The toxicological effects of oxybenzone, an active ingredient in suncream personal care products, on prokaryotic alga Arthrospira sp. and eukaryotic alga Chlorella sp.

Aquatic Toxicology 216 (2019) 105295

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The toxicological effects of oxybenzone, an active ingredient in suncream personal care products, on prokaryotic alga Arthrospira sp. and eukaryotic alga Chlorella sp.

T

Xin Zhonga,b, Craig A. Downsc, Xingkai Chea,d, Zishan Zhanga,d, Yiman Lia,b, Binbin Liua, ⁎ ⁎ Qingming Lia,b, , Yuting Lia,d, , Huiyuan Gaoa,d a

State Key Laboratory of Crop Biology, China College of Horticulture Science and Engineering, Shandong Agricultural University, China Haereticus Environmental Laboratory, P.O. Box 92, Clifford, VA, 24533, USA d College of Life Sciences, Shandong Agricultural University, China b c

ARTICLE INFO

ABSTRACT

Keywords: Oxybenzone Photosynthesis Electron transport Respiration Oxidative stress Algae

Oxybenzone (OBZ; benzophenone-3, CAS# 131-57-7) is a known pollutant of aquatic and marine ecosystems, and is an ingredient in over 3000 personal care products, as well as many types of plastics. The aim of this study is to explore the different toxicities of OBZ on an eukaryotic (Chlorella sp.) and a prokaryotic algae (Arthrospira sp.). OBZ is a photo-toxicant, with all observed toxicities more sever in the light than in the dark. Cell growth and chlorophyll inhibition were positively correlated with increasing OBZ concentrations over time. Twenty days treatment with OBZ, as low as 22.8 ng L−1, significantly inhibited the growth and chlorophyll synthesis of both algae. Both algae were noticeably photo-bleached after 7 days of exposure to OBZ concentrations higher than 2.28 mg L−1. Relatively low OBZ concentrations (0.228 mg L−1) statistically constrained photosynthetic and respiratory rates via directly inhibiting photosynthetic electron transport (PET) and respiration electron transport (RET) mechanisms, resulting in over production of reactive oxygen species (ROS). Transmission and scanning electron microscopy showed that the photosynthetic and respiratory membrane structures were damaged by OBZ exposure in both algae. Additionally, PET inhibition suppressed ATP production for CO2 assimilation via the Calvin-Benson cycle, further limiting synthesis of other biomacromolecules. RET restriction limited ATP generation, restricting the energy supply used for various life activities in the cell. These processes further impacted on photosynthesis, respiration and algal growth, representing secondary OBZ-induced algal damages. The data contained herein, as well as other studies, supports the argument that global pelagic and aquatic phytoplankton could be negatively influenced by OBZ pollution.

1. Introduction Oxybenzone (OBZ, benzophenone-3, CAS#131-57-7) is one of the most widely used active constituents in suncream and other Pharmaceutical and Personal Care Products (PPCPs) because of its ability to absorb photons in a wide range of the ultraviolet (UV) light spectrum (including UVA and UVB). (Aguirre et al., 1992; Emonet et al., 2001; Kasichayanula et al., 2007). Recently, the U.S. Environmental Protection Agency and other national and international governing bodies have recognized OBZ as an environmental contaminant of emerging concern (Blitz and Norton, 2008). A number of countries and regions with coral reefs, including Palau, Hawaii, Aruba, Bonaire, Mexico, and the City of Key West, started prohibiting the importation, ⁎

sale and use of OBZ-containing products (e.g., Hawaii Law 104 2018; Palau 2018 Chapter 12, Title 24, Palau National Code; City of Key West, Ordinance No. 19-03). Industries such as resorts, dive shops, airlines and cruise ships, and local and recreational retailers have implemented policies that stops the sale of OBZ-products, and encourages the sale and use of safer alternatives that have less of an environmental impact, such as sun-wear UPF clothing and non-nanosized mineral sunscreens, as a means of mitigating the detrimental impacts of sunscreen pollution (Downs et al., 2016). The ecotoxicity of OBZ and its threats to wildlife have recently garnered worldwide attention and concern (Wood, 2018). The ecological toxicity of OBZ has had a long history with model laboratory species, such as rodents, fish, invertebrates and algae (Sieratowicz

Corresponding authors at: State Key Laboratory of Crop Biology E-mail addresses: [email protected] (Q. Li), [email protected] (Y. Li).

https://doi.org/10.1016/j.aquatox.2019.105295 Received 26 July 2019; Received in revised form 5 September 2019; Accepted 5 September 2019 Available online 06 September 2019 0166-445X/ © 2019 Elsevier B.V. All rights reserved.

