Freshwater systems in the Anthropocene: why we need to evaluate microplastics in the context of multiple stressors

Introduction

Microplastics are a contaminant of emerging concern in freshwater ecosystems (Lambert and Wagner 2018; Rochman and Hoellein 2020). They can cause a range of adverse effects in freshwater biota at multiple levels of biological organization, due in part to their inherent complexity (e.g., size, polymer, morphology) (Rochman et al. 2019; Bucci et al. 2020). Studies that investigate microplastics as a single stressor (e.g., Li et al. 2024; Malinowski et al. 2023; Schür et al. 2023) have demonstrated that microplastics are impacting freshwater ecosystems. However, freshwater ecosystems are impacted by more than just microplastics, which current research often fails to incorporate.

In addition to microplastics, freshwater ecosystems are impacted by anthropogenic stressors including climate change (Carpenter et al. 1992; Perkins et al. 2010), pollutant loading (Amoatey and Baawain 2019; Peters et al. 2013), nutrient loading (Smith 2003) and invasive species (Gallardo et al. 2016; Dextrase and Mandrak 2006), which could be better incorporated into microplastic research (Figure 1). Each of these stressors individually impact freshwater ecosystems with effects ranging from loss of biodiversity (Habibullah et al. 2022; IPCC 2022), to degraded habitat (Paul and Meyer 2001) and altered ecosystem functioning (Everard and Moggridge 2012). Despite this, few microplastic studies incorporate the reality that freshwater ecosystems are impacted by not just microplastics, but rather many anthropogenic stressors.

Figure 1. A simplified schematic of multiple anthropogenic stressors (left) that can impact an ecosystem (center) and be monitored across multiple levels of biological organization (right).

Using a multi-stressor lens to understand the effects of microplastics in freshwater ecosystems is critical in understanding how these stressors may also impact other environments, e.g., terrestrial ecosystems. Freshwater ecosystems serve as the integral connection between terrestrial and aquatic environments; rivers in particular serve as the connection between terrestrial, freshwater, and marine ecosystems (Likens and Bormann 1974; Krause et al. 2019). Given this, and that freshwater ecosystems are viewed as sentinels (e.g., the canary in the coal mine) with respect to anthropogenic perturbations (Woodward et al. 2010; Perkins et al. 2010), it is an important environment to assess multiple stressor impacts.

Despite the significant progress in our understanding of the sources, fate, and effects of microplastics in the last decade, knowledge gaps remain regarding how microplastics impact aquatic ecosystems in the context of multiple stressors, particularly at higher levels of biological organization, and how this data may be used to inform monitoring priorities and management decisions across aquatic and terrestrial ecosystems, alike. Here, we argue that researchers should (1) study microplastics as inherent multiple stressors by evaluating the physical and chemical effects; and (2) take an ecosystem level approach in investigating the effects of microplastics with other anthropogenic stressors present in the environment (e.g., metals, industrial chemicals) under varying environmental conditions (e.g., temperature, water chemistry). Last, we outline research priorities and recommendations to facilitate ecotoxicological assessments that will better encompass the multidimensionality of microplastics with other multiple stressors as environmental conditions (e.g., climate) continue to change. By taking a holistic approach and evaluating microplastics as a multi-stressor and in the presence of other anthropogenic stressors, we can move toward improved conservation and management policies and practices.

Microplastics as a physical and chemical stressor

Microplastics (<5 mm; Arthur et al. 2009) are inherently complex, varying in toxicity across physical and chemical characteristics (Rochman et al. 2019), whose nature and interactions remain misunderstood. While it has become clear that certain morphologies, sizes, polymer types, and additives may lead to varying effects (Zimmermann et al. 2020; Strungaru et al. 2019; Bucci et al. 2020; da Costa et al. 2023), most studies do not design experiments to evaluate these characteristics in combination; thus, failing to consider the inherent complexity of microplastics.

