Letcher, R.J R Dietz

Bytingvisk, J T M Ciesielski

Gabrielsen P E Drevnick

Jenssen, B.M M Levin

Ecotoxicity effects of oil Water Accommodated Fractions and oil Water Accommodated Fractions + Dispersant on cold environments: Acarita tonsa based bioassays and microbial community dynamics as monitoring tools

 Prof. Jasmine Nahrgang (UiT) for her invaluable help in the collection of water and sediment samples

AUTHORISATION OF THE THESIS SUPERVISOR FOR ITS PRESENTATION

Mr/Ms. Maren Ortiz Zarragoitia__________with National ID Card num.16061890R In his/her capacity as Supervisor of the Doctoral Thesis:

Ecotoxicity effects of oil Water Accommodated Fractions and oil Water Accommodated Fractions + Dispersant on cold environments: Acarita tonsa based bioassays and microbial community dynamics as monitoring tools completed within the Doctoral Programme Marine Environment and resources by the PhD student Mr/Ms. Tamer Hafez , having reviewed the SIMILARITY REPORT issued by the tool provided for this purpose by the university, hereby authorises the presentation of the aforementioned Doctoral

Thesis, given that it fulfils the conditions necessary for its viva.

In

PHD STUDENT

Signed_____________________

This work was funded by:

• Basque Government grant to consolidated research groups (IT1302-19).

•

European Union Horizon 2020 Research and Innovation Programme under Grant Agreement No. 679266 (GRACE project).

Table of Contents General Abstract

Oil pollution is a concerning problem in the world seas, especially in cold environments or polar regions where remediation procedures are difficult and the ecosystems are highly vulnerable. After an oil spill, wave dynamics and other weathering factors introduce soluble oil hydrocarbons, mainly, polycyclic aromatic hydrocarbons (PAHs) in the water column, creating oil Water Accommodated Fractions (WAFs). As a form of remediation, chemical dispersants, among others, are added to assist in oil biodegradation processes.

However, chemical dispersants may have adverse effects on marine organisms. The objective of this thesis is to estimate the effects to marine organisms of oil WAF, with or without the addition of a chemical dispersant, in cold or sub-polar conditions, using copepod based bioassays and changes in native microbial communities. Bioassays using the model organism Acartia tonsa, showed that naphthenic North Atlantic (NNA) oil WAF was less toxic than the WAFs prepared from distilled oils (IFO 180 and Marine gas oil), which exhibited toxicity toward the copepods considering endpoints such as the mortality, reproduction and transcription levels of genes involved in the antioxidative and xenobiotic metabolism pathways. The addition of the chemical dispersant Finasol OSR52 to NNA oil WAF increased the toxicity toward copepods. Acartia tonsa exposed to NNA WAF with dispersant showed low fecundity, high mortality and altered gene transcription patterns, further highlighting the toxicity effects of the chemical dispersant. Upon examining the effects of NNA WAF and NNA WAF with dispersant on microbial communities, using microcosm experimental scenarios, Finasol OSR52 dispersant did not improve bacterial PAHs biodegradation rates. However, NNA WAF with dispersant contributed to changes in the microbial community dynamics by selecting certain bacterial taxa in samples of water and sediments from sub-Arctic regions artificially spiked with the WAF. Results showed that microbial communities with initial hydrocarbon degrading bacteria due to history of chronic hydrocarbon exposure (legacy effects), were the main drivers of the degradation rate of PAHs in water and in sediment.

In conclusion, integrating both copepod based in vivo toxicity bioassays and studying the dynamics of microbial communities offers a broad picture of the effects and responses that may cause an oil spill scenario and the addition of chemical dispersants in sensitive environments.

Résumé

La pollution par les hydrocarbures est un problème émergent dans les mers du monde, en particulier dans les environnements froids où les procédures d'assainissement sont difficiles. Après un déversement de pétrole, la dynamique des vagues et d'autres facteurs d'altération introduisent la solubilisation des hydrocarbures, en particulier des hydrocarbures aromatiques polycycliques (HAP) dans la colonne d'eau, créant des fractions d'hydrocarbures solubilisés dans l'eau (WAF). Après une marée noire, des stratégies de lutte impliquent l'addition de dispersant afin de favoriser la biodégradation.

Cependant, des études ont montré que les dispersants ont des effets néfastes sur les organismes marins. L'objectif de cette thèse est d'étudier les effets des hydrocarbures altérés par le temps (WAF) dans les environnements froid, en focalisant sur la faune marine à l'aide de bio-essais in vivo et sur les communautés microbiennes. De plus, il est important d'étudier les effets toxicologiques de l'ajout de dispersant chimique au WAF et son efficacité dans la dégradation des hydrocarbures. Les essais toxicologiques in vivo, réalisés en utilisant Acartia tonsa comme organisme modèle, ont montré que les WAF préparées à partir de pétroles distillés (IFO 180 et Marine diesel) présentaient une toxicité élevée envers le copépode considérant les paramètres de létalité, de reproduction et indicateurs moléculaires Tels que les modèles de transcription des gènes. Ces résultats suggèrent que la composition chimique des WAF obtenues à partir de pétroles distillés ont contribué à une toxicité supérieure à celle des WAF obtenues à partir de pétrole brut.

Cependant, l'ajout de dispersant dans les WAF obtenues à partir de pétrole brut a augmenté la toxicité envers Acartia tonsa. En outre, Acartia tonsa exposé au WAF contenant un dispersant a également montré une faible fécondité, une mortalité élevée et des profils d'expression génique affectés, soulignant davantage les effets toxique des dispersants. Après avoir examiné les effets du WAF et du WAF avec dispersant sur les communautés microbiennes dans des expérimentations en microcosmes, le dispersant ajouté au WAF n'a pas amélioré les taux de biodégradation. Cependant, le WAF avec dispersant a contribué à des changements dans la structure des communautés microbiennes et leur dynamique, à la fois dans l'eau et dans les sédiments, résultant en la sélection de certains taxons. Les résultats ont montré que les communautés microbiennes possédant des microorganismes dégradant les hydrocarbures, en raison d'une exposition chronique aux hydrocarbures (effet héritage), étaient responsables des taux de dégradation élevés des HAP dans l'eau et dans les sédiments. En conclusion, l'intégration à la fois des essais de toxicité in vivo et de l'étude de la dynamique des communautés microbiennes a ouvert la voie pour comprendre le devenir du pétrole, déversé dans l'environnement froid, après leur altération. Nous avons également évalué si l'utilisation traditionnelle de dispersant atténuait les effets du déversement de pétrole.

Resumen

La contaminación por petróleo es un problema importante en los océanos y mares, especialmente en ambientes fríos y regiones polares, donde los procesos de remediación son más complejos y los ecosistemas son muy vulnerables. Durante un vertido de petróleo diversos factores meteorológicos y el movimiento de las olas favorecen la integración de los hidrocarburos hidrofílicos en la columna de agua, siendo el producto resultante la conocida como fracción acomodada del petróleo en el agua (WAF), que suele estar compuesta principalmente, aunque no solo, por hidrocarburos aromáticos policíclicos (HAPs). Históricamente como forma de remediación ante los vertidos de petróleo se ha utilizado la incorporación de dispersantes químicos que favorecen la degradación del petróleo. Sin embargo, diversos estudios han demostrado que los dispersantes químicos pueden tener efectos adversos sobre los organismos marinos. El objetivo de este proyecto de Tesis doctoral ha sido estimar el posible impacto del WAF de un petróleo nafténico del Atlántico Norte (NNA), con o sin la adición del dispersante químico Finasol OSR52, mediante bioensayos in vivo con copépodos y el estudio de alteraciones en comunidades microbianas. Los bioensayos con el organismo modelo Acartia tonsa demostraron que el NNA WAF fue menos tóxico que los WAFs obtenidos a partir de destilados de fuel oil (IFO 180 y Gasóleo de uso marítimo), que mostraron una alta toxicidad a distintos niveles de organización biológica (transcripción génica, reproducción y supervivencia) en los copépodos. Al añadir el dispersante químico el NNA WAF fue más tóxico para los copépodos en términos de alteración de la transcripción génica, la reproducción y la mortalidad. En el caso de las comunidades microbianas, al examinar en condiciones de microcosmos los efectos del NNA WAF en solitario y NNA WAF con dispersante, se pudo observar que el dispersante no favoreció la biodegradación bacteriana de los compuestos procedentes del NNA WAF. Sin embargo, sí alteró las dinámicas de las

General Introduction

The Arctic and Sub-Arctic marine ecosystems

The Arctic and sub-Arctic environments are regions that lie beyond 50 o North (Figure 1). These environments are characterized seasonally by periods of midnight sun and complete day darkness and the warmest months do not exceed 10 o C [START_REF] Hoberg | Northern hostparasite assemblages. History and biogeography on the borderlands of episodic climate and environmental transition[END_REF].

The Arctic and sub-Arctic ecosystems are unique in the extreme conditions they present for living organisms. In winter, the top layer of the ocean freezes, creating sea ice. Sea ice can range from paper-thin ice, which melts very quickly, to incredibly thick ice that can be 3 m thick and can survive for many years. Although its surface is mostly covered with ice-sheets throughout the year, Arctic and sub-Arctic environments are teeming with life. [START_REF] Hoberg | Northern hostparasite assemblages. History and biogeography on the borderlands of episodic climate and environmental transition[END_REF].

These ecosystems consist of two thousand algal species, tens of thousands of microbes and approximately five thousand species of animals [START_REF] Meltofte | Appearance of the Pacific diatom Neodenticula seminae in the northern Nordic Seas -An indication of changes in Arctic sea ice and ocean circulation[END_REF]. Like any other ecosystem, these ecosystems are organized in complex food webs with interactions between microbes, phytoplankton, zooplankton, macro-invertebrates, fish, seabirds and mammals (Figure 2). Similar to other marine environments, Arctic and sub-Arctic regions are dominated by microbial communities, which are responsible for carbon, energy and nutrient cycling. Representatives of the three domains of life, Bacteria, Archaea and Eukarya are present among those communities. Changes in microbial communities largely depend on seasonality and different niches (Pedrós-Alió et al., 2015).

Microbial biodegradation rates in Arctic and sub-Arctic environments

Cold adapted bacteria, which are known to thrive in temperatures as low as 0ºC, are widely distributed at temperatures below 5ºC [START_REF] Margesin | Biological decontamination of oil spills in cold environments[END_REF]. However, temperature is a major contributor to the biodegradation rates of nutrients and other elements. Atlas et al (1998) demonstrated that biodegradation rates were several folds faster in 25ºC than in 5ºC. Therefore, in Arctic and sub-Arctic environments, biodegradation rates in winter are significantly slower than in summer [START_REF] Siron | Environmental factors influencing the biodegradation of petroleum hydrocarbons in cold seawater[END_REF]. However, at subzero temperatures, slow biodegradation still takes place until -6ºC [START_REF] Rike | In situ biodegradation of hydrocarbons in arctic soil at sub-zero temperatures -Field monitoring and theoretical simulation of the microbial activation temperature at a Spitsbergen contaminated site[END_REF]. At such low temperatures, microbial communities need to have specific adaptations for survival and metabolism. For example, when temperature falls, cytoplasmic membrane fluidity decreases and membrane associated metabolic processes are slower [START_REF] Rike | In situ biodegradation of hydrocarbons in arctic soil at sub-zero temperatures -Field monitoring and theoretical simulation of the microbial activation temperature at a Spitsbergen contaminated site[END_REF]. Consequently, cold adapted microorganisms (Psychrophilic) have modifications of membrane lipids, which increase the fluidity of the cytoplasmic membrane (Gounot and Russel, 1999). Cryo protective compounds, such as trehalose, can be used as a source of energy to maintain viability at freezing temperatures [START_REF] Kandror | Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures[END_REF].

Planktonic life in Arctic and sub-Arctic marine environments

Phytoplankton typically live in the euphotic zone, which varies from a few meters to 100 m depth where the sunlight, in certain cases, is still strong enough to allow photosynthesis to take place. The primary production of phytoplankton and algae in the Arctic Sea usually peaks during the spring season, when sunlight penetrates the water column.

During the polar night, planktonic algae stop growing for lack of light. After thawing of ice, the nutritious elements in the water are enriched by the addition of seawater and various organisms that were trapped in the sea ice and by input brought by the currents and coastal run-off [START_REF] Meltofte | Appearance of the Pacific diatom Neodenticula seminae in the northern Nordic Seas -An indication of changes in Arctic sea ice and ocean circulation[END_REF]. When the light returns, the marine life reawakens and the food chain starts to function once more [START_REF] Meltofte | Appearance of the Pacific diatom Neodenticula seminae in the northern Nordic Seas -An indication of changes in Arctic sea ice and ocean circulation[END_REF]. The plant life provides energy for primary consumers such as zooplankton communities, which are food sources for several species of fish and marine mammals (Soreide et al., 2010).

Zooplankton are key intermediaries in marine food webs, including those in Arctic and sub-Arctic regions. Zooplankton transfer the energy from primary producers to fish and higher consumers. By exerting heavy grazing pressure on the microalgal primary producers, large herbivorous copepods have the capacity to grow in a few weeks producing huge biomass at the end of the feeding season (Falk-Petersen et al. 2009). The Arctic and sub-Arctic copepod communities are composed of large Calanus copepods and the small copepod species (Oithona sp., Triconia sp., Pseudocalanus spp. and Microcalanus spp.). Understanding the biogeographic distributions and range shifts of these copepods is a key prerequisite for predicting ecosystem shifts and community reorganization under the rapid changes of the Arctic system.

In the Arctic and sub-Arctic environments, zooplankton communities are dominated by the Calanus genus [START_REF] Ashjian | Annual cycle in abundance, distribution, and size in relation to hydrography of important copepod species in the western Arctic Ocean. Deep-Sea Res Part I[END_REF]. Examples of native Calanus species in the Arctic are Calanus glacialis and Calanus hyperboreus (Daase, 2013). Examples of newly introduced Arctic Calanus species are Calanus finmarchicus and Calanus marshallae [START_REF] Scott | Lipids and life strategies of Calanus finmarchicus, Calanus glacialis and Calanus hyperboreus in late autumn, Kongsfjorden, Svalbard[END_REF]. Members of the Calanus genus have larger body masses than other groups of copepods. Arctic copepods have the ability to store huge lipid reserves, mostly as wax esters, which consist of up to 60% of their total body weight at the end of the feeding season (Falk Peterson et al., 2009). The wax esters in Calanus contain omega-3 polyunsaturated fatty acids, which are obtained from dinoflagellates. These fatty acids are important components of the lipid membranes in all animals (Falk Peterson et al., 2009, Soreide, 2010). Arctic copepods are a vital food source for many different organisms such as the Arctic cod Boreogadus saida, the Pacific herring Clupea pallasii, the bowhead whale Balaena mysticetus, different species of seabirds, the amphipods Themisto libellula and gelatinous zooplankton (Ctenophores) (Darnis et al., 2012).

Global change, growing shipping activity and natural resources exploitation may increase invasion risk of alien species in Arctic and sub-Arctic ecosystems. Recently the presence of invasive temperate copepod species such as Eurytemora carolleeae, Eurytemora affinis, Centropages typicus and Acartia tonsa in Arctic waters has been described. The invasive success of these groups has been attributed to their generalist (eurytolerant and omnivorous) and R-selected life-history characteristics and thus, suppose a threat to native copepod species [START_REF] Goldsmit | Screening for High-Risk Marine Invaders in the Hudson Bay Region, Canadian Arctic[END_REF].

Threats to the Arctic and sub-Arctic marine environments

Arctic marine ecosystems are extremely sensitive to alterations provoked by human activities. Severe pressure from highly exploitative commercial fisheries, contamination from dumping, long-range atmospheric transfer of pollutants, noise pollution, oil and mineral exploitation, climate and sea temperature changes, ozone depletion, increased vessel traffic, and other factors directly or indirectly affect equilibrium of Arctic ecosystems (Clarke and Harris, 2003;[START_REF] Huntington | A preliminary assessment of threats to arctic marine mammals and their conservation in the coming decades[END_REF][START_REF] Jenssen | Marine pollution: the future challenge is to link human and wildlife studies[END_REF][START_REF] Jenssen | Endocrine-disrupting chemicals and climate change: A worst-case combination for Arctic marine mammals and seabirds?[END_REF].

Over the last two decades, the global average temperature has increased by 0.6°C in the Arctic (Bockris, 2010), provoking seasonal retreats of ice sheets in the western Arctic [START_REF] Stroeve | Arctic sea ice decline: Faster than forecast[END_REF]. Models have predicted that by 2050, the Arctic will be ice-free in the summer [START_REF] Arzel | Sea ice evolution over the 20th and 21st centuries as simulated by current AOGCMs[END_REF]. A warmer seawater temperature in the Arctic as well as the shrinking coverage of ice sheets has significantly affected the whole Arctic ecosystem and its biota [START_REF] Karcher | Arctic warming: Evolution and spreading of the 1990s warm event in the Nordic seas and the Arctic Ocean[END_REF]. During the spring, phytoplankton blooms in the Arctic sea, boosting the primary productivity in the region. However, a second phytoplankton bloom has started to appear in the fall, which is a common feature in warmer oceans [START_REF] Ardyna | Recent Arctic Ocean sea ice loss triggers novel fall phytoplankton blooms[END_REF]. This extra bloom disturbs the carbon cycle in the ocean and the food web. These conditions are shifting Arctic ecosystems to become more like temperate ecosystems. As a result, temperate species shift more northward toward the Arctic and subarctic environments. Recently, the presence of the Pacific native diatom Neodenticula seminae has been reported in the Nordic Sea [START_REF] Meltofte | Appearance of the Pacific diatom Neodenticula seminae in the northern Nordic Seas -An indication of changes in Arctic sea ice and ocean circulation[END_REF]. In the North Sea, species such as the Atlantic cod Gadus morhua, the common sole Solea solea and the scaldfish Arnoglossus laterna have shifted their habitats northward to higher latitudes (Perry et al.;2005).

In addition, the Arctic environment is vulnerable to acidification caused by the high solubility of atmospheric carbon dioxide in cold waters [START_REF] Steinacher | Imminent ocean acidification in the Arctic projected with the NCAR global coupled carbon cycle-climate model[END_REF]. This acidification promotes changes in calcium deposits, negatively affecting Arctic invertebrates such as zooplankton pteropods [START_REF] Orr | Oil dispersant increases PAH uptake by fish exposed to crude oil[END_REF], sea urchins [START_REF] Fabry | Impacts of ocean acidification on marine fauna and ecosystem processes[END_REF] and cold-water corals [START_REF] Langdon | Effect of elevated pCO2 on photosynthesis and calcification of corals and interactions with seasonal change in temperature/ irradiance and nutrient enrichment[END_REF].

The Arctic marine environment is highly sensitive to chemical pollution such as oil spills and urban and industrial sewage discharges, as ice cover makes cleanup difficult or impossible. Also, fauna are susceptible since oil degrades especially slowly in cold climates. Spilled oil enters marine animals' using different processes (ingestion, respiration, absortion, food) and poses a direct and lethal threat to marine life (Dietz et al., 2019;Jørgensen et al., 2019).

Oil pollution in the Arctic and sub-Arctic environments

With the increasing demand for fossil fuels, oil extraction and transport in the oceans have significantly increased during the last decade. Consequently, the risks of oil spill incidents are escalating rapidly. Between 2010 and 2018, 59 major oil spills occurred worldwide, resulting in the release of approximately 163,000 tons of oil into the marine environment (ITOPF 2019). Currently, it has been estimated that the Arctic and sub-Arctic seas contain up to 13% of the undiscovered oils in the world (Camus et al 2017).

The biggest oil spill incident in history was during the 1991 Gulf War, where 6 million barrels (818.000 -1,091.000 tons) were deliberately spilled in the Gulf of Kuwait [START_REF] Saadoun | Impact of oil spills on marine life[END_REF]. Another major and recent oil spill incident was the explosion of the Deepwater Horizon oil-drilling rig in 2010, which released at least 4 million barrels (560.000 -585.000 tones) in the Gulf of Mexico (Redmond et al., 2011). The best-known example of an oil spill in cold environments was the Exxon Valdez oil spill near Alaska, which occurred in 1989. A volume of 41.6 million liters (37.000 tons) of North Slope heavy oil was spilled near island shorelines (Atlas, 2011). Storms that occurred after the spill incident greatly dispersed the oil to cover 15% of the shoreline along Prince William Sound and the Gulf of Alaska [START_REF] Short | Estimate of oil persisting on the beaches of Prince William Sound 12 years after the Exxon Valdez oil spill[END_REF]. This incident had a great impact on the fish and invertebrate species in the area. For example, there were greater abnormality rates among Pacific herring, Clupea pallasi, larvae that hatched in oiled areas compared with non-impacted areas (Faksness et al., 2020). The amphipod populations impacted by this oil spill did not recover in terms of population size until 1995 (Falk Peterson, 2009).

Microbial ecology of oil degradation in Arctic and sub-Arctic ecosystems

In cold or polar environments, bioavailability of hydrocarbons decreases due to the solidification of the substrates [START_REF] Lo Giudice | Cold-adapted bacteria from a coastal area of the Ross Sea (Terra Nova Bay, AntArctica): linking microbial ecology to biotechnology[END_REF]. Little is known about ecological succession of microbial communities at low temperature after the introduction of pollutants such as oil. However, using next generation sequencing, thoroughly studying aftermath of Deepwater Horizon, helped in understanding how oil contamination can result in shifts in the resident microbial communities (King et al., 2014). Specialization disturbance hypothesis can explain microbial succession patterns shortly after an oil spill, where after disturbances, generalist microbes are favored while specialists are selected against [START_REF] Heritier-Robbins | Beach sand oil spills select for generalist microbial populations[END_REF]. Once an oil spill occurred, early responders often consisted of generalist communities, which are rapidly stimulated due to their tolerance to severe disturbances (Rodrigues et al., 2015). However, during the depletion of resources, resident microbial communities are shifted toward more specialist's communities, which are adapted to carbon-limited conditions or are capable of utilizing alternative energy sources.

However, several bacterial taxa have capabilities for hydrocarbon biodegradation at low temperature. For example, Gamma protobacteria, especially from the genera of Colwellia and Oleispera showed significant increases in abundance after the enrichment of crude oil and ammonium phosphate to Antarctic seawater under pack of ice [START_REF] Yakimov | Crude oil-induced structural shift of coastal bacterial communities of rod bay (Terra Nova Bay, Ross Sea, Antarctica) and characterization of cultured cold-adapted hydrocarbonoclastic bacteria[END_REF]. Genera of the psychrotolerant Rhodococcus isolates showed ability to degrade alkanes and PCBs at temperatures ranging from 4º to 35ºC (De Domenico et al., 2004).

In addition, Rhodococcus bacteria have shown to be able to produce biosurfactants [START_REF] Yakimov | Structural characterization of lichenysin A components by fast atom bombardment tandem mass spectrometry[END_REF]. In fact, biosurfactants play important roles in bacterial biodegradation capabilities by allowing bacteria to interact with the droplets of hydrocarbons [START_REF] Lo Giudice | Cold-adapted bacteria from a coastal area of the Ross Sea (Terra Nova Bay, AntArctica): linking microbial ecology to biotechnology[END_REF]. Biosurfactants are considered as a valuable alternative to commercially designed dispersants in the solubilization and dispersion of the hydrocarbons, because biosurfactants are easily biodegradable and do not accumulate in the environment (Desai Banat, 1997).

Aside from temperature, other factors also contribute to the biodegradation rates of oil in the environment. For example, availability of nutrients plays an important role in the microbial biodegradation rates of hydrocarbons in the environment. Nutrients, such as phosphorus and nitrogen, are crucial elements needed for the oil degraders [START_REF] Xu | Petroleum hydrocarbon-degrading bacteria for the remediation of oil pollution under aerobic conditions: A perspective analysis[END_REF]. More than 150 g of nitrogen and 30 g of phosphorus are consumed to convert 1 Kg of hydrocarbons in bacterial cells [START_REF] Ron | Enhanced bioremediation of oil spills in the sea[END_REF]. The initial bacterial communities with hydrocarbon degradation capabilities highlight the biodegradation rates of the communities. In an unpolluted site, hydrocarbon-degrading communities comprise 0.1% of the total microbial communities, while sites with history of oil pollution have up to 100% of hydrocarbon degraders [START_REF] Atlas | Microbial degradation of petroleum hydrocarbons: an environmental perspective[END_REF].

Calanoids, the most abundant order of copepods

The order Calanoida has 40 families of both freshwater and saltwater copepods. Calanoids are the most abundant copepods in the planktonic communities, making from 55% to 95% of the total planktonic populations in a given environment (Mauchline, 1998). Calanoids main distinguishable feature are the two tall antennae protruding from the opposite sides of the copepod cephalosome. Such antennae are usually as long as the whole body. In addition, Calanoids have five pairs of legs used for swimming. Calanoids are considered food for many important commercial fish, making them of commercial importance for culturing. Most calanoids are considered herbivores feeding on small phytoplankton [START_REF] Naganuma | Calanoid copepods: Linking lower-higher trophic levels by linking lower-higher Reynolds numbers[END_REF].

Calanoid life cycle has four major stages: egg stage, nauplius stage, copepodite stage and adult stage (Figure 3). Generally, calanoids have six naupliar stages. In the first naupliar stage, the newly hatched nauplius are around 0.1 mm long, they develop maxillopodian eyes, which are simple centered eyes with several photoreceptors. Naupliar stages undergo six stages of molts until the copepodite stages. There are five copepodite stages, in which the organisms lose the naupliar maxillopodian eye and develop until it closely resembles the adult copepod shape. Finally, copepods will reach the final adult stage and become sexually mature, capable of reproduction [START_REF] Trujillo Ortiz | Life cycle of the marine calanoid copepod Acartia californiensis Trinast reared under laboratory conditions[END_REF]. Calanoid copepods have both of their sexes reproductively active throughout the year. During mating, a male will grasp the females by their antennae and males will deposit spermatophores onto female urosomes to fertilize the eggs. Upon fertilization, eggs are released from the urosomes. Additionally, males can mate consequently with several females [START_REF] Halsband | Reproductive cycles of dominant calanoid copepods in the North Sea[END_REF].

Acartia tonsa, an invasive and generalistic species

Acartia tonsa (Dana 1849) is a species from the order Calanoida, family Acartiidae, present in coastal and estuarine areas. Its adult size ranges from 1.1 to 1.3 mm, and it is sexually dimorphic (Pastorinho et al., 2003). A. tonsa has a wide geographical distribution, covering several regions of the North Atlantic, Indian and Pacific oceans, the North Sea, Mediterranean Sea and recently the Arctic Sea (Figure 4

(a)

2018). A. tonsa is a great example of temperate species that are shifting towards Arctic and sub-Arctic environments. It tolerates a wide range of salinity (from 1 to 32 psu) and temperature (1 to 32°C), making it a suitable invasive species that is capable of living in many environments (Holste and Peck 2006;[START_REF] Villate | Chapter 5: Impact of the invasive species Acartia tonsa on the distribution of autochthonous Acartiidae species in estuaries of the Bay of Biscay[END_REF]Wooton et al., 2018).

Recently, Acartia tonsa has been introduced to the North Sea through aquaculture or through shipping activities [START_REF] Streftaris | Globalisation in marine ecosystems: The story of non-indigenous marine species across European seas[END_REF]. In addition, Acartia tonsa has become one of the most abundant copepod species in the boreal coastal waters and estuaries (Caudill et al., 2004). A. tonsa produces highly resistant diapause eggs as well as eggs that can be induced into quiescence, and this trait has probably played an important role in its wide distribution (Jensen and Carroll, 2010). Acartia tonsa females are larger and live longer than males. Females are distinguished from males by having broader urosome and thicker antennae (Figure 5) [START_REF] Sabatini | The developmental stages (copepodids I to VI) of Acartia tonsa Dana, 1849 (Copepoda, Calanoida)[END_REF].

Acartia tonsa females are broadcast spawners, meaning they do not carry their own eggs as other copepod species and spawn them as soon as the eggs are mature (Marcus et al., 2007). Females release around 18 to 50 eggs per brood and can produce up to 718 eggs during their lifetime (Mauchline, 1998). In Acartia, the ovary is situated at the dorsal side of the females. Oocytes of all developmental stages are present in females, indicating that oocyte maturation is a continuous process [START_REF] Niehoff | Life history strategies in zooplankton communities: The significance of female gonad morphology and maturation types for the reproductive biology of marine calanoid copepods[END_REF]. The level of maturation of the oocytes increases with the distance from the ovary, with mature oocytes close to the genital segment ready for spawning [START_REF] Niehoff | Life history strategies in zooplankton communities: The significance of female gonad morphology and maturation types for the reproductive biology of marine calanoid copepods[END_REF]. The constant egg spawning abilities make Acartia tonsa females a suitable candidate for toxicological studies in respect of reproduction capabilities.

