Controlled experimental aquarium system for multi-stressor investigation: carbonate chemistry ...

23 Pages·2013·0.84 MB·English

BiogeosciencesDiscuss.,10,3431–3453,2013 D
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www.biogeosciences-discuss.net/10/3431/2013/ c BGD
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doi:10.5194/bgd-10-3431-2013 s
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aquarium system for
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multi-stressor
Controlled experimental aquarium system D
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for multi-stressor investigation: carbonate ss E.E.Bockmonetal.
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chemistry, oxygen saturation, and P
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| Abstract Introduction
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E. E. Bockmon1, C. A. Frieder2, M. O. Navarro2, L. A. White-Kershek3, and is Conclusions References
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A. G. Dickson1 uss Tables Figures
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1MarinePhysicalLaboratory,ScrippsInstitutionofOceanography,UniversityofCalifornia,San n
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Diego,9500GilmanDrive,LaJolla,CA92093-0244,USA a J I
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2IntegrativeOceanographyDivision,ScrippsInstitutionofOceanography,Universityof e
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California,SanDiego,9500GilmanDrive,LaJolla,CA92093-0218,USA
3DepartmentofBiologicalSciences,CaliforniaStateUniversity,SanMarcos,333TwinOaks |
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ValleyRoad,SanMarcos,CA92096,USA D
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Received:28January2013–Accepted:12February2013–Published:25February2013 u
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Correspondenceto:E.E.Bockmon([email protected]) io
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PublishedbyCopernicusPublicationsonbehalfoftheEuropeanGeosciencesUnion. a
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Abstract D
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As the field of ocean acidification has grown, researchers have increasingly turned to s
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laboratory experiments to understand the impacts of increased CO2 on marine or- ion 10,3431–3453,2013
ganisms. However, other changes such as ocean warming and deoxygenation are P
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5 occurring concurrently with the increasing CO2 concentrations, complicating the an- er Experimental
thropogenic impact on organisms. This experimental aquarium design allows for in- aquarium system for
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dependent regulation of CO2 concentration, O2 levels, and temperature in a controlled multi-stressor
environmenttostudytheimpactsofmultiplestressors.Thesystemhastheflexibilityfor Dis investigation
a wide range of treatment chemistry, seawater volumes, and study organisms. Control c
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oftheseawaterchemistryisachievedbyequilibrationofachosengasmixturewithsea- s E.E.Bockmonetal.
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waterusingaLiqui-Cel® membranecontactor.Includedasexamples,twoexperiments n
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performed using the system have shown control of CO between approximately 500– a
2 p
−1 ◦ e TitlePage
1400µatmandO from80–240µmolkg .Temperaturehasbeenmaintainedto0.5 C r
2
◦
orbetterintherangeof10–17 C.Onaweeklongtimescale,controlresultsinvariability
| Abstract Introduction
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in pH of less than 0.007pH units and in oxygen concentration less than 3.5µmolkg .
15 D
Longer experiments, over a month, have been completed with reasonable but less- is Conclusions References
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ened control, still better than 0.08 pH units and 13µmolkg−1 O . The ability to study u
2 ss Tables Figures
the impacts of multiple stressors in the laboratory simultaneously, as well as indepen- io
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dently,willbeanimportantpartofunderstandingtheresponseofmarineorganismsto P
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a high-CO world. p
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1 Introduction Back Close
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Duetoincreasinganthropogeniccarbondioxideintheatmosphere,theamountofinor- cu FullScreen/Esc
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ganic carbon in the oceans is increasing and the pH decreasing, a process commonly s
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known as ocean acidification (Caldeira and Wickett, 2003). In the past decade, ocean n Printer-friendlyVersion
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acidification has received increasing attention from the scientific community, particu- a
25 p InteractiveDiscussion
larlytheimpactoftheexpectedchangesincarbonatechemistryonorganisms(Doney e
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et al., 2009a). Experimental studies in the laboratory are an important part of this re- D
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search (Doney et al., 2009b). While significant insight has been gained from these c BGD
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laboratory experiments, it has also become clear that the questions are not simple to s
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answer,andthatreasonablecontroloftheexperimentalconditionscanbechallenging. n
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In 2010 EPOCA published a Guide to best practices for ocean acidification research a
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anddatareporting (Riebeselletal.,2010)whichincludesseveralrelevantchaptersfor e Experimental
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setting up experimental aquaria with the intent to control carbonate chemistry. Various aquarium system for
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options for modifying carbonate chemistry are suggested, including bubbling or mix- multi-stressor
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ing high-CO2 water, which changes dissolved inorganic carbon (DIC) at constant total is investigation
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alkalinity (A ), or adding acids and bases, or carbonate and bicarbonate, potentially u
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changing both AT and DIC. Some discussion has surrounded these various methods, sio E.E.Bockmonetal.
