Effects of salinity and acetaminophen on egestion rate and movement of Poecilia reticulata

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Elevated salinity can decrease organismal survival and growth and can alter the toxicity of pollutants. Pharmaceuticals, such as acetaminophen, enter freshwaters through non-point sources and from hospital and wastewater treatment plants effluent. Available research is limited on the combined effects of elevated salinity and acetaminophen on freshwater organisms. Our study evaluated the effects of acetaminophen (350 µg L − 1 ) and salinity (680 mg L − 1 ) on Poecilia reticulata over 96 hours. Egestion rate (mg cm − 1 h − 1 ) and movement parameters were measured following exposure. We observed significant changes in egestion rate under salinity likely due to impaired osmoregulatory mechanisms and no effect on movement. Further, there was no interaction between salinity and acetaminophen; thus, salinity did not affect acetaminophen toxicity at the concentrations tested. Our findings highlight the need of establishing threshold of salinity and pharmaceuticals to protect freshwater ecosystems and to help predict ecological impacts on aquatic organisms. saltwater intrusion pharmaceuticals salinity acetaminophen freshwater ecosystems Poecilia reticulata Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ecosystem services, including drinking water, irrigation, food, and recreation, rely on healthy freshwater environments. However, these ecosystems are increasingly threatened by human activities (Venâncio et al., 2019 ). Specifically, agriculture and road salt usage have altered the concentration and composition of ions in water bodies (Cañedo-Argüelles et al., 2013 ; Cañedo-Argüelles et al., 2019 ; Meybeck & Helmer, 1989 ; Williams, 2001 ). Additionally, because of global climate change, rising sea levels allows saltwater to intrude into groundwater and coastal freshwater systems (Velasco et al., 2019 ; Venâncio et al., 2019 ). Shifts in environmental salinity disrupt organisms' metabolism and water balance, leading to osmotic imbalances (Velasco et al., 2019 ; Kefford et al., 2002 ). Organisms exposed to elevated salinity may allocate energy from growth and movement into protective mechanisms to maintain homeostasis. This redirection in energy can affect fitness, altering ecosystem structure and functions such as food webs and nutrient cycling (Berger et al., 2019 ; Cañedo-Argüelles et al., 2019 ; Velasco et al., 2019 ). Velasco et al. ( 2019 ) reported that salinity alone reduced growth, increased mortality, and altered metabolic rates in various species including Boeckella hamata (freshwater copepod), Cherax quadricarinatus (redclaw crayfish), Crassostrea virginica (Eastern oyster), Daphnia pulex (water flea), Gammarus roeseli (amphipod). Salinity can also modify pollutant toxicity, as observed in saltwater ecosystems (DeLorenzo et al., 2009 ), and influence species' sensitivity to contaminants (Velasco et al., 2019 ). For example, aldicarb becomes more toxic to rainbow trout in saline water (El-Alfy & Schlenk, 1998 ; El-Alfy et al., 2001 ), while hypersaline conditions protect juvenile trout from dietary seleno-L-methionine toxicity (Schlenk et al., 2003 ). Climate change adds complexity to the interactions between salinity and other stressors, with effects varying based on whether the interaction is additive, antagonistic, or synergistic. While interactions between salinity, temperature, and metals are well studied, the combined impact of salinity and pharmaceuticals on freshwater organisms remains underexplored (Cañedo-Argüelles et al., 2019 ). Pharmaceuticals are detected in the environment at concentrations ranging from ng L − 1 to µg L − 1 (Kola & Landis, 2004 ; Van Boeckel et al., 2014 ; Bradley et al., 2020 ). Their presence is associated to the increasing demand for human and veterinary drugs; further, due to incomplete removal by wastewater treatment plants, pharmaceuticals can accumulate in aquatic ecosystems (Gros et al., 2010 ; Zur et al., 2018, Adeleye et al., 2022 ). Additionally, runoff from fields treated with animal waste fertilizer introduces pharmaceuticals into water bodies (Nikolaou et al., 2007 ). These contaminants are commonly detected in aquatic environments worldwide (Nikolaou et al., 2007 ; Ferguson et al., 2013 ; Taylor, 2015 ; Ngqwala & Muchesa, 2020 ; Ortúzar et al., 2022 ). Acetaminophen (paracetamol), widely used for its antipyretic and analgesic properties (Blough et al., 2011; Keaveney et al., 2020 ), has been detected in surface and marine waters (Guerra et al., 2014 ; Kolpin et al., 2002 ; Nödler et al., 2014 ). High concentrations of acetaminophen (> 500 µg/L) have been detected in hospital and wastewater treatment plant effluent (Kumar et al. 2019 ; Kosma et al. 2020 ; Pharms UBA 2021; Liu et al. 2023 ). Once in aquatic ecosystems, acetaminophen can negatively impact organisms. For example, studies have reported altered kidney tubule morphology in zebrafish (Galus et al., 2013 ), as well as kidney, gill, and liver damage, reduced oxygen consumption, and impaired swimming performance in rainbow trout (Choi et al., 2018 ). Additionally, acetaminophen affects reproduction processes in invertebrates by blocking prostaglandin synthesis, gamete maturation, and spawning (Solé et al., 2010 ). However, acetaminophen toxicity is influenced by environmental conditions as indicated by Correia et al. ( 2016 ) who reported different oxidative stress levels in Ruditapes philippinarum under varying salinity concentrations. Our research evaluates the effects of individual and combined salinity and acetaminophen on Poecilia reticulata movement and egestion. P. reticulata is an ideal organism for laboratory studies due to its hassle-free maintenance and its use in multiple research fields including ecotoxicology (Doleželová et al., 2008 ), behavior (Lukas et al., 2021 ), gene expression (Fraser et al., 2011 ). We examined egestion rate and movement as indicators of fish fitness that can further influence ecological dynamics. For example, reduced egestion can limit nutrient availability (Conley et al., 2009 ) and altered movement can impact predator avoidance and feeding behavior (Covich et al., 1994 ; Justice and Bernot, 2017). We hypothesize that individual and combined elevated salinity and acetaminophen will decrease egestion rates and alter movement potentially through mechanisms involving oxidative stress and neurotoxicity (Almeida and Nunes 2019 ) affecting the digestive system, as well as energetic trade off from egestion and movement to maintenance of osmotic balance. Methods Chemicals Sodium chloride (NaCl, Sigma-Aldrich, CAS# 1647-14-5) was used to mimic salinization of water sources. NaCl concentrations were prepared by dissolving salt in the control medium (dechlorinated, aerated freshwater). Only fresh solutions were used for toxicity assays. Acetaminophen (Sigma-Aldrich, CAS# 103-90-2) was used to simulate pharmaceutical pollution in water sources. A stock solution was prepared and stored at 4°C. Aliquots of the stock solution were added to the corresponding treatment to achieve a target nominal concentration of 350 µg L -1 . Experimental setup Poecilia reticulata were purchased from Carolina Biological Supply and acclimated for 14 days in a 110 L glass aquarium in aerated dechlorinated water under a 16:8 photoperiod and room temperature (22.2 °C). Fish were fed Aqueon® commercial fish flakes three times per week ad libitum. The mean length of P. reticulata was 16.44 ± 2.16 mm. Ten replicates were assigned to each treatment (N = 40): control, elevated salinity (680 mg L -1 ; SAL), acetaminophen (350 μg L -1 ; ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Beginning a week before experimentation, half of the organisms were acclimated to elevated salinity. Twenty fish were placed in a glass aquarium filled with 48.6 L of water. NaCl (4.86 g) was added daily for 5 days, increasing the salinity by 500 mg L -1 for a total value of 680 mg L -1 . After acclimatization, 10 replicate organisms were randomly selected for each treatment (SAL and SAL × ACE). Each replicate was housed in a 300 mL glass container with the respective treatment solution. Containers were covered with aluminum foil, with small holes on the top. Environmentally relevant concentrations of acetaminophen (350 µg L -1 ) and salinity (680 mg L -1 ) were selected to represent concentrations detected in freshwater ecosystems where organisms are exposed to pharmaceuticals (Pharms UBA, 2021) and saltwater intrusion (Stockwell et al., 2011). Organisms were exposed to treatments for 96 hours. After 48 hours, each organism was fed 0.01 g fish flakes (Aqueon®). Excess food was removed after 15 minutes. A 100% water change and treatment renewals were completed