Bioavailability of Pyrene Associated with Suspended Sediment of

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Environmental Science & Technology

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Bioavailability of Pyrene Associated with Suspended Sediment of Different Grain Sizes to

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Daphnia magna as Investigated by Passive Dosing Devices

3 4

Xiaotian Zhang, Xinghui Xia∗, Husheng Li, Baotong Zhu, Jianwei Dong

5

School of Environment, Beijing Normal University, State Key Laboratory of Water

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Environment Simulation/Key Laboratory of Water and Sediment Sciences of Ministry of

7

Education, Beijing 100875, China

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∗

Corresponding author. Tel.: +86 10 58805314; fax: +86 10 58805314. E-mail address: [email protected] (X. Xia). 1

ACS Paragon Plus Environment


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Abstract

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Suspended sediment (SPS) is widely present in rivers around the world. However, the

11

bioavailability of hydrophobic organic compounds (HOCs) associated with SPS is not well

12

understood. In this work, the influence of SPS grain size on the bioavailability of

13

SPS-associated pyrene to Daphnia magna was studied using a passive dosing device, which

14

maintained a constant freely dissolved pyrene concentration (Cfree) in the exposure systems.

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The immobilization and protein as well as enzymatic activities of Daphnia magna were

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investigated to study the bioavailability of SPS-associated pyrene. With Cfree of pyrene ranging

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from 20.0-60.0 µg L-1, the immobilization of Daphnia magna in the presence of 1 g L-1 SPS

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were 1.11-2.89 times that in the absence of SPS. The immobilization caused by pyrene

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associated with different grain size SPS was ordered as 50-100 µm> 0-50 µm> 100-150 µm.

20

When pyrene Cfree was 20.0 µg L-1, the immobilization caused by pyrene associated with

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50-100 µm SPS was 1.42 and 2.43 times that with 0-50 µm and 100-150 µm SPS, respectively.

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The protein and enzymatic activities of Daphnia magna also varied with the SPS grain size.

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The effect of SPS grain size on the bioavailability of SPS-associated pyrene was mainly due to

24

the difference in SPS ingestion by Daphnia magna and SPS composition, especially the

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organic carbon type, among the three size fractions. This study suggests that not only the

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concentration but also the size distribution of SPS should be considered for the development of

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biological effect database and establishment of water quality criteria for HOCs in natural

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waters.

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Keyword: suspended sediment, PAHs, toxicity, bioavailability, passive dosing device, freely

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dissolved concentration

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Table of Contents

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1. Introduction

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Hydrophobic organic compounds (HOCs) can exhibit toxicity to organisms and threaten

46

human health by accumulation in biota through the food chain.1 Once HOCs enter rivers, they

47

tend to be associated with suspended sediment (SPS) or deposit sediment and concentrate in

48

these media due to their hydrophobic and lipophilic properties.2-4 The characteristics of SPS

49

and deposit sediment exert significant influence on the distribution and fate of HOCs.4-6

50

Especially for turbid rivers, SPS is an important carrier of pollutants which plays an important

51

role in controlling the behavior and bioavailability of HOCs, such as polycyclic aromatic

52

hydrocarbons (PAHs) with high octanol-water partition coefficients.7,8

53

Generally, the concentration of HOCs in water phase which can pass through the 0.45 µm

54

or 0.50 µm filter membrane is used to conduct water quality assessment.9 The toxicity of HOCs

55

in water phase is also investigated to provide valid data base for the establishment of water

56

quality criteria.10, 11 However, some researchers have found that the particle-associated HOCs

57

are partly bioavailable to organisms and cause toxic effects.12,

58

revealed that the SPS enhanced the toxicity of phenanthrene to filter-feeding invertebrate

59

Daphnia magna (D. magna) compared to that in the absence of SPS with identical freely

60

dissolved phenanthrene in water. There is evidence that the diffusion of PAHs through aquatic

61

boundary layer can be enhanced by the presence of PAH binding particles15, suggesting that the

62

exposure of HOCs to aquatic organisms might be increased by enhanced passive diffusion

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through integument because of the presence of SPS-associated HOCs. As a result, the toxicity

64

of SPS-associated HOCs should not be ignored, especially for turbid rivers with high SPS

65

concentrations because the content of SPS-associated HOCs in those rivers might be large

66

enough to affect the growth and reproduction of aquatic organisms.

13

Our previous research14

67

Meanwhile, plenty of research suggested that the sorption and desorption of HOCs will

68

be significantly influenced by particle size. For example, Xia et al. 8 reported that the sorption

69

and desorption of PAHs had a significant relationship with sediment particle size, and fine

70

particles with large specific surface areas sorbed a greater amount of PAHs. Wang and Keller 16

71

found that the partitioning and desorbability of two hydrophobic pesticides (atrazine and 4


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17

72

diuron) in a soil-water system are soil particle-size dependent. Cui et al.

reported that the

73

biodegradation rate of pyrene sorbed on sediments by Mycobacterium vanbaalenii increased

74

with sediment particle size. These research results suggest that the bioavailability of HOCs

75

sorbed on particles to aquatic organisms might vary with the particle size. In addition, the

76

ingestion of particles by organisms might be related to the particle size18, exerting influences

77

on particle-associated HOC bioaccessibility to aquatic organisms. Consequently, it is

78

hypothesized that the bioavailability of HOCs associated with SPS to aquatic organisms might

79

depend on the SPS grain size.

