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Journal of Hazardous Materials

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Toxicity of benzophenone-3 and its biodegradation in a freshwatermicroalga Scenedesmus obliquusSang-Hun Leea, Jiu-Qiang Xionga,b,*, Shaoguo Rub, Swapnil M. Patilc, Mayur B. Kuradec,Sanjay P. Govindwarc, Sang-Eun Ohd, Byong-Hun Jeonc,*a Department of Environmental Science, Keimyung University, 42601 Daegu, South Koreab College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Chinac Department of Earth Resources and Environmental Engineering, Hanyang University, Seoul 04763, South KoreadDepartment of Biological Environment, Kangwon National University, 192-1 Hyoja-2-dong, Gangwondo, Chuncheon 200-701, South Korea

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Editor: R. Debora

Keywords:MicroalgaeBiodegradationBenzonphenone-3UV filtersToxicity

A B S T R A C T

Environmental contamination by benzophenone-3 has gained attention because of its frequent occurrence andadverse environmental impact. Studies investigating the toxicity and removal mechanisms, along with its de-gradation pathway in microalgae are still rare. In this study, the ecotoxicity of benzophenone-3 on Scenedesmusobliquus was assessed through dose-response test, risk quotient evaluation, and changes of microalgal bio-chemical characteristics and gene expression. The calculated risk quotients of benzophenone-3 were>1, im-plying its high environmental risk. Expression of the ATPF0C and Tas genes encoding ATP-synthase and oxi-doreductase was significantly increased in S. obliquus after exposure to benzophenone-3, while that of Lhcb1 andHydA genes was reduced. When exposed to 0.1−3mg L−1 benzophenone-3, 23–29 % removal was achieved byS. obliquus, which was induced by abiotic removal, bioadsorption, bioaccumulation and biodegradation.Metabolic fate analyses showed that biodegradation of benzophenone-3 was induced by hydroxylation, andmethylation, forming less toxic intermediates according to the toxicity assessment of the identified products.This study provides a better understanding of the toxicity and metabolic mechanisms of benzophenone-3 inmicroalgae, demonstrating the potential application of microalgae in the remediation of benzophenone-3 con-taminated wastewater.

https://doi.org/10.1016/j.jhazmat.2020.122149Received 3 November 2019; Received in revised form 30 December 2019; Accepted 19 January 2020

⁎ Corresponding authors.E-mail addresses: ysxjq2014@hanyang.ac.kr (J.-Q. Xiong), bhjeon@hanyang.ac.kr (B.-H. Jeon).

Journal of Hazardous Materials 389 (2020) 122149

Available online 20 January 20200304-3894/ © 2020 Elsevier B.V. All rights reserved.

T

1. Introduction

Clean water is a crucial element for all living organisms. Numerouscontaminants of emerging concern have significantly impacted waterquality, reducing the availability of clean water resources(Schwarzenbach et al., 2006). Among these contaminants, ultraviolet(UV) filters have gained research attention worldwide. UV filters areactive ingredients in sunscreens and cosmetics that prevent the pene-tration of UV sunlight. These compounds are similarly found in pro-ducts that protect against photodegradation and have been frequentlydetected in the environment (Langford et al., 2015). These compoundshave been found to contaminate surface water, groundwater, waste-water, seawater, sediments (Ramos et al., 2016), and discharge fromsewage treatment plants (Sánchez-Quiles and Tovar-Sánchez, 2015),negatively impacting both the ecosystems and aspects of human health.For example, these compounds have been demonstrated to affect en-docrine signaling, alter sex hormone balance, and impede the re-productive ability of wild-living animals (Schlumpf et al., 2004). UVfilters inhibit the growth of crustaceans, marine invertebrates, andalgae, even presenting at low concentrations (1 μg L−1–1mg L−1) (Maoet al., 2017). UV filters are readily accumulated in the liver tissue ofFranciscana dolphin, showing uptake concentrations up to 782 ng g−1

(Gago-Ferrero et al., 2013).Benzophenone-3 (BP-3) is one of the frequently found UV filters in

environment, with a maximum found concentration of 3.95mg L−1 inwastewater (Ramos et al., 2016). The proportion of BP-3 in cosmeticsand other products, such as insecticides and plastic bags, ranges from 5% to 10 %. These values are consistent across countries like Japan,Korea, Australia, China, Europe, and U.S.A., indicative of its commer-cial utilization worldwide (Sánchez-Quiles and Tovar-Sánchez, 2015).Continuous consumption of BP-3 will result in its successive dischargeto and contamination of the environment. Therefore, there is an urgentneed to comprehensively analyze its eco-toxicological effects and de-velop efficient treatment strategies. Various approaches including oxi-dation of BP-3 using UV/H2O2 processes, adsorption, and biodegrada-tion in soil have been developed (Luo et al., 2019; Charaabi et al.,2019). Incomplete mineralization and high costs associated with theseapproaches have restricted their utilization in large-scale applications,resulting in the continued need for better technologies.Microalgae-facilitated remediation of BP-3 contamination is of

