Given the societal concern about the presence of nano- and microplastics in the environment, our nescience with respect to in situ effects is disturbing. Data on long-term implications under ecologically realistic conditions are particularly important for the risk assessment of nano- and microplastics. Here, we evaluate the long-term (up to 15 months) effects of five concentrations of nano- and microplastics on the natural recolonization of sediments by a macroinvertebrate community. Effects were assessed on the community composition, population sizes and species diversity. Nano- and microplastics adversely affected the abundance of macroinvertebrates after 15 months, which was caused by a reduction in the number of Naididae at the highest concentration (5% plastic per sediment dry weight). For some other taxa, smaller but still significant positive effects were found over time, altogether demonstrating that nano- and microplastics affected the community composition.

The aim of this study was to evaluate the effects of NP and MP on a benthic macroinvertebrate community located in an outdoor experimental ditch, for a long exposure time of up to 15 months. Trays containing natural sediment mixed with NP or MP at concentrations of 0, 0.005, 0.05, 0.5, and 5% plastic per sediment dry weight were embedded in the sediment of a ditch that contained a well-characterized donor community. This community is typical for standing water systems such as ditches, canals, ponds, and lakes. Deposition and accumulation of NP and MP may occur in such systems, rendering their benthic communities to be particularly exposed to these particles. Spherical polystyrene NP with an average size of 96.3 ± 1.85 nm and irregular polystyrene MP fragments with sizes ranging from 20 to 516 μm were used for the NP and MP treatments, respectively. Each NP and MP concentration was prepared in quadruplicate, and concentrations were selected on the basis of measured environmental concentrations in the Rhine river shore sediments, which were up to 1 g kg −1 (0.1% plastic per sediment dry weight) ( 13 ). The two lowest concentrations used in the present study (0.005 and 0.05%) can therefore be considered environmentally realistic ( 13 ). After 3 and 15 months of colonization, trays were retrieved and species were identified and counted. The contribution of plastic type, exposure time, and concentration plus the interaction of time and concentration, but also by block (spatial variation) and the interaction of block with type of the plastic particles, were evaluated for the effect on abundance of benthic macroinvertebrates, number of taxa, Shannon diversity index (H), and the number of individuals of 21 taxa for both NP and MP treatments separately. We provide long-term community effect thresholds for freshwater benthic macroinvertebrates and compare them with environmental concentrations measured in freshwater sediments.

The ability of freshwater benthic macroinvertebrates to ingest MP depends on species characteristics such as their feeding habit or developmental stage ( 5 , 6 ), as well as on plastic particle properties and environmental conditions ( 7 ). Single-species laboratory studies have found that the ingestion of MP by freshwater benthic macroinvertebrates can cause adverse effects ( 8 – 10 ), which also seems to differ among species. For instance, a reduction in the growth of the amphipod Gammarus pulex was found after a 28-day exposure to polystyrene MP, while five other benthic macroinvertebrates were not affected under the same experimental conditions ( 6 ). Over time, these differences in sensitivity to MP particles may lead to changes in the community structure, triggering disproportionate responses ( 11 ). For instance, reductions in the abundance of shredders, such as the amphipod G. pulex, have shown to affect detritus processing ( 12 ). Consequently, changes in benthic community structure can have negative consequences for the functioning of ecosystems ( 12 ). However, single-species laboratory tests cannot offer the ecological realism required to detect such ecological implications. After all, they lack the ecological processes that drive community change in the long term, such as community interactions, temperature and light variations, flow dynamics, seasonality, aging, and reproduction. Therefore, the effects of MP should be evaluated under field conditions and for much longer time periods to take all these processes into account.

Nanoplastics (NP), with a size smaller than 0.1 μm, and microplastics (MP), with a size between 0.1 μm and 5 mm, comprise the smallest particle fraction of plastic debris globally ( 1 ). Although the accumulation of NP and MP is currently a major concern ( 1 ), studies addressing their effects on single species are scarce, and nothing is known about their long-term effects at the community level ( 1 – 3 ). Freshwaters are particularly affected as sediments are known to accumulate NP and MP due to the vicinity of sources and due to aggregation and biofouling processes and subsequent settling, which create hot spot areas that might pose a risk for benthic organisms ( 4 ).