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et al., 2011; Alsalhi et al., 2012; Blüthgen et al., 2012; Laplante et al., 2018; Mao et al., 2017). These studies were concerned with classical ecotoxicological parameters, but also examined cellular and physiological-level changes that ultimately demonstrated OBZ’s role as an endocrine and neurological disruptor, as well as a factor that can induce development and birth defects (Jannesson et al., 2010; Coronado et al., 2008). OBZ can have a significant impact to coral genomic integrity, pathomorphology, endocrine disruption, as well as assault the symbiosis between the coral host and its algal symbiont, inducing coral bleaching (Danovaro et al., 2008; Downs et al., 2016). OBZ is almost ubiquitous in oceanic and marine areas, as well as in navigable lakes and rivers (Balmer et al., 2005; Li et al., 2007; Zwiener et al., 2007; Rodil et al., 2012; Agüera et al., 2013; Tsui et al., 2017; Downs et al., 2016; Mandaric et al., 2017). The half-life of OBZ in receiving waters is between 90 days and 2.3 years (Vione et al., 2013). Because of its pervasiveness in aquatic habitats and its lipophilicity, OBZ easily accumulates in a variety of aquatic organisms, such as coral, fish, mollusks, arthropods, cetaceans, and is the most pervasive personal care product chemical to contaminate industrialized societies (Sieratowicz et al., 2011). OBZ is highly toxic to both higher plants and algae (Zhong et al., 2019; Mao et al., 2018; Chen et al., 2017; Mao et al., 2017). Its prevalence in the environment, especially in pelagic systems, begs the question of the impact of OBZ on plankton. In the past 20 years, there have been radical declines and community structure shifts in pelagic and global phytoplankton (Gregg and Conkright, 2002; Boyce et al., 2010). OBZ has been hypothesized as a factor in this chemical pollution of phytoplankton, but its role has been speculative because adequate physiological and toxicological data do not exist and is only beginning to be explored. Aquatic and marine environments are usually the places most easily polluted by OBZ. In addition, algae are the first plants in which the biological enrichment and for processes such as bio- magnification occur. Mao et al. showed that both eukaryotic algae and prokaryotic algae could absorb OBZ in culture solution, resulting in a decrease in chlorophyll content and the inhibition of algal growth (Mao et al., 2017, 2018). However, regardless of whether OBZ is harmful to the photosynthesis and respiration of prokaryotes and eukaryotes, some questions remain unanswered: will the differences in structure between eukaryotic and prokaryotic algae affect the influence of OBZ on their photosynthesis and respiration? What causes the difference between the dark effect and light effect of OBZ on algae? If OBZ damages the microstructure of the photosynthetic and respiratory apparatus, what are the mechanisms that give rise to these pathomorphologies? In this study, we aim to answer these questions by examining the responses of photosynthesis, respiration, photosynthetic electron transport (PET) of thylakoid membranes, cellular and subcellular structure of algal cells, reactive oxygen species contents, growth rates and chlorophyll contents of both a eukaryotic alga (Chlorella sp.) and a prokaryotic alga (Arthrospira sp.) under different OBZ treatment regimens. We also document the direct and secondary damages to the algae caused by OBZ.

medium (pH = 7.3 ± 0.1, 25 °C ± 1 °C) (Rippka et al., 1979). The culture of Chlorella sp. was kept on a shaking table that shook for 15 min every 1 h at 75 r.p.m. Arthrospira sp. was cultured in Zarrouk medium (Zarrouk, 1966; Vonshak et al., 1982). The culture of Arthrospira sp.was kept on a shaking table that shook for 5 min every 2 h at 75 r.p.m. The OBZ used in this research is manufactured by Sigma-Aldrich Company (USA). Since OBZ is not water soluble, it was first dissolved in a small amount of absolute ethyl alcohol; then, 10 μL OBZ solutions with different concentration were put in the culture media, while 10 μL of alcohol was put in the culture media as control. Two groups of OBZ treatments were used in this study. In the first group, the final OBZ concentration in the culture medium was 0 ng.L−1, 22.8 ng L−1, 114 ng L−1, 228 ng L−1, 2.28 μg L−1, 22.8 μg L−1, 228 μg L−1, respectively. The chlorophyll contents of the algae treated with different OBZ concentration were measured every 4 days. In the second group, the final OBZ concentration in the culture medium was: 0 mg L−1, 0.228 mg L−1, 2.28 mg L−1, 11.4 mg L−1, respectively. The algal growth rate and Chlorophyll (Chl) content were measured once per day, and the photosynthetic parameters, ROS content, ATP content, PET, scanning electron microscope (SEM) parameters, and transmission electron microscope (TEM) parameters were measured after 7 days of treatment. 2.2. Measurement of algal dry weight (DW) Because the Arthrospira sp. is filamentous algae, it is hard to count the cell number of it. So we used dry weight of the algae to reflect the biomass/cell growth of the algae (Liang et al., 2009). The DW of the algae at different growth periods was detected by putting a 50-mL algal suspension in a centrifuge tube and then centrifuged the suspension at 10 000 g for 10 min. The pellet was later suspended in 1 mL of algal growth medium and then transferred onto a filter paper that had been dried to a constant weight in advance. The filter paper with the algal pellet was dried in a 60 °C oven for 1 h until it reached a constant weight and was later weighed with an analytical balance (Sartorius, Germany). Each treatment was replicated 5 times. 2.3. Measurement of Chl content A 5-mL algal suspension was centrifugated at 10 000 g for 5 min, and the pellet was used to extract Chl with 5 mL of 80% acetone. The assay method was according to that of Porra (2002). The decrease of the Chl content per culture volume (mg L−1) was used to reflect the OBZ inhibition on algal growth and Chl content, while the decrease of the Chl content per dry weight (mg g−1DW) was used to reflect the inhibition of Chl synthesis and bleaching of Chl caused by OBZ. To verify that the OBZ did have bleaching effect on algae under growing light, we did an comparative experiment under weak light (10 μmol m−2 s −1) and under normal growing light (35 μmol.m−2 s −1 ), if the Chl content under both the weak light (10 μmol m−2 s −1) and growing light (35 μmol m−2 s −1) decreased, but the later decreased greater than that under weak light (10 μmol m−2 s1), it is reasonable to say that the decrease of Chl content under growing light is caused by both inhibition of Chl synthesis and bleaching of Chl.

