Contributions of Abiotic and Biotic Processes to the Aerobic Removal

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Contributions of abiotic and biotic processes to the aerobic removal of phenolic endocrine-disrupting chemicals in a simulated estuarine aquatic environment Lihua Yang, Qiao Cheng, Nora FY Tam, Li Lin, Weiqi Su, and Tiangang Luan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b06196 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 23, 2016

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Environmental Science & Technology

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Contributions of abiotic and biotic processes to the aerobic

2

removal of phenolic endocrine-disrupting chemicals in a simulated

3

estuarine aquatic environment Lihua Yang 1, 2, Qiao Cheng 1, Nora FY Tam 2, Li Lin 3, Weiqi Su 1, Tiangang Luan 1, *

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1

MOE Key Laboratory of Aquatic Product Safety, Guangdong Provincial Key

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Laboratory of Marine Resources and Coastal Engineering, School of Marine

7

Sciences, Sun Yat-Sen University, Guangzhou 510275, China

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2

Hong Kong SAR, China

9 10

3

13

MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China

11

12

Department of Biology & Chemistry, City University of Hong Kong, Kowloon,

*

Corresponding author. Tel: +86 2084112958; fax: +86 2084113785; E-mail address: [email protected]

14 15

1

ACS Paragon Plus Environment


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Abstract

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The contributions of abiotic and biotic processes in an estuarine aquatic environment

18

to the removal of four phenolic endocrine-disrupting chemicals (EDCs) were

19

evaluated through simulated batch reactors containing water-only or water-sediment

20

collected from an estuary in South China. More than 90% of the free forms of all four

21

spiked EDCs were removed from these reactors at the end of 28 days under aerobic

22

conditions, with the half-life of 17α-ethynylestradiol (EE2) longer than those of

23

propylparaben (PP), nonylphenol (NP) and 17β-estradiol (E2). The interaction with

24

dissolved oxygen contributed to NP removal and was enhanced by aeration. The PP

25

and E2 removal was positively influenced by adsorption on suspended particles

26

initially, whereas abiotic transformation by estuarine-dissolved matter contributed to

27

their complete removal. Biotic processes, including degradation by active aquatic

28

microorganisms, had significant effects on the removal of EE2. Sedimentary inorganic

29

and organic matter posed a positive effect only when EE2 biodegradation was

30

inhibited. Estrone (E1), the oxidizing product of E2, was detected, proving that E2 was

31

removed by the naturally occurring oxidizers in the estuarine water matrixes. These

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results revealed that the estuarine aquatic environment was effective in removing free

33

EDCs, and the contributions of abiotic and biotic processes to their removal were

34

compound specific.

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2


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Endocrine-disrupting chemicals (EDCs), important groups of toxic organic

38

contaminants, have attracted increasing attention in recent years. Exposure to

39

exogenous EDCs, even at low concentrations (ng/L), could have potential harmful

40

impacts on the hormonal control, sexual differentiation, reproductive success, and

41

community structure of aquatic organisms and their offspring. 1-4 Phenolic compounds,

42

such as the natural estrogens 17β-estradiol (E2) and estrone (E1), synthetic estrogen

43

17α-ethynylestradiol (EE2), and industrial compounds, nonylphenol (NP) and

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propylparaben (PP), are the main representative EDCs that have been widely detected

45

in different environmental matrixes around the world.

46

EDCs are controlled by their fate and behavior in the environment. Significant

47

removal of E1, E2 and EE2 from river water, 11 E2, E1 and NP from river sediment, 12-14

48

or E2, EE2 and NP from water-sediment systems

49

suggesting that natural aquatic environments are efficient systems for the removal of

50

EDCs.

Introduction

5-10

15-19

The occurrence and risk of

have been widely reported,

51

The elimination and transportation of EDCs in the environment are controlled by

52

abiotic and biotic processes/factors. Biotic processes, such as biodegradation by

53

aquatic and/or sedimentary microorganisms, controlled the removal of E2,

54

22

55

microorganisms enriched from the field environment to degrade NP,

56

26

57

processes, such as sorption of E2, E1 and EE2 in sterilized water or sediment were

58

found to play a significant role in the removal of EDCs. 15, 27 Photo-degradation of E2

59

was also important in water matrixes.

60

influenced by abiotic factors, such as total organic carbon content,

61

oxygen (DO) content,

62

pollutants of the sampling site

63

specific EDC compounds, such as octanol/water partitioning coefficients (log Kow) 15,

64

27

EE2

21-24

and NP in a simulated natural environment.

14, 23, 24

20-24

E1,

20,

The ability of 14, 25

E1 and EE2

was also demonstrated by laboratory experiments. On the other hand, abiotic

17, 18, 30

28, 29

Additionally, the removal of EDCs was

biochemical oxygen demand (BOD), 13, 23

12, 13

15

dissolved

salinity,

15, 31

and the physico-chemical characteristics of

and the isomer structure.32 Although some studies have reported the removal of 3



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spiked EDCs in a simulated environment by biodegradation or adsorption, most of the

66

published data focused on a single removal process. In the natural environment, the

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existence of various biotic and abiotic processes makes the removal of EDCs

68

complicated. However, the role of each process, and the interactions among different

69

processes, have seldom been explored. In addition, instead of a single pollutant,

70

mixed pollutants are commonly found in the natural environment, which merits the

71

detailed clarification of compound-specific removal processes.

72

Estuarine ecosystems near mega-cities receive the direct input of different classes

73

of pollutants from domestic and industrial waste and exhibit the characteristics of high

74

organic matter (OMs) and suspended particles, abundant microorganisms, fluctuations

75

in dissolved oxygen content and the wide occurrence of EDC pollution.

