The current study demonstrates the potential acute toxicity of non-functionalized nano- polystyrene beads to three different clones of Daphnia longispina species complex. If ecotoxicity global harmonized hazard identification system (GHS) or classification and labelling of products (CLP) would be applied to these particles, the 50 nm beads would qualify to very toxic to the aquatic environment (GHS & CLP acute ecotoxicity class 1, EC50 below 1 mg.L− 1), while the 100 nm beads would be considered less toxic (GHS aquatic toxic class 2, EC10 between < 10 mg.L− 1). It is worth to mention that GHS (Nations., 2011) and CLP (Counci, 2008) were designed to address intrinsic toxicity of dissolved chemicals and that effect concentrations reported here may be underestimated compared to actual exposure likely being lower than nominal concentration (Fig. 1). Notwithstanding, the current results place 50 nm polystyrene beads amongst the most acutely ecotoxicity category of materials in global and European chemicals regulatory frameworks.
Those particles were not dissolved and it is unclear whether the observed effects could be attributable to intrinsic ecotoxicity in a traditional sense. Thus, the next paragraphs explore three main insights from this experiment under the lenses of fundamental ecotoxicological principles.
Variability Across Daphnia Clones
The sensitivity across clones varied c.a. 10-fold or more between Amme 51 and Amme 3 within a given exposure-time. Such intra-specific variability in sensitivity is compatible with previous studies that have investigated (dissolved) chemical ecotoxicity and inter-clonal variation in D. magna. For instance, Barata et al. (Barata et al., 1998) found that differences in cadmium tolerance among D. magna clones within populations were up to 10-fold, whereas differences among naturally genetic different populations could be higher.
Intraspecies variability of ecotoxicological responses within Daphnia after nanoplastic exposure has not been investigated. Howerver, two studies included multiple Daphnia clones in their assessments of toxicity of larger plastic particles (microplastics), showing that these responses are genetically variable (Imhof et al., 2017), even within a single population (Sadler et al., 2019). Specifically, there was a high inter-clonal variation in gene and protein expression in exposed three clonal lines of D. magna (Imhof et al., 2017), and differences in life-history and immune responses of exposed D. magna clones (Sadler et al., 2019). There is conclusive evidence of the genetic distance amongst the morphologically and eco-physiological diverse D. longispina clones studied here (i.e. Amme 12, Amme 3, Amme 51. galeata × longispina) (Griebel et al., 2015). Thus, genetic variation in responses to plastic particle exposure helps to explain some of the contradictory results among Daphnia studies based on single genotypes (reviewed in (Samadi et al., 2022)). This clone-specific nature of Daphnia responses to nanoplastics demonstrates that there might be potential for evolutionary selection and adaptations of populations in contaminated sites. Nevertheless, these results strongly advocate for incorporating genetic variation into assessments of the impact of plastic particle exposure
A 2-fold Decrease in Particle Size Associated to up to 1000s-fold Increase in Toxicity
There is evidence that the decrease in size of plastic particles is accompanied by increase in reactivity and occurrence of chemical-like effects (de Souza Machado et al., 2018). In the present study, the decrease in particle diameter from 100 to 50 nm represented an approximately ∼100-fold increase in toxicity for the clone Amme 51, and a typical ∼10-fold increase in toxicity for Amme 3 and Amme 12 in EC50s. In fact, differences across particle sizes and clones in EC50s are about more than 450-fold (Imhof et al., 2017; Rist et al., 2017; Schwarzer et al., 2022; Takeshita et al., 2022). Kögel et al. reviewed that the evidence points towards the tendency of larger negative impact by smaller plastic particles compared to larger ones, from the nm to triple-digit µm size range (Kögel et al., 2020). Adjusting the exposure amount for the different conditions of the size comparisons by mass by most of the existing studies, leads to three orders of magnitude higher particle numbers for each order of magnitude smaller particle diameter (Kögel et al., 2020).
Altogether, the current results support than several 1000s-fold difference on sensitivity to a particles of a same polymer matrix and chemistry can be expected across studies with the same species when intra-clonal and particle size variability are not addressed.
Considering Exposure to Plastic Mass or Particle Number
The patterns above-discussed are evident if the exposure metric to determine effect concentrations is mass-based, which is the default metric for hazard identification of materials in ecotoxicology (Nations., 2011; OECD, 2004). Nevertheless, most environmental reports of microplastic contamination are based on particle numbers (counts) (Du et al., 2021; Horton et al., 2017). Therefore, there are questions on whether exposure to plastic mass, particle number, or other metrics of exposure are ecotoxicological relevant variables for the assessment of micro-nanoplastic effects.
If exposure of Daphnia is measured in terms of particle numbers it cannot be consistently asserted whether larger particles are indeed less toxic for all clones (Fig. 3B). In fact, for clones Amme 12 and Amme 3 no significant difference could be observed between 50 nm or 100 nm beads. This suggests similar potency of these individual particles in triggering toxicity for both clones, with ≈ 1015 particles L− 1 as the levels for ecotoxicological effects. Nevertheless, less particles of 50 nm beads were required to trigger similar effects on Amme 51 comparted to the 100 nm beads. Despite significant, at the same scale, the difference in Amme 51 sensitivity to the two particle sizes considering particle number-based effect concentration (104- fold) is smaller than the difference considering mass-based effect concentrations (106- fold). That suggests that normalization of exposure by particle number provides some explanatory power of the particle toxicity, but mass-based inference is still relevant. Such results are coherent to the review of Kögel et al., which did not conclude whether the size or the sheer particle number has the largest impact on the effect (Kögel et al., 2020).
Particles with the same shape were investigated here. Therefore, limited insights can be obtained for some other potentially relevant metrics for micro-nanoparticle as they are redundant to ECmass−based or ECparticle number−based. For instance, particle volume is linearly related to particle mass. Similarly, particle surface area co-varies with the product of particle mass, diameter and particle numbers (Fig. 3C). Further studies with particles of varying shape would be needed to elucidate the relationship amongst these exposure metrics.
Thus, the current results provide evidence that there may be a physical-like component of the micro-nano particle toxicity that can be explained by normalizing exposure values by counts of single particle numbers. However, the exposure metric based on plastic mass seems to provide better differentiation on toxicity across particles, which suggests the existence of some chemical-like (intrinsic) effects.