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Levels and trends of polycyclic aromatic hydrocarbons in the Indo-Pacific humpback dolphins from the Pearl River Estuary (2012–2017)
Marine Pollution Bulletin 131 (2018) 693–700
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Baseline
Levels and trends of polycyclic aromatic hydrocarbons in the Indo-Pacific humpback dolphins from the Pearl River Estuary (2012–2017) Duan Guia, Lingli Zhanga, Fengping Zhana, Wen Liua, Xinjian Yua, Laiguo Chenb, Yuping Wua,
T ⁎
a
Zhuhai Key Laboratory of Marine Bioresources and Environment, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Zhuhai 519000, China b Urban Environment and Ecology Research Center, South China Institute of Environmental Sciences (SCIES), Ministry of Environmental Protection, Guangzhou 510655, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polycyclic aromatic hydrocarbons Indo-Pacific humpback dolphin (Sousa chinensis) Spatiotemporal trends
We investigated the levels and trends of the 16 USEPA priority PAHs in the blubber of 37 Indo-Pacific humpback dolphins sampled during the period 2012–2017 from the Pearl River Estuary (PRE), China. Σ16PAHs concentrations (17.6–6080 ng g−1 wet weight) were at median level compared to dolphin species worldwide. Humpback dolphins affiliated with the hotspots of PAHs, had significantly higher levels of Σ16PAHs than individuals from the other areas in the PRE. Moreover, dolphins stranded on the coast of Lingdingyang are significantly more contaminated by Σ16PAHs than those in the West-four region of the PRE, which appears to reflect the heterogeneous distribution of PAHs in the environment. A marked decline in blubber Σ16PAHs levels is observed over the studied period, with the control of a range of confounding factors. The trend is strongly and statistically significant (p < 0.0001), indicating that the loading of PAHs are gradually being reduced.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, which pose a significant health threat to human and wildlife due to their highly estrogenic, carcinogenic and mutagenic toxicity (Ravindra et al., 2008; Ololade, 2010). PAHs are mainly formed by anthropogenic activities (e.g., petroleum products and sources of the incomplete combustion of organic matter in industrial operations, waste incineration, power plants, vehicle engines and biomass burning). High PAH levels were found in the environment of developing countries, due to the rapid urbanization process in recent years. (Barra et al., 2007; El Deeb et al., 2007; Nikolaou et al., 2009; Kwach and Lalah, 2009). The Pearl River Estuary (PRE), being the second largest river mouth in China in terms of water and sediment discharge, serves the main pathway of terrestrial anthropogenic pollutants to the South China Sea, including PAHs (Yuan et al., 2015). Although many researches have investigated the source, distribution and fate of PAHs in the PRE (Mai et al., 2002; Wang et al., 2007; Yuan et al., 2015), there is a general lack of information of PAH accumulation in higher trophiclevel predators. It is widely acknowledged that higher trophic-level aquatic organisms have a certain capacity of metabolizing PAHs. (Nfon et al., 2008; Takeuchi et al., 2009). However, some studies showed that the metabolism ability of PAHs by cetaceans is limited (Fossi et al., 1999). A positive relationship between PAH concentrations in captive killer whale blood samples and dietary intake of fish has been reported
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Corresponding author at: No. 135, Xingang Xi Road, Guangzhou 510275, China. E-mail address: [email protected] (Y. Wu).
https://doi.org/10.1016/j.marpolbul.2018.04.058 Received 3 December 2017; Received in revised form 19 April 2018; Accepted 25 April 2018 0025-326X/ © 2018 Elsevier Ltd. All rights reserved.
(Formigaro et al., 2014). Therefore, excess intake of PAHs by cetaceans will lead to a bioaccumulation of the pollutant in their blubber. The PRE harbours the largest population of Indo-Pacific humpback dolphins (Sousa chinensis) in the world, which are long-term resident in the PRE year-round (Jefferson and Karczmarski, 2001; Jefferson and Hung, 2004; Chan and Karczmarski, 2017). It has been reported that the number of PRE humpback dolphins may be declining by approximately 2.46% annually, resulting in a reduction of approximately 74% of the current population numbers within three generations of lifespan (about 60 years) (Huang et al., 2012; Karczmarski et al., 2016). As longterm residents and apex predators with a long lifespan, humpback dolphins are vulnerable to bioaccumulation of persistent organic pollutants (POPs) (Bossart, 2011; Fair et al., 2010; Moon et al., 2011). There has been mounting evidence of a link between high accumulation of contaminants and population decline or suppression of population recovery for several cetacean species (Schwacke et al., 2002; Jepson et al., 2016). Regarding the remarkably high ratio of neonatal mortality and immune system abnormalities observed in the stranded dolphins from the PRE, it has long been suspected that anthropogenic contaminants are contributing to the population decline of humpback dolphins in the PRE (Parsons and Jefferson, 2000; Parsons, 2004; Jefferson et al., 2006), which are known to be exposed to an excessively high level of contaminant mixtures, e.g.,
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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]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 analytical procedures followed those described in Mai et al. (2002) and Leung et al. (2005). Approximately 0.5 g of a blubber sample was mixed with anhydrous Na2SO4, spiked with 2.5 ng of each recovery internal standard (naphthalene-d8, acenanaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12), and then extracted with 3:1 (v:v) hexane:dichloromethane (DCM) in an automated Soxhlet apparatus. Subsequently, the extract was evaporated to 10 mL, in which an aliquot (1.0 mL) was taken for gravimetric determination of the lipid content, while the rest of the extract was subjected to purification by BioBeads SX-3-packed gel permeation chromatography (GPC) using a 1:1 (v:v) mixture of hexane:DCM as the eluent at a constant flow rate of 3 mL min−1. The first 70 mL of the eluent, which contained most of the lipids, was discarded. The following 130 mL volume, which contained all the target analytes that were co-extracted with the fat, was collected. The eluent obtained in the last step was evaporated to approximately 2.0 mL and was further purified by passing through a glass column (1 cm i.d.) 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 (m‑terphenyl‑d14) were added to the final eluate prior to instrumental analysis. The target 16 USEPA PAHs were analyzed using 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 electronic ionization mode (EI) with selected-ion monitoring (SIM) acquisition. The GC was operated in the splitless injection mode using 1 μL injection volumes. 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. Helium was used as the carrier gas. The flow rate was set at 1.0 mL min−1. All data were subjected to strict quality assurance/quality control (QA/QC). An internal calibration method with five-point calibration curves is used for the quantitative determination of individual PAHs. A method blank, 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 in every batch of 10 samples were analyzed together with our samples. The relative standard deviation (RSD) for the repeated samples is < 5%. The concentrations in the procedural blanks never exceeded the three-fold values of the method detection limit (MDL), which ranged from 0.35 ng g−1 wet weight (ww) to 4.5 ng g−1 ww for all analyzed individual compounds. The relative difference between duplicate samples was below 15% for all target analytes. Recovery of surrogate internal standards ranged from 51% to 114% for naphthalene-d8, acenanaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12. Concentrations of POP classes were not corrected for recoveries since recoveries were within an established acceptable range. Statistical analyses were conducted using R (Ver 3.4.1) (R Core Team, 2017). Due to the small sample size, we used geometric mean to estimate the median of samples. A permutation-based Mann–Whitney rank-sum test was employed to ascertain significant differences between two groups using a Monte Carlo simulation to sample all possible permutations. A non-parametric version of ANOVA followed by a Tukey's honestly significant difference test was used to examine the differences between maturity and sex groups. Spearman's rank correlation coefficient was used to analyze potential relationships between
