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Spatio-temporal trends of heavy metals in Indo-Pacific humpback dolphins (Sousa chinensis) from the western Pearl River Estuary, China Duan Gui, Riqing Yu, Leszek Karczmarski, Yulong Ding, Haifei Zhang, Yong Sun, Mei Zhang, and Yuping Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05566 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 12, 2017

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Environmental Science & Technology

1

Spatio-temporal trends of heavy metals in

2

Indo-Pacific

3

chinensis) from the western Pearl River

4

Estuary, China

5

Duan Gui a, Ri-Qing Yu b, Leszek Karczmarski c, *, Yulong Ding a, Haifei Zhang a,

6

Yong Sun a, Mei Zhang a, Yuping Wu a, *

7 8

a

9

Innovation Center, Zhuhai Key Laboratory of Marine Bioresources and Environment,

10

Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering,

11

School of Marine Sciences, Sun Yat-Sen University, Guangzhou, 510275, China

12

b

Department of Biology, University of Texas at Tyler, Tyler, TX 75799, USA

13

c

The Swire Institute of Marine Sciences, School of Biological Sciences, The

14

University of Hong Kong, Cape d’Aguilar, Shek O, Hong Kong

humpback

dolphins

(Sousa

South China Sea Bio-Resource Exploitation and Utilization Collaborative

15

16 17 18

Keywords: Indo-Pacific humpback dolphins; Heavy metals; Ecotoxicology; Pearl

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River Estuary 1 ACS Paragon Plus Environment


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ABSTRACT: We assessed the spatio-temporal trends of the concentrations of 11

21

heavy metals (HMs) in the liver and kidney of Indo-Pacific humpback dolphins

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(Sousa chinensis) from western Pearl River Estuary (PRE) during 2004-2015. The

23

hepatic levels of Cr, As and Cu in these dolphins were among the highest reported for

24

cetaceans globally, and the levels of Zn, Cu and Hg were sufficiently high to cause

25

toxicological effects in some of the animals. Between same age-sex groups, dolphins

26

from Lingdingyang were significantly more contaminated with Hg, Se and V than

27

those from the West-four region, while the opposite was true for Cd. Generalized

28

additive mixed models showed that most metals had significant but dissimilar

29

temporal trends over a 10-year period. The concentrations of Cu and Zn increased

30

significantly in recent years, corresponding to the high input of these metals in the

31

region. Body-length-adjusted Cd levels peaked in 2012, accompanied by the highest

32

annual number of dolphin stranding events. In contrast to the significant decrease in

33

HM levels in the dolphins in Hong Kong waters (the eastern reaches of the PRE), the

34

elevated metal exposure in the western PRE raises serious concerns.

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INTRODUCTION

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The second largest estuarine system in China in terms of water and sediment

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discharge, the Pearl River Estuary (PRE) receives high contaminant loads from across

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one of the most densely populated and rapidly developing region of China

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Pearl River Delta. The PRE serves also as a habitat for the largest known (putative)

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population of Indo-Pacific humpback dolphins (Sousa chinensis)

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large-scale urban and industrial developments of the Pearl River Delta in recent

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decades, the range and intensity of human-induced pressures on humpback dolphins

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in PRE waters are of a scale rarely found in other small cetacean populations

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elsewhere 6. Recent demographic study indicate a declining population trend of ~2.5%

45

per annum, suggesting that ~74% of the current population numbers may likely be

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lost within the lifespan of three generations (~60 years) 7. Preliminary line-transect

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surveys in the western part of the PRE also suggest declining sighting rates 8. High

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body burden of contaminants are increasingly linked to population decline or slow

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population recovery of several cetacean species 9-11, largely due to effects of pollutants

50

on the reproduction, endocrine and immune systems disruption. Humpback dolphins

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in the PRE are currently exposed to an alarmingly high level of Persistent Organic

52

Pollutants (POPs)

53

been empirically addressed.

1, 2

, the

3-5

. Due to

12-15

. However, their exposure to heavy metals (HMs) has never

54

HMs are regarded as powerful tracers of the eco-physiological status of marine

55

mammals. For instance, significantly higher hepatic Hg and Zn levels were found in

56

harbor porpoises that died from infectious diseases than in those that died from

57

physical trauma

58

Hg and the redistribution of Zn in the liver caused by infection. In the tissue of

59

healthy cetaceans, Zn is regularly correlated with Cu due to their antagonistic

16

. This pattern might be due to the immunosuppressive toxicity of



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behavior (both bind to metallothioneins). However, a high intake of anthropogenic Zn

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can substantially alter the Zn/Cu ratio, reflecting a severe homeostasis disturbance

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linked to emaciation, disease and mortality in porpoises

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that

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health-compromised harbor porpoises, indicating that Zn concentration can be used as

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an index of deteriorating health in these animals due to exposure to POPs

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metals reflect the diet of marine mammals, such as Cd and As 16, 19-22. In cetaceans, Cd

67

is absorbed mainly by feeding on cephalopods, as cephalopods contain mostly the

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bioavailable form of Cd 16.

