1. Introduction

Crustaceans provide ideal biomarkers for monitoring water quality because of their key role in the food chain and susceptibility to environmental stressors [1, 2]. A diverse group of crustacea is Decapoda comprising marine and freshwater species. Procambarus clarkii is a non-native crustacean to Egypt that lives in remarkable abundance in the Nile and its distributaries. Lately, P. clarkii has emerged as the most economically significant freshwater crustacean species in numerous nations. It is a rich source of high-quality proteins that contain all of the essential amino acids needed for human nutrition [3, 4]. A metabolite of hydrolyzable phenolic tannins, pyrogallol (PG), is found in many plant tissues. Its industrial uses raise issues about how it might affect a variety of aquatic organisms [5]. Aquatic species experience increased stress due to PG-induced changes in water chemistry, which affect pH and impair respiratory function [6, 7]. Our previous works on PG have demonstrated several alterations in the catfish (Clarias gariepinus). These deteriorations include decreased red blood cells, hemoglobin, hematocrit, white blood cells, thrombocytes, and large and small lymphocytes and deteriorated biochemical parameters [8]; injury in the liver and spleen and immunotoxicological effects [9]; histological alterations in the intestine, kidney, and muscles; induced oxidative stress; and altered antioxidant defense responses [10], as well as impaired reproductive and endocrine markers [11].

Microplastics (MPs) can adhere and absorb aquatic contaminants due to their hydrophobic characteristics and large surface area [12, 13]. In addition, MPs serve as transmitter for other pollutants [12], therefore exacerbating their impact. Previous works demonstrated that exposure of aquatic animals to MPs can cause oxidative stress [14], decreases swimming competence [15], and deteriorates hemato-biochemical parameters and antioxidant variables [16, 17]. Furthermore, the bioaccumulation of MPs [18], histopathological alterations [19], gastrointestinal dysfunction [20], and altered hematology, histology, and endoplasmic reticulum [21] are reported in the fish exposed to MPs. MPs have been shown to accumulate in the soft tissues of crustaceans, such as decapods. Decapods exposed to MPs in a laboratory setting may experience a number of adverse effects [22]. Zhang et al. [23] assessed the amount of MP contamination in crayfish (P. clarkii) water, sediment, and particular tissues. In the crustacean Litopenaeus vannamei, the toxic impact of MPs was evaluated [24].

Literature and environmental data show that massive volumes of contaminants are produced annually and released into ecosystems. In aquatic environments, a variety of environmental contaminants are usually present and can combine and impact aquatic life [25–27]. A large body of studies have investigated the combined effect of MPs and other pollutants and their impact on different taxa. Coexposure to MPs and chromium(VI) synergistically and negatively affected Pomatoschistus microps [28]. Also, MPs increase the toxicity effects of cadmium on Danio rerio [29] and Cyprinus carpio [12]. For the narrow-clawed crayfish (Pontastacus leptodactylus), Zeidi et al. [30] evaluated the individual and combined impacts of CuSO₄ and polyethylene MPs. The impact of combined MPs and cadmium on duck pancreas was evaluated by Sun et al. [31].

