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Biochemical effects of polypropylene microplastics on red tilapia (Oreochromis niloticus) after individual and combined exposure with boron


Toxicity of single pollutants or microplastics (MPs) on organisms have been widely reported. However, their combined toxicity with boron has not been investigated. This study examined effects of individual polypropylene microplastics (PP-MPs) or mixed PP-MPs and boron on biochemical biomarkers in red tilapia (Oreochromis niloticus). O. niloticus were exposed for 21 days to pristine PP-MPs concentrations (10 or 100 mg/L), concentrations of boron alone (30 or 70 mg/L), and identical concentrations of boron in the presence of PP-MPs in laboratory aquaria. Results showed that higher concentrations of individual PP-MPs lead to significantly decreased acetylcholinesterase (AChE) in the brain and malondialdehyde (MDA) in fish liver. In contrast, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione (GSH) were significantly increased in fish liver exposed to higher concentrations of individual PP-MPs. Mixed concentrations of boron and PP-MPs significantly decreased AChE, GSH, and MDA activity in fish. In contrast, mixed concentrations of boron and PP-MPs significantly increased CAT, SOD, and GPx activity in fish. Findings highlight that PP-MPs may increase adverse effects of boron in O. niloticus. We present evidence that individual MPs in long-term exposure have a significant impact on biomarker responses in O. niloticus.


In recent decades, the demand for the use of plastics in a variety of industries has continuously grown [9, 28, 44]. Plastics are estimated to reach up to 54% (by mass) of anthropogenic waste materials discharged into the environment due to overuse and inappropriate management [22]. Microplastics (MPs; ranging from 1 μm to 5 mm) [16] are ubiquitous in all matrices of the environment [4], including seas [46], sediments [34], rivers [10, 31], soils [29, 61], and airs [1]. Ingestion of different MPs has been shown to be hazardous to a number of species in laboratory experiments, ranging from invertebrates to fish [14]. For example, Hanachi et al. [21] found that Zebrafish (Danio rerio) exposed to combined polyethylene terephthalate (PET) and abamectin for 96h exhibited alterations on glutathione (GSH) content, glutathione peroxidase (GPX), and superoxide dismutase (SOD) activity. Likewise, Ding et al. [12] found that red tilapia (Oreochromis niloticus) exposed to three sizes of PS-MPs (0.3, 5, and 70 − 90 μm) for 14 days had significant effects on oxidative stress in fish.

Biomarker responses have been applied to evaluate effects of environmental stressors on fish [50]. Some laboratory studies have reported MPs uptake in freshwater organisms including fish [13, 27], water flea (Daphnia magna) [24], and zebra mussel (Dreissena polymorpha) [35]. However, information on the mixed effects of MPs and chemical contaminants in freshwater fish is still limited. Zhang et al. [60] showed interactive effects of polystyrene (PS) MPs and roxithromycin on activities of cytochrome P450 (CYP) enzymes [7-ethoxyresorufin o deethylase (EROD) and 7-benzyloxy-4-trifluoromethyl-coumarin Odibenzyloxylase (BFCOD) O. niloticus.

Boron (including borates, boric acid, and boric oxide) can be found in rocks, soils, seawater and fresh water and is considered an essential micro-mineral [47]. Boron is also widely used in agricultural (e.g., fertilizer) and industrial applications (e.g., glass and antifreeze ingredients) [48]. Land-based anthropogenic activities can allow the release of boron into aquatic receiving environments from fertilizers, pesticides, and detergents [49]. Adverse effects on fish from boron contamination, including hematological, serum and DNA damage in Nile tilapia and Rainbow Trout (Oncorhynchus mykiss) have been reported by Acar et al. [2] and ÖZ et al. [41], respectively.

MPs may have a synergistic effect on organism health when combined with other pollutants, or they may serve as a transport vector for environmental pollutants. However, there is still a lack of understanding about the potential for pollutants to be incorporated into aquatic organisms by MP consumption [11]. To our knowledge, no study is available to investigate the combined effect of PP-MPs and boron on biomarker responses on organisms. The interaction of MPs and chemical contaminants has shown conflicting findings, with several studies reporting increased or decreased toxicity of mixed MPs with organic or inorganic chemicals [8, 18, 27]. For example, Karbalaei et al. [27] reported that PS-MPs increased toxicity of chlorpyrifos to O. mykiss. In another study, Guven et al. [18] found that MPs do not enhance the acute toxicity effects of pyrene on predatory performance of barramundi (Lates calcarifer).

