FOR OSRI USE ONLY Oil Spill Recovery Institute OSRI PROPOSAL Grant Application NUMBER

DATE RECEIVED

PROJECT TITLE Sensitivity of Arctic embryos and larvae to oil; delayed impacts on juvenile growth and lipid condition.

EMPLOYER INDENTIFICATION NUMBER RFP TITLE (EIN) or (TIN) N/A See attached cover sheets by institution

DATE SUBMITTED # OF COPIES IS THIS AWARD

09/26/2017 1 [X] NEW RENEWAL NAME AND ADDRESS OF ORGANIZATION NAME AND ADDRESS OF PERFORMING TO WHICH AWARD SHOULD BE MADE ORGANIZATION IF DIFFERENT

See attached cover sheets by institution Same

IS AWARDEE ORGANIZATION (Check All That Apply)

UNIVERSITY AGENCY NON-PROFIT FOR-PROFIT INDIVIDUAL

SMALL BUSINESS MINORITY BUSINESS WOMEN-OWNED BUSINESS CHECK APPROPRIATE BOX(ES) for area to which the proposal applies

FELLOWSHIP SMALL AWARD < $25,000 MEDIUM AWARD $25,000 to <$100,000.

LARGE AWARD $100,000 or greater.

REQUESTED AMOUNT PROPOSED DURATION (1- REQUESTED $53,194 (OSU) 60 MONTHS) STARTING DATE $24,072 (NOAA) 12 November 1, 2017

NAMES Telephone Electronic Mail Address

PRINCIPAL INVESTIGATOR Benjamin J. Laurel 541-867-0197 [email protected] CO-PRINCIPAL INVESTIGATOR Louise A. Copeman & John Incardona BUSINESS OFFICER

To the best of my knowledge and belief, all data in this application/pre-application are true and correct, the document has been duly authorized by the governing body of the applicant and the applicant will comply with the attached assurances if the grant is awarded. Typed Name of Authorized Representative Title Telephone No. See attached cover sheets by institution See attached cover sheets by institution Signature of Authorized Representative Date Signed 9/25/17

*SUBMISSION OF SOCIAL SECURITY NUMBERS IS VOLUNTARY AND WILL NOT AFFECT THE ORGANIZATION’S ELIGIBILITY FOR AN AWARD. OSRI Form 10/16 PROJECT SUMMARY

Arctic cod (Boreogadus saida) are important components of Arctic food webs, channeling lipid- rich energy between plankton and higher trophic levels such as marine mammals and seabirds. Arctic cod have physiology adapted for growing and storing energy in their cold-water environment, but environmental conditions that reduce growth or energy allocation (e.g., temperature, disease, toxins) will likely reduce their overwintering survival in their 1st year of life. The objective of this project is to quantify the delayed impacts of embryonic oil exposure on growth and energetic condition (lipid content) of later larval and early juvenile stages of Arctic cod. By addressing the non-lethal chronic effects of embryonic oil exposure to later life stages in Arctic cod this project will provide an ecologically meaningful measure of injury assessment for this species. Further, this project will deliver information on how low dose oil-exposure (100 μg oil/L) affects Arctic cod quality as a forage fish for higher trophic levels. This research directly addresses OSRI’s mission by providing measurable damage assessment characteristics in Arctic cod (lipid content, growth rate). This will help scientists, industry and Arctic communities understand the delayed long-range effects of low-dose embryonic exposure to a key-stone fish species and indirectly to the health of the Arctic marine ecosystem. Measurements and techniques developed in this project could be widely adapted to other important gadids in the sub-Arctic region such as walleye ( chalcogrammus) or (Gadus macrocephalus), the two most commercially important ground fisheries in the USA. This proposed research capitalizes and directly builds on recent live- experiments on Arctic cod within NOAA-AFSC (~$900,000) and $200,000 of new 2016-17 investment by NOAA ORR towards embryonic oil exposure experiments on Arctic cod.

PROJECT DESCRIPTION

Background: Arctic cod (Boreogadus saida) is a federally managed species in the Chukchi and Beaufort Seas with an abundant, wide-spread distribution throughout the circumpolar Arctic region (NPFMC 2009). Arctic cod have a pelagic life-history and episodically occupy coastal zones (Craig et al. 1982) and serve an essential component of polar food webs by transferring energy from the plankton to upper trophic levels such as marine mammals, birds, and other fish (Fig. 1, Whitehouse et al. (2014). Significant changes in the abundance and distribution of Arctic cod will likely be catastrophic, leading to widespread food web changes, particularly in ice- obligate species. As a key-stone species, the loss of Arctic cod will likely lead to a reorganization of the Figure 1. The mid-trophic level fish trophic community structure in the Arctic (Bluhm & assemblage of Arctic cod forms a Gradinger 2008). critical link in the food web (from Moore & Stabeno 2015). The population dynamics of Arctic cod are poorly understood, but the overwintering period is considered a critical period for most marine fish species in Arctic regions. Juvenile fish must develop, grow and store lipid reserves rapidly during their first year to minimize predation and maximize overwintering survival (Sogard 1997, Copeman et al. 2008). This is especially important in the Arctic where the summers are short and prolonged winter-spring temperatures are <0 °C (Bouchard & Fortier 2008, 2011). High levels of lipid storage at the end of the short and pulsed summer feeding period are essential for Arctic fish species to survive harsh overwintering conditions of sub-zero temperatures and scarce food resource lasting up to nine months. Therefore, environmental conditions that reduce growth or energy storage in the form of lipids may reduce recruitment to the adult population by way of size- and energy-dependent overwintering survival. Despite this knowledge very few toxicology experiments have followed the long-term growth of fish and potential recruitment dynamic impacts caused by embryological exposure to oil (Heintz et al. 2000). Increased interest in offshore oil development and the presence of higher amounts of ship traffic in the Arctic threaten poorly understood ecologically important marine species. Determining possible injuries to Arctic cod from oil exposure is critical to predicting and determining the impacts of an oil spill on this keystone species and the Arctic ecosystem, which aligns with OSRI’s mission to understand the effects of spilled oil on Arctic environments. In 2017, funding from NOAA-ORR and in kind support from NOAA-NMFS (AFSC and NWFSC), supported the first embryonic exposure experiment of Arctic cod to low concentrations of crude oil (controls, 100, 300, 900 ug/L whole oil exposure equivalent to 1 – 9 ug/L total PAH). Embryos were exposed to environmentally relevant concentrations of oil for a short 3 day period (~5% of their egg stage duration) using a new continuous generated oil dispersion laboratory (Fig 2., SINTEF methods; Nordtug et al. (2011)).

