**1. Introduction**

Polycyclic aromatic hydrocarbons (PAHs) are a group of over 100 different organic compounds generated by natural events or anthropogenic activities. PAHs predominantly originate from anthropogenic processes, especially from incomplete combustions of organic fuels. Certain naturally occurring processes, such as volcanic eruptions and forest fires, contribute to the increase of these organic compounds in the environment. In Santos Bay and Estuary, the anthropogenic contributions to PAHs in sediments resulted of about 99% (i.e., concentrations varied from 79.6 for uninhabited area to 15,389.1 ng/g for area located in the proximity of industries [1]).

PAHs are formed by two or more fused benzene rings, and their toxicity depends on the number of benzene rings [2–4]. Because of their low water solubility and hydrophobicity, in the water column, PAHs tend to associate with suspended particulate matter and are

**Citation:** Albarano, L.; Serafini, S.; Toscanesi, M.; Trifuoggi, M.; Zupo, V.; Costantini, M.; Vignati, D.A.L.; Guida, M.; Libralato, G. Genotoxicity Set Up in *Artemia franciscana* Nauplii and Adults Exposed to Phenanthrene, Naphthalene, Fluoranthene, and Benzo(k)fluoranthene. *Water* **2022**, *14*, 1594. https://doi.org/ 10.3390/w14101594

Academic Editors: François Gagné, Stefano Magni and Valerio Matozzo

Received: 2 April 2022 Accepted: 13 May 2022 Published: 16 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

eventually deposited in sediments, where their degradation is very slow [5]. The level of PAHs in the water column is closely linked to the level of PAHs in sediments [6,7]. In fact, PAHs concentration in the water column increase with increased concentration in surface sediment [6,7]. Although they are not very soluble in water, their concentration in the water column remains stable for a long time, thus representing a great problem for biota and consequently for human health [8–10]. Specifically, the half-lives of lowmolecular-weight PAHs (naphthalene, acenaphthene, fluorene, and phenanthrene) ranged from approximately 3 to 8 days, whereas half-lives of high-molecular-weight PAHs (pyrene, chrysene, benzo[a]pyrene, dibenz[a,h]anthracene) ranged from 73 to 1780 days [11–13].

For these reasons, the European Water Framework Directive 2000/60/EC (WFD) was developed aiming to achieve and ensure good ecological and chemical water status [14]. The list of monitored pollutants has recently been updated with a new daughter Directive (2013/39/EU) to identify a number of emerging chemicals of concern, including non-polar organic substances (e.g., PAHs and PCB) and polar compounds (e.g., pharmaceuticals and pesticides) [15].

In aquatic environments, PAHs can have several toxic effects, such as immunotoxicity, embriotoxicity, and cardiotoxicity, especially impacting fish, benthic organisms, and other marine vertebrates [16–19]. Five different PAHs (naphthalene (NAP), phenanthrene (PHE), fluoranthene (FLT), fluorene (FLR), pyrene (PYR), and hydroxypyrene), known to be potentially toxic, inhibited and reduced the larval development and growth of both *Mytilus galloprovincialis* and *Paracentrotus lividus*, whereas NAP was able to impact the embryos and larval stages of *Ciona intestinalis* [16]. The benzo(a)anthracene (BaA), one of the most toxic PAHs, showed higher toxicity on crustaceans *Daphnia magna* (LC50 = 4.3 μg/L) and *Ceriodaphnia reticulata* (LC50 = 4.7 μg/L) than that displayed *Artemia salina* at conceivable concentrations in the environment (from 1 to 32 μg/L) [20,21]. At the same manner, PHE and FLT were able to impact the survival of *D. magna* (LC50 = 50 and 10 mg/kg, respectively), *Hyalella azteca* (LC50 = 15 and 5 mg/kg, respectively), and *Chiromonus riparius*(LC50 = 20 and 15 mg/kg, respectively) [22]. After PYR, FLT, and anthracene (ANT) exposure, *D. magna* and *Artemia salina* crustaceans displayed higher sensibility than those registered for the mosquito *Aedes aegypti*, the amphibian *Rana pipiens,* and the fish *Pimephales promelas* [23].

