Acute exposure to high concentrations of polystyrene nanoparticles induces genotoxicity in Daphnia pulex

TL;DR

Exposure to high concentrations of polystyrene nanoparticles harms water fleas by causing DNA damage, reduced movement, and increased death.

Contribution

This study demonstrates genotoxic effects of polystyrene nanoparticles in Daphnia pulex using the comet assay.

Findings

Immobilisation of daphnids increased with higher PSNP concentrations and longer exposure times.

DNA damage was significantly observed after 24 hours at 200 mg/L and after 48 hours at 100 and 200 mg/L.

Hopping frequency decreased at PSNP concentrations above 100 mg/L after 24 hours and above 50 mg/L after 48 hours.

Abstract

Micro- and nanoplastics (MNPs) are known to detrimentally impact a wide range of aquatic species, inducing mortality, decreased growth, a reduction in offspring production and increase in reactive oxygen species in their tissues. However, the genotoxic impact of MNPs in freshwater organisms remains understudied. In the present study we investigate the genotoxic impact of acute exposure to polystyrene nanoparticles (PSNPs) in Daphnia pulex using the comet assay, alongside immobilisation rate and hopping frequency. Daphnids were exposed to 100 nm PSNPs for 24 and 48 h at concentrations between 10 and 200 mg/L. Immobilisation increased with PSNPs concentration and exposure time, while hopping frequency among surviving daphnids decreased at concentrations above 100 mg/L after 24 h, and above 50 mg/L after 48 h. Comet assay results showed increasing DNA damage with concentration and exposure time, with significant DNA damage after 24 h of exposure to 200 mg/L PSNPs, and after 48 h of exposure to 100 and 200 mg/L. These results suggest that exposure to MNPs increases daphnid mortality, impacts behaviour, and induces genotoxicity. The incorporation of the comet assay into freshwater monitoring methods alongside existing techniques could further understanding of the full impact of MNP pollution upon ecosystem health.

The online version contains supplementary material available at 10.1007/s11356-026-37490-9.

Figures

Immobilisation data from 24- and 48-h exposures to varying concentrations of 100 nm polystyrene nanoparticles. Asterisks denote significantly (p < 0.05) elevated immobilisation relative to the negative control of each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction. No immobilisation was reported for the 24 h negative control or 48 h positive control conditions

Hopping frequency data from 24 and 48 h exposures to varying concentrations of 100 nm polystyrene nanoparticles. Asterisks denote significant (p < 0.05) differences relative to the negative control in each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction

Mean tail DNA percentage results from 24 and 48 h exposures. Asterisks denote significant (p < 0.05) differences relative to the negative control in each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction

Full text

Introduction

The presence of micro- and nanoplastics (MNPs) in aquatic systems is cause for increasing concern (Anderson et al. 2016; Ha And Yeo 2018), with a growing number of studies detailing harm caused to aquatic organisms at both an individual and community level (de Sá et al. 2018), particularly as a result of their ingestion (Au et al. 2017; Li et al. 2018). The impact of MNPs have been particularly well-studied in the context of marine organisms, with studies indicating increased mortality, reduced fertility, disturbed feeding behaviour, and inflammation in a wide variety of marine organisms expose to MNPs (Jeong et al. 2024; Zolotova et al. 2022). MNPs can be found in the guts and other tissues of filter feeder species such as mussels (Li et al. 2020) as well as consumer species at higher trophic levels (Nelms et al. 2018). It has consequently been suggested that trophic transfer of MNPs consumed from aquatic and other sources could pose a threat to human health (Carbery et al. 2018). Despite the abundance of literature regarding MNPs in a marine context, and indeed despite the fact that freshwater and diadromous species constitute approximately 41% of aquatic food consumption (FAO 2025), the impact of MNP pollution in freshwater ecosystems was, until recently, relatively understudied (Ding et al. 2018).

Abundance of MNPs in freshwater bodies is correlated with human population density (Eriksen et al. 2013), with wastewater treatment plants constituting an important vector (Li et al. 2018), however they can also be spread to remote regions far beyond population centres by aerial transport (Lasee et al. 2017). A review by Ashrafy et al. (2023) suggested that the most MNPs found in aquatic environments consist of only a handful of polymer types including polyethylene, polyethylene terephthalate, polyamide, polypropylene, polystyrene, polyvinyl alcohol, and polyvinyl chloride. There is much variation in MNP concentration, type, and other properties, both between sites and temporally at individual sites (Stanton et al. 2020) however polystyrene is a particularly commonly-found plastic type in freshwater, being the third most common after polyethylene and polypropylene, with environmental particle concentrations ranging up to 1 × 10^5^ particles/L (Koelmans et al. 2019; Nugnes et al. 2022a).

