A Method for Estimating Uptake and Produdion Rates for Urea in
Seawater using
Urea and
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Dennis A. Hansell and JohnJ. Coering
bnstitute of Marine Ssiense, Sshssl of Fisheries and Ocean Sciences, University of Alaska - Fairbanks, Fairbank, Ad< 99775-9088, USA
Hansell, D. A., and 6.6. Coering. 1989. A method for estimating uptake and production rates for urea in seawater
using [j4"N]ureaand ["Clurea. Can. J. Fish. Aquat. Sci. 46: 19&202.
Improved estimates of the rates of urea production and uptake by natural populations of phytoplankton were
method is
made after determining the change in "kl-atom % enrichment of urea during incubations. A [14C]~rea
described by which the change in enrichn~entis measured. Estimates of uptake rates are increased (relative to
uptake rates determined without correction for isotope dilution) by up to 83% using a 15Naccumulation model
and by >216%%using a 15Ndisappearance model, A discrepancy exists between [15N]urearemoved from the
aqueous phase and l5N accumulated in the particulate phase at stations occupied in the northeastern Bering Sea.
The ability to find in the particulate fraction the l5N removed fron-8 solution as [lSN]ureawas improved by 72%
following removal of the >269-p+mparticulate fraction. This corresponded to only a 4% reduction in the concentration of chlorophyll a and a 37% reduction in the concentration of particulate N. Removal of microzooplankton may have improved the efficiency of urea-kl retention by phytoplankton.
Des estin-sationsarn4lior6es du taux de production et d'absorption d'uree par des p~pulationsphfloplanctsniques
naturelies ont 6t6 obtenues apr&s d4terminatisn de la variation en pourcentage de l'enrichissernent en l5N-atomique de I'uree au coun de p5r"sdes d'incubation. Une mkthsde de ["Clurke est d6crite qui prmet de mesurer
cette variation. Les estimations des taux d'absorption sont augrnentees (relativement aux taux d2bsorption determin6s sans correction pour la dilution isotopique) de 83 % au maximum grace & un rnodele d%ccumulation du
15N, et de plus de 210 % avec un rnd&lede disparition du 9 4 . II exisfe un k a r t entre la 1SN-ur4e
extraite de la
phase aqueuse et le
accuumkll dans les particules aux stations visitks dans la partie nord-est de la mer de
Bering. La eapacit6 de retracer dans des particules le "N retir6 de solution sous forme de lN-urtie a 6t$ arn4liorek
de 7% % apres lkxtraction de la fraction des particuEes sup4rieures 3 20 pm. Cela correspond 3 une rauction
de seulement 4 % dans la concentration de chlorophylle a et 3 une r6duction de 37 % dans la concentration de
particules N.t'extraction du microzooplancton a pu am4liomr I'efficacite de la retention de i'azote contenu dans
IPur6epar le phytopBancton.
Received April 25, 1988
Accepted October 5, 7 988
QJ9712)
T
he nitrogen cycle in the marine environment is still far
from being fully understood. Several models have been
developed that attempt to describe the pathways sf the
nitrogen nutrients most important to phpopl&ton (i.e. nitrate,
oniurn, and urea); however, because of me~odslogicd
problems, the models have not been horougMy tested.
Urea is an endproduct of hekrotrophic nitrogen metabolism
that can be utilized by phytopldton when excreted into quatic
environments. Previous studies of urea utilization by phyton in marine ecosystems (McCxhy 19'92; Hmey md
Caperon 19%; Estimsen 1983; Harrison et d. 1985; Rice
et d. 1985) have k e n unable to accurately report the rate of
m a - N removd from solution md accumulation into the p a ticulate fraction because of the effects sf isotope dilution sn
rate dekminations. Further, there has not been a rigorous
determination of the urea production rate (Maet d. 1985)
other thm for individual srgmisms (EppBey d d. 1973; Smith
and m i t k d g e $977), due to an inability to account for isotope
dilution of the nutrient tracer ([B5N]ma)during incubation.
'Contribution No. 707, Institute of Marine Science, University of
Alaska S.
We have developed a method combining CM4C]wea a d
["Nlwa with isotope dilution methods that provides simdtaneous estimates sf the rates of urea removal from so%ution(disappearance uptake rates), urea accumulation into the particulate
fraction (accmulation uptake rates), and heterobophic urea
production. n i s method builds upon the work sf others in the
areas sf aquatic nitrogen uptake md reminerdization (Bugdde
and Gmring 196'9; Blackburn 1979; Caperon et d. 19'99;
Glibert et d. 11982).
