Potential association between exposure to legacy ...

Science of the Total Environment 643 (2018) 785–792

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Potential association between exposure to legacy persistent organic pollutants and parasitic body burdens in Indo-Pacific finless porpoises from the Pearl River Estuary, China Duan Gui a, Jingwen He a, Xiyang Zhang a, Qin Tu a, Laiguo Chen b, Kangkang Feng c, Wei Liu c, Bixian Mai d, Yuping Wu a,⁎ a Zhuhai Key Laboratory of Marine Bioresources and Environment, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Sun Yat-Sen University, Zhuhai 519000, China b Urban Environment and Ecology Research Center, South China Institute of Environmental Sciences (SCIES), Ministry of Environmental Protection, Guangzhou 510655, China c Guangdong Jiangmen Chinese White Dolphin Provincial Nature Reserve, Jiangmen 529000, China d State Key Laboratory of Organic Geochemistry and Guangdong Key Laboratory of Environmental Protection and Resources Utilization, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• ∑DDTs concentrations in blubber of N. phocaenoides from the Pearl River Estuary are among the highest in cetaceans globally. • A high prevalence of nematode parasites (mostly lungworms) were found in our samples. • Only the concentrations of p,p’-DDT and DDDs were significantly higher in diseased than in healthy ones. • Contrasted accumulation pattern of DDTs was found between animals with different health status. • First time to describe a significant positive correlation between parasitic infections and DDT exposure in cetaceans.

a r t i c l e

i n f o

Article history: Received 3 May 2018 Received in revised form 19 June 2018 Accepted 20 June 2018 Available online xxxx Editor: Adrian Covaci Keywords: Indo-Pacific finless porpoises Persistent organic pollutants Infectious diseases

⁎ Corresponding author. E-mail address: [email protected] (Y. Wu).

https://doi.org/10.1016/j.scitotenv.2018.06.249 0048-9697/© 2018 Elsevier B.V. All rights reserved.

a b s t r a c t A high prevalence of infectious diseases (mostly lungworms) is found in finless porpoises (genus Neophocaena) in the coastal waters of China, which is one of the most dichlorodiphenyltrichloroethane (DDT)-polluted areas worldwide, while its association with contaminant exposure remains undetermined. To address this gap, we investigated blubber levels of polychlorinated diphenyls (PCBs), organochlorine pesticides (OCPs) and polycyclic aromatic hydrocarbons in Indo-Pacific finless porpoises (Neophocaena phocaenoides) stranded in the Pearl River Estuary (PRE) of China. In the post-mortem examinations, lungworms (Halocercus species) were found to be the most common parasites, with a high density observed in lungs and bronchi. Severe infections by nematode parasites were also found in the uterus (Cystidicola species), intestine (Anisakis typica) and muscle (A. typica). For all the pollutant compounds analyzed, only the concentrations of p,p′-DDT, p,p′dichlorodiphenyldichloroethane (DDD) and o,p′-DDD were significantly higher in porpoises died of infectious diseases than in the “healthy” individuals (died from physical trauma). Contrasted accumulation pattern of DDTs and their metabolites was found between animals with different health status. The proportion of p,p′DDT in ΣDDTs was higher than that of p,p′-dichlorodiphenyldichloroethylene (DDE) in diseased animals,

