Stranded false killer whales, Pseudorca crassidens, in Southern South America reveal potentially dangerous silver concentrations

Marine Pollution Bulletin 145 (2019) 325–333

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Stranded false killer whales, Pseudorca crassidens, in Southern South America reveal potentially dangerous silver concentrations

T

Iris Cáceres-Saeza, , Daniela Harob, Olivia Blankc, Anelio Aguayo-Lobod, Catherine Dougnace, Cristóbal Arredondof, H. Luis Cappozzoa, Sergio Ribeiro Guevarag ⁎

a

Consejo Nacional de Investigaciones Científicas y Técnicas, Museo Argentino de Ciencias Naturales Bernardino Rivadavia, Avenida Ángel Gallardo 470, C1405DJR Buenos Aires, Argentina b Centro Bahía Lomas, Universidad Santo Tomas, Avenida Costanera 01834, Punta Arenas, Chile c Clínica Veterinaria Timaukel y Centro de Rehabilitación de Aves Leñadura (CRAL), José Pithon 01316, Punta Arenas, Chile d Instituto Antártico Chileno (INACH), Plaza Muñoz Gamero 1055, Punta Arenas, Chile e Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Av. Santa Rosa 11735, La Pintana, Santiago, Chile f Tarukari, Non-Government Organization, Chile g Laboratorio de Análisis por Activación Neutrónica, Centro Atómico Bariloche, Avenida Bustillo 9500, Bariloche, Argentina

ARTICLE INFO

ABSTRACT

Keywords: Odontocetes Strandings Toxic metal Organs South Atlantic Ocean

Silver (Ag) is a non-essential metal known to bioaccumulate in aquatic organisms. We determined Ag concentrations in five false killer whales stranded in South America. Silver concentrations (in dry weight basis) range as 6.62–10.78 μg g−1 in liver, 0.008–7.41 μg g−1 in spleen, 0.004–5.71 μg g−1 in testis, 0.757–1.69 μg g−1 in kidney, 0.011–0.078 μg g−1 in lung and < 0.01–0.038 μg g−1 in muscle, whereas in the single samples of uterus and ovary were 0.051 and 0.023 μg g−1; respectively. Overall, Ag concentration in liver and kidney exceeded the cetacean toxic thresholds, proposed as “unhealthy concentrations” and “critically dangerous” in liver and kidney. These results warrant further eco-toxicological studies, to examine biological effects of elevated silver levels for individuals and to assess the species' conservation status with respect to marine pollution.

1. Introduction

likely affect the false killer whales. The International Union for Conservation of Nature (IUCN) classifies P. crassidens as “Data Deficient” on its Red List of Threatened Species and is listed in Appendix II of CITES (Taylor et al., 2008). In view of that, further research is required to assess the pollution status of the false killer whale. The increased human activity in recent decades has accelerated inputs of pollutants, including metals, to the marine environment. Silver (Ag) is a natural constituent of Earth's crust and it has been extensively used by humans in jewelry, utensils, monetary currency and explosives (Luoma, 2008; Ratte, 1999; Saeki et al., 2001). During the last decades of the 20th century, large amounts of Ag contained in waste from electronic industries, photography, and a variety of pharmaceutical products were disposed (Luoma et al., 1995; Luoma, 2008). Silver was the most important antimicrobial compound before the introduction of antibiotics in the 1940s and is still used in a wide range of medical applications because of its antibacterial properties and low toxicity to human cells (Mijnendonckx et al., 2013). Nowadays, release of silver nanoparticles (AgNPs) linked to the extended use in commercial textiles and personal care products is considerable (Yu et al., 2013;

The false killer whale is a large odontocete that inhabits oceans worldwide, mainly tropical and semitropical regions and does not range into latitudes higher than 50° in either hemisphere (Stacey et al., 1994; Baird, 2008). False killer whales occur offshore and in coastal waters around archipelagos, travelling in large pods, of 20 to 50 members (Baird, 2008). In the Southwestern South Atlantic Ocean, the species has been found in the Strait of Magellan, Malvinas Islands and the Beagle Channel (Koen Alonso et al., 1999; Goodall et al., 2008). In the Chilean region seven strandings have been documented and sightings made mostly along the north, central and south coast of Chile (Oporto et al., 1994, Aguayo-Lobo et al., 1998; Flores et al., 2003; revised by Haro et al., 2015). Commonly stranded individuals showed evidence of parasite infestations (Dougnac, unpublished data), and exposure to heavy metals, which could eventually lead to a low health condition (Mouton et al., 2015; Hansen et al., 2016; Cáceres-Saez et al., 2018). Predicted impacts of the global climate change (Learmonth et al., 2006) and other anthropogenic disturbances such as marine pollution also

⁎

Corresponding author. E-mail addresses: [email protected], [email protected] (I. Cáceres-Saez).

https://doi.org/10.1016/j.marpolbul.2019.05.047 Received 16 November 2018; Received in revised form 14 May 2019; Accepted 21 May 2019 Available online 03 June 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

