**1. Introduction**

Plastics comprise a wide range of synthetic or semi-synthetic organic compounds, usually polymers with a high molecular mass [1]. For many decades, their production and use has continued to increase worldwide, and many studies reported their increasing presence in natural environments [2–4]. Discarded plastic wastes can directly or indirectly enter the environment, and degrade due to solar radiation, mechanical forces, and the biological activity of organisms to smaller-sized pieces, including micro- (MPs, particles smaller than 5 mm) and nanoplastics (NPs, particles smaller than 1 μm, e.g., [5]; or smaller than 0.1 μm, [6]). In addition, primary nanoparticles originating from engineered materials (e.g., personal health care products) can be found. Among the MPs and NPs found in marine and freshwater environments, polystyrene (PS) is one of the most common types [7]. NPs pollution is an issue of special concern because of its unique features that include: (i) their colloidal properties, (ii) their tendency to aggregate, (iii) their high surface area to the volume ratio, resulting in a high ability to absorb and release chemicals, and (iv) the ability to penetrate cell membranes [8]. Because of their small size, adequate quantitative analytical techniques are not currently available to assess the NP concentrations in the environment [9]. While MPs' presence in marine and freshwater systems has been studied

**Citation:** Maszczyk, P.; Kiersztyn, B.; Gozzo, S.; Kowalczyk, G.; Jimenez-Lamana, J.; Szpunar, J.; Pijanowska, J.; Jines-Muñoz, C.; Zebrowski, M.L.; Babkiewicz, E. Combined Effects of Polystyrene Nanoplastics and Enrofloxacin on the Life Histories and Gut Microbiota of *Daphnia magna*. *Water* **2022**, *14*, 3403. https://doi.org/10.3390/w14213403

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 22 August 2022 Accepted: 24 October 2022 Published: 27 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for several years, NPs have been detected in the North Atlantic Gyre only recently [10]. It is assumed that NP concentrations are even 1014 times higher than those currently measured for MPs [6].

Several studies have indicated that many organisms ingest NPs or absorb them on their surfaces, e.g., in [11–13], and that because of their small sizes and colloidal properties, they may cross biological barriers [14,15], negatively affecting the organisms. The negative effects depend on the particle type, size, density, charge, and origin (primary or secondary), and this may be related to mechanical (e.g., adhering to external surfaces hindering the mobility) and chemical effects [8]. In addition, chemical harmfulness results from the presence of additives that have the potential to leach into the environment, causing damage to organisms. Among the most common additives are plasticisers, which may affect life history and morphology in *Daphnia magna* [16], flame retardants that may cause induced significant sublethal chronic toxicity to *D. magna* [17], antioxidants that may reduce the hatching rates, increase the malformation rates and decrease the length of calcified vertebrae [18], and UV stabilisers that recently have been revealed to cause potential immune dysfunction [19]. Moreover, chemical harmfulness may be due to the particles that can act as a carrier for other co-occurring pollutants, resulting in organisms' accumulation of harmful hydrophobic substances from the surrounding water [13,15].

The ability of NPs, due to their high surface area to volume ratio, to adsorb, concentrate, and act as a vector of toxic pollutants can modify the environmental impact of the latter. In fact, it has been revealed that most of the combined toxic effects are not simply additive, but rather synergistic or antagonistic [20]. On the other hand, NPs may decrease the toxicity of other pollutants by absorbing and then agglomerating them to form larger particles, reducing the ease of uptake by organisms [21–23]. Additionally, their presence may cause an enhancement of toxicity occurring from the on-surface pre-concentration ("The Trojan horse effect") [22,24]. Although many ecotoxicological studies suggest that realistic environmental concentrations of micro- and nanoplastics may not induce significant detrimental effects on marine organisms nor threaten their survival [13,25], the co-exposure to NPs and other associated contaminants/stressors could exacerbate their effects [26–28].

A significant group of such pollutants are antibiotics, which are detectable in surface waters, including rivers, lakes, and seas [13], in the ng L−<sup>1</sup> up to μg L−<sup>1</sup> range, exceeding sometimes the predicted no-effect environmental concentration [29]. Their extensive and irregular use has induced multifaceted adverse impacts in recent years, such as the propagation of multi-drug-resistant bacteria, antibiotic-resistant bacteria (ARB), and antibiotic-resistant genes (ARGs) in the aquatic environment [30,31]. Their antibacterial impacts are not strain-specific; thus, while the pathogenic bacteria are killed, some bacteria which are beneficial for organisms' health are also targeted, which may cause several adverse effects, such as an intestinal flora imbalance [32,33]. In addition, different classes of antibiotics have been shown to be toxic to organisms at different trophic levels, such as algae, bacteria, crustaceans, and fish [34,35].

Among the most widely used antibiotics are fluoroquinolones (FQs), which are broadspectrum synthetic antibiotics commonly used in human and veterinary medicine [36,37] and in agriculture and aquaculture [38]. Among FQs, enrofloxacin is used to prevent and treat a broad spectrum of gram-positive and -negative bacterial infections in livestock. Due to the spread of antibiotic resistance [39], it is listed among the compounds that can be considered to be of a high ecotoxicological concern [40]. It is usually detected in the effluents of municipal sewage plants and the related aquatic environments in the range of ng and μg L−<sup>1</sup> [41,42] or even in extreme cases in mg L−<sup>1</sup> [43]. Other examples concern their concentrations in surface waters (up to 248 ng L−<sup>1</sup> [44]), in groundwater [45], and up to 7.7 mg kg−<sup>1</sup> in sediments [46].

Some published studies have investigated the combined effect of NPs and antibiotics on cyanobacteria [47], algae [48], bivalvia [49], and fish [23,24,50,51]. However, there are no reviews yet on other aquatic organisms, including planktonic animals such as *D. magna*, a keystone species in the food webs of fishless ponds. The combined effect of the stressors

may be different for different organisms. It is important to build experimental datasets using a range of different organisms to quantify and predict the factors and mechanisms responsible for the pattern under different contexts. The endpoint of many published studies has focused mainly on the effect at the molecular level: the integrated biomarkers response, antioxidant indexes, gene expression, and histological symptoms [24,52] rather than at the organismal level, e.g., as the combined effect on the life history traits and gut microbiota.

The present work aimed to test several hypotheses concerning the single and combined effects of polystyrene NPs and enrofloxacin on the selected life history traits of *D. magna* as well as the metabolic and taxonomic diversity of the bacterial community in their intestinal tracts. **First**, the presence of each of the stressors results in decreasing *Daphnia's* body size and reproductive parameters. The effect of enrofloxacin differs in the presence and absence of NPs. **Second**, as the NPs presence increases, the enrofloxacin presence decreases the metabolic rate of the gut microbiota of *Daphnia*. **Third**, the metabolic fingerprint measured as the relative use of various carbon sources is different in the presence of each of the stressors on its own and combined. **Finally**, those stressors affect *Daphnia's* taxonomic diversity in the gut microbiota. On the whole, there is an interaction in the effect of both stressors.