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removal of EDCs in this aquatic system is very complicated due to the existence of

77

various biotic and abiotic processes/factors in the estuarine water and sediment. The

78

published studies on EDC removal in estuarine environments have focused only on a

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single process, such as sorption of steroid estrogens to solid phases from the

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Blackwater estuary

81

Estuary. 36 To date, there has been one report regarding plant effluent showing that the

82

biodegradation process by fortified algae for NP removal was more important than the

83

sorption process by suspended particles at 500 ± 200 mg TSS/L.30 The controlling

84

processes responsible for EDC removal, the underlying mechanisms and their

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interactive effects have seldom been investigated, especially in estuarine

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environments. The evaluation of the contribution of each biotic/abiotic process in the

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removal of a specific EDC under the conditions of mixed pollutants, as well as the

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underlying mechanisms and the interactive effects, is a unique approach.

15

10, 33-35

The

and photochemical degradation of EE2 in the Acushnet River

89

The present study, based on simulated batch reactors containing water-only or

90

water-sediment under aerobic conditions, aims to (i) understand the contribution and

91

interactions of different abiotic and biotic processes/factors in a simulated estuarine

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environment on the removal of four representative EDCs; (ii) identify the dominant

93

process controlling the removal of each EDC compound and its influencing factors;

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and (iii) determine the occurrence and controlling removal processes/factors of the 4


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metabolite E1 from E2 transformation.

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Material and methods

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Chemicals and reagents. Standards, including those for propylparaben (PP, 99+%),

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technical nonylphenol (NP, 99+%), 17α-ethinylestradiol (EE2, 99+%), 17β-estradiol

100

(E2, 99+%), and estrone (E1, 99+%), were purchased from Aldrich Chemistry

101

Corporation (Milwaukee, WI). The chemical structures and physical-chemical

102

properties are shown in Table S1. Internal standards, including NP-d4 and EE2-d4 were

103

obtained from Sigma-Aldrich (St. Louis, MO, USA). The derivatization reagent

104

N,O-Bis (trimethylsilyl) trifluoroacetamide with 1% of trimethylchlorosilane

105

(BSTFA/TMCS) was obtained from Supelco (Supelco Park, PA). The fluorescent

106

stain 4',6-diamidino-2-phenylindole (DAPI), HPLC grade acetone and methanol were

107

all from Sigma-Aldrich (St. Louis, MO, USA). Pyridine (AR) and sodium azide

108

(NaN3) were from Guangzhou Chemical Reagent Factory (Guangzhou, China).

109 110

Experimental design. In March of 2012, estuarine water and sediment samples

111

were taken from the Humen Estuary (Figure S1, 22。48’02’’N; 113。36’12’’E), one of

112

the most heavily contaminated estuaries in the Pearl River Delta, South China. 10, 33 A

113

total of 100 L of the water samples from a depth of 10-15 cm below the surface were

114

collected from three sites distributed in a triangular configuration (adjacent distance

115

of approximately 1.5 km) in this estuary using a glass collector. The samples were

116

placed in 4 L brown glass bottles. Surface sediments (approximately 20 L) from the

117

top 15 cm were also collected from the same sampling site using a stainless steel grab

118

sampler.

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Round glass columns (height: 40 cm; i.d.: 15 cm; volume: 10 L) were used as the

120

reactors in the laboratory for the different treatment groups; each glass column

121

contained 3 L of water-only or a mixture of 2.5 L water and 1 L wet sediment

122

(moisture content = 49.3%). The experimental reactors were set according to the

123

OECD 308 guideline.

37

To study the contribution of abiotic and biotic processes, as 5



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well as the significance of the controlling factors involved, seven treatment groups

125

with estuarine water-only (groups ① and ②) and estuarine water-sediment system

126

(groups ⑤, ⑥, ⑦, ⑧ and ⑨) were set in triplicate (Table 1).

127

No sterilized estuarine water (EW) or estuarine sediment (ES) was used after

128

mixing without any treatment. The sterilized water (EWS) was sterilized by

129

autoclaving at 120°C for 30 min twice and the sterilized sediment (ESS) was

130

sterilized by autoclaving at 120°C for 60 min for 4 times. NaN3 at a concentration of 1

131

mg/L was added to all the sterilized water groups (once per week) to ensure a sterile

132

condition was maintained during the experiment. The addition of 1 mg/L NaN3 did

133

not have a significant effect on EDC removal in our previous study. The organic

134

matter removed sediment (ESM) was achieved by heating in a furnace at 550°C for 5

135

hours. None of the experimental set-ups in Table 1 were shaken during the incubation,

136

but the set-ups were gently aerated by an air pump with an aeration speed of 0.9

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L/min to maintain the oxic condition.

138

After acclimatization for a week, each group was spiked with a mixture of EDCs

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(PP, NP, EE2 and E2, each at a concentration of 100 µg/L) in water. During the 28-day

140

incubation, the columns were wrapped in aluminum foil to prevent photolysis. The

141

presence

142

4',6-diamidino-2-phenylindole (DAPI) staining technique. In brief, the water sample

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was filtered through a black 0.22 µm polycarbonate Millipore filter, stained by DAPI

144

and observed using an epifluorescence microscope (Leica DM5000B, Germany) with

145

blue excitation light (450–490 nm).

146

was analyzed by a Microtract S3500 laser particle size analyzer (Microtract Inc.,

147

USA). The temperature, dissolved oxygen, pH and salinity were measured regularly

148

by a multi-parameter water analyzer (YSI, USA). All measurements were performed

149

in triplicate. At day 0, 1, 3, 7, 14 and 28, a 10 mL water sample was collected from

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each group after thorough mixing for the determination of EDCs and metabolite E1 in

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the free form.

of

aquatic

microflora

38

was

checked

by

the

standard

The particle size distribution of the sediment

152

6


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Table 1 Estuarine water–only or water-sediment groups, and their processes and factors involved in EDC removal Gp

①

Abbreviation

EW

Water

Sediment

treatment

treatment

Not sterilized a

Processes involved e

Contribution of specific controlling factors

-

Biotic and abiotic

Microbes in estuarine water (①-②)

-

Abiotic only

Suspended particles (②-③) Dissolved matrixes (③-④)

②

EWS

Sterilized

③

EWF

Filtrate b

-

Abiotic only

④

PW

Pure water

-

Abiotic only

⑤

EW-ES

Not sterilized

Not sterilized

Biotic and abiotic in both water and sediment

Sediment addition (⑤-①); Microbes in sediment (⑤-⑥)

