Engineered nanomaterials are often designed to have a certain reactivity, functionality or biological effect. As discussed, nano- and microplastics often stem from unintentional anthropogenic rather than engineered processes. Even when they are intentionally produced, they are not as such intended to be biologically active. Certain polymer additives may, however, have the purpose of, for example, preventing biotic or abiotic degradation. For both engineered nanomaterials and nano- and microplastics, it is therefore useful to consider their intended use and properties when evaluating their potential environmental risk. Engineered nanomaterials that are intended to have biocidal effects are likely to be more toxic to non-target organisms than materials intended to be inert. Similarly, plastic particles containing biocidal additives, plasticisers or flame retardants are likely to be more environmentally hazardous, as these substances may leach out of the polymer matrix.
One effect mechanism is being highlighted as important for both engineered nanomaterials and nano- and microplastics, namely, physical interactions between the particle and the organisms [
43]. This includes inflammation and interference with the energy balance caused by uptake of particles into the gut, thereby limiting food uptake. Different types of engineered nanomaterials, as well as nanoplastics, have been observed to adhere to the surface of microalgae, potentially causing a physical shading effect on a cellular level [
44]. Physical effects of microplastics on marine organisms have been reviewed recently [
45], and mechanisms that have been described as potentially relevant include blockage of the digestive system, abrasion of tissues, blockage of feeding appendages of invertebrates, embedment in tissues, blockage of enzyme production, reduced feeding stimulus, nutrient dilution, decreased growth rates, lower steroid hormone levels and impaired reproduction. Table
1 presents an overview of effects in response to the physical particle properties that have been observed in different species.
Table 1
Examples of biological effects observed in aquatic organisms after exposure to engineered nanoparticles or nano- and microplastics
Molecular/cellular level
|
Oxidative stressa
Inhibition of photosynthesis (shading)b
| DNA damage and differential gene expressionl
Cellular stress response and impaired metabolismm
|
Tissue level
|
Histopathological changesc
Transfer into cellsd
| Tissue damagen
Transfer into tissueso
|
Organ/organismal level
|
Morphological malformatione
Decreased swimming velocitiesf
Increased mucus productiong
Toxic effects of released ionsh
Decreased growth rates and biomass productioni
Moulting inhibitionj
Impaired mobilityk
| Impaired respirationp
Impaired feedingq
Impaired development and reproductionr
Decreased growth rates and biomass productions
Behavioural changest
Increased mortalityu
|
For nanomaterials, size-dependent changes in effects are of particular interest. The whole purpose of nanotechnology is to take advantage of the novel properties that come with a smaller size. For engineered nanomaterials, this involves, for example, the novel catalytic effects of some materials on the nanoscale including gold (Au), titanium dioxide (TiO
2) and cerium dioxide (CeO
2). As larger-sized (bulk) materials, these are relatively inert, but with decreasing particle size and increasing surface area, they become reactive. Therefore, as particle size decreases, there is a tendency for toxicity to increase, even if the same material is relatively inert in its corresponding bulk (micron-sized) form [
85]. In addition, the small size of engineered nanomaterials may enable their uptake into tissues and cells [
49]. Observed biological effects of engineered nanomaterials in aquatic organisms include oxidative stress, inhibition of photosynthesis, tissue damage, impaired growth and development, behavioural changes and increased mortality (Table
1).
Similarly, the question for nano- and microplastics is therefore: Is it likely that a decrease in size will make them more hazardous? To answer this question, we will examine the two main causes for concern: novel properties and ingestion by organisms (and potential subsequent transfer into tissues). The novel properties that would occur for smaller-sized polymer particles are linked to their increased surface-to-volume ratio. With decreasing particle size, a larger fraction of the molecules will be present on the surface of the particle. As the surface is where interactions with the surrounding environment take place, this can lead to an increase in chemical reactions and biological interactions. For example, smaller particles may (on a mass basis) have a larger adsorption capacity compared to larger particles [
86], which in turn is of relevance for the vector effects. The second concern relates to the potential to cross biological barriers. Nanosized particles, such as nanoplastics, are potentially more hazardous due to their easier uptake into tissues and cells [
2]. Depending on particle size, different uptake routes into organisms are also involved. For example, the freshwater crustacean
Daphnia magna normally catches prey (mainly algae) in the size range 0.4–40 μm [
87,
88]. For particles or agglomerates that are within this size range, uptake can occur through active filtration, and at the same time unwanted particles can be rejected. Particles smaller than the preferred size are not actively taken up by the animals, but may instead enter the organisms through ‘drinking’ of the surrounding water, resulting in non-selective, uncontrolled uptake. Depending on the feeding strategies of specific aquatic organisms and their ability to actively select their food source, they may be able to regulate their uptake of microplastics, whereas nanoplastics may enter the organisms unintentionally.
