Chronic Exposure of Marine Medaka (Oryzias melastigma) to 4,5

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Chronic exposure of marine medaka (Oryzias melastigma) to 4,5-dichloro-2n-octyl-4-isothiazolin-3-one (DCOIT) reveals its mechanism of action in endocrine disruption via the hypothalamus-pituitary-gonadal-liver (HPGL) axis Lianguo Chen, Weipeng Zhang, Rui Ye, Chenyan Hu, qiangwei wang, Frauke Seemann , Doris Wai Ting Au, Bingsheng Zhou, John P. Giesy, and Pei-Yuan Qian Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01137 • Publication Date (Web): 01 Apr 2016 Downloaded from http://pubs.acs.org on April 3, 2016

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 34

Environmental Science & Technology

1

Chronic

2

4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) reveals its mechanism of

3

action in endocrine disruption via the hypothalamus-pituitary-gonadal-liver

4

(HPGL) axis

exposure

of

marine

medaka

(Oryzias

melastigma)

to

5 6

Lianguo Chen †, Weipeng Zhang †, Rui Ye §, Chenyan Hu ‡, Qiangwei Wang ⌘, Frauke

7

Seemann §, Doris W.T. Au §, Bingsheng Zhou ⌘, John P. Giesy #, Pei-Yuan Qian †,*

8 9

†

HKUST Shenzhen Research Institute and Division of Life Science, Hong Kong

10

University of Science and Technology, Clear Water Bay, Hong Kong SAR, China

11

§

12

and

13

Hong Kong SAR, China

14

⌘

15

Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China

16

#

17

S7N 5B3, Canada

State Key Laboratory in Marine Pollution, Department of Biology and Chemistry, ‡

School of Energy and Environment, City University of Hong Kong, Kowloon,

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of

Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK

18 19

* Corresponding author:

20

Dr. Pei-Yuan Qian

21

Tel: 0852-2358-7331

22

Fax: 0852-2358-1559

23

E-mail: [email protected]

24

1

ACS Paragon Plus Environment


Page 2 of 34

25

ABSTRACT

26

In this study, marine medaka (Oryzias melastigma) were chronically exposed for 28

27

days

28

4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) (0, 0.76, 2.45 and 9.86 µg/L),

29

the active ingredient in commercial antifouling agent SeaNine 211. Alterations of the

30

hypothalamus-pituitary-gonadal-liver (HPGL) axis were investigated across diverse

31

levels of biological organization to reveal the underlying mechanisms of its endocrine

32

disruptive effects. Gene transcription analysis showed that DCOIT had positive

33

regulatory effects mainly in male HPGL axis with lesser extent in females. The

34

stimulated steroidogenic activities resulted in increased concentrations of steroid

35

hormones, including estradiol (E2), testosterone (T), and 11-KT-testosterone (11-KT),

36

in the plasma of both sexes, leading to an imbalance in hormone homeostasis and

37

increased E2/T ratio. The relatively estrogenic intracellular environment in both sexes

38

induced the hepatic synthesis and increased the liver and plasma content of

39

vitellogenin (VTG) or choriogenin. Furthermore, parental exposure to DCOIT

40

transgenerationally impaired the viability of offspring, as supported by a decrease in

41

hatching and swimming activity. Overall, the present results elucidated the estrogenic

42

mechanisms along HPGL axis for the endocrine disruptive effects of DCOIT. The

43

reproductive impairments of DCOIT at environmentally realistic concentrations

44

highlights the need for more comprehensive investigations of its potential ecological

45

risks.

46

KEYWORDS: Antifouling; SeaNine 211; Fish; Estrogenic activity; Reproductive

47

fitness

to

environmentally

realistic

48

2


concentrations

of

Page 3 of 34


49

INTRODUCTION

50

In the marine environment, biofouling is referred to as the undesired colonization of

51

marine organisms on anthropogenic surfaces, resulting in tremendous economic costs

52

and ecological disturbances each year.1,2 To prohibit the occurrence of biofouling,

53

immersed surfaces are usually coated with a layer of antifouling paint that

54

incorporates biocidal products. The gradual release of antifouling biocides from the

55

coat repels or kills nearby biofoulers, thus serving as a protector of the surfaces.3

56

Since the definitive ban of organotin compounds as antifouling additives due to their

57

bioaccumulative potential and negative endocrine effects, a variety of booster biocides,

58

including Irgarol 1051, Diuron, zinc pyrithione (ZnPT), chlorothalonil, and SeaNine

59

211, have been used alternatively in combination with cuprous oxide to prevent

60

biofouling.3,4

61

SeaNine 211, which contains 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT)

62

as the bioactive ingredient, has been proposed to be an environmentally acceptable

63

alternative with regard to its fast degradation in the marine environment.5 However,

64

the large-scale application of SeaNine 211 as an antifouling agent eventually leads to

65

coastal pollution worldwide.4 For example, in Spain marinas, accumulated levels of

66

DCOIT were detected greater than 3.3 µg/L,6 which is much higher than the 0.1 ng/L,

67

concentration previously shown to greatly delay embryogenesis in the sea urchin

68

Anthocidaris crassispina.7 Additional research using chronically exposed marine

69

medaka (Oryzias melastigma) has reported endocrine-disrupting effects of DCOIT at

70

a concentration of 2.55 µg/L, demonstrating that DCOIT functions as an estrogen

71

mimic to cause an imbalance in the steroid hormone ratio and to induce the

72

production of vitellogenin (VTG).8,9 Furthermore, previous research demonstrates that

73

chronic exposure of the mummichog Fundulus heteroclitus to 1 µg/L SeaNine 211 for 3



74

28 days induces apoptosis in testicular germ cells, indicating an impairment of

75

reproductive function in males.10 Therefore, DCOIT pollution along the coast and its

76

toxicity to non-target organisms support the occurrence of non-negligible hazards to

77

the marine environment, arguing for a more comprehensive assessment of the

78

associated environmental risks.