Lipids in Acartia tonsa

In copepods, lipids, such as polyunsaturated fatty acids, are an important source for the metabolism of molecules responsible for regulating cell differentiation, egg production and hatching [START_REF] Acheampong | Food availability effects on reproductive strategy: the case of Acartia tonsa (Copepoda: Calanoida)[END_REF]. Copepods cannot produce enough essential fatty acids (EFAs) to cover their basic biochemical needs. Therefore, the quality of EFA source (i.e phytoplankton) plays an important role in copepods growth, development and egg production (Bi et al., 2020). Compared to other genus of copepods, Acartia sp. are known to have low amounts of storage lipids (Lee et al., 2006). Acartia copepods lack the wax esters in comparison to higher latitude calanoids. Consequently relying on its small lipid reserve consisting of triacylglycerol (TAG) for their temporary energy needs (Lee et al., 2006[START_REF] Werbrouck | Temperature impact on the trophic transfer of fatty acids in the congeneric copepods Acartia tonsa and Acartia clausi[END_REF]. However, when a lipid rich food source is available for Acartia tonsa, the original lipid storage levels can rapidly be magnified up to 60 factors [START_REF] Werbrouck | Temperature impact on the trophic transfer of fatty acids in the congeneric copepods Acartia tonsa and Acartia clausi[END_REF], which will be highly invested in egg production (Bi et al., 2020). [START_REF] Hazzard | Egg production of the copepod Acartia tonsa in Florida Bay: role of fatty acids in the nutritional composition of the food environment[END_REF] have demonstrated that adults of Acartia tonsa contained fatty acids up to 40% of the total lipids; high proportions of these fatty acids are omega-3. Therefore, Acartia tonsa is considered as a valuable live feed for several species of fish as its omega-3 profile exceeds that of enriched rotifers and enriched Artemia (Marcus et al., 2007).

Acartia tonsa, a model organism for toxicological assays

Acartia tonsa has been considered for toxicity assays of marine pollution since 1977 (Gorbi et al 2012). The International Organization for Standardization (ISO)

recommended Acartia tonsa as a model organism for acute, chronic and semi chronic bioassays to test for the toxicity of several contaminants (Gorbi et al., 2012). Recently, OECD, 2007) for studying full life cycle with sexually reproducing Acartia tonsa. Its wide distribution, short life cycle, continuous ability to produce eggs and the ability to be easily cultured in lab conditions make A. tonsa a suitable candidate for toxicological studies (Bellas et al., 2007). Acartia tonsa females can produce dormant eggs, which can be stored at 3ºC for up to four months (Vitielo et al., 2016). Additionally, Acartia tonsa has shown sensitivity under a range of different contaminants and toxins (Andersen et al., 1999[START_REF] Bielmyer | Toxicity of silver, zinc, copper, and nickel to the copepod Acartia tonsa exposed via a phytoplankton diet[END_REF][START_REF] Pinho | Physiological effects of copper in the euryhaline copepod Acartia tonsa: Waterborne versus waterborne plus dietborne exposure[END_REF]Sahlmann et al., 2019). Several endpoints have been studied in Acartia tonsa (Table 1) ranging from mortality to reproductive assays.

Acartia

Crude oil and Water Accommodated Fractions (WAFs)

Crude oil contains a mixture between different compounds such as hydrocarbons, heterocyclic and alkylated compounds and some heavy metals [START_REF] Saadoun | Impact of oil spills on marine life[END_REF]. Crude oils are mainly classified into three categories: Paraffinic, Naphthenic and Aromatic [START_REF] Simanzhenkov | Crude oil chemistry[END_REF]. In addition, the types of crude oils are usually named after their properties. For example, sweet crude oil contains low amounts of sulfur while sour crude oil contains high sulfur content (Petro Online, 2014). Light and heavy crude oils depend on the oil density. Light crude oil has a high percentage of light hydrocarbons, which can be inexpensively recovered during distillation. Heavy crude oil contains high concentrations of bitumen, nickel and sulfur. Compared to light crude oil, heavy crude oil requires more refining for good quality oil products (Petro Online, 2014).

After an oil spill, weathering processes and wave dynamics in the water column can introduce the oil soluble hydrocarbons into the water column and thus be more bioavailable to marine organisms [START_REF] Barron | Evaluation of fish early lifestage toxicity models of chronic embryonic exposures to complex polycyclic Polycyclic Aromatic Hydrocarbons in Food[END_REF]. These Water-soluble fractions, called water accommodated fractions (WAF) contain harmful aromatic hydrocarbons such as BTEX (benzene, toluene, ethylbenzene and xylenes) and polycyclic aromatic hydrocarbons (PAHs) (Jiang et al., 2010). However, the chemical composition of WAF is mainly dependent on the crude oil and the weathering conditions (OECD, 2019). In WAF, PAHs are mostly two to five rings hydrocarbons, higher rings PAHs are difficult to dissolve in water for their high lipophilic ability (Jiang et al., 2010).

The fate of oil spill pollution in the cold environments

Upon the introduction of oil to the water column, it faces several changes in its physical and chemical properties. Oil faces weathering processes such as spread, evaporation, dissolution, emulsification, dispersion, absorption, sedimentation, photo oxidation and biological degradation (Figure 6) (Sun et al., 2019). However, oil spills behave differently in the cold environment compared to temperate environments. For example, during high production periods, polynyas or opening in ice packs are very susceptible to oil pollution because oil slicks can be concentrated in the small shallow areas. Low temperature leads to increase in viscosity of oil, reduced evaporation of volatile compounds and increased water solubility (Yang et al., 2009). In addition, the different hydrocarbon constituents in oil behave differently after an oil spill. Aromatic hydrocarbons are more soluble than the aliphatic hydrocarbons with the same molecular weight and can be washed out preferentially (Prince et al., 2002). Biodegradation of linear alkanes is more preferential than branched alkanes (Prince et al., 2002). Consequently, the natural process of oil biodegradation is very slow, and thus, untreated oil slicks remain in the environment for more than 50 years. The dynamic movement of ice packs combined with the high residence time of the oil particles in the water would have a significant effect on the albedo effect on ice sheets (Van angelen, 2012). In addition, several weathering processes of oil, such as degradation and dissolution, are highly influenced by low temperatures (Brandvik et al., 2008).

Oil cleanup techniques

Oil slick cleanups can be conducted using different techniques. For example, sorbent-like materials such as polypropylene are used to absorb the oil from the water [START_REF] Bayat | Oil spill cleanup from sea water by sorbent materials[END_REF].

Booms are buoyant barriers, which limit the spread of oil without changing the physical and chemical properties of it [START_REF] Dave | Remediation Technologies for Marine Oil Spills: A Critical Review and Comparative Analysis[END_REF]. Another technique involves the controlled burning of the spilled oil to decrease the amount of oil in the water (Mullin, 2003). One well-known technique is "bio-stimulation", which was used after the Exxon Valdez oil spill. This process involves adding nitrogen-containing fertilizers to the water, which will stimulate hydrocarbon biodegradation bacteria and speed up the biodegradation process (Atlas, 2011).

One of the preferred methods in several countries as a form of remediation after oil spills is the addition of dispersants in order to break down the oil particles and thus increase their surface area for more efficient bacterial biodegradation (George-Ares, 2000).

The use of chemical dispersants and their effects on bacterial oil biodegradation rates

Dispersants are surfactants that are made of anionic and nonionic molecules, which orient the oil water interface and reduce interfacial tension (Ramachandran et al., 2004).

Consequently, dispersants facilitate the formation of small mixed oil surfactant micelles (Ramachandran et al., 2004), increasing the bioavailability of oil for bacterial biodegradation (Sun et al., 2019). Dispersion of oil in the water surface reduces the potential impact on shorelines and coastal ecosystems [START_REF] Personna | Dual effects of a dispersant and nutrient supplementation on weathered Endicott oil biodegradation in seawater[END_REF]. However, several studies have shown that chemical dispersants have harmful effects on the environment because they facilitate the introduction of PAHs to the water column, making them more bioavailable to marine organisms (Yamada et al., 2003). In addition, several studies have shown that dispersants have toxic effects on several species of crustaceans, fish and mollusks [START_REF] Wise | A review of the toxicity of chemical dispersants[END_REF].

An example of a widespread use of dispersants was during the Deepwater Horizon oil spill accident, where 2.1 million gallons of the dispersant, Corexit 9500, were applied at the water surface and seafloor (Techtman et al., 2017). Although the use of dispersants is widely used as a form of emergency response, the efficiency of dispersants to enhance biodegradation rates is still debated (Sun et al., 2019). For example, Techtmann et al (2017) saw slight increase in biodegradation rates of oil dispersed with the dispersant Corexit 9500 at both 5 and 25ºC. In addition, [START_REF] Venosa | Biodegradability of dispersed crude oil at two different temperatures[END_REF] also observed slight increase in biodegradation at 5ºC and rapid increase of biodegradation rates at 20ºC of oil dispersed with Corexit 9500. In contrast, Kleindienst et al (2015) illustrated that microorganisms select the hydrocarbons in dispersant as a carbon substrate and consequently attenuate biodegradation. Additionally, experiments with chemically enhanced WAF (CEWAF) showed that dispersant stimulates and selects the dispersant degrading bacteria against the more effective hydrocarbon degraders (Kleindienst et al., 2015[START_REF] Kleindienst | Ability of chemical dispersants to reduce oil spill impacts remains unclear[END_REF]. Therefore, further research is definitely needed for understanding the effects of dispersant on oil biodegradation rates.

Polycyclic Aromatic Hydrocarbons (PAHs) and their effects on marine zooplankton and microbial communities

PAHs are aromatic hydrocarbons formed by the fusion of two or more benzene rings [START_REF] Haritash | Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review[END_REF]. The common forms of PAHs are shown in Figure (7). PAHs are usually formed from the incomplete combustion of organic matter such as burning of fossil fuels or coal and during the subjection of several organic processes (EFSA, 2008). Natural sources of PAHs can be as well forest fires, oil seeps and volcanic eruptions [START_REF] Haritash | Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review[END_REF]. Several forms of PAHs are considered carcinogenic, mutagenic, genotoxic and can bio-accumulate in marine organisms (Pane et al., 2005). A number of PAHs are currently listed in the Water Framework Directive (WFD) among the 33 top priority hazardous substances (Boonen et al., 2020). In the marine environment, two to four rings PAHs are persistent in the water column and are highly soluble (Bellas et al., 2007). Therefore, naphthalene, fluorene, phenanthrene and anthracene have significant toxicity towards organisms [START_REF] Arias | Bioaccumulation of PAHs in marine zooplankton: an experimental study in the copepod Pseudodiaptomus marinus[END_REF]. High molecular weight PAHs, such as those with more than four rings, are known carcinogens, but do not have long lasting threat to the marine environment due to their extreme low solubility in water [START_REF] Albers | The compositions of wax esters, triacylglycerols and phospholipids in Arctic and Antarctic copepods: evidence of energetic adaptations[END_REF]. PAHs are hydrophobic in nature and thus, tend to accumulate in the fatty tissues of aquatic biota [START_REF] Hsieh | Environmental effects on the bioaccumulation of PAHs in marine zooplankton in Gaoping coastal waters, Taiwan: Concentration, distribution, profile, and sources[END_REF]Lee et al., 2006). Therefore, PAHs accumulating in marine plankton have a big risk to the whole marine biota due to the transfer of PAHs to high trophic levels in the food chain [START_REF] Hsieh | Environmental effects on the bioaccumulation of PAHs in marine zooplankton in Gaoping coastal waters, Taiwan: Concentration, distribution, profile, and sources[END_REF].

In some copepods, PAHs such as naphthalene are excreted through feces [START_REF] Berrojalbiz | Accumulation and cycling of polycyclic aromatic hydrocarbons in zooplankton[END_REF]. PAHs with more rings such as fluoranthene and pyrene may accumulate in zooplankton for long term [START_REF] Mitra | Macondo-1 well oil-derived polycyclic aromatic hydrocarbons in mesozooplankton from the northern Gulf of Mexico[END_REF]. Copepod eggs are high in lipid content;

they are therefore at great risk to accumulate PAHs. Some PAHs, such as phenanthrene, fluoranthene, and chrysene, were reported to be bio-accumulated in the eggs of Acartia tonsa, suggesting the transfer of PAHs to the next generation (Almeda et al., 2013). In addition to PAHs lipophilicity, PAHs have shown to be interfering with the fluidity of the cell membrane [START_REF] Van Brummelen | Enrichment of polycyclic aromatic hydrocarbons in forest soils near a blast furnace plant[END_REF]. On a cellular level, PAHs damage the DNA by forming PAH-DNA adducts (Xue et al., 2005) and enhance the generation of reactive oxygen species (ROS) during their metabolism (Patri et al., 2009)

Cellular metabolism of PAHs in marine copepods

In aquatic organisms, the cellular metabolism of xenobiotic compounds is sub-divided into three phases [START_REF] Ikenaka | Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species: Unique pyrene metabolism and species specificity observed in fish species[END_REF]. At phase I, xenobiotics undergo biotransformations through oxidation or reduction processes. At phase II, conjugation reactions produce polar derivatives, which are more easily to be excreted than the parent compound. At phase III, metabolites are excreted from the cell through membrane proteins, such as the ATP binding cassette transporters (ABC) [START_REF] Ikenaka | Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species: Unique pyrene metabolism and species specificity observed in fish species[END_REF]. In vertebrates, CYP1A1 and CYP1B1 family proteins are some of the responsible for the metabolism of PAHs under phase I (Shimada, 2003;Han et al., 2017). Glutathione S-transferases (GSTs), among others, are important isoenzymes, which are involved in the phase II detoxification system, limit oxidative damage and are involved in several conjugation reactions (Zhou et al., 2020). Several studies have demonstrated that several phase II conjugation metabolites present in invertebrates have not yet been confirmed in vertebrates, suggesting specific pathways in invertebrates [START_REF] Ikenaka | Glucose-sulfate conjugates as a new phase II metabolite formed by aquatic crustaceans[END_REF].

In vertebrates, xenobiotic compounds such as PAHs binds to the aryl hydrocarbon receptor (AhR) which activates the expression of CYP1A1 gene. However, AhR-CYP metabolism pathway is a source of reactive intermediate metabolites, which are classified as carcinogenic (Nerbert and Dalton, 2006). For example, oxidative metabolites such as diol epoxide covalently bind to DNA and may cause several adverse effects such as cancer in vertebrates (Boonen et al., 2020). PAHs toxicity effects and CYP1A1 metabolic pathways on marine vertebrates has been well studied [START_REF] Kim | Dose-and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBTexposed intertidal copepod Tigriopus japonicus[END_REF]Ryu et al., 2009).

However, AhR-CYP1A pathway is still poorly understood in marine invertebrates, as CYP1 gene family has not been found in invertebrate genomes yet (Han et al., 2017).

Nevertheless, some CYP genes described in marine invertebrates have shown to be involved in signaling pathways similar to AhR-CYP1A in vertebrates [START_REF] Kim | Dose-and time-dependent expression of aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) in PCB-, B[a]P-, and TBTexposed intertidal copepod Tigriopus japonicus[END_REF].

A PAH-AhR-CYP signaling pathway for marine copepods was proposed by Han et al (2017) (Figure 8). Inside the cytosol, the PAHs can cause oxidative stress by generating ROS and damage the DNA in the nucleus. Inactive AhR usually forms protein complexes with molecular chaperones, such as HSP90, and other proteins. Activated AhR binds with PAHs and translocate to the nucleus. In the nucleus, the PAH-AhR dimerizes with the transcriptionally active aryl hydrocarbon receptor nuclear translocator (AhRNT) and binds to the promoter region of AhR regulated genes. As CYP1A family genes are yet to be found in marine copepods, other genes from the CYP family such as those of the clade 3, cyp3024 and cyp3027, are found in Tigropus japonicus and Paracyclopina nana has been suggested to have similar functions (Han et al., 2017).

Figure 8: Proposed PAH-AhR-CYP signaling pathway in marine copepods (Han et al., 2017).

Microbial degradation pathways of PAHs

Microbes have developed strategies to degrade PAHs under aerobic and anaerobic conditions (Figure 9). Under aerobic conditions, ring hydroxylating dioxygenases enzymes (RHDs) directly incorporates two oxygen molecules into an aromatic ring, forming cis-dihydrodiol (Duran and Cravo Laureau, 2016). Using dehydrogenase enzymes, cis-dihydrodiol is converted to catechol, which then undergo an aromatic ring fission into aliphatic products. At this stage, aliphatic products can enter metabolic pathways via the tricarboxylic acid cycle (Cernigilia, 1992).

However, under anaerobic conditions, PAH molecules are activated through the carboxylation of the aromatic rings using a carboxylase enzyme [START_REF] Rabus | Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment[END_REF].

Additionally, another activation mechanism is via methyl transferase, which methylates the aromatic ring. The methylation process is then followed by naphthyl-2-methylsuccinate synthase, which catalyzes the addition in fumarate (Meckenstock and Moutakki, 2011). However, in both mechanisms mentioned, the degradation pathways proceeds through beta-oxidation after the oxidation with coenzyme A (Meckenstock and Moutakki, 2011).

PAH

State of the art

Oil spill pollution has become an emerging problem in the world seas. This issue has bigger impact on cold environments due to the harsh conditions these environments possess. Weathered oil from oil spills, in the form of Water Accommodated Fractions, introduce PAHs in the water column and have detrimental effects on the marine fauna inhabiting these environments. As a form of bioremediation, chemical dispersants are added to the oil in order to break down the oil slick into smaller droplets and therefore facilitate microbial biodegradation of oil. However, the use of dispersant as a form of remediation have shown to have detrimental effects on marine fauna.

Due to its sensitivity to toxins and being a crucial link in the marine food chain, the copepod Acartia tonsa has proved to be a suitable organism to toxicologically investigate the effects of several toxins. Objectives 1) To assess the toxicity profiles of WAF prepared from two different types of distilled oils and a naphthenic North Atlantic crude oil using copepods Acartia tonsa, as experimental organism.

2) To investigate the toxic effects of WAF prepared from naphthenic North Atlantic oil single or in combination with a commercial chemical dispersant using the model organism Acartia tonsa.

3) To describe the changes produced by the exposure to naphthenic North Atlantic oil WAF with and without dispersant on native microbial communities in water and in sediment samples from subarctic areas.

4) To determine whether the addition of a commercial chemical dispersant to a naphthenic North Atlantic oil WAF improves the biodegradation rates of PAHs in subarctic water or sediment experimental samples and changes its toxicity to Acartia tonsa copepods Chapter 1: Application of a biological multilevel response approach in the copepod Acartia tonsa for toxicity testing of three oil Water Accommodated Fractions

This chapter has been presented in the 39th international symposium on Halogenated Persistent Organic Pollutants (DIOXIN 2019). Kyoto Japan

This chapter has been published as: Barron et al., 2003). Several environmental factors, such as temperature and light, influence the degree of bioavailability and toxicity of PAHs in WAF (Lyons et al., 2011;[START_REF] Lee | Photo-oxidation and Photo-toxicity of Crude and Refined Oils[END_REF]. At low temperatures, PAHs show low solubility and reduced bioavailability, but at high temperatures, both low and high molecular weight hydrocarbons can be introduced in the water column (Lyons et al., 2011). Photo-oxidation of PAHs results in the production of free radicals that react with oxygen to generate toxic reactive oxygen species (ROS) [START_REF] Lee | Photo-oxidation and Photo-toxicity of Crude and Refined Oils[END_REF]. Several members of the superfamily of cytochrome P450 genes participate in the metabolism and biotransformation of organic chemicals, including PAHs, and are considered biomarkers for WAF and oil exposure in aquatic animals [START_REF] Snyder | Cytochrome P450 enzymes belonging to the CYP4 family from marine invertebrates[END_REF]Rhee et al., 2013). The role of the aryl-hydrocarbon receptor (AhR) dependent pathway and the activity of cytochrome P450 enzymes (CYPs) in the detoxification of PAHs is well characterized in vertebrates, but poorly described in invertebrates (Han et al., 2017;[START_REF] Nelson | Cytochrome P450 diversity in the tree of life[END_REF].

PAHs are known to have a deleterious impact on cellular metabolism and functions [START_REF] Allan | Impact of the deepwater horizon oil spill on bioavailable polycyclic aromatic hydrocarbons in gulf of Mexico coastal waters[END_REF]Han et al., 2014). Inside the cell, the metabolism of PAHs generates ROS (Hannam et al., 2009) and an imbalance between generation of ROS and ROS neutralization via antioxidants or oxyradical scavenging systems. An imbalance of these processes in the cell triggers oxidative stress ( [START_REF] Valavanidis | Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants[END_REF] that can result in DNA damage, oxidation of biomolecules and cell death [START_REF] Martins | Comparative DNA damage and oxidative effects of carcinogenic and non-carcinogenic sedimentbound PAHs in the gills of a bivalve[END_REF][START_REF] Wilk | Polycyclic aromatic hydrocarbons-induced ROS accumulation enhances mutagenic potential of T-antigen from human polyomavirus JC[END_REF].

Consequently, the expression of several genes involved in antioxidant defenses represents a valuable tool for understanding the toxicity response mechanisms against oxidative stress. Antioxidant enzymes (e.g Catalase and glutathione transferase (GST)) [START_REF] Kim | Effect of heavy metals on the antioxidant enzymes in the marine ciliate Euplotes crassus[END_REF], molecular chaperones (e.g. heat shock proteins) and iron storage proteins (i.e.

ferritin) have been used as biomarkers of ROS induced responses in aquatic organisms [START_REF] Tarrant | Molecular physiology of copepodsfrom bio markers to transcriptomes and back again[END_REF] ).

The calanoid copepod, Acartia tonsa, is a cosmopolitan species inhabiting temperate regions and is usually abundant in coastal and estuarine areas [START_REF] Cervetto | Feeding behaviour and migrations in a natural population of the copepod Acartia tonsa[END_REF].

Copepods are considered ecologically crucial to the marine food web, serving as a primary food source for secondary consumers, and exerting trophic effects on primary producers such as phytoplankton (Gorbi et al. 2012). The International Organization for Standardization (ISO) recommended the use of Acartia tonsa for the evaluation of lethality and toxicity of several contaminants (Gorbi et al., 2012), since it is sensitive to different types of contaminants and toxins (Andersen et al., 1999[START_REF] Bielmyer | Toxicity of silver, zinc, copper, and nickel to the copepod Acartia tonsa exposed via a phytoplankton diet[END_REF][START_REF] Pinho | Physiological effects of copper in the euryhaline copepod Acartia tonsa: Waterborne versus waterborne plus dietborne exposure[END_REF]Sahlmann et al., 2019). Thus, Acartia tonsa is considered a valuable model organism for understanding pollutants' effects on the marine zooplankton communities.

Few studies have investigated the effects of oil WAF on Acartia tonsa (Avila et al., 2010, Faksness et al., 2014), mainly focusing on lethality without concurrently considering different sublethal endpoints. The toxic effects of WAF on several other copepod species with respect to mortality, development, egg production, and motility have been established [START_REF] Cowles | Effects of exposure to sublethal concentrations of crude oil on the copepod Centropages hamatus -I. Feeding and egg production[END_REF]Bejarano, 2006;Jiang et al., 2012;Hansen, 2013 and[START_REF] Nelson | Cytochrome P450 diversity in the tree of life[END_REF]Lee et al., 2013). Exposure to dispersed oil alters the lipid sac volume in the cold-water copepod Calanus finmarchicus (Hansen et al., 2016), leading the authors to conclude that changes in lipid content due to contaminant exposition might have adverse effects on the reproduction capabilities in copepods. It has been shown that fatty acid composition in Acartia tonsa influenced egg hatching success and egg production rates [START_REF] Broglio | Effect of heterotrophic versus autotrophic food on feeding and reproduction of the calanoid copepod Acartia tonsa: Relationship with prey fatty acid composition[END_REF][START_REF] Cavallo | Lipid storage patterns in marine copepods: environmental, ecological, and intrinsic drivers[END_REF], but to date, the effects of oil WAF on the lipid composition in Acartia tonsa are poorly understood.

A potential role in PAH metabolism for genes belonging to the CYP family has been suggested in copepods. Several genes of CYP3 clan showed differential transcription profiles after exposure to individual PAHs and WAF [START_REF] Hansen | Suppression subtractive hybridization library prepared from the copepod Calanus finmarchicus exposed to a sublethal mixture of environmental stressors[END_REF]2008;Han et al., 2014[START_REF] Han | Developmental retardation, reduced fecundity, and modulated expression of the defensome in the intertidal copepod Tigriopus japonicus exposed to BDE-47 and PFOS[END_REF]Han et al., and 2017)). A role of genes in the CYP 3 clan in PAH and oil metabolism has been suggested in marine copepods, such as Tigriopus japonicus and Paracyclopina nana (Han et al., 2014 and[START_REF] Saadoun | Impact of oil spills on marine life[END_REF].

In order to better understand the effects of oil WAF on Acartia tonsa copepods, we studed the effects of three oil WAF (crude and refined) with different water solubility and bioaccumulative characteristics. We selected a Naphtenic North Atlantic crude oil (NNA) and two refined oils: Intermediate Fuel Oil 180 (IFO 180) and a commercial Marine gas oil (MGO). NNA and IFO 180 have low dispersion potential and a low concentration of water-soluble components [START_REF] Hansen | Acute exposure of water soluble fractions of marine diesel on Arctic Calanus glacialis and boreal Calanus finmarchicus: Effects on survival and biomarker response[END_REF]. In contrast, MGO, commonly used as fuel for different types of ships, has low viscosity and high volatility, which facilitate its dispersion in the marine environment [START_REF] Hansen | Acute exposure of water soluble fractions of marine diesel on Arctic Calanus glacialis and boreal Calanus finmarchicus: Effects on survival and biomarker response[END_REF]. We applied a biological multilevel response approach to integrate effects on lethality, fecundity (egglaying), total lipid content and transcription levels of genes related to stress responses and PAH metabolism. In addition, potential transgenerational effects on egg hatching success were assessed.

Materials and Methods

Selected Oils and preparation of Water Accommodated Fractions (WAF)

The three oils used for WAF preparation were NNA, IFO 180, and commercial MGO.

NNA oil is a naphthenic-based light crude oil characterized by a low aromatic content, a low wax content, a low viscosity index (299 cP at 2° C), and a density of 0.900 g/ml . It is also rich in branched and cyclic saturated hydrocarbons (Katsumiti et al., 2019). IFO 180 is a blend of heavy fuel oil and gas oil composing approximately of 88% residual oil and 12% distillate oil. It is characterized by a very high viscosity (180 cSt at 50° C), high density >0.967 g/ml , and has a maximum sulfur content of up to 3.5% (Johann et al., 2020). IFO 180 is mainly used in the machinery and engines of large shipping vessels (Alexander et al., 2016). MGO is used in the engines of small ships and fast boats. It is a blend between gas oil and heavy fuel oils and is highly volatile, with low viscosity (2-4 cSt at 40° C), low density 0.856 g/ml containing up to 60% aromatic hydrocarbons [START_REF] Hansen | Acute exposure of water soluble fractions of marine diesel on Arctic Calanus glacialis and boreal Calanus finmarchicus: Effects on survival and biomarker response[END_REF]. Oils were provided by the Norwegian University of Science and Technology (NTNU; Trondheim, Norway), within the framework of the EU-funded project GRACE (Integrated oil spill response actions and environmental effects) (Jørgensen et al., 2020).

The WAF were prepared according to the protocol by Singer et al. (2000) for lowenergy WAF with slight modifications. Briefly, oil was added to 0.2 µm filtered seawater in 4 L glass bottles with bottom tap, at a final loading concentration of 5 g oil per 1 L of seawater. The water-oil mixture was continuously stirred at 800 rpm (lowenergy) using a magnetic stirrer without forming any vortex for 40 h. The stirring was conducted at 10°C under dark conditions to avoid photo oxidation and degradation of oil elements. Finally, 3.5 L of WAF were carefully extracted from the bottles using the tap, avoiding the presence of non-diluted oil, and serial dilutions of each WAF were immediately prepared using clean seawater.

Copepod culture

Copepods, Acartia tonsa (Plentzia Marine Station, PiE-UPV/EHU), were maintained in a 10 L tank filled with 0.2 µm filtered seawater at 18°C, a salinity of 30 practical salinity unit (PSU) and a constant photoperiod of 16/8 h light/dark cycle. Copepods were fed every two days using a 1:1 mixture of the brown microalgae Isochrysis galbana and green microalgae Tetraselmis chuii. To prevent potential cannibalism, recently hatched nauplii were transferred from the main tank into a smaller 5 L tank until adulthood.