Zn

levels

were

positively

correlated

17

. Previous studies showed

with

high

PCB

burden

in

18, 19

. Some

69

As humpback dolphins occupy high trophic level in the marine food chain,

70

bioaccumulate throughout their long life-span, and, off the China coast, often live in

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close proximity of high concentration of anthropogenic pollution sources, they can be

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considered a good bio-indicator of the level of persistent pollutants in the marine

73

environment, including HMs

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the contamination levels in their environment, which in turn can facilitate the

75

identification of populations that face the greatest risks of metal toxicity and,

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subsequently, develop necessary management and conservation practices.

23-26

. Metal concentrations in dolphin tissue may reflect

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The PRE has been severely polluted by anthropogenic HMs 2, some of which are

78

highly persistent and bioaccumulative. The HMs in the PRE originate mainly from

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eight terrestrial anthropogenic sources, including four that run into Lingdingyang and

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other neighboring estuaries (referred to as the West-four PRE region) that flow

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directly into the South China Sea

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in the environmental level of HMs in Lingdingyang and the West-four PRE region

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were previously reported 2. Waste waters directly discharged into the PRE from

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industrial factories and metal-containing fish feed widely used in marine aquaculture

2, 27

(Figure 1). Geographic and temporal variation


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zones are the major sources of HMs in this region 28, 29. In recent years, there has been

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a notable acceleration of economic development in the western Pearl River Delta

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(Zhongshan, Zhuhai and Jiangmen city), including a number of marine constructions,

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such as reclamation projects, port development, dredging of shipping channels, and

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building of bridges in the Lingdingyang waters

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alarming amounts of HMs to enter into the suspension

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dolphins to elevated levels of bioaccumulative metals.

92

30

. Offshore engineering can cause 31, 32

, exposing the PRE

Although the temporal trends of HM levels have previously been examined in 33

93

cetaceans within Hong Kong-administered waters

, no study on the dolphins from

94

the rest of the PRE has yet been performed. Given that > 90% of the known habitat of

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the PRE humpback dolphins is in waters west of the Hong Kong Special

96

Administrative Region (denoted as the western PRE in this study) 6, it is important to

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examine the long-term trend of the dolphin exposure to HM in the western PRE. The

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previous study conducted in Hong Kong (HK) waters 33 reported that the HM levels in

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humpback dolphins decreased significantly in the past decade, which was associated

100

with the decline of the HM concentrations in HK waters due to strict pollution control

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by the local government and the relocation of industries to mainland China

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However, the western PRE is more contaminated by HMs due to the proximity to the

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HM pollution sources of the river outlets from mainland China 2. In this study, the

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current levels and spatio-temporal trends of HMs were comprehensively investigated,

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for the first time, in the Indo-Pacific humpbak dolphins stranded in the western PRE

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(2004-2015), including Lingdingyang and the West-four PRE region, which are close

107

to the river outlets and pollution sources in the PRE. Our analyses include

108

mother-fetus pairs which to the best of our knowledge is the first use of such data. We

109

also analyzed the relationship between HMs and POPs to determine whether Zn can

34

.



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be used as an indicator of harmful effects of POP exposure in humpback dolphins,

111

which has previously been seen in other cetacean species 17, 19.

112 113

MATERIALS AND METHODS

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Sample collection. Between 2004 and 2015, liver (n = 69) and kidney (n = 63)

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tissue samples were collected from dolphin carcasses stranded on the shore of western

116

Lingdingyang (mostly off the coast of Zhuhai, denoted as ZH) and the West-four PRE

117

region (mainly found off the coast of Jiangmen, denoted as JM). Of those, 57 were

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paired samples (liver and kidney tissue collected from the same stranded individual;

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Figure 1). The physiographic features of the PRE and patterns of coastal hydrology

120

make the stranded carcasses unlikely to be transported from Lingdingyang to the

121

West-four PRE region and vice versa

122

study suggests that there is a limited exchange of individuals, if any, between

123

Lingdingyang to the West-four PRE region (L. Karczmarski and Y. Wu, study in

124

progress). Therefore, in this study the ZH and JM samples were treated as from the

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Lingdingyang and West-four region, separately.

35

. Furthermore, ongoing photo-identification

126

Tissue samples were collected from each specimen during dolphin necropsies.