There has been significant progress in understanding the physical effects of microplastics, i.e., morphology and size. Previous research has established simpler morphologies like spheres, for example, are less toxic than complex morphologies like fibers and fragments (e.g., Ziajahromi et al. 2017; Qiao et al. 2019a; Cole et al. 2019; Danopoulos et al. 2022). Further, particle size influences toxicity (Thornton Hampton et al. 2022) as well as the toxicokinetics/dynamics in exposed organisms (Coffin et al. 2022; Thornton Hampton et al. 2022b). Understanding the mechanisms of toxicity and biological fate could be furthered by investigating mixtures of different morphologies and sizes.

Polymer type is a chemical characteristic of microplastics that may influence toxicity due to its chemical constituents, e.g., monomers (Lithner et al. 2011), yet there is a lot we still don’t know about the effects of different polymer types. Some studies have experimentally tested hypotheses about how effects vary among different polymers types, and have found differences in observed effects (e.g., Zimmermann et al. 2020). Despite this, the majority of studies often focus on a few polymer types that are most easily accessible for research, namely polyethylene (PE) and polystyrene (PS; Thornton Hampton et al. 2022) and omit other polymer types with that are commonly found in the environment, such as polyvinyl chloride (PVC) and polyurethane (PU) (PlasticsEurope 2022; Zimmermann et al. 2020). While these studies have allowed us to understand the physiological and ecological impacts of specific microplastic polymers across different levels of biological organization, integration of other polymers and polymer mixtures will improve our understanding of the impacts of microplastics as a multi-stressors.

Additive chemicals are another chemical aspect of microplastics that should be considered more when assessing toxicity as they can illicit adverse effects (e.g., Tian et al. 2021; Chibwe et al. 2021; Zimmermann et al. 2019). Plastic additives provide specific properties such as flexibility, color, UV protection, and durability (Hahladakis et al. 2018; Hermabessiere et al. 2017), can account for nearly 60% of the total mass of the material (Net et al. 2015) and often are not bound to the polymeric matrix; thus, leaching into the environment (Hermabessiere et al. 2017). In the environment, these additives can be independently toxic (Catrouillet et al. 2021; Fauser et al. 2022, 2020; Tian et al. 2021). Biological effects may differ according to polymer type due to their varied chemical constituents (Groh et al. 2023; Hahladakis et al. 2018; Zimmermann et al. 2019). For example, styrenic compounds that are often found in polystyrene (PS) are known endocrine disruptors (Lithner et al. 2011), whereas additives such as flame retardants or UV-filters, which are found in a variety of polymer types, have been shown to have a range of toxicological effects, such as oxidative stress and cytotoxicity (Zimmermann et al. 2019). Given that additives are used commercially in microplastic production, they should be considered a co-contaminant when evaluating microplastic toxicity.

Effects of microplastics with other environmental stressors

While microplastics will impact ecosystems as a multiple stressor, they also interact with other anthropogenic stressors present in a system (e.g., climate change, increased salinity; Figure 1). The combined effects of these stressors may result in different ecological outcomes than when assessed in combination. Thus, in addition to being evaluated as a multi-stressor on its own, microplastics should be evaluated in relation to other ecological stressors present within the Anthropocene (e.g., climate change).

Microplastics co-occur with other emerging contaminants of concern such as pharmaceuticals, agricultural chemicals (e.g., pesticides; Bhagat et al. 2020), POPs (Rodrigues et al. 2019), and flame retardants (Lambert and Wagner 2018) whose combined effects to freshwater ecosystems should be considered (Figure 1). It is well established that these environmental contaminants have their own suite of toxic effects. For example, pharmaceuticals can lead to endocrine disruption in fish (e.g., estrogenic compounds from wastewater effluent, Kidd et al. 2007) while pyrethroid insecticides can lead to neurotoxicity in vertebrates (Vijverberg and van den Bercken 1990). In addition to co-occurring with microplastics, these anthropogenic contaminants may be transported by microplastic when they sorb to the surface of the plastic. While this is an important phenomenon that may contribute to the toxicity of microplastics and other stressors (see Lohmann (2017), Gouin (2021) and Koelmans (2022), a deep dive into this discussion is outside the scope of this paper. However, acknowledging the presence, potential transport, and toxicity of other chemical contaminants in an ecosystem, is important to disentangle the impacts of microplastics from those of other chemical contaminants in the environment (Campanale et al. 2020).

