In contrast to studies that assign microplastics an important role as pollutant vector, this experimental study found no evidence for this. Having the same total mass of BPA in the system, even fewer daphnids were immobilized in the presence of PA particles than without. This was despite that clear evidence could be provided for sorption of BPA to the particles and ingestion of the PA particles by the daphnids.
4.1. Study Design with Predefined Conditions
Recently, there was a call for more complex experimental setups with scenarios likely encountered in the environment for the risk assessment of microplastics [3
]. Including only selected predefined parameters under well controlled laboratory conditions in the setup of this study provided the opportunity to focus on a more mechanistic understanding of single relevant aspects. In many previous studies, the potential vector effect of microplastics was analyzed by measuring uptake rates of the pollutants assessed by tissue concentrations as proxy [22
]. Other studies analyzed pollutant effects, e.g., biomarker activity or histopathological changes [42
]. This study focused on analyzing how the acute effects of a pollutant are modified by microplastics, rather than measuring uptake rates and tissue concentrations of the pollutant within the daphnids. Experimental results on immobilization in daphnids and analytical measurements of BPA in water were complemented by calculations of the mass distribution of BPA in the test system.
Sorption behavior of pollutants to microplastics is crucial for their potential vector effect. Batch experiments showed that PA particles are an intermediate strong sorbent for BPA with fast sorption equilibration. Shaking the mixtures for 48 h prior to testing assured that sorption processes of BPA to PA particles were in an equilibrium state. In most previous studies, microplastics loaded with organic pollutants were directly fed to the test animals without reaching sorption equilibrium in the test system before exposure [34
]. Even if microplastic pollutant mixtures have been pre-equilibrated prior to exposure to ensure sorption of the pollutant to microplastics in some studies, dilution of the mixtures led to a non-equilibrium state at the beginning of exposure [32
]. While this non-equilibrium state is also a relevant environmental scenario with contaminated microplastics emitted into the aqueous system, e.g., via point sources, sorption equilibrium for microplastics and pollutants can be expected for microplastics being in the environment for a longer time [3
], and allows estimation of the contaminant distribution within the experimental setup without additional kinetic studies. In marine systems, the majority of microplastics are expected to be in the environment for 2–4 years at, or close to sorption equilibrium. Rivers were shown to act as an emission compartment of microplastics ending up in the oceans [13
]. Being close to emission sources (e.g., wastewater treatment plants), microplastics in freshwater systems might be more abundant than in the oceans. Shorter residence times of microplastics in freshwater systems could lead to a smaller proportion of microplastics at, or close to sorption equilibrium. Time to reach equilibrium depends on properties of the microplastic material, the pollutant, and characteristics of the water [3
]. Sorption capacity is influenced by the properties of the microplastic material itself (e.g., size, polymer type, shape) and of the pollutants (e.g., physicochemical characteristics, hydrophobicity [24
]). Fast sorption is expected for pollutants like hydrophobic organic pollutants (HOC) and for small microplastics [3
]. Sorption equilibrium within 48 h for BPA to PA particles is relatively fast. The equilibrium partition coefficient indicates similar sorption characteristics, like sorption of phenanthrene to polyvinylchloride in seawater with equilibrium sorption within 24 h [26
]. Log KPA,w
of 3.22 corresponds to a log Kow
of BPA of 3.4 measured in an earlier study [57
], indicating that hydrophilicity of BPA is a good estimate for sorption capacity of BPA to PA particles. The partition coefficient (103
) is within the range of HOC sorption to microplastics in seawater (102
]). Competitive sorption by other pollutants and leaching additives influence sorption behavior as well, but were not analyzed in this study [59
4.2. Exposure Experiments with Mixtures of BPA and PA Particles
Besides sorption behavior, also organism dependent factors need to be considered for the potential vector effect of microplastics, i.e., uptake of microplastics, as well as conditions and processes within the organism. Two possible uptake pathways for BPA were included in the experiments: direct uptake by BPA dissolved in water, and vector-based uptake by ingestion of PA particles loaded with BPA. Microplastics tested in most studies were the only uptake pathway for the pollutants [34
]. Other media, e.g., water, prey and detritus, which were shown to also hold a fraction of the pollutants, have not been included [3
]. Since daphnids are organisms living in the water column, an important uptake pathway of nutrients, but also pollutants, is water. This is why water was selected as an additional uptake pathway for BPA in the simplified exposure scenario of this study. Non-suspended microplastics (e.g., aggregated at the water surface or settled) were discussed to reduce interactions of test organisms, leading to reduced effects of microplastic associated pollutants [54
]. In this study, grazing by daphnids on settled PA particles from the bottom of the test beakers was observed. We consider high ingestion rates of microplastics by the daphnids, because intestines were observed to be filled up with PA particles within the first 24 h until the end of the test. Grazing with high uptake rates ensured availability of PA particles, which could then potentially act as vector for BPA. Daphnids were also able to egest PA particles. Quantification of exact ingestion and egestion rates was beyond the focus of this study, but would allow getting a deeper understanding of the processes within the daphnids. The scope of furthermore refined studies could be to analyze, e.g., if the time span of the microplastic particles within the daphnids is related to sorption and desorption kinetics.
