Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy

MPB-07424; No of Pages 11 Marine Pollution Bulletin xxx (2016) xxx–xxx

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Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy Carolyn S. Bentivegna a,⁎, Chelsea R. DeFelice a, Wyatt R. Murphy b a b

Department of Biological Science, Seton Hall University, 400 South Orange Avenue, South Orange, NJ 07079, United States Department of Chemistry and Biochemistry, Seton Hall University, 400 South Orange Avenue, South Orange, NJ 07079, United States

a r t i c l e

i n f o

Article history: Received 1 September 2015 Received in revised form 17 December 2015 Accepted 16 January 2016 Available online xxxx Keywords: Menhaden PAH Excitation–emission matrix spectroscopy Raw fish oil Hurricane Sandy

a b s t r a c t The impact of Hurricane Sandy (October 29, 2012) on PAH exposure was investigated in adult Atlantic menhaden (Brevoortia tyrannus) collected along the NJ coast. Collections were made in August, September and/or October of 2011, 2012 and 2013. PAHs were monitored in raw fish oil using excitation–emission matrix (EEM) spectroscopy. Results showed that raw fish oils had relatively high levels of high molecular weight, PAH-like compounds (173 to 24,421 ng/mL) compared to values reported for bile in other species. EEM profiles resembled that of crude oil and excluded matrix interference by some common biological molecules that also fluoresce. Concentrations and EEM profiles varied by collection; however, collection ship, month, year and fish size did not account for the data. Replicates showed that fish from the same catch had similar PAH exposure. Overall, Hurricane Sandy did not alter body burdens of PAHs in raw fish oil of menhaden. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Hurricanes and major storms can disturb and redistribute sediment reservoirs of legacy contaminants such as polycyclic aromatic hydrocarbons (PAHs) (Mitra et al., 2009). PAHs typically enter the aquatic environment from crude oil spills and the burning and/or processing of fossil fuels (Neff, 1979). High molecular weight PAHs (4–5 rings) can remain in the environment for years due to chronic pollution (Myers et al., 2008) or episodic oil spills (Jewetta et al., 2002). Hurricane Sandy's storm surge on October 29, 2010 was strongest in the NJ/NY Harbor area. This area has a long history of PAH contamination resulting from urbanization (Crawford et al., 1994). Major sources of PAHs to the harbor are from coal tar, creosote, and motor oil (Valle et al., 2007). A study following the World Trade Center collapse (2001–2004) shows that many PAH sediment and tissue concentrations exceed the national 75th percentile established by the Mussel Watch Program, indicating that the Hudson River Estuary is one of the most PAH contaminated areas in the country (Lauenstein and Kimbrough, 2007). This level of contamination is associated with impaired reproductive health in killifish (Fundulus heteroclitus) collected in the same region (Bugel et al., 2010). In addition, some PAHs are toxic to fish and have been associated with genotoxicity (Harvey et al., 1999, Aas et al., 2000a), neoplasms (Vogelbein et al., 1999) and tumor prevalence (Pinkney et al., 2001). ⁎ Corresponding author. E-mail addresses: [email protected] (C.S. Bentivegna), [email protected] (C.R. DeFelice), [email protected] (W.R. Murphy).

Hurricane Sandy may have suspended and redistributed PAHs from urban waterways into NJ/NY coastal ecosystems. Given the high levels of PAHs and documented health effects, the potential increase in PAH bioavailability to marine organisms is of concern. Menhaden is an oily marine fish inhabiting estuaries and coastlines of the Atlantic Ocean and Gulf of Mexico (GOM) (Ahrenholz, 1991). They are an important prey species as well as economically valuable (Franklin, 2007). Menhaden support the purse-seine and bait industries with landings reaching 131,000 metric tons in 2014 (NOAA, 2015). The main uses of menhaden are for fish oil and animal feed. Recent evidence shows PAH-like compounds in the raw fish oil of menhaden (Pena et al., 2015). Particulates derived from weathered petrogenic PAH mixtures were found in the heart and stomach muscularis of GOM menhaden, likely leading to localized cellular damage from chronic release of bioavailable PAHs (Millemann et al., 2015). Contaminated aquatic animals can be a vector for PAHs entering the human food chain. Fish oils and proteins contribute to Persistent Organic Pollutants (POPs) such as PAHs and polychlorinated biphenyls in animal feed and other food products (Yebra-Pimentel et al., 2013, 2014). PAHs and their hydroxylated metabolites were detected in infant food (Rey-Salgueiro et al., 2009). PAH levels in menhaden raw fish oil before and after Hurricane Sandy may reflect the extent of the storm's effect on contaminant availability and transport. Having this knowledge is important for managing the health of NJ's marine species and food industries. In environmental samples, molecules detected by fluorescence are called fluorescent aromatic compounds (FACs). Methods for detecting FACs include fixed wavelength fluorescence (Aas et al., 2000b), synchronous fluorescence scanning (Lin et al., 1994), high performance

http://dx.doi.org/10.1016/j.marpolbul.2016.01.023 0025-326X/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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liquid chromatography with fluorescence detection (HPLC-F) (Kreitsberg et al., 2010, Rey-Salgueiro et al., 2009) emission matrix/emission matrix (EEM) spectroscopy (Elcoro et al., 2014, Ferretto et al., 2014). The excitation/emission wavelengths for detecting most FACs overlap making it difficult to distinguish the presence of one specific PAH from another. Therefore, compounds detected using fluorescence are referred to as “PAH-like”. However, FAC methods have an advantage over more reference driven methods such gas chromatography-mass spectroscopy (GCMS). FACs methods can detect the wide variety of substituted and unsubstituted PAH compounds and PAH metabolites typically found in natural samples (Ariese et al., 1993). Research shows that PAH exposure can be monitored in the aquatic environment using fish tissues (Kreitsberg et al., 2010, Murawski et al., 2014) and bile (Lin et al., 1994, Fuentes-Rios et al., 2005). FACs in bile have been successfully used as biomarkers of PAH exposure in marine (Beyer et al., 2010; Vuorinen et al., 2006; Lima et al., 2008), estuarine (Ferreira et al., 2006) and freshwater systems (Cormier et al., 2000; Barra et al., 2001). However, fish can metabolize PAHs and thereby reduce their body burdens (Collier and Varanasi, 1991, Ferreira et al., 2006). The PAHs are metabolized by cytochrome P450 enzyme systems and excreted into bile and urine (Kreitsberg et al., 2010, Trisciani et al., 2011). Metabolism of PAHs may account for the failure of FACs in bile to discriminate between reference and contaminated sites in some cases (Collier et al., 1996, Jewetta et al., 2002). Raw fish oil may be an alternative to bile. Its hydrophobic nature may allow greater accumulation and retention of PAHs over time. Research in GOM and NJ menhaden shows PAHs in whole body and GI tract and heart tissues as detected by fluorescence and GCMS (Bentivegna et al., 2015, Millemann et al., 2015). This makes the oily menhaden a promising model organism for biomonitoring PAHs in the environment. The goal of this work was to assess the levels of PAH-like compounds in raw fish oil of Atlantic menhaden (Brevoortia tyrannus) before and after Hurricane Sandy. Atlantic menhaden were collected along the NJ coast in August, September and/or October of 2011, 2012 and 2013. The null hypothesis was that raw fish oils collected in 2013 would be similar to those collected in 2011 and 2012. PAHs were detected using spectroscopy. Results showed crude-oil like PAHs in most but not all collections of menhaden. Concentrations were relatively high, ranging up to 20,000 ng/mL. Their presence was not associated with the size of the fish nor the month or year of collection. Therefore, Hurricane Sandy did not increase levels of PAHs in menhaden raw fish oil analyzed 10 months after the event. 2. Materials and methods 2.1. Menhaden collection Adult Atlantic menhaden were collected off the coast of NJ. Collection sites are show in the Fig. 1, and dates of collection are given in Table 1 (see Results). Most collections were from boats of the PurseSeine Industry obtained by staff of NJ Fish and Wildlife. Ships included Evening Star (ESNJ), Eva Marie (EMNJ), Briana Louise (BLNJ) and Sea Huntress (SHNJ). Other procedures for collecting fish were gill net and trawling. Fish obtained by gill net were collected by staff of NJ Fish and Wildlife (FWNJ). Those obtained by trawling were collected by staff of the Virginia Institute of Marine Science (VIMS). Replicate raw fish oil samples were prepared from each collection when possible. Each separate sample contained raw oil from three or more fish combined. For example, ten fish were collected from the ship, Evening Star, on 10/8/2011. Five of the fish were used to prepare one sample, and another five fish were used to prepare the second sample. These two samples were considered replicates of the collection. However, for some collections, there were insufficient numbers of fish or the raw oil recovery was too low to obtain two separate samples. Tissues from at least three fish were combined to make each sample. Usually 5 fish were used to make each sample.

