http://lod.bco-dmo.org/id/dataset/557253
eng; USA
utf8
dataset
Highest level of data collection, from a common set of sensors or instrumentation, usually within the same research project
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
2015-04-29
ISO 19115-2 Geographic Information - Metadata - Part 2: Extensions for Imagery and Gridded Data
ISO 19115-2:2009(E)
Carbonate chemistry, shell growth, and respiration data from laboratory experiments on California mussel larvae condcuted at the Hatfield Marine Science Center, Newport, OR in 2013
2015-04-29
publication
2015-04-29
revision
BCO-DMO Linked Data URI
2015-04-29
creation
http://lod.bco-dmo.org/id/dataset/557253
George G. Waldbusser
Oregon State University
principalInvestigator
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
publisher
Cite this dataset as: Waldbusser, G. G. (2015) Carbonate chemistry, shell growth, and respiration data from laboratory experiments on California mussel larvae condcuted at the Hatfield Marine Science Center, Newport, OR in 2013. Biological and Chemical Oceanography Data Management Office (BCO-DMO). (Version 29 April 2015) Version Date 2015-04-29 [if applicable, indicate subset used]. http://lod.bco-dmo.org/id/dataset/557253 [access date]
Carbonate chemistry, shell growth, and respiration data from laboratory experiments on California mussel larvae. Dataset Description: <p>Carbonate chemistry, shell growth, and respiration data from laboratory experiments on California mussel larvae (<em>Mytilus californianus</em>). Experiments were conducted in the Hatfield Marine Science Center, Newport, OR.</p>
<p><strong>Related publications:</strong><br />
Waldbusser et al. 2015 (<em>in press</em>). Ocean acidification has multiple modes of action on bivalve larvae. PLOSOne.</p> Methods and Sampling: <p>Methods below from Waldbusser et al. 2015 (<em>in press</em>). Ocean acidification has multiple modes of action on bivalve larvae. PLOSOne.</p>
<p><strong>Carbonate chemistry measurements</strong><br />
Discrete samples were taken for carbonate chemistry at three time points: 1) following decarbonation but prior to manipulation, 2) immediately prior to filling BOD bottles from gas-impermeable bags, and 3) from each BOD bottle at the termination of the development experiment. Treatment data presented are from the samples in step #2 above. Seawater samples at the termination of the experiment were taken by placing a siphon tube into the BOD bottles, with its submerged end covered with a 37 micron Nitex screen to prevent removal of larvae. The siphon was started and the initial seawater flow discarded before sample collection with minimal aeration in a 350 ml amber glass bottle. All seawater samples collected in 350 ml amber glass bottles were preserved with 30 ml of saturated HgCl<sub>2</sub>, and sealed with polyurethane-lined metal crimp caps. P<sub>CO2</sub> and DIC analyses were carried out via gas equilibration and stripping, respectively, followed by infrared detection, as in Bandstra et al. 2006 and Hales et al. 2005, modified for discrete samples. Standards for P<sub>CO2</sub> and DIC encompassed the complete range of values in this study which are outside the range of typical modern-ocean seawater. To compute the complete carbonate chemistry we used, Millero 2010 carbonic acid dissociation constants with temperature and salinity dependencies (which capture the Lueker et al. 2000 seawater constants), Dickson 1990 constants for boric acid, and Millero 1995 water dissociation constants.</p>
<p><strong>Proportion Normal</strong><br />
At termination of the development experiment (48 hours post-fertilization) and after seawater samples had been taken, larvae were concentrated in a known volume of seawater and three replicate samples were collected from each BOD bottle and stored in 20 ml vials and preserved with buffered (pH 8.1-8.2) formalin. The average number of total larvae per replicate vial from each BOD bottle was 79 (±20), with a range from ~ 50 to ~100.</p>
<p>The proportion of normally developed larvae 48 hours post fertilization was evaluated under an inverted microscope, with normal development noted as a completely formed D-shaped shell with a straight hinge and the presence of tissue inside the shell. We previously found that velum extrusion was sometimes related to the speed and strength of additions of buffered formalin; therefore, we scored larvae as normal if larvae showed minor velum extrusion and normally developed shells.</p>
<p><strong>Shell Growth</strong><br />
