The synergistic effect of mono and co-exposure of microplastic suspensions on Daphnia magna’s survival, population density, reproduction rate & swimming behavior.

The synergistic effect of mono and co-exposure of microplastic suspensions on Daphnia magna’s survival, population density, reproduction rate & swimming behavior. | 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 synergistic effect of mono and co-exposure of microplastic suspensions on Daphnia magna’s survival, population density, reproduction rate & swimming behavior. Hassan Shafiq, Hira Amjad, Imran Hashmi, Mariam Ali This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3545738/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Microplastics represent a pervasive contaminant in aquatic ecosystems, posing substantial risks to aquatic biota. This study sought to elucidate the toxicological impacts of three prevalent plastic polymers high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polypropylene (PP) in both mono and co-exposure scenarios on Daphnia. Employing a range of toxicological endpoints, investigation assessed Daphnia magna's ingestion, bioaccumulation, population density, swimming behavior, and reproduction rate in response to microplastics measuring 0–32 µm, which were synthesized in laboratory using virgin polymer beads . The findings revealed that synergistic effects of microplastics in a co-exposure medium pose a greater hazard to the organism than mono-exposure to individual microplastics (PP, LDPE, and HDPE). In co-exposure scenerio, LC50 value decreased to 77 mg/L, compared to 120, 123, and 109 mg/L for PP, LDPE, and HDPE, respectively. Daphnia magna exhibited a more pronounced response to co-exposure, characterized by reduced survival rates, increased microplastic ingestion, reduced reproduction rates and population densities. Furthermore, co-exposure scenarios led to increased erratic swimming movements relative to mono-exposure, with immobility and energy deficiency observed across all exposure types, albeit with a greater magnitude in co-exposure settings. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plastics, which were once considered to be a remarkable product discovery has now caused irreversible damage to the environment (Shim and Thomposon 2015 ). The plastics are generated through the polymerization reactions of non-renewable resource-derived monomers like ethylene, propylene, and styrene (Lim 2021 ). Plastics are categorized based on their chemical structures and properties, with notable types including polyethylene, polypropylene, polyvinyl chloride (PVC), and polystyrene (Borrelle et al. 2020 ). The “Microplastics” are defined as plastic fragments measuring less than 5mm in size in any direction (Khan and Ali 2023 ). Microplastics are produced through the degradation of larger plastic objects or intentionally manufactured for usage in diverse products such as cosmetics, cleaning agents, and textiles (Köster and Paffenhöfer 2022 ). Dominant microplastics in lakes and oceans include polypropylene, polyethylene and polystyrene (Bashir and Hashmi 2022 ). The presence of microplastics in the environment has raised several concerns as an emerging contaminant, encompassing environmental impact, human health implications, resource depletion, and contributions to climate change (Cózar et al. 2014 ). Microplastics present a substantial chemical risk to marine and freshwater environments (Desforges et al. 2015 ). They possess the ability to adsorb and accumulate toxic pollutants, including persistent organic pollutants (POPs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals, from the surrounding water (Mataji et al. 2020 ). Microplastics have been shown to exert toxic effects on zooplankton, which are fundamental aquatic organisms in the marine food chain and their trophic transfer through the marine food web has been confirmed by many studies (Cole et al. 2011 ). The zooplankton species, Daphnia magna , is commonly employed as a test organism in ecotoxicology studies due to its sensitivity to water quality changes and its vital role as a food source for many aquatic organisms (Rist et al. 2017 ). Daphnia magna exhibits specific characteristics, such as size range of 1 to 5 mm, cyclic parthenogenesis, filter-feeding behavior using branched antennae, and a relatively short lifespan of a few weeks to a few months (Lampert 2006 ; Schrank et al. 2019 ). Notably, transparency of Daphnia magna enables researchers to observe internal organs and physiological processes (Yin et al. 2023 ). Several studies have investigated toxic impacts of microplastics on Daphnia magna , revealing alterations in swimming behaviour, feeding rate, reproduction rate, tissue accumulation, and increased susceptibility to other environmental pollutants. Cole et al. ( 2013 ) demonstrated that exposure to microplastics induced changes in swimming behaviour, feeding rates, and reproductive rates of zooplanktons, particularly copepods and Daphnia magna . Ellis et al. ( 2020 ) found that Daphnia magna may ingest microplastics, leading to their accumulation in tissues over time, which may negatively impact the organism's health and survival. Furthermore, Pacheco et al. ( 2018 ) analysed that microplastic exposure enhanced the toxicity of other pollutants, such as heavy metals, to Daphnia magna , indicating interactive effects between microplastics and co-occurring environmental stressors. De Felice et al. ( 2019 ) investigated the polystyrene microplastics effects on Daphnia magna ’s phototactic and swimming behaviour along with reproduction, it was observed that microplastics affect behavioral traits of Daphnia magna leading to potentially harmful consequences on population dynamics of this zooplanktonic species. This study focuses on evaluating the toxicological impacts of both single microplastic type (mono-exposure) and mixed multiple microplastic types (co-exposure) on various aspects of Daphnia magna . The parameters of research include survival rate, swimming behavior, ingestion behavior, population density, and reproduction rate. Through these experiments, objective is to gain insights into the toxicity mechanisms associated with microplastic exposure in a realistic context and address existing research gaps in this field. Materials & Methods Test organism & culture conditions The Daphnia magna starter culture for this study was procured from a fish hatchery near Rawal Dam, Islamabad. To culture Daphnia magna , a medium of pure groundwater (1100 feet) from Islamabad, Pakistan was used, which was tested for residual chlorine (Not detected) to avoid toxicity towards the organisms. The medium had a pH range of 7–8 and dissolved oxygen (DO) levels of 6.5 mg/L (Yin et al. 2023 ). The culture conditions included maintaining a temperature of 20–22°C, following a 12-hour light/dark cycle. Chlorella vulgaris, a single-celled algae, procured from Plant virology lab, Atta-ur-Rahman School of Applied Biosciences (ASAB), NUST was provided as a food source for Daphnia magna at a concentration of 1–2 mg/L, with feeding occurring every 24 hours (Sarma and Nandini, 2006 ). The final culture density of Daphnia magna was approximately 1–2 daphnids/ml, which was considered suitable for laboratory experiments (Lee et al. 2019 ). Microplastics Preparation Crystalline PP, HDPE and LDPE beads, trademarked under Marlex, were purchased for the preparation of microplastics (Woo et al. 2021 ). The beads were rinsed with ethanol and allowed to air dry before undergoing a melting procedure. For PP, beads were melted at 80°C for 5–10 minutes in a glass petri dish (Dimensions 100x15 mm) on a hotplate. Similarly, HDPE and LDPE were heated at 65°C and 55°C, respectively, for 5–10 minutes. These temperatures were kept below the melting point of the plastics to prevent fumes, pre-oxidation, and chemical degradation (Xu et al. 2019 ). The melted plastics were shaped into rectangular pallets, with the help of a glass stirrer, to facilitate their crushing process ergonomically. A stainless-steel foot filer, typically used to remove dead skin, was used to crush the plastic pallets. The rough graded surface of the foot filer produced plastic debris. This procedure was performed for all three types of microplastics. The plastic debris obtained from filing wer subjected to a sieving process using ASTM Standard Sieve No. 635 (32 µm) and No. 450 (20 µm) (American Standard Test Sieve Series - Endecotts). The plastic debris were washed with ethanol, and a mixture of crushed microplastic debris in deionized water was obtained (Woo et al. 2021 ). The mixture was sonicated at 30°C for 10 minutes to prevent agglomeration. Filtration was carried out using a 32 µm sieve, resulting in a solution of microplastics < 32 µm. The solution was further filtered using a 0.45 µm glass fiber filter paper to collect the microplastics. The plastic particles were washed, dried at room temperature, and subjected to physico-chemical characterization. In this study, microplastics ranging from 0 to 32 µm were used for all experiments. Physico-Chemical Characterization of Microplastics The microplastics prepared were of irregular shape, mostly fragments, fibers were also present. The color of microplastics was transparent due to the transparent color of crystalline beads used. The density of each microplastic polymer was 0.91, 0.94 and 0.90 g/cm 3 respectively for PP, LDPE, HDPE as mentioned on the label (Chanpiwat and Damrongsiri 2021 ). To validate the polymer and size FT-IR and particle size analyzer was used. Physical Characterization by Particle Size Analyzer The size of microplastic particles was determined using a Horiba LA-300 Particle Size Analyzer, which has a measurement range of 0.1 to 600 µm. Each type of microplastic (PP, LDPE, HDPE) was tested separately in a wet medium, specifically deionized water (Huang et al. 2022 ). The PP, HDPE and LDPE microplastics were found to be within the desired range of 0–32 µm, as depicted in Fig. 1 . The average mean size of