ASSIMILATION OF ORGANIC MATTER

BY ZOOPLANKTON'

Robert J. Conover
Woods Hole OceanographicInstitution,
Woods Hole, Massachusetts02543

ABSTRACT
A methodfor directmeasurementof percentageof assimilationof organicmatterby
zooplanktonthat does not requirethe quantitativerecoveryof feces and can be adapted
foruse in the fieldas well as in laboratory
investigations
is described.If it is assumedthat
onlythe organicfractionof the food ingestedby a zooplanktonanimal is affectedby the
digestiveprocess,it can be shownthatpercentageof utilizationis definedas
U'l=f ( E')( ))]X 100,
whereF' is the ash-freedryweight: dryweightratio (fractionof organicmatter)in the
ingestedfood,and E' is the same ratioin a representative
sampleof feces.
The methodcomparesfavorably,as a measureof assimilation, with directmeasurement
of the quantityof organicmatterlostfromfood duringpassage throughthe digestivetract.

INTRODUCTION of zooplanktonfeces,a process that can be

Digestionand assimilationare vitalto the difficulteven in laboratoryexperimentsbe-
transferof energy from one trophic level cause pellets are frequentlytorn open or
to the next. These processes also influence reingested by actively feeding animals.
the distributionand compositionof particu- Particles of foreign matter and "pseudo-
late organic matter at all levels in the feces," formed from uningested organic
oceans. Little is known about the forma- matterconcentratedby feedingmovements
tion of particulatedetritusor of its impor- of the copepod's appendages, are difficult
tance in sustainingthe animals contributing to separate fromtrue feces.
to its formation.A methodfordetermining The method described in this paper re-
the fractionof energyremoved by the di- quires neitherthe quantitativerecoveryof
gestiveprocesseach timeparticulatematter feces nor knowledgeof the amountof food
passes throughan animal is needed. eaten if only the percentage assimilatedis
Previous estimates of percentage of as- required; hence, the method can be used
similationby zooplanktonrange from6 to in the field almost as readily as in the
99% (Conover 1964). Different species laboratory.
feeding on differentfoods under different The authorwould like to thankR. R. L.
experimentalconditionsno doubt account Guillard who supplied the algal cultures
forsome of the variability,but the methods and R. F. Vaccaro who made bacterial
used to measure assimilationmay not al- counts on the fecal pellets. Technical as-
ways give comparableresults. The quantity sistancewas providedby Thomas Renshaw,
of a radioisotopeaccumulated in the body JohnWoodcock, Robin Walden, and Bar-
of an animal fed labeled food is not a bara Steinberg.
measure of assimilation unless it is cor- METHODS
rected for losses due to recycling. On the
The method depends on the assumption
other hand, the directmeasurementof as-
that only the organic component of the
similationrequiresthe quantitativerecovery
food is significantlyaffectedby the diges-
tive process. If this assumptionis correct,
1ContributionNo. 1719 fromthe Woods Hole it is necessary only to obtain the ratio of
OceanographicInstitution.This researchwas sup-
ported by National Science Foundation Grants ash-freedryweightto dryweight (fraction
G-23134 and GB-3120. of organicmatter) fora sample of food and
338
 ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 339
a sample of feces to calculate percentage Food samples for dry weight determina-
of assimilation,U', using the equation tion were filtered onto tared, glass-fiber
filters. Samples and seawater blanks were
u~ [(1Et)(F'J x 100, (1) washed with isotonic ammonium formate
to remove adventitioussalts and dried at
where F' and E' are the requiredratiosfor 60-70C for at least 12 hr. Blank filters
the food and excreta, respectively. The generallyshowed a small weightloss or no
derivation of this equation is as follows. change; as there was no evidence of
Food assimilatedis definedas that amount nitrogencontamination,complete sublima-
of the food ingested which is taken in tion of the ammoniumformatemust have
throughmembranesof the digestiveorgans occurred.Filtersand sampleswere weighed
of an animal. Then, percentageof assimila- on a quartz helix microbalance (Micro-
tion, U', may be definedas chemical Specialties Co., Berkeley,Califor-
nia) or on a Mettlersingle pan microgram
U'=(I/ )xlOO, (2) balance. Weightof sample was determined
by differencefromthe filtertare,aftercor-
where I is the quantityof organic matter rectingforany weightchange in the blank.
ingested,and N is the quantityexcretedin Ash-freedry weight was sometimesde-
the feces. Thus, I is equal to the total par- termined by difference from a second
ticulate matteringested (F) minus its ash weighing after ignitingfiltersin a muffle
(Af) and N equal to the total particulate furnaceat 450C, but an alternativemethod
matterexcreted(E) minusits ash (Ae), so gave more reproducibleresults. When suf-
thatequation (2) can be rewritten ficientfood materialwas available, a thick
U'= [(F At) (E e)_ x 100. (3) pad was firstdeposited on a Millipore'
filterof suitable porosityand washed with
However, Af is assumed to be equal to Ae; ammonium formate. A portion was then
hence, (3) becomes carefullyscraped fromthe filterwitha dull
spatula,dried,weighed,ashed, and weighed
U' = (F-A-)x 100l . (4) again.
Fecal pellets were collected in several
F' and E', the ash-free dry weight: dry ways, dependingon the type of experiment
weightratios foringestedfood and forthe and food source. If feeding was heavy,
feces,can now be written
sufficient materialcould be picked up with
F' = F ' a fine-bore squeeze bulb pipette in 5 or 10
(5)
min. When feces were scarce, they were
and separatedfromthe food cultureby filtration
El = onto a suitable bolting cloth (usually No.
E (6)
25 Nytex). Sometimes pellets were col-
Since Af=Ae, equations (5) and (6) can lected in the field with a suspended trap
be rearrangedso that made froman 8-inch(20 cm) plasticfunnel
fittedwith a shell vial in which the sample
F- Af (7) accumulated. Feces thus collected were
(1- F')'
and comparedwith a surfacesample of particu-
late mattertaken at the timethe trapswere
E (1-E) (8) set out. Alternatively,zooplankton were
permittedto feed on natural particulate
Substitutingthesevalues forF and E, equa- matterand feces were collected by pipette
tion (4) reduces to or filtrationas describedabove. Field sam-
ples were frozenand returnedto the labora-
(I, -FE')( P) toryfor analysis.
340 ROBERT J. CONOVEA