80

Keeping dissolved HOC concentration constant is a great challenge for laboratory tests.

81

Traditional test results are always compromised by chemical losses and adverse effects of the

82

excessive renewal of exposure media on test organisms. In recent years, the passive dosing

83

system19, 20 has enabled defined and constant freely dissolved HOC exposure in a simpler and

84

less materially costly way than flow-through systems, which is suitable for aquatic toxicity

85

tests or bioconcentration tests.

86

In the present study, we chose pyrene as a typical HOC to study the bioavailability of

87

HOCs associated with different grain size SPS to D. magna, which was widely used as a

88

standard testing organism to study the toxicity of PAHs. The passive dosing devices were made

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to control the freely dissolved concentration of pyrene in the exposure systems, and the effect

90

of pyrene associated with SPS of different grain sizes on the immobilization and enzymatic

91

activities of D. magna was investigated. Both the freely dissolved concentration, a measure for

92

the bioavailable fraction in bioassays, and the toxicity endpoints were used to reflect the

93

bioavailability of SPS-associated pyrene to the organisms. The influencing mechanisms of SPS

94

grain size on the bioavailability of SPS-associated pyrene were explored through analyzing the

95

ingestion of SPS by D. magna as well as the sorption/desorption of pyrene sorbed on SPS with

96

different grain sizes. In addition, the importance of SPS grain size in the bioavailability

97

assessment of HOCs in natural waters was discussed.

98

2. Materials and Methods

99

2.1 Chemicals and materials 5



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Pyrene (solid powder) was purchased from AccuStandard Inc. (New Haven, USA).

101

Di-fluoro-biphenyl used as recovery standard and meta-terphenyl used as internal standard

102

were obtained from J&K Scientific Ltd. (Beijing, China). Dichloromethane (GC-MS grade),

103

methanol (HPLC grade), hexane (HPLC grade), and acetone (HPLC grade) were purchased

104

from J&K Scientific Ltd. (Beijing, China). Ethanol, KCl, CaCl2, NaHCO3, and MgSO4

105

(analytical grade) were purchased from Beijing Chemical Reagents Company (Beijing, China).

106

Polydimethylsiloxane (PDMS) pre-polymer and catalyst used to make passive dosing devices

107

were purchased from Dow Corning Co. Ltd. (Shanghai, China). All glassware was soaked in a

108

10% HNO3 solution (analytical grade, Beijing Chemical Reagents) for 24 h followed by

109

washed with tap water and ultrapure water (each beaker for three times) to eliminate the

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inorganic ion residues in glassware which might affect the living of D. magna, afterwards dried

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in an oven (DHG-9070A, Shanghai, China) at 105 ℃ and heated in a muffle furnace

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(KSW-5-12A, Tianjin, China) at 350-400 ℃ for 5 h.

113

2.2 Sediment sampling and characterization

114

The surface sediment was collected at depths of 0-10 cm using a pre-cleaned grab sampler

115

at the Xiaolangdi Hydrological station in the middle reach of the Yellow River in September of

116

2013. The samples were placed in a 4℃ cooler and transported to the laboratory. Then they

117

were dried in a freeze-drier (LGJ-12, Beijing, China) and sieved through a 100-mesh sieve

118

(150 µm). According to the results of our research about the size distribution of SPS in the

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Yellow River, the percentages of SPS in the size fractions of 0-50 µm, 50-100 µm, and > 100

120

µm were 75.6%, 16.8%, and 7.6%, respectively. This was similar to the SPS size distribution of

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the river Danube reported by Haun et al. 21. Therefore, the sediment sample was divided into

122

three sieve fractions (including 100-150 µm, 50-100 µm, and 0-50 µm) with 140-mesh (100 µm)

123

and 270-mesh (50 µm) sifters, to study the effect of SPS grain size on the bioavailability of

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pyrene associated with SPS. Then the samples were respectively placed in centrifuge tubes

125

with 50 mL pure water, shaken for 24 h, and centrifuged at 4000 rpm (JW-3021HR, Beijing,

126

China) for 5 minutes. After that, the supernatant was discarded, which was done twice to

127

remove the dissolved organic carbon (DOC) of sediment particles. The DOC in the supernatant 6


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was measured with a Shimadzu TOC analyzer (Kyoto, Japan), and the result showed that it was

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lower than the detection limit after washed twice. The organic carbon content of the treated

130

sediment samples were measured using an elemental analyzer (Vario El, Elementar

131

Analysensysteme GmbH, Germany). The total sixteen PAHs sorbed on SPS after washed twice

132

were extracted and detected with the method described in section 2.6.2. The physiochemical

133

properties of sediment samples are listed in Table 1.