immediately after feeding. This study was conducted in compliance with ethical guidelines for animal research and was approved by the Institutional Review Board and Institutional Animal Care and Use Committee (IRB-IACUC) at North Carolina Wesleyan University. Egestion One organism and a small volume of water were transferred to a petri dish with a ruler underneath (Fig. 1). Images were taken with an Apple iPhone 6S placed ~30 cm above and parallel to the petri dish and uploaded to ImageJ software for length measurements. The line segment tool was used to measure the length of each fish, from the head to the base of the tail. Feces collected after 96-hour exposure were transferred to pre-weighed tin foil and dried in a convection oven (Quincy Lab 10GC) at 60 °C for 24 hours. After drying, weight difference was measured using a precision scale (Ohaus Pioneer®). Egestion rate was calculated as the weight of feces and standardized with fish length over the exposure time (mg cm -1 h -1 ). Movement Individual fish were placed in a white plastic bucket (20 L, 30 cm diameter) filled with 3 L of water that matched the respective treatments (freshwater or saltwater). An Apple iPhone 6S was placed above the bucket to record fish movement. Fish were given one minute to acclimatize, and their movement was recorded for analyses for the next one minute. Video files were used to measure fish movement using ANY-MazeTM (v4.99). Two zones were designated in the apparatus: center zone (314 cm²) and outer zone (392 cm²; Fig. 2). Four movement variables were selected to measure changes in fish movement: total distance traveled, mean speed, number of rotations, and number of movements in/out of the center zone (Table 1). Data Analysis Egestion and continuous movement variables (total distance traveled and mean speed) were assessed using a linear regression model with categorical predictors of treatment. Normality of egestion and continuous movement variables were assessed using the Shapiro-Wilks Test. Homogeneity of variance was checked using Bartlett's Test. Count movement variables (number of rotations and movement in and out of center) were analyzed using generalized linear models (GLMs) with the Poisson error structure or negative binomial error structure if data were over dispersed. Statistical analyses were conducted in R v4.2.1 (R Core Team 2022), with alpha set to 0.05 for all tests. Linear regressions were performed using the lm() function, generalized linear models were performed using the glm() function, and pairwise comparisons of all models were performed using the emmeans() function in the emmeans v1.10.4 (Length 2024). Results Control treatment water parameters were salinity (180 mg/L), conductivity (220 µS/cm), pH (7.28), and total dissolved solids (TDS, 156 ppm). Elevated salinity treatment water parameters were salinity (680 mg/L), conductivity (1370 µS/cm), pH (7.3), and TDS (891 ppm). Egestion rates followed a normal distribution across all treatments (p > 0.05), and Bartlett's test confirmed equal variance (K² = 4.35, df = 3, p = 0.226). Egestion rates ranged from 0.0002 to 0.0029 mg cm -1 h -1 . Mean egestion rates for SAL (0.0019 ± 0.0005 mg cm -1 h -1 ) and SAL × ACE (0.0020 ± 0.0008 mg cm -1 h -1 ) were nearly double those of control (0.0012 ± 0.0004 mg cm -1 h -1 ) and ACE (0.0010 ± 0.0005 mg cm -1 h -1 ). Egestion was significantly different (Fig. 3) between the control and SAL (t = -4.838, p < 0.001) as well as between control and SAL × ACE (t = -3.443, p = 0.008). Significant differences were also found between SAL and ACE (t = 3.399, p = 0.009) and between ACE and SAL × ACE (t = -4.838, p < 0.001). Total distance travel ranged from 0 m (immobile) to 2.8 m across treatments. Control (1.46 ± 0.92 m) and SAL × ACE (1.51 ± 0.61 m) traveled similar distances. SAL exhibited the highest mean distance (1.76 ± 0.43 m), while ACE had the lowest (1.15 ± 0.65 m). Total distance traveled was normally distributed across treatments ( p > 0.05) with equal variance (K² = 3.29, df = 3, p = 0.350). Mean speed ranged from 0 m/s (immobile) to 0.047 m/s. Control (0.024 ± 0.020 m/s) and SAL × ACE (0.025 ± 0.010 m/s) had similar speeds. SAL had the highest speed (0.029 ± 0.010 m/s), while ACE had the lowest (0.019 ± 0.010 m/s). Mean speed was normally distributed across all treatments ( p > 0.05), with equal variance across treatments (K² = 3.35, df = 3, p = 0.342). No significant differences were observed in total distance or speed across treatments (Fig. 4 - 5). For count-based movement parameters, normality was not required. Movements in and out of the center zone were over-dispersed (z = 3.082, p = 0.001), so a Negative Binomial GLM was applied. Movements ranged from 0 to 12. Elevated salinity treatments showed higher movements (SAL: 6.5 ± 3.17; SAL × ACE: 4.7 ± 2.36) compared to control (2.6 ± 2.88) and ACE (3.6 ± 3.53). However, there were no statistically significant differences in movement in and out of the center zone across treatments ( p > 0.05). Rotations were not over-dispersed (z = -3.409, p = 0.999) and were analyzed using a Poisson GLM. Rotations ranged from 0 to 4. Control and SAL × ACE recorded 2.4 ± 1.43 and 2.4 ± 1.07 rotations, respectively, while SAL had the highest count (3.0 ± 0.94) and ACE the lowest (1.9 ± 1.29). No statistically significant differences in the number of rotations were found across treatments (p > 0.05). Discussion Egestion Egestion rates were significantly higher in fish exposed to SAL compared to control ( p < 0.001) and ACE ( p = 0.009), as well as in SAL × ACE compared to control ( p = 0.008) and ACE ( p < 0.001). These results suggest that elevated salinity influences digestive function, potentially through increased ion uptake and impairment of the enteric nervous system (Ern et al., 2014). Under elevated salinity, fish may allocate more energy toward maintaining osmotic balance, either by excreting excess ions or adapting to increased internal salinity (Cañedo-Argüelles et al., 2013; Hallali et al., 2018; Venâncio et al., 2019). Higher sodium chloride concentrations in digestive tissues can stimulate digestion and egestion, as observed in Gambusia affinis (mosquitofish), where metabolism and nitrogen excretion changed under acute salinity exposure (Uliano et al., 2010). Although all fish in our study were fed the same amount of food, variations in ingestion were observed. Increased energy demands for osmotic regulation may lead to higher food intake, potentially explaining differences in egestion. Similar mechanisms have been reported in other species, where energy consumption varies with salinity, including Ictalurus punctatus and Carassius auratus (Altinok and Grizzle, 2004), Allenbatrachus grunniens (Walsh et al., 2004), Gambusia affinis and Danio rerio (Uliano et al., 2010), Perca fluviatilis (Ern et al., 2014), Oreochromis mossambicus , Oncorhynchus mykiss , and Anguilla anguilla (Kültz et al., 2015), O. niloticus (Hallali et al., 2018), and C. auratus , Clarias gariepinus , and O. niloticus (Lee et al., 2022). However, these responses are species-dependent, and further research is needed to determine the specific effects of elevated salinity on ingestion and egestion in Poecilia reticulata . Movement Fish exposed to SAL and SAL × ACE showed higher movement counts in and out of the center zone compared to control and ACE, but these differences were not statistically significant ( p > 0.05). Greater movement across zones could indicate increased exploratory behavior or an attempt to avoid unfavorable conditions, including predators. While Cañedo-Argüelles et al. (2013) reported that increased salinity triggered movement associated with predator avoidance, our study did not find a statistically significant effect. Mean speed, movement frequency, and rotations also appeared slightly higher under SAL and SAL × ACE, but these differences were not statistically significant ( p > 0.05). In contrast, fish exposed to ACE exhibited reduced movement compared to SAL and control treatments, with lower rotations and movement counts, although these differences were not statistically significant ( p > 0.05). Reduced movement under ACE exposure may be linked to physiological stress associated with acetaminophen metabolism. Acetaminophen exposure has been linked to oxidative stress (Letelier et al., 2011; Correia et al., 2016), which can impair neuromuscular function and reduce overall activity. As acetaminophen is metabolized, it produces N-acetyl-p-quinone-imine (NAPQI), a reactive intermediate that is detoxified by glutathione-S-transferase (GST). At high concentrations, GST becomes saturated, leading to NAPQI accumulation, oxidative stress, and depletion of cellular energy reserves (Letelier et al., 2011; Vermot et al., 2021). This oxidative imbalance, along