Effects of Microplastics (Nylon) on the Growth and Blood Profile of Oreochromis niloticus

Water pollution has become a global issue in both developed and developing nations (Qadri and Bhat, 2020). The quality of water is affected by number of pollutants including mining, agriculture, hazardous chemicals and urbanization (Asmath et al., 2022). Plastics have ubiquitous applications in nearly every aspect of modern life which break down into smaller particles in marine settings, the bulk of regularly used plastics are unfortunately not biodegradable and cause harm when they enter food chains (Pothiraj et al., 2023). Plastic manufacturing and use in daily life have increased more than ever before due to its low cost, lightweight, recyclable nature and durability (Almohana et al., 2022). The outbreak of the COVID-19 pandemic has recently triggered a surge in public apprehension regarding plastic waste and underscored the pressing necessity to address the problem of plastic pollution. The global consumption of 7.8 billion pairs of plastic polymer medical gloves and 129 billion facial masks every month has placed significant strain on plastic waste management (Eraslan et al., 2023).
       
Microplastics are particles having a diameter of less than 5 mm (Weis, 2020). In aquatic ecosystems, MPs have the potential to act as vectors for the collection and transportation of heavy metals and organic pollutants, leading to the bioaccumulation of contaminants and toxicants (Khalid et al., 2021). Significant amounts of microfibers from clothing can also be released into the environment during the wearing of garments, not just after washing. Putting on clothing can also produce a sizable number of microfibers. Nylon-based microplastics have been found in a variety of freshwater and aquatic habitats. Microfibers are released from the laundry washer during the washing process and end up in sewage systems and municipal wastewater networks. They are the most common kind of microplastics in freshwater, wastewater and the ocean, according to several studies (Le et al., 2022).
       
Fishing gear that is made of nylon netting and monofilament line is easily lost at sea, which adds to the entanglement of marine life. Specifically, ghost fishing nets account for over 10% of plastic waste in the ocean (Koziol et al., 2022). Fish are an essential source of high-quality protein for humans. Chemical contaminants rapidly alter the morphology, tissue and biochemistry of aquatic animals, especially in the liver, kidneys and gills. Fish naturally carry bacteria and other factors that cause spoilage, leading to financial losses and health risks for consumers (Guetarni and Labdi, 2023). Tilapia are omnivores, consuming detritus, biofilms, aquatic plants, small invertebrates and phytoplankton and they can be raised intensively on low-protein diets. Studies on freshwater tilapia (Oreochromis niloticus) have examined their dispersion, bioaccumulation and metabolic responses to pollutants (Cuevas-Rodríguez  et al., 2024).
       
Hematological factors play a crucial role in understanding and investigating fish health status indicators that respond to alterations in diet, illnesses and the quality of the water. Numerous diseases, environmental variables and even husbandry issues have been linked to significant losses in aquaculture operations (Chen and Luo, 2023). Behavior, habitat and climate can also have an impact on hematological parameters. PCV, Hb concentration, erythrocyte indices (MCV, MCH, MCHC), RBCs and WBCs count are among the parameters measured during hematology evaluation of fish (Ruby et al., 2022).
       
Growth is a multifaceted and environmentally influenced trait characterized as a somatic function that reflects the interplay between dietary composition and quality, intake, utilization and an organism’s physiological processes (Triantaphyllopoulos et al., 2020). Plastic particles, being non-digestible, accumulate within the stomach, causing hunger reduction and ultimately resulting in starvation, thereby adversely affecting the organism’s growth rate (McHale and Sheehan, 2024).

Microplastics are prevalent polluants in aquatic environments and can be ingested by fish, causing poisoning, diseases and even death (Zheng et al., 2022). This study aimed to access the impact of nylon microplastics ingestion by fish. The growth performance and hematology of Oreochromis niloticus were carefully assessed under controlled conditions in an aquarium. The experiment was conducted during 2022-2024 at the Zoology Department, Laboratory of Riphah International University Faisalabad, Pakistan.
 