The overall effects of NP on the abundance of macroinvertebrates, the number of taxa, the Shannon diversity index (H), and the abundance of Naididae did not differ significantly from those for MP. However, when comparing the means between the two plastic types per concentration-time combination in one hypothesis test, a significant difference was found for Valvata (GLM; NP betweenplastictypes , P = 0.03) and G. albus (GLM; NP betweenplastictypes , P = 0.006). For Orthocladiinae and H. complanatus, the difference in effects between plastic types had P values of 0.08 and 0.05.

Besides Naididae, NP concentration had a significant positive effect on the number of Valvata over time (GLM; NP conc , P = 0.02) (fig. S2). Tukey multiple comparisons test showed, however, no significant differences among NP concentrations per time point. NP also had a significant positive effect on the number of Orthocladiinae (GLM; NP conc , P = 0.02) (fig. S3). As for Valvata, no significant differences among concentrations were found per time point. MP had a significant positive effect on the number of individuals of Hippeutis complanatus (GLM; MP conc , P = 0.03) and Gyraulus albus (GLM; MP conc , P = 0.002) (figs. S4 and S5). Again, no significant differences among NP or MP concentrations were found per time point. For the 16 other taxa analyzed, no effects of NP or MP concentrations were found.

When categorizing the number of benthic macroinvertebrates found in trays by class (fig. S1), it appears that this reduction in macroinvertebrate abundance at the highest NP and MP concentrations is mainly caused by the class Clitellata, which mostly consisted of Naididae worms (tables S1 and S2). Again, here, both NP and MP concentrations had a significant negative effect on the abundance of this family of worms (GLM; NP conc , P = 0.008; MP conc , P = 0.008) ( Fig. 4 ). Just like for the macroinvertebrate abundance, the number of Naididae did not differ among concentrations after 3 months of exposure for both NP and MP. After 15 months, the number of Naididae at the highest NP concentration (5%) was significantly lower than at the second highest concentration (0.5%) and the lowest concentration (0.005%) (Tukey; NP15 5/0.5 , P = 0.04; NP15 5/0.005 , P = 0.001). After 15 months, the number of Naididae at the highest MP concentration (5%) was significantly lower than at the second highest concentration (0.5%) (Tukey; MP15 5/0.5 , P = 0.01).

NP and MP concentrations had significant negative effects on the total abundance of macroinvertebrates, which is the sum of all individuals of all taxa found in trays [GLM (Generalized Linear Models); NP conc , P = 0.04; MP conc , P = 0.03] ( Fig. 1 ). Multiple comparison analysis performed for each time point revealed no significant differences among concentrations after 3 months of exposure for both NP and MP. After 15 months, however, the abundance of macroinvertebrates at the highest NP concentration (5%) was significantly lower than at the second highest concentration (0.5%) and the lowest concentration (0.005%) (Tukey; NP15 5/0.5 , P = 0.03; NP15 5/0.005 , P = 0.002). After 15 months, the abundance of macroinvertebrates at the highest MP concentration (5%) was significantly lower than the second highest MP concentration (0.5%) (Tukey; MP15 5/0.5 , P = 0.02). In contrast to these results, NP and MP concentrations did not affect the number of taxa ( Fig. 2 ) (GLM; NP conc , P = 0.34; MP conc , P = 0.31) nor the Shannon diversity index (H) (GLM; NP conc , P = 0.56; MP conc , P = 0.57) ( Fig. 3 ).

DISCUSSION

After 15 months, the total abundance of macroinvertebrates, the number of taxa, and the number of Naididae worms were significantly higher than those after 3 months for both NP and MP treatments, confirming the colonization of the trays over time as intended. In contrast, the Shannon diversity index (H) significantly decreased over time for both NP and MP treatments, probably due to a higher abundance of the family Naididae, which dominated all trays except for the ones with the highest NP and MP concentration (5%). A higher diversity at the highest NP and MP concentration (5%) can be observed (Fig. 3), although effects of NP and MP on the Shannon diversity index (H) were not statistically significant. It is possible that a decrease in the abundance of only one taxon, i.e., the Naididae, might not have been sufficient in this period of time to obtain statistically significant effects on the Shannon diversity index (H), given that all other species affect the index as well. It cannot be ruled out that effects on diversity would become significant after a prolonged exposure. The spatial variation (block) had a significant influence on the total abundance of macroinvertebrates, the number of Naididae, and the Shannon diversity index (H), revealing that the distribution of the organisms along the ditch was not entirely homogeneous. This, however, is considered part of the targeted ecological realism of the experimental design.