with increased reactive oxygen species, can damage cellular structures, exacerbate lipid peroxidation, and impair ATP production (Antunes et al., 2013; Correia et al., 2016). While oxidative stress is a known effect of acetaminophen exposure (Letelier et al., 2011; Correia et al., 2016), our study did not find statistically significant reductions in movement parameters, suggesting that the impact of acetaminophen on fish behavior may vary depending on concentration, exposure duration, and species sensitivity. Acetaminophen exposure has been associated with behavioral and physiological changes in other species, including decreased swimming speeds in Oncorhynchus mykiss (rainbow trout; Choi et al., 2018), increased lipid peroxidation in Mytilus galloprovincialis (marine mussels; Solé et al., 2010), and reduced heartbeat rates in Clarias gariepinus (African catfish larvae; Erhunmwunse et al., 2021). However, lack of statistical significance in our study suggests that movement responses are species-specific that vary with exposure conditions. Conclusions Salinity and acetaminophen influenced egestion and movement in P. reticulata . Elevated salinity significantly increased egestion, likely due to overstimulation of the enteric nervous system via enhanced digestion and corresponding egestion. Although movement was higher under salinity, differences were not statistically significant, suggesting potential species specific responses. We observed lower movement with acetaminophen exposure, however this effect was not statistically significant. This may be a response to oxidative stress, changes in energy allocation, or neuromuscular disruption as a result of acetaminophen metabolism. Overall, environmental stressors that affect adaptability to changing conditions can reduce species survival and disrupt ecosystem dynamics. Our findings highlight the need to examine the combined effects of pharmaceuticals and abiotic stressors (e.g., salinity, pH, temperature) across varying exposure durations. Thus, our research can contribute to more accurate ecological risk assessments and mitigation strategies for freshwater ecosystems. Declarations Ethical Statement Funding: No funding was received for this study. Conflict of Interest: The authors declare no conflict of interest. Ethical Approval: Ethical approval was obtained from the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) at North Carolina Wesleyan University. Informed Consent: Not applicable, as this study did not involve human participants. Author Contributions: All authors contributed equally to the research design, data collection, analysis, and manuscript preparation. Data Availability Statement: The data supporting this study is available in the supplementary material. References Adeleye AS, Xue J, Zhao Y, Taylor AA, Zenobio JE, Sun Y, Zhu Y (2022) Abundance, fate, and effects of pharmaceuticals and personal care products in aquatic environments. 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S Afr J Sci 116(7-8):1-7. https://doi.org/10.17159/sajs.2020/5730 Nikolaou A, Meric S, Fatta D (2007) Occurrence patterns of pharmaceuticals in water and wastewater environments. Anal Bioanal Chem 387:1225-1234. https://doi.org/10.1007/s00216-006-1035-8 Nödler K, Voutsa D, Licha T (2014) Polar organic micropollutants in the coastal environment of different marine systems. Mar Pollut Bull 85(1):50-59. https://doi.org/10.1016/j.marpolbul.2014.06.024 Ortúzar M, Esterhuizen M, Olicón-Hernández DR, González-López J, Aranda E (2022) Pharmaceutical pollution in aquatic environments: a concise review of environmental impacts and bioremediation systems. Front Microbiol 13:869332. https://doi.org/10.3389/fmicb.2022.869332 Pharms UBA Umwelt Bundesamt (2021) Database - Pharmaceuticals in the environment. Umwelt Bundesamt . Available from: https://www.umweltbundesamt.de/en/database-pharmaceuticals-in-the-environment. Accessed January 1, 2025 R Core Team (2024) R: A language and environment for statistical computing. R Foundation for Statistical Computing , Vienna, Austria. Available from: https://www.R-project.org/ Schlenk D, Zubcov N, Zubcov E (2003) Effects of salinity on the uptake, biotransformation, and toxicity of dietary seleno-L-methionine to rainbow trout. Toxicol Sci 75(2):309-313. https://doi.org/10.1093/toxsci/kfg184 Solé M, Shaw JP, Frickers PE, Readman JW, Hutchinson TH (2010) Effects on feeding rate and biomarker responses of marine mussels experimentally exposed to propranolol and acetaminophen. Anal Bioanal Chem 396:649-656. https://doi.org/10.1007/s00216-009-3182-1 Stockwell CA, Purcell KM, Collyer ML, Janovy J (2011) Effects of salinity on Physa acuta , the intermediate host for the parasite Posthodiplostomum minimum : implications for the translocation of the protected white sands pupfish. Trans Am Fish Soc 140(5):1370-1374. https://doi.org/10.1080/00028487.2011.620499 Taylor D (2015) The pharmaceutical industry and the future of drug development. Issues Environ Sci Technol 1:1-33. https://doi.org/10.1039/9781782622345-00001 Uliano E, Cataldi M, Carella F, Migliaccio O, Iaccarino D, Agnisola C (2010) Effects of acute changes in salinity and temperature on routine metabolism and nitrogen excretion in gambusia (Gambusia affinis) and zebrafish (Danio rerio). Comp Biochem Physiol A Mol Integr Physiol 157(3):283-290. https://doi.org/10.1016/j.cbpa.2010.07.019 Van Boeckel TP, Gandra S, Ashok A, Caudron Q, Grenfell BT, Levin SA, Laxminarayan R (2014) Global antibiotic consumption 2000 to 2010: an analysis of national pharmaceutical sales data. Lancet Infect Dis 14(8):742-750. DOI: 10.1016/S1473-3099(14)70780-7 Velasco J, Gutiérrez-Cánovas C, Botella-Cruz M, Sánchez-Fernández D, Arribas P, Carbonell JA, Pallarés S (2019) Effects of salinity changes on aquatic organisms in a multiple stressor context. Philos Trans R Soc B 374(1764):20180011. https://doi.org/10.1098/rstb.2018.0011 Venâncio C, Castro BB, Ribeiro R, Antunes SC, Abrantes N, Soares AMVM, Lopes I (2019) Sensitivity of freshwater species under single and multigenerational exposure to seawater intrusion. Philos Trans R Soc B 374(1764):20180252. https://doi.org/10.1098/rstb.2018.0252 Vermot A, Petit-Härtlein I, Smith SM, Fieschi F (2021) NADPH oxidases (NOX): an overview from discovery, molecular mechanisms to physiology and pathology. Antioxidants 10(6):890. https://doi.org/10.3390/antiox10060890 Walsh PJ, Wei Z, Wood CM, Loong AM, Hiong KC, Lee SML, Ip YK (2004) Nitrogen metabolism and excretion in Allenbatrachus grunniens (L): effects of variable salinity, confinement, high pH and ammonia loading. J Fish Biol 65(5):1392-1411. https://doi.org/10.1111/j.0022-1112.2004.00538.x Williams WD (2001) Salinization: unplumbed salt in a parched landscape. Water Sci Technol 43(4):85-91. https://doi.org/10.2166/wst.2001.0186 Żur J, Piński A, Marchlewicz A, Hupert-Kocurek K, Wojcieszyńska D, Guzik U (2018) Organic micropollutants paracetamol and ibuprofen—toxicity, biodegradation, and genetic background of their utilization by bacteria. Environ Sci Pollut Res 25:21498-21524. https://doi.org/10.1007/s11356-018-2517-x Tables Table 1. List of names and descriptions of movement parameters selected for analysis. Acronyms and zones were defined for the purposes of this experiment. Descriptions were retrieved from the complete list of ANY-maze measures (ANY-maze, 2023). Name Description Measurement Total Distance Traveled Reports the total distance that the animal traveled during the test. meters, m Mean Speed Reports the mean speed of the animal during a test. meters per second, m/s Rotations Reports the number of times the animal's body completed an entire rotation of 360°. count Number of Entries and exits to the zone Counts the number of times the animal entered and exit the zone. count Table 2. Poecilia reticulata were exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Egestion rate (mg/cm/h) was determined after standardizing fecal mass (dried and weighed feces) with fish length (measured with ImageJ). Standard deviation between parentheses. Treatment Mean Egestion Rate (mg/cm/h) Control 0.0012 (0.0004) ACE 0.0010 (0.0005) SAL 0.0019 (0.0005) SAL x ACE 0.0020 (0.0008) Table 3. Poecilia reticulata were exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE; n = 10 each). Each replicate was placed in an isolated apparatus to record fish movement for two minutes. Video files were uploaded to a laptop, where the outer/center zones were defined, and movement parameters were determined using ANY-MazeTM (v4.99). Standard deviation between parentheses. Treatment Mean distance Traveled (m) Mean speed (m/s) Mean Movements in/out of Center Zone (#) Mean Rotations (#) Control 1.46 (0.92) 0.02 (0.02) 2.6 (2.88) 2.4 (1.43) ACE 1.15 (0.65) 0.02 (0.01) 3.6 (3.53) 1.9 (1.29) SAL 1.76 (0.43) 0.03 (0.01) 6.5 (3.17) 