The Effect of Chronic Microplastic Exposure Towards the Growth, Biochemical Responses and Histological Changes of the Juvenile Sea Cucumber Holothuria Scabra

The Effect of Chronic Microplastic Exposure Towards the Growth, Biochemical Responses and Histological Changes of the Juvenile Sea Cucumber Holothuria Scabra | 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 Effect of Chronic Microplastic Exposure Towards the Growth, Biochemical Responses and Histological Changes of the Juvenile Sea Cucumber Holothuria Scabra Sarah Syazwani Shukhairi, Nurzafirah Mazlan, Nur Nashrah Abd Rahman, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4412255/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jun, 2025 Read the published version in Environmental Science and Pollution Research → Version 1 posted 5 You are reading this latest preprint version Abstract Microplastics (MPs), are minuscule plastic particles less than 5 millimeters in size, originating from the degradation of larger plastic debris. They are found in various sources and posing a significant threat to marine ecosystems. Sea cucumber Holothuria scabra is a high value commercial species of sea cucumber. They are also crucial in maintaining a clean seabed and recycling nutrients in the ocean ecosystem. This research aimed to investigate the toxicity effects of microplastics on the well-being of juvenile sea cucumber H. scabra . Over 60 days treatment period, polymethylmethacrylate MPs were exposed to the juvenile sea cucumber diet at concentrations of 0.6 MPs/g, 1.2 MPs/g and 10 MPs/g to observe changes in their growth, biochemical responses, and histological alteration. The mean weight, weight gain percentage and specific growth rate exhibited significant differences (p < 0.05) with the control group displaying the highest SGR value of 1.22 ± 0.35%. Mortality was observed in treatment 2 and 3, respectively. Notably, a disruption in enzyme assays was also observed (p < 0.05). The findings of growth rates and biochemical responses were further supported by histological observation, uncovering injuries and loss of cellular components in respiratory trees and intestines. This study enhance our understanding of the toxicity mechanism associated with MPs in filter-feeding organisms. Echinoderms Microplastics Holothuria scabra PMMA Chronic Toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Plastic waste serves as a clear indicator of the global consequences of anthropogenic activities. The inherent hazard of these materials lies predominantly in their original polymers, additives, and byproducts despite their plastic characteristics. Degraded plastics exhibit characteristics such as fragility, discoloration, dullness, brittleness, flakiness, and powdery texture (Andrady, 2011 ). Following their release, into the environment, these particles undergo gradual fragmentation into smaller debris, often referred to as microplastics due to the influence of biological, chemical, and mechanical factors. Rivers, stormwater runoff, and sewage effluents serve as significant pathways for the transport of plastic debris to the ocean, as highlighted by Lebreton et al., ( 2017 ). Microplastics (MPs), ranging in size less than 5 mm, have become prevalent in oceans globally, spanning from surface to sediments. The MPs abundance has been discovered in surface water and sediments in Malaysia (Khalik et al., 2018 ; Noik et al., 2015 ; Sarijan et al., 2018 ). There is a growing realization that MPs are contaminating seafood as a result of their uptake in habitats, processing, or packaging (Cox et al., 2019 ). Mohsen et al., ( 2023 ) reported the presence of microplastics in canned, instant, and salt-dried sea cucumber products with an average of 0 to 4 MPs per individual in their findings. In 50 canned fish samples obtained from Iranian hypermarkets, a total of 128 microplastics were identified (Akhbarizadeh et al., 2020 ). Microplastics were found in green-lipped mussel Perna canaliculus from six out of nine locations in New Zealand, with abundances ranging from 0 to 1.5 particles per mussel (Webb et al., 2019 ). Holothurians are marine invertebrates that are members belonging to Phylum Echinodermata and are also referred to as sea cucumbers. These deposit feeders are often found on the seabed, utilizing their ability to convert the habitat’s substrate into a food source (Manuputty et al., 2019 ). Many sea cucumbers are commercially harvested and dried for human consumption or medical purposes, particularly in Asian nations. Sea cucumber offers an exceptional nutritional profile that includes vitamins, minerals, and amino acids as well as pharmacological actions which include wound healing, anti-inflammatory, and antioxidant (Bordbar et al., 2011 ; Shi et al., 2016 ). Holothuria scabra is one of the rare tropical species that prefers ordinary coastal areas to coral reefs and muddy sand habitats. Due to their daily burrowing cycle and non-selective feeding behaviours, sea cucumbers generally move sluggishly and often bury themselves in the sediment. They inadvertently consume MPs as they use their tentacles to collect sediments, which eventually leads to an increase in the intake of plastics. As the increased demand and value of sea cucumber products pose a significant threat to wild populations, sea cucumber mariculture offers a pathway for the restoration of wild stocks (Purcell et al., 2014 ). Cultivation of sea cucumbers primarily takes place in ponds and coastal shallow waters, regions susceptible to pollution from environmental contaminants such as MPs. In their natural habitat, sea cucumbers fulfill their nutrient requirements by consuming and digesting algae and organic debris in sediments, including those contaminated with microplastics (Purcell et al., 2014 ). This ecological dynamic also affects sea cucumbers in pond cultivation, given that the sediments used are sourced from the natural environment, an inevitable factor contributing to potential exposure to MPs. Research findings indicate various adverse impacts of MPs on sea cucumbers. Ingestion of MPs negatively impacts weight and growth, affecting the digestive system, gut microbiota, and altered biochemical assays (Iwalaye et al., 2020 ; Mohsen et al., 2019 , 2020 ). Histological analysis also showed abnormalities in the respiratory tree caused by the penetration of MPs (Mohsen et al., 2021b ). While studies have detected microplastics in sea cucumbers, the extent of their toxicological impact remains unclear. The physiological, and biochemical functions and the organ tissue structure of sea cucumbers may be adversely affected due to the microplastic intake. Toxicological data for the benthic filter feeders, including sea cucumbers, are still under assessment and have yet to be fully published, given the potential differences in plastic polymers and their toxicities compared to fishes and other marine species. The growth rate, biochemical enzymes, and histological structures will be measured after the sea cucumbers were exposed to different microplastic concentrations treatments for 60 days, contributing to a better understanding of the response mechanisms and adaptation strategies of H. scabra to hypoxia stress induced by microplastics. 2. Materials and Methods 2.1 Experimental Animal The experiment was performed in the Integrated Multi-Trophic Aquaculture (IMTA) hatchery at Borneo Marine Research Institute, Universiti Malaysia Sabah, Sabah, Malaysia. Sea cucumber H. scabra (n = 36) were obtained from a hatchery,located in Tuaran, Sabah, Malaysia with an average weight of 15 to 18 g. The study animals were acclimated in a water recirculation tank supplied with aeration and fed with sargassum powder for one week before the experiment. 2.2 Experimental Setup Polymethylmethacrylate (PMMA-MPs) used for construction sites were obtained from the Crustacean Hatchery of Universiti Malaysia Sabah and was prepared using a grinder and sieved using an 870-micrometer sieve. The polymers of MPs were identified using Fourier Transform Infrared Spectroscopy (FTIR). The experimental animals were divided into four groups (including control)with three replicates. The stocking density was four animals per m 3 . The animal feed was composed of Sargassum powder at 3% biomass. H. scabra were exposed to the diet that was mixed with MPs in three treatments of 0.6 MPs/g (Treatment 1), 1.2 MPs/g (Treatment 2) and 10 MPs/g (Treatment 3) for 60 days to investigate the effects of chronic PMMA-MPs exposure on the growth and physiological status of juvenile sea cucumber (Mohsen et al., 2021b ). 2.3 Rearing Conditions The juvenile sea cucumbers were distributed in tanks provided with aeration. The water temperature was 29 to 30°C, which was the optimum temperatures for the growth of sea cucumbers. The pH was 7 to 8 and the salinity was 33 to 34 ppt. Four juvenile sea cucumbers were stocked per tank in a 1 m³ tank. During the experiment, sea cucumbers were fed once a day and the 50% water exchange was done daily. 2.4 Growth Rate Evaluation The impact of dietary exposure to PMMA-MPs on the growth rate of juvenile sea cucumber was evaluated according to Bai et al. ( 2018 ) and Mohsen et al. ( 2021b ). The juvenile sea cucumbers’ weight was calculated at the start and end of the experiments by shedding excess water off the animals of the same size until they reached a constant weight. Sea cucumbers were weighed two days after the last meal on day 60. Weight gain percentage and specific growth rate (SGR) were calculated according to the following equations: Weight Gain Percentage (%) = (Final Weight -Initial Weight) \(x\) 100 Specific Growth Rate (SGR) = \(100 x \frac{\left(InFW-InIW\right)}{T}\) where IW and FW were the initial and final body weights of sea cucumbers, and T was the duration of the experiment (Bai et al., 2018 ; Mohsen et al., 2021b ). 2.5 Enzyme Assay Analysis The enzymes activities were studied to examine the physiological condition of H. scabra after prolonged PMMA-MPs exposure. Coelomic fluid was collected in test tubes and stored at -80°C until further analysis (Mohsen et al., 2021b ). The immune, digestive, and oxidative enzymes were evaluated spectrophotometrically using commercial kits. Acid phosphatase (ACP) activities were determined spectrophotometrically at a wavelength of 405 nm with the Acid Phosphatase Activity assay kit (p-nitrobenzene phosphate (PNPP) method) obtained from ElabScience (United States). Digestive enzyme lipase was measured at 710 nm using commercial kits (Macklin (China)). malondialdehyde (MDA) and superoxide dismutase (SOD) were measured at 532 nm and 600 nm respectively using kit produced from Macklin (China). 2.6 Histological Analysis The respiratory trees and the intestines of the juvenile sea cucumber H. scabra were removed carefully from the animal and fixed into a 10% neutral buffered formalin solution. All the tissues underwent tissue processing and embedded in paraffin blocks. Then, the samples were sectioned into 5 µm thickness and stained with Haematoxylin and Eosin (H & E) staining (Lei et al., 2018 ). The slides were examined under a microscope with an attached camera (Mohsen et al., 2021b ). 2.7 Statistical Analysis One-way ANOVA followed by Tukey’s test for multiple comparisons was used to compare the specific growth rate and enzyme activities between four experimental treatments. Differences were accepted as significant if p < 0.05. Values are presented as mean ± standard error (SE). All statistical analysis were performed with software SPSS version 29. 