Fish collection and acclimatization
 
Oreochromis niloticus were obtained from the Govt fish hatchery in Faisalabad. Fish were kept in glass aquariums and allowed to acclimate in laboratory conditions for 7 days. Air stones were placed for aeration. Fish were fed on commercially prepared meal twice a day. Water quality parameters and oxygen levels were monitored weekly.
 
Sample preparation
 
Polyamide nylon microplastics were purchased from the chemical market in Faisalabad in the form of irregular-shaped particles. For sample preparation, the microplastics were mixed with tap water at a concentration of 2.5 g microplastics per liter (2.5 g MP/L) and mixed using an electric stirrer. For each experiment, additional dilutions were made from this initial solution to set up the required treatments.
 
Microplastics exposure
       
Healthy fingerlings were randomly chosen and divided into four groups. The length and weight of the fish were measured prior to transfer into the experimental aquariums. The fish density was maintained at 12 fish per tank for each group. The first group (T0) were treated as the control, while the remaining three groups (T1, T2 and T3) were exposed to varying concentrations of nylon microplastics, 100 mg/L, 150 mg/L and 200mg/L respectively, for a period of 28-day.
 
Growth performance assessment
 
The growth of fish was evaluated by calculating growth parameters such as condition factor, weight gain, absolute weight gain, absolute length growth, specific growth rate and survival rate.
 
Condition factor
 

 
Weight gain (%)
 

 

 
Absolute weight gain (AWG)
 

 

 
Absolute length growth (ALG)
 

  

 
Specific growth rate (SGR)
 

 

  
Survival rate (%)
 

  

 
 

Hematology analysis
 
Hematological parameters were accessed by CBC test which showed following indices such as red blood cell count, white blood cell count, hemoglobin concentration and hematocrit level. The mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and mean corpuscular volume were calculated by using the following formulas:
 
Red blood cells count
 
Blood was drawn into an erythrocyte pipette, diluted with hayem solution and thoroughly shaken to determine the number of RBCs. Counting of the cells was performed under ´ 250 magnifications. Final values of red blood cells count were calculated by using following formula:
 

      

    
White blood cells count
 
White blood cells were calculated by taking blood into white blood cells pipette then the blood was diluted with white blood cells diluting fluid and mixed well. A few minutes were given to the cells to settle. The WBCs white blood cell counting chamber was filled with blood and the huge squares in each of the chamber’s four corners were counted. The final count of WBCs was determined using the following formulas:
 

Hemoglobin
 
To determine hemoglobin, blood was drawn into a tube that contained the Drabkin reagent. The pipette was given multiple rinses with the reagent before being let to settle for a short while. The absorbance of hemoglobin solution was measured at 530–550 nm to compare the known hemoglobin content and unknown sample to a reagent. Final hemoglobin concentrations were calculated by using the following formula:
 

 

 
Hematocritÿ
 
Hematocrit% was calculated by taking blood into a heparinized hematocrit pipette. The pipette was then allowed to centrifuge for few minutes. Using reading equipment, capillary tubes were used to record the hematocrit volume. The percentage of blood cells in the entire volume was used to estimate the hematocrit value. The following formulas were used to estimate the MCH,
Mean corpuscular hemoglobin: 
 

 

 
Mean corpuscular hemoglobin concentration:
 

 

 
Mean corpuscular volume:
 

 

 
Statistical analysis
 
The study was conducted under a completely randomized design (CRD). The one-way ANOVA was used to examine the effects of different concentrations of microplastics.

Nile tilapia were exposed to different concentrations of microplastics nylon, T0 (control group), T1 (100 mg/L MPs), T2 (150 mg/L MPs) and T3 (200 mg/L MPs) respectively, for 28 days. The maximum values of average weight and weight gain were observed in T1 exposed to the 100 mg/L concentration of nylon microplastics while T3 showed minimum average weight and weight gain as compared to control group. However, T2 showed similar values as T1. Maximum values of specific growth rate were observed T1 which was nearly similar to control group, while T3 showed minimum values of specific growth rate in nile tilapia. Maximum values of Average length and length gain also showed the similar results with significant lower values as compare to control and T1. Condition factor showed normal values in T1 while T3 showed minimum values of condition factor in nile tilapia. 
       