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 ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 341

Once a sample was collected, it was ex- introducea significantchange in the weight
amined under the dissectingmicroscopein of the ash during the relativelybrief pas-
a Syracuse staining dish and any foreign sage throughthe gut of a planktonicherbi-
materialremoved. If care is taken to keep vore.
experimentalglassware free fromdust and To see if copepods mightcause the dis-
otherparticulatecontaminants,this task is solution of silicate from diatom shells
greatly simplified. The sample was then crushed in feeding,three sets of Calanus
swirled gently,causing the pellets to ac- hyperboreuswere each placed in a richcul-
cumulate near the centerof the dish. Ex- ture of Thalassiosirafluviatiliscontaininga
cess waterwas siphonedoffleaving a paste knownamountof dissolvedsilicate,and an
of pellets or, alternatively,large pellets additionalthreesets were placed in filtered
were collected into a bolus and directlyre- seawater of known silicate content. Ap-
moved fromthe water. In eithercase, pel- propriatecontrolswithoutanimals were in-
letswere transferred as a lump to the folded cluded. At the end of 46 hr in the dark,
edge of several thicknessesof hard filter therewas no differencein the dissolvedsili-
paper (S and S sharkskin)and were washed cate level (as determinedby the methodof
by dropping ammoniumformateon them Mullin and Riley 1955) in the containers
with a capillary tube; each drop was al- with animals and those without,whether
lowed to soak intothe paper beforeadding diatoms were presentor not. Because nu-
the next. Finally the bolus was transferred merous fecal pellets were present in the
to a small tared platinumpan for drying, containerswithboth copepods and T. fluvi-
weighing,and ashing. As littleas 20 ,ugof atilis, the animals must have been feeding
pellets could be used to obtain the neces- actively.
saryratio with an errorof usually less than On several occasionsattemptswere made
15% in the final calculationof assimilation. to recover fecal material quantitativelyto
In laboratory experiments,Stage V or compare a direct measurementof organic
female Calanus hyperboreus,either from matter assimilated with the ratio method.
the Gulf of Maine or nearby slope water, If the fecal pellets were compact,with lit-
were used as experimentalanimals. Food tle damage due to recapture,and if there
organisms were chiefly bacteria-freecul- was no tendencyfor clumps of uningested
tures of marine diatoms and flagellates particulatematterto be formed,agreement
fromthe Woods Hole Oceanographic In- betweenthemethodswas good withdiatom
stitutioncollection or natural phytoplank- food (T.
fluviatilis). An exampleof another
ton assemblages. The experimentswere run
experimentwith C. hyperboreususing the
from12 to 24 hr in 1-gallon(4 liter), poly-
dinoflagellateExuviella sp. as a food source
ethylenecontainerswith small populations
of animals (20-35) in 2 liters of culture is shown in Table 1. The results of this
or in pint (0.5 liter) containerswith one experiment are particularlysignificantbe-
animal to about 200 ml of medium. cause they suggest that the ratio method
may give a reasonable estimateof percent-
EVALUATION OF THE METHOD age of assimilationeven when the food or-
ganismdoes not containa large fractionof
A major fractionof the food of herbivor-
ous copepods such as Calanus finmarchicus relativelyinertsilica.
consistsof diatoms (Marshall 1924; Lebour BACTERIA IN THE FECES
1922). Diatoms have a high ash content,
chiefly silica (hydrated SiO2) (Parsons, The feces of higher animals are known
Stephens,and Strickland1961). Although to contain many bacteria that play an im-
Lewin (1961) showed that diatom shells portantrole in the digestiveprocess. Noth-
dissolved fasterat high temperatures,high ing is known regardingthe intestinalflora
pH, and aftertreatment withacid or chelat- of the zooplankton,but large numbers of
ing agents,dissolutionwould probably not bacteria could contributesignificantlyto
342 ROBERT J. CONOVER