growing interest, as it is solar-driven, environment-friendly, and cost-effective. This approach has been implemented for the efficient removalof various contaminants like nutrients, heavy metals, organic con-taminants associated with the production of biofuel and other valuableproducts (Bai and Acharya, 2016; Norvill et al., 2016). Microalgae areancient microorganisms and are widely distributed across diverse ha-bitats (Subashchandrabose et al., 2011). These microorganisms cansurvive extreme conditions by shifting their metabolism from auto-trophic to mixotrophic types, depending on the availability of nutrients.This metabolic flexibility allows microalgae to acclimatize to new en-vironments (Xiong et al., 2017a). Microalgae-mediated removal of or-ganic contaminants such as those found in pharmaceuticals, steroids,and phenols, has been investigated. For example, microalgae-basedbiotechnology efficiently treated pharmaceutical wastewaters by re-moving 92 % of 81 different compounds on average (Villar-Navarroet al., 2018). In another study, microalgae were found to remove 20–99% of seven pharmaceutical contaminants from wastewater (Matamoroset al., 2016). Toxicity analyses of contaminants by microalgae has beendemonstrated to be a reliable method by the Organization for EconomicCo-operation and Development (OECD) (OECD TG 201, 2011). Whilemany early studies focused on the effects of BP-3 on microalgal growth,investigation into its environmental toxicity, removal efficiency andmechanisms, and biodegradation pathways by microalgae are rare.In this study, a thorough toxicological analysis of BP-3 according to

its effects on microalgal growth, risk quotients analysis, changes ofmicroalgal biochemical characteristics and gene expression was

conducted. The treatment efficiency and removal mechanism of BP-3was investigated during the microalgal cultivation. A metabolicpathway of BP-3 by microalgae was proposed for the first time. Acuteand chronic toxicity of the identified degradation intermediates wascomprehensively investigated. This study showed a potential utilizationof microalgae-based biotechnology for an efficient treatment of BP-3polluted wastewater.

2. Materials and method

2.1. Growth inhibition assay and assessment of environmental risk quotientsfor benzophenone-3

The inoculum of S. obliquus used in the following experiments wasprepared by cultivating the microalgae in flasks containing sterilizedBold’s Basal Medium (BBM, 150mL) with shaking and continuous il-lumination (a fluorescent light that yields a photon flux of45−50 μmol photon m−2 s−1). Cultures were grown for one week at150 rpm, 27 °C. Benzophenone-3 (oxybenzone, (2-Hydroxy-4-methox-yphenyl)-phenylmethanone, 99 %) was obtained from Sigma-Aldrich(St. Louis, MO, USA). A growth inhibition assay was performed in250mL-capacity flasks containing different concentrations of BP-3 (0,0.1, 0.5, 1, 2, 3 mg L−1) in sterilized BBM (150mL) and microalgalinoculum (1.0 %, Vinoculum/Vmedia). Cultivation conditions were as de-scribed above, except for the light condition which was set to light/darkcycle of 16/8 h, respectively. The absorbance the microalgal inoculumwas 1.0 at a 680 nm optical density (OD 680 nm), which is equivalent toa microalgae biomass of 0.014 g L−1.Dry cell weight (DCW) was determined by drying 10mL of the

microalgal suspension filtered by a pre-weighed Whatman filter paper(GF-52) at 105 °C for 24 h. Filter papers were weighed again afterdrying, and the difference in weight was calculated as DCW. The half-maximum effective concentration (EC50) is used to indicate the toxicityof various contaminants, and can be calculated as the relationship be-tween pollutant and relative growth inhibition. The specific growth rate(SGR) of S. obliquus was determined using according to an equation(OECD TG 201, 2011);

= N lnNt t

SGR ln 2 0

2 0 (1)

where N2 is DCW at time t2 and N0 is DCW at time t0.The pigment content of S. obliquus was extracted and its absorbance

was calculated using a previously determined method (Kurade et al.,2016). In brief, cell pellets were collected from 10-mL cultures bycentrifugation at 4000 rpm for 300 s, followed by resuspension in 10mLof 90 % methanol. Samples were treated at 60 °C for 10min and left toreach ambient temperature. The absorbance of extracted pigments wasdetermined using a spectrophotometer (at 665, 652 and 470 nm, Hach,USA).The environmental risk of the pollutants has been predicted ac-

cording to their risk quotients (RQs), which are the ratios of measuredenvironmental concentration (MEC) and predicted no-effect con-centration (PNEC) (Xiong et al., 2017b). If an RQ value is> 1, thepotential risk of the target contaminant is considered to be significant.The MEC of BP-3 in surface water, wastewater, and seawater accordingto the literature was used to calculate the RQs to anticipate the po-tential environmental risk of BP-3 in unfavorable environmental con-ditions. The PNEC can be calculated by dividing the half-maximumeffective concentration and an assessment factor of 1000 (Commissionof the European communities, 1996). Von der Ohe et al. suggestedvalues of PNECs can be derived by dividing the half-maximum effectiveconcentration (EC50) by 1000, according to the toxicity data of 500classical and emerging organic contaminants (Von der Ohe et al.,2011). RQ values were determined using the following equation:

S.-H. Lee, et al. Journal of Hazardous Materials 389 (2020) 122149

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=RQ MECPNEC (2)

where, MEC is maximum environmental concentration of BP-3(Table 2), and PNEC is predicted no-effect concentration.

2.2. Changes in gene expression of Scenedesmus obliquus

Microalgal cell pellets (100mg) were collected by centrifugation(4000 rpm, 4 °C, 10min) for control, low concentration (0.1mg L−1 BP-3), and high concentration (3mg L−1 BP-3) treatment conditions after10 days of cultivation. Harvested cells were flash-frozen in liquid ni-trogen and total RNA was extracted using the RNeasy plant kit(Qiagen). cDNA synthesis and gene expression analysis were carried outusing one-step real-time polymerase chain reaction (RT-PCR) in a re-action volume of 50 μL containing 2× SYBR RT-PCR premixture(25 μL), primers (10 μM, 1 μL), diluted total RNA (10 ng, 2 μL),SuperScript III RT/Platinum Taq mix (2 μL), and nuclease-free water(19 μL). The primer sequences of ATPFOC, Lhcb1 (Wang et al., 2019),and α-tubulin have been referred to earlier reports (Zhang et al., 2017).Primers for TaS and HydA were designed according to the mRNA se-quences in the National Center for Biotechnology Information (NCBI)database (Table S1). RT-PCR reactions were performed on a StepOnePlus qRT-PCR System (Applied Biosystems) with the following tem-perature gradients: 55 °C for 30min, 94 °C for 2min, 40 cycles of 94 °Cfor 15 s, 58 °C for 30 s, and 68 °C for 30 s. Analyses were performed intriplicate for all samples, and negative controls consisted of RT-PCRreactions without reverse transcriptase. The transcript levels for thegenes investigated were normalized to the reference gene (α-tubulin),and then evaluated according to the 2-△△Ct method, as follows;

△△Ct=(Cttarget gene− Ctα-tubulin)test – (Cttarget gene− Ctα-tubulin)control(3)

2.3. Removal kinetics and mechanisms of benzophenone-3 by Scenedesmusobliquus

Removal kinetics of BP-3 (0.1, 0.5, 1, 2, 3 mg L−1) by S. obliquuswere investigated. A control experiment was conducted with the sameamount of BP-3 but without microalgal inoculum to monitor abiotic BP-3 elimination. Two milliliter samples were withdrawn after 0, 2, 4, 6, 8,and 10 days of culturing, and the supernatant of the microalgae solu-tion was used to measure the residual BP-3 concentration after cen-trifugation. To identify the removal mechanism of BP-3 (bioadsorption,bioaccumulation, and biodegradation), ten milliliter of microalgalsuspension was acquired at day 10. Cell pellets were harvested bycentrifugation at 4000 rpm (5min) and resuspended in 10mL of dis-tilled water. Pellets were washed three times to completely removeadsorbed BP-3. The amount of adsorbed BP-3 was determined bymeasuring BP-3 concentration in the resuspended microalgal solution,while bioaccumulated BP-3 was calculated by measuring the amountextracted from microalgae pellets. BP-3 extraction from cell pellets wasperformed as previously described (Mao et al., 2017). Briefly, 10mLmicroalgae solution was withdrawn after cultivation for 10 days andcentrifuged. The supernatant was decanted and cell pellets extractedusing 5mL of dichloromethane:methanol (1:2 v/v). The supernatantwas then recovered and analyzed to estimate the amount of bioaccu-mulated BP-3. The biodegradation percentage (Pb) of BP-3 by S. ob-liquus was calculated using the following equation;

=P A A A A AA

(%) ( ) 100b t r d a c

t (4)

where At is the initial BP-3 concentration (at day 0), Ar is the residualBP-3 concentration, Ad is the adsorbed amount of BP-3, Aa is the abioticremoval of BP-3, and Ac is the bioaccumulated amount of BP-3.The removal kinetics of BP-3 were analyzed using a first order

model as follows;