127

Only two individuals (ZH-SC-73 and ZH-SC-74) were stranded alive and were

128

sampled immediately after their death, while the other carcasses were moderately

129

decomposed (code 3). The two life-stranded individuals (ZH-SC-73 and ZH-SC-74)

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were subsequently identified (matched to the photo-ID catalogue at the University of

131

Hong Kong) as dolphins that were previously seen in HK waters and therefore their

132

tissue were excluded from the western PRE samples. Morphological and biological

133

data, including total length, weight and sex, were collected before necropsy. Total

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length was measured as the straight-line length from the tip of the upper jaw to the 6 ACS Paragon Plus Environment

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fluke notch. The sex of each individual was determined by inspecting the reproductive

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organs, and if this was not possible in the field, it was subsequently determined by

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DNA analysis. Organs were packed in clean plastic storage bags and frozen at -20˚C

138

for further analysis.

139

Sample Preparation and Chemical Analysis. Samples were freeze-dried

140

(Freeze-drying system, Labconco, Kansas City, Missouri, USA) for 48 h to a constant

141

weight and then ground to powder using an automatic agate mortar (Mixer Mill MM

142

400, Retsch, Germany). The mean ratio between the dry weight (d.wt.) and wet

143

weight (w.wt.) was 0.29 for liver and 0.23 for kidney. Approximately 0.2 g of dry

144

tissue sample was digested in a Teflon digestive vessel with a mixture of 2 ml of

145

deionized water (18.2 MΩ·CM, Milli-Pore, USA) and 6 ml of nitric acid (Suprapur®

146

quality, Sigma, USA) in a microwave digestion oven (XT-9912, XinTuo, Shanghai,

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China). The resulting digests were cooled and filtered through disposable syringe

148

filter discs (0.45 µm pores, Jing Teng, China). The final solution was diluted with

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deionized water to a volume of 50 ml.

150

The concentrations of 11 metals (V, Cr, Mn, Ni, As, Zn, Cu, Cd, Se, Pb and Hg)

151

were measured following the method of Gui et al., (2014) 36 using inductively coupled

152

plasma mass spectrometry (ICP-MS) (Agilent 7700X, USA). Four elements, Sc, Ge,

153

In and Bi, were used as internal standards to correct the matrix effects and

154

instrumental drift of the ICP-MS.

155

Quality assurance/quality control (QA/QC) was performed using TORT-2

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(lobster hepatopancreas, NCRC Canada) as the standard reference material, and was

157

treated under the same conditions as the dolphin tissue samples. The results were in

158

good agreement with the certified values, with metal recoveries ranging from 82% to

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115%. A method blank, spiked blank (with a known standard solution), matrix spike 7 ACS Paragon Plus Environment


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(a known amount of target analyte standard solution spiked into dolphin tissue), and

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sample duplicate were included in the batch of 18 samples. The method blanks were

162

below the detection limit. The relative difference of replicate analyses between

163

duplicate samples was less than 15% for all target analytes. The method detection

164

limits (MDLs) (µg L-1 dw) were 0.0015, 0.0275, 0.0304, 0.0253, 0.0017, 0.0628,

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0.0028, 0.0026, 0.2526, 0.0024, and 0.0036 for V, Cr, Mn, Ni, As, Zn, Cu, Cd, Se, Pb,

166

and Hg, respectively. The concentrations of most metals were above the detection

167

limits. All concentrations below the detection limit were set to a value of half the

168

detection limit for the statistical analyses.

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Data Analysis. Trace element levels in this study are presented as µg g-1 dry

170

weight (dw), and the dw values are used in all statistical analyses. For comparison

171

with previously published studies, the dw data were converted into wet weight (ww)

172

basis using a conversion factor of 0.27 and 0.35 for liver and kidney tissues,

173

respectively. Because most of the data (metal concentrations and body lengths) did not

174

meet the assumption of a normal distribution, as determined by probability plots

175

(Quantile−Quantile (Q−Q) plots), the geometric mean and interquartile range were

176

used to describe the skewed distributions of the trace element concentrations.

177

Permutation-based statistical tests were applied to test for significant differences

178

between groups using Monte Carlo simulation to sample all possible permutations.

179

Gender, maturity, body length and habitat are potential sources of variation of trace

180

element levels in cetaceans. The ZH and JM samples were separately grouped

181

according to their maturity and sex: adult male (AM), adult female (AF), juvenile

182

male (JM), and juvenile female (JF). Then, a non-parametric version of ANOVA was

183

used to examine the differences between maturity and gender groups for each study

184

site. Spearman’s rank correlation coefficient was used to analyze potential linear 8 ACS Paragon Plus Environment

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relationships between trace element concentrations and body length.