TABLE 2. Numberof bacteriain fecalpelletsproducedby C. hyperboreus

Stage V feedingon different
foods

No. No.
Date Temp. of No. bacteria/ bacteria/mg
(1963) Food incubation bacteria/ mg dry wt organcw
(C) fecal pellet of pellet ani wt-
>K10-6 1-
26 Sept Thalassiosirafluviatilis ca. 25 150
ca. 25 400
9 Oct T. fluviatilis ca. 25 1,200
16 Oct T. fluviatilis ca. 25 2,700 4.4 13.5
Dunaliellasp. ca. 25 4,520 6.4 10.5
24 Oct T. fluviatilis ca. 25 1,800 2.3 6.45
Skeletonemacostatum ca. 25 1,500 2.5 6.25
Coscinodiscussp. ca. 25 625 0.9 1.82
T. fluviatilis 4 1,200 1.5 4.3
S. costatum 4 300 0.5 1.25
Coscinodiscus sp. 4 375 0.53 1.1
31 Oct T. fluviatilis ca. 25 250 0.5 1.8
T. fluviatilis
(after3 days
at roomtemperature) ca. 25 3,440
S. costatum ca. 25 1,480 2.4 5.7
S. costatum(after3 days
at roomtemperature) ca. 25 1,590
Coscinodiscus sp. ca. 25 730 0.65 2.35
Coscinodiscussp. (after3
days at roomtempera-
ture) ca. 25 11,000
7 Nov T. fluviatilis ca. 25 580 1.35 4.45
21 Nov T. fluviatilis ca. 25 580 0.88 2.23
T. fluviatilis ca. 25 370 0.56 1.42
T. fluviatilis ca. 25 510 0.775 1.95

the quantityof organicmatterin the feces umeof 1 /3, thehighestconcentrations

or break down some of the unassimilated
organic matter after defecation. Newell
(1965) has shownthatfreshfecesproduced
by the deposit-feedingmolluscs Hydrobia
ulvae and Macoma balthica were relatively
high in organiccarbon and low in nitrogen,
but afterseveral days of incubationthe ni-
trogencontentin the pellets rose and the
carbon level fell slightly.Apparentlybac-
teria were using fecal carbon as an energy
source and utilizing some source of dis-
solved nitrogento formtheirown body sub-
stance.
To obtain an approximate quantitative
estimateof bacterial abundance, C. hyper-
boreus feces were ground,suitablydiluted,
and plated on seawater agar enrichedwith
beef extractand bacto-peptone.The counts
are summarizedin Table 2. Assumingeach
bacteriumhas a densityof one and a vol-
ob-
served,2,700 bacteria per pellet forThalas-
and 4,520 forDunaliella
siosirafluviatilis
(16 October 1963), would represent as
much as (13.5 x 106/109)x 100 = 1.35and
1.05%,respectively, of the totalorganicmat-
ter presentin the feces only if the bacteria
contained no water or ash. It is doubtful
thatall bacteriain thefeceswere favoredby
the medium used, but even if counts were
low by an order of magnitude, bacteria
would seldom have contributedmore than
a few per cent to the total organic content
of the feces. When pellets were incubated
at 4C (the temperatureat which most of
the assimilationexperimentswere carried
out) the total countwas lower than at 25C,
and the incubationperiodhad to be at least
doubled beforecolonies were large enough
to count. When assimilationexperiments
are run at low temperaturesfor 24 hr or
 ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 343

