The Impact of Salinity on Copper-Induced Toxicity in Palaemon spp.: Effects on Survival, Morphological Deformities, and Toxicity Indicators

The Impact of Salinity on Copper-Induced Toxicity in Palaemon spp.: Effects on Survival, Morphological Deformities, and Toxicity Indicators | 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 The Impact of Salinity on Copper-Induced Toxicity in Palaemon spp.: Effects on Survival, Morphological Deformities, and Toxicity Indicators Charrel A. Williams, Murty S. Kambhampati, Rayan Demery This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5672506/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 6 You are reading this latest preprint version Abstract Heavy metal contamination, resulting from pollution, presents serious threats to aquatic species and has far-reaching consequences for ecosystem health. This study investigates the acute toxicity of copper (Cu) on grass shrimp ( Palaemon spp. ), a key species in North American estuaries. We exposed shrimp to a range of copper concentrations (0, 0.25, 0.50, 1.0, and 2.0 ppm) and salinities (1, 5, 10, and 20 ppt) over periods of 3, 6, 9, 12, 24, 48, 72, and 96 hours. We hypothesized that exposure to 1.0 ppm Cu and 10 ppt salinity would reduce copper toxicity; but contrary to our expectations, optimal shrimp survival occurred at 20 ppt salinity and 0.25 ppm Cu. Copper solutions were prepared using CuSO₄·5H₂O, and toxicity was monitored using indicators such as mortality, abdominal curvature, discoloration, and mobility. Water quality remained stable throughout the study, with dissolved oxygen consistently at 8.675 ± 0.187 ppm, pH at 6.88 ± 0.088, and temperature at 28.75 ± 0.244ºC. Copper toxicity increased at lower salinities, with the highest mortality and quickest onset observed at 1 ppt. Mortality was lowest at 0.25 ppm Cu, while 2.0 ppm Cu induced the highest mortality across all salinities, supporting a dose-response relationship. LC 50 values increased with salinity, with the highest survival rates occurring at 20 ppt. These findings highlight the protective role of higher salinity in mitigating copper toxicity, emphasizing the need for further research on the long-term ecological consequences of copper contamination in estuarine ecosystems. Toxicology Salinity Morphological deformities LC50 Metal bioavailability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Aquatic pollution is primarily driven by contaminants released from industrial, agricultural, and urban stressors. These pollutants enter water systems through processes such as chemical leaching, soil erosion, and direct discharge of industrial effluents [ 1 ]. Both intentional and unintentional human activities exacerbate this issue, introducing a variety of toxic substances such as heavy metals, chemicals, and industrial byproducts into aquatic environments [ 1 ]. In the United States alone, approximately 190 million pounds of metallic ions are released into water sources annually [ 2 ]. This accumulation of pollutants disrupts aquatic habitats, threatens biodiversity, and undermines ecosystem stability. Like other non-biodegradable heavy metals, such as mercury (Hg), zinc (Zn), and cadmium (Cd), copper (Cu) plays a crucial role in metabolic functions. These metals typically enter organisms through contaminated food, water, or air, leading to potentially harmful bioaccumulation [ 3 , 4 ]. Furthermore, pollution sources-waste dumps, mines, landfills, and sewage outlets- significantly contribute to environmental contamination, posing risks to ecosystems [ 4 , 5 ]. While copper is essential in trace amounts for enzyme catalysis and ATP production, excessive accumulation disrupts cellular processes by binding to proteins and nucleic acids [ 6 , 7 ]. When metal concentrations surpass an organism's tolerance levels, they interfere with critical biological functions, jeopardizing both individual health and overall ecosystem stability [ 6 , 7 ]. Bioaccumulation occurs when an organism's uptake of a substance exceeds its capacity to excrete it, resulting in profound physiological disruptions. Salinity is a key determinant in modulating the toxicity of copper, in brackish water species. As seen in the blue crab ( Callinectes sapidus ), where varying salinity levels influence enzyme activity and ammonia excretion [ 8 ]. Similarly, research on Fundulus heteroclitus showed that the highest sensitivity to copper (Cu) occurred at salinities between 0.018 ppt and 0.294 ppt. While tolerance increased at intermediate salinities around 10 ppt, a pattern linked to differences in osmoregulatory physiology [ 6 ]. To further elucidate these effects, acute toxicity trials are commonly employed to assess visible physiological and morphological alterations [ 9 ]. Copper's ability to shift between its oxidation states (most common being Cu⁺ and Cu²⁺) plays a significant role in facilitating redox reactions. These reactions can lead to the formation of free radicals, which are highly reactive molecules with unpaired electrons. The generation of free radicals can cause oxidative damage to cellular components, such as lipids, proteins, and DNA. For instance, copper exposure has been shown to induce noticeable changes, including increased redness and discoloration in shrimp [ 10 ], as well as the development of external lesions in fish species [ 11 ]. By inducing oxidative stress, normal cellular function is disrupted due to there being an imbalance between free radicals and antioxidants within a cell [ 12 ]. Elevated copper concentrations in aquatic environments disrupt ion regulation, particularly by affecting chloride cells in fish gills, which are crucial for maintaining ionic balance [7,13,14,]. Furthermore, copper concentrations in wastewater have been reported to range from approximately 2.5 mg/L to 10,000 mg/L. Indicating that in areas where runoff occurs, concentrations can become significantly elevated upsetting a habitat’s biota [ 5 ]. As it accumulates within the aquatic organism’s tissues, it interferes with processes like energy production, reproduction, and survival. Grass shrimp, the Palaemon spp. , are ideal model organisms for studying copper toxicity in aquatic environments [ 15 ]. Native to estuaries of the Northern America, these euryhaline shrimp are highly adaptable with the ability to tolerate a broad range of salinity conditions from freshwater to hypersaline [ 16 , 17 ]. This tolerance makes them particularly valuable for assessing how variations in water chemistry, such as