Marine Pollution Bulletin 145 (2019) 325–333

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Deycard et al., 2017). Silver toxicity is highly dependent on its chemical species, and a large number of Ag species are found in aquatic systems. The most toxic form is regarded as Ag+ (Ratte, 1999) although lipophilic AgCl molecules enter easily into the cell (Tappin et al., 2010). Chemical properties of Ag allow its uptake via membrane ion transporters into cells (Luoma, 2008). For example, in freshwater free ionic Ag is one of the most toxic commonly occurring species and is present in traceable quantities. The ionic Ag is highly persistent and toxic to bacteria, invertebrates and fishes, and showed a tendency to bioaccumulate in organisms (Luoma et al., 1995; Ratte, 1999). In marine ecosystems, the formation of chloro-complexes enhances the bioavailability and toxicity of Ag to the biota (Ratte, 1999; López-Serrano et al., 2014); thereby Ag can enter into marine food webs (Wang et al., 2014). The mechanism of Ag uptake into a cell is not completely known although it is assumed that it enters the cells by active transport (Ratte, 1999). Once Ag is absorbed by the body it interacts with proteins, DNA and RNA by binding to sulfhydryl, amino, carboxyl and phosphate groups (ATSDR, 1990; Drake and Hazelwood, 2005). Silver may cause negative effects on ocean top predators, and marine mammals are particularly thought to suffer potentially detrimental impacts of chronic Ag exposure (Wang et al., 2014; Chen et al., 2017; Li et al., 2018a). Even though Ag toxicity is currently a major concern, it has also been of interest due to its interaction with essential elements such as selenium (Se). The ability of marine mammals to withstand large concentrations of toxic metals is partly due to the protective role of Se (Becker et al., 1995; Mackey et al., 1995; Ikemoto et al., 2004a; Kunito et al., 2004) and the interaction has been documented in odontocetes and pinnipeds (Wöshner et al., 2001a, 2001b; Saeki et al., 2001; Seixas et al., 2009a, 2009b). The aim of our study was to assess the Ag distribution and concentration in stranded false killer whales at the Subantarctic waters of Strait of Magellan in South America. The specific goals were to: 1) analyze concentrations of Ag in liver, kidney, muscle, lung, spleen and reproductive tissues samples; 2) review and compare with published literature on Ag bioaccumulation in marine mammals worldwide; and 3) to determine the Se to Ag molar ratios in each tissue to determine the potential protective effect of Se against Ag toxicity.

spleen, skeletal muscle, testis, and uterus. All samples of organs and tissues (liver, kidney, spleen, lung, skeletal muscle, testis, ovary and uterus) were stored at −80 °C after field necropsy. Before the analysis, 10 g of each tissue were freeze-dried for at least 72 h and then homogenized to a fine powder. Aliquots ranging in mass from 100 to 150 mg were placed in quartz ampoules and sealed for irradiation. The Ag concentrations were determined by Instrumental Neutron Activation Analysis (INAA) (Cáceres-Saez et al., 2013). Quality control analysis was performed using the Certified Reference Materials NRCC DORM-2 (dogfish muscle), which were processed under the same conditions as the odontocete tissue samples. The quality control results were satisfactory (measured value = 0.0342 ± 0.0052 μg g−1; certificated value = 0.041 ± 0.013 μg g−1). Silver concentrations [Ag] are expressed in dry weight (DW) basis. In the current study descriptive statistics and graphics were used to present [Ag] per tissue across individuals, and its concentrations were expressed as mean, and standard deviation in parenthesis. 3. Results and discussion 3.1. Silver concentration and tissue distribution Silver concentrations in tissues of stranded false killer whales showed high variability among the specimens (Table 1). The maximum [Ag] recorded were in liver (6.62 to 10.78 μg g−1), followed by spleen (0.008 to 7.41 μg g−1), testis (0.004 to 5.71 μg g−1), kidney (0.757 to 1.69 μg g−1), uterus (0.051 μg g−1), lung (0.011 to 0.078 μg g−1), ovary (0.023 μg g−1) and muscle (< 0.01 to 0.038 μg g−1) (Table 1). This tissue accumulation pattern is consistent with the results of dietary uptake under laboratory conditions (Van der Zande et al., 2012). Outcomes of such type of experiments based on a short-term exposure can be different from the lasting dietary Ag exposure in wild animals. However, a comparable distribution pattern has been reported for other studies regarding diverse tissues of marine mammals (Agusa et al., 2011a, 2011b). The uptake pathway of marine mammal exposure to toxic metals is by food ingestion (Bowles, 1999; Becker et al., 1995; Dehn et al., 2006; Savery et al., 2013). Therefore, the main Ag exposure in the false killer whale is likely to be through feeding of contaminated prey. Higher [Ag] in the liver of specimens is consistent with the liver's role as the main organ of protein metabolism, storage and detoxification in mammals (Mackey et al., 1995; Saeki et al., 2001; Kunito et al., 2004). Silver, as other heavy metals, possesses a high affinity for thiol-groups (-SH) of amino acids in proteins, and tends to accumulate in hepatic tissue. Once absorbed by the gastrointestinal tract into the circulatory system, Ag interacts with essential elements and binds to other components of the blood plasma (Barbier et al., 2005). During the portal circulation, Ag combines with biomolecules such as serum albumin and enzymatic proteins (i.e., metallothioneins, MTs; Drake and Hazelwood, 2005), being accumulated in the liver. The hepatocytes can retain some Ag compounds (Ikemoto et al., 2004a, 2004b), while other chemical fractions remain in the circulatory system reaching all other organs (Loeschner et al., 2011; Van der Zande et al., 2012). Hepatic [Ag] in marine mammals may reflect lifetime exposure because Ag can be deposited and stored in the liver neutralizing its toxic effect (i.e., storage in detoxified forms, such as inorganic granules or binding to MTs; Becker et al., 1995; Ikemoto et al., 2004a, 2004b; Kunito et al., 2004; Nakazawa et al., 2011). Since Ag is characterized by a strong affinity for several amino acids, it forms organic compounds with proteins containing a large proportion of sulphydric groups. This leads to Ag storage after reaction with sulfur ligands S2− (Martoja et al., 1988). Lastly, Ag is sequestered in a stable chemical form (biomineralized) as Ag2S, which becomes localized in extracellular cytoplasmic components (i.e. basement membranes) and macrophageous cells (Baldi et al., 1988; Martoja et al., 1988; Kunito et al., 2004; Nakazawa et al., 2011).