Chemical analysis of the PAH content

The PAH composition and concentrations of the different types of WAF were determined by GC/MS analysis. A mixed standard solution of 16 PAHs was used [Norwegian Standard (NS 9815: S-4008-100-T): phenanthrene (Phe), anthracene (An), fluoranthene and vent pressure (7 psi). This desorption unit was coupled in an Agilent 6890 gas chromatograph also coupled to an Agilent 5975 mass spectrometer system (Agilent Technologies, USA). The Mass spectrometer (MS) was operated in selected ion monitoring (SIM) to quantify target compounds. An Agilent DB-5MS+DG column with the following dimensions 30 m x 0.25 mm and 0.25 μm was used. Analytes were separated using the following conditions: helium as carrier gas (1.3 mL/min); ion source transfer line and quadrupole analyzer temperatures maintained at 300, 230, and 150ºC, respectively. The following temperature program was used for target PAHs and lineal hydrocarbons: 170 ºC for 5 min; ramp at 30ºC min -1 to 260 ºC; ramp at 8 ºC/min to 300 º C and hold 15 min. Three replicates per WAF were analyzed and concentrations calculated at the ng/L level.

(Fluo), pyrene (Pyr), 11 H-benzo[a]fluorene (11H-B[a]F), 11 H-benzo[b]fluorene (11H- B[b]F), benz[a]anthracene (B[a]A), chrysene (Chry), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (D[a,h]A), benzo[g,h,i]perylene (B[g,h,i]P)]

Lethal toxicity assays 96 h

Lethality tests were conducted according to the international standard for water quality determination of acute lethal toxicity to marine copepods (ISO14669). Thirty adult copepods were evenly distributed among six replicates, 5 copepods in each container.

During the assay duration, the copepods were not fed, and no sex preference was considered when selecting adult copepods. Copepods in each replicate were exposed to the contaminant for 96 h in 250 mL glass beakers containing 50 mL of 0% (control), 10%, 30%, 50%, 70% and 100% WAF diluted in seawater. Fifty percent WAF+seawater volume was replaced every 48 h. Each day of exposure, replicates were checked for mortality, and dead copepods were counted and removed. A copepod was considered dead if it did not move shortly after a gentle stimulation using a pipette. The LC50 values obtained from the lethal toxicity assays determined the low, medium and high concentrations to be further used in the sub-lethal endpoints assays (Table 1).

Fecundity and egg hatching assays

Twenty adult gravid females were selected under a 10X magnification dissecting microscope. Additionally, five adult males were included in each treatment to maintain female fertility throughout the experiment (Holste et al., 2006). The fecundity assay was performed similarly to the exposure methods described by Olsen et al. (2013), with some modifications in exposure time and methods. The fecundity assay was divided into three consecutive stages: exposure, recovery and individual egg collection.

In the exposure stage, copepods were placed into 300 mL glass beakers filled with 150 mL of the corresponding WAF dilution. Copepods were exposed in a semi-static condition for a duration of seven days. Every two days, 50% of the WAF+seawater volume was renewed. Containers were cleaned every day to remove fecal pellets, dead algae and any dead copepods . Throughout the experiment, copepods were fed daily with the algae Tetraselmis chuii at a concentration of 3000 cells per mL. In the recovery stage, the surviving females were transferred to clean, WAF-free seawater, for four days. Water was changed every two days, and copepods were fed daily with Tetraselmis chuii as in the exposure stage. This experimental procedure allows the identification of delayed effects on exposed female reproductive capacity to mimic environmental realistic scenarios of point oil spills or discharges.

In the fecundity egg collection stage, each surviving female was individually transferred to an incubation chamber with clean seawater for three days. Incubation chambers were divided into two compartments, spawning chamber and brooding chamber, similar to the device proposed by Kleppel et al (1998). The upper spawning chamber consisted of a plastic tube (50 mL, 10 cm in diameter), which housed an individual female for egg laying. A mesh of 200 µm was attached at the tube's base to prevent the females from entering the bottom compartment (brooding chamber) where eggs were deposited. The copepods were fed daily as in the previous stages. Each day, eggs were counted, carefully collected by pipetting, and transferred to individual vials containing seawater to allow them to hatch. Only females producing two or more eggs during the three days of the fecundity test stage were considered reproductive females. After 48 h, the unhatched eggs were considered unviable.

Total lipid quantification using Nile red lipophilic dye

We used a slightly modified protocol from Tingaud et al. (2011) andJordao et al. (2015) to quantify fecund females' total lipid content utilizing Nile red lipophilic dye. The working solution of Nile red dye (Sigma Aldrich) was prepared by dissolving the dye in acetone at a concentration of 334 µg/L. Briefly, adult females (n=10) were maintained and exposed to WAF for 96 h as described for the fecundity assay. Two doses for each WAF (medium and high, Table 1) were analyzed. After the exposure, copepods were rinsed with seawater for 5 minutes and then transferred to 20 mL of seawater mixed with 29 µL of Nile red dye working solution. The adult females were stained for one hour at room temperature in darkness. For lipid level quantification in adult females, the stained copepods were washed with seawater for 5 minutes. Then, each copepod was placed into a vial with 200 mL isopropanol and homogenized using a tissue homogenizer (Precellys 24 homogenizer, Bertin Instruments, France) at 2500 vibrations per second for 45 seconds. The homogenate was centrifuged at 12000 rpm for 2 minutes. Then, 150 mL of the extract was pipetted in a black 96 well microplate for fluorescence determination, and the plate was read at a wavelength of 530/590 nm excitation/emission using a fluorimeter (Biotek Cytation 5 imaging reader, USA).

A histological and histochemical study of lipid distribution in copepod tissues was carried out in parallel to detect which tissue/organ showed the highest accumulation pattern for lipids. The procedure and results are presented in the (Supplementary Materials chapter 1, figure S1) and annex section.

Gene transcription analysis

Exposure experiments

A total of 175 adult copepods were exposed to 150 mL of WAF for 48 h. Exposure concentrations were the same as described in section 2.6. Adult copepods were divided into five replicates per treatment (35 copepods in each replicate). The copepods were fed daily with Tetraselmis chuii, and no gender preferences were taken during copepod collection for exposure. After 48 h of exposure, the copepods were transferred to cryovials filled with RNA later solution (AMBION, USA) and stored at -80ºC until extraction.

RNA extraction and cDNA synthesis

RNA was extracted using RNeasy Minikit (Qiagen,Germany) following the manufacturer protocol. Total RNA was treated with DNase-1 using the DNA-free kit (Invitrogen, USA)

to remove genomic DNA. RNA integrity and concentration were measured using the Bioanalyzer 2100 (Agilent Technologies, USA). All RNA samples obtained a RIN score higher than 7, suitable for downstream qPCR analysis (Fleige et al., 2006). RNA purity was determined by measuring sample absorbance ratios of 260/280 nm using a UVspectrophotometer (Epoch, Biotek, USA). All samples had acceptable ratios between 1.8-2.3. cDNA was synthesized from purified RNA using the AffinityScript Multiple

Temperature cDNA synthesis kit (Agilent Technologies, USA) with random primers and following the manufacturer's protocol.

Primer design

Catalase, ferritin and members of gst, hsp90, hsp70 families genes were targeted as 1). Specific primers to amplify fragments of hsp70, hsp90 and ferritin genes were obtained from Nilsson et al (2014). βactin gene was used for transcription normalization as previously proposed (Zhou et al. 2020), as it showed low coefficient of variability (CV < 5%). The identity of targeted genes was confirmed by sequencing (General Genomics Service Sequencing and Genotyping Unit, SGIker, UPV/EHU). Primer sequences and the primers' amplicon regions' multi-sequence alignments are presented in Table 2 and (Supplementary Materials, chapter 1, Figure S2) respectively. Amplification efficiency for each primer pairs was calculated by conducting a 1:2 serial dilution curve generated from pooled experimental samples. All primer pairs showed amplification efficiency between 90% and 100%. Current study

RT-qPCR

Real-time quantitative PCR was conducted using FastStart SYBR Green fluorescence dye (ROCHE Diagnostics, Switzerland) with ROX passive dye included in the mix. The reaction medium (25 µL) consisted of 12.5 µL FastStart SYBR Green master mix, 0.3 µL forward primer (800 nM), 0.3 µL reverse primer (800 nM) and 3 µL diluted cDNA template (1:5 v/v). qPCR was performed in 384 optical well plates (Applied Biosystems, USA) using ViiA 7 Real-Time PCR system (Applied Biosystems, USA). The amplification conditions were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, and then 1 minute of annealing at the corresponding Tm for each gene (Table 2). A dissociation curve was performed after the PCR cycles to determine the product specificity and check the absence of primer dimers. All qPCR assays were conducted in triplicates, and a non-template control (NTC) was included for each plate.

Integrated Biological Response (IBR)/n index

The selected endpoints reflect a hierarchy of biological organization from molecular to an individual level. The percentage of reproductive females, egg hatching success, total lipid levels, and cyp3026b and hsp70 transcription levels were used to develop an integrated biomarker response index (IBR). The IBR constitutes a practical and robust tool to assess the responsiveness using multiple levels of biological responses [START_REF] Broeg | Indices for the assessment of environmental pollution of the Baltic Sea coasts: Integrated assessment of a multi-biomarker approach[END_REF]. Standardized values of each biological response were calculated and plotted using star plots according to [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF]. IBR index was calculated by summing up the triangular surfaces of the star plots (IBR= ∑Ai) according to the procedure discussed by [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF] with the modifications suggested by [START_REF] Devin | The integrated biomarker response revisited: Optimization to avoid misuse[END_REF], which avoids the biomarkers order bias in the IBR calculation. Since the IBR index is directly dependent on the number of biomarkers in the data set, the obtained IBR index value was divided by the number of biomarkers used [START_REF] Broeg | Indices for the assessment of environmental pollution of the Baltic Sea coasts: Integrated assessment of a multi-biomarker approach[END_REF]Lehtonen, 2006, Marigomez et al., 2013). To facilitate data representation of the different concentrations of the selected endpoints for IBR/n index, only medium and high doses of each WAF type were only used in the star plot visualization.

Statistical data analysis

Copepod mortality at different times (48, 72, 96 h) from lethal toxicity assays were analyzed using the PROBIT regression model (Vincent et al., 2008) to calculate the LC50 of the different WAF types. Significant differences among LC50 values were determined based on the absence of 95% confidence interval limits overlap. Data from egg production rate, egg-hatching success, Nile red fluorescence and gene expression ratios was initially checked for normal distribution using Kolmogorov-Smirnov test, while homogeneity of variances were tested using Levene´s test . One-way ANOVA analyzed data showing normal distribution. Significant differences were further analyzed using Dunnett´s posthoc test to identify differences among treated and control groups. Data showing nonhomogenous variance but normal distribution was analyzed using Welch one-way ANOVA, followed by the Games-Howell post hoc test. Data showing non-normal distribution were analyzed using the non-parametric Kruskal-Wallis test, followed by Mann-Whitney post hoc test. All these analyses were performed with the aid of the statistical package IBM-SPSS v26. The relationship between the count data of the number of reproductive females and WAF doses was analyzed by a modified Fisher exact test (Freeman Hamilton test), due to the small sample size and the contingency table larger than 2x2 (VassarStats, [START_REF] Lowry | VassarStats: Website for Statistical Computation[END_REF]Lowry -2019)). The association between both variables was determined by a linear regression coefficient (IBM SPSS v26). Egg hatching success during individual fecundity tests was calculated as follows: (number of hatched egg per female/total number of eggs produced per female) x100. Finally, relative gene transcription levels were determined using the Pfaffl method (Pfaffl et al., 2001). The transcript levels were log2 transformed and statistical differences between control and treatments were determined using one-way ANOVA. The Dunnet post-hoc test was further used to determine the statistical difference compared to control (IBM SPSS v26).

Z-test was used to determine significant differences in the IBR/n indices among the experimental groups

Results

WAF chemical composition

The three studied types of WAF contained mostly (99%) low-molecular-weight PAHs (< 3 rings), with naphthalene being predominant (Table 3). Among the 16 quantified PAHs, high-molecular-weight PAHs (> 4 rings) were below the detection limit for all three WAF. NNA WAF showed the highest content of total PAHs, particularly naphthalene (Table 3). IFO 180 WAF had the lowest total PAH concentration.

H-benzo[a]fluorene (11H-B[a]F), 11 H-benzo[b]fluorene (11H-B[b]F), chrysene (Chry), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (D[a,h]A), benzo[g,h,i]perylene (B[g,h,i]P)]

WAF Lethal toxicity

The lethal toxicity assays showed that WAF toxicity to adult Acartia tonsa was different according to the fuel oil. The MGO WAF showed to be the most toxic, while the NNA was the least toxic to copepods (Table 4). For MGO WAF, LC50 were as low as 34%, 10% and 8 % after 48, 72 and 96 h, respectively. The IFO 180 WAF had the second most lethal effect to adult Acartia tonsa with LC50 of 74%, 52%, and 40% after 48, 72 and 96 h, respectively. The NNA WAF showed the least lethal effect with LC50 of 74% after 72 hours and 52% after 96 h, and no lethal effects on the first 48 h. Noteworthy, the three types of WAF showed LC50 at 72 h and 96 h significantly lower than their respective LC50 at 48 h.

Effect of WAF on fecundity

Upon investigating reproductive capabilities, the exposure of female copepods to IFO 180 WAF and MGO WAF produced a significant reduced the proportion of individuals laying eggs (Figure 1). The proportion of reproductive females was negatively associated with the doses of IFO 180 WAF (Fisher exact test p < 0.0005, a = -23) and MGO WAF (Fisher exact test p < 0.05, a = -17.4). The effect was very pronounced with the high IFO 180 WAF dose, with only two females laying eggs. Therefore, the high dose of IFO 180 WAF was removed from further statistical analysis. No significant difference was observed between the egg production rates of control fecund females and the fecund females exposed to NNA WAF, IFO 180 WAF or MGO WAF (Figure 2). However, MGO WAF at high dose negatively affected the viability of produced eggs (Figure 3) compared to controls, suggesting a disruption in the egg production process.

Effect of WAF on total lipid

Ovaries were demonstrated as a vital accumulation tissue for lipids in female Acartia tonsa copepods (see Supplementary Material , chapter 1, Figure S1). We quantified the lipid content using the Nile red lipophilic dye to give further insights into how exposure to the three types of WAF affected the amount of accumulated lipids in females.

Overall, the collected data was highly variable in all treatments and control (Figure 4).

Nevertheless, Nile red fluorescence values of the copepods exposed to the high MGO WAF dose were significantly lower than those observed in the control copepods (Figure 4c), indicating a loss in total lipid levels.

Effect of WAF on gene expression

Changes in transcription levels of the studied genes indicated toxic effects of tested WAF in Acartia tonsa (Figure 5). Down-regulation was observed for hsp70 transcription levels in copepods exposed to WAF, being statistically significant for copepods exposed to the medium dose of IFO 180 WAF. In contrast, cyp3026b up-regulation was observed in copepods exposed to the high dose of IFO 180 WAF condition. Statistical significant differences were absent for cat, gst, hsp 90 and ferritin. 3026b (cyp3026b). Transcription levels are represented in log2 scale (mean ± standard error). Asterisks denote statistically significant differences from control according to one-way ANOVA followed by Dunnett´s post hoc-test (p<0.05).

IBR/n index

Discussion

In this study, a multilevel biological response approach was applied to assess the toxicity of three different oil WAF in the copepod Acartia tonsa. Adult mortality, female fecundity, egg hatchability and individual total lipid levels were analyzed in copepods exposed to a light crude oil (NNA) and two refined oils (IFO 180 and MGO). Besides, transcriptional responses of members of gene families involved in ROS metabolism (cat and gst), stress and iron homeostasis (hsp70, hsp90, and ferritin), and xenobiotic Odontesthes argentinensis, exhibited LC50 of 5.86% after 96 h exposure to MGO WAF (Rodrigues et al., 2010). Similarly, several studies showed that the observed toxicity of MGO on marine fish and crustaceans is due to the highly volatile BTEX compounds (benzene, toluene, ethylbenzene, and xylene) in the WAF (Gonzalez-Doncel et al. 2008;[START_REF] Neff | Effects of weathering on the toxicity of three offshore Australian crude oils and a diesel fuel to marine animals[END_REF]. Indeed, WAF from crude oil derivatives (i.e., MGO) has higher BTEX content and additives (e.g., phenols and heterocyclic compounds containing toxic nitrogen and sulfur) than WAF from crude oils [START_REF] Saeed | Chemical composition of the watersoluble fraction of the leaded gasolines in seawater[END_REF]Rodrigues et al., 2010). It has been reported that MGO WAF prepared by mixing at 1:40 (oil : water) ratio, contained BTEX compounds up to 1008 µg/L. Therefore, aside from PAHs, further chemical analysis of other types of hydrocarbons is necessary to better understand the

WAF toxicity profiles

Several studies in copepods highlighted a decrease in the egg production rates due to toxic effects of oil WAF compounds (Hansen et al., 2015 and2017;Olsen 2013). For example, copepods of the genus Calanus exposed up to 5.5 mg/L naphthenic crude oil, that is similar to the NNA crude oil tested in the present study, with mechanically and chemically dispersed conditions, showed reduced egg and nauplii production (Hansen et al., 2015). The copepod Paracartia grani showed decreased egg production under exposure to high doses of naphthalene (Calbet et al., 2007). Accordingly, the results of the present study demonstrated that both IFO 180 WAF and MGO WAF showed lower percentage of egg-laying females than the control. Indeed, only two females laid eggs in the high dose of IFO 180.

The egg production rate of fecund females did not vary among the studied WAF conditions and controls, suggesting a possible compensatory mechanism in exposed fecund female reproductive capacity. A compensatory effect in egg-laying was also described for the harpacticoid Nitocra spinipes exposed to musk fragrances (Breitholtz et al., 2003). In Calanus glacialis, a reduction in the number of reproductive females was described after exposure to WAF prepared with 10 µg/L crude oil without affecting the cumulative produced egg number, suggesting that egg-laying was at a normal rate, similar to controls (Jensen and Carroll, 2010), as shown in the present study for Acartia tonsa.

Similarly, C. finmarchicus females exposed to 16.5 mg/L arctic crude oil WAF showed a reduction in the number of egg-laying females, but the number of produced eggs in the exposed females was not different from that of control females (Olsen et al., 2013).

Furthermore, as shown in the present study, the four-day recovery period was not enough for the Acartia tonsa females to recover from the observed toxicity effects, suggesting a long-term effect of the exposure conditions. This observation is consistent with effects detected in Calanus copepods, which showed signs of recovery after exposure to oil in fecundity after 20 days in clean water (Hansen et al., 2015 and2017).

The females exposed to the high dose of MGO WAF showed lower lipid content levels than the non-exposed control females. Linking both lipid and egg viability endpoints suggested that the short exposure to MGO WAF affected egg development, lipid metabolism and lipid accumulation in females. In copepods, lipids sacs, when present, are often close to developing oocytes (Lee et al., 2006;[START_REF] Hansen | Maternal polycyclic aromatic hydrocarbon (PAH) transfer and effects on offspring of copepods exposed to dispersed oil with and without oil droplets[END_REF]. We have shown that in Acartia tonsa, the ovary is the main organ for lipid accumulation. Thus, it is likely that the disruption of lipid levels caused by exposure to MGO WAF resulted in decreasing the percentage of egg-laying females and egg-hatching success. Such observation is consistent with previous works on Calanus finmarchicus females showing that exposure to naphthenic crude oil reduced lipid sac size and egg-laying rate. In addition, egg hatching was negatively affected by maternal transfer of PAHs in lipid sacs [START_REF] Hansen | Maternal polycyclic aromatic hydrocarbon (PAH) transfer and effects on offspring of copepods exposed to dispersed oil with and without oil droplets[END_REF]. In the marine copepod Centropages hamatus, long-lasting effects on egg viability were detected after oil WAF exposure [START_REF] Cowles | Effects of exposure to sublethal concentrations of crude oil on the copepod Centropages hamatus -I. Feeding and egg production[END_REF], suggesting that the biosynthetic pathway involved in oogenesis was affected.

Although the expression patterns of catalase, gst and hsp90 genes in oil WAF exposed copepods were not significantly different from those of non-exposed control copepods, the medium dose of IFO 180 showed a significant downregulation of the selected form of heat shock protein 70 gene (hsp70). Several studies showed up-regulation of some members in the heat shock proteins family in response to environmental pollutants [START_REF] Yoshimi | Activation of a stress-induced gene by insecticides in the midge, Chironomus yoshimatsui[END_REF][START_REF] Rhee | Heat shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to environmental toxicants[END_REF]. However, fluoranthene, cadmium, nonylphenol, and octylphenol have shown to inhibit the expression of heat shock proteins (Werner et al., 1997;[START_REF] Rhee | Heat shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to environmental toxicants[END_REF], as shown in the present work. Noteworthy, the sublethal concentrations of IFO 180 WAF contained fluoranthene, suggesting that fluoranthene might be involved in the observed hsp70 downregulation. We observed that the cyp3026b gene was up-regulated in Acartia tonsa after exposure to IFO 180 WAF. Such response was consistent with previous studies reporting that genes from the cytochrome P450 (cyp) clan 3 showed up-regulation in the copepods T. japonicus and P. nana after exposure to crude oil WAF (Han et al., 2014[START_REF] Han | Developmental retardation, reduced fecundity, and modulated expression of the defensome in the intertidal copepod Tigriopus japonicus exposed to BDE-47 and PFOS[END_REF](Han et al., and 2017)). Although little is known about the regulation of the cyp genes of the PAHs detoxification process in invertebrates, there is some evidence of their involvement in the PAHs detoxification pathway (Han et al., 2014[START_REF] Han | Developmental retardation, reduced fecundity, and modulated expression of the defensome in the intertidal copepod Tigriopus japonicus exposed to BDE-47 and PFOS[END_REF](Han et al., and 2017)). Indeed, several core sequence motifs (e.g., xenobiotic response element, aryl-hydrocarbon response element, and estrogen response element) were described in the promoter regions of cyp clan 3 members of T. japonicus and P. nana (Han et al., 2014;[START_REF] Saadoun | Impact of oil spills on marine life[END_REF]2017). We advocate that the forms of hsp70 and cyp3026b genes used in this study can be suitable biomarker genes for estimating the toxic effect of WAF exposure because they showed early responses after oil WAF exposure conditions. The other assessed genes involved in ROS and stress dependent responses (hsp90, ferritin, gst, and catalase) showed variability in their transcription profiles, but did not differ to levels determined in controls. Future transcriptomic studies such as, RNA-seq, can provide a further comprehensive view of changes in gene expression.

Conclusions

In

Chapter 2: Toxicity effects of a Naphthenic North Atlantic Crude Oil Water Accommodated Fraction with and without Dispersant on Acartia tonsa This chapter has been presented in the 19th international symposium on Pollutant Responses in Marine Organisms (PRIMO). Matsuyama, Japan

This chapter is being prepared to be published as:

Abstract

Zooplankton, such as copepods, plays a key role in the marine food web, being the primary consumers of phytoplankton and a rich energy source for other invertebrates and vertebrates. Alterations in copepod populations after oil spills have been reported at several places in temperate waters. In this study, we investigated the effects on mortality, fecundity and gene expression of a naphthenic crude oil Water Accommodated Fraction (WAF) prepared alone or in combination with a commercial dispersant (Finasol OSR 52)

on the model species Acartia tonsa. Toxicity assays (96 h) were carried out on adult copepods and LC50 values were calculated. Gravid females were exposed to WAF or NNA WAF+Dispersant for 7 days, then transferred to clean water for 3 days (to mimic an exposure and recovery scenario) to assess effects on fecundity. The reproductive endpoints investigated were: egg laying, egg production rate, egg hatching success and the number of productive females. In addition, relative transcript levels of key genes involved in oxidative stress and xenobiotic metabolism were determined. According to the lethality test, the WAF +Dispersant mixture was the most lethal toward copepods. In terms of egg production rate, female copepods showed the highest disrupted fecundity after exposure to NNA WAF+Dispersant conditions. However, neither the percentage of fecund females nor the egg hatching rate after maternal exposure were affected by WAF or NNA WAF+Dispersant exposure. In addition, 10% WAF+Dispersant treatment showed significant transcription level of hsp70 gene. Principal component analysis (PCA)

showed that WAF treatment and 10% NNA WAF+Dispersant had different transcription pattern than that of control. In conclusion, results show that NNA WAF+Dispersant was more toxic toward Acartia tonsa than WAF alone.

Introduction

Overexploitation of oil resources resulted in the boost of the maritime traffic, expansion of oil extraction platforms and the construction of new oil transportation pipelines.

Consequently, there is an increasing risk of oil spill accidents towards the marine environment. During an oil spill, the oil slick can be mitigated with several cleanup techniques such as the in-situ burning of oil (Mullins et al., 2003), introduction of nitrogen containing fertilizers to stimulate the biodegradation bacteria on site (Atlas, 2011) and using sorbent like materials such as polypropylene to absorb oil slick [START_REF] Bayat | Oil spill cleanup from sea water by sorbent materials[END_REF]. Another technique to emulsify the oil slick during spill accidents is the introduction of chemical dispersant agents. Dispersants split the oil into smaller droplets, increasing the surface area of the oil vesicles and making the oil more bioavailable (Kliendienst et al., 2015). The increased surface area and bioavailability facilitate bacterial biodegradation of the oil droplets (Sun et al., 2019). For example, more than 7 million liters of chemical dispersants have been used during the Deepwater Horizon well blowout in order to stimulate the bacterial biodegradation of oil (Kleindienst et al., 2015).

However, several studies have demonstrated that dispersant is toxic for several marine organisms (Mitchell and Holdway, 2000;Lee et al., 2013;[START_REF] González-Penagos | Alterations in the Gut Microbiota of Zebrafish (Danio rerio) in Response to Water-Soluble Crude Oil Components and Its Mixture With a Chemical Dispersant[END_REF]Kastumiti et al., 2019).

Water Accommodated Fractions (WAF) are hydrocarbon fractions with low molecular weight that with adequate wave dynamics and weathering processes will be soluble in water. Hydrocarbons in WAF are more bioavailable for marine organisms (Barron, 2003).

WAF contains harmful aromatic hydrocarbons such as benzene, toluene, xylenes and polycyclic aromatic hydrocarbons (PAHs). PAHs have deleterious effects on cellular metabolism and function (Han et al., 2014). For example, PAHs form DNA damage by forming DNA adducts which cause cancer (Xue et al., 2005). In addition, PAHs increase the generation of reactive oxygen species (ROS) in organisms, which result in oxidative damage on lipid, nucleic acids and proteins (Dvorak et al., 2021). Consequently, cells respond to this oxidative stress through the activation of antioxidant enzymes, which interact with the ROS and protecting the cells from oxidative stress [START_REF] Kim | Effect of heavy metals on the antioxidant enzymes in the marine ciliate Euplotes crassus[END_REF].

Copepod dominated zooplankton play a vital role in the food chain of marine communities by transferring energy from primary producers (phytoplankton) to secondary and tertiary consumers (i.e., planktivorous fish) (Ustun, 2014). Because of the copepod's ecological importance, any disruption in their populations have adverse effects on the whole food chain cycle and therefore disrupt the entire web fauna (Darnis et al., 2012). Similar to any other organisms, copepods are vulnerable to contaminants that are released in oil spills as water-soluble fractions. The effect of water-soluble oil fractions on selected species of copepods in terms of mortality, development, egg production and motility is well established (Bejarano, 2006;Avila et al., 2010;Lee et al., 2013). In copepods, several genes, such as glutathione S transferases (GST) catalase, and Heat shock proteins have been considered as valuable biomarkers for cellular stress due to ROS (Zhou et al.,2020;Hansen et al., 2008). Additionally, members of the superfamily of cytochrome P450 genes have been considered as valuable biomarkers for WAF and oil exposure due to their roles in the biotransformation of a wide range of compounds (Rhee et al., 2013). Until now, little is known about cytochrome P450 detoxification functions in copepods. However, some cyp genes, members of the clan 3, have been shown to be WAF sensitive in Tigriopus japonicus and Paracyclopina nana (Han et al., 2014).

This work aims to investigate the toxicity profile of WAF and WAF with dispersant (NNA WAF+ Dispersant) using the model organism Acartia tonsa. Acartia tonsa short life cycle, easy maintenance and the high continuous ability to produce eggs, make it a suitable candidate for toxicological assays (Gorbi et al., 2012). Cosequently, Acartia tonsa has been studied after exposure to different contaminants and showed sensitivity to several toxins (Bellas et al., 2007[START_REF] Pinho | Physiological effects of copper in the euryhaline copepod Acartia tonsa: Waterborne versus waterborne plus dietborne exposure[END_REF]Lauer et al., 2010). Freshly prepared Water accommodated fraction from a Naphthenic North Atlantic sea oil was tested mimicking an oil spill scenario. In addition, the commercial dispersant Finasol OSR 52 was added to the WAF was also tested as a measure of remediation procedure after an oil spill.