Polystyrene MNPs can be taken up and accumulate in terrestrial and aquatic organisms (Ziani et al. 2023), upon which they can cause lacerations and inflammation within organisms’ digestive tracts (Carbery et al. 2018) and subsequently translocate into other tissues (Browne et al. 2008). As MNPs are of little to no nutritional value, they can be retained within an organism and digested for prolonged periods of time at high energetic cost (Wright et al. 2013). They can additionally physically block the digestive tracts of smaller animals such as juvenile fish and zooplankton including cladocerans, resulting in reduced food uptake or even starvation (Canniff And Hoang 2018; Jovanović, 2017). MNPs including polystyrene MNPs can be genotoxic, which is to say they can damage genetic information within organisms (Tang 2025a). This can include both primary genotoxicity – the direct inducement of DNA strand breaks via physical interaction – as well as secondary genotoxicity which can result from a diverse array of mechanisms, often involving the generation of reactive oxygen species (ROS) (Alijagic et al. 2022), resulting in interference with cellular processes such as gene expression and regulation (Marcellus et al. 2024), and cell replication (Roda et al. 2020).

Some mechanisms of MNP-induced genotoxicity can be species-dependent. For example, exposure of human cell lines to MNPs has been shown to activate p53 – a gene involved in tumour suppression that can be used as an indicator of genotoxicity (Alijagic et al. 2024) – whereas in mussels MNP exposure has been shown to suppress p53 (Brandts et al. 2018) despite genotoxicity being induced. Indeed, this suppression could contribute to MNP-induced genotoxicity in mussels, the occurrence of which is well-documented (Tang 2025a). It has been suggested that genotoxic pollutants can pose a threat to biodiversity and ecosystem stability by affecting health and fitness in individual organisms (Ellwanger et al. 2025), and this is particularly true of keystone species, which play an important role within the ecosystems they inhabit. Disrupted populations of keystone species can therefore have cascading effects across ecosystems.

MNPs, including polystyrene MNPs, have additionally been shown to induce genotoxicity in freshwater organisms including many species of fish (Araújo et al. 2022; Hamed et al. 2020; Menezes et al. 2024) and select invertebrates. For example, two studies (Nugnes et al. 2022a, 2022b) detected significant genotoxicity in the freshwater invertebrate species Ceriodaphnia dubia following 24-h exposures to 1 µm polystyrene microparticles. An understanding of the genotoxic potential of MNPs offers insights into the initiating event of observed effects on freshwater organisms (Gong et al. 2023; Lencioni et al. 2020) at the individual and community level following environmental exposure to MNPs.

Within MNPs, nanoplastics are regarded as more strongly toxic than microplastics in general, able to more readily cross barriers to penetrate tissues, as well as being more bioavailable (Tang 2025b). Indeed, their ability to directly enter cells and interact with genetic material may make them especially potent in this regard relative to microplastics (Tang 2025a). There is therefore a need, from an ecotoxicological perspective, to investigate the genotoxic impact of MNPs and particularly nanoplastics, especially for keystone species. Acute toxicity studies, though not always as able to determine true risk as chronic or multigenerational studies in the context of ecotoxicology, can nonetheless provide important insights into the hazards posed by toxicants and the mechanisms by which they do harm on chronic timescales.

Daphnia are a genus of cladoceran considered to be keystone species in many freshwater habitats such as ponds and lakes, often acting as the main primary consumer of algal species and constituting an important food source for predator species (Ebert 2022). They are highly sensitive to environmental pollutants, commonly used as a model species (Miner et al. 2012; Reilly et al. 2023) and used routinely for freshwater quality testing on both acute and chronic timescales (OECD 2012, 2004). Acute daphnid immobilisation in particular is a very common endpoint for biomonitoring purposes.To date, research has shown that daphnid immobilisation – a commonly-used proxy for mortality – increases with exposure to higher MNP concentrations, and can be dependent on the size of MNPs, with smaller MNP particle sizes tending to correspond to heightened daphnid immobilisation and other toxic effects relative to larger particles (An et al. 2021; Pikuda et al. 2023; Rehse et al. 2016).