3.0 (0.94) SAL × ACE 1.51 (0.61) 0.03 (0.01) 4.7 (2.36) 2.4 (1.07) Supplementary Files ResultsBIO.xlsx Cite Share Download PDF Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Biologia → Version 1 posted Editorial decision: Minor revisions 23 Jun, 2025 Reviewers agreed at journal 17 Mar, 2025 Reviewers invited by journal 17 Mar, 2025 Editor assigned by journal 21 Feb, 2025 First submitted to journal 20 Feb, 2025 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6067329","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430084024,"identity":"4ba428f5-c8a6-4aaf-81b6-501e01822b74","order_by":0,"name":"Alyssa Brookhart","email":"","orcid":"","institution":"North Carolina Wesleyan College","correspondingAuthor":false,"prefix":"","firstName":"Alyssa","middleName":"","lastName":"Brookhart","suffix":""},{"id":430084025,"identity":"54ebe3dc-652e-4ae0-b7aa-8355cede2cc0","order_by":1,"name":"Jason Doll","email":"","orcid":"","institution":"Francis Marion University","correspondingAuthor":false,"prefix":"","firstName":"Jason","middleName":"","lastName":"Doll","suffix":""},{"id":430084026,"identity":"7591d17f-bbbd-47aa-b62e-1389650793ab","order_by":2,"name":"Daniel Elias","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIie3PoQ6DMBCA4eIJnrdAVZHyIDMlTZhhfgLRme0ZeIjZ6SNNwJDVNsFQMzv2AsvKEjSHW7J+qThxfy4lxPN+EZAACEndFJwAm8yvmBO5KVHLjBB1ClR41Ox6Ue5Kle5Wk7gvuAr7Qdz63CVtcZBrSQJl0tTnQVBwSSAVItHPqanfd0G1xSamJPCSwKjBXonNI4GpFZwad4Vj/hJpYUdesYzqvR2nKl1PFvl3k2PXZ9mWZc/zvD/zAUr1URcr/kdoAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4257-6804","institution":"North Carolina Wesleyan College","correspondingAuthor":true,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Elias","suffix":""}],"badges":[],"createdAt":"2025-02-19 23:45:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6067329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6067329/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11756-025-02005-3","type":"published","date":"2025-07-23T15:56:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79257295,"identity":"981a3f94-c644-4cdc-8ed1-6ca03ba1c586","added_by":"auto","created_at":"2025-03-26 09:05:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12177,"visible":true,"origin":"","legend":"\u003cp\u003eA single \u003cem\u003ePoecilia reticulata\u003c/em\u003e was transferred to a petri dish containing sufficient water to ensure animal welfare. A ruler was placed beneath the dish for scale calibration. Images were captured and analyzed using ImageJ, with the line segment tool used to measure fish length from the head to the base of the tail after calibrating the scale with the ruler.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/5f36cded8ba836633312a670.png"},{"id":79258836,"identity":"70422f92-44c8-470e-b373-94d5b40fa14d","added_by":"auto","created_at":"2025-03-26 09:13:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13345,"visible":true,"origin":"","legend":"\u003cp\u003eNotional diagram of zones designated in the apparatus used to track fish movement: the center zone (314 cm²) and outer zone (392 cm²). ANY-Maze software was used to set zones within the uploaded video files. Fish movement parameters were analyzed according to the zone boundaries to make inferences about changes in fish behavior.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/a49caae38791a63a6412cffd.png"},{"id":79257297,"identity":"a8095eb3-8e6e-468e-a916-7767e653a932","added_by":"auto","created_at":"2025-03-26 09:05:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003e were exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Egestion rate (mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) was determined after standardizing fecal mass (dried and weighed feces) with fish length (measured with ImageJ). After 96 hours of exposure, SAL treatments induced significant changes in fish egestion rates (p = 0.006).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/1365c3fbe966aac2848db8cb.png"},{"id":79257303,"identity":"299ed7fb-c587-4c53-8024-3eaa9863d670","added_by":"auto","created_at":"2025-03-26 09:05:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003ewere exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Total distance (m) was determined using ANY-MazeTM (v4.99). After 96 hours of exposure, there was no significant difference across treatments.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/d4a089daf7b995b1b5798e44.png"},{"id":79259537,"identity":"224db49a-d228-485d-9da2-6d258bcb3454","added_by":"auto","created_at":"2025-03-26 09:21:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":64039,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003ewere exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Mean speed was determined using ANY-MazeTM (v4.99). After 96 hours of exposure, there was no significant difference across treatments.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/409ce1d282f55b874aca09bd.png"},{"id":79258840,"identity":"b5e59e50-3789-44b0-8072-7943eef16558","added_by":"auto","created_at":"2025-03-26 09:13:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":72515,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003ewere exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Outer/center zones were defined and movements across zones were determined using ANY-MazeTM (v4.99). Results indicated SAL treatments induced a significant increase in the number of movements in/out of the center zone (p = 0.0157).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/a3face34b47c870050c6aa0e.png"},{"id":79257305,"identity":"a8afe3ba-d7ea-46d1-85e8-03b4235c8b64","added_by":"auto","created_at":"2025-03-26 09:05:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":55539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003ewere exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL × ACE). Rotations were determined using ANY-MazeTM (v4.99). After 96 hours of exposure, there was no significant difference across treatments.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/3c0485bf6ab91e35c30ad7a0.png"},{"id":87756556,"identity":"b4eb5e3d-ebfe-467d-be64-f76d6ea724c7","added_by":"auto","created_at":"2025-07-28 16:01:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":966728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/5a47797b-c9d4-4fbe-9be4-ebf41a079431.pdf"},{"id":79258837,"identity":"2fd6eb56-3514-4b6d-9a51-727d222dc38c","added_by":"auto","created_at":"2025-03-26 09:13:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15403,"visible":true,"origin":"","legend":"","description":"","filename":"ResultsBIO.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6067329/v1/e77cd598b28b89a57d1f5dbb.xlsx"}],"financialInterests":"","formattedTitle":"Effects of salinity and acetaminophen on egestion rate and movement of Poecilia reticulata","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEcosystem services, including drinking water, irrigation, food, and recreation, rely on healthy freshwater environments. However, these ecosystems are increasingly threatened by human activities (Ven\u0026acirc;ncio et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Specifically, agriculture and road salt usage have altered the concentration and composition of ions in water bodies (Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meybeck \u0026amp; Helmer, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Williams, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Additionally, because of global climate change, rising sea levels allows saltwater to intrude into groundwater and coastal freshwater systems (Velasco et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ven\u0026acirc;ncio et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Shifts in environmental salinity disrupt organisms' metabolism and water balance, leading to osmotic imbalances (Velasco et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kefford et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Organisms exposed to elevated salinity may allocate energy from growth and movement into protective mechanisms to maintain homeostasis. This redirection in energy can affect fitness, altering ecosystem structure and functions such as food webs and nutrient cycling (Berger et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Velasco et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eVelasco et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that salinity alone reduced growth, increased mortality, and altered metabolic rates in various species including \u003cem\u003eBoeckella hamata\u003c/em\u003e (freshwater copepod), \u003cem\u003eCherax quadricarinatus\u003c/em\u003e (redclaw crayfish), \u003cem\u003eCrassostrea virginica\u003c/em\u003e (Eastern oyster), \u003cem\u003eDaphnia pulex\u003c/em\u003e (water flea), \u003cem\u003eGammarus