*3.2. Preliminary Adsorption Experiments*

Before starting the adsorption experiments, control samples (20 mg HNT/Fe3O4 or HNT, 10 mL tap water), not containing OFL, were shaken for 24 h at room temperature. Then, the supernatants were magnetically separated for the pH measurement and analyzed by UV-vis spectrophotometer and HPLC-FD to check the instrumental baseline.

A pH value of 7.7–7.8, similar to that of natural waters, was measured in all samples, thus no additional pH adjustment was performed.

The background noise level was satisfactory for the commercial HNT, HNT/Fe3O4-C, and HNT/Fe3O4-H. On the contrary, HNT/Fe3O4-SG was rinsed with EtOH in an ultrasonic bath for 10 min, centrifuged for 5 min at 4000 rpm, separated, and dried at 50 ◦C for 1.5 h. The washing step was repeated twice to obtain a good signal-to-noise ratio.

### *3.3. Isotherm and Kinetic Studies*

The behavior of the three magnetic HNT composites was evaluated through thermodynamic and kinetic experiments carried out under controlled conditions (see Section 2.3.1) and compared with the commercial HNT.

Adsorption isotherms are commonly used to describe the adsorption process in terms of maximum uptake and the relationship between the amount of adsorbed analyte (*qe*) and its concentration in solution at equilibrium ( *Ce*).

To fit the experimental data, the Langmuir and Freundlich models were considered.

As shown in Figure 7, the Langmuir model gave the best fitting of the experimental data.

Figure 7 shows that all materials were able to adsorb the antibiotic, although the maximum adsorption capacities were quite different. In detail, the highest value, 45 mg g<sup>−</sup>1, was obtained for HNT/Fe3O4-H, while the lowest value was obtained for HNT/Fe3O4-C, which was equal to 23 mg g<sup>−</sup>1. The HNT/Fe3O4-SG sample had an intermediate value of 31 mg g<sup>−</sup>1, close to the commercial HNT (30 mg g<sup>−</sup>1). This trend can be due to both the different amount of HNT present in the samples, ranging from about 30% in HNT/Fe3O4-C to more than 80% in HNT/Fe3O4-SG (see Table 2), and to the possible presence of some carbonaceous component related to the glucose added during HNT/Fe3O4-H synthesis. In fact, as reported by Tian et al. [30], the carbon/organic groups formed on the HNTs not only favor the Fe3O4 nanoparticle nucleation, but also may improve the analyte adsorption. On

the contrary, no difference in the adsorption mechanism was observed among all materials. The Langmuir model, which describes a monolayer coverage, gives the best fitting of the experimental data, as confirmed by the good correlation coefficient R<sup>2</sup> and χ2 values.

**Figure 7.** Adsorption profiles Langmuir (—) and Freundlich ( ... ) for Ofloxacin (OFL) on HNT (- black), HNT/Fe3O4-C (• red), HNT/Fe3O4-H ( blue) and HNT/Fe3O4-SG ( green) (Experimental conditions: Sorbent 20 mg, 10 mL OFL tap water solution from 25 to 200 mg <sup>L</sup>−1, RSD < 10%).

The experimental *qmax* values of HNT/Fe3O4-C, HNT/Fe3O4-H, and HNT/Fe3O4-SG were in agreemen<sup>t</sup> with the calculated ones, and fell within the OFL adsorption range reported in the literature for other clays, i.e., 3.2 mg g<sup>−</sup><sup>1</sup> on kaolinite [54], 160.8 mg g<sup>−</sup><sup>1</sup> on calcined Verde-lodo bentonite clay [55]).

The isothermal parameters calculated by dedicated software are listed in Table 4.


**Table 4.** Isotherm parameters for OFL adsorption onto HNT, HNT/Fe3O4-C, HNT/Fe3O4-H, and HNT/Fe3O4-SG.

Concerning the kinetic aspect, quantitative adsorption occurred in less than five minutes in the presence of all the magnetic composites. As shown in Figure 8, a satisfactory fitting is obtained by applying the pseudo-second-order model, thus, considering a chemisorption process. For commercial HNT, the adsorption was instantaneous, thus, it was not possible to discriminate between the two models.

**Figure 8.** Kinetic profiles (pseudo-first-order (—), pseudo-second-order ( ... )) for OFL onto HNT (- black), HNT/Fe3O4-C (• red), HNT/Fe3O4-H ( blue) and HNT/Fe3O4-SG ( green) (Experimental conditions: sorbent 20 mg, 10 mL tap water, OFL initial concentration 20 mg <sup>L</sup>−1, RSD < 10%).