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whereas an opposite pattern was shown for “healthy” ones. While this study is the first to describe a significant positive correlation between parasitic diseases and high levels of DDTs in cetaceans, the direction of causality cannot be determined in our data: either a parasitic infection affected the porpoises' ability to metabolize DDTs, resulting in high levels of p,p′-DDT in their blubber, or the pollutant burden rendered them more susceptible to parasitic infection. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Persistent organic pollutants (POPs) are resistant to environmental degradation, bioaccumulative as they move through food webs and have detrimental effects on biota (United Nations Environment Programme, 2001). Odontocetes (toothed whales, dolphins and porpoises) are prone to accumulate high levels of POPs due to their high trophic-level feeding, large deposits of fat, relatively long life-span and a relatively low capacity to metabolize and excrete POPs (Law, 2014; Tanabe et al., 1994; Aguilar et al., 2002). Currently, alarmingly high levels of legacy POPs are still found in populations inhabiting coastal regions with significant industrial and agricultural development (Jepson and Law, 2016). For example, legacy industrial-use polychlorinated biphenyls (PCBs) showed a static or even increasing trend in European odontocetes in recent years (Jepson et al., 2016). Numerous experimental and epidemiological studies have indicated that PCBs are associated with increases in the risk of cancer and infectious diseases, population declines and low or zero reproduction rates in populations of cetaceans and pinnipeds (Helle et al., 1976; Hall et al., 2006; Hickie et al., 2007; Buckman et al., 2011; Bull et al., 2006; Jepson et al., 2005; Murphy et al., 2015). However, no such relationship has yet been established for organochlorine pesticides (OCPs), e.g., dichlorodiphenyltrichloroethanes (DDTs), even though DDTs have higher reproductive transfer rates than PCBs, (Borrell et al., 1995; Mckenzie et al., 1996) causing serious toxic effects to the newborns. DDTs are still in use in certain regions of developing countries, such as South Africa and China, mainly for malaria control and dicofol production, respectively (Van den Berg, 2009). Our previous studies have shown that DDT levels in cetaceans from these regions are high enough to cause concern (Gui et al., 2014, 2016). Indo-Pacific finless porpoise (Neophocaena phocaenoides) is one of the two residential cetacean species in the Pearl River Estuary (PRE) (Jefferson et al., 2002a). The PRE is reported to be one of the most DDT-polluted areas in the world (Fu et al., 2003; Ying et al., 2009). Sources of DDTs in the PRE include the river discharge of technical DDTs and dicofol from agricultural areas, as well as emissions from anti-fouling paints on ships in PRE coastal waters (Guo et al., 2008; Qiu et al., 2005). Neophocaena phocaenoides is recently included in Appendix I of the Convention on International Trade in Endangered Species (CITES) (https://www.cites.org/eng/app/appendices.php) and is identified as “vulnerable” by IUCN (Wang and Reeves, 2017). The highest number of finless porpoises sighted per year in the PRE was 260 individuals during the period 2013–2014, which is consequently the minimum estimate for the size of this population (Jefferson et al., 2002a). However, there were on average approximately 30 cases of dead finless porpoise strandings that occurred in HK waters each year during the period 2004–2013, (http://www.afcd.gov.hk/english/conservation/con_mar/con_mar_fin/ con_mar_fin_fin/con_mar_fin_fin_rep_from.html) which raises serious concern about their survival and health. It has long be suspected that the apparent high ratio of mortality and diseases seen among the newborn finless porpoises and Indo-Pacific humpback dolphins (Sousa chinensis) from the PRE are linked to the high levels of pollutants found in their tissues, particularly DDTs (Gui et al., 2014, 2017; Ramu et al., 2005; Yeung et al., 2009; Chan, 1998; Minh et al., 1999; Lam et al., 2009; Jefferson et al., 2002b; Jefferson et al., 2006; Parsons, 2004). Our objective was to investigate the relationship between exposures to legacy POPs, including PCBs (21 congeners), 10 groups of OCPs and 16

United States Environmental Protection Agency (USEPA) priority polycyclic aromatic hydrocarbons (PAHs) and postmortem findings of infectious diseases in the Indo-Pacific finless porpoises that were stranded and necropsied from the PRE between 2007 and 2016. Significant and positive correlation between percentages of DDT metabolites and total DDT burden in whales suggests intensification of the dehydrochlorinative and differential excretive functions at higher pollutant levels (Borrell and Aguilar, 1987). Thus, we also aim to examine variations of dichlorodiphenyldichloroethylene (DDE) proportion between the diseased and “healthy” finless porpoises to provide insights into the influence of diseases on pollutant metabolism. 2. Materials and methods 2.1. Sample collection and post-mortem examination Of the nearly 30 N. phocaenoides strand along the shore of the PRE from Zhuhai to Jiangmen between 2007 and 2016, 19 were necropsied and sampled for blubber tissues (Fig. 1) within the collaborative cetacean stranding network program run by the Pearl River Estuary Chinese White Dolphin Reserve, Jiangmen Guangdong Chinese White Dolphin Provincial Nature Reserve and Sun Yat-Sen University. In addition, three N. phocaenoides and one narrow-ridged finless porpoise (Neophocaena asiaorientalis) were collected from the adjacent regions of the PRE along the shore of China (Table 1). Gross post-mortem examinations were carried out on all samples, using the protocols described in Law et al. (2006). When nematodes were found, we counted their number, measured their size, and identified to species using both morphological and genetic methods (detailed in the Supporting Information). Histopathological examination was carried out on representative individuals which are freshly dead (code 2) or early moderately decomposed (code 3) (n = 14) to assist in cause of death analysis. Infected lungs were examined visually for worms and histologically for lesions consistent with verminous pneumonia. Tissues were fixed in 10% neutral buffered formalin and submitted to the Sun Yatsen University Histopathology Center for evaluation. Sex was determined by observing the reproductive organs, and if sex could not be determined in the field, it was determined by DNA analysis. Age was estimated from the growth layer groups in the dentine of teeth (Jefferson, 2000; Myrick et al., 1983). The sexual maturity of the animals was determined by examining the reproductive organs. If it could not be determined in the field, body length was used to estimate the age class. The adult porpoises were categorized as either females with body lengths 137 cm and longer or males with body lengths 138 cm and longer (Jefferson et al., 2002c). The maturity and sex composition were Juvenile Females (JF = 7, 30.4%), Juvenile Males (JM = 5, 21.7%), Adult Females (AF = 4, 17.4%), and Adult Males (AM = 7, 30.4%). Organs were packed in clean plastic storage bags and frozen at −20 °C for further analysis. 2.2. Chemical analysis The analyzed compounds were 21 PCB congeners (CB28, CB37, CB52, CB77, CB81, CB101, CB105, CB114, CB118, CB123, CB126, CB153, CB138, CB128, CB156, CB157, CB167, CB169, CB180, CB188 and CB209); ΣDDTs (dichlorodiphenyltrichlorethanes, o,p′-DDT, p,p′-DDT,

