#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           	-	bivalves_17C_C0_05	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_005.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_06	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_006.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_07	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_007.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_08	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_008.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_09	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_010.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_10	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_011.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C0_11	Factor:bivalves_17C_C0	RAW_FILE_NAME=bivalves_012.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_01	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_013.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_02	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_014.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_03	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_015.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_04	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_016.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_05	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_017.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_06	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_018.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_07	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_019.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_08	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_020.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_09	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_021.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_10	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_022.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C5_11	Factor:bivalves_17C_C5	RAW_FILE_NAME=bivalves_024.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_01	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_025.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_02	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_026.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_03	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_027.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_04	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_028.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_05	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_029.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_06	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_030.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_07	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_031.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_08	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_032.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_09	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_033.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_10	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_034.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_11	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_035.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C25_12	Factor:bivalves_17C_C25	RAW_FILE_NAME=bivalves_036.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_01	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_037.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_02	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_038.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_03	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_039.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_04	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_040.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_05	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_041.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_06	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_042.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_07	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_043.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_08	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_044.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_09	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_045.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_10	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_046.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C125_11	Factor:bivalves_17C_C125	RAW_FILE_NAME=bivalves_047.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_01	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_049.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_02	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_050.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_03	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_051.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_04	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_052.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_05	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_053.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_06	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_054.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_07	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_055.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_08	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_056.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_09	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_057.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_10	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_058.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_17C_C625_11	Factor:bivalves_17C_C625	RAW_FILE_NAME=bivalves_059.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_01	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_061.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_02	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_062.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_03	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_063.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_04	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_064.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_05	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_065.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_06	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_066.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_07	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_067.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_08	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_069.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_09	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_070.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C0_10	Factor:bivalves_21C_C0	RAW_FILE_NAME=bivalves_071.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_01	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_073.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_02	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_074.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_03	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_075.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_04	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_077.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_05	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_078.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_06	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_079.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_07	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_080.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_08	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_081.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_09	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_083.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C5_10	Factor:bivalves_21C_C5	RAW_FILE_NAME=bivalves_084.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_01	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_085.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_02	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_086.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_03	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_087.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_04	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_088.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_05	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_089.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_06	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_093.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_07	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_094.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_08	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_095.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C25_09	Factor:bivalves_21C_C25	RAW_FILE_NAME=bivalves_096.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_01	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_097.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_02	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_098.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_03	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_099.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_04	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_100.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_05	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_101.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_06	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_102.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_07	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_105.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_08	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_107.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C125_09	Factor:bivalves_21C_C125	RAW_FILE_NAME=bivalves_108.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C625_01	Factor:bivalves_21C_C625	RAW_FILE_NAME=bivalves_109.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C625_02	Factor:bivalves_21C_C625	RAW_FILE_NAME=bivalves_110.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C625_03	Factor:bivalves_21C_C625	RAW_FILE_NAME=bivalves_111.raw
SUBJECT_SAMPLE_FACTORS           	-	bivalves_21C_C625_04	Factor:bivalves_21C_C625	RAW_FILE_NAME=bivalves_112.raw
SUBJECT_SAMPLE_FACTORS           	-	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