2. Materials and methods 2.1. Plant materials The eukaryotic alga Chlorella sp. and prokaryotic cyanobacterium Arthrospira sp. were used as research materials which were cultured in 100 mL Duran® Erlenmeyer narrow-neck flasks (Sigma-Aldrich Company, USA). Both Chlorella sp. and Arthrospira sp. were incubated in a climate chamber, the chamber was supplied with white LED lights (the range of the light wavelength is from 380 nm∼780 nm). The culture of Chlorella sp. and Arthrospira sp. was illuminated under light (photon flux density PFD) of 30–35 μmol m−2 s−1 (380nm∼780 nm wavelength without UV light) with a 12 h:12 h (light:dark) photoperiod under a 25 °C ambient temperature. Chlorella sp. was cultivated in BG11

2.4. Measurement of photosynthesis, respiration and PET Photosynthesis, respiration and PET were measured with an Oxytherm oxygen electrode (Hansatech, UK). A volume of 1.8 mL of treated algae and 0.2 mL of 500 mM NaHCO3 were put into the reaction vessel, the respiratory rates were recorded after the O2 uptake became stable in the dark, a light of 500 μmol.m−2.s −1 intensity was switched on, and the photosynthetic rate was recorded after the O2 release rate became stable again. The O2 absorbing rate in the presence of 1.8 mL of reaction agent (50 mM HEPES/KOH(PH 7.6), 100 mM sorbitol, 10 mM 2

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KCl, 10 mM MgCl2, 5 mM NH4Cl), 0.2 mL of algae and 50 μL of 0.1 mM methylviologen (MV) under a 500 μmol m−2 s−1 light intensity was measured as PET via the whole electron transport chain (Brandle et al., 1977; Zhong et al., 2019).

2.8. SEM and TEM images The algal cells after treated with different OBZ concentrations for 7 days were centrifugated at 3000 g for 10 min. The deposits were used to prepare the slices for TEM and SEM, the method was according to Zhao et al. (2013). The pellet were fixed with 2.5% glutaraldehyde for over 24 h, and then used to prepare the slices for TEM and SEM. For TEM observation, the samples firstly washed with phosphate buffer (0.1 M, pH 7.2) for 3 times, and then postfixed with 1% osmium tetroxide in phosphate buffer for 2 h to fix. After fixing, the samples were dehydrated in the resin and then were sectioned to explore the potential changes in intracellular organelles with energy dispersive spectroscopy and electron diffraction (EDS, INCA100, Oxfordshire, UK). For SEM observation, the samples, after fixed, were washed for 3 times with a phosphate buffer (0.1 M, pH 7.2), and then dried in a vacuum freeze dryer, the cell surface morphology was observed using a SIGMA 300 thermal field emission SEM (Carl Zeiss, Germany).”

