Histopathological Damage and Mortality in Rainbow Trout Exposed to Microplastics from Disused Tires

Rubber pollution is one of the fastest-growing environmental concerns today, due to the proliferation of products made with this synthetic material, which is present not only in the automotive industry, but also in sectors such as footwear, construction, and the manufacturing of industrial products [1, 2]. Synthetic rubber, highly durable and resistant, presents a great advantage in terms of functionality, but, at the same time, poses a serious environmental challenge, since its decomposition is extremely slow [3-5]. Tires, as one of the most widely used rubber products, contribute significantly to the generation of solid waste, known as end-of-life tires (EDT), which have become a major source of pollution [6, 7].

Each year, global production of end-of-life tires exceeds 1.5 billion units, and more than 3.5 million tons of discarded tires are generated in Europe alone, reflecting the global extent of the problem [8-10]. Globally, end-of-life tires not only occupy vast spaces in landfills but also contribute to the release of microplastics into the environment, one of the most persistent and difficult-to-manage forms of pollution [11, 12]. The lack of efficient solutions for the proper recycling of these materials has increased pollution in both terrestrial and aquatic bodies [13, 14].

End-of-life tires, when exposed to environmental conditions such as friction wear, temperature variations, and rain, release small fragments that break down into microplastics [15]. These fragments are easily transported by water to rivers, lakes, and oceans, where they can be ingested by aquatic organisms, seriously altering the health of species and aquatic ecosystems [16]. Microplastics derived from tires contain not only synthetic rubber but also dangerous chemical compounds such as mineral oils, which, when released into water, aggravate the pollution of water bodies [17-19].

Synthetic rubber released from end-of-life tires not only physically affects aquatic organisms when ingested but also carries a series of harmful chemical compounds that increase the toxicity of water. These compounds include heavy metals, additives, and other substances derived from tire manufacturing processes. These contaminants affect aquatic species at the cellular level, causing negative effects such as digestive obstructions, endocrine disruptions, and, in more severe cases, the death of exposed organisms [20, 21].

The impact of microplastics on aquatic ecosystems has gained great relevance in the scientific community in recent years [22].

These pollutants, largely derived from the wear and tear of end-of-life tires, not only affect the aquatic species that ingest them but also alter the physical and chemical properties of aquatic ecosystems. The accumulation of microplastics in sediments and aquatic organisms generates changes in key parameters such as pH and water quality, disrupting the homeostasis of aquatic ecosystems [23, 24]. In the long term, the presence of these fragments can contribute to the biomagnification of contaminants through the food chain, directly or indirectly affecting species that consume these microplastics. Although the full effects of this contamination are not yet fully understood, scientific evidence indicates that microplastics pose a significant threat to aquatic biodiversity, generating negative impacts on the health of organisms and the functionality of aquatic ecosystems [25-27]. This is shown by the study presented by the study [28] on the toxicity of tire wear-derived pollutants, particularly 6PPD-quinone and nanoplastics (PS-NPs), in fish such as zebrafish. This study showed that exposure to 6PPD-quinone, both alone and in combination with PS-NPs, significantly altered fish locomotor behavior, increasing traveled distance, speed, and time spent in specific areas of the tank, indicating a neurological impact. The results suggest that the effects of combined exposure to 6PPD-quinone and PS-NPs are more intense than when the pollutants are administered separately. However, no significant damage was observed in liver or intestinal tissues, indicating that while the effects are noticeable at the behavioral level, severe morphological damage is not present in the short term. Furthermore, transcriptomic analysis revealed alterations in genes related to lipid and cholesterol metabolism, which could affect cellular integrity and mitochondrial function. This gap in knowledge about the interactions between contaminants highlights the need for more in-depth research, such as that potentially conducted with rainbow trout, to determine the long-term impacts on the health of aquatic ecosystems.

