Introduction
In the context of modern industrialization, plastics are widely used in all aspects of daily life because they are inexpensive, lightweight, malleable, and chemically stable. Today, the world produces more than 300 million tons of plastics annually, of which more than 20% enter the environment as pollutants and are subsequently degraded into microplastic particles by light, heat, wind, acid, and salt, gradually accumulating in the environment. Due to their microscopic size and high mobility, microplastics (MPs, < 5mm) threaten ecosystems and biological health.1–3
Researchers have found MPs within specimens of more than 1,300 species of fish, birds, and mammals, including in human tissues and organs.4 Studies have confirmed that MPs can directly affect animal growth, reproduction, and metabolism through various toxicity mechanisms, including histopathological damage, intestinal flora disruption, oxidative stress, DNA damage, genotoxicity, reproductive toxicity, and neurotoxicity.5 Moreover, MPs can indirectly affect metabolic and synthetic processes such as glucose metabolism, lipid metabolism, and energy metabolism, which further affect the growth and development of organisms.6 In addition, toxic substances such as persistent organic pollutants (POPs) and heavy metals adsorbed in MPs or contained in the MPs themselves can be released into the aquatic environment continuously, with toxic effects on aquatic organisms.7
In recent years, MPs have been widely reported to be found in marine, estuarine, river, lake, reservoir, and other aquatic environments. MPs from land-based sources and the impact of aquaculture production activities8will inevitably threaten the fisheries environment. Studying the pollution characteristics of MPs and their effects on aquatic animals and aquaculture systems is of great significance to the scientific response to MPs and to guarantee the green development of aquaculture. In current studies, research on MPs has mostly focused on the toxic effects on model organisms, while aquaculture fish are less frequently studied. Common carp (Cyprinus carpio) is one of the world’s most widely bred freshwater fish, with nearly 1.9 million tons produced in 2020 and accounting for more than 50% of inland aquaculture production as of 2022.9 Amino acids play a crucial role in the growth and development of fish, and there are no reports on the effect of MPs on amino acid metabolism in carp culture. This experiment was conducted to investigate the effects of polystyrene microplastics (PS-MPs) on the growth performance, immunity and antioxidant ability, and amino acid metabolism of carp, to provide some references for the healthy aquaculture of carp and environmental protection.
Materials and Methods
Experimental materials
Common carp used in the experiment were purchased from a fishery firm in Zhengzhou City, Henan Province. They were around 8 months old and uniform in size [body mass (24.8 ± 1.9) g, body length (10.1 ± 0.5) cm]. They were kept in acrylic pools (60 cm × 40 cm × 50 cm). 5 μm mono-dispersed PS-MPs were purchased from Jiangsu Zhichuan Science and Technology Co, Ltd.
Experimental design
During this experiment, the carp were kept in the aquaculture laboratory of Henan University of Science and Technology. The rearing conditions were kept stable, with 60 L of culture water, oxygen solubility ≥ 5 mg/L, water pH 7.5~8.0, water temperature 26±1°C, and daily light time of 14 h. After 2 weeks of acclimatization, 180 healthy and active carp were selected as the experimental fish, which were randomly divided into four concentrations (0, 50, 100, and 200 μg/L) of PS-MPs groups, three replicates were set up for each group, and 15 experimental fish were used in each parallel unit, accumulating 12 pools. The PS-MPs exposure experiment lasted for 15 days.
Experimentation procedure
The experimental fish were fed twice a day (9:00 and 16:00) amounting to 3% of the carp’s body weight. The activity of carp was observed daily, and food residues and feces were removed in time. The water was changed once every 24 h, and the amount of water changed was one-fourth (15 L) of the culture water, directly add the corresponding amount of MPs suspension, and then disperse it by supplement water, aiming to make it evenly distributed as far as possible During the experimental process, the fish in the pool were weighed every 7 days to adjust the subsequent feeding amount. No mortality was observed throughout the experiment. The experimental process complied with the requirements of the Experimental Animal Management and Ethical Welfare Committee of Henan University of Science and Technology.
Sampling
After 15 days of PS-MPs exposure, the carp were fasted for 24 h and then sampled for analysis. For sampling, carp were put into the water containing 100 mg/L MS-222 anesthetic in advance, followed by rapid dissection, removal of hepatopancreas tissues into prepared tissue tubes, placed in liquid nitrogen for quick-freezing, and then frozen in -80°C refrigerator for the detection of alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), superoxide dismutase (SOD), malondialdehyde (MDA), peroxidase (POD), catalase (CAT), total protein (TP) and amino acid content.
