Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints

STOTEN-21642; No of Pages 8 Science of the Total Environment xxx (2016) xxx–xxx

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Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints J. Raimundo a,b,⁎, F. Ruano a, J. Pereira a, M. Mil-Homens a,b, P. Brito a,b, C. Vale b, M. Caetano a a b

IPMA - Portuguese Institute of Sea and Atmosphere, Rua Alfredo Magalhães Ramalho, 6, 1495-006 Lisbon, Portugal CIIMAR, Marine and Environmental Research Center, Rua dos Bragas, 289, 4050-123 Porto, Portugal

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Short-term runoff event leads to sudden octopus mortality. • Histology results showed severe lesions in the cells of the digestive gland. • Pb isotopes as tracers for the rapid and high freshwater/runoff material input • Different Pb isotopic signatures pointing to a change in Pb source with runoff • Pb isotopes proved to be an adequate tool to confirm the cause of death.

a r t i c l e

i n f o

Article history: Received 14 October 2016 Received in revised form 16 December 2016 Accepted 17 December 2016 Available online xxxx Editor: D. Barcelo Keywords: Octopus Digestive gland Pb isotopes Rainfall Runoff

a b s t r a c t Octopus vulgaris is a sedentary organism that inhabits coastal waters being exposed to anthropogenic compounds. Lead concentration in coastal environments reflects many processes and activities namely weathering, industrial and domestic discharges, and atmospheric deposition. Since lead isotopic composition is little affected by kinetic processes occurring between source and sink, its signature has been used to identify different Pb sources. After a short-term heavy rainfall, hundreds of octopus appeared dead in two Portuguese coastal areas. Histopathology and Pb levels and its stable isotopes were determined in tissues, such as digestive gland, of stranded octopus and compared to alive specimens, sediments and runoff material from the same areas. Histology results showed severe damage in stranded octopus tissues suggesting that death was probably associated to multiple organ failure linked to hypertrophy and exudates input. In addition, Pb in stranded specimens reach concentrations up to one order of magnitude above the levels reported for alive octopus. Pb isotopic signatures in stranded organisms were closer to runoff material pointing to a similar origin of Pb. In summary, the results in this study showed that a short-term runoff event might change abruptly the salinity leading to the disruption of the osmoregulation function of octopus and consequently leading to its death. The analyses of stable isotopic Pb signature in octopus tissues corroborate these results and points to a change in the Pb source due to runoff after the storm water event. Pb stable isotopes in octopus proved to be an adequate tool to confirm the cause of death and linking it to the environment conditions. © 2016 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding author at: IPMA - Portuguese Institute of Sea and Atmosphere, Rua Alfredo Magalhães Ramalho, 6, 1495-006 Lisbon, Portugal. E-mail address: [email protected] (J. Raimundo).

Coastal environments are dynamic and complex ecosystems, and spatial-temporal variability associated with natural processes may

http://dx.doi.org/10.1016/j.scitotenv.2016.12.121 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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mask the effect of anthropogenic pressures. Most of the existing large cities are located near the coast. Runoff from urban areas after heavy rain events, also known as storm water, often transport high concentrations of metals (Walker et al., 1999; Morrisey et al., 2003). Urban storm water runoff is known as an important source of contaminants to receiving waters (Wu et al., 1996, 1998; Sansalone and Buchberger, 1997; Davis et al., 2001). The sources of metals in urban storm water runoff are numerous and the metal release mechanisms complex (Davis et al., 2001), being transported attached to solids or dissolved. In urban environments worldwide, anthropogenic Pb originates from a variety of sources including vehicular exhaust, leaded paint, and industrial emissions. The identification of the potential sources to the environment is of extreme importance. The study of stable Pb isotopes provides a powerful tool in tracing Pb sources. The Pb isotope ratios vary with local geology and proximity to anthropogenic inputs, as well as temporally as pollution sources change. The ratios between isotopes provide an identification of different Pb sources, which have distinct isotopic signatures (Labonne et al., 1998, 2001). Physic-chemical and biological fractionation processes do not significantly alter these isotopic lead ratios. There are four stable Pb isotopes. The 204Pb isotope is non-radiogenic, while 206Pb, 207Pb and 208Pb result from the decays of 238U, 235U and 232Th, respectively (Labonne et al., 1998). Pb isotopes have been successfully applied in various studies using different matrices. For instance, Hoven et al. (1999) used isotopes ratios to distinguish between recent anthropogenic Pb inputs from background Pb in estuaries. In sediment cores, isotope Pb ratios were used as fingerprint of human activities, suggesting a change in Pb sources related to the increase of anthropogenic activities (Mil-Homens et al., 2013). The same authors also demonstrate that sediments contaminated with Pb were not constrained to estuarine-coastal areas and upper part of submarine canyons, but also to deeper parts of the Portuguese margin. By means of comparing Pb isotopes in sediments and organisms, the bioavailability of Pb to ragworms was assessed (Philippe et al., 2008). The same approach was used with octopus from different areas, and authors found that Pb isotopes could be used to distinguish octopus populations (Raimundo et al., 2009). The Pb isotope ratios were also done in biomonitoring studies to determine the potential Pb sources to small cetaceans in European waters (Caurant et al., 2006). Monitoring programs and research lines are focused in estuaries and coastal areas affected by relevant local emissions of contaminants (e.g. Islam and Tanaka, 2004). In recent years, mitigation measures have been implemented in various systems and, in particular, the Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD) persuade managers and politicians to take actions to improve the quality status of transitional and coastal waters. The MSFD includes contaminants in commercial fishes as a key descriptor in line with the recognition of fish suitability for assessing the environmental status of marine regions (e.g. Van der Oost et al., 2003). Metal accumulation in fish tissues tends to be proportional to levels in water and food, although highly influenced by the biological role of the elements in metabolic mechanisms (Leland and Kuwabara, 1985). Cephalopods represent an essential link in marine trophic chains. O. vulgaris have a short life span of 12 to 18 months, high metabolic rates and inhabit at coastal waters. This species is normally distributed on rocky, sandy and muddy bottoms (Mangold, 1983). Several studies have pointed that metal accumulation in octopus digestive gland can reflect elemental origin (Bustamante et al., 1998; Nessim and Riad, 2003; Raimundo et al., 2004; Napoleão et al., 2005; Seixas et al., 2005). In some cases, geographical variations of metal availability can overcome the biological differences (Nessim and Riad, 2003). Concentrations of Pb were showed to present contrasting geographic patterns in digestive gland of specimens collected in the Portuguese coast (Raimundo et al., 2004; Napoleão et al., 2005; Seixas and Pierce, 2005). After a period of a heavy rainfall, hundreds of octopus died in two Portuguese coastal areas adjacent to rivers. Their arms presented symptoms of exposure to freshwater. The aims of this study were: (i) to