5.1 Nano- and Microplastics in Standard Ecotoxicity Tests
In the quest to determine the environmental risk posed by nano- and microplastics, laboratory-based experiments need to be carried out which analyse the effects of the particles under well-defined conditions. The number of controlled laboratory studies investigating the effects of nano- and microplastics on freshwater organisms is steadily increasing, and many different impacts have been observed – extending from the molecular and cellular to the physiological level (see Table
1). These include inflammation, disruption of lipid and amino acid metabolism, lower growth rates, decreased feeding rates, behavioural changes, impairment of reproduction and increased mortality [
62,
64,
75,
79,
81,
82]. When studies involving marine organisms are also taken into account, the number and variety of biological effects of nano- and microplastics that have been found are even greater.
However, most effect studies differ greatly with respect to the parameters used, for example, particle type (different polymers, sizes, shapes, presence of chemicals), test species, exposure duration, exposure concentration and response variables. This makes it difficult to compare results between studies and hampers reproducibility. It can, therefore, be advantageous to apply standardised tests, which come with a number of benefits as they ensure controlled and reproducible test designs and inter-laboratory comparability. Another advantage of standardised ecotoxicity tests is the extensive knowledge base resulting from decades of testing the effects of chemicals on selected model organisms. For ecotoxicology of nanomaterials, this has been highlighted as a motivation for using standardised short-term tests as a starting point for gaining an insight into the fate and bioavailability of engineered nanomaterials in the environment [
89]. By using a well-defined test system and a fully defined synthetic medium, other test parameters can be varied individually and in a controlled manner, thereby providing an insight into specific processes and mechanisms [
90].
However, the use of standard test guidelines also comes with some potential disadvantages, especially for testing of particles. For freshwater systems, a commonly used species is the freshwater flea
Daphnia magna, for which the OECD has developed two standard tests: an acute immobilisation test (48 h) (OECD TG 202) and a chronic reproduction test (21 days) (OECD TG 211). These tests were originally developed for soluble chemicals. Since particles show very different behaviours to soluble chemicals, it is challenging to apply the same test set-ups. Even so, some studies have used these standard tests to investigate the effects of nano- and microplastics. Casado et al. [
91] conducted an acute immobilisation test with 55 and 110 nm polyethyleneimine PS beads and reported EC
50 values of 0.8 mg/l and 0.7 mg/l, respectively. The same test with 1 μm PE beads resulted in an EC
50 value of 57.4 mg/l [
83]. This huge difference could be a consequence of the different polymer types and sizes used in the studies, but it might also indicate that mortality is not a very sensitive biological response when it comes to plastic particles. Finally, it may be indicative of a problem that has been highlighted for tests with engineered nanomaterials: That reproducibility and data interpretation in standard ecotoxicity tests with particles, rather than soluble chemicals, are challenged by the dynamic nature of particles suspended in aqueous media [
90]. Particle properties and behaviour may change as a function of time or as a result of interactions with test organisms and emitted biomolecules (e.g. exudates) [
7]. It has therefore been recognised as essential in the work with engineered nanomaterials to conduct a particle and exposure characterisation before and during a laboratory test [
26,
92]. This includes an analysis of the size, shape, surface area and surface chemistry of the tested particle, as well as aggregation/agglomeration, sedimentation and dissolution behaviour in the test system, thereby providing information on exposure in both qualitative and quantitative terms. Furthermore, appropriate ways of dispersing the particles in aquatic media have been highlighted as an important area of future test method development [
93]. The rationale behind thorough characterisation and carefully considered sample preparation methods relates to data interpretation and avoidance of the introduction of test artefacts. Such activities are currently rarely undertaken in the work with nano- and microplastics, but should be included in order to gain an insight into the behaviour of the particles in exposure media and the resulting influence on their interaction with test organisms.
Another aspect that needs to be taken into account is the leaching of molecules from particles. For engineered nanomaterials, work is ongoing within the OECD to develop test guidelines for investigating the dissolution of metal ions from metal-containing nanomaterials [
94]. In the case of plastic particles, the leaching of chemicals from the polymer matrix (e.g. additives or monomers) and the release of adhered co-pollutants can influence the test results. Appropriate test methods are therefore needed to investigate the actual release of plastic additives from nano- and microplastics under relevant conditions (media, temperature, pH, etc.), and a control for the effects of chemicals and released additives or adhered pollutants needs to be included as a reference.
Transformation processes, such as oxidation/reduction, interaction with macromolecules, light exposure and biological transformation, can significantly influence the integrity, behaviour and persistence of nanomaterials in aquatic media [
95‐
97]. Depending on the specific conditions, dissolution and degradation can be enhanced or reduced. Enhanced dissolution may result in increased toxicity of, for example, metal and metal oxide nanomaterials. At the same time it may cause a gradual decrease in particle size [
97]. For nano- and microplastics, aging/weathering processes should also be accounted for as they may change particle properties (e.g. surface chemistry, polarity and density) and enhance fragmentation. It should be emphasised that a complete degradation of plastic particles under realistic environmental conditions has not yet been demonstrated [
6,
98,
99]. While aging is potentially important for nanomaterials, and for nano- and microplastics, in the environment, the relevant aging processes and kinetics may differ. Based on current knowledge, nano- and microplastics may have a higher core persistence and lower release of soluble compounds than certain engineered nanomaterials (especially metal and metal oxide nanomaterials such as ZnO and Ag). However, this is clearly an area of future research – for both nanomaterials and nano- and microplastics. Aging is currently not incorporated in standard ecotoxicity test protocols, but has been proposed for engineered nanomaterials [
100]. There are also indications that aging of plastic particles can influence biological effects [
79]. This aspect should therefore generally be considered in the future development of ecotoxicological tests for particle testing.