2. Material and methods 2.1. Specimens and sample collection Specimens of false killer whales under study were obtained from a mass stranding at Strait of Magellan, Chile, (52°39′12″S–70°19′57″W), South America in 2013 (Haro et al., 2015). Tissue samples (liver = 3, kidney = 5 (including > 15–20 renules, with both medulla and cortex tissue), spleen = 4, lung = 5, skeletal muscle = 5, testis = 3, ovary = 1 and uterus = 1) from two females and three males of mature false killer whales were collected and analyzed. 2.2. Physiological data and sample analysis The sex of the specimens was determined by external examination of genitalia during the field necropsy. Following Kasuya (1986), the sexual maturity is reached between 3.40 and 3.80 m in females and between 3.96 and 4.30 m in males of total body length (TBL). Both sexes reach sexual maturity within 8 and 14 years (Ferreira et al., 2014). According to TBL, the specimens under study were classified as mature animals, therefore constituting a same age-group. This is an important issue to assess representativeness when studying few individuals. The organs of specimens showed no apparent pathological conditions in the macroscopic evaluation during necropsy. However, all individuals had no stomach content suggesting a fasting period. In addition, the specimens presented high load of gastro-intestinal parasites indicating a poor health condition (Blank comm. pers.). The organs and tissues studied here are kidney, liver, lung, ovary, 326

327

Ag Se/Ag Se/(0.5Ag + Hg) Ag Se/Ag Se/(0.5Ag + Hg) Ag Se/Ag Se/(0.5Ag + Hg) Ag Se/Ag Se/(0.5Ag + Hg) Ag Se/Ag Se/(0.5Ag + Hg) Ag Se/Ag Se/(0.5Ag + Hg)

Measurement

sna, sample not available. The analytical uncertainty is reported after ‘ ± ’. Se/(0.5Ag + Hg), Se to 0.5Ag + Hg molar ratio.

Mean(SD)

4.25

4.10

FKW#5 ♂

FKW#7 ♀

4.43

FKW#4 ♀

4.96

5.29

FKW#1 ♂

FKW#6 ♂

TBL (m)

Specimen code

0.015 ± 0.008 1974 1.10 0.008 ± 0.001 6165 1.01 0.007 ± 0.001 9489 1.03 0.038 ± 0.003 3906 1.01 0.017(0.01) 5384(3227) 1.04(0.04)

< 0.01

Muscle

Organ/tissue

8.92(2.12) 63.5(20.6) 0.95(0.08)

10.78 ± 0.61 58.4 0.99 6.62 ± 0.39 86.3 0.85 sna

9.37 ± 0.53 45.93 0.99 sna

Liver 1.690 ± 0.100 109 1.88 1.39 ± 0.091 177 1.58 0.76 ± 0.048 187 1.53 1.23 ± 0.071 129 1.57 1.47 ± 0.09 256 1.31 1.31(0.35) 172(57.1) 1.57(0.20)

Kidney

Table 1 Specimens of false killer whales (P. crassidens), concentration of silver (μg g−1 DW) and the molar ratios per tissues.

0.014 ± 0.002 5553 1.09 0.011 ± 0.002 4680 1.63 0.078 ± 0.008 3614 0.69 0.075 ± 0.005 2213 0.70 0.058 ± 0.005 5179 0.77 0.047(0.03) 4248(1351) 0.97(0.40)

Lung

0.020 ± 0.003 13,115 0.96 3.40 ± 0.330 75.1 0.86 7.41 ± 0.430 115 0.92 2.71(3.52) 6083(6967) 1.05(0.29)

0.008 ± 0.002 11,028 1.48 sna

Spleen

1.94(3.26) 2931(4825) 3.47(1.10)

0.004 ± 0.001 8500 4.34 5.71 ± 0.980 5.57 2.23 –

0.121 ± 0.018 288 3.84 –

Testicle

sna

–

0.023 ± 0.002 2479 1.88 –

–

Ovary

0.051 ± 0.004 8234 1.10

–

–

sna

–

Uterus

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Marine mammals possess reniculate kidneys in structure, made up of hundreds of reniculi, unlike those of terrestrial mammals (except the polar bears). Their kidneys have an increased medullary thickness necessary to produce highly concentrated urine accomplished via hormonal regulation, which is especially important for mammals in a hyperosmotic environment (Ortiz, 2001). Kidneys of the false killer whale exhibited accumulation of Ag, although their mean concentration was lower than those measured in liver and spleen tissues (Table 1). Experimentally, mammal kidneys filter Ag through the glomeruli and then reabsorbed by the proximal tubules in reniculi (Barbier et al., 2005; Kurasaki et al., 2000). Li et al. (2018a) suggested that kidneys act as a transit organ for Ag metabolism in odontocetes, hence accumulating high concentrations of metals. Concerning the spleen, as the major and peripheral lymphoid organ in mammals, it has several functions in red blood cell homeostasis, in metabolism and the endocrine system (Tarantino et al., 2013). The spleen is normally rich in amino acids due to its role in hemoglobin recycling from senescent erythrocytes from the circulatory system, and develops immune function acting in antigen surveillance and antibody assembly in mammals. Therefore, it can be assumed that Ag accumulation is a result of circulatory metabolism. Accordingly, exposure experiments under laboratory conditions showed that Ag is largely accumulated in the spleen, as well as in the liver and bone marrow (Gammill et al., 1950). A recent assessment of the metabolic profiles of tissues and fluids from experimental animal models showed that AgNPs circulate in the bloodstream, and are readily taken up by different tissues. The organs showing a higher accumulation of Ag were the liver, spleen and kidneys, followed by the heart and lungs (Jarak et al., 2017), in agreement with our results. Among other the tissues analyzed in our study, skeletal muscle constitutes an important tissue for long-term monitoring purposes. It exhibited lower [Ag] than liver, spleen and renal tissues (Table 1), a pattern that was observed in previous studies of marine mammals (Wöshner et al., 2001a, 2001b; Agusa et al., 2011a, 2011b). Lung exhibited low [Ag] among tissues analyzed. This might reflect another possible way for Ag intake. The inhalation of Ag compounds has been reported in humans (Takenaka et al., 2001; Drake and Hazelwood, 2005) and air breathing is another potential route of Ag exposure in cetaceans (Savery et al., 2013). The source could be Ag emissions into the environment that then bind to suspended particles (i.e. aerosols), which can be transported long distances through atmosphere (Ratte, 1999). Silver can enter to the marine mammals' body through the skin, although this pathway is uncertain due the fact that Ag has not been shown to attain cells by passive diffusion (Ratte, 1999). The passive diffusion across lipid membranes is the main process for neutral organic substances. However, the lipophilicity of metals is usually low, and they might accumulate in organisms via different pathways. The uptake of metals shows complex internal dynamics at specific channels in cell membrane, active transport or endocytosis (Simkiss and Taylor, 1989). To date, several studies have included Ag measurements in the skin of cetaceans, and showed that this organ exhibits lower levels in comparison with those in internal tissues analyzed (Yang et al., 2002; O'Hara et al., 2008; Cáceres-Saez et al., 2017). In addition, an assessment of Ag through skin biopsies of sperm whales (Physeter macrocephalus) and Southern right whales (Eubalaena australis) was reported (Savery et al., 2013; Martino et al., 2013). Savery et al. (2013) established a global baseline of Ag level in skin biopsies of sperm whales of 16.9 μg g−1 WW (wet weight). The authors proposed that skin acts as a Ag reservoir organ where it can be slowly removed by periodic sloughing of the epidermis. Furthermore, it has been observed that [Ag] in fur and hair of marine mammals are relatively high in comparison with internal tissues. Silver concentrations in hair suggest that it is available to the growing pile via blood during annual molting. Thus, such physiological processes are considered a primary pathway of Ag excretion in pinnipeds and phocids (Nielsen, 1987; Saeki et al., 2001; Agusa et al., 2011a, 2011b). The blood metal concentrations represent a

