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MB Sample ID: SA245803
| Local Sample ID: | bivalves_21C_C5_02 |
| Subject ID: | SU002545 |
| Subject Type: | Invertebrate |
| Subject Species: | Ruditapes philippinarum |
| Taxonomy ID: | 129788 |
| Height Or Height Range: | length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm |
| Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002545 |
| Subject Type: | Invertebrate |
| Subject Species: | Ruditapes philippinarum |
| Taxonomy ID: | 129788 |
| Height Or Height Range: | length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm |
| Gender: | Male and female |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| bivalves_21C_C5_02 | SA245803 | FL030860 | bivalves_21C_C5 | Factor |
Collection:
| Collection ID: | CO002538 |
| Collection Summary: | Ruditapes philippinarum clams were collected at the Ria de Aveiro, a shallow coastal system located on the Northwest Atlantic coast of Portugal. Individuals of similar size (length: 3.81 ± 0.42 cm; and width: 3.06 ± 0.51 cm) were selected. For depuration and acclimation to laboratory conditions, all clams were maintained in artificial seawater for 10 days (salinity: 30 ± 1, Tropic Marin® SEA SALT, from Tropic Marine Center), under continuous aeration, constant temperature (17 ± 1 ºC) and a natural photoperiod. Artificial seawater was renewed every 2-3 days and clams were fed every 2-3 days with Algamac Protein Plus (150,000 cells/animal/day) after the 3rd day. After depuration, the organisms were subjected to a chronic toxicity test for 28 days, consisting of exposure to five different EE2 concentrations (Sigma-Aldrich, purity ≥ 98%, MW = 296.40 g/mol, 1 mg/L stock solution in ultrapure water): 0 (control group), 5, 25, 125 and 625 ng/L. To assess the effects of a warming scenario on the impacts of EE2, the experiments were carried out at 17 ± 1 °C (control; mean temperature of sampling area during September: 16 - 19 °C) and at 21 ± 1 °C (worst-case climate change scenario, IPCC, 2021). The aquaria were placed in distinct climatic rooms for each temperature. To reach 21 °C, the temperature was raised by 2 °C, every 2–3 days. For each concentration level and temperature, 12 samples were considered: 4 individuals per aquarium and 3 aquaria per treatment. In each aquarium, a total of 3 L of artificial seawater (salinity: 30 ± 1), continuous aeration, and a natural photoperiod were used. The exposure medium for each condition was renewed weekly, after which EE2 concentration levels were re-established. Mortality was checked daily and found to be null. At the end of the 28-day exposure period, the clams were frozen in liquid nitrogen and stored at – 80 ºC. |
| Collection Protocol Filename: | Bivalves_Experimental_Procedure.docx |
| Sample Type: | Tissue |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002557 |
| Treatment Summary: | Sea warming and 17-α-ethinylestradiol exposure. After depuration, the organisms were subjected to a chronic toxicity test for 28 days, consisting of exposure to five different EE2 concentrations: 0 (control group), 5, 25, 125 and 625 ng/L. To assess the effects of a warming scenario on the impacts of EE2, the experiments were carried out at 17 ± 1 °C (control; mean temperature of sampling area during September) and at 21 ± 1 °C (worst-case climate change scenario). The aquaria were placed in distinct climatic rooms for each temperature. To reach 21 °C, the temperature was raised by 2 °C, every 2–3 days. For each concentration level and temperature, 12 samples were considered: 4 individuals per aquarium and 3 aquaria per treatment. In each aquarium, a total of 3 L of artificial seawater (salinity: 30 ± 1), continuous aeration, and a natural photoperiod were used. The exposure medium for each condition was renewed weekly, after which EE2 concentration levels were re-established. Mortality was checked daily and found to be null. At the end of the 28-day exposure period, the clams were frozen in liquid nitrogen and stored at – 80 ºC. |
| Treatment Protocol Filename: | Bivalves_Experimental_Procedure.docx |
| Treatment Compound: | Sea warming and 17-α-ethinylestradiol exposure |
| Treatment Dose: | Clams were exposed to five different EE2 concentrations: 0 (control group), 5, 25, 125 and 625 ng/L. To assess the effects of a warming scenario on the impacts of EE2, the experiments were carried out at 17 ± 1 °C (control) and at 21 ± 1 °C (worst-case climate change scenario). |
Sample Preparation:
| Sampleprep ID: | SP002551 |
| Sampleprep Summary: | Metabolite extraction was performed using a water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et al., 2007). Briefly, the clams´ soft tissue (0.15 g per sample) was ground with a pestle and mortar, in liquid nitrogen, and then transferred to a microtube, followed by the addition of cold methanol (600 µL), ultrapure water (128 µL) and chloroform (300 µL). The mixture was vortexed, left in ice for 10 min and centrifuged (2,500 g, 4 °C, 10 min). The top layer was transferred into a microtube to which chloroform (300 µL) and water (300 µL) were added. The mixture was vortexed and centrifuged (2,500 g, 4 °C, 10 min). The upper layer (aqueous) was transferred into vials, dried in a centrifugal vacuum concentrator (UNIVAP 100H) and stored at −80 °C until NMR analysis. |
| Sampleprep Protocol Filename: | Bivalves_Experimental_Procedure.docx |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Water/methanol/chloroform method, as described in (Hines, Oladiran, Bignell et al., 2007) |
| Extract Storage: | -80℃ |
| Sample Resuspension: | The dried polar extracts of clam samples were resuspended in 600 μL of sodium phosphate buffer (0.1 M in D2O, 99.96% D, pH 7.4, containing 0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, TSP-d4, chemical shift referencing). The mixture was vortexed and centrifuged (16,000 g, 10 min, room temperature) and 550 μL were transferred into 5 mm NMR tubes. |
| Sample Spiking: | 0.5 mM sodium salt of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP-d4), as a chemical shift reference. |
Analysis:
| MB Sample ID: | SA245803 |
| Analysis ID: | AN004006 |
| Laboratory Name: | Metabolomics group |
| Analysis Type: | NMR |
| Analysis Protocol File: | Bivalves_Experimental_Procedure.docx |
| Software Version: | TopSpin 3.2 and Amix 3.9.14 |
| Operator Name: | Joao A. Rodrigues |
| Detector Type: | Bruker Avance III 500 MHz spectrometer |
| Data Format: | fid, 1r |
NMR:
| NMR ID: | NM000263 |
| Analysis ID: | AN004006 |
| Instrument Name: | Bruker AVANCE III 500 spectrometer |
| Instrument Type: | FT-NMR |
| NMR Experiment Type: | 1D-1H |
| Spectrometer Frequency: | 500 MHz |
| NMR Probe: | TXI probe |
| NMR Solvent: | D2O |
| NMR Tube Size: | 5 mm NMR tubes |
| Shimming Method: | Topshim |
| Pulse Sequence: | noesypr1d |
| Water Suppression: | presat |
| Pulse Width: | 90-degree |
| Receiver Gain: | 203 |
| Temperature: | 298 K |
| Number Of Scans: | 256 |
| Dummy Scans: | 8 |
| Acquisition Time: | 2.34 s |
| Relaxation Delay: | 3 s |
| Spectral Width: | 7,002.8 Hz |
| Num Data Points Acquired: | 32 k |
| Line Broadening: | 0.3 Hz |
| Zero Filling: | 64 k |
| Baseline Correction Method: | manual |
| Chemical Shift Ref Std: | 0 ppm for TSP-d4 |
| NMR Results File: | 4._Bivalves_results_data.txt UNITS:ppm |