Immobilization of daphnids was analyzed as an experimental endpoint to directly determine the influence of microplastics on pollutant toxicity. The pairwise comparison of immobilization rates with the same nominal concentration of BPA directly compares scenarios with the same overall mass of BPA without taking the distribution of BPA into account. The increase of immobilization rates in a dose-dependent manner for nominal concentrations follows the known acute toxicity pattern for BPA. The same dose-dependent pattern for BPA, in combination with microplastics but with overall lower immobilization rates, shows that the presence of PA particles reduced immobilization in daphnids.
Water seems to be the most bioavailable fraction, as hypothesized. Analytical measurements showed that decreased immobilization for BPA in combination with microplastics was associated with lower BPA concentrations in water compared to BPA alone. Sorption of BPA to PA particles led to lower actual concentrations of BPA in water, already during preparation of test solutions. Sorption of PCB to microplastics (PE, 10–180 μm) with dilution of PCB in water was shown to compensate possible vector effects, contributing to bioaccumulation of PCB in lugworms [38
]. That sorption of phenanthrene to microplastics (unplasticized polyvinyl chloride, 200–250 μm) can lead to reduced effect rates of the compound was shown by biomarker activity in zebra fish larvae [60
]. Vector-based uptake by ingestion was excluded, because larvae did not develop mouthparts yet. Larvae interacted with microplastics only by dermal contact. Even though PA particles loaded with a fraction of the pollutant were ingested by the daphnids, this vector-based uptake of the pollutant does not seem to compensate reduced uptake from water. The overall effect of the pollutant seems to depend mainly on the amount of dissolved pollutant in water, if sorption equilibrium is assumed.
While comparing the same nominal concentrations of BPA clearly showed a reduction of immobilization in the presence of PA particles, it cannot be excluded that a fraction of BPA causing immobilization was associated to PA particles. How much the single fractions (water, microplastics) contributed to the overall effect of BPA can be addressed by comparing EC50 values. EC50 values were calculated with immobilization rates and actual concentrations of BPA measured by HPLC in water. If only BPA dissolved in water is determining the rate of immobilization, EC50 values of BPA alone, and BPA in combination with microplastics, can be expected to be in the same range. A lower EC50 for BPA in combination with microplastics would indicate higher sensitivity of daphnids to BPA if PA particles are present. Although the EC50 for BPA in combination with microplastics is lower than for BPA alone, the overlapping confidence intervals indicate no significant difference. Thus, we consider the contribution of PA particles as a source for BPA to the overall immobilization rate to be negligibly small.
Tissue concentrations of pollutants were measured in most studies to analyze the potential vector effect of microplastics. While we did not measure internal conditions, including tissue concentrations, calculations based on physicochemistry give an indication for the overall uptake of BPA. Calculations on the mass distribution of BPA indicate that not only less BPA is partitioning into water, but also, less BPA is distributed into the daphnids. Lower calculated body burden with BPA corresponds to observed lower immobilization rates. In adult zebra fish tissue, concentrations of silver (Ag) were reduced, if Ag could sorb to microplastics during 96 h incubation prior to exposure [54
]. In another study, whole body concentrations of phenanthrene in daphnids were not different between treatments with microplastics or phenanthrene alone [22
]. Only nanoplastics enhanced phenanthrene uptake in this study, which stresses the bigger vector potential of plastics below micro scale.