Dichlorodiphenyltrichloroethanes (DDTs), Polybrominated diphenyl ethers (PBDEs) and butyltin, etc. (Gui et al., 2014; Gui et al., 2017; Parsons, 2004; Ramu et al., 2005; Schwacke et al., 2002; Kajiwara et al., 2006). Significant increasing trends of alternatives of conventional POPs, e.g., hexabromocyclododecane (HBCD) and perfluorobutane sulfonate (PFBS), were reported in the blubber of dolphin and porpoise from Hong Kong waters over the past decade (Yeung et al., 2009; Zhu et al., 2014; Lam et al., 2016; Ruan et al., 2018). However, the baseline data about PAH exposures of the humpback dolphins from the western PRE (west of Hong Kong) is still lacking, despite that one study has reported PAH levels in the humpback dolphins in Hong Kong waters (Leung et al., 2005). Understanding differences of pollutant exposure in different communities of animals have important implications for risk assessment as well as conservation and management strategies. The main source of PAHs in the PRE is riverine input (Luo et al., 2004, Yuan et al., 2015). The mixing process of riverine input freshwater and marine tidal current can influence the spatial distribution and circulation of PAHs in the PRE, which creates Turbidity Maximum Zone (TMZ) for the deposition of PAHs and other organic pollutants. It is well-documented that the coastal region off Qi'ao Island and Macau are PAH hotspot formed by TMZ, in which the sediments and waters are more contaminated by PAHs than those in any other area in the PRE (Mai et al., 2002; Wang et al., 2007; Luo et al., 2004; Yuan et al., 2015). It is also reported that PAH levels in the west-four region of the PRE is significantly lower than that in the eastern four outlet region (Lingdingyang), due to the marked difference in hydrodynamic conditions between the two main outlet regions. A previous study showed that levels of most halogenated organic pollutants in fish from the PRE have significantly declined from 2008 to 2013, indicating the effectiveness of regulations and source controls in substantively reducing inputs of these contaminants to the PRE (Sun et al., 2015). However, whether these changes can influence PAH levels in the PRE humpback dolphins is still unknown. Significant temporal trends in PAH burdens have been reported in marine vertebrates, mainly after a large amount of petroleum was discharged into marine systems due to the oil spill (Marsili et al., 2001), while no study has reported the time trend of PAH burden in the PRE humpback dolphins. The present study assessed the concentrations of the 16 United States Environmental Protection Agency (USEPA) prior PAHs in the blubber of stranded humpback dolphins collected from the PRE during 2012–2017. Generalized additive mixed models (GAMs) were fitted to the data to test for possible temporal trends and whether the temporal trend is confounded by region, age class, sex, body length and lipid content. To our knowledge, this is the first study of temporal trend of PAH levels in cetaceans from the PRE. Blubber samples were obtained from 37 Indo-Pacific humpback dolphins that were stranded between 2012 and 2017 from the PRE (Fig. 1) within the collaborative stranding program run by the Guangdong Pearl River Estuary Chinese White Dolphin Reserve, Guangdong Jiangmen Chinese White Dolphin Provincial Nature Reserve and Sun Yat-Sen University. The states of decomposition for the stranding dolphin carcasses were defined using the protocols described in Law et al. (2006). All humpback dolphins used in this study were freshly dead (code 2) or moderately decomposed (code 3). Sex was determined by observing the reproductive organs, and if sex could not be determined in the field, it was determined by DNA analysis. The sexual maturity of the animals was determined by examining the reproductive organs. According to Jefferson et al. (2012), physical maturity of the Indo-Pacific humpback dolphins occurs at lengths between approximately 238 and 249 cm, while calves had body lengths of < 130 cm. Therefore, the adult humpback dolphins were categorized as individuals with body lengths 238 cm and longer and calves with body lengths 130 cm and shorter. Blubber samples were firstly packed in aluminum foil and then placed in clean plastic storage bags and frozen at −80 °C for further analysis. The analyzed compounds were the 16 USEPA prior PAHs, including 694
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Fig. 1. (A) Map for the stranding location of Indo-Pacific humpback dolphins on the coast of the Pearl River Estuary (n = 37). (B) Boxplot for the comparison of ∑16PAH levels (wet weight) in the blubber of humpback dolphins stranded at different sites.
PAH congeners were detected in our samples, despite that the detection rates of high molecular weight PAHs in blubber samples were generally below 90%. Blubber concentrations of Σ16PAHs in the Indo-Pacific humpback dolphins in this study ranged from 17.6 to 6080 ng g−1 ww, with a geometric mean of 2400 ng g−1 ww (Table 1). Comparing to the previous study regarding blubber PAH burdens in the Indo-Pacific humpback dolphins, the mean levels of PAHs in the PRE humpback dolphins were similar with those reported in Zhuhai and Hong Kong dolphins (Leung et al., 2005), which are 1-fold lower than those in Xiamen samples (Table 2). Compared to other coastal cetaceans in Asia and Australia, the mean levels of PAHs in our samples were 10-fold higher than those in the narrow-ridged finless porpoises (Neophocaena asiaeorientalis) stranded in Korean waters (Moon et al., 2011), and is an
concentrations of different PAH congeners and between concentrations of total PAHs and each PAH congener with body length. To assess the temporal trend and the other potentially confounding factors on the concentrations of total and different cyclic groups of PAHs, we fitted a generalized additive model (GAM) to the data using the mgcv library (Wood, 2011) in R (R Core Team, 2017). GAM is a smoothing equivalent of generalized linear modeling (GLM), which has been proven useful for depicting the time trends of pollutant levels in cetaceans (Law et al., 2012; Jepson et al., 2016; Gui et al., 2017). Body length, sex, age class, and stranding area were considered as potential covariates in the models. PAH levels in this study are expressed on a wet weight (ww) basis. The results were blank subtracted but not corrected with recovery rates, since the recovery rates were in the established range. All the sixteen
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Table 1 Summary statistics (geometric mean with range) by year, age class and region of the concentrations of PAHs (ng g−1 wet weight) in the blubber of Indo-Pacific humpback dolphins from the Pearl River Estuary sampled between 2012 and 2017.
2012 (n = 11) 2013 (n = 5) 2014 (n = 9) 2015 (n = 4) 2016 (n = 3) 2017 (n = 5) Calf (n = 8) Juvenile/adult (n = 29) Regions off Qi'ao island and Macau (n = 9) All (n = 37)
∑16PAHs
Di-cyclic
Tri-cyclic
Tetra-cyclic
Penta-cyclic
Hexa-cyclic
4740 (3500–6080) 4030 (2870–5710) 3040 (2100–4560) 2770 (2250–3380) 868 (418–1460) 346 (17.6–1290) 4590 (2620–6080) 2010 (17.6–5670) 4840 (3950–5670) 2400 (17.6–6080)
462 (135–1550) 395 (212–667) 391 (317–538) 370 (275–486) < 10 (< 1–389) < 10 (< 1–640) 480 (169–1550) < 10 (< 1–750) 535 (426–667) < 10 (< 1–1550)
4000 (3000–4930) 3450 (2350–4970) 2510 (1680–3980) 2290 (1860–2840) 644 (310–1360) 134 (5.61–835) 3830 (2200–4970) 1460 (5.61–4930) 4070 (3250–4930) 1800 (5.61–4970)
332 (96.2–626) 439 (329–737) 361 (244–589) 316 (264–421) 103 (53.8–248) < 5 (< 5–180) 345 (96.2–737) < 5 (< 5–538) 485 (445–532) < 5 (< 5–737)
<1 <1 <1 <1 4.21 4.79 <1 <1 <1 <1
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1
(< 1–17.7) (< 1–8.81) (< 1–12.3) (< 1–22.6) (< 1–11.5) (< 1–17.7) (< 1–22.6) (< 1–8.81) (< 1–22.6)
(< 1−123)
(< 1–123) (< 1–123) (< 1–123)
high aqueous solubility, accounted for 99.8% of total PAH concentrations. Among these, Phenanthrene are the most abundant PAH isomer, ranging from < 10 to 3275 (mean = 1770) ng g−1 ww. This composition pattern was in accordance with previous studies of PAHs in the blubber of bottlenose dolphins from the Canary Islands (García-Álvarez et al., 2014a, 2014b). Phenanthrene, a compound of crude oil, is more readily absorbed by marine organism than the HMW PAH isomer, benzo[a]pyrene (Nisbet and Lagoy, 1992). Although the toxicity induced by Phenanthrene is considered to be lower than that of benzo[a] pyrene, the high accumulation of Phenanthrene is potentially dangerous for the health of these dolphins. Di and tetra-cyclic PAHs accounted for 13.2% and 9.98% of the total PAH concentrations analyzed in this study, respectively. Among the 4-rings PAHs, Fluoranthene and Pyrene were detected in over 90% of total blubber samples, whereas the rest 4-rings PAHs and higher molecular weight PAHs were not detected in most of our samples. PAHs have two main environmental sources, including petrogenic (LMW) and pyrolytic origins (HMW). The ratios of two thermodynamic stable PAH isomer pair, Phe/Ant and Flu/Pyr have been widely used to infer the sources of PAHs. As illustrated in Fig. 2, the ratios of Phe/Ant and Flu/Pyr demonstrated that PAHs in the blubbers of the PRE dolphins are originated from both pyrolytic and petrogenic sources (Phe/ Ant > 10 and Flu/Pyr > 1). Previous studies on sediment and fish also reported that PAHs in the PRE originates from both petrogenic and pyrogenic sources, including vehicular emissions, coal combustion and coke ovens (Peng et al., 2008; Sun et al., 2016; Liu et al., 2017).