PP particles was 31 µm, followed by LDPE particles with a size of 12 µm and HDPE particles with a size of 26 µm. Compositional Characterization by ‘Fourier-transform infrared’ spectroscopy Experimental design and procedure The experiment was designed to investigate the effects of mono exposure and co exposure of microplastics on various parameters of Daphnia magna . To assess the toxicological effects, exposure medium was prepared, and pre-exposure conditions were established. Daphnia magna individuals were transferred from the culture medium to a 2 L glass beaker filled with deionized water. Prior to microplastic exposure, Daphnia magna were starved for a minimum of 4 hours to ensure they would readily consume microplastics, as microplastics often resemble food particles (Canniff and Hoang 2018 ). For the mono-exposure experiments , 20 adult Daphnia magna were placed in 80 ml beakers containing 50 ml of deionized water with microplastics. The concentrations of microplastics for each polymer type (PP, HDPE, LDPE) were 30, 60, 90, 120, and 150 mg/L, with a control group for each type. The microplastic fragments were weighed and added to 500 ml of deionized water, followed by sonication to disperse any agglomerates. During the transfer of solution to beakers, stock suspension was agitated to ensure thorough mixing of the microplastics. In case of co-exposure experiment , a mixture containing 33% of each microplastic polymer (PP, HDPE, LDPE) was prepared to simulate a natural environment where multiple types of microplastics are present, predominantly in fragmented form. Solutions with microplastic concentrations of 30, 60, 90, 120, and 150 mg/L were created for co-exposure experiments. This approach adds novelty to the research by considering the combined presence of different microplastic types. To assess the survival of Daphnia magna in both exposure scenarios, a 48-hour acute toxicity test was conducted following Test No. 202: Daphnia magna Sp. Acute Immobilisation Test (2004). Each exposure unit was provided with 0.1 mg of feed. The survival rate, indicated by the LC50 value, was determined using Probit Analysis (Na et al. 2021 ). In order to study ingestion and bioaccumulation of microplastics in Daphnia magna , similar concentrations of microplastics were used for both mono and co-exposure experiments. For mono-exposure, microplastics were dyed with Nile red fluorescent dye to visualize their ingestion by Daphnia magna . A dye solution of 0.1 mg/10 ml was prepared using acetone, and the microplastics were added to the dye, filtered, and thoroughly washed to remove excess dye (Frydkjær et al. 2017 ). The fluorescently labeled microplastics were used in suspensions ranging from 30 to 150 mg/L. 20 Daphnia magna individuals were exposed to these suspensions for 120 hours (5 days) and analyzed under a fluorescent microscope (Optika B-350) to observe microplastic ingestion. To determine the bioaccumulation of microplastics in both exposure scenarios, 40% of the population (8 Daphnia magna ) from each exposure beaker was taken. The draft digestion method with a slight modification was employed, using 50% nitric acid to digest the Daphnia magna for 3 hours at a temperature of 50 o C. This process was repeated for both exposure groups. The digested solution was diluted with deionized water, filtered through a glass fiber filter paper, and dried at room temperature (Canniff and Hoang 2018 ). For mono-exposure, filter papers containing digested Daphnia magna were directly examined under a stereomicroscope, and microplastics were quantified by counting the particles per organism. In case of co-exposure, although microplastics were present, their specific types could not be identified. The filter paper containing the microplastics was subjected to ATR-FTIR analysis to observe the type of plastic accumulated in Daphnia magna at different concentrations, while the number of microplastic particles was quantified (Gerdes et al. 2019 ). To calculate the reproduction rate per organism per day, total number of neonates produced in mono or co-exposure groups were divided by the number of adult Daphnia magna in the same treatment replicate on the same day. This value was then divided by the number of days to obtain the reproduction rate. Representing the reproduction rate as "RR," the number of neonates on day "x" as "N (x) ," and the number of alive adult Daphnia magna on day "x" as "A (x) ." Mathematically, the reproduction rate may be expressed as: RR = N (x) / A (x Eq: 1 To determine if there were significant differences between treatments, the average cumulative number of neonates produced by surviving adults over a 5-day period was calculated. Additionally, average reproductive rate per day for each treatment was calculated and compared (Imhof et al. 2017 ). Population density was calculated by counting the number of Daphnia magna in a sample of water. Population density can also be observed visually using a colony counter when the sample is poured into a petri dish (Guilhermino et al. 2021 ). To assess swimming behavior , 1-minute-long videos were recorded for each replicate in both exposure types. The swimming patterns, such as hopping and sinking, cruising, and vertical swimming, were visually noted. The videos were analyzed using a software called AnimApp, specifically designed for insect tracking. AnimApp is a python based tracking module. The software tracked the swimming behavior of the organisms and identified changes in their swimming patterns, which can indicate stress in Daphnia magna. The threshold in AnimApp was adjusted to accurately detect the organisms, and based on the tracking data, a plot was generated (Rao et al. 2019 ). Results Daphnia magna survival The results of the present study demonstrated that survival of Daphnia magna decreased as the concentration of microplastics increased in both sets of experiments. Specifically, LC50 values for mono exposure were found to be 120 mg/L for PP, 107 mg/L for LDPE, and 123 mg/L for HDPE. However, when considering the synergistic effect of microplastics, LC50 value decreased to 77 mg/L. (Fig. 3 ). The mortality to concentration (Fig. 4 ) demonstrates that co-exposure of microplastics caused maximum mortality of Daphnia magna i.e 15 individuals followed by PP (13), HDPE (12) and LDPE (12) respectively. Ingestion of microplastics by Daphnia magna The ingestion of microplastics in Daphnia magna increased with increase in concentration of microplastics in both of the experiments. The mono exposure of microplastics had a relatively smaller number of particles inside the Daphnia magna than co-exposure which showed that synergistic effect of microplastics was causing more accumulation of microplastics. Figure 5 , shows the ingestion of microplastics in Daphnia magna . FT-IR results of ingested microplastics revealed that at lower concentration LDPE was the abundant microplastic type accumulated in Daphnia magna . When concentration increased to 120 mg/L, HDPE was more pronounced followed by PP and LDPE whereas in case of 150 mg/L concentration PP was most abundantly present inside Daphnia magna followed by HDPE and LDPE. As reported in Table 1 . Table 1 Microplastics abdudance in Daphnia magna calculated by FT-IR analysis Conc. (mg/L) Microplastics present Abundance 0 None None 30 LDPE, HDPE, PP LDPE > PP > HDPE 60 LDPE, HDPE, PP LDPE > PP > HDPE 90 LDPE, HDPE, PP LDPE > HDPE > PP 120 LDPE, HDPE, PP HDPE > PP > LDPE 150 LDPE, HDPE, PP PP > HDPE > LDPE Daphnia magna swimming behavior In this study, healthy Daphnia magna specimens were collected from each replicate of both mono and co-exposure experiments. These specimens were observed and recorded under a 4X magnification for a duration of 1 minute, and videos were created for further analysis using the AnimApp software. The results of the mono-exposure experiments revealed that the mobility and swimming pattern of Daphnia magna were negatively affected as the concentration of microplastics increased. The movement span of Daphnia magna was reduced in all three types of mono exposures. In case of co-exposure, synergistic effect of microplastics was found to be significant. This was evident from the increased hopping and sinking behavior of Daphnia magna , indicating extreme stressful conditions. As the concentration of microplastics increased, a decrease in velocity and an increase in immobility were observed in the co-exposure group, but not in the mono-exposure group (as shown in Fig. 6). This was observed that the normal behavior of vertical swimming and cruising of the organisms from the top to the bottom of the beakers was absent at higher concentrations. Daphnia magna reproduction rate and population density On the 5th day of exposure, reproduction rate was calculated for both mono and co-exposure experiments. In the mono exposures, reproduction rate remained relatively constant but gradually decreased with increasing concentration of microplastics. However, in case of co-exposure, synergistic effect of microplastics had a significant impact on reproduction rate. It was observed that reproduction rate in co-exposure group was reduced to half compared to the normal single type microplastic exposure, as indicated in Table 2 . Furthermore, population density was found to decrease with an increase in microplastic concentration. Although low population size played a role in determining the population density, it was evident that the visible number of organisms in all the exposure setups was reduced when cross-checked using a colony counter. Table 2 Reproduction rate, as calculated on 5th day. Exposure Conc. (mg/L) PP LDPE HDPE Co-exposure 0 2.2 2.2 2.2 2.1 30 2 1.85 1.57 1.5 60 2 1.83 1.5 1.3 90 1.7 1.6 1.2 1.3 120 1.5 1.5 1 0 150 1 1 1 0 Average 1.64 1.556 1.254 0.82 Discussion Daphnia magna survival The LC50 results of the study showed that mono exposure of microplastics of all three types (PP, HDPE, LDPE) resulted in death of 50% of Daphnia magna population at certain concentrations of co and