A C

B

Figure 2. The experimental set-up for embryonic exposure of Arctic cod to Alaskan crude oil showing: (A) the large flow-through cold water tanks housing exposure units and tubing from the SINTEF dosing system, (B) a close-up of the surface in glass exposure tanks demonstrating the dose- dependent slick formation and (c) a schematic of the exposure unit showing embryos exposed to a surface slick for 3 days followed by a 4 day wash-out period.

Results from this short embryonic exposure indicated a clear dose-response effect in larval phenotypes, particularly in cardiac and craniofacial defects that have been described in other fish species (Fig. 3a, Incardona et al. (2005)). Exposed embryos also had reduced size-at-hatch (Fig. 3b) and reduced initial growth during the first 2 weeks after yolk absorption.

Fig 3. The morphology at hatch shows a clear dose response demonstrate with increased level of edema around the yolk and high rates of facial deformities in the 900 and 300 μg oil/L water treatment. Further, a significant effect of oil dosage on standard length at hatch was measured and explained 94% of the variance in size-at-hatch.

Characterizing the energetic status of fish can be done in numerous ways (e.g., Fulton’s K, hepatosomatic index) but is most accurately achieved by measuring the amount of lipids in different body tissues. Lipids are a limiting nutrient in cold-water marine food webs (Litzow et al. 2006) and are particularly restrictive to growth and survival at the onset of first-feeding (Sargent et al. 1999) and during the juvenile stages of marine gadid development (Copeman et al. 2008, Copeman et al. 2009). The major lipid classes in fish include triacylglycerols (TAG), sterols (ST) and phospholipids (PL) (Parrish 1998). TAG is the major storage lipid class in gadids while PL and ST are important components of cellular membranes. However, recently PL has also been demonstrated to be an important energy source for eggs, larvae as well as low-lipid juveniles ((Copeman et al. 2008, Laurel et al. 2010). Improvements in fish condition in Atlantic herring (Clupea harengus), (Gadus morhua) and Pacific cod have previously been attributed to elevated total lipid, TAG density and TAG/ST ratios (Lochmann et al. 1995, Copeman & Laurel 2010). As such, these detailed lipid metrics provide an accurate reflection of overall fish condition, and given their near instantaneous tissue incorporation (Amara et al. 2007, Copeman & Laurel 2010), are well-suited for assessing the impacts of environmental conditions on fish performance and health.

Fig 4. Total storage lipids (μg/individual, yolk lipids) in Arctic cod larvae at fertilization (day-0), post-exposure (day 26) and at hatch (day 44) in larvae exposed to the 900, 300, 100 μg oil/L water treatment plus a control. Data represent the mean ± SE of four replicate tanks. Significant differences at P<0.05, ANOVA with Tukey’s Pairwise comparison.

Lipid analyses has been completed on Arctic cod eggs at fertilization, eggs from each treatment post exposure and on larvae at 100% hatch. Total lipids and lipid classes were not significantly different between oil treatments in eggs post-exposure. However, at hatch larvae showed significant differences in their lipid class composition. TAG levels (see Fig 4) were elevated in larvae at hatch in the high dose treatments and decreased in a dose dependent manner to significantly lower levels in controls. Elevated triacylglycerols in oil-exposed larvae indicated an inability of these larvae to utilize yolk lipid stores. Further, decreased trends in phospholipids at hatched indicated that they were mobilizing body membrane lipids for basic metabolic function, and finally elevated levels of free fatty acids (metabolic intermediates) in high dosed larvae indicated that larvae were showing signs of disruption of normal lipid metabolic pathways (data only shown for triacylglycerols, Copeman unpublished data, Fig 4.). These measures of lipids in larvae following embryonic exposure agree with published accounts in Haddock that have shown disruption of lipid metabolic pathways as indicated by transcriptomic data (Sørhus et al. 2017).

Collectively, morphometric, growth and lipid data indicate that Arctic cod embryos are very sensitive to oil exposure, showing toxic effects at the lowest dose tested; approximately 1 ppb total PAHs. However, several questions remain: 1) Are the non-lethal effects of embryonic exposure to oil apparent at later feeding life stages and 2) how do embryonic injuries impact the survival potential and ecosystem services provided by Arctic cod via reduced growth and lipid content in the late summer and early fall?

The NOAA-AFSC has collected samples and continues to hold Arctic cod juveniles exposed to control and low dose conditions (1 ug/L PAH) in separate, quadruplicate tanks at the Hatfield Marine Science Center (HMSC). We propose to determine and quantify the possible delayed impacts on growth and condition (total lipids and lipid classes) on these fish from first-feeding stages through to the juvenile stage prior to overwintering. This represents a unique value-added opportunity that would capitalize on nearly 5 years of broodstock development and live-animal research on Arctic cod working towards carrying out the first oil exposure experiment on Arctic cod embryos (~$1,100,000 investment by NOAA, NPRB and ORR).