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Fig. 1. Map of the distribution of the finless porpoises stranded in the Pearl River Estuary. The size and color of the pie-chart represent the sample size and maturity-sex group, respectively. The numbers indicate the eight main outlets where the Pearl River flows into the South China Sea, including Yamen (1), Hutiaomen (2), Jitimen (3), Modaomen (4), Hengmen (5), Hongqimen (6), Jiaomen (7) and Humen (8).

o,p′-DDD, p,p′-DDD, o,p′-DDE, and p,p′-DDE); HCHs (hexachlorocyclohexanes, including α-HCH, β-HCH, and γ-HCH); CHLs (chlordanes, including cis-chlordane and trans-chlordane); mirex; HCB (hexachlorobenzene); heptachlor; aldrin; pentachlorobenzene; dieldrin; endrin; and 16 PAHs (polycyclic aromatic hydrocarbons)

designated priority pollutants by the United States Environmental Protection Agency (EPA), including naphthalene (Nap), acenaphthylene (Acy), acenaphthene (Ace), fluorine (Fl), phenanthrene (Phe), anthracene (Ant), fluoranthene (Flu), pyrene (Pyr), benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), benzo[k]

Table 1 Descriptive statistics (geometric mean and range) for blubber concentrations (ng g−1 lw) of POPs in Indo-Pacific finless porpoises from the Pearl River Estuary and other coastal regions of China during the period 2007–2016. Pearl River Estuary (n = 19)

∑DDTs p,p′-DDE o,p′-DDE p,p′-DDT o,p′-DDT p,p′-DDD o,p′-DDD HCB Mirex ∑PCBs ∑PAHs

JM (n = 4)

JF (n = 6)

AM (n = 7)

AF (n = 2)

74,700 (19500–163,000) 33,200 (6840–88,600) 451 (103–904) 22,600 (6770–44,400) 3780 (1150–6870) 12,300 (4090–22,900) 1070 (565–1560) 85.5 (56.5–132) 146 (70.4–314) 1660 (598–3530) 887 (633–1200)

24,100 (2900–102,000) 7460 (1060–23,000) 0 (0–372) 9020 (869–46,900) 1010 (109–3640) 5500 (781–26,300) 464 (84.9–2140) 29.2 (8.46–79.5)⁎

98,600 (48400–746,000) 41,400 (14700–416,000) 686 (217–5590) 30,000 (10700–173,000) 5570 (2470–30,000) 16,400 (6650–113,000) 1680 (674–8390) 83.4 (27.4–219) 138 (68.1–293) 1580 (591–4300) 495 (286–1300)

36,100 (24800–52,700) 17,600 (13300–23,300) 292 (195–436) 7820 (5310–11,500) 1360 (954–1930) 7970 (4550–14,000) 858 (488–1510) 178 (159–200)⁎

36.6 (4.63–152) 379 (117–1120) 894 (584–1350)

NA is the abbreviation of “not analyzed”. a Narrow-ridged finless porpoise (Neophocaena asiaorientalis). ⁎ Significant difference identified by ANOVA (p b 0.05).

