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Environmental Pollution 250 (2019) 503e510

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Environmental Pollution

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Antibiotics in corals of the South China Sea: Occurrence, distribution,bioaccumulation, and considerable role of coral mucus*

Ruijie Zhang a, b, Kefu Yu a, *, An Li b, Yinghui Wang a, Xueyong Huang a

a Guangxi Laboratory on the Study of Coral Reefs in the South China Sea; Coral Reef Research Center of China, School of Marine Sciences, Guangxi University,Nanning, 530004, Chinab Environmental and Occupational Health Sciences, School of Public Health, University of Illinois at Chicago, Chicago, 60612, USA

a r t i c l e i n f o

Article history:Received 2 December 2018Received in revised form30 March 2019Accepted 6 April 2019Available online 13 April 2019

Keywords:Coral reefCoral tissueCoral mucusAntibioticsBioaccumulation

* This paper has been recommended for acceptance* Corresponding author.

E-mail address: kefuyu@gxu.edu.cn (K. Yu).

https://doi.org/10.1016/j.envpol.2019.04.0360269-7491/© 2019 Elsevier Ltd. All rights reserved.

a b s t r a c t

Manmade antibiotics are emerging organic pollutants widely detected in the marine environment. In thisstudy, 14 out of 19 target antibiotics were detected in corals collected from coastal and offshore regions inthe South China Sea. The average total antibiotic concentrations (

P19ABs) in the two regions were

similar: 28 ng/g for coastal corals and 31 ng/g for offshore corals, based on dry tissue weight (dw).Fluoroquinolones (FQs) were predominant antibiotics in the coastal corals (mean

PFQs: 18 ng/g dw),

while sulfonamides (SAs) predominated in the offshore corals (meanP

SAs: 23 ng/g dw). However, coralsliving in coastal regions tend to excrete more mucus than corals in offshore habitat. We found 53% byaverage of

P19ABs in the mucus of the coastal corals; while in offshore corals, most antibiotics (88% by

average) were accumulated in the tissues. In addition, the tissue-mucus mass distribution differs amongindividual antibiotics. Sulfonamides were mainly accumulated in tissues while fluoroquinolones werepresent mainly in mucus. The results of this study suggest that mucus played an important role in thebioaccumulation of antibiotics by corals. It may resist the bioaccumulation of antibiotics by coral tissue,especially for the coastal corals. Additionally, corals were compared with other marine biotas in the studyarea and found to be more bioaccumulative towards antibiotics.

© 2019 Elsevier Ltd. All rights reserved.

1. Introduction

Antibiotics are pharmaceuticals widely used in humans andanimals. In China, the total consumption of 36 common antibioticswas 92,700 tonnes in 2013 (Zhang et al., 2015). More than half(53,800 tonnes) has entered the environment due to their wastefuluses, incompletemetabolism in human and animals, and inefficientremoval during wastewater treatment (Kim and Carlson, 2007;Lindberg et al., 2005; McArdell et al., 2003; Zhang et al., 2015).Designed for bactericidal toxicity, antibiotics are often complex inmolecular structure. They differ widely in physicochemical prop-erties, and many are ionizable and highly soluble in water. Similarto the cases of other chemicals of emerging concerns, our knowl-edge on the behavior and impact of antibiotics in natural waters islimited.

Reef building corals are marine invertebrates with calcium

by Prof. Dr. Klaus Kümmerer.

carbonate skeleton and soft tissue that exudes mucus. The lipidcontent of the soft tissue varies widely among coral species, and upto 58% of the total dry weight of the tissue could be lipid (Imbs,2013). The mucus layer is vital to corals, as it interfaces betweencoral epithelium and seawater, aids heterotrophic feeding andsediment cleansing, and acts against various environmentalstresses. However, coral mucus has not been well defined andcharacterized (Brown and Bythell, 2005). Multitudinous chemicalpollutants such as crude oil and oil dispersants, nutrients, heavymetals, pesticides and herbicides, and traditional persist organicpollutants are known to negatively affect corals (Ali et al., 2011; El-Sikaily et al., 2003; El Nemr et al., 2004; Glynn et al., 1984; Khaledet al., 2003; Ko et al., 2014; Miao et al., 2000; Ramos et al., 2004;Sabourin et al., 2013; Shafir et al., 2007; Szmant, 2002). Because oftheir antibacterial properties, antibiotics may cause unpredictableeffects on the coral reefs ecosystem (Sweet et al., 2014). Little isknown on the bioaccumulation of various pollutants, especiallythose of emerging concerns, by corals as well as the roles played bytheir mucus and tissue.