Fig. 5 Experimental design included the portion proposed to OSRI on feeding larval which follows the hatch period at day 45 post-fertilization.

Approach and Methods: Low dose oil-exposed Arctic cod and control fish were held in replicate tanks at the HMSC 6 months following embryonic exposure for a total of ~170 past the time of oil exposure (see Fig. 5). Fish have been sampled for growth (non-lethal) at 1-month intervals under identical environmentally controlled conditions.

Whole animal lipid class content will be assessed at 5 critical growth periods in the early life history: late larval (May), early summer larval- juvenile transition (June & July), late summer juvenile (August) and fall pre-wintering juvenile (September).

Fish were sampled at each time-step in the experiment for morphometrics (image analyses), dry mass (DWT), total lipids and lipid class analyses. Samples ranged from 2 and 5 composite samples/tank for DWT at each monthly time-step in the experiments (5 time-steps x 2 treatments x 4 replicates/treatment x 3 samples/replicate ~120 DWT samples). At each time-step we also imaged larvae for body depth, standard length and eye diameter (~300 images). Further, at each time-step a composite sample ranging from 50 at day 66 to 5 individuals per tank at day 156 were sampled for lipid analyses. Decreased numbers of individuals were required for lipid analyses as larvae increased in mass. During the last sampling date, 8 individual fish were sampled for lipid analyses from each tank to account for individual variation with variable growth rates and energy storage at day 193 (See Fig. 5). This resulted in a total of 100 lipid samples collected for Arctic cod over the 5 feeding sampling periods. Lipid samples are currently stored at -80°C in chloroform under nitrogen until extraction (Copeman et al. 2008, Copeman et al. 2009).

Lipid analyses: We will use total lipids and lipid class analyses to assess the condition of larval and juvenile stages (i.e., quantitative lipid storage) in response low dosage and control treatment conditions (Fig. 5). Tissues will be homogenized in chloroform and methanol and total lipids will be extracted according to Parrish (1987) using a modified Folch procedure (Folch et al. 1956). Lipid classes will be determined using thin layer chromatography with flame ionization detection (TLC/FID) with a MARK V Iatroscan (Iatron Labratories, Tokyo, Japan) and modified from those methods described by (Lu et al. 2008). Extracts will be spotted on duplicate silica gel coated Chromarods and a three-stage development system was used to separate lipid classes (lipid classes quantified were: wax esters, triacylglyerols, free fatty acids, sterols and polar lipid, as in Copeman et al. (2016)). Following the last development, rods will be scanned using Peak Simple software (ver. 3.67, SRI Inc.) and the signal detected in millivolts will be quantified using lipid standards (Sigma, St Louis, MO, USA). A specific triacylglycerol standard purified from walleye pollock liver will be used Copeman et al. (2016). Lipid classes will be expressed both in relative (% of total lipids) and absolute amounts (lipid per WWT, mg/g).

Growth will be assessed on both length (mm SL) and weight (dry mass) will be developed for each species using a 3-parameter polynomial function as per growth models. Tank replicates will be used as the level of observation for models of total lipid and lipid classes in each species. Tank replicates will also be used to test for statistical differences in the Generalized Linear Models.

*Note, all samples are currently archived at -80°C in sampling periods that precede the proposed funding timeline of the project.

RESEARCH TEAM COORDINATION (If applicable) Linkages between current field and modeling efforts: This proposal integrates with the broader ecosystem assessment and field effort (Arctic IES Phase II; NPRB 5 yr Arctic Integrative Ecosystem Research Program (IERP)) by focusing on environmental effects on varying ontogenetic stages of Arctic cod. The laboratory data this proposal will provide additional mechanistic understanding of varying size-at-age and condition of specimens sampled from different water masses in the field. Moreover, this proposal uses laboratory reared with known environmental histories that can opportunistically be used to parameterize models and validate tools used in complementary components of the Arctic cod research in Alaska. For example, larval and juvenile Arctic cod growth parameters are currently being used by Dr. Franz Mueter at UAF to develop biophysical transport models for Arctic cod in the Chukchi and Beaufort Seas using the Hedstrom/Danielson 3-D ocean-ice circulation model. Bioenergetic parameters from this proposal also have direct use in FEAST models (Forage Euphasids Abundance in Space and Time), a size-based bioenergetics model used in the Bering Sea IERP. Additional whole-body or tissue component samples from experiments can be provided to other research collaborators at the discretion of OSRI. Specific links already include lipid/calorimetry energetics as part of the mid-trophic proposal by NOAA-AFSC and future investigations of gene expression, genotyping, RNA/DNA validation, respiration studies and cellular physiology.

Expected outcomes and deliverables: Successful completion of the proposed experiments will provide the first information on the bioenergetic impact of low dosage oil exposure on Arctic cod during their early life history. These data alongside ongoing work on early life history growth and lipid allocation in Arctic cod during summer and winter. These include North Pacific Research Board (NPRB) projects #1228 (http://projects.nprb.org/#metadata/e9cb3579-4fd4-493c-b9d7-58885930b218/project) and #1403 (http://projects.nprb.org/#metadata/744fc858-9df4-453b-ba77-f1cf29958e66/project) and 2016- 2020 Arctic IERP. Together, these data will provide information on whether oil-impacted fish can survive their first overwintering in the Arctic. The data from this project is anticipated to result in a peer-reviewed manuscripts on such effects. Each of the PIs and collaborators of this project has an established track record of publishing under previously funded projects on bioenergetics and/or oil impacts in early life stages of marine fish.