Results from the Analysis of Variance (ANOVA) showed that the weight gain of nile tilapia were significantly reduced in treatment T3 as compared to other experimental groups and the average weight and length growth of nile tilapia showed significant variations in T3 as compare to other treatments. A progressive reduction in the length gain of nile tilapia was also observed in T3. T1 showed nearly similar values as compared to control group T0, while T2 showed no any significance changes with comparison to T1. However, slight differences were observed in condition factor and specific growth rate among the fish exposed to different concentrations of nylon microplastics. The survival probability of nile tilapia was not affected by microplastics nylon exposure in all groups (Table 1).


 
Hematological analysis
 
All hematological parameters were interactively affected by the higher concentrations of nylon microplastics. Hematological parameters in the blood were estimated by the CBC test. Normal values of red blood cells, hemoglobin, hematocrit was observed in treatment T0, while T1 showed nearly equal values of red blood cells as compared to T0. However, T2 showed slight differences from T0 and T1. T3 showed significant reduced values from all other treatments. MCH, MCV and platelets values also showed similar trends in all treatments. However, a significant raise in MCHC and white blood counts seen in T3 as compared to other treatments, while T1 and T2 showed similar values with slight differences in comparison to control group T0. Lymphocytes, monocytes and eosinophils also showed maximum values in T3 while nearly similar values in all other treatment groups.
       
Results from the Analysis of Variance (ANOVA) showed that RBCs, platelets, hemoglobin, hematocrit MCH and MCV significantly decreased in treatment T3 as compared to the control group T0, while the WBCs and MCHC levels significantly increased in treatment T3 compared to the control group T0 (Table 2).


       
Growth parameters are crucial for assessing the growth performance of fish.  Comparison of the three treatments (T1, T2 and T3) with the control group (T0) showed significant differences to high concentrations of microplastics. While low concentrations showed negligible effects. In high concentrations, reduced weight could stem from inadequate digestion, as plastic particles, being indigestible, occupy the stomach, diminishing hunger and potentially causing starvation, consequently affecting the organism’s weight negatively. The consumption of microplastics can cause disturbances to fish metabolism by altering the bloodstream’s triglyceride and cholesterol ratios. The results on the specific growth rate of Oreochromis niloticus similarly showed this pattern, with treatment T3 having the lowest specific growth rate and control group T0 having the greatest growth rates. In contrast to the control group, T1 and T2 displayed comparable specific growth rates.
       
The observed effects may be attributed to stress induced by poor water chemistry, which creates a stressful environment leading to disturbances in hormone levels, ultimately impacting growth and immune functions negatively. Microplastics possess chemical properties that can inhibit or diminish the production of digestive enzymes, resulting in poor assimilation and subsequently reduced or stunted growth. Ouyang et al., (2021) reported similar findings in their experiment with common carp, where exposure to different concentrations of MPs over 30 days resulted in a significant reduction in SGR at higher doses, indicating a delayed effect on growth post-microplastic exposure.
       
The control group T0 showed the maximum length gain whereas treatment T3 showed the lowest length gain. In contrast to the control group, T1 and T2 displayed comparable length increase rates. Mtega et al., (2023) reported similar findings in their experiments with nile tilapia. The ingestion of microplastics induced stress in Nile tilapia, as evidenced by various clinical signs and a delay in proper growth. While there were no mortalities observed among the Nile tilapia, their responses to the presence of microplastics in the aquarium indicated adverse effects on their well-being.
       