⑥

EW-ESS

Not sterilized

Sterilized

⑦

EW-ESM

Not sterilized

⑧

EWS-ESS

Sterilized a

⑨ 154 155 156 157 158 159 160 161

EWS-ESM

Sterilized

a

c

Biotic and abiotic in water and only abiotic in sediment

Sterilized sediment (⑥-①)

OM removal d

Biotic and abiotic in water and only abiotic in sediment

Inorganic matter in sediment (⑦-①)

Sterilized c

Abiotic in both water and sediment

Sterilized sediment (⑧-②)

Abiotic in both water and sediment

Inorganic matter in sediment (⑨-②)

OM removal

d

a

:Water was sterilized by autoclaving at 120oC for 30 min, twice; NaN3 at a concentration of 1 mg/L was added to all sterilized water groups (once per week) to ensure a sterilization condition was maintained during the experiment; b : Water was filtrated by 0.22 µm pore size membrane; c : Sediment was sterilized by autoclaving at 120oC for 60 min, 4 times; d : Organic matters (OMs) were removed by heating at 550oC for 5 hours and sediment was also sterilized under this treatment; e : Abiotic processes in water and sediment include hydrolysis, evaporation, precipitation, adsorption, abiotic transformation by suspended or dissolved inorganic and organic matters, biotic processes mainly imply biodegradation by microorganisms in estuarine water, sediment, or both; all groups were gently aerated at 0.9 L/min by air pumps to maintain aerobic conditions throughout the experiment.

162

7



163

To disentangle the influencing parameters and the contribution of the biotic and abiotic

164

processes/factors in the estuarine water related to EDC removal, an additional experiment

165

was performed in June of 2015. In addition to the EW and EWS treatments, two more

166

treatments, including pure water (PW, group ④) and filtrate from estuarine water (EWF,

167

group ③ ) were also prepared. The details of the treatments are shown in Table 1.

168

Additionally, treatments investigating other factors for EDC removal were also designed,

169

including natural light, and no air supply in pure water (PW-NA) and estuarine water

170

(EW-NA). For NP removal, another oxygen-poor water group was designed by gentle

171

purging with nitrogen for one hour and sealed with a cap. All treatment groups were set in

172

triplicate and spiked with mixed EDCs. On days 0, 1, 3, 7 and 14, the water was sampled for

173

the determination of EDC and environmental parameters.

174 175

Analytical methods. According to our previous research, EDCs existed mostly in free 10

176

fractions in this estuarine water column.

177

also focused on their free fraction.

178

conducted by fully automated direct solid phase microextraction (SPME) in combination with

179

the headspace derivatization method.

180

and EE2-d4 were filtered using a syringe with a glass fiber filter (GF/F 0.7 µm, Whatman),

181

then directly extracted using polyacrylate (PA) fiber (Supelco Inc. USA) and headspace

182

derivatization by BSTFA/TMCS. In addition, EDCs adsorbed on glassware were rinsed with

183

acetone twice, dried with nitrogen, and then subjected to in situ derivatization by

184

BSTFA/TMCS. Volatilized EDCs in the enclosed air were extracted by headspace SPME for

185

10 hours immediately after EDC spiking.

11-30

Additionally, EDC removal in previous studies

The analysis of free EDCs in the present study was

39, 40

In brief, 10 mL water samples spiked with NP-d4

186

An Agilent 7890A gas chromatograph coupled with a 5975C mass spectrometer (GC-MS)

187

was used for the EDC quantification. The selected ions monitoring (SIM) mode for the target

188

compounds were as follows: NP-d4 (m/z 183/296), EE2-d4 (m/z 429/444), NP (m/z 221/292),

189

PP (m/z 193/252), E1 (m/z 342/257), E2 (m/z 416/285) and EE2 (m/z 425/440). An internal

190

standard method with NP-d4 for NP and PP, and EE2-d4 for E1, E2 and EE2 was used to

191

quantify the concentrations of free EDCs in the samples. The limits of detection (LODs) and

192

limits of quantification (LOQs) for the five detected EDCs were 0.95-2.43 ng/L and 3.20-8.10 8


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ng/L, respectively, and the recoveries ranged from 73.95% to 118.0%.

194

195

Results and discussion

196

Characterization of water and sediment samples. The concentrations of the total organic

197

matter (TOMs) in both the sterilized and non-sterilized sediment were approximately 0.36%.

198

The sterilized and non-sterilized sediment exhibited similar texture with 24.3 ± 7.2% sand,

199

62.1 ± 8.4% silt and 13.6 ± 4.4% clay, toward median grain size of 47.3 ± 14.4 µm (n = 10).

200

On the other hand, the OMs removed sediment had different particle sizes, that is, 64.8 ± 6.9%

201

sand, 32.5 ± 6.0% silt and 2.7 ± 1.0% clay, and a higher size distribution (median grain size:

202

140.7 ± 22.5 µm, n = 10) than those with OMs. No live cells were found in the sterilized

203

water, sterilized sediment and OMs-removed sediment. The concentration of suspended

204

particles in water was 195.2±15.1 mg/L. The temperature, pH and salinity were measured

205

regularly, and the mean and standard deviation values of three replicates were 25 ± 1°C, 7.34

206

± 0.5, and 7.37 ± 0.25‰, respectively. The dissolved oxygen concentrations in the aerated

207

groups, non-aerated groups and oxygen-poor water group were 10.4 ± 2.8, 6.8 ± 1.2 and 0.8 ±

208

0.2 mg/L, respectively.

209

The original concentrations of EDCs in the water samples collected directly from the field

210

were 269.3 ± 50.6 ng/L PP, 480.9 ± 44.2 ng/L NP, 2.3 ± 0.9 ng/L E2 and 20.5 ± 6.9 ng/L EE2,

211

and the values in the sediment (based on dry mass) were 598.3 ± 47.8 ng/g PP, 1778.8 ±

212

130.1 ng/g NP, 17.5 ± 5.3 ng/g E2 and 14.7 ± 7.2 ng/g EE2. After EDC spiking, the dissolved

213

concentration of each EDC at day 0 was determined, and no significant difference was found

214

among these reactors; the measured concentrations of PP, NP, E2 and EE2 were 132.9 ± 37.9,

215

109.1 ± 43.9, 105.7 ± 20.8, 122.8 ± 24.3 µg/L, respectively. The amounts of all four spiked

216

EDCs adsorbed on glassware varied from 1.1‰ to 2.4‰, and the values in the enclosed air

217

were less than 0.1‰.