Climate change is another aspect of the Anthropocene that is interacting with microplastics to modulate ecological effects in aquatic ecosystems. Some calls have been made to incorporate this reality in the microplastics field however, relatively few studies have experimentally tested this (e.g., Kolomijeca et al. 2020). Some studies have tested the multi-stressor effects of climate change and microplastics on a variety of organisms (e.g., Yang et al. 2020; O’Brien et al. 2022; Lins et al. 2022) shedding light on how aspects of climate change (e.g., CO₂ and temperature) modulate biotic endpoints including individual growth (Yang et al. 2020), motility, survival (Lins et al. 2022), and species interactions (O’Brien et al. 2022). Given the urgency in understanding the impacts of climate change across a range of environments, studies should incorporate aspects of climate change when assessing ecological effects of microplastics.

Finally, microplastic studies should seek to understand the effects of microplastics and other stressors at higher levels of biological organization. Experimental studies have shown that the effects of microplastics can span multiple levels of biological organization (Bucci et al. 2020), including at the community (e.g., biodiversity, species composition; Green et al., 2016; Redondo-Hasselerharm et al. 2020), population (e.g., abundance; Bosker et al. 2019), individual (e.g., survival, growth; Bucci et al. 2022; Silva et al. 2019), and sub-organismal (e.g., inflammation, oxidative stress; Qiao et al. 2019b) levels. However, studies tend to focus on individual, or population-level impacts, ignoring higher-level impacts. Clements and Rohr (2009) propose to treat contaminants as a predator, with direct and indirect impacts to individuals and trophic levels to elucidate effects at higher levels of biological organization; thus, improving community and ecosystem level effect assessments. This approach could be taken with microplastic studies to incorporate the complexity of microplastics themselves and their interactions with other stressors (e.g., chemicals, climate change, invasive species) to predict ecosystem-level effects.

A need to apply novel approach methods to monitor microplastics in a multi-stressor world

Addressing the multi-stressor impacts of microplastics with other stressors is no easy task but is nonetheless crucial to informing monitoring and management (Figure 2). To address this, new analytical, genomic, and design approaches are needed to better understand the toxicity of microplastics in the context of multiple stressors. Because most proposed monitoring programs focus on the physical characteristics of microplastics with only recent calls to include the chemical characteristics (Hamilton et al. 2022) researchers will need to re-imagine microplastic experiments in this multi-stressor view. Additionally, leveraging new technologies will facilitate better evaluation of the physical and chemical effects of microplastics through a multi-stressor lens, improving our understanding of the impacts at multiple biological levels of organization, ultimately improving monitoring and management decisions.

Figure 2. Flow chart describing (1) microplastics in the context of multiple stressors; (2) novel and analytical approaches to assess microplastics in the context of multiple stressors; and (3) research opportunities needed to assess microplastics as a multiple stressor in order to inform robust, evidence-based monitoring and management policies.

To facilitate multi-stressor assessments of microplastics with other anthropogenic stressors, experimental designs should be re-imagined. To assess the effects of multiple stressors, multiple experiment units are required to tease apart the effects of individual stressors. Logistically, including multiple experimental units can be challenging due to space, time, and human-power constraints. Ecotoxicologists should work together with other experts (e.g., engineers) to design instruments to facilitate multi-stressor work. For example, Nguyen et al. (2018) present a well-plate platform designed to test effects of climate change and other anthropogenic stressors on aquatic organisms. This platform was later used to investigate the multi-stressor effect of microplastics and climate change on algae (Yang et al. 2020; Guo et al. 2021), plants (O’Brien et al. 2022), and invertebrates (Lins et al. 2022). Technologies like these will help to facilitate multi-stressor testing of microplastics with other stressors.