Daphnia exposure to MNPs can also result in effects on sublethal endpoints including daphnid reproduction, growth, behaviour, and community function (Pikuda et al. 2022; Schwarzer et al. 2022; Yin et al. 2023). Schwarzer et al. (2022) Among these sublethal endpoints, daphnid behaviours such as swimming behaviour can be acutely sensitive to toxicant exposure. Daphnids swim by rhythmically beating their second antennae to move with the characteristic hopping motion that gives Daphnia their common name – water fleas – and assessing the frequency of these antennae beats (hopping frequency) can be a valuable method by which to assess the toxicological impact on swimming behaviour (Bownik 2017). Swimming behaviour can have a significant impact on trophic relationships, affecting the rate of predation as faster hopping behaviour can be associated with escape and avoidance responses, however conversely, reducing hopping frequency can serve to make daphnids less easily detectable by predator species (Bhattacharjee et al. 2013; Bownik 2017).

In addition to more commonly used sublethal endpoints, exposure to MNPs is known to induce inflammation and ROS buildup in daphnids (Esterhuizen et al. 2023), and interfere with the expression of genes that can play a role in repairing DNA damage (Imhof et al. 2017). Both of these can result in DNA strand breaks in a diverse range of species (Sottile and Nadin, 2018; Tang 2025a, b), and indeed it has been shown that MNP exposure for the related cladoceran species Ceriodaphnia dubia results in both ROS production and genotoxicity (Nugnes et al. 2022a). However, the extent to which MNPs induce genotoxicity in Daphnia remains unexplored.

Given the need to investigate the genotoxic impact of MNPs on freshwater keystone species and the lack of direct evidence regarding MNP-induced strand breaks in Daphnia, this study therefore aims to apply the comet assay to Daphnia pulex to directly detect DNA strand breaks resultant of acute exposure to high concentrations of MNPs. In doing so, the usefulness of the comet assay as an endpoint as an indicator of toxicity in Daphnia pulex relative to other endpoints – immobilisation and hopping frequency – will be assessed. To our knowledge, this is the first time that the comet assay has been applied to Daphnia pulex.

Methods

Experimental setup

Daphnia pulex were sourced from Blades Biological (Daphnia pulex Water Fleas c300, SKU: LZJ 345). Species identity was confirmed visually using a stereoscopic microscope. Stock daphnids were maintained in 1L vessels of aerated commercially available mineral water and fed with one Daphnia pellet (Blades Biological, SKU: DTS 125) and 1 mL stock (5 × 10^5^ cells/mL) Scenedesmus quadricauda (Blades Biological, SKU: LZA 235) culture per week. The polystyrene nanoparticles – henceforth referred to as polystyrene nanoparticles (PSNPs) – used as the test article were purchased from Sigma-Aldrich (Buchs, Switzerland) as an aqueous suspension (10% weight/volume; 1.82 × 10^17^ particles/mL), and were characterised by the manufacturer as 100 nm diameter micro particles based on polystyrene in aqueous suspension with a particle specific gravity of 1.05 g/cm^3^, and a solid content of 10% WT.

Individual daphnids were removed from the stock daphnid vessel and pooled in 50 mL vessels, with four replicate pools for each treatment condition each containing a minimum of 9 individuals, with treatment conditions containing a minimum of 36 individuals in total. All vessels were made up to 10 mL with mineral water prior to the addition of daphnids, and each pool was fed with 200 µL stock Scenedesmus quadricauda immediately prior to exposure. Vessels were not aerated for the duration of the exposures.

As a negative control, one group of four pools was not exposed to PSNPs. As a positive control, one group was treated with 10 µM hydrogen peroxide. This concentration of hydrogen peroxide was selected with the aim of inducing detectable genotoxicity among the treated daphnids, as previously demonstrated by Pellegri et al. (2014) and Pellegri et al. (2020) in their studies applying the comet assay to Daphnia magna. The remaining groups were exposed to PSNPs at the following concentrations: 10, 20, 50, 100 and 200 mg/L (1.82 × 10^10^, 3.64 × 10^10^, 9.09 × 10^10^, 1.82 × 10^11^ and 3.64 × 10^11^ particles/L respectively). The procedure was then repeated across two timepoints, with exposures lasting 24 and 48 h. These PSNP concentrations were chosen to increase almost exponentially and thus observe a wide variety of conditions, similar to a study by Rehse et al., (2016), who investigated immobilisation of Daphnia magna exposed to 1 µm microplastics for 24, 48, 72 and 96 h. However, pre-tests showed high immobilisation at even low PSNP concentration after 72 h of exposure, therefore only 24- and 48-h timepoints were selected for the final experimental design. A total of 293 individuals were used for the 24-h exposure, and 274 for the 48-h exposure.