Hematology and hemolymph chemical indices are sensitive, significant, and promising bioindicators to assess the health of invertebrates [32]. Thus, these biomarkers are frequently employed in ecotoxicological research. According to Jia et al. [33], the hematopoietic tissue of decapod crustaceans is a thin layer of tissue that covers the dorsal side of the stomach and is made up of lobules that are packed with hemocyte precursors [34]. Hemocytes, a type of immune cell found in the circulation of crustaceans, are vital for cytotoxicity, immunological response, wound healing, and infection prevention via phagocytosis [35, 36]. Granular, semigranular, and hyaline (without granules) hemocytes were the three forms of hemocytes found in crayfish [37]. The criteria and types of hemocytes were scored in P. clarkii and exposed to different stressors, in both natural habitats and experimental settings [38]. Therefore, changes in the number and type of hemocytes indicate a physiological reaction to water pollution and chemical environmental stress [39, 40]. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in freshwater crayfish have not been the subject of much research [41]; therefore, measuring their levels is important, particularly when conducting toxicological studies. Yang et al. [42] evaluated the activities of the hepatopancreas damage indicators, AST and ALT, as biomarkers for thermal stress in the crayfish (Chernobyl destroyer). Moreover, the amount of protein in the hemolymph is an important biochemical indicator of health [43]. Assessment of antioxidant enzymes can reveal the state of oxidative stress and antioxidant capacity in aquatic species [11, 44–46]. Glutathione (GSH) is essential for detoxification because it neutralizes free radicals brought on by oxidative stress and aids in their sequestration [47, 48]. The appearance of the hepatopancreas varies greatly between species of crustaceans and is influenced by the surrounding environment [49]. Therefore, examining histopathological changes yields valuable information for evaluating the effects of stressors and toxicants on aquatic species. Histological changes in aquatic crustacean species have been linked to exposure to both organic and inorganic pollution [50–53]. This study completes our [54] recent examination on crayfish exposed to the two chemicals being studied. During our work [54], neurological indicators including acetylcholinesterase and nitric oxide were examined, as well as the activity of serum lysozyme, phenoxide, and acid phosphatase. Additionally, intestinal histological changes were evaluated. The detrimental effects of MPs and PG on red swamp crayfish (P. clarkii) were investigated in this study both separately and in combination. The selected biomarkers were assessed in order to clarify the synergistic interaction between the two chemicals under evaluation.

2. Material and Methods

The present study is an integral part of our most recent investigation [54] on crayfish exposed to MPs and PG. The standard protocol employed in this study is by the Animal Use and Care Committee of the Faculty of Science, Assiut University, Egypt (Protocol No. 28/2024/038).

3. Results

3.1. Hemocyte Count

The hemocyte count following exposure to PG and MPs is presented in Figure 1. The count of granular hemocytes significantly decreased after exposure to PG (p < 0.0001), MPs (p < 0.0001), and the combined treatment (PG + MPs) (p < 0.01) compared to the control group (Figure 1). Similarly, the semigranular hemocyte count significantly decreased after exposure to PG (p < 0.0001), MPs (p < 0.001), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 1). The total hemocyte count also showed a significant decrease following exposure to PG (p < 0.0001), MPs (p < 0.0001), and the combined treatment (PG + MPs) (p < 0.001) compared to the control group (Figure 1).

3.2. Hepatopancreatic Parameters

The hepatopancreatic function parameters following exposure to PG and MP were presented in Figure 2. AST significantly increased after exposure to PG (p < 0.0001), MPs (p < 0.001), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 2). Similarly, AST showed a significant increase after exposure to PG (p < 0.01), MPs (p  < 0.05), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 2). Total protein also exhibited a significant increase after exposure to PG (p < 0.0001), MPs (p < 0.0001), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 2).

3.3. Antioxidant Defense Enzymes

The effects of PG and MPs on the antioxidant defense enzymes of crayfish are shown in Figure 3. SOD and MDA were notably decreased after exposure to PG (p < 0.0001), MPs (p < 0.0001), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 3). Additionally, CAT was significantly reduced after exposure to PG (p < 0.01), MPs (p < 0.05), and the combined treatment (PG + MPs) (p < 0.05) compared to the control group (Figure 3). Conversely, GSH and TAC showed significant increases following exposure to PG (p < 0.0001), MPs (p < 0.0001), and the combined treatment (PG + MPs) (p < 0.0001) compared to the control group (Figure 3).

3.4. Histological Examination

The hepatopancreas of the control crayfish featured normal hepatopancreatic tubules with well-organized structures (Figure 4A). On the other hand, the sublethal exposure to PG and MPs, both separately and together, had a histopathological impact on the hepatopancreas tissues. Individually, PG posed dilation in tubule lumen associated with vacuolation (Figure 4B). As well as PG, MPs potentially caused multiple vacultions, in addition to severe degenerations in the hepatopancreatic tublues (Figure 4C). The coexposure to PG and MPs (Figure 4C) exhibited multiple histological alcerations in the hepatopancreas including eosinophilic deposits, completely damaged and ruptured tubules, hemocytic infiltrations, and degenerated tubule margins.