evaluate the impact of freshwater inputs in octopus after a high rainfall; and (ii) to trace the mechanism that caused the octopus death. These objectives were tested through: i) histological alterations in octopus tissues; and ii) determination of Pb concentrations and Pb stable isotopic signature in digestive gland, sediments and runoff particles. All biological and environmental data from the high runoff period was compared to data collected from “dry” environmental conditions. Digestive gland was selected since bioaccumulation studies have reported that storage of Pb in cephalopods occurs mainly in this tissue (e.g. Miramand and Bentley, 1992; Nessim and Riad, 2003; Seixas and Pierce, 2005; Bustamante et al., 2008; Raimundo and Vale, 2008). Although the application of Pb stable isotopes is scarce in marine organisms, their importance as a tool for environmental studies has been showed (e.g. Caurant et al., 2006; Philippe et al., 2008; Raimundo et al., 2009). 2. Material and methods 2.1. Samples Seventy five common octopuses, Octopus vulgaris, were collected in two areas (Matosinhos and Cascais) located in the western Portuguese coastal zones that receive the discharge of the two major rivers of the Iberian Peninsula, Tagus and Douro, respectively (Fig. 1). Specimens were captured in two contrasting environmental conditions: i) octopuses were caught alive in November 2009; ii) dead octopuses were caught in January 2010, after a short period of heavy rains and runoff, hereafter identified has “Stranded”. Table 1 gives the number of specimens, gender proportion (males:females) and mantle length (ML, mm) and weight (g). Specimens were stored in individual plastic bags and frozen

Fig. 1. Location of the two areas of capture of Octopus vulgaris in the Portuguese Coast: Matosinhos and Cascais.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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Table 1 Number (n), male:female proportions (M:F), total weight (g) and mantle length (ML, mm) of octopus captured alive and stranded in the coastal areas of Matosinhos and Cascais. Coastal area

Condition

n

M:F

Total weight (g)

ML (mm)

Matosinhos

Alive

31

14:17

Stranded

16

8:8

Alive

11

6:5

Stranded

17

9:8

914 (578–1433) 205 (86–1576) 1300 (805–2440) 173 (27–588)

143 (125–170) 65 (45–88) 140 (120–195) 65 (40–90)

Cascais

(− 80 °C) to minimize mobilization of metals among organs/tissues (Martin and Flegal, 1975). In the laboratory, digestive gland was totally removed under partially defrost conditions without rupture of the tissue, freeze-dried, grounded and homogenised. Six stranded (n = 3) and alive (n = 3) octopus were used for histological analysis. The top 5-cm layer of sediments were sampled in 2009 at Matosinhos (n = 5) and Cascais (n = 4), onboard of the RV Noruega using a Van-Veen grab. The runoff particles resulted associated with the rains of January 2010 was collected from two gully pots located below the highway of a bridge crossing the Tagus estuary (30 km from Cascais). Sediments and runoff-material were oven-dried to constant weight at 40 °C, sieved through a 2-mm mesh and grounded with an agate mortar. 2.2. Analytical methodology 2.2.1. Sample pre-treatment Samples of digestive gland (≈200 mg) were digested with a mixture of HNO3 (sp, 65% v/v) and H2O2 (sp, 30% v/v) at 60 °C for 12 h, 100 °C for 1 h and 1 h at 80 °C (Ferreira et al., 1990). Two mineralization procedures were used for sediment and runoff material samples. Digestion for Al quantification using HF (sp, 40% v/v), Aqua Regia (HCl36%:HNO3-65%; 3:1) and H3BO4 (Rantala and Loring, 1975). A second digestion for analysis of Pb concentration and stable Pb isotopes by using the first step of the previous method, evaporated to near dryness and elute with HNO3 (double-distilled) and Milli-Q water (18.2 MΩ cm) (Caetano et al., 2007). Procedural blanks were prepared using the same analytical procedure and reagents, and included within each batch of 10 samples. 2.2.2. Methods Aluminium (Al) was analysed in sediments and runoff material samples by flame atomic absorption spectrometry (Perkin Elmer AA100) with a nitrous oxide-acetylene flame and concentrations determined