order of magnitude lower than the Australian humpback dolphins (Sousa sahulensis) and/or Australian snubfin dolphins (Orcaella heinsohni) in the Great Barrier Reef, Australia (Cagnazzi et al., 2013). The highest levels of PAHs ever reported in cetacean worldwide were found in the Mediterranean cetaceans (Marsili et al., 2001), which have cause toxicological stress in these animals. PAH levels in this study were far below than the levels of PAHs in Mediterranean cetaceans (Marsili et al., 2001), but are similar with those found in the blubber tissue of the wild bottlenose dolphins in the estuarine areas from the southeastern United States (Fair et al., 2010). Although the concentrations of PAHs found in the blubber samples in this study are relatively low compared to the previous studies (García-Álvarez et al., 2014a, 2014b), the potential toxicological impact of PAHs still needs caution. Marine mammals can detoxify and eliminate petroleum hydrocarbons via their mixed function oxidase enzyme systems. Thus, PAHs are generally not highly accumulated in tissues of marine mammals as organochlorinate hydrocarbons. However, when PAHs are found, it may indicate an overload of the mixed function oxidase system. Besides, the metabolic products of PAHs may be bioaccumulative and can induce toxic effects (Brunström et al., 1991). Martineau et al. (2002) suggested a linkage between exposure to excessively high levels of PAHs and cancers in Beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada. Moreover, the different analytical methods of PAHs in samples across studies in cetaceans must be taken with caution in the comparison. Lower molecular weight PAHs (di, tri and tetra-cyclic), which had
Table 2 Comparison of blubber concentrations of PAHs in cetaceans from different regions around the world. Locations
Dolphin species
Sampling year (s)
Sample type
Sample number
Total PAHs (ng g−1 wet weight)
Range
NO. of PAHs
Reference
Zhuhai and Jiangmen, China Zhuhai, China Xiamen, China Hong Kong, China Korean coastal waters Great Barrier Reef, Australia Mediterranean Sea, Italy
Sousa chinensis
2012–2017
Stranded
37
2400
17.6–6080
16
This study
2761.6 6751 3274.6 160b 51,035 ± 5227b 32,296 ± 3152b 9052.5 ± 21,304
– Max = 8530 2263.1–4663.2 4.8-432 12,552–86,711 8682–61,921 228.60–83,662
Sousa chinensis
2003 2002–2004 2002–2004 2003 2009–2010
Stranded Stranded Biopsy Stranded Stranded
a
15
Leung et al., 2005
16 16
Moon et al., 2011 Cagnazzi et al., 2013
14
Marsili et al., 2001
García-Álvarez et al., 2014a García-Álvarez et al., 2014b Fair et al., 2010
1993–1996
Biopsy
Canary Islands, Spain
Finless porpoise Sousa sahulensis Orcaella heinsohni Balaenoptera physalus Stenella coeruleoalba Tursiops truncatus
1 4 5 52 18 17 23
1997–2011
Stranded
25 25
36,205 ± 41,107 788.8 ± 1409b
199.40–198,368 75.3–5761.33
16
Canary Islands, Spain
Tursiops truncatus
2003–2011
Biopsy
64
15,932 ± 10,233b
1394–42,577
16
Charleston, US Indian River Lagoon, US
Tursiops truncatus
2003–2005
Biopsy
17 11
3010b 1316b
10–9140 10–1200
45c
NA: not analyzed. a 16 EPA PAHs excluding benz[a]anthracene. b ng g−1 lipid weight. c 45 PAH parent and homologs and 12 PAH metabolite compounds. 696
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Fig. 2. Scatter plot of the PAH congener ratios Phe/Ant vs Flu/Pyr in the blubber samples of Indo-Pacific humpback dolphins from waters off the Pearl River Estuary (n = 37). Table 3 Generalized additive mixed modeling results for temporal trends in PAH levels in blubber of Indo-Pacific humpback dolphins from the Pearl River Estuary, China. Model summarizes the following information: sample size (N), %deviation explained (%dev), Akaike Information Criterion (AIC) value, and effects of explanatory variables, with associated probabilities (p). Only the PAH levels with significant time trends and the explanatory variables with significant effects are reported. PAH data
N
%dev
AIC
Year
Σ16PAHs Σ16PAHs Σ16PAHs Σ16PAHs Σ16PAHs
37 37 28a 29b 23c
82 81.7 79.1 81.4 75.5
599 599 456 467 372
F = 99.2, F = 87, p F = 38.6, F = 81.4, F = 64.8,
a b c
p < 0.0001 < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
Body length
Age class
Stranding site
p < 0.05 – – – –
– p < 0.05 – – –
p p p p –
< < < <
0.05 0.05 0.05 0.05
Individuals from the coastal region near Macau and Qi'ao island were excluded in the dataset. Calves were excluded. Calves and individuals from the coastal region near Macau and Qi'ao island were excluded.