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To test whether there were significant time trends in the metal concentrations, we

187

fitted generalized additive mixed models (GAMs) for all data using the mgcv library 37

188

in R 38. GAM is a smoothing equivalent of generalized linear modelling (GLM). This

189

approach has proven useful for interpreting the time trends of pollutant levels in

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cetaceans 10, 39, which are usually non-linear. We used cubic regression splines for the

191

smoothing. In modelling the temporal variation of the metal concentrations, all

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possible subsets of covariates, including sampling year, body length, gender and

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habitat, were used to determine the best model, in which all remaining explanatory

194

variables were significant, the value for the Akaike information criterion (AIC) was

195

the lowest, and there were no obvious patterns in the residuals. The natural log

196

transformation (log1p function in R) of the metal concentrations was used to

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normalize the frequency distribution of the data. After transformation, the trace

198

element concentration data were approximately normal. Therefore, a Gaussian

199

distribution with identity link was used. For liver tissue, a sufficient sample size was

200

available for each year between 2004 and 2015 for the Zhuhai samples, except for

201

2006, whereas sufficient sample sizes for kidney tissue were obtained by combining

202

the Zhuhai and Jiangmen samples. Therefore, we used the Zhuhai samples for the

203

time trend analysis for liver tissue, whereas we combined the Zhuhai and Jiangmen

204

samples for the kidney tissue analysis. The results were considered significant when p

205

< 0.05.

206 207

RESULTS AND DISCUSSION

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Overall Distribution and Potential Toxic Risk of Metals. Descriptive data for

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the hepatic and renal concentrations of each metal analyte are shown in Table 1. For 9 ACS Paragon Plus Environment


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all samples as well as the groups from Zhuhai, Jiangmen and Zhanjiang, Zn and Cu

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were the dominant metals. The mean hepatic levels of these two metals were

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comparable with those measured in the humpback dolphins from HK waters

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both were higher than those in cetaceans from other regions of the world

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(Table 2). A similar pattern was observed for arsenic, for which the mean

215

concentration in this study was at least twice that known from cetaceans elsewhere in

216

the world

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the PRE appears to be of natural origin 2. For most samples, excluding mother-fetus

218

pairs, the hepatic levels of essential metals (Zn and Cu) were generally within the

219

range of normal regulation of these metals in marine mammals (80–400 µg g-1 dw for

220

Zn and 12 to 120 µg g-1 dw for Cu) 49, although there were exceptions. Ten individuals

221

(15% of all samples) had hepatic Zn levels below the normal range, while the Zn

222

concentrations in two mature females far exceeded the upper range (616 and 1300 µg

223

g-1 dw). High levels of Zn in the liver suggest a connection to infectious or

224

inﬂammatory processes that could have occurred in these individuals before they

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stranded

226

threshold (range 133–519 µg g-1 dw), all of them were from young individuals with

227

body length ranging from 76 cm to 120 cm.

22, 42-48

, except Hong Kong

40, 41

40, 41

and

22, 42-48

(Table 2). It is noteworthy that arsenic in

17

. The Cu concentrations in 15 liver samples (21.7%) were above the

228

Chromium, an essential metal in low doses, is also a known human carcinogen

229

and genotoxicant. The mean hepatic Cr level in our samples was at least twice the

230

value of those found in marine mammals elsewhere (Table 2), including bottlenose

231

dolphins that died from an unusual mortality event in the Gulf of Mexico 42. This high

232

concentration of Cr in the PRE dolphins may be due to an excessive intake of Cr

233

because of serious Cr pollution in the PRE environment. The Cr levels in the waste

234

waters released into the PRE by many industrial factories are far above the maximum 10 ACS Paragon Plus Environment

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permitted concentration 28, and the highest concentration of Cr in Spiny-head croaker

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(Collichthys lucidus), one of the preferred prey of the PRE humpback dolphins 50, was

237

~48 times higher than the edible standard for humans 51. Cr is widely used in fish feed

238

in mariculture zones in the PRE to promote fish growth

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dolphin prey, shrimp contain the highest level of Cr 52, while Cr concentrations in fish

240

and cephalopods are relatively low 53. It is possible, therefore, that shrimp serve as the

241

primary source of Cr for the PRE dolphins. Globally, the highest levels of Cr in

242

cetaceans were found in the Gulf of Mexico, which was caused by the Deepwater

243

Horizon oil spill crisis, as Cr is a key component in crude oils 26. The epidermal Cr

244

concentrations in the Gulf whales (range: 8.0 to 294 µg g-1 dw) were higher than

245

previous records reported in marine mammals

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one-third of the liver and kidney samples had Cr concentrations within the range

247

reported for the cetaceans in the Gulf of Mexico

248

however, to identify the environmental source and route of Cr, which is accumulating

249

at alarmingly high levels in the PRE dolphins.