with the standard addition method. Aluminium is one of the major constituents of fine-grained aluminosilicate metal-bearing particles (Windom et al., 1989). Trace elements exhibit a high affinity to the elevated specific surface area of particles (Mil-Homens et al., 2014). Because Al is a major constituent of clay minerals, and remains relative stable under post-depositional early diagenesis in sediments, it is commonly used as a proxy of fine particles (Windom et al., 1989). Due to that, Al was selected as a reference element for normalizing metal concentrations. Total Pb concentration and stable Pb isotopes (206Pb, 207Pb and 208Pb) were determined in the same samples but in separate runs. It was used quadruple ICP-MS (Thermo Elemental, X-Series) equipped with a Peltier Impact bead spray chamber and a concentric Meinhard nebulizer. A 7-points calibration within a range of 1 to 100 μg L−1 was used to quantify total Pb concentration. The precision and accuracy of the Pb concentration measurements, determined through repeated analysis of reference materials (BCSS-1 and MESS-3 for sediment and TORT-1 and TORT-2 for organisms), using 115In as internal standard, was better than 2%. Procedural blanks always accounted for b 1% of the total Pb in the samples. The Pb isotopes 206Pb, 207Pb and 208Pb were determined in all samples. A certified reference material NIST-SRM981 was analysed every two samples, corrections for mass fractionation. The Pb isotopic composition of procedural blanks did not significantly influence the 206Pb/207Pb and 206Pb/208Pb ratios measured in all samples. The coefficients of variation of the NIST-SRM981 reference material obtained in between-batch external quality control were 0.37% for 206 Pb/207Pb and 0.22% for 206Pb/208Pb ratios. 2.3. Histology Pieces of mantle, arms and digestive gland were collected from each animal. This material was processed for histology analysis following the protocol adopted by the IPMA laboratory of pathology for molluscs (Howard and Smith, 1983). Briefly, samples were fixed in seawater formalin (10% v/v), embedded in paraffin blocks and cut in a microtone in

Fig. 2. Weight (g) and mantle length (ML, mm) relationships for alive (♦) and stranded (○) Octopus vulgaris from Matosinhos and Cascais.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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Fig. 3. Normal dermis of the mantle (A). Evident oedematous disruptions are observed in the structures of inner and outer tunic and in the basal lamina caused by the abundant infiltration of exudates (B).

3-μ tissue sections. Paraffin embedded sections were deparaffinised in xylene and hydrate to water by carrying the slides preparations through descending concentrations of ethyl alcohol. Hydrated slides were prepared with Hematoxylin and Eosin staining method, mounted and set aside the cover slide to dry.

(Moreno et al., 2014; Faraj and Bez, 2007); (2) rainfall period occurred close to the recruitment season. Stranded specimens showed, in general, swelling bodies with turgid consistencies. Their arms and mantle were contracted and rigid losing the normal flaccid appearance of the post-mortem conditions commonly observed on fished individuals.

2.4. Statistical analysis

3.2. Octopus samples - histopathology

Prior to statistical analyses, metal concentrations were tested for normality and equality of variances. Non-compliance with parametric ANOVA assumptions led to employment of the Kruskal-Wallis H (KWH) non-parametric test, which was used to evaluate the existing differences between concentrations in alive and stranded specimens and between areas. Differences between areas were also search for sediments. The significance levels used for statistical analyses was always α = 0.05. Statistical analyses were performed using the STATISTICA 6.0 (Statsoft) software.

The histopathology of the connective tissues surrounding mantle and arm muscles of stranded octopus showed severe lesions in the collagen fibbers that integrated the inner and outer tunic of the mantle structure, as well as in the basal lamina that supports the dermis. The large disruption and destruction observed in those structures seems to be caused by the presence of large featureless spaces fulfilled with exudates. These also separated and destroyed the support structure of the organ and increased the spaces between the different layers of the skin (Fig. 3). Notwithstanding the apparent integrity of the muscular tissue of arms, fibbers were also observed to be disrupted and widely separated. The digestive gland has a tubular structure divided in 3 to 5 lobes formed by coelomic epithelium and collagen reticular fibbers, separated by a thin membranous tunic (Mangold and Bidder, 1989) (Fig. 4 A). A generalised and massive disaggregation of their inner components was evident on affected specimens (Fig. 4 B, C). The internal parenchyma destruction with a dispersion of their cellular components due to the enlargement of the interstitial spaces (Fig. 4 B) was most likely caused by the presence of an exudate that induces the physical expansion of the interstitial spaces and the disruption of the tissular structure of the organ. Some integrity of the collagen fibrous components remains. The digestive cells are hypertrophied showing an augmented