mono exposure. This indicates that in realistic scenarios where multiple types of microplastics are present in aquatic environments, plastics may be more harmful at lower concentrations. Isinibilir and co researchers (2022) concluded in their study that survival of Daphnia magna was affected by microplastics of a single type, but higher concentrations with algae had a greater decline on their survival. Similarly, Gerdes et al. ( 2019 ) found that PET microplastics had a low survival rate with an LC50 value of 160 mg/L for Daphnia magna . In comparison, current study reported LC50 values ranging from 107 to 123 mg/L for individually exposed microplastics, while synergistic effect of microplastic mixtures had a lower LC50 value of 77 mg/L. This indicates that mixture of microplastics is more lethal at lower concentrations, posing a greater threat to Daphnia magna and potentially disrupting food chain, as they are an important food source for predators. It's important to note that LC50 values are just one measure of toxicity, and other factors such as growth, reproduction, and behavior may also be affected by microplastics. Additionally, current study used irregularly sized microplastic fragments, which mimic the dominant form of microplastics found in marine and freshwater environments. Na et al. ( 2021 ) found that microplastic fragments alone were 80 times more lethal in acute experiments compared to regularly sized microplastics. Ingestion of microplastics by Daphnia magna. The digestion procedure confirmed the ingestion of microplastics in both exposures. In terms of mono exposure, highest ingestion rate was observed for HDPE microplastics, with an average of 8.1 particles per Daphnia magna organism, followed by PP and LDPE with 7 and 6.3 particles respectively. Canniff and Hoang ( 2018 ) found that there exists direct relationship between concentration and number of particles ingested, although the values were relatively small (0.8-2 microplastics per Daphnia magna ) due to larger particle size (63–75 µm) and lower concentration exposures. microplastics in real environmental conditions is needed, rather than relying solely on synthetic lab-based environments. They observed that Daphnia magna readily ingests both types of microplastics at all concentrations. The ingestion of microplastics provides evidence that the primary food source for predators accumulates plastic, and there is a potential for these organisms to transfer microplastics in the food chain at the foundational level (Fabricant et al. 2021 ). Ingestion of irregularly shaped microplastics leads to adsorption of microplastic particles in the gut of Daphnia magna , this fact is supported by the study conducted on regular and irregular shaped microplastics in which there was reduced or no egestion of irregularly sized microplastics than the regular microplastics (Frydkjær et al. 2017 ). Daphnia magna swimming behavior The swimming pattern of Daphnia magna is an indicator of how the organism is behaving in different environments. There are typically three types of patterns exhibited by Daphnia magna normally in aquatic ecosystem. The vertical swimming and cruising of the organism from the top to bottom of beakers is generally considered as a normal behavior but increase in hopping and sinking movements or decrease in swimming speed, staying towards the bottom of beakers is an indicator of stressful condition to the organism. The swimming behavior of Daphnia magna is frequently studied as an indicator of stress. In this study, swimming behavior of Daphnia magna was analyzed using AnimApp and visual observation. Mono exposures to microplastics resulted in reduced swimming speed, increased immobility, and decreased filterability. The increased effects were observed for LDPE, followed by PP and HDPE. Previous studies have investigated the effects of specific microplastic types on Daphnia magna swimming behavior. Magester et al. ( 2021 ) found that exposure to PP microplastics (1-1000 µm) significantly decreased swimming velocity and increased erratic movements. Similarly, Na et al. ( 2021 ) reported a significant decrease in swimming velocity and increased turning behavior in Daphnia magna exposed to HDPE microplastics (10-1000 µm). Choi et al. ( 2018 ) investigated the effects of LDPE microplastics (1-100 µm) and found a significant decrease in swimming velocity and increased turning behavior in Daphnia magna . No previous studies have investigated the impact of microplastics co-exposure on the swimming behavior of Daphnia magna. This study represents the first attempt to examine the combined effects of different types of microplastics on the swimming behavior of Daphnia magna. The researchers observed that Daphnia magna exhibited spinning and erratic movements when exposed to microplastics at concentrations up to 90 mg/L. However, when the concentration exceeded this threshold, co-exposure to various types of microplastics caused the organisms to become immobile, a phenomenon not observed in single exposures. Complete immobility indicates that the organisms experience extreme stress and lack the energy to move, likely due to the interference of microplastics with their appendages and chemosensory organs. The primary factor contributing to the inability to swim was found to be the size of the microplastic particles, with smaller particles leading to greater immobility. These findings align with a previous study conducted by Yide and colleagues, who also reported similar findings. Co-exposure also severely impairs the ability of Daphnia magna to move and detect food, as reported by He et al. ( 2023 ), Galloway et al. ( 2017 ), and Junaid et al. ( 2023 ). Daphnia magna reproduction rate and population density Daphnia magna exhibit high reproductive capacity, with population doubling or even tripling within 3–4 days. Optimal environmental conditions enhance the reproduction of Daphnia magna . In this study, reproduction rate was considered as a measure to assess organism behavior under stressful conditions. The reproduction rate was determined by counting neonates and adult Daphnia magna within the exposure solution, following the method by (Huang et al. 2022 ). The normal reproduction rate was 2.2, while PP, LDPE, and HDPE exhibited reproduction rates of 1.6, 1.5, and 1.3 respectively. Among the mono exposures, HDPE had the lowest reproduction rate, followed by LDPE and PP. Study conducted by Canniff in 2018 reported an average reproduction rate of 1.2 for polyethylene, which is consistent with similar trend observed in this study, possibly due to smaller size (32 µm) of microplastic fragments used (Huang et al. 2022 ; Imhof et al. 2017 ; Schür et al. 2022 ). In case of co-exposure to microplastics, reproduction rate was 0.8, thrice lower than normal rate. Microscopic observations revealed that Daphnia magna had brood chambers containing 2–3 neonates, but they were unable to release them due to stressful conditions and unfavorable environment for neonate laying. The gut of Daphnia magna was filled with microplastics, leading to continuous starvation and hampered movement, which were induced by synergistic effect of microplastics. A study published by Huang et al. ( 2022 ) observed effects of polystyrene microplastics in the Daphnia magna, it was concluded that the reproduction rate was significantly reduced along with delayed development of eggs, reduced body lengths and feeding rates, although the concentrations varied but similar trend is observed in current study. Energy reserves depleted more rapidly in co-exposure scenario compared to mono exposures. Several studies have investigated the effects of microplastics on Daphnia magna reproduction. One study found that exposure to microplastics reduced fecundity of Daphnia magna , resulting in fewer offspring per brood (Jemec et al. 2016 ). Another study found that exposure to microplastics altered the sex ratio of offspring, with a higher proportion of males being produced (Yuan et al. 2018 ). The population density of Daphnia magna decreased over a 5-day period, primarily due to frequent organism deaths. Smaller population size led to more rapid declines, but co-exposure resulted in a sharper reduction in population density as compared to mono exposures, confirming the greater impairment caused by the synergistic effects of microplastics. Similar results were reported by Schrank et al. ( 2019 ). Conclusion and Suggestions In conclusion, Daphnia magna readily consume microplastics, mistaking them for food. Co-exposure to multiple types of microplastics resulted in more pronounced toxicological effects on Daphnia magna , impacting various biological parameters. The presence of PP, HDPE, and LDPE in a mixture reduced reproduction rate, limited swimming activity, ingestion and egestion, population density, and increased immobility compared to Daphnia magna exposed to a single type of microplastic. Co-exposure also led to higher particle ingestion, rapid depletion of energy reserves, and increased stress levels. Further studies should investigate different concentrations and combinations of microplastic polymers to better understand their effects under various environmental conditions. Additionally, research on the pathways of microplastics in the food chain is necessary. Declarations Acknowledgements The experiments were performed at Environmental Toxicology Lab, Institute of Environmental sciences and Engineering (IESE), NUST. The study was supported by National University of Science and Technology (NUST), Islamabad, Pakistan. Auhtor Contributions HA, IH conceived the theoretical framework. 