For test method developments, the field of ecotoxicity testing of nanomaterials has benefitted from the availability of reference materials (e.g. NIST Standard Reference Materials) and representative industrial nanomaterials (such as those from the JRC Nanomaterials Repository). Such materials are valuable for analytical method validation and for conducting comparable inter-laboratory and inter-species studies. The field of ecotoxicity testing of nano- and microplastics would similarly benefit from the establishment of sources of well-characterised, industrially and environmentally relevant materials of various sizes and compositions.
The applicability of current standard ecotoxicity tests has been questioned for engineered nanomaterials. Development of new test guidelines and guidance is under discussion, for example, within the OECD [
101]. The same concerns apply to testing of nano- and microplastics: They represent a specific challenge due to their dynamic nature in environmental media, resulting in, for example, differences in relevant exposure routes (through food or other active uptake routes, grazing on sedimented materials, etc.), as well as potentially different effect mechanisms. Soluble molecules can be taken up into aquatic organism by diffusion and then distributed within the organism based on partitioning, e.g. to lipid tissues. Cellular uptake of soluble chemicals generally relates to passage of biological membranes, mainly through passive diffusion or active uptake, such as transport through ion channels or carrier-mediated transport [
102]. In the tissues, they can act non-specifically, leading to narcosis, or specifically by inhibiting or affecting certain biological processes. In comparison, particle distribution is not governed by diffusion and partitioning. Uptake of particles by organisms depends on mechanisms such as feeding rather than molecular diffusion. On a cellular level, particles may be taken up through processes such as phagocytosis. Effects will therefore most likely differ from those of soluble chemicals. An essential aspect is therefore to determine sensitive biological endpoints for the exposure to particles, potentially moving away from the current standard test organisms. A limited number of response variables and test species can be seen as a disadvantage of standardised tests. Based on the argument above, it may further be claimed that ‘no effect’ in a standard test does not imply a lack of ecological impact of nano- and microplastics, as these tests may not cover the most sensitive endpoints and test species for particle exposure.
As mentioned, effects of microplastics have been observed on a molecular, cellular and physiological level (see Table
1). When performing ecotoxicity testing, the aim is to establish a dose-response relationship based on the underlying assumption that effects are strongly dependent on exposure dose/concentration and time. For engineered nanomaterials, however, an inverse relationship has been observed between concentration and agglomerate size, meaning that with higher particle concentrations, particles tend to form larger agglomerates [
103]. High concentrations of engineered nanomaterials have also been linked to effects that are not due to an actual toxic response, but rather caused by an overloading of the test organisms with engineered nanomaterials, causing physical inhibition [
8]. Testing of low, environmentally relevant particle concentrations during short exposure times may, however, not be sufficient to detect effects when using endpoints on a physiological level. Before an organism shows impairment to, for example, its reproduction or survival, multiple changes must take place on a cellular level. Cellular responses may therefore be more sensitive to microplastic particle stress compared with whole-organism responses. On this level, however, we are dealing with a complex network and huge number of reactions, which makes it challenging to find and define a meaningful, reliable set of response variables. If cellular responses are to be used as indicators of the potentially hazardous properties of nano- and microplastics, more research is needed to develop suitable (standard) test methods. Another option for testing the toxicity of relatively low concentrations of particles is chronic effect studies, as chronic endpoints can prove more sensitive than acute ecotoxicity. An added benefit of testing lower concentrations is that particle agglomeration/aggregation is reduced, leading to more stable exposure.
One major criticism of current nano- and microplastic ecotoxicity studies is their lack of realism and environmental relevance when selecting test parameters [
104]. Pristine particles with a clearly defined, homogenous chemical composition are most often applied in laboratory tests. This is in sharp contrast to the particles present in the environment, which undergo transformation processes, potentially influencing their morphology, and, in the case of plastic, often contain various additives. This trade-off between environmental realism and standardised test conditions is not a dilemma that is unique to testing of particles [
105]. It should be kept in mind that different testing paradigms inform different scientific and regulatory questions. In standard ecotoxicity, applying simplified test systems and often synthetic media, test parameters can more easily be controlled and modified one by one in order to gain deeper insight into the mechanisms of toxicity and particle uptake [
89]. They are also developed to ensure data comparability and study repeatability. For example, data generated following OECD Test Guidelines and Good Laboratory Practice are considered to satisfy the criteria for Mutual Acceptance of Data and can be used for regulatory assessment purposes in all OECD member states, ideally minimising testing efforts and use of test animals [
106]. More environmentally realistic studies can, on the other hand, provide case- and site-specific information on the effects of particle pollution under specific environmental conditions. They may also provide more realistic information with regard to the combined effects of multiple environmental stressors and their interactions with plastic particles. Standard ecotoxicity tests and more environmentally realistic studies should therefore be seen as complementary tools of equal importance but potentially addressing different questions of scientific and regulatory relevance.