The effects of the WAF and WAF+Dispersant to copepods will be studied using different biological endpoints. Lethality will be primarily investigated by calculating the lethal concentration (LC50). Afterward, reproductive capabilities after exposure to sub lethal concentrations will be studied through investigating different endpoints such as: the number of reproductive females, the egg reproduction rate and the percentage of successfully hatched eggs. Finally, studying different transcriptome levels of genes related to PAH exposure and oxidative stress will highlight the exposure effects at molecular level.

Materials and methods

Preparation of the Water Accommodated Fractions (WAF)

Two conditions of WAF were prepared for the exposure experiments: (299 cP at 2°) and a density of 0.900 g/ml. In addition, it is rich in branched and cyclic aromatic hydrocarbons (Katsumiti et al., 2019). The dispersant selected for this study was

Finasol OSR 52 (Total energies, France). Finasol OSR 52 is a second generation dispersant composed of a complex mixture of surfactants and petroleum distillates (Katsumiti et al., 2019). Detailed information regarding the composition of Finasol OSR 52 dispersant is presented in Table 1 (Total energies, France ).

20-25%

Hydrocarbons, C11-C14,n-alkanes, isoalkanes, cyclics,<2% aromatics

15-20%

(2-methoxymethylethoxy) propanol

15-20%

Carboxylic acids, di, C6-12 cmpds, with ethanolamine, boric acid cmpd with ethanolamine 0-2% Ethanolamine 0-1% Preparation of WAF was similar to the protocol described by Singer et al (2000) but with slight modifications. Briefly, crude oil was added to filtered seawater at a ratio of 5 g of oil to every 1 liter of water. The oil, water mixture was stirred for 42 hour at 10° C and under dark conditions to avoid photo oxidation of oil elements using a magnetic stirrer without creating vortex. Serial dilutions were prepared for the WAF. For NNA WAF+Dispersant, the commercial dispersant Finasol OSR 52 was added first to the oil at a ratio of 1 g of dispersant to every 10 g of crude oil, gently mixed, then WAF was produced as mentioned before.

Copepod culture

Different generations of Acartia tonsa were obtained from a continuous culture established for six months in the aquaria facility of PiE (UPV/EHU). Copepods were cultured in a 10 L tank with 0.1 µm filtered seawater at a temperature of 18°C, salinity of 30 Practical salinity unit and constant photoperiod of 16:8 light/dark cycle. Copepods were daily fed with a mixture of the brown micro algae Isochrysis galbana and the green microalgae Tetraselmis chuii at a ratio of 1:1

Lethal toxicity assays 96 hours:

Lethality tests were conducted according to the international standard for water quality, for the determination of acute lethal toxicity to marine copepods, (ISO14669). For each condition of WAF, 30 adult copepods were exposed in 250 ml glass concentrations containing 50 ml of filtered seawater and WAF conditions for up to 96 hours. Lethal assays were done in triplicates and total of five dilutions were assessed in each WAF condition. The tested WAF doses were: 10%, 30%, 50%, 70% and 100%. NNA WAF+Dispersant doses were initially considered to be the same for the NNA WAF.

However, the first experiment showed 100% lethality of the NNA WAF +Dispersant at 30% dose. As a result, the selected doses for NNA WAF+Dispersant assay were 5%, 10%, 15% ,20% and 25%

Each day of exposure, replicates were checked for mortality and dead copepods were removed. A copepod was considered dead if it didn't move shortly after a gentle stimulation using a pipette. Similar to the works done by Jiang et al. (2012) copepods

were not fed during the exposure period and no gender preference was considered when selecting adult copepods for the experiment. The LC50 results from the toxicity assays determined the sub lethal concentrations to be used in further assays.

Fecundity and egg hatching assays:

Exposure doses which were selected ranged from low to very high sub lethal concentrations. For NNA WAF, the selected exposure doses were 10%, 30% and 50%

NNA WAF+Dispersant exposure were 7.5%, 12.5% and 20% . In each treatment, 20 adult females were selected under a dissecting microscope. In addition, five adult males were included in each treatment to assist in maintaining female fertility throughout the whole experiment (Holste and Peck, 2006). The fecundity assay was divided into three consecutive stages: exposure stage, recovery stage and individual female fecundity test stage. This allows testing for the delayed effects caused by exposure scenarios.

In the exposure stage, females were transferred to 300 ml glass tanks filled with the different WAF conditions and left for seven days. Each day, eggs and dead copepods were removed. Every two days, 150 ml of the experimental tanks was replaced and tanks cleaned from fecal pellets and dead algae. Copepods were fed daily with the algae Tetraselmis chuii throughout the whole experiment at a concentration of 3000 cells per ml. During the recovery stage, the surviving copepods were gently transferred to filtered clean seawater for four days. Similar to the procedure during the exposure stage, the eggs were daily removed and water was replaced every two days.

In the individual fecundity experimental stage, the surviving females were transferred to incubating chambers containing filtered clean seawater for three days. Incubation chambers were constructed similar to the device proposed by Kleppel et al (1998). The system was divided into two chambers (spawning chamber and brooding chamber). The upper spawning chamber was a 10 cm in diameter plastic tube, which housed one female for egg production. A 200 µm mesh was attached at the base of the tube to prevent female individuals from entering the bottom compartment where eggs are stored. Each day, eggs were counted and copepods were fed at the same algal concentration mentioned before.

Recently laid eggs from each treatment were pipetted to individual petri dishes containing seawater to allow to hatch. Hatched eggs were counted and unhatched eggs after 48 hours were considered non viable.

Gene transcription analysis

Exposure experiments

Adult copepods were exposed in five replicates, with each replicate containing 35 adult copepods exposed to 150 ml of NNA WAF or NNA WAF+Dispersant solutions for 48 h.

Two exposure doses were chosen per WAF condition: NNA WAF (25% and 50%), NNA WAF+Dispersant (10% and 20%) . The copepods were fed daily with Tetraselmis chuii at a concentration of 3000 cells per ml, and no gender preferences were considered during copepod collection for exposure. After 48 h of exposure, the copepods were transferred to cryovials filled with RNA later solution (AMBION, USA) and stored at -80ºC until RNA extraction.

RNA extraction and cDNA synthesis

RNA was extracted using RNeasy Minikit (Qiagen, Germany) following the manufacturer protocol. Total RNA was treated with DNase-1 using the DNA-free kit (Invitrogen, USA) to remove traces of genomic DNA. RNA integrity and concentration

were measured using the Bioanalyzer 2100 (Agilent Technologies, USA). All RNA samples obtained a RIN score higher than 7, suitable for downstream qPCR analysis (Fleige et al., 2006). RNA purity was determined by measuring sample absorbance ratios of 260/280 nm using a UV-spectrophotometer (Epoch, Biotek, USA). All samples had ratios between 1.8-2.3. cDNA was synthesized from purified RNA using the AffinityScript Multiple Temperature cDNA synthesis kit (Agilent Technologies, USA)

with random primers and following the manufacturer's protocol. Current study

RT-qPCR

Real-time quantitative PCR was conducted using FastStart SYBR Green fluorescence dye (ROCHE Diagnostics, Switzerland) with ROX passive dye included in the mix. The reaction medium (25 µL) consisted of 12.5 µL FastStart SYBR Green master mix, 0.3 µL forward primer (800 nM), 0.3 µL reverse primer (800 nM) and 3 µL diluted cDNA template (1:5 v/v). qPCR was performed in 384 optical well plates (Applied Biosystems, USA) using ViiA 7 Real-Time PCR system (Applied Biosystems, USA). The amplification conditions were 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 second, and then 1 minute of annealing at the corresponding Tm for each gene (Table 2). A dissociation curve was performed after the PCR cycles to determine the product specificity and check the absence of primer dimers. All qPCR assays were conducted in triplicates, and a non-template control (NTC) was included for each plate.

Data analysis

Mortality of copepods at different timeframes (48 ,72, 96 hours) from the lethal toxicity assay were analyzed using PROBIT analysis (Vincent, 2008) Finally, relative gene transcription levels were determined using the Pfaffl method (Pfaffl et al., 2001). The transcript levels were log2 transformed and statistical differences among experimental groups and their respective controls were determined using one-way ANOVA. The Dunnet post-hoc test was further used to determine the statistical difference compared to control (IBM SPSS v26). A principal component analysis (PCA) for transcription results integration was performed using PAST statistical software version 4.03 (Hammer et al 2020)

Results

WAF and WAF+Dispersant lethal toxicity

The PROBIT analysis ( Letter superscript denote significant difference based on the absence of confidence interval overlap

Effect of WAF and WAF+Dispersant on fecundity

After the end of the exposure and recovery periods, adult females were individually placed in incubation chambers for three days to check the female reproductive capabilities. Females producing more than 2 eggs during the three days of the experimentation period were the only ones considered reproductive. Chi-square analysis showed that there was no significant association between the percentage of reproductive females and the doses of NNA WAF or NNA WAF+Dispersant (figure 1), suggesting that exposure did not affect the ability of laying eggs in adult females.

The number of surviving and reproductive females after exposure and recovery stage are presented in Figure 2. The egg production rate of females exposed to NNA WAF (Figure 3a) showed a slight dose dependent reducing trend, although not significant. In contrast, NNA WAF +Disp 34% (24 -100) a 31% (24 -55) a 21% ( 11.7 -63.5) a females exposed to 20% NNA WAF+Dispersant had a significant reduction in egg production rate compared to other experimental groups (p<0.05). None of NNA WAF and NNA WAF+Dispersant did significantly affect egg hatching (Figure 3).

Oil

Effect of WAF and WAF+Dispersant on gene transcription levels

High variability in relative gene transcription levels was observed. According to the expression patterns of the biomarkers, NNA WAF treatments and the 10% treatment of NNA WAF+Dispersant showed to have transcription levels different from the control.

Antioxidant enzyme coding genes, gst and cat, showed noticeable down regulation in the 10% NNA WAF+Dispersant treatment (Figure 4 a & b). In addition, hsp70, showed significant transcription downregulation, although non-significant, in the 10% NNA WAF + Dispersant treatment (Figure 4,d). Hsp70 also showed downregulation in expression levels at 25% NNA WAF treatment. Ferritin, hsp 90 and cyp 3026b did not show any significant changes expression patterns compared to controls (Figure 4). The principal component analysis explained the variability of combined transcription levels with two components. Both axes explained 91% of the total variance, with the first axis explaining 73% of the variance, while the second axis explained 18% of the variance.

Correlation scores between the biomarker genes and the prinicipal components(Supplimentary Materials, chapter 2, Table S1) showed that Hsp70, ferritin, catalase and gst have the highest positive correlation with the first component. Whereas .

4.Discussion:

In this study, we investigated the toxicity of NNA WAF with and without the addition of a commercial dispersant (Finasol OSR 52). NNA WAF+Dispersant had the most negative effect on the copepod Acartia tonsa. Such toxicity was highlighted by the low LC50, the low egg production rate at high doses and the changes in gene expression patterns such as the significant downregulation of hsp 70 gene. However, NNA WAF showed to have a slight, non-statistically significant, response regarding some of the reproductive and molecular endpoints.

During the last decades, the use of dispersants showed its efficiency in dispersing oil slicks into small droplets and minimize the oil slick area in several oil spill incidents, thus facilitating natural oil degradation [START_REF] Noirungsee | Influence of oil, dispersant, and pressure on microbial communities from the Gulf of Mexico[END_REF]. However, it has been shown that the toxicity of dispersants to organisms can be due to the effects of surface active components on the biological membrane of organisms. In addition, exposure to dispersants has been described to cause the disruption of respiratory cells resulting from electrolytic and/ or osmotic imbalance [START_REF] Wolfe | Influence of dispersants on the bioavailability and trophic transfer of petroleum hydrocarbons to larval topsmelt (Atherinops affinis)[END_REF]. Oil dispersed with chemicals can be more toxic than oil or dispersant alone as dispersants increase the concentration WAF+Dispersant had 96 hours LC50 values as low as 45% whereas the 96 hours LC50

of WAF was higher than 100% dose (Lee et al.,2013). [START_REF] Almeida | Ingestion and sublethal effects of physically and chemically dispersed crude oil on marine planktonic copepods[END_REF] showed that Acartia tonsa exposed to dispersant treated emulsions had higher mortality than adults exposed for 48 hours to crude oil emulsion alone. Additionally, dispersant added to crude oil emulsions decreased Acartia tonsa's egg production rates and affected naupilar development [START_REF] Almeida | Ingestion and sublethal effects of physically and chemically dispersed crude oil on marine planktonic copepods[END_REF]. Aside from direct toxicity, oil + dispersant cause narcosis and affect the feeding behaviors of the adult female copepods, causing decrease in egg production rates as a consequence WAF with and without dispersant did not have significant effect on the number of fecund females, as most of the females in all NNA WAF treatments produced eggs. In addition, considering the production of viable eggs, the eggs obtained from almost all exposed adult females showed high percentage of hatching success. Olsen et al. (2013) also observed high egg hatching success from Calanus finmarchicus exposed to dispersed crude oil droplets. Our results suggest that the WAF conditions did not have a significant effect on the female capability to produce viable eggs. However, further transgenerational studies are needed to investigate whether offsprings develop normally. Bejarano et al (2006) demonstrated with a full life cycle experiment on the meiobenthic copepod Amphiascus tenuiremis that up to 50% WAF from light crude oil had detrimental effects on the development time and fecundity of the exposed females. Our results also highlight that the WAF conditions mainly affected the eggs production rates. In addition, the significant reduction of egg production rate in NNA WAF+Dispersant exposure in the individual fecundity stage even after the four days recovery period, suggest that the toxic damage from the exposure stage can be long-termed and persistent. Such toxic effects after exposure period highlights that the toxicity is still persistent even after the WAF remediation from the environment or after the migration of the copepods from the WAF polluted area during for example vertical migration (Calbet et al., 2007).

Studying the co-expression of different genes involved in oxidative stress and xenobiotic metabolism provides a valuable insight on gene co-regulation patterns in Acartia tonsa in response to the exposure to NNA WAF or NNA WAF+Dispersant. PCA showed that both NNA WAF and NNA WAF+Dispersant have distinct expression profiles from each other (Figure 5), suggesting thus different cellular toxicity effects. The co-downregulation patterns exhibited in 10% NNA WAF +Dispersant might suggest a decrease in the cellular antioxidant machinery, which probably result in the over accumulation of ROS in cells (Dvorak at al., 2021). Similar to our findings, the intertidal copepod, Tigripus japonicus, showed significant downregulation in the expression levels of gst after exposure to 6.25 µM of PCB (Lee et al., 2006).

Interestingly, the transcription patterns of NNA WAF+Dispersant exposed adults were showing a non monotonic response, in which the effects of the high dose cannot predict the effects of low doses [START_REF] Bicho | Effects of Ag nanomaterials (NM300K) and Ag salt (AgNO3) can be discriminated in a full life cycle long term test with Enchytraeus crypticus[END_REF]. We hypothesize that the exposure to 20%

NNA WAF+Dispersant dose had narcotic effects on Acartia tonsa, resulting to low metabolism and therefore affected reproduction capabilities and gene transcription levels.

Similar to the results we observed, PAHs in WAF was shown to narcotize the copepod O. davisae that result in severely impaired behavioral and reproductive responses [START_REF] Barata | Predicting single and mixture toxicity of petrogenic polycyclic aromatic hydrocarbons to the copepod Oithona davisae[END_REF]. Several contaminants, such as fluoranthene, cadmium, nonylphenol, and octylphenol, inhibit the expression of heat shock proteins, which consequently reduce the cellular cyto-protective mechanisms (Werner et al., 1997;[START_REF] Rhee | Heat shock protein (Hsp) gene responses of the intertidal copepod Tigriopus japonicus to environmental toxicants[END_REF]. Compared to WAF alone, dispersant increase the bioavailability of petroleum hydrocarbons in water (Yamada et al., 2003). Therefore, we speculate that the addition of dispersant increased the bioavailable concentrations of PAHs, such as fluoranthene, possibly resulting to hsp70 downregulation. However, to fully understand the toxicity effects of dispersants chemical analysis of WAF +Dispersant of the available PAHs is needed .

Conclusions

In conclusion, WAF+Dispersant had marked effects on the adult Acartia tonsa at different biological levels. As shown, WAF+Dispersant had detrimental effects on Acartia tonsa in respect of mortality, reproduction capabilities and caused cellular stress. Therefore, we speculate that exposure to WAF+Dispersant disrupt the population of Acarita tonsa by inhibiting reproduction, which might disrupt the whole food chain. Transgenerational studies will provide further information on the effects of contamination on the different generation of Acartia tonsa after exposure.

Chapter 3: Microbial community dynamics in Crude oil Water Accommodated Fraction and Water Accommodated Fraction + Dispersant contaminated cold seawater

This chapter has been presented in the 30th SETAC Europe online annual meeting

This chapter is being prepared to be published as: specific bacterial taxa probably involved in PAHs degradation, and iii) the effects of

Introduction

Cold areas, such as the sub arctic area, are increasingly threatened by human activities due to climate change effects opening novel sea lines (Farrington, 2014). Particularly, the Arctic seas are exposed to oil-related activities presenting a high risk of oil spill accidents [START_REF] Vergeynst | Biodegradation, Photo-oxidation, and Dissolution of Petroleum Compounds in an Arctic Fjord during Summer[END_REF]. Among hydrocarbon compounds, polycyclic aromatic hydrocarbons (PAHs) are the most toxic, being of global concern due to their persistence in the environment, and their toxicity towards a wide range of organisms [START_REF] Patel | Polycyclic Aromatic Hydrocarbons: Sources, Toxicity, and Remediation Approaches[END_REF]. In addition, the fate of PAHs in marine ecosystems is largely dependent on microbial activities and abiotic weathering processes (Duran and Cravo-Laureau, 2016).

The abiotic weathering processes, including mechanical actions (e.g. wave movements)

and chemical reactions (e.g. photo-oxidation), favor PAHs solubility, especially low molecular weight PAHs (LMW-PAHs), increase the hydrocarbon bioavailability and thus toxicity (Duran and Cravo-Laureau, 2016;[START_REF] Neff | Ecological risk assessment of polycyclic aromatic hydrocarbons in sediments: Identifying sources and ecological hazard[END_REF]. In order to simulate weathering processes, scientists are using water-accommodated fractions (WAF), which contain harmful aromatic hydrocarbons and aliphatic compounds, PAHs being the most toxic compounds (Jiang et al., 2012). WAF is considered as useful model for studying oil spills dynamics, determining the fate of solubilized hydrocarbons, their toxicity and degradation by microorganisms (Faksness et al., 2020).

In response to oil spill accidents, several intervention approaches have been developed

for clean up processes, including bioremediation, the use of dispersants, and mechanical removal [START_REF] Hazaimeh | Bioremediation perspectives and progress in petroleum pollution in the marine environment: a review[END_REF]. The cost of oil spill clean up is difficult to evaluate because it depends on several factors including the type of the oil, the environmental conditions, the nation, and the incident site [START_REF] Prendergast | Assessing the performance and cost of oil spill remediation technologies[END_REF]. The clean up cost has been evaluated for the Exxon Valdez, the Prestige, and the Deepwater Horizon oil spills was around 3, 12, and 63 billon dollars respectively, both combining several strategies including the use of dispersant, bioremediation and mechanical recovery [START_REF] Dave | Remediation Technologies for Marine Oil Spills: A Critical Review and Comparative Analysis[END_REF]. It has been shown that the clean up strategies can be ranked in increasing order of cost as follow: bioremediation (including natural attenuation, bio-stimulation by fertilizer addition, and bio-augmentation by addition of microorganisms), in-situ burning, use of dispersants, and mechanical and manual recovery [START_REF] Etkin | Arctic and Marine Oil spill Program Technical Seminar[END_REF][START_REF] Prendergast | Assessing the performance and cost of oil spill remediation technologies[END_REF]. Thus, bioremediation, involving microbial activities, is considered as a less costly, highly effective, and environmental-friendly approach in comparison to the other remediation procedures [START_REF] Ali | Bioremediation of soils saturated with spilled crude oil[END_REF][START_REF] Hazaimeh | Bioremediation perspectives and progress in petroleum pollution in the marine environment: a review[END_REF]. It has been estimated that cleaning costs can be reduced by about 30 to 50% by bioremediation approaches [START_REF] Lü | Bioremediation: The New Directions of Oil Spill Cleanup[END_REF]. In addition, the application of chemical and physical treatments after oil spills are difficult to implement under certain situations, such as large areas covered by spilled oil, and bad weather conditions [START_REF] Kumar | Novel and cost-effective technologies for hydrocarbon bioremediation[END_REF][START_REF] Ventikos | A high-level synthesis of oil spill response equipment and countermeasures[END_REF]. Therefore, bioremediation is then the most suitable strategy [START_REF] Alvernia | The Use of Bioremediation Technology on Oil-Contaminated Soil[END_REF].

The use of dispersants is a strategy implemented to stimulate the microbial degradation by increasing hydrocarbon solubility and thus bioavailability (George-Ares et al., 2000;Yamada et al., 2003), but unfortunately increasing also toxicity [START_REF] Baca | Net environmental benefit analysis (EBA) of dispersed oil on nearshore tropical ecosystems derived from the 20 year "TROPICS" filed study[END_REF].

Indeed, several studies have shown that dispersant have adverse toxic effects on marine organisms (Lee et al., 2013;[START_REF] Cohen | Toxicity of Three Oil Spill Remediation Techniques to the Australian Bass Macquaria novemaculeata[END_REF]Mitchell and Holdway, 2000), including benthic organisms (Cuny et al., 2015) and microbial communities [START_REF] Brakstad | Biodegradation of dispersed Macondo oil in seawater at low temperature and different oil droplet sizes[END_REF][START_REF] Mcfarlin | Biodegradation of Crude Oil and Corexit 9500 in Arctic Seawater[END_REF]Garneau et al., 2016;Kleindienst et al., 2015). However, only few studies have investigated the effect of dispersant on PAHs microbial degradation at low temperature [START_REF] Pan | Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp. S-07[END_REF]. In cold environments, the low temperature and the seasonal variation of nutrient availability limit PAHs biodegradation by microorganisms [START_REF] Vergeynst | Biodegradation, Photo-oxidation, and Dissolution of Petroleum Compounds in an Arctic Fjord during Summer[END_REF][START_REF] Brakstad | Natural and Stimulated Biodegradation of Petroleum in Cold Marine Environments[END_REF]. Therefore, further knowledge on microbial hydrocarbon degradation in cold conditions is required in order to develop adapted microbial management strategies for fighting oil spills. In addition, industrialization and urbanization introduce PAHs into coastal areas (Duran et al., 2015), resulting in chronic PAHs pollution threatening the human health (Duran and Cravo-Laureau, 2016;[START_REF] Lv | Human impacts on polycyclic aromatic hydrocarbon distribution in Chinese intertidal zones[END_REF]. Such chronic PAHs pollution allows the adaptation of microbial communities exhibiting PAHs degradation capabilities (Bordenave et al., 2004;[START_REF] Louati | The roles of biological interactions and pollutant contamination in shaping microbial benthic community structure[END_REF]Stauffert et al., 2014), which constitute the legacy of the microbial communities.

We hypothesize that microbial communities have their own PAHs degradation capacity at low temperature, which probably depend whether or not they have been adapted to the presence of PAHs and to the presence of dispersant. In order to verify the hypothesis, we investigated the effect of WAF, prepared in presence of dispersant or not, on water from two cold environments, one oil-polluted and the other pristine, in microcosm experiment during 21 days, focusing on PAHs because they are the most toxic compounds in WAF.

The study focused on three main objectives: i) determine the PAHs degradation capacities of the bacterial communities assessed by GC-MS analysis, and how the addition of dispersant affects PAHs degradation capabilities ii) evaluate the resulting toxic effect of the introduction of WAF and WAF+dispersant by toxicity assays on the copepod Acartia tonsa, and iii) characterize microbial community changes in the water column identifying microbial taxa linked with PAHs degradation by 16S rRNA gene Illumina sequencing.

The comparison of microbial communities' behavior from oil-contaminated site to that from pristine site allowed the characterization of the microbial legacy effect, which was more important that the presence of dispersant in shaping the organization of microbial community. Our study provides useful information for the development of strategies for the management of microbial communities in response to an oil-spill.

Materials and methods

Sampling sites

Sediment and water samples were collected from two sites, a pristine and an oilcontaminated site. The oil-contaminated site was located in a harbor in the middle of Tromso, Norway (69°39'08.7"N 18°57'50.2"E) characterized with busy boat activity.

Whereas the pristine site located in a sound in Tisnes, Norway (69°36'24.4"N 18°49'56.3"E) with little anthropogenic influences nearby. Samples were collected from the coast at a shallow depth, stored in polypropylene containers at 4 °C. The containers were then shipped to Pau, France at constant temperature of 4 °C.

Water accommodated fractions preparation

Two types of water accommodated fractions were prepared for the microcosm experiments: 1) crude oil WAF, and 2) dispersant added to WAF (WAF+dispersant). The soluble fractions were made from an arctic crude oil provided by Norwegian university of science and technology (NTNU). Naphthenic North Atlantic (NNA) is a naphthenic based light crude oil with a low aromatic hydrocarbons content, low wax content, low viscosity index (299 cP at 2 °C) and density of (0.900 g mL -1 ) [START_REF] Hafez | Application of a biological multilevel response approach in the copepod Acartia tonsa for toxicity testing of three oil Water Accommodated Fractions[END_REF]. The crude oil is characterized by high amount of polycyclic aromatic hydrocarbons (Katsumiti et al., 2019). Preparation of WAF was similar to the protocol described by Singer et al., (2000) but with slight modifications. Briefly, in 8 L glass bottles, crude oil was added to artificial seawater of salinity 30 ppt at a ratio of 5 g of oil per 1 L water. The oil water mixture was stirred for 42 h at 10 °C and under dark conditions using a magnetic stirrer without creating vortex. For WAF+dispersant, the dispersant Finasol OSR 52 (Total energies company, France) was added first to the oil at a ratio of 1 g of dispersant per 10 g of crude oil, gently mixed, then the WAF+dispersant was produced following the same procedure than for WAF.

Microcosm set up

For each microcosm, 200 g of sediment, 500 mL of collected water and 500 mL of WAF were added to a 2 L sterile glass flasks covered with filter rubber mesh tap to allow gas exchange. Control, WAF and WAF+dispersant microcosms were prepared in triplicate with water and sediments collected from either the pristine or the contaminated sites. Thus a total of (3 control + 3 WAF + 3 WAF+dispersant) x 2 sites = 18 microcosms were prepared. All microcosms were incubated at 4 °C for 3 days before the addition of either WAF or WAF+dispersant to allow acclimatization. After addition of WAFs, the microcosms were incubated for 21 days at 4 °C in the dark to prevent photosynthesis and photoreactions. Every day, each microcosm was gently mixed to allow the resuspension of particles and oxygenation. Water samples were collected after 0, 7, 14, and 21 days for microbial and PAHs analyses. In addition, samples at day 0 and day 21 were collected for copepods toxicity assays. The samples for microbial analysis were collected by filtration (100 mL water) on 0.22 m membrane under vacuum. The filters were stored at -80 °C until analysis.

Chemical analysis of the PAHs content

The Analytes were separated using the following conditions: carrier gas, helium (1.3 mL min - 1 ); transfer line, ion source and quadrupole analyser temperatures were maintained at 300, 230 and 150 ºC, respectively. The following temperature program was used for target sequences. The total number of sequences for each sample obtained after denoising is presented in Supplementary Materials (Table S1). Denoised sequences were assigned with taxonomic affiliation using RNA gene reference database SILVA version 132 (Quast et al., 2013). In addition, singletons were filtered. Finally, sequences were rarefied according to the lowest number of total sequences observed in samples.

Rarefactioncurves are presented in (Supplementary Materials, chapter3, Figure S1). An abundance table with the total available amplicon sequence variant (ASVs) was produced removing those with less than 250 counts (representing about 1% of total counts per sample). The complete dataset was deposited in the NCBI Sequence Read Archive (SRA) database (SUB11018949) under the Bioproject (PRJNA802112).

Copepod mortality assays and gene transcription assays

Total of 30 adults of the copepod Acartia tonsa were selected under a dissection microscope. Adult copepods were placed in 75 mL water samples collected from the microcosms for 96 hours. Copepods were not fed during exposure and no gender preference was considered when collecting the copepods for the experiments. Everyday, copepod mortality was checked and dead copepods were removed. A copepod was considered dead when is not moving after a gentle stimulation with a pipette. After 96 hours, the surviving copepods were collected and placed in cryotubes filled with RNA later (Invitrogen, USA). The pooled copepods were homogenized using beads tissue homogenizer (Precellys 24 tissue homogenizer, France) with the following program: 45 secs at 6500 rpm, then 2 min on ice, then another 6500 rpm. RNA was extracted using RNA easy minikit (Qiagen, Germany) following the manufacture protocol. The extracted RNA quantity and quality were observed using 2100 bioanalyzer (Agilent technologies, USA). RNA from the extracted samples showed to have minimum Rin score of 7, indicating intact extracted RNA. In addition, RNA purity was determined by measuring samples' absorbance ratios of 260/280 nm using a spectrophotometer (Epoch, Biotek, USA). All samples had acceptable ratios between 1.8 and 2.3. cDNA was synthesized from purified RNA using the AffinityScript Multiple Temperature cDNA synthesis kit (Agilent Technologies, USA) with random primers and following the manufacturer´s protocol. In addition, cDNA concentration was quantified using Quant -it Oil green stain (Invitrogen, USA).