Immobilisation and hopping frequency

After 24- and 48- hours, immobilised daphnids in each pool were counted and discarded. Individuals were considered to be immobilised if they did not respond to gentle disturbance with a Pasteur pipette. Hopping frequency of surviving daphnids was assessed separately to the main experiment after additional 24- and 48-h exposures. Due to the difficulties associated with assessing hopping frequency with the naked eye, each test vessel was recorded for a duration of thirty seconds. This approach allowed for more precise measurement of movement. The video footage was then replayed in slow motion, enabling clear identification and counting of individual hops. The total number of hops observed during the 30-s recording was doubled to calculate the hopping frequency as hops per minute for each surviving Daphnia pulex.

Genotoxicity

The comet assay was used to assess genotoxicity. It is a quick and sensitive method of detecting DNA strand breaks in a wide variety of organisms (Dhawan et al. 2009), and its usefulness for detecting genotoxicity in both lab and field settings with Daphnia magna has been previously demonstrated (Gomes et al. 2018; Pellegri et al. 2020, 2014), making it a good choice of assay to detect genotoxicity in this study.

Daphnids were removed from their vessels using a Pasteur pipette and placed on filter paper to remove excess water. They were then quickly and gently picked up using metal forceps and placed in a 1.5 mL Eppendorf tube containing 200 µL of phosphate-buffered saline (PBS) and 20 mM EDTA solution. 100 µL of glass microspheres with a mean diameter of 0.4mm were added before each tube was subjected to a rapid shock lasting 1 s in a dental amalgamator device (4300 oscillations per minute, 3M CapMix Capsule Mixing Device) to dissociate haemolymph cells (Pellegri et al. 2014).

The resultant suspension containing the cells was then filtered through a 50 µm mesh and centrifuged at 45 × g for 5 min. Most of the supernatant was removed and the pellet resuspended. 25µL of this reduced volume was suspended in 250 µL 1% low melting point agarose (LMA) and mixed via inversion. 25µL of combined cell suspension and LMA was then spread across microscope slides previously dipped in 1% normal melting point agarose (NMA). Slides were allowed to solidify at 4 °C for 10 min before a second layer of 25 µL 1% LMA was added in order to eliminate the “edge effect” of distorted comets appearing close to the edge of the gel (Koppen et al. 2017). Slides were then placed in a lysis solution (2.5 M NaCl, 100 mM Na_2_ EDTA, 1% Triton X-100, 10% DMSO, pH 10) at 4 °C for 1 h, rinsed briefly in distilled water and placed in an alkaline unwinding/electrophoresis solution (200 mM NaOH, 1 mM EDTA, pH > 13) at 4 °C for a further 10 min. Electrophoresis was then conducted in the same solution (1V/cm, 230mA, 20 min). After electrophoresis, slides were neutralised in PBS, fixed in ethanol at 4 °C and air dried.

Slides were stained with SYBR Gold (3µL SYBR Gold Nucleic Acid Stain 10,000X concentrate in DMSO, 90 mL PBS) at room temperature in darkness. Stained slides were then examined under a Thermo Scientific Invitrogen EVOS FL Auto 2 Imaging System with a GFP filter cube (Excitation: 470 nm; Emission: 535 nm). The tail DNA percentage was scored for every imaged cell, with a minimum of 150 nuclei per treatment condition scored using CometScore 2.0 automatic comet scoring software. The scoring process involved manually identifying and selecting comets before identifying the centre of the comet head.

Statistical analysis

Datasets for all endpoints were tested for homoscedasticity and normality using the Levene’s and Shapiro–Wilk tests respectively. All tests failed the assumptions of the ANOVA, so the Kruskall-Wallis H test was used, followed by a Dunn’s post hoc pairwise comparison test. For immobilisation, the percentage immobilisation of each pool was used for the analysis. For hopping frequency, the hops per minute were calculated for ten daphnids per treatment condition. For genotoxicity, the tail DNA percentage was calculated for each cell imaged from at least 150 nuclei per treatment condition. All statistical analysis was performed using R-Statistics (R Core Team, 2023) with the packages ICSNP (Nordhausen et al. 2023), mvtnorm (Genz and Bretz. 2009), and ICS (Nordhausen et al. 2008). The level of significance for hypothesis testing was considered to be p < 0.05. Effect sizes were estimated using eta-squared (η^2^), representing the proportion of total variance explained by MNP exposure. LC_50_ values for immobilisation after 48 h were calculated using Finney’s probit analysis spreadsheet calculator Version 2021 (Mekapogu 2021).