29

. Among the humpback

26

. In our study, approximately

26

. Further research is needed,

250

Regarding non-essential metals, the Hg level in one individual (ZH-SC-04, 1110

251

µg g-1 dw) reached the range indicating hepatic damage, that was established for

252

marine mammals by Wagemann and Muir (1984) (400–1600 µg g-1 dw) 54, whereas 19

253

PRE dolpins had levels that could alter the functional activity of bottlenose dolphin

254

leukocytes (40 µg g-1 dw) 55. As an antagonist against the toxicity of several HMs, Se

255

is both an essential and toxic element, depending on its concentration

256

individual ZH-SC-04 also had the greatest concentration of Se in the liver (356 µg g-1

257

dw), which was much higher than values previously reported in cetaceans (< 120 µg

258

g-1 dw)

259

second highest renal level of the toxic metal Cd, which suggested that its health was

56

. The

56

. Furthermore, this individual had also the highest hepatic level and the



260

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comprised.

261

All metals analyzed in this study showed significant positive correlations (r =

262

0.33-0.89, p < 0.01) between the paired liver and kidney samples (n = 57, excluding

263

the mother-fetus pairs), except for Pb (Figure S1). For the entire data set, the median

264

concentrations of all metals were considerably higher in the liver than in the kidney,

265

except for Cd. This distribution pattern agrees with the general accumulation profiles

266

of metals in marine mammals 49.

267 268

Based on the results of toxicological inference, heavy metal exposure in the PRE poses a potential threat to the PRE dolphins.

269

Potential Maternal Metal Transfer Based on Analysis of the Mother-Fetus

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Pairs. The analysis of two mother-fetus pairs collected from the ZH site represents the

271

first report of metals in the liver and kidney of mother-fetus pairs of humpback

272

dolphins. The body lengths of the two fetuses were 76 cm and 99 cm, compared to the

273

average body length of 101 cm at birth

274

essential and non-essential metals were found in the liver and kidney tissues, with

275

levels comparable to those reported in the fetuses of other cetacean species

276

The concentrations of non-essential metals, including Hg, Cd, Pb, V and As, in the

277

livers and kidneys of the fetuses were far lower than those in their mothers, suggesting

278

limited transplacental transfer of these toxic metals (Table 1).

57

. In the two fetuses, similar profiles of

18, 48, 58

.

279

Despite the small sample size, the hepatic and renal Hg concentrations in the

280

paired mothers and fetuses were correlated; the ratio of the Hg concentrations

281

between mother and fetus were 52 and 66 for the liver and 17.5 and 21.5 for the

282

kidney. A similar pattern was reported for short-beaked common dolphins (Delphinus

283

delphis) 58. For the essential metals, Zn, Cu and Se combined accounted for more than

284

95% of the total concentration of all analyzed metals in the livers and kidneys of the 12 ACS Paragon Plus Environment

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fetuses, with considerably higher values than their mothers. Similar findings were

286

previously reported for striped dolphins (Stenella coeruleoalba) and short-beaked

287

common dolphins 48, 58. The largest difference was observed for hepatic Cu, for which

288

the concentrations were 15-20 times higher in the fetuses than in the mothers,

289

indicating an effective maternal transfer and bioaccumulation of Cu in the fetuses.

290

Compared to Hg, Se had a much higher concentration in the liver and kidney of the

291

fetuses, which could alleviate the detrimental effects of Hg by forming Hg-Se

292

compounds.

293

One of the mother dolphins exhibited an exceptionally high level of Mn in the

294

kidney (87-fold the median level of the overall population without the mother-fetus

295

pairs), in accordance with the elevated hepatic Mn level reported in a pregnant Dwarf

296

sperm whale from the waters of Taiwan 59. The concentrations of Cr in the liver and

297

kidney and Pb in the kidney of this individual were also high in comparison to the

298

overall population. However, abnormally high levels of essential (Mn) and

299

non-essential (Cr and Pb) metals were not found in her fetus, suggesting that the

300

maternal transfer of these metals to the offspring was not significant.

301

Variation of Metal Accumulation Related to Sex, Maturity, Body Length, and

302

Habitat, and Interelement Correlations. Generally, no significant sex-specific

303

difference was observed for most metals in the liver and kidney, in agreement with

304

previous studies showing no sex difference in metal accumulation in marine mammals

305

60

306

was significantly higher in females (n = 12) than males (n = 20, Figure S2), whereas

307

that of Mn was significantly higher in JM adult females (n = 4) than in adult males (n

308

= 3, Figure S3).

