Individual and combined toxicities of fluorescent polystyrene nanoplastics and chromium(III) nitrate nonahydrate in Artemia salina

Abstract

There is a growing apprehension on the toxic effects of various pollutants on marine ecosystems. This study was aimed to investigate the influence of the combination of 200 nm fluorescent polystyrene nanoplastics (F-PSNPs) and chromium(III) nitrate nonahydrate (CNN) on their toxicity in Artemia salina, a marine crustacean. The interaction between CNN and F-PSNPs in natural seawater resulted in the formation of micron-sized particles. This interaction also caused a decrease in F-PSNP fluorophore intensity. Reduced residual CNN concentration within the mixture indicated CNN binding to F-PSNPs. Acute toxicity tests were conducted on Artemia salina using different concentrations of F-PSNPs alone, CNN alone, and the F-PSNPs + CNN complex. The study assessed the potential toxicity of these emerging contaminants by examining the mortality rates, hatching success, morphological changes, and biochemical alterations in Artemia salina. Exposure to the F-PSNPs + CNN complex resulted in a decreased hatching success, an increased mortality rate, elevated levels of reactive oxygen species, catalase, lipid peroxidase, and superoxide dismutase, and reduced total protein content, and the independent action model suggested an additive toxic effect of the complex. Significant differences were noted between the impact of the complex and the individual. However, the accumulation of these particles in organisms may affect the food chain. These findings underscore the potential environmental risks associated with the concurrent exposure of aquatic organisms to nanoplastics and other co-contaminating heavy metals.

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Environmental significance

Microplastics and nanoplastics are two emerging contaminants that endanger aquatic ecosystems and the environment. Nanoplastics are released into the environment after being handled improperly. These nanoplastics have the potential to carry other co-pollutants to nonpoint sources. The majority of the aquatic species cannot tolerate large concentrations of chromium(III) nitrate nonahydrate (CNN), despite it being an essential element. The primary link between chromium(III) nitrate nonahydrate's (CNN) toxicity and its capacity to produce hydroxyl radicals. Recent research has predominantly focused on the combined toxic effects of co-pollutant adsorption in marine environments. The interaction between Artemia salina and the complex of fluorescent polystyrene nanoplastics (F-PSNPs) and chromium(III) nitrate nonahydrate (CNN) was examined for the first time. Even though both compounds are known to be harmful when used alone, this study explicitly investigated how they work together and whether they could increase particle-mediated toxicity.

1 Introduction

Alarmingly, over 90% of global marine debris consists of plastic waste, which creates significant water pollution and severely threatens marine ecosystems.¹ Micro-/nano-plastic pollution has recently become a major global environmental concern in ocean waters.² Plastic production surged from 200 million tons in 2002 to 311 million tons in 2014, with estimates suggesting it could reach 33 billion tons by 2050.³ Plastic particles, often derived from various industrial and household products, frequently degrade into smaller pieces called micro-/nano-plastics.⁴ When exposed to UV radiation, weathering, and biodegradation, plastic waste breaks down into nanoplastics (NPs: <1 μm) and microplastics (MPs: 5 mm to 1 μm).⁵ Micro-/nano-plastics (MNPs) efficiently transport heavy metals⁶ across various ecosystems owing to their small size, large surface-to-volume ratio, high surface hydrophobicity, and enhanced reactivity.

Micro-/nano-plastics can infiltrate the circulatory water system and accumulate in the digestive tracts of aquatic life.⁷ The buildup of plastics has been linked to numerous detrimental effects on marine species, ranging from physical injuries to impaired growth and reproductive incapability.⁸ MNP fragments can attach to algae and plankton and subsequently be consumed by various aquatic organisms, including invertebrates, bivalves, fish, and shrimp.⁹ In addition to toxicity concerns, nanoplastics can carry additional pollutants, such as heavy metals, by adsorbing them on their surfaces.^10,11 Furthermore, owing to their extensive surface area and high hydrophobicity, microplastics can serve both as a direct threat and a carrier for other contaminants that penetrate biological barriers such as tissues and organs, resulting in combined toxic effects on marine species.¹²

Polystyrene, a plastic derived from the styrene (vinyl benzene) monomer, is renowned for its insulating properties and lightweight. However, its excessive production, use, and disposal present significant environmental challenges. This versatile material is utilized in various applications, particularly in packaging.¹³ In 2012, global polystyrene production reached approximately 32.7 million tons.¹⁴ Polystyrene nanoplastics (PS-NPs) ERCs at 10 μg L⁻¹ and 100 μg L⁻¹ are considered environmentally significant due to their potential ecological impact.¹⁵ The Environmental Protection Agency (EPA) has established that a concentration of 300 ppm (1000 mg m⁻³) of styrene is acceptable for chronic exposure. Levels exceeding this threshold could pose health risks. In the polymer industry, styrene concentrations typically remain below 20 ppm.¹⁶ The styrene monomer in polystyrene is carcinogenic and may pose serious risks to aquatic organisms.¹⁷ It has been found that polystyrene microbeads (20–50 μm) cause significant injuries to many marine organisms including crabs, fish, mussels, sea urchins, and microalgae. Because of its lightweight nature, polystyrene is easily transportable and may breach ocean barriers to organisms.^8,18

In recent decades, environmental heavy metal concentrations have exceeded acceptable limits, endangering living organisms. Heavy metal chromium (Cr) occurs naturally and is the 17th most prevalent element in the earth's crust.¹⁹ While necessary in small quantities for plants and animals, Cr becomes a significant environmental pollutant at higher levels. Natural processes and human activities have introduced Cr into soil, air, and water, causing widespread contamination.²⁰ When food lands and drinking water are contaminated, Cr can quickly enter the food chain and harm every living creature, either directly or through indirect damage. Cr is also hazardous to plants, causing delays in seed germination, damaged roots, decreased root development, reduced biomass and height, disruption of photosynthetic activities, low grain, chlorosis, membrane damage, necrosis, low grain production, and plant death.²¹ The WHO recommends a maximum total Cr concentration of 50 μg L⁻¹ in drinking water.

High quantities of chromium are intimately associated with pollution caused by humans, especially in the vicinity of tannery discharges, where levels can reach 40 mg of chromium(III) per litre. Notably, the amount of chromium found in ocean water is substantially lower than that in freshwater bodies like lakes and rivers. Total chromium levels in suspended materials and sediments across various water bodies can range dramatically from 1 to 500 mg kg⁻¹. In Canada and the USA, the concentration of total chromium in soils and surface materials ranges from 1 to as much as 2000 mg kg⁻¹, with a geometric mean hovering around 40 mg kg⁻¹. These data underscore the significant environmental impact of chromium contamination and highlight the urgent need for monitoring and remediation efforts.²²

Heavy metal contamination poses a serious ecological and health risk. This research focuses on chromium [Cr], which the Environmental Protection Agency has classified as one of the most common and dangerous contaminants.²³ Cr was used as a soluble reference chemical for toxicity assessment in Artemia salina.²⁴ Cr is predominantly stable in two oxidation states: hexavalent Cr [Cr(VI)] and trivalent Cr [Cr(III)]. In contrast to Cr(III), Cr(VI) is poisonous, persistent, and soluble in saltwater.²⁵ It enters saltwater by natural processes [soil and rock leaching] or human activities [industrial sources].^26,27

The presence of nanoparticles (NPs) and heavy metals in marine environments raises urgent ecological concerns.²⁸ This simultaneous occurrence of NPs and Cr, fueled by industrial effluents and urban runoff, poses significant risks that have not yet been thoroughly investigated.²⁹ Exploring the interactions between NPs and Cr is crucial since they can amplify toxicity in marine organisms. Research shows that NPs can change the bioavailability and mobility of heavy metals, impacting their toxicity levels.³⁰ NPs can be complex with chromium species, potentially mitigating or exacerbating toxicity.³¹ Moreover, NPs might facilitate Cr uptake in marine life by serving as carriers that penetrate biological membranes, and these interactions pose a serious threat to aquatic biodiversity and coastal ecosystem stability.³² NPs may increase Cr toxicity by enhancing solubility and facilitating cellular uptake.³³ Moreover, NP–Cr complexes can induce oxidative stress and disrupt cellular homeostasis in marine organisms.³⁴ Understanding the combined toxicity of NPs and Cr is essential for assessing environmental risks and developing effective mitigation strategies. This study aims to fill this critical gap by investigating the interactive effects of NPs and Cr on marine biota, focusing on toxicity mechanisms, bioaccumulation, and ecological implications.