Results and discussion

Observations

PSNPs were observed to remain in the water column immediately after exposure and throughout the experiment. Neither clumping, aggregation nor uneven distribution were noted in the water column, however clumping was observed around daphnids, particularly at higher concentrations, which may have been resultant of the filtered PSNPs among daphnids’ appendages resulting in higher concentrations than those found in the water column.

Ingestion of PSNPs was confirmed by observation of gut discoloration in daphnids treated with higher concentrations of PSNPs under a stereoscopic microscope. The guts of daphnids from the positive and negative control groups were translucent and pale green, whereas guts of treated daphnids were paler or vividly white, especially at higher concentrations, indicating ingestion of the white PSNPs (Fig. S1, Supplementary material). Ingestion was likely inadvertent, resultant of non-selective feeding behaviour. Daphnids are unable to distinguish between some MNPs and algal cells (Chen et al. 2020; Nugnes et al. 2022b), however existing literature to date has largely focused on investigating the effects of MNPs of comparable size to algal cells rather than smaller particles which may interact differently. Consequently, the effects of plastic particles below 1 µm on daphnids are relatively understudied compared to larger microplastics, however they are generally thought to exhibit greater toxicity (Pikuda et al. 2023).

Immobilisation

Significantly elevated rates of immobilisation were found at both timepoints and across almost all PSNP exposure groups (Fig. 1) (Table S1, Supplementary material). The percentage immobilisation for the positive control groups (treated with 10 µM hydrogen peroxide) was very low compared to the PSNP-exposed groups, with no immobilisation at all in the 48-h positive control group. The effect size, as estimated by eta-squared, was large across both 24- and 48-h timepoints (η2 = 0.74 and η2 = 0.6 respectively), suggesting a strong correlation between PSNP exposure and immobilisation.Fig. 1. Immobilisation data from 24- and 48-h exposures to varying concentrations of 100 nm polystyrene nanoparticles. Asterisks denote significantly (p < 0.05) elevated immobilisation relative to the negative control of each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction. No immobilisation was reported for the 24 h negative control or 48 h positive control conditions

The rate of immobilisation was both dose- and time-dependent, increasing with PSNP concentration across both timepoints. There was a markedly higher rate of immobilisation at 48 h versus 24 h (Fig. 1), with pools exposed to the highest dose – 200 mg/L – seeing a mean of 37% immobilisation after 24 h and 70% after 48 h (Table S2, Supplementary material). Immobilisation for the negative controls were low, with no immobilisation recorded after 24 h, and only a single individual in one pool found immobilised after 48 h.

Immobilisation rates in this study were higher than were found in a study by (Rehse et al., (2016), who investigated immobilisation of Daphnia magna exposed to 1 µm and 100 µm polyethylene microparticles at very similar concentrations to those investigated in the present study. This is consistent with the suggestion that smaller particle sizes can induce higher immobilisation in daphnids (An et al. 2021; Frankel et al. 2020; Pikuda et al. 2023), however the use of Daphnia pulex rather than D. magna means the results cannot be directly compared**.** We observed lower immobilisation than that observed in more directly comparable studies by Lin et al. (2019a, b) and Lin et al. (2019a, b), who examined Daphnia magna exposed to 100 nm polystyrene particles, reporting LC_50_ (with 95% confidence intervals) of 5 (3–6) mg/L and 5.24 (4.47–6.13) mg/L respectively after 48 h, whereas our results indicated a much lower toxicity in Daphnia pulex after the same period, with an LC_50_ (with 95% confidence intervals) of 53.11 (22.98–122.72) mg/L. This suggests a sharply lower toxicity in our results than that reported by the aforementioned papers, however our results align with the wider picture of daphnid toxicity shown by Pikuda et al. (2023), whose review of micro- and nanoplastic toxicity indicates a tendency for smaller particles to be more readily bioavailable to daphnids and consequently induce greater toxicity at lower concentrations. It is possible that Daphnia pulex exhibit greater sensitivity to MNPs than Daphnia magna, however comparative studies would be required to confirm this.