Further, novel analytical approaches such as non-target screening, suspect screening, and effect-directed analysis, can be used to identify microplastics and other environmental contaminants, which will be critical to monitoring the effects of microplastics in nature. New developments in analytical chemistry involving high resolution mass spectrometers, (i.e., non-target and suspect screening) can enable the screening of a broad range of compounds in a sample (e.g., biota or plastics; Ballesteros-Gómez et al. 2016; Fries et al. 2022). These techniques often aim to identify contaminants rather than quantify them, but can also result in qualitatively identifying certain compounds (Schymanski et al. 2015; Fries et al. 2022). Regardless, non-target and suspect screening is an important first-step in identifying specific chemicals and microplastics in a complex mixture and matrix. Often combined with nontarget screening techniques, effect-directed analyses use toxicity assays to determine whether a sample with a stressor mixture exhibits a toxic effect. Here, the complexity of a sample is reduced and potential compounds that may illicit a toxic effect are identified. These approaches have been applied to plastics to a limited extent (Schönlau et al. 2019) and offer possibilities of addressing toxicity and chemical identification in a multi-stressor mixture.

New genomic tools can be used to determine contaminant burdens and effects at different levels of biological organization. For example, Zahaby et al. (2021) developed a toxicogenomics approach (ToxChip) to determine oil-related contaminant burdens in the livers of seabirds in the Canadian Arctic and successfully distinguished between the two distinct colonies of seabirds based on the expression of genes associated with trace element and polycyclic aromatic compound exposure (Zahaby et al. 2021). Furthermore, a recent study by Giroux et al. (2023) utilized eRNA/eDNA metabarcoding approaches to successfully detect community-level impacts of nanoplastics in benthic estuarine ecosystems. Here, Giroux et al. (2023) produced dose-response relationships of nanoplastics at a community level; thus, underscoring the importance of assessing and monitoring community-level effects. Additionally, RNA sequencing approaches can be used to understand the underlying mechanism of toxicity of microplastics and other stressors. RNAseq can be used to screen for differential expression of genes for organisms that have been exposed to varying stressors or stressor levels (e.g., Waller et al. 2019; Bertucci et al. 2017; Shaw et al. 2007). Through this analysis, candidate genes that are differentially expressed are identified and gene expression changes (up or downregulation) can be measured. Ultimately, by utilizing RNAseq, effects from one stressor can be disentangled from another, assuming that the gene response is specific to one stressor, and ultimately improve ecosystem level monitoring of multi-stressor effects.

Last, to facilitate the multi-stressors assessment of microplastics with other anthropogenic stressors, engineering technologies can be used to reimagine toxicity experiments. To assess the effects of multiple stressors, multiple experiment units are required to tease apart the effects of individual stressors. Logistically, including multiple experimental units is challenging due to space, time, and human-power constraints. Ecotoxicologists should work together with engineers to design instruments to facilitate multi-stressor work. For example, Nguyen et al. (2018) present a well-plate platform designed to test effects of climate change and other anthropogenic stressors on aquatic organisms. This platform was later used to investigate the response of algae (Yang et al. 2020; Guo et al. 2021), plants (O’Brien et al. 2022), and invertebrates (Lins et al. 2022) to climate change with microplastics. Technologies like these will help to facilitate multi-stressor testing of microplastics with other stressors.

Conclusion

Terrestrial and aquatic ecosystems are inextricably linked together, and are impacted by multiple stressors. Current ecotoxicology approaches must be adapted in order to incorporate the complexity of stressors that ecosystems are subject to in the Anthropocene. Here, we have argued that microplastics should be studied as multiple stressors; first on their own by incorporating physico-chemical aspects of microplastics into study designs and second, by considering their interactions with other anthropogenic stressors that may modulate effects at multiple levels of biological organization. Last, we highlight the need to embrace novel chemical, genomic, and other approaches to develop robust and consistent monitoring efforts across biological levels of organization. By implementing these changes, using these new techniques, and improving study design, monitoring and management of microplastics and other environmental stressors will be improved.

Author contributions

RKG and BMH contributed equally to the conception and production of this manuscript.