salinity changes and copper concentrations, affect organism health [ 18 ]. Studies by Manyin and Rowe [ 13 , 19 ] have demonstrated that Palaemon species are highly sensitive to copper toxicity. Exposure results in metabolic disruptions, inhibited growth, and impaired physiological functions, reinforcing their effectiveness as bioindicators of copper contamination in aquatic ecosystems. Given their role in aquatic food webs and sensitivity to metal pollution, this shrimp provide reliable insights into the broader toxicological impacts of copper exposure [ 20 ]. To evaluate the specific effects of copper exposure on grass shrimp, the lethal concentration (LC 50 ) of copper was measured in this study. This measure is crucial for understanding the susceptibility of aquatic species to copper and the potential risks posed by elevated copper concentrations. The study examined copper toxicity across varying concentrations (0–2 ppm) and salinities (1–20 ppt). The findings will enhance our understanding of how environmental factors, such as salinity, interact with metal pollution. Further contributing to our knowledge of metal toxicity mechanisms in aquatic ecosystems. This research is critical for informing conservation efforts and environmental policies aimed at protecting aquatic life from the detrimental effects of pollutants. Materials and Methods Saltwater solutions at concentrations of 1, 5, 10, and 20 ppt were prepared, with 10 liters of each solution placed in 20-gallon glass aquaria tanks and aerated for 7–10 days to stabilize the media. Water quality parameters—dissolved oxygen (DO), pH, and temperature—were monitored weekly and maintained at stable conditions. Temperature was measured using a thermometer for one-minute intervals, pH was assessed using pH strips, and dissolved oxygen was measured with a LaMotte water test kit, with values averaged from three measurements per aquarium. Thirty 5x5-inch fish bowls were cleaned using a 10 mL solution of < 1% hydrochloric acid, rinsed three times with distilled water, and air-dried upside down for 48 hours. Copper sulfate (CuSO₄·5H₂O) was used to prepare copper concentrations of 0.25, 0.5, 1.0, and 2.0 ppm. The control group, 0 ppm, contains no added copper. These concentrations were chosen to reflect environmental copper contamination levels and to assess varying toxicity thresholds. Approximately 330 mL of each copper-saline solution was added to each bowl. Grass shrimp (2.0–2.5 cm in length) were obtained from a local aquarium supplier, initially kept in a 1 ppt saline medium, and were morphologically consistent with members of the genus Palaemon . However, species-level identification was not conducted. We recognize that different Palaemon species may exhibit varying salinity tolerances which could influence copper sensitivity. The 1 ppt saline medium was treated as control solution for salinity in experimental trials. Prior to experimentation, the shrimp were acclimated overnight in the specific salinity conditions (1 ppt, 5 ppt, 10 ppt, or 20 ppt) of the testing aquaria tanks. This ensured proper adaption to each subject’s respective salinity level before exposure to the copper treatments. The following morning, four shrimp were placed in each bowl, in triplicates for each concentration (12 shrimp total per ppt/ppm treatment). Shrimp were monitored for LC 50 , behavioral changes, and morphological deformities at 0, 3, 6, 9, 12, 24, 48, 72, and 96 hours. A 1 mm mesh was placed over each bowl to prevent shrimp from potentially escaping. This procedure was repeated for both experimental trials: the first trial involved 1 ppt and 5 ppt salinities, and the second trial involved 10 ppt and 20 ppt salinities. The relationship between copper concentrations and LC 50 across salinities was analyzed using Two-way Analysis of Variance (ANOVA) to assess the significance of salinity as a determinant for toxicity. Results and Discussion Physio-chemical factors of water (media): Elevated salinity reduces the bioavailability of copper by enhancing ionic competition. Higher concentrations of sodium (Na⁺) and chloride (Cl⁻) ions compete with copper ions (Cu²⁺) for binding sites on cell membranes, thus limiting copper uptake [21]. Furthermore, chloride ions promote the formation of copper-chloride complexes, which are less bioavailable than free copper ions [22]. As a result, the toxicity of copper is mitigated in high salinity environments, as the concentration of bioavailable copper ions decreases, reducing their harmful effects on aquatic organisms [16, 21, 23]. Our findings align with previous studies [14, 24] which suggest that copper toxicity is closely linked to the osmoregulatory capacity of aquatic organisms. This osmoregulatory ability plays a crucial role in maintaining internal solute balance and preventing adverse physiological effects. Grass shrimp are eurythermal organisms, capable of surviving in temperatures ranging from 5°C to 38°C (41°F to 101°F), with an optimal range between 18°C and 25°C (65°F to 77°F) [16, 25]. They have been observed in waters with dissolved oxygen concentrations between 6 and 11 ppm [16, 25]. Throughout the study, water quality parameters remained stable, with dissolved oxygen averaging 8.675 ± 0.187 ppm, pH at 6.88 ± 0.088, and temperature at 28.75 ± 0.244ºC; conditions within the range suitable for Palaemon spp . Morphological Deformities: As salinity increased, subjects were noticeably more active. Those in the 20 ppt salinity medium displayed fewer morphological deformities, such as discoloration, reduced mobility, and a lack of response to external stimuli (gentle agitation of the bowl) than other media. This trend was also consistent across all copper concentrations. Subjects in higher Cu concentrations (1.0 ppm and 2.0 ppm) displayed a redder hue at the time of death, than those in lower concentrations (0.25 ppm and 0.50 ppm). Copper exposure in Litopenaeus vannamei led to significant declines in growth, immune function, and gut microbiota composition, with higher concentrations causing hepatopancreatic damage and decreased hemocyte counts [26]. At concentrations of 1 mg Cu²⁺ L⁻¹, copper also disrupted antioxidant enzyme activities and altered the diversity of intestinal microbes, highlighting its toxic effects on the shrimp's overall health [26]. Similarly, exposure to copper sulphate in Macrobrachium lamarrei caused severe behavioral disruptions, including equilibrium loss, excessive mucus secretion, and increased mortality [27]. LC 50 and Rates of