= +C kt lnCln t 0 (5)

where, C0 is the initial amount of BP-3 at day 0, Ct is the residual BP-3concentration at time t, k is the removal rate constant (d−1), and t isremoval period in days.The concentration of BP-3 was analyzed by high-performance liquid

chromatography (HPLC) with a UV–vis detector (Waters 2695, 2487),equipped with a C18 column (250mm×4.6mm, 5 μm pore size),which measures BP-3 at 288 nm. The temperature of the column was setto 30 °C, and the rate of mobile phase flow was 0.8 mLmin−1.Acetonitrile and water were used as the mobile phase at a ratio of 70:30(v/v). Two standard curves were prepared, the first with a concentra-tion range of 0.02-0.12mg L−1 BP-3 and an injection volume of 50 μL,and the second with a concentration range of 0.2−4mg L−1 BP-3 andan injection volume of 20 μL.The intermediates were isolated by ethyl acetate at the end of each

experiment. Briefly, fifty milliliter of supernatant from microalgal cul-tures were collected by centrifugation (4000 rpm for 5min) followed byfiltration, then mixed overnight with an equal volume of ethyl acetate.The ethyl acetate layer was carefully collected and evaporated overanhydrous Na2SO4. Residues were dissolved in methanol and 1 μL ofthis solution was injected into a gas chromatography-mass spectrometer(GC–MS, 7890B, 5977B, Agilent, USA) with an HP-5ms capillarycolumn (30m×250 μm×0.25 μm) (Agilent J&W, USA) and helium asa carrier gas (flow rate 1.0mLmin−1) at a split ratio of 5:1. The tem-perature gradient of the oven and full-scan mass spectra were set aspreviously described (Xiong et al., 2019). National Institute for Stan-dard Technology’s (NIST) mass spectral library was used for identifi-cation of the metabolites.

2.4. Toxicity assessment of microalgal degradation intermediates

Acute and chronic toxicity of the identified intermediates recoveredafter the microalgal degradation of BP-3 were evaluated using theEcological Structure Activity Relationships (ECOSAR) Program (version1.11). The ECOSAR system is developed by United States EnvironmentalProtection Agency to calculate the values of the half-maximum effectiveconcentrations (EC50) and half-maximum inhibition concentrations(IC50) of diverse contaminants on different model organisms (https://www.epa.gov/tsca-screening-tools/ecological-structure-activity-relation-ships-ecosar-predictive-model). The toxicity levels of pollutants can becategorized into four levels: “very toxic” with EC50<1mg L−1, “toxic”with EC50=1−10mg L−1, “harmful” with EC50=10−100mg L−1,and “non-harmful” with EC50 > 100mg L−1 toward aquatic organismsusing the technical guidance document (Commission of the Europeancommunities, 1996).

2.5. Statistical analysis

The data presented in this study are the mean values and standarddeviations from experiments performed in triplicate. Statistical sig-nificance (p < 0.05) of the data was obtained by one-way analysis ofvariance with a Tukey-Kramer multiple comparison test in GraphPadPrism 5.0.

3. Results and discussion

3.1. Toxicity of benzophenone-3 on Scenedesmus obliquus and its riskassessment

The effect of BP-3 on S. obliquus indicates that low concentrations ofBP-3 (< 0.5mg L−1) had a negligible effect on the tested microalga,while the microalgal dry cell weight gradually declined with exposureto 1−3mg L−1 BP-3 (Fig. 1A). Relative growth inhibition was observedby 14, 16 and 23 % with exposure to 1, 2 and 3mg L−1 BP-3,

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respectively, when compared to the control at day 10. To our knowl-edge, the maximum found concentration of BP-3 is 3.95mg L−1 inwastewater (Ramos et al., 2016). Our findings suggest that environ-mental contamination of BP-3 at such concentrations would sig-nificantly inhibit the growth of microalgae, thereby decreasing micro-bial diversity by causing lethal effects to sensitive species. The effect ofBP-3 on the specific growth rate (SGR) of S. obliquus showed that thegreatest inhibitory effect of BP-3 on growth rate occurred on day 4,with values of 0.19 - 0.36 for 0–3mg BP-3 L−1 (Fig. 1B). Microalgaehave the capacity to recover from the effects of BP-3 treatment, whichhas been demonstrated according to the improved specific growth rate.A linear relationship between BP-3 and relative growth inhibition wasobserved, and the EC50 of BP-3 at 96 h was calculated to be 3.64mg L−1

according to the regression equation (y=11.652ln(x)+ 34.946,R2=0.94), where y represents relative growth inhibition and x is theSGR. Based on our findings, BP-3 can be classified as contaminant toxic