Integration with existing projects and reliance on other sources of data: The strength of this proposal is that it is both self-contained yet integrates with existing components of toxicology and bioenergetics research on Arctic cod within NOAA and academic institutions. All specimens proposed for use in laboratory experiments have already been collected and are currently available for the proposed research. Husbandry protocols (egg, larval, juvenile and adult) are also established, with the majority of samples already archived for analysis in this project. As such, there is relatively low risk in completing experiments and obtaining fish samples for this project. This project most directly links with the 2016-17 ARD support for embryonic exposures on Arctic cod but will also support cross-benefit laboratory research within NOAA, NPRB and Oregon State University examining causes and consequences of annual variability of size-at-age and condition of juvenile Arctic cod in the Chukchi and Beaufort Sea. This proposal will provide additional data on environmental impacts on growth/lipids in this species.

Project Management: The assembled team has combined expertise in live-animal experiments, lipids, and modeling while having a collaborative network in place to integrate with outside field teams to address climate change impacts in the Arctic on important fish species. Lead PI Laurel and co- investigator Copeman have a proven record of successful collaboration on bioenergetics in Alaskan gadids (including Arctic cod), which has resulted in 15 peer-reviewed publications in the past 5 years. Co-investigator John Incardona (NOAA-NWFSC) is a developmental biologist/toxicologist who has worked on environmental teratogens and gene-environment interactions since 1997. In addition, the collaborators have an established track-record in ecotoxicology and laboratory oil exposures of marine fish (Sonnich Meier (Norway IMR), Trond Nordtug (Norway SINTEFF), Nathaniel Scholz (NOAA-NWFSC)).

Ben Laurel will be the overall project lead and will be fully responsible for project coordination and ensuring the project’s overall success. He will supervise all experimental work closely and integrate bioenergetic data to assess delayed injury effects of oil on juvenile Arctic cod. He has >15 years of experience researching the effects of temperature on the vital rates of larval and juvenile marine fish in the Arctic as well as the North Pacific and North Atlantic. He has led field and laboratory research involving graduate students and technical staff under funding from NPRB, NOAA and NSERC Canada. He has experience in several interdisciplinary projects, including the Canadian Healthy Oceans Network (CHONE) and NPRB’s BSIERP and Gulf of Alaska IERP. Louise Copeman has used lipids/FAs for >10 years to determine condition and food web linkages in cold-water marine larval and juvenile fish. She brings to the project extensive experience in nutritional live-feeding studies in the Atlantic and Pacific and has published multiple studies on the effects of essential fatty acids (EFAs) in marine larval fish and invertebrates. She is also a PI on the current Arctic IERP project funded by NPRB. Dr. Copeman will be responsible for lipid analyses, sampling, metadata and preparation of manuscripts. John Incardona is an expert on environmental pollution impacts on fish heart performance and developmental toxicity in marine fish species. He has an established publication record and will be responsible for integrating ecological metrics from this proposal within an ecotoxicology perspective.

All investigators and collaborators are currently involved with research on egg/larval exposures of Arctic cod embryos, thereby facilitating coordination and communication for this project. As needed, outside coordination with the remaining collaborators will be achieved through annual in-person meetings in conjunction with bi-month conference call meetings.

Benjamin J. Laurel, PhD

Contact information Fisheries Behavioral Ecology Program, Alaska Fisheries Science Center NOAA Fisheries, Hatfield Marine Science Center, 2030 SE Marine Science Dr, Newport OR 97365 USA PHONE: (541) 867-0197, FAX: (541) 867-0136, E-MAIL: [email protected]

Education B.A. (Hons.), St. Olaf College (1994); M.Sc., Memorial University (1998); Ph.D., Memorial University (2003).

Employment Research Fisheries Biologist, NOAA-AFSC (2005-Present); Postdoctoral Researcher, Department of Fisheries and Oceans - Newfoundland (2003-2005).

Research Interests Early life history fish physiology and behavior, thermal ecology, juvenile fish habitat, connectivity, bioenergetics.

Publications (5 most recent) Laurel BJ, Cote D, Gregory RS, Rogers L, Olsen E (2017) Recruitment signals in juvenile cod surveys depend on thermal growth conditions. Can J Fish Aquat Sci. 74(4): 511-523, DOI: 10.1139/cjfas-2016-0035 Laurel BJ, Spencer M, Iseri P, Copeman L (2017) Temperature-dependent growth as a function of size and age in juvenile Arctic cod (Boreogadus saida) ICES Journal of Marine Science, 74: 1614–1621 Copeman LA, Laurel BJ, Spencer M, Sremba A (2017) Temperature impacts on lipid allocation among juvenile gadid species at the Pacific Arctic−Boreal interface: an experimental laboratory approach. Marine Ecology Progress Series 566:183-198. DOI: 10.3354/meps12040 Laurel BJ, Spencer M, Iseri P, Copeman L (2016) Temperature-dependent growth and behavior of juvenile Arctic cod (Boreogadus saida) and co-occurring North Pacific gadids. Polar Biology DOI :10.1007/s00300-015-1761-5 Laurel BJ, Knoth BA, Ryer CH (2016) Growth, mortality, and recruitment signals in age-0 gadids settling in coastal Gulf of Alaska. ICES Journal of Marine Science 73: 2227–2237. doi.org/10.1093/icesjms/fsw039