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Nat Ecol Evol, 1(5). https://doi.org.10.1038/S41559-017-0116 Gerdes Z, Hermann M, Ogonowski M, Gorokhova E (2019) A novel method for assessing microplastic effect in suspension through mixing test and reference materials. Sci Rep, 9(1), 1–9. https://doi.org/10.1038/s41598-019-47160-1 Guilhermino L, Martins A, Cunha S, Fernandes JO (2021) Long-term adverse effects of microplastics on Daphnia magna reproduction and population growth rate at increased water temperature and light intensity: Combined effects of stressors and interactions. Sci Total Environ, 784, 147082. https://doi.org/10.1016/j.scitotenv.2021.147082 He S, Tong J, Xiong W, Xiang Y, Peng H, Wang W, Zeng G et al (2023) Microplastics influence the fate of antibiotics in freshwater environments: Biofilm formation and its effect on adsorption behavior. J Hazard Mater, 442, 130078. Huang Z, Hu B, Wang H. (2022) Analytical methods for microplastics in the environment: a review. Environ Chem Lett, 21(1), 383–401. https://doi.org/10.1007/S10311-022-01525-7 Imhof HK, Rusek J, Thiel M, Wolinska J, Laforsch C (2017) Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level? PLoS ONE, 12(11). https://doi.org/10.1371/JOURNAL.PONE.0187590 Isinibilir M, Svetlichny L, Mykitchak T, Türkeri EE., Eryalçın KM, Doğan O, Kideys AE et al (2020) Microplastic consumption and its effect on respiration rate and motility of Calanus helgolandicus from the Marmara Sea. Front Mar Sci, 7, 603321. Jemec A, Horvat P, Kunej U, Bele M, Kržan A (2016) Uptake and effects of microplastic textile fibers on freshwater crustacean Daphnia magna. Environ Pollut, 219, 201–209. https://doi.org/10.1016/J.ENVPOL.2016.10.037 Junaid M, Liu S, Chen G, Liao H, Wang J (2023) Transgenerational impacts of micro(nano)plastics in the aquatic and terrestrial environment. J Hazard Mater, 443. https://doi.org/10.1016/J.JHAZMAT.2022.130274 Khan D, Ali SA (2023) On the novel process of pristine microplastic bio-fragmentation by zebrafish (danio rerio). Arch Environ Contam Toxicol. https://doi.org/10.1007/S00244-023-00987-2/FULLTEXT.HTML Köster M, Paffenhöfer GA (2022) Feeding of marine zooplankton on microplastic fibers. Arch Environ Contam Toxicol, 83(2), 129–141. https://doi.org.10.1007/S00244-022-00948-1/FULLTEXT.HTML Lampert W (2006) Daphnia: Model herbivore, predator and prey. Polish J Ecol. http://pubman.mpdl.mpg.de/pubman/faces/viewItemOverviewPage.jsp?itemId=escidoc:1506950 Lee BY, Choi BS, Kim MS, Park JC, Jeong CB, Han J, Lee JS (2019) The genome of the freshwater water flea Daphnia magna: A potential use for freshwater molecular ecotoxicology. Aquat Toxicol, 210, 69–84. https://doi.org/10.1016/J.AQUATOX.2019.02.009 Lim XZ (2021) Microplastics are everywhere - but are they harmful? Nature, 593(7857), 22–25. https://doi.org/10.1038/D41586-021-01143-3 Magester S, Barcelona A, Colomer J, Serra T (2021) Vertical distribution of microplastics in water bodies causes sublethal effects and changes in Daphnia magna swimming behaviour. Ecotoxicol Environ Saf, 228, 113001. https://doi.org/10.1016/j.ecoenv.2021.113001 Mataji A, Taleshi MS, Balimoghaddas E (2020) Distribution and characterization of microplastics in surface waters and the Southern Caspian sea coasts sediments. Arch Environ Contam Toxicol, 78(1), 86–93. https://doi.org/10.1007/S00244-019-00700-2/FULLTEXT.HTML Na J, Song J, Achar JC, Jung J (2021) Synergistic effect of microplastic fragments and benzophenone-3 additives on lethal and sublethal Daphnia magna toxicity. J Hazard Mater, 402(September 2020), 123845. https://doi.org/10.1016/j.jhazmat.2020.123845 Pacheco A, Martins A, Guilhermino L (2018) Toxicological interactions induced by chronic exposure to gold nanoparticles and microplastics mixtures in Daphnia magna. Sci Total Environ, 628–629, 474–483. https://doi.org/10.1016/j.scitotenv.2018.02.081 Rao SR, Olechnowicz SWZ, Krätschmer P, Jepson JEC, Edwards CM, Edwards JR (2019) Small animal video tracking for activity and path analysis using a novel open-source multi-platform application (animapp). Sci Rep, 9(1). https://doi.org/10.1038/S41598-019-48841-7 Rist S, Baun A, Hartmann NB (2017) Ingestion of micro- and nanoplastics in Daphnia magna – Quantification of body burdens and assessment of feeding rates and reproduction. Environ Pollut, 228, 398–407. https://doi.org/10.1016/j.envpol.2017.05.048 Sarma SS, Nandini S (2006) Review of recent ecotoxicological studies on cladocerans. J Environ Sci Health B. 2006;41(8):1417-30. doi: 10.1080/03601230600964316. PMID: 17090502. Schrank I, Trotter B, Dummert J, Scholz-Böttcher BM, Löder MGJ, LaforschC (2019) Effects of microplastic particles and leaching additive on the life history and morphology of Daphnia magna. Environ Pollut, 255. https://doi.org.10.1016/j.envpol.2019.113233 Schür C, Beck J, Lambert S, Scherer C, Oehlmann J , Wagner M (2022) Effects of microplastics mixed with natural particles on Daphnia magna populations. BioRxiv, 2022.05.04.490562. https://doi.org/10.1101/2022.05.04.490562 Shim WJ, Thomposon RC (2015) Microplastics in the Ocean. Arch Environ Contam Toxicol. 69(3). doi:10.1007/S00244-015-0216-X/FULLTEXT.HTML Sirui Huang, Ruitong J, Nicholas J, Hua D, Wenhui H, Juan-Ying L, Lei Su (2023) Accumulation and re-distribution of microplastics via aquatic plants and macroalgae - A review of field studies, Mar Environ Res, Volume 187, https://doi.org/10.1016/j.marenvres.2023.105951. Test No. 202: Daphnia sp. Acute Immobilisation Test. (2004). https://doi.org/10.1787/9789264069947-EN Veerasingam S, Ranjani M, Venkatachalapathy R, Bagaev A, Mukhanov V, Litvinyuk D, Mugilarasan M, Gurumoorthi K, Guganathan L, Aboobacker VM, Vethamony P (2020) Contributions of Fourier transform infrared spectroscopy in microplastic pollution research: A review. Environ Sci Technol, 51(22), 2681–2743. https://doi.org/10.1080/10643389.2020.1807450 Woo H, Seo K, Choi Y, Kim J, Tanaka M, Lee KH, Choi J (2021) Methods of analyzing microsized plastics in the environment. Appl Sci (Switzerland), 11(22). https://doi.org.10.3390/app112210640 Xu JL, Thomas KV, Luo Z. Gowen AA (2019) FTIR and Raman imaging for microplastics analysis: State of the art, challenges and prospects. Trends in Analyti Chem, 119, 115629. https://doi.org.10.1016/J.TRAC.2019.115629 Yin J, Long Y, Xiao W, Liu D, Tian Q, Li Y, Liu C, Chen L, Pan Y (2023) Ecotoxicology of microplastics in Daphnia: A review focusing on microplastic properties and multiscale attributes of Daphnia. Ecotoxico Environ Saf, 249. https://doi.org.10.1016/j.ecoenv.2022.114433 Yuan S, Li H, Dang Y, Liu C (2018) Effects of triphenyl phosphate on growth, reproduction and transcription of genes of Daphnia magna. Aqu Toxico, 195, 58–66. https://doi.org.10.1016/J.AQUATOX.2017.12.009 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-3545738","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":245812581,"identity":"7674c4a6-9967-4b40-82e4-3f5e0a4697a1","order_by":0,"name":"Hassan Shafiq","email":"","orcid":"","institution":"NUST: National University of Sciences and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Shafiq","suffix":""},{"id":245812582,"identity":"2a7e6b7a-3a4d-40f6-a2ce-1cb7e2d603c3","order_by":1,"name":"Hira Amjad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYBACAzBms+FBF0wgpCUNrkWCKC0MDGyH4QKEtZizn04o+FF2Xoaf//AB5sK2ujoG9uZtEow70nBqsezJ3WDYc+42j+SMtATmmW2HJRh4jpVJMJ7Jwe2wA7kbDHjbbvMY3OAxYOZtOyDBIJFjJsHYVoFby/m3Gwz/tp3jsT9//gNQS50Eg/wbAlpu5G4wBhrOY8CQwwDUwgy0hQekBY/DbrzdYCxzLplH4kaawWGec4cl23jSii0Sz+D2vsH53G2Gb8rs7Pn7Dz98zFNWx8/PfnjjjY87knFqAQI2AxjrAJgLIhIb8OlgYH6AKcaIX8soGAWjYBSMLAAAJgBOmjkG9EwAAAAASUVORK5CYII=","orcid":"","institution":"NUST: National University of Sciences and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hira","middleName":"","lastName":"Amjad","suffix":""},{"id":245812583,"identity":"e8392f74-53c2-4590-aaa4-7c20a1af4fd9","order_by":2,"name":"Imran Hashmi","email":"","orcid":"","institution":"NUST: National University of Sciences and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Imran","middleName":"","lastName":"Hashmi","suffix":""},{"id":245812584,"identity":"d2549767-f068-414c-9cfd-d659b17f4520","order_by":3,"name":"Mariam Ali","email":"","orcid":"","institution":"NUST: National University of Sciences and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mariam","middleName":"","lastName":"Ali","suffix":""}],"badges":[],"createdAt":"2023-11-02 16:09:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3545738/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3545738/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46050116,"identity":"8d1ed2f4-fe76-4ee8-87b6-312a587a1caa","added_by":"auto","created_at":"2023-11-07 23:49:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49150,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size analysis results showing size ranges of PP, LDPE and HDPE.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/5a3594daf51165bcc0684e15.png"},{"id":46050824,"identity":"043d5f33-72fb-4514-ac39-7b8ede22e06d","added_by":"auto","created_at":"2023-11-07 23:57:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315927,"visible":true,"origin":"","legend":"\u003cp\u003eInfrared Spectrums of PP, LDPE, HDPE analyzed by FT-IR in ATR mode.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/0d78c4c9afb09badeaedbdb7.png"},{"id":46050118,"identity":"a884b7aa-f847-4c1c-b0e9-85ef750b932d","added_by":"auto","created_at":"2023-11-07 23:49:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":45345,"visible":true,"origin":"","legend":"\u003cp\u003eLC50 values expressed in mg/L for all exposure types after 48 hours of exposure.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/cfef6845391a83f710b9336c.png"},{"id":46050115,"identity":"a8145dd8-6873-4d5b-9a95-a18652dfeb80","added_by":"auto","created_at":"2023-11-07 23:49:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39901,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of Daphnia magna in response to different exposures to microplastics\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/52730d869d91a42968ad0fd4.png"},{"id":46050823,"identity":"3c621c86-248e-4928-9c37-d7ff3036c422","added_by":"auto","created_at":"2023-11-07 23:57:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":60114,"visible":true,"origin":"","legend":"\u003cp\u003eParticles per Daphnia analyzed and calculated in all sets of microplastic exposures.