The South China Sea (SCS) has a water surface area of 3 millionkm2 and surrounded by seven countries. It is home to the most

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510504

diverse and well-developed coral reefs in the world. In the SCS,coral reefs extensively distributed in coastal and offshore regions.Over the past 50 years, the coral reefs have suffered a dramaticdecline due to climate changes, destructive fishing practices,coastal development, and release of toxic chemicals (Burke et al.,2002; UNEP, 2004; Yu, 2012). In 2009 alone, an estimated 193tonnes of antibiotics were discharged into the SCS (Xu et al., 2013).In our recent study, 13 antibiotics were detected in surface waterfrom coastal and offshore coral reef regions (CRRs) of the SCS(Zhang et al., 2018c). The occurrence of antibiotics in corals has notbeen previously reported for the SCS or any other parts of the globalmarine environment.

We hypothesized that antibiotics have accumulated to detect-able levels in corals of the SCS, and that different coral species andcoral components, such as tissue and mucus, have different bio-accumulation potentials for antibiotics. In this study, 40 coralspecimens from the SCS CRRs were collected and analyzed for 19antibiotics. The coral samples belong to 7 families, 14 genera, and26 species. Each coral sample was separated into tissue and mucus.The antibiotics included sulfonamides and synergists, fluo-roquinolones, macrolides, and chloramphenicols. To our knowl-edge, this is the first report on man-made antibiotics in corals.

Fig. 1. Sampling location in the South China Sea. The three coastal coral reef regions (CRRs)CRRs include Xisha Islands and Nansha Islands. Xisha Islands sampling sites include Yongxin(SJ), Xian'e Reef (XE), and Xinyi Reef (XY).

2. Materials and methods

2.1. Study areas and sample collection

Three coastal and two offshore CRRs were selected as studyareas (Fig. 1). The location information, sampling time, and waterquality data are given in Supporting Information (SI) Table S1. Thethree coastal CRRs included Daya Bay (DY), Weizhou Island (WZ),and Sanya Luhuitou (SY). They all are famous tourist areas and theirecosystems have been heavily affected by human activities. DayaBay is located in the Pearl River Delta Economic Zone, which isdensely populated and known for aquaculture. Coral reefs in DayaBay are sparsely distributed in a simple community structure, witha narrow growth zone and few coral species (Chen et al., 2009). Itslive coral cover decreased from 77% to 15% between 1983/1984 and2008 (Chen et al., 2009). Weizhou Island has 17,000 residents andabout 600,000 visitors per year in recent years (Wang et al., 2016).Between 1991 and 2010, live coral cover around Weizhou Islanddecreased from 60% to 18% and from 80% to 8% in the south-easternand south-western regions, respectively (Wang et al., 2016). TheSanya Luhuitou fringing reef is in the Sanya National Coral ReefsNatural Reserve. More than 230,000 tonnes of sewage were pro-duced every day by the city of Sanya; one-third of the sewage is

include Daya Bay (DY), Weizhou Island (WZ) and Sanya Luhuitou (SY). The two offshoreg Island (YX) and Langhua Reef (LH). Nansha Islands sampling sites include Sanjiao Reef

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510 505

released untreated into the Sanya Bay north of the Luhuitou Reeftract (Xu, 2015). The coral reefs in this region have also declinedseverely since the 1960s (Shi et al., 2010; Zhao et al., 2012).

The offshore CRRs include Yongxing Island (YX) and LanghuaReef (LH) from the Xisha Islands, and Sanjiao Reef (SJ), Xian'e Reef(XE) and Xinyi Reef (XY) from the Nansha Islands (Fig. 1). YongxingIsland is an administration centre of Sansha City with about 1000residents and 2000 floating population per year (Zhang et al.,2018c). The other four reefs have no permanent residents. Theyare approximately 300e1200 km from Hainan Island and sufferedless from human activity than the coastal regions.

A total of 40 coral samples, including 24 from coastal sites and16 from offshore sites, were collected from May to October 2015and in May 2016 by divers using a hammer and chisel. Their sizeswere from approximately 5 cm� 5 cme10 cm� 10 cm. The sam-ples were placed in polyethylene bags, which were put in a coolerwith ice, immediately after they were taken out of seawater. Thenthe samples were transferred to a �20 �C freezer in the boat in 1 h.All the samples were transported to our laboratory with the freezerand transferred to a�80 �C freezer. Table S2 summarizes the family,genus, and species information for the collected coral samples.

2.2. Analytical procedures

Nineteen antibiotics were targeted in this study (Table 1). Theyincluded seven sulfonamide antibiotics and one synergisttrimethoprim (all together are abbreviated SAs), five fluo-roquinolone antibiotics (FQs), four macrolide antibiotics (MLs), andtwo chloramphenicol antibiotics (CAPs). Selected physicochemicalproperties and molecular structures of the target antibiotics areprovided in Tables S3 and S4, respectively. Four isotope-labelledcompounds (13C, D3-erythromycin, 13C6-sulfamethoxazole, D5-norfloxacin, 13C3-caffeine) were used as analytical surrogates. The

Table 1Antibiotic concentrations in seawater and coral samples from the South China Sea.