Publications (5 relevant) Copeman LA, Laurel BJ, Sremba A, Klinck K, Heintz R, Vollenweider J, Boswell, K (2015) Ontogenetic variability in the lipid content of saffron Cod ( gracilis) from the Western Arctic and Northern Chukchi. Polar Biology DOI: 10.1007/s00300-015-1792-y Laurel BJ, Copeman LA, Parrish CC (2012) Role of temperature on lipid/fatty acid composition in Pacific cod (Gadus macrocephalus) eggs and unfed larvae. Marine Biology 159: 2025- 2034. Laurel BJ, Hurst TP, Ciannelli L (2011) An experimental examination of temperature interactions in the atch-mismatch hypothesis for Pacific cod larvae. Canadian Journal of Fisheries and Aquatic Sciences. 68: 51-61. Laurel BJ, Copeman LA, Parrish C, Hurst TP (2010) The ecological significance of lipid/fatty acid synthesis in developing eggs and unfed larvae of Pacific cod (Gadus macrocephalus). Marine Biology 157:1713-1724. Laurel, B.J., Copeman, L.A., Hurst, T.P., Davis, M.W. (2008) The role of temperature on the growth and survival of early and late hatching Pacific cod larvae (Gadus macrocephalus). Journal of Plankton Research. 30: 1051-1060.

Lead Funding (past 5 years)

2016-17 $142,000 NOAA-ARD Co-PI’s: Allan, Incardona, Scholz 2016-17 $85,000 NOAA/HEPR Co-PI’s: Heinz, Copeman, Ryer, Hurst. Overwintering in juvenile walley pollock 2014-17 $217,000 NPRB Co-PI’s: Copeman. Pacification of the Arctic 2014 $69,000 NOAA/HEPR Co-PI’s: Copeman, Ryer. Thermal habitat of snow crab 2013 $68,000 NOAA/HEPR Co-PI’s: Copeman, Ryer. Optimal thermal habitats of gadids in Alaskan waters 2012-16 $178,487 NPRB Co-PI’s: Copeman, Heintz. Arctic cod in a warming ocean 2012 42,800 NOAA/HEPR Co-PI: Stoner. The role of benthic habitat in larval rock sole settlement dynamics (yr2)

PI Collaborators – Last 4 years Louise Copeman – Oregon State University Cliff Ryer – NOAA/NMFS/AFSC Robert Gregory – DFO Canada Newfoundland* Lauren Rogers – IMR Norway Esben Olsen – University of Oslo Halvor Knutsen – University of Oslo Ron Heintz – NOAA / NMFS / AFSC David Cote – Parks Canada Newfoundland Brian Knoth - NOAA / NMFS / AFSC Ian Bradbury – DFO Canada – Halifax, NS Franz Mueter – University of Alaska Fairbanks Seth Danielson – University of Alaska Fairbanks Lorenzo Ciannelli – Oregon State University Thomas Helser – NOAA / NMFS / AFSC Michael Davis – NOAA / NMFS / AFSC Janet Duffy-Anderson – NOAA/NMFS/AFSC Ed Farley – NOAA / NMFS / AFSC Allan Stoner – NOAA / NMFS / AFSC* Kevin Boswell – Florida *Also served in an advisory role Louise A. Copeman, PhD

Louise A. Copeman Assistant Professor (Senior Research), College of Earth, Ocean and Atmospheric Sciences (CEOAS), Oregon State University Tel: (541) 867-0165 Hatfield Marine Science Center, FAX: (541) 867-0292 Newport, OR 97365 Email: [email protected]

Professional Preparation B.Sc., 1996 Biology (Hons) Memorial University of Newfoundland M.Sc., 2001 Aquaculture Memorial University of Newfoundland Ph.D., 2011 Marine Ecology Memorial University of Newfoundland Postdoctoral, 2011-13 Marine Ecology CIMRS, Oregon State University

Appointments July 2013- Current Assistant Professor, CEOAS, Oregon State University (OSU) May 2012-July 2013 Research Associate, CIMRS, OSU 2010-May 2012: Post-Doctoral Research Associate, CIMRS, OSU 2006-2010: Research Assistant, Alaskan Fisheries, NOAA 2001-2003: Research Assistant, Dr. C. Parrish, Lipid Chemistry Laboratory, Memorial University, NL Canada.

Research Interests My research in marine ecology resides at the interface of marine chemistry and biology in the area of marine lipids research. Lipids are important biochemical components of marine food webs as they are carbon-rich and provide a concentrated source of energy. Through the analyses of lipid classes as well as fatty acid and sterol biomarkers I am able to explore food quality and dietary carbon sources for marine consumers in both temperate and Arctic marine environments. I use chemical biomarkers to understand predator-prey relationships and to facilitate an understanding of larger scale oceanographic processes. These combined approaches further our understanding of how climate change and oceanographic processes at lower trophic levels may affect food quality and condition of zooplankton, fish and ultimately top end marine predators.

Laboratory Facilities In 2011 Louise Copeman established and continues to manage the Marine Lipids Laboratory at the Hatfield Marine Sciences Center. It is a multiple user facility that represents collaboration between NOAA and OSU Scientists. Equipment grants and long-term equipment loans now support a laboratory package that is >$ 200,000 for GC and TLC-FID capabilities. Since 2011 this laboratory has serviced over 20 external supported (6 NOAA) research projects.