218

The high concentrations of EDCs at day 0 after spiking could be possible in some natural

219

environments, such as point-source pollution, “hot spots” of pollution, industrial discharges,

220

etc. For instance, the NP concentrations in industrial wastewater treatment ranged from

221

30-400 µg/L.

41

Pharmaceutical pills, such as EE2 and E2 at 1000 µg/piece (Data from 9



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222

website) could be involuntarily discarded into the water body. Additionally, these EDC

223

concentrations should not have any adverse effect on microorganisms and their

224

biodegradation as the reported toxic concentration of some EDCs was higher than 100 µg/L.

225

For example, NP induced significant cytotoxic effects on S. cerevisiae cells at 50 mg/L.

226

The lowest observation effect concentration (LOEC) for PP to bacteria ranged from 0.9 to 4.5

227

mg/L.

228

100 µg/L mixed EDCs in a mangrove water /sediment system.

43

42

In our previous research, the number of bacteria was not affected by the addition of

229 230

Removal of free EDCs in a simulated estuarine aquatic environment. To understand

231

how much EDCs could be removed in a real estuarine environment, non-sterilized estuarine

232

water-only (EW) and water with the addition of non-sterilized sediment (EW-ES) were

233

compared (Table 2). The removal percentage at a definite time was calculated based on the

234

percentage ratio of the difference to the time zero concentration. The actual measured time

235

zero concentration, instead of the nominal spiked concentration, was used in all calculations.

236

The contributions of the factors, such as natural light and no air supply on the EDC removal

237

in the simulated EW group are summarized in Figure S2. Natural light did not have any

238

significant effect on EDC removal in EW, although a slight improvement on the NP removal

239

was observed at day 1. On the other hand, the treatment without aeration (i.e., no air supply

240

group) posed a significantly negative effect on the removal of all four EDCs at the initial

241

phase of day 1 and 3, but then weakened to no obvious effect at day 7 and afterwards. The

242

phenomenon of an initially limited oxygen supply inhibiting EDC removal in natural water

243

was in accordance with previous studies. 30, 44-48

244

The final removal percentages of all the spiked EDCs in the free form were 93.5-100% in

245

EW and EW-ES, respectively, at the end of the 28-day experiment. The first-order decay

246

model was well fitted for the removal of spiked EDCs in the free form with a correlation

247

coefficient (R2) that ranged from 0.81 to 0.96 (Table 2). The calculated rate constants (K) of

248

the removal of free EDCs followed the declining order of PP, NP, E2 > EE2. The calculated

249

half-lives (t1/2) for the spiked EDCs were consistent in both EW and EW-ES (p > 0.05),

250

evidenced by PP at 0.68±0.21 d and 1.34±0.78 d, NP at 2.36±1.76 d and 1.31±0.51 d, E2 10


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at 3.12±2.25 d and 1.28 ± 0.67 d, respectively. The t1/2 was longer for EE2 than for PP, NP

252

and E2 with 15.21±3.18 d in EW and 13.05±0.80 d in EW-ES.

253

The removal of E2, NP and EE2, but not PP, have been widely reported in simulated aquatic 16-19

254

environments under aerobic conditions in the published literature.

255

among the three EDCs found in the present study were comparable to the order established in

256

previous work; that is, NP, E2 > EE2. 16, 18, 19 The t1/2 for E2 was comparable to those reported

257

in previous studies, which ranged from 0.24 d to 1.4 d. 16-19 Regarding the t1/2 for NP and EE2,

258

the reported values varied among the studies. For example, the t1/2 value in the present study

259

for NP was comparable to the 0.42 d in river water-sediment slurries but shorter than at 7 d in

260

the aquifer material

261

the present study was longer than the 5 d in seawater

262

water-sediment slurries

263

marine sediment.

264

the tested matrixes, such as the sources, chemical composition, biomass, OMs content and

265

pollution status, as well as the variations in the experimental design, such as the sample

266

volume, water to sediment ratio and incubation conditions. The removal behavior of each

267

EDC compound in the natural aquatic environment was the result of the interactive effect of

268

the complex biotic and abiotic processes in the matrixes.

19

18

and 5.8 d in the marine sediment.

17

19

The removal rates

The reported t1/2 value for EE2 in 16

and 0.29 - 1.1 d in river

but shorter than 81 d in the aquifer material

18

and > 20 d in the

These variations in t1/2 values were most likely due to the differences in

269 270

Biotic removal of free EDCs in estuarine water. The difference in the removal

271

percentages (%) between EW and EWS in the present study was considered biotic removal

272

by estuarine water microbes (EW-Microbes), which are shown in Figure 1. Compared with

273

the limited EE2 removal in the sterilized groups (Figure S3), the contribution of the

274

EW-Microbes on EE2 was in the range of 13.3% to 19.7% at a lag phase of day 1 to 7, which

275

increased notably to 62.9% at day 14, then remained at the same level until day 28. The

276

biotransformation of EE2 by bacteria,

277

bacterial cultures enriched from the field

278

also reported to play an important role in the attenuation of EE2 in surface water.

279

findings indicated the role of EW-Microbes on EE2 removal was significant and worthy of

49

microalgae, 26, 52

50

, white rot fungus,

51

and mixed

were widely reported. Stream biofilms were

11


23

These


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280

further research in the areas of in situ bioremediation by indigenous microorganisms in

281

natural estuarine environments.