TABLE 3. Assimilation
of naturalorganicmatter

Cruise Date LocationDet Animals Depth %

(N lat) (W long) (in) assimilation

Gosnold 40 20 Mar 64 42050' 69050' Meganyctiphanes surface 65.0

norvegica
Crawford106 8-9 Apr 64 42?50' 69050' mixedzooplankton 10 81.2
Crawford 106 13-14 Apr 64 41055.2' 65014.2' mixedzooplankton surface 32.5
Crawford106 13-14 Apr 64 420261 67?10' mixedzooplankton 100 72.8
Crawford 106 15-16 Apr 64 43024' 67048' mixedzooplankton surface 45.9
Crawford 106 15-16 Apr 64 43024' 67048' mixedzooplankton surface 3(6.7
Crawford 106 15-16 Apr 64 43024' 67048' mixedzooplankton surface 65.2
Crawford 106 17-18 Apr 64 420 12' 69039' mixedzooplankton 10 72.6
Crawford106 17-18 Apr 64 42012' 69039' mixedzooplankton 50 79.6
Crawford 106 17-18 Apr 64 42012' 69039' mixedzooplankton 100 87.1
Gosnold 44 8-9 July 64 42050' 69050' mixedzooplankton surface 75.7
Gosnold 44 8-9 July 64 42050' 69?50' mixedzooplankton surface 79.5
Gosnold 44 8-9 July 64 42050' 69050' mixedzooplankton 10 74.3
Gosnold 44 8-9 July 64 42o5OQ 69050' mixedzooplankton 50 72.7
Gosnold 44 8-9 July 64 42050' 69050' mixedzooplankton 100 88.7
Gosnold 44 10-11 May 64 41055' 69014' mixedzooplankton surface 38.6
Gosnold 44 10-11 May 64 41o55' 69?14' mixedzooplankton 50 78.1
Gosnold 44 10-11 May 64 41o55' 69014' mixedzooplankton 100 92.1
Gosnold68 14-15 May 65 42011.3' 69039' mixedzooplankton surface 54.1
Gosnold 68 13-15 May 65 39050.3' 69033.3' mixedzooplankton surface 40.7
Gosnold 68 13-15 May 65 39050.3' 69033.3' C. hyperboreus surface 43.8
Gosnold 71 10 May 65 39046' 69044.5' Euchirella rostrata surface 17.7
Gosnold71 11-12 May 65 42015.6' 69041.0' mixedzooplankton surface 80.6
Gosnold 71 12 May 65 420?17.6E' 69036.7' mixedzooplankton surface 40.0
Gosnold 71 12 May 65 42017.6' 69?36.7' mixedzooplankton surface 58.2
Gosnold 71 12 May 65 420 16.5' 69042' C. hyperboreus surface 69.5

less, bacteria in the feces probably have tion.On severaloccasions,feceswere found

little quantitativeeffecton the estimation to contain a smaller ash fractionthan the
of percentageof assimilation. particulatematteroccurringin the water;
but away fromthe coast where the inor-
ASSIMILATION OF ORGANIC MATTER
ganic particulatefractionmightbe affected
IN NATURE by material of terrestrialorigin,field esti-
Assuming that the ash content of nat- mates of assimilation were usually com-
ural particulate organic matter is unaf- parable to laboratorymeasurements(Table
fected by the digestive process, it should 3, cf. Table 1).
be possible to determinethe percentageof When traps were used to collect feces
assimilationfroma natural population by from differentdepths, the ash contentin
the zooplankton if representativesamples pellets from deeper water was always
of uneaten organic matterand feces pro- higher,indicatinga larger percentage as-
duced fromit are available. Corner (1961) similated there than in the surface water
presentedevidence thatCalanus helgoland- (Table 3). The values derived fromfeces
icus preferentially selectedthe organicfrac- captured at 50 or 100 m should be consid-
tionof the particulatematterin naturalsea- ered maximumestimates,because some par-
wateroffPlymouth,England. If particulate ticulatematterat these depthsprobablyhas
matterwas selected on the basis of its or- already passed throughthe intestineof an-
ganic content,a serious errorwould be in- otheranimal or undergonepartial degrada-
troduced into the calculation of assimila- tion by bacteriabeforeingestionand so has
344 ROBERT J. CONOVER