2.2. Chemicals and standards All samples were prepared in 75% aqueous ethanol (EtOH). This was prepared by diluting the stock 95% EtOH (KOPEC 190 proof) with water, 18 MΩ resistivity, obtained from a Milli-Q Integral 5 purification unit (EMD Millipore). PAH standards were obtained from Sigma-Aldrich: 2-naphthol, CAS# 135-19-3 (HNA), 9-phenanthrol, CAS# 484-17-3 (HPH), 1-hydroxypyrene, CAS# 5315-79-1 (HPY), benzo (a) pyrene-7d (deuterated hydrogen at the 7th position), CAS# 68041-22-5 (BaP), and benzo (b) fluoranthene CAS# 205-99-2 (BbFLAN). Other standards were also obtained from Sigma-Aldrich: bovine serum albumin, CAS# 9048-46-8 (albumin), retinoic acid (metabolite of vitamin A), CAS# 302-79-4, α-tocopherol (vitamin E), CAS# 59-02-9 and Nicotinamide Adenine Dinucleotide (NADH), CAS# 58-68-4. The concentrations analyzed using EEM spectroscopy varied depending on the fluorescence intensity (quantum yield) of individual compounds. Concentrations of PAHs were 200 ng/mL of HNA, HPY and BbFLAN and 500 ng/mL of HPH and BaP. Concentrations of standards were based on achieving similar levels of fluorescence intensity: albumin (1000 ng/mL); vitamin E (5000 ng/mL); retinoic acid (25,000 ng/mL) and NADH (100,000 ng/mL). A sample of crude oil was tested as a reference matrix containing the large variety of PAH-like compounds likely found in fish if exposed to fossil fuels. The sample tested was from the riser pipe of the DeepWater Horizon oil rig collected on April 24, 2010. The sample was prepared by spiking 2 μL of crude oil into 998 μL of 75% EtOH in a microcentrifuge tube. The mixture was vortexed for one minute continuously and then centrifuged for 20 min at 13,000 rpm and 4 °C (PrismR microcentrifuge). The fluorescence spectrum of the supernatant was collected. 2.3. Extraction of PAHs from raw fish oil Fish were measured for fork length and weight and given a unique tracking number. Raw fish oil was prepared as described by Pena et al. (2015). Fish skin and filet were combined and centrifuged to produce three layers- fish oil, an aqueous layer and fish meal (Fig. 2). The resulting fish oil was centrifuged again. An aliquot of sample (1 mL) was placed in a 1.5 mL microcentrifuge tube. It was centrifuged for 5 min at 13,000 rpm (PrismR microcentrifuge) and 4 ⁰C. Samples separated as follows: 1) A viscous oil layer formed at the top, 2) a more aqueous layer formed underneath it (bottom layer), and 3) a protein pellet formed at the bottom of the tube. The volumes of top and bottom layers varied by samples and were recorded. An aliquot (50 μL) from each layer was combined with 950 μL of 75% EtOH. The mixture was vortexed continuously for 1 min and then centrifuged for 20 min at 13,000 rpm and 4 °C. The supernatant was removed for fluorescence analysis. 2.4. Fluorescence analysis Raw fish oils were analyzed on a Fluorolog 3 (Horiba), equipped with single excitation and emission monochromators and a non-ozone producing 450 W xenon arc lamp source. A Hamamatsu 928 side on photomultiplier tube was used to collect the emitted photons, and yielded a signal in photon counts per second (CPS). Lamp variations were corrected by measuring the lamp excitation intensity in microamps as a function of wavelength with a reference photodiode. Emission spectra were corrected for instrument artifacts with correction files provided by the manufacturer. Samples were analyzed in semi-micro 1 mL sample volume fused silica cuvettes (Starna) with 1 cm excitation pathlengths. The excitation scans were from 260 to 400 nm and emission scans were from 320 to 480 nm. These wavelengths were previously established using PAH standards listed in Section 2.2. The fluorescence intensities (color on the contour maps) values were represented in CPS/μA. The Fluorolog 3 provided 3D contour maps showing excitation scans for multiple emission wavelengths.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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Fig. 1. Menhaden were collected off the coast of New Jersey during 2011, 2012 and 2013 in the fishing vessel locations represented by numbers. Map provided by NJ Fish and Wildlife.

The contour map separated fluorescence compounds based on their optimal excitation and emission wavelengths. Scan parameters were: integration time = 0.1 s, Rayleigh Masking 1st and 2nd order, Masking slit width = 2 nm, Increment data points of 5 nm, excitation and emission slits = 1.00 nm band pass, excitation monochromator blaze 330 nm, and emission monochromator blase 500 nm. Figures of contour maps were generated by SigmaPlot version 13.

2.5. Percent recovery Percent recovery of PAHs was determined by spiking HPY into a subset of samples according to Pena et al. (2015). Samples tested were ESNJ 9/4/2012, BLNJ 9/7/2012 replicate 2, VIMS 10/12/2012 and FWNJ 10/3/ 2012. The upper and lower layers of fish oil were spiked independently. Samples generated for percent recovery were: 1) upper layer spiked,

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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Table 1 Information on menhaden collected. Collection date, map location (Fig. 1), collection source, fish weight and fork length and number of fish used to make each oil sample are provided. PS = collection from purse-seine vessel; VIMS = vessel out of Virginia Institute of Marine Science that was trawling off shore; NJFW = menhaden collected by NJ Fish & Wildlife staff using a gill net. Collection date

Map

Collection source (method)

Map

Replicate

Fish per replicate

Fish Weight (g)

Fish length (mm)

Ratio weight/length

8/31/2011

621

ESNJ (PS)

621

8/31/2011

621

EMNJ (PS)

621

10/8/2011

621

ESNJ (PS)

621

10/8/2011

621

EMNJ (PS)

621

9/4/2012 9/7/2012

621 621

ESNJ (PS) BLNJ (PS)

621 621

9/12/2012

621

EMNJ (PS)

621

10/12/2012

614

VIMS (trawl)

614

8/20/2013 8/21/2013

394 621

SHNJ (PS) BLNJ (PS)

394 621

10/3/2013

396

NJFW (gill net)