3. Results 3.1 Growth Rate Evaluation Juvenile sea cucumber H. scabra were fed with MPs in their diet for 60 days. The mean weight, weight gain percentage and specific growth rate differed significantly in the control group compared to the treatment groups as shown in Table 1 . The mean weight (mean ± SE) of sea cucumber H. scabra decreases as the concentration of MPs increased with significant difference (p < 0.05, p = 0.01) as shown in Fig. 1 . The weight gain percentage (mean ± SE) of sea cucumber H. scabra showed decrease between treatment groups compared to control groups with significant difference (p < 0.05, p = 0.032) as shown in Fig. 2 . Figure 3 depicted specific growth rate (SGR) (mean ± SE) of sea cucumber H. scabra with decreases significantly (p < 0.05, p = 0.03) as the MPs concentration increased. The survival rate was 100% for control and Treatment 1 group, however, 75% for Treatment 2 and 3 with 3 animals loss, respectively. Unfortunately, there were several sea cucumbers from Treatment 2 and Treatment 3 showed signs of skin lesions disease before 2 days of starvation period and evisceration was also observed. Figure 1 Mean weight (mean ± SE) of sea cucumber H. scabra . *Significant difference (p < 0.05, p = 0.01) Figure 2 Weight gain percentage (mean ± SE) of sea cucumber H. scabra. * Significant difference (p < 0.05, p = 0.032) Figure 3 Specific growth rate (SGR) (mean ± SE) of sea cucumber H. scabra . *Significant difference (p < 0.05, p = 0.03) Table 1 The initial body weight, final body weight, weight gain percentage and specific growth rate of Holothuria scabra (mean ± SE) Treatments Initial W Final W Weight Gain Percentage SGR (% d) Control 15.37 ± 0.87 32.13 ± 3.20 16.76 ± 0.35 1.22 ± 0.35 Treatment 1 14.03 ± 0.94 28.79 ± 2.24 14.76 ± 0.35 1.20 ± 0.35 Treatment 2 15.04 ± 2.27 23.98 ± 1.78 8.94 ± 0.22 0.77 ± 0.22 Treatment 3 16.48 ± 1.16 21.65 ± 1.13* 5.17 ± 0.13* 0.47 ± 0.13* 3.2 Enzyme Assay Analysis Acid phosphatase (ACP) levels in juvenile H. scabra were significantly higher in the Treatment 3 (p < 0.05, p = < 0.001) compared to the control group over 60 days of the treatment period (Fig. 4 ). Figure 4 Activity of immune enzyme acid phosphatase (ACP) of sea cucumber Holothuria scabra after 60 days of microplastics treatments (mean ± SE). *Significant difference (p < 0.05, p = < 0.001) Lipase levels in these juvenile H. scabra exhibited a slight increase in the treatment groups compared to the control groups, as depicted in Fig. 5 with a significant difference. Figure 5 Activity of digestive enzyme lipase of sea cucumber Holothuria scabra after 60 days of microplastics treatments (mean ± SE). *Significant difference (p < 0.05, p = 0.022) Superoxide dismutase (SOD) activity in Fig. 6 demonstrated an increase in the treatment groups. However, the difference did not reach statistical significance. Figure 6 Activity of oxidative enzyme superoxide dismutase (SOD) of sea cucumber Holothuria scabra after 60 days of microplastics treatments (mean ± SE). *Significant difference (p < 0.05, p = 0.047) Malondialdehyde (MDA) exhibited a slight increase between the control and treatment groups, with a significant difference observed in Fig. 7 . Figure 7 Activity of oxidative enzyme malondialdehyde (MDA) of sea cucumber Holothuria scabra after 60 days of microplastics treatments (mean ± SE). *Significant difference (p < 0.05, p = 0.037) 3.3 Histological Analysis Histological examination revealed histopathological changes in the sea cucumber H. scabra inhabiting PMMA-MPs-contaminated environments. Figure 8 shows the damage sustained by H. scabra intestinal tract following 60 days of exposure to PMMA-MPs. The gastrointestinal tract of H. scabra was characterized by coelomic epithelial lining, a longitudinal muscular layer, loose connective tissues, and well-arranged pseudostratified mucosal epithelium, as observed in Fig. 8 (A). In Fig. 8 (B), the intestinal structure showed no noticeable difference in Treatment 1. Exposure to higher concentrations of MPs in Treatment 2 and Treatment 3 resulted in diminished connective tissue in the intestines (Fig. 8 (C) and (D)). Furthermore, the pseudostratified mucosal epithelium in Treatment 3 was damaged and disorganized, and the coelomic epithelial lining disintegrated, as observed in Fig. 8 (D). Figure 8 Histological sections in the gastrointestinal tract of Holothuria scabra after 60 days of MPs treatments. (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g). CE – Coelomic Epidermis, ML – Muscular Layer, CT – Connective Tissues, ME – Mucosal Epithelium. [Magnification − 10x, Scale Bar − 50 µm] Figure 9 shows the detrimental impact of 60 days of exposure to PMMA-MPs sustained by H. scabra respiratory tree. Figure 9 (A) illustrates the epithelium of the respiratory tree cavity in H. scabra, comprising coelomic epithelium, muscular layer, connective tissue, and lining epithelium. The collagen fibers in the connective tissue were loose and well organized with a visible central cavity of the lining epithelium. In Treatment 1, there were no apparent histological changes, as depicted in Fig. 9 (B). However, with higher PMMA-MPs concentration, visible changes have been observed in Fig. 9 (C) and (D), particularly in the connective tissues and muscular layer, which becomes thinner and disintegrate. The lining epithelium was disorganized, the coelomic epithelial layer was damaged and vacuolation was apparent in Treatment 3 (Fig. 9 (D)), illustrating the loss of cell components due to penetration of PMMA-MPs. Figure 9 Histological sections in the respiratory tree of Holothuria scabra after 60 days of MPs treatments (x20). (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g) CE - Coelomic Epidermis, ML - Muscular Layer, CT- Connective Tissues, LE - Lining Epithelium. [Magnification − 20x, Scale Bar − 20 µm] 4. Discussion MPs are recognized as hazardous pollutants primarily due to their widespread presence and persistence in various habitats. The absorption of chemicals onto the plastic surface, resulting in a complex mixture of pollutants available to marine species, is facilitated by the high surface area to volume ratio of small particles and their non-polar surface (Sheela et al., 2021 ). The toxicity of MPs varies primarily depending on their size, as smaller particles can penetrate organisms more deeply. The density of MPs particles influences their position in the water column and their potential interaction with organisms, with denser or contaminated polymers posing a particular threat to benthic species (Desforges et al., 2015 ). Sea cucumbers typically feed on sediments enriched with organic matter, especially in regions where a high concentration of MPs particles builds up. The specific growth rate (SGR) in sea cucumber H. scabra from three different PMMA-MPs treatment for 60 days of rearing period is presented in Table 1 . In Fig. 1 , the mean final weight of juveniles in control was higher (32.13 g ± 3.20) compared to treatment groups after 60 days (p < 0.05, p = 0.01). The weight gain percentage in control group was highest (16.76% ± 0.35) as shown in Fig. 2 . The specific growth rate of H. scabra differed significantly (p < 0.05, p = 0.03) among treatments as shown in Fig. 3 . The highest SGR value was obtained from control group at 1.22 ± 0.35%, while the lowest value was found in Treatment 3 at 0.47 ± 0.13% as depicted in Table 1 . The findings demonstrated a significant impact of PMMA-MPs concentration on the sea cucumber’s final weight, specific growth rate, and survival rate. This is consistent with research by Liu et al. ( 2022 ), which showed that polystyrene nanoplastics and MPs adversely impact the final dry weight and weight gain of sea cucumber Apostichopus japonicus . However, Mohsen et al. ( 2021b ) reported that microfibres did not have a significant effect on the survival rate and growth of sea cucumber A. japonicus , contradicting our current results. Previous studies have reported adverse effects of MPs ingestion on the growth of various other marine animals. The weight gain and body length growth of carp Cyprinus carpio larvae were suppressed by MPs made of polyvinyl chloride (PVC) (Xia et al., 2020 ). According to Bringer et al. ( 2021 ), seven days of exposure to MPs resulted in delayed growth retardation of oyster pediveliger larvae Crassostrea gigas for up to 28 days. In the control group with no PMMA-MPs treatment, sea cucumber experienced optimal growth conditions with minimal stress since there were no inhibitory effects on growth. The occurrence of biofilm development in the tanks of the control group also suggested the availability of extra food and nutrients such as microalgal and microbial cells, supporting the growth of juvenile sea cucumbers by facilitating easy access to nutrition in the enriched substrate (Rodrigues et al., 2023 ). In Treatment 3, sea cucumbers are likely to experience more adverse effects because higher concentrations may lead to physiological stress and metabolic disturbance significantly impacting their growth. While smaller MPs have a higher likelihood of entering the animal’s tissue, the larger MPs that are found in the animal feeds may result in lower ingestion rate values and energy digestibility, ultimately impacting growth performance (Shi et al., 2015 ). The overall survival of H. scabra was 100% in control and Treatment 1, whereas it decreased to 75% in Treatment 2 and Treatment 3, experiencing the loss of 3 animals, respectively. The loss in Treatment 2 and Treatment 3 was linked to skin ulceration and lesion disease. White spots developed on the body wall, rapidly affecting the entire integument, ultimately resulting in evisceration and fatalities. This could be due to the contaminated tanks whereby Treatment 2 and Treatment 3 had higher PMMA-MPs concentrations. The presence of additional PMMA-MPs in the sediment led to environmental pollution, causing hypoxia stress in sea cucumbers themselves and potentially contributing to the development of this disease. This environmental factor caused the outbreak of cutaneous disease within the group since sea cucumbers are highly susceptible to sudden changes in external conditions. According to Huang et al. ( 2020 ), the accumulation of MPs can disrupt the homeostasis of animals as they persist in the body over an extended period, thereby elevating energy consumption and leading to malnutrition, which can potentially cause severe damage to organisms. Sea cucumbers depend on their cellular and humoral innate immune responses when they are under hypoxic stress and are attacked by pathogens (Xue et al., 2015 ). These responses play a vital role in identifying and expelling invading microbes, as well as in repairing tissue. Acid phosphatase (ACP) activities was used as indicators for evaluating the immune status of H. scabra. ACP, as an intracellular lysosomal enzyme, involved in the immunological response, where it is responsible to eliminate and digest microorganisms and foreign substances (Yan et al., 2014 ). As the concentration of PMMA-MPs in the ingested feed of juveniles increased, Fig. 4 depicted a rise in ACP activity with significant difference. This elevation suggested a concentration-dependent response. The observed disruption in ACP was likely attributed to increasing number of PMMA-MPs in the ingested sediment, which in turn triggered immune responses. This observation aligns with previous research, as noted by Mohsen et al. ( 2021b ), where significant fluctuations in ACP activity were observed in the juvenile and adult sea cucumber A. japonicus , with decreased in