The study [29], which investigated microplastic (MP) contamination in commercial tilapia feeds in Bangladesh, highlighted the presence of MPs in all samples analyzed, with grower feeds showing the highest contamination (2150 ± 70.71 MPs/kg). Fibers were the predominant morphotype (85%), and the most common polymers were polypropylene (38.74%) and polyethylene (33.61%). The polymer hazard index (PHI) was high, especially due to polyvinyl chloride (PVC), which poses a significant health risk to tilapia and human consumers. MPs mainly originate from feed ingredients, packaging materials, and wear and tear on processing machinery. Although the pollution loading index (PLI) values were low, indicating minor contamination, the results show that MP contamination remains a risk in aquaculture. The study suggests reducing the use of single-use plastics in animal feed production and conducting further studies to assess the toxicological effects of MPs, especially in different geographic regions. It also calls for greater regulation and oversight of commercial animal feed production to mitigate this problem.

Likewise, there is the study [30] analyze microplastic pollution in rivers and its interaction with freshwater fish, highlighting the spatial and temporal dynamics of these contaminants. The study describes how microplastic input sources affect their concentration and composition in rivers, influencing their distribution throughout the river system. Microplastics interact with fish primarily through the ingestion of particles that are mistaken for food. Furthermore, the article analyzes the physical and toxic effects of microplastics on fish species, with special attention to environmentally relevant concentrations. The results suggest that microplastics can accumulate in fish, affecting their physiology and behavior, and that benthic species are especially susceptible due to the high concentration of microplastics in riverbed sediments. However, it was observed that the behavior of microplastics varies according to their size, density, and polymer type, which affects their retention and translocation in aquatic organisms. Despite the impacts found, the authors suggest that pollutant management could be improved through better monitoring and control of microplastic sources in river ecosystems, which would allow for more accurate data on their influence on aquatic species and ecosystem health.

To understand the specific toxicological effects of end-of-life tire fragments (EDT) in aquatic ecosystems, toxicological bioassays play a fundamental role. These experimental assays allow the toxicity of microplastics released into water to be assessed by observing parameters such as mortality, behavior, and histological changes in exposed organisms. Bioassays are essential for obtaining quantitative and qualitative data on the effects of microplastic pollution, facilitating the identification of toxicity levels and understanding the mechanisms of damage to exposed species [31-33]. In this context, the rainbow trout (Oncorhynchus mykiss) has been used as an experimental model in various investigations due to its high sensitivity to aquatic pollutants, its ability to bioaccumulate toxic substances and its relevance in both scientific research and aquaculture [34].

Rainbow trout is widely recognized as a model organism in ecotoxicological studies due to its biological characteristics, which make it especially sensitive to aquatic pollutants. This species has demonstrated a remarkable capacity to bioaccumulate contaminants, making it an ideal tool for assessing the toxicity of various substances, including microplastics derived from end-of-life tires [35, 36]. Furthermore, rainbow trout are of considerable economic importance to the aquaculture industry, reinforcing their relevance for studies seeking to assess the environmental impacts of pollutants. As a species commonly used in bioassays, rainbow trout allows for the simulation of the impact of microplastics on aquatic species in general, providing data that can be extrapolated to other species that may be exposed to similar pollutants [37].

To evaluate the acute toxic effects of EDT in juvenile Oncorhynchus mykiss, considering mortality, digestive bioaccumulation, and hepatic histopathological damage during 96 hours of exposure. The methodology will include fish mortality analysis and histological evaluation of internal organs to determine the median lethal dose (LD50), as well as other harmful effects caused by exposure. This approach will allow for an accurate measurement of the toxic effects of microplastics and provide relevant data for establishing safe exposure limits for aquatic organisms, in order to prevent negative impacts on the health of species and the stability of aquatic ecosystems.

3.1 Results, acclimatization, and preparation of fragments

Phase I constituted the methodological starting point for the study, ensuring controlled conditions for both the model organisms (Oncorhynchus mykiss) and the contaminants (disused tire fragments – DUT). A simulated aquatic environment was established on an experimental scale with homogeneous and monitored physicochemical conditions, which was essential to guarantee the reliability of the experiment. In parallel, the DUT fragments were processed and characterized morphologically and compositionally. These parameters are essential to ensure that subsequent toxicological tests are performed with homogeneous and comparable inputs, as shown in Table 3, Figure 6, Table 4, and Figure 7, respectively.

