are still derived from petroleum, their production contributes to fossil fuel depletion, environmental contamination, and greenhouse gas emissions. Growing awareness of these impacts has led to tighter regulatory scrutiny. In the European Union, recently revised legislation now requires surfactants to achieve at least 60% biodegradability within 28 days, following OECD and ISO test standards, and has introduced stricter labeling obligations for substances posing risks to human or environmental health (Council of European Parliament, 2024; Mohr et al., 2024 ). Although the United States has not enacted SDS-specific restrictions under the Toxic Substances Control Act (TSCA), the Environmental Protection Agency monitors surfactants through its new chemicals program, requiring hazard evaluations for novel applications (Borotkanics and Locke, 2017 ; Cowan-Ellsberry et al., 2014 ). These evolving policies highlight increasing recognition of the ecological risks posed by common surfactants and underscore the importance of extending ecotoxicological studies to less-characterized groups such as aquatic invertebrates. Feng et al. ( 2023 ) conducted a detailed assessment of sodium dodecyl sulfate (SDS) toxicity in the freshwater planarian Dugesia japonica . The authors reported a progressive decline in LC₅₀ values over 96 hours, indicating a time-dependent increase in lethality. At sublethal concentrations, SDS exposure triggered oxidative stress responses, with alterations in antioxidant enzyme activity and lipid peroxidation, and induced genotoxic effects as revealed by reduced genomic template stability. Molecular analyses further demonstrated dysregulation of genes involved in apoptosis, cell-cycle progression, DNA repair, and neoblast proliferation, suggesting that SDS interferes with key cellular pathways necessary for regeneration and genomic maintenance (Feng et al., 2023 ). Together, these findings emphasize that SDS exerts a broad spectrum of toxic effects on planarians, ranging from oxidative imbalance to disruption of fundamental processes of stem cell biology, underscoring its ecological risk to freshwater invertebrates. A recent investigation by Santos et al. ( 2024 ) reported that sodium dodecylbenzene sulfonate (SDBS), a widely used anionic surfactant, can cause pronounced histological and physiological alterations in zebrafish ( Danio rerio ). After 96 hours of exposure to environmentally relevant concentrations (0.25–0.5 mg/L), the authors described structural impairment of the gill lamellae, circulatory disturbances, and changes in mucus and chloride cell abundance. At the higher concentration, superoxide dismutase (SOD) activity was reduced, while catalase (CAT) activity remained unaffected. Hematological changes, including increased neutrophil and lymphocyte counts, further indicated that short-term exposure to SDBS is sufficient to trigger oxidative stress and immune-related responses, underscoring its ecological risk (Santos et al., 2024 ). Concerns about mercury pollution remain central to discussions on aquatic toxicants because of its persistence, capacity to accumulate in food webs, and broad toxicological effects (Sunderland and Selin, 2013 ; Zhang et al., 2016 ). While mercury is naturally present in the environment, human activities, including industrial processes, mining operations, and agricultural practices, have markedly intensified its release into aquatic ecosystems. Within its various chemical forms, mercuric chloride (HgCl₂) deserves particular attention since it dissolves readily in water and persists over time, increasing the likelihood of exposure to aquatic organisms (Fernandes Azevedo et al., 2012 ). Inorganic mercury salts, including HgCl₂, are highly reactive due to their strong interaction with thiol (-SH) groups present in proteins and enzymes. This reactivity disrupts cellular metabolism and weakens antioxidant defenses, creating conditions that favor oxidative damage (Carvalho et al., 2008 ; Farina et al., 2011 ). In aquatic invertebrates, exposure to HgCl₂ has been linked to neurochemical and physiological disturbances, such as inhibition of acetylcholinesterase (AChE), disruption of ion homeostasis, and the generation of reactive oxygen species that promote lipid peroxidation, protein oxidation, and DNA lesions (Mela et al., 2007 ). Even when present at sublethal levels, mercury can compromise behavioral and reproductive traits, while long-term exposure reduces growth and survival (Singh Rathore and Khangarot, 2003 ). In planarians specifically, mercury has been shown to impair neoblast activity, slow down regenerative processes, and alter sensory-driven behaviors like phototaxis and chemotaxis, indicating simultaneous impacts on stem cell dynamics and neural circuits (Agata et al., 2007 ; Saló and Baguñà, 2002 ). Acute toxicity tests in freshwater organisms further demonstrate the high potency of HgCl₂, with 96-h LC₅₀ values ranging from a few µg/L to the low mg/L scale, underlining both its environmental risk and capacity to bioaccumulate in aquatic systems (Boudou and Ribeyre, 1989 ; Camargo and Martinez, 2007 ). Mercury occurs in several chemical forms, including elemental (Hg⁰), inorganic species (Hg 2+ , HgS), and organic derivatives such as methylmercury and ethylmercury. Among these, methylmercury is of particular concern because it is efficiently absorbed and progressively concentrated through aquatic food webs, reaching high levels in top predators (Buckman et al., 2017 ; Chen et al., 2024 ; De Almeida Rodrigues et al., 2019 ). After entering aquatic systems, mercury can be incorporated into organisms both directly, by diffusion, and indirectly, through trophic transfer. These processes contribute to toxic outcomes such as oxidative imbalance, inhibition of key enzymes, and impairment of neural function (De Almeida Rodrigues et al., 2019 ). Mercury is recognized as a highly toxic contaminant with no known biological function. Its presence in aquatic systems originates both from natural processes and from human activities such as mining, industrial emissions, and agriculture. Once released, mercury persists due to its chemical stability and capacity to form soluble species that are readily assimilated by organisms. In aquatic environments, microbial activity can transform inorganic mercury into methylmercury (CH 3 Hg + ), a more toxic and bioavailable form. Methylmercury accumulates progressively through trophic levels, leading to higher concentrations in predatory fish. Consequently, fish may carry elevated mercury burdens even in regions with low inorganic mercury in the water, provided that environmental conditions favor microbial methylation. The present study aimed to assess the toxicological effects of SDS and HgCl 2 on the freshwater planarian Girardia tigrina , an established model in ecotoxicology due to its high sensitivity to pollutants, robust regenerative capacity, and ecological relevance in freshwater ecosystems (Bach et al., 2016 ; Calevro et al., 1998 ; Hagstrom et al., 2016 ; Pestana and Ofoegbu, 2021 ; Rawls et al., 2011 ). Specifically, this study evaluated the median lethal concentration (LC 50 ), alterations in locomotor behavior, reproductive performance, tissue glycogen content, and AChE activity in response to SDS and HgCl₂ exposure. By investigating these endpoints, the research aims to elucidate the sublethal and chronic effects of both toxicants on freshwater invertebrate, thereby contributing to a broader understanding of their ecological impact and supporting more informed environmental risk assessments and conservation strategies. Materials and Methods Chemicals Sodium dodecyl sulfate (SDS, ≥ 99%), mercuric chloride (HgCl₂, ≥ 99.5%), acetylthiocholine iodide (ACTh, ≥ 98%), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, ≥ 98%), bovine serum albumin (BSA, ≥ 98%, Fraction V), ethylenediaminetetraacetic acid disodium salt (EDTA, ≥ 99%), and Triton X-100 (laboratory grade) were purchased from Sigma Chemical (St. Louis, MO, USA) and Merck (Darmstadt, Germany). All other reagents were of analytical grade. Planaria Husbandry The freshwater planarian Girardia tigrina was employed as the model organism throughout this investigation. A stable breeding colony has been maintained since 2017 under controlled laboratory conditions (20 ± 1°C) in the BioTox (Laboratory of Experimental Biochemistry and Toxicology). The culture was sustained in ASTM-recommended water. The nutritional regimen consisted of weekly ad libitum feeding sessions with bovine liver, limited to a 2-hour duration to prevent water quality degradation. Immediately following each feeding, the entire culture medium was replenished. This maintenance protocol aligns with previously described standards for the species (Dean and Duncan, 2020 ; Schaly and Braguini, 2024 ; Torres-Da Matta et al., 1989 ). Toxicity and Behavioral Assessments Chemical Exposure and Experimental Design SDS (10–300 µg/L) and HgCl₂ (10–100 µg/L) were dissolved in purified water. Concentration ranges were selected based on (1) literature-reported sublethal/lethal effects on freshwater invertebrates and (2) preliminary tests with Girardia tigrina to establish no-observed-mortality and complete-mortality thresholds. Eight G. tigrina individuals were exposed to 100 mL test solution in Petri dishes (125 × 20 mm) at 20 ± 1°C in darkness (96 h, unfed). Mortality was recorded at 24-h intervals. LC₅₀ values were derived via Probit analysis. No combined SDS + HgCl₂ tests were conducted (synergistic/antagonistic effects unassessed). Identical sublethal concentrations (5, 10, 25 µg/L) were used for chronic assays to: ( i ) Ensure exposures remained below LC₅₀ thresholds, and ( ii ) Standardize response comparisons between toxicants. Evaluation of planarian locomotor velocity Planarian locomotor velocity (pLmV) was assessed following the method of Raffa and Valdez ( 2001 ). Individual planarians were placed in 60 × 10 mm Petri dishes containing APW and positioned over graph paper with 0.5 cm² grids. Locomotion was quantified as the total number of grid lines crossed during a 5-minute observation period (Raffa and Valdez, 2001 ), recorded using a high-speed NEOCoolcam® webcam with a varifocal zoom lens. Planarians were pre-incubated with SDS or HgCl₂ for either 24 h (acute test) or 7 days (chronic test) at concentrations of 5, 10, and 25 µg/L. Each concentration group consisted of a separate set of planarians (n = 9), and no individual was used more than once in the experiment. Reproductive Toxicity Assay Reproductive performance was assessed in sexually mature G. tigrina (average body length 1.2 ± 0.1 cm). Groups of nine animals were randomly allocated to glass Petri dishes (125 × 20 mm) containing 100 mL of planarian water supplemented with SDS or HgCl₂ at nominal concentrations of 5, 10, or 25 µg/L. Exposures lasted four weeks, during which reproductive activity was monitored at weekly intervals. Two