79

Despite accumulating evidence for the adverse effects of DCOIT on endocrine

80

homeostasis and reproductive function, to date, the underlying molecular mechanism

81

has remained unclear. In the present study, the marine medaka (O. melastigma), an

82

increasingly used marine toxicological model, was chronically exposed to

83

environmentally realistic concentrations of DCOIT (0, 1, 3 and 10 µg/L) for 28 days.

84

After exposure, alterations across diverse biological organization levels (e.g., gene

85

transcriptions in each tissue, plasma steroid hormone levels, VTG and choriogenin

86

protein content, hepatic and gonadal histology, as well as individual fitness) were

87

examined based on the hypothalamus-pituitary-gonadal-liver (HPGL) axis. Shotgun

88

proteomics were also conducted to profile differential proteins in the plasma. In

89

addition, following parental exposure, transgenerational effects on the viability of the

90

larval offspring were also monitored.

91 92

MATERIAL AND METHODS

93

Chemicals

94

The 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) with a purity >99% was

95

purchased from Waterstone Technology (Carmel, IN, USA). The dimethyl sulfoxide

96

(DMSO) used to make the stock solutions of DCOIT was of high-performance liquid

97

chromatography (HPLC) grade (Sigma-Aldrich, St. Louis, MO, USA). HPLC-grade

98

solvents were used to measure the DCOIT concentrations in seawater. Other 4


Page 4 of 34

Page 5 of 34


99

chemicals were of analytical grade.

100

Fish Maintenance and DCOIT Exposure

101

Four-month-old adult marine medaka (females: 0.21 ± 0.01 g; males: 0.19 ± 0.01 g)

102

were cultured and exposed at 24 ± 0.5°C on a 14 h: 10 h light/dark cycle in fully

103

aerated, charcoal-filtered artificial seawater (salinity: 28%), as previously described.9

104

Prior to chemical exposure, the marine medaka were first randomly divided into 25-L

105

tanks containing 20 L of seawater (20 males and 20 females per tank) and acclimated

106

in this stable environment for two weeks. Afterwards, three replicate tanks were

107

randomly assigned to each exposure group, and the adult medaka were exposed to

108

various nominal concentrations of DCOIT (0, 1, 3 and 10 µg/L) for 28 days in a

109

semi-static system with daily seawater replenishment. The final content of DMSO

110

was 0.001% in all groups. The selection of exposure concentrations was based on a

111

previous report in which DCOIT pollution greater than 3.3 µg/L is detected in

112

seawater around marinas in Spain.6 Thus, the DCOIT concentrations used in this study

113

are environmentally realistic. After exposure, the medaka were anesthetized with

114

0.03% MS-222. Tissues, including brain, gonads, liver, and blood, were sampled,

115

immediately frozen in liquid nitrogen, and stored at -80°C until analysis.

116

HPLC Monitoring of DCOIT Concentrations in Seawater

117

During the exposure, the concentrations of DCOIT were monitored regularly on

118

weekly interval after the replenishment of seawater. A 600-mL aliquot of seawater

119

was collected from each tank (n = 3) and extracted three times with 200 mL of

120

dichloromethane as previously described.8 After pooling the bottom layer, the

121

dichloromethane was dried in a rotary evaporator. The residues were reconstituted in

122

100 µL of methanol for chemical measurement using a reverse-phase HPLC system

123

(Waters 2695) equipped with a Phenomenex Luna C18 column and a photodiode array 5



124

detector. A 50-µL aliquot was injected using an autosampler, and the flow rate of the

125

mobile phase, consisting of 40% water and 60% acetonitrile, was set at 1 mL/min for

126

the isocratic elution. The DCOIT content was calculated using the peak area at 210

127

nm (DCOIT retention time: 34 min) against the established standard curves. The

128

recovery efficiency of DCOIT was 87.6% with a detection limit of 2 µg/mL.

129

Evaluation of Reproductive Endpoints

130

During the 28-day exposure, spawned eggs were collected and counted every day.

131

Cumulative egg production per day per female was calculated after exposure to detect

132

changes in fecundity. Eggs spawned during the final three days were collected to

133

determine the egg weight, total protein content according to the Bradford method, and

134

lipid content.11 In addition, the hatching rate, malformation rate, and mortality rate

135

were monitored in clear water without DCOIT until 18 days post-fertilization (dpf). At

136

10 dpf, 10 unhatched embryos were selected (n = 3) and preserved in TRIzol reagent

137

(Invitrogen, Carlsbad, CA, USA) for the gene transcription analysis. The locomotor

138

activity of medaka larvae at 18 dpf (n = 20) was also examined either under 15-min

139

continuous light or in response to photoperiod stimuli (dark-light-dark-light, 5 min

140

each period) using a ZebraLab behavior monitoring station (ViewPoint Life

141

Sciences).12

142

Quantitative Real-Time PCR (qPCR) Assay

143

After exposure, tissues were collected to evaluate gene transcription (i.e., adult brain,

144

gonad, liver, and unhatched embryos at 10 dpf) and preserved in TRIzol reagent at

145

-80°C. Five brains, livers, and testes from each tank were pooled together as one

146

replicate, with two ovaries as one replicate (n = 3). RNA extraction and purification,

147

first-strand cDNA synthesis, and qPCR assays were performed as previously

148

described.12 The primer sequences for the target genes (Table S1 in Supporting 6


Page 6 of 34

Page 7 of 34


149

Information (SI)) were adopted from the literature or designed using Primer3 software

150

(http://frodo.wi.mit.edu/). The qPCR analysis was conducted using the QuantStudio

151

12K Flex system (Applied Biosystems). The gene transcription levels were

152

normalized to that of 18S ribosomal RNA (18S rRNA), which remained unchanged in

153

response to DCOIT. Calculations were conducted using the 2−∆∆T method.

154

Plasma Steroid Hormone Measurements

155

Blood pool collected from the caudal vein of 10 fish of the same sex was considered

156

one replicate (n = 3 per group). After a 10-min centrifugation at 15000 × g at 4°C, the

157

plasma supernatant was transferred to a new tube and purified by extraction to

158

measure steroid hormones, including estradiol (E2), testosterone (T), and

159

11-keto-testosterone (11-KT), according to the instructions provided with the

160

commercial immunoassay kits (Cayman Chemical Company, Ann Arbor, MI, USA;

161

detection limits of 19 pg/mL, 6 pg/mL, and 1.3 pg/mL for E2, T, and 11-KT,

162

respectively).