85.2 (84.1–86.4) 984 (958–1010) 1840

Yangjiang AF (n = 1)

Shanwei JF (n = 1)

Xiamen AF (n = 1)

Bohaia JM (n = 1)

11,800 6450 68 2650 590 1800 217 98.9 67 431 NA

129,000 66,700 814 28,900 4920 25,800 2290 228 104 1360 NA

48,600 23,900 330 12,200 2460 8900 817 243 80.7 560 NA

28,000 14,000 291 7570 1960 3640 541 253 26.7 647 NA

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fluoranthene (BkF), benzo[a]pyrene (BaP), indeno[1,2,3-cd]pyrene (InP), dibenzo[a,h]anthracene (DBahA) and benzo[g,h,i]perylene (BghiP). The chemical compounds listed above were analyzed according to methods detailed in Gui et al. (2014, 2016). Briefly, approximately 0.5 g of a blubber sample was mixed with anhydrous Na2SO4, spiked with 2.5 ng of each recovery internal standard (13C-PCB141, 13C-hexachlorobenzene, naphthalene-d8, acenanaphthene-d10, phenanthrened10, chrysene-d12, and perylene-d12), and then extracted with 3:1 (v: v) hexane:dichloromethane (DCM) in a Soxhlet apparatus for 24 h. Subsequently, the extract was concentrated to 10 mL using a rotary vacuum evaporator. Then, an aliquot (1.0 mL) was taken for gravimetric determination of the lipid content, while the lipidic material in the rest of the extract was cleaned using a Bio-Beads S-X3-packed gel permeation chromatography (GPC) column, eluting with a 1:1 (v:v) mixture of hexane:DCM. The fraction was further purified by two types of chromatography columns. For PCBs, a glass column (1 cm i.d.) was packed from bottom to top with 1 cm of anhydrous sodium sulfate, 10 cm of acidic silica gel, and 1 cm of anhydrous sodium sulfate. For OCPs and PAHs, a glass column (1 cm i.d.) was packed from bottom to top with 1 cm of anhydrous sodium sulfate, 6 cm of alumina, 10 cm of silica gel, and 1 cm of alumina. A known quantity of internal standards (13C-PCB138 and 13Chexamethylbenzene) were added to the final eluate prior to instrumental analysis. We used a gas chromatograph (GC, Agilent 7890, USA) coupled with a capillary column (DB-5MS with dimensions 60 m × 0.25 mm × 0.25 μm) (J&W Scientific, USA) and a mass spectrometer (MS) detector (Agilent 5975, USA) in electron impact ionization (EI) mode with selected-ion monitoring (SIM) acquisition. These are the same parameters for GC–MS-EI-SIM listed by Gui et al. (2014, 2016). Briefly, the GC was operated in the splitless injection mode using 1 μL injection volumes. The column oven temperature for the detection of PCBs was programed as follows: 80 °C held for 2 min, increased at 4 °C per minute to 290 °C and held for 6 min, and increased at 20 °C per minute to the final temperature of 310 °C and held for 5 min. The column oven temperature for the detection of OCPs was programmed as follows: 70 °C held for 2 min, increased at 3 °C per minute to 270 °C and held for 5 min, and increased at 5 °C per minute to the final temperature of 300 °C and held for 10 min. The column oven temperature for the detection of PAHs was programmed as follows: 70 °C held for 2 min, increased at 15 °C per minute to 180 °C and held for 2 min, increased at 5 °C per minute to the final temperature of 240 °C and held for 2 min, and increased at 3 °C per minute to the final temperature of 315 °C and held for 2 min. The temperatures of the transfer line, injector interface, and ion source were set at 300 °C, 290 °C and 230 °C, respectively. The temperature of the quadrupole analyzer was 150 °C. Helium was used as the carrier gas. The flow rate was set at 1.0 mL min−1. 2.3. Quality assurance/quality control (QA/QC) Our quality assurance/quality control (QA/QC) analysis followed the criteria established by Mai et al. (2002). In every batch of 18 samples, we analyzed a blank sample spiked with a known standard solution, a matrix spike sample (a known amount of target analyte standard solution spiked into pre-extracted blubber), and a sample duplicate using the same procedure as for our samples. The method detection limit (MDL), defined as a signal/noise ratio (S/N) = 3, ranged from 0.35 ng g−1 lipid weight (lw) to 4.5 ng g−1 lw for all analyzed individual compounds. The concentrations in the procedural blanks never exceeded the MDLs for all compounds. The matrix spike recovery rates of the target analytes ranged from 83.2% to 110%, with a relative significant difference (RSD) lower than 11.4%. The relative difference between duplicate samples was below 15% for all target analytes. Recovery rates of surrogate internal standards ranged from 85.5% to 111% for 13C-PCB141 and 13C-hexachlorobenzene, while for naphthalene-d8, acenanaphthene-d10, phenanthrene-d10, chrysene-