134

2.3 Preparation of passive dosing dishes

135

The passive dosing dishes were prepared according to our previous research.22 In detail, a

136

total of 12 ± 0.01 g mixture of PDMS pre-polymer and the matched catalyst (10:1, weight)

137

were added to each 60 mm-diameter glass culture dish with a maximum volume of 40 mL,

138

which was sufficient to control the freely dissolved concentration of pyrene in 300 mL test

139

water.23 These dishes were then vacuumed to eliminate trapped air and subsequently placed in

140

an oven at 110°C for 48 h to complete the curing. Cured dishes were soaked in methanol for 72

141

h to remove impurities and oligomers and were then rinsed with Milli-Q water three times

142

before pyrene loading. The loading solution was made by dissolving 0, 0.3, 2.0, 3.0, 4.0, 6.0 g

143

pyrene in glass bottles containing 1 L methanol respectively, and less than 0.25 mL acetone

144

was added as the co-solvent which would not cause confusing effect. Then the bottles were

145

ultra-sonicated in an ultrasonic apparatus (KQ5200, Kunshan, China) for 6 h. After that, 300

146

mL of loading solutions were transferred to 500 mL beakers, respectively in which three

147

PDMS dishes were placed. The loading process lasted for 72 h with loading solutions refreshed

148

every 24 h. After that, loaded dishes were rinsed with Milli-Q water three times, and then they

149

were respectively placed in a series of beakers containing 300 mL of artificial freshwater

150

(AFW) for at least 48 h to dose the AFW with pyrene, which were used as the exposure

151

solutions. AFW was prepared according to the guideline of Organization for Economic

152

Cooperation and Development for the testing of chemical.24 The details about that have been

153

described in our previous study.14

154 155

For the loading procedure, the partition coefficient of pyrene between PDMS and methanol (MeOH) (KP/M, L kg-1) was calculated as follows: 7



C K P/ M = P CM

156

157 158

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(1)

For the dosing procedure, the partition coefficient of pyrene between AFW and PDMS (KA/P, kg L-1) was calculated as follows:

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KA/P =

CA CP

(2)

160

where CM is the pyrene concentration in the methanol loading solution (µg L-1); CP is the

161

pyrene concentration in PDMS paint coats (µg kg-1); CA is the pyrene concentration in dosed

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AFW (µg L-1). Thus, the partition coefficient of pyrene between MeOH and AFW (KM/A) could

163

be calculated with the following equation:

164

KM/A =

CM CA

(3)

165

The partition coefficient (KM/A) was obtained by linear regression between the pyrene

166

concentration in MeOH loading solution and AFW. According to the results shown in Figure

167

S1 in Supporting Information (SI), the KM/A value for pyrene was 100875. Based on the value

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of KM/A, the concentration of pyrene in AFW can be deduced from that in MeOH and vice

169

versa.

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2.4 Establishment of passive dosing exposure systems

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According to the value of KM/A, the loading solution should be 2.0175, 4.0350, and 6.0525

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g L-1 to obtain three freely dissolved concentrations of pyrene in exposure system, including

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20.0 µg L-1, 40.0 µg L-1, and 60.0 µg L-1, respectively. One liter of loading solution was needed

174

to hold 12 PDMS dishes for exposure test at each pyrene level. The loading solutions were

175

prepared by dissolving 2.0175 g, 4.0350 g, and 6.0525 g pyrene in glass bottles containing 1 L

176

methanol to obtain the above mentioned concentrations respectively. The loading process was

177

the same as that described in section 2.3.

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2.5 Biological experiment

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2.5.1 Effect of SPS grain size

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The tests were conducted in 500 mL glass beakers containing 300 mL exposure solution. 8


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One prepared PDMS dish loaded with pyrene was placed upside down on a V-shaped glass rod

182

in each beaker to prevent the SPS from depositing on PDMS layer (Figure S2). The Cfree of

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pyrene in AFW maintained by the PDMS dishes were 0.00, 20.0, 40.0, and 60.0 µg L-1,

184

respectively. Then 0.3 g sediment sample was added in each beaker to obtain 1 g L-1 SPS in test

185

water system, and the beakers were covered by parafilm and placed on magnetic stirrers at

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room temperature for 48 h, which was long enough for pyrene to get equilibrium between SPS

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and water according to our preliminary experiment results. After that, the stir bars were taken

188

out and 30 D. magna, at the age of 6-24 h old, were placed into each beaker. Then they were

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maintained in a temperature-monitored artificial climate incubator (RXZ-500B, Beijing, China)

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with a 16:8 (light:dark) photoperiod at 20 ± 1℃ for 48 h. The solution was gently stirred with

191

the V-shaped glass rod every 2 h to keep the sediments in suspension. In addition, the control

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experiments were conducted in the absence of SPS. Each experimental treatment was

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conducted in triplicate.