Numemus experiments have been pedomed to determine
nitrogen uptake rates. Following the work of Dugdale and
Goering (1967), absolute uptake rates (p) were cdculated h m
accumulation sf I5Ninto the particulate fraction:
(1) p = (atom 5% 'a5N%6,,aPN)lR*t
where atom % I5Nx, = BSN-at~m
96 in the particulate sample
adjusted to atom 96 excess by subtracting the natural abundance
sf
((8.365%), PN = particulate nitrogen content of the s m ple, R is the I5N-atom % e ~ c h m n of
t the substrate at the
beginning of the incubation, md t is the incubation duration.
The model assumed a constant
enrichment of the substrate
during the incubation.
Can. 9. Fish. &pal. Sci., Vol. 46, 6989
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Blackbum (1979) md Caperon et al. (1979) developed identied linear differential equation models (here referred to as the
BIackbum-Capron model) for the simultmeous determination
nium disappearance and production rates. The Blackron model does n d assume a consmt value for 15N
enrichment of the substrate, and thus, the initial and find values
must be measured. The production rate (D) is cdculated as
where F is the natural abundance of I5N, C is the substrate
concentration, and the subscripts 0 md t refer to initid and final
ce uptake rate (U) is cdculated as
where S is the concentration of [15N]ureain the incubation bottle
at the start of the incubation. The final enrichment of f a5N]urea
(R,) is calculated as
Accumulation uptake rates corrected for isotope dilution (PI
are calculated according to Equation 4. The urea production
rate ( D ) md disappearance uptake rate (U) me cdculated from
the Blackbum-Caperon model using Equations 2 and 3,
respectively.
At two stations in the northeastern Bering Sea, water was
collected from the depth of penetration of 50% of the incident
photosynthetically active radiation as determined with a Liwr
EI-f 85 deck unit and a EI-192S underwater sensor. The water
was prefiltered though a 333-pm Nitex screen into three 2.27L plycarbonate bottles screened with a neutral density filter
(Perforated Products, lnc., nickel screens) to simulate the in
sita light intensity. Within an hour of s m p l h g the concentratim of urea was determined by an automated diacetyl monoxime method (Whitldge et ale 119811, after which 99 atom %
[15N]urea(Icon Services, Inc.) was added to each of the samples. The final 15N-atom76 enrichment of urea in each smple
was less than 5%. In addition, approximately 27 x
~ g Dual-Eakl Model
at urea-N labeled with lT (1.054 MBq-pg-at urea-N - I; New
apron and Laws (1985) equations have not
England Nuclear Research Products) was added to each of the
been used for estimating rates of urea uptake and production
samples. After mixing, a 125-I& aliquot was removed from
because R , the IW-atom % enrichment of urea, could not be
each bottle and the remainder was incubated on deck in Plexmeasured. This problem has k e n overcome by inoculation of
iglas incubators cooled with flowing surface seawater.
samples with both [15N]urea md [lT]urea. The [lW]ma is
The 1 2 5 - d diquot was filtered though a 0.6-pm W a r n a n
used in the traditional sense to determine the "N-atom 96 excess
quartz filter. From the filtrate, triplicate 20-IT& diquots were
in the particulate fraction. Because the [l
solution is
removed for the determination of the initid concentration of
g that isoremoved at the same rate as the [l5N]we
urea (C,). To remove [14C]C0, produced during urea metabotopic ~ s c ~ ~ n a tisi negligible),
on
its activity provides a mealism, the remaining filtrate ( 5 M 0 EL)was acidified with
sure of the quantify of [B5N]urearemaining in solution. Deter0.5 mL of 1 N HCl and then bubbled with N, gas for 15 min.
mination of the fractionalchange in f 'Tlurea enrichent d ~ n g Based on experimentation with [14C]HC0,- in our laboratory,
the incubation provides a memure of the fiactiond change in
this technique removed more than 99% of the dissolved inorlSN-atom% enrichment.
ganic carbon from solution. To obtain time-zero activity (A,),
Enrichment of ['%]urea in water is calculated as
5 rnL of the degassed water was added to a scintillation vial
containing 115 d of Scinti Verse I. Counting was performed
on a B e c h a n ES-1WC liquid scintillation counter. Twentyminute counts gave a counting e m r of 1-1.596.