roeseli\u003c/em\u003e (amphipod). Salinity can also modify pollutant toxicity, as observed in saltwater ecosystems (DeLorenzo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and influence species' sensitivity to contaminants (Velasco et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, aldicarb becomes more toxic to rainbow trout in saline water (El-Alfy \u0026amp; Schlenk, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; El-Alfy et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), while hypersaline conditions protect juvenile trout from dietary seleno-L-methionine toxicity (Schlenk et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Climate change adds complexity to the interactions between salinity and other stressors, with effects varying based on whether the interaction is additive, antagonistic, or synergistic. While interactions between salinity, temperature, and metals are well studied, the combined impact of salinity and pharmaceuticals on freshwater organisms remains underexplored (Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePharmaceuticals are detected in the environment at concentrations ranging from ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Kola \u0026amp; Landis, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Van Boeckel et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Bradley et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Their presence is associated to the increasing demand for human and veterinary drugs; further, due to incomplete removal by wastewater treatment plants, pharmaceuticals can accumulate in aquatic ecosystems (Gros et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zur et al., 2018, Adeleye et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, runoff from fields treated with animal waste fertilizer introduces pharmaceuticals into water bodies (Nikolaou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). These contaminants are commonly detected in aquatic environments worldwide (Nikolaou et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ferguson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Taylor, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ngqwala \u0026amp; Muchesa, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ort\u0026uacute;zar et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcetaminophen (paracetamol), widely used for its antipyretic and analgesic properties (Blough et al., 2011; Keaveney et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), has been detected in surface and marine waters (Guerra et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kolpin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; N\u0026ouml;dler et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). High concentrations of acetaminophen (\u0026gt;\u0026thinsp;500 \u0026micro;g/L) have been detected in hospital and wastewater treatment plant effluent (Kumar et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kosma et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pharms UBA 2021; Liu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Once in aquatic ecosystems, acetaminophen can negatively impact organisms. For example, studies have reported altered kidney tubule morphology in zebrafish (Galus et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), as well as kidney, gill, and liver damage, reduced oxygen consumption, and impaired swimming performance in rainbow trout (Choi et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, acetaminophen affects reproduction processes in invertebrates by blocking prostaglandin synthesis, gamete maturation, and spawning (Sol\u0026eacute; et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, acetaminophen toxicity is influenced by environmental conditions as indicated by Correia et al. (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) who reported different oxidative stress levels in \u003cem\u003eRuditapes philippinarum\u003c/em\u003e under varying salinity concentrations.\u003c/p\u003e \u003cp\u003eOur research evaluates the effects of individual and combined salinity and acetaminophen on \u003cem\u003ePoecilia reticulata\u003c/em\u003e movement and egestion. \u003cem\u003eP. reticulata\u003c/em\u003e is an ideal organism for laboratory studies due to its hassle-free maintenance and its use in multiple research fields including ecotoxicology (Doleželov\u0026aacute; et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), behavior (Lukas et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), gene expression (Fraser et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). We examined egestion rate and movement as indicators of fish fitness that can further influence ecological dynamics. For example, reduced egestion can limit nutrient availability (Conley et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and altered movement can impact predator avoidance and feeding behavior (Covich et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Justice and Bernot, 2017). We hypothesize that individual and combined elevated salinity and acetaminophen will decrease egestion rates and alter movement potentially through mechanisms involving oxidative stress and neurotoxicity (Almeida and Nunes \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) affecting the digestive system, as well as energetic trade off from egestion and movement to maintenance of osmotic balance.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eChemicals\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSodium chloride (NaCl, Sigma-Aldrich, CAS# 1647-14-5) was used to mimic salinization of water sources. NaCl concentrations were prepared by dissolving salt in the control medium (dechlorinated, aerated freshwater). Only fresh solutions were used for toxicity assays. Acetaminophen (Sigma-Aldrich, CAS# 103-90-2) was used to simulate pharmaceutical pollution in water sources. A stock solution was prepared and stored at 4\u0026deg;C. Aliquots of the stock solution were added to the corresponding treatment to achieve a target nominal concentration of 350 \u0026micro;g L\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperimental setup\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePoecilia reticulata\u003c/em\u003e were purchased from Carolina Biological Supply and acclimated for 14 days in a 110 L glass aquarium in aerated dechlorinated water under a 16:8 photoperiod and room temperature (22.2 \u0026deg;C). Fish were fed Aqueon\u0026reg; commercial fish flakes three times per week \u003cem\u003ead libitum.\u003c/em\u003e The mean length of \u003cem\u003eP. reticulata\u003c/em\u003e was 16.44 \u0026plusmn; 2.16 mm. Ten replicates were assigned to each treatment (N = 40): control, elevated salinity (680 mg L\u003csup\u003e-1\u003c/sup\u003e; SAL), acetaminophen (350 \u0026mu;g L\u003csup\u003e-1\u003c/sup\u003e; ACE), and combined elevated salinity and acetaminophen (SAL \u0026times; ACE). Beginning a week before experimentation, half of the organisms were acclimated to elevated salinity. Twenty fish were placed in a glass aquarium filled with 48.6 L of water. NaCl (4.86 g) was added daily for 5 days, increasing the salinity by 500 mg L\u003csup\u003e-1\u003c/sup\u003efor a total value of 680 mg L\u003csup\u003e-1\u003c/sup\u003e. After acclimatization, 10 replicate organisms were randomly selected for each treatment (SAL and SAL \u0026times; ACE). Each replicate was housed in a 300 mL glass container with the respective treatment solution. Containers were covered with aluminum foil, with small holes on the top. Environmentally relevant concentrations of acetaminophen (350 \u0026micro;g L\u003csup\u003e-1\u003c/sup\u003e) and salinity (680 mg L\u003csup\u003e-1\u003c/sup\u003e) were selected to represent concentrations detected in freshwater ecosystems where organisms are exposed to pharmaceuticals (Pharms UBA, 2021) and saltwater intrusion (Stockwell et al., 2011). Organisms were exposed to treatments for 96 hours. After 48 hours, each organism was fed 0.01 g fish flakes (Aqueon\u0026reg;). Excess food was removed after 15 minutes. A 100% water change and treatment renewals were completed immediately after feeding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was conducted in compliance with ethical guidelines for animal research and was approved by the Institutional Review Board and Institutional Animal Care and Use Committee (IRB-IACUC) at North Carolina Wesleyan University.