andthedifferencesincarbonatechemistryhavebeenevaluatedandfoundtobesmall n
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(Schulz et al., 2009). Despite this, bubbling is recommended as the “first choice” be- a
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cause it “exactly mimics carbonate chemistry changes occurring in the years to come” r
(Gattuso et al., 2010). However, bubbling can lead to difficulties in sustaining phyto-
15 | Abstract Introduction
plankton cultures (Shi et al., 2009), so often header tanks are used for equilibration to
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eliminate the impact of bubbling on the experimental organism. is Conclusions References
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A variety of experimental systems have been designed and used, with varying de- uss Tables Figures
grees of success, by researchers interested in control over carbonate chemistry in the io
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20 laboratory. Several experiments have been performed by bubbling seawater directly Pa J I
with gas mixtures created by combining pure CO with ambient or CO -stripped air to p
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create an elevated partial pressure of CO (p(CO )) (Miller et al., 2009; Talmage and r J I
2 2
Gobler, 2009). One published system bubbles a custom gas mixture while monitoring |
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p(CO2) (Fangue et al., 2010), and another uses acid additions to create constant pH D
25 seawater as determined by spectrophotometric measurements (McGraw et al., 2010). isc FullScreen/Esc
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Although the field has come a long way in a short time, gaps in knowledge and in ex- s
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ecution of some studies still exist (Gattuso and Lavigne, 2009). Most published meth- io
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ods manipulate small volumes of water, limiting experimental organism biomass. They P
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also generally modify header tanks that then supply seawater to the culture vessels, pe InteractiveDiscussion
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ratherthandirectmodification.Useofamoreprecise,reliable,andflexibleexperimen- D
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talaquariumdesign,suchasthatpresentedhere,shouldhelptoelucidateandconfirm c BGD
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some of the biological responses to ocean acidification. s
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In addition to changes in the carbon parameters, there are other expected impacts n
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of anthropogenic climate change on the ocean. Temperatures in the upper 300m of a
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the oceans are rising (Lyman et al., 2010), and changes in stratification have occurred e Experimental
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(Palaciosetal.,2004).Oxygensaturationisexpectedtodecreaseastheoceanswarm, aquarium system for
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stratify,andabsorbcarbon(Keelingetal.,2010;Shafferetal.,2009).Predictionsofthe multi-stressor
consequencesofoceanacidificationmustconsidersynergisticeffectsbetweenchang- Dis investigation
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ing inorganic carbon parameters and changes to these other variables. Multi-stressor, u
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or multi-variable, interactions have gained a lot of attention recently as researchers sio E.E.Bockmonetal.
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have begun to examine the many simultaneous impacts that climate change will have
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on organisms (Boyd, 2011; Po¨rtner et al., 2005). However, a review of marine climate a
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change papers found that most were single-factor experiments, most often focusing r
on acidification alone (Wernberg et al., 2012). In particular, the need for experiments
15 | Abstract Introduction
focusing on the combination of CO and O has been identified (Melzner et al., 2012;
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Po¨rtneretal.,2005).OxygenandpHarestrongly,positivelycorrelatedinsystemsdom- is Conclusions References
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inated by photosynthesis and respiration, as has been documented recently in coastal uss Tables Figures
upwelling systems (Frieder et al., 2012; Paulmier et al., 2011). The implications for or- io
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20 ganisms simultaneously experiencing low pH and low oxygen seawater are only just Pa J I
startingtobeinvestigated.TheabilitytomodifyCO andO levelsindependentlyinan p
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experimentallaboratory,inadditiontotemperature,willbecriticaltounderstandingthe r J I
response of organisms that live in natural environments with these multiple stressors. |
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PresentedhereistheMultipleStressorExperimentalAquariumatScripps(MSEAS) D
25 designed to address some of the challenges facing the scientific community regard- isc FullScreen/Esc
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ing ocean acidification research and to facilitate the study of organisms under future s
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ocean scenarios. The aim of this study was to develop a system capable of indepen- io
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dent manipulation and control of the inorganic carbon chemistry, oxygen levels, and P
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temperature of the seawater in each tank. Additionally, the system is designed with pe InteractiveDiscussion
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flexibility so that it may be adapted for a variety of marine organisms and life stages. D
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Finally, this study demonstrates the stability and accuracy of the system by presenting c BGD
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data from two separate experiments. s
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2 Methods p
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aquarium system for
5 2.1 Carbon parameter control |
multi-stressor
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Achieving good control in an experimental laboratory setting is difficult, in part be- is investigation
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cause the carbonate chemistry is complicated. Factors affecting control of the carbon- u
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atechemistryincludegasexchange,temperatureinfluences,anddirectmodificationby io
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the study organisms through processes such as photosynthesis, respiration, and cal- P