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/10e41cba0af1d8c426cadbdc.png"},{"id":46050121,"identity":"8ae06e56-09b0-4489-99f0-dfde8b641a2c","added_by":"auto","created_at":"2023-11-07 23:49:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":312520,"visible":true,"origin":"","legend":"\u003cp\u003eAnimApp generated figures showing swimming span of Daphnia magna in various exposure types\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/64151d1831ce39ef24214d51.png"},{"id":46050120,"identity":"1997bac0-dd49-45f2-a569-aa683125ce3c","added_by":"auto","created_at":"2023-11-07 23:49:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":348807,"visible":true,"origin":"","legend":"\u003cp\u003eMicroplastics presence in gut of Daphnia magna at 4X magnification\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/ebd3d8445365c7fb20e57fd1.png"},{"id":46591865,"identity":"c6ccdffd-720b-4053-8f42-4d25bda51f98","added_by":"auto","created_at":"2023-11-16 22:44:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1259687,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3545738/v1/ebb31d04-a226-4808-b85e-81a9cd2c2592.pdf"}],"financialInterests":"","formattedTitle":"The synergistic effect of mono and co-exposure of microplastic suspensions on Daphnia magna’s survival, population density, reproduction rate \u0026amp; swimming behavior.","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlastics, which were once considered to be a remarkable product discovery has now caused irreversible damage to the environment (Shim and Thomposon \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The plastics are generated through the polymerization reactions of non-renewable resource-derived monomers like ethylene, propylene, and styrene (Lim \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Plastics are categorized based on their chemical structures and properties, with notable types including polyethylene, polypropylene, polyvinyl chloride (PVC), and polystyrene (Borrelle et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u0026ldquo;Microplastics\u0026rdquo; are defined as plastic fragments measuring less than 5mm in size in any direction (Khan and Ali \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microplastics are produced through the degradation of larger plastic objects or intentionally manufactured for usage in diverse products such as cosmetics, cleaning agents, and textiles (K\u0026ouml;ster and Paffenh\u0026ouml;fer \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dominant microplastics in lakes and oceans include polypropylene, polyethylene and polystyrene (Bashir and Hashmi \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The presence of microplastics in the environment has raised several concerns as an emerging contaminant, encompassing environmental impact, human health implications, resource depletion, and contributions to climate change (C\u0026oacute;zar et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicroplastics present a substantial chemical risk to marine and freshwater environments (Desforges et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). They possess the ability to adsorb and accumulate toxic pollutants, including persistent organic pollutants (POPs), polycyclic aromatic hydrocarbons (PAHs), and heavy metals, from the surrounding water (Mataji et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Microplastics have been shown to exert toxic effects on zooplankton, which are fundamental aquatic organisms in the marine food chain and their trophic transfer through the marine food web has been confirmed by many studies (Cole et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe zooplankton species, \u003cem\u003eDaphnia magna\u003c/em\u003e, is commonly employed as a test organism in ecotoxicology studies due to its sensitivity to water quality changes and its vital role as a food source for many aquatic organisms (Rist et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eDaphnia magna\u003c/em\u003e exhibits specific characteristics, such as size range of 1 to 5 mm, cyclic parthenogenesis, filter-feeding behavior using branched antennae, and a relatively short lifespan of a few weeks to a few months (Lampert \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Schrank et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Notably, transparency of \u003cem\u003eDaphnia magna\u003c/em\u003e enables researchers to observe internal organs and physiological processes (Yin et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have investigated toxic impacts of microplastics on \u003cem\u003eDaphnia magna\u003c/em\u003e, revealing alterations in swimming behaviour, feeding rate, reproduction rate, tissue accumulation, and increased susceptibility to other environmental pollutants. Cole et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated that exposure to microplastics induced changes in swimming behaviour, feeding rates, and reproductive rates of zooplanktons, particularly copepods and \u003cem\u003eDaphnia magna\u003c/em\u003e. Ellis et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) found that \u003cem\u003eDaphnia magna\u003c/em\u003e may ingest microplastics, leading to their accumulation in tissues over time, which may negatively impact the organism's health and survival. Furthermore, Pacheco et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) analysed that microplastic exposure enhanced the toxicity of other pollutants, such as heavy metals, to \u003cem\u003eDaphnia magna\u003c/em\u003e, indicating interactive effects between microplastics and co-occurring environmental stressors. De Felice et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) investigated the polystyrene microplastics effects on \u003cem\u003eDaphnia magna\u003c/em\u003e\u0026rsquo;s phototactic and swimming behaviour along with reproduction, it was observed that microplastics affect behavioral traits of \u003cem\u003eDaphnia magna\u003c/em\u003e leading to potentially harmful consequences on population dynamics of this zooplanktonic species.\u003c/p\u003e \u003cp\u003eThis study focuses on evaluating the toxicological impacts of both single microplastic type (mono-exposure) and mixed multiple microplastic types (co-exposure) on various aspects of \u003cem\u003eDaphnia magna\u003c/em\u003e. The parameters of research include survival rate, swimming behavior, ingestion behavior, population density, and reproduction rate. Through these experiments, objective is to gain insights into the toxicity mechanisms associated with microplastic exposure in a realistic context and address existing research gaps in this field.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTest organism \u0026amp; culture conditions\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eDaphnia magna\u003c/em\u003e starter culture for this study was procured from a fish hatchery near Rawal Dam, Islamabad. To culture \u003cem\u003eDaphnia magna\u003c/em\u003e, a medium of pure groundwater (1100 feet) from Islamabad, Pakistan was used, which was tested for residual chlorine (Not detected) to avoid toxicity towards the organisms. The medium had a pH range of 7\u0026ndash;8 and dissolved oxygen (DO) levels of 6.5 mg/L (Yin et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The culture conditions included maintaining a temperature of 20\u0026ndash;22\u0026deg;C, following a 12-hour light/dark cycle. Chlorella vulgaris, a single-celled algae, procured from Plant virology lab, Atta-ur-Rahman School of Applied Biosciences (ASAB), NUST was provided as a food source for \u003cem\u003eDaphnia magna\u003c/em\u003e at a concentration of 1\u0026ndash;2 mg/L, with feeding occurring every 24 hours (Sarma and Nandini, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The final culture density of \u003cem\u003eDaphnia magna\u003c/em\u003e was approximately 1\u0026ndash;2 daphnids/ml, which was considered suitable for laboratory experiments (Lee et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMicroplastics Preparation\u003c/h2\u003e \u003cp\u003eCrystalline PP, HDPE and LDPE beads, trademarked under Marlex, were purchased for the preparation of microplastics (Woo et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The beads were rinsed with ethanol and allowed to air dry before undergoing a melting procedure. For PP, beads were melted at 80\u0026deg;C for 5\u0026ndash;10 minutes in a glass petri dish (Dimensions 100x15 mm) on a hotplate. Similarly, HDPE and LDPE were heated at 65\u0026deg;C and 55\u0026deg;C, respectively, for 5\u0026ndash;10 minutes. These temperatures were kept below the melting point of the plastics to prevent fumes, pre-oxidation, and chemical degradation (Xu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe melted plastics were shaped into rectangular pallets, with the help of a glass stirrer, to facilitate their crushing process ergonomically. A stainless-steel foot filer, typically used to remove dead skin, was used to crush the plastic pallets. The rough graded surface of the foot filer produced plastic debris. This procedure was performed for all three types of microplastics.