where E is the [lT]wea e ~ c h e n tA, is the activity of the
Samples were incubated for 6--8 h. =or to filtration, a 125counted smple, V is the vdume of the counted sample, SA is
d diquot was removed and treatd as at 'To to determine the
the specific activity of the d i o l a k l e d m a , a d C is the conconcentration of urea (C,) and the activity of ['Tlwea (A,). The
centration of urea. From the enrichment of ['Tqurea, deterremaining 2.02 E was filtered onto a grecombusted (450°C)
at times To md Tt9the fractional change (PP) in enrich0.6-pm quartz filter md rinsed with filtered seawater. The filment is cdculated as
ters were dried at 550°C for 24 h sand transported in a desiccator
to the laboratory where they were csmbusted at 800°C for 4 h
with appoximtely 1 g of Cuprox in evacuated and seded
where Eo and Et are the hitid and find enrichments.
quartz
tubes. Particulate nitrogen (PN) was determined from
The initid EHN]weaenrichment (R,) is calculated from the
pressure
of N, I n e a s ~ ~
with
d an MKS type 122A absolute
the
concentration of urea (CJ determined f i r the addition of
pressure trmsducer after breaking the sealed qumtz tubes in a
[15N]wea.Thus, Co is the sum of ambient and added urea. By
high-vacuum manifold connected to an AEI MS20 dud colleccdculating R, in this way, we have negated the effect of a change
tor mass spectrometer. Isotope ratio mdyses were also perin the concentration of urea on the enrichment that may occur
formed with the mass spectrometer. The coefficient of variation
in the incubation b t l e s
the period between the recovery
for the ratio of N, mass to transducer voltage (determination of
of the water from the collectim b t l e s and the addition of
PN)
and the standard deviation for isotope ratio mdysis (natural
nutrients. Hence:
abundance samples) were typically <0.5% and 0.00 11 atom %,
respectively.
Glibert et al. (1982) combined the concepts of the Blackbum-Caperon and Dugdale md Goering (1967) models for calculating W, uptake and production rates. Accumulation
uptake rates were cdcdated after measuring the accumulation
of [a5N]NH,+into the particulate fiaction and the changes in
"N enrichment of the NH,'. Laws (1985) presented a formula
for cdculating accumulation uptake rates (P) that requires
determination of the Blackbm-Caperon disapparance uptake
rate (U):
+
At m additional station (Station 30931, the particulate phase
was size-fractionatedto evaluate better the fate of the EBWNjurea.
Prior to incubation, water was passed though 202- or 20-pm
Nitex screens or a 3.0-prn Nucleopore polycarbnate membrane. The cMorophy!% a concentratjron of each fraction was
determined by fluorometry (Strickland and Pasons 1972) using
a Turner Designs model 10 fluorometer.
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Results and Discussion
Accumulation uptake rates (Table I), uncorrected for isotope
dilution (p), were calculated with 8, (in excess of natural abundance) using Equation 1. Estimates of uptake rates, when corrected for isotope dilution, were increased by averages of 25
and 83% (Stations 2 101 a d 2 120, respectively) with the accumulation model (Equation 4) md by averages of 265 and 2 10%
(Stations 2 1011 and 2 120, respectively) with the disappearance
m d e l (Equation 3). The large coefficient of variation (CV)
associated with replicate estimates of accumulation rates from
Station 2 120 (41%) was caused by an anomalously low PN and
atom % excess in replicate 2. Replicates 1 and 3 are in very
g o d agreement, with a CV of <O. 1%. Station 2 101 exhibited
a CV of 8% for accumulation uptake rates. Coefficients of variation for urea production rates were 15 md 40% for Stations
2 120 and 2 181, respectively.
The "5N data was evaluated for mass balance using "4C activity in the aqueous phase as an indication of [lSNjurearemaining
in solution. The "N that had disappeared from the dissolved
['SN]weapool was consistently greater than the amount of 19N
that had accumulated in the particulate fraction (Table 2). On
average, 58% (Station 2101) and 37% (Station 2120) of
[lSNfurearemoved from solution could not be accounted for in
the particulate phase. The missing
may have accumulated
as "N-labeled dissolved organic nitrogen (DON) or NH4+. Possible avenues include phytopldton leakage md
losses, such as ~ooplariktonexcretion, leakage, and loss of soluble plant material at the mouth pats.
Observations of dissolved inorganic nitrogen disappamce
in excess of PN accumulation are not new. Dugdale md Wikemon (1986) found t a t 66% of the disappearnee of N&+ md
76% sf the disappearance of NO, - at the 50% light penetration
depth off Peru was accounted for by lSN accumulation in tihe
particulate fraction. Price et d.(11985) reparted that the change
in concentratispa sf dissolved inorganic nitrogen md urea was
consistently greater &an the change in PN in frontal waters off
British Columbia.