d12, and perylene-d12, the rates were between 81% and 115%. Concentrations of POP classes were not corrected for recoveries since recoveries were within an established acceptable range. 2.4. Data analysis POP levels in this study are presented as ng g−1 lipid weight (lw). Log10(1 + x) transformation was used on the data to normalize the frequency distribution since most of the data (contaminant concentrations and body lengths) did not meet the assumption of a normal distribution. Due to the small sample size, a one-way permutation test based on 9999 Monte-Carlo resamplings was employed to ascertain significant differences between two groups. A permutational non-parametric version of ANOVA followed by a Tukey's honestly significant difference test was performed to examine the differences between maturity and sex groups using the function aovp() in package lmPerm (Wheeler, 2010). Spearman's rank correlation coefficient was used to analyze potential linear relationships between POP concentrations and body length. Principal component analysis (PCA) by Euclidean distances was used to reveal clustering variables for each different group of POP mixtures. Statistical analyses were all conducted using R (Ver 3.1.0) (R Core Team, 2014). 3. Results and discussion Descriptive statistics, including the number of individuals, geometric mean and range stratified by site, maturity, and sex, are shown in Table 1. Of all the compounds analyzed, the majority of OCP analytes, CB77, CB81 and CB169 were not detected in N70% of the samples, and thus are not reported here. Those compounds that were present had detectable concentrations in 100% of the samples. 3.1. Contaminant levels and association with biological factors Compared to the historical records of blubber DDT levels that have been reported in cetaceans on a global scale, the observed DDT levels in this study (Table 1) fell within the third highest tier (30,000–100,000 ng g−1 lipid) out of five classifications evaluated in bottlenose dolphins worldwide, 4 whereas the levels in this study were above the highest range measured in harbor porpoises (N50 μg g−1 lipid) (Jepson et al., 2016) and were higher than that reported in bottlenose dolphins stranded in South African waters between 2005 and 2009 (34 μg g−1 lipid) (Gui et al., 2016). The geometric mean concentration of DDTs in the adult male (AM) porpoises in this study was similar to the mean levels reported in the finless porpoises stranded in HK waters in 1996 (100 μg g−1 lipid) (Minh et al., 1999) and was one-fold lower than that found in the PRE AM humpback dolphins stranded on the coastline of the western bank of the PRE during 2007–2013 (175 μg g−1 lipid) (Gui et al., 2014). Compared to S. chinensis, which inhabits waters closer to the coasts that are heavily contaminated by pollutants from riverine input, N. phocaenoides in the PRE appears to avoid the brackish waters in the estuary (Jefferson et al., 2002a). Thus, the habitat of finless porpoises is less contaminated by DDTs, which explains marked lower levels of DDTs in finless porpoises as compared to the humpback dolphins. For ∑21PCBs, the geometric mean concentrations in AM individuals in this study (1580 ng g−1 lw, Table 1) was generally 1–2 orders of magnitude lower than that reported in cetaceans from industrial regions of Europe (19.0–617 μg g−1 lw), (Jepson et al., 2016) and are far below than the toxicity threshold established for marine mammals (Kannan et al., 2000). In fact, the production and usage of PCBs in the Pearl River Estuary and adjacent waters were of minor importance on a global scale (Breivik et al., 2002). No difference was found in the levels of HCBs and mirex between the PRE finless porpoise and humpback dolphins (mean = 1790 ng g−1 ww), (Gui et al., 2014) reflecting an equilibrium distribution of these compounds in the PRE. The concentrations of the