163

Measurement of VTG Content in Liver and Plasma

164

The livers of five fish of the same sex were pooled together as one replicate (n = 3).

165

The liver was homogenized on ice in 0.5 mL of saline (0.9% sodium chloride) and

166

centrifuged at 12,000 × g for 10 min at 4°C. The liver supernatant and plasma were

167

then used to determine the VTG content according to the manual provided with the

168

ELISA kit (Biosense Laboratories, Bergen, Norway), with a detection limit of 0.05

169

ng/mL. After incubation with a VTG-specific antibody labeled with horseradish

170

peroxidase, color development was conducted with 3,3′,5,5′-tetramethylbenzidine

171

(TMB) substrate, and the absorbance was read at 510 nm. The VTG content in liver

172

and plasma was quantified against the standard curves (R2 = 0.993) as ng/mg protein.

173

Hepatic and Gonadal Histology 7



Page 8 of 34

174

After DCOIT exposure, potential morphological changes in the liver and gonads were

175

examined using the whole-fish sectioning method.13 Five male or female fish from

176

each group were fixed after otolith removal and gas release from the swim bladder.

177

Whole medaka were then dehydrated in a methanol gradient, cleared with chloroform

178

and finally embedded in melted paraffin for subsequent serial sectioning at 5 µm

179

using a rotary microtome (Leica RM2125, Germany). The sections were stained with

180

hematoxylin and eosin (H&E) and examined for morphological abnormalities.

181

Oocytes

182

postvitellogenic phases) were counted according to previous description.14

183

Shotgun Proteomics Profiling of Plasma

184

Blood pooled from ten fish of the same sex after exposure to nominal 0 or 10 µg/L

185

DCOIT was regarded as one biological replicate (n = 3). After centrifugation at 15000

186

× g for 10 min at 4°C, the plasma supernatant was transferred and dried in a Speedvac.

187

The protein pellets were reconstituted in 100 µL of buffer (8 M urea and 40 mM

188

HEPES, pH 7.4) and quantified using the RC-DC assay (Bio-Rad, Hercules, CA,

189

USA). Next, a 100-µg aliquot of each sample was loaded into a 12% SDS-PAGE gel

190

for electrophoresis. The protein bands in the gel were visualized by Coomassie

191

staining. After excision of the protein bands, the gel was cut into small pieces and

192

destained for subsequent in-gel reduction with dithiothreitol (DTT), alkylation with

193

iodoacetamide (IAA) and tryptic digestion (10 µg/mL; Promega, Madison, WI).15 The

194

peptides in the gel pieces were extracted, dried in a Speedvac, and redissolved in 20

195

µL of 0.1% formic acid for shotgun proteomics analysis using a Thermo Scientific

196

LTQ Velos platform (Thermo Fisher Scientific, Bremen, Germany).16 The MS data

197

generated in .mgf format were searched against the protein database for Japanese

198

medaka (Oryzias latipes) using Mascot version 2.3 software (Matrix Sciences Ltd.,

in

each

oogenesis

phase

(i.e.,

previtellogenic,

8


vitellogenic

and

Page 9 of 34


199

London,

UK).

The

search

criteria

were

as

follows:

trypsin

digestion;

200

carbamidomethylation (cysteine) for fixed modifications; protein N-terminal

201

acetylation, oxidation (methionine) and peptide N-terminal pyroglutamate formation

202

for variable modifications; 1.0 Da for precursor and 0.2 Da for fragments. Only one

203

missed cleavage was allowed, and the false discovery rate (FDR) threshold was

204

dynamically set at 1% for each biological replicate. Counts of spectral matches were

205

summed to indicate the protein intensity. At least two counts for each protein were

206

included in any two out of the three biological replicates.

207

Statistical Analysis

208

To analyze the plasma proteomics data, a one-tailed independent-sample t-test was

209

used to filter the differentially expressed proteins if a significant difference was found

210

in the means between the control group and the 10 µg/L DCOIT group. One-way

211

analysis of variance (ANOVA) followed by the post hoc LSD test was applied for the

212

other data to identify significant differences between the control group and the groups

213

exposed to DCOIT. The Shapiro-Wilk test and Levene's test were performed,

214

respectively, to examine the normality of the data and the homogeneity of variances.

215

Data were log-transformed if necessary. Non-parametric analysis were conducted if

216

data could not meet the normality even after transformation. Statistical analysis were

217

performed using SPSS v13.0 software (SPSS, Chicago, IL, USA). All values were

218

expressed as the mean ± SEM, and the significance criterion was set at P < 0.05.

219 220

RESULTS

221

Actual Concentrations of DCOIT in Seawater

222

The actual concentrations of DCOIT in seawater measured by HPLC were 0.76 ± 0.02,

223

2.45 ± 0.17 and 9.86 ± 0.60 µg/L, respectively, for the nominal 1, 3 and 10 µg/L 9



224

DCOIT groups. No DCOIT was detected in the control group.

225

Adult Growth and Reproductive Success

226

The body weights of both male and female fish increased marginally but significantly

227

after exposure to 2.45 µg/L DCOIT (SI Table S2). An increase in the hepatosomatic

228

index (HSI = liver weight/body weight × 100) was also observed in adult medaka of

229

both sexes exposed to DCOIT (Table S2).

230

There were no significant differences in egg production during the exposure,

231

although a general decrease was observed in the exposure groups (Table S3). Parental

232

exposure resulted in a significant decrease by 14.4% in total protein deposition in

233

each egg and a remarkable delay in the offspring hatching rate despite the unchanged

234

transcriptions of the hatching enzymes choriolysin H and L (Table S3).