to aquatic organisms (Commission of the European communities,1996). Earlier studies focusing on BP-3 toxicity to microalgae aresummarized in Table 1, with EC50 values of BP-3 ranging from 0.36 to3.64mg L−1 for the different species tested. These differential effectsmay be due to differences in species sensitivity to BP-3. The BP-3-mediated growth inhibition observed in our study correlates with pre-vious observations. For example, BP-3 can significantly decrease thegrowth rate of S. vacuolatus (Rodil et al., 2009), and Mao et al. de-monstrated that BP-3 can disrupt the growth of both green microalgaand cyanobacteria (Mao et al., 2017). Microalgal growth can be dis-rupted due to overproduction of reactive oxygen species (ROS) whichare easily formed through the disrupted electron transport activities ofchloroplasts, mitochondria and the plasmamembrane, causing severeoxidative damages to cell apparatus (Esperanza et al., 2019). Increasedcytoplasmic and mitochondrial membrane potential and ROS level ofChlamydomonas reinhardtii have been observed with exposure to BP-3(Esperanza et al., 2019). Alternatively, growth inhibition may becaused by disruption to photosynthetic activities and energy transduc-tion (Sieratowicz et al., 2011).RQ values for BP-3 in surface water, wastewater, and seawater were

calculated to predict its potential environmental risks (Table 2). Thisrevealed that RQ values were 1.87, 1092, and 0.91 for surface water,wastewater, and seawater, respectively. High RQs values indicated thatBP-3 presents a high risk to aquatic organisms, and although the RQvalue for seawater is 0.91 (< 1), it is likely that this value will increaseto more harmful levels with continuous consumption and disposal ofBP-3. The RQ calculated in this study indicate an urgent need for BP-3contamination to be addressed, and highlights the importance of effi-cient treatment technologies.

3.2. Changes in photosynthetic pigments

Changes in S. obliquus photosynthetic pigment content were as-sessed (Fig. 2). Total chlorophyll (TChl) content decreased by 8.2, 7.9,and 10.4 % at 1, 2 and 3mg L−1 BP-3, respectively (Fig. 2A). Perox-idation of thylakoid lipids and disruption to the photosystem II com-plexes decrease the chlorophyll content (Ding et al., 2017). DecreasedTChl content is indicative of disrupted photosynthetic activities in S.obliquus in response to BP-3 as chlorophyll embedded in the thylakoidmembranes of chloroplasts plays important roles in photosynthesis forboth light harvesting and energy transfer. The endocrine disruptioneffect of BP-3 may also be responsible for the interruption of photo-synthetic electron flow and disrupt photosynthetic activities (Mao et al.,2017). A slight decrease in the ratio of chlorophyll a (Chl a) andchlorophyll b (Chl b) was also observed (Fig. 2A). Chl a is the primarypigment that converts radiant energy into chemical energy. Chl b is anaccessory pigment, widening the spectrum for light absorption. Chl balso transfers light energy to Chl a. The reduced ratio of these twopigments is consistent with decreased TChl content. Insignificantchanges in TChl content, as well as a lack of change to the Chl a:Chl bratio, may indicate tolerance and recovery capacity of microalgae (Dinget al., 2017; Xiong et al., 2016).The carotenoid content of S. obliquus increased by 4.43 %, and de-

creased by 1.8 %–5.31 % with exposure to 0.1–0.5 mg L−1, and1−3mg L−1 BP-3, respectively (Fig. 2B). Increased carotenoid content

Fig. 1. Effect of benzophenone-3 concentrations on microalgal growth in termsof (A) the dry cell weight (DCW), and (B) specific growth rate of S. obliquus after10 days of cultivation. Error bars represent standard error of the mean (n= 3).Columns with different letters indicate significant differences (p < 0.05) be-tween control and treatment.

Table 1Summary of ecotoxicological effects of benzophenone-3 on microalgae in terms of growth inhibition, obtained in this study and compared with previously reportedwork.

Algal species EC50 of BP-3 (mg L−1) Major focus of the study Refs.

Chlamydomonas reinhardtii 240 h EC50= 1.85 Ecotoxicological evaluation (Mao et al., 2017)Microcystis aeruginosa 240 h EC50= 2.46Scenedesmus vacuolatus 24 h EC50= 0.36 Ecotoxicological evaluation (Rodil et al., 2009)Desmodesmus subspicatus 72 h EC50= 0.96 Ecotoxicological evaluation (Sieratowicz et al., 2011)Scenedesmus obliquus 96 h EC50= 3.64 Ecotoxicological evaluation, biodegradation, metabolic pathway This study

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serves to protect cells from oxidative damage, as carotenoids have areknown to neutralize excited triplet chlorophyll and reduce lipid per-oxidation components, subsequently inhibiting chain reactions and/orquenching accumulated intracellular ROS (Jahns and Holzwarth,2012). Higher concentrations of pollutants cause significant damage toorganelles and disrupt metabolic activity, which can decrease car-otenoid content (Chen et al., 2008). It has been observed that the TChlcontent of the Chlamydomonas reinhardtii and Microcystis aeruginosadecrease with exposure to BP-3. Conversely, the carotenoid content ofM. aeruginosa increases with increasing concentrations of BP-3 (Maoet al., 2017).