Antibiotics Coastal seawater Offshore seawater

(ng/L) (n¼ 15) (ng/L) (n¼ 2)

Name Abbrev D.R. a Mean± SD b D.F. Mean± SD

Sulfadiazine SDZ 87% 0.12± 0.089 0% ndc

Sulfapyridine SPD 40% 0.054± 0.1 0% ndSulfamethazine SMZ 60% 0.081± 0.1 50% 0.26± 0.37Sulfamethoxazole SMX 67% 0.52± 0.5 0% ndSulfadimethoxine SDM 27% 0.022± 0.048 0% ndSulfacetamide SAAM 0% nd 0% ndSulfathiazole STZ 0% nd 0% ndTrimethoprim TMP 60% 0.19± 0.27 0% ndSulfonamides and synergist ∑SAs 100% 0.98 ± 0.94 50% 0.26 ± 0.37Ofloxacin OFX 0% nd 0% ndEnoxacin ENX 0% nd 0% ndNorfloxacin NOX 0% nd 0% ndCiprofloxacin CIX 0% nd 0% ndEnrofloxacin ENR 0% nd 0% ndFluoroquinolones ∑FQs 0% nd 0% ndDehydrated erythromycin DETM 100% 0.57± 0.33 100% 0.071± 0.0Azithromycin AZM 20% 0.12± 0.29 0% ndClarithromycin CTM 100% 0.34± 0.37 100% 0.035± 0.0Roxithromycin RTM 100% 1.01± 0.78 100% 0.17± 0.00Macrolides ∑MLs 100% 2.03 ± 1.30 100% 0.28 ± 0.00Florfenicol FF 100% 0.95± 0.63 100% 0.16± 0.23Chloramphenicol CAP 53% 0.40± 0.50 0% ndChloramphenicols ∑CAPs 100% 1.35 ± 0.88 50% 0.16 ± 0.23All target antibiotics ∑ABs 100% 4.36 ± 1.71 100% 0.71 ± 0.60

a Detection rates.b All the “nd” values were regard as zero in the calculation of mean and standard devc Not detected.d All the “nd” values in the fish were not included in the calculation of Log BAF.

purchasing sources and the handling procedures of all the chem-icals andmaterials used in this work are summarized in Text S1 andTable S3.

Procedures of processing coral samples are illustrated in Fig. S1.Briefly, coral samples were unfreezed firstly, then coral tissues andmucus were separated from the skeleton using a Waterpik (UltraWater Flosser, Jiebi Limited, China) with 1 L synthetic seawater.Coral tissues were separated from the water through settlementand filtering (0.7 mm, GFF filter, Whatman®, England). Coral mucuswas in the filtrate, which looked opaque and colloidal. The antibi-otics in the filtrate were analyzed using the same method forseawater as described previously (Zhang et al., 2018c). Briefly, 0.2 gNa2EDTA was added to each filtrate, which then underwent solidphase extraction (SPE) with Oasis® HLB cartridge (6mL, 500mg,Waters). The cartridges were eluted using 3� 2mL methanol. Theextract was concentrated under nitrogen flow to about 20 mL, andreconstituted to 1mL 10% methanol in water before instrumentalanalysis. The weight or volume of mucus in the filtrate was difficultto quantify; therefore, the antibiotic concentrations in mucus werereported based on the dry weight (dw) of the tissues, whichsecreted the mucus.

Of the 40 coral samples, 27 had almost all their tissues washedout with the mucus by the Waterpik, leaving little on the skeleton.For the other 13 corals (12 out of 19 Favosites corals and one Fun-giidae), some gel-like tissue remained on the skeleton afterWaterpik flushing. This part of the tissue was then collected using atweezers (Fig. S1). The tissues flushed-by-Waterpik and taken-by-tweezers were extracted and analyzed separately. All the tissueswere freeze-dried, weighed, and homogenized. To extract thetarget antibiotics from coral tissues, our method previouslydeveloped for biota samples was used in this work (Zhang et al.,2018a). In brief, tissue samples (approximately 200mg dw) wereextracted by ultrasonic extraction with a mixture of methanol and

Coastal coral Offshore coral Log BAFs ww

(ng/g dw) (n¼ 24) (ng/g dw) (n¼ 16) Coastal coral Offshore coral

D.R. Mean± SD D.R. Mean± SD Mean± SD d Mean± SD

42% 1.42± 2.64 100% 9.74± 5.72 3.31± 0.46 4.45± 0.3942% 0.22± 0.38 nd nd 3.01± 0.70 /46% 0.92± 1.45 100% 4.50± 1.77 3.41± 0.73 3.35± 0.4742% 0.40± 0.57 81% 3.14± 3.26 2.28± 0.83 3.62± 0.550% nd 0% nd / /0% nd 0% nd / /0% nd 0% nd / /46% 0.91± 1.43 100% 5.66± 3.09 3.51± 0.91 4.21± 0.5279% 3.88 ± 5.31 100% 23.0 ± 11.0 2.55 ± 1.01 3.8 ± 0.4313% 0.70± 2.32 nd nd 3.08± 1.10 /8.3% 0.10± 0.37 6.3% 0.35± 1.39 2.06± 0.06 3.12100% 8.27± 13.6 6.3% 2.44± 9.74 3.00± 0.61 3.8538% 8.40± 36.0 50% 1.43± 3.40 3.35± 0.98 2.9± 0.684.2% 0.48± 2.38 6.3% 2.11± 8.45 3.45 4.51100% 18.0 ± 38.6 50% 6.33 ± 20.0 2.68 ± 0.76 2.38 ± 0.86