Shell size was determined by taking photographs of all sampled larvae scored as normally developed using an inverted transmission microscope (Jena Sedival 250-CL coupled to a FujiFilm Digital SLR S5) at 50x magnification and measuring shell lengths (longest axis parallel to shell hinge) on size calibrated images (ImageJ v 1.42). Shell lengths were measured for a total of 4639 normally developed individual larvae. We only measured size of normally developed larvae to prevent bias in the size estimates (poorly or undeveloped larvae are always smaller) and to prevent conflation of developmental and growth effects in interpreting results.</p>
<p><strong>Respiration Rate</strong><br />
Respiration rates were measured for 48-h larvae that were first pooled from triplicate BOD bottles of each seawater treatment, after samples had been taken from each BOD bottle for determination of development and water chemistry analysis. Larvae were stocked at an estimated concentration of 500 larvae in each of five replicate 2 ml, solid-capped GC vials filled with the same seawater treatment as they had experienced during development in the BOD bottles. Larvae were concentrated and enumerated prior to stocking such that the larvae suspension constituted less than 10% of the volume added to each 2 ml vial. The desired concentration of 500 larvae per vial was chosen to yield the strongest and most consistent respiration signal while minimizing the effect of respiration on ambient carbonate chemistry within each treatment. Preliminary analyses showed that respiration rates under normal conditions were unaffected by larval densities between 200 and 800 larvae per vial (data not shown). Fluorescent oxygen-sensitive sensor spots (5 mm planar oxygen-sensitive spots, PSt3, PreSens, Germany) were pre-attached to the base of each vial before seawater or larvae were added. Oxygen measurements were made using the Fibox 3 (PreSens, Germany) that utilizes a fiber-optic cable to transmit and receive light from a sensor spot through the glass vial, allowing oxygen measurements to be taken in a non-destructive manner and without opening the vials. The vials were filled with warmed (18 degrees C) seawater siphoned from seawater contained in the same impermeable bags as that used to fill the BOD bottles, followed with additions of chloramphenicol and 10 ppm ampicillin to control bacterial respiration. Five control vials (without additions of larvae) per seawater treatment allowed correction of larval respiration measurements for background bacterial respiration. Larval respiration rates were approximately 2 – 3 times those of background bacterial rates. Following larval additions, solid caps with PTFE liners were tightly screwed onto each vial, taking care to eliminate any bubbles from the vials. Vials were held on their sides submerged in a temperature-controlled seawater bath to maintain an experimental temperature of 18<sup> </sup>degrees C. Oxygen measurements on all 60 vials were taken every 2 hours over the first 6 hours of incubation (including time 0, initiation of respiration measurements), allowing linear regression of four time-points per treatment. We used a 6-hour incubation period because longer periods resulted in reduced respiration rates.&nbsp; The slopes of the regressions were corrected for background bacterial respiration determined in the control vials. Once respiration rate measurements were completed, larvae from each vial were preserved, counted and respiration rates expressed per larva (abnormal plus normal). The total average number of larvae per vial was 457 +/- 89 (1 standard deviation).</p>
<p><strong>Initiation of Feeding</strong><br />
To assess the impact of water treatments on development of feeding organs and processes, we determined the proportion of larvae that ingested fluorescent beads at 44 hours post fertilization (initiation of feeding; IF). Preliminary experiments showed that at 44 hours post-fertilization ≥50% of <em>M. californianus</em> larvae began feeding when reared in natural seawater at 18 degrees C. Fertilized eggs were stocked at 10 ml<sup>-1</sup> in triplicate 20 ml solid-capped, sealed vials completely filled (no head space) with seawater treatments, at the same time as larvae were stocked in BOD bottles. After addition of fertilized eggs, 2µm Fluorescbrite<sup>®</sup> yellow beads (excitation maxima at 441nm and emission maxima at 485nm; Polysciences Inc., Warrington, PA) were added to the vials at a concentration of 20 beads ul<sup>-1</sup>. Larval density effects, including the density used here, on microsphere availability were found to be negligible during preliminary experiments. Although these are higher than found in natural systems, for short-term, experimental comparisons our larval and bead densities did not appear to skew results. At 44 hour post-fertilization, the experiment was terminated by adding 40 ul of 10% buffered formalin (pH ~8.1-8.2) to the vials. A minimum of 20 larvae per replicate vial were later examined under epifluorescent microscopy for the presence or absence of beads within their guts. The proportion of larvae feeding was then determined as the ratio of larvae that had at least 1 bead in their gut to the total # of larvae counted.</p>