Measurement of parameters related to carp growth performance
Survival rate (SR), Weight gain rate (WGR), feed conversion ratio (FCR), hepatopancreas somatic indices (HSI), and visceral index (VSI) of carp were calculated according to the following formulae.
SR (%) = Final fish count / Initial fish count × 100%
WGR (%) = (Wt - W0) / W0 × 100%
FCR = Feed consumption / (Wt - W0)
HSI (%) = (Wh / Wt) × 100%
VSI (%) = (Wv / Wt) × 100%.
W0 and Wt are the initial and final body weight of fish (g) at the beginning and end of the experiment; feed consumption refers to the total amount of feed (dry weight); Wh is the weight of fish’s hepatopancreas (g); Wv is the weight of fish’s viscera (g).
Detection of antioxidant and immune-related parameters
MDA contents and AKP, ACP, LZM, SOD, POD and CAT activity levels were determined by using relevant test kits produced by Nanjing Jiancheng Institute of Biological Engineering, and the specific operation was referred to the instruction manual of the relevant kits.10
Determination of amino acid content in hepatopancreas
The amino acid content in carp hepatopancreas was determined using a gas chromatography-tandem mass spectrometry (GC-MS/MS) metabolomics technical method, and the steps included sample collection, sample pretreatment, metabolite derivatization, and detection. An appropriate amount of sample was taken into an EP tube, and a pre-cooled methanol-acetonitrile mixture was added, shaken for 1 min, and sonicated for 10 min, after which the sample was allowed to stand at -20℃. After standing, the samples were centrifuged for 10 min, and 100 μL of the supernatant from the centrifugation was removed and transferred to a wide lined tube to be swished dry and subjected to a two-step derivatization process. Thereafter, 80 μL methicillin hydrochloride pyridine solution (concentration 15 mg/mL) was added to a glass vial. After that, vortex shaking was performed for 2-min and the oxidation reaction was carried out in a shaking incubator at 37°C for 60-min. After the oxidation reaction, the samples were removed, and 80 μL of BSTFA derivatization reagent and 20 μL of hexane were added. Vortex shaking was continued for 2-min and the reaction was carried out at 70°C for 60-min. Finally, the samples were removed and left at room temperature for 30-min, followed by GC-MS metabolomics analysis, and the concentrations of the target substances in the test samples were calculated based on the standard curves, which were converted to obtain the actual content of the target substances in the samples.
Statistics analysis
The data obtained from the experiments were expressed as mean ± standard error of the mean (SEM), and all data were subjected to One-way ANOVA using SPSS 27.0 software. GraphPad Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA) and OriginPro 2024 (OriginLab Inc., USA) were used for graphing, and comparisons between groups were made using the Least Significant Difference (LSD) method, and t-test was employed for comparisons with P < 0.05 indicating a significant difference between groups.
Results
Effects of PS-MPs on growth indexes of common carp
After 15 days of PS-MPs exposure, the carp did not die during the whole process. Compared with the control group, carp in 100 and 200 μg/L PS-MPs groups demonstrated lower WGR and HSI and higher FCR (P < 0.05). Carp exposed to 50 μg/L PS-MPs did not affect WGR, FCR, and HSI, but significantly reduced VSI. A significantly lower VSI was also found in carp exposed to 100 μg/L PS-MPs compared to CK (P < 0.05). There was no significant change in VSI between 200 μg/L group and CK (Fig. 1).
Effects of PS-MPs on the activity of immune-related enzymes of common carp
Compared with the control group, the activities of ACP and AKP in the experimental groups were significantly lower (P < 0.05), and there was no significant difference between the experimental groups, while the content of LZM was significantly higher (P < 0.05) and was highest in 100 μg/L groups, with a significant difference within the groups (P < 0.05) (Fig. 2).