circulatory exposure over previous days and are dependent on prey consumed. Silver has similar behavior as other chalkofile elements, with high affinity to -SH group in cysteine showing longer biological half-life (Mason and Jenkins, 1995; Kunito et al., 2004). A small fraction of this metal can be excreted via the gastrointestine (Zeisler et al., 1993). In mammals, Ag is excreted via the bile and feces, and only < 1% of Ag intake is absorbed and retained within internal tissues (Nielsen, 1987; Eisler, 2000). However, Wood et al. (2002) suggest that > 30% of the Ag accumulated in aquatic organisms could be eliminated later. 3.2. Silver levels among marine mammals Diverse monitoring programs have determined Ag accumulation among stranded and/or bycaught mammal species to monitor possible adverse effects, to gain insights into their health condition, and to establish safe levels within the oceans (Fig. 1a, b; listed in Supplementary Table S1). Trace metal concentrations in marine mammal tissues varies in relation to feeding habits (Byrne et al., 1985; Dehn et al., 2006), but is also a function of their excretion/detoxification capacities (Saeki et al., 2001; Kunito et al., 2004; Nakazawa et al., 2011). As well, several life history factors (i.e. age-class, body size, reproductive status, and nutritive condition, among others) can be related to the intra- and interspecific variations in element concentrations, together with ecological and geographical variables (Becker et al., 1995; Bowles, 1999; Agusa et al., 2011a; Mackey et al., 2003; Reed et al., 2015; Romero et al., 2017). These factors have to be considered when analyzing contaminant concentrations and their potential effects. Mackey et al. (1996) suggested that Ag levels in marine mammals livers generally range from 0.01 to 1 μg g−1 WW (conversion from WW to DW can be estimated by multiplying WW by 4, due to tissue water content). Our study of false killer whales showed high hepatic [Ag] with a maximum of 10.78 μg g−1 and a mean value of 8.92 μg g−1 (2.70 and 2.23 μg g−1 WW; respectively). According to Bowles (1999), odontocetes (or toothed whales) are commonly linked to a higher chemical risk category than their counterparts' baleen whales (filter feeding), due to their position at the top of the food web. The diet of odontoceti includes larger and higher trophic level fishes and squids. Baleen whales are filter feeders consuming small fishes and crustaceans, feeding at lower levels. Low [Ag] have been reported in species such as the bowhead whale (Balaenoptera mysticetus) (Byrne et al., 1985; Wöshner et al., 2001a; Dehn et al., 2006; Rosa et al., 2008) and the gray whale (Eschrichtius robustus) (Varanasi et al., 1994; Tilbury et al., 2002; Dehn et al., 2006), in contrast with odontocetes such as the striped dolphin (Stenella coeruleoalba) (Kunito et al., 2004), the bottlenose dolphin (Tursiops truncatus) (Méndez-Fernandez et al., 2014) and the Risso's dolphin (Grampus griseus) (Li et al., 2018a) (Fig. 1a, b). Until now, little is known about the diving behavior of cetaceans in the wild. However, deep diving organisms which feed predominantly on cephalopods are exposed to greater concentrations of toxic elements such as Ag and cadmium (Cd) because these elements are naturally enriched in this prey (Bustamante et al., 1998; Lahaye et al., 2006). Cephalopods are key source of Cd and Ag for diverse odontocetes (Becker et al., 1995; Lahaye et al., 2006), and those with teutophagous habits generally tend to accumulate higher concentrations of both elements than piscivorous feeders. Mean hepatic [Ag] in false killer whales was 8.9(2.1) μg g−1 being higher than other odontocetes at the Southern Hemisphere (Fig. 1a, b). For example, in bycaught specimens of estuarine dolphins (Sotalia guianensis), Atlantic spotted dolphins (Stenella frontalis) and Franciscana dolphins (Pontoporia blainvillei) from coasts of Brazil, South Atlantic Ocean, concentrations of Ag were 0.79(0.92) μg g−1 DW (Seixas et al., 2009a), 1.5 μg g−1 DW (Kunito et al., 2004) and 2.4(4.1) μg g−1 DW (Kunito et al., 2004); respectively. Besides, [Ag] in bottlenose dolphins (T. truncatus), common dolphins (Delphinus delphis) and melon-headed whales (Peponocephala electra) stranded in Australia were < 4.0 μgg−1 328