<p><strong>References</strong><br />
Bandstra L, Hales B and Takahashi T. High-frequency measurements of total CO2: method development and first oceanographic observations. Mar Chem. 2006;100: 24-38.</p>
<p>Hales B, Chipman D, and Takahashi T. High-frequency measurement of partial pressure and total concentration of carbon dioxide in seawater using microporous hydrophobic membrane contactors. Limnol Oceanogr Methods. 2005;2: 356-364.</p>
<p>Millero FJ. Carbonate constants for estuarine waters. Mar Freshwater Res. 2010;61: 139-142.</p>
<p>Lueker TJ, Dickson AG and Keeling CD. Ocean pCO2 calculated from dissolved inorganic carbon, alkalinity, and equations for K-1 and K-2: validation based on laboratory measurements of CO2 in gas and seawater at equilibrium. Mar Chem. 2000;70: 105-119.</p>
<p>Dickson AG. Standard potential of the reaction - Agcl(S)+1/2h-2(G)=Ag(S)+Hcl(Aq) and the standard acidity constant of the ion Hso4- in synthetic sea-water from 273.15-K to 318.15-K. J Chem Thermodyn. 1990;22: 113-127.</p>
<p>Millero FJ. Thermodynamics of the carbon-dioxide system in the oceans. Geochim Cosmochim Ac. 1995;59: 661-677.</p>
Funding provided by NSF Division of Ocean Sciences (NSF OCE) Award Number: OCE-1041267 Award URL: https://www.nsf.gov/awardsearch/show-award?AWD_ID=1041267
completed
George G. Waldbusser
Oregon State University
541-737-8964
Oregon State University 104 COAS Admin. Bldg.,
Corvallis
OR
97331
USA
waldbuss@coas.oregonstate.edu
pointOfContact
asNeeded
Dataset Version: 29 April 2015
Unknown
taxon
omega_trtmnt
PCO2_trtmnt
total_alk
DIC
PCO2
HCO3
CO3
pH
omega_A
prop_norm
prop_norm_stdev
shell_length
shell_length_stdev
respir_rate
respir_rate_stdev
prop_feeding
prop_feeding_stdev
biological oxygen demand (BOD) bottles
epifluorescent microscope
non-dispersive infra-red (NDIR) gas analyzer
Inverted transmission microscope
theme
None, User defined
taxon
treatment
No BCO-DMO term
dissolved inorganic Carbon
Partial pressure of CO2
bicarbonate concentration [HCO3]-
Carbonate concentration [CO3]2-
pH
Aragonite Saturation State
standard deviation
length
featureType
BCO-DMO Standard Parameters
Bottle
Fluorescence Microscope
Gas Analyzer
Inverted Microscope
instrument
BCO-DMO Standard Instruments
Waldbusser_HMSC
service
Deployment Activity
Hatfield Marine Science Center, Newport, OR
place
Locations
otherRestrictions
otherRestrictions
Access Constraints: none. Use Constraints: Please follow guidelines at: http://www.bco-dmo.org/terms-use Distribution liability: Under no circumstances shall BCO-DMO be liable for any direct, incidental, special, consequential, indirect, or punitive damages that result from the use of, or the inability to use, the materials in this data submission. If you are dissatisfied with any materials in this data submission your sole and exclusive remedy is to discontinue use.
Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES): Ocean Acidification (formerly CRI-OA)
https://www.nsf.gov/funding/pgm_summ.jsp?pims_id=503477
Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES): Ocean Acidification (formerly CRI-OA)
NSF Climate Research Investment (CRI) activities that were initiated in 2010 are now included under Science, Engineering and Education for Sustainability NSF-Wide Investment (SEES). SEES is a portfolio of activities that highlights NSF's unique role in helping society address the challenge(s) of achieving sustainability. Detailed information about the SEES program is available from NSF (https://www.nsf.gov/funding/pgm_summ.jsp?pims_id=504707).
In recognition of the need for basic research concerning the nature, extent and impact of ocean acidification on oceanic environments in the past, present and future, the goal of the SEES: OA program is to understand (a) the chemistry and physical chemistry of ocean acidification; (b) how ocean acidification interacts with processes at the organismal level; and (c) how the earth system history informs our understanding of the effects of ocean acidification on the present day and future ocean.
Solicitations issued under this program:
NSF 10-530, FY 2010-FY2011
NSF 12-500, FY 2012
NSF 12-600, FY 2013
NSF 13-586, FY 2014
NSF 13-586 was the final solicitation that will be released for this program.