Mortality: At 96 hours, replicates at 1 ppt exhibited more rapid rates of mortality at higher copper concentrations compared to those at 5 ppt, 10 ppt, and 20 ppt salinities. At 20 ppt, mortality rates were lower than at the other salinity levels with LC 50 values increasing as salinity decreased (mortality rate: 20 ppt < 10 ppt < 5 ppt < 1 ppt). Copper toxicity increased as salinity decreased, with higher salinity levels reducing the metal’s potency. At 2.0 ppm Cu, the LC 50 threshold was reached within 48 hours for all salinity replicates as shown in Figure 5. However, at 0.25 ppm Cu, mortality rates were lower at 96 hours compared to higher copper concentrations (mortality rate of Cu: 0.25 ppm < 0.50 ppm < 1.0 ppm < 2.0 ppm). Among all salinity levels, higher copper concentrations were associated with higher mortality rates, with 2.0 ppm being the most toxic. After 96 hours, subjects exposed to 0.25 ppm Cu had the lowest mortality rates (see Figure 2). Mortality increased more rapidly for those exposed to 1.0 and 2.0 ppm Cu compared to those exposed to 0.25 and 0.50 ppm Cu. In most replicates at all salinity levels, 1.0 ppm and 2.0 ppm Cu exceeded the LC 50 threshold (see Figures 4 and 5). Statistical Analysis Table 1. Two-way ANOVA Test Results: P-value vs. F-Critical Value Exposure Time Interval (Hours) Interactions Between Salinity and Copper Concentrations P-value F-Critical Value 0 0** 2.003459 3 0.000000524** 2.003459 6 0.000053** 2.003459 9 0.00000131** 2.003459 12 0.000000303** 2.003459 24 0.012655** 2.003459 48 0.166288 2.003459 72 0.193488 2.003459 96 0.103501 2.003459 Table 1: The p-values for each exposure time along with the corresponding F-critical value are shown. The F-critical value remains constant at 2.003459 for each exposure time, and the p-values indicate whether the null hypothesis should be rejected or not based on the threshold of 𝛼=0.05. Exposure times of 0, 3, 6, 9, 12, and 24 hours (**) exhibit significant interactions between salinity and copper, whereas 48, 72, and 96 hours do not. The null hypothesis is rejected for exposure times of 3, 6, 9, 12, and 24 hours (p-values < α = 0.05), suggesting that exposure time significantly influences shrimp survival. This implies that the effect of copper on shrimp survival varies with exposure time. In contrast, for 48, 72, and 96 hours (p-values > α = 0.05), the null hypothesis is not rejected, indicating no significant impact on survival at these longer exposure times. Conclusion This study emphasizes the significant role of salinity in modulating copper (Cu) toxicity in grass shrimp, with higher salinity levels (10 ppt and 20 ppt) reducing copper bioavailability, improving shrimp activity, reducing morphological deformities, and lowering mortality rates. However, contrary to our initial hypothesis, shrimp exposed to 1.0 ppm Cu at 10 ppt salinity did not show the anticipated improvements in health. Future research should aim to better understand the mechanisms driving copper absorption and tissue accumulation in varying salinity conditions. By utilizing advanced techniques such as Inductively Coupled Plasma (ICP) Spectrometry to quantify copper concentrations in shrimp tissues, a deeper insight can be had into the bioaccumulation processes and toxicity thresholds. Additionally, investigating the long-term effects of copper exposure at different salinities on reproductive health, deformities, growth, and overall population dynamics will be crucial to assessing the ecological risks posed by copper in marine environments. This comprehensive approach will help refine our understanding of copper toxicity in marine organisms and contribute to the development of more effective water quality management strategies. Declarations Acknowledgements and Funding We greatly appreciate the summer internships provided by the US Department of Education - Minority Science and Engineering Improvement Program - Renaissance In STEM Education (MSEIP-RISE) Project (Grant number P120A220005) to carry out this research project successfully. Ethics, Consent to Participate, and Consent to Publish This study involved invertebrate animals ( Palaemon spp .) and did not require ethical approval under institutional or regulatory guidelines. All experiments were conducted following established laboratory protocols to ensure the welfare of the shrimp. Consent to participate and consent to publish are not applicable. Conflict of Interest Statement The authors declare that they have no competing interests. Data Availability Statement All data generated and analyzed during this study are included in this published article and its supplementary information files. Author Contribution C.W. wrote the main text and prepared figures 1-5 and table 1 shown within manuscript, while M.K. gave advisement and instruction. R.D. and M.K. assisted C.W. in the experimental process. References Singh, V., Singh, N., Rai, S. N., Kumar, A., Singh, A. K., Singh, M. P., Sahoo, A., Shekhar, S., Vamanu, E., & Mishra, V. (2023). 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(2024). Effect of heavy metals, cadmium & copper on freshwater prawn, Macrobrachium lamarrei (Crustacea-Decapoda). International Journal of Entomology Research. 9. 32-36. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Reviews received at journal 28 Apr, 2025 Reviewers agreed at journal 28 Apr, 2025 Reviewers invited by journal 28 Apr, 2025 Editor assigned by journal 28 Apr, 2025 Submission checks completed at journal 26 Apr, 2025 First submitted to journal 24 Apr, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5672506","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":449207876,"identity":"48fbe5ca-8543-473d-ad83-dfa134b43c48","order_by":0,"name":"Charrel A. 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The results establish a baseline survival rate, serving as a reference for comparison with copper-treated groups.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/3236fefada148a52c331ba8c.png"},{"id":81631313,"identity":"d4db8ee9-e07e-4995-ab80-e09fcbffb361","added_by":"auto","created_at":"2025-04-29 11:33:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51179,"visible":true,"origin":"","legend":"\u003cp\u003eShrimp mortality rates under 0.25 ppm copper exposure across salinity conditions. Mortality remained relatively low compared to higher copper concentrations, with the highest mortality observed at 96 hours in 5 ppt salinity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/075569fc6073b23596abd0e2.png"},{"id":81630943,"identity":"70fdfaad-088d-4055-8194-8c1302674b2a","added_by":"auto","created_at":"2025-04-29 11:25:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":22633,"visible":true,"origin":"","legend":"\u003cp\u003eThe mortality rates of shrimp exposed to 0.50 ppm copper across salinity conditions. By 48 hours all salinity levels except for 20 ppt, reached the LC\u003csub\u003e50 \u003c/sub\u003ethreshold suggesting that 20 ppt is the most tolerable.