3. Results 3.1. Biological parameters Alive captured octopus in Matosinhos (median ML 143 mm) and Cascais (median ML 140 mm) were larger than the stranded ones (median ML 65 mm in both areas) (Table 1). A strong relation was obtained between weight (g) and ML (mm) including both stranded and alive octopus (Fig. 2). This relationship suggests that stranded individuals were growing in the same proportion weight vs. length as the specimens collected alive. Two factors may be the main cause for this biometric difference: (1) the habitat preference of recruits and adults/sub-adults

Fig. 4. Normal parenchyma of the digestive gland (A). Massive destruction of the internal parenchyma with a dispersion of their cellular components, due to the abundant presence of exudates in inter-cellular spaces (B). The most important cellular components of the tissue, the digestive cells, are hypertrophic and its shape changes to spherical with a vacuolated cytoplasm (arrows) (C).

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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significantly lower (p b 0.05) than the Pb isotopic ratios in alive individuals. 3.4. Lead concentrations and lead isotopic signature in sediments Lead concentration in sediment samples varied within a broader interval in Cascais (34–94 μg g−1) than in Matosinhos (20–25 μg g−1) (Fig. 7A). Differences between sites were less marked in Al concentrations, 3.7–6.9% in Cascais and 5.1–6.8% in Matosinhos. Normalization of Pb to Al, which is currently used to minimize the grain-size effects (Windom et al., 1989), indicates significantly (p b 0.05) higher Pb/Al ratios in Cascais (6.2–21 × 10−4) than in Matosinhos (3.2–4.6 × 10−4) (Fig. 7B). Accordingly, sediments from Matosinhos showed significantly (p b 0.05) higher 206Pb/207Pb ratios (1.189–1.207) than sediments from Cascais (1.167–1.178) (Fig. 8). No significant differences were observed for 206Pb/208Pb between areas (0.479–0.482 in Cascais and 0.480–0.485 in Matosinhos). Fig. 5. Box-plot presenting the median, 25 and 75 percentiles, minimum and maximum, Kruskal-Wallis test (KW-H) and p-values of Pb concentrations (μg g−1, dry weight) in the digestive gland of Octopus vulgaris captured alive (white boxes) and stranded (grey boxes) in the two areas of the Portuguese coast, Matosinhos and Cascais.

vacuolated cytoplasm. Their typical pyramidal shape (Westermann and Schipp, 1998) was transformed into a spherical shape. Large quantities of isolated cells are widely dispersed, together with haemocytes (Fig. 4 C).

3.5. Lead concentrations and lead isotopic signature in runoff-derived material Runoff-derived material presented a broad interval of Pb concentrations (27–85 μg g−1), as observed in Cascais, and a lower Al content (1.9–2.1%). Consequently, Pb/Al ratios (13–40 × 10−4) were higher in that material than in the sediments from Cascais and Matosinhos. Lead isotopic ratios were less radiogenic than in sediments, 1.117– 1.154 for 206Pb/207Pb and 467–0.474 for 206Pb/208Pb. 4. Discussion

3.3. Lead concentrations and lead isotopic signature in octopus 4.1. Deleterious effect caused by freshwater Median Pb concentrations in alive octopus were higher in Cascais (6.8 μg g−1) than in Matosinhos (3.1 μg g−1) (Fig. 5). Stranded octopus presented similar median Pb concentrations in the two areas, 13 and 11 μg g− 1, respectively, being one order of magnitude above (p b 0.05) the ones obtained for the alive octopus from both areas. Median Pb isotopic ratios in alive octopus were significantly (p b 0.05) higher in Matosinhos (1.179 and 0.481 for 206Pb/207Pb and 206 Pb/208Pb, respectively) than in Cascais (1.172 and 0.478 for 206 Pb/207Pb and 206Pb/208Pb, respectively) (Fig. 6). The same pattern was observed in stranded specimens, 1.176 for 206Pb/207Pb and 0.479 for 206Pb/208Pb in Matosinhos and 1.168 for 206Pb/207Pb and 0.476 for 206 Pb/208Pb in Cascais. Isotopic signature of stranded individuals was

Strong rain periods cause the input of suspended particulate matter load and high volume of fresh water into the adjacent coastal waters (Vale and Sundby, 1987; Caetano and Vale, 2003). A high precipitation event occurred just before the stranded octopus were found. The average rainfall increased from 43 mm to 216 mm within two weeks (www.ipma.pt). Under these conditions, the death of the octopus was presumably related to the rapid decrease of salinity, since cephalopods are known to show a high sensitivity to salinity conditions. It is known that freshwater is potentially lethal to cephalopods, for which their bare skin is a weak barrier between the internal hypersaline environment and the external freshwater environment (e.g. Sobrino et al.,

