**2. Materials and Methods**

Six NEPs, namely three sunscreens, SUN1 (nTiO2 + nZnO), SUN2–3 (nTiO2); body cream CA1 (nTiO2 + nAg); sanitiser; SAN1 (nAg); and socks SK1 (nTiO2 + nAg) were purchased from South African retailers. The physicochemical properties of the ENMs incorporated in the six NEPs varied and were previously reported [41]. Briefly, SUN1 contained elongated nTiO2, and angular nZnO particles sized 14 × 62 nm and 35 × 38 nm, respectively. SUN2 and SUN3 contained angular-shaped nTiO2 with a size range of

20–28 × 27–32 nm and 20–28 × 27–32 nm, respectively. Near-spherical nAg particles with the size range of 22–37 nm were observed in SAN1. CA1 and SK1, respectively, contained nTiO2 particles sized at 8 × 53 nm (elongated) and 32–203 × 48–135 nm (angular), the NEPs also contained near-spherical nAg ranging 18–28 nm. The particles were negatively charged, and the nTiO2 phase was rutile or anatase. The total amounts of ENMs differed between NEPs [41].

### *2.1. Procedures for ENMs Release*

The procedures used to investigate the release of ENMs from NEPs differed and are described in Sections 2.1.1 and 2.1.2. In all instances, the procedures were adapted to simulate conditions (but not fully replicate actual life cycle stages) that promote ENMs release from the NEPs. However, some stages applicable during the use of current NEPs (e.g., application of sunscreen or sanitiser to the skin, prior wearing of socks) were eliminated as the primary focus of the study was the analytical determination of the potential for ENMs release. Furthermore, simple (or standard) aqueous media were preferred to avoid physicochemical complexation, which occurs when using complex media and uncharacterised commercial detergents [42]; however, release evaluations in complex media with greater environmental realism are the cornerstone of nanotechnology risk assessment and are recommended for future studies as analytical capabilities and access advances. All investigations were carried out in triplicate.

### 2.1.1. Release of ENMs from Suncreen 1–3 (SUN1–3) and Body Cream 1 (CA1)

The release of ENMs from SUN1–3 and CA1 followed a slightly modified protocol of Botta et al. [43]. Briefly, 2 g of SUN1–3 and CA1 were aged in 180 mL of release media for 48 h at 25 ◦C under darkness (plastic beakers were capped and covered with heavy-duty aluminum foil) and under illumination: −6000 lux (uncapped transparent plastic beakers). The SUN1–3 ENMs were released in Milli-Q water (18 M Ω·cm), freshwater, seawater or swimming pool water (S1.1), while the CA1 ENMs were released using Milli-Q water only; the properties of the release media are given in S1.1. ENMs were released by agitating the suspension at 400 rpm for 48 h, and the sample volume was maintained by continuously adding aqueous media throughout the 48 h. Agitation was stopped after 48 h, and the samples were allowed to settle for another 48 h, a step that caused sedimentation that resulted in two phases (surface suspension and sediment); the overall duration of ENMs release was 96 h. The two phases were separated by sampling 150 mL of stable suspension; the sediments were not disturbed during sampling. The stable suspensions were prepared for product-released ENMs analysis (Section 2.2).

### 2.1.2. Release of ENMs from Sock1 (SK1) and Sanitiser1 (SAN1)

The release of ENMs from SK1 was undertaken by adapting previously developed methods [44,45]. Briefly, areas (spots/regions) of the sock material marketed and experimentally confirmed to be incorporated with nAg and nTiO2 [41] were cut from SK1 samples and transferred into 1 L glass bottles, washed with 200 mL of the release media (Milli-Q water, tap water, and sodium dodecyl sulfate as a detergent). The detergent media was prepared in two ways: (i) sodium dodecyl sulfate 1, which was prepared in Milli-Q water, while (ii) sodium dodecyl sulfate 2 was prepared in tap water. The samples were washed by shaking at 350 rpm at 40 ◦C for 12 h (2 washes). After the final washing cycle, the fabrics were removed from the washing water, and the samples were prepared for analysis (Section 2.2).

For SAN1, ENMs were released following a slightly modified method of Benn et al. [46] and Mackevica et al. [47]. Briefly, 1 mg/L of the sample was prepared in Milli-Q water and ENMs released by agitating at 350 rpm at 40 ◦C for 24 h. After ENMs' release, samples were prepared for analysis (given in Section 2.2).

SUN1 (nZnO), CA1 (nAg), SAN1 (nAg), and SK1 (nAg) were incorporated with ENMs that are relatively soluble. The released ions were recovered from the release media through sequential filtration. The samples were sequentially filtered using Amicon® Ultra-15 30 K centrifugal filters (30000 MWCO, Merck, South Africa), followed by further centrifugation using Amicon® Ultra-15 3 K centrifugal filter devices (3000 MWCO, Merck, South Africa) for 30 min at 10,000 rpm for each filtration step. The released ions (filtrates from the 3 K centrifugal filter device) were quantified (Section 2.2.3).

### *2.2. Physicochemical Properties of Product-Released ENMs*