Effects of PS-MPs on antioxidant indices in the hepatopancreas of common carp
Compared with the control group, the MDA content of the carp in 50 and 100 μg/L PS-MPs groups was significantly higher (P < 0.05), while significantly lower (P < 0.05) in the 200 μg/L PS-MPs group; the POD activity of carp in the experimental groups was significantly lower (P < 0.05); the CAT activity of carp in the experimental group increased significantly (P < 0.05) with increasing PS-MPs concentration, and there was no significant difference between the 100 and 200 μg/L PS-MPs groups; the SOD activity of carp in the experimental group were all significantly higher (P < 0.05), and the SOD, POD, and CAT activity were all basically concentration dependent (Fig. 3).
Effects of PS-MPs on amino acid metabolism in the hepatopancreas of common carp
The absolute quantification of the metabolites of the samples was counted, and the results after analyzing the amino acid species with significant differences are shown in Fig. 4. From the results of the experiment, it can be seen that after exposure to PS-MPs, the content of methionine, threonine, leucine and isoleucine in the experimental groups appeared to be significantly reduced compared with that of the control group (P < 0.05); the phenylalanine content were significantly decreased (P < 0.05) in the 100 μg/L PS-MPs group, and no significant changes occurred in 50 and 200 μg/L PS-MPs groups; the lysine content did not change significantly in 50 and 100 μg/L PS-MPs groups, and a significant increase (P < 0.05) was observed in the 200 μg/L PS-MPs group.
Correlation analysis of amino acid content with growth, immunity and antioxidant indexes
Pearson correlation analysis was used to analyze the relationship between carp amino acid content with growth, immunity, and antioxidant indexes (Fig. 5). The results showed that WGR was negatively related to Lys (P < 0.05). FCR positively linked with Lys (P < 0.05). HSI and VSI had a significant and positive correlation with Met, Thr, Leu, Ile, and Phe (P < 0.05). ACP, AKP and POD positively correlated with the contents of Met, Thr, Leu and Ile (P < 0.01); MDA negatively and significantly correlated with the contents of Lys and Phe (P < 0.05).
Discussion
Effects of PS-MPs on the growth and amino acid metabolism of common carp
WGR is an important indicator of fish growth, and its decrease may mean a disturbance in the feeding, digestive, or absorptive processes in fish.11 In 100 and 200 μg/L PS-MPs groups, the WGR was significantly reduced, indicating that PS-MPs (> 100 μg/L) reduced fish growth. The phenomenon can be explained from two aspects. Firstly, the accumulation of PS-MPs particles in the digestive tract of carp, occupied the space of the food, resulting in the inability of carp to obtain sufficient nutrients.12 Secondly, PS-MPs may also affect the activity of digestive enzymes and the structure of intestinal microbial communities in carp,13 which in turn may affect their nutrient absorption and utilization. Moreover, the increase of FCR is also a phenomenon of concern. The FCR reflects the efficiency of feed utilization in the growth process of fish. In this study, the FCR was significantly higher in 100 and 200 μg/L PS-MPs groups, indicating that carp need to consume more feed to obtain the same weight gain. This may be due to the presence of PS-MPs reducing the appetite of carp or affecting their ability to digest and absorb the feed, which in turn interferes with energy metabolism.6 In addition, changes in HSI and VSI provided clues about the effects of PS-MPs on the internal organs of carp. The decrease in HSI may mean that the functions of hepatopancreas are affected, such as detoxification and metabolism may be decreased.14 A decrease in VSI indicates that the weight or function of internal organs is affected. These changes may further affect the health status and viability of the carp. It is noteworthy that there was no significant change in the WGR and FCR of carp in the 50 μg/L PS-MPs group, which may be related to the concentration effect of PS-MPs. However, this does not mean that there is no potential risk to carp from low concentrations of PS-MPs, as long-term exposure to low concentrations of PS-MPs may still have chronic effects on common carp.