Significant correlations were found between concentrations of total PAHs and each PAH congeners (spearman r = 0.52–0.98) (Fig. S2). Σ16PAHs were strongly and significantly correlated with the levels of Fl, Phe, Ace, Ant, Flu and Pyr, and were moderately and significantly correlated with the levels of Nap and Acy. The strongest correlation was found between Flu and Pyr. Summary statistics (geometric mean with range) by region and year for the concentration of Σ16PAHs and PAHs with different rings were given in Table 1. We fitted a GAM to the whole dataset to investigate the effects of year, region, body length, sex, age class and lipid content on the concentrations of total and different cyclic groups of PAHs in the blubber of humpback dolphins. The final model was selected in which all the remaining explanatory variables have a significant effect, the deviance explained is higher than 50% and the AIC score is the lowest. The summarized results of the GAMs by the analysis of variance table were given in Table 3. As shown in the table, sex and lipid content have no significant effect in any cases on PAH levels, while the remaining variables (year, region, and body length/age class) are all statistically significant (p < 0.05). As expected, dolphin samples stranded near the two PAH hotspots (Qi'ao island and Macau) had statistically significantly higher levels of PAHs than those stranded in other regions of the PRE, indicating that stranding site has a significant confounder effect with other explanatory variables (Table 3). However, it is also possible that the foraging area/ habitat of the dolphins and where the stranding occurred may be very different, which needs further investigations. The spatial difference in
Furthermore, dolphin samples in this study showed much higher Phe/ Ant ratios (8–26) than did marine fish from the Daya Bay (0.98–4.9) (Sun et al., 2016), with the lowest ratios found in sediments in the PRE. This could be due to the greater water solubility of phenanthrene compared to anthracene, thus phenanthrene can be more readily absorbed by marine organisms than anthracene (Martinez et al., 2004). Fig. S2 illustrated that the concentrations of Σ16PAHs or individual PAH congeners were not significantly correlated with body lengths (spearman correlation test, p > 0.05). Similar pattern was also found in finless porpoises from Korean coastal waters (Moon et al., 2011). Despite no significant difference was found among the different sex and maturity groups, the median levels of Σ16PAHs in the juvenile dolphins are apparently higher than those in the adult samples (Fig. S1 of the Supplementary material; “S” indicates tables and figures in the Supplementary material). No significant difference in the levels of individual PAH compound between male and female samples were shown. We further compared PAH levels between the calf (body lengths < 137 cm according to Jefferson et al., 2012) and juvenile/ adult (body lengths longer than 137 cm) samples. Significantly higher levels of Σ16PAHs and/or individual PAH congeners were found in calves compared to juvenile/adult group (Table 1), probably due to incomplete development of metabolic system in calves. Previous studies have found a high neonatal mortality rate among all stranded humpback dolphin samples in Hong Kong waters (Parsons, 2004; Jefferson et al., 2006; Jefferson et al., 2012), which is possibly related to the high concentration of POPs in dolphin tissues. 697
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Fig. 3. Temporal trends in blubber concentrations of ∑16PAHs (wet weight) in Indo-Pacific humpback dolphins stranded in the Pearl River Estuary (PRE) from 2012 to 2017 using the different datasets. (A) All samples (n = 37) were used. (B) Dolphin samples stranded in the coastal region near Macau and Qi'ao island were excluded in the analysis (n = 28). (C) Calf individuals in the whole samples were excluded (n = 29). (D) Calves and individuls which were stranded in the coastal region near Macau and Qi'ao island were excluded in the analysis (n = 23). For (A) and (C), dolphins stranded in the coastal region near PAH hotspot (Macau and Qi'ao island) are illustrated by green dots, while red dots denote individuals stranded in the other regions in the PRE. For (B), green dots indicate dolphin calf, while red dots denote juvenile/adult dolphins. In all cases, the decreasing trend of ∑16PAH levels is highly statistically significant (p < 0.0001), against the null hypothesis of no trends. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and exposure to disease among PRE humpback dolphins may increase lipid tissue metabolism and release contaminants into blood and other vital organs, resulting in an increase of their risk of health effects. In our samples, lipid content has a weak and negative effect on the blubber PAH levels (wet weight) (spearman correlation test, r = −0.369, p = 0.024), indicating that the overall reduction of PAH concentrations was not related to the decrease in lipid content in blubber samples from 2012 to 2017. A number of recent studies have investigated the temporal trends of contaminants accumulated in cetacean tissues from the PRE (including Hong Kong waters). Significant increases of the levels of hexabromocyclododecane (HBCD) and perfluorobutane sulfonate (PFBS) were observed in the dolphin and porpoise from Hong Kong waters over the past decade, which could be contributed to the replacement of conventional halogenated flame retardants (HFRs) and perfluoroalkyl substances (PFASs) by alternatives and the increasing usage of these alternatives following the restriction of some conventional POPs (Yeung et al., 2009; Zhu et al., 2014; Lam et al., 2016; Ruan et al., 2018). For organochlorine pesticides, hexachlorocyclohexanes (HCHs) levels in finless porpoises from Hong Kong waters decreased significantly from the year 1990 to 2001, while no declining trend was observed for DDTs and PCBs, suggesting prolonged exposure of these compounds for the PRE cetaceans (Ramu et al., 2006). Despite the marked decline of PAH levels in blubbers of PRE humpback dolphin in recent years, further investigation is needed to address the health effects of chronic exposure of humpback dolphins to even low concentrations of PAHs. As dolphins are a sentinel species, the potential impacts of PAH exposure to
PAH levels in stranded dolphins from different site may reflect the contamination status in their habitat. As shown in Table 1, substantial variations of PAHs concentrations in dolphin samples in this study were found according to the sampling year, with the mean levels ranging from 346 ng g−1 ww in year 2017 to 4740 ng g−1 ww in year 2012. The blubber PAH levels in all samples showed a marked decline from 2012 to 2017 (Fig. 3A). The decreasing trend is strongly and statistically significant (p < 0.0001) against the null hypothesis of no trend (Table 3). Besides, body length/age class also have significant effects on the concentrations of Σ16PAHs (Table 3). To control the confounding effect of stranding site, we excluded the nine dolphin samples from Qi'ao island and Macau in the GAM analysis. The result shows that the remaining explanatory variables (stranding year and body length or age class) still have significant effects on the concentrations of PAHs in the model, indicating that the temporal trend of PAH accumulation in dolphin blubber was not confounded by sampling site (Fig. 3B). We further excluded all the calf samples in the dataset (n = 23) (Fig. 3C, D), for which the GAM results were similar as those based on the whole dataset, indicating that the overall reduction of PAH accumulation is not due to unequal sampling of the levels of over the period of this study. This may reflect changes in local industrial structure and/or source controls of PAHs in the Pearl River Delta. Tissue contaminant concentrations in blubber of humpback dolphins may also be related to differences in body condition, as blubber serves as the primary site for lipophilic contaminants (e.g., PCBs, DDTs and PAHs) as well as for metabolic energy storage (Yordy et al., 2010). Reduced body condition due to commercially overfishing, habitat loss 698
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dolphins can be correlated to both river and human health.