endpoints were quantified: fecundity, expressed as the number of cocoons deposited per worm per week, and fertility, defined as the proportion of cocoons that hatched successfully. Additionally, the time required for cocoon hatching was recorded. Reproductive indices followed the approach described by Knakievicz et al. ( 2006 ), with minor adjustments in exposure duration and sampling schedule (Knakievicz et al., 2006 ). Planarian Regeneration Assays Prior to experimentation, planarians were starved for seven days to standardize their metabolic state. This study evaluated two distinct regenerative processes: head regeneration and ocellus regeneration. For each treatment condition, including control, 5, 10, and 25 µg/L groups, a sample size of six to nine animals per concentration was utilized. Following amputation, the planarians were continuously exposed to one of three concentrations of either SDS or HgCl₂ for the entire duration of the regeneration period. In the head regeneration assay, animals were decapitated transversely, and only the posterior fragments were retained for observation. For the ocellus regeneration assay, the right ocellus was surgically excised using a 31-gauge insulin needle to ensure minimal adjacent tissue damage, a technique adapted from established methodology (Deochand et al., 2016 ). Regenerative progress was documented daily through imaging under a light microscope for 14 consecutive days. Imaging for head regeneration commenced immediately post-amputation (day 0) and continued through day 14. For ocellus regeneration, imaging began on day 2 and continued through day 14, with a pre-surgery baseline image captured for each animal to enable subsequent scale normalization. To facilitate consistent image acquisition, planarians were transferred to 60 × 10 mm Petri dishes featuring a uniform dark background; this minimized their light-aversive movement and optimized contrast. Before each imaging session, animals were rinsed and placed in fresh artificial planarian water (APW) to eliminate potential chemical interference from the exposure solutions. All acquired images were subjected to quality control and quantitatively analyzed using ImageJ software (version 1.51, NIH, USA). For both regenerative assays, a regeneration index was calculated to quantify outcomes. This index was defined as the area of the regenerated structure (either the cephalic blastema or the new ocellus) relative to the total body area of the animal. To ensure statistical robustness and account for individual size variation, each measurement was performed on three independent replicates per animal and the resulting data were normalized. This approach allowed for consistent and equitable comparisons across all experimental groups. Biochemical Analysis Acetylcholinesterase Assay (E.C. 3.1.1.7) Acetylcholinesterase (AChE) activity was quantified according to the spectrophotometric method of Ellman et al. ( 1961 ), with modifications. The assay is based on the enzymatic hydrolysis of the substrate acetylthiocholine (ACh) by AChE present in the sample. This reaction yields thiocholine, which subsequently reacts with the colorimetric agent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to produce the yellow anion 5-thio-2-nitrobenzoate. The rate of formation of this product, which is proportional to the enzyme activity, was monitored by measuring the absorbance at 412 nm (Ellman et al., 1961 ). Planarians exposed to SDS or HgCl₂ at concentrations of 5, 10, and 25 µg/L for 24 hours and 7 days were homogenized in an ice-cold 20 mM Tris-HCl buffer (pH adjusted) containing 1 mM EDTA. The homogenate was centrifuged at 12,000 × g for 20 minutes at 4°C using a refrigerated centrifuge (Loccus® model L3024R). The resulting supernatant was collected and used as the enzyme source for the assay. The reaction was initiated by adding the supernatant to a cuvette containing a working solution of DTNB (0.75 mM) and acetylthiocholine (9 mM) as the substrate, yielding a final reaction volume of 3 mL. Enzyme activity was calculated from the linear rate of absorbance change and is expressed as units of hydrolyzed acetylcholine per milligram of protein (U × mg protein⁻¹), where one unit corresponds to the amount of enzyme required to hydrolyze one micromole of substrate per minute under the specified assay conditions. Protein determination The protein content was quantified by the method of Bradford (Bradford, 1976 ), using bovine serum albumin as a standard. Statistical Analysis Data were analyzed using ANOVA, and Dunnett's test was used to compare treatments with the control group. The p values < 0.05 were considered statistically significant. The statistical analysis was performed using the Prism 8 statistical software (GraphPad, San Diego, US). Results The LC₅₀ values determined for SDS and HgCl₂ in Girardia tigrina revealed a progressive decrease over time, indicating a clear dose- and time-dependent toxicity pattern (Table I). For HgCl₂, the LC 50 declined from 252.58 µg/L at 24 hours to 229.16 µg/L at 96 hours, while SDS exhibited a slight decrease over the same period, with a final LC₅₀ of 45.27 µg/L at 96 hours. These findings suggest cumulative effects and reinforce the importance of considering exposure duration in toxicity assessments. Accordingly, sublethal concentrations below the LC 50 were selected for subsequent experiments to evaluate regeneration, reproduction, locomotion, and enzymatic activity without confounding mortality effects. These sublethal concentrations were then used to assess functional endpoints, including planarian locomotion, allowing evaluation of behavioral effects without confounding mortality. Planarian locomotion was significantly inhibited by both SDS (Fig. 1 A and B) and HgCl₂ (Fig. 2 A and B), as shown by pLmV measurements, respectively. After 24 hours of SDS exposure (Fig. 1 A), locomotor inhibition reached 26.8%, 32.1%, and 33.2% at 5, 10, and 25 µg/L, respectively. Statistical analysis (two-way ANOVA) confirmed a significant effect of concentration, locomotion time, and their interaction. Similar effects were observed after 7 days (Fig. 1 B), though inhibition levels were slightly reduced, suggesting possible adaptive mechanisms over time. HgCl₂ exposure also impaired locomotion, with reductions of 43.3%, 33.9%, and 33.5% at the same concentrations after 24 hours (Fig. 2 A), and corresponding decreases of 7.8%, 14.2%, and 16.5% after 7 days (Fig. 2 B). Significant effects of concentration, time, and their interaction were again confirmed. These results underscore a concentration- and time-dependent inhibition of planarian locomotion by both compounds, with evidence suggesting partial tolerance or compensatory responses following prolonged exposure. Reproductive parameters were also markedly affected. SDS exposure led to substantial fecundity inhibition (Fig. 3 A) of 74.1%, 79.8%, and 82.1%, and fertility reductions (Fig. 3 B) of 91.4%, 90.1%, and 100% at 5, 10, and 25 µg/L, respectively. Moreover, cocoon hatching time increased by 16.6% and 46.2% at 5 and 10 µg/L, respectively, with no viable cocoons observed at 25 µg/L (Fig. 3 C). Similarly, HgCl₂ exposure resulted in fecundity reductions (Fig. 4 A) of 89.1%, 79.1%, and 96.6%, and fertility declines (Fig. 4 B) of 94%, 88.4%, and 97.2% at the same concentrations. Hatching times were prolonged by 24.1% and 40.6% at 5 and 10 µg/L, respectively, while no viable cocoons were detected at 25 µg/L (Fig. 4 C). These findings confirm that both compounds severely compromise planarian reproductive capacity and development, posing ecological risks. Building on the observed reproductive impairments, head regeneration assays were conducted to further evaluate the sublethal effects of SDS and HgCl₂ on planarian physiology. Head regeneration in planarians was significantly affected by SDS exposure (Fig. 5 A) from day 3 to day 6 (p < 0.05). During this period, inhibition rates reached 50.8% and 53.8% at 10 and 25 µg/L, respectively, on day 3; 45.4% and 63.6% at 10 and 25 µg/L on day 4; 43.5% at 25 µg/L on day 5; and 31.4% and 48.6% at 10 and 25 µg/L on day 6. No significant effects were detected between days 7 and 10. From day 11 to day 14, SDS once again significantly inhibited regeneration (p < 0.05). Inhibition levels were 15.9%, 22.2%, and 31.8% at 5, 10, and 25 µg/L, respectively, on day 11; 17.6% and 33.8% at 5 and 25 µg/L on day 12; 20%, 29.3%, and 36% at 5, 10, and 25 µg/L on day 13; and 22.7%, 32.9%, and 40.9% at 5, 10, and 25 µg/L on day 14. These findings indicate that SDS impairs regenerative capacity in a concentration- and time-dependent manner, with effects more evident at higher concentrations. In contrast, HgCl₂ exposure (Fig. 5 B) impaired head regeneration throughout the entire experimental period (days 1–14, p < 0.05). On day 1, inhibition reached 85.7% at 10 and 25 µg/L, followed by 75% at the same concentrations on day 2. No significant effects were observed on day 3, but inhibition resumed on day 4 with 54.5% and 50% at 10 and 25 µg/L, respectively. Subsequent values included 21.8% and 52.2% at 10 and 25 µg/L on day 5; 40% and 60% on day 6; 36.8% and 55.3% on day 7; 22.5% and 40% on day 8; 36.2% at 25 µg/L on day 9; and 33.3% at 25 µg/L on day 10. From days 11 to 14, concentration-dependent effects were consistently observed, with inhibition rates of 20.6%, 27%, and 36.5% at 5, 10, and 25 µg/L on day 11; 20.6%, 32.4%, and 35.3% on day 12; 20%, 33.3%, and 30.7% on day 13; and 17%, 36.4%, and 37.5% on day 14, respectively. These results demonstrate that HgCl₂ exerts an earlier and more sustained inhibitory effect on planarian head regeneration compared to SDS. Planarian eye regeneration was significantly impaired by SDS exposure in a time- and concentration-dependent manner (Fig. 6 A). While regeneration progressed steadily in the control group, animals exposed to SDS displayed marked delays in ocellus formation, particularly at concentrations of 10 and 25 µg/L. On day 4, eye regeneration was reduced by 55.5%, 62.0%, and 71.8% in the 5, 10, and 25 µg/L SDS groups, respectively, compared to controls. This inhibitory effect persisted and intensified over time, with reductions reaching 60.5%, 61.3%, and 67.1% on day 14. Notably, even the lowest concentration tested (5 µg/L) led to a significant reduction of 60.5% in regeneration by the final day. A two-way repeated measures ANOVA confirmed the significant main effects of both treatment concentration (p = 0.0049) and regeneration time ( p = 0.0056), along with a significant interaction between these factors ( p = 0.0154), indicating that SDS modulates regeneration dynamics across time. Although the early stages (day 2) showed slightly increased values in some treated groups, possibly reflecting transient compensatory responses, these were not sustained. These findings reveal that SDS, a common surfactant and environmental contaminant, substantially disrupts regenerative capacity