Previous studies in aquatic animals have suggested that fish growth performance is closely associated with amino acids.15 The liver is an important organ in carrying out amino acid metabolism. Thus, we measured the amino acid content of the liver and analyzed the relationship between the amino acid content and the growth indexes of carp. The effects of the feed intake of aquatic animals and its deficiency will lead to a decline in growth performance, which has been reported in various fish species, such as carp, catfish (Silurus asotus), and flounder (Pleuronectiformes), and with the increase of Thr level, the feed intake of these fishes are reduced (Liu. et al., 2024; Yu-Wen et al.16). Leu is essential for maintaining various physiological functions in the body, and appropriate levels of Leu were found to promote growth and protein synthesis in largemouth bass (Micropterus salmoides).17 Met, Thr, Leu, Ile, and Phe are essential amino acids, and the sole source of these essential amino acids was from the diets. A decrease in the liver’s amino acid content may indicate a potential deficiency in the intake of these amino acids. The study on rainbow trout (Oncorhynchus mykiss) has also indicated that dietary Leu, Ile, and Thr levels positively correlated with Leu, Ile, and Thr contents in the liver.18 In this study, the contents of Met, Thr, Leu, and Ile in carp showed a significant decrease after PS-MPs exposure, which reflected a deficiency in intake of these amino acids and would affect the normal amino acid metabolism and protein synthesis process of the carp. It can adversely affect the normal growth and development of carp, which was reflected in the significant changes in the WGR, FCR, HSI, and VSI, thus hindering the growth of the carp. Correlation analysis also showed a positive correlation between HSI and VSI with the content of Met, Thr, Leu, Ile, and Phe. Similarly, the study on golden pompano (Trachinotus ovatus)19 reported that VSI and HSI elevated with increasing Ile (13.2-18.2 g/kg dry feed). In addition, PS-MPs also affected Phe metabolism. Phe plays an important role in growth and development, as well as in the production of neurotransmitters, hormones and proteins, which in turn are involved in metabolic processes such as glucose metabolism and fat metabolism in the body. Lack of Phe can lead to mental confusion, depression, memory loss, and low energy levels.20
Effects of PS-MPs on immunity, antioxidant, and amino acid metabolism in common carp
ACP and AKP, as key enzymes closely related to organismal immunity, are involved in various metabolic processes in the body and are important biological indicators for assessing the damage caused by exogenous substances to fish.21 ACP enables cells to recognize foreign substances and accelerates phagocytosis and degradation of foreign substances by phagocytes. AKP is related to nutrient absorption and transport and can act as an important detoxification system in the body. In this study, the experimental groups’ ACP and AKP activities showed a significant decreasing trend with the increase of PS-MPs concentration after 15 days of exposure. This suggests that PS-MPs inhibited the activities of ACP and AKP in the carp, and the decrease in their activities may induce an inflammatory response, which in turn may affect the immune function of the carp organism. Similar results to the present experiment were obtained in an exposure experiment to PVC-MPs in carp, where corresponding changes in the activity of ACP, AKP, LZM, and the expression levels of a range of immune-related genes were observed.22 LZM is a key defense molecule of the innate immune system, produced in vivo mainly by neutrophils and macrophages, which can mediate the defense mechanism against exogenous pathogen infection through the complement system and macrophages, and its activity can be affected by a variety of factors such as temperature, acidity and alkalinity, and heavy metal ions.23 LZM content in the carp of experimental groups was significantly higher, indicating that the immune response of the organism plays a role under the stress of PS-MPs. These results indicate that PS-MPs can significantly disrupt the homeostasis of relevant immune-enzymes in the carp, produce immunotoxicity, and lead to the development of immune stress.24
MDA is a product of lipid peroxidation and can be used as an indicator of the degree of oxidative damage in the body. SOD, CAT and POD are common antioxidant enzymes in the body and have the ability to scavenge free radicals and protect the cells from oxidative damage.25 The MDA content of experimental groups was significantly different compared with the control group, indicating that PS-MPs stress disturbed the reactive oxygen species balance in the body of carp and caused oxidative stress in the carp. MDA content in 50 and 100 μg/L PS-MPs groups was significantly higher, but the MDA content of the 200 μg/L PS-MPs group was significantly lower, suggesting that the carp’s immune system played a role in inhibiting PS-MPs-induced oxidative stress, perhaps by inducing an increase in the activity of antioxidant enzymes, which is in line with the results of the relevant studies on the effect of PS-MPs on crucian carp (Carassius auratus).26 The results of this experiment showed that both SOD and CAT activity increased significantly with the increase of PS-MPs concentration, and the enzyme activity was also at its highest value at the highest concentration of PS-MPs, but the MDA content was significantly reduced, which may suggest that the activities of these two antioxidant enzymes in the carp were activated after being stressed by PS-MPs, which reduced the content of MDA, and thus came to cope with the challenge of oxidative stress. Similar results were obtained in a study where PVC-MPs were applied to common carp.27 POD is an important enzymatic antioxidant that scavenges hydrogen peroxide radicals and other toxic molecules from cells and protects them from oxidative damage. In the results of this experiment, the POD activity in the carp of experimental groups was significantly reduced compared with the control group, and the lowest POD activity was found in the high-concentration group, indicating that the increase in the concentration of PS-MPs exacerbated the oxidative stress response in the carp, and the POD activity was not enough to scavenge the increase in the amount of reactive oxygen species in the carp, which led to the decrease in its activity.