2.5. Assessment of the Chl fluorescence transient and fluorescence quench Different effects of OBZ on the algae in the dark or under light were assessed using algae at the logarithmic growth phase. Two groups of algae were treated with 0 or 45.6 mg L−1 OBZ in the dark for 24 h. Then, one group of the algae treated with 0 or 45.6 mg L−1 OBZ was placed under a PFD of 200 μmol m-2 s−1, while another group was kept in the dark. After treatment under light or in the dark for 2 h, 1.5 mL of algae was added to a 1.5 mL tip-bottom centrifuge tube to be centrifugated at 2 000 g for 1 min, after which the pellet completely covered the bottom of the tube. The tubes containing the algae pellet from different treatments were first dark adapted for 20 min. And then the fluorescence transients and the fluorescence quench analysis were performed with a modified dark leaf clip. The bottom half of the commercial leaf clip was taken off, and the 1.5 mL sharp-bottom centrifugal tube containing the algae pellet was closely put into the hole of the upper part of the leaf clip, the sensor of fluorimeter covers the upper clip for measurement. The fluorescence transients were detected using Handy-PEA, a portable continuous-excitation fluorimeter (Hansatech, UK). The fluorescence transient was assessed by exposing the dark-adapted algae to 1 s of saturated light (3 000 μmol.m−2.s−1) using a Handy-PEA. The standardized OJIP curves was calculated according to: Vt curve=(FtFo)/(Fm-Fo) (Strasser et al., 2000). The ratio of inactivated reaction centers =(Fj-Fo)/(Fp-Fo) (Dai et al., 2010). The fluorescence quench analysis was performed using an FMS-2 pulse-modulated fluorimeter (Hansatech, UK) (Zhang et al., 2011). The maximum PSII efficiency (Fv/Fm) was measured using an FMS-2 pulsemodulated fluorimeter by exposing the dark-adapted algae to 1 s of saturated light (8 000 μmol m−2 s−1) provided by the FMS-2. The algae from both the dark and light treatments with different OBZ contents were then irradiated under 200 μmol m−2 s−1 for 3 min. The tube containing algae pellet were exposed to 200 μmol m−2 s−1 actinic light provided by the FMS-2 for 30 s, the light stable-state fluorescence (Fs) was recorded after 30 s of irradiation, and a 0.8 s saturated light of 8 000 μmol m−2 s−1 was applied to gain the maximum fluorescence in the light-adapted state (Fm’). Then, the actinic light was switched off for 3 s, and the minimum fluorescence in the light-adapted state (Fo’) was assessed after 3 s of far-red light illumination. The fluorescence parameters were computed as follows:

2.9. Measurement of immediate inhibition of thylakoid PET by different OBZ concentrations The thylakoid membrane extract of the prokaryotic algae was prepared according to Zhong et al. (2019) with some modification. All the operations were performed in the dark. A 50-mL algal suspension without OBZ treatment was centrifugated at 10 000 g for 10 min, the centrifuged algae were rapidly homogenized with liquid nitrogen and 5 mL of grinding buffer (0.4 M sorbitol, 5 mM EDTA, 5 mM MgCl2, 10 mM NaHCO3, 20 mM Tricine/NaOH (pH 8.4), and 0.5% (w/v) fatty acid-free BSA) with mortar and pestle. The suspension was centrifuged at 3000 g for 3 min at 4°C and the pellet was suspended in 2 mL resuspension buffer (0.3 M sorbitol, 2.5 mM EDTA, 5 mM MgCl2, 10 mM NaHCO3, 20 mM HEPES/KOH (pH 7.8), and 0.5% (w/v) fatty acid-free BSA), the suspension was then centrifuged at 3000 g for 3 min at 4℃. The pellet was resuspended in 5 mL hypotonic buffer (2.5 mM EDTA, 5 mM MgCl2, 10 mM NaHCO3, 20 mM HEPES/KOH (pH 7.8), and 0.5% (w/v) fatty acid-free BSA) and then the resuspension were centrifugation at 3000 g for 5 min at 4℃. The pellet was re-suspended in 1.0 mL resuspension buffer and stored on ice in the dark. The thylakoid membrane were only used within one hour. The PET activities were determined using an Oxytherm oxygen electrode system according to Zhong et al. (2019). 2.10. Measurement of immediate inhibition of RET activities by different OBZ concentrations Because prokaryotic algae do not have true mitochondria, their RET chain is distributed in plasma and thylakoids (Molitor and Peschek, 1986); thus, the extraction method is different from that of mitochondria in eukaryotic plants. The method used in this study was based on that of Molitor and Peschek (1986), with some modification. The 50-mL algal suspension without OBZ treatment was centrifugated at 10 000 g for 10 min, and the centrifuged algae was rapidly homogenized with liquid nitrogen and 4 mL of extracted solution (0.4 M.L−1 mannitol, 1 mM L−1 EGTA, 10 mM L−1 Tris-HCl (pH 7.4), 0.1% BSA and 1% PVP40) with mortar and pestle. The homogenate was centrifugated at lowspeed spin (1 000 g) for 10 min to discard intact cells and coarse cell debris which are deposited at the bottom of the tube; the suspension was centrifugated later at 18 000 g for 12 min to concentrate the mitochondrial membrane. After washing the sediments with the extracted solution, the suspension was then centrifuged at 10 000 g for 5 min, eventually the sediments were suspended with mannitol buffer (0.4 M.L−1 mannitol, 10 mM.L−1 Tris-HCl (pH 7.4) and 1 mM.L−1 EGTA). The extracting procedure was accomplished within 1 h. The extracts were then preserved in ice and used within 30 min. The measurement of RET was performed using an Oxygen electrode system following the protocol of Zhong et al. (2019).