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Acknowledgements The research was supported by the National Natural Science Foundation of China (31500433 and 41576128), the Natural Science Foundation of Guangdong Province (2017A030308005) in China, the Ocean Park Conservation Foundation Hong Kong (MM01.1718 and AW03.1617) and the Sousa chinensis Conservation Action Project, China. 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.marpolbul.2018.04.058. References Barra, R., Castillo, C., Torres, J.P.M., 2007. Polycyclic aromatic hydrocarbons in the South American environment. Rev. Environ. Contam. Toxicol. 191, 1. Bossart, G.D., 2011. Marine mammals as sentinel species for oceans and human health. Vet. Pathol. 48 (3), 676. Brunström, B., Broman, D., Näf, C., 1991. Toxicity and EROD-inducing potency of 24 polycyclic aromatic hydrocarbons (PAHs) in chick embryos. Arch. Toxicol. 65 (6), 485–489. Cagnazzi, D., Fossi, M.C., Parra, G.J., Harrison, P.L., Maltese, S., Coppola, D., et al., 2013. Anthropogenic contaminants in Indo-Pacific humpback and Australian Snubfin dolphins from the central and southern great barrier reef. Environ. Pollut. 182 (6), 490. Chan, S.C.Y., Karczmarski, L., 2017. Indo-pacific humpback dolphins (Sousa chinensis) in Hong Kong: modelling demographic parameters with mark-recapture techniques. PLoS One 12 (3). Deeb, K.Z.E., Said, T.O., Naggar, M.H.E., Shreadah, M.A., 2007. Distribution and sources of aliphatic and polycyclic aromatic hydrocarbons in surface sediments, fish and bivalves of Abu Qir Bay (Egyptian Mediterranean sea). Bull. Environ. Contam. Toxicol. 78 (5), 373–379. Fair, P.A., Adams, J., Mitchum, G., Hulsey, T.C., Reif, J.S., Houde, M., et al., 2010. Contaminant blubber burdens in Atlantic bottlenose dolphins (Tursiops truncatus) from two southeastern us estuarine areas: concentrations and patterns of PCBs, pesticides, PBDEs, PFCs, and PAHs. Sci. Total Environ. 408 (7), 1577–1597. Formigaro, C., Henríquez-Hernandez, L.A., Zaccaroni, A., Garcia-Hartmann, M., Camacho, M., Boada, L.D., ... Luzardo, O.P., 2014. Assessment of current dietary intake of organochlorine contaminants and polycyclic aromatic hydrocarbons in killer whales (Orcinus orca) through direct determination in a group of whales in captivity. Sci. Total Environ. 472, 1044–1051. Fossi, M.C., Casini, S., Marsili, L., 1999. Nondestructive biomarkers of exposure to endocrine disrupting chemicals in endangered species of wildlife. Chemosphere 39 (8), 1273. García-Álvarez, N., Boada, L.D., Fernández, A., Zumbado, M., Arbelo, M., Sierra, E., ... Luzardo, O.P., 2014a. Assessment of the levels of polycyclic aromatic hydrocarbons and organochlorine contaminants in bottlenose dolphins (Tursiops truncatus) from the Eastern Atlantic Ocean. Mar. Environ.Res. 100, 48–56. García-Alvarez, N., Martín, V., Fernández, A., Almunia, J., Xuriach, A., Arbelo, M., ... Luzardo, O.P., 2014b. Levels and profiles of POPs (organochlorine pesticides, PCBs, and PAHs) in free-ranging common bottlenose dolphins of the Canary Islands, Spain. Sci. Total Environ. 493, 22–31. Gui, D., Yu, R., Xuan, H., Qin, T., Chen, L., Wu, Y., 2014. Bioaccumulation and biomagnification of persistent organic pollutants in Indo-pacific humpback dolphins (Sousa chinensis) from the Pearl River estuary, China. Chemosphere 114 (22), 106–113. Gui, D., Yu, R., Karczmarski, L., Ding, Y., Zhang, H., Yong, S., et al., 2017. Spatiotemporal trends of heavy metals in Indo-pacific humpback dolphins (Sousa chinensis) from the western pearl river estuary, China. Environ. Sci. Technol. 51 (3), 1848. Huang, S.L., Karczmarski, L., Chen, J., Zhou, R., Lin, W., Zhang, H., et al., 2012. Demography and population trends of the largest population of Indo-Pacific humpback dolphins. Biol. Conserv. 147 (1), 234–242. Jefferson, T.A., Hung, S.K., 2004. A review of the status of the Indo-Pacific humpback dolphin (Sousa chinensis) in Chinese waters. Aquat. Mamm. 30 (1), 149–158. Jefferson, T.A., Karczmarski, L., 2001. Sousa chinensis. Mamm. Species 655 (655), 1–9. Jefferson, T.A., Hung, S.K., Lam, P.K.S., 2006. Strandings, mortality and morbidity of Indo-pacific humpback dolphins in Hong Kong, with emphasis on the role of organochlorine contaminants. J. Cetacean Res. Manag. 8, 181–193. Jefferson, T.A., Hung, S.K., Robertson, K.M., Archer, F.I., 2012. Life history of the IndoPacific humpback dolphin in the Pearl River estuary, southern China. Mar. Mamm. Sci. 28 (1), 84–104. Jepson, P.D., Deaville, R., Barber, J.L., Aguilar, Àlex, Borrell, A., Murphy, S., et al., 2016. PCB pollution continues to impact populations of orcas and other dolphins in
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Baseline
Levels and trends of polycyclic aromatic hydrocarbons in the Indo-Pacific humpback dolphins from the Pearl River Estuary (2012–2017) Duan Guia, Lingli Zhanga, Fengping Zhana, Wen Liua, Xinjian Yua, Laiguo Chenb, Yuping Wua,
T ⁎
a
Zhuhai Key Laboratory of Marine Bioresources and Environment, Guangdong Provincial Key Laboratory of Marine Resources and Coastal Engineering, School of Marine Sciences, Zhuhai 519000, China b Urban Environment and Ecology Research Center, South China Institute of Environmental Sciences (SCIES), Ministry of Environmental Protection, Guangzhou 510655, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Polycyclic aromatic hydrocarbons Indo-Pacific humpback dolphin (Sousa chinensis) Spatiotemporal trends
We investigated the levels and trends of the 16 USEPA priority PAHs in the blubber of 37 Indo-Pacific humpback dolphins sampled during the period 2012–2017 from the Pearl River Estuary (PRE), China. Σ16PAHs concentrations (17.6–6080 ng g−1 wet weight) were at median level compared to dolphin species worldwide. Humpback dolphins affiliated with the hotspots of PAHs, had significantly higher levels of Σ16PAHs than individuals from the other areas in the PRE. Moreover, dolphins stranded on the coast of Lingdingyang are significantly more contaminated by Σ16PAHs than those in the West-four region of the PRE, which appears to reflect the heterogeneous distribution of PAHs in the environment. A marked decline in blubber Σ16PAHs levels is observed over the studied period, with the control of a range of confounding factors. The trend is strongly and statistically significant (p < 0.0001), indicating that the loading of PAHs are gradually being reduced.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, which pose a significant health threat to human and wildlife due to their highly estrogenic, carcinogenic and mutagenic toxicity (Ravindra et al., 2008; Ololade, 2010). PAHs are mainly formed by anthropogenic activities (e.g., petroleum products and sources of the incomplete combustion of organic matter in industrial operations, waste incineration, power plants, vehicle engines and biomass burning). High PAH levels were found in the environment of developing countries, due to the rapid urbanization process in recent years. (Barra et al., 2007; El Deeb et al., 2007; Nikolaou et al., 2009; Kwach and Lalah, 2009). The Pearl River Estuary (PRE), being the second largest river mouth in China in terms of water and sediment discharge, serves the main pathway of terrestrial anthropogenic pollutants to the South China Sea, including PAHs (Yuan et al., 2015). Although many researches have investigated the source, distribution and fate of PAHs in the PRE (Mai et al., 2002; Wang et al., 2007; Yuan et al., 2015), there is a general lack of information of PAH accumulation in higher trophiclevel predators. It is widely acknowledged that higher trophic-level aquatic organisms have a certain capacity of metabolizing PAHs. (Nfon et al., 2008; Takeuchi et al., 2009). However, some studies showed that the metabolism ability of PAHs by cetaceans is limited (Fossi et al., 1999). A positive relationship between PAH concentrations in captive killer whale blood samples and dietary intake of fish has been reported
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Corresponding author at: No. 135, Xingang Xi Road, Guangzhou 510275, China. E-mail address: [email protected] (Y. Wu).
https://doi.org/10.1016/j.marpolbul.2018.04.058 Received 3 December 2017; Received in revised form 19 April 2018; Accepted 25 April 2018 0025-326X/ © 2018 Elsevier Ltd. All rights reserved.