adults and increased in juveniles. The developmental stage of sea cucumber appears to influence activities of enzymes. Research involving other aquatic species also support the relationship between MPs exposure and ACP activity. For instance, exposure to 50 or 500 µg L-1 MPs containing cadmium showed considerably increased ACP activity in discus fish Symphysodon aequifasciatus (Wen et al., 2018 ). In a study particularly with pearl oyster Pinctada fucata martensii , the ACP activity of 1.5 mg/L and 15 mg/L polyvinyl chloride (PVC) MPs treatment groups were also increased (Lu et al., 2024 ). Additionally, the enzyme activity of ACP in brine shrimp Artemia franciscana increased with the increasing of MPs concentration after 14 days exposure period (Han et al., 2021 ). The responsiveness of ACP to environmental stimuli in sea cucumbers is evident, including hypoxic stress caused by oxygen deficiency and dietary supplementation of biofloc and fulvic acid (Chen et al., 2018 ; Dou & Wu, 2023 ; Huo et al., 2018 ). The ability of marine species to utilize ingested nutrients from food relies on the activities of digestive enzymes in their intestinal tract such as lipase. The activity of digestive enzymes not only signify the digestive process, but also function as a biological indicator of the growth and health of the animal (Li et al., 2014 ). In comparison to the control with treatment groups, Fig. 5 showed increased in lipase activity with a significant difference in the digestive tract of sea cucumber as the concentration of PMMA-MPs increased. This finding was consistent with a study on A. stichopus , where no significant changes were observed at different concentrations of polyethylene terephthalate (PET) MPs (particle size: 0.5–45 µm, 2–200 µm and 20–300 µm) (Zhang et al., 2023 ). As highlighted by Khan et al. ( 2017 ), the enzyme can degrade plastics polymer polyurethane (PU), suggesting that the lipase present in sea cucumber may play a role in lipid metabolism associated with MPs. This enzymatic activity could be linked to a stress response and detoxification mechanism to adapt to changes in environmental conditions. In contrast, polystyrene (PS) microspheres, a type of MPs had a substantial impact on lipase level of thick shell mussels Mytilus coruscus throughout the experiment (Wang et al., 2020 ). According to Susanto Barus et al. ( 2023 ), the activity of lipase in hard clam Paphia undulata was also significantly affected by PS-MPs treatments, either alone or in combination with heavy metals like copper and lead. Other than that, environmental conditions such as water temperature do influence lipase level in A. japonicus , with increases reaching it peaks at 20°C (Sun et al., 2018 ). It is well recognized that MPs can be hazardous by generating free radicals, which harm cellular macromolecules and ultimately alter the physiology and biochemistry (Alimba & Faggio, 2019 ). Oxidative stress arises from an imbalance between oxidants and antioxidants in favour of oxidants, thereby disrupting redox signalling, cellular control and cellular damage. This imbalance occurs when the production of reactive oxygen species (ROS) overwhelms the antioxidant defense mechanisms. The excessive formation of ROS can lead to cellular damages. Superoxide dismutase (SOD) served as the primary defense in the antioxidant system, catalyzed superoxide anion free radicals produced by activated coelomocytes to hydrogen peroxide (H 2 O 2 ) or oxygen (O 2 ) to protect cells from injuries. In the current experiment, the observed increase yet the difference did not reach statistical significance in SOD level in treatment groups indicated heightened oxidative stress, likely to be linked to a rapid rebound in oxygen uptake and consumption, along with an accelerated rate of reactive oxygen species (ROS) regeneration as shown in Fig. 6 . Gu et al. ( 2023 ) noted that SOD activity in A. stichopus exhibited significant elevation in the initial exposure of PS-nanoplastics, however, it decreases as exposure time is prolonged suggesting the exhaustion of antioxidant defence. Various aquatic animals have been reported to experience disturbance in antioxidant enzymes due to MPs. Exposure to PS-MPs induces an increase in SOD activity in mussels, which reflects an adaptive response to mitigate oxidative stress and protect tissue from damage (Paul-Pont et al., 2016). In a study conducted by Ding et al. ( 2018 ), increased activity of anoxidative enzyme SOD in the liver of freshwater fish red tilapia Oreochromis niloticus was observed during exposure to MPs. Additionally, earlier studies have also demonstrated that sea cucumbers also change SOD levels in response to environmental stressors such as temperature changes and salinity fluctuations. Higher temperature was associated with increased SOD levels, as reported by (Kamyab et al., 2017 ; Wang et al., 2008 ; Yunwei et al., 2007 ). The SOD level increased in 35% salinity treatment in the first three hours of treatment and 25% salinity treatment exhibited a similar response for the first 72 hours (Wang et al., 2008 ). Antioxidant activities particularly SOD in H. scabra play a crucial role in maintaining the oxidation-antioxidation equilibrium of sea cucumbers, reflecting the immune condition of animals. This suggests prolonged exposure to PMMA-MPs can weaken the antioxidant defense of H. scabra and increase susceptibility to infections, further impacting survival and growth rates. When organisms encountering environmental stress, the balance of ROS was broken, resulting to lipid peroxidation. Malondialdehyde (MDA) is a toxic byproducts of lipid peroxidation, specifically the breakdown of polyunsaturated fatty acids by ROS. It serves as a dependable indicator of oxidative stress, known to cause damage to the cell membrane (Ayala et al., 2014 ). MDA is widely acknowledged as a biomarker for oxidative stress across many health disorders. Increased in the activity of the tested antioxidant enzyme MDA were observed. Although the effects were statistically significant, there was a trend for higher MDA activities in treatment 3, compared to control and other treatment groups, as shown in Fig. 7 . This suggest that an increased oxidative stress brought on by PMMA-MPs exposure consequently promotes inflammation and cellular damage, potentially resulting in the breakdown of lipids. An excess of MDA level can occur when the antioxidant system is overworked, there may be an insufficient capacity to prevent lipid peroxidation. Similar response has been observed after MPs ingestion in previous studies. Mohsen et al ( 2021b ) reported that A. japonicus showed disruption of MDA levels in both juvenile and adult after 60 days of exposure. Lu et al. ( 2024 ) have also found out that MDA content in pearl oyster Pinctada fucata martensii was significantly higher in 15 mg/L dose PVC-MPs group compared to control group. High level of MDA in freshwater fish is said to induce oxidative stress due to presence of MPs (Atamanalp et al., 2022 ). Histological examination revealed histopathological changes in the sea cucumber H. scabra inhabiting MPs-contaminated environments. The elevated levels of SOD and MDA observed in this study provide supporting evidence for the histological changes of the intestines and respiratory tree, suggesting that oxidative stress induced by MPs was taking place in these tissues. Figure 8 and Fig. 9 show the damage sustained by H. scabra 's gastrointestinal tract and respiratory tree following 60 days of exposure to PMMA-MPs. Sea cucumber’s capacity to digest and absorption of nutrients from their feed relies significantly on the effectiveness of their gastrointestinal tract. The gastrointestinal tract of H. scabra exhibited distinct features, including coelomic epithelial lining, a longitudinal muscular layer, loose connective tissues, and well-arranged pseudostratified mucosal epithelium, as depicted in Fig. 8 (A). Following exposure to PMMA-MPs, histopathological examination uncovered changes in an inflammatory response and impaired integrity of the epithelial barrier in the intestine of sea cucumber. Disintegration of coelomic epithelial lining, reduced connective tissues and disorganized arrangement of pseudostratified mucosal epithelium were observed in Fig. 8 . Irregularly shaped MPs can cause physical damage to the intestinal tract due to sharp edges (Cole et al., 2019 ). Thus, this confirms that PMMA-MPs caused intestinal damage and inflammation, and damaged the intestinal folds. Impaired intestinal structure, particularly the mucus layer, would likely to affect immune function and raise the risk of intestinal inflammation since the intestinal epithelium serves as a crucial barrier between the immune system and external environment (Zhao et al., 2019 ). The degradation observed in intestine tissue suggests weak functions of digestion and absorptive functions under environmental stress, potentially impacting growth. Furthermore, intestinal tract is notably sensitive to stressors, leading to a various alterations including changes of normal protective microflora and reduced integrity of the intestinal epithelium (Zhao et al., 2019 ). Studies have demonstrated that exposure to MPs can induce damage to the tissue morphology of aquatic animals. The main intestinal damage in zebrafish Danio rerio included inflammation, bowel wall thinning, epithelial damage cracking of villi and splitting of enterocytes after exposure to MPs (Lei et al., 2018 ; Qiao et al., 2019 ). According to Zheng et al. ( 2022 ), the observed shedding of microvilli, increased in vacuolization and goblet cells, cytoplasmic damage dispersion and the loosening of connective tissue collectively signify that the exposure to PS-MPs has induced notable histopathological alterations in the intestines of the octopus Amphioctopus fangsio . The respiratory tree in H. scabra serve as its unique respiratory organ, facilitating gas exchange between water and coelomic fluid (Gao & Yang, 2015 ). Additionally, they play a role in osmotic regulation and provide space for excretion of metabolized byproducts. PMMA-MPs have the potential to enter the coelomic fluid of sea cucumber through the respiratory tree, where it could accumulate in the coelomic fluid with no way out since haemal system of sea cucumber is a closed system (Mohsen et al., 2020 ). The respiratory tree was composed coelomic epithelium, muscular layer, connective tissue and lining epithelium as shown in Fig. 9 (A). The collagen fibers in the connective tissue were loose and well organized with a visible central cavity of the lining epithelium. In our current study, a clear indication of loss of cell components, vacuolation and damaged of cell layer was apparent in treatment 2 (Fig. 9 (C) and treatment 3 (Fig. 9 (D) after 60 days of treatments. According to Mohsen et al. ( 2021 a), abnormal changes in respiratory tree tissues including erosion of epithelium, vacuolation and injuries in all tissue layers of A. japonicus were observed due to MPs exposure. Damages of respiratory tree were also induced by environmental stressor such as mercury and salinities variation (Geng et al., 2016 ; Telahigue et al., 2020 ). PMMA MPs ingestion highlights the implications of MPs in sea cucumber H. scabra , as demonstrated by the complex interactions between growth performance, immune responses, oxidative stress and histopathological changes. These findings will help to further assess the toxicity studies of MPs and emphasized the need for effective mitigation strategies to address the current issues of MPs pollution in the marine environments as well as to improve the health of marine species and ecosystem. However, understanding environmental factors which may influence MPs toxicity, is crucial in developing effective conservation and management strategies to mitigate the impacts of MPs pollution in marine environment. 