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cification. Another obstacle is that measurement of the carbon parameters is neither p
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simplenorinexpensive(Dicksonetal.,2007;Dickson,2010).Theacid-basechemistry r
in clean seawater can be described using known equilibrium constants given salinity, | Abstract Introduction
temperature, pressure and the total boron/salinity ratio, together with two other mea- D
sured parameters (typically A DIC, p(CO ), and/or pH). As only two parameters are is Conclusions References
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15 needed to characterize the system in an aquarium setting, controlling two of these ss Tables Figures
parameters will result in complete control over the carbonate chemistry. io
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Controlling two parameters at once is not accomplished easily. However, by assum- Pa J I
ing a constant AT only a single parameter needs to be controlled continually (Dickson, pe
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2010). Constant A is a reasonable assumption for the seawater system that supplies
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MSEAS, thus achieving control of one carbon parameter (Fig. 1). Over a three-year |
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period,theobservedrangeofA fortheseawatersystemwas50µmolkg−1 Thiswould D
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change pH calculated from A and constant p(CO ) by less than 0.01 pH units. More c FullScreen/Esc
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oftenA issimilarfromonedaytothenext,showinglargechangesovermonths,rather s
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thandays.ConsequentlytheassumptionofconstantAT wouldonlyleadtosmallerrors n Printer-friendlyVersion
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25 in the understanding of the carbonate chemistry when paired with controlled p(CO2), ap InteractiveDiscussion
and thus serves as a reasonable assumption for this experimental system. e
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However, some care must be taken to ensure that the assumption of constant A D
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remains true in an aquarium setting. Organisms continually modify the chemistry of c BGD
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their environment. For A this usually means calcification or the assimilation and rem- s
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ineralizationofothernutrientsandions(forfurtherdiscussionseeWolf-Gladrowetal., n
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2007). To maintain A levels throughout the duration of the experiment, some action a
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must be taken to counteract these modifications. The most straightforward solution is e Experimental
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theadditionofnewseawatertothetanks,whichreplenishesA .Sufficientoverturning, aquarium system for
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depending on the organism and biomass in the aquaria, will guarantee that the A in multi-stressor
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each tank reflects the assumed constant AT of the seawater system. is investigation
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Alkalinity is particularly useful as a control variable for carbonate chemistry in an u
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ocean acidification experimental laboratory because it is not affected by changes in sio E.E.Bockmonetal.
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temperature and is conservative with respect to mixing. Most importantly, the addition
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or removal of CO (and O ) gas from seawater does not change A . This allows for a
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modificationofthetotalamountofCO intheseawatertocontrolasecondparameter, r
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withoutinvalidatingtheassumptionofconstantA .Inthissetup,thesecondparameter
15 T | Abstract Introduction
is controlled by reacting a gas of a particular CO and O content with seawater in
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awaythatallowsadesiredp(CO )andoxygenpercentsaturationtobeachieved.This is Conclusions References
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equilibrationofaknowngaswithseawaterhasthesameresultingcarbonatechemistry uss Tables Figures
as if bubbling had been used to modify the seawater sample. io
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20 2.2 Apparatus ap J I
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To achieve the desired chemistry in MSEAS, a gas mixture is equilibrated with sea-
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water using a Membrana Liqui-Cel 2.5×8 Extra-Flow membrane contactor with ×50 Back Close
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fiber for each aquarium (Fig. 2). The desired gas composition (N2, O2, CO2) is mixed isc FullScreen/Esc
from individual gas cylinders using Omega® mass flow controllers (FMA 5418 0–5 us
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25 SLM, FMA 5411 0–2 SLM, and FMA 5402 0–10sccm, r™espectively). The mass flow ion Printer-friendlyVersion
controllers are operated by a laptop running NI LabVIEW software with communica- P
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tion using a voltage generating NI 9265 4-Channel Analog Output Module combined p InteractiveDiscussion
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with an NI USB-9162 Single Module Carrier . Mass flow controller function is moni- is
™ c BGD
tored using a NI USB-6210 Multifunction DAQ (Fig. 2). Mixing individual gases gives u
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the user complete control over both the CO2 and O2 concentrations. After the three sio 10,3431–3453,2013
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gases mix in the desired proportions, the line is split, providing an identical gas mix-
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ture to two or more replicates. Currently the system is designed with two sets of three a
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mass flow controllers, allowing for two treatment levels. A submerged MARINELAND® er Experimental
aquarium system for
Maxi-Jet 400 Power Head pumps seawater from each treatment tank through a 5µm |
multi-stressor
filter and then through the tank’s associated Liqui-Cel membrane contactor. The gas
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mixture is introduced to the Liqui-Cel in the opposite direction, maximizing equilibra- is investigation
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10 tion. The newly equilibrated seawater is returned to the corresponding treatment tank ss E.E.Bockmonetal.