(1) PSII maximum photochemical efficiency, Fv/Fm=(Fm-Fo)/Fm (Maxwell and Johnson, 2000) (2) The actual photochemical efficiency under light, φPSII= (Fm’-Fs)/ Fm’ (Genty et al., 1989). 2.6. Measurement of ROS content The 50-mL algal suspension was centrifugated at 10 000 g for 10 min. The centrifuged algae was ground with pre-cold grinding extracting solution (50 mM phosphate buffer solution/PH 7.8 for O2− and MDA, anhydrous acetone for H2O2) and liquid nitrogen by using mortar and pestle. The O2- production rate was measured according to Schneider and Schlegel (1981). The measurement of H2O2 content followed that of Freguson et al. (1983). The measurement of malondialdehyde (MDA) content followed that of Heath and Packer (1968). 2.7. Measurement of ATP content The ATP content was measured using a luciferase assay according to (Maclean and Luo, 2004). 3

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2.11. Statistical analysis

3.2. The influence of OBZ treatments on the photosynthesis, respiration and PET of Chlorella sp. and Arthrospira sp.

The standard deviation (SD) was calculated according to the formula: n i=1

s=

ˆ x

(x i

n

The OBZ treatment significantly reduced the photosynthetic and respiratory rates of Chlorella sp. and Arthrospira sp. after 7 days of treatment, and the degree of inhibition of photosynthesis and respiration was similar between Chlorella sp. and Arthrospira sp., which increased with the increase in OBZ concentration (Fig. 3). OBZ significantly inhibited the PET (from PSII to PSI) of Chlorella sp. and Arthrospira sp. after 7 days of treatment (Fig. 4).

x )2 1

The standard error (SE) was calculated according to the formula:

=

s n

Significant difference was computed with SPSS 11 (SPSS Inc., Chicago, IL, USA).

3.3. The influence of OBZ treatments on the ATP content of Chlorella sp. and Arthrospira sp.

3. Results

The ATP content per unit DW decreased with the increase in OBZ concentration (Fig. 5).

3.1. The influence of OBZ treatments on the growth and Chl content of Chlorella sp. and Arthrospira sp.

3.4. The influence of different OBZ treatments on the ROS content in Chlorella sp. and Arthrospira sp.

The Chl content per culture volume not only reflects Chl content of algae but also reflects the growth of algae (Aruga and Monsi, 1963). The results demonstrated that even the lowest content of OBZ (22.8 ng.L−1) used in this study significantly decreased Chl contents and the growth of both algae after 20 days of OBZ treatment. And the decrease of chlorophyll content and cell growth were positively correlated to OBZ concentrations over time (Fig. 1). In order to shorten OBZ treatment time to explore the mechanism of OBZ damage on algae, we used higher OBZ concentration (0.228 mg L−1, 2.28 mg L−1 and 11.4 mg L−1) in the following experiments (Fig. 2). The growth of the eukaryotic alga and prokaryotic alga was significantly inhibited with the increase in treatment time. When the algae were treated with 0.228 mg L−1, 2.28 mg L−1 or 11.4 mg L−1 OBZ for 7 days, the growth of Chlorella sp. was approximately 90%, 78%, and 65% of that in the 0 mg L−1 group, respectively, while the growth of Arthrospira sp. was approximately 94%, 80%, and 37% of that in the 0 mg L−1 group. Significant inhibition of algal growth by the treatment with a high OBZ concentration (11.4 mg L−1) was observed after approximately two days. The high OBZ concentration (11.4 mg L−1) significantly inhibited the logarithmic growth of Arthrospira sp. The Chl content per unit DW of Chlorella sp. decreased significantly with the increase in OBZ concentration and treatment time after five days of treatment (Fig. 2. D) (, while that of Arthrospira sp. significantly decreased only under the high OBZ concentration treatment (11.4 mg L−1) (Fig. 2. H). In the early stage (1–4 days) of OBZ treatment, the inhibition of algae by OBZ was mainly observed as a reduction in the growth rate, while in the later stage (5–7 days), the Chl content per unit DW of Chlorella sp. significantly decreased, indicating that OBZ began to cause death and bleaching of the algae.