Primer design and Quantitative Real-time PCR (qRT-PCR)

In order to evaluate the effects of PAHs exposure in Acartia tonsa, the gene encoding the multifunctional enzyme glutathione S-transferase (gst) has been selected as a biomarker because its expression has been shown to be responsive to PAHs exposure in Acartia tonsa (Zhou et al., 2020). The primers for the amplification of gst genes were designed from conserved regions observed by aligning Acartia tonsa genome (PRJEB20069) with

Tortanus forcipatus (KT755427.1) and Acartia pacifica (KT754520.1) using Clustal omega (Madiera et al., 2019). Hybridization Taqman Probes were designed according to the sequenced amplicons obtained from the gst primers. For the quantitative PCR, the reaction mixture (25 µL) consisted of 12.5 µL Taqman gene expression master mix (Applied Biosystems, USA), 0.3 µL (800 nM) forward and reverse primers, 0.25 µL hybridization probe, 8.2 µL water and 3 µL cDNA template, undiluted. Quantitiative PCR was performed in 384 optical well plates (Applied Biosystems, USA) using ViiA 7 Real-Time PCR system (Applied Biosystems, USA). The amplification conditions were 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and then 1 min. of annealing at 60 °C.

Statistical analysis

Analysis of the bacterial community dynamics such as alpha diversity indices, bacterial abundances at the phylum level, were conducted on R software using RAM package (Chen et al., 2018). Correspondence analysis (CA), canonical correspondence analysis (CCA), permutational multivariate analysis of variance (PERMANOVA), and similarity percentages analysis (SIMPER) were performed using PAST statistical software version 4.03 (Hammer et al., 2001).

Linear discriminant analysis effect size (LEfSe, Segata et al., 2011) PAHs. Finally, the network modules were generated with "greedy modularity optimization mode" and the contaminated and pristine networks were visualized using cytoscape [START_REF] Shannon | Cytoscape: a software environment for integrated models of biomolecular interaction networks[END_REF]. Finally, Acartia tonsa gene transcript levels were normalized with the amount of input cDNA using formula adapted from the ΔΔCT normalization method (Rojo-Bartolome et al., 2016).

Results and Discussion

PAHs degradation and toxicity

The initial water accommodated fractions (WAF) contained similar total PAHs concentration whether they were prepared with or without dispersant, and irrespective of the water origin. They exhibited similar PAHs composition (unpaired t test p > 0.05) containing mainly naphthalene and derivatives (acinaphtalene and acenaphtlene, > 50 µg L -1 ), while the content of high molecular weight PAHs (> 3 rings) was below 1 µg L -1 .

For both contaminated and pristine control microcosms the mortality at time 0 and time 21 days was below 15 %. However, the WAF+dispersant showed higher toxicity with 100% copepod (Acartia tonsa) mortality whereas the WAF exhibited 20% copepod mortality (Figure 1), showing the toxic effect of the WAF+dispersant. It has been shown

that WAF+dispersant has high toxicity toward marine organisms compared to WAF (Lee et al., 2013), especially toward Acartia tonsa (Rattes de [START_REF] Almeida | Ingestion and sublethal effects of physically and chemically dispersed crude oil on marine planktonic copepods[END_REF]. In addition, dispersed oil droplets have been shown to be more toxic than WAF due to the bioavailability of low soluble and high molecular weight PAHs [START_REF] Berrojalbiz | Accumulation and cycling of polycyclic aromatic hydrocarbons in zooplankton[END_REF].

During microcosm incubation, the PAHs content decreased drastically, faster in microcosms prepared with water from the contaminated site, with removal of almost all total PAHs (96%) within 7 days while with pristine site water the complete PAHs removal (98%) was obtained after 14 days (Figure 1). Such discrepancy on PAHs content decrease according to the origin of water suggests that biodegradation participate to PAHs removal, consistent with the fact that abiotic removal is limited at low temperature as the volatility of low molecular weight PAHs decreases increasing their viscosity [START_REF] Atlas | Effects of Temperature and Crude Oil Composition on Petroleum Biodegradation[END_REF]. Such observation is further supported by studies showing hydrocarbon biodegradation at low temperature [START_REF] Gomes | Biodegradation of water-accommodated aromatic oil compounds in Arctic seawater at 0 °C[END_REF][START_REF] Ribicic | Microbial communities in seawater from an Arctic and a temperate Norwegian fjord and their potentials for biodegradation of chemically dispersed oil at low seawater temperatures[END_REF]. Noteworthy, the PAHs degradation rate was not increased by the presence of dispersant (Figure 1a), probably due to the bioavailability of solubilized PAHs in WAF [START_REF] Liu | Chemical Composition and Potential Environmental Impacts of Water-Soluble Polar Crude Oil Components Inferred from ESI FT-ICR MS[END_REF][START_REF] Berrojalbiz | Accumulation and cycling of polycyclic aromatic hydrocarbons in zooplankton[END_REF]. Accordingly with PAHs removal, the toxicity at the end of the experiment (21 days), assessed by Acartia tonsa mortality, was similar in all conditions (unpaired t test p > 0.05), close to that observed at the beginning of the experiment in WAF (Figure 1b) without dispersant.

Microbial communities characterization and composition

After trimming, the retained 296,386 sequences were rarefied to 3,224 sequences per samples, corresponding to the lowest sequences number in a sample. The retained sequences were then dispatched within 4,024 ASVs (Supplementary Materials, chapter 3, Table S1). The rarefaction curves for the different conditions indicated that the sequencing depth was sufficient to reveal the microbial diversity (Supplementary Materials, chapter 3, Figure S1). Nevertheless, despite several attempts, it was not possible to characterize the microbial communities from contaminated water at day 14 under the WAF+dispersant condition, and at days 14 and 21 under the WAF condition, probably because the microbial abundance was low. We hypothesize that the depletion of hydrocarbon (Figure 1) in water caused the collapse of microbial communities inhabiting the contaminated water at day 14 in WAF and WAF+dispersant. Hydrocarbons available in WAF and in WAF+dispersant probably represent the main carbon source for the growth of oil degraders [START_REF] Xu | Petroleum Hydrocarbon-Degrading Bacteria for the Remediation of Oil Pollution Under Aerobic Conditions: A Perspective Analysis[END_REF]. However, in the case of WAF+dispersant, the added hydrocarbons and dispersant represent an easily degradable carbon source for prokaryotes rapidly consumed (after 7 days, Figure 1) corresponding to a priming effect usually described after addition of fresh bioavailable carbon source [START_REF] Pascault | Stimulation of Different Functional Groups of Bacteria by Various Plant Residues as a Driver of Soil Priming Effect[END_REF].

Then after adaptation (around 21 days, Figure 1), microbial community use less bioavailable carbon source present on the water.

The Shannon index was between 2.95 and 4.26, increasing in pristine water during the experiment while decreasing in polluted water (Figure 2, and Supplementary Materials, chapter3, Table S1). However, according to one way ANOVA, no significant differences were observed among Shannon indices during the experiment in all conditions.

The microbial communities were dominated by Proteobacteria (65% -25%),

Bacteroidetes (60% -10%), Firmicutes (40% -5%) and Nanoarchaeaota (20% -1%) phyla, while Cyanobacteria, Actinobacteria, Fusobacteria, Thaumarchaeota, phyla were present at lower abundance, less than 10% (Figure 2). Noteworthy, changes in microbial community composition were observable during the incubation period characterized by increasing abundance of Bacteroidetes with concomitant decreasing of Proteobacteria and Firmicutes abundances irrespective of the condition and the origin of water (Figure 2). However, it can be noticed that at the beginning of the experiment, the Bacteroidetes abundance was lower in the pristine water but reached similar abundance to that observed in contaminated water at the end of the experiment. Such observation suggested that members of the phyla Proteobacteria, Bacteroidetes and Firmicutes play crucial roles in PAHs degradation within the microbial community. These phyla have been described to be dominant in hydrocarbon-enriched environments [START_REF] Kwon | Aerobic Hydrocarbon-Degrading Bacteroidetes 4[END_REF]. For example, during the Deepwater Horizon oil spill accident, members of Bacteriodetes became the major phyla in the water column (Redmond and Valentine, 2012), contributing to 3% of the total hydrocarbon degradation activity [START_REF] Kwon | Aerobic Hydrocarbon-Degrading Bacteroidetes 4[END_REF]. Crisafi et al. (2016) demonstrated that at 4 °C for 90 days, oil enriched samples favored several hydrocarbon degrader strains belonging to Gammaproteobacteria and Alphaproteobacteria, being capable to degrade up to 80% of the available hydrocarbons. In addition, Firmicutes showed to be important components in the bacterial communities composition from samples from western Siberia oil fields [START_REF] Frank | Water-Injected and Pristine Oil Reservoirs[END_REF]. In fact, several members of the phyla Firmicutes and Proteobacteria are considered to be opportunistic heterotroph (Nguyen-Lopez Lozano et al., 2013) that probably explains the increase abundance of such phyla after the addition of WAF and WAF+dispersant.

Comparison of microbial communities

The correspondence analysis (CA) separated the microbial communities of pristine water apart from those of contaminated water into two main clusters along the axis 1 that explained 19% of the data distribution (Figure 3), indicating that the microbial community structures were different according to the site. Noteworthy, the microbial communities of both sites were separated, according to time within each main cluster, along the axis 2 explaining 13% of the data distribution despite the missing samples of polluted water at days 14 and 21 (Figure 3). Such observation further confirmed the modifications of microbial communities during the experiment, which corresponds to the ecological succession relating the microbial plasticity for the adaptation to the environmental conditions (Bordenave et al., 2004). The ecological succession has been reported during PAHs-bioremediation processes [START_REF] Cravo-Laureau | Role of environmental fluctuations and microbial diversity in degradation of hydrocarbons in contaminated sludge[END_REF] as well as in environments with the presence of various pollutants, including metals [START_REF] Giloteaux | Three-year survey of sulfate-reducing bacteria community structure in Carnoulès acid mine drainage (France), highly contaminated by arsenic[END_REF], nanoparticles [START_REF] Brito | Effect of CuO nanoparticles over isolated bacterial strains from agricultural soil[END_REF], pesticides [START_REF] Vercraene-Eairmal | Diuron biotransformation and its effects on biofilm bacterial community structure[END_REF], and even during niche colonization in volcano [START_REF] Medrano-Santillana | Bacterial diversity in fumarole environments of the Paricutín volcano, Michoacán (Mexico)[END_REF]. Additionally, similarity percentage analysis (SIMPER) revealed differences between the microbial community composition according to the conditions (control, WAF, and WAF+dispersant) with dissimilarity between 38% and 54% at the beginning of the experiment, and above 55% for the rest of the experiment. The dissimilarity was higher between control and WAF+dispersant (60% < SIMPER > 80%) than between control and WAF (32% < SIMPER > 62%), revealing the effect of both WAF and WAF+dispersant on the microbial communities. These observations were supported by permutation analysis of variance (PERMANOVA) indicating that the origin of water was the main factor explaining data distribution followed by time and then the conditions (Supplementary Materials, chapter 3, Table S2). The initial composition of the microbial communities plays important roles in shaping the responses toward oil contamination as observed [START_REF] Ortmann | Initial community and environment determine the response of bacterial communities to Dispersant and oil contamination[END_REF][START_REF] Al-Jawasim | Synergistic effect of crude oil plus dispersant on bacterial community in a louisiana salt marsh sediment[END_REF]. Linear discriminant analysis (LDA) effect size (LEfSe) revealed taxa significantly more abundant according to the condition for both pristine and contaminated waters (Figure 4 and Supplementary Material, chapter 3, Figure S2) explaining the observed differences between the microbial communities. Taxa found significantly more abundant in a condition have been proposed as bioindicator (Segata et al., 2011). More bioindicators for contamination were observed for contaminated water than for pristine water while, in contrast, more bioindicators were detected for the control in the pristine water (Figure 4).

Such observation indicates that due to legacy effects, the contaminated water microbial community was adapted to the presence of hydrocarbon. Such legacy effect has been described in temperate aquatic environments [START_REF] Bourhane | Microbial diversity alteration reveals biomarkers of contamination in soil-river-lake continuum[END_REF]Ben Salem et al., 2019;Bordenave et al., 2008;Duran et al., 2015;[START_REF] Misson | Chemical multi-contamination drives benthic prokaryotic diversity in the anthropized Toulon Bay[END_REF] but not yet in cold environments.

The abundance distribution of the identified bioindicators by LEfSe according to the treatment (control, WAF, and WAF+dispersant) further revealed the specificity of taxa to the treatment (Figure 4a). For the pristine water, seven bioindicators were exclusive for the WAF+dispersant (Figure 4a) while no exclusive ASVs were found for the other conditions. It is likely that most of taxa associated to the bioindicators for pristine water were able to cope with the presence of contaminant as they are equally distributed within The most striking observation was that among the bioindicators taxa, Colwellia was exclusively found in WAF+dispersant treatment for contaminated water. Such observation was in agreement with previous studies showing that Colwellia was detected in oil containing cold seawater (Garneau et al., 2016), dominating bacterial communities after the Deepwater Horizon oil spill, and presenting the capacity to biodegrade aromatic hydrocarbons [START_REF] Mason | Single-cell genomics reveals features of a Colwellia species that was dominant during the Deepwater Horizon oil spill[END_REF]. Additionally, Colwellia has been shown to benefit from the presence of dispersant (Kliendiest et al., 2015).

Noteworthy, several ASVs belonging to Fusibacter, genus characterized by the ability to degrade low molecular weight PAHs [START_REF] Folwell | Exploring the capacity for anaerobic biodegradation of polycyclic aromatic hydrocarbons and naphthenic acids by microbes from oil-sands-process-affected waters[END_REF], were identified as contamination bioindicator (WAF and WAF+dispersant) in contaminated and pristine waters (Figure 4b). Interestingly, it was demonstrated that Fusibacter dominated the bacterial community after dispersant addition in oiled sediments, probably by using it as carbon source (Calderon et al., 2018). Most of the ASVs revealed as contamination bioindicators were related to taxa known as hydrocarbon degraders such as Moritella [START_REF] Bagi | Naphthalene biodegradation in temperate and arctic marine microcosms[END_REF], Paraperlucidibaca [START_REF] Brakstad | Natural and Stimulated Biodegradation of Petroleum in Cold Marine Environments[END_REF]. Similarly, the psychrophilic

Shewanella have shown pyrene degradation potential (Rathour et al., 2018), some strains have even been isolated from Arctic Sea PAHs-rich sediment [START_REF] Dong | Distribution of PAHs and the PAH-degrading bacteria in the deep-sea sediments of the high-latitude Arctic Ocean[END_REF]. In addition, it is important to note that two ASVs belonging to the Candidatus Woesearchaeota archaeal phylum (ASV 314 and ASV 318) were identified as exclusive bioindicators for control contaminated water, suggesting that they were sensitive to the addition of WAF and WAF+dispersant.

The relation between prokaryotic taxa and PAHs toxicity

Canonical correspondence analysis (CCA) assisted in visualizing how microbial taxa interacted with the PAHs toxicity through Acartia tonsa mortality and the downregulation of gst transcription levels. For microcosms prepared with pristine site water, the first two axes of the CCA explained 99% of the observed variance (axis 1: 72% and axis 2: 27%; Figure 5a). Axis 1 was positively explained by PAHs concentration and Acartia tonsa mortality but negatively explained by the downregulation levels of gst gene. Such observation indicated the correlation between PAHs concentrations and toxicity.

Microbial communities in microcosm prepared with WAF+dispersant (at t0) were strongly correlated with the mortality and the concentration of PAHs (Figure 5a). These positively correlated with the gst gene expression levels (Figure 5a).

For microcosms prepared with water from contaminated site (Figure 5b), the CCA explained 98% of the variance (axis 1: 87%, axis 2: 11%). The PAHs concentrations and Acartia tonsa mortality were positively explained by the first axis while gst gene downregulation levels were negatively explained by the second axis (Figure 5b). In general, the communities of WAF and WAF+dispersant microcosms at t0 were correlated with PAHs concentrations and with copepod mortality. However, the communities at microcosm treatments at time 21 correlated with the gst gene down transcription patterns.

The abundance of several ASVs showed strong positive correlation with copepod mortality such as Fusibacter (ASV 73, ASV 58, ASV 93, ASV 67, ASV 26, ASV 39, ASV 11, and ASV 74), an ASV of Moritella (ASV 33), and an ASV from the family Rhodobacteraceae (ASV 42). In addition, the abundance of some ASVs was correlated with the concentration of PAHs. These ASVs were affiliated to Marinifilum (ASV 83, ASV 30, ASV 2, ASV 68, ASV 48, ASV 89, and ASV 9), Rhodobacteraceae family (ASV 64), and Colwellia (ASV 88). In both pristine and contaminated microbial communities, Fusibacter, showed to be an important taxa capable to survive the toxicity of dispersant. Currently, Fusibacter is yet to be directly linked to PAHs biodegradation (Kappel et al., 2014). However, we hypothesize that strains belonging to Fusibacter, among the other correlated ASVs identified, played an important role in reducing the toxicity of dispersant. The mineralization of dispersant (e.g. Corexit EC9500A) by a consortium of marine microbes has been shown to be faster than that of crude oil at low temperature [START_REF] Lindstrom | Biodegradation of petroleum hydrocarbons at low temperature in the presence of the dispersant Corexit 9500[END_REF].

Several ASVs correlated with gst gene expression belonged to Marinifilum, Lutibacter, Salinirepens, Polaromonas, Colwellia. These correlations probably result from the reduction of dispersant toxicity, suggesting the sensitivity of these taxa toward the toxicity of dispersant. It is likely that such taxa, stimulated after 21 days of microcosm experiment, were initially out competed by the opportunistic taxa, which were rapidly stimulated by the dispersant at the beginning. Then, later on the experiment, such stimulated taxa were replaced upon the depletion of the carbon source provided from dispersant. Dispersant have shown to be able to alter bacterial communities by stimulating some taxa while inhibiting others [START_REF] Yoshida | Microbial responses using denaturing gradient gel electrophoresis to oil and chemical Dispersant in enclosed ecosystems[END_REF].

Although the toxicity of the dispersant decreased during the experiment (Figure 1), the WAF+dispersant microcosms still had deleterious sub-lethal effects towards Acartia tonsa at day 21 as observed by the down expression of the gst gene. The gst genes are known to be involved in antioxidant mechanisms capable to detoxify several xenobiotics and protect the cell from oxidative stress (Lee et al., 2006). The low expression levels of gst gene observed in exposed Acartia tonsa suggests a decrease in the cellular antioxidant machinery resulting probably in the over accumulation of ROS in cells (Dvořák et al., 2021). However, further studies are needed to understand the significance of transcript downregulation of gst gene in Acartia tonsa. Bacterial communities of microcosms prepared with pristine site water, (b) bacterial communities of microcosms prepared with contaminated water. Blue numbers denote the number of ASV, black names denote the microcosm communities: t(n): time (day) sample is collected from the microcosm, w, wd, and c: WAF, WAF+dispersant, and control respectively.

Ecological association network

In order to characterize the microbial interactions occurring in response to WAF or WAF+dispersant exposures, a molecular ecological network analysis was performed.

Networks describing strong correlations (spearman correlation threshold at 0.910 and 0.950; p value < 0.05) were obtained (Supplementary Materials, chapter 3, Table S4).

Pristine communities' network showed more density by having 171 total nodes and 468 links, while contaminated communities network showed 112 total nodes and 225 links.

However, R 2 of power law fitted better the pristine site network (0.68) compared to contaminated site network (0.48), indicating that pristine site fits better as a scale free network, where fewer taxa accounts for the majority of interactions. However, both networks exhibited average path distance of 3.925 for contaminated and 4.296 for pristine and similar average clustering coefficient (0.307 contaminated, 0.306 pristine). Network indices from both networks were higher than the random network indices, suggesting that both networks exhibited small world features as described for microbial communities (Jeanbille et al., 2016 a,b). Such result indicated that the networks are highly clustered and nodes are related [START_REF] Watts | Collective dynamics of 'small-world' networks[END_REF]. However, no association was found between ASV and PAHs concentrations (Mantel test, for pristine (r = 0.007318, P = 0.375) and contaminated (r = -0.02143, P = 0.625) networks). Nevertheless, the networks, for both pristine and contaminated waters, were modular. Contaminated site network yielded 17 modules with modularity value of 0.65 while pristine site network yielded 14 modules with modularity value of 0.60. A plot was constructed from the ASVs connectivity within the modules (Zi) and ASVs connectivity among other modules (Pi).

According to Zi and Pi values, the roles of ASVs in modules were classified as: 1)

Peripheral, 2) Connectors, 3) Module hubs, 4) Network Hubs [START_REF] Liang | Long-term oil contamination alters the molecular ecological networks of soil microbial functional genes[END_REF]. The threshold for these classifications were selected at Zi = 2.5 and Pi = 0.62 as previously described by Guimera and Amaral (2005).

In general, most of the ASVs in both networks were peripherals, and therefore considered as specialists (Figure 6a). In contaminated network, there are distinct modules with little interconnection, evidenced by the ASVs with low Pi value. In contrast, pristine network showed with more interconnected modules, evidenced by the several ASVs with high Pi value (Figure 6a). In addition, several connector ASVs were identified in pristine network, suggesting the complexity of the ecological interactions within the network.

Connectors are generalist species, which are capable to connect different modules [START_REF] Liang | Long-term oil contamination alters the molecular ecological networks of soil microbial functional genes[END_REF]. The connector taxa identified belonged to Flavobacterium (ASV 49) genus, The networks, for microbial communities from microcosms of both pristine and contaminated sites, showed a modularization according to time (Figure 6b &c). Indeed, most interactions were observed at t14 and t21 days for the pristine network (Figure 6a), and at t0 for the contaminated site network (Figure 6b). Such observation further supports the ecological succession observed with the CCA (Figure 5). Interestingly, one module in the pristine network clustered ASVs showing high bacterial abundances in WAF+dispersant treatments, indicating that microbial community composition changes in response to the presence of dispersant. Among the ASVs in this module, 7 ASVs belonged to the genus Fusibacter, further showing the importance of such genus in the microbial community dynamics, and four ASVs were connector nodes (ASV 321, ASV 277, ASV 153, and ASV 290).

Interestingly, some taxa showed noticeable patterns in the networks, indicating their ecological role on the microbial communities in either WAF or WAF+dispersant microcosms. For example, in pristine network, four small modules exclusively belonged to three taxa: Fusibacter, Shewanella and Polarimonas, and in contaminated water network (Figure 6c), several small modules included Fusibacter at t0. These small modules, which are lacking co-occurring interactions, suggest that such taxa could be exhibiting other type of interactions with the other communities, such as competition. At t0, ASVs affiliated to Marinifilium genus showed high node degree in the network of contaminated water, suggesting that Marinifilium is an important key taxon.

The ASVs identified as bioindicators by LEfSe were found in specific modules within the contaminated water network at time 7 days, further suggesting that these ASVs are important for the organization of microbial assemblage. Such observation was in agreement with a previous study showing that soil with a previous history of contamination has fewer interactions than pristine soil [START_REF] Sheng | History of petroleum disturbance triggering the depth-resolved assembly process of microbial communities in the vadose zone[END_REF]. It is likely that the selection pressure exerted by the presence of contaminant reduces diversity, and thus the interconnectivity among taxa.

Figure 6: Molecular Ecological Network Analysis of microbial communities interactions at t0,t7,t14 and t21 (days) of the microcosms from pristine and contaminated site. (a) ASV distribution according to their topological features: within module connectivity (Zi) and among module connectivity (Pi). (b) Pristine microbial community and (c) contaminated microbial community networks. The networks were constructed considering the microbial communities from all the samples collected during the microcosm experiment from either pristine or contaminated sites. Blue lines are positive interactions (Cooccurrence). Purple nodes are bioindicator taxa identified by LEfSe for WAF+dispersant treatment. Light blue nodes are bioindicator taxa identified for WAF treatment. White and Grey nodes are bioindicator taxa identified for control in pristine and contaminated networks respectively. Taxa names mentioned on the networks denote taxa with observable effects on communities upon the introduction of WAF or WAF+dispersant.

Conclusions

In cold environments, microbes are not dormant, active microbial communities were observed in response to the addition of WAF and WAF+dispersant. Our results showed that the legacy effect plays a vital role in shaping the microbial response toward WAF and WAF+dispersant. Adapted microbial communities presented higher removal rates allowing the identification of several WAF stimulated taxa, but the contribution of dispersant to the PAHs removal is still questionable. Network analysis demonstrated that the microbial interactions patterns were time dependent, but the presence of dispersant also changed the microbial community structure, probably by stimulating taxa with potential biodegradation capabilities. We demonstrated that WAF+dispersant rapidly stimulated Firmucutes (Fusibacter), which consequently affected the microbial communities' dynamics. The PAHs removal decreased the toxicity against marine organisms (such as copepods). We assume that the toxicity decrease was due to PAHs and dispersant removal by bacteria affiliated to specialized taxa, such as Fusibacter and Colwellia. In conclusion, this study provides new insights on the behavior of water microbial communities at 4 °C in response to WAF and WAF+dispersant simulating an oil spill scenarios and a bioremediation efforts, respectively.

Zhou, Z., Wang, B., Zeng, S., Gong, Z., Jing, F., & Zhang, J. 2020. Glutathione Stransferase (GST) genes from marine copepods Acartia tonsa: cDNA cloning and mRNA expression in response to 1,2-dimethylnaphthalene. Aquatic Toxicology. https://doi.org/10.1016/j.aquatox.2020.105480

Chapter 4: Cold sediment microbial community shifts in response to Crude oil Water Accommodated Fraction and Water Accommodated Fraction + Dispersant

This chapter is being prepared to be published as: with oil spill accidents (Farrington, 2014). Hydrocarbon degradation via microbial communities is often considered as the most cost effective remediation technique [START_REF] Chen | Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by composting: Applications, microbes and future research needs[END_REF]. However, because low temperatures slow down the microbial activities, especially the hydrocarbon degradation functions, the bioremediation is hindered in cold environments (Yang et al., 2009). In addition, low temperatures change the physical and chemical properties of oil by increasing its viscosity favoring the sorption of harmful compounds, such as polycyclic aromatic hydrocarbons (PAHs), to organic matter (Crisafi et al., 2016). It is thus crucial to develop remediation techniques for mitigating the adverse oil spill effects adapted to cold environments.

After an oil spill, the fate of the oil in the environment depends on several weathering processes such as evaporation, dispersion, dissolution, emulsification, biodegradation, photo oxidation and sedimentation (Sun et al., 2019;Duran and Cravo-Laureau, 2016).

For example, low molecular weight hydrocarbons (LMWH) from crude oil solubilize in the water column with sufficient wave dynamics. Such process result in the formation of water accommodated fractions (WAF), which contain harmful aromatic hydrocarbons, polycyclic aromatic hydrocarbon (PAHs), and aliphatic compounds (Jiang et al., 2012).

Because of their bioavailability, the compounds in WAF are very toxic for several aquatic species [START_REF] References Abbriano | Deep water horizon oil spill: A review of the planktonic response[END_REF].

In case of an oil spill, the introduction of dispersants is often used to stimulate the bioremediation processes. Dispersants are introduced to the oil slick to reduce the surface tension of the oil, facilitating the microbial degradation processes [START_REF] Suja | Role of EPS, dispersant and nutrients on the microbial response and MOS formation in the subarctic Northeast Atlantic[END_REF].

Dispersants have been applied as early as the Exxon Valdez oil spill accident in Alaska (Etkin et al., 2003), and extensively used during the Deepwater Horizon oil spill accident, where 2.1 millions gallons of the dispersant Corexit 9500 were spread at the water surface and seafloor (Techtman et al., 2017). The efficiency of dispersants to improve hydrocarbon degradation by microbial communities has been under debate (Techtman et al., 2017, Kleindiest et al., 2015, Rahsepar et al., 2016), while their harmful effect has been shown on various marine organisms (Almeda et al., 2013, George Ares et al., 2000), including benthic micro-and macro-fauna (Cuny et al., 2015;Duran et al., 2015).