396

1 2 1 2 1 2 1 2 1 1 2 1 2 1 2 1 1 2 1

5 3 5 5 5 5 5 3 10 5 5 8 4 5 4 6 4 4 6

325 ± 44 387 ± 55 312 ± 73 315 ± 15 447 ± 54 403 ± 92 357 ± 33 375 ± 71 325 ± 30 290 ± 33 299 ± 47 364 ± 22 390 ± 31 395 ± 42 379 ± 26 335 ± 63 316 ± 17 298 ± 16 399 ± 55

261 ± 12 271 ± 10 268 ± 10 277 ± 18 265 ± 11 268 ± 19 254 ± 12 255 ± 18 257 ± 9 246 ± 12 255 ± 18 271 ± 6 280 ± 11 274 ± 14 263 ± 4 265 ± 12 255 ± 3 247 ± 14 274 ± 11

1.25 ± 0.15 1.42 ± 0.16 1.17 ± 0.28 1.14 ± 0.12 1.68 ± 0.15 1.50 ± 0.31 1.41 ± 0.16 1.46 ± 0.19 1.26 ± 0.08 1.18 ± 0.08 1.17 ± 0.10 1.34 ± 0.06 1.39 ± 0.06 1.44 ± 0.10 1.44 ± 0.09 1.26 ± 0.18 1.24 ± 0.11 1.24 ± 0.06 1.45 ± 0.17

2) upper layer unspiked, 3) lower layer spiked, 4) lower layer unspiked and 5) 500 ng/mL spike only in 75% EtOH. Percent recovery was calculated by summing fluorescence signals at excitation (Ex) wavelength emission (Em) wavelength pairs of Ex345/Em385 and Ex345/Em410 and using the formula below. Percent Recovery ¼

ðspiked layer signal  unspiked layer signalÞ  100 HPY Spike only Signal

Percent recoveries of the upper layers of the four fish oils above were 42, 29, 59, and 46, respectively, with an average ± standard deviation (SD) of 44 ± 12. Percent recoveries of the lower layers were 62, 59, 76, and 58, respectively, with an average ± SD of 64 ± 9. 2.6. Calculation of PAH-like compound concentration Concentrations of PAH-like compounds were calculated from Ex/Em values (z values) of the major peaks in EEM scans. The major Ex/Em peak varied for some fish oils and likely represented differences in

FAC composition. Concentrations were calculated using a HPY standard curve. Concentrations of HPY were 1, 5, 10, 50, 100 and 500 ng/mL in 75% EtOH. Fluorescence intensity for the curve was measured at Ex360/Em430. This wavelength pair overlapped with the major peaks of most fish oils. The HPY standard curve line equation was y = 2942.4× + 21,679 with R2 = 0.9905. The limit of detection (LOD) and limit of quantification (LOQ) were 1.86 ng/mL (2.3 × 104 CPS/μA) and 6.19 ng/mL (4.8 × 104 CPS/μA), respectively. The LOD was calculated as the y intercept plus three times the standard error. The LOQ was the y intercept plus ten times the standard error (MacDougall et al., 1980). Using the HPY standard curve, y-values (CPS/μA) for each fish oil layer were converted to x-values (ng/mL) with the regression line. The concentration was multiplied by 1 mL (volume of supernatant) leaving the amount of ng in each extracted aliquot of fish oil layer. The amount was corrected for percent recovery determined separately for each layer (see above). The amount was then divided by the volume (mL) of each layer generated when 1 mL of raw fish was centrifuged. This resulted in the ng/mL of PAH-like compounds in each layer. The upper and lower layers were summed together for the total concentration of PAH-like compounds in 1 mL of raw fish oil.

2.7. Statistics

Fig. 2. Picture of menhaden tissues and liquid layers following the initial (A) and second (B) centrifugation steps. A: The top layer had the consistency of oil, while the lower layer had the consistency of water. Minced filet and skin were at the bottom. B: The top layer was more viscous than the lower layer. In between the layers was whitish matter.

One way ANOVA followed by Tukey posthoc test was used to calculate significant differences between PAH-like compounds in raw fish oils and several collection parameters. Parameters included ship collecting the menhaden, month of collection, and year of collection. One way ANOVA followed by Tukey post hoc test was also used to determine any differences in fish weight/length ratios for each month and year of collection. Groups were considered significantly different if p ≤ 0.05. Data was tested for normality using the Shapiro–Wilk Test. Data for each parameter above was found to be normally distributed, p N 0.05. Independent Samples T-test with 2 tailed distribution was used to compare fish weight/length ratios for menhaden collected in August of 2011 or 2013 as there were only 2 groups. Student's paired T-test with 2 tailed distribution was used to compare PAH-like compounds in the upper and lower layers of raw fish oils. Significant differences had p ≤ 0.05. The relationship between fish size (ratio weight/ length) and PAH-like compounds was determined using Pearson correlation, 2-tailed test. Significant correlations had p ≤ 0.05. All statistics were performed using IBM SPSS.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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3. Results 3.1. Menhaden collection Atlantic menhaden used in this study were largely collected by purse seine ships fishing outside the Delaware Bay in the Cape May, NJ area (Fig. 1). Menhaden are migratory fish; therefore, any contaminants found represent the collection region at large. The fish were frozen after collection and remained frozen until they were thawed and dissected for fish oil preparation. Multiple collections of menhaden were carry out from 2011, 2012 and 2013 (Table 1). The menhaden sampled in this study were considered to be adult fish based on the mean fork lengths (246–280 mm) of the composited fish (Higham and Nicholson). Statistical analyses (one ANOVA, Tukey post hoc) for every year collected showed that the menhaden in October were larger than those in August (p = 0.001) and September (p = 0.003) based on weight to length ratios. The October fish were heavier but not longer making the ratio significantly higher than for the other two months. Statistical analyses of weight to length ratios of fish from 2011 and 2012 were compared with those from 2013 in order to assess the impact of Hurricane Sandy. Results showed no significant differences in size between menhaden collected in August 2011 (n = 16) with those collected in August of 2013 (n = 14), p = 0.980 (Independent Samples T-test). Also, there were no significant differences in size between menhaden collected in October of 2011 (n = 8) and 2012 (n = 9) with those in October of 2013 (n = 6), p = 0.966 (One way ANOVA, Tukey posthoc). Although sample size was small, it appeared that Hurricane Sandy did not affect fish size. 3.2. Standards of FACs Multiple standards were analyzed by EEM spectroscopy in order to establish what types of compounds were present in fish oils. A wide variety of compounds are capable of fluorescing including some naturally occurring ones likely to be found in a biological matrix such as fish oil. Naturally occurring compounds tested included albumin, vitamin E, retinoic acid and nicotinamide adenine dinucleotide (NADH). Albumin is a common protein. It contains amino acids such as tryptophan and tyrosine that have aromatic structures that allow proteins to fluoresce. Results showed the maximum fluorescence intensity of albumin at Ex280/Em340 (Fig. 3). Vitamin E (α-tocopherol) and vitamin A are hydrophobic compounds associated with fish oil. Retinoic acid is a metabolite of vitamin A. The maximum fluorescence intensity of vitamin E was at Ex290/Em350. Retinoic acid showed two major peaks, one at Ex345/Em480 and one at Ex360/Em480. NADH had its major peak at Ex340/Em450. The EEM analysis of crude oil in 75% EtOH showed a broad spectrum of fluorescence. Fluorescence intensity ranged from Ex320/Em340 to NEx400/Em480. Maximum intensity was between Ex340/Em380 and Ex360/Em410. PAH standards showed different EEMS scans (Fig. 3). HNP is a 2 aromatic ring molecule with a hydroxyl group at the C-2 position. It had a major excitation peak at Ex285/Em360 and a minor one at Ex340/Em360). Note that the major peak of HNP overlapped with those of albumin and vitamin E, which would make it difficult to distinguish one from the others in a mixture. HPH is a 3 aromatic ring molecule with a hydroxyl group at the C-9 position. It had a major peak bEx260/Em385 and a minor one at Ex300/Em385. HPY is a 4 aromatic ring molecule with a hydroxyl group at the C-1 position. It had multiple peaks at emissions 390 and 410 nm. The major excitation peaks for these emissions ranged from 270 to 290 nm and from 340 to 360 nm. BaP is a 5 aromatic ring molecule. Multiple peaks occurred at emissions 405, 425 and 455 nm. For these emissions, excitations ranged between 260 and 295 nm and between 360 and 390 nm. BbFLAN is also a 5 aromatic ring molecule; however, one ring is 5 instead of 6 membered. It had multiple excitation peaks at Em440. The major excitation peak was at 265 nm, minor ones ranged between 285 and 360 nm. Note