Fig. 6. Box-plot presenting the median, 25 and 75 percentiles, minimum and maximum, Kruskal-Wallis test (KW-H) and p-values of 206Pb/207Pb (A) and 206Pb/208Pb (B) ratios in the digestive gland of Octopus vulgaris captured alive (white boxes) and stranded (grey boxes) in the two areas of the Portuguese coast, Matosinhos and Cascais.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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Fig. 7. Box-plot presenting the median, 25 and 75 percentiles, minimum and maximum, Kruskal-Wallis test (KW-H) and p-values of Pb concentrations (μg g−1, dry weight; A), Pb/Al (×10−4; B) in the surface sediments collected in the two areas of the Portuguese coast, Matosinhos and Cascais.

2002; Vidal et al., 2014). Nonetheless, it has been questioned whether under natural conditions the input of freshwater alone would be sufficient to kill the animals, particularly considering that they are highly mobile. The main tissue structures of stranded octopus were compromised, showing severe lesions in the collagen fibbers as well as in the cells of the digestive gland. This suggests that death was likely due to multiple organ failure, related to hypertrophy and exudates that correlate with the extreme environmental alterations. The octopus skin is one of the most important interfaces between the organism and the aquatic environment for the respiratory function, water flux and ionic regulation (Budelmann et al., 1997). The skin is extremely permeable to water, which are classified as osmoconformers (Larsen et al., 2014) for their lack of ability to counter changes in the ionic status of the environment. It takes time to promote the adjustment of the whole body when changes in environment are rapid, and the ionic haemolymph composition tends to be nearly identical to the seawater (Larsen et al., 2014). In this situation, the main organs became swelled due to an intense tissular oedema caused by large amounts of exudates fluid inside the tissues. The histopathology of octopus tissues support this hypothesis since images of different organs (Figs. 3 and 4) showed that the main tissue structures were severely compromised. The accumulation of fluid

in octopus tissues, that likely was the immediate cause of their death, may concomitantly lead to the input of other solutes. In fact, strong rainfall periods cause the afflux of contaminated fresh water. The sources of metals in urban storm water runoff are numerous and the metal release mechanisms complex (Davis et al., 2001). The identification of potential sources of pollutants to the environment is of extreme importance. 4.2. Sudden Pb accumulation Lead concentrations in the digestive gland of alive Octopus vulgaris captured in the two areas ranged within the intervals observed in previous works in the Portuguese coast (Raimundo et al., 2008, 2009, 2010). The geographical differences observed for Pb concentrations in the digestive gland of octopus, higher in Cascais, are in line with the distinct levels registered in the sediments from the two areas (Fig. 7 A and B). This response to the environmental availability is consistent with findings of other investigations with cephalopods (Bustamante et al., 1998; Koyama et al., 2000; Raimundo et al., 2004; Napoleão et al., 2005). Cascais is located at the mouth of the Tagus estuary, one of the largest in Europe. Tagus receives loads of several trace elements in its river basin, which are derived from industrial discharges, urban

Fig. 8. Box-plot presenting the median, 25 and 75 percentiles, minimum and maximum, Kruskal-Wallis test (KW-H) and p-values of 206Pb/207Pb (A) and 206Pb/208Pb (B) ratios in the surface sediments collected in the two areas of the Portuguese coast, Matosinhos and Cascais.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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Fig. 9. Relationships between 206Pb/207Pb and 206Pb/208Pb ratios in urban run-off material ( ), sediments (□), alive (○) and stranded (Δ) Octopus vulgaris captured in Matosinhos (A) and Cascais (B).

effluents, and diffuse sources (Figuéres et al., 1985; Vale, 1990; Canário et al., 2005). High levels of Zn, Cu, Pb and Hg have been reported in sediments (Vale et al., 2008) and suspended particulate matter (Vale, 1990; Canário et al., 2008). The improvement of the technologies used in wastewater treatment plants, environmental control policies and the closing down of some industries resulted in a decrease input of trace elements to the coastal area (Raimundo et al., 2011). Octopus from Matosinhos were collected in the coastal area under the influence Douro estuary, which have a high freshwater input. A recent overview of Mil-Homens et al. (2014) showed that trace elements in sediments from Douro estuary are dominated by the lithology of their drainage basin; while at Tagus, a clear anthropogenic signal was evident because of urban, harbour and industrial activities. One of the most striking results observed in this study is the elevated Pb concentrations in stranded specimens, which were up to one order of magnitude above the levels reported for alive octopus captured in the Portuguese coast (Raimundo et al., 2008, 2009, 2010). The increase of Pb bioaccumulation only pointed to an enhancement in the environment however metal origin could not be identified. Results do not evidence whether the accumulation occurred while the animals were alive or after they died. Since there is evidence of the accumulation of fluids throughout the body of the animals including in internal organs, it appears that the enhanced Pb bioaccumulation initialized when the animals were still alive. Most likely, that the process occurred simultaneously with the exposure of the animals to extreme low salinity values, but likely continued after death. Although the noteworthy increase of Pb was concomitant to salinity change our results do not allow to conclude if the metal accumulation also contributed to the disruption of the osmoregulatory process. Since the Pb isotopic signature can point the origin of Pb, it was used as a tool to trace run-off material and the entrance of freshwater into octopus cells. The less radiogenic Pb isotopic signature of stranded octopus compared to bottom sediments and alive specimens, points to accumulation of Pb from a different source. The Pb isotopic signature in particulate material collected in gully pots showed lower 206Pb/207Pb ratios. Particles that constitute this material derived from urban and industrial runoff (road dust). Another source of Pb that still needs to be considered is “unleaded” gasoline that contains a concentration of this metal just below 13 mg of Pb per litre (e.g. Blackwood et al., 2003). In cities with high number of cars as the metropolitan area of European cites (Lisbon has 3.5 million inhabitants and 672 cars per thousand of inhabitants), the atmospheric input of anthropogenic Pb should be considered. The three isotope plots (Fig. 9) shows that alive octopus have higher Pb signatures (less radiogenic), while stranded organism showed lower Pb isotope ratios closer to the runoff material (collected in the gully pots). These results point to a significant input of anthropogenic Pb to the estuary that was consequently transported to the coastal area. This Pb became available to biota and

