La Direcció General de la Policia

P.S. Carvalho, UFPE - Universidade Federal de Pernambuco / Zoología Polycyclic aromatic hydrocarbons (PAHs) are oil derived compounds known for their toxicity to aquatic organisms. Estuarine regions are frequently contaminated with PAHs as a result of urbanization processes and industrial activities, including the oil productive chain. This study aimed to evaluate PAH biliary bioconcentration and biochemical effects in the fish Eugerres brasilianus sampled along four estuaries in the state of Pernambuco, northeastern Brazilian coast. Fish were sampled in Aquirindá river, Formoso River Estuarine System (AR-FRES), Massangana river, inside Suape Estuarine Complex (MA-SEC), Barra de Jangada Estuarine System (BJES) and Bacia do Pina Estuarine Complex (BPEC). Fish bile samples were analyzed using fixed wavelength fluorescence to estimate equivalent concentrations of the PAHs naphthalene, phenanthrene and chrysene. Liver samples were analyzed for activities of biotransformation enzymes

Ethoxyresorufin-O-deethylase (EROD), and glutathione S-transferase (GST),

antioxidant defense enzymes catalase (CAT) and glutathione reductase (GR), and acetylcholinesterase (AChE). Bile PAHs and biochemical biomarkers in fish sampled during an annual cycle in AR-RFES and MA-SEC indicated similar bile PAH concentrations and enzymatic activity levels between these estuaries, despite the different anthropogenic activity patterns. Suape Estuarine Complex includes a developing industrial port complex, while Rio Formoso Estuarine System is within a low population density area focused mostly on tourism. Fish sampled in the two other estuarine systems near Recife metropolitan area, BJES and BPEC, showed chrysene equivalent bile concentrations between 13 x and 19 x higher than AR-FRES, respectively. EROD, GST and CAT activities were also increased in BJES and BPEC, reaching 30 x for EROD, and approximately 2 x for GST and CAT when compared to AR-FRES. Higher PAH bioconcentration and enzymatic induction in E. brasilianus fish from BJES and BPEC indicate that these fish are spending energy to biotransform and excrete these contaminants, which may have consequences to their growth and survival in such regions. The results indicate that BJES and BPEC receive a greater input of PAHs, associated with the higher population density and anthropogenic activities in these regions. The parameters used will be useful for monitoring of these estuarine systems, especially Suape Estuarine Complex, which is under a rapid urbanization and industrialization process.

MO006

Bioaccumulation of Sulfur and Nitrogen Containing Hydrocarbons in Petroleum Substances

T. Parkerton, ExxonMobil Biomedical Sciences Inc. / Toxicology & Environmental Science; A. Bleich, ExxonMobil Biomedical Sciences Inc; J. Butler, ExxonMobil Biomedical Sciences, Inc / Environmental Toxicology and Chemistry Laboratory; C.A. Sutherland, ExxonMobil Biomedical Sciences, Inc. / Toxicology and Environmental Science; A.D. Redman, Exxon Mobil Biomedical Sciences / Toxicology and Environment Science Division; M. Lampi, ExxonMobil Biomedical Sciences, Inc.

A recent analytical effort to characterize the composition of petroleum substances from different categories indicated that a variety of sulfur and nitrogen heterocyclic compounds were detected in the range of 0.01-3.24% and < 0.01-0.08%, respectively. Given the limited information available on the bioaccumulation potential of these substance classes, a dietary bioaccumulation study with rainbow trout was performed. Representative compounds with log KOW values > 4.2 from five classes (sulfides, thiols, thiophenes, carbazoles and acridines) were investigated along with a positive control (hexachlorobenzene). Test compounds were administered simultaneously in the diet at a 2% body weight ration per day to minimize growth dilution and limit vertebrate use. Chemical and lipid analysis were performed on diet and fish tissues at different sampling times. Results were used to calculate substance-specific lipid-normalized biomagnification factors, dietary assimilation efficiencies and growth-corrected eliminated rates. Comparison of experimental results to model predictions for non-metabolizable chemicals was used to infer the role of tissue and gut biotransformation in mitigating observed bioaccumulation. This study provides new data to inform bioaccumulation assessments of heterocyclic compounds and to support development of quantitative structure-property relationships for improved bioaccumulation prediction of sulfur and nitrogen containing compounds.

