Polycyclic aromatic hydrocarbons (PAHs) in mussels (Modiolus capax) from sites with increasing anthropogenic impact in La Paz Bay, Gulf of California

Regional Studies in Marine Science 33 (2020) 100948

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Polycyclic aromatic hydrocarbons (PAHs) in mussels (Modiolus capax) from sites with increasing anthropogenic impact in La Paz Bay, Gulf of California Nefertiti Taydé Roldán-Wong a , Karen A. Kidd b ,1 , Bertha Patricia Ceballos-Vázquez a,b , ∗ Alma Rosa Rivera-Camacho a , Marcial Arellano-Martínez a,b , a

Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas. Av. Instituto Politécnico Nacional s/n, Col. Playa Palo de Santa Rita. C.P. 23096. La Paz, Baja California Sur, Mexico b Canadian Rivers Institute & Biology Department, University of New Brunswick, 100 Tucker Park Road, P.O. Box 5050, Saint John, New Brunswick, Canada, E2L 4L5

article

info

Article history: Received 27 March 2019 Received in revised form 6 November 2019 Accepted 7 November 2019 Available online 11 November 2019 Keywords: Bivalves Biomonitors Marine pollution Petrogenic hydrocarbons Benzo(a)pyrene

a b s t r a c t The composition, distribution and source of 16 polycyclic aromatic hydrocarbons (PAHs) in surface sediments and tissues of the mussel Modiolus capax from three sites with increasing anthropogenic impact in La Paz Bay on the Gulf of California, were determined. Levels of total PAHs in sediments (18.9-94.5 ng/g dw) were below the effect range low for marine sediments at all sites. PAHs in mussels (147.01-271.09) were higher than in sediments and similar to mussels from other moderately contaminated sites. The origin of PAHs was predominantly petrogenic, attributed to port activities and a thermoelectric plant. The mussels of Las Pacas and Pichilingue showed the lowest condition indexes and some organisms with the presence of benzo(a)pyrene in their tissues, which is among the most toxic hydrocarbons and may represent a risk to human health. However, this study only represents a baseline for future studies, which are necessary to dismiss risks to human health and the ecosystem. © 2019 Published by Elsevier B.V.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are among the most common pollutants in coastal marine environments, especially in the most densely populated and urbanized areas (Hylland, 2006; Balcıoğlu, 2016). PAHs can occur naturally, however, most come from anthropogenic sources such as the combustion or spills of fossil fuels (petrogenic), incomplete combustion of organic matter (pyrolytic) and degradation of biogenic precursors (diagenesis) (Neff, 2002; Hylland, 2006; Balcıoğlu, 2016). In environmental studies, PAHs are considered priority pollutants since they act as potential endocrine, genotoxic, carcinogenic and mutagenic disruptors (IARC, 2002; Mastandrea et al., 2005; Balcıoğlu, 2016). Bivalve molluscs, especially mussels, are considered good biomonitors of environmental contamination because of their ∗ Corresponding author at: Instituto Politécnico Nacional, Centro Interdisciplinario de Ciencias Marinas. Av. Instituto Politécnico Nacional s/n, Col. Playa Palo de Santa Rita. C.P. 23096. La Paz, Baja California Sur, Mexico. E-mail addresses: [email protected] (N.T. Roldán-Wong), [email protected], [email protected] (K.A. Kidd), [email protected] (B.P. Ceballos-Vázquez), [email protected] (A.R. Rivera-Camacho), [email protected] (M. Arellano-Martínez). 1 Present address: Department of Biology & School of Geography and Earth Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada. L8S 4K1. https://doi.org/10.1016/j.rsma.2019.100948 2352-4855/© 2019 Published by Elsevier B.V.

sedentary life mode, broad distribution, high abundance, filtering feeding mode, high tolerance to environmental changes and their ability to accumulate high concentrations of chemicals in their tissues (e.g. Webster et al., 2006; Sureda et al., 2011; Olenycz et al., 2015). In particular, the mussel Modiolus capax is a bivalve of commercial importance along the Mexican Pacific coast, including the Gulf of California. This species inhabits coastal environments to a maximum depth of 50 m, on hard and soft substrates (rocks, stones, mud) (Poutiers, 1995), and its distribution includes La Paz Bay, considered a low pollution area (RodríguezCastañeda et al., 2006; Ruelas-Inzunza et al., 2013). However, this bay is under increasing influences from wastewater discharges and port activities from the City of La Paz, capital of the State of Baja California Sur, which has increasing population growth due, mainly, to its growing tourism. Although it is well known that these activities increase the levels of PAHs in the marine environment (Latimer and Zheng, 2003), the only such assessment was done over two decades ago in Pichilingue Port (Botello et al., 2002). The main objective of this study was to examine the levels of PAHs in the bivalve M. capax and in superficial sediments from three sites with different levels of anthropogenic activity in La Paz Bay.

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2. Material and methods 2.1. Sample collection During March 2014, samples of surficial marine sediments and mussels M. capax were collected from three sites in La Paz Bay: (a) Pichilingue, a port with traffic of deep draft vessels, (b) Punta Prieta, a site near a thermoelectric plant, a hydrocarbon warehouse and a marina, and (c) Las Pacas, a fishing area without major anthropogenic activities (Fig. 1). Sediment samples were collected by free diving from the subtidal zone (2–4 m depth) by scarping the surface sediment using a plastic spoon (maximum depth of 10 cm). All collected sediment samples were placed in a clean bag (seawater filtered, and all organisms removed), mixed, refrigerated, transported to the laboratory and frozen at −80 ◦ C until sample processing. All sediment collections were performed in the same way at the three sampling sites (one sample per site). The mussels M. capax were collected at the three sites during sediment sampling (between 34 and 35 per site), the shell surfaces were washed with clean seawater at the time of collection, and the organisms were placed in clean plastic bags, refrigerated, and transported to the laboratory. Shell length (±1 mm), shell height (±1 mm), total weight (±0.1 g) and shell weight (±0.1 g) were measured for each mussel. The condition index was calculated as the relative ratio (percentage) between the wet weight without shell and total weight (Mouneyrac et al., 2008). Soft tissues were removed from the shells and frozen at −80 ◦ C until sample processing. 2.2. Sample processing Soft tissues and sediments were lyophilized (Labconco FreeZone 12) and ground using a pre-cleaned mortar and pestle. Percent moistures were determined for mussels. PAH analyses were done using 10 g of dried sediments or tissues per sample; to achieve adequate masses, mussels were pooled within sites (n = 6 pooled samples/site, between 5 to 6 mussels per pool). Analyses were done following US EPA’s standard testing protocol 8270C (US EPA, 1996) and all solvents were Optima grade (Fisher Scientific). Tissues and sediments were extracted using an Accelerated Solvent Extraction system (ASE 3009; Dionex), with 50:50 dichloromethane (DCM): hexane, and then cleaned up using a gel permeation column (J2 Scientific Automated Gel Permeation System) and 50:50 DCM: hexane. Samples were then concentrated into hexane using a Büchi rotavapor and nitrogen evaporator and then solvent exchanged to isooctane (final volume of 1 mL). The concentrated extracts were run on a gas chromatograph-mass spectrometer (Agilent 6890/5975B GC–MS) using a DB-5 column (60 m × 0.25 mm × 0.25 µm; Agilent J&W) in single ion monitoring mode and quantified using internal standard calibration. Method detection limits (MDLs) were determined by running 8 low level spike samples (5x higher than the expected MDL) through the entire process. The t-value (n = 8, 95%) was multiplied by the standard deviation of the 8 runs to determine the MDL for each PAH. The MDL of the total PAHs was determined by taking the square root of the sum of squares of the individual PAH MDLs. The 16 priority PAHs (Keith, 1979) were quantified and included: naphthalene, acenaphthene, acenaphthylene, anthracene, fluorene, phenanthrene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(k)fluoranthene, benzo(b)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, and benzo(g,h,i)perylene. Data are reported as individual PAHs and values above MDLs were summed for total PAHs as the average value obtained from sediments replicates (between <0.01 and 0.01 = 0.005). Data below the detection limit are reported as <0.01.

QA/QC procedures for each batch of samples included a method blank (MB; Ottawa Sand, Fisher Scientific), a method spike (MS; sand and 100 µL spike SK20), a certified reference material (CRM; NIST SRM 1941b Organics in Marine Sediments) and a sample duplicate. For each PAH and each internal standard, a target ion was used for quantification and 2 others were used as qualifier ions, and these spectra was compared against scans of a pure standard, confirmed on the NIST library, to avoid false positives. Surrogates (nitrobenzene-d5, 2-fluorobiphenyl and pterphenyl; SPEX Certiprep, Metuchen, NJ, USA) were added to samples prior to extraction (recoveries averaged 92.4 ± 23.3%; SD). CRM recoveries averaged 90.8 ± 19.0% (SD; n = 3) across individual PAHs. MDLs were <0.01 mg/kg for individual PAHs and were <0.04 mg/kg for total PAHs. 2.3. Data processing and statistical analyses To determine which hydrocarbons represent a greater contribution to total PAHs (Σ PAHs) by site, their relative importance was calculated (% Importance = [average concentration of each PAH in sediments or mussels * 100]/Σ PAHs). The probable origin (pyrogenic or petrogenic) of the PAHs was determined according to pattern in mussel tissues of each site, for this purpose the following isomeric relationships were calculated: fluoranthene/pyrene; fluoranthene/(fluoranthene + pyrene); chrysene/benzo(a)anthracene; and benzo(a)anthracene/(benzo(a) anthracene + chrysene) (Baumard et al., 1998; Yunker et al., 2002; Soclo et al., 2008). The resulting values were compared with the ranges reported in the literature. To determine if there were differences in size, weight, condition index or concentration of PAHs among the mussels of each locality, a one-way analysis of variance (one-way ANOVA; level of significance al p < 0.005; factor: locality) was applied for each variable. All statistical analyses were carried out using STATISTICA 6.0 (StatSoft, Inc. Tulsa, OK, USA). The concentrations of PAHs in sediments could not be compared statistically because only one sample of sediment from each site was processed. 3. Results 3.1. Biometric variables The biometric data and condition index of M. capax from each sampling site are shown in Table 1. The mussels from Pichilingue presented larger sizes and weights, while the organisms of Las Pacas were the smallest. The mussels of Punta Prieta showed the highest condition index. 3.2. Concentration and composition of polycyclic aromatic hydrocarbons Sediment levels of PAHs and dominant congeners differed among the three sites in this study. At Las Pacas, only phenanthrene, pyrene and chrysene were found at values of 6.95, 6.77 and 5.92 ng/g dw, respectively (Table 2). A similarly low number of PAHs – fluorene, phenanthrene and naphthalene only – were found at the Pichilingue site at levels just above the MDLs. In contrast, 8 PAHs were measured in sediments from Punta Prieta, the most contaminated site, and they ranged in concentrations from 5.86 to 17.02 ng/g dw, with TPAHs of 94.54 ng/g dw. In contrast to the sediments, higher levels and numbers of PAH congeners were found in the mussels but they varied among animals both within and among sites (Table 2). Most mussels tended to have naphthalene, fluoranthene, pyrene, and chrysene, with pyrene at the highest levels of all these individual PAHs (average concentrations from 80.66 to 159.89 ng/g dw). Some animals also

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Fig. 1. Sampling sites for surficial marine sediments and mussels Modiolus capax in La Paz Bay, Gulf of California: two sites were near the City of La Paz (Pichilingue and Punta Prieta) and one was away from any major anthropogenic activities (Las Pacas)

had phenanthrene (17.73–34.88 ng/g dw), benzo(a)anthracene (22.45–24.26 ng/g dw) and benzo(a)pyrene (57.46–97.64 ng/g dw) in two of the three sampling sites. Total PAHs in mussels ranged from 97 to 209 ng/g dw at Las Pacas, 37 to 355 ng/g dw at Punta Prieta and 205 to 321 ng/g dw at Pichilingue. Interestingly the site with the highest sediment levels did not also have the highest PAHs in mussels. 3.3. Percentage importance of polycyclic aromatic hydrocarbons The percentage importance of each PAH in sediments and mussels from each location is shown in Fig. 2 along with the level of carcinogenicity according to the International Agency for Research on Cancer (IARC, 2002). The PAH that showed the highest contribution across sediments and mussels of all locations was pyrene (17 to 44%). Naphthalene was the most abundant in sediments from Pichilingue (40%), phenanthrene in sediments from Las Pacas and Pichilingue (35 and 31%, respectively) and benzo(a)pyrene in mussels from Las Pacas and Pichilingue (24%, in both cases). The PAHs with higher percentage importance are classified as less carcinogenic, except for benzo(a)pyrene, which is the PAH with the highest carcinogenicity, in mussels. 3.4. Origin of PAHs by sampling site The isomeric relationships between PAHs in mussels are shown in Table 3. Most relations showed values related with a petrogenic origin, except for chrysene/benzo(a)anthracene in Las Pacas and benzo(a)anthracene/(benzo(a)anthracene + chrysene) in Las Pacas and Punta Prieta, where the values corresponded to a pyrolytic origin.

4. Discussion The concentrations of PAHs in sediments (18–94 ng/g dw) in the three sampling sites were below the effect range low for marine and estuarine sediments (4022 ng/g dw) (Long et al., 1995). According to this, the risk of toxic effects on the marine biota in these areas is low or unlikely. The highest values were found in sediments of Punta Prieta, which can be attributed to its proximity to a thermoelectric plant and a fuel storage facility. The mussels M. capax had total PAH values that were between 1 and 17 times higher than sediments, with mean concentrations ranging from 147 to 271 ng/g dw (Table 2). These levels correspond to moderately contaminated sites according to the classification of Baumard et al. (1998) for mussels, and are within the range reported in bivalves that live near urban areas and ports, such as Ruditapes philippinarum, Mytilus galloprovincialis and Crassostrea gigas in Japan (134–450 ng/g dw) (Onozato et al., 2016); M. galloprovincialis in the Mediterranean (25–390 ng/g dw) (Baumard et al., 1998); and R. philippinarum, Perna viridis and M. edulis in China (276–939 ng/g dw) (Fang et al., 2009; Haiqing et al., 2009). In fact, these levels are also in accordance with those found in bivalves from different locations (up to 339 sites) in the Gulf of Mexico, Central and South America, and the USA coast, measured by the ‘‘NOAA’s National Status & Trends’’ and the ‘‘International Mussel Watch’’ programs, where most sites showed concentrations ranging between 77 and 1100 ng/g dw (Sericano et al., 1995; O’Connor, 2002). However, these large-scale studies showed higher total concentrations due to the presence of higher average concentrations of high molecular weight hydrocarbons (24–770 ng/g dw), (O’Connor, 2002) compared to our results (33–51 ng/g dw, Table 2). Our results differ considerably from what was observed by Botello et al. (2002) in C. palmula from Pichilingue (Total average

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Table 1 Biometric variables and condition index of Modiolus capax from three sites in La Paz Bay, Gulf of California. Mean values ±standard deviation. Letters (a, b, c) indicate significant differences between sites (p > 0.005). N = number of organisms. Biometric variables

Sampling sites Las Pacas

Punta Prieta

Pools

Shell length (cm)

5.0 5.3 5.2 5.4 5.3 5.3

± ± ± ± ± ±

0.6 0.4 0.4 0.3 0.4 0.4

Shell height (cm)

9.0 9.4 9.4 9.1 9.2 9.1

± ± ± ± ± ±

0.7 0.9 0.7 0.4 0.9 0.7

Total weight (g)

65.0 64.7 62.4 59.1 75.6 59.4

± ± ± ± ± ±

25.2 19.9 20.4 18.3 21.7 25.5

Soft tissues weight (g)

11.6 12.1 13.7 11.8 12.1 11.9

± ± ± ± ± ±

3.1 1.7 4.0 1.9 2.3 1.8

Condition index

19.4 19.6 23.4 20.8 18.4 23.4

± ± ± ± ± ±

6.6 3.9 8.1 4.7 10.1 10.4

N

Mean

Pools

5.3 ± 0.4a

5.5 5.3 5.3 5.5 5.0 5.4

9.2 ± 0.7a

9.9 ± 0.6 10.0 ± 0.7 9.9 ± 0.6 10.0 ± 0.5 10.0 ± 0.3 9.9 ± 0.7

64.4 ± 21.1a

84.0 88.1 89.9 84.7 86.4 87.4

± ± ± ± ± ±

16.8 13.5 24.8 19.4 8.8 18.6

12.2 ± 2.4a

18.8 18.2 18.9 19.4 21.0 19.3

± ± ± ± ± ±

5.0 5.2 3.0 3.2 1.7 3.5

20.8 ± 7.4ab

22.5 20.6 22.0 23.4 24.5 22.6

± ± ± ± ± ±

4.2 3.9 5.0 4.2 3.6 4.6

± ± ± ± ± ±

Pichilingue

0.5 0.3 0.3 0.3 0.4 0.5

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Mean

Pools

5.3 ± 0.4a

5.7 5.7 5.4 5.6 5.6 5.6

9.9 ± 0.5b

10.7 ± 0.3 9.9 ± 0.8 9.8 ± 0.7 9.9 ± 0.6 10.4 ± 0.8 10.1 ± 0.7

10.1 ± 0.7b

86.7 ± 16.7b

144.1 123.2 109.6 115.9 126.4 120.9

123.1 ± 24.6c

19.2 ± 3.6b

25.0 20.6 23.1 22.8 21.5 20.5

± ± ± ± ± ±

4.8 5.9 3.4 3.9 4.5 3.4

22.1 ± 4.4c

22.5 ± 4.1a

17.4 16.5 21.6 19.8 17.0 17.4

± ± ± ± ± ±

1.8 2.2 5.0 1.9 1.1 3.5

18.2 ± 3.1b

± ± ± ± ± ±

Mean 0.2 0.2 0.4 0.4 0.5 0.3

± ± ± ± ± ±

5.6 ± 0.3b

29.2 21.0 21.6 19.9 23.0 29.7

35

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Table 2 Total and individual average concentrations (ng/g dw) of polycyclic aromatic hydrocarbons (PAHs) in sediments (n = 1/site) and mussels Modiolus capax (n = 6 pools/site) from three sites in La Paz Bay, Gulf of California (mean ±SD). Letters (a, b, c) indicate the significant differences between sites (p < 0.005). Values below the detection limit are reported as <0.01. Values without standard deviation indicate that the hydrocarbon was only detected in one of the six sample pools. PAHsa

Sampling site Las Pacas

Lower molecular weight

Higher molecular weight

Σ Low molecular weight Σ High molecular weight Σ PAHs a

Punta Prieta

Pichilingue

Sediments

Mussels

Sediments

Mussels

Sediments

Mussels

Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Fluoranthene Pyrene

<0.01 <0.01 <0.01 <0.01

3.06 ± 1.21a 4.45 ± 0.04 <0.01 <0.01 17.73 ± 2.14a 14.98 ± 2.23a 80.66 ± 8.22a

<0.01 <0.01 <0.01 5.86 10.27 17.02 15.92

21.79 ± 13.30c <0.01 15.30 <0.01 <0.01 28.17 ± 10.23b 116.26 ± 47.48b

7.56 <0.01 <0.01 5.40 5.94 <0.01 <0.01

14.07 ± 2.19b <0.01 <0.01 <0.01 34.88 ± 5.93b 31.50 ± 2.85b 159.89 ± 36.63c

Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd) pyrene Benzo(g,h,i)perylene

<0.01 5.92 <0.01 <0.01 <0.01 <0.01 <0.01

19.41 ± 10.86 22.86 ± 0.93a 2.24 11.19 57.46 <0.01 <0.01

9.72 14.29 <0.01 <0.01 <0.01 11.71 9.76

22.45 29.65 ± 12.30b <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01

<0.01 36.59 ± 7.51c <0.01 <0.01 87.64 <0.01 <0.01

13.72 5.92 19.64

99.98 ± 4.15 47.04 ± 35.60 147.01 ± 38.47

49.06 45.48 94.54

140 ± 94.77 33.39 ± 15.17 173.39 ± 106.03

18.9 <0.01 18.90

219.90 ± 38.08 51.20 ± 30.85 271.09 ± 47.52

6.95 <0.01 6.77

Anthracene and dibenzo(a, h)anthracene are not shown as their values were below the limit of detection in all samples.

concentration 22,860 ng/g dw). This author reported levels most commonly found in bivalves from highly polluted coastal areas near to large cities, industries and maritime traffic, or in animals exposed to large oil spills. The concentrations reported by Botello et al. (2002) are closer to those found in M. edulis from Shanghai, China (>3000 ng/g dw) (Fung et al., 2004), of P. viridis from Victoria Harbor, Hong Kong (up to 22,858 ng/g dw) (Fang et al.,

2009), and of Saxidomus giganteus, M. edulis and Clinocardium nuttallii from British Columbia, Canada (up to 3,980 ng/g dw) (Thompson et al., 2017). The differences between total PAHs in our study and those of Botello et al. (2002) may be due to reduced inputs of PAHs at this site in recent years or differences in habitat use by the two species of mussels.

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Fig. 2. Carcinogenicity (gray scale in the outer layer) and average percentage importance (radially towards each name) of individual polycyclic aromatic hydrocarbons (PAHs) relative to total PAHs in sediments (solid line) and mussels Modiolus capax (dotted line) from three locations in La Paz Bay, Gulf of California. *Classification of carcinogenicity according the International Agency for Research on Cancer (IARC, 2002). Table 3 Isomeric relationships between polycyclic aromatic hydrocarbons (PAHs) present in Modiolus capax tissues from three locations in La Paz Bay, Gulf of California. LMW: low molecular weight, HMW: high molecular weight. The values attributable to petrogenic origin are highlighted in bold.

Mussels Las Pacas Punta Prieta Pichilingue

Fluoranthene/ pyrene

Fluoranthene/ (fluoranthene + pyrene)

Chrysene/ benzo(a) anthracene

Benzo(a)anthracene/ (benzo(a)anthracene + chrysene)

Σ LMW/Σ HMW

0.19 0.24 0.20

0.16 0.20 0.16

0.94 1.32 –

0.51 0.43 –

2.24 4.24 5.10

<0.40c >0.50c

>1b <1b

<0.20c >0.35c

>1d <1d

Value range by source Petrogenic <1a Pyrogenic >1a a

Baumard et al. (1998). Soclo et al. (2008). c Yunker et al. (2002). d Zhang et al. (2008). b

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N.T. Roldán-Wong, K.A. Kidd, B.P. Ceballos-Vázquez et al. / Regional Studies in Marine Science 33 (2020) 100948

In the present study, the most abundant PAHs found in sediments and mussels from all the localities were those of low molecular weight, especially naphthalene, phenanthrene, fluoranthene and pyrene (Fig. 2). These compounds degrade more rapidly than those of high molecular weight (Simó et al., 1997), especially in regions with high temperatures and long photoperiods, as is the case of La Paz Bay, because they undergo photo-oxidation (Mastandrea et al., 2005). This could explain why some PAHs that were detected in mussels were not found in the sediments at the same sites (Table 2, i.e. naphthalene, acenaphthylene, acenaphthene and fluoranthene), since they could have been degraded in the sediment but bioaccumulated by the mussels. Additionally, bivalves tend to present higher bioaccumulation of low molecular weight PAHs (Thompson et al., 2000; Thorsen et al., 2004). On the other hand, some hydrocarbons were detected in sediments, but not in the mussels at the same location (Table 2, i.e. fluorene, indene(1,2,3-d)pyrene and benzo(g, h, i)perylene), which has been reported in other bivalves (Oros and Ross, 2005; Arias et al., 2009) and can be attributed to the low bioavailability of PAHs present in sediments (Pruell et al., 1987). Regarding the most abundant hydrocarbons, naphthalene represented 40% of the total PAHs in sediments of Pichilingue and was found at levels 10 times higher in mussels of Punta Prieta (9.3%) than in those of Las Pacas (1.3%). Likewise, phenanthrene and fluoranthene were present in higher concentrations in mussels of Punta Prieta and Pichilingue than in those of Las Pacas (Table 2). These three hydrocarbons are commonly associated with petrogenic sources, naphthalene is associated with gasoline discharges, and phenanthrene and fluoranthene are related to diesel discharges and motor oils (Neff, 2002). Our results indicate discharges of unburned petroleum products in Punta Prieta and Pichilingue, which is also confirmed by the isomeric relationships of PAHs in mussels (Table 3). Pyrene was the most representative hydrocarbon by percentage and the most abundant in all locations (Fig. 2), especially in mussels of Pichilingue. This hydrocarbon is generally associated pyrolytic sources, especially the incomplete combustion of fossil hydrocarbons typical of port activities and refineries (Bayona et al., 1993). The high concentrations of pyrene in Pichilingue can be attributed then to the high maritime activity and traffic of deep draft vessels in the area. Regarding the risks to human health, most of the PAHs measured have low carcinogenic potential except for benzo(a)pyrene, which has been listed as a category 1 human carcinogen (highest level) by the International Agency for Research on Cancer (IARC, 2002) (Fig. 2). This hydrocarbon is present in most sources of contamination by PAHs and is used as a reference for environmental and public health studies because of its extended genotoxic, mutagenic and carcinogenic effects (Mastandrea et al., 2005). Our results showed that benzo(a)pyrene represents 24% of the total PAHs detected in mussels from Las Pacas and Pichilingue (Fig. 2); however, it is important to note that, this hydrocarbon was detected in only one of the six pools (between five and six organisms of the 34 to 35 of each site), while the rest of the samples were below of the detection limit. Notwithstanding, the European Regulation 208/2005/EC fixed the maximum admissible concentrations for benzo(a)pyrene in bivalve molluscs at 10 ng/g ww (EC, 2005), and we found mean levels of 57.46 and 87.64 ng/g dw in mussels from Las Pacas and Pichilingue respectively (Table 2), which equals to 9.65 and 16.46 ng/g in wet weight (wet weight values were calculated using the moisture content and the moisture factor of each sample). Studies conducted with other bivalves have reported lower values of benzo(a)pyrene: between 3.5 and 7.62 ng/g dw in Crassostrea iridiscens from Oaxaca, Mexico (Botello et al., 1995); between <0.1 and 40 ng/g dw in Crassostrea virginica from GA, USA

(Senthil Kumar et al., 2008); between 11 and 33 ng/g dw in R. philippinarum, M. galloprovincialis, and C. gigas from the Pacific Coast of Japan (Onozato et al., 2016); while, other studies have reported higher levels in heavily polluted areas: between 79.9 and 313.3 ng/g dw, and monthly averages of up to 967.1 ng/g dw in P. viridis from Hong Kong (Fang et al., 2009), where the authors considered a possible risk to human health. The mussels of Pichilingue were the largest, while those of Las Pacas the smallest (Table 1), although this site presented lower levels of contamination. This can be attributed to the higher fishing pressure in Las Pacas, which is known to cause a decrease in the maximum sizes of bivalves and other organisms (Enberg et al., 2012; Munroe et al., 2016). Additionally, the mussels of these two localities had lower condition index than those of Punta Prieta, indicating a lower general overall health. This can be attributed to the fact that the sampling area in Las Pacas is exposed to seasonal freshwater runoff, which can cause sudden changes in the salinity of the area and thereby affect the growth of these bivalves, as has been reported for Mytilus edulis (Hiebenthal et al., 2012; Riisgård et al., 2012). Another factor that could be related to the low condition index is the presence of high concentrations of benzo(a)pyrene, which was detected in higher concentrations than in Punta Prieta and as mentioned above, it is the PAH of greatest toxicity among the hydrocarbons examined herein. However, more studies are needed to evaluate whether this or other contaminants have a negative effect on the health of bivalves in La Paz Bay. 5. Conclusions The levels of PAHs found in sediments and mussels M. capax of the three study sites indicate that La Paz Bay is moderately contaminated by PAHs and the composition suggests local, mainly petrogenic sources related to port activities. The detection of high levels of benzo(a)pyrene in a single sample of organisms from both, Las Pacas and Pichilingue (where the mussels showed the lowest condition indexes) is an alert that should be investigated since it is considered one of the most toxic PAHs. Nevertheless, this study only represents a baseline for future studies, which are necessary to dismiss concerns about risks to human health and the ecosystem. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was made possible through funding from the Natural Sciences and Engineering Research Council of Canada Discovery grant, the Canada Research Chairs program, the Canadian Rivers Institute, the Mexican projects 2023 and 20195375 of the Secretaría de Investigación y Posgrado (SIP), and grants from COFAA, Mexico, EDI, Mexico, and SNI-CONACyT, Mexico. We thank Angella Mercer, PTech for her help with the laboratory analysis. References Arias, A.H., Spetter, C.V., Freije, R.H., Marcovecchio, J.E., 2009. Polycyclic aromatic hydrocarbons in water, mussels (Brachidontes sp. Tagelus sp.) and fish (Odontesthes sp.) from Bahia Blanca Estuary Argentina. Estuar. Coast. Shelf Sci. 85 (1), 67–81. http://dx.doi.org/10.1016/j.ecss.2009.06.008. Balcıoğlu, E.B., 2016. Potential effects of polycyclic aromatic hydrocarbons (PAHs) in marine foods on human health: a critical review. Toxin Rev. 35 (3–4), 98–105. http://dx.doi.org/10.1080/15569543.2016.1201513.

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