a higherash contentthan the surfacesam- laboratorythe ratio method gave reason-

ple used for the calculations. ably consistentestimatesof percentage of
assimilationof organicmatterby zooplank-
DISCUSSION ton,whetherthe food consistedof the dia-
To determineassimilationaccuratelyby tom T. fluviatilis,with high silica ash con-
the ratio method described in this paper, tent, or the dinoflagellateExuviella, with
organic matter and ash must be ingested a relativelylow ash, and results obtained
by the zooplanktonin the same proportions fromnatural populations agreed well with
as they occur in the naturalfood. Alterna- those from controlled laboratory experi-
tive methods would also yield inaccurate ments.
resultsif organicmaterialwas lost fromthe Whatever the errorin the ratio method,
particulatefractionwithoutbeing ingested; the resultsobtained also comparefavorably
othermethodsgenerallyalso require quan- with some earlierestimatesfromthe litera-
titative recovery of feces. Beklemishev ture (see Table 1, Conover 1964). Because
(1954) and Cushing (1955) observedcope- only organicmatteractually removedfrom
pods ingestingthe cell contentsbut not the feces (ratherthan the fractionappear-
the tests of large diatoms. Corner (1961) ing in the tissues of the animals) is con-
found that C. helgolandicus removed a sidered, there is no confusion using the
higher percentage of particulate organic ratio method whether assimilation or
than of inorganicmatterfromnatural sea- growthefficiencyis being measured (as in
water taken at the entrance to Plymouth many experiments involving radioactive
Sound, England. Because the samplinglo- tracers). The quantitativerecoveryof feces
cation was near land, a significantportion (a laborious process and subject to error)
of the uningested inorganic matter could is usuallynecessaryto measure assimilation.
have been terrigenous.On the otherhand, S. M. Haq (personal communication)has
C. hyperboreuscompletelyingestedthe test recentlyshown that the copepod Metridia
of a large Coscinodiscus but probably lost longa does not consistentlyformfecal pel-
some of the cell sap during the feeding letsbut defecatesa portionof itsundigested
process (Conover 1966). About 15% of the food in small, unrecognizable fragments.
carbonremovedfroma cultureof T. fluvia- When large quantities of feces are to be
tilis by the feeding of C. hyperboreuswas recovered by filtrationor centrifugation,
returnedto the medium as dissolved or- care must be taken to keep food cultures
ganic matter, presumably some through uncontaminatedby dust,lint,and otherfine
particulate matter of atmospheric origin
leakage from cells damaged during the
thatquicklyaccumulateson glassware,and
feedingprocess and the remainderthrough
so on. Cotton plugs, used to keep algal
excretionby the animals (Hellebust and cultures
free frombacterial contamination,
Conover, unpublisheddata). contributeconsiderablyto particulatecon-
At present,the effectof selective inges- tamination. Although precautions should
tion of organic matter or the leakage of always be takento limitthe introductionof
organic matterfrom crushed cells on the foreigncontaminants,an adequate sample
determinationof assimilationby the ratio for determinationof assimilation by the
methodcannotbe assessed. If zooplankton ratio method can be recoveredby sorting
ingesta higherproportionof organic mat- under a microscopeeven if a mistake has
ter than inorganic in their food, as sug- been made. The measurementof the as-
gested by Corner,the ratio method would similationof individualcompoundsor com-
give a deceptivelylow estimateof assimila- ponentsof organicmattersuch as nitrogen
tion, while if the reverse were true, as or phosphorusis also facilitatedby relating
suggested by the preliminaryexperiments them to the ash fraction.The method has
of Hellebust and Conover, assimilation the added advantage of simplicityand low
would appear too high. However, in the equipmentcost.
 ASSIMILATION OF ORGANIC MATTER BY ZOOPLANKTON 345

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