To further evaluate the apparent loss of lSNfrom the paticulate phase, particulate material was size-fractionated and, following incubation, the resulting "'N distribution evaluated
(Table 2). Although the <20-pm fraction had only a 4% reduction in chlorophyll a concentration relative to the <200-ym
fraction, there was a 72% improvement in our abiiity to account
for "5N removed from solution as [l5Nurea. A 33% reduction
in the concentration of chlorophyll s (<3-pm fraction) provided nas further improvement in the recovery of "N. These
results point to the importance of the >20-pm fraction in
effecting the loss of "N. Perhaps large-celled phytsplmkton
(>20 pm) are especially prone to leak DON a d NH4+, or
Ilraicrozoopl&ton (>20 pm) play a significant role in causing
a gazer-induced loss of nitrogen from the plant cells.
Filtration though the 20-pm screen reduced the coneentration of PN by 37%. Much of the PN removed must be dehtd;
however, a portion also must be composed of m i c r s z q I d t o n
Can. 9. Fish. Aquae. Sci., Vo&.46, 1989
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TABLE2. Fraction of [15N]urea removed from the dissolved phase which is unaccounted for in the
particulate phase (m).Stations 2101 md 2120 are replicated at one depth, while Station 3093 is s i x fractionated (<2W, <20, and <3 pm). So md S, = initid a d find concentrations (pg-at N-L-l) of
[15N]urea;St is estimated fmm the fractional reduction in [lT]wea activity. hitid (Ao) md find (A3
[lT]nrea activities are expressed in Bq, [15N],, is the ratio of 15N to total N in the partisdate phase
(minus O.W367), PN is the particulate nitrogen concen$ration(pg-at N-%-I),Ch1 is the ch1orophylH a
concentration (y~goL-I)md FR is cdculatd as [(S,- St) - (fiNk-PN)].[(So-SJ] - l .
Replicate
$
(A,-A,).Ao-B
S,
Station 2101
1
2
3
0.009
0.009
0.009
0.515
0.552
0.506
0.
0.0040
0.
md the removal of these grazers improved the nitrogen retend the nmotion efficiency of the phytoplankton. The ~ m o v of
plankton fraction (3-20 pm) provided no further improvement
in recovery of I5N, suggesting hat the mechanism controlling
the retention of urea-N in the <20-pm size class is co
b u g h o u t that size class. Possible mechanisms for the apparent loss of 15N from the <20-pm fraction include grazerinduced and self-induced leakage of 15Nas DON md NE&+ by
the plant cells, analytical errors, and fractionation of [14C]urea
and [15N]ureaduring uptake (thus providing m e m r in our estimates of the remaining p o l of dissolved [lSN]wea).
We are confident in ow mdyticd ability to precisely measure the lSNatom 5% excess sf the PN, the nitrogen content of
activity of the aqueous phase. These measthe PN, and the
urements, and a howkdge of the hitid concentration of
[a5N]urea,provide the only vdues required for this mass bda c e analysis. Whereas some fractionation of ['Tlurea md
[BSN]ureaduring uptake must exist, we doubt that it could be
responsible for the noteworthy lack of accountability of 15N.
This argument is supported by noting the improved accountability of 15N in the <20-pm fraction, which still contained
96% of h e concentration of chlorophyll a, over that of the
<2W-pm fraction.
Plant- and grazer-induced leakage is, then, the most likely
cause for loss of
in the <28-pm size class. Plant-induced
leakage must account for the bdk of the 1 1-1296 of 15Nremoved
from the urea p o l ha could not be accounted for in the <20md <3-pm particulate phases (Table 2) because there was no
improvement in 15Nrecovery in the <3-pm size class relative
to the <28-pm fraction, while there must have been a reduction in the grazing stress following this fractionation.
In the <2W-g%rnsamples, 40% of the ISN removed from
solution as urea could not be accounted for in the particulate
p o l . If we extend the approximate 10% loss due to plant leakage in the <20-pm &action to h e entire <200-pm plant population, the remaining 30% loss e m be attributed to grazing
stress. This implies that most of the assimilated urea-N is going
into a persistent soluble pool, which only slowly goes to a garticulate fom. The soluble pool is readily lost during grazing.