The control group T0 showed the highest condition factor values, while treatment T3 showed the lowest condition factor values. In contrast to the control group, T1 and T2 displayed condition factor values that were similar. The decrease in condition index could stem from diminished energy reserves, potentially caused by reduced feed intake observed in T3. Since the condition factor reflects energy reserves, alterations in feeding rate and type may influence changes in this index. The findings from this study regarding condition factors align with the research conducted by Mizra et al., (2017), who investigated the microplastic content across different feeding types. Their results revealed that omnivorous fish exhibited higher levels of microplastic fibers compared to herbivores and carnivores.
       
Hematological indices are dependable markers for evaluating fish health (Ruby et al., 2022). The results of this study suggest that, in comparison to the control group and the other two treatments, Oreochromis niloticus treated with microplastic nylon exhibited significantly lower hematological indices in T3. Red blood cell counts, hemoglobin, hematocrit, platelets, MCH and MCV were considerably higher in treatment T0, while T1 and T2 were comparable to those of the control group. However, T3 showed considerably lower values of these indices. Hamed et al., (2019) found a drop in O. niloticus’s RBC, Hct and Hgb levels. Similar to this, Vijayaraghavan et al., (2022) observed a drop in RBC count following exposure to PVC microplastics in research on Etroplus suratensis. According to Iheanacho and Odo (2020), changes in hemoglobin concentrations, hematocrit values and erythrocyte content can all be used as markers of a fish’s defensive systems against stress brought on by harmful environmental conditions. Microplastics can accumulate in the digestive and circulatory systems after exposure. This can be harmful and cause a drop in hematological parameters like RBCs, Ht and Hb. The lower hematocrit percentage in the high-concentration treatment may be attributed to increased erythropoiesis, leading to blood lysis. These results align with the research conducted by Lee et al., (2023), who found that exposure to different contaminants in a lab setting reduced the prevalence of peripheral RBCs, Hb and Hct in fish. These findings are also aligned with the investigations by Abdel Zaher et al. (2023) who observed reductions in hematological parameters among mice and fish exposed to polyethylene and polyamide microplastics, respectively. Furthermore, Hamed et al., (2019) also documented a decrease in hematological indices (RBC, HGB, HCT, MCH and PLT) in Nile tilapia exposed to MPs. Additionally, a decrease in the hematological indices (RBC, HGB and HCT) in Nile tilapia exposed to microplastics.
       
White blood cell counts were considerably higher in treatment T3 as compared to the control group T0, while T1 and T2 showed similar values with comparison to T0. Variations were observed in the counts of lymphocytes, monocytes and eosinophils among all treatment groups (T1, T2 and T3). As reported by Hasan et al., (2023), the white blood cells increased significantly with exposure to microplastic polyamide. Moreover, animals’ immune systems and defense systems may be impacted by the absorption of microplastics, which could change their overall health. This may be the result of chemicals found in microplastics that are hazardous or physically obstruct the digestive system, decreasing the absorption of nutrients and delaying the distribution of energy. The results align with the observations of Sinha et al., (2022), who noted comparable patterns in Labeo rohita exposed to fenvalerate, indicating activated immune systems in the exposed fish as opposed to the control group.

This study concludes by highlighting the negative impacts of microplastics on the hematological and growth performance of the freshwater fish species Oreochromis niloticus. Water pollution, exacerbated by the widespread use of plastics, poses significant threats to aquatic ecosystems and human health. The experiment revealed that exposure to microplastics, even at relatively low concentrations can impairs the growth rates of fish while high concentrations can lead to drastic reductions in weight gain and specific growth rates. The observed effects on growth parameters are attributed to various factors, including inadequate digestion, disruptions in fish metabolism and stress induced by poor water chemistry. Furthermore, microplastics build up in fish tissues, changing hematological parameters as hematocrit, hemoglobin and RBCs count. These changes indicate potential toxicity and immune system disturbances resulting from microplastic exposure.

The authors would like to thank those who contributed to the completion of this research. We would like to thank the Riphah International University Faisalabad, Pakistan, for offering facilities and resources for this project.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal Care Riphah International University Faisalabad.

The authors declare that there is no conflicts of interest.