235

Parental exposure to DCOIT also resulted into reduced viability of the larval

236

offspring (Figure 1). Under continuous light, medaka larvae became lethargic, and

237

their swimming activity decreased to 1.7 mm/s in the 9.86 µg/L DCOIT group

238

compared with 2.1 mm/s in the control group (Figure 1A). In addition, in response to

239

photoperiod transition stimuli, the larval offspring of parents exposed to 2.45 and 9.86

240

µg/L DCOIT did not respond vigorously to the dark-to-light transition, whereas larvae

241

from the 0.76 µg/L exposure group maintained a hyperactive response and exhibited a

242

delayed decrease in swimming speed in response to the dark-to-light switch compared

243

with the sharp decrease in swimming speed observed in control larvae (Figure 1B).

244

The average swimming speed of the larvae in each photoperiod was consistently

245

decreased in response to 9.86 µg/L DCOIT (Figure 1C).

246

Changes in Gene Transcriptions along the HPGL Axis

247

In the male brain exposed to DCOIT, transcription of gonadotropin releasing hormone

248

(mGnRH) was significantly up-regulated, accompanied by increased transcript levels 10


Page 10 of 34

Page 11 of 34


249

of gonadotropin α (GTHα) and androgen receptor (ARα) (Table S4). In the male testis,

250

the transcription levels of the three subtypes of estrogen receptor (ERα, ERβ and ERγ)

251

were decreased. The transcriptions of genes involved in the steroidogenic pathway

252

(i.e., steroidogenic acute regulatory protein [Star], 20,22-desmolase [CYP11a],

253

3β-hydroxysteroid dehydrogenase [3βHSD] and cytochrome P450 11b [CYP11b])

254

were

255

dehydrogenase (17βHSD), which showed a 0.5-fold decrease (Table S4). Similarly,

256

transcriptions of ERα and ERβ were also significantly down-regulated in response to

257

9.86 µg/L DCOIT in the male liver, while VTG-1 and VTG-2 were up-regulated (Table

258

S4).

consistently

up-regulated,

with the exception of

17β-hydroxysteroid

259

In the female brain, DCOIT exposure had no effect on the transcription levels of

260

the genes examined in this study (Table S4). Two genes important for steroidogenesis,

261

17βHSD and cytochrome P450 19a (CYP19a), were significantly up-regulated in the

262

DCOIT-treated female ovary (Table S4). Increased transcription of VTG-2 by 2.0-fold

263

and 1.6-fold was also observed in the female liver exposed to 2.45 and 9.86 µg/L

264

DCOIT, respectively (Table S4).

265

Levels of Steroid Hormones in Plasma

266

A significant increase in the level of T by 1.9-fold was detected in the plasma of males

267

exposed to 0.76 µg/L DCOIT; E2 was also elevated in the plasma of males in all

268

exposure groups (Figure 2A), resulting in 2.3-fold, 5.0-fold, and 4.4-fold increases in

269

the E2/T ratio in the 0.76, 2.45, and 9.86 µg/L DCOIT groups, respectively (Figure

270

2C). In female plasma, the levels of three steroid hormones (i.e., T, E2 and 11-KT)

271

consistently increased after DCOIT exposure (Figure 2B). Consequently, the E2/T

272

ratio in female plasma also increased by 2.4-fold in the 9.86 µg/L DCOIT group

273

(Figure 2C). 11



274

VTG Content in Liver and Plasma

275

In the male liver, DCOIT exposure led to an increase in VTG content by 2.2-fold,

276

2.3-fold, and 3.0-fold for the 0.76, 2.45, and 9.86 µg/L groups, respectively (Figure 3).

277

Accordingly, the content of VTG in male plasma concomitantly increased by 2.9-fold

278

and 2.5-fold in the 2.45 and 9.86 µg/L exposure groups, respectively (Figure 3).

279

Regarding VTG content in the female liver, a marginal but significant increase by

280

1.4-fold was observed in the 9.86 µg/L DCOIT group, while the levels of VTG

281

remained unchanged in female plasma (Figure 3).

282

Hepatic and Gonadal Histology

283

No obvious morphological abnormalities were observed in male livers following

284

DCOIT exposure (SI Figure S1C, E, G and I). However, compared with the control

285

female liver (Figure S1D), mild vacuolization was observed in the female liver

286

exposed to 2.45 and 9.86 µg/L DCOIT (Figure S1H and J).

287

Histological observations of the male testis and female ovary did not reveal any

288

apparent differences between the control group and the exposure groups. Additionally,

289

exposure to DCOIT did not significantly modify the respective percentage of oocytes

290

in each oogenesis phase (Figure S2).

291

Proteomics Profiling of Medaka Plasma

292

Shotgun proteomics analysis identified differentially expressed proteins in male

293

plasma after exposure to 9.86 µg/L DCOIT (Figure 4A). Of particular concern were

294

the identification of differentially expressed proteins that participate in several

295

biological processes such as lipid transport, vitellogenesis, immune response,

296

coagulation and fibrinolysis, iron metabolism, blood pressure regulation, and neuronal

297

injury. Two apolipoproteins (B and Ea) that participate in lipid transport exhibited a

298

concomitant decrease (Figure 4A). Consistent with the ELISA results, proteomics 12


Page 12 of 34

Page 13 of 34


299

profiling also showed that the VTG content in male plasma increased significantly in

300

response to DCOIT exposure (Figure 4A). Abundance of proteins associated with the

301

immune response, including complement component proteins, S-antigen, and alpha-1

302

antitrypsin, were significantly altered, indicating an inhibition of immune function in

303

the male fish. Together with a mild decrease in plasminogen abundance for the

304

degradation of fibrin, remarkable decreases were detected for coagulation factor X

305

and fibronectin 1b, which play crucial roles in blood coagulation. Ceruloplasmin,

306

which participates in the oxidation of Fe2+ into Fe3+, demonstrated lower protein

307

levels in male plasma exposed to DCOIT, which yielded less Fe3+ and accounted for

308

the simultaneous diminished protein abundance of transferrin for Fe3+ binding and

309

transport (Figure 4A). In addition, in male plasma, DCOIT exposure increased the

310

expression of angiotensinogen protein for the regulation of blood pressure but

311

decreased the expression of visinin-like 1a protein, a marker of neuronal injury

312

(Figure 4A).