4. Discussion

Fish and shellfish have been the subject of most experimental studies on the effects of MPs on aquatic life rather than crustaceans. On the other hand, there have been relatively few investigations on how PG affects various aquatic taxa, particularly aquatic crustaceans. Fortunately, we have sought to address the combined toxicity of PG and MPs in our recent study [54], which has partially filled a gap. Accordingly, the current findings are substantially supported by our previous research on PG [8, 10, 11], suggesting that coexposure of PG with MPs synergistically and potentially impacted the crayfish [54]. It is commonly known that MPs have a significant ability to absorb organic compounds in addition to their individual effects [68]. Therefore, MPs serve as carriers of other pollutants, and this characteristic contributes to their cotoxicity. On the other hand, aquatic invertebrates may have a better absorption route once contaminants are carried and absorbed by MPs, enhancing the bioavailability upon consumption [69–74]. The results of the current investigation indicated that PG and MPs could have an impact on the evaluated biomarkers, both independently and significantly combined. The PG- and MP-damaging impact on hemocyte count observed here implies the toxic potential of both chemicals. In accordance, MPs have been found to affect immune system functionality by decreasing the count of hemocytes, modifying the oxidative system, respiring, and increasing energy consumption in bivalves, due to physiological changes [75]. The coexposure to MPs and cadmium induces oxidative stress, and fibrosis in duck pancreas was evaluated by Sun et al. [31]. There was a appositive correlation between the adsorption capacity of bisphenol A on PVC-MP and PVC-MP concentration [76]. Our findings [54] on the effects of PG and MPs on crayfish consistently showed that both PG and MPs, either alone or in combination, reduced immunological parameters (LYZ, Phx, and ACP) and neurotoxic markers (AchE and NO). This implies the potential adsorption capacity of MPs, therefore their toxicity [69]. Supporting study has recently demonstrated that the growth and photosynthetic activities of Microcystis aeruginosa were more severely reduced when PS-MPs were added to 8 mg/L PG [77]. When stressed or diseased, a crayfish loses a significant portion of its hemocytes and their concentration in the blood drops [78]. In other words, crustaceans have rapid fluctuations in the number of circulating hemocytes in response to pathogen infection and injury [79]. In the current study, the hemocyte count decreased significantly after exposure to PG and MPs or their combination. Other crustaceans exposed to different stresses and toxicants have also been reported to have lower hemocyte numbers. Comparable to this, Qin et al. [80] observed a drop in the number of hemocytes in the freshwater crab Sinopotamon henanense following cadmium exposure. Our results are in accordance with Belek et al. [2] on the crayfish (Astacus leptodactylus) following exposure to perfluorooctane sulfonate for 21 days. Similar results of hemocyte count were reported by Zhang et al. [81] on P. clarkii after a 21-day dietary exposure to MPs. Comparing the current hemocyte count with other comparable research suggests that their random fluctuation is a reflection of environmental conditions, chemical and physicochemical stress, and natural cycles [82]. Furthermore, MPs and PG altered serological parameters (protein, AST, and ALT) and oxidation indicators (SOD, GSH, and TAC). In the current study, the level of total protein decreased significantly due to exposure to PG and MPs. These observations agree well with Oreochromis niloticus exposed to MPs [83] and C. gariepinus which treated with PG [8]. Javed et al. [84] stated that increased immunological response and tissue healing will require more protein production. In this regard, MPs changed enzymatic markers of lipid and energy metabolism and caused tissue damage in critical organs such as the digestive gland and the gills of the bivalves Crassostrea giga [85]. AST and ALT enzymes are indicators of the health of the decapod hepatopancreas [41, 86]. Our findings on the altered levels of AST and ALT imply impaired balance in these markers. The results of Zeidi et al. [30] on the crayfish Pontastacus leptodactylus exposed to both single and combined effects of CuSO₄ and MPs support the current findings. Also, Yang et al. [42] reported a dramatic elevation in AST and ALT measured from the hepatopancreas of the crayfish, Cherax destructor, following exposure to different temperatures. The assessed oxidation indicators, SOD, GSH, TAC, CAT, and MDA, showed a substantial decrease following single and exposure to PG MPs (0.05 ≥ p < 0.0001) when compared to control P. clarkii. The parameters employed in this work to evaluate the existence of oxidative stress have been demonstrated previously to be particular biomarkers that alter in response to various harmful substances in a range of aquatic species, including crayfish [48, 55].