Daphnid immobilisation is a widely-used endpoint in biomonitoring. The mechanisms by which microplastic-induced immobilisation in daphnids occur are currently understudied, however it is known that MNPs can induce inflammation in daphnids (Wang and Wang 2023) and numerous other species (Agrawal et al. 2024; Carbery et al. 2018; Jin et al. 2018). They additionally reduce starvation resistance in some organisms (Kholy And Naggar 2022; Shen et al. 2021; Wright et al. 2013), induce protein breakdown in mussels similar to the effects of prolonged starvation (Shang et al. 2021), and induce degradation of amino acid metabolites in Daphnia magna, which affects energy metabolism (Wang et al. 2022). It is recognised that low food availability increases the acute toxicity of MNPs to daphnids (Ghosh et al. 2025), and it was for this reason that daphnids were well-fed in this study. Thus, if the PSNP-induced immobilisation observed in this study was due to starvation, then it suggests ingested PSNPs may have prevented nutrient uptake from ingested algae, or else inducing daphnids to feed at a reduced rate rather than due to a low availability of food. Future studies could investigate whether MNP exposure induces starvation in Daphnia by analysing biomarkers associated with starvation despite the presence of adequate food.

Hopping frequency

PSNP exposure had a significant impact on hopping frequency after both 24 h (Kruskal–Wallis, χ^2^(5) = 37.28, p < 0.001) and 48 h (Kruskal–Wallis, χ^2^(5) = 32.06, p < 0.001). Hopping frequency was significantly reduced (p < 0.05) in daphnids exposed to 100 mg/L and 200 mg/L at the 24-h timepoint, and all concentrations apart from 20 mg/L at the 48-h timepoint (Table. S3, Supplementary material). The effect of PSNP exposure was likewise significantly different between the two timepoints, with a greater effect apparent after 48 h than after 24 h (2-way ANOVA, F_1, 18_ = 24.332, p < 0.001). The effect size as estimated by eta-squared was large across both 24- and 48-h timepoints (η2 = 0.6 and η2 = 0.5 respectively), suggesting a strong correlation between PSNP exposure and changed hopping frequency.

The effect of PSNP exposure on hopping frequency was again both dose- and time-dependent, with an overall trend towards reduced hopping frequency at higher PSNP concentrations. PSNP-exposed daphnids exhibited reduced hopping frequency, particularly at higher concentrations and after 48 h of exposure (Fig. 2).Mean hopping rate for the negative control daphnids were similar across both timepoints at 118.76 hops per minute (hpm) with a standard deviation ± 12.06 after 24 h and 123.22 ± 20.03) hpm after 48 h, while daphnids exposed to the highest concentration of PSNPs exhibited a mean hopping rate of 86.53 ± 12.47 hpm after 24 h and 58.03 ± 12.38 hpm after 48 h (Table S4, Supplementary material). This suggests a cumulative effect over time upon hopping frequency, similar to that exerted upon immobilisation rates.Fig. 2. Hopping frequency data from 24 and 48 h exposures to varying concentrations of 100 nm polystyrene nanoparticles. Asterisks denote significant (p < 0.05) differences relative to the negative control in each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction

Daphnid hopping behaviour can be influenced in complex ways by toxicants. An elicited escape response can induce a higher rate of hopping (Lovern et al. 2007), (Brewer et al. 1999) while exposure to other toxicants including neurotoxic compounds can reduce hopping frequency can reduce hopping frequency (Bownik 2017). As with immobilisation, the effect of MNP exposure on daphnids’ hopping frequency can be more pronounced with smaller MNP particle sizes (Pikuda et al. 2022) It is not clear from our results whether the reduced hopping frequency at higher concentrations was induced by the same mechanisms that resulted in increased immobilisation. However, there was no significant change in hopping frequency in daphnids exposed to up to 50 mg/L PSNPs after 24 h, despite this inducing significant immobilisation relative to the negative control condition. This may indicate that the reduced hopping frequency in response to PSNP exposure was induced by different mechanisms to those which resulted in immobilisation, though more research would be required to confirm this.

Our results were in contrast with a study by Pikuda et al. (2022) in which Daphnia magna exposed to 50 mg/L 20 nm carboxylated polystyrene nanoparticles for 21 days exhibited slightly increased hopping frequency, rather than the decrease observed at higher concentrations in our results. Daphnids are known to increase hopping frequency to escape from perceived danger (Lovern et al. 2007) and decreased hopping frequency can be a means by which they avoid detection by predators, however the observed response in our study may not be deliberate in that sense, but rather a result of reduced energy reserves due to PSNP buildup in the gut preventing normal feeding and causing starvation (Magester et al. 2021). If reduced hopping frequency results in reduced predator ability to detect daphnids as prey (Bhattacharjee et al. 2013), then daphnid exposure to MNPs could affect predator–prey dynamics in complex ways, with cascading effects on species at higher trophic levels.