Fig. 3. Excitation Emission Matrix scans of standards. Standards included biological compounds likely to be found in raw fish oil: albumin as a representative protein (1000 ng/mL), vitamin E (5000 ng/mL), retinoic acid as a metabolite of vitamin A (25,000 ng/mL) and NADH (100,000 ng/mL). Crude oil (5 μL) was extracted into 1 mL of 75% EtOH. PAH standards included HNP and BbFLAN at 200 ng/mL and HPH, HPY and BaP at 500 ng/mL. All standards were in 75% EtOH. Note that the legends vary in fluorescence intensity.

that the major peak of NADH (Ex340/Em450) overlapped with the minor peak (Ex360/Em440) of BbFLAN. However, the concentration of NADH needed to obtain fluorescence intensity similar to that of BbFLAN

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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was 500 times greater. It is important to note that the fluorescence quantum yields for the PAHs were generally 0.1 or greater, while most of the biological fluorophores had quantum yields of 1 × 10−3 or less (Ridley, personal communication). Comparison of the crude oil spectra to that of PAH standards showed a quenching of excitations at short wavelengths (i.e. blue shifted). The majority of crude oil fluorescence was associated with the long excitation wavelengths (i.e. red shifted) found for HPY and BaP. 3.3. EEM of fish oil layers Centrifugation of raw fish oil usually resulted in two layers: a more viscous upper layer (with the consistency of oil) and a bottom, more water-like layer. The lower layer dissolved into 75% EtOH and was therefore aqueous in nature. The upper layer separated when added to 75% EtOH and was therefore oily in nature. Both layers were extracted and analyzed by EEM spectroscopy. Spectra of the upper and lower layers individually and combined are shown in Fig. 4. The upper layer of VIMS R2 (replicate 2 of VIMS 10/12/2012 collection) contained levels of PAH-like compounds similar to those in the lower layer. Both had fluorescence intensities of 1x106 CPS/μA. The lower layer appeared to contain more of the high molecular weight (HMW) compounds. This was indicated by the red shift of emission from approximately Em425 in the upper layer to Em450 in the lower one. This differed in the second fish oil example, ESNJ R2. The upper layer contained less PAH-like compounds than the

lower layer, approximately 1 × 105 versus 8 × 105 CPS/μA, respectively. The EEMS scan of each appeared to contain HMW PAHs with maximum fluorescence at Ex375/Em450. The presence of HMW PAHs in the more aqueous, lower layer indicated that fluorescent compounds in raw fish oils were likely hydroxylated metabolites of PAHs. Both fish oil layers were spiked with HPY (100 ng/mL) prior to centrifugation in order to assess percent recovery (see Methods) as well as the signal of HPY in this medium. Results showed that HPY excitation peaks in the upper layer were red shifted. PAH-like compounds found in the upper layer (1.5 × 104 CPS/μA at Ex360/Em430) were obscured in the graph due to the intensity of the HPY spike (1 × 106 CPS/μA). PAH-like compounds in the lower layer had intensities (1 × 106 CPS/ μA) similar to that of spike; and therefore, did not stand out from the sample. Combining spectra for the upper and lower layer showed the two red shifted excitation peaks for the HPY standard, one at Ex345/Em390 and one at Ex345/Em410. Results of spiked EEMS scans demonstrated that the presence of HPY could account for one component of the raw fish oil spectra and that the blue shifted excitations seen in the standard were quenched by both the upper and lower layers of the fish oil matrix. 3.4. EEM of raw fish oils EEMS scans of raw fish oils indicated different types of PAH-like compounds in different collections (Fig. 5), indicating the presence of

Fig. 4. Examples of EEMS scan construction from raw fish oil layers with and without HPY spike. EEMS scans of raw fish oils were constructed by combining values (CPS/μA) of upper and lower layers. Results showed that the lower layer usually had more PAH-like compounds than the upper layer. For spiked samples, HPY (100 ng/mL) was added to raw fish oil prior to its separation into two layers. Note the absence of Ex260 fluorescence (see Fig. 3) due to excimer formation. Also note that the HPY peak (Ex340/Em410) was within the region of EEMS scans of unspiked fish oils. EEM values of each layer were adjusted to the same fluorescence intensity for comparison purposes. R = replicate.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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Fig. 5. EEMS scans of raw fish oils collected in 2011, 2012 and 2013. Scans are of combined layers. Sample location, month of collection, and replicate number (R) are provided. One example of each replicate is provided. For comparison, fluorescence intensities of scans were adjusted to 1 × 106 or greater. Note that fluorescence intensities as well as location of the strongest fluorophores varied between scans. This indicated different levels and types of PAH-like compounds, respectively.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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different types of PAH-like compounds in menhaden. For example, the major peak for EMNJ Aug. 2011 R1 was very red shifted ranging from Ex370/Em440 to Ex400/Em480. Based on PAH standards this indicated the presence of 5 ring PAHs. On the other hand, the major peak for BLNJ Aug. 2013 R1 was blue shifted ranging from Ex340/Em400 to Ex360/ Em440. This indicated the presence of 4 ring PAHs. Although collections varied, replicates were often similar in fluorescence range (not all data shown). For example, replicate 2 of VIMS Oct. 2012 (Fig. 4) can be compared to replicate 1 of the same collection (Fig. 5). The major peaks in VIMS Oct. 2012 R1 and R2 both ranged from Ex340/Em400 to Ex370/ Em460. For ESNJ Aug. 2011, the major peaks were also similar ranging from Ex360/Em460 to Ex390/Em470 for both replicate 1 (Fig. 5) and 2 (Fig. 4). This finding indicated that fish collected by the same ship usually had similar types of PAH-like compounds. There was no consistent EEMS scan for a particular collection year. For example, ESNJ Oct. 2011 R2 had a similar range of fluorescence as VIMS Oct. 2012 R1, and FWNJ Oct. 2013. The influence of collection month was harder to determine. EEMS scans of the Sep. 2012 collections were similar: the major peak ranged from Ex360/Em430 to Ex380/Em460 for all three. However, EEMS scans for the two Aug. 2011 collections were more red-shifted than the BLNJ Aug. 2013 R1 collection. The SHNJ Aug. 2013 collection showed no detectable PAH-like compounds. 3.5. Concentrations of PAH-like compounds in raw fish oils Concentrations of PAH-like compounds were calculated for each of the layers and combined to represent 1 mL of the raw fish oil (Table 2). Results showed concentrations of PAH-like compounds ranging from non-detect (b LOD) to 20,421 ng/mL. Most concentrations ranged from 1459 to 9038 ng/mL. Fish captured at the same time (same ship and date) had similar levels of PAH-like compounds. Six of the eight replicates had levels within 2× of one another. One was within 3 × (EMNJ 8/31/2011), and the other within 5 × (EMNJ 10/3/2011). Table 2 also showed that the lower, more aqueous layer had significantly more PAH-like compounds than the upper, more oil like layer, p b 0.001 (Student's paired t-test). Concentrations of PAH-like compounds were compared to parameters that might account for variations in exposure (Table 2). Menhaden were collected in August, September and October. One way ANOVA was used to compare collections by month and showed that there was no statistical differences between groups, p = 0.236. Statistical analyses