MO007

Biochemical biomarkers and histopathology in juvenile Solea senegalensis for early warning assessment of marine ecosystem health

T. Briaudeau, University of the Basque country UPVEHU; A. Alves Dos Santos, University of the Basque country UPVEHU / CBET Research Group Dept Zoology and Animal Cell Biology Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE; G. Guerrero Limón, University of the Basque country UPVEHU; I. Marigomez, Euskal Herriko Unibertsitatea / Zoology & Animal Cell Biology (Sci & Tech Fac); I. Zorita, FUNDACION AZTI; M. Soto, University of the Basque Country / Zoology and Animal Cell Biology, Research Centre for Experimental Marine Biology and Biotechnology PIEUPVEHU; U. Izagirre, University of the Basque Country UPV/EHU / CBET Research Group Dept Zoology and Animal Cell Biology Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE

Human originated contaminants can appear diluted in estuarine and marine waters or accumulate in sediments. Chemical analysis provides key data on toxicant levels but gives limited inputs on their potential biological effects. The combination of biological responses with chemical data is essential to improve the assessment of environmental pollution. In this context, the use of benthic species for the assessment of biological effects of marine pollution is crucial for marine environment monitoring. In the Bay of Biscay, the common flatfish Solea sp., is candidate to be recognized as sentinel species in pollution monitoring programmes. The present study uses juvenile Solea sp. (23.24± 1.22cm standard length) exposed to contamination conditions to better understand toxicity processes involved based on biochemical biomarkers and histopathology. Solea senegalensis was exposed to three different experimental set ups: (a) contaminated sediments; waterborne metal (Cd) and (c) waterborne organic pollutant (Benzo(α)pyrene). A battery of biochemical biomarkers was analysed in samples of liver and brain: Catalase, glutathione S-transferase, acetylcholinesterase and superoxide dismutase. Exposure to contaminated sediments led to reduction of catalase, glutathione S-transferase and superoxide dismutase activities and induction of acetylcholinesterase activities. Exposure to waterborne toxicants provoked a reduction of catalase and glutathione S-transferase activities. Biochemical biomarkers in sole were sensitive enough to differentiate degree of response after three days of exposure. Histopathological responses were detected after long-term exposures showing higher prevalence of liver alterations such as hyperaemia, melanomacrophage centres and necrosis. The present laboratory experiments helped characterizing the impact of pollution in sole at different levels of biological organization and different time scales. Work funded by Spanish MINECO (CTM2012-40203-C02-01 and PhD fellowship to T.B.), University of the Basque Country– UPV/EHU (UFI 11/37) and Basque Government through Consolidated Research Groups fellowship (IT810-B).

MO008

BIOMARKER AND GENE TRANSCRIPTION VARIABILITY IN PERCH IN REFERENCE SITES USED FOR BIOMONITORING STUDIES

L. Förlin, N. Asker, University of Gothenburg / Department of Biological and Environmental Sciences; M. Töpel, University of Gothenburg / Department of Marine Sciences; T. Österlund, Chalmers University of Technology / Mathematical Sciences; J. Parkkonen, J. Sturve, University of Gothenburg / Department of Biological and Environmental Sciences

Perch (Perca fluviatilis) has been used in biological effect monitoring of point sources in Sweden for many years, for example in studies of effects of industrial effluents. Since 1988, perch has also annually been included in a program for integrated coastal fish monitoring in three reference sites along the Swedish east coast, sites characterized by no or minor local anthropogenic influences. Long term studies of reference sites offer the possibility to follow the natural variability of physiological and biochemical endpoint (i.e. biomarkers) as this defines the changes of relevance in polluted sites. Using a set of physiological and biochemical endpoints (i.e. biomarkers) clear time trends for “early warning” signs of impaired health are noted in the perch from these three reference sites possibly as a result of increased baseline pollution. The data sets also show relatively large variations between years. To further investigate these time trends and to identify additional temporal variation in biological parameters, global gene transcription studies using RNA sequencing was performed. Perch collected in 2010 and 2014 were selected as they showed variation in several biomarkers such as the activity of the