Numerous secondary producers, including commercially important fish and cetaceans, rely on zooplankton as a crucial food source.³⁵ Zooplankton primarily filters large volumes of surface waters for feeding, often contaminated with micro-/nano-plastics. This increases the likelihood of micro-/nano-plastics ingestion and exposure for higher trophic-level species, such as fish.³⁶ The invertebrate zooplankton, brine shrimp (Artemia salina), plays a vital role in the energy transfer within food chains across various seawater ecosystems, from lakes to oceans. As a nonselective filter feeder, brine shrimp filter substantial amounts of water hourly, making them more susceptible to pollutant exposure than other aquatic organisms.³⁷Artemia salina is frequently employed as a model organism in ecotoxicology studies investigating different environmental contaminants because of its susceptibility to many environmental stresses, especially toxins.³⁸ Environmental impact assessment of pollutants in marine ecosystems utilizes ecotoxicology analysis. Bio-indicators are essential for these analyses because of their ability to detect very low contaminant levels and their responsiveness to environmental stress.³⁹ The examined the effects of nanoparticles on biochemical markers in brine shrimp.⁴⁰

Nanoplastics are known to adsorb other co-pollutants, which can facilitate their entry into organisms and potentially exacerbate toxic effects, as seen in other co-pollutant systems. They highlight significant biological and biochemical changes. A growing body of research examines the toxicity of heavy metals and F-PSNPs in aquatic organisms. This might be the first report on the investigation of the combined toxicity of Fluorescent Polystyrene Nanoplastics (F-PSNPs) and Chromium(III) Nitrate Nonahydrate (CNN) in Artemia salina, a marine invertebrate model widely used in ecotoxicological assessments. While both pollutants have been studied individually, their interactive toxicological effects remain relatively unexplored. Our findings reveal that CNN adsorbs onto the surface of F-PSNPs, significantly altering their physicochemical properties, as confirmed through atomic absorption spectroscopy (AAS), dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, and fluorescence quenching. This interaction enhances CNN's bioavailability, resulting in greater toxicity than either pollutant alone. Notably, we observed significant reductions in hatching success, increased mortality, elevated oxidative stress markers, and various biochemical disruptions in A. salina. These outcomes suggest that the F-PSNPs + CNN complex exerts additive toxic effects, as demonstrated through the application of the Independent Action (IA) model and quantification using Relative Interaction (RI) indices, marking a novel approach in nanoparticle-heavy metal interaction studies. Experimental concentrations were selected based on environmentally relevant levels of Cr(III), particularly those near tannery waste discharge sites, thereby enhancing the ecological relevance of our findings. The study emphasizes that nanoplastics can act as carriers for co-pollutants, increasing their uptake and exacerbating toxicity, a pattern seen in other pollutant systems as well. Even minimal adsorption of CNN onto F-PSNPs was found to modify nanoparticle behavior, increasing organismal exposure and risk over prolonged periods. Our toxicological assessments, including hatching rate, mortality, biochemical markers, and morphological alterations, consistently indicated oxidative stress, characterized by increased reactive oxygen species (ROS) production and compromised antioxidant defense systems. These cellular and molecular disturbances may pose threats to higher trophic levels. However, several research gaps still exist, particularly regarding the combined effects of multiple pollutants, along with long-term and transgenerational impacts, as well as the specific molecular pathways involved. Most current studies are conducted in controlled laboratory settings, highlighting the need for field-based investigations to accurately assess real-world ecological risks. Future research focusing on bioaccumulation, trophic transfer, and chronic exposure is essential for developing effective mitigation strategies against nanoparticle-associated pollution in the aquatic ecosystem.

2 Materials and methods

Fluorescent polystyrene nanoplastics (F-PSNPs) 200 nm in size were procured from Polyscience Inc., while chromium(III) nitrate nonahydrate (CNN) was acquired from Sigma Aldrich in India. SRL Chemicals Pvt. Ltd supplied potassium dichromate (K₂Cr₂O₇) for purchase. Sigma-Aldrich provided sodium carbonate (Na₂CO₃) and 2′,7′-dichlorofluorescein diacetate. In India, Hi-Media Pvt. Ltd provided hydroxylamine hydrochloride, Triton X-100, and dimethyl sulfoxide. We acquired nitroblue tetrazolium chloride (NBT) and hydrogen peroxide (H₂O₂) (30% w/v) from SDFCL in India. We acquired Artemia salina cysts from Ocean Star International Inc. in the United States.

2.1 Characterization and preparation of individual and complex

The initial stock preparation entailed the dispersion of F-PSNPs (200 nm) at 100 mg L⁻¹ and CNN at 100 mg L⁻¹ in Milli-Q water. The experiment was conducted utilizing diverse individual and complex concentrations. The specific concentrations comprised F-PSNPs at 10 mg L⁻¹ and CNN at 5, 10, 15, and 20 mg L⁻¹. In this study, we examined the interaction between F-PSNPs and CNN, keeping F-PSNPs constant at 10 mg L⁻¹ and varying CNN concentrations at 10 + 5, 10 + 10, 10 + 15, and 10 + 20 mg L⁻¹. The mixtures were incubated for 48 hours at 180 rpm to promote interaction. Following incubation, we assessed the toxicity and key characteristics of the complex, providing valuable insights into its potential applications.

2.2 Characterization of individual nanoparticles and complex

The dimensions of the particles and the stability of F-PSNPs and CNN dispersions were assessed using zeta potential measurements using an SZ100 Nanoparticle Analyzer from Horiba Scientific, Japan. An FTIR-JASCO 6800 Fourier transform infrared Spectrometer was utilized to investigate the compounds and their complexes within the range of 4000 cm⁻¹ to 400 cm⁻¹. The physical architecture of F-PSNPs, CNN, and their complexes was analyzed using a Thermo Fisher FEI-Quanta 250 FEG Field Emission Scanning Electron Microscope (FE-SEM), which provided high magnification and elemental analysis by EDX. A Cora 5001 dual Raman spectrometer, including a 785 nm laser with a 450-mW power output, produced by Anton Paar, was employed to examine both complex and individual samples treated with F-PSNPs. The Atomic Absorption Spectrophotometer (AAS) from PerkinElmer, India, was utilized to analyze CNN residual concentrations. The Jenway 6850 UV/Vis spectrophotometer was employed to measure UV spectrophotometric absorbance. A JASCO Model FP-8300 spectrofluorometer, with a 150 W xenon lamp and a 5 nm slit width, was employed to measure the fluorescence intensity and detect peak variations in F-PSNPs following interaction with CNN.