313

In female plasma, the identified differential proteins were involved in biological

314

processes including lipid transport, zonagenesis, immune response, and blood

315

coagulation. An increased abundance of apolipoproteins for lipid transport was

316

observed in female plasma in response to DCOIT treatment (Figure 4B). The content

317

of choriogenin for formation of the egg envelop increased in female plasma after

318

DCOIT exposure (Figure 4B), although there were no significant changes in VTG

319

content. DCOIT exposure decreased the content of complement component proteins

320

responsible for certain immune responses (Figure 4B). Differential proteins associated

321

with blood coagulation (i.e., coagulation factor X, fibrinogen, and antithrombin) were

322

consistently up-regulated in the plasma of females exposed to DCOIT (Figure 4B).

323 13



324

DISCUSSION

325

In teleosts, there is a complicated regulatory network referred to as the HPGL axis

326

that is supposedly involved in the maintenance of endocrine homeostasis and

327

reproductive function. Along the HPGL axis, tissues encompassing the hypothalamus,

328

pituitary, gonads, liver, and blood interact vigorously with one another to achieve the

329

dynamically homeostatic endocrine system. The results of the present study provided

330

an integrative perspective of the endocrine-disrupting effects of DCOIT in both male

331

(Figure 5) and female medaka (Figure 6).17 Thus, it is conceivable that a generalized

332

positive regulatory effect was activated throughout the HPGL axis in response to

333

DCOIT treatment in both male and female fish (Figures 5 and 6). A sex-dependent

334

response was also clearly observed in which male medaka appeared to be more

335

susceptible to DCOIT stress, consistent with a previous report wherein a suite of

336

biomarkers was used to assess the relative sensitivity of the sexes.8 The distinct

337

susceptibility of male and female fish against chemical stress addresses the important

338

consideration of sex-specific responses in ecological risk assessment. In addition, the

339

present results showed that parental exposure to DCOIT could transgenerationally

340

impair offspring viability, as supported by the delayed hatching and lethargic larvae.

341

As the initial step in the HPGL network, GnRH is a releasing hormone that is

342

synthesized and secreted by the hypothalamus, which plays central roles in the

343

subsequent synthesis of the gonadotropins, follicle-stimulating hormone (FSH) and

344

luteinizing hormone (LH), in the pituitary gland.18,19 In the present study, chronic

345

exposure to DCOIT induced a significant up-regulation of mGnRH rather than sGnRH,

346

another subunit examined, probably as a consequence of the physiologically closer

347

correlation of mGnRH with endocrine regulation in marine medaka.14 Moreover,

348

increased transcription of mGnRH would stimulate the synthesis of gonadotropins in 14


Page 14 of 34

Page 15 of 34


349

the pituitary, together with the concomitant up-regulation of the GTHα subunit for

350

gonadotropins. The simultaneous up-regulation of mGnRH and GTHα suggested that

351

DCOIT had a positive regulatory effect on the HPGL axis in the male brain (Figure 5).

352

As a response to the estrogenic environment induced by DCOIT, the up-regulation of

353

ARα transcription in male brain would adaptively sensitize the perception to T via the

354

positive feedback loop of HPGL axis. However, compared with the positive responses

355

observed in males, no significant changes in response to DCOIT were detected in the

356

female hypothalamus or pituitary (Figure 6), suggesting a sex-specific responsiveness.

357

The higher susceptibility of male endocrine axis to endocrine disruptors may be due to

358

the lower basal concentration of estrogen while the female has greater capacity of

359

compensatory regulation.20

360

The increased synthesis and release of gonadotropins is expected to stimulate the

361

steroidogenic pathway in the gonad via their transport in the blood and binding to

362

respective receptors.21 Despite the unmodified transcripts of follicle stimulating

363

hormone receptor (FSHR) and luteinizing hormone receptor (LHR), an increase in

364

steroidogenic activities was observed herein in both the male testis and the female

365

ovary in response to DCOIT; however, this effect was much greater in males (Figures

366

5 and 6). Some of the gene transcriptions along the HPGL axis did not show

367

concentration-dependent responses, which may be explained by the differential

368

sensitivities of tissues or the overwhelming toxic stress.21 Considering that the

369

steroidogenesis pathway is responsible for steroid hormone production (e.g., E2, T and

370

11-KT), stimulation of its activity is likely to disrupt the levels and balance of

371

hormone homeostasis. The present findings demonstrated that DCOIT exposure

372

significantly increased the levels of steroid hormones in both male and female plasma

373

as a consequence of active steroidogenesis. The consequent increases in the E2/T ratio 15



374

Page 16 of 34

support the estrogenic effect of DCOIT in both sexes of marine medaka.8,9

375

Following its diffusion into the nucleus and binding to the ER, relatively increased

376

levels of E2 compared with T would trigger vitellogenesis and zonagenesis processes

377

to support formation of the egg yolk and envelope during oogenesis,22 eventually

378

yielding higher contents of VTG and choriogenin in the liver and plasma, as observed

379

in the present study. A nonlinearity between the levels of VTG mRNA and VTG

380

protein was observed herein, which may be attributed to the altered translational rates,

381

post-translational

382

pollutants.23,24 Based on the observed increase in the HSI value and the vacuolization

383

phenomenon, excessive production of VTG and choriogenin would likely lead to the

384

accumulation of lipid droplets and hypertrophy of hepatocytes.25,26 However,

385

compared with the general positive responses observed along the HPGL axis, the

386

transcriptions of subtypes of ER (ERα, ERβ and ERγ) were differentially

387

down-regulated in the male testis and liver but without changes in the male brain and

388

female tissues, which could be attributed to the tissue- and sex-specific expressions

389

because of the auto-regulatory capabilities of the ER subtypes against estrogen

390

mimics to reduce the responsiveness of target tissues to abnormally elevated hormone

391

levels.27

modifications

or

protein

degradations

by

environmental

392

The histological examination revealed that oogenesis-related processes were not

393

blocked in the female ovary, as verified by the mildly but nonsignificantly decreased

394

fecundity. The incongruity between molecular events and egg production is supposed