Boron could adsorb on MPs, because B(OH)3 attaches to dissolved organic matter through surface complexations [53]. A recent study on the interaction between boron and MPs in aquatic environments showed boron adsorption capacity on aged PVC, aged PS, PVC, and PS. In addition, on aged PVC, 35.9% of the boron desorbed in the simulated gut of warm-blooded animals [53]. Another study also showed amino-modified polystyrene (PS-NH2) and excess boron inhibited the growth of Microcystis aeruginosa [59].

The Canadian federal government has called for more research to improve our understanding of the ecotoxicological impacts of MPs [15]. Thus, these important knowledge gaps must be address to help inform government strategies to reduce adverse effects of environmental MP pollution [52]. Very limited studies examined the effects of MPs and boron on aquatic environments and organisms [53, 59]. This study is the second research to investigate the combined effects of MPs and boron on aquatic organisms. To improve our understanding on impacts of individual and mixed PP-MPs and boron exposure, this study used multiple biomarkers in O. niloticus including a nervous system enzyme acetylcholinesterase (AChE) in the brain to assess potential neurotoxicity; cytochrome P450 (CYP) enzymes (EROD, and BFCOD) in the liver of O. niloticus to assess metabolic disturbances; and an antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), GPX, GSH, and malondialdehyde (MDA) in the liver of O. niloticus to assess potential oxidative damage. The main objectives of this study were to: (1) examine the effect of pristine PP-MPs exposure on enzymatic activities in liver of O. niloticus; and, (2) investigate whether PP-MPs change impacts of boron on enzymatic activities in liver of O. niloticus.

Materials and methods

Sources of MPs

MPs used in this study were pristine polypropylene pellets purchased from commercial MP pellets Takht-e-Jamshid company. Original purity of PP was frozen in liquid nitrogen and crushed with a 0.5 mm sieve in an Ultra Centrifugal Mill ZM 200 (Germany). PP-MPs mixed with ethanol were pipetted onto aluminum stubs and gold sputtered, and considered in a scanning electron microscope (SEM; VEGA3 TESCAN; Czech Republic) for morphological observations (Fig. 1). Image J software was used to analyze the SEM image to determine the MPs particle size distributions. Sizes ranged from 5.12 to 398 μm, with 82% of particles < 100 μm, 13% between 100 and 250 μm, 3% between 250 and 300 μm, and 2% > 300 μm. MP composition was confirmed using Fourier transform infra-red spectroscopy (FTIR, Bruker tensor 27, Germany).

Fig. 1
figure 1

Scanning electron microscope (SEM) image of virgin polypropylene (PP) fragments used by this experiment

Experimental design

Fish samples

Tilapia is one of the most widely used farmed fish worldwide for aquaculture, marketability and stable market prices, and about ten species including Nile, blue, Mozambique tilapia, and red tilapia are the most commercially important species [51]. O. niloticus were bred from brood stock fish in a sterilised farm that used UV-treated water in tanks and the environment was cleaned with detergent and sterilised with 70% ethanol. Larvae were initially fed ad libitum with newly hatched Artemia nauplii, 4 times/day for 2 weeks. Fish were then fed 5–10% of body weight two times/day with fish pellets (crude protein: 38–40%). Early juveniles of tilapia were transported to the fish biology laboratory and acclimatized for 2 weeks at 28.5 ℃ in UV-treated water in a 2000 L fiberglass tank in photoperiod 12:12 light:dark. During the acclimatization period, no mortality was observed.

