Effects of three zinc-containing sunscreens on development of purple sea urchin (Strongylocentrotus purpuratus) embryos

Highlights

• Zinc-sunscreens interfere with sea urchin skeletal formation and axial development.

• Zinc-sunscreens are internalized by sea urchin embryos in a dose-dependent manner.

• Exposure to sunscreens reduces sea urchin multidrug-resistant transporter activity.

Abstract

The growing popularity of physical sunscreens will lead to an increased release of ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations may be released into marine environments by use of sunscreens. The extent of the consequences of this addition of Zn to the ocean are not fully understood. We investigated the effects of materials released by ZnO- sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos incubated in various concentrations of Zn (0.01, 0.05, 0.1, 0.5, and 1 mg/L), the sources of which included zinc-containing compounds: ZnO and zinc sulfate (ZnSO₄); and ZnO sunscreens: All Good, Badger, and Raw Elements brands. Based on EC₅₀ values, ZnO-containing sunscreens were slightly, but not significantly, more toxic than ZnO and ZnSO₄, suggesting that sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn-exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescence confocal microscopy. The concentration of Zn²⁺ internalized by the developing embryos correlated positively with the concentration of Zn in seawater. Additionally, exposure to both ZnO sunscreens and ZnO and ZnSO₄ at 1 mg/L Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is one of the first studies documenting ZnO-containing sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms.

Introduction

Topical sunscreen products have been in use for almost 100 years. Physical or mineral, sunscreens contain inorganic ultraviolet (UV) filters, generally titanium dioxide (TiO₂) or zinc oxide (ZnO) that reflect and scatter sunlight, whereas chemical sunscreens contain organic UV filters including avobenzone, oxybenzone, and octocrylene, and absorb easily into the skin and blood stream (Matta et al., 2019). Unlike traditional chemical UV filters, physical UV filters sit on top of the skin (Stiefel and Schwack, 2015), a characteristic that increases the potential for physical sunscreens to be released into the environment during use. Little research exists on the amount of sunscreen transferred from people to the environment; however, one study on a chemical sunscreen found that over a twenty-minute submersion period about 25% of applied sunscreen ingredients are released into the water (Danovaro et al., 2008). Additionally, sampling of waters off Majorca Island have found that midday, when sunlight and consequently sunscreen application are expected to be at a maximum, concentrations of common sunscreen ingredients were greater than 60–90 percent of background values (Tovar-Sánchez et al., 2013).

Physical ZnO sunscreens are available in both nano and non-nano (bulk) zinc formulas. Recently, health and environmentally-conscious consumers have begun to question the safety of nanotechnologies. Though studies examining topical toxicity have found that nano-metals are not more damaging to humans than non-nano metals (Burnett and Wang, 2011; Lademann et al., 2006; Moezzi et al., 2012; Nohynek et al., 2007), public distrust of nanotechnology continues to contribute to the growing popularity of bulk ZnO sunscreens. Moreover, additional studies have demonstrated that nano zinc oxide (nZnO) has unique toxicities on marine invertebrates specifically due to their small particle size (Fairbairn et al., 2011; Manzo et al., 2013; Miglietta et al., 2011; Wu et al., 2015). Still, no studies have investigated the effects of sunscreen-related bulk ZnO that may be discharged into the environment.

Release of heavy-metal Zn from natural sources is exceeded by introduction from anthropogenic sources. Currently, domestic wastewater is one of the greatest sources of anthropogenic heavy-metal Zn introduction into aquatic environments with 21,000–58,000 metric tons per year (Roney et al., 2005). Zn²⁺ can also enter wastewater and the environment through the use of personal care products (PCPs) (Díaz-Cruz and Barceló, 2009; Giokas et al., 2007). Many PCPs including sunscreens, skin cream, and make-up products contain ZnO UV filters, which could be washed from the body during bathing or swimming (Nohynek et al., 2010). The removal of dissolved metals from wastewater is becoming more efficient (Barakat, 2011; Gunatilake, 2015); for example, a technique called Activated Sludge Biomass can remove 86.5% of Zn²⁺ from wastewater (Ahmad et al., 2010). Another technique using microalgae to remove heavy metals, reduced the amount of Zn in wastewater by 94.1% (Chan et al., 2013). As these processes become more efficient, other, unregulated, zinc sources such as direct release from ocean bathers will become responsible for larger percentages of marine ZnO pollution.