282

Although biodegradation of E2 and NP by microalgae, 14, 23, 24

53-55

fungus,

56-59

and

283

microorganisms from field water

284

EW-Microbes for the removal of PP, NP and E2 in the present study was limited. This

285

phenomenon was due to the removal percentages of PP, NP and E2 in the sterilized estuarine

286

water (EWS) treatment (without microorganisms) were high at day 1 (more than 40%) and

287

reached almost 100% at day 14. There are conflicting findings related to EDC removal in

288

sterilized water, such as, significant losses of NP

289

seawater, whereas an opposite phenomenon showed that E2 and NP persisted in sterilized

290

groundwater.

291

removal,60,

292

responsible for EDC removal in a complex estuarine matrix have seldom been reported. The

293

limited biodegradation of PP, NP and E2 in estuarine water in the present study indicated that

294

the abiotic processes might be more dominant than the biotic ones. Based on the present

295

findings and the reports in the literature, the presumed abiotic processes should include

296

hydrolysis, evaporation, precipitation, adsorption, abiotic transformation by suspended or

297

dissolved inorganic and organic matter. However, the controlling process responsible for the

298

removal of each EDC merits further investigation because the contribution of each process

299

remains unclear.

61

11, 24

has been frequently reported, the contribution of

19

and E2

16

were observed in sterilized

It is clear that both biotic and abiotic processes could contribute to EDC

but their contribution varied among the EDCs. The controlling processes

300 301

Abiotic process of free NP removal in EWS. The removal percentage of the free NP in

302

the treatment groups and the contribution of each factor were calculated and are shown in

303

Figure 2. The removal percentage of NP varied from 35.6% to 58.0% in pure water without

304

an air supply (PW-NA), but increased to a higher percentage (80.1% to 98.7%) in pure water

305

with an air supply (PA), indicating that 40.1% to 51.2% removal in pure water was

306

contributed by aeration.

307

The abiotic processes in pure water are complex and might include photodegradation,

308

hydrolysis, evaporation, precipitation, adsorption on glassware, etc. All these processes might

309

contribute to influence NP removal. The photodegradation in the present study was minimal 12


Page 13 of 35


310

as the flasks were wrapped by aluminum foil. The precipitation of the EDCs in the present

311

study could also be ignored as the spiked concentration of 100 µg/L was far lower than the

312

solubility data shown in Table S1. Montgomery-Brown et al. (2003, 2008) emphasized that

313

NP hydrolysis could be neglected because NP was persistent in anaerobic water.

314

vapor pressures presented in Table S1 shows that NP exhibited higher volatilization than E2

315

and EE2, suggesting volatilization might contribute to NP removal. Previous studies reported

316

that NP was detected in atmospheric particulate matter. 64 Ying et al. (2003) proposed that the

317

initial rapid loss of NP in sterilized seawater might be caused by volatilization.

318

volatilization and co-distillation of a branched NP isomer was also found to occur in lake

319

water/sediment system.

320

glassware in the present study were lower than 0.25%, indicating the contribution of these

321

abiotic processes could be ignored. Thus, the possible mechanism contributing to NP removal

322

might be the interaction of NP with dissolved oxygen. In the further experiment, the removal

323

percentage of NP in the oxygen-poor water group ranged from 8.0% at day 1 to 25.0% at day

324

14, which was significantly lower than the removal percentages in the aerated and

325

non-aerated pure water. The DO-dependent removal percentage indicated that the NP removal

326

in water was controlled by the reaction of oxygen, whereas the reaction mechanism and its

327

influencing factors were rarely reported. Montgomery-Brown et al. (2008) also reported that

328

highly oxygenated conditions in the dark favored NP removal by the cleavage of the benzylic

329

carbon and the formation of tertiary alcohols (ROHs) via ipso-hydroxylation. 63

65

62, 63

19

The

The

However, the amounts lost by evaporation and adsorption on

330 331

Abiotic process of free PP and E2 removal in EWS. Both PP and E2 persisted in pure

332

water (PW) but were significantly removed in filtrate from estuarine water (EWF) (Figure 2).

333

Compared with PW, EWF had more complicated estuarine dissolved matrixes (EDMs)

334

containing colloids, aggregates, and dissolved inorganic and organic matter. The

335

contributions of EDMs on PP and E2 removal were significantly increased by the treatment

336

time, from 0.6-2.9% at day 1 to 77.2-89.6% at day 14.

337

It was estimated that the sorption percentages associated with aquatic colloids were

338

between 15 and 30% for E2. 66 However, the average E2 concentration sorbed on colloids was 13



Page 14 of 35

67

339

low and comparable to that in the dissolved phase in a constructed wetland.

340

organic matter (DOM) with a heterogeneous mixture of aromatic and aliphatic structures

341

attached by various functional groups is another important component in water. DOM derived

342

from effluent, river and lake water,

343

E2 removal in water. The abiotic transformation of E2 in the presence of vegetable matter was

344

demonstrated.

345

through the formation of reactive oxygen species (ROS), such as hydroxyl radicals (•OH) was

346

also reported.

347

aeration of humic acids,

348

non-humic fractions, such as protein and carbohydrates accounted for 40–60% and 20–40%

349

of DOMs, respectively. 66 It is possible that abiotic transformation by hydroxyl radicals from

350

estuarine dissolved matter might contribute to PP and E2 removal in the present study.

45, 70, 71

72

68

and dissolved organic matter surrogates

69

Dissolved

could affect

The degradation of PP in the presence of Fe(III)-citrate complexes

Recently, dark production of •OH by aeration of anoxic lake water, 74, 75

73

and

was recorded. In surface waters, humic substances and

351

Subtracting the removal percentages of EWF, the major process contributing to EDC

352

removal in the EWS group was adsorption by suspended inorganic and organic particles. The

353

contribution of suspended particles in the EWS group accelerated the initial removal

354

percentages of PP and E2 from day 1 to day 3 or day 7, but did not have any significant

355

effects on PP removal at day 7 to 14 and E2 removal at day 14 (Figure 2). EDCs were

356

reported to have high binding capacity to aquatic particles with sorption coefficients

357

depending on the Kow values of the selected EDCs,

358

particle size. 27 The sorption of E2 with suspended minerals was irreversible when intercalated

359

into the interlayer spaces of montmorillonite, but could be reversible when the binding was at

360

the external surfaces of goethite and kaolinite.