309

. However, the median hepatic Cu concentration in samples of juveniles from ZH

In the ZH samples, that hepatic levels of V, Cd, Pb, Hg and Se increased 13 ACS Paragon Plus Environment


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310

significantly with increasing maturity. Cu was an exception, with a significantly

311

higher level in juvenile females (JF) than adults (Figure S2). In the JM samples, only

312

Mn exhibited a significant difference among the four maturity and sex groups, with

313

significantly higher hepatic concentrations in the AF than the JM (Figure S3). In the

314

kidney, As, Cd, Hg and Se exhibited significantly higher concentrations in ZH adult

315

dolphins than in juveniles, whereas the concentration of Cu was significantly higher in

316

juvenile males than in adult females (Figure S4). In the JM samples, no significant

317

difference was observed in the renal concentrations between adult and juvenile

318

dolphins (Figure S5).

319

Consistent with the ANOVA results, significant positive correlations were

320

observed between body length and the V, Mn, As, Se, Hg, Cd and Pb concentrations in

321

the liver of ZH dolphins, with Spearman’s rank coefficients ranging from 0.4 (As) to

322

0.83 (Cd) (Figure S6). As expected, the hepatic and renal Cu concentrations were

323

significantly negatively correlated with body length in the ZH samples, in accordance

324

with most previous analyses of the relationship between Cu and body length in marine

325

mammals 61. For the JM samples, only the V, Se, Hg and Cd levels were significantly

326

positively correlated with body length. In the kidney samples, the As, Cd, Hg, Se

327

concentrations were significantly positively correlated with the body length of the ZH

328

samples, while only Hg and Cd were correlated with body length in the JM samples.

329

Significant site-related differences were found for Cd (one-way test, p < 0.001),

330

Hg (p < 0.01), Se (p < 0.01) and V (p < 0.05). Juveniles from Lingdingyang had

331

significantly higher mean hepatic levels of Hg, Se and V than those from the

332

West-four PRE region. However, the mean Cd levels in the livers of juveniles and in

333

the kidneys of both juvenile and adult dolphins from the West-four PRE region were

334

significantly higher than those in the same maturity groups from Lingdingyang 14 ACS Paragon Plus Environment

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335 336

(details about the comparison method are provided in the Supporting Information). Cetacean species that feed primarily on cephalopods are expected to accumulate 18, 62

337

higher levels of Cd than those feeding on fish

. Although humpback dolphins

338

primarily feed on fish, they also eat cephalopods, as documented in some areas, e.g.,

339

HK waters

340

unknown. Therefore, further investigations are needed to test whether there are

341

differences between the prey of humpback dolphins from Lingdingyang and the

342

West-four PRE region.

50

. However, the diet of humpback dolphin in the western PRE remains

343

Temporal Trends. In the liver tissue, statistically significant temporal trends

344

were found for all HMs, excluding V and Pb, whereas statistically significant trends

345

for renal samples were observed only for Hg, Cd, Cr, Se, Mn, As and Ni (detailed

346

results of the GAMs are summarized in Table S1). The effects of time on metal

347

bioaccumulation are plotted in Figure 2 along with factors such as body length, at

348

their mean value.

349

A rapid increase in the Zn concentration from approximately 2012 onward

350

(Figure 2) was observed in samples from Lingdingyang. Because Zn does not

351

accumulate with age, an elevated Zn concentration in the liver of marine mammals is

352

generally assumed to be an index of acute-phase protein synthesis rather than a direct

353

result of elevated environmental levels 63, 64. Previous research showed that Zn levels

354

were positively associated with a high body burden of polychlorinated biphenyls

355

(PCBs) in harbor porpoises 19, indicating that hepatic Zn levels are an indicator of the

356

harmful effects of PCB exposure for cetaceans. In our case, the hepatic Zn levels in

357

this study were moderately positively correlated with dichlorodiphenyltrichloroethane

358

(DDT) blubber levels (Spearman’s r = 0.48, p < 0.01) and weakly positively

359

correlated with those of PCBs (Spearman’s r = 0.33, p < 0.05) (Figure 3). Therefore, 15 ACS Paragon Plus Environment


Page 16 of 38

360

we propose that a significant exposure of the PRE dolphins to POPs is related to an

361

increasing number of disease-caused mortalities, as opposed to a trauma-related death

362

of otherwise healthy individuals, resulting in an elevated Zn concentration in recent

363

years. During post-mortem examination, diseases such as lung abnormality and

364

tumors were frequently observed in the autopsy and histological analysis of the

365

stranded dolphins, with an increasing trend in recent years (personal observation).

366

The liver Cu concentrations showed a stable increasing trend throughout the

367

entire period (Figure 2), which reflects an excessive intake of Cu by this species. Cu

368

is included in commercial fish feed as a fish-growth promoter, leading to intensive

369

loading of Cu in the PRE sediment due to fish farming

370

antifouling paints are widely applied to the aquaculture cages and nets in the PRE to

371

prevent the growth of marine biofouling organisms, such as bivalves

372

amounts of Cu may dissolve from the antifouling paints and lead to a serious

373

contamination of the PRE dolphins.