The ROS content in algal cells significantly increased in response to OBZ treatment, and the MDA content also rose with the increase in OBZ concentration (Fig. 6), indicating that the peroxidation of the cell membrane was aggravated by the over-accumulated ROS. 3.5. Effects of OBZ on the cell morphology and ultra-microstructure of Chlorella sp. and Arthrospira sp. Compared with the control algae, after treated with 2.28 mg L−1 OBZ for 7 days, Chlorella sp. exhibited cell deformation and membrane damage (Fig. 7.C, D), and Arthrospira sp. exhibited abnormal structure and a number of abnormally broken cells (Fig. 7. G, H). Compared with the control algae, after treated with 2.28 mg L−1 OBZ for 7 days, the algal cells exhibited a severely damaged structure (Fig. 8). The membrane structures of chloroplasts and mitochondria were destroyed in Chlorella sp. cells; the structure of thylakoids in Arthrospira sp. cells was destroyed. 3.6. The immediate inhibition of PET and RET in Arthrospira sp. by different OBZ concentrations The PET of thylakoids and the RET in the cells of Arthrospira sp. were immediately inhibited by the addition of different OBZ concentrations to the reaction medium. The lowest (0.228 mg L−1) OBZ concentration used in this experiment significantly restricted PET and RET. The degree of inhibition of PET increased greatly with increasing OBZ concentration, and the highest OBZ concentration (45.6 mg L−1) used in this experiment almost completely restricted PET. However, no significant differences in the inhibition of RET were observed between OBZ treatments (Fig. 9).

Fig. 1. The time course of OBZ effects on the Chl content (per culture volume) in Chlorella sp. (A) and Arthrospira sp. (B). Different lowercase letters above each time point indicate significant differences between OBZ treatments (p < 0.05). Values are the means ± SEs (n = 9). 4

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Fig. 2. The influence of OBZ treatments on the growth and Chl content in Chlorella sp. and Arthrospira sp. A: Growth of Chlorella sp. after 7 days of treatment with different OBZ concentrations. B: The influence of OBZ treatments on the DW of Chlorella sp. C: The influence of OBZ treatments on the Chl content per culture volume of Chlorella sp. D: The influence of OBZ treatments on the Chl content per unit DW of Chlorella sp. E: Growth of Arthrospira sp. after 7 days of treatment with different OBZ concentrations. F: The influence of OBZ treatments on the DW of Arthrospira sp. G: The influence of OBZ treatments on the Chl content per culture volume of Arthrospira sp. H: The influence of OBZ treatments on the Chl content per unit DW of Arthrospira sp. Different lowercase letters at each time point represent significant differences between OBZ treatments (p < 0.05). Values are the means ± SEs (n = 9).

Fig. 3. After treatment with OBZ for 7 days, OBZ effects on the photosynthetic and respiratory rates of Chlorella sp. (A, C) and Arthrospira sp. (B, D) cell. Different lowercase letters above each bar represent significant differences between treatments (p < 0.05). Values are the means ± SDs (n = 3).

Fig. 4. After OBZ treatment for 7 days, the influence of different OBZ treatments on the PET in Chlorella sp. (A) and Arthrospira sp. (B) cell. Different lowercase letters above each bar represent significant differences between treatments (p < 0.05). Values are the means ± SDs (n = 3).

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Fig. 5. After treatment with OBZ for 7 days, the influence of different OBZ treatments on the ATP content in Chlorella sp. (A) and Arthrospira sp. (B). Different lowercase letters above each bar represent significant differences between treatments (p < 0.05). Values are the means ± SDs (n = 3).

3.7. Influence of OBZ on photosynthetic behavior of Arthrospira sp. under light and in the dark

compared to that in the dark. It indicated that the normal photosynthetic electron transport was seriously blocked by OBZ treatment under light, restricting the linear electron transport rate and leading to closure of PSII reaction center, which is also supported by the increase in the J point of OJIP curve. Although the values in the control algae also decreased, the OBZ treatment greatly magnified the decreases (Fig. 10.A–D), indicating that the OBZ treatment caused more severe photoinhibition or photodamage of algae under light than in the dark.

To verify whether OBZ is more harmful to algae under light, Arthrospira sp. was treated with OBZ (45.6 mg L−1) under 200 μmol.m2 −1 .s PFD (the range of the wave length is 380 nm∼780 nm) or in the dark. Although algae normally live in relative lower light environments, in order to shorten the test period, reduce the secondary damage to algae caused by long-term treatment, and facilitate observation of the direct inhibition of OBZ on photosynthetic performance, we used “highlight + short-term treatment” in this experiment. The results demonstrated that after 2 h of light treatment, no significant changes were observed in the maximum photochemical efficiency (Fv/Fm), the actual photochemical efficiency in the light (ΦPSII) which reflects the electron transport efficiency of PSII, the fluorescence transients and the number of inactive reaction centers of algae treated with OBZ in the dark, however, the values significantly declined in the algae exposed to light