Genotoxicity

Genotoxicity in the comet assay can be quantified by analysing the tail DNA percentage of nucleoids, with a greater percentage of DNA in the tail relative to the head indicating a greater number of strand breaks which give the broken fragments greater freedom to migrate through the agarose gel towards the anode. Significant (p < 0.05) genotoxicity – as defined by differences in tail DNA percentage – was detected between the negative control group and the group exposed to 200 mg/L PSNPs after 24 h, as well as between the negative control group and the groups exposed to 100 mg/L and 200 mg/L PSNPs after 48 h (Figure S2, Supplementary material; Table S5, Supplementary material). Significant genotoxicity was not detected at lower concentrations (Fig. 3), however it was detected between the negative and positive control groups for both timepoints. Temporal variation was detected, with post hoc Dunn’s test pairwise comparisons showing significant (p < 0.05) differences in % tail DNA between the 24- and 48-h timepoints for all corresponding groups apart from the positive controls.Fig. 3. Mean tail DNA percentage results from 24 and 48 h exposures. Asterisks denote significant (p < 0.05) differences relative to the negative control in each timepoint, as determined by a Kruskal–Wallis test and post hoc Dunn’s test with Bonferroni correction

Across both timepoints the effect was small and overall not as sensitive to the presence of the PSNPs as daphnid immobilisation or hopping frequency, although there was a larger effect at the 48-h timepoint than at 24 h. The negative control conditions for the 24- and 48-h timepoints exhibited significantly different levels of genotoxicity, with a greater percentage of DNA found in the tails of negative control daphnids after 48 h (Table S6, Supplementary material). This could be due to the fact that adult daphnids of variable age were used, as it has been demonstrated that DNA damage accumulates with age in Daphnia magna (Constantinou et al. 2019). As neonates exhibit the lowest level of baseline DNA damage, future research should ensure the use of neonates in daphnid genotoxicity testing. Despite this limitation, the significantly increased level of genotoxicity after 48 h of exposure to 100 mg/L PSNPs but not 24 h nevertheless implies cumulative genotoxicity resultant of the longer exposure. Thus, future studies which investigate genotoxicity following chronic exposure to MNPs may demonstrate greater sensitivity at lower concentrations.

Meanwhile, the comet assay was more sensitive than the immobilisation test to the presence of the positive control substance – hydrogen peroxide – across both timepoints, with significant genotoxicity detected in response to the positive control but no significant immobilisation detected at either timepoint. This was expected, as hydrogen peroxide was chosen as a positive control substance intended to elicit a strong genotoxic response at a concentration that would not induce significant immobilisation (Pellegri et al. 2020, 2014). Hydrogen peroxide is a ROS that can build up in cells if antioxidant defence mechanisms are disrupted (Das 2023) and can induce DNA strand breaks through direct interaction with DNA (Mahaseth And Kuzminov 2016), so the presence of significant DNA damage after a 24-h exposure was expected and had indeed been previously shown by (Pellegri et al. 2014).

The toxicity of plastic particles is known to be dependent on a wide range of factors, including their size, shape, concentration and chemical composition (Sun et al. 2021). Immobilisation and DNA damage were detected in Daphnia pulex resultant of acute exposure to pristine 100 nm PSNPs, however the pristine nature of nanoparticles used in this study may differ from MNPs more commonly found in the environment, which can often be weathered and found in combination with other pollutants (Duan et al. 2021), both factors which can affect the impact of MNP pollution (Liu et al. 2020). Artificially weathered MNPs have been shown to exhibit higher toxicity than pristine particles including increased genotoxicity, but reduced inflammatory potential in vitro (Völkl et al. 2022). Thus, the pristine nature of the PSNPs could alter their genotoxic impact relative to particles of similar size and composition found in freshwater systems and make them an imperfect comparison. Future work could consider the genotoxic impact of weathered MNPs and MNPs as vectors of other genotoxic pollutants to help elucidate the specific mechanisms of MP-induced genotoxicity in daphnids.

Direct interaction of DNA with MNPs is capable of damaging DNA when particles are small enough to pass through the nuclear membrane (González-Acedo et al. 2021; Liu et al. 2021), however damage is more commonly resultant of secondary effects resulting from the buildup of ROS (Shi et al. 2022; Xuan et al. 2023) including alterations to DNA replication or repair capacity (Kadac-Czapska et al. 2024). The increased genotoxicity between 24- and 48-h timepoints could be due to a range of factors, including the aforementioned buildup of ROS in cells, which is thought to constitute a major cause of DNA strand breaks. This occurs due to the oxidising of DNA bases and free radical attacks on the DNA sugar-phosphate backbone (Juan et al. 2021) as well as the disruption of antioxidant defence mechanisms (Imhof et al. 2017; Das 2023), or the interference with DNA repair pathways (Brandts et al. 2018; Estrela et al., 2021; Imhof et al. 2017; Sun et al. 2021). Thus, PSNP-induced genotoxicity may not be caused by direct interaction between PSNPs and DNA, but rather by secondary mechanisms in response to aggravation caused by PSNPs including inflammation and damage to mitochondria (Das 2023).