Table 3. Conditioning of organisms and conditions of the experimental enclosure

Figure 6. Acclimatization of fingerlings in the laboratory 1

Table 4. Conditioning of organisms and conditions of the experimental enclosure

Figure 7. Acclimatization of fingerlings in the laboratory 2

Furthermore, to understand the nature of the observed toxicity, a detailed chemical characterization of the tire fragments used in this study was performed.

Table 3 shows that the conditions of the experimental enclosure remained within the limits recommended by the OECD (2019), which validates the correct acclimatization process of the organisms. The temperature (12.3 ℃) and dissolved oxygen (8.1 mg/L) guarantee an optimal environment for the model species, minimizing possible stress effects unrelated to exposure to the contaminant. Likewise, the distribution of the 180 fingerlings in six aquariums made it possible to maintain an adequate population density to avoid aggressive behavior or stress due to overcrowding, which strengthens the reproducibility and reliability of the bioassays.

Table 4 shows that the NFU fragments used in the study exhibit homogeneous physical and chemical characteristics. Their average size of 2 mm and angular shape ensure adequate contact surface area with the fish's digestive tract, maximizing physical interaction and potential toxicity. Negative buoyancy ensures that the fragments remain submerged, increasing the likelihood of ingestion by fish during exposure. Finally, the use of techniques such as standardized sieving and FTIR spectroscopy for characterization ensures that the material used in subsequent experimental phases is replicable and scientifically validated.

3.2 Results of toxicological bioassays with NFU fragments

During the 96-hour acute toxicological bioassays, juvenile trout (Oncorhynchus mykiss) were exposed to five concentration levels of EDT. The objective was to determine the evolution of cumulative mortality as a function of exposure time, following the OECD 203 protocol. Mortality observations were performed every 12 hours. As shown in Table 5, mortality was progressive in the treatments with the highest concentrations, while no deaths were recorded in the control group. The results are complemented by those illustrated in Figure 8, corresponding to the experimental system installation model.

Figure 8. Percentage of cumulative mortality in juvenile trout by treatment

In Table 5, C-T5 (50 mg/L) shows a mortality rate of 71.43% at 96 hours, which is consistent with the data and allows for the calculation of CL₅₀.

Table 5. Cumulative mortality (%) by treatment

C-T4 (25 mg/L) shows a mortality rate of 28.57% at 96 hours, confirming that higher concentrations result in higher mortality.

C-T1 (0 mg/L) shows a mortality rate of 0%, confirming that there was no external interference.

In Table 6, the highest density was observed for C‑T3 (1.0633 g/mL), while the lowest was for C‑T5 (0.8189 g/mL). This suggests that treatments with intermediate concentrations could be associated with greater accumulation of biomass or digestive residues before filtration.

Table 6. Sample mass, volume, and density after digestion with 10% KOH

The highest density was observed for C‑T3 (1.0633 g/mL), while the lowest was for C‑T5 (0.8189 g/mL).

In Table 7, the results suggest that treatments with intermediate concentrations could be associated with a greater accumulation of biomass or digestive waste before filtration.

Table 7. Particle counts after digestion with 10% KOH

3.3 Results of digestive tract analysis and detection of NFU fragments

Following exposure of aquatic organisms to fragments of end-of-life tires (EDTs), a physiological and anatomical evaluation was performed to verify the presence, accumulation, and histological impact of these contaminants. This phase was structured in two key parts: (a) digestive analysis with alkaline digestion to quantify retained particles, and (b) histological analysis of the liver to assess the internal damage caused by the treatments.

