potential methodological influences on the determination

AQUATIC BIOLOGYAquat Biol

Vol. 25: 61–73, 2016doi: 10.3354/ab00660

Published August 10

INTRODUCTION

Dense populations of wild and cultured bivalvemolluscs can play an important role in coastal marineecosystems through the provision of ecological serv-ices and possibly by way of negative environmentaleffects (Shumway 2011). This role is largely linked tothe population’s capacity to capture suspended par-

ticulate matter through suspension feeding (Cran-ford et al. 2011). Particle clearance rate (CR), the volume of water cleared of particles of a designatedsize in a given time, is commonly measured using theindirect method. This method was used to measureCRs reported in 92% of the 133 peer-reviewed publi-cations reviewed by Cranford et al. (2011). The indi-rect approach is based on determining the rate of

© The authors and Fisheries and Oceans Canada 2016. OpenAccess under Creative Commons by Attribution Licence. Use,distribution and reproduction are un restricted. Authors andoriginal publication must be credited. Publisher: Inter-Research · www.int-res.com

*Corresponding author: [email protected]

Potential methodological influences on the determination of particle retention efficiency by

suspension feeders: Mytilus edulis and Cionaintestinalis

Peter J. Cranford1,*, Tore Strohmeier2, Ramon Filgueira3, Øivind Strand2

1Department of Fisheries and Oceans, Bedford Institute of Oceanography, 1 Challenger Dr., Dartmouth, Nova Scotia B2Y 4A2, Canada

2Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway3Marine Affairs Program, Dalhousie University, 1355 Oxford St., Halifax, Nova Scotia B3H 1R2, Canada

ABSTRACT: The retention efficiency (RE) of suspension-feeding bivalve molluscs depends on par-ticle size and is generally assumed to decline below a maximum retention of particles larger than 3to 7 μm. Previous suggestions that the RE spectrum of mussels Mytilus edulis can exhibit variability,possibly as a result of physiological regulation, have been attributed to artifacts associated with theindirect method. The possibility that variable physical properties of seston particles and/or miscal-culations can result in inaccurate RE measurements was examined using 3 methodologies (static,flow-through and a new approach based on the static method) and 3 particle sources (naturalseston, algal cell monocultures and clay). Measurements obtained with the static method varied de-pending on the selected sampling interval. However, this artifact can be removed using frequentsampling and a regression analysis approach. Accurate RE measurements can be obtained with theflow-through method when feeding behaviour is flow independent. For all particle suspensionsand methods, mussels from the study site in Lysefjord, Norway, had a maximum RE for particles>8–11 μm (1 to 5 September 2015). The RE for smaller particles declined gradually, with 50–60%retention of 4 μm particles and 30–40% retention of 2 μm particles. Differences in the RE sizespectra of mussels and tunicates Ciona intestinalis, collected and measured at the same site, furtherindicated that RE was not influenced by potentially confounding methodological factors. Assump-tions regarding the RE spectrum of bi valves have contributed to many conclusions on their ecosys-tem interactions. The reliability of clearance rate measurements obtained using the indirect methodcan only be assured if the effective retention of tracer particles is confirmed and not assumed.

KEY WORDS: Filter feeding · Retention efficiency · Clearance rate · Mytilus edulis · Ciona intestinalis · Physiological regulation · Feeding mechanism

OPENPEN ACCESSCCESS

Aquat Biol 25: 61–73, 2016

removal of particles from a set volume or constantflow. In both cases, CR is a function of the rate thatwater is pumped through the gill (pumping rate, PR)and the proportion of suspended particles that areretained by the feeding apparatus (retention effi-ciency, RE). Coughlan (1969) outlined the fundamen-tal assumptions of the indirect ap proach for estimatingCR, including the requirement that the organismretain tracer particles of a predetermined size rangewith known efficiency (usually 100%). Williams(1982) subsequently showed that the ob served rate ofparticle removal cannot easily be trans lated into a CRwhen RE is less than 100%. Knowledge on the rela-tionship between RE and particle size is thereforeessential for determining accurate CRs and subse-quently for interpreting ecosystem interactions withbivalve populations.

The particle RE of suspension-feeding bivalve molluscs clearly depends on particle size, and thisrelationship has been described as an asymptoticincrease to maximum retention at particle diametersbetween 3 and 7 μm (e.g. Haven & Morales-Alamo1970, Vahl 1972, Jørgensen 1974, Møhlenberg &Riisgård 1978, Palmer & Williams 1980, Lucas et al.1987, Riisgård 1988). Particle retention in Mytilusedulis is reported to rapidly decrease below approxi-mately 4 μm (Møhlenberg & Riisgård 1978), and thisempirical model has become firmly established inthe literature. However, several field studies havereported distinctly different RE size distributions.Strohmeier et al. (2012) showed that blue mussels M.edulis in a Norwegian fjord exhibited seasonallyvariable particle retention size spectra, with a grad-ual increase in RE from small to large particles andmaximum RE at sizes between 7 and 35 μm. Changesin the RE maxima appeared to coincide with changesin the ambient particle size distribution. In addition,RE often declined as particle size exceeded the REmaxima. Similar results with seston diets wereobserved in studies of mussel filtration by Lucas et al.(1987) and Rosa et al. (2015) and have been reportedfor other bivalve species (Stenton-Dozey & Brown1992, Barillé et al. 1993). The divergence of resultsbetween laboratory and field studies raises uncer-tainty regarding the relative contribution of potentialtrophic resources to bivalve growth and suggests thata widely accepted methodological assumption fordetermining feeding rates (effective retention of par-ticles larger than 3–7 μm) may not always be valid.