(Formigaro et al., 2014). Therefore, excess intake of PAHs by cetaceans will lead to a bioaccumulation of the pollutant in their blubber. The PRE harbours the largest population of Indo-Pacific humpback dolphins (Sousa chinensis) in the world, which are long-term resident in the PRE year-round (Jefferson and Karczmarski, 2001; Jefferson and Hung, 2004; Chan and Karczmarski, 2017). It has been reported that the number of PRE humpback dolphins may be declining by approximately 2.46% annually, resulting in a reduction of approximately 74% of the current population numbers within three generations of lifespan (about 60 years) (Huang et al., 2012; Karczmarski et al., 2016). As longterm residents and apex predators with a long lifespan, humpback dolphins are vulnerable to bioaccumulation of persistent organic pollutants (POPs) (Bossart, 2011; Fair et al., 2010; Moon et al., 2011). There has been mounting evidence of a link between high accumulation of contaminants and population decline or suppression of population recovery for several cetacean species (Schwacke et al., 2002; Jepson et al., 2016). Regarding the remarkably high ratio of neonatal mortality and immune system abnormalities observed in the stranded dolphins from the PRE, it has long been suspected that anthropogenic contaminants are contributing to the population decline of humpback dolphins in the PRE (Parsons and Jefferson, 2000; Parsons, 2004; Jefferson et al., 2006), which are known to be exposed to an excessively high level of contaminant mixtures, e.g.,
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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]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 analytical procedures followed those described in Mai et al. (2002) and Leung et al. (2005). Approximately 0.5 g of a blubber sample was mixed with anhydrous Na2SO4, spiked with 2.5 ng of each recovery internal standard (naphthalene-d8, acenanaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12), and then extracted with 3:1 (v:v) hexane:dichloromethane (DCM) in an automated Soxhlet apparatus. Subsequently, the extract was evaporated to 10 mL, in which an aliquot (1.0 mL) was taken for gravimetric determination of the lipid content, while the rest of the extract was subjected to purification by BioBeads SX-3-packed gel permeation chromatography (GPC) using a 1:1 (v:v) mixture of hexane:DCM as the eluent at a constant flow rate of 3 mL min−1. The first 70 mL of the eluent, which contained most of the lipids, was discarded. The following 130 mL volume, which contained all the target analytes that were co-extracted with the fat, was collected. The eluent obtained in the last step was evaporated to approximately 2.0 mL and was further purified by passing through a glass column (1 cm i.d.) 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 (m‑terphenyl‑d14) were added to the final eluate prior to instrumental analysis. The target 16 USEPA PAHs were analyzed using 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 electronic ionization mode (EI) with selected-ion monitoring (SIM) acquisition. The GC was operated in the splitless injection mode using 1 μL injection volumes. 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. Helium was used as the carrier gas. The flow rate was set at 1.0 mL min−1. All data were subjected to strict quality assurance/quality control (QA/QC). An internal calibration method with five-point calibration curves is used for the quantitative determination of individual PAHs. A method blank, 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 in every batch of 10 samples were analyzed together with our samples. The relative standard deviation (RSD) for the repeated samples is < 5%. The concentrations in the procedural blanks never exceeded the three-fold values of the method detection limit (MDL), which ranged from 0.35 ng g−1 wet weight (ww) to 4.5 ng g−1 ww for all analyzed individual compounds. The relative difference between duplicate samples was below 15% for all target analytes. Recovery of surrogate internal standards ranged from 51% to 114% for naphthalene-d8, acenanaphthene-d10, phenanthrene-d10, chrysene-d12, and perylene-d12. Concentrations of POP classes were not corrected for recoveries since recoveries were within an established acceptable range. Statistical analyses were conducted using R (Ver 3.4.1) (R Core Team, 2017). Due to the small sample size, we used geometric mean to estimate the median of samples. A permutation-based Mann–Whitney rank-sum test was employed to ascertain significant differences between two groups using a Monte Carlo simulation to sample all possible permutations. A non-parametric version of ANOVA followed by a Tukey's honestly significant difference test was used to examine the differences between maturity and sex groups. Spearman's rank correlation coefficient was used to analyze potential relationships between
Dichlorodiphenyltrichloroethanes (DDTs), Polybrominated diphenyl ethers (PBDEs) and butyltin, etc. (Gui et al., 2014; Gui et al., 2017; Parsons, 2004; Ramu et al., 2005; Schwacke et al., 2002; Kajiwara et al., 2006). Significant increasing trends of alternatives of conventional POPs, e.g., hexabromocyclododecane (HBCD) and perfluorobutane sulfonate (PFBS), were reported in the blubber of dolphin and porpoise from Hong Kong waters over the past decade (Yeung et al., 2009; Zhu et al., 2014; Lam et al., 2016; Ruan et al., 2018). However, the baseline data about PAH exposures of the humpback dolphins from the western PRE (west of Hong Kong) is still lacking, despite that one study has reported PAH levels in the humpback dolphins in Hong Kong waters (Leung et al., 2005). Understanding differences of pollutant exposure in different communities of animals have important implications for risk assessment as well as conservation and management strategies. The main source of PAHs in the PRE is riverine input (Luo et al., 2004, Yuan et al., 2015). The mixing process of riverine input freshwater and marine tidal current can influence the spatial distribution and circulation of PAHs in the PRE, which creates Turbidity Maximum Zone (TMZ) for the deposition of PAHs and other organic pollutants. It is well-documented that the coastal region off Qi'ao Island and Macau are PAH hotspot formed by TMZ, in which the sediments and waters are more contaminated by PAHs than those in any other area in the PRE (Mai et al., 2002; Wang et al., 2007; Luo et al., 2004; Yuan et al., 2015). It is also reported that PAH levels in the west-four region of the PRE is significantly lower than that in the eastern four outlet region (Lingdingyang), due to the marked difference in hydrodynamic conditions between the two main outlet regions. A previous study showed that levels of most halogenated organic pollutants in fish from the PRE have significantly declined from 2008 to 2013, indicating the effectiveness of regulations and source controls in substantively reducing inputs of these contaminants to the PRE (Sun et al., 2015). However, whether these changes can influence PAH levels in the PRE humpback dolphins is still unknown. Significant temporal trends in PAH burdens have been reported in marine vertebrates, mainly after a large amount of petroleum was discharged into marine systems due to the oil spill (Marsili et al., 2001), while no study has reported the time trend of PAH burden in the PRE humpback dolphins. The present study assessed the concentrations of the 16 United States Environmental Protection Agency (USEPA) prior PAHs in the blubber of stranded humpback dolphins collected from the PRE during 2012–2017. Generalized additive mixed models (GAMs) were fitted to the data to test for possible temporal trends and whether the temporal trend is confounded by region, age class, sex, body length and lipid content. To our knowledge, this is the first study of temporal trend of PAH levels in cetaceans from the PRE. Blubber samples were obtained from 37 Indo-Pacific humpback dolphins that were stranded between 2012 and 2017 from the PRE (Fig. 1) within the collaborative stranding program run by the Guangdong Pearl River Estuary Chinese White Dolphin Reserve, Guangdong Jiangmen Chinese White Dolphin Provincial Nature Reserve and Sun Yat-Sen University. The states of decomposition for the stranding dolphin carcasses were defined using the protocols described in Law et al. (2006). All humpback dolphins used in this study were freshly dead (code 2) or moderately decomposed (code 3). Sex was determined by observing the reproductive organs, and if sex could not be determined in the field, it was determined by DNA analysis. The sexual maturity of the animals was determined by examining the reproductive organs. According to Jefferson et al. (2012), physical maturity of the Indo-Pacific humpback dolphins occurs at lengths between approximately 238 and 249 cm, while calves had body lengths of < 130 cm. Therefore, the adult humpback dolphins were categorized as individuals with body lengths 238 cm and longer and calves with body lengths 130 cm and shorter. Blubber samples were firstly packed in aluminum foil and then placed in clean plastic storage bags and frozen at −80 °C for further analysis. The analyzed compounds were the 16 USEPA prior PAHs, including 694
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Fig. 1. (A) Map for the stranding location of Indo-Pacific humpback dolphins on the coast of the Pearl River Estuary (n = 37). (B) Boxplot for the comparison of ∑16PAH levels (wet weight) in the blubber of humpback dolphins stranded at different sites.
PAH congeners were detected in our samples, despite that the detection rates of high molecular weight PAHs in blubber samples were generally below 90%. Blubber concentrations of Σ16PAHs in the Indo-Pacific humpback dolphins in this study ranged from 17.6 to 6080 ng g−1 ww, with a geometric mean of 2400 ng g−1 ww (Table 1). Comparing to the previous study regarding blubber PAH burdens in the Indo-Pacific humpback dolphins, the mean levels of PAHs in the PRE humpback dolphins were similar with those reported in Zhuhai and Hong Kong dolphins (Leung et al., 2005), which are 1-fold lower than those in Xiamen samples (Table 2). Compared to other coastal cetaceans in Asia and Australia, the mean levels of PAHs in our samples were 10-fold higher than those in the narrow-ridged finless porpoises (Neophocaena asiaeorientalis) stranded in Korean waters (Moon et al., 2011), and is an
concentrations of different PAH congeners and between concentrations of total PAHs and each PAH congener with body length. To assess the temporal trend and the other potentially confounding factors on the concentrations of total and different cyclic groups of PAHs, we fitted a generalized additive model (GAM) to the data using the mgcv library (Wood, 2011) in R (R Core Team, 2017). GAM is a smoothing equivalent of generalized linear modeling (GLM), which has been proven useful for depicting the time trends of pollutant levels in cetaceans (Law et al., 2012; Jepson et al., 2016; Gui et al., 2017). Body length, sex, age class, and stranding area were considered as potential covariates in the models. PAH levels in this study are expressed on a wet weight (ww) basis. The results were blank subtracted but not corrected with recovery rates, since the recovery rates were in the established range. All the sixteen
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Table 1 Summary statistics (geometric mean with range) by year, age class and region of the concentrations of PAHs (ng g−1 wet weight) in the blubber of Indo-Pacific humpback dolphins from the Pearl River Estuary sampled between 2012 and 2017.