As shown in Table 6, the digestion process allowed the samples to be reduced to an optimal state for sieving and microscopic analysis. Table 7 and Figure 9 then quantify the particles retained in the fish's digestive system, which is complemented by the series of Figures 10 to 14, which progressively demonstrate liver damage.

The results of the bioassay shown in Figure 15 demonstrate a clear progression of liver damage in rainbow trout as NFU increases. In the control group (0 mg/L), no liver damage was observed, with a score of 0, indicating healthy tissue. At 6.25 mg/L, the liver showed mild damage, with early signs of stress such as mild vascular congestion, resulting in a score of 1. At 12.5 mg/L, the damage became more pronounced, with the appearance of fat vacuoles and cell swelling, yielding a score of 2, indicating moderate liver damage. At 25 mg/L, severe liver dysfunction was observed, with greater congestion and altered hepatocytes, resulting in a score of 3. Finally, at 50 mg/L, the most severe damage occurred, with necrosis, loss of liver architecture, and significantly altered hepatocytes, reaching a score of 4. This dose-dependent increase in liver damage clearly reflects the toxicity of NFU fragments, with higher concentrations causing progressively more severe damage to liver tissue.

Figure 15. Semi-quantitative liver damage scores in rainbow trout

3.4 Results of statistical analysis and calculation of LC50-96 h

The toxicological evaluation of Oncorhynchus mykiss exposed to synthetic rubber fragments for 96 hours was based on statistical analysis of cumulative mortality by concentration. A completely randomized design was used with five treatments (0, 6.25, 12.5, 25, and 50 mg/L) and three replicates per concentration, recording data at 24, 48, 72, and 96 hours. Normality tests (Shapiro-Wilk), homogeneity of variance (Levene), and statistical significance (ANOVA or Kruskal-Wallis) were applied with an α = 0.05 level.

As shown in Table 8, at 96 hours, the 25 mg/L and 50 mg/L treatments showed the highest mortality rates, at 28.57% and 71.43%, respectively. The 50 mg/L treatment exceeded the 50% threshold, allowing the LC50 to be calculated at 96 hours. The lowest lethal concentration observed (LOEC) was identified at 6.25 mg/L, the lowest concentration with an observable lethal effect, while the NOEC corresponds to the control group.

Table 8. Mortality of Oncorhynchus mykiss at different times of exposure, because of four treatments (mg/L) and a synthetic rubber control

At 24 h, there was a complete absence of mortality in all treatments (0%), suggesting a lag phase in the compound's toxic action. No immediate lethal stress was observed, which may be attributed to a delay in hepatic biotransformation or interaction with target organs.

At 48 h, a transient sublethal response began in treatments ≥12.5 mg/L, with a mean mortality rate of up to 9.52 ± 16.49% (C-T4). However, the differences were not statistically significant (p = 0.67), which is consistent with interindividual heterogeneity and the initial physiological tolerance threshold.

At 72 h, an increasing pattern was detected, with 14.28% at 25 mg/L and 19.04% at 50 mg/L, but without exceeding the critical lethal threshold of 50%. Therefore, the LC50-96h value remains ND (Not Determined). Here, p = 0.10 indicates a significant developing trend, suggesting imminent cumulative cellular damage, especially in the liver, as confirmed by histological observations in later phases.

At 96 h, the C-T4 and C-T5 treatments recorded mortality rates of 28.57 ± 8.24% and 71.43 ± 14.29%, respectively, with confirmed statistical significance (p = 0.04, Kruskal-Wallis test). This confirms chronic sublethal toxicity associated with inflammatory processes, oxidative stress, and disruption of osmotic homeostasis.

LOEC (<6.75 mg/L): This implies that even concentrations close to the lowest threshold induce observable toxic effects. This is of great concern in environmental contexts where there is prolonged exposure to NFU microfragments.

NOEC (50 mg/L): This value, as it corresponds to the highest treatment, should be interpreted with caution, as the statistical significance at 96 h calls into question its actual validity in long-term studies. The use of the term "NOEC" here is only applicable to the first 72 h.