may be a severe environmental issue during this type of episodic event. The fraction of anthropogenic Pb accumulated in the digestive gland of octopus was calculated according to the equation by Sundby et al. (2005): 206

Pb=207 PbðstrandedÞ ¼ x206 Pb=207 Pbðrunoff Þ þ ð1−xÞ206 Pb=207 PbðaliveÞ

where, 206Pb/207Pb(stranded) is the median 206Pb/207Pb ratio in the digestive gland of stranded octopus, 206Pb/207Pb(runoff) is the median ratio found for the road dust material and 206Pb/207Pb(alive) is the median 206 Pb/207Pb ratio in the digestive gland of alive octopus. Accumulated anthropogenic Pb reached a maximum of 40% in the digestive gland of stranded octopus, suggesting the high input of Pb through runoff material associated with an elevated accumulation by octopus. In summary, short-term and extreme runoff event changed abruptly the salinity causing the disruption of the osmoregulation function of octopus and consequently leading to its death. This event also introduced high amounts of anthropogenic Pb in the environment that was rapidly accumulated in octopus tissues. Remarkably, levels of this metal in digestive gland increased 10 folds in a daytime scale. The Pb isotope signature in digestive gland of alive and stranded octopus confirmed that the cause of cell disruption was the freshwater derived from run-off event. Lead isotopes proved to be an adequate tool to link the input of Pb in the marine environment with this metal entrance in the cells. The study also raise the question: can the accumulation of Pb (and other solutes) occur with minor inputs of freshwater? This process may explain the erratic increase of contaminants found in same populations or groups of organisms. Acknowledgments Authors wish to acknowledge Pedro da Conceição e Sílvia Lourenço for their help with the sampling. Joana Raimundo acknowledge the grant by “Fundação para a Ciência e a Tecnologia” (SFRH/BPD/91498/ 2012). References Blackwood, D.M., Flemming, K.E., Wilkie, M.A., Nugent, J., 2003. Unleaded gasoline compositions. U.S. Patent 2003/0173250 A1, Washington, USA. Budelmann, B.U., Schipp, R., von Boletzky, S., 1997. Cephalopoda. In: Mollusca, A. (Ed.), Microscopic Anatomy of InvertebratesIn: Harrison, F.W., Kohn, A. (Eds.), vol. 6. Wiley-Liss, New York, pp. 119–414. Bustamante, P., Cherel, Y., Caurant, F., Miramand, P., 1998. Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean. Polar Biol. 19, 264–271. Bustamante, P., González, A.F., Rocha, F., Miramand, P., Guerra, A., 2008. Metal and metalloid concentrations in the giant squid Architeuthis dux from Iberian waters. Mar. Environ. Res. 66, 278–287. Caetano, M., Vale, C., 2003. Trace-elemental composition of seston and plankton along the Portuguese coast. Acta Oecol. 24, S341–S349.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121