in planarians, underscoring its potential ecological risk even at environmentally relevant concentrations. Ocellus regeneration was markedly inhibited by HgCl₂ exposure in a concentration- and time-dependent manner (Fig. 6 B). Statistically significant reductions were detected as early as day 2 across all concentrations ( p < 0.05), with the most dramatic effects observed at 10 and 25 µg/L. By day 14, eye regeneration was inhibited by 80.6%, 92.7%, and 93.5% in the 5, 10, and 25 µg/L groups, respectively, when compared to controls. Even at the lowest concentration tested (5 µg/L), a progressive decline in regenerative ability was evident, culminating in a near-complete inhibition by the end of the experimental period. A two-way repeated measures ANOVA confirmed significant main effects of both concentration ( p = 0.0048) and time ( p = 0.0045), as well as a strong interaction between them ( p = 0.0081), indicating that the impact of mercury on regeneration intensifies over time. These findings highlight the potent inhibitory effects of mercury on neuro-regeneration and suggest that planarians represent a sensitive invertebrate model for detecting sublethal developmental toxicity in aquatic systems. Regarding AChE activity, both toxicants elicited significant and temporally dynamic effects (Fig. 7 A and B). SDS exposure resulted in a consistent increase in AChE activity across time, with significant stimulation at all concentrations after 24 hours (32.6%, 63.0%, and 80.3%) and a partial attenuation after 7 days (Fig. 7 A). Notably, no significant effect was observed at 5 µM after 7 days, while significant increases persisted at 10 and 25 µM. This sustained AChE activation may reflect an adaptive neurochemical response to SDS. HgCl₂ induced an increase in AChE activity after 24 hours at all tested concentrations (33.3%, 79.5%, and 40.8% increases), followed by a notable decrease after 7 days (reductions of 40.6%, 33.7%, and 35.1%) (Fig. 7 B). This biphasic response suggests an initial stimulatory phase potentially due to enzymatic upregulation, followed by compensatory downregulation or enzymatic inhibition. In summary, SDS and HgCl₂ exerted significant, concentration- and time-dependent effects on planarian survival, locomotion, reproduction, regeneration, and enzymatic activity. The observed outcomes suggest complex toxicodynamic interactions, with both compounds posing potential ecological threats due to their persistent and multifactorial impacts on freshwater invertebrates. These findings underscore the importance of evaluating sublethal endpoints and temporal patterns to fully understand pollutant risks in aquatic ecosystems. Discussion Based on the results presented, a comprehensive evaluation can be made regarding the sublethal toxic effects of the surfactant SDS and the inorganic salt HgCl₂ in the freshwater planarian G. tigrina . These organisms have been extensively employed as invertebrate models in environmental toxicology due to their ecological relevance, high sensitivity to pollutants, and remarkable regenerative capacity (Best and Morita, 1982 ; Kang et al., 2023 ). Our results demonstrate that the toxicity of both SDS and HgCl₂ is cumulative, as evidenced by the progressive decline in their LC₅₀ values over the 96-hour exposure period. This time- and dose-dependent pattern aligns with findings reported for other toxicants in aquatic invertebrates (Li, 2008 ; Simão et al., 2021 ). Notably, SDS exhibited a significantly greater acute toxicity than HgCl₂ (96-h LC 50 of 45.27 µg/L versus 229.16 µg/L, respectively), suggesting a more rapid mechanism of action or enhanced tissue penetration efficiency for the surfactant. Significant alterations in planarian locomotion underscore the neurotoxic potential of both SDS and HgCl₂. Impaired movement observed after 24 hours, with partial recovery after 7 days, may reflect neurophysiological compensation or adaptive mechanisms. The more pronounced initial effect of HgCl₂ relative to SDS may be attributed to mercury’s affinity for sulfhydryl (-SH) groups, which play essential roles in neuronal protein function (Aschner et al., 2010 ; Katðno et al., 1977 ). These findings highlight the utility of locomotor behavior as an early and sensitive biomarker of neurotoxicity. The reproductive capacity of G. tigrina was severely compromised by both contaminants, as indicated by marked reductions in fecundity and fertility, coupled with extended cocoon hatching durations. This collective impairment points to a profound disruption of core reproductive mechanisms, which could entail the dysregulation of endocrine pathways, modifications in gene expression profiles, or diminished gamete health (Heath et al., 2012 ; Mojica-Vázquez et al., 2019 ). The failure to produce any viable cocoons at the highest concentrations (25 µg/L) highlights a severe threat to population sustainability in contaminated environments. Distinct regenerative impairment profiles emerged for each toxicant. SDS exposure resulted in a delayed inhibitory effect on head regeneration, becoming significant only after day 3, which implies a slower disruption of processes like cell proliferation or differentiation. Mercury, conversely, exhibited a more immediate and aggressive toxicity, inhibiting regeneration from day 2 onward and achieving nearly complete suppression by the end of the observation period. This potent effect is likely linked to HgCl₂'s ability to induce oxidative stress and epigenetic alterations (Kalafatić et al., 2004 ), potentially damaging the neoblast stem cell population vital for regeneration (Sengupta et al., 2015 ). The heightened sensitivity of eye regeneration to mercury further underscores its specific neurotoxic potency. The contrasting AChE activity profiles underscore the distinct neurotoxic mechanisms of each compound. The biphasic response (initial increase followed by inhibition) induced by HgCl₂ implies a complex neurochemical imbalance, potentially stemming from inflammatory processes or altered gene regulation (De Oliveira et al., 2012 ; Xia et al., 2022 ). In contrast, the sustained enzymatic activation observed with SDS exposure suggests a state of persistent cholinergic stress or an adaptive compensatory response (Biswas et al., 2025 ; Meshorer and Soreq, 2002 ; Toiber and Soreq, 2005 ). These substrate-specific biochemical disruptions provide a plausible mechanism underlying the locomotor deficits recorded in the behavioral assays. A key limitation of this study is its focus on the individual toxic effects of SDS and HgCl₂. The potential for interactive effects, whether synergistic, additive, or antagonistic, remains unexplored, as binary or more complex mixture assays were not performed. Given the ubiquitous co-occurrence of surfactants and heavy metals in polluted aquatic systems, particularly downstream from urban and industrial effluents, research into combined exposures constitutes a critical next step. Such studies are essential for developing a more environmentally realistic risk assessment and for deciphering the complex toxicological mechanisms that emerge from pollutant interactions. Notwithstanding this limitation, our findings clearly show that both SDS and HgCl₂ can disrupt a suite of critical physiological functions in G. tigrina , from behavior and reproduction to cellular regeneration and neurochemistry. The persistence of these compounds in aquatic environments underscores their potential to threaten biodiversity at sublethal concentrations. This work further solidifies the status of planarians as sensitive bioindicators for environmental toxicology. Specifically, their unparalleled regenerative capability provides a powerful, integrative model for probing the initial cellular and systemic injuries inflicted by environmental contaminants before these effects manifest at higher levels of biological organization. While this study provides a multifaceted toxicity assessment, several constraints should be noted. Our laboratory-based approach, though controlled, simplifies the dynamic variables present in natural freshwater habitats. The exclusion of combinatorial exposures with other pollutants precludes insight into potential interactive effects. Furthermore, while our selected endpoints (locomotion, reproduction, regeneration, AChE) are ecologically relevant, the incorporation of molecular analyses, such as oxidative stress or gene expression biomarkers, would offer deeper mechanistic understanding. The focus on a single species ( G. tigrina ) under acute exposure scenarios also cautions against extrapolating these results to diverse aquatic taxa or to the consequences of long-term, chronic pollution. Investigations encompassing these dimensions are needed to refine ecological risk predictions. Conclusion This study demonstrated that both the anionic surfactant SDS and the inorganic salt HgCl₂ exhibit pronounced, multifaceted, and time-dependent toxicity in Girardia tigrina . Both contaminants affected multiple biological levels, including locomotor behavior, reproductive performance, tissue regeneration, and enzymatic activity, highlighting systemic and cellular impacts. Locomotor inhibition indicated potential neurotoxic effects, with partial recovery after prolonged exposure, suggesting adaptive or compensatory mechanisms. Reproductive endpoints were severely impaired, with significant reductions in fecundity and fertility, delayed cocoon hatching, and absence of viable offspring at the highest concentrations, underscoring potential risks to population sustainability in contaminated environments. Regeneration assays revealed distinct toxic profiles for each compound: SDS induced delayed regenerative responses, whereas HgCl₂ caused early and sustained inhibition, implicating disruption of neoblast stem cell activity and neurogenic processes essential for regeneration. AChE activity exhibited complex, compound-specific responses, reflecting neurochemical dysregulation that may partially explain the observed behavioral and regenerative impairments. Collectively, these findings underscore the capacity of persistent pollutants to interfere with multiple physiological and biochemical functions in freshwater invertebrates, even at sublethal concentrations. The results reinforce the value of planarians as sensitive and informative models for environmental toxicity assessment, enabling the detection of subtle effects that precede mortality. Future studies should extend these approaches to other freshwater invertebrate species, in order to refine and broaden the ecological relevance of the findings. These findings have direct implications for ecotoxicology and environmental management, as they suggest that the release of petroleum-derived synthetic surfactants and heavy metals should be closely monitored and regulated. Moreover, the observed multifactorial effects highlight the need for future studies involving combined exposures, reflecting environmentally realistic scenarios to elucidate potential synergistic or antagonistic interactions among pollutants and to develop more effective mitigation strategies for the conservation of aquatic ecosystems. Declarations Acknowledgements Acknowledgments to Universidade Estadual do Centro-Oeste (UNICENTRO) for providing laboratory space and equipment at the Experimental Biochemistry and Toxicology Laboratory (BioTox), which supported the research conducted for the development of this paper. Ethical Approval Not applicable. Consent to Participate Not applicable. Consent to Publish Not applicable. Competing Interests The authors declare no conflicts of interest. Funding This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ Contributions All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by V.E.S.A and W.L.B.. The first draft of the manuscript was written by V.E.S.A and W.L.B. and all authors commented on previous versions of the manuscript. All authors read and approved final manuscript. 