29

. Furthermore, Cu-based

29

. Thus, large

374

The Cd concentrations in the liver and kidney samples peaked in 2012 (Figure 2

375

and Figure S7), the year in which the construction of the Hong Kong-Zhuhai-Macau

376

Bridge began in Zhuhai

377

dolphins was significantly higher than the annual number in the PRE in the previous 8

378

years. Resuspension of metal-contaminated sediments caused by marine engineering

379

construction works can result in a rapid increase in metal bioavailability to marine

380

organisms

381

An environmental monitoring study reported that the Cd levels in the PRE increased

382

almost twofold from 2011 to 2012, at a rate higher than the rates of Cu and Zn

383

Further study is needed to assess the interactions between metal sediment disturbance

384

and the accumulation of Cd in PRE dolphins. For Cr, Mn and Ni, the maximum levels

65

. In 2012, the number of recorded stranding of humpback

66

. Cd is easily biomagnified in cetaceans through the marine food webs.

67

.


Page 17 of 38


385

occurred in 2009 (n = 6), and the concentrations have since decreased, which may be

386

due to the efficient control of the pollution sources of these metals in the PRE (Figure

387

2). As shown in Figure 3G and H, the temporal trends for Hg and Se remained

388

relatively unchanged, while an apparent decrease was observed for As.

389

The elevated metal levels found in Indo-Pacific humpback dolphins stranded in the

390

western PRE could be due to their location near the river outlets and pollution sources

391

of mainland China. Compared to the generally decreasing trends of metal levels in

392

dolphins in HK waters 33, the increasing levels of metals found in this study should be

393

seen as a cause of concern, as more than 90% of the known habitat of the PRE

394

humpback dolphins is located in waters west of Hong Kong. A recent study indicates

395

that the demographic trajectory of the PRE dolphins is highly sensitive to fluctuations

396

in adult survival and the availability of suitable habitat 68. Both habitat quantity and

397

quality matters, and under the ever growing anthropogenic pressure, securing the

398

latter is becoming increasingly important

399

elsewhere in Asia and beyond

400

contaminant bioaccumulation in the PRE dolphins are still lacking, especially in

401

western PRE. Further assessment of current and future exposure levels and long-term

402

monitoring of pollutants in dolphins and other marine biota in the western PRE should

403

be given high regional priority.

6, 68

, as reported for humpback dolphins

69-72

. However, long-term studies of temporal trends of



404

Page 18 of 38

FIGURES

405 406

Figure 1. Sampling locations of stranded Indo-Pacific humpback dolphins in the Pearl

407

River Estuary, China. The colors represent the tissue types analyzed in each dolphin.

408

The numbers indicate the eight main outlets where the Pearl River flows into the

409

South China Sea, including Yamen (1), Hutiaomen (2), Jitimen (3), Modaomen (4),

410

Hengmen (5), Hongqimen (6), Jiaomen (7) and Humen (8).


Page 19 of 38


411 412

Figure 2. Temporal trends of the trace elements in the liver samples of the

413

PRE-stranded Indo-Pacific humpback dolphins during 2004-2015 with the other

414

predictor variables held at their mean. The solid line represents the smoothed trend

415

from a generalized additive model fitted to the data. The gray strip represents the 95%

416

bootstrapped confidence intervals.

417



Page 20 of 38

418 419

Figure 3. Spearman correlation matrix between hepatic metal concentrations and

420

blubber persistent organic pollutant levels of Indo-Pacific humpback dolphins with

421

correlation coefficient and significance levels denoted.


Page 21 of 38


422

423

TABLES

424 425 426

Table 1. Heavy metal concentrations (geometric mean with interquartile range, µg g-1 dry weight) in the liver a and kidney b of Indo-Pacific humpback dolphins from the Pearl River Estuary and Zhanjiang (ZJ) sampled between 2004 and 2015. ZH represents Zhuhai samples collected on the coast of Lingdingyang. JM represents Jiangmen samples collected on the coast of the West-four PRE region. Fetus1 Zn Cu Se Hg Mn Cr As Ni Cd

Mother1

Fetus2

Mother2

ZJ (n = 1)

JM (n = 13a, 12b)

ZH (n = 53a*, 48b)

ALL (n = 69a, 63b)

Liver

184.88

120.53

76.33

25.79

139

185 (128-231)

136 (108-184)

149 (121-204)

Kidney

89.79

80.79

35.51

40.72

145

96.5 (75.8-135)

99.9 (89.9-123)

104 (89.3-130)

Liver

334.76

15.87

519.23

32.77

8.78

32.8 (21.6-40.2)

41.2 (18.5-66)

36.5 (19-42.6)

Kidney

18.42

11.12

66.55

20.18

13.9

17 (13.3-21.4)