Although the effects of WAF, with or without dispersant addition, has been studied on the water microbial community [START_REF] Brakstad | Biodegradation of Water-Accommodated Fractions and Dispersed Oil in the Seawater Column[END_REF][START_REF] Nelson | Cytochrome P450 diversity in the tree of life[END_REF][START_REF] Personna | Dual effects of a dispersant and nutrient supplementation on weathered Endicott oil biodegradation in seawater[END_REF], little is known about the effect of WAF hydrocarbons on the benthic microbial community after their sedimentation (Cuny et al., 2015;Duran et al., 2015). It is thus crucial to understand the effects of WAF, with or without dispersant addition, on benthic microbial communities and activity, in order to propose adapted mitigation procedures, especially in cold environment. Most studies analyzing the effect of hydrocarbons on microbial community were performed at the DNA level providing information on the genetic potential but hide the real microorganisms at work, i.e. the active microbial community [START_REF] Stauffert | Impact of Oil on Bacterial Community Structure in Bioturbated Sediments[END_REF](Stauffert et al., , 2014)), because the DNA extracts contain dormant taxa and non growth active cells [START_REF] Argudo | Responses of resident (DNA) and active (RNA) microbial communities in fluvial biofilms under different polluted scenarios[END_REF]. As RNA, involved in metabolic processes, is stable in active cells [START_REF] Argudo | Responses of resident (DNA) and active (RNA) microbial communities in fluvial biofilms under different polluted scenarios[END_REF], several studies have performed the analysis at the RNA level providing information on the active microbial communities modifications in response to the presence of oil in microbial mats, (Bordenave et al., 2008(Bordenave et al., , 2004) and marine sediments [START_REF] Stauffert | Impact of Oil on Bacterial Community Structure in Bioturbated Sediments[END_REF], 2014[START_REF] Terrisse | Variation of oxygenation conditions on a hydrocarbonoclastic microbial community reveals Alcanivorax and Cycloclasticus ecotypes[END_REF]. The analysis of active microbial communities (RNA level) is particularly well adapted to follow the microbial dynamic in short course microcosm experiment (Cravo-Laureau and Duran, 2014).

The objective of this study was to investigate the effects of WAF, prepared with or without dispersant addition, on total (DNA level) and active (RNA level) microbial communities inhabiting either pristine or contaminated cold marine sediments in microcosm experiment maintained at 4° C. We specifically investigated 1) the dynamics of both total and active microbial communities, 2) the legacy effect comparing pristine and contaminated sediments communities, and 3) the PAHs biodegradation potential by predicting functional pathways.

Materials and methods

Sampling site

Water and sediment samples were collected from pristine and contaminated sites. The contaminated site was located in a harbor in the middle of Tromso, Norway (69°39'08.7"N 18°57'50.2"E) characterized with busy boat activity. The pristine site was located in a sound in Tisnes, Norway (69°36'24.4"N 18°49'56.3"E) with little anthropogenic influences. Sediment were collected by hands with a sterile spatula, and then stored in polypropylene containers at 4° C. Water and sediment containers were shipped to Pau, France at constant temperature of 4° C.

Water accommodated fractions preparation

Two types of water accommodated fractions (WAF) were prepared for the microcosm experiments: 1) WAF from crude oil, and 2) WAF+dispersant from crude oil added with dispersant. The WAFs were made from a Naphthenic North Atlantic (NNA) crude oil provided by the Norwegian University of science and technology (NTNU). This arctic crude oil, naphthenic based light crude oil, is characterized by a low aromatic and wax contents, a low viscosity index (299 cP at 2° C) and a density of 0.900 g/ml. It is rich in branched and cyclic aromatic hydrocarbons (Katsumiti et al., 2019). Preparation of WAF was similar to the protocol described by Singer et al., (2000) but with slight modifications.

Briefly, in 8 L glass bottles, crude oil was added to artificial seawater of salinity 30 ppt at a ratio of 5 gram of oil per 1 L water. The oil water mixture was stirred for 42 hour, at 10 ° C under dark conditions, using a magnetic stirrer without creating vortex. For WAF+dispersant, the dispersant FINASOL ® OSR 52 (Total Energies Fluids) was added first to the oil at a ratio of 1 gram of dispersant per 10 grams of crude oil, gently mixed, then the WAF was produced following the same procedure.

Microcosm set up

For each microcosm 500 mL of water, 500 mL of WAF, and 200 g of sediment were added to a 2 L sterile glass flasks covered with filter rubber mesh tap to allow gas exchange. Each microcosm condition was performed in triplicates for a total number of 18 microcosms: 3 controls for pristine sediment, 3 controls for contaminated sediment, 3

WAF treatments with pristine sediment, 3 WAF treatments with contaminated sediment, 3 WAF dispersant treatments with pristine sediment and 3 WAF dispersant treatments with contaminated sediment (Figure 1). The sediment were homogenized and dispatched in the 2 L bottles (9 containing pristine sediments and 9 containing contaminated sediments). The different conditions were applied after 3 days acclimatization at 4 ° C by adding the different type of WAF. Microcosms were maintained for 21 days at 4 ° C in the dark to prevent the growth of photosynthetic organisms. Every day, the microcosms were gently shaken to allow the suspension of particles and for homogenization of sediments. Sub-samples of sediments were collected after 0, 7, 14, 21 days of incubation for microbial analysis and PAHs analysis. Sub-samples were stored at -80° C until analysis.

Chemical analysis

Extraction of organic compounds

The extraction of organic compounds was carried out in a closed MARX 6 microwave (CEM, USA) with temperature controller. For each sample, about 1.0 gram of freezedried sediment sample was loaded into the Teflon lined extraction vessel and 15 mL of acetone was added. In addition, 25 μL (5 μg L -1 ) of 6 deuterated PAHs solution in acetone that acted as surrogate was added. Three blank samples were used in the analysis. The microwave extraction program consisted of 5 minutes reaching a temperature of 110° C for 15 minutes. Once samples were extracted, the extracts were filtered (Millipore PTFE, 0.45μm), evaporated and reconstituted in 1 mL of hexane.

Extract cleaning up stage

Standard solution containing all 16 PAHs was prepared in n-hexane. The extracts were loaded on top of 1 g of Florisil ® cartridges previously conditioned with 8 mL of n-hexane. Elution was performed with 12 mL of a mixture of hexane: toluene (80:20). These 12 mL fractions were evaporated using a stream of nitrogen and at a constant temperature (40° C) until dryness. About 1 mL of hexane was then added, filtered again and stored in chromatographic vials until GC MS analysis.

Extract chemical analysis

The 16 PAHs were quantified using GC-MS / MS according to [START_REF] Navarro | Comparison of solid phase extraction, saponification and gel permeation chromatography for the clean-up of microwave-assisted biological extracts in the analysis of polycyclic aromatic hydrocarbons[END_REF].

Briefly, the extracted compounds were analyzed on a 6890 N Agilent gas

Total DNA and RNA Co-extraction

From each sub-sample, genomic DNA and RNA transcripts were co-extracted. First, the total RNA was extracted using RNeasy PowerSoil total RNA isolation kit (Qiagen, Germany) following the extraction protocol. Then, microbial DNA was further eluted from the RNA spinning column kit using the RNeasy PowerSoil DNA elution kit (Qiagen, Germany). RNA was quantified using Quant it RNA BR kit (Theremofisher scientific, USA), and DNA was quantified using Quant it dsDNA HS kit (Theremofisher scientific, USA).

cDNA RT and DNA amplification

Extracted RNA were initially treated with DNase (Invitrogen, USA) to remove genomic DNA carried over from the extraction procedure. DNase treated RNA were reverse transcribed using M-MLV reverse transcriptase (Invitrogen, USA) using random hexamers and following the manufacturer protocols. The 16S rRNA gene (DNA level)

and transcript (RNA (cDNA) level) were amplified using the universal prokaryotic primers U515-532-GTGYCAGCMGCCGCGGTA and U909-928-CCCCGYCAATTCMTTTRAGT targeting the V4-V5 region of the 16S rRNA gene (Wang and Qian 2009). The reaction medium (25 μL) consisted of 12.5 μL AmpliTaq Gold 360 Master Mix (Invitrogen, USA), 1 μL of forward and reverse primers at concentration of (20 μM), 5.5 μL water and 5 μL undiluted DNA template (10 ng). The polymerase chain reaction was performed using the following cycling conditions: 10 minutes at 95 ° C, followed by 30 cycles of: 30 seconds at 95 ° C, 30 seconds at 60 ° C

and 40 second at 72 ° C, followed by a final step of 10 minutes at 72 ° C. Successful amplification of the PCR was checked on 2% agarose gel electrophoresis, stained with ethidium bromide. PCRs were performed in triplicates and were eventually pooled for further sequencing.

Amplicon sequencing and bioinformatics analysis

The 16S rRNA (gene and transcript) barcoding was performed using NGS Illumina MiSeq at the GenoToul platform (Toulouse, France) by sequencing the V4-V5 region of the 16S rRNA gene. The obtained demultiplexed sequences were treated with Qiime 2 software (Caporaso et al., 2010) according to Quince et al., (2009). Briefly, DNA and cDNA sequences were trimmed and then denoised using DADA2 pipeline integrated in the Qiime2 software to remove chimeric sequences. The total number of sequences for each sample obtained after denoising is presented in (Supplementary Materials, Table S1). Denoised sequences were assigned with taxonomic affiliation using RNA gene reference database SILVA version 132 at 97% similarity threshold (Quast et al., 2013) and singletons were then removed. Finally, rarefaction was performed to the minimum read number observed (4,396 sequences) in a sample. The rarefaction curves are presented in Figure S1 in supplementary materials. An abundance table with the total available amplicon sequence variant (ASVs) was produced removing those with less than 250 counts (representing about 1% of total counts per sample). The complete dataset was deposited in the NCBI Sequence Read Archive (SRA) database (SUB10611368) under the Bioproject (PRJNA777560).

Statistical analysis

Statistical analysis on the bacterial community dynamics, alpha diversity indices and total bacterial relative abundances, were conducted on R software using RAM package (Chen et al., 2018). Procrustes analysis on DNA and RNA abundances was conducted using vegan package (Okansen et al., 2020). The RNA:DNA ratio was calculated by dividing RNA abundances by DNA abundance of the same taxa.

Linear discriminant analysis effect size (LEfSe, Segata et al., 2011) was performed with galaxy (http://huttenhower.sph.harvard.edu/galaxy/). Briefly, non-parametric Kruskal Wallis was used to detect taxa with significant abundances (p-value < 0.05). Biological consistency was conducted using pair wise Wilcoxon test (p-value < 0.05). Finally, LDA with threshold score of 3.0 and 1000 bootstraps was applied. The RNA:DNA ratio of Taxa with the highest LDA scores were plotted overtime.

Non metric Multi Dimensional Scaling (nNMDS), Canonical correspondence analysis (CCA), and PERMANOVA were performed by PAST statistical software v4.03 (Hammer et al., 2020). The analyses were based on Bray Curtis dissimilarity index.

Tax4fun2 R package [START_REF] Asshauer | Tax4Fun: Prediction of functional profiles from metageno 16S rRNA data[END_REF]) was used to predict the gene functions of the ASVs classified from the RNA abundance table. The Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used for the annotation of the functional predicted genes (Kanehisa and Goto 2000). Only predicted genes involved in PAHs biodegradation pathways were selected for the analysis.

Finally, heatmaps and clustered correlations were generated from functional genes using CIMminer (http://discover.nci .nih.gov/cimminer/), based on Pearson correlation. Only significant correlations (Pearson correlation > 0.7 and p-value < 0.05) were considered

Results and Discussion

PAH removal

The concentration of total PAHs was below 3 µg per gram of dry sediment in the majority of samples, the highest PAH concentration being measured in contaminated sediment containing WAF+dispersant (Figure 2,b). PAH concentration increased in control from contaminated site during the experiment, suggesting that the PAHs present in the sediment become more available under the experimental conditions. In the WAF+dispersant treatments, the total PAH concentration significantly decreased during the experiment with pristine sediment (p < 0.01), being close to 0 at day 21 (Figure 2,a).

In presence of dispersant (WAF+dispersant), the PAH concentration decrease was notable from day 7. Additionally, the proportion of low molecular weight PAHs (LMW-PAHs, 2 -3 rings such as naphthalene, acenaphthene, acenaphthylene, fluorene, phenanthrene, and anthracene) decreased during the experiment, whereas that of high molecular weight PAHs (HMW-PAHs, > 3 rings such as fluoranthene, pyrene, and chrysene) increased (Figure 2,c & d). This observation was made for both sediments (pristine and contaminated) irrespective of the treatment (WAF and WAF+dispersant), but the proportion of HMW-PAHs was higher in sediments from the contaminated site, confirming the chronic pollution (Figure 2,c). The ratio LMW-PAHs/HMW-PAHs decreasing during the experiment substantiated the biodegradation of PAHs, as LMW-PAHs are more susceptible to microbial degradation [START_REF] Olayinka | Polycyclic Aromatic Hydrocarbons in Sediment and Health Risk of Fish, Crab and Shrimp Around Atlas Cove, Nigeria[END_REF]. It is likely that PAHs were removed by biotic processes considering the conditions (at 4° C in the dark, limiting abiotic processes) under which the experiment was conducted, although abiotic processes could not be excluded.

Microbial community structure and dynamics

After trimming, a total of 631,207 reads was obtained for sediment samples (Supplementary Material, chapter 4, Table S1). Rarefaction curves indicated that sufficient sequences were obtained to estimate the microbial diversity (Supplementary Material, chapter 4, Figure S1). Interestingly, the total community (DNA level analysis)

showed more observed ASVs than the active community (RNA level analysis),

suggesting that just a portion of the total bacterial communities was active during the experiment.

The total (DNA) microbial communities were dominated by Proteobacteria (60%-50%)

and Bacteroidetes (35%-25%) phyla (Figure 3,a). Cyanobacteria, Actinobacteria, Planctomycetes, Nanoarchaeaeota phyla were observed at low abundance. Several bacterial genera from the phylum Proteobacteria, such as Marinobacter, Oleispira, Rhodobacter, have shown PAHs degradation potentials at 4 ° C (Crisafi et al., 2016). In general, the abundance of the different phyla did not showed noticeable changes during the experiment, except members of the Cyanobacteria phylum decreasing during the experiment in all treatments, probably because of the incubation in the dark. In addition, one way ANOVA showed no significant differences among the samples in respect of species richness (Shannon diversity index).

The Procrustes analysis showed significant differences between total (DNA) and active (RNA) microbial communities from pristine sediment (P=0.4, M 2 = 0.94, permutations 999) while total and active communities were similar (P = 0.04, M 2 = 0.8, permutations 999) with microbial communities from contaminated sediments (Supplementary Material, chapter 4, Figure S2). It is likely that the microbial communities from contaminated sediments were well adapted to the presence of PAHs explaining that total and active microbial communities were similar. In contrast, for the microbial community from pristine sediment the presence of WAF and WAF+dispersant probably activated hydrocarbon-degrading bacteria, representing only a proportion of the total community.

Thereafter, the ratio RNA to DNA was used to compare the active microbial communities between samples. Additionally, the comparison of the behavior of microbial community from contaminated sediment to that of pristine sediment highlighted the legacy effect of the PAHs-adapted microbial community inhabiting the contaminated sediment. The legacy effect, initially reported for microbial communities adapted to the presence of hydrocarbon [START_REF] Paissé | Structure of bacterial communities along a hydrocarbon contamination gradient in a coastal sediment[END_REF] et al., 2020). In contrast, ASVs identified as bio-indicators in WAF treatments were related to the Gram-negative Thermoanaerobacteraceae and to Bacteroidales, which have been described to be able to degrade PAHs [START_REF] Marozava | Anaerobic degradation of 1-methylnaphthalene by a member of the Thermoanaerobacteraceae contained in an iron-reducing enrichment culture[END_REF][START_REF] Miller | Oil hydrocarbon degradation by caspian sea microbial communities[END_REF]. It is also important to note that an ASV (ASV 1178) belonging to the Desulfobulbaceae family was identified as bio-indicator for both WAF and WAF+dispersant treatments in comparison to the control (Figure 4a &b), suggesting that this ASV was active in both conditions that reveal its capacity to thrive in different environmental conditions. This result was consistent with the fact that members of the Desulfobulbaceae have been found abundant in oil-contaminated marine sediment with the capacity to degrade hydrocarbon anaerobically under sulfate reducing conditions (Matturo et al., 2017;[START_REF] Beazley | Microbial community analysis of a coastal salt marsh affected by the Deepwater Horizon oil spill[END_REF]. The presence of active ASVs related to

Bacteroidales and Desulfobulbaceae significantly abundant on both WAF treatments suggested that anaerobic processes could be involved in hydrocarbon degradation.

For pristine sediment, only one ASV belonging to the Gammaproteobacteria class was identified as bio-indicator for WAF+dispersant treatment (Figure 4a). Regarding the WAF treatment, LEfSe identified 8 bio-indicator taxa with an ASV related to the cyanobacteria Microcystacae family showing the highest LDA score. The other ASVs identified as WAF bio-indicators belonged to genera related to Gammaproteobacteria and Alphaproteobacteria, which have been detected in coastal salt march after the Deepwater Horizon oil spill [START_REF] Beazley | Microbial community analysis of a coastal salt marsh affected by the Deepwater Horizon oil spill[END_REF]. ASVs related to Desulfobulbaceae were also revealed as bio-indicator of WAF treatment (Figure 4d) indicating that anaerobic processes participate to PAH degradation.

The fact that large number of bio-indicator taxa was identified in contaminated sediment than in pristine sediment further supported that hydrocarbon-adapted microorganisms inhabit the contaminated sediment. It has been shown that the constant exposure to anthropogenic pressures, such as oil spillage, select microbial communities with taxa able to degrade PAHs (Storey et al., 2018), or at least well adapted to the presence of PAHs (Jeanbille et al., 2016a,b).

Dynamic of the active bacterial communities in response to WAF and WAF+dispersant

The ASVs with the highest LDA score for each treatment were selected to follow the dynamic change of their abundance pattern during the experiment (Figure 5). The highest change of the RNA:DNA ratio was observed for the cyanobacteria Microcystaceae, decreasing overtime. This can be explained by the lack of light during the experiment, inhibiting photosynthesis and therefore inhibiting the growth of photosynthetic bacteria.

Interestingly, the ASV 974 Gammaproteobacteria, a bio-indicator for WAF+dispersant in pristine sediment, showed more than five folds RNA:DNA increase at the seventh day followed by decrease in RNA:DNA for the rest of the experiment. Also, Colwellia (ASV 366) showed RNA:DNA rapid increase until day 7. However, unlike

Gammaproteobacteria, Colwellia was detected in the active community after day 7, suggesting its resilience. Other selected ASVs, (Candidatus Alysiosphaera, Gammaproteobacteria incertae sedis, Bacteroidales, Flavobacteriaceae) showed a delayed increase in the RNA:DNA ratio after 14 days of the microcosm experiment.

Gammaproteobacteria and Colwellia ASVs showed active patterns similar to those observed for opportunistic taxa with r-strategy (Storey et al., 2018). The r-strategists have high and rapid growth rates, capable to colonize environment with high resource availability. However, r-strategists have low competitive capability and lack stability [START_REF] Vadstein | K-selection as microbial community management strategy: A method for improved viability of larvae in aquaculture[END_REF]. In the case of Gammaproteobacteria, the initial growth is probably stimulated by the hydrocarbon enrichment (WAF or the WAF+dispersant), but then they collapse probably because of the depletion of carbon source. In contrast, the delayed response in RNA:DNA ratio, exhibited by Candidatus Alysiosphaera, Gammaproteobacteria incertae sedis, Bacteroidales, Flavobacteriaceae, follows the growth K-strategist taxa. The K-strategist taxa exhibit slow growth, evidenced in peak active communities after 14 days or more (Figures 5a & c & d). They eventually succeed the collapse of r-strategist because they are better competitor for resources [START_REF] Vadstein | K-selection as microbial community management strategy: A method for improved viability of larvae in aquaculture[END_REF].

Relationship between the predicted functional community and the concentrations of PAHs

The Tax4fun2 inferred functional genes in the active microbial communities allowed to understand the degradation patterns observed in response of WAF or WAF+dispersant enrichment.

Among the 8426 functional genes predicted using Tax4fun2, we selected the genes linked to PAH degradation pathways (Supplementary materials, chapter 4, Table S2). For the anaerobic PAH degradation pathway, the genes (naphthoate synthetase, 1,4-dihydroxy-6-naphthoate synthase, 1,4-dihydroxy-2-naphthoyl-CoA hydrolase) were found more abundant with similar distribution in all treatments (p-value > 0.05), supporting that anaerobic processes participate to PAH degradation. For the aerobic PAH degradation pathway, the 18 more abundant predicted genes, mostly involved in naphthalene degradation pathways (only six were involved in degradation pathways of PAHs with more than 2 rings), exhibited different distribution according to the treatment. Canonical

Correspondence Analysis (CCA) showed that the aerobic PAH degradation functional communities were linked with the concentrations of the different types of PAHs (Figure 6).

For pristine sediment (Figure 6a), both CCA axes explained 74% of the total variance observed (axis 1: 45% and axis 2: 29%). The CCA demonstrated that the first axis correlated with 2-3 ring PAHs (fluorene, naphthalene, acenaphthene, and acenaphthylene), whereas PAHs with more rings (anthracene, fluoranthene, phenanthrene, and pyrene) were negatively correlated. The functional microbial community in WAF and WAF+dispersant microcosms at time zero were associated with naphthalene and acenaphthylene, while at time 14 and time 21 they were associated with pyrene and chrysene, as the microbial community from WAF treatment at time 14. The functional communities of time 21 WAF and control were associated with anthracene.

Control sample at time 14 was highly variable from the rest of the samples and therefore it was removed from the CCA analysis.

For contaminated sediment (Figure 6b), both CCA axes explained 72% of the total variance observed (axis 1: 41% and axis 2: 31%). The first axis was positively correlated with naphthalene and its derivatives (acenaphthene and acenaphthylene), while the second axis was negatively correlated with PAHs with more rings (fluorene, pyrene, anthracene, fluoranthene, phenanthrene). Functional communities of control and WAF at time 7 and time 14 respectively were associated with LMW-PAHs of two rings (naphthalene, acenaphthene, acenaphthylene), as WAF+dispersant functional communities of times 0, 7 and 14. Whereas functional communities of WAF+dispersant and WAF at time 21 and control at time 7 were associated to PAHs with more than two rings (fluorene, pyrene, anthracene, fluoranthene, and phenanthrene). The observed correlation could be due to the slow microbial degradation of HMW-PAH of four aromatic rings (Garneau et al., 2016). In addition, degraders of HMW-PAH are not commonly in high abundance in the environment (Rathour et al., 2018), therefore it would take some time to stimulate such taxa to abundances adequate for noticeable biodegradation processes.

PAH concentration correlations with predicted functional genes involved in PAH aerobic degradation

In order to understand the dynamics of aerobic PAH metabolisms within bacterial communities, correlation analyses between the predicted functional genes and PAH concentrations were performed (Table 7). The PAH degradation pathways are complex and involve several metabolic steps. Generally, PAH degradation pathways under oxic conditions are initialized through the hydroxylation or deoxygenation of PAHs through the catalysis of oxygenases. PAHs are then transformed into several key intermediate metabolites such as protocatechuate, salicylate, gentisate and catechol, which are then metabolized through central pathways [START_REF] Cao | Genomic and metabolic analysis of fluoranthene degradation pathway in Celeribacter indicus P73[END_REF].

The microbial community from the pristine site in WAF+dispersant microcosms According to the correlation patterns, it is likely that the functional communities from pristine sediment were enriched with active populations involved in LMW-PAHs biodegradation pathways. The addition of WAF+dispersant stimulated taxa with functional genes involved in LMW-PAHs degradation pathways. The observed strong correlation patterns of functional communities in pristine sediment suggested that contamination is a strong driver stimulating rapidly the active bacteria communities that results on bacterial structure shifts as shown by [START_REF] Coelho | Unraveling the interactive effects of climate change and oil contamination on laboratory-simulated estuarine benthic communities[END_REF]. In contrast, the microbial community from contaminated sediment was not stimulated as observed for that inhabiting the pristine sediment probably because it exhibited functions indicating that it was adapted to the chronic presence of the hydrocarbon. Although our results

showed that active microbial community exhibited promising predicted functional patterns, it is important to highlight that such functional genes are predicted and not directly measured. Therefore, more research is needed to determine enzymatic activities involved in PAH degradation pathways to fully understand these pathways.

Conclusions

This study highlighted the behavior of microbial communities in sediments upon the introduction of WAF and WAF+dispersant at 4° C. The initial microbial communities showed to be an important player on the fate of PAHs in sediment. Sediment with a chronic history of contamination owned taxa responsive to the PAHs in the WAF. In addition, dispersant showed to have impact on the total and active microbial community dynamics, especially for those inhabiting pristine sediment. Throughout the course of the experiment, active taxa followed an ecological succession degrading first LMW-PAHs and then HMW-PAHs, possibly because the degradation of HMW-PAHs is challenging.

However, the difference in microbial communities between the two types of sediment also highlighted the different biodegradation capabilities in the sediments revealing the legacy effect for the microbial community inhabiting the contaminated sediment.

Dispersant added to pristine sediment showed to stimulate PAH biodegradation pathways of LMW-PAHs such as naphthalene and its derivatives. Whereas in contaminated sediment, both WAF with and without dispersant stimulated HMW-PAHs biodegradation pathways. We conclude that dispersant can be of importance in improving microbial degradation capabilities in cold environment sediments. This is especially the case in sediments without history of contamination pressures such as pristine sediments. Through the different bioassays performed and endpoints studied, the copepod Acartia tonsa has shown to be a valuable toxicological tool in understanding the effects of contaminants on the marine environments. The different levels of endpoints used in this thesis has further demonstrated how cheap and valuable can toxicity assays be in order to rapidly diagnose lethal and sub lethal toxicity effects of environmental contaminants. In addition, Acartia tonsa geographic distribution and its tolerance to a wide range of temperature and salinities allow this species to be a valuable toxicological candidate for testing contamination in different environments such as tropical, temperate and cold environments.

Water Accommodated Fractions prepared from a crude oil and two types of distilled oils (IFO 180 and marine gas oil) showed to have different chemical compositions and consequently different toxicity effects on the copepod Acartia tonsa. NNA WAF had the highest concentration of total PAH, mainly naphthalene. Whereas, distilled oils WAF have high concentrations of phenanthrene. Although PAHs compositions contribute to the observed toxicity profiles, it has been shown that additives added to oil derivatives (e.g. phenol and sulfur) play a major role in the toxicity toward Acartia tonsa (Rodrigues et al., 2010). WAF prepared from distilled oils (marine gas oil and IFO180) was the most toxic in respect of mortality and reproductive capabilities. In the case of marine gas oil, both histology and Nile red imaging have shown that lipid deposits in Acartia tonsa are majorly accumulated in eggs and ovaries. At the molecular level, exposure to IFO 180 WAF showed to initiate cellular damage, evidenced by the early significant expression patterns of CYP 3026b and heat shock protein 70 genes. In addition, our results have demonstrated that these genes act as valuable biomarkers for assessment of early cellular damage due to exposure. Although of the high naphthalene content, WAF from the NNA crude oil showed very little toxicity effects on Acartia tonsa, further suggesting that distilled oils have more toxic effects than the crude oil. Results have shown that the toxicity effects after exposure to WAF from distilled oils are highly focused on offspring production, therefore affecting the copepod population through the inability of producing viable offspring. This decrease in reproduction capabilities can potentially has detrimental effects on the food web as Acartia tonsa are dominant components in their ecosystems (Mauchline, 1998). However, further research on transgenerational effects of distilled oils of WAF will provide wider picture on the effects on the population of Acartia tonsa.

Dispersant added to Water Accommodated Fraction (WAF) has shown to have toxic effects on Acartia tonsa . WAF with dispersant had caused high mortality rates, affected the rates of egg production and affected the transcription levels. It is likely that the addition of the dispersant have more detrimental effects on marine fauna than the WAF alone. As illustrated from the in vivo and microcosm experiments, we have a good understanding on the toxicity effects of WAF+ Dispersant. Although we observed that the initial concentrations of PAHs in WAF+ Dispersant in the microcosms were not significantly higher than PAHs concentrations in WAF, WAF +Dispersant has been shown to increase the bioavailability of PAHs in the water column [START_REF] Milinkovitch | Effects of dispersed oil exposure on the bioaccumulation of polycyclic aromatic hydrocarbons and the mortality of juvenile Liza ramada[END_REF]. However, as demonstrated in chapter 3, the initial high toxicity exhibited by the WAF+ dispersant is not long lasting. The microcosm study has shown that after 21 days, the toxicity of dispersant has noticeably decreased due to the possible microbial degradation of the dispersant and PAHs. However, at day 21, potential sub-lethal toxicity was still present as shown by the downregulation of the antioxidant gene gst in the exposed Acartia tonsa. The biodegradation of the dispersant was due to the rapid stimulation of several opportunistic taxa, such as Colwellia and Fusibacter, which have

shown to be able to use dispersant as carbon source for growth (Kleindiest et at., 2015;Calderon et al., 2018).