The high levels of GSH resulted from PG and MPs imply its central role in modulating oxidative stress. This may be explained by the fact that GSH can act as a scavenger on its own and is affected by environmental factors; hence, it may be essential for regulating the redox cellular balance [87, 88]. Consistently, elevated GSH expression suggests a potential prognostic marker and appears to be related to the clinical prognosis of individuals with breast cancers [89]. Moreover, we found increased levels of CAT and MDA in the serum of C. gariepinus during our previous investigation [11], which assessed the effects of sublethal toxic doses of PG (1, 5, and 10 mg/L for 15 days).

The main histological changes in the hepatopancreas included necrosis, vacuolization, dissolved hepatic epithelial cells, tubular degeneration, tubule loss, and lesions. In addition to nutrition absorption, storage, vitellogenesis during growth, and ovarian development [90], the hepatopancreas is thought to be the main organ involved in detoxification in P. clarkii, similar to the liver in vertebrates [91]. This role is comparable to that of the vertebrate liver, which serves as the site of detoxification; therefore, histopathological changes in the hepatopancreas are a useful tool for assessing the general health of crustaceans [92]. In this context, the hepatopancreas has been reported to accumulate higher levels of antibiotic residues than other tissues [93, 94]. The current findings are consistent with previous histological alterations in aquatic crustaceans including crayfish. For example, Benli [95] observed necrosis in tubule cells of the hepatopancreas of A. leptodactylus subjected to the pesticide etofenprox. Furthermore, the histology of the crayfish gut in our most recent investigation showed significant intestinal damage, including tissue tearing, necrosis, and epithelial disarray; additionally, PG and MPs jointed exposure exacerbated these effects [54]. Also, previously, Cheng et al. [96] demonstrated hepatopancreatic structural damage (degeneration of the hepatic ducts and tubular vacuolization in epithelial cells) due to joint toxicity of erythromycin and cadmium in mitten crab (Eriocheir sinensis), which is consistent with and supportive of the current results.

These results offered compelling evidence that P. clarkii could experience varied degrees of hepatopancreatic damage induced by PG and MPs, either separately or in combination.

Totally, the current outcomes imply the ability of aquatic organisms, including crayfish, to accumulate PG and MPs in their tissues, which could be harmful to individuals consuming aquatic food contaminated with these chemicals. Time and concentration seemed to have an impact on the degree of hemo-biochemical changes and pathological damage.

5. Conclusion

The results imply that PG and MPs in the freshwater environment may accumulate within the tissues of crayfish. Furthermore, PG and MPs provide synergistic toxicity to crayfish. Hemocyte count, biochemical measurements, and antioxidant variables demonstrated substantial responses in accordance with the hepatopancreatic pathological anomalies after exposure to PG and MPs. The current study elucidated that the possible toxicity of MPs in raising the toxicity of PG was revealed by the increase/decrease in the measured parameters and the histopathological changes in the synergy ratio.

Ethics Statement

Studies were approved by the Research Ethics Committee of the Molecular Biology Research and Studies Institute (MB-2024-39-Z), Assiut University, Assiut, Egypt. All methods were carried out following the relevant regulations and ARRIVE guidelines. The ethics committee of Assuit University, Assuit, Egypt, authorized the experimental setup and fish handling in accordance with the OIE criteria for the use of animals in research.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Rashad E.M. Said, Mohamed Hamed, and Alaa El-Din H. Sayed contributed to conceptualization, methodology, and investigation. Rashad E.M. Said, Mohamed Hamed, Walaa M. Shaalan, and Alaa El-Din H. Sayed contributed to visualization and formal analysis. Rashad E.M. Said, Mohamed Hamed, Walaa M. Shaalan, Heba Allah M. Elbaghdady, and Alaa El-Din H. Sayed contributed to writing–original draft, and writing–review and editing. Alaa El-Din H. Sayed contributed to supervision. All authors have read and approved the final version of the manuscript.

Funding

This research received no external funding.