**1. Introduction**

The benefits related to the specific properties of engineered nanomaterials (ENMs) have led to an increase in their use and production, raising particular attention to their environmental behavior and toxicological impact. The wide disparity in estimates of industrial production of ENMs demonstrates an important uncertainty in the quantification of ENM discharges into the environment [1–3]. Nevertheless, early studies have predicted that both terrestrial and aquatic environments are likely to be exposed to ENMs [1,4]. In the case of European surface waters, modeled concentrations of nanoparticles (NPs) were found to exceed 0.18 ng.L−<sup>1</sup> and 150 ng.L−<sup>1</sup> for Ag and ZnO NPs, respectively, in the 10% most exposed rivers, likely corresponding to rivers located near large cities and downstream river systems [5]. ENMs can be transformed before entering the environment,

**Citation:** Stetten, L.; Mackevica, A.; Tepe, N.; Hofmann, T.; von der Kammer, F. Towards Standardization for Determining Dissolution Kinetics of Nanomaterials in Natural Aquatic Environments: Continuous Flow Dissolution of Ag Nanoparticles. *Nanomaterials* **2022**, *12*, 519. https://doi.org/10.3390/ nano12030519

Academic Editor: Vivian Hsiu-Chuan Liao

Received: 19 December 2021 Accepted: 1 February 2022 Published: 2 February 2022

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for instance if they are released from wastewaters treatment plants [6]. Moreover, the discharge of pristine ENMs in natural environments should also be considered, since ENMs can be released through accidental events, weathering, or directly from the use of consumer products [7–10].

### *1.1. Dissolution of Nanoparticles in Aquatic Environments*

Dissolution is emphasized as one of the most important mechanisms governing the fate of ENMs in aquatic environments. The oxidative dissolution of Ag NPs has been largely studied in aquatic media [11–18]. For example, Li and Lenhart, (2012) [18] showed that the dissolution of Ag NPs and the release of Ag+ is significantly limited in natural river waters. It has been argued that aggregation of Ag NPs may hamper dissolution due to a substantial decrease in the specific surface area [19]. However, several studies found that dissolution of Ag NPs is dependent on the particle size rather than aggregation [14,20]. For regulatory testing of ENMs dissolution rates, this information is important since up to now it was seen as a requirement to bring ENMs into a stable dispersion for dissolution testing to avoid effects of agglomeration. Extrinsic parameters such as pH, ionic strength, inorganic/organic ligands, and temperature play also an important role. Gondikas et al. [21] demonstrated that dissolution of citrate- and PVP-capped Ag NPs increases in the presence of cysteine. In contrast, several studies showed that Ag NP dissolution is inhibited in the presence of humic and fulvic acids or in the presence of natural organic matter (NOM) with high sulfur and nitrogen content [17,22]. This points to a more complex process of ENMs dissolution than only the complexation of the released cations by NOM, lowering the free ion activity and thereby enhancing the apparent solubility and dissolution rate. Finally, pH is also an important factor controlling the dissolution of Ag NPs, being enhanced at low pH values [23–25].

For soluble ENMs such as Ag NPs, it has been established that the aqueous forms (e.g., dissolved ions or aqueous complexes) are to a large extent causing the toxic effects exerted by these ENMs [26–29]. Aqueous solubility and dissolution kinetics are thus essential for (eco-)toxicological hazard testing and risk assessment strategies and are a requested information for registration of ENMs at regulatory bodies such as the European Chemicals Agency (ECHA) [30]. Determining the dissolution rates of ENMs is particularly important in determining risk/hazard since the rate of release of toxic ions prior to interaction with ligands may be more important than equilibrium concentrations, and the lifetime of the particulate form will determine the exposure to this ENM. However, high degree of uncertainty remains when it comes to understanding dissolution kinetics of ENMs in aquatic environments, mostly due to the lack of studies using aqueous media representing the heterogeneity of the world's aquatic environments, and adapted experimental setups.