5 Relevant Publications Copeman LA, Laurel BJ, Spencer M, Sremba A (2017) Temperature impacts on lipid allocation among gadid species at the Pacific Arctic–Boreal interface: an experimental laboratory approach. Marine Ecology Progress Series 566:183–198. Copeman LA, Laurel BJ, Sremba A, Klinck K, Heintz R, Vollenweider J, Boswell K (2016) Ontogenetic variability in the lipid content of saffron Cod (Eleginus gracilis) from the Western Arctic and Northern Chukchi. Polar Biology 39 (6): 1109–1126 Laurel BJ, Spencer M, Iseri P, Copeman LA (2016) Temperature-dependent growth and behavior of juvenile Arctic cod (Boreogadus saida) and co-occuring North Pacific gadids. Polar Biology 39 (6): 1127–1135. Copeman LA, Laurel BJ, Parrish CC (2013) Effect of temperature and tissue type on fatty acid signatures of two species of North Pacific juvenile gadids: A laboratory feeding study. Journal of Experimental Marine Biology and Ecology 448, 188-196 Copeman LA, Parrish CC, Gregory RS, Wells J (2008) Decreased lipid storage in juvenile Atlantic cod (Gadus morhua) during settlement in cold-water eelgrass habitat. Marine Biology 154(5): 823-832

5 Other Publications Laurel BJ, Copeman LA, Spencer M, Iseri P (2017) Temperature-dependent growth as a function of size and age in juvenile Arctic cod (Boreogadus saida). ICES J Mar Sci. DOI: 10.1093/icesjms/fsx028. Copeman LA, Laurel BJ (2010) Experimental evidence of fatty acid limited growth and survival in Pacific cod (Gadus macrocephalus) larvae. Marine Ecology Progress Series. 412:259-272. Copeman LA, Parrish CC, Gregory RS, Jamieson, RE, Well J, Whiticar MJ (2009) Fatty acid biomarkers in coldwater eelgrass meadows: elevated terrestrial input to the food web of age-0 Atlantic cod (Gadus morhua). Marine Ecology Progress Series 386:237-251. Copeman LA, Daly, B, Eckert GL, Swingle J. 2014. Storage and utilization of lipid classes and fatty acids during the early ontogeny of blue king crab, Paralithodes platypus. Aquaculture 424: 86-94. Copeman LA, Parrish CC, Brown JA, and Harel M (2002) Effects of DHA, EPA and AA on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (Limanda ferruginea); a live food enrichment experiment. Aquaculture 210: 185- 204.

Collaborations in the last 48 months Brodeur R (NWFSC, NOAA, Newport, OR), Daly B (AFSC, NOAA, Kodiak, AK), Davis M (AFSC, NOAA, Newport, OR), Eckert G (University of Alaska), Gregory RS (DFO Canada, St. John’s), Hurst TP (AFSC, NOAA, Newport, OR), Langdon C (COMES, OSU), Laurel BJ (AFSC, NOAA, Newport, OR), Parrish CC (Memorial University of Newfoundland, Canada), Peterson W (NWFSC, NOAA, Newport, OR), Ryer C (AFSC, NOAA, Newport, OR), Snelgrove P (Memorial University of Newfoundland), Stoner A (AFSC, NOAA, Newport, OR), Jessica Miller (Oregon State University).

BIOGRAPHICAL SKETCH Provide the following information for the key personnel in the order listed for Form Page 2. Follow the sample format for each person. DO NOT EXCEED FOUR PAGES.

NAME POSITION TITLE

John P. Incardona Supervisory Research Toxicologist

EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.) INSTITUTION AND LOCATION DEGREE YEAR(s) FIELD OF STUDY (if applicable) Indiana University, Bloomington, IN BS 1988 Honors Biology Case Western Reserve University, Cleveland, OH PhD 1995 Genetics Case Western Reserve University, Cleveland, OH MD 1996 University of Washington, Seattle, WA Post-doc 1996-2001 Developmental Biology

A. 5 Most Recent Peer-reviewed Publications 1. Brette F, Shiels HA, Galli GL, Cros C, Incardona JP, Scholz NL, Block BA. (2017) A Novel Cardiotoxic Mechanism for a Pervasive Global Pollutant. Scientific Reports 7:41476 2. Sørhus E, Incardona JP, Karlsen Ø, Linbo T, Sørensen L, Nordtug T, van der Meeren T, Thorsen A, Thorbjørnsen M, Jentoft S, Edvardsen RB, Meier S. Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development. (2016) Scientific Reports 6:31058 3. Sørhus E, Incardona JP, Furmanek T, Goetz GW, Scholz NL, Meier S, Edvardsen RB, Jentoft S. (2017) Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish. eLife 10.7554/eLife.20707 4. Incardona JP, Scholz NL. The influence of heart developmental anatomy on cardiotoxicity-based adverse outcome pathways in fish. (2016) Aquatic Toxicology 177:515-525 5. Sørhus E, Incardona JP, Furmanek T, Jentoft S, Meier S, Edvardsen RB. Developmental transcriptomics in Atlantic haddock: Illuminating pattern formation and organogenesis in non- model vertebrates. (2016) Developmental Biology 411:301-313.

B. 5 other Relevant Peer-reviewed Publications 6. Edmunds RC, Gill JA, Baldwin DH, Linbo TL, French BL, Brown TL, Esbaugh AJ, Mager EM, Stieglitz J, Hoenig R, Benetti D, Grosell M, Scholz NL, Incardona JP. Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi. (2015) Scientific Reports 5:17326 7. Edmunds RC, Gill JA, Baldwin DH, Linbo TL, French BL, Brown TL, Esbaugh AJ, Mager EM, Stieglitz J, Hoenig R, Benetti D, Grosell M, Scholz NL, Incardona JP. Corresponding morphological and molecular indicators of crude oil toxicity to the developing hearts of mahi mahi. (2015) Scientific Reports 5:17326