5. Conclusion and Recommendation In conclusion, the exposure of sea cucumber H. scabra to PMMA-MPs has significant implications for its growth, survival and overall health. The study revealed that different concentrations of PMMA-MPs led to variations in the specific growth rate and survival rate, exhibited significant differences (p < 0.05) with the control group displaying the highest SGR value of 1.22 ± 0.35% and Treatment 3 has the lowest SGR value (0.47 ± 0.13). This also emphasize the potential risks posed by MPs, including environmental stress, skin ulceration and lesions, which may result in evisceration and fatalities. Mortality was observed in Treatment 2 and 3, respectively. Additionally, the study highlighted the impact of MPs on immune responses and oxidative stress which underscore the damage caused at the cellular level. A disruption in enzyme assays was observed (p < 0.05) in acid phosphatase, lipase, superoxide dismutase and malondialdehyde. Higher concentrations of PMMA-MPs caused histological changes towards the intestines and respiratory trees of sea cucumber H. scabra . This study signifies the first exploration into the toxicity of MPs specifically targeted at H. scabra . Further work is required to enhance better understanding the risks associated to various types MPs polymers and their interaction with other environmental stressors. It is important to improve general wellbeing and productivity of sea cucumber population as well as the marine ecosystem, which are currently facing the impacts of MPs pollution. Declarations Ethical Approval This study was performed in line with animal ethics approved by the Animal Ethics Committee Universiti Malaysia Sabah (AEC UMS) (AEC 0023/2022) and Sabah Biodiversity Center Access License (SaBC) (JKM/MBS.1000-2/2 JLD. 17 (136). Consent to Participate This study involved the use of animal tissue and has been approved for animal ethics (AEC 0023/2022) as stated in the ‘Ethical Approval’ section above. Consent to Publish Not applicable Author Contribution Conceptualization: Nurzafirah Mazlan and Sarah Syazwani; Formal analysis: Nurzafirah Mazlan, Sarah Syazwani; Funding acquisition: Nurzafirah Mazlan; Methodology and Data Collection: Sarah Syazwani Shukhairi, Nurzafirah Mazlan, Nur Nashrah Abd Rahman, Muhammad Nor Afdall Nazahuddin and Amir Syazwan Shawel; Supervision: Nurzafirah Mazlan; Writing - original draft preparation: Sarah Syazwani; Writing - review & editing: Nurzafirah Mazlan and Vijay Subbiah Kumar Funding Geran Bantuan Penyelidikan Pascasiswazah (UMSGreat) (Project Code: GUG0561-1/2022) from the Research Management Centre of Universiti Malaysia Sabah provided the funding for this study. Competing Interest The authors confirm that all authors and this study have no competing interests. Acknowledgments The authors would like to thank the lab assistance from Universiti Malaysia Sabah (UMS) for the assistance provided during this research. 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Borneo","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Nor Afdall","lastName":"Nazahuddin","suffix":""},{"id":313156967,"identity":"0347d39d-e8aa-4e69-a990-312a8923d0a0","order_by":4,"name":"Amir Syazwan Shawel","email":"","orcid":"","institution":"University of Malaysia Sabah Borneo Marine Research Institute: Universiti Malaysia Sabah Institut Penyelidikan Marin Borneo","correspondingAuthor":false,"prefix":"","firstName":"Amir","middleName":"Syazwan","lastName":"Shawel","suffix":""},{"id":313156968,"identity":"692769b2-1cad-4576-ae06-57142fac1f31","order_by":5,"name":"Vijay Subbiah Kumar","email":"","orcid":"","institution":"Universiti Malaysia Sabah","correspondingAuthor":false,"prefix":"","firstName":"Vijay","middleName":"Subbiah","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2024-05-13 09:36:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4412255/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4412255/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-025-36559-1","type":"published","date":"2025-06-04T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59166799,"identity":"4008ac31-4fb1-4ffa-9288-111efe8259c5","added_by":"auto","created_at":"2024-06-27 07:34:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMean weight (mean ± SE) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. *Significant difference (p\u0026lt;0.05, p=0.01)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/1f5475c586239a58204848d4.png"},{"id":59166711,"identity":"1a9880a1-bad6-4be9-8441-138203be9946","added_by":"auto","created_at":"2024-06-27 07:26:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWeight gain percentage (mean ± SE) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. scabra. *\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eSignificant difference (p\u0026lt;0.05, p=0.032)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/401a483d585344cf438bcf7a.png"},{"id":59166800,"identity":"46086555-5f00-4212-a529-e51d6f14690d","added_by":"auto","created_at":"2024-06-27 07:34:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":26878,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecific growth rate (SGR) (mean ± SE) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. *Significant difference (p\u0026lt;0.05, p=0.03)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/a55ae3fff31b7fa2d18705d4.png"},{"id":59166715,"identity":"c5e1fc5d-a357-496f-9874-6e5af74a2021","added_by":"auto","created_at":"2024-06-27 07:26:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of immune enzyme acid phosphatase (ACP) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 60 days of microplastics treatments (mean±SE). *Significant difference (p\u0026lt;0.05, p= \u0026lt;0.001)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/339a42b9c41b5b8e4e82f911.png"},{"id":59166717,"identity":"aa842e79-73fa-4991-93e9-c62cb4b2bc20","added_by":"auto","created_at":"2024-06-27 07:26:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":30290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of digestive enzyme lipase of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 60 days of microplastics treatments (mean±SE). *Significant difference (p\u0026lt;0.05, p= 0.022)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/f8e497942b93ab9bf43b7c18.png"},{"id":59166712,"identity":"5651b0e6-b2c1-4c67-9688-21569f146efb","added_by":"auto","created_at":"2024-06-27 07:26:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of oxidative enzyme superoxide dismutase (SOD) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 60 days of microplastics treatments (mean±SE). *Significant difference (p\u0026lt;0.05, p= 0.047)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/f6d9172c41425a4729ad76a6.png"},{"id":59166714,"identity":"ac14c8e9-8458-4819-a40b-912a960323c7","added_by":"auto","created_at":"2024-06-27 07:26:29","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivity of oxidative enzyme malondialdehyde (MDA) of sea cucumber \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eafter 60 days of microplastics treatments (mean±SE). *Significant difference (p\u0026lt;0.05, p= 0.037)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/8223346f33c5cec6dbc39a6d.png"},{"id":59166718,"identity":"04cfd0a0-a9ec-4a66-99b3-3b52504f4875","added_by":"auto","created_at":"2024-06-27 07:26:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":602298,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological sections in the gastrointestinal tract of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 60 days of MPs treatments. (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g). CE – Coelomic Epidermis, ML – Muscular Layer, CT – Connective Tissues, ME – Mucosal Epithelium. [Magnification - 10x, Scale Bar - 50 µm]\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/59d5c0d40abb47b56769a721.png"},{"id":59166716,"identity":"b54ccf7c-24a6-42b5-b3fd-3b08a9dfd04c","added_by":"auto","created_at":"2024-06-27 07:26:29","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":602319,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological sections in the respiratory tree of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHolothuria scabra\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 60 days of MPs treatments (x20). (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g) CE - Coelomic Epidermis, ML - Muscular Layer, CT- Connective Tissues, LE - Lining Epithelium. [Magnification - 20x, Scale Bar - 20 µm]\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/0b9b6286f37f681507672913.png"},{"id":84242718,"identity":"757f61d4-1b31-4daa-a1d6-3a49c8bf07d1","added_by":"auto","created_at":"2025-06-09 16:11:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3278894,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4412255/v1/2b2fd19c-19cf-4356-a7dd-f283d75d1225.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe Effect of Chronic Microplastic Exposure Towards the Growth, Biochemical Responses and Histological Changes of the Juvenile Sea Cucumber Holothuria Scabra\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlastic waste serves as a clear indicator of the global consequences of anthropogenic activities. The inherent hazard of these materials lies predominantly in their original polymers, additives, and byproducts despite their plastic characteristics. Degraded plastics exhibit characteristics such as fragility, discoloration, dullness, brittleness, flakiness, and powdery texture (Andrady, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Following their release, into the environment, these particles undergo gradual fragmentation into smaller debris, often referred to as microplastics due to the influence of biological, chemical, and mechanical factors. Rivers, stormwater runoff, and sewage effluents serve as significant pathways for the transport of plastic debris to the ocean, as highlighted by Lebreton et al., (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Microplastics (MPs), ranging in size less than 5 mm, have become prevalent in oceans globally, spanning from surface to sediments. The MPs abundance has been discovered in surface water and sediments in Malaysia (Khalik et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Noik et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sarijan et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). There is a growing realization that MPs are contaminating seafood as a result of their uptake in habitats, processing, or packaging (Cox et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mohsen et al., (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) reported the presence of microplastics in canned, instant, and salt-dried sea cucumber products with an average of 0 to 4 MPs per individual in their findings. In 50 canned fish samples obtained from Iranian hypermarkets, a total of 128 microplastics were identified (Akhbarizadeh et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Microplastics were found in green-lipped mussel Perna canaliculus from six out of nine locations in New Zealand, with abundances ranging from 0 to 1.5 particles per mussel (Webb et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHolothurians are marine invertebrates that are members