PI Meetings:
1st U.S. Ocean Acidification PI Meeting(March 22-24, 2011, Woods Hole, MA)
2nd U.S. Ocean Acidification PI Meeting(Sept. 18-20, 2013, Washington, DC)
3rd U.S. Ocean Acidification PI Meeting (June 9-11, 2015, Woods Hole, MA – Tentative)
NSF media releases for the Ocean Acidification Program:
Press Release 10-186 NSF Awards Grants to Study Effects of Ocean Acidification
Discovery Blue Mussels "Hang On" Along Rocky Shores: For How Long?
Discovery nsf.gov - National Science Foundation (NSF) Discoveries - Trouble in Paradise: Ocean Acidification This Way Comes - US National Science Foundation (NSF)
Press Release 12-179 nsf.gov - National Science Foundation (NSF) News - Ocean Acidification: Finding New Answers Through National Science Foundation Research Grants - US National Science Foundation (NSF)
Press Release 13-102 World Oceans Month Brings Mixed News for Oysters
Press Release 13-108 nsf.gov - National Science Foundation (NSF) News - Natural Underwater Springs Show How Coral Reefs Respond to Ocean Acidification - US National Science Foundation (NSF)
Press Release 13-148 Ocean acidification: Making new discoveries through National Science Foundation research grants
Press Release 13-148 - Video nsf.gov - News - Video - NSF Ocean Sciences Division Director David Conover answers questions about ocean acidification. - US National Science Foundation (NSF)
Press Release 14-010 nsf.gov - National Science Foundation (NSF) News - Palau's coral reefs surprisingly resistant to ocean acidification - US National Science Foundation (NSF)
Press Release 14-116 nsf.gov - National Science Foundation (NSF) News - Ocean Acidification: NSF awards $11.4 million in new grants to study effects on marine ecosystems - US National Science Foundation (NSF)
SEES-OA
largerWorkCitation
program
A mechanistic understanding of the impacts of ocean acidification on the early life stages of marine bivalves
https://osprey.bco-dmo.org/project/2167
A mechanistic understanding of the impacts of ocean acidification on the early life stages of marine bivalves
<p><em>Extracted from the NSF award abstract:</em></p>
<p>The shift in the carbonate chemistry of marine waters, as a result of direct anthropogenic CO2 addition and climate-driven changes in circulation, poses a threat to many organisms. A rapidly expanding body of literature has shown that increasing levels of carbonic acid and decreasing carbonate ion levels will have deleterious effects on many marine organisms; however little is known about the mode of action of these changes in water chemistry on marine bivalves. Many marine organisms, particularly bivalves, depend critically on the production of calcium carbonate mineral, and this material becomes thermodynamically unstable under more acidic conditions. The actual mineral precipitation, however, takes place within interstitial volumes intermittently separated from ambient seawater by biological membranes. Therefore, abiotic relationships between solid phase minerals and seawater thermodynamics are oversimplified representations of the complex interplay among seawater chemistry, bivalve physiology, and shell growth processes.</p>
<p>In this integrative, multi-disciplinary project we will develop and apply novel experimental approaches to elucidate fundamental physiological responses to changes in seawater chemistry associated with ocean acidification. The four primary objectives of this project are to: 1) develop a novel experimental approach and system capable of unique combinations of pCO2, pH, and mineral saturation state (Ω), 2) conduct short-term exploratory experiments to determine bivalve responses to different carbonate system variables, 3) conduct longer-term directed studies of the integrated effects of different carbonate system variables over early life history of bivalves, and 4) compare these biological responses among a group of bivalve species that differ in shell mineralogy and nativity to the periodically acidified upwelling region of the Pacific Northwest coast of North America. By isolating the effects of different components of the carbonate system on the early life stages of marine bivalves, e.g. does an oyster larvae respond more strongly to pCO2 or mineral saturation state?, we can begin to identify the mechanisms behind bivalve responses as well as understand how these organisms survive in transiently corrosive conditions.</p>
<p>Laboratory based experiments on three primary taxa (oyster, mussel, clam) having native and non-native species pairs to Oregon’s coastal waters: oysters <em>Ostrea lurida </em>and <em>Crassostrea gigas</em>; mussels <em>Mytilus califonianus</em> and <em>Mytilus galloprovincialis</em>; and clams <em>Macoma nasuta</em> and <em>Ruditapes philippinarum</em>, will allow for species comparisons among different shell mineralogy, microstructure, life-history, and adaptability. High-precision pCO2 and dissolved inorganic carbon (DIC) instruments will be used in experiments to control and properly constrain the carbonate chemistry. A compliment of response variables will be measured across the early life stages of these species that include tissue acid-base balance, shell mineralogy and chemistry, respiration rate, and behavior. Additionally, our emphasis will be placed on observation of development, growth, and shell structure by directly linking observational data with other measured response data. An adaptive strategy using short-term experiments to determine the most salient variables in the carbonate system to manipulate in longer-term studies is being employed. This approach allows us to evaluate acute effects, mimicking diurnal changes to carbonate variables often found in coastal areas, and integrated chronic effects mimicking a more gradual acidification due to the rise in atmospheric CO2.</p>
Mechanisms of bivalve response to acidification
largerWorkCitation
project
eng; USA
biota
oceans
Hatfield Marine Science Center, Newport, OR
2015-04-29
Coastal and estuarine waters of Oregon, U.S.A.