8. Esbaugh AJ, Mager EM, Stieglitz JD, Hoenig R, Brown TL, French BL, Linbo TL, Lay C, Forth H, Scholz NL, Incardona JP, Morris JM, Benetti DD, Grosell M. The effects of weathering and chemical dispersion on Deepwater Horizon crude oil toxicity to mahi-mahi (Coryphaena hippurus) early life stages. (2016) Science of the Total Environment 543:644-651 9. Jung JH, Kim M, Yim UH, Ha SY, Shim WJ, Chae YS, Kim H, Incardona JP, Linbo TL, Kwon JH. Differential Toxicokinetics Determines the Sensitivity of Two Marine Embryonic Fish Exposed to Iranian Heavy Crude Oil. (2015) Environmental Science and Technology 49:13639- 13648. 10. Incardona JP, Carls MG, Holland L, Linbo TL, Baldwin DH, Myers MS, Peck KA, Rice SD, and Scholz NL. Very low embryonic crude oil exposures cause lasting cardiac defects in salmon and

B. Competitive Grants Awarded

Project Title: Population-level impacts of acute and latent toxicity effects in oil exposed herring and Arctic cod Funding source: NOAA National Ocean Service, Office of Response and Restoration Co-investigators: Nathaniel Scholz, NOAA/NWFSC; Ben Laurel, NOAA/AFSC-Newport Dates of support: FY16-18 Direct costs annually: $250,000 – 300,000 50% effort

Project Title: Population-level impacts of acute and latent toxicity effects in oil exposed herring and Arctic cod Funding source: Prince William Sound Regional Citizens’ Advisory Council Co-investigators: Nathaniel Scholz, NOAA/NWFSC; Ben Laurel, NOAA/AFSC-Newport Dates of support: FY16-18 Direct costs annually: $75,000 50% effort

Project Title: EGGTOX: Unraveling the mechanistic effects of crude oil toxicity during early life stages of cold-water marine teleosts Funding source: Research Council of Norway Co-investigators: Sonnich Meier, Institute of Marine Research – Norway; Nathaniel Scholz, NOAA/NWFSC Dates of support: FY17-19 Direct costs total: NOK11,588,200 10% effort

Project Title: Reduced recruitment of Pacific herring due to oil spill impacts on early development, growth and disease resistance Funding source: North Pacific Research Board Co-investigators: Louise Copeman, Oregon State University Dates of support: FY18 Direct costs annually: $208,000 50% effort

Grant Application - OSRI

REFERENCES CITED Amara R, Meziane T, Gilliers C, Hermell G, Laffargue P (2007) Growth and condition indices in juvenile sole Solea solea measured to assess the quality of essential fish habitat. Mar Ecol-Prog Ser 351:201-208 Bluhm BA, Gradinger R (2008) Regional variability in food availability for arctic marine mammals. Ecol Appl 18:77-96 Bouchard C, Fortier L (2008) Effects of polynyas on the hatching season, early growth and survival of polar cod Boreogadus saida in the Laptev Sea. Mar Ecol-Prog Ser 355:247-256 Bouchard C, Fortier L (2011) Circum-arctic comparison of the hatching season of polar cod Boreogadus saida: A test of the freshwater winter refuge hypothesis. Prog Oceanogr 90:105-116 Copeman LA, Laurel BJ (2010) Experimental evidence of fatty acid limited growth and survival in Pacific cod larvae. Mar Ecol-Prog Ser 412:259-272 Copeman LA, Laurel BJ, Boswell KM, Sremba ALand others (2016) Ontogenetic and spatial variability in trophic biomarkers of juvenile saffron cod (Eleginus gracilis) from the Beaufort, Chukchi and Bering Seas. Polar Biol 39:1109-1126 Copeman LA, Parrish CC, Gregory RS, Jamieson RE, Wells J, Whiticar MJ (2009) Fatty acid biomarkers in coldwater eelgrass meadows: elevated terrestrial input to the food web of age-0 Atlantic cod Gadus morhua. Mar Ecol-Prog Ser 386:237-251 Copeman LA, Parrish CC, Gregory RS, Wells JS (2008) Decreased lipid storage in juvenile Atlantic cod (Gadus morhua) during settlement in cold-water eelgrass habitat. Mar Biol 154:823-832 Craig PC, Griffiths WB, Haldorson L, McElderry H (1982) Ecological studies of Arctic cod (Boreogadus saida) in Beaufort Sea coastal waters, Alaska. Can J Fish Aquat Sci 39:395-406 Folch J, Less M, Sloane Stanley GH (1956) A simple method for the isolation and purification of total lipids from animal tissues. The Journal of Biological Chemistry 22:497-509 Heintz RA, Rice SD, Wertheimer AC, Bradshaw RF, Thrower FP, Joyce F, Short JW (2000) Delayed effects on growth and marine survival of pink salmon Oncorhynchus gorbuscha after exposure to crude oil during embryonic development. Mar Ecol-Prog Ser 208:205-216 Incardona JP, Carls MG, Teraoka H, Sloan CA, Collier TK, Scholz NL (2005) Aryl Hydrocarbon Receptor–Independent Toxicity of Weathered Crude Oil during Fish Development. Environmental Health Perspectives 113:1755-1762 Laurel BJ, Copeman LA, Hurst TP, Parrish CC (2010) The ecological significance of lipid/fatty acid synthesis in developing eggs and newly hatched larvae of Pacific cod (Gadus macrocephalus). Mar Biol 157:1713-1724 Litzow MA, Bailey KM, Prahl FG, Heintz R (2006) Climate regime shifts and reorganization of fish communities: the essential fatty acid limitation hypothesis. Mar Ecol-Prog Ser 315:1-11 Lochmann SE, Maillet GL, Frank KT, Taggart CT (1995) Lipid class composition as a measure of nutritional condition in individual larval Atlantic cod (Gadus Morhua). Can J Fish Aquat Sci 52:1294-1306 Lu YH, Ludsin SA, Fanslow DL, Pothoven SA (2008) Comparison of three microquantity techniques for measuring total lipids in fish. Can J Fish Aquat Sci 65:2233-2241 Nordtug T, Olsen AJ, Altin D, Overrein I, Storøy W, Hansen BH, De Laender F (2011) Oil droplets do not affect assimilation and survival probability of first feeding larvae of North-East Arctic cod. Science of The Total Environment 412:148-153 NPFMC (2009) North Pacific Fishery Management Council. 2009 (second edition). Navigating the North Pacific Council Process. Parrish CC (1987) Separation of aquatic lipid classes by chromarod thin-layer chromatography with measurement by Iatroscan flame ionization detection. Can J Fish Aquat Sci 44:722-731 Parrish CC (1998) Lipid biogeochemistry of plankton, settling matter and sediments in Trinity Bay, Newfoundland. I. Lipid classes. Org Geochem 29:1531-1545 Sargent J, McEvoy L, Estevez A, Bell G, Bell M, Henderson J, Tocher D (1999) Lipid nutrition of marine fish during early development: current status and future directions. Aquaculture 179:217-229 Sogard SM (1997) Size-selective mortality in the juvenile stage of teleost fishes: A review. Bulletin of Marine Science 60:1129-1157 Sørhus E, Incardona JP, Furmanek T, Goetz GWand others (2017) Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish. eLife 6:e20707