Fish exposure experiment

A total of 60 early juveniles O. niloticus (mean weight ± SD: 24.15 ± 9.21 g, mean total length ± SD: 8.91 ± 1.56 cm) were randomly distributed among 100 L glass aquaria (Seven fish per aquarium, one aquarium per treatment) 1 week prior to the exposure. A farmwork of this study is shown in Fig. 2. According to OECD guideline for testing chemicals, a minimum of seven fish must be used at each treatment and in the controls and no test tank replication is required [38]. Protocols for exposure, sampling and methods were approved by Laboratory Animal Center. Boron (purity: 99%, size: 44 µm), was selected as the pollutant model, purchased from Nanomaterial Powders, Turkey. Boron concentrations were below reported median lethal concentration (LC50) values for O. niloticus (141.42 mg/L) [2]. Boron concentrations were prepared from a boron standard stock diluted in boric acid (H3BO3, Merck This solution was prepared by dissolving 0.571 g of boric acid, then dried beforehand at 50 ℃ until constant weight, in 500 mL double distilled water and made up to 1 L. Suspension of PP-MPs was prepared by adding MPs particles (0.1 g) in Milli-Q water (1 L), then the bottle was placed for 30 min in an ultrasonic bath (Elma Schmidbauer GmbH, Singen, Germany) to achieve a homogeneous suspension. Chronic toxicity was conducted to identify adverse effects of individual PP-MPs, boron, and mixed boron and PP-MPs concentrations within 21 days exposure according to OECD guidelines [38] and modified by Karami et al. [26]. Treatments included: negative control (NC; no added PP particles), nominal PP-MP concentrations (10 mg/L PP-MPs, 100 mg/L PP-MPs), nominal boron concentrations (30 mg/L boron, 70 mg/L boron), and mixed PP-MP and boron concentrations (30 mg/L boron + 10 mg/L PP-MPs, 70 mg/L boron + 10 mg/L PP-MPs, 30 mg/L boron + 100 mg/L PP-MPs, 70 mg/L boron + 100 mg/L PP-MPs). Concentrations of PP-MPs tested in this study (i.e., 10 and 100 mg/L) were within reported ranges found in previous studies [3, 19]. For combination of boron and PP-MPs, target concentrations of boron (30 mg/L boron, 70 mg/L boron) were loaded to MPs (10 mg/L PP-MPs, 100 mg/L PP-MPs) in cleaned glass tube, then tubes were incubated in shaker for 28 h [27]. Water quality of experiment was as follows: temperature 28.5 ± 0.1 ℃, pH 7.2 ± 0.3, and dissolved oxygen 7.16 ± 0.3 mg/L. To reduce MP aggregation, glass aquaria were aerated gently with two air stones attached to the up and down parts of the aquaria. Fish were fed once a day at 2% of body weight during the 21-day exposure time. Water in aquaria was changed every 24 h with UV-treated water spiked with appropriate boron/PP-MP concentrations. No mortality was observed during exposure. The fish were fasted for 24 h prior to sampling to prevent vomiting during sampling. After a 21-day exposure, five fish per treatment were euthanized with clove oil, weighed and measured. Livers and brains were quickly sampled, washed in 0.15 M KCl, weighed, and then frozen in liquid nitrogen, and stored at – 80 °C until analysis of enzymatic activity.

Fig. 2
figure 2

Framework of the study for 21-day exposure of individual polypropylene microplastics (PP-MPs), boron, and mixed boron and PP-MPs concentrations

Analysis of enzymatic activity

Liver and brain of each sample were homogenized by 0.15 M KCl, 0.1 M Tris–HCl at pH 7.4, and centrifuged (10,000 ×g; 25 min) at 4 °C. The remaining supernatant was collected for determination of enzymatic activity using a microplate reader (Biotek, USA). Analysis of AChE (in brain), CAT, SOD, GPx, GST, MDA activities and protein content were performed according to Diagnostic Reagent Kits (Comin Biotechnology Co., Ltd., Suzhou, China) according to manufacture instruction. All treatments were repeated three times. CAT activity of fish samples was assayed according to the method of ammonium vanadate–molybdate. A unit of CAT enzyme activity defined by catalytic degradation of 1 nmol H2O2 per minute. The xanthine oxidase method (hydroxylamine method) was used to measure SOD activity, and the absorbance was read at 550 nm. The dithio-binitrobenzoic acid method was used to measure the GPx activity, and the absorbance was read at 412 nm. GST activity was determined by measuring the substrates of 1-chloro2,4-dinitrobenzene (CDNB) and glutathione (GSH), and the absorbance was read at 340 nm. The MDA level was measured by the thiobarbituric acid method in the absorbance at 532 nm and 600 nm. EROD and BFCOD activities of the livers were quantified based on the method described by Mayeaux and Winston [37] with a slight modification [13]. The excitation and emission filters for EROD and BFCOD activities were set at 530 and 585 nm, respectively. Briefly, liver homogenate (10 mL), buffer (30 mL), and 40 mmol/L alkyl-substituted resorufin in Tris buffer (12.5 mL) were mixed and the reactions were initiated by adding 10 mmol/L NADPH (10mL). Reactions were stopped by adding ice-cold methanol (150 mL). For quantification, the fluorescence values were compared to authentic resorufin standards.