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEgestion\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOne organism and a small volume of water were transferred to a petri dish with a ruler underneath (Fig. 1). Images were taken with an Apple iPhone 6S placed ~30 cm above and parallel to the petri dish and uploaded to ImageJ software for length measurements. The line segment tool was used to measure the length of each fish, from the head to the base of the tail. Feces collected after 96-hour exposure were transferred to pre-weighed tin foil and dried in a convection oven (Quincy Lab 10GC) at 60 \u0026deg;C for 24 hours. After drying, weight difference was measured using a precision scale (Ohaus Pioneer\u0026reg;). Egestion rate was calculated as the weight of feces and standardized with fish length over the exposure time (mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMovement\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIndividual fish were placed in a white plastic bucket (20 L, 30 cm diameter) filled with 3 L of water that matched the respective treatments (freshwater or saltwater). An Apple iPhone 6S was placed above the bucket to record fish movement. Fish were given one minute to acclimatize, and their movement was recorded for analyses for the next one minute. Video files were used to measure fish movement using ANY-MazeTM (v4.99). Two zones were designated in the apparatus: center zone (314 cm\u0026sup2;) and outer zone (392 cm\u0026sup2;; Fig. 2). Four movement variables were selected to measure changes in fish movement: total distance traveled, mean speed, number of rotations, and number of movements in/out of the center zone (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData Analysis \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEgestion and continuous movement variables (total distance traveled and mean speed) were assessed using a linear regression model with categorical predictors of treatment. Normality of egestion and continuous movement variables were assessed using the Shapiro-Wilks Test. Homogeneity of variance was checked using Bartlett\u0026apos;s Test. Count movement variables (number of rotations and movement in and out of center) were analyzed using generalized linear models (GLMs) with the Poisson error structure or negative binomial error structure if data were over dispersed. Statistical analyses were conducted in R v4.2.1 (R Core Team 2022), with alpha set to 0.05 for all tests. Linear regressions were performed using the lm() function, generalized linear models were performed using the glm() function, and pairwise comparisons of all models were performed using the emmeans() function in the emmeans v1.10.4 (Length 2024).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eControl treatment water parameters were salinity (180 mg/L), conductivity (220 \u0026micro;S/cm), pH (7.28), and total dissolved solids (TDS, 156 ppm). Elevated salinity treatment water parameters were salinity (680 mg/L), conductivity (1370 \u0026micro;S/cm), pH (7.3), and TDS (891 ppm).\u003c/p\u003e\n\u003cp\u003eEgestion rates followed a normal distribution across all treatments (p \u0026gt; 0.05), and Bartlett\u0026apos;s test confirmed equal variance (K\u0026sup2; = 4.35, df = 3, \u003cem\u003ep\u003c/em\u003e = 0.226). Egestion rates ranged from 0.0002 to 0.0029\u0026nbsp;mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e. Mean egestion rates for SAL (0.0019 \u0026plusmn; 0.0005\u0026nbsp;mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) and SAL \u0026times; ACE (0.0020 \u0026plusmn; 0.0008\u0026nbsp;mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) were nearly double those of control (0.0012 \u0026plusmn; 0.0004\u0026nbsp;mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e) and ACE (0.0010 \u0026plusmn; 0.0005\u0026nbsp;mg cm\u003csup\u003e-1\u003c/sup\u003e h\u003csup\u003e-1\u003c/sup\u003e). Egestion was significantly different (Fig. 3) between the control and SAL (t = -4.838, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) as well as between control and SAL \u0026times; ACE (t = -3.443, \u003cem\u003ep\u003c/em\u003e = 0.008). Significant differences were also found between SAL and ACE (t = 3.399, \u003cem\u003ep\u003c/em\u003e = 0.009) and between ACE and SAL \u0026times; ACE (t = -4.838, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eTotal distance travel ranged from 0 m (immobile) to 2.8 m across treatments. Control (1.46 \u0026plusmn; 0.92 m) and SAL \u0026times; ACE (1.51 \u0026plusmn; 0.61 m) traveled similar distances. SAL exhibited the highest mean distance (1.76 \u0026plusmn; 0.43 m), while ACE had the lowest (1.15 \u0026plusmn; 0.65 m). Total distance traveled was normally distributed across treatments (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) with equal variance (K\u0026sup2; = 3.29, df = 3,\u003cem\u003e\u0026nbsp;p\u003c/em\u003e = 0.350).\u003c/p\u003e\n\u003cp\u003eMean speed ranged from 0 m/s (immobile) to 0.047 m/s. Control (0.024 \u0026plusmn; 0.020 m/s) and SAL \u0026times; ACE (0.025 \u0026plusmn; 0.010 m/s) had similar speeds. SAL had the highest speed (0.029 \u0026plusmn; 0.010 m/s), while ACE had the lowest (0.019 \u0026plusmn; 0.010 m/s). Mean speed was normally distributed across all treatments (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05), with equal variance across treatments (K\u0026sup2; = 3.35, df = 3, \u003cem\u003ep\u003c/em\u003e = 0.342). No significant differences were observed in total distance or speed across treatments (Fig. 4 - 5).\u003c/p\u003e\n\u003cp\u003eFor count-based movement parameters, normality was not required. Movements in and out of the center zone were over-dispersed (z = 3.082, \u003cem\u003ep\u003c/em\u003e = 0.001), so a Negative Binomial GLM was applied. Movements ranged from 0 to 12. Elevated salinity treatments showed higher movements (SAL: 6.5 \u0026plusmn; 3.17; SAL \u0026times; ACE: 4.7 \u0026plusmn; 2.36) compared to control (2.6 \u0026plusmn; 2.88) and ACE (3.6 \u0026plusmn; 3.53). However, there were no statistically significant differences in movement in and out of the center zone across treatments (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eRotations were not over-dispersed (z = -3.409, \u003cem\u003ep\u003c/em\u003e = 0.999) and were analyzed using a Poisson GLM. Rotations ranged from 0 to 4. Control and SAL \u0026times; ACE recorded 2.4 \u0026plusmn; 1.43 and 2.4 \u0026plusmn; 1.07 rotations, respectively, while SAL had the highest count (3.0 \u0026plusmn; 0.94) and ACE the lowest (1.9 \u0026plusmn; 1.29). No statistically significant differences in the number of rotations were found across treatments (p \u0026gt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eEgestion\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEgestion rates were significantly higher in fish exposed to SAL compared to control (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and ACE (\u003cem\u003ep\u003c/em\u003e = 0.009), as well as in SAL \u0026times; ACE compared to control (\u003cem\u003ep\u003c/em\u003e = 0.008) and ACE (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). These results suggest that elevated salinity influences digestive function, potentially through increased ion uptake and impairment of the enteric nervous system (Ern et al., 2014).\u003c/p\u003e\n\u003cp\u003eUnder elevated salinity, fish may allocate more energy toward maintaining osmotic balance, either by excreting excess ions or adapting to increased internal salinity (Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al., 2013; Hallali et al., 2018; Ven\u0026acirc;ncio et al., 2019). Higher sodium chloride concentrations in digestive tissues can stimulate digestion and egestion, as observed in \u003cem\u003eGambusia affinis\u003c/em\u003e (mosquitofish), where metabolism and nitrogen excretion changed under acute salinity exposure (Uliano et al., 2010).