0
BCO-DMO catalogue of parameters from Carbonate chemistry, shell growth, and respiration data from laboratory experiments on California mussel larvae condcuted at the Hatfield Marine Science Center, Newport, OR in 2013
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
http://lod.bco-dmo.org/id/dataset-parameter/557263.rdf
Name: taxon
Units: dimensionless (text)
Description: <p>Name of species studied.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557264.rdf
Name: omega_trtmnt
Units: dimensionless (text)
Description: <p>Saturation state (omega Aragonite) treatment condition. Four treatment levels were used encompassing a range from ~0.5 to 5.0; targets were 0.50, 1.0, 2.0, 4.0. Treatment values for saturation state of aragonite are noted as "a" to "d" denoting the lowest to highest values.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557265.rdf
Name: PCO2_trtmnt
Units: dimensionless (text)
Description: <p>Partial pressure of carbon dioxide (PCO2) treatment condition. Four treatment levels were used encompassing a range from ~200 to 2500 microatmospheres (uatm); targets were 200, 400, 800, 2500. Treatment values for PCO2 are noted as Low (L), Mid-Low (ML), Mid_High (MH), and High (H).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557266.rdf
Name: total_alk
Units: micromoles per kilogram (umol/kg)
Description: <p>Total alkalinity.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557267.rdf
Name: DIC
Units: micromoles per kilogram (umol/kg)
Description: <p>Dissolved inorganic carbon (DIC).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557268.rdf
Name: PCO2
Units: micromoles per kilogram (umol/kg)
Description: <p>Partial pressure of carbon dioxide (PCO2).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557269.rdf
Name: HCO3
Units: micromoles per kilogram (umol/kg)
Description: <p>Bicarbonate ion (HCO3).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557270.rdf
Name: CO3
Units: micromoles per kilogram (umol/kg)
Description: <p>Carbonate ion (CO3).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557271.rdf
Name: pH
Units: pH scale.
Description: <p>pH.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557272.rdf
Name: omega_A
Units: dimensionless
Description: <p>The saturation state of seawater with respect to aragonite.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557273.rdf
Name: prop_norm
Units: dimensionless (proportion)
Description: <p>The proportion of normally developed larvae 48 hours post fertilization.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557274.rdf
Name: prop_norm_stdev
Units: dimensionless (proportion)
Description: <p>Standard deviation of the proportion of normally developed larvae.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557275.rdf
Name: shell_length
Units: micrometers (um)
Description: <p>Shell length (longest axis parallel to shell hinge) as measured on size calibrated images.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557276.rdf
Name: shell_length_stdev
Units: micrometers (um)
Description: <p>Standard deviation of shell length.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557277.rdf
Name: respir_rate
Units: picomoles O2 per hour per individual (pmol O2 /hr ind)
Description: <p>Respiration rate (see acquisition description for methods). Once respiration rate measurements were completed, larvae from each vial were preserved, counted and respiration rates expressed per larva (abnormal plus normal).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557278.rdf
Name: respir_rate_stdev
Units: picomoles O2 per hour per individual (pmol O2 /hr ind)
Description: <p>Standard deviation of respiration rate.</p>
http://lod.bco-dmo.org/id/dataset-parameter/557279.rdf
Name: prop_feeding
Units: dimensionless (proportion)
Description: <p>The proportion of larvae feeding determined as the ratio of larvae that had at least 1 bead in their gut to the total # of larvae counted (see acquisition description for methods).</p>
http://lod.bco-dmo.org/id/dataset-parameter/557280.rdf
Name: prop_feeding_stdev
Units: dimensionless (proportion)
Description: <p>Standard deviation of the proportion of larvae feeding.</p>
GB/NERC/BODC > British Oceanographic Data Centre, Natural Environment Research Council, United Kingdom
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
Unavailable
508-289-2009
WHOI MS#36
Woods Hole
MA
02543
USA
info@bco-dmo.org
http://www.bco-dmo.org
Monday - Friday 8:00am - 5:00pm
For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
1859
https://datadocs.bco-dmo.org/file/8XXgykXIBX6m9L/cal_mussels.csv
cal_mussels.csv
Primary data file for dataset ID 557253
download
https://osprey.bco-dmo.org/dataset/557253/data/download
download
onLine
dataset
<p>Methods below from Waldbusser et al. 2015 (<em>in press</em>). Ocean acidification has multiple modes of action on bivalve larvae. PLOSOne.</p>