and the gas flows to waste (Fig. 2). It is the continuous recirculation and equilibration io
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of the treatment seawater that enables such precise control of the CO and O levels. P
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Temperatureofeachtankismaintainedwithatitaniumcoilthroughwhichtemperature- p
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controlled water flows from a Thermo Scientific NESLAB RTE 7 Refrigerated Bath.
Thesystemdesignallowstheseawaterineachaquariumtobeexchangedinaflow- | Abstract Introduction
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through mode, where raw seawater is continuously added at a slow rate, replenishing D
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nutrients,andmaintainingA levels.Theresultantincreasedvolumeofseawaterover- c
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flows, removing organism waste. This rate of overturning must be optimized, and will ss Tables Figures
beorganismandbiomassdependent.Thesystemcanalsoberunasaclosedsystem, ion
20 with no input of seawater, although changes in AT quickly become a concern. Pa J I
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Althoughthesystemwasoriginallydesignedwithalarge,50Ltankinmind,thesize e
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of the treatment tank is easily exchangeable. Several of the experiments performed
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have used much smaller volumes, to fit the experimental organism better, and to max-
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imize control over the chosen parameters. The size of the tank, and therefore the vol- D
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umeofwaterneedingtobeequilibrated,mustbechosenforeachexperimentalorgan- c FullScreen/Esc
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ism. Smaller volumes of water will recirculate through the membrane and equilibrate s
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with the gas more often, leading to better control. n Printer-friendlyVersion
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EquilibrationbetweenthegasandseawaterusingtheLiqui-Celisdoneseparatelyfor a
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eachreplicate,sothereisnomixingoftreatedseawater:eachtankismodifieddirectly e
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andindependently.Theonlysharedpartofanyreplicateisthegascompositionandthe D
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originalsourcewater,eliminatingmanyofthepseudoreplicationproblemsthatcontinue c BGD
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to plague other ocean acidification aquarium experimental designs (Wernberg et al., s
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2012; Havenhand et al., 2010). In addition, because of the independent equilibration n
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foreachreplicate,thereisnoconcernoverinferenceofthecarbonatechemistrybased a
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on measurements made in a header tank. e Experimental
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One of the main advantages of MSEAS, is that it allows the user to choose any de- aquarium system for
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sired CO and O composition, within a large range. The CO level of the gas mixture multi-stressor
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can be chosen by the user to be any value between 0 and 5000ppm, although the is investigation
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maximum p(CO ) of any experiment performed to date is approximately 1500µatm. u
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Barry et al. (2010) includes suggestions of CO2 levels for ocean acidification labora- sio E.E.Bockmonetal.
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tory experiments; the recommendation for a two treatment system is for one treatment
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near a “present-day” atmospheric value of 385ppm and the other at a “future” value a
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of 750ppm. MSEAS is well suited to perform experiments at these specified values, r
but also has the advantage of flexibility in terms of its target CO . This is especially
15 2 | Abstract Introduction
valuable,inthatiteasilyallowsinvestigationofenvironmentsthatarenotatequilibrium
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withtheatmosphere.Forexample,thecoastalregionofwesternNorthAmericaexperi- is Conclusions References
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encesupwellingevents,inwhichseawateralreadyeleveatedinCO2 flowsontopartsof uss Tables Figures
the continental shelf (Feely et al., 2008). For studying impacts of anthropogenic ocean io
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20 acidification on organsims living in elevated CO2 environments such as this, an incre- Pa J I
mental increase to the known elevated value is recommended, using increases simi- p
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lar to those between the original recommended treatment values (385 and 750ppm) r J I
(Barryetal.,2010).Thisiseasilyaccomplishedusingthedescribedaquariumsystem. |
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3 Assessment and discussion u
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Theusefulnessandcapabilityofthissystemisexhibitedbytheexperimentsperformed n Printer-friendlyVersion
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to date. Two of these experiments are described below as examples of the stability a
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that can be maintained – one a week long, the other lasting longer than a month. Both er
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experiments modified the CO and O of the seawater for each treatment and were D
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performed at different temperatures. Discrete samples for A , pH, and O were taken c BGD
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daily during the experiments and analyzed at SIO. A samples were poisoned with s
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saturated mercuric chloride and stored for later analysis which was done by open-cell n
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titration. Discrete pH samples were analyzed spectrophotometrically on the same day a
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as sampling. Values are reported at the in situ temperature and on the total pH scale. e Experimental
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Discrete oxygen samples were pickled immediately and analyzed a few days later by aquarium system for
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Winkler titration. Temperature was monitored every five minutes in all tanks by HOBO multi-stressor
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Pendant® Temperature/Light Data Loggers. is investigation
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3.1 Experiment M7 s E.E.Bockmonetal.