also showed no differences between years, p = 0.168. This indicated that Hurricane Sandy did not alter PAH-like compounds in 2013 menhaden. Although most collections were in the area outside Delaware Bay, the region designated as 621 was very broad. Therefore, PAH-like compounds might be associated with the specific locations where particular ships fished. Statistical analyses showed no significant differences between ships, p = 0.806. The weight to length ratio was the final parameter considered. Pearson's correlation showed no significant relationship between fish size and PAH-like compounds, p = 0.131. Overall, PAH-like compounds were similar in terms of EEMS scans and raw fish oil concentrations for menhaden collected at the same time; however, the concentrations could not be attributed to collection ship, month, year or fish size. 4. Discussion 4.1. Effects of hurricanes Hurricanes are natural disasters likely to increase in frequency with global climate change, yet literature on the impact of coastal hurricanes on wild aquatic organisms is limited. PAHs are ubiquitous, anthropogenic contaminants that continue to be a concern (Peterson et al., 2003). Studies show that following Hurricane Katerina redistribution of near shore sediments was associated with 10× higher levels of the PAH, pyrene, in surface sediments (Mitra et al., 2009). BaP equivalents in the water column and suspended sediments also increased temporarily following Katrina (Weston et al., 2010). These findings indicate that availability of contaminants may change due to hurricanes and modulate body burdens in exposed organisms. This is supported by a study of eastern oyster (Crassostrea virginica). Tissue analyses, prior to and after Hurricanes Katrina and Rita, show increases in heavy metals and reductions in LMW PAHs but no changes in HMW PAHs (Johnson et al., 2009). On the other hand, PAHs in bile of Atlantic croaker (Micropogonias undulates) and bigeye tuna (Thunnus obesus) were no greater one month following Katrina than previously reported (Krahn et al., 2005). The primary purpose of this work is to investigate changes in PAH loads in Atlantic menhaden (B. tyrannis) that may have occurred due to Hurricane Sandy. We employ a relatively new technology, EEM spectroscopy, that provides a profile of fluorescent contaminants such as PAHs and uses a novel biological matrix, raw fish oil, which may provide a better measure of PAH body burdens than bile.

Table 2 Concentrations of PAH-like compounds in raw fish oil. Data provided include collection, replicate, wavelength pair with major peak (Ex/Em), amount (ng) of PAH-like compound in upper or lower layer of oil calculated from HPY standard curve, volume (mL) of layer, concentrations (ng/mL) in upper and lower layers as well as in the 1 mL of raw fish oil (combined) from which layers were derived. The average ± SD for all collections in the same year is also provided. ND = not detected, fluorescence intensity was below the LOQ, 6.19 ng/mL. Collection

ESNJ 8/31/2011 EMNJ 8/31/2011 ESNJ 10/8/2011 EMNJ 10/8/2011 ESNJ 9/4/2012 BLNJ 9/7/2012 EMNJ 9/12/2012 VIMS 10/12/2012 SHNJ 8/20/2013 BLNJ 8/21/2013 FWNJ 10/3/2013

Replicate

1 2 1 2 1 2 1 2 1 1 2 1 2 1 2 1 1 2 1

Ex/Em

370/450 370/450 385/455 385/455 360/430 360/430 370/450 370/450 370/450 370/450 360/430 370/450 370/450 360/430 360/430 360/430 345/415 345/415 360/430

Combined

Year

ng

Upper layer mL

ng/mL

ng

Lower layer mL

ng/mL

ng/mL

Ave (SD)

32 68 882 731 7 ND ND ND 575 186 15 1579 568 519 644 ND ND ND 4

0.50 0.40 0.15 0.50 0.99 0.99 0.98 0.75 0.15 0.15 0.50 0.10 0.25 0.30 0.10 1.00 0.50 0.40 0.50

316 546 2645 7306 131 0 0 0 1726 559 151 3159 2839 3116 1287 ND 0 0 39

761 378 350 1312 203 173 316 292 273 196 260 211 379 347 431 ND 426 409 397

0.50 0.60 0.85 0.50 0.01 0.01 0.02 0.25 0.85 0.85 0.50 0.90 0.75 0.70 0.90 0.00 0.50 0.60 0.50

7607 4536 5944 13116 203 173 316 1459 4645 3326 2597 3797 5683 4861 7751 ND 4257 4912 3971

7923 5082 8590 20421 333 173 316 1459 6371 3885 2748 6955 8521 7976 9038 0 4257 4912 4010

5537 (6933)

6499 (2375)

3295 (2226)

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

C.S. Bentivegna et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

4.2. Concentrations of PAH-like compounds in raw fish oils Researchers have used EEM spectroscopy to detect PAHs and pesticides in marine water samples (Ferretto et al., 2014) as well as PAHs from water collected following the DeepWater Horizon oil spill (Zhou et al., 2013). Previous work in our laboratory developed a method for measuring PAHs in raw fish oil (Pena et al., 2015) and heart tissue (Millemann et al., 2015) of menhaden using fluorescence. We applied this procedure to Atlantic menhaden collected before and after Hurricane Sandy. Results for EEMS scans were converted into concentrations of PAH-like compounds using the most intense region of fluorescence for a particular sample (Ex340–385/Em415–450) and a standard curve for HPY. Concentrations ranged from non-detect to 20,421 ng/mL (Table 2). The median for all samples was 4912 ng/mL. These concentrations were generally higher than those found in bile of dab (Limanda limanda) and flounder (Platichthys flesus) collected in the Baltic Sea, b0.7 to 838 ng/mL (Kammann, 2007). Other studies monitoring PAHs in bile reported concentrations in units of ng/g wet weight. Therefore, units of ng/mL were converted to ng/g given that the average weight ± SD of 1 mL of raw fish oil was 1.33 ± 0.05 g. Using these units, raw fish oil concentrations ranged from non-detect to 15,341 ng/g with a median of 3690 ng/g. These values were also high compared to other species. Maximum values found in bile were 300 ng/g for cod (Gadus morhua), 300 ng/g for salmon (Clupea harengus), 1000 ng/g for flounder (P. flesus) and 1200 ng/g for eelpout (Zoarces viviparus) (Vuontisjarvi et al., 2004). Raw fish oil concentrations (median 3690 ng/g) were more similar to those of Atlantic croaker (Micropogonias undulatus) collected in the Gulf of Mexico before and after Hurricane Katrina (Krahn et al., 2005). PAHs were measured using HPLC-F at Ex380/Em430. These wavelengths were similar to those used in the present study to calculate HPY-like concentrations. BaP equivalents were reported in the range of 1400 to 7500 ng/g bile before and 620 to 3100 ng/g bile after Katrina. 4.3. Effects of Hurricane Sandy on PAH-like compounds in menhaden raw fish oil Raw fish oil samples showed different EEM profiles and concentrations of PAH-like compounds indicating various types and levels of contaminant exposure. Several parameters were investigated to determine if any might account for these differences. They included the month and year of collection, ship collecting the fish and fish size. The statistical difference between years was p = 0.168; therefore, results did not show that Hurricane Sandy altered the concentrations of PAHs in menhaden. Pena et al. (2015) presented EEM scans for Atlantic menhaden collected in September and October of 2010 from the same purse seine ships as those used in the present study. The paper showed the development of the extraction method, which was only modified in the present study by centrifuging the raw fish oil into layers. EEM scans for the three 2010 collections showed very similar profiles to those presented here (Fig. 5). The raw fish oil profiles of 2010 followed an approximate 45 degree angle from Ex340/Em410 to Ex370/Em460. This matched the profiles of ESNJ Oct. 2011 R2, VIMS Oct. 2012 R1 and FWNJ Oct. 2013. Therefore, the PAH mixture within menhaden raw fish oil appeared consistent across 4 years of monitoring in the Delaware Bay region during September and October. Results of the present study showed that Hurricane Sandy did not appear to influence PAH levels. However, EEM profiles showed differences in types of PAHs in fish oil samples. For example, EMNJ Aug. 2011 R1 had its major peak at Ex385/Em455, while ESNJ Oct. 2011 had its major peak at Ex360/Em430. This indicated exposure to different sources of PAHs. PAH-like compounds found in raw fish oil appeared to be primarily HMW. Emission wavelengths began at approximately 410 nm and continued out to N 480 nm. LMW PAHs such as HNP (2 ring) and HPH (3 ring) had lower emission wavelengths of 355 and 385 nm, respectively. While those of HPY, BaP and BbF