2.2.1 Analysis of UV-visible absorption spectra. A Jenway 6850 UV/vis spectrophotometer was employed to evaluate UV spectrophotometric absorption. The concentration of F-PSNPs was maintained at 10 mg L⁻¹, while CNN concentrations were adjusted to 5, 10, 15, and 20 mg L⁻¹ for spectral analysis. Wavelengths between 190 and 400 nm were used to record the absorption spectra. F-PSNP individuals served as a control. To enhance adsorption, F-PSNPs and CNN samples were agitated at 180 rpm for 48 hours. After incubation, spectra were collected to measure the quantity of CNN that was adsorbed.

2.2.2 Interaction studies of fluorescence intensity of F-PSNPs with CNN. Samples were placed in a cuvette within a temperature-regulated cell holder. To determine the fluorescence intensity, CNN was added to F-PSNPs (kept at 10 mg L⁻¹) at concentrations of 5, 10, 15, and 20 mg L⁻¹ and agitated for 48 h. Measurements were conducted using a thermostatic cuvette holder and a 10 mm rectangular quartz cell. Spectral measurements were performed using the following parameters: excitation and emission spectra slit widths of 2.5 nm and 5 nm, respectively; λ_ex = 228 nm; λ_em = 260–400 nm; scan speed = 500 nm min−1; detector voltage = 240 V; and data pitch = 1 nm.

2.2.3 Resonance light scattering (RLS) investigation. At 298 K, with Δλ = 0, RLS spectra were recorded in the synchronous mode for both F-PSNPs and CNN complex, spanning a 200–800 nm range. The other parameters were consistent with those applied in the fluorescence emission spectra. This approach was employed to uncover intricate relationships between F-PSNPs and CNN.

2.2.4 Three-dimensional (3D) fluorescence spectral analysis. The spectrofluorometric investigation analysed the excitation and emission spectra of F-PSNPs at 10 mg L⁻¹ as a control, alongside various CNN concentrations. Excitation and emission wavelengths used in the study ranged from 200 to 400 nm and 250 to 600 nm, respectively. All other parameters were maintained as in the fluorescence emission spectroscopy. Colour surface maps and contour view plots were used to display the results for F-PSNPs and F-PSNPs + CNN complexes visually.

2.2.5 Evaluation of toxicity in both individual and complex particles.
2.2.5.1 Maintenance of Artemia salina culture. Natural seawater, constantly aerated for 24–48 hours and irradiated with light at 13 001×, developed Artemia salina cysts. Biological pollutants were removed from seawater using Whatman 0.45 μm filter paper, and the culture medium was maintained at a constant temperature of 28 °C throughout the investigation. We used the nauplii from Artemia salina cysts for the subsequent tests.
2.2.5.2 Hatching rate of Artemia salina. In each 12-well plate, 10 Artemia salina cysts were introduced to determine the hatching percentage. Standard methods were used to examine the hatching rate of Artemia salina cysts exposed to F-PSNPs, CNN, and the F-PSNPs + CNN complex. CNN was added at 5, 10, 15, and 20 mg L⁻¹ to the cysts, whereas F-PSNPs were always 10 mg L⁻¹. F-PSNPs were held at 10 mg L⁻¹ and combined with CNN at 5, 10, 15, and 20 mg L⁻¹ for the combinatorial impact. Untreated cysts were used as controls in all duplicate tests. Since cysts become 2nd instar nauplii after 48 hours, the hatching rate was monitored.

The formula for hatching percentage:

HT % = (A/(B + A)) × 100 (1)

In this context, HT denotes the percentage of hatched cysts, A indicates the number of hatched cysts, and B refers to the number of decapsulated cysts. The analysis focused on assessing the impact of various treatments on the hatching rate of Artemia salina cysts.

2.2.5.3 Mortality percentage of Artemia salina. Each beaker contains 10 nauplii for the experiments. The toxicity of F-PSNP was assessed at 10 mg L⁻¹, while the toxicity of CNN was examined at 5, 10, 15, and 20 mg L⁻¹. F-PSNPs were maintained at 10 mg L⁻¹, while CNN was introduced to Artemia salina at 10 + 5, 10 + 10, 10 + 15, and 10 + 20 mg L⁻¹ to determine the complex toxicity. Control nauplii were not treated with F-PSNPs or CNN. The mortality rate was recorded in triplicates at 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours. The LC₅₀ value was calculated for each sample.
2.2.5.4 Bioaccumulation of F-PSNPs and CNN in Artemia salina. A phase contrast microscope was used to view the morphological changes in Artemia salina cysts following particle treatment. A Leica Microsystems Germany microscope was used to see the Artemia salina nauplii at a magnification of 40× after it was put independently on a glass slide cleaned of greasy. Images were captured to record the alterations in morphology and the intake of particles.
2.2.5.5 Biochemical assays. The nauplii were subjected to several doses of F-PSNPs, CNN, and their combination as described above. After 48 hours, they were washed with deionized water and homogenized using 0.5 M phosphate buffer (pH 7.5). The obtained supernatant was used for the following bioassays after the resulting samples were centrifuged for ten minutes at 13 000 rpm.

The Bradford technique was used to measure the protein levels in both the experimental and control groups. Bovine serum albumin was used as the standard reference while recording the absorbance values at 595 nm.

This method used DCFH-DA (dichloro-dihydro-fluorescein diacetate) to assess reactive oxygen species (ROS). A direct correlation exists between the quantity of ROS produced within the cells and the intensity of DCFH fluorescence. Then, 80 μl of supernatant and 20 μl of DCFH-DA solution were combined for this, and the combination was then left to incubate for 30 minutes at room temperature (28 °C) without contact with light. A JASCO fluorescence spectrophotometer FP8300 was then used to measure the production of ROS at an excitation wavelength of 485 nm and an emission range of 510–560 nm.

A key component of our research is measuring catalase (CAT) activity in the presence of oxidative stress. Catalase, an essential enzyme that accelerates the conversion of hydrogen peroxide into water and oxygen, is present in all aerobic organisms. PBS served as the control in this evaluation, which involved mixing 800 μl of hydrogen peroxide solution with 200 μl of sample supernatants. A UV-visible spectrophotometer (EVALUATION 220, Thermo Scientific) was then used to measure the absorbance for three minutes at 240 nm.

Assessing the superoxide dismutase (SOD) activity assists in estimating the antioxidant capacity of a biological system. First, 70 μl of recovered supernatant, 50 mM of Na₂CO₃ buffer (pH 10), 96 mM of NBT, 0.6% of Triton X-100, and 20 mM of hydroxylamine hydrochloride were all put in that order to a 24-well plate to carry out this experiment. For 20 minutes, this mixture was exposed to visible light with a set wavelength. The reaction mixture's absorbance at 560 nm was then determined using a BIORAD microplate reader.

The malondialdehyde (MDA) test is the most widely used method for researching lipid peroxidation in biological systems. The thiobarbituric acid (TBA) test is a dependable technique used in this assay to evaluate thiobarbituric acid-reactive substances (TBARS). Lipid peroxidation may be reliably detected by MDA, a byproduct of lipid hydroperoxides, whereas TBARS indicates oxidative stress. First, 400 μl of a 0.25% TBA/TCA mixture was well mixed with 100 μl of supernatant for this experiment, which was then incubated for 60 minutes at 95 °C. Following the incubation period, the tubes were briefly submerged in a cold bath, and the supernatant was collected and quantified at 532–600 nm.

