#METABOLOMICS WORKBENCH jvaorodrigues1980_20230126_075438 DATATRACK_ID:3706 STUDY_ID:ST002456 ANALYSIS_ID:AN004006 PROJECT_ID:PR001584 VERSION 1 CREATED_ON January 27, 2023, 1:12 am #PROJECT PR:PROJECT_TITLE 1H NMR metabolomics applied to assess the metabolic response of Ruditapes PR:PROJECT_TITLE philippinarum clams to sea warming and 17-α-ethinylestradiol exposure PR:PROJECT_TYPE 1H NMR metabolomics to study the effects of warming conditions and exposure to PR:PROJECT_TYPE 17-α-ethinylestradiol (EE2) on the polar metabolome of Ruditapes philippinarum PR:PROJECT_TYPE clams PR:PROJECT_SUMMARY Hormones correspond to one of the most important classes of PhACs and are PR:PROJECT_SUMMARY usually classified as endocrine-disrupting chemicals (EDCs), due to their PR:PROJECT_SUMMARY ability to alter endocrine system functions, resulting in known adverse health PR:PROJECT_SUMMARY effects on non-target organisms. Among the known EDCs, 17-α-ethinylestradiol PR:PROJECT_SUMMARY (EE2) is characterized by high estrogenic potency, chemical stability, and a PR:PROJECT_SUMMARY tendency to accumulate in aquatic biota. This hormone reaches the environment PR:PROJECT_SUMMARY mainly through human and animal excretion, with conventional WWTP processes not PR:PROJECT_SUMMARY fully managing its removal from treated effluents. Bivalves are excellent model PR:PROJECT_SUMMARY organisms to assess the toxicity of several environmental stressors. Alongside PR:PROJECT_SUMMARY the impacts of pollutants, organisms in coastal ecosystems are also subjected to PR:PROJECT_SUMMARY climate change-related factors, such as temperature rise. Water temperature may PR:PROJECT_SUMMARY increase up to 4 °C by 2100 and this is expected to severely impact several PR:PROJECT_SUMMARY aspects of marine organisms’ biology. In addition, these environmental changes PR:PROJECT_SUMMARY are also likely to affect the sensitivity of organisms to pollutants and, hence, PR:PROJECT_SUMMARY pollutants toxicity. In this study, a comprehensive untargeted 1H NMR PR:PROJECT_SUMMARY metabolomics strategy was applied to measure the metabolic impact of sea PR:PROJECT_SUMMARY warming, in tandem with exposure to EE2, on Ruditapes philippinarum clams. The PR:PROJECT_SUMMARY clams were exposed to different EE2 concentrations, either at 17 °C as control PR:PROJECT_SUMMARY temperature or at 21 °C (representing a 4 °C increase, which corresponds to PR:PROJECT_SUMMARY the worst-case warming scenario). The obtained data added important knowledge, PR:PROJECT_SUMMARY unveiling individual metabolic effects of temperature rise and synergetic PR:PROJECT_SUMMARY effects upon EE2 exposure, and paving the way for the definition of new PR:PROJECT_SUMMARY metabolic markers for the monitoring of environmental stressors. PR:INSTITUTE University of Aveiro PR:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry PR:LABORATORY Metabolomics group PR:LAST_NAME Gil PR:FIRST_NAME Ana M. PR:ADDRESS University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, PR:ADDRESS Portugal PR:EMAIL agil@ua.pt PR:PHONE +351234370707 PR:FUNDING_SOURCE This work was developed within the CICECO-Aveiro Institute