detoxification enzyme CYP1A (EROD), plasma levels of vitellogenin, markers for oxidative stress, white blood cells count and gonad sizes. The RNA sequencing study identified approximately 3800 genes that were differentially expressed in the five sexually mature female perch collected in 2010 compared to the five individuals from 2014. Also principal component analysis (PCA) using all sequenced transcripts identified large differences in gene transcription as individual perch collected during the separate time points were clearly divided into two groups. Gene Ontology enrichment analysis showed that the differentially expressed genes were involved in biological processes such as innate immune

responses, response to toxic substance, response to hypoxia and cholesterol biosynthetic process. In conclusion, differences in immune system parameters and

responses to exposure of toxic substances have now been verified on two different biological levels (mRNA and protein) in perch collected in 2010 as well as 2014. Additional biological processes having temporal variation have been identified compared to the previous measurements of biomarkers.

MO009

Cellular and tissue-level biomarkers in mussels (Mytilus edulis) sampled in two different study areas in the Northern Atlantic

D.B. Fernández, ESTACIÓN MARINA DE PLENTZIA. UPV/EHU / Departamento de Zoología y Biología Celular Animal; U. Izagirre, University of the Basque country UPVEHU / CBET Research Group Dept Zoology and Animal Cell Biology Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE; X. Lekube, PiEUniversity of the Basque Country; I. Marigomez, Euskal Herriko Unibertsitatea / Zoology & Animal Cell Biology (Sci & Tech Fac); B. Zaldibar, University of the Basque Country UPV/EHU / Department of Zoology and Animal Cell Biology. Centre of Experimental Marine Biology & Biotechnology; M. Soto, University of the Basque Country / Zoology and Animal Cell Biology, Research Centre for Experimental

Marine Biology and Biotechnology PIEUPVEHU

Biomarker approach has been widely used in mussel monitoring programs for several years. However, up to know it has not been commonly used in high latitude study areas. In order to establish reference values of cellular and tissue-level biomarkers in the Northern Atlantic Ocean, mussels of two sizes (small, 2-3 cm; large, 3.5-4.5 cm) from selected polluted (commercial harbor & ports, WWTP dumping area) and reference sites in Tromsø (69º 40´N) and Trondheim (63º 26´N) were sampled in early autumn of 2016 and late summer 2017. Different tissue-level biomarkers including cell type composition (VvBAS) in digestive gland

epithelium, structural changes of digestive alveoli (MLR/MET), relative proportion of digestive and connective tissue (CTD) and histopathological alterations in the digestive gland were measured. In addition, lipofuscin and neutral lipid accumulation, lysosomal membrane stability (LMS) and structural changes in the endo-lysosomal system (LSC) of digestive cells were also determined. Higher VvBAS values were recorded in polluted sites than in mussels from reference sites in both study areas. Moreover, mussels from impacted sites exhibited enhanced atrophy of the digestive alveoli (high MLR/MET values) and retraction of digestive diverticula resulting in apparently higher relative extent of interstitial connective tissue (high CTD ratio). Regarding inflammatory responses, parasitic burden and atresia, higher weighed prevalence values than in the reference site were recorded in the two polluted sites from Trondheim. Differences between the two mussel sizes were recorded in parasitic burden, large mussels exhibiting a higher level of parasitization than small mussels. Lipofuscin accumulation was higher and neutral lipid accumulation lower in the polluted sites than in reference sites in both study areas. Lysosomal biomarkers were different between the two sizes. Overall, all biomarkers respond similarly in both study areas indicating the suitability of the selected biomarkers in order to be applied in the Northern Atlantic

Ocean.Acknowledgements: Work funded by, EU GRACE Project (Grant Agreement Number 679266), Basque Government (IT810-13) and UPV/EHU (UFI 11/37).