Strohmeier et al. (2012) suggested that the season-ally variable RE of mussels in nature may indicatea high degree of physiological control of particle capture. However, the absence of a known particle

capture mechanism that would fully explain all theseresults has contributed to a focus on identifying alter-native explanations, particularly methodological errors(Rosa et al. 2015). Strohmeier et al. (2012) noted thatRE artifacts could stem from the improper use of particle-counting instrumentation, the method fordata standardization, flow variations in feedingchambers, and the disaggregation of flocculated par-ticles in feeding chambers. In addition, Rosa et al.(2015) suggested that artifacts may result from non-spherical seston particles confounding the measure-ment of particle size, the escape of motile cells fromcapture, and/or mathematical happenstance. Pres -ently, no definitive conclusion has been reached onwhether or not RE is physiologically controlled orvaries simply as a result of methodological artifacts.

The present study investigated possible sources oferror in RE and CR methodologies with the objectiveof providing recommendations on approaches thatensure a high degree of measurement accuracy. Dataob tained with these methods were also used to deter-mine if the retention of seston particles by M. edulisconforms to the traditionally accepted RE size distri-bution (see above) or to some alternate relationshipwith particle size.

Variations in particle shape can influence the deter -mination of equivalent spherical diameter (ESD) byparticle analyzers and thereby cause errors in thecalculation of the RE size distribution. For example,a long particle may be effectively captured despitehaving a relatively small ESD. The possibility ofmethodological artifacts arising from variations in theshape of different particle sources in natural sestonwas addressed by comparing RE size distributionsobtained for natural seston suspensions with thosefrom reference diets containing particles with uni-form properties across their size spectra (marine clayand algal cell monocultures). In the absence of a sig-nificant particle shape effect, the RE size distributionfor all experimental particle suspensions should besimilar. The potential effect on RE measurementsfrom variability in the physical properties of sestonparticles was also addressed by examining particleretention by species with different feeding mecha-nisms. Methodological artifacts introduced in the cal-culation of RE by the physical properties of differentparticles should consistently arise for a given particlesuspension regardless of the particle capture mecha-nism. Published RE size distributions for M. edulis(cirri and cilia particle capture anatomy) and the vasetunicate Ciona intestinalis (mucociliary net) haveboth been characterized as including a rapid declinein RE below a particle size threshold (e.g. Randløv &

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Cranford et al.: Accuracy of retention efficiency measurements

Riisgård 1979 and references in this section for mussels). Although the threshold size for maximal REvaries between these species, the shape of the REsize distribution should follow the same general pat-tern for both species if methodological errors wereinfluencing the measurements. In the absence ofmethodological errors, changes in the shape of theRE size distributions of mussels, relative to tunicates,would reveal a higher degree of flexibility in particleretention. The relatively simple mucus net of C.intestinalis may be expected to produce a typicalsieve retention response, with RE rapidly decliningbelow the pore size of the net, while the more com-plex feeding apparatus of mussels may facilitate amore variable response (e.g. Ward et al. 1998, Stroh -meier et al. 2012). All of the methodological valida-tions and comparisons described herein were con-ducted using disaggregated particle suspensions toprevent errors that would occur if the mussel, tuni-cate or experimental apparatus was altering the am -bient particle size distribution by means other thanan active particle capture mechanism.

Measurement accuracy was assessed by compar-ing results obtained using 3 methods (static, flow-through and a new approach based on the staticmethod) and 3 different particle suspensions (naturalseston, algal cell monocultures and clay). In addition,a revised static method was developed that employs arigorous sampling approach designed to statisticallyassess the reliability of all measurements. RE resultsobtained with these different methods were comparedto assess the inherent assumption that they providesimilar results. The flow-through approach strictlyrequires that the suspension is not recirculated by thesuspension feeder(s) and that the results are not flow-dependent (Riisgård 1977), while the static methodexponential model is expected to account for recircu-lation (Coughlan 1969). Both approaches are assumedto give an equivalent standardized RE size spectra,despite the fact that the flow-through method pro-vides a near-instantaneous particle retention re sponse,while the static method provides a cumula tive re -sponse after several passes of the suspension throughthe filtration apparatus.

MATERIALS AND METHODS

Study site, test organisms and particle suspensions

RE measurements for blue mussels Mytilus edulisand vase tunicates Ciona intestinalis were performedbetween 1 and 5 September 2015 at a field station

(Måkastein) located near the head of the Lysefjord(59° 03.24 N, 6° 837.77 E). Lysefjord is located on thesouthwestern coast of Norway and is approximately40 km long and 0.5 to 2 km wide, with a depth rangefrom 13 m at the outer sill to a maximum of 460 m.The mean tidal range is 0.4 m. Seasonal variationsin suspended particle matter concentrations at thestudy site average 3.1 mg l−1 (SD = 1.2; Strohmeier etal. 2015). Mussels and tunicates were collected at thestudy site by divers from approximately 7 m depth on22 July 2015. Mussels were initially graded to a shelllength range of 49 to 62 mm, and both species werethereafter held at 7 m depth at Måkastein to accli -matize to local conditions. Mussels and tunicatesselected for feeding experiments ranged between 52and 58 mm shell length and 74 to 96 mm tunic height,respectively. Three days prior to conducting feedingexperiments, Velcro strips were fixed to the base ofthe tunic with cyanoacrylate glue to allow each tuni-cate to be positioned within feeding chambers.

A variety of particle suspensions were employed inthis study, including 5 species of cultured algal cells,natural seston pumped from 7 m depth at Måkastein,and clay particles of a size distribution similar to theseston diet but containing particles of uniform shape.The algal species were chosen from available culturestocks at the Austevoll Research Station to includeoval and pennate shapes based on microscopic meas-urements. The cultured stocks included Thalassiosirapseudonana (4.75 μm modal size based on laser particle counter analysis described below), Chaeto-ceros calcitrans (7.25 μm), Skeletonema costatum(14.25 μm), Tetraselmis suecica (12.75 μm) and C.muelleri (6.75 μm). Length:width ratios measured bymicroscopy ranged from 1.5 (C. muelleri) to 2.6 (S.costatum). The clay was obtained from a naturalglaciomarine deposit composed principally of kaolin-ite and illite that was initially dry-sieved at 63 μm.The unfiltered seawater for ex periments with naturalseston was supplied using a shallow well jet pump.High sheer forces and rapid pressure changes withinthe pump ensured that a disaggregated seston sus-pension was delivered to all experimental chambers.The clay and algal cell suspensions served as controlsfor the potential effects of variable particle shapes inthe seston on instrument calculations of ESD and anyensuing effects on particle retention by mussels.