belonging to Phylum Echinodermata and are also referred to as sea cucumbers. These deposit feeders are often found on the seabed, utilizing their ability to convert the habitat\u0026rsquo;s substrate into a food source (Manuputty et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Many sea cucumbers are commercially harvested and dried for human consumption or medical purposes, particularly in Asian nations. Sea cucumber offers an exceptional nutritional profile that includes vitamins, minerals, and amino acids as well as pharmacological actions which include wound healing, anti-inflammatory, and antioxidant (Bordbar et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Shi et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eHolothuria scabra\u003c/em\u003e is one of the rare tropical species that prefers ordinary coastal areas to coral reefs and muddy sand habitats. Due to their daily burrowing cycle and non-selective feeding behaviours, sea cucumbers generally move sluggishly and often bury themselves in the sediment. They inadvertently consume MPs as they use their tentacles to collect sediments, which eventually leads to an increase in the intake of plastics. As the increased demand and value of sea cucumber products pose a significant threat to wild populations, sea cucumber mariculture offers a pathway for the restoration of wild stocks (Purcell et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Cultivation of sea cucumbers primarily takes place in ponds and coastal shallow waters, regions susceptible to pollution from environmental contaminants such as MPs. In their natural habitat, sea cucumbers fulfill their nutrient requirements by consuming and digesting algae and organic debris in sediments, including those contaminated with microplastics (Purcell et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This ecological dynamic also affects sea cucumbers in pond cultivation, given that the sediments used are sourced from the natural environment, an inevitable factor contributing to potential exposure to MPs. Research findings indicate various adverse impacts of MPs on sea cucumbers. Ingestion of MPs negatively impacts weight and growth, affecting the digestive system, gut microbiota, and altered biochemical assays (Iwalaye et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mohsen et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Histological analysis also showed abnormalities in the respiratory tree caused by the penetration of MPs (Mohsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile studies have detected microplastics in sea cucumbers, the extent of their toxicological impact remains unclear. The physiological, and biochemical functions and the organ tissue structure of sea cucumbers may be adversely affected due to the microplastic intake. Toxicological data for the benthic filter feeders, including sea cucumbers, are still under assessment and have yet to be fully published, given the potential differences in plastic polymers and their toxicities compared to fishes and other marine species. The growth rate, biochemical enzymes, and histological structures will be measured after the sea cucumbers were exposed to different microplastic concentrations treatments for 60 days, contributing to a better understanding of the response mechanisms and adaptation strategies of \u003cem\u003eH. scabra\u003c/em\u003e to hypoxia stress induced by microplastics.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Experimental Animal\u003c/h2\u003e \u003cp\u003eThe experiment was performed in the Integrated Multi-Trophic Aquaculture (IMTA) hatchery at Borneo Marine Research Institute, Universiti Malaysia Sabah, Sabah, Malaysia. Sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;36) were obtained from a hatchery,located in Tuaran, Sabah, Malaysia with an average weight of 15 to 18 g. The study animals were acclimated in a water recirculation tank supplied with aeration and fed with sargassum powder for one week before the experiment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental Setup\u003c/h2\u003e \u003cp\u003ePolymethylmethacrylate (PMMA-MPs) used for construction sites were obtained from the Crustacean Hatchery of Universiti Malaysia Sabah and was prepared using a grinder and sieved using an 870-micrometer sieve. The polymers of MPs were identified using Fourier Transform Infrared Spectroscopy (FTIR). The experimental animals were divided into four groups (including control)with three replicates. The stocking density was four animals per m\u003csup\u003e3\u003c/sup\u003e. The animal feed was composed of Sargassum powder at 3% biomass. \u003cem\u003eH. scabra\u003c/em\u003e were exposed to the diet that was mixed with MPs in three treatments of 0.6 MPs/g (Treatment 1), 1.2 MPs/g (Treatment 2) and 10 MPs/g (Treatment 3) for 60 days to investigate the effects of chronic PMMA-MPs exposure on the growth and physiological status of juvenile sea cucumber (Mohsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Rearing Conditions\u003c/h2\u003e \u003cp\u003eThe juvenile sea cucumbers were distributed in tanks provided with aeration. The water temperature was 29 to 30\u0026deg;C, which was the optimum temperatures for the growth of sea cucumbers. The pH was 7 to 8 and the salinity was 33 to 34 ppt. Four juvenile sea cucumbers were stocked per tank in a 1 m\u0026sup3; tank. During the experiment, sea cucumbers were fed once a day and the 50% water exchange was done daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Growth Rate Evaluation\u003c/h2\u003e \u003cp\u003eThe impact of dietary exposure to PMMA-MPs on the growth rate of juvenile sea cucumber was evaluated according to Bai et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and Mohsen et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). The juvenile sea cucumbers\u0026rsquo; weight was calculated at the start and end of the experiments by shedding excess water off the animals of the same size until they reached a constant weight. Sea cucumbers were weighed two days after the last meal on day 60.\u003c/p\u003e \u003cp\u003eWeight gain percentage and specific growth rate (SGR) were calculated according to the following equations:\u003c/p\u003e \u003cp\u003eWeight Gain Percentage (%) = (Final Weight -Initial Weight) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(x\\)\u003c/span\u003e\u003c/span\u003e 100\u003c/p\u003e \u003cp\u003eSpecific Growth Rate (SGR) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(100 x \\frac{\\left(InFW-InIW\\right)}{T}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003ewhere IW and FW were the initial and final body weights of sea cucumbers, and T was the duration of the experiment (Bai et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mohsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Enzyme Assay Analysis\u003c/h2\u003e \u003cp\u003eThe enzymes activities were studied to examine the physiological condition of \u003cem\u003eH. scabra\u003c/em\u003e after prolonged PMMA-MPs exposure. Coelomic fluid was collected in test tubes and stored at -80\u0026deg;C until further analysis (Mohsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). The immune, digestive, and oxidative enzymes were evaluated spectrophotometrically using commercial kits.\u003c/p\u003e \u003cp\u003eAcid phosphatase (ACP) activities were determined spectrophotometrically at a wavelength of 405 nm with the Acid Phosphatase Activity assay kit (p-nitrobenzene phosphate (PNPP) method) obtained from ElabScience (United States). Digestive enzyme lipase was measured at 710 nm using commercial kits (Macklin (China)).\u003c/p\u003e \u003cp\u003emalondialdehyde (MDA) and superoxide dismutase (SOD) were measured at 532 nm and 600 nm respectively using kit produced from Macklin (China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Histological Analysis\u003c/h2\u003e \u003cp\u003eThe respiratory trees and the intestines of the juvenile sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e were removed carefully from the animal and fixed into a 10% neutral buffered formalin solution. All the tissues underwent tissue processing and embedded in paraffin blocks. Then, the samples were sectioned into 5 \u0026micro;m thickness and stained with Haematoxylin and Eosin (H \u0026amp; E) staining (Lei et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The slides were examined under a microscope with an attached camera (Mohsen et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical Analysis\u003c/h2\u003e \u003cp\u003eOne-way ANOVA followed by Tukey\u0026rsquo;s test for multiple comparisons was used to compare the specific growth rate and enzyme activities between four experimental treatments. Differences were accepted as significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). All statistical analysis were performed with software SPSS version 29.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Growth Rate Evaluation\u003c/h2\u003e \u003cp\u003eJuvenile sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e were fed with MPs in their diet for 60 days. The mean weight, weight gain percentage and specific growth rate differed significantly in the control group compared to the treatment groups as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The mean weight (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e decreases as the concentration of MPs increased with significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.01) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The weight gain percentage (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e showed decrease between treatment groups compared to control groups with significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.032) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicted specific growth rate (SGR) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e with decreases significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.03) as the MPs concentration increased. The survival rate was 100% for control and Treatment 1 group, however, 75% for Treatment 2 and 3 with 3 animals loss, respectively. Unfortunately, there were several sea cucumbers from Treatment 2 and Treatment 3 showed signs of skin lesions disease before 2 days of starvation period and evisceration was also observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eMean weight (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber\u003c/b\u003e \u003cb\u003eH. scabra\u003c/b\u003e. \u003cb\u003e*Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.01)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eWeight gain percentage (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber\u003c/b\u003e \u003cb\u003eH. scabra. *\u003c/b\u003e\u003cb\u003eSignificant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.032)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eSpecific growth rate (SGR) (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of sea cucumber\u003c/b\u003e \u003cb\u003eH. scabra\u003c/b\u003e. \u003cb\u003e*Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.03)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe initial body weight, final body weight, weight gain percentage and specific growth rate of \u003cem\u003eHolothuria scabra\u003c/em\u003e (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial W\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal W\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeight Gain Percentage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSGR\u003c/p\u003e \u003cp\u003e(% d)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e32.