MO010

Cytotoxicity of the WAF of naphthenic North Sea crude oil with and without dispersant in hemocytes of the marine mussel Mytilus galloprovincialis (L.)

G. Nicolussi, University of the Basque Country / CBET Research Group, Dept. Zoology and Animal Cell Biology, Science and Technology Faculty and Plentzia Marine Station, University of the Basque Country (UPV/EHU). Basque Country, Spain; A. Katsumiti, University of the Basque Country / CBET Research Group, Dept. Zoology and Animal Cell Biology Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE; D. Bilbao, University of the Basque country (UPV/EHU) / IBeA Research Group, Dept. Analytical Chemistry; N. Etxebarria, University of the Basque Country UPV/EHU / Plentzia Marine Station (PiE-UPV/EHU) & Dep Analytical Chemistry; M.P. Cajaraville, University of the Basque Country / CBET Research Group, Dept. Zoology and Animal Cell Biology Faculty of Science and Technology and Research Centre for Experimental Marine Biology and Biotechnology PIE Oil pollution coming from accidental oil spills and from activities related to oil production and transport pose a risk to marine ecosystems. Impact of oil spills may vary depending on environmental parameters such as temperature. There is a need to develop efficient tools to assess the risks of oil pollution and of oil spill response strategies, such as the use of dispersants. The aim of this work was to apply an in

vitro approach using hemocytes of the marine mussel Mytilus galloprovincialis as a

model to evaluate the toxicity of the water accommodated fraction (WAF) of naphthenic North Sea crude oil produced at different temperatures (10, 15 and 20°C) with and without the dispersant Finasol OSR 52. In order to evaluate the contribution of the dispersant on the toxicity of the WAF plus dispersant, the cytotoxicity of the dispersant alone was also tested. Primary cultures of hemocytes were exposed in glass covered microplates to different dilutions of WAF (0.25, 2.5, 25, 50 and 100%) with and without the dispersant and to the dispersant alone at the same concentrations present in the WAF dilutions of oil plus dispersant (1.25, 12.5, 125, 250 and 500 mg/L). After 24 h exposure, cytotoxicity (MTT test) and ROS production were measured. WAF was moderately cytotoxic to mussel hemocytes. WAF produced at different temperatures showed similar cytotoxicity to hemocytes. A slight but significant decrease in cell viability occurred at 25, 50 and 100% WAF (produced at 10 and 15°C) and at 50 and 100% WAF (produced at 20°C). These results suggest that relevance of temperature of WAF production on its cytotoxicity is limited. WAF caused a significant induction of ROS production in hemocytes, indicating occurrence of oxidative stress. When tested alone, the dispersant caused a slight but significant decrease in cell viability at the two highest concentrations. However, WAF produced with dispersant at the three different temperatures was not toxic to hemocytes. These results appear to indicate that the dispersant efficiently reduced the toxicity of the crude oil WAF in the selected cell model. Overall, the in vitro toxicity testing approach in mussel hemocytes could be used as a rapid screening tool for environmental risk assessment of oil spills and oil response strategies in the marine environment. *Funded by EU H2020 GRACE project (679266), Spanish MINECO NACE project (CTM2016-81130-R), Basque Government (consolidated research group IT810-13) and UPV/EHU (UFI 11/37).

MO011

Determination of inorganic cations and amines in wastewater, surface water,

and neutralizing amine solutions by IC coupled with a single quadrupole MS

T. Christison, Thermo Fisher Scientific / Strategic Ion Chromatography Applications; G. Ellison, ThermoFisher Scientific / Chromatography and mass spectrometry division; T. Cross, Thermo Fisher Scientific; J. Rohrer, Thermo Fisher Scientific / Chromatography and mass spectrometry division