Can. J. Fish. @at. Sci.,Vob. 46, 1989
WX,, P
0.W93
0.W100
0.00097
2.119
1 . 9
2.051
CM
15N,;PN
0.0519'7
0 0 5 1
0.00199
0.57
0.61
0.57
-
Glibert et ale (1985), in discussing the implications of missing 15N,suggested that it is important to determine whether the
uptake of nitrogen by phytoplAton is reported more xeurately by the disappearance of 15Nfrom the dissolved phase s r
by h e accumulation of 15Nin the patieralate phase. n i s is a
distinction between gross md net nitrogen uptake by the phytoplankton if we c m attribute most of the urea removal to plant
cells. Plants pmbably play the most significant role in urea consumption, since bacterial utilization of urea is not thought to
occur at significant rates in oceanic water (Weeler and Kirchman 1986) and it is unlikely that abiotic pathways for loss of
urea are important. As seen here, the difference between the
disappearance md accumulation uptake rates c m provide
insight ts the relative importance of plant- and grazer-induced
loss of nitrogen.
This method is, in fact, subject to underestimates of the
mount of I5Nmissing from the combined posl of PN and m a N. ['TIDOC may be released into the water, causing an overestimate of the mount of [15N]ureain solution. lf this process
is important for the autotrophs in the <20-pm fraction, the
apparent improvement in accountabiliq of '94 would be misleading. In other high-latitude waters, <f 0% of the urea-C
taken up by the plants was incorporated into particulate matter,
with the rest disposed sf as i1T]C8, (Harrison et al. 1985).
During saaa incubdions, appmximately 50% of the [ B T ] m a
was removed from solution. If we assume that 18% of this carbon is incorporated into the organic fraction and that 58% of
the assimilated cabon is lost as DOC, then 2.5% of the "C
initially in solution would be returned as [MC]BOC. The
[14C]D0C returned would cause a 4% underestimate of the
mount of lSNmissing from the p o l of PN and dissolved urea.
Using ow data from Station 2101 (replicate 1) and assuming
that 2.5% of the urea-C removed from solution is reintroduced
as iBT]DOC,we calculate that the accumulation md disappearance uptake rates of urea-N would be underestimated by
2.7 md 5 5 % md t h a the production rate of urea-N would be
underestimated by 6.3% .
Calculating urea uptake rates based on rates of disappearance
provides values that average 140% greater than those calculated
for rates of aa=eumulationfor northeastern Bering Sea data. Gli20 1
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k r t et d. (1982) presented data (their tables 2 and 3) where
onium disappmmce uptake rates exceeded accumulation
uptake rates by an average of 197%. The ratio sf the rate of
m a production to the rate of urea uptake is dso greatly affected
by the model used for calculation of uptake rates. For Station
2%20, the production to uptake ratios are 1.8 t.ind 1.I , respectively, for the accmulation a d & s a p p a a c e models.
Our results show that the in situ production sf urea is approximately q u d to the consumption of urea (i.e. dynamic steady
state) md that a portion of the urea-N that is consumed must
F
x r e ~ n e r d i z e dquickly. Our data suggest that to describe more
completely the cycling of ma-N, models must include a dissolved organic pool, as has k e n recognized for NH, (E&oche
a d H&ssn 1987).
Mor studies of the uptake of urea by phytopldton have not
accounted for isotope dilution during incubation. Studies sf urea
regeneration have been limited in the same way a d have
depnded on mass balances md d e t e ~ n a t i o n sof uptake rates
uncorrected for isotope dilution. Our method provides improved
md direct estimates of in situ urea production a d uptake rates.
Trace addition of labeled substrate is achievable with this
method, md as a conquence, changes in subswate concenmtion and fluxes we not significt.int. The method is pedomed
with relative ease in h s h or m e water and requires minimal
effort beyond that needed for csnventiond uptake experiments
which do not account for isotope dilation.
+
n i s research was hnded by the Division of P01a h o g a n s of the
National Sdence Foundation under Grant No. DPP8N5286. We thank
S. W d e n , S. Hemichs, E. Brown, md R. Day for reviewing the
gg%m~scp.ipe.
B. Hmma, aipn,E. LAWS.1979.
ay, Hawaii, measwed by a 15N
C
DUGBALE,R. C., m J. J.
foms of nitrogen in p
ium excretion
dilution tech-
Uptake of new md regenerated
ity. L i m l . Bceaogr. 1%: 196-
206.
DUGDALE,R. C., AND E WLERS~N.1986. m e US2 of "N to I I X 8 8 U E
nitrogen uptake in eutrophic m a n s ; experimentalconsi&HEations. Li~aanol.
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BPPEY, R. W.,E. N. FIG^, El. L. V E ~ I C Ka , ~ M.
a M.
~ MULL^. 1973. A
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, M.,E b ~ s e ~ aJ.nJ. ,M ~ ~ T H r' Y
n M.
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marine plankton. L h o l . &eanog. 27: 639-650.
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imewts b n resolved? L h d , Bcemogr. 30:
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rates of N&+ turnover in
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