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTreatment 1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e14.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e28.79\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTreatment 2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e15.04\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.98\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTreatment 3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e16.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Enzyme Assay Analysis\u003c/h2\u003e \u003cp\u003eAcid phosphatase (ACP) levels in juvenile \u003cem\u003eH. scabra\u003c/em\u003e were significantly higher in the Treatment 3 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the control group over 60 days of the treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e \u003cb\u003eActivity of immune enzyme acid phosphatase (ACP) of sea cucumber\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of microplastics treatments (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). *Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLipase levels in these juvenile \u003cem\u003eH. scabra\u003c/em\u003e exhibited a slight increase in the treatment groups compared to the control groups, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e with a significant difference.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003eActivity of digestive enzyme lipase of sea cucumber\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of microplastics treatments (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). *Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.022)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) activity in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e demonstrated an increase in the treatment groups. However, the difference did not reach statistical significance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e \u003cb\u003eActivity of oxidative enzyme superoxide dismutase (SOD) of sea cucumber\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of microplastics treatments (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). *Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.047)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMalondialdehyde (MDA) exhibited a slight increase between the control and treatment groups, with a significant difference observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u003cb\u003eActivity of oxidative enzyme malondialdehyde (MDA) of sea cucumber\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of microplastics treatments (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE). *Significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.037)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Histological Analysis\u003c/h2\u003e \u003cp\u003eHistological examination revealed histopathological changes in the sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e inhabiting PMMA-MPs-contaminated environments. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the damage sustained by \u003cem\u003eH. scabra\u003c/em\u003e intestinal tract following 60 days of exposure to PMMA-MPs. The gastrointestinal tract of \u003cem\u003eH. scabra\u003c/em\u003e was characterized by coelomic epithelial lining, a longitudinal muscular layer, loose connective tissues, and well-arranged pseudostratified mucosal epithelium, as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (A). In Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (B), the intestinal structure showed no noticeable difference in Treatment 1. Exposure to higher concentrations of MPs in Treatment 2 and Treatment 3 resulted in diminished connective tissue in the intestines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (C) and (D)). Furthermore, the pseudostratified mucosal epithelium in Treatment 3 was damaged and disorganized, and the coelomic epithelial lining disintegrated, as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e \u003cb\u003eHistological sections in the gastrointestinal tract of\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of MPs treatments. (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g). CE \u0026ndash; Coelomic Epidermis, ML \u0026ndash; Muscular Layer, CT \u0026ndash; Connective Tissues, ME \u0026ndash; Mucosal Epithelium. [Magnification \u0026minus;\u0026thinsp;10x, Scale Bar \u0026minus;\u0026thinsp;50 \u0026micro;m]\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e shows the detrimental impact of 60 days of exposure to PMMA-MPs sustained by \u003cem\u003eH. scabra\u003c/em\u003e respiratory tree. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (A) illustrates the epithelium of the respiratory tree cavity in H. scabra, comprising coelomic epithelium, muscular layer, connective tissue, and lining epithelium. The collagen fibers in the connective tissue were loose and well organized with a visible central cavity of the lining epithelium. In Treatment 1, there were no apparent histological changes, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (B). However, with higher PMMA-MPs concentration, visible changes have been observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (C) and (D), particularly in the connective tissues and muscular layer, which becomes thinner and disintegrate. The lining epithelium was disorganized, the coelomic epithelial layer was damaged and vacuolation was apparent in Treatment 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (D)), illustrating the loss of cell components due to penetration of PMMA-MPs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e \u003cb\u003eHistological sections in the respiratory tree of\u003c/b\u003e \u003cb\u003eHolothuria scabra\u003c/b\u003e \u003cb\u003eafter 60 days of MPs treatments (x20). (A) Control (B) Treatment 1 (0.0003 g) (C) Treatment 2 (0.0005 g) (D) Treatment 3 (0.0042 g) CE - Coelomic Epidermis, ML - Muscular Layer, CT- Connective Tissues, LE - Lining Epithelium. [Magnification \u0026minus;\u0026thinsp;20x, Scale Bar \u0026minus;\u0026thinsp;20 \u0026micro;m]\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMPs are recognized as hazardous pollutants primarily due to their widespread presence and persistence in various habitats. The absorption of chemicals onto the plastic surface, resulting in a complex mixture of pollutants available to marine species, is facilitated by the high surface area to volume ratio of small particles and their non-polar surface (Sheela et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The toxicity of MPs varies primarily depending on their size, as smaller particles can penetrate organisms more deeply. The density of MPs particles influences their position in the water column and their potential interaction with organisms, with denser or contaminated polymers posing a particular threat to benthic species (Desforges et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Sea cucumbers typically feed on sediments enriched with organic matter, especially in regions where a high concentration of MPs particles builds up.\u003c/p\u003e \u003cp\u003eThe specific growth rate (SGR) in sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e from three different PMMA-MPs treatment for 60 days of rearing period is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the mean final weight of juveniles in control was higher (32.13 g\u0026thinsp;\u0026plusmn;\u0026thinsp;3.20) compared to treatment groups after 60 days (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.01). The weight gain percentage in control group was highest (16.76% \u0026plusmn; 0.35) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The specific growth rate of \u003cem\u003eH. scabra\u003c/em\u003e differed significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;=\u0026thinsp;0.03) among treatments as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest SGR value was obtained from control group at 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35%, while the lowest value was found in Treatment 3 at 0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13% as depicted in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The findings demonstrated a significant impact of PMMA-MPs concentration on the sea cucumber\u0026rsquo;s final weight, specific growth rate, and survival rate. This is consistent with research by Liu et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), which showed that polystyrene nanoplastics and MPs adversely impact the final dry weight and weight gain of sea cucumber \u003cem\u003eApostichopus japonicus\u003c/em\u003e. However, Mohsen et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) reported that microfibres did not have a significant effect on the survival rate and growth of sea cucumber \u003cem\u003eA. japonicus\u003c/em\u003e, contradicting our current results. Previous studies have reported adverse effects of MPs ingestion on the growth of various other marine animals. The weight gain and body length growth of carp \u003cem\u003eCyprinus carpio\u003c/em\u003e larvae were suppressed by MPs made of polyvinyl chloride (PVC) (Xia et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to Bringer et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), seven days of exposure to MPs resulted in delayed growth retardation of oyster pediveliger larvae \u003cem\u003eCrassostrea gigas\u003c/em\u003e for up to 28 days. In the control group with no PMMA-MPs treatment, sea cucumber experienced optimal growth conditions with minimal stress since there were no inhibitory effects on growth. The occurrence of biofilm development in the tanks of the control group also suggested the availability of extra food and nutrients such as microalgal and microbial cells, supporting the growth of juvenile sea cucumbers by facilitating easy access to nutrition in the enriched substrate (Rodrigues et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In Treatment 3, sea cucumbers are likely to experience more adverse effects because higher concentrations may lead to physiological stress and metabolic disturbance significantly impacting their growth. While smaller MPs have a higher likelihood of entering the animal\u0026rsquo;s tissue, the larger MPs that are found in the animal feeds may result in lower ingestion rate values and energy digestibility, ultimately impacting growth performance (Shi et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe overall survival of \u003cem\u003eH. scabra\u003c/em\u003e was 100% in control and Treatment 1, whereas it decreased to 75% in Treatment 2 and Treatment 3, experiencing the loss of 3 animals, respectively. The loss in Treatment 2 and Treatment 3 was linked to skin ulceration and lesion disease. White spots developed on the body wall, rapidly affecting the entire integument, ultimately resulting in evisceration and fatalities. This could be due to the contaminated tanks whereby Treatment 2 and Treatment 3 had higher PMMA-MPs concentrations. The presence of additional PMMA-MPs in the sediment led to environmental pollution, causing hypoxia stress in sea cucumbers themselves and potentially contributing to the development of this disease. This environmental factor caused the outbreak of cutaneous disease within the group since sea cucumbers are highly susceptible to sudden changes in external conditions. According to Huang et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the accumulation of MPs can disrupt the homeostasis of animals as they persist in the body over an extended period, thereby elevating energy consumption and leading to malnutrition, which can potentially cause severe damage to organisms.