3.3. Gene expression in Scenedesmus obliquus under benzophenone-3 stress

Significant changes in S. obliquus functional genes (ATPFOC, Lhcb1,TaS, HydA) were observed under BP-3 stress (Fig. 3). ATPFOC encodesthe subunit C of ATP-synthase. ATPFOC gene expression increased by21 % compared to controls at 0.1mg L−1 BP-3, and decreased by 38 %at 3mg L−1 BP-3. Enhanced activity of Microcystis aeruginosa ATP-synthase in the presence of amoxicillin at low concentrations has been

reported (Liu et al., 2015). Significantly decreased ATP-synthase ac-tivity in Pseudokirchneriella subcapitata upon exposure to a mixture ofvarious pharmaceutical contaminants at high concentrations also hasbeen demonstrated (Esperanza et al., 2019). The observed changes inATPFOC levels were consistent with our growth inhibition data, whichshowed there was a slight effect of BP-3 on microalgal growth whenadministered at low concentrations, and a significant decrease at highconcentrations. There is 5 % and 42 % inhibition in the expression ofLhcb1 gene at 0.1 and 3mg L−1 BP-3, respectively. Lhcb1 plays essentialrole in the conversion of photons to energy and biomass, and protectionfrom photooxidative damage. Decreased Lhcb1 expression may indicatethat photosynthetic activities are disrupted in the presence of BP-3,which correlates with our measurements of total chlorophyll. The HydAgene encodes iron hydrogenase and was significantly inhibited by 18 %and 74 % at 0.1 and 3mg BP-3 L−1, respectively. HydA is linked to thephotosynthetic electron transport chain, and its expression is inhibitedby BP-3, indicating a disruption in photosynthetic activity (Florin et al.,2001). TaS mediates the expression of S. obliquus oxidoreductase B,which was negligibly affected at 0.1mg L−1 BP-3 and was significantlyelevated by 29 % at 3mg L−1. Oxidoreductase is the key enzyme fordegradation of various organic contaminants (García-Rodríguez et al.,2015). Consistent with our data, increased expression of oxidoreductasegene has been observed in S. obliquus exposed to bisphenol A (Wanget al., 2017).

3.4. Removal efficiency of benzophenone-3 and its mechanism

Removal kinetics of microalgae were measured in response to0.1−3mg L−1 BP-3 (Fig. 4). S. obliquus removed 23.3–28.5 % of BP-3after 10 day of cultivation. The removal kinetics of BP-3 by S. obliquuswere fitted into a first order kinetic model, which demonstrated that thekinetic removal rate constant (k, d−1) ranged from 0.023–0.32 d−1,

Table 2Risk quotients (RQs) of benzophenone-3 calculated from the ratio between measured environmental concentrations (MECs) and predicted no effect concentrations(PNECs) for S. obliquus.

96 h EC50 of BP-3 PNECa Surface water Wastewater Seawater RQ for surface water RQ for wastewater RQ for seawaterMEC(μg L−1) MEC(μg L−1) MEC(μg L−1)

3.64mg L-1 3.64 μg L-1 6.812b 3975c 3.3d 1.87 1092 0.91

a Ref. Xiong et al. (2019).b Ref. Tsui et al. (2014).c Ref. Ramos et al. (2016).d Ref. Sánchez-Quiles and Tovar-Sánchez (2015).

Fig. 2. Effect of benzophenone-3 on (A) the total chlorophyll (TChl), and ratiobetween chlorophyll a (Chl a) and chlorophyll b (Chl b) and (B) carotenoidcontent of S. obliquus after 10 days of cultivation. Error bars represent thestandard error of the mean (n= 3). Columns with different letters indicatesignificant differences (p < 0.05) between the control and treatment.

Fig. 3. Effect of benzophenone-3 on the expression of ATPF0C, Lhcb, TAS, HydAgenes of S. obliquus after 10 days of cultivation. Control: microalgae withoutexposure to BP-3; 0.1 mg L−1: microalgae treated with 0.1mg L−1 BP-3;3mg L−1: microalgae treated with 3mg L−1 BP-3. Error bars represent thestandard error of the mean (n=3). Columns with different letters indicatesignificant differences (p < 0.05) between the control and treatment.

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and the degradation half-life (T1/2, d) was calculated to be 22.0–30.5days (Table 3). Higher removal rate constants have been observed withlower exposure concentrations of BP-3 since less toxic effects on S.obliquus have been demonstrated with decreased amount of BP-3. Theremoval mechanisms of BP-3 by S. obliquus were investigated at 0.5 and2mg L−1 BP-3 using mass balance analysis (Table 4). A 3–10 % removalof 0.1−3mg BP-3 L−1 was observed in abiotic controls, which is