Physical UV filters can be found in sediment as well as water samples and can have a long life span in these environments, which vary depending on location, intensity of recreational activity, and the weather, with maximum concentrations released during warmer weather (Botta et al., 2011; Giokas et al., 2007). Similarly, aging and breaking down of physical sunscreen containing TiO₂ in an aqueous environment releases significant amounts of titanium into the aquatic habitat (Botta et al., 2011). For example, Botta et al. (2011) estimated that in reef areas, 36–56 tons of TiO₂ were released from sunscreens; however, no such study could be found for ZnO release from sunscreen in aqueous environments.

The toxicity of a sunscreen to aquatic organisms is mostly dependent on the solubility of the sunscreen ingredients in seawater (Giokas et al., 2007). Attempts have been made to reduce the solubility and consequently the toxicity of ZnO through iron doping. While this was shown to reduce its cytotoxicity in cell culture (George et al., 2009), 10% iron-doped ZnO was found to be just as toxic as non-doped ZnO to sensitive marine embryos in a study by Fairbairn et al. (2011).

One component that allows embryos to cope with exposure to xenobiotics, such as heavy metals, are multidrug resistance (MDR) transporters (Cole et al., 2013; Hamdoun et al., 2004). These efflux transporters belong to the ATP-binding cassette (ABC) transporter family and play a protective role during early sea urchin embryo development (Litman et al., 2001). If they are inactivated or preoccupied with removing large quantities of a toxicant from the embryos’ cells, then other ions or compounds may build up to toxic levels within the embryos. Exposure to nZnO (Wu et al., 2015) and other heavy metals in both nano and non-nano forms (Achard et al., 2004; Torres-Duarte et al., 2017) has been shown to impair MDR activity in developing sea urchin embryos.

The ecotoxicity of ZnO is attributed to its solubility into Zn²⁺. For marine invertebrate embryos, Zn²⁺ is known to cause animalization, a disruption in the development of the animal-vegetal axis (Timourian, 1968) where endomesoderm formation is prevented (Poustka et al., 2007). This and other abnormalities have been observed in sea urchins developing in water contaminated with heavy metals (Kobayashi and Okamura, 2004). Sea urchins are sensitive to Zn and many common abnormalities of sea urchin embryos exposed to Zn compounds are known (Fairbairn et al., 2011; Kobayashi and Okamura, 2004; Manzo et al., 2013; Miglietta et al., 2011; Timourian, 1968; Wu et al., 2015). The sensitivity and availability of sea urchins, along with the wide body of existing research on development of their early life stages, make them a good model organism for examining specific effects of topical sunscreens on marine organisms.

For this study, purple sea urchin, Strongylocentrotus purpuratus, embryos were cultured in varying concentrations and formulations of bulk (non-nano) ZnO sunscreen solutions or either ZnSO₄ or ZnO as positive controls. We evaluated the experimental embryos for morphological abnormalities and documented their relative concentrations of internalized Zn²⁺. We also examined the differential impacts related to stage of embryo at exposure to sunscreen components. Because sea urchin embryos are planktonic and can be found in the water column of shallow coastal oceans, they may be exposed to increased Zn concentrations at various stages of development. In order to capture the full range of Zn toxicity, it is important to know if the effects of Zn exposure are dependent on sea urchin embryos’ developmental stage. Additionally, this study investigated the effect of these sunscreen ingredients on the activity of MDR transporters to understand the potential implications on the developmental physiology of the embryos. We hypothesized that commercially available bulk ZnO sunscreens release materials into marine environments that have a negative effect on the development of S. purpuratus embryos. Embryos exposed to sunscreen solutions were expected to develop abnormally and contain higher than average concentrations of internalized Zn²⁺. The internalized Zn²⁺ was predicted to decrease MDR transporter effectiveness.