The calculated kinetic parameters are shown in Table 5.

**Table 5.** Kinetic parameters for OFL adsorption onto HNT, HNT/Fe3O4-C, HNT/Fe3O4-H, and HNT/Fe3O4-SG.


3.3.1. Ofloxacin Removal from Real Waters Samples

Magnetic HNTs were also tested under environmental conditions, i.e., μg L−<sup>1</sup> OFL concentration, tap and river waters, WWTP effluent (see Table S2 for the physicochemical parameters).

An amount of 20 mg of each material was suspended in 10 mL of each water sample, river water and WWTP effluent samples spiked with 10 μg L−<sup>1</sup> OFL (*C*0) and shaken for 24 h. Then, the suspensions were magnetically separated and the supernatants were filtered on a 0.22 μm nylon syringe filter before HPLC-FD analysis to quantify the drug content (*Ce*).

The removal efficiency (*R*%) was calculated according to Equation (3):

$$R\% = \frac{\mathcal{C}\_0 - \mathcal{C}\_\varepsilon}{\mathcal{C}\_0} \times 100\tag{7}$$

where *C*0 is the initial OFL concentration and *Ce* is the OFL concentration in solution at the equilibrium.

The obtained results were reported in Figure 9.

**Figure 9.** OFL removal (%) from tap and river water samples and effluent from WWTPS with HNT, HNT/Fe3O4-C, HNT/Fe3O4-H, and HNT/Fe3O4-SG (Experimental conditions: sorbent 20 mg, 10 mL tap water, OFL initial concentration 10 μg <sup>L</sup>−1, *n* = 3, RSD < 10%).

The investigated HNT/Fe3O4 composites gained an antibiotic removal ≥90% despite different aqueous matrix constituents and other potential contaminants. The different amount of Fe3O4 in each composite did not affect the adsorption process; on the contrary, the Fe3O4 percent in HNT/Fe3O4-C, higher than in HNT/Fe3O4-H and HNT/Fe3O4-SG, favored its complete magnetic recovery from the media after the use with no additional centrifugation step.

### 3.3.2. Reusability and Post-Use Characterization of HNT/Fe3O4-C

Among the investigated magnetic HNTs, the HNT/Fe3O4-C sample ensured a quantitative OFL removal in different real water samples and excelled for its magnetic properties. For these reasons, its reusability was explored.

The HNT/Fe3O4-C sample was suspended in 10 mL tap water containing OFL 10 μg L−1. After 1 h, HNT/Fe3O4-C was magnetically separated, and the supernatant was analyzed by HPLC-FD. Then the recovered sorbent material was suspended for a second time in 10 mL tap water samples containing OFL 10 μg L−1. After 1 h contact, the suspended material was magnetically separated, and the OFL concentration in the solution was measured. A third cycle was carried out following the same procedure.

Figure 10 shows the adsorbed OFL percentage after each adsorption cycle. The adsorbed antibiotic amount slightly decreased from 95% after the first use to 75% after the third one.

This trend may be ascribed to a small loss of material during its magnetic separation from the sample solution and not to matrix interference, as XRPD analysis demonstrates.

The recovered sorbent material after three adsorption cycles was analyzed by XRPD and compared to the synthesized HNT/Fe3O4-C sample. The two diffraction patterns (Figure S6) are really comparable, confirming the sorbent material does not undergo degradation processes with use.

### 3.3.3. Acute Toxicity Test with *Daphnia magna*

For the toxicity test, a single concentration, equal to 0.2 g L−<sup>1</sup> of HNT, Fe3O4, and HNT/Fe3O4-C was tested. This concentration reflected a potential residual amount of each material in waters after depollution treatment.

All the individuals efficiently ingested the administered materials over 48 h of exposure (Figure 11), as shown by their presence in the digestive tract of exposed individuals.

**Figure 11.** Individuals of *D. magna* showing their digestive tract full of HNT (**a**), Fe3O4 (**b**), and HNT/Fe3O4-C (**c**) after 48 h of exposure to 0.2 g L−<sup>1</sup> (10 mg/50 mL) for each material. Scale bar = 500 μm.

No mortality occurred in the control group. Despite the ingestion of all the materials, the 48 h exposure to 0.2 g L−<sup>1</sup> of HNT and HNT/Fe3O4-C did not induce the mortality of any daphnid, while the viability of the individuals included in the Fe3O4 experimental group was slightly decreased compared to the corresponding control, accounting for the 96 ± 9%.