The efficiency of dispersant in improving biodegradation rates has been widely debated in literature. Our results participate in better understanding the role of dispersant on the biodegradation rates after introducing oil under low temperature. In water, our microcosms results did not provide clear evidence that the dispersant improved the PAH biodegradation rates as the concentration of PAHs in WAF and WAF+ Dispersant were similar. In fact, at low temperature, the addition of dispersant to oil has shown to initially inhibit biodegradation rates and lower the degradation rates of aromatic hydrocarbon [START_REF] Foght | Effect of the dispersant Corexit 9527 on the microbial degradation of Prudhoe Bay oil[END_REF]Rahespar et al., 2016).

Our results support the fact that the primary factor influencing the biodegradation rates is the initial composition of bacterial communities that own PAH-degrading taxa (Storey et al., 2018). Compared to microbial communities inhabiting pristine water communities, those inhabiting contaminated water contained more hydrocarbon degrading taxa. This corresponds to the legacy effect explaining the presence of adapted microbial populations to the presence of PAH, indicating that the whole microbial community is "PAHimmunized" with the ability to respond rapidly after WAF addition. However, WAF+ Dispersant has clearly shown to play a key role in shaping the dynamics of bacterial communities by stimulating several opportunistic taxa, which exerted ecological pressure on other bacterial populations. However, to fully understand the biodegradation rates in water in response to the addition of WAF of WAF+ Dispersant, further work is still needed by studying the bacterial communities at the functional level through the analysis of RNA and DNA sequences. Unfortunately, we were not able to obtain RNA from water samples probably because of low microbial biomass in cold temperature, constituting a limitation in this thesis.

In sediments, the concentrations of PAHs were significantly lower than in the water column. Several reasons could explain these differences in concentrations. For example, more time was needed for further sedimentation of PAHs from the water column. In addition, PAHs could have been lost due to evaporation during the freeze dry pre treatment [START_REF] Law | Polycyclic aromatic hydrocarbons (PAH)-Problems and progress in sampling, analysis and interpretation[END_REF], a problem with wet sediments extractions. In aquatic environments, dispersants can speed up the sedimentation of suspended particles and can assist in the formation of oil mineral aggregates in the water column , therefore promoting the transfer of PAHs into marine sediment [START_REF] Cai | Effects of oil dispersants on photodegradation of parent and alkylated anthracene in seawater[END_REF]. In addition, the strong adhesion force between oil and the sediment decrease the bioavailability of PAHs, thus reducing the bacterial biodegradation rates [START_REF] Ansari | Study the Microbial Communities' Changes in Desert and Farmland Soil After Crude Oil Pollution[END_REF]Louvado et al., 2019).

Our study has demonstrated that the integrative study of both total and active bacterial communities through both DNA and RNA sequencing assisted in understanding the functional microbial communities, which are involved in PAHs degradation in response to the addition of WAF and WAF+Dispersant.

Interestingly, in pristine sediment, the abundance of active communities corresponding to different functional genes of PAH degradation pathways demonstrated that biodegradation mechanisms occurred in sediment in response to the introduction of dispersant. In pristine and contaminated sediments, functional communities demonstrated that low molecular weight PAHs, such as naphthalene, are degraded faster than heavier PAHs with more than 3 rings. In fact, we hypothesize that legacy effect is an important contributor to the responses of microbial communities toward WAF. Low response of functional taxa to WAF and WAF+Dispersant is due to the legacy effects resulting in the high abundance of hydrocarbon degrading and dispersant resistant taxa in the contaminated sediment (Louvado et al., 2019;Paisse et al., 2010).

Conclusions:

1) Different types of oil have showed different toxicological effects in exposed Acartia tonsa copepods. WAF prepared from marine gas oil showed to have detrimental effects on mortality, fecundity, total lipid content and egg hatching success to copepods. The combination of such endpoints demonstrates that the sub lethal concentrations of marine gas oil WAF affected the egg production capabilities of female copepods. IFO 180 WAF showed detrimental effects on mortality, fecundity and transcription levels of selected genes involved in ROS and xenobiotic metabolism. Therefore, WAF prepared with distilled oils showed to have more toxic effects than WAF prepared from NNA crude oil.

2) Compared to the exposure to NNA WAF alone, the exposure of Acartia tonsa to WAF+ Dispersant had detrimental effects on the survival and the egg production rates in females and downregulated key genes in the ROS and xenobiotic metabolism,. This indicates that the use of chemical dispersants of the FINASOL OSR52 type as a form of remediation after an oil spill could have detrimental effect on the marine fauna.

3) Studying endpoints at different biological levels, ranging from molecular to reproductive endpoints, demonstrated that the copepod Acartia tonsa is a valuable model organism suitable for studying the effects of contaminants.

4) In the water column at 4°C, the structures of bacterial communities are not static, they are actively changing overtime due to the addition of WAF with and without the dispersant. WAF+Dispersant had great impact on the structure of the bacterial communities. The presence of dispersant stimulates certain taxa, which can play a role in driving the community structure. However, there is no enough evidence that dispersant improve the biodegradation rates of PAHs in the water column. Additionally, the toxicity of WAF+ Dispersant to organisms is acute but not long lasting. The dispersant can stimulate taxa able to degrade the dispersant, and consequently decrease the dispersant toxicity.

5) The initial structure of the bacterial communities plays a key role in PAHs removal in both sediment and water column. Microbial communities of cold areas with historical exposure to chronic contamination contain bacterial taxa capable of biodegradation of PAHs.Whereas bacterial communities from pristine site, not exposed to PAHcontamination, exhibited slower removal rates, indicating that pristine sites are sensitive to oil spills.

6) Analysis of RNA from bacterial communities from cold areas paved the way to understand the functional communities in sediment upon the introduction of WAF and WAF+ Dispersant. Dispersant showed to play a role stimulating active communities in pristine sediment, suggesting the possible involvement of several taxa in biodegradation due to dispersant. On the other hand, functional communities in contaminated sediment showed that legacy effects contributed in shaping the microbial response towards WAF and WAF+ Dispersant Thesis:

Bioassays using the marine copepod Acartia tonsa in combination with a biological multilevel approach demonstrated that refined oils are more toxic to copepods than naphthenic crude oil. The addition of the chemical dispersant, Finasol OSR 52 provokes more prominent toxic effects in copepods. This raise the environmental concern on the application of these compounds in oil spill scenarios. Moreover, at 4°C, microbial biodegradation rates are more influenced by the initial composition of bacterial communities than by the addition of dispersant. However, dispersant has shown to affect the microbial community dynamics by favoring certain microbial taxa than others.

.

  /MS. Tamer Hafez TITLE OF THE THESIS: Ecotoxicity effects of oil Water Accommodated Fractions and oil Water Accommodated Fractions + Dispersant on cold environments: Acarita tonsa based bioassays and microbial community dynamics as monitoring tools After having witnessed the completion of the viva by the author and their response to any objections and/or suggestions made, the Panel appointed by the Postgraduate Commission of the University of the Basque Country to examine the Doctoral Thesis indicated above, meeting on the indicated date, agreed ______________ to award the following grade: unanimously or by majority vote DISTINCTION / MERIT / PASS / FAILViva language(s) (in the event of there being more than one language, please specify the percentage of the thesis defended in each)

•

  University of the Basque Country (UPV/EHU) and University of Pau and the Adour Region (UPPA) cotutelle pre-doctoral grant program 2017. "If learning the truth is the scientist's goal, then he must make himself the enemy of all that he reads" -Al Hasan Ibn el Haytham فالواجب النا علي حقائق ال معرفة غرضه كان العلوم،إذا كتب في ظر جعل ي أن ، فيه ينظر ما لكل خصما نفسه أ الهيثم بن حسن ل -

Figure 1 :

 1 Figure 1: Distribution and theoretical borders of Arctic and sub-Arctic environments (Hoberg et al.,

Figure 2 :

 2 Figure 2: Schematic representation of the Arctic and sub-Arctic marine food web, showing the key position (red square) of microbial and planktonic communities (adapted from Darnis et al., 2012).

Figure 3 :

 3 Figure 3: Calanoid life cycle: (a) egg hatching stage, (b) 6 naupliar stages (N1-N6), (c) 5 copepodite stages (C1-C5),(d) adult stage. Source: http://www.sahfos.ac.uk/TaxonManual/figures/image/ARTHROPODA/callifecycl.JPG

  )(Wooton et al.,

Figure 4 :

 4 Figure 4: Geographic distribution of Acartia tonsa (red dots). Source: World Register of Marine Species (https://www.marinespecies.org/aphia.php?p=taxdetails&id=345943#distributions).

Figure 5 :

 5 Figure 5: Sexual dimorphism in Acartia tonsa. (a) Female Acartia tonsa; it shows round urosome fused to the main body. (b) Male Acartia tonsa; the urosome is more straight than in females. Arrow denotes urosomes.

  tonsa has been included by the Italian Ministry of Environment and by the Government of Canada in the list of species used for the toxicological evaluation of sediment pollution (Carotenuto et al., 2020). The Organization for Economic Co operation and Development (OECD) has one validated ecotoxicity test (test number 79;

Figure 6 :

 6 Figure 6: Different weathering effects facing oil after an oil spill (ITOPF, 2019).

Figure 7 :

 7 Figure 7: Most common PAHs conformations with different numbers of aromatic rings[START_REF] Haritash | Biodegradation aspects of Polycyclic Aromatic Hydrocarbons (PAHs): A review[END_REF]

Figure 9 :

 9 Figure 9: Main activation mechanisms of the PAH biodegradation pathways in microbes(Duran and Cravo Laureau, 2016)

  and dibenzo[a,e]pyrene (D[a,e]P). Passive samplers of poly dimethylsiloxane polymer coat (Gerstel GmbH, Germany), with 10 mm in length and 0.5 mm film thick, were introduced in aqueous samples (35 mL) and stirred during 315 minutes. Once the extraction step was over, samplers were cleaned with Milli-Q water in order to eliminate seawater and dried with a paper tissue. Passive samplers were desorbed using a commercial thermal desorption TDS-2 unit (Gerstel GmbH) connected to a CIS-4 injector (Gerstel GmbH) with the following conditions: desorption time (10 min), desorption temperature (300º C), desorption flow (23 mL min -1 ), cryo-focusing temperature(-50ºC)

  biomarkers for the ROS response. A CYP gene from clan 3 (cyp3026b) was targeted as a biomarker for PAH metabolism. Primers for catalase (cat), cyp3026b and gst were designed de novo from conserved regions in closely related crustacean species and scaffolds from the Acartia tonsa genome project (Jorgensen et al. 2019) (Accession number PRJEB20069). Clustal Omega (Madiera et al 2019) was used for multiple sequence alignment. For cyp3026b, Acartia tonsa genome sequence LS054218.1 was aligned with the gene sequences from Tigriopus japonicus (KF639998.1) and Eurytemora affinis (XM 023493699.1). For catalase, the genome sequence LS054460.1 was aligned with sequences from Calanus finmarchicus (EL965956.1) and Eurytemora affinis (XM023472406.1). For gst, the genome sequence LS085988.1 was aligned with Tortanus forcipatus (KT755427.1) and Acartia pacifica (KT754520.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Proportion of reproductive (egg producing) females after exposure to different doses of a) NNA WAF, b) IFO 180 WAF and c) MGO WAF. X-axis represents the selected sub-lethal WAF doses. A negative association between number of reproductive females and doses in IFO 180 WAF (Fisher exact test p < 0.0005, a = -23) and MGO WAF (Fisher exact test p < 0.05, a = -17.4) was detected. C: control. Only two females produced eggs in high IFO WAF treatment, therefore, high IFO 180 WAF dose was not included in the statistical analysis

Figure 4 :

 4 Figure 4: Total lipid content corresponding to the percentage of Nile red fluorescence intensity compared to control (100%) after 96 hours of exposure in a) NNA WAF, b) IFO 180 WAF and c) MGO WAF. Xaxis represent the selected sub-lethal WAF doses. Asterisk denotes significant difference from control after one-way Welsh ANOVA, followed by Games-Howell post hoc test (p<0.05). C: control. Graphs show mean ± standard error.

Figure 5 :

 5 Figure 5: Relative gene transcription levels (RQ) for a) Catalase (cat), b) Glutathione S-transferase (gst), c) Heat shock protein 90 (hsp90), d) Heat shock protein 70 (hsp70), e) Ferritin and f) Cytochrome P450

Figure 6 (Figure 6 :

 66 Figure 6 (a-f) shows star plots for each experimental group. The surface value inside the

  ) were investigated. As expected, throughout the different endpoints studied, MGO WAF caused the most detrimental effects on the copepods considering mortality, percentage of fecund females, egg hatching success, and total lipid content. Conversely, MGO WAF produced relatively mild effects on gene expression levels. IFO 180 WAF was also toxic to copepods, resulting in increased mortality, a decreased percentage of fecund females, and significant effects on gene expression compared to control. In contrast, NNA WAF was the least toxic considering mortality, reproductive, lipid endpoints and the gene expression changes. The high doses of both IFO 180 and MGO WAF had a significantly higher IBR/n index than the other WAF conditions, demonstrating the toxicity of IFO180 and MGO to Acartia tonsa.It was not surprising to observe MGO toxicity, as previous studies have highlighted the toxicity of MGO WAF to marine organisms. The larvae of the marine Pejerrey

  1) Freshly prepared WAF, 2) Dispersant added to WAF (WAF+ Dispersant). The soluble fractions were made from a Naphthenic North Atlantic crude oil provided by the Norwegian University of Science and Technology (NTNU) (Trodheim Norway) within the framework of the GRACE project. Naphthenic North Atlantic oil (NNA) is a naphthenic based light crude oil. It is characterized by a low aromatic content, a low wax content, a low viscosity index

  to calculate the LC 50 of different WAF conditions. Data from egg production rate and egg hatching success were initially checked for normality using Kolmogrov-Smirnov test. Numbers of eggs produced during the exposure phase and the egg production rate of females (eggs Females -1 day -1 ) during the individual female fecundity stage were analyzed by one way ANOVA. Significant differences were further analyzed using Dunnett's and Tukey Kramer post-hoc tests (IBM SPSS v26). Percentage of reproductive females was analyzed by Chi-square test. Egg hatching success during individual fecundity test was calculated as follows: (number of hatched eggs per female /Total number produced per female) x100; average results per treatment were then analyzed using one -way ANOVA.

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Percentage of egg producing females after the 7 days exposure to NNA WAF (blue) and NNA WAF+Dispersant (orange). Females having more than 2 eggs were considered as a reproductive female. Fisher exact test showed no significant association between the reproductive females and the NNA WAF & NNA WAF+Dispersant

  hsp 90 and cyp3026b were negatively correlated with the first component. Ferritin and , negatively correlated with the second component, while gst, hsp90, cyp3026b and catalase were positively correlated with the second component. Upon looking at the expression patterns of the different genes (Figure5), the PCA showed that the two WAF treatment groups were clustered together, suggesting similar expression patterns and different from control. However, 10% NNA WAF +Dispersant treatment showed to be different from control, WAF treatments and 20% NNA WAF+Dispersant.Interestingly, treatment of 20% NNA WAF +Dispersant was closely clustered with control, suggesting similar transcription patterns.

Figure 4 :Figure 5 :

 45 Figure 4: Relative gene transcription levels (RQ) for a) Glutathione S-transferase (gst), b) Catalase (cat), c) Ferritin, d) Heat shock protein 70 (hsp70), e) Cytochrome P450 3026b (cyp3026b) and f) Heat shock protein 90 (hsp90). Purple bar is control, blue is NNA WAF doses and orange NNA WAF+Dispersant doses. Transcription levels are represented in log2 scale (mean ± standard error). Asterisks denote statistically significant differences from the corresponding control according to one-way ANOVA followed by Dunnett´s post hoc-test (p<0.05) NNA WAF NNA WAF+D NNA WAF NNA WAF+D

  in the water column(Lyons et al., 2011).Lee et al (2013) observed a similar pattern of lethality when studying the effects of crude oil WAF with and without the addition of Corexit 9500 on the rock pool copepod Trigriopus japonicus.

  PAHs composition and concentrations of WAF and WAF+dispersant were determined by GC/MS analysis. A mix standard solution of 16 PAHs was used [Norwegian Standard (NS 9815: S-4008-100-T): phenanthrene (Phe), anthracene (An), fluoranthene (Fluo), pyrene (Pyr), 11 H-benzo[a]fluorene (11H-B[a]F), 11 Hbenzo[b]fluorene (11H-B[b]F), benz[a]anthracene (B[a]A), chrysene (Chry), benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P), benzo[e]pyrene (B[e]P), indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (D[a,h]A), benzo[g,h,i]perylene (B[g,h,i]P)]and dibenzo[a,e]pyrene (D[a,e]P). Stir bars (poly dimethylsiloxane polymer coated twisters, supplied by Gerstel GmbH, Germany) with 10 mm length and 0.5 mm film thick were used. The twisters were introduced in aqueous samples (35 mL) and stirred during 315 minutes. Then, twisters were cleaned with Milli-Q water, in order to eliminate seawater, and dried with paper tissue. Twisters were desorbed using a commercial thermal desorption TDS-2 unit (Gerstel GmbH) connected to a CIS-4 injector (Gerstel GmbH) with the following conditions: desorption time (10 min), desorption temperature (300 ºC), desorption flow (23 mL min -1 ), cryo-focusing temperature (-50 ºC) and vent pressure (7 psi). This desorption unit was coupled in an Agilent 6890 gas chromatograph also coupled to an Agilent 5975 mass spectrometer system (Agilent Technologies, USA). The Mass spectrometer (MS) was operated in selected ion monitoring (SIM) for quantification of target compounds. An Agilent DB-5MS+DG column with the following dimensions 30 m x 0.25 mm and 0.25 μm was used.

Figure 1 :

 1 Figure 1: PAHs removal in a) WAF+dispersant and b) WAF microcosms. Numbers above points denote percentage of mortality of Acartia tonsa after 96 hours exposure at microcosm water samples obtained at day 0 and day 21. Colors of numbers represent the origin of water. N=3 Error bars = ±1 Standard error

Figure 2 :

 2 Figure 2: Prokaryotic composition (relative abundance) at the phylum level in pristine and contaminated waters for the three treatments (control, WAF, WAF+dispersant) during the incubation. Number above each bar corresponds to the Shannon index.

Figure 3 :

 3 Figure 3: Correspondence analysis (CA) comparing microbial communities from pristine (square) and oil-polluted waters (dots) in the three treatments (controls, WAF, WAF+dispersant), at different incubation time. Light blue, Control; Purple, WAF; Black, WAF+dispersant. Numbers indicate the incubation time (days).

  control and contaminated treatments, but only the WAF+dispersant treatment provides favorable conditions for the development of specific microorganisms able to use hydrocarbon and/or dispersant as carbon source. The seven bioindicators exclusive for the WAF+dispersant include ASVs belonging to Fusibacter (ASV 75, ASV 2, and ASV 154), Flavobacterium (ASV 49), Psychrobacter (ASV 71), Psychromonas (ASV 106), and Shewanella (ASV 161). For the contaminated water, the bioindicator ASVs were specifically distributed according to the conditions (Figure 4b), showing abundance above 50% in their respective condition. The ASVs specifically identified for the WAF+dispersant treatment were members of the Proteobacteria phylum, belonging to Colwellia (ASV 14, ASV 232, and ASV 7), Marinomonas (ASV 86, ASV 126, and ASV 73), Moritella (ASV 95), Sulfitobacter (ASV 139), Paraperlucidibaca (ASV 118, ASV 179, and ASV 292) genera. The exclusive bioindicators for the WAF treatment included ASVs belonging to Bacteroidales (ASV 189), Cyanobacteria (ASV 243), Arcobacter (ASV 63), Sulfurovum (ASV 244), Moritella (ASV 36), Marinifilium (ASV 145), and Fusibacter (ASV 181) genera. Interestingly, some bioindicator ASVs were equally distributed between WAF and WAF+dispersant treatments suggesting that they respond to the presence of hydrocarbons. These bioindicator ASVs belong to Caedibacteraceae (ASV 21) family, Moritella (ASV 109), Olispera, (ASV 89), Fusibacter (ASV 182, ASV 131, and ASV 291), and Marinifilium (ASV 201) genera.

Figure 4 :

 4 Figure 4: Ternary plots showing the abundance of bioindicator taxa identified by LEfSe in microcosms prepared with water from (a) pristine and (b) contaminated sites. Numbers refer to ASV taxa. Bioindicator taxa identified for WAF+dispersant treatments are purple dots, for WAF are light blue dots, and for Control are black dots.

  microbial communities were dominated by ASVs belonging mainly to the Firmicutes Fusibacter (ASV 58, ASV 97, ASV 42, ASV 46, ASV 96, ASV 93, ASV 91, ASV 22, ASV 28, ASV 19, and ASV 81), Proteobacteria, Burkholderiaceae family (ASV 50, ASV 26, and ASV 38), and Actinobacteria, Microbacteriaceae family (ASV 7). Stress levels in copepods, measured by the transcription of gst gene, were positively correlated with WAF+dispersant samples collected after 21 days, in which ASVs affiliated to Flavobacterium (ASV 74), Salinirepens (ASV 4), and Psychrobacter (ASV 20) were

Figure 5 :

 5 Figure 5: Canonical correspondence analysis (CCA) showing correlation between the top 100 most abundant ASVs and the sum of concentration of PAHs, expression levels of gst gene in Acartia tonsa

3

  ASVs of uncultured bacteria from the order Rickettsiales (ASV 259, ASV 224, and ASV 174), Paraperlucidibacta (ASV 227), ASV from class Nitrososphaeria (ASV 321), an ASV from family Cyanobiaceae (ASV 153), and 2 ASV from Fusibacter (ASV 37 and ASV 290) genus.

  Hafez, T., Ortiz-Zarragoitia, M ,Cravo-Laureau C, Cagnon C, Duran R .,2022. Cold sediment microbial community shifts in response to Crude oil Water Accommodated Fraction and Water Accommodated Fraction + Dispersant (In preparation)1. Introduction:The constant polar ice cap melting has open novel marine areas where oil extraction and transportation has dramatically increased threatening such cold environments (e.g. arctic)

Figure 1 :

 1 Figure 1: Scheme of the microcosm experiment set up. The incubation was performed at 4 ° C for 21 days at dark.

  chromatography coupled to a 5973 N Agilent mass spectrometer (Agilent Technologies, USA) with a 7683 Agilent auto-sampler. For the analysis, 2 μL of the sample were injected in the splitless mode at 270 ° C into a 30 m × 0.25 mm × 0.25 μm HP-5 capillary column. The temperature program used for the chromatographic separation was as follows: 60 ° C for 2 min, temperature increase at 10 ° C min -1 to 290 ° C where it was finally held for 10 min. The carrier gas was helium (C-50) and was kept at a constant flux of 1.5 mL min -1 . The mass spectrometer was operated in the electron impact ionisation mode and the energy of the electrons was kept at 70 eV. The interface was kept at 300 ° C and the ionisation source and the quadrupole at 230 and 150 ° C, respectively.

Figure 2 :

 2 Figure 2: PAHs degradation during the microcosm experiment. (a,b) Concentrations of sum of PAHs in pristine (a) and contaminated (b) sediments. (c,d) Relative abundance of low molecular weight (LMW) and high molecular weight (HMW) PAHs in pristine (c) and contaminated (d) sediments. The incubation was performed at 4 ° C for 21 days at dark. Grey = control, Orange = WAF, Green = WAF+dispersant. Darker bars denote (HMW) PAHs while lighter bars denote (LMW) PAHs. The experiment was performed in replicates (n=3). Error bars shows standard error.

  and other contaminants(Ben Salem et al., 2019) in temperate marine sediments, is thus also observable for microbial communities inhabiting cold environments.The comparison of total microbial communities (DNA) by nNMDs, based on Bray Curtis dissimilarity index revealed the modification of the microbial communities during the experiment for both pristine and contaminated sediments(Figure 3, b & c). Noteworthy, the microbial communities of the control showed less modification during the experiment than those of the WAF and WAF+dispersant treatments (Figure3, b & c).At time 0 with pristine sediment, total microbial communities clustered together, suggesting similar communities at the beginning of the experiment (Figure3, b). Then, changes were observed within the microbial communities according to the treatments during the experiment. Particularly, for microbial communities from pristine sediment, the presence of dispersant resulted in changes more important than WAF alone. This observation suggests that upon the introduction of dispersant to pristine sediment, continuous microbial succession in the total communities was occurring overtime.In general, according to RNA:DNA ratios (Figure3d & e), microcosms' active communities are not showing distinctive clustering. The nMDS shows that in pristine and contaminated sediment, the samples of the same treatment are distant from each other, therefore changes in active microbial community is observed overtime in the three treatments. However, in contaminated sediment, microbial communities (both total and active) showed an important shift in the WAF+dispersant treatment at time 14. In addition, PERMANOVA based on Bray Curtis dissimilarity index only showed significant difference between contaminated and pristine sediments in respect of total (DNA) and active (RNA:DNA) communities (PERMANOVA p < 0.0001), demonstrating the significant difference between the bacterial communities from pristine sediment and that from contaminated sediment. Dispersed oil with Corexit dispersant has been demonstrated to trigger bacterial communities' shifts in sediment after 30 days of incubation[START_REF] Al-Jawasim | Synergistic effect of crude oil plus dispersant on bacterial community in a louisiana salt marsh sediment[END_REF].[START_REF] Doyle | Rapid Formation of Microbe-Oil Aggregates and Changes in Community Composition in Coastal Surface Water Following Exposure to Oil and the Dispersant Corexit[END_REF] observed rapid changes within the microbial composition in surface water upon exposure to chemically enhanced WAF with Corexit dispersant, which was explained by the presence of hydrocarbon degraders in the environment chronically exposed to hydrocarbons. In contrast,Louvado et al., (2019) observed that the microbial community structure from hydrocarbon-chronically polluted sediment was not significantly affected by the addition of dispersant because of the legacy effect, preconditioning the microbial community to contamination.

Figure 3 :

 3 Figure 3: Bacterial communities structures and dynamics during the microcosm experiment. a) Relative abundances of total bacterial communities at the phylum level in the three treatments (controls, WAF, WAF+dispersant) overtime. Number above each bar denotes Shannon diversity index (n=3). (b,e) Comparison of bacterial communities by non metric Multi Dimensional Scaling (NMDS) based on Bray Curtis Dissimilarity index. (b,c) total bacterial communities (DNA level). (d,e) active bacterial communities (ratio RNA to DNA).

Figure 4 :

 4 Figure 4: Comparison of bacterial communities by LEfSe based on linear discriminant analysis (LDA) revealing taxa with RNA:DNA ratio significantly different from control in the microcosms of (a) WAF+dispersant, pristine (b) WAF, pristine (c) WAF+dispersant, contaminated (d) WAF, contaminated

Figure 5 :

 5 Figure 5: Dynamics of RNA:DNA ratios of microbial bio-indicators identified by LEfSe during the incubation period of microcosms prepared by: (a) WAF+dispersant, pristine; (b) WAF, pristine; (c) WAF+dispersant, contaminated; (d) WAF, contaminated

Figure 6 :

 6 Figure 6: Canonical Correspondence analysis (CCA) between the selected functional genes in active bacterial communities and the concentrations of the different PAHs. (a) Pristine sediment, (b) Contaminated sediment.

  exhibited 15 functional genes showing strong positive correlations (0.80 < Pearson correlation < 0.99) with 2/3 rings PAHs (naphthalene, acenaphthene, fluorene, and acenaphthylene), while in WAF treatment, 6 functional genes showed strong positive correlation (0.80 < Pearson correlation < 0.99) with naphthalene and acenaphthylene (Figure 7 a). The microbial community from contaminated sediment exhibited several functional genes showing strong positive correlation (0.74 < Pearson correlation < 0.99) with the concentrations of PAHs (Figure 7 b). The 4,5 dihydroxylphalate decarboxylase gene showed high positive correlation (0.74 < Pearson correlation < 0.99) with all PAHs except chrysene in WAF+dispersant treatments (Figure 7 b). However, in the WAF treatments, 4,5 dihydroxylphalate decarboxylase gene was strongly positively correlated (0.74 < Pearson correlation < 0.99) with all PAHs except naphthalene and its derivatives. The 4,5 dihydroxylphalate is known as a major intermediate metabolite in the microbial degradation of PAHs such as fluoranthene, fluorene and phenanthrene (Kasai et al., 2019).Additionally, seven functional genes in WAF+dispersant treatments were strongly positively correlated with chrysene, suggesting the availability of active taxa involved in the degradation pathways of chrysene. Four more functional genes were positively correlated (0.70 < Pearson correlation < 0.85) with PAHs with more than 2 rings (pyrene, fluoranthene, anthracene, and phenanthrene). Interestingly, in WAF treatments, (acenaphthylene and acenaphthene) showed strong correlation (0.80 < Pearson correlation < 0.99) with 4 genes. Whereas, PAHs with more rings were positively correlated (0.70 < Pearson correlation < 0.85) with 3 different genes producing intermediate metabolites (dihydroxylphalate, protochatecuate, catechol). Thus, our result demonstrates that the active functional communities from contaminated sediment are efficient to degrade PAH with more than 2 rings.