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/b007b297c2a22deb93591d34.png"},{"id":81630942,"identity":"a8ff756b-8274-4c0c-ab51-a2ed4cd0ad51","added_by":"auto","created_at":"2025-04-29 11:25:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49669,"visible":true,"origin":"","legend":"\u003cp\u003eThe mortality rates of shrimp exposed to 1.0 ppm copper across time intervals and salinity conditions. All subjects in the 1 ppt salinity treatment reached 100% mortality by 12 hours, while all shrimp in the 5 ppt salinity treatment reached 100% mortality by 24 hours. Mortality at 10 ppt and 20 ppt salinities increased more gradually, with the LC\u003csub\u003e50\u003c/sub\u003e threshold being exceeded by 48 hours.\u0026nbsp;\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/82034945e2f38163fd92553a.png"},{"id":81630945,"identity":"45243e13-122a-405c-8521-a89cff719aaf","added_by":"auto","created_at":"2025-04-29 11:25:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22181,"visible":true,"origin":"","legend":"\u003cp\u003eThe highest copper concentration (2.0 ppm) resulted in the fastest mortality rates across all salinity levels. By 48 hours, all replicates surpassed the LC\u003csub\u003e50\u003c/sub\u003e threshold, confirming that higher copper concentrations are more lethal especially at lower salinity levels.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/0b1c0d365131ec9f0ff6af71.png"},{"id":81633826,"identity":"38aa2de0-0089-4690-83f2-a6f94b814174","added_by":"auto","created_at":"2025-04-29 11:57:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":738318,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5672506/v1/85d823bb-b369-41be-af19-2709f17afdc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Impact of Salinity on Copper-Induced Toxicity in Palaemon spp.: Effects on Survival, Morphological Deformities, and Toxicity Indicators","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAquatic pollution is primarily driven by contaminants released from industrial, agricultural, and urban stressors. These pollutants enter water systems through processes such as chemical leaching, soil erosion, and direct discharge of industrial effluents [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Both intentional and unintentional human activities exacerbate this issue, introducing a variety of toxic substances such as heavy metals, chemicals, and industrial byproducts into aquatic environments [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In the United States alone, approximately 190\u0026nbsp;million pounds of metallic ions are released into water sources annually [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This accumulation of pollutants disrupts aquatic habitats, threatens biodiversity, and undermines ecosystem stability.\u003c/p\u003e \u003cp\u003eLike other non-biodegradable heavy metals, such as mercury (Hg), zinc (Zn), and cadmium (Cd), copper (Cu) plays a crucial role in metabolic functions. These metals typically enter organisms through contaminated food, water, or air, leading to potentially harmful bioaccumulation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, pollution sources-waste dumps, mines, landfills, and sewage outlets- significantly contribute to environmental contamination, posing risks to ecosystems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. While copper is essential in trace amounts for enzyme catalysis and ATP production, excessive accumulation disrupts cellular processes by binding to proteins and nucleic acids [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. When metal concentrations surpass an organism's tolerance levels, they interfere with critical biological functions, jeopardizing both individual health and overall ecosystem stability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bioaccumulation occurs when an organism's uptake of a substance exceeds its capacity to excrete it, resulting in profound physiological disruptions.\u003c/p\u003e \u003cp\u003eSalinity is a key determinant in modulating the toxicity of copper, in brackish water species. As seen in the blue crab (\u003cem\u003eCallinectes sapidus\u003c/em\u003e), where varying salinity levels influence enzyme activity and ammonia excretion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, research on \u003cem\u003eFundulus heteroclitus\u003c/em\u003e showed that the highest sensitivity to copper (Cu) occurred at salinities between 0.018 ppt and 0.294 ppt. While tolerance increased at intermediate salinities around 10 ppt, a pattern linked to differences in osmoregulatory physiology [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. To further elucidate these effects, acute toxicity trials are commonly employed to assess visible physiological and morphological alterations [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCopper's ability to shift between its oxidation states (most common being Cu⁺ and Cu\u0026sup2;⁺) plays a significant role in facilitating redox reactions. These reactions can lead to the formation of free radicals, which are highly reactive molecules with unpaired electrons. The generation of free radicals can cause oxidative damage to cellular components, such as lipids, proteins, and DNA. For instance, copper exposure has been shown to induce noticeable changes, including increased redness and discoloration in shrimp [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], as well as the development of external lesions in fish species [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. By inducing oxidative stress, normal cellular function is disrupted due to there being an imbalance between free radicals and antioxidants within a cell [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eElevated copper concentrations in aquatic environments disrupt ion regulation, particularly by affecting chloride cells in fish gills, which are crucial for maintaining ionic balance [7,13,14,]. Furthermore, copper concentrations in wastewater have been reported to range from approximately 2.5 mg/L to 10,000 mg/L. Indicating that in areas where runoff occurs, concentrations can become significantly elevated upsetting a habitat\u0026rsquo;s biota [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. As it accumulates within the aquatic organism\u0026rsquo;s tissues, it interferes with processes like energy production, reproduction, and survival.