361

contribution of adsorption on suspended particles was initially positive for PP and E2 removal

362

but then weakened to no effect. This phenomenon indicated that this contribution could be

363

reversible, which might occur when PP and E2 removal in water is controlled by their

364

dominant processes, such as abiotic removal by estuarine dissolved matter.

15, 76

77, 78

particulate organic carbon,

5, 76

and

The present study found that the

365 366

E1 occurrence and controlling processes. Figure 3 showed the aqueous free concentration

367

(µg/L) of the metabolite E1 in each group (a) and the contribution by each process/factor in

368

estuarine water based on the concentration differences (b). Lower E1 concentration in PW 14


Page 15 of 35


369

(1.0-2.2 µg/L) was consistent with the lower removal percentage of E2 in PW, whereas the

370

higher E1 concentrations in EWF and EWS were consistent with the higher removal

371

percentages of E2 in these groups. The calculated contributions of the increased E1

372

concentrations by estuarine dissolved matter were consistent with the significant contribution

373

of estuarine dissolved matter on E2 removal as mentioned above. Additionally, the E1

374

concentration in the EW group increased from the lower initial value to a peak of 47.5 µg/L at

375

day 3, but gradually decreased to 5.3 µg/L at day 14. Compared to the EWS group, the

376

calculated contribution by estuarine microbes on the E1 concentration increased to 47.5 µg/L

377

at day 3, but then decreased to 53.5 µg/L at day 14, implying that E1 would be further

378

removed because of the biodegradation by aqueous active microorganisms. E1 biodegradation

379

by mixed bacterial cultures enriched from the field was significant. 26 Stream biofilms were

380

also reported to play an important role in the attenuation of E1 in surface waters.

381

present findings supported the importance of biodegradation by aqueous active microbes in

382

E1 removal in estuarine water.

383

In addition to biodegradation,

11

23

The

E2 could also be chemically oxidized to E1 by converting

384

the hydroxyl group at position 17 to a carbonyl group. 79 Some naturally occurring materials,

385

including iron (III), humic acid and vegetable matter

386

which rapidly removed E2 from water by oxidation. In the present study, these naturally

387

oxidizing agents in sterilized estuarine water might also oxidize E2 to E1 and contribute to the

388

removal of E2. This finding needs to be confirmed by more in-depth research.

45, 70

were potential oxidizing agents,

389 390

Contribution by inorganic matters addition. After the OMs were removed, the estuarine

391

sediment was mainly inorganic minerals composed of sand, silt and clay, which were

392

considered inorganic matter derived from the sediment (ES-IM). The contributions of ES-IM

393

on the removal of PP, NP and E2 in both EWS and EW groups, and EE2 in EW group were

394

similar. The removal effects were positive during the initial 3 days, but became insignificant

395

at day 7 or 14 and thereafter (Figure 4). The contribution of ES-IM on EE2 removal in EWS

396

was different from that in EW, with slightly positive effects initially (approximately 4.4%),

397

which gradually increased to 34.7-45.6% at day 7 and remained at these levels until the end

398

of the experiment. 15



399

ES-IM might act on EDC removal in two main and opposite ways. One is the adsorption of

400

EDCs on inorganic matter, which would enhance EDC removal from water. Evidence for this

401

mechanism has been provided by the published reports that clays were effective sorbents for

402

E2,

403

acid,

404

another main factor responsible for EDC removal.

405

effects of ES-IM on the removal percentages of the EDCs at day 1 or 3 were the synthetic

406

results of these two main opposite effects. However, these effects weakened and became

407

insignificant from day 3 onwards as the influences of other controlling factors in water, such

408

as the reaction of dissolved oxygen on NP removal, were more obvious. There was evidence

409

that EDC sorption was rapid and most likely occurred at external surfaces and could be

410

reversible.

411

ES-IM in EWS became persistently positive.

78

EE2 and E1. 80

77

The other effect is the interaction of ES-IM with OMs, such as humic

which would inhibit the removal of EDCs from water because OMs interaction was

80

66, 69

The significant but inconsistent

As the removal percentage of EE2 in sterilized water was limited, the effects of

412 413

Contribution by sterilized sediment addition. The contribution of the addition of

414

sterilized sediment (ESS) on EDC removal in EWS and EW were calculated (Figure 5). EWS

415

contained sedimentary inorganic minerals and sedimentary organic matter, including humic

416

acids and OMs released from dead or lysed microorganisms. Their contributions to the

417

removal of PP, NP and E2 in both groups and EE2 in EW were initially positive, but the

418

effects became insignificant as the experiment progressed. This result was the same as the

419

effect of ES-IM (Figure 4), which also suggested that these effects could be reversible and

420

could weaken with time because of the dominant processes in water controlling the EDC

421

removal. It was found that the association between NP and humic acids occurred rapidly and

422

was reversible.

423

in EW (approximately -23%) was observed at day 14. This negative effect might result from

424

the inhibited effect of ESS acting on EW-Microbes, whose contribution to EE2 removal in the

425

EW group was high at 62% at day 14 as shown in Figure 1(a). On the other hand, the positive

426

contribution of ESS to EE2 removal in EWS lasted throughout the study, ranging from 9.0 to

427

23.8%.

428

81

Additionally, a significantly negative contribution of ESS on EE2 removal

The results obtained from these simulated microcosms under the effect of estuarine water 16


Page 16 of 35

Page 17 of 35


429

differed somewhat from the traditional opinion that sorption onto sediment played an

430

important role in EDC removal. For example, the sorption of estrogen onto sediments

431

correlated with the total organic carbon content.

432

followed the order of NP >> EE2 > E2.

433

the composition of sedimentary organic carbon were the important parameters determining

434

the sorption of the target EDCs onto the sediments.

435

coefficient values were associated with smaller particle size and higher organic carbon

436

content.

437

processes in estuarine water, the contributions of the sedimentary inorganic and organic

438

matters to the removal of free EDCs were limited.