Particle analysis

Analysis of all particle suspensions was performedusing a PAMAS field laser particle counter (Mod -

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Aquat Biol 25: 61–73, 2016

el S4031GO) containing an HCB-LD-50/50 light-scattering sensor. This instrument provides particlecounts (numbers) within a potential interval of detec-tion between 1 and 200 μm (32 size channels). Theanalyzer was initially set to determine the particlesize distribution spanning a total range from 1 to32 μm (studies with the flow-through method) butwas reduced to a detection range from 1 to 16.5 μmfor the remainder of the experiments. The PAMASwas factory-calibrated according to ANSI/NFPAT2.9.6 R1-1990 and ASTM F658-80 for 17 sizes of certified latex beads spanning the full range of theinstrument (1200 μm). The largest standard deviationdetected during calibration for particle sizes inthe range 1 to 40 μm (12 different sizes used) was0.52 μm. Instrument calibration was confirmed atthe study site using certified particle size standards(Beckman Coulter latex beads; nominal 2, 5 and10 μm diameter). The PAMAS was used for both discrete sample analysis (10 ml water samples) andtime-series analysis of particle suspensions. The lat-ter was accomplished by programming the instru-ment to pump water from feeding chambers at setintervals (more details in the next subsection).

Retention efficiency

Static method

Prior to starting each experiment, 3 mussels or 7tunicates were transferred from a holding tank toclear PVC feeding chambers (13 cm height and10 cm diameter; 1000 ml volume) supplied with flow-ing unfiltered seawater from 7 m depth. The tuni-cates were attached to Velcro strips on the side of thechamber, while the mussels rested on a perforatedshelf on the base of the chambers. Water withinchambers was continually mixed by aeration in aninternal airlift tube that pumped water from belowthe shelf up to the surface. This recirculationapproach results in a low degree of turbulence. Anidentical chamber without any suspension feedersserved as a control for sedimentation. The suspen-sion feeders were initially left undisturbed for ca. 1 h,until observed to be actively feeding (open siphons).The water flow to the chamber was then terminated.For experiments with natural seston, the PAMAS wasimmediately set to withdraw 10 ml water samplesfrom the chamber at 30 s intervals, to determine theparticle size distribution, and then to return the waterto the chamber as the next sample was acquired. Forstudies with suspensions containing clay or algae

cells, a small volume (<10 ml) of a concentrated par-ticle stock suspension was added to the unfilteredseawater in each chamber immediately after the sea-water flow was terminated. The particles wereallowed to mix for 1 min, and then the PAMAS wasstarted for semi-continuous analysis, as for the natu-ral seston. A Turner Cyclops chlorophyll a fluorome-ter, inserted through the top of the static chambers,was used to monitor the decline in microalgal con-centrations throughout each experiment. The incu-bation was terminated when the fluorescence signaldeclined to a constant value, which generally re -quired 30 min. These fluorometer data were notemployed in the following RE calculations, as they donot provide information on fluorescent particle size.

A suspension feeder pumping at a constant rate ina static chamber will remove particles at a rate that isa function of the concentration of particles in thechamber and the capacity of the filter feeder to retainthese particles (RE). As the suspension feeder contin-uously removes particles, the concentration (C) in thechamber will decrease in an exponential form, result-ing in a linear decline in lnC over time (Coughlan1969). Semi-continuous (30 s interval) particle count-ing with the PAMAS analyzer monitored this pro-gressive particle reduction and the RE for each parti-cle size, and for any given elapsed time, REsize,t isproportional to:

REsize,t ∝ ln(Csize,0 /Csize,t) (1)

where Csize,t and Csize,0 are the concentrations of par-ticles of a given size at times t and 0, respectively. Forall measurements, a check was made to confirm thatthe decline in lnC was linear over time (i.e. confirmassumption of constant pumping rate). REsize,t valueswere standardized across the full range of particlesizes measured by setting the highest REsize to 1 (designated as REmax) and proportionately adjustingREsize for the other particle size bins (Møhlenberg &Riisgård 1978). Standardized REsize values are re -ported below as REstd. RE values obtained by Eq. (1)represent the cumulative particle capture responseof the suspension feeder(s) in the chamber over anelapsed time ranging from 30 s to 30 min. RE wasmeasured over a range of elapsed times to determineif the standardized RE for each particle suspensionwas dependent on feeding time.

The precision of values obtained using Eq. (1) isrelated to the variability in particle counts measuredduring just 2 sampling times (start and end). Toincrease measurement precision and to better assessthe quality of RE measurements, the exponentialdecay of particles in feeding chambers was analyzed

64


using an approach that utilizes all the time-seriesdata simultaneously to provide a single REsize valuefor each diet treatment. Particle depletion in a staticchamber is represented as a continuous function thatfollows (from Coughlan 1969):

(2)

where C is the concentration of particles and λ is theexponential decay constant, which in turn is a func-tion of PR, RE and the chamber volume (V). Given thedependence of RE on particle size, λ is size-specific:

λsize = λsize (PR,REsize,V) (3)

Given that PR and V are assumed to be constantduring the feeding trials and independent of particlesize, λsize can be used as an indicator to compare REamong particles of different size. Therefore, Eq. (2)can be integrated for each particle size:

Csize,t = Csize,0 × e–λsizet (4)

Applying logarithms:

lnCsize,t – lnCsize,0 = –λ sizet (5)

This linear form of the equation was applied to thetime-series data collected with the PAMAS for eachparticle size, and λsize was calculated as the slope ofthe linear regression between lnCsize and the elapsedtime. It was assumed that the maximum λsize corre-sponds to particles that can be effectively retained(i.e. 100% after standardization). Accordingly, thefollowing relationship was used to standardize RE foreach particle size:

(6)

where λmax is the maximum λsize obtained in Eq. (5).This results in a RE scale ranging from 0 to 1, repre-senting particle sizes that are not retained and re-tained at maximum efficiency, respectively. The r2 ofthe regression between lnCsize and elapsed time wasused as a standardized means of assessing the effectof temporal variability in (1) particle counts on thesemeasurements and (2) pumping rate of bivalves. Tominimize the effect of this analytical error or behav-ioural change, regressions with r2 below 0.95 wereconsidered to represent a condition where the REsize

value obtained was of low precision because of ex-cessive variability in particle counts between sam-pling times or changes in pumping behaviour. Settingsuch a high r2 threshold represents an extremely con-servative approach (i.e. sta tistically robust) to ensur-

ing the high precision of RE values calculated by thismethod. Residuals were also visually analyzed to discard bias across elapsed time.

Flow-through method

The flow-through method for measuring particleCR and RE is intended to simulate more natural conditions experienced by sessile suspension feedersthan the static method. The feeding chambers em -ployed were the same as described by Strohmeier etal. (2009, 2012). In brief, the internal dimensions of thechambers were 3.8 cm wide, 19.5 cm long and 8.1 cmhigh, with an internal design that constrains recircu-lation of water past the suspension feeders when anacceptable flow speed is provided. RE was measuredfor mussels exposed to the natural seston using40 separate flow-through chambers. Four additionalchambers were left empty to control for any effect thechamber may have on the outflow particle concen -tration. The seawater supply to all chambers waspumped from 7 m depth (15 m total depth at seawaterinlet) into a header tank. The flow rate to each cham-ber from the header tank was controlled by individualvalves, and the flow rate exiting each chamber wasmeasured at the chamber outlets immediately beforebeginning any measurements. The bivalves were leftundisturbed in chambers with flowing seawater atleast 1 h to resume feeding before sampling waterfrom the outlet of the chambers. Water samples col-lected from each chamber outlet were analyzed forparticle concentration and size distribution using thePAMAS particle analyzer. Three 10 ml subsamplesfrom each water sample were analyzed using the PAMAS, and mean particle counts for each suspensionfeeder were used to calculate size-specific RE (32 particle size channels) according to:

REsize = 1 − (Ce / Cc) (7)

where Cc is the particle count exiting the controlchamber and Ce is the count in water exiting the sus-pension feeder chamber. CRs were calculated as theproduct of the flow rate to the chamber and the RE ofparticles averaged over the 3.75 to 7.75 μm sizerange. Suitable flow rates for the chambers were pre-viously identified by Strohmeier et al. (2009; 1114 lh−1 for mussels of similar size as used in the presentstudy) to ensure that measured CRs are flow inde-pendent. However, additional tests were conductedto further investigate the possibility of flow effects onRE. For this assessment, 80 measurements were con-ducted with mussels using flow rates between 1 and

Ct

C= −λdd

= λλ

REsizesize

max

65

Aquat Biol 25: 61–73, 2016

50 l h−1. All RE measurements were standardizedas for the static method; REsize was expressed as afraction of REmax.

Statistical analysis

Curve fitting and regression analysis were per-formed with SigmaPlot Version 12 (Systat Software).One-way ANOVA comparisons were performed at α =0.05 with Systat Version 13 software (SPSS). Priorto hypo thesis testing, the data were screened for nor-mality and homo scedasticity by examining normalprobability and residual plots (Wil kinson et al. 1996).

RESULTS

Characterization of experimental particle suspensions

Average particle size distributions for the naturalseston and seston/ clay mixtures are shown in Fig. 1.The seston from 7 m depth at the study site consistedprimarily of particles smaller than 16 μm, with asteady logarithmic decline in particle counts acrossthe size spectra (Fig. 1a). The volume concentrationpeaked at approximately 5 μm and rapidly declinedfor particles larger than 11 μm (Fig. 1b). Addition ofthe stock clay to the unfiltered seawater (referencesuspension) resulted in a uniform increase in particlecounts for all size bins, with counts increasing by anaverage of 35% (Fig. 1a). The clay additions had littleeffect on the shape of the seston volume distribution(Fig. 1b). Particle counts in the algal cell diet treat-ments were between 7 and 12 times higher than forthe natural seston diets. The median size (ESD) of the5 microalgal species measured with the PAMAS wassimilar to median cell lengths obtained by micro scopy(difference in median size averaged 1.0 ± 1.1 μm SD).

RE based on the static method

Water temperature and salinity during the studyperiod were 12.7 ± 0.5°C (mean ± SD) and 27.2 ± 1.6,respectively. RE size distributions of mussels feedingon natural seston are shown in Fig. 2. The standard-ized RE results (REstd) based on Eq. (1) were cal -culated for 5 sampling intervals ranging from 1 to16 min. The longest time span was chosen after pre-liminary observations showed that the decline in lnCremained linear for this period. The standardization

of these data assumed that maximum RE occurredwithin the size range 1.25 to 11.25 μm. The reason fordeciding on this range is described below. Althoughthe shape of the REstd size distribution was generallysimilar regardless of the time between initial andfinal samples, RE increased for most particle sizeswith increasing elapsed time (Fig. 2a). Short sam-pling intervals (<2 min) resulted in relatively lowRE across the size spectra and higher variability com-pared with longer intervals (Fig. 2a). Longer sam-pling intervals tended to reduce the measured REmax,and an elapsed time of 16 min indicated maximumretention for particles larger than approximately 8 to9 μm. RE decreased gradually below this threshold toan average of 42% for the 2.25 μm size bin (Fig. 2a).The average RE for 3 to 4 μm particles was approxi-mately 60%.