2.2.5.6 Statistical analysis. The standard error and mean of the triplicate data were reported for each test. A two-way ANOVA with Bonferroni's post-test and a one-way ANOVA with Bonferroni's Multiple Comparison test were conducted to assess the statistical significance of the data, with a significance level of P < 0.05, P < 0.01, P < 0.001. Furthermore, a graphical depiction of the differences between the treatment groups was examined using Prism software. The impact of F-PSNPs on CNN toxicity was investigated using an independent action (IA) model. It was established how the F-PSNPs and the CNN related to one another. Based on the toxicity percentages induced by F-PSNPs and CNN separately, eqn (2) was used to determine the mixture's expected toxicity (C_EXP).In this equation, A and B denote the individual toxicities of F-PSNPs and CNN, respectively.

The reported combined toxicities for CNN and F-PSNPs are represented by C_obs. Eqn (3) illustrates how the inhibition ratio (RI) was used to evaluate the interaction between F-PSNPs and the CNN.

3 Results and discussion

3.1 Dynamic light scattering

Dynamic light scattering (DLS) is a widely recognized technique for measuring the hydrodynamic dimensions of nanoparticles and assessing their aggregation or agglomeration state within a liquid medium.⁴¹ The findings align with our earlier observations, which showed that this distinct aggregation pattern of PS NPs in NSW and RSW, with microscale aggregates at 900–1000 nm, was validated by the size distributions based on intensity from DLS data.⁴² In ASW, both with and without Cr(VI), the effective diameter of nano-TiO₂ was measured at various time points, revealing a slight increase in nano-TiO₂ agglomeration when Cr(VI) was present.⁴³ The hydrodynamic diameters of the F-PSNPs and PSNP-CNN combinations were assessed using DLS. The pristine F-PSNPs displayed a hydrodynamic size of 239.9 nm and 246.3 served as the control. The sizes of F-PSNPs + CNN complexes in seawater with varying concentrations after 24 hours were measured to be 291.3 nm for 10 + 5 mg L⁻¹, 715 nm for 10 + 10 mg L⁻¹, 1308.9 nm for 10 + 15 mg L⁻¹, and 1520.3 nm for 10 + 20 mg L⁻¹, as shown in Fig. 1(A). Additionally, the F-PSNPs + CNN complex exhibited mean hydrodynamic sizes after 48 hours in seawater as follows: the concentration of 10 + 5 mg L⁻¹ showed 2184.8 nm, 10 + 10 mg L⁻¹ showed 2812.6 nm, 10 + 15 mg L⁻¹ showed 3073.7 nm, and 10 + 20 mg L⁻¹ showed 4304.6 nm, respectively (Fig. 1(B)). Fig. 1 illustrates the results, which showed that the complexes' hydrodynamic diameters were greater than those of the individual F-PSNPs.

Fig. 1 Hydrodynamic diameters of control F-PSNPs with Milli-Q water and F-PSNPs + CNN complexes with varied concentrations after 24 (A) and 48 (B) hours. Z-average particle size (nm) and mean ± SD of three trials are presented. X represents the concentration, and Y represents the Z-average.

3.2 Zeta potential

To comprehend the behaviour of nanoparticles (NP) in complicated environmental situations, the zeta (ζ) potential (mV) was evaluated as a critical indication.⁴⁴ When the zeta potential of a suspension is more than +30 mV or less than −30 mV, it is generally regarded as stable. The dividing line between stable and unstable suspensions typically lies at these values.⁴⁵ The zeta potential of F-PSNPs in seawater is −32 mV, indicating an instability in this environment. The zeta potentials of the F-PSNPs + CNN complex in seawater were measured at different concentrations of CNN. As the concentration of CNN increased with a constant F-PSNP concentration of 10 + 5 mg L⁻¹, the zeta potential showed −38.1 mV. At a 10 + 10 mg per L F-PSNP concentration, the zeta potential was measured at −20.6 mV, and at a 10 + 15 mg L⁻¹ concentration, it was −16 mV. At the highest concentration of 10 + 20 mg L⁻¹, the zeta potential was measured at −9 mV. These results indicate that increasing the concentration of CNN in F-PSNPs leads to decreasing stability compared to lower concentrations of CNN shown in Table S1.† Previous studies reported seawater has surface charges measurements showed that seawater had a zeta potential of ζ = −6.7 ± 0.5 mV. Dilutions of seawater yielded zeta potentials of ζ = −9.31, −9.32, and −7.99 mV, respectively. So, it increases the values after the dilution of seawater.⁴⁶ Ionic species' binding to the NP surface changes the charge and causes this rise in negative charge. Numerous additional researchers have documented these results, emphasizing that the abundance of ionic species in saltwater causes PSNPs to have an enhanced negative charge.⁴⁷ Research conducted by⁴⁸ revealed that surface charge significantly influenced NP accumulation. The zeta potential serves as an approximation of the nanoparticle's surface charge, which is heavily concentration-dependent. However, current methodologies struggle to accurately capture this relationship.⁴⁹

3.3 Fourier transform infrared spectroscopy

The FTIR spectra of F-PSNPs, CNN, and complex are displayed in Fig. 2. The PSNP peaks at particular wavelengths, such as 625–970 cm⁻¹ (C–H out of phase bend), 880–1000 cm⁻¹ (C–O stretch), 1300–1380 cm⁻¹ (CH₂ bending), 1550–1750 cm⁻¹ (C=O), 2800–3000 cm⁻¹ (C–H stretch aliphatic), 2800–3060 cm⁻¹ (C–H), and 3610–3645 cm⁻¹ (hydroxyl), like those found in an earlier investigation, are shown.⁵⁰ An antisymmetric O–Cr–O stretching vibration is responsible for the 959 cm⁻¹ band, whereas the symmetrical stretching mode of the Cr–O bond is linked to the 892 cm⁻¹ peak, as demonstrated by earlier research with comparable findings in 953 cm⁻¹ and 875 cm⁻¹ identifying the presence of chromium.^51,52 Similar results were observed in earlier investigations demonstrating the presence of Cr(OH)₃·3H₂O in the band at 1557 cm⁻¹, which may be the consequence of O–H stretching vibration in the short O–H–O bonds of Cr(OH)₃·3H₂O.^53,54 The complex particles show the major peak of CNN presence in the peak of 985 cm⁻¹ 883 cm⁻¹ 1560 cm⁻¹ as the similar result was found in the individual CNN as that result denotes the CNN binding in the upper layer of F-PSNPs, so that the major peak shows CNN.

Fig. 2 Fourier transform infrared spectra: F-PSNPs are indicated in brown line (A), CNN in green line (B), and F-PSNPs + CNN in orange line (C).

3.4 Field emission scanning electron microscope

FESEM is the most effective technology for observing and magnifying surface features such as texture, porosity, and structure. Understanding the adsorption process necessitates comprehending this knowledge.⁵⁵Fig. 3(A) depicts the morphological composition of individual F-PSNPs as a control. When F-PSNPs interacted with CNN, their initially smooth surface became rough. Energy-dispersive X-ray spectroscopy (EDX) confirmed this alteration and showed that F-PSNPs and CNN bind together. The formation of F-PSNPs + CNN complexes occurred due to particles attaching to exterior surfaces. Analysis revealed that the combination of F-PSNPs and CNN resulted in larger particle dimensions (Fig. 3(B)). As previously mentioned the incorporation of EPS into CuSO₄·5H₂O led to modifications in the shape of PS Cu NPs and an increase in particle dimensions.⁵⁶ Earlier research indicated that Cd metal particles may have completely enveloped the PSNs' surface, diminishing the laser's effect on their form, whereas uncoated PSNs typically undergo deformation when subjected to laser treatment.⁵⁷

Fig. 3 Field emission scanning electron microscopic images and EDX spectra of control F-PSNPs (200 nm nanoparticles) (A) and F-PSNPs + CNN complex (B).