Although significant genotoxicity was found at high concentrations, our results differed from studies by Nugnes et al. (2022a, 2022b), who found significant DNA damage after 24 h in Ceriodaphnia dubia even at far lower concentrations than those used in this study. The lower genotoxicity observed in this study should be considered in the context of lower acute immobilisation compared to that recorded by Nugnes et al. (2022a) and could additionally be due to the sensitivities of the different test species being used, the different size of MNP particles to which they were exposed, or methodological differences in carrying out the comet assay.

This study has investigated the effects of NPs at far higher concentrations than those typically found in the environment in terms of particles/L, with the lowest concentration of MNPs used in this study approximately 10,000 times higher than those found in the environment (Koelmans et al. 2019). This study and others (Nugnes et al. 2022b; Pellegri et al. 2020) have demonstrated that daphnids exhibit a genotoxic response to toxicants on short timescales. However, in the environment, exposures to MNPs are often chronic or even multi-generational as a result of long-term water pollution and will often involve far lower concentrations of MNPs than those investigated in this study. Although this study provides a demonstration of MNP toxicity in Daphnia pulex, further investigation at ecologically-relevant concentrations and on longer timescales is required.

Lower concentrations on longer timescales are already known to impact Daphnia in a variety of ways (Procházková et al. 2024), and the detection of MNP-induced genotoxicity in Daphnia has ecological implications, as genotoxicity in aquatic invertebrates can indicate community-level stress which may wreak cascading effects on wider ecosystems (Lencioni et al. 2020). The genotoxic impact could work to further compound the potential cascading impact of MNP-induced changes to daphnid behaviour as demonstrated by our results regarding hopping frequency. As previously discussed, changes to swimming behaviour could affect predator–prey dynamics (Bhattacharjee et al. 2013), with cascading effects on species at higher trophic levels. Further investigation into the long-term toxicity of MNPs upon daphnids at the individual and community level is required to gain a more complete understanding of the ecological implications.

This study carries implications for human health. (Pellegri et al. 2020) demonstrated elevated genotoxicity in Daphnia magna after exposure to water collected near wastewater treatment plants. Wastewater treatment plants constitute a major vector by which MNPs enter some freshwater systems, as they are difficult to entirely remove from effluent (Li et al. 2018). The presence of MNPs in effluent could therefore contribute to the genotoxicity of treated wastewater, which can be directly used for irrigating crops for human consumption (Ofori et al. 2021). Our results therefore lend further cause for the inclusion of genotoxicity bioassays in freshwater quality testing.

Conclusion

This study’s primary purpose was to investigate the impact of high concentrations of PSNPs upon Daphnia pulex. It was found that genotoxicity was induced in response to PSNP exposure, but that the genotoxic response was less pronounced than that of immobilisation or hopping behaviour. However, lower concentrations could induce DNA damage to both individuals and populations of daphnids on a longer time frame than the acute exposures investigated in this study. Microplastic-induced DNA damage could have long-lasting effects, with unpredictable consequences on both daphnid populations and wider ecosystems. Thus, further work including chronic exposures, multigenerational studies and trophic transfer studies is required to investigate such long-term impacts.The detection of genotoxicity resultant of MNP exposure adds to the usefulness of the daphnid comet assay as part of a battery of bioassays for the assessment of water quality, as suggested by (Pellegri et al. 2020). While immobilisation and swimming behaviour endpoints provide strong and sensitive early-warning indicators of toxicity, MNP-induced DNA damage has the potential to persist beyond the exposure period and to contribute to adverse community- and ecosystem-scale impacts (Gong et al. 2023; Lencioni et al. 2020)(Gong et al. 2023; Lencioni et al. 2020). The low cost and resource-intensiveness of immobilisation and swimming behaviour makes them ideal for biomonitoring, however wider incorporation of the comet assay into freshwater monitoring methods alongside such existing endpoints could further understanding of the full impact of micro- and nanoplastic pollution upon ecosystem health.

Supplementary Information

Below is the link to the electronic supplementary material.ESM 1(DOCX 1.23 MB)