2012 (n = 11) 2013 (n = 5) 2014 (n = 9) 2015 (n = 4) 2016 (n = 3) 2017 (n = 5) Calf (n = 8) Juvenile/adult (n = 29) Regions off Qi'ao island and Macau (n = 9) All (n = 37)
∑16PAHs
Di-cyclic
Tri-cyclic
Tetra-cyclic
Penta-cyclic
Hexa-cyclic
4740 (3500–6080) 4030 (2870–5710) 3040 (2100–4560) 2770 (2250–3380) 868 (418–1460) 346 (17.6–1290) 4590 (2620–6080) 2010 (17.6–5670) 4840 (3950–5670) 2400 (17.6–6080)
462 (135–1550) 395 (212–667) 391 (317–538) 370 (275–486) < 10 (< 1–389) < 10 (< 1–640) 480 (169–1550) < 10 (< 1–750) 535 (426–667) < 10 (< 1–1550)
4000 (3000–4930) 3450 (2350–4970) 2510 (1680–3980) 2290 (1860–2840) 644 (310–1360) 134 (5.61–835) 3830 (2200–4970) 1460 (5.61–4930) 4070 (3250–4930) 1800 (5.61–4970)
332 (96.2–626) 439 (329–737) 361 (244–589) 316 (264–421) 103 (53.8–248) < 5 (< 5–180) 345 (96.2–737) < 5 (< 5–538) 485 (445–532) < 5 (< 5–737)
<1 <1 <1 <1 4.21 4.79 <1 <1 <1 <1
<1 <1 <1 <1 <1 <1 <1 <1 <1 <1
(< 1–17.7) (< 1–8.81) (< 1–12.3) (< 1–22.6) (< 1–11.5) (< 1–17.7) (< 1–22.6) (< 1–8.81) (< 1–22.6)
(< 1−123)
(< 1–123) (< 1–123) (< 1–123)
high aqueous solubility, accounted for 99.8% of total PAH concentrations. Among these, Phenanthrene are the most abundant PAH isomer, ranging from < 10 to 3275 (mean = 1770) ng g−1 ww. This composition pattern was in accordance with previous studies of PAHs in the blubber of bottlenose dolphins from the Canary Islands (García-Álvarez et al., 2014a, 2014b). Phenanthrene, a compound of crude oil, is more readily absorbed by marine organism than the HMW PAH isomer, benzo[a]pyrene (Nisbet and Lagoy, 1992). Although the toxicity induced by Phenanthrene is considered to be lower than that of benzo[a] pyrene, the high accumulation of Phenanthrene is potentially dangerous for the health of these dolphins. Di and tetra-cyclic PAHs accounted for 13.2% and 9.98% of the total PAH concentrations analyzed in this study, respectively. Among the 4-rings PAHs, Fluoranthene and Pyrene were detected in over 90% of total blubber samples, whereas the rest 4-rings PAHs and higher molecular weight PAHs were not detected in most of our samples. PAHs have two main environmental sources, including petrogenic (LMW) and pyrolytic origins (HMW). The ratios of two thermodynamic stable PAH isomer pair, Phe/Ant and Flu/Pyr have been widely used to infer the sources of PAHs. As illustrated in Fig. 2, the ratios of Phe/Ant and Flu/Pyr demonstrated that PAHs in the blubbers of the PRE dolphins are originated from both pyrolytic and petrogenic sources (Phe/ Ant > 10 and Flu/Pyr > 1). Previous studies on sediment and fish also reported that PAHs in the PRE originates from both petrogenic and pyrogenic sources, including vehicular emissions, coal combustion and coke ovens (Peng et al., 2008; Sun et al., 2016; Liu et al., 2017).
order of magnitude lower than the Australian humpback dolphins (Sousa sahulensis) and/or Australian snubfin dolphins (Orcaella heinsohni) in the Great Barrier Reef, Australia (Cagnazzi et al., 2013). The highest levels of PAHs ever reported in cetacean worldwide were found in the Mediterranean cetaceans (Marsili et al., 2001), which have cause toxicological stress in these animals. PAH levels in this study were far below than the levels of PAHs in Mediterranean cetaceans (Marsili et al., 2001), but are similar with those found in the blubber tissue of the wild bottlenose dolphins in the estuarine areas from the southeastern United States (Fair et al., 2010). Although the concentrations of PAHs found in the blubber samples in this study are relatively low compared to the previous studies (García-Álvarez et al., 2014a, 2014b), the potential toxicological impact of PAHs still needs caution. Marine mammals can detoxify and eliminate petroleum hydrocarbons via their mixed function oxidase enzyme systems. Thus, PAHs are generally not highly accumulated in tissues of marine mammals as organochlorinate hydrocarbons. However, when PAHs are found, it may indicate an overload of the mixed function oxidase system. Besides, the metabolic products of PAHs may be bioaccumulative and can induce toxic effects (Brunström et al., 1991). Martineau et al. (2002) suggested a linkage between exposure to excessively high levels of PAHs and cancers in Beluga whales (Delphinapterus leucas) from the St. Lawrence Estuary, Canada. Moreover, the different analytical methods of PAHs in samples across studies in cetaceans must be taken with caution in the comparison. Lower molecular weight PAHs (di, tri and tetra-cyclic), which had
Table 2 Comparison of blubber concentrations of PAHs in cetaceans from different regions around the world. Locations
Dolphin species
Sampling year (s)
Sample type
Sample number
Total PAHs (ng g−1 wet weight)
Range
NO. of PAHs
Reference
Zhuhai and Jiangmen, China Zhuhai, China Xiamen, China Hong Kong, China Korean coastal waters Great Barrier Reef, Australia Mediterranean Sea, Italy
Sousa chinensis
2012–2017
Stranded
37
2400
17.6–6080
16
This study
2761.6 6751 3274.6 160b 51,035 ± 5227b 32,296 ± 3152b 9052.5 ± 21,304
– Max = 8530 2263.1–4663.2 4.8-432 12,552–86,711 8682–61,921 228.60–83,662
Sousa chinensis
2003 2002–2004 2002–2004 2003 2009–2010
Stranded Stranded Biopsy Stranded Stranded
a
15
Leung et al., 2005
16 16
Moon et al., 2011 Cagnazzi et al., 2013
14
Marsili et al., 2001
García-Álvarez et al., 2014a García-Álvarez et al., 2014b Fair et al., 2010
1993–1996
Biopsy
Canary Islands, Spain
Finless porpoise Sousa sahulensis Orcaella heinsohni Balaenoptera physalus Stenella coeruleoalba Tursiops truncatus
1 4 5 52 18 17 23
1997–2011
Stranded
25 25
36,205 ± 41,107 788.8 ± 1409b
199.40–198,368 75.3–5761.33
16
Canary Islands, Spain
Tursiops truncatus
2003–2011
Biopsy
64
15,932 ± 10,233b
1394–42,577
16
Charleston, US Indian River Lagoon, US
Tursiops truncatus
2003–2005
Biopsy
17 11
3010b 1316b
10–9140 10–1200
45c
NA: not analyzed. a 16 EPA PAHs excluding benz[a]anthracene. b ng g−1 lipid weight. c 45 PAH parent and homologs and 12 PAH metabolite compounds. 696
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Fig. 2. Scatter plot of the PAH congener ratios Phe/Ant vs Flu/Pyr in the blubber samples of Indo-Pacific humpback dolphins from waters off the Pearl River Estuary (n = 37). Table 3 Generalized additive mixed modeling results for temporal trends in PAH levels in blubber of Indo-Pacific humpback dolphins from the Pearl River Estuary, China. Model summarizes the following information: sample size (N), %deviation explained (%dev), Akaike Information Criterion (AIC) value, and effects of explanatory variables, with associated probabilities (p). Only the PAH levels with significant time trends and the explanatory variables with significant effects are reported. PAH data
N
%dev
AIC
Year
Σ16PAHs Σ16PAHs Σ16PAHs Σ16PAHs Σ16PAHs
37 37 28a 29b 23c
82 81.7 79.1 81.4 75.5
599 599 456 467 372
F = 99.2, F = 87, p F = 38.6, F = 81.4, F = 64.8,
a b c
p < 0.0001 < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
Body length
Age class
Stranding site
p < 0.05 – – – –
– p < 0.05 – – –
p p p p –
< < < <
0.05 0.05 0.05 0.05
Individuals from the coastal region near Macau and Qi'ao island were excluded in the dataset. Calves were excluded. Calves and individuals from the coastal region near Macau and Qi'ao island were excluded.