3.5 Atomic absorption spectrometry

The goal of the study was to use AAS to calculate the residual CNN concentration in the F-PSNPs + CNN Complex. While maintaining a constant F-PSNP concentration of 10 mg L⁻¹, CNN concentrations were varied to 5, 10, 15, and 20 mg L⁻¹. After 48 hours of incubation, F-PSNPs were separated using a 0.1 μm pore size particle filter and a syringe filter. The CNN remaining in the F-PSNPs + CNN mixture was subsequently measured, resulting in values of 3.21, 7.42, 10.63, and 13.85 mg L⁻¹. These results suggest that the residual concentration indicates CNN adsorption onto F-PSNPs. Similar results were reported in previous studies during the period for which Cr adsorbed over nano-TiO₂.⁴³

3.6 Raman spectroscopy

Raman spectroscopy provides a quick and non-destructive technique for examining predominantly carbon-based materials shown in Fig. 4. The diagram shows the Raman spectra of F-PSNPs, CNN, and the F-PSNPs + CNN mixture after exposure to gamma radiation. Distinctive polystyrene signals are evident at several wavenumbers: 1002 cm⁻¹ (aromatic ring breathing), 1032 cm⁻¹ (C–H bending), 1604 cm⁻¹ (the most intense band, indicating C=C aromatic ring stretching), and 624 cm⁻¹ (ring deformation). These spectral characteristics, which align with earlier studies, verify the existence of polystyrene in the analyzed samples.⁵⁸ A characteristic peak at 1048 cm⁻¹ is observed in the CNN spectra, signifying the occurrence of symmetric N–O stretching in nitrate.⁵⁹ Similar results are noted in the F-PSNPs + CNN complex, which also show a peak at 1048 cm⁻¹. This correspondence implies that the spectral data demonstrate that CNN has been attached to the F-PSNPs surface.

Fig. 4 Raman spectra: F-PSNP + CNN complex represented in pink line (A), F-PSNPs in blue line (B), and CNN in orange line (C).

3.7 Analysis of UV-visible absorption spectra

An analytical method that is often employed in many different domains is UV-vis absorption spectroscopy. CNN was exposed to F-PSNPs for 48 hours at a time. According to the analysis of the UV-vis absorbance spectra, CNN absorbance gradually rises with concentration and F-PSNP incubation duration. The F-PSNPs control sample shows a λ_max of 228, while the CNN exhibits a peak near 202 λ_max. After the interaction, the F-PSNPs + CNN complex demonstrates the same lambda max as CNN, with peaks indicating CNN presence on the F-PSNPs surface despites in Fig. 5(A). This observation suggests that CNN adsorption onto F-PSNPs occurs through physical or van der Waals forces and non-specific binding. The change in absorption spectra might be the consequence of f-PP chain aggregation brought on by Cr(VI), which is consistent with observations that were previously reported in the literature.⁶⁰ This observation suggests that f-PP and Cr(VI) have interacted. The ESIPT emission of f-PP is related to one wavelength of fluorescence, whereas the normal form is associated with another. This dual-emission was quenched after the introduction of Cr(VI). Considering that solvents have a major impact on inter-ESPT reactions, we investigated the fluorescence behaviour of f-PP in different solvent environments.⁶¹ Chromium(III) ions can interact with the functional groups on polystyrene nanoplastics, forming complexes that result in fluorescence quenching. For example, a study demonstrated that polystyrene-based Excited-State Intramolecular Proton Transfer (ESIPT) fluorescent polymeric probes exhibited significant fluorescence quenching when interacting with chromium(VI) ions. This quenching occurs due to the formation of non-fluorescent complexes. Additionally, chromium(III) nitrate can cause polystyrene nanoplastics to aggregate, altering their surface properties and further contributing to fluorescence quenching.⁶¹ Research has shown that polystyrene nanoplastics can aggregate in the presence of various electrolytes and proteins, which affects their fluorescence characteristics. The interaction and binding of chromium ions may lead to their adsorption onto the nanoplastics surface, thereby changing the fluorophore and reducing its emission efficiency.

Fig. 5 UV-vis spectra of standard samples of CNN and interacted samples of the F-PSNPs + CNN complex (A). UV-fluorescence spectra: the intensity of the F-PSNPs in the complex was reduced with increasing concentration of CNN (B). Resonance light scattering (RLS) spectra of the F-PSNPs + CNN complex (C).

3.8 Interaction studies of the fluorescence intensity of F-PSNPs with CNN

Studies have demonstrated that the interaction between CNN and F-PSNPs can reduce a compound's fluorescence intensity. Fig. 5(B) shows the fluorescence emission spectra of F-PSNPs with and without CNN activated. F-PSNPs' intrinsic fluorescence intensity decreased in a concentration-dependent manner when a quencher complex (F-PSNPs + CNN) was formed. The control F-PSNPs exhibit an emission peak at 317 nm. The quenching process induced a blue shift in the maximum emission; at varying concentrations, the F-PSNPs + CNN complex displayed shifts from 317 nm to 308 nm, while the F-PSNPs control exhibited a fluorescence intensity at 421. The F-PSNPs + CNN complex shows diminished fluorescence intensity relative to CNN concentration: 10 + 5 mg L⁻¹ gives 272, 10 + 10 mg L⁻¹ yields 240, 10 + 15 mg L⁻¹ produces 221, and 10 + 20 mg L⁻¹ 199 exhibits hypochromic effects. The hypsochromic shift noted in this study indicates a lower intensity level of F-PSNPs. Consequently, the fluorescence quenching experiments suggest that the quenching effect on F-PSNPs is due to the introduction of CNN. Similar results were reported in earlier studies the quenching method generated a blue shift in maximum emission from 328 nm to 307 nm (HSA) and 339–337 nm (BSA), which interacted with the CLA-PSNPs.⁶²

3.9 Resonance light scattering (RLS) investigation

The resonant light scattering (RLS) spectra of F-PSNPs + CNN complex were examined by researchers to evaluate their aggregation status, as shown in Fig. 5(C). According to the findings, the F-PSNPs + CNN complex's RLS intensity increases in tandem with the concentration. Notably, throughout this process, the peak position remained constant. This observation suggests that the interaction between F-PSNPs and CNN leads to the creation of an F-PSNPs + CNN complex. Previous experiments yielded similar results: when it interacts with other particles, it clumps.⁶²