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(Em385, Em410, and Em440, respectively) were in the same range as those of raw fish oils. PAH-like compounds in menhaden differed from those found in marine water. Ferretto et al. (2014) found LMW PAH profiles for water collected from hydrocarbon contaminated harbors in the Mediterranean Sea. Major peaks had emission wavelengths at 330 and 350 nm. Zhou et al. (2013) also found a LMW PAH profile for water collected near the DWH oil spill. This suggested that menhaden retained primarily HMW PAHs in their body oil following exposure. The month of collection was considered as it might influence the levels and sources of PAHs. Menhaden are filter feeders, so changes in plankton populations and dissolved organic matter might have affected PAH exposure. PAHs from crude oil have been found to adsorb to mesozooplankton (Mitra et al., 2012), and researchers have found seasonal differences in bile PAHs (Kammann, 2007). In the present study, PAH-like concentrations were not significantly different between collection months, p = 0.236. However, types of PAHs visualized in EEM profiles did vary by month. For example, 3 of 4 October scans had major peaks at Ex350 nm, while 5 of 6 August/September scans had major peaks at Ex360 nm or higher. This represented greater body burdens of HMW versus LMW PAHs in August/September fish. It was interesting that the two raw fish oils from August 2013 had either undetectable PAHs or an EEM scan similar to those for October. This suggested that levels and types of PAHs were modified in 2013 versus 2011 and 2012, but the number of collections were too few to support any impact of Hurricane Sandy in this regard. Differences might have reflected normal variation of body burdens. The ships collecting menhaden were considered as a factor influencing PAH levels. Most ships collected in the region designated as 621, which is a broad area, and a particular ship might favor specific locations (Fig. 1). Results showed no significant differences in PAH levels for ships, p = 0.806. However, it was interesting that EEM scans for replicate samples were similar (Figs. 4 and 5). Replicates were separate raw fish oils prepared from different fish collected at the same time by the same ship. This finding indicated that fish from a particular school were exposed to similar levels and types of PAH-like compounds; and therefore, the sample was representative of the catch. Overall, EEM scans provided a profile of HMW PAH-like compounds in menhaden that represented the mixture of fluorescent compounds to which the menhaden in a particular school had been exposed. 4.4. Matrix interference and effects Fluorescence found in raw fish oils could not be attributed to other biological compounds. Examples of compounds likely to be abundant in a biological matrix such as raw fish oil included polymeric nucleic acids (DNA, RNA), nucleic acid containing compounds (nicotinamide adenine dinucleotide: NADH), aromatic amino-acids in protein and some hydrophobic vitamins such as vitamin A (retinols) and vitamin E (α-tocopherol). Nucleic acids have been found to absorb at 267 nm and emit at 327 nm (Vaya et al., 2010). These wavelengths would have been blue shifted outside the area associated with raw fish oil scans. Vitamin E had its major peak at Ex290/Em330, while albumin (a representative protein) had its major peak at EX280/EM340. These peaks overlapped with each other and HNP, which had its major peak at Ex285/Em360. They did not overlap with the major peak of HPH, Ex b 260/Em385. Therefore, vitamin E and albumin could be confounding factors for the detection of HNP. NADH had its major peak at Ex340/ Em455. This peak was within the range of BbFLAN, particularly if BbFLAN formed excimers. However, the emission of NADH was more red shifted than the major peaks of raw fish oil, and a concentration 500X greater was necessary to achieve similar fluorescence intensity. Overall, common biological molecules did not account for EEM profiles of raw fish oils. The EEM profile for the crude oil sample was similar to that for raw fish oils. It had the same 45 degree angle; although, the range was somewhat lower from Ex320/Em360 to Ex380/Em430. This likely reflected a

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

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higher proportion of LMW PAHs. In addition, when HPY was spiked into raw fish oil, its major peak overlapped with the most intense fluorescence wavelengths of fish oils (Fig. 4). This supported previous GCMS data (Bentivegna et al., 2015, Millemann et al., 2015) that the fluorescence signal seen in fish oils were crude oil-like PAHs. An interesting finding related to the matrix was that the two layers of raw fish oil had different concentrations of PAH-like compounds (Table 2). Raw fish oil was originally centrifuged with the intent of pelleting protein and improving duplicates. The formation of two layers in most samples was unexpected. Further work is necessary in order to ascertain the chemical make-up of the layers; however, both layers likely contained fatty acids. This was evidenced by HPY excimer formation in both layers, which happens in the presence of micelles (Fig. 4, HPY spiked sample). Higher PAH concentrations in the lower layer indicated the presence of hydrophilic metabolites. Hydrophilic metabolites of PAHs were found in bile (Deshpande et al., 2002, Vuontisjarvi et al., 2004, Kammann, 2007) and in fish urine (Kreitsberg et al., 2010). Their presence in urine supported distribution of ingested PAHs to body oil of fish. If raw fish oil contained primarily PAH metabolites, then analyzes of parent and alkylated PAHs by GCMS might underrepresent PAH body burdens. 4.5. Summary Raw fish oils samples were prepared from Atlantic menhaden collected at various times during the two years prior and one year after Hurricane Sandy. EEM scans showed various concentrations and types of FACs, which were determined to be PAH-like compounds. This was based on 1) exclusion of common fluorescent compounds found in biological samples, 2) similarity of raw fish oil scans to crude oil scans, 3) spikes of HPY that fluoresced in the same region as raw fish oils and 4) GCMS confirmation that menhaden tissues with similar EEM scans contained PAHs. Concentrations of PAH-like compounds in raw fish oils were high compared to those in bile of other fish species. This indicated that raw fish oil might accumulate and retain more PAHs than bile. Alternatively, there might have been species differences. EEM scans of the more aqueous layer of raw fish oils indicated the presence of HMW, hydrophilic metabolites of PAHs. The presence of metabolites might result in underestimation of PAHs in fish using standard GCMS techniques, which depend on parent and alkylated compounds as references. The concentrations and types of PAH-like compounds could not be attributed to changes in PAH availability following Hurricane Sandy. This was consistent with the limited number of reports pertaining to the impact of hurricanes on PAH body burdens in other pelagic species (Krahn et al., 2005); however, benthic species may be more susceptible to sediment-derived PAHs (Johnson et al., 2009). Results did show that Atlantic menhaden collected for this study were contaminated with PAH-like compounds. Research has shown that contaminants in menhaden can be trophically transferred to predators (Candelmo et al., 2010) and commercial products (Rey-Salgueiro et al., 2009; Yebra-Pimentel et al., 2013, 2014). Taken together, results indicated that urban contaminants in fish collected along the NJ shore deserve future study. Acknowledgments The authors appreciate the assistance of the staff of NJ Fish and Wildlife Service and the Virginia Institute of Marine Science, who assisted in the collection of menhaden. They also appreciate the financial support of the Departments of Chemistry and Biochemistry and Biological Sciences for purchasing and maintaining the fluorimeter. The authors wish to recognize the work of the reviewers whose recommendations improved this paper. This work is part of the US Geological Survey's response to Hurricane Sandy funded by the Disaster Relief Appropriations Act of 2013 (PL 113-2).