Data analysis

All data were checked for normality (Shapiro–Wilks test) and homoscedasticity (Levene's test) prior to analysis. Data of enzyme activities were analyzed using one-way ANOVAs and If ANOVA indicate a significant difference (p ˂ 0.05), treatments were compared by Tukey multiple comparison tests. Two-way ANOVA with interaction was used to compare effects of boron in absence and presence of PP-MPs (main factors: boron concentrations and presence of PP-MPs). Data were analyzed with IBM SPSS Statistics (V. 23).


Biomarkers in O. niloticus exposed to individual PP-MPs

As shown in Fig. 3, obvious reduction was observed in the AChE activity in fish brains exposed to high concentration of PP-MPs (p < 0.05). In addition, AChE activity has no significant difference in the lower concentration of PP-MPs and control group (p > 0.05). PP-MPs do not change activity of CAT, SOD, and GPx (p > 0.05) except in higher concentration of MPs that SOD and GPx activity was increased in fish liver (p < 0.05; Fig. 4a–c). Both individual MPs significantly increased GSH activity in fish liver (p < 0.05; Fig. 4d). MDA contents was significantly decreased in higher concentration of individual PP-MPs (p < 0.05; Fig. 5). No significant difference was observed in EROD and BFCOD activity of fish liver in all treatments of PP-MPs (p > 0.05; Fig. 6a, and b).

Fig. 3
figure 3

Change in AChE activity in the brain of fish following various treatments with PP-MPs and boron over 21-day exposure duration. Error bars indicate ± SD (n = 5). Bars surmounted with different letters are statistically different (p < 0.05, Tukey multiple comparison tests)

Fig. 4
figure 4

Change in a CAT, b SOD, c GPx, and d GSH activity of fish liver following various treatments with PP-MPs and boron over 21-day exposure duration. Error bars indicate ± SD (n = 5). Bars surmounted with different letters are statistically different (p < 0.05, Tukey multiple comparison tests)

Fig. 5
figure 5

MDA content of fish liver following various treatments with PP-MPs and boron over 21-day exposure duration. Error bars indicate ± SD (n = 5). Bars surmounted with different letters are statistically different (p < 0.05, Tukey multiple comparison tests)

Fig. 6
figure 6

Change in a) EROD and b) BFCOD activity of fish liver following various treatments with PP-MPs and boron over 21-day exposure duration. Error bars indicate ± SD (n = 5). Bars surmounted with different letters are statistically different (p < 0.05, Tukey multiple comparison tests)

Biomarkers in O. niloticus exposed to individual boron

AChE activity decreased in higher concentration of boron compared to control and lower concentration of boron in fish brain (p < 0.05; Fig. 2). CAT activity was significantly increased in both concentrations of boron in comparison with control group of fish liver (p < 0.05; Fig. 3a). In SOD activity, no significant differences were observed in in both concentrations of boron and control group of fish liver (p > 0.05; Fig. 3b). There was no significant difference in GPx activity among boron concentrations and control group (p > 0.05; Fig. 3c). Higher concentration of born significantly increased GSH activity (p < 0.05; Fig. 3d) and decreased MDA contents in fish liver (p < 0.05; Fig. 4). No significant difference was observed in EROD and BFCOD activity of fish liver in all treatments of boron (p > 0.05; Fig. 5a and b).