3.10 Three-dimensional (3D) fluorescence spectral analysis

The presence of new peaks, variations in the fluorescence intensity of preexisting peaks, or shifts in excitation and emission wavelengths can all be signs of interactions. Fig. 6 shows two main peak categories: Rayleigh scattering peaks (λ_ex = λ_em) and Fluorescence peaks (peaks 1 and 2). An additional peak (peak a) signifies the interaction of the F-PSNPs + CNN complex. The F-PSNPs control sample exhibits a wide range for peaks 1 and 2, without any extra peaks. The F-PSNPs + CNN complex shows a decrease in the peak intensity depending on various concentrations. As CNN concentration rises, peaks 1 and 2 decrease, and an extra peak (a, b) appears at all concentrations except 5 mg L⁻¹. These changes in peak intensity can be explained by the interacted complexes.⁶² The spectrum of the F-PSNPs peak 1 shows excitation and emission wavelengths (λ_ex/λ_em) of 216/487 nm. In the presence of CNN, the F-PSNPs + CNN complex at various concentrations demonstrates the following spectra: at 10 + 5 mg L⁻¹, (λ_ex/λ_em: 224/488 nm); at 10 + 10 mg L⁻¹, (λ_ex/λ_em: 224/488 nm); at 10 + 15 mg L⁻¹, (λ_ex/λ_em: 224/484 nm); and at 10 + 20 mg L⁻¹, (λ_ex/λ_em: 224/484 nm). For complex peak 2, all concentrations show spectra at (λ_ex/λ_em: 262/482 nm), while the F-PSNPs peak 2 shows (λ_ex/λ_em: 262/486 nm). The intensity of peak 1 significantly decreased in the presence of CNN, accompanied by a reduction in the Stokes' shift (Δλ) value, indicating a maximum blue shift. The intensity of the F-PSNPs is noted as 10 000. For the complex at a concentration of 10 + 5 mg L⁻¹, the peak intensities for peaks 1 and 2 are (3130, 1643). At 10 + 10 mg L⁻¹, the intensities for peaks 1 and 2 are (1313, 1396). At 10 + 15 mg L⁻¹, they are (1665, 1856), and at 10 + 20 mg L⁻¹, the intensities are (1351, 1369).^62,63 Additional spectral characteristic parameters are provided in accompanying Table S2.†

Fig. 6 Three-dimensional fluorescence spectra of control sample of F-PSNPs (A) and F-PSNPs + CNN complex 10 + 5 mg L⁻¹ (B), 10 + 10 mg L⁻¹ (C), 10 + 15 mg L⁻¹ (D), and 10 + 20 mg L⁻¹ (E).

3.11 Hatching rate of Artemia salina percentage

Multiple aquatic animals have been shown to eat nanoplastics, which are prevalent in marine environments. The harmful effects of NPs on Artemia salina, a suitable indicator species for hazardous contamination, were therefore investigated in this work.⁶⁴ As shown in Fig. 7(A). Following both individual and complex treatment with F-PSNPs/CNN, the hatching rate of Artemia salina cysts was assessed. All particles showed a substantial change in hatching rate (p < 0.001) when compared to the control. The F-PSNPs concentration of 10 mg L⁻¹, no hatching was observed at 6 hours. However, the hatching rate increased to 35% at 12 hours, 75% at 24 hours, and 99.66% at 48 hours, showing a significant difference from the control at all time-points except 6 hours. Similarly, at CNN 5 mg L⁻¹, the hatching rate was 35% at 12 hours, 75% at 24 hours, and 99.66% at 48 hours, with results comparable to F-PSNPs and no significant difference observed at 6 hours across all treated concentrations of individual and complex particles. For higher CNN concentrations, the hatching rates varied. At CNN 10 mg L⁻¹, the hatching rate was 30% at 12 hours, 65% at 24 hours, and 99% at 48 hours. CNN 15 mg L⁻¹ showed a similar trend with 30% at 12 hours, 65% at 24 hours, and a slightly lower 95% at 48 hours. At CNN 20 mg L⁻¹, the hatching rate was 30% at 12 hours, 60% at 24 hours, and 95% at 48 hours.

Fig. 7 Hatching percentage of Artemia salina in the presence of F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component represented and the combination of F-PSNPs and CNN (A). Mortality percentage of Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component represented and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

In the F-PSNPs (10 mg L⁻¹) + CNN (5 mg L⁻¹) complex, the hatching rate was 20% at 12 hours, increasing to 60% at 24 hours and 80% at 48 hours. For F-PSNPs (10 mg L⁻¹) + CNN (10 mg L⁻¹), the hatching rate remained at 20% at 12 hours, then reached 55% at 24 hours and 60% at 48 hours. Similarly, in the F-PSNPs (10 mg L⁻¹) + CNN (15 mg L⁻¹) group, the hatching rate was 20% at 12 hours, 50% at 24 hours, and 60% at 48 hours. The lowest hatching rate was observed in the F-PSNPs (10 mg L⁻¹) + CNN (20 mg L⁻¹) group, with 15% at 12 hours, 40% at 24 hours, and 55% at 48 hours. Compared to individual treatments, the complex particles exhibited a lower hatching percentage due to their higher toxicity levels, which led to a delayed hatching process. The complex of F-PSNPs/CNN had a more marked effect on cell membranes, resulting in a higher drop in the hatching rate than the individual particles, whereas the individual particles had a less detrimental influence on the hatching rate at various doses (p < 0.001).

In earlier research, it was seen that as the quantity of each particle went up, the hatching rate steadily went down.⁶⁵ Similar results were seen in the hatching rate, which constantly went down as the concentration of each particle went up. This interaction enhances their ability to reach particular cells, organs, or the nucleus, potentially leading to bioaccumulation and increased risks.⁶⁶ These suspended particles might stop free-floating Artemia cysts from hatching in the suspension medium.⁶⁷