66

b

Particle size (µm)

0 2 4 6 8 10 12 14 16 180.00

0.02

0.04

Rel

ativ

e vo

lum

e co

ncen

trat

ion

(µl l

–1)

0.06

0.08

0.10

Par

ticle

co

unt

(par

ticle

s 10

ml–

1)

100

101

102

103

104

105

106

aSeston Seston and clay

Fig. 1. Particle size distributions of the natural seston (closedcircles) and seston/clay mixture (open circles) used in feed-ing experiments with mussels and tunicates. Size distribu-tions are provided based on (a) particle numbers per 10 mlsample and (b) volume concentrations relative to the totalparticle volume in suspension (μl l–1). Mean (±SD; n = 5) val-ues are shown for each particle suspension sampled from

static chambers at the start of feeding experiments


Unlike the traditional approach to calculating REfrom the static method (Eq. 1), RE results obtainedusing Eq. (6) (referred to hereafter as the regressionmethod) utilized all the time-series data (30 s sam-pling intervals over a total period of 15 min) to char-acterize the retention of particles in each size bin.Mean results obtained using this method showed asimilar relationship between RE and particle size(Fig. 2b) as observed for the traditional method witha 16 min sampling interval (Fig. 2a), albeit with lowervariability. The results of regression analysis of therelationship between lnCsize and sampling time arealso shown in this figure, and the r2 value declinedbelow 0.95 for particles larger than 11.25 μm. The

relatively high variability of particle counts in largersize bins decreased the curve fit to a level where theprecision of REstd results was too heavily influencedby temporal variability in the relatively low particlecounts to provide precise results. For this reason, allRE measurements reported for the seston diet werelimited to the 1.25 to 11.25 μm size range (Fig. 2).

Mussels exposed to the reference particle suspen-sion, containing a mixture of natural seston and clayparticles, exhibited similar REstd spectra (Fig. 3) asdescribed in this subsection for the pure seston sus-pension (Fig. 2). As with the seston diet, the resultsobtained using Eq. (1) show that REstd progressivelyincreased for most particle sizes as the elapsed timein the chamber increased (Fig. 3a). The regressionmethod identified the size range between 2.25 and11.25 μm as providing precise values for this particlesuspension (r2 ≥ 0.95), and REstd declined steadilybelow a mean REmax at 11.25 μm (Fig. 3b). Curvesdescribing an exponential rise to a maximum were fitto mussel REstd data for each particle suspension andprovided the following equations:

Seston (Fig. 2b): REstd = 1.067 (±0.048) {1 − e[−0.227 (±0.025) × Size]}; r2 = 0.985, p < 0.0001 (8)

Reference (Fig. 3b): REstd = 1.145 (±0.050) {1 − e[−0.197 (±0.019) × Size]}; r2 = 0.987, p < 0.0001 (9)

where values in parentheses are the 95% CI for eachcoefficient. Despite the close fit of these data to theregression model, coefficient CIs overlapped for bothdiets, indicating that the addition of clay to naturalseston had no apparent effect on the size-specificREstd response of mussels. The retention of the 5 algalcell suspensions by mussels (Fig. 4), calculated usingthe regression method, followed a similar relation-ship with particle size as for the seston (Fig. 2) andreference diets (Fig. 3).

The REstd responses of Ciona intestinalis, averagedfor both the seston and reference diets, are shownin Fig. 5. As with the mussels, the traditional cal -culation approach gave markedly different resultsdepending on the length of the sampling interval(Fig. 5a). The regression approach indicated thatmeasurement precision was high for particle sizesbetween 1.25 and 11.25 μm and that over 95% of par-ticles larger than approximately 5 μm were retained(Fig. 5b). REstd declined below this size to an averageof 71% retention for 1.25 μm particles. The followingequation described the relationship between REstd

and particle size for this species:

67

0 2 4 6 8 10 12 14 16 18

RE

std

0.0

0.2

0.4

0.6

0.8

1.0

1 min 2 min 4 min 8 min

a

16 min

Particle size (µm)

0 2 4 6 8 10 12 14 16 18

RE

std

and

r2

0.0

0.2

0.4

0.6

0.8

1.0 br 2

REstd

Fig. 2. Standardized retention efficiency (REstd; mean ± SD;n = 5) of natural seston particles by Mytilus edulis as meas-ured by the static method using (a) traditional data analysis(Eq. 1 in text based on 5 standard elapsed times: 1, 2, 4, 8and 16 min) and (b) time-series regression method (Eq. 6 intext), showing r2 values; only REstd values with an associatedr2 ≥ 0.95 are displayed to control for errors from excessive

variability in particle counts

Aquat Biol 25: 61–73, 2016

REstd = 0.961 (±0.010) {1 − e[−0.970 (±0.075) × Size]}; r2 = 0.937, p < 0.0001 (10)

Comparison of Eqs. (8−10) (and Figs. 2−5) showsthat the tunicates retained a larger fraction of all particle sizes below approximately 8 μm than themussels.

RE based on flow-through method

Mussel RE values averaged across the 3.75 to7.75 μm size range of seston particles (selected basedon traditional assumptions regarding maximum RE)declined exponentially with increasing flow speed(decreasing water residence time) in the feedingchambers (Fig. 6). Although this range of flows andRE values can occur under natural conditions, CRmeasurements obtained with this method are only

68

0 2 4 8 106

Cell size (µm)12

RE

STD

0.0

0.2

0.4

0.6

0.8

1.0

Chaetoceros muelleri Tetraselmis suecica Skeletonema costatum Chaetoceros calcitrans Thalassiosira pseudonana

RE = 1.243 (1 – e )(–0.146 x Size)

r 2 = 0.988

Fig. 4. Standardized retention efficiency (REstd) of algal cellcultures by Mytilus edulis measured using the static cham-ber method and time-series regression analysis (Eq. 6 in text)