\u003c/p\u003e\n\u003cp\u003eAlthough all fish in our study were fed the same amount of food, variations in ingestion were observed. Increased energy demands for osmotic regulation may lead to higher food intake, potentially explaining differences in egestion. Similar mechanisms have been reported in other species, where energy consumption varies with salinity, including \u003cem\u003eIctalurus punctatus\u003c/em\u003e and \u003cem\u003eCarassius auratus\u003c/em\u003e (Altinok and Grizzle, 2004), \u003cem\u003eAllenbatrachus grunniens\u003c/em\u003e (Walsh et al., 2004), \u003cem\u003eGambusia affinis\u003c/em\u003e and \u003cem\u003eDanio rerio\u003c/em\u003e (Uliano et al., 2010), \u003cem\u003ePerca fluviatilis\u003c/em\u003e (Ern et al., 2014), \u003cem\u003eOreochromis mossambicus\u003c/em\u003e, \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e, and \u003cem\u003eAnguilla anguilla\u003c/em\u003e (K\u0026uuml;ltz et al., 2015), \u003cem\u003eO. niloticus\u003c/em\u003e (Hallali et al., 2018), and \u003cem\u003eC. auratus\u003c/em\u003e, \u003cem\u003eClarias gariepinus\u003c/em\u003e, and \u003cem\u003eO. niloticus\u003c/em\u003e (Lee et al., 2022). However, these responses are species-dependent, and further research is needed to determine the specific effects of elevated salinity on ingestion and egestion in \u003cem\u003ePoecilia reticulata\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMovement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFish exposed to SAL and SAL \u0026times; ACE showed higher movement counts in and out of the center zone compared to control and ACE, but these differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Greater movement across zones could indicate increased exploratory behavior or an attempt to avoid unfavorable conditions, including predators. While Ca\u0026ntilde;edo-Arg\u0026uuml;elles et al. (2013) reported that increased salinity triggered movement associated with predator avoidance, our study did not find a statistically significant effect. Mean speed, movement frequency, and rotations also appeared slightly higher under SAL and SAL \u0026times; ACE, but these differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eIn contrast, fish exposed to ACE exhibited reduced movement compared to SAL and control treatments, with lower rotations and movement counts, although these differences were not statistically significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05). Reduced movement under ACE exposure may be linked to physiological stress associated with acetaminophen metabolism. Acetaminophen exposure has been linked to oxidative stress (Letelier et al., 2011; Correia et al., 2016), which can impair neuromuscular function and reduce overall activity. As acetaminophen is metabolized, it produces N-acetyl-p-quinone-imine (NAPQI), a reactive intermediate that is detoxified by glutathione-S-transferase (GST). At high concentrations, GST becomes saturated, leading to NAPQI accumulation, oxidative stress, and depletion of cellular energy reserves (Letelier et al., 2011; Vermot et al., 2021). This oxidative imbalance, along with increased reactive oxygen species, can damage cellular structures, exacerbate lipid peroxidation, and impair ATP production (Antunes et al., 2013; Correia et al., 2016).\u003c/p\u003e\n\u003cp\u003eWhile oxidative stress is a known effect of acetaminophen exposure (Letelier et al., 2011; Correia et al., 2016), our study did not find statistically significant reductions in movement parameters, suggesting that the impact of acetaminophen on fish behavior may vary depending on concentration, exposure duration, and species sensitivity. Acetaminophen exposure has been associated with behavioral and physiological changes in other species, including decreased swimming speeds in \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e (rainbow trout; Choi et al., 2018), increased lipid peroxidation in \u003cem\u003eMytilus galloprovincialis\u003c/em\u003e (marine mussels; Sol\u0026eacute; et al., 2010), and reduced heartbeat rates in \u003cem\u003eClarias gariepinus\u003c/em\u003e (African catfish larvae; Erhunmwunse et al., 2021). However, lack of statistical significance in our study suggests that movement responses are species-specific that vary with exposure conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSalinity and acetaminophen influenced egestion and movement in \u003cem\u003eP. reticulata\u003c/em\u003e. Elevated salinity significantly increased egestion, likely due to overstimulation of the enteric nervous system via enhanced digestion and corresponding egestion. Although movement was higher under salinity, differences were not statistically significant, suggesting potential species specific responses. We observed lower movement with acetaminophen exposure, however this effect was not statistically significant. This may be a response to oxidative stress, changes in energy allocation, or neuromuscular disruption as a result of acetaminophen metabolism.\u003c/p\u003e \u003cp\u003eOverall, environmental stressors that affect adaptability to changing conditions can reduce species survival and disrupt ecosystem dynamics. Our findings highlight the need to examine the combined effects of pharmaceuticals and abiotic stressors (e.g., salinity, pH, temperature) across varying exposure durations. Thus, our research can contribute to more accurate ecological risk assessments and mitigation strategies for freshwater ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e No funding was received for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e Ethical approval was obtained from the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) at North Carolina Wesleyan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent:\u003c/strong\u003e Not applicable, as this study did not involve human participants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e All authors contributed equally to the research design, data collection, analysis, and manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data supporting this study is available in the supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdeleye AS, Xue J, Zhao Y, Taylor AA, Zenobio JE, Sun Y, Zhu Y (2022) Abundance, fate, and effects of pharmaceuticals and personal care products in aquatic environments. \u003cem\u003eJ Hazard Mater\u003c/em\u003e 424:127284. https://doi.org/10.1016/j.jhazmat.2021.127284\u003c/li\u003e\n\u003cli\u003eAlmeida F, Nunes B (2019) Effects of acetaminophen in oxidative stress and neurotoxicity biomarkers of the gastropod \u003cem\u003ePhorcus lineatus\u003c/em\u003e. \u003cem\u003eEnviron Sci Pollut Res\u003c/em\u003e 26:9823-9831. https://doi.org/10.1007/s11356-019-04349-1 \u003c/li\u003e\n\u003cli\u003eAltinok I, Grizzle JM (2004) Excretion of ammonia and urea by phylogenetically diverse fish species in low salinities. \u003cem\u003eAquaculture\u003c/em\u003e 238(1-4):499-507. https://doi.org/10.1016/j.aquaculture.2004.06.020\u003c/li\u003e\n\u003cli\u003eAntunes SC, Freitas R, Figueira E, Gon\u0026ccedil;alves F, Nunes B (2013) Biochemical effects of acetaminophen in aquatic species: edible clams \u003cem\u003eVenerupis decussata\u003c/em\u003e and \u003cem\u003eVenerupis philippinarum\u003c/em\u003e. \u003cem\u003eEnviron Sci Pollut Res\u003c/em\u003e 20:6658-6666. https://doi.org/10.1007/s11356-013-1784-9 \u003c/li\u003e\n\u003cli\u003eBerger E, Fr\u0026ouml;r O, Sch\u0026auml;fer RB (2019) Salinity impacts on river ecosystem processes: a critical mini-review. \u003cem\u003ePhilos Trans R Soc B\u003c/em\u003e 374(1764):20180010. https://doi.org/10.1098/rstb.2018.0010\u003c/li\u003e\n\u003cli\u003eBlough ER, Wu M (2011) Acetaminophen: beyond pain and fever-relieving. \u003cem\u003eFront Pharmacol\u003c/em\u003e 2:72. https://doi.org/10.3389/fphar.2011.00072 \u003c/li\u003e\n\u003cli\u003eBradley PM, Journey CA, Button DT, Carlisle DM, Huffman BJ, Qi SL, Van Metre PC (2020) Multi-region assessment of pharmaceutical exposures and predicted effects in USA wadeable urban-gradient streams. \u003cem\u003ePLoS One\u003c/em\u003e 15(1):e0228214. https://doi.org/10.1371/journal.pone.0228214\u003c/li\u003e\n\u003cli\u003eCa\u0026ntilde;edo-Arg\u0026uuml;elles M, Kefford BJ, Piscart C, Prat N, Sch\u0026auml;fer RB, Schulz CJ (2013) Salinisation of rivers: an urgent ecological issue. \u003cem\u003eEnviron Pollut\u003c/em\u003e 173:157-167. https://doi.org/10.1016/j.envpol.2012.10.011\u003c/li\u003e\n\u003cli\u003eCa\u0026ntilde;edo-Arg\u0026uuml;elles M, Kefford B, Sch\u0026auml;fer R (2019) Salt in freshwaters: causes, effects and prospects\u0026mdash;introduction to the theme issue. \u003cem\u003ePhilos Trans R Soc B\u003c/em\u003e 374(1764):20180002. https://doi.org/10.1098/rstb.2018.0002\u003c/li\u003e\n\u003cli\u003eChoi E, Alsop D, Wilson JY (2018) The effects of chronic acetaminophen exposure on the kidney, gill, and liver in rainbow trout (\u003cem\u003eOncorhynchus mykiss\u003c/em\u003e). \u003cem\u003eAquat Toxicol\u003c/em\u003e 198:20-29. https://doi.org/10.1016/j.aquatox.2018.02.007\u003c/li\u003e\n\u003cli\u003eConley DJ, Paerl HW, Howarth RW, Boesch DF, Seitzinger SP, Havens KE, Likens GE (2009) Controlling eutrophication: nitrogen and phosphorus. \u003cem\u003eScience\u003c/em\u003e 323(5917):1014-1015. 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Antioxidants 10(6):890. https://doi.org/10.3390/antiox10060890\u003c/li\u003e\n\u003cli\u003eWalsh PJ, Wei Z, Wood CM, Loong AM, Hiong KC, Lee SML, Ip YK (2004) Nitrogen metabolism and excretion in Allenbatrachus grunniens (L): effects of variable salinity, confinement, high pH and ammonia loading. J Fish Biol 65(5):1392-1411. https://doi.org/10.1111/j.0022-1112.2004.00538.x\u003c/li\u003e\n\u003cli\u003eWilliams WD (2001) Salinization: unplumbed salt in a parched landscape. Water Sci Technol 43(4):85-91. https://doi.org/10.2166/wst.2001.0186 \u003c/li\u003e\n\u003cli\u003eŻur J, Piński A, Marchlewicz A, Hupert-Kocurek K, Wojcieszyńska D, Guzik U (2018) Organic micropollutants paracetamol and ibuprofen\u0026mdash;toxicity, biodegradation, and genetic background of their utilization by bacteria. Environ Sci Pollut Res 25:21498-21524. https://doi.org/10.1007/s11356-018-2517-x \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. List of names and descriptions of movement parameters selected for analysis. Acronyms and zones were defined for the purposes of this experiment. Descriptions were retrieved from the complete list of ANY-maze measures (ANY-maze, 2023).