id ed

−s

te

hi

w

d Pi olp lo hi t Pi wh n[1 lo al 8] tw e Pi ha [17 lo le ] Py P gm i t w [18 Py y lot w hal ] g kil h e[ Py my ler ale 9] gm kil wh [14 y ler ale ] sp w [1 R erm hale 5] is R so' wh [16] is s a R ou R so's dol le[9 p R gh− isso do hin ] ou to 's lp [9 h So gh− oth do in[ ] w to ed lph 15] er ot d in by he ol [1 's d ph 9] b d in Sp eak olph [22 ot ed in ] Sp ted wh [15 ot do al ] e St ted lph [9] rip do in [ St ed lph 19] rip do in [ St ed lph 15] rip do in e lp [1 W Stri d d hin 4] hi pe olp [1 te d 1 So −s do hin ] ut ide lp [21 he d hin ] rn do [1 se lph 6] Ba a o in[ i tt 9 Be kal er[3 ] s Be ard ea 6] ar ed l[30 d C ed sea ] as pi se l[2] H an al[3 ar se 1 ] b H or al[3 ar se 0 bo a ] l[2 N r or H se 9 ] t a N her rp al[3 or n se 4 th fu al ] N er r [3 or n se 3 th fu al ] er r [2 n se 8] fu a R r s l[29 in e ] R ged al[3 in g s 0] R ed eal in s [2 g e ] R ed al[3 in s e 5 Sp ged al[ ] St ott se 31] el ed al[ l St er s se 31] el e al[ le a 3 r s lio 1] ea n[ li 29 W on ] Po alr [32 la us ] Po r be [27 la ar ] Po r be [37 la ar ] Po r be [38 la ar ] Po r be [35 la a r ] Po r be [39 la ar ] r b [4 ea 0] r[4 0]

c

ifi

Pa c

(µ gg1)DW Bo w Bo hea w d Bo hea wh w d ale Bo hea wh [1] w d ale Bo hea wh [2] w d ale Bo hea wh [3] w d ale he w [4 ad ha ] w le Fi ha [5] n l G wh e[6 ra a ] y l G wh e[9 ra a ] At y l G wh e[7 la r n At tic M ay ale ] la sp in wh [8 nt o k a ] e i Ba c s tted w le[5 ird pot do hal ] 's ted lp e[9 be d hin ] ak olp [1 Be ed hin 1] lu wh [20 g a Be a w le[ ] h 1 Be luga ale 6] lu w [17 ga h Bl Be w ale ] ai lu ha [2 nv ille Be ga le[2 ] lu w 3 ' Bo s be ga hal ] tt a w e[4 Bo leno ked hal ] tt se w e[5 B len d ha ] C ott ose olp le[9 om le h ] m nos dol in[1 er e ph 3] so do in co n's lph [14 m d i ] co m olp n[1 m on hi 5] co mo do n[1 m n d lph 0] m o in o lp [9 D n d hin ] al ol [1 p l D D po hin 3] wa a rp [1 ll Fa D rf s po oise 4] w r p ls a e po [2 e rf rm i 5] s ki s lle p w e[ Fr r w erm ha 16] le an h w [ a Fr cis le[ ha 26] an ca Th le[ Fr cis na is s 15] an ca do tu Fr cis na lph dy] an ca do in[ Fr cis na lph 11] an ca do in[ ci na lph 20 sc d in ] Fr ana olp [16 as d hin ] e o G r's lph [24] ui d in a o G na lph [24] ui d in a o [ G na lph 15] Lo ui d in an ol [1 ng H ar a ph 9] Lo −b ng ea H bor dol in[2 −b ke ar p ph 0] ea d c bo orp in[1 ke om r p oi 1] d m o se M co on rpo [14 i el m on m do se[ ] −h on lph 18 ea do in ] de lp [11 d hin ] w [1 ha 2 le ] [1 3]

(µ gg1)DW

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Marine Pollution Bulletin 145 (2019) 325–333

a

1e+02

log(SD)

1e+00 2

0

−2

−4

1e−02

b

1e+02

log(SD) 2

1e+00 1

0

−1

−2

−3

1e−02

Fig. 1. part a), and b). Hepatic silver concentrations (μg gr−1 DW) in marine mammals' worldwide. The red-cross data indicate the level measured for false killer whales (P. crassidens) in our study. Species are grouped in Familia's according one colour: Mysticeti (red), Odontoceti (blue), Mustelidae (green), Pinnipedia/Phocidae (violet) and Ursidae (orange). The critically dangerous threshold of hepatic silver is depicted by black dashed line (after Chen et al., 2017). log(sd), denotes colourscale varying from lower to higher standard deviation range. The numbers following the common name of species correspond to the author's reference (listed in Supplementary Table S1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

DW (Law et al., 2003), and common dolphins (Delphinus capensis) from New Zealand with 2.68(1.22) μg g−1 DW (Stockin et al., 2007), being all of them lower than those in the false killer whales analyzed here. Moreover, hepatic [Ag] determined in the present study were higher

than levels found in small odontocetes at Northern Atlantic Ocean, such as the Iberian Peninsula, including common dolphins (Delphinus delphis) 0.21(0.21) μg g−1 WW, harbor porpoises (Phocena phocena) 1.04(1.06) μg g−1 WW, striped dolphins (Stenella coeruleoalba)