Inorganic cation and amine determinations are important to assess salt build-up in amine neutralizing solutions, or to meet regulatory discharge compliance from petroleum and municipal treatment plants wastewater. Additionally, municipal water plants require cation determinations to monitor secondary water characteristics. In the petroleum industry, alkanolamines (monoethanolamine, diethanolamine, and methyldiethanolamine) are used routinely to prevent corrosion during transportation to the refinery or to remove sour gases during the refining process. Processing plants require accurate analytical methods to characterize and determine the next refining steps needed for oil and gas products received from various oil, gas, and fracking wells and to meet wastewater discharge requirements. Ion chromatography coupled to a single quadrupole mass spectrometer (IC-MS) is an ideal and economical way to determine and confirm cations and amines. Here we demonstrate cation, alkylamine, and alkanolamine determinations in amine neutralizing solutions, amine wastewater, municipal wastewater samples, drinking, and surface water samples by cation-exchange separation followed by suppressed conductivity and mass spectrometry detections in a serial configuration. Cations, alkylamines, and alkanolamines were determined in full scan from m/z 18 to 250 and individual SIMs as bare ions and when further sensitivity is needed, as their hydrated adducts. Unlike earlier IC-MS methods for cation determinations, the new single quadrupole MS used in these experimentsrequired no organic solvent for desolvation. Limit of Detections were single digit or double digit µg/L for most analytes. The experiments showed that typically sodium, ammonium, and primary amines were the primary contaminants in the scrubbing amines.

MO012

Distribution and ecological risk assessment of palm stearin in coastal marine environments of Hong Kong after an accidental pollution in Pearl River Estuary, South China

G. Zhou, R.W. Lai, R.C. Sham, C. Lam, The University of Hong Kong / The Swire Institute of Marine Science and School of Biological Sciences; K. Yeung, J.C. Astudillo, The University of Hong Kong; K.K. Ho, The University of Hong Kong / The Swire Institute of Marine Science and School of Biological Sciences; M.M. Yung, J.K. Yau, The University of Hong Kong; K.M. Leung, The University of Hong Kong / The Swire Institute of Marine Science

On 3 August 2017, two container vessels collided in the Pearl River Estuary, southwest of Hong Kong, leading to release over 1,000 tonnes of palm stearin into adjacent waters. About 200 tonnes of the stearin reached south coasts of Hong Kong after two days. However, toxicity of palm stearin to marine organisms is unknown. This study, therefore, aimed to examine its contamination levels in seawater, sediment and animal samples collected from seven locations along the south coasts of Hong Kong; determine its toxicities to selected marine organisms including microalgae (Isochrysis galbana and Chaetoceros gracilis), the copepod (Tigriopus

japonicus), the artemia (Artemia franciscanas), and the fish (Oryzias melastigma),

and derive interim water quality guidelines (WQG) of the palm stearin and thereby assess its ecological risks to local marine ecosystems. Samples of the palm stearin, surface seawater, sediment and three intertidal gastropods were collected twice (within seven days and four months after the incident) in six locations along the south coast of Hong Kong. Fatty acids in these samples were detected using gas chromatography-mass spectrometry. Standard toxicity tests were conducted with the selected species. The results showed that all seawater samples collected from the six sites were heavily contaminated by the palm stearin after one week of the accidental pollution. We also found that although the palm stearin had little effects on marine animals, it was toxic to microalgae species as reflected by growth inhibition. Its toxic mechanisms on the microalgae may be associated with its adsorption onto microalgal cells, and reudction of light prenetration to the cells due to obstruction from the stearin and palm oil. At present, we are generating additional toxicity data on other microalgal species (Thalassiosira weissflogii and

Tetraselmis suecica). Using all toxicity data generated from this study, we will

ultimately determine an interim WQG for the palm stearin, and use this WQG to assess its ecological risks to local marine ecosystems. This study represents the first comprehensive investigation on the ecological risk of the palm stearin in the world, and the results will facilitate informed decision-making by the environmental authority.

MO013

Ecological impacts of larvicidal oil on the marine ecosystem: implications on its management

K. Yeung, The University of Hong Kong; K.M. Leung, The University of Hong Kong / The Swire Institute of Marine Science

Mosquitoes are some of the most influential insects to mankind in the world due to their ability to transmit diseases to humans, resulting in millions of deaths every year. Prevention of mosquito-borne diseases and elimination of mosquitoes are important to protect human health. Among different methods of eliminating