25.3 (19-33.2)

23.2 (17.1-32.1)

Liver

11.12

36.3

20.41

27.98

92.4

13.8 (4.75-31.1)

10.6 (4.46-28.5)

11.1 (4.36-31.1)

Kidney

3.23

7.63

6.47

13.18

20.4

9.41 (6.2-11.2)

6.56 (4.32-9.45)

7.18 (4.66-10.3)

Liver

1.02

68.2

0.05

2.64

217

11.3 (1.1-41.7)

4.88 (0.99-36.2)

6.52 (1.01-41.7)

Kidney

0.33

6.17

0.02

0.45

26

2.82 (0.943-11.1)

1.06 (0.344-6.01)

1.47 (0.389-6.97)

Liver

2.58

7.84

5.61

14.83

6.88

10.3 (7.81-16.2)

9.47 (6.43-14.7)

9.81 (6.67-15.7)

Kidney

2.1

3.16

2.78

498.5

3.48

5.92 (3.24-9.46)

5.92 (3.27-8.75)

5.67 (3.48-8.94)

Liver

2.11

2.96

0.87

203

1.58

4.38 (1.63-15.3)

3.54 (0.926-9.52)

3.54 (1.18-9.76)

Kidney

1.72

2.11

0.53

134

0.687

3.81 (0.911-19.7)

4.25 (1.55-12.1)

4.04 (1.07-13.9)

Liver

0.29

1.41

0.77

2.66

2.73

1.31 (0.886-2.35)

1.23 (0.615-1.93)

1.28 (0.681-2.16)

Kidney

0.27

1.08

0.73

4.15

2.06

1.39 (0.917-1.98)

0.931 (0.461-1.5)

1.02 (0.683-1.66)

Liver

0.027

0.026

1.23

1.27

0.098

0.423 (0.167-0.851)

0.302 (0.118-0.694)

0.328 (0.118-0.801)

Kidney

0.223

0.035

0.67

13.05

0.11

0.291 (0.066-1.31)

0.336 (0.167-0.776)

0.311 (0.147-0.856)

Liver

0.003

0.643

0.01

1.72

0.643

0.256 (0.063-0.725)

0.095 (0.012-0.604)

0.122 (0.026-0.63)

21

ACS Paragon Plus Environment


Pb V

427

*

Page 22 of 38

Kidney

0.001

4.462

0.001

6.95

4.49

2.77 (1.31-6.76)

0.214 (0.008-3.77)

0.411 (0.01-3.91)

Liver

0.119

0.466

0.27

0.9

0.628

0.374 (0.215-0.581)

0.282 (0.218-0.573)

0.299 (0.215-0.611)

Kidney

0.001

0.235

0.09

70.51

4.77

0.198 (0.099-0.38)

0.269 (0.154-0.434)

0.273 (0.144-0.43)

Liver

0.039

0.681

0.08

0.93

0.478

0.261 (0.121-0.477)

0.209 (0.092-0.468)

0.222 (0.108-0.469)

Kidney

0.032

0.082

0.09

3.42

0.279

0.088 (0.04-0.126)

0.114 (0.068-0.203)

0.107 (0.06-0.196)

The sex was unknown for one individual; a liver sample; b kidney sample

428

22

ACS Paragon Plus Environment

Page 23 of 38

429


Table 2 Comparison of the mean concentrations of heavy metals (µg g-1 dw) in the livers of cetaceans from different regions. Species

Region

n

V

Cu

Zn

As

Se

Hg

Cd

Cr

Pb

Ni

Mn

Reference

Sousa chinensis

Zhuhai

53

0.325

90.1

167

2.81

17.5

30.4

0.359

12.5

0.465

0.611

12.3

This study

Sousa chinensis

Jiangmen

13

0.571

59.8

254

1.71

47.5

122

2.24

8.76

0.562

0.789

12.8

This study

Sousa chinensis

Zhuhai

2

NA

143

194

38.5

14.3

75.1

0.21

1.00

0.26

0.29

13.6

40

Sousa chinensis

Hong Kong

20

NA

89.8

193

0.83

24.4

41.4

0.39

1.73

0.43

0.43

8.04

40

Sousa chinensis

Hong Kong

11

NA

14.1

100.1

2.3

23.25

141.7

2.18

0.06

3.59

0.38

n/a

41

Neophocaena phocaenoides

Hong Kong

27

NA

92.7

223

1.24

81.4

129.2

1.81

2.74

0.24

0.71

10.8

40

Tursiops truncatus

The Gulf of Mexico

10

NA

NA

NA

NA

73.6

182

1.72

4.16

2.72

NA

8.04

42

Delphinus delphis

Northwest Iberian Peninsula

98

Sousa chinensis - ACS Publications - American Chemical Society

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