0 2 4 6 8 10 12 14 16 18

RE

std


a

16 min

Particle size (µm)0 2 4 6 8 10 12 14 16 18

RE

std

and

r2

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

b

r 2

REstd

Fig. 3. Same as Fig. 2 except that the standardized retentionefficiency (REstd; mean ± SD; n = 4) of mussels is for a dietthat consisted of a mixed suspension of seston and clay

particles

a

Particle size (µm)0 2 4 6 8 10 12 14 16 18

RE

std

and

r2

0.0

0.2

0.4

0.6

0.8

1.0 b

r2REstd

0 2 4 6 8 10 12 14 16 18

RE

std

0.0

0.2

0.4

0.6

0.8

1.0


Fig. 5. Same as Fig. 2 except that standardized retention effi-ciency (REstd; mean ± SE; n = 3) is for tunicates Ciona intesti-nalis exposed to natural seston (n = 1) and mixtures of seston

and clay particles (n = 2)


considered to be accurate when independent of flowrate. Individual mussel CRs, calculated from RE val-ues measured at the different flows, were observedto be dependent on flow at relatively low and highspeeds (Fig. 5). The CR data were grouped over 7flow ranges (0 to 5, 5 to 7.5, 7.5 to 10, 10 to 15, 15 to20, 20 to 25 and 25 to 30 l h−1), and normal probabilityplots showed that a normal distribution was presentonly for the 3 groups with flow rates between 7.5 and20 l h−1. Skewed distributions and heteroscedasticityof CR data were detected for the remaining flowranges (Fig. 5). One-way ANOVA showed no sig -nificant difference in mean CR values measured forthe 7.5 to 10, 10 to 15 and 15 to 20 l h−1 flow ranges(df = 28, F-ratio = 0.748, p = 0.483). This flow-independent range provided an average CR of 4.5 lh−1 (SD = 0.8) for mussels averaging 54.8 mm (SD =1.3) shell length.

The effect of flow rate on the REstd size distributionis shown in Fig. 6. These calculations assume that theREmax for the seston suspension occurred within thesame size range as identified for the static method(1.25 to 11.25 μm). As with the static method, theshape of the REstd size distribution varied with thetime the mussels had to clear particles passingthrough the chamber (i.e. residence time). Highestresidence times occur during the flow dependencyphase (0 to 7.5 l h−1) and resulted in relatively highretention of all particle sizes below the REmax (Fig. 6).Reducing residence time resulted in a reduction inthe efficiency of mussels to retain the majority ofavailable seston particles (Figs. 1 & 7). The increasedvariability in REstd during the flow inhibition phase(>20 l h−1) reflected the relatively small difference inparticle concentrations between the control and mus-sel chambers. At these high flows, the effect of mus-sel feeding on seston concentration was approachingthe range of variability in seston concentrations incontrol chambers.

Eq. (8) was also plotted in Fig. 7 for comparison ofREstd results obtained for natural seston using boththe flow-through and static chamber methods. Bothapproaches showed a gradual decline in REstd belowan REmax within the 9 to 11.25 μm size bins. However,the static method indicated a slightly faster decline inREstd with reducing particle size and a lower reten-

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RE

0.0

0.2

0.4

0.6

0.8

1.0

0 10 40 5020 30

Flow rate (l h–1)

CR

(l h

–1)

0

1

2

3

4

5

6

7

A B C

RE

a

b

= 0.999 e –(0.0711 x Flow)

r 2 = 0.854

Fig. 6. Relationships between the flow rate to feeding cham-bers (flow-through method) and the (a) unstandardized re-tention efficiency (RE) and (b) clearance rate (CR) of naturalseston by Mytilus edulis. The labels and lines (drawn byeye) on the CR plot indicate (A) flow dependency, (B) flow

independency and (C) feeding inhibition phases

0 2 4 8 106

Particle size (µm)12

RE

std

0.0

0.2

0.4

0.6

0.8

1.0

0 to 7.5 l h–1 (n = 30) 7.5 to 20 l h–1 (n = 31) 20 to 30 l h–1 (n = 60) Static method

Fig. 7. Mean (SD) standardized retention efficiency (REstd) ofnatural seston particles by Mytilus edulis measured by theflow-through method using the indicated flow rate ranges.The broken line summarizes REstd data obtained with thetime-series static method (Eq. 8 in text). Sample size (n) is

shown for each flow rate range

Aquat Biol 25: 61–73, 2016

tion of particles smaller than 4 μm (Fig. 7). However,even for these small particle sizes, there was con -siderable overlay in RE results obtained using bothmethods (Figs. 2 & 7).

DISCUSSION

Blue mussels Mytilus edulis in Lysefjord, Norway,displayed maximum RE for particles larger than 8 to11 μm. The retention of smaller particles declinedslowly, with 50 to 60% retention of 4 μm particles and30 to 40% retention of 2 μm particles. This RE sizedistribution was maintained for mussels fed naturalseston, mixtures of seston and clay, and algal cellmonocultures. These observations indicate that theestablished relationship between RE and particlesize for this species (see ‘Introduction’) does not con-sistently apply across the broad biogeographic distri-bution of this species. The CRs of mussels, whichaveraged 4.5 l h−1 for the flow-independent phase,were measured using the commonly accepted ap -proach of assuming 100% RE for particles larger thanapproximately 4 μm (Cranford et al. 2011). Ratherthan being completely retained, 4 μm particles wereactually retained at 61 to 86% efficiency, indicatingan incorrect use of the indirect method (Williams1982). Recalculation based on the measured deple-tion of particles in the 8.25 to 11.25 μm size rangeyielded a 20% increase in average CR (5.6 l h−1; SD =1.5). It is therefore recommended that the RE size dis-tribution be routinely determined to identify the sizerange of effectively retained particles prior to calcu-lating bivalve CRs with the indirect method.