\u003c/p\u003e \u003cp\u003eGrass shrimp, the \u003cem\u003ePalaemon spp.\u003c/em\u003e, are ideal model organisms for studying copper toxicity in aquatic environments [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Native to estuaries of the Northern America, these euryhaline shrimp are highly adaptable with the ability to tolerate a broad range of salinity conditions from freshwater to hypersaline [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This tolerance makes them particularly valuable for assessing how variations in water chemistry, such as salinity changes and copper concentrations, affect organism health [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies by Manyin and Rowe [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] have demonstrated that \u003cem\u003ePalaemon\u003c/em\u003e species are highly sensitive to copper toxicity. Exposure results in metabolic disruptions, inhibited growth, and impaired physiological functions, reinforcing their effectiveness as bioindicators of copper contamination in aquatic ecosystems. Given their role in aquatic food webs and sensitivity to metal pollution, this shrimp provide reliable insights into the broader toxicological impacts of copper exposure [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo evaluate the specific effects of copper exposure on grass shrimp, the lethal concentration (LC\u003csub\u003e50\u003c/sub\u003e) of copper was measured in this study. This measure is crucial for understanding the susceptibility of aquatic species to copper and the potential risks posed by elevated copper concentrations. The study examined copper toxicity across varying concentrations (0\u0026ndash;2 ppm) and salinities (1\u0026ndash;20 ppt). The findings will enhance our understanding of how environmental factors, such as salinity, interact with metal pollution. Further contributing to our knowledge of metal toxicity mechanisms in aquatic ecosystems. This research is critical for informing conservation efforts and environmental policies aimed at protecting aquatic life from the detrimental effects of pollutants.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eSaltwater solutions at concentrations of 1, 5, 10, and 20 ppt were prepared, with 10 liters of each solution placed in 20-gallon glass aquaria tanks and aerated for 7\u0026ndash;10 days to stabilize the media. Water quality parameters\u0026mdash;dissolved oxygen (DO), pH, and temperature\u0026mdash;were monitored weekly and maintained at stable conditions. Temperature was measured using a thermometer for one-minute intervals, pH was assessed using pH strips, and dissolved oxygen was measured with a LaMotte water test kit, with values averaged from three measurements per aquarium.\u003c/p\u003e \u003cp\u003eThirty 5x5-inch fish bowls were cleaned using a 10 mL solution of \u0026lt;\u0026thinsp;1% hydrochloric acid, rinsed three times with distilled water, and air-dried upside down for 48 hours. Copper sulfate (CuSO₄\u0026middot;5H₂O) was used to prepare copper concentrations of 0.25, 0.5, 1.0, and 2.0 ppm. The control group, 0 ppm, contains no added copper. These concentrations were chosen to reflect environmental copper contamination levels and to assess varying toxicity thresholds. Approximately 330 mL of each copper-saline solution was added to each bowl.\u003c/p\u003e \u003cp\u003eGrass shrimp (2.0\u0026ndash;2.5 cm in length) were obtained from a local aquarium supplier, initially kept in a 1 ppt saline medium, and were morphologically consistent with members of the genus \u003cem\u003ePalaemon\u003c/em\u003e. However, species-level identification was not conducted. We recognize that different \u003cem\u003ePalaemon\u003c/em\u003e species may exhibit varying salinity tolerances which could influence copper sensitivity. The 1 ppt saline medium was treated as control solution for salinity in experimental trials. Prior to experimentation, the shrimp were acclimated overnight in the specific salinity conditions (1 ppt, 5 ppt, 10 ppt, or 20 ppt) of the testing aquaria tanks. This ensured proper adaption to each subject\u0026rsquo;s respective salinity level before exposure to the copper treatments. The following morning, four shrimp were placed in each bowl, in triplicates for each concentration (12 shrimp total per ppt/ppm treatment). Shrimp were monitored for LC\u003csub\u003e50\u003c/sub\u003e, behavioral changes, and morphological deformities at 0, 3, 6, 9, 12, 24, 48, 72, and 96 hours. A 1 mm mesh was placed over each bowl to prevent shrimp from potentially escaping. This procedure was repeated for both experimental trials: the first trial involved 1 ppt and 5 ppt salinities, and the second trial involved 10 ppt and 20 ppt salinities. The relationship between copper concentrations and LC\u003csub\u003e50\u003c/sub\u003e across salinities was analyzed using Two-way Analysis of Variance (ANOVA) to assess the significance of salinity as a determinant for toxicity.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003ePhysio-chemical factors of water (media):\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eElevated salinity reduces the bioavailability of copper by enhancing ionic competition. Higher concentrations of sodium (Na⁺) and chloride (Cl⁻) ions compete with copper ions (Cu\u0026sup2;⁺) for binding sites on cell membranes, thus limiting copper uptake [21]. Furthermore, chloride ions promote the formation of copper-chloride complexes, which are less bioavailable than free copper ions [22]. As a result, the toxicity of copper is mitigated in high salinity environments, as the concentration of bioavailable copper ions decreases, reducing their harmful effects on aquatic organisms [16, 21, 23]. Our findings align with previous studies [14, 24] which suggest that copper toxicity is closely linked to the osmoregulatory capacity of aquatic organisms. This osmoregulatory ability plays a crucial role in maintaining internal solute balance and preventing adverse physiological effects. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGrass shrimp are eurythermal organisms, capable of surviving in temperatures ranging from 5\u0026deg;C to 38\u0026deg;C (41\u0026deg;F to 101\u0026deg;F), with an optimal range between 18\u0026deg;C and 25\u0026deg;C (65\u0026deg;F to 77\u0026deg;F) [16, 25]. \u0026nbsp;They have been observed in waters with dissolved oxygen concentrations between 6 and 11 ppm [16, 25]. Throughout the study, water quality parameters remained stable, with dissolved oxygen averaging\u003c/p\u003e\n\u003cp\u003e8.675 \u0026plusmn; 0.187 ppm, pH at 6.88 \u0026plusmn; 0.088, and temperature at 28.75 \u0026plusmn; 0.244\u0026ordm;C; conditions within the range suitable for \u003cem\u003ePalaemon\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological Deformities:\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs salinity increased, subjects were noticeably more active. Those in the 20 ppt salinity medium displayed fewer morphological deformities, such as discoloration, reduced mobility, and a lack of response to external stimuli (gentle agitation of the bowl) than other media. This trend was also consistent across all copper concentrations. \u0026nbsp;Subjects in higher Cu concentrations (1.0 ppm and 2.0 ppm) displayed a redder hue at the time of death, than those in lower concentrations (0.25 ppm and 0.50 ppm).\u003c/p\u003e\n\u003cp\u003eCopper exposure in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e led to significant declines in growth, immune function, and gut microbiota composition, with higher concentrations causing hepatopancreatic damage and decreased hemocyte counts [26]. At concentrations of 1 mg Cu\u0026sup2;⁺ L⁻\u0026sup1;, copper also disrupted antioxidant enzyme activities and altered the diversity of intestinal microbes, highlighting its toxic effects on the shrimp\u0026apos;s overall health [26]. Similarly, exposure to copper sulphate in \u003cem\u003eMacrobrachium lamarrei\u003c/em\u003e caused severe behavioral disruptions, including equilibrium loss, excessive mucus secretion, and increased mortality [27]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC\u003csub\u003e50\u003c/sub\u003e and Rates of Mortality:\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 96 hours, replicates at 1 ppt exhibited more rapid rates of mortality at higher copper concentrations compared to those at 5 ppt, 10 ppt, and 20 ppt salinities. At 20 ppt, mortality rates were lower than at the other salinity levels with LC\u003csub\u003e50\u0026nbsp;\u003c/sub\u003evalues increasing as salinity decreased (mortality rate: 20 ppt \u0026lt; 10 ppt \u0026lt; 5 ppt \u0026lt; 1 ppt). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCopper toxicity increased as salinity decreased, with higher salinity levels reducing the metal\u0026rsquo;s potency. At 2.0 ppm Cu, the LC\u003csub\u003e50\u003c/sub\u003e threshold was reached within 48 hours for all salinity replicates as shown in Figure 5. However, at 0.25 ppm Cu, mortality rates were lower at 96 hours compared to higher copper concentrations (mortality rate of Cu: 0.25 ppm \u0026lt; 0.50 ppm \u0026lt; 1.0 ppm \u0026lt; 2.0 ppm). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong all salinity levels, higher copper concentrations were associated with higher mortality rates, with 2.0 ppm being the most toxic. After 96 hours, subjects exposed to 0.25 ppm Cu had the lowest mortality rates (see Figure 2). Mortality increased more rapidly for those exposed to 1.0 and 2.0 ppm Cu compared to those exposed to 0.25 and 0.50 ppm Cu. In most replicates at all salinity levels, 1.0 ppm and 2.0 ppm Cu exceeded the LC\u003csub\u003e50\u003c/sub\u003e threshold (see Figures 4 and 5). \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eStatistical Analysis \u0026nbsp;\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 1.\u003c/em\u003e\u003c/strong\u003e Two-way ANOVA Test Results: P-value vs. F-Critical Value \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"378\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eExposure\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTime Interval\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(Hours)\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 254px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInteractions Between \u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eSalinity and Copper Concentrations\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-value\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eF-Critical\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eValue\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.000000524** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.000053** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.00000131** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e12\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.000000303** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e24\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.012655** \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e48\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.166288 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e72\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.193488 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e96\u0026nbsp;\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 129px;\"\u003e\n \u003cp\u003e0.103501 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e2.003459 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 1: The p-values for each exposure time along with the corresponding F-critical value are shown. The F-critical value remains constant at 2.003459 for each exposure time, and the p-values indicate whether the null hypothesis should be rejected or not based on the threshold of 𝛼=0.05. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExposure times of 0, 3, 6, 9, 12, and 24 hours (**) exhibit significant interactions between salinity and copper, whereas 48, 72, and 96 hours do not. The null hypothesis is rejected for exposure times of 3, 6, 9, 12, and 24 hours (p-values \u0026lt; \u0026alpha; = 0.05), suggesting that exposure time significantly influences shrimp survival. This implies that the effect of copper on shrimp survival varies with exposure time. In contrast, for 48, 72, and 96 hours (p-values \u0026gt; \u0026alpha; = 0.05), the null hypothesis is not rejected, indicating no significant impact on survival at these longer exposure times. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study emphasizes the significant role of salinity in modulating copper (Cu) toxicity in grass shrimp, with higher salinity levels (10 ppt and 20 ppt) reducing copper bioavailability, improving shrimp activity, reducing morphological deformities, and lowering mortality rates. However, contrary to our initial hypothesis, shrimp exposed to 1.0 ppm Cu at 10 ppt salinity did not show the anticipated improvements in health. Future research should aim to better understand the mechanisms driving copper absorption and tissue accumulation in varying salinity conditions. By utilizing advanced techniques such as Inductively Coupled Plasma (ICP) Spectrometry to quantify copper concentrations in shrimp tissues, a deeper insight can be had into the bioaccumulation processes and toxicity thresholds. Additionally, investigating the long-term effects of copper exposure at different salinities on reproductive health, deformities, growth, and overall population dynamics will be crucial to assessing the ecological risks posed by copper in marine environments. This comprehensive approach will help refine our understanding of copper toxicity in marine organisms and contribute to the development of more effective water quality management strategies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements and Funding\u003c/h2\u003e\n\u003cp\u003eWe greatly appreciate the summer internships provided by the US Department of Education - Minority Science and Engineering Improvement Program - Renaissance In STEM Education (MSEIP-RISE) Project (Grant number P120A220005) to carry out this research project successfully. \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eEthics, Consent to Participate, and Consent to Publish \u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThis study involved invertebrate animals (\u003cem\u003ePalaemon spp\u003c/em\u003e.) and did not require ethical approval under institutional or regulatory guidelines. All experiments were conducted following established laboratory protocols to ensure the welfare of the shrimp. Consent to participate and consent to publish are not applicable. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConflict of Interest Statement\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eData Availability Statement \u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in this published article and its supplementary information files. \u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eC.W. wrote the main text and prepared figures 1-5 and table 1 shown within manuscript, while M.K. gave advisement and instruction. R.D. and M.K. assisted C.W. in the experimental process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSingh, V., Singh, N., Rai, S. N., Kumar, A., Singh, A. K., Singh, M. P., Sahoo, A., Shekhar, S., Vamanu, E., \u0026amp; Mishra, V. (2023). Heavy Metal Contamination in the Aquatic Ecosystem: Toxicity and Its Remediation Using Eco-Friendly Approaches. Toxics, 11(2), 147. https://doi.org/10.3390/toxics11020147 \u003c/li\u003e\n\u003cli\u003eU.S. Environmental Protection Agency. (2024, March 20). Water releases by chemical \u0026amp; industry. U.S. EPA. https://www.epa.gov/trinationalanalysis/water-releaseschemicalindustry \u003c/li\u003e\n\u003cli\u003eGautam, P. K., Gautam, R. K., Banerjee, S., Chattopadhyaya, M. C., \u0026amp; Pandey, J. D. (2016). Heavy metals in the environment: fate, transport, toxicity and remediation technologies. \u003cem\u003eNova Sci Publishers\u003c/em\u003e, \u003cem\u003e60\u003c/em\u003e, 101-130. \u003c/li\u003e\n\u003cli\u003eBriffa, J., Sinagra, E., \u0026amp; Blundell, R. (2020). 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Copper toxicity in aquatic ecosystem: A Review. Int J Fish Aquat Stud 2023;11(4):134-138. DOI: https://doi.org/10.22271/fish.2023.v11.i4b.2835 \u003c/li\u003e\n\u003cli\u003eGuerreiro Gomes, E., da Silva Freitas, L., Everton Maciel, F. \u003cem\u003eet al.\u003c/em\u003e Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab \u003cem\u003eCallinectes sapidus\u003c/em\u003e. \u003cem\u003eEcotoxicology\u003c/em\u003e 28, 781\u0026ndash;789 (2019). https://doi.org/10.1007/s10646-019-02073-7 \u003c/li\u003e\n\u003cli\u003eKousar, S., \u0026amp; Javed, M.A. (2012). 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Bioenergetic effects of aqueous copper and cadmium on the grass shrimp, Palaemonetes pugio. \u003cem\u003eComparative Biochemistry and Physiology \u003cem\u003eCtoxicology \u0026amp; Pharmacology\u003c/em\u003e, \u003cem\u003e150\u003c/em\u003e(1), 65\u0026ndash;71. https://doi.org/10.1016/j.cbpc.2009.02.007 \u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eTavares-Dias, M. (2021). Toxic, physiological, histomorphological, growth performance and antiparasitic effects of copper sulphate in fish aquaculture. \u003cem\u003eAquaculture\u003c/em\u003e, \u003cem\u003e535\u003c/em\u003e, 736350. https://doi.org/10.1016/j.aquaculture.2021.736350 \u003c/li\u003e\n\u003cli\u003eSergio Ch\u0026aacute;zaro-Olvera, Growth, Mortality, and Fecundity of \u003cem\u003ePalaemonetes Pugio\u003c/em\u003e from a Lagoon System Inlet in the Southwestern Gulf of Mexico, \u003cem\u003eJournal of Crustacean Biology\u003c/em\u003e, Volume 29, Issue 2, 1 April 2009, Pages 201\u0026ndash;207, https://doi.org/10.1651/08-3055R.1 \u003c/li\u003e\n\u003cli\u003eKey, P. B., Wirth, E. F., \u0026amp; Fulton, M. H. (2006). A Review of Grass Shrimp, \u003cem\u003ePalaemonetes\u003c/em\u003e spp., as a Bioindicator of Anthropogenic Impacts. \u003cem\u003eEnvironmental Bioindicators\u003c/em\u003e, \u003cem\u003e1\u003c/em\u003e(2), 115\u0026ndash;128. https://doi.org/10.1080/15555270600685115 \u003c/li\u003e\n\u003cli\u003eFanning, D. D., Wicksten, M. K., \u0026amp; Schulze, A. (2024). Taxonomic and Genetic Diversity in \u003cem\u003ePalaemon\u003c/em\u003e Species (Decapoda: Caridea: Palaemonidae) of the Northern Gulf of Mexico. \u003cem\u003eDiversity\u003c/em\u003e, \u003cem\u003e16\u003c/em\u003e(9), 543. https://doi.org/10.3390/d16090543 \u003c/li\u003e\n\u003cli\u003eGretchen K., Bullington, B.B., DeCarlo, C.A., Chalk, S.J., \u0026amp; Smith, K. (2012), The Effects of Salinity on Acute Toxicity of Zinc to Two Euryhaline Species of Fish, \u003cem\u003eFundulus heteroclitus\u003c/em\u003e and \u003cem\u003eKryptolebias marmoratus\u003c/em\u003e, \u003cem\u003eIntegrative and Comparative Biology\u003c/em\u003e, Volume 52, Issue 6, December 2012, Pages 753\u0026ndash;760, https://doi.org/10.1093/icb/ics045 \u003c/li\u003e\n\u003cli\u003eManyin, T., \u0026amp; Rowe, C. L. (2010). Reproductive and life stage-specific effects of aqueous copper on the grass