of Materials project PR:FUNDING_SOURCE (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) financed by national funds PR:FUNDING_SOURCE through the FCT/MEC (PIDDAC). We are also grateful to the Portuguese National PR:FUNDING_SOURCE NMR Network (PTNMR), supported by FCT funds as the NMR spectrometer used is part PR:FUNDING_SOURCE of PTNMR and partially supported by Infrastructure Project No. 022161 PR:FUNDING_SOURCE (co-financed by FEDER through COMPETE 2020, POCI and PORL, and the FCT through PR:FUNDING_SOURCE PIDDAC). This work was also financially supported by the project BISPECIAl: PR:FUNDING_SOURCE BIvalveS under Polluted Environment and ClImate chAnge (POCI-01-0145-FEDER- PR:FUNDING_SOURCE 028425) funded by FEDER, through COMPETE2020 - Programa Operacional PR:FUNDING_SOURCE Competitividade e Internacionalização (POCI), and by national funds (OE), PR:FUNDING_SOURCE through FCT/MCTES. Mónica G. Silva benefited from Research Grant (MSc) PR:FUNDING_SOURCE (BI/CESAM/0043_2019/POCI-01-0145-FEDER-028425) under the project BISPECIAl: PR:FUNDING_SOURCE BIvalveS under Polluted Environment and ClImate PR:FUNDING_SOURCE change PTDC/CTA-AMB/28425/2017 (POCI-01-0145-FEDER-028425). #STUDY ST:STUDY_TITLE 1H NMR metabolomics applied to assess the metabolic response of Ruditapes ST:STUDY_TITLE philippinarum clams to sea warming and 17-α-ethinylestradiol exposure ST:STUDY_TYPE 1H NMR metabolomics to study the effects of warming conditions and exposure to ST:STUDY_TYPE 17-α-ethinylestradiol (EE2) on the polar metabolome of Ruditapes philippinarum ST:STUDY_TYPE clams ST:STUDY_SUMMARY In this study, a comprehensive untargeted 1H NMR metabolomics strategy was ST:STUDY_SUMMARY applied to measure the metabolic impact of sea warming, in tandem with exposure ST:STUDY_SUMMARY to EE2, on Ruditapes philippinarum clams. The clams were exposed to five ST:STUDY_SUMMARY different EE2 concentrations: 0 (control group), 5, 25, 125 and 625 ng/L; either ST:STUDY_SUMMARY at 17 °C as control temperature or at 21 °C (representing a 4 °C increase, ST:STUDY_SUMMARY which corresponds to the worst-case warming scenario). The obtained data added ST:STUDY_SUMMARY important knowledge, unveiling individual metabolic effects of temperature rise ST:STUDY_SUMMARY and synergetic effects upon EE2 exposure, and paving the way for the definition ST:STUDY_SUMMARY of new metabolic markers for the monitoring of environmental stressors. ST:INSTITUTE University of Aveiro ST:DEPARTMENT CICECO – Aveiro Institute of Materials, Department of Chemistry ST:LABORATORY Metabolomics Group- CICECO ST:LAST_NAME Rodrigues ST:FIRST_NAME Joao A. ST:ADDRESS University of Aveiro, Campus Universitario de Santiago, 3810-193 Aveiro, ST:ADDRESS Portugal ST:EMAIL joao.e.a.rodrigues@gmail.com ST:PHONE 00351963481841 ST:NUM_GROUPS 10 ST:TOTAL_SUBJECTS 103 ST:STUDY_COMMENTS This work was developed within the CICECO-Aveiro Institute of Materials project ST:STUDY_COMMENTS (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) financed by national funds ST:STUDY_COMMENTS through the FCT/MEC (PIDDAC). We are also grateful to the Portuguese National ST:STUDY_COMMENTS NMR Network (PTNMR), supported by FCT funds as the NMR spectrometer used is part ST:STUDY_COMMENTS of PTNMR and partially supported by Infrastructure Project No. 022161 ST:STUDY_COMMENTS (co-financed by FEDER through COMPETE 2020, POCI and PORL, and the FCT through ST:STUDY_COMMENTS PIDDAC). This work was also financially supported by the project BISPECIAl: ST:STUDY_COMMENTS BIvalveS under Polluted Environment and ClImate chAnge (POCI-01-0145-FEDER- ST:STUDY_COMMENTS 028425) funded by FEDER, through COMPETE2020 - Programa Operacional ST:STUDY_COMMENTS Competitividade e Internacionalização (POCI), and by national funds (OE), ST:STUDY_COMMENTS through FCT/MCTES. Mónica G. Silva benefited from Research Grant (MSc) ST:STUDY_COMMENTS (BI/CESAM/0043_2019/POCI-01-0145-FEDER-028425) under the project BISPECIAl: ST:STUDY_COMMENTS BIvalveS under Polluted Environment and ClImate ST:STUDY_COMMENTS change PTDC/CTA-AMB/28425/2017 (POCI-01-0145-FEDER-028425). #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Ruditapes philippinarum SU:TAXONOMY_ID 129788 SU:HEIGHT_OR_HEIGHT_RANGE length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - bivalves_17C_C0_01 Factor:bivalves_17C_C0 RAW_FILE_NAME=bivalves_001.raw SUBJECT_SAMPLE_FACTORS - bivalves_17C_C0_02 Factor:bivalves_17C_C0 RAW_FILE_NAME=bivalves_002.raw SUBJECT_SAMPLE_FACTORS - bivalves_17C_C0_03 Factor:bivalves_17C_C0 RAW_FILE_NAME=bivalves_003.raw SUBJECT_SAMPLE_FACTORS - bivalves_17C_C0_04 Factor:bivalves_17C_C0 RAW_FILE_NAME=bivalves_004.raw SUBJECT_SAMPLE_FACTORS - 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bivalves_21C_C625_05 Factor:bivalves_21C_C625 RAW_FILE_NAME=bivalves_113.raw SUBJECT_SAMPLE_FACTORS - bivalves_21C_C625_06 Factor:bivalves_21C_C625 RAW_FILE_NAME=bivalves_114.raw SUBJECT_SAMPLE_FACTORS - bivalves_21C_C625_07 Factor:bivalves_21C_C625 RAW_FILE_NAME=bivalves_115.raw SUBJECT_SAMPLE_FACTORS - bivalves_21C_C625_08 Factor:bivalves_21C_C625 RAW_FILE_NAME=bivalves_117.raw SUBJECT_SAMPLE_FACTORS - bivalves_21C_C625_09 Factor:bivalves_21C_C625 RAW_FILE_NAME=bivalves_119.raw #COLLECTION CO:COLLECTION_SUMMARY Ruditapes philippinarum clams were collected at the Ria de Aveiro, a shallow CO:COLLECTION_SUMMARY coastal system located on the Northwest Atlantic coast of Portugal. Individuals CO:COLLECTION_SUMMARY of similar size (length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm) were CO:COLLECTION_SUMMARY selected. For depuration and acclimation to laboratory conditions, all clams CO:COLLECTION_SUMMARY were maintained in artificial seawater for 10 days (salinity: 30 ± 1, Tropic CO:COLLECTION_SUMMARY Marin® SEA SALT, from Tropic Marine Center), under continuous aeration, CO:COLLECTION_SUMMARY constant temperature (17 ± 1 ºC) and a natural photoperiod. Artificial CO:COLLECTION_SUMMARY seawater was renewed every 2-3 days and clams were fed every 2-3 days with CO:COLLECTION_SUMMARY Algamac Protein Plus (150,000 cells/animal/day) after the 3rd day. After CO:COLLECTION_SUMMARY depuration, the organisms were subjected to a chronic toxicity test for 28 days, CO:COLLECTION_SUMMARY consisting of exposure to five different EE2 concentrations (Sigma-Aldrich, CO:COLLECTION_SUMMARY purity ≥ 98%, MW = 296.40 g/mol, 1 mg/L stock solution in ultrapure water): 0 CO:COLLECTION_SUMMARY (control group), 5, 25, 125 and 625 ng/L. To assess the effects of a warming CO:COLLECTION_SUMMARY scenario on the impacts of EE2, the experiments were carried out at 17 ± 1 °C CO:COLLECTION_SUMMARY (control; mean temperature of sampling area during September: 16 - 19 °C) and CO:COLLECTION_SUMMARY at 21 ± 1 °C (worst-case climate change scenario, IPCC, 2021). The aquaria CO:COLLECTION_SUMMARY were placed in distinct climatic rooms for each temperature. To reach 21 °C, CO:COLLECTION_SUMMARY the temperature was raised by 2 °C, every 2–3 days. For each concentration CO:COLLECTION_SUMMARY level and temperature, 12 samples were considered: 4 individuals per aquarium CO:COLLECTION_SUMMARY and 3 aquaria per treatment. In each aquarium, a total of 3 L of artificial CO:COLLECTION_SUMMARY seawater (salinity: 30 ± 1), continuous aeration, and a natural photoperiod CO:COLLECTION_SUMMARY were used. The exposure medium for each condition was renewed weekly, after CO:COLLECTION_SUMMARY which EE2 concentration levels were re-established. Mortality was checked daily CO:COLLECTION_SUMMARY and found to be null. At the end of the 28-day exposure period, the clams were CO:COLLECTION_SUMMARY frozen in liquid nitrogen and stored at – 80 ºC. CO:COLLECTION_PROTOCOL_FILENAME Bivalves Experimental Procedure CO:SAMPLE_TYPE Tissue CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Sea warming and 17-α-ethinylestradiol exposure. After depuration, the organisms TR:TREATMENT_SUMMARY were subjected to a chronic toxicity test for 28 days, consisting of exposure to TR:TREATMENT_SUMMARY five different EE2 concentrations: 0 (control group), 5, 25, 125 and 625 ng/L. TR:TREATMENT_SUMMARY To assess the effects of a warming scenario on the impacts of EE2, the TR:TREATMENT_SUMMARY experiments were carried out at 17 ± 1 °C (control; mean temperature of TR:TREATMENT_SUMMARY sampling area during September) and at 21 ± 1 °C (worst-case climate change TR:TREATMENT_SUMMARY scenario). The aquaria were placed in distinct climatic rooms for each TR:TREATMENT_SUMMARY temperature. To reach 21 °C, the temperature was raised by 2 °C, every 2–3 TR:TREATMENT_SUMMARY days. For each concentration level and temperature, 12 samples were considered: TR:TREATMENT_SUMMARY 4 individuals per aquarium and 3 aquaria per treatment. In each aquarium, a TR:TREATMENT_SUMMARY total of 3 L of artificial seawater (salinity: 30 ± 1), continuous aeration, TR:TREATMENT_SUMMARY and a natural photoperiod were used. The exposure medium for each condition was TR:TREATMENT_SUMMARY renewed weekly, after which EE2 concentration levels were re-established. TR:TREATMENT_SUMMARY Mortality was checked daily and found to be null. At the end of the 28-day TR:TREATMENT_SUMMARY exposure period, the clams were frozen in liquid nitrogen and stored at – 80 TR:TREATMENT_SUMMARY ºC. TR:TREATMENT_PROTOCOL_FILENAME Bivalves Experimental Procedure TR:TREATMENT_COMPOUND Sea warming and 17-α-ethinylestradiol exposure TR:TREATMENT_DOSE Clams were exposed to five different EE2 concentrations: 0 (control group), 5, TR:TREATMENT_DOSE 25, 125 and 625 ng/L. To assess the effects of a warming scenario on the impacts TR:TREATMENT_DOSE of EE2, the experiments were carried out at 17 ± 1 °C (control) and at 21 ± 1 TR:TREATMENT_DOSE °C (worst-case climate change scenario). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite extraction was performed using a water/methanol/chloroform method, as SP:SAMPLEPREP_SUMMARY described in (Hines, Oladiran, Bignell et al., 2007). Briefly, the clams´ soft SP:SAMPLEPREP_SUMMARY tissue (0.15 g per sample) was ground with a pestle and mortar, in liquid SP:SAMPLEPREP_SUMMARY nitrogen, and then transferred to a microtube, followed by the addition of cold SP:SAMPLEPREP_SUMMARY methanol (600 µL), ultrapure water (128 µL) and chloroform (300 µL). The SP:SAMPLEPREP_SUMMARY mixture was vortexed, left in ice for 10 min and centrifuged (2,500 g, 4 °C, 10 SP:SAMPLEPREP_SUMMARY min). The top layer was transferred into a microtube to which chloroform (300 SP:SAMPLEPREP_SUMMARY µL) and water (300 µL) were added. The mixture was vortexed and centrifuged SP:SAMPLEPREP_SUMMARY (2,500 g, 4 °C, 10 min). The upper layer (aqueous) was transferred into vials, SP:SAMPLEPREP_SUMMARY dried in a centrifugal vacuum concentrator (UNIVAP 100H) and stored at −80 °C SP:SAMPLEPREP_SUMMARY until NMR analysis. SP:SAMPLEPREP_PROTOCOL_FILENAME Bivalves Experimental Procedure SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et SP:EXTRACTION_METHOD al., 2007) SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION The dried polar extracts of clam samples were resuspended in 600 μL of sodium SP:SAMPLE_RESUSPENSION phosphate buffer (0.1 M in D2O, 99.96% D, pH 7.4, containing 0.5 mM sodium salt SP:SAMPLE_RESUSPENSION of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, TSP-d4, chemical shift SP:SAMPLE_RESUSPENSION referencing). The mixture was vortexed and centrifuged (16,000 g, 10 min, room SP:SAMPLE_RESUSPENSION temperature) and 550 μL were transferred into 5 mm NMR tubes. SP:SAMPLE_SPIKING 0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4), as a SP:SAMPLE_SPIKING chemical shift reference. #ANALYSIS AN:ANALYSIS_TYPE NMR AN:LABORATORY_NAME Metabolomics group AN:OPERATOR_NAME Joao A. Rodrigues AN:DETECTOR_TYPE Bruker Avance III 500 MHz spectrometer AN:SOFTWARE_VERSION TopSpin 3.2 and Amix 3.9.14 AN:ANALYSIS_PROTOCOL_FILE Bivalves Experimental Procedure AN:DATA_FORMAT fid, 1r #NMR NM:INSTRUMENT_NAME Bruker AVANCE III 500 spectrometer NM:INSTRUMENT_TYPE FT-NMR NM:NMR_EXPERIMENT_TYPE 1D-1H NM:SPECTROMETER_FREQUENCY 500 MHz NM:NMR_PROBE TXI probe NM:NMR_SOLVENT D2O NM:NMR_TUBE_SIZE 5 mm NMR tubes NM:SHIMMING_METHOD Topshim NM:PULSE_SEQUENCE noesypr1d NM:WATER_SUPPRESSION presat NM:PULSE_WIDTH 90-degree NM:RECEIVER_GAIN 203 NM:TEMPERATURE 298 K NM:NUMBER_OF_SCANS 256 NM:DUMMY_SCANS 8 NM:ACQUISITION_TIME 2.34 s NM:RELAXATION_DELAY 3 s NM:SPECTRAL_WIDTH 7,002.8 Hz NM:NUM_DATA_POINTS_ACQUIRED 32 k NM:LINE_BROADENING 0.3 Hz NM:ZERO_FILLING 64 k NM:BASELINE_CORRECTION_METHOD manual NM:CHEMICAL_SHIFT_REF_STD 0 ppm for TSP-d4 NM:NMR_RESULTS_FILE 4._Bivalves_results_data.txt UNITS:ppm #END