Despite the influences of time and spatial variation (block) on the total abundance of macroinvertebrates and the abundance of Naididae worms, effects of NP and MP particles were detectable. Community effects for other inert particles, such as activated carbon and multiwalled carbon nanotubes, have been previously detected using a similar setup (14, 15). For instance, a lower abundance of Lumbriculidae worms and Pisidiidae clams was found after 15 months of exposure to activated carbon via natural sediment (14). To the best of our knowledge, this is the first time that effects of NP and MP are demonstrated in a setting with such a high level of natural ecological variability (i.e., diurnal and seasonal variation and spatial variation) and for an exposure time longer than 3 months. To our knowledge, community effects have only been reported for MP in one earlier study, which exposed a marine benthic community to polylactic acid (80 μg/liter) and high-density polyethylene MP for 3 months using outdoor mesocosms (16). MP affected the abundance of periwinkles Littorina sp. and isopods Idotea balthica and the biomass of the clam Scrobicularia plana and the lugworm Arenicola marina (16). In the present study, differences were observed over time, especially for the Naididae worms, where the abundance increase allowed distinguishing differences among treatments. The number of Naididae increased by a factor of 13 (from 37 to 466) and 70 (from 8 to 531) in the NP and MP controls, respectively, while it only increased by a factor of 2 (from 90 to 160) and 30 (from 9 to 279) at the highest concentration in a period of 1 year. For the other taxa affected by the exposure to NP or MP, differences between 3 and 15 months were much smaller, and their abundance was always below 40 individuals per tray, which makes the conclusions less evident than for Naididae.

The detected community effects of NP and MP could affect ecosystem functions. For instance, the burrowing activity of worms causes mixing of particles and chemicals in the sediment top layer, facilitates the oxidation of organic matter, and reduces minerals in the sediment, thereby mobilizing nutrients and sulfide-bound heavy metals from the sediment back to the water layer (17, 18). In addition, worms are an easy and nutritious prey for fish and other benthic invertebrates in the system (17). This implies that these functions could be impaired due to the reduction in the abundance of Naididae worms observed here.

It has been hypothesized that for NP, different and probably more severe effects may be anticipated than for MP, due to a higher chance of translocation, systemic uptake, and subsequent particle toxicity effects (19, 20). For MP, mainly physical effect modes of action have been suggested (1, 7). The effects of NP on the abundance of macroinvertebrates, the number of taxa, the Shannon diversity index (H), and the abundance of Naididae did not differ significantly from those for MP. The similarity observed here relates to the effect thresholds and to the identity of the primarily affected species, i.e., worms. We have no conclusive explanation for this similarity; however, plausible explanations can be provided. For instance, upon aging, biofouling, encapsulation, and aggregation of the smallest particles in the sediment (21, 22), they could lose behaviors that specifically relate to the submicrometer scale, rendering them more similar to larger MP particles. Formation of hetero-aggregates between the NP and sediment particles could strongly reduce differences in bioavailability, uptake, and particle-specific effects, such that only the general effect of loss of habitat quality due to dilution of food remains. Accordingly, the simultaneous presence of natural particles is essential when evaluating the effects of NP and MP on benthic macroinvertebrates (6, 7, 23).

As mentioned, this study was designed to detect community-level impacts, and therefore, we are not able to demonstrate the exact mechanism that caused the lower abundance of Naididae worms. MP ingestion has been previously demonstrated for Tubifex worms, which belong to the family Naididae (24). In a study by Hurley et al. (24), Tubifex worms were able to ingest MP fragments with a size between 50 and 4500 μm contained in natural sediment and were found to retain MP for longer time periods than other sediment components. A reduction in food intake due to the dilution of organic matter in the sediment, together with the uptake and longer retention of MP by the Naididae worms, could have caused a depletion of energy reserves over time, as previously found in laboratory tests for other benthic invertebrates (6, 9, 25). For these worms, this energy depletion might have taken longer than for other benthic invertebrates, as the exposure of Tubifex worms to the same polystyrene MP fragments used in the present study using standard chronic laboratory bioassays did not cause any effects on their survival, growth, nor feeding activity (6). Therefore, exposure time seems to be an important factor to take into account when evaluating the ecologically relevant effects of MP. Standard laboratory tests might not be sufficient to detect NP and MP effects for all organisms. When it comes to NP, filter feeders were found to be able to ingest NP particles alone or as aggregates with natural particles (26). Aggregates were more likely to be ingested than NP alone, leading to a reduction in species feeding activity.