Biomarkers in O. niloticus exposed to mixed concentrations of boron and PP-MPs

Similar to the higher concentration of individual PP-MPs, three mixed concentrations of boron and PP-MPs (30 mg/L boron + 100 mg/L PP-MPs, 70 mg/L boron + 10 mg/L PP-MPs, 70 mg/L boron + 100 mg/L PP-MPs) significantly decreased AChE activity in fish brains (p < 0.05; Fig. 2). Higher concentration of PP-MPs and boron was significantly increased CAT activity in fish liver (p < 0.05; Fig. 3a), while no changed observed in other individual and mixed concentrations. Similar to higher concentration of individual PP-MPs, both concentration of boron mixed with higher concentration of PP-MPs were significantly increased SOD activity in fish liver compared to control groups (p < 0.05; Fig. 3b). GPx activity was increased in 30 mg/L boron + 100 mg/L PP-MPs, 70 mg/L boron + 10 mg/L PP-MPs, and 70 mg/L boron + 100 mg/L PP-MPs (p < 0.05; Fig. 3c). In contrast, GSH activity was decreased in 30 mg/L boron + 100 mg/L PP-MPs, 70 mg/L boron + 10 mg/L PP-MPs, and 70 mg/L boron + 100 mg/L PP-MPs (p < 0.05; Fig. 3d). All mixed concentrations of PP-MPs and boron were significantly decreased MDA contents in fish (p < 0.05; Fig. 4). EROD was significantly increased in mixed concentrations of PP-MPs with higher concentration of boron (p < 0.05; Fig. 5a). However, BFCOD activity was significantly increased in all mixed treatments of PP-MPs and boron (p < 0.05; Fig. 5b).


Cholinesterases (ChE) belong to a family of enzymes that hydrolyze acetylcholine into choline and acetic acid, which can block acetylcholine metabolism causing acetylcholine to accumulate in the synaptic cleft, causing nerve impulse transmission to be disrupted [36]. Neurotoxic effects of individual MPs and combined MPs with other contaminants have been widely reported [5, 6, 40, 58]. The activity of AChE in the brain was measured to indicate impacts of individual PP-MPs, boron, and mixed boron and PP-MPs on neural activity. In this study, significant reductions found in AChE activity in the brain of O. niloticus in mixed contaminants concentrations, suggesting that mixed PP-MPs and boron can suppress catalytic capacity of this enzyme and lead to neurotoxicity in fish. In fact, AChE activity inhibition due to individual MPs, contaminants, and MPs load other contaminants has been found in various marine organisms, such as brain juvenile seabass (Dicentrarchus labrax) [6], larvae zebrafish (Danio rerio) [6], liver benthic crustacean (Eriocheir sinensis) [58], and liver mice [58]. Oliveira et al. [39] showed individual polyethylene microplastics (1–5 μm) and combined MPs with pyrene were able to inhibit AChE activity in common goby (Pomatoschistus microps).

SOD, CAT, and GPx endogenous antioxidant defense systems, are important for fish health by scavenging, neutralizing, and/or detoxifying ROS. Both CAT and SOD act as first antioxidant defense enzymes that eliminate ROS induced by xenobiotics. SOD can also catalyze partitioning of superoxide into hydrogen peroxide (H2O2) and molecular oxygen (O2). Any subsequent H2O2 can be removed by CAT to nonharmful products [43]. Changes of CAT activity in individual concentration of boron and mixed concentration of boron and PP-MPs showed MPs and boron led to CAT activation, probably due to the antioxidative response to increased H2O2 production. Some studies showed individual MPs and combined with other contaminants increased CAT and SOD activity in fish [20, 54, 55]. For example, CAT and SOD activity were significantly increased in Channa argus exposed to 80 nm and 0.5 μm PS-MPs (200 μg/L), and Cd (50 μg/L)[54].

GPx aids in the conversion of peroxides into less-toxic hydroxyl compounds and prevents the accumulation of ROS [57]. The higher GPX activity in individual PP-MPs and mixed PP-MPs/boron might have been the result of de novo synthesis, which was potentially induced by increased oxidative stress of liver in response to the contaminants [17] and required further investigation. Similarly, increased GPx activity in the marine copepod (Paracyclopina nana) with 20 μg/mL PS-MPs (0.05 μm) [25] and juvenile guppy (Poecilia reticulata) with low and high concentration of PS-MPs (32 − 40 μm)[23] were observed. In a study conducted by Magara et al. [33], GPx activity increased in combined exposures of polyethylene MPs and fluoranthene in fish gills.

GSH antioxidant enzymes can also play a major role in the maintenance of redox status. The GSH level increased in O. niloticus exposed to individual PP-MPs and higher concentration of boron, suggesting a protective response of fish to MPs concentrations. Such an increase in GSH may show the activation of the glutathione-dependent system of antioxidant defense caused by MPs [55]. However, the GSH content was reduced by mixed PP-MPs and boron, probably due to an antagonistic interaction between PP-MPs and boron. In addition, Wen et al. [55] reported that the mixture of PS-MPs and cadmium resulted in a decreased GSH content in discus fish (Symphysodon aequifasciatus).