3.12 Mortality percentage of Artemia salina

Fig. 7(B) shows that all treatment concentrations of F-PSNPs exhibited no significant differences from the control groups until 24 hours post-treatment. At 48 hours, a 13% mortality rate was observed. A significant difference (P < 0.001) was noted at concentrations of 5 mg L⁻¹ and 10 mg L⁻¹ after 24 hours, whereas no significant mortality was observed at 6 and 12 hours. In Artemia salina exposed to CNN, a significant change (P < 0.001) was observed across all treatment periods compared to the control. At 15 mg L⁻¹, the mortality was 3.33% at 6 hours, increasing to 40% at 72 hours. At 20 mg L⁻¹, the mortality was 6.67% at 6 hours, rising to 56.67% at 72 hours. An association between F-PSNPs and CNN-treated Artemia salina was noted, showing significant differences (p < 0.001) at higher doses and increased mortality at all intervals. The study revealed that mortality was contingent on contact time and increased with it. When compared with individual F-PSNPs, CNN, and the complex, a significant difference (p < 0.001) was observed after interaction with Artemia salina. In the F-PSNPs (10 mg L⁻¹) + CNN (5 mg L⁻¹) treatment group, Artemia salina exhibited a mortality rate of 3.33% at 6 hours, increasing to 10% at 12 hours, 16.67% at 24 hours, 20% at 48 hours, and 30% at 72 hours. At a concentration of F-PSNPs (10 mg L⁻¹) + CNN (10 mg L⁻¹), the mortality was 6.67% at 6 hours, significantly increasing over time to reach 40% at 72 hours. In the F-PSNPs (10 mg L⁻¹) + CNN (15 mg L⁻¹) group, the mortality was 10% at 6 hours and increased to 60% at 72 hours. The highest mortality was observed in the F-PSNPs (10 mg L⁻¹) + CNN (20 mg L⁻¹) group, where the death rate was 13.33% at 6 hours, rising sharply to 83.33% at 72 hours. The individual and combination treatments also increased the mortality rate with the increase in contaminant concentration. The mortality rates for individual particles in the F-PSNPs were less than 50%, which indicates that there were no available LC₅₀ values. By contrast, the individual particles of the CNN exhibited mortality rates greater than 50%. The mortality for Artemia salina is 15.6 mg L⁻¹, while the LC₅₀ value for the F-PSNPs + CNN complex is 10 mg L⁻¹. Compared to the control group, a greater concentration of 100 mg L⁻¹ polystyrene microplastics had a significant effect on growth during the instar 1 and 2 stages.¹² In current studies, the concentration of F-PSNPs is 10 mg L⁻¹, so it shows that the toxic level has a less significant effect on Artemia salina. Because they may more readily breach the biological barrier and enter the organism's tissues, cells, or nuclei, smaller nanoparticles are more hazardous to A. salina than larger ones.⁶⁸ Comparable findings were observed in earlier research where Cr(NO₃)₃·9H₂O, Cr³⁺ exhibited no toxic effects on A. clause within the examined concentration range (0 to 17 mg L⁻¹). The mortality rate of test subjects was not significantly higher (up to 5%) than the control group.⁶⁹ Present investigations indicate that toxicity becomes apparent only when the CNN concentration is increased to 20 mg per L per individual. Furthermore, as the complex's concentration rises, additives' toxicity of additives becomes evident, aligning with the independent action model. Cr(VI) exhibits high toxicity, stability, and solubility in seawater²⁵ compared to Cr(III). Similar data that have been previously reported,⁴³ showed that the combination of nano-TiO₂ and Cr(VI) might inhibit the synergistic effect of the mixed compounds through Cr(VI) adsorption onto nano-TiO₂, resulting in an antagonistic impact in Artemia salina. An independent action model was used to validate the combined toxicity of F-PSNPs and CNN (Table S3†). In Artemia salina, the RI values exhibited additive effects when paired with an F-PSNP concentration of 10 mg L⁻¹ and CNN at different concentrations. Independent action modelling was conducted to understand the interaction between CNN and F-PSNPs when they were co-exposed. The relative interaction (RI) values calculated for the mixture of CNN and F-PSNPs are presented along with their corresponding nature of interaction. It was observed that the RI values increased with higher concentrations of CNN, a trend that was noted only with the addition of F-PSNPs. Regardless of the obtained Relative Interaction (RI) values, whether they are less than or greater than 1, the nature of the interaction between F-PSNPs and CNN has been deemed additive. This determination is based on the statistical insignificance (p > 0.05) of the results. Therefore, the toxicity patterns observed for the mixture align with the predictions made by independent action modelling.⁷⁰ According to earlier research, mixed groups acted in an aggressive manner, which was reflected in the death rate.⁴³

3.13 Bioaccumulation of F-PSNPs and CNN in Artemia salina

Brine shrimp are phagotrophic filter feeders that consume suspended particles of the right size continuously, regardless of the makeup of the particles.⁴⁰ The intestine is the main target for marine creatures, particularly zooplankton.⁷¹ Research has identified the accumulation of particles in Artemia salina's digestive system as a primary cause of mortality and toxicity. Artemia salina nauplii were subjected to different concentrations of CNN individual and F-PSNPs + CNN complex. Morphological changes were observed microscopically after 48 hours. The control group and those treated with F-PSNP, CNN, and F-PSNPs + CNN complex showed no signs of trauma or damage, while individual F-PSNPs and CNN particles caused harm to Artemia salina. However, particle accumulation was observed in the gut region of all treated groups except the control. In the F-PSNPs + CNN complex, the fluorescence intensity of F-PSNPs in the Artemia salina gut region decreased with increasing CNN concentration, as evident in Fig. 8. The aggregation of nanoparticles in the gut hinders excretion and enhances toxicity.⁴⁴ According to earlier research, nanoparticles may be consumed by Meta nauplii and Artemia salina in their first and second instars, and the buildup of nanoparticles in different physiological tissues allows for the transmission of particles from Artemia salina to fish further up the aquatic food chain.⁷² The primary component determining a variety of toxicological effects in organisms is the intake of aggregated particles.⁷³ Furthermore, a study conducted by⁷⁴ revealed a notable build-up of PS NPs in the digestive system. Research has revealed damage to epithelial cells exposed to polystyrene microplastics, suggesting that gut breakdown in brine shrimp may compromise digestive function by decreasing energy metabolism and nutrient absorption. The bioavailability might cause oxidative stress and cellular injury.¹² Reactive oxygen species have been recognized as markers of oxidative stress.⁷⁵ Inflammation and apoptosis can be triggered by oxidative stress, which is caused by excessive ROS formation.⁷⁶ Based on our research, there may be health problems associated with the increased levels of ROS in the guts of Artemia salina caused by F-PSNPs and CNN buildup.

Fig. 8 Microscopic images of Artemia salina show the accumulation of individual and complex particles. Nauplii exposed to 200 nm F-PSNPs exhibited no morphological damage. Individual CNN particles accumulated in the gut region, while the presence of complex F-PSNPs with CNN affected the accumulation based on CNN concentration, leading to decreased fluorescence intensity in F-PSNPs.

3.14 Total protein

Considerable alterations in the overall protein composition of tissues signify disturbances in regular cellular and metabolic processes.⁷⁷ Raised reactive oxygen species (ROS) can damage proteins, lipids, and nucleic acids, resulting in genetic instability, compromised membrane function, and changed cell signalling, among other detrimental consequences.⁷⁷ To determine the impact of nanoparticles (NPs) and their complexes with MH on aquatic species, it is important to measure and analyze metabolic alterations in the test animals. Significant variations in tissue total protein levels may indicate abnormalities in normal cellular and metabolic processes.⁶⁵ Test animals' changes in the total protein content of metabolic modifications can be used to evaluate and estimate the effects of F-PSNPs and their complexes with CNN on aquatic species of Artemia salina. The disruption of normal cellular and metabolic functioning is indicated by a significant drop in tissue protein levels. The total protein content of Artemia salina was considerably (P < 0.0001) reduced after exposure to F-PSNPs and CNN, as Fig. 9 illustrates. Because of interactions with the cell membrane, changed cellular responses, and metabolism, all treated particles had a lower protein content. A significant reduction in protein content was seen in the F-PSNP/CNN complex-treated groups when compared to the control group. Differences were found between individual groups (p < 0.001). Nonetheless, the long-term deposition of these particles significantly affects the organisms' general health and protein composition. Artemia salina, developmental delays and cell death can impact total protein levels. Research indicates that environmental stressors, such as exposure to heavy metals, may increase the total protein content in these organisms. This increase is thought to result from the shrimp's stress response, which could involve the production of stress proteins. Total protein levels in Artemia salina are linked to developmental delays and higher rates of cell death, especially under stressful environmental conditions. These changes may reflect the organism's adaptive responses, including the upregulation of stress-related proteins, to mitigate adverse effects.⁷⁸

Fig. 9 Total protein content of Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component (A), and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

3.15 Reactive oxygen species

Reactive oxygen species (ROS) generation plays a crucial role in the toxicity induced by nanomaterials.⁷⁹ These highly toxic ROS can damage cells and biomolecules, leading to oxidative stress.⁸⁰ Apoptosis caused by ROS generation, substantial reductions in cell viability, modifications to membrane integrity, and reduced cell function are common toxic processes shared by animal, microbial, algal, plant, and human cells.⁸¹ The physicochemical properties of the nanomaterials, including their chemical composition, surface charge, and particle size, affect the ROS response.⁸² The formation of ROS is thought to be a crucial sign of toxic consequences in Artemia salina.⁷² Catalytic reduction of Cr(VI) to Cr(III) is known to be linked to the production of ROS within cells.⁸³ The produced ROS can trigger antioxidant defense mechanisms, including superoxide dismutase (SOD) and catalase (CAT), to shield cells from oxidative damage.⁴³ Similar findings indicate that ROS generation rises with the hazardous amount. The levels of Reactive Oxygen Species (ROS) in Artemia salina following exposure to several concentrations of F-PSNPs, CNN, and a combination of both are depicted in Fig. 10. The graph's indication of the fluorescence intensity and the ROS activity were exactly proportionate. The results indicated that, when applied separately, F-PSNPs and CNN significantly affected the production of reactive oxygen species (ROS) in cells, with a p < 0.001 level of significance for ROS activity. Furthermore, a dose-dependent increase in ROS activity was seen in the upper range of F-PSNPs + CNN complexes after exposure to the F-PSNP + CNN complexes, and this increase was significant at the p < 0.001 level. In comparison to the control, ROS generation was considerably greater (p < 0.001) in the individual and complex exposure situations.