<p><strong>Carbonate chemistry measurements</strong><br />
Discrete samples were taken for carbonate chemistry at three time points: 1) following decarbonation but prior to manipulation, 2) immediately prior to filling BOD bottles from gas-impermeable bags, and 3) from each BOD bottle at the termination of the development experiment. Treatment data presented are from the samples in step #2 above. Seawater samples at the termination of the experiment were taken by placing a siphon tube into the BOD bottles, with its submerged end covered with a 37 micron Nitex screen to prevent removal of larvae. The siphon was started and the initial seawater flow discarded before sample collection with minimal aeration in a 350 ml amber glass bottle. All seawater samples collected in 350 ml amber glass bottles were preserved with 30 ml of saturated HgCl<sub>2</sub>, and sealed with polyurethane-lined metal crimp caps. P<sub>CO2</sub> and DIC analyses were carried out via gas equilibration and stripping, respectively, followed by infrared detection, as in Bandstra et al. 2006 and Hales et al. 2005, modified for discrete samples. Standards for P<sub>CO2</sub> and DIC encompassed the complete range of values in this study which are outside the range of typical modern-ocean seawater. To compute the complete carbonate chemistry we used, Millero 2010 carbonic acid dissociation constants with temperature and salinity dependencies (which capture the Lueker et al. 2000 seawater constants), Dickson 1990 constants for boric acid, and Millero 1995 water dissociation constants.</p>
<p><strong>Proportion Normal</strong><br />
At termination of the development experiment (48 hours post-fertilization) and after seawater samples had been taken, larvae were concentrated in a known volume of seawater and three replicate samples were collected from each BOD bottle and stored in 20 ml vials and preserved with buffered (pH 8.1-8.2) formalin. The average number of total larvae per replicate vial from each BOD bottle was 79 (±20), with a range from ~ 50 to ~100.</p>
<p>The proportion of normally developed larvae 48 hours post fertilization was evaluated under an inverted microscope, with normal development noted as a completely formed D-shaped shell with a straight hinge and the presence of tissue inside the shell. We previously found that velum extrusion was sometimes related to the speed and strength of additions of buffered formalin; therefore, we scored larvae as normal if larvae showed minor velum extrusion and normally developed shells.</p>
<p><strong>Shell Growth</strong><br />
Shell size was determined by taking photographs of all sampled larvae scored as normally developed using an inverted transmission microscope (Jena Sedival 250-CL coupled to a FujiFilm Digital SLR S5) at 50x magnification and measuring shell lengths (longest axis parallel to shell hinge) on size calibrated images (ImageJ v 1.42). Shell lengths were measured for a total of 4639 normally developed individual larvae. We only measured size of normally developed larvae to prevent bias in the size estimates (poorly or undeveloped larvae are always smaller) and to prevent conflation of developmental and growth effects in interpreting results.</p>
<p><strong>Respiration Rate</strong><br />
Respiration rates were measured for 48-h larvae that were first pooled from triplicate BOD bottles of each seawater treatment, after samples had been taken from each BOD bottle for determination of development and water chemistry analysis. Larvae were stocked at an estimated concentration of 500 larvae in each of five replicate 2 ml, solid-capped GC vials filled with the same seawater treatment as they had experienced during development in the BOD bottles. Larvae were concentrated and enumerated prior to stocking such that the larvae suspension constituted less than 10% of the volume added to each 2 ml vial. The desired concentration of 500 larvae per vial was chosen to yield the strongest and most consistent respiration signal while minimizing the effect of respiration on ambient carbonate chemistry within each treatment. Preliminary analyses showed that respiration rates under normal conditions were unaffected by larval densities between 200 and 800 larvae per vial (data not shown). Fluorescent oxygen-sensitive sensor spots (5 mm planar oxygen-sensitive spots, PSt3, PreSens, Germany) were pre-attached to the base of each vial before seawater or larvae were added. Oxygen measurements were made using the Fibox 3 (PreSens, Germany) that utilizes a fiber-optic cable to transmit and receive light from a sensor spot through the glass vial, allowing oxygen measurements to be taken in a non-destructive manner and without opening the vials. The vials were filled with warmed (18 degrees C) seawater