08 88% 1.55± 1.45 38% 0.70± 1.30 2.32± 0.74 3.37± 0.6638% 0.47± 0.97 nd nd 2.79± 0.39 /

03 46% 0.47± 0.99 13% 0.07± 0.18 2.21± 0.52 3.5± 0.604 88% 3.89± 7.73 94% 1.23± 1.56 2.23± 0.90 2.82± 0.661 96% 6.39 ± 9.90 94% 1.99 ± 2.02 2.27 ± 0.64 2.7 ± 0.63

0% nd 0% nd / /0% nd 0% nd / /0% nd 0% nd / /100% 28.2 ± 43.7 100% 31.4 ± 24.9 2.57 ± 0.64 3.23 ± 0.44

iation (SD).

Fig. 2. Concentrations of antibiotic groups in (A) coral (tissue and mucus combined)and (B) seawater, at coastal and offshore coral reef regions.

PSAs is the sum of 8

sulfonamides and synergists,P

FQs is the sum of 5 fluoroquinolones,P

MLs is the sumof 4 macrolides, and

PCAPs is the sum of 2 chloramphenicols. The “nd” means “not

detected”. The columns show the averages with error bars representing one standarddeviation for four groups of targeted antibiotics.

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510506

0.1M acetic acid in water (50:50, v/v). The resulted solutions werecleaned-up and solid phase extracted with SAX/PSA (6mL, 200 mg/200mg, CNWBOND) and HLB cartridges (6mL, 500mg, Waters) intandem. Subsequent steps for the tissue and skeleton samples,including elution from the cartridges, concentration, reconstitu-tion, were the same as that used for seawater and Waterpikfiltrates.

The target antibiotics and the surrogates in all the samples wereanalyzed using Agilent 1290 ultra-high-performance liquid chro-matography coupled with Agilent 6460 triple quadrupole massspectrometry (UHPLC-MS/MS) (Zhang et al., 2018a; Zhang et al.,2018c). The operational parameters of the UHPLC-MS/MS aredescribed in Text S2 along with Tables S5 and S6.

2.3. Quality control

Procedural blanks (Milli-Q water) were included with eachbatch of samples to check for possible contaminations. None of thetarget antibiotics were detected in the blanks. Prior to SPE extrac-tions of seawater and filtrate samples, each samplewas spiked witha known amount of surrogates 13C, D3-erythromycin, 13C6-sulfa-methoxazole, D5-norfloxacin, 13C3-caffeine. For coral tissue sam-ples, these surrogates were added before ultrasonic extraction. Therecoveries of the four surrogates ranged from 62% to 85%, and 60%e82%, for filtrate and coral tissue, respectively. The reported con-centrations were not adjusted by surrogate recoveries. Instru-mental quantitative limits (IQLs) were determined to be the lowestcalibration concentration resulting in a signal-to-noise ratio (S/N)� 10 (Luo et al., 2011). They ranged from 0.039 to 1.32 ng/mL(Table S6). Method quantitative limits (MQLs) were calculated us-ing IQLs and the samples weight or volume. TheMQLs of the filtrateand coral tissue ranged from 0.039 to 1.32 ng/L, and 0.195e6.6 ng/g,respectively.

3. Results and discussion

3.1. Occurrence and spatial distribution of antibiotics in corals

In the study, antibiotic concentration in the coral is the sum oftheir concentrations in tissue and mucus. The concentrations intissue and mucus are additive because both are based on the dryweight of the tissue. Method of calculation is described in Text S3.

Fourteen of the 19 target antibiotics were detected in the coastalcorals, while 11 in the offshore corals (Tables 1 and S7). The sum ofthe target antibiotics (

P19ABs) in the offshore corals averaged

31.4± 24.9 ng/g dw, and that in the coastal corals averaged28.2± 43.7 ng/g dw. These two averages are not statisticallydifferent (t-test, p¼ 0.798). However, the relative abundance ofdifferent antibiotics differs largely between corals from the coastaland offshore CRRs (Fig. 2). The antibiotics that were detected in>80% of the coastal corals included an FQ (norfloxacin) and twoMLs (dehydrated erythromycin and roxithromycin). In offshorecorals, four SAs (sulfadiazine, sulfamethazine, sulfamethoxazole,and trimethoprim) and one ML (roxithromycin) were widespreadwith detection rate >80%. The average

PSAs in the offshore corals

(23.0± 11.0 ng/g dw) was about six time higher than that(3.9± 5.3 ng/g dw) in the coastal corals (t-test, p¼ 0.000); while theaverage

PFQs and

PMLs in the coastal corals were both higher

than those in the offshore corals (t-test, p¼ 0.22 and 0.044,respectively). In offshore corals,

PSAs were obviously higher than

PFQs (t-test, p¼ 0.007) and

PMLs (t-test, p¼ 0.000); while

PFQs

were higher than the other two groups (t-test, p> 0.05, not sig-nificant) in the coastal corals.