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16 PAHs in this study (286–1840 ng g−1 lw) were lower than those reported in the humpback dolphins in Zhuhai and HK waters (2761–3274 ng g−1 ww), (Leung et al., 2005) reflecting the heterogeneous distribution of PAHs in the PRE (Mai et al., 2002; Yuan et al., 2015). The highest geometric mean concentration of DDTs was observed for AM porpoises from the PRE (98,600 ng g−1 lw) and was almost 2fold higher than that in AF porpoises. This result may be explained by the potential for gravid females to offload their pollutant burdens through gestation and lactation. JM porpoises from the PRE also showed a 2-fold higher geometric mean concentration of DDTs than JF porpoises, whereas the average body lengths of animals from those two groups were close. The maturity and gender-related variation of concentrations of PCBs and mirex was consistent with that of DDTs, except that the geometric concentration of PCBs and mirex is slightly higher in JMs than in AMs. For HCB, the geometric mean concentration of HCB was found to be highest in the AF group, which was significantly higher than that in the JF group and was one-fold higher than that in JMs and AMs (Table 1). This distinctive accumulation pattern may indicate the low maternal transfer rate of HCB in finless porpoises. HCB is more volatile but less lipophilic than PCBs and DDTs (Bacci, 1994; Tanabe et al., 1984). It has been reported that the transplacental transfer rate of HCB between striped dolphin (Stenella coeruleoalba) mother-fetus pairs is one-fold higher than that of DDTs and PCBs (Tanabe et al., 1982). Otherwise, other influencing factors, such as recent acute exposure, diseases and reproductive failure, may also make them unable to pass these compounds to their offspring, resulting in the relatively higher HCB levels in adult females of finless porpoises. Further research is needed with larger sample size to test these hypotheses. Like DDTs, PCBs and mirex, the geometric mean concentration in JMs was almost two-fold higher than that in the JF group. Significant positive associations were found between the blubber concentration of PCB209 and body length (Spearman's r = 0.49, p b 0.05) and between HCB and age (Spearman's r = 0.49, p b 0.05) (Fig. S5). PAHs showed no significant trend with maturity, sex or body lengths, with higher levels found in juveniles than adults. The highest blubber concentrations of DDTs and PCBs in this study were both found in an adult male individual (JMNP131206, the number refers to the stranding date of this animal, which is December 6, 2013) stranded in a freshwater river (a tributary of the Pearl River) along an approximately 50 km stretch of river in the upstream portion of the outlet (Fig. 1). Ecological surveys have reported that finless porpoises in the PRE were seldom found in the brackish waters near the coast, which is the main habitat of the Indo-Pacific humpback dolphins (Jefferson et al., 2002a). Thus, the stranding of this porpoise was very unusual. The DDT levels in JMNP131206 were at least 4-fold higher than those in other samples. This porpoise had a body length of 170 cm, which is the longest body length ever reported in this species, to the best of our knowledge. The lipid-normalized concentrations of ΣDDTs and ΣPCBs in this individual were 9.7- and 2.6-fold higher than the geometric mean lipid-normalized concentrations of ΣDDTs and ΣPCBs in the disease group of finless porpoises in this study, respectively, which are high enough to impair the health of this individual. However, no information about parasite infection was available for this individual. 3.2. Composition profiles of POP congeners The general composition of DDTs in the blubber of PRE-stranded porpoises was p,p′-DDE (mean = 40.8%) N p,p′-DDT (mean = 32.5%) N p,p′-DDD (mean = 19.4%) N N o,p′-DDT (mean = 4.95%) N o,p′-DDD (mean = 1.87%) N o,p′-DDE (mean = 0.69%). CB153 (mean = 27.5%) was the major PCB congener, followed by CB180 (mean = 21.4%) CB101 (mean = 12.4%) and CB118 (mean = 10.9%). CB153 has been reported as the most abundant PCB in marine mammals from different parts of the globe (Aguilar et al., 2002). The higher contribution of these compounds is related to their predominance in commercial

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mixtures (e.g., Aroclor), to their resistance to degradation and to their high lipophilicity and biomagnification potentials (Aguilar et al., 2002). Significant differences in the levels and composition of PAHs were shown between the finless porpoises sampled during the periods 2008–2009 (n = 3) and 2011–2013 (n = 10), with significantly higher levels of PAHs found in the latter samples (mean = 1059 ng g−1 lw) in comparison to those sampled earlier (mean = 292 ng g−1 lw). The samples collected between 2011 and 2013 were mainly composed of low molecular weight (2- and 3-ringed) PAHs (mean = 73.1%), while in the samples collected in 2008–2009, high molecular weight (4- and 6 + -ringed) PAHs (mean = 59.9%) were the dominant isomers. There are two main sources of PAHs in the environment, namely petrogenic and pyrolytic origins, which can be inferred by the ratios of two thermodynamically stable PAH isomer pairs, Phe/Ant and Flu/Pyr (Karacik et al., 2009; Sun et al., 2016). As shown in Fig. S6, the ratios of Phe/Ant and Flu/Pyr in finless porpoises ranged from 0.77 to 0.90 and from 0.98 to 1.64, respectively, indicating that PAHs likely originated from both pyrolytic sources (Phe/Ant b10 and Flu/Pyr N 1). 3.3. Contaminants and health association In the post-mortem examinations, lungworms (identified as Halocercus species, Fig. S1) were found to be the most common parasites, with a high density observed in lungs and bronchi of 5 animals. Severe infections by nematode parasites were also found in the uterus (identified as Cystidicola species), intestine (identified as Anisakis typica, Fig. S2) and muscle (identified as A. typica, Fig. S2) of three other animals (detailed description of the morphological and molecular identification was provided in the Supporting Information). The histopathological results suggested that the ultimate cause of death for these animals was verminous pneumonia (Fig. S3 and S4), while these porpoises showed a poor nutrition state. In comparison with the diseased animals (AM = 4, AF = 1, JF = 2 and JM = 1), there were several individuals with a good nutrition state (thicker blubber layer), in which no disease or parasite was found during the post-mortem examinations. The cause of death for these finless porpoises was likely acute physical trauma (lesions caused by fishing nets were found in the skin, indicating that they were bycaught during fishery activities). Thus, these samples can be considered “healthy” (AM = 2, AF = 1, JF = 2, JM = 1). The geometric mean concentrations of p,p′-DDT, p,p′-DDD and o,p′-DDD were significantly higher in samples with infectious diseases than in the “healthy” samples (permutation-based one-way test, p b 0.05), whereas no significant difference was found for the rest of POPs analyzed (Table 2). A high occurrence of lungworms (identified as Halocercus pingi) has been reported in stranded finless porpoises from HK waters (10 in 32 individuals) (Parsons and Jefferson, 2000) and those living off the