4. Discussion This research revealed that OBZ had severe toxicological effects on both prokaryotic and eukaryotic algae. Even as low as 22.8 ng.L−1 OBZ concentration significantly decreased the Chl content and inhibited the growth of the both algae. For the first time, we observed that OBZ significantly restricted photosynthesis and respiration in both prokaryotic and eukaryotic algae and exacerbated the photoinhibition of the Fig. 6. After treatment with OBZ for 7 days, the effects of different OBZ treatments on the rate of O2− production in Chlorella sp. (A) and Arthrospira sp. (B) on the H2O2 content in Chlorella sp. (C) and Arthrospira sp. (D) and on the MDA content in Chlorella sp. (E) and Arthrospira sp. (F). Different lowercase letters above each bar represent significant differences between treatments (p < 0.05). Values are the means ± SDs (n = 3).

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Fig. 7. The effects of OBZ treatments on the cell morphology of Chlorella sp. and Arthrospira sp. after treatment with OBZ for 7 days. Morphology of Chlorella sp. cells of the control algae (A, B) and in algae treated with 2.28 mg L−1 OBZ for 7 days (C, D). Morphology of Arthrospira sp. cells of the control algae (E, F) and of the algae treated with 2.28 mg L−1 OBZ for 7 days (G, H).

it also led to overaccumulation of ROS, resulting in ROS damage, the all above finally inhibited the growth of the algae. Although we recently found that OBZ instantly inhibited the PET and RET in cells of a eukaryotic plant (cucumber) (Zhong et al., 2019), the influence of OBZ on the PET and RET in cells of a prokaryotic plant has not yet been tested because the structures of the photosynthetic and respiratory apparatus are very different from those in a eukaryotic plant. In this studies we first verified that OBZ could instantly inhibit the PET and RET in cells of prokaryotic algae (Arthrospira sp.) (Fig. 9) as well, and the inhibition phenomenon in the prokaryotic algae was similar to that in the eukaryotic plant (cucumber) (Zhong et al., 2019). The structure of the eukaryotic algae was generally the same as the higher plant cucumber, while the prokaryotic algae was actually cyanobacteria and in which the structures of photosynthetic and respiratory apparatus are very different from those in eukaryotic plants. So, the results indicated that the OBZ could directly inhibit the PET in both prokaryotic and eukaryotic plants. The inhibition of PET and RET was directly responsible for restriction of the production of NADPH and ATP via photosynthesis and respiration, which was in accordance with the observation that the ATP content significantly decreased in the OBZ-treated algae. ATP is involved in all power-required metabolisms such as activation of Rubisco and the Calvin-Benson cycle. A restriction of ATP synthesis will reduce Rubisco activation and carboxylation efficiency (Zielinski et al., 1989; Portis et al., 2008). Restricting the synthesis of NADPH and ATP will inevitably inhibit CO2 assimilation via the Calvin-Benson cycle. And it also limits a series of vital activities in plants which require energy, including the process of Chl synthesis and so on. Therefore, we think that the inhibition of PET and RET by OBZ is the mechanism of photosynthesis and respiration inhibition and is also responsible for the damage to both algae. Normally, the electrons transport through the whole electron transport chain and will finally reduce NADP to NADPH at the end of PSI. However, the restriction of PET from PSII to PSI by OBZ will increase amount of the electrons that cannot be used by PSI, which will cause closure of PSII reaction centers, resulting in excessive excitation energy. The excessive excitation energy may transfer the exciting energy to O2 to form ROS such as 1O2 (singlet oxygen). In addition, it has been known that the activity of Rubisco in chloroplasts effectuates both production and consumption of O2, the accumulation of excited electrons will inevitably encounter O2 to over produce ROS (Karpinski et al., 1999). The restriction of PET by OBZ will lead to a rise in excessive excitation energies, which will result in the overproduction of ROS (Karpinski et al., 1999). The over-accumulation of ROS due to the inhibition of PET will further damage the photosynthetic apparatus and cell structure. In addition, when respiration is restricted, the reducing energy cannot be consumed via RET, so the extra ROS will be produced via the respiration chain (Moller, 2001). The significant inhibition of RET also contributed to the overproduction of ROS in the algae. Many studies on other plants have demonstrated that the over-accumulation of ROS damages the plasma membrane, proteins, nucleic acid and other life macromolecules

Fig. 8. The effects of OBZ treatments on the ultra-microstructure of Chlorella sp. (B) and Arthrospira sp. (D) after treatment with 2.28 mg L−1 OBZ for 7 days and the ultra-microstructure of Chlorella sp. (A) and Arthrospira sp. (C) in the control group. Chlp: chloroplast; N: cell nucleus; S: starch grain; M: mitochondrion; CW: cell wall; Thyl: thylakoid.