### *1.2. Experimental Methods to Study the Dissolution of Nanoparticles in Aquatic Environments*

When it comes to understanding the fate of ENMs in aquatic environments, a limiting factor is the lack of standardized experimental procedures to study the specific endpoints (solubility, dissolution rate, chemical transformation, hetero-agglomeration). Standardized procedures would allow easier comparison between different ENMs and forms of the same ENM, enable grouping and read-across between nanoforms, as well as the production of more reliable data that can be implemented in geochemical models. International regulatory bodies, such as ECHA, the Organization for Economic Cooperation and Development (OECD) and the International Organization for Standardization (ISO), have argued that these points should be addressed [31]. To date, several methods have been applied for dissolution testing of ENMs [16]. However, there is no standardized OECD guideline for dissolution testing of ENMs other than the guidance in OECD GD 318 [32,33]. The OECD TG 105 [34] has been evaluated as not applicable for ENMs [35] and OECD GD 29 has been identified as being in principle applicable with some adjustments for determining solubility [36,37]. Nanomaterial-specific OECD test guidelines for dissolution rate testing

in environmental media would thus be beneficial for generating data that are regulatory relevant and reliable [38].

Generally, dissolution-testing methods can be divided into static batch and continuous flow/flow-through approaches. Batch testing leads to a direct measure of the ENM solubility in a specific aqueous medium. Moreover, when using ultra-trace analytical techniques, it is possible to measure very low ion concentrations. However, it has several limitations, especially when dealing with particles that are rapidly dissolving. For instance, the ultra-centrifugation or centrifugal ultrafiltration of rapidly dissolving particles can lead to an overestimation of the dissolution, because of the dissolution of the particles during the centrifugation process. To the opposite, the dissolved fraction could be underestimated using filtration techniques if the dissolved ions are adsorbed to the ultrafiltration membrane or the walls of the filtration device. Furthermore, batch experiments are performed under static conditions (i.e., limited supply of exposure medium) and may lead to a change of the medium composition, due to ENMs dissolution and/or the re-precipitation of secondary species. The challenges associated with the batch systems can be overcome by using a flow-through/continuous flow setup. The continuous flow dissolution systems are considered to be more appropriate for dissolution rate measurements and present a more environmentally realistic system. It allows for working with high liquid-to-solid ratios (realistic high dilution of the ENM), and when using appropriate analytical instrumentation, it allows for the detection of low levels of dissolved species. Because of a constant and almost unlimited provision of the medium, the back reactions are limited and low electrolyte concentrations in the exposure medium can be used to mimic the chemical composition of natural waters. These results can finally serve as inputs into environmental fate modeling. The flow-through setup has been described in ISO TR 19057 [39]. For instance, Koltermann-Juelly et al. [40] studied the dissolution rates of 24 types of nanomaterials in phagolysosomal simulant fluids, and Bove et al. [31] investigated dissolution of Ag NPs in an in vitro assay simulating conditions which likely occur in human digestion. More recently, Keller et al. [41] applied continuous flow systems to study the dissolution of BaSO4 in phagolysosomal and lung lining fluids. These studies demonstrated the advantage of using continuous flow rather than static incubations to investigate and compare the dissolution rates of ENMs. Nevertheless, when adapted to simulate ENMs dissolution in specific biological media, these studies applied relatively low flow rates and high loadings of ENMs, which resemble elevated solid-to-liquid ratios and would not be translatable to environmentally relevant conditions.

Here, we aim to provide guidance and recommendations for nanomaterials dissolution testing and dissolution rates determination under environmentally relevant conditions. Based on a survey of surface water hydrochemistry, we outline the environmental concentration ranges of key parameters to be considered in studying ENMs dissolution in natural aquatic environments. We present the proof of concept of a continuous flow setup suitable to investigate dissolution kinetics at low ENMs concentrations, relevant for natural environments. The results obtained on Ag NPs exposed to artificial waters demonstrate the suitability of the continuous flow dissolution method to determine dissolution rates of ENMs under non-equilibrium environmentally relevant conditions. This work will aid in the standardization of a continuous flow dissolution approach adapted to study ENMs dissolution kinetics in natural aquatic environments, and supports the ongoing development towards OECD dissolution guidelines.