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SDS Time (h) Equation LC 50 (µg/L) 95% confidence intervals 24 y = 306.8x – 514.7 49.42 37.84 53.83 48 y = 322.1x – 541.6 49.77 38.11 54.45 72 y = 298x – 497.8 48.67 35.4 0 53.70 96 y = 287.8x – 472.7 45,27 25.94 53.09 HgCl 2 24 y = 409.8x – 979.5 252.58 217.27 264.85 48 y = 361.7x – 858.4 243.82 214.78 252.35 72 y = 338x – 796.1 234.49 194.54 248.31 96 y = 340.7x – 799.1 229.16 170.22 250.61 Data represent one of 3 independent replicates; each performed in triplicate. 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. 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Data represent the mean ± SD of 3 independent experiments, each performed in triplicate.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/3a62d98bb62a4853c8298320.jpg"},{"id":92544375,"identity":"ceaad78f-e835-4b5a-b267-e62d2429977a","added_by":"auto","created_at":"2025-09-30 19:56:23","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":783471,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of HgCl₂ on planarian locomotion after 24 hours (A) and 7 days (B). Data represent the mean ± SD of 3 independent experiments, each performed in triplicate.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/236768ba88d4951898435029.jpg"},{"id":92544362,"identity":"3f3be0ae-724d-421d-9940-5cc22b1bc653","added_by":"auto","created_at":"2025-09-30 19:56:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":850826,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of SDS on planarian reproduction. (A) Fecundity; (B) Fertility, and (C) Days until cocoons hatching. Data are the mean ± standard deviation of 3 experiments.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/7ed8bf98a8357be18ebef727.jpg"},{"id":92543718,"identity":"908e9f7b-2759-4424-8169-753a33d493f9","added_by":"auto","created_at":"2025-09-30 19:48:19","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":819227,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of HgCl\u003csub\u003e2\u003c/sub\u003e on planarian reproduction. (A) Fecundity; (B) Fertility, and (C) Days until cocoons hatching. Data are the mean ± standard deviation of 3 experiments.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/93b2473d3ccdbbf7bcdbc281.jpg"},{"id":92543719,"identity":"38c39d52-4abb-4e8e-bc67-0ff5228bf052","added_by":"auto","created_at":"2025-09-30 19:48:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1174154,"visible":true,"origin":"","legend":"\u003cp\u003eRegeneration index of planarian head during exposition to SDS (A) and HgCl\u003csub\u003e2\u003c/sub\u003e (B) concentrations. Day 0 represents the cut head day. Data are means ± S.D. of 3 experiments in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/5de5216d4d33f72664eaf8d5.jpg"},{"id":92543722,"identity":"154fef74-39a7-44a1-9df3-9bae830228c2","added_by":"auto","created_at":"2025-09-30 19:48:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":904669,"visible":true,"origin":"","legend":"\u003cp\u003eRegeneration index of planarian ocellus after exposition to SDS (A) and HgCl\u003csub\u003e2\u003c/sub\u003e (B) concentrations. Day 2 represents the second day after right ocellus was surgically removed. Data are means ± S.D. of 3 experiments in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/cc4a4d1e2511f779c6ed3a8a.jpg"},{"id":92543729,"identity":"64c63778-84aa-4fee-b2d5-4d8d0168e44e","added_by":"auto","created_at":"2025-09-30 19:48:20","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":727397,"visible":true,"origin":"","legend":"\u003cp\u003eAChE activity after exposition to SDS (A) and HgCl2 (B) per 24 h and 7 days. Data are mean ± S.D. of 3 experiments in triplicate. *** \u003cem\u003ep = 0,002; \u003c/em\u003e****\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/caf0a37fd627d79831b89621.jpg"},{"id":101297042,"identity":"8949e11c-49b3-4ceb-89b8-23d9f512d9e6","added_by":"auto","created_at":"2026-01-28 09:24:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6846810,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7457039/v1/008a31bd-e7bc-4de9-9605-4e03d7dbbf83.pdf"}],"financialInterests":"","formattedTitle":"Comparative Sublethal Toxicity of Sodium Dodecyl Sulfate and Mercuric Chloride in the Freshwater Planarian Girardia tigrina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSynthetic contaminants with long environmental half-lives, particularly anionic surfactants and heavy metals, have raised increasing concern due to their ubiquity in aquatic ecosystems and their ability to produce both acute and chronic biological effects. Among them, sodium dodecyl sulfate (SDS) is one of the most widely used surfactants, present in detergents, cosmetics, pharmaceuticals, and industrial formulations. In aquatic organisms, SDS can compromise membrane integrity, interfere with enzymatic pathways, and promote oxidative imbalance (Cruz-de-Carvalho et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Gibson et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Likewise, mercuric chloride (HgCl₂), a soluble inorganic mercury compound, is highly toxic and readily enters food webs, where it may accumulate and exert effects ranging from reproductive failure to neurotoxicity and oxidative damage (Saturday, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Urbano et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vaidya and Mehendale, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). While SDS is predominantly introduced into surface waters through wastewater effluents, HgCl₂ inputs are more often associated with industrial and mining activities (Banaee, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ge et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite their different chemical properties, both substances are capable of producing severe impacts on aquatic invertebrates. However, their comparative and combined effects on sensitive model species such as planarians remain poorly investigated.\u003c/p\u003e\u003cp\u003eThe large-scale production of synthetic surfactants, currently exceeding 15\u0026nbsp;million tons annually, reflects their economic importance but also their potential ecological burden, as most are derived from fossil-based feedstocks (Fung et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Their amphiphilic structure, consisting of hydrophilic and hydrophobic moieties, allows them to associate with biological membranes, disturbing cell homeostasis. At relatively low concentrations, SDS (C₁₂H₂₅OSO₃⁻ Na⁺; sodium lauryl sulfate) can increase membrane permeability by interacting with proteins and lipids. With rising concentrations, progressive extraction of phospholipids destabilizes membranes, ultimately leading to structural collapse, impaired function, and cell death (Qiao et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Stewart et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Thomas and White, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). These properties, together with its strong foaming capacity, complicate removal during wastewater treatment and account for its frequent detection as a pollutant with ecotoxicological relevance (Najim et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ying, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGlobal demand for synthetic surfactants continues to rise, with market projections indicating growth from nearly USD 45\u0026nbsp;billion in 2021 to more than USD 80\u0026nbsp;billion by 2030 (Zargar and Srivastava, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Because most of these compounds are still derived from petroleum, their production contributes to fossil fuel depletion, environmental contamination, and greenhouse gas emissions. Growing awareness of these impacts has led to tighter regulatory scrutiny. In the European Union, recently revised legislation now requires surfactants to achieve at least 60% biodegradability within 28 days, following OECD and ISO test standards, and has introduced stricter labeling obligations for substances posing risks to human or environmental health (Council of European Parliament, 2024; Mohr et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although the United States has not enacted SDS-specific restrictions under the Toxic Substances Control Act (TSCA), the Environmental Protection Agency monitors surfactants through its new chemicals program, requiring hazard evaluations for novel applications (Borotkanics and Locke, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cowan-Ellsberry et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These evolving policies highlight increasing recognition of the ecological risks posed by common surfactants and underscore the importance of extending ecotoxicological studies to less-characterized groups such as aquatic invertebrates.\u003c/p\u003e\u003cp\u003eFeng et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) conducted a detailed assessment of sodium dodecyl sulfate (SDS) toxicity in the freshwater planarian \u003cem\u003eDugesia japonica\u003c/em\u003e. The authors reported a progressive decline in LC₅₀ values over 96 hours, indicating a time-dependent increase in lethality. At sublethal concentrations, SDS exposure triggered oxidative stress responses, with alterations in antioxidant enzyme activity and lipid peroxidation, and induced genotoxic effects as revealed by reduced genomic template stability. Molecular analyses further demonstrated dysregulation of genes involved in apoptosis, cell-cycle progression, DNA repair, and neoblast proliferation, suggesting that SDS interferes with key cellular pathways necessary for regeneration and genomic maintenance (Feng et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Together, these findings emphasize that SDS exerts a broad spectrum of toxic effects on planarians, ranging from oxidative imbalance to disruption of fundamental processes of stem cell biology, underscoring its ecological risk to freshwater invertebrates.