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The immobilization (%) of D. magna was monitored every 12 h and the immobility was

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considered to have happened when D. magna was unable to swim within 15 s after shaking the

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V-shaped glass rod in beakers. At the end of test, the alive D. magna in each beaker were

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collected and the wet weights were obtained. Then the protein contents and enzymatic

198

activities of D. magna were determined and the Cfree of pyrene was measured. The SPS in each

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treatment was collected through filtering the water with a 0.45 µm membrane and then

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freeze-dried for 48 h to measure the pyrene associated with SPS.

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2.5.2 Effect of SPS composition

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As the composition of SPS varied with its grain size, the toxicity of spiked pyrene sorbed

203

on different components of SPS was studied to analyze the effect mechanism of SPS size on

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the toxicity of SPS-associated pyrene. Since the toxicity of spiked pyrene sorbed on SPS with

205

the grain size of 50-100 µm was the highest among the three grain sizes (section 3.2), the

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50-100 µm SPS was selected as a case to study the mechanism. The 50-100 µm SPS sample

207

containing amorphous organic carbon (AOC), black carbon (BC) and mineral (sample 1) was

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heated at 375℃ for 24 h to obtain the sample containing black carbon (BC) and mineral 9



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(sample 2); another part of the original sample was combusted at 700℃ for 3 h to obtain the

210

sample containing mineral (sample 3). The native pyrene sorbed on these three samples were

211

extracted and detected with the method described in section 2.6.2. The toxicity of pyrene

212

associated with these three SPS samples was tested with the procedure as above mentioned.

213

To study the effect of SPS itself (including background pollutants sorbed on SPS) on the

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immobilization of D. magna, the relationship between SPS concentration and immobilization

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was investigated without spiked pyrene. The original sediment collected from the Yellow River

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was used in this experiment, and the tested SPS concentration ranged from 0 to 25 g L-1.

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2.5.3 Ingestion of SPS by D. magna

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To study the ingestion of SPS by D. magna, another exposure test was conducted by

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exposing one hundred D. magna to the systems with 20.0 µg L-1 pyrene and 1 g L-1 SPS of

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three different grain sizes. After exposure for 48 h, seventy five alive D. magna in each beaker

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were collected, washed with ultrapure water to remove the SPS attached to the external surface

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of D. magna, and touch-dried by filter paper. They were dried in a freeze drier to constant

223

weight for 48 h and then kept in a drier to room temperature. After that, the dry weight of the D.

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magna was measured using a balance with an accuracy of 0.00001 g. The control tests were

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conducted without SPS. Each experimental treatment was conducted in triplicate. Because the

226

D. magna were in the same size before the tests, the dry weight of SPS ingested by D. magna

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was calculated by subtracting the dry weight of D. magna in the control group without SPS

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from that in the test group with SPS.

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2.6 Biochemical and chemical analysis

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2.6.1 Analysis of enzymatic activity

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The total superoxide dismutase (T-SOD), peroxidase (POD), and catalase (CAT) activities

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of D. magna were analyzed in this study. The collected alive D. magna were placed in a 2 mL

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centrifuge tube which contained 1.5 mL 0.05 M Tris–HCl buffer on ice (pH = 8.2). Then the

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organisms were disrupted with an ultrasonic tissue destructor (Sonics Uibra Cell VCX 105) in

235

the ice bath to obtain homogenates, which was then centrifuged at 10000 rpm at 4 ℃ for 20 min. 10


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The supernatant was used for the determination of the protein content and enzymatic activities,

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which were measured using the reagent kit, purchased from Nanjing Jiancheng Bioengineering

238

Institute, according to the manufacturer’s instructions.25 The results of the enzymatic activities

239

are given in units of U per milligram of protein (U mg-1).26

240

2.6.2 Analysis of freely dissolved and SPS-associated pyrene

241

The freely dissolved pyrene in water was measured using solid-phase microextraction a

passive

sampling

technique.27

242

(SPME),

The

preparation

and

usage

of

243

polydimethylsiloxane-coated glass fibers can be found in our previous research.28 The details of

244

the measurement are shown in SI. For the determination of pyrene sorbed on SPS, the pyrene

245

were extracted using elution of hexane-dichloromethane (1:1 volume) with an accelerated

246

solvent extractor (Dionex ASE300) and analyzed with GC/MS.5 The detailed procedures are

247

described in SI.

248

2.7 Quality assurance and quality control

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The correlation coefficient for pyrene calibration curve with GC/MS was higher than 0.99.

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The detection limit of pyrene was 0.10 µg L-1 for GC/MS. The recovery of pyrene on SPS was

251

83.3 ± 4.5%. The measured freely dissolved pyrene concentrations in AFW maintained by the

252

PDMS dishes were consistent with the calculated values (20.0, 40.0, and 60.0 µg L-1) based on

253

KM/A (Table S1). During the exposure experiments, the mass variation of PDMS was less than

254

0.038%, which would not affect the control capacity of PDMS dishes on the Cfree of pyrene in

255

water. The variation of Cfree of pyrene in the water phase was within 0.18% during the exposure

256

experiments and the water environmental condition was stable: the pH was 7.04 ± 0.05 and

257

dissolved oxygen was 8.02 ± 0.11 mg L-1.