Fig. 10 ROS production in Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component (A), and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

3.16 Superoxide dismutase (SOD)

Superoxide dismutase (SOD) units produced in Artemia salina following treatment with the individual and complexes are depicted in Fig. 11. Within cells, the amount and activity of hazardous reactive oxygen species (ROS) are regulated by several enzyme defence mechanisms, including SOD.⁴³ Molecular oxygen and hydrogen peroxide are produced more easily when superoxide radicals are converted with the help of SOD. According to this study, species exposed to NPs and complexes generated higher levels of ROS than control species. To prevent oxidative damage to cells, antioxidant defense systems such as SOD and catalase (CAT) may be triggered. Thus, an increase in SOD activity was expected in the therapy groups as a preventive strategy. The SOD level was significantly higher in the group treated with F-PSNPs and CNN than in the control group (p < 0.001). Earlier research indicated a decrease in SOD activity, potentially resulting from enzyme degradation caused by excessive cellular ROS.⁸⁴ However, recent studies demonstrate an elevated SOD state, which is contingent on ROS production. A reduction in enzymatic activity may lead to self-catalysed oxidative damage.⁸⁵ Furthermore, when looking at the individual and complex, the F-PSNP + CNN complex revealed a significant difference (p < 0.001) from the control.

Fig. 11 SOD activity of Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component (A), and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

3.17 Catalase activity

Antioxidant defence against reactive oxygen species (ROS) is mainly carried out by catalase (CAT), This process mitigates cellular toxicity by decreasing H₂O₂, an essential precursor of the most detrimental reactive oxygen species.⁸⁶ Additionally, the imbalance of superoxide causes H₂O₂ and oxygen to be produced, activating CAT.⁴³ Following treatment with F-PSNPS, CNN, and a complex, the quantity of catalase in Artemia salina is displayed in Fig. 12. The individually treated groups exhibited a wide range of catalase production, leading to an increase significant in catalase synthesis (p < 0.001). Significant catalase production was shown by all particles in comparison to the control (P < 0.0001). The combination of F-PSNPs and CNN interaction showed a comparable rise in catalase activity, enhancing the CAT enzyme in a synergistic manner (p < 0.001) compared with the control. The complex exhibited significantly higher catalase enzyme production than the individually treated groups (p < 0.001).

Fig. 12 Catalase activity of Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component (A), and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

3.18 Lipid peroxidation (LPO)

Lipid peroxidation (LPO) compromises the structural integrity of cell membranes and results in the loss or modification of functional proteins and DNA, culminating in the formation of compounds such as malondialdehyde (MDA).⁶⁵ Assessing MDA levels can provide valuable information regarding LPO activity in response to contaminants.⁸⁷ In the presence of Artemia salina, all three samples, namely individual F-PSNPs and CNN particles and the F-PSNPs + CNN particle complex, showed a substantial increase in LPO. This rise was considerably different compared to the control groups (p < 0.001). Furthermore, the F-PSNPs + CNN particle complex exhibited significantly higher (p < 0.001) LPO activity than either individual F-PSNPs or CNN particles shown in Fig. 13.

Fig. 13 Lipid peroxidation (LPO) of Artemia salina upon exposure to F-PSNPs at a constant concentration of 10 mg L⁻¹, with CNN exposed to various concentrations of the individual component (A), and the combination with various concentrations of F-PSNPs and CNN (B). The results are presented as mean ± S.E.M. The significance level is shown by the asterisks, which indicate a significant change when compared to the control (p < 0.05*, p < 0.01**, and p < 0.001***).

4 Conclusion

A comprehensive investigation into the uptake, detrimental effects, and biochemical consequences of fluorescent polystyrene nanoplastics (F-PSNPs) and their associations with chromium(III) nitrate nonahydrate (CNN) on the sea crustacean Artemia salina has uncovered a complex array of factors contributing to environmental degradation. The study confirmed a monolayer coverage of CNN on F-PSNPs, substantiated through multiple analytical methods including DLS, zeta potential measurement, FESEM, and reduced fluorescence intensity due to CNN interaction. Toxicological findings highlighted the impacts of F-PSNPs and CNN at different concentrations. A significant negative outcome was a substantial reduction in Artemia salina cyst hatching rates when subjected to F-PSNPs and their CNN complexes, indicating compromised reproductive ability essential for sustaining populations and ecological equilibrium. Moreover, concentration-dependent increases in mortality were observed in acute toxicity tests on Artemia salina exposed to these particles and complexes. Morphological studies revealed particle accumulation in Artemia salina exposed to F-PSNPs and their CNN complexes, suggesting that the hastening of normal growth and developmental processes could have enduring negative consequences on population dynamics and ecosystem functioning. Biochemical analyses further elucidated the underlying toxicity mechanisms, showing significant alterations in total protein content, Reactive oxygen species (ROS) generation, enhanced antioxidant enzyme activities, SOD, CAT, and lipid peroxidation (LPO) levels. The presence of CNN, a heavy metal co-contaminant, on the F-PSNP surface further amplified their toxicity, potentially leading to bioaccumulation in organisms. In conclusion, the research underscores the individual and combined harmful effects of F-PSNPs and their CNN complexes on aquatic organisms. These results emphasized the urgent need for effective measures to reduce nanoplastics pollution and mitigate its adverse impacts on ecosystem health and biodiversity. Many environmental agencies have strict limits on chromium discharge. Reducing chromium pollutants encourages better waste treatment methods, leading to more sustainable industrial processes. Industries may adopt eco-friendly substitutes or improved treatment processes, leading to innovation in pollution control.

Abbreviation

F-PSNPs	Fluorescent polystyrene nanoplastics
CNN	Chromium(III) nitrate nonahydrate
MNPs	Micro nanoplastics
ROS	Reactive oxygen species
SOD	Superoxide dismutase
LPO	Lipid peroxidation
FE-SEM	Field emission-scanning electron microscopy
DLS	Dynamic light scattering
FTIR	Fourier transform infrared
RLS	Resonance light scattering
AAS	Atomic absorption spectroscopy

Data availability

Data will be made available upon request.

Author contributions

Mahalakshmi Kamalakannan: investigation, methodology, data curation, writing – original draft, writing – review & editing; John Thomas: writing – review & editing; Natarajan Chandrasekaran: conceptualization, supervision, resources, project administration, funding acquisition, writing – review & editing.

Conflicts of interest

The authors declare that there is no conflict of interest.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5em00251f

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