Effect-Directed Analysis of Toxicants in Sediment with Combined

Apr 27, 2017 - Polydimethylsiloxane served as passive dosing matrix for midge bioassays. The fractions showing abnormal enzymatic response were subjec...
0 downloads 0 Views 508KB Size
Subscriber access provided by University of Colorado Boulder

Article

Effect-directed analysis of toxicants in sediment with combined passive dosing and in vivo toxicity testing Hongxue Qi, Huizhen Li, Yanli Wei, W. Tyler Mehler, Eddy Y. Zeng, and Jing You Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

Environmental Science & Technology

1

Effect-directed analysis of toxicants in sediment with combined passive

2

dosing and in vivo toxicity testing

3

Hongxue Qia,c,e, Huizhen Lib, Yanli Weia,b, W. Tyler Mehlerd, Eddy Y. Zengb, Jing

4

Youb,*

5

a

State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China

6 7

b

School of Environment, Guangzhou Key Laboratory of Environmental Exposure and Health,

8

and Guangdong Key Laboratory of Environmental Pollution and Health, Jinan University,

9

Guangzhou 510632, China

10

c

College of Chemistry and Chemical Engineering, Jinzhong University, Jinzhong 030619, China

11

d

School of Biosciences, Centre for Aquatic Pollution Identification and Management, The

12 13

University of Melbourne, Parkville, Victoria, 3010, Australia e

University of Chinese Academy of Sciences, Beijing 10049, China

14 15

1 ACS Paragon Plus Environment

Environmental Science & Technology

16

ABSTRACT

17

Identifying key toxicants in sediment is a great challenge, particularly if non-target

18

toxicants are involved. To identify the contaminants responsible for sediment toxicity to

19

Chironomus dilutus in Guangzhou reach of the Pearl River in South China, passive dosing and in

20

vivo toxicity testing were incorporated into effect-directed analysis (EDA) to account for

21

bioavailability. Fractionation of sediment extracts was performed with gel permeation

22

chromatography and reverse phase liquid chromatography sequentially. Polydimethylsiloxane

23

served as passive dosing matrix for midge bioassays. The fractions showing abnormal enzymatic

24

response were subject to a non-target analysis, which screened out 15 candidate toxicants. The

25

concentrations of the screened contaminants (log-based organic carbon normalized) in sediments

26

of 10 sites were compared to sediment toxicity (10-d and 20-d mortality and 10-d enzymatic

27

response) to C. dilutus using correlation analyses. The results suggested that oxidative stress

28

induced by cypermethrin, dimethomorph, pebulate and thenylchlor may have in part caused the

29

observed toxicity to C. dilutus. The present study shows that EDA procedures coupled with

30

passive dosing and in vivo toxicity testing can be effective in identifying sediment-bound

31

toxicants, which may pose high risk to benthic organisms but are not routinely monitored and/or

32

regulated. The findings of the present study highlight the importance of incorporating

33

environmentally relevant approaches in assessing sediment heavily impacted by a multitude of

34

contaminants, which is often the case in many developing countries.

35

2 ACS Paragon Plus Environment

Page 2 of 28

Page 3 of 28

36

Environmental Science & Technology

INTRODUCTION

37

Sediment serves as a sink for a battery of hydrophobic contaminants in aquatic

38

environment and deteriorated sediment quality due to multiple stressors has been reported

39

worldwide, especially in rivers draining through large cities.1,2 In the case of complex mixtures,

40

the identification of toxicants causing ecotoxicological effects is critical for effectively selecting

41

sediment management measures. Toxicity identification evaluation (TIE) and effect-directed

42

analysis (EDA) are the two most widely used approaches for diagnosing causes of sediment

43

toxicity.3,4

44

In the TIE work, researchers mainly focused on characterizing the contaminant classes

45

causing toxicity, i.e., organics, metals and ammonia, then estimating the toxicity contribution of

46

individual contaminants on the target lists (i.e., a pre-chosen list of chemicals to measure), and

47

eventually identifying the toxicants causing adverse effects.4,5 This approach, however, often

48

fails to find the main toxicants when the toxicants are not present in the list of target analytes.6

49

Alternatively, EDA techniques have been proposed to find organic toxicants by using analytical

50

techniques to fractionate test samples for both chemical analyses and biological tests and confirm

51

the identity of key toxicants in the toxic fractions without solely relying on the target lists.3,6 As

52

such, the EDA method is not limited to analyzing chemicals in specified target lists, but rather

53

screens for contaminants of unknown identity under the guidance of the bioassay, providing a

54

way to discover toxicants which are not monitored and/or regulated.3,6-8

55

Traditional EDA is mainly guided by cell-based in vitro bioassays, which are easy to

56

achieve high-throughput analysis, however it lacks ecological relevance and ignores the

57

bioavailability and toxicokinetic process of the contaminants.8-10 The use of in vivo toxicity