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"617\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 319px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasurement\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eTotal Distance Traveled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 319px;\"\u003e\n \u003cp\u003eReports the total distance that the animal traveled during the test.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003emeters, m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eMean Speed\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 319px;\"\u003e\n \u003cp\u003eReports the mean speed of the animal during a test.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003emeters per second, m/s\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eRotations\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 319px;\"\u003e\n \u003cp\u003eReports the number of times the animal\u0026apos;s body completed an entire rotation of 360\u0026deg;.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003ecount\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 173px;\"\u003e\n \u003cp\u003eNumber of Entries and exits to the zone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 319px;\"\u003e\n \u003cp\u003eCounts the number of times the animal entered and exit the zone.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003ecount\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. \u003cem\u003ePoecilia reticulata\u003c/em\u003e were exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL \u0026times; ACE). Egestion rate (mg/cm/h) was determined after standardizing fecal mass (dried and weighed feces) with fish length (measured with ImageJ). Standard deviation between parentheses.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"294\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 174px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Egestion Rate (mg/cm/h)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 174px;\"\u003e\n \u003cp\u003e0.0012 (0.0004)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eACE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 174px;\"\u003e\n \u003cp\u003e0.0010 (0.0005)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eSAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 174px;\"\u003e\n \u003cp\u003e0.0019 (0.0005)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 120px;\"\u003e\n \u003cp\u003eSAL x ACE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 174px;\"\u003e\n \u003cp\u003e0.0020 (0.0008)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. \u003cem\u003ePoecilia reticulata\u003c/em\u003e were exposed to varying conditions for 96 hours: control, elevated salinity only (SAL), acetaminophen only (ACE), and combined elevated salinity and acetaminophen (SAL \u0026times; ACE; n = 10 each). Each replicate was placed in an isolated apparatus to record fish movement for two minutes. Video files were uploaded to a laptop, where the outer/center zones were defined, and movement parameters were determined using ANY-MazeTM (v4.99). Standard deviation between parentheses.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"611\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean distance Traveled (m)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 93px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean speed (m/s)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 184px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Movements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ein/out of Center Zone (#)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 113px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean Rotations (#)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e1.46 (0.92)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0.02 (0.02)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003e2.6 (2.88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.4 (1.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003eACE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e1.15 (0.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0.02 (0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003e3.6 (3.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e1.9 (1.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003eSAL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e1.76 (0.43)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0.03 (0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003e6.5 (3.17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e3.0 (0.94)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 86px;\"\u003e\n \u003cp\u003eSAL \u0026times; ACE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 135px;\"\u003e\n \u003cp\u003e1.51 (0.61)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e0.03 (0.01)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 184px;\"\u003e\n \u003cp\u003e4.7 (2.36)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 113px;\"\u003e\n \u003cp\u003e2.4 (1.07)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"saltwater intrusion, pharmaceuticals, salinity, acetaminophen, freshwater ecosystems, Poecilia reticulata","lastPublishedDoi":"10.21203/rs.3.rs-6067329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6067329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHigh salinity, from agricultural activities, road salt runoff, and climate change, is a problem affecting freshwater ecosystems. Elevated salinity can decrease organismal survival and growth and can alter the toxicity of pollutants. Pharmaceuticals, such as acetaminophen, enter freshwaters through non-point sources and from hospital and wastewater treatment plants effluent. Available research is limited on the combined effects of elevated salinity and acetaminophen on freshwater organisms. Our study evaluated the effects of acetaminophen (350 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and salinity (680 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) on \u003cem\u003ePoecilia reticulata\u003c/em\u003e over 96 hours. Egestion rate (mg cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and movement parameters were measured following exposure. We observed significant changes in egestion rate under salinity likely due to impaired osmoregulatory mechanisms and no effect on movement. Further, there was no interaction between salinity and acetaminophen; thus, salinity did not affect acetaminophen toxicity at the concentrations tested. Our findings highlight the need of establishing threshold of salinity and pharmaceuticals to protect freshwater ecosystems and to help predict ecological impacts on aquatic organisms.\u003c/p\u003e","manuscriptTitle":"Effects of salinity and acetaminophen on egestion rate and movement of Poecilia reticulata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 09:04:55","doi":"10.21203/rs.3.rs-6067329/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-06-23T10:27:41+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-17T20:25:30+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-17T18:18:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-21T12:35:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biologia","date":"2025-02-20T09:02:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biologia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biol","sideBox":"Learn more about [Biologia](http://link.springer.com/journal/11756)","snPcode":"11756","submissionUrl":"https://www.editorialmanager.com/biol/default2.aspx","title":"Biologia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"40e1eb17-f580-4924-9f03-8a99605872c2","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T15:58:41+00:00","versionOfRecord":{"articleIdentity":"rs-6067329","link":"https://doi.org/10.1007/s11756-025-02005-3","journal":{"identity":"biologia","isVorOnly":false,"title":"Biologia"},"publishedOn":"2025-07-23 15:56:53","publishedOnDateReadable":"July 23rd, 2025"},"versionCreatedAt":"2025-03-26 09:04:55","video":"","vorDoi":"10.1007/s11756-025-02005-3","vorDoiUrl":"https://doi.org/10.1007/s11756-025-02005-3","workflowStages":[]},"version":"v1","identity":"rs-6067329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6067329","identity":"rs-6067329","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}