Figure 7 :

 7 Figure 7: Clustering correlation analysis showing the correlation between predicted functional genes and the concentration of the different PAHs for bacterial communities from (a) WAF+dispersant, pristine (b) WAF, pristine (c) WAF+dispersant, contaminated (d) WAF, contaminated. Correlation and cluster analysis are based on Pearson correlation. Red denote positive correlation while blue denote negative correlation

Figure S1 :

 S1 Figure S1: (a) Longitudinal section of a fecund female stained with hematoxylin and eosin. Circle represents ovary with eggs at different developmental stages. Scale bar: 250 µm. D: dorsal side, V: ventral side, H: head. (b) Fluorescence microscope image of a fecund female stained with Nile Red. Bright red spots (in circle) represent high lipid deposit areas around the ovaries. Scale bar: 500 µm. D: dorsal side, V: ventral side, H: head.

Figure S2 :

 S2 Figure S2: Multi sequence alignment (a) cyp3026b (b) catalase (c) gst with homologs of other species. Grey areas denote amplicon regions.

Figure S2 :

 S2 Figure S2: LEfSe based on Linear Discriminant analysis (LDA) of taxa with abundances ratio significantly different from control. a) WAF+ Dispersant contaminated water. b) WAF Contaminated sediment. water. c) WAF pristine water. d) WAF+ Dispersant pristine

Figure S1 :

 S1 Figure S1: Rarefaction curves in respect of a) time, b) sediment type, c) treatment, d) Active (RNA) and total (DNA) communities.

Figure S2 :

 S2 Figure S2: Procrustes analysis of total communities (DNA) and Active communities (RNA) using Non metric MDS. A) NMDS for Contaminated sediment, b) NMDS for pristine sediment.



  Incubate the reaction at 65 C for 5 minutes  The reaction was cooled for 10 minutes at room temperature.  Each of the following component were added to the reaction for a final volume of 20 µL: o 2 µL 10X Affinity Script RT buffer o 0.8 µL dNTP mix o 0.5 µL RNase block ribonuclease inhibitor o 1 µL Affinity Script Multiple Temperature RT  The reaction was incubated at 25 C for 10 minutes, then incubated at 55 C for 1 hour. Finally, the reaction was terminated by incubating at 70C for 15 minutes. I feel my PhD was a long journey. In this trip, I met a lot of wonderful people and I have great memories with them. I wouldn't have completed this journey without the help and support (technically and emotionally) from a lot of people. First of all, I have to give my sincere gratitude to both of my advisors: Maren, no words can explain my appreciation for everything you did for me throughout my PhD and my masters. Throughout the seven years you were my mentor, I learned from you a lot. I will always be in debt for your continuous help and for always encouraging me to move forward. In addition, thank you for always having the patience with my constant requests and corrections. Robert, Thank you for everything! You were always available for me and were always supportive. I will always remember being in your office in Pau for long hours designing the microcosm experiments. Even later after I returned to Spain, thank you very much for the several online meetings and your constant support. I would like to thank my fellow collegues in PiE and in Leioa. Thank you Tiffany, Dennis, Esther, Laura, Erik, Nerea, Anthony, Ander, Nagore and Javi. I will not forget the funny and the great memories we have together: the long coffee breaks, the Baetz and kebab commun missil on Friday and your tolerance hearing me singing Katty Perry Fireworks. Thanks making me feel like home. In addition, I wish for the new PhD students, Mar, Markel and Joyanta best of luck on their PhD journey! You guys will do great! I cannot forgot to mention my gratitude for all the PiE staff professors and researchers. Thank you Irune, Carlos, Urtzi, Mutri, and all for your continuous help and support during my PhD. Especially, tolerating my naggings while doing experiments, asking for lab equipments or finishing some paperworks. Additionally, my deepest thanks to Oihane and Eider for their generous help and patience in teaching me several molecular biology techniques.

Table 1 :

 1 An overview of some toxicological studies using Acartia tonsa as a model organism.

	Contaminants	Concentration range	Exposure period	Endpoints studied		References
	Flouranthene,Phenanthrene,	Flu: 200-800 nM	48 hours	Egg	production	rate,	Bellas et al 2007
	Pyrene	Phe:200-1800 nM		lethality, recruitment rate
		Py: 40-640 nM
	Water Accommodated Fraction	0-100% WAF	48 hours	Lethality		Faksness et al 2015
	(WAF) from crude oils
	Nickel and Nickel nanoparticles	5-50 mg/L	7 days, 4 days, 48 hours	Egg hatching, naupliar	Zhou et al 2016
		1-10 mg/L		mortality,egg production
				rate
	Dimethylnapht-halene	0.54-57.358 µg/L	24, 48,72, 96 hours	GST gene expression		Zhou et al 2020
				levels
	hhAgNO3	5-178 µg/L	48 hours	Mortality, concentration of	Pedroso et al 2007
				magnesium, chloride,
				sodium / dry weight,
				ATPase activity
	Polyethylene Microbeads	0.5-2.5 µg/L	48 hours	Mortality		Syberg et al 2017
	Copper	0-160 µg/L	6 days	Egg production rate, egg	Lauer et al
				hatching rate
	Nitro and polycyclic synthetic	0.6-5.0 mg/L	5 days	Mortality,	larval	Wollenberger et al 2003
	musks			development
	ZnO nanoparticles	50-300 µg/L	7 days	Reproduction, mortality	Jarvis et al
	Ag, Zn, Cu, Ni	5.4-15.4 µg/gm	7 days	Reproduction, mortality	Bielmyer et al 2006
	Scrubber discharge water	0-100%	24 hours	Feeding, reproduction	Koski et al

Table 1 :

 1 Selected doses of WAF of studied oils in the different analyzed biological endpoints

	NNA WAF	IFO 180 WAF	MGO WAF

Low Medium High Low Medium High Low Medium High

	Reproductive	10%	30%	50% 10%	25%	40% 3%	5%	7%
	assays
	Lipid levels	-	25%	50%	-	20%	40%	-	5%	7%
	Gene	-	25%	50%	-	20%	40%	-	5%	7%
	transcription
	levels

NNA: Naphthenic North Atlantic; IFO 180: Intermediate fuel oil 180; MGO: Marine gas oil.

(-): not tested

Table 2 :

 2 Primer sequences and melting temperature (Tm) for all the genes analyzed in this study.

	Gene	Primer sequences 5' to 3'	Tm (°C)	References
	hsp70	Fw: TTCAATGATTCACAGAGACAAGC	57.1	Nilsson et al.,
		Rv:TCCTTGTGATGTTAAGACCAGCTAT	59.7	2014
	hsp90	Fw:GTCACATCCCAGTATGGTTGG	59.8	Nilsson et al.,
		Rv :CCATGGTGGAGGTGTCACGG	63.5	2014
	ferritin	Fw:ACGCTTGCACTGATAATCCA	55.3	Nilsson et al.,
		Rv :AGTTCTACCGTGACGCATCC	59.4	2014
	β-actin	Fw:TTGGGTATGGAGTCCTGTGG	59.4	Zhou et al.,
		Rv :CCTGGATACATAGTGGTGCC	59.4	2020
	cyp3026b Fw:TGCCATTGGCATCCACCATGAC	62.1	Current study
		Rv:AGCCTCCAGCAAAGCAAACCTC	62.1
	gst	Fw:TGCTTGATTCACTTCTACAAGAGA	57.6	Current study
		Rv:GTCACCATCAACAACAGTTGGA	58.4
	cat	Fw:GGAGAGAATCCCAGAAAGAGTG	60.3
		Rv:ACAGTCCAATTTCCCCTCCC	59.4

Table 3 :

 3 Concentrations

of the most prevalent PAHs in the three different types of 100% WAF prepared at 10°C. All concentrations are presented at ng/L (mean values ± SD; n=3). PAHs below detection limit for the three tested oils were: 11

  and dibenzo[a,e]pyrene (D[a,e]P).

	PAH	# of	NNA	IFO 180	MGO
		rings
	Naphthalene	2	19295 ± 759	8441±660	11213± 1166
	Acenaphthene	2	347±25	965±3	362±37
	Fluorene	2	1008±3	909±115	1161±95
	Phenanthrene	3	1115±15	1968±63	1953±71
	Anthracene	3	LDL	132±18	LDL
	Fluoranthene	3	LQL	23±2.3	25±3
	Pyrene	4	141±33	56±2	37±3
	Benzo[a]anthracene	4	LQL	29±3	LQL
	∑ PAHs		21934±7141	12524±2861	14759±4080

LDL: Low detection limit. LQL: Lower than quantification limit.

Table 4 :

 4 Lethal concentrations (LC50) for the adult Acartia tonsa exposed to the different WAF after 48, 72, 96 hours expressed as percentage of WAF diluted in seawater. The upper and lower limits of 95% confidence interval are indicated in parenthesis. Different superscript letters in each row denote significant differences among times, based on the absence of confidence interval overlap. Different superscript numbers in each column denote significant differences between oils, based on the absence of confidence interval overlap.

	Oil WAF	48 h	72 h	96 h
	NNA	>100% a 1	74% b 1	52% b 1
		(95-176)	(61-94)	(40-65)
	IFO180	74% a 2	52% b 1	40% b 1
		(67-81)	(45-61)	(32-49)
	MGO	34% a 3	10% b 2	8% b 2
		(29-42)	(0.3-16)	(0-13)

  However, in the case of high doses of MGO WAF, the females produced non-viable eggs, which could be explained because of altered lipid levels in exposed females. In addition, transcriptional levels of the forms of Cyp3026band hsp 70 used in this study could be potential biomarkers of oil WAF exposure in Acartia tonsa. Metabolic, reproductive and transgenerational toxic effects detected in copepods exposed to refined oil WAF raise concerns about the risks of copepod communities in regions with high maritime activities such as harbors and high maritime traffic routes. Such areas can have concentations of PAHs at similar levels to those used in this study. Disruption in the copepod population can strongly impact the whole food web by transferring the effects to the polluted ecosystem's higher trophic levels.

conclusion, the results of the present work demonstrated that exposure to IFO 180 and MGO WAF had harmful effects on Acartia tonsa. The NNA showed less toxicity in comparison to refined oil WAF. The biological multi level approach applied in the present study, highlights the importance of integrating the effects elicited by environmental pollutants at different biological levels. The combination of multilevel biological endpoints provided a comprehensive picture of how oils (crude and refined) affect Acartia tonsa individuals. Suppression in egg spawning was observed in treated females, but the egg-producing females still produced eggs at rates similar to controls, probably as a result of unknown compensatory mechanisms.

Table 1 :

 1 Chemical composition of dispersant Finasol OSR 52 obtained from Total energies, France

	Chemical name	Percentage weight
	Docusate sodium

  Amplification efficiency for each primer pairs was calculated by conducting a 1:2 serial dilution curve generated from pooled experimental samples. All primer pairs showed amplification efficiency between 90% and 100%.

	2.5.3 Primer design
	Catalase (cat), glutathione S transferase (gst), heat shock protein 90 (hsp90) , heat shock
	protein 70 (hsp70), and ferritin genes were targeted as biomarkers of cellular oxidative
	stress and PAH metabolism. Cytochrome p 450 gene (cyp) from clan 3 (cyp3026b), a
	2020), showing in the current experiment a low coefficient of variability (CV < 5%). The
	identity of all targeted genes was confirmed by sequencing (General Genomics Service

biomarker for PAH metabolism, was analyzed. Primers for cat, cyp3026b and gst were designed de novo from conserved regions in closely related crustacean species and scaffolds from the Acartia tonsa genome project (Jorgensen et al., 2019) (Accession number PRJEB20069). Clustal Omega

(Madiera et al., 2019)

was used for multiple sequence alignment. For cyp3026b, Acartia tonsa genome sequence LS054218.1 was aligned with the gene sequences from Tigriopus japonicus (KF639998.1) and Eurytemora affinis (XM 023493699.1). For catalase, the genome sequence LS054460.1 was aligned with sequences from Calanus finmarchicus (EL965956.1) and Eurytemora affinis (XM023472406.1). For gst, the genome sequence LS085988.1 was aligned with Tortanus forcipatus (KT755427.1) and Acartia pacifica (KT754520.1). Specific primers to amplify fragments of hsp70, hsp90 and ferritin genes were obtained from

Nilsson et al (2014)

. βactin gene was used for transcription normalization as previously proposed

(Zhou et al.

Sequencing and Genotyping Unit, SGIker, UPV/EHU). Primer sequences and the primers' amplicon regions' multi-sequence alignments are presented in Table

2 and

(Supplementary Materials, chapter 1, figure

S2

) respectively.

Table 2 :

 2 Primer sequences and melting temperature (Tm) for all the genes analyzed in this study.

	Gene	Primer sequences 5' to 3'	Tm (°C)	References
	hsp70	Fw: TTCAATGATTCACAGAGACAAGC	57.1	Nilsson et al.,
		Rv:TCCTTGTGATGTTAAGACCAGCTAT	59.7	2014
	hsp90	Fw:GTCACATCCCAGTATGGTTGG	59.8	Nilsson et al.,
		Rv :CCATGGTGGAGGTGTCACGG	63.5	2014
	ferritin	Fw:ACGCTTGCACTGATAATCCA	55.3	Nilsson et al.,
		Rv :AGTTCTACCGTGACGCATCC	59.4	2014
	β-actin	Fw:TTGGGTATGGAGTCCTGTGG	59.4	Zhou et al.,
		Rv :CCTGGATACATAGTGGTGCC	59.4	2020
	cyp3026b Fw:TGCCATTGGCATCCACCATGAC	62.1	Current study
		Rv:AGCCTCCAGCAAAGCAAACCTC	62.1
	gst	Fw:TGCTTGATTCACTTCTACAAGAGA	57.6	Current study
		Rv:GTCACCATCAACAACAGTTGGA	58.4
	cat	Fw:GGAGAGAATCCCAGAAAGAGTG	60.3
		Rv:ACAGTCCAATTTCCCCTCCC	59.4

  Table3) showed that the mixture of NNA WAF+Dispersant had the highest lethality on adults Acartia tonsa. LC50 values were as low as 34%, 31% and

	21 % of NNA WAF+Dispersant dose during the 48, 72, 96 hours, respectively. Compared
	to the NNA WAF+Dispersant mixture, NNA WAF showed to have lower lethality on
	Acartia tonsa adults. LC50 values for NNA WAF were above 100% at 48 hours, and 74
	% at 72 hours and 52% at 96 hours.

Table 3 :

 3 Lethal concentration values (LC50) determined at 48, 72, 96 hours with adult Acartia tonsa exposed to the different WAF conditions (n=30). Number in parenthesis denote 95% confidence intervals.

  Abbasian, F., Lockington, R., Megharaj, M., Naidu, R., n.d. The Biodiversity Changes in the Microbial Population of Soils Contaminated with Crude Oil. Curr. Microbiol. 72. https://doi.org/10.1007/s00284-016-1001-4 Abbriano, R.M., Carranza, M.M., Hogle, S.L., Levin, R.A., Netburn, A.N., Seto, K.L., Snyder, S.M., Franks, P.J.S., 2011. Deep water horizon oil spill: A review of the planktonic response. Oceanography. https://doi.org/10.5670/oceanog.2011.80

Table S1 :

 S1 Correlation scores of the different biomarker genes with the first two components of the PCA

		PC 1	PC 2
	GST	0.79904	0.56739
	Ferritin	0.78983	-0.16592
	HSP70	0.95128	-0.26854
	HSP90	-0.62442	0.66488
	Cyp3026b	-0.70163	0.50327
	cat	0.85422	0.35021

Table S3 :

 S3 Major topological characteristics of (a) empirical networks generated for pristine and contaminated site. (b) Random networks generated for pristine and contaminated site.

	Community	RMT	Total	Total	R 2	Average	Average	Average	Modularity
		threshold	nodes	links	power	Degree	Path	Clustering	And
					law	(avgKK)	distance	Coefficient	number of
							(GD)	(avg CC)	nodes
	Contaminated 0.950	112	225	0.448 4.018	3.925		0.307	0.65 (17)
	Pristine	0.910	171	468	0.663 5.474	4.296		0.308	0.60 (14)
	Community	Average		Average			Modularity
			Path distance	Clustering	and number of
				(GD)		Coefficient		nodes
						(avg CC)
	Contaminated 3.356 +/-0.098 0.074 +/-0.016 0.439 +/-0.011
	Pristine	3.197 +/-0.032 0.043 +/-0.008 0.385 +/-0.009

Table S1 :

 S1 denoising Features and alpha diversites for all samples used in this study

		input	filtered	denoised	merged	non-	Shannon	Chao
						chimeric
	DNA SEQUENCES
	RD-t0wcc1	33507	26431	23303	11720	9583	461.4643	5.886716

Table S2 :

 S2 Predicted functional genes selected for this study and their function in the PAH degradation pathways.

	Predicted gene	function	References
	naphthalene 1,2-dioxygenase	Naphthalene/ Phenanthrene	Parales
	ferredoxin component	Degradation Pathway,	2003
		initial breakdown
	naphthalene 1,

2-dioxygenase subunit alpha

	Naphthalene degradation	Parales
	pathway, initial breakdown	2003
	naphthalene 1

,2-dioxygenase ferredoxin reductase component

	salicylate hydroxylase	Naphthalene and other	Jouanneau
		HMW PAHs	2007
	alcohol dehydrogenase	Naphthalene and other	Haritash
		PAHs	2009
	alcohol dehydrogenase, propanol-	Naphthalene and other	Haritash
	preferring	PAHs	2009
	4,5-dihydroxyphthalate decarboxylase Metabolite for	Kasai et al
		flouranthene, flourene	2019
		phenanthrene
		biodegradation pathways
	catechol 1,2-dioxygenase	Pyrene degradation	Singh et al
		pathway, intermediate in	2013
		PAH pathway
	catechol 2,3-dioxygenase	Pyrene degradation	Singh et al
		pathway, intermediate in	2013
		PAH pathway
	protocatechuate 4,5-dioxygenase,	Pyrene, Phenanthrene	Thomas et
	alpha chain	degradation pathways,	al 2016
		intermediate in PAH
		pathway
	protocatechuate 4,5-dioxygenase, beta	Pyrene, Phenanthrene	Thomas et
	chain	degradation pathways,	al 2016
		intermediate in PAH
		pathway
	protocatechuate 3,4-dioxygenase, beta	Pyrene, Phenanthrene	Thomas et
	subunit	degradation pathways,	al 2016
		intermediate in PAH
		pathway
	protocatechuate 3,4-dioxygenase,	Pyrene, Phenanthrene
	alpha subunit	degradation pathways,
		intermediate in PAH
		pathway
		Naphthalene degradation	Parales
		pathway, initial breakdown	2003
	cis-1,2-dihydro-1,2-	Naphthalene degradation	Denome
	dihydroxynaphthalene	pathway	1994
	/dibenzothiophene dihydrodiol
	dehydrogenase
	1,2-dihydroxynaphthalene dioxygenase Naphthalene degradation	Patel 1980
		pathway, initial breakdown
	1,3,6,8-tetrahydroxynaphthalene	Naphthalene degradation	Ghimire et
	synthase	pathway	al 2008
	2-hydroxychromene-2-carboxylate	Naphthalene degradation	Denome
	isomerase	pathway	1994
	trans-o-hydroxybenzylidenepyruvate	Naphthalene degradation	Eaton 1994
	hydratase-aldolase	pathway

State of the art, Hypothesis and Objectives

Results and Discussion

Acknowledgments

This work was funded by the Basque Government (grant to consolidated research groups IT1302-19) and the European Union (GRACE project, EU horizon 2020 grant No 679266). The University of the Basque Country and the Université de Pau et des Pays de l'Adour supported the first author with a cotutelle (co-supervision) doctoral grant. We want to thank the Norwegian University of Science and Technology (NTNU; Trondheim, Norway) for providing us with the oils used in the experiments. We want to thank Prof. Jasmine Nahrgang (UiT) for her invaluable help in the collection of the water and sediment samples from Trømso for the microbiome studies, and the technical and human support provided by SGIKER (UPV/EHU ERDE/ EU) for the chemical analysis of water in this study. Acknowledgments Acknowledgements Acknowledgements 233 I cannot forget to thank my fellow PhD students and staff in the IBEAS lab at the University of Pau. Thank you Zeina, Joyce, Kevin, Jonathan, Ivan, Eva, Manu, Elise for making my stay in IBEAS great and memorable. Initially, I thought I was going to have hard time during my stay in Pau to adapt, but you guys proved otherwise! You guys made my stay in Pau very memorable, thank you. Que, thank you for always being supportive and being on my side for all these years! Of course, I want dedicate my thesis to my mother and brother, for all their continuous support throughout my PhD journey. No words can describe my gratitude. You were always encouraging me to go ahead no matter what. In addition, I dedicate this thesis in loving memory of my late father.

 Dr. Ionan Marigomez, Coordinator of the Marine Environment and Resources doctorate program, for providing me this opportunity to do my PhD in the UPV/EHU.  Dr. Miren Cajaraville, Head of the CBET research group, for her guidance and help.

General Discussion

Abstract

Among the strategies for combating oil spills, dispersants are used to increase the bioavailability of hydrocarbons promoting then microbial degradation. During oil weathering process, harmful soluble hydrocarbons, such as polycyclic aromatic hydrocarbons (PAHs), are introduced into the water column, resulting in water accommodated fraction (WAF). Such weathering process can be influenced by the presence of dispersants increasing PAHs solubility, toxicity and biodegradability.

However, little is known on how dispersants affect microbial communities and their degradation capacities, especially in cold environment where low temperature decreases microbial activity and thus hydrocarbon degradation. In this study, we investigated the microbial community dynamics in cold water contaminated by WAF prepared from crude oil with or without a commercial dispersant (Finasol OSR52).

The WAFs, prepared with Naphthenic North Atlantic crude oil, were used to contaminate seawater from Norwegian cold sites, one oil-contaminated and the other pristine. The WAF-contaminated seawaters were maintained in microcosms at 4°C for 21 days. The content of PAHs and microbial compositions (16S rRNA gene sequencing) were determined at days 0, 7, 14 and 21. In addition, the 96 hours toxicity assay with adult Acartia tonsa revealed WAFs toxicity at days 0 and 21.

The toxicity of WAF mixtures, with and without dispersant, against Acartia tonsa was reduced during the experiment from high at day 0 to low at day 21. The observed toxicity reduction was not accompanied by an increase of PAHs removal. The water from the oilcontaminated site showed the highest PAHs removal revealing legacy effect (presence of microorganisms adapted to PAHs). Additionally, our results reveal: i) the plasticity of the microbial community to adapt to the presence of PAHs and dispersant, ii) the presence of PAHs and lineal hydrocarbons: 170 ºC for 5 min; ramp at 30 º C min -1 to 260 ºC; ramp at 8 ºC min -1 to 300 ºC and hold 15 min.

DNA extraction and 16S rRNA gene PCR amplification

The membrane filters were cut into small pieces then homogenized using metal beads.

The total DNA was extracted using power soil total DNA isolation kit (Qiagen, Germany) following the extraction protocol recommended by manufacturer. DNA quantity was determined using Quant it dsDNA HS kit (Theremofisher scientific, USA). The 16S rRNA gene was amplified using the universal prokaryotic primers U515-532-GTGYCAGCMGCCGCGGTA and U909-928-CCCCGYCAATTCMTTTRAGT targeting the V4-V5 region of the 16S rRNA gene (Wang and Qian 2009). The reaction medium (25 µL) consisted of 12.5 μL AmpliTaq Gold 360 Master Mix (Invitrogen, USA), 1 µL of forward and reverse primer at concentration of (20 µM), 5.5 µL water and 5 µL DNA template (10 ng). The polymerase chain reaction was performed using thermocycler (Applied biosystems, USA) with the following cycling conditions: 10 min at 95 °C followed by 30 cycles of: 30 s at 95 °C, 30 s at 60 °C and 40 s at 72 °C, followed by a final step of 10 min at 72 °C. Successful amplification of the PCR was checked on 2% agarose gel electrophoresis, stained with ethidium bromide. PCRs were performed in triplicates and then pooled for further sequencing.

Amplicon sequencing and bioinformatics analysis

The 16S rRNA gene barcoding was performed using NGS Illumina MiSeq at the GenoToul platform (Toulouse, France). Demultiplexed sequences obtained were treated with Qiime2 software (Quantitative insights into microbial ecology; Caporaso et al., 2010) according to Quince et al. (2009). Briefly, sequences were trimmed and then denoised using DADA2 pipeline integrated in the Qiime2 software to remove chimeric

Abstract:

In cold environment, the low temperature slows down microbial metabolisms, especially the biodegradation processes of hydrocarbons, which are often stimulated by the addition of dispersants in oil spill disasters. In this study, we investigated the effects of

Conclusions and thesis

Supplementary Materials

Supplementary Materials

Chapter 3:

Acartia tonsa intensive culturing:

Good practices of culturing and maintenance are crucial for a good supply of Acartia tonsa for experimentation. In literature, several methods of intensive culturing of Acartia tonsa has been suggested (Marcus et al 2007[START_REF] Medina | Static-renewal culture of Acartia tonsa (Copepoda: Calanoida) for ecotoxicological testing[END_REF]. In this section, we discuss the culturing practices followed for Acartia tonsa production. Acartia tonsa were initially purchased from an aquaculture distributer, either in the form of dormant eggs or adults. Adults purchased were first acclimatized to temperature and salinity of the laboratory. Copepods were cultured in 20 liters plastic tanks with gentle aeriation.

Copepods were fed every 2 days with 1:1 algae of Tetraselmis chuii and Isochrysis galbana. at a concentration of 3000 cells per ml.

To avoid of adults consuming the newly hatched nauplii, nauplii were collected from the main tank to smaller culturing tank every weeks. The early stage naupli were feed only with Isochrysis galbana for the first week. Once copepods reach late copepodite stages, they were returned to the main stock tank.

A simple, small siphon was made to be used for copepod collection with gentle force and water removal (figure a). Depending on the function, small tips made with 200 µm or 45 µm mesh (figure b) to be attach to the siphon. Siphon with 45 um mesh tip was used to remove water from the culturing tanks for cleaning without removing adult and naupli.

To collect naupli, the 200 um tip was used.

Nile red in vivo staining of Adult Acartia tonsa:

Based  Remove all water using fine tip glass Pasteur pipettes (note: there could be water droplets on the walls of the tube that must also be removed);  Add 0.3 ml of isopropanol and put on ice;

Extraction and quantification  Extraction can be made immediately or samples can be conserved on ice in the dark for a few hours… but the quantification must be done just after extraction;

Histological visualization of adult females

 About 30 Female copepods were introduced in 1.5 mL Eppendorf tubes filled with warm liquid 2.5% agarose and placed in a warm water bath at 40ºC.

 Copepods were left to suspend through the liquid agarose for a few minutes and then the tubes were placed on ice to solidify the gel.

 After solidification, the gel containing copepods was pushed out of the tube and cut into 1 cm thick gel cores.

 The gel cores were placed in histology cassettes and fixed with Bouin solution for 8 hours at 4ºC.

 After fixation, the samples were rinsed with 70% ethanol and processed for paraffin embedding using the ASP300 Automatic tissue processor (Nussloch, Germany).

 Samples were formed into histology blocks and sectioned into 5 µm thick sections using an automated microtome (Leica RM225, Germany).

 Sections were mounted on glass slides and stained using hematoxylin and eosin (Leica Autostainer ST5010, Germany).

RNA Extraction in Acartia tonsa using RNA easy mini kit:

 Upon exposure, copepods were filtered through a 200 µm mesh for collection and placed in cryotubes filled with RNA later (Invitrogen,USA) and stored in -80 c

 Using a small needle with syringe, RNA later was carefully removed without removing copepods in the process.

 The cryto tubes containing the copepods were filled with RLT lysis buffer with beads for tissue homogenization  Copepod were homogenized using a Precelis tissue disruptor at the following settings: 45 seconds disruptions twice, then place samples on ice for a minute then tissue disruption for 45 seconds twice again.

 Samples were centrifuged for 3 minutes at maximum speed and supernatant was collected in new tubes  1 volume of 70% ethanol was added to the supernatant and mixed well by pipetting.

 700 µL of samples were pipetted in RNeasy spin column placed in 2ml collection tube and centrifuged at 8000 G for 15 seconds. To get maximum yield, this step was repeated with the remainder of the supernatant from the previous step using the same spin column.

 700 µL of RW1 buffer was added to the spin column and centrifuged at 8000 G for 15 seconds. Then the flow through was discarded