258

The sensitivity of D. magna to the standard toxicant, potassium dichromate (the reference

259

compound) was determined and the EC50 (24 h) was 0.90 mg L-1, which was within the normal

260

range of 0.50-1.2 mg L-1.24 The PDMS coat without loaded pyrene did not show any effect on

261

the growth and survival of D. magna in the absence or presence of SPS, indicating that the

262

cured polydimethlsiloxane material was biocompatible. 11



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3 Results and discussion

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3.1 Effect of SPS itself without spiked pyrene on the immobilization of D. magna

265

According to the results shown in Table 1, Table S2 and Table S3, the native pyrene

266

sorbed on the SPS with different grain sizes and compositions were below the detection limit,

267

and the sixteen kinds of PAHs were less than 15.2 ng g-1 and accounted for less than 0.21% of

268

the spiked pyrene associated with SPS in the exposure tests. Therefore, the background PAHs

269

would not exert significant influence on the sorption/desorption of pyrene in the exposure

270

systems.

271

As shown in Figure S3, the relationship between SPS concentration and immobilization of

272

D. magna fit the sigmoid curve very well (R2=0.996, p < 0.05), which was often used to reflect

273

the concentration-effect relationship of mixture toxicity.29,

274

themselves and the pollutants sorbed on SPS might cause mixture toxicity to D. magna.

275

However, the immobilization of D. magna in the presence of SPS with concentration below 1 g

276

L-1 was less than 3.3% after exposure for 48 h, which was significantly lower than that in the

277

test groups in the presence of 1 g L-1 SPS with spiked pyrene (p < 0.05). Furthermore, the

278

concentration of SPS used in the present study was fixed at 1 g L-1 in each treatment. Therefore,

279

the effect of SPS itself on the immobilization of D. magna was very low in the present study.

280

To reflect the effect of spiked pyrene associated with SPS on the immobilization of D. magna,

281

the results of test groups with SPS of different grain sizes and compositions have been

282

corrected by respectively subtracting the immobilization caused by those SPS without spiked

283

pyrene.

284

3.2 Effect of pyrene associated with different grain size SPS on the immobilization of D.

285

magna

30

This suggested that the SPS

286

In the absence of SPS, the immobilization of D. magna increased with the exposure time

287

and pyrene concentration in the water (Figure 1). A significant linear correlation was observed

288

between pyrene concentration and D. magna immobilization for 36 h (p < 0.05) and 48 h

289

exposure (p < 0.01). The median effect concentration of freely dissolved pyrene causing 50%

290

immobilization of D. magna (EC50) was 0.077 mg L-1 at 36 h and 0.055 mg L-1 at 48 h, which 12


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291


were comparable to the values (0.029-0.054 mg L-1 at 48 h) reported by Nikkilä et al. 31.

292

As shown in Figure 2, with freely dissolved pyrene concentration ranging from 20.0 to

293

60.0 µg L-1, the immobilization of D. magna in the presence of 1 g L-1 SPS with different grain

294

sizes was 1.11-1.63 times and 1.22-2.89 times that in the absence of SPS (p < 0.01) after

295

exposure for 36 h and 48 h, respectively. This indicates that the pyrene associated with SPS

296

exerted toxic effects on D. magna. In this study, the Cfree of pyrene was kept constant by PDMS

297

in water systems with or without SPS, and DOC was not detected in these systems. Therefore,

298

the difference in immobilization of D. magna in the water between presence and absence of

299

SPS was caused by the SPS-associated pyrene. As shown in Figure 2, the toxicity of pyrene

300

sorbed on SPS varied with SPS grain size; the greatest toxicity was induced by the 50-100 µm

301

size SPS, followed by 0-50 µm and 100-150 µm size SPS. For instance, when the Cfree of

302

pyrene was 20.0 µg L-1, the immobilization caused by pyrene associated with 50-100 µm SPS

303

was 1.42 and 2.43 times that associated with 0-50 µm and 100-150 µm SPS, respectively after

304

exposure for 48 h. As shown in Table 2, the contribution ratios of SPS-associated pyrene to the

305

immobilization of D. magna caused by total pyrene in the exposure systems differed among the

306

three grain sizes of SPS, with a decreasing order of 50-100 µm > 0-50 µm > 100-150 µm. For

307

example, when the Cfree of pyrene was maintained at 40.0 µg L-1, the contribution ratios were

308

48.9%, 42.5%, and 36.5% for the three grain size SPS, respectively after exposure for 48 h.

309

The immobilization of D. magna caused by SPS-associated pyrene increased with the Cfree

310

of pyrene significantly after exposure for 36h or 48 h (p < 0.01) (Figure S4). For instance, after

311

exposure for 48 h, the immobilization caused by pyrene associated with 100-150 µm SPS

312

increased from 11.7% to 30.0% when the Cfree of pyrene increased from 20.0 µg L-1 to 60.0 µg

313

L-1. This was due to the fact that the amount of SPS-associated pyrene increased with the Cfree

314

of pyrene (Table S2). In contrast, the contribution of SPS-associated pyrene to the

315

immobilization of D. magna caused by total pyrene decreased with increasing Cfree of pyrene

316

(Table 2). In the presence of 50-100 µm size SPS when the Cfree of pyrene increased from 20.0

317

µg L-1 to 60.0 µg L-1, the contribution ratio decreased from 38.5% to 22.9% after exposure for

318

36 h and decreased from 65.4% to 42.1% after exposure for 48 h. Therefore, it infers that the

319

contribution of SPS-associated pyrene to toxic effects on aquatic organisms caused by total 13



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320

pyrene in water system would increase with the decrease of pyrene concentration in water

321

phase.