\u003c/p\u003e\u003cp\u003eA recent investigation by Santos et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that sodium dodecylbenzene sulfonate (SDBS), a widely used anionic surfactant, can cause pronounced histological and physiological alterations in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e). After 96 hours of exposure to environmentally relevant concentrations (0.25\u0026ndash;0.5 mg/L), the authors described structural impairment of the gill lamellae, circulatory disturbances, and changes in mucus and chloride cell abundance. At the higher concentration, superoxide dismutase (SOD) activity was reduced, while catalase (CAT) activity remained unaffected. Hematological changes, including increased neutrophil and lymphocyte counts, further indicated that short-term exposure to SDBS is sufficient to trigger oxidative stress and immune-related responses, underscoring its ecological risk (Santos et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConcerns about mercury pollution remain central to discussions on aquatic toxicants because of its persistence, capacity to accumulate in food webs, and broad toxicological effects (Sunderland and Selin, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While mercury is naturally present in the environment, human activities, including industrial processes, mining operations, and agricultural practices, have markedly intensified its release into aquatic ecosystems. Within its various chemical forms, mercuric chloride (HgCl₂) deserves particular attention since it dissolves readily in water and persists over time, increasing the likelihood of exposure to aquatic organisms (Fernandes Azevedo et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eInorganic mercury salts, including HgCl₂, are highly reactive due to their strong interaction with thiol (-SH) groups present in proteins and enzymes. This reactivity disrupts cellular metabolism and weakens antioxidant defenses, creating conditions that favor oxidative damage (Carvalho et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Farina et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In aquatic invertebrates, exposure to HgCl₂ has been linked to neurochemical and physiological disturbances, such as inhibition of acetylcholinesterase (AChE), disruption of ion homeostasis, and the generation of reactive oxygen species that promote lipid peroxidation, protein oxidation, and DNA lesions (Mela et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Even when present at sublethal levels, mercury can compromise behavioral and reproductive traits, while long-term exposure reduces growth and survival (Singh Rathore and Khangarot, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In planarians specifically, mercury has been shown to impair neoblast activity, slow down regenerative processes, and alter sensory-driven behaviors like phototaxis and chemotaxis, indicating simultaneous impacts on stem cell dynamics and neural circuits (Agata et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sal\u0026oacute; and Bagu\u0026ntilde;\u0026agrave;, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Acute toxicity tests in freshwater organisms further demonstrate the high potency of HgCl₂, with 96-h LC₅₀ values ranging from a few \u0026micro;g/L to the low mg/L scale, underlining both its environmental risk and capacity to bioaccumulate in aquatic systems (Boudou and Ribeyre, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Camargo and Martinez, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMercury occurs in several chemical forms, including elemental (Hg⁰), inorganic species (Hg\u003csup\u003e2+\u003c/sup\u003e, HgS), and organic derivatives such as methylmercury and ethylmercury. Among these, methylmercury is of particular concern because it is efficiently absorbed and progressively concentrated through aquatic food webs, reaching high levels in top predators (Buckman et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; De Almeida Rodrigues et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). After entering aquatic systems, mercury can be incorporated into organisms both directly, by diffusion, and indirectly, through trophic transfer. These processes contribute to toxic outcomes such as oxidative imbalance, inhibition of key enzymes, and impairment of neural function (De Almeida Rodrigues et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMercury is recognized as a highly toxic contaminant with no known biological function. Its presence in aquatic systems originates both from natural processes and from human activities such as mining, industrial emissions, and agriculture. Once released, mercury persists due to its chemical stability and capacity to form soluble species that are readily assimilated by organisms. In aquatic environments, microbial activity can transform inorganic mercury into methylmercury (CH\u003csub\u003e3\u003c/sub\u003eHg\u003csup\u003e+\u003c/sup\u003e), a more toxic and bioavailable form. Methylmercury accumulates progressively through trophic levels, leading to higher concentrations in predatory fish. Consequently, fish may carry elevated mercury burdens even in regions with low inorganic mercury in the water, provided that environmental conditions favor microbial methylation.\u003c/p\u003e\u003cp\u003eThe present study aimed to assess the toxicological effects of SDS and HgCl\u003csub\u003e2\u003c/sub\u003e on the freshwater planarian \u003cem\u003eGirardia tigrina\u003c/em\u003e, an established model in ecotoxicology due to its high sensitivity to pollutants, robust regenerative capacity, and ecological relevance in freshwater ecosystems (Bach et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Calevro et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Hagstrom et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pestana and Ofoegbu, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Rawls et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Specifically, this study evaluated the median lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e), alterations in locomotor behavior, reproductive performance, tissue glycogen content, and AChE activity in response to SDS and HgCl₂ exposure. By investigating these endpoints, the research aims to elucidate the sublethal and chronic effects of both toxicants on freshwater invertebrate, thereby contributing to a broader understanding of their ecological impact and supporting more informed environmental risk assessments and conservation strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eChemicals\u003c/h2\u003e\u003cp\u003eSodium dodecyl sulfate (SDS, \u0026ge;\u0026thinsp;99%), mercuric chloride (HgCl₂, \u0026ge;\u0026thinsp;99.5%), acetylthiocholine iodide (ACTh, \u0026ge;\u0026thinsp;98%), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, \u0026ge;\u0026thinsp;98%), bovine serum albumin (BSA, \u0026ge;\u0026thinsp;98%, Fraction V), ethylenediaminetetraacetic acid disodium salt (EDTA, \u0026ge;\u0026thinsp;99%), and Triton X-100 (laboratory grade) were purchased from Sigma Chemical (St. Louis, MO, USA) and Merck (Darmstadt, Germany). All other reagents were of analytical grade.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlanaria Husbandry\u003c/h3\u003e\n\u003cp\u003eThe freshwater planarian \u003cem\u003eGirardia tigrina\u003c/em\u003e was employed as the model organism throughout this investigation. A stable breeding colony has been maintained since 2017 under controlled laboratory conditions (20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) in the BioTox (Laboratory of Experimental Biochemistry and Toxicology). The culture was sustained in ASTM-recommended water. The nutritional regimen consisted of weekly ad libitum feeding sessions with bovine liver, limited to a 2-hour duration to prevent water quality degradation. Immediately following each feeding, the entire culture medium was replenished. This maintenance protocol aligns with previously described standards for the species (Dean and Duncan, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Schaly and Braguini, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Torres-Da Matta et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eToxicity and Behavioral Assessments\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eChemical Exposure and Experimental Design\u003c/h2\u003e\u003cp\u003eSDS (10\u0026ndash;300 \u0026micro;g/L) and HgCl₂ (10\u0026ndash;100 \u0026micro;g/L) were dissolved in purified water. Concentration ranges were selected based on (1) literature-reported sublethal/lethal effects on freshwater invertebrates and (2) preliminary tests with \u003cem\u003eGirardia tigrina\u003c/em\u003e to establish no-observed-mortality and complete-mortality thresholds. Eight \u003cem\u003eG. tigrina\u003c/em\u003e individuals were exposed to 100 mL test solution in Petri dishes (125 \u0026times; 20 mm) at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C in darkness (96 h, unfed). Mortality was recorded at 24-h intervals. LC₅₀ values were derived via Probit analysis. No combined SDS\u0026thinsp;+\u0026thinsp;HgCl₂ tests were conducted (synergistic/antagonistic effects unassessed). Identical sublethal concentrations (5, 10, 25 \u0026micro;g/L) were used for chronic assays to: (\u003cem\u003ei\u003c/em\u003e) Ensure exposures remained below LC₅₀ thresholds, and (\u003cem\u003eii\u003c/em\u003e) Standardize response comparisons between toxicants.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEvaluation of planarian locomotor velocity\u003c/h3\u003e\n\u003cp\u003ePlanarian locomotor velocity (pLmV) was assessed following the method of Raffa and Valdez (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Individual planarians were placed in 60 \u0026times; 10 mm Petri dishes containing APW and positioned over graph paper with 0.5 cm\u0026sup2; grids. Locomotion was quantified as the total number of grid lines crossed during a 5-minute observation period (Raffa and Valdez, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), recorded using a high-speed NEOCoolcam\u0026reg; webcam with a varifocal zoom lens. Planarians were pre-incubated with SDS or HgCl₂ for either 24 h (acute test) or 7 days (chronic test) at concentrations of 5, 10, and 25 \u0026micro;g/L. Each concentration group consisted of a separate set of planarians (n\u0026thinsp;=\u0026thinsp;9), and no individual was used more than once in the experiment.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eReproductive Toxicity Assay\u003c/h2\u003e\u003cp\u003eReproductive performance was assessed in sexually mature \u003cem\u003eG. tigrina\u003c/em\u003e (average body length 1.