27

18

15

The sorption coefficients in the sediment

The hydrophobic properties of the target EDCs and

82

In the bed sediments, higher partition

In the present study, under the dominant contribution of the abiotic and biotic

439 440

Contribution by addition of non-sterilized sediment. From the difference in the removal

441

percentages between EW and EW-ES, the contribution of non-sterilized sediment (ES)

442

addition in EW was deduced (Figure 6). Similar to the contribution of ES-IM and ESS, the

443

addition of ES posed no obvious, or a slightly positive, effect on EDC removal in the initial

444

period of the experiment, but the effects became insignificant thereafter. Similar to the

445

contribution of ESS, a significantly negative effect of ES on the EE2 removal percentage was

446

also found, approximately -38% at day 14. Consequently, the removal difference between the

447

groups with ES and ESS was the biotic removal of the sedimentary microbes (ES-Microbes)

448

(Figure S4). This effect was limited for PP and was slightly negative for NP, E2 and EE2

449

initially, but at the end of the experiment there were no effects.

450

Previous published literature related to the biodegradation of EDCs in sediment focused

451

mainly on two aspects. One was the degradation ability of microorganisms cultured from the

452

field sediment, 14, 25 and the other was the mini-size laboratory microcosms with the design of

453

a large ratio of sediment to water, such as 1-10 g of river/marine sediment and 0-45 mL of

454

river/marine water,

455

synthetic media.

456

laid more emphasis on the sediment effect, and both led to the exaggeration of the

457

biodegradation of EDCs by sedimentary microbes. The present results showed that under the

458

influence of estuarine water, the effect of biodegradation by ES-Microbes on EDC removal

32

11-13, 18, 19, 25

or 10-100 g of river/marine sediment with 5-70 mL of

The former separated microbes from the real environment and the latter

17



459

was limited because the removal of EDCs in these reactors was controlled by the dominant

460

processes/factors in estuarine water, such as the biodegradation by estuarine microbes on EE2

461

removal.

462 463

Environmental significance. Estuarine aquatic systems are often the repository of EDC

464

contaminants due to the upstream discharge of EDCs-containing effluent from industries,

465

households and even wastewater treatment plants. The collected water and sediment in the

466

present estuary were representative with important compositions of abundant microbes,

467

dissolved matter and suspended particles, and also displaying typical environmental

468

parameters, such as temperature, DO, pH, salinity and light. Thus, the present results could be

469

generalizable to all estuarine environments with similar environmental conditions. This study

470

provided direct evidence of the powerful capacity of aquatic systems, especially estuarine

471

water, to remove free EDCs under aerobic conditions. We also demonstrated that the removal

472

behavior and controlling factors were compound-specific under mixed pollutants. NP

473

exhibited greater removal in aerated pure water. The removal of PP and E2 was mainly

474

controlled by the abiotic processes of estuarine aquatic matrixes, whereas EE2 removal was

475

due to biodegradation by estuarine aquatic microbes. In sterilized water with limited EE2 and

476

E1 removal, the addition of inorganic and organic estuarine sediment matter resulted in

477

significantly positive effects. Different from previous reports, we found that the

478

biodegradation by sedimentary microorganisms under the effect of estuarine water on these

479

EDCs was limited.

480

It has been proposed that the self-purification ability of a water body could effectively

481

relieve its pollution problem; however, this requires time, as discharges of EDCs, especially

482

for EE2, in real environments, continue and these pollutants could be accumulated. It is

483

essential to develop efficient methods for in situ remediation in the estuarine environment.

484

How to improve the abiotic removal processes of PP, NP and E2, and the biodegradation of

485

EE2 and E1 remain challenging tasks for future investigation. The present study provided

486

scientific evidence of the effectiveness of an estuarine environment in removing free EDCs

487

and identified the EDC compound-specific contribution of each abiotic or biotic process.

488

Based on these findings, it is possible to develop better control processes to tackle the 18


Page 18 of 35

Page 19 of 35


489

pollution problem in estuarine ecosystems, such as air supply for NP removal, addition of

490

free radical generators for PP and E2 removal and bioremediation for EE2 removal. Moreover,

491

the contributions of specific components to EDC removal, their characteristics and the

492

bioavailability of their transformation products should be clarified in detail in the future.

493

Because estuaries are subject to variations of dissolved oxygen and NP, removal of EDCs was

494

found to be enhanced by aeration; further studies of the effects of dissolved oxygen and the

495

removal of EDCs under anaerobic conditions are required.

496

497

Supporting Information

498

Structures of EDCs, sampling location, removal by contribution of no air supply and

499

natural light, removal of free EE2 by abiotic processes, contribution of sedimentary microbes

500

on EDC removal. These materials are available free of charge via the Internet at

501

http://pubs.acs.org.

502

503

Acknowledgements

504

This research was financially supported by a research grant from CityU (Account No.

505

7004394), the National Science Foundation of China (NSFC Project No. 40903046,

506

21277177), Hongkong Scholar Program (XJ2011048), Pearl River Nova Program of

507

Guangzhou (No. 2011J2200052) and Foundation for High-level Talents in Higher Education

508

of Guangdong Province, China.

509

510

511 512 513 514 515 516 517

1. 2. 3.

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25



Figure captions

750 751 752

Figure 1 Removal percentages (%) of free EDCs in sterilized and non-sterilized estuarine

753

water-only and the contribution by estuarine microbes. (a) EE2, (b) PP, (c) NP and

754

(d) E2; EWS: sterilized estuarine water-only; EW: non-sterilized estuarine

755

water-only; Contribution by estuarine microbes was obtained from the removal

756

percentage differences (%) between EW and EWS.

757

Figure 2 Removal percentages (%) of free EDCs in pure water, filtrate from estuarine water,

758

sterilized and non-sterilized estuarine water-only (left-hand panels) and the

759

contributions by each process including aeration, estuarine dissolved matters and

760

suspended particles (right-hand panels). PW-NA: pure water without air supply;

761

PW: pure water; EWF: filtrate from estuarine water; EWS: sterilized estuarine

762

water-only; Contribution by aeration in pure water was obtained from the removal

763

percentage differences (%) between PW and PW-NA. Contribution by dissolved

764

matters was obtained from the removal percentage differences (%) between EWF

765

and PW; Contribution by suspended particles was obtained from the removal

766

percentage differences (%) between EWS and EWF.