siphoned from seawater contained in the same impermeable bags as that used to fill the BOD bottles, followed with additions of chloramphenicol and 10 ppm ampicillin to control bacterial respiration. Five control vials (without additions of larvae) per seawater treatment allowed correction of larval respiration measurements for background bacterial respiration. Larval respiration rates were approximately 2 – 3 times those of background bacterial rates. Following larval additions, solid caps with PTFE liners were tightly screwed onto each vial, taking care to eliminate any bubbles from the vials. Vials were held on their sides submerged in a temperature-controlled seawater bath to maintain an experimental temperature of 18<sup> </sup>degrees C. Oxygen measurements on all 60 vials were taken every 2 hours over the first 6 hours of incubation (including time 0, initiation of respiration measurements), allowing linear regression of four time-points per treatment. We used a 6-hour incubation period because longer periods resulted in reduced respiration rates.&nbsp; The slopes of the regressions were corrected for background bacterial respiration determined in the control vials. Once respiration rate measurements were completed, larvae from each vial were preserved, counted and respiration rates expressed per larva (abnormal plus normal). The total average number of larvae per vial was 457 +/- 89 (1 standard deviation).</p>
<p><strong>Initiation of Feeding</strong><br />
To assess the impact of water treatments on development of feeding organs and processes, we determined the proportion of larvae that ingested fluorescent beads at 44 hours post fertilization (initiation of feeding; IF). Preliminary experiments showed that at 44 hours post-fertilization ≥50% of <em>M. californianus</em> larvae began feeding when reared in natural seawater at 18 degrees C. Fertilized eggs were stocked at 10 ml<sup>-1</sup> in triplicate 20 ml solid-capped, sealed vials completely filled (no head space) with seawater treatments, at the same time as larvae were stocked in BOD bottles. After addition of fertilized eggs, 2µm Fluorescbrite<sup>®</sup> yellow beads (excitation maxima at 441nm and emission maxima at 485nm; Polysciences Inc., Warrington, PA) were added to the vials at a concentration of 20 beads ul<sup>-1</sup>. Larval density effects, including the density used here, on microsphere availability were found to be negligible during preliminary experiments. Although these are higher than found in natural systems, for short-term, experimental comparisons our larval and bead densities did not appear to skew results. At 44 hour post-fertilization, the experiment was terminated by adding 40 ul of 10% buffered formalin (pH ~8.1-8.2) to the vials. A minimum of 20 larvae per replicate vial were later examined under epifluorescent microscopy for the presence or absence of beads within their guts. The proportion of larvae feeding was then determined as the ratio of larvae that had at least 1 bead in their gut to the total # of larvae counted.</p>
<p><strong>References</strong><br />
Bandstra L, Hales B and Takahashi T. High-frequency measurements of total CO2: method development and first oceanographic observations. Mar Chem. 2006;100: 24-38.</p>
<p>Hales B, Chipman D, and Takahashi T. High-frequency measurement of partial pressure and total concentration of carbon dioxide in seawater using microporous hydrophobic membrane contactors. Limnol Oceanogr Methods. 2005;2: 356-364.</p>
<p>Millero FJ. Carbonate constants for estuarine waters. Mar Freshwater Res. 2010;61: 139-142.</p>
<p>Lueker TJ, Dickson AG and Keeling CD. Ocean pCO2 calculated from dissolved inorganic carbon, alkalinity, and equations for K-1 and K-2: validation based on laboratory measurements of CO2 in gas and seawater at equilibrium. Mar Chem. 2000;70: 105-119.</p>
<p>Dickson AG. Standard potential of the reaction - Agcl(S)+1/2h-2(G)=Ag(S)+Hcl(Aq) and the standard acidity constant of the ion Hso4- in synthetic sea-water from 273.15-K to 318.15-K. J Chem Thermodyn. 1990;22: 113-127.</p>
<p>Millero FJ. Thermodynamics of the carbon-dioxide system in the oceans. Geochim Cosmochim Ac. 1995;59: 661-677.</p>
Specified by the Principal Investigator(s)
<p>BCO-DMO Processing:<br />
- Replaced blanks (missing data/not measured) with 'nd' to indicate 'no data'.<br />
- Modified parameter names to conform with BCO-DMO naming conventions.<br />
- Added taxon column.</p>
<p>&nbsp;</p>
Specified by the Principal Investigator(s)
asNeeded
7.x-1.1
Biological and Chemical Oceanography Data Management Office (BCO-DMO)
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508-289-2009
WHOI MS#36
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For questions regarding this resource, please contact BCO-DMO via the email address provided.