\u003c/p\u003e \u003cp\u003eThe plastic debris obtained from filing wer subjected to a sieving process using ASTM Standard Sieve No. 635 (32 \u0026micro;m) and No. 450 (20 \u0026micro;m) (American Standard Test Sieve Series - Endecotts). The plastic debris were washed with ethanol, and a mixture of crushed microplastic debris in deionized water was obtained (Woo et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The mixture was sonicated at 30\u0026deg;C for 10 minutes to prevent agglomeration. Filtration was carried out using a 32 \u0026micro;m sieve, resulting in a solution of microplastics\u0026thinsp;\u0026lt;\u0026thinsp;32 \u0026micro;m. The solution was further filtered using a 0.45 \u0026micro;m glass fiber filter paper to collect the microplastics. The plastic particles were washed, dried at room temperature, and subjected to physico-chemical characterization. In this study, microplastics ranging from 0 to 32 \u0026micro;m were used for all experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhysico-Chemical Characterization of Microplastics\u003c/h2\u003e \u003cp\u003eThe microplastics prepared were of irregular shape, mostly fragments, fibers were also present. The color of microplastics was transparent due to the transparent color of crystalline beads used. The density of each microplastic polymer was 0.91, 0.94 and 0.90 g/cm\u003csup\u003e3\u003c/sup\u003e respectively for PP, LDPE, HDPE as mentioned on the label (Chanpiwat and Damrongsiri \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To validate the polymer and size FT-IR and particle size analyzer was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePhysical Characterization by Particle Size Analyzer\u003c/h2\u003e \u003cp\u003eThe size of microplastic particles was determined using a Horiba LA-300 Particle Size Analyzer, which has a measurement range of 0.1 to 600 \u0026micro;m. Each type of microplastic (PP, LDPE, HDPE) was tested separately in a wet medium, specifically deionized water (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The PP, HDPE and LDPE microplastics were found to be within the desired range of 0\u0026ndash;32 \u0026micro;m, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The average mean size of PP particles was 31 \u0026micro;m, followed by LDPE particles with a size of 12 \u0026micro;m and HDPE particles with a size of 26 \u0026micro;m.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCompositional Characterization by \u0026lsquo;Fourier-transform infrared\u0026rsquo; spectroscopy\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and procedure\u003c/h2\u003e \u003cp\u003eThe experiment was designed to investigate the effects of mono exposure and co exposure of microplastics on various parameters of \u003cem\u003eDaphnia magna\u003c/em\u003e. To assess the toxicological effects, exposure medium was prepared, and pre-exposure conditions were established. \u003cem\u003eDaphnia magna\u003c/em\u003e individuals were transferred from the culture medium to a 2 L glass beaker filled with deionized water. Prior to microplastic exposure, \u003cem\u003eDaphnia magna\u003c/em\u003e were starved for a minimum of 4 hours to ensure they would readily consume microplastics, as microplastics often resemble food particles (Canniff and Hoang \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the \u003cem\u003emono-exposure experiments\u003c/em\u003e, 20 adult \u003cem\u003eDaphnia magna\u003c/em\u003e were placed in 80 ml beakers containing 50 ml of deionized water with microplastics. The concentrations of microplastics for each polymer type (PP, HDPE, LDPE) were 30, 60, 90, 120, and 150 mg/L, with a control group for each type. The microplastic fragments were weighed and added to 500 ml of deionized water, followed by sonication to disperse any agglomerates. During the transfer of solution to beakers, stock suspension was agitated to ensure thorough mixing of the microplastics.\u003c/p\u003e \u003cp\u003eIn case of \u003cem\u003eco-exposure experiment\u003c/em\u003e, a mixture containing 33% of each microplastic polymer (PP, HDPE, LDPE) was prepared to simulate a natural environment where multiple types of microplastics are present, predominantly in fragmented form. Solutions with microplastic concentrations of 30, 60, 90, 120, and 150 mg/L were created for co-exposure experiments. This approach adds novelty to the research by considering the combined presence of different microplastic types.\u003c/p\u003e \u003cp\u003eTo assess the \u003cem\u003esurvival of Daphnia magna\u003c/em\u003e in both exposure scenarios, a 48-hour acute toxicity test was conducted following Test No. 202: \u003cem\u003eDaphnia magna\u003c/em\u003e Sp. Acute Immobilisation Test (2004). Each exposure unit was provided with 0.1 mg of feed. The survival rate, indicated by the LC50 value, was determined using Probit Analysis (Na et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn order to study \u003cem\u003eingestion and bioaccumulation\u003c/em\u003e of microplastics in \u003cem\u003eDaphnia magna\u003c/em\u003e, similar concentrations of microplastics were used for both mono and co-exposure experiments. For mono-exposure, microplastics were dyed with Nile red fluorescent dye to visualize their ingestion by \u003cem\u003eDaphnia magna\u003c/em\u003e. A dye solution of 0.1 mg/10 ml was prepared using acetone, and the microplastics were added to the dye, filtered, and thoroughly washed to remove excess dye (Frydkj\u0026aelig;r et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The fluorescently labeled microplastics were used in suspensions ranging from 30 to 150 mg/L. 20 \u003cem\u003eDaphnia magna\u003c/em\u003e individuals were exposed to these suspensions for 120 hours (5 days) and analyzed under a fluorescent microscope (Optika B-350) to observe microplastic ingestion.\u003c/p\u003e \u003cp\u003eTo determine the \u003cem\u003ebioaccumulation of microplastics\u003c/em\u003e in both exposure scenarios, 40% of the population (8 \u003cem\u003eDaphnia magna\u003c/em\u003e) from each exposure beaker was taken. The draft digestion method with a slight modification was employed, using 50% nitric acid to digest the \u003cem\u003eDaphnia magna\u003c/em\u003e for 3 hours at a temperature of 50\u003csup\u003eo\u003c/sup\u003eC. This process was repeated for both exposure groups. The digested solution was diluted with deionized water, filtered through a glass fiber filter paper, and dried at room temperature (Canniff and Hoang \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor mono-exposure, filter papers containing digested \u003cem\u003eDaphnia magna\u003c/em\u003e were directly examined under a stereomicroscope, and microplastics were quantified by counting the particles per organism. In case of co-exposure, although microplastics were present, their specific types could not be identified. The filter paper containing the microplastics was subjected to ATR-FTIR analysis to observe the type of plastic accumulated in \u003cem\u003eDaphnia magna\u003c/em\u003e at different concentrations, while the number of microplastic particles was quantified (Gerdes et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo calculate the \u003cem\u003ereproduction rate\u003c/em\u003e per organism per day, total number of neonates produced in mono or co-exposure groups were divided by the number of adult \u003cem\u003eDaphnia magna\u003c/em\u003e in the same treatment replicate on the same day. This value was then divided by the number of days to obtain the reproduction rate.\u003c/p\u003e \u003cp\u003eRepresenting the reproduction rate as \"RR,\" the number of neonates on day \"x\" as \"N\u003csub\u003e(x)\u003c/sub\u003e,\" and the number of alive adult Daphnia magna on day \"x\" as \"A\u003csub\u003e(x)\u003c/sub\u003e.\"\u003c/p\u003e \u003cp\u003eMathematically, the reproduction rate may be expressed as:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRR\u0026thinsp;=\u0026thinsp;N\u003csub\u003e(x)\u003c/sub\u003e / A\u003csub\u003e(x\u003c/sub\u003e Eq: 1\u003c/h2\u003e \u003cp\u003eTo determine if there were significant differences between treatments, the average cumulative number of neonates produced by surviving adults over a 5-day period was calculated. Additionally, average reproductive rate per day for each treatment was calculated and compared (Imhof et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003ePopulation density\u003c/em\u003e was calculated by counting the number of \u003cem\u003eDaphnia magna\u003c/em\u003e in a sample of water. Population density can also be observed visually using a colony counter when the sample is poured into a petri dish (Guilhermino et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo assess \u003cem\u003eswimming behavior\u003c/em\u003e, 1-minute-long videos were recorded for each replicate in both exposure types. The swimming patterns, such as hopping and sinking, cruising, and vertical swimming, were visually noted. The videos were analyzed using a software called AnimApp, specifically designed for insect tracking. AnimApp is a python based tracking module. The software tracked the swimming behavior of the organisms and identified changes in their swimming patterns, which can indicate stress in Daphnia magna. The threshold in AnimApp was adjusted to accurately detect the organisms, and based on the tracking data, a plot was generated (Rao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eDaphnia magna survival\u003c/h2\u003e\n \u003cp\u003eThe results of the present study demonstrated that survival of Daphnia magna decreased as the concentration of microplastics increased in both sets of experiments. Specifically, LC50 values for mono exposure were found to be 120 mg/L for PP, 107 mg/L for LDPE, and 123 mg/L for HDPE. However, when considering the synergistic effect of microplastics, LC50 value decreased to 77 mg/L. (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe mortality to concentration (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) demonstrates that co-exposure of microplastics caused maximum mortality of \u003cem\u003eDaphnia magna\u003c/em\u003e i.e 15 individuals followed by PP (13), HDPE (12) and LDPE (12) respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eIngestion of microplastics by Daphnia magna\u003c/h2\u003e\n \u003cp\u003eThe ingestion of microplastics in \u003cem\u003eDaphnia magna\u003c/em\u003e increased with increase in concentration of microplastics in both of the experiments. The mono exposure of microplastics had a relatively smaller number of particles inside the \u003cem\u003eDaphnia magna\u003c/em\u003e than co-exposure which showed that synergistic effect of microplastics was causing more accumulation of microplastics. Figure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, shows the ingestion of microplastics in \u003cem\u003eDaphnia