Changes in MDA levels, indicate ROS production and intense oxidation, accompanied by severe damage to cell structure and function [56]. Kim et al. [30] found that SOD activity was negatively correlated with MDA levels, owing to the ability of SOD to metabolize and neutralize ROS. This study showed an increase in SOD activity in O. niloticus liver following exposure of individual and mixed PP-MPs and boron treatments. Similarly, MDA content was inhibited in O. niloticus liver in this study. Zhang et al. (2021) reported single and combined effects of phenanthrene and PS-MPs also showed negative correlation of SOD activity and MDA levels in the clam (Mactra veneriformis). Although this study demonstrated that exposure to individual and mixed MPs and boron treatments increases levels of intracellular ROS and, induced oxidative stress, more studies are required to improve our understanding of the biological effects of individual MPs or combined effects with other environmental stressors on aquatic organisms.

Both EROD and BFCOD activities in fish liver showed an increase in mixed concentrations of PP-MPs and boron, indicating disturbance of fish metabolism, but the disturbance mechanism is still unknown. Increase in CYP enzyme activity could be attributed to a period of contaminant adaptation [13]. No significant effects of individual PP-MPs on CYP enzyme activity observed in this study corroborates similar studies that showed CYP enzymes, such as EROD activity, were not sensitive to MPs exposure (e.g., [7, 32, 42]). In contrast to our study, Pannetier et al. [45] reported ingestion of individual MPs increased EROD activity in Japanese medaka. Conflicting results of EROD activity and CYP enzyme metabolism in organisms exposed to MPs pollution required further studies.

In General, MPs may exhibit different effects on O. niloticus, particularly when combined with other pollutants. CAT, SOD, GPx, EROD, and BFCOD activity showed similar results, with a significant increase observed in combined MPs and boron treatments. In contrast, AChE, GSH activity and MDA content decreased in combined MPs and boron treatments. Induced perturbations in antioxidant enzymes observed in this study, may suggest that aquatic organisms activate their antioxidant defense systems to cope with oxidative stress induced by mixed MPs and boron exposure. Recent study showed the charges on MPs affected boron adsorption on MPs and the aggregation of MPs with algal (Microcystis aeruginosa) cells, showing that the charge on MPs is a dominant factor influencing the combined effects of MPs and excess boron on M. aeruginosa [59]. Previous studies have also shown individual or mixed MPs combined with contaminants may induce complex, or even contradictory responses in fish due to the complex suite of contaminants or polymers used in exposure studies. Therefore, further studies are required to verify long-term or short-term biochemical responses in fish exposed to mixed MPs and other contaminants.


This study shows that individual PP-MPs, boron, and mixed PP-MPs and boron exposure induces complex biochemical response in O. niloticus. However, biochemical effects were observed more in mixed PP-MPs and boron treatments. Inhibition of AChE activity suggests potential neurotoxicity of mixed MPs and contaminants in fish. Alterations in biochemical biomarkers highlight that under oxidative stress from individual MPs and in combination with contaminant exposure, antioxidative enzymatic systems could be activated and hamper oxidative damage from occurring in fish. Further studies are required to expand our knowledge on the biological effects of individual MPs or combined effects with other environmental stressors on aquatic organisms.

Availability of data and materials

All data are publicly available, with sources described in the manuscript and supplementary material.


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The authors would like to acknowledge Dr. Abbas Abdollahi for his critical review and valuable suggestions.


This study was not supported by any internal or external funding agencies.

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JY: investigation, review and editing. SK: conceptualization, writing original draft, visualization, formal analysis, investigation. KS: investigation, SMH: formal analysis, review and editing, AFA: formal analysis, review and editing, TRW: review and editing.

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Correspondence to Jian Yang or Samaneh Karbalaei.

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Yang, J., Karbalaei, S., Hussein, S.M. et al. Biochemical effects of polypropylene microplastics on red tilapia (Oreochromis niloticus) after individual and combined exposure with boron. Environ Sci Eur 35, 71 (2023).

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