395

to result from the complexity of HPGL axis and regulatory network as well as the

396

timing of spawning pattern.28 However, transgenerational effects due to parental

397

DCOIT exposure were imposed on medaka offspring and the capacity to produce

398

viable offspring was reduced, as mainly manifested by a strong inhibition of hatching 16


Page 17 of 34


399

and reduced swimming activity (Figure 6), both of which have been frequently used

400

as sensitive indicators of offspring viability following exposure to toxins.12,29,30

401

Previous research has shown that altered expression of the hatching enzyme

402

choriolysin could be a major contributor to the abnormal hatching rate.30 However, in

403

the present study, no differential transcriptions of the two choriolysin enzymes were

404

observed in the medaka embryo after DCOIT treatment. In addition to the gradual

405

hydrolysis of the chorion by choriolysin enzymes at the time of hatching, vigorous

406

activity of medaka larvae inside the chorion is also a prerequisite to facilitate the

407

ability of the larvae to rupture the hard eggshell.31,32 Therefore, although no

408

morphological malformations of the larvae were observed among the groups, the

409

lethargic state of the larval offspring in response to parental DCOIT exposure may

410

account for the decreased hatching rate.

411

In summary, our results systematically demonstrated that chronic exposure of

412

marine medaka to environmentally realistic concentrations of DCOIT stimulated a

413

battery of positive responses along the HPGL axis, ranging from an initial positive

414

regulation in the hypothalamus and pituitary and active gonadal steroidogenic

415

synthesis, consequently increasing the steroid hormone levels and E2/T ratio, to the

416

activated synthesis in the liver and release into the plasma of VTG and choriogenin.

417

The estrogenic activities of DCOIT were applicable to both male and female marine

418

medaka; however, a clear sex-specific response to DCOIT stress was identified based

419

on the greater susceptibility of male fish. In addition, parental DCOIT exposure at

420

environmentally realistic concentrations exerted hazardous transgenerational effects

421

on offspring viability. Overall, given the coastal pollution reported for DCOIT,

422

mounting evidence supporting the adverse effects of DCOIT on aquatic organisms

423

highlights the need for a systematic evaluation of the environmental risks of DCOIT, 17



424

with a special focus on its endocrine-disrupting effects. The differential sensitivity of

425

male and female fish in response to chemical toxicity should be taken into

426

consideration during a comprehensive ecological risk assessment. Furthermore, given

427

the coastal pollution and scarcity of toxicological information for the other antifouling

428

compounds, the present study also inspires more mechanistic research to bridge the

429

gap and advocates systematic evaluation of environmental risks of any new

430

antifoulant prior to marketing.

431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 18


Page 18 of 34

Page 19 of 34


447

Figure captions

448

Figure 1. Changes in larval average swimming activity under continuous 15-min light

449

(A), swimming trace in response to photoperiod stimuli (B) and average speed in each

450

lighting period (C) after parental exposure to various concentrations of DCOIT (0,

451

0.76, 2.45 and 9.86 µg/L). Values represent the mean ± SEM of twenty larvae and

452

significant difference between control and DCOIT groups is indicated by *P < 0.05

453

and **P < 0.01.

454

Figure 2. Imbalanced plasma hormone levels (testosterone, T; estradiol, E2;

455

11-keto-testosterone, 11-KT) in the male (A) and female (B) medaka and the

456

subsequent changes in hormone ratios (C) after exposure to various concentrations of

457

DCOIT (0, 0.76, 2.45 and 9.86 µg/L). Values represent the mean ± SEM of three

458

replicates and significant difference between control and exposure groups is indicated

459

by *P < 0.05, **P < 0.01 and ***P < 0.001.

460

Figure 3. ELISA measurement showing the VTG content changes in the liver and

461

plasma from male and female medaka after exposure to various concentrations of

462

DCOIT (0, 0.76, 2.45 and 9.86 µg/L). Values represent the mean ± SEM of three

463

replicates and significant difference between control and exposure groups is indicated

464

by *P < 0.05 and **P < 0.01.

465

Figure 4. Plasma shotgun proteomics profiling the differentially expressed proteins in

466

male (A) and female (B) after 28-days exposure to 0 and 9.86 µg/L DCOIT. Three

467

biological replicates are included and values represent the average spectral counts of

468

three measurements.

469

Figure 5. Overview of the changes in male medaka across entire HPGL axis

470

integrating the interactive map between tissues (i.e., hypothalamus, pituitary, gonad,

471

liver and blood). Each index comprises the responses of DCOIT exposure groups 19



472

(0.76, 2.45 and 9.86 µg/L) and the color intensity indicates the extent of changes

473

relative to the control group. Green coloring stands for down-regulation and red for

474

up-regulation. The gene transcriptions are shown italic to distinguish changes in other

475

biological organizations.

476

Figure 6. Overview of the changes in female medaka across entire HPGL axis

477

integrating the interactive map between tissues (i.e., hypothalamus, pituitary, gonad,

478

liver and blood). Each index comprises the responses of DCOIT exposure groups

479

(0.76, 2.45 and 9.86 µg/L) and the color intensity indicates the extent of changes

480

relative to the control group. Green coloring stands for down-regulation and red for

481

up-regulation. The gene transcriptions are shown italic to distinguish changes in other

482

biological organizations.

483 484 485 486 487 488 489 490 491 492 493 494 20


Page 20 of 34

Page 21 of 34


495

SUPPORTING INFORMATION AVAILABLE

496

Figure S1 shows the morphological changes in the liver in male and female fish,

497

showing the development of vacuolization in female liver. Figure S2 is the summary

498

of oocyte percentages at each phase (previtellogenic, vitellogenic and postvitellogenic

499

oocyte). Table S1 lists the genes primers for qPCR. Table S2 shows the growth and

500

condition factors of adult medaka. Table S3 shows the effects of parental DCOIT

501

exposure on offspring viability. Table S4 is the results of gene transcription involved

502

in the endocrine disruption of DCOIT. This information is available free of charge via

503

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

504 505 506 507

ACKNOWLEDGEMENTS

508

This work was supported by grants from the Natural Science Foundation of China (#

509

41576140) and from China Ocean Mineral Resources Research and Development

510

Association (COMR-RDA12SC01) to PY Qian.