pointOfContact
biological oxygen demand (BOD) bottles
biological oxygen demand (BOD) bottles
PI Supplied Instrument Name: biological oxygen demand (BOD) bottles Instrument Name: Bottle Instrument Short Name:Bottle Instrument Description: A container, typically made of glass or plastic and with a narrow neck, used for storing drinks or other liquids.
epifluorescent microscope
epifluorescent microscope
PI Supplied Instrument Name: epifluorescent microscope PI Supplied Instrument Description:Epifluorescent microscopy was used to determine the proportion of larvae feeding. Instrument Name: Fluorescence Microscope Instrument Short Name: Instrument Description: Instruments that generate enlarged images of samples using the phenomena of fluorescence and phosphorescence instead of, or in addition to, reflection and absorption of visible light. Includes conventional and inverted instruments. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB06/
non-dispersive infra-red (NDIR) gas analyzer
non-dispersive infra-red (NDIR) gas analyzer
PI Supplied Instrument Name: non-dispersive infra-red (NDIR) gas analyzer PI Supplied Instrument Description:PCO2 and DIC analyses were carried out via gas equilibration and stripping, respectively, followed by infrared detection, as in Bandstra et al. (Reference: Bandstra L, Hales B and Takahashi T. High-frequency measurements of total CO2: method development and first oceanographic observations. Mar Chem. 2006;100: 24-38. doi:10.1016/j.marchem.2005.10.009) Instrument Name: Gas Analyzer Instrument Short Name:Gas Analyzer Instrument Description: Gas Analyzers - Instruments for determining the qualitative and quantitative composition of gas mixtures.
Inverted transmission microscope
Inverted transmission microscope
PI Supplied Instrument Name: Inverted transmission microscope PI Supplied Instrument Description:Shell size was determined by taking photographs of all sampled larvae scored as normally developed using an inverted transmission microscope (Jena Sedival 250-CL coupled to a FujiFilm Digital SLR S5) at 50x magnification and measuring shell lengths (longest axis parallel to shell hinge) on size calibrated images (ImageJ v 1.42). Instrument Name: Inverted Microscope Instrument Short Name: Instrument Description: An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane University (then named the Medical College of Louisiana).
Inverted microscopes are useful for observing living cells or organisms at the bottom of a large container (e.g. a tissue culture flask) under more natural conditions than on a glass slide, as is the case with a conventional microscope. Inverted microscopes are also used in micromanipulation applications where space above the specimen is required for manipulator mechanisms and the microtools they hold, and in metallurgical applications where polished samples can be placed on top of the stage and viewed from underneath using reflecting objectives.
The stage on an inverted microscope is usually fixed, and focus is adjusted by moving the objective lens along a vertical axis to bring it closer to or further from the specimen. The focus mechanism typically has a dual concentric knob for coarse and fine adjustment. Depending on the size of the microscope, four to six objective lenses of different magnifications may be fitted to a rotating turret known as a nosepiece. These microscopes may also be fitted with accessories for fitting still and video cameras, fluorescence illumination, confocal scanning and many other applications. Community Standard Description: http://vocab.nerc.ac.uk/collection/L05/current/LAB05/
Deployment: Waldbusser_HMSC
Waldbusser_HMSC
OSU-HMSC
OSU-HMSC
laboratory
Waldbusser_HMSC
George G. Waldbusser
Oregon State University
OSU-HMSC
OSU-HMSC
laboratory