Ambient seawater samples were collected at the same timewithcorals. The water sample information and measured water quality

parameters are summarized in Tables S1 and S2. The occurrence ofantibiotics in the seawater samples were reported previously(Zhang et al., 2018c). The data obtained for the coral sampling sitesare included in Table 1 and Fig. 2 in order to compare with those ofthe corals. Twelve antibiotics were detected in coastal water andfive in offshore water. The

P19ABs in the coastal water averaged

4.36 ng/L, which was significantly higher than the average of0.71 ng/L found in the offshore water (t-test, p¼ 0.004). The anti-biotic groups

PSAs,

PMLs, and

PCAPs were all higher in the

coastal water than that in the offshore water (p¼ 0.132, 0.000, and0.004, respectively). In general, less antibiotics were detected inseawater than corals. In particular, the five FQs that were detectedin corals were not found in the seawater samples. However, neitherof the two CAPs (florfenicol and chloramphenicol), which werewidely detected in coastal seawater, were found in corals.

Comparing among coral families (Fig. S2), the averageP

19ABs incorals from the coastal sites is in the rank order of Acroporidae(71.8± 85.8 ng/g dw, n¼ 4)> Poritidae (61.5± 73.4 ng/g dw,n¼ 3)> Faviidae (11.1± 5.77 ng/g dw, n¼ 13). The rank order in theoffshore corals is Faviidae (46.1± 34.9 ng/g dw, n¼ 6)> Pocillopor-idae (29.6± 14.3 ng/g dw, n¼ 3)> Acroporidae (25.7± 11.0 ng/g dw,n¼ 3)> Fungiidae (14.7± 7.76 ng/g dw, n¼ 3). However, the dif-ferences among coral families are not statistically significant (t-test,all the p values> 0.05). The limited number of samples may haveweakened the power of the comparisons.

We converted the antibiotic concentrations expressed in drytissue wight to in wet weight by applying the correction factorstaking into account the moisture conctent of the coral tissues(Table S8). The antibiotic concentrations in the corals werecompared with those in other biotas of the SCS in Table S9.

PSAs,

PFQs and

PMLs were noticeably higher in the corals than in the

coral reef fish muscle from the same study sites (Xisha Islands andWeizhou Island) (Zhang et al., 2018b). Additionally, the concen-trationsmeasured in this study are also higher than the levels in thecultured organisms (shrimp, oyster, crab) frommariculture areas in

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510 507

the Beibu Gulf, the SCS(Zhang et al., 2018a). The highestP

SAs,P

FQs andP

MLs in the corals were equal to or lower than those inthe cultured organisms surrounding Hailing Island, the SCS (Chenet al., 2015). Comparing with other chemicals in corals of the SCS,the individual antibiotic levels in the corals of this work (nd-87.9 ng/g ww; nd-181 ng/g dw) were similar to those of some in-dividual UV filtering compounds used in personal care products(range of average concentrations: nd-31.8 ng/g ww) from theHongkong CRRs (Tsui et al., 2017) individual PAHs levels from theKenting CRRs (range of average concentrations: nd-236 ng/g dw)and the Sanya CRRs (range of average concentrations: nd-272 ng/gdw) (Ko et al., 2014; Xiang et al., 2018).

3.2. Tissue-mucus distribution of antibiotics in corals

Weusedmass fractions of antibiotics in tissue (ftissue) andmucus(fmucus) in the whole coral to describe tissue-mucus distribution ofantibiotics in coral. For the same antibiotics in the same coralsamples, ftissue þ fmucus¼ 1. The average fmucus values are shown inFig. 3 for three groups of antibiotics and

P19ABs. In offshore corals,

ftissue averaged 0.88± 0.13 and fmucus averaged 0.12± 0.13 forP

19ABs (Fig. 3). We noticed that the mucus layer is much thinner inthe offshore coral samples than that of the coastal corals. This is notsurprising because the secretion of mucus is often less in cleanerambient waters that are farther away from land-based pollutionsources (Brown and Bythell, 2005). Coastal corals secrete moremucus than offshore coral in response to the heavier environmentalstress (Bessell-Browne et al., 2017; Brown and Bythell, 2005;Huettel et al., 2006). The average fmucus of

P19ABs was 0.53± 0.34

in coastal corals, which is significantly higher than that in theoffshore corals (t-test, p¼ 0.000) (Fig. 3). This observationdemonstrated that mucus played considerable role in the accu-mulation of antibiotics by coastal corals.