21



Page 22 of 34

511

REFERENCES

512

(1) Callow, M. E.; Callow, J. A. Marine biofouling: A sticky problem. Biologist 2002,

513

49 (1), 1–5.

514

(2) Dobretsov, S.; Dahms, H. U.; Qian, P. Y. Inhibition of biofouling by marine

515

microorganisms and their metabolites. Biofouling 2006, 22 (1), 43–54.

516

(3) Qian, P. Y.; Chen, L.; Xu, Y. Mini-review: Molecular mechanisms of antifouling

517

compounds. Biofouling 2013, 29 (4), 381–400.

518

(4) Konstantinou, I. K.; Albanis, T. A. Worldwide occurrence and effects of

519

antifouling paint booster biocides in the aquatic environment: A review. Environ. Int.

520

2004, 30 (2), 235–248.

521

(5) Jacobson, A. H.; Willingham, G. L. Sea-nine antifoulant: an environmentally

522

acceptable alternative to organotin antifoulants. Sci. Total Environ. 2000, 258 (1–2),

523

103–110.

524

(6) Martínez, K.; Ferrer, I.; Barceló, D. Part-per-trillion level determination of

525

antifouling pesticides and their by products in seawater samples by off-line

526

solid-phase

527

chromatography–atmospheric pressure chemical ionization mass spectrometry. J.

528

Chromatogr. A 2000, 879 (1), 27–37.

529

(7) Kobayashi, N.; Okamura, H. Effects of new antifouling compounds on the

530

development of sea urchin. Mar. Pollut. Bull. 2002, 44 (8), 748–751.

531

(8) Chen, L.; Ye, R.; Xu, Y.; Gao, Z.; Au, D. W. T.; Qian, P. Y. Comparative safety of

532

the antifouling compound butenolide and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one

533

(DCOIT) to the marine medaka (Oryzias melastigma). Aquat. Toxicol. 2014, 149,

534

116–125.

extraction

followed

by

high-performance

22


liquid

Page 23 of 34


535

(9) Chen, L.; Sun, J.; Zhang, H.; Au, D. W. T.; Lam, P. K. S.; Zhang, W.; Bajic, V. B.;

536

Qiu, J. W.; Qian, P. Y. Hepatic proteomic responses in marine medaka (Oryzias

537

melastigma)

538

[5-octylfuran-2(5H)-one] or 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT).

539

Environ. Sci. Technol. 2015, 49 (3), 1851–1859.

540

(10) Ito, M.; Mochida, K.; Ito, K.; Onduka, T.; Fujii, K. Induction of apoptosis in

541

testis of the marine teleost mummichog Fundulus heteroclitus after in vivo exposure

542

to the antifouling biocide 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (Sea-Nine 211).

543

Chemosphere 2013, 90 (3), 1053–1060.

544

(11) Fossati, P.; Prencipe, L. Serum triglycerides determined colorimetrically with an

545

enzyme that produces hydrogen peroxide. Clin. Chem. 1982, 28 (10), 2077–2080.

546

(12) Chen, L.; Yu, K.; Huang, C.; Yu, L.; Zhu, B.; Lam, P. K.; Lam, J. C.; Zhou, B.

547

Prenatal transfer of polybrominated diphenyl ethers (PBDEs) results in developmental

548

neurotoxicity in zebrafish larvae. Environ. Sci. Technol. 2012, 46 (17), 9727–9734.

549

(13) Kong, R. Y. C.; Giesy, J. P.; Wu, R. S. S.; Chen, E. X. H.; Chiang, M. W. L.; Lim,

550

P. L.; Yuen, B. B. H.; Yip, B. W. P.; Mok, H. O. L.; Au, D. W. T. Development of a

551

marine fish model for studying in vivo molecular responses in ecotoxicology. Aquat.

552

Toxicol. 2008, 86 (2), 131–141.

553

(14) Kinoshita, M.; Murata, K.; Naruse, K.; Tanaka, M. Medaka: Biology,

554

Management, and Experimental Protocols; Wiley-Blackwell: Ames, IA, 2009.

555

(15) Shevchenko, A.; Tomas, H.; Havliš, J.; Olsen, J. V.; Mann, M. In-gel digestion

556

for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 2006,

557

1 (6), 2856–2860.

chronically

exposed

to

antifouling

23


compound

butenolide


558

(16) Liu, X.; Hu, Y.; Pai, P. J.; Chen, D.; Lam, H. Label-free quantitative proteomics

559

analysis of antibiotic response in Staphylococcus aureus to oxacillin. J. Proteome Res.

560

2014, 13 (3), 1223–1233.

561

(17) Zhang, X.; Hecker, M.; Jones, P. D.; Newsted, J.; Au, D.; Kong, R.; Wu, R. S. S.;

562

Giesy, J. P. Responses of the medaka HPG axis PCR array and reproduction to

563

prochloraz and ketoconazole. Environ. Sci. Technol. 2008, 42 (17), 6762–6769.

564

(18) Han, X. B.; Lei, E. N. Y.; Lam, M. H. W.; Wu, R. S. S. A whole life cycle

565

assessment of waterborne PBDEs on gene expression profile along the

566

brain–pituitary–gonad axis and in the liver of zebrafish. Mar. Pollut. Bull. 2011, 63

567

(5–12), 160–165.

568

(19) Sun, L.; Zuo, Z.; Chen, M.; Chen, Y.; Wang, C. Reproductive and

569

transgenerational toxicities of phenanthrene on female marine medaka (Oryzias

570

melastigma). Aquat. Toxicol. 2015, 162, 109–116.

571

(20) Zhang, X.; Hecker, M.; Park, J. W.; Tompsett, A. R.; Newsted, J.; Nakayama, K.;

572

Jones, P. D.; Au, D.; Kong, R.; Wu, R. S.; et al. Real-time PCR array to study effects

573

of chemicals on the Hypothalamic-Pituitary-Gonadal axis of the Japanese medaka.

574

Aquat. Toxicol. 2008, 88 (3), 173–182.