Significant correlations were found between concentrations of total PAHs and each PAH congeners (spearman r = 0.52–0.98) (Fig. S2). Σ16PAHs were strongly and significantly correlated with the levels of Fl, Phe, Ace, Ant, Flu and Pyr, and were moderately and significantly correlated with the levels of Nap and Acy. The strongest correlation was found between Flu and Pyr. Summary statistics (geometric mean with range) by region and year for the concentration of Σ16PAHs and PAHs with different rings were given in Table 1. We fitted a GAM to the whole dataset to investigate the effects of year, region, body length, sex, age class and lipid content on the concentrations of total and different cyclic groups of PAHs in the blubber of humpback dolphins. The final model was selected in which all the remaining explanatory variables have a significant effect, the deviance explained is higher than 50% and the AIC score is the lowest. The summarized results of the GAMs by the analysis of variance table were given in Table 3. As shown in the table, sex and lipid content have no significant effect in any cases on PAH levels, while the remaining variables (year, region, and body length/age class) are all statistically significant (p < 0.05). As expected, dolphin samples stranded near the two PAH hotspots (Qi'ao island and Macau) had statistically significantly higher levels of PAHs than those stranded in other regions of the PRE, indicating that stranding site has a significant confounder effect with other explanatory variables (Table 3). However, it is also possible that the foraging area/ habitat of the dolphins and where the stranding occurred may be very different, which needs further investigations. The spatial difference in
Furthermore, dolphin samples in this study showed much higher Phe/ Ant ratios (8–26) than did marine fish from the Daya Bay (0.98–4.9) (Sun et al., 2016), with the lowest ratios found in sediments in the PRE. This could be due to the greater water solubility of phenanthrene compared to anthracene, thus phenanthrene can be more readily absorbed by marine organisms than anthracene (Martinez et al., 2004). Fig. S2 illustrated that the concentrations of Σ16PAHs or individual PAH congeners were not significantly correlated with body lengths (spearman correlation test, p > 0.05). Similar pattern was also found in finless porpoises from Korean coastal waters (Moon et al., 2011). Despite no significant difference was found among the different sex and maturity groups, the median levels of Σ16PAHs in the juvenile dolphins are apparently higher than those in the adult samples (Fig. S1 of the Supplementary material; “S” indicates tables and figures in the Supplementary material). No significant difference in the levels of individual PAH compound between male and female samples were shown. We further compared PAH levels between the calf (body lengths < 137 cm according to Jefferson et al., 2012) and juvenile/ adult (body lengths longer than 137 cm) samples. Significantly higher levels of Σ16PAHs and/or individual PAH congeners were found in calves compared to juvenile/adult group (Table 1), probably due to incomplete development of metabolic system in calves. Previous studies have found a high neonatal mortality rate among all stranded humpback dolphin samples in Hong Kong waters (Parsons, 2004; Jefferson et al., 2006; Jefferson et al., 2012), which is possibly related to the high concentration of POPs in dolphin tissues. 697
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Fig. 3. Temporal trends in blubber concentrations of ∑16PAHs (wet weight) in Indo-Pacific humpback dolphins stranded in the Pearl River Estuary (PRE) from 2012 to 2017 using the different datasets. (A) All samples (n = 37) were used. (B) Dolphin samples stranded in the coastal region near Macau and Qi'ao island were excluded in the analysis (n = 28). (C) Calf individuals in the whole samples were excluded (n = 29). (D) Calves and individuls which were stranded in the coastal region near Macau and Qi'ao island were excluded in the analysis (n = 23). For (A) and (C), dolphins stranded in the coastal region near PAH hotspot (Macau and Qi'ao island) are illustrated by green dots, while red dots denote individuals stranded in the other regions in the PRE. For (B), green dots indicate dolphin calf, while red dots denote juvenile/adult dolphins. In all cases, the decreasing trend of ∑16PAH levels is highly statistically significant (p < 0.0001), against the null hypothesis of no trends. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and exposure to disease among PRE humpback dolphins may increase lipid tissue metabolism and release contaminants into blood and other vital organs, resulting in an increase of their risk of health effects. In our samples, lipid content has a weak and negative effect on the blubber PAH levels (wet weight) (spearman correlation test, r = −0.369, p = 0.024), indicating that the overall reduction of PAH concentrations was not related to the decrease in lipid content in blubber samples from 2012 to 2017. A number of recent studies have investigated the temporal trends of contaminants accumulated in cetacean tissues from the PRE (including Hong Kong waters). Significant increases of the levels of hexabromocyclododecane (HBCD) and perfluorobutane sulfonate (PFBS) were observed in the dolphin and porpoise from Hong Kong waters over the past decade, which could be contributed to the replacement of conventional halogenated flame retardants (HFRs) and perfluoroalkyl substances (PFASs) by alternatives and the increasing usage of these alternatives following the restriction of some conventional POPs (Yeung et al., 2009; Zhu et al., 2014; Lam et al., 2016; Ruan et al., 2018). For organochlorine pesticides, hexachlorocyclohexanes (HCHs) levels in finless porpoises from Hong Kong waters decreased significantly from the year 1990 to 2001, while no declining trend was observed for DDTs and PCBs, suggesting prolonged exposure of these compounds for the PRE cetaceans (Ramu et al., 2006). Despite the marked decline of PAH levels in blubbers of PRE humpback dolphin in recent years, further investigation is needed to address the health effects of chronic exposure of humpback dolphins to even low concentrations of PAHs. As dolphins are a sentinel species, the potential impacts of PAH exposure to
PAH levels in stranded dolphins from different site may reflect the contamination status in their habitat. As shown in Table 1, substantial variations of PAHs concentrations in dolphin samples in this study were found according to the sampling year, with the mean levels ranging from 346 ng g−1 ww in year 2017 to 4740 ng g−1 ww in year 2012. The blubber PAH levels in all samples showed a marked decline from 2012 to 2017 (Fig. 3A). The decreasing trend is strongly and statistically significant (p < 0.0001) against the null hypothesis of no trend (Table 3). Besides, body length/age class also have significant effects on the concentrations of Σ16PAHs (Table 3). To control the confounding effect of stranding site, we excluded the nine dolphin samples from Qi'ao island and Macau in the GAM analysis. The result shows that the remaining explanatory variables (stranding year and body length or age class) still have significant effects on the concentrations of PAHs in the model, indicating that the temporal trend of PAH accumulation in dolphin blubber was not confounded by sampling site (Fig. 3B). We further excluded all the calf samples in the dataset (n = 23) (Fig. 3C, D), for which the GAM results were similar as those based on the whole dataset, indicating that the overall reduction of PAH accumulation is not due to unequal sampling of the levels of over the period of this study. This may reflect changes in local industrial structure and/or source controls of PAHs in the Pearl River Delta. Tissue contaminant concentrations in blubber of humpback dolphins may also be related to differences in body condition, as blubber serves as the primary site for lipophilic contaminants (e.g., PCBs, DDTs and PAHs) as well as for metabolic energy storage (Yordy et al., 2010). Reduced body condition due to commercially overfishing, habitat loss 698
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dolphins can be correlated to both river and human health.
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