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Caetano, M., Fonseca, N., Cesário, R., Vale, C., 2007. Mobility of Pb in salt marshes recorded by total content and stable isotopic signature. Sci. Total Environ. 380, 93–101. Canário, J., Vale, C., Caetano, M., 2005. Distribution of monomethylmercury and mercury in surface sediments of the Tagus Estuary (Portugal). Mar. Pollut. Bull. 50, 1121–1145. Canário, J., Vale, C., Nogueira, M., 2008. The pathway of mercury in contaminated waters determined by association with organic carbon (Tagus Estuary, Portugal). Appl. Geochem. 23, 519–528. Caurant, F., Aubail, A., Lahaye, V., Van Canneyt, O., Rogan, E., López, A., Addink, M., Churlaud, C., Robert, M., Bustamante, P., 2006. Lead contamination of small cetaceans in European waters - the use of stable isotopes for identifying the sources of lead exposure. Mar. Environ. Res. 62, 131–148. Davis, A.P., Shokouhian, M., Ni, S., 2001. Loading estimates of lead, copper, cadmium, and zinc in urban runoff from specific sources. Chemosphere 44, 997–1009. Faraj, A., Bez, N., 2007. Spatial considerations for the Dakhla stock of Octopus vulgaris: indicators, patterns, and fisheries interactions. ICES J. Mar. Sci. 64, 1820–1828. Ferreira, A., Cortesão, C., Castro, O., Vale, C., 1990. Accumulation of metals and organochlorines in tissues of the oyster Crassostrea angulata from the Sado Estuary. Sci. Total Environ. 97/98, 627–639. Figuéres, G., Martin, J.M., Meybeck, M., Seyler, P., 1985. A comparative study of mercury contamination in the Tagus Estuary (Portugal) and major French Estuaries (Gironde, Loire, Rhône). Estuary and coastal shelf. Science 20, 183–203. Hoven, H.M., Gaudette, H.E., Short, F.T., 1999. Isotope ratios of 206Pb/207Pb in eelgrass, Zostera marina, indicate sources of Pb in an estuary. Mar. Environ. Res. 48, 377–387. Howard, D., Smith, C., 1983. Histological techniques for marine bivalve mollusks. NOAA Technical Memorandum NMFS - F/NEC -25 Woods Hole, MA 02543 (June 83, 97pp). Islam, M.S., Tanaka, M., 2004. Impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis. Mar. Pollut. Bull. 48, 624–649. Koyama, J., Nanamori, N., Segawa, S., 2000. Bioaccumulation of waterborne and dietary cadmium by oval squid, Sepioteuthis lessoniana, and its distribution among organs. Mar. Pollut. Bull. 40, 961–967. Labonne, M., Othman, D.B., Luck, J.-M., 1998. Recent and past anthropogenic impact on a Mediterranean lagoon: lead isotope constraints from mussel shells. Appl. Geochem. 13, 885–892. Labonne, M., Othman, D.B., Luck, J.-M., 2001. Pb isotopes in mussels as tracers of metal sources and water movements in a lagoon (Thau Basin, S. France). Chem. Geol. 181, 181–191. Larsen, E.H., Deaton, L.E., Onken, H., O'Donnell, M., Grosell, M., Dantzler, W.H., Weihrauch, D., 2014. Osmoregulation and excretion. Commun. Phys. 4, 405–573. Leland, H.V., Kuwabara, J.S., 1985. Trace metals. In: Rand, G.M. (Ed.), Fundamentals of Aquatic Toxicology, Effects, Environmental Fate and Risk Assessment. Taylor and Francis, Washington, pp. 374–415. Mangold, K., 1983. Octopus vulgaris. In: Boyle, P. (Ed.), Cephalopod Life Cycles, Species Accounts vol. 1. Academic Press, United Kingdom, pp. 335–364. Mangold, K., Bidder, A.M., 1989. L'appareil digestif et la digestion. In K. Mangold. Céphalopodes. In P.P. Grassé, Traité de Zoologie Tome V. Masson, Paris, pp. 321–373. Martin, J., Flegal, A., 1975. High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc. Mar. Biol. 30, 51–55. Mil-Homens, M., Caetano, M., Costa, A.M., Lebreiro, S., Richter, T., de Stigter, H., Trancoso, M.A., Brito, P., 2013. Temporal evolution of lead isotope ratios in sediments of the central Portuguese margin: a fingerprint of human activities. Mar. Pollut. Bull. 74, 274–284. Mil-Homens, M., Vale, C., Raimundo, J., Pereira, P., Brito, P., Caetano, M., 2014. Major factors influencing the elemental composition of surface estuarine sediments: the case of 15 estuaries in Portugal. Mar. Pollut. Bull. 84, 135–146. Miramand, P., Bentley, D., 1992. Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel. Mar. Biol. 114, 407–414. Moreno, A., Lourenço, S., Pereira, J., Gaspar, M.B., Cabral, H.N., Pierce, G.J., Santos, A.M.P., 2014. Essential habitats for pre-recruit Octopus vulgaris along the Portuguese coast. Fish. Res. 152, 74–85. Morrisey, D.J., Turner, S.J., Mills, G.N., Williamson, R.B., Wise, B.E., 2003. Factors affecting the distribution of benthic macrofauna in estuaries contaminated by urban runoff. Mar. Environ. Res. 55, 113–136.

Napoleão, P., Pinheiro, T., Sousa Reis, C., 2005. Elemental characterization of tissues of Octopus vulgaris along the Portuguese coast. Sci. Total Environ. 345, 41–49. Nessim, R.B., Riad, R., 2003. Bioaccumulation of heavy metals in Octopus vulgaris from coastal waters of Alexandria (Eastern Mediterranean). Chemical. Ecology 19, 275–281. Philippe, S., Leterne, C., Lesourd, S., Courcot, L., Haack, U., Caillaud, J., 2008. Bioavailability of sediment-borne lead for ragworms (Hediste diversicolor) investigated by lead isotopes. Appl. Geogr. 23, 2923–2944. Raimundo, J., Vale, C., 2008. Partitioning of Fe, Cu, Zn, Cd and Pb concentrations among eleven tissues of Octopus vulgaris from the Portuguese coast. Cienc. Mar. 34, 297–305. Raimundo, J., Caetano, M., Vale, C., 2004. Geographical variation and partition of metals in tissues of Octopus vulgaris along the Portuguese coast. Sci. Total Environ. 325, 71–81. Raimundo, J., Vale, C., Duarte, R., Moura, I., 2008. Sub-cellular partitioning of Zn, Cu, Cd and Pb in the digestive gland of native Octopus vulgaris exposed to different metal concentrations (Portugal). Sci. Total Environ. 390, 410–416. Raimundo, J., Vale, C., Caetano, M., Cesário, R., Moura, I., 2009. Lead isotope signature in digestive gland of Octopus vulgaris and sediments from the Portuguese coast. Aquat. Biol. 6, 25–30. Raimundo, J., Costa, P.M., Vale, C., Costa, M.H., Moura, I., 2010. Metallothioneins and trace elements in digestive gland, gills, kidney and gonads of Octopus vulgaris. Comp. Biochem. Physiol. C 152, 139–146. Raimundo, J., Pereira, P., Caetano, M., Cabrita, M.T., Vale, C., 2011. Decrease of Zn, Cd and Pb concentrations in marine fish species over a decade as response to reduction of anthropogenic inputs: the example of Tagus estuary. Mar. Pollut. Bull. 62, 2854–2858. Rantala, R., Loring, D., 1975. A rapid determination of 10 elements in marine suspended matter by atomic absorption spectrophotometry. Atom Absorp. News 16, 51–52. Sansalone, J.J., Buchberger, S.G., 1997. Characterization of solid and metal element distributions in urban highway stormwater. Water Sci. Technol. 36, 155–160. Seixas, S., Pierce, G., 2005. Vanadium, rubidium and potassium in Octopus vulgaris (Mollusca: Cephalopoda). Sci. Mar. 69, 215–222. Sobrino, I., Silva, L., Bellido, J.M., Ramos, F., 2002. Rainfall, river discharges and sea temperature as factors affecting abundance of two coastal benthic cephalopod species in the Gulf of Cádiz (SW Spain). Bull. Mar. Sci. 71, 851–865. Sundby, B., Caetano, M., Vale, C., Gobeil, C., Luther, G., Nuzzio, D., 2005. Root-induced cycling of lead in salt marsh sediments. Environ. Sci. Technol. 39, 2080–2086. Vale, C., 1990. Temporal variations of particulate metals in the Tagus River Estuary. Sci. Total Environ. 97/98, 137–154. Vale, C., Sundby, B., 1987. Suspended sediment fluctuations in the Tagus estuary on semidiurnal and fortnightly time scales. Estuarine and coastal shelf. Science 25, 495–508. Vale, C., Canário, J., Caetano, M., Lavrado, J., Brito, P., 2008. Estimation of anthropogenic quantities of elements in surface sediments of the Tagus Estuary (Portugal). Mar. Pollut. Bull. 56, 1364–1367. van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 13, 57–149. Vidal, E.A.G., Villanueva, R., Andrade, J.P., Gleadall, I.G., Iglesias, J., Koueta, N., Rosas, C., Segawa, S., Grasse, B., Franco-Santos, R.M., Albertin, C.B., Caamal-Monsreal, C., Chimal, M.E., Edsinger-Gonzales, E., Gallardo, P., Le Pabic, C., Pascual, C., Roumbedakis, K., Wood, J., 2014. Cephalopod culture: current status of main biological models and research priorities. Adv. Mar. Biol. 67, 1–98. Walker, W.J., McNutt, R.P., Maslanka, C.K., 1999. The potential contribution of urban runoff to surface sediments of the Passaic river: sources and chemical characteristics. Chemosphere 38, 363–377. Westermann, B., Schipp, R., 1998. Cytological and enzyme-histochemical investigations on the digestive organs of Nautilus pompilius (Cephalopoda, Tetrabranchiata). Cell Tissue Res. 293, 327–336. Windom, H.L., Schropp, S.J., Calder, F.D., Ryan, J.D., Smith, R.G., Burney, L.C., Lewis, F.G., Rawlinson, C.H., 1989. Natural trace-metal concentrations in estuarine and coastal marine-sediments of the Southeastern United-States. Environ. Sci. Technol. 23, 314–320. Wu, J.S., Holman, R.E., Dorney, J.R., 1996. Systematic evaluation of pollutant removal by urban wet detention ponds. J. Environ. Eng. ASCE 122, 983–988. Wu, J.S., AlIan, C.A., Saunders, C.J.A., Evett, J.B., 1998. Characterization and pollutant loading estimation for highway runoff. J. Environ. Eng. ASCE 124, 584–592.

Please cite this article as: Raimundo, J., et al., Abnormal mortality of octopus after a storm water event: Accumulated lead and lead isotopes as fingerprints, Sci Total Environ (2016), http://dx.doi.org/10.1016/j.scitotenv.2016.12.121