Table 2 Descriptive statistics (geometric mean and range) for blubber concentrations (ng g−1 lw) of POPs in finless porpoises with different health status as characterized based on dissected sample.

∑DDTs p,p′-DDT o,p′-DDT DDTs/∑DDTs p,p′-DDE o,p′-DDE DDEs/∑DDTs p,p′-DDD o,p′-DDD DDDs/∑DDTs ∑PCBs HCB Mirex

Diseased (n = 8)

“Healthy” (n = 6)

p-Value

69,500 (24800–163,000) 24,200 (5310–50,000) 3650 (954–9030) 0.403 (0.253–0.494) 24,800 (10100–88,600) 390 (129–928) 0.363 (0.228–0.548) 12,800 (4550–26,300) 1220 (488–2290) 0.203 (0.148–0.278) 1180 (591–3530) 52.8 (20.6–159) 104 (57.6–314)

33,700 (11800–80,800) 9440 (2650–20,400) 1560 (590–4550) 0.329 (0.27–0.56) 14,800 (3760–42,400) 197 (53.3–657) 0.445 (0.216–0.575) 5810 (1800–13,000) 546 (217–1340) 0.189 (0.151–0.233) 805 (118–2470) 89.8 (24.5–194) 73.8 (4.63–293)

0.09 0.035⁎ 0.07 0.201 0.276 0.192 0.221 0.043⁎ 0.041⁎

⁎ Significance at p b 0.05.

0.503 0.45 0.203 0.656

790

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Chinese Yellow/Bohai Sea coast, (Wan et al., 2017) suggesting that these animals are particularly vulnerable to this nematode parasite. It was reported that neonatal finless porpoises have a remarkably high ratio of lungworm diseases (7 in 10), indicating that these parasites can be transferred to the neonate before birth or during lactation (Parsons and Jefferson, 2000). The immune systems of neonatal finless porpoises are underdeveloped, which makes them particularly susceptible to parasite infection. DDTs are well-known for immunosuppressive toxicity in animals (Busbee et al., 1999). One study showed that high levels of DDTs and PCBs were associated with decreased lymphocyte response in the blood of free-ranging bottlenose dolphins (Lahvis et al., 1995). There existed convincing evidence showing that elevated levels of PCBs were associated with increasing risks of infectious diseases in harbor porpoises from European waters, (Hall et al., 2006) while no such association was found for DDTs in cetaceans. One study has shown a linkage between carcinoma and DDT exposure in California sea lions, which is possibly due to the immunosuppressive effects of the DDTs, making the sea lions susceptible to carcinoma-inducing viruses (Randhawa et al., 2015). Higher (but not significant) DDT and HCH levels were found in finless porpoises with “poor” health (e.g., as evidenced by thinner than normal blubber layer, heavy parasite loads and/or serious chronic pathological conditions) as compared to the normal ones in Hong Kong waters (the eastern boundary of the PRE) (Jefferson et al., 2002b). Thus, our study represents the first time that the occurrence of parasite infections in cetaceans has been linked to the high levels of DDTs to which they were exposed.