algae, which resulted in the overproduction of ROS, further damage to cell structure, bleaching and growth restriction. In this study, we aim to investigate the toxicology effect of OBZ itself on algae and its mechanism to damage algae without the interaction of OBZ with UV light, so all of the results in this experiment were those without influence of the interaction between UV light and OBZ. The results of this study will provide evidence for people to further realize the damage of OBZ on plants without UV light. We also documented the direct and secondary damages to the algae caused by OBZ. Our research demonstrated that the damage to photosynthetic behaviors caused by OBZ was greatly exacerbated under light, which was supported by the facts that the Fv/Fm and ΦPSII markedly decreased and the number of inactive reaction centers markedly increased under light (Fig. 10). The significant rise of the inactivated reaction centers indicated by an increase in the number of non-QB-reducing reaction centers due to significant increase in irreducible QB number in PSII reaction centers, restricting electron transfer from QA to QB, increasing the closure of PSII reaction centers. The observation that the “J” point greatly increased in the OJIP curves (Fig. 10. C) by the OBZ treatment under light further supports the suggestion that the number of non-QB-reducing reaction centers increased. The damage of photosynthesis reduced ATP supply and synthesis of carbohydrates, causing carbon starvation and limiting series of life metabolic activities in algae, and 7

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Fig. 9. The immediate restriction of thylakoid PET (A) and RET (C) in cells of Arthrospira sp. by OBZ. The figures indicate the O2 absorption traces of controls and cells treated with different OBZ concentrations. Arrow on each trace line indicates when the different OBZ concentration was added into the reaction medium. The relative PET (B) and RET (D) of Arthrospira sp. after treatment with different OBZ concentrations. The rates of PET and RET before treatment were considered as 100%, whereas that treated with different OBZ concentrations was considered as the ratio of the control. Different lowercase letters above each bar indicate significant differences in PET or RET between the controls and OBZ treatments (p < 0.05). Values are the means ± SDs (n = 5).

(Blokhina et al., 2003; Río, 2015). The observations via TEM and SEM verified that OBZ damaged the structures of the photosynthetic apparatus and membrane systems of the algal cells (Figs. 7 and 8). We think that this damage occurred via the over-accumulation of ROS caused by the inhibition of photosynthesis; thus, we believe that this damage was secondary damage to the algae caused by OBZ. Impairment of photosynthesis apparatus will inevitably aggravtes the inhibition of photosynthesis, resulting in a positive feedback loop. The over-accumulation of ROS finally led to the bleaching or even death of the algae (Figs. 2, 7 and 8). We think that this over-accumulation of ROS is also the mechanism by which OBZ bleaches coral under light. Even as low as 22.8 ng.L−1, OBZ content significantly decreased Chl content. The decrease in Chl content may involve the inhibition of Chl synthesis, inhibition of algae growth and photo-bleaching of Chl by OBZ treatment. Our experiment under low light (10 μmol.m−2.s−1 PFD) and growth condition light (35 μmol.m−2.s−1 PFD) demonstrated that the decrease in Chl content under 35 μmol.m−2.s−1 PFD was significantly greater that that under 10 μmol.m−2.s−1 PFD (Fig. 1 in supporting information), which indicated that the decrease in Chl content includes photo-bleaching and inhibition of Chl synthesis caused by OBZ treatment.

However, the OBZ concentration found in some parts of the ocean is well above this value (Downs et al., 2016). Therefore, the OBZ pollution of some waters is likely an important reason for coral bleaching and death, and its destruction of algae is an important factor threatening aquatic ecosystems. The findings of the research are helpful to understand the mechanism of OBZ damage to marine organisms, and they provide useful evidence to support limiting the use of OBZ-containing products and protecting aquatic ecosystems. Although our research verified that OBZ could directly inhibit the PET and RET of the algae, there are a series of electron transport donors and acceptors in the photosynthetic and respiratory electron transport chain. However, we have not located the exact inhibiting site and the inhibiting mechanism yet. In addition, we have not located the inhibiting site in respiration chain neither. Furthermore, whether or not the OBZ has different inhibition effect and mechanism on other algae are also worth to explore. To clarify molecular mechanism of OBZ inhibition on different algae needs more cooperating studies from scientists in different fields. And as to whether the interaction between UV light and OBZ will have different effect on plant from OBZ, and what the ratio of concentration/illumination intensity of OBZ and the UV

Fig. 10. The photosynthetic behaviors of Arthrospira sp. treated with 45.6 mg L−1 OBZ in the dark and under light for 2 h. A: Maximum photochemical efficiency, Fv/ Fm. B: Actual photochemical efficiency in the light, ΦPSII. C: The fluorescence transients. D: The number of inactive reaction centers, (Fj-Fo)/(Fm-Fo). 8

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light will cause damage on plants, it also needs more studies to be clarified.

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