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 cm). Groups of nine animals were randomly allocated to glass Petri dishes (125 \u0026times; 20 mm) containing 100 mL of planarian water supplemented with SDS or HgCl₂ at nominal concentrations of 5, 10, or 25 \u0026micro;g/L. Exposures lasted four weeks, during which reproductive activity was monitored at weekly intervals. Two endpoints were quantified: fecundity, expressed as the number of cocoons deposited per worm per week, and fertility, defined as the proportion of cocoons that hatched successfully. Additionally, the time required for cocoon hatching was recorded. Reproductive indices followed the approach described by Knakievicz et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), with minor adjustments in exposure duration and sampling schedule (Knakievicz et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlanarian Regeneration Assays\u003c/h3\u003e\n\u003cp\u003ePrior to experimentation, planarians were starved for seven days to standardize their metabolic state. This study evaluated two distinct regenerative processes: head regeneration and ocellus regeneration. For each treatment condition, including control, 5, 10, and 25 \u0026micro;g/L groups, a sample size of six to nine animals per concentration was utilized. Following amputation, the planarians were continuously exposed to one of three concentrations of either SDS or HgCl₂ for the entire duration of the regeneration period.\u003c/p\u003e\u003cp\u003eIn the head regeneration assay, animals were decapitated transversely, and only the posterior fragments were retained for observation. For the ocellus regeneration assay, the right ocellus was surgically excised using a 31-gauge insulin needle to ensure minimal adjacent tissue damage, a technique adapted from established methodology (Deochand et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Regenerative progress was documented daily through imaging under a light microscope for 14 consecutive days. Imaging for head regeneration commenced immediately post-amputation (day 0) and continued through day 14. For ocellus regeneration, imaging began on day 2 and continued through day 14, with a pre-surgery baseline image captured for each animal to enable subsequent scale normalization.\u003c/p\u003e\u003cp\u003eTo facilitate consistent image acquisition, planarians were transferred to 60 \u0026times; 10 mm Petri dishes featuring a uniform dark background; this minimized their light-aversive movement and optimized contrast. Before each imaging session, animals were rinsed and placed in fresh artificial planarian water (APW) to eliminate potential chemical interference from the exposure solutions. All acquired images were subjected to quality control and quantitatively analyzed using ImageJ software (version 1.51, NIH, USA).\u003c/p\u003e\u003cp\u003eFor both regenerative assays, a regeneration index was calculated to quantify outcomes. This index was defined as the area of the regenerated structure (either the cephalic blastema or the new ocellus) relative to the total body area of the animal. To ensure statistical robustness and account for individual size variation, each measurement was performed on three independent replicates per animal and the resulting data were normalized. This approach allowed for consistent and equitable comparisons across all experimental groups.\u003c/p\u003e\n\u003ch3\u003eBiochemical Analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eAcetylcholinesterase Assay (E.C. 3.1.1.7)\u003c/h2\u003e\u003cp\u003eAcetylcholinesterase (AChE) activity was quantified according to the spectrophotometric method of Ellman et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1961\u003c/span\u003e), with modifications. The assay is based on the enzymatic hydrolysis of the substrate acetylthiocholine (ACh) by AChE present in the sample. This reaction yields thiocholine, which subsequently reacts with the colorimetric agent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) to produce the yellow anion 5-thio-2-nitrobenzoate. The rate of formation of this product, which is proportional to the enzyme activity, was monitored by measuring the absorbance at 412 nm (Ellman et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1961\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePlanarians exposed to SDS or HgCl₂ at concentrations of 5, 10, and 25 \u0026micro;g/L for 24 hours and 7 days were homogenized in an ice-cold 20 mM Tris-HCl buffer (pH adjusted) containing 1 mM EDTA. The homogenate was centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 minutes at 4\u0026deg;C using a refrigerated centrifuge (Loccus\u0026reg; model L3024R). The resulting supernatant was collected and used as the enzyme source for the assay.\u003c/p\u003e\u003cp\u003eThe reaction was initiated by adding the supernatant to a cuvette containing a working solution of DTNB (0.75 mM) and acetylthiocholine (9 mM) as the substrate, yielding a final reaction volume of 3 mL. Enzyme activity was calculated from the linear rate of absorbance change and is expressed as units of hydrolyzed acetylcholine per milligram of protein (U \u0026times; mg protein⁻\u0026sup1;), where one unit corresponds to the amount of enzyme required to hydrolyze one micromole of substrate per minute under the specified assay conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eProtein determination\u003c/h2\u003e\u003cp\u003eThe protein content was quantified by the method of Bradford (Bradford, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), using bovine serum albumin as a standard.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using ANOVA, and Dunnett's test was used to compare treatments with the control group. The \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. The statistical analysis was performed using the Prism 8 statistical software (GraphPad, San Diego, US).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe LC₅₀ values determined for SDS and HgCl₂ in \u003cem\u003eGirardia tigrina\u003c/em\u003e revealed a progressive decrease over time, indicating a clear dose- and time-dependent toxicity pattern (Table I). For HgCl₂, the LC\u003csub\u003e50\u003c/sub\u003e declined from 252.58 \u0026micro;g/L at 24 hours to 229.16 \u0026micro;g/L at 96 hours, while SDS exhibited a slight decrease over the same period, with a final LC₅₀ of 45.27 \u0026micro;g/L at 96 hours. These findings suggest cumulative effects and reinforce the importance of considering exposure duration in toxicity assessments. Accordingly, sublethal concentrations below the LC\u003csub\u003e50\u003c/sub\u003e were selected for subsequent experiments to evaluate regeneration, reproduction, locomotion, and enzymatic activity without confounding mortality effects. These sublethal concentrations were then used to assess functional endpoints, including planarian locomotion, allowing evaluation of behavioral effects without confounding mortality.\u003c/p\u003e\u003cp\u003ePlanarian locomotion was significantly inhibited by both SDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B) and HgCl₂ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B), as shown by pLmV measurements, respectively. After 24 hours of SDS exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), locomotor inhibition reached 26.8%, 32.1%, and 33.2% at 5, 10, and 25 \u0026micro;g/L, respectively. Statistical analysis (two-way ANOVA) confirmed a significant effect of concentration, locomotion time, and their interaction. Similar effects were observed after 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), though inhibition levels were slightly reduced, suggesting possible adaptive mechanisms over time. HgCl₂ exposure also impaired locomotion, with reductions of 43.3%, 33.9%, and 33.5% at the same concentrations after 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and corresponding decreases of 7.8%, 14.2%, and 16.5% after 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Significant effects of concentration, time, and their interaction were again confirmed. These results underscore a concentration- and time-dependent inhibition of planarian locomotion by both compounds, with evidence suggesting partial tolerance or compensatory responses following prolonged exposure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eReproductive parameters were also markedly affected. SDS exposure led to substantial fecundity inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) of 74.1%, 79.8%, and 82.1%, and fertility reductions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) of 91.4%, 90.1%, and 100% at 5, 10, and 25 \u0026micro;g/L, respectively. Moreover, cocoon hatching time increased by 16.6% and 46.2% at 5 and 10 \u0026micro;g/L, respectively, with no viable cocoons observed at 25 \u0026micro;g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Similarly, HgCl₂ exposure resulted in fecundity reductions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) of 89.1%, 79.1%, and 96.6%, and fertility declines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) of 94%, 88.4%, and 97.2% at the same concentrations. Hatching times were prolonged by 24.1% and 40.6% at 5 and 10 \u0026micro;g/L, respectively, while no viable cocoons were detected at 25 \u0026micro;g/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings confirm that both compounds severely compromise planarian reproductive capacity and development, posing ecological risks. Building on the observed reproductive impairments, head regeneration assays were conducted to further evaluate the sublethal effects of SDS and HgCl₂ on planarian physiology.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHead regeneration in planarians was significantly affected by SDS exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) from day 3 to day 6 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). During this period, inhibition rates reached 50.8% and 53.8% at 10 and 25 \u0026micro;g/L, respectively, on day 3; 45.4% and 63.6% at 10 and 25 \u0026micro;g/L on day 4; 43.5% at 25 \u0026micro;g/L on day 5; and 31.4% and 48.6% at 10 and 25 \u0026micro;g/L on day 6. No significant effects were detected between days 7 and 10. From day 11 to day 14, SDS once again significantly inhibited regeneration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Inhibition levels were 15.9%, 22.2%, and 31.8% at 5, 10, and 25 \u0026micro;g/L, respectively, on day 11; 17.6% and 33.8% at 5 and 25 \u0026micro;g/L on day 12; 20%, 29.3%, and 36% at 5, 10, and 25 \u0026micro;g/L on day 13; and 22.7%, 32.9%, and 40.9% at 5, 10, and 25 \u0026micro;g/L on day 14. These findings indicate that SDS impairs regenerative capacity in a concentration- and time-dependent manner, with effects more evident at higher concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, HgCl₂ exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) impaired head regeneration throughout the entire experimental period (days 1\u0026ndash;14, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On day 1, inhibition reached 85.7% at 10 and 25 \u0026micro;g/L, followed by 75% at the same concentrations on day 2. No significant effects were observed on day 3, but inhibition resumed on day 4 with 54.5% and 50% at 10 and 25 \u0026micro;g/L, respectively. Subsequent values included 21.8% and 52.2% at 10 and 25 \u0026micro;g/L on day 5; 40% and 60% on day 6; 36.8% and 55.3% on day 7; 22.5% and 40% on day 8; 36.2% at 25 \u0026micro;g/L on day 9; and 33.3% at 25 \u0026micro;g/L on day 10. From days 11 to 14, concentration-dependent effects were consistently observed, with inhibition rates of 20.6%, 27%, and 36.5% at 5, 10, and 25 \u0026micro;g/L on day 11; 20.6%, 32.4%, and 35.3% on day 12; 20%, 33.3%, and 30.7% on day 13; and 17%, 36.4%, and 37.5% on day 14, respectively. These results demonstrate that HgCl₂ exerts an earlier and more sustained inhibitory effect on planarian head regeneration compared to SDS.