Different factors have been discussed to influence desorption of pollutants within organisms between ingestion and egestion of microplastics, i.e., pre-exposure with pollutants, biological conditions, and processes. The concentration gradient in this study was not influenced by pre-experimental BPA burden in daphnids, because the daphnids have not been exposed to BPA before exposure. When organisms have already accumulated pollutants in their body, remobilization of the pollutant from microplastics can be expected to be lower because of smaller concentration gradients. Ingestion of relatively clean microplastics is discussed to reduce pollutant burden in organisms, if partition coefficients are higher for the plastic material [61
]. Three week exposure of lobsters with microplastics loaded with PCBs and incorporated in food had no effect on PCB concentrations in tail tissue [43
]. The lobsters had been pre-exposed to PCBs prior to experiments in the environment, which resulted in a smaller concentration gradient compared to clean organisms. After a depuration phase of one week with ingestion of clean microplastics, PCB concentrations were the same, indicating no cleaning effect of microplastics.
Based on experimental evidence, physiological conditions in the gut, like gut surfactants, pH, and temperature, were discussed to enhance remobilization of absorbed pollutants on microplastics [25
]. Faster desorption rates were found only for warm-blooded organisms. Model-based studies on marine organisms hypothesize that desorption of organic pollutants from microplastics is negligible, even if physiological factors are included [37
]. Interactions between organismal tissue and ingested microplastics loaded with pollutants also depend on gut passage time. Higher remobilization rates of pollutants from microplastics can be expected for longer gut passage times. Gut passage time in daphnids for food particles is relatively short, with egestion within minutes [62
]. Thus, remobilization from loaded microplastics which pass through the digestive system might be limited. Smaller microplastics which are able to pass tissue or even cell barriers, might be of more importance for acting as vectors, while bigger microplastics can be egested more easily [22
]. In addition to microplastics in the intestinal tract of daphnids, translocation of PA particles within the body, like observed for 1 μm microplastics [21
], cannot be excluded.
Besides tissue concentration, also the location of a pollutant within the body and depuration was shown to be influenced by microplastics. A bigger proportion of Ag was found to be located in the intestines in the presence of microplastics in zebra fish, although overall Ag concentration was lower compared to exposure without microplastics [54
]. Gut content was not separated from organismal tissue for analysis. This is why the higher proportion could be due to microplastics still carrying Ag, rather than higher concentrations of Ag in organismal tissue.
Similar to the results of this study, the role of microplastics as vector seemed small as soon as other uptake pathways than microplastics were included in recent studies. In a sediment-living marine worm, PCB uptake from microplastics was lower than from sediment [41
]. Gut solubilization potential was relatively low compared to natural material, i.e., wood and biochar, indicating the limited role of microplastics in pollutant transfer. In marine mussels, a mixture of fluoranthene (Flu), microplastics (PS, mix of 2 and 6 μm), and food algae did not change the concentration of Flu in digestive glands after seven days, compared to Flu and algae without microplastics [42
]. By incubating Flu with microplastics and food algae prior to exposure, different uptake pathways were included in this study (water, microplastics, algae). A fraction of Flu which was held by algae was transferred to microplastics during incubation, due to the higher partition coefficient. During a seven day period without any exposure, depuration was lower if mussels had been receiving mixtures, including microplastics, beforehand. Negative effects on detoxification and impairment of the filter activity were discussed as reasons. Also, remaining microplastics loaded with Flu could not be excluded. While concentrations of a pollutant within the organism indicate uptake associated to microplastics, it is necessary to also analyze specific effects of the pollutant on the organism. Even if pollutants desorb from microplastics, a negative effect only manifests if the pollutant reaches the target tissue. Tissue concentrations of a pollutant, especially the whole body burden, do not necessarily reflect the extent of a net pollutant effect. Studies including effects of pollutants, e.g., toxicity, can help to identify the actual influence of microplastics on organisms. Interestingly, toxic effects of Flu on mussels were enhanced for treatments including microplastics, although concentrations of Flu in tissue were not different [42
]. More histopathlogical damage and higher activity of antioxidant markers were found.
Besides studies showing low evidence for microplastics as carriers for pollutants, there are also reports about enhanced pollutant body burden and negative effects after ingestion of loaded microplastic [32
]. Different conclusions have been made about the role of microplastics as vector, because of different outcomes of experimental studies. Experimental approaches in these studies differed a lot, but re-evaluation including equilibrium sorption showed that most studies indicate no, or only low relevance for microplastics as pollutant vector [3
]. In a recent study, model and experimental approaches were combined to analyze the vector effect of microplastics (PE) for PCB on marine lugworms [38
]. Uptake fluxes from all exposure pathways were quantified to comply with environmental relevant exposure conditions. Experimental and model approaches both go along with the general results of our study, that the role of microplastics as vector for organic pollutants is small.