In terms of amino acid metabolism, Met, Leu, Ile, and Thr as essential amino acids in fish, play an important role in enhancing antioxidant and immune function. Thr is an important nutritional fortifier in the animal body and has the effect of improving immune functions.28 Ile can significantly enhance the physical barrier function of fish gills and intestines and improve the phagocytosis and bactericidal efficacy of phagocytes, thus enhancing the non-specific immunity of fish.29 In this study, correlation analysis showed a positive correlation between ACP, AKP, and POD with the content of Met, Thr, Leu, indicating that PS-MPs induced oxidative stress and immune response in the body of carp and affected the related amino acid metabolism process, and interfered with the synthesis or catabolism of some amino acids, affecting the balance of amino acid metabolism in carp, which was associated with a variety of pathological and physiological development processes. Similarly, the experiment found that high levels of Leu could enhance the activities of intestinal amylase, promote hepatic and pancreatic glycolysis and gluconeogenesis, as well as hepatic lipid catabolism, and reduce inflammation when Ile is deficient or in excess, cells may be at risk of oxidative damage.30
Our results align with previous research on marine species, which also reports adverse effects such as reduced growth, oxidative stress, and immune suppression due to MPs exposure. However, our study highlights that freshwater species may exhibit distinct physiological responses, potentially due to differences in habitat conditions and species-specific sensitivities. These findings underscore the broader relevance of MPs impacts across aquatic ecosystems and emphasize the need for tailored mitigation strategies in both marine and freshwater aquaculture systems. To address MPs exposure in aquaculture, our findings suggest that integrating advanced filtration technologies, such as membrane filtration or biofilters, could effectively reduce microplastic contamination in water sources. Additionally, modifying feed practices, such as using microplastic-free feed or incorporating natural binders to reduce PS-MP uptake, could further mitigate risks. These strategies, informed by our results, could enhance the sustainability and resilience of aquaculture systems globally.
Conclusions
In summary, PS-MPs significantly affect the common carp’s growth indicators, immune and antioxidant enzyme activities, and amino acid metabolism. These changes may negatively impact their health condition and nutritional value, consequently reducing the economic value generated by common carp farming. Therefore, this study emphasizes that the potential threat of PS-MPs pollution to fish health and consumer safety needs to be urgently addressed. The control of PS-MPs levels in the aquatic environment during common carp culture is a key direction for future research. To mitigate these risks, it is recommended that aquaculture industries adopt stricter monitoring and filtration systems to reduce microplastic contamination in water sources. Additionally, policymakers should consider implementing regulations to limit plastic waste and promote sustainable practices to safeguard aquatic ecosystems and the economic viability of fish farming.
Acknowledgments
This work was supported by the National Natural Science Foundation Youth Foundation. (Grant numbers: 32202952).
Authors’ Contribution
Conceptualization: Pengcheng Li (Equal), Ping Sun (Equal). Methodology: Pengcheng Li (Equal), Deshan Chen (Equal), Pengfei Xie (Equal). Formal Analysis: Pengcheng Li (Equal), Deshan Chen (Equal), Pengfei Xie (Equal). Investigation: Pengcheng Li (Equal), Deshan Chen (Equal), Pengfei Xie (Equal). Writing – original draft: Pengcheng Li (Lead). Writing – review & editing: Weijun Chen (Equal), Lei Han (Equal), Feng Yang (Equal), Ping Sun (Equal). Funding acquisition: Weijun Chen (Equal), Ping Sun (Equal). Resources: Ping Sun (Lead). Supervision: Ping Sun (Lead).
Competing of Interest – COPE
No competing interests were disclosed.
Ethical Conduct Approval – IACUC
This experiment complies with the requirements of the Experimental Animal Management and Ethical Welfare Committee of Henan University of Science and Technology.
Informed Consent Statement
All authors and institutions have confirmed this manuscript for publication.
Data Availability Statement
All are available upon reasonable request.