magna\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eFT-IR results of ingested microplastics revealed that at lower concentration LDPE was the abundant microplastic type accumulated in \u003cem\u003eDaphnia magna\u003c/em\u003e. When concentration increased to 120 mg/L, HDPE was more pronounced followed by PP and LDPE whereas in case of 150 mg/L concentration PP was most abundantly present inside \u003cem\u003eDaphnia magna\u003c/em\u003e followed by HDPE and LDPE. As reported in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMicroplastics abdudance in \u003cem\u003eDaphnia magna\u003c/em\u003e calculated by FT-IR analysis\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConc. (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMicroplastics present\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAbundance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE, HDPE, PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLDPE\u0026thinsp;\u0026gt;\u0026thinsp;PP\u0026thinsp;\u0026gt;\u0026thinsp;HDPE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e60\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE, HDPE, PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLDPE\u0026thinsp;\u0026gt;\u0026thinsp;PP\u0026thinsp;\u0026gt;\u0026thinsp;HDPE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e90\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE, HDPE, PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eLDPE\u0026thinsp;\u0026gt;\u0026thinsp;HDPE\u0026thinsp;\u0026gt;\u0026thinsp;PP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e120\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE, HDPE, PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eHDPE\u0026thinsp;\u0026gt;\u0026thinsp;PP\u0026thinsp;\u0026gt;\u0026thinsp;LDPE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e150\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLDPE, HDPE, PP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ePP\u0026thinsp;\u0026gt;\u0026thinsp;HDPE\u0026thinsp;\u0026gt;\u0026thinsp;LDPE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eDaphnia magna swimming behavior\u003c/h2\u003e\n \u003cp\u003eIn this study, healthy \u003cem\u003eDaphnia magna\u003c/em\u003e specimens were collected from each replicate of both mono and co-exposure experiments. These specimens were observed and recorded under a 4X magnification for a duration of 1 minute, and videos were created for further analysis using the AnimApp software.\u003c/p\u003e\n \u003cp\u003eThe results of the mono-exposure experiments revealed that the mobility and swimming pattern of \u003cem\u003eDaphnia magna\u003c/em\u003e were negatively affected as the concentration of microplastics increased. The movement span of \u003cem\u003eDaphnia magna\u003c/em\u003e was reduced in all three types of mono exposures.\u003c/p\u003e\n \u003cp\u003eIn case of co-exposure, synergistic effect of microplastics was found to be significant. This was evident from the increased hopping and sinking behavior of \u003cem\u003eDaphnia magna\u003c/em\u003e, indicating extreme stressful conditions. As the concentration of microplastics increased, a decrease in velocity and an increase in immobility were observed in the co-exposure group, but not in the mono-exposure group (as shown in Fig. 6). This was observed that the normal behavior of vertical swimming and cruising of the organisms from the top to the bottom of the beakers was absent at higher concentrations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eDaphnia magna reproduction rate and population density\u003c/h2\u003e\n \u003cp\u003eOn the 5th day of exposure, reproduction rate was calculated for both mono and co-exposure experiments. In the mono exposures, reproduction rate remained relatively constant but gradually decreased with increasing concentration of microplastics. However, in case of co-exposure, synergistic effect of microplastics had a significant impact on reproduction rate. It was observed that reproduction rate in co-exposure group was reduced to half compared to the normal single type microplastic exposure, as indicated in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Furthermore, population density was found to decrease with an increase in microplastic concentration. Although low population size played a role in determining the population density, it was evident that the visible number of organisms in all the exposure setups was reduced when cross-checked using a colony counter.\u003c/p\u003e\n \u003cp\u003e\u003c/p\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eReproduction rate, as calculated on 5th day.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eExposure Conc. (mg/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLDPE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHDPE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCo-exposure\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eAverage\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.64\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.556\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.254\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.82\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDaphnia magna survival\u003c/h2\u003e \u003cp\u003eThe LC50 results of the study showed that mono exposure of microplastics of all three types (PP, HDPE, LDPE) resulted in death of 50% of \u003cem\u003eDaphnia magna\u003c/em\u003e population at certain concentrations of co and mono exposure. This indicates that in realistic scenarios where multiple types of microplastics are present in aquatic environments, plastics may be more harmful at lower concentrations.\u003c/p\u003e \u003cp\u003eIsinibilir and co researchers (2022) concluded in their study that survival of \u003cem\u003eDaphnia magna\u003c/em\u003e was affected by microplastics of a single type, but higher concentrations with algae had a greater decline on their survival. Similarly, Gerdes et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found that PET microplastics had a low survival rate with an LC50 value of 160 mg/L for \u003cem\u003eDaphnia magna\u003c/em\u003e. In comparison, current study reported LC50 values ranging from 107 to 123 mg/L for individually exposed microplastics, while synergistic effect of microplastic mixtures had a lower LC50 value of 77 mg/L. This indicates that mixture of microplastics is more lethal at lower concentrations, posing a greater threat to \u003cem\u003eDaphnia magna\u003c/em\u003e and potentially disrupting food chain, as they are an important food source for predators. It's important to note that LC50 values are just one measure of toxicity, and other factors such as growth, reproduction, and behavior may also be affected by microplastics. Additionally, current study used irregularly sized microplastic fragments, which mimic the dominant form of microplastics found in marine and freshwater environments. Na et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that microplastic fragments alone were 80 times more lethal in acute experiments compared to regularly sized microplastics.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIngestion of microplastics by Daphnia magna.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe digestion procedure confirmed the ingestion of microplastics in both exposures. In terms of mono exposure, highest ingestion rate was observed for HDPE microplastics, with an average of 8.1 particles per \u003cem\u003eDaphnia magna\u003c/em\u003e organism, followed by PP and LDPE with 7 and 6.3 particles respectively. Canniff and Hoang (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that there exists direct relationship between concentration and number of particles ingested, although the values were relatively small (0.8-2 microplastics per \u003cem\u003eDaphnia magna\u003c/em\u003e) due to larger particle size (63–75 µm) and lower concentration exposures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003emicroplastics in real environmental conditions is needed, rather than relying solely on synthetic lab-based environments. They observed that \u003cem\u003eDaphnia magna\u003c/em\u003e readily ingests both types of microplastics at all concentrations. The ingestion of microplastics provides evidence that the primary food source for predators accumulates plastic, and there is a potential for these organisms to transfer microplastics in the food chain at the foundational level (Fabricant et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ingestion of irregularly shaped microplastics leads to adsorption of microplastic particles in the gut of \u003cem\u003eDaphnia magna\u003c/em\u003e, this fact is supported by the study conducted on regular and irregular shaped microplastics in which there was reduced or no egestion of irregularly sized microplastics than the regular microplastics (Frydkjær et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDaphnia magna swimming behavior\u003c/h2\u003e \u003cp\u003eThe swimming pattern of \u003cem\u003eDaphnia magna\u003c/em\u003e is an indicator of how the organism is behaving in different environments. There are typically three types of patterns exhibited by \u003cem\u003eDaphnia magna\u003c/em\u003e normally in aquatic ecosystem. The vertical swimming and cruising of the organism from the top to bottom of beakers is generally considered as a normal behavior but increase in hopping and sinking movements or decrease in swimming speed, staying towards the bottom of beakers is an indicator of stressful condition to the organism. The swimming behavior of \u003cem\u003eDaphnia magna\u003c/em\u003e is frequently studied as an indicator of stress. In this study, swimming behavior of \u003cem\u003eDaphnia magna\u003c/em\u003e was analyzed using AnimApp and visual observation. Mono exposures to microplastics resulted in reduced swimming speed, increased immobility, and decreased filterability. The increased effects were observed for LDPE, followed by PP and HDPE. Previous studies have investigated