Grant Application - OSRI

Whitehouse GA, Aydin K, Essington TE, Hunt GL (2014) A trophic mass balance model of the eastern Chukchi Sea with comparisons to other high-latitude systems. Polar Biol 37:911-939

Grant Application - OSRI

BUDGET JUSTIFICATION- NOAA-AFSC Start date is November 1, 2017 and end date is October 30, 2018.

Total Amount requested by NOAA-AFSC for this project: $24,072

Personnel/Salaries: No funding is requested for NOAA personnel/salaries.

Travel: $1,884 Travel funds are requested for 3 roundtrips between Newport, OR and Seattle, WA via car for PI Laurel (2 trips) and co-PI Incardona (1 trip). The purposes of travel will be to oversee sampling, exchange data/tissues samples, and presentation/coordination of proposed research on Arctic cod. Total estimated costs are $1,884.

Equipment: $0

No Equipment is requested

Supplies/Commodity/Other: $2,500

A total of $2,500 is requested to cover supplies associated with preserving, preparing, measuring and analyzing samples (e.g., gloves, glassware, foils, chemicals).

Other: $0

Contractual/Consultants: $18,400 A total of $18,400 is requested to pay for a 2.5 Month contract technician@$26/hr+20$ overhead via an approved NOAA contracting agency (e.g., Lynker contracting). The technician will assist with husbandry, sampling and tissue preparation within the wet and dry lab components of the project.

Indirect Costs: $1,288

NOAA-AFSC has an indirect cost of 7% on all contracts < $150,000.

Total direct costs amount to $22,784 Total indirect costs amount to $1,288 Total NOAA-AFSC funds requested from OSRI are $24,072

Grant Application - OSRI

BUDGET JUSTIFICATION- OREGON STATE UNIVERSITY Sensitivity of Arctic cod embryos and larvae to oil; delayed impacts on juvenile growth and lipid condition. –Oregon State University

Start date is November 1, 2017 and end date is October 30, 2018.

Total Amount requested by Oregon State University for this project: $53,194

Personnel/Salaries:

PI Copeman will devote 2.25 month to this project in FY18. Dr. Copeman will be responsible for supervising the lipid class analyses of Arctic cod in relation to the energy loss of control and low-dose exposed fish. She will contribute to writing peer-review papers with the other PIs on the energetics and lipid metabolism of Arctic cod larval previously exposed to oil during embryological development.

Personnel/Fringe Benefits:

Fringe benefits (OPE) rates were calculated using Oregon State University Budget Guidelines.

Personnel Expense Details: Time devoted Overtime Personnel cost Fringe Fringe cost Year Title/Name to project rate per month rate per month FY18 Louise 2.25 months $0 $6,013 0.59 $3,548 Copeman

Totals 2.25 $13,529 $7,982

Total salary and fringe costs are $21,511.

Travel: No Travel is requested.

Equipment:

No Equipment is requested

Supplies/Commodity/Other: $14,675

Laboratory services for lipid class analyses on Arctic cod tissues completed through the Marine Lipids laboratory at the CIMRS (Cooperative Institute for Marine Resources Studies at Oregon State University): https://fees.oregonstate.edu/Public/BrowseExternal.aspx.

Grant Application - OSRI

We are budgeting for:

100 Arctic cod samples analyzed for Lipid Class Analyses: Fee #11350 - $60 per Sample for a total of $6,000.

100 Arctic cod samples dissected and processed for Total Lipid Extraction: Fee #11310 - $80 per sample for a total of $ 8,000.

Therefore, we require total for supplies and laboratory service fees for 100 samples of $14,000.

Other: $300 per month of computer time is requested for each month that Dr. Copeman is involved with is project (2.25 months). Costs are based on prorated charges for the CEOAS computer network, including network management costs, and workstation maintenance based on PI FTE.

Total other funds requested is $675.

Contractual/Consultants: No contracts are requested $0.

Indirect Costs:

Oregon State University’s federally negotiated indirect cost rate is 47% of modified total direct costs.

Total direct costs amount to $36,187 Total indirect costs amount to $17,007 Total requested from OSRI is $53,194

Total Amount requested by Oregon State University for this project is: $53,194