The present study investigated possible sources oferror in 2 basic approaches for estimating RE (staticand flow-through methods) with the purpose of vali-dating their performance in studies with natural ses-ton and to recommend any methodological improve-ments. The RE size distribution measured using thestatic method was shown to be dependent on theselected sampling interval. RE data obtained usingthe static method showed that the retention of mostparticle sizes increased with the time the musselsand tunicates were exposed to the particle suspen-sion. This apparent time dependency in RE is notindicative of a biological response but reflects accu-mulated mathematical errors related to variability inthe concentration decay and the apparent size ofmaximum particle retention (REmax). Both parametersare more precisely determined as the differencebetween C0 and Ct increases with time. Given thatstandardization of individual measurements is based

on REmax, values obtained after a relatively long ex -posure time (16 min in the present study) should pro-vide the most reliable measure of the true RE sizedistribution. The decline in lnC remained linear overthis time period in the current study, but this require-ment should be checked prior to any calculationsof RE by the static method. A consequence of thisrequirement is that the static method is unsuitable formeasuring rapid fluctuations in particle capture ratesand efficiencies.

The availability of automated particle counting in -strumentation facilitates a more statistically robustapproach for measuring RE and CRs using the staticmethod. The regression approach developed in thecurrent study utilized an average of 30 samplingperiods to calculate the RE of particles in each of32 size bins over short (30 s) sampling intervals. Thislarge sample size facilitates the high precision ofmeasurements, while setting an r2 threshold at 0.95ensures that only accurate results are presented. Aregression approach has been used previously toensure the linear decay of particle concentrations instatic chambers (Bayne et al. 1999) and to calculateCRs (Riisgård & Seerup 2003). However, the auto-mated high-frequency sampling and particle analy-sis approach reported herein, combined with Eq. (6),represents a novel method for estimating RE thatensures a high degree of measurement accuracy andstatistical precision.

Standardized RE results based on the modifiedstatic method were generally similar to those ob -tained with the flow-through method. The REmax

occurred within the same 8 to 11 μm size range, andretention declined to approximately 60% for 4 μmparticles. Some variation in the REstd spectrum wasobserved to occur for the different flow rates, withrelatively high RE measured at low flows (<7.5 l h−1)and highly variable efficiencies measured at highflows (>20 l h−1; Fig. 6). The flow-through methodis known to provide flow-dependent feeding rateresponses because of flow bypass and/or inhibition ofthe bivalve pump at high flows and recirculation atlow flows (e.g. Filgueira et al. 2006). Consequently,CR and RE results obtained using this method areonly reliable for the flow independency phase, whichwas between 7.5 and 20 l h−1 (Fig. 6) for the chambergeometry employed in the current study. Recircula-tion at low flows will result in the flow-through cham-ber behaving more like a static chamber, in whichparticles are depleted exponentially during the resi-dence time of water in the chamber. The assumedlinear difference in particle concentrations exitingthe control and experimental chambers (Eq. 7) is

70


therefore not valid for low flows, resulting in erro-neous RE measurements. The high variability in REvalues measured at high flows resulted, at least inpart, from the magnitude of particle removal by thesuspension feeder approaching the analytical errorfor estimating particle concentrations.

Rosa et al. (2015) reported that 4 μm and largerpolystyrene beads were retained with maximum effi-ciency by mussels and suggested that lower RE val-ues, such as reported herein for particles ≥4 μm, arethe result of methodological errors related to severalpotentially confounding factors (see ‘Introduction’).These re sults are in contrast to those of Dunphy et al.(2006), who showed that oysters retained 6 μm poly-styrene beads at a significantly lower efficiency than15 μm beads. Oysters have been reported to exhibit asimilar RE size spectra as mussels (Møhlenberg &Riisgård 1978). The present study used a natural par-ticle source, with uniform physical properties acrossthe particle size distribution, to control for possiblesources of error in RE measurements caused by vari-ations in particle shape and surface properties. Theuse of polystyrene beads was considered less suit-able as a reference suspension for the following rea-sons. First, the reference particles should ideallyinclude a continuous particle size distribution thatmimics the seston under study and which can beadded to the seston for simultaneous particle analysisusing the same instrumentation. Mathematical arti-facts may arise if RE responses to reference and ex -perimental particles are measured and/or standard-ized separately. Second, polystyrene microspheresare inherently hydrophobic and strongly attract eachother. A surface charge, added during their synthe-sis, and/or the use of a surfactant is required to facil-itate their dispersion in aqueous media. AlthoughRosa et al. (2013) showed that the surface charge ofthe fluorescent beads employed was similar to othermarine particles, this charge is insufficient for col-loidal stability, and a surfactant is employed to main-tain particle dispersion. Addition of dispersed beadsto seawater in feeding chambers or directly into theinhalant siphon dilutes the surfactant, and the hydro -phobic beads regain a high affinity for attraction/aggregation and attachment to surfaces, includingbivalve feeding structures. In addition, surfactants ofthe type employed to ensure microsphere dispersionare known to inhibit the CRs of M. edulis (Ostroumov& Widdows 2006) and may result in an artificial feed-ing response.

The present study employed a glaciomarine clay toserve as a reference diet. The particle size distribu-tion of the clay was very similar to that of the seston

at the study site (Fig. 1). Previous particle aggrega-tion tests with this same clay source resulted in noflocs being formed even after 1 wk in a flocculator(Milligan 1996). Although this same source of claywill flocculate at relatively high concentrations understatic conditions, concentrations below 30 mg l−1 didnot begin to form flocs for several hours (Kranck1980). The aggregation of clay particles during therelatively short period within feeding chambers wastherefore expected to be minimal. Previous compar-isons of average CR responses of M. edulis to sestonparticles and mixed suspensions of seston and natu-ral marine inorganic matter showed no significantdifferences (Bayne et al. 1987). However, CRs maybe expected to decline at clay concentrations greaterthan employed in the present study (Widdows et al.1979). Average RE size distributions measured formussels exposed to both the seston and reference(seston/clay mixture) suspensions were very similar,and the regression equation coefficients describingthe RE responses to these 2 treatments were within95% CIs. It was therefore concluded that the re -ported RE responses of mussels to the natural sestonparticles available during this study are not causedby artifacts from confounding physical attributesof constituent particles but accurately reflect theresponse of mussels under the conditions at the studysite. However, particle shape effects on RE cannotalways be discounted. Under conditions where theseston is dominated by lengthy phytoplankton cellsor detritus, the variable orientation of these particlesas they pass the instrument detector will result inestimates of particle sizes that do not adequatelyreflect their capacity to be retained by a suspensionfeeder.