767

Figure 3 Free E1 concentration (µg/L) in pure water, filtrate from estuarine water, sterilized

768

and non-sterilized estuarine water-only (a) and contribution by each of the

769

processes, including estuarine dissolved matters, suspended particles and active

770

microbes (b). PW: pure water; EWF: filtrate from estuarine water; EWS: sterilized

771

estuarine water-only; EW: non-sterilized estuarine water-only. Contribution by

772

dissolved matters was obtained from the removal percentage differences (%)

773

between EWF and PW; Contribution by suspended particles was obtained from the

774

removal percentage differences (%) between EWS and EWF. Contribution by

775

estuarine microbes was obtained from the removal percentage differences (%)

776

between EW and EWS.

777

Figure 4 Contribution of sedimentary inorganic matters (ES-IM) to the removal of free EDCs

778

by the addition of ES-IM to sterilized estuarine water (EWS) (a) and to

779

non-sterilized estuarine water (EW) (b). Contribution by ES-IM was obtained from

780

the removal percentage differences (%) between ES-IM addition in EWS

781

(EW-ESIS) and EWS (a) and between ES-IM addition in EW (EW-ESIS) and EW

782

(b). 26


Page 26 of 35

Page 27 of 35


783

Figure 5 Contribution of sterilized estuarine sediment (ESS) to the removal of free EDCs in

784

estuarine water by the addition of ESS to sterilized estuarine water (EWS) (a) and

785

to non-sterilized water (EW) (b). Contribution by ESS was obtained from the

786

removal percentage differences (%) between ESS addition in EWS (EWS-ESS) and

787

EWS (a) and between ES-IM addition in EW (EW-ES) and EW (b).

788

Figure 6 Contribution of non-sterilized estuarine sediment (ES) to the removal of free EDCs

789

by the addition of ES to non-sterilized water (EW). Contribution by ES was

790

obtained from the removal percentage differences (%) between ES addition in EW

791

(EW-ES) and EW.

792 793 794 795

27



Page 28 of 35

796 797

798 100

PP removal percentage (%)

EE2 removal percentage (%)

100

80

60

40

20

80

60

40

20

0

0 0

7

14

21

0

28

7

21

28

Time (Day)

Time (Day)

799

14

(a) EE2

(b) PP

800

100

E2 removal percentage (%)

NP removal percentage (%)

100 80 60 40 20

80

60

40

20 0

0 0

801 802 803 804

7

14

21

0

28

7

14

Time (Day)

Time (Day)

(c) NP

(d) E2

Figure 1

805 28


21

28

Page 29 of 35


PW-NA EWF

PW EWS

Aeration in pure water Estuarine dissolved matters Estuarine suspended particles

100 Contribution in NP removal percentage (%)

60

NP removal percentage (%)

80

60

40

20

0 0

7

40 20 0 -20 -40 -60 -80

14

0

Time (Day)

806

7

14

Time (Day)

100 Contribution in PP removal percentage (%)

PP removal percentage (%)

100 80 60 40 20 0 0

7

40

20

0 0

14

Contribution in E2 removal percentage (%)

E2 removal percentage (%)

80 60 40 20 0

-20

0

7

7

14

Time (Day)

100

809

60

Time (Day)

807

808

80

80

60

40

20

0 0

14

7 Time (Day)

Time (Day)

Figure 2

810 29


14


PW

EWF

EWS

Page 30 of 35

EW

80

Concentration (µg/L)

60

40

20

0 0

7

14

Time (Day)

811

(a) Free E1 concentration in different groups

812

Estruarine dissolved matters Estuarine suspended paricles Estuarine microbes

80

Contribution in E1 concentration (µg/L)

60 40 20 0 -20 -40 -60 0

7

Time (Day)

813 814

(b) Contribution by each of the processes

815

Figure 3 30


14

Page 31 of 35


816

Day 1

Day 3

Day 7

Day 14

Day 28

Removal percentage difference (%)

60 40 20 0 -20 PP

NP

E2

EE2

-40 Compounds -60

(a) Contribution in EWS

817

Day 1

Day 3

Day 7

Day 14

Day 28


60

40

20

0 PP -20

818 819

NP

E2 Compounds

(b) Contribution in EW

Figure 4

820

31


EE2


Day 1

Day 3

Day 7

Day 14

Page 32 of 35

Day 28


50 40 30 20 10 0 -10

PP

NP

E2

EE2

Compounds -20 (a) ESS in EWS

821


Day 1

823

Day 7

Day 14

Day 28

30 20 10 0 -10 -20 -30

822

Day 3

40

PP

NP

E2 Compounds

(b) ESS in EW

Figure 5

824

825

32


EE2

Page 33 of 35


826

827

Day 1

Day 3

Day 7

Day 14

Day 28


40 30 20 10 0 -10 PP

NP

E2

-20 -30 -40 Compounds

828 829

-50

Figure 6

830

33


EE2


831 832 833

Abstract art

Industrial compounds

Natural estrogen

Synthetic estrogen

Pure water Filtrate from estuarine water Sterilized estuarine water Non-sterilized estuarine water

Propylparaben Nonylphenol

17β-estradiol

17α-ethynylestradiol

Organic matters removed sediment Sterilized sediment

Spiked

834

Pure water- Filtrate from only estuarine water

Aerated

Non-sterilized sediment

Estuarine water-only

Estuarine water-sediment system

34


Page 34 of 35

Industrial compounds

Natural estrogen

Synthetic estrogen

Pure water

Filtrate from estuarine water PageEnvironmental 35 of 35 Science & Technology Sterilized estuarine water

Non-sterilized estuarine water Propylparaben

Nonylphenol

17β-estradiol

17α-ethynylestradiol

Organic matters removed sediment Sterilized sediment

Spiked

Aerated

Non-sterilized sediment

ACS Paragon Plus Environment Pure water- Filtrate from only estuarine water

Estuarine water-only

Estuarine water-sediment system

Contributions of Abiotic and Biotic Processes to the Aerobic Removal

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