the effects of specific microplastic types on \u003cem\u003eDaphnia magna\u003c/em\u003e swimming behavior. Magester et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that exposure to PP microplastics (1-1000 µm) significantly decreased swimming velocity and increased erratic movements. Similarly, Na et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported a significant decrease in swimming velocity and increased turning behavior in \u003cem\u003eDaphnia magna\u003c/em\u003e exposed to HDPE microplastics (10-1000 µm). Choi et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) investigated the effects of LDPE microplastics (1-100 µm) and found a significant decrease in swimming velocity and increased turning behavior in \u003cem\u003eDaphnia magna\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNo previous studies have investigated the impact of microplastics co-exposure on the swimming behavior of Daphnia magna. This study represents the first attempt to examine the combined effects of different types of microplastics on the swimming behavior of Daphnia magna. The researchers observed that Daphnia magna exhibited spinning and erratic movements when exposed to microplastics at concentrations up to 90 mg/L. However, when the concentration exceeded this threshold, co-exposure to various types of microplastics caused the organisms to become immobile, a phenomenon not observed in single exposures. Complete immobility indicates that the organisms experience extreme stress and lack the energy to move, likely due to the interference of microplastics with their appendages and chemosensory organs. The primary factor contributing to the inability to swim was found to be the size of the microplastic particles, with smaller particles leading to greater immobility. These findings align with a previous study conducted by Yide and colleagues, who also reported similar findings. Co-exposure also severely impairs the ability of Daphnia magna to move and detect food, as reported by He et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Galloway et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), and Junaid et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eDaphnia magna reproduction rate and population density\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDaphnia magna\u003c/em\u003e exhibit high reproductive capacity, with population doubling or even tripling within 3–4 days. Optimal environmental conditions enhance the reproduction of \u003cem\u003eDaphnia magna\u003c/em\u003e. In this study, reproduction rate was considered as a measure to assess organism behavior under stressful conditions. The reproduction rate was determined by counting neonates and adult \u003cem\u003eDaphnia magna\u003c/em\u003e within the exposure solution, following the method by (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The normal reproduction rate was 2.2, while PP, LDPE, and HDPE exhibited reproduction rates of 1.6, 1.5, and 1.3 respectively. Among the mono exposures, HDPE had the lowest reproduction rate, followed by LDPE and PP. Study conducted by Canniff in 2018 reported an average reproduction rate of 1.2 for polyethylene, which is consistent with similar trend observed in this study, possibly due to smaller size (32 µm) of microplastic fragments used (Huang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Imhof et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Schür et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn case of co-exposure to microplastics, reproduction rate was 0.8, thrice lower than normal rate. Microscopic observations revealed that \u003cem\u003eDaphnia magna\u003c/em\u003e had brood chambers containing 2–3 neonates, but they were unable to release them due to stressful conditions and unfavorable environment for neonate laying. The gut of \u003cem\u003eDaphnia magna\u003c/em\u003e was filled with microplastics, leading to continuous starvation and hampered movement, which were induced by synergistic effect of microplastics. A study published by Huang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) observed effects of polystyrene microplastics in the Daphnia magna, it was concluded that the reproduction rate was significantly reduced along with delayed development of eggs, reduced body lengths and feeding rates, although the concentrations varied but similar trend is observed in current study. Energy reserves depleted more rapidly in co-exposure scenario compared to mono exposures. Several studies have investigated the effects of microplastics on \u003cem\u003eDaphnia magna\u003c/em\u003e reproduction. One study found that exposure to microplastics reduced fecundity of \u003cem\u003eDaphnia magna\u003c/em\u003e, resulting in fewer offspring per brood (Jemec et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Another study found that exposure to microplastics altered the sex ratio of offspring, with a higher proportion of males being produced (Yuan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe population density of \u003cem\u003eDaphnia magna\u003c/em\u003e decreased over a 5-day period, primarily due to frequent organism deaths. Smaller population size led to more rapid declines, but co-exposure resulted in a sharper reduction in population density as compared to mono exposures, confirming the greater impairment caused by the synergistic effects of microplastics. Similar results were reported by Schrank et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"Conclusion and Suggestions","content":"\u003cp\u003eIn conclusion, \u003cem\u003eDaphnia magna\u003c/em\u003e readily consume microplastics, mistaking them for food. Co-exposure to multiple types of microplastics resulted in more pronounced toxicological effects on \u003cem\u003eDaphnia magna\u003c/em\u003e, impacting various biological parameters. The presence of PP, HDPE, and LDPE in a mixture reduced reproduction rate, limited swimming activity, ingestion and egestion, population density, and increased immobility compared to \u003cem\u003eDaphnia magna\u003c/em\u003e exposed to a single type of microplastic. Co-exposure also led to higher particle ingestion, rapid depletion of energy reserves, and increased stress levels. Further studies should investigate different concentrations and combinations of microplastic polymers to better understand their effects under various environmental conditions. Additionally, research on the pathways of microplastics in the food chain is necessary.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were performed at Environmental Toxicology Lab, Institute of Environmental sciences and Engineering (IESE), NUST. The study was supported by National University of Science and Technology (NUST), Islamabad, Pakistan.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuhtor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHA, IH conceived the theoretical framework. HA provided technical support on the microplastic experiments. HS planned and carried out the experiments, and analyzed data. HS wrote the manuscript with input from MA, and in consultation with IH, HA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmerican Standard Test Sieve Series (ASTM) - Endecotts. (n.d.). Retrieved August 1, 2022, from https://www.endecotts.com/faq/sieves-and-calibration/american-standard-test-sieve-series-astm/\u003c/li\u003e\n\u003cli\u003eBashir A and Hashmi I (2022) Detection in influx sources and estimation of microplastics abundance in surface waters of Rawal Lake, Pakistan. Heliyon. 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Aqu Toxico, 195, 58\u0026ndash;66. https://doi.org.10.1016/J.AQUATOX.2017.12.009\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3545738/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3545738/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eMicroplastics represent a pervasive contaminant in aquatic ecosystems, posing substantial risks to aquatic biota. This study sought to elucidate the toxicological impacts of three prevalent plastic polymers high-density polyethylene (HDPE), low-density polyethylene (LDPE), and polypropylene (PP) in both mono and co-exposure scenarios on Daphnia. Employing a range of toxicological endpoints, investigation assessed Daphnia magna's ingestion, bioaccumulation, population density, swimming behavior, and reproduction rate in response to microplastics measuring 0–32 µm, which were synthesized in laboratory using virgin polymer beads\u003c/em\u003e. \u003cem\u003eThe findings revealed that synergistic effects of microplastics in a co-exposure medium pose a greater hazard to the organism than mono-exposure to individual microplastics (PP, LDPE, and HDPE). In co-exposure scenerio, LC50 value decreased to 77 mg/L, compared to 120, 123, and 109 mg/L for PP, LDPE, and HDPE, respectively. Daphnia magna exhibited a more pronounced response to co-exposure, characterized by reduced survival rates, increased microplastic ingestion, reduced reproduction rates and population densities. Furthermore, co-exposure scenarios led to increased erratic swimming movements relative to mono-exposure, with immobility and energy deficiency observed across all exposure types, albeit with a greater magnitude in co-exposure settings.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"The synergistic effect of mono and co-exposure of microplastic suspensions on Daphnia magna’s survival, population density, reproduction rate \u0026amp; swimming behavior.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-07 23:49:21","doi":"10.21203/rs.3.rs-3545738/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e9853c5-19d3-410a-9441-11aac43bfde8","owner":[],"postedDate":"November 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-12-29T01:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-07 23:49:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3545738","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3545738","identity":"rs-3545738","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}