322

The theoretical dissolved concentration of pyrene (Ceffective, µg L-1) required to produce the

323

observed immobilization of D. magna in systems with SPS could be calculated according to

324

the linear relationship between D. magna immobilization and pyrene concentration in the

325

absence of SPS shown in Figure 1. The bioavailable fraction of SPS-associated pyrene (FSPS, %)

326

could be calculated as follows:

327

F SPS =

C effective - C free × 100 % 0.001 ×C SPS

(4)

328

where CSPS is the pyrene concentration on SPS phase (µg kg-1) and 0.001 is the SPS

329

concentration in exposure systems (kg L-1). As shown in Table 2, the bioavailable fractions of

330

SPS-associated pyrene were related to the SPS sizes and the 50-100 µm size SPS had the

331

highest bioavailable fraction, followed by 0-50 µm and 100-150 µm size fractions. For example,

332

when the Cfree of pyrene was 20.0 µg L-1, the bioavailable fractions were 27.0%, 18.0%, and

333

15.0% for the SPS sizes of 50-100 µm, 0-50 µm, and 100-150 µm, respectively according to

334

the toxicity data after exposure for 48 h.

335

3.3 Effect of pyrene associated with different grain size SPS on protein contents and

336

enzymatic activities of D. magna

337

The protein content of D. magna has proved to be susceptible to organic and inorganic

338

toxicants.32, 33 Furthermore, many researchers have used the activities of antioxidant enzymes,

339

including CAT, POD, and T-SOD, to describe the oxidative damage mechanisms of D.

340

magna.34-36 In the present research, the protein content of D. magna increased initially and then

341

decreased with the exposure level of pyrene, with that in the medium Cfree group (40.0 µg L-1)

342

being the highest (Figure 3). De Coen and Janssen

343

low concentration (< 1.8 mg L-1) caused a significant increase in protein content of D. magna

344

while exhibited a decrease at a higher concentration (> 3.2 mg L-1). In addition, the protein

345

contents of D. magna in the test groups with SPS were different from those in the control group

346

without SPS. For example, when the Cfree of pyrene was 40.0 µg L-1, the protein content in the

37

reported similar results that lindane at a

14


Page 15 of 31


347

test groups with 1 g L-1 SPS was significantly higher than those in the control group, and the

348

SPS of 50-100 µm size produced greatest enhancement among the three SPS size fractions,

349

with the protein content being twice that of the control group. This might be because D. magna

350

resist the stress of low concentration pyrene through enhancing the protein content.

351

As shown in Figure S5, in the absence of SPS, the CAT and POD activities of D. magna

352

were greatly influenced by Cfree of pyrene. They increased initially and then decreased with the

353

exposure Cfree of pyrene. For POD, its activity was stimulated at a lower Cfree of pyrene (20.0

354

µg L-1) while inhibited at a higher Cfree of pyrene (> 40.0 µg L-1), and the activity could not be

355

detected when the Cfree of pyrene reached 60.0 µg L-1. Since D. magna can counteract its

356

oxidative damage through the antioxidant system directly participating in detoxification

357

reactions with simultaneous and sequential action of relative enzymes

358

and POD activities might alleviate the oxidative stress to D. magna caused by a low freely

359

dissolved pyrene. In addition, the presence of SPS intensified the influence of freely dissolved

360

pyrene concentration on the CAT and POD activities of D. magna. For example, when the Cfree

361

of pyrene was maintained at 20.0 µg L-1, the CAT activity of D. magna in the presence of

362

50-100 µm SPS increased by 62.2% compared to the control group in the absence of SPS.

363

When the Cfree of pyrene was maintained at 40.0 µg L-1, the CAT activity of D. magna in the

364

presence of three grain size SPS decreased by 39.4%-40.4% compared to the control group.

365

The POD activity in the presence of SPS was also significantly different from those in the

366

absence of SPS (Figure S5). For T-SOD, its activity in D. magna decreased with increasing

367

exposure Cfree of pyrene, and the activity in the presence of SPS was generally lower than those

368

in the absence of SPS. The above results suggest that the pyrene associated with SPS has

369

exerted influence on the physiological and biochemical characteristics of D. magna.

370

3.4 Influencing mechanisms of SPS size on the bioavailability of SPS-associated pyrene

371

38

, the increased CAT

Our previous study has shown that the SPS could be ingested by D. magna14, and Gophen 39

have reported that the size of particles found in the gut of D. magna were larger

372

and Geller

373

than the smallest mesh sizes of the filters of D. magna. In this study, the microphotograph of D.