575

(21) Liu, C.; Yu, L.; Deng, J.; Lam, P. K. S.; Wu, R. S. S.; Zhou, B. S. Waterborne

576

exposure to fluotelomer alcohol 6:2 FTOH alters plasma sex hormone and gene

577

transcription in the hypothalamic-pituitary-gonadal (HPG) axis of zebrafish. Aquat.

578

Toxicol. 2009, 93 (2–3), 131–137.

579

(22) Arukwe, A.; Goksøyr, A. Eggshell and egg yolk proteins in fish: Hepatic proteins

580

for the next generation: Oogenetic population, and evolutionary implications of

581

endocrine disruption. Comp. Hepatol. 2003, 2 (4), 1–21.

24


Page 24 of 34

Page 25 of 34


582

(23) de Wit, M.; Keil, D.; van der Ven, K.; Vandamme, S.; Witter, E.; de Coen, W. An

583

integrated transcriptomic and proteomic approach characterizing estrogenic and

584

metabolic effects of 17α-ethinylestradiol in zebrafish (Danio rerio). Gen. Comp.

585

Endocrinol. 2010, 167 (2), 190–210.

586

(24) Pillai, S.; Behra, R.; Nestler, H.; Suter, M. J. -F.; Sigg, L.; Schirmer, K. Linking

587

toxicity and adaptive responses across the transcriptome, proteome, and phenotype of

588

Chlamydomonas reinhardtii exposed to silver. Proc. Natl. Acad. Sci. USA 2014, 111

589

(9), 3490–3495.

590

(25) Du, Y.; Shi, X.; Liu, C.; Yu, K.; Zhou, B. Chronic effects of water-borne PFOS

591

exposure on growth, survival and hepatotoxicity in zebrafish: A partial life-cycle test.

592

Chemosphere 2009, 74 (5), 723–729.

593

(26) Schwaiger, J.; Spieser, O. H.; Bauer, C.; Ferling, H.; Mallow, U.; Kalbfus, W.;

594

Negele, R. D. Chronic toxicity of nonylphenol and ethinylestradiol: haematological

595

and histopathological effects in juvenile Common carp (Cyprinus carpio). Aquat.

596

Toxicol. 2000, 51 (1), 69–78.

597

(27) Marlatt, V. L.; Martyniuk, C. J.; Zhang, D.; Xiong, H.; Watt, J.; Xia, X.; Moon, T.;

598

Trudeau, V. L. Auto-regulation of estrogen receptor subtypes and gene expression

599

profiling of 17β-estradiol action in the neuroendocrine axis of male goldfish. Mol.

600

Cell. Endocrinol. 2008, 283 (1–2), 38–48.

601

(28) Saunders, D. M. V.; Podaima, M.; Wiseman, S.; Giesy, J. P. Effects of the

602

brominated flame retardant TBCO on fecundity and profiles of transcripts of the

603

HPGL-axis in Japanese medaka. Aquat. Toxicol. 2015, 160, 180–187.

604

(29) Kane, A. S.; Salierno, J. D.; Gipson, G. T.; Molteno, T. C.; Hunter, C. A

605

video–based movement analysis system to quantify behavioral stress responses of fish.

606

Water Res. 2004, 38 (18), 3993–4001. 25



607

(30) Wu, X.; Huang, Q.; Fang, C.; Ye, T.; Qiu, L.; Dong, S. PFOS induced precocious

608

hatching of Oryzias melastigma-From molecular level to individual level.

609

Chemosphere 2012, 87 (7), 703–708.

610

(31) Lema, S. C.; Schultz, I. R.; Scholz, N. L.; Incardona, J. P.; Swanson P. Neural

611

defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2, 2′, 4,

612

4′-tetrabromodiphenyl ether (PBDE 47). Aquat. Toxicol. 2007, 82 (4), 296–307.

613

(32) Ong, K. J.; Zhao, X.; Thistle, M. E.; Maccormack, T. J.; Clark, R. J.; Ma, G.;

614

Martinez-Rubi, Y.; Simard, B.; Loo, J. S.; Veinot, J. G.; Goss, G. G. Mechanistic

615

insights into the effect of nanoparticles on zebrafish hatch. Nanotoxicology 2014, 8

616

(3), 295–304.

617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 26


Page 26 of 34

Page 27 of 34


Average Swimming Speed (mm/s)

A 2.5 2.0

*

1.5 1.0 0.5 0.0 0

0.76

2.45

9.86

Exposure Concentrations (µg/L)

632

0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

B 4.0 Swimming Trace

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Dark

Light

Light

Dark

Photoperiod Stimuli

633

C 4.0

0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

Average Swimming Speed (mm/s)

3.5 3.0 2.5 2.0

*

*

1.5

*

1.0

**

0.5 0.0 Dark

634 635

Light

Dark

Light

Photoperiod Stimuli

Figure 1 27



Steroid Hormone Levels (pg/mg protein)

A 700

0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

600 500 400

**

300 200 100

** *** **

0

T

636

E2

11-KT

Steroid Hormone Levels (pg/mg protein)

B 600

0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

500 400

*

300

*

200 100

* ** 11-KT

0

T

637

Hormone Ratio (as % of values in control)

C

639

E2

0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

7 6

***

5

***

4 3

*

***

2 1 0 E2/T

638

Page 28 of 34

E2/11-KT

E2/T

E2/11-KT

Female

Male

Figure 2 28


Page 29 of 34


0 µg/L 0.76 µg/L 2.45 µg/L 9.86 µg/L

14000 12000

VTG Content (ng/mg protein)

10000 8000 6000 4000

*

2000

** 40

* *

20 0 640

** * Liver

Plasma

Liver

Female

Male

641 642 643 644 645 646 647 648 649 650 651 652 653 654

Plasma

Figure 3 29



30


Page 30 of 34

Page 31 of 34


Figure 4

31



Figure 5

32


Page 32 of 34

Page 33 of 34


1 2

Figure 6 33



3

Table of Contents (TOC) Art Antifouling Ready Degradation

Low Toxicity Effective Green

High Activity

34


Page 34 of 34

Chronic Exposure of Marine Medaka (Oryzias melastigma) to 4,5

Recommend Documents