Pollution of the water environment has been a major threat to the survival of aquatic habitats. For many species, the aquatic environment is a vital resource on which they depend almost entirely for growth, reproduction, and survival [1]. Currently, plastic pollution in the water environment has become an important global concern. At the second United Nations Environment Assembly in 2015, plastic pollution was listed as the second most significant scientific issue in the field of environmental and ecological sciences [2]. As the world's largest producer and user of plastics, China is also experiencing severe plastic pollution in its water environment [3]. Under the action of UV, chemical, physical, and other external forces, larger plastics gradually break down into small plastic fragments, referred to as microplastics (MPs), with particle sizes less than 5 mm [4]. MPs are characterized by their small size, large specific surface area, and strong ability to adsorb pollutants. As they continue to decompose and degrade in the environment, their particle size decreases, leading to a larger specific surface area and an increasing ability to adsorb pollutants [5]. Due to their nonbiodegradable nature, MPs are widely found in aquatic environments, where they are ingested by aquatic animals and passed along the food chain, resulting in inestimable losses to the aquaculture industry. MPs that accumulate in fish move through their cells to the circulatory or lymphatic systems, dispersing throughout their bodies [6]. MPs affect the reproductive system of aquatic animals. Polystyrene microplastics (PS-MPs) induce apoptosis in the zebrafish reproductive system [7], and their reproductive toxicity is closely linked to oxidative damage [8,9]. Studies have demonstrated that MPs hinder the development of sperm and ova in zebrafish [10], while also altering energy allocation and reducing resources dedicated to reproduction [11]. Tigriopus japonicus mistakenly ingests microplastics as food, resulting in reduced food intake, inhibited digestion, and undernutrition [12]. These factors may contribute to the declining fertility of aquatic organisms. The toxicity of MPs is influenced by their particle size. For instance, MPs with a diameter of 20 μm can enter the liver and kidneys of mice via blood transport [13]. Additionally, MPs with a diameter of 5 μm are capable of crossing the blood-testis barrier and reaching the testicular tissue of mice, which can lead to cellular inflammation and a decrease in sperm motility, ultimately affecting the reproductive capacity of the mice [14]. Polystyrene is commonly used in the manufacture of disposable plastic lunch boxes, foam boxes, and foam insulation materials and has become a common model particle for studying biological effects. Therefore, MPs were used as the study material in this experiment.

Numerous studies have found that MPs are important carriers of pollutants in water, such as heavy metals [15], persistent organic pollutants (POPs) [16,17], pesticides [18] and environmental endocrine disrupting chemicals (EDCs) [19]. Exposure of organisms to both MPs and other environmental contaminants can lead to the accumulation of organic contaminants on the surface of microplastics, potentially causing toxicological interactions. In a study conducted, zebrafish larvae were exposed to a combination of polybrominated diphenyl ethers (PBDEs) and nanoplastics, resulting in the development of morphological deformities such as pericardial edema, swollen yolk sacs, and curved tails. Combined exposure also led to a higher level of developmental toxicity in zebrafish larvae compared to exposure to PBDEs alone [20].

EDCs, also known as environmental hormones, are a group of compounds with hormone-like biological effects and are widely found in natural, seminatural, and man-made environments [21]. EDCs enter the organism and cause endocrine imbalance by affecting the formation, secretion, transport, and metabolism of sex hormones in the organism, thus affecting the growth, development, and reproduction of the organism [22]. 17α-Methyltestosterone (MT) is an exogenous androgen that is an EDC and is extremely harmful to plants and animals. MT disrupts the reproductive systems of Gobiocypris rarus [23] and Pseudorasbora parva [24], inhibits germ cell maturation, and regulates gonadal development by mediating the KISS/GPR54 system on the hypothalamic-pituitary-gonadal (HPG) axis [25].

This paper aims to investigate the effects of MT/PS-MPs on zebrafish morphology, key genes for steroid hormone synthesis, hormone content, sperm quality, and cross-generational toxicity to investigate the mechanism of MT/PS-MPs in zebrafish and to search for biomarkers that are sensitive to MT/PS-MPs to provide a theoretical basis for early warning of MT/PS-MPs in the environment.