The difference in tissue-mucus distribution among coral fam-ilies was not evident except that most (13 out of 16) Faviidaeaccumulated more than half antibiotics in tissue. For all coastalcorals in the families of Poritidae, Acroporidae and Pocilloporidae,fmucus is greater than ftissue; while corals in the same families butcollected from offshore sites had more

P19ABs in the tissues than

in the mucus (Fig. S3). In conclusion, the mass distribution of an-tibiotics in corals betweenmucus and tissue largely depends on theamount of the mucus secreted by the coral.

The tissue-mucus mass distribution differs among individualantibiotics. The

PSAs were mainly accumulated in tissues. Espe-

cially, the average ftissue ofP

SAs in the offshore corals was up to0.96± 0.05. The

PFQsmainly occurred inmucus. Their fmucus in the

coastal and offshore corals averaged 0.89± 0.26 and 0.79± 0.39,respectively.

PMLs had an average ftissue of 0.56± 0.41 in the

coastal corals; while in the offshore coralP

MLs was higher inmucus than in the tissue, with an average fmucus of 0.82± 0.40.

Fig. 3. Tissue-mucus distribution of antibiotics in corals. The fmucus is the mass fractionof antibiotics in the mucus of the coral. The columns show the averages with error barsrepresenting one standard deviation for three groups and the sum of targetedantibiotics.

Coral mucus is composed of mucins along with a variety ofdissolved and/or particulate organic matter excreted by coral.Mucus protects the coral tissue by acting as a physical barrier forxenobiotics to reach over the epidermis (Johnston and Rohwer,2007). With thicker mucus layer in the costal corals, more tar-geted antibiotics were accumulated in the mucus than in the tissue.Additionally, coral mucus accumulates particulate matters fromambient environment. Chemical pollutants absorbed on the par-ticulate matter may enter coral mucus through sorption on theirhost particles. This was demonstrated for aromatic hydrocarbons(Neff and Anderson, 1981). Among the antibiotics involved in thiswork, FQs could have higher tendency of sorption on particle sur-faces due to their planar quinolone core structure. Sorption of FQsonto suspended particles and sediments has been demonstrated(Kim and Carlson, 2007; Tolls, 2001; Yang et al., 2010) and sorptionmay be an important route of FQ accumulation in coral mucus. Theresults of this work may also imply that the coral tissue tends toaccumulate SAs. Upon uptake from water or ingestion, SAs such assulfamethoxazole and trimethoprim were found to be well absor-bed in the intestinal tracts but not efficientlymetabolized in aquaticorganisms, such as crustaceans, mollusks, and fish (Chair et al.,1996; Connors et al., 2013; Klopman et al., 2002; Meshi and Sato,1972; Nouws, 1985; Touraki et al., 1999). It was also reported thatSAs biomagnified in marine food web of the Laizhou Bay, NorthChina (Liu et al., 2017). Future studies on the mechanisms ofaccumulation by corals and their components are warranted.

However, it should be noted that the approach used to collectmucus in this study is not the best way to have puremucus. The factthat the samples were frozen and thawed would indicate sometissue lysis and thus the ‘filtrate’ is likely a mixture of mucus, de-natured protein and other cellular debris that was able to passthrough the 0.7 mm filter. Therefore, it may have limited the accu-racy oft tissue-mucus distribution of antibiotics in the corals.

3.3. Tissues flushed-by-waterpik and taken-by-tweezers

After pressurized flushing by the Waterpik, 13 coral samples (12Faviidae and 1 Fungiidae) had a portion of tissues left on theirskeletons, which had to be taken by a tweezer. These corals hadrelatively high proportion of coenosarc as well as larger and deepercuplike structure. Microscope analysis revealed abundant presenceof zooxanthellae, which is photosynthetic algae living in coral tis-sues, in the tissue flushed-by-Waterpik but little in the tissuetaken-by-tweezers (Fig. S5). Most of zooxanthellae with polyp mayhave been flushed down by the Waterpik, while parts of coenosarcclosely adhere to the skeleton were left in the skeleton.

For 10 of the 13 corals, the quantities of the tissues taken-by-tweezers were less than those flushed-by-Waterpik (Figs. S5eA).More importantly, we found that the concentrations of

P19ABs in

the tissues taken-by-tweezers were about six times higher than inthe tissues flushed-by-Waterpik (Figs. S5eB). By average, 64% and54% in the coastal and offshore coral samples, respectively, werefound in tissues taken-by-tweezers (Fig. 4).

3.4. Bioaccumulation of antibiotics in corals

In this work, bioaccumulation factors (BAFs, in L/kg) werecalculated by dividing the antibiotic concentrations (ng/g ww tis-sue) in whole coral by that in the ambient seawater (ng/L) and thenmultipling by 1000. For four areas (Weizhou Island, Daya Bay, Sanyaand Xisha Islands), the average antibiotic concentrations in theseawater of each area were used, respectively (Zhang et al., 2018c).For Nansha Islands, the concentrations in seawater of the XishaIslands were used because the seawater samples of the NanshaIslands were lost during sampling thus the data are not available.