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0.3(0.3) μg g−1 WW, and bottlenose dolphins (Tursiops truncates) 0.2(0.19) μg g−1 WW (Méndez-Fernandez et al., 2014). Also, false killer whales showed mean hepatic [Ag] higher than pilot whales (Globicephala melas) from the Northeastern Atlantic Ocean with 0.13(0.21) μg g−1 WW (Méndez-Fernandez et al., 2014) and from Cape Cod, USA, North Atlantic Ocean with 0.163(0.114) μg g−1 WW (Becker et al., 1995) (Fig. 1a, b). Both delphinids are largely pelagic feeders and are found in tropical and temperate waters of ocean basins. Previous reports of stranded odontocetes at the east coast of England and Wales, Northern Atlantic Ocean, range from 0.05 to 5.5 μgg−1 WW, including pilot whales (G. melas), Sowerby's beaked whales (Mesoplodon bidens), pygmy sperm whales (Kogia breviceps) and Blainville's beaked whales (Mesoplodon densirostris), and also misticetes, such as fin whales (Balaenoptera phisalus) and minke whales (Megaptera novaenglidae) (Law et al., 2001) (Fig. 1a, b). As our findings show, the mean hepatic [Ag] in false killer whales was higher than those concentrations measured in baleen whales, like the bowhead whale (B. mysticetus) with a concentration of 1.215(0.235) μg g−1 DW (Tilbury et al., 2002) and 0.392(0.549) μg g−1 DW (Dehn et al., 2006); and the gray whale (E. robustus) with concentrations ranging between 0.039 and 0.196 μg g−1 DW (Mackey et al., 1996), and between 0.214 and 0.399 μg g−1 DW (Krone et al., 1999). On the other hand, hepatic [Ag] of false killer whales was in the range of studies conducted on stranded odontocetes from the coasts of Taiwan, North Pacific Ocean, such as Fraser's dolphins (Lagenodelphis hosei) 6.96(6.87) μg g−1 DW, pygmy sperm whales (Kogia spp.) 9.32(6.08) μg g−1 DW, rough-toothed dolphins (Steno bredanensis) 11.89(6.13) μg g−1 DW, spotted dolphins (Stenella attenuata) 12.62(6.28) μg g−1 DW, pygmy killer whales (Feresa attenuata) 12.95(6.64) μg g−1 DW, bottlenose dolphin (T. truncatus) 9.27(5.6) μg g−1 DW, Risso's dolphin (Grampus griseus) 5.87(10.80) μg g−1 DW (Chen et al., 2017; Li et al., 2018a), and also the Baird's beaked whale (Berardius bairdii) 14 μg g−1 DW (Nakazawa et al., 2011) from Japan, North Pacific Ocean. However, Fig. 1a shows that mean hepatic [Ag] of false killer whales lies at the low end of the average [Ag] found in livers of a large odontocete species such as the beluga whale (Delphinapterus leucas). Only beluga whales from the Arctic Alaska are among the extremely high values (5.93–107.4 μg g−1 WW; Becker et al., 1995) measured worldwide (this findings are discussed in detail elsewhere, Becker et al., 1995; Mackey et al., 1995, 1996; Wöshner et al., 2001a; Dehn et al., 2006). A recent study of stranded Fraser's dolphin from Taiwan showed the exceptive high level of [Ag] ever measured in liver as 726.11 μg g−1 DW (or 181.5 μg g−1 WW, Chen et al., 2017) (Fig. 1a). The authors pointed a marked Ag pollution in recent decades in the Northwestern Pacific Ocean, suggesting that specimens inhabiting those marine environments could have severe Ag pollution linked to disease symptoms (Chen et al., 2017). Hence, body accumulation should be monitored to record the exposure degree of populations. Finally, there is available information on [Ag] in other aquatic mammals such as marine carnivores; and due the relevance of this potentially toxic element a comparison was made (Fig. 1b). In this regard, mean [Ag] in the liver of false killer whale was higher than those in other furry mammals including sea otters (Enhydra lutris) 1.6 μg g−1 DW (Kannan et al., 2006); pinnipeds like the Steller sea lion (Eumetopias jubatus) with mean values of 0.30 μg g−1 DW (Sydeman and Jarman, 1998), 1.44 μg g−1 DW (Saeki et al., 2001), Northern fur seals (Callorhinus ursinus) with 0.8 μg g−1 DW (Saeki et al., 2001), and 0.69 μg g−1 DW (Ikemoto et al., 2004), and also in phocids such as harbor seals (Phoca vitulina) with 0.84 μg g−1 DW (Saeki et al., 2001), 1.2 μg g−1 DW (Agusa et al., 2011a), harp seal (Phoca groenlandica) 0.85 μg g−1 DW (Agusa et al., 2011b), ringed seals (Pusa hispida) with 0.48 μg g−1 DW (Wöhsner et al., 2001b), 2.2 μg g−1 DW (Dehn et al., 2005), and Baikal seals (Pusa sibirica) 0.04 μg g−1 DW (Ikemoto et al., 2004a, 2004b). Other high marine predators such as the polar bear (Ursus maritimus) from Canada and Alaska showed low [Ag] 0.33 μg g−1 DW (Kannan et al., 2007), 0.47 μg g−1 DW (Norstrom et al., 1986),