consistent with previously reported data (Mao et al., 2017). Bioaccu-mulated BP-3 at 0.5mg L−1 accounted for 0.09 % of the total removedBP-3, while it increased to 7.03 % in the 2mg L−1 condition. Thepercentage of bioadsorption increased from 0.13 % to 9.98 % when BP-3 concentration increased from 0.5mg L−1 to 2mg L−1. Mass balanceanalysis indicated that biodegradation was the major mechanism of BP-3 clearance, accounting for 96.66 and 74.38 % of the total removal of0.5 and 2mg L−1 BP-3, respectively. Higher biodegradation ratio in0.5 mg L−1 BP-3 than 2mg L−1 can be explained by the less toxicity oflower concentrations of BP-3 (Fig. 1A). Meanwhile, the amount ofbioaccumulated and adsorbed BP-3 in S. obliquus significantly increasedfrom lower concentration to higher concentrations, which may explainthe toxicity of high concentrations of BP-3 on S. obliquus since increasedintracellular BP-3 levels lead to the over-production of ROS (Ding et al.,2017). Furthermore, BP-3 can interfere with endocrine function inmicroalgae (Mao et al., 2017). The remediation efficiency of differentemerging contaminants by microalgae, including pharmaceuticals andendocrine disruptors, has been demonstrated (Ding et al., 2017).Increased intracellular bioaccumulation of BP-3 by S. obliquus was

10.63 and 40.10 μg/g at 0.5 and 2mg L−1 BP-3, respectively. BP-3 isreadily accumulated because it has high logKow value of 3.79. Maoet al. also found BP-3 can be taken up by the green alga Chlamydomonasreinhardtii and the cyanobacterium Microcystis aeruginosa, whichshowed M. aeruginosa had a greater overall uptake compared to C. re-inhardtii (Mao et al., 2017). Bioaccumulation of emerging contaminantssuch as levofloxacin (Xiong et al., 2017a) and ibuprofen by variousmicroalgae has also been previously demonstrated (Ding et al., 2017).

3.5. Biodegradation pathway of benzophenone-3 by Scenedesmus obliquus

Identification of the intermediates after microalgal remediation is ofgreat importance because this can illustrate the metabolic mechanisminvolved in the degradation of contaminants. The identified inter-mediates can be matched with data from chemical toxicity libraries tounderstand their potential environmental effects. A degradationpathway for BP-3 was proposed according to the identified inter-mediates using gas chromatography – mass spectrometry (Fig. 5). Themass spectra of the identified BP-3 metabolites are shown in Table S2.Based on our findings, we propose that BP-3 degradation by S. obliquuscan occur through ring cleavage, hydroxylation, and methylation,forming three byproducts after 10 days of cultivation. Intermediate 1(P1) was formed through ring cleavage and hydroxylation, which wasthen further hydroxylated and methylated into intermediate 2 (P2).Intermediate 3 (P3) was generated by methylation of BP-3. Variouspathways for the degradation of organic pollutants by microalgae havebeen reported. For example, ibuprofen can be degraded through hy-droxylation, acylation, demethylation, and glucuronidation by Naviculasp. (Ding et al., 2017), the major mechanisms of bisphenol A destruc-tion by Desmodesmus sp. were hydroxylation, oxidation, and methyla-tion (Wang et al., 2017), and biodegradation of natural and syntheticsteroid hormones by S. obliquus and C. pyrenoidosa was caused by side-chain breakdown, hydroxylation, and de/hydrogenation (Peng et al.,2014).Degradation of organic pollutants by microalgae is a very complex

process, which normally begins with hydroxylation, oxidation, or re-duction reactions mediated by enzymes such as mono/dioxygenase or

Fig. 4. (A) Kinetic analysis of benzophenone-3 removal by S. obliquus and (B)its total removal (%) during 10 days of cultivation. Error bars represent thestandard error of the mean (n= 3).

Table 3Kinetic parameters of benzophenone-3 degradation during 10 days of cultiva-tion.

BP-3 concentration mg L−1 k (d−1) T1/2 (d) R2

0.1 0.0315 22.00 R²= 0.940.5 0.025 27.73 R²= 0.941 0.0281 24.67 R²= 0.902 0.0227 30.54 R²= 0.883 0.024 28.88 R²= 0.89

k – kinetic removal rate constant (day−1).T1/2 – removal half-life (day).R2 – correlation coefficient.

Table 4Mass balance analysis of benzophenone-3 removal by S. obliquus showing biodegradation, bioaccumulation, bioadsorption and abiotic process and its kineticparameters of BP-3 degradation after 10 days of cultivation.