374

magna after exposure tests also demonstrated that the SPS with different grain sizes could enter 15



Page 16 of 31

375

the alimentary canal of D. magna (Figure 4 and Table S4). The ingestion activity provided a

376

chance of contact between digestive system and SPS, which enhanced the bioaccessibility of

377

SPS-associated pyrene. Yang et al. 40 and Heinlaan et al. 41 suggested that the alimentary canal

378

of D. magna might be the target organ for chemical toxicants. In this case, the SPS-associated

379

pyrene had opportunities to contact with the possible target organ of D. magna, leading to the

380

enhanced immobilization of D. magna compared to the systems in the absence of SPS.

381

Furthermore, it took time for the pyrene sorbed on ingested SPS to be desorbed and enhance

382

the tissue concentration; then produce toxicity in the alimentary canal of D. magna. This was

383

demonstrated by the fact that the difference in immobilization of D. magna between the

384

presence and absence of SPS as well as the difference among the three SPS grain size fractions

385

increased with exposure time (Figure 2).

386

According to the microphotograph shown in Figure 4, the amount of ingested SPS in the

387

alimentary canal of D. magna was ordered as 50-100 µm > 0-50 µm > 100-150 µm grain size

388

fractions. In addition, as shown in Table S4, the dry weight of ingested SPS by seventy five D.

389

magna were 2.39, 2.06, and 0.14 mg for 50-100 µm, 0-50 µm, and 100-150 µm grain size SPS,

390

respectively. This might be explained by the difference in intake and retention of SPS in the

391

alimentary canal of D. magna among different grain size fractions during the exposure. Among

392

the three size fractions, it might be most difficult for the SPS of 100-150 µm grain size to pass

393

through the filter and enter the alimentary canal of D. magna, leading to their least ingestion by

394

D. magna. The SPS of the other two grain size fractions (0-50 µm and 50-100 µm) might enter

395

alimentary canal of D. magna more easily, 42, 43 but the SPS of 0-50 µm might also be easy to

396

be eliminated from the gut of D. magna. Harbison and McAlister

397

particles were easier to be removed from the stomach of Cyclosalpa (Tunicata, Thaliacea) than

398

coarse particles. This resulted in less retention of the 0-50 µm grain size SPS in the alimentary

399

canal of D. magna than the 50-100 µm SPS.

44

reported that the fine

400

Furthermore, BC has a higher sorption capacity for HOCs than AOC, and the HOCs

401

sorbed on BC are more difficult to be desorbed.45-47 In this study, the bioavailability of pyrene

402

sorbed on three components of 50-100 µm size SPS was calculated using the method described

403

in 3.2 section (detailed information for calculation is described in SI). According to the results 16


Page 17 of 31


404

shown in Table S5, the bioavailable fractions of AOC-associated, BC-associated, and

405

mineral-associated pyrene to D. magna were 42.5%, 7.50%, and 12.5%, respectively. It means

406

that the pyrene associated with AOC is easier to be desorbed by digestive fluid of D. magna.

407

Meanwhile, the SPS of 50-100 µm grain size had the highest AOC content, suggesting that it

408

contained more pyrene associated with AOC than the other two SPS grain size fractions (Table

409

1). Therefore, the bioavailable pyrene sorbed on SPS of 50-100 µm grain size might be the

410

most among the three SPS grain size fractions. Furthermore, as mentioned above, the retention

411

of 50-100 µm grain size SPS in the D. magna was the most, leading to that the desorption

412

amount of pryene associated with the 50-100 µm grain size SPS by digestive fluid of D. magna

413

was the most among the three SPS grain size fractions. Consequently, at the same Cfree of

414

pyrene, the toxicity enhancement to D. magna caused by pyrene associated with different grain

415

size SPS was ordered as 50-100 µm > 0-50 µm > 100-150 µm. In addition, the presence of SPS

416

might cause enhanced diffusion of pyrene from water to D. magna48 and the enhancement

417

might vary with the grain size and composition of SPS, and this might be another reason for

418

the difference in SPS-associated pyrene toxicity among the three size fractions. Besides,

419

although the content of pyrene sorbed on SPS of 50-100 µm grain size was not the highest

420

among the three grain size fractions (Table S2), the enhancement of toxicity caused by pyrene

421

associated with SPS of 50-100 µm grain size to D. magna was the highest among the three

422

fractions, and the bioavailable fraction of pyrene sorbed on 50-100 µm SPS was also the

423

highest (Table 2). This indicated that the bioavailability of SPS-associated pyrene depends not

424

only on the pyrene content sorbed on SPS but also on the SPS grain size and composition as

425

well as SPS ingestion by organisms.

426

3.5 Importance of SPS in the risk assessment and water quality criteria establishment of

427

HOCs

428

According to the results obtained in the present research, the SPS-associated pyrene was

429

partly bioavailable and exerted toxicity to D. magna regardless of the SPS grain size and

430

composition. Furthermore, the contribution ratio of SPS-associated pyrene to total toxic effects

431

of pyrene on D. magna increased with decreasing freely dissolved concentration of pyrene, and 17



432

the influence of SPS grain size on the contribution ratio also increased with the decrease of

433

pyrene concentration.