\u003c/p\u003e\u003cp\u003ePlanarian eye regeneration was significantly impaired by SDS exposure in a time- and concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). While regeneration progressed steadily in the control group, animals exposed to SDS displayed marked delays in ocellus formation, particularly at concentrations of 10 and 25 \u0026micro;g/L. On day 4, eye regeneration was reduced by 55.5%, 62.0%, and 71.8% in the 5, 10, and 25 \u0026micro;g/L SDS groups, respectively, compared to controls. This inhibitory effect persisted and intensified over time, with reductions reaching 60.5%, 61.3%, and 67.1% on day 14. Notably, even the lowest concentration tested (5 \u0026micro;g/L) led to a significant reduction of 60.5% in regeneration by the final day. A two-way repeated measures ANOVA confirmed the significant main effects of both treatment concentration (p\u0026thinsp;=\u0026thinsp;0.0049) and regeneration time (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0056), along with a significant interaction between these factors (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0154), indicating that SDS modulates regeneration dynamics across time. Although the early stages (day 2) showed slightly increased values in some treated groups, possibly reflecting transient compensatory responses, these were not sustained. These findings reveal that SDS, a common surfactant and environmental contaminant, substantially disrupts regenerative capacity in planarians, underscoring its potential ecological risk even at environmentally relevant concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOcellus regeneration was markedly inhibited by HgCl₂ exposure in a concentration- and time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Statistically significant reductions were detected as early as day 2 across all concentrations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with the most dramatic effects observed at 10 and 25 \u0026micro;g/L. By day 14, eye regeneration was inhibited by 80.6%, 92.7%, and 93.5% in the 5, 10, and 25 \u0026micro;g/L groups, respectively, when compared to controls. Even at the lowest concentration tested (5 \u0026micro;g/L), a progressive decline in regenerative ability was evident, culminating in a near-complete inhibition by the end of the experimental period. A two-way repeated measures ANOVA confirmed significant main effects of both concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0048) and time (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0045), as well as a strong interaction between them (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0081), indicating that the impact of mercury on regeneration intensifies over time. These findings highlight the potent inhibitory effects of mercury on neuro-regeneration and suggest that planarians represent a sensitive invertebrate model for detecting sublethal developmental toxicity in aquatic systems.\u003c/p\u003e\u003cp\u003eRegarding AChE activity, both toxicants elicited significant and temporally dynamic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and B). SDS exposure resulted in a consistent increase in AChE activity across time, with significant stimulation at all concentrations after 24 hours (32.6%, 63.0%, and 80.3%) and a partial attenuation after 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Notably, no significant effect was observed at 5 \u0026micro;M after 7 days, while significant increases persisted at 10 and 25 \u0026micro;M. This sustained AChE activation may reflect an adaptive neurochemical response to SDS. HgCl₂ induced an increase in AChE activity after 24 hours at all tested concentrations (33.3%, 79.5%, and 40.8% increases), followed by a notable decrease after 7 days (reductions of 40.6%, 33.7%, and 35.1%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This biphasic response suggests an initial stimulatory phase potentially due to enzymatic upregulation, followed by compensatory downregulation or enzymatic inhibition.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, SDS and HgCl₂ exerted significant, concentration- and time-dependent effects on planarian survival, locomotion, reproduction, regeneration, and enzymatic activity. The observed outcomes suggest complex toxicodynamic interactions, with both compounds posing potential ecological threats due to their persistent and multifactorial impacts on freshwater invertebrates. These findings underscore the importance of evaluating sublethal endpoints and temporal patterns to fully understand pollutant risks in aquatic ecosystems.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBased on the results presented, a comprehensive evaluation can be made regarding the sublethal toxic effects of the surfactant SDS and the inorganic salt HgCl₂ in the freshwater planarian \u003cem\u003eG. tigrina\u003c/em\u003e. These organisms have been extensively employed as invertebrate models in environmental toxicology due to their ecological relevance, high sensitivity to pollutants, and remarkable regenerative capacity (Best and Morita, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Kang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results demonstrate that the toxicity of both SDS and HgCl₂ is cumulative, as evidenced by the progressive decline in their LC₅₀ values over the 96-hour exposure period. This time- and dose-dependent pattern aligns with findings reported for other toxicants in aquatic invertebrates (Li, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Sim\u0026atilde;o et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Notably, SDS exhibited a significantly greater acute toxicity than HgCl₂ (96-h LC\u003csub\u003e50\u003c/sub\u003e of 45.27 \u0026micro;g/L versus 229.16 \u0026micro;g/L, respectively), suggesting a more rapid mechanism of action or enhanced tissue penetration efficiency for the surfactant.\u003c/p\u003e\u003cp\u003eSignificant alterations in planarian locomotion underscore the neurotoxic potential of both SDS and HgCl₂. Impaired movement observed after 24 hours, with partial recovery after 7 days, may reflect neurophysiological compensation or adaptive mechanisms. The more pronounced initial effect of HgCl₂ relative to SDS may be attributed to mercury\u0026rsquo;s affinity for sulfhydryl (-SH) groups, which play essential roles in neuronal protein function (Aschner et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kat\u0026eth;no et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). These findings highlight the utility of locomotor behavior as an early and sensitive biomarker of neurotoxicity.\u003c/p\u003e\u003cp\u003eThe reproductive capacity of \u003cem\u003eG. tigrina\u003c/em\u003e was severely compromised by both contaminants, as indicated by marked reductions in fecundity and fertility, coupled with extended cocoon hatching durations. This collective impairment points to a profound disruption of core reproductive mechanisms, which could entail the dysregulation of endocrine pathways, modifications in gene expression profiles, or diminished gamete health (Heath et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mojica-V\u0026aacute;zquez et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The failure to produce any viable cocoons at the highest concentrations (25 \u0026micro;g/L) highlights a severe threat to population sustainability in contaminated environments.\u003c/p\u003e\u003cp\u003eDistinct regenerative impairment profiles emerged for each toxicant. SDS exposure resulted in a delayed inhibitory effect on head regeneration, becoming significant only after day 3, which implies a slower disruption of processes like cell proliferation or differentiation. Mercury, conversely, exhibited a more immediate and aggressive toxicity, inhibiting regeneration from day 2 onward and achieving nearly complete suppression by the end of the observation period. This potent effect is likely linked to HgCl₂'s ability to induce oxidative stress and epigenetic alterations (Kalafatić et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), potentially damaging the neoblast stem cell population vital for regeneration (Sengupta et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The heightened sensitivity of eye regeneration to mercury further underscores its specific neurotoxic potency.\u003c/p\u003e\u003cp\u003eThe contrasting AChE activity profiles underscore the distinct neurotoxic mechanisms of each compound. The biphasic response (initial increase followed by inhibition) induced by HgCl₂ implies a complex neurochemical imbalance, potentially stemming from inflammatory processes or altered gene regulation (De Oliveira et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xia et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, the sustained enzymatic activation observed with SDS exposure suggests a state of persistent cholinergic stress or an adaptive compensatory response (Biswas et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Meshorer and Soreq, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Toiber and Soreq, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These substrate-specific biochemical disruptions provide a plausible mechanism underlying the locomotor deficits recorded in the behavioral assays.\u003c/p\u003e\u003cp\u003eA key limitation of this study is its focus on the individual toxic effects of SDS and HgCl₂. The potential for interactive effects, whether synergistic, additive, or antagonistic, remains unexplored, as binary or more complex mixture assays were not performed. Given the ubiquitous co-occurrence of surfactants and heavy metals in polluted aquatic systems, particularly downstream from urban and industrial effluents, research into combined exposures constitutes a critical next step. Such studies are essential for developing a more environmentally realistic risk assessment and for deciphering the complex toxicological mechanisms that emerge from pollutant interactions.