References Aas, E., Baussant, T., Balk, L., Liewenborg, B., Andersen, O.K., 2000a. PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod. Aquat. Toxicol. 51, 241–258. Aas, E., Beyer, J., Goksøyr, A., 2000b. Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation. Biomarkers 5, 9–23. Ahrenholz, D.W., 1991. Population biology and life history of the North American menhadens, Brevoortia spp. Mar. Fish. Rev. 53, 3–19. Ariese, F., Kok, S.J., Verkaik, M., Gooijer, C., Velthorst, N.H., Hofstraat, J.W., 1993. Synchronous fluorescence spectrometry of fish bile: a rapid screening method for the biomonitoring of PAH exposure. Aquat. Toxicol. 26, 273–286. Barra, R., Sanchez-Hernandez, J.C., Orrego, R., Parra, O., Gavilan, J.F., 2001. Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss. Chemosphere 45, 439–444. Bentivegna, C.S., Cooper, K.R., Olson, G., Pena, E.A., Millemann, D.R., Portier, R.J., 2015. Chemical and histological comparisons between Brevoortia sp. (menhaden) collected in Fall 2010 from Barataria Bay, LA and Delaware Bay, NJ following the Deep Water Horizon (DWH) oil spill. Mar. Environ. Res. 112 (Part A), 21–34. Beyer, J., Jonsson, G., Ported, C., Krahn, M.M., Ariese, F., 2010. Analytical methods for determining metabolites of polycyclic aromatic hydrocarbon (PAH) pollutants in fish bile: a review. Environ. Toxicol. Pharmacol. 30, 224–244. Bugel, S.M., White, L.A., Cooper, K.R., 2010. Impaired reproductive health of killifish (Fundulus heteroclitus) inhabiting Newark Bay, NJ, a chronically contaminated estuary. Aquat. Toxicol. 96, 182–193. Candelmo, A.C., Deshpande, A., Dockum, B., Weis, P., Weis, J.S., 2010. The effect of contaminated prey on feeding, activity, and growth of young-of-the-year bluefish, Pomatomus saltatrix, in the laboratory. Estuar. Coasts 33, 1025–1038. Collier, T.K., Varanasi, U., 1991. Hepatic activities of xenobiotic metabolizing enzymes and biliary levels of xenobiotics in English sole (Parophrys vetulus) exposed to environmental contaminants. Arch. Environ. Contam. Toxicol. 20, 462–473. Collier, T.K., Krone, C.A., Krahn, M.M., Stein, J.E., Chan, S.L., Varanasi, U., 1996. Petroleum exposure and associated biochemical effects in subtidal fish after the Exxon Valdez oil spill. Am. Fish. Soc. Symp. 18, 671–683. Cormier, S.M., Lin, E.L.C., Fulk, F., Subramanian, B., 2000. Estimation of exposure criteria values for biliary polycyclic aromatic hydrocarbon metabolite concentrations in white suckers (Catostomus commersoni). Environ. Toxicol. Chem. 19, 1120–1126. Crawford, D.W., Bonnevie, N.L., Gillis, C.A., Wenning, R.J., 1994. Historical changes in the ecological health of the Newark Bay Estuary, New Jersey. Ecotoxicol. Environ. Saf. 29, 276–303. Deshpande, A.D., Huggett, R.J., Halbrook, R.A., 2002. Polycyclic aromatic hydrocarbon metabolites in the bile of a territorial benthic fish, oyster toadfish (Opsanus tau) from the Elizabeth River, Virginia. Arch. Environ. Contam. Toxicol. 42, 43–52. Elcoro, A.S., de Juan, A., García, J.A., Durana, N., Alonso, L., 2014. Comparison of secondorder multivariate methods for screening and determination of PAHs by total fluorescence spectroscopy. Chemom. Intell. Lab. Syst. 132, 63–74. Ferreira, M., Moradas-Ferreira, P., Reis-Henriques, M.A., 2006. The effect of long-term depuration on phase I and phase II biotransformation in mullets (Mugil cephalus) chronically exposed to pollutants in River Douro Estuary, Portugal. Mar. Environ. Res. 61, 326–338. Ferretto, N., Tedetti, M., Guigue, C., Mounier, S., Redon, R., Goutx, M., 2014. Identification and quantification of known polycyclic aromatic hydrocarbons and pesticides in complex mixtures using fluorescence excitation–emission matrices and parallel factor analysis. Chemosphere 107, 344–353. Franklin, H.B., 2007. The Most Important Fish in the Sea. Island Press/Shearwater books, Washington, DC, USA. Fuentes-Rios, D., Orrego, R., Rudolph, A., Mendoza, G., Gavilan, J.F., Barra, R., 2005. EROD activity and biliary fluorescence in Schroederichthys chilensis (Guichenot 1848): biomarkers of PAH exposure in coastal environments of the South Pacific Ocean. Chemosphere 61, 192–199. Harvey, J.S., Lyons, B.P., Page, T.S., Stewart, C., Parry, J.M., 1999. An assessment of the genotoxic impact of the sea empress oil spill by the measurement of DNA adducts levels in selected invertebrate and vertebrate species. Mutagenesis 441, 103–114. Jewetta, S.C., Dean, T.A., Woodin, B.R., Hoberg, M.K., Stegeman, J.J., 2002. Exposure to hydrocarbons 10 years after the Exxon Valdez oil spill: evidence from cytochrome P4501A expression and biliary FACs in near shore demersal fishes. Mar. Environ. Res. 54, 21–48. Johnson, W.E., Kimbrough, K.L., Lauenstein, G.G., Christensen, J., 2009. Chemical contamination assessment of Gulf of Mexico oysters in response to hurricanes Katrina and Rita. Environ. Monit. Assess. 150, 211–225. Kammann, U., 2007. PAH metabolites in bile fluids of dab (Limanda limanda) and flounder (Platichthys flesus) — spatial distribution and seasonal changes. Environ. Sci. Pollut. Res. 14, 102–108. Krahn, M.M., Ylitalo, G.M., Collier, T.K., 2005. Analysis of Bile of Fish Collected in Coastal Waters of the Gulf of Mexico Potentially Affected by Hurricane Katrina to Determine Recent Exposure to Polycyclic Aromatic Compounds (PACs). National Marine Fisheries Service, Northwest Fisheries Science Center, Seattle. Kreitsberg, R., Zemit, I., Freiberg, R., Tambets, M., Tuvikene, A., 2010. Responses of metabolic pathways to polycyclic aromatic compounds in flounder following oil spill in the Baltic Sea near the Estonian coast. Aquat. Toxicol. 99, 473–478. Lauenstein, G.G., Kimbrough, K.L., 2007. Chemical contamination of the Hudson–Raritan Estuary as a result of the attack on the World Trade Center: analysis of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in mussels and sediment. Mar. Pollut. Bull. 54, 284–294.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023

C.S. Bentivegna et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx Lima, D., Santos, M.M., Ferreira, A.M., Micaelo, C., Reis-Henriques, M.A., 2008. The use of the shanny Lipophrys pholis for pollution monitoring: a new sentinel species for the northwestern European marine ecosystems. Environ. Int. 34, 94–101. Lin, E.C., Cormier, S.M., Racine, R.N., 1994. Synchronous fluorometric measurement of polycyclic aromatic hydrocarbons in the bile of brown bullhead. Environ. Toxicol. Chem. 13, 707–715. MacDougall, D., Crummett, W.B., et al., 1980. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal. Chem. 52, 2242–2249. Millemann, D.R., Portier, R.J., Olson, G., Bentivegna, C.S., Cooper, K.R., 2015. Particulate accumulations in the vital organs of wild Brevoortia patronus from the northern Gulf of Mexico after the Deepwater Horizon oil spill. Ecotoxicology 24, 1831–1847. Mitra, S., Kimmel, D.G., Snyder, J., Scalise, K., McGlaughon, B.D., Roman, M.R., Jahn, G.L., Pierson, J.J., Brandt, S.B., Montoya, J.P., Rosenbauer, R.J., Lorenson, T.D., Wong, F.L., Campbell, P.L., 2012. Macondo-1 well oil-derived polycyclic aromatic hydrocarbons in mesozooplankton from the northern Gulf of Mexico. Geophys. Res. Lett. 39, L01605. http://dx.doi.org/10.1029/2011GL049505. Mitra, S., Lalicata, J.J., Allison, M.A., Dellapenna, T.M., 2009. The effects of Hurricanes Katrina and Rita on seabed polycyclic aromatic hydrocarbon dynamics in the Gulf of Mexico. Mar. Pollut. Bull. 58, 851–857. Murawski, S.A., Hogarth, W.T., Peebles, E.B., Barbeiri, L., 2014. Prevalence of external skin lesions and polycyclic aromatic hydrocarbon concentrations in Gulf of Mexico fishes, post-Deepwater Horizon. Trans. Am. Fish. Soc. 143, 1084–1097. Myers, M.S., Anulacion, B.F., French, B.L., Reichert, W.L., Laetz, C.A., Buzitis, J., Olson, O.P., Sol, S., Collier, T.K., 2008. Improved flatfish health following remediation of a PAHcontaminated site in Eagle Harbor, Washington. Aquat. Toxicol. 88, 277–288. Neff, J.M., 1979. Polycyclic aromatic hydrocarbons in the aquatic environment. sources, fates and biological effects. Applied Science Publishers LTD., London. NOAA: National Marine Fisheries Service, March 2015. Forecast for the 2015 Gulf and Atlantic menhaden purse-seine fisheries and review of the 2014 fishing season. Sustainable Fisheries Branch, NMFS Beaufort, NC. Pena, E.A., Ridley, L.M., Murphy, M.R., Sowa, J.R., Bentivegna, C.S., 2015. Detection of polycyclic aromatic hydrocarbons (PAHs) in raw menhaden fish oil using fluorescence spectroscopy: method development. Environ. Toxicol. Chem. 34, 1946–1958. Peterson, C.H., Rice, S.D., Short, J.W., Esler, D., Bodkin, J.L., Ballachey, B.E., Irons, D.B., 2003. Long term ecosystem response to the Exxon Valdez oil spill. Science 302, 2082–2086. Pinkney, A.E., Harshbarger, J.C., May, E.B., Melancon, M.J., 2001. Tumor prevalence and biomarkers of exposure in brown bullheads (Ameiurus nebulosus) from the tidal Potomac river, USA, watershed. Environ. Toxicol. Chem. 20, 1196–1205.

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Rey-Salgueiro, L., Martínez-Carballo, E., García-Falcón, M.S., González-Barreiro, C., SimalGándara, J., 2009. Occurrence of polycyclic aromatic hydrocarbons and their hydroxylated metabolites in infant foods. Food Chem. 115, 814–819. Trisciani, A., Corsi, I., Torre, C.D., Perra, G., Focardi, S., 2011. Hepatic biotransformation genes and enzymes and PAH metabolites in bile of common sole (Solea, Linnaeus, 1758) from an oil contaminated site in the Mediterranean sea: a field study. Mar. Pollut. Bull. 62, 806–814. Valle, S., Panero, M.A., Shor, L., 2007. Pollution prevention and management strategies for polycyclic aromatic hydrocarbons in the New York/New Jersey harbor. New York Academy of Sciences, New York, pp. 1–171. Vaya, I., Gustavsson, T., Miannay, F.A., Douki, T.D., 2010. Fluorescence of natural DNA: from the femtosecond to the nanosecond time scales. J. Am. Chem. Soc. 132, 11834–11835. Vogelbein, W.K., Fournie, J.W., Cooper, P.S., Van Veld, P.A., 1999. Hepatoblastomas in the mummichog, Fundulus heteroclitus (Linnaeus), from a creosote-contaminated environment: a histologic, ultrastructural, and immunohistochemical study. J. Fish Dis. 22, 419–431. Vuontisjarvi, H., Keinanen, J., Vuorinen, P.J., Peltonen, K., 2004. A comparison of HPLC with fluorescence detection and fixed wavelength fluorescence methods for the determination of polycylic aromatic hydrocarbon metabolites in fish bile. Polycycl. Aromat. Compd. 24, 333–342. Vuorinen, P.J., Keinanen, M., Vuontisjarvi, H., Barsien, J., Broeg, K., Forlin, L., Gercken, J., Kopecka, J., Kohler, A., 2006. Use of biliary PAH metabolites as a biomarker of pollution in fish from the Baltic Sea. Mar. Pollut. Bull. 53, 479–487. Weston, J., Warren, C., Chaudhary, A., Emerson, B., Argote, K., Khan, S., Willett, K.L., 2010. Use of bioassays and sediment polycyclic aromatic hydrocarbon concentrations to assess toxicity at coastal sites impacted by Hurricane Katrina. Environ. Toxicol. Chem. 29, 1409–1418. Yebra-Pimentel, I., Fernández-González, R., Martínez-Carballo, E., Simal-Gándara, J., 2014. Optimization of purification processes to remove polycyclic aromatic hydrocarbons (PAHs) in polluted raw fish oils. Sci. Total Environ. 470–471, 917–924. Yebra-Pimentel, I., Martínez-Carballo, E., Regueiro, J., Simal-Gándara, J., 2013. The potential of solvent-minimized extraction methods in the determination of polycyclic aromatic hydrocarbons in fish oils. Food Chem. 139, 1036–1043. Zhou, Z., Guo, L., Shiller, A.M., Lohrenz, S.E., Asper, V.L., Osburn, C.L., 2013. Characterization of oil components from the Deepwater Horizon oil spill in the Gulf of Mexico using fluorescence EEMS and PARAFAC techniques. Mar. Chem. 148, 10–21.

Please cite this article as: Bentivegna, C.S., et al., Excitation–emission matrix scan analysis of raw fish oil from coastal New Jersey menhaden collected before and after Hurricane Sandy, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.01.023