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A week-long experiment was performed on mussel larvae (Mytilus gallorprovincialis) a
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with the described system – one treatment of “ambient” pH and oxygen levels, typical e TitlePage
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of a California coastal upwelling environment, and the other with low pH and oxygen
| Abstract Introduction
levels. For this experiment, the treatment tanks were round 7.5L buckets with lids.
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The larvae were protected from the flowing seawater recirculation by containment in
15 is Conclusions References
a smaller nested bucket that freely exchanged seawater with the main tank. All treat- cu
ments were held at an average temperature of 17.2◦C. Alkalinity varied only slightly ss Tables Figures
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over the week, and consequent control of pH and oxygen levels were very good (Ta- n
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ble 1 & Fig. 3). a J I
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20 In addition to the discrete sampling, a Honeywell Durafet pH sensor was used r J I
continuously throughout the experiment to monitor the carbonate chemistry on short
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timescales, switching between tanks daily. The Durafet sensor data in Fig. 4 shows Back Close
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day-long variability in pH beginning each day when sensor’s location was changed. is
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Four consecutive examples are given, one from each tank. Some fluctuation is seen, u
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25 possibly a respiration signal. This fluctuation is possible due to the somewhat passive io
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approachtocontrollingthecarbonatechemistryinthecurrentsystemdesign.Thecon- P
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stantcompositiongasmixtureissuppliedtoeachLiqui-Cel,butthereisnowaytomea- p InteractiveDiscussion
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sure and counteract the influence of outside factors on the carbonate chemistry of the r
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seawater. Fluctuations such as those seen in Fig. 4 could be dampened if a more ac- D
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tiveapproachtopHcontrolwastaken,usinginformationgatheredbychemicalsensors c BGD
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inthetanksasfeedbacksignalstothegasmixingsystem.DurafetpHsensorsarewell s
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suitedtomonitorthecarbonatechemistry,andforoxygen,anoptodethatmeasuresin n
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real time would be appropriate. Based on the information from these sensors, the gas a
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compositionsuppliedtotheLiqui-Celscouldbecontinuallyadjustedtocompensatefor e Experimental
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any divergences from the desired seawater pH and oxygen levels. The adjustments aquarium system for
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to the gas mixture would help dampen or eliminate the small diurnal signal seen, but multi-stressor
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would also eliminate any large changes in seawater chemistry that might happen over is investigation
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the course of the experiment. u
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3.2 Experiment S32 n
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The system can be used on much longer time scales than a week, demonstrated by e TitlePage
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a32-dayexperimentinvestigatingtheimpactsofvariedpHandoxygenlevelsonsquid
| Abstract Introduction
embryos (Doryteuthis opalescens). The experiment was performed in square 50L in-
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sulated tanks, with the squid egg capsules attached to the bottom. Some turbulence
15 is Conclusions References
wascausedbytherecirculationoftheseawaterforequilibration.TargetpHandoxygen cu
levels were chosen based on values recorded at a location near Scripps (Nam et al., ss Tables Figures
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2011).Forthisexperiment,lowpHandhighoxygenlevelswerepairedinonetreatment, n
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and high pH and low oxygen levels in the other. This is in contrast to experiment M7 a J I
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whichpairedlowoxygenandlowpH,anddemonstratesthesystemflexibilityandinde- e
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pendent control of chosen seawater chemistry. The longer duration of this experiment
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reflectsagrowingneedinthescientificcommunitytounderstandtheeffectsofchronic Back Close
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exposure to low pH on organisms. Results from discrete samples indicate adequate is
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control for a successful biological experiment, even over this extended period (Table 2 u
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25 & Fig. 5). However, there are clear discrepancies from target values and both gradual io
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and abrupt changes during the experiment, some of which are easily explained.
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Thecontroloftheseawaterchemistryinthissystemisbasedonthemolefractionof p InteractiveDiscussion
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CO and O in the gas that is supplied to the Liqui-Cel for equilibration. Any changes r
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