\u003c/p\u003e\u003cp\u003eNotwithstanding this limitation, our findings clearly show that both SDS and HgCl₂ can disrupt a suite of critical physiological functions in \u003cem\u003eG. tigrina\u003c/em\u003e, from behavior and reproduction to cellular regeneration and neurochemistry. The persistence of these compounds in aquatic environments underscores their potential to threaten biodiversity at sublethal concentrations. This work further solidifies the status of planarians as sensitive bioindicators for environmental toxicology. Specifically, their unparalleled regenerative capability provides a powerful, integrative model for probing the initial cellular and systemic injuries inflicted by environmental contaminants before these effects manifest at higher levels of biological organization.\u003c/p\u003e\u003cp\u003eWhile this study provides a multifaceted toxicity assessment, several constraints should be noted. Our laboratory-based approach, though controlled, simplifies the dynamic variables present in natural freshwater habitats. The exclusion of combinatorial exposures with other pollutants precludes insight into potential interactive effects. Furthermore, while our selected endpoints (locomotion, reproduction, regeneration, AChE) are ecologically relevant, the incorporation of molecular analyses, such as oxidative stress or gene expression biomarkers, would offer deeper mechanistic understanding. The focus on a single species (\u003cem\u003eG. tigrina\u003c/em\u003e) under acute exposure scenarios also cautions against extrapolating these results to diverse aquatic taxa or to the consequences of long-term, chronic pollution. Investigations encompassing these dimensions are needed to refine ecological risk predictions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that both the anionic surfactant SDS and the inorganic salt HgCl₂ exhibit pronounced, multifaceted, and time-dependent toxicity in \u003cem\u003eGirardia tigrina\u003c/em\u003e. Both contaminants affected multiple biological levels, including locomotor behavior, reproductive performance, tissue regeneration, and enzymatic activity, highlighting systemic and cellular impacts. Locomotor inhibition indicated potential neurotoxic effects, with partial recovery after prolonged exposure, suggesting adaptive or compensatory mechanisms. Reproductive endpoints were severely impaired, with significant reductions in fecundity and fertility, delayed cocoon hatching, and absence of viable offspring at the highest concentrations, underscoring potential risks to population sustainability in contaminated environments.\u003c/p\u003e\u003cp\u003eRegeneration assays revealed distinct toxic profiles for each compound: SDS induced delayed regenerative responses, whereas HgCl₂ caused early and sustained inhibition, implicating disruption of neoblast stem cell activity and neurogenic processes essential for regeneration. AChE activity exhibited complex, compound-specific responses, reflecting neurochemical dysregulation that may partially explain the observed behavioral and regenerative impairments.\u003c/p\u003e\u003cp\u003eCollectively, these findings underscore the capacity of persistent pollutants to interfere with multiple physiological and biochemical functions in freshwater invertebrates, even at sublethal concentrations. The results reinforce the value of planarians as sensitive and informative models for environmental toxicity assessment, enabling the detection of subtle effects that precede mortality. Future studies should extend these approaches to other freshwater invertebrate species, in order to refine and broaden the ecological relevance of the findings.\u003c/p\u003e\u003cp\u003eThese findings have direct implications for ecotoxicology and environmental management, as they suggest that the release of petroleum-derived synthetic surfactants and heavy metals should be closely monitored and regulated. Moreover, the observed multifactorial effects highlight the need for future studies involving combined exposures, reflecting environmentally realistic scenarios to elucidate potential synergistic or antagonistic interactions among pollutants and to develop more effective mitigation strategies for the conservation of aquatic ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eAcknowledgments to Universidade Estadual do Centro-Oeste (UNICENTRO) for providing laboratory space and equipment at the Experimental Biochemistry and Toxicology Laboratory (BioTox), which supported the research conducted for the development of this paper.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eEthical Approval\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthors\u0026rsquo; Contributions\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by V.E.S.A and W.L.B.. The first draft of the manuscript was written by V.E.S.A and W.L.B. and all authors commented on previous versions of the manuscript. All authors read and approved final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eAvailability of materials and data are available through the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAgata K, Saito Y, Nakajima E (2007) Unifying principles of regeneration I: Epimorphosis versus morphallaxis. 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LC\u003csub\u003e50\u003c/sub\u003e for SDS and HgCl\u003csub\u003e2\u003c/sub\u003e in planarian \u003cem\u003eG. tigrina\u003c/em\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eSDS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTime (h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eEquation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eLC\u003csub\u003e50\u003c/sub\u003e (µg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% confidence intervals\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 306.8x – 514.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e49.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e37.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.83\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 322.1x – 541.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e49.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e38.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e54.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 298x – 497.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e48.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e35.4 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.70\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 287.8x – 472.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e45,27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e25.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e53.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003e\u003cstrong\u003eHgCl\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 409.8x – 979.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e252.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e217.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e264.85\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 361.7x – 858.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e243.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e214.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e252.35\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 338x – 796.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e234.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e194.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e248.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003ey = 340.7x – 799.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e229.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e170.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e250.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eData represent one of 3 independent replicates; each performed in triplicate.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","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":"Sodium dodecyl sulfate, Mercuric chloride, Sublethal toxicity, Freshwater planarian, Girardia tigrina, Behavioral toxicity, Regeneration","lastPublishedDoi":"10.21203/rs.3.rs-7457039/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7457039/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSynthetic chemicals with high environmental persistence, including anionic surfactants and heavy metals, pose significant risks to freshwater ecosystems. This study evaluated the sublethal toxicity of sodium dodecyl sulfate (SDS) and mercuric chloride (HgCl₂) on the freshwater planarian \u003cem\u003eGirardia tigrina\u003c/em\u003e, a widely used ecotoxicological model. Median lethal concentrations (LC₅₀) were determined over 96 hours, revealing a time- and dose-dependent toxicity, with SDS (LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;45.27 \u0026micro;g/L) exhibiting higher acute toxicity than HgCl₂ (LC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;229.16 \u0026micro;g/L). Sublethal concentrations (5, 10, 25 \u0026micro;g/L) were used to assess locomotor behavior, reproduction, regeneration, and acetylcholinesterase (AChE) activity. Both SDS and HgCl₂ impaired locomotion in a concentration- and time-dependent manner, with partial recovery after prolonged exposure, indicating potential neurophysiological compensation. Reproductive performance was severely affected, with reduced fecundity and fertility, prolonged cocoon hatching times, and complete inhibition of viable cocoons at higher concentrations. Regeneration assays revealed that SDS delayed head and eye regeneration primarily after day 3, whereas HgCl₂ induced rapid and sustained inhibition from day 2 onward. AChE activity exhibited compound-specific alterations: HgCl₂ induced a biphasic response (initial stimulation followed by suppression), whereas SDS caused sustained activation. These multifaceted effects indicate disruption of neurochemical balance, reproductive physiology, and stem cell-mediated regeneration. Collectively, the findings highlight the ecological risks of SDS and HgCl₂, emphasizing the sensitivity of planarians as sentinel species for detecting sublethal toxicological effects in freshwater environments. This study underscores the importance of evaluating behavioral, reproductive, regenerative, and biochemical endpoints to support comprehensive environmental risk assessments and inform regulatory policies on emerging pollutants.\u003c/p\u003e","manuscriptTitle":"Comparative Sublethal Toxicity of Sodium Dodecyl Sulfate and Mercuric Chloride in the Freshwater Planarian Girardia tigrina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 19:48:14","doi":"10.21203/rs.3.rs-7457039/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"38fe65e4-ae93-46bb-81fb-fea096454c04","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-26T11:40:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-30 19:48:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7457039","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7457039","identity":"rs-7457039","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}