\u003c/p\u003e \u003cp\u003eSea cucumbers depend on their cellular and humoral innate immune responses when they are under hypoxic stress and are attacked by pathogens (Xue et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These responses play a vital role in identifying and expelling invading microbes, as well as in repairing tissue. Acid phosphatase (ACP) activities was used as indicators for evaluating the immune status of H. scabra. ACP, as an intracellular lysosomal enzyme, involved in the immunological response, where it is responsible to eliminate and digest microorganisms and foreign substances (Yan et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs the concentration of PMMA-MPs in the ingested feed of juveniles increased, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e depicted a rise in ACP activity with significant difference. This elevation suggested a concentration-dependent response. The observed disruption in ACP was likely attributed to increasing number of PMMA-MPs in the ingested sediment, which in turn triggered immune responses. This observation aligns with previous research, as noted by Mohsen et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e), where significant fluctuations in ACP activity were observed in the juvenile and adult sea cucumber \u003cem\u003eA. japonicus\u003c/em\u003e, with decreased in adults and increased in juveniles. The developmental stage of sea cucumber appears to influence activities of enzymes. Research involving other aquatic species also support the relationship between MPs exposure and ACP activity. For instance, exposure to 50 or 500 \u0026micro;g L-1 MPs containing cadmium showed considerably increased ACP activity in discus fish \u003cem\u003eSymphysodon aequifasciatus\u003c/em\u003e (Wen et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In a study particularly with pearl oyster \u003cem\u003ePinctada fucata martensii\u003c/em\u003e, the ACP activity of 1.5 mg/L and 15 mg/L polyvinyl chloride (PVC) MPs treatment groups were also increased (Lu et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, the enzyme activity of ACP in brine shrimp \u003cem\u003eArtemia franciscana\u003c/em\u003e increased with the increasing of MPs concentration after 14 days exposure period (Han et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The responsiveness of ACP to environmental stimuli in sea cucumbers is evident, including hypoxic stress caused by oxygen deficiency and dietary supplementation of biofloc and fulvic acid (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dou \u0026amp; Wu, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Huo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ability of marine species to utilize ingested nutrients from food relies on the activities of digestive enzymes in their intestinal tract such as lipase. The activity of digestive enzymes not only signify the digestive process, but also function as a biological indicator of the growth and health of the animal (Li et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In comparison to the control with treatment groups, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e showed increased in lipase activity with a significant difference in the digestive tract of sea cucumber as the concentration of PMMA-MPs increased. This finding was consistent with a study on \u003cem\u003eA. stichopus\u003c/em\u003e, where no significant changes were observed at different concentrations of polyethylene terephthalate (PET) MPs (particle size: 0.5\u0026ndash;45 \u0026micro;m, 2\u0026ndash;200 \u0026micro;m and 20\u0026ndash;300 \u0026micro;m) (Zhang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As highlighted by Khan et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the enzyme can degrade plastics polymer polyurethane (PU), suggesting that the lipase present in sea cucumber may play a role in lipid metabolism associated with MPs. This enzymatic activity could be linked to a stress response and detoxification mechanism to adapt to changes in environmental conditions. In contrast, polystyrene (PS) microspheres, a type of MPs had a substantial impact on lipase level of thick shell mussels \u003cem\u003eMytilus coruscus\u003c/em\u003e throughout the experiment (Wang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to Susanto Barus et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the activity of lipase in hard clam \u003cem\u003ePaphia undulata\u003c/em\u003e was also significantly affected by PS-MPs treatments, either alone or in combination with heavy metals like copper and lead. Other than that, environmental conditions such as water temperature do influence lipase level in \u003cem\u003eA. japonicus\u003c/em\u003e, with increases reaching it peaks at 20\u0026deg;C (Sun et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well recognized that MPs can be hazardous by generating free radicals, which harm cellular macromolecules and ultimately alter the physiology and biochemistry (Alimba \u0026amp; Faggio, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Oxidative stress arises from an imbalance between oxidants and antioxidants in favour of oxidants, thereby disrupting redox signalling, cellular control and cellular damage. This imbalance occurs when the production of reactive oxygen species (ROS) overwhelms the antioxidant defense mechanisms. The excessive formation of ROS can lead to cellular damages.\u003c/p\u003e \u003cp\u003eSuperoxide dismutase (SOD) served as the primary defense in the antioxidant system, catalyzed superoxide anion free radicals produced by activated coelomocytes to hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) or oxygen (O\u003csub\u003e2\u003c/sub\u003e) to protect cells from injuries. In the current experiment, the observed increase yet the difference did not reach statistical significance in SOD level in treatment groups indicated heightened oxidative stress, likely to be linked to a rapid rebound in oxygen uptake and consumption, along with an accelerated rate of reactive oxygen species (ROS) regeneration as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Gu et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) noted that SOD activity in \u003cem\u003eA. stichopus\u003c/em\u003e exhibited significant elevation in the initial exposure of PS-nanoplastics, however, it decreases as exposure time is prolonged suggesting the exhaustion of antioxidant defence. Various aquatic animals have been reported to experience disturbance in antioxidant enzymes due to MPs. Exposure to PS-MPs induces an increase in SOD activity in mussels, which reflects an adaptive response to mitigate oxidative stress and protect tissue from damage (Paul-Pont et al., 2016). In a study conducted by Ding et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), increased activity of anoxidative enzyme SOD in the liver of freshwater fish red tilapia \u003cem\u003eOreochromis niloticus\u003c/em\u003e was observed during exposure to MPs. Additionally, earlier studies have also demonstrated that sea cucumbers also change SOD levels in response to environmental stressors such as temperature changes and salinity fluctuations. Higher temperature was associated with increased SOD levels, as reported by (Kamyab et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Yunwei et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The SOD level increased in 35% salinity treatment in the first three hours of treatment and 25% salinity treatment exhibited a similar response for the first 72 hours (Wang et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Antioxidant activities particularly SOD in \u003cem\u003eH. scabra\u003c/em\u003e play a crucial role in maintaining the oxidation-antioxidation equilibrium of sea cucumbers, reflecting the immune condition of animals. This suggests prolonged exposure to PMMA-MPs can weaken the antioxidant defense of H. scabra and increase susceptibility to infections, further impacting survival and growth rates.\u003c/p\u003e \u003cp\u003eWhen organisms encountering environmental stress, the balance of ROS was broken, resulting to lipid peroxidation. Malondialdehyde (MDA) is a toxic byproducts of lipid peroxidation, specifically the breakdown of polyunsaturated fatty acids by ROS. It serves as a dependable indicator of oxidative stress, known to cause damage to the cell membrane (Ayala et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). MDA is widely acknowledged as a biomarker for oxidative stress across many health disorders. Increased in the activity of the tested antioxidant enzyme MDA were observed. Although the effects were statistically significant, there was a trend for higher MDA activities in treatment 3, compared to control and other treatment groups, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. This suggest that an increased oxidative stress brought on by PMMA-MPs exposure consequently promotes inflammation and cellular damage, potentially resulting in the breakdown of lipids. An excess of MDA level can occur when the antioxidant system is overworked, there may be an insufficient capacity to prevent lipid peroxidation. Similar response has been observed after MPs ingestion in previous studies. Mohsen et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) reported that \u003cem\u003eA. japonicus\u003c/em\u003e showed disruption of MDA levels in both juvenile and adult after 60 days of exposure. Lu et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) have also found out that MDA content in pearl oyster \u003cem\u003ePinctada fucata martensii\u003c/em\u003e was significantly higher in 15 mg/L dose PVC-MPs group compared to control group. High level of MDA in freshwater fish is said to induce oxidative stress due to presence of MPs (Atamanalp et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHistological examination revealed histopathological changes in the sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e inhabiting MPs-contaminated environments. The elevated levels of SOD and MDA observed in this study provide supporting evidence for the histological changes of the intestines and respiratory tree, suggesting that oxidative stress induced by MPs was taking place in these tissues. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e show the damage sustained by \u003cem\u003eH. scabra\u003c/em\u003e's gastrointestinal tract and respiratory tree following 60 days of exposure to PMMA-MPs.