BP-3 concentration mg L−1 Mass balance of BP-3 removal (%)

Biodegradation Bioaccumulation Bioadsorption Abiotic removal

0.5 96.66 ± 2.56 0.09 ± 0.00 0.13 ± 0.01 3.12 ± 0.452 74.38 ± 1.32 7.03 ± 1.03 9.98 ± 1.63 8.64 ± 3.2

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cytochrome P450 enzymes (Xiong et al., 2018). Cytochrome P450 de-grades contaminants by heterolytic cleavage of dioxygen, resulting inC-, N- or S-hydroxylation of the substrate. This renders a compoundmore hydrophilic, thereby enhancing bioavailability. Subsequent con-jugation of contaminants occurs through phase-II enzymes, such asglutathione S-transferase. Finally, the intermediates formed in phases Iand II are differentially transferred into vacuoles in phase III. The ex-pression of various degradation enzymes, such as mono(di)oxygenase,oxidoreductase (Foflonker et al., 2016), and methyltransferase, havebeen studied in microalgae (Wang et al., 2017). Here, we demonstrate

that there is a significant increase in the expression of oxidoreductasegenes when S. obliquus is exposed to BP-3, which may be responsible forthe production of BP-3 intermediates after microalgal degradation.Transcriptomic and proteomic analyses are needed to gather more in-formation about the enzymes and genes involved in microalgae de-toxification and degradation of organic contaminants.

3.6. Toxicity assessment of microalgal degradation intermediates

Toxicity evaluation of the degradation intermediates is necessary

Fig. 5. Proposed degradation pathway of benzophenone-3 by S. obliquus.

Fig. 6. Toxicity assessment of the de-gradation intermediates on fish,daphnia and green algae after micro-algal degradation using EcologicalStructure Activity Relationships(ECOSAR) Program (version 1.11). (A)Acute toxicity of P1, P2 and P3 on fish,(B) Acute toxicity of P1, P2 and P3 ondaphnia, (B) Acute toxicity of P1, P2and P3 on green algae, (D) Chronictoxicity of P1, P2 and P3 on fish, (E)Chronic toxicity of P1, P2 and P3 ondaphnia, and (F) Chronic toxicity of P1,P2 and P3 on green algae.

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since the treatment can produce the compounds that are even moretoxic than their parent compound. In current study, acute and chronictoxicity of the identified intermediates on fish, daphnia and green algaeafter microalgal degradation has been assessed using the ECOSARprogram (Fig. 6). The ECOSAR system can predict the IC50/EC50 valuesof the compounds without standard substances by grouping structurallysimilar organic chemicals with available data of experimental toxicity,and programming of a classification scheme to identify the most re-presentative class for new or untested chemicals. The values of the IC50/EC50 of P1, P2, and P3 on the model organisms have shown in Table S3.The toxicity of the identified intermediates on fish, daphnia, and algaewas in the order of P1 < P2 < P3 < BP-3, P2 < P1 < P3 < BP-3,and P1 < P3 < P2 < BP-3, respectively. In all the cases, BP-3 possessthe highest toxicity compared to its degradation products. The P3 hashigher toxicity on fish and daphnia, P2 was the most toxic product ongreen algae, and P1 was found to be the least toxic on three modelorganisms. These findings showed that the toxicity of degradation in-termediates significantly decreased after microalgal treatment, whichindicated the environmental feasibility of microalgal degradation of BP-3.

4. Conclusion

The green microalga, S. obliquus can withstand high concentrationsof BP-3. The environmental risk quotients of BP-3 under unfavorableconditions in surface water, wastewater and seawater was determined,and implied that the ecotoxicological effects of BP-3 are of significantconcern. Bioaccumulation and biodegradation of BP-3 by S. obliquuswere found. Three intermediates were formed through enzymatic re-actions such as hydroxylation, and methylation. Toxicity of degradationintermediates was significantly decreased compare to parent com-pounds. In all, this study provides the mechanisms of toxicity, removaland metabolism of BP-3 in microalgae, demonstrating the microalgae-based biotechnology for the efficient remediation of wastewater con-taminated by UV filters such as BP-3. Further in-depth transcriptomic,proteomic, and metabolomic studies of BP-3-treated microalgae arerequired to gain a better understanding of detoxification mechanisms ofmicroalgae against organic contaminants.

Declaration of Competing Interest

The authors declare no competition of interests.

CRediT authorship contribution statement

Sang-Hun Lee: Conceptualization, Funding acquisition, Resources,Methodology, Validation, Formal analysis, Investigation, Visualization,Writing - original draft, Writing - review & editing. Jiu-Qiang Xiong:Conceptualization, Funding acquisition, Resources, Methodology,Validation, Formal analysis, Investigation, Visualization, Writing - ori-ginal draft, Writing - review & editing. Shaoguo Ru: Validation,Visualization, Writing - review & editing. Swapnil M. Patil: Validation,Visualization, Writing - review & editing.Mayur B. Kurade: Resources,Investigation, Writing - review & editing. Sanjay P. Govindwar:Resources, Investigation, Writing - review & editing. Sang-Eun Oh:Resources, Writing - review & editing. Byong-Hun Jeon: Funding ac-quisition, Resources, Validation, Supervision, Project administration,Writing - review & editing.

Acknowledgment

This research was supported by the Research Grant of KeimyungUniversity in 2017–2018.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in theonline version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122149.

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