58

testing with benthic organisms is more environmentally realistic than cell-based assays for 3 ACS Paragon Plus Environment

Environmental Science & Technology

59

identifying sediment-bound toxicants. In addition, ignoring bioavailability may bias the

60

estimation of toxicity of HOCs in sediment and provide false conclusions as to the suspected

61

toxicants, which calls for the development of bioavailability-based EDA methods.8-12 Passive

62

dosing techniques with polydimethylsiloxane (PDMS) have been shown to successfully maintain

63

constant concentrations of hydrophobic contaminants in water.13-16 During the bioassays, PDMS

64

serve as a partitioning delivery system to transfer chemical mixtures into water, acting as a

65

surrogate for sediment organic carbon (OC).17,18 Therefore, a EDA procedure combined with

66

passive dosing and in vivo bioassays can take chemical bioavailability into account, improving

67

the accuracy in diagnosing causes of sediment toxicity in aquatic system containing complex

68

mixtures, such as urban rivers.

69

The Pearl River flows through Guangzhou, which is the largest city in South China.

70

Various sediment-bound contaminants have been detected in Guangzhou reach of the Pearl

71

River, including polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers, pesticides

72

and metals.2,5,19 Current-use pesticides in sediment, particularly pyrethroid insecticides, were

73

deemed as the principal causes of the mortality to benthic invertebrates in urban tributaries of the

74

Pearl River based on TIE methods.2,5 It was noted that urban sediments, such as those in

75

Guangzhou reach of the Pearl River were quite complex with the presence of various pollutants

76

many of which were of unknown identity. Similar to its tributaries, sediment-bound pyrethroids

77

played a role in the toxicity to benthic invertebrates in Guangzhou reach of the Pearl River, yet

78

their toxicity contribution was relatively small.20 Meanwhile, the concentrations of other

79

routinely monitored contaminants (e. g., metals) appeared to not contributing to sediment toxicity

80

in this river.2 These results suggested the need for exploration of non-target contaminants, which

81

would contribute to the observed adverse effects in benthic organisms. 4 ACS Paragon Plus Environment

Page 4 of 28

Page 5 of 28

82

Environmental Science & Technology

The aims of the present study were to develop and validate an EDA method in combination

83

with passive dosing and in vivo testing using Chironomus dilutus (abnormal enzymatic response

84

as the endpoint) for diagnosing causes of sediment toxicity, particularly for identifying organic

85

toxicants which are not targeted in common chemical analyses. The applicability of the method

86

was validated by a thorough evaluation of sediment toxicity and identification of key toxicants in

87

a complex aquatic system (using Guangzhou reach of the Pearl River as an example).

88 89 90

MATERIALS AND METHODS Experimental Design. An environmentally relevant EDA method for sediment-bound

91

toxicants was developed and the stepwise procedures are shown in Figure 1. The experiments

92

were separated into two parts: screening bioassays and EDA development. The screening

93

bioassays evaluated sediment toxicity to C. dilutus collected from various sites along Guangzhou

94

reach of the Pearl River. The EDA method was first developed with sediment from a

95

representative site (P4), including sediment extraction, fractionation, bioassays and chemical

96

screening, and the confirmation of potential toxicants was conducted with all sediments.

97

In the screening bioassays, 10 sediment samples were collected in Guangzhou Reach of the

98

Pearl River (Figure S1; “S” represents figures and tables in the Supporting information

99

thereafter) and sediment toxicity was evaluated using 1st (20-d chronic testing) and 3rd instar C.

100

dilutus (10-d acute testing) as part of the screening bioassays (more information can be found in

101

the Supporting information and Cheng et al.20). In short, five of the 10 sediments exhibited acute

102

lethality to the midges compared with the controls, and all of the sediments caused significant

103

chronic toxicity to C. dilutus (Table S1). In addition, enzymatic activities in the surviving midges

104

were significantly altered compared with the control, indicating ubiquitous sublethal toxicity in 5 ACS Paragon Plus Environment

Environmental Science & Technology

105

the study area. A previous study measured current-use pesticides (pyrethroids, organophosphates

106

and fipronil) and heavy metals in the sediments and evaluated their toxicity contributions20. Only

107

pyrethroids (mostly cypermethrin) were correlated to sediment toxicity, which, however, can

108

explain only a small portion of the observed toxicity.

109

To better explain the observed sediment toxicity, an EDA method using passive dosing and

110

in vivo testing with C. dilutus was developed and used to diagnose other potential causes of

111

toxicity besides the target analytes. The EDA procedure includes sediment extraction,

112

fractionation, passive dosing, in vivo toxicity testing and non-target chemical analysis and is

113

complex and laborious. Thus, only the sediments from site P4, which exhibited moderate

114

mortality (37 ± 4%) to the midges, was chosen to test the EDA procedure. This site is adjacent to

115

Chebei Creek, which is a well-studied urban tributary of the Pearl River and polluted by a variety

116

of contaminants including pyrethroids and fipronils.5,12,19 After the candidate toxicants in

117

sediment P4 were screened by the EDA procedure, their concentrations were determined in the

118

remaining nine sediments and the toxicity contributions of these contaminants were assessed

119

using linear correlation and canonical correlation analyses. Accordingly, key toxicants were

120

identified in the sediments. Further information for each step of the EDA procedure is detailed

121

below.