The comparison of RE responses by M. edulis andCiona intestinalis provides further evidence of theaccuracy of the reported measurements. As noted inthe ‘Introduction’, possible methodological artifactsthat could potentially give an erroneous impressionof an individual’s RE spectrum should consistentlyarise regardless of the suspension feeder being stud-ied. However, the tunicates were observed to have amarkedly different RE spectrum (Fig. 5) than musselsand retained >70% of particles larger than 1 μm. TheRE spectra of C. intestinalis has been reported previ-ously based on laboratory studies with diets consist-ing of marine flagellates (Randløv & Riisgård 1979),algal cell cultures and bacterioplankton (Jørgensenet al. 1984). The present study with natural sestonconfirms the conclusion from those studies that fil -tration through the tunicate mucus net consistentlyresults in the effective retention of particles as small

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Aquat Biol 25: 61–73, 2016

as 1 to 2 μm. The filtration mechanism of mussels isconsiderably more complex, and the exact mecha-nisms of particle capture are still controversial(reviewed in Ward et al. 1998). Numerous factors, inaddition to particle size, have been shown to stronglyinfluence particle capture without any methodologi-cal explanation (reviewed by Ward & Shumway2004). The capture of particles takes place upstreamof a current produced by the beating of lateral cilia/cirri on the ctenidial filaments. The laterofrontalcilia/cirri have been proposed to act as mechanicalsieves, such that particle RE is somewhat correlatedwith the degree of development of these structures(Jørgensen et al. 1984, Riisgård 1988, Silverman et al.1996). However, Ward et al. (1998) suggested thatthis particle impaction mechanism is not possible atthe applicable Reynolds numbers. These authors pro-posed a hydrosol filtration theory in which particlesare captured on the ctenidial filaments throughdirect interception with the frontal ciliary tracts.They suggest that interspecific differences in particleRE are associated with the flow patterns that are pro-duced by the type of latero frontal cilia/cirri present.They also proposed that an individual bivalve mightadjust feeding rate and efficiency by altering theamount of water passing directly through the interfil-amentary spaces through the regulation of vorticalflow patterns set up by the beating of the latero -frontal cirri. A mechanical sieving mechanism is notcompatible with our observations of a slow decline inRE below the size of maximum retention. In addition,previous observations of a seasonally variable REsize distribution (Strohmeier et al. 2012, Rosa et al.2015) lend support to the theory of a hydrosol filtra-tion mechanism in bivalves, as described by Ward etal. (1998).

Strohmeier et al. (2012) reported that relativelysmall particles were occasionally captured moreeffectively than larger ones. The present study ex -amined the precision of RE measurements acrossthe ambient particle size spectra and showed thatthe high variability of particle concentrations in sizebins larger than 11.2 μm consistently reduced theprecision of RE values for those sizes. Measure-ments obtained for relatively large particle sizeswere shown to be of low reliability and are notreported (Figs. 2−5 & 7). However, measurementprecision is not strictly a function of particle size butwill depend on regional and seasonal variations inseston concentrations. It is therefore not possible,based on the results of the current study, to pre -clude the possi bility of reduced RE of relativelylarge particles.

CONCLUSIONS

The results of the present study showed that the REsize distribution of mussels feeding on natural sestondoes not always conform to the traditionally assumedmodel. This result, which was confirmed using 3 REmethodologies and 3 types of particle suspensions,cannot be attributed to potential methodological artifacts. Both the static and flow-through methodsprovided comparable results when the inherent as -sumptions were verified. Selection of an appropriatesampling interval is critical to providing accurateresults with the static method, while the proper use ofthe flow-through method requires that feeding be -haviour and particle availability in feeding chambersis not flow dependent, as has previously been re -ported. Standardization of RE data can be problem-atic for all methods given that mathematical errorswill result from inaccurately identifying the particlesize at which RE is most efficient (REmax). A newapproach—based on the static method—is described,that provides a statistical analysis of the reliability ofall results. This time-series regression approachovercomes the problem of time dependency in previ-ous measurements and provides a more rigorousmeans of identifying REmax. Results obtained usingthis new approach show that the reliability of REmeasurements is greatly reduced when the ambientparticle concentration is low.

The previous assumption of an unchanging RE sizespectrum for each bivalve species has contributed tonumerous conclusions on bivalve trophic resourcesand food partitioning between species, as well as onthe ecological services and environmental effects ofthe biofiltration activities of bivalve populations.Rather than continuing to assume 100% RE for allparticles larger than 4 μm, as a prerequisite for meas-uring mussel CRs with the indirect method, it is firstnecessary to confirm the size range of effective reten-tion. Extrapolation of individual CRs to dense popu-lations of wild and cultured bivalves will greatlycompound any methodological error. While the focusof this study was on mussels, any suspension feederthat filters particles by means other than mechanicalsieving may exhibit similar variability in the sizedistri bution of particles retained by the feeding apparatus.

Acknowledgements. The work was carried out as part of 2projects funded by the Research Council of Norway: ‘Trac-ing phytoplankton grazed by mussels using molecular meth-ods to identify preys and improve modeling’ (TRAPH; Pro-ject No. 225213) and ‘Carrying capacity of native low-

72


trophic resources for fish feed ingredients—the potential oftunicate and mussel farming’ (CARLO; Project No. 234128).We thank Sissel Andersen, Cathinka Krogness and HenriceJansen for providing cultured algal cells and Samuel Ras-trick for support and facilities at the study site. We alsothank Evan Ward and Maria Rosa for valuable discussionsduring the preliminary design of this study.

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Editorial responsibility: Judith Grassle, New Brunswick, New Jersey, USA

Submitted: January 20, 2016; Accepted: June 30, 2016Proofs received from author(s): August 2, 2016

potential methodological influences on the determination

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