Fig. 5. Bioaccumulation factors (BAFs) of antibiotics in corals from the South China Sea.The full names of individual antibiotics are given in Table 1. A chemical is “bio-accumulative” if its log BAF is greater than 3.7 (red dotted line), and “potentiallybioaccumulative” if log BAF is between 3.3 (blue dotted line) to 3.7. (For interpretationof the references to colour in this figure legend, the reader is referred to the Webversion of this article.)

Fig. 4. Distribution of antibiotics in the coral tissues flushed-by-Waterpik and taken-by-tweezers. The size of the pies and data under the pies represent average concen-tration of

P19ABs in coral tissues.

Fig. 6. Relationship between bioaccumulation factors (BAFs) in the corals and pH-adjusted octanol-water distribution coefficient (D) of detected antibiotics in calcifiedfluid of corals (pH¼ 8.55).

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510508

TheMQLs were used in the BAF calculations if the concentrations inseawater were below the MQL. The BAFs were not calculated if theconcentrations in corals were below the MQL.

Calculated BAFs are given in Table 1 and shown in Fig. 5. Ac-cording to European Chemicals Agency, a chemical is “bio-accumulative” if its BAF is greater than 5000 L kg�1 (log BAF 3.7),and “potentially bioaccumulative” if BAF is between 2000 (log BAF3.3) to 5000 L kg�1 in biota (European Chemicals Agency, 2012). Bythese criteria, twelev antibiotics presented bioaccumulative (logBAF> 3.7 in a few or many corals of this study. The log BAFs of theSAs, FQs, and MLs in the corals range from 1.04 to 5.11, 1.98 to 5.54,and 0.40 to 4.27, respectively. In general, the offshore corals pre-sented higher bioaccumulation ability to SAs than to other anti-biotic groups, while the coastal corals did to FQs. Based on theavailable data (Table S9), the BAFs for corals are generally higherthan those for coral fish (log BAFs: �0.34 to 4.22) from the samearea (Zhang et al., 2018b), the crab, shrimp, and oyster (logBAFs: �0.29 to 4.22, most <3.3) from the Beibu Gulf of the SCS, thecultured crab, shrimp, oyster and fish (log BAFs: 0.30 to 3.81) fromHailing Bay of the SCS, the fresh water organisms (crab, river snail,shrimp, lobster, fish, turtle and bird) (log BAFs <4.22) in Baiyang-dian Lake and in the fish of the Haihe River of North China(2.45e3.65) (Chen et al., 2018; Gao et al., 2012; Li et al., 2012; Zhanget al., 2018a). In conclusion, corals have stronger bioaccumulativeabilities to antibiotics than many other biotas. The structure ofcorals features large surface area (Yonge, 1940) and mucus withhigh content of particulate organic matter (Huettel et al., 2006),which may contribute to their strong bioaccumulative abilities.

Many antibiotics are polar molecules and contain ionizablefunctional groups. According to previous studies (Fu et al., 2009;Meredith-Williams et al., 2012), pH-corrected octanol-waterpartition coefficient (D) is useful for estimating the uptake ofionizable compounds by biota, as it accounts for difference in par-titioning of the neutral and ionic species of a molecule at aparticular pH value. Strong positive linear correlations were foundbetween log D and logarithm bioconcentration factors (BCFs) forsome pharmaceuticals in freshwater shrimp (Gammarus pulex) andwater boatman (Notonecta glauca) (Meredith-Williams et al., 2012).A significant positive correlation was observed between trophicmagnification factors of the sulfonamides and fluoroquinolonesand their log D values in the marine food web of Laizhou Bay, NorthChina (Liu et al., 2017). In this study, we calculated the log D(Table S3) using the following equations presented by Fu et al.(2009):

fn ¼ 11þ 10iðpKa�pHÞ (1)

D ¼ fn$KowðneutralÞ þ ð1� fnÞ$KowðionÞ (2)

log KowðionÞ ¼ logKowðneutralÞ � 3:5 (3)

where i is 1 for bases (FQs and MLs) or �1 for acids (SAs), and fn isthe fraction of the compound in neutral form. pKa is the negativelogarithm of the acid dissociation constant, and the pKa values forindividual antibiotics are presented in Table 1. The pH of 8.55 forcalcified fluid of corals by McCulloch et al. (2012) was used with eq.[1].