0.64 μg g−1 DW (Rush et al., 2008) and 0.68 μg g−1 DW (Wöshner et al., 2001b) in contrast with level reported here for the false killer whale (Fig. 1a, b). Silver accumulation in the fur and body hair of marine mammals have suggested that molting strategy is considered the principal pathway for Ag excretion in mammals, (as other heavy metal such as mercury, Hg) (Saeki et al., 2001; Savery et al., 2013; Agusa et al., 2011a, 2011b). 3.3. Interaction of Ag with other elements Silver, as well as Hg, is known to interact with Se in the bloodstream and their toxicity is reduced by that interaction (Sasakura and Suzuki, 1998; Ralston and Raymond, 2010). Previous studies have established that Ag can bind to Se, copper (Cu), MTs and other high weight molecules (HWM) in liver of marine mammals (Becker et al., 1995; Ikemoto et al., 2004a, 2004b; Kunito et al., 2004; Agusa et al., 2008; Seixas et al., 2009a; Nakazawa et al., 2011). Silver is chemically similar to Cu, and is able to hamper the metabolism and transport of Cu (Saeki et al., 2001). Selenium is essential, because it constitutes an antioxidant as an integral part of the glutathione peroxidase (GPX), which plays an important role in maintaining the proper function and structure of red blood cells, as well as eliminating organic peroxides (Baldi et al., 1988; Drake and Hazelwood, 2005). Silver has been known to inhibit GPX, transported into the bile and depleting the amount of reduced GPX available for biochemical pathways (Ridlington and Whanger, 1981; Sugawara and Sugawara, 1984). In particular, Ag interaction with Se differs from other Se–metal relationships because Ag can induce symptoms of Se-deficiency in animals with vitamin E deficiency by forming Ag‑selenium complexes. That may reduce the effectively available Se for normal cellular processes (Nielsen, 1987; Hammond and Beliles, 1980; Becker et al., 1995); therefore, high [Ag] may affect radical scavenging enzyme systems (Becker et al., 1995). In the liver of marine mammals, Ag is detoxified through the formation of Ag2Se and Ag2S (sulfur) complexes (Ikemoto et al., 2004a, 2004b; Nakazawa et al., 2011). According to Whanger (1985), Se detoxifies Ag by forming Ag2Se, meanwhile Ag may compete also with Hg for binding to Se (Becker et al., 1995; Saeki et al., 2001). It has been suggested that Ag2Se (which is possibly the more stable Agcompound) is primarily accumulated in nuclear and mitochondrial fractions of hepatocytes as showed the chemical extraction technique by Kunito et al. (2004). Additionally, the interaction of Ag with Se in experimental animals has been proposed through the correlation of [Se] to (0.5 [Ag] + [Hg]) on a molar basis (Nakazawa et al., 2011). Because other elements than Ag can form different compounds with Se, particularly Hg, may compete with Ag for binding sites on Se, therefore, the molar ratio should be computed also considering Hg contents in the proportion of stable Se compound (Ikemoto et al., 2004a, 2004b; Kunito et al., 2004; Agusa et al., 2008). Accordingly, we examined both molar relationships of [Se] and (0.5 [Ag] + [Hg]), and [Se] and [Ag] to assess the protective effect of Se against Ag toxicity in the false killer whale. The concentrations of Hg and Se were simultaneously determined with [Ag] in the same sample tissues of stranded specimens by INAA (Cáceres-Saez et al., 2018). Results showed that [Se] to [Ag] molar ratio in false killer whales were largely higher than 1 in all tissues analyzed, and displayed great variability among them ranging in liver from 45.9 to 86.3, in kidney from 109 to 256, in lung from 2213 to 5553, in spleen from 75 to 13,115, in muscle from 1974 to 9489, in testicle from 5.57 to 8500; and 2479 in ovary and 8234 in uterus. These values are consistent with an excess of Se for metal detoxification, which is important for the potential amelioration of adverse effects due to Hg exposure and to maintain Se-dependent processes. On the other hand, the [Se] to (0.5[Ag] + [Hg]) molar ratio were ≤1 in tissues analyzed, specifically, the liver and lung presented average values of 0.95 and 0.97; respectively (Table 1). A decrease in molar ratios (together with Hg) is observed, probably limiting the protective effect of Se within tissues. 330

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3.4. Toxicological significance of Ag

induce oxidative stress and tissue injury in mammals. Particularly, the production of the reactive oxygen species might have adverse effect on the immune system. Our results are consistent with Ag accumulation in the false killer whales studied, and exposure to contaminated marine environment during their life might affect their health provided that [Ag] reached levels above the critically dangerous threshold. Nevertheless, additional research is necessary to define whether the levels present in these tissues pose a health hazard to this species.