122

Sediment Extraction and Fractionation. To obtain sediment extracts for EDA analysis,

123

ultrasound-assisted microwave extraction (UAME) was performed using a CW-2000 UAME

124

extractor (Xintuo Company, Shanghai, China).21 In brief, 40 g of freeze-dried sediment was

125

extracted twice with 100 mL of a mixture of hexane and acetone (1꞉1, v/v). The extraction was

126

carried out for 6 min with ultrasound and microwave power at 50 and 100 W, respectively. After

127

decanting the extract, the extraction was repeated with an additional 50 mL of fresh extraction 6 ACS Paragon Plus Environment

Page 6 of 28

Page 7 of 28

Environmental Science & Technology

128

solution. The extracts were combined, filtered through a Whatman 0.45 µm filter, evaporated to

129

near dryness, and solvent exchanged to 2 mL of dichloromethane.

130

The sediment extract was first fractionated using gel permeation chromatography (GPC)

131

with a Bio-beads S-X3 column (300 mm × 20 mm) and dichloromethane as the mobile phase at a

132

flow rate of 5 mL min-1.22 Four fractions were collected at the time intervals of 0–4–8–18–27

133

min, and were solvent-exchanged to methanol for midge toxicity testing using the passive dosing

134

method.

135

Two of the GPC fractions exhibited toxicity (G3 and G4) (Figure 2), which were

136

combined, solvent exchanged to methanol and further fractionated for the second round of EDA

137

analysis. Reverse phase liquid chromatography (RPLC; Lab-Tech Corporation, China) with a

138

C18 semi-preparative column (150 mm × 10 mm, 10 µm) was used for the fractionation and the

139

mobile phase was consisted of methanol and water (with an initial composition of 50꞉50 and then

140

increasing to 100 % methanol at a flow rate of 3 mL min-1).23 A total of nine RPLC fractions

141

were collected (F1–F9) over a period of 45 min at 5-min intervals.

142

On the basis of the toxicity results of these nine fractions, the third round of fractionations

143

was carried out for the toxic F5 and F6 fractions (Figure 2). Again, the two fractions were

144

combined and further separated using RPLC, resulting in five fractions that were collected every

145

2 min (2F1–2F5). The individual fractions were then solvent-exchanged to hexane and methanol

146

for chemical analysis and toxicity testing, respectively. Passive dosing method was applied for all

147

toxicity tests in the EDA procedure.

148

Passive Dosing and Midge Toxicity Testing. The PDMS films used for passive dosing

149

procedures were made from a MDX4-4210 Bio-Medical grade elastomer kit (Dow Corning

150

(China) Holding Company Limited, Shanghai, China), in accordance to the manufacturer's 7 ACS Paragon Plus Environment

Environmental Science & Technology

151

instruction. The method to prepare PDMS films24 was modified from a previously developed

152

method by Mayer and Holmstrup.25 In brief, PDMS pre-polymer and catalyst (10꞉1) were

153

thoroughly mixed to cast into a film and cured at 23 °C for 72 h. The thickness of PDMS film

154

was 0.25 mm (± 0.1 mm) with a density of 1.11 g cm-3. The impurities and oligomers in the

155

cured films were removed using three sequential ultrasonic extractions with methanol and three

156

rinses using Milli-Q water. The films were then cut into small pieces (2 × 4 cm) before use.

157

The fractions after GPC and RPLC separations were individually loaded onto the PDMS

158

films. The loading of chemical mixture to the films was achieved by shaking methanol-water

159

solutions of the extracts in beakers containing the films at 220 rpm for 48 h. Reconstituted water

160

was gradually added to the solution to drive the contaminants into the PDMS film.24,26 For

161

dosing sediment extracts containing the mixtures with unknown identity into water, it was crucial

162

to normalize the concentrations of contaminants in PDMS to sediment OC-based concentrations.

163

Li et al.27 suggested that the partitioning coefficient of a chemical between OC and PDMS was

164

independent of its hydrophobicity and was a constant at approximately 2.1. Accordingly, the

165

mass of PDMS used in the passive dosing was 2.1 times the mass of sediment OC and detailed

166

calculations are presented in the Supporting information. After loading, the PDMS films were

167

placed into the reconstituted water and equilibrated for 48 h by stirring at 660 rpm to release the

168

contaminants in PDMS into the aqueous phase. This passive dosing procedure has been validated

169

with a series of polychlorinated biphenyls (PCBs) with log Kow values ranging from 5.35–7.42

170

and the results showed that the equilibrium time for all test PCBs was