\u003c/p\u003e \u003cp\u003eSea cucumber\u0026rsquo;s capacity to digest and absorption of nutrients from their feed relies significantly on the effectiveness of their gastrointestinal tract. The gastrointestinal tract of H. scabra exhibited distinct features, including coelomic epithelial lining, a longitudinal muscular layer, loose connective tissues, and well-arranged pseudostratified mucosal epithelium, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e (A). Following exposure to PMMA-MPs, histopathological examination uncovered changes in an inflammatory response and impaired integrity of the epithelial barrier in the intestine of sea cucumber. Disintegration of coelomic epithelial lining, reduced connective tissues and disorganized arrangement of pseudostratified mucosal epithelium were observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Irregularly shaped MPs can cause physical damage to the intestinal tract due to sharp edges (Cole et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, this confirms that PMMA-MPs caused intestinal damage and inflammation, and damaged the intestinal folds. Impaired intestinal structure, particularly the mucus layer, would likely to affect immune function and raise the risk of intestinal inflammation since the intestinal epithelium serves as a crucial barrier between the immune system and external environment (Zhao et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The degradation observed in intestine tissue suggests weak functions of digestion and absorptive functions under environmental stress, potentially impacting growth. Furthermore, intestinal tract is notably sensitive to stressors, leading to a various alterations including changes of normal protective microflora and reduced integrity of the intestinal epithelium (Zhao et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies have demonstrated that exposure to MPs can induce damage to the tissue morphology of aquatic animals. The main intestinal damage in zebrafish \u003cem\u003eDanio rerio\u003c/em\u003e included inflammation, bowel wall thinning, epithelial damage cracking of villi and splitting of enterocytes after exposure to MPs (Lei et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Qiao et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). According to Zheng et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), the observed shedding of microvilli, increased in vacuolization and goblet cells, cytoplasmic damage dispersion and the loosening of connective tissue collectively signify that the exposure to PS-MPs has induced notable histopathological alterations in the intestines of the octopus \u003cem\u003eAmphioctopus fangsio\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe respiratory tree in \u003cem\u003eH. scabra\u003c/em\u003e serve as its unique respiratory organ, facilitating gas exchange between water and coelomic fluid (Gao \u0026amp; Yang, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, they play a role in osmotic regulation and provide space for excretion of metabolized byproducts. PMMA-MPs have the potential to enter the coelomic fluid of sea cucumber through the respiratory tree, where it could accumulate in the coelomic fluid with no way out since haemal system of sea cucumber is a closed system (Mohsen et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The respiratory tree was composed coelomic epithelium, muscular layer, connective tissue and lining epithelium as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (A). The collagen fibers in the connective tissue were loose and well organized with a visible central cavity of the lining epithelium. In our current study, a clear indication of loss of cell components, vacuolation and damaged of cell layer was apparent in treatment 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (C) and treatment 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e (D) after 60 days of treatments. According to Mohsen et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea), abnormal changes in respiratory tree tissues including erosion of epithelium, vacuolation and injuries in all tissue layers of \u003cem\u003eA. japonicus\u003c/em\u003e were observed due to MPs exposure. Damages of respiratory tree were also induced by environmental stressor such as mercury and salinities variation (Geng et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Telahigue et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePMMA MPs ingestion highlights the implications of MPs in sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e, as demonstrated by the complex interactions between growth performance, immune responses, oxidative stress and histopathological changes. These findings will help to further assess the toxicity studies of MPs and emphasized the need for effective mitigation strategies to address the current issues of MPs pollution in the marine environments as well as to improve the health of marine species and ecosystem. However, understanding environmental factors which may influence MPs toxicity, is crucial in developing effective conservation and management strategies to mitigate the impacts of MPs pollution in marine environment.\u003c/p\u003e"},{"header":"5. Conclusion and Recommendation","content":"\u003cp\u003eIn conclusion, the exposure of sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e to PMMA-MPs has significant implications for its growth, survival and overall health. The study revealed that different concentrations of PMMA-MPs led to variations in the specific growth rate and survival rate, exhibited significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the control group displaying the highest SGR value of 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35% and Treatment 3 has the lowest SGR value (0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13). This also emphasize the potential risks posed by MPs, including environmental stress, skin ulceration and lesions, which may result in evisceration and fatalities. Mortality was observed in Treatment 2 and 3, respectively. Additionally, the study highlighted the impact of MPs on immune responses and oxidative stress which underscore the damage caused at the cellular level. A disruption in enzyme assays was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in acid phosphatase, lipase, superoxide dismutase and malondialdehyde. Higher concentrations of PMMA-MPs caused histological changes towards the intestines and respiratory trees of sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e. This study signifies the first exploration into the toxicity of MPs specifically targeted at \u003cem\u003eH. scabra\u003c/em\u003e. Further work is required to enhance better understanding the risks associated to various types MPs polymers and their interaction with other environmental stressors. It is important to improve general wellbeing and productivity of sea cucumber population as well as the marine ecosystem, which are currently facing the impacts of MPs pollution.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with animal ethics approved by the Animal Ethics Committee Universiti Malaysia Sabah (AEC UMS) (AEC 0023/2022) and Sabah Biodiversity Center Access License (SaBC) (JKM/MBS.1000-2/2 JLD. 17 (136).\u003c/p\u003e\n\u003cp\u003eConsent to Participate\u003c/p\u003e\n\u003cp\u003eThis study involved the use of animal tissue and has been approved for animal ethics (AEC 0023/2022) as stated in the \u0026lsquo;Ethical Approval\u0026rsquo; section above.\u003c/p\u003e\n\u003cp\u003eConsent to Publish\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAuthor Contribution\u003c/p\u003e\n\u003cp\u003eConceptualization: Nurzafirah Mazlan and Sarah Syazwani; Formal analysis: Nurzafirah Mazlan, Sarah Syazwani; Funding acquisition: Nurzafirah Mazlan; Methodology and Data Collection: Sarah Syazwani Shukhairi, Nurzafirah Mazlan, Nur Nashrah Abd Rahman, Muhammad Nor Afdall Nazahuddin and Amir Syazwan Shawel; Supervision: Nurzafirah Mazlan; Writing - original draft preparation: Sarah Syazwani; Writing - review \u0026amp; editing: Nurzafirah Mazlan and Vijay Subbiah Kumar\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eGeran Bantuan Penyelidikan Pascasiswazah (UMSGreat) (Project Code: GUG0561-1/2022) from the Research Management Centre of Universiti Malaysia Sabah provided the funding for this study.\u003c/p\u003e\n\u003cp\u003eCompeting Interest\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all authors and this study have no competing interests.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the lab assistance from Universiti Malaysia Sabah (UMS) for the assistance provided during this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkhbarizadeh, R., Dobaradaran, S., Nabipour, I., Tajbakhsh, S., Darabi, A. 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Fish and Shellfish Immunology, 93, 369\u0026ndash;379. https://doi.org/10.1016/J.FSI.2019.07.073\u003c/li\u003e\n\u003cli\u003eZheng, J., Li, C., \u0026amp; Zheng, X. (2022). Toxic Effects Of Polystyrene Microplastics On The Intestine Of Amphioctopus fangsiao (Mollusca: Cephalopoda): From Physiological Responses To Underlying Molecular Mechanisms. Chemosphere, 308, 136362. https://doi.org/10.1016/j.chemosphere.2022.136362\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Echinoderms, Microplastics, Holothuria scabra, PMMA, Chronic Toxicity","lastPublishedDoi":"10.21203/rs.3.rs-4412255/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4412255/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMicroplastics (MPs), are minuscule plastic particles less than 5 millimeters in size, originating from the degradation of larger plastic debris. They are found in various sources and posing a significant threat to marine ecosystems. Sea cucumber \u003cem\u003eHolothuria scabra\u003c/em\u003e is a high value commercial species of sea cucumber. They are also crucial in maintaining a clean seabed and recycling nutrients in the ocean ecosystem. This research aimed to investigate the toxicity effects of microplastics on the well-being of juvenile sea cucumber \u003cem\u003eH. scabra\u003c/em\u003e. Over 60 days treatment period, polymethylmethacrylate MPs were exposed to the juvenile sea cucumber diet at concentrations of 0.6 MPs/g, 1.2 MPs/g and 10 MPs/g to observe changes in their growth, biochemical responses, and histological alteration. The mean weight, weight gain percentage and specific growth rate exhibited significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with the control group displaying the highest SGR value of 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35%. Mortality was observed in treatment 2 and 3, respectively. Notably, a disruption in enzyme assays was also observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The findings of growth rates and biochemical responses were further supported by histological observation, uncovering injuries and loss of cellular components in respiratory trees and intestines. This study enhance our understanding of the toxicity mechanism associated with MPs in filter-feeding organisms.\u003c/p\u003e","manuscriptTitle":"The Effect of Chronic Microplastic Exposure Towards the Growth, Biochemical Responses and Histological Changes of the Juvenile Sea Cucumber Holothuria Scabra","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 07:26:24","doi":"10.21203/rs.3.rs-4412255/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-01-04T02:38:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-06-11T16:50:08+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-11T13:08:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T04:31:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-05-19T23:42:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9ab0ed31-ab27-4bd4-8d05-cce12748c093","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T16:07:09+00:00","versionOfRecord":{"articleIdentity":"rs-4412255","link":"https://doi.org/10.1007/s11356-025-36559-1","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2025-06-04 15:57:39","publishedOnDateReadable":"June 4th, 2025"},"versionCreatedAt":"2024-06-27 07:26:24","video":"","vorDoi":"10.1007/s11356-025-36559-1","vorDoiUrl":"https://doi.org/10.1007/s11356-025-36559-1","workflowStages":[]},"version":"v1","identity":"rs-4412255","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4412255","identity":"rs-4412255","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}