According to the Agency for Toxic Substances and Disease Registry, health impacts of Ag in vertebrates may have effects on the brain, heart and reproductive system (ATSDR, 1990). Silver can induce adverse effects such as fatty degeneration of liver and kidney, irritation of the respiratory and intestinal tract, and alterations in blood cells (Drake and Hazelwood, 2005). The chronic exposure to Ag of humans causes permanent discoloration of skin (argyria) or eyes (argyrosis) (Beer et al., 2012). This metal is deposited in the perivascular regions of skin as black granules, which contain Ag2Se and/or S complexes (Drake and Hazelwood, 2005) as a potential result of Ag detoxification process (Nakazawa et al., 2011). Of particular concern, is the extended use of AgNPs, which have prevalence in agriculture, medicine and personal care products around the world, which may affect natural environments due to their high volume of production (Yu et al., 2013; Deycard et al., 2017). The AgNPs showed to induce several deleterious effect in experimental laboratory animals, such as hepatic alterations, nephritis and bile-duct hyperplasia. The severity of hepatic lesions correlates with [Ag] in liver (Mahdy et al., 2014). Moreover, in vitro studies have demonstrated genotoxicity and DNA damage in mammalian linage cells with exposure to AgNP (Asharani et al., 2009; Mahdy et al., 2014). Chronic low-level Ag exposure may suppress immunity and could be a contributing factor in health outcomes and weakness to infections in the organisms. Concerning wild animals, it has been hypothesized, that [Ag] might range between 0.01 and 72.6 μg ml−1 in blood plasma of stranded odontocetes (Li et al., 2018a). In connection with that, Li et al. (2018b) presented the first evidence of AgNPs on the cytotoxicity and immunotoxicity of cetacean's leukocytes. A novel study proposed an unhealthy (or intermediate) and critically dangerous [Ag] for odontocetes through a study in the North Pacific Ocean, which can be a useful criterion for assessing Ag poisoning in marine mammals. Following Chen et al. (2017), the unhealthy (or intermediate) Ag levels proposed are the following: for liver, 4.45(5.36) μg g−1 DW; for kidney, 0.33(0.30) μg g−1 DW; for muscle, 0.09(0.11) μg g−1 DW; and for lung, 0.06(0.02) μg g−1 DW; whereas, the critically dangerous [Ag] proposed are: for liver, 6.15(7.57) μg g−1 DW; for kidney, 0.56(0.34) μg g−1 DW; for muscle; 0.14(0.16) μg g−1 DW; and for lung, 0.06(0.04) μg g−1 DW. A comparison of tissue contaminant concentrations of Ag to toxicological threshold levels established for cetaceans indicates that Ag in false killer whales is clearly above, or close to the levels where a range of physiological adverse effects can be triggered. In particular, the false killer whale specimens analyzed here (see values in Table 1) exceeded these hepatic and renal toxic thresholds. In addition, [Ag] in tissue samples of spleen and testis are high, suggesting that they could be above critical levels. Silver concentrations in muscle and lung range in the critically dangerous threshold level (Table 1). Furthermore, Li et al. (2018a) have investigated Ag deposition in hepatic and renal tissues together with lesions presented in stranded odontocetes from Taiwan, North Pacific Ocean. Among hepatic histochemical analyses, a nonspecific reactive hepatitis, congestion, vacuolar degeneration and hyaline inclusions in hepatocyte were observed in individuals with higher (or critically dangerous), intermediate (unhealthy) and even baseline [Ag]. As well, vacuolar degeneration and hyaline droplets in the proximal renal tubular epithelium of kidneys were also found in individuals with high, intermediate and baseline levels. Though, the authors stated that there were no correlations between the Ag deposition and both liver and kidney lesions of cetaceans (Li et al., 2018a). In this context, there is no direct evidence that Ag accumulation induces injuries in organs of cetaceans, and experiments with wild individuals are not allowed to be performed under controlled pollutant exposure. Therefore, more investigation is necessary to produce Ag metabolic profile and eco-toxicity outcomes, for a comprehensive evaluation of negative impact on wild populations with the aim to be able to connect pollution and diseases to cetacean's mortalities. Kannan et al. (2006) indicate that exposure to toxic contaminants, as well as infections, can

4. Conclusions Our study has revealed high Ag accumulation in diverse tissues (liver, kidney, muscle, lung, spleen, testis, ovary and uterus) of false killer whales in the Southern Hemisphere. The distribution of Ag among different tissues was similar to those in previous studies of marine mammals around the world, with the liver having the highest concentrations. A number of physiological and environmental factors may influence Ag bioaccumulation in tissues of false killer whales. We suggest that the main exposure route for Ag pollution is occurring through ingestion of prey items. The feeding behavior based on Ag-rich predatory fishes and also cephalopods together with the complexity of marine food web are key factors accountable for bioaccumulation of toxic metals in long-lived cetaceans. In general, [Ag] of this individuals were higher than those reported in odontocetes from other marine areas of South America and concentration values varied within ranges measured in species worldwide (aside from the high values of beluga whales in the Arctic). The analysis suggests that stranded false killer whales in the Strait of Magellan an area, which connects South Atlantic and South Pacific Oceans could be exposed to Ag accumulation, yet no evidence is available on the sources or marine areas where these animals have been contaminated. Considering also its distribution and possible migratory movements may not be typical for these latitudes. The molar Se to Ag ratio varied among tissues and was largely higher than 1 in all tissues analyzed, the values suggested an excess of Se for cell metabolic processes. The Se to (0.5Ag + Hg) molar ratio close to, or equal 1, suggest that individuals presented a deficiency of Se against the toxicity of Ag and Hg. However, a decrease in molar ratios of Se to (0.5Ag + Hg) - considering the interaction of both metals depicted a limited protective effect of Se besides detoxification. Overall, [Ag] found in the analyzed false killer whales exceeded the new proposed toxicological levels in odontocetes for hepatic, renal, muscle and lung tissues. This is of particular concern in view of the numerous stressors that this species faces and which may lead to stranding events. Therefore, it is essential to continuously monitor these populations and implement an approach that includes the assessment of pollutants over time, together with other health indicators (e.g. diseases and pathogens) in stranded animals. In this context toxic metal levels can help to determine the degree of contamination within these organisms, and support to future conservation plans. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.marpolbul.2019.05.047. Acknowledgements We are grateful for help of volunteers who provided their assistance during the stranding episode Nathasja Haddow, Tamara Martínez, Constanza Cifuentes, Carolina Calabro, Benjamín Cáceres, Ricardo Matus, Susana Sáez and Manuel Cáceres. Our appreciation to the Director of INACH and the colleagues of Laboratory “Jorge Berguño” of INACH. Special thanks to Professor PhD Ian Hogg (School of Science, The University of Waikato, New Zealand) for language revision and to PhD Fabio Machado (MACN – CONICET, Argentina) for their helpful support with figure editing. The access to stranded animals was assisted by officers of the Armada de Chile, Teniente Claudio Zúñiga and Ramón Aguilar, Capitán René Rojas and Jared Yánez, and Mr. Ivelic, the manager of Estancia 5 de Enero. Research permit CITES 331

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N°16CL000003WS for the cetacean sample collection was provided by SERNAPESCA, Punta Arenas, Chile. All the samples were imported under research permit issued by the Ministerio de Ambiente y Desarrollo Sustentable de la Nación, Departamento de Biodiversidad y Dirección de Fauna Silvestre, Argentina (License N°2680428/16). We appreciate both reviewers, and the journal editor for thoughtful feedback on the earlier draft of manuscript.

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