We found a significantly negative correlation between log BAFsof the detected antibiotics and their log D values in the corals(Fig. 6). It was opposite to the previously reported positive corre-lations between log BCFs and log D for some aquatic organisms,such as fish, shrimp and water boatman (Fu et al., 2009; Meredith-Williams et al., 2012). Different from BCFwhich describes uptake ofchemicals dissolved in water due to passive partitioning, BAF takesinto account of all accumulation routes including ingestion of foodand particulate matter to which chemicals adhere. In this study, theantibiotics in corals should come from water, coral food (mainlyzooplankton), and particles caught by coral tentacles or in sheets ofmucus. In addition, the values of fn and log D of the SAs aregenerally lower but the FQs and MLs are higher in calcified fluid ofcorals (pH¼ 8.55) than in typical animal cytosol (pH¼ 7.4) andintestinal tract of marine fish (pH¼ 7.5) (Fu et al., 2009; Liu et al.,

R. Zhang et al. / Environmental Pollution 250 (2019) 503e510 509

2017; Meredith-Williams et al., 2012). Furthermore, the pH effecton speciation between neutral and ionic form differs among anti-biotics of different pKa's. For example, at pH 8.55, almost all sul-fathiazole (STZ) would deprotonate thus be ionic (fn¼ 0.03), while84% of ofloxacin (OFX) will remain neutral (Table S3). The neutralform of organics tends to have higher bioaccumulation potentialthan their ionized counterpart, given their order-of-magnitudehigher Kow values (equation [3]). Roughly speaking, the log Dvalues of SAs are generally lower than the log D values of FQs andMLs at pH 8.55 (Table S3); however, the opposite is true when pH is7.4 or 7.5. Besides the intake of particulate matter, the oppositeeffects of varying pH on neutral-ionic speciation for organic acidsand organic bases may help explain the contrasted trends in cor-relations of BCF or BAF with log D, between aquatic animals andreef-building corals. The cytosol pH of zooplankton should betypical animal cytosol pH 7.4 and their BCFs to antibiotics shouldpositive correlate to the log D at pH 7.4, i.e., SAs with higher log Dshould show higher BCFs while FQs andMLs with lower log D showlower BCFs. But for corals at pH 8.55, SAs have lower log Dwhile FQsand MLs have higher log D. If just taking account the uptake ofantibiotics dissolved in water, coral may have lower BAFs for SAsand higher BAFs for FQs and MLs. It was opposite to the facts.Therefore, coral ingestion should play an important role in coralbioaccumulating antibiotics.

Fu et al. reviewed the BCFs of ionizing organic compounds in fishand established BCF regression models for acids and bases based onthe pKa and log Kow(Fu et al., 2009). In this study, we predicted logBCF values of the target antibiotics according to Fu's equations,which are presented with SI Fig. S6. The calculated BCFs werecompared with the measured BAFs. The results showed that ourmeasured BAF values were higher by 2e4 log units than the pre-dicted BCF values (Fig. S6), suggesting that coral may have strongpassive partitioning uptake from water, and/or dietary intake ofantibiotics by corals greatly outweighs their passive partitioninguptake.

It should be noted that the BAF calculations abovewere based ondata obtained from one-time sampling in a dynamic water envi-ronment (Tsui et al., 2017). Difficulties in quantifying the mass andvolume of coral mucus in the filtrates may have also brought un-certainties to the calculated BAFs. Nonetheless, this study is the firstattempt to estimate BAFs for antibiotics in corals and their com-ponents. The results indicate clearly that corals accumulate mostantibiotics targeted in this work.

4. Conclusion

Most of target antibiotics (14/19) were detected in the coralsamples from the CRRs in the South China Sea, with the averageconcentrations ranging from 0.07 ng/g dw to 9.74 ng/g dw. Theaverage

P19ABs in the coastal and offshore CRRs were 28 ng/g dw

and 31 ng/g dw, respectively. They are not statistically different.However, the average

P19ABs in the coastal seawater (4.36 ng/L)

was significantly higher than in the offshore seawater (0.71 ng/L).Coastal corals may secret more mucus than offshore corals becauseof poor water quality in coastal regions. Therefore, more than halfP

19ABs in the corals were in mucus for the coastal corals while 11%for the offshore corals. Coral mucus played the role of resistingbioaccumulation of antibiotics by coral tissue especially in thecoastal CRR. Therefore,

P19ABs in the coastal coral tissue was even

lower than in the offshore coral tissue althoughP

19ABs in thecoastal seawater was obviously higher than in the offshorewater. Inaddition, coral tissue and mucus had different accumulation abili-ties to different antibiotic groups, i.e., sulfonamides were mainlyaccumulated in tissues while fluoroquinolones were presentmainly in mucus. In the last, corals were compared with other

marine biota in the study area, and found to be more bio-accumulative towards antibiotics. Their strong bioaccumulationability may be caused by their large surface area.

Acknowledgments

This work was supported by the National Natural ScienceFoundation of China (91428203, 41463011 and 41673105), theGuangxi scientific projects (Nos. 17129063 CE, AA17204074,2015GXNSFBA139185 and 2016GXNSFAA380011), the BaGuiFellowship from Guangxi Province of China (2014BGXZGX03),China Postdoctoral Science Foundation (2016M602614) and theGuangxi Postdoctoral Special Fund. We would also like to thankZhenjun Qin for assistance with identifying coral species. ThankJiying Pei, Ruiling Zhang, Minwei Han and Weibin Zeng for assis-tance with sample treatment and analysis.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.envpol.2019.04.036.

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