PCA performed with all DDT variables illustrated that the “healthy” individual samples form a firm cluster as illustrated in Fig. 2, whereas the diseased animals form a separate cluster (AF individual were excluded since gravid female can offload their pollutant burden through gestation and lactation (Aguilar et al., 1999)). 3.4. Influence of diseases on DDT metabolism In the porpoises with heavy parasite burden, the mean ratio of (p,p′DDT + o,p′-DDT)/ΣDDTs was higher than that of (p,p′-DDE + o,p′DDE)/ΣDDTs, while an opposite pattern was observed in the “healthy” group (Table 2). A previous study (Borrell and Aguilar, 1987) has shown that the proportion of DDEs in blubber of whales was significantly and positively correlated with total DDT burden (200–3000 ng g−1 lw), indicating intensification of metabolism and excretion of DDTs (p,p′-DDT + o,p′DDT) at higher levels. Although there was a significant positive correlation between parasitic diseases and high levels of DDTs in our samples, the direction of causality cannot be determined in our data: either a parasitic infection affected the porpoises' ability to metabolize DDTs, resulting in high levels of p,p′-DDT in their blubber, or the pollutant burden rendered them more susceptible to parasitic infection. Regarding the high prevalence of parasites observed in neonate finless porpoises, it is possible that infectious diseases impaired the health of the neonates and thus weakened their dehydrochlorinative and excretive functions on p,p′-DDT, resulting in higher accumulation of this compound,

0.6

Diseased ppDDT

“Healthy”

ppDDD opDDD

PC2 (8.8%)

0.3

opDDT 0.0

−0.3

opDDE ppDDE −0.50

−0.25

0.00

0.25

PC1 (87.07%) Fig. 2. Principal component analysis plot based on the concentrations of six DDT congeners in the blubber of finless porpoises with known health status (n = 12, adult female samples were excluded).

D. Gui et al. / Science of the Total Environment 643 (2018) 785–792

which is the main component of technical DDT mixtures (77.1%) (Kannan et al., 1995). In the opposite way, high levels of DDTs can weaken the immune system of the neonates, making it easier for them to be parasitized. An in vitro study on beluga whale leukocytes has shown that p,p′-DDT, rather than p,p′-DDE, can significantly reduce the proliferative response of beluga splenocytes, (Guise, 1998) which increases the weight of evidence for the causal relationship between high levels of p,p′-DDT and infectious diseases in finless porpoises. Several studies indicated that the incidence of infectious diseases in marine mammals has significantly increased over the past decades, (Gulland and Hall, 2007; Van Bressem et al., 2009; Desforges et al., 2016) whereas cumulative epidemiologic evidence suggests that exposure to elevated levels of legacy (Hall et al., 2006) and/or emerging (Kannan et al., 2006) POPs plays a key role in reducing host resistance to parasitic infections. Despite the small sample size, this study is the first to report a significant positive correlation between parasitic infections and high levels of DDTs in cetaceans. Nevertheless, many other persistent organic pollutants that were not analyzed in this study may also be responsible for the parasitic infections in the PRE porpoises, such as polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs) and perfluoroalkyl substances (PFASs). Lam et al. (2016) reported that concentrations of perfluorooctane sulfonate (PFOS) and many perfluorinated carboxylic acids (PFCAs) in liver samples of finless porpoises from Hong Kong waters were among the highest records in marine mammals globally, with 4% hepatic levels of PFOS exceeded the tentative critical concentration values associated with hepatic toxicity to mammals. A significantly increasing trend of alternative halogenated flame retardants (HFRs) was also found in finless porpoise carcasses recovered in Hong Kong between 2003 and 2012, which may also serve as a potential threat to the health of PRE cetaceans (Zhu et al., 2014). Further epidemiological studies by employing more samples and analyzing more known and unknown POP compounds are urgently needed to clarify the relationships more robustly. DDTs are now widely applied throughout Southeast Asia in agriculture (particularly by oil palm plantations) (Gupta, 2012; Leong et al., 2007), causing insecticide laden run-off to contaminate the estuaries and nearshore waters, which are important habitats for finless porpoises and other nearshore populations of small cetaceans, like the now critically endangered Irrawaddy dolphin (Kannan et al., 2005) and the Indo-Pacific humpback dolphins (Gui et al., 2014). Further work is needed to better understand the effect threshold and dose– response relationship of DDTs in marine mammals, which has important conservation implications. Acknowledgments The research was supported by the Natural Science Foundation of Guangdong Province (2017A030308005) in China, the National Natural Science Foundation of China (31500433, 41576128 and 41230639), the Ocean Park Conservation Foundation Hong Kong (AW03.1617 and MM01.1718) and the S. chinensis Conservation Action Project of the Administration of Ocean and Fisheries of Guangdong Province, China (2017). We sincerely thank the Guangdong Pearl River Estuary Chinese White Dolphin Reserve and Guangdong Jiangmen Chinese White Dolphin Provincial Nature Reserve for the assistance of stranding dolphin report and sample collection. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2018.06.249. References Aguilar, A., Borrell, A., Pastor, T., 1999. Biological factors affecting variability of persistent pollutant levels in cetaceans. J. Cetacean Res. Manag. 1, 83–116.

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