shrimp, \u003cem\u003ePalaemonetes pugio\u003c/em\u003e. \u003cem\u003eMarine Environmental \u003c/em\u003e \u003cem\u003eResearch\u003c/em\u003e, 69(3), 152-157. https://doi.org/10.1016/j.marenvres.2009.09.006 \u003c/li\u003e\n\u003cli\u003eChaplin-Ebanks, S. A., \u0026amp; Curran, M. C. (2007). Prevalence of the bopyrid isopod Probopyrus pandalicola in the grass shrimp, Palaemonetes pugio, in four tidal creeks on the South Carolina-Georgia coast. \u003cem\u003eThe Journal of parasitology\u003c/em\u003e, \u003cem\u003e93\u003c/em\u003e(1), 73\u0026ndash;77. https://doi.org/10.1645/GE-3537.1 \u003c/li\u003e\n\u003cli\u003eSch\u0026uuml;ck, M., \u0026amp; Greger, M. (2023). Salinity and temperature influence removal levels of heavy metals and chloride from water by wetland plants. \u003cem\u003eEnvironmental science and pollution research international\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(20), 58030\u0026ndash;58040. https://doi.org/10.1007/s11356023-26490-8 \u003c/li\u003e\n\u003cli\u003eMartins, C.deM., Barcarolli, I. F., de Menezes, E. J., Giacomin, M. M., Wood, C. M., \u0026amp; Bianchini, A. (2011). Acute toxicity, accumulation and tissue distribution of copper in the blue crab Callinectes sapidus acclimated to different salinities: in vivo and in vitro studies.\u003cem\u003eAquatic toxicology (Amsterdam, Netherlands)\u003c/em\u003e, \u003cem\u003e101\u003c/em\u003e(1), 88\u0026ndash;99. https://doi.org/10.1016/j.aquatox.2010.09.005 \u003c/li\u003e\n\u003cli\u003eIbrahim, M., \u0026amp; Minghetti, M. (2022). Effect of chloride concentration on the cytotoxicity, bioavailability, and bioreactivity of copper and silver in the rainbow trout gut cell line, RTgutGC. \u003cem\u003eEcotoxicology (London, England)\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(4), 626\u0026ndash;636. https://doi.org/10.1007/s10646-022-02543-5 \u003c/li\u003e\n\u003cli\u003eLee, J. A., Marsden, I. D., \u0026amp; Glover, C. N. (2010). The influence of salinity on copper accumulation and its toxic effects in estuarine animals with differing osmoregulatory strategies. \u003cem\u003eAquatic toxicology (Amsterdam, Netherlands)\u003c/em\u003e, \u003cem\u003e99\u003c/em\u003e(1), 65\u0026ndash;72.https://doi.org/10.1016/j.aquatox.2010.04.006 \u003c/li\u003e\n\u003cli\u003eAnderson, G. 1985. Species profiles: life histories and environmental requirements of Coastal fishes and invertebrates (Gulf of Mexico)\u0026mdash;grass shrimp. Biological Report 82(11.35), March 1985 TR EL-82\u0026ndash;4, U.S. Fish and Wildlife Service, U.S. Department of the Interior \u003c/li\u003e\n\u003cli\u003eQian, D., Xu, C., Chen, C., Qin, J. G., Chen, L., \u0026amp; Li, E. (2020). Toxic effect of chronic waterborne copper exposure on growth, immunity, anti-oxidative capacity and gut microbiota of Pacific white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. \u003cem\u003eFish \u0026amp; Shellfish Immunology, 100\u003c/em\u003e, 445-455. https://doi.org/10.1016/j.fsi.2020.03.018 \u003c/li\u003e\n\u003cli\u003eAhmed, Aman \u0026amp; Lodhi, Samiksha. (2024). Effect of heavy metals, cadmium \u0026amp; copper on freshwater prawn, Macrobrachium lamarrei (Crustacea-Decapoda). International Journal of Entomology Research. 9. 32-36.\u003c/li\u003e\n\u003c/ol\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":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Toxicology, Salinity, Morphological deformities, LC50, Metal bioavailability","lastPublishedDoi":"10.21203/rs.3.rs-5672506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5672506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeavy metal contamination, resulting from pollution, presents serious threats to aquatic species and has far-reaching consequences for ecosystem health. This study investigates the acute toxicity of copper (Cu) on grass shrimp (\u003cem\u003ePalaemon spp.\u003c/em\u003e), a key species in North American estuaries. We exposed shrimp to a range of copper concentrations (0, 0.25, 0.50, 1.0, and 2.0 ppm) and salinities (1, 5, 10, and 20 ppt) over periods of 3, 6, 9, 12, 24, 48, 72, and 96 hours. We hypothesized that exposure to 1.0 ppm Cu and 10 ppt salinity would reduce copper toxicity; but contrary to our expectations, optimal shrimp survival occurred at 20 ppt salinity and 0.25 ppm Cu. Copper solutions were prepared using CuSO₄\u0026middot;5H₂O, and toxicity was monitored using indicators such as mortality, abdominal curvature, discoloration, and mobility. Water quality remained stable throughout the study, with dissolved oxygen consistently at 8.675\u0026thinsp;\u0026plusmn;\u0026thinsp;0.187 ppm, pH at 6.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.088, and temperature at 28.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.244\u0026ordm;C. Copper toxicity increased at lower salinities, with the highest mortality and quickest onset observed at 1 ppt. Mortality was lowest at 0.25 ppm Cu, while 2.0 ppm Cu induced the highest mortality across all salinities, supporting a dose-response relationship. LC\u003csub\u003e50\u003c/sub\u003e values increased with salinity, with the highest survival rates occurring at 20 ppt. These findings highlight the protective role of higher salinity in mitigating copper toxicity, emphasizing the need for further research on the long-term ecological consequences of copper contamination in estuarine ecosystems.\u003c/p\u003e","manuscriptTitle":"The Impact of Salinity on Copper-Induced Toxicity in Palaemon spp.: Effects on Survival, Morphological Deformities, and Toxicity Indicators","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 11:24:59","doi":"10.21203/rs.3.rs-5672506/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-04-28T15:15:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271366288319809816249020011177771774041","date":"2025-04-28T15:09:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-28T08:19:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-28T08:18:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-26T13:33:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Toxicology","date":"2025-04-24T23:42:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa4f4654-1962-4286-b6d6-5866c9f27b28","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2025-04-30T12:32:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 11:24:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5672506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5672506","identity":"rs-5672506","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}