## 4 Discussion  
This is the first study we are aware of to examine the impact of tyre particle exposure on ecosystem functioning in a model estuarine ecosystem ex situ. During the duration of the experiment, exposure to sedimentary tyre particles did not lead to any significant differences in nutrient cycling or primary productivity, with limited impact on meiofaunal community structure and secondary productivity. There was a notable pattern regarding nitrate, with negative fluxes (indicating an uptake from the sediment) from mesocosms treated with tyre particles and positive fluxes (indicating nitrate release from the sediment) from control mesocosms, but this was not statistically significant, likely due to low statistical power. Microplastics decreased fluxes of nitrogen in mesocosms with oysters and infaunal assemblages [39]. Tyre particle exposure-driven alterations in _S. plana_ burrowing have been demonstrated [16], which may lead to changes in nutrient flux. It is also probable that changes to microbial communities from exposure to microplastics can result in changes in nutrient fluxes (as found by [PERSON] et al. [40]), but further research is needed to explore direct and indirect mechanisms. Whilst estuarine macrofauna have been documented to ingest tyre particles [16], the size of tyre particles used in this study ranged from 30 to 350 \(\mu\)m, and are likely too large for meiofauna to ingest; therefore, any impacts would be more likely due to exposure to leaching tyre particle chemicals or indirect effects from the macrofauna, such as changes in predation and food availability. Additionally, the burial activity of _S. plana_ is impacted by the presence of tyre particles [16], and this may directly or indirectly affect meiofaunal communities due to reduced bioturbation. Further studies are required to validate and further examine these findings. Organic (e.g., polycyclic aromatic hydrocarbons, PAHs, and benzothiazoles) and inorganic (e.g., heavy metals) chemical additives can leach out of tyre particles when they are exposed to water [41]. These leachates have been shown to be toxic to several marine invertebrates [21; 22; 23; 24] and microalgae [25; 26] yet are currently untested on meiofauna. Opportunistic meiofaunal taxa, including Nematoda and Polychaeta, have been found in increased densities in urban estuaries with high concentrations of PAHs [42]. Similarly, some meiofauna can thrive in the presence of heavy metals [43; 44]. Figure 3: nMDS plot showing the similarity of community biomass composition between treatments based on Gower’s distance, grouped by treatment type; 95% confidence ellipses for each treatment are coloured as symbols. C is control (0%), L is low (0.2%), M is medium (1%), and H is high (5%). Taxa names indicate which taxa are responsible for differences between groups. We did not observe a significant impact of tyre particle exposure on the microhypothesis and only a limited impact on the meiofauna. This may be due to the ability of meiofauna to tolerate tyre leachates, but the lack of effect on microphytobenthos, which generally appear to be highly sensitive to leachates [25], suggests there may be an alternative explanation. Indirect effects may also play an important role in masking the effects of tyre particle exposure. For example, _S. plana_ has been shown to reduce its feeding in response to tyre particle exposure [16]. If this is the case, then we would expect an increase in microphytobenthos; yet, our study revealed no such effect. Our experimental set up allowed for the constant replacement of seawater in the mesocosms, therefore making the conditions more reflective of moving bodies of water, like estuaries, where the water is constantly being replaced. Therefore, the leachate concentrations may have been lower than they would be in a static set up. Most tyre leachate studies focus on the toxicity within a closed system, whereas this study demonstrates results using continuously flowing water with environmentally relevant concentrations of tyre particles.