To the best of our knowledge, few studies have been carried out so far to investigate direct toxic effects of individual PAHs on *A. franciscana*, but no work has been conducted to establish the possible changes in expression levels of genes after organic compounds exposure. Rojo-Nieto et al. [24] established that mixtures of ten PAHs (naphthalene, acenaphthene, phenanthrene, fluoranthene, fluorene, pyrene, anthracene, benzo(a)pyrene, benzo(a)anthracene, and chrysene) found in sediment samples from the Bay of Algeciras did not have impact on survival of *A. franciscana* using passive dosing. Similarly, the passive dosing of three PAHs (toluene, 1-methylnaphthalene, and phenanthrene) did not impact the hutching of cysts [25].

The crustacean *A. franciscana* has been considered as a model species to investigate the ecotoxicological response of marine invertebrates to environmental pollutants [26–28]. The main advantage of this species is that nauplii can be hatched as needed from commercially available durable cysts to avoid the maintenance of laboratory cultures as required for many model species used in ecotoxicity tests. In any case, these tests (namely "Toxkit") employing dormant stages ("cryptobiotic eggs") have the same efficacy and sensitivity as tests with cultured animals [29]. Moreover, the embryo hatches and grows rapidly in laboratory conditions (the nauplius stage is reached in 24 h), and the small body size permits to conduct tests in small beakers or even plates. In addition, *Artemia* is a eurialine organism with large adaptability to a range of salinities (5–300 PSU) and temperatures (6–40 ◦C) [30]. However, *Artemia* models revealed several disadvantages due to a limited sensitivity towards a wide range of substances in comparison to other species so that the possibility to underestimate potential effects may occur [26,31]. In fact, in the recent years, the use of this crustacean in ecotoxicology has become increasingly rare [31]. For these reasons, in this work, we are interested in giving a new life to this model organism by proposing

genotoxicity as a new endpoint. Since changes of gene expression induced by some toxicants may be very subtle and differences of animal reactivity between experimental groups may not be noticed by simple observations, the genotoxicity could be considered a good approach providing more detailed toxicological information. Therefore, the use of *A. franciscana* for evaluating the molecular aspects that are on the base of toxicological effects could confirm this branchiopod crustacean as a good biological model.

Thus far, few studies investigated the stress response of *Artemia* spp. through the evaluation of key genes involved in larval growth, molting, stress, and detoxification processes [32–37]. In this work, as well as evaluating PHE, NAP, FLT, and BkF acute (24 h–48 h-LC50) toxicity on nauplii and adults by measuring survival, we defined for the first time the molecular response of PAHs toxicity. In particular, after 48 h under sublethal exposure for both tested life stages, the effect on several key genes involved in stress response (*hsp26*, *hsp60*, *hsp70*, *COXI,* and *COXIII*) was assessed. In addition, the impact on developmental genes (*HAD-like*, *tcp*, *UCP2,* and *CDC48*) was also evaluated for nauplii.

Sediment can be the final main sink and source of PAHs and genotoxicity can represent an easy and fast screening method for their ranking [38–40]. Prior to direct PAHs contaminated sediment investigation, we decided to highlight the sensitivity of genotoxicity endpoint in *A. franciscana* from spiked saltwater solutions.

In this study, we tested the PHE, NAP, FLT, and benzo(k)fluoranthene (BkF) toxicity on embryos and adults of the branchiopod crustacean *A. franciscana* Kellog 1906, using environmental concentrations (from 0.025 to 10 mg/L, from 0.36 to 2.3 × 102 mg/L, from 0.41 to 3.9 × 102 mg/L, and from 0.025 to 9.4 × 101 mg/L for NAP, PHE, FLT, and BkF, respectively) detected in polluted sediments subjected to various pollution sources [38].