434

High SPS concentrations exist in many rivers around the world, and the SPS in rivers has

435

great difference in grain size distribution.49 In addition, the HOCs in natural environment are

436

generally at low concentration levels (ng L-1 or µg L-1).50, 51 As a result, according to the

437

present research, the toxicities caused by SPS-associated HOCs might account for a

438

considerable proportion of the total HOC toxicities to organisms in natural rivers. Besides, the

439

partition coefficients of HOCs between SPS and water as well as their organic

440

carbon-normalized distribution coefficients will vary with SPS grain size, and this has been

441

demonstrated by the results of pyrene shown in Table S2. Therefore, the SPS grain size will

442

influence the content and bioavailability of HOCs associated with SPS; not only the

443

concentration but also the grain size of SPS should be taken into account in the

444

eco-environment risk assessment of HOCs.

445

The aquatic animals are widely used to evaluate the toxicity of chemicals for the

446

development of biological effect database as well as establishment of water quality criteria. In

447

aquatic habitats, animals can ingest SPS in different grain size fractions 14, 52, and desorption of

448

SPS-associated HOCs in their body depends on the organism species with different feeding

449

habits, body grain size, and life span.53-55 It means that the bioaccessibility and bioavailabilty

450

of SPS-associated HOCs are dependent on biological species. Therefore, not only the

451

concentration and grain size distribution of SPS but also the living habits of aquatic animals

452

should be taken into account for the development of biological effect database as well as

453

establishment of water quality criteria. Further work should be carried out to study the

454

comprehensive influences of these biotic and abiotic factors on HOC bioavailability and

455

toxicity in natural waters.

456

Acknowledgements

457

Special thanks to Biru Zhu (School of Life Science, Beijing Normal University, Beijing,

458

China) for her collaboration. This study was supported by the National Science Foundation for

459

Distinguished Young Scholars (No. 51325902), National Science Foundation for Innovative 18


Page 18 of 31

Page 19 of 31


460

Research Group (No. 51421065), and the National Natural Science Foundation of China (No.

461

51079003).

462

Supporting Information Additional experimental details and results are available free of

463

charge via the Internet at http://pubs.acs.org.

464 465 466

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467

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71-79.

24


Page 24 of 31

Page 25 of 31

618 619 620


Table 1 Physiochemical properties of the washed sediment samples (Mean ± standard deviation, n=3) SPS size fraction

0-50 µm

TOC (%)

0.246±0.021

0.162±0.013

0.123±0.012

BC (%)

0.168±0.010

0.082±0.009

0.074±0.006

AOC (%)

0.078±0.008

0.080±0.007

0.049±0.005

0.316

0.492

0.397

∑16 PAHs (ng g )

16.1

15.2

11.3

Pyrene (ng g-1)

nd

nd

nd

AOC/TOC -1

621 622 623

50-100 µm

100-150 µm

TOC means total organic carbon; BC means black carbon; AOC means amorphous organic carbon, and it is calculated as following equation: AOC = TOC – BC; nd means lower than the detection limit.

25



624 625

Table 2 Bioavailability of pyrene sorbed on SPS to the D. magna

SPS size (µm)

626

Page 26 of 31

Cfree of pyrene (µg L-1)

Contribution ratio of pyrene Bioavailable fraction sorbed on SPS to the total of pyrene sorbed on immobilization (%) SPS (%) 36 h

48 h

36 h

48 h

control

20.0

0.00

0.00

0.00

0.00

0-50

20.0

33.3

57.1

17.8

18.0

50-100

20.0

38.5

65.4

23.6

27.0

100-150

20.0

20.0

43.8

12.7

15.0

Control

40.0

0.00

0.00

0.00

0.00

0-50

40.0

20.0

42.5

24.4

23.2

50-100

40.0

33.3

48.9

32.9

33.9

100-150

40.0

14.3

36.5

21.8

24.2

control

60.0

0.00

0.00

0.00

0.00

0-50

60.0

18.2

37.7

19.9

22.3

50-100

60.0

22.9

42.1

30.4

30.7

100-150 60.0 10.0 35.3 25.6 25.8 Control means without SPS and each treatment group contains 1 g L-1 SPS.

26


Page 27 of 31


Figure captions Figure 1 Effect of freely dissolved pyrene concentration on the immobilization of D. magna in the absence of SPS. Data are mean ± standard deviation (n=3). Figure 2 Relationship between the freely dissolved pyrene concentration and immobilization of D. magna in the presence of SPS with different grain sizes. Control means without SPS. Data are mean ± standard deviation (n=3). Figure 3 Total protein content of D. magna (mg g-1, wet weight) in the absence or presence of 1 g L-1 SPS with different grain